J Neurosurg 121:423–431, 2014 ©AANS, 2014
Sensory recovery after cell therapy in peripheral nerve repair: effects of naïve and skin precursor-derived Schwann cells Laboratory investigation Antos Shakhbazau, Ph.D.,1,2 Chandan Mohanty, M.D.,1 Ranjan Kumar, M.Sc.,1–3 and Rajiv Midha, M.D., F.R.C.S.C.1,2 Department of Clinical Neuroscience, Faculty of Medicine, 2Hotchkiss Brain Institute, and 3Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada
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Object. Cell therapy is a promising candidate among biological or technological innovations sought to augment microsurgical techniques in peripheral nerve repair. This report describes long-term functional regenerative effects of cell therapy in the rat injury model with a focus on sensory recovery. Methods. Schwann cells were derived from isogenic nerve or skin precursor cells and injected into the transected and immediately repaired sciatic nerve distal to the injury site. Sensory recovery was assessed at weeks 4, 7, and 10. Axonal regeneration was assessed at Week 11. Results. By Week 10, thermal sensitivity in cell therapy groups returned to a level indistinguishable from the baseline (p > 0.05). Immunohistochemistry at 11 weeks after injury showed improved regeneration of NF+ and IB4+ axons. Conclusions: The results of this study show that cell therapy significantly improves thermal sensation and the number of regenerated sensory neurons at 11 weeks after injury. These findings contribute to the view of skin-derived stem cells as a reliable source of Schwann cells with therapeutic potential for functional recovery in damaged peripheral nerve. (http://thejns.org/doi/abs/10.3171/2014.5.JNS132132)
Key Words • cell therapy • Schwann cells • skin precursors • stem cells • peripheral nerve • regeneration • thermal sensitivity
T
raumatic injury results in significant peripheral nerve damage in 2.8% of trauma patients, often leading to a long-term disability.32 Although the peripheral nervous system is commonly considered to possess a certain regenerative potential, existing microsurgical techniques have reached a plateau in terms of functional recovery.4 Further advances are expected from biological or technological innovations, such as improved nerve conduits, cell and gene therapy, electric stimulation, infusion of neurotrophic factors, and other techniques.3,11,24,39,41,43
Abbreviations used in this paper: CMAP = compound muscle action potential; IB4 = isolectin B4; NCV = nerve conduction velocity; nSC = nerve Schwann cell; PBS = phosphate-buffered saline; PFA = paraformaldehyde; SC medium = Schwann cell medium; SKP = skin precursor; SKP-SC = SKP-derived Schwann cell.
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In cell-based therapeutic approaches for peripheral nerve regeneration, Schwann cells are among the most appropriate candidates, being the myelinating glial cells in the peripheral nervous system and therefore key contributors to axonal regeneration and remyelination after injury.23,30 Guided by injury signals, Schwann cells divide and migrate to provide pathways for axonal regrowth by their alignment in the so-called bands of Büngner.15 Schwann cells also contribute to regeneration by secreting signaling molecules, cytokines, components of the extracellular matrix, cell adhesion molecules, and trophic factors.8,44 The therapeutic potential of autologous Schwann cells can be limited by lengthy expansion and the need This article contains some figures that are displayed in color online but in black-and-white in the print edition.
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A. Shakhbazau et al. to sacrifice a healthy nerve, resulting in neuropathic pain and donor site morbidity, and several groups have succeeded in deriving Schwann cells from bone marrow mesenchymal stem cells, adipose stem cells, and skin precursor (SKP) cells.2,9,10,17,21,26,42 Prior in vivo experiments from our group and others show some histological improvement after stem cell/Schwann cell therapies; however, the evidence for functional peripheral nerve recovery remains limited.16,47,48 In the present study, we aimed to evaluate the potential of SCs derived from the isogenic nerve and skin precursors for improved sensory recovery in a standard sciatic nerve transection model.
