Validation of a simple and inexpensive method for the quantitation of infarct in the rat brain (original) (raw)
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Morphometric evaluation of brain infarcts in rats and gerbils
Journal of Pharmacological Methods, 1986
The Levine rat prepartion, the gerbil stroke model, and appropriate control animals were used to determine if the 2,3,5-triphenyltetrazolium chloride (lTC) would selectively identify noninfarcted versus infarcted cerebral tissue. The lTC is frequently used to quantify infarcted myocardial tissue and has been shown to have great specificity, reproducibility, and efficacy. The TTC produces a red product upon reaction with the respiratory enzymes (dehydrogenases) present in noninfarcted tissues. Irreversibly damaged tissues, lacking dehydrogenases, do not form red reaction products. Six gerbil brains and seven rat brains were incubated with the lTC, and the unreacted areas were macroscopically identified. The brains were fixed and sectioned for routine hematoxylin and eosin staining to determine the specificity of the lTC. The TTC was found to react selectively only with noninfarcted cerebral tissue. The gross brain sections were evaluated by macroscopic morphometric analysis, and the unreacted area was always ipsilateral to ligation and correlated with histologic identification of infarct. The brains from neurologically intact animals demonstrated neither macroscopic nor histological evidence of infarction. This technique allows macroscopic quantification of infarct size by planimetry. The average area of infarct for the neurologically impaired rats was 34.7% and it was 31.4% for the impaired gerbils. The percentage of surface area of each infarcted slice was found to correlate with the severity of the neurologic deficit. We conclude that the TTC staining is effective for macroscopically delineating cerebral infarcts in rats and gerbils, thus permitting quantification of infarct size.
Neuroscience Letters, 2008
The mouse model of transcranial permanent occlusion of the middle cerebral artery (tpMCAO) is widely used in stroke research. Here we quantified infarct size using a conventional histological method at several post-ischaemic times, going beyond the commonly analysed period of up to 2 days, following artery occlusion. Two different mouse strains, which are widely used for pharmacological studies of neuroprotection and for genetic engineering, were used. A drill whole was made into the skull of anaesthetised mice and ischaemia was induced by electrocoagulation of the middle cerebral artery. In both mouse strains tested (C57Black/6 and NMRI), the measured infarct volumes decreased significantly during the first days after tpMCAO. Notably, 13 days after surgery, ischaemic and sham-operated animals had indistinguishably small lesions, which where in the range of only 5% of the infarct size on day 2 post-ischaemia. The standard method of calculating oedema and shrinkage correction provided no sufficient explanation for this significant decrease in infarct volume. There was, however, evidence that structural changes in the residual ipsilateral hemisphere may compromise the significance of results arising from the method of calculating oedema and shrinkage correction. In conclusion, our study indicates that the pronounced and fast, time-dependent decrease in histologically defined infarct volume can compromise results when studying the lasting neuroprotective effects of potential drugs.
Translational stroke research, 2015
Evaluating infarct volume is the primary outcome for experimental ischemic stroke studies and is a major factor in determining translation of a drug into clinical trials. Numerous algorithms are available for evaluating this critical value, but a major limitation of current algorithms is that brain swelling is not appropriately considered. The model by Lin et al. is widely used, but overestimates swelling within the infarction, yielding infarct volumes which do not reflect the true infarct size. Herein, a new infarct volume algorithm is developed to minimize the effects of both peri-infarct and infarct core swelling on infarct volume measurement. 2,3,5-Triphenyl-2H-tetrazolium chloride-stained brain tissue of adult rats subjected to middle cerebral artery occlusion was used for infarct volume analysis. When both peri-infarct swelling and infarction core swelling are removed from infarct volume calculations, such as accomplished by our algorithm, larger infarct volumes are estimated ...
