Hiroshi Yao

Local cerebral glucose utilization and cytoskeletal proteolysis as indices of evolving focal ischemic injury in core and penumbra

To ascertain the tempo of progression to irreversible injury in focal ischemia, we subjected halothaneanesthetized Sprague–Dawley rats to photochemically induced distal middle cerebral artery occlusion (dMCAO) combined with permanent ipsilateral and 1 h contralateral common carotid artery occlusions. Head temperature was maintained at 36°C. At times centered at either 1.5 or 3 h post-dMCAO, the rate of local glucose metabolism (lCMRgl) was measured by 2-deoxyglucose autoradiography, and cytoskeletal proteolysis was assessed regionally by an immunoblotting procedure to detect spectrin breakdown products. At 1.5 h (n = 5), the cortical ischemic core was already severely hypometabolic (lCMRgl 15.5 ± 10.8 μmol 100 g−1 min−1, mean ± SD), whereas the cortical penumbral zone was hypermetabolic (69.0 ± 9.7). (The lumped constant was verified to be unchanged by methylglucose studies.) Neutral red pH studies at this time point showed that both the core and penumbral zones were equally acidotic. By 3 h post-dMCAO (n = 6), lCMRgl in the penumbral zone had fallen to low levels (15.4 ± 2.2 μmol 100 g−1 min−1) equal to those of the ischemic core (16.7 ± 4.5). Correspondingly, spectrin breakdown in the ischemic core was advanced at both 2 and 3.5 h post-dMCAO (36 ± 18% and 33 ± 18% of total spectrin, respectively), whereas in the penumbral zone spectrin breakdown was less extensive and more highly variable at both times (22 ± 23% and 29 ± 16%). We conclude that irreversible deterioration of the ischemic core, as evidenced by the onset of local cytoskeletal proteolysis, begins within 2 h of middle cerebral artery occlusion. In the ischemic penumbra, the transition from glucose hyper- to hypometabolism occurs by 3.5 h and is associated with a milder and more variable degree of spectrin breakdown.

Photothrombotic Middle Cerebral Artery Occlusion in Spontaneously Hypertensive Rats: Influence of Substrain, Gender, and Distal Middle Cerebral Artery Patterns on Infarct Size

BACKGROUND AND PURPOSEnTo analyze the effects of substrain and gender differences in spontaneously hypertensive rats (SHR) and distal middle cerebral artery (MCA) branching patterns on infarct size, we compared infarct volumes produced by photothrombotic distal MCA occlusion using SHR/Kyushu and SHR/Izumo (Izm).nnnMETHODSnTwenty-four male and 8 female SHR/Kyushu, 15 male and 5 female SHR/Izm, and 6 male Wistar-Kyoto rats (WKY)/Izm (5 to 7 months old) were subjected to photothrombotic distal MCA occlusion, and infarct volumes were determined.nnnRESULTSnAlthough blood pressure levels were essentially the same between the two substrains of hypertensive rats, infarct volumes were significantly larger in the SHR/Kyushu substrain than in SHR/Izm of either sex (P<0.001); infarct volumes in male and female SHR/Kyushu were 83.8+/-11.7 and 58.5+/-9.2 mm3, and those in male and female SHR/Izm were 61.5+/-10.7 and 34.8+/-7.9 mm3, respectively (values are mean+/-SD). Male SHR/Kyushu that had simple Y-shaped MCA showed smaller infarcts (75.8+/-14.6 mm3, n=11) than those with more branching (regular) MCA (93.2+/-19.1, n=13), the difference being significant (P=0.022). Male SHR/Izm with simple distal MCA also produced smaller infarctions than those with regular MCA (51.0+/-3.7 versus 68.9+/-8.7 mm3, P=0.0004).nnnCONCLUSIONSnPhotothrombotic occlusion of distal MCA in hypertensive rats provides a simple and reproducible model of focal ischemia. Most importantly, this study emphasizes the substantial variabilities in infarct sizes caused by the differences in substrains of SHR, gender, and distal MCA patterns.

Failure of MK-801 to reduce infarct volume in thrombotic middle cerebral artery occlusion in rats.

