Chronic vagus nerve stimulation in Crohn's disease: a 6-month follow-up pilot study.

The vagus nerve (VN) is a link between the brain and the gut. The VN is a mixed nerve with anti-inflammatory properties through the activation of the hypothalamic-pituitary-adrenal axis by its afferents and by activating the cholinergic anti-inflammatory pathway through its efferents. We have previously shown that VN stimulation (VNS) improves colitis in rats and that the vagal tone is blunted in Crohn's disease (CD) patients. We thus performed a pilot study of chronic VNS in patients with active CD. Seven patients under VNS were followed up for 6 months with a primary endpoint to induce clinical remission and a secondary endpoint to induce biological (CRP and/or fecal calprotectin) and endoscopic remission and to restore vagal tone (heart rate variability). Vagus nerve stimulation was feasible and well-tolerated in all patients. Among the seven patients, two were removed from the study at 3 months for clinical worsening and five evolved toward clinical, biological, and endoscopic remission with a restored vagal tone. These results provide the first evidence that VNS is feasible and appears as an effective tool in the treatment of active CD.

Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway.

BACKGROUND: The brain and the gut communicate bidirectionally through the autonomic nervous system (ANS). The vagus nerve (VN), a major component of the ANS, plays a key role in the neuro-endocrine-immune axis to maintain homeostasia through its afferents (through the activation of the hypothalamic pituitary adrenal axis and the central ANS) and through its efferents (i.e. the cholinergic anti-inflammatory pathway; CAP). The CAP has an anti-TNF effect both through the release of acetylcholine at the distal VN acting on macrophages and through the connection of the VN with the spleen through the splenic sympathetic nerve. Vagus nerve stimulation (VNS) of vagal afferents at high frequency (20-30 Hz) is used for the treatment of drug-resistant epilepsy and depression. Low-frequency (5 Hz) VNS of vagal efferents activates the CAP for an anti-inflammatory effect that is as an anti-TNF therapy in inflammatory diseases were TNF is a key cytokine as represented by experimental sepsis, postoperative ileus, burn-induced intestinal barrier injury, colitis. However, both vagal afferents and efferents are activated by VNS. PURPOSE: The objective of this review was to explore the following: (i) the supporting evidence for the importance of VNS in epilepsy (and depression) and its mechanisms of action, (ii) the anti-inflammatory characteristics of the VN, (iii) the experimental evidence that VNS impact on inflammatory disorders focusing on the digestive tract, and (iv) how VNS could potentially be harnessed therapeutically in human inflammatory disorders such as inflammatory bowel diseases, irritable bowel syndrome, postoperative ileus, rheumatoid arthritis as an anti-inflammatory therapy.

Anti-inflammatory effect of vagus nerve stimulation in a rat model of inflammatory bowel disease.

Vagus nerve stimulation of afferents is used as an adjunctive treatment for drug-resistant epilepsy and depression. In addition, anti-inflammatory properties of vagus nerve stimulation have been reported in various experimental models of inflammation but not in colitis. These effects are thought to be mediated via peripheral release of acetylcholine from the vagus and subsequent activation of macrophages. Our aim was to evaluate in rats the anti-inflammatory effects of chronic vagus nerve stimulation on colonic inflammation. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Vagus nerve stimulation (left cervical) was performed in freely moving animals 3 h per day for five consecutive days. Assessment of colonic inflammation was obtained using physiological (e.g. body weight, temperature and locomotor activity) parameters, macroscopical (area of lesions), histological, and biological parameters (e.g. myeloperoxidase activity, cytokine and cytokine-related mRNAs), both at the level of the damaged colon and the colon immediately above. A global multivariate index of colitis was then generated for a better characterization of colonic inflammation. Vagus nerve stimulation reduced the degree of body weight loss and inflammatory markers as observed above the lesion by histological score and myeloperoxidase quantification. This anti-inflammatory effect was also demonstrated by the improvement of the multivariate index of colitis. These data argue for an anti-inflammatory role of vagus nerve stimulation chronically performed in freely moving rats with colitis and provide potential therapeutic applications for patients with inflammatory bowel diseases.

Therapeutic efficacy of intra-cochlear administration of methylprednisolone after acoustic trauma caused by gunshot noise in guinea pigs.

