Evaluation of cockerel spermatozoa viability and motility by a novel enzyme based cell viability assay.

1. The results of spermatozoa assessment by the WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) assay, flow cytometry (FC) or computer-assisted sperm analysis (CASA) were compared. 2. Different live/killed ratios of cockerel semen were serially diluted to 120, 60, and 30 × 106 cells/ml, and each sample was analysed by (1) WST-8 assay at 0, 10, 20, 30, 40, 50, 60 min, (2) viability with FC, and (3) motility with CASA. 3. The WST-8 reduction rate was closely correlated with spermatozoa viability and motility. The optimal semen concentration for the WST-8 assay was 120 × 106 cells/ml, and the standard curves for spermatozoa viability and motility predictions, respectively, were yviability60 = 162.8x + 104.96 (R2 = 0.9594) after 60 min of incubation and ymotility40 = 225.09x + 96.299 (R2 = 0.8475) after 40 min of incubation. 4. It was concluded that the WST-8 assay is useful for the practical evaluation of cockerel spermatozoa viability and motility. Compared to FC and CASA, the WST-8 assay does not require expensive and complex instrumentation in the lab. Furthermore, one well of the WST-8 reaction can be used to predict spermatozoa viability and motility at the same time, which all lead it to be efficient and economical for semen quality assessment.

The role of TRPC6 in seizure susceptibility and seizure-related neuronal damage in the rat dentate gyrus.

Transient receptor potential canonical channel-6 (TRPC6) forms Ca(2+)-permeable non-selective cation channels in neurons. Although TRPC6 plays an important role in neurite outgrowth and neuronal survival during development, TRPC6 expression profiles available to identify distinctive hippocampal neuronal damage and hippocampal excitability in epilepsy are less defined. As compared to normal animals, TRPC6 expression was down-regulated in chronic epileptic rats showing spontaneous recurrent seizures. TRPC6 knockdown increased seizure susceptibility, excitability ratio and paired-pulse inhibition in the dentate gyrus (DG) of normal animals. Furthermore, TRPC6 knockdown promoted programmed neuronal necrosis in dentate granule cells, but prevented it in CA1 and CA3 neurons following status epilepticus. The present data suggest for the first time that TRPC6 may inhibit seizure susceptibility and neuronal vulnerability in the rat DG.

ETB receptor-mediated MMP-9 activation induces vasogenic edema via ZO-1 protein degradation following status epilepticus.

The blood-brain barrier (BBB) is formed by the endothelial cells with specialized tight junctions (TJs) lining the blood vessels and astroglial endfeet surrounding the blood vessels. Although BBB disruption during brain insults leads to vasogenic edema as one of the primary steps in the epileptogenic process, little is known about the molecular and physiological events concerning vasogenic edema formation. In the present study, status epilepticus (SE) changed the expressions and subcellular localizations of TJ proteins (claudin-5, occludin and zonula occludens-1 (ZO-1)) in endothelial cells of the rat piriform cortex. Among TJ proteins, the alteration in ZO-1 expression was relevant to endothelin B (ETB) receptor-mediated endothelial nitric oxide synthase (eNOS) activation, which increased matrix metalloproteinase-9 (MMP-9) activity. Indeed, BQ788 (an ETB receptor antagonist) effectively attenuated SE-induced vasogenic edema by inhibiting eNOS-mediated MMP-9 activation and ZO-1 protein degradation in endothelial cells, although astroglial endfeet were detached from endothelial cells. Therefore, we suggest that SE-induced ETB receptor/eNOS-mediated MMP-9 activation may lead to impairments of endothelial cell function via TJ protein degradation, which are involved in vasogenic edema formation independent of perivascular astroglial functions.

PARP1 activation/expression modulates regional-specific neuronal and glial responses to seizure in a hemodynamic-independent manner.

Poly(ADP-ribose) polymerase-1 (PARP1) plays a regulatory role in apoptosis, necrosis and other cellular processes after injury. Status epilepticus (SE) induces neuronal and astroglial death that show regional-specific patterns in the rat hippocampus and piriform cortex (PC). Thus, we investigated whether PARP1 regulates the differential neuronal/glial responses to pilocarpine (PILO)-induced SE in the distinct brain regions. In the present study, both CA1 and CA3 neurons showed PARP1 hyperactivation-dependent neuronal death pathway, whereas PC neurons exhibited PARP1 degradation-mediated neurodegeneration following SE. PARP1 degradation was also observed in astrocytes within the molecular layer of the dentate gyrus. PARP1 induction was detected in CA1-3-reactive astrocytes, as well as in reactive microglia within the PC. Although PARP1 inhibitors attenuated CA1-3 neuronal death and reactive gliosis in the CA1 region, they deteriorated the astroglial death in the molecular layer of the dentate gyrus and in the stratum lucidum of the CA3 region. Ex vivo study showed the similar regional and cellular patterns of PARP1 activation/degradation. Taken together, our findings suggest that the cellular-specific PARP1 activation/degradation may distinctly involve regional-specific neuronal damage, astroglial death and reactive gliosis in response to SE independently of hemodynamics.

