# data[].attributes.body
(Clemons & Seeman, 2016)

# data[].attributes.body
Individual: guinea pigs are active 20 or more hours per day with short period of sleep. (for singly housed)

No distinct circadian pattern but avoid intense light.

Aggression: In response to potential threats, attend postures can be observed with a frozen position with the front legs extended and eyes and ears forward.

Communication within the species: Guinea pig vocalizations

Social: Primary physical contact between adult guinea pigs is huddling; little or no grooming among individuals except at mating or at mothers rearing young.

(Clemons & Seeman, 2016)

# data[].attributes.body
FULL references

# data[].attributes.body
Short, squat animal with a blunt head and short neck.

Body is square with a rounded, tailless rump.

Mouth is triangular with a split upper lip.

External ears are large, oval, and sparsely haired.

Adult guinea pigs have 20 teeth.

Both sexes have a single pair of abdominal mammae.

(Clemons & Seeman, 2016)

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PHYSIOLOGICAL (CARDIOVASCULAR & RESPIRATORY) PARAMETERS OF THE GUINEA PIG

# data[].attributes.subject
PHYSIOLOGICAL (CARDIOVASCULAR & RESPIRATORY) PARAMETERS OF THE GUINEA PIG

# data[].attributes.body
(Clemons & Seeman, 2016)

# data[].attributes.body
In 1907 - Vitamin C was discovered using guinea pigs as guinea pigs just like humans, require Vitamin C in its diet.

In 1882 - The bacterium, Mycobacterium tuberculosis causing tuberculosis (TB) was discovered by Robert Koch, a German scientist.

In 1919 - Acquired immunity was discovered based on the immune reactions of guinea pigs in response to inoculation with blood from people with yellow fever.

(Understanding Animal Research, n.d.)

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Standardized guinea pig model for Q fever vaccine reactogenicity

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Standardized guinea pig model for Q fever vaccine reactogenicity

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Laurie A. Baeten,

Brendan K. Podell,

Ann E. Sluder,

Anja Garritsen,

Richard A. Bowen,

Mark C. Poznansky

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Abstract

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls. An initial comparative study used viable C. burnetii to compare the routes of sensitizing exposure of guinea pigs (intranasal vs intraperitoneal), evaluation of two time points for antigen challenge (21 and 42 days) and an assessment of two routes (intradermal and subcutaneous) of challenge using the ruminant vaccine Coxevac as the antigenic control. Animals sensitized by intraperitoneal exposure exhibited slightly larger gross reactions than did those sensitized by intranasal exposure, and reactions were more pronounced when skin challenge was performed at 42 days compared to 21 days post-sensitization. The intradermal route proved to be the optimal route of reactogenicity challenge. Histopathological changes at injection sites were similar to those previously reported and a scoring system was developed to compare reactions between groups receiving vaccine by intradermal versus subcutaneous routes. Based on the comparative study, a standardized protocol for assessment of vaccine reactogenicity in intranasally-sensitized animals was tested in a larger confirmatory study. Results suggest that screens utilizing a group size of n = 3 would achieve 90% power for detecting exposure-related reactogenic responses of the magnitude induced by Coxevac using either of two outcome measures.

Citation: Baeten LA, Podell BK, Sluder AE, Garritsen A, Bowen RA, Poznansky MC (2018) Standardized guinea pig model for Q fever vaccine reactogenicity. PLoS ONE 13(10): e0205882. https://doi.org/10.1371/journal.pone.0205882

Editor: Victor C. Huber, University of South Dakota, UNITED STATES

Received: May 22, 2018; Accepted: October 3, 2018; Published: October 12, 2018

Copyright: © 2018 Baeten et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: All funding for this study was provided by the United States Defense Threat Reduction Agency ( www.dtra.mil) through contract HDTRA1-15-C-0020 to Massachusetts General Hospital (MCP Principal Investigator). Under the prime contract to MGH, subcontract funding for aspects of the study was provided to Colorado State University (RAB subcontract Principal Investigator) and Innatoss Laboratories BV (AG subcontract Principal Investigator). The funding agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AG is an employee of Innatoss Laboratories BV. Innatoss Laboratories BV provided support in the form of salary for author AG, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the 'author contributions' section.

Competing interests: AG is an employee and shareholder of Innatoss Laboratories BV, which produces diagnostic tests for bacterial infections including Q fever. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have declared that no competing interests exist.

Introduction

Q fever is a zoonotic disease most often caused by exposure to infected ruminants and is prevalent in individuals with occupational exposures. Recent disease outbreaks in the Netherlands [ 1] and diagnosis among military personnel deployed to endemic regions [ 2- 7] have highlighted this as a re-emerging infectious disease. The causative agent Coxiella burnetii, an obligate intracellular organism, is difficult to cultivate and diagnostic testing is inconsistent/unreliable in both humans and animals [ 8].

The four Q fever vaccines that have been developed for use in humans exhibit variable efficacy [ 9]. Furthermore, all four vaccines have been found to have significant side effects in vaccinees with prior sensitization to C. burnetii, variably resulting in fever, malaise and inoculation site granulomatous reactions [ 10- 12]. These reactions are thought to be induced by a delayed hypersensitivity reaction to phase I LPS [ 12, 13]. As such there is a need for development of alternative effective vaccines that do not cause such hypersensitivity reactions in individuals previously exposed to C. burnetii.

In the evaluation of new vaccine candidates, a critical component is the ability to assess the potential for induction of hypersensitivity reactions. Guinea pig models have been described for the evaluation of vaccination reactions for the current vaccines in use [ 14- 19]; the majority of these studies used inactivated bacterial suspensions to immunize guinea pigs prior to skin testing. Here we describe a refinement of the guinea pig model for evaluation of hypersensitivity reactions to C. burnetii antigens, undertaken during the course of an ongoing vaccine development project [ 20]. In a comparative study of selected protocol variables, we evaluated guinea pig sensitization by different routes of infection with live C. burnetii, tested two routes of antigen challenge, and assessed the time course of reactogenic responses following antigen challenge. We employed Coxevac, a whole cell vaccine approved for annual use in ruminants in Europe and reported to induce granulomatous reactions after repeated use in cattle [ 21], as a standardized antigen control to probe the responses of sensitized and unsensitized animals to different doses of vaccine antigen challenge. Based on the results of the comparative study, we defined a standardized protocol that was tested in a larger confirmation study to establish a framework for evaluation of the reactogenic potential of future Q fever vaccine candidates in the guinea pig model.

Materials and methods

Materials

Bacteria.

C. burnetii Nine Mile strain ( Cb9M) phase 1 RSA 411 (lot 3662550) was obtained from BEI Resources (Manassas, VA, USA). The phase 1 status of the stock prior to propagation was confirmed by the supplier by confirming reactivity with hyperimmune serum. An axenic bacterial working stock was prepared by a single expansion passage in acidified citrate cysteine medium (ACCM-2) pH 4.75 (Sunrise Science Products, San Diego, CA, USA). Propagation was as described [ 22], seeding cell free culture media with 1 x 10 ⁶ genome equivalents (ge) of the Cb9M BEI Resources stock. Flasks were incubated for nine days on a shaker (75 rpm) at 37˚C in a 2.5% O ₂, 5% CO ₂ environment. Bacteria were recovered by centrifugation at 14,000 x g for 30 min, resuspended in sucrose phosphate buffer [ 23] and stored at -80˚C. The working stock was found to contain 2.17 x 10 ⁷ ge per μL as determined by qPCR (see below). Infectivity of the working stock was confirmed in both mice (A/J) and guinea pigs. Reproducible induction of anti- Coxiella antibodies in guinea pigs following inoculation with the working stock at a standardized infective dose of 10 ⁶ ge [ 24] was confirmed prior to initiation of reactogenicity studies.

Vaccine.

The vaccine, Coxevac, was obtained from CEVA Sante Animale (Lot 0101Eg1A, Libourne, France). This formalin inactivated phase I C. burnetii corpuscular antigen formulation is preserved with thiomersal and marketed for annual use in ruminants. Dosing in this study was based upon the assumption that Coxevac was standardized at 100 μg of antigen per mL [ 25]. The vaccine was determined to contain 86 μg of protein per mL (BCA protein assay, Pierce Biotechnology, Rockford, IL, USA) and qPCR indicated that the vaccine contained 1.3 x 10 ⁸ ge per mL.

Experimental animals and ethics statement

Guinea Pigs.

Female Dunkin-Hartley Crl:HA guinea pigs (300 g/35-42 days of age) were purchased from Charles River Laboratories, Wilmington, MA, USA. All guinea pigs were maintained under biosafety level 3 conditions in isolator cages (Smart Flow, Techniplast, Westchester, PA, USA) at the Regional Biocontainment Laboratory, Colorado State University, Fort Collins, Colorado, USA. Animals were provided water and guinea pig chow ad libitum. Dried fruit and hay pellets were offered for enrichment. Clinical evaluations were made daily on each animal throughout the study period to detect changes in body weight, body condition, behavior and activity level. Body temperature was documented at four-hour intervals by temperature recorders (Thermochron iButton, Maxim Integrated Products, Inc., Sunnyvale, CA, USA) implanted in the abdominal cavity via flank incision.

Animal research protocols were reviewed and approved by Colorado State University Institutional Animal Care and Use Committee (protocol number 16-6844A) and all activities were conducted in accordance with university and federal regulations.

Methods

Quantitative PCR (qPCR).

Genomic DNA was extracted from samples using standard techniques (QIAamp DNA mini blood kit, Qiagen, Valencia, CA, USA) and qPCR was performed to detect C. burnetii targets using LSI VetMax Coxiella burnetii Absolute Quantification kit (Life Technologies, Lissieu, France).

Serology.

Serological status was determined by ELISA using the Q Fever antibody test kit (IDEXX Laboratories Inc., Westbrook, ME, USA) with the secondary antibody replaced with peroxidase conjugated protein A/G at 1:10,000 dilution (Pierce Biotechnology, Rockford, IL, USA); the kit substrate is 3,3',5,5'-tetramethylbenzidine (TMB). Results are presented as optical density at 450 nm (OD ₄₅₀). Antibody titers were also assessed by an indirect fluorescent antibody assay (IFA) using Q fever IFA substrate slides (Focus Diagnostics, Cyprus, CA, USA); staining methods as described by Kersh [ 26] were modified for use with guinea pig sera. Briefly, dilutions of sera (1:16 and 1:256) were added to wells on slides pre-coated with C. burnetii Phase I and Phase II and binding was assessed following reaction with DyLight 488 fluorochrome conjugated goat anti-guinea pig IgG (H+L) at 1:1,000 dilution (Thermo Fisher Scientific, Rockford, IL, USA). Titers ≥ 1:128 were considered positive for anti- Coxiella antibodies.

Guinea pig sensitization.

Animals were chemically restrained (ketamine 40 mg/kg and xylazine 5 mg/kg, intraperitoneal (i.p.)) and inoculated with Cb9M by intranasal (i.n.) or i.p. administration with a target dose of 10 ⁶ ge [ 24] in a 100 μL volume of saline. Guinea pigs in the unsensitized control group received saline (100 μL) either i.p or i.n. Blood samples were collected from chemically restrained animals by venipuncture at days 0, 21, and 42 and at termination (day 28, 42, 49 or 63) for serum collection and evaluation of serological status.

Guinea pig antigen challenge and reactogenicity assessment.