Standardization of the Simple Methodology for Experimentally Induced Ischemic Stroke in Rat Models
scienceline publication, 2023
Stroke is a globally significant and devastating disease that requires prompt treatment. Animal models are commonly used to investigate stroke therapy, often through experimentally induced ischemic stroke (EIIS). However, challenges arise in implementing EIIS in animal models. The current study aimed to present a simple EIIS methodology for animal models. A total of 60 male Sprague-Dawley rats were randomly divided into five groups, namely Group 1 (sham-operated), Groups 2 to 5 (EIIS groups) with different duration of common carotid artery (CCA) ligation, including 1, 2, 4, and 8 hours, respectively. The ligation was performed on the CCA and its branches. Before the experiment, the rats were anesthetized, and the incision area was shaved and disinfected. The sagittal ventral midline was incised, with neck muscles retracted to expose the right CCA. The occlusion was performed on three sides of a carotid artery (common, external, and internal) using a simple interrupted suture. The occlusion of blood flow using ligation was performed at different times depending on the groups. After that, the CCA ligations were re-perfused by cutting the suture knot. The brain and blood were collected on days 1 and 7 after reperfusion. The results indicated that 4 and 8 hours of CCA ligation significantly impacted the general condition and neuro-deficit score. Moreover, 4 and 8 hours of CCA ligation could induce ischemic stroke by its capacity to cause infarction within the brain parenchyma and increase the platelet-to-white blood cell ratio, C-reactive protein, and De Ritis ratio. In contrast, 1 and 2 hours of CCA ligation did not significantly affect the observed parameters. It can be concluded that the EIIS using 4 and 8 hours of CCA ligation can be applied to induce ischemic stroke in rat models with consistent impacts on general conditions, neuro-deficit, hematology, and serology.
Identification of Ischemic Regions in a Rat Model of Stroke
PLoS ONE, 2009
Background: Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia.
Acta neurochirurgica. Supplement, 2016
The primary measure for experimental stroke studies, infarct volume, can be affected by brain swelling. The algorithm by Lin et al. was developed to correct for brain swelling, however, the correction is not adequate. This chapter presents a new infarct volume algorithm that more appropriately corrects for brain hemisphere volume changes (swelling and stunted growth). Fifty-one adult rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO). Forty-four P10 rat pups were sacrificed 48 h after hypoxia-ischemia (HI). Infarct volumes for 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) stained brains were calculated using our algorithm and that of Lin and colleagues. For MCAO animals, the algorithm of Lin et al. computed smaller infarct volumes than those of our algorithm. For HI animals, Lin et al.'s algorithm's infarct volumes were greater than those of our algorithm. For sham animals, Lin et al.'s algorithm's computed infarct volumes were significantly diff...
International Journal of Stroke, 2012
Background Strokes have both ischemic and hemorrhagic components, but most studies of experimental stroke only address the ischemic component. This is likely because investigations of hemorrhagic transformation are hindered by the lack of methods based on unbiased principles for volume estimation. Aims We evaluated different methods for estimating the volume of infarcts, hemorrhages, after embolic middle cerebral artery occlusion with or without thrombolysis. Methods An experimental thromboembolytic rat model was used in this study. The rats underwent surgery and were placed in two groups. Group 1 was treated with saline, and group 2 was treated with 20 mg/kg recombinant tissue plasminogen activator to promote intracerebral hemorrhages. Stereology, semiautomated computer estimation, and manual erythrocyte counting were used to test the precision and efficiency of determining the size of the infarct and intracerebral hemorrhage. Results No differences were observed in the infarct volume or amount of bleeding when comparing the three methods of volume estimation. Although semiautomated computer estimation and manual erythrocyte counting provided similar results as the stereological measurements, the stereological method was the most efficient and advantageous. Conclusions We found that stereology was the superior method for quantification of hemorrhagic volume, especially for rodent petechial bleeding, which is otherwise difficult to measure. Our results suggest the possibility of measuring both the ischemic and the hemorrhagic components of stroke, two parameters that may be differentially regulated when therapeutic regimens are tested.
