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Doctor of Philosophy (PhD)
Dr. John C. Gensel
Spinal cord injuries (SCI) are debilitating and life altering events that can lead to permanent motor and sensory loss. SCI outcomes are impacted by both clinical factors such as blood alcohol content (BAC) at the time of injury as well as biological factors like the lipid-rich myelin debris that accumulates in the injury site. Both clinical and biological factors contribute to SCI recovery, impacting neuroinflammation, locomotor recovery, and histopathology. The purpose of the studies described here is to investigate the role of acute alcohol intoxication and intracellular lipid processing pathways on SCI outcomes in a rodent model.
An elevated BAC is found in a third of SCI cases. Clinical and preclinical studies report variable effects of alcohol/ethanol intoxication on patient outcomes and functional recovery after SCI. Few studies to date have investigated the neuropathological consequences of ethanol intoxication at the time of SCI or the reciprocal effect of SCI on ethanol metabolism. Therefore, we combined a pre-clinical model of acute ethanol intoxication and experimental contusion SCI to investigate their interactive effects in female mice. We first investigated the effect of SCI on ethanol metabolism and found that SCI does not alter ethanol metabolism. However, we did find that isoflurane anesthesia significantly slowed ethanol metabolism independent of SCI. We also determined how acute ethanol intoxication at the time of SCI alters locomotor recovery and lesion pathology. We observed a detrimental effect of ethanol on tissue sparing after SCI.
Lipid-rich myelin debris accumulation can also impact SCI outcomes. After SCI, monocyte-derived macrophages (MDMs) infiltrate the lesion to aid in cellular debris clearance. However, this response is emerging as a double-edged sword. Lingering debris inhibits repair and plasticity while debris clearance by infiltrating MDMs induces a proinflammatory and damaging shift in phenotype. Clearance of lipid-rich myelin debris causes MDMs to resemble proinflammatory foam cells phenotypically and morphologically. Foam cells are lipid-laden macrophages with numerous lipid droplets (LDs) that form when excessive lipid uptake overwhelms lipid metabolism. Foam cells persist chronically in the injured spinal cord and may contribute to the sustained proinflammatory lesion environment. We investigated the role of two proteins, cytosolic phospholipase-A2 (cPLA2) and Perilipin 2 (Plin2), in MDMs after SCI and interrogated how manipulation with two different tools alters locomotor recovery and tissue pathology. cPLA2 is a key enzyme in the production of arachidonic acid metabolites and drives the potentiation of a proinflammatory phenotype observed when MDMs are exposed to purified myelin in vitro. To interrogate the role of MDM cPLA2 in vivo, we used hematopoietic stem cell transplantation to generate chimeric mice that lacked cPLA2 in circulating leukocytes. We found that chimeric cPLA2 KO mice had no changes in locomotor recovery or tissue pathology compared to controls. Plin2 is a master regulator of intracellular lipid trafficking and may play a role in foamy MDM phenotype after SCI. Plin2 is highly expressed in MDMs acutely after injury and Plin2 deficient mice display improved remyelination in other models of CNS injury. Therefore, we generated a tamoxifen-inducible Cre/Flox system to specifically delete Plin2 from infiltrating MDMs prior to SCI. We developed a method to quantify lipid droplet volume and density in the MDM-rich lesion core using Oil-Red O staining and scanning confocal microscopy. Plin2 deletion did not alter MDM lipid droplet size but did alter cell morphology in the MDM-rich lesion core.
Collectively, these data show that both extrinsic clinical factors and intrinsic genetic manipulations can alter SCI outcomes. Further, we identified novel tools and models for the evaluation of both clinical (ethanol) and biological (lipid droplets) processes involved in SCI pathophysiology.
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I was supported by the National Institute on Alcohol Abuse and Alcoholism T32 (5T32AA027488): Interdisciplinary Training in Alcohol Research from August 2020 to August 2022
I was supported by the National Institute of Neurological Disorders and Stroke F30 ( 1F30NS129251-01) The Role of Perilipin 2 in Macrophages after Experimental Spinal Cord Injury from August of 2022 to August of 2023
My work was also supported by The Craig H. Neilsen Foundation Grant (651996) The role of lipid dysfunction in spinal cord injury pathophysiology from August 2020 to August of 2023
My work was also supported by the National Institute of Neurological Disorders and Stroke R01 (1R01NS116068-01A1) Macrophage Depletion Therapy for Spinal Cord Injury from August 2021 to August 2023
Glaser, Ethan, "Clinical and Biological Factors Determine Spinal Cord Injury Outcomes: Liquor to Lipids" (2023). Theses and Dissertations--Physiology. 64.
Available for download on Friday, February 16, 2024