The therapeutic efficacy of cochlear infusion of methylprednisolone (MP) after an impulse noise trauma (170dB SPL peak) was evaluated in guinea pigs. The compound action potential threshold shifts were measured over a 14 days recovery period after the gunshot exposure. For each animal, one of the cochlea was perfused directly into the scala tympani with MP during 7 days via a mini-osmotic pump, whereas the other cochlea was not pump-implanted. The functional study of hearing was supplemented by histological analysis. Forty eight hours after the trauma, significant differences between auditory threshold shifts in the implanted and non-implanted ears were observed for frequencies above 8kHz. At day 7, the difference was significant for only one frequency and no difference was observed after 14 days recovery. Cochleograms showed that the hair cell losses were significantly lower in the MP treated ears. This work indicates that direct infusion of MP into perilymphatic space accelerates hearing recovery, reduces hair cell losses after impulse noise trauma but does not limit permanent threshold shifts.

Spectrum of neural electrical activity in guinea pig cochlea: effects of anaesthesia regimen, body temperature and ambient noise.

Spectral analysis of electric noise recorded from the round window of the cochlea is thought to represent the summed spontaneous activity of the auditory nerve. It has been postulated that it could provide a possible tinnitus index. Because experimental conditions could change this neural activity, the effect of anaesthesia regimen, body temperature and ambient noise on the spectrum of spontaneous neural noise (SNN) were investigated in guinea pig cochlea. SNN was studied in awake guinea pigs and after anaesthesia with pentobarbital (P), xylazine/ketamine (XK) or xylazine/tiletamine-zolazepam (XTZ). Body temperature varied gradually from 33 to 41 degrees C under XK regimen. In awake animals, broadband noise was generated with intensity varying from 0 to 50 dB. The SNN consisted in a broad peak at approximately 900 Hz. With ambient broadband noise, it increased exponentially with the sound level with no shift in frequency. Soon after anaesthetic induction, the lowest frequencies were constantly decreased, and gradually the 900 Hz peak either increased moderately (P) or dropped steeply (XTZ) or remained unchanged (XK). Peak frequency increased linearly with body temperature whereas the amplitude reached a maximum at around 39.5 degrees C. In conclusion, these data indicate that experimental conditions such as anaesthesia regimen, body temperature and ambient noise modify the spontaneous neural outflow of the cochlea and must be taken into account when studying SNN.

Nitric oxide voltammetric measurements in the rat brain after gamma irradiation.

The effects of a lethal gamma irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude (+120%) 24 h later. A dose-effect relationship from 2 to 15 Gy for gamma-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca(++)-independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after gamma-ray exposure. These results demonstrate that a supralethal whole-body irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome.

IL-1beta, TNFalpha and IL-6 induction in the rat brain after partial-body irradiation: role of vagal afferents.

PURPOSE: To evaluate the central nervous system neuroimmune and inflammatory responses during the prodromal phase of the acute irradiation syndrome in rat brains after partial-body exposure (head-protected) and to investigate the potential neural signalling pathways from the irradiated periphery to the non-irradiated brain. MATERIAL AND METHODS: The study included four groups of rats: one irradiated group and one sham irradiated group, each containing non-vagotomized and vagotomized rats. In vagotomized rat groups, the subdiaphragmatic vagal section surgery was carried out 45 days before the irradiation exposure. The rats were partial-body irradiated with the head shielded with (60)Co gamma-rays to a dose of 15 Gy. They were sacrificed 6 h after the end of exposure. The hypothalamus, hippocampus, thalamus and cortex were then collected, and the concentrations of IL-1beta, TNFalpha and IL-6 in each were measured by ELISA assays. RESULTS: Six hours after irradiation, IL-1beta levels had increased in the hypothalamus, thalamus and hippocampus, and TNFalpha and IL-6 levels had increased significantly in the hypothalamus. Vagotomy before irradiation prevented these responses. CONCLUSIONS: It was concluded that the hypothalamus, hippocampus, thalamus and cortex react rapidly to peripheral irradiation by releasing pro-inflammatory mediators. The results also show that the vagus nerve is one of the major ascending pathways for rapid signalling to the brain with respect to partial body irradiation.

Effect of heavy ions on neuro-endocrine regulations.

During American and Russian short and long-term space flights neuroimmune dysregulations have been observed in man and rats for up to three months after the return. During Extra-Vehicular Activity, radiation exposure risk is greater to elicit short and/or long-term deleterious effects on the functional capacity of the neuroimmune system. In order to assess the effects of high LET events on neuroimmune networks, our preliminary ground-based study was to investigate brain inflammatory responses in mouse after low dose radiation exposure with high LET particles (12C, 95MeV/u, 42 mGy). Plasma corticosterone levels were rapidly (6 hours) increased by two-fold, then decreased 24 hours post-irradiation. At 3 days plasma corticosterone and ACTH concentrations were also two- to three-fold increased. Plasma ACTH levels were still elevated up to seven days to two months. Furthermore immune functions are under current assessment. The results of this study should allow a greater understanding of the effects of high LET particles on neuroimmune system.

