Inhibition of Nkcc1 promotes axonal growth and motor recovery in ischemic rats.

Bumetanide is a selective inhibitor of the Na+-K+-Cl--co-transporter 1(NKCC1). We studied whether bumetanide could affect axonal growth and behavioral outcome in stroke rats. Adult male Wistar rats were randomly assigned to four groups: sham-operated rats treated with vehicle or bumetanide, and ischemic rats treated with vehicle or bumetanide. Endothelin-1 was used to induce focal cerebral ischemia. Bumetanide administration (i.c.v.) started on postoperative day 7 and continued for 3 weeks. Biotinylated dextran amine (BDA) was injected into the right imotor cortex on postoperative day 14 to trace corticospinal tract (CST) fibers sprouting into the denervated cervical spinal cord. Nogo-A, NKCC1, KCC2 and BDNF in the perilesional cortex and BDA, PSD-95 and vGlut1 in the denervated spinal cord were measured by immunohistochemistry and/or Western blot. Behavioral outcome of rats was assessed by the beam walking and cylinder tests. The total length of CST fibers sprouting into the denervated cervical spinal cord significantly increased after stroke and bumetanide further increased this sprouting. Bumetanide treatment also decreased the expressions of NKCC1 and Nogo-A, increased the expressions of KCC2 and BDNF in the perilesional cortex and enhanced the synaptic plasticity in the denervated cervical spinal cord after cerebral ischemia. The behavioral performance of ischemic rats was significantly improved by bumetanide. In conclusion, bumetanide promoted post-stroke axonal sprouting together accompanied by an improved behavioral outcome possibly through restoring and maintaining neuronal chloride homeostasis and creating a recovery-promoting microenvironment by overcoming the axonal growth inhibition encountered after cerebral ischemia in rats.

Forced limb-use enhanced neurogenesis and behavioral recovery after stroke in the aged rats.

Constraint-induced movement therapy (CIMT) after stroke enhances not only functional reorganization but also structural plasticity of the brain in the adult rats. We examined whether forced limb-use which mimicked CIMT could influence ischemia-induced neurogenesis, apoptosis and behavioral recovery in the aged rats. Aged rats were divided into a sham group, an ischemia group, and an ischemia group with forced limb-use. Focal cerebral ischemia was induced by injection of endothelin-1. Forced limb-use began on post-stroke day 7 by fitting a plaster cast around the unimpaired upper limbs of rats for 3 weeks. Behavioral recovery was evaluated by tapered/ledged beam-walking test on postoperative day 32. The expression of doublecortin, neuronal nuclei, glial fibrillary acidic protein and Iba-1 were measured by single or double immunohistochemistry, and apoptosis was measured by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay. The production of neuroblasts in the subventricular zone (SVZ) was significantly increased after stroke. Forced limb-use enhanced the proliferation of newborn neurons in the SVZ, as well as increased the long-term survival of newborn neurons. Furthermore, forced limb-use suppressed apoptosis and improved the motor functions after stroke in the aged rats. Forced limb-use exerted few effects on inflammation. Neither the number nor dendritic complexity of newborn granule cells in the hippocampus was affected by forced limb-use. Forced limb-use is effective in enhancing neurogenesis and behavioral recovery after stroke even in the aged rats.

Neurodegeneration and cellular stress in the retina and optic nerve in rat cerebral ischemia and hypoperfusion models.

