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.

Transport Reversal during Heteroexchange: A Kinetic Study.

It is known that secondary transporters, which utilize transmembrane ionic gradients to drive their substrates up a concentration gradient, can reverse the uptake and instead release their substrates. Unfortunately, the Michaelis-Menten kinetic scheme, which is popular in transporter studies, does not include transporter reversal, and it completely neglects the possibility of equilibrium between the substrate concentrations on both sides of the membrane. We have developed a complex two-substrate kinetic model that includes transport reversal. This model allows us to construct analytical formulas allowing the calculation of a "heteroexchange" and "transacceleration" using standard Michaelis coefficients for respective substrates. This approach can help to understand how glial and other cells accumulate substrates without synthesis and are able to release such substrates and gliotransmitters.

Proximal tibial strain in medial unicompartmental knee replacements: A biomechanical study of implant design.

As many as 25% to 40% of unicompartmental knee replacement (UKR) revisions are performed for pain, a possible cause of which is proximal tibial strain. The aim of this study was to examine the effect of UKR implant design and material on cortical and cancellous proximal tibial strain in a synthetic bone model. Composite Sawbone tibiae were implanted with cemented UKR components of different designs, either all-polyethylene or metal-backed. The tibiae were subsequently loaded in 500 N increments to 2500 N, unloading between increments. Cortical surface strain was measured using a digital image correlation technique. Cancellous damage was measured using acoustic emission, an engineering technique that detects sonic waves ('hits') produced when damage occurs in material. Anteromedial cortical surface strain showed significant differences between implants at 1500 N and 2500 N in the proximal 10 mm only (p < 0.001), with relative strain shielding in metal-backed implants. Acoustic emission showed significant differences in cancellous bone damage between implants at all loads (p = 0.001). All-polyethylene implants displayed 16.6 times the total number of cumulative acoustic emission hits as controls. All-polyethylene implants also displayed more hits than controls at all loads (p < 0.001), more than metal-backed implants at loads ≥ 1500 N (p < 0.001), and greater acoustic emission activity on unloading than controls (p = 0.01), reflecting a lack of implant stiffness. All-polyethylene implants were associated with a significant increase in damage at the microscopic level compared with metal-backed implants, even at low loads. All-polyethylene implants should be used with caution in patients who are likely to impose large loads across their knee joint.

L-DOPA Uptake in Astrocytic Endfeet Enwrapping Blood Vessels in Rat Brain.

Astrocyte endfeet surround brain blood vessels and can play a role in the delivery of therapeutic drugs for Parkinson's disease. However, there is no previous evidence of the presence of LAT transporter for L-DOPA in brain astrocytes except in culture. Using systemic L-DOPA administration and a combination of patch clamp, histochemistry and confocal microscopy we found that L-DOPA is accumulated mainly in astrocyte cell bodies, astrocytic endfeet surrounding blood vessels, and pericytes. In brain slices: (1) astrocytes were exposed to ASP(+), a fluorescent monoamine analog of MPP(+); (2) ASP(+) taken up by astrocytes was colocalized with L-DOPA fluorescence in (3) glial somata and in the endfeet attached to blood vessels; (4) these astrocytes have an electrogenic transporter current elicited by ASP(+), but intriguingly not by L-DOPA, suggesting a different pathway for monoamines and L-DOPA via astrocytic membrane. (5) The pattern of monoamine oxidase (MAO type B) allocation in pericytes and astrocytic endfeet was similar to that of L-DOPA accumulation. We conclude that astrocytes control L-DOPA uptake and metabolism and, therefore, may play a key role in regulating brain dopamine level during dopamine-associated diseases. These data also suggest that different transporter mechanisms may exist for monoamines and L-DOPA.

GABAergic pathway in a rat model of chronic neuropathic pain: modulation after intrathecal transplantation of a human neuronal cell line.

