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TISSUE-SPECIFIC STEM CELLS

Spinal GABAergic Transplants Attenuate Mechanical Allodynia in a Rat Model of Neuropathic Pain

^aCell Restoration Laboratory, Departments of Anatomy and Neurobiology and Surgery (Neurosurgery), Dalhousie University, Halifax, Nova Scotia, Canada;
^bDepartment of Anaesthesia, Queen's University, Kingston, Ontario, Canada;
^cPharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada

Key Words. Neural precursor cell • Transplantation • Pain • Inhibition

Correspondence: Ivar Mendez, M.D., Ph.D., Division of Neurosurgery, Queen Elizabeth II Health Sciences Centre, Room 3806, 1796 Summer Street, Halifax, Nova Scotia B3H 4H7, Canada. Telephone: 902-473-7046; Fax: 902-473-3343; e-mail: mendez{at}dal.ca

Received on May 1, 2007; accepted for publication on July 31, 2007.

First published online in STEM CELLS EXPRESS  August 16, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Injury to the spinal cord or peripheral nerves can lead to the development of allodynia due to the loss of inhibitory tone involved in spinal sensory function. The potential of intraspinal transplants of GABAergic cells to restore inhibitory tone and thus decrease pain behaviors in a rat model of neuropathic pain was investigated. Allodynia of the left hind paw was induced in rats by unilateral L5– 6 spinal nerve root ligation. Mechanical sensitivity was assessed using von Frey filaments. Postinjury, transgenic fetal green fluorescent protein mouse GABAergic cells or human neural precursor cells (HNPCs) expanded in suspension bioreactors and differentiated into a GABAergic phenotype were transplanted into the spinal cord. Control rats received undifferentiated HNPCs or cell suspension medium only. Animals that received either fetal mouse GABAergic cell or differentiated GABAergic HNPC intraspinal transplants demonstrated a significant increase in paw withdrawal thresholds at 1 week post-transplantation that was sustained for 6 weeks. Transplanted fetal mouse GABAergic cells demonstrated immunoreactivity for glutamic acid decarboxylase and GABA that colocalized with green fluorescent protein. Intraspinally transplanted differentiated GABAergic HNPCs demonstrated immunoreactivity for GABA and β-III tubulin. In contrast, intraspinal transplantation of undifferentiated HNPCs, which predominantly differentiated into astrocytes, or cell suspension medium did not affect any behavioral recovery. Intraspinally transplanted GABAergic cells can reduce allodynia in a rat model of neuropathic pain. In addition, HNPCs expanded in a standardized fashion in suspension bioreactors and differentiated into a GABAergic phenotype may be an alternative to fetal cells for cell-based therapies to treat chronic pain syndromes.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Injury to the central or peripheral nervous system can lead to the development of allodynia, an abnormal sensory state in which innocuous stimuli gain the ability to evoke intense pain. It has been estimated that 64% with spinal cord injuries alone suffer with intractable chronic pain symptoms [1–4]. Clinically, allodynia is triggered by peripheral substrates, which normally are not involved in pain transmission, from low-threshold mechanoreceptive primary afferent fibers that mediate light tactile sensory function [5]. Chronically, allodynia affects patients' physical and emotional well-being such that their quality of life is compromised and ability to work is impaired [1–4].

Current treatment options for allodynia are suboptimal [6, 7]. A variety of pharmacological agents have been used, including opioids, nonsteroidal anti-inflammatory drugs, anticonvulsants, and tricyclic antidepressants, but clinical trials have demonstrated that their efficacy is limited and their use can be associated with deleterious side effects, such as the development of tolerance and addiction [6, 8–15]. Although alternative drug delivery systems, such as indwelling intrathecal catheters, address some of those issues, their use is accompanied by other disadvantages, such as mechanical malfunction, infection, tissue compression and fibrosis by the catheter tubing, and the need for routine surgical maintenance [16]. Neurosurgical therapies such as spinal cord and cortical stimulation hold promise, but up to 40% of patients do not experience sufficient pain relief [17–21]. Although, consequently, psychological and occupational therapies remain the mainstay of symptomatic treatments for neuropathic pain [22], patients suffering from allodynia often have a poor quality of life and are disabled by their pain. The ideal therapy for neuropathic pain disorders such as allodynia should address the underlying pathophysiological etiology of the pain syndrome rather than the general symptoms of the pain or its temporal nature [6, 7, 22].

