Increased spinal c-Fos expression with noxious and non-noxious peripheral stimulation after severe spinal contusion.

The effects of severe contusive spinal cord injury (SCI), at thoracic level 8 (T8), on lumbar c-Fos expression in the spinal cord was investigated. As hypothesized, chronic SCI has a significant effect on expression of c-Fos in the dorsal spinal sensory areas with noxious and innocuous peripheral stimulation of the sciatic nerve. This alteration to stimulation effects was measured using counts of c-Fos immunoreactive cells in the dorsal horn of the L5 lumbar spinal cord in injured animals at 90 days post-injury and in uninjured controls. The number of c-Fos immunoreactive cells increased in SCI rats only after noxious peripheral stimulation (electrical and chemical) suggesting a general increase in excitability in spinal pathways (central sensitization) associated with chronic SCI. These altered responses may represent a functional anatomical reorganization of spinal cord circuitry leading to increased dorsal horn c-Fos expression as a response to severe chronic contusive damage to the spinal cord sensory pathways.

Metal-backed versus all-polyethylene unicompartmental knee arthroplasty: Proximal tibial strain in an experimentally validated finite element model.

OBJECTIVES: Up to 40% of unicompartmental knee arthroplasty (UKA) revisions are performed for unexplained pain which may be caused by elevated proximal tibial bone strain. This study investigates the effect of tibial component metal backing and polyethylene thickness on bone strain in a cemented fixed-bearing medial UKA using a finite element model (FEM) validated experimentally by digital image correlation (DIC) and acoustic emission (AE). MATERIALS AND METHODS: A total of ten composite tibias implanted with all-polyethylene (AP) and metal-backed (MB) tibial components were loaded to 2500 N. Cortical strain was measured using DIC and cancellous microdamage using AE. FEMs were created and validated and polyethylene thickness varied from 6 mm to 10 mm. The volume of cancellous bone exposed to < -3000 µÎµ (pathological loading) and < -7000 µÎµ (yield point) minimum principal (compressive) microstrain and > 3000 µÎµ and > 7000 µÎµ maximum principal (tensile) microstrain was computed. RESULTS: Experimental AE data and the FEM volume of cancellous bone with compressive strain < -3000 µÎµ correlated strongly: R = 0.947, R2 = 0.847, percentage error 12.5% (p < 0.001). DIC and FEM data correlated: R = 0.838, R2 = 0.702, percentage error 4.5% (p < 0.001). FEM strain patterns included MB lateral edge concentrations; AP concentrations at keel, peg and at the region of load application. Cancellous strains were higher in AP implants at all loads: 2.2- (10 mm) to 3.2-times (6 mm) the volume of cancellous bone compressively strained < -7000 µÎµ. CONCLUSION: AP tibial components display greater volumes of pathologically overstrained cancellous bone than MB implants of the same geometry. Increasing AP thickness does not overcome these pathological forces and comes at the cost of greater bone resection.Cite this article: C. E. H. Scott, M. J. Eaton, R. W. Nutton, F. A. Wade, S. L. Evans, P. Pankaj. Metal-backed versus all-polyethylene unicompartmental knee arthroplasty: Proximal tibial strain in an experimentally validated finite element model. Bone Joint Res 2017;6:22-30. DOI:10.1302/2046-3758.61.BJR-2016-0142.R1.

Hyperglycemia reduces functional expression of astrocytic Kir4.1 channels and glial glutamate uptake.

