Rt-PA causes a significant increase in endogenous u-PA during experimental focal cerebral ischemia

The aim of this study was to investigate the effects of different doses of exogenous recombinant human tissue plasminogen activator (rt-PA) on the endogenous cerebral plasminogen-plasmin system in focal ischemia in rats. Ischemia was induced using the suture model. Each group of rats (n = 6) received either treatment (0.9, 9 or 18 mg rt-PA/kg body weight) or saline (control group) at the end of ischemia; a sham-operated group was added. The activity of the plasminogen activators was measured by casein-dependent plasminogen zymography. In the cortex urokinase (u-PA) rose from sham (no ischemia), 91 +/- 7% to ischemia, 176 +/- 10% (P < 0.005). Increasing rt-PA doses led to further significant (P < 0.001) cortical u-PA activation which was maximal at 18 mg: 249 +/- 13%. An extreme increase in the u-PA activity was observed in the basal ganglia to 1019 +/- 22% (P < 0.001). This increase was further aggravated by higher rt-PA doses (18 mg, 1236 +/- 15%; P < 0.001). The t-PA level did not change I3R24 during (3 h ischemia followed by reperfusion for 24 h); however, during low and moderate doses of rt-PA, endogenous t-PA was reduced. In conclusion, while ischemia leads to a significant increase in u-PA, mainly in the basal ganglia, t-PA is not altered. Increasing doses of rt-PA lead to a further elevation of u-PA. Thus, u-PA seems to play a major role in the endogenous plasminogen activator system following focal cerebral ischemia.     

Matrix metalloproteinase induction by EMMPRIN in experimental focal cerebral ischemia

Focal cerebral ischemia leads to the gradual disruption of the extracellular matrix. A key role in the turnover of the extracellular matrix is played by the system of matrix metalloproteinases (MMPs). In this study we describe changes of the MMP inducer protein (EMMPRIN) following experimental cerebral ischemia (induced for 3 h and followed by 24 h reperfusion, suture model) in rats. Extracellular EMMPRIN was measured by Western blot of the ischemic and nonischemic basal ganglia and cortex separately. Compared with the contralateral nonischemic area, the ischemic hemisphere showed a significant increase in EMMPRIN: basal ganglia, 158% +/- 4% (P < 0.05); cortex, 128% +/- 25% (P < 0.05). Immunohistochemistry was used to localize EMMPRIN on cerebral microvessels. EMMPRIN-positive microvascular structures were quantified by automatic morphometric video-imaging analysis and a significant increase in the number of cerebral microvessels staining positive for EMMPRIN in the ischemic basal ganglia was shown. The significant loss of microvascular basal lamina antigen collagen type IV in ischemic cortex and basal ganglia was calculated by Western blot. Measured by gelatin zymography, we demonstrated an MMP-2 and MMP-9 increase in the ischemic brain regions (P < 0.05). For the first time the MMP activation system EMMPRIN was shown to be relevant in cerebral ischemia. These results raise the possibility that the increased expression of EMMPRIN, the increase in MMPs and the damage of the basal lamina following cerebral ischemia are connected and part of a network of related changes.     

Plasminogen activation in experimental permanent focal cerebral ischemia

BACKGROUND: Previous experimental work using in situ zymography has shown very early increased plasminogen activation in ischemic regions after 3 h of ischemia with and without reperfusion. The objective of the present study was to evaluate the time course and extent of plasminogen activation in long-term permanent focal cerebral ischemia. MATERIAL AND METHODS: The middle cerebral artery in male Fisher rats was irreversibly occluded by electrocoagulation. Duration of ischemia was 48, 72, and 168 h. Occlusion was controlled in vivo by MRI at day 2. Plasminogen activation was detected by in situ zymography of 10 microm cryosections with an overlay containing plasminogen and the plasmin substrate caseine. Areas of plasminogen activation were compared to structural lesions (immunohistochemical loss of microtubule-associated protein 2; MAP 2). RESULTS: Compared to controls, increased plasminogen activation was observed in the basal ganglia and the cortex of the ischemic hemisphere after 48, 72, and 168 h (affected area of basal ganglia: 44.5+/-21.9, 70.1+/-2.3 and 66.6+/-2.8%, respectively; affected area of cortex: 63.4+/-9.8, 67.7+/-0.7 and 64.0+/-3.7%, respectively). The duration of ischemia had no significant influence on the extent of plasminogen activation. Areas of increased plasminogen activation significantly overlapped with and exceeded areas of MAP 2 loss (P<0.005). DISCUSSION: Permanent focal cerebral ischemia leads to increased plasminogen activation in ischemic regions. This plasminogen activation remains elevated at persistent levels over days. It may contribute to extracellular matrix (ECM) disruption, secondary hemorrhage, and brain edema in subacute stages of ischemic stroke.     