One of the hypotheses to explain the development of allodynia after injury involves the pathological loss of inhibition in the spinal cord. Under normal conditions, the synaptic transmission of the low-threshold afferents is modulated by GABA and glycine released from interneurons in the dorsal horn of the spinal cord [23]. GABA is present in high concentrations within the superficial layers of the spinal cord (laminae I to III) where nociceptive sensory processing is predominant [24–30]. Following spinal cord or peripheral nerve injury, there is an apparent loss of GABAergic inhibitory interneurons in the spinal cord [7, 31–34]. Thus, it is conceivable that a loss of inhibitory modulation could enhance the synaptic efficacy between A- fibers and pain-signaling pathways, leading to sustained excitatory synaptic transmission in the dorsal horn in a manner that permits misunderstanding of low-threshold input as pain [34, 35].

Previous studies support the hypothesis that loss of spinal cord GABA-mediated inhibitory tone is responsible for allodynia. For example, pain behaviors in allodynic rats are attenuated by spinal cord stimulation, which enhances spinal GABA levels [17–19, 21]. Hyperalgesia and allodynia consequent to peripheral nerve or spinal cord injuries are ameliorated by intrathecal administration of GABA [36], GABA_A, or GABA_B receptor agonists [37–40] and exacerbated by GABA_A or GABA_B receptor antagonists [37, 38, 40–43]. Transgenic mice that lack the β₃ subunit of the GABA_A receptor [44] or GABA_B1 or GABA_B2 receptors [45, 46] exhibit spontaneous hyperalgesia. Thus, it may be possible to augment GABA inhibitory tone by transplantation of GABAergic cells. Previous studies have shown that subarachnoid implantation of adrenal medullary [47–54] or serotonergic cells [55–57] or cells bioengineered to secrete brain-derived neurotrophic factor (BDNF) [58, 59] or GABA [60, 61] can reduce abnormalities in sensory function that are observed following nerve injury, but it is not clear whether these cells integrate with the host in a manner that enables them to respond to changes in the neuronal microenvironment.

The present study investigated whether transplantation of GABAergic cells directly into the spinal cord to increase inhibitory tone can produce a sustained decrease in pain behaviors in the nerve root ligation model of allodynia in the rat. Potential clinical application of this strategy requires the availability of a reliable source of clinical-grade GABAergic cells. Human neural precursor cells (HNPCs) are attractive candidates for such strategies because of their properties of self-renewal and multipotentiality [62–65] and the ability to efficiently generate large quantities of HNPCs in a standardized, reproducible manner using suspension bioreactors [66–72]. Therefore, it was also investigated whether bioreactor-expanded HNPCs, differentiated to a GABAergic phenotype prior to transplantation, could be used as an alternative to fetal GABAergic cells as a standardized source of clinical grade cells for spinal cord transplants.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Animals and Study Design
Twenty-six female Wistar rats (Charles River Laboratories, Saint Constant, Quebec, Canada, http://www.criver.com) weighing 175–200 g were used and housed in pairs in a temperature/humidity-controlled room on a 12-hour light/dark cycle with access to food and water ad libitum when behavioral tests were not being performed. The experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care.

Allodynia of the left hind paw was induced by unilateral L5 and L6 nerve root ligations (described below). Postligation, rats were randomly assigned to treatment groups and received intraspinal transplants of fetal striatal primordia cells derived from transgenic green fluorescent protein (GFP) mice (n = 7), HNPCs differentiated into a GABAergic phenotype (n = 7), undifferentiated HNPCs (n = 7), or cell suspension medium only (n = 5). Mechanical sensitivity was assessed pre- and postlesion and 1, 2, 4, and 6 weeks post-transplantation using von Frey filaments.

Cell Culture
Striatal primordia tissue was harvested from embryonic day 15 transgenic mice that constitutively expressed GFP. Both the medial and lateral eminences of the striatal primordia were dissected in Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT, http://www.hyclone.com) and hibernated at 4°C for one night in 10 ml of a low-sodium, phosphate-buffered, calcium-free hibernation medium [73]. A cell suspension was prepared as previously described [73] with a final concentration of 200,000 cells per microliter in 0.05% DNase/DMEM, with viability exceeding 98% as determined by the trypan blue dye exclusion method.

Cells from the fetal GFP mouse striatal primordia prepared for transplantation were plated on 13-mm poly-L-lysine-coated glass coverslips in 24-well plates at a density of 100,000 cells per milliliter in a medium containing 2% B27 (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 1% N2 (Gibco, Grand Island, NY, http://www.invitrogen.com), 1% penicillin-streptomycin (HyClone), and 1% fetal bovine serum (HyClone) in DMEM. The cells were fed every 3 days by replacing 50% of the medium with fresh medium. After 7 days, the cells were fixed with 4% paraformaldehyde and processed for immunocytochemistry.