Methods Cell Isolation and Culture
Schwann cells were isolated from sciatic nerves of P2 Lewis rats according to established protocols22,47 with minor modifications. Briefly, sciatic nerves were excised, stripped of the epineurium, and cut into 1-mm2 pieces. Nerve segments were placed on poly-d-lysine–coated 35mm culture dishes in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.25 mg/ ml Fungizon for 3 days, allowing fibroblast migration out from the nerve. The medium was then changed to serumfree DMEM containing 1% N2, 10 ng/ml heregulin-1b, 4 mM forskolin (Schwann cell medium [SC medium]) for another 3 days to stimulate Schwann cell outgrowth. At Day 6, fragments were removed from the dish and explanted onto another 35-mm dish in SC medium with 2.5% FBS. The explant procedure was repeated until little outgrowth of fibroblasts was observed, then the explants were discarded and all dishes containing Schwann cells were purified in serum-free SC medium. Purity of nerve Schwann cell (nSC) cultures was assessed by p75 and GFAP immunolabeling and a heregulin exclusion test.35 Skin-derived precursor (SKP) cells were obtained from dermis of postnatal Day 2 Lewis rats and cultured according to published protocols.6,46,47 Briefly, skin from the dorsal torso was minced in HBSS (Gibco) on ice, incubated for 45 minutes in 0.1% collagenase XI at 37°C, dissociated mechanically, washed in cold DMEM, and passed through a 40-mm cell strainer. Filtrate was centrifuged at 1200 rpm, and the pellet was triturated and resuspended in culture medium (DMEM:F12 3:1, 100 U/ ml penicillin and 100 mg/ml streptomycin, 0.25 mg/ml Fungizon, 1× B27 supplement, 20 ng/ml EGF, and 40 ng/ ml bFGF [all from Gibco]). Skin-derived precursor cells were cultured and passaged as undifferentiated spherical aggregates at 37°C in a humidified atmosphere containing 5% CO2/95% air. The progenitor state of the cells within the aggregates was confirmed with nestin immunolabeling. To induce differentiation toward Schwann cells, aggregates were triturated and replated on poly-d-lysine/laminin– coated culture dishes (Corning) in SC medium (DMEM/ F12 supplemented with 100 U/ml penicillin and 100 mg/ ml streptomycin, 0.25 mg/ml Fungizon, 4 mM forskolin, 10 ng/ml heregulin-1b, and 1% N2 supplement [Gibco]), as described.6,40 Media were changed every 3–4 days. 424
Following 1–2 weeks incubation, cells appearing under phase contrast to have bipolar Schwann cell morphology were isolated with cloning cylinders and their fate specification was confirmed with GFAP and p75-NTR antibody staining. Cultures were further enriched for p75-NTR– positive cells by means of fluorescence activated cell sorting (FACS) using flow cytometer FACSAria II (Beckton Dickinson) and expanded in the SC medium until ~ 95% purity was achieved. These cells were subsequently referred to as SKP-derived Schwann cells, or SKP-SCs. SKP-SCs grow as adherent monolayers in static cultures. Both nSCs and SKP-SCs were then routinely maintained on plastic tissue culture flasks and plates (Falcon) at 37°C in a humidified atmosphere containing 5% CO2/95% air. In Vitro Myelination
To confirm the myelinating properties of cultured cells, SKP-SCs and nSCs were seeded onto the neurite network formed by 1-week culture of dorsal root ganglia from postnatal (P10) Lewis rats in Neurobasal medium containing B27 supplement, 10% FBS, 50 ng/ml NGF, 1% penicillin/streptomycin and 0.25 mg/ml Fungizon. Assay was performed in 8–well chamber slides (Nunc) coated with Matrigel. Host Schwann cells from dorsal root ganglia were killed by the addition of 7 mM cytosine arabinoside. Nerve Schwann cells (nSCs) and SKP-SCs were labeled with CellTracker CM-DiI (Invitrogen) prior to seeding. Co-cultures were maintained in SC medium containing 50 ng/ml NGF, and ascorbic acid (50 mg/ ml, Sigma) was added to induce myelination.42 The coculture was maintained for 2 weeks, then fixed with 2% paraformaldehyde (PFA) in PBS, blocked/permeabilized with 2% BSA/ 0.3% Triton-X100 in PBS and stained for myelin basic protein (MBP) with secondary antibodies with an emission wavelength of 488 nm. Animals
A total of 27 male Lewis rats weighing 225–250 g (Charles River) were used in this study (as detailed below). Animals were maintained in a temperature- and humidity-controlled environment with a 12-hour light/dark cycle. Food (Purina) and water were available ad libitum. Surgical interventions were carried out under inhalation anesthetic (Isoflurane, 99.9%, Halocarbon Laboratories), and pain control was provided by means of intraperitoneal or oral administration of buprenorphine (30 mg/kg). Surgical procedures were carried out aseptically, and standard microsurgical techniques were used with an operating microscope (Wild M651; Wild Leitz). Animals were killed at endpoint under deep anesthesia using an overdose of intracardiac Euthanol (Bimeda-MTC). All efforts were made to minimize suffering and animal numbers by using appropriate protocols. The protocol was approved and monitored by the University of Calgary animal care committee and adhered strictly to guidelines set by the Canadian Council on Animal Care.
