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From the Departments of *Anesthesiology and Pathology, Emory University School of Medicine, Atlanta, Georgia.
Address correspondence and reprint requests to Marie Csete, MD, PhD, Emory Anesthesiology Labs, 1462 Clifton Rd. NE, Room 420, Atlanta GA 30322. Address e-mail to marie.csete{at}emoryhealthcare.org.
Abstract
BACKGROUND: Stem cells mediate neuroprotection in a variety of nervous system injury models. In this study, we evaluated a potential role for stem cells in pain therapies. Marrow mononuclear cells containing mixed stem cell populations were used because of wide experience with these cells in experimental and clinical transplantation.
METHODS: After sciatic nerve chronic constriction injury (CCI), adult male Sprague Dawley rats were treated with freshly isolated marrow mononuclear cells (107 cells in 0.5 mL IV) from the same strain, or with carrier. The major end points of analysis were thermal and mechanical hypersensitivity using paw withdrawal latency (PWL) to a calibrated heat source and paw withdrawal response to von Frey filaments, evaluated by a blinded investigator.
RESULTS: Marrow transplantation did not prevent pain, and 5 days after CCI all animals were equivalently lesioned. However, 10 days after CCI, rats that received marrow transplants demonstrated paw withdrawal response and PWL patterns indicating recovery from pain, whereas untreated rats continued to have significant pain behavior patterns. For example, PWL values for marrow-treated animals were similar to baseline pre-CCI values (P = 0.54) but significantly shorter latency to withdrawal indicative of continuing pain was seen in untreated rats compared with pre-CCI values (P < 0.001).
CONCLUSIONS: These studies suggest that stem or progenitor cell-mediated therapies may be useful for the treatment of pain after nerve injury, and deserve further study to elucidate the mechanisms of analgesia.
Stem cells have demonstrated utility in limiting neuronal damage in a wide variety of experimental neurologic injuries, including stroke, Parkinson’s disease, traumatic brain injury, spinal cord injury, and peripheral nerve damage (1,2). A variety of stem cells from marrow (sorted directly or expanded in vitro) have been used in these models. Adult stem cell therapies for human neurologic diseases are under study (3). Despite the association of pain with these neurologic injuries, pain is rarely an end point of such studies. Animal models of pain, such as chronic constriction injury (CCI), have not been used to study the role of stem cells in modulating pain after neurologic injury. Here we used the CCI model to investigate the ability of infused marrow mononuclear cells to change the pattern of pain behavior after injury.
METHODS
All procedures were conducted after Institutional Animal Care and Use Committee approval, and rats were housed and handled according to National Institute of Health guidelines for the care of laboratory rats.
Isolation of Marrow Mononuclear Cells
Bone marrow mononuclear cells were isolated from hindlimbs of three rats and pooled. Two-month old (donor) male Sprague Dawley rats were euthanized using CO2 inhalation. Using sterile technique, the hindlimbs were skinned, and amputated at the hip. Under a sterile tissue culture hood, tissues were removed from the femur and tibia. The ends of the bones were cut with sterile scissors, and bone marrow flushed, using an 18-guage needle, into Dulbecco’s Modified Eagle Medium supplemented with penicillin/streptomycin and l-glutamine. Cells were separated by gradient centrifugation with Histopaque (Sigma, St. Louis, MO) at a density of 1.803 g/mL. The buffy coat containing mononuclear cells was removed and washed, and then resuspended in iced phosphate buffered saline (PBS). Cells were counted manually using a hemacytometer.
