Browsing by Author "Jones, Nathan"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item Evaluation of a bi-modal therapy in a model of cerebral ischemia(2024-03-21) Hernandez, Katherine; Jones, Nathan; Ortega, SterlingPurpose: Ischemic strokes are a significant contributor to cardiovascular-related deaths in the U.S. Currently, pharmacological interventions are limited to alteplase, an FDA-approved thrombolytic agent, or a surgical procedure known as a thrombectomy. However, their effectiveness is limited by a narrow therapeutic window and incomplete reperfusion rates. Our research explores an alternative strategy centered around PNU-120596 (PNU), a positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (a7 nAChR), which has shown promise in previous studies in a male rat stroke model. The a7 nAChR is expressed in various central nervous system cells, and studies suggest that PNU directly promotes neuroprotection in these cells. Furthermore, research indicates increased expression of the a7 nAChR in immune cells, specifically in activated inflammatory CD4 T-cells, a cell crucial in stroke studies. We hypothesize that PNU is a bi-modal therapeutic agent, addressing neuronal loss and inflammatory responses, presenting a unique advantage. Methods: This study aims to provide insight into PNU's efficacy and regulatory role in stroke-induced inflammatory CD4 T-cells. The study will assess PNU treatment's efficacy in promoting stroke recovery in transient middle cerebral artery (tMCAO) mice, a murine stroke model, and investigate how PNU modulates stroke-induced inflammatory CD4 T-cells, examining their suppression, neuronal survival, and alleviation of the disease using mixed cortical cultures. Mice that had undergone the tMCAO procedure were administered 20mg/kg of PNU immediately after reperfusion, and brain tissue was collected 24 and 72 hours post-tMCAO for infarct analysis. Spleen and cervical lymph nodes were collected at the same time points for flow cytometry analysis. Results: PNU reduces stroke infarct when administered immediately after tMCAO, and brain tissue is collected 24 hours post-tMCAO. However, protection is lost when the same dose is administered, and brain tissue is collected 72 hours post-tMCAO. No significant changes in levels of CD4 T-cell percentages were seen in the spleen or cervical lymph nodes at both time points. Conclusion: This study suggests that PNU reduces infarct volume immediately 24 hours post-tMCAO, but the protection is lost at 72 hours post-tMCAO. However, subsequent studies are necessary to assess the efficacy of PNU at longer time points with different dosages.Item In-Vitro Assessment of Cortical Repair Induced by Branched-chain Amino Acid Treatment(2024-03-21) Mathew, Ezek; Jones, Nathan; Dickerman, Rob; Ortega, SterlingPurpose: Traumatic Brain Injury (TBI) refers to a constellation of pathologies resulting from mechanical damage to cortical tissue. The neurological sequala of such injuries can be devastating, and definitive treatment does not exist at this time. Branched-chain Amino Acid (BCAA) treatment has demonstrated neuroprotective effects in clinical literature and in various animal models of TBI. However, there is a lack of in-vitro literature referencing the repair capacity of BCAA administration after neuronal injury, particularly in the context of TBI. To fill this gap in knowledge, a scratch assay was repurposed for use in cortical culture, to assess the repair capacity of BCAA treatment. Methods: Mouse-derived Mixed Cortical Culture (MCC) cells were extracted and seeded in 24 well plates. A scratch assay was performed, where a vertical scratch was drawn across each well in a reproducible manner with a 200 uL pipette tip. This procedure is meant to recapitulate aspects of mechanical damage induced by TBI on cortical tissue. Subsequently, images were taken immediately post-injury (0 hour time point), at 24 hour, and at 48 hour time points post-scratch to quantify the area of scratch unfilled by cells. Various dose concentrations of BCAA were tested in comparison to control (media only) and vehicle control (water). Test conditions included the customary BCAA ratio, which is a 2:1:1 mix of leucine, isoleucine, and valine; additionally, a 1:1:1 ratio of leucine, isoleucine, and valine was also tested. Results: At 48 hours post-scratch, significant differences were found in open wound area when comparing the media only control to 10 uM (p < 0.01), 30 uM (p < 0.01), 300 uM (p < 0.001), and 1000 uM (p < 0.01) of the 2:1:1 BCAA dose. Significant differences were also found in the wound area when comparing the water vehicle control to 10 uM (p < 0.05), 30 uM (p < 0.01), 300 uM (p < 0.01), and 1000 uM (p < 0.01) of the 2:1:1 BCAA dose. No significant differences were found in the open wound area when comparing the controls and BCAA doses, at the 24 hour time point. Of note, no significant differences were found between control and treatment with the 1:1:1 ratio of leucine, isoleucine, and valine at any time point. Conclusion: BCAA treatment at the 2:1:1 ratio was seen to accelerate injury recovery at various dose concentrations, as quantified by open wound area after scratch injury was induced. This cell culture model demonstrates the importance of BCAA ratios. While this aligns with animal models and clinical literature, this is the first in-vitro assessment of BCAA repair capacity, in the context of cortical culture. Future studies will be undertaken to further elucidate the constituents of the repair mechanism.Item Investigating an early source of IFN-gamma production post large vessel occlusion in mice(2024-03-21) Sharif, Safia; Hernandez, Katherine; Jones, Nathan; Ortega, SterlingIn the United States, strokes are one of the leading causes of death- with ischemic strokes being the most common. The etiology by which ischemic strokes occur may be due to large vessel occlusion. Typically, this process occurs by which a thrombus or embolus occludes a major vessel within the brain leading to reduced blood flow (ischemia) precipitating damage to brain tissue. Large vessel occlusion is a critical event, in which a rapid response is required to ensure the survival of brain tissue. It is understood that IFN-gamma is involved in the production of various inflammatory mediators, however, in the context of the stroke, it is not known where the IFN-gamma production is sourced from early in the stroke cascade. This study investigates the production of IFN-gamma in the context of a large vessel occlusion in mice. In vitro and in vivo studies were utilized to (1) compare the production of IFN-gamma amongst large vessel pre-occlusion, large vessel post-occlusion, and a distal vascular site (2) examine the direct relationship between oxygen-glucose deprivation applied to mixed cortical cultures and naïve lymphocytes (3) investigate the source of IFN-gamma production. Collected data was analyzed by utilizing flow cytometry. Findings revealed that in comparison to the pre-occlusion, the post-occlusion sample of blood contained increased levels of IFN-gamma. Mixed cortical cultures were examined under ischemic-like conditions and demonstrated increased IFN-gamma production from naïve cells. Evidence also suggested that the most likely source of IFN-gamma production stems from macrophages. IFN-gamma could potentially be an important modulator in the exacerbation of brain parenchymal damage, especially in the context of post-large vessel occlusion.