Thimmasettappa Thippeswamy

Glial‐mediated neuroprotection: Evidence for the protective role of the NO‐cGMP pathway via neuron–glial communication in the peripheral nervous system

The NO‐cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)‐deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co‐culture of neonatal DRG neurons with either Schwann cells pre‐treated with an NO donor or a membrane‐permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO‐sGC‐cGMP‐mediated NGF and NT3 synthesis by Schwann cells protect neurons.

Inhibition of neuronal nitric oxide synthase results in neurodegenerative changes in the axotomised dorsal root ganglion neurons : evidence for a neuroprotective role of nitric oxide in vivo

In axotomised adult rat dorsal root ganglion (DRG), many neurons show a marked increase in expression of neuronal nitric oxide synthase (nNOS). It has been established that NO functions as a neuron-glial signalling molecule by generating cGMP in glia cells that surround the neuron in DRG. Furthermore, in cultures of dissociated DRG deprived of nerve growth factor, many neurons expressed nNOS and cGMP and subsequently died if either enzymes activity was inhibited suggesting that NO-cGMP pathway could be neuroprotective in stressed DRG neurons. This has now been tested in vivo. It was found, 10 days after sciatic axotomy that nNOS was expressed in 36% of DRG neurons in the L5 and L6 ganglia giving rise to the damaged nerve, compared with 6% in contralateral ganglia. Almost all nNOS neurons and 24% of non-nNOS neurons expressed c-Jun in their nuclei. Ten days following axotomy, treatment with the relatively selective nNOS-blocker, 1-(2-trifluoromethylphenyl) imidazole (TRIM), caused morphology changes in approximately 50% of neurons that consisted of vacuolations, blebbing and highly irregular cell boundaries. Sham operated, TRIM treated, nerve-sectioned, vehicle treated, and controls did not show these changes. These observations further support the view that NO could be neuroprotective in some injured/stressed primary sensory neurons.

Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non‐nNOS neurons. Hence, NO or the NO–cGMP cascade appears to have a neuroprotective action in trophic factor‐deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l‐arginine completely abolished this effect in almost all nNOS neurons and 8‐bromo‐cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase‐9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO‐cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase‐3 and ‐9 activation.

The Roles of Nitric Oxide in Dorsal Root Ganglion Neurons

Abstract: The functions of nitric oxide (NO) in primary somatosensory neurons are reviewed. During the early development of these neurons the neuronal isoform of nitric oxide synthase (nNOS) is expressed during neurite extension. As their axons extend peripherally and start to take up nerve growth factor (NGF), nNOS starts to disappear from the majority of these neurons. A small number (less than 5%) continue to express nNOS, and for this small population NO may have a role in synaptic transmission. Following peripheral nerve section in adult rats, nNOS is reexpressed in many small peptidergic DRG neurons that have been axotomized. At the same time cGMP synthesis is increased in satellite glia cells. From culture studies, it was established that NGF negatively regulates nNOS synthesis in DRG neurons and that block of NO production leads to neuronal death. Further recent data is reviewed that supports the view that NO has a neuroprotective action preventing loss of DRG neurons and facilitates regeneration.

Seizure-Induced Oxidative Stress in Temporal Lobe Epilepsy

An insult to the brain (such as the first seizure) causes excitotoxicity, neuroinflammation, and production of reactive oxygen/nitrogen species (ROS/RNS). ROS and RNS produced during status epilepticus (SE) overwhelm the mitochondrial natural antioxidant defense mechanism. This leads to mitochondrial dysfunction and damage to the mitochondrial DNA. This in turn affects synthesis of various enzyme complexes that are involved in electron transport chain. Resultant effects that occur during epileptogenesis include lipid peroxidation, reactive gliosis, hippocampal neurodegeneration, reorganization of neural networks, and hypersynchronicity. These factors predispose the brain to spontaneous recurrent seizures (SRS), which ultimately establish into temporal lobe epilepsy (TLE). This review discusses some of these issues. Though antiepileptic drugs (AEDs) are beneficial to control/suppress seizures, their long term usage has been shown to increase ROS/RNS in animal models and human patients. In established TLE, ROS/RNS are shown to be harmful as they can increase the susceptibility to SRS. Further, in this paper, we review briefly the data from animal models and human TLE patients on the adverse effects of antiepileptic medications and the plausible ameliorating effects of antioxidants as an adjunct therapy.