Diabetics are at risk for a number of serious health complications including an increased incidence of epilepsy and poorer recovery after ischemic stroke. Astrocytes play a critical role in protecting neurons by maintaining extracellular homeostasis and preventing neurotoxicity through glutamate uptake and potassium buffering. These functions are aided by the presence of potassium channels, such as Kir4.1 inwardly rectifying potassium channels, in the membranes of astrocytic glial cells. The purpose of the present study was to determine if hyperglycemia alters Kir4.1 potassium channel expression and homeostatic functions of astrocytes. We used q-PCR, Western blot, patch-clamp electrophysiology studying voltage and potassium step responses and a colorimetric glutamate clearance assay to assess Kir4.1 channel levels and homeostatic functions of rat astrocytes grown in normal and high glucose conditions. We found that astrocytes grown in high glucose (25 mM) had an approximately 50% reduction in Kir4.1 mRNA and protein expression as compared with those grown in normal glucose (5mM). These reductions occurred within 4-7 days of exposure to hyperglycemia, whereas reversal occurred between 7 and 14 days after return to normal glucose. The decrease in functional Kir channels in the astrocytic membrane was confirmed using barium to block Kir channels. In the presence of 100-µM barium, the currents recorded from astrocytes in response to voltage steps were reduced by 45%. Furthermore, inward currents induced by stepping extracellular [K(+)]o from 3 to 10mM (reflecting potassium uptake) were 50% reduced in astrocytes grown in high glucose. In addition, glutamate clearance by astrocytes grown in high glucose was significantly impaired. Taken together, our results suggest that down-regulation of astrocytic Kir4.1 channels by elevated glucose may contribute to the underlying pathophysiology of diabetes-induced CNS disorders and contribute to the poor prognosis after stroke.

Distribution of aromatic L-amino acid decarboxylase mRNA in mouse brain by in situ hybridization histology.

Aromatic L-amino acid decarboxylase (AAAD) is the second enzyme in the sequence leading to the synthesis of catecholamines or serotonin. Antisense riboprobes for aromatic L-amino acid decarboxylase mRNA were used to map the gene in mouse brain by in situ hybridization. The substantia nigra, the ventral tegmental nucleus, the dorsal raphe nucleus, the locus coeruleus, and the olfactory bulb contained the highest signal for AAAD mRNA. After treatment with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the signal disappeared in the substantia nigra, decreased somewhat in the ventral tegmental area, and remained unchanged in the dorsal raphe nucleus. Hypothalamic and cerebellar Purkinje neurons known to contain histidine decarboxylase or glutamic acid decarboxylase, respectively, were unlabeled by the probes. However, neurons in the deep layers of the frontal cortex, many thalamic nuclei, and the pyramidal neurons of the hippocampus were lightly to moderately labeled for mouse AAAD mRNA. The presence of AAAD message in these neurons suggests that the enzyme has functions other than that for the synthesis of the classical biogenic amine neurotransmitters.

Lumbar transplants of immortalized serotonergic neurons alleviate chronic neuropathic pain.

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to non-permissive temperature. Brain-derived neurotrophic factor (BDNF) induced the serotonergic phenotype and increased the survival of RN46A cells in vitro. After transfection of the rat BDNF gene into RN46A cells, an autocrine BDNF-secreting cell line, 46A-B14, was isolated and transplanted into the rat CNS. Transplanted 46A-B14 cells had increased survival and enhanced serotonin (5HT) synthesis compared to 46A-V1 cells, RN46A cells transfected with vector-alone. When 46A-B14 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after a chronic constriction injury (CCI) of the sciatic nerve, they survived longer than 6 weeks on the pia mater. Furthermore, the tactile and cold allodynia and thermal hyperalgesia induced by CCI was significantly reduced during a 4-6- week period. The maximal effect occurred 1 week after transplantation. 46A-V1 cells, transplanted after CCI, did not survive beyond 2-3 weeks and had no effect on the allodynia and hyperalgesia induced by CCI. Acute intrathecal injection of the 5HT receptor antagonist methysergide decreased the antinociceptive effects of the 46A-B14 cells to pre-transplant levels. These data suggest that a chronically applied, low local dose of serotonin near the dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters such as serotonin, in a model of chronic pain offers a novel approach to pain management.

Lumbar transplant of neurons genetically modified to secrete brain-derived neurotrophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction.

