Validation of a simple and inexpensive method for the quantitation of infarct in the rat brain

Abstract

A gravimetric method was evaluated as a simple, sensitive, reproducible, low-cost alternative to quantify the extent of brain infarct after occlusion of the medial cerebral artery in rats. In ether-anesthetized rats, the left medial cerebral artery was occluded for 1, 1.5 or 2 h by inserting a 4-0 nylon monofilament suture into the internal carotid artery. Twenty-four hours later, the brains were processed for histochemical triphenyltetrazolium chloride (TTC) staining and quantitation of the schemic infarct. In each TTC-stained brain section, the ischemic tissue was dissected with a scalpel and fixed in 10% formalin at 0ºC until its total mass could be estimated. The mass (mg) of the ischemic tissue was weighed on an analytical balance and compared to its volume (mm 3 ), estimated either by plethysmometry using platinum electrodes or by computer-assisted image analysis. Infarct size as measured by the weighing method (mg), and reported as a percent (%) of the affected (left) hemisphere, correlated closely with volume (mm 3 , also reported as %) estimated by computerized image analysis (r = 0.88; P < 0.001; N = 10) or by plethysmography (r = 0.97-0.98; P < 0.0001; N = 41). This degree of correlation was maintained between different experimenters. The method was also sensitive for detecting the effect of different ischemia durations on infarct size (P < 0.005; N = 23), and the effect of drug treatments in reducing the extent of brain damage (P < 0.005; N = 24). The data suggest that, in addition to being simple and low cost, the weighing method is a reliable alternative for quantifying brain infarct in animal models of stroke.

The Ginkgo biloba extract, EGb 761, fails to reduce brain infarct size in rats after transient, middle cerebral artery occlusion in conditions of unprevented, ischemia‐induced fever

“…The possible influence of fever in masking the efficacy of EGb 761 in the present study is similar to our previous observation that the anticonvulsant lamotrigine failed to reduce infarct size when given alone. However, when lamotrigine was combined with dipyrone (given 90 and 30 min before MCAO, respectively) there was a significant reduction in infarct size, and core temperature was maintained normothermic during the intra-ischemic periods (Schilichting et al, 2004). This agrees with previous studies using lamotrigine under normothermia achieved by physical means during the intra-ischemic period (Smith and Meldrum, 1995;Yang et al, 1998).…”

“…The groups receiving dipyrone or a sham operation were the same as those used in the first experiment, since the treatment regimen was not changed between the two experiments. Further, the results observed in these groups reproduced our previous data very well (Schilichting et al, 2004).…”

“…Despite this, however, the neuroprotective effect of EGb 761 reported by others was also obtained under conditions of spontaneous breathing, without monitoring pCO 2 and pH levels (Clark et al, 2001;Ünal et al, 2001). In addition, the anesthesia procedure used here was the same as that used when a neuroprotective effect of lamotrigine plus dipyrone was found (Schilichting et al, 2004), a result which reproduces the neuroprotective effect of lamotrigine observed in rats anesthetized with halothane/O 2 and supported by mechanical ventilation Meldrum, 1995, Yang et al, 1998).…”

Transient Bilateral Common Carotid Artery Occlusion (tBCCAO) of Rats as a Model of Global Cerebral Ischemia

“…The mitochondria in ischemic brain is damaged and LDH exits the cell into the plasma. Damaged cells will be white because of the decline or even disappearance of intracellular LDH enzymes 29,30 The time of observation of ischemic tissue using the TTC method varied greatly, starting from 60 minutes after the induction of ischemia up to 12-24 hours after the induction 31 Rekabi et al (2015) showed that tBCCAO for 30 minutes followed by 1-hour of reperfusion gave rise to infarct areas in TTC staining. The duration of observation of permanent brain damage using the TTC method varied, at the latest two -three hours after the occurrence of ischemia, while other studies reported a period of around 12-24 hours.…”

Transient middle cerebral artery occlusion in rats as an experimental model of brain ischemia

“…The dimension is of the infarct zone have frequently been used as a primary indicator of the extension of cerebral ischemic damage in animal models. The infarction volume can be identified by histochemical methods, such as those employed in the study, through the use of triphenyltretrazolium chloride (TTC) 7,9,22 . There is evidence that when injuries are measured by TTC they appear smaller than with magnetic resonance imaging, probably due to the influence of tissue edema .…”