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Name the collective structure where Botox exerts its action and identity specific components (minimum of four) within the structure.

Botox affects the events that occur in the Neuromuscular junction which is the site for the communication of an action potential. This structure contains a synapse region where communication is initiated between neurons and target cells. The synaptic cleft in the synapse region divides the two cells apart where chemical messengers such as neurotransmitters can communicate across. The Synaptic end bulbs are the neural part of the region containing hundreds of synaptic vesicles (Tortora et al., 2019).

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Outline the role that calcium plays in the initiation of a muscle action potential.

Calcium ions are the first to realise at the onset of a contraction, they then bind to troponin in the sarcoplasm reticulum by Ca2+ active transport pumps which moves to tropomyosin. Calcium ions move into the cytosol and contraction is initiated. Increasing and decreasing amounts of Ca2+ level in the sarcoplasm starts and stops the sliding of thin filaments. Calcium ultimately governs the role of a muscle action potential (Tortora et al., 2019).

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How does Botulinum Neurotoxin A prevent the initiation of a muscle action potential at the structure identified in Q. 1? (3 marks) (75 words)

Botox affects the Neuromuscular junction which is the site for the communication of an action potential by preventing the initiation of a muscle action potential by blocking exocytosis of synaptic vesicles in the neuromuscular junction. As a result, Botox binds and blocks acetylcholine receptors so that it can’t be realised, and ion channels do not open resulting to muscle paralysis (Tortora et al., 2019).

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Outline the role that sodium plays in the production of a muscle action potential.

When a nerve impulse induces a muscle action potential, it does so in 4 steps; it is step 3 which generates this potential. During this stage, sodium is released down its electrochemical gradient, generating a positively charged environment within the muscle fibre. This prompts the muscle action potential, subsequently triggering the action potential to travel down the sarcolemma and into the transverse tubules. The sarcoplasmic reticulum then releases its stored calcium into the sarcoplasm, causing the muscle fibre to contract (Tortora et al., 2019).

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Botulinum Neurotoxin A can also be used for dystonic conditions. Define muscle tone and outline the mechanism by which muscles maintain it. Identify which tonic disorder can be treated by Botox.

Muscle tone can be defined as prolonged tension within the muscle due to reflexive contractions of its motor units. Muscles are able to maintain their tone as a result of motor units continuously shifting from a state of active to inactive, ensuring that muscles remain firm, whilst simultaneously inhibiting force which will produce a significant amount of movement. A tonic disorder which can be treated by botox is Hypertonia (Tortora et al., 2019).

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Describe the process of calcification.

Calcification (mineralisation), is when calcium phosphate, calcium hydroxide and other mineral salts combine to form crystals, known as hydroxyapatite within bone tissue. They are then deposited into the structural framework of the extracellular matrix, where they solidify. For this process to occur, the presence of collagen fibres are required. The salts crystallise within the spaces of collagen fibres and when these spaces become full, the crystals then assemble themselves amongst these collagen fibres (Tortora et al., 2019).

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Choose one principal effect of ageing on Francine’s bone tissue which may have contributed to her fracture. Describe the effect and identify a possible consequence /disease that may occur as a result.

Deterioration of bone mineral density is a principle effect of ageing. As this usually leads to bone fragility and weakness, it most likely contributed to Francine’s fracture. A possible consequence that may occur as a result of decreased bone density, is osteoporosis. This is particularly true for post-menopausal women (Tortora et al., 2019).

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Healing a fracture can sometimes take months. Provide a detailed rationale as to why this occurs.

Bones generally have a substantial blood supply, but healing can take months to repair as minerals such as calcium and phosphorus are distributed gradually through the process. Also, bone cells grow and reproduce slowly as well resulting in a gradual healing process (Tortora et al., 2019).

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Francine underwent a bone mineral density [BMD] test. She was diagnosed with osteoporosis. Outline three (3) factors that may contribute to porous bones.

Ageing contributes to porous bones. The principle effect of ageing is demineralisation, a loss of calcium from bones and decreased production of the ECM proteins (mostly collagen fibres), which make bones brittle and thus more susceptible to fracture.

Gender is another contributing factor. For women, bone loss increases quickly after menopause when oestrogen levels decrease. Oestrogen perseveres bone; therefore, a fall in oestrogen levels typically results in bone loss.

Other risk factors for developing osteoporosis include an inactive lifestyle (Tortora et al., 2019).

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Diet plays an important role in the ongoing management of Francine’s condition. Choose three (3) nutrients that impact the skeletal system and describe how they contribute to bone metabolism and repair.

Many vitamins and minerals play a vital role in bone regeneration. Vitamin A is required for the action of osteoblasts during the remodelling  phase of the bone repair. Another valuable nutrient is vitamin C. Vitamin C is used for collagen synthesis which is the main bone protein. Vitamin D also helps build and repair bone by increasing calcium absorption. An inadequate level of Vitamin D contributes to faulty calcification and therefore slows down bone growth (Tortora et al., 2019).

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Guidelines for online discussion (Lancaster 2017)

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Guidelines for online discussion (Lancaster 2017)

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It is required that all group members comply with with the following netiquette rules. Please agree to the following guidelines by commenting 'I agree' below.

*Permission to comment on answers if one has a different suggestion.

*Permission to edit wording and word count on particular answers.

*Contact partner before submitting assignment to ensure both parties are happy with the final result.

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Here are 14 great tips for student netiquette. The ideas come from university and college students.

We ran a short-essay competition to find the best ways of showing excellent online etiquette. The top answers were used to form a set of rules or guidelines for internet manners.

The word netiquette is a combination of 'net' (from internet) and 'etiquette'. It means respecting other users' views and displaying courtesy when posting your views to online discussion groups.

1. Be accurate with factual information

Misleading others is obviously a major breach of online etiquette. This is true even if it's unintentional.

Check facts before providing information or giving advice. Provide sources. If you're not an expert on a topic, maybe you shouldn't be acting like you are.

Providing inaccurate information degrades the quality of discussion. It can also have serious real-world consequences if it's accepted and acted upon in some way. Everyone needs to take care when informing others.

I encountered top rated answers such as "international students get basically zero financial aid from American institutions", which was obviously not true, as I later understood when I received my scholarship.

~ Jamal Amir

2. Extend your good nature online

The digital world is an increasingly important part of our lives. We should be our best selves there too. The manners our parents taught us apply everywhere.

Do you think you're generally a good person? Are you pleasant, polite and well-mannered? Most people would answer "Yes" or "Most of the time". If someone says "Hello", you say "Hi" back. If a stranger asks for directions, you try and tell them the best way to go.

But what about online? Are you still that same considerate, nice person when you're in an online forum (including social media)?

Think back to when you learned how to be a good human being. When you donated your hard-earned money, cut grass for your older neighbor, or sent a card to a friend who was having a bad week. Embody the rules of life that you've learned and are continuing to perfect, and use them online.

~ Amanda

3. Use internet resources ethically

Online study resources should be used to support learning, not replace it.

When you take unethical shortcuts, such as downloading assignment answers or hiring an essay writer, you deny yourself learning opportunities. While there may be a short-term gain, there are also long term costs. If you graduate, it will be with a weaker skill base.

By accessing and studying from completed homework and tests without a professor's permission, you are cheating.

~ Tryssa de Ruyter

4. Promote healthy discussions

To get the most out of online forums, a useful netiquette guideline is to promote healthy discussion.

You can help your online community by posing questions, sharing experiences, providing positive feedback, asking follow-up questions, and referring to information sources. Being a positive contributor is better than being a critic, troll or other negative force.

Lead by example and the discussion will snowball into a useful web of ideas and answered questions.

~ Delaney Wright

5. Ignore inflammatory comments by trolls

It's generally best to ignore trolls. These are internet users who try to bait other users into a reaction.

Trolls might be honest in what they're saying, or they could be sarcastic or deliberately dishonest. You can tell a troll by the inflammatory nature of their statements. They want to stir up negative emotions and responses.

Responding to a troll is usually not a good move, even if your response is well argued and compelling. Save your time and energy for more positive interactions.

Ignore the troll as the troll is mainly looking for a response and attention so when it does not get it, it will be neutralized... how many people do you see arguing with themselves?

~ Brandon

6. Respect others as equals

Show a little respect and humility online.

Think - that idiot who wrote the opinion you completely disagree with is a human being. They have feelings and experiences. They may believe passionately in what they're saying. And they may actually be right.

Even if you're feeling dismissive or knowledgable or whatever, inject respect into your writing. That's just being fair to others.

Don't be an e-jerk. You wouldn't be rude in everyday conversations at home, at school or at work - so why change your shining personality when you're online?

~ Coralie

7. You're here to learn, not dictate

While we all like to think that our opinion matters, you'll gain more from internet forums by approaching them as a learner.

When everyone is trying to express their view rather than hearing from others, forums become noisy, crowded with posts, and disjointed. A more polite and effective path is to adopt a listening mode. Read posts carefully, ask questions, and write something only if it offers value to the discussion.

Read to understand and not to respond. If you examine conversations that take place on any social media site or forum you choose to use, it's apparent that a lot often gets lost in translation.

~ Chris

8. Remember, your words are permanent

Be careful with what you post online. Once it's out there, you may not be able to get it back.

Tatoos are seen as risky and dangerous because they're permanent. Once the ink is injected, it's pretty much there for the rest of your life. With the internet, what you write may be there for eternity.

Anything is possible with information that reaches the internet. It can go viral. It will be stored somewhere and may be findable through search. What you post can easily affect your relationships and career, if not now then possibly in the future. So always be at least a little cautious in your online interactions.

Your digital words are a record, in a permanent, unalterable ledger, forever bound to your online persona... never do anything online that you wouldn't want your parents finding out about.

~ Liam Lambert

9. Pause before you post

It's worth taking a moment to reflect before hitting the send button.

When you're using a computer, you're normally clicking, and scrolling and typing all over the place. Most things are done quickly. But one time when it's important to slow down is when you're about to post something online for all the world to see. Pause and reflect for a second. Are you truly comfortable with what you're sending?

A good rule of thumb is asking yourself: would I be happy to let my Nan see what I have written? If not, perhaps it's inappropriate.

~ Emily

10. Make your point in a nice way

Write in a way to get the kind of reaction you want. A little thoughtfulness, strategy and netiquette can go a long way in online discussions.

Your first draft of an online post is unlikely to be your best. Are you disagreeing with someone in a flippant way? Have you misinterpreted what they really meant? Will you put people off with the tone of your text.

Writing in a constructive way is a great habit to get into. Your online communications will turn out much better.

Ask probing questions for clarity, avoid making assumptions or jumping to conclusions... If you disagree with someone, think "how can I express my disagreement without being disrespectful or antagonistic?"

~ Ashley

11. Use anonymity responsibly

While anonymity frees up online conversations, your ability to hide much of yourself shouldn't be misused.

It's a measure of your character that you conduct yourself with class all the time, even when you think no one will trace your online activity back to you personally.

Good behaviour helps others, improves your online experience and ultimately will help you become a better communicator. Conversely, bad behaviour catches up with people eventually. Trust in karma.

Anonymity is what distances users from their cyber counterparts, and has in turn stimulated the advent of cyber bullying.

~ Jill

12. Write like you'd speak

Write naturally as if you were talking to someone right in front of you. Generally, the text will be easier to understand and relate to.

While it's helpful to put thought into what you write, you generally don't want the product to appear laboured. Dense and unnatural writing can be difficult to follow and could well be a conversation stopper. So try to sum up your thoughts in a fluent, natural way.

If you try to force it, your message may come across as shrewd or too formal... read aloud what you are writing so you can determine how the person reading it may feel, react, or respond.

~ Jeremy Marroquin

13. Good writing is good manners

When posting text for others to see, try to write well. While rushing may save you a little time, think of the poor reader. They don't want to waste time trying to read something unclear or incomprehensible.

And remember that many people might see your work. In a busy student forum, it could be dozens or hundreds of students. On a popular website or for a viral post, it could be many thousands. All these readers are inconvenienced by poor writing. Good writing is, therefore, an essential part of netiquette.

Use language with skill. Urgency and laziness in typing or formulating communications is still plain old bad manners, and polite conversation is impossible with an inebriate :-).

~ Catherine Lockley

14. Clarity is essential

Clear writing is essential to convey proper meaning online. Have you ever tried to sarcasm or dry wit in an email? It seldom works. That's because tone of voice and body language are lost in text-based communication.

Unfortunately, short texts have their limitations. You need to be accurate and very clear to avoid misunderstandings when writing online.

Some online conversations descend into piles of useless text that fail to convey any meaningful information... use more specific language in text.

~ Katie Marie Lawles

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Netiquette - Are You a Responsible Online Contributor?

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Here are 14 great tips for student netiquette. The ideas come from university and college students. We ran a short-essay competition to find the best ways of showing excellent online etiquette. The top answers were used to form a set of rules or guidelines for internet manners.

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Name the two (2) thyroid hormones affected by the above disease and identify the cells that produce them.

The two thyroid hormones affected by hashimoto’s disease are thyroxine (T4) and triiodothyronine (T3) which are produced in the thyroid gland by the follicular cells (Tortora et al., 2019).

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Effect of Exercises on Quality of Life in Patients with Postmenopausal Osteoporosis(Koveska et al,2019).

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Abstract

Keywords: Postmenopausal osteoporosis, Exercises, Quality of life

Introduction

Osteoporosis is a systemic skeletal disease characterised by a low bone mineral density and microarchitectural deterioration of bone with a consequent decrease of its density and increased risk of fractures [ 1]. According to the data of the World Health Organization (WHO), osteoporosis is on the fourth place after cardiovascular diseases, cancer and stroke. Approximately 30% of all postmenopausal women in the United States of America and Europe have PO. Ageing of populations worldwide will be responsible for a major increase in the incidence of osteoporosis in postmenopausal women [ 2]. The first clinical manifestation very often is vertebral fractures, which can happen during normal activities such as stairs climbing, weight bearing etc. Age, deformity as kyphosis, disturbance of balance and non-specific back pain, increases the risk of a fall, for a new fracture, poor quality of life, disability and high mortality [ 3], [ 4].

Treatment of these patients has to be comprehensive and include both, pharmacological and non-pharmacological interventions. Drug therapy reduces the risk of fracture by influencing the mechanism of bone remodelling. These drugs decrease the risk of vertebral fracture by 30-70%, hip fractures by 20-40% and non-vertebral fractures by 15-20% [ 5], [ 6]. Non-pharmacological interventions include specific physical exercises for osteoporosis to improve muscle strength and balance, decrease pain, and improve quality of life [ 7]. In general, exercises are simple, do not require large financial costs and specific expensive equipment, and can be done at home. Exercises for osteoporosis are designed so that they enable better spinal stability and posture [ 8], [ 9], [ 10]. In the literature, only a few previous randomised studies have been published about the effect of exercises and physical activity on quality of life in osteoporotic patients. These studies have applied diverse methodologies and have presented controversial results [ 11], [ 12]. In our country, no such study has been conducted so far. Also, the questionnaire about the quality of life in postmenopausal osteoporotic patients has been applied for the first time in our country.

Our study aimed to evaluate the effectiveness of exercises for osteoporosis on quality of life in patients with postmenopausal (PO).

Material and Method

This was a randomised blind, one-sided trial, which included 92 patients with postmenopausal osteoporosis (PO) diagnosed and treated at the Institute of Physical Medicine and Rehabilitation in Skopje, R. Macedonia. The inclusion criterion was diagnosed osteoporosis. Diagnostic criterion was taken from total t score -1.5 SD to -2.5 SD, determined with x-ray DXA densitometry. The survey was approved by the Ethics Committee for people research at the Faculty of Medicine, Ss. Cyril and Methodius University, the Republic of Macedonia (Num.03-124/2, approved 22.03.2015year), and each of the patients was previously informed about the research from the researcher and signed informed consent to participate in it. Exclusion criteria were: secondary osteoporosis, fever, lumbar sciatica, pacemaker, cardiorespiratory instability, arrhythmia, malignant disease, neurological diseases.