High-dose methylprednisolone treatment in experimental focal cerebral ischemia

Previous studies using steroids for experimental focal stroke have demonstrated conflicting results, possibly related to dose used or ischemic models employed. In this study we examined high-dose methylprednisolone treatment following permanent and temporary focal cerebral ischemia in the rat. Focal stroke was induced in spontaneously hypertensive rats by permanent right common carotid and either permanent or 3 h of temporary middle cerebral artery (MCA) occlusion. Methylprednisolone (105 mg/kg) was administered intra-arterially. Infarct volume was measured at 24 h after permanent and temporary MCA occlusion. Cerebral edema was determined by measuring right and left hemispheric volumes and water content 24 h after permanent MCA occlusion in one experiment. Methylprednisolone, whether administered in divided doses over 12 h (n = 15 in each group) or a single bolus (n = 9 per group), had no effect on infarct volume after permanent MCA occlusion. Methylprednisolone treatment also had no influence on cerebral edema (n = 9 per group). In two different experiments, methylprednisolone given in divided doses over 12 h (n = 11, n = 25) after temporary MCA occlusion decreased infarct volume (P < 0.05) by 20% compared with saline controls (n = 10, n = 25). High dose methylprednisolone decreased infarct volume following temporary, but not permanent, focal ischemia. The benefit suggests that high dose methylprednisolone may prove useful clinically if reperfusion can be established with thrombolytic agents. Furthermore, the differential treatment effect in the setting of comparable ischemic insults implies that different modifiable biochemical processes may be present during temporary but not permanent focal ischemia, thus providing indirect evidence for reperfusion injury.     

Superoxide dismutase activity in experimental focal cerebral ischemia

The activity of the free radical scavenger, superoxide dismutase, was studied in focal cerebral ischemia produced in Mongolian gerbils (Meriones unguiculatus) by occluding the right common and left external carotid arteries under halothane anesthesia. After recovery from anesthesia animals were classified according to their neurologic symptoms. Five animals exhibiting neurologic symptoms such as hemiparesis and rolling seizures were reanesthetized 120 min after vascular occlusion and their brains frozen in situ with liquid nitrogen. A series of 20-micron-thick coronal sections was cut in a cryostat; pictorial representations of tissue pH, ATP, and glucose were obtained using fluorescent and bioluminescent techniques. Using a highly sensitive bioluminescent technique, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and Mn-superoxide dismutase (Mn-SOD) activities were then measured in samples from both ischemic and nonischemic regions of the remaining tissue block. Cu,Zn-SOD and Mn-SOD activities were, respectively, 13.9 +/- 0.7 X 10(3) units/g and 5.4 +/- 0.3 X 10(3) units/g in the nonischemic tissue, and 13.2 +/- 0.6 X 10(3) units/g and 5.0 +/- 0.2 X 10(3) units/g within the ischemic tissue. Thus focal cerebral ischemia does not lead to a global decrease in SOD activity, as observed by others after heart and liver ischemia.     

Leukocyte-endothelium interactions during permanent focal cerebral ischemia in mice.

The contribution of leukocyte infiltration to brain damage after permanent focal cerebral ischemia and the underlying molecular mechanisms are still unclear. Therefore, the aim of this study was to establish a mouse model for the visualization of leukocytes in the cerebral microcirculation in vivo and to investigate leukocyte-endothelial interaction (LEI) after permanent middle cerebral artery occlusion (MCAO). Sham-operated 129/Sv mice showed physiologic LEI in pial venules as observed by intravital fluorescent microscopy. Permanent focal cerebral ischemia induced a significant increase of LEI predominantly in pial venules. The number of rolling and adherent leukocytes reached 36.5 +/- 13.2/100 microm x min and 22.5 +/- 7.9/100 microm x min, respectively at 120 minutes after MCAO (P = 0.016 vs. control). Of note, rolling and adherent leukocytes were also observed in arterioles of ischemic animals (7.3 +/- 3.0/100 microm x min rolling and 3.0 +/- 3.6/100 microm x min adherent). Capillary density was not different between groups. These results demonstrate that leukocytes accumulate in the brain not only after transient but also after permanent focal cerebral ischemia and may therefore contribute to brain damage after stroke without reperfusion.     

