Progressive cardiorespiratory dysfunction in Kv1.1 knockout mice may provide temporal biomarkers of pending sudden unexpected death in epilepsy (SUDEP): The contribution of orexin.

OBJECTIVE: Immediately preceding sudden unexpected death in epilepsy (SUDEP), patients experienced a final generalized tonic-clonic seizure (GTCS), rapid ventilation, apnea, bradycardia, terminal apnea, and asystole. Whether a progressive pathophysiology develops and increases risk of SUDEP remains unknown. Here, we determined (a) heart rate, respiratory rate, and blood oxygen saturation (SaO2 ) in low-risk and high-risk knockout (KO) mice; and (b) whether blocking receptors for orexin, a cardiorespiratory neuromodulator, influences cardiorespiratory function mice or longevity in high-risk KO mice. METHODS: Heart rate and SaO2 were determined noninvasively with ECGenie and pulse oximetry. Respiration was determined with noninvasive airway mechanics technology. The role of orexin was determined within subject following acute treatment with a dual orexin receptor antagonist (DORA, 100 mg/kg). The number of orexin neurons in the lateral hypothalamus was determined with immunohistochemistry. RESULTS: Intermittent bradycardia was more prevalent in high-risk KO mice, an effect that may be the result of increased parasympathetic drive. High-risk KO mice had more orexin neurons in the lateral hypothalamus. Blocking of orexin receptors differentially influenced heart rate in KO, but not wild-type (WT) mice. When DORA administration increased heart rate, it also decreased heart rate variability, breathing frequency, and/or hypopnea-apnea. Blocking orexin receptors prevented the methacholine (MCh)-induced increase in breathing frequency in KO mice and reduced MCh-induced seizures, via a direct or indirect mechanism. DORA improved oxygen saturation in KO mice with intermittent hypoxia. Daily administration of DORA to high-risk KO mice increased longevity. SIGNIFICANCE: High-risk KO mice have a unique cardiorespiratory phenotype that is characterized by progressive changes in five interdependent endpoints. Blocking of orexin receptors attenuates some of these endpoints and increases longevity, supporting the notion that windows of opportunity for intervention exist in this preclinical SUDEP model.

The effect of astragaloside IV on JAK2-STAT6 signalling pathway in mouse model of ovalbumin-induced asthma.

Asthma is a chronic inflammatory lung disease of the airway; the incidence and prevalence of asthma remain high worldwide. Astragaloside IV (AS-IV) is the main active constituent of Astragalus membranaceus. Accumulating evidence suggests that AS-IV possesses anti-inflammatory and anti-asthmatic ability, but the potential molecular mechanism is required to further clarify. In this study, the anti-asthmatic effects of AS-IV on mice with ovalbumin (OVA)-induced allergic inflammation were analysed. We analysed airway hyperresponsiveness (AHR), numbers of inflammatory cells, inflammation situation in lung tissue and cytokines level in bronchoalveolar lavage fluid (BALF) between OVA-induced mice with and without AS-IV treatment. Moreover, we explored the possible signalling pathway behind the anti-asthmatic effects. Our results revealed that AS-IV treatment ameliorates airway inflammation and AHR in an OVA-induced asthma model. Besides, AS-IV treatment inhibits the interleukin (IL)-4, -5 and -13 production, and further study indicated that AS-IV treatment downregulates the expression level of p-JAK2/p-STAT6 proteins. Taken together, the present study suggested that the inhibitory effects of AS-IV on asthma therapy are at least partially involved in inhibiting the JAK2/STAT6 signalling pathway.

Molecular and genetic association of interleukin-6 in tacrine-induced hepatotoxicity.

