Jianya Ma

Enlarged Infarcts in Endothelial Nitric Oxide Synthase Knockout Mice are Attenuated by Nitro-L-Arginine:

Infarct size and vascular hemodynamics were measured 24 h after middle cerebral artery (MCA) occlusion in mice genetically deficient in the endothelial nitric oxide synthase (eNOS) isoform. eNOS mutant mice developed larger infarcts (21%) than the wild-type strain when assessed 24 h after intraluminal filament occlusion. Moreover, regional CBF values recorded in the MCA territory by laser-Doppler flowmetry were more severely reduced after occlusion and were disproportionately reduced during controlled hemorrhagic hypotension in autoregulation experiments. Unlike the situation in wild-type mice, nitro-L-arginine superfusion (1 mM) dilated pial arterioles of eNOS knockout mice in a closed cranial window preparation. As noted previously, eNOS mutant mice were hypertensive. However, infarct size remained increased despite lowering blood pressure to normotensive levels by hydralazine treatment. Systemic administration of nitro-L-arginine decreased infarct size in eNOS mutant mice (24%) but not in the wild-type strain. This finding complements published data showing that nitro-L-arginine increases infarct size in knockout mice expressing the eNOS but not the neuronal NOS isoform (i.e., neuronal NOS knockout mice). We conclude that NO production within endothelium may protect brain tissue, perhaps by hemodynamic mechanisms, whereas neuronal NO overproduction may lead to neurotoxicity.

Prolonged Therapeutic Window for Ischemic Brain Damage Caused by Delayed Caspase Activation

Apoptotic cell death is prominent in neurodegenerative disorders, such as Alzheimers disease and Huntingtons disease, and is found in cerebral ischemia. Using a murine model of delayed cell death, we determined that cleavage of zDEVD-amino-4-trifluoromethyl coumarin (zDEVD-afc) in brain homogenate, a measure of caspase activation, increased initially 9 hours after brief (30 minutes) middle cerebral artery occlusion along with caspase-3p20 immunoreactive cleavage product as determined by immunoblotting. zDEVD-afc cleavage activity was blocked by pretreatment or posttreatment with the caspase-inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl-ketone (zDEVD-fmk), and ischemic damage was reduced when the drug was injected up to 9 hours after reperfusion. The protection was long lasting (21 days). Hence, the period before caspase activation defined the therapeutic opportunity for this neuroprotective agent after mild ischemic brain injury. Prolonged protection after caspase inhibition plus the extended treatment window may be especially relevant to the treatment of neurodegenerative disorders.

Synergistic effects of caspase inhibitors and MK‐801 in brain injury after transient focal cerebral ischaemia in mice

Excitotoxic and apoptotic mechanisms have been implicated in the pathophysiology of cerebral ischaemia. Both MK‐801, an NMDA receptor antagonist, or peptide inhibitors of the caspase family (z‐VAD.FMK and z‐DEVD.FMK), protect mouse brain from ischaemic cell damage. In this study, we examined whether these drugs which act via distinct mechanisms, afford even greater neuroprotection when given in combination following 2 h MCA occlusion (filament model) and 18 h reperfusion. Given alone as pretreatment, MK‐801 (1, 3 and 5 mg kg−1, but not 0.3 mg kg−1, i.p.) decreased infarct size by 34–75%. When injected 1 h after occlusion and before reperfusion, 3 mg kg−1 reduced injury but not when administered 1 h after reperfusion. Pretreatment with a subthreshold dose of MK‐801 (0.3 mg kg−1) plus a subthreshold dose of z‐VAD.FMK (27 ng) or z‐DEVD (80 ng) significantly decreased infarct size by 29 and 30%, respectively, and enhanced neurological function. Administering a subthreshold dose of z‐VAD.FMK (27 ng) or z‐DEVD.FMK (80 ng) as pretreatment extended the time window for MK‐801 (3 mg kg−1) by 2 h from 1 h before reperfusion to at least 1 h after reperfusion. Pretreating with a subthreshold dose of MK‐801 (0.3 mg kg−1) extended the time window for z‐DEVD.FMK (480 ng) from 1 h after reperfusion to at least 3 h after reperfusion. We conclude that caspase inhibitors which putatively block apoptotic cell death and inhibit cytokine production and the NMDA antagonist MK‐801 act synergistically and prolong their respective therapeutic windows in cerebral ischaemia.

L-NA-Sensitive rCBF Augmentation during Vibrissal Stimulation in Type III Nitric Oxide Synthase Mutant Mice

Regional cerebral blood flow (rCBF) was studied in type III nitric oxide (NO) synthase (endothelial, eNOS) mutant and wild type mice during mechanical whisker stimulation before and after nitro-L-arginine (L-NA) superfusion using the closed cranial window technique. rCBF increased equally in cortical barrel fields in both strains during stimulation, as measured by laser Doppler-flowmetry, and was inhibited by L-NA superfusion (1 mM) in both groups. Hence, coupling of blood flow and metabolism appears neuronal NOS- (nNOS) but not eNOS-dependent in cortical barrel fields of the mouse.

Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia.

We tested the hypothesis that combined use of trophic factors and caspase inhibitors increases brain resistance to ischaemia in mice. Intracerebroventricular administration of bFGF (>10 ng) 30 min after MCA occlusion decreased infarct size and neurological deficit in a dose‐dependent manner following 2 h ischemia and reperfusion (20 h). Combined administration of the subthreshold doses of bFGF (3 ng) and caspase inhibitors (z‐VAD.FMK, 27 ng or z‐DEVD.FMK, 80 mg) reduced infarct volume by 60%, and reduced neurological deficit. Treatment with a subthreshold dose of bFGF (3 ng) extended the therapeutic window for z‐DEVD.FMK (480 ng) from 1 to 3 h after reperfusion. Caspase‐3 activity in the ischaemic brain was increased 30 min and 2 h after reperfusion but, was significantly reduced in bFGF‐treated animals by 29 and 16%, respectively. Caspase‐3 activity was not reduced by a direct bFGF effect because addition of bFGF (10 nM – 2 μM) did not decrease recombinant caspase‐3 activity, in vitro. Our data show that combining caspase inhibitors and bFGF lengthens the treatment window for the second treatment, plus lowers the dosage requirements for neuroprotection. These findings are important because low doses of caspase inhibitors or bFGF reduce the possibility of side effects plus extend the short treatment window for ischaemic stroke.