Hypersensitivity testing was modified from that described by Wilhelmsen and Waag [ 14]. At day 21 or 42 post-infection, guinea pigs were chemically restrained (ketamine 40 mg/kg and xylazine 5 mg/kg, i.p.) and the dorsal lumbar surface was shaved. Subcutaneous (s.c.) and intradermal (i.d.) inoculations were made in 100 μL volumes with saline or vaccine at 1, 2, or 4 μg. The inoculation sites were evaluated at 8 hours post-injection and daily thereafter for 7 or 21 days. The diameter of erythema and thickness of induration at each inoculation site were measured with calipers. At 7 or 21 days post-challenge, inoculation sites were excised from euthanized animals by punch biopsy (12mm AcuPunch, Acuderm Inc., Ft. Lauderdale, FL, USA) and organs (heart, lung, liver, spleen) were collected in 10% formalin for paraffin embedding and sectioning. Sections were stained with hematoxylin and eosin for histopathological analysis. The extent of histological lesions in each of two non-adjacent skin biopsy sections from i.d. and s.c. inoculation sites were evaluated by a single blinded pathologist (BKP) in their entirety to develop a scoring system on a graded scale of 0-5 ( Table 1). The entire slide set was then re-evaluated in a blinded manner to obtain final scores. All sections were deemed appropriate for scoring as all contained targeted areas of dermis and subcutaneous tissue.

Statistics.

Statistical analyses were performed using GraphPad PRISM (version 5). Group mean comparisons using one-way ANOVA with Tukey's multiple comparison test or unpaired t test (one-tailed) were used to evaluate differences among groups. Unless otherwise noted P values of ≤0.05 were considered statistically significant.

Estimates of statistical power and group size requirements were made using computational tools available from the MGH Biostatics Center ( http://hedwig.mgh.harvard.edu/sample_size/size.html) and the University of Iowa Department of Statistics and Actuarial Science (Lenth, R.V., Java Applets for Power and Sample Size, Retrieved 21 September 2018; http://www.stat.uiowa.edu/~rlenth/Power).

Results

Comparative study of bacterial and antigen exposure routes

A comparative study was designed to evaluate reactogenic responses in the guinea pig model using different routes of sensitizing exposure to C. burnetii and of antigen challenge. Guinea pigs (n = 24) were randomly assigned to three groups ( Fig 1). Experimental animals were inoculated with a standardized dose of a defined working stock of Cb 9M by either intranasal (i.n.) or intraperitoneal (i.p.) administration (n = 8 per group). Guinea pigs in the unsensitized control group (n = 8) received saline either i.p or i.n. (n = 4 each route). At day 21 or 42 post-inoculation (p.i.), antigen challenge inoculations were made subcutaneously (s.c.) or intradermally (i.d.) as described in the Methods (n = 4 at each challenge time point). Each animal received inoculations of saline and Coxevac vaccine at 1, 2, and 4 μg at distinct challenge sites on the back. Two animals per challenge time point were terminated and tissues were harvested for histopathology at 7 days and at 21 days post-challenge (p.c.) (days 28 and 42 p.i. for animals challenged at day 21 p.i.; days 49 and 63 p.i. for animals challenged at day 42 p.i.). Reactions to antigen challenge were assessed by daily inspection for erythema and induration and by histopathology of challenge site skin biopsies taken at termination, as described in the Methods.

Clinical responses to inoculation with Cb9M

Anti- C. burnetii immune responses were confirmed in all guinea pigs inoculated with Cb9M by detection of antibody prior to vaccine antigen challenge (day 21 or 42 p.i.). No anti- C. burnetii antibody was detected by ELISA in guinea pig sera at day 0 (mean OD ₄₅₀ 0.118 ± 0.034) or in saline-inoculated guinea pigs at the time of antigen challenge (day 21 or 42 p.i.; group mean OD ₄₅₀ 0.156 ± 0.046). In contrast, high levels of antibody were detected in serum collected from Cb9M-inoculated animals. At day 21 p.i. serum antibody levels were characterized by mean OD ₄₅₀ values of 3.616 ± 0.198 for the i.p. group and 3.391 ± 0.087 for the i.n. group. At day 42 p.i., mean OD ₄₅₀ values were 3.593 ± 0.425 for the i.p. group and 3.847 ± 0.052 for the i.n. group. No significant differences in OD ₄₅₀ values (p = 0.625) were noted between inoculation routes or days post-challenge ( Fig 2), although OD ₄₅₀ values near the maximum detection limit for the instrumentation used for this assay (4.600) limited discrimination between robustly responding groups. Antibody titers, as measured by IFA, were found to be ≥ 1:256 in all C. burnetii- inoculated guinea pigs.

Despite the antibody responses indicating bacterial exposure, no apparent changes in guinea pig behavior or disease symptoms were noted following inoculation with Cb9M. The group mean body weight at day 0 was 416.5 gm (range 320-480 g). Guinea pigs inoculated by the i.p. route experienced a 4% drop in weight (mean 28 g; range 5-60 g) between days 0-7 p.i., which was statistically different from weights of saline-inoculated control guinea pigs in the same time window (p value = 0.003) ( Fig 3). During this time the i.n. inoculated group did not experience weight loss, and though they exhibited less weight gain than did saline controls the differences were not statistically significant. Body temperature, as determined by i.p. iButton readings, generally remained within the normal range throughout the study; the group baseline mean body temperature was 39.2˚C compared to the published normal range of 38-40˚C [ 27]. Transient body temperature spikes were noted between days 4 and 8 in the i.p. inoculated group (mean 40.2˚C; range 38.9-41.6˚C). Mean group body temperatures remained within the normal range for the i.n. inoculation group, with two of four individuals experiencing a transient spike (< 4 hours) up to 41.5˚C between days seven and ten. Body temperatures remained within the normal range for all animals following antigen challenge.

Intradermal and subcutaneous challenge reactions

Sensitization of Cb9M-inoculated animals to Coxiella antigens was confirmed using the formalin-inactivated phase I whole cell vaccine Coxevac as a standardized challenge antigen. Responses to both s.c. and i.d. antigen challenge were assessed. Gross abnormalities were not observed at the s.c. inoculation sites in animals until day 10 p.c., with some reactions persisting until day 21 p.c. These reactions appeared as minor subcutis swelling with no erythema or induration noted. The i.d. vaccine challenge sites in Cb9M-inoculated animals developed detectable erythema and induration at all doses of vaccine tested ( Fig 4), generally within eight hours after inoculation. Reactions were most consistent at 2 and 4 μg doses of vaccine, and most severe (visibly darker) at day 42 p.i. challenge inoculation sites relative to the day 21 p.i. challenge sites ( Table 2). In general, i.d. reaction size in Cb9M-inoculated animals reached full extent by three to seven days p.c. and persisted until at least day 21 p.c. (the longest time point assessed; Figs 4 and 5; S1 Fig). Gross reactions at i.d. challenge sites were also noted in some saline control animals, though visible erythema generally decreased in these animals by day 21 p.c. ( S1 Fig). No reactions were observed to challenge with a control saline solution in either saline- or C. burnetii-sensitized animals.

Histological changes

Visceral organ lesions in sensitized animals were limited to a few microgranulomas in the liver and sporadic hepatocellular necrosis. Splenic lesions were limited to increased neutrophils without tissue destruction. No significant differences in organ lesions were noted between animals sensitized using the i.p. and i.n. inoculation routes. Histopathological evaluation of guinea pig skin reactions at vaccine inoculation sites utilized scoring criteria based upon the cellular composition, distribution of inflammation, overall size of the inflammatory response, infiltration of surrounding tissue, vascular sequelae and presence/absence of tissue injury and/or necrosis ( Table 1 and Fig 6). Inflammation was observed at both intradermal and subcutaneous vaccine inoculation sites in both unsensitized and Cb9M-sensitized guinea pigs ( Fig 7). Inflammation was more severe in Cb9M-sensitized animals, and at intradermal challenge sites compared to subcutaneous challenge sites, consistent with the observed levels of gross erythema and induration. The degree of inoculation site inflammation was proportional to vaccine dose, with very little difference noted between 2 and 4 μg doses ( Table 3 and Fig 8). The inflammation was more severe overall in guinea pigs sensitized by the i.p. route compared to the i.n. route. In sensitized animals microabscesses were more frequently observed at day 21 p.c. than at day 7 p.c. ( Fig 7), consistent with ongoing inflammatory tissue damage. Among the unsensitized guinea pigs, there was limited histopathological difference between skin samples collected at day 7 p.c. and day 21 p.c. indicating that vaccine alone can induce a granulomatous inflammation that fails to resolve within the time frame evaluated but does not result in progressive tissue damage.

Reproducibility of standardized protocol using i.n.-sensitization

The results of the initial comparative study indicated that detectable sensitization to Coxiella antigens was achieved using both i.p. and i.n. infection routes, with i.p.-sensitized animals showing more pronounced reactions compared to saline control animals ( Fig 5 and Fig 8). The i.n. route is a better mimic of the natural route of exposure leading to antigen sensitization and vaccine reactogenicity in humans. Therefore, an expanded replicate study was performed to confirm the reproducibility of guinea pig sensitization following i.n. exposure, and to estimate the group size that would be required to detect a hypothesized difference in reaction severity between control and i.n.-sensitized animals. Preliminary estimates made using the limited data available from the comparative study suggested that a group size of 4-12 animals would have 80% power to detect a difference between control and i.n.-sensitized animals, depending on the effect size hypothesized. Based on these estimates and the capacity of the BL3 facility for housing an extended guinea pig study, the replicate study utilized group sizes of n = 9 for Cb 9M-sensitized animals and n = 4 for saline control animals.

Antigenic challenge was standardized as 2 μg Coxevac administered i.d. 42 days after infection. Reactions were assessed 7 days post-challenge, when erythema was expected to have reached full extent but before milder reactions may have begun to resolve. As in the initial study, reactions to antigenic challenge were observed in all Cb9M-sensitized and some saline control guinea pigs ( Fig 9). No reactions were observed in response to negative control saline challenge. Reactions were more severe and consistent in the Cb 9M-sensitized animals than in the control animals, as measured both by extent of erythema (P = 0.002) and by histopathology (P = 0.002). Based on these data, group sizes of n = 3 would have ≥90% power to detect a difference in mean reaction severity between control and sensitized animals of the magnitude observed in this study, at a significance level of 0.05 in a one-sided t test, using either erythema or histological score as an outcome measure.

Discussion

Guinea pigs have been used for Coxiella infection models since the identification of this pathogen in the early 1950s. Guinea pigs are also used as the animal model of choice for the evaluation of several other hypersensitivity reactions such as contact dermatitis [ 28], responses to respiratory allergens and irritants [ 29], drug reactions [ 30] and reaction to other infectious disease agents [ 31, 32].

The term reactogenicity was coined by the Federal Drug Administration to describe the expected adverse vs unexpected (local and systemic) reactions noted during clinical trials of drugs, vaccines, and other pharmaceuticals [ 33]. In most cases, these reactions were secondary to adjuvants and/or were short term reactions with no long term sequelae. Reactions were scored 0-4 with regard to fever, injection site pain, induration and erythema [ 34]. In the case of C. burnetii vaccines, granulomatous reactions are common and can persist for several weeks [ 35, 36].

The reactogenicity model described here using viable organism for sensitization and a ruminant vaccine as an antigenic control exhibited similar reactions to those reported by others [ 14, 15]. Our findings confirm reactogenicity is not strain dependent as skin reactions to Coxevac ( Cb9M) were similar to those reported using antigen preparation from the Henzerling and Ohio strains [ 16] and the California 76 strain [ 18]. In addition, the skin lesions observed in guinea pigs indicate that Coxevac gives comparable reactions to those reported for Q-Vax [ 14, 15], the vaccine currently available for use in humans (Seqirus, Victoria, Australia).

