Radon Inhalation Protects Against Transient Global Cerebral Ischemic Injury in Gerbils

Although brain disorders are not the main indication for radon therapy, our previous study suggested that radon inhalation therapy might mitigate brain disorders. In this study, we assessed whether radon inhalation protects against transient global cerebral ischemic injury in gerbils. Gerbils were treated with inhaled radon at a concentration of 2,000 Bq/m³ for 24 h. After radon inhalation, transient global cerebral ischemia was induced by bilateral occlusion of the common carotid artery. Results showed that transient global cerebral ischemia induced neuronal damage in hippocampal CA1, and the number of damaged neurons was significantly increased compared with control. However, radon treatment inhibited ischemic damage. Superoxide dismutase (SOD) activity in the radon-treated gerbil brain was significantly higher than that in sham-operated gerbils. These findings suggested that radon inhalation activates antioxidative function, especially SOD, thereby inhibiting transient global cerebral ischemic injury in gerbils.     

The effects of peripheral leukocytes on the hippocampal neuronal changes in transient global ischemia and unilateral cerebral hemispheric infarction.

The participation of activated leukocytes and subsequent production of chemical mediators has been well accepted in the pathophysiology of hypoxic-ischemic injury. This study was performed to see the effects of leukocytes on hippocampal neuronal damage in transient global ischemia induced by 10-min occlusion of bilateral common carotid arteries (CCAs) with reperfusion for various times, and in complete unilateral ischemia induced by 24-hr ligation of left CCA. Leukopenia was induced by intraperitoneal injection of cyclophosphamide for 4 days. The results showed that hippocampal neuronal damages were worse at 6-hr reperfusion in leukopenic experimental group than in the control group. In comparison, 24-hr and 3-day reperfusion leukopenic groups showed less numbers of damaged neurons and milder changes. The 5-day reperfusion group showed inconsistent changes. Unilateral CCA occlusion showed extensive infarction in 83.3% of gerbils in the control group, compared to 25% of gerbils in the experimental group (p<0.05). These results strongly suggest that the number of peripheral leukocytes were closely related to the development of delayed neuronal damage of hippocampus in transient global ischemia and the incidence of infarction induced by 24-hr unilateral CCA ligation.     

Dependency of human neck reflex responses on the bandwidth of pseudorandom anterior-posterior torso perturbations

Anoxic brain injury resulting from cardiac arrest is responsible for approximately two-thirds of deaths. Recent evidence suggests that increased oxygen delivered to the brain after cardiac arrest may be an important factor in preventing neuronal damage, resulting in an interest in hyperbaric oxygen (HBO) therapy. Interestingly, increased oxygen supply may be also reached by application of normobaric oxygen (NBO) or hyperbaric air (HBA). However, previous research also showed that the beneficial effect of hyperbaric treatment may not directly result from increased oxygen supply, leading to the conclusion that the mechanism of hyperbaric prevention of brain damage is not well understood. The aim of our study was to compare the effects of HBO, HBA and NBO treatment on gerbil brain condition after transient forebrain ischemia, serving as a model of cardiac arrest. Thereby, we investigated the effects of repetitive HBO, HBA and NBO treatment on hippocampal CA1 neuronal survival, brain temperature and gerbils behavior (the nest building), depending on the time of initiation of the therapy (1, 3 and 6 h after ischemia). HBO and HBA applied 1, 3 and 6 h after ischemia significantly increased neuronal survival and behavioral performance and abolished the ischemia-evoked brain temperature increase. NBO treatment was most effective when applied 1 h after ischemia; later application had a weak or no protective effect. The results show that HBO and HBA applied between 1 and 6 h after ischemia prevent ischemia-evoked neuronal damage, which may be due to the inhibition of brain temperature increase, as a result of the applied rise in ambient pressure, and just not due to the oxygen per se. This perspective is supported by the finding that NBO treatment was less effective than HBO or HBA therapy. The results presented in this paper may pave the way for future experimental studies dealing with pressure and temperature regulation.     

