Intraventricular ascorbic acid administration decreases hypoxic-ischemic brain injury in newborn rats.

Neuronal cell damage following hypoxic-ischemic (HI) brain injury is partly caused by production of free radicals and reactive oxygen species (ROS). Ascorbic acid (AA) is a potent antioxidant, which scavenges various types of ROS. Some studies have shown that it is neuroprotective, however, the issue is still controversial. In this study, we examined the effect of intraventricular AA administration on immature HI brain using the Rice-Vannucci model. After unilateral carotid artery ligation under isoflurane anesthesia, 7-day-old rat pups received varying concentrations of AA (0.04, 0.2, 1 and 5 mg/kg) by intraventricular injection and were exposed to 8% oxygen for 90 min. Vehicle controls received an equal volume of phosphate saline buffer. We assessed the neuroprotective effect of AA at 7 days post-HI. The percent brain damage measured by comparing the wet weight of the ligated side of hemisphere with that of contralateral one was reduced in both 1 and 5 mg/kg groups but not in either 0.04 or 0.2 mg/kg groups compared to vehicle controls (5 mg/kg 16.0 +/- 4.3%, 1 mg/kg 10.9 +/- 5.0%, vs. controls 36.7 +/- 3.6%, P < 0.05). Macroscopic evaluation of brain injury revealed the neuroprotective effect of AA in both 1 and 5 mg/kg groups (5 mg/kg 1.1 +/- 0.4, 1 mg/kg 0.4 +/- 0.3, vs. controls 2.9 +/- 0.3, P < 0.05). Western blots of fodrin on the ligated side also showed that AA significantly suppressed 150/145-kDa bands of fodrin breakdown products, which suggested that AA suppressed activation of calpain. Neuropathological quantitative analysis of cell death revealed that 1 mg/kg of AA injection significantly reduced the number of necrotic cells in cortex, caudate putamen, thalamus and hippocampus CA1, whereas that of apoptotic cells was only reduced in cortex. These findings show that intraventricular AA injection is neuroprotective after HI in immature rats.

Calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis.

MDL 28170 is a CNS-penetrating calpain inhibitor, and we examined the effects of MDL 28170 on hypoxic-ischemic brain injury in immature brain using the Rice-Vannucci model. Immediately after hypoxic exposure, 24 mg/kg of MDL 28170 was injected intraperitoneally as an initial dose, followed by 12 mg/kg every 4 h for a total dose of 60 mg/kg over 12 h post-HI. A vehicle control group received peanut oil injection instead. Macroscopic evaluation of brain injury revealed the neuroprotective effect of MDL 28170 after 12 h post-HI. Neuropathological quantitative analysis of cell death showed that MDL 28170 significantly decreased the number of necrotic cells in all the examined regions except for cingular cortex, and the number of apoptotic cells in caudate putamen, parietal cortex, hippocampus CA1, and laterodorsal thalamus. Western blots showed that MDL 28170 suppressed 145/150 kDa subunits of alpha-spectrin breakdown products (SBDP) in cortex, hippocampus, thalamus, and striatum, and also 120-kDa subunit of SBDP in all regions except for striatum. This suggests that MDL 28170 inhibited activation of calpain and caspase-3, respectively. Our results indicate that post-hypoxic MDL 28170 injection is neuroprotective in HI newborn rat brain by decreasing both necrosis and apoptosis. SBDP expression also suggests that MDL 28170 injection inhibits both calpain and caspase-3 activation after HI insult.

Prolonged hypothermia protects neonatal rat brain against hypoxic-ischemia by reducing both apoptosis and necrosis.

Although hypothermia is an effective treatment for perinatal cerebral hypoxic-ischemic (HI) injury, it remains unclear how long and how deep we need to maintain hypothermia to obtain maximum neuroprotection. We examined effects of prolonged hypothermia on HI immature rat brain and its protective mechanisms using the Rice-Vannucci model. Immediately after the end of hypoxic exposure, the pups divided into a hypothermia group (30 degrees C) and a normothermia one (37 degrees C). Rectal temperature was maintained until they were sacrificed at each time point before 72h post HI. Prolonged hypothermia significantly reduced macroscopic brain injury compared with normothermia group. Quantitative analysis of cell death using H&E-stained sections revealed the number of both apoptotic and necrotic cells was significantly reduced by hypothermia after 24h post HI. Hypothermia seemed to decrease the number of TUNEL-positive cells. Immunohistochemistry and Western blot showed that prolonged hypothermia suppressed cytochrome c release from mitochondria to cytosol and activation of both caspase-3 and calpain in cortex, hippocampus, thalamus and striatum throughout the experiment. These results showed that prolonged hypothermia significantly reduced neonatal brain injury even when it was started after HI insult. Our results suggest that prolonged hypothermia protects neonatal brain after HI by reducing both apoptosis and necrosis.

Ascorbic acid protects the newborn rat brain from hypoxic-ischemia.

Ascorbic acid (AA) is a potent antioxidant, and its neuroprotective effect has not been established yet. Using the Rice-Vannucci model, we examined the effect of AA on hypoxic-ischemic (HI) injury in the immature rat brain. Under isoflurane anesthesia, 7-day-old rat pups received 750 mg/kg of AA by intraperitoneal injection just before hypoxic exposure; 8% oxygen for 90 min. Vehicle controls received an equal volume of saline. AA decreased a macroscopic brain injury score at 48 and 168 h post-HI compared with vehicle controls (48 h post-HI, AA 1.38+/-0.45 vs. controls 2.94+/-0.24, p<0.05; 168 h post-HI, 1.13+/-0.44 vs. 2.50+/-0.25, p<0.05). AA injection significantly decreased the number of both necrotic and apoptotic cells in cortex, caudate putamen, thalamus and hippocampus, and also seemed to reduce the number of TUNEL-positive cells. Western blot analysis showed that AA significantly suppressed 150/145 kDa subunits of alpha-fodrin breakdown products (FBDP) in cortex, striatum, thalamus and hippocampus at 24 and 48 h post-HI, and also 120 kDa subunit of FBDP in all examined regions except for thalamus, which indicated that AA injection inhibited both calpain and caspase-3 activation. Western blot analysis of nitrotyrosine failed to show inhibition of free radical production by AA, however, our results show that AA inhibits both necrotic and apoptotic cell death and that AA is neuroprotective after HI in immature rat brain.