Elevated Intracranial Pressure and Cerebral Edema following Permanent MCA Occlusion in an Ovine Model

Introduction

Malignant middle cerebral artery (MCA) stroke has a disproportionately high mortality due to the rapid development of refractory space-occupying cerebral edema. Animal models are essential in developing successful anti-edema therapies; however to date poor clinical translation has been associated with the predominately used rodent models. As such, large animal gyrencephalic models of stroke are urgently needed. The aim of the study was to characterize the intracranial pressure (ICP) response to MCA occlusion in our recently developed ovine stroke model.

Materials and Methods

30 adult female Merino sheep (n = 8–12/gp) were randomized to sham surgery, temporary or permanent proximal MCA occlusion. ICP and brain tissue oxygen were monitored for 24 hours under general anesthesia. MRI, infarct volume with triphenyltetrazolium chloride (TTC) staining and histology were performed.

Results

No increase in ICP, radiological evidence of ischemia within the MCA territory but without space-occupying edema, and TTC infarct volumes of 7.9+/-5.1% were seen with temporary MCAO. Permanent MCAO resulted in significantly elevated ICP, accompanied by 30% mortality, radiological evidence of space-occupying cerebral edema and TTC infarct volumes of 27.4+/-6.4%.

Conclusions

Permanent proximal MCAO in the sheep results in space-occupying cerebral edema, raised ICP and mortality similar to human malignant MCA stroke. This animal model may prove useful for pre-clinical testing of anti-edema therapies that have shown promise in rodent studies.     

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 ¹. Because of the ease of genetic manipulation in mice, these models have also been developed in this species ^2-3.Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume ⁴. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

Neuronal Ca2+-activated K+ channels limit brain infarction and promote survival

Neuronal calcium-activated potassium channels of the BK type are activated by membrane depolarization and intracellular Ca(2+) ions. It has been suggested that these channels may play a key neuroprotective role during and after brain ischemia, but this hypothesis has so far not been tested by selective BK-channel manipulations in vivo. To elucidate the in vivo contribution of neuronal BK channels in acute focal cerebral ischemia, we performed middle cerebral artery occlusion (MCAO) in mice lacking BK channels (homozygous mice lacking the BK channel alpha subunit, BK(-/-)). MCAO was performed in BK(-/-) and WT mice for 90 minutes followed by a 7-hour-reperfusion period. Coronal 1 mm thick sections were stained with 2,3,5-triphenyltetrazolium chloride to reveal the infarction area. We found that transient focal cerebral ischemia by MCAO produced larger infarct volume, more severe neurological deficits, and higher post-ischemic mortality in BK(-/-) mice compared to WT littermates. However, the regional cerebral blood flow was not significantly different between genotypes as measured by Laser Doppler (LD) flowmetry pre-ischemically, intra-ischemically, and post-ischemically, suggesting that the different impact of MCAO in BK(-/-) vs. WT was not due to vascular BK channels. Furthermore, when NMDA was injected intracerebrally in non-ischemic mice, NMDA-induced neurotoxicity was found to be larger in BK(-/-) mice compared to WT. Whole-cell patch clamp recordings from CA1 pyramidal cells in organotypic hippocampal slice cultures revealed that BK channels contribute to rapid action potential repolarization, as previously found in acute slices. When these cultures were exposed to ischemia-like conditions this induced significantly more neuronal death in BK(-/-) than in WT cultures. These results indicate that neuronal BK channels are important for protection against ischemic brain damage.     

Elevated production of 20-HETE in the cerebral vasculature contributes to severity of ischemic stroke and oxidative stress in spontaneously hypertensive rats