Surgical Procedures
Surgery was performed unilaterally, in the right hindlimb, in all animals. The sciatic nerve was exposed at J Neurosurg / Volume 121 / August 2014
Sensory recovery after cell therapy in peripheral nerve repair mid-thigh level after splitting the biceps femoris muscle and transected ~ 1 cm proximal to the trifurcation with a small segment removed to create a 5-mm gap. All animals received an immediate entubulation repair within a silicon tube, inner diameter 1.5 mm, secured with 9-0 Prolene microsutures at each end, leaving a 5-mm gap between the proximal and distal stumps, using previously reported techniques.19,38 Immediately following repair, 10 ml of nSCs (n = 6) or SKP-SCs (n = 5) or carrier medium (n = 6) was injected using a 10-ml Hamilton syringe with a 30-gauge needle into the segment of transected sciatic nerve immediately distal to the graft, giving a total of 1 × 106 cells. Schwann cells were labeled 20 minutes prior to injection with CellTracker CM-DiI (Invitrogen) according to the manufacturer’s guidelines. Briefly, cells were trypsinized from culture vessels and resuspended to 1 × 106 cells/ml in medium containing 1 mM DiI staining solution. Following incubation for 20 minutes in the dark, cells were washed 3 times in DMEM and stored on ice until injection. To monitor cell/medium delivery, 0.1% FastGreen was added to the cells and medium immediately before injection. In a non-repair group (n = 5), severed nerve was not repaired and no injections were made. In a sham-surgery group (n = 5), nerve was exposed but not operated. Wounds were closed in layers, and the animals were housed as described above with appropriate analgesic administration (4 days), until they were killed at the 11-week time point. Electrophysiology
Electrophysiological recovery was assessed by measuring compound muscle action potential (CMAP) amplitude and latency. Electrical conductivity across the injury site was evaluated by stimulating the sciatic nerve proximally (at the sciatic notch) and measuring the CMAP amplitude evoked in the gastrocnemius muscle 5 and 8 weeks following the surgery. Animals were anesthetized with isoflurane as described above, stimulating bipolar electrodes were inserted percutaneously into the sciatic notch proximal to the nerve repair site, and a supramaximal stimulus was delivered to the sciatic nerve. Recording electrodes were placed longitudinally into the gastrocnemius muscle, and the ground electrode was placed subcutaneously in the base of the tail. Recordings were obtained using a VikingQuest electrodiagnostic system (Nicolet). The distance between the electrodes was measured, and latencies were used to calculate nerve conduction velocity (NCV).
Thermal Sensitivity Testing
Sensory recovery of the experimental animals was evaluated by analyzing the responses of injured and contralateral intact hindlimbs to thermal stimulus by the Hargreaves plantar test according to published methods.13,52,53 The animals were placed in a Plexiglas chamber atop a transparent glass surface that housed a radiant heat source and acclimatized for 5 minutes. The heat source was placed directly under the plantar surface of the hind paw and activated, starting a digital timer. The time count stopped when the paw was withdrawn from the heat source, indicating the paw withdrawal latency (seconds). J Neurosurg / Volume 121 / August 2014
Three or four evaluations for each paw were performed by an observer blinded to the nature of the groups. Intervals of 5 minutes were allowed between the readings to prevent paw sensitization, and withdrawal latency of the injured (right) limb was normalized to the uninjured side to generate latency ratio. Baseline levels were established 1 week before the injury/cell injection. Sensitivity testing resumed 4 weeks after surgery and was also performed at weeks 7 and 10 postinjury. Target Muscle Weight
As the animals were killed at the 11-week time point as described above, the gastrocnemius muscles were immediately exposed and dissected bilaterally. Muscles were explanted accurately to prevent excessive bleeding, and the wet muscle weights were recorded. Muscle weights were normalized to the contralateral side to account for individual differences between animals.