Current understanding of chronic pain points a decrease in level of the inhibitory neurotransmitter GABA, in the spinal dorsal horn, leading to an imbalance between excitatory and inhibitory pathways. A subcloned derivative of the human NT2 cell line (hNT2.17) which, after neuronal differentiation, secretes different inhibitory neurotransmitters such as GABA and glycine has been recently isolated. In this study, we have investigated the effect of this new cell line on peripheral nerve injury induced by chronic constriction (CCI) and notably the effect on the cellular GABAergic pathway. Our data show that the decrease in GABA expression in the spinal dorsal horn of injured animals is concomitant with a decline of its synthetic enzyme GAD67-Ir and mRNA but not GAD65. Interestingly, in transplanted animals we observed a strong induction of GAD67 mRNA with one week after graft, which is followed by a recovery of GAD67 and GABA Ir. This effect paralleled a reduction of hindpaw hypersensitivity and thermal hyperalgesia induced by CCI. These results suggest that hNT2.17 GABA cells can modulate neuropathic pain after CCI certainly by minimizing the imbalance and restoring the cellular GABAergic pathway.

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

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.

Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells.

Tandem-pore domain (2P-domain) K+-channels regulate neuronal excitability, but their function in glia, particularly, in retinal glial cells, is unclear. We have previously demonstrated the immunocytochemical localization of the 2P-domain K+ channels TASK-1 and TASK-2 in retinal Müller glial cells of amphibians. The purpose of the present study was to determine whether these channels were functional, by employing whole-cell recording from frog and mammalian (guinea pig, rat and mouse) Müller cells and confocal microscopy to monitor swelling in rat Müller cells. TASK-like immunolabel was localized in these cells. The currents mediated by 2P-domain channels were studied in isolation after blocking Kir, K(A), K(D), and BK channels. The remaining cell conductance was mostly outward and was depressed by acid pH, bupivacaine, methanandamide, quinine, and clofilium, and activated by alkaline pH in a manner consistent with that described for TASK channels. Arachidonic acid (an activator of TREK channels) had no effect on this conductance. Blockade of the conductance with bupivacaine depolarized the Müller cell membrane potential by about 50%. In slices of the rat retina, adenosine inhibited osmotic glial cell swelling via activation of A1 receptors and subsequent opening of 2P-domain K+ channels. The swelling was strongly increased by clofilium and quinine (inhibitors of 2P-domain K+ channels). These data suggest that 2P-domain K+ channels are involved in homeostasis of glial cell volume, in activity-dependent spatial K+ buffering and may play a role in maintenance of a hyperpolarized membrane potential especially in conditions where Kir channels are blocked or downregulated.

Quantitative measurement of serotonin synthesis and sequestration in individual live neuronal cells.

Synthesis and subsequent sequestration into vesicles are essential steps that precede neurotransmitter exocytosis, but neither the total neurotransmitter content nor the fraction sequestered into vesicles have been measured in individual live neurons. We use multiphoton microscopy to directly observe intracellular and intravesicular serotonin in the serotonergic neuronal cell line RN46A. We focus on how the relationship between synthesis and sequestration changes as synthesis is up-regulated by differentiation or down-regulated by chemical inhibition. Temperature-induced differentiation causes an increase of about 60% in the total serotonin content of individual cells, which goes up to about 10 fmol. However, the number of vesicles per cell increases by a factor of four and the proportion of serotonin sequestered inside the vesicles increases by a factor of five. When serotonin synthesis is inhibited in differentiated cells and the serotonin content goes down to the level present in undifferentiated cells, the sequestered proportion still remains at this high level. The total neurotransmitter content of a cell is, thus, an unreliable indicator of the sequestered amount.

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.

Grafts of immortalized chromaffin cells bio-engineered to improve met-enkephalin release also reduce formalin-evoked c-fos expression in rat spinal cord.