BACKGROUND: Tacrine, an anticholinesterase used to treat Alzheimer's disease (AD), leads to an increase in serum alanine aminotransferase (ALT) levels. The factors determining individual susceptibility are largely unknown. The purpose of this study was to investigate genetic predisposition. METHODS: Rats were administered single dose tacrine (3-40 mg/kg). After 6 and 24 h, hepatic gene expression was determined using the affymetrix rat U34A microarray. On the basis of the gene expression data, the IL6 gene was identified as a potential candidate for tacrine transaminitis susceptibility. Sixty-nine patients with AD on tacrine with or without transaminitis were genotyped for 17 IL6 polymorphisms. RESULTS: Serum aspartate aminotransferase levels in rats increased after tacrine (40 mg/kg) administration. Forty-six and 29 genes showed significant upregulation at 6 and 24 h, respectively, after administration, including the IL-6-regulated acute phase response genes alpha2-macroglobulin, fibronectin and haptoglobin. Five of the 17 IL6 polymorphisms studied in AD patients showed an association (P<0.05) with transaminitis [ALT>2 x upper limit of normal (ULN)]. An association existed between maximum ALT and area under curve for ALT over 15 weeks and an intronic polymorphism (P<0.01) and a 3'-variable nucleotide tandem repeat (P<0.05). Multilocus haplotype analysis showed one haplotype (which included the -597A, -572G, -174G and variable nucleotide tandem repeat-D alleles) had a frequency of 0.1 in patients with ALT values >2 x ULN, whereas it was absent in patients with ALT less than 2 x ULN (P=0.0093, Pcorrected=0.049). CONCLUSION: The IL6 genotype may act as a predisposing factor for tacrine transaminitis. This, however, requires further confirmatory functional studies. The role of acute dosing rodent models in identifying candidate genes associated with drug-induced liver injury in man deserves further study.

Central cardiovascular effects of tacrine in the conscious dog: a role for catecholamines and vasopressin release.

Centrally acting cholinergic agents are currently reported to increase blood pressure in various species through the stimulation of muscarinic cholinoceptors. Moreover, several cardiovascular adverse effects have been reported from clinical studies. The aim of this study was to investigate the effects of tacrine, an acetylcholinesterase inhibitor which has been reported to have therapeutic potential in Alzheimer's disease, on blood pressure and two vasopressor systems (sympathetic and vasopressinergic) in Beagle dogs. Intravenous (i.v.) tacrine (2 mg kg(-1)) induced, in conscious and anesthetized dogs, an increase in systolic and diastolic blood pressure, accompanied by bradycardia. This increase was dose-dependent with a peak effect at 1.5 min following administration. Tacrine also induced an increase in noradrenaline, adrenaline and vasopressin plasma levels. Pretreatment with the muscarinic receptor antagonist, atropine (2 mg kg(-1), i.v.), abolished the pressor response to i.v. injection of tacrine while pretreatment with the peripheral muscarinic receptor antagonist, methylscopolamine (0.2 mg kg(-1), i.v.), did not alter the increase in blood pressure. Similarly, noradrenaline and adrenaline changes in plasma levels were not modified by methylscopolamine but were abolished by atropine pretreatment. A similar tendency although not significant was observed for vasopressin plasma levels. The present results demonstrate that in dogs, tacrine (2 mg kg(-1), i.v.) stimulates central muscarinic cholinoceptors to increase blood pressure through activation of the two components of the sympathetic nervous system (i.e., neuroneuronal noradrenergic and the neurohormonal adrenergic pathways) as well as through increasing noradrenaline, adrenaline and vasopressin plasma levels.

Cytotoxic and cytostatic effects of arecoline on oral mucosal fibroblasts.

Betel quid (BQ) chewing shows strong correlation to the incidence of oral submucous fibrosis and oral cancer in Taiwan. Arecoline, the main areca alkaloid, is considered to be one of the etiologic factors in BQ. To elucidate the role(s) of arecoline in the pathogenesis of BQ chewing related oral mucosal lesions, we used oral mucosal fibroblasts to study the effects of serum concentration, cell density, and incubation time on the cytotoxic response to arecoline. At a concentration less than 0.2 mM, arecoline was not cytotoxic to oral mucosal cells after 1, 3, and 6 days of incubation. After 3 days of incubation, the cytotoxic and cytostatic effects of arecoline became evident when the cells were exposed to higher concentrations of arecoline (0.2 mM) and serum (10% FCS). Exposure of cells (1 x 10(4) cells/well) to 0.2 mM of arecoline in 0.5% FCS for 3 and 6 days led to a 20% and 23% decrease, respectively, in the cell number, whereas exposure of cells (1 x 10(4) cells/well) to 0.2 mM arecoline in 10% FCS led to a 38% and 53% decrease, respectively, in cell number. At a higher cell density (5 x 10(4) and 1 x 10(5) cells/well), 0.2 mM arecoline led to less cytotoxicity (38% and 21% of decreasing in cell number, respectively) after 6 days of incubation. Our results indicated that arecoline was not mitogenic to oral mucosal fibroblasts, and that the cytotoxic and cytostatic effects of arecoline on oral mucosal fibroblasts could be modulated by the changes in the cell density, serum concentrations, and incubation time.