Compared efficacy of diazepam or avizafone to prevent soman-induced electroencephalographic disturbances and neuropathology in primates: relationship to plasmatic benzodiazepine pharmacokinetics.

We performed an experiment to characterize the toxicity of soman in cynomolgus monkeys in which organophosphorus intoxication was followed by treatment with either the current three-drug therapy atropine/pralidoxime/diazepam or a combination of atropine/pralidoxime/avizafone, avizafone being the water soluble prodrug of diazepam. Clinical, electrophysiological, and histological approaches were combined. When benzodiazepines were injected at the similar molar dose of 0.7 micromol/kg, the protection against soman toxicity was better with the atropine/ pralidoxime/diazepam combination than with the atropine/pralidoxime/avizafone one. Pharmacokinetic studies demonstrated that this difference of efficacy could be explained by a lower plasmatic load of diazepam obtained after injection of avizafone at 0.7 micromol/kg, compared to the administration of diazepam at the same molar dose. Moreover, after injection of avizafone, plasmatic levels of diazepam were achieved faster and declined more rapidly than after administration of diazepam. Compared to diazepam given at a dose of 0.7 micromol/kg, injection of 1 micromol avizafone/kg gave a similar plasmatic load of benzodiazepine, but with a lower time to maximum plasma concentration (tmax) and a higher maximum plasma concentration (Cmax) for plasmatic diazepam. We therefore went on to demonstrate that administration of the atropine/pralidoxime/avizafone combination at a dose 1 micromol benzodiazepine/kg to intoxicated monkeys afforded electrophysiological and histological protection similar to that obtained after administration of atropine/pralidoxime/diazepam at a dose of 0.7 micromol diazepam/kg. Reflections on the possible incorporation of avizafone in three-drug emergency treatment are presented.

Review of the value of gacyclidine (GK-11) as adjuvant medication to conventional treatments of organophosphate poisoning: primate experiments mimicking various scenarios of military or terrorist attack by soman.

Today, organophosphorus nerve agents are still considered as potential threats in both military or terrorism situations. These agents act as potent irreversible inhibitors of acetylcholinesterase in both central and peripheral nervous systems. Conventional treatment of organophosphate poisoning includes the combined administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). However, numerous studies have demonstrated that the excitatory amino acid glutamate also plays a prominent role in the maintenance of organophosphate-induced seizures and in the subsequent neuropathology especially through an overactivation of the N-methyl-D-aspartate (NMDA) receptor subtype. Contrary to other non-competitive NMDA antagonists successfully tested in rodents exposed to organophosphate, gacyclidine is a novel antiNMDA compound which is in the process of approval for human use in France for neurotraumatology. This review summarizes the therapeutic value of gacyclidine as a complement to the available emergency treatment against severe organophosphate poisoning. Previous data obtained from experiments on primates in several scenarios mimicking military or terrorist attacks, using soman as the nerve agent, were used. Primates pretreated with pyridostigmine and receiving conventional emergency therapy at the first signs of poisoning survive. However, only gacyclidine is able to ensure complete management of nerve agent poisoning for rapid normalization of EEG activity, clinical recovery and neuroprotection. Gacyclidine also ensures optimal management of severe nerve agent poisoning in animals neither pretreated nor receiving emergency therapy likewise during an unexpected exposure. However, this beneficial effect is obtained provided that medical intervention is conducted rapidly after intoxication. Globally, the current lack of any other NMDA receptor antagonist suitable for human use reinforces the therapeutic value of gacyclidine as a central nervous system protective agent for the treatment of OP poisoning.

Acute soman poisoning in primates neither pretreated nor receiving immediate therapy: value of gacyclidine (GK-11) in delayed medical support.

Organophosphorus (OP) nerve agents are still used as warfare and terrorism compounds. Classical delayed treatment of victims of organophosphate poisoning includes combined i.v. administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). The objective of this study was to evaluate, in a realistic setting, the therapeutic benefit of administration of GK-11 (gacyclidine), an antiglutamatergic compound, as a complement to the above therapy against organophosphate poisoning. Gacyclidine was injected (i.v.) in combination with atropine/diazepam/pralidoxime at man-equivalent doses after a 45- or 30-min latency period to intoxicated primates (2 LD50). The effects of gacyclidine on the animals' survival, electroencephalographic (EEG) activity, signs of toxicity, recovery after challenge and central nervous system histology were examined. The present data demonstrated that atropine/diazepam/pralidoxime alone or combined with gacyclidine did not prevent signs of soman toxicity when treatment was delayed 45 min after poisoning. Atropine/diazepam/pralidoxime also did not control seizures or prevent neuropathology in primates exhibiting severe signs of poisoning when treatment was commenced 30 min after intoxication. However, in this latter case, EEG recordings revealed that additional treatment with gacyclidine was able to stop soman-induced seizures and restore normal EEG activity. This drug also totally prevented the neuropathology observed 5 weeks after soman exposure in animals treated with atropine/diazepam/pralidoxime alone. Overall, in the case of severe OP-poisoning, gacyclidine represents a promising adjuvant therapy to the currently available polymedication to ensure optimal management of organophosphate poisoning in man. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication. However, it should always be kept in mind that, in the case of severe OP-poisoning, medical intervention must be conducted as early as possible.