Reactogenic responses of Cb9M-sensitized guinea pigs to antigen challenge were relatively stable overall to changes in several protocol variables, using two measures of response (visible extent of erythema and histopathological scoring). Our study provides additional information with respect to routes of sensitization with viable organism and challenge with Coxevac. In the context of model standardization, the i.p. route is likely to deliver a more consistent dose of organism compared to the i.n. route. However, the i.n. route is a better mimic of the natural route of exposure, and we demonstrated that a standardized i.n. dose of viable C. burnetii reproducibly sensitized animals to subsequent vaccine antigen challenge.

The i.d. challenge route is preferable to the s.c. route in a standardized screening model as the gross reactions are more easily detected and more consistent following i.d. challenge. The i.d. route also mimics the pre-vaccination screening currently used in humans [ 15, 37]. In our initial comparative study, consistent reactions were observed when the Coxevac antigen challenge was administered either 21 or 42 days after the sensitizing infection, suggesting that there is an operational window within which a reactogenicity screening protocol can be standardized to accommodate the work flow of a given program. In our confirmatory study we administered antigen challenge at day 42 p.i., as reactions were generally more pronounced at this challenge time in the comparative study.

A range of Coxevac vaccine doses elicited detectable reactions in Cb9M-sensitized guinea pigs, with visible erythema peaking by 3-7 days post-challenge and persisting throughout a 21-day post-challenge monitoring period. More variable reactions were observed in some unsensitized control guinea pigs, with any visible erythema generally diminishing by 7-10 days post-challenge. Histological evaluation of challenge site biopsies revealed generally mild inflammation in some control animals, with little difference between biopsies taken at day 7 and day 21 post-challenge. In contrast, challenge site biopsies from Cb 9M-sensitized animals exhibited pronounced inflammation at day 7 p.c., with more extensive inflammation and the presence of microabscesses observed at day 21 p.c., consistent with ongoing inflammatory tissue damage. These results suggest that when testing novel vaccine candidates, assessment at day 7 p.c. should allow detection of acute reactions prior to resolution of milder reactions, while examination at longer post-challenge time points can identify sustained and progressive reactions.

The standardized protocol utilized in our confirmatory study is designed to be used in initial evaluation of the reactogenicity potential of novel Q fever vaccine candidates or vaccine components. The results of the confirmatory study suggest that screens utilizing group sizes of n = 2 or n = 3 would achieve 80% or 90% power, respectively, to detect exposure-related reactogenic responses of the magnitude induced in intranasally-sensitized guinea pigs by i.d. antigen challenge with 2 μg Coxevac. Milder reactions observed in an initial assessment of a novel vaccine would need to be further confirmed with a larger follow up study. Characterization of any observed reactogenic effects in the guinea pig model can inform preclinical and clinical vaccine development decisions. For example, if a novel vaccine elicits comparable reactions in Coxiella-sensitized and control animals, an assessment of lower vaccine doses in control and sensitized animals can be performed to determine if the vaccine is differentially reactogenic or a general irritant. If a vaccine candidate induces a reaction by day 7 p.c. in the standardized protocol described here, further evaluation would be of interest to determine if the reaction resolves more rapidly than does the reaction to Coxevac, since an efficacious vaccine with shorter-lived side effects than those observed with whole cell vaccines could still have value.

Supporting information

S1 Fig. Daily zone of erythema at intradermal challenge sites after day 42 challenge.

Data are shown as the zone of erythema (mm) at intradermal vaccine injection sites after day 42 p.i. challenge, grouped by challenge dose and route of sensitization with C. burnetii Nine Mile (Cb9M) (i.p., intraperitoneal; i.n., intranasal) or saline controls. Measurements are presented as group mean (n = 2) from day 0 to day 21 post-challenge.
https://doi.org/10.1371/journal.pone.0205882.s001
(TIF)

Acknowledgments

The authors wish to thank Nicole Marlenee, Stephanie Porter, Danielle Adney, Paul Gordy, Airn Hartwig, the Q-VaxCelerate consortium members and the CSU Experimental Pathology Facility for technical assistance in the study. The following reagent was obtained through BEI Resources, NIAID, NIH: Coxiella burnetii, Strain Nine Mile Phase I RSA/411, NR-134. We thank Ceva Biomune for providing the Coxevac vaccine.

References

1. 27. Terril LA, Clemons DJ. The Laboratory Guinea Pig. Boca Raton, FL: CCR Press; 1998.

2. 34. DMID Interventional Protocol Template. In: Diseases NIoAaI, editor. Version 5.0 ed: National Institute of Health; 2011.

# data[].attributes.attachment_link.title
Standardized guinea pig model for Q fever vaccine reactogenicity

# data[].attributes.attachment_link.description
Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates.

# data[].attributes.body
Q fever is a zoonotic disease due to the exposure to the  causative agent Coxiella burnetii. The current four Q fever vaccines contain significant adverse effects and thus an alternative vaccine is required to be developed to avoid such hypersensitivity reactions in individuals previously exposed to C. burnetii.
* Reactogenicity: describe the expected adverse vs unexpected (local and systemic) reactions noted during clinical trials of drugs, vaccines, and other pharmaceuticals

Animals were chemically restrained (ketamine 40 mg/kg and xylazine 5 mg/kg, intraperitoneal (i.p.)) and inoculated with the vaccine Coxevac (Cb 9M) by intranasal (i.n.) or i.p. administration with a target dose of 10⁶ ge in a 100 μL volume of saline.

Guinea pigs under control group received saline (100 μL) either i.p or i.n.

Blood samples were collected from chemically restrained animals by venipuncture at days 0, 21, and 42 and at termination (day 28, 42, 49 or 63) for serum collection and evaluation of serological status.

Findings:

- Reactogenicity is not strain dependent as skin reactions to Coxevac (Cb9M).
- Skin lesions observed in guinea pigs indicate that Coxevac gives comparable reactions to those reported for Q-Vax, the vaccine currently available for use in humans.
- When testing novel vaccine candidates, assessment at day 7 p.c. should allow detection of acute reactions prior to resolution of milder reactions, whereas examination at longer post-challenge time points can identify sustained and progressive reactions.
- Characterization of any observed reactogenic effects in the guinea pig model can inform preclinical and clinical vaccine development decisions.
(Baeten, 2018)

# data[].attributes.headline
A Guinea Pig Model of Airway Smooth Muscle Hyperreactivity Induced by Chronic Allergic Lung Inflammation: Contribution of Epithelium and Oxidative Stress

# data[].attributes.subject
A Guinea Pig Model of Airway Smooth Muscle Hyperreactivity Induced by Chronic Allergic Lung Inflammation: Contribution of Epithelium and Oxidative Stress

# data[].attributes.attachment_link.readable_text
Luiz Henrique César Vasconcelos 1†, Maria da Conceição Correia Silva 1†,

Alana Cristina Costa ²,

Giuliana Amanda de Oliveira ², Iara Leão Luna de Souza ¹, Fernando Ramos Queiroga ¹, Layanne Cabral da Cunha Araujo ³, Glêbia Alexa Cardoso ⁴,

Renato Fraga Righetti 5,6, Alexandre Sérgio Silva 4,7,

Patrícia Mirella da Silva 1,8,

Carla Roberta de Oliveira Carvalho 3,9, Giciane Carvalho Vieira ¹⁰,

Iolanda de Fátima Lopes Calvo Tibério ⁵,

Fabiana de Andrade Cavalcante 1,11 and

Bagnólia Araújo da Silva 1,12*

Programa de Pós graduação em Produtos Naturais e Sintéticos Bioativos, Centro de Ciências da Saúde, Universidade Federal da Paraíba, João Pessoa, Brazil

Graduação em Farmácia, Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal da Paraíba, João Pessoa, Brazil

Programa de Pós graduação em Ciências (Fisiologia Humana), Instituto de Ciências Biológicas, Universidade de São Paulo, São Paulo, Brazil

Programa Associado de Pós graduação em Educação Física, Universidade Federal da Paraíba/Universidade do Pernambuco, João Pessoa, Brazil

Faculdade de Medicina FMUSP, Universidade de São Paulo, São Paulo, Brazil

Hospital Sírio Libanês, São Paulo, Brazil

Departamento de Educação Física, Centro de Ciências da Saúde, Universidade Federal da Paraíba, João Pessoa, Brazil

Departamento de Biologia Molecular, Centro de Ciências da Saúde, Universidade Federal da Paraíba, João Pessoa, Brazil

Departamento de Biofísica e Fisiologia, Instituto de Ciências Biológicas, Universidade de São Paulo, São Paulo, Brazil

Departamento de Morfologia/Centro de Ciências da Saúde/Universidade Federal da Paraíba, João Pessoa, Brazil

Departamento de Fisiologia e Patologia/Centro de Ciências da Saúde/Universidade Federal da Paraíba, João Pessoa, Brazil

Departamento de Ciências Farmacêuticas/Centro de Ciências da Saúde/Universidade Federal da Paraíba, João Pessoa, Brazil

Asthma is a heterogeneous disease of the airways characterized by chronic inflammation associated with bronchial and smooth muscle hyperresponsiveness. Currently, different murine models for the study of asthma show poor bronchial hyperresponsiveness due to a scarcity of smooth muscle and large airways, resulting in a failure to reproduce smooth muscle hyperreactivity. Thus, we aimed to standardize a guinea pig model of chronic allergic lung inflammation mimicking airway smooth muscle hyperreactivity observed in asthmatics (Asth). Animals were randomly divided into a control group (Ctrl), which received saline (0.9% NaCl), and the Asth group, subjected to in vivo sensitization with ovalbumin (OVA) nebulization. Morphological analysis was performed by hematoxylin-eosin staining. Bronchial hyperresponsiveness was evaluated by nebulization time in the fifth, sixth, and seventh inhalations (NT5-7) and tracheal isometric contractions were assessed by force transducer. Total antioxidant capacity was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and protein expression by Western blot. Histologically, the Asth group developed peribronchial cellular infiltrate, epithelial hyperplasia and smooth muscle thickening. After the fourth nebulization, the Asth group developed bronchial hyperreactivity. The trachea from the Asth group contracted after in vitro stimulation with OVA, differing from the Ctrl group, which showed no response. Additionally, airway smooth muscle hyperreactivity to carbachol and histamine was observed in the Asth group only in intact epithelium preparations, but not to KCl, and this effect was associated with an augmented production of reactive oxygen species. Moreover, lung inflammation impaired the relaxant potency of isoproterenol only in intact epithelium preparations, without interfering with nifedipine, and it was found to be produced by transforming growth factor-β negative modulation of β adrenergic receptors and, furthermore, big-conductance Ca ²⁺-sensitive K⁺ channels. These effects were also associated with increased levels of phosphatidylinositol 3-kinases but not extracellular signal-regulated kinases 1/2 or phosphorylation, and augmented α-actin content as well, explaining the increased smooth muscle mass. Furthermore, pulmonary antioxidant capacity was impaired in the Asth group. Therefore, we developed a standardized and easy-to-use, reproducible guinea pig model of lung inflammation that mimics airway smooth muscle hypercontractility, facilitating the investigation of the mechanisms of bronchial hyperresponsiveness in asthma and new therapeutic alternatives.

