Protective and regenerative responseendogenously induced in the ischemic brain1

Kazuo Kitagawa, Masayasu Matsumoto, and Masatsugu Hori

Abstract: Neuronal cells are highly vulnerable to ischemic insult. Because adult neurons are highly differentiated andcannot self-propagate, loss of neurons often results in functional deficits in mammalian brains. However, it has recentlybeen shown that neurons and neuronal circuits exhibit protective and regenerative responses in a rodent model of ex-perimental ischemia. At first, neurons respond by producing several protective proteins such as heat shock proteins(HSPs) after sublethal ischemia and then acquire tolerance against a subsequent ischemic insult (ischemic tolerance).Once neurons suffer irreversible injury, two repair processes, neurogenesis and synaptogenesis, are endogenously in-duced. Neuronal stem and (or) progenitor cells can proliferate in two brain areas in adult animals: the subventricularzone and the subgranular zone in the dentate gyrus. After ischemic insult, these stem (progenitor) cells proliferate anddifferentiate into neurons in the dentate gyrus of the hippocampus. Reactive synaptogenesis has been also observed inthe injured brain following a period of long-term infarction, but it is unclear if it can compensate for disconnected cir-cuits. Understanding the molecular mechanism underlying these protective and regenerative responses will be importantin developing a new strategy for aimed at the augmentation of resistance against ischemic insult and the replacement ofinjured neurons and neuronal circuits.

Key words: ischemic tolerance, neurogenesis, synaptogenesis.

Kitagawa et al.Résumé: Les cellules neuronales sont très vulnérables à un accident ischémique. Étant donné que les neurones adultessont hautement différenciés et qu’ils ne peuvent se diviser, la perte de neurones entraîne souvent un déficit fonctionneldans le cerveau des mammifères. Toutefois, des travaux récents ont démontré que les neurones et les circuits neuronauxont des réponses protectrices et régénératrices dans un modèle d’ischémie expérimentale chez des rongeurs. D’abord,les neurones répondent et produisent plusieurs protéines protectrices, telles que les protéines du stress (HSP), après uneischémie sublétale et ils acquièrent une tolérance à un accident ischémique ultérieur (tolérance ischémique). Quand lesneurones ont subi une lésion irréversible, deux processus de réparation, la neurogenèse et la synaptogenèse, sont induitsde manière endogène. On a constaté que les cellules souches/progénitrices prolifèrent dans deux régions du cerveau desanimaux adultes : la zone subventrale et la zone subgranulaire du corps godronné. Après un accident ischémique, cescellules souches/progénitrices prolifèrent et se différencient dans les neurones du corps godronné de l’hippocampe. Ona aussi observé une synaptogénèse réactive dans le cerveau lésé après un infarctus de longue durée, mais on ignore en-core si elle peut compenser le circuit disconnecté. La compréhension des mécanismes moléculaires sous-jacents à cesréponses protectrices et régénératrices sera importante pour mettre au point une nouvelle stratégie visant à augmenter larésistance à un accident ischémique et à remplacer les circuits neuronaux et les neurones lésés.

Mots clés: tolérance ischémique, neurogenèse, synaptogenèse.

[Traduit par la Rédaction] 265

Introduction

Neurons are the mammalian cells most vulnerable toischemic insult. The excitotoxicy caused by the glutamatereleased during ischemia is believed to play an importantrole in this ischemic vulnerability (Sheardown et al. 1990),but the precise molecular mechanisms underlying ischemic

neuronal death remain unclear. Several pathophysiologicalprocesses such as apoptosis (Ninatori et al. 1995), free radi-cal formation (Kitagawa et al. 1990a), calcium overload(Choi 1995), and microcirculatory disturbances (del Zoppoet al. 1991) have been discussed, however, there have beenfew effective strategies for managing patients withcerebrovascular disease. To develop a new strategy forstroke management, we need to understand how neuronalcells respond to ischemic insult in terms of survival and re-pair. We discuss the following in this review: (i) ischemictolerance as an adaptive stress response of neuronal cells;(ii ) neurogenesis as a replacement response to neuronal in-jury; and (iii ) reactive synaptogenesis as a repair response ofdisconnected synapses.

