Characterisation Of The Membrane Transport Of Pilocarpine In Cell Suspension Cultures Of Pilocarpus Microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.

Neuroprotective Effects of Diazepam, Carbamazepine, Phenytoin and Ketamine after Pilocarpine-induced Status Epilepticus

Cell damage and spatial localization deficits are often reported as long-term consequences of pilocarpine-induced status epilepticus. In this study, we investigated the neuroprotective effects of repeated drug administration after long-lasting status epilepticus. Groups of six to eight Wistar rats received microinjections of pilocarpine (2.4 mg/mu l, 1 mu l) in the right dorsal hippocampus to induce a status epilepticus, which was attenuated by thiopental injection (35 mg/kg, i.p.) 3 hrs after onset. Treatments consisted of i.p. administration of diazepam, ketamine, carbamazepine, or phenytoin at 4, 28, 52, and 76 hr after the onset of status epilepticus. Two days after the treatments, rats were tested in the Morris water maze and 1 week after the cognitive tests, their brains were submitted to histology to perform haematoxylin and eosin staining and glial fibrillary acidic protein (GFAP) immunofluorescence detection. Post-status epilepticus rats exhibited extensive gliosis and cell loss in the hippocampal CA1, CA3 (70% cell loss for both areas) and dentate gyrus (60%). Administration of all drugs reduced cell loss in the hippocampus, with best effects observed in brains slices of diazepam-treated animals, which showed less than 30% of loss in the three areas and decreased GFAP immunolabelling. Treatments improved spatial navigation during training trials and probe trial, with exception of ketamine. Interestingly, in the probe trial, only diazepam-treated animals showed preference for the goal quadrant. Our data point to significant neuroprotective effects of repeated administration of diazepam against status epilepticus-induced cell damage and cognitive disturbances.

Bilateral Anterior Thalamic Nucleus Lesions Are Not Protective against Seizures in Chronic Pilocarpine Epileptic Rats

Aims: To investigate whether anterior thalamic nucleus (AN) lesions are protective against spontaneous recurrent seizures in the chronic phase of the pilocarpine model of epilepsy. Methods: Two groups of rats were treated with bilateral AN radiofrequency thalamotomies or sham surgery 2 weeks after pilocarpine-induced status epilepticus. After the lesions, animals were videotaped from the 2nd to the 8th week after status epilepticus (total 180 h). Results: During the 6 weeks of observation, no differences in the frequency of spontaneous seizures were found between animals that had bilateral AN lesions (n = 26; 3.1 +/- 0.6 seizures per animal) and controls (n = 25; 3.0 +/- 0.6 seizures per animal; p = 0.8). Conclusions: We conclude that AN thalamotomies were not effective in reducing the frequency of seizures during the chronic phase of the pilocarpine model of epilepsy. Copyright (C) 2009 S. Karger AG, Basel

Expression of vitamin D receptor mRNA in the hippocampal formation of rats submitted to a model of temporal lobe epilepsy induced by pilocarpine

Vitamin D (VD), is a steroid hormone with multiple functions in the central nervous system (CNS), producing numerous physiological effects mediated by its receptor (VDR). Clinical and experimental studies have shown a link between VD dysfunction and epilepsy. Along these lines, the purpose of our work was to analyze the relative expression of VDR mRNA in the hippocampal formation of rats during the three periods of pilocarpine-induced epilepsy. Male Wistar rats were divided into five groups: (1) control group; rats that received saline 0.9%, i.p. and were killed 7 days after its administration (CTRL, n = 8), (2) SE group; rats that received pilocarpine and were killed 4 h after SE (SE, n = 8), (3) Silent group-7 days; rats that received pilocarpine and were killed 7 days after SE (SIL 7d, n = 8), (4) Silent group-14 days; rats that received pilocarpine and were killed 14 days after SE (SIL 14d, n = 8), (5) Chronic group; rats that received pilocarpine and were killed 60 days after the first spontaneous seizure, (chronic, n = 8). The relative expression of VDR mRNA was determined by real-time PCR. Our results showed an increase of the relative expression of VDR mRNA in the SIL 7 days, SIL 14 days and Chronic groups, respectively (0.060 +/- 0.024; 0.052 +/- 0.035; 0.085 +/- 0.055) when compared with the CTRL and SE groups (0.019 +/- 0.017; 0.019 +/- 0.025). These data suggest the VDR as a possible candidate participating in the epileptogenesis process of the pilocarpine model of epilepsy. (C) 2008 Elsevier Inc. All rights reserved.

