TY - JOUR
AU - Kim, Dong-Wook
AB - Abstract We investigated the therapeutic potential of mouse ESC-derived gamma-amino butyric acid (GABA)ergic neurons (∼74% of total neurons in vitro) to reduce neuropathic pain following spinal cord injury (SCI) in rats. Spinal cord hemisection at the T13 segment, which is used as a rat SCI pain model, induced tactile hypersensitivity of the hind paw, as evidenced by decreased paw withdrawal thresholds in response to von Frey filaments, and also induced hyperexcitability of wide dynamic range neurons in the lumbar spinal cord in response to natural cutaneous stimuli. At 2 weeks posthemisection, GABAergic neurons (500,000 cells) were transplanted into the subarachnoid space of the spinal lumbar enlargement via a modified lumbar puncture technique. The transplantation of GABAergic neurons led to long-term attenuation of hemisection-induced tactile hypersensitivity and neuronal hyperexcitability as compared with vehicle-treated controls. These attenuations were reversed by the application of bicuculline and CGP52432, GABA-A and GABA-B receptor antagonists, respectively, but not by application of the serotonergic receptor antagonist methylsergide, indicating a specific restoration of spinal GABAergic inhibition. Histological data from sections of the lumbar cord in grafts demonstrated that 43.5% of surviving engrafted cells were neurons and located densely in the lower-medial portion of the dorsal funiculi in the spinal white matter. Among the observed neurons, 26.2% were GABAergic. The results suggest that subarachnoid transplantation of ESC-derived GABAergic neurons appear to restore spinal GABAergic inhibitory tone and can be a promising strategy to treat SCI-induced pain. ESC, GABAergic neuron, Spinal cord injury, Neuropathic pain, Transplantation Introduction Spinal cord injury (SCI) results in a devastating loss of motor function below the level of the lesion. In many cases, SCI is also accompanied by chronic central pain syndrome, such as allodynia (pain elicited by normally nonpainful stimulation) and hyperalgesia (exaggerated pain evoked by noxious stimulation) [1, 2]. As increased excitability of spinal dorsal horn neurons has been observed in animal studies following SCI, several underlying mechanisms of SCI-induced pain have been proposed. These include removal of inhibitory influences involving gamma-amino butyric acid (GABA), serotonin, and opioids [3–6], an increase in glutamatergic excitatory effects via activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors [7–9], and anatomical reorganization in the spinal cord with sprouting of central terminals of primary afferent neurons [10, 11]. In this study, we focused on the effect of GABAergic inhibition in SCI-induced pain. Studies using animal models of SCI provide evidence indicating that changes in the spinal GABAergic system occur and contribute to central pain following SCI. After spinal ischemic injury in the rat, the behavioral and neuronal hypersensitivities are associated with reduced GABA immunoreactivity in the spinal cord and can be attenuated by activation of GABA receptors [12, 13]. Similar observation has been reported in studies of rats with spinal hemisection injury [6]. Thus, augmentation of spinal GABAergic inhibitory tone by increasing spinal GABA levels could lead to attenuation of SCI-induced central pain. Previous studies provide evidence that a cell transplantation strategy that allows a long-term supply of GABA is one of the most useful means to treat pain following injury to the peripheral or central nervous system. Transplantation of immortalized fetal cells bioengineered to secrete GABA into the subarachnoid space shows an improvement of pain-like responses induced by injury to the peripheral nerve [14, 15]. A subarachnoid transplant of the carcinoma-derived neuronal cell line that secretes GABA attenuates pain-like responses following SCI [16]. A similar finding was reported by a study that performed spinal transplantation of GABAergic cells derived from human neuronal stem cells and demonstrated attenuation of pain-like responses following peripheral nerve injury and long-term survival of some GABAergic neurons around the transplanted site [17]. Although these previous studies provide evidence demonstrating the usefulness of spinal transplantation of GABAergic neurons in attenuation of injury-induced pathological pain, many of the mechanisms regarding the analgesic effects of GABAergic transplantation have not been explained. In our study, we attempted to develop GABAergic neurons from mouse ESCs for the treatment of SCI-induced pain. ESCs are proposed as a good source for cell therapy because of their properties of self-renewal and pluripotency and the ability to generate large quantities of transplantable cells [18, 19]. We induced differentiation of mouse ESCs into GABAergic neurons, and then transplanted these cells into the subarachnoid space of the spinal cord via a lumbar puncture (LP) in rats with SCI-induced tactile hypersensitivity to examine the effects of transplanted cells. After transplantation, we performed the behavioral test to investigate whether SCI-induced hypersensitivity is attenuated. We then performed not only functional analysis to ensure that the attenuation of hypersensitivity is due to the restoration of spinal GABAergic function but also histological analysis to evaluate the survival rate of engrafted GABAergic neurons. Materials and Methods Differentiation of GABAergic Neurons from Mouse ESCs and In Vitro Characterization PA6 cells (Riken, Tsukuba, Japan) and undifferentiated mouse ESCs (J1; ATCC, Manassas, VA, USA) were maintained as described previously [20]. Mouse ESCs were differentiated into a GABAergic phenotype based on the modification of methods described by Barberi et al. [21] and Kawasaki et al. [22] (see Supporting Information). Immunocytochemistry, semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and high-performance liquid chromatography (HPLC) analysis for GABA secretion were performed as described in Supporting Information Methods. Experimental Designs for Transplantation Studies Adult male Sprague-Dawley rats (170–200 g, Harlan