Telencephalon-derived HNPCs were harvested from a fetal brain (10 weeks gestational age) using protocols developed in our center [74, 75]. The tissue was rinsed four times in 0.05% DNase/Pharmaceutical Production Research Facility (PPRF)-h2 medium. PPRF-h2 medium was generated by supplementing PPRF-m4 medium (which includes DMEM/Ham's F-12 medium, 1:1 [Invitrogen, Carlsbad, CA, http://www.invitrogen.com], 5 mM HEPES [Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com], 0.6% glucose [Sigma-Aldrich], 1.73 g/L sodium bicarbonate [Sigma-Aldrich], 2.0 mM glutamine [Invitrogen], 0.023 g/L insulin [Sigma-Aldrich], 20 nM progesterone [Sigma-Aldrich], 9.0 mg/L putrescine [Sigma-Aldrich], 0.025 g/L transferrin [Sigma-Aldrich], 30 nM sodium selenite [Sigma-Aldrich], and 20 µg/L epidermal growth factor [Peprotech, Rocky Hill, NJ, http://www.peprotech.com]) with 10 µg/L leukemia inhibitory factor (Chemicon, Temecula, CA, http://www.chemicon.com), 1.0 µmol/l dehydroepiandrosterone (Steraloids, Newport, RI, http://www.steraloids.com), and 20 µg/L basic fibroblast growth factor (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). The telencephalon tissue was then incubated in 0.25% trypsin-EDTA at 37°C for 20 minutes, rinsed four times in 0.05% DNase/PPRF-h2, and mechanically dissociated using a 1-ml and a 200-µl Eppendorf pipetter until a uniform cell suspension was achieved. HNPCs were subsequently cultured in a 25-cm³ culture plate (passage level 0) in PPRF-h2 expansion medium. A 50% medium exchange was performed every 4 days. After 14 days, the aggregates were harvested and centrifuged at 1,500 rotations per minute for 5 minutes in expansion medium. The supernatant was discarded and the pellet was incubated in 0.25% trypsin (HyClone) at 37°C for 20 minutes. The cells were rinsed twice with 0.05% DNase/PPRF-h2, triturated into a single-cell suspension using a 1-ml Eppendorf pipetter, and then cultured again in expansion medium (passage level 1).

At the end of passage level 1, the aggregates were transferred to one T-75 flask containing 12 ml of PPRF-h2 medium (passage level 2). After 5 days in culture, the aggregates were harvested and enzymatically dissociated using 0.25% trypsin-ethylenediaminetetraacetic acid, and the resulting single-cell suspension was inoculated into six T-25 flasks (stationary cultures), each containing 5 ml of PPRF-h2 medium (passage level 3). After 14 days, the cells were harvested and enzymatically dissociated. Cell counts revealed a cell fold increase of more than 13.7 and an average viability of 91% ± 1%. The resulting single-cell suspension was inoculated into duplicate 125-ml bioreactors each containing 100 ml of PPRF-h2 medium (passage level 4). After 14 days in suspension culture, the aggregates were harvested and cryopreserved in PPRF-h2 with 10% dimethyl sulfoxide. Unless otherwise noted, all cultures were inoculated at 10⁵ cells per milliliter using a single-cell suspension, incubated at 37°C and 5% CO₂, and had 40% of their spent medium replaced with fresh medium every 5 days. Cell counts were performed using trypan blue dye exclusion.

For transplantation, the cryopreserved undifferentiated bioreactor-expanded cells were thawed and then passaged two additional times in stationary tissue culture flasks. The resulting neurospheres were rinsed four times in 0.05% DNase/DMEM and then incubated for 20 minutes in 0.25% trypsin at 37°C. The neurospheres were rinsed in 0.05% DNase/DMEM another four times prior to being mechanically triturated into a single-cell suspension using a 1-ml Eppendorf pipetter. The cells were centrifuged at 1,000 rotations per minute for 5 minutes, and the pellet was suspended in 0.05% DNase/DMEM to make a cell suspension for transplantation with a concentration of 200,000 cells per microliter and viability exceeding 99%.