Patients were randomly assigned to three groups: the first group of patients with exercises and physical modalities (gr. I), the second group with exercises (gr. II), and the third control group of patients who did not practice exercises (gr. III). At the beginning of the study, the first group (gr. I) received physical modalities (interferent currents and magnetic therapy) for 3 weeks, each day with a weekend break. Physical modalities were given for the treatment of pain. Exercise program consisted of: respiratory exercises, active exercises and exercises for strengthening the paravertebral muscles, active exercise for maintaining the range of motion of the joints of upper and lower extremities and spine, exercises for strengthening the muscles of the upper and lower extremities, isometric exercises for strengthening the abdominal muscles and exercises for balance. For weight-bearing exercises, the weight was determined by the functional abilities of the patients, 1 (one) kilogram at the most. Exercises were performed 3 times per week; each exercise was repeated for 5 to 8 times. Patients regularly took bisphosphonates, calcium and vitamin D. The follow-up period lasted for one year. Quality of life was evaluated with the specifically designed questionnaire for quality of life-Qualeffo-41 at the beginning and the end of the investigation.

Qualeffo-41 is a specially designed questionnaire approved by the International Association for Osteoporosis for measuring the quality of life in patients with postmenopausal osteoporosis [ 13]. The questionnaire consists of 41 items and includes 5 domains: pain, physical function, social activities, general health perception and mental function. Total score (TS) and domain scores (DS) are calculated by the following formulas:

Total score along with domain scores was standardised to a 100-point scale; score 0 shows that the patient has no problems, that is, the quality of life is excellent, whereas score 100 shows maximum problems, that is, poor quality of life. All patients independently filled in the questionnaire at the beginning and the end of the investigation.

The statistical analysis of the obtained data was made in the statistical program SPSS for Windows 17.0. Qualitative data were presented with absolute and relative numbers; the quantitative data were shown by the measures of descriptive statistics (mean ± SD, median with IQR). Cronbach's alpha was used to determine the degree of internal consistency of the questions in the five areas of the Quality of Life Questionnaire in postmenopausal women (QUALEFFO-41). For comparing the three analysed groups of patients, nonparametric and parametric methods for independent samples were used (Chi-square test, and Kruskal-Wallis test. The values of p < 0.05 were statistically significant.

Results

Patients were with a mean age of 60.64±6.7 years; the youngest patient was 43 years old, and the oldest 73 years. The largest number/percentage of examined patients was at the age of 60 to 69 years-53 (57.61%). According to the level of education, patients with completed high school predominated (48.91%) ().

Table 1

Patients of the three groups did not differ significantly in terms of menopausal status (p = 0.3). Patients without physical agents and exercises (gr.I), insignificantly rarely from patients in the other two groups provided anamnestic data for early menopause (36.67%, 46.88%, 56.67% respectively). The presence of risk factors for osteoporosis was 65.63% of patients treated with physical agents and exercises (gr.I), 80% of patients treated with exercise (gr.II), and 73.33% of patients without physical therapy and exercises (gr.III),. Statistical analysis was non-significant, between the three groups, for the frequency of risk factors for PO (p = 0.44). The difference was also non-significant in terms of the number of risk factors present (p = 0.7). The analysis of the three groups compared to the present comorbidity showed that 71.88% of patients treated with physical agents and exercises (gr.I), 56.67% of patients treated with exercise (gr.II), and 76.67% of patients without physical therapy and exercises (gr.III), had accompanying chronic conditions. The tested difference between subjects with and without co-morbidity, and depending on how the disease was treated, was statistically non-significant (p = 0.22) ().

Table 2

When comparing the results of QUALEFFO-41 obtained at the beginning and at the end of our study for each group longitudinally, we noticed a significant improvement in all domains in the first and in the second group of patients, whereas in the third group, who did not practice exercises, there were no significant changes in the domains, except in the social life that showed a substantial impairment.

We found a statistically significant difference among the three groups of patients in the average score for the domain pain at the end of the investigation (p < 0.000). However, this significant difference was due to the significantly higher average score in the control group compared to the group treated with physical agents and exercises (59.3 ± 21.3 vs 40.87 ± 20.6 p=0.004), and the group treated with exercises alone (59.3 ± 21.3 vs 31.0 ± 23.2 p < 0.0001). This was mainly a result of the regular practising of exercises by patients in the first and the second group. We can conclude that at the end of the follow-up period, patients who did not perform exercises had substantially diminished the quality of life regarding presence and severity of pain in comparison with the other two groups of patients ().

Table 3

On the final check-up, a significantly different average score for the domain physical activities among the three groups was observed (p < 0.0001). Post hoc analysis confirmed a significant difference between the control and the first group (41.8 ± 19.3 vs 19.95 ± 13.3; p < 0.0001), and between the control and the second group (41.8 ± 19.3 vs 19.99 ± 15.4; p < 0.0001). After one-year follow-up, patients from the control group, who did not practice exercises for osteoporosis, showed substantially diminished the quality of life regarding physical activities when compared to the other two groups of patients (). On the final check-up, the difference was statistically significant (p < 0.0001) for the domain social life, which was due to the significantly higher average score for this domain between the control and the first group (67.06 ± 27.9 vs 34.58 ± 19.9, p < 0.0001), and between the control and the second group (67.06 ± 27.9 vs 27.65 ± 21.64, p < 0.0001). The comparison of the quality of life among the three groups from the aspect of social functioning at the end of the investigation, showed that patients from the control group, who did not practice exercises, had a significantly larger number of problems in performing social activities when compared to the other two groups, (). Significant differences were observed (p < 0.000) in the domain of perception of their general health at the end of the investigation. On the last check-up, the average score for the general health perception in the group treated with interferential currents, magnet and exercises was 45.88 ± 22.1, in the group performing exercises 41.5 ± 21.9, and in the control group, who did not practice exercises, the score was significantly higher (78.2 ± 21.2). At the end of the follow-up period, patients who did not practice exercises rated their general health condition as significantly poorer compared to the patients from the other two groups ().

The three groups of patients had non-significantly different quality of life regarding their mental functioning, both on admission (p = 0.3) and at the end of the follow-up (p = 0.3), indicating that the method of treatment of primary osteoporosis had no significant impact on their mental functions ().

The comparative analysis of total average scores showed that, on admission, there were no significant differences in quality of life among the three groups of patients (p = 0.1), whereas, at the end of the follow-up, significant differences were registered (p < 0.0001). Post hoc analysis showed a significant difference in total average score between the control and the first group and between the control and the second group (p = 0.00011). The average total score on the last check-up was 28.86 ± 8.4 for the first group, 26.98 ± 11.8 for the second group, and significantly higher for the third group (47.43 ± 11.75) () ().

Table 4

Discussion

There are many general questionnaires for measuring the quality of life to evaluate the general health condition; however, none of them is as specific for PO as is the Qualeffo-4. Over the last decade, its application in assessing the quality of life in patients with OP has been emphasised due to its specificity, coherence and extensiveness. Osteoporosis (OP) can negatively influence the quality of life, thus limiting and restricting the performance of everyday activities. Chronic pain as a result of PO might lead to depression, anxiety, frustration and social isolation. Practising exercises have become an important intervention in increasing the self-confidence in women in performing their activities and tasks. Regular practising of exercises in women with PO has a positive effect on the general health condition, social inclusion, self-respect, better mood and conscience for better body shape and has decreased depression, anxiety and fear from falls.

Our study has shown that all patients with PO demonstrated a significant improvement in the quality of life in all domains of the questionnaire for one year. This was supported by the results obtained for the average scores in all domains when we compared them at the beginning and the end of the study. It means that patients with back pain have a poor quality of life. Also, the other domains such as physical functioning, social life, general health perception and mental function showed a substantial improvement at the end of the study. This is also supported by the result in the total Qualeffo-41 score, which at the end of the study was significantly increased. In the last 10 years, a small number of studies has been conducted that examined the influence of exercises on quality of life in patients with OP for one year by applying the Qualeffo-41 questionnaire.

Similar to our study is the investigation of Evstigneeva et al., which demonstrated that after 12 months of performing exercises for PO the questionnaire about the quality of life showed a significant improvement in the quality of life in patients. Total Qualeffo-41 score after 12 months was significantly better in the group of patients who practised exercises (44.2 ± 7.5) compared to the control group (56.6 ± 9.4), p < 0.0001.

In another study, a general questionnaire about the quality of life in patients with PO was applied. Patients were followed for 13 months and were divided into three groups: the first group practised exercises 3 times per week, the second 2 times per week and the third, control group, did not perform exercises. Their results were similar to our results. The total score about the quality of life in both groups who practiced exercises showed a significant improvement (p < 0.0001); gr. 1 (at the beginning 330.2 ± 22.02 and at the end 369.05 ± 1.5) and gr. 2 (at the beginning 313.3 ± 22.01 and at the end 348.8 ± 22.6), but not in the control group (at the beginning 312.3 ± 35.09 and at the end 311.4 ± 35.7) [ 14].

The largest number of investigations is with a shorter follow-up period, and usually, the type of the exercises and their influence on the quality of life in patients with PO has been compared. For example, one study compared the effect of the three different types of exercises on the back pain and quality of life in an adult population with low bone density. A total of 98 women participated in the study, divided into three groups. The investigation lasted for 6 months. The results showed no significant difference among the groups in the total score of Qualeffo-41 questionnaire [ 15].

The study conducted by Schröder et al. comprised 45 patients with PO who were assigned to two groups: the first group had the usual exercise program for PO, and the second group had exercises similar to our program. The total score for the quality of life in both groups showed improvement; in the first group (at the beginning 26.0 ± 11.2 and the end 23.9 ± 10.0; p = 0.766) and in the second group (at the beginning 29.7 ± 9.8 and the end 21.8 ± 8.1; p < 0.001), but the changes in the second group were statistically significant. In three months, there was an improvement in all domains on the Qualeffo-41 questionnaire in both groups, with higher significance in the second group [ 16].

The study of Schröder et al. covering three months and the study of Liu-Ambrose TY et al., covering six months have shown that even for a shorter period the exercises for PO improved the quality of life in patients with osteoporosis.

Similar results have been demonstrated in the study of Bennell et al., presenting a significant pain decrease and a significant improvement in the domain of physical functioning even though patients practised the exercises only for three months [ 17], which was not the case in our study.

One of the advantages of exercises is that they do not represent a burden to the home budget and they can be performed at home. They can yield good results as it was presented in the investigation of Papaioannou A et al., where the questionnaire about the quality of life showed a significant improvement after a 12-month follow-up period [ 18]. It has to be emphasised that practising exercises should be a continuous process in which patients are actively involved.

Exercises should be practised regularly because with advancing age the muscle tissue is decreased as well as the strength of the muscles and physical abilities. Therefore, exercises maintain physical condition, mobility and social life and hence contribute to a better quality of life.

In conclusion, the exercise program for osteoporosis has significantly improved the quality of life in patients after one year of practising in all four domains: pain, physical activities and mobility, social activities and perception about general health condition (p < 0.0001). The role of exercises in the treatment of patients with postmenopausal osteoporosis is undisputable.

Footnotes

Funding: This research did not receive any financial support

Competing Interests: The authors have declared that no competing interests exist

References

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Effect of Exercises on Quality of Life in Patients with Postmenopausal Osteoporosis - Randomized Trial

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Keywords: Postmenopausal osteoporosis, Exercises, Quality of life Osteoporosis is a systemic skeletal disease characterised by a low bone mineral density and microarchitectural deterioration of bone with a consequent decrease of its density and increased risk of fractures [ 1]. According to the data of the World Health Organization (WHO), osteoporosis is on the fourth place after cardiovascular diseases, cancer and stroke.

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Group chat room for T1M2.

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Describe the mechanism of action of thyroid hormones from release into the blood stream through to the response of the target cell.

Thyroid hormones are produced from iodine and tyrosine inside thyroglobulin (TGB).

T3 and T4 are both lipid soluble, so they diffuse through the plasma membrane and into the interstitial fluid the hormones are then transported in the blood bound to plasma proteins, through thyroxine-binding globulin (TGB). T4 is secreted in larger amounts than T3 but T3 is a lot more potent than T4. Additionally, when T4 enters a body cell most of it is converted into T3 via subsequent removal of one iodine (Tortora et al., 2019).

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Are paraspinous intramuscular injections of botulinum toxin a (BoNT-A) efficient in the treatment of chronic low-back pain? A randomised, double-blinded crossover trial(Cogńe et al 2017)

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Abstract

Background

Treatment for patients with chronic low-back pain (LBP) is a public health issue. Intramuscular injections of botulinum toxin A (BoNT-A) have shown an analgesic effect on LBP in two previous randomized controlled studies. The objective of the study was to verify the efficacy of paravertebral injections of BoNT-A in patients with LBP.

Methods

Patients were included in this phase 3 randomized double-blinded trial comparing the efficacy of BoNT-A versus placebo in a crossover study on LBP. Both groups received 200 units of BoNT-A in paravertebral muscles or a placebo, and vice versa at Day 120. The main judgment criterion was LBP intensity 1 month after the injections, evaluated by using a visual pain scale (VAS). Secondary assessment criteria included: LBP intensity 90 and 120 days after injection day; number of days when an allowed antalgic oral treatment was needed in between each evaluation; functional disability measured by the Quebec Back Pain Disability Scale; quality of life; inability to work; patient satisfaction in relation to the treatment's effect; spinal mobility; and strength of spinal muscles, measured by isokinetic technique.

Results

Nineteen patients completed the study. There was no significant difference between the groups' average LBP during the last 8 days at Day30 ( p = 0.97). There was no significant difference between the two groups regarding the secondary assessment criteria ( p > 0.05).

Conclusions

Injections of BoNT-A in the paravertebral muscles were not found to be effective to relieve chronic LBP. The limits of the study are that the dose of BoNT-A used was lower than in other studies, and that the limited number of patients included may explain the negative results.

Trial registrations

Identifiers: NCT03181802. Unique Protocol ID: CHUBX2003.

Keywords: Low-back pain, Botulinum toxin, Disability, Function, Quality of life, Work

Background

Chronic low-back pain (LBP) is a public health problem that concerns 5 to 7% of the general occidental population [ 1] and has a significant impact on the quality of life of its sufferers [ 2].

Since the significance of lumbar stiffness in relation to contraction of the erector spinae muscles has been linked to the level of intensity of LBP [ 3], the lumbar erector spinae muscles have become a therapeutic target. Many recent arguments purport that paravertebral muscles have a predominant pathogenic role in perpetuating chronic back pain. During spinal movements, paravertebral muscles' activity, recorded by electromyography, show abnormalities in subjects with low-back pain compared to subjects without LBP. A decrease in the power ratio between the erector spinae and flexor spinal muscles, measured by isokinetic techniques, is associated with chronic low-back pain. Finally, the significance of lumbar stiffness in relation to the erector spinae muscles contracting is linked to the level of intensity of low-back pain [ 3]. Local muscular treatments have already been tried such as physiotherapy, massage, infrared therapy and botulinum toxin A (BoNT-A) [ 4- 6].

In addition to its muscle-relaxing effect, local intramuscular injections of BoNT-A have also shown an analgesic effect on pain related to dystonia, tension headaches, myofascial pain syndrome and chronic neck pain [ 7- 11]. This effect is usually reversible after 3 months. Foster et al. [ 4] used BoNT-A A for its peripheral muscle relaxant action as a local intramuscular treatment of chronic LBP. This double-blinded, placebo, controlled trial in 31 patients showed that paravertebral administration of BoNT-A in patients with chronic LBP relieved pain and improved function at 3 and 8 weeks after treatment. Machado et al. [ 6] showed also in a randomized controlled trial that BoNT-A injections relieved pain and improved quality of life of 19 patients at 4 weeks. Further open studies have been performed to value the efficacy of BoNT-A in patients with chronic LBP [ 12- 15] but all of them aimed to establish predictive factors of pain relief, and the efficacy was limited to 3 months. A Cochrane meta-analysis [ 16] concluded that "there was low quality evidence in the short term, and very low quality in the intermediate term, that BoNT-A injections reduced pain intensity more effectively than saline injections in participants with LBP" and that "there was very low quality evidence that BoNT-A injections compared to corticosteroid injections could reduce chronic LBP intensity in the short term".