Laboratories, Seoul, Korea) were used and weighed at the time of surgery. The rats were housed in groups of four, provided with food and water ad libitum under a 12-hour light/dark cycle, and allowed to acclimate for a week before surgery and behavioral testing. All animal experiments were carried out in accordance with NIH regulations for animal care and approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine. Rats initially received a spinal hemisection injury as described in Supporting Information Methods, followed 2 weeks later by lumbar subarachnoid injection with either GABAergic neurons (n = 45) or vehicle (Dulbecco's modified phosphate buffered saline (DPBS), n = 87). For a control group (n = 71), sham-operations were performed exactly as the hemisection and transplantation but without the actual cord hemisection or a cell/vehicle injection. Behavioral testing for mechanical sensitivity of the hind paws was evaluated by measuring paw withdrawal thresholds (PWTs) on the application of a von Frey filament as described in Supporting Information Methods once before and then weekly after the spinal hemisection until the 12-week test period was complete. We did not observe teratoma formation or anaplasia or any complication by infection in any of the transplanted animals. However, autophagic behavior (mild self-inflicted injury on hindlimb) was observed in some rats that received cell transplantation (5 of 45 rats) with a similar incidence in vehicle-injected control rats (10 of 87 rats). As we had not been able to examine the pain-like response on injured feet of rats with autophagic behavior, those rats were excluded from further analysis in the present study. Behavioral testing, neuronal activity recording, and cell counting were performed by investigators blinded to the animal treatments. Cell Transplantation into Spinal Cord Two weeks after spinal hemisection, rats were subjected to GABAergic transplantation or vehicle injection. Transplantation was performed using a modified LP technique described previously by Hylden and Wilcox [23]. Under enflurane anesthesia, a small longitudinal incision was made in the skin over the T13-L1 spinous processes. A 26-gauge needle attached to a 25-μl Hamilton syringe was held at an angle of about 30 degrees above the vertebral column, and positioned to one side of the T13 or L1 spinous process. The needle was slipped into the groove between the T13 and L1 vertebrae, and carefully advanced into the intervertebral space as the angle of the needle decreased to about 10 degrees. Proper placement of the needle in the lumbar subarachnoid space was confirmed at the time of entry by a sudden loss of resistance and a brief twitch of the muscles underneath the skin of the lateral region of the hind limb on the side of the needle entry. GABAergic neurons (500,000 cells/10 μl) were then administered over a 2-minute period. The syringe was left in place for 1-minute and slowly withdrawn to avoid any outflow of the cell suspension. The incised skin was sutured and the rat was returned to its cage. The control rats received vehicle only. All rats received cyclosporine (5 mg/kg, i.p.) every day beginning 2 days prior to transplantation and until they were sacrificed. Cell Counting and Analysis The total number of surviving engrafted cells was estimated by examining differences in the amount of 4′,6-diamidino-2-phenylindole (DAPI)-staining nuclei in the lumbar spinal segments (L3-L5) between cell-transplanted rats and vehicle-injected rats. The nuclear counting and nuclear diameter measurement were performed on digitally photographed images of the entire sectioned cord area of a given stained section using an image analysis program (MetaMorph v. Seven.17, Molecular Devices, Sunnyvale, CA, USA). As nuclear counts were made with the sections selected from every 10th section, the total number of cells in the lumbar spinal segments was obtained by multiplying the number of counted nuclei by 10. In addition, as some nuclei could be split by a sectioning knife and thus the same nucleus could be seen in two adjacent sections, the nuclear count could have been higher than the true cell number. As a result, corrections were made for split nuclei and calculations of the total true cell numbers based on the formulae described by Königsmark [24], N = n × t/(t + 2a) and a = sqrt [r2 − (k/2)2]; where N = estimated number of cells, n = counted number of cells, t = section thickness in μm, r = nuclear radius in μm, and k = 1. The proportion of surviving neuronal or GABAergic cells that were presumably derived from mouse ESCs was examined using coronal sections stained with anti-M6, which specific for a membrane glycoprotein of the central neurons of mice [25], as well as anti-GABA antibody, and DAPI. For this purpose, 25 sections were selected randomly from the stained sections that were obtained from the transplanted spinal cord area (five sections per each of five rats in each group). Images were captured with a confocal microscope (Olympus-Led, Japan) and subjected to cell counting. Cell counts were performed using a 100 × 100 μm2 counting frame with a height of 10 μm, which was placed over the area distributed by engrafted cells for cell transplanted rats and the corresponding area for vehicle-injected rats. As engrafted cells were detected evenly in the medial portions on both sides of the dorsal funiculi of the spinal white matter, cell counts were not performed separately for the ipsilateral and contralateral lesion sides. Statistical Analysis To determine the differences between each treatment group on a given testing day, data were analyzed using a Mann-Whitney rank sum test or a Kruskal Wallis analysis of variance (ANOVA) followed by Dunn's test for multiple comparisons. The differences from baseline within the given treatment group were analyzed using Friedman repeated measures ANOVA followed by Dunn's test for multiple comparisons. Bonferroni corrections were made. The Mann-Whitney rank-sum test for unmatched pairs was employed for the comparison of two different groups. The Wilcoxon signed rank test for matched pairs was used for the comparison with pretreatment baseline control values. p < .05 was considered