Glucocorticoids are popular hormones to measure in both biomedical and ecological studies of stress. Many assumptions used to interpret glucocorticoid results are derived from biomedical data on humans or laboratory rodents, but these assumptions often fail for wild animals under field conditions. We discuss five common assumptions often made about glucocorticoids in ecological and conservation research that are not generally supported by the literature. (1) High acute elevations of glucocorticoids indicate an animal in distress. In fact: because glucocorticoids are needed to survive stressors, elevated concentrations often reflect adequate coping. (2) Low glucocorticoid concentrations indicate a healthy animal. In fact: because glucocorticoids are important in responding to stressors, low glucocorticoid concentrations might indicate the lack of adequate coping. (3) Sustained elevated glucocorticoids indicate chronically stressed animals. In fact: glucocorticoid concentrations by themselves have no predictive value in diagnosing chronic stress. (4) Glucocorticoids mobilize energy to survive short‐term stressors such as predator attacks. In fact: glucocorticoids' primary impact on energy regulation is to remove glucose transporters from cell surfaces. Not only is this process too slow to provide short‐term energy, but glucocorticoid‐induced increases in glucose reflect decreased, not increased, glucose utilization. (5) Glucocorticoid measurements in non‐blood tissues (e.g., feces, hair, feathers, etc.) are equivalent to blood concentrations. In fact: these alternative tissues present imperfect reflections of blood concentrations, and it is blood concentrations that interact with receptors to evoke biological change. In summary, proper consideration of these common assumptions will greatly aid in interpreting glucocorticoid data from ecological and conservation studies.

Among species, larger animals tend to live longer than smaller ones, however, the opposite seems to be true for dogs—smaller dogs tend to live significantly longer than larger dogs across all breeds. We were interested in the mechanism that may allow for small breeds to age more slowly compared with large breeds in the context of cellular metabolism and oxidative stress. Primary dermal fibroblasts from small and large breed dogs were grown in culture. We measured basal oxygen consumption (OCR), proton leak, and glycolysis using a Seahorse XF96 oxygen flux analyzer. Additionally, we measured rates of reactive species (RS) production, reduced glutathione (GSH) content, mitochondrial content, lipid peroxidation (LPO) damage and DNA (8-OHdg) damage. Our data suggests that as dogs of both size classes age, proton leak is significantly higher in older dogs, regardless of size class. We found that all aspects of glycolysis were significantly higher in larger breeds compared with smaller breeds. We found significant differences between age classes in GSH concentration, and a negative correlation between DNA damage in puppies and mean breed lifespan. Interestingly, RS production showed no differences across size and age class. Thus, large breed dogs may have higher glycolytic rates, and DNA damage, suggesting a potential mechanism for their decreased lifespan compared with small breed dogs.

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.

Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration (AMD), in which local inflammation and hyperactivity of the complement pathway have been implicated in its pathophysiology. This study explores whether any surrogate biomarkers are specifically associated with GA. Plasma from subjects with GA, intermediate dry AMD and non-AMD control were evaluated in 2 cohorts. Cohort 1 was assayed in a 320-analyte Luminex library. Statistical analysis was performed using non-parametric and parametric methods (Kruskal-Wallis, principal component analysis, partial least squares and multivariate analysis of variance (MANOVA) and univariate ANCOVAs). Bioinformatic analysis was conducted and identified connections to the amyloid pathway. Statistically significant biomarkers identified in Cohort 1 were then re-evaluated in Cohort 2 using individual ELISA and multiplexing. Of 320 analytes in Cohort 1, 273 were rendered measurable, of which 56 were identified as changing. Among these markers, 40 were identified in univariate ANCO-VAs. Serum amyloid precursor protein (sAPP) was analyzed by a separate ELISA and included in further analyses. The 40 biomarkers, sAPP and amyloid-β (Aβ) (1-42) (included for comparison) were evaluated in Cohort 2. This resulted in 11 statistically significant biomarkers, including sAPP and Aβ(1-40), but not Aβ(1-42). Other biomarkers identified included serum proteases-tissue plasminogen activator, tumor-associated trypsinogen inhibitor, matrix metalloproteinases 7 and 9, and non-proteases-insulin-like growth factor binding protein 6, AXL receptor tyrosine kinase, omentin, pentraxin-3 and osteopontin. Findings suggest that there is a preferential processing of APP to Aβ(1-40) over Aβ(1-42), and