Experimental cerebral ischemia induces a stress response in neuronal and non-neuronal cells. In the present study we aimed to evaluate detailed cellular stress responses and neurodegenerative changes in the retinas in rat focal cerebral ischemia and hypoperfusion models involving invasive vascular manipulations. Independent groups of adult male Wistar rats were subjected to i) transient middle cerebral artery occlusion (tMCAO), ii) permanent middle cerebral artery occlusion (pMCAO), iii) cortical photothrombosis of the sensorimotor cortex using Rose Bengal dye or iv) bilateral common carotid artery occlusion (BCCAO). Rats were killed, and their eyes with the optic nerves enucleated and processed for histology, immunohistochemistry for neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), hypoxia-inducible factor 1alpha (HIF-1alpha), c-fos, alphaB-crystallin, heat shock protein (HSP) 27, HSP60 and HSP70, and detection of DNA defragmentation. The total number of the retinal ganglion cell layer (RGCL) neurons and GFAP-immunoreactive astrocytes located in the nerve fiber layer were estimated using unbiased stereological counting. Our findings indicate that although permanent and transient MCAO does not cause detectable morphological alterations in the retina or optic nerve, it evokes ischemic stress as revealed by HIF-1alpha and HSPs expression in the RGCL neurons and reactive gliosis in the Müller cells. Severe neurodegenerative changes in the retina and optic nerve of the BCCAO rats are accompanied by a significant increase in immunoreactivities for the c-fos, HSP27 and HSP70 as compared with the sham-operated animals. The retinas from the ipsilateral side of the Rose Bengal model showed a significant decrease in the total number of NeuN-positive neurons in the RGCL as compared with the contralateral ones. However, these eyes did not differ between each other in the HSPs and HIF-1alpha expression or in the GFAP-immunoreactivity of the Müller cells. In conclusion, our data suggest differential expression of various HSPs in the retina and possibly their distinct roles in the cerebral ischemia-mediated stress response and neurodegeneration.

Epileptogenesis after cortical photothrombotic brain lesion in rats.

We investigated epileptogenesis after cortical photothrombotic stroke induced with Rose Bengal dye in adult Sprague-Dawley rats. To detect spontaneous seizures, video-electroencephalograms were recorded at 2, 4, 6, 8, and 10 months for 7-14 days (24 h/day). At the end, spatial and emotional learning and memory were assessed using the Morris water-maze and fear-conditioning test, respectively, and the brains were processed for histologic analysis. Seizures were detected in 18% of rats that received photothrombosis. The average seizure frequency was 0.39 seizures per recording day and mean seizure duration was 117 s. Over 60% of seizures occurred during the dark hours. Rats with photothrombotic lesions were impaired in the water-maze (P<0.05) but not in the fear-conditioning test as compared with controls. Histology revealed that lesion depth varied from cortical layers I to VI in photothrombotic rats with epilepsy. Epileptic rats had light mossy fiber sprouting in the inner molecular layer of the dentate gyrus both ipsilateral and contralateral to the lesion. This study extends the current understanding of epileptogenesis and functional impairment after cortical lesions induced by photothrombosis. Our observations support the hypothesis that photothrombotic stroke in rats is a useful animal model for investigating the mechanisms of post-stroke epileptogenesis.

Enriched environment enhances transplanted subventricular zone stem cell migration and functional recovery after stroke.

Stroke patients suffer from severe impairments and significant effort is under way to develop therapies to improve functional recovery. Stem cells provide a promising form of therapy to replace neuronal circuits lost to injury. Indeed, previous studies have shown that a variety of stem cell types can provide some functional recovery in animal models of stroke. However, it is unlikely that replacement therapy alone will be sufficient to maximize recovery. The aim of the present study was to determine if rodent stem cell transplants combined with rehabilitation resulted in enhanced functional recovery after focal ischemia in rats. Middle cerebral artery occlusion was induced by injection of the vasoconstrictive peptide endothelin-1 adjacent to the middle cerebral artery. Seven days after stroke the rats received adult neural stem cell transplants isolated from mouse subventricular zone or vehicle injection and then subsequently were housed in enriched or standard conditions. The rats in the enriched housing also had access to running wheels once a week. Enriched housing and voluntary running exercise enhanced migration of transplanted stem cells toward the region of injury after stroke and there was a trend toward increased survival of stem cells. Enrichment also increased the number of endogenous progenitor cells in the subventricular zone of transplanted animals. Finally, functional recovery measured in the cylinder test was facilitated only when the stem cell transplants were combined with enrichment and running exercise 7 days after the transplant. These results suggest that the ability of transplanted stem cells in promoting recovery can be augmented by environmental factors such as rehabilitation.