to be statistically significant. Results Differentiation of Mouse ESCs We generated GABAergic neurons from mouse ESCs by coculturing them with PA6 feeder cells exhibiting stromal cell-derived inducing activity (SDIA) with modification of previous methods (see Materials and Methods) [20–22]. After 14 days of differentiation, most ESCs were differentiated into neuronal cells (Tuj1-positive cells/total cells; 81% ± 4%), and 74.1% ± 4.5% of them exhibited immunoreactivity for GABA (Fig. 1A–1C). A small fraction of 5-hydroxytryptamine (5-HT)-positive neurons (serotonergic neurons, 7% ± 2.3% of total neurons) and tyrosine hydroxylase (TH)-positive neurons (6% ± 3.7% of total neurons) were also observed (Fig. 1D, 1E). Semiquantitative RT-PCR analysis showed that sequential application of signaling molecules (e.g., Sonic hedgehog and fibroblast growth factor 8 (FGF8), d9-11) and neurotrophic factors (brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), d11-13) during differentiation enhanced the expression of genes involved in the synthesis and vesicular transport of GABA (Fig. 1F). The level of high-K+-induced GABA release in the medium examined by HPLC analysis was 4.46 ± 0.55 pmol/μl per a million cells (at differentiation day 14, mean ± SEM, n = 4). These results indicate that most of the ESCs were specifically differentiated into functional GABAergic neurons by SDIA and the treatment with signaling molecules. 1 Open in new tabDownload slide GABAergic differentiation from mouse ESCs. Mouse ESCs were cocultured with PA6 feeder cells for 14 days. During differentiation, basic-fibroblast growth factor (bFGF), Sonic Hedgehog (Shh), neurotrophin 4 (NT4), and brain-derived neurotrophic factor (BDNF) were sequentially added to the differentiation media to induce GABAergic neurons. (A–C): Most mouse ESCs were efficiently differentiated into Tuj1-positive neuronal cells (A), ∼74% of which were also immunolabeled with anti-GABA antibody (B, C). (D, E): Dopaminergic ([D], ∼6%) and serotonergic ([E], ∼7%) neurons were also detected in differentiated cells. (F): Reverse-transcriptase polymerase chain reaction analysis shows the enhanced expression of markers involved in GABA synthesis (GAD65 and GAD67) and vesicular transport (VIAAT) after GABAergic differentiation as compared with differentiation by SDIA-only. Scale bar = 25 μm. Abbreviations: GABA, gamma-amino butyric acid; GAD, glutamate decarboxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SDIA, stromal cell-derived inducing activity; SM, signaling molecules; VIAAT, vesicular inhibitory amino acid transporter. 1 Open in new tabDownload slide GABAergic differentiation from mouse ESCs. Mouse ESCs were cocultured with PA6 feeder cells for 14 days. During differentiation, basic-fibroblast growth factor (bFGF), Sonic Hedgehog (Shh), neurotrophin 4 (NT4), and brain-derived neurotrophic factor (BDNF) were sequentially added to the differentiation media to induce GABAergic neurons. (A–C): Most mouse ESCs were efficiently differentiated into Tuj1-positive neuronal cells (A), ∼74% of which were also immunolabeled with anti-GABA antibody (B, C). (D, E): Dopaminergic ([D], ∼6%) and serotonergic ([E], ∼7%) neurons were also detected in differentiated cells. (F): Reverse-transcriptase polymerase chain reaction analysis shows the enhanced expression of markers involved in GABA synthesis (GAD65 and GAD67) and vesicular transport (VIAAT) after GABAergic differentiation as compared with differentiation by SDIA-only. Scale bar = 25 μm. Abbreviations: GABA, gamma-amino butyric acid; GAD, glutamate decarboxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SDIA, stromal cell-derived inducing activity; SM, signaling molecules; VIAAT, vesicular inhibitory amino acid transporter. Hemisection-Induced Outcomes Are Restored by Transplantation of GABAergic Neurons We investigated whether an intrathecal administration of GABAergic neurons derived from mouse ESCs into the subarachnoid space at the lumbar spinal segment could reverse the hypersensitivity in the hind limb that was induced after spinal hemisection at the T13 segment. We explored the time course of changes in PWTs of the ipsilateral hind paw in response to von Frey filament stimuli for rats that received spinal hemisection followed by GABAergic transplantation (n = 45) or vehicle injection (n = 87), and those that received sham-operations (n = 71). As no significant differences in changes of PWTs after hemisection or cell-administration were observed between both hind paws (data not shown), data from the ipsilateral hind paw were used. After 2 weeks of hemisection, PWTs of the affected hind paw significantly decreased as compared with the prehemisection state and PWTs of the sham-operated controls (p < .05; Fig. 2A). At this time point, a subarachnoid injection with either GABAergic neurons or vehicle was performed. The cell transplantation resulted in a reversal of decreased PWTs, which was significantly beyond post-transplantation week 5 as compared with PWTs of vehicle-injected rats (p < .05). This reversal reached its maximum on post-transplantation week 6 and remained at its maximum level until the 10th week. The rats that received vehicle did not show a significant change in PWT over the entire test period (Fig. 2A). 2 Open in new tabDownload slide Changes in tactile sensitivity (A) and the evoked activity of WDR neurons (B) after spinal cord hemisection and GABAergic neuron transplantation. (A): Unilateral hemisection led to tactile hypersensitivity of the affected hind paw by showing decreased paw withdrawal thresholds (≈2). This hypersensitivity was attenuated following subarachnoid administration of GABAergic neurons. (B): The evoked activities of spinal wide dynamic range (WDR) dorsal horn neurons to three natural cutaneous stimuli (brush, pressure, pinch) increased in hemisected rats that received DPBS vehicle against the sham-operated controls. The evoked activities of WDR neurons in hemisected rats that received GABAergic neurons were reduced to levels similar to response levels in the sham-operated controls. Bar = mean ± SEM. Numbers in parentheses indicate the number of rats used for the examination. *, p < .05 versus hemisected rats that received DPBS vehicle in (A) and versus both hemisected rats that received GABAergic neurons and sham-operated rats in (B). Abbreviations: DPBS, Dulbecco's modified phosphate buffered saline; Hemi, hemisection; stim, stimulation. 