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A brief schematic of the Methods

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Observe functional connectivity in organoids --> expose to varying amounts of Ag-NP and duration times

Use dye or retroviral staining

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Use mouse model --> easy to observe various life stages, reaches adulthood at 4 weeks

Expose mice to various doses (more than 5 micr/ml) and time-periods (extending 24hrs) of Ag-NPs

Conduct behavioural testing for motor skills

Rotarod: tests motor coordination, balance

Open field test: locomotor activity

Sacrifice mice and perform histological markings to study neuronal and circuitry response

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1. Asharani, P. V., Wu, Y. L., Gong, Z., & Valiyaveettil, S. (2008). Toxicity of silver nanoparticles in zebrafish models. Nanotechnology, 19(25), 255102.

2. Braydich-Stolle, L., Hussain, S., Schlager, J. J., & Hofmann, M. C. (2005). In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicological sciences, 88(2), 412-419.

3. Catsakis, L. H., & Sulica, V. I. (1978). Allergy to silver amalgams. Oral Surgery, Oral Medicine, Oral Pathology, 46(3), 371-375.

4. Chen, X., & Schluesener, H. J. (2008). Nanosilver: a nanoproduct in medical application. Toxicology letters, 176(1), 1-12.

5. Chou, C. C., Hsiao, H. Y., Hong, Q. S., Chen, C. H., Peng, Y. W., Chen, H. W., & Yang, P. C. (2008). Single-walled carbon nanotubes can induce pulmonary injury in mouse model. Nano Letters, 8(2), 437-445.

6. Drake, P. L., & Hazelwood, K. J. (2005). Exposure-related health effects of silver and silver compounds: a review. Annals of Occupational Hygiene, 49(7), 575-585.

7. Gottesman, R., Shukla, S., Perkas, N., Solovyov, L. A., Nitzan, Y., & Gedanken, A. (2010). Sonochemical coating of paper by microbiocidal silver nanoparticles. Langmuir, 27(2), 720-726.

8. Navarro, E., Baun, A., Behra, R., Hartmann, N. B., Filser, J., Miao, A. J., ... & Sigg, L. (2008). Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology, 17(5), 372-386.

9. Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., ... & Behra, R. (2008). Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environmental science & technology, 42(23), 8959-8964.

10. Oberdörster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Kreyling, W., & Cox, C. (2004). Translocation of inhaled ultrafine particles to the brain. Inhalation toxicology, 16(6-7), 437-445.

11. Rossi, E. M., Pylkkänen, L., Koivisto, A. J., Vippola, M., Jensen, K. A., Miettinen, M., ... & Vanhala, E. (2009). Airway exposure to silica coated TiO2 nanoparticles induces pulmonary neutrophilia in mice. Toxicological Sciences, kfp254.

12. Rungby, J., & Danscher, G. (1983). Localization of exogenous silver in brain and spinal cord of silver exposed rats. Acta neuropathologica, 60(1-2), 92-98.

13. Sondi, I., & Salopek-Sondi, B. (2004). Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. Journal of colloid and interface science, 275(1), 177-182.

14. Wan, A. T., Conyers, R. A., Coombs, C. J., & Masterton, J. P. (1991). Determination of silver in blood, urine, and tissues of volunteers and burn patients. Clinical Chemistry, 37(10), 1683-1687.

15. Zhang, Y., Ferguson, S. A., Watanabe, F., Jones, Y., Xu, Y., Biris, A. S., ... & Ali, S. F. (2013). Silver nanoparticles decrease body weight and locomotor activity in adult male rats. Small, 9(9‐10), 1715-1720.

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Previous studies have demonstrated in vitro AgNP exposure to mouse germline stem cells (Braydich-Stolle 2005 O ) as well as oral administration in vivo rats (Kim 2008 O) resulting in increases cellular apoptosis and inflammatory responses, respectively.

Following exposure, through functional tests employed on intact animal models, behavioural impairment in adult rats manifested in lower levels of physical activity and mobility as well as drastic reduction in body weight (Zhang 2013).

It has also previously been shown that AgNPs are capable of intruding BBB and building up heterogeneously in brain regions such as cerebellum,  large motorneurons in brainstem and spinal cord accumulating higher levels quicker compared to diencephalic and telencephalic regions that required more extensive exposure (Rungby and Danscher 1983).

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High environmental Ag-NP levels have been shown to affect other species such as plants, algae, and fungi (Navarro 2008), vertebrates such as zebra fish (Asharani 2008) and even micro-organisms/bacteria such as E. Coli (Sondi 2004) and Chlamydomonas reinhardtii (Navarro 2008).

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Chronic exposure to silver can cause permanent bluish-gray skin (argyria) or eye (argyrosis) discolouration where most cases have seen to rise due to exposure to soluble form silver.

Other deleterious effects include liver and kidney damage as well as eye, respiratory, skin and intestinal irritation. Metallic silver appears to pose minimal risk to health (Drake 2005).

Effect on red blood cells: Silver ions hold high affinity for thiol groups in the liver and by binding to reduced glutathione they get transported into bile, lowering amount of available glutathione for biochemical reactions which is vital for structure and function of red blood cells (Drake 2005).

