An improved version of embolic model of brain ischemic injury in the rat

We describe a cuff-type electrode specifically designed for recording from, and electrical stimulation of, cut nerves in acute experiments on small animals. Unlike existing designs of cuff electrodes, it is simple to manufacture, inexpensive and takes little time to implant. The electrode was tested on the hypoglossal, phrenic, recurrent laryngeal, and superior laryngeal nerves in anesthetized rats. It provides satisfactory signal-to-noise ratios (3.0+/-0.8 (mean+/-S.D.)) for hypoglossal and 5.4+/-2.1 for phrenic nerve activity and stable recording over the time course of a typical acute experiment. It eliminates or minimizes the problems with recording stability and space availability associated with conventional hook-type electrodes, and reduces experiment preparation time. This should facilitate neurophysiological experiments on small rodents involving complex protocols that include recording from, and/or stimulation of, multiple nerves.     

A focal embolic model of cerebral ischemia in rats: introduction and evaluation

Cerebral thromboembolism is the most common type of acute stroke in the clinical setting. In the present study, we have described a focal embolic model of cerebral ischemia in rat. Cerebral ischemic injury in two different sizes was induced by injection of two different volumes of pre-formed clots into the middle cerebral artery (MCA). Neurological deficits were assessed at 1 and 24 h, respectively, after the MCA embolization. The brain infarction was evaluated with 2,3,5-triphenyltetrazolium chloride (TTC) staining at 72 h following embolic stroke. The incidence of gross hemorrhage was also identified on the TTC-stained brain sections. This study consisted of two groups. In the first group 10 microl of pre-formed clot was injected into MCA (n=10), and in the second group 5 microl of clot was injected (n=10). Embolizing a pre-formed clot resulted in an infarction in the territory irrigated by the MCA. Embolization with the different volume of clots resulted in different sizes of brain infarction, 32.1+/-2.9% (mean+/-S.E.) in the 10-microl group versus 23+/-4.5% (mean+/-S.E.) in the 5-microl group (P<0.05). The infarction size as well as neurological deficits correlated well with the volume of the clot injected. These results thus show that with the procedure described here a reliable and reproducible ischemic injury is produced in the brain. The model is relevant to thromboembolic ischemia in patients, and may offer a useful tool to investigate mechanisms underlying ischemic brain injury. It may also be used to test thrombolytic agents in ischemic brain injury.     

A model of embolic cerebral ischemia in the rat

Experimental studies of embolic cerebral ischemia using the rat are limited by variability in the location, size, and frequency of lesions produced. A technique is described herein which improves the reliability of an established model. Eight male Sprague-Dawley rats underwent injection of the cervical internal carotid artery with 0.1 ml of 1-h-old fragmented autologous blood clot through an external carotid artery cannula. The pterygopalatine artery was ligated prior to embolization. At killing 2 h after embolization, clot was observed in the proximal middle cerebral and posterior cerebral arteries in all animals. Areas of reduced blood flow at 2 h postembolization were assessed by digital image processing of iodo-[¹⁴C]antipyrine autoradiographic images. No-flow and low-flow areas were measured for each of approximately 25 serial brain sections with a computerized bit-pad. Volumes were calculated and lesions localized by anatomical reconstructions. No animal sustained a hemorrhagic lesion. One animal sustained only a very small area of ischemia in the internal capsule. Of the remaining seven, all had large regions of ischemia in the middle cerebral distribution involving cortex and basal ganglia. Posterior cerebral involvement was observed in six of the seven animals as well. The contralateral hemisphere was unaffected. Volume values could be calculated for primary vascular distributions. Most variability occurred in the pattern of posterior cerebral involvement. The technique described produces a relatively consistent region of ischemia in the middle and posterior cerebral artery distributions in the rat and is a useful model for the study of cerebral ischemia.     

A novel embolic stroke model resembling lacunar infarction following proximal middle cerebral artery occlusion in beagle dogs

It is estimated that lacunar infarcts account for 25% of all ischemic strokes, but its exact etiology is still on debating. The existing controversies include whether the embolisms can indeed cause lacunar stroke in humans or animal models. We hypothesized that lacunar infarction can be induced by the proximal middle cerebral artery (MCA) segmental occlusion involving the orifices of lenticulostriate arteries in animal models, which have abundant distal cerebral collateral anastomosis. Our work here establishes a proximal MCA occlusion model using thrombi (autologous blood clots about 1.7 mm in diameter and 5 mm in length) in 8 beagle dogs, evaluates the progression of ischemic lesions at 30 min interval within 6 h after embolization using the diffusion weighted imaging (DWI), and discusses the potential mechanisms of lacunar infarction. Our results indicate that the left proximal MCAs can be successfully occluded in all dogs using interventional single-thrombus method. The small solitary or multiple ischemic lesions shown in DWI were observed in the deep brain area, with the mean detecting time of 1.21 ± 0.45 h using DWI and diameter of 6.62 ± 0.60mm in 6h-DWI after procedure. In conclusion, our method established an ischemic model which can recapitulate the radiologic and histologic changes in lacunar infarcts, suggesting that emboli can cause lacunar infarcts in animal model.     

