Viruses and the immune system: their roles in seizure cascade development

Viral encephalitis affects approximately 7.5 people/100 000 and carries a high rate of morbidity and mortality. Most patients with viral encephalitis will develop some form of seizure during the infectious process, and of those who survive encephalitic disease, approximately 4–20% will develop epilepsy. Arthropod-borne (arbo)viruses are the leading cause of viral encephalitis in the world today, with between 10% and 35% of patients infected with these viruses displaying some form of seizure. Several neurotropic DNA viruses, including Herpes and cytomegalovirus also commonly cause seizures in infected patients. In the clinical setting, the cause of seizures seen during viral encephalitis is usually attributed to acute febrile responses. However, it has become apparent that the mechanisms behind seizure generation during viral encephalitis are likely to be much more complicated. For example, CD4⁺ and CD8⁺ T cells possibly through their secretion of interferon-γ, appear to play an important role in determining neuronal responses when challenged with kainic acid. In addition, the ability of the human immunodeficiency virus, transactivating protein to modulate NMDA signaling possibly triggering seizures, highlights the fact that elements of the antiviral response and even virally derived proteins are capable of directly manipulating neuronal function. Understanding the complex relationships between the CNS, the immune system, and invading pathogens is a critical step in understanding the pathogenesis of seizures seen during viral infections and informing the development of novel therapies.     

Expression of functional Kir6.1 channels regulates glutamate release at CA3 synapses in generation of epileptic form of seizures

The Kir6.1 channels are a subtype of ATP-sensitive inwardly rectifying potassium (K_ATP) channels that play an essential role in coupling the cell's metabolic events to electrical activity. In this study, we show that functional Kir6.1 channels are located at excitatory pre-synaptic terminals as a complex with type-1 Sulfonylurea receptors (SUR1) in the hippocampus. The mutant mice with deficiencies in expressing the Kir6.1 or the SUR1 gene are more vulnerable to generation of epileptic form of seizures, compared to wild-type controls. Whole-cell patch clamp recordings demonstrate that genetic deletion of the Kir6.1/SUR1 channels enhances glutamate release at CA3 synapses. Hence, expression of functional Kir6.1/SUR1 channels inhibits seizure responses and possibly acts via limiting excitatory glutamate release.     

Benefit of nicorandil using an immunologic murine model of experimental colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory condition with an unknown etiology. Nicorandil, a potassium channel opener, has been used for many years for the treatment of angina. Recently, it has been shown that nicorandil possesses some novel traits such as anti-apoptotic, gastroprotective, free radical scavenging, and anti-inflammatory properties. Therefore, we set out to examine the possible beneficial effect of nicorandil in a rat model of IBD. Colitis was induced by rectal administration of 2,4,6-trintrobenzene sulphonic acid (TNBS) into rats. Groups of animals used in this study were sham, control, and exposure to dexamethasone, nicorandil, glibenclamid (a pure adenosine triphosphate sensitive potassium channel (KATP) blocker), or nicorandil plus glibenclamid. Drugs were administered by gavage and animals were sacrificed after 7 days. Biochemical markers, including TNF-α and IL-1β, ferric reducing/antioxidant power (FRAP), myeloperoxidase (MPO) activity and thiobarbitoric acid-reactive substance (TBARS), were measured in the homogenate of colonic tissue. Results indicate that nicorandil significantly reduces macroscopic and histological damage induced by TNBS. Nicorandil diminishes MPO activity and levels of TBARS, TNF-∢, and IL-1β in damaged colonic tissue with a concomitant increase in FRAP value (P<0.01). These effects were not reversed by coadministration of glibenclamide. In conclusion, nicorandil is able to ameliorate experimental IBD with a dose in which it does not show any anti-hypertensive effect, and the mechanism of which is partially or totally independent from KATP channels. It is hypothesized that nitric oxide donation and free-radical scavenging properties of nicorandil upregulate endothelial nitric oxide synthase may be responsible for this phenomenon. These findings suggest that nicorandil can be useful in treatment of IBD, although further investigations are needed to elucidate the mechanisms involved.     

Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis

Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H(2)O(2), NO production, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H(2)O(2) production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-alpha, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival.     

