George D. Pappas

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.

Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar gamma-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the alpha(3) subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln-integrin interactions and the consequent decrease of cytoskeleton protein turnover.

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.

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy.

Reelin is a glycoprotein (∼400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.

Reelin function in neural stem cell biology

In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, α3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients.

Neural tissue engineering: Adrenal chromaffin cell attachment and viability on chitosan scaffolds

This study introduces chitosan-based matrices as cell substrates for bovine chromaffin cell attachment in transplantation procedures. Chitosan ([1-->4] linked 2-amino-2-deoxy-beta-D-glucopyranose), having structural similarity to glycosaminoglycans, was modified using several proteins (collagen, albumin and gelatin) to increase surface area and improve biocompatibility. In vitro, collagen-blended chitosan (CC) matrices were found to attach more readily to chromaffin cells than to gelatin- or albumin-blended matrices. Morphological evidence showed that the chromaffin cells attached to CC substrates integrated well with the hydrogel matrix and survived for at least two weeks, under in vivo culture conditions. The chromaffin cells within chitosan scaffolds also survived for at least two weeks in vitro and after subarachnoid grafting to rats.

Oxytocin receptors in brain cortical regions are reduced in haploinsufficient (+/−) reeler mice

Abstract Objective: Both oxytocin (OT) and reelin are particularly significant during development and the absence of either may interfere with normal brain development. In addition, reelin is critical to the development of the GABAergic system and GABA modulates the release of OT. Availability of the reelin haploinsufficient (+/−) reeler mouse (HRM) provides a model for examining the role of reelin in the development of the OT system and especially in the expression of the OT receptor (OTR). Methods: In this study we used immunocytochemistry and in situ hybridization in HRM versus wild-type (+/−) mice (WTM) to quantify OTR abundance in regions of the brain cortex. Results: Our findings reveal that the oxytocin receptor (OTR), measured either by immunohistochemistry or in situ hybridization, is significantly lower in HRM. Areas showing significant deficits included the piriform cortex, neocortex, retrosplenial cortex and certain regions of the hippocampus. Conclusion: Both reelin and OT play a role in regulating affect and mood. Down-regulation of reelin has been strongly correlated with schizophrenia and it is proposed that HRM may serve as a model for neural deficits seen in both schizophrenia and autism. We report that HRM show regionally specific reductions in OTRs, especially in cortical areas, which previously have been implicated in social memory and cognitive functions. These findings offer support for the more general hypothesis that down-regulation of reelin, of either genetic or epigenetic origin, through associated reductions in the OTRs, contributes to the deficiencies in social behavior that are characteristic of both schizophrenia and autism.

Relief of intractable cancer pain by human chromaffin cell transplants: Experience at two medical centers

In addition to its possible role as a replacement source in CNS degenerative diseases, neural transplantation may be used to augment the normal production of neuroactive substances. Our laboratory at the University of Illinois at Chicago has shown, in both acute and chronic pain models, that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as the donor source since they produce high levels of both opioid peptides and catecholamines, substances which reduce pain sensitivity when injected locally into the spinal subarachnoid space. The analgesia produced by these transplants probably results from the release of both opioid peptides and catecholamines since it can be blocked or attenuated by both opiate and adrenergic antagonists. Studies indicate that even over long periods there is no apparent development of tolerance. Promising results have been obtained in preliminary clinical studies using allografts of adrenal medulla to relieve cancer pain. This clinical review encompasses results at two Medical Centers-University of Illinois at Chicago and University Paul Sabatier, Toulouse, France-in assessing efficacy of subarachnoid adrenal medullary transplantation for alleviating cancer pain. Our clinical and autopsy data strongly support our previous laboratory studies, i.e., that chromaffin cell transplants into the subarachnoid space represent a promising new approach to the alleviation of chronic pain. It is suggested that further clinical studies are now warranted.

Short-term immunosuppression enhances long-term survival of bovine chromaffin cell xenografts in rat CNS.

Xenogeneic donors, a largely untapped resource, would solve many of the problems associated with the limited availability of human donor tissue for neural transplantation. Previous work in our laboratory has revealed that xenografts of isolated bovine chromaffin cells survive transplantation into the periaqueductal gray (PAG) of immunosuppressed adult rats. Electron microscopic analysis reveals that graft sites contain healthy chromaffin cells, but do not contain host immune cells typical of graft rejection. The aim of the current study was to assess the necessary conditions for long-term survival of bovine chromaffin cell xenografts in the central nervous system (CNS). In particular, the need for short-course vs. permanent immunosuppressive therapy with cyclosporine A (CsA) for the long-term survival of grafted bovine chromaffin cells was addressed. Grafts from animals receiving continuous CsA treatment for either 3, 6, or 12 wk contained large clumps of dopamines-β-hydroxylase (DBH) positive cells in contrast to the few surviving cells observed in nonimmunosuppressed animals. In addition, grafts from animals that had CsA treatment terminated at 3 or 6 wk contained similarly large clumps of DBH-positive cells. Furthermore, short-term immunosuppression (3 wk) appeared to enhance the long-term survival of grafted cells, since clumps of DBH staining cells could still be positively identified in the host PAG at least 1 yr after transplantation. Complete rejection of graft tissue depends on several factors, such as blood–brain barrier integrity, the presence of major histocompatability complex (MHC) antigens in either the host or graft, and the status of the host immune system. By using a suspension of isolated bovine chromaffin cells, potential MHC antigen presenting cells, such as endothelial cells, are eliminated. In addition, CsA treatment may negate the immunologic consequences of increased blood–brain barrier permeability following surgical trauma by attenuating the host cell mediated response. In summary, long-term survival of isolated chromaffin cell xenografts in the rat CNS may be attained by a short-term course of CsA.