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Effects of acute and chronic corticosterone treatments in the American Bullfrog wound healing

Cite this dataset

Madelaire, Carla (2023). Effects of acute and chronic corticosterone treatments in the American Bullfrog wound healing [Dataset]. Dryad. https://doi.org/10.5061/dryad.c2fqz61dg

Abstract

Glucocorticoid (GC) release is triggered by adverse stimuli that activate hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis. Glucocorticoids may enhance or suppress immune functions depending on the level of elevation. In this study, we investigated the effects of transient and chronic increases of CORT on the wound healing of the American bullfrog. Frogs were submitted to a daily transdermal hormonal application that acutely elevated CORT plasma levels, or vehicle as a control. Other frogs were surgically implanted with a silastic tube filled with CORT that resulted in chronic elevation of CORT plasma levels or received empty implants as a control. A dermal biopsy was performed to create a wound and was photographed every 3 days. Individuals treated with transdermal CORT started healing faster than their control 32 days after the biopsy. Frogs that received CORT implants tended to heal slower than control subjects. Plasma bacterial killing ability (BKA) was not affected by treatment, which reinforces the constitutive nature of this innate immune trait.  By the end of the experiment, frogs from the acute CORT treatment had smaller wounds compared to those receiving the CORT-filled implants, highlighting the differential effects of acute (immunoenhancing) and chronic (immunosuppressive) elevation of CORT plasma levels.

Methods

Animal maintenance and experimental groups

Forty adult male American bullfrogs (Lithobates catesbeianus) were bought from a local animal farm in the state of São Paulo, Brazil (Ranário Santa Clara, Santa Isabel, SP – 23°18’13.0”S 46°12’21.5”W) and brought into laboratory conditions at the University of São Paulo in October 2017 (Week 0). Animals were kept in individual plastic boxes (43.0 x 28.5 x 26.5 cm) inside a climatic chamber (FITOTRON 011 – Eletro-lab, São Paulo/SP/Brazil) with controlled photoperiod (12:12 LD, lights on 6 a.m.) and temperature (22±0.5°C.) Each individual box was equipped with a small container with water, and animals were weekly fed with 10 cockroaches (Nauphoeta cinerea). Seven days of habituation were given before experiments began.

Animals were weighed (0.01g) and had their snout-vent length (SVL) measured (0.01mm). Animals were randomly separated into four different groups of 10 animals each: 1) Transdermal application of CORT solution, 2) Transdermal application of sesame oil, 3) CORT implant and 4) Empty implants. We calculated body condition index as the residuals of the linear regression between mass as the dependent variable and the SVL as the independent variable and performed an ANOVA to ensure that groups did not differ in body condition at the begging of the experiment (F3,36 = 0.47, P = 0.71).

Corticosterone transdermal application and implant surgery

The day after basal blood sampling (Week 1, Day 8), animals were subjected to experimental treatments (Week 1, Day 9). Group 1 (control sesame oil) received a transdermal application of sesame oil (10µL), Group 2 (transient elevation of CORT) received CORT solution using sesame oil as the vehicle (2µg/10µL) aiming for achieving CORT plasma levels similar to what males experience during advertisement calling (Mendonça et al. 1985). Daily transdermal applications were carried out (1h after lights-off, at 7 p.m.) by placing a single drop of either treatment onto the subject's back using a micropipette (Assis et al. 2017; Madelaire et al. 2019) for 53 days (Day 9–63). During treatments, animals were not handled (application performed inside the box) and a red flashlight was used to minimize disturbance (Jessop et al. 2014).

Surgically implanted capsules are an effective method for sustaining high CORT plasma levels for longer periods (Falso et al. 2015), which can test the link between the chronic elevation of CORT and a decrease in immune response in anurans (Falso et al. 2015). The implants preparation consisted in cutting pieces (1cm) of silastic tubing (Perfimed®, Brazil; 1.5 mm inner diameter, and 2mm outer diameter), sealing one end with nontoxic silicone glue (Pulvitec Polystic Ref. 106612). Half of the implants were filled with crystalline corticosterone (Sigma - 27840) and all implants were washed with ethanol (70%) and allowed to dry overnight to sterilize them. Group 3 (empty implants) was surgically implanted with two empty silastic tubes and Group 4 (CORT implants) was implanted with two silastic tubes containing CORT (Falso et al. 2015). The implant surgery consisted of locally anesthetize animals with (Benzocaine 1%), making a superficial cut on animals’ dorsum (2.5 mm) with an ophthalmic scissor, sliding the implant below the skin, and closing the incision externally with tissue adhesive (Ethyl-2-Cyanoacrylate, Epiglu®). To avoid possible infections, procedures were performed with sterilized gloves that were changed for each animal. Additionally, animals were fasted for 24h after the surgery.