Although stress can cause overall damage to the genome, it is currently unknown whether normal background damage to DNA varies throughout the annual cycle. If DNA damage did vary seasonally, it would have major implications on environmental-genomic interactions. We measured background DNA doublestranded breaks using the neutral comet assay in five tissues (nucleated red blood cells, abdominal fat, hippocampus, hypothalamus, and liver) in four cohorts of house sparrows (Passer domesticus): free-living summer, captives on a summer light cycle, free-living winter, and captives on a winter light cycle. The experiment was designed to answer three questions: (1) Is red blood cell DNA damage representative of other tissues? (2) Is DNA damage in captive birds representative of DNA damage in free-living birds? (3) Does DNA damage show seasonality?We found that (1) blood is a representative tissue, (2) captive animals are representative of free-living animals, and (3) DNA damage is higher in the summer than in the winter. These data indicate that red blood cells can be an index of DNA damage throughout the body and that background levels of DNA damage show substantial seasonal variation. The latter result suggests the possibility that underlying molecular mechanisms of DNA damage and/or repair also change seasonally.

The reactive scope model was created to address two major unanswered questions in stress physiology: how and when does the adaptive acute stress response turn into harmful chronic stress? Previous studies suggest that immunoenhancement should occur in reactive homeostasis (acute stress) and immunosuppression should occur in homeostatic overload (chronic stress). We used this dichotomy of immune function to further elucidate the transition from acute to chronic stress by treating house sparrows (Passer domesticus) with different intensities of chronic stress and then monitoring their immune function. By varying the number of stressors given per day and the length of chronic stress bouts over a period of 6 months, we produced four treatment groups: high, medium, and low stress, and captivity‐only. We tracked immunity through the bacterial killing assay and monitored healing of a 4 mm skin biopsy punch. We hypothesized that higher‐stress birds would repair their skin more slowly and have lower bacterial killing capacity. The opposite was true—high‐stress birds initially repaired their skin fastest. Additionally, all birds dramatically reduced bacterial killing capacity after the biopsy and increased food‐derived uric acid, suggesting increased energy acquisition and a shift in immune resources to a more immediate concern (healing). Once healing finished, only the high‐stress birds were unable to recover circulating immune function, suggesting that the combination of high stress and an immune challenge pushed these birds into homeostatic overload. Prioritizing healing over other immunological processes might be the best defense for a bird in its natural habitat.

One of the biggest unanswered questions in the field of stress physiology is whether variation in chronic stress intensity will produce proportional (a gradient or graded) physiological response. We were specifically interested in the timing of the entrance into homeostatic overload, or the start of chronic stress symptoms. To attempt to fill this knowledge gap we split 40 captive house sparrows (Passer domesticus) into four groups (high stress, medium stress, low stress, and a captivity-only control) and subjected them to six bouts of chronic stress over a 6-month period. We varied the number of stressors/day and the length of each individual bout with the goal of producing groups that would experience different magnitudes of wear-and-tear. To evaluate the impact of chronic stress, at the start and end of each stress bout we measured body weight and three plasma metabolites (glucose, ketones, and uric acid) in both a fasted and fed state. All metrics showed significant differences across treatment groups, with the high stress group most frequently showing the greatest changes. However, the changes did not produce a consistent profile that matched the different chronic stress intensities. We also took samples after a prolonged recovery period of 6 weeks after the chronic stressors ended. The only group difference that persisted after 6 weeks was weight—all differences across groups in metabolites recovered. The results indicate that common blood metabolites are sensitive to stressors and may show signs of wear-and-tear, but are not reliable indicators of the intensity of long-term chronic stress. Furthermore, regulatory mechanisms are robust enough to recover within 6 weeks post-stress.

Tropical birds have a 'slower pace of life', with lower rates of whole-animal metabolism, smaller metabolically active organs and lower cellular metabolic rates than their temperate counterparts. Oxidative stress is a physiological mechanism that may dictate differing life-histories such as those found between tropical and temperate birds. Oxygen is required to make ATP, resulting in the production of reactive oxygen species (ROS). If left unchecked, ROS can structurally alter proteins, induce mutations in DNA and damage structural lipids. To combat accumulating oxidative damage, organisms have evolved an elaborate and costly antioxidant system that serves to sequester ROS before they wreak cellular havoc. We examined whether oxidative stress would differ between tropical and temperate birds. We used isolated primary dermal fibroblasts and pectoralis muscle tissue for measurements. We measured four aspects of oxidative stress in primary fibroblasts -reduced glutathione (GSH) concentration, ROS production, mitochondrial content and lipid peroxidation (LPO) damage. We found no significant differences in the four variables between temperate and tropical birds. In muscle tissue, we measured catalase (CAT), superoxide dismutase (SOD) glutathione peroxidase (GPx) activity, peroxyl and hydroxyl scavenging capacity and LPO damage. We found that peroxyl scavenging capacity was significantly higher in tropical birds compared with temperate birds.