Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination

We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.     

Diffusion-weighted magnetic resonance imaging: rapid and quantitative detection of focal brain ischemia.

We examined serial changes of diffusion- (DWI) and T2-weighted (T2WI) magnetic resonance images 30 minutes to 3 hours after intraluminal suture occlusion of the middle cerebral artery (MCA) in eight rats and after sham occlusion in four. We correlated the abnormal areas on DWI and T2WI with postmortem areas of infarction determined by 2,3,5-triphenyltetrazolium chloride (TTC), 24 hours after the operation. The 30-minute DWI in each MCA-occluded rat demonstrated increased signal intensity in the ipsilateral MCA territory, while T2WI showed no changes. At 3 hours, the ipsilateral DWI signal intensity increased further and the area of abnormality slightly increased. In some animals, the 3-hour T2WI disclosed an area of hyperintensity significantly smaller than that seen on the 30-minute DWI. TTC staining demonstrated an extensive MCA infarction in all rats with permanent MCA occlusion, confirmed by hematoxylin and eosin staining. The percent infarcted area of coronal brain sections, as determined by TTC staining, correlated significantly with areas on similar DWI sections at both 30 minutes and 3 hours. Sham-occluded control animals did not display any changes on DWI, T2WI, or TTC staining. The present study suggests that DWI is a very sensitive modality for detecting early ischemic brain injury, being highly correlated with post-mortem area of infarction, and may be useful to assess pharmacologic intervention.     

Focal cerebral infarction in the rat: II. Neuropathological study and local cerebral blood flow pattern

We have recently reported that middle cerebral artery (MCA) occlusion in the rat produces a uniform pattern of cerebral ischemia in an acute phase. This study was done to determine if this model is also useful for quantitative evaluation of infarction size in a chronic phase. [Methods] Sprague-Dawley rats were anesthetized with halothane and left MCA was occluded via transretro-orbital approach. The following studies were done. Neuropathological study was done one week after MCA occlusion. After perfusion fixation, the brain was cut into 6 coronal slices and stained sections were examined. Local cerebral blood flow patterns were observed by 14C-iodoantipyrine autoradiographic technique 1, 2, and 5 days after the occlusion. [Results] Neuropathological studies invariably showed infarct in the cortex and the lateral part of the basal ganlia. The ratio of the infarct to the total areas of both hemispheres in 6 coronal sections was 14.05 +/- 2.66% (Mean +/- SD) in MCA occluded animals (N = 14) and 0.59 +/- 0.46% in sham operated animals (N = 12). Relative to the contralateral hemisphere, marked reduction in CBF was seen in the territory of the MCA and moderate reductions were also seen in the surrounding areas. The same pattern of increased CBF as previously reported was also seen in the ipsilateral substantia nigra and globus pallidus 1, 2, and 5 days after the occlusion. These results indicate the usefulness of this chronic focal cerebral infarction model in the evaluation of infarction.     

利用激光散斑成像技术观察尤瑞克林对脑梗死大鼠脑血流的影响

目的 利用激光散斑成像技术研究尤瑞克林对大鼠脑梗死后局部脑血流的影响.方法 成年雄性SD大鼠24只,线栓法制备大鼠永久性大脑中动脉梗死模型.激光散斑成像系统观测缺血半球皮质及大脑中动脉供血区血流,2,3,5-三苯基氯化四氮唑(TTC)染色法测定脑梗死体积,并进行神经功能评分.结果 皮质及大脑中动脉供血区血流在大剂量组第1天及第2天给药后均有明显改善,部分大脑皮质血管增粗,血流速度加快,小剂量组及生理盐水组无明显变化,脑缺血48 h后,大、小剂量尤瑞克林组及生理盐水组的梗死体积分别为10.14%±3.02%,25.99%±3.90%,27.10%±3.32%,大剂量组与生理盐水组比较差异有统计学意义(F=61.14,P<0.01),小剂量组与生理盐水组比较差异无统计学意义.缺血后4 h,大剂量组神经功能损伤明显改善,小剂量组及生理盐水组无明显改变,36 h各组间的神经功能评分差异无统计学意义.结论 尤瑞克林可以减少大鼠局灶性脑缺血后梗死体积,延缓神经功能损伤,其作用可能与促进侧支循环的开放,增加大脑皮质和缺血区血流有关.     

Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats

We examined the neuroprotective action of a standardized extract of Ginkgo biloba leaves (EGb 761) in permanent and transient middle cerebral artery (MCA) occlusion models in Sprague-Dawley rats. Forty-four animals were given either EGb 761 (50-200 mg/kg) or vehicle intraperitoneally, 1 hr before permanent MCA occlusion, to evaluate the dose-response effects. An additional 58 animals received EGb 761 (200 mg/kg) or vehicle, 0.5- 4 hr after permanent MCA occlusion, for establishing the therapeutic window. Delayed treatment was also employed in 110 animals treated with either EGb 761 (100-200 mg/kg) or vehicle at 2-3 hr following transient focal cerebral ischemia induced by MCA occlusion for 2 hr. Neurobehavioral scores were determined 22-24 hr after permanent MCA occlusion and either 3 or 7 days after transient MCA occlusion, and brain infarction volumes were measured upon sacrifice. Local cortical blood flow (LCBF) was serially measured in a subset of animals receiving EGb 761 (100-200 mg/kg) or vehicle, 0.5 hr and 2 hr after permanent and transient MCA occlusion, respectively. Relative to vehicle-treated controls, rats pretreated with EGb761 (100 and 200 mg/kg) had significantly reduced infarct volumes, by 36% and 49%, respectively, and improved sensory behavior (P < 0.05). Delayed treatment with EGb 761 also significantly reduced brain infarction, by 20-29% and 31%, when given up to 2 and 3 hr following transient and permanent MCA occlusion, respectively, whereas improved neurobehavioral scores were noted up to 2 hr after the onset of MCA occlusion (P < 0.05). LCBF was significantly improved in the ipsilateral cortex following the EGb 761 treatment, whereas a higher dose showed a more sustained effect. In conclusion, EGb 761 protected against transient and permanent focal cerebral ischemia and was effective after a prolonged reperfusion period even when therapy is delayed up to 2 hr. This neuroprotection may be at least partially attributed to the beneficial effects of selectively improved LCBF in the area at risk of infarction.     

Dynamic susceptibility contrast-enhanced perfusion MR imaging at 1.5 T predicts final infarct size in a rat stroke model

The purpose of the present animal experiment was to determine whether source images from dynamic susceptibility contrast-enhanced perfusion weighted imaging (DSC-PWI) at a 1.5T MR scanner, performed early after photochemically induced thrombosis (PIT) of cerebral middle artery (MCA), is feasible to predict final cerebral infarct size in a rat stroke model. Fifteen rats were subjected to PIT of proximal MCA. T2 weighted imaging (T2WI), diffusion-weighted imaging (DWI), and contrast-enhanced PWI were obtained at 1 h and 24 h after MCA occlusion. The relative lesion size (RLS) was defined as lesion volume/brain volume x 100% and measured for MR images, and compared with the final RLS on the gold standard triphenyl tetrazolium chloride (TTC) staining at 24 h. One hour after MCA occlusion, the RLS with DSC-PWI was 24.9 +/- 6.3%, which was significantly larger than 17.6 +/- 4.8% with DWI (P < 0.01). At 24 h, the final RLS on TTC was 24.3 +/- 4.8%, which was comparable to 25.1 +/- 3.5%, 24.6 +/- 3.6% and 27.9 +/- 6.8% with T2WI, DWI and DSC-PWI respectively (P > 0.05). The fact that at 1 h after MCA occlusion only the displayed perfusion deficit was similar to the final infarct size on TTC (P > 0.05) suggests that early source images from DSC-PWI at 1.5T MR scanner is feasible to noninvasively predict the final infarct size in rat models of stroke.     

Protective effects of treadmill training on infarction in rats

This study was undertaken to determine the protective effects of treadmill training on brain ischemic lesions caused by middle cerebral artery (MCA) occlusion in male rats. Rats were divided into four groups: control, 1-week treadmill pre-training, 2-week treadmill pre-training, and 4-week treadmill pre-training. Cerebral infarction was induced by MCA occlusion for 60 min, followed by reperfusion. After 24 h, rats were killed and brain slices were then stained to assess lesion size. Treadmill training at least for 2 weeks can reduce the infarction size and edema caused by MCA occlusion (P<0.01). The present study provides evidence that treadmill training reduces ischemic brain damage in an animal model of cerebral ischemia.     