Seizures are associated with brain injury severity in a neonatal model of hypoxia–ischemia

Hypoxia–ischemia is a significant cause of brain damage in the human newborn and can result in long-term neurodevelopmental disability. The loss of oxygen and glucose supply to the developing brain leads to excitotoxic neuronal cell damage and death; such over-excitation of nerve cells can also manifest as seizures. The newborn brain is highly susceptible to seizures although it is unclear what role they have in hypoxic-ischemic (H/I) injury. The aim of this study was to determine an association between seizures and severity of brain injury in a piglet model of perinatal H/I and, whether injury severity was related to type of seizure, i.e. sub-clinical (electrographic seizures only) or clinical (electrographic seizures+physical signs). Hypoxia (4% O₂) was induced in anaesthetised newborn piglets for 30 min with a final 10 min period of hypotension; animals were recovered and survived to 72 h. Animals were monitored daily for seizures both visually and with electroencephalogram (EEG) recordings. Brain injury was assessed with magnetic resonance imaging (MRI), ¹H-MR spectroscopy (¹H-MRS), EEG and by histology (haematoxylin and eosin). EEG seizures were observed in 75% of all H/I animals, 46% displayed clinical seizures and 29% sub-clinical seizures. Seizure animals showed significantly lower background amplitude EEG across all post-insult days. Presence of seizures was associated with lower cortical apparent diffusion coefficient (ADC) scores and changes in ¹H-MRS metabolite ratios at both 24 and 72 h post-insult. On post-mortem examination animals with seizures showed the greatest degree of neuropathological injury compared to animals without seizures. Furthermore, clinical seizure animals had significantly greater histological injury compared with sub-clinical seizure animals; this difference was not apparent on MRI or ¹H-MRS measures. In conclusion we report that both sub-clinical and clinical seizures are associated with increased severity of H/I injury in a term model of neonatal H/I.     

Differential effects and changes of ceruloplasmin in the hippocampal CA1 region between adult and aged gerbils after transient cerebral ischemia

In this study, we examined the differential effects and changes of ceruloplasmin between adult and aged gerbil hippocampus after transient forebrain ischemia. Ceruloplasmin in the hippocampal CA1 region of adult and aged gerbils was significantly changed after ischemia/reperfusion. Whereas, it was not significantly changed in the CA2/3 region compared to the CA1 region after ischemia. Ceruloplasmin immunoreactivity and its protein level in aged gerbil CA1 region were higher than those in adult gerbil CA1 region. Ceruloplasmin in the CA1 region was highest in adult gerbils and aged gerbils at 24h and 12h after transient ischemia, respectively. At these time points, strong ceruloplasmin immunoreactivity was observed in CA1 pyramidal cells. Thereafter, ceruloplasmin was decreased with time after ischemia. Four days after ischemia/reperfusion, ceruloplasmin immunoreactivity in both adult and aged gerbils was expressed in astrocytes in the CA1 region. Ceruloplasmin treatment in adult ischemic gerbils showed strong protective effect against ischemic damage in CA1 pyramidal cells compared to that in aged ischemic gerbils. We conclude that ceruloplasmin early increases in the aged gerbil CA1 region compared to that of the adult gerbil CA1 region may be associated with the earlier induction of reactive oxygen species, and ceruloplasmin shows strong neuroprotective effects in adults compared to those in aged gerbils.     

Poor outcome after hypoxia-ischemia in newborns is associated with physiological abnormalities during early recovery. Possible relevance to secondary brain injury after head trauma in infants.

