Research report
Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia–ischemia in rats

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Abstract

It is well known that neonatal hypoxic–ischemic brain injury leads to mental retardation and deficits in cognitive abilities such as learning and memory in human beings. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic–ischemic brain injury in neonatal rats has been recently reported. However, the effect of Epo on cognitive abilities in the hypoxic–ischemic brain injury model is unknown. The aim of this study is to investigate the effects of Epo on learning-memory, behavior and neurodegeneration induced by hypoxia–ischemia. Seven days old Wistar Albino rat pups have been used in the study (n=28). Experimental groups in the study were: (1) saline-treated hypoxia–ischemia group, (2) Epo-treated (i.p., 1000 U/kg) hypoxia–ischemia group, (3) sham-operated group, (4) control group. In hypoxia–ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was induced for 2.5 h. Epo was administered as a single dose immediately after the hypoxia period. When pups were 22 days old, learning experiments were performed using Morris water maze. On the 20th week, when brain development is accepted to be complete, learning experiments were repeated. Rats were then perfused and brains removed for macroscopic and microscopic evaluation. Epo treatment immediately after hypoxic–ischemic insult significantly improved long-term neurobehavioral achievements when tested during the subsequent phase of brain maturation and even into adulthood. Histopathological evaluation demonstrated that Epo also significantly diminished brain injury and spared hippocampal CA1 neurons. In conclusion, Epo administrated as a single dose immediately after neonatal hypoxic–ischemic insult provides benefit over a prolonged period in the still developing rat brain. Since the wide use of Epo in premature newborns, this agent may be potentially beneficial in treating asphyxial brain damage in the perinatal period.

Introduction

Hypoxic–ischemic brain injury in the human perinatal period can result from several pathological processes such as premature placental detachment or other complications of pregnancy and delivery and is an important cause of neonatal mortality and subsequent sequelae such as cerebral palsy, mental retardation, learning disability, and epilepsy [4], [43], [44]. Despite the fact that perinatal asphyxia closely corresponds to experimental models of cerebral hypoxia–ischemia, where successful neuroprotective interventions were introduced, presently no agent has been proven useful to ameliorate the chronic sequelae of perinatal asphyxia in the clinical setting [44]. Therefore, to explore the protective agents against hypoxic–ischemic brain injury is one of the most urgent tasks in pharmacological and clinical studies.

The most widely used and accepted animal model of perinatal asphyxia is a modification of the Levine preparation described by Rice et al. [29] which includes a combination of ischemia, obtained by unilateral occlusion of carotid artery, followed by exposure to hypoxia in 7-day-old rats. Neurodevelopmental stage of 7-day-old rats corresponds to that of newborn infants. [31], [40]. This animal model represents a useful tool for studying potential neuroprotective strategies capable of preventing or limiting the perinatal hypoxic–ischemic injury in humans. However, any pharmacological treatment, besides being effective in reducing neuronal degeneration of the lesioned area, should also improve the long-lasting functional alterations resulting from the brain damage. One candidate agent which may have an impact on this condition is erythropoietin (Epo).

Epo is a hematopoietic cytokine which has recently been shown to be expressed in the nervous system [12], [18], [19], [20], [26], [36]. Erythropoietin receptor signaling is required for normal brain development [49]. The expression of Epo and its receptor in the nervous system are modulated by hypoxia and metabolic insult [35], [36], [37]. Recent in vitro studies suggest that Epo has neuroprotective effects against various insults such as glutamate-induced excitotoxicity, serum deprivation, hypoxia, and growth factor deprivation [25], [27], [35]. It has also been shown that Epo possesses neuroprotective action in animal models of global and focal cerebral ischemia in adult rodents [5], [6], [7], [32], [33], [35]. We have recently reported the beneficial neuroprotective effect of Epo in neonatal rat model of hypoxic–ischemic brain injury [23]. However, the long-term beneficial effect of Epo on behavioral deficits and neuronal injury in this model is not known. The ameliorative effect of Epo on ischemia-induced cognitive impairment in adult animals has been shown various studies [8], [32], [33]. It is well known that human neonatal hypoxic–ischemic encephalopathy results in mental retardation and learning abnormalities [30]. Therefore, the aim of this study was to test whether Epo treatment immediately after hypoxic–ischemic insult in neonatal rats would reduce brain damage and minimize long-lasting functional deficits throughout brain maturation and into adulthood.

Section snippets

Animals

This study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory and approved by the Animal Care and Use Committee of the Dokuz Eylul University, School of Medicine. Wistar rats with dated pregnancies were maintained at the same center and housed in individual cages with free access to water and laboratory chow. Twenty-eight offsprings delivered spontaneously were reared with their dams until the time of initial experimentation at 7 days of postnatal age

Effects of Epo on Morris water maze performance

The results of the present study showed that neonatal hypoxic–ischemic brain injury induced significant long-lasting cognitive deficits throughout brain maturation and adulthood in rats. As shown in Fig. 2A, the mean latency to find the platform declined progressively during the period of P22–25 in all animals. However, the saline-treated hypoxic–ischemic rats had longer escape latencies throughout the training days than sham-operated rats and normal control animals. Epo treatment significantly

Discussion

The aim of the present study was to investigate whether Epo treatment immediately after neonatal hypoxic–ischemic insult limits morphologic damage and improves long-lasting neurobehavioral deficits. Residual brain volume was found to be significantly greater by Epo treatment at 20 weeks after the insult. In addition, Epo treatment significantly diminished long-term spatial memory deficits, known to depend mainly upon hippocampal damage. These data demonstrate that a single dose of Epo

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