Hypertension is a major risk factor for stroke, but the factors that contribute to the increased incidence and severity of ischemic stroke in hypertension remain to be determined. 20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to be a potent constrictor of cerebral arteries, and inhibitors of 20-HETE formation reduce infarct size following cerebral ischemia. The present study examined whether elevated production of 20-HETE in the cerebral vasculature could contribute to the larger infarct size previously reported after transient middle cerebral artery occlusion (MCAO) in hypertensive strains of rat [spontaneously hypertensive rat (SHR) and spontaneously hypertensive stroke-prone rat (SHRSP)]. The synthesis of 20-HETE in the cerebral vasculature of SHRSP measured by liquid chromatography-tandem mass spectrometry was about twice that seen in Wistar-Kyoto (WKY) rats. This was associated with the elevated expression of cytochrome P-450 (CYP)4A protein and CYP4A1 and CYP4A8 mRNA. Infarct volume after transient MCAO was greater in SHRSP (36+/-4% of hemisphere volume) than in SHR (19+/-5%) or WKY rats (5+/-2%). This was associated with a significantly greater reduction in regional cerebral blood flow (rCBF) in SHR and SHRSP than in WKY rats during the ischemic period (78% vs. 62%). In WKY rats, rCBF returned to 75% of control following reperfusion. In contrast, SHR and SHRSP exhibited a large (166+/-18% of baseline) and sustained (1 h) postischemic hyperperfusion. Acute blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016; 1 mg/kg) reduced infarct size by 59% in SHR and 87% in SHRSP. HET0016 had no effect on the fall in rCBF during MCAO but eliminated the hyperemic response. HET0016 also attenuated vascular O2*- formation and restored endothelium-dependent dilation in cerebral arteries of SHRSP. These results indicate the production of 20-HETE is elevated in the cerebral vasculature of SHRSP and contributes to oxidative stress, endothelial dysfunction, and the enhanced sensitivity to ischemic stroke in this hypertensive model.     

High-soy diet decreases infarct size after permanent middle cerebral artery occlusion in female rats

Estrogen is a powerful neuroprotective agent in rodent models of ischemic stroke. However, in humans, estrogen treatment can increase risk of stroke. Health risks associated with hormone replacement have led many women to consider alternative therapies including high-soy diets or supplements containing soy isoflavones, which act as estrogen receptor ligands to selectively mimic some of estrogen's actions. We hypothesized that a high-soy diet would share the neuroprotective actions of estrogen in focal cerebral ischemia. Female Sprague-Dawley rats were ovariectomized and divided into three groups: isoflavone-free diet + placebo (IF-P), isoflavone-free diet + estradiol (IF-E), or high-soy diet + placebo (S-P). Two weeks after being placed on diets, rats underwent left permanent middle cerebral artery occlusion (MCAO). Reductions in ipsilateral cerebral blood flow were equivalent across groups ( approximately 50%). Twenty-four hours later neurological deficit was determined, and brains were collected for assay of cerebral infarct by TTC staining. In the IF-P rats MCAO produced a 50 +/- 4% cerebral infarct. Estrogen and high-soy diet both significantly reduced the size of the infarcts to 26 +/- 5% in IF-E rats and to 37 +/- 5% in S-P rats. Analysis at five rostro-caudal levels revealed that estrogen treatment was slightly more effective at reducing infarct size than high soy diet. Overall neurological deficit scores at 24 h correlated with infarct size; however, there were no statistically significant differences among the treatment groups. These data show that 2 wk of a high-soy diet is an effective prophylactic strategy for reducing stroke size in a rat model of focal cerebral ischemia.     

Mechanism of Protection by Soluble Epoxide Hydrolase Inhibition in Type 2 Diabetic Stroke

Inhibition of soluble epoxide hydrolase (sEH) is a potential target of therapy for ischemic injury. sEH metabolizes neuroprotective epoxyeicosatrienoic acids (EETs). We recently demonstrated that sEH inhibition reduces infarct size after middle cerebral artery occlusion (MCAO) in type 1 diabetic mice. We hypothesized that inhibition of sEH would protect against ischemic injury in type 2 diabetic mice. Type 2 diabetes was produced by combined high-fat diet, nicotinamide and streptozotocin in male mice. Diabetic and control mice were treated with vehicle or the sEH inhibitor t-AUCB then subjected to 60-min MCAO. Compared to chow-fed mice, high fat diet-fed mice exhibited an upregulation of sEH mRNA and protein in brain, but no differences in brain EETs levels were observed between groups. Type 2 diabetic mice had increased blood glucose levels at baseline and throughout ischemia, decreased laser-Doppler perfusion of the MCA territory after reperfusion, and sustained larger cortical infarcts compared to control mice. t-AUCB decreased fasting glucose levels at baseline and throughout ischemia, improved cortical perfusion after MCAO and significantly reduced infarct size in diabetic mice. We conclude that sEH inhibition, as a preventative treatment, improves glycemic status, post-ischemic reperfusion in the ischemic territory, and stroke outcome in type 2 diabetic mice.     