An alpha(2)-adrenergic antagonist, atipamezole, facilitates behavioral recovery after focal cerebral ischemia in rats.

Previous studies suggest that enhanced noradrenergic neurotransmission promotes functional recovery following cerebral lesions. The present study investigated whether systemic administration of an alpha(2)-adrenergic antagonist, atipamezole, facilitates recovery following transient focal cerebral ischemia in rats. The effect of atipamezole therapy on recovery from ischemia was compared with the effect of enriched-environment housing in rats. Ischemia was induced by occlusion of the right middle cerebral artery (MCA) for 120 min using the intraluminal filament model. Daily atipamezole treatment (1 mg/kg, subcutaneously) was started on day 2 after ischemia induction and drug administration stopped after 10 days. Another group of rats was housed in an enriched environment from day 2 following ischemia induction until the end of the experiment. Several different behavioral tests were used to measure functional recovery during the 26 days following the induction of focal cerebral ischemia. There was improved performance in the limb-placing test from the beginning of atipamezole treatment to day 8, and in wheel-running in the foot-slip test on days 2 and 4. Enriched-environment housing facilitated recovery in the foot-slip test in a later phase of the test period (days 8 to 10). Discovery of a hidden platform in a water-maze task was also facilitated in rats housed in the enriched environment, but this was probably due to the increased swimming speed of these rats. The present data suggest that the alpha(2)-adrenergic antagonist, atipamezole, facilitates sensorimotor recovery after focal ischemia, but has no effect on subsequent water-maze tests assessing spatial learning and memory, when assessed 11 days after the cessation of drug administration.

Enriched-environment housing increases neuronal Fos-staining in the dentate gyrus after a water maze spatial learning task.

The present study examined whether housing in an enriched environment affects hippocampal function in responding to the challenge of a spatial water maze task in naive rats and following transient global ischemia. The enriched-environment housing was used for 11 days and was instituted the day after the induction of 20-min ischemia. Thereafter, the rats were tested in the water maze. The function of hippocampal neurons was assessed by Fos-immunostaining in ischemic and sham-operated rats 3 h after water maze testing. Rats housed in an enriched environment had an increased number of Fos-positive neurons per section in the granule cell layer of the dentate gyrus compared to rats housed individually in standard cages. This increase was observed in both ischemic and sham-operated rats. The experimental groups showed no differences in the number of Fos-positive cells in different hippocampal areas when the rats were placed in the enriched environment for the same period without the learning task. These results suggest that the number of neurons responding with altered gene expression in the dentate gyrus is increased in rats housed in an enriched environment following training in a water maze task. The altered gene expression is also preserved in ischemic rats.

Selegiline combined with enriched-environment housing attenuates spatial learning deficits following focal cerebral ischemia in rats.

Selegiline (l-deprenyl) is an irreversible monoamine oxidase B (MAO-B) inhibitor that is suggested to have neuroprotective and neuronal rescuing properties. The present study investigated whether systemic administration of selegiline facilitates behavioral recovery after transient focal cerebral ischemia in rats using a combination of different behavioral tests (limb placing, foot slip, water maze, and Montoya's staircase test) to measure the outcome of recovery. Selegiline (0.5 mg/kg, SC) or 0.9% NaCl was administered once a day, beginning on the second day after induction of ischemia and continuing for 30 days. Selegiline administration combined with enriched-environment housing attenuated ischemia-induced spatial learning deficits in a water-maze task and enhanced performance of both the contralateral affected and ipsilateral nonaffected forelimbs in a staircase test. Selegiline administration alone was not beneficial in any of the tests. Subsequent histologic examination revealed that the infarct volumes were not different between the experimental ischemic groups. Thus, these results suggest that selegiline combined with enriched-environment housing reduces behavioral and cognitive deficits without affecting infarct size.