"Secondary hypoxia/ischemia" (i.e. regional impairment of oxygen and substrate delivery) results in secondary deterioration after traumatic brain injury in adults as well as in children and infants. However, detailed analysis regarding critical physiological abnormalities resulting from hypoxia/ischemia in the immature brain, e.g. acid-base-status, serum glucose levels and brain temperature, and their influence on outcome, are only available from non-traumatic experimental models. In recent studies on hypoxic/asphyxic cardiac arrest in neonatal piglets, we were able to predict short-term outcome using specific physiologic abnormalities immediately after the insult. Severe acidosis, low serum glucose levels and fever after resuscitation were associated with an adverse neurologic recovery one day after the insult. The occurrence of clinically apparent seizure activity during later recovery increased mortality (epileptic state), and survivors had greater neocortical and striatal brain damage. Brain damage after transient hypoxia/ischemia and "secondary brain injury" after head trauma may have some mechanistic overlap, and these findings on physiological predictors of outcome may also apply to pathologic conditions in the post-traumatic immature brain. Evaluation of data from other models of brain injury will be important to develop candidate treatment strategies for head-injured infants and children and may help to initiate specific studies about the possible role of these physiological predictors of brain damage in the traumatically injured immature brain.     

Critical levels of extracellular glutamate mediating gerbil hippocampal delayed neuronal death during hypothermia: brain microdialysis study.

When the brain temperature was lowered by 2 degrees C from normothermic temperature, a protective effect on postischemic neuronal death was exhibited and levels of extracellular glutamate were attenuated to about half of those at normothermic brain temperature in the gerbil hippocampus. Hypothermia has been reported to confer a protective effect on ischemia-induced delayed neuronal death. The present study was carried out to quantify this protective effect of hypothermia on the degree of alteration in extracellular release of glutamate during ischemia and the final histopathological outcome in the hippocampus. Extracellular glutamate levels were measured by microdialysis. In gerbils whose brain temperature was maintained at normothermia (37 degrees C), glutamate increased during ischemia and the early period of recirculation (by 15-fold), and CA1 neurons were consistently damaged. In animals whose brain temperature was maintained at 35 or 33 degrees C during ischemia, the release of glutamate was significantly attenuated to half or a quarter, respectively, at 37 degrees C. In animals whose brain temperature was maintained at 31 degrees C during ischemia, the release of glutamate was slightly lower than that at 33 degrees C. No CA1 ischemic neuronal damage was seen in 60% of gerbils at 35 degrees C and none was seen in any gerbils at 33 and 31 degrees C. In animals whose brain temperature was maintained at 39 degrees C during ischemia, the release of glutamate was slightly higher than that at 37 degrees C, and a high mortality rate of animals (75%) was observed. Our results reinforce other recent evidence suggesting that one of the mechanisms by which lowering of the brain temperature by only a few degrees during ischemia exerts a protective effect in the hippocampus, involves the reduction of ischemia-induced glutamate release.     

Dynamic induction of the long pentraxin PTX3 in the CNS after limbic seizures: evidence for a protective role in seizure-induced neurodegeneration

Pentraxin 3, a prototypic long pentraxin, is induced by proinflammatory signals in the brain. Inflammatory cytokines are rapidly induced in glia by epileptic activity. We show that pentraxin 3 immunoreactivity and mRNA are enhanced in the rat forebrain above undetectable control levels by limbic seizures with a dual pattern of induction. Within 6 h from seizure onset, pentraxin 3 immunoreactivity was increased in astrocytes. Eighteen to 48 h later, specific neuronal populations and leucocytes were strongly immunoreactive only in areas of neurodegeneration. This staining was abolished when neuronal cell loss, but not seizures, was prevented by blocking N-methyl-D-aspartate receptors. Pentraxin 3 -/- mice had a more widespread seizure-related neuronal damage in the forebrain than their wild-type littermates although both groups had similar epileptic activity. Our results provide evidence that pentraxin 3 is synthesized in brain after seizures and may exert a protective role in seizure-induced neurodegeneration.     

DSP4 treatment worsens hippocampal pyramidal cell damage after transient ischemia.