Long-term changes in cerebral blood flow in patients with atherothrombotic and embolic brain infarction

Cerebral blood flow (CBF) was measured twice at years interval using xenon-133 inhalation technique in patients with atherothrombotic and embolic supratentorial brain infarction. The purpose of the study was to elucidate factors influencing long-term change in CBF in two subtypes of brain infarction of different mechanisms. Those patients were excluded from the study, who had bilateral hemispheric lesions, significant arterial lesion in the contralateral carotid axis and recurrent stroke before the second measurement of CBF. Of 46 patients studied, 23 (17 men and 6 women) were classified as atherothrombotic and 23 (15 men and 8 women) as embolic infarction based on the diagnostic criteria reported earlier. Their age at onset was 60.6 +/- 8.9 years old (mean +/- S.D.) for atherothrombotic patients, and 60.2 +/- 11.0 years old for embolic patients. The first measurement of CBF was performed between 31 and 87 days and the second measurement between 13 and 99 months after onset. In atherothrombotic group, mean hemispheric CBF (mCBF) of the affected side tended to be higher in patients examined at intervals shorter than 30 months, but tended to decrease in those at longer interval. The multivariate stepwise regression analysis indicated only "the interval between 2 measurements" to have a significant effect on reduction of mCBF, although in addition to the interval (p less than 0.05), infarct-size (p less than 0.01) and change in PaCO2 (p less than 0.05) were suggested to be possible factors by single regression analysis. In embolic group, an increase in hematocrit had a significant effect on reduction of mCBF, even when being evaluated with multivariate stepwise regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved test of neurological dysfunction following transient focal cerebral ischemia in rats

The Adhesive Removal (sticky-tape) test is a commonly used test of somatosensory dysfunction following cerebral ischemia in rats. This test requires several days of pre-training prior to surgery, which can be time consuming. We present our results with an improved version of the sticky-tape test. Male Wistar rats were subjected to either sham surgery (n = 4) or right middle cerebral artery occlusion (rMCAo) using an intraluminal filament (n = 9), followed by a 10-day survival period. On post-operative days (POD) 1, 3, 7, and 10 animals underwent both the conventional sticky-tape test (CST) with measurement of the time to remove the stimulus (trs), as well as a modified sticky-tape test (MST), in which a non-removable tape sleeve was placed around the animal's paw. Time spent attending to this stimulus (tas) was recorded. Despite 3 days of pre-training, animals undergoing baseline CST still exhibited marked variability in pre-operative baseline test performance (trs range 1-60s). In contrast, animals undergoing MST for the first time demonstrated nearly uniformly excellent performance (% tas range 91.5-98.5% of the 30s testing period). Although, affected (left) limb performance on both CST (6.8-fold increase in trs on POD 1 compared to baseline) and MST (100% decrease in tas on POD 1 compared to baseline) was markedly altered by rMCAo, CST performance declined bilaterally, and no significant differences in the ratio of affected (left) and unaffected (right) limb performance between sham-operated and rMCAo animals were observed at any time point. In contrast, the ratio of left to right performance on the MST was significantly different at all time points (P<0.01). In conclusion, we present a simple modification of the widely used Adhesive Removal test and provide evidence that this test can accurately assess neurological dysfunction in rodents, not only with minimal pre-training, but also with improved localization of the side of injury.     

Dietary nonprotein calories and cerebral infarction size in rats.

BACKGROUND AND PURPOSE: Conventional diets may cause hyperglycemia in patients with neurological injuries. The purpose of this study was to examine the effect on the severity of cerebral infarction of replacing carbohydrates as the primary dietary source of nonprotein calories. METHODS: Sixty-nine Long-Evans rats were either fasted for 24 hours, fed isocaloric amounts of a control diet containing 51.5% of the calories as carbohydrates, or fed one of five experimental diets before middle cerebral artery occlusion for 45 minutes. In the experimental diets, 60% of the carbohydrate calories were replaced with one or more of the following substrates: 1,3-butanediol, triacetin, tributyrin, and long- and medium-chain triglycerides. RESULTS: The plasma glucose concentration in the fasted animals was 6.4 +/- 1.1 mumol/ml. In the animals receiving the control diet, which contained the greatest number of carbohydrate calories, plasma glucose was 9.1 +/- 1.4 mumol/ml. The 1,3-butanediol diet resulted in an intermediate plasma glucose concentration averaging 7.8 +/- 1.3 mumol/ml. Plasma beta-hydroxybutyrate levels were elevated in the fasted group and with the 1,3-butanediol diet. Plasma acetate levels were increased with the diets supplemented with triacetin. The smallest infarct volume (53 +/- 43 mm3) was found in the fasted group and the largest (162 +/- 56 mm3) in the control diet group. Infarct volumes that were significantly smaller were found with the 1,3-butanediol diet (98 +/- 41 mm3) and with the triacetin/tributyrin diet (105 +/- 53 mm3). The volume of the infarct was directly related to the plasma glucose concentration before ischemia (n = 69, r = 0.47, p less than 0.01), but not to plasma lactate, ketone body, or acetate levels. CONCLUSIONS: It may be possible to develop a diet for patients with neurological injuries using noncarbohydrate calorie sources, such as 1,3-butanediol, triacetin, or tributyrin, that would supply systemic caloric and protein requirements without the adverse effect of conventional diets.     

