We previously showed that palmitic methyl ester (PAME) and stearic acid methyl ester (SAME) are simultaneously released from the sympathetic ganglion and PAME possesses potent vasodilatory properties which may be important in cerebral ischemia. Since PAME is a potent vasodilator simultaneously released with SAME, our hypothesis was that PAME/SAME confers neuroprotection in rat models of focal/global cerebral ischemia. We also examined the neuroprotective properties of Solutol HS15, a clinically approved excipient, because it possesses similar fatty acid compositions as PAME/SAME. Asphyxial cardiac arrest (ACA, 6min) was performed 30mins after PAME/SAME treatment (0.02mg/kg, IV). Solutol HS15 (2 ml/kg, IP) was injected chronically for 14 days (once daily). Histopathology of hippocampal CA1 neurons was assessed 7 days after ACA. For focal ischemia experiments, PAME, SAME, or Solutol HS15 was administered following reperfusion after 2 hrs of middle cerebral artery occlusion (MCAO). 2,3,5-triphenyltetrazolium staining of the brain was performed 24hrs after MCAO and the infarct volume was quantified. Following ACA, the number of surviving hippocampal neurons was enhanced by PAME (68%), SAME (69%), and Solutol HS15 (68%)-treated rats as compared to ACA only-treated groups. Infarct volume was decreased by PAME (83%), SAME (68%), and Solutol HS15 (78%) as compared to saline (vehicle) in MCAO-treated animals. PAME, SAME, and Solutol HS15 provide robust neuroprotection in both paradigms of ischemia. This may prove therapeutically beneficial since Solutol HS15 is already administered as a solublizing agent to patients. With proper timing and dosage, administration of Solutol HS15 and PAME/SAME can be an effective therapy against cerebral ischemia.

Cerebral ischemia causes cerebral blood flow (CBF) derangements resulting in neuronal damage by enhanced protein kinase C delta (δPKC) levels leading to hippocampal and cortical neuronal death after ischemia. Contrarily, activation of εPKC mediates ischemic tolerance by decreasing vascular tone providing neuroprotection. However, whether part of this protection is due to the role of differential isozymes of PKCs on CBF following cerebral ischemia remains poorly understood. Rats pretreated with a δPKC specific inhibitor (δV1-1, 0.5 mg/kg) exhibited attenuation of hyperemia and latent hypoperfusion characterized by vasoconstriction followed by vasodilation of microvessels after two-vessel occlusion plus hypotension. In an asphyxial cardiac arrest (ACA) model, rats treated with δ V1-1 (pre- and postischemia) exhibited improved perfusion after 24 h and less hippocampal CA1 and cortical neuronal death 7 days after ACA. On the contrary, εPKC-selective peptide activator, conferred neuroprotection in the CA1 region of the rat hippocampus 30 min before induction of global cerebral ischemia and decreased regional CBF during the reperfusion phase. These opposing effects of δ v. εPKC suggest a possible therapeutic potential by modulating CBF preventing neuronal damage after cerebral ischemia.

We previously showed that inhibition of protein kinase C delta (PKCd) improves brain perfusion 24 hours after asphyxial cardiac arrest (ACA) and confers neuroprotection in the cortex and CA1 region of the hippocampus 7 days after arrest. Therefore, in this study, we investigate the mechanism of action of PKCd-mediated hypoperfusion after ACA in the rat by using the two-photon laser scanning microscopy (TPLSM) to observe cortical cerebral blood flow (CBF) and laser Doppler flowmetry (LDF) detecting regional CBF in the presence/absence of dV1-1 (specific PKCd inhibitor), nitric oxide synthase (NOS) substrate (L-arginine, L-arg) and inhibitor (N o -Nitro-L-arginine, NLA), and nitric oxide (NO) donor (sodium nitroprusside, SNP). There was an increase in regional LDF and local (TPLSM) CBF in the presence of dV1-1 þ L-arg, but only an increase in regional CBF under dV1-1 þ SNP treatments. Systemic blood nitrite levels were measured 15 minutes and 24 hours after ACA. Nitrite levels were enhanced by pretreatment with dV1-1 30 minutes before ACA possibly attributable to enhanced endothelial NOS protein levels. Our results suggest that PKCd can modulate NO machinery in cerebral vasculature. Protein kinase C delta can depress endothelial NOS blunting CBF resulting in hypoperfusion, but can be reversed with dV1-1 improving brain perfusion, thus providing subsequent neuroprotection after ACA. Keywords: asphyxial cardiac arrest; middle cerebral artery occlusion; neuroprotection; palmitic acid methyl ester; stearic acid methyl ester INTRODUCTIONWe previously showed that inhibition of protein kinase C delta (PKCd) via dV1-1 can increase perfusion 24 hours after asphyxial cardiac arrest (ACA). Global cerebral ischemia (via ACA) causes derangement of cerebral blood flow (CBF) responsible for neuronal cell death in the CA1 region of the hippocampus as well as the cortex.1 Owing to the overall decrease in CBF after ischemia, neuronal cell death 1 can occur in major regions of the brain responsible for learning, memory, and cognitive function.2 It is thought that during global ischemia, PKCd (a novel PKC) levels are elevated causing PKCd to translocate to the nucleus (activation) resulting in cellular damage. In the normal brain, PKCd levels are nominal whereas global ischemia can cause activation/translocation of PKCd.3 Inhibition of PKCd (via specific inhibitor of PKCd, dV1-1) can cause a revival of CBF 24 hours after ischemia to counteract hypoperfusion or low CBF found to be suppressed after cardiac arrest from 38% to 65%. 1,4 We previously showed that pretreatment of dV1-1 before ACA can enhance perfusion 24 hours after ischemia, resulting in improved neuronal survival in the hippocampal CA1 and cortex regions in our rat model of ACA.1 Here, we sought out to define the specific mechanism(s) of how inhibition of PKCd can alleviate these pathologies. The possible endothelium and endothelial-mediated nitric oxide synthase (eNOS) involvement as a target for PKCd relating to general circulation was first reported by Monti et al.