Differential effect of the alpha2-adrenoceptor antagonist, atipamezole, in limb-placing task and skilled forepaw use following experimental stroke.

PURPOSE: Tbc present study compared tbc effect of chronic administration of the selective alpha2-adrenoceptor antagonist, atipamezole, on performance in behavioral tests that differ in motoric complexity in two experimental stroke models. METHODS: Transient occlusion (120 min) of the middle cerebral artery (MCA) using the intraluminal method was used to produce corticostriatal infarcts and permanent occlusion of distal MCA by electrocoagulation was used to produce cortical infarcts. Chronic atipamezole treatment (1 mg/kg, s.c., once per day) was started 2 days after ischemia induction and continued until the end of the experiment, 35 days after ischemia induction. Behavioral performance of the operated rats was assessed 30 min after drug administration using the limb-placing test and Montoya's staircase test. RESULTS: Atipamezole facilitated spontaneous recovery in the limb-placing task particularly in rats subjected to transient MCA occlusion. The analysis of retrieved pellets in Montoya's staircase test suggests that there is no recovery (Time effect, P > 0.05) in the use of the impaired forelimb (contralateral-to-lesion) following transient MCA occlusion, whereas there was some recovery following permanent MCA occlusion (Time effect, P < 0.001). The impairment was bilateral in rats subjected to transient MCA occlusion. Atipamezole treatment did not affect the use of the impaired forelimb to retrieve pellets following transient MCA occlusion, but there was a tendency to facilitate impaired forelimb use following permanent MCA occlusion (Time*Treatment interaction, P = 0.086). CONCLUSION: Transient occlusion of the MCA produced a severe, long lasting, and bilateral deficit in skilled forelimb use. Permanent occlusion of the distal MCA was associated with less severe impairment, which was alleviated to some extent by administration of atipamezole. This is in contrast to spontaneous recovery and recovery-enhancing effects of atipamezole in the limb-placing test, particularly in the transient MCA occlusion model.

MAO-B inhibition by a single dose of l-deprenyl or lazabemide does not prevent neuronal damage following focal cerebral ischaemia in rats.

The present study investigated the effect of postischaemic infusion of an irreversible monoamine oxidase B (MAO-B) inhibitor, l-deprenyl, an equipotent dose of a reversible MAO-B inhibitor, lazabemide, or 0.9% NaCl on infarct volumes following focal cerebral ischaemia in rats. The drug doses (0.3 mg/kg) were selected to induce selective MAO-B inhibition (45-55%), but not MAO-A inhibition. The infarct volumes in the cortex or in the striatum did not differ between the experimental groups 72 hr after transient occlusion of the middle cerebral artery, which suggests that during ischaemia/reperfusion, suppressed oxidative stress by partial MAO-B inhibition or MAO-B independent mechanisms such as induction of trophic factors, does not protect against ischaemia/reperfusion damage.

Predictors and clinical significance of declining plasma dehydroepiandrosterone sulfate in old age.

