Contrasting effects of transdermal and implant corticosterone treatments in the American bullfrog wound healing.

Glucocorticoid (GC) release is triggered by adverse stimuli that activate the hypothalamus-pituitary-adrenal/interrenal 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 increase of corticosterone (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 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 with those receiving the CORT-filled implants, highlighting the differential effects of acute (immunoenhancing) and chronic (immunosuppressive) elevation of CORT plasma levels. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Fibroblasts as an experimental model system for the study of comparative physiology.

Mechanistic evaluations of processes that underlie organism-level physiology often require reductionist approaches. Dermal fibroblasts offer one such approach. These cells are easily obtained from minimally invasive skin biopsy, making them appropriate for the study of protected and/or logistically challenging species. Cell culture approaches permit extensive and fine-scale sampling regimes as well as gene manipulation techniques that are not feasible in vivo. Fibroblast isolation and culture protocols are outlined here for primary cells, and the benefits and drawbacks of immortalization are discussed. We show examples of physiological metrics that can be used to characterize primary cells (oxygen consumption, translation, proliferation) and readouts that can be informative in understanding cell-level responses to environmental stress (lactate production, heat shock protein induction). Importantly, fibroblasts may display fidelity to whole animal physiological phenotypes, facilitating their study. Fibroblasts from Antarctic Weddell seals show greater resilience to low temperatures and hypoxia exposure than fibroblasts from humans or rats. Fibroblast oxygen consumption rates are not affected by temperature stress in the heat-tolerant camel, whereas similar temperature exposures depress mitochondrial metabolism in fibroblasts from rhinoceros. Finally, dermal fibroblasts from a hibernator, the meadow jumping mouse, better resist experimental cooling than a fibroblast line from the laboratory mouse, with the hibernator demonstrating a greater maintenance of homeostatic processes such as protein translation. These results exemplify the parallels that can be drawn between fibroblast physiology and expectations in vivo, and provide evidence for the power of fibroblasts as a model system to understand comparative physiology and biomedicine.

Effects of corticosterone treatment and wound healing on reproductive traits of American bullfrogs.

During reproductive season, calling anuran males display high testosterone (T) and episodically high corticosterone (CORT) plasma levels, which are positively associated with higher calling rates and immunocompetence. However, exposure to constant stress stimuli can result in chronically elevated CORT levels, possibly leading to inhibition of reproductive and immune activity. Reproduction and immune responses are energetically expensive, so when an animal is immunologically challenged, a tradeoff might be expressed, with CORT potentially mediating it. Our aim was to test how episodic and chronic CORT treatments, alongside wound healing, would affect reproduction in American bullfrog males (Lithobates catesbeianus). Forty animals were divided in four groups: Episodic CORT (daily transdermic application of CORT), placebo (daily transdermic application of sesame oil), chronic CORT (subcutaneous CORT silastic implants), and sham control (subcutaneous empty silastic implants). One week after treatments began, animals were punctured in the leg with a biopsy needle and the wound was photographed after 45 days to determine wound healing status (WS). Blood samples were collected throughout the experiment to measure CORT and T plasma levels. After animal euthanasia, testes were dissected, fixed, and analyzed histologically to determine spermatogenic activity (germinative cyst [GmC] morphometrics). As expected, the episodic CORT treatment had no effect on T plasma levels or spermatogenic activity. On the other hand, chronic CORT treatment reduced GmC morphometric traits, indicating suppression of reproduction, although T levels were not altered. In addition, animals from sham control and chronic CORT treatments with higher T levels presented higher WS, which indicates an immune-enhancing T effect.