Transplantation of adrenal medullary tissue for terminal cancer pain has been tested clinically, but this approach is not practical for routine use because of the shortage of organ donors and lack of tissue homogeneity. As a first alternative step, we have generated immortalized chromaffin cells over-expressing opioid peptides, namely met-enkephalin. Rat chromaffin cells have been genetically modified with vectors containing expression cassettes with either synthetic met-enkephalin or pro-enkephalin gene coding regions, fused with the nerve growth factor signal peptide for secretion. After stable transfection and differentiation in vitro, met-enkephalin and pro-enkephalin cells had higher met-enkephalin immunoreactivity and secreted met-enkephalin levels, compared to control cells containing the expression vector only. In the formalin hindpaw-injection model, 15 days after subarachnoid transplant of cells, grafts of met-enkephalin and pro-enkephalin cells significantly reduced the number of formalin-evoked c-fos immunoreactive spinal neurons in the spinal cord, compared to grafts of vector-alone chromaffin cells. The use of such expandable cell lines, for chronic spinal delivery of opiates, could offer an attractive and safe alternative strategy based on ex vivo gene therapy for the control of opioid-sensitive chronic pain.

[Functional interaction between nicotinic cholinergic receptors and Na, K-ATPase in the skeletal muscles]. / Funktsional'naia sviaz' nikotinovykh kholinoretseptorov i Na, K-ATFazy v skeletnoi myshtse.

Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.

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.

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord.

Changing the levels of neurotrophins in the spinal cord micro-environment after nervous system injury has been proposed to recover normal function, such that behavioral response to peripheral stimuli does not lead to chronic pain. We have investigated the effects of recombinant adeno-associated viral (rAAV)-mediated over-expression of brain-derived neurotrophic factor (BDNF) in the spinal cord on chronic neuropathic pain after unilateral chronic constriction injury (CCI) of the sciatic nerve. The rAAV-BDNF vector was injected into the dorsal horn at the thirteenth thoracic spinal cord vertebra (L(1) level) 1 week after CCI. Allodynia and hyperalgesia induced by CCI in the hindpaws were permanently reversed, beginning 1 week after vector injection, compared with a similar injection of a control rAAV-GFP vector (green fluorescent protein) or saline. In situ hybridization for BDNF demonstrated that both dorsal and ventral lumbar spinal neurons contained an intense signal for BDNF mRNA, at 1 to 8 weeks after vector injection. There was no similar BDNF mRNA over-expression associated with either injections of saline or rAAV-GFP. These data suggest that chronic neuropathic pain is sensitive to early spinal BDNF levels after partial nerve injury and that rAAV-mediated gene transfer could potentially be used to reverse chronic pain after nervous system injuries in humans.

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.

Kir6.1 is the principal pore-forming subunit of astrocyte but not neuronal plasma membrane K-ATP channels.

ATP-sensitive potassium channels (K-ATP channels) directly couple the energy state of a cell to its excitability, are activated by hypoxia, and have been suggested to protect neurons during disturbances of energy metabolism such as transient ischemic attacks or stroke. Molecular studies have demonstrated that functional K-ATP channels are octameric protein complexes, consisting of four sulfonylurea receptor proteins and four pore-forming subunits which are members of the Kir6 family of inwardly rectifying potassium channels. Here we show, using specific antibodies against the two known pore-forming subunits (Kir6.1 and Kir6.2) of K-ATP channels, that only Kir6.1 and not Kir6.2 subunits are expressed in astrocytes. In addition to a minority of neurons, Kir6.1 protein is present on hippocampal, cortical, and cerebellar astrocytes, tanycytes, and Bergmann glial cells. We also provide ultrastructural evidence that Kir6.1 immunoreactivity is primarily localized to distal perisynaptic and peridendritic astrocyte plasma membrane processes, and we confirm the presence of functional K-ATP channels in Bergmann glial cells by slice-patch-clamp experiments. The identification of Kir6.1 as the principal pore-forming subunit of plasma membrane K-ATP channels in astrocytes suggests that these glial K-ATP channels act in synergy with neuronal Kir6.2-mediated K-ATP channels during metabolic challenges in the brain.

Engraftment of serotonergic precursors enhances locomotor function and attenuates chronic central pain behavior following spinal hemisection injury in the rat.

