Pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury

Abstract

A previous study from our lab has shown that the polyphenol-rich pomegranate juice can protect the neonatal mouse brain against hypoxic-ischemic (H-I) injury when given to mothers in their drinking water. To test the hypothesis that this protection is due to the polyphenols in the juice, we studied the effects of the pomegranate polyphenol extract in the same neonatal H-I model. To further explore the role of a specific polyphenol in neonatal H-I we investigated the effects of resveratrol. The neuroprotective effects of resveratrol have been demonstrated in adult models of stroke, but had not previously been examined in neonates. We show that pomegranate polyphenols and resveratrol reduce caspase-3 activation following neonatal H-I. Resveratrol reduced caspase-3 activation when given before the injury but not when given 3 h after the injury. In addition to preventing caspase-3 activation, resveratrol also reduced calpain activation. Finally, we show that resveratrol can protect against tissue loss measured at 7 days after the injury. These and other recent findings suggest that polyphenols should be further investigated as a potential treatment to decrease brain injury due to neonatal H-I.

Figure 1. Pomegranate polyphenol extract protects against caspase-3 activation following neonatal H-I

Female mice had their drinking water replaced with either sugar water (vehicle, VEH) or sugar water containing the pomegranate polyphenol extract (PPE) during pregnancy and following delivery. Pups from these dams were subjected to neonatal H-I at P7 and caspase-3 activity was measured in the left (L) and right (R) hippocampus at 24 hours following the injury by DEVD cleavage assay. Pups of dams drinking PPE (n=25) had significantly less caspase-3 activation in the left hippocampus than pups of dams drinking sugar water (VEH, n=21). ** = p = 0.0024 comparing PPE vs. vehicle-treated mice.

Figure 2. Resveratrol protects the neonatal brain against H-I induced caspase-3 activation in a time and dose dependent manner

At different time points pre- or post-injury, littermate mouse pups were injected interperitoneally with vehicle (DMSO) or resveratrol at high (20 mg/kg), medium (200 μg/kg) or low (2 μg/kg) doses. Caspase-3 activation was measured as DEVD cleavage activity in the injured hippocampus at 24 hours following neonatal H-I injury given at P7. The number of animals in each treatment group is indicated inside each bar in the graph: N=55 pups were given either resveratrol or vehicle at 24 hours pre-injury, N=74 pups were given resveratrol or vehicle 10 minutes before injury, and N=64 mice were treated with resveratrol or vehicle 3 hours post injury. There is no difference in DEVD cleavage activity in the non-injured hippocampus, data not shown for clarification. If injected at 24 hours pre-hypoxia, resveratrol protected against caspase-3 activation in the hippocampus in a dose dependent manner, when compared to littermate mice injected with vehicle (A). The high and the medium dose of resveratrol significantly reduce the amount of caspase-3 activation while the low dose of resveratrol did not provide significant protection. Similarly, if injected 10 minutes before hypoxia resveratrol reduces caspase-3 activation at high and medium doses, but not at the low dose (B). However, when injected at 3 hours post injury, resveratrol does not reduce caspase-3 activity at any of the doses utilized (C). * = p < 0.05 and ** = p < 0.01 resveratrol vs. vehicle injected littermates.

Figure 3. Resveratrol reduces caspase-3 and calpain cleavage of spectrin following neonatal H-I

Spectrin cleavage products were investigated at 24 hours after injury in mice injected with 20 mg/kg resveratrol or vehicle at 10 minutes before exposure to hypoxia and after carotid ligation. Pooled lysates from 4 different animals were run in each lane. Injured (left, L) hippocampus or non-injured (right, R) were run side by side on SDS PAGE and probed with antibodies against spectrin. Spectrin is cleaved by calpain to give rise to a p150 and a p145 band and by caspase-3 to give rise to a p120 band. These bands are strongly present in lysates from vehicle injected mice but to a much lesser extent in lysates from resveratrol injected mice. Relative intensity of the p150 and p145 bands was calculated by comparison to the intensity of the tubulin loading control. The intensities of the p150 and p145 bands were significantly reduced in the mice that had received resveratrol injection, suggesting that calpain activation is prevented by resveratrol. *** = p < 0.001 resveratrol vs. vehicle by t-test.

Figure 4. Resveratrol decreases the amount of tissue loss at P14

P7 mice were injected with resveratrol or vehicle following carotid ligation and 10 minutes before hypoxia and tissue loss was assessed at 7 days post injury. This is a good measure of overall brain injury following neonatal H-I. Percentage tissue loss was determined by comparing the area of the brain region in the left and right hemisphere through several serial coronal brain sections as described in the methods section. When compared to mice injected with vehicle, mice receiving resveratrol were significantly protected against tissue loss in the hippocampus and striatum. Resveratrol also appears to protect the cortex but this protection is not statistically significant (p= 0.1). ** = p < 0.01 and *** = p < 0.001 resveratrol vs. vehicle injected littermates.

Figure 5. Resveratrol protects the neonatal rat brain against caspase-3 activation following H-I injury

Rats underwent neonatal H-I at P7 and were injected with 20 mg/kg resveratrol or vehicle following carotid ligation and 10 minutes before hypoxia (A) or 3 hours after hypoxia (B). DEVD cleavage activity was measured at 24 hours post injury in hippocampal lysates. As seen in the mice, rats that received resveratrol before injury are protected against caspase-3 activation following neonatal H-I (A). Due to a higher incidence of no detectable caspase-3 activation following neonatal H-I in rats, a larger number of rats were enrolled in this study to determine whether there was a statistical difference between resveratrol (n=18) and vehicle (n=16) treated rats.* = p < 0.05 resveratrol vs. vehicle injected littermates. When administered 3 hours after hypoxia, there was no significant difference in tissue loss between rats that received resveratrol (n=19) vs. vehicle (n=18) (B).