The analgesic potential of intraventricular polymer-encapsulated adrenal chromaffin cells in a rodent model of chronic neuropathic pain.

Adrenal chromaffin cells reportedly produce analgesic effects when implanted in the periaqueductal gray and the intrathecal space near the spinal cord. Chromaffin cells implanted in the cerebral ventricles may also produce analgesic effects, and the availability of the cerebral ventricles as a potential implant site could be advantageous for some patients. In fact, some of the first patients were implanted in the intraventricular site, even though the analgesic potential of that site had never been demonstrated. The present study was conducted to assess the analgesic potential of intraventricular, polymer-encapsulated calf adrenal chromaffin cells in the Bennett model. Sciatic nerve ligations produced substantial, long-lasting pain-related behaviors. However, there was no evidence that polymer-encapsulated adrenal chromaffin cells implanted in the cerebral ventricles produce analgesic effects in this model of chronic neuropathic pain.

Intrathecal polymer-encapsulated bovine adrenal chromaffin cells fail to produce analgesic effects in the hotplate and formalin test.

Numerous publications have reported that adrenal chromaffin cells implanted in the lumbar intrathecal space produce analgesic effects in rodent models of pain. The present study was intended to replicate and extend on the results of those previous publications. This study was conducted to determine: (1) if analgesic effects of polymer-encapsulated intrathecal adrenal chromaffin cells could be detected in the more sensitive low temperature hot-plate test without using nicotine to stimulate chromaffin cell output, (2) if a dose-response curve can be produced in the hot-plate and formalin tests with different numbers of adrenal chromaffin cells (0K, 120K, or 330K), (3) if cell viability and/or the magnitude of analgesic effects are affected by differences in implant site (i.e. thoracic versus lumbar implant site), and (4) the magnitude of the analgesic effects of adrenal chromaffin cells relative to doses of systemic morphine. No analgesic effects related to chromaffin cells were detected in the present study in the formalin test or in the hot-plate test, even with nicotine stimulation, regardless of the number of cells implanted or the intrathecal implant site. The reliability and sensitivity of the behavioral measures to the effects of mild analgesics are supported by the fact that consistent differences in performance between individual rats could be detected, the effects of fairly modest doses of nicotine and morphine were detectable, and consistent dose-response curves were evident with respect to both nicotine and morphine. Device performance appeared to be within the range of previous studies that reported analgesic effects of encapsulated adrenal chromaffin cells. It is unclear why analgesic effects of adrenal chromaffin cells were not detected in the present study.

Intrathecal implants of bovine chromaffin cells alleviate mechanical allodynia in a rat model of neuropathic pain.

Intrathecal implants of adrenal chromaffin cells are known to release analgesic substances such as catecholamines and opioid peptides. In the present study, bovine chromaffin cells were encapsulated in a permselective polymer membrane which protects the cells from the host immune system and allows grafting of xenogeneic cells without immunosuppression. The effects of such implants were evaluated on the pain behavior resulting from a chronic constrictive injury (CCI) of the rat sciatic nerve. Sprague-Dawley rats with a unilateral lesion were implanted in the lumbar subarachnoid space and tested for mechanical/thermal allodynia and hyperalgesia. A significant reduction in pain was observed after mechanical non-nociceptive stimulation in animals implanted with chromaffin cells. Furthermore, these animals showed decreased signs of spontaneous pain. However, response to thermal non-noxious stimuli or to painful mechanical stimuli was not significantly decreased. Abundant clusters of viable chromaffin cells intensely labeled with the anti-tyrosine hydroxylase antibodies were observed in the retrieved implants. These results establish the analgesic efficacy of intrathecal encapsulated chromaffin cells in a chronic pain model of nerve injury. Immunoprotected allo- or xenogeneic chromaffin cells acting as 'mini pumps' continuously delivering neuroactive substances could be a useful therapy for patients suffering from neuropathic pain.

Long-term alleviation of allodynia-like behaviors by intrathecal implantation of bovine chromaffin cells in rats with spinal cord injury.