Neuroprotective activity of tiagabine in a focal embolic model of cerebral ischemia

Gamma aminobutyric acid (GABA) agonists have been shown to have neuroprotective effects when used after focal or global cerebral ischemia. In this study, we evaluated the neuroprotective effects of a GABA re-uptake inhibitory agent, tiagabine, on focal ischemic brain injury in an embolic model in rats. Tiagabine, injected at 1 h after embolization, significantly reduced brain infarction volume, measured with 2,3,5-triphenyltetrazolium chloride (TTC) histological assay. There were varying degrees of neuroprotective effect exhibited in the other experimental groups however this did not reach significance. These results suggest that tiagabine is neuroprotective when administrated at an early period after the ischemic brain injury.     

Intracerebral hemorrhage after experimental embolic infarction. Anticoagulation

Embolic stroke was induced in rabbits using autologous blood clot. One hour after stroke, animals received heparin anticoagulation (AC) for five hours (acute AC) or five days (chronic AC). Animals received excessive AC (partial thromboplastin time greater than 3.0 times control), adequate AC (partial thromboplastin time, 1.2 to 2.5 times control), or saline. After the animals were killed, the brains were examined for macroscopic evidence of intracerebral hemorrhage. There was no significant increase over control in the incidence or severity of hemorrhage in any of the four treatment groups. The data suggest that heparin AC does not promote intracerebral hemorrhage after experimental embolic stroke.     

Quantitative effects of cerebral infarction on spatial learning in rats.

Outcome following stroke is difficult to measure because the behavioral response to infarction is variable. We hypothesized that cognitive function, such as spatial learning, may be a reproducible and sensitive outcome variable. We developed an animal model of multifocal cerebral ischemia in order to study the effects of infarction on learning. To cause ischemia, several hundred microspheres were injected into the internal carotid arteries of rats. After ischemia, behavior was measured using a global rating and a Morris water maze. Postmortem serial brain sections were stained and the size of the infarctions was measured. We found that intracerebral microspheres caused cortical infarction and an impairment of spatial learning. This impairment was not due to occlusion of the internal carotid artery and was not found in animals who received a sham injection of saline. The degree of learning impairment was not correlated with the volume density of the infarctions or with the volume density of the remaining cerebral hemisphere. The learning impairment clearly differentiated normal from lesioned animals, and the impairment was probably due to a delay in acquisition of spatial information rather than a defect in retention or retrieval. Measurement of learning deficit after cerebral ischemia is an efficient and sensitive method for evaluating new stroke treatments and possibly for exploring structure function relationships.     

A new model of embolic stroke produced by photochemical injury to the carotid artery in the rat

We report a new model of embolic stroke in the rat, based on endothelial disruption and platelet aggregation in the carotid artery, which leads to distal embolization and focal brain infarction. The common carotid artery was irradiated for 6.5 minutes with the focused beam of an argon laser operated at a wavelength of 514.5 nm with a peak power of 2 W and an average power of 400 mw. Ipsilateral cerebral infarcts, ranging in size from 0.1 to 1.7 mm, were produced by platelet emboli in 12 of 13 rats. A total of 44 infarcts were observed in the 12 rats: 29 in the cortex, 6 in the hippocampus, 5 in the thalamus, and 4 in the basal ganglia. Scanning electron microscopy identified platelet aggregates in the carotid artery and in a deep cortical arteriole 50 minutes after the photochemical lesion. Twenty-four hours after the experiment, scanning electron microscopy of the carotid artery revealed damaged endothelium but few remaining adherent platelets. More intense laser irradiation in 8 rats, leading to carotid occlusion, produced an infarct in only 1. This new model can be used to study the acute and chronic pathological changes in the brain associated with platelet embolism.     

Effects of tissue type plasminogen activator in embolic versus mechanical models of focal cerebral ischemia in rats.