Expression of basic fibroblast growth factor receptor messenger RNA in the periinfarcted brain tissue

The present study examined whether expression of basic fibroblast growth factor receptor (bFGFR) messenger ribonucleic acid (mRNA) was upregulated by focal ischemia. We have studied the in situ hybridization autoradiography for bFGFR mRNA in the rat model of middle cerebral artery (MCA) occlusion. Male Wistar rats were used for occlusion of the left MCA, and were sacrificed 1, 3, 7 and 14 days after MCA occlusion. In situ hybridization was performed on the brain sections of these animals and sham controls by using 35S-labeled antisense and sense (control) RNA probes for rat bFGFR. Expression of bFGFR mRNA was observed in the periinfarcted area of the rats within 1-14 days after MCA occlusion. Expression was evident in the whole hemisphere of the infarcted side, especially at 1 and 3 days after ischemia, but no expression was detected in the contralateral side. On microautoradiograms, the signals of bFGFR mRNA were detected in both neurons and non-neural cells located in the periinfarcted area. Upregulation of bFGFR mRNA detected in the periinfarcted brain tissue suggests that receptor-mediated action of bFGF may be related to preservation of neurons injured by ischemia.     

Spatial relations of dorsal anastomoses and lesion border after middle cerebral artery occlusion

Occlusion of the middle cerebral artery invariably results in infarction of tissue in stroke-prone spontaneously hypertensive rats (SHRSP). To determine if the lesion border extends beyond the territory of the occluded middle cerebral artery or if the lesion enlarges with time after the occlusion, spatial relations of the lesion and the primary anastomosing collateral branches were investigated. Measurements were made 1 day (n = 8) or 21 days (n = 8) after occlusion in 5-8-week-old SHRSP brains marked by triphenyltetrazolium chloride (TTC) or tissue atrophy. After 1 day of occlusion, the border between TTC-marked and -unmarked tissue was parallel to, and without spatial displacement from, the medial border of infarcted tissue measured 21 days after the occlusion. Thus, the TTC border accurately localizes the medial border of ischemic tissue that progresses to atrophy. The lesion border was 1.16 +/- 0.04 mm downstream from the anastomoses, and the mean distance was not significantly different in frontal, parietal, or rostral occipital regions or between the 2 groups of rats. Thus, a small but significant amount of tissue between the anastomoses and the lesion border was protected against infarction after middle cerebral artery occlusion in SHRSP. After 21 days of occlusion, the dorsal anastomoses were enlarged, bilaterally symmetric in position but not size, and without displacement from the anastomoses in 1-Day rats. Large-diameter anastomoses were further from the lesion than small-diameter anastomoses in both groups of rats, thus indicating that protection is greater near large anastomoses than near small ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

An anatomical and pathological evaluation of middle cerebral artery occlusion in rats

Adult male Sprague-Dawley rats (n = 87) weighing 350-400 g were used for studying the anatomy of the horizontal segment of middle cerebral artery and infarct area after occlusion of the artery. In the experimental group (n = 27) middle cerebral artery was coagulated 3-4 mm length from the origin of the lateral striate arteries to the inferior cerebral vein and divided. Control rats (n = 20) had all the surgical procedures except occlusion. Another group of rats (n = 40) were used to determine the anatomical variations of middle cerebral artery after intracarotid carbon black injection. Five major patterns of middle cerebral artery were observed and two of them were major and constituted 92.5% of rats. Twenty-four hours after middle cerebral artery occlusion, all animals were neurologically evaluated. On the third day after occlusion the brains were stained with 2%, 2,3,5-triphenyltetrozolium chloride. The area of infarction was assessed by computerized analysis method. In our study after determining the variations of the middle cerebral artery and its branches in our strain of rats, we were able to achieve 92.5% grade III and IV infarcted area.     

Neurological deficit and cerebral infarction after temporary middle cerebral artery occlusion in unanesthetized cats

Forty-four unanesthetized cats underwent temporary middle cerebral artery (MCA) occlusion with an implanted, externally controlled balloon cuff occluder. The occlusion was reversed to allow reperfusion of the MCA after 2 min to 24 hr of ischemia. Fourteen cats had temporary occlusions lasting 2 min to 3 hr; their neurological deficits improved or resolved after reperfusion, and brain sections showed only scattered microscopic areas of necrosis. After a 4-hr occlusion, five of nine cats (55%) recovered completely within 24 hr; two had persistent deficit when sacrificed, 10 days later, and each had a circumscribed infarct. All 18 cats undergoing 5-, 6-, 8-, and 24-hr occlusions sustained permanent neurological deficits. Three 3-hr occlusions at 2-day intervals in three cats resulted in permanent deficits and infarcts that were 25% larger than those after single 8-hr occlusions. Ten cats underwent permanent MCA occlusion; three deteriorated neurologically and died, and the survivors showed no improvement. Infarcts after 5-, 6-, and 8-hr occlusions followed by reperfusion were 66% smaller (p less than 0.05) than those after permanent occlusion; reperfusion after 24 hr of occlusion did not reduce infarct size. Hemorrhagic infarction occurred after two permanent occlusions, but after only one 5-hr temporary occlusion. The results obtained with this method of temporary regional ischemia indicate that restoration of flow after 1-8 hr, but not after 24 hr, of MCA occlusion resulted in less severe neurological deficit and smaller infarcts than did permanent occlusion. The infarct size correlated with the duration of MCA occlusion (p less than 0.05) rather than with the degree of deficit during occlusion.     

