Human C-reactive protein increases cerebral infarct size after middle cerebral artery occlusion in adult rats.

Human C-reactive protein (CRP), the classic acute phase plasma protein, increases in concentration after myocardial infarction and stroke. Human CRP binds to ligands exposed in damaged tissue and can then activate complement and its proinflammatory functions. In contrast, rat CRP, which binds to similar ligands, does not activate complement. In the present study, systemic complement depletion with cobra venom factor in adult rats subjected to middle cerebral artery occlusion did not affect cerebral infarct size, indicating that circulating complement does not contribute to injury in this model. However, we have previously reported that administration of human CRP to rats undergoing coronary artery ligation caused a marked increase in size of the resulting myocardial infarction, associated with codeposition of human CRP and rat complement in the infarcts. In the present study, we show that adult rats subjected to middle cerebral artery occlusion and then treated with human CRP similarly developed significantly larger cerebral infarcts compared with control subjects receiving human serum albumin. Human CRP can thus contribute to ischemic tissue damage in the brain as well as in the heart, and inhibition of CRP binding may therefore be a promising target for tissue protective acute therapeutic intervention in stroke as well as in myocardial infarction.     

Iron intake increases infarct volume after permanent middle cerebral artery occlusion in rats

Experimental and clinical data suggest an important role of iron in cerebral ischaemia. We measured infarct volume and analysed the oxidative stress, and also the excitatory and inflammatory responses to brain injury in a rat stroke model after an increased oral iron intake. Permanent middle cerebral artery occlusion (MCAO) was performed in ten male Wistar rats fed with a diet containing 2.5% carbonyl iron for 9 weeks, and in ten control animals. Glutamate, interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were determined in blood samples before and at 2, 4, 6, 8, 24 and 48 h after MCAO, and thiobarbituric acid reaction substances (TBARS) were analysed at 48 h. Infarct volume was measured at 48 h by image analysis on brain slices stained with 1% TTC. Tissue iron was measured by atomic absorption spectrophotometry. Infarct volume was 66% greater in the iron fed rats than in the control group (178±49 mm³ versus 107±53 mm³, P<0.01). Significant higher levels of glutamate, IL-6 and TNF-α were observed in the group with iron intake (peak values were obtained at 6, 8 and 4 h, respectively). Iron-fed animals also showed significantly higher levels of TBARS than those receiving a normal diet (6.52±0.59 vs. 5.62±0.86 μmol/l, P=0.033) Liver iron stores (3500±199 vs. 352±28 μg Fe/g, P<0.0001), but not brain iron stores (131 vs. 139 μg Fe/g, P=0.617), were significantly higher in the iron fed rats group. These results suggest that iron intake is associated with larger infarct volumes after MCAO in the rat. This effect seems to be associated with higher oxidative stress, excitotoxicity and inflammatory responses.     

Iron intake increases infarct volume after permanent middle cerebral artery occlusion in rats

The effects of exercise on the topography of movement representations and blood vessel density within the rat forelimb motor cortex was examined. Adult male rats were allocated to either a Voluntary eXercise (VX) or Inactive Condition (IC). VX animals were housed for 30 days with unlimited access to running wheels while IC animals were housed in standard laboratory cages. VX animals exhibited a progressive increase in the distance traveled per day and ran an average of 58.3 km across the 30-day training period. Microelectrode stimulation was used to derive high resolution maps of the forelimb representations within the motor cortex of animals from both conditions. No significant differences in the area of either distal (wrist/digit) or proximal (elbow/shoulder) movement representations were found between VX and IC animals. However, VX animals did have a significantly greater density of blood vessels within layer V of the forelimb motor cortex. These results demonstrate that increases in forelimb motor activity sufficient to induce cortical angiogenesis does not alter the topography of forelimb movement representations within forelimb motor cortex.     

