The intricate interplay between different brain cell types is crucial to understanding neural pathophysiological states. This project aims to investigate the effects of the treatment of GHK on glial activity and inflammation using organotypic brain slice cultures. GHK improves tissue regeneration and exhibits anti-inflammatory effects, promoting neural protection. Slices taken from mice mirror the human-brain microenvironment, allowing a better understanding of neuronic health in pathological states. They also preserve the 3-D architecture of our brain, maintaining the intricacies between diverse cell types. First, the brain is collected from an euthanized mouse and rinsed in PBS, then sectioned into 100µm slices to culture, where they are exposed to different levels of GHK. Brain tissue samples are fixed in formalin to preserve cellular structure and stored in PBS. The tissue is embedded in paraffin to support stable sectioning using a microtome, allowing precise slicing into 4µm thick sections for analysis. By employing immunohistochemistry and histological techniques, insights into therapeutic strategies with the comparison of tissue cultures are shown. IHC looks at activated microglia(using IBA1), astrocytes(using GFAP), chronic inflammation(MCP1), and synaptic activity (synaptophysin) and characterizes neurons with cresyl violet staining. MCP1 levels are expected to decrease with GHK treatment. For microglia, there might be a reduction in their activated, proinflammatory state; astrocytes may show a shift towards reduced reactivity, shifting toward a homeostatic role in maintaining brain tissue stability and function. Synaptic activity is expected to improve. Neuronal health is predicted to be preserved, with enhanced structural stability and reduced signs of cellular stress. These results will help demonstrate the potential of GHK in mitigating chronic inflammation and promoting neuronal health. By revealing how GHK influences glial function and neuronal health, this research could pave the way for novel interventions targeting the improvement of neuronal health. 

Resilience to aging is a critical biological process that precedes age-related declines in physiological function. Defined as an organism’s ability to respond to physical stress and return to equilibrium quickly despite increasing age, wound healing can help in the evaluation of resilience by indicating the efficiency of repairing physical tissue damage to the body by generating new tissue. Previous correlations between resilience to aging and wound healing suggest that mice with higher rates of wound closure have less cognitive impairment and increased grip strength. GHK-Cu, a peptide that has been shown to accelerate wound healing and skin repair, has been used in topical treatments. Current studies have shown that systemic administration of the GHK-Cu peptide improves cognitive performance in aging mice and lowers inflammation levels. Understanding this role in resilience mechanisms could provide valuable insight into more complex interventions such as multiplexing. A cohort of 20 male 18-month-old C57BL/6 mice was used. Ten randomly chosen mice received daily intraperitoneal doses of GHK-Cu, while ten controls received saline for 19 days. After 3 days, a 2 mm ear punch was taken from both ears. Wound closure was measured on day 3 and day 19 by photographing the biopsy area and analyzing images with ImageJ software. After final measurements, cognitive performance and correlation to wound healing was assessed via phenotypic assays. Tissues surrounding the wound were then stained using immunohistochemistry. TNF-α and VEGF antibodies evaluated inflammation and cell growth respectively and were quantified using QuPath. The GHK-Cu peptide during the experiment showed enhanced wound healing from the physical stressor, suggesting a promising therapeutic strategy to improve recovery from injuries and cognition abilities in aging populations. The findings from this study may inform translational strategies to enhance wound healing and quicker recovery from tissue injury in aging and age-related diseases.

Age-related cognitive decline (ARCD) is very common and increases the risk for severe neurodegenerative conditions such as Alzheimer's disease. Treatment of ARCD can delay and lead to the cure of age-related diseases, but there is a lack of clinically proven drugs. One option is the naturally occurring peptide GHK (glycyl-L-histidyl-L-lysine), which readily forms a complex with copper (II). GHK is a key ingredient in anti-aging skin creams and regulates astrocytes through TGF-β and the SMAD pathway. As synaptic signaling decreases with age, this study investigates GHK-Cu's impact on synaptic function in middle-aged mice as a potential treatment for ARCD. Male and female C57BL/6 mice aged 20-22 months were treated with either the GHK-Cu peptide or saline as a control through intraperitoneal (IP) injection for five days. A spatial navigation learning task, the Box Maze, was utilized to analyze cognitive function by assessing the memory and learning of the mice on their last day of treatment. After the brain tissue samples were processed, synaptic function was assessed by performing immunohistochemistry (IHC) with Synaptophysin and PSD95 antibodies as molecular markers of pre- and post-synaptic integrity. The tissue slides were rehydrated, incubated with the antibodies overnight, and stained. After, the presence of antibodies was seen through microscopic examination and photographed for QuPath image analysis. Preliminary results of the Box Maze behavioral assay reveal the treated mice had increased cognitive function, memory, and learning capacity, which signals alleviated symptoms of ARCD. It is predicted that this increased resilience to ARCD will also be observed in the brain through the increased presence of Synaptophysin and PSD95 antibodies in the treated tissues compared to the control cohort. These results will show that short-term treatment of the GHK-Cu peptide will improve cognitive function and synaptic function, providing a potential treatment for ARCD and neurodegenerative diseases.

