Investigating the effects of acute and chronic stress on DNA damage.

A hallmark of the vertebrate stress response is a rapid increase in glucocorticoids and catecholamines; however, this does not mean that these mediators are the best, or should be the only, metric measured when studying stress. Instead, it is becoming increasingly clear that assaying a suite of downstream metrics is necessary in stress physiology. One component of this suite could be assessing double-stranded DNA damage (dsDNA damage), which has recently been shown to increase in blood with both acute and chronic stress in house sparrows (Passer domesticus). To further understand the relationship between stress and dsDNA damage, we designed two experiments to address the following questions: (1) how does dsDNA damage with chronic stress vary across tissues? (2) does the increase in dsDNA damage during acute stress come from one arm of the stress response or both? We found that (1) dsDNA damage affects tissues differently during chronic stress and (2) the hypothalamic-pituitary-adrenal axis influences dsDNA damage with acute stress, but the sympathetic-adreno-medullary system does not. Surprisingly, our data are not explained by studies on changes in hormone receptor levels with chronic stress, so the underlying mechanism remains unclear.

Neophobia, but not perch hopping, is sensitive to long-term chronic stress intensity.

To further elucidate the role that wear-and-tear plays in the transition from acute to chronic stress, we manipulated the intensity and duration of applied chronic stress to determine if behavior would respond proportionately. We brought wild house sparrows into captivity and subjected them to high-stress, medium-stress, low-stress, or captivity-only. We varied the number of stressors per day and the duration of stress periods to vary wear-and-tear, and thus the potential to exhibit chronic stress symptoms. The behaviors we assessed were neophobia (the fear of the new; assessed via food approach latency) and perch hopping (activity). We predicted that our birds would show proportionate decreases in neophobia and activity throughout a long-term chronic stress paradigm. Our results indicate that neophobia is sensitive to the intensity of chronic stress, however, the birds became more neophobic, which was the opposite of what we expected. Conversely, perch hopping did not differ across treatment groups and is thus not sensitive to the intensity of chronic stress. Together, these data show that different behavioral measurements are impacted differently by chronic stress.

A mathematical representation of the reactive scope model.

Researchers have long sought to understand and predict an animal's response to stressful stimuli. Since the introduction of the concept of homeostasis, a variety of model frameworks have been proposed to describe what is necessary for an animal to remain within this stable physiological state and the ramifications of leaving it. Romero et al. (Horm Behav 55(3):375-389, 2009) introduced the reactive scope model to provide a novel conceptual framework for the stress response that assumes an animal's ability to tolerate a stressful stimulus may degrade over time in response to the stimulus. We provide a mathematical formulation for the reactive scope model using a system of ordinary differential equations and show that this model is capable of recreating existing experimental data. We also provide an experimental method that may be used to verify the model as well as several potential additions to the model. If future experimentation provides the necessary data to estimate the model's parameters, the model presented here may be used to make quantitative predictions about physiological mediator levels during a stress response and predict the onset of homeostatic overload.

Varying intensities of chronic stress induce inconsistent responses in weight and plasma metabolites in house sparrows (Passer domesticus).

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.

Long term stability of corticosterone in feathers.

Measuring corticosterone in feathers allows researchers to make long-term, retrospective assessments of physiology with non-invasive sampling. To date, there is little evidence that steroids degrade within the feather matrix, however this has yet to be determined from the same sample over many years. In 2009, we made a pool of European starling (Sturnus vulgaris) feathers that had been ground to a homogenous powder using a ball mill and stored on a laboratory bench. Over the past 14 years, a subset of this pooled sample has been assayed via radioimmunoassay (RIA) 19 times to quantify corticosterone. Despite high variability across time (though low variability within assays), there was no effect of time on measured feather corticosterone concentration. In contrast, two enzyme immunoassays (EIA) produced higher concentrations than the samples assayed with RIA, though this difference is likely due to different binding affinities of the antibodies used. The present study provides further support for researchers to use specimens stored long-term and from museums for feather corticosterone quantification, and likely applies to corticosteroid measurements in other keratinized tissues.

House sparrows prioritize skin repair over constitutive innate immunity during long-term chronic stress.

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.

Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes.

In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.

Background DNA damage is higher in summer than winter in both free-living and captive birds.

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 double-stranded 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.

Maternal Responses in the Face of Infection Risk.

