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

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.

[Evaluation of the rewarding effects of drugs by conditioned place preference (CPP) paradigm: properties of volatile organic solvents and uncontrolled newly-abused drugs].

The conditioned place preference paradigm has been used to evaluate the motivational effects of drugs. The primary motivational effects of a drug are conditioned to prominent environmental stimuli of a conditioned place preference box, and after several conditionings, animals acquire secondary motivational effects of drugs. If the drug experience produces a positive effect, it is expected that animals will spend more time in the drug-paired place. This technique only requires that the animals carry out a simple operation to approach or avoid the drug-paired place. Place conditioning procedures have been used to assess rewarding and aversive properties of drugs. Abuse of volatile organic solvents among young people is a serious social issue in Japan. Organic solvents are cheap and relatively easy to obtain; hence, they carry the risk of becoming "gate-way drugs" for users. We developed an airtight conditioned place preference system for drug inhalation. Using this system, we found that toluene inhalation produced a rewarding effect in mice. The conditioned place preference paradigm has been used as a tool to delineate the neurochemical substrates mediating the rewarding effect of drugs. This method may be of great help in evaluating the abuse potential of substances (e.g., volatile organic solvents and uncontrolled newly-abused drugs) in animals.

[Neurochemical mechanisms for development of psychological dependence on volatile organic solvents].

Abuse of volatile organic solvents among youth remains a major social problem. Organic solvents are cheap and relatively easy to obtain, so they carry the risk of becoming a "gateway drug" for users. The effect of repeated inhalation of toluene on subsequent responses to other drugs of abuse is unclear. In the present study, we investigated the effect of toluene inhalation on methamphetamine-induced behavioral change using a newly developed sealed inhalation shuttlebox. The influence of the cyclic AMP response element binding (CREB) protein expression following toluene inhalation was also examined. Mice were exposed to toluene or air once daily for five days. Methamphetamine produced significant hyperlocomotion in air-exposed mice. This stimulatory effect of methamphetamine was significantly enhanced following repeated inhalation of toluene. Furthermore, repeated toluene inhalation increased the levels of CREB proteins in the limbic forebrain. The present study demonstrated that adaptation of the adenylate cyclase system following repeated toluene inhalation might be involved in the expression of behavioral sensitization to subsequent methamphetamine administration. Inhalant abuse could thus be associated with the risk of other substances of abuse.

[Role of the monoamine system in the brain on the development of psychological dependence on toluene].

Abuse of volatile organic solvents among youth remains a major social problem in Japan. Organic solvents are cheap and relatively easy to obtain, so they carry the risk of becoming a so-called "gate-way drug" for users. Psychological dependence assessment systems have been established for drug inhalation using the conditioned place preference (CPP) procedure. We found toluene produced the rewarding effect in this new CPP system. The mesolimbic dopamine pathway, which includes dopaminergic neurons in the ventral tegmental area (VTA) of the midbrain and their targets in the limbic forebrain, especially the nucleus accumbens (NAC), is one of the most important substrates for the development of psychological dependence on drugs such as stimulants, cocaine, and heroin. Recently, it has indicated that the VTA-NAC pathway (monoamine system) may play an important role of the expression of psychological dependence on the volatile organic solvent toluene. Clarification of organic solvent's mechanism for the development of psychological dependence focusing on the monoamine system can be exploited for the new medicine and useful treatments for dependence on toluene.

Rehabilitative training tasks improve spatial learning impairment in the water maze following hypoxic-ischemic insult in neonatal rats.