Background and Purpose We examined the effects of the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 using a newly developed stroke model of thrombotic distal middle cerebral artery occlusion under conditions of carefully controlled head temperature. Methods Male Sprague-Dawley rats were treated with 1 mg/kg of MK-801 or saline before the induction of ischemia. An argon laser-activated dye laser (562 nm) was used to cause thrombotic distal middle cerebral artery occlusion. In experiments 1 and 2, the single laser beam (20 mW) was separated into three beams. Each beam was positioned onto the distal middle cerebral artery at three sites along the vessel. The photosensitizing dye rose bengal (20 mg/kg) was administered intravenously over 2 minutes; the three points were then irradiated for 3 minutes. In experiment 3, higher power of the laser (three separate irradiations using a single beam of 20 mW) was used. The ipsilateral common carotid artery was occluded permanently, and the contralateral carotid artery was occluded for 60 minutes. Head temperature was controlled at 36°C in experiment 1 and not controlled in experiments 2 and 3. Three days after the ischemic insult, brains were perfusion-fixed and infarct volumes were determined. Results Head temperature was mildly hypothermic (34–35°C before ischemia, with a further decrease of 1-2°C during the initial 60 minutes of ischemia) in experiment 2. However, no differences were observed in head temperature between the MK-801-treated and control groups. Cortical infarct volume in experiment 1 was 89±29 mm3 (mean±SD) in the treated group, which was not different from the control value of 84±40 mm3. Infarct volumes were smaller (58±35 mm3 and 54±14 mm3) in the control groups of experiments 2 and 3, respectively. However, MK-801 also failed to reduce infarct volumes in experiments 2 and 3. Conclusions MK-801 is not effective in this stroke model of focal thrombotic infarction under conditions of either controlled (normothermic) or uncontrolled (mildly hypothermic) head temperature.

Inhibition of Na+/H+ exchanger reduces infarct volume of focal cerebral ischemia in rats

Activation of Na+/H+ exchanger (NHE) may have an important role in ischemic cell death by means of intracellular overload of Na(+) and Ca(2+). Recent evidence has suggested that inhibitors of NHE have protective effects on myocardial ischemia both in vivo and in vitro. In this study, we tested the hypothesis that FR183998, an inhibitor of NHE, reduces infarct volume produced by focal cerebral ischemia in rats. We used 20 male spontaneously hypertensive rats. Either FR183998 (1 mg/kg; n=10), or vehicle (n=10) was given intravenously to the rats and the distal middle cerebral artery of each animal was occluded using a photothrombotic technique. We measured regional cerebral blood flow using laser-Doppler flowmetry throughout the experiments. After 3 days, infarct volume was measured in each animal group. To estimate the brain edema, we also calculated the cortical volume in both hemispheres. The infarct volume in the FR183998-treated group (82+/-8 mm(3), mean+/-S.E.M.) was significantly smaller than that in the control group (115+/-12 mm(3)) (P=0.034). The cortical volume of the occluded side in the FR183998-treated group (359+/-7 mm(3)) tended to be smaller than that in the control group (378+/-9 mm(3)) (P=0.116). The regional cerebral blood flow and physiological variables during ischemia were not significantly different between the two groups throughout the experiments. These results suggest that inhibition of NHE by FR183998 may have beneficial effects in reducing infarct volume and brain edema during cerebral ischemia. Thus, NHE may play an important role in the development of neuronal damage during acute cerebral ischemia.

L-arginine does not improve cortical perfusion or histopathological outcome in spontaneously hypertensive rats subjected to distal middle cerebral artery photothrombotic occlusion

