Elsevier

Experimental Neurology

Volume 226, Issue 2, December 2010, Pages 285-292
Experimental Neurology

Sex differences in the benefits of rehabilitative training during adolescence following neonatal hypoxia–ischemia in rats

https://doi.org/10.1016/j.expneurol.2010.09.002Get rights and content

Abstract

Much effort and many resources are being devoted to rehabilitative programs for children with disabilities caused by neonatal hypoxic–ischemic encephalopathy without clear evidence of the efficacy of such programs. We recently reported that rehabilitative training tasks during adolescence improve spatial learning impairment following neonatal hypoxic–ischemic injury in rats without histological improvement. In the present study we focused on sex differences. Wister rat pups were exposed to a unilateral hypoxic–ischemic insult at 7 days of age. Six weeks after hypoxia–ischemia, rehabilitative training tasks were started. The tasks consisted of the plus maze, the eight-arm radial maze, and the choice reaction time task. Sixteen weeks after the insult, the water maze task was performed to evaluate spatial learning ability. Afterwards, we morphologically examined brain injury. Our rehabilitative training significantly improved swimming time and length in females (P < 0.01) but not in males. Likewise, the training ameliorated infarct areas in the injured cerebral hemisphere in females but not in males (P < 0.01). These results suggest that it may be important to develop and evaluate cognitive rehabilitation programs for children with brain injury on the basis of gender.

Research Highlights

►Rehabilitative training during adolescence following neonatal hypoxia–ischemia. ►The training improved spatial learning in female but not in male rats. ►The training histologically ameliorated the brain injury in females but not in males. ►Sex stratification is important in rehabilitation programs for brain injury.

Introduction

Perinatal hypoxic–ischemic encephalopathy (HIE) is the single most important problem in neonatal neurology (Aicardi, 1998). Children with severe neonatal HIE typically die or develop severe impairments such as cerebral palsy, mental retardation, and epilepsy (van Handel et al., 2007). A population-based cohort study showed that among late teenagers who survived moderate neonatal HIE, 81% have cognitive dysfunctions and 30% have cerebral palsy (Lindström et al., 2006). Hypothermia is the only therapy proven to be effective in clinical trials for full-term neonates with HIE (Shankaran et al., 2005, Gluckman et al., 2005), and currently no therapeutic drug is available for perinatal HIE. Therefore, rehabilitative training is the only hope for children with neurological sequelae from neonatal HIE once the acute phase subsides. Vast resources are being devoted to rehabilitative training and special education, as well as medical care, for these patients. Rehabilitation can offer hope, but it can also become a burden for the patients as well as society. Despite the importance of this issue, there is no conclusive evidence of the efficacy of cognitive rehabilitation in children with acquired brain injury (Limond and Leeke, 2005). A randomized control clinical trial with a long-term follow-up period would be ideal; however, there are methodological difficulties in this field of study. Therefore, using an animal model is a viable alternative. The effects of rehabilitative training tasks on cognitive deficits have been extensively studied in adult stroke patients as well as in animal models of adult stroke. In contrast, similar studies of children with brain injury, either clinical (Limond and Leeke, 2005) or animal studies (Ikeda et al., 2006, Pereira et al., 2007), are scarce.

We have previously reported long-term learning impairment following neonatal HIE in a rat model (Ikeda et al., 2001) and have shown that rehabilitative training tasks ameliorate special learning impairment in the same model (Ikeda et al., 2006). We have focused on rehabilitative training during adolescence because the effects on cognitive functions of rehabilitative interventions in children remain unclear, particularly for adolescents. Additionally, practitioners and caregivers of children with cognitive deficits are often worried about whether to continue their training during adolescence, which is a time when children with disabilities are finishing school and available social resources become limited. We discovered a form of late-onset slowly progressing brain damage in rats that starts around 5 weeks after neonatal HI insult by evaluating neurobehavioral functions, histological analysis, and magnetic resonance imaging (Mishima et al., 2005). These findings indicated that neonatal HI-induced damage in children is not a stable disorder but instead a disease that slowly progresses in later stages of life, such as adolescence (5–7 weeks of age in a rat's life span). Our work further showed that rehabilitation in this period of life may be valuable. Although neurological sequelae from neonatal HIE are commonly considered to be stable, not progressive, children with those disabilities face many difficulties as they grow older. The survivors of moderate HIE without cerebral palsy show age-appropriate levels of intellectual ability on standardized testing when they are 7 to 9 years of age (van Handel et al., 2007), and the majority of these survivors have cognitive problems when they are 15 to 19 years of age (Lindström et al., 2006). These data indicate that the cognitive functions of HIE survivors may deteriorate during adolescence.