MitoK(ATP) opener,diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat

We investigated effects of diazoxide, a selective opener of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels, against brain damage after middle cerebral artery occlusion (MCAO) in male Wistar rats. Diazoxide (0.4 or 2 mM in 30 microl saline) or saline (sham) was infused into the right lateral ventricle 15 min before MCAO. Neurological score was improved 24 h later in the animals treated with 2 mM diazoxide (13.8 +/- 0.7, n = 13) compared with sham treatment (9.5 +/- 0.2, n = 6, P < 0.01). The total percent infarct volume (MCAO vs. contralateral side) of sham treatment animals was 43.6 +/- 3.6% (n = 12). Treatment with 2 mM diazoxide reduced the infarct volume to 20.9 +/- 4.8% (n = 13, P < 0.05). Effects of diazoxide were prominent in the cerebral cortex. The protective effect of diazoxide was completely prevented by the pretreatment with 5-hydroxydecanoate (100 mM in 10 microl saline), a selective blocker of mitoK(ATP) channels (n = 6). These results indicate that selective opening of the mitoK(ATP) channel has neuroprotective effects against ischemia-reperfusion injury in the rat brain.     

Caffeic acid phenethyl ester ameliorates cerebral infarction in rats subjected to focal cerebral ischemia

The effects of caffeic acid phenethyl ester (CAPE), an antioxidant derived from propolis, on the infarct volume elicited by focal cerebral ischemia were studied on Long-Evans rats. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of bilateral common carotid arteries (CCA) for 60 min. The rats were sacrificed 24 h later and serial brain slices of 2 mm thickness were taken and stained for the measurement of infarct area. CAPE was administered intravenously 15 min before MCA occlusion. Pretreatment of CAPE (0.1, 1 and 10 microg/kg) significantly reduced the total infarct volume from 169.6 +/- 14.5 mm3 (control) to 61.0 +/- 24.1 mm3 (0.1 microg/kg CAPE), 47.4 +/- 9.1 mm3 (1 microg/kg CAPE), and 42.4 +/- 8.7 mm3 (10 microg/kg CAPE), respectively. Plasma nitric oxide (NO) content was significantly increased in rats subjected to focal cerebral ischemia. It is concluded that CAPE possesses neuroprotective properties in focal cerebral ischemia injury in rats possibly through its antioxidant effect and/or via the upregulation of NO production.     

Nox2 oxidase activity accounts for the oxidative stress and vasomotor dysfunction in mouse cerebral arteries following ischemic stroke

Background and Purpose

Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase.

Methods

Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2^-/-) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N ^ω-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting.

Results

Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2^-/- mice. In WT mice, L-NAME-induced constriction was reduced by ∼50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2^-/- mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ∼1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2^-/- mice. Vascular CD45 levels were unchanged by ischemia-reperfusion.

Conclusions

Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase.     

小鼠脑缺血再灌注模型稳定性研究

目的:探讨影响小鼠脑缺血再灌注模型成功率,梗死体积以及行为学评分稳定性的因素。方法:采用昆明小鼠75只,体重为20-23 g,随机分为5组,比较在不同长度的线栓以及不同进栓深度的条件下对小鼠脑缺血再灌注模型成功率,梗死体积以及行为学评分的稳定性的影响。同时术中行脑血流监测,比较各组小鼠大脑中动脉脑血流下降的差异。结果:规格1组,模型成功率为40%,梗死体积为(16.7±9.3)%,神经功能缺损评分(NSS):7.2±2.4,大脑中动脉(MCA)血流下降百分比:(86.9±4.2)%;规格2组,模型成功率为46.7%,梗死体积百分比为(19.2±11.6)%,NSS:8.8±2.5,MCA血流下降百分比:(87.4±3.8)%;规格3组,模型成功率为33.3%,梗死体积百分比为(16.6±9.6)%,NSS:8.2±2.6,MCA血流下降百分比:(88.3±3.4)%;规格4组,模型成功率为86.7%,梗死体积百分比为(23.4±2.2)%,NSS:13.9±1.3,MCA血流下降百分比:(87.5±3.5)%。结论:1.小鼠脑缺血再灌注模型稳定性关键因素在于线栓能对后交通动脉(PComA)和大脑前动脉(ACA)起始段形成有效栓塞。2.小鼠大脑中动脉血流监测并不能作为评价小鼠脑缺血再管注模型成功与否的主要依据。     

Effect of combined therapy with hyperbaric oxygen and antioxidant on infarct volume after permanent focal cerebral ischemia