Studying the therapeutic effect of paravertebral injections of BoNT-A requires further studies to confirm the reported short-term therapeutic effect and to determine potential predictive factors of efficacy.

Objectives of this trial

Main objective: To evaluate the analgesic effect 1 month after a single injection of 200 IU of BoNT-A in 10 bilateral paravertebral intramuscular points for treating chronic LBP.

Secondary objectives

To evaluate the analgesic effect of paravertebral injections of BoNT-A 3 months after its administration in chronic LBP sufferers.

To measure the impact of paravertebral injections of 200 IU of BoNT-A in a single administration on lumbar stiffness and on spinal extensor muscle strength in patients with chronic LBP.

To search for predictive factors of the analgesic effect of BoNT-A injections.

Materials and methods

This study was a randomized, double-blinded, placebo-controlled phase 3 trial comparing BoNT-A Type A injections (Botox) to a placebo in patients with chronic LBP (Level 2, OCEBM Levels of Evidence Working Group*, "The Oxford 2011 Levels of Evidence"). This superiority trial obtained support from the French Hospital Clinical Research Project (PHRC).

The number of participants included in the study was similar as those included in the previous study (see [ 4]), that showed a strong positive effect of BoNT-A injections on LBP. Furthermore, the design of our study (i.e. a crossover) increased the power of the statistical analysis. In this context, 60 inclusions were planned (30 in each group). Nevertheless, regular intermediary analyses were planned by an independent scientific committee, to ensure that the trial did not present any secondary effect, or that we could conclude in an intermediary step that BoNT-A was inefficient in pain relief. After obtaining a similar number of injections than Foster, the study was stopped by the scientific committee, because there was no trend in pain relief.

Ethics, consent and permissions

This trial obtained the approval of a French ethics Committee (2003/02) and all participants received an information note and gave their written informed consent. The clinical trial registration number was: Identifiers: NCT03181802, Unique Protocol ID: CHUBX2003.

Population

The patients included were consulted by Physical and Rehabilitation Medicine spinal pathology specialists at the University Hospital of Bordeaux, met the eligibility criteria, and volunteered to participate in the study.

Inclusion criteria were: LBP defined as a pain located between the thoracic lumbar hinge and the gluteal sulcus, where pain had evolved over a period of 6 months despite well conducted medical treatment, self-assessed lumbar pain intensity over 50 mm long on a visual analogue scale of 100 mm (0 = no pain; 100 = maximal pain), having been on sick leave for 60 or more days in the year preceding the inclusion (in order to include patients with high consequences of chronic low-back pain on their work), the same long-term chronic pain treatment for at least 6 weeks, and a paravertebral painful point pressure.

Exclusion criteria were: age under 18 or over 55 years (to avoid secondary causes of low back pain, like spinal tumor), ongoing pregnancy or breast-feeding, a neuromuscular pathology (myasthenia gravis, amyotrophic lateral sclerosis, myopathy, polymyositis), aminoglycoside treatment at the time of inclusion, skin infection at injection points, diabetes and alcoholism (in order to avoid other etiologies of chronic pain), a history of injecting BoNT-A A, anticoagulation treatment, sciatica, suspected spinal inflammatory disorder (spondylitis, inflammatory rheumatism, tumoral pathology), a failed back surgery syndrome (when surgery failed to relieve low-back pain), incapacity to stand, cardiorespiratory deficiency which does not allow the isokinetic exploration of the spinal muscles, cognitive disorders limiting patient participation, conflicts of interest owing to existing pain (unconsolidated work accident, ongoing damage compensation). Spine infection, tumour or trauma had been specifically excluded by an MRI done by all patients before the inclusion in the present study. Some of risk factors associated with going from acute low-back pain to chronic low-back pain are linked to the socio-professional context, notably with the job dissatisfaction [ 17, 18]. Furthermore, 2 studies [ 17, 19] showed that there was a significant positive association between a damage compensation and chronic incapacity. In general, patients with unconsolidated work accident or ongoing damage compensation have a higher probability to be at risk of chronic disease; they also have a lower probability to positive response to treatment in general. That is why we excluded them from the study. We measured it by asking to each participant: "are you currently in an unconsolidated work accident?" and "are you currently ongoing a damage compensation?". As a High Authority of Health in France (l'Agence Nationale d'Accréditation et d'Evaluation en Santé, Diagnostic, Prise en charge et suivi des malades atteints de lombalgie chronique, Décembre 2000) classified the beginning of a LBP after the age of 55 as an " alert sign ", we excluded them from the study.

No patient was allowed to take opiates during the time of the study, and facet joint injections were also not permitted during the study period. Physiotherapy programs offered during the study period were isometric exercises and core muscle strengthening exercises one or twice per week (usual physiotherapy in chronic low-back pain, that patients made before the study, and which was not modified during the study).

Experimental procedure

Task

The design of this study was a crossover. The subjects were randomized into two groups and successively received the two treatments of the study: patients in group 1 received intramuscular paravertebral injections of BoNT-A during the first sequence of treatment, then a placebo during the second sequence of treatment 120 days later; patients in group 2 received a placebo during the first sequence of treatment, then intramuscular paravertebral injections of BoNT-A during of the second sequence of treatment 120 days later. The crossover was performed 120 days after the inclusion in the study, because most patients with initial improvement induced by BoNT-A injections reported in previous studies [ 12] that the beneficial effect waned at four months.

A paper table of randomization was used by the pharmacist at the University Hospital of Bordeaux (block randomization with block size of 6). The pharmacist who performed the randomization was blinded to the patient's characteristics.

Therapeutic procedure: For each group, the injected solution was prepared by the hospital pharmacist in order that both the patients and the injectors were blinded to the nature of the injected solution. The treatments compared were: 200 IU of BoNT-A diluted in 4 ml of physiological saline injected intramuscularly in the paravertebral lumbar muscles, versus 4 ml of physiological saline injected intramuscularly in the paravertebral lumbar muscles (placebo). The injector administered the solution in 10 intramuscular puncture points (0.4 mL/point) equally distributed from L1 to L5, bilaterally. The site of injection was detected by electromyography using the injection needle. No complementary pain treatment was prescribed after the injections.

Follow-up: patients were examined at inclusion Days 0, 30, 90, 120 (Day of the crossover), 150, 210 and 240, i.e., D0, D30, D90 and D120 after both sessions of injection. The follow-up was done in person. Patients were blinded throughout the entire study.

Measures

The main judgment criterion was the level of LBP intensity at D30 (when the maximal effect of BoNT-A injections is anticipated). Pain intensity was measured on a horizontal visual analogue scale 100 mm long, with " no pain " written on one end and " maximum pain " on the other (0 = no pain; 100 = maximal pain). The question asked was: "How was the intensity of your LBP over the last 8 days?" To consider the pain decrease as clinically significant, we used the guidelines of Pham et al. [ 20], who suggested that a change of 40 mm could be clinically significant.

Secondary judgment criteria

Initial pain was detailed as follows: Immediate average LBP was recorded on VAS at the first injection (D0). Average pain intensity over the last week and the last month were also recorded at D0, with the same horizontal visual analogue scale.

Lumbar pain intensity at D90 and D120 was measured on a horizontal visual analogue scale 100 mm long, with " no pain " written on one end and "maximum pain" on the other. The question asked was: "How was the intensity of your LBP over the last 8 days?" (0 = no pain; 100 = maximal pain).

The number of days when oral pain treatment (antalgic or non-steroid anti-inflammatory, opiates were not permitted) between evaluation times was taken. Days when treatment was taken were noted as they occurred by the patient in a calendar, which was distributed at D0. We thought that a change of 25% would be significant.

Functional disability related to LBP was measured by the Quebec Back Pain Disability Scale at each evaluation time. The higher the score (/100), the higher the disability. We considered as determined by Ostelo et al. [ 21] 20 points of change of the Quebec score as clinically significant.

Quality of life was measured at each evaluation time on a horizontal visual analogue scale 100 mm long. The question asked was: "In your opinion, how was your quality of life over the last month?" (0 = no impact to 100 = major deterioration). We considered as clinically significant a change of 0.2 standard deviation (small change), 0.5 standard deviation (moderate change) and 0.8 standard deviation (large change) [ 22, 23].

Inability to work was measured by a compendium of data indicating the number of sick leave days due to LBP in the 8 months preceding inclusion and during follow-up. A change of 25% was considered as clinically significant.

Patient satisfaction regarding the effect of the treatment was measured on a horizontal visual analogue scale 100 mm long at each evaluation time. The question asked at each evaluation was: "In your opinion, how is the overall efficacy of the treatment that you have received?"(0 = no efficacy; 100 = high efficacy). A change superior than 50% was considered as clinically significant.

Spinal mobility was measured at each evaluation time by using Schober & Macrae's test (Miller 1984). Two lines were drawn 10 cm above the postero superior iliac spine and 5 cm below the postero superior iliac spine. The distances in a standing position and in anteflexion were measured. A difference less than 4 cm was considered as a spine stiffness.

Spinal muscle strength was measured by flexion and extension isokinetic technique at a speed of 60° per second before the injections, at D30, D120, D150 and D240. A variation of strength up to 20% or a reversal of the flexor/extensor ratio was considered as clinically significant.

MODIC classification of discopathy and Hadar classification of the rector spinae muscles were based on MRI performed in the previous year. The MODIC measures are divided in 3 classes: [ 24]: there were type 1 (inflammatory phase), type 2 (fatty phase) and type 3 (marked sclerosis adjacent to the endplates). We collected the data in order to look for predictive factors for efficacy of BoNT-A.

Tolerance to BoNT-A injections was studied by actively asking at each visit for possible side effects (pain at injection points, sensation of general weakness, falling, nausea, diplopia, dry mouth).

Statistical analysis

Comparisons were made by a paired Student t-test after verifying the conditions of validity of the test (normal distribution, homogeneous variances). The Chi square test was used in order to compare the gender distribution of the two groups. Paired t-tests and Chi square tests were performed on cumulative data from 19 patients following placebo (19 patients) and BoNT-A injections (19 patients) after a crossover. Linear regression analysis was also planned. Risk of type 1 error was α = 5% at each statistical analysis. To run the statistical analyses, we used the Excel software, version 15.32. The statistician who decided the kind of statistics used was blinded. The author who made the statistical analyses was not blinded, but he/she did not compile the data into the statistical software.

Results

The group who began the injections with BoNT-A was named group 1; the group who began the injections with a placebo was named group 2. As planned, in order to increase the power of the statistical analysis of the crossover, we pooled post-BoNT-A follow-up and post-placebo follow-up. The group with BoNT-A injections was named group A and the group with placebo injections was named group B. The follow-up of groups A and B was performed at D30, D90 and D120 following each injection time.

In this study, 19 patients were approached and eligible to the study. No patient declined participation in the study. The inclusion period was about 23 months. All patients included were randomized in one of the two groups. Nine of them received BoNT-A at D0, 10 of them received placebo at D0. In the BoNT-A group (group 1), all patients were followed at D30, D90, all of them received placebo at D120, were followed at D150, D210 and D240 and completed the trial. In the placebo group (group 2), one patient was lost during the follow-up at D90 and one patient was lost during the follow-up at D210; 8 patients received BoNT-A at D120, all of them were followed at D150, D210 and D240 and completed the trial. We excluded the 2 lost patients from the statistical analysis, because they did not benefit from the 2 injections (BoNT-A and placebo). Patients' distribution is presented in Fig. .

Description of the population at baseline (standard deviations are noted in parentheses) (Table )

Table 1

The group who initially received BoNT-A was named group 1; the group who initially received the placebo was named group 2. The group 1 contained 6 women and 3 men, and the group 2 contained 10 women (Chi square = 3.96, p = 0.047). There was no significant difference concerning the mean age of group 1 (38.1(±5.94)) and group 2 (38.2(±10.27)) ( p = 0.98). The mean usual spinal pain intensity of group 1 was 59.33 mm (±15.71) and the one of group 2 was 58.70 (±15.89) ( p = 0.93). The usual root pain intensity did not differ between groups either ( p = 0.26) (mean pain intensity in group 1: 42.89 mm (±26.98); mean pain intensity in group 2: 28.40 mm (±27.16)). The mean pain intensity during the last month was 63.11 mm (±25.70) in group 1 and 66.70 mm (±24.50) in group 2 ( p = 0.76); the mean pain intensity during the last 8 days was 67.67 mm (±22.37) in group 1 and 57.50 mm (±25.63) in group 2 ( p = 0.37). There was no significant difference concerning the mean Quebec initial score between group 1 (52.56 mm (±11.64)) and group 2 (51.70 mm (±16.55)) ( p = 0.90). There was no significant difference concerning the mean disability during the last month between group 1 (7.44 mm (±12.99)) and group 2 (13.4 mm (±14.55)) ( p = 0.36); but the disability during the last 8 months was higher in the group 2 (151.6 mm (±96.56)) than in the group 1 (58.22 mm (±82.29)) ( p = 0.03). The quality of life at inclusion was estimated at 76.56 mm (±16.41) for group 1 and at 65.00 mm (±17.80) for group 2 ( p = 0.16). There was no significant difference concerning the number of days with painkillers or anti-inflammatories between group 1 (19.67 days (±13.44)) and group 2 (14.1 days (±12.57)) ( p = 0.36). In group 1, 4 patients had a right-, 3 had a left- and 2 had a bilateral paravertebral painful point pressure; in group 2, 4 patients had a right- and 6 patients had a bilateral paravertebral painful point pressure. In group 1, 8 patients and 6 patients of group 2 had a stiffness ( p = 0.17). The Schober's test was measured at 4.22 cm (±1.30) for group 1 and 3.95 cm (±1.77) for group 2 ( p = 0.71). The hand-ground distance was about 28.60 cm (±13.60) in group 1 and 20.60 (±15.60) for group 2 ( p = 0.25). The mean number of localization of spinal pain was 3.13 (±1.46) in group 1 and 3.60 (±1.84) in group 2 ( p = 0.55), and the mean number of localization of paravertebral pain was 5.00 (±1.51) in group 1 and 4.60 (±2.32) in group 2 ( p = 0.67). No patients presented a Lasegue sign at the inclusion; 2 patients presented a pseudo-Lasegue sign in group 1 and 5 presented a pseudo-Lasegue sign in group 2 ( p = 0.30). Only one patient in group 2 presented a disco-radicular conflict ( p = 0.34). The isokinetic evaluation revealed a maximum strength at 115.33n/m (±58.63) in group 1 and at 114.44n/m (±37.63) in group 2 ( p = 0.97); the endurance was calculated at 89.11n/m (±62.50) in group 1 and 77.33n/m (±53.86) in group 2 ( p = 0.67); the flexors/extensors ratio at 60° was calculated at 123.63% (±37.56) in group 1 and 119.36 (±49.51) in group 2 ( p  = 0.84). Population at baseline is described in Table .

Table 2

Number of patients (n) A/B

Mean group A

Standard deviation group A

Mean group B

Standard deviation group B

Level of LBP intensity

Between-group comparisons are presented in Table .

Functional disability related to LBP evaluated by Quebec back pain disability scale (figure )

Estimated impact of LBP on quality of life (figure )

Isokinetic maximum strength

There was no significant difference concerning the isokinetic maximum strength between group 1 and group 2 at D30 ( p = 0.34), at D120 ( p = 0.30) and at D150 ( p = 0.11). There was a significant difference concerning the isokinetic maximum strength between group 1 and group 2 at D240 ( p  = 0.04).

There was no significant difference concerning the isokinetic maximum strength between group A and group B at D30 ( p = 0.70) and at D120 ( p  = 0.70).

Isokinetic endurance

There was no significant difference concerning the isokinetic endurance between group A and group B at D30 ( p = 0.76) and at D120 ( p  = 0.65).

Isokinetic maximum force ratio flexors/extensors

There was no significant difference concerning the isokinetic maximum force measured by the radio flexors/extensors at 60° between group 1 and group 2 at D30 ( p  = 0.90), at D120 (0.89), at D150 (0.08), at D240 (0.19).