Immunohistochemistry
At 11 weeks following surgery and cell implantation, nerve segments immediately distal to the injury site were excised, removed carefully from silicon tubes, and fixed overnight in 2% PFA in PBS. Samples were washed three times in PBS, cryoprotected in 30% sucrose, and embedded in optimal cutting temperature (OCT) compound (Sakura Fine technical Co.). Longitudinal sections 12 mm in length were cut with a cryostat (Leica Microsystems Inc.) at −23°C and mounted on Superfrost slides (Fisher Scientific). Sections were blocked/permeabilized with 2% BSA/0.3% Triton-X100 in PBS and incubated overnight in primary antibody. Antibodies used were antineurofilament NF200 (1:500, from Sigma) and FITC-IB4 (1:100, from Vector Laboratories). Following 3 washes with PBS, NF200-labeled slides were incubated with secondary antibody (Alexa Fluor anti–mouse IgG 488) for 3 hours. Slides were then washed and cover-slipped using Fluorosave reagent (Calbiochem) and viewed under a fluorescence microscope (Olympus BX51). Omission of primary or secondary antibody was used as negative control for the staining process. On average, 5 or 6 sections were stained for each regenerating nerve, and in each of these sections, a line was drawn perpendicular to the center of the section and antibody-positive profiles crossing this line were counted and summed, similar to previously described methods.18
Statistical Analysis
Differences between groups were analyzed using a 1-way ANOVA with a post hoc t-test. Statistical significance was accepted at p < 0.05 for pairwise comparisons. All results are presented as the mean ± SEM
Results Cells
Our first aim was to confirm phenotypic stability of nSCs and SKP-SCs after isolation from nerves or differentiation from dermis stem cells. Both cell types retained 425
A. Shakhbazau et al. characteristic Schwann cell morphology and were positive for p75 neurotrophin receptor, a canonical Schwann cell marker,30,47 after purification and expansion in culture (Fig. 1 and B). An in vitro myelination assay further demonstrated that both nSCs and SKP-SCs formed robust and abundant myelination profiles when seeded on dorsal root ganglion neurite network, evidence that purification and in vitro culture did not impair Schwann cells’ ability to myelinate (Fig. 1C and D). In the case of SKP-SCs, in vitro myelination testing also further confirmed successful differentiation of SKP cells into functional Schwann cell analogs. Expanded cells were next used for injections into transected rat sciatic nerves. Surgeries
All operated animals survived the surgeries, the operations were well tolerated, and wounds healed without signs of infection, pain, or discomfort. A total of 1 × 106 cells or 10 μl of medium alone was used for injection into transected sciatic nerve, as described in Methods. The intraoperative spread of cell/medium injection was monitored with Fast Green and was typically observed from the injection site to 1–1.2 cm distal, confined within the nerve with no significant leakage outside. Graft repair remained intact and regenerating cables formed in all repair groups across the prior injury and repair site,
characterized by areas of irregular fibers of connective tissue, invading macrophages, Schwann cells, debris, and regenerating axons. Interestingly, DiI-positive cells were observed 11 weeks postsurgery in both the nSC and the SKP-SC injection groups. This may not necessarily be a characteristic of cell survival (since the lipophilic dye may dilute when cells divide or myelinate37), but the finding definitely suggests that DiI CellTracker has a potential for effective long-term labeling (Fig. 1E and F). Electrophysiology
After the surgery the electrophysiological parameters (CMAP amplitude and NCV) were decreased at Weeks 5 and 8 in all injury groups in comparison with findings in the sham-surgery group, with the non-repair group predictably showing the lowest values. The cell therapy groups demonstrated a trend toward improved electrophysiological recovery, with the SKP-SC group being the only repair group to reach significant difference in NCV from non-repair by Week 8 after injury (p < 0.05). In agreement with our previous data,47 peak CMAP amplitude measurements also revealed a trend for improved recovery in the SKP-SC group (Fig. 2). However, the advantage of this group’s amplitude versus other operated groups did not reach statistical significance in this study.