Chronic delivery of anti-nociceptive molecules by means of cell grafts near the pain processing centers of the spinal cord is a newly developing technique for the treatment of neuropathic pain. The rat neuronal cell line, RN33B, derived from E13 rat brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat brain-derived neurotrophic factor cDNA (BDNF), and the BDNF-synthesizing cell line, 33BDNF.4, was isolated. The 33BDNF.4 cells synthesized mature BDNF protein at permissive temperature (33 degrees C), when the cells were proliferating, and during differentiation at non-permissive temperature (39 degrees C) in vitro. The bio-active BDNF protein was also secreted by the cells during both growth conditions, as measured by ELISA analysis of BDNF content and secretion. The bio-activity of the BDNF in 33BDNF.4 cell conditioned media was assessed by neurite outgrowth from E15 dorsal root ganglion (DRG) cultures. A control cell line, 33V1, transfected with the vector alone, did not synthesize or secrete any significant BDNF at either growth condition. Both cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hindpaw. When 33BDNF.4 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord and the 33BDNF.4 cells continued to synthesize BDNF in vivo. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7 week period after grafts of 33BDNF.4 cells. The maximal effect on chronic pain behaviors with the BDNF grafts occurred 2-3 weeks after transplant and the anti-nociceptive effects of the BDNF cell grafts was permanent. Transplants of the control 33V1 cells had no effect on the allodynia and hyperalgesia induced by CCI and these cells did not synthesize BDNF in vivo. These data suggest that a chronically applied, low local dose of BDNF supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver anti-nociceptive molecules, such as BDNF, in a model of chronic pain offers a novel approach to pain management and such 'biologic minipumps' can be developed for safe use in humans.

Comparison of the effects of remoxipride and raclopride on nigrostriatal and mesolimbic dopaminergic neuronal activity and on the secretion of prolactin and alpha-melanocyte-stimulating hormone.

Raclopride and remoxipride, which are reported to be selective dopamine-D2 antagonists, are currently under clinical investigation as antipsychotic drugs. The present study compared the relative abilities of these two drugs to alter the activity of nigrostriatal and mesolimbic dopaminergic neurons, and plasma levels of hormones originating from the anterior and intermediate lobes of the pituitary in male rats. Although raclopride was consistently more potent than remoxipride, both drugs produced dose- and time-related increases in concentrations of 3,4-dihydroxyphenylacetic acid in the striatum and nucleus accumbens, which contain terminals of nigrostriatal and mesolimbic dopaminergic neurons, respectively. Both drugs also caused significant dose- and time-related increases in plasma levels of prolactin, but only raclopride increased plasma levels of alpha-melanocyte-stimulating hormone (alpha-MSH). These results suggest that although raclopride and remoxipride are both classified as D2 receptor antagonists they can be distinguished from one another by their relative ability to block the inhibitory dopaminergic control of alpha-MSH from melanotrophs in the intermediate lobe of the rat pituitary.

Remoxipride and raclopride differentially alter the activity of incertohypothalamic dopaminergic neurons.

The effects of two dopaminergic (DA) antagonists, raclopride (S-(-)-3,5-dichloro-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy- 6-methoxybenzamide(+)-tartrate) and remoxipride (S(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2, 6-dimethoxybenzamide hydrochloride monohydrate), were compared on the DA receptor-mediated regulation of incertohypothalamic and nigrostriatal DA neurons. Both drugs produced dose- and time-related increases in concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, which contains the terminals of the nigrostriatal DA neurons. On the other hand, raclopride but not remoxipride increased concentrations of DOPAC in the medial zona incerta and dorsomedial hypothalamic nucleus, regions that contains cell bodies and terminals, respectively, of incertohypothalamic DA neurons. These results suggest that raclopride blocks a population of DA receptors that regulates the activity of incertohypothalamic DA neurons, whereas remoxipride does not.

Serotonergic neural precursor cell grafts attenuate bilateral hyperexcitability of dorsal horn neurons after spinal hemisection in rat.