Over-expression of laminin correlates to recovery of vasogenic edema following status epilepticus.

In the present study, we addressed the question of whether the up-regulation of laminin expression represents the astroglio-vascular responses to status epilepticus (SE) in the rat brain to better understand the role of vasogenic edema in epileptogenic insult. In the hippocampus, vasogenic edema was observed in the hippocampus 12h after SE when astroglial degeneration was undetected. Vasogenic edema in the hippocampus was more severe in the CA1 region where astroglial loss was absent than in the dentate gyrus showing astroglial degeneration. In the piriform cortex (PC), vasogenic edema was accompanied by appearance of astroglial degeneration 12h after SE. Laminin expression in the hippocampus and the PC was increased 3 days and 4 days after SE, respectively. Laminin expression was up-regulated in the hippocampus and the PC with concomitant reduction of SMI-71 (the endothelial barrier antigen) expression. Four weeks after SE, laminin expression was reduced in vessels showing strong SMI-71 expression within vasogenic edema lesion. Inhibition of SE-induced vasogenic edema formation by BQ788 effectively prevented laminin over-expression. Therefore, our findings indicate that laminin over-expression may be one of consequences from vasogenic edema rather than astroglial loss, and that laminin over-expression may promote migration of astrocytes to damaged or newly generated vessels to repair brain-blood barrier (BBB) disruption accompanied by the reconstruction of endothelial barrier.

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission.

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27(Kip1) expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27(Kip1)-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.

Reduction in heat shock protein 90 correlates to neuronal vulnerability in the rat piriform cortex following status epilepticus.

In the present study, we addressed the question of whether the distinct patterns of heat shock protein (HSP) 70 and HSP90 expressions in the brain region represents the regional specific responses to status epilepsticus (SE) in an effort to better understand the role of HSPs in epileptogenic insult. HSP70 immunoreactivity was increased in CA3 pyramidal cells as well as dentate granule cells at 12h-1week after SE. HSP70 immunoreactivity was transiently increased in neurons within the piriform cortex (PC) following SE. Linear regression analysis showed no correlation between the intensity of NeuN and that of HSP70. In contrast to HSP70, HSP90 immunoreactivity was decreased in CA1-3 pyramidal cells at 4days-4weeks after SE. In addition, HSP90 immunoreactivity was decreased in PC neurons at 12h-4weeks after SE. linear regression analysis showed a direct proportional relationship between the intensity of NeuN and that of HSP90. Therefore, these findings suggest that HSP90 degradation may be closely related to neuronal vulnerability to SE insult.

F-actin depolymerization accelerates clasmatodendrosis via activation of lysosome-derived autophagic astroglial death.

Clasmatodendrosis is an irreversible astroglial degenerative change, which includes extensive swelling and vacuolization of cell bodies and disintegrated and beaded processes. Since alteration in F-actin level influences on the formation of vacuoles/vesicles during exocytosis/endocytosis in astrocytes, we investigated whether F-actin polymerization involves clasmatodendrosis in the rat hippocampus following status epilepticus (SE). In the present study, vacuoles in clasmatodendrotic astrocytes showed LAMP-1 and LC3-II (a marker for autophagy) immunoreactivity. These findings reveal that clasmatodendrosis may be lysosome-derived autophagic astroglial death. Jasplakinolide (an F-actin stabilizer) infusion significantly decreased the size and the number of medium/large-sized vacuoles in each clasmatodendritic astrocyte accompanied by enhancement of phalloidin signals, as compared to vehicle-infusion. In contrast, latrunculin A (an F-actin-depolymerizing agent) infusion increased the size and the number of medium/large-sized vacuoles, which were dissociated adjacent to cell membrane. Therefore, our findings suggest that F-actin stabilization may inhibit lysosome-derived autophagic astroglial death during clasmatodendrosis.

ReLA/P65-serine 536 nuclear factor-kappa B phosphorylation is related to vulnerability to status epilepticus in the rat hippocampus.