Chronic Constriction Injury
We used the CCI model (4) because of its historic utility in the evaluation of treatment modalities for neuropathic pain. General anesthesia of 2-month-old male Sprague Dawley rats was induced with pentobarbital 40 mg/kg IP, and supplemented with doses of 2–4 mg/kg as necessary. Sterile technique was used for the surgery. Skin incision extended from the left sciatic notch to the distal thigh, and then blunt dissection of the subcutaneous tissue was used to expose the biceps femoris, which was incised at the sciatic notch. The fibers of the biceps femoris were spread, Mayo scissors placed through the muscle, and a 2-cm cut made in the muscle toward the patella. The muscle was further freed with blunt dissection, and the sciatic nerve was freed from investing fascia. Left sciatic nerves (n = 12) were constricted with 4 4–0 Chromic sutures, placed 1 mm apart in the center of the nerve, and tightened until the nerve appeared indented under a dissecting microscope. The incisions were closed with 3-0 Vicryl suture (Ethicon, Piscataway NJ). Right sciatic nerves were mobilized in precisely the same way, but not constricted. This manipulation of the right side creates equivalent soft tissue and muscle injury as does surgery on the left side, but leaves the nerve purposefully unconstricted for comparison (control) studies (5). After bilateral incisions were closed, but before the rats were awake, half the rats (randomly assigned) received freshly isolated mononuclear cells from marrow IV (107 cells/0.5 mL PBS) via tail vein, and half received 0.5 mL PBS. The animals were recovered on a warming blanket under a heat lamp, and returned to the animal facility once they were ambulatory and eating. Within a few hours of surgery, one rat in each group died. The remaining rats were killed on day 10 after behavioral testing. Blood was collected by ventricular puncture and stored for later analysis. The sciatic nerves were paraffin-embedded for histologic analysis.
Pain Behavioral Testing
Thermal and mechanical hypersensitivity, the major end points of analysis, were measured bilaterally before CCI on three occasions, and at 5 and 10 days later by an investigator blinded to treatment groups. Thermal sensitivity using paw withdrawal latency (PWL) to a calibrated heat source was measured with an automated sensor. PWL values for the lesioned limb (left) are expressed as a percentage of the values for the right limb [(PWLL/PWLR) x 100]. Mechanical allodynia was assayed by paw withdrawal response (PWR) to von Frey filaments as previously reported (4). Each filament was applied (until it bent or the animal withdrew) from below at about 1 cm from the heel of the animal, starting with the 4.08-g filament. Testing was continued using increasing weight filaments until the rat withdrew from the filament, or a force of 6.65 g was reached. The bending force from three tests was used as the withdrawal threshold. Five sets of tests were done on each hindpaw at each session postoperatively. These results were compared with the grand mean of three baseline preoperative tests.
Data are expressed as mean ± sd. Measurement sets contain an n of five per group, and three time-points. Data were tested for both homogeneity of variance and normalcy of distribution. Based on these tests, intragroup temporal comparisons of PWL and PWR were made using repeated measures analysis of variance (ANOVA) or Friedman’s repeated measures ANOVA on ranks followed by a post hoc Student-Newman-Keuls correction for multiple comparisons. A Student’s t-test for independent means was used for intergroup comparisons at each time point. Intergroup comparisons in PWR measures at each time were assessed using either a Student’s t-test for independent means or a Mann–Whitney ranked sum test as appropriate. A P value (
level) of <0.05 was considered the minimum for rejection of the null hypothesis.
Hematoxylin and Eosin Stains of Nerve Sections
Nerve sections (8 µm) were deparaffinized then rehydrated, stained for 2 min in Harris’s hematoxylin (Sigma), then rinsed in tap water. The sections were dipped in ammonia water, rinsed with tap water, then stained in eosin for 5 min, followed by three dips in 95% ethanol. Nerve areas were measured using National Institute of Health Image (freely downloadable software), for normalization of cell counts. Nuclei were counted in the nerve fascicles (not in surrounding perineurium) in three sections for each nerve. Nuclear counts, normalized to area, were compared between groups by using Student’s t-test.
Nissl Staining of Nerve Sections
Nissl solution was made by dissolving 0.5 g cresyl echt violet acetate in 100 mL distilled water, and the solution filtered. Sciatic nerve sections (8-µm) were deparaffinized and rehydrated to distilled water (8 sections per nerve per animal). The sections were then dipped in Nissl solution for 2 min, and washed with distilled water. Before clearing with xylene, the sections were photographed for archiving. Photographs of the sections (40x fields) were printed on paper, and degenerated fibers circled. The percentage of degenerated nerve fibers in the field was counted by hand, and compared between groups using 
2 testing.