We recently reported that hypoxic-ischemic (HI) insult to the brain of 7-d-old rats resulted in a slowly progressive learning and memory disability, which started at around 5 wk after HI, a time frame that is representative of human adolescence. The purpose of the present study was to examine whether physical or mental exercises can prevent this late-onset, slowly progressing disability. Wistar rats were subjected to left carotid ligation followed by 2 h of hypoxic stress (8% O2 and 92% N(2) at 33 degrees C). Sham-control rats were subjected to the same procedure without ligation and hypoxic stress. Six weeks after the HI, the animals were divided into four groups: pretraining control, no training control, pretraining HI, and no training HI groups. We used the plus maze, eight-arm radial maze, and choice reaction time task as the rehabilitative training. Sixteen weeks after the HI, the water maze task was performed over 5 d to evaluate spatial learning ability; thereafter, cerebral morphology of the animals was examined. There were no differences in swimming length and latency between the pretraining control and no training control groups. Swimming length and latency in the pretraining HI group were significantly shorter and swifter than those in the no training HI group. The infarct areas on the left cerebral hemisphere were equivalent between pretraining HI and no training HI groups at each sectional slice. Rehabilitative training tasks prevented the neonatal HI-induced late-onset slowly progressive learning and memory disability.

Short-term administration of a new free radical scavenger, edaravone, is more effective than its long-term administration for the treatment of neonatal hypoxic-ischemic encephalopathy.

BACKGROUND AND PURPOSE: Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a new free radical scavenger that is used for the treatment of adult acute cerebral infarction in Japan. We examined the effect of edaravone on the optimal duration of treatment, the long-term effect on the brain, and the effect on learning and memory disability in a rat model of neonatal hypoxic-ischemic encephalopathy. METHODS: Seven-day-old Wistar rats were subjected to left common carotid artery ligation then 2 hours of hypoxic-ischemic insult or sham operation. Edaravone was administered intraperitoneally (9 mg/kg) after hypoxic-ischemic insult every 24 hours for 2, 5, or 10 consecutive days. The neuroprotective effect of edaravone was evaluated by behavioral test and histological analysis. RESULTS: Two-day treatment with edaravone significantly gave protection to the learning and memory capability, as well as morphological recovery compared with control rats. Five-day treatment showed morphological improvement but no behavioral improvement. In contrast, 10-day treatment did not show either morphological or behavior improvement. CONCLUSIONS: These findings indicate that edaravone is a promising candidate as a treatment of choice for neonatal hypoxic-ischemic encephalopathy, when its use is limited to the acute phase after hypoxia-ischemia.

Implantation of encapsulated glial cell line-derived neurotrophic factor-secreting cells prevents long-lasting learning impairment following neonatal hypoxic-ischemic brain insult in rats.

OBJECTIVE: Implantation of encapsulated glial cell line-derived neurotrophic factor-secreting cells into brain parenchyma reduces histological brain damage following hypoxic-ischemic stress in neonatal rats. We examined the effect of glial cell line-derived neurotrophic factors on long-term learning and memory impairment and morphological changes up to 18 weeks after hypoxic-ischemic stress in neonatal rats. STUDY DESIGN: Baby hamster kidney cells were transfected with expression vector either including (glial cell line-derived neurotrophic factor-hypoxic-ischemic group; n = 10) or not including (control-hypoxic-ischemic group; n = 8) human glial cell line-derived neurotrophic factor cDNA, encapsulated in semipermeable hollow fibers, and implanted into the left brain parenchyma of 7-day-old Wistar rats. Two days after implantation the rats received hypoxic-ischemic stress, and their behavior was then examined in several learning tasks: the 8-arm radial maze, choice reaction time, and water maze tasks, which examine short-term working memory, attention process, and long-term reference memory, respectively. The rats were killed 18 weeks after the hypoxic-ischemic insult for evaluation of brain damage. Two additional control groups were used: the control group (n = 15), which underwent no treatment, and the glial cell line-derived neurotrophic factor group (n = 6), which underwent implantation of the glial cell line-derived neurotrophic factor capsule but did not undergo hypoxic-ischemic stress. RESULTS: The decrease in the size of the cerebral hemisphere was significantly less in the glial cell line-derived neurotrophic factor-hypoxic-ischemic group, compared with the control-hypoxic-ischemic group, and improved performance was observed in all three tasks for the glial cell line-derived neurotrophic factor-hypoxic-ischemic group: for the control-hypoxic-ischemic group versus the glial cell line-derived neurotrophic factor-hypoxic-ischemic group, respectively, in the 8-arm radial maze test, average number of correct choices was 6.2 +/- 0.1 versus 6.9 +/- 0.1 ( P < .01); in the choice reaction time test, average reaction time for a correct response was 2.35 +/- 0.1 seconds versus 1.97 +/- 0.09 seconds ( P < .01); in the water maze test, average swimming length was 1120.0 +/- 95.2 cm versus 841.6 +/- 92.1 cm ( P < .01). All results for the glial cell line-derived neurotrophic factor group were similar to those for the control group. CONCLUSION: Glial cell line-derived neurotrophic factor treatment is effective in not only reducing brain damage but also inhibiting learning and memory impairment, following hypoxic-ischemic insult in neonatal rats. No adverse effects in learning and memory tests were observed in the glial cell line-derived neurotrophic factor group.