Although nuclear factor-kappa B (NF-κB) is essential for neuron survival and its activation may protect neuron against oxidative-stresses or ischemia-induced neurodegeneration, NF-κB activation can contribute to inflammatory reaction and apoptotic cell death after brain injury and stroke. However, there are little data concerning the specific pattern of NF-κB phosphorylations in neuronal damage/survival induced by status epilepticus (SE). In the present study, NF-κB phosphorylation showed the cellular specific pattern in responses to SE. p52-S865, p52-Ser869, p65-Ser276, p65-Ser311, p65-Ser468, and p65-Ser529 NF-κB phosphorylation was significantly decreased in the CA1 and CA3 pyramidal cells vulnerable to SE, although neuronal specific nuclear antigen immunoreactivity was strongly detected. In contrast, p65-Ser536 NF-κB phosphorylation was enhanced in these neurons accompanied by TUNEL- and Fluoro-Jade B 244signals. These findings serve as the first comprehensive description of the cellular specific distribution of NF-κB phosphorylation in response to pilocarpine-induced SE in the rat hippocampus, and suggest that enhancement in p65-Ser536 NF-κB phosphorylation may be closely relevant to neuronal vulnerability to SE, while others may be involved in neuronal survival.

The roles of fractalkine/CX3CR1 system in neuronal death following pilocarpine-induced status epilepticus.

Although fractalkine is one of chemokines involved in mediation of neuronal/microglial interaction, it is not known whether fractalkine/CX3CR1-mediated pathogenesis occurs in the rat brain following epileptogenic insults. In order to elucidate the roles of the fractalkine/CX3CR1 system in microglial activation and neurodegeneration induced by status epilepticus (SE), we investigated changes in fractalkine/CX3CR1 system within the rat hippocampus following SE. In non-SE induced animals, fractalkine and CX3CR1 immunoreactivity was detected in neurons and microglia, respectively. Following SE, fractalkine immunoreactivity was transiently increased in neurons and astrocytes. CX3CR1 immunoreactivity was also transiently detected in neurons (particularly in CA1 pyramidal cells). Intracerebroventricular infusions of recombinant rat fractalkine aggravated SE-induced neuronal damage, while fractalkine IgG or CX3CR1 IgG infusion alleviated it, compared to saline-infused animals. These findings suggest that fractalkine/CX3CR1 system may play an important role in SE-induced neuronal damages via neuron-microglial interactions.

Association of CHRM2 polymorphisms with severity of alcohol dependence.

The cholinergic muscarinic 2 receptor (CHRM2) gene has been considered a candidate gene for the alcohol dependence in that it might underpin certain risk factors for this condition. This study examined variations in the CHRM2 between the patients with alcohol dependence and population controls in Korean and explored the associations between CHRM2 polymorphisms and severity of symptoms in the patients with alcohol dependence. One hundred and fifty-five patients with alcohol dependence, defined by the Alcohol Use Disorders Identification Test (AUDIT) and the Alcohol Dependence Scale (ADS) to measure the severity of symptoms, and one hundred and ninety-five population controls were drawn in the study. Three single nucleotide polymorphisms (SNPs) of CHRM2 were genotyped using the TaqMan assay and analyzed with the severity of symptoms of alcohol dependence. We found that although SNP rs324650 showed marginal association with the risk of alcohol dependence (P = 0.03), the significance of the result was not sustained after multiple corrections. SNP rs1824024 was significantly associated with the AUDIT and ADS scores in patients (P = 0.005 and 0.003, respectively). These findings suggested that the muscarinic acetylcholine function might be related not with alcohol dependence itself but with the severity of alcohol dependence in Korean population.

The protective effects of interleukin-18 and interferon-γ on neuronal damages in the rat hippocampus following status epilepticus.

To elucidate whether interleukin-18 (IL-18) or interferon-γ (IFN-γ) participates in neurodegeneartion, we investigated the changes in IL-18 and IFN-γ systems within the rat hippocampus following status epilepticus (SE). In non-SE induced animals, IL-18, IL-18 receptor α (IL-18Rα), IFN-γ and IFN-γ receptor α (IFN-γRα) immunoreactivity was not detected in the hippocampus. Following SE, IL-18 immunoreactivity was increased in CA1-3 pyramidal cells as well as dentate granule cells. IL-18 immunoreactivity was also up-regulated in astrocytes and microglia/macrophages. IL-18Rα immunoreactivity was detected in astrocytes and microglia/macrophages. IFN-γ immunoreactivity was detected only in astrocytes within all regions of the hippocampus. IFN-γRα immunoreactivity was increased in neurons as well as astrocytes. Intracerebroventricular infusions of recombinant rat IL-18 or IFN-γ alleviated SE-induced neuronal damages, while neutralization of IL-18, IFN-γ or their receptors aggravated them, as compared to saline-infused animals. These findings suggest that astroglial-mediated IFN-γ pathway in response to IL-18 induction may play an important role in alleviation of SE-induced neuronal damages.