When animals are sick, their physiology and behavior change in ways that can impact their offspring. Research is emerging showing that infection risk alone can also modify the physiology and behavior of healthy animals. If physiological responses to environments with high infection risk take place during reproduction, it is possible that they lead to maternal effects. Understanding whether and how high infection risk triggers maternal effects is important to elucidate how the impacts of infectious agents extend beyond infected individuals and how, in this way, they are even stronger evolutionary forces than already considered. Here, to evaluate the effects of infection risk on maternal responses, we exposed healthy female Japanese quail to either an immune-challenged (lipopolysaccharide [LPS] treated) mate or to a healthy (control) mate. We first assessed how females responded behaviorally to these treatments. Exposure to an immune-challenged or control male was immediately followed by exposure to a healthy male, to determine whether treatment affected paternity allocation. We predicted that females paired with immune-challenged males would avoid and show aggression towards those males, and that paternity would be skewed towards the healthy male. After mating, we collected eggs over a 5-day period. As an additional control, we collected eggs from immune-challenged females mated to healthy males. We tested eggs for fertilization status, embryo sex ratio, as well as albumen corticosterone, lysozyme activity, and ovotransferrin, and yolk antioxidant capacity. We predicted that immune-challenged females would show the strongest changes in the egg and embryo metrics, and that females exposed to immune-challenged males would show intermediate responses. Contrary to our predictions, we found no avoidance of immune-challenged males and no differences in terms of paternity allocation. Immune-challenged females laid fewer eggs, with an almost bimodal distribution of sex ratio for embryos. In this group, albumen ovotransferrin was the lowest, and yolk antioxidant capacity decreased over time, while it increased in the other treatments. No differences in albumen lysozyme were found. Both females that were immune-challenged and those exposed to immune-challenged males deposited progressively more corticosterone in their eggs over time, a pattern opposed to that shown by females exposed to control males. Our results suggest that egg-laying Japanese quail may be able to respond to infection risk, but that additional or prolonged sickness symptoms may be needed for more extensive maternal responses.

The Effect of a Combined Fast and Chronic Stress on Body Mass, Blood Metabolites, Corticosterone, and Behavior in House Sparrows (Passer domesticus).

One aspect of the Reactive Scope Model is wear-and-tear, which describes a decrease in an animal's ability to cope with a stressor, typically because of a period of chronic or repeated stressors. We investigated whether wear-and-tear due to chronic stress would accelerate a transition from phase II to phase III of fasting. We exposed house sparrows (Passer domesticus) to three weeks of daily fasts combined with daily intermittent repeated acute stressors to create chronic stress, followed by two weeks of daily fasts without stressors. We measured circulating glucose, ß-hydroxybutyrate (a ketone), and uric acid in both fasted and fed states. We expected birds to be in phase II (high fat breakdown) in a fasted state, but if wear-and-tear accumulated sufficiently, we hypothesized a shift to phase III (high protein breakdown). Throughout the experiment, the birds exhibited elevated ß-hydroxybutyrate when fasting but no changes in circulating uric acid, indicating that a transition to phase III did not occur. In both a fasted and fed state, the birds increased glucose mobilization throughout the experiment, suggesting wear-and-tear occurred, but was not sufficient to induce a shift to phase III. Additionally, the birds exhibited a significant decrease in weight, no change in corticosterone, and a transient decrease in neophobia with chronic stress. In conclusion, the birds appear to have experienced wear-and-tear, but our protocol did not accelerate the transition from phase II to phase III of fasting.

Common myths of glucocorticoid function in ecology and conservation.

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.

Plasma biomarkers of the amyloid pathway are associated with geographic atrophy secondary to age-related macular degeneration.

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 ANCOVAs. Serum amyloid precursor protein (sAPP) was analyzed by a separate ELISA and included in further analyses. The 40 biomarkers, sAPP and amyloid-ß (Aß) (1-42) (included for comparison) were evaluated in Cohort 2. This resulted in 11 statistically significant biomarkers, including sAPP and Aß(1-40), but not Aß(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 Aß(1-40) over Aß(1-42), and a potential role for the carboxylase activity of the γ-secretase protein, which preferentially splices sAPPß to Aß(1-40). Other markers are associated with the breakdown and remodeling of the extracellular matrix, and loss of homeostasis, possibly within the photoreceptor-retinal pigment epithelium-choriocapillaris complex. These data suggest novel disease pathways associated with GA pathogenesis and could provide potential novel targets for treatment of GA.

Cellular metabolism and oxidative stress as a possible determinant for longevity in small breed and large breed dogs.

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.

Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival.

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.