Recent studies in the rat have suggested that hippocampal norepinephrine can regulate the amount of damage seen after transient forebrain ischemia. We used the gerbil to study the role of norepinephrine in ischemic damage. Using tyrosine hydroxylase immunocytochemistry and chemical measurements of norepinephrine, we determined that the gerbil hippocampus has a similar but topographically different norepinephrine innervation than the rat. Brains from gerbils treated with 100 mg/kg of N-(2-chloroethyl)-N-methyl-2-bromobenzylamine (DSP4) had 60% less norepinephrine than saline-treated controls, similar to the effect of the drug in rats. We administered DSP4 to gerbils two weeks before exposing them to 5 min of bilateral carotid artery occlusion. Animals treated with DSP4 and subjected to ischemia had worse pyramidal cell loss in the CA3 and CA4 regions than saline-treated ischemic controls. CA1 pyramidal cell loss (about 90%) was severe in both the saline- and DSP4-treated animals. These data provide further evidence that norepinephrine can regulate the neuronal death in the hippocampal formation after transient forebrain ischemia. Furthermore, this is the first demonstration of that regulation in the gerbil and suggests that noradrenergic input to the hippocampus may be important in ischemia in other species besides the rat.     

Epileptic tolerance is associated with enduring neuroprotection and uncoupling of the relationship between CA3 damage,neuropeptide Y rearrangement and spontaneous seizures following intra-amygdala kainic acid-induced status epilepticus in mice

Brief, non-harmful seizures can activate endogenous protective programmes which render the brain resistant to damage caused by prolonged seizure episodes. Whether protection in epileptic tolerance is long-lasting or influences the subsequent development of epilepsy is uncertain. Presently, we investigated the relationship between hippocampal pathology, neuropeptide Y rearrangement and spontaneous seizures in sham- and seizure-preconditioned mice after status epilepticus induced by intra-amygdala kainate. Seizure-induced neuronal death at 24 h was significantly reduced in the ipsilateral hippocampal CA3 and hilus of tolerance mice compared to sham-preconditioned animals subject to status epilepticus. Damage to the CA3-hilus remained reduced in tolerance mice 21 days post-status. In sham-preconditioned mice subject to status epilepticus correlative statistics showed there was a strong inverse relationship between CA3, but not hilar, neuron counts and the number of spontaneous seizures. A strong positive association was also found between neuropeptide Y score and spontaneous seizure count in these mice. In contrast, there was no significant association between spontaneous seizure count and CA3 neuron loss or neuropeptide Y rearrangement in the tolerance mice. These data show that tolerance-conferred neuroprotection is long-lasting and that tolerance disrupts the normal association between CA3 damage, synaptic rearrangement and occurrence of spontaneous seizures in this model     

Subcortical morphological correlates of impaired clock drawing performance

The present study investigates the neurological protective effects of edaravone against global brain ischemia. Gerbils were treated with edaravone (3 mg/kg; i.p.) 30 min before transient forebrain ischemia, which was induced by occluding the bilateral common carotid artery for 5 min. The effects of edaravone were examined by measuring neuronal damage and behavioral deficits. Hexanoyl-lysine adduct (HEL) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), oxidative stress markers, were also examined to assess the anti-oxidative effects of edaravone. Edaravone treatment significantly inhibited both lipid and DNA oxidative damage 72 h after ischemia, and decreased neuronal damage. Edaravone also significantly reduced the locomotor activity deficit 72 h after ischemia and improved memory impairment. These findings suggest that edaravone inhibits oxidative stress and attenuates neuronal damage induced by transient forebrain ischemia in gerbils and which may contribute to improvements in behavioral deficits.     

c-Fos, N-methyl-d-aspartate receptor 2C, GABA-A-alpha1 immonoreactivity, seizure latency and neuronal injury following single or recurrent neonatal seizures in hippocampus of Wistar rat