Reproducibility of cerebral cortical infarction in the wistar rat after middle cerebral artery occlusion

Although middle cerebral artery (MCA) occlusion in the rat is often used to study focal cerebral ischemia, the model of ischemia affects the size and reproducibility of infarction. The purpose of this experiment was to methodically examine different preparations to determine the optimum focal cerebral ischemia model to produce a reproducible severe ischemic injury. Eighty-two Wistar rats underwent either 1 hour, 3 hour, or permanent MCA occlusion combined with no, unilateral, or bilateral common carotid artery artery (CCA) occlusion. Three days after ischemia, the animals were prepared for tetrazolium chloride assessment of infarction size. One-hour MCA occlusion produced a coefficient of variation (CV) of 200% with an infarction volume of 20.3+/-10.5 mm(3). Adding unilateral or bilateral CCA occlusion resulted in a CV of 134% and 101%, respectively. Three-hour MCA occlusion combined with bilateral CCA occlusion decreased the CV to 58% with a cortical infarction volume of 82.6+/-12.1 mm(3), P<05, compared with 1-hour MCA occlusion with or without CCA occlusion. Permanent MCA occlusion combined with 3 hours of bilateral CCA occlusion resulted in a CV of 47% with a cortical infarction volume of 89.6+/-16.0 mm(3). These results indicate that 3-hour MCA occlusion combined with bilateral CCA occlusion provide consistently a large infarction volume after temporary focal cerebral ischemia.     

Limitations of tetrazolium salts in delineating infarcted brain

Summary Tetrazolium salts, histochemical indicators of mitochondrial respiratory enzymes, have been used by some pathologists to detect infarcts in myocardium. We explored the utility of this technique in detecting experimental brain infarcts and report our findings. Infarcts were produced in cats, gerbils, and rats by unilateral temporal and permanent cerebral vessel occlusion. After various time periods the animals were killed, and their brains were reacted with 2,3,5, triphenyl, 2H-tetrazolium chloride (TTC). The experimental and contralateral hemispheres were examined by light and electron microscopy. The TTC-stained tissue was correlated with histology. In some situations the histological condition of the tissue correlated well with the TTC staining results. Brain regions supplied by temporarily occluded vessels and judged infarcted by light and electron microscopy did not stain. In these regions less than 6% of the mitochondria were intact. In brain tissue from animals with permanent vessel occlusion (no reflow) mitochondria were intact despite the fact that other cellular organelles, such as nuclei, were destroyed. TTC stained such mitochondria and as a result could not distinguish infarcted brain in complete ischemia situations (no reflow). Another draw back to this staining procedure was 36 h after infarction macrophages with intact mitochondria would replace damage neurons and be stained. Under ideal conditions though this technique can detect irreversibly damaged brain as early as 2.5 h after artery occlusion.This work was completed under partial support from the following grants: NS-10828-07 from the NINCDS and HL-28152-01, from the Heart and Lung Branch of the United States Public Health Service     

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.     

使用"夹心TTC染色法"可显著提高脑缺血模型中梗死灶的检测效率

目的 优化红四氮唑(TTC)染色法,以提高对脑缺血模型中梗死灶的位置及面积的精确判定程度.方法 采用线栓法建立Balb/c小鼠的大脑中动脉阻塞(MCAO)模型,缺血5h后断头取脑,利用"夹心TTC染色法"对梗死灶进行判定.结果 传统TTC染色仅有单侧脑片染色效果较好而另侧染色效果很差,相比之下,"夹心TTC染色法"染出的脑片整体颜色鲜红、均一,两侧染色程度均十分良好,能够明确判定出梗死灶.结论 优化后的"TTC夹心染色法"适合于梗死灶位置以及面积的精确判定,可显著提高脑缺血模型中梗死灶的检测效率.     

Mexiletine and magnesium independently, but not combined, protect against permanent focal cerebral ischemia in Wistar rats.