The potential of nitric oxide (NO) to influence positively or negatively the outcome of mechanically induced focal cerebral ischemia is still controversial. Recent evidence suggests that NO of vascular origin, whether synthesized from exogenously administered L-arginine (L-Arg) or from NO donor compounds, is beneficial but that of neuronal origin is not. However, the therapeutic potential of NO to ameliorate stroke induced by arterial thrombosis has not been reported. We assessed the therapeutic effect of L-Arg administration in spontaneously hypertensive rats (SHR) subjected to permanent photothrombotic occlusion of the distal middle cerebral artery (dMCA). The ipsilateral carotid artery was left unligated to enhance L-Arg delivery into the putative penumbral region. Local CBF (LCBF) was assessed at 30 min by the [14C]iodoantipyrine technique (n = 9), while histological infarct volumes and index of peripheral ischemic cell change were determined at 3 days (n = 7). Rats (n = 9) given 300 mg/kg L-Arg at 18 and 3 h before photothrombotic dMCA occlusion and at 5 min afterward displayed no significant differences in LCBF compared with animals (n = 8) injected with water (the carrier vehicle) and similarly irradiated. Infarct volumes were also similar, being 37.0 ± 9.7 mm3 (SD) in the vehicle-treated and 49.1 ± 17.2 mm3 (SD) in the L-Arg-treated groups (both n = 7), as were assessments of ischemic neuronal density in the penumbra. In contrast, L-Arg administered intravenously in a dose of 300 mg/kg to nonischemic SHR (n = 5) increased cortical CBF by ∼75% during a 70-min observation period. We conclude that thrombotic processes superimposed upon cerebral ischemia may facilitate tissue reactions that offset the potentially beneficial effect of L-Arg, and this caveat must be considered when proposing L-Arg for clinical treatment of focal thrombotic stroke.

Substrain Differences, Gender, and Age of Spontaneously Hypertensive Rats Critically Determine Infarct Size Produced by Distal Middle Cerebral Artery Occlusion

Abstract1. The present work discussed the effects of substrain or genetic differences, gender, and age of the rat on infarct size produced by distal middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats (SHR).2. In SHR/Kyushu, infarct volume was significantly larger than that of SHR/Izm, while blood pressure levels were essentially the same between the two substrains. Although SHR-SP/Izm had a higher blood pressure than SHR/Kyushu, infarct volumes were the same between SHR/Kyushu and SHR-SP/Izm. These results suggest the presence of blood pressure- independent factors which affect the infarct size after MCAO.3. Estrogen accounted the large part of greater tolerability against focal brain ischemic injury in female compared with male SHR.4. We found age-related vulnerability to focal cerebral ischemia in female SHR. This age-related vulnerability in aged female SHR was unrelated to the blood levels of sex hormones such as estrogens and progesterone.5. Finally, we emphasized the importance of reproducible and least invasive focal ischemia models in stroke research.

Ischemia-Induced Release of Amino Acids in the Hippocampus of Aged Hypertensive Rats

We have recently demonstrated the age-related vulnerability of hippocampal neurons to 20-min forebrain ischemia in spontaneously hypertensive rats (SHR). In the present study, we investigated the effect of aging on the release of amino acids in the hippocampus during transient cerebral ischemia for 20 min. Concentrations of extracellular amino acids and cerebral blood flow in the CA1 subfield were examined by an in vivo brain dialysis technique and a hydrogen clearance method, respectively, in adult (5–7 month) and aged (19–23 month) female SHR. During cerebral ischemia by bilateral carotid artery occlusion, cerebral blood flow to the hippocampus decreased to 20% of the resting values in both groups. After recirculation, both groups showed delayed hypoperfusion which was more prominent in the aged SHR. In the adult rats, concentrations of both aspartate and glutamate increased to ∼8-fold of the resting values during ischemia. The elevation of these excitatory amino acids in the adult SHR was not significantly different from that in the aged rats. In contrast, the concentration of taurine increased 26-fold in the adult SHR but only 16-fold in the aged rats. Changes in other amimo acids were not different between the two groups. These results indicate that an imbalance of excitatory and inhibitory amino acids, e.g., smaller release of taurine, during ischemia may, at least in part, contribute to the age-related vulnerability of hippocampal neurons to transient cerebral ischemia in SHR.