Plasma protein extravasation induced in the rat dura mater by stimulation of the parasympathetic sphenopalatine ganglion.

It has been proposed that migraine could result from a neurogenic inflammation of the dura mater. According to this theory, inflammation could be initiated by an axon reflex of nociceptive nerve fibers, but the trigger of this axon reflex remains poorly understood. Previous works have shown that parasympathetic agonists can activate mast cells and/or sensory C-fibers, inducing pain and inflammation. The aim of the present work was to determine whether the activation of intracranial parasympathetic nerve fibers could trigger an inflammatory mechanism within the rat dura mater. Activation of the intracranial parasympathetic system was achieved by electrical stimulation of the sphenopalatine ganglion (SPG). The development of a neurogenic inflammation was estimated either by microscopic examination or by quantitative measurement of plasma protein extravasation (PPE) in the dura. To determine the respective roles of the parasympathetic and sensory innervations, two groups of rats were pretreated either with atropine or with capsaicin. Stimulation of the SPG induced a PPE increase of about 200% in the stimulated side on the dura mater. Extravasated material was mainly concentrated around small blood vessels. This extravasation was significantly reduced by capsaicin pretreatment and completely abolished by atropine. Infusion of carbachol in the common carotid artery induced PPE in the ipsilateral dura comparable to that induced by electrical stimulation of the SPG. This extravasation was also blocked by atropine infusion. These data indicate for the first time that the parasympathetic nervous system can trigger a neurogenic inflammation in the dura via muscarinic cholinergic receptors. Sensory C-fibers seem to play a role in this phenomenon. With respect to the potential autonomic imbalance described in the etiology of various types of vascular headaches, such a mechanism could be important in inducing attacks.

Alteration in neurofilament axonal transport in the sciatic nerve of the diisopropyl phosphorofluoridate (DFP)-treated hen.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester that produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hens 7-14 days after a single s.c. dose of 1.7 mg/kg. In this study, hens were treated with a single dose of DFP (1.7 mg/kg, s.c.) 24 hr after [35S]methionine injection into the sacrolumbar region of their spinal cord, and killed 3, 7, 14, or 27 days post-DFP treatment. The rates of transport of labeled high (NF-H), medium (NF-M), and low (NF-L) molecular weight neurofilaments, and tubulin were faster in DFP-treated birds than in controls after 3 days. Subsequently, the rate of transport of these proteins started falling, so that the peaks of labeled proteins in control and DFP-treated hens were overlapping after 7 days. At 14 days, the peaks of NF-H, NF-M, and NF-L in treated hens were distinctly behind the corresponding peaks in control hens. This was again followed by an increase in transport of NF-H and NF-L, but not of NF-M, so that the labeled NF-H and NF-L showed the same pattern in control and treated hens after 27 days. The transient decrease in NF-H and NF-L axonal transport rate, and recovery correlated in a temporal manner with the previously reported increase of Ca2+/calmodulin-dependent protein kinase-mediated phosphorylation of neurofilament proteins and inhibition of calpain activity in the sciatic nerve in OPIDN. Proteinase inhibition has been reported recently to result in enhanced phosphorylation of neurofilaments in some cells. The present study suggests that the enhanced phosphorylation of neurofilaments by DFP-increased Ca2+/calmodulin-dependent protein kinase activity may be contributing toward alteration in NF axonal transport and the development of OPIDN.

Tremulous jaw movements produced by acute tacrine administration: possible relation to parkinsonian side effects.