Blood sampling

Five blood samples were obtained in a 5-week interval. Blood was collected through cardiac puncture using heparinized 1mL syringed and 23Gx1/2” needles at: 8 days after habituation in the laboratory conditions which we considered as baseline (Week 1, day 8 of the experiment), 6, 13, 20, and 27 days after implant surgery and beginning of the CORT transdermal application. The repeated blood draws were performed to ensure that CORT plasma levels were still elevated for Group 4 (CORT implant) and remained similar to baseline for Group 1 (control sesame oil) and Group 3 (empty implant). All blood samples were collected at 8 p.m., 1h after the transdermal delivery, within a 3-minute interval between individuals. Blood samples were centrifuged (4 min at 604 g) in order to separate the plasma, which was then aliquoted into separate microtubes and stored at a -80°C freezer for further quantification of CORT plasma concentrations and BKA.

Validation of hormonal transdermal application

Unexpectedly, CORT plasma levels for animals that received transdermal application of CORT solution (2ug/10ul) (Group 2) were already low 1h after the transdermal delivery. To validate that CORT plasma levels increased after the transdermal treatment; 5 additional individuals were subjected to the same transdermal application of CORT solution (2ug/10ul). Blood samples were obtained 15 and 30 min after transdermal delivery.

Dermal biopsy and wound healing rate analysis

The wound was performed 6 days after the hormonal treatment started (week 2, day 15 of the experiment). A sterile 4 mm diameter punch (Kruuse – Cat. Nº 273691) was used in the dorsum of the right hind limb, after locally applying an anesthetic (10% benzocaine). Wounds were superficial, removing only the skin layer without damaging the muscle tissue below. To assess the change in the healing over time, the wound area was photographed every three days and ending on day 61 of the experiment (Week 8). Animals were not sedated and the handling to obtain the photographs was kept under three minutes. Digital images of the wounds were taken using a stereomicroscopy DMLB (Leica) (1.6x). All images were analyzed using the QWin Lite 3.1 image analyzer software by a person that was blind to the aim of the study and the treatment of each animal. The healing wound percentage was calculated as [(wound area/initial wound area)*100].

Quantification of corticosterone

The hormone was initially extracted from 10–30ul of plasma with ether according to (Lima et al. 2020; Figueiredo et al. 2021; Barsotti et al. 2021). CORT was determined using EIA kits with an assay sensitivity of 17pg/ml (CORT number 501320, Cayman Chemical), according to the manufacturer’s instructions and previous studies conducted with several anuran species, including the same species in this study (Lima et al. 2020; Figueiredo et al. 2021). The mean values for intra and inter-assay variation were 8.87% and 6.60%, respectively.

Bacterial killing ability (BKA) assay

To evaluate the effects of the treatments on the BKA, we used the plasma obtained weekly from all groups. The BKA assay was conducted according to (Lima et al. 2020) and (Figueiredo et al. 2021). Plasma samples were diluted (1:20) in amphibian Ringer's solution (10µl plasma: 190µl Ringer) and mixed with 10µl of Aeromonas hydrophila working solution (2.5 x 107 microorganisms/1000uL). The positive control was 10µl of bacteria working solution in 200µl of Ringer's solution, and the negative control contained 210µl of Ringer's solution. Samples and controls were incubated for 60 minutes at 37°C. After incubation, 500µl of tryptic soy broth was added to each sample and mixed. Then, 300µl of each sample was transferred (in duplicate) to a 96-well microplate and incubated at 37ºC for 1h. The optical density of the samples was measured hourly in a plate spectrophotometer (wavelength: 595nm) totaling four readings. The BKA was considered at the beginning of the bacterial exponential growth phase and calculated according to the formula: 1 - (optical density of sample / optical density of positive control), which represents the proportion of killed microorganisms in the samples compared to the positive control.

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Funding

São Paulo Research Foundation, Award: 2014/16320-7

São Paulo Research Foundation, Award: 2018/00406-0

Coordenação de Aperfeicoamento de Pessoal de Nível Superior, Award: 443649/2020-0