There was no significant difference concerning the isokinetic maximum force measured by the radio flexors/extensors at 60° between group A and group B at D30 ( p = 0.16) and at D120 ( p  = 0.72).

Within-group comparisons

There was no significant difference in group A and in group B concerning the pain intensity between D0 and D120 ( p = 0.58 for group A and p  = 0.70 for group B).

Symmetric carryover effect

There was a symmetric carryover effect between group 1 and group 2 concerning the main judgement criterion, i.e. pain intensity at D30.

Adverse effects

The adverse effects were actively asked at each visit. No patients declared an adverse effect during the present study. No complications were experienced in this study.

Discussion

This randomized controlled trial did not find any advantage for injections of BoNT-A versus placebo in the paravertebral muscles of patients with LBP at 30, 90 et 120 days with regard to pain relief, functional disability, sick leave, quality of life, consumption of oral antalgics, spinal flexibility and isokinetic strength or endurance. Indeed, there was no significant difference between the two groups regarding the main criterion, i.e., average lumbar pain over the last 8 days at D30 ( p = 0.97), nor was there any significant difference between the two groups regarding secondary judgment criteria ( p > 0.05, see Table ).

Our results differ largely from those of two previous studies [ 4, 6]. Since LBP is a complex phenomenon involving heavy lifting, twisting and trauma which is sometimes work-related [ 25] psychological factors [ 26], smoking, alcoholism, biomechanical and psychosocial professional factors, the difference in results could be due to differences in the populations of the two studies. Indeed, in a previous study [ 4], 3 patients had a discectomy compared to none in our study. In addition, no patient had any MRI evidence of acute disc pathology in the two previous studies [ 4, 6], whereas 6 patients in our study had a MODIC 1. Furthermore, in our study, only 3 male patients were included. Nevertheless, there was no difference between groups concerning the gender. Furthermore, the present literature is not uniform about the role of the gender on the chronicity of low-back pain [ 27, 28].

In our study, we used 200 units of Botox for bilateral injections. But we assume that the negative results of our study could also be secondary to the lower dose of botulinum toxin A used on each injection point compared to Foster et al.'s study. Indeed, we decided to inject bilaterally the paravertebral muscles, because there is usually a bilateral injury in both paravertebral muscles after an acute low back-pain, which leads to chronic low-back pain [ 29]. More precisely, we think that LBP could be secondary to an over-activity of muscles compensating multifidus' atrophy. The dose used in Machado et al.'s study (1000UI of Dysport in case of bilateral injections) was also superior to ours, which could explain the difference between the results.

Reporting a negative study is still an interesting point, because the efficiency of BoNT-A on chronic low-back pain is still not proved at this time, and because it could make reconsider on one part in researchers' further studies about BoNT-A and LBP, and one the other part in clinicians using BoNT-A injections for chronic LBP.

The strength of this trial is its randomized, controlled crossover design. A limitation is the small sample. Nevertheless, the number of patients treated was similar to that in previous studies [ 4, 6, 14], which showed a strong positive effect of BoNT-A on LBP. While some differences between the groups became apparent before the crossover (hand-ground distance, Schober's test, isokinetic measures), they disappeared when the data were aggregated after the crossover (group A and group B), perhaps owing to variations due to the size of groups 1 and 2. Indeed, group 1 demonstrated more spine stiffness and less strength than group 2, a finding that was unexpected.

The main result of the study is the absence of any significant difference or trend to feel pain relief with injections of BoNT-A A compared to placebo injections. A larger sample of patients now needs to be studied in order to identify those who would benefit most from BoNT-A injections for LBP.

Conclusions

Botulinum toxin injections did not show any efficacy in relieving pain in patients with chronic low-back pain in this randomised controlled trial using a cross-over. Result is in contradiction with the existing literature. With 200UI of Botox injected bilaterally, we did not find any pain relief. But this negative result could also be explained by the lower dose used compared to other studies, and by the low number of patients included. Nevertheless, this negative result could be useful being included in a meta-analysis.

Acknowledgements

The authors thank Briane Marie Sheperd, Ray Cooke and Anna Moraitis for helping with the English syntax. The authors thank also Dr. Jean-François Knebel for his help with the statistical analyses.

This work was supported by the French Hospital Clinical Research Project (PHRC).

Funding

The authors thank the Allergan company for co-funding this study (co-financing with 13,700 euros) within the French Hospital Clinical Research Project (PHRC Project) framework (42,000 euros). The Allergan company did not interfere with the collection, the analysis and the interpretation of data, nor with the writing of the manuscript.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Authors' contributions

MC made the statistical analysis and wrote the paper. HP and AC included some patients, helped for the interpretation of the statistical analyses and were involved in drafting the manuscript. MDS helped for promoting the study, included the patients and helped for the statistical analysis. DL helped for designing and promoting the study. She was also involved in drafting the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.

Notes

Ethics approval and consent to participate

This trial obtained the approval of a French ethics Committee (2003/02) (Comité Sud-Ouest et Outre-mer III, consent obtained in February 2003) and all participants received an information note and gave their written informed consent.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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# data[].attributes.attachment_link.title
Are paraspinous intramuscular injections of botulinum toxin a (BoNT-A) efficient in the treatment of chronic low-back pain? A randomised, double-blinded crossover trial

# data[].attributes.attachment_link.description
Treatment for patients with chronic low-back pain (LBP) is a public health issue. Intramuscular injections of botulinum toxin A (BoNT-A) have shown an analgesic effect on LBP in two previous randomized controlled studies. The objective of the study was ...

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Name the specific neuronal component that is affected in the disease and describe its
associated structure (including the neuroglia involved).

The myelin sheath is destructed in Multiple Sclerosis. A protein and lipid based covering, the myelin sheath insulates the axon, helping to increase the rate at which nerve conductions travel. Neuroglia involved include Schwann Cells within the PNS and Oligodendrocytes which are found within the CNS (Tortora et al., 2016).

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Outline how damage to the above component affects the functioning of the neuron and why
axons in the CNS display minimal regeneration after injury.

Damaged myelin sheath can lead to scaring and the formation of plaque. The neuron is no longer insulated and the conduction of electrical impulses are thus unable to travel along the nerve cells. Regeneration is difficult since there is no neurolemma (a protective axon covering which is responsible for regrowth), present (Tortora et al., 2016).

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Compare and contrast continuous and saltatory conduction of the nerve impulse. Answer must
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The synthetic thyroid hormone replaces the deficient production of the endogenous thyroid
hormone. Discuss two actions of thyroid hormone within the body.

Increased body growth and increased BMR are two actions of thyroid hormone within the body. Body growth is accelerated in conjunction with hGH and insulin; this also increases growth in both the nervous and skeletal system. Additionally, cellular oxygen helps to produce ATP, increasing BMR and metabolism of carbohydrates, lipids and protein (Tortora et al., 2016).

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Hormonal axes are pathways that direct neuroendocrine function. Identify the relevant axis, in
this case, and describe the associated control and regulation of thyroid hormones. Include the
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The HPA, HPG, and HPT hormonal axes are the three major pathways in which the hypothalamus and pituitary direct neuroendocrine function of the thyroid gland. The thyroid hormone exerts a negative feedback control over the hypothalamus as well as the pituitary, which ultimately governs the secretion of both TRH from hypothalamus and TSH from the pituitary gland. The thyroid follicles also contain follicle cells, which are involved in the secretion of the thyroid hormones thyroxine T4 and triiodothyronine T3; also involved are para-follicular cells. It are these cells which secrete calcitonin. Additionally, specific hormones are synthesised from iodine and tyrosine within thyroglobulin. These thyroid hormones assist in the regulation and utilisation of oxygen, help to increase metabolic rate, enhance cellular metabolism, and regulate basal metabolic rate, which coincides with human growth and development (Tortora et al, 2016).

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Many individuals with MS experience impairment of proprioception and vibrational sensation. Name and describe the function of each of the components of the somatosensory pathways.

Somatosensory pathways communicate information from receptors to the cerebral cortex involve three neuron groups first order, second order, and third order neuron. The first order neuron manages impulses from receptors to the brain and spinal cord. The second order neuron directs impulses from the brain stem to spinal cord to the thalamus. The third order neuron conducts impulses from the thalamus to the same side of the primary somatosensory area of the cortex
(Tortora et al., 2019).

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Which specific somatosensory pathway is affected when an individual with MS experiences the loss of the sensations identified in Q. 4? Name two (2) specific areas in the cerebral cortex that may be affected when experiencing loss of vibrational sensation and proprioception and provide rationale for answer.

The somatosensory pathway that is affected is the first order neuron. The parietal lobe and postcentral gyrus lobe of the cerebral cortex would be impacted as it is the area that receives somatic sensory input from different regions throughout the body. As loss of tactile sensations and proprioception that has occurred affects the trigeminothalamic pathway from occurring to the cerebral cortex
(Tortora et al., 2019).

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Thyroidectomy Versus Medical Management for Euthyroid Patients

With Hashimoto Disease and Persisting Symptoms

A Randomized Trial(Guldvog et al., 2019).

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The effect of inflammation and its reduction on brain plasticity in multiple sclerosis: MRI evidence (Tomassini et al., 2016).

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Valentina Tomassini

Corresponding Author

Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University School of Medicine, United Kingdom

Cardiff University Brain Research Imaging Centre (CUBRIC), Cardiff University School of Psychology, United Kingdom

IRCCS Fondazione Santa Lucia, Rome, Italy

The first two authors contributed equally to this work.

Correspondence to: Dr. Valentina Tomassini, MD, PhD, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University School of Medicine, University Hospital of Wales, Heath Park, CF14 4XN Cardiff, United Kingdom. E‐mail: tomassiniv@cardiff.ac.uk Search for more papers by this author

Alessandro d'Ambrosio

Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University School of Medicine, United Kingdom

Cardiff University Brain Research Imaging Centre (CUBRIC), Cardiff University School of Psychology, United Kingdom

Department of Medical, Surgical, Neurological, Metabolic and Aging Sciences, Second University of Naples, Italy

The first two authors contributed equally to this work.
Search for more papers by this author

First published:

Abstract

Brain plasticity is the basis for systems‐level functional reorganization that promotes recovery in multiple sclerosis (MS). As inflammation interferes with plasticity, its pharmacological modulation may restore plasticity by promoting desired patterns of functional reorganization. Here, we tested the hypothesis that brain plasticity probed by a visuomotor adaptation task is impaired with MS inflammation and that pharmacological reduction of inflammation facilitates its restoration. MS patients were assessed twice before (sessions 1 and 2) and once after (session 3) the beginning of Interferon beta (IFN beta), using behavioural and structural MRI measures. During each session, 2 functional MRI runs of a visuomotor task, separated by 25‐minutes of task practice, were performed. Within‐session between‐run change in task‐related functional signal was our imaging marker of plasticity. During session 1, patients were compared with healthy controls. Comparison of patients' sessions 2 and 3 tested the effect of reduced inflammation on our imaging marker of plasticity. The proportion of patients with gadolinium‐enhancing lesions reduced significantly during IFN beta. In session 1, patients demonstrated a greater between‐run difference in functional MRI activity of secondary visual areas and cerebellum than controls. This abnormally large practice‐induced signal change in visual areas, and in functionally connected posterior parietal and motor cortices, was reduced in patients in session 3 compared with 2. Our results suggest that MS inflammation alters short‐term plasticity underlying motor practice. Reduction of inflammation with IFN beta is associated with a restoration of this plasticity, suggesting that modulation of inflammation may enhance recovery‐oriented strategies that rely on patients' brain plasticity. Hum Brain Mapp 37:2431-2445, 2016. © 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.

Abbreviations

BOLD

Blood oxygen level dependent

CNS

Central Nervous System

EDSS

Expanded Disability Status Scale

FSL

FMRIB Software Library

T25‐FW

Timed 25 Foot Walk

FOV

Field of View

Gd

Gadolinium

GM

Grey matter

9‐HPT

9 Hole Peg Test

IFN

Interferon

MNI

Montreal Neurological Institute

fMRI

functional MRI

MS

Multiple Sclerosis

PASAT

Paced Auditory Serial Addition Test

ROIs

Regions of interest

SE

Standard error

TE

Echo Time

TF

Thumb flexion

TR

Repetition Time

VBM

Voxel‐based morphometry

WI

Weighted image

INTRODUCTION

The neural reorganization that mitigates the functional impact of damage [Enzinger and Fazekas, 2015] and promotes recovery in multiple sclerosis (MS) is based on brain plasticity [Tomassini et al., 2012a] that is the dynamic reorganization of brain function in response to experience (e.g., skill learning) [Doyon and Benali, 2005; Nudo 2013] or damage [Nudo, 2013]. Advanced neuroimaging shows that clinically meaningful functional changes occur during and after an active phase of MS [Lee et al., 2000; Mezzapesa et al., 2008; Pantano et al., 2002, 2002] or in the presence of preserved [Hulst et al., 2012] or fully recovered behaviour [Reddy et al., 2000]. These patterns of functional reorganisation differ from those of healthy volunteers even when recovery interventions drive performance improvements in the patients [Tomassini et al., 2012b]. While disability 2005]. With this functional compensation, damage‐related altered patterns of functional reorganisation occur in MS, even early during the course of the disease [Pantano et al., 2002], evidence suggests that MS inflammatory damage independently contributes to them [Lee et al., 2000; Reddy et al., 2002]. The effects of inflammation and its modulation on brain plasticity, however, remain largely unexplored, hampering the development of effective strategies enhancing recovery of function in MS patients [Tomassini et al., 2012a]. per se can sustain these abnormal patterns [Reddy et al.,

Indeed, experimental findings suggest that immune mediators, chronically over‐expressed in the MS inflammatory process, can affect the brain mechanisms underlying functional recovery by interfering with biochemical interactions, from a neuronal to a neurovascular level. Firstly, inflammation can influence the synaptic transmission, impairing the ability to encode and retain information [Di Filippo et al., 2014; Hauss‐Wegrzyniak et al., 2002; Min et al., 2009]. Electrophysiological studies support this mechanism [Mori et al., 2014; Nistico et al., 2013] and link it to clinically significant deficits [Mori et al., 2011]. Secondly, inflammation can damage connections between brain regions, hindering the spatial and temporal coherence of signal transmission [Allan and Rothwell, 2001; Di Filippo et al., 2008] necessary for plasticity to effectively drive recovery [Robertson and Murre, 1999]. Indeed, systems‐level plasticity relies on long‐range connections between brain regions. Damage to these connections occurs in MS [He et al., 2009; Petsas et al., 2013] and induces aberrant neural connectivity among widely distributed brain regions [Rocca et al., 2009]. Thirdly, inflammation can alter the neurovascular coupling [Girouard and Iadecola, 2006], a fundamental function of the brain that is the balance between energy demand imposed by neural activity and substrate delivery through blood flow, by interfering with the biochemical milieu that regulates the vasoactive chemicals supporting the normal neurovascular interactions.

As inflammation can interfere with brain plasticity in MS, its pharmacological modulation may restore plasticity by promoting desired patterns of brain functional reorganization. In this proof of concept study, we use advanced imaging methods to test whether brain plasticity is impaired as a result of MS inflammation and if a pharmacological reduction of inflammation through Interferon (IFN) beta may restore this plasticity. IFN beta reduces inflammatory activity in MS through a modulation of the pro‐inflammatory environment in the central nervous system (CNS) [Whitaker, 1994] that contributes to altered plasticity [Di Filippo et al.. 2014]. In patients with MS, the effects of IFN beta on MRI disease activity can be detected 4 weeks after the beginning of high dose IFN beta [De Stefano et al., 2012], suggesting that an early anti‐inflammatory effect can be expected in patients.

To probe plasticity and its changes over time, we used practice of a simple motor task that induces short‐term motor adaptation [Morgen et al., 2004], i.e., changes in sensorimotor cortical excitability and/or in the location or extent of cortical representations following repeated task execution that represent an optimized recruitment of the sensory‐motor network with practice [Butefisch et al., 2000; Classen et al., 1998; Hagenbeek et al., 2007]. The repetitive performance of simple and unskilled, but over‐learned movements can cause representational changes in MS patients that can be measured using functional MRI (fMRI) [Mancini et al., 2009]. Here, we hypothesised that training of these simple movements, requiring no fine hand coordination and very little strength, and inducing short‐term motor adaptation, caused brain functional changes that (i) were detectable in the MS patients, (ii) were altered as a result of inflammation and (iii) could be restored with immunomodulation.

