DOI: 10.5061/dryad.c2fqz61dg
title: Effects of acute and chronic corticosterone treatments in the American Bullfrog wound healing

description: 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.
description: <strong>Animal maintenance and
 experimental groups</strong> Forty adult male American bullfrogs
 (<em>Lithobates catesbeianus</em>) 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
 (<em>Nauphoeta cinerea</em>). 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 (F<sub>3,36</sub> = 0.47, P = 0.71).
 <strong>Corticosterone
 transdermal application and implant surgery</strong> 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.
 <strong>Blood sampling
 </strong> 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. <strong>Validation of hormonal
 transdermal application </strong> 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. <strong>Dermal biopsy and
 wound healing rate analysis</strong> 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]. <strong>Quantification of
 corticosterone</strong> 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. <strong>Bacterial killing
 ability (BKA) assay </strong> 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
 <em>Aeromonas hydrophila</em> working solution (2.5 x
 10<sup>7</sup> 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
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