Ischemic tolerance

More than two decades ago, the phenomenon of

Can. J. Physiol. Pharmacol.79: 262–265 (2001) © 2001 NRC Canada

262

DOI: 10.1139/cjpp-79-3-262

Received July 5, 2000. Published on the NRC Research PressWeb site on February 28, 2001.

K. Kitagawa,2 M. Matsumoto, and M. Hori. Division ofStrokology, Department of Internal Medicine andTherapeutics (A-8), Osaka University Graduate School ofMedicine, 2-2 Yamadaoka, Suita-city, Osaka 565-0871, Japan.

1This paper has undergone the Journal’s usual peer reviewprocess.

2Author for correspondence(e-mail: kitagawa@medone.med.osaka-u.ac.jp).

“thermotolerance” was discovered in HeLa cells subjected tohyperthermic stress (Gerner and Schneider 1974). Severalstudies using microinjections of a specific antibody againstHSP72 (Riabowol et al. 1988) and competitive inhibitorsagainst the expression of HSP72 (Johnston and Kucey 1988)have clarified the close relationship between thermo-tolerance and the induction of stress proteins, especiallyHSP72. Likewise, several studies proved that neurons exhib-ited the induction of HSP72 gene and related proteins invivo after cerebral ischemia (Vass et al. 1988; Nowak 1991).The fact that ischemia can cause the HSP72 expression inneurons has led us to expect that a mild ischemic insult(non-lethal but strong enough to induce stress proteins) maymake neurons more resistant to a subsequent ischemic in-jury. In transient global ischemia in gerbils, a 5-min is-chemic episode consistently resulted in delayed neuronaldeath in the hippocampal CA1 sector (Kirino 1982); a 2-minischemic episode depleted high energy phosphates and in-duced HSP72 after recirculation, but rarely caused neuronaldeath. When we introduced a 2-min ischemic period morethan 24 h prior to a second 5-min ischemic insult, we ob-served a dramatic protective effect against neuronal deathwhich should have been obvious following 5 min ofischemia (Kitagawa et al. 1990b). This ischemic tolerancewas found in both global and focal ischemia models and inboth gerbils and rats (Kirino et al. 1991; Chen et al. 1996a;Glazier et al. 1994). A recent paper suggested that ischemictolerance may be observed in stroke patients (Weih et al.1999).

While the ischemic pretreatment must be strong enough toinduce HSP72 for the acquisition of tolerance, HSP72 ex-pression does not seem to explain the temporal profile(Kirino et al. 1991) or cumulative effect of ischemic toler-ance (Kitagawa et al. 1997). Sublethal ischemia inducesgene induction of other HSP family members such as man-ganese superoxide dismutase (SOD) (Kato et al. 1995), c-jun(Sommer et al. 1995), and basic fibroblast growth factor(FGF) (Sakaki et al. 1996). In addition, phosphorylation ofthe transcription factor cyclic-AMP response element bind-ing protein (CREB) was observed after sublethal ischemia(Mabuchi et al. 1999). Several genes such as brain derivedneurotrophic factor (BDNF) and HSP72 have a CRE elementin their promotor area, therefore CRE-mediated gene expres-sion may play a crucial role for acquisition of ischemic tol-erance. However, ischemic insult has been also shown toinduce gene expression of neurotoxic or pro-apoptotic mole-cules such as interleukin 1β (IL-1β) (Davies et al. 1999), tu-mor necrotizing factorα (TNF α) (Gregersen et al. 2000),and Bax (Chen et al. 1996b). Therefore, gene expression af-ter ischemia would represent either protective or detrimentalresponses according to the severity of the insult. The bio-chemical changes after ischemic insult may be also impor-tant for neuronal survival. It has been reported that tolerantneurons showed inhibition of intracellular uptake of calciumions (Ohta et al. 1996) and recovery of protein synthesis(Nakagomi et al. 1993). Therefore, ischemic tolerance maybe a complex phenomenon in which several factors increaseor diminish the effect.