Bioreactor-expanded HNPCs were differentiated into a GABAergic phenotype based on the methods described by Laeng and colleagues [76]. Neurospheres were dissociated into a single-cell suspension as described and then cultured in the presence of BDNF and valproic acid. After 1 week of differentiation, the cells were collected and then centrifuged at 1,000 rotations per minute for 5 minutes. A single-cell suspension was made, and the trypan blue dye exclusion method was used to ascertain the viability of the HNPCs in suspension. A final concentration of 200,000 cells per microliter was prepared, with viability exceeding 98%.

To determine their default differentiation pattern in vitro, HNPCs were plated on 13-mm poly-L-lysine-coated glass coverslips in 24-well plates at a density of 100,000 cells per milliliter in a differentiation medium consisting of 2% B27, 1% N2, and 1% penicillin-streptomycin in DMEM. The cultures were fed every 3 days by replacing 50% of the medium with fresh medium. After 7 days, the cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) in preparation for immunocytochemistry.

Surgical Procedures
Animals underwent a left hemilaminotomy at L5/6 and ligation of the left L5 and L6 nerve roots distal to the dorsal root ganglion but proximal to the joining of these nerves with L4 using 3-0 silk thread [77] under a 0.2 ml per 100 g of body weight dose of the ketamine-xylazine-acepromazine anesthetic mixture (60 mg/kg ketamine hydrochloride [MTC Pharmaceuticals, Cambridge, ON, Canada], 1.2 mg/kg xylazine [Bayer Canada, Inc, Etobicoke, ON, Canada, http://www.bayer.ca], 1.6 mg/kg acepromazine maleate [Wyeth Pharmaceuticals, Markham, ON, Canada, http://www.wyeth.com] in 0.9% saline]. Ten days postligation, animals received transplants of either 200,000 fetal striatal primordia GFP cells, HNPCs differentiated into a GABAergic phenotype, undifferentiated HNPCs, or cell suspension medium only into the spinal cord. The cells were implanted in the midline at the L1 level, 1.0 mm below the surface of the spinal cord, using a glass capillary with an outer opening diameter of 50 µm attached to a Hamilton microsyringe (Hamilton Co., Reno, NV, http://www.hamiltoncompany.com). The 1.0-µl deposit of cells or medium only was performed over 5 minutes, and the microcapillary was left in place for an additional 5 minutes. All animals received cyclosporine (10 mg/kg, intraperitoneal; Novartis International, Basel, Switzerland, http://www.novartis.com) daily beginning 2 days prior to transplantation and until they were sacrificed. No morbidity or mortality was associated with either the nerve root ligations or the transplantation procedures in any animals.

Behavioral Assessment
Mechanical sensitivity in the rats' hind paws was assessed using von Frey filaments (Stoelting Company, Wood Dale, IL, http://www.stoeltingco.com) pre- and postlesion and 1, 2, 4, and 6 weeks post-transplantation, as described by Pitcher et al. [78]. The same investigator performed the test for all animals at all time points and was observed by another investigator to ensure consistency. Both investigators were blinded to the treatment. The testing platform consisted of a transparent plastic box with 1.5-mm diameter holes in a 5-mm grid of perpendicular rows in its base through which von Frey filaments were applied to the central region of the plantar surface of the hind paws. Application of a series of hairs ranging from 0.008 to 300 g was performed to determine the left and right hind paw withdrawal thresholds. The animals were habituated to the testing box for at least 15 minutes, and testing began once exploratory behavior ceased and all four paws were in contact with the platform. In ascending order, von Frey filaments were applied. Only complete removal of the hind paw from the platform accompanied by at least one other behavior to indicate mechanical sensitivity, such as vocalization, head turning to the stimulus, or grooming of the hind paw, was counted as a withdrawal response [57, 79, 80], to exclude the segmental response of hyperreflexia [57, 81]. The filaments were applied five times at 5-second intervals. The paw withdrawal threshold was determined as the force in grams of the filament that caused hind paw withdrawal for four of five consecutive applications. Once the paw withdrawal threshold was determined for the left hind paw, the procedure was repeated for the right hind paw. The entire test was repeated the following day for both hind paws.

Hind limb motor function was assessed in an open field (5 x 5 feet) using a locomotor rating scale [82]. Rats were observed by two trained observers for 5 minutes and scored prelesion, postlesion, and 1, 2, 4, and 6 weeks post-transplantation.