MATERIALS AND METHODS

Participants and Study Design

Right‐handed patients with a diagnosis of MS according to the revised McDonald Criteria [Polman et al., 2005] and eligible to start treatment with IFN beta were recruited at Sapienza University of Rome. Steroid administration and relapse within 3 months of study entry were exclusion criteria. Patients were assessed using behavioural and MRI measures twice before (at session 1, week −6 ± 1, and at session 2, baseline) and once after (at session 3, week +12 ± 1) the beginning of IFN beta 1a 44 mcg (Rebif®, Merck Serono) subcutaneously administered 3 times weekly. The interval between sessions was chosen in order to balance the need to avoid a delay in treatment initiation for eligible patients, considered unethical, with the necessity to allow for a sufficient period of time for the drug to manifest an effect on MRI activity [De Stefano et al., 2012; Pozzilli et al., 1996]. Age‐ and sex‐matched healthy volunteers were assessed behaviourally and with MRI at session 1 only. Figure 1 reports the details of the study design and measurements.

Participants' written informed consent was obtained according to the Declaration of Helsinki and the protocol approved by the local Ethics Committee.

Behavioural Measures and Statistical Analysis

During each session, participants were assessed using the Nine Hole Peg Test (9‐HPT), the Timed 25‐Foot Walk (T25‐FW) and the Paced Auditory Serial Addition Test (PASAT) 2s and 3s that are part of the MS Functional Composite (Cutter, Brain 1999). Patients were also assessed using the Expanded Disability Status Scale (EDSS) score [Kurtzke, 1983].

We used two‐tailed unpaired t‐test to investigate between‐group differences in age and behavioural measures. We used chi‐square test to assess sex differences between groups. To investigate changes in behavioural measures across sessions in patients we used repeated measures ANOVA followed by two‐tailed paired t‐tests that identified changes between pairs of sessions. For all the statistical tests, differences were considered significant at P  ≤ 0.05. Values are reported as mean ± standard error (SE), unless stated otherwise.

MRI Acquisitions and Analysis

Structural and functional MRI measurements were acquired using a Siemens Magnetom Verio 3T/70cm bore magnet (Siemens healthcare, Siemens, Germany). Analysis was carried out using tools from the FMRIB Software Library (FSL) ( www.fmrib.ox.ac.uk/fsl) [Smith et al., 2004] and software for lesion volume quantification (Jim 5.0 software, Xinapse System, Leicester, UK; http://www.xinapse.com). All brain functional activations were labelled using the Montreal Neurological Institute (MNI) Structural Atlas, Harvard‐Oxford Structural Atlas, the Juelich Histological Atlas, the Cerebellar Atlas in MNI and the Oxford Thalamic Connectivity Probability Atlas ( http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html).

Structural MRI

Patients underwent conventional MRI acquisitions that included T1‐weighted images (WIs) [Repetition Time (TR) = 550 ms; Echo Time (TE) = 9.8 ms; Field of View (FOV) =240 mm; matrix = 320 × 320; 25 axial slices, 4 mm thickness; 30% gap], acquired 5 minutes after the administration of Gadolinium (Gd) to detect the presence and quantify the number of Gd‐enhancing lesions; proton density and T2‐WIs (TR= 3,320 ms; TE1= 10 ms; TE2= 103 ms; FOV= 220 mm; matrix= 384 × 384; 25 axial slices; 4 mm thickness; 30% gap) to quantify T2‐hyperintense lesions; high resolution 3D T1‐WIs (2 acquisitions of 1 mm voxel size MPRAGE sequence with TR= 1,900 ms; TE= 2.93 ms; flip angle= 9°; FOV= 260 mm; matrix= 256 × 256; 176 sagittal slices; 1 mm thickness; no gap) to quantify grey matter (GM) volume. Healthy volunteers underwent 3D T1‐WIs only at session 1.

A trained researcher (F.T.) assessed the presence of Gd‐enhancing lesions on the post‐contrast T1‐WIs and calculated the volume of T2‐hyperintense lesions on T2‐WIs using a semiautomated technique (Jim 5.0 software, Xinapse System, Leicester, UK; http://www.xinapse.com). The statistical significance of the difference in the proportion of Gd‐positive vs. Gd‐negative scans was calculated using a non‐parametric binomial test with a significance level of P ≤ 0.05. 3D T1‐WIs were analyzed with FSL‐Voxel‐Based Morphometry (VBM) [Ashburner and Friston, 2000; Good et al., 2001], incorporating non‐linear registration and correction for local expansion or contraction to produce the GM template that was used as a covariate in the fMRI analysis to control for local differences in GM volume between patients and controls at session 1.

Functional MRI

Motor task fMRI was acquired in MS patients in the three scanning sessions and in healthy volunteers in session 1. Whole brain functional images with blood oxygen level dependent (BOLD) contrast were obtained using gradient‐echo echo‐planar imaging (TR/TE = 3,000/30ms, 64x64 matrix, 50 transverse interleaved 3 mm slices, FOV 192 mm, flip angle 89°). Fieldmaps were also generated (TR = 488ms, TE1 = 4.92ms, TE2 = 7.38ms, 3 mm voxel size, 36 3‐mm interleaved transverse slices, FOV 250 mm, 64 × 64 matrix, flip angle 60°) for each individual to aid registration.

The motor task consisted of the repetition of directionally specific, voluntary, visually cued thumb flexion (TF) movements before (run 1) and after (run 2) 25 minutes of TF training [Morgen et al., 2004]. Participants performed 30‐second blocks of TF alternated with 30‐second blocks of rest. A colour fixation cross, flashing between orange (TF) and white (thumb relaxing with return to resting position) every 0.5 seconds, was used as cue to obtain a rate of 1 Hz TF. A 3‐second instruction of either "Flexion" or "Rest" condition preceded each block. The flashing cross was present throughout both conditions. Task instructions and pacing were visually presented using a MRI‐compatible stimulus‐presentation system with the use of goggles (VisuaStim Digital system from Resonance Technology Inc, Northridge, California). A researcher monitored the execution of the task to ensure consistency throughout the experiment.

The fMRI data analysis tested (a) within‐ and between‐group changes in brain activation associated with training at session 1; (b) changes in training‐related functional responses with IFN beta in the patients. Analysis was carried out using FEAT of FSL. The direction of functional contrasts is indicated in the text as vs., e.g. run 1 vs. run 2 indicates run 1 ‐ run 2 and run 2 vs. run 1 indicates run 2 ‐ run 1.

First‐level analysis ( step1) was conducted in each participant and in each run to identify the main effect of the TF task. Data pre‐processing included motion correction, brain extraction, spatial smoothing (Gaussian kernel of 5‐mm full width at half maximum), high‐pass temporal filter (100‐s cut off) and correction for field inhomogeneities through fieldmap‐based echo‐planar imaging unwarping. Non‐linear registration from high‐resolution T1 structural to MNI standard brain space was carried out. The time series was analyzed using a general linear model approach with local autocorrelation correction. The canonical gamma variate hemodynamic response function was used. One explanatory variable (along with the temporal derivative) specified the onset and duration of the task periods to identify the mean effect associated with the task for both run 1 and run 2. Mixed effects, within session, higher‐level (group) analyses were conducted in patients and in controls [Beckmann et al., 2003] to identify the main effect of the task for run 1 and for run 2. Group Z statistical images were thresholded using clusters determined by Z > 2.3 and a cluster‐extent corrected significance threshold of P = 0.05 [Forman et al., 1995; Friston et al., 1994; Worsley et al., 1992].

Fixed effects, within‐session, higher‐level (group) analyses ( step 2) were conducted in patients and in controls [Beckmann et al., 2003] to identify within‐subject between‐run signal changes in functional responses. Within‐session between‐run percent signal changes in functional responses represented the practice‐related change in brain activity that was our imaging marker of training‐related brain plasticity [Morgen et al., 2004].

Mixed effects, within‐session, higher‐level (group) analyses ( step 3) to investigate the mean between run signal changes in both groups and to compare groups were conducted for session 1 [Beckmann et al., 2003] with automatic outlier de‐weighting [Woolrich, 2008]. GM partial volume information based on the individual structural images was added to the model as a voxel‐dependent covariate. Group Z statistical images were thresholded using clusters determined by Z > 2.3 and a cluster‐extent corrected significance threshold of P  = 0.05.

Mixed effects, between‐session, within‐group paired higher‐level (group) analysis ( step 4) was carried out with automatic outlier de‐weighting to detect changes in between‐run fMRI signal differences between sessions 2 and 3 in the patients. Group Z statistical images were thresholded using clusters determined by Z > 2.3 and a cluster‐extent corrected significance threshold of P = 0.05. Between‐run (session 2 vs. session 3) percent signal change in functional responses for the trained movements was considered as representing brain plasticity modulated by IFN beta anti‐inflammatory effects, an assumption discussed further below.

Considering the longitudinal design of the study and the absence of a placebo patient group, it not being ethically acceptable to withhold IFN beta treatment from a group of eligible patients, we expected that between‐session differences in between‐run changes in functional activity could be related to both a drug effect and a time effect not caused by the drug, but reflecting the natural history of reduction in MRI detectable active inflammation [Ciccarelli et al., 1999]. Therefore, for the purposes of exploring these effects in our data, without statistical testing, in the Discussion, we plotted the results of the between‐run changes in functional response using the fMRI signal extracted from the functionally defined, anatomically constrained, regions of interest (ROIs) within the cortical areas of the session 2 vs. session 3 group analysis (from step 4). Anatomical constraints were applied by multiplying the masks of the significant cortical areas (motor, parietal, temporal and visual cortices) from the MNI Structural Atlas with the binarized functional maps, thresholded using clusters determined by Z  > 2.3.

RESULTS

Demographic and Clinical Characteristics

A total of 26 MS patients and 22 healthy volunteers were recruited. The demographic and clinical characteristics of the patients and healthy volunteers are reported in Table 1. Of the 26 patients who entered the study and completed the first assessment, 24 underwent all the scanning sessions. Two patients dropped out: one, after session 1, due to pregnancy and the other, after session 2, due protocol violation. In the 24 patients who completed the study, the mean ± SE overall study duration was 128.7 ± 4.8 days, with 45.2 ± 2.0 days between sessions 1 and 2 and 84.5 ± 4.2 days between sessions 2 and 3. During the study, there was no significant change in the EDSS scores or in the mean T25‐FW. A significant improvement in the mean 9‐HPT was observed for the dominant hand ( F = 9.0, df = 1.9, P = 0.001) between sessions 1 and 2 (17.7 ± 0.4 vs. 16.8 ± 0.4, P = 0.02), and for the non‐dominant hand ( F = 3.6, df = 1.5, P = 0.05) between sessions 2 and 3 (18.5 ± 0.5 vs. 17.8 ± 0.5, P = 0.03). The PASAT 3s ( F = 15.2, df = 1.9, P < 0.0001) showed a significant change between sessions 1 and 2 (43.1 ± 1.9 vs. 49.2 ± 1.9, P < 0.0001), while the PASAT 2s ( F = 10.4, df = 1.8, P < 0.0001) significantly changed both between sessions 1 and 2 (32.4 ± 2.6 vs. 35.8 ± 2.3, P < 0.0001) and between sessions 2 and 3 (35.8 ± 2.3 vs. 38.9 ± 2.8, P  < 0.0001). None of the patients received steroids or experienced onset of new symptoms or worsening of previously reported symptoms during the study period.

Patients (n=26)

Controls (n=22)

P

T2 hyperintense lesion volume (mm ³)

2863.6 ± 531.1

-

-

Chi‐square test.

Values are reported as mean ± SE, unless indicated otherwise. The 9‐HPT and T25‐FW are expressed as the mean of two trials. Significance is tested using two‐tailed unpaired t test, unless stated otherwise. Abbreviations: EDSS = Expanded Disability Status Scale; Gd+ MRI scans= scan with at least one gadolinium‐enhancing lesion; 9‐HPT= 9‐hole peg test; PASAT= Paced Auditory Serial Addition Test; SE = standard error; T25‐FW= timed 25‐foot walk.

Structural MRI Results

The initial structural MRI characteristics of the 26 patients who entered the study are reported in Table 1. Fourteen out of 26 (54%) MS patients during session 1 and 11 out 25 (44%) during session 2 had MRI scans with at least one Gd‐enhancing lesion, with no significant difference in the number of Gd‐positive vs. Gd‐negative scans ( P = 0.5). At session 3 (on IFN beta), the number of Gd‐active scans (3 out of 24) reduced by 72% compared to session 2, with a significant difference in the number of Gd‐positive vs. Gd‐negative scans ( P  = 0.001).

Functional MRI Results

The fMRI analysis was performed on 26 patients at session 1 and on 24 patients for sessions 2 and 3 of the study. All participants performed the functional task and training appropriately and completed the experimental protocol. In session 1, during run 1 (step 1 of fMRI analysis), the TF task engaged a large bilateral cluster of regions including the primary motor, premotor, posterior parietal and occipital cortices, as well as the basal ganglia and the cerebellum in both groups (Table 2). This cluster of regions was more extended in patients than in controls, including the left primary somatosensory cortex and the right V3 area in patients (Table 2).

Patients (n=26)

Controls (n=22)

MNI Coordinates

MNI Coordinates

Localization of clusters is in Montreal Neurological Institute (MNI) Standard Brain Space. Z score of the peak voxel is reported for each cluster showing the main effect of the thumb flexion task in patients and in controls (random effects, Z > 2.3, P < 0.05, corrected). Thalamic and cerebellar regions connected to specific cortical regions are reported in brackets.

Abbreviations: PMd = dorsal premotor cortex; SMC = supplementary motor cortex; M1 = primary motor cortex; S1 = primary somatosensory cortex; IPL = intraparietal lobule; SPL = superior parietal lobule; V = visual cortex; R = right; L = left.

The within‐group between‐run (run 1 vs. run 2) changes in fMRI responses during session 1 (step 3 of fMRI analysis) are reported in Fig. 2 and in Table 3 for both patients and healthy controls. These changes involved a cluster of regions that included the prefrontal, premotor, primary sensorimotor, posterior somatosensory and visual cortices, as well as the basal ganglia and the cerebellum. During session 1, patients showed a greater between‐run (run 1 vs. run 2) signal reduction than controls in fMRI activity of secondary visual areas (V2 and V4) and cerebellum (lobule V‐VI) (Fig. 2 and Table 3).

Training‐related signal changes (run 1 vs. run 2) during session 1

Patients (n=26)

Controls (n=22)

MNI Coordinates

MNI Coordinates

Training‐related signal changes in sessions 2 and 3 in patients only

Session 2 (n=24)

Session 3 (n=24)

MNI Coordinates

MNI Coordinates

Localization of clusters is in Montreal Neurological Institute (MNI) Standard Brain Space. Z score of the peak voxel is reported for each cluster showing the effect of thumb flexion training in patients and in controls during session 1 and in patients only during session 2 vs. 3 (random effects, Z > 2.3, P < 0.05, corrected). Thalamic and cerebellar regions connected to specific cortical areas are reported in brackets.

Abbreviations: BA = Broca's area; FP = frontal pole; IC = insular cortex; IPL = inferior parietal lobule; ITG = inferior temporal gyrus; PMd = dorsal premotor cortex; PMv = ventral premotor cortex; M1 = primary motor cortex; S1 = primary somatosensory cortex; V = visual cortex; R = right; L = left.

There was an increase in fMRI responses during session 1 (run 2 vs. run 1) that involved the right angular gyrus and posterior cingulate gyrus bilaterally in the patients, and the right inferior parietal lobule and cingulate gyrus bilaterally in the healthy volunteers, with no significant between‐group difference (data not shown).