Immunohistochemical Analysis of T Cells and Macrophages in Nerve Sections
After deparaffinization, 8-µm sections of sciatic nerve were fixed in acetone for 10 min at room temperature, then air dried. The sections were extensively washed in PBS containing 0.05% Tween-20. Primary antibody against the ß T-cell receptor (1:300 dilution of clone R73, BD Biosciences, San Jose CA) was incubated in PBS with 3% fetal bovine serum and 2% bovine serum albumin for 1 h at room temperature, and then washed with PBS. AlexaFluor 568 goat anti-mouse IgG (Molecular Probes, Eugene OR) was incubated at 1:400 in the same conditions as primary antibody, and then washed with PBS. The sections were mounted with Vectashield (Vector Labs, Burlingame, CA) and photoarchives collected.
RESULTS
All rats, regardless of treatment group, developed significant thermal hyperalgesia by day 5. Thermal hyperalgesia did not resolve in PBS-treated rats by day 10, but completely resolved in marrow-treated rats (P < 0.001 for control rats compared to preoperatively indicating continuing pain and, P = 0.54 for marrow-treated animals, indicating no difference from baseline measures, Fig. 1). Similarly all animals developed significant mechanical allodynia by day 5, but only those that received marrow transplants showed resolution of mechanical hypersensitivity on day 10. Control animals continued to demonstrate painful behavior (P < 0.003 for control animals compared to preoperatively, P = 0.15 for marrow-treated rats, Fig. 2). These data show that the marrow infusion did not prevent pain, as all the animals appeared to be equally lesioned at day 5, using assays for thermal and mechanical hypersensitivity. Though the rats were indistinguishable at day 5, marrow-treated rats had resolution of pain behaviors by day 10, suggesting that marrow augments and or speeds recovery from CCI-mediated pain.
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Gross histology of the CCI-lesioned nerves showed that they were hypercellular, with thickened perineuria and epineuria, compared to unlesioned (right) nerves (Fig. 3). We counted the number of nuclei in the nerves in three sections each of right sided nerves, CCI nerves after marrow, and CCI nerves without marrow, normalized to area. The cellular identity of nuclei on hematoxylin and eosin stain sections is not known. As expected, CCI (left) nerves contained significantly more nuclei than right sciatic nerves (mean ± se): 3.0 ± 0.5 vs 1.9 ± 0.2 per unit area, P = 0.02. There was no significant difference in the number of nuclei in CCI-lesioned nerves from marrow-treated rats versus from untreated rats: 3.5 ± 1.0 vs 2.6 ± 0.2 per unit area, P = 0.20. Similarly the difference in nuclear number in the sham surgery side nerves of marrow-treated rats was not different from that of untreated rats: 1.8 ± 0.2 vs 2.0 ± 0.3 per unit area, P = 0.33.
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The histologic changes on the CCI side were consistent with Wallerian degeneration. No morphologic differences between marrow-treated and PBS-treated nerves were discernable by a neuropathologist. However, in order to better quantify the degree of damage, nerve sections were Nissl-stained (a standard technique for assessing neuron integrity). The number of intact nerves (uniform staining in cross-section), and degenerating or degenerated nerves were counted using one Nissl-stained section per nerve per rat, and the total numbers pooled. Degenerating nerves on the unlesioned side, as expected, were rare. On the CCI-lesioned side of marrow-treated rats, nerves collectively contained 13.0% degenerated fibers, while PBS-treated nerves after CCI collectively had 12.4% degenerated fibers. This preliminary data suggest that the degree of Wallerian degeneration was not the reason for differences in pain between marrow-treated and PBS-treated rats.
We used an antibody against mature T cells for an initial evaluation of the sciatic nerve infiltrate, but there was considerable background staining of nerve fibers despite multiple attempts to optimize the stain. Using these admittedly non-optimal stains, we counted distinctly positive cells in 4–6 random high power fields from three nerve sections of each CCI-lesioned nerve. There was no significant difference in the total numbers of positive cells in CCI nerves from PBS-treated versus marrow-treated rats (3.6 ± 0.6 vs 2.7 ± 0.4, mean ± se, P = 0.14).