Pretreatment with sublethal ischemia is highly effectivefor the induction of tolerance in experimental animals, butthe procedure can not be applied clinically. However, several

other metabolic stressors such as hyperthermia (Kitagawa etal. 1991), oxygen radical stress (Ohtsuki et al. 1992), mito-chondrial inhibition (Riepe et al. 1997), and spreading de-pression (Matsushima et al. 1996; Yanamoto et al. 1998) caninduce tolerance in neurons against subsequent ischemic in-sult (cross tolerance). Induction of cortical spreading depres-sion was shown to induce the induction of immediate earlygenes such as c-fos (Ikeda et al. 1994) and cyclooxygenase-2 (Miettinen et al. 1997) and to induce tolerance againstboth global and focal ischemia (Matsushima et al. 1996;Yanamoto et al. 1998). Because spreading depression nevercauses neuronal damage, induction of membrane deporaliz-ation may be one of the practical applications of ischemictolerance. Several pharmacological agents have been shownto induce tolerance in the brain against ischemia. Both pre-treatment with IL-1β (Ohtsuki et al. 1996), TNFα(Nawashiro et al. 1997), and lipopolysaccharide (LPS)(Tasaki et al. 1997) can protect the brain from subsequentischemia. The precise mechanisms underlying the protectiveeffects of these treatments are unknown, however, pharma-cological induction of tolerance could be valuable in a clini-cal setting because of the ease of application.

Neurogenesis and synaptogenesis

It has been long believed that neurons do not proliferatein the adult mammalian brain. However, accumulating evi-dence indicates that neuronal stem and (or) progenitor cellsare found in two areas of the hippocampus, the sub-ventricular zone (SVZ) and the subgranular zone (SGZ)(Johansson et al. 1999; Doetsch et al. 1999; Suhonen et al.1996). Several physiological and pathological stimuli havebeen shown to modify neurogenesis. Neurogenesis increasesin the SVZ of animals subjected to enriched environmentsand running or learning behaviors (Kempermann et al.1997), while increasing steroid hormone levels decreaseneurogenesis (Cameron and Gould 1994). Epileptic stimuli(Parent et al. 1997) and ischemic insult (Liu et al. 1998) havebeen shown to increase neurogenesis in the SGZ. After a briefperiod of forebrain ischemia, both CA1 and CA4 neurons areselectively degenerated within four days. Because the peak ofincreased neurogenesis in the SGZ is 7–10 days afterischemia, it is unclear which stimuli are responsible for en-hancing neurogenesis, the ischemic insult or the existence ofdamaged neurons. Nevertheless, an immunohistochemicalstudy using antibody against Musashi-1 (Msi) (Sakakibara etal. 1996) demonstrated that proliferating cells in the SGZ af-ter ischemia are neuronal stem and (or) progenitor cells andthey differentiate into neurons four weeks later (Yagita et al.1999). More work is required to determine what functionalor synaptogenic roles these newly formed neurons will play.

Once neuronal cell bodies are destroyed, presynaptic ter-minals undergo Wallerian degeneration within several days.When the target neurons make important functional connec-tions, the reconstruction of neuronal circuits may contributetremendously to functional recovery. In the hippocampus,where neuronal circuits have been extensively examined, re-active synaptogenesis following deafferentation has beenshown to occur after entorhinal lesion (Frotscher et al.1997). The use of an immunohistochemical reaction forpresynaptic terminal proteins such as synapsin I allowed us

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to evaluate reactive syanptogenesis semi-quantitatively afterischemia (Kitagawa et al. 1992). Furthermore, Stroemer etal. (1995) demonstrated neocortical neural sprouting andsynaptogenesis after cerebral infarction. Basic FGF andD-amphetamine have been shown to stimulate neuronal sprout-ing and enhance functional recovery after middle cerebralartery occlusion in rats (Kawamata et al. 1997; Stroemer etal. 1998). Therefore, the strategy for enhancing sprouting inclinical stroke may facilitate recovery of neuronal functionin patients. It will be some time before these physiologicalor pharmacological stimuli can be applied in the clinic to in-duce endogenous neurogensis and (or) resynaptogenesis inpatients recovering from stroke.

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