Dexamethasone prevents long-lasting learning impairment following a combination of lipopolysaccharide and hypoxia-ischemia in neonatal rats.

OBJECTIVE: There are no established therapies for preventing or rescuing perinatal infection or inflammation-induced perinatal brain damage. We administered dexamethasone (DEX), a synthetic corticosteroid anti-inflammatory drug, to neonatal rats in a model of such damage induced by a combination of lipopolysaccharide (LPS) and hypoxia-ischemia (HI), which produces characteristic histologic and behavioral abnormalities. STUDY DESIGN: Four hours after the injection of LPS (1 mg/kg, i.p.), 7-day-old Wistar rat pups were subjected to unilateral HI for 1 hour according to Levine's procedure. Injections of 0.5 mg/kg of dexamethasone (DEX-treated group, n = 15) or saline (saline-treated group, n = 15) were given 4 hours before HI. A sham-operated control group received neither LPS nor HI (n = 15). We chose rats of this age because their stage of brain maturation is similar to the human neonate. Over the 7 to 16 weeks after treatment, a choice reaction time (CRT) task was used for assessment of attention processes in each group, an 8-arm radial maze task was used to test short-term memory, and a water maze task was used to test long-term memory. In the CRT task, the reward food was released when the tested animal correctly pressed a lever on the side of an illuminating lamp. The correct and incorrect lever pressings were counted. In the 8-arm radial maze task, rats were allowed to move freely, seeking a reward of food placed at the end of 1 arm. An error was defined as the choice of an arm that had already been visited. In the water maze, rats had to swim to seek a concealed platform as aversive escape motivation. At 19 weeks, the rats were euthanized, the brain was removed, sectioned coronally, and the volume of each part was measured. RESULTS: The striatum, cortex, and hippocampus showed reductions in volume in the saline-treated group (42.7%, 49.2%, and 34.9% decreases compared with the sham-operated controls, respectively), but this was not observed in the DEX-treated group. All learning and memory processes were impaired with the combination of LPS and HI treatment, but these deficits were almost completely prevented by DEX treatment. CONCLUSION: Dexamethasone is a promising candidate for prevention of infection and inflammation-induced perinatal brain damage. The impact of dexamethasone identifies potential therapeutic pathways once the mechanism of dexamethasone's protection is determined.

Hypoxia-ischemic insult in neonatal rats induced slowly progressive brain damage related to memory impairment.

The present study was designed to determine potential associations between the brain damage induced by hypoxic-ischemic (HI) insult and spatial learning impairment in an eight-arm radial maze task. We first determined the pathological outcomes after 2, 5, 9, and 17 weeks of recovery following the HI insult. The results show that the brain damage progressed from 2 up to 17 weeks of recovery. To clarify the time course of the brain damage changes, we investigated the histological changes of the same individual with magnetic resonance imaging (MRI) after 5, 9, and 57 weeks of recovery following the HI insult. The MRI changes were similar to the histological changes, and the brain damages were exacerbated in the contralateral hemisphere after 57 weeks of recovery following the HI insult. To investigate whether alteration in brain function was correlated with MRI and histological changes, the rats were made to find their way through an eight-arm radial maze was performed at either 7th or 16th weeks of recovery. According to the results, the spatial learning impairments of rats in the maze starting at 16 weeks of recovery were more severe than those at 7 weeks of recovery, indicating that the impairments were progressive and depended on the degree of brain damage. The results of the present study are the first demonstration that the evolutional and specific brain damage following the HI insult is slowly and progressively exacerbated to the contralateral hemisphere and rats who experience the HI are at risk for showing a late impairment of brain function.