Cerebral palsy and mental retardation are more common in males than in females (Johnston and Hagberg, 2007). While sexual dimorphism has long been recognized in adult stroke both in humans and animal models (Hurn et al., 2005), it has not drawn much attention in animal experiments in neonatal HIE until the last decade. One study has shown that no sex differences in morphological outcome are observed in neonatal rodents with HI (Zhu et al., 2006). Moreover, reduced sensitivity to ischemic injury in females has been attributed to circulating estradiol, a factor that is not relevant to pre-pubertal animals (Hurn et al., 2005). Therefore, most research groups, including ours, designed studies without sex stratification in neonatal HI (Rice et al., 1981, Tsuji et al., 2004). In practice, clinicians in NICUs have long been aware that baby boys and girls are quite different with regard to susceptibility to various diseases and immaturity. Clinical studies have demonstrated that among children born preterm, there are sex differences in cognition (Marlow et al., 2005) and cerebral volume (Reiss et al., 2004) at 6 years and 8 years of age, respectively. A good example demonstrating the importance of sex stratification is the contrasting results from two randomized trials for indomethacin prophylaxis in preterm infants. A trial without sex stratification showed no improvement in the rate of neurosensory impairment and cognitive delay (Schmidt et al., 2001). In contrast, the study with sex stratification showed higher verbal scores at 3 to 8 years of age in boys, but not in girls (Ment et al., 2004). Recent animal studies demonstrated that there are sex differences in neonatal HIE (Bona et al., 1998, Hagberg et al., 2004). As for the effects of environmental stimulations and/or rehabilitative training for animals subjected to neonatal HI, there is, to the best of our knowledge, only one report so far showing sex differences (Pereira et al., 2008).

The purpose of the present investigation is to determine 1) whether the effects of rehabilitative training during adolescence on cognitive function differ substantially based on sex and, if so, 2) whether they are accompanied by morphological alterations in a rat model of neonatal HIE.

Section snippets

Hypoxic–ischemic insult in rats

Seven-day-old Wister rat (Japan SLC, Hamamatsu, Japan) pups (day of birth = day 0) were prepared for surgery. All experiments were performed in accordance with protocols approved by the Experimental Animal Care and Use Committee of the National Cerebral and Cardiovascular Center and Fukuoka University. Under ether anesthesia, the left carotid artery was ligated. After surgery, the rat pups were allowed to recover for 1 to 2 h separated from the dam. Hypoxic exposure was achieved by placing

Water maze

First, we examined spatial learning ability using the water maze in sham-surgery control rats. Figs. 2A, C, and E show three parameters (swimming time, length, and speed, respectively) of each block of three trials and the temporal changes during 5 consecutive days of trials. Swimming times and lengths became shorter on the second day, meaning that the rats learned to get to the hidden platform more quickly. Swimming speeds remained the same for the 5-day experimental period despite sex or

Discussion

We have previously reported that brain damage in a rat model of neonatal HI insult is partially ameliorated by rehabilitative training tasks starting at 5 weeks of life, a time frame that represents human adolescence (Ikeda et al., 2006). The tasks consisted of the plus maze, the eight-arm radial maze, and the choice reaction time task. The training tasks improved the impairment of spatial learning abilities in the water maze at adulthood with no amelioration of morphological brain damage. In

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (JSPS KAKENHI 20590552) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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