Dehydroepiandrosterone sulfate (DHEAS) was measured in random persons of three age cohorts (75, 80 and 85 years, N=271) at five-year intervals in order to find out predictors and significance of declining DHEAS in old age. The mean values decreased from 2.88 micromol/L to 2.39 micromol/L in men (p<0.001), and from 1.93 micromol/L to 1.73 micromol/L in women (p<0.05) at entry. Strong correlations were found between the baseline levels of DHEAS and those measured after five years both in men (r=0.727, p<0.001) and women (r=0.605, p<0.001), and the changes in DHEAS were associated with DHEAS levels at entry (r=-0.418, p<0.05). Baseline DHEAS was higher (2.47 micromol/L vs 2.05 micromol/L, p<0.05) and the decline more pronounced (-0.50 micromol/L vs 0.20 micromol/L, p<0.05) in the healthy subjects than in those suffering from diseases at entry, but the percentage five-year decline was similar (-6.5% and -5.2%) in both groups. The five-year decline in DHEAS was predicted neither by the baseline levels of risk indicators, e.g., serum lipids, body mass index, electrocardiographic, nor echocardiographic findings at entry. The age-and gender-adjusted baseline levels of DHEAS predicted neither mortality nor cognitive decline with 5- and 10-year follow-up periods. The 5-year decline in DHEAS was significant (p<0.05) in the subjects who died or developed cognitive decline during the subsequent 5-year follow-up. However, the changes did not differ significantly from those with favorable prognosis. The data indicate that the decline in DHEAS is primarily a gender-specific aging phenomenon, and only partly a consequence of actual diseases and frailty.

The neuroprotective effects of (-)deprenyl in the gerbil hippocampus following transient global ischemia.

(-)Deprenyl (selegeline) is a monoamine oxidase B (MAO-B) inhibitor, but it also exerts several effects independent of MAO-B inhibition. For example, it has been shown to improve neuronal survival in different neurodegenerative models. In the present study, we have tested whether (-)deprenyl attenuates the neuronal damage in the hippocampus that is induced in a model of transient global ischemia in gerbils. (-)Deprenyl was administered 1) at a low daily dose starting two weeks before occlusion, 2) at a single high dose administered 3h after occlusion, or 3) at a low daily dose for one or two weeks after occlusion. A nonsignificant trend of reduced neuronal damage in the hippocampal CA1 area was seen in all experimental groups treated with (-)deprenyl, regardless of the timing of treatment. The results together with previous evidence suggest that (-)deprenyl may protect CA1 neurons from ischemia-induced delayed death by several possible mechanisms, including the suppression of oxidative stress and apoptotic processes.

Behavioral effects of the alpha(2)-adrenoceptor antagonist, atipamezole, after focal cerebral ischemia in rats.

The present study characterized the behavioral effects of the selective alpha(2)-adrenoceptor antagonist, atipamezole, in a rat model of focal cerebral ischemia. Atipamezole (1 mg/kg, s.c.) or desipramine (5 mg/kg, i.p.), a noradrenaline reuptake blocker, was administered either as a single injection 2 days after ischemia induction or for 10 days thereafter (subacute administration). A subacute atipamezole treatment given 30 min before behavioral assessment improved performance in the limb-placing test (days 5, 7, 9, and 11) and in the foot-slip test (days 3 and 7), but not in the beam-walking test. There was no difference between experimental groups in behavioral performance following a single administration of atipamezole or following single or subacute administration of desipramine. The drug treatments did not attenuate the impairment of spatial cognitive performance of ischemic rats in the Morris water-maze test. These results suggest that repeated use-dependent release of noradrenaline by atipamezole facilitates the sensorimotor recovery following focal cerebral ischemia in rats.

Transgenic mice overexpressing truncated trkB neurotrophin receptors in neurons show increased susceptibility to cortical injury after focal cerebral ischemia.

It has been suggested that the increased production of endogenous BDNF after brain insults supports the survival of injured neurons and limits the spread of the damage. In order to test this hypothesis experimentally, we have produced transgenic mouse lines that overexpress the dominant-negative truncated splice variant of BDNF receptor trkB (trkB.T1) in postnatal cortical and hippocampal neurons. When these mice were exposed to transient focal cerebral ischemia by occluding the middle cerebral artery for 45 min and the damage was assessed 24 h later, transgenic mice had a significantly larger damage than wild-type littermates in the cerebral cortex (204 +/- 32% of wild-type, P = 0.02), but not in striatum, where the transgene is not expressed. Our results support the notion that endogenously expressed BDNF is neuroprotective and that BDNF signaling may have an important role in preventing brain damage after transient ischemia.