Spinal cord injury (SCI) results in abnormal locomotor and pain syndromes in humans. T13 spinal hemisection in the rat results in development of permanent mechanical allodynia and thermal hyperalgesia partially due to interruption of descending inhibitory modulators such as serotonin (5-HT). We hypothesize that lumbar transplantation of nonmitotic cells that tonically secrete antinociceptive and trophic compounds will reduce the pain-like behavior and enhance locomotor recovery after SCI. We used RN46A-B14 cells, a conditionally immortalized (SV40tsTag) rat neuronal cell line derived from E13 raphe bioengineered to secrete both 5-HT and BDNF in vitro at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Three groups (n = 72) of 30-day-old male Sprague-Dawley rats were spinally hemisected at T13 and allowed 4 weeks for adequate recovery of locomotor function and development of allodynia and hyperalgesia. Immunosuppressed animals received either lumbar RN46A-B14 (n = 24) or control RN46A-V1 (n = 24) empty-vector transplants or no cell (n = 24) transplant. HPLC analysis of media and CSF demonstrated increases of both in vitro and in vivo 5-HT levels at 28 days in RN46A-B14 animals. ELISA demonstrated BDNF secretion in vitro and in vivo by RNA46A-B14 cells. Locomotor function (BBB scale) and nociceptive behaviors measured by paw withdrawals to von Frey filaments, radiant heat, and noxious pin stimuli were tested for 4 weeks posttransplant. Animals receiving RN46A-B14 cells demonstrated significantly improved locomotor function and reductions in both fore- and hindlimb mechanical allodynia and thermal hyperalgesia compared to controls receiving RN46A-V1 or no transplants. These effects were modulated by the 5-HT antagonist methysergide and reuptake inhibitor fluvoxamine. Bromodeoxyuridine and 5-HT immunoreactivity confirmed cell survival and graft location 4 weeks posttransplantation. These results support the therapeutic potential of bioengineered serotonin-secreting cell lines in reducing chronic central pain following spinal cord injury.

Subdural engraftment of serotonergic neurons following spinal hemisection restores spinal serotonin, downregulates serotonin transporter, and increases BDNF tissue content in rat.

Spinal hemisection injury at T13 results in development of permanent mechanical allodynia and thermal hyperalgesia due to interruption and subsequent loss of descending inhibitory modulators such as serotonin (5-HT) and its transporter (5-HT(T)). We hypothesize that lumbar transplantation of non-mitotic cells that tonically secrete 5-HT and brain-derived neurotrophic factor (BDNF) will restore alterations in 5-HT and 5-HT(T) systems within the spinal dorsal horn. We used an immortalized rat neuronal cell line derived from E13 raphe (RN46A-B14) which is shown to secrete 5-HT and BDNF in vitro and in vivo. Three groups (n=35) of 30 day old male Sprague-Dawley rats were spinally hemisected at T13 and 28 days later received either lumbar RN46A-V1 control empty-vector (n=15) or RN46A-B14 (n=15) intrathecal grafts, or no transplant. Twenty-eight days following transplantation, animals were perfused and tissue examined for changes in 5-HT, 5-HT(T), and BDNF at the site of transplantation or at lumbar enlargements (L5). Immunohistochemistry revealed that RN46A-B14, but not RN46A-V1 cells, increased 5-HT tissue staining at L5 in the dorsal white matter as well as in superficial dorsal horn laminae I and II on both ipsilateral and contralateral sides, results confirmed by ELISA. Transplantation of RN46A-B14 cells significantly reduced ipsilateral 5-HT(T), upregulated after injury. Significantly increased levels of BDNF were also observed after RN46A-B14 transplantation but were not localized to particular spinal laminae. These results are consistent with recovery of locomotor function and reductions in chronic pain behaviors observed behaviorally after RN46A-B14 transplantation and supports the pragmatic application of cell-based therapies in correcting damaged circuitry after spinal cord injury.

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).