Elsevier

Neuroscience Research

Volume 29, Issue 1, September 1997, Pages 37-47
Neuroscience Research

Cortical spreading depression induces long-term alterations of BDNF levels in cortex and hippocampus distinct from lesion effects: implications for ischemic tolerance

https://doi.org/10.1016/S0168-0102(97)00069-2Get rights and content

Abstract

Cortical spreading depression (CSD) protects hippocampal and cortical neurons from an otherwise lethal ischemic insult delivered days later. The present study was undertaken to evaluate changes in the expression of BDNF following CSD, distinct from lesion effects and its possible involvement in delayed ischemic tolerance. CSD was elicited by KCl application and a cortical lesion was made by hyperosmolar NaCl application. BDNF mRNA was examined by in situ hybridization and Northern blot up to 7 days post-CSD. BDNF protein content was measured by ELISA. In the cortex, BDNF protein was mildly elevated despite minimal increases of mRNA in the NaCl lesion group. CSD specifically up-regulated BDNF mRNA at 4 h, followed by a delayed secondary increase at 2–3 days. BDNF protein exhibited smaller biphasic increases at 24 h and 3–7 days post-CSD which were significantly higher than the NaCl lesion group. In the hippocampus, BDNF protein levels showed a delayed decrease in both groups independent of mRNA changes, but CSD specifically delayed this decrease. Thus, CSD can alter BDNF levels independent of lesion effects. The increased BDNF following CSD in the cortex is consistent with the involvement of BDNF in cortical ischemic tolerance. BDNF could not, however, be directly related to ischemic tolerance in the hippocampus.

Introduction

Brief, sublethal ischemia induces tolerance to later lethal ischemia in hippocampal neurons (Kitagawa et al., 1990, Kirino et al., 1991). Similar ischemic tolerance induced in both hippocampal and cortical neurons has been produced by prior induction of cortical spreading depression (CSD) (Kawahara et al., 1995, Kobayashi et al., 1995, Matsushima et al., 1996). CSD manifests as slowly propagating depolarization waves with increased extracellular glutamate (Fabricius et al., 1993), resembling anoxic depolarization (Hansen and Zeuthen, 1981), but does not result in irreversible neuronal damage even when elicited repetitively (Nedergaard and Hansen, 1988). Induction of CSD 24 h or 3 days prior to an ischemic insult protects cortical neurons from cell death (Kobayashi et al., 1995, Matsushima et al., 1996). In hippocampal neurons, which are not directly exposed to depolarization waves, protection was observed at 3 days, but not 1 or 7 days, after CSD (Kawahara et al., 1995).

Repetitive CSD induces a rapid increase of brain-derived neurotrophic factor (BDNF) mRNA in the cortex (Kokaia et al., 1993). BDNF, a member of the neurotrophin family of neurotrophic factors (Barde et al., 1982, Leibrock et al., 1989), is expressed by neurons in many regions of the adult central nervous system, with particularly high levels in the hippocampus and cortex (Ernfors et al., 1990, Maisonpierre et al., 1990). A wide variety of insults such as kainic acid seizures (Zafra et al., 1990, Wetmore et al., 1994), limbic seizures (Isackson et al., 1991), ischemia (Lindvall et al., 1992, Tsukahara et al., 1994), and hypoglycemia (Lindvall et al., 1992) increase BDNF mRNA in the brain, suggesting that BDNF may contribute to endogenous defense mechanisms. BDNF has been shown to protect neurons in vitro against glutamate toxicity (Lindholm et al., 1993, Cheng and Mattson, 1994), glucose deprivation (Cheng and Mattson, 1994), as well as against ischemia (Beck et al., 1994, Tsukahara et al., 1994) and axotomy-induced injury in vivo (Morse et al., 1993).

Because BDNF mRNA increases rapidly after CSD (Kokaia et al., 1993), and BDNF exerts clear effects on many classes of CNS neurons (Lindsay et al., 1994), alterations in endogenous BDNF may be involved in the neuroprotective effect of CSD against subsequent ischemia. In this paper we attempt to clarify the protective role of BDNF by examining BDNF mRNA and protein levels in the cortex and hippocampus for 7 days after repetitive CSD, which includes the period of increased tolerance to ischemia and compared them with levels after hyperosmolar cortical lesion without CSD.

Section snippets

Subjects

Male Sprague–Dawley rats weighing 270–330 g were used as subjects in this experiment (Harlan Sprague Dawley, Indianapolis, IN). Rats were maintained on a 12:12 h light:dark cycle with food and water available ad libitum. All animal-related procedures were conducted in accordance with a protocol approved by the Animal Care and Use Committee, NINDS.

Induction of cortical spreading depression

Animals were anesthetized with 1.5% halothane in a 30/70% O2/N2O mixture, and the tail artery was cannulated for monitoring blood pressure and for

Incidence of CSD and physiological parameters

Application of 5 M KCl to the dura over the occipital cortex elicited repetitive waves of spreading depression (SD) in all rats (Fig. 1), with an average number of 10.0±3.0 (mean±S.D.; Table 1). Application of 5 M NaCl did not induce any depolarization waves during the recording period (Fig. 1). There were no statistically significant differences in physiological parameters (blood pressure, pH, PaCO2, PaO2, and base excess) between the KCl and NaCl groups (Table 1).

Northern blot analysis

Northern blot hybridization

Post-CSD alterations in BDNF

The present study was conducted to examine changes in BDNF mRNA and protein in the cortex and hippocampus following cortical spreading depression, and to relate these changes to CSD-induced ischemic tolerance. We found that BDNF mRNA and protein both increased biphasically in the cortex in a manner distinct from any hyperosmolar lesion effect. The biphasic increases correspond temporally to periods of relative protection from cortical ischemic damage following CSD (Kobayashi et al., 1995,

Acknowledgements

We wish to thank Dr Michael Brenner for his technical advice and valuable suggestions, Pat Holst and Dr Susan Radka for assistance with the BDNF ELISA, Ning Cai for histological assistance, Jaeyoung Cho for assistance in preparing probes for in situ hybridization, and Christl Ruetzler for her valuable assistance in coordinating many aspects of this project.

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