Voltammetric measurement of blood nitric oxide in irradiated rats.

UNLABELLED: PURPOSE. To investigate the effect of blood nitric oxide (NO) as a mediator of the neurovascular syndrome in rats following gamma-irradiation. MATERIAL AND METHODS: Using a voltametric method together with a carbon fibre based sensor, NO measurements were carried out in sham-irradiated and irradiated animals either in blood from the abdominal aorta or in blood samples from the heart. RESULTS: In in vitro conditions, properties of the probe were not altered by the ionizing radiation. Significant increases of +17% and +25.6% were observed in the voltametric signal height at 90 min and 24 h respectively after a 15 Gy gamma-ray exposure. These effects were followed on days 3 and 4 by a progressive decrease in the signal height of 7% and 18% respectively. Dose-effect relationships were observed at 90 min and 24 h after exposure to gamma-rays in the range of 3-15 Gy. Finally, the NO dependence on the measured voltametric signal was controlled by using inhibitors of the NO synthase (NOS) and by performing nitrate assays. CONCLUSIONS: Specific blood NO voltametric measurements are possible. Functional changes associated with NO after gamma-ray exposure are discussed.

Nerve agent poisoning in primates: antilethal, anti-epileptic and neuroprotective effects of GK-11.

Organophosphorus nerve agents are still in use today in warfare and as terrorism compounds. Classical emergency treatment of organophosphate poisoning includes the combined administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). However, recent experiments with primates have demonstrated that such treatment, even when administered immediately after organophosphate exposure, does not rapidly restore normal electroencephalographic (EEG) activity and fails to totally prevent neuronal brain damage. The objective of this study was to evaluate, in a realistic setting, the therapeutic benefit of administration of GK-11 (gacyclidine), an antiglutamatergic compound, as a complement to the available emergency therapy against organophosphate poisoning. GK-11 was injected at a dose of 0.1 mg/kg (i.v) after a 45-min latency period to heavily intoxicated (8 LD50) primates. Just after intoxication, man-equivalent doses of one autoinjector containing atropine/pralidoxime/diazepam were administered. The effects of GK-11 were examined on survival, EEG activity, signs of toxicity, recovery after challenge and central nervous system histology. The present data demonstrate that treatment with GK-11 prevents the mortality observed after early administration of classical emergency medication alone. EEG recordings and clinical observations also revealed that GK-11 prevented soman-induced seizures and motor convulsions. EEG analysis within the classical frequency bands (beta, theta, alpha, delta) demonstrated that central activity was totally restored to normal after GK-11 treatment, but remained profoundly altered in animals receiving atropine/pralidoxime/diazepam alone. GK-11 also markedly accelerated clinical recovery of soman-challenged primates. Lastly, this drug totally prevented the neuropathology observed 3 weeks after soman exposure in animals treated with classical emergency treatment alone. GK-11 represents a promising adjuvant therapy to the currently available emergency polymedication to ensure optimal management of organophosphate poisoning in man. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication.

Efficacy of atropine/pralidoxime/diazepam or atropine/HI-6/prodiazepam in primates intoxicated by soman.

We performed an experiment to characterize the toxicity of soman in cynomolgus monkeys when the organophosphorus intoxication was followed by a treatment with either the three-drug therapy atropine/pralidoxime/diazepam or the association atropine/HI-6/prodiazepam. Clinical, electrophysiological and histological approaches were combined. Our data demonstrate that the protection afforded against soman toxicity was better with the combination atropine/HI-6/prodiazepam compared to atropine/pralidoxime/diazepam. This was observed transiently in term of vigilance and respiratory function of intoxicated animals, but particularly in term of their EEG- and ECG disturbances. Moreover, compared to those treated with atropine/pralidoxine/diazepam, animals treated with atropine/ HI-6/prodiazepam recovered slightly sooner and did not exhibit prostration 2 days after intoxication although their rapidity of movements was not totally restored. The final recovery observed 3 weeks after intoxication was similar for the two groups. The value of the combination of atropine/HI-6/prodiazepam vs atropine/pralidoxime/diazepam to counteract soman toxicity was also confirmed in term of brain neuroprotection since greater lesions were observed with the second three drug treatment three weeks after intoxication.