Potential roles of D-serine and serine racemase in experimental temporal lobe epilepsy.

To confirm the roles of D-serinergic gliotransmission in epilepsy, we investigated the relationship between spatiotemporally specific glial responses and the D-serine/serine racemase system in mesial temporal structures following status epilepticus (SE). In control animals, D-serine and serine racemase immunoreactivities were detected mainly in astrocytes. After SE, D-serine and serine racemase immunoreactivities were increased in astrocytes. Double-immunofluorescence study revealed that up-regulation of serine racemase immunoreactivity was relevant not to D-serine immunoreactivity but to nestin or vimentin immunoreactivity. Neither D-serine nor serine racemase was found in naïve or reactive microglia. In addition, phosphorylated N-methyl-D-aspartate (NMDA) receptor subunit 1 (pNR1-Ser896) immunoreactivity in the hippocampus was increased compared with controls. Increased D-serine immunoreactivity showed direct correlation with the phosphorylation of Ser896 of NR1. Given the findings of our previous study, these findings suggest that D-serine and serine racemase in astrocytes may play roles in neuronal hyperexcitability via a cooperative activation of NMDA receptors. Furthermore, serine racemase may be involved in migration and differentiation of immature astrocytes, which is relevant to reactive astrogliosis.

Differential expressions of aquaporin subtypes in astroglia in the hippocampus of chronic epileptic rats.

In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus.

Three-dimensional anatomy of the temporal bone in normal mice.

This study was performed to determine the three-dimensional (3D) structure of the murine temporal bone and to provide a survey atlas of the temporal bone structures in mice. The temporal bones of adult BALB/c mice were examined and 3D high-resolution reconstructions of the temporal bone were obtained using a micro-CT system. Using the system described here, the bony labyrinth and membranous labyrinth could be investigated in a non-destructive manner. The turning rate of the cochlea was two (human rate: two and a half). The shapes of the superior and posterior semicircular canals were more flexed than those in humans. The malleus manubrium was directed anteriorly and was thin and fan-shaped like a Persian sword. The size of the incus relative to the malleus was smaller than that in the human ossicles. The 3D reconstruction of murine temporal bone described in this study provides anatomical information that will be useful in future studies using mouse model.

Enhanced pyridoxal 5'-phosphate synthetic enzyme immunoreactivities do not contribute to GABAergic inhibition in the rat hippocampus following pilocarpine-induced status epilepticus.

To comprehend the role of pyridoxal 5'-phosphate (PLP) in epilepsy or seizure, we investigated whether the expressions of two PLP synthetic enzymes (pyridoxal kinase, PLK; pyridoxine-5'-phosphate oxidase, PNPO) are altered in the hippocampus and whether changes in paired-pulse responses in the hippocampus are associated with altered PLP synthetic enzyme expressions following status epilepticus (SE). PLK and PNPO immunoreactivities were significantly increased in the rat hippocampus accompanied by reductions in paired-pulse inhibition at 1 day and 1 week after SE. Four weeks after SE, PLK and PNPO immunoreactivities in dentate granule cells were similar to those in control animals, while their immunoreactivities were markedly reduced in Cornu Ammonis 1 (CA1) pyramidal cells due to neuronal loss. Linear regression analysis identified a direct proportional relationship between PLK/PNPO immunoreactivity and normalized population spike amplitude ratio in the dentate gyrus and the CA1 region as excluded the data obtained from 4 weeks after SE. These findings indicate that the upregulation of PLK and PNPO immunoreactivities in principal neurons may not be involved in gamma-aminobutyric acid (GABA)ergic inhibition, but rather in enhanced excitability during epileptogenic periods.

Anti-glutamatergic effect of riluzole: comparison with valproic acid.

