Jacqueline Sagen

Loss of gaba-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts

Abnormal pain-related behaviour that accompanies peripheral nerve injury may be the result of altered spinal neuronal function. The long-term loss of inhibitory function by GABA neurons in particular may be a mechanism by which abnormal neural hyperactivity occurs, leading to exaggerated sensory processing following nerve injury. In order to assess this, changes in spinal GABA immunoreactivity at several time points following constriction nerve injury were quantified in parallel with behavioural assessments of abnormal sensory responses to noxious and innocuous stimuli. In addition, the effects of spinal adrenal medullary transplants were determined since previous findings have demonstrated alleviation of behavioural pain symptoms by such transplants. In response to unilateral sciatic nerve injury, GABAergic profiles normally found in lumbar dorsal horn laminae I-III significantly decreased. The decrease was apparent three days following ligation, particularly on the side ipsilateral to the nerve injury. By two weeks, no GABAergic profiles could be seen, with the deficit appearing in the spinal dorsal horn both ipsilateral and contralateral to the unilateral peripheral nerve injury. Marked decreases in GABA-immunoreactive profiles persisted for at least up to five weeks post-injury, with partial restoration occurring by seven weeks. However, even at seven weeks, losses in GABA-immunoreactive profiles persisted in the dorsal horn ipsilateral to peripheral nerve injury. These findings were comparable in animals receiving control striated muscle transplants. In contrast, adrenal medullary transplants markedly reduced the loss in GABA-immunoreactive profiles at all time-points examined. In addition, GABA-immunoreactive profile levels were normalized near that of intact animals by five to seven weeks following nerve injury in animals with adrenal medullary transplants. Parallel improvements in sensory responses to innocuous and noxious stimuli were also observed in these animals. The results of this study indicate that peripheral nerve injury can result in severe losses in spinal inhibitory mechanisms, possibly leading to exaggerated sensory processes in persistent pain states. In addition, adrenal medullary transplants may provide a neuroprotective function in promoting recovery and improving long-term survival of GABAergic neurons in the spinal dorsal horn which have been damaged by excitotoxic injury.

Effect of intrathecally administered noradrenergic antagonists on nociception in the rat

Recent evidence suggests that some groups of noradrenergic neurons found in the brainstem have axonal connections in the spinal cord dorsal horn and may be involved in the control of pain sensitivity. Such evidence includes the demonstration that intrathecal injection of noradrenergic agonists increases nociceptive threshold (hypoalgesia). The present studies examined whether the descending noradrenergic system is tonically active and, if so, what noradrenergic receptor subtypes mediate the actions of endogenously-released norepinephrine. These studies involved the measurement of nociceptive threshold before and after the intrathecal injection of noradrenergic antagonists having different relative affinities for alpha-noradrenergic receptor subtypes. The intrathecal administration of alpha-noradrenergic antagonists produced a dose-dependent decrease in nociceptive threshold (hyperalgesia). This finding is consistent with the proposal that tonically-active bulbospinal noradrenergic neurons modulate the processing of nociceptive information in the spinal cord. The potency and duration of the hyperalgesia was correlated with the relative potency of the antagonists for the alpha-2 noradrenergic receptor. The relative potencies were as follows: yohimbine greater than phentolamine greater than WB 4101 greater than prazosin. Thus, endogenous norepinephrine which is tonically released from bulbospinal axon terminals may interact preferentially with noradrenergic receptors of the alpha-2 type.

Evidence for pain modulation by pre- and postsynaptic noradrenergic receptors in the medulla oblongata

Activation of neurons in nucleus raphe magnus (NRM) produces hypoalgesia which most likely results from inhibition of spinal cord pain transmission pathways. Previous reports from this laboratory suggest that noradrenergic (NA) neurons modulate the activity of NRM neurons. More specifically, NA projections to NRM neurons appear to be inhibitory since iontophoretically applied norepinephrine (NE) inhibits the activity of NRM neurons. Furthermore, blockade of NA receptors in the NRM by the microinjection of alpha-adrenergic antagonists produces potent analgesia. Thus, the NA input to the NRM appears to increase pain sensitivity by tonically inhibiting NRM neurons. Pharmacological and physiological studies have differentiated alpha-adrenergic receptors into alpha-1 and alpha-2 subtypes. The present study was designed to examine the nature of the alpha-adrenergic receptor subtypes in the NRM and their role in the modulation of pain sensitivity. The results of these experiments are consistent with the classical model of postsynaptic alpha-1 receptors and presynaptic alpha-2 receptors which modulate NE release. Both the alpha-1 antagonist, prazosin, and the alpha-2 agonist, clonidine, produced an increase in nociceptive threshold. Conversely, both the alpha-1 agonist, phenylephrine, and the alpha-2 antagonist, yohimbine, produced a decrease in nociceptive threshold. Thus, in the region of the NRM, both presynaptic alpha-2 and postsynaptic alpha-1 noradrenergic receptors may be involved in the modulation of nociception.