Nerve growth factor inhibits the expression of nitric oxide synthase in neurones in dissociated cultures of rat dorsal root ganglia

In dissociated cultures of DRG derived from 15-day-old rats the numbers of neurones expressing immunocytochemically detectable quantities of the neuronal isoform of nitric oxide synthase (nNOS) was determined and the effects of different culture media examined. The availability of NGF in the cultures was found to be a critical determinant of nNOS expression. In a serum-rich media (SRM) supplemented with NGF, 24% of the neurones expressed nNOS compared with 72% in the absence of added NGF and the presence of an antibody to NGF (t-test, P < 0.0001). Cultures grown in a defined media (DM) developed poorly and many neurones died, these cultures also showed poor growth of other cell types. Immunostaining for NGF revealed that some of the non-neuronal cells produce NGF and that this would be predicted to contribute to the survival of the neurones. In cultures in which neurones were dying most of the surviving neurones expressed nNOS suggesting it may have a survival promoting function.

Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies.

NO‐cGMP mediated galanin expression in NGF‐deprived or axotomized sensory neurons

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5′ proximal promoter fragment (− 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF‐deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above‐mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF‐deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF‐deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant‐negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO‐cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up‐regulation of galanin, and for DRG neuron survival and regeneration after axotomy.

Advantages of Repeated Low Dose against Single High Dose of Kainate in C57BL/6J Mouse Model of Status Epilepticus: Behavioral and Electroencephalographic Studies

A refined kainate (KA) C57BL/6J mouse model of status epilepticus (SE) using a repeated low dose (RLD) of KA (5 mg/kg, intraperitoneal; at 30 min intervals) was compared with the established single high dose (SHD) of KA (20 mg/kg, intraperitoneal) model. In the RLD group, increased duration of convulsive motor seizures (CMS, Racine scale stage ≥3) with a significant reduction in mortality from 21% to 6% and decreased variability in seizure severity between animals/batches were observed when compared to the SHD group. There was a significant increase in the percentage of animals that reached stage-5 seizures (65% versus 96%) in the RLD group. Integrated real-time video-EEG analysis of both groups, using NeuroScore software, revealed stage-specific spikes and power spectral density characteristics. When the seizures progressed from non-convulsive seizures (NCS, stage 1–2) to CMS (stage 3–5), the delta power decreased which was followed by an increase in gamma and beta power. A transient increase in alpha and sigma power marked the transition from NCS to CMS with characteristic ‘high frequency trigger’ spikes on the EEG, which had no behavioral expression. During SE the spike rate was higher in the RLD group than in the SHD group. Overall these results confirm that RLD of KA is a more robust and consistent mouse model of SE than the SHD of KA mouse model.

Immediate Epileptogenesis after Kainate-Induced Status Epilepticus in C57BL/6J Mice: Evidence from Long Term Continuous Video-EEG Telemetry.

The C57BL/6J mouse as a model of seizure/epilepsy is challenging due to high mortality and huge variability in response to kainate. We have recently demonstrated that repeated administration of a low dose of kainate by intraperitoneal route can induce severe status epilepticus (SE) with 94% survival rate. In the present study, based on continuous video-EEG recording for 4-18 weeks from epidurally implanted electrodes on the cortex, we demonstrate that this method also induces immediate epileptogenesis (<1-5 days post-SE). This finding was based on identification of two types of spontaneous recurrent seizures; behavioral convulsive seizures (CS) and electrographic nonconvulsive seizures (NCS). The identification of the spontaneous CS, stage 3-5 types, was based on the behaviors (video) that were associated with the EEG characteristics (stage 3-5 epileptiform spikes), the power spectrum, and the activity counts. The electrographic NCS identification was based on the stage 1-2 epileptiform spike clusters on the EEG and their associated power spectrum. Severe SE induced immediate epileptogenesis in all the mice. The maximum numbers of spontaneous CS were observed during the first 4-6 weeks of the SE and they decreased thereafter. Mild SE also induced immediate epileptogenesis in some mice but the CS were less frequent. In both the severe and the mild SE groups, the spontaneous electrographic NCS persisted throughout the 18 weeks observation period, and therefore this could serve as a chronic model for complex seizures. However, unlike rat kainate models, the C57BL/6J mouse kainate model is a unique regressive CS model of epilepsy. Further studies are required to understand the mechanism of recovery from spontaneous CS in this model, which could reveal novel therapeutic targets for epilepsy.