Estrogen effects on pilocarpine-induced temporal lobe epilepsy in rats

Objetive: To evaluate the effects of conjugated equine estrogens (CEE) on the pilocarpine-induced epilepsy in rats. Study design: 40 female rats were divided into: GPC (positive control) presented ""status epilepticus"" (SE) induced by pilocarpine; GOC(ovariectomized control) only castrated; GNC (negative control) received only saline solution; GPE received pilocarpine, presented SE, castrated and received 50 mu g/kg CEE treatment; GPV received pilocarpine, castrated and received propylene glycol (vehicle). The animals were monitored by a video system. At the end of observation, the brains removed for later histologic analysis using Neo-Timm and Nissl methods. Results: The GPE presented a reduction in number of seizures compared to GPV. The Neo-Timm analysis showed that GPV had greater sprouting of mossy fibers, with a denser band in the area of the dentate gyrus hilum compared to GPE. On Nissl staining, GPE showed evident neuronal loss in the CA3 area. GPV presented loss in CA1 and dentate gyrus. Conclusion: Estrogen may have a protecting effect on the central nervous system. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Comparative neuroanatomical and temporal characterization of FluoroJade-positive neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine in Wistar rats

The aims of this study were to characterize the spatial distribution of neurodegeneration after status epilepticus (SE) induced by either systemic (S) or intrahippocampal (H) injection of pilocarpine (PILO), two models of temporal lobe epilepsy (TLE), using FluoroJade (FJ) histochemistry, and to evaluate the kinetics of FJ staining in the H-PILO model. Therefore, we measured the severity of behavioral seizures during both types of SE and also evaluated the FJ staining pattern at 12, 24, and 168 h (7 days) after the H-PILO insult. We found that the amount of FJ-positive (FJ+) area was greater in SE induced by S-PILO as compared to SE induced by H-PILO. After SE induced by H-PILO, we found more FJ+ cells in the hilus of the dentate gyrus (DG) at 12 h, in CA3 at 24 h, and in CA1 at 168 h. We found also no correlation between seizure severity and the number of FJ+ cells in the hippocampus. Co-localization studies of FJ+ cells with either neuronal-specific nuclear protein (NeuN) or glial fibrillary acidic protein (GFAP) labeling 24 h after H-PILO demonstrated spatially selective neurodegeneration. Double labeling with FJ and parvalbumin (PV) showed both FJ+/PV+ and FJ+/PV- cells in hippocampus and entorhinal cortex, among other areas. The current data indicate that FJ+ areas are differentially distributed in the two TLE models and that these areas are greater in the S-PILO than in the H-PILO model. There is also a selective kinetics of FJ+ cells in the hippocampus after SE induced by H-PILO, with no association with the severity of seizures, probably as a consequence of the extra-hippocampal damage. These data point to SE induced by H-PILO as a low-mortality model of TLE, with regional spatial and temporal patterns of FJ staining. (C) 2010 Elsevier B.V. All rights reserved.

Study of spontaneous recurrent seizures and morphological alterations after status epilepticus induced by intrahippocampal injection of pilocarpine

Epileptic seizures are clinical manifestations of neuronal discharges characterized by hyperexcitability and/or hypersynchrony in the cortex and other subcortical regions. The pilocarpine (PILO) model of epilepsy mimics temporal lobe epilepsy (TLE) in humans. In the present study, we used a more selective approach: microinjection of PILO into the hilus of the dentate gyrus (H-PILO). Our main goal was to evaluate the behavioral and morphological alterations present in this model of TLE. Seventy-six percent of all animals receiving H-PILO injections had continuous seizures called status epilepticus (SE). A typical pattern of evolution of limbic seizures during the SE with a latency of 29.3 +/- 16.3 minutes was observed using an analysis of behavioral sequences. During the subsequent 30 days, 71% of all animals exhibited spontaneous recurrent seizures (SRSs) during a daily 8-hour videotaping session. These SRSs had a very conspicuous and characteristic pattern detected by behavioral sequences or neuroethological analysis. Only the animals that had SE showed positive Neo-Timm staining in the inner molecular layer of the dentate gyrus (sprouting) and reduced cell density in Ammon`s horn pyramidal cell subfield CA1. However, no correlation between the intensity of sprouting and the mean number and total number of SRSs was found. Additionally, using Fluoro-Jade staining, we observed neurodegeration in the hilus and pyramidal cell subfields CA3 and CM 24 hours after SE. These data indicate that H-PILO is a reliable, selective, efficient, low-mortality model that mimics the acute and chronic behavioral and morphological aspects of TLE. (C) 2010 Elsevier Inc. All rights reserved.