Adrenal medullary implants in the rat spinal cord reduce nociception in a chronic pain model.

&NA; Previous work in this laboratory has indicated that the transplantation of adrenal medullary tissue into the subarachnoid space of the rat spinal cord can reduce pain sensitivity to acute noxious stimuli, particularly following stimulation by nicotine. This most likely results from the stimulated release of opioid peptides and catecholamines from the transplanted chromaffin cells. However, chronic pain models may more closely resemble human clinical pain, and the arthritic rat model has been used for screening potential therapeutic strategies. The purpose of the present study was to assess the potential for adrenal medullary tissue implanted into the spinal subarachnoid space to alleviate chronic pain. Adrenal medullary tissue was implanted into adjuvant‐in‐duced arthritic rats, and changes in body weight and vocalization responses were monitored over the 10 week course of the disease. Results indicate that the severe weight reduction normally associated with this inflammatory arthritis was attenuated by adrenal medullary, but not control, implants. In addition, vocalizations were reduced in animals implanted with adrenal medullary, but not control tissue following nicotine stimulation. This reduction was blocked by the opiate antagonist, naloxone, and partially attenuated by the alpha‐adrenergic antagonist, phentolamine. Together, these results suggest that the transplantation of adrenal medullary tissue into the subarachnoid space of the spinal cord may provide a local source of opioid peptides and catecholamines for the reduction of chronic pain.

Adrenal medullary tissue transplants in the rat spinal cord reduce pain sensitivity.

Adrenal chromaffin cells contain and release several neuroactive substances which induce analgesia when injected directly into the spinal cord (e.g. opioid peptides and catecholamines). Furthermore, the release of these substances can be induced by nicotine. In order to determine whether adrenal medullary tissue transplanted to the spinal cord can produce alterations in pain sensitivity, pieces of dissected rat adrenal medulla were placed in the subarachnoid space of rat spinal cords. Stimulation by a low dose of nicotine induced potent analgesia in animals with adrenal medullary transplants, but not in animals with control transplants. Furthermore, this analgesia was reversed to pre-nicotine levels by the opiate antagonist naloxone. Thus adrenal medullary transplants in the spinal cord may provide a permanent and locally available source of opioid peptides for the relief of intractable pain.

Reduced pain-related behavior by adrenal medullary transplants in rats with experimental painful peripheral neuropathy.

&NA; Adrenal medullary transplants in the spinal subarachnoid space, by providing a continual source of opioid peptides and catecholamines, offer a potentially important adjunct in the management of chronic pain. While previous studies have shown that this approach is effective against high‐intensity phasic stimuli, adrenal medullary implants need to be evaluated against long‐term and abnormal pain syndromes before transplantation can be used for human chronic pain. Using a recently developed model of painful peripheral neuropathy, the effects of adrenal medullary chromaffin cells transplanted into the subarachnoid space was evaluated. Peripheral mononeuropathy was induced by loosely tying 4 ligatures (4‐0 chromic gut) around the right sciatic nerve. This procedure produces various pain symptoms including allodynia, hyperalgesia and dysesthesia. Rats were given either adrenal medullary tissue or control striated muscle transplants. Animals with adrenal medullary tissue transplants showed markedly decreased allodynia to innocuous cold as early as 1 week post‐transplantation. In addition, hyperalgesia to a noxious thermal stimulus was eliminated by adrenal medullary, but not control, transplants. Touch‐evoked allodynia was only slightly reduced by adrenal medullary transplants. In addition, indicators of spontaneous pain appeared reduced in animals with adrenal medullary transplants. These findings indicate that adrenal medullary transplants may be effective in reducing neuropathic pain.

Adrenal medullary transplants increase spinal cord cerebrospinal fluid catecholamine levels and reduce pain sensitivity

Abstract: Previous work in this laboratory has shown that adrenal medullary transplants into the spinal cord subarachnoid space can reduce pain sensitivity. This analgesia most likely results from the release of neuroactive substances, particularly catecholamines and opioid peptides, from the transplanted cells into the CSF of the spinal cord, since it can be attenuated or blocked by α‐adrenergic or opiate antagonists. The purpose of the present study was to more directly measure the release of catecholamines from adrenal medullary transplants in the spinal cord CSF using a spinal superfusion technique. CSF samples from rats with 6‐month‐old transplants were assayed for catecholamines using HPLC with electrochemical detection. Results indicated that norepinephrine levels were increased threefold, and epinephrine levels nearly 100‐fold, in animals with adrenal medullary transplants compared with control transplanted animals. There was no apparent increase in dopamine levels. Furthermore, the increased levels of total catecholamines were correlated with decreased pain sensitivity. Results of this study indicate that adrenal medullary transplants can survive for long periods in the rat spinal CSF and continue to release high levels of catecholamines. Together, the release of catecholamines and opioid peptides from adrenal medullary transplants may provide the ideal combination for the reduction of pain.