Immunohisto- and Immunocytochemistry
Seven weeks after transplantation, the rats received an anesthetic overdose and were transcardially perfused with 300 ml of ice-cold 0.1 M PB followed by 300 ml of ice-cold 4% paraformaldehyde in 0.1 M PB. The spinal cords were extracted, postfixed in 4% paraformaldehyde in 0.1 M PB for 24 hours, and then cryoprotected in 30% sucrose in 0.1 M PB at 4°C for 24 hours. Spinal cords were embedded in 10% gelatin (Sigma-Aldrich) in 0.1 M PB and immersed in 4% paraformaldehyde in 0.1 M PB for 24 hours and then in 30% sucrose in 0.1 M PB for 48 hours. Longitudinal sections (40 µm) of the spinal cord were cut serially on a freezing microtome.

Immunohisto- and immunocytochemistry were performed on the spinal cord sections and cultured cells, respectively. Spinal cord sections or cultured cells were rinsed three times for 5 minutes each in 0.1 M PB, treated for 1 hour in 5% normal goat serum and 0.3% Triton X-100 in 0.1 M PB, and then incubated for 16 hours at room temperature in a solution of 5% normal goat serum/0.3% Triton X-100 in 0.1 M PB that also contained rabbit anti-GABA (1:1,000; Sigma-Aldrich), rabbit anti-glutamic acid decarboxylase 65/67 (GAD 65/67; 1:1,000; Chemicon), rabbit anti-glial fibrillary acidic protein (GFAP; 1:2,000; Sigma-Aldrich), mouse anti-human nuclei (1:1,000; Chemicon), rabbit anti-microtubule-associated protein (MAP2; 1:1,000; Chemicon), rabbit or chicken anti-βIII tubulin (TUJ1; 1:1,000; Chemicon), rabbit anti-GFP (1:1,000; Chemicon), rabbit anti-nestin (1:1,000; Chemicon), rabbit anti-tyrosine hydroxylase (1:2,500; Pel-Freez, Rogers, AK, http://www.invitrogen.com), rabbit anti-serotonin (1:1,000; Chemicon), or rabbit anti-Ki67 (1:1,000). After incubation, the sections or cells were washed three times for 5 minutes each in 0.1 M PB and then incubated for 1 hour in diluent containing secondary antibody (goat anti-rabbit Alexa 488 [Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com], 1:300; goat anti-mouse Alexa 555 [Molecular Probes], 1:300; or goat anti-chicken Alexa 633 [Molecular Probes], 1:300). After washing the sections or cells in 0.1 M PB, the sections were mounted on gelatin-coated slides, and the sections and cells were coverslipped using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

Stereology for cultured and transplanted cells was assessed using Stereo Investigator (MicroBrightField Inc., Williston, VT, http://www.mbfbioscience.com) by two investigators who were blind to the treatment of the cells and animals, respectively. Each coverslip of cultured cells (133 mm²) was imaged at a magnification of x20 and then analyzed using the fractionator stereological probe. Cell counts were performed at 10 randomly selected sites (100 x 100 µm) within each culture well. In addition, cells in four wells were analyzed for each immunocytochemical stain. The total number of surviving transplanted human nuclei-immunoreactive or colabeled human nuclei- and Ki67-immunoreactive cells in the grafts was estimated stereologically using the optical fractionator probe, which consisted of a 90 x 90 µm counting frame with a height of 11.5 µm, on every fourth human nuclei-immunostained section throughout the grafted area. The sampling grid (250 x 250 µm) and counting grids were randomly placed in the grafted area. The section thickness was estimated every fifth dissector measurement and then averaged for each section.

Statistical Analyses
Paw withdrawal thresholds for the left hind paw (raw and normalized) were compared with the scores for the right hind paw for each animal at each time point and presented as the mean ± SEM. These raw and normalized paw withdrawal thresholds before and after lesioning and transplantation, as well as the number of surviving transplanted cells, number of Ki67-immunoreactive transplanted cells, and graft volumes, were assessed for within- and between-group differences at p < .05 using a two-way analysis of variance followed by Bonferroni's post hoc test.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Fetal Striatal Primordia Culture
Fetal striatal primordia cells that were cultured for 7 days differentiated predominantly into GAD 65/67-immunoreactive cells (87.23% ± 3.10%) (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Fetal striatal primordia cells derived from GFP mice cultured for 7 days demonstrated immunoreactivity for GAD 65/67 in vitro. Scale bar = 50 µm. Abbreviations: GAD 65/67, glutamic acid decarboxylase 65/67; GFP, green fluorescent protein.