Hemisection of the rat spinal cord at thoracic level 13 provides a model of spinal cord injury that is characterized by chronic pain attributable to hyperexcitability of dorsal horn neurons. Presuming that this hyperexcitability can be explained in part by interruption of descending inhibitory modulation by serotonin, we hypothesized that intrathecal transplantation of RN46A-B14 serotonergic precursor cells, which secrete serotonin and brain-derived neurotrophic factor, would reduce this hyperexcitability by normalizing the responses of low-threshold mechanoreceptive, nociceptive-specific, and multireceptive dorsal horn neurons. Three groups (n=45 total) of 30-day-old male Sprague-Dawley rats underwent thoracic level 13 spinal hemisection, after which four weeks were allowed for development of allodynia and hyperalgesia. The three groups of animals received transplants of no cells, 10(6) RN46A-V1 (vector-only) or 10(6) RN46A-B14 cells at lumbar segments 2-3. Electrophysiological experiments were done two weeks later. Low-threshold mechanoreceptive, nociceptive-specific, and multireceptive cells (n=394 total) were isolated at depths of 1-300 and 301-1000 micro in the lumbar enlargement. Responses to innocuous and noxious peripheral stimuli were characterized, and analyses of population responses were performed. Compared with normal animals, dorsal horn neurons of all types in hemisected animals showed increased responsiveness to peripheral stimuli. This was true for neurons on both sides of the spinal cord. After hemisection, the proportion of neurons classified as multireceptive cells increased, and interspike intervals of spontaneous discharges became less uniform after hemisection. Transplantation of RN46A-B14 cells restored evoked responses to near-control levels, normalized background activity, and returned the proportion of multireceptive cells to the control level. Restoration of normal activity was reversed with methysergide.These electrophysiological results corroborate anatomical and behavioral studies showing the effectiveness of serotonergic neural precursors in correcting phenomena associated with chronic central pain following spinal cord injury, and provide mechanistic insights regarding mode of action.

Immunocytochemical and in situ hybridization studies of the expression and distribution of three subunits of a complex with N-methyl-D-aspartate receptor-like properties.

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

Characterization of acid-sensitive ion channels in freshly isolated rat brain neurons.

Transient proton-activated currents induced by rapid shifts of the extracellular pH from 7.4 to < or =6.8 were recorded in different neurons freshly isolated from rat brain (hypoglossal motoneurons, cerebellar Purkinje cells, striatal giant cholinergic interneurons, hippocampal interneurons, CA1 pyramidal neurons and cortical pyramidal neurons) using whole-cell patch clamp technique. Responses of hippocampal CA1 pyramidal neurons were weak (100-300 pA) in contrast to other types of neurons (1-3 nA). Sensitivity of neurons to rapid acidification varied from pH(50) 6.4 in hypoglossal motoneurons to 4.9 in hippocampal interneurons. Proton-activated currents were blocked by amiloride (IC(50) varied from 3.6 to 9.5 microM). Reversal potential of the currents was close to E(Na), indicating that the currents are carried by sodium ions. The data obtained suggest that the proton-activated currents in the neurons studied are mediated by acid-sensitive ion channels. Strong acidification (pH<4) induced biphasic responses in all neuron types: the transient current was followed by a pronounced sustained one. Sustained current was not blocked by amiloride and exhibited low selectivity for sodium and cesium ions. Slow acidification from pH 7.4 to 6.5 did not induce detectable whole-cell currents. At pH 6.5, most of the channels are desensitized and responses to fast pH shifts from this initial level are decreased at least 10 times. This suggests that slow acidification which is well known to accompany some pathological states should rather desensitize than activate acid-sensitive ion channels and depress their function. Our results provide evidence for a widespread and neuron-specific distribution of acid-sensitive ion channels in the brain. The large amplitudes and transient character of currents mediated by these channels suggest that they could contribute to fast neuronal signaling processes.

Useful cell lines derived from the adrenal medulla.

Five approaches for the preparation of adrenal chromaffin cell lines have been developed. Initially, continuous chromaffin lines were derived from spontaneous pheochromocytoma tumors of the medulla, either from murine or human sources, such as the rat PC12 cell line and the human KNA and KAT45 cell lines. Over the last few decades, more sophisticated molecular methods have allowed for induced tumorigenesis and targeted oncogenesis in vivo, where isolation of specific populations of mouse cell lines of endocrine origin have resulted in model cells to examine a variety of regulatory pathways in the chromaffin phenotype. As well, conditional immortalization with retroviral infection of chromaffin precursors has provided homogeneous and expandable chromaffin cells for transplant studies in animal models of pain. This same strategy of immortalization with conditionally expressed oncogenes has been expanded recently to create the first disimmortalizable chromaffin cells, with an excisable oncogenic cassette, as might be envisioned for the creation of human chromaffin cell lines. Eventually, as we increase our understanding of regulating the phenotypic fate of chromaffin cells in vitro, stem or progenitor adrenal medullary cell lines will be derived as an alternative source for expansion and clinical use.