Previous work has shown that cholinomimetic drugs induce "vacuous" or non-directed jaw movements in rats. In the present study, five experiments were conducted to provide a pharmacological, anatomical and behavioral characterization of tacrine-induced vacuous jaw movements. In the first experiment, tacrine produced vacuous chewing in a dose-related manner in a range of 1.25 mg/kg to 1.0 mg/kg. This effect was reduced, also in a dose-related manner, by the co-administration of the muscarinic antagonist scopolamine in a range of 0.125 to 1.0 mg/kg, but not by N-methylscopolamine. The fourth experiment examined the effect of scopolamine (2.5 to 10.0 micrograms) injected into the ventrolateral striatum on vacuous jaw movements induced by 5.0 mg/kg tacrine. Intrastriatal injections of scopolamine completely blocked tacrine-induced jaw movements. The fifth experiment utilized a slow-motion videotaping system to analyze the temporal characteristics of vacuous chewing induced by 5.0 mg/kg tacrine. The vast majority of the movements occurred in rapid "bursts," and analysis of interresponse times (i.e., the time between each jaw movement) showed that most of the jaw movements occurred within a local frequency range of 3 to 7 Hz. Thus, tacrine-induced jaw movements are reduced by antimuscarinic treatment, and most of these movements occur in the parkinsonian tremor frequency range. Tremulous jaw movements induced by tacrine in rats appear to share some characteristics with Parkinsonian tremor.

Effects of acute and repeated clozapine injections on cholinomimetic-induced vacuous jaw movements.

Three studies were undertaken to investigate the effects of the atypical neuroleptic clozapine on the vacuous jaw movements induced by cholinergic stimulation in rats. In the first experiment, acute clozapine injections (4.0-16.0 mg/kg) produced a dose-related suppression of the vacuous jaw movements induced by 0.4 mg/kg physostigmine. In the second experiment, acute injections of clozapine (2.0-16.0 mg/kg) also suppressed vacuous jaw movements induced by 4.0 mg/kg pilocarpine in a dose-related manner. The third experiment was designed to compare the effects of acute and repeated administration of 16.0 mg/kg clozapine. In this experiment, there were three groups: one that received 4.0 mg/kg pilocarpine, a second group that received pilocarpine plus an acute injection of 16.0 mg/kg clozapine, and a third group that received injections of 16.0 mg/kg clozapine for 14 consecutive days, including the final day in which they also were injected with pilocarpine. For the third experiment, animals were assessed for the sedative effects of clozapine as well as vacuous jaw movements. The results indicated that either acute or repeated injections of 16.0 mg/kg clozapine reduced vacuous jaw movements relative to rats that received pilocarpine alone, and the two clozapine-treated groups did not differ from each other. The sedation ratings indicated that acute injections of 16.0 mg/kg clozapine produced substantial drowsiness and sedation, whereas rats that had received clozapine for 14 days did not show substantial sedation. These results indicate that clozapine can suppress cholinomimetic-induced vacuous jaw movements. The suppressive effects of clozapine on pilocarpine-induced vacuous jaw movements do not show tolerance within the 14-day period of repeated injections, whereas the sedative effects of clozapine do show tolerance. Thus, these results demonstrate that the suppression of pilocarpine-induced vacuous jaw movements by clozapine is not merely an artifact of clozapine-induced sedation. Because pilocarpine-induced vacuous jaw movements share some characteristics with human parkinsonian symptoms, the present results are consistent with previous reports indicating that repeated injections of clozapine produce anti-parkinsonian effects.

A central cholinergic link in the cardiovascular effects of the benzodiazepine receptor partial inverse agonist FG 7142.

Previous work demonstrated that systemic administration of the benzodiazepine receptor (BZR) partial inverse agonist beta-carboline FG 7142 (FG) augments the cardiovascular response to non-signal stimuli, similar to the effects of an aversive context. Analysis of the parasympathetic and sympathetic contributions to the effects of FG prompted the hypothesis that increases in central cholinergic activity mediates the potentiation of the cardioacceleratory response by FG. Consistent with this hypothesis, the present experiments demonstrate: (a) intracerebroventricular (ICV) infusion of the cholinergic receptor agonist carbachol mimics the response-potentiating effects of FG; (b) this effect of carbachol was blocked by ICV co-administration of the muscarinic antagonist atropine; (c) ICV infusions of atropine blocked the potentiation of the cardioacceleratory response by systemically administered FG, but did not alter the basal response to the stimulus; and (d) 192 IgG-saporin-induced lesions of basal forebrain cholinergic neurons prevented the FG-induced potentiation of the cardioacceleratory response, again without altering the basal cardiac response. These data strongly support the hypothesis that the effects of FG on cardiac reactivity are mediated via an activation of central muscarinic cholinergic mechanisms.