Combination treatment of neonatal rats with hypoxia-ischemia and endotoxin induces long-lasting memory and learning impairment that is associated with extended cerebral damage.

OBJECTIVE: We assessed the long-term effects of perinatal hypoxia-ischemia and endotoxin on attention and short- and long-term memory in neonatal rats with the use of behavioral tasks and brain histologic results. STUDY DESIGN: Four hours after injections of lipopolysaccharide (1 mg/kg, intraperitoneally) or saline solution, 7-day-old Wistar rat pups were subjected to unilateral hypoxia-ischemia for 1 hour. We studied 4 groups: controls (n = 43 rats), lipopolysaccharide alone (n = 12 rats), hypoxia-ischemia alone (n = 29 rats), and combined lipopolysaccharide + hypoxia-ischemia treatment (n = 34 rats). Seven to 16 weeks after the treatment, we measured attention with a choice reaction time task, short-term memory with an 8-arm radial maze task, and long-term memory with a water maze task. At 19 weeks of age, the brain was removed, fixed, and sectioned coronally; and the volume of each part was measured. RESULTS: A loss of volume in the hippocampus was observed in the lipopolysaccharide, hypoxia-ischemia, and lipopolysaccharide + hypoxia-ischemia groups; a loss of striatum was observed in the hypoxia-ischemia and lipopolysaccharide + hypoxia-ischemia groups, but loss of cortex was observed only in the lipopolysaccharide + hypoxia-ischemia group. The lipopolysaccharide, hypoxia-ischemia, and lipopolysaccharide + hypoxia-ischemia groups showed significantly poorer performance (attention deficit) than controls in the choice reaction time task. Correct choices decreased, and error increased in the lipopolysaccharide + hypoxia-ischemia group compared with the other groups in the radial maze task, which shows short-term memory impairment. Swimming distance was significantly greater in the hypoxia-ischemia and lipopolysaccharide + hypoxia-ischemia groups than in the other 2 groups in the water maze test, which shows long-term memory impairment. CONCLUSION: Combined lipopolysaccharide and hypoxia-ischemia treatment synergistically induced short-term memory impairment that is associated with loss of cortical volume.

Effects of hypothermia and hyperthermia on attentional and spatial learning deficits following neonatal hypoxia-ischemic insult in rats.

We previously reported that rats exposed to neonatal hypoxic-ischemic (HI) insult showed selective and long-lasting learning and memory impairments in the plus maze, 8-arm radial maze, choice reaction time (CRT) task, and water maze, and that they showed severe brain injury to areas such as parietal cortex, hippocampus, striatum and thalamus. In this study, we examined the effects of hypothermia and hyperthermia on learning and memory deficits following neonatal HI insult. Seven-day-old Wistar rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8% O2/92% N2) under three different temperature conditions: 27 degrees C (hypothermia), 33 degrees C (normothermia) and 37 degrees C (hyperthermia) in temperature-controlled chambers. Hypothermia significantly reduced attentional deficits in the CRT task and spatial learning deficits in the water maze, and protected against severe brain injury in comparison with the control temperature. On the other hand, hyperthermia aggravated the behavioral deficits and brain injury. These outcomes clearly show that temperature regulation during HI insult plays an important role in the induction of behavioral and histological changes following neonatal HI insult in rats.

Local administration of glial cell line-derived neurotrophic factor improves behavioral and histological deficit of neonatal Erb's palsy in rats.