Increased levels of Met-enkephalin-like immunoreactivity in the spinal cord CSF of rats with adrenal medullary transplants.

Recent work in our laboratory has demonstrated that the transplantation of adrenal medullary tissue into the spinal cord subarachnoid space can reduce pain sensitivity, particularly following nicotinic stimulation. This analgesia most likely results from the release of opioid peptides from the implanted chromaffin cells since it is blocked by the opiate antagonist naloxone. The purpose of the present study was to more directly measure opioid peptide release from adrenal medullary implants in the spinal cord using spinal cord superfusions. Basal levels of Met-enkephalin-like immunoreactivity (MELI) in spinal cord superfusates of animals with adrenal medullary implants was twice that in animals with control implants. The injection of nicotine further increased MELI release in adrenal medullary, but not control implanted animals. Both the basal MELI levels and the MELI levels following nicotine were correlated with reduced pain sensitivity in animals with adrenal medullary implants. Morphological studies revealed good long-term survival of grafted chromaffin cells. Results of this study suggest that it is possible to increase opioid peptide levels and concomitantly decrease pain sensitivity by the transplantation of adrenal medullary tissue into the spinal cord subarachnoid space.

Transplantation of microencapsulated bovine chromaffin cells reduces lesion-induced rotational asymmetry in rats.

Surrounding bovine chromaffin cells by a semipermeable membrane may protect the transplanted cells from a host immune response and shield them from the inflammatory process resulting from the surgical trauma. Encapsulation of the chromaffin cells was achieved by interfacial adsorption of a polycation on a polyanionic colloid matrix in which the chromaffin cells were entrapped. Basal and potassium-evoked release of catecholamines from encapsulated bovine chromaffin cells was analyzed over a 4-week period in vitro. Norepinephrine and dopamine release remained constant over time whereas epinephrine release significantly decreased. The chromaffin cells also retained the capacity for depolarization-elicited catecholamine release 4 weeks following the encapsulation procedure. Morphological analysis revealed the presence of intact chromaffin cells with well-preserved secretory granules. Striatal implantation of chromaffin cell-loaded capsules significantly reduced apomorphine-induced rotation compared to empty polymer capsules in animals lesioned with 6-hydroxydopamine for at least 4 weeks. Intact chromaffin cells expressing tyrosine hydroxylase and dopamine-beta-hydroxylase were observed in all capsules implanted in the striatum for 4 weeks. The assessment of the clinical potential of transplanting encapsulated adrenal chromaffin cells of either allo- or xenogeneic origin for Parkinsons disease will require long-term behavioral studies. The present study suggests, however, that the polymer encapsulation procedure may offer an alternative to adrenal autografts as a source of dopaminergic tissue.

Transplantation of encapsulated bovine chromaffin cells in the sheep subarachnoid space: A preclinical study for the treatment of cancer pain

Chromaffin cells have been shown to release a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides. The allogeneic transplantation of chromaffin cells in the subarachnoid space has been shown to alleviate pain in various rodent models and possibly in terminal cancer patients. Because of the shortage of human cadaver donor tissue, we are investigating the possibility of transplanting xenogeneic cells in polymer capsules. In this technique, cells are surrounded by a permselective synthetic membrane whose pores are suitably sized to allow diffusion of nutrients, neurotransmitters and growth factors, but restrict the diffusion of the large molecules of the immune system and prevent contact with immunocompetent cells. The encapsulation technique therefore allows transplantation of xenogeneic tissue between species as well as retrieval of transplanted cells. Previously we have reported that encapsulated bovine chromaffin cells survive and alleviate pain in various rodent models. The purpose of the present study was to assess the feasibility of implanting a human sized device in a large animal model. Adrenals from 5 calves were surgically removed; chromaffin cells were isolated from these glands using a collagenase-based digestion-filtration technique. Cells were loaded into acrylic-based tubular (5 cm long, 920 μm wide) permselective capsules attached to silicone tethers. The capsules were maintained in vitro for at least 7 days following the encapsulation procedure. Nicotine evoked release was analyzed in a defined subgroup from each batch. One capsule was then implanted using a guiding cannula system in the lumbar subarachnoid space of each sheep for 4 (n = 5) and 8 (n = 1) wk. All capsules were retrieved intact by gentle pulling on the silicone tether. Except for one capsule, the evoked catecholamine release of the retrieved capsules was in the same range as that of other capsules from the same cohort that had been maintained in vitro. All retrieved capsules were devoid of host cell reaction. Clusters of viable cells dispersed in an alginate immobilizing matrix were observed throughout all the implanted capsules. This study demonstrates the feasibility of transplanting functional encapsulated xenogeneic chromaffin cells into the cerebrospinal fluid of a large animal model using a capsule of appropriate dimensions for human implants. We believe that these results suggest the appropriateness of human clinical trials in patients suffering from refractory terminal cancer pain.