Introduction

Asthma is a chronic inflammatory disease of the airways in which many different innate and adaptive cells of the immune system act together with epithelial cells to promote excessive mucus production, airway wall remodeling, smooth muscle thickening and lumen narrowing ( Lambrecht and Hammad, 2015), in addition to bronchial hyperresponsiveness, characterized as an increase in airway smooth muscle sensitivity and responsiveness to bronchoconstrictors ( Baraldo et al., 2014) as well as a decrease in response to bronchodilator stimuli ( Brehm et al., 2015).

Bronchial hyperresponsiveness results from some structural changes in airway morphology due to the inflammatory process ( Amin, 2015). There is a strong relationship between Th2 cytokines and the pathognomonic airway changes characteristic of asthma remodeling. Among these changes, the most prominent feature is the increase in smooth muscle mass ( Carroll et al., 1993) due to hypertrophy and hyperplasia, as well as the differentiation and migration of mesenchymal cells to the smooth muscle cell layer ( Schmidt et al., 2003; Hirst et al., 2004; Bergeron et al., 2009). These phenomena are responsible for the increased contractility in asthma, the main causal factor of bronchial hyperresponsiveness ( Lauzon and Martin, 2016).

Furthermore, early and late reactions in acute asthma involve changes in the control of airway smooth muscle function of neurogenic and non-neurogenic origin. Additionally, epithelial damage, thickening of the mucosa due to local vasodilation and edema and mucous secretions in the airway lumen are all involved in the development of airway hyperresponsiveness ( Bousquet et al., 2004).

Asthma is also characterized by an overload of reactive oxygen species (ROS), which causes oxidative stress and changes in the functions of various components of the respiratory system ( Morwood et al., 2005; Bishopp et al., 2017). The main ROS involved are superoxide anion (O ₂⁻), hydroxyl radicals (OH⁻) and hydrogen peroxide (H ₂O ₂), which lead to the formation of another variety of ROS ( Loxham et al., 2014). In addition, asthma alters the mediators responsible for regulating airway smooth muscle tone, namely agonists, such as acetylcholine (ACh), histamine, prostaglandin D ₂ (PGD ₂), neurokinins and cysteinyl leukotrienes (cysLTs), which act by pharmacomechanical coupling in which binding to pharmacological receptors activates the phospholipase C effector system, leading to increased cytosolic Ca ²⁺ levels and smooth muscle contraction ( Pelaia et al., 2008; Alexander et al., 2013). This event can also be activated by electromechanical coupling, dependent on membrane depolarization and the opening of voltage-sensitive Ca ²⁺ channels ( Bolton, 1979).

Several asthma animal models have been established to investigate relevant pathological mechanisms. Mice have traditionally been used to elucidate the inflammatory mechanisms of respiratory diseases; however, mediators that regulate smooth muscle tone and determine bronchial responsiveness in these animals differ considerably from those found in humans ( Canning, 2003). In addition, murine pulmonary structure is characterized by large and sparse airways in smooth muscle tissue ( Zosky and Sly, 2007) as well as less responsiveness to several bronchoconstrictor stimuli implicated in the pathophysiology of asthma, such as histamine, cysLTs, neurokinins, and prostanoids, including PGD ₂ and thromboxane A ₂ (TxA ₂) ( Canning, 2003). Differently, guinea pigs display greater proximity to humans in relation to smooth muscle response and autonomic reflex. They also release mainly histamine and cysLTs in response to mast cell stimulation, as well as showing a pharmacological receptor profile approaching humans,' which explains the great similarity of responses after exposure to drugs ( Ressmeyer et al., 2006).

Although there are many animal models of asthma, most of these models focus on acute inflammation based on allergen exposure for a short period of time ( McMillan and Lloyd, 2004). In addition, many models fail to reproduce human airway hyperresponsiveness characteristics ( Goldie et al., 1986).

Therefore, this study was conducted to develop a standardized guinea pig model of chronic allergic lung inflammation that mimics the characteristics of contractile hyperreactivity and relaxant hyporesponsiveness of airway smooth muscle, similar to that occurring in human asthma, thus providing a useful tool for the study of bronchial hyperresponsiveness mechanisms and the search for new antiasthmatic drugs.

Materials and Methods

Animals

Male and female adult guinea pigs ( Cavia porcellus), weighing 300-500 g, were obtained from the Bioterium Professor Thomas George of Universidade Federal da Paraíba (UFPB). The animals were maintained on a 12-h light-dark cycle (lights on from 6 am to 6 pm), under controlled ventilation and temperature (21 ± 1°C) with free access to food (Presence ®) and water. The experimental procedures were performed following the guidelines for the ethical use of animals in applied etiology studies ( Sherwin et al., 2003), the Brazilian Guide for the Production, Maintenance or Use of Animals in Teaching or Scientific Research Activities, from Conselho Nacional de Controle de Experimentação Animal (CONCEA) ( Silva et al., 2016), and approved by the Ethics Committee on Animal Use of UFPB (Protocol No. 0410/13).

Chemicals

Calcium chloride (CaCl ₂), magnesium sulfate (MgSO ₄), potassium chloride (KCl) and sodium chloride (NaCl) were purchased from Vetec Química Fina Ltda. (Brazil). Glucose and sodium bicarbonate (NaHCO ₃) were obtained from Dinâmica (Brazil). Hydrochloric acid (HCl), potassium monobasic phosphate (KH ₂PO ₄) and sodium hydroxide (NaOH) were purchased from Nuclear (Brazil). Solutions of these substances, except for glucose, NaCl, and NaHCO ₃, were prepared in distilled water and kept under refrigeration.

1,1-Diphenyl-2-picrylhydrazyl (DPPH), apocynin, arachi donic acid (AA), carbamylcholine hydrochloride (carbachol, CCh), dithiothreitol, eosin, ethylenediaminetetraacetic acid (EDTA), formaldehyde, histamine dihydrochloride, iberiotoxin (IbTX), isoproterenol, Mayer's hematoxylin, methane hydroxymethylamine (Tris), nifedipine, ovalbumin (OVA) (grade V), tempol and Tween 20 were purchased from Sigma-Aldrich (Brazil). Catalase was obtained from Cayman Chemical (Brazil) and saline (0.9% NaCl) was from Fresenius Kabi LTDA (Brazil). The carbogen mixture (95% O ₂ and 5% CO ₂) was acquired from White Martins (Brazil).

All reagents used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Bradford's reagent, Laemmli's buffer, sodium fluoride; sodium pyrophosphate, sodium vanadate and Triton X 100 were purchased from Bio-Rad (Richmond, CA). Nitrocellulose membranes and kits for chemiluminescence detection were obtained from Amersham (United Kingdom). Anti-ERK1/2, anti-SOD and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States), anti-PI3K antibody from Abcam plc. (United Kingdom) and biotinylated goat anti-rabbit secondary antibody from Vector Laboratories (Burlingame, CA, United States).

The reagents used in the immunohistochemical analysis, including ABC Kit by Vectastain PK-6102 (anti-mouse) and the chromogen 3,3′-diaminobenzidine (3,3′-DAB), were purchased from Dako (United States). Harris hematoxylin was obtained from Merck.

Induction of Chronic Allergic Lung Inflammation in Guinea Pig

The animals were randomly divided into two groups (5-8 animals/group): control (Ctrl) and chronic allergic lung inflammation (Asth). For nebulization, the guinea pigs were individually placed in a closed polyacrylic box coupled to an ultrasonic nebulizer. Soon after, they were nebulized with a saline solution of OVA for a maximum of 15 min or until the onset of respiratory distress, defined as the presence of sneezing, coryza, coughing and/or drawing of the thoracic wall; Ctrl received only saline. The time that the guinea pigs remained in nebulization was defined as inhalation time.

The protocol consisted of seven inhalations performed over a period of 4 weeks at 96-h intervals between each inhalation, with OVA concentration being increased (1 to 5 mg/mL) to avoid tolerance. In the first four inhalations, the guinea pigs were subjected to 1 mg/mL OVA; in the fifth and sixth inhalations, the animals received OVA 2.5 mg/mL; and in the seventh inhalation, OVA concentration was increased to 5 mg/mL. At 72 h after the last inhalation, the animals were euthanized by cervical dislocation followed by sectioning of cervical vessels for experimentation (Figure 1). The control animals were subjected to the same inhalation procedure but received only saline (adapted from Tibério et al., 1997; Angeli et al., 2008; Pigati et al., 2015).

Figure 1. Protocol for induction of chronic allergic lung inflammation in guinea pigs. OVA: ovalbumin.

Evaluation of Chronic Allergic Lung Inflammation Effects on Lung and Bronchial Morphology of Guinea Pigs

The animals were euthanized by guillotine, and the lung and the right extrapulmonary bronchus were isolated, fixed in 10% formaldehyde for 24 h. The tissues were then subjected to standard histological procedures as follows: (1) dehydration with an increasing alcohol series of 70% for 24 h, 80, 96, and 100% (three baths) for 1 h each; (2) clearing in 100% xylene:alcohol (1:1) for 1 h, followed by two baths in pure xylene for 1 h each; and (3) embedding in paraffin by passing the sample through two baths of liquid paraffin (heated to 50°C) for 1 h each. Then, the samples were embedded in new paraffin. The blocks obtained were cut to 5 μm thick and stained with Mayer's hematoxylin/eosin ( Howard et al., 2004). For a panoramic analysis of the histological section, the slides were analyzed at ×40 (1500 μm) and ×100 (600 μm) magnification.

The aspects evaluated were bronchial wall structure, tissue integrity and cell migration. For each experimental group, 5 slides of 5 different animals were analyzed. The photomicrographs of the slides were captured with a camera coupled to a light microscope. The images were calibrated using the Motic Plus program according to the objectives used: 4× and 10×. The histological analysis of the slides was performed by a trained operator who qualitatively analyzed the histological parameters and then evaluated them statistically by score (below).

1. Preserved histoarchitecture of the airways with the absence of perivascular and peribronchiolar cellular infiltration;

2. Mild degree: an increase of less than 25% compared to control;

3. Moderate degree: an increase of 25 to 49% compared to control;

4. Pronounced degree: an increase of 50 to 75% compared to control; and

5. Very pronounced degree: an increase of more than 75% compared to control.

Evaluation of Chronic Allergic Lung Inflammation Effects on Guinea Pig Nebulization Time

The time elapsed for guinea pigs to begin showing signs of respiratory distress during inhalation time, which lasted up to a maximum of 15 min, was assessed, as well as the development of respiratory restrictions such as dyspnea or apnea ( Tibério et al., 1997). Comparisons were then made between the two groups.

Preparation of Guinea Pig Trachea

The animals were euthanized as described in Section "Evaluation of Chronic Allergic Lung Inflammation Effects on Lung and Bronchial Morphology of Guinea Pigs." The trachea was immediately removed and cleaned of fat and connective tissue; the tracheal rings (2-3 cm) were immersed in Krebs solution and bubbled with carbogen mixture (95% O ₂ and 5% CO ₂). The Krebs solution composition was (mM): NaCl (118.0), KCl (4.55), MgSO ₄ (5.7), KH ₂PO ₄ (1.1), CaCl ₂ (2.52), NaHCO ₃ (25.0) and glucose (11.0), with pH adjusted to 7.4. To record isometric contractions, tracheal segments were suspended in steel rods in organ baths (6 mL), connected to a force transducer (TIM 05), attached to an amplifier (AECAD04F) and connected to an A/D converter in a computer running AQCAD ® software (São Paulo, Brazil). The system contained a thermostatic pump model BT 60 that controlled the organ bath temperature. The trachea resting time was 60 min in a preload tension of 1 g (baseline). During the organ resting phase, the solution was changed every 15 min to avoid metabolite accumulation ( Tschirhart et al., 1987).