Increased intracellular calpain detection in experimental focal cerebral ischemia

Calpains are intracellular proteinases whose proteolytic activity is directed mainly against the cytoskeleton and regulatory proteins. We studied the presence of calpain by immunohistochemistry in a rat model of reversible focal cerebral ischemia (3 h) at various times of reperfusion. The numbers of calpain-positive cells on the ischemic side were compared with the non-ischemic side. In controls only 2 +/- 1% cells were positive, whereas the cortex of the ischemic vs the non-ischemic side showed 88 +/- 3% vs 13 +/- 4% calpain-positive cells (p < 0.001), and the basal ganglia 47 +/- 3% vs 13 +/- 4% (p < 0.01) after 3 h ischemia and 24 h reperfusion. This is the first demonstration of elevated intracellular levels of calpains in areas of cerebral ischemia. Longer reperfusion resulted in an increase in calpain positivity.     

Autoregulation of cerebral blood flow in experimental focal brain ischemia

The relationship between systemic arterial pressure (SAP) and neocortical microcirculatory blood-flow (CBF) in areas of focal cerebral ischemia was studied in 15 spontaneously hypertensive rats (SHRs) anesthetized with halothane (0.5%). Ischemia was induced by ipsilateral middle cerebral artery/common carotid artery occlusion and CBF was monitored continuously in the ischemic territory using laser-Doppler flowmetry during manipulation of SAP with I-norepinephrine (hypertension) or nitroprusside (hypotension). In eight SHRs not subjected to focal ischemia, we demonstrated that 0.5% halothane and the surgical manipulations did not impair autoregulation. Autoregulation was partly preserved in ischemic brain tissue with a CBF of greater than 30% of preocclusion values. In areas where ischemic CBF was less than 30% of preocclusion values, autoregulation was completely lost. Changes in SAP had a greater influence on CBF in tissue areas where CBF ranged from 15 to 30% of baseline (9% change in CBF with each 10% change in SAP) than in areas where CBF was less than 15% of baseline (6% change in CBF with each 10% change in SAP). These findings demonstrate that the relationship between CBF and SAP in areas of focal ischemia is highly dependent on the severity of ischemia. Autoregulation is lost in a gradual manner until CBF falls below 30% of normal. In areas without autoregulation, the slope of the CBF/SAP relationship is inversely related to the degree of ischemia.     

Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies

The purposes of this review are to clarify the effect of hypothermia therapy on focal cerebral ischemia in rats, and to consider the relevancy of its application to human focal cerebral ischemia. Since 1990, 26 reports confirming the brain-protecting effect of hypothermia in rat focal cerebral ischemia models have been published. Seventy-four experimental groups in these 26 reports were classified as having transient middle cerebral arterial occlusion (MCAO) with mild hypothermia (group A; 43 groups), permanent MCAO with mild hypothermia (group B; 14 groups), permanent MCAO with deep hypothermia (group C; 8 groups) and transient or permanent MCAO with mild hyperthermia (group D; 9 groups). The results were evaluated as the % infarct volume change caused by hypothermia or hyperthermia compared with the infarct volume in normothermic animals. The effectiveness was confirmed in 36 (83%) of the 43 groups in group A, 10 (71%) of the 14 in group B, and six (75%) of the eight in group C. The infarct volume of eight of the nine groups in group D was markedly aggravated. The percent infarct volume change was 55.3% +/- 27.1% in group A, 57.6% +/- 24.7% in group B, 60.8% +/- 45.5% in group C, and 189.7% +/- 89.4% in group D. For effective reduction of the infarct volume, hypothermia should be started during ischemia or within 1 h, at latest, after the beginning of reperfusion in the rat transient MCAO model. However, it is not clear whether this neuroprotective effect of hypothermia can also be observed in the chronic stage, such as several months later. Keeping the body temperature normothermic in order to avoid mild hyperthermia seems to be rather important for not aggravating cerebral infarction. Clinical randomized studies on the efficacy of mild hypothermia for focal cerebral ischemia and sophisticated mild hypothermia therapy techniques are mandatory.     

Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats

Using a modification of Karnovsky's Mn²⁺/diaminobenzizine (DAB) technique, we examined the production of superoxide anion (O⁻₂) in the vascular lumen following transient occlusion and reperfusion of the left middle cerebral artery (MCA) in Sprague–Dawley rats. The MCA was occluded for 2 h using an intraluminal suture method. Zero, 15, 30, and 60 min after reperfusion, animals were perfused transcardially with buffer containing Mn²⁺ and DAB, and brain samples were prepared for light and electron microscopic examination. The amber reaction deposits of O⁻₂ were observable to the naked eye along the major cerebral vessels of the ischemic hemisphere after each reperfusion period. Upon microscopic examination the deposits were revealed to be within arterial, capillary, and venular lumen. The amount of reaction deposits in the ischemic hemisphere corresponded to the duration of reperfusion. The formation of O⁻₂ was suppressed when the perfusate contained superoxide dismutase and when either Mn²⁺ or DAB was omitted, confirming that the reaction products produced are due to the enhanced production of O⁻₂. These results show that there is a progressive increase in intraluminal O⁻₂ during reperfusion following an ischemic insult which may participate in the aggravation of cerebral damage.     

大鼠急性脑缺血后Hephaestin的表达变化

目的 研究大鼠急性局灶性脑缺血后皮质和纹状体Hephaestin表达的变化。方法 线栓法制备大鼠急性大脑中动脉阻塞(MCAO)再灌注模型,在再灌注后的不同时间点应用免疫组化、图像分析以及SDS-PAGE Werstern blot方法检测缺血侧皮质和纹状体的表达变化。结果 MCAO再灌注后大鼠出现大脑中动脉梗死的神经系统损害体征,TTC染色有白色梗死区。Hephaestin在正常大鼠的皮质、纹状体有表达,在急性脑缺血再灌注后12h缺血侧皮质、纹状体表达明显增加并持续到再灌注后48h,在24h时到达高峰(P<0.01),至1周时表达较正常明显减少(P<0.01)。结论 大鼠急性脑缺血再灌注后Hephaestin的表达出现明显的变化,在急性脑缺血的病理生理变化中可能起着重要的作用。     

Selective platelet deposition during focal cerebral ischemia in cats

Platelet deposition in the microcirculation may play a role in focal cerebral ischemia. We investigated platelet deposition in selected parts of the cat brain after temporary middle cerebral artery occlusion. Ten anesthesized cats were given autologous indium-111-labeled platelets and chromium-51-labeled erythrocytes. The right middle cerebral artery was occluded with miniature aneurysm clips for 3 hours via a transorbital approach; blood pressure was reduced concomitantly to decrease the collateral circulation. Removal of the clips initiated a 45-minute period of normotensive reperfusion. After sacrifice, the brain was removed and sectioned for comparison of right- versus left-hemisphere platelet deposition. Platelets were selectively deposited in the territory of the occluded right middle cerebral artery. Significant deposition was found in the caudate nucleus, internal capsule, parietal cortex, and the centrum semiovale. Our findings support the evidence that platelets are deposited in the microvasculature during temporary severe focal cerebral ischemia.     

Sphingomyelin changes in rat cerebral cortex during focal ischemia

Abstract

To better define the sphingolipid metabolism during focal brain ischemia, levels of ceramide, sphingomyelin, cerebroside and gangliosides were determined in rat cerebral cortex during focal ischemia produced by occlusion of the middle cerebral artery. Sphingomyelin began to decrease at 2 hours of ischemia and continued to decrease for 96 hours. In contrast, ceramide increased at 6 hours and increased to 4.2-fold at 96 hours after ischemia, and the fatty acid composition of ceramide was solely nonhydroxylated fatty acid similar to sphingomyelin. Hydroxylated fatty acid-linked cerebroside decreased at 6 hours of ischemia, whereas any significant decrease of nonhydroxylated fatty acid-linked cerebroside didn't occur for 96 hours of ischemia. There were no measurable changes in the levels of gangliosides. These results suggested that ceramide was produced in the cerebral cortex by the breakdown of sphingomyelin during early ischemia. [Neurol Res 1996; 18: 337-341]     

Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia.