2 Open in new tabDownload slide Changes in tactile sensitivity (A) and the evoked activity of WDR neurons (B) after spinal cord hemisection and GABAergic neuron transplantation. (A): Unilateral hemisection led to tactile hypersensitivity of the affected hind paw by showing decreased paw withdrawal thresholds (≈2). This hypersensitivity was attenuated following subarachnoid administration of GABAergic neurons. (B): The evoked activities of spinal wide dynamic range (WDR) dorsal horn neurons to three natural cutaneous stimuli (brush, pressure, pinch) increased in hemisected rats that received DPBS vehicle against the sham-operated controls. The evoked activities of WDR neurons in hemisected rats that received GABAergic neurons were reduced to levels similar to response levels in the sham-operated controls. Bar = mean ± SEM. Numbers in parentheses indicate the number of rats used for the examination. *, p < .05 versus hemisected rats that received DPBS vehicle in (A) and versus both hemisected rats that received GABAergic neurons and sham-operated rats in (B). Abbreviations: DPBS, Dulbecco's modified phosphate buffered saline; Hemi, hemisection; stim, stimulation. To provide the neural basis for mechanical hypersensitivity, the evoked activity to natural cutaneous stimuli was recorded from spinal WDR dorsal horn neurons. As shown in Figure 2B, the response of WDR dorsal horn neurons to three different stimuli (brush, pressure, pinch) significantly increased in hemisected rats that received vehicle (n = 32) compared to the sham-operated controls (n = 36; p < .05). Unlike these increased responses, the evoked responses to all three stimuli of WDR neurons in hemisected rats with engrafted GABAergic neurons (n = 38) were significantly reduced to levels close to those observed in the sham-operated control (p < .05). These results provide evidence that spinal transplantation of GABAergic neurons could reduce the exaggerated signals evoked from WDR neurons by peripheral tactile stimulation in rats with spinal hemisection injury. Attenuation of Hemisection-Induced Outcomes by Cell Transplantation Is Due To a Restoration of Spinal GABAergic Inhibition To verify that the attenuation of tactile hypersensitivity and hyperexcitability of spinal WDR neurons following cell transplantation results from a restoration of impaired spinal GABAergic inhibition, pharmacological activation and inhibition of spinal GABA receptors were investigated. In sham-operated rats, an intrathecal administration of GABA-A receptor antagonist, bicuculline, induced a significant decrease in PWTs at doses of 3 and 15 nmol in a dose-dependent manner as compared with the PBS-treated control (Fig. 3A, a; p < .05), and the decrease began at 30 minutes postadministration and lasted over 120 minutes. In hemisected rats that showed the tactile hypersensitivity of the hind paw, intrathecal administration of the GABA-A receptor agonist, muscimol, significantly increased PWTs at doses of 2.5 and 5 nmol compared with the control (Fig. 3A, b; p < .05), and the increase lasted over 150 minutes. Similar results were obtained with intrathecal administration of GABA-B receptor antagonist (CGP52432, Fig. 3A, d) or agonist (baclofen, Fig. 3A, e). Interestingly, for hemisected rats that received engrafted GABAergic neurons, in which PWT values returned close to those for sham-operated rats, intrathecal application of bicuculline (15 nmol) or CGP52432 (15 nmol) significantly decreased PWTs (Fig. 3A, c and f; p < .05) with a stronger effect by bicuculline than CGP52432. 3 Open in new tabDownload slide Effects of inhibition or activation of spinal GABA receptors on tactile sensitivity (A) and the evoked activity of WDR neurons (B). (Aa): In sham-operated rats, intrathecal administration of GABA-A receptor antagonist bicuculline induced tactile hypersensitivity of the hind paw by decreasing PWTs in a dose-dependent manner. (Ab): An intrathecal administration of GABA-A receptor agonist muscimol attenuated tactile hypersensitivity in hemisected rats that received DPBS vehicle by increasing PWTs in a dose-dependent manner. (Ac): In hemisected rats that received GABAergic neurons, in which PWT values returned close to those of sham-operated rats, intrathecally administered bicuculline (15 nmol) induced tactile hypersensitivity. (Ad–Af): Similar results were obtained with intrathecal administration of GABA-B receptor antagonist or agonist, CGP52432 or baclofen, respectively. (Ba): The evoked activities of spinal WDR neurons to brush, pressure, and pinch in sham-operated rats increased following topical application of bicuculline (15 nmol). (Bb): In hemisected rats that received DPBS vehicle and showed enhanced responses of WDR neurons, topically applied muscimol (5 nmol) suppressed WDR neuronal responses. (Bc): In hemisected rats that received GABAergic neurons, a topical application of bicuculline (15 nmol) increased the evoked activities of WDR neurons, similar to the case of sham-operated rats. (Bd–Bf): Similar results were obtained with topically applied GABA-B receptor antagonist or agonist. Bar = mean ± SEM. Numbers in parentheses indicate the number of rats used for the examination. *, p < .05 versus PBS-treated group in (A) versus predrug baseline in (B). Abbreviations: DPBS, Dulbecco's modified phosphate buffered saline; PBS, phosphate buffered saline; WDR, wide dynamic range. 