Neuroprotection by the alpha2-adrenoceptor agonist, dexmedetomidine, in rat focal cerebral ischemia.

The present study was undertaken to explore the possible neuroprotective effect of the selective alpha2-adrenoceptor agonist, dexmedetomidine in a rat model of focal cerebral ischemia. The effect of dexmedetomidine (9 microg kg(-1)) on infarct volume was assessed and compared to that of glutamate receptor antagonists cis-4(phosphonomethyl)-2-piperidine carboxylic acid (CGS-19755) (20 mg kg(-1)) or 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) (50 mg kg(-1)). Dexmedetomidine decreased total ischemic volume by 40% in the cortex (P<0.05) compared to NaCl-treated control rats, whereas NBQX reduced the infarct by 73% in the cortex (P<0.001) and by 43% in the striatum (P<0.01). Dexmedetomidine infusion was associated with some minor degree of hyperglycemia and hypotension. Drug-induced kidney changes were only seen in NBQX-treated rats. These results suggest that dexmedetomidine reduced ischemic volume despite causing a minor increase in blood glucose concentrations and hypotension. Its neuroprotective efficacy was better than that produced by CGS-19775, and dexmedetomidine was safer with respect to kidney toxicity when compared to NBQX.

Systemic administration of atipamezole, a selective antagonist of alpha-2 adrenoceptors, facilitates behavioural activity but does not influence short-term or long-term memory in trimethyltin-intoxicated and control rats.

The present study used trimethyltin (TMT)-intoxicated rats as a model for the behavioural syndrome seen after neuronal damage to the limbic system. Behavioural assessments indicated increased locomotor activity and reduced number of groomings in an open-arena task in TMT-intoxicated (6.6 mg/kg as a free base) rats, as has been found previously. A novel finding was the severe deficit in swimming to a visible platform in the water maze task, with reduced swimming speed at the beginning of the training period. During the reacquisition phase of a radial arm maze task, TMT-intoxicated rats made more short-term and long-term memory errors, and their behavioural activity was increased in comparison with controls. The administration of atipamezole (300 micrograms/kg), a selective antagonist of alpha 2-adrenoceptors, enhanced locomotor activity compared to saline-treated rats, but these effects did not differ between the TMT group and their controls. Atipamezole did not enhance short-term or long-term memory in either TMT or control groups. Taken together, the present data indicate that TMT intoxication is a model for global dementia rather than for a specific loss of relational memory. Previous studies on the neurochemical effects of TMT and the alleviation or prevention of neurotoxicity of TMT are reviewed.

Fentanyl decreases beta-CIT binding to the dopamine transporter.

Evidence from animal studies suggest that centrally acting opiates increase synaptic dopamine (DA) concentration. However, the interaction between mu-opioid receptors and the DA system is unclear. We report here an effect of fentanyl on striatal [123I]beta-CIT binding to the DA transporter in a patient and in rats. A female patient underwent [123I]beta-CIT single-photon emission tomography (SPET) study after intrathecal injection of fentanyl for her back pain. After a 2-week drug-free period, the SPET study was repeated. In the experimental study, male Wistar rats were treated with fentanyl either acutely (50 micrograms/kg, i.p.) before imaging study or subacutely for 4 days (10 micrograms/kg, twice a day, i.p.). Brain planar imaging was performed at 3.5 hours after an intravenous injection of [123I]beta-CIT with gamma camera with a pinhole collimator. In a female patient, [123I]beta-CIT binding in the basal ganglia was decreased by 37% during fentanyl as compared to the binding after 2-week drug-free period. Similarly in rats, acute fentanyl treatment decreased [123I]beta-CIT binding to the striatum by 30% as compared to that of with the control rats. After subacute administration of fentanyl, no significant difference was observed compared to the control group. According to the present data, fentanyl decreases [123I]beta-CIT binding in the basal ganglia both in human and rats, suggesting that opiates possibly directly affect DA reuptake.