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. 

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.