OBJECTIVE: We sought to evaluate from a behavioral and histological viewpoint the effect of local administration of glial cell line-derived neurotrophic factor (GDNF) on neonatal preganglionic Erb's palsy in rats. METHODS: The Erb's palsy model was produced by transecting the anterior and posterior roots of the left C5-C7 nerves of 7-day-old rats. The rats were divided into GDNF-treated (n = 10) and vehicle-treated groups (n = 11). After we transected the roots, contact in the proximal and distal stumps of the transected nerves was maintained, and the transected point and the entire intraspinal portion of the transected roots were enclosed by Gelfoam soaked with 10 micro g GDNF or saline. The behavioral evaluation consisted of a foot-fault test and a forepaw muscle strength test, all of which were performed from the third to the seventh weeks after the operation. Seven weeks after the operation, all rats were killed, the number of anterior horn cells was counted at C5-C7, and the differences on each side were compared. RESULTS: In the vehicle-treated group, the foot-fault test indicated an abnormality in forelimb function on the root transection side. In the GDNF-treated group, however, significant improvement in forelimb function was observed on the basis of the foot-fault test results obtained in the third to sixth weeks after the operation. In the histological evaluation, the number of anterior horn cells from the side in which the operation took place in the vehicle-treated group was significantly less than that taken from the contralateral side at each segment. In the GDNF-treated group, however, there was no difference in any of the segments, regardless of the side from which they were taken. CONCLUSION: Local administration of GDNF in a neonatal preganglionic Erb's palsy model resulted in significant improvement in deficits on the basis of behavioral and histological evaluations.

The pharmacological characterization of attentional processes using a two-lever choice reaction time task in rats.

Activating the noradrenergic and cholinergic systems is known to enhance attentional processes, while stimulating dopaminergic, serotonergic, and GABAergic systems suppresses them. The objective of the present study was to investigate the pharmacological characterization in the attentional processes of a two-lever choice reaction time (CRT) task using different centrally acting drugs. We designed seven parameters in this task: the correct response (CR) rate; error response rate; nonresponse (NR) rate; differential reinforcement of other behavior (DRO) responses; number of incorrect lever pressings during both the intertrial interval and DRO periods; the mean CRT of CR; and activity during 30 trials. The compounds produced different profiles at each dose. 1) Facilitative and disruptive effects on attentional processes occurred with changes in CRT alone. Scopolamine (0.1 mg/kg) and prazosin (0.3-1 mg/kg) prolonged the CRT, whereas methamphetamine (0.3 mg/kg) shortened the CRT. 2) Attentional deficits occurred with abnormal behavior showing premature response or perseverative behavior. Scopolamine (0.2-1 mg/kg), methamphetamine (3 mg/kg), delta(9)-tetrahydrocannabinol (10 mg/kg), and MK-801 (0.1-0.3 mg/kg) produced a marked increase in the number of total lever pressings. 3) Motor function deficits rather than attentional deficits occurred. 8-OH DPAT (1 mg/kg) and muscimol (1 mg/kg) produced a decrease in CR and an increase in NR with a marked decrease in activity and prolonged the CRT. Activating noradrenergic alpha(1) receptors was found to enhance the attentional processes, while blocking muscarinic receptors, alpha(1) receptors, and NMDA receptors, and stimulating cannabinoid receptors and the dopaminergic systems impaired the attentional processes in the two-lever CRT task.

Development of a novel experimental rat model for neonatal pre-ganglionic upper brachial plexus injury.

A neonatal upper brachial plexus injury, referred to as Erb's palsy, is a serious obstetric problem. Some surgical methods are used to treat this injury, but they are inadequate. To seek new treatments for Erb's palsy, we used a model for cervical preganglionic root transection in neonate rats and evaluated the behavioral and histological compatibility of this model with Erb's palsy. Two groups were used in this study. In the group, receiving the Erb operation, the left anterior and posterior roots of spinal vertebra C5-C7 were transected at the preganglionic level, and the results were compared with those of a group that received a sham operation. In the group, receiving the Erb operation, walking difficulties and behavioral abnormalities were observed. These observations were noted on the side where the transection took place, and the problems were attributed to proximal muscle weakness in the forelimb. Additionally, the forepaw grip was not impaired. Furthermore, in this group, the number of anterior horn cells in the cervical cord on the transected side was significantly lower than that on the contralateral side (P < 0.001). The results of this study indicate that the model fulfills the criteria for the clinical symptoms of Erb's palsy and that it may also serve as a new method for enabling treatment of the condition.