3 Open in new tabDownload slide Effects of inhibition or activation of spinal GABA receptors on tactile sensitivity (A) and the evoked activity of WDR neurons (B). (Aa): In sham-operated rats, intrathecal administration of GABA-A receptor antagonist bicuculline induced tactile hypersensitivity of the hind paw by decreasing PWTs in a dose-dependent manner. (Ab): An intrathecal administration of GABA-A receptor agonist muscimol attenuated tactile hypersensitivity in hemisected rats that received DPBS vehicle by increasing PWTs in a dose-dependent manner. (Ac): In hemisected rats that received GABAergic neurons, in which PWT values returned close to those of sham-operated rats, intrathecally administered bicuculline (15 nmol) induced tactile hypersensitivity. (Ad–Af): Similar results were obtained with intrathecal administration of GABA-B receptor antagonist or agonist, CGP52432 or baclofen, respectively. (Ba): The evoked activities of spinal WDR neurons to brush, pressure, and pinch in sham-operated rats increased following topical application of bicuculline (15 nmol). (Bb): In hemisected rats that received DPBS vehicle and showed enhanced responses of WDR neurons, topically applied muscimol (5 nmol) suppressed WDR neuronal responses. (Bc): In hemisected rats that received GABAergic neurons, a topical application of bicuculline (15 nmol) increased the evoked activities of WDR neurons, similar to the case of sham-operated rats. (Bd–Bf): Similar results were obtained with topically applied GABA-B receptor antagonist or agonist. Bar = mean ± SEM. Numbers in parentheses indicate the number of rats used for the examination. *, p < .05 versus PBS-treated group in (A) versus predrug baseline in (B). Abbreviations: DPBS, Dulbecco's modified phosphate buffered saline; PBS, phosphate buffered saline; WDR, wide dynamic range. In recording experiments assessing the evoked activity of spinal WDR neurons to cutaneous mechanical stimuli, drug effects were observed as similarly as those obtained from PWTs responses. A topical application of bicuculline (15 nmol) or CGP52432 (15 nmol) onto the lumbar spinal cord significantly increased the evoked responses of WDR neurons in sham-operated rats when compared with preapplication baseline (Fig. 3B, a and d; p < .05). Also, with a topical application of muscimol (5 nmol) or baclofen (25 nmol), a considerable decrease of WDR neuronal responses was found in hemisected rats that showed increased evoked responses of WDR neurons (Fig. 3B, b and e; p < .05). For hemisected rats that received engrafted GABAergic neurons, as expected, bicuculline or CGP52432 application significantly increased the evoked responses of WDR neurons (Fig. 3B, c and f; p < .05) as similar to the case of sham-operated rats. Collectively, our behavioral and electrophysiological data suggest that the reduction of spinal hemisetion-induced pain-like responses through subarachnoid transplant of GABAergic neurons is attributed to functional recovery of inhibitory controls onto spinal nociceptive circuitry via a restoration of the spinal GABAergic system. The Decrease of Pain-Like Responses by Cell Transplantation Is Unlikely Due To the Restoration of the Spinal Serotonergic System To see if the descending serotonin (5-HT) inhibitory system is restored in the spinal cord after cell transplantation, as our cell culture contain a small fraction of serotonergic neurons (Fig. 1E), we investigated inhibition and activation of spinal 5-HT receptors. In sham-operated rats, an intrathecal administration of 5-HT receptor antagonist methylsergide induced a significant decrease in PWTs at doses of 50 and 100 nmol in a dose-dependent manner compared with PBS-treated control rats (Fig. 4A, a; p < .05). In hemisected rats, intrathecal administration of 5-HT significantly increased PWTs dose-dependently (120-240 nmol) compared with the controls (Fig. 4A, b; p < .05). However, interestingly, no significant changes in PWTs were induced by intrathecal administration of methylsergide (100 nmol) in hemisected rats given cell transplantation as compared with the controls (Fig. 4A, c). Our data show that the spinal serotonergic system is involved in the modulation of spinal nociceptive transmission in both naïve rats and rats with spinal hemisection injury; however, it is unlikely that the decrease of tactile hypersensitivity that is observed in hemisected rats given cell transplantation is due to the restoration of the spinal serotonergic system. 4 Open in new tabDownload slide Effects of inhibition or activation of spinal 5-HT receptors on tactile sensitivity (A) and the evoked activity of WDR neurons (B). (Aa): In sham-operated rats, an intrathecal administration of 5-HT receptor antagonist methysergide induced tactile hypersensitivity of the hind paw by decreasing PWTs in a dose-dependent manner. (Ab): In hemisected rats, intrathecal administration of 5-HT attenuated tactile hypersensitivity by increasing PWTs in a dose-dependent manner. (Ac): In hemisected rats that received GABAergic neurons, in which PWT values returned close to those of sham-operated rats, intrathecally administered methylsergide (100 nmol) did not induce tactile hypersensitivity. (Ba): The evoked activities of spinal WDR neurons in response to brush, pressure, and pinch in sham-operated rats increased following topical application of methylsergide (100 nmol). (Bb): In hemisected rats that showed enhanced responses of WDR neurons, topically applied 5-HT (240 nmol) suppressed WDR neuronal responses. (Bc): In hemisected rats given cell transplantation, a topical application of methylsergide (100 nmol) produced no significant changes in the evoked activities of WDR neurons. Bar = mean ± SEM. Numbers in parentheses indicate the number of rats used for the examination. *, p < .05 versus PBS-treated group in (A) and versus predrug baseline in (B). Abbreviations: DPBS, Dulbecco's modified phosphate buffered saline; Hemi, hemisection; HT, hydroxytryptamine; PBS, phosphate buffered saline. 