Between‐run changes in fMRI activity in the patients for sessions 2 and 3 (step 4 of fMRI analysis) are reported in Fig. 3 and in Table 3. There was a reduction in the activation of cortical areas (run 1 vs. run 2) across sessions. Between sessions 2 (before IFN beta) and 3 (on IFN beta), patients showed a reduction of the between‐run signal change in the secondary visual areas (V2), as well, as in motor, temporal and parietal cortical areas (Fig. 3 and Table 3). There was no increase in the activation of cortical regions (run 1 vs. run 2) across sessions (session 3 vs. session 2).

DISCUSSION

Our results suggest that MS inflammation alters plasticity underlying short‐term training of a simple motor task [Morgen et al., 2004], as reflected in abnormally greater practice‐related fMRI signal changes in the patients compared to healthy volunteers that were over and above structural differences in GM volume. Indeed, in session 1, patients showed a greater between‐run reduction than controls in fMRI activity of secondary visual areas and cerebellum (Fig. 2). This larger practice‐induced signal change in visual areas and in functional connected regions [Rizzolatti and Luppino, 2001] was reduced in the patients in session 3 compared with session 2 (Fig. 3), i.e., while on IFN beta, suggesting that a reduction of inflammation with IFN beta is accompanied by a restoration of systems‐level brain plasticity in the patients.

MS inflammatory Activity on MRI and Its Changes With IFN Beta Treatment

Not all the MS patients who entered the study showed Gd‐enhancing lesions in their brains at the time of the baseline MRI scan. However, neuropathological evidence demonstrates that inflammatory activity typically spreads well beyond lesions visible on conventional MRI [Moll et al., 2011]. Indeed, conventional MRI is able to capture only part of the pathological processes underlying MS damage [Barkhof, 2002]. Also, Gd‐enhancing lesions represent a snapshot of inflammatory activity and thus their absence in the context of the natural history of the disease can reflect the dynamics associated with blood brain barrier alteration and repair [Tomassini and Palace, 2009].

While on IFN beta treatment, patients with Gd‐activity at session 1 showed a reduction in MRI disease activity. Evidence suggests that this reduction, observed after about 12 weeks of treatment, can already reflect the anti‐inflammatory effect of IFN beta [De Stefano et al., 2012; Pozzilli et al., 1996], thus offering a model to study the effect of rapid‐onset modulation of inflammation on brain plasticity.

The Effect of the Motor Task and Training on Brain Functional Responses at Session 1

The motor task used for motor training consisted of the repetition of visually cued, directionally specific, simple thumb movements. As expected, this training led to changes in functional responses of a wide set of regions involved in visuomotor integration and motor execution both in patients and in controls [Morgen et al., 2004].

Training‐dependent reductions in brain activity were also different between groups and more pronounced in the patients in secondary visual areas (V2 and V4) and in cortical cerebellar regions connected to the primary and premotor cortices [O'Reilly et al., 2010]. This cluster of regions is involved in higher visuomotor control.

V2 and V4 are part of the ventral visual pathway that allows recognition of an object's characteristics for appropriate selection of motor plans [Fogassi et al., 2001; Schubotz and von Cramon, 2003]. V2 is involved in the storage of memory for object recognition and in the conversion of short‐term into longer‐term memories of objects [Lopez‐Aranda et al., 2009]. Although both V2 and V4 can be modulated by attention, V4 shows more pronounced changes in the spatial profile of its receptive fields with attention [Moran and Desimone, 1985] and thus is involved in encoding of stimulus salience. It is tuned for recognition of object features of intermediate complexity, like simple geometric shapes. Our task initially relied on external cues to drive the pace of thumb movements. As the participants practised the task, the need for attentional engagement when performing the visually cued movements will have decreased, while the representation of the cue pacing the task would have been stored as longer‐term memory of the object and incorporated in the internal sensorimotor model of the body‐environment interactions. The greater reduction in functional signal in visual regions in the patients could be explained by the greater effort spent in attending to the task at first exposure, before training, or, alternatively, by the more pronounced need to re‐build an internal model of body‐environment interactions with training on the task.

This functional difference in visual areas can be part of a long‐range modulation of higher motor control areas. Indeed, among the wide range of cortical connections, the cerebellum has connections with the visual cortices from lobules V and VI that represent primary sensorimotor zones [O'Reilly et al., 2010]. These zones contain overlapping functional connectivity maps for domain‐specific motor, somatosensory, visual and auditory cortices. The connections between cerebellar lobule V and VI and the extrastriate visual areas reflect the importance of visual information in motor control, as the cerebellum is essential to calibrate the relationship between visual and somatosensory/motor information. Indeed lobule V and VI are relevant for skill acquisition [Tomassini et al., 2011] and visuomotor adaptation [Della‐Maggiore et al., 2009]. The regions showing more pronounced reductions in functional responses with training in the patients were localised in lobule V and VI bilaterally, suggesting that the need for higher motor control based on external sensory feedback was greater at first exposure to the task or decreased more pronouncedly with practice, as an intrinsic modulation within the cerebellum set up accurately an acquired pattern of movements, possibly in concert with activation of motor‐related cortical regions [Halsband and Lange, 2006].

The Effect of Pharmacological Modulation of Inflammation on Brain Plasticity

At session 1, patients showed a training‐related reduction in the functional signal of regions involved in higher visuomotor control that was more pronounced than the reduction observed in healthy volunteers. We interpreted these results as an expression of training‐related re‐building of an internal model of the body‐environment interactions in the patients that required progressively lower levels of motor control in order to set up accurately an acquired pattern of movements. Indeed, this larger practice‐induced signal change in visual areas and in functionally connected regions was reduced in the patients in session 3 compared with session 2. Specifically, we observed a reduction in training‐related signal changes in secondary visual areas and in connected posterior parietal and temporal regions. These regions encompass the ventral and dorsal streams that provide detailed representation of the visual world transformed into coordinates for skilled motor behaviour [Goodale and Milner, 1992]. Interestingly, between‐session changes in training‐dependent functional responses were observed also in the right primary sensorimotor cortices. The involvement of the right sensorimotor cortices (ipsilateral to the trained hand) has been associated with functional reorganisation and recovery in MS [Tomassini et al., 2012b]. Indeed, an increase in functional responses of the ipsilateral primary sensorimotor cortices has been associated with increasing levels of hand disability (Reddy H et al, Brain 2002), while a reduction in the functional signal of these same regions has been observed with training‐related improvements of hand movements [Tomassini et al., 2012a]. In this study, a reduction in functional changes between session 2 and 3 in these regions argues in favour of reduced involvement of the ipsilateral cortex in training‐related changes during pharmacological intervention and suggests that modulation of inflammation may influence the brain's functional connectivity at a systems‐level, possibly restoring patterns of normal activity [Pantano et al., 2002, 2005].

The influence of inflammation and its modulation on brain plasticity is expected on the basis of experimental evidence [Di Filippo et al., 2008]. Indeed, inflammatory molecules can interact with plasticity at a synaptic level [Di Filippo et al., 2014]. Many immune‐signalling molecules are expressed in the healthy brain and regulate synaptic function and plasticity [Pribiag and Stellwagen, 2014]. Also, neurons and glial cells can strictly interact [Perea et al., 2009]. The interaction between the immune and the nervous systems in the regulation of synaptic plasticity is needed for the physiologic development of neural circuitries [Wake et al., 2013] and for the regulation of plasticity and learning‐dependent synapse formation [Parkhurst et al., 2013]. However, during active neuroinflammation, microglial cells become activated and many immune molecules are released in higher concentrations with detrimental effects on the induction of brain plasticity. The brains of patients with MS are characterized by marked signs of immune cells activation/infiltration [Frohman et al. 2006], including activation of microglial cells, even in normal appearing white matter [van Horssen et al., 2012]. Thus, it is conceivable that the neuroinflammatory environment that characterizes MS may interfere with the neural circuits that subserve systems‐level plasticity. Experimental models of neuroinflammation support this immune‐neural interference and extend the concept of altered immune‐neural interactions to dysfunctional behaviour. The release of reactive oxygen species and pro‐inflammatory cytokines, along with the infiltration of immune cells and the activation of astrocytes and microglia, has been associated with abnormalities in synaptic plasticity [Di Filippo et al., 2014] and this altered synaptic transmission has been shown to parallel an impaired ability to encode and retain spatial information [Hauss‐Wegrzyniak et al., 2002; Kim do et al., 2012; Min et al., 2009], suggesting that inflammation‐related impairment in plasticity can also have behavioural consequences.

Training‐dependent changes in functional responses between session 2 and session 3 paralleling the reduction in MRI disease activity with IFN beta suggest a drug‐related effect of modulated inflammation on plasticity (Fig. 4). The ROIs that are selected on the basis of a difference between sessions 2 and 3 are regions involved in motor training. These regions show little difference between sessions 1 and 2 and a more pronounced difference between sessions 2 and 3, with IFN beta treatment. Should between‐session changes reflect a session effect, they would be expected to be more pronounced during the first phase of the study, between sessions 1 and 2, which was also shorter than the intervention phase and thus more prone to repetition effects. Also, values from session 3 in the patients are similar to those of the controls suggesting a restoration of function in those regions that could result from reduction in inflammatory activity.

In this study, healthy volunteers were assessed only once, at session 1. A longitudinal assessment of functional changes in healthy controls was not included because the normal state of the brain did not allow us to explore changes in functional responses with changes in inflammation and we did not expect any biologically meaningful sustained effects of 25 minutes of motor training to carry over between imaging sessions 6 ‐ 12 weeks apart in the healthy controls.

Limitations of the Study

We studied only one aspect of systems‐level brain plasticity that reflects the adaptation of functional responses with motor training. However, this type of plasticity is the basis of a resilient, yet adaptable behaviour and is probed in systems relevant for visuomotor integration, thus proving important and informative for rehabilitation [Bastian, 2008].

The time frame studied here is limited. While this restricts our ability to observe further and wider changes in training‐dependent plasticity with longer‐term modulation of inflammation, it allows us to pinpoint the anti‐inflammatory treatment effect without introducing the confounding factor of irreversible disease‐related structural changes that could affect the fMRI data interpretation.

The absence of an untrained group of patients makes it difficult to rule out completely the possibility that the difference between runs in each scanning session could be due to the passage of time rather than to effect of 25‐minute training. However, considering previous evidence on the successful induction of changes in functional responses using a similar adaptation task [Morgen et al., 2004], we assume that the training accounts for most of the between‐run difference in this study.

The patients included in the study had shown some clinical or sub‐clinical activity that made them eligible to start a disease modifying treatment, thus making the inclusion of a matched patient group acting as untreated control unethical. However, the lack of a patient group that could act as an untreated control makes it difficult to disentangle the treatment from the time effect, i.e., the effect of drug from the effect of a spontaneous reduction of active inflammation on the fMRI signal changes. One could argue that the observed changes in systems‐level plasticity are not necessarily related in all the patients to the anti‐inflammatory effects of IFN beta, which was used here to promote a rapid‐onset and consistent reduction in inflammatory activity in the patients [De Stefano et al., 2012] that paralleled training‐related changes in functional responses. Indeed, a reduction in inflammatory activity resulting from spontaneous evolution of MS lesions may have contributed to these functional changes [Tomassini and Palace, 2009]. However, should these changes have occurred as a result of natural history, they would still support our research hypothesis concerning the role of inflammation in altering plasticity.

The difficulty in interpreting the fMRI changes as being related to the effects of the drug derives from the potential session effect, i.e., the passage of time. However, the presence of 3 time points in the study design allows us to make predictions of signal changes related to the effects of drug or sessions. Here, we infer a drug effect on the assumption that changes associated with session effect on the functional signal would appear most strongly in the session 1 to session 2 differences, due to practice effects expected to occur more pronouncedly closer to the first exposure to the task and reducing with repeated exposure. We suggest, therefore, that the greater between‐run changes in fMRI activity for session 2 vs. 3 would be mainly due to the effect of reduced inflammation (Fig. 4). Under this reasoning, the different length of the two study phases would help to distinguish between session effect and the effect of modulated inflammation, i.e., the longer interval between session 2 and 3 made the functional changes less prone to session effect and thus more likely to highlight a drug effect. Therefore, we interpret the changes seen between sessions 2 and 3 as largely due to the effect of modulated inflammation, but we cannot completely exclude the potential influence of a residual session effect.

A specific interaction between drug‐induced immunomodulation and mechanisms of synaptic plasticity mediated by local modulation of cytokines and growth factors may have also contributed further to changes in systems‐level plasticity. Indeed, IFN beta has been shown to restore deficits of synaptic plasticity in MS patients [Mori et al., 2012] and this effect may be mediated by a modulation of the inflammatory milieu, with changes in the cytokine network that interfere with or favour processes of synaptic plasticity [del Rey et al., 2013; Khairova et al., 2009].

Finally, we cannot exclude that the changes during IFN beta treatment in brain functional responses could reflect a direct effect of IFN beta on mechanisms of brain plasticity. Although IFN beta only partially crosses the blood‐brain barrier, if directly administered on brain slices, it has the potential to modulate glutamate neurotransmission [Di Filippo et al., 2016], opening the possibility of a direct modulatory effect of the drug on plasticity, which is independent from its anti‐inflammatory action.

CONCLUSION

Our results suggest that MS inflammation alters short‐term plasticity underlying motor training as reflected in greater practice‐related fMRI signal changes in the patients in the pre‐IFN beta phase when compared to healthy volunteers. Reduction of inflammation with IFN beta seems to restore this plasticity in the patients, at least within the range of damage and disability studied here, suggesting that modulation of MS inflammation can enhance recovery‐oriented strategies that rely on this plasticity.

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Abstract

Brain plasticity is the basis for systems‐level functional reorganization that promotes recovery in multiple sclerosis (MS). As inflammation interferes with plasticity, its pharmacological modulation may restore plasticity by promoting desired patterns of functional reorganization. Here, we tested the hypothesis that brain plasticity probed by a visuomotor adaptation task is impaired with MS inflammation and that pharmacological reduction of inflammation facilitates its restoration. MS patients were assessed twice before (sessions 1 and 2) and once after (session 3) the beginning of Interferon beta (IFN beta), using behavioural and structural MRI measures. During each session, 2 functional MRI runs of a visuomotor task, separated by 25‐minutes of task practice, were performed. Within‐session between‐run change in task‐related functional signal was our imaging marker of plasticity. During session 1, patients were compared with healthy controls. Comparison of patients' sessions 2 and 3 tested the effect of reduced inflammation on our imaging marker of plasticity. The proportion of patients with gadolinium‐enhancing lesions reduced significantly during IFN beta. In session 1, patients demonstrated a greater between‐run difference in functional MRI activity of secondary visual areas and cerebellum than controls. This abnormally large practice‐induced signal change in visual areas, and in functionally connected posterior parietal and motor cortices, was reduced in patients in session 3 compared with 2. Our results suggest that MS inflammation alters short‐term plasticity underlying motor practice. Reduction of inflammation with IFN beta is associated with a restoration of this plasticity, suggesting that modulation of inflammation may enhance recovery‐oriented strategies that rely on patients' brain plasticity. Hum Brain Mapp 37:2431-2445, 2016. © 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.

Abbreviations

INTRODUCTION

The neural reorganization that mitigates the functional impact of damage [Enzinger and Fazekas, ] and promotes recovery in multiple sclerosis (MS) is based on brain plasticity [Tomassini et al., ] that is the dynamic reorganization of brain function in response to experience (e.g., skill learning) [Doyon and Benali, ; Nudo ] or damage [Nudo, ]. Advanced neuroimaging shows that clinically meaningful functional changes occur during and after an active phase of MS [Lee et al., ; Mezzapesa et al., ; Pantano et al., ]. With this functional compensation, damage‐related altered patterns of functional reorganisation occur in MS, even early during the course of the disease [Pantano et al., ] or in the presence of preserved [Hulst et al., ] or fully recovered behaviour [Reddy et al., ]. These patterns of functional reorganisation differ from those of healthy volunteers even when recovery interventions drive performance improvements in the patients [Tomassini et al., ]. While disability per se can sustain these abnormal patterns [Reddy et al., ], evidence suggests that MS inflammatory damage independently contributes to them [Lee et al., ; Reddy et al., ]. The effects of inflammation and its modulation on brain plasticity, however, remain largely unexplored, hampering the development of effective strategies enhancing recovery of function in MS patients [Tomassini et al., ].