Comparison of the effects of pilocarpine and cevimeline on salivary flow

Objective: The aim of the present study was to compare the effect of low-dose pilocarpine and cevimeline as stimulants for salivary flow in healthy subjects. Methods: In this cross-over clinical trial with a 1-week washout period, 40 male volunteers were submitted to an oral dose of pilocarpine 1% (Salagen (TM)) -60 mu g kg(-1) body-weight (Group 1) or Cevimeline (Evoxac (TM)) -30 mg (Group 2). Saliva samples were collected and the salivary flow rate was measured (ml min(-1)) at baseline and 20, 40, 60, 80, 140 and 200 min after administration of drugs. In addition, salivary secretion was also measured under mechanical stimulation to observe salivary gland function. Results: The data were analyzed by Friedman and Wilcoxon signed-rank tests (significance level = 5%). Pilocarpine and cevimeline significantly increased salivary flow 140 min after intake. There was a significant higher secretion with cevimeline 140 and 200 min after administration. There were no differences seen among subjects in the salivary glands function by mechanical stimulation. Conclusion: Both drugs showed efficacy in increasing the salivary flow in healthy volunteers, but cevimeline was more effective than pilocarpine.

Temporal changes in mRNA expression of the brain nutrient transporters in the lithium-pilocarpine model of epilepsy in the immature and adult rat.

The lithium-pilocarpine model mimics most features of human temporal lobe epilepsy. Following our prior studies of cerebral metabolic changes, here we explored the expression of transporters for glucose (GLUT1 and GLUT3) and monocarboxylates (MCT1 and MCT2) during and after status epilepticus (SE) induced by lithium-pilocarpine in PN10, PN21, and adult rats. In situ hybridization was used to study the expression of transporter mRNAs during the acute phase (1, 4, 12 and 24h of SE), the latent phase, and the early and late chronic phases. During SE, GLUT1 expression was increased throughout the brain between 1 and 12h of SE, more strongly in adult rats; GLUT3 increased only transiently, at 1 and 4h of SE and mainly in PN10 rats; MCT1 was increased at all ages but 5-10-fold more in adult than in immature rats; MCT2 expression increased mainly in adult rats. At all ages, MCT1 and MCT2 up-regulation was limited to the circuit of seizures while GLUT1 and GLUT3 changes were more widespread. During the latent and chronic phases, the expression of nutrient transporters was normal in PN10 rats. In PN21 rats, GLUT1 was up-regulated in all brain regions. In contrast, in adult rats GLUT1 expression was down-regulated in the piriform cortex, hilus and CA1 as a result of extensive neuronal death. The changes in nutrient transporter expression reported here further support previous findings in other experimental models demonstrating rapid transcriptional responses to marked changes in cerebral energetic/glucose demand.

Effect of pilocarpine mouthwash on salivary flow

Pilocarpine is a cholinergic agonist that increases salivary flow and has been used to treat xerostomia. Oral intake is the most frequent route of administration. Adverse effects are dose-dependent and include sudoresis, facial blushing and increased urinary frequency. The objective of the present study was to evaluate the effects of topical pilocarpine solutions as mouthwashes on salivary flow and their adverse effects on healthy subjects. Forty volunteers received 10 ml 0.5, 1 and 2% pilocarpine solutions or 0.9% saline in a randomized, double-blind, placebo-controlled manner. Salivation was measured before and 45, 60 and 75 min after mouth rinsing for 1 min with 10 ml of saline or pilocarpine solutions. Vital signs were measured and ocular, gastrointestinal and cardiovascular symptoms, anxiety and flushing were estimated using visual analog scales. There was a dose-dependent increase in salivation. Salivation measured after 1 and 2% pilocarpine (1.4 ± 0.36 and 2.22 ± 0.42 g, respectively) was significantly (P<0.001) higher than before (0.70 ± 0.15 and 0.64 ± 0.1 g), with a plateau between 45 and 75 min. Cardiovascular, visual, gastrointestinal and behavioral symptoms and signs were not changed by topical pilocarpine. Mouth rinsing with pilocarpine solutions at concentrations of 1 to 2% induced a significant objective and subjective dose-dependent increase in salivary flow, similar to the results reported by others studying the effect of oral 5 mg pilocarpine. The present study revealed the efficacy of pilocarpine mouthwash solutions in increasing salivary flow in healthy volunteers, with no adverse effects. Additional studies on patients with xerostomia are needed.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 µg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 µg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 µg) injected into the MnPO prior to pilocarpine attenuated (100%) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 µg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 µg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO.