View larger version (83K): [in this window] [in a new window]   Figure 2. Photomicrographs of human telencephalon-derived neural precursor cells grown in 125-ml suspension bioreactors. Cells are shown at 1 hour postincubation (PI) (A), 4 days PI (B), 10 days PI (C), and 14 days PI (D). A single-cell suspension generated by enzymatically dissociating HNPC aggregates was inoculated at a density of 100,000 cells per milliliter into duplicate 125-ml bioreactors, each containing 100 ml of Pharmaceutical Production Research Facility-h2 medium. The bioreactors were incubated in a humidified environment at 37°C and 5% CO2. The cultures were fed every 5 days by removing 40% of the spent medium and replacing it with fresh medium. The expanded fetal telencephalon-derived neural precursor neurospheres expanded in suspension bioreactors demonstrated immunoreactivity for nestin (E). Scale bars = 125 µm (A), 250 µm (B–D), and 200 µm (E).

View larger version (36K): [in this window] [in a new window]   Figure 3. Bioreactor-expanded HNPCs demonstrated multipotentiality in vitro. HNPCs differentiated into cells immunoreactive for nestin (A) and cells with immunohistochemical profiles of astrocytes (B), neurons (C), and GABAergic cells (D). Cells that were differentiated into a GABAergic phenotype prior to transplantation demonstrated immunoreactivity predominantly for GABA (E) and TUJI (F). Scale bar = 50 µm (A, C–F), 100 µm (B). Abbreviations: GFAP, glial fibrillary acidic protein; HNPC, human neural precursor cell; HuN, human nuclei; TUJ1, βIII tubulin.

Behavioral Studies
Unilateral L5–6 nerve root ligations induced a reproducible hypersensitivity to mechanical stimulation of the plantar aspect of the left hind paw within 1 week (Fig. 4A, 4B). Following ligation, the paw withdrawal thresholds for the left hind paw decreased significantly from more than 10 g to less than 2 g (p < .001), whereas that for the right hind paw remained unchanged.

View larger version (29K): [in this window] [in a new window]   Figure 4. Intraspinal transplantation of GABAergic cells derived from either the fetal mouse striatum or differentiated bioreactor-expanded HNPCs significantly attenuated mechanical allodynia by 1 week post-transplantation that was sustained for the duration of the experiment (A, B). represents a statistically significant difference at p < .001 compared with prelesion paw withdrawal scores, which are expressed as the mean score for the left hind paw (A) or the mean score for the left paw normalized for each animal's right hind paw (B). * represents a statistically significant difference at p < .001 compared with postlesion paw withdrawal scores. Abbreviations: GFP, green fluorescent protein; HNPC, human neural precursor cell.

In Vivo Histological Analyses
Grafts of fetal striatal primordia cells revealed the presence of viable grafts composed of numerous GFP-positive cell bodies and distinct fibers (Fig. 5) in both the dorsal horn and more ventral portions of the spinal cord. Confocal microscopy confirmed that some GFP-positive cells colocalized with GAD 65/67 and GABA. GFP-positive cells were seen aggregating around the transplant site, as well as several millimeters rostral and caudal to the injection site (Fig. 5D). The transplantation injection cannula tract could be visualized in the spinal gray matter, but the transplanted cells did not disrupt the anatomy of either the spinal gray or white matter.

View larger version (30K): [in this window] [in a new window]   Figure 5. Fetal mouse GFP striatal primordia cells transplanted into the spinal cord of allodynic rats demonstrated robust graft survival and maintained immunoreactivity for GAD 65/67 up to 7 weeks post-transplantation (A–D) and migrated several millimeters away from the injection site in the spinal gray matter (D). Scale bars = 500 µm (A), 50 µm (B), 20 µm (C), and 400 µm (D). * = transplantation injection site. Abbreviations: cc, central canal; GAD 65/67, glutamic acid decarboxylase 65/67; GFP, green fluorescent protein.

View larger version (52K): [in this window] [in a new window]   Figure 6. Intraspinally transplanted human neural precursor cells differentiated into a GABAergic phenotype prior to transplantation into the spinal cords of allodynic rats maintained a GABAergic and neuronal immunohistochemical phenotype up to 7 weeks post-transplantation (A) and did not differentiate into astrocytes (B). Some cells demonstrated immunoreactivity for Ki67 (C), but no tumor formation was observed in any transplanted animals. The grafts appeared as dense clusters of cells in the spinal gm and wm (D). Scale bars = 50 µm (A), 25 µm (B), 20 µm (C), and 100 µm (D). Abbreviations: GFAP, glial fibrillary acidic protein; gm, gray matter; HuN, human nuclei; TUJ 1, βIII tubulin; wm, white matter.