Oxidative stress generated during stroke is a critical event leading to blood-brain barrier (BBB) disruption with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of thrombolytic reperfusion. In this study, the authors demonstrate that ischemia-reperfusion-induced BBB disruption in mice deficient in copper/zinc-superoxide dismutase (SOD1) was reduced by 88% ( P < 0.0001) and 73% ( P < 0.01), respectively, after 3 and 7 hours of reperfusion occurring after 1 hour of ischemia by the inhibition of matrix metalloproteinases. Accordingly, the authors show that local metalloproteinase-generated proteolytic imbalance is more intense in ischemic regions of SOD1 mice than in wild-type litter mates. Moreover, active in situ proteolysis is, for the first time, demonstrated in ischemic leaking capillaries that produce reactive oxygen species. By showing that oxidative stress mediates BBB disruption through metalloproteinase activation in experimental ischemic stroke, this study provides a new target for future therapeutic strategies to prevent BBB disruption and potentially reperfusion-triggered intracerebral hemorrhage.     

Calpain inhibitor A-558693 in experimental focal cerebral ischemia in rats

OBJECTIVES: Calpains are intracellular proteases, which are activated in various cerebral injuries. We studied the expression of mu-calpain in a model of focal cerebral ischemia/reperfusion and the efficacy of the calpain inhibitor A-558693. METHODS: A transient occlusion of the middle cerebral artery was produced in male Wistar rats by using the suture model with 3 hours of ischemia and 24 hours of reperfusion. Six animals were given the calpain inhibitor and six animals were treated with placebo. The infarct size was determined by the loss of the calpain substrate microtubule-associated protein-2 (MAP-2) immunohistochemistry using volumetry in serial slices of the brains. Furthermore mu-calpain positive-stained cells were detected by immunohistochemistry and western blotting. RESULTS: In placebo-treated animals the mu-calpain expression was significantly increased in the ischemic hemisphere compared with the contralateral non-ischemic hemisphere (88.6 versus 10.5% in the basal ganglia, 60.7 versus 10.7% in the cortex, p < 0.001, respectively) with a subsequent loss its substrate MAP-2. However, the use of the calpain inhibitor A-558693 did not significantly change the mu-calpain expression, nor significantly reduce the infarct volume. DISCUSSION: The present data indicate that mu-calpain proteolysis plays an important role in the chain of events following cerebral ischemia. However, the calpain inhibitor A-558693 failed to prevent these changes.     

大鼠局部脑缺血再灌流的实验研究

采用大鼠局部脑缺血再灌流模型，研究了大鼠脑缺血６ｈ、９ｈ和缺血６ｈ再灌流３ｈ脑梗塞体积，脑含水量，能量代谢，丙二醛（ＭＤＡ）和超氧化物歧化酶（ＳＯＤ）的变化。结果：脑缺血６ｈ、９ｈ可以造成严重的脑梗塞和脑水肿，ＡＴＰ含量和ＳＯＤ活性显著降低，乳酸和ＭＤＡ含量显著增加，和对照组相比均有显著差异（Ｐ＜０．０５或Ｐ＜０．００１）。再灌组和缺血两组比较，脑梗塞体积，脑水肿无明显差别（Ｐ＞０．０５），ＡＴＰ、乳酸、ＳＯＤ和ＭＤＡ均有不程度的改善。提示，大鼠局部脑缺血超过６ｈ可造成严重的脑损伤，并随缺血时间的再延长，脑损伤变化趋于平缓。再灌后，脑损伤未见明显加重。     

Dexanabinol; a novel neuroprotective drug in experimental focal cerebral ischemia

The permanent middle cerebral artery occlusion (PMCAO) model was used to investigate the cerebroprotective effects of the synthetic cannabinoid, dexanabinol (HU-211). Dexanabinol is a noncompetitive N-methyl-D-aspartate antagonist, with antioxidant and anti-TNFalpha properties. Twenty hypertensive rats were subjected to PMCAO. Eight were given 4 mg/kg dexanabinol, i.v., 1 h after PMCAO, eight received vehicle and four were not injected Five rats underwent sham surgery. Infarct volumes were assessed, as well as TNFalpha concentrations and NOS activity in brain homogenates. Dexanabinol significantly decreased infarct volumes. It also significantly lowered TNFalpha levels in the ipsilateral hemisphere although not to the level of sham operated rats. No effect could be demonstrated on NOS activity. In conclusion, dexanabinol may be a pluripotent cerebroprotective agent.     