To evaluate the long-term effects of single or recurrent prolonged neonatal seizures on seizure threshold and neuronal activity in the brain, a novel "twist" seizure was induced by coupling early-life flurothyl-induced seizures with later exposure to pentylenetetrazol. The authors assigned six neonatal rats for each group: the single-seizure group (SS), the recurrent-seizure group (RS) and the control group. At postnatal day 46, seizure threshold was examined using pentylenetetrazol, and then the brain slices were evaluated with thionine staining, in situ end labeling and immunohistochemical studies. The Results showed that the rats in SS and RS groups all had reduced latencies to develop generalized tonic seizures induced by PTZ compared with controls (P<0.01). Morphologic changes, cell loss and apoptotic cells were observed only in those of RS group. Significant fos and NR2C-immunoreactive positive cells were seen in hippocampus of rats in both SS and RS groups compared with controls (P<0.01). A significant decrease in the number of GABA-A-alpha1 immunoreactive positive neurons was detected in hippocampus in rats of SS and RS groups compared with the controls (P<0.01). We conclude that neonatal rats subjected to prolonged seizures have pronounced long-term effects on seizure threshold and neuronal neurophysiological activity in the brain. Obvious neuronal injury, however, was only seen in rat with recurrent-seizures. Subtle brain damage might occur in rats experiencing single prolonged neonatal seizures.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.     

Gliomas and seizures

Glial neoplasms account for nearly 50% of all adult primary brain tumors. They originate from glial cells in the brain and/or spinal cord and include low-grade diffuse astrocytomas, anaplastic-astrocytomas, and glioblastomas. Of all brain tumors, glioblastoma multiforme (GBM) is the most aggressive and is characterized by rapid glial cell growth, resistance to radio- and chemo- therapies, and relentless infiltration and spreading throughout the central nervous system (CNS). In glioblastomas, primary tumor growth and CNS invasion are associated with the activation of complex structural molecular and metabolic changes within the tumor tissue, which profoundly affect the surrounding neuronal networks and may in part explain induction of epilepsy. In fact, epileptic seizures are very common among patients with glial tumors, reaching nearly 50% in glioblastoma patients and almost 90% in low-grade astrocytomas. The overall hypothesis presented here discusses the possibility that the aberrant tumor cell metabolism may act directly on neuronal network, and this leads to seizure susceptibility. Further invasion and growth of the malignant glial cells exacerbate this initial pathologic state which promotes recurrent seizures (epileptogenesis).     

Effects of aniracetam on extracellular levels of transmitter amino acids in the hippocampus of the conscious gerbils: an intracranial microdialysis study

The effects of aniracetam on extracellular amino acid levels in the hippocampus of conscious gerbils, with or without transient cerebral ischemia/reperfusion, were measured by microdialysis and reverse phase-high performance liquid chromatography. Increased extracellular levels of aspartate and glutamate that were observed in the hippocampus of conscious gerbils during transient global forebrain ischemia were reversed by aniracetam. In contrast, the level of extracellular gamma-aminobutyric acid was increased, while taurine was maintained at a higher level than other amino acids by administration of aniracetam (100 mg/kg, p.o.) 60 min before ischemia. Further, in contrast to ischemic animals, administration of aniracetam (100 mg/kg, p.o.) enhanced the release of glutamate and aspartate in the normal gerbil hippocampus. The results suggest that these effects might be due to a partial calcium agonist activity of aniracetam, and that the effects of aniracetam on amino acid levels might be a mechanism of protection against delayed neuronal death in the ischemic hippocampus, thereby improving memory dysfunction induced by ischemia/reperfusion.     

Protective effect of topiramate against hippocampal neuronal damage after global ischemia in the gerbils

The purpose of this study was to examine whether topiramate would reduce neuronal damage after transient global ischemia in the gerbils because topiramate blocks voltage sensitive sodium channels and non-N-methyl-D-aspartate receptors and enhances gamma-aminobutyric acid-mediated inhibitory transmission. Both common carotid arteries were occluded for 3 min with microaneurysmal clips. The gerbils were treated with topiramate (50, 100, or 200 mg/kg, i.p.) immediately after ischemia. Neuronal cell damage in the hippocampal CA1 region was evaluated quantitatively 7 days after ischemia. Topiramate at the dose of 50 mg/kg failed to reduce hippocampal neuronal damage. However, topiramate when administered at the dose of 100 or 200 mg/kg significantly reduced hippocampal neuronal damage in dose-dependent manner (P<0.001 and P<0.0005, respectively). These results suggest that topiramate has a neuroprotective effect against neuronal damage following global ischemia in the gerbils.     

