Ischemic preconditioning (IPC) is the neuroprotective phenomenon in which a brief ischemic episode induces genetic reprogramming of affected brain cells, causing physiological changes that minimize cell death from subsequent, sustained ischemic insult. We hope to characterize the cellular and molecular mechanisms of IPC. Our lab previously has shown that IPC is dependent on Toll-Like Receptor 4 (TLR4) and involves the production of interferon stimulated genes (ISGs) in microglia. Following ischemia, damaged neurons release endogenous TLR4 agonists called damage-associated molecular patterns (DAMPS), which induce interferon (IFN) release by microglia. The IFNs initiate a biochemical signaling cascade that activates transcription factors which bind to the interferon-stimulated response element (ISRE), promoting ISG transcription which alters the immunological function and phenotype of microglia, mediating neuroprotection. My project is to confirm the cell signaling mechanism by which hypoxic/hypoglycemic (H/H) conditions induce ISG expression in vitro. To accomplish this, I am using a reporter assay to detect ISRE activation with luciferase protein expression, looking specifically at the effects of H/H experimental stimulations on ISRE activity in microglia from the N9 cell line. I am first optimizing a transfection protocol using green fluorescent protein (GFP) as a reporter. I will use the optimized protocol for experiments, transfecting microglia with DNA encoding the luceriferase enzyme linked to the ISRE promoter, then incubating the cells under H/H conditions, and finally using a plate reader to quantify fluorescence. We anticipate that H/H stimulation will result in increased luminescence in wild-type microglia, and we would interpret these results as indicating that ISG expression in H/H-stimulated microglia is mechanistically due to transcriptional activation at ISRE sites. Identifying the genetic actors in IPC will reveal intracellular targets for pharmaceuticals that can mimic this neuroprotection. We can then administer these medications pre-emptively to patients at risk for stroke or emergently to acute stroke patients.

Infection occurs in about 30% of patients with stroke and is associated with worse outcomes. Data suggest that certain antibiotics used to treat post-stroke infection may be neurotoxic (fluoroquinolones) and others may be neuroprotective (β-lactams). We are studying the outcome of rats who are subjected to ischemic stroke then pulmonary infection. Rats are treated with either ceftiofur (β-lactam class), enrofloxacin (fluoroquinolone class) or normal saline and outcome determined by a series of behavioral tests administered over the ensuing four weeks. The behaviour tests include the rat's ability to stay atop a rotating drum (rotarod), ability to retain their footing while walking on a grated surface (foot fault test), and tendency to turn in circles (neurological score). Outcome of rats treated with enrofloxacin and ceftiofur will be compared to the outcome of rats treated with normal saline. Outcome that is better than in saline treated rats would suggest the antibiotic is neuroprotective, while outcome worse than in saline treated animals would suggest that the antibiotic is neurotoxic. The results of the study could have immediate and direct applications to clinical care as it could influence the antibiotic choice in patients who develop post-stroke infection.

Prenatal Cocaine Exposure (PCE) can cause movement disorders and developmental problems in affected children. Statistic from Substance Abuse and Mental Health Services Administration stated that over 4% of pregnant women in the United States use illicit drugs, and up to 1% of pregnant women use cocaine. Investigations in the Bamford Lab have shown that PCE causes long-lasting abnormalities in corticostriatal activity. By using slice preparation techniques and Marianas imaging system, we quantify GABA interneuron density within the striatum in 30 and 60-day-old mice that are exposed to cocaine in utero. Lhx6-GFP BAC transgenic mice are used as they express enhanced green fluorescent protein (GFP) in the GABA interneurons which makes imaging analysis more precise and accurate. From our findings, we have found that there is a decreased population of GABA neurons in the striatum than wild type mice. PCE inhibits the tangential migration of GABA interneurons, which results in a reduction of GABAergic neurons within the striatum. The decreased GABA neurons may contribute to the structural and functional deficits in the exposed offsprings. This research will also help future studies to investigate changes in GABA interneuron complementing in the prenatal period, apoptosis, and changes in dendritic spine density in hopes for better understanding of the mechanisms and effects of prenatal drugs use.

Stroke is the fourth leading cause of death and the leading cause of long-term disability in the United States. Microglia are the resident immune cells in the central nervous system and play critical roles in stroke pathophysiology. During ischemic stroke, cytokines known as interferons (IFNs) are released from CNS cells and bind to microglial cell surface receptors. This binding initiates a signaling cascade that leads to increased expression of Interferon-Stimulated Genes (ISGs). We hypothesize that increased expression of ISGs contributes to post-stroke neuroprotective pathways. There are three types of IFNs, and Type 1 includes IFNs α and β, which are well-known immunomodulatory factors. Type 2 IFNs include IFNγ, a prototypical pro-inflammatory activator. Type 3 IFNs (IFNλs) are the most recently discovered and least well characterized. IFNλs are effective in treating several viral illnesses and they are better tolerated (fewer side effects) than Type 1 IFNs. We have recently shown that Type 1 IFNs can induce increases in ISG expression in microglia. My project goal was to characterize the ability of IFNλs to induce ISG expression in microglia and compare their pharmacological potency to that of Type 1 IFNs. To do this, I stimulated primary microglia with varying dosages of IFNλ3 and for varying amounts of time. I extracted RNA and prepared protein lysates from the stimulated cells. ISG expression was quantified by qRT-PCR and immune (Western) blotting. I also collected conditioned medium and assayed for the release of specific ISG chemokines, such as CCL5 and CXCL10 using ELISA. I obtained dose response curves and time course data. Our results showed robust up-regulation of multiple ISG mRNA transcripts and increases in release of specific chemokines following stimulation of microglia with IFNλ3. These studies will help us continue to characterize important cellular and molecular targets for pharmacological intervention in stroke.