Hypothermia decreased the expression of heat shock proteins in neonatal rat model of hypoxic ischemic encephalopathy

Abstract

Hypothermia (HT) is a well-established neuroprotective strategy against neonatal hypoxic ischemic encephalopathy (HIE). The overexpression of heat shock proteins (HSP) has been shown to provide neuroprotection in animal models of stroke. We aimed to investigate the effect of HT on HSP70 and HSP27 expression in a neonatal rat model of HIE. Seven-day-old rat pups were exposed to hypoxia for 90 min to establish the Rice-Vannucci model and were assigned to the following four groups: hypoxic injury (HI)-normothermia (NT, 36 °C), HI-HT (30 °C), sham-NT, and sham-HT. After temperature intervention for 24 h, the mRNA and protein expression of HSP70 and HSP27 were measured. The association between HSP expression and brain injury severity was also evaluated. The brain infarct size was significantly smaller in the HI-HT group than in the HI-NT group. The mRNA and protein expression of both HSPs were significantly greater in the two HI groups, compared to those in the two sham groups. Moreover, among the rat pups subjected to HI, HT significantly reduced the mRNA and protein expression of both HSPs. The mRNA expression level of the HSPs was proportional to the brain injury severity. Post-ischemic HT, i.e., a cold shock attenuated the expression of HSP70 and HSP27 in a neonatal rat model of HIE. Our study suggests that neither HSP70 nor HSP27 expression is involved in the neuroprotective mechanism through which prolonged HT protects against neonatal HIE.

Keywords: Heat shock protein; Hypothermia; Hypoxic ischemic encephalopathy; Newborn infant.

Fig. 1

Trend of rectal temperature in the NT groups (rectangles) and the HT groups (triangles) (*p < 0.001 by Student’s t test)

Fig. 2

The ratio of the ipsilateral infarct area to the contralateral hemispheric area in the four study groups. The size of the infarct was significantly greater in the HI-HT group (triangles) than in the HI-NT group (inverted triangles)

Fig. 3

Protein expression of HSP70 and HSP27 in the ipsilateral hemisphere. a HSP70 expression was significantly greater in both the HI groups, as compared to the sham-NT group that demonstrated little or no HSP70 expression (*p < 0.001). Hypothermia decreased the post-ischemic HSP70 expression in the neonatal rat brain (**p < 0.01). b HSP27 expression was greater in the two HI groups than in the sham-NT groups (*P < 0.001). HSP27 expression was lower in the HI-HT group than in the HI-NT group (†p < 0.05). Constitutional expression of HSP27, which was not influenced by HT, was observed in the sham groups. OD optical density

Fig. 4

The relative mRNA expression levels of HSP70 and HSP27 using real-time PCR of the homogenates of the ipsilateral hemisphere. After HI, the mRNA expression of HSP70 and HSP27 was significantly increased (*p < 0.001). HT significantly decreased the mRNA expression of HSP70 (A) (**p < 0.05) and HSP27 (B) (^† p < 0.01) at 24 h after HI. Data were normalized to GAPDH

Fig. 5

Scatterplot of protein and mRNA expression of HSP70 and HSP27 versus the infarct size in 7-day-old rats treated with either 24 h HT (triangles) or NT (rectangles) after hypoxic ischemic injury. The size of the brain infarct was significantly correlated with the magnitude of mRNA expression of HSP70 (a) and HSP27 (b), but not with their protein expression levels (c and d). Regression lines are only drawn for the HI-NT group. The expression level of HSP was normalized to that of the Sham-NT group