After the stabilization period, the tracheal rings were contracted with 1 μM CCh and isometric tension was recorded. When a stable contraction was attained, 0.1 mM arachidonic acid was added to the organ bath to confirm the presence of epithelium by the presence of arachidonic acid-induced relaxation equal to or greater than 50% of maximal tension. In some tracheal rings, the luminal surface was gently rubbed with Krebs-moistened cotton tip to remove the epithelial layer. The absence of epithelium was confirmed when arachidonic acid-induced relaxation was absent or lower than 10% of maximal tension ( Tschirhart et al., 1987).

Contractile Reactivity Measurement

Evaluation of Contractile Response to Ovalbumin in Guinea Pig Trachea in the Presence of Functional Epithelium

The trachea was assembled as previously described. After the stabilization period, when baseline remained constant, the intact epithelium tracheal rings were stimulated with 10 μg/mL OVA ( Schultz, 1910; Dale, 1913; Chand and Eyre, 1978; Jones et al., 1988), and the contraction amplitude was compared between the two groups. The maximum mean amplitude (measured in gram-force of tension) obtained for the control group was set as 100%, and the Asth group had their E max calculated by comparing their maximum amplitude with that of the control. All the contraction experiments were unpaired.

Evaluation of Contractile Response to CCh, Histamine, and KCl in Guinea Pig Trachea in Both the Presence and Absence of Functional Epithelium

The trachea was assembled as described in Section "Preparation of Guinea Pig Trachea." After the evaluation of epithelium integrity, a cumulative concentration-response curve was obtained to CCh (10 nM to 0.1 mM), a muscarinic receptor agonist ( Ouedraogo and Roux, 2014), histamine (10 nM to 3 mM), a histaminergic receptor agonist ( Ouedraogo and Roux, 2014) or KCl (1 mM to 0.3 M), a contractile agent of electromechanical mechanism ( Du et al., 2006).

In some experiments, the curves to CCh were obtained in the absence and presence of the following: 10 μM apocynin, an inhibitor of NADPH oxidase ( Sutcliffe et al., 2012), and 1 mM tempol, a superoxide dismutase mimetic ( De Boer et al., 2001) after 30 min incubation; and 100 IU/mL catalase ( Rhoden and Barnes, 1989) after 10 min incubation.

Contractile reactivity was evaluated on the basis of the maximum effect (E max) and the negative logarithm of the molar concentration of a substance that produced 50% of its maximal effect (pEC ₅₀) of the contractile agent, calculated from the concentration-response curves obtained. The maximum mean amplitude (measured in gram-force of tension) obtained for the control group was set at 100%, and the other groups had their E max calculated by comparing their maximum amplitude with that of the control. The comparisons were made only between preparations with and without epithelium, not making cross-comparisons (e.g., Ctrl without epithelium vs. Asth with epithelium). For the comparison between the preparations in the absence and presence of pharmacological tools, each group had its own control, referred to as 100%. All the contraction experiments were unpaired. All experiments were performed using the middle third of the trachea, avoiding the initial and final thirds, to prevent the effect of differences in the expression of receptors in the tracheal tissue, as described by Daniel et al. (1986) and Leff (1988).

Relaxant Reactivity Measurement

The trachea was assembled as described in Section "Preparation of Guinea Pig Trachea." After the epithelium integrity evaluation, we induced a contraction with 1 μM CCh and performed a cumulative concentration-response curve to isoproterenol (1 pM to 3 μM), a β-adrenergic receptors agonist ( Naline et al., 1994), or nifedipine (10 pM to 1 mM), a Ca V blocker ( Ahmed et al., 1985).

In some experiments, the curves to isoproterenol were obtained in the absence and presence of 10 μM SB431542, a type 1 TGF-β receptor antagonist ( Inman et al., 2002), after 60 min incubation, and 0.1 μM IbTX, a selective BK Ca blocker ( Yagi et al., 2002), after 20 min incubation.

Relaxant reactivity was expressed as the reverse percentage of the initial contraction force elicited by CCh and evaluated on the basis of pEC ₅₀ and E max, calculated from the obtained concentration-response curves.

Evaluation of Antioxidant Activity in Plasma and Lung

Plasma and Lung Tissue Preparation

After euthanasia, blood was collected by cardiac puncture. Next, the blood was placed in test tubes containing EDTA to obtain the plasma. In addition, lungs were homogenized with KCl (1:1). The samples were then centrifuged at 1198 × g for 10 min, and the supernatant was transferred to Eppendorf tubes and stored at -20°C ( Okafor et al., 2011; Silva et al., 2012).

Total Antioxidant Capacity Analysis

The procedure was based on the colorimetric analysis of DPPH reduction ( Brand-Williams et al., 1995). Briefly, 1.25 mg DPPH were dissolved in 100 mL of ethanol and the solution kept under refrigeration and protected from light. DPPH solution, 2 mL, and 100 μL of the sample were added to proper centrifuge tubes. The tubes were vortexed and allowed to stand for 30 min. They were then centrifuged at 7489 × g at 20°C for 15 min, and the supernatant was read in a spectrophotometer at 515 nm (Biospectro, model SP-220/Brazil). Results were expressed as the percentage of oxidation inhibition: AOA = 100 - [(DPPH⋅R) S/(DPPH⋅R) W × 100]. Where (DPPH⋅R) S and (DPPH⋅R) W correspond to the concentration of DPPH⋅ remaining after 30 min, measured in the sample (S) and blank (B) prepared with distilled water.

Western Blot Analysis

Lung samples were collected, homogenized in a protein extraction buffer (100 mM Tris, 10% sodium dodecyl sulfate, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA and 10 mM sodium orthovanadate) and boiled for 5 min. The tissue extracts were then centrifuged at 17,530 × g at 4°C for 40 min. Protein determination of the supernatants was performed by the Bradford method (Bio-Rad Laboratories, Hercules, CA, United States). The proteins were treated with Laemmli sample buffer containing 200 mM dithiotreitol. The aliquots (50 μg) were subjected to 6.5% SDS-10% PAGE. Electro-transfer of proteins from the gel to a nitrocellulose membrane was performed for 2 h at 120 V in a Bio-Rad miniature transfer apparatus ( Towbin et al., 1992).

The membranes were blocked for 2 h at room temperature with blocking solution (5% skim milk). The nitrocellulose membranes were incubated overnight at 4°C with primary antibodies anti-p85 PI3K (catalog number #4228, Cell Signaling, MA, United States), anti-ERK 1/2 (catalog number sc-292838, Santa Cruz, Dallas, TX, United States) or anti-p-ERK 1/2 (catalog number sc-81492, Santa Cruz, Dallas, TX, United States) diluted in blocking buffer with 3% albumin added, and then washed for 30 min in buffer without milk. The blots were subsequently incubated with peroxidase-conjugated secondary antibody for 1 h. Immunoblot visualization was carried out with the enhanced chemiluminescence system (Amersham Biosciences). The immunoblots were scanned and quantified using ImageJ software (imagej.net/Downloads).

Imunohistochemical for α-Actin Analysis

The lungs were obtained and paraffin-embedded, as described in Section " Evaluation of Chronic Allergic Lung Inflammation Effects on Lung and Bronchial Morphology of Guinea Pigs." The tissue sections were deparaffinized, rehydrated and washed three times for 10 min each with 3% H ₂O ₂ 10 V to inhibit endogenous peroxidase activity. Actin was detected with anti-α-actin antibody (dilution: 1:600; Santa Cruz Biotechnology, CA, United States - cod. SC-22613) for the detection of human smooth muscle actin markers (Dako). Sections of experimental and control (positive and negative) slides were incubated overnight with primary antibody. The following day, antigen retrieval for the smooth muscle actin marker was performed at high temperature in a citrate solution at pH 6.0 for 30 min. After this treatment, the sections, still immersed in the same solution, were cooled to room temperature for 20 min, washed in PBS three times and incubated with a secondary antibody using the ABC Kit by Vectastain (PK-6102, anti-mouse). The chromogen used was 3,3′-DAB (Dako). Finally, the sections were washed in water and counterstained with Harris hematoxylin (Merck). Optical density was used to evaluate the volume fractions of α-actin. Images were captured using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany), a digital camera (Leica DFC420 Leica Microsystems) and the image analysis software Image-Pro Plus 4.5 (Media Cybernetics, Bethesda, MD, United States). This software allowed a threshold of color shades to be developed. These shades represented the positive areas quantified in the previously determined area. Ten fields at ×400 magnification were evaluated per animal, and positive areas in the airway were expressed as a percentage of the total airway area ( Camargo et al., 2018).

Statistical Analysis

Results were expressed as the mean and standard error of the mean (SEM) and statistically analyzed using Student's t-test for single comparison or one-way analysis of variance (ANOVA) followed by the Tukey's post-test for multiple comparisons. Values were significantly different when p < 0.05. All data were analyzed by GraphPad Prism ® version 5.01 (GraphPad Software Inc., San Diego, CA, United States).

Results

Evaluation of Chronic Allergic Lung Inflammation Effects on Lung and Bronchial Morphology of Guinea Pigs

The histological analysis allowed an evaluation of the morphological structure of blood vessels, intra-and extrapulmonary bronchi, bronchioles and pulmonary alveoli (Figure 2, n = 5).

Figure 2. Photomicrographs of lung from guinea pigs in Ctrl and Asth groups, demonstrating infiltration of inflammatory cells in the peribronchial region and score of peribronchial cellular infiltrate (A), bronchial smooth muscle and score of muscle layer thickness (B), and bronchial epithelium and score of epithelial hypertrophy (C). Cellular infiltrate (red arrows), intrapulmonary bronchial smooth muscle (blue arrows), and bronchial epithelium (green arrows). Hematoxylin/eosin, ×40 (1500 μm) and ×100 (600 μm). The columns and vertical bars represent the mean and SEM, respectively ( n = 5). Student's t-test, ∗p < 0.05 (Ctrl vs. Asth). Ctrl: control group; Asth: chronic allergic lung inflammation group.

At ×40 magnification, the histological sections of the lungs obtained from Ctrl showed a normal histological appearance and preserved pulmonary histoarchitecture. Meanwhile, the lungs from Asth ( p < 0.05) showed large amounts of peribronchiolar and perivascular inflammatory cell infiltration and absence of infiltrates in the pulmonary alveoli (Figure 2A, n = 5). In addition, images at ×100 confirmed with greater structural detail the alterations seen at ×40. In this case, epithelial hyperplasia and intrapulmonary bronchial smooth muscle thickness were increased in animals in Asth ( p < 0.05), compared to Ctrl (Figure 2B, n = 5).

The histological sections of the extrapulmonary bronchi at ×40 revealed that animals in Asth displayed bronchial smooth muscle development due to hypertrophy and/or hyperplasia ( p < 0.05), compared to the histological sections of Ctrl (Figure 2C, n = 5).

Evaluation of Chronic Allergic Lung Inflammation Effects on Guinea Pig Nebulization Time

The inhalation period of 900 s was standardized for guinea pigs in Ctrl (Table 1, n = 8). In the first four nebulizations, the animals in Asth had similar nebulization times as Ctrl. However, chronic allergic lung inflammation reduced the times of the fifth (456.4 ± 96.7 s), sixth (400.8 ± 51.0 s) and seventh (273.3 ± 26.6 s) nebulizations in relation to Ctrl (900 s) ( p < 0.05) (Table 1, n = 8).