Glutamate antagonist MK-801 attenuates incomplete but not complete infarction in thrombotic distal middle cerebral artery occlusion in Wistar rats

The purpose of this study was to investigate the effects of a non-competitive N-methyl-D-aspartate antagonist, MK-801, on incomplete infarction (selective neuronal necrosis), a zone of which had been found previously in a thrombotic distal middle cerebral artery (MCA) occlusion model in Wistar rats. Male Wistar rats were treated with 1 mg/kg of MK-801 or saline 30 min before MCA occlusion. Laser irradiation with intravenous administration of Rose Bengal dye was used to cause thrombotic distal MCA occlusion. The ipsilateral common carotid artery was occluded permanently and the contralateral carotid artery for 60 min. Head temperature was controlled at 36 degrees C. Cerebral blood flow (CBF) was determined with laser-Doppler flowmetry. Three days after the ischemic insult, brains were perfusion-fixed and volumes of cortical (complete and incomplete) infarction were determined. There were no significant differences in physiological variables or CBF between the two groups. Volumes of complete infarction were equivalent between the two groups (94.9 +/- 15.6 mm3 and 91.6 +/- 14.0 mm3 in the control and MK-801 treated groups, respectively). In MK-801 treated group, the volume of incomplete infarction was reduced by 44% (6.4 +/- 1.7 mm3 vs. 3.6 +/- 2.1 mm3 in control and MK-801 treated groups, respectively, P < 0.05). Although the zone responsive to MK-801 was small in this thrombotic MCA occlusion model, our present study revealed that MK-801 has a beneficial effect on the tissue zone containing selective neuronal alterations (incomplete infarction). Our results support the concept that this drug is effective in the area of less severe ischemia.

Subclinical Cerebral Abnormalities in Chronic Kidney Disease

BACKGROUND AND PURPOSEnImpaired kidney function or chronic kidney disease (CKD), as measured by estimated glomerular filtration rate (eGFR), is associated with incident stroke risk. However, few studies have examined the relationship between CKD and subclinical cerebral abnormalities.nnnMETHODSnWe examined 675 elderly subjects (mean age 69.9 years), who were living independently at home without apparent dementia, using magnetic resonance imaging. Serum creatinine values, measured by the enzymatic method, were used for the Japanese equation of eGFR.nnnRESULTSnSubclinical lacunar infarction, deep white matter lesions, and periventricular hyperintensities were detected in 88 (13.0%), 240 (35.6%) and 158 (23.4%) of the 675 participants, respectively. In the forward stepwise method of logistic analysis, age (OR 2.081/10, 95% CI 1.541-2.810), hypertension (OR 3.656, 95% CI 2.184-6.119), diabetes mellitus (OR 1.961, 95% CI 1.007-3.820), alcohol intake (OR 2.130, 95% CI 1.283-3.535), and eGFR <45 ml/min/1.73 m(2) were significant factors concerning subclinical lacunar infarction. CKD defined as eGFR <60 ml/min/1.73 m(2) was not significantly associated with subclinical lacunar infarction. Decreased eGFR was not a significant factor associated with white matter lesions (WMLs). Age (OR 2.781/10, 95% CI 2.252-3.435), hypertension (OR 1.746, 95% CI 1.231-2.477), diabetes mellitus (OR 1.854, 95% CI 1.070-3.213), but not eGFR were significant factors concerning WMLs.nnnCONCLUSIONSnThe present study showed that community-dwelling elderly subjects with late stage 3 CKD were at high risk for prevalent subclinical lacunar infarction. The identification of CKD-specific modifiable risk factors for SBI and WMLs is of increased importance for prevention of subclinical brain ischemic lesions.

Therapeutic time window for YAG laser-induced reperfusion of thrombotic stroke in hypertensive rats.

We examined the novel YAG laser-induced reperfusion method in the photothrombotic middle cerebral artery (MCA) occlusion model in spontaneously hypertensive rats. In the 1u2009h ischemia group, infarct volume was significantly reduced to 41.1u2009±u200915.6u2009mm3 compared with 81.6u2009±u200918.3u2009mm3 in the no-reperfusion group. There were no significant differences in infarct volume among 2u2009h or 3u2009h ischemia and no-reperfusion groups. Three of six rats in the 3u2009h ischemia group showed hemorrhagic infarction. Our present results showed that recirculation must be instituted within 2u2009h of MCA occlusion to get beneficial effects in our model, supporting the concept of a narrow therapeutic time window for intervention in ischemic stroke.