Adrenal chromaffin cells produce analgesic substances, such as catecholamines and enkephalins, and intrathecal (i.t.) implantation of either allografted adrenal tissue or xenogenic chromaffin cells produce antinociception in animals. We evaluated the analgesic effect of bovine chromaffin cells in a model of central pain in which rats exhibit chronic allodynia-like behavior after photochemically induced ischemic spinal cord injury. Bovine chromaffin cells or endothelial cells were injected i.t. onto the lumbar spinal cord and their effects on mechanical and cold allodynia-like behaviors were studied for up to 8 weeks. The chronic allodynia-like behavior was stable for months without signs of remission and i.t. implantation of human endothelial cells did not alleviate the chronic allodynia-like behavior for the entire observation period. In contrast, 2 weeks after i.t. implantation of bovine chromaffin cells, the mechanical allodynia was abolished in the spinally injured rats, and the enhanced response to cold stimuli was significantly reduced. The overall effects were significant up to 8 weeks after i.t. implantation, although the anti-allodynic effect decreased towards the end of the observation period. No signs of side-effects were noted after i.t. implantation. The allodynia-like state was temporarily restored by naloxone (0.5 mg/kg) or phentolamine (0.3 mg/kg) injected intraperitoneally. Immunohistochemical examination revealed that tyrosine hydroxylase (TH)-positive chromaffin cells could be identified adjacent to the spinal cord up to 4 weeks after i.t. implantation, whereas at 8 weeks the TH-positive cells were sparse. It is concluded that bovine chromaffin cells stay viable in rat spinal cord for a considerable period of time after i.t. administration and alleviate chronic allodynia-like behavior in spinally injured rats, possibly through activation of opioid and alpha-adrenoceptors. The present results further document a new therapeutic approach for the treatment of chronic neuropathic pain.

Immunoisolating encapsulation of intrathecally implanted bovine chromaffin cells prolongs their survival and produces anti-allodynic effect in spinally injured rats.

We have previously reported that intrathecal (i.t.) implantation of bovine chromaffin cells has an anti-allodynic effect in a rat model of mechanical and cold allodynia-like neuropathic pain after spinal cord injury. The technique of encapsulation of the cells by a semipermeable membrane has been developed recently. The present study was undertaken to investigate the effects of encapsulated bovine chromaffin cells on the allodynia-like pain in the same model. Capsules with bovine chromaffin cells or control capsules were implanted in the spinal subarachnoidal space in rats. Their response in behavioural tests were recorded for 2 months. At termination, the capsules were explanted and examined morphologically with tyrosine hydroxylase immunohistochemistry. The mechanical allodynia was totally abolished from week 2 after implantation of the cells and throughout the 8-week test period. The abnormal cold response was also attenuated in about half of the animals. The threshold to acute nociceptive stimulation was not affected. Eight weeks after implantation, 60-80% of the encapsulated chromaffin cells were still tyrosine hydroxylase positive. No effects were observed with control capsules. The results indicate that spinal implantation of encapsulated xenogeneic chromaffin cells may be useful in treating some refractory painful states associated with spinal cord injury. Immunoisolation of chromaffin cells by a semipermeable membrane may inhibit immunorejection, prolong the survival of the cells and enhance their anti-allodynic effect. Copyright 1998 European Federation of Chapters of the International Association for the Study of Pain.

Polymer-encapsulated PC-12 cells demonstrate high-affinity uptake of dopamine in vitro and 18F-Dopa uptake and metabolism after intracerebral implantation in nonhuman primates.

Intracranial implantation of polymer-encapsulated PC-12 cells has been shown to improve motor behavioral performance in animal models of Parkinson's disease. The purpose of this blinded study was to examine whether such improvement is associated with the active uptake and metabolism of dopamine precursors by intracerebrally implanted polymer-encapsulated PC-12 cells. In an in vitro experiment we demonstrate that 3H-dopamine uptake by PC-12 cells was 10(8) fmol/min x 10(6) cells, and that this uptake can be specifically blocked 88% by the addition of 10nM of nomifensine. In the in vivo experiments, polymer-encapsulated PC-12 cells were implanted in four MPTP-treated monkeys into the left deep parietal white matter (R1) or left striatum (R2-4). A fifth MPTP-treated monkey (R5) served as a control and received left striatal implants of empty capsules. 18-F-Dopa Positron Emission Tomography (PET) imaging was performed on each monkey before and after implantation surgery by blinded investigators. PET images obtained 5-13 wk after implantation demonstrated well delineated focal areas of high 18F-dopa uptake in R1, R2, and R4. The focal area of high 18F-dopa uptake in R1 precisely coregistered on a brain magnetic resonance image to the site of implantation. R3 (in whom the polymer-encapsulated PC-12 cells demonstrated poor cell survival upon explantation) and R5 (empty capsules) failed to demonstrate any area of increased 18F-dopa uptake in their PET images. Histological examination of the host brain revealed no sprouting of dopaminergic nerve terminals around the implantation sites of the polymer-encapsulated PC-12 cells. These results indicate that the previously noted behavioral improvement after intrastriatal implantation of polymer encapsulated PC-12 cells is at least in part due to their highly specific uptake and metabolism of dopamine precursors. Furthermore, these data suggest that polymer-encapsulated PC-12 cells can store, reuptake, and functionally replenish dopamine and therefore, may be an effective treatment for Parkinson's disease.