Fig. 1. Representative images of nSCs (A, C, and E) and SKP-SCs (B, D, and F). A and B: p75 immunoprofiling of nSCs and SKP-SCs in culture: co-localization with nuclear stain Hoechst confirms population purity. C and D: In vitro myelination of dorsal root ganglion neurites by DiI-labeled nSCs and SKP-SCs. E and F: In vivo survival of DiI-labeled nSCs and SKP-SCs in the regenerating cable at Week 11 after injection. Original magnification ×100. Bar = 50 µm.
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Fig. 2. Electrophysiological parameters of nerve regeneration at Week 8 time point: NCV and normalized CMAP amplitude. *p < 0.05 vs non-repair group.
Thermal Sensitivity
In our sciatic nerve transection model, all injury groups developed, by Week 4, a significant decrease in sensitivity to thermal stimulation in the injured hind paw (p < 0.01) compared with baseline levels. This hypoalgesic state remained significantly higher compared with baseline in non-repair and medium groups at all time points analyzed and until the end of experiment (p < 0.01). However, cell therapy with 1 × 106 of either nSCs or SKP-SCs markedly reversed thermal responses to a normalgesic level, which was no longer significantly different from the baseline (p > 0.05) by Week 10 (Fig. 3).
Target Muscle Weights
To analyze potential myotrophic effects of cell therapy in our injury model, we weighed the gastrocnemius muscles 11 weeks after surgery and normalized them to their respective contralateral (left) sides to compare with the denervated controls (non-repair group) and shamoperated animals. Weights of the muscles in all repair groups were approximately 41%–44% of the uninjured side, significantly (p < 0.001) higher than in the non-repair group (12% of normal). The differences between the repair groups were, however, not significant (Fig. 4).
Fig. 3. Thermal perception recovery in the experimental groups (injured right limb normalized to contralateral limb). The nSC and SKP-SC groups demonstrate steady return to the normalgesic state by Week 10 (no significant difference from baseline, p > 0.05), while the medium and non-repair groups values remain significantly hyposensitive. **p < 0.01 versus baseline.
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Fig. 4. Gastrocnemius muscle weights at Week 11 (normalized to uninjured side).
Axonal Regeneration
To anatomically confirm our findings on sensory recovery, regenerated nerves were excised at the termination time point (Week 11), fixed in PFA, and sectioned longitudinally, as described above. Axonal outgrowth was analyzed distal to the injury site by immunohistochemistry for isolectin B4 (IB4) as a robust and specific marker of the sensory neuron subpopulation and for neurofilament NF200 as a more general marker. Both NF200 and IB4 immunohistochemistry revealed prominent regenerative improvement in both cell therapy groups (30%–70% increase in NF+ profiles and 70%–110% in sensory-specific IB4 staining in cell therapy groups compared with the medium alone group, Fig. 5).
Discussion
Cell therapies with Schwann cells and other cell types have gained increased attention in recent years as a potent tool to improve the outcomes of nerve recovery beyond those achieved with the current gold standard— direct microsurgical repair.25,29,33,34,49,55 It has been found that pre-differentiation of stem cells into the Schwann cell phenotype in vitro prior to injection results in remarkably better outcomes compared with the use of undifferentiated SKPs or mesenchymal stem cells.16,47,48 In previous studies, we and others showed that SKPs, predifferentiated in vitro into Schwann cells (SKP-SCs), survived in vivo and promoted robust axonal regrowth both in vitro and in acute and chronic models of rodent nerve injury.5,47,51 In the present study, we therefore moved on to assess the effects of nSC and SKP-SC therapy on the functional sensory recovery of an injured limb. Transection (Grade V) nerve injury results in a dramatic deficit in both motor and sensory functions of the injured limb.1,29,54 Observed changes in thermal responses vary from hyperalgesia in less severe to hypoalgesia in more severe injuries.14 In our study, non-repair and medium-treated groups predictably became hypoalgesic after injury and remained so until the end of the experiment (as late as Week 10 postsurgery). Present time points for the regeneration analysis were based on our earlier observations of peripheral nerve regeneration after transection injury.19,20,47 In sharp contrast to the medium injection group, animals that received cell therapy showed by 427
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Fig. 5. A: Quantitative analysis of mean (± SEM) numbers of axonal profiles stained for NF200 and IB4 at the Week 11 time point. The values obtained in the cell therapy groups are significantly different from those obtained in the medium group. *p < 0.05, **p < 0.01. B and C: Representative images of IB4 staining after nSC (B) and medium (C) injections. D and E: Representative images of NF200 staining after SKP-SC (D) and medium (E) injections. Original magnification ×100. Bar = 50 µm.