The aim of the present study was to evaluate the efficiency of combination of hyperbaric oxygen (HBO) and an antioxidant on permanent focal cerebral ischemia. Male Wistar rats underwent permanent middle cerebral artery occlusion (MCAO). Then, animals were randomly assigned to one of four groups: the control group (n=9) received no treatment, HBO group (n=9) was treated for 90 min at 2.5 absolute atmosphere for 3 days, the U-74389G group (n=8) received single U-74389G injection (3 mg/kg), the HBO + U-74389G group (n=8) received both HBO and U-74389G treatments. Treatments were initiated within the first 10 min after MCAO. After 3 days, the infarct volumes in rat brains were measured. The infarct ratios were 25.6+/-6.5 % for the control group, 21.9+/-6.4 % for the HBO group, 15.7+/-5.7 % for U-74389G group and 12.5+/-3.8 % for HBO + U74389G group. The infarct volumes were significantly reduced in rats treated with U-74389G (p<0.05) and combination therapy (p<0.05). HBO failed to reduce infarct volume significantly. We concluded that 1) U-74389G is more beneficial than HBO on permanent MCAO in rats, and 2) a combined therapy failed to significantly improve infarct volume more than either single treatment.     

Does nitric oxide contribute to iron-dependent brain injury after experimental cerebral ischaemia?

Experimental and clinical data suggest that iron has a key role in cerebral ischaemia. We measure infarct volume and analyse the nitric oxide responses to brain injury in rat stroke model after increased oral iron intake. Permanent middle cerebral artery occlusion (MCAO) was performed in a group of 20 male Wistar rats, 10 of which were fed with a control diet and 10 of which were fed with iron-enriched diet containing 2.5% carbonyl iron for 9 weeks. L-arginine and nitric oxide metabolites were determined in blood samples before and at 2, 6, 8 and 48 h after MCAO. Infarct volume, thiobarbituric acid reaction substances (TBARS) and tissue iron were measured at 48 h. Infarct volume was 66% greater in the iron-fed rats than in the control group. Iron-fed animals showed significantly higher levels of TBARS. Liver iron stores (3500 +/- 199 vs 352 +/- 28 microg Fe/g, p<0.0001) but not brain iron stores (131 +/- 11 vs 139 +/- 8 microg Fe/g, p=0.617), were significantly higher in the iron-fed group. L-arginine levels were slightly lower in iron-fed rats and decreased significantly in both groups at 6 and 8 hours after MCAO. The levels of the stable end products of NOS (NOx = nitrite + nitrate) were significantly higher in iron-fed rats before MCAO (16.2 +/- 2.2 vs. 9.6 +/- 0.8 micromol x L(-1), p<0.05), with a further increase during the six first hours after MCAO in both groups. These results suggest that the iron overload that increases both superoxide and nitric oxide production leads to peroxynitrite formation, thus enhancing brain damage.     

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% ± 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet’s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.     

Pro-Inflammatory Mediators and Apoptosis Correlate to rt-PA Response in a Novel Mouse Model of Thromboembolic Stroke

Background

A recent study suggests that patients with persistent occlusion of the middle cerebral artery (MCA) following treatment with recombinant tissue plasminogen activator (rt-PA) have better outcomes than patients with MCA occlusion not receiving rt-PA. We performed a study to elucidate possible mechanisms of this finding in a new model of thromboembolic stroke closely mimicking human pathophysiology.

Methods

Thromboembolic stroke was induced by local injection of thrombin directly into the right MCA of C57 black/6J mice. Rt-PA was administered 20 and 40 min after clot formation. The efficiency of rt-PA to induce thrombolysis was measured by laser Doppler. After 24 h, all animals were euthanized and interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase (MMP)-9, Caspase-3, hsp 32 and hsp 70 protein levels were investigated by immunofluorescence. Presence of hemorrhage was verified and infarct volume was measured using histology.

Results

Thrombin injection resulted in clot formation giving rise to cortical brain infarction. Early rt-PA treatment starting at 20 min after the clot formation resulted in 100% recanalization. However, rt-PA-induced thrombolysis dissolved the clot in only 38% of the animals when administered 40 min after clot formation. Protein levels of IL-6, TNF-α, MMP-9, Caspase-3, hsp 32 and hsp 70 were increased after MCAO, whereas treatment with rt-PA attenuated the expressions of inflammatory markers in those animals where the thrombolysis was successful. In addition, the infarct size was significantly reduced with rt-PA treatment compared to non-treated MCAO, regardless of whether MCA thrombolysis was successful.

Conclusions

The present study demonstrates a clear correlation of the protein expression of inflammatory mediators, apoptosis and stress genes with the recanalization data after rt-PA treatment. In this model rt-PA treatment decreases the infarct size regardless of whether vessel recanalization is successful.     