Alzheimer’s disease (AD) is a neurodegenerative disorder that disrupts memory, thinking, and behavior. It is the most common type of dementia and occurs with increasing frequency with increasing age. Transgenic AD mouse models have not predicted clinical efficacy because neurodegeneration occurs rapidly at a young age, so an aging environment is not a factor. To address this, an adeno-associated viral vector model of AD (AAV-AD) containing a green fluorescent-induction marker (GFP) was created to deliver pathogenic proteins Aβ-42 and P301L tau to neurons of old mice. The AAV capsid was engineered to have an affinity for neurons. Analysis of the model demonstrated successful expression of Aβ-42 and P301L tau in neurons in the brains of old mice when the vector constructs were administered intravenously (IV). However, it has yet to be shown whether the AAV-AD vector has off-target effects in systemic organs like the liver. Characteristic AD pathology does not naturally occur outside the brain. Therefore, this project was designed to determine if the AAV-AD vector became established in hepatic cells. Paraffin-embedded tissues were obtained from 27-month-old C57BL/6 male and female mice infected with the AAV-AD or sham vector for 3 months. Immunohistochemistry (IHC) was used to examine expression of GFP, Aβ-42, P301L tau, MCP-1 inflammatory cytokine, and yH2AX DNA-damage response. Images were taken using digital microscope software, and quantified through an open-source digital image software. Age-related histopathology lesion scores from H&E-stained brain and liver were compared with IHC stains. The expectation is there will be little evidence of AAV-AD proteins but incremental increases in inflammatory and DNA-damage proteins proportional to histopathology lesion scores. These observations would help validate translational efficacy of the AAV-AD mouse model for preclinical testing of pharmaceuticals to treat or prevent AD.

Organ slice cultures present a promising alternative to cell culture to study biological processes in-vitro by maintaining the integrity of interactions between different cell types. A need for a model that can be used to investigate cell interactions becomes apparent when studying the impact of stress, due to its effect on many pathways. Resilience, which decreases with aging, is defined as the ability to respond to stress. This project aims to investigate the impact of a chemical stressor to study resilience in aging C57BL/6 mice. Organ slice cultures were prepared from thin slices of the brain and the chemotherapy drug, cyclophosphamide (Cyp), was added to represent an immune response. After 2 weeks, tissues were fixed and embedded in wax blocks to make tissue slides. Immunohistochemistry (IHC) assays were performed to evaluate the impact of Cyp on microglia, astrocytes, and chronic inflammation. These particular markers were chosen for IHC analysis for their role in the immune response. It is anticipated that Cyp will induce a stress response in the brain slice cultures and increase chronic inflammation, and activated microglia and astrocyte counts compared to the control group. The results from this study will provide information about the ability to recover from a chemical stressor while improving the protocol for culturing brain organ slices to reduce the number of animals used in research. Developing stress tests is important to be able to identify at-risk individuals that may require early intervention to reduce the likelihood of cognitive decline with aging.

Sleep deprivation (SD) is a pervasive issue linked to significant cognitive and neurological impairments, affecting billions of people. SD accelerates markers of aging, but some individuals exhibit resilience to its effects. SD response is indicative of resilience. Identifying factors that promote SD resilience may inform interventions to enhance resilience. Studies have shown that SD alters gene expression in rodents, yet it remains uncertain which changes are specific to homeostasis. Previous rodent studies examined the effects of single day SD. Our study increases the duration to five days and separates mice into high and low responders, providing a novel insight into SD responses. This establishes a valuable evaluation of resilience for aging interventions. Female mice in the treatment group were sleep deprived through continuously stirring them during sleep periods. Control and treated mice were then subjected to the box-maze assay to evaluate relative learning rates and cognitive impairment. High performance in the box maze was designated as a high responder, and vice versa. Mice were then euthanized, and the hippocampus was isolated. The transcriptomes of control and treated mice were analyzed via mRNA sequencing. Analyzing transcriptomes of control, high, and low responder mice showed distinct changes in expression of key physiological and biochemical phenotypes. Genes known to be associated with SD were isolated and examined separately regardless of difference. Overall, high degrees of similarity were observed in control and high responders to SD, while low responders had the greatest changes in comparison to the latter groups. These experiments provide an efficient, robust platform to study the biochemical effects of SD, offering attractive insights for frameworks to quickly evaluate therapeutic strategies aimed at enhancing resilience to aging,

Aging is characterized by functional decline and increased disease susceptibility, making it essential to study its mechanisms for developing treatments. However, human-based research presents ethical, logistical, and financial challenges, leading to the use of animal models such as rodents and non-human primates. House crickets (Acheta domesticus) offer a promising alternative due to their short lifespan, well-defined organ systems, and ease of rearing. Based on lifespan and age-related decline, a 4-week-old cricket corresponds to a young adult mouse (~3 months) and a human in their 20s, while a 10-week-old cricket is comparable to a geriatric mouse (~24 months) and a human in their 70s. This study examines age-related cognitive decline in crickets using the Y-maze, a widely used cognitive assessment tool in rodents. The Y-maze measures spontaneous alternation, defined as the frequency of sequential entries into three different arms, divided by total arm entries. A higher alternation rate indicates better working memory and decision-making ability, while a lower rate suggests cognitive deficits. Previous experiments showed a significant age-related decline in alternation (p = 0.019), with geriatric crickets exhibiting lower rates than young adult crickets, suggesting age-related cognitive decline. However, the single 10-minute trial design may have introduced confounds such as fatigue or habituation, potentially skewing results.To improve data reliability, a refined Y-maze protocol will implement a two-phase trial. Crickets at 4, 6, 8, and 10 weeks (10 males, 10 females per group) will undergo a 5-minute test phase followed by a 5-minute main trial. One-way ANOVA will compare alternation percentages across age groups, while two-way ANOVA will assess sex-related differences. This study provides a clearer understanding of cognitive function across age groups, strengthening the validity of house crickets as a model for aging research and laying the groundwork for further translational studies.