Methionine sulfoximine reduces cortical infarct size in rats after middle cerebral artery occlusion

The methionine analogue methionine sulfoximine was administered to 10 rats 24 hours before occlusion of the proximal left middle cerebral artery. Three days later the rats were decapitated and the brain infarct volumes were compared with those in 10 control rats that received saline before middle cerebral artery occlusion. The mean volume of the infarct in the cerebral cortex was reduced by 33% in the group treated with methionine sulfoximine (p less than 0.01). This protective effect may be mediated by a presynaptic mechanism; methionine sulfoximine profoundly inhibits brain glutamine synthetase, thereby interrupting the astrocyte-neuron glutamate shuttle and impairing neuronal glutamate release. Methionine sulfoximine also increases brain glycogen stores, and this increased energy reserve may benefit penumbral tissue during the peri-infarct period. Further study of the mechanisms by which methionine sulfoximine decreases infarct volume could lead to new therapeutic approaches for stroke.     

Clentiazem reduces infarct size in rabbit middle cerebral artery occlusion

We assessed the value of pretreatment with clentiazem (8-chlorodiltiazem), a diltiazem derivative with cerebroselective properties, on the consequences of surgical occlusion of the middle cerebral artery via a transorbital approach in 38 rabbits. Nineteen rabbits received 1.7 (n = 5), 5 (n = 8), or 15 (n = 6) mg/kg clentiazem orally four times a day for 24 hours before and 48 hours after occlusion. Upon sacrifice, a segment of the right middle cerebral artery distal to the occlusion and a corresponding segment from the nonoccluded left middle cerebral artery were mounted on myographs for in vitro study of their reactivity to histamine, acetylcholine, serotonin, norepinephrine, and electrical stimulation of intramural sympathetic nerves. Morphometric measurements of 2,3,5-triphenyltetrazolium chloride-stained brain slices permitted us to estimate infarct volume. Pretreatment with 1.7, 5, and 15 mg/kg clentiazem significantly reduced infarct volume (p less than 0.05, p less than 0.01, and p less than 0.01, respectively). Mean infarct volume of the 15 mg/kg-treated group was only 4% that of the untreated group. There were no postoperative deaths in any treated group compared with a death rate of 36% in the untreated group. Mean values for vascular smooth muscle contractility to histamine and relaxation to acetylcholine were significantly enhanced in vessels from treated rabbits. These studies indicate that pretreatment with clentiazem offers cerebral protection and significantly reduces infarct volume as well as arterial wall damage beyond the occlusion.     

Kynurenate does not reduce infarct size after middle cerebral artery occlusion in spontaneously hypertensive rats

The effects of the excitatory amino acid receptor antagonist, kynurenate, were investigated in spontaneously hypertensive rats after middle cerebral artery occlusion. Kynurenate did not significantly modify either the infarct volume, measured 48 h after occlusion, or the neurological score. The absence of a neuroprotective effect of kynurenate in this study, which contrasts with results in normotensive rats, is suggested to be due to impaired collateral circulation in spontaneously hypertensive rats.     

Testosterone increases and estradiol decreases middle cerebral artery occlusion lesion size in male rats

This study was undertaken to determine the effects of estrogen and testosterone on cerebral ischemic lesion size induced by middle cerebral artery (MCA) occlusion in male rats. Rats were gonadectomized and treated with testosterone, estrogen, or testosterone plus estrogen filled Silastic® pellets. The animals were divided into 6 groups: intact, intact+estrogen (E2), castrate, castrate+testosterone (T), castrate+E2, and castrate+T+E2. One week after treatment, cerebral ischemia was induced by MCA occlusion for 40 min, followed by reperfusion. After 24 h, rats were sacrificed and slices were then stained to assess lesion size. The presence of testosterone increased and the removal of testosterone decreased lesion size. A strong positive correlation (r²=0.922) between plasma testosterone concentrations and ischemic lesion size was observed. Estradiol treatment reduced ischemic area. In summary, the present study provides evidence that testosterone exacerbates and estrogens ameliorate ischemic brain damage in an animal model of cerebral ischemia.     

Middle cerebral artery perfusion pressure in cerebrovascular occlusive disease

We measured the MCAP (middle cerebral artery pressure)/MSBP (mean systemic blood pressure) ratio in 76 patients who underwent an EIAB (extracranial-intracranial arterial bypass). Patients were divided into groups on the basis of angiographic findings. We found a definite correlation between increasing angiographic cerebral vascular occlusive disease and lower MCAP/MSBP ratios. Six of 32 patients with a preoperative neurologic deficit demonstrated mild but definite postoperative neurologic improvement. The mean MCAP/MSBP ratio in these six patients was significantly lower than that for the remainder of this group (p less than .05). Finally, postoperative filling of the middle cerebral artery territory through the graft was found to correlate in an inverse linear relationship with MCAP/MSBP. Evidence is presented that hypoperfusion produced by occlusion of major cranial vessels plays an important role in temporary and permanent neurological deficits seen in patients with cerebrovascular disease.     