View larger version (62K): [in this window] [in a new window]   Figure 7. Undifferentiated human neural precursor cells transplanted into the spinal cord differentiated predominantly into astrocytes (A, B) and maintained immunoreactivity for nestin (C, D); some cells demonstrated immunoreactivity for Ki67 (E), but no tumor formation was observed in the spinal cords of the transplanted animals (F). Scale bars = 100 µm (A), 25 µm (B, D), 50 µm (C, E), and 200 µm (F). Abbreviations: GFAP, glial fibrillary acidic protein; gm, gray matter; HuN, human nuclei; wm, white matter.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
To determine whether intraspinally transplanted GABAergic cells could be used in the treatment of allodynia, we used a rat model of neuropathic pain in which L5 and L6 spinal nerve root ligation induced profound and persistent tactile allodynia in the ipsilateral hind paw [77, 83]. Previous studies investigating cell-based therapies for chronic pain syndromes have deposited cells onto the subarachnoid space [47–51, 55, 56, 59, 60, 61, 84]; however, the reduction of pain behaviors has not been always consistent or sustained [49, 53, 85, 86]. In the present study, we demonstrate that transplantation of GABAergic cells directly into the spinal cord produces a reduction in mechanical allodynia by 1 week post-transplantation, with peak reductions observed at 4 weeks that persisted even after 6 weeks following transplantation. Transplantation of cells into the spinal cord parenchyma allows the cells to be better targeted directly to areas of pathology where dysfunctional nociceptive processing occurs [86, 87]. In addition, we have demonstrated that HNPCs, expanded in a standardized fashion in suspension bioreactors, can be differentiated into an apparent GABAergic phenotype in vitro, maintain this phenotype in vivo, and may be used as an alternative to fetal GABAergic cells in a cell restoration strategy for allodynia.

The present findings support the notion that GABAergic inhibition in the spinal cord contributes to nociceptive processing [42, 88]. Intraspinally transplanted GABAergic cells may have decreased pain behaviors by restoring GABAergic inhibitory input after inhibitory interneurons are permanently lost in the ipsilateral spinal dorsal horn laminae I to III after spinal nerve injury [31]. These transplanted cells may have been able to restore GABAergic function to the spinal cord dorsal horn to reduce the hyperexcitability that develops in dorsal horn projection neurons. This is consistent with previous studies that transplanted immortalized fetal rat cells bioengineered to secrete GABA into the subarachnoid space of allodynic rats [60, 61] where tactile and thermal allodynia and hyperalgesia were significantly attenuated. The transplanted cells were thought to modulate ascending pain signals by secreting GABA [60, 61]. Increased production of GABA in the lumbar dorsal root ganglia, with a resulting increase in GABA levels in the dorsal horn, was also a postulated mechanism for behavioral improvement in allodynic rats with either unilateral spinal nerve ligation or thoracic hemisection spinal cord injuries that received subcutaneous injections of nonreplicating herpes simplex virus vectors containing the human GAD 67 gene into the plantar surface of the hind paw [89, 90]. The allodynic rats demonstrated a significant increase in mechanical thresholds beginning 1 week postinoculation [89, 90] that correlated with a significant increase in the amount of GAD 67 protein in the lumbar dorsal root ganglia compared with sham-inoculated controls [89].

In other studies, trophic factors secreted by transplanted cells have been hypothesized to contribute to behavioral improvement by protecting or limiting the death of endogenous spinal cord inhibitory interneurons after peripheral or spinal cord injury [31, 58, 59]. Neural stem and precursor cells have also been shown to secrete trophic factors that regulate the fate of neighboring cells [91–99]. We have demonstrated that telencephalon-derived bioreactor-expanded HNPCs enhance the survival of cotransplanted fetal rat dopaminergic neurons in a rat model of Parkinson disease, presumably by secreting neurotrophic factors, such as glial-derived neurotrophic factor or sonic hedgehog [100]. In the current study, however, it is less likely that trophic factors potentially secreted by the transplanted HNPCs were responsible for the behavioral improvement, since only the differentiated HNPCs, which were GABAergic, and not the undifferentiated HNPCs, which became predominantly astrocytic after transplantation, conferred attenuation of pain behavior.

Alternatively, the trophic effects of intraspinally transplanted GABAergic cells may not be related to the secretion of neurotrophins but instead to the neuroprotective effects of GABA on spinal cord inhibitory interneurons. A GABA-mediated neuroprotective effect has been shown in various animal models of ischemia, in which acute GABA therapy limited and reversed cell death [101–104]. In our study, GABAergic cells were transplanted 10 days following spinal nerve injury, at which time it has been shown that a reduction in GABA-immunoreactive neuronal profiles in laminae I to III is apparent but incomplete [31]. Since it is only by 3 weeks post-nerve injury that GABAergic profiles become nearly completely absent in the superficial dorsal horn, there are still GABAergic neurons in the spinal cord dorsal horn that may be rescued by GABA secreted by transplanted fetal striatal primordial or differentiated HNPCs [31].

We have demonstrated that bioreactor-expanded HNPCs differentiated into a GABAergic phenotype can be used as an alternative to fetal GABAergic tissue. Unlike expansion of HNPCs in standard stationary culture flasks, which are not amenable to scaled-up production of large numbers of HNPCs, suspension bioreactors can efficiently generate clinically relevant quantities of cells in a reproducible manner under standardized, computer-controlled conditions in which parameters such as oxygen concentration, osmolarity, and pH can be maintained at optimal levels [66, 67, 69–72]. Mammalian neural stem cells can be expanded for extended periods of time using a specialized serum-free expansion medium without any loss of defining stem cell characteristics, such as multipotentiality and self-renewal, as determined by karyotype analyses and immunohistochemistry [67, 68, 105], and without altering the cell cycle kinetics of the stem cells [106].

Ultimately, differentiation of neural precursor cells into a GABAergic phenotype prior to transplantation may be crucial to avoid the potential deleterious side effects associated with the transplantation of undifferentiated stem cells [107, 108]. Neural stem and precursor cells have typically differentiated into glia after transplantation into nonneurogenic regions of the central nervous system, such as the spinal cord, because of the predominance of glial restrictive cues [109–114]. The bioreactor-expanded HNPCs used in our study differentiated into a phenotype immunoreactive for both GABA and TUJ1 in vitro that was maintained even up to 7 weeks after transplantation into the rat spinal cord. In previous studies in which undifferentiated neural stem cells were transplanted into rodent models of spinal cord injury, astrocytic differentiation of transplanted stem cells was associated with a lack of recovery of motor function, as well as the development of allodynia in the forepaws [107, 108]. Although a small proportion of transplanted HNPCs seen here maintained proliferative potential as indicated by Ki67 immunoreactivity, there was no evidence of tumor formation, which is consistent with observations of other studies using HNPCs [93, 115–119].

Although our study demonstrated significant improvement of pain behaviors after intraspinal transplantation of GABAergic cells, it may be necessary to recapitulate the normal spinal processing of nociception that occurs using neurotransmitter systems additional to GABA to obtain complete recovery. For example, adenosine receptor agonists have been shown to synergistically suppress tactile hypersensitivity in allodynic rats when combined with GABA receptor agonist administration, and administration of both GABA and adenosine receptor antagonists abolished the effects of spinal cord stimulation in abolishing allodynic pain [17–19]. Transplantation of cells that secrete serotonin [55–57], galanin [84], or catecholamines or opioid peptides [47–54, 85, 87] have also been shown to significantly improve pain behaviors in animal models of neuropathic pain. Although this effect may have been related to a GABAergic effect by restoration of GABA immunoreactivity to the spinal cord [31, 58], it was also thought to be related to the secretion of non-GABAergic substances as well. Thus, a transplantation strategy that is dependent upon restoration of GABAergic tone to the spinal cord but also involves transplantation of cells of other phenotypes may be necessary for a more complete amelioration of chronic pain.

	CONCLUSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The present study supports the use of intraspinal transplantation of GABAergic cells as a novel strategy for attenuating chronic pain. These GABAergic cells may be from a fetal source, but as a practical alternative, HNPCs expanded under controlled conditions and differentiated into a GABAergic phenotype could be used. However, use of these cells will require further surveillance to ensure that the behavioral effects are persistent and without adverse effects over the long term. This transplantation strategy may be especially relevant following spinal cord injury, in which the majority of patients suffer from disabling chronic pain.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The technical assistance of Alia Mukhida, Damaso Sadi, Ruperto Ulalia, Cindy Leopold, Matthew Lewington, and Angela Gamouras is greatly appreciated. This study was supported, in part, by research grants from AO North America and the Capital Health Research Foundation (to K.M.), the Canadian Anaesthesiologists' Society (to B.M. and M.H.), the Stem Cell Network (to L.A.B.), and the Natural Sciences and Engineering Research Council of Canada (to B.B., A.S., and L.A.B.). K.M. acknowledges the generous support of an Izaak Walton Killam Memorial Scholarship and a Dalhousie Medical Research Foundation Clinical Research Fellowship. The GFP mice were kindly provided by Dr. H. Robertson.

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