Riluzole, an anti-amyotrophic lateral sclerosis drug, known to decrease presynaptic glutamate release, is viewed as a candidate supplementary medication for epilepsy. In the present study, we compared the effects of riluzole and valproate (VPA) in the pilocarpine-induced limbic seizure model and in the gamma-hydroxybutyrate lactone (GBL)-induced absence seizure model. We applied immunohistochemical study for vesicular transporter 1 (VGLUT1) and extracellular recording in the rat dentate gyrus of both pilocarpine- and GBL-induced seizure models to measure effects of riluzole and VPA. Both VPA and riluzole treatments reduced VGLUT1 immunoreactivity. Riluzole treatment completely inhibited pre-ictal spikes and spike-wave discharges in the pilocarpine- and GBL-induced epilepsy models, whereas VPA partially inhibited these phenomena. In both seizure models, the anti-epileptic effects of VPA and riluzole are basically related to anti-glutamatergic (reducing field excitatory postsynaptic potential slope and excitability ratio), not GABAergic (paired-pulse inhibition) effect. Riluzole was more effective at reducing seizure activity in both epilepsy models than VPA. These results suggest that riluzole is a potential antiepileptic drug with activity against limbic seizure and absence seizure.

Transient ischaemia affects plasma membrane glutamate transporter, not vesicular glutamate transporter, expressions in the gerbil hippocampus.

In the present study, we investigated expressions of vesicular glutamate transporter (VGLUT) and of the plasma membrane glutamate transporters [glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC-1)] in the gerbil hippocampus following transient ischaemia. The expressional levels and distribution patterns of VGLUT immunoreactivities were unaltered until 3 days after ischaemic-insults. However, VGLUT-2 immunoreactivity in the CA1 region was reduced at 4 days after ischaemia due to delayed neuronal death. In addition, both GLT-1 and GLAST immunoreactivities in the CA1 region were enhanced at 30 min - 12 h after ischaemia-reperfusion and their expression began to reduce at 24 h after ischaemia-reperfusion. In contrast, EAAC-1 immunoreactivity was transiently reduced in the CA1 region at 30 min after ischaemia, re-enhanced at 3-12 h after ischaemia, and re-reduced at 24 h after ischaemia. These findings suggest that malfunctions of plasma membrane glutamate transporters, not of VGLUT, may play an important role in the elevation of extracellular glutamate concentration following ischaemic insults.

Immunohistochemical localization of glutamate in the gerbil main olfactory bulb using an antiserum directed against glutamate.

Information on the localization and the roles of glutamate in the nervous system is becoming valuable because the axon terminals of the olfactory sensory neurons and the synapses of the mitral and tufted output cells appear to be glutamatergic. In this study, we have analysed the distribution of glutamate immunoreactivity in the main olfactory bulb (MOB) of the Mongolian gerbil using an antiserum directed against glutamate. Glutamate immunoreactivity in the MOB was present in the olfactory nerve layer (Onl), glomerular layer (GL), external plexiform layer (EPL) and mitral cell layer (ML), but not in the granule cell layer (GCL). Glutamate immunoreactivity detected in the Onl was thought to be terminal ramifications of glomeruli. Some neurons in the periglomerular region showed glutamate immunoreactivity. In the EPL, glutamate immunoreactivity was found in some neuronal somata (tufted cells) and processes. In addition, mitral cells in the ML were labelled by the glutamate antibody. The pattern of glutamate immunoreactivity in the mitral cells was similar to that in the tufted cells. In brief, glutamate in the gerbil MOB is the neurotransmitter used by primary afferents and output neurons.

Reduced calcium binding protein immunoreactivity induced by electroconvulsive shock indicates neuronal hyperactivity, not neuronal death or deactivation.

Calcium-binding proteins (CBPs), such as parvalbumin and calbindin D-28k, are useful markers of specific neuronal types in the CNS. In recent studies, expression of CBPs may be indicative of a deactivated neuronal state, particularly epilepsy. However, it is controversial whether altered expression of CBPs in the hippocampus practically indicate neuronal activity. Therefore, the present study was performed to investigate the extent of profiles of expression of CBPs in the rat hippocampus affected by several episodes induced by electroconvulsive shock. In the present study, following electroconvulsive shock expression of CBPs were reduced in the hippocampus in a stimulus-dependent manner, and recovered to the control level at 6 h after electroconvulsive shock. However, paired-pulse responses of the dentate gyrus were transiently impaired by electroconvulsive shock, and immediately normalized to baseline value. In addition, effects of electroconvulsive shock on expression of CBPs and paired-pulse responses were prevented by pretreatment of vigabatrin. These findings suggest that reduced expression of CBPs induced by seizure activity may be indicative of hyperactivity of CBP positive neurons, which is a practical consequence of the abnormal discharge, and that they may play an important role in regulating seizure activity.