Week 10 clear signs of return to the normalgesic state according to a standard Hargreaves plantar test. The SKP-SC group showed maximum recovery potential, which was, however, closely followed by the nSC group. Reduced response latencies in the cell therapy groups were paralleled by increased signal conduction velocity, as showed by standard electrophysiological tests.19,47 Normalized thermal response latencies for the cell therapy groups were restored to levels indistinguishable from the pre-surgery baseline, but did not extend beyond normal thermal response to hyperalgesic state. 428
To complement our sensory response data histologically, immunohistochemical analysis was performed on nerve longitudinal sections using IB4 from Griffonia (Bandeiraea) simplicifolia, a long-known marker for sensory neurons in the peripheral nervous system.31,45 IB4 binds Ret tyrosine kinase7 which controls a number of ion channels (Nav1.8, Nav1.9, Trp family) and receptors (delta opioid, Mrg family) critical for the detection and transduction of sensory signals.12 IB4 was ranked the most specific and predictive marker of nociceptive C fibers, while NF200 is expressed in both A and C fibers.36 J Neurosurg / Volume 121 / August 2014
Sensory recovery after cell therapy in peripheral nerve repair Our immunohistochemical data suggest that cell therapy is highly beneficial for the successful regrowth of sensory IB4+ neurons, being (especially in the case of SKP-SCs) a part of a more general regenerative improvement (Fig. 5). This histoanatomical finding is well in line with our observations on electrophysiological and thermal sensation recovery. Our data on target muscle recovery/preservation suggest that cell therapies with either nSCs or SKP-SCs in the present injury model are apparently not additive to microsurgical repair. This is in contrast to our earlier findings in a chronic denervation model50 and the work of others,16 but the difference can be explained by the more severe degree of injury in the chronic model and in the work of Keilhoff et al.,16 leading to a more pronounced deficit in the no-cell group. Ipsilateral/contralateral ratios in Keilhoff’s cell therapy groups remained around 40%, consistent with our study (Fig. 4). Of note, cell therapy did not show any improvement in mechanical sensitivity (von Frey filament test) in our injury model (our unpublished observations; interestingly, thermal but not mechanical sensory recovery was also shown in a neuropathic pain model53). Assessment of skilled locomotion using the ladder-rung technique19,27,28 also did not reveal a significant additive effect of cell therapy after sciatic nerve transection, but we found a noticeable improvement with SKPSC treatment in a tibial nerve repair model.20 Observed differences in recovery between small and large peripheral nerves may lead to a better understanding of regenerative potential span for stem cell therapies. Contribution of nSCs and SKP-SCs to the regenerative process may result from a variety of effects, being very likely a convergence of many. The most obvious effects would be a competent myelination of the regenerating axons, together with the enhancement of Wallerian degeneration and accelerated clearance of inhibitory debris.30 Injected nSCs and SKP-SCs may also release a variety of growth factors and other molecules supportive for axonal regeneration and the remodeling of the microenvironment. SKP-SCs were previously shown by our lab to produce increased amounts of NGF, NT-3 and BDNF.47 This may also imply the cooperation of exogenous nSCs and SKP-SCs with the host cells. Schwann cells and their stem cell–derived counterparts may recruit into the injury site cells of other types that participate in debris clearance and tissue reconstruction and revascularization.48,49 The major novel findings in our study are: 1) Cell therapy with the use of nSCs and SKP-SCs is highly additive to (enhances) the regrowth of IB4+ sensory neurons as well as general regeneration of NF+ axons. 2) SKP-SCs and naïve Schwann cells facilitate paw sensory recovery in the regenerating sciatic nerve in an 11-week time frame. 3) SKP-SCs support functional recovery in peripheral nerve to an extent equal to or higher than achieved with nSCs. Our data therefore add to the evidence for significant potential of SKP-SCs as a tool for cell therapies in reconstructive neurosurgery. Acknowledgments We thank Joey Grochmal, Jeff Biernaskie, Bhagat Singh, and Laura Craig for their kind assistance.