MRI tissue characterization of experimental cerebral ischemia in rat
Journal of Magnetic Resonance Imaging, 2003
To extend the ISODATA image segmentation method to characterize tissue damage in stroke, by generating an MRI score for each tissue that corresponds to its histological damage. After preprocessing and segmentation (using ISODATA clustering), the proposed method scores tissue regions between 1 and 100. Score 1 is assigned to normal brain matter (white or gray matter), and score 100 to cerebrospinal fluid (CSF). Lesion zones are assigned a score based on their relative levels of similarities to normal brain matter and CSF. To evaluate the method, 15 rats were imaged by a 7T MRI system at one of three time points (acute, subacute, chronic) after MCA occlusion. Then they were killed and their brains were sliced and prepared for histological studies. MRI of two or three slices of each rat brain (using two DWI (b = 400, b = 800), one PDWI, one T2WI, and one T1WI) was performed, and an MRI score between 1 and 100 was determined for each region. Segmented regions were mapped onto the histology images and scored on a scale of 1-10 by an experienced pathologist. The MRI scores were validated by comparison with histology scores. To this end, correlation coefficients between the two scores (MRI and histology) were determined. Experimental results showed excellent correlations between MRI and histology scores at different time points. Depending on the reference tissue (gray matter or white matter) used in the standardization, the correlation coefficients ranged from 0.73 (P < 0.0001) to 0.78 (P < 0.0001) using the entire dataset, including acute, subacute, and chronic time points. This suggests that the proposed multiparametric approach accurately identified and characterized ischemic tissue in a rat model of cerebral ischemia at different stages of stroke evolution. The proposed approach scores tissue regions and characterizes them using unsupervised clustering and multiparametric image analysis techniques. The method can be used for a variety of applications in the field of computer-aided diagnosis and treatment, including evaluation of response to treatment. For example, volume changes for different zones of the lesion over time (e.g., tissue recovery) can be evaluated.
Morphometric study of focal cerebral ischemia in rats: a stereological evaluation
Brain Research, 1995
An efficient, unbiased stereological method for estimating and evaluating volumetric parameters of cortical infarction is presented. Long-Evans rats were subjected to a standard procedure for achieving focal cerebral ischemia, consisting of permanent ligation of the right middle cerebral artery and temporary occlusion of both common carotid arteries. Animals were killed 24 h later by perfusion fixation and the brains were embedded in celloidin, serially sectioned at 60/zm and stained with Cresyl violet. All cases showed identifiable areas of infarction of variable extent within the territory of the right middle cerebral artery. Involvement of allocortical or subcortical structures was variable and in three cases there were small foci of infarct in the contralateral hemisphere. The absolute volumes of the infarcted tissue and of the spared cortex of both hemispheres were obtained by means of an unbiased estimator of volume based on Cavalieri's principle. The best estimate of the actual amount of infarcted cortex was found to be given by the volume ratio between the spared cortex of the right (infarcted) hemisphere and the total cortex of the left (control) hemisphere. This approach avoided the error introduced by the accompanying edema and decreased the observed interanimal variance. Sample size predictions for l~herapeutic-pharmacological trials show that relatively few animals would be needed using the model of ischemia and quantitative morphometry presented here to detect a 30-40% change in infarct volume.
Journal of Applied Animal Research, 2008
Kheradpezhouh, E., Miri, R., Noorafshan, A., Panjehshahin, M.R. and Mehrabani, D. 2008. A new method of brain staining with triphenyltetrazolium chloride to determine the infarct size in rats. J. Appl. Anim. Res., 33: 149-152. To show the accuracy of 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining method, in a case-control study, 10 rats were divided into two groups, an ischemia-induced (experimental) and a control group. The brains of both groups were stained with the new method. The complete staining of the brain of all rats in the control group confirmed the efficacy of this method. Besides, in the experimental group, the method distinguished between the viable and dead tissues and could demonstrate the borders and the size of the infarction too.