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors.

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter gamma-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1-8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.

Only early intervention with gamma-aminobutyric acid cell therapy is able to reverse neuropathic pain after partial nerve injury.

Pharmacological treatment for neuropathic pain, although often effective for brief periods, can result in intractable persistent pain with certain patients. Cell therapy for neuropathic pain is a newly developing technology useful for an examination of enhanced normal sensory function after nerve injury with the placement of cells near the spinal cord, and grafts of immortalized cells bioengineered to chronically supply the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) have been used to reverse the chronic pain behaviors. However, it is not known whether there is a therapeutic window for the use of intervention with cell therapy after partial nerve injury. To investigate whether neuropathic pain is sensitive to the timing of placement of cell grafts, neuronal cells bioengineered to synthesize GABA were transplanted in the lumbar subarachnoid space one to four weeks after unilateral chronic constriction injury (CCI) of the sciatic nerve and sensory behaviors were evaluated before and after CCI and transplants. Both thermal hyperalgesia and tactile allodynia were reversed when transplants were placed either one or two weeks after partial nerve injury, compared to maintenance of these behaviors with the injury alone. However, if GABA cells were placed any later than 2 weeks after nerve injury, such intervention was ineffective to reverse the thermal and tactile hypersensitivities induced by the injury. This suggests that altered spinal GABA levels may contribute to the early development of chronic neuropathic pain and that early intervention with cellular therapy to restore GABA may prevent the development of that pain.

Kir subfamily in frog retina: specific spatial distribution of Kir 6.1 in glial (Müller) cells.

We show by immunocytochemistry in frog retina that most members of the Kir subfamily are expressed in specific neuronal compartments. However, Kir 6.1, the pore-forming subunit of K(ATP) channels, is expressed exclusively in glial Müller cells. Müller cell endfeet display strong Kir 6.1 immunolabel throughout the retina, whereas the somata are labeled only in the retinal periphery. This spatial pattern is similar to that of Kir 4.1, of the ratio of inward to outward K+ currents, and of spermine/spermidine immunoreactivity. We suggest that the co-expression of Kir 4.1 and Kir 6.1 subunits may enable the cells to maintain their high K+ conductance and hyperpolarized membrane potentials both at high ATP levels (Kir 4.1) and during ATP deficiency (Kir 6.1).

Initial characterization of the transplant of immortalized chromaffin cells for the attenuation of chronic neuropathic pain.

Cultures of embryonic day 17 (E17) rat adrenal and neonatal bovine adrenal cells were conditionally immortalized with the temperature-sensitive allele of SV40 large T antigen (tsTag) and chromaffin cell lines established. Indicative of the adrenal chromaffin phenotype, these cells expressed immunoreactivity (ir) for tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines. At permissive temperature in vitro (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffin-like morphology, and contain detectable TH-ir. At nonpermissive temperature in vitro (39 degrees C), these cells stop proliferating and express increased TH-ir. When these immortalized chromaffin cells were transplanted in the lumbar subarachnoid space of the spinal cord I week after a unilateral chronic constriction injury (CCI) of the rat sciatic nerve, they survived longer than 7 weeks on the pia mater around the spinal cord and continued to express TH-ir. Conversely, grafted chromaffin cells lost Tag-ir after transplant and Tag-ir was undetectible in the grafts after 7 weeks in the subarachnoid space. At no time did the grafts form tumors after transplant into the host animals. These grafted chromaffin cells also expressed immunoreactivities for the other catecholamine-synthesizing enzymes 7 weeks after grafting, including: dopamine-beta-hydroxylase (DbetaH) and phenylethanolamine-N-methyltransferase (PNMT). The grafted cells also expressed detectable immunoreactivities for the opioid met-enkephalin (ENK), the peptide galanin (GAL), and the neurotransmitters y-aminobutyric acid (GABA) and serotonin (5-HT). Furthermore, after transplantation, tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI were significantly reduced during a 2-8-week period, related to the chromaffin cell transplants. The maximal antinociceptive effect occurred 1-3 weeks after grafting. Control adrenal fibroblasts, similarly immortalized and similarly transplanted after CCI, did not express any of the chromaffin antigenic markers, and fibroblast grafts had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that embryonic and neonatal chromaffin cells can be conditionally immortalized and will continue to express the phenotype of primary chromaffin cells in vitro and in vivo; grafted cells will ameliorate neuropathic pain after nerve injury and can be used as a homogeneous source to examine the mechanisms by which chromaffin transplants reverse chronic pain. The use of such chromaffin cell lines that are able to deliver antinociceptive molecules in models of chronic pain after nerve and spinal cord injury (SCI) offers a novel approach to pain management.

Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain.

The use of cell lines utilized as biologic "minipumps" to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme for GABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17 cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33 degrees C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39 degrees C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vector alone, contained no GAD67 or GABA at either temperature. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hind paw. When 33G10.17 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7-week period after grafts of 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 2-3 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers a novel approach to pain management.

Electrophysiology and pharmacology of neurons of the medial zona incerta: an in vitro slice study.

The electrical properties and chemical sensitivities of neurons located in the medial zona incerta (ZI), a region containing a clustering of dopaminergic cell bodies known as the A13 cell group, were studied in Sprague-Dawley female rats in the in vitro slice preparation. Extracellular single cell recordings were made from 473 neurons located in the medial ZI. The neurons generally displayed a slow and regular firing pattern with a mean firing rate of 4.7 impulses/s. Some neurons exhibited an orthodromic response to electrical stimulation of the ventromedial nucleus of the hypothalamus (VMH). The response consisted of a short-term, variable-latency increase in activity, indicating a predominantly excitatory input from the VMH to the medial ZI. The chemical sensitivity of medial ZI neurons was tested by micropressure application of various neurotransmitters and peptides. Dopamine and serotonin were found to inhibit the spontaneous firing in many of the neurons tested, whereas none of the peptides tested consistently produced a short-term change in firing rate. Over half (51%) of the cells that were orthodromically excited by VMH stimulation were also inhibited by the exogenous application of dopamine. Since many of the cells in the medial ZI are dopaminergic and are believed to be autoregulated, the present findings indicate that a portion of the VMH neurons that project to the medial ZI may be synapsing on dopaminergic neurons.

A single intrathecal injection of GABA permanently reverses neuropathic pain after nerve injury.

To investigate whether neuropathic pain is sensitive to spinal GABA levels, GABA was injected intrathecally after nerve injury and sensory behaviors were evaluated. Both thermal and tactile hypersensitivities were permanently reversed at the highest doses of GABA. However, if GABA was injected any later than 2-3 weeks after nerve injury, it was ineffective to prevent such hypersensitivity. This suggests that altered spinal GABA levels contribute to the induction phase of chronic neuropathic pain and that early intervention to restore GABA may prevent the development of that pain.

The sigma ligand rimcazole activates noradrenergic neurons projecting to the paraventricular nucleus and increases corticosterone secretion in rats.

Intraperitoneal injection of rimcazole, a sigma ligand, into male rats increased plasma concentrations of corticosterone in a dose- and time-related fashion. Concurrently, rimcazole increased concentrations of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the paraventricular nucleus (PVN) of the hypothalamus, suggesting that the drug activates noradrenergic neurons terminating in this nucleus. This latter suggestion was confirmed by the finding that rimcazole also increased concentrations of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) in the PVN. Pentazocine, a sigma ligand was without effect per se, but blocked the ability of rimcazole to increase concentrations of MHPG in the PVN and corticosterone in plasma. Taken together, these results suggest that rimcazole activates noradrenergic neurons projecting to the PVN via a mechanism involving sigma binding sites, and this action may be responsible for the ability of this drug to increase secretion of corticosterone.