The neuroprotective effect of mexiletine (Mex), a potent Na(+) channel blocker which decreases neuronal energy demands and prevents energy depletion during ischemia, was evaluated in Wistar rats subjected to permanent middle cerebral artery (MCA) occlusion. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium (TTC)-stained brain sections. Pretreatment with Mex resulted in a significant infarct volume reduction when administered intraperitoneally, either at the dosage of 50 or 60 mg/kg, 1 hr before MCA occlusion (P < 0.05). Delayed treatment with Mex (50 mg/kg) also had neuroprotective effects when given at 0.5 hr (< 0.05), but not 2-4 hr, after MCA occlusion. Intraarterial administration of MgSO(4) (90 mg/kg), in combination with Mex at 60 mg/kg, showed no additive neuroprotective effect, although each agent independently reduced the MCA occlusion-induced infarction volume (P < 0.05). Our results indicate that a single, acute administration of Mex is neuroprotective against permanent focal cerebral ischemia, but perhaps chronic administration is needed to establish a more effective therapeutic window beyond 0.5 hr. Moreover, the present in vivo data do not favor a combined use of Mg(2+) with Mex for limiting ischemic injury in the brain, since these agents caused cardiopulmonary suppression, which may have led to the loss of the neuroprotective effect of each agent independently.     

Mild focal cerebral ischemia in the rat. The effect of local temperature on infarct size

We aimed at investigating a new model of mild focal cerebral ischemia in rats with repeated, noninvasive magnetic resonance scanning combined with histology. Magnetic resonance imaging yielded information about infarct development enabling us to test the putative growth of the infarct over time. The effect of local temperature at the occlusion site in this model was furthermore tested. Thirty-three Wistar rats were subjected to 30 min of simultaneous common carotid artery and distal middle cerebral artery occlusion or sham treatment. Animals were magnetic resonance-scanned repeatedly between day one and day 14 after surgery, then sacrificed, and paraffin brain sections stained. All animals scanned 24 h after reperfusion showed an area of edema in the affected cortex, which later was identified as an infarct. Animals with a temperature of 33.9 +/- 1.5 degrees C at the MCA site (hypothermic) showed smaller infarcts (14.4 +/- 10 mm3) than animals with normothermic local temperature (36.7 +/- 0.2 degrees C, 57.7 +/- 26.4 mm3). Infarct size was maximal on day 3 after ischemia but decreased as edema subsided. Infarct volumes from histology and magnetic resonance imaging correlated well. The model reproducibly yielded cortical infarcts, which did not grow after edema had subsided. Local temperature had a considerable effect on final infarct size.     

去骨瓣减压术对大鼠局灶性脑缺血保护作用的有效时间窗

目的 :探讨去骨瓣减压术对大鼠局灶性脑缺血保护作用的时间窗。方法 :改良Koizumi法制作脑缺血动物模型 ,然后在不同时间点行去骨瓣减压术 ,观察 7d内体重、神经功能和生存率的变化 ,第 7天时取脑行氯化 2 ,3 ,5 三苯基四氮唑 (TTC)染色 ,计算梗死灶面积和体积。结果 :脑缺血后 6、12、2 4h行去骨瓣减压术可显著缩小梗死体积 ,6、12h减压可显著改善神经功能 ,其中以 6h减压的效果最好。结论 :去骨瓣减压术对大鼠局灶性脑缺血具有保护作用 ,其有效时间窗为脑缺血后 2 4h以内     

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.     

Triphenyltetrazolium chloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion

Triphenyltetrazolium chloride (TTC) was used to delineate ischaemic lesions in the rat brain at various times following middle cerebral artery occlusion. A comparison was made of TTC staining by immersion and perfusion techniques and conventional light microscopy. The lesions were quantified by measuring the ischaemic area at the sections corresponding to 7 mm in front of the AO line (atlas of Konig and Klippel). In animals examined 24 h after middle cerebral artery occlusion (MCAO), the area of infarction was 17.4 +/- 1.3 mm2 on the TTC perfused slices and 17.6 +/- 1.6 mm2 on the TTC immersed slices (mean +/- SEM). By contrast, there was a marked difference between the two TTC methods when tissues were examined at shorter intervals after artery occlusion. In the TTC-perfused animals, there was no significant difference between the mean areas of infarction measured at 5-20 min, 3-4 h, or 24 h post occlusion. Immersion in TTC, however, failed to reveal any consistent ischaemic damage when applied at the earlier post-occlusion times. Conventional histopathology demonstrated minimal lesions at 5-20 min but at 4 h or more the lesions were not significantly different from those demonstrated by TTC perfusion. TTC immersion staining can, thus, only be used as a reliable marker of cerebral ischaemia damage with post-occlusion survival periods of 24 h. TTC perfusion staining gives results not significantly different from histopathology at 4 h or more post-occlusion but at earlier intervals than 24 h it differs significantly from TTC immersion staining.(ABSTRACT TRUNCATED AT 250 WORDS)