Decreased antiviral immune response within the central nervous system of aged mice is associated with increased lethality of West Nile virus encephalitis

West Nile virus (WNV) is an emerging pathogen that causes disease syndromes ranging from a mild flu‐like illness to encephalitis. While the incidence of WNV infection is fairly uniform across age groups, the risk of lethal encephalitis increases with advanced age. Prior studies have demonstrated age‐related, functional immune deficits that limit systemic antiviral immunity and increase mortality; however, the effect of age on antiviral immune responses specifically within the central nervous system (CNS) is unknown. Here, we show that aged mice exhibit increased peripheral organ and CNS tissue viral burden, the latter of which is associated with alterations in activation of both myeloid and lymphoid cells compared with similarly infected younger animals. Aged mice exhibit lower MHCII expression by microglia, and higher levels of PD1 and lower levels of IFNγ expression by WNV‐specific CD8⁺ T cells in the CNS and CD8⁺CD45⁺ cells. These data indicate that the aged CNS exhibits limited local reactivation of T cells during viral encephalitis, which may lead to reduced virologic control at this site.     

Type 1 diabetes exacerbates blood–brain barrier alterations during experimental epileptic seizures in an animal model

The aim of this study was to perform the effects of diabetes on the permeability of the blood–brain barrier (BBB) during pentylenetetrazole (PTZ)‐induced epileptic attacks. For this propose, the animals were divided into four groups. These groups contained were intact, PTZ‐treated, diabetic and PTZ‐treated diabetic individuals, respectively. To evaluate the functioning of the BBB, Evans blue was used as a BBB permeability indicator, and the expressions of zonula occludens‐1 and glial fibrillary acidic protein involving the functioning of the BBB were determined immunohistochemically. Also, the changes in the release of serum tumour necrosis factor‐alpha and interleukin‐10 and interleukin‐12 were studied by using enzyme‐linked immunosorbent assay method. BBB permeability in the seizures under diabetic conditions showed a considerable increase (p < 0·01) in all of the brain we studied. The immunoreactive staining intensity of zonula occludens‐1 and glial fibrillary acidic protein was found reduced in the brain regions of diabetic rats (p < 0·01). However, the serum level of tumour necrosis factor‐alpha increased in diabetes and diabetes + PTZ groups, and the serum level of interleukin‐12 increased significantly in all experimental groups (p < 0·05). In conclusion, diabetes dramatically increases BBB damage during epileptic seizures, and it may be derived from an elevation of paracellular passage. Copyright © 2015 John Wiley & Sons, Ltd.     

Role of K(ATP) channels in protection against neuronal excitatory insults

ATP-sensitive K⁺ (K_ATP) channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of K_ATP channels during energy deficits in the CNS induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of K_ATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. In this review, we bring together recent studies about what kinds of molecules are needed to build and regulate arrays of K_ATP channel functions in the CNS neurons. We propose a model to explain how K_ATP channel activation regulates glutamate release from the pre-synaptic terminals and how this regulation protects against ischemic neuronal injury and epilepsy.     

The inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis

Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.     

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.     

Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.³²P-labelled cells have been stimulated with 100 M glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.Abbreviations (NMDA) N-methyl-D-aspartate - (PKC) protein kinase C - (EAA) excitatory aminoacids - (GAMSA) -D-glutamylaminomethylsulfonate - (MK801) (+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine - (TPA) phorbol 12-myristate 13-acetate - (MARCKS) myristoylated alanine-rich C kinase substrate - (GAP-43) growth-associated protein-43 - (SDS) sodium dodecyl sulfate - (PAGE) polyacrylamide gel electrophoresis - (H7) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - (DIV) days in vitro     

Role of VEGF in an experimental model of cortical micronecrosis

Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke.After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis.     

Cyclosporine lowers seizure threshold in an experimental model of electroshock-induced seizures in Munich-Wistar rats

We have developed a model of cyclosporin A (CsA) central nervous system toxicity in the Munich-Wistar rat in which CsA, 20 mg/kg/day i.p., produces significant EEG abnormalities and mortality. In the present study we used cohorts of Munich-Wistar rats to assess effects of CsA on the threshold for tonic-clonic electroshock-induced seizures. Rat cohorts were begun on cremephore, CsA-10 mg/kg/day, or CsA-20 mg/kg/day. On day 7 and day 14 of the dosing protocol, cohorts of animals were exposed to maximal electroshock (MES) using a minimal staircase method within each cohort. Multiple logistic regression models were used to determine differences between groups on the relative odds of producing a MES-induced seizure while controlling for other variables. Seizure threshold was significantly affected by shock amperage and body weight, but not by SUN, creatinine, bilirubin, sodium, potassium, weight loss or day the shock was delivered. The odds ratios of seizure induction in the CsA-treated groups versus placebo group were 1.91 for CsA-10 mg/kg/day and 3.63 for CsA 20-mg/kg/d, both statistically significant. These results suggest that cyclosporine lowers seizure threshold and probably increases susceptibility to seizures, the etiology of which may be multifactorial clinically.     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

