Brain injury and protective effects of hypothermia using triphenyltetrazolium chloride in neonatal rat

Abstract

Colorless 2,3,5-triphenyltetrazolium chloride (TTC) is reduced by enzymes in functioning mitochondria to a red-colored compound, and has been used to differentiate areas of viable tissue from areas of infarction in adult animals. TTC was used to study the central nervous system protective effects of hypothermia on the neonatal rat exposed to hypoxia and ischemia. The effect of hypothermia on survival and weight gain was also determined. Seven-day-old Wistar rats with right carotid artery ligation were exposed to 3 hours of 8% oxygen and maintained at either 37°C (n = 22) or 30°C (n = 18). The survivors were sacrificed 2 days later and brain slices exposed to TTC. These slices were photographed and the percentage of damage to the right brain was estimated gravimetrically from the stained and unstained areas of enlarged images. The mean weight gains were 4.2 ± 1.2 gm in the 30°C group and −1.0 ± 2.8 gm in the 37°C group ( P < .001). The survival in the 37°C group was 77% and in the 30°C group 100% ( P < .025). The mean percentage damage to the right side of the brain in the 37°C group was 45.5% (range: 0–87.5%); there was no detectable damage in any of the 30°C group pups ( P < .0001). In our study, TTC proved to be a rapid and simple method for assessing central nervous system injury in the neonatal rat. This study also confirms that moderate hypothermia is protective against hypoxic-ischemic brain injury.