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Disclosure This research was supported by CIHR grant #163322; by the Center for Excellence in Nerve Regeneration (partnership between the Hotchkiss Brain Institute, University of Calgary, and Integra LifeSciences); and by Alberta Innovates-Health Solutions. Author contributions to the study and manuscript preparation include the following. Conception and design: Shakhbazau, Midha. Acquisition of data: Shakhbazau, Mohanty, Kumar. Analysis and interpretation of data: Shakhbazau. Drafting the article: Shakhbazau. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Shakhbazau. Statistical analysis: Shakhbazau. Administrative/technical/material support: Midha. Study supervision: Midha. References 1. Alant JD, Kemp SW, Khu KJ, Kumar R, Webb AA, Midha R: Traumatic neuroma in continuity injury model in rodents. J Neurotrauma 29:1691–1703, 2012 2. Amoh Y, Li L, Campillo R, Kawahara K, Katsuoka K, Penman S, et al: Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves. Proc Natl Acad Sci U S A 102:17734–17738, 2005 3. Aronson JP, Mitha AP, Hoh BL, Auluck PK, Pomerantseva I, Vacanti JP, et al: A novel tissue engineering approach using an endothelial progenitor cell-seeded biopolymer to treat intracranial saccular aneurysms. Laboratory investigation. J Neurosurg 117:546–554, 2012 4. Belkas JS, Shoichet MS, Midha R: Axonal guidance channels in peripheral nerve regeneration. Oper Tech Orthop 14:190– 198, 2005 5. Biernaskie J, Sparling JS, Liu J, Shannon CP, Plemel JR, Xie Y, et al: Skin-derived precursors generate myelinating Schwann cells that promote remyelination and functional recovery after contusion spinal cord injury. J Neurosci 27:9545–9559, 2007 6. Biernaskie JA, McKenzie IA, Toma JG, Miller FD: Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nat Protoc 1:2803– 2812, 2006 7. Boscia F, Esposito CL, Casamassa A, de Franciscis V, Annunziato L, Cerchia L: The isolectin IB4 binds RET receptor tyrosine kinase in microglia. J Neurochem 126:428–436, 2013 8. Brushart TM: Nerve Repair. Oxford: Oxford University Press, 2011 9. Dezawa M: Central and peripheral nerve regeneration by transplantation of Schwann cells and transdifferentiated bone marrow stromal cells. Anat Sci Int 77:12–25, 2002 10. Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H: Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci 14:1771–1776, 2001 11. Eggers R, Hendriks WT, Tannemaat MR, van Heerikhuize JJ, Pool CW, Carlstedt TP, et al: Neuroregenerative effects of lentiviral vector-mediated GDNF expression in reimplanted ventral roots. Mol Cell Neurosci 39:105–117, 2008 12. Franck MC, Stenqvist A, Li L, Hao J, Usoskin D, Xu X, et al: Essential role of Ret for defining non-peptidergic nociceptor phenotypes and functions in the adult mouse. Eur J Neurosci 33:1385–1400, 2011 13. Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32:77–88, 1988 14. Huang C, Zou W, Lee K, Wang E, Zhu X, Guo Q: Different symptoms of neuropathic pain can be induced by different degrees of compressive force on the C7 dorsal root of rats. Spine J 12:1154–1160, 2012 15. Ide C: Peripheral nerve regeneration. Neurosci Res 25:101– 121, 1996