Cellular delivery of human CNTF prevents motor and cognitive dysfunction in a rodent model of Huntington's disease.

The delivery of ciliary neurotrophic factor (CNTF) to the central nervous system has recently been proposed as a potential means of halting or slowing the neural degeneration associated with Huntington's disease (HD). The following set of experiments examined, in detail, the ability of human CNTF (hCNTF) to prevent the onset of behavioral dysfunction in a rodent model of HD. A DHFR-based expression vector containing the hCNTF gene was transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting hCNTF were transplanted bilaterally into rat lateral ventricles. Eight days later, the same animals received bilateral injections of quinolinic acid (QA, 225 nmol) into the previously implanted striata. A third group received sham surgery (incision only) and served as a normal control group. Bilateral infusions of QA produced a significant loss of body weight and mortality that was prevented by prior implantation with hCNTF-secreting cells. Moreover, QA produced a marked hyperactivity, an inability to use the forelimbs to retrieve food pellets in a staircase test, increased the latency of the rats to remove adhesive stimuli from their paws, and decreased the number of steps taken in a bracing test that assessed motor rigidity. Finally, the QA-infused animals were impaired in tests of cognitive function-the Morris water maze spatial learning task, and the delayed nonmatching-to-position operant test of working memory. Prior implantation with hCNTF-secreting cells prevented the onset of all the above deficits such that implanted animals were nondistinguishable from sham-lesioned controls. At the conclusion of behavioral testing, 19 days following QA, the animals were sacrificed for neurochemical determination of striatal choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) levels. This analysis revealed that QA decreased striatal ChAT levels by 35% and striatal GAD levels by 45%. In contrast, hCNTF-treated animals did not exhibit any decrease in ChAT levels and only a 10% decrease in GAD levels. These results support the concepts that implants of polymer-encapsulated hCNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage, produce extensive behavioral protection as a result of that neuronal sparing, and that this strategy may ultimately prove relevant for the treatment of HD.

Neuroendocrine and cardiovascular effects of serotonin: selective role of brain angiotensin on vasopressin.

Central serotonin (5-HT) and angiotensin (ANG II) stimulate arginine vasopressin (AVP), oxytocin (OT), and adrenocorticotropin (ACTH) secretion and increase blood pressure. Studies were conducted in conscious rats to determine whether neuroendocrine activation by 5-HT requires a brain angiotensinergic intermediate pathway. In the first study, ANG II formation was inhibited by the angiotensin-converting enzyme inhibitor enalapril before injection of the 5-HT releaser/uptake inhibitor d-fenfluramine. Fenfluramine (2 mg/kg ip) stimulated AVP, OT, corticosterone, and prolactin (PRL) secretion (P<0.01). Enalapril (60 mg/l in drinking water for 4 days and 10 mg/kg ip 2 h before the rats were killed) inhibited only the AVP response (P<0.01) to d-fenfluramine. In the second study, the effect of intracerebroventricular injection of the 5-HT2A/2C antagonist LY-53857 (10 microgram), or the ANG II AT1 antagonist DuP-753 (10 microgram), on intracerebroventricular 5-HT (10 microgram)-stimulated AVP, OT, ACTH, PRL, renin secretion, mean arterial pressure (MAP) and heart rate (HR) was tested. LY-53857 inhibited the AVP, OT, and ACTH responses to 5-HT (P<0.01), whereas DuP-753 inhibited only the AVP response (P<0.01). Intraventricular injection of 5-HT increased MAP and decreased HR. The MAP response was not affected by LY-53857 or DuP-753, and at no time did MAP decline below starting levels. The decreased HR was inhibited by LY-53857 but not by DuP-753. These results demonstrate that 5-HT-induced AVP secretion is mediated selectively via brain angiotensinergic mechanisms by way of the AT1 receptor.