A reproducible experimental model of focal cerebral ischemia in the cat

In the past experimental methods used for producing focal cerebral ischemia have had considerable difficulty with regard to reproducibility of the size of the infarcted region. In this study we have developed an experimental model which enables us to consistently produce focal regions of cerebral ischemia (resulting in infarction) which vary little in size in a number of animals. Thirty-seven cats (3–4 kg b. wt.) anesthetized with chlorolose and urethane were used. Physiologic monitoring and adjustments maintained arterial food values as follows: pCO₂ 27–35 Torr, pO₂ 100–150 Torr, pH ± 7.4, glucose 200 mg%, hematocrit > 25. The left middle cerebral artery was exposed via a transorbital approach and occluded for 1–2 h with and without left and/or both carotid artery occlusion. Sixteen hours following the ischemic episode, the animals were sacrificed and sections of fresh brain tissue were processed for vital staining using 1% tetrazolium solution. With this method normal brain areas appear dark red, ischemic regions (without infarction) appear gray and irreversibly infarcted areas appear pinkish-white. The volumetric dimensions of the lesioned area were measured using a planimeter. The same tissue was also evaluated histologically by means of standard histopathologic techniques on paraffin-embedded material. Infarcted areas as delineated macroscopically by the tetrazolium correlated well with the light microscopic findings. Ten animals subjected to a 2-h occlusion of the left middle cerebral artery (LMCA) and both carotid arteries resulted in a reproducible infarct which was 3.2 ± 0.7 ml in volume. This represents 13.3 ± 2.9 % of the total volume of both cerebral hemispheres (above the level of the inferior colliculus). This experimental procedure was used to yield reproducible regions of focal cerebral ischemia in a subsequent study involving the use of flourocarbon emulsion to reverse the ischemic process and prevent permanent neurological damage with infarction. Neurological manifestations of permanent cerebral ischemia were assessed using a clinical scale developed in this model.     

Characterizing the diffusion/perfusion mismatch in experimental focal cerebral ischemia

Diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI) can rapidly detect lesions in acute ischemic stroke patients. The PWI volume is typically substantially larger than the DWI volume shortly after onset, that is, a diffusion/ perfusion mismatch. The aims of this study were to follow the evolution of the diffusion/ perfusion mismatch in permanent and 60- minute temporary focal experimental ischemia models in Sprague-Dawley rats using the intraluminal middle cerebral artery occlusion (MCAO) method. DWI and arterial spin-labeled PWI were performed at 30, 60, 90, 120, and 180 minutes after occlusion and lesion volumes (mm(3)) calculated At 24 hours after MCAO, and infarct volume was determined using triphenyltetrazolium chloride staining. In the permanent MCAO group, the lesion volume on the ADC maps was significantly smaller than that on the cerebral blood flow maps through the first 60 minutes after MCAO; but not after 90 minutes of occlusion. With 60 minutes of transient ischemia, the diffusion/perfusion mismatch was similar, but after reperfusion, the lesion volumes on ADC and cerebral blood flow maps became much smaller. There was a significant difference in 24- hour infarct volumes between the permanent and temporary occlusion groups.     

Role of matrix metalloproteinases in apoptosis after transient focal cerebral ischemia in rats and mice

The involvement of matrix metalloproteinases (MMPs) in cerebral ischemia-induced apoptosis was investigated in a model of transient focal cerebral ischemia in rats treated intracerebroventricularly (i.c.v.) with 4-((3-(4-phenoxylphenoxy)propylsulfonyl)methyl)-tetrahydropyran-4-carboxylic acid N-hydroxy amide, a broad spectrum non-peptidic hydroxamic acid MMP inhibitor, and in MMP-9-deficient mice. Our results showed that MMP inhibition reduced DNA fragmentation by 51% (P < 0.001) and cerebral infarct by 60% (P < 0.05) after ischemia. This protection was concomitant with a 29% reduction of cytochrome c release into the cytosol (P < 0.005) and a 54% reduction of calpain-related alpha-spectrin degradation (P < 0.05), as well as with an 84% increase in the immunoreactive signal of the native form of poly(ADP) ribose polymerase (P < 0.01). By contrast, specific targeting of the mmp9 gene in mice did reduce cerebral damage by 34% (P < 0.05) but did not modify the apoptotic response after cerebral ischemia. However, i.c.v. injection of MMP-9-deficient mice with the same broad-spectrum inhibitor used in rats significantly reduced DNA degradation by 32% (P < 0.05) and contributed even further to the protection of the ischemic brain. Together, our pharmacological and genetic results indicate that MMPs other than MMP-9 are actively involved in cerebral ischemia-induced apoptosis.