Chronic parasympathetic sectioning decreases regional cerebral blood flow during hemorrhagic hypotension and increases infarct size after middle cerebral artery occlusion in spontaneously hypertensive rats.

Regional cerebral blood flow (rCBF) during controlled hemorrhagic hypotension (140-20 mm Hg) was assessed 10-14 days after chronic unilateral sectioning of parasympathetic and/or sensory fibers innervating pial vessels in spontaneously hypertensive rats (SHR). rCBF was measured in the cortical barrel fields bilaterally by laser Doppler blood flowmetry. Immunohistochemistry of middle cerebral artery (MCA) whole mount preparations was used to verify the surgical lesion. During hemorrhagic hypotension, rCBF was equivalent on the two sides in shams, after selective sensory denervation, or in parasympathetically sectioned animals exhibiting small decreases (less than or equal to 30%) in immunoreactive vasoactive intestinal peptide (VIP)-containing fibers. After chronic parasympathetic denervation, decreases in perfusion pressure were accompanied by greater reductions in rCBF on the lesioned side; changes in vascular resistance were also attenuated on that side. The rCBF response to hypercapnia (PaCO2 50 mm Hg), however, was symmetrical and robust. To examine the effects of impaired neurogenic vasodilation on the pathophysiology of cerebral ischemia, infarct size was measured 24 h following tandem MCA occlusion in denervated animals. Infarction volume was larger after selective parasympathetic sectioning (sham, 156 +/- 27 vs. 196 +/- 32 mm3, respectively) but only in those denervated animals demonstrating greater than or equal to 40% decrease in immunoreactive VIP-containing fibers within the ipsilateral MCA. Lower than expected blood flow/perfusion pressure in the cortex distal to an occluded blood vessel may relate the observed blood flow responses to the occurrence of larger cortical infarcts in parasympathetically denervated animals. If true, the findings suggest a novel role for neurogenic vasodilation in the pathophysiology of cerebral ischemia and in rCBF regulation within the periinfarction zone.     

Sigma receptor activation reduces infarct size at 24 hours after permanent middle cerebral artery occlusion in rats

The only available treatment for embolic stroke is recombinant tissue plasminogen activator, which must be administered within three hours of stroke onset. We examined the effects of 1,3-di-o-tolyguanidine (DTG), a high affinity sigma receptor agonist, as a potential treatment for decreasing infarct area at delayed time points. Rats were subjected to permanent embolic middle cerebral artery occlusion (MCAO) and allowed to recover before receiving subcutaneous injections of 15 mg/kg of DTG at 24, 48, and 72 hours. At 96 hours the rats were euthanized, and brains harvested and sectioned. Infarct areas were quantified at the level of the cortical/striatal and cortical/hippocampal regions in control (MCAO-only) and DTG treated animals using a marker for neurodegeneration, Fluoro-Jade. DTG treatment significantly reduced infarct area in both cortical/striatal and cortical/hippocampal regions by >80%, relative to control rats. These findings were confirmed by immunohistochemical experiments using the neuronal marker, mouse anti-neuronal nuclei monoclonal antibody (NeuN), which showed that application of DTG significantly increased the number of viable neurons in these regions. Furthermore, DTG blocked the inflammatory response evoked by MCAO, as indicated by decreases in the number of reactive astrocytes and activated microglia/macrophages detected by immunostaining for glial fibrillary acidic protein (GFAP) and binding of isolectin IB4, respectively. Thus, our results demonstrate that the sigma receptor-selective agonist, DTG, can enhance neuronal survival when administered 24 hr after an ischemic stroke. In addition, the efficacy of sigma receptors for stroke treatment at delayed time points is likely the result of combined neuroprotective and anti-inflammatory properties of these receptors.     