Tissue type plasminogen activator (tPA) can be effective therapy for embolic stroke by restoring cerebral perfusion. However, a recent experimental study showed that tPA increased infarct size in a mouse model of transient focal ischemia, suggesting a possible adverse effect of tPA on ischemic tissue per se. In this report, the effects of tPA in two rat models of cerebral ischemia were compared. In experiment 1, rats were subjected to focal ischemia via injection of autologous clots into the middle cerebral artery territory. Two hours after clot injection, rats were treated with 10 mg/kg tPA or normal saline. Perfusion-sensitive computed tomography scanning showed that tPA restored cerebral perfusion in this thromboembolic model. Treatment with tPA significantly reduced ischemic lesion volumes measured at 24 hours by >60%. In experiment 2, three groups of rats were subjected to focal ischemia via a mechanical approach in which a silicon-coated filament was used intraluminally to occlude the origin of the middle cerebral artery. In two groups, the filament was withdrawn after 2 hours to allow for reperfusion, and then rats were randomly treated with 10 mg/kg tPA or normal saline. In the third group, rats were not treated and the filament was not withdrawn so that permanent focal ischemia was present. In this experiment, tPA did not significantly alter lesion volumes after 2 hours of transient focal ischemia. In contrast, permanent ischemia significantly increased lesion volumes by 55% compared with transient ischemia. These results indicate that in these rat models of focal cerebral ischemia, tPA did not have detectable negative effects. Other potentially negative effects of tPA may be dependent on choice of animal species and model systems.     

丁咯地尔对大鼠脑梗死的影响

目的 :观察丁咯地尔对大鼠脑梗死的保护及治疗作用 ,并探讨其机制。方法 :应用线栓法阻塞大鼠大脑中动脉制成大鼠脑梗死模型。结果 :丁咯地尔 10、2 0mg/kg均能减少梗死面积 ,降低缺血脑组织钙含量。结论 :丁咯地尔对大鼠脑梗死具有治疗作用 ,其机制可能与降低缺血脑组织钙含量有关     

光化学法诱导大鼠海马梗死的一种新模型

缺血性脑血管疾病是危害人类健康的常见病、多发病,患者常出现学习记忆障碍并持久存在,而海马是大脑中参与学习、记忆的重要核团.光化学法作为近年来制备脑血栓模型的一种新方法,应用于海马梗死的制备尚未见报道.本研究即探索应用光化学法原理制备海马局灶性梗死模型的方法,并对模型进行梗死体积测定,以寻求最适的光敏剂浓度,从而建立满意、稳定的局灶性海马梗死模型.     

Liposome-entrapped superoxide dismutase reduces cerebral infarction in cerebral ischemia in rats

We studied the role of superoxide radicals in the pathogenesis of ischemic brain injury using a model of focal cerebral ischemia in 102 rats and liposome-entrapped CuZn-superoxide dismutase, which can penetrate the blood-brain barrier and cell membranes efficiently. The bolus intravenous administration of 25,000 units of liposome-entrapped CuZn-superoxide dismutase elevated superoxide dismutase activities in the blood and brain 1, 2, 8, and 24 hours later as well as in the ischemic hemisphere and contralateral cortex. Determined 24 hours after right middle cerebral and bilateral common carotid artery occlusion by the lack of staining for mitochondrial dehydrogenase activity with 2,3,5-triphenyltetrazolium chloride, infarct sizes were reduced by 33%, 25%, and 18% in the anterior, middle, and posterior brain slices, respectively, by treatment with liposome-entrapped CuZn-superoxide dismutase. Our data demonstrate that superoxide radicals are important determinants of infarct size following focal cerebral ischemia and that liposome-entrapped CuZn-superoxide dismutase may have pharmacologic value for the treatment of focal cerebral ischemic injury.     

Heparin therapy in embolic cerebral infarct. A retrospective study

Heparin therapy in acute stroke is a controversial issue. It is uncertain, whether heparin has a therapeutic or preventive effect in the early phase of the stroke. From 1984-1989, 1095 patients with acute ischemic stroke were treated, 141 (12.9%) of whom received heparin within 3 days of stroke onset. The mean duration of heparin anticoagulation was 10 days. In 28 cases (20%), heparin was used as antithrombotic agent (25/28 patients suffered a basilar artery occlusion, of whom 22 died). In 113 cases (80%), heparin was used in embolic stroke to prevent recurrence (24% cardioembolic stroke, 54% arterio-arterial embolism, and 22% embolism of unknown etiology). The rate of recurrent stroke in the early phase was 13% with a persistent deficit in 5.3%. The results are comparable with those of other trials reported in the literature. Only 2 patients had an anticoagulation-related haemorrhage with clinical deterioration. Heparin anticoagulation in acute stroke is a low-risk therapy, but its preventive value has not yet been demonstrated.