In-vivo genotoxicity of the alkaloid drug pilocarpine nitrate in bone marrow cells and male germ cells of mice.

Pilocarpine nitrate, an alkaloid drug of plant origin induces spindle disfunction in bone marrow cells of mice. Further studies were carried out to investigate its mutagenic effects in somatic and germ cells of mice by assessing chromosome aberrations at mitotic metaphase and as micronuclei in bone marrow cells and sperm-shape abnormality in cauda epididymides. The dose and time yield effects of the drug were investigated. The statistically significant results that were obtained for both chromosomal aberrations and micronucleus test but not for the sperm-shape abnormality test, indicated the genotoxicity of this compound in somatic cells but not in germ cells.

Salivary secretion and histopathological effects after single administration of the muscarinic agonist SNI-2011 in MRL/lpr mice.

The salivary secretion and the histopathological effects after administration of a single dose of cis-2-methylspiro (1,3-oxathiolane-5,3') quinuclidine hydrochloride hemihydrate (SNI-2011) were monitored in adult, male and female MRL/lpr mice, C57BL/6J mice and ICR mice. SNI-2011 (1-10 mg/kg, i.p.), dose-dependently increased the secretion of saliva in MRL/lpr mice. The flow rate decreased gradually over the course of 60 min. The total volume of saliva, secreted in response to SNI-2011, was significantly higher in male than in female MRL/lpr mice, but there was no significant difference in this parameter between male and female C57BL/6J and ICR mice. Degeneration, apparent as atrophy and necrosis of serous cells in MRL/lpr mice, was reversed by treatment with SNI-2011 (3 and 6 mg/kg). These results suggest that SNI-2011 could be useful in the treatment of xerostomia in patients with Sjögren's syndrome.

Effects of sustained low-level muscarinic agonism in rats.

Sustained, low level muscarinic activity was induced in rats by feeding the muscarinic agonist and experimental drug candidate CI-969 at 50, 100 and 200 mg/kg body weight/day for 4 wk. Except for urine staining, clinical signs typical of acute high-dose exposure to muscarinic agonists were not observed. A dose-related suppression of body weight gain approached 60% at the high dose, but no significant effects on haematology or clinical chemical parameters were observed after 4 wk of exposure. Corneal opacities with histopathological features including neovascularization, acanthosis and stromal proliferation were observed in a dose-related fashion in both sexes at 100 and 200 mg/kg/day. Hypertrophy of the Harderian and lacrimal glands also occurred, probably as an adaptive response to sustained muscarinic activity. Lacrimal gland concentrations of the muscarinic agonist were in the range of pmol/mg tissue and therefore significant direct exposure of cornea to the compound through the tears was discounted. The presence of corneal muscarinic receptors was investigated to determine whether opacities could be related to direct, receptor-mediated events in the cornea; however, no specific binding of the muscarinic receptor radioligand [3H]quinuclidylbenzilate was detected. Because muscarinic agonist-induced opacities can be inhibited by scopolamine, the apparent lack of muscarinic receptors in the cornea indicates that the opacities are not a direct effect, but are instead secondary to muscarinic events at another site. To our knowledge, this is the first report of corneal opacities induced by a muscarinic agonist.

Relevance of drug-melanin interactions to ocular pharmacology and toxicology.

In melanocytes, the biosynthesis of L-dopa derived indole polymer, melanin, is accelerated by tyrosinase and related enzymes. The brown to black pigment is characterized by a stable free-radical property. In humans, a pigment dependent slow onset of ocular actions of ephedrine, atropine, cocaine, pilocarpine and related medications was observed. Extensive accumulation of drugs by melanin appears to be the most important factor governing the long term therapeutic/toxicological activities. Drugs crossing placental circulation are localized in the mouse fetal eye. Thus, drugs exhibit a high binding capacity for melanin containing tissues. Studies on synthetic melanin and melanin granules also indicated a high binding capacity of many therapeutic classes of drugs, including psychotropics. In addition to the liposoluble property of the molecule, there is a definite relationship between chemical structure and the affinity of drugs for melanin. For example, the affinity of chlorpromazine for melanin is higher than that of chlorprothixene. NMR studies, with soluble melanins indicate that there is a steric preference among ephedrine enantiomers. A high binding capacity indicates that more than two molecules of (-)-ephedrine may complex with one indole unit of melanin. Ocular drug development calls for the study of qualitative and quantitative aspects of drug-melanin interaction.