Table 1. Mean time of contact (s) of guinea pigs with ovalbumin from the first to fourth (1-4), fifth (5), sixth (6) and seventh (7) nebulizations.

No signs of respiratory distress were observed in guinea pigs in Ctrl. In contrast, animals in Asth exhibited coughing, sneezing, dyspnea and thoracic drainage, especially after the seventh nebulization.

Contractile Reactivity Measurement

Evaluation of the Contractile Response to Ovalbumin in Guinea Pig Trachea in the Presence of Functional Epithelium

The guinea pig trachea of Ctrl had no contractile response to OVA stimulation (E max = 1.8 ± 0.8%) (Figure 3). Differently, chronic allergic lung inflammation caused a significant contractile response (E max = 100%) ( p < 0.05) (Figure 3).

Figure 3. Representative records of guinea pig trachea response to stimulation with 10 μg/mL OVA in Ctrl (A) and Asth (B). CCh: carbachol; AA: arachidonic acid; W: washout; OVA: ovalbumin; Ctrl: control group; Asth: chronic allergic lung inflammation group.

Evaluation of Contractile Response to CCh, Histamine, and KCl in Guinea Pig Trachea in the Presence and Absence of Functional Epithelium

In Ctrl, guinea pig trachea contracted in response to the addition of cumulative concentrations of CCh (1 nM to 0.1 mM) in both the presence (E max = 100%; pEC ₅₀ = 6.63 ± 0.10) and absence of functional epithelium (E max = 100%; pEC ₅₀ = 6.85 ± 0.09). Chronic allergic lung inflammation increased the contractile efficacy of CCh (E max = 185.3 ± 16.1%) ( p < 0.05), but it did not alter its potency (pEC ₅₀ = 6.74 ± 0.03) in the presence of functional epithelium; meanwhile, in the absence of epithelium there was no difference in either the efficacy (E max = 89.2 ± 9.8%) or potency of CCh (pEC ₅₀ = 6.62 ± 0.07), compared to Ctrl (Figure 4A, n = 5).

Figure 4. Cumulative concentration-response curves to CCh (A), histamine (B) and KCl (C) in guinea pig trachea in Ctrl and Asth, both in the presence and absence of functional epithelium. The symbols and vertical bars represent the mean and SEM, respectively ( n = 5). ANOVA one-way followed by Tukey's post-test: ∗p < 0.05 [Ctrl E (+) vs. Asth E (+)]. Ctrl: control group; Asth: chronic allergic lung inflammation group; E (+): epithelium present; E (-): epithelium absent.

Moreover, guinea pig trachea from Ctrl contracted in response to the addition of cumulative concentrations of histamine (10 nM to 3 mM) in both the presence (E max = 100%; pEC ₅₀ = 5.05 ± 0.04) and absence of functional epithelium (E max = 100%; pEC ₅₀ = 5.80 ± 0.15). Chronic allergic lung inflammation also increased histamine contractile efficacy in the presence of epithelium (E max = 132.6 ± 5.9%) ( p < 0.05) but did not alter its potency (pEC ₅₀ = 5.11 ± 0.12). In the absence of epithelium, there was no difference in contractile efficacy (E max = 102.2 ± 11.4%) or potency (pEC ₅₀ = 5.80 ± 0.15) of histamine between the two groups.

Intragroup comparison of the agonists' effects showed that, in Ctrl and Asth, there was a difference in histamine contractile potency between the tracheal rings with and without epithelium; in this case, the agonist was more potent in the absence of epithelium, about 6.4-fold in Ctrl and 4.2-fold in Asth ( p < 0.05) (Figure 4B, n = 5).

Furthermore, guinea pig trachea in Ctrl contracted in response to the addition of cumulative concentrations of KCl (1 mM to 0.3 M) in both the presence (E max = 100%; pEC ₅₀ = 1.60 ± 0.01) and absence of functional epithelium (E max = 100%; pEC ₅₀ = 1.89 ± 0.08). Chronic allergic lung inflammation did not alter the contractile effectiveness or potency of KCl, either in the presence (E max = 119.0 ± 14.2%; pEC ₅₀ = 1.56 ± 0.03) or absence of functional epithelium (E max = 107,6 ± 11.5%; pEC ₅₀ = 1.73 ± 0.07). However, in Ctrl, the potency of KCl was 1.8-fold higher in the absence of epithelium ( p < 0.05), not differing in Asth (Figure 4C, n = 5).

Evaluation of ROS Participation in the Alterations Induced by Chronic Allergic Lung Inflammation

In Ctrl, the cumulative concentration-response curve to CCh (0.1 nM to 30 μM) (E max = 100%; pEC ₅₀ = 6.82 ± 0.06) was not altered in the presence of apocynin, either in relation to efficacy (E max = 115.6 ± 9.4%) or potency (pEC ₅₀ = 6.52 ± 0.13), in guinea pig trachea in the presence of epithelium. In contrast, in the presence of tempol, the contraction curve to CCh (0.1 nM to 30 μM) was 3.2-fold shifted to the right (pEC ₅₀ = 6.30 ± 0.07) ( p < 0.05), with no alteration in the contractile effectiveness of the agonist (E max = 99.4 ± 12.3%). Similarly, in the presence of catalase, the contractile potency of CCh (0.1 nM to 0.1 mM) was reduced 4-fold (pEC ₅₀ = 6.23 ± 0.09) ( p < 0.05), with no effect on its efficacy (E max = 116.6 ± 11, 9%) (Figure 5A, n = 5).

Figure 5. Cumulative concentration-response curves to CCh in guinea pig trachea with epithelium in Ctrl (A) and Asth (B), in both the absence and presence of apocynin, tempol or catalase. The symbols and vertical bars represent the mean and SEM, respectively ( n = 5). ANOVA one-way followed by Tukey's post-test: ∗p < 0.05 [Ctrl E (+) vs. Asth E (+)]. Ctrl: control group; Asth: chronic allergic lung inflammation group; E (+): epithelium present.

In guinea pigs with chronic allergic lung inflammation (Asth), there were no changes in either efficacy or potency of CCh (0.1 nM to 0.1 mM) (E max = 100%; pEC ₅₀ = 6.35 ± 0.11) induced by apocynin (E max = 105.6 ± 11.7%; pEC ₅₀ = 6.54 ± 0.11) in guinea pig trachea in the presence of epithelium. Similarly, in the presence of tempol, the contraction curve to CCh (0.1 nM to 0.1 mM) was not modified (E max = 101.0 ± 11.7%; pEC ₅₀ = 6.62 ± 0.27), but the potency of CCh (0.1 nM to 0.1 mM) was reduced about 3-fold in the presence of catalase (E max = 102.4 ± 9.1%; pEC ₅₀ = 5.99 ± 0.06) ( p < 0.05) (Figure 5B, n = 5).

Relaxant Reactivity Measurement

Evaluation of the Relaxant Response to Isoproterenol and Nifedipine in Guinea Pig Trachea in the Presence and Absence of Functional Epithelium

In Ctrl, isoproterenol (0.1 nM to 1 μM) relaxed the guinea pig trachea pre-contracted with 1 μM CCh in both the presence (E max = 135.7 ± 9.1%; pEC ₅₀ = 7.79 ± 0.10) and absence of functional epithelium (E max = 115.9 ± 8.7%; pEC ₅₀ = 7.81 ± 0.09). Chronic allergic lung inflammation did not alter the relaxant efficacy of isoproterenol in either the presence (E max = 124.4 ± 6.6%) or absence of functional epithelium (E max = 118.2 ± 7.3%). However, agonist relaxant potency was reduced about 3.5-fold in Asth (pEC ₅₀ = 7.24 ± 0.09) ( p < 0.05) compared to Ctrl in the presence but not absence of epithelium (pEC ₅₀ = 7.89 ± 0.12) (Figure 6A, n = 5).

Figure 6. Cumulative concentration-response curves to isoproterenol (A) and nifedipine (B) in guinea pig trachea pre-contracted with 1 μM CCh in Ctrl and Asth, in both the presence and absence of functional epithelium. The symbols and vertical bars represent the mean and SEM, respectively ( n = 5). Ctrl: control group; Asth: chronic allergic lung inflammation group; E (+): epithelium present; E(-): epithelium absent.

In addition, nifedipine (1 pM to 1 mM) relaxed the guinea pig trachea in Ctrl pre-contracted with 1 μM CCh in both the presence (E max = 138.2 ± 8.3%; pEC ₅₀ = 4.54 ± 0.15) and absence of functional epithelium (E max = 132.9 ± 7.2; pEC ₅₀ = 4.73 ± 0.10). Chronic allergic lung inflammation did not alter the efficacy or relaxant potency of nifedipine, either in the presence (E max = 122.8 ± 8.8%; pEC ₅₀ = 4.54 ± 0.15) or absence of functional epithelium (E max = 123.9 ± 10.4%; pEC ₅₀ = 4.71 ± 0.23). There was no difference in the efficacy or relaxant potency of nifedipine between the preparations with and without epithelium in either Ctrl or Asth (Figure 6B, n = 5).

Evaluation of the Participation of Transforming Growth Factor β (TGF-β) and Big Conductance Ca²⁺-Sensitive K⁺ Channels (BKCa) in the Alterations Induced by Chronic Allergic Lung Inflammation

In Ctrl, the relaxation curve of isoproterenol (0.1 nM to 1 μM) (E max = 135.7 ± 9.1%; pEC ₅₀ = 7.79 ± 0.10) was not altered by SB431542 (E max = 132.4 ± 7.8%; pEC ₅₀ = 7.96 ± 0.08), but its potency was reduced (pEC ₅₀ = 7.39 ± 0.06) and its efficacy increased (E max = 175.7 ± 11.0%) by IbTX ( p < 0.05) (Figure 7A, n = 5). However, in animals with chronic allergic lung inflammation, the relaxation curve of isoproterenol (0.1 nM to 1 μM) (E max = 120.7 ± 5.2%; pEC ₅₀ = 7.24 ± 0.09) was potentiated about 7-fold by SB431542 (pEC ₅₀ = 8.38 ± 0.08) ( p < 0.05), with no change in its relaxant efficacy (E max = 108.9 ± 5.6%); and furthermore, both potency and efficacy were unaltered by IbTX (E max = 137.5 ± 3.9%; pEC ₅₀ = 7.44 ± 0.12) (Figure 7B, n = 5).

Figure 7. Cumulative concentration-response curves to isoproterenol in guinea pig trachea with epithelium pre-contracted with 1 μM CCh in Ctrl (A) and Asth (B), in both the absence and presence of SB431542 or IbTX. The symbols and vertical bars represent the mean and SEM, respectively ( n = 5). Ctrl: control group; Asth: chronic allergic lung inflammation group; E (+): epithelium present. IbTX: iberiotoxin.

Evaluation of Antioxidant Activity in Plasma and Lung

Total antioxidant capacity in plasma was similar in Ctrl (23.6 ± 3.6%) and Asth (19.8 ± 3.2%). Meanwhile, in the lung, it was decreased from 83.0 ± 4.2% (Ctrl) to 59.8 ± 6.6% in Asth ( p < 0.05) (Figure 8, n = 5).

Figure 8. Measurement of oxidation inhibition in plasma and lung in Ctrl and Asth. The columns and vertical bars represent the mean and SEM, respectively ( n = 5). Ctrl: control group; Asth: chronic allergic lung inflammation group.