DISCUSSION
Our experience in evaluating pharmacologic therapies for neuropathic pain in the CCI model highlights the striking analgesic results obtained here after marrow infusions. Such complete reversal of tactile and thermal hyperalgesia by pharmacologic intervention is generally not seen in this model. For example, treatment of CCI-lesioned rats with the antiepileptic zonisamide improved thermal hypersensitivity (to a lesser extent than did marrow infusion), and improved mechanical hypersensitivity only at highly sedating doses (and again, to a lesser extent than did marrow infusion) (6). Nonetheless, recent evaluation of zonisamide in patients with neuropathic pain suggests that it may benefit at least a subset of these patients (7,8).
The mechanism underlying the effectiveness of marrow infusions for pain in this study is unknown. However, neurologic improvement after stem cell transplants in experimental central nervous system injuries such as stroke may provide some insight into the results obtained here. First, stem cells have a surprising ability to home to sites of injury and ischemia using chemokine signals (9). The neuroprotective effects of transplanted stem cells are thought to result from a combination of antiinflammatory, proangiogenic, and neurotrophic factors produced by transfused cells (10), rather than from extensive de novo regeneration from transplanted stem cells. In addition, neurotrophic factors such as glial cell line derived neurotrophic factor, produced by stem and progenitor cells, can mediate potent antioxidant effects (11).
Similar multimodal mechanisms may explain the effectiveness of marrow in the CCI model, since neuropathic pain has both an inflammatory (12) and an ischemic component (13), and a variety of neurotrophic factors can ameliorate neuropathic pain. These include glial cell line derived neurotrophic factor (14) and erythropoietin (15). Because the infused marrow population is mixed, and its secreted products therefore heterogeneous, the analgesic effect seen here is likely multifactorial, and is the subject of ongoing work. In addition, it will be of interest to isolate the stem cell populations that mediate the analgesic effect. Marrow contains not only hematopoietic stem cells, but pluripotent side population cells, stromal stem cells that have not been well characterized, mesenchymal stem cells and endothelial progenitors, making the identification of the operant stem-cell population a fairly complex undertaking.
Further investigation into the inflammatory component of pain relief in this model is also warranted. Our T-cell characterization does not distinguish between proinflammatory or antiinflammatory cells, for example. Depletion of proinflammatory T cells can dramatically reduce pain in this model (16). Furthermore, the rat strain used in these studies (Sprague Dawley) is considered to be outbred, though Sprague Dawley to Sprague Dawley marrow transplantation is tolerated without immunosuppression and results in marrow chimerism in longer-term experiments (17). Nonetheless, the specific nature of the host immunologic reaction may have contributed to our findings. Thus, manipulation of the immune response with immunosuppressants may also lead to an understanding of the results presented here.
Our results may also be model-specific, and it will be important to study the effects of bone marrow or other stem cell transplants in different models of neuropathic pain. For example, a recent study (18) suggests that bone marrow transplantation can exacerbate established peripheral neuropathy in a murine model of diabetes. In this case, transplanted marrow cells fused with peripheral neurons, initiating neuronal apoptosis.
In summary, this pilot study is the first examining the use of primary, nontransformed progenitor cell transplants for the treatment of pain in the CCI model. Our results suggest that mobilization of marrow into the circulation using autologous cells may benefit patients with pain due to nerve injury. Furthermore, pain evaluations should be more fully incorporated into the analysis of the many ongoing clinical studies of marrow stem cell and mesenchymal stem cell transplants for a variety of human diseases. Carefully controlled animal and clinical trials will be essential to evaluate the potential of more characterized marrow-derived stem cell populations for the treatment or prevention of chronic neuropathic pain.
Footnotes
Accepted for publication December 22, 2006.
Supported by the Emory University Department of Anesthesiology and NIA PO1 AG386003609.
REFERENCES
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