Indeed, experimental findings suggest that immune mediators, chronically over‐expressed in the MS inflammatory process, can affect the brain mechanisms underlying functional recovery by interfering with biochemical interactions, from a neuronal to a neurovascular level. Firstly, inflammation can influence the synaptic transmission, impairing the ability to encode and retain information [Di Filippo et al., ; Hauss‐Wegrzyniak et al., ; Min et al., ]. Electrophysiological studies support this mechanism [Mori et al., ; Nistico et al., ] and link it to clinically significant deficits [Mori et al., ]. Secondly, inflammation can damage connections between brain regions, hindering the spatial and temporal coherence of signal transmission [Allan and Rothwell, ; Di Filippo et al., ] necessary for plasticity to effectively drive recovery [Robertson and Murre, ]. Indeed, systems‐level plasticity relies on long‐range connections between brain regions. Damage to these connections occurs in MS [He et al., ; Petsas et al., ] and induces aberrant neural connectivity among widely distributed brain regions [Rocca et al., ]. Thirdly, inflammation can alter the neurovascular coupling [Girouard and Iadecola, ], a fundamental function of the brain that is the balance between energy demand imposed by neural activity and substrate delivery through blood flow, by interfering with the biochemical milieu that regulates the vasoactive chemicals supporting the normal neurovascular interactions.

As inflammation can interfere with brain plasticity in MS, its pharmacological modulation may restore plasticity by promoting desired patterns of brain functional reorganization. In this proof of concept study, we use advanced imaging methods to test whether brain plasticity is impaired as a result of MS inflammation and if a pharmacological reduction of inflammation through Interferon (IFN) beta may restore this plasticity. IFN beta reduces inflammatory activity in MS through a modulation of the pro‐inflammatory environment in the central nervous system (CNS) [Whitaker, ] that contributes to altered plasticity [Di Filippo et al.. 2014]. In patients with MS, the effects of IFN beta on MRI disease activity can be detected 4 weeks after the beginning of high dose IFN beta [De Stefano et al., ], suggesting that an early anti‐inflammatory effect can be expected in patients.

To probe plasticity and its changes over time, we used practice of a simple motor task that induces short‐term motor adaptation [Morgen et al., ], i.e., changes in sensorimotor cortical excitability and/or in the location or extent of cortical representations following repeated task execution that represent an optimized recruitment of the sensory‐motor network with practice [Butefisch et al., ; Classen et al., ; Hagenbeek et al., ]. The repetitive performance of simple and unskilled, but over‐learned movements can cause representational changes in MS patients that can be measured using functional MRI (fMRI) [Mancini et al., ]. Here, we hypothesised that training of these simple movements, requiring no fine hand coordination and very little strength, and inducing short‐term motor adaptation, caused brain functional changes that (i) were detectable in the MS patients, (ii) were altered as a result of inflammation and (iii) could be restored with immunomodulation.

MATERIALS AND METHODS

Participants and Study Design

Right‐handed patients with a diagnosis of MS according to the revised McDonald Criteria [Polman et al., ] and eligible to start treatment with IFN beta were recruited at Sapienza University of Rome. Steroid administration and relapse within 3 months of study entry were exclusion criteria. Patients were assessed using behavioural and MRI measures twice before (at session 1, week −6 ± 1, and at session 2, baseline) and once after (at session 3, week +12 ± 1) the beginning of IFN beta 1a 44 mcg (Rebif®, Merck Serono) subcutaneously administered 3 times weekly. The interval between sessions was chosen in order to balance the need to avoid a delay in treatment initiation for eligible patients, considered unethical, with the necessity to allow for a sufficient period of time for the drug to manifest an effect on MRI activity [De Stefano et al., ; Pozzilli et al., ]. Age‐ and sex‐matched healthy volunteers were assessed behaviourally and with MRI at session 1 only. Figure 1 reports the details of the study design and measurements.

Participants' written informed consent was obtained according to the Declaration of Helsinki and the protocol approved by the local Ethics Committee.

Behavioural Measures and Statistical Analysis

During each session, participants were assessed using the Nine Hole Peg Test (9‐HPT), the Timed 25‐Foot Walk (T25‐FW) and the Paced Auditory Serial Addition Test (PASAT) 2s and 3s that are part of the MS Functional Composite (Cutter, Brain 1999). Patients were also assessed using the Expanded Disability Status Scale (EDSS) score [Kurtzke, ].

We used two‐tailed unpaired t‐test to investigate between‐group differences in age and behavioural measures. We used chi‐square test to assess sex differences between groups. To investigate changes in behavioural measures across sessions in patients we used repeated measures ANOVA followed by two‐tailed paired t‐tests that identified changes between pairs of sessions. For all the statistical tests, differences were considered significant at P  ≤ 0.05. Values are reported as mean ± standard error (SE), unless stated otherwise.

MRI Acquisitions and Analysis

Structural and functional MRI measurements were acquired using a Siemens Magnetom Verio 3T/70cm bore magnet (Siemens healthcare, Siemens, Germany). Analysis was carried out using tools from the FMRIB Software Library (FSL) ( www.fmrib.ox.ac.uk/fsl) [Smith et al., ] and software for lesion volume quantification (Jim 5.0 software, Xinapse System, Leicester, UK; http://www.xinapse.com). All brain functional activations were labelled using the Montreal Neurological Institute (MNI) Structural Atlas, Harvard‐Oxford Structural Atlas, the Juelich Histological Atlas, the Cerebellar Atlas in MNI and the Oxford Thalamic Connectivity Probability Atlas ( http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html).

Structural MRI

Patients underwent conventional MRI acquisitions that included T1‐weighted images (WIs) [Repetition Time (TR) = 550 ms; Echo Time (TE) = 9.8 ms; Field of View (FOV) =240 mm; matrix = 320 × 320; 25 axial slices, 4 mm thickness; 30% gap], acquired 5 minutes after the administration of Gadolinium (Gd) to detect the presence and quantify the number of Gd‐enhancing lesions; proton density and T2‐WIs (TR= 3,320 ms; TE1= 10 ms; TE2= 103 ms; FOV= 220 mm; matrix= 384 × 384; 25 axial slices; 4 mm thickness; 30% gap) to quantify T2‐hyperintense lesions; high resolution 3D T1‐WIs (2 acquisitions of 1 mm voxel size MPRAGE sequence with TR= 1,900 ms; TE= 2.93 ms; flip angle= 9°; FOV= 260 mm; matrix= 256 × 256; 176 sagittal slices; 1 mm thickness; no gap) to quantify grey matter (GM) volume. Healthy volunteers underwent 3D T1‐WIs only at session 1.

A trained researcher (F.T.) assessed the presence of Gd‐enhancing lesions on the post‐contrast T1‐WIs and calculated the volume of T2‐hyperintense lesions on T2‐WIs using a semiautomated technique (Jim 5.0 software, Xinapse System, Leicester, UK; http://www.xinapse.com). The statistical significance of the difference in the proportion of Gd‐positive vs. Gd‐negative scans was calculated using a non‐parametric binomial test with a significance level of P  ≤ 0.05. 3D T1‐WIs were analyzed with FSL‐Voxel‐Based Morphometry (VBM) [Ashburner and Friston, ; Good et al., ], incorporating non‐linear registration and correction for local expansion or contraction to produce the GM template that was used as a covariate in the fMRI analysis to control for local differences in GM volume between patients and controls at session 1.

Functional MRI

Motor task fMRI was acquired in MS patients in the three scanning sessions and in healthy volunteers in session 1. Whole brain functional images with blood oxygen level dependent (BOLD) contrast were obtained using gradient‐echo echo‐planar imaging (TR/TE = 3,000/30ms, 64x64 matrix, 50 transverse interleaved 3 mm slices, FOV 192 mm, flip angle 89°). Fieldmaps were also generated (TR = 488ms, TE1 = 4.92ms, TE2 = 7.38ms, 3 mm voxel size, 36 3‐mm interleaved transverse slices, FOV 250 mm, 64 × 64 matrix, flip angle 60°) for each individual to aid registration.

The motor task consisted of the repetition of directionally specific, voluntary, visually cued thumb flexion (TF) movements before (run 1) and after (run 2) 25 minutes of TF training [Morgen et al., ]. Participants performed 30‐second blocks of TF alternated with 30‐second blocks of rest. A colour fixation cross, flashing between orange (TF) and white (thumb relaxing with return to resting position) every 0.5 seconds, was used as cue to obtain a rate of 1 Hz TF. A 3‐second instruction of either "Flexion" or "Rest" condition preceded each block. The flashing cross was present throughout both conditions. Task instructions and pacing were visually presented using a MRI‐compatible stimulus‐presentation system with the use of goggles (VisuaStim Digital system from Resonance Technology Inc, Northridge, California). A researcher monitored the execution of the task to ensure consistency throughout the experiment.

The fMRI data analysis tested (a) within‐ and between‐group changes in brain activation associated with training at session 1; (b) changes in training‐related functional responses with IFN beta in the patients. Analysis was carried out using FEAT of FSL. The direction of functional contrasts is indicated in the text as vs., e.g. run 1 vs. run 2 indicates run 1 ‐ run 2 and run 2 vs. run 1 indicates run 2 ‐ run 1.

First‐level analysis ( step1) was conducted in each participant and in each run to identify the main effect of the TF task. Data pre‐processing included motion correction, brain extraction, spatial smoothing (Gaussian kernel of 5‐mm full width at half maximum), high‐pass temporal filter (100‐s cut off) and correction for field inhomogeneities through fieldmap‐based echo‐planar imaging unwarping. Non‐linear registration from high‐resolution T1 structural to MNI standard brain space was carried out. The time series was analyzed using a general linear model approach with local autocorrelation correction. The canonical gamma variate hemodynamic response function was used. One explanatory variable (along with the temporal derivative) specified the onset and duration of the task periods to identify the mean effect associated with the task for both run 1 and run 2. Mixed effects, within session, higher‐level (group) analyses were conducted in patients and in controls [Beckmann et al., ] to identify the main effect of the task for run 1 and for run 2. Group Z statistical images were thresholded using clusters determined by Z > 2.3 and a cluster‐extent corrected significance threshold of P = 0.05 [Forman et al., ; Friston et al., ; Worsley et al., ].

Fixed effects, within‐session, higher‐level (group) analyses ( step 2) were conducted in patients and in controls [Beckmann et al., ] to identify within‐subject between‐run signal changes in functional responses. Within‐session between‐run percent signal changes in functional responses represented the practice‐related change in brain activity that was our imaging marker of training‐related brain plasticity [Morgen et al., ].

Mixed effects, within‐session, higher‐level (group) analyses ( step 3) to investigate the mean between run signal changes in both groups and to compare groups were conducted for session 1 [Beckmann et al., ] with automatic outlier de‐weighting [Woolrich, ]. GM partial volume information based on the individual structural images was added to the model as a voxel‐dependent covariate. Group Z statistical images were thresholded using clusters determined by Z > 2.3 and a cluster‐extent corrected significance threshold of P  = 0.05.

Mixed effects, between‐session, within‐group paired higher‐level (group) analysis ( step 4) was carried out with automatic outlier de‐weighting to detect changes in between‐run fMRI signal differences between sessions 2 and 3 in the patients. Group Z statistical images were thresholded using clusters determined by Z > 2.3 and a cluster‐extent corrected significance threshold of P = 0.05. Between‐run (session 2 vs. session 3) percent signal change in functional responses for the trained movements was considered as representing brain plasticity modulated by IFN beta anti‐inflammatory effects, an assumption discussed further below.

Considering the longitudinal design of the study and the absence of a placebo patient group, it not being ethically acceptable to withhold IFN beta treatment from a group of eligible patients, we expected that between‐session differences in between‐run changes in functional activity could be related to both a drug effect and a time effect not caused by the drug, but reflecting the natural history of reduction in MRI detectable active inflammation [Ciccarelli et al., ]. Therefore, for the purposes of exploring these effects in our data, without statistical testing, in the Discussion, we plotted the results of the between‐run changes in functional response using the fMRI signal extracted from the functionally defined, anatomically constrained, regions of interest (ROIs) within the cortical areas of the session 2 vs. session 3 group analysis (from step 4). Anatomical constraints were applied by multiplying the masks of the significant cortical areas (motor, parietal, temporal and visual cortices) from the MNI Structural Atlas with the binarized functional maps, thresholded using clusters determined by Z  > 2.3.

RESULTS

Demographic and Clinical Characteristics

A total of 26 MS patients and 22 healthy volunteers were recruited. The demographic and clinical characteristics of the patients and healthy volunteers are reported in Table 1. Of the 26 patients who entered the study and completed the first assessment, 24 underwent all the scanning sessions. Two patients dropped out: one, after session 1, due to pregnancy and the other, after session 2, due protocol violation. In the 24 patients who completed the study, the mean ± SE overall study duration was 128.7 ± 4.8 days, with 45.2 ± 2.0 days between sessions 1 and 2 and 84.5 ± 4.2 days between sessions 2 and 3. During the study, there was no significant change in the EDSS scores or in the mean T25‐FW. A significant improvement in the mean 9‐HPT was observed for the dominant hand ( F = 9.0, df = 1.9, P = 0.001) between sessions 1 and 2 (17.7 ± 0.4 vs. 16.8 ± 0.4, P = 0.02), and for the non‐dominant hand ( F = 3.6, df = 1.5, P = 0.05) between sessions 2 and 3 (18.5 ± 0.5 vs. 17.8 ± 0.5, P = 0.03). The PASAT 3s ( F = 15.2, df = 1.9, P < 0.0001) showed a significant change between sessions 1 and 2 (43.1 ± 1.9 vs. 49.2 ± 1.9, P < 0.0001), while the PASAT 2s ( F = 10.4, df = 1.8, P < 0.0001) significantly changed both between sessions 1 and 2 (32.4 ± 2.6 vs. 35.8 ± 2.3, P < 0.0001) and between sessions 2 and 3 (35.8 ± 2.3 vs. 38.9 ± 2.8, P  < 0.0001). None of the patients received steroids or experienced onset of new symptoms or worsening of previously reported symptoms during the study period.

Chi‐square test.

Values are reported as mean ± SE, unless indicated otherwise. The 9‐HPT and T25‐FW are expressed as the mean of two trials. Significance is tested using two‐tailed unpaired t test, unless stated otherwise. Abbreviations: EDSS = Expanded Disability Status Scale; Gd+ MRI scans= scan with at least one gadolinium‐enhancing lesion; 9‐HPT= 9‐hole peg test; PASAT= Paced Auditory Serial Addition Test; SE = standard error; T25‐FW= timed 25‐foot walk.

Structural MRI Results

The initial structural MRI characteristics of the 26 patients who entered the study are reported in Table 1. Fourteen out of 26 (54%) MS patients during session 1 and 11 out 25 (44%) during session 2 had MRI scans with at least one Gd‐enhancing lesion, with no significant difference in the number of Gd‐positive vs. Gd‐negative scans ( P = 0.5). At session 3 (on IFN beta), the number of Gd‐active scans (3 out of 24) reduced by 72% compared to session 2, with a significant difference in the number of Gd‐positive vs. Gd‐negative scans ( P  = 0.001).

Functional MRI Results

The fMRI analysis was performed on 26 patients at session 1 and on 24 patients for sessions 2 and 3 of the study. All participants performed the functional task and training appropriately and completed the experimental protocol. In session 1, during run 1 (step 1 of fMRI analysis), the TF task engaged a large bilateral cluster of regions including the primary motor, premotor, posterior parietal and occipital cortices, as well as the basal ganglia and the cerebellum in both groups (Table 2). This cluster of regions was more extended in patients than in controls, including the left primary somatosensory cortex and the right V3 area in patients (Table 2).

Localization of clusters is in Montreal Neurological Institute (MNI) Standard Brain Space. Z score of the peak voxel is reported for each cluster showing the main effect of the thumb flexion task in patients and in controls (random effects, Z > 2.3, P < 0.05, corrected). Thalamic and cerebellar regions connected to specific cortical regions are reported in brackets.

