Research report
Experimental subarachnoid hemorrhage induces changes in the levels of hippocampal NMDA receptor subunit mRNA

https://doi.org/10.1016/j.molbrainres.2005.02.023Get rights and content

Abstract

NMDA receptors may play a crucial role in nerve cell death following subarachnoid hemorrhage (SAH). Changes in NMDA receptor-mediated transmission appear before neuronal death in rodent models of transient ischemia, and NMDA receptor function is known to be dependent on subunit composition. Here, we have investigated whether mRNA expression of the NMDA receptor subunits is altered in the hippocampal formation 3–5 h following experimental SAH, and correlated these early alterations to subsequent delayed cell death. SAH was induced by intraluminal perforation of the internal carotid artery intracranially, and cerebral blood flow (CBF) was bilaterally monitored by laser-Doppler flowmetry. Early changes in NMDA receptor subunit mRNA and early nerve cell death were analyzed at 3–5 h after SAH, and delayed nerve cell death was analyzed at 2–7 days after SAH. Duration of ipsilateral CBF reduction below 30% of baseline (CBF30) was predictive of ipsilateral delayed nerve cell death in the CA1 2–7 days after SAH. At CBF30 > 9 min, we found downregulation of mRNA for NR2A, NR2B, and NR3B at 3–5 h after SAH, whereas the levels of NR1 mRNA were unaffected. The downregulation of NR2A and NR2B mRNA may result in a reduced NMDA receptor function. Such reduction may be sufficient to provide neuroprotection in the dentate gyrus, where no cell death appears, but insufficient to rescue neurons in the hippocampus proper following SAH.

Introduction

Subarachnoid hemorrhage (SAH) represents 5–10% of all stroke incidents, and is a major cause of permanent disability and death in adult humans [38]. In rodents, SAH leads to nerve cell death in the brain, including the hippocampal formation [18], [22], [29], [39], which is especially vulnerable to ischemia [17], [30]. In fact, SAH results in a decrease in cerebral blood flow (CBF) in the acute phase [2], [29], [39].

After SAH, there is a massive release of glutamate [3], [28], [31] which may cause an excessive Ca2+ influx through NMDA receptors resulting in cell death. However, it is not known whether SAH produces alterations in the NMDA receptors. Nor is it known whether the expression of individual NMDA receptor subunits is affected by SAH. There are seven different types of NMDA receptor subunits, NR1, NR2A–D, and NR3A–B [1], [25]. Both glutamate and glycine are required to activate NMDA receptors; the glycine site resides on the NR1 subunit and the glutamate site on the NR2 subunits [19]. Hence, both NR1 and NR2 subunits are required to achieve functional NMDA receptors. The NR3 subunits confer a reduced Ca2+ influx when coexpressed with NR1 and NR2 subunits in vitro [5], [6], [20], [21], [25], [35], but their regulation or functional role in vivo is little known [8]. Hence, the relative expression of these subunits may affect the functional properties of the NMDA receptors and thereby enhance or diminish SAH-induced cell death.

In the present work, we have investigated whether experimental SAH causes changes in the expression of NMDA receptor subunits and how such changes correlate to subsequent cell death in the hippocampal formation.

Section snippets

Induction of SAH

All animal treatments were approved by the Northern Committee for Animal Experimentation, Stockholm, Sweden. Thirty-three male Sprague–Dawley rats (B & K Universal AB, Sollentuna, Sweden), weighing 300–400 g, were endotracheally intubated and artificially ventilated with 70% N2O and 30% O2. Surgery was performed under 1% halothane (Fluothane®, Zeneca, Gothenburg, Sweden) and body temperature was maintained at 37.5 ± 0.5 °C. The tail artery was cannulated to obtain blood samples and to monitor

Changes in CBF after SAH

The reduction in CBF induced by the vascular rupture varied greatly and it was generally more pronounced on the ipsilateral side (Fig. 1). CBF30 was 4.5 (1, 17) min (median, 1st, and 3rd quartiles) on the ipsilateral side and 2 (0, 11) min on the contralateral side (P < 0.05 by Wilcoxon's paired test, N = 33).

Analysis of neuronal cell death in the hippocampal formation

At 2–7 days after SAH in the moderate hypoperfusion group, a massive neuronal damage appeared in the ipsilateral CA1 and CA3, but not in the dentate gyrus or on the contralateral side.

Discussion

Following experimental SAH, a CBF-dependent early decrease in the gene expression of specific NMDA receptor subunits was observed in the hippocampal formation. Since the subunit composition of NMDA receptors are known to affect its function [23], [25], this finding suggests that NMDA receptor function is altered by SAH, which may have implications for subsequent neuronal death.

We found ipsilateral, but not contralateral, CBF30 to correlate to mortality and delayed cell death, in line with

Acknowledgments

We thank Britt Meijer for excellent technical assistance. This work was supported by the Swedish Research Council (M-12593), the Magn. Bergvall's Foundation, Swedish Society for Medical Research, the Stroke Foundation, and Funds of the Karolinska Institutet.

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    These authors contributed equally to this work.

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