Elsevier

Brain Research

Volume 1019, Issues 1–2, 3 September 2004, Pages 178-188
Brain Research

Research report
Spreading depression induces long-lasting brain protection against infarcted lesion development via BDNF gene-dependent mechanism

https://doi.org/10.1016/j.brainres.2004.05.105Get rights and content

Abstract

Preconditioning the rat brain with spreading depression for 48 h induces potent ischemic tolerance (infarct tolerance) after an interval of 12–15 days, consequently reducing the infarcted lesion size in the acute phase following focal cerebral ischemia. However, persistence of the morphological and functional neuroprotection has not yet been proven. We tested whether tolerance-derived neuroprotection against focal cerebral ischemia persists or merely delays the progress of cerebral infarction. Prolonged spreading depression was induced in mice by placing a depolarized focus with intracerebral microinfusion of KCl for 24 h; after intervals of 3, 6, 9 or 12 days, temporary focal ischemia was imposed. In the analysis of the infarcted lesion volume 24 h after ischemia, groups with 6 or 9 day interval demonstrated significantly smaller lesion volume compared to time-matched vehicle control group (P=0.002). Significant reduction in cerebral infarction was also observed at the chronic phase, namely 14 days after ischemia (33% reduction) (P=0.021) accompanied with less severe neurological deficits (38% reduction) (P=0.020). Using this technique, we also investigated if the mice with targeted disruption of a single BDNF allele (heterozygous BDNF-deficient mice) can gain the same potency of tolerance as the wild mice. In the result on infarcted lesion volumes following temporary focal ischemia, potent tolerance developed in the wild type (35% reduction) (P=0.007) but not in the heterozygous BDNF-deficient mice (<19% reduction) (P=0.155), indicating that BDNF expression level following spreading depression is contributing to infarct tolerance development.

Introduction

Survivability of neurons increases after receiving specific types of pathophysiological stresses such as anoxia/hypoxia [12], [54]. Specific stresses that have been identified to increase neuronal survivability against ischemia in vivo (ischemic tolerance [35]) include hyperthermia [11], [36], sublethal transient forebrain ischemia [34], [35], temporary focal ischemia [16], epileptic seizures [51], and a brief period (1–2 h) of spreading depression (SD) [30], [36]. It had been reported that SD preconditioning for 1 h induced ischemic tolerance after 3 days to protect the rat hippocampus from global ischemia-induced delayed neuronal death [30]; however, SD for 2 h did not protect the brain from severe focal ischemia in rats (unpublished data). Since SD, which exerts a fully reversible depolarization, is essentially nonlethal for normal neurons, and is known to impart less stress than anoxic or ischemic depolarization [1], we tested 48 h of SD as preconditioning to enhance tolerance. By preconditioning the rat brain with SD for 48 h, we enhanced ischemic tolerance to severe temporary focal ischemia (infarct tolerance), with a consequential reduction of infarcted lesion size [64]. Tissue damage was limited to a pinpoint (<2 mm) focus when KCl was used to induce SD [64]. SD preconditioning did not induce heat shock protein 70, a stress protein commonly induced by ischemic depolarization stress [67]. However, in this study, the volume of cerebral infarction was only analyzed at the acute phase, 48 h after ischemia, and therefore the persistence and functional outcome of the neuroprotection derived by the achieved tolerance has not yet been proven [64]. In the first part of the present study, denoted as (1), we demonstrate that the tolerance following 24 h of SD actually exerts persistent brain protection, rather than merely delaying the development of an infarcted lesion, by measuring the cerebral infarction size and the functional outcome at the chronic phase, using a newly established focal cerebral ischemia model in mice [70]. Since most studies with genetically altered samples are done in mice, switching the experimental animals from rats to mice confers an advantage for future study.

When designing the new infarct tolerance model in mice, we needed to adjust the duration of SD preconditioning according to animal size. Because of the different biological rates [45], physiological times [10], and pathophysiological times (different speed in the development of cerebral infarction) [70], we cautiously estimated the stress imposed by 48 h of SD application in rats to be equivalent to 24 h of SD application in mice.

In terms of the molecular mechanisms of infarct tolerance, involvement of BDNF production during the interval period has been suggested, since the increase of BDNF protein levels in the brain accompanying delayed increase of the BDNF-like immunoreactivity is correlated with the development of infarct tolerance following a prolonged period of SD in rats [67]. However, an etiologic role of BDNF in infarct tolerance development has not been conclusively proven by these results alone. Clarifying and utilizing the endogenous molecular mechanisms that are active during infarct tolerance development may lead to the development of a new therapeutic intervention for ischemic stroke. In the second part of the present study, denoted as (2), we demonstrate that homozygous BDNF alleles are required for infarct tolerance development, by confirming the inability to induce potent tolerance in mice with targeted disruption of a single BDNF allele (heterozygous BDNF-deficient mice) [14].

Section snippets

Experimental animals

(1) One hundred and fifteen male C57BL/6J (normal) mice (8–9 weeks old, 24–26 g, Clea Japan, Osaka, Japan), and (2) 95 (male or female) heterozygous BDNF-deficient. BDNF (+/−) mice (n=50) or their wild-type littermates BDNF (+/+) (n=45) (STOCK BDNFtm1Jae, originating in C57BL/6, which was back-crossed with C57BL/6J strain, 4–6 months old, 28–32 g, Jackson Laboratories, Bar Harbor, MA) [14] were used. BDNF knockout mice were genotyped into heterozygous BDNF-deficient or wild type by PCR

DC potential in the mouse brain

Recurrent waves of cellular depolarization (SDs) were observed as repetitive spikes in DC potential during KCl pump implantation. The spike wave duration (a single negative DC deflection) was 17±5.2 s and the interval between the SDs was 56±39 min during KCl infusion in genetically normal mice (Fig. 1). In the vehicle pump-implanted animals, no SD wave was detected (data not shown).

Analyses of infarction volumes

After prolonged KCl infusion, the actual infarction volume corrected by edema (infarct index) was gradually

Discussion

In experiment (1), it was demonstrated that following a prolonged period of SD, mouse brains achieve infarct tolerance after an interval of 6–9 days. Morphological brain protection persists for 14 days until the infarcted lesion is completely liquefied. The neurological deficits in the chronic phase following focal ischemia are less severe in the prolonged SD-preconditioned group than those in the vehicle-preconditioned group. Preconditioning with prolonged SD does not affect the resting rCBF

Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. We acknowledge the valuable assistance of Yamada Ayako, Okuno Atsuko, Inoue Ayako, Yasuda Tomoko, Kurimoto Mihoko, and Nishii Naoto (Nihon Kohden Kansai) in this study.

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