Elsevier

Brain Research Bulletin

Volume 79, Issue 6, 14 August 2009, Pages 351-357
Brain Research Bulletin

Research report
Post-ischemic hypothermia for 24 h in P7 rats rescues hippocampal neuron: Association with decreased astrocyte activation and inflammatory cytokine expression

https://doi.org/10.1016/j.brainresbull.2009.03.011Get rights and content

Abstract

Hypothermia is an effective method for reducing the neuronal damage induced by hypoxia–ischemia (HI) but the underlying mechanism remains unclear. To investigate the effects of post-HI hypothermia on the developing brain, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2 h. They were divided into a hypothermia group (rectal temperature 32–33 °C for 24 h) and a normothermia group (36–37 °C for 24 h) immediately after hypoxia–ischemia. Animals were sacrificed at 12, 24 and 72 h for gene analysis and 0, 1, 3 and 7 days for protein analysis after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The hypothermia group had more neuronal nuclei (NeuN) positive neurons and lower levels of glial fibrillary acidic protein (GFAP) mRNA and immunoreactivity in the hippocampus CA1 region than the normothermia group. Real-time PCR showed no significant difference in glial cell line-derived neurotrophic factor (GDNF) mRNA expression in the hippocampus in the two groups at various time points after HI. However, GDNF protein level was significantly increased in the hypothermia group. On the other hand, mRNA and protein levels of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were dramatically decreased in the hypothermia compared with the normothermia group. The present findings highlight an apparent association between inhibition of hippocampal neuron loss by hypothermia and decreased astrocytosis and inflammatory cytokine release after hypoxia–ischemia in the developing brain.

Introduction

Neonatal hypoxic–ischemic encephalopathy, caused by perinatal asphyxia, remains an important contributor to perinatal mortality and long-term neurological impairments in term and preterm survivors [13], [21]. The mechanisms underlying hypoxic–ischemic brain damage are only partially understood but are commonly suggested to include excitotoxicity, and induction of secondary apoptosis and inflammation [8]. Previous studies have shown that hypoxia–ischemia (HI) results in a cascade of biochemical events and pathological reactions that lead to neuronal death in the hippocampus CA1 region [9], [25].

Astrocytes are the most numerous cell types in the central nervous system (CNS). They provide structural, metabolic and trophic support to neurons and modulate synaptic activity [39]. There is considerable evidence that astrocyte-conditioned media support the survival of neurons in vitro[12], [18]. Long-term recovery after brain injury, through neurite outgrowth, synaptic plasticity, or neuron regeneration, is influenced by astrocyte surface molecule expression and trophic factors such as glial cell line-derived neurotrophic factor (GDNF). Impairment in these astrocyte functions during HI or other insults can critically influence neuron survival. Moreover, the inevitable consequence of ischemic infarct is glial scar formation, which, in the long-term, prevents axonal growth and impedes recovery [26]. Besides, a previous study has shown that astrocytes are the major source of interleukin-6 (IL-6) in CNS injury and inflammation [16]. Reactive astrocytes release inflammatory cytokines such as tumor necrosis factor α (TNF-α) and IL-6 through a carrier-dependent mechanism, which can result in sustained modulatory action on neighboring neurons [6], [42]. These cytokines are thought to be involved in ischemic encephalopathy, as TNF-α and IL-6 are elevated in infants with hypoxic–ischemic encephalopathy [5], [35].

Hypothermia is effective in reducing brain injury and improving behavioral recovery in animal experiments [27] and is also safe and effective in clinical settings [15], [19]. Previous studies have shown that mild hypothermia preserves neurons and reduces astrocyte proliferation in animal models of lethal hemorrhage, transient forebrain ischemia, and asphyxial cardiac arrest [3], [17], [23]. It has been shown recently that post-ischemic hypothermia reduces IL-18 expression and suppresses microglial activation accompanied by decreased loss of MAP-2 immunoreactivity in the cortex [14]. Nevertheless, it is still unclear if mild hypothermia can attenuate neuron loss in the hippocampus of hypoxic–ischemia injured immature rat brain, and the corresponding changes of astrocyte proliferation and its related inflammatory factors response.

In the present study, we investigated whether hypothermia neuroprotection after hypoxia–ischemia in the neonatal rat was associated with changes in astrocyte activation and expression of astrocyte-associated inflammatory factors.

Section snippets

Animal models

This study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Committee on Ethics in the Care and Use of Laboratory Animals in Shanghai. Sprague–Dawley rats were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences. Neonatal HI was induced in rats at postnatal day 7, according to the method described by Rice et al. [33]. Seven-day-old pups of either sex were

Hypothermia reduced infarct volume and neuron loss after HI in hippocampus CA1 region

Nissl staining (Fig. 2A and B) revealed a significant reduction in the infarct volume of animals treated with hypothermia in comparison with normothermia (Fig. 2G). Histologically, the injured hippocampus appeared to be well preserved (Fig. 2B and b) in animals treated with hypothermia as compared to normothermia. In the latter, the injured hippocampal tissue appeared to be relatively deformed with great cell loss in the CA1 region (Fig. 2a, yellow arrowhead). To assess the effects of

Discussion

In the present study we found that the reduction in volume of infarct and neuronal loss in the hypothermia treated rats brain after hypoxia–ischemia was accompanied by a marked reduction of astrocytosis and of TNF-α and IL-6 mRNA and protein levels in the ipsilateral hippocampus. Contrary to our initial hypothesis there was no increase in GDNF mRNA expression in the hypothermia group up to 72 h after HI compared with normothermia. Although there was a significant increase in GDNF protein at 24

Conflict of interest

There is no conflict of interest about this manuscript.

Acknowledgement

This study was supported by a grant from National Natural Science Foundation of China (No. 30672256).

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