Blockade of N- and Q-type Ca2+ channels inhibit K(+)-evoked [3H]acetylcholine release in rat hippocampal slices.

In the present study, we examined the contribution of specific Ca2+ channels to K(+)-evoked hippocampal acetylcholine (ACh) release using [3H]choline loaded hippocampal slices. [3H]ACh release was Ca(2+)-dependent, blocked by the nonspecific Ca2+ channel blocker verapamil, but not by blockade of L-type Ca2+ channels. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTx GVIA; 250 nM) inhibited [3H]ACh release by 44% and the P/Q-type Ca2+ channel blocker omega-agatoxin IVA (omega-Aga IVA; 400 nM) inhibited [3H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM omega-Aga IVA was necessary to inhibit [3H]ACh release. omega-Conotoxin MVIIC (omega-CTx-MVIIC) was used after first blocking N-type Ca2+ channels with omega-CgTx GVIA (1 microM). Under these conditions, 500 nM omega-CTx-MVIIC led to a nearly maximal inhibition of the omega-CgTx GVIA-insensitive [3H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca2+ channels to these toxins, this study indicates that N- and Q-type Ca2+ channels primarily mediate K(+)-evoked hippocampal [3H]ACh release.

Studies on the role of K+, Cl- and Na+ ion permeabilities in the acetylcholine release enhancing effects of linopirdine (DuP 996) in rat cortical slices.

Linopirdine is a compound being assessed for value in reversing the dementia associated with Alzheimer's disease. The drug improves learning and memory performance in several rodent behavioral paradigms. Its proposed mechanism of action is the enhancement of neurotransmitter release, but the molecular site which mediates this action is unknown. The present study examines the possible involvement of channels which mediate the movement of K+, Cl- and Na+, which are important to the polarization state of excitable membranes, in the release-enhancing effects of linopirdine. Linopirdine causes a shift in the K+ dose response for the release of tritium from cerebral cortical slices preloaded with [3H]choline ([3H]acetylcholine (ACh) released) to the left, consistent with the blockade of a site, such as a K+ channel, involved in dampening the response of neuronal terminals to depolarizing stimuli. Linopirdine does not appear to act at aminopyridine-sensitive K+ channels, inasmuch as the aminopyridines and linopirdine have different patterns of effects regarding [3H]ACh release. Tetraethylamonium (TEA), on the other hand, does share common effects with linopirdine. TEA enhances K(+)-evoked [3H]ACh release to as much as 620% of control without affecting basal efflux of the neurotransmitter. Experiments using the muscarinic agonist carbachol indicate that linopirdine does not affect the cholinergic autoreceptor influence on [3H]ACh release. Linopirdine also does not appear to act at tetrodotoxin-sensitive Na+ channels. The anion channel blocker 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and Cl(-)-deficient media do not affect the level of linopirdine release enhancement.

The role of serotonergic neurons in intravenous hypertonic saline-induced secretion of vasopressin, oxytocin, and ACTH.

This study tested the effect of brain serotonin (5-HT) depletion on the secretion of oxytocin (OT), vasopressin (VP), and adrenocorticotropin (ACTH) due to an osmotic load. The 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) was used to deplete brain 5-HT. The OT, VP, and ACTH osmotic sensitivity (slope of delta[OT]/delta[Osm]) and the osmotic threshold (X intercept of delta[OT]/delta[Osm]) were evaluated. Depletion of brain 5-HT decreased the OT osmotic sensitivity by > 80% (p < 0.001) without changing the OT osmotic threshold. Brain 5-HT depletion had no effect on the VP osmotic sensitivity and increased the VP osmotic threshold from 287.8 +/- 1.5 to 293.1 +/- 2.0 mOsm/kg (p < 0.05). The plasma ACTH increase due to infusion of hypertonic saline was not affected by brain 5-HT depletion. Brain 5-HT depletion significantly (p < 0.01) decreased the pituitary content of OT and VP by 38 and 32%, respectively, without changing ACTH content. These results provide evidence for a functional role of serotonergic neurons in osmoregulation of plasma and pituitary concentration of OT and VP, but not ACTH.

