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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.

Angiotensin II (Ang II) activates cytosolic phospholipase A2α and releases arachidonic acid (AA) from tissue phospholipids. AA metabolites mediate or modulate one or more renocardiovascular effects of this peptide and have been implicated in hypertension. Since AA release is the rate limiting step in eicosanoid production, it is possible that cPLA2α might play a central role in the development of Ang II-induced hypertension. To test this hypothesis, we investigated the effect of Ang II infusion for 13 days by micro-osmotic pumps (700 ng/kg/min), on systolic blood pressure and associated pathophysiological changes in wild type (cPLA2α+/+) and cPLA2α-/- mice. Ang II infusion increased systolic blood pressure in cPLA2α+/+ but not in cPLA2α-/- mice. Ang II induced increase in systolic blood pressure was also abolished by the AA metabolism inhibitor, 5,8,11,14-eicosatetraenoic acid in cPLA2α+/+ mice. Ang II infusion in cPLA2α+/+ mice increased cardiac and renal cPLA2 activity, resulted in cardiovascular and renal dysfunction, caused cardiovascular remodeling, endothelial dysfunction, increased vascular reactivity and compromised renal hemodynamics in cPLA2α+/+ mice; these changes were diminished in cPLA2α-/- mice. Ang II also increased cardiac and renal infiltration of F4/80+ macrophages and CD3+ T lymphocytes, caused cardiac fibrosis and produced cardiovascular and renal oxidative stress and end organ damage, in cPLA2α+/+ but not cPLA2α-/- mice. Infusion of Ang II increased cardiac ER stress and activity of ERK1/2 and cSrc in cPLA2α+/+, but not cPLA2α-/- mice. These data suggest that Ang II-induced hypertension and associated renocardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of AA and the generation of pro-hypertensive eicosanoids.