Western Blot Analysis

Compared to Ctrl, PI3K expression levels (0.64 ± 0.11 a.u.) were increased in Asth (1.34 ± 0.18 a.u.) ( p < 0.05). Conversely, ERK 1/2 (0.92 ± 0.06 a.u) and p-ERK (2.80 ± 0.56 a.u.) levels were not altered in the animals by lung inflammation (0.93 ± 0.07 and 2.67 ± 0.68 a.u., respectively) (Figure 9, Supplementary Figure 1, n = 4).

Figure 9. Expression of PI3K (A), ERK 1/2 (B), and p-ERK 1/2 (C) proteins in the lung of guinea pigs in Ctrl and Asth. The columns and vertical bars represent the mean and SEM, respectively ( n = 5). a.u.: arbitrary units. Ctrl: control group; Asth: chronic allergic lung inflammation group; PI3K: phosphatidylinositol-3-kinase; ERK 1/2: extracellular regulated kinase 1/2; p-ERK 1/2: phosphorylated ERK 1/2.

Immunohistochemical for α-Actin Analysis

There were differences in smooth muscle-specific α-actin present in the airway walls between groups. We found increased α-actin content in the airways of the lung inflammation group (11.0 ± 0.53%) compared to the control group (4.32 ± 0.38%) ( p < 0.05) (Figure 10).

Figure 10. Representative photomicrographs of guinea pig airways immunostained with anti-α-actin antibody in Ctrl (A) and Asth (B) groups, and its content (%). The columns and vertical bars represent the mean and SEM, respectively ( n = 5). ×40 (1500 μm). Ctrl: control group; Asth: chronic allergic lung inflammation group.

Discussion

In this work, a model of chronic allergic lung inflammation based on repeated and prolonged exposure of the guinea pig respiratory tract to OVA was standardized, with generation of smooth muscle hypercontractility in response to antigenic challenge, muscarinic and histaminergic receptor stimulation, and relaxant hyporesponsiveness to β-adrenergic agonist, dependent on epithelium-induced release of mediators, ROS generation, reduction in pulmonary antioxidant capacity associated with a decrease in SOD levels, as well as increased PI3K and ERK 1/2 expression and smooth muscle α-actin content. In this sense, the major contribution of this work is the characterization of a model, already used in scientific studies, that reproduces the characteristics of remodeling, smooth muscle hyperreactivity and thickening of the airways. In addition, since this model produces increased airway resistance ( Nakashima et al., 2008; Possa et al., 2012), these findings may be associated with in vivo bronchial hyperresponsiveness data already described in previous works.

The acute and chronic phases of asthma have different functional and tissue characteristics, that is structural remodeling, due to extensive and repeated animal exposure to inhaled antigenic agents, the marker of the disease chronicity ( Westerhof et al., 2001). This pathophysiological event is more related to clinical asthma ( Tibério et al., 1997). In this sense, a model of chronic allergic lung inflammation was reproduced, on the basis of OVA inhalation in guinea pigs at increasing concentrations for 4 weeks to obtain an experimental model that mimics the increased contractile reactivity associated with lower airway smooth muscle relaxant responsiveness, providing a valuable tool in studies of asthma pathophysiology and in new pharmacological approaches as well.

By morphological analysis, a peribronchial infiltrate of inflammatory cells was observed, besides some characteristics of tissue remodeling, namely epithelial cell hyperplasia and thickening of the bronchial smooth muscle layer, probably due to the hyperplasia and/or hypertrophy process (Figures 2A-C). These findings agree with similar occurrences found in allergic asthma in humans ( Cohen et al., 2007; Bergeron et al., 2009). Also, since persistent inflammation, subsequent remodeling and bronchial hyperesponsiveness have a strong cause and effect relationship and are cardinal characteristics in asthma, these findings are of clinical relevance ( Montuschi and Barnes, 2011).

Type I hypersensitivity mediates many of the asthmatic reactions, including airway hyperresponsiveness triggered by antigenic stimulation. This mechanism is related to the capture of immunoglobulins E (IgE) by mast cells, dendritic cells and eosinophils, which release the contractile agents histamine, cys-LTs (LTC ₄, D ₄, and E ₄), contractile prostanoids prostaglandin D ₂ and F 2α (PGD ₂ and PGF 2α, respectively) and thromboxane A ₂ (TXA ₂), thus causing bronchial obstruction ( Fireman, 2003). Therefore, the allergenic stimulation of sensitized animals by OVA inhalation is an effective method for evaluating airway hyperresponsiveness.

Animals with lung inflammation had a shorter time in contact with the antigen in the fifth, sixth and seventh inhalations, since this time was sufficient to trigger the respiratory stress signals resulting from the acute response after antigenic exposure, consistent with a profile of bronchial hyperresponsiveness (Table 1), which is corroborated by previous data ( Tibério et al., 1997; Pigati et al., 2015). This bronchial hyperresponsiveness may occur due to increased smooth muscle mass, the release of mediators from autonomic nerve endings or innate immune cells. This confirms the last hypothesis that guinea pig trachea showed contractile response to in vitro OVA stimulation (Figure 3) on the basis of the Schulz-Dale reaction, in which smooth muscle in an antigenically sensitized animal contracts after exposure to antigen by releasing contractile mediators from microenvironmental cells ( Schultz, 1910; Dale, 1913; Shaukat et al., 2015).

The primary function of airway smooth muscle cells is to regulate the tone and narrowing of the airways, controlling intrapulmonary ventilation and consequently the ventilation perfusion ratio ( Mitchell, 2009; Ouedraogo and Roux, 2014). The ability to modify smooth muscle length in experiments with isolated organs is useful in demonstrating important features of airway smooth muscle contractile function. In this sense, contradictory results are observed when studies evaluating the contractile response of smooth muscle to different constrictor agents in different animal models of asthma are summarized. Hypercontractility was observed for histamine ( Bai, 1991), cholinergic stimuli ( Matusovsky et al., 2016) and KCl ( Chesné et al., 2015). However, hypocontractility has also been observed for histamine ( Whicker et al., 1988) and cholinergic stimuli ( Goldie et al., 1986). This variability can be explained by the phenotypic differences of asthma, the experimental model adopted, and the tissue preparation as well ( Wright et al., 2013).

In view of the smooth muscle role in bronchial hyperresponsiveness, we investigated guinea pig trachea reactivity in response to contractile stimuli. In guinea pigs with lung inflammation compared to non-sensitized animals, there was an increase in tracheal reactivity to CCh and histamine but not to KCl, only in the presence of functional epithelium (Figures 4A-C). This indicates a greater participation of pharmacomechanical coupling in hypercontractility of the guinea pig trachea, without alteration in the electromechanical component, where this effect depends on epithelial functionality. Moreover, contractile reactivity increased more for CCh than histamine results from cholinergic stimulation being more pronounced in the central airways, such as the trachea (Leff et al., 1988).

It has been demonstrated that the concomitant reduction of airway hyperresponsiveness and smooth muscle mass are correlated, suggesting that such structural changes could explain the functional change in smooth muscle contractile responsiveness ( Danek et al., 2004). Thus, these data corroborate the previous morphological ones.

It was observed that the greatest amplitude of contraction to CCh occurred only in the preparations with intact epithelium. It is well described in the literature that epithelial damage resulting from allergic inflammation results in epithelial mediator production, such as TGF-β ( Davies, 2009), which mediates smooth muscle hypertrophy in asthma and promotes faster and more sustained contraction ( Boxall et al., 2006). These cells are also a source of contractile factors that contribute to bronchoconstriction, such as LTB ₄, LTC ₄, LTD ₄, LTE ₄, and PGD ₂ ( Montuschi, 2008; Santini et al., 2016). Since the animals were subjected to chronic stimulation with OVA, which leads to tissue structural and functional changes, it is reasonable to say that the increase in the contractile response was related to the epithelium effect because of its release of hypertrophic mediators.

Additionally, airway smooth muscle function is modulated by ROS. H ₂O ₂ and increased molecular oxygen levels induce contraction of guinea pig trachea ( Rhoden and Barnes, 1989) and promote augmented reactivity to contractile agonists, such as ACh and serotonin ( Henricks and Nijkamp, 2001). It has also been reported that 8-isoprostane, a marker of oxidative stress, formed by lipid peroxidation, contributes to the greater activity of the airway smooth muscle. In addition, this marker has been found in high concentrations in exhaled breath condensate of asthmatic patients ( Montuschi et al., 2010).

Therefore, we have shown that apocynin, an inhibitor of NADPH oxidase ( Sutcliffe et al., 2012), did not alter the cumulative curve of CCh in guinea pig trachea preparations in the Ctrl and Asth groups, indicating that O ₂⁻ from NADPH oxidase does not play an important role in the modulation of the contractile tone of guinea pig trachea, regardless of whether there is a profile of allergic lung inflammation. In contrast, the SOD mimetic tempol ( De Boer et al., 2001) reduced the contractile potency of CCh in animals of the Ctrl but not Asth group. This suggests that ROS, especially O ₂⁻, is increased because of lung inflammation, since tempol was unable to prevent its action in the tracheal smooth muscle, but that this increase would occur through pathways independent of NADPH oxidase, such as xanthine oxidase and decoupled endothelial nitric oxide (eNOS), which are also responsible for the formation of the O ₂⁻. Moreover, although catalase, an enzyme that converts H ₂O ₂ to water and molecular oxygen ( Rhoden and Barnes, 1989), reduced the contractile potency of CCh about 4-fold in Ctrl, the efficacy of catalase in reducing the potency of CCh was lower in guinea pigs with lung inflammation, indicating an increase in H ₂O ₂ production triggered by the inflammatory process (Figures 5A,B).

Indeed, Mátyás et al. (2002) showed that superoxide anion, hydroxyl radicals, and hydrogen peroxide cause the tracheal smooth muscle to contract, the latter being the ROS responsible for the greatest increase in muscle tone. In addition, the effects of these free radicals are contained by antioxidant defenses present in the intact epithelium, and epithelial damage potentiates the contractile effect of these ROS. Therefore, it is reasonable to infer that an imbalance between ROS and antioxidant defenses favors airway smooth muscle damage. However, the role of oxidative stress was not directly investigated by measuring biomarkers of oxidative stress (e.g., H ₂O ₂ or nitrates reflecting peroxynitrite production), so this is a limitation of the study.

Airway hyperresponsiveness is also related to a lower release of smooth muscle relaxants, such as adrenaline ( Barnes, 1986), vasoactive intestinal peptide ( Ben-Jebria et al., 1993) and prostaglandin E ₂ and I ₂ (PGE ₂ and PGI ₂, respectively) ( Van Schoor et al., 2000). Desensitization of β ₂ adrenergic receptors is also associated with increased smooth muscle responsiveness to contractile stimuli in asthma ( Barnes, 1995).

The relaxant airway smooth muscle responsiveness evaluation in animal models of asthma has shown hyporresponsiveness to theophylline and β ₂ agonists ( Nesmith et al., 2014). In agreement with these data, we observed a reduction in the relaxant potency of isoproterenol in guinea pig trachea in the Asth group, compared to Ctrl, in an epithelium-dependent manner (Figure 6A). In contrast, there were no differences in either efficacy or potency between Ctrl and Asth in nifedipine relaxant response (Figure 6B), indicating that lung inflammation did not promote alterations in the activity of Ca V, which was confirmed by the previous KCl cumulative contraction data.