Systemic and local tolerability of ophthalmic drug formulations. An update.

All eye drops raise problems of local tolerance, but with variable frequencies. They can induce pain on instillation, allergic reactions, delayed healing, punctate keratitis, disturbances of lacrimal secretion, disturbances of accommodation (especially the parasympathomimetics) and local pigmentation after prolonged use. Corticosteroids are associated with 2 major risks: chronic glaucoma and cataract, initially reversible if treatment is stopped. There is still a major risk of corneal herpes with corticosteroids. It is important to be aware of these local problems as they are responsible for poor patient compliance. The systemic effects essentially concern the agonists and antagonists of the autonomic nervous system. beta-Blocker eye drops can cause bronchospasm, heart failure, syncope and psychiatric disorders, especially at high doses and with nonselective beta-blockers. These consequences are usually related to failure to comply with the prescribing precautions. alpha-Adrenergic agonists, which exert dose-dependent effects, can induce hypertensive crises or angina attacks. Apart from patients at risk (children under the age of 30 months and the elderly), parasympathomimetics cause few systemic adverse effects; anticholinesterases, which have curare-like properties, are contraindicated for 6 weeks before general anesthesia. In the very young and the very old, atropinic eye drops carry a risk of cardiovascular collapse and neuropsychiatric disturbances. Problems may also occur with other classes of drugs such as anti-infectives, antispectics, anti-inflammatories and contact lens products. Nevertheless, it is clear that this form of treatment is generally very well tolerated in relation to the volume of eye drops prescribed by ophthalmologists each day.

Assessment of the acute acrylonitrile-induced neurotoxicity in rats.

Acrylonitrile (VCN) is an aliphatic nitrile which is used extensively in manufacturing of synthetic fibers, plastics, and rubber. Although the neurotoxicity of VCN is recognized, no thorough characterization of this effect has been reported. Current studies were designed to quantitatively characterize the acute phase of VCN-induced cholinomimetic neurotoxicity, and to determine the effects of dose, route of administration, and atropine on such toxicity. Administration of a single gavage or subcutaneous doses of 20, 40, or 80 mg VCN/kg to male Sprague-Dawley rats causes two distinctive phases of acute neurotoxic effects. Signs observed in the early phase had a rapid onset, and were cholinomimetic in nature. They included salivation, lacrimation, chromodacryorrhea, polyuria, miosis, vasodilatation in face, ears and extremities, increased gastric secretion, and diarrhea. A late phase developed hours after VCN dosing, and the toxic signs included depression, convulsions, and respiratory failure followed by death at high doses. These results revealed that the cholinomimetic toxicity induced by VCN was dose related regardless of the route of administration. In another study, rats were pretreated with atropine (1 mg/kg, IP) prior to VCN (40 mg/kg) in order to investigate the role of the cholinergic system. Atropine protected rats against VCN-induced cholinomimetic neurotoxicity, suggesting possible involvement of the cholinergic system. Finally, this work provides essential basic information for studying the biochemical, pharmacological, and neurological basis of VCN-induced neurotoxicity in the rat.

Human erythrocyte as a model for investigating muscarinic agonists and antagonists.

1. Human erythrocyte muscarinic receptor is further classified as M1 subtype by its binding of [3H]pirenzepine (a specific M1 antagonist). Conversely, binding of [3H]piperidinyl AF-DX116 (a specific M2 antagonist) is not detected. 2. There are high correlations between the binding efficacies of 25 known or putative cholinergic agents using either human erythrocyte membrane or mouse caudate homogenate. Also the in vitro data corresponds satisfactorily with in vivo data measuring the protective effect of these compounds against organophosphate poisoning. 3. The human erythrocyte membrane is an efficient model for investigating cholinergic agonists and antagonists.