4 Open in new tabDownload slide Effects of inhibition or activation of spinal 5-HT receptors on tactile sensitivity (A) and the evoked activity of WDR neurons (B). (Aa): In sham-operated rats, an intrathecal administration of 5-HT receptor antagonist methysergide induced tactile hypersensitivity of the hind paw by decreasing PWTs in a dose-dependent manner. (Ab): In hemisected rats, intrathecal administration of 5-HT attenuated tactile hypersensitivity by increasing PWTs in a dose-dependent manner. (Ac): In hemisected rats that received GABAergic neurons, in which PWT values returned close to those of sham-operated rats, intrathecally administered methylsergide (100 nmol) did not induce tactile hypersensitivity. (Ba): The evoked activities of spinal WDR neurons in response to brush, pressure, and pinch in sham-operated rats increased following topical application of methylsergide (100 nmol). (Bb): In hemisected rats that showed enhanced responses of WDR neurons, topically applied 5-HT (240 nmol) suppressed WDR neuronal responses. (Bc): In hemisected rats given cell transplantation, a topical application of methylsergide (100 nmol) produced no significant changes in the evoked activities of WDR neurons. Bar = mean ± SEM. Numbers in parentheses indicate the number of rats used for the examination. *, p < .05 versus PBS-treated group in (A) and versus predrug baseline in (B). Abbreviations: DPBS, Dulbecco's modified phosphate buffered saline; Hemi, hemisection; HT, hydroxytryptamine; PBS, phosphate buffered saline. In the evoked WDR neuronal responses, a topical application of methylsergide (100 nmol) onto the lumbar spinal cord dramatically increased the evoked responses of WDR neurons in sham-operated rats as compared with the predrug baseline (Fig. 4B, a; p < .05). With a topical application of 5-HT (240 nmol) in hemisected rats in which evoked WDR neuronal responses were enhanced, a significant decrease of evoked neuronal responses was observed compared with the predrug baseline (Fig. 4B, b; p < .05). However, in consistent with PWT data, no significant changes of evoked WDR neuronal responses were observed in hemisected rats with receiving cell transplantation when methylsergide (100 nmol) was applied (Fig. 4B, c). Survival of GABAergic Neurons in the Spinal Cord To estimate the survival of engrafted GABAergic cells in the spinal cord after cell transplantation, cell counting was performed on coronal sections of the lumbar spinal cord after staining with anti-M6 (a neuronal marker for grafted mouse cells), anti-GABA antibodies, and DAPI. Sections were obtained from hemisected rats that received either engrafted GABAergic neurons (n = 5) or vehicle (n = 5). M6-positive cells were mostly observed in lumbar spinal segments (L3–L5). Immunohistochemical analysis indicated that M6-positive cells were detected over a length of 2.34 ± 0.62 mm (mean ± SEM) in the rostro-caudal direction, a depth of 0.90 ± 0.12 mm from the dorsal surface, and a width of 0.49 ± 0.14 mm in the form of a slender-oval shape, with the greatest number of cells around the site of cell injection. A representative stained section is shown in Figure 5. M6-positive mouse neuronal cells were detected densely in the lower-medial portion of the dorsal funiculi in the spinal white matter of the rats (rectangle in Fig. 5A). GABA-immunoreactive cells were distributed evenly throughout the M6-positive cells (Fig. 5B). Mouse ESC-derived GABAergic neurons are clearly visible at high magnification (Fig. 5C). M6/TH double-positive cells were occasionally observed (Fig. 5D, < 0.1%) and 5-HT-positive cells were detected but represented less than 1% of the total M6-positive cells (Fig. 5E), which were also evenly distributed throughout the engrafted cells. In addition, there were some Nestin-positive cells among the engrafted cells (Fig. 5F), indicating that some transplanted cells did not fully differentiate into mature neuronal cells. 5 Open in new tabDownload slide Fate of donor cells in the rat spinal cord after subarachnoid cell transplantation. (A): Distribution of transplanted cells in coronal rat spinal cord sections stained with DAPI and anti-M6 and anti-GABA antibodies near the site of cell injection. Most donor cells are densely located in the lower medial portion of dorsal funiculi of the spinal white matter. (B): High-power images of areas surrounded by boxes shown in (A). The cells that are triple-labeled with DAPI and anti-M6 and anti-GABA antibodies are GABAergic cells derived from engrafted mouse ESCs. (C): Confocally scanned-layer image of ESC-derived GABAergic neurons (GABA in cytoplasm, green; M6 in cell membrane, red) confirming that GABAergic neurons were donor derived. (D–F): Other types of cells from donor cells were also observed in the spinal cord that received cell transplantation; (D) TH-positive neuron (green), (E) serotonergic neurons (green), (F) Nestin-positive neural precursors (green), which still did not fully differentiate into the mature neuronal cells. Some of these were proliferative (Ki67 [red]-/Nestin [green]-double positive). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GABA, gamma-amino butyric acid; HT, hydroxytryptamine. 5 Open in new tabDownload slide Fate of donor cells in the rat spinal cord after subarachnoid cell transplantation. (A): Distribution of transplanted cells in coronal rat spinal cord sections stained with DAPI and anti-M6 and anti-GABA antibodies near the site of cell injection. Most donor cells are densely located in the lower medial portion of dorsal funiculi of the spinal white matter. (B): High-power images of areas surrounded by boxes shown in (A). The cells that are triple-labeled with DAPI and anti-M6 and anti-GABA antibodies are GABAergic cells derived from engrafted mouse ESCs. (C): Confocally scanned-layer image of ESC-derived GABAergic neurons (GABA in cytoplasm, green; M6 in cell membrane, red) confirming that GABAergic neurons were donor derived. (D–F): Other types of cells from donor cells were also observed in the spinal cord that received cell transplantation; (D) TH-positive neuron (green), (E) serotonergic neurons (green), (F) Nestin-positive neural precursors (green), which still did not fully differentiate into the mature neuronal cells. Some of these were proliferative (Ki67 [red]-/Nestin [green]-double positive). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GABA, gamma-amino butyric acid; HT, hydroxytryptamine. The cell counting data demonstrated the mean difference in the numbers of DAPI-positive cells present within counting frames between the cell-transplanted group and the control group as 567 ± 46.2 per section (Table 1). Meanwhile, in the counting frames on 25 stained sections from hemisected rats given GABAergic grafts, the numbers of M6-immunoreactive and M6 plus GABA-immunoreactive cells were 246.7 ± 49.8 and 64.9 ± 4.8, respectively. Therefore, the proportion of neuronal cells among surviving engrafted cells 10 weeks after transplantation are estimated as ∼43.5%, and ∼26.2% of the neuronal cells are GABAergic. Table 1 Incidence of cells stained with anti-M6 and anti-GABA antibodies among engrafted cells in the lumbar spinal segment of the rat Open in new tab Table 1 Incidence of cells stained with anti-M6 and anti-GABA antibodies among engrafted cells in the lumbar spinal segment of the rat Open in new tab To estimate the total amount of surviving engrafted cells within the lumbar spinal cord, the nucleus counts were made with DAPI-stained sections from hemisected rats transplanted with GABAergic neurons and control rats. The total cell numbers in the lumbar enlargement, which were estimated from nuclear counts and correction factors as described in Materials and Methods, were 1,324,130 ± 416,176 for grafted rats and 1,061,934 ± 328,851 for control rats. The difference between these values can be considered as the number of surviving engrafted cells within the lumbar spinal cord 10 weeks after mouse cell transplantation, which was 262,196 ± 115,547. Based on this estimation, we speculated that there are ∼114,055 ESC-derived neurons (43.5% of surviving cells) and ∼29,882 GABAergic neurons (26.2% of ESC-derived neurons). These GABAerigic neurons may play a crucial role in the restoration of the impaired spinal inhibitory GABAergic system. Discussion There is some evidence indicating that the spinal GABAergic system plays a critical role in the development and maintenance of chronic pain states following an injury of the peripheral and central nervous system. The GABA-immunoreactivity in the spinal dorsal horn decreases once peripheral nerve and spinal ischemic injuries occur [12, 26]. The spinal administration of GABA-A receptor antagonist, bicuculline, induces hyperexcitability of spinal dorsal horn neurons in naïve rats [27]. Following central and peripheral nerve injury, the mechanical allodynia is attenuated by intrathecal administration of GABA receptor agonists [6, 13, 28]. Based on these observed data, one can suggest that the hypofunction of spinal GABAergic inhibition is involved in pathological pain states that develop due to peripheral nerve and/or spinal cord injuries. These previous findings have inspired several studies to investigate cell-based therapies for chronic pain syndromes [14, 16, 17, 29]. Besides one study that used uncommitted neural precursors [29], most studies have used neuronal cells exhibiting GABAergic phenotypes that were derived from various cell sources to be transplanted into the chronic pain model. Although these studies present promising data such as the behavioral recovery of pain symptoms and histological evidence to support cell engraftment, they do not clearly show whether the behavioral recovery after cell transplantation is due to the restoration of spinal inhibitory tone or the provision of neurotrophic/protective factors into the microenvironment of the host tissue. In our study, using an animal model of SCI, we focused on determining whether transplantation of GABAergic neurons derived from ESCs effectively reduces pain-like responses induced by SCI and to verify the possibility of cell replacement therapy using ESCs for the treatment of SCI-induced pain-like responses. The generation of GABAergic neurons from pluripotent stem cells (e.g., ESCs or induced pluripotent stem cells [iPS cells]) will provide a potentially unlimited supply of grafts to use in practical cell-based therapies. Therefore, developing techniques for the generation and transplantation of GABAergic neurons using ESCs or iPS cells will play an important role in widening the possibility of therapeutic applications. Here, we demonstrate that subarachnoid transplantation of GABAergic cells at the lumbar spinal segment results in long-term attenuation of hemisection-induced tactile hypersensitivity and neuronal hyperexcitability. More importantly, we provide novel evidence, via pharmacological and physiological assessments that such attenuation is mainly due to the restoration of spinal GABAergic inhibitory tone. According to our data, both the excitability of dorsal horn neurons and tactile sensitivity of hind paws are clearly enhanced when the cord is hemisected, whereas such enhancement is reduced by GABAergic transplantation (Fig. 2), just like by the intrathecal administration of both GABA-A and GABA-B receptor agonists (Fig. 3A, b and e; Fig. 3B, b and e), even for a long period (∼10 weeks). Previous reports demonstrated that GABA-A receptors are present on dorsal horn neurons and both GABA-A and GABA-B receptors are expressed on primary afferent terminals [30, 31]. Thus, diminished GABA release by spinal hemisection injury reduces both presynaptic (influencing synaptic input) and postsynaptic (modulating dorsal horn neuron excitability) GABA-A as well as GABA-B receptor-mediated inhibition, resulting in hemisection-induced tactile hypersensitivity. Meanwhile, activation of both GABA-A and GABA-B receptors reduces hemisection-induced tactile hypersensitivity probably via decreased responsiveness of dorsal horn neurons (Fig. 3A, b and e; Fig. 3B, b and e), which may result from decreased transmitter release and neuronal hyperpolarization. In addition, our data demonstrate that the long-term attenuation of tactile hypersensitivity and neuronal hyperexcitibility through cell transplantation is reversed by antagonism of spinal GABA-A as well as GABA-B receptors with a stronger effect by GABA-A receptor antagonist than GABA-B receptor antagonist (Fig. 3A, c and f; Fig. 3B, c and f). These data suggest that GABA-A receptors on both afferent terminals and dorsal horn neurons may be more involved than GABA-B receptors on afferent terminals in the restoration of spinal GABAergic system after cell transplantation. Additionally, we have examined whether transplanted cells have exerted antinociceptive effects via the restoration of spinal serotonergic system, because we have found that some serotonergic neurons (∼7%) exist in neuronal populations that are differentiated from ESCs (Fig. 1E) and it is also well established that the descending serotonergic inhibitory pathways participate in the inhibition of pain transmission at the