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Due to their antimicrobial and anti-inflammatory effects, AgNPs have been incorporated into wound dressings such as burn creams (Wan et al., 1991), surgical instruments such as catheters and acupuncture needles, dental amalgams (Drake 2005), bone prostheses and contraceptive devices, household products and textiles (Chen and Schulesener 2008) and food packaging to lengthen product shelf life (Gottesman 2011) as well as jewelry (Catsakis and Sulica, 1978)

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Intratracheal application of carbon nanotubes (CNTs) into male ICR mice was capable of inducing pulmonary injury via alveolar macrophage activation, chronic inflammatory response, pulmonary granuloma formation and oxidative stress (Chou 2008)

Exposure of mice abdominal cavity to CNTs also induced inflammation and granuloma formation (Poland 2008). Inhalation of titanium dioxide (TiO2) nanoparticle aerosols also induced neutrophilic pulmonary inflammation (Rossi 2010).

Moreover, carbon (13C) isotope ultrafine particles have been sown to accumulate onto olfactory mucosa of the nasopharyngeal region of the respiratory tract and thereon translocate to CNS via olfactory nerve (Oberdorster 2004).

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This past decade has faced massive boom in nanotechnology and developing engineered materials (ENM). ENM are incorporated into medical devices and products (ie: bandages) and drug delivery systems.

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https://ehp.niehs.nih.gov/121-a220/

https://www.epa.gov/aboutepa/about-national-exposure-research-laboratory-nerl

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7 surprising facts about silver nanoparticles and health

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Subscribe: http://bit.ly/riskbites Silver nanoparticles have been hitting the headlines for a few years now, both as a smart new technology for preventing bacterial infection, and as a potential new health and environmental threat. How much do you know this technology and its pros and cons? Check out these seven facts -- they may surprise you.

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Ag exposure causes NSCs to release reactive oxygen species (ROS).

(A) DCF fluoresces in response to oxidative conditions in NSCs. Significant, dose-dependent levels of DCF observed after 24-hour, 5 μg/ml Ag exposure.

(B) 8-oxo-dG is another marker for DNA oxidation and higher levels in NSC medium indicates oxidative stress. Significant levels were observed after 24-hour, 5 μg/ml Ag exposure.

(B/C&D) After issuing an anti-oxidant - acetyl-L-carnitine - caused levels of 8-oxo-dG as well as pro-apoptotic marker/protein Bax to decrease to levels similar to control.

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Another important role for NSCs is their ability to proliferate. This was measured by (A/B) EdU-Alexa Fluor staining and (C/D) DAPI counterstaining. Control is on the left and 24 hours of 5 μg/ml Ag exposure is on the right. Panels (E/F) show the colocalization of EdU+DAPI and the significant reduction of proliferating NSCs.

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What is a neural stem cell? Narrated by Dr. Sam Weiss

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Uploaded by stemcellfoundation on 2015-03-17.

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Silver nanoparticles, effects on environment and health

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This animation is an introduction to the world of nanoparticles. We explain what nanoparticles are and why silver nanoparticles could be of concern for the environment and health.

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Conduct long-term NSC damage exposure and study

Observe neurons in vivo during developmental stages

Further study of mitochondrial origination of apoptotic factors

Look at axon terminals to observe chemical signals released to neighboring neurons

Study other antioxidant derivatives used to attenuate Ag-NP affects

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Main findings correspond to to other similar studies!

Ag-Np induced cytotoxicity and increase in ROS species also found in Drosophila germ line stem cell http://www.nature.com/articles/srep20632

Vulnerability of mitochondiral pathway through which Ag-NPs induce cytoxicity http://www.mdpi.com/1422-0067/17/10/1603/htm

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1. The authors did not justify use of NSCs (as opposed to in vivo mechanisms for example)

2. The real life applications are questionable.

1. Dose dependence of 5 μg/ml causing cytotoxic damage in NSCs - significant in humans

2. Time/Duration significance of 24 hours found in NSCs applicable in humans?

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Increase in LDH release and decrease in mitochondrial activity indicate cytotoxicity

Increased BAX protein expression and TUNEL-positive cells also indicate apoptosis

Acetyl-L-carnitine blocks Ag-NP induced NSC cell death

Toxic effect of Ag-NP related to mitochondrial membrane integrity and function

ROS is significant in Ag-NP induced neurodegeneration

Ag-NPs directly negatively affect NSC proliferation

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https://docs.google.com/presentation/d/14F200XRBeGnOVkk2zCM85qyQgHEENM3KGAWhmg5epZo/edit?usp=sharing

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NSCs undergo cytotoxicity after 24 hours of Ag exposure at 5 μg/ml.

(A) LDH is an enzyme found in viable cells with intact membranes. LDH levels indicate leakage into NSC medium. Significant increase of LDH after 5 μg/ml in a dose-dependent manner.

(B) MTT is an indicator for metabolic mitochondrial activity. Lower levels reflect cytotoxic events within the cell. Significant levels of MTT were observed after 5 μg/ml of Ag exposure.

(C) and (D) reflect the number of cells that undergo apoptosis after 5 μg/ml of Ag exposure compared to controls via TUNEL-Assay. (E) demonstrates this via graph.