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34. Raheja A, Suri V, Suri A, Sarkar C, Srivastava A, Mohanty S, et al: Dose-dependent facilitation of peripheral nerve regeneration by bone marrow-derived mononuclear cells: a randomized controlled study. Laboratory investigation. J Neurosurg 117:1170–1181, 2012 35. Rahmatullah M, Schroering A, Rothblum K, Stahl RC, Urban B, Carey DJ: Synergistic regulation of Schwann cell proliferation by heregulin and forskolin. Mol Cell Biol 18:6245–6252, 1998 36. Ruscheweyh R, Forsthuber L, Schoffnegger D, Sandkühler J: Modification of classical neurochemical markers in identified primary afferent neurons with Abeta-, Adelta-, and C-fibers after chronic constriction injury in mice. J Comp Neurol 502:325–336, 2007 37. Shakhbazau A, Kawasoe J, Hoyng SA, Kumar R, van Minnen J, Verhaagen J, et al: Early regenerative effects of NGFtransduced Schwann cells in peripheral nerve repair. Mol Cell Neurosci 50:103–112, 2012 38. Shakhbazau A, Shcharbin D, Bryszewska M, Kumar R, Wobma HM, Kallos MS, et al: Non-viral engineering of skin precursor-derived Schwann cells for enhanced NT-3 production in adherent and microcarrier culture. Curr Med Chem 19:5572–5579, 2012 39. Shakhbazau A, Shcharbin D, Petyovka N, Goncharova N, Seviaryn I, Kosmacheva S, et al: Non-virally modified human mesenchymal stem cells produce ciliary neurotrophic factor in biodegradable fibrin-based 3D scaffolds. J Pharm Sci 101:1546–1554, 2012 40. Shakhbazau A, Shcharbin D, Seviaryn I, Goncharova N, Kosmacheva S, Potapnev M, et al: Dendrimer-driven neurotrophin expression differs in temporal patterns between rodent and human stem cells. Mol Pharm 9:1521–1528, 2012 41. Shakhbazau AV, Goncharova NV, Kosmacheva SM, Kartel NA, Potapnev MP: Plasticity of human mesenchymal stem cell phenotype and expression profile under neurogenic conditions. Bull Exp Biol Med 147:513–516, 2009 (Erratum in Bull Exp Biol Med 147:667, 2009) 42. Shea GK, Tsui AY, Chan YS, Shum DK: Bone marrow-derived Schwann cells achieve fate commitment—a prerequisite for remyelination therapy. Exp Neurol 224:448–458, 2010 43. Singh B, Xu QG, Franz CK, Zhang R, Dalton C, Gordon T, et al: Accelerated axon outgrowth, guidance, and target reinnervation across nerve transection gaps following a brief electrical stimulation paradigm. Laboratory investigation. J Neurosurg 116:498–512, 2012 44. Snyder EY, Senut MC: The use of nonneuronal cells for gene delivery. Neurobiol Dis 4:69–102, 1997 45. Tannemaat MR, Boer GJ, Eggers R, Malessy MJ, Verhaagen J: From microsurgery to nanosurgery: how viral vectors may help repair the peripheral nerve. Prog Brain Res 175:173– 186, 2009 46. Toma JG, Akhavan M, Fernandes KJ, Barnabé-Heider F, Sadikot A, Kaplan DR, et al: Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 3: 778–784, 2001 47. Walsh S, Biernaskie J, Kemp SW, Midha R: Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration. Neuroscience 164:1097– 1107, 2009 48. Walsh S, Midha R: Practical considerations concerning the use of stem cells for peripheral nerve repair. Neurosurg Focus 26(2):E2, 2009 49. Walsh S, Midha R: Use of stem cells to augment nerve injury repair. Neurosurgery 65 (4 Suppl):A80–A86, 2009 50. Walsh SK, Gordon T, Addas BM, Kemp SW, Midha R: Skinderived precursor cells enhance peripheral nerve regeneration following chronic denervation. Exp Neurol 223:221–228, 2010 51. Walsh SK, Kumar R, Grochmal JK, Kemp SW, Forden J, Mid-
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Sensory recovery after cell therapy in peripheral nerve repair ha R: Fate of stem cell transplants in peripheral nerves. Stem Cell Res (Amst) 8:226–238, