Research reportIschemic “cross” tolerance in hypoxic ischemia of immature rat brain
Introduction
The phenomenon of ischemic tolerance has been extensively studied and results indicate that giving sublethal cerebral ischemia can induce a prominent neuroprotective effect against subsequent severe cerebral ischemia 20, 25, 35. This phenomenon has been studied closely in relation to the expression of heat shock proteins, in particular hsp72 2, 27. The expression of hsp72 has recently been discovered to be initiated via the adenosine A1 receptor and ATP-sensitive potassium channel during transient forebrain ischemia 16, 19 and has also been shown to be induced under hyperthermic conditions in vivo 4, 23, 26. Hyperthermic preconditioning has also been reported to have a mild neuroprotective effect against subsequent transient forebrain ischemia [8].
Though the previous reports suggest that stress proteins can confer thermotolerance in many types of cell, it is clear that cells have other mechanisms in addition to the well-studied heat shock response that help them to survive a wide variety of potentially lethal stresses. For example, hyperthermia protects animals against death caused by endotoxin injection [17] and chemical inhibition of succinic dehydrogenase can precondition mitochondrial oxidation [33]. Studies on such a “cross” tolerance phenomenon may help to reveal an unknown common pathway for the induction of tolerance. From the viewpoint of therapeutic approach, it would be beneficial to find a non-toxic drug to induce tolerance and protect the brain against a variety of injuries.
Cerebral palsy caused by brain damage during 10 months of pregnancy includes periventricular leukomalacia [3], hemorrhagic lesions [15] and hydrocephalus [10]. Unlike the situation in the adult population, it may be possible to pretreat the full-term neonate in the hope of limiting this cerebral damage. Pathophysiological mechanisms and response of neuroprotective agents in immature brain are different from mature brain [9]; however, previous studies of ischemic tolerance and expression of heat shock proteins have been performed mostly in mature brain. The type of brain damage and its mechanisms are known to depend on the maturity of the brain and that tolerance of anoxia decreases with maturity [31]. The development of neuronal network, synapse formation and neurotransmitter-related enzyme activity is still incomplete at 7-day postnatal rat brain [34]. The aspect of development of non-neuronal cells is quite different from the mature one; myelination of the white matter is underway and the number of astrocytes is considerably low. In the present study, we have examined the ischemic tolerance phenomenon using a neonatal model of hypoxic ischemia (H-I). With this model, it has been reported that hypoxic stress induces tolerance 13, 18, 29 but that the expression of heat shock proteins, as well as mRNA, after H-I is lower than in adult 11, 12, 22. The effects of heat stress in vivo in neonatal rats are not known yet. In the present study, therefore, we have studied different two sublethal stresses, hypoxia and hyperthermia, with respect to neuroprotection from subsequent H-I injury in immature brain.
Section snippets
Animal preparation
Seven-day-old rat pups were used and all the experiments were conducted according to the Guidelines for Animal Experimentation at the Kobe University School of Medicine. The birth day of the animals was defined as day 0 and animals were used on day 7 (weights 12–18 g). For the induction of H-I, the right common carotid artery was ligated with 9-0 nylon suture in two places and then cut between the ligations. Light anesthesia by halothane was used for the surgery. It has been reported that
Results
Fig. 1A and B demonstrated the representative histology of the section at the each level. In the control group, the number of the animals with histological damage was 19 out of 23 (83%). The necrotic area was clearly distinguished from the intact area under high-power magnification due to shrunken dark neurons and loss of normal cortical layers. By the preconditioning with hypoxia for 3 or 1.5 h, the numbers of rats with histological damage were 3 of 20 (15%) and 15 of 23 (65%), respectively.
Discussion
There have been a number of studies recently showing an ischemic tolerance induced by prior sublethal ischemia. With transient forebrain ischemia in adult brain, preconditioning by sublethal ischemia for a few minutes can rescue almost all of the neuronal deaths in CA1 hippocampus in subsequent severe forebrain ischemia 20, 25, 35. Most of the previous studies suggested that the expression of heat shock proteins in CA1 region, in particular hsp72, is playing a key role in the protection of
Acknowledgements
The authors thank Takashi Kokunai, MD, for expert technical support and Abesh K. Bhattacharjee, MD and Elizabeth Jansen, PhD, in the preparation of this manuscript. This study was supported, in part, by the Grant-In-Aid for Scientific Research (nos. 9557119 and 10671300) from the Ministry of Education, Science, Sports and Culture.
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