Effect of Trp53 gene deficiency on brain injury after neonatal hypoxia-ischemia

Abstract

Hypoxia-ischemia (HI) can result in permanent life-long injuries such as motor and cognitive deficits. In response to cellular stressors such as hypoxia, tumor suppressor protein p53 is activated, potently initiating apoptosis and promoting Bax-dependent mitochondrial outer membrane permeabilization. The aim of this study was to investigate the effect of Trp53 genetic inhibition on injury development in the immature brain following HI. HI (50 min or 60 min) was induced at postnatal day 9 (PND9) in Trp53 heterozygote (het) and wild type (WT) mice. Utilizing Cre-LoxP technology, CaMK2α-Cre mice were bred with Trp53-Lox mice, resulting in knockdown of Trp53 in CaMK2α neurons. HI was induced at PND12 (50 min) and PND28 (40 min). Extent of brain injury was assessed 7 days following HI. Following 50 min HI at PND9, Trp53 het mice showed protection in the posterior hippocampus and thalamus. No difference was seen between WT or Trp53 het mice following a severe, 60 min HI. Cre-Lox mice that were subjected to HI at PND12 showed no difference in injury, however we determined that neuronal specific CaMK2α-Cre recombinase activity was strongly expressed by PND28. Concomitantly, Trp53 was reduced at 6 weeks of age in KO-Lox Trp53 mice. Cre-Lox mice subjected to HI at PND28 showed no significant difference in brain injury. These data suggest that p53 has a limited contribution to the development of injury in the immature/juvenile brain following HI. Further studies are required to determine the effect of p53 on downstream targets.

Keywords: brain injury; cell death; hypoxia-ischemia; mitochondria; p53.

Figure 1. Brain injury assessment of Trp53 WT (+/+) and Het (+/−) mice 7 days following 50 min HI at PND 9

MAP-2 tissue loss A. volume tissue loss B. MBP loss C. and neuropathology scores D. were assessed at 8 levels of the brain in Trp53 WT (black bars; n=21) and Trp53 het (grey bars; n=16). MAP-2 stained images at posterior levels of the brain from Trp53 WT +/+ E(i). and Trp53 het +/− E(ii). Mean ± SEM. * p< 0.05, ** p < 0.01, **** p<0.0001. Cortex (Cx), Hippocampus (Hipp), posterior hippocampus (P. Hipp), Striatum (Stri), Thalamus (Thal). Scale bar represents 2 mm.

Figure 2. Brain injury assessment of Trp53 WT (+/+) and Het (+/−) mice 7 days following 60 min HI at P9

MAP-2 tissue loss A. volume tissue loss B. and MBP loss C. were assessed at 8 levels of the brain in Trp53 WT (black bars; n=20) and Trp53 het (grey bars; n=29). Mean ± SEM.

Figure 3. β-gal expression in the brains of CaMK2α Cre - Rosa26 reporter mice

CaMK2α Cre mice were bred with ROSA26 reporter mice; brains were collected at postnatal day (PND) 7 A. PND 14 B. PND 19 C. and PND29 D. B-gal staining is representative of cre-recombinase activity, which occurs from PND 19. Scale bar represents 1000 μm.

Figure 4. Trp53 in situ hybridization on brains from WT Lox Trp53^+/+, KO Lox Trp53^f/+ and Trp53 KO

In situ hybridization of Trp53 in WT Lox Trp53^+/+ A-D. KO Lox Trp53^f/+ E-H. and traditional Trp53 KO I, J. at postnatal day (PND) 7 A, E. PND 12 B, F. 6 weeks of age C, G. and [higher magnification D, H.]. Traditional Trp53 KO I. higher magnification J. shows no Trp53 staining. Scale bars represent 500 μm.

Figure 5. Brain injury assessment of WT Lox Trp53^+/+ and KO Lox Trp53^f/+ mice 7 days following 40 min HI at PND 28

MAP-2 tissue loss A. volume tissue loss B. MBP loss C. and neuropathology score D. were assessed in WT Lox Trp53^+/+ (black bars; n=8) and KO Lox Trp53^f/+ (grey bars; n=16). Mean ± SEM. Cortex (Cx), Hippocampus (Hipp), Striatum (Stri), Thalamus (Thal).