M ₃ receptors crucially influence the function of these receptors by activation of protein kinase C (PKC) and direct phosphorylation of β ₂ receptors, as well as G rms protein, promoting receptor decoupling with its G protein, as well as desensitization ( Meurs et al., 2001); in addition to phosphorylation by a G-protein receptor kinase (GRK), the β-adrenoceptor kinase (BARK), amplifying the desensitization of these receptors ( Boterman et al., 2006). Additionally, ACh acting via M ₂ receptors counteracts the bronchodilator actions of β ₂ agonists by reducing the cAMP levels due to inhibition of adenylate cyclase (AC) ( Montuschi and Ciabattoni, 2015). Such processes explain the well-known attenuation of the efficacy of β-agonists during episodes of severe bronchoconstriction ( Meurs et al., 2008). Furthermore, TGF-β, a key growth factor involved in some remodeling features, and released by cholinergic stimulation ( Oenema et al., 2013) has been shown to reduce the number of β-adrenoceptors in human tracheal smooth muscle cells ( Nogami et al., 1994), so this process is closely related to the lower relaxant responsiveness due to the inactivation of the G s/BK Ca pathway, either by β-adrenergic desensitization, by TGF-β or M ₃-PKC, or by negative modulation of these channels via the pathway triggered by cholinergic stimulation of M ₂-G iβγ/BK Ca ( Kume, 2015).

We then showed that the relaxation curve of isoproterenol in control animals was not modified by the antagonist of type I TGF-β receptors as expected, since the antagonist has no effect in the absence of agonist. However, the potency of the agonist was decreased by the BK Ca blocker, as expected since in non-asthmatic airway smooth muscle, relaxation via β-receptors is also mediated via the opening of BK Ca ( Kume, 2013), but also had its maximum response increased, probably due to a compensatory mechanism. Furthermore, in the trachea of the Asth group, the antagonist recovered the relaxant potency of isoproterenol, indicating that signaling mediated via TGF-β RI leads to desensitization of β-receptors (Figures 7A,B). These data are confirmed by previous research which showed that lung inflammation produces a greater release of TGF-β in the lungs ( Possa et al., 2012). Additionally, the BK Ca blocker did not modify the relaxation curve, indicating that this inability is due to the fact that these channels are already poorly activated because of lung inflammation, contributing to the observed less responsiveness of the relaxant.

In summary, we found that as lung inflammation increased the contractility of guinea pig trachea, it also reduced the relaxant reactivity of this organ by mechanisms related to ROS generation and the release of TGF-β. Confirming the influence of ROS, we showed that allergic inflammation caused a decrease in the antioxidant capacity of lungs, but not in the plasma, indicating local pulmonary damage rather than systemic damage (Figure 8).

As demonstrated, lung inflammation promoted thickening of the smooth muscle layer, which may have occurred due to hypertrophy and/or hyperplasia. These cell growth processes are dependent on two independent signaling pathways that regulate the growth of airway smooth muscle cells, phosphatidylinositol-3-kinase (PI3K) and extracellular regulated kinase 1/2 (ERK 1/2) ( Gosens et al., 2008). These pathways can be switched on by the activation of G protein-coupled receptors or tyrosine kinase receptors by contractile mediators or growth factors, such as the TGF-β ( Hoshino et al., 2001). Thus, we showed that pulmonary expression of PI3K was increased in the Asth group, but not total or phosphorylated ERK 1/2, confirming the participation of this kinase in pulmonary inflammation, which could be associated with hypertrophy/hyperplasia induced by allergic lung inflammation (Figure 9).

It is well established that airway inflammation and cholinergic stimulation promote the release of TGF-β, which induces airway remodeling ( Oenema et al., 2013), which, in turn, induces increased expression of smooth muscle contractile proteins such as α-actin, smooth muscle myosin (SM-myosin) and myosin light chain kinase (MLCK; Fairbank et al., 2008). Accordingly, α-actin content has been shown to be increased almost 3-fold by pulmonary inflammation (Figure 10), indicating that thickening of the smooth muscle layer is related to observed contractile hyperresponsiveness. Given the mechanisms investigated, we can assume that this increase is due to the participation of the epithelium, which acts by releasing ROS and TGF-β.

Unlike the current models for the study of asthma in animals, the model provided here is characterized by smooth muscle hypercontractility due to ROS and growth factor release, as well as activation of PI3K. Furthermore, this work will promote advances in asthma research on bronchial hyperresponsiveness and will also contribute to the search for new therapeutic strategies in this field.

Author Contributions

LV and MS performed pharmacological experiments, analyzed data, completed the literature review and designed the manuscript. AC and GO engaged in the pharmacological experiments. IS participated in pharmacological experiments and prepared the manuscript. FQ and PdS involved in histological evaluation and data acquisition. LA and CC participated in the acquisition of protein expression data. GC and AS performed the antioxidant capacity experiments, and analyzed and interpreted the data. RR and IT carried out the immunohistochemical assays. GV performed the histological analysis. FC and BS involved in the design of the study, interpretation of data and paper review.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors thank CAPES and CNPq for the financial support, UFPB and PPgPNSB for experimental support, José Crispim Duarte, Luís C. Silva and Roberta N. Parentoni, from UFPB, for providing technical assistance. Dr. A. Leyva (United States) helped with English editing of the manuscript.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2018.01547/full#supplementary-material

References

Silva, A. S., Paim, F. C., Santos, R. C., Sangoi, M. B., Moresco, R. N., Lopes, S. T., et al. (2012). Nitric oxide level, protein oxidation and antioxidant enzymes in rats infected by Trypanosoma evansi. Exp. Parasitol. 132, 166-170. doi: 10.1016/j.exppara.2012.06.010

# data[].attributes.attachment_link.title
A Guinea Pig Model of Airway Smooth Muscle Hyperreactivity Induced by Chronic Allergic Lung Inflammation: Contribution of Epithelium and Oxidative Stress

# data[].attributes.attachment_link.description
Asthma is a heterogeneous disease of the airways characterized by chronic inflammation associated with bronchial and smooth muscle hyperresponsiveness. Currently, different murine models for the study of asthma show poor bronchial hyperresponsiveness due to a scarcity of smooth muscle and large airways, resulting in a failure to reproduce smooth muscle hyperreactivity.

# data[].attributes.headline
Allergies & Respiratory Disease Research

# data[].attributes.subject
Allergies & Respiratory Disease Research

# data[].attributes.body
Guinea pigs have been used in asthma research for decades due to the anatomical and physiological similarities between human and guinea pig airways.
(Adner et al., 2020)

Asthma is a airway or respiratory disease characterized by chronic inflammation associated with bronchial and smooth muscle hyperreactivity.

Different rodent models for asthma study fails to reproduce bronchial and smooth muscle hyperreactivity due to a scarcity of smooth muscle and large airways.

Guinea pig is then used as the model of chronic allergic lung inflammation mimicking airway smooth muscle hyperreactivity observed in asthmatics (Asth).

Guinea pigs were randomly divided into a control group (Ctrl), which received saline (0.9% NaCl), and the Asth group, subjected to in vivo sensitization with ovalbumin (OVA) nebulization.

Findings:

- Lung inflammation increased the contractility of guinea pig trachea
- Lung inflammation also reduced the relaxant reactivity of the trachea due to the mechanisms of ROS generation and TGF-β release.
- Allergic inflammation decreased antioxidant capacity of lungs without affecting the plasma, indicating local pulmonary damage rather than systemic damage.
- Guinea pig model has the edge against other rodent models as it characterized by smooth muscle hypercontractility due to ROS and growth factor release, as well as activation of phosphatidylinositol-3-kinase (PI3K).

(Vasconcelos et al., 2019)

# data[].attributes.headline
Pharmaceutical otoprotection strategies to prevent impulse noise-induced hearing loss

# data[].attributes.subject
Pharmaceutical otoprotection strategies to prevent impulse noise-induced hearing loss

# data[].attributes.attachment_link.provider_name
Scitation: Acoustical Society of America

# data[].attributes.attachment_link.title
Pharmaceutical otoprotection strategies to prevent impulse noise-induced hearing loss

# data[].attributes.attachment_link.description
One of the ongoing challenges for hearing researchers is successful protection of the ear from noise injury. For decades, the most effective methods have been based on modifying the acoustic proper...

# data[].attributes.body
Guinea pigs are highly utilized in studies of:
- pathological auditory system
- after damage due to noise exposure
- ototoxic drug treatment
(Naert et al.,2019)

Antioxidants as otoprotectants against noise-induced hearing loss (NIHL)

Astragaloside IV is a type of traditional Chinese herb which possesses antioxidant properties, has been tested in guinea pigs as an otoprotectant against impulse noise.

Guinea pigs were exposed to the noise of 15 shots from a 7.62 mm Chinese Army 81–1 assault rifle at a distance of 35 cm, creating an exposure level of 176 dB pSPL.

The guinea pigs treated with astragaloside IV (200 mg/kg/day for three days by intragastric administration) showed lower threshold shifts of 10–15 dB compared to control animals treated with saline.

It is proven that antioxidants is capable of improving recovery from impulse noise exposure.

(Bielefeld et al., 2019)

# data[].attributes.body
Normal hearing range: Guinea pig (150 to 50 000 Hz), Human (20–20 000 Hz)

Best hearing range: Guinea pig (8000 to 16 000 Hz), Human (1000 to 4000 Hz)

(Bielefeld et al., 2019)

# data[].attributes.headline
Major Uses in Biomedical Science Research

# data[].attributes.subject
Major Uses in Biomedical Science Research

# data[].attributes.body
Guinea pigs are mainly used in:

Auditory/Hearing

Allergies and respiratory diseases

Nutritional research

Safety testing

(Understanding Animal Research, n.d.)

# data[].attributes.body
American guinea pig: Smooth-coated, shorthaired breed (outbred stock in lab) that’s one of the most popular, and one of the oldest recognized by the American Cavy Breeders Association. (Clemons & Seeman, 2016)

# data[].attributes.headline
BASIC BIOLOGIC PARAMETERS OF THE GUINEA PIG

# data[].attributes.subject
BASIC BIOLOGIC PARAMETERS OF THE GUINEA PIG

# data[].attributes.body
(Clemons & Seeman, 2016)

# data[].attributes.body
The name may be related to the price (one guinea) of the animal in the English markets.
Carcasses in the South American marketplace resembled small suckling pigs, but it is more likely that
the vocalizations of the animals sounded like the squeals of pigs.
The names may come from different languages:
In Portuguese, for example, they are called 'Porchitas da India' ('little pigs from India'), and in Latin they are called C. porcellus (Latin for 'small pig').                          (Clemons & Seeman, 2016)

# data[].attributes.body
Originally from South America, particularly the Andes, the modern guinea pig is a descendant of Cavia cutleri.  (Clemons & Seeman, 2016)

# data[].attributes.body
Duncan-Hartley and Hartley are the short-haired albino animals with several coat colours.
(Clemons & Seeman, 2016)

# data[].attributes.body
There were 5 types of stock or strain that are often used in research:
Outbred stocks (:)
1) American or English guinea pig
2) Duncan-Hartley
3) Hartley
Inbred strains (/)
4) Strain 2
5) Strain 13
(Clemons & Seeman, 2016)

# data[].attributes.body
External anatomy of the adult male guinea pig.
Males are generally larger than females
(Clemons & Seeman, 2016)

# data[].attributes.body
External anatomy of the adult female guinea pig. Note the vaginal closure membrane. (Clemons & Seeman, 2016)

# data[].attributes.body
Domain: Eukaryota

Kingdom: Metazoa

Phylum: Chordata

Subphylum: Vertebrata

Class: Mammalia

Order: Rodentia

Suborder: Hystricomorpha

Family: Caviidae

Genus: Cavia

Species: Cavia porcellus

(Higman, 2019)