Absence-like seizures in adult rats following pilocarpine-induced status epilepticus early in life

Administration of pilocarpine causes epilepsy in rats if status epilepticus (SE) is induced at an early age. To determine in detail the electrophysiological patterns of the epileptogenic activity in these animals, 46 Wistar rats, 7-17 days old, were subjected to SE induced by pilocarpine and electro-oscillograms from the cortex, hippocampus, amygdala, thalamus and hypothalamus, as well as head, rostrum and vibrissa, eye, ear and forelimb movements, were recorded 120 days later. Six control animals of the same age range did not show any signs of epilepsy. In all the rats subjected to SE, iterative spike-wave complexes (8.1 ± 0.5 Hz in frequency, 18.9 ± 9.1 s in duration) were recorded from the frontal cortex during absence fits. However, similar spike-wave discharges were always found also in the hippocampus and, less frequently, in the amygdala and in thalamic nuclei. Repetitive or single spikes were also detected in these same central structures. Clonic movements and single jerks were recorded from all the rats, either concomitantly with or independently of the spike-wave complexes and spikes. We conclude that rats made epileptic with pilocarpine develop absence seizures also occurring during paradoxical sleep, showing the characteristic spike-wave bursts in neocortical areas and also in the hippocampus. This is in contrast to the well-accepted statement that one of the main characteristics of absence-like fits in the rat is that spike-wave discharges are never recorded from the hippocampal fields.

Comparison of sweat rate during graded exercise and the local rate induced by pilocarpine

Centrally stimulated sweat rate produced by graded exercise until exhaustion was compared to the local sweat rate induced by pilocarpine, often used as a sweating index for healthy individuals. Nine young male volunteers (22 ± 4 years) were studied in temperate environment in two situations: at rest and during progressive exercise with 25 W increases every 2 min until exhaustion, on a cycle ergometer. In both situations, sweating was induced on the right forearm with 5 ml 0.5% pilocarpine hydrochloride applied by iontophoresis (1.5 mA, 5 min), with left forearm used as control. Local sweat rate was measured for 15 min at rest. During exercise, whole-body sweat rate was calculated from the body weight variation. Local sweat rate was measured from the time when heart rate reached 150 bpm until exhaustion and was collected using absorbent filter paper. Pharmacologically induced local sweat rate at rest (0.4 ± 0.2 mg cm-2 min-1) and mean exercise-induced whole-body sweat rate (0.4 ± 0.1 mg cm-2 min-1) were the same (P > 0.05) but were about five times smaller than local exercise-induced sweat rate (control = 2.1 ± 1.4; pilocarpine = 2.7 ± 1.2 mg cm-2 min-1), indicating different sudorific mechanisms. Both exercise-induced whole-body sweat rate (P < 0.05) and local sweat rate (P < 0.05) on control forearm correlated positively with pilocarpine-induced local sweat rate at rest. Assuming that exercise-induced sweating was a result of integrated physiological mechanisms, we suggest that local and whole-body sweat rate measured during graded exercise could be a better sweating index than pilocarpine.

Assessment of the progressive nature of cell damage in the pilocarpine model of epilepsy

Pilocarpine-induced (320 mg/kg, ip) status epilepticus (SE) in adult (2-3 months) male Wistar rats results in extensive neuronal damage in limbic structures. Here we investigated whether the induction of a second SE (N = 6) would generate damage and cell loss similar to that seen after a first SE (N = 9). Counts of silver-stained (indicative of cell damage) cells, using the Gallyas argyrophil III method, revealed a markedly lower neuronal injury in animals submitted to re-induction of SE compared to rats exposed to a single episode of pilocarpine-induced SE. This effect could be explained as follows: 1) the first SE removes the vulnerable cells, leaving behind resistant cells that are not affected by the second SE; 2) the first SE confers increased resistance to the remaining cells, analogous to the process of ischemic tolerance. Counting of Nissl-stained cells was performed to differentiate between these alternative mechanisms. Our data indicate that different neuronal populations react differently to SE induction. For some brain areas most, if not all, of the vulnerable cells are lost after an initial insult leaving only relatively resistant cells and little space for further damage or cell loss. For some other brain areas, in contrast, our data support the hypothesis that surviving cells might be modified by the initial insult which would confer a sort of excitotoxic tolerance. As a consequence of both mechanisms, subsequent insults after an initial insult result in very little damage regardless of their intensity.