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.¹ in 1992 and further characterized by Zhang et al. in 1997². Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.     

A surgical model of permanent and transient middle cerebral artery stroke in the sheep

Background

Animal models are essential to study the pathophysiological changes associated with focal occlusive stroke and to investigate novel therapies. Currently used rodent models have yielded little clinical success, however large animal models may provide a more suitable alternative to improve clinical translation. We sought to develop a model of acute proximal middle cerebral artery (MCA) ischemic stroke in sheep, including both permanent occlusion and transient occlusion with reperfusion.

Materials and Methods

18 adult male and female Merino sheep were randomly allocated to one of three groups (n = 6/gp): 1) sham surgery; 2) permanent proximal MCA occlusion (MCAO); or 3) temporary MCAO with aneurysm clip. All animals had invasive arterial blood pressure, intracranial pressure and brain tissue oxygen monitoring. At 4 h following vessel occlusion or sham surgery animals were killed by perfusion fixation. Brains were processed for histopathological examination and infarct area determination. 6 further animals were randomized to either permanent (n = 3) or temporary MCAO (n = 3) and then had magnetic resonance imaging (MRI) at 4 h after MCAO.

Results

Evidence of ischemic injury in an MCA distribution was seen in all stroke animals. The ischemic lesion area was significantly larger after permanent (28.8%) compared with temporary MCAO (14.6%). Sham animals demonstrated no evidence of ischemic injury. There was a significant reduction in brain tissue oxygen partial pressure after permanent vessel occlusion between 30 and 210 mins after MCAO. MRI at 4 h demonstrated complete proximal MCA occlusion in the permanent MCAO animals with a diffusion deficit involving the whole right MCA territory, whereas temporary MCAO animals demonstrated MRA evidence of flow within the right MCA and smaller predominantly cortical diffusion deficits.

Conclusions

Proximal MCAO can be achieved in an ovine model of stroke via a surgical approach. Permanent occlusion creates larger infarct volumes, however aneurysm clip application allows for reperfusion.     

Early Treatment with Poly(ADP-Ribose) Polymerase-1 Inhibitor (JPI-289) Reduces Infarct Volume and Improves Long-Term Behavior in an Animal Model of Ischemic Stroke

In patients with stroke and neurodegenerative diseases, overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) causes harmful effects by inducing apoptosis, necrosis, neuroinflammation, and immune dysregulation. The current study investigated the neuroprotective effect of a novel PARP-1 inhibitor, JPI-289, in an animal model of ischemic stroke. A transient middle cerebral artery occlusion (tMCAO, 2 h) model was used to determine the therapeutic effect and the most effective dose and time window of administration of JPI-289. We also investigated the long-term outcomes of treatment with JPI-289 by diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) MRI and by measuring neurological function at 24 h, 7 days, and 28 days after MCAO. The most effective dose and time window of administration of JPI-289 was 10 mg/kg administered 2 h after MCAO with reperfusion. Twenty-four hours after MCAO, infarct volume was reduced by 53% and the number of apoptotic cells was reduced by 56% compared with control. JPI-289 also reduced infarct volume by 16% in the permanent MCAO model. In an MRI-based study, initial infarct volume, as measured using DWI, was similar in the control and JPI-289-treated groups. However, infarct volume and brain swelling were significantly reduced in the group treated with JPI-289 (2 h) at 24 h and 7 days after MCAO. Neurological functions also improved in the group treated with JPI-289 (2 h) until 28 days after MCAO. Inhibition of PARP-1 has neuroprotective effects (reduction of infarct volume and brain swelling) in both tMCAO and pMCAO models of ischemic stroke.     

Cyclosporin A Aggravates Electroshock-Induced Convulsions in Mice with a Transient Middle Cerebral Artery Occlusion

1. To test whether an ischemic insult increases the susceptibility to cyclosporine A (CsA)-induced neurotoxicity, we examined the effect of CsA on the minimal electroshock-induced convulsions in mice treated with a transient middle cerebral artery occlusion (MCAO) for a short period (2 h). 2. This MCAO produced small to mid-sized infarcted regions in the cerebral hemisphere with increasing post-operative days. In MCAO mice, CsA (30 mg/kg, i.p.) elevated the incidence of minimal electroshock-induced convulsions to 90-100% over that in sham mice (20-30%) at 1-7 days but not 14 days post-surgery. 3. In light of these findings, the possibility that CsA increases the risk of convulsions in patients with cerebral infarction and/or at an early stage following focal cerebral ischemia would have to be considered.