Enhanced serotonergic transmission stimulates oxytocin secretion in conscious male rats.

The involvement of serotonin (5-HT) in oxytocin secretion was investigated in this study. Pharmacologic agents that influence serotonergic transmission were administered to conscious unrestrained male rats 30 min prior to sacrifice and plasma oxytocin concentration was measured by radioimmunoassay. The d- and l-stereoisomers of the 5-HT releaser fenfluramine significantly increased plasma oxytocin in a dose-dependent manner. Oxytocin secretion was more potently stimulated by d-fenfluramine than by l-fenfluramine. The 5-HT releaser p-chloroamphetamine also increased plasma oxytocin. The following 5-HT agonists increased plasma oxytocin concentration: the 5-HT1&2 agonist m-chlorophenyl-piperazine [10-20 mg/kg intraperitoneally (i.p.)], the 5-HT1C&2 agonist 1-(2,5-dimethoxy-4-l-phenyl)-2-aminopropane (0.5-2.0 mg/kg i.p.) and the 5-HT1&2 agonist 1-piperazinyl-6-chloropyrazine (10 mg/kg i.p.). In contrast, the 5-HT1AB agonist 5-methoxy-3-(1,2,3,4-tetrahydro-4-pyridinyl)-1H-indole (0.2-5.0 mg/kg i.p.) did not increase oxytocin secretion. Pretreatment with the 5-HT1C&2 antagonist, 6-(2-(4-[bis(4-fluorophenyl)methylene]-1-piperidinyl)ethyl)-7-methyl-5H- thiazolo(3(1)2-a)pyrimidin-5-one [2.5 mg/kg subcutaneously (s.c.)], 60 min before injection of 1-piperazinyl-6-chloropyrazine attenuated, but did not completely block, 1-piperazinyl-6-chloropyrazine-induced secretion of oxytocin. Both low and high (0.01 and 0.1 mg/kg s.c.) doses of 6-(2-(4-[bis(4-fluorophenyl)methylene]-1-piperidinyl)ethyl)-7-methyl-5H- thiazolo(3(1)2-a)pyrimidin-5-one or the 5-HT2 antagonist spiperone inhibited the 1-(2,5-dimethoxy-4-l-phenyl)-2-aminopropane-induced increases in plasma oxytocin. These studies provide evidence that enhanced serotonergic transmission stimulates oxytocin secretion and that 5-HT2 receptors contribute to this effect.

Effects of immunoneutralization of corticotropin-releasing hormone on ultradian rhythms of plasma adrenocorticotropin.

ACTH, like other anterior pituitary peptide hormones, is secreted episodically and demonstrates both circadian and ultradian rhythms. CRH is the major regulator of ACTH release from the pituitary corticotroph. To determine the dependence of ACTH ultradian rhythms on CRH, passive immunoneutralization was used to block the activity of endogenous CRH in rats with indwelling venous catheters. Blood was sampled at 2- and 15-min intervals while blood volume was replaced. Plasma ACTH was measured by RIA. Time-series analysis of plasma ACTH concentrations was performed with PULSAR and Cluster Analysis. The 2 min data demonstrated secretory bursts approximately every 20 min. CRH immunoneutralization had no effect on the frequency of these pulses, but significantly reduced their amplitude. This was the case for raw data as well as data in which lower frequency variation had been filtered out. The 15 min data demonstrated pulsatile secretion, with a secretory episode approximately every 100 min. This lower frequency rhythm was also observed when high frequency components were filtered out of the 2 min data series. Analysis of the 15 min and the filtered 2 min time series showed this rhythm to be almost totally ablated by CRH immunoneutralization. These results suggest that CRH is responsible for amplitude modulation of an underlying CRH-independent rhythm and that through intermittent amplitude modulation of this rhythm a lower frequency rhythm is generated. Comparison between treatment groups of pulses identified by PULSAR or Cluster Analysis yielded similar results, but the programs were discordant with each other. This is the first in vivo evidence of pulsatile ACTH secretion independent of CRH, the first report demonstrating that different ultradian rhythms of ACTH may be regulated by different mechanisms, and the first comparison of PULSAR and Cluster Analysis on plasma ACTH time series.