Real-time spike detection in EEG signals using the wavelet transform and a dedicated digital signal processor card.

This paper describes a complete real-time system for EEG signal analysis. Specific software and hardware have been designed to provide biologists with an efficient tool, which allows a complete study of the different states of vigilance as well as the paroxysmal activities. The analysis method which is based on the wavelet transform is first presented and compared to the standard spectral approach. The dedicated digital signal processor card, based on the Motorola 96002 processor chip, that has been designed to support real-time acquisition and real-time processing of EEG signals is then presented. We finally illustrate the proposed method by processing real EEG signals of rats, and show that it opens up new prospects in the domain of EEG-based diagnosis. We propose a new representation, called globalization, that provides a global view and better detection of paroxysmal activities.

Stimulated release of acetylcholinesterase in rat striatum revealed by in vivo microspectrophotometry.

The microspectrophotometric technique allows a direct in vivo measurement of brain extracellular acetylcholinesterase. An optical probe associated with electrodes for stimulation was implanted in striatum of anaesthetized rats to determine the effects of neuronal excitation on the acetylcholinesterase activity. Electrical stimulations induced a reversible increase in acetylcholinesterase activity of about 30 to 50%, with a recovery to baseline occurring after 1 or 2 h. Furthermore, iterative electrical stimulation induced a progressive fading of this phenomenon. An enhancement of acetylcholinesterase activity was also observed by stimulations with potassium injections through a canal of the probe. These results suggest mainly an intracellular origin of the released enzyme and estimate its contribution at about 40% of the whole extracellular enzyme activity.

Influence of the radioprotective agent WR 2721 on the striatal acetylcholinesterase activity in the rat.

The radioprotective thiophosphate S-2(3 amino-propyl-amino) phosphorothioic acid (WR 2721) induced an early reduction of striatal acetylcholinesterase activity followed by an increase, when intraperitoneally injected to rats, although it does not cross the blood-brain barrier. These results were obtained using an original technique which allows the measurements in the same animal for several days. Transient general oxidative metabolism inhibition might affect the extra-cellular enzyme amount or its activity.

Rapid postmortem decrease in the ectocellular acetylcholinesterase activity in rat striatum as assessed by in vivo microspectrophotometry.

The acetylcholinesterase (AChE) activity in striatum rat was determined before and shortly after death using the in vivo microspectrophotometric method. This technique allowed us to monitor the Ellman colorimetric reaction directly inside the brain using an optical probe implanted in a live animal and to determine locally the AChE activity. Whatever the cause of the animals death, we observed a drastic postmortem decrease of the AChE activity of about 35-50%, 10 min after death. We have verified that the postmortem decrease of brain temperature or pH and postmortem optical properties changes could only explain a fraction of the AChE activity fall (16%). This phenomenon seems to be related to events strictly localized at the cellular level, since local injection of cyanide at the measuring site promotes a decrease of the enzymatic activity (40%) close to the levels observed after death. The origin of this rapid postmortem fall of the AChE activity is discussed. The technical properties of the microspectrophotometric method exclude a decrease of the ectocellular pool of enzyme after death. Our results allow us to envisage the existence of an in vivo endogenous regulation of the AChE activity which disappears shortly after death.

Sleep alterations in experimental street rabies virus infection occur in the absence of major EEG abnormalities.

Brain electrical activity and sleep organization were investigated in chronically implanted mice during street rabies virus infection. Continuous EEG recordings showed no gross electrical abnormalities until a few hours before the fatal issue. In contrast, alterations of sleep stages were observed at an early stage during the course of rabies virus infection, at a time when clinical signs were absent. Quantification by spectral analysis showed that the main feature was the early decrease of REM-sleep stages and the increase of the duration of waking stages. Neuromuscular disorders which could occur early were also observed during the disease. Comparison of these data with those obtained from fixed rabies virus infection shows that in the latter the EEG recordings demonstrated early alterations and a progressive deterioration with disappearance of both sleep and waking stages, which were replaced by a pathological sleep stage. In order to evaluate the potential role of the host-specific immune response in promoting brain electrophysiological alterations, EEG recordings and spectral analysis were also performed in cyclophosphamide-treated mice. Street rabies virus-infected and immunosuppressed mice showed identical physiopathological changes as those observed in immunocompetent mice. The implication of these viral-induced electrophysiological alterations in the context of the pathogenic mechanisms of rabies virus is discussed.