Comparative study of four antifungal drugs in an experimental model of murine cryptococcosis

A comparative study among amphotericin B, 5-fluorocytosine, itraconazole and fluconazole in the treatment of experimental cryptococcosis in mice, was carried out.Seventy male Balb C mice were inoculated intraperitoneally with 10⁷ cells of Cryptococcus neoformans var. neoformans. They were divided in 7 groups of 10 animals each one: 1) treated with fluconazole by gavage at a daily dose of 16 mg/kg; 2) treated with itraconazole by gavage at a daily dose of 16 mg/kg; 3) treated with 5-fluorocytosine by gavage at a daily dose of 300 mg/kg; 4) treated with amphotericin B intraperitoneally at a dose of 6 mg/kg every other day; 5) control animals receiving polietilenglicol 200 by gavage; 6) control animals receiving distilled water by gavage and 7) control animals receiving sterile distilled water by intraperitoneal route. All the treatments started 5 days after the challenge inoculation and they were given for 2 weeks.The following parameters were taken into account: survival time, macroscopic aspect of the organ after the complete autopsy, microscopic investigation of yeasts in brain, lungs, spleen and liver, histopathology studies of these organs, the colony forming units per gram and massive seeding of brain and lungs.The survival index of the different groups was the most efficient method to measure the antifungal compounds activity. Amphotericin B increased significantly the animals survival and modified the histopathologic response in the studied organs. The colony forming units and the massive seeding in brain and lung showed that this antifungal agent is unable of producing the biological cure of this experimental model.The remaining drugs assayed did not promote important modifications in the histopathologic picture as well as in the tissues cultures. However, the three drugs increased the animals survival. In this aspect, fluconazole proved to be slightly superior.     

Regional brain serotonin synthesis is increased in the olfactory bulbectomy rat model of depression: an autoradiographic study

Serotonin synthesis rates were evaluated using alpha-[14C]methyl-l-tryptophan (alpha-MTrp) autoradiographic methods in olfactory bulbectomized (OBX) rats. They were significantly (p < 0.05) increased in the frontal (50%) and parietal (40%) cortices, superior olive (over 30%), and the substantia nigra (30%) in the OBX rats as compared to the sham operated animals. There were also increases in 5-hydroxytryptamine (5-HT) synthesis in some limbic areas: the cingulate (32%), the medial forebrain bundle (58%), the hippocampus (13-25%) and the thalamus (22-40%). The largest increase in 5-HT synthesis after OBX was observed in the sensory-motor cortex (67%). 5-HT synthesis rates were significantly decreased in the dorsal and medial raphe nuclei, but there was no significant change the ventral tegmental area and the locus coeruleus following OBX. These results indicate that olfactory bulbectomy causes an imbalance in 5-HT synthesis in some projection areas by disproportionally increasing 5-HT synthesis rates in specific brain regions and making more 5-HT available for neurotransmission. This imbalance in 5-HT synthesis and the subsequent elevation of tissue 5-HT may be responsible for the creation of non-physiological circuitry which may, in part, be reflected in the symptoms resembling human depression.     

Synaptosomal glutamate uptake in a model of experimental cerebral ischemia

In the present investigation we studied the synaptosomal uptake of glutamate in brain omogenate of Mongolian gerbils submitted to bilateral common carotid occlusion, with and without subsequent return of blood flow. The results show that glutamate uptake after ischemia is reduced by about 35% The damage appears to be persistent, since return of blood flow restores uptake only slightly. The membrane alterations occurring in ischemia could explain the persistence of glutamate transporter impairment. Besides the blockade of NMDA receptors, the stimulation and/or the protection of the uptake systems for glutamate could be of help in preventing neuronal ischemic damage.     

Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region

West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8) and 10(8.5) plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.     

Role of boundary conditions in an experimental model of epithelial wound healing

Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.