spinal level [32–35]. In our data, unlike the GABA receptor antagonist, methylsergide, the serotonin receptor antagonist did not show a significant effect on tactile sensitivity (Fig. 4A, c) or neuronal excitability (Fig. 4B, c) in rats showing behavioral recovery after cell transplantation. In addition, serotonergic neurons constituted the minority of the engrafted cells within the spinal cords of hemisected rats that received cell transplantation (<1% of M6-posivite cells). These results strongly suggest that the behavioral recovery obtained through cell transplantation in our experiment is mainly due to a restoration of the spinal GABAergic system rather than recovery of the spinal serotonergic system. Previous studies have argued that intrathecal injection for cell transplantation might not be adequate for consistent and sustained engraftment [17], probably because grafted cells were deposited onto the subarachonoid space and survival rates of transplanted cells were very low (∼1%) [16]. Therefore, a suggestion has been made to encourage direct transplantation into the spinal cord parenchyma, allowing the cells to settle down and function properly and consistently. However, injecting cells directly into the parenchyma will also require laminectomy and secondary injury by an injection needle can occur. To reduce such distress during the transplantation procedure, we took an advantage of modified LP method (see Materials and Methods). LP was originally well known to extract the cerebrospinal fluid or applying drugs locally to the spinal cord [36]. Recently, LP was also reported as a less invasive method for cell transplantation into the spinal cord [37–39]. Our histological data demonstrate that cells delivered by the LP method were crowded in the lower medial portion of the dorsal funiculi of the spinal white matter, even though lots of cells were still flocked together over the spinal cord surface (Fig. 5A). It was surprising that cells transplanted intrathecally by the LP method migrated into the spinal parenchyma through the dorsal midline fissure and integrated in the white matter of the dorsal column in a similar manner to those cells that were transplanted through a direct injection method. We assume that ESC-derived GABAergic cells migrate and integrate into the host parenchyma through the small pit generated on the spinal cord surface during the cell injection procedure. However, we unfortunately did not observe any M6-positive cells in the dorsal gray matter, and have no evidence that donor cells directly interact with host neurons through synaptic connection. Although we did not show a direct interaction at the single-cell level, we here provide electrophysiological data demonstrating that the enhancement of response activities of spinal WDR neurons by spinal hemisection was considerably reduced to levels of those in the sham-operated control following cell transplantation and that such restoration was hampered by the GABA receptor antagonist treatment. From these results, we suggest that GABAergic neurons in the medial portion of the dorsal funiculi, which are probably derived from transplanted cells, may secrete GABA tonically into the dorsal horn region to produce a tonic inhibition of pain signal transmission that is strengthened after spinal hemisection. Another interesting finding in our study was that antinociceptive effects from cell transplantation started in the third week and reached a peak point (about 80% of the fully recovered level) at 6 weeks after transplantation (Fig. 2A), whereas the recovery in previous studies has been observed within a week after transplantation [16, 17, 29]. Such a latent period for functional integration is because it takes some time for cells engrafted via LP to migrate and integrate into the host spinal cord. Moreover, this implicates that the indirect effects of cell transplantation such as neurotrophic/protective effects, have a minor influence on the recovery of pain-like response in our experiments. Conclusion In conclusion, our data suggest not only that the loss of the spinal GABAergic system following SCI is a major cause of pain but also that the restoration of the GABAergic system through transplantation of cells differentiated from ESCs can be a promising strategy to treat the pain induced by SCI. The development of cell therapy to relieve SCI-induced pain using ESCs will increase the possibility of therapeutic application of pluripotent stem cells such as ESCs and iPS cells. Acknowledgements We thank Mr. H.S. Park and Mr. S. Cho for technical assistance and Dr. J. B. Seo and S. Y. Kim at Korea Basic Science Institute for helping us with HPLC analysis. This work was supported by grants from the Stem Cell Research Center of the 21st Century Frontier Research Program (SC1110 and SC4140) funded by the Ministry of Education, Science and Technology, Republic of Korea. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: D.-S.K.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; S.J.J.: collection and/or assembly of data, data analysis and interpretation; T.S.N.: conception and design, data analysis and interpretation; Y.H.J.: collection and/or assembly of data; D.R.L.: collection and/or assembly of data; J.S.L.: collection and/or assembly of data; J.W.L.: conception and design, financial support, data analysis and interpretation, manuscript writing and final approval of manuscript; D.-W.K.: conception and design, financial support, data analysis and interpretation, manuscript writing and final approval of manuscript. First published online in STEM CELLS EXPRESS September 16, 2010. Disclosure of potential conflicts of interest is found at the end of this article. Telephone: 82-2-2228-1709; Fax: 82-2-393-0203 Telephone: 82-2-2228-1703; Fax: 82-2-393-0203 Copyright © 2010 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Transplantation of GABAergic Neurons from ESCs Attenuates Tactile Hypersensitivity Following Spinal Cord Injury
JF - Stem Cells
DO - 10.1002/stem.526
DA - 2010-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/transplantation-of-gabaergic-neurons-from-escs-attenuates-tactile-leaOWc7FLK
SP - 2099
EP - 2108
VL - 28
IS - 11
DP - DeepDyve
ER -