Borna disease virus induces acute fatal neurological disorders in neonatal gerbils without virus- and immune-mediated cell destructions

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that is known to cause neurological disturbances in various animal species. Our previous experiment demonstrated that neonate gerbils develop an acute fatal neurological disease following infection with BDV, Virology 282, 65-76). The study suggested that BDV directly causes functional damage of neuronal cells resulting in the lethal disorder in neonatal gerbils. To extend this finding, we examined whether BDV can induce neurological diseases in the absence of virus- and immune-mediated cell destruction, by using cyclosporine A (CsA)-treated neonatal gerbils. Although CsA completely suppressed specific antibody production and brain inflammation in the infected gerbil brains, the fatal neurological disorder was not inhibited by the treatment. Furthermore, we demonstrated that CsA treatment significantly decreased brain levels of cytokines, except interleukin (IL)-1 beta, in the infected gerbils. These results suggested that BDV replication, as well as brain cytokines, at least IL-1 beta, rapidly induces fatal disturbances in gerbil brain. We demonstrate here that BDV exhibits a unique neuropathogenesis in neonatal gerbil that may be pathologically and immunologically different from those in two other established rodent models, rats and mice. With this novel rodent model of virus infection it should be possible not only to examine acute neurological disturbances without severe neuroanatomical and immunopathological alterations but also to analyze molecular and cellular damage by virus replication in the central nervous system.     

Repeated brief epileptic seizures by pentylenetetrazole cause neurodegeneration and promote neurogenesis in discrete brain regions of freely moving adult rats

Recurrent epileptic seizures are known to provoke various forms of cellular reorganization in the brains of humans and experimental animals. However, little is known about the mechanism of neuronal cell death resulting from epileptic seizures elicited by GABA antagonists. In the present study, we explored the effect on the central nervous systems of freely moving adult rats, of repeated brief epileptic seizures induced by systemic injection of pentylenetetrazole, a GABA-A receptor antagonist. Starting with minor convulsions, repeated epileptic seizures elicited a progressive increase in seizure severity, culminating in the fully kindled state. Histological examination showed that the epileptic seizures caused overt neuronal cell death in the limbic system, including the hippocampus and amygdala, and its adjoining cortex. During the recurrent epileptic seizures, neurogenesis occurred in the subgranular zone of the hippocampus, the subventricular zone of the lateral ventricle, and the amygdala. This type of pentylenetetrazole-induced neurogenesis was seen at an early stage of epileptogenesis in some regions in which massive cell loss was not evident. This suggests that neurogenesis is not a secondary consequence of neuronal cell death, but rather an independent effect of recurrent epileptic seizures.     

Synchronization Implies Seizure or Seizure Implies Synchronization?

Epileptic seizures are considered as abnormally hypersynchronous neuronal activities of the brain. The question is “Do hypersynchronous neuronal activities in a brain region lead to seizure or the hypersynchronous activities take place due to the progression of the seizure?” We have examined the ECoG signals of 21 epileptic patients consisting of 87 focal-onset seizures by three different measures namely, phase synchronization, amplitude correlation and simultaneous occurrence of peaks and troughs. Each of the measures indicates that for a majority of the focal-onset seizures, synchronization or correlation or simultaneity occurs towards the end of the seizure or even after the offset rather than at the onset or in the beginning or during the progression of the seizure. We have also briefly discussed about a couple of synchronization dependent seizure termination mechanisms. Our conclusion is synchronization is an effect rather than the cause of a significant number of pharmacologically intractable focal-onset seizures. Since all the seizures that we have tested belong to the pharmacologically intractable class, their termination through more coherent neuronal activities may lead to new and effective ways of discovery and testing of drugs.