Reduction of infarct volume by magnesium after middle cerebral artery occlusion in rats

The effects of magnesium, an endogenous inhibitor of calcium entry into neurons, upon ischemic brain damage were investigated using a well-characterized model of focal cerebral ischemia in rats. Infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride transcardiac perfusion 48 h after middle cerebral artery (MCA) occlusion. The area of ischemic damage was quantified by image analysis in coronal sections taken every 0.5 mm. MgCl2 (1 mmol/kg) was injected intraperitoneally just after MCA occlusion and again 1 h later. Posttreatment with MgCl2 (16 control and 16 treated rats) significantly reduced the cortical infarct volume. Compensation for the hyperglycemic effect of MgCl2 with insulin (17 rats) further reduced the infarct volume in the neocortex. No systemic effects of either treatment could account for the observed neuroprotection.     

Middle cerebral artery occlusion in rats studied by magnetic resonance imaging

Ischemia due to middle cerebral artery occlusion was studied in 29 rats from 1 to 24 hours after occlusion using magnetic resonance imaging. Images were made before and after the injection of a superparamagnetic iron oxide compound, AMI-25. Subtraction images demonstrated the region of perfusion deficit as early as 1 hour after occlusion, earlier than conventional T2-weighted images. The area of altered perfusion detected by this technique (subtraction imaging after AMI-25 administration) correlated with that demonstrated by iodoantipyrine autoradiography. Since this magnetic resonance technique can be used to serially estimate the location and size of the ischemic area, the technique can be an important adjunct to metabolic studies of focal ischemia using magnetic resonance spectroscopy. The technique may have clinical applications as well.     

A novel AMPA receptor antagonist, YM872, reduces infarct size after middle cerebral artery occlusion in rats

The neuroprotective effect of YM872 ([2.3-dioxo-7-(1H-imidazol-1-yl) 6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate), a novel α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist with improved water solubility, was examined in a rat focal cerebral ischemia model. Rats were subjected to permanent middle cerebral artery (MCA) occlusion using the intraluminal suture occlusion method for 24 h. YM872 was intravenously infused for 4 h (20 and 40 mg/kg/h) or 24 h (10 and 20 mg/kg/h), starting 5 min after the MCA occlusion, to investigate the effect of prolonged duration of the treatment on infarct volume. In the 4 h infusion study, YM872 reduced the cortical infarct volume by 48% at a dose of 40 mg/kg/h. YM872 did not significantly reduce the infarct at 20 mg/kg/h for 4 h. In the 24 h infusion study, however, YM872 markedly reduced the cortical infarct volume by 62%, even at 20 mg/kg/h. The present study indicates that the neuroprotective effect of YM872 is enhanced by extending the duration of treatment, and demonstrates the benefit of the prolonged treatment with AMPA antagonists following focal cerebral ischemia. YM872, a highly water soluble compound, is applicable to investigate the role of AMPA receptors in ischemic models without concern about nephrotoxicity and could be useful in the treatment of human stroke.     

Modified permanent middle cerebral artery occlusion rat model aiming to reduce variability in infarct size

In animal cerebral infarct experiments, the most important aspect is to produce consistent infarct size and localization. In an attempt to improve the conventional middle cerebral artery (MCA) coagulation technique, we developed a new animal model using a microclip to reduce variability in infarct size. Male Sprague-Dawley rats were subjected to right MCA occlusion. The animals were divided into two groups; conventional MCA occlusion group (Group 1; n = 9) and modified clip occlusion group (Group 2; n = 9). In Group 2, the proximal portion of MCA was occluded by applying a small clip just proximal to the olfactory nerve, and the MCA from the clipped position to the position just proximal to the level of the inferior cerebral vein was electrocoagulated using a bipolar diathermy in the same manner as in Group 1. In other words, the only difference between these two groups was the manner of occlusion of the most proximal portion of the MCA. Rats were killed 24 hours after the stroke-inducing surgery, and infarct volume was determined by an image analysis program following staining with 2,3,5-triphenyltetrazolium chloride. The cortical infarct volumes were 51.0 +/- 13.8% in Group 1 and 46.3 +/- 6.2% in Group 2. The scattering of cortical infarct volume was significantly small in Group 2 (p=0.0176). The differences in scattering of striatal and total infarct volumes did not reach statistical significance. The present results demonstrated that the new MCA occlusion model using a clip significantly reduces the variability in cortical infarct volume, solving the problems of the model using coagulation alone. That permanent MCA occlusion model using a clip is an excellent method that produces more consistent and reproducible infarction.     