Abbreviations: PMd = dorsal premotor cortex; SMC = supplementary motor cortex; M1 = primary motor cortex; S1 = primary somatosensory cortex; IPL = intraparietal lobule; SPL = superior parietal lobule; V = visual cortex; R = right; L = left.

The within‐group between‐run (run 1 vs. run 2) changes in fMRI responses during session 1 (step 3 of fMRI analysis) are reported in Fig. 2 and in Table 3 for both patients and healthy controls. These changes involved a cluster of regions that included the prefrontal, premotor, primary sensorimotor, posterior somatosensory and visual cortices, as well as the basal ganglia and the cerebellum. During session 1, patients showed a greater between‐run (run 1 vs. run 2) signal reduction than controls in fMRI activity of secondary visual areas (V2 and V4) and cerebellum (lobule V‐VI) (Fig. 2 and Table 3).

Training‐related signal changes (run 1 vs. run 2) during session 1

Patients (n=26)

Controls (n=22)

MNI Coordinates

MNI Coordinates

Training‐related signal changes in sessions 2 and 3 in patients only

Session 2 (n=24)

Session 3 (n=24)

MNI Coordinates

MNI Coordinates

Localization of clusters is in Montreal Neurological Institute (MNI) Standard Brain Space. Z score of the peak voxel is reported for each cluster showing the effect of thumb flexion training in patients and in controls during session 1 and in patients only during session 2 vs. 3 (random effects, Z > 2.3, P < 0.05, corrected). Thalamic and cerebellar regions connected to specific cortical areas are reported in brackets.

Abbreviations: BA = Broca's area; FP = frontal pole; IC = insular cortex; IPL = inferior parietal lobule; ITG = inferior temporal gyrus; PMd = dorsal premotor cortex; PMv = ventral premotor cortex; M1 = primary motor cortex; S1 = primary somatosensory cortex; V = visual cortex; R = right; L = left.

There was an increase in fMRI responses during session 1 (run 2 vs. run 1) that involved the right angular gyrus and posterior cingulate gyrus bilaterally in the patients, and the right inferior parietal lobule and cingulate gyrus bilaterally in the healthy volunteers, with no significant between‐group difference (data not shown).

Between‐run changes in fMRI activity in the patients for sessions 2 and 3 (step 4 of fMRI analysis) are reported in Fig. 3 and in Table 3. There was a reduction in the activation of cortical areas (run 1 vs. run 2) across sessions. Between sessions 2 (before IFN beta) and 3 (on IFN beta), patients showed a reduction of the between‐run signal change in the secondary visual areas (V2), as well, as in motor, temporal and parietal cortical areas (Fig. 3 and Table 3). There was no increase in the activation of cortical regions (run 1 vs. run 2) across sessions (session 3 vs. session 2).

DISCUSSION

Our results suggest that MS inflammation alters plasticity underlying short‐term training of a simple motor task [Morgen et al., ], as reflected in abnormally greater practice‐related fMRI signal changes in the patients compared to healthy volunteers that were over and above structural differences in GM volume. Indeed, in session 1, patients showed a greater between‐run reduction than controls in fMRI activity of secondary visual areas and cerebellum (Fig. 2). This larger practice‐induced signal change in visual areas and in functional connected regions [Rizzolatti and Luppino, ] was reduced in the patients in session 3 compared with session 2 (Fig. 3), i.e., while on IFN beta, suggesting that a reduction of inflammation with IFN beta is accompanied by a restoration of systems‐level brain plasticity in the patients.

MS inflammatory Activity on MRI and Its Changes With IFN Beta Treatment

Not all the MS patients who entered the study showed Gd‐enhancing lesions in their brains at the time of the baseline MRI scan. However, neuropathological evidence demonstrates that inflammatory activity typically spreads well beyond lesions visible on conventional MRI [Moll et al., ]. Indeed, conventional MRI is able to capture only part of the pathological processes underlying MS damage [Barkhof, ]. Also, Gd‐enhancing lesions represent a snapshot of inflammatory activity and thus their absence in the context of the natural history of the disease can reflect the dynamics associated with blood brain barrier alteration and repair [Tomassini and Palace, ].

While on IFN beta treatment, patients with Gd‐activity at session 1 showed a reduction in MRI disease activity. Evidence suggests that this reduction, observed after about 12 weeks of treatment, can already reflect the anti‐inflammatory effect of IFN beta [De Stefano et al., ; Pozzilli et al., ], thus offering a model to study the effect of rapid‐onset modulation of inflammation on brain plasticity.

The Effect of the Motor Task and Training on Brain Functional Responses at Session 1

The motor task used for motor training consisted of the repetition of visually cued, directionally specific, simple thumb movements. As expected, this training led to changes in functional responses of a wide set of regions involved in visuomotor integration and motor execution both in patients and in controls [Morgen et al., ].

Training‐dependent reductions in brain activity were also different between groups and more pronounced in the patients in secondary visual areas (V2 and V4) and in cortical cerebellar regions connected to the primary and premotor cortices [O'Reilly et al., ]. This cluster of regions is involved in higher visuomotor control.

V2 and V4 are part of the ventral visual pathway that allows recognition of an object's characteristics for appropriate selection of motor plans [Fogassi et al., ; Schubotz and von Cramon, ]. V2 is involved in the storage of memory for object recognition and in the conversion of short‐term into longer‐term memories of objects [Lopez‐Aranda et al., ]. Although both V2 and V4 can be modulated by attention, V4 shows more pronounced changes in the spatial profile of its receptive fields with attention [Moran and Desimone, ] and thus is involved in encoding of stimulus salience. It is tuned for recognition of object features of intermediate complexity, like simple geometric shapes. Our task initially relied on external cues to drive the pace of thumb movements. As the participants practised the task, the need for attentional engagement when performing the visually cued movements will have decreased, while the representation of the cue pacing the task would have been stored as longer‐term memory of the object and incorporated in the internal sensorimotor model of the body‐environment interactions. The greater reduction in functional signal in visual regions in the patients could be explained by the greater effort spent in attending to the task at first exposure, before training, or, alternatively, by the more pronounced need to re‐build an internal model of body‐environment interactions with training on the task.

This functional difference in visual areas can be part of a long‐range modulation of higher motor control areas. Indeed, among the wide range of cortical connections, the cerebellum has connections with the visual cortices from lobules V and VI that represent primary sensorimotor zones [O'Reilly et al., ]. These zones contain overlapping functional connectivity maps for domain‐specific motor, somatosensory, visual and auditory cortices. The connections between cerebellar lobule V and VI and the extrastriate visual areas reflect the importance of visual information in motor control, as the cerebellum is essential to calibrate the relationship between visual and somatosensory/motor information. Indeed lobule V and VI are relevant for skill acquisition [Tomassini et al., ] and visuomotor adaptation [Della‐Maggiore et al., ]. The regions showing more pronounced reductions in functional responses with training in the patients were localised in lobule V and VI bilaterally, suggesting that the need for higher motor control based on external sensory feedback was greater at first exposure to the task or decreased more pronouncedly with practice, as an intrinsic modulation within the cerebellum set up accurately an acquired pattern of movements, possibly in concert with activation of motor‐related cortical regions [Halsband and Lange, ].

The Effect of Pharmacological Modulation of Inflammation on Brain Plasticity

At session 1, patients showed a training‐related reduction in the functional signal of regions involved in higher visuomotor control that was more pronounced than the reduction observed in healthy volunteers. We interpreted these results as an expression of training‐related re‐building of an internal model of the body‐environment interactions in the patients that required progressively lower levels of motor control in order to set up accurately an acquired pattern of movements. Indeed, this larger practice‐induced signal change in visual areas and in functionally connected regions was reduced in the patients in session 3 compared with session 2. Specifically, we observed a reduction in training‐related signal changes in secondary visual areas and in connected posterior parietal and temporal regions. These regions encompass the ventral and dorsal streams that provide detailed representation of the visual world transformed into coordinates for skilled motor behaviour [Goodale and Milner, ]. Interestingly, between‐session changes in training‐dependent functional responses were observed also in the right primary sensorimotor cortices. The involvement of the right sensorimotor cortices (ipsilateral to the trained hand) has been associated with functional reorganisation and recovery in MS [Tomassini et al., ]. Indeed, an increase in functional responses of the ipsilateral primary sensorimotor cortices has been associated with increasing levels of hand disability (Reddy H et al, Brain 2002), while a reduction in the functional signal of these same regions has been observed with training‐related improvements of hand movements [Tomassini et al., ]. In this study, a reduction in functional changes between session 2 and 3 in these regions argues in favour of reduced involvement of the ipsilateral cortex in training‐related changes during pharmacological intervention and suggests that modulation of inflammation may influence the brain's functional connectivity at a systems‐level, possibly restoring patterns of normal activity [Pantano et al., ].

The influence of inflammation and its modulation on brain plasticity is expected on the basis of experimental evidence [Di Filippo et al., ]. Indeed, inflammatory molecules can interact with plasticity at a synaptic level [Di Filippo et al., ]. Many immune‐signalling molecules are expressed in the healthy brain and regulate synaptic function and plasticity [Pribiag and Stellwagen, ]. Also, neurons and glial cells can strictly interact [Perea et al., ]. The interaction between the immune and the nervous systems in the regulation of synaptic plasticity is needed for the physiologic development of neural circuitries [Wake et al., ] and for the regulation of plasticity and learning‐dependent synapse formation [Parkhurst et al., ]. However, during active neuroinflammation, microglial cells become activated and many immune molecules are released in higher concentrations with detrimental effects on the induction of brain plasticity. The brains of patients with MS are characterized by marked signs of immune cells activation/infiltration [Frohman et al. ], including activation of microglial cells, even in normal appearing white matter [van Horssen et al., ]. Thus, it is conceivable that the neuroinflammatory environment that characterizes MS may interfere with the neural circuits that subserve systems‐level plasticity. Experimental models of neuroinflammation support this immune‐neural interference and extend the concept of altered immune‐neural interactions to dysfunctional behaviour. The release of reactive oxygen species and pro‐inflammatory cytokines, along with the infiltration of immune cells and the activation of astrocytes and microglia, has been associated with abnormalities in synaptic plasticity [Di Filippo et al., ] and this altered synaptic transmission has been shown to parallel an impaired ability to encode and retain spatial information [Hauss‐Wegrzyniak et al., ; Kim do et al., ; Min et al., ], suggesting that inflammation‐related impairment in plasticity can also have behavioural consequences.

Training‐dependent changes in functional responses between session 2 and session 3 paralleling the reduction in MRI disease activity with IFN beta suggest a drug‐related effect of modulated inflammation on plasticity (Fig. 4). The ROIs that are selected on the basis of a difference between sessions 2 and 3 are regions involved in motor training. These regions show little difference between sessions 1 and 2 and a more pronounced difference between sessions 2 and 3, with IFN beta treatment. Should between‐session changes reflect a session effect, they would be expected to be more pronounced during the first phase of the study, between sessions 1 and 2, which was also shorter than the intervention phase and thus more prone to repetition effects. Also, values from session 3 in the patients are similar to those of the controls suggesting a restoration of function in those regions that could result from reduction in inflammatory activity.

In this study, healthy volunteers were assessed only once, at session 1. A longitudinal assessment of functional changes in healthy controls was not included because the normal state of the brain did not allow us to explore changes in functional responses with changes in inflammation and we did not expect any biologically meaningful sustained effects of 25 minutes of motor training to carry over between imaging sessions 6 ‐ 12 weeks apart in the healthy controls.

Limitations of the Study

We studied only one aspect of systems‐level brain plasticity that reflects the adaptation of functional responses with motor training. However, this type of plasticity is the basis of a resilient, yet adaptable behaviour and is probed in systems relevant for visuomotor integration, thus proving important and informative for rehabilitation [Bastian, ].

The time frame studied here is limited. While this restricts our ability to observe further and wider changes in training‐dependent plasticity with longer‐term modulation of inflammation, it allows us to pinpoint the anti‐inflammatory treatment effect without introducing the confounding factor of irreversible disease‐related structural changes that could affect the fMRI data interpretation.

The absence of an untrained group of patients makes it difficult to rule out completely the possibility that the difference between runs in each scanning session could be due to the passage of time rather than to effect of 25‐minute training. However, considering previous evidence on the successful induction of changes in functional responses using a similar adaptation task [Morgen et al., ], we assume that the training accounts for most of the between‐run difference in this study.

The patients included in the study had shown some clinical or sub‐clinical activity that made them eligible to start a disease modifying treatment, thus making the inclusion of a matched patient group acting as untreated control unethical. However, the lack of a patient group that could act as an untreated control makes it difficult to disentangle the treatment from the time effect, i.e., the effect of drug from the effect of a spontaneous reduction of active inflammation on the fMRI signal changes. One could argue that the observed changes in systems‐level plasticity are not necessarily related in all the patients to the anti‐inflammatory effects of IFN beta, which was used here to promote a rapid‐onset and consistent reduction in inflammatory activity in the patients [De Stefano et al., ] that paralleled training‐related changes in functional responses. Indeed, a reduction in inflammatory activity resulting from spontaneous evolution of MS lesions may have contributed to these functional changes [Tomassini and Palace, ]. However, should these changes have occurred as a result of natural history, they would still support our research hypothesis concerning the role of inflammation in altering plasticity.

The difficulty in interpreting the fMRI changes as being related to the effects of the drug derives from the potential session effect, i.e., the passage of time. However, the presence of 3 time points in the study design allows us to make predictions of signal changes related to the effects of drug or sessions. Here, we infer a drug effect on the assumption that changes associated with session effect on the functional signal would appear most strongly in the session 1 to session 2 differences, due to practice effects expected to occur more pronouncedly closer to the first exposure to the task and reducing with repeated exposure. We suggest, therefore, that the greater between‐run changes in fMRI activity for session 2 vs. 3 would be mainly due to the effect of reduced inflammation (Fig. 4). Under this reasoning, the different length of the two study phases would help to distinguish between session effect and the effect of modulated inflammation, i.e., the longer interval between session 2 and 3 made the functional changes less prone to session effect and thus more likely to highlight a drug effect. Therefore, we interpret the changes seen between sessions 2 and 3 as largely due to the effect of modulated inflammation, but we cannot completely exclude the potential influence of a residual session effect.

A specific interaction between drug‐induced immunomodulation and mechanisms of synaptic plasticity mediated by local modulation of cytokines and growth factors may have also contributed further to changes in systems‐level plasticity. Indeed, IFN beta has been shown to restore deficits of synaptic plasticity in MS patients [Mori et al., ] and this effect may be mediated by a modulation of the inflammatory milieu, with changes in the cytokine network that interfere with or favour processes of synaptic plasticity [del Rey et al., ; Khairova et al., ].

Finally, we cannot exclude that the changes during IFN beta treatment in brain functional responses could reflect a direct effect of IFN beta on mechanisms of brain plasticity. Although IFN beta only partially crosses the blood‐brain barrier, if directly administered on brain slices, it has the potential to modulate glutamate neurotransmission [Di Filippo et al., ], opening the possibility of a direct modulatory effect of the drug on plasticity, which is independent from its anti‐inflammatory action.

CONCLUSION

Our results suggest that MS inflammation alters short‐term plasticity underlying motor training as reflected in greater practice‐related fMRI signal changes in the patients in the pre‐IFN beta phase when compared to healthy volunteers. Reduction of inflammation with IFN beta seems to restore this plasticity in the patients, at least within the range of damage and disability studied here, suggesting that modulation of MS inflammation can enhance recovery‐oriented strategies that rely on this plasticity.

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# data[].attributes.attachment_link.title
The effect of inflammation and its reduction on brain plasticity in multiple sclerosis: MRI evidence

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Valentina Tomassini Corresponding Author Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University School of Medicine, United Kingdom Cardiff University Brain Research Imaging Centre (CUBRIC), Cardiff University School of Psychology, United Kingdom IRCCS Fondazione Santa Lucia, Rome, Italy The first two authors contributed equally to this work.