Hyperglycaemia and infarct size in animal models of middle cerebral artery occlusion: systematic review and meta-analysis

Poststroke hyperglycaemia (PSH) is common, has an unclear pathophysiology, and is associated with poor outcomes. Animal studies report conflicting findings. We systematically reviewed the effects of hyperglycaemia on infarct volume in middle cerebral artery occlusion (MCAO) models, generating weighted mean differences between groups using random effects models summarised as effect size (normalised to control group infarct volume as 100%) and 95% confidence interval. Of 72 relevant papers, 23 reported infarct volume. Studies involved 664 animals and 35 distinct comparisons. Hyperglycaemia was induced by either streptozotocin (STZ, 17 comparisons, n=303) or dextrose (18 comparisons, n=356). Hyperglycaemic animals had infarcts that were 94% larger, but STZ was associated with significantly greater increase in infarct volumes than dextrose infusion (140% larger versus 48% larger). In seven studies, insulin did not significantly reduce infarct size and results were heterogeneous. Although hyperglycaemia exacerbates infarct volume in MCAO models, studies are heterogeneous, and do not address the common clinical problem of PSH because they have used either the STZ model of type I diabetes or extremely high glucose loads. Insulin had a nonsignificant and significantly heterogeneous effect. Further studies with relevant models may inform clinical trial design.     

Recombinant tissue plasminogen activator reduces infarct size after reversible thread occlusion of middle cerebral artery in mice

It has been suggested that tissue plasminogen activator (tPA), which is widely used for the thrombolytic treatment of stroke, exhibits neurotoxic side effects. To test this hypothesis, mice exposed to 90 min nonthrombotic middle cerebral artery thread occlusion were treated with 10 mg/kg recombinant tPA (rt-PA) at 15 min after the onset of vascular occlusion. Measurements of blood flow, infarct volume, brain swelling and neurological performance revealed faster recirculation and a significant reduction of ischemic injury in rt-PA-treated animals. These data are at variance with previous reports on tPA neurotoxicity and demonstrate, on the contrary, that tPA protects the brain even after non-thrombotic vascular occlusion.     

Middle cerebral artery occlusion resulting from hypereosinophilic syndrome

Hypereosinophilic syndrome (HES) is a rare disorder that can cause ischemic stroke. We present a patient with middle cerebral artery (MCA) occlusion resulting from HES. Transarterial thrombolysis resulted in MCA recanalization and adjuvant therapy may have contributed to stabilization of the underlying HES in our patient.     

Endovascular suture occlusion of the middle cerebral artery in rats: Effect of suture insertion distance on cerebral blood flow,infarct distribution and infarct volume

Abstract

Sprague-Dawley rats anesthetized with isoflurane, underwent MCA occlusion by intraluminal 3-0 suture insertionl either 22 mm (n =8) or 18 mm (n =6) beyond the CCA bifurcation or were sham-operated as controls (n =3) for autoradiographic analysis of cerebral blood flow. Infarct volume was measured 24 hours after the onset of ischemia (22 mm, n =11; 18 mm, n =10); neurological examinations were performed at 6 and 24 hours. Cerebral blood flow in the MCA distribution was significantly lower in the 22 mm suture insertion group than in the 18 mm group (p < 0.05). The total infarct volume was significantly larger (197± 15 versus 135± 19 mm³, p < 0.05) and the coefficient of variance was significantly smaller (23.8% versus 43.9%, p < 0.05) in the 22 mm group. Border zone regions of medial caudoputamen and dorsolateral cortex were often spared in the 18 mm group but never in the 22 mm group. The neurological deficit was more severe in the 22 mm group at 24 hours (p < 0.05), but not at 6 hours. The greater blood flow reduction and the less variable histological damage in dorsolateral cortex of watershed area between the middle and anterior cerebral arteries) and the greater histological damage in medial caudate in the 22 mm group further characterizes this focal ischemia model for two potential applications: 22 mm insertion for studies requiring extensive and reproducible infarcts; 18 mm insertion for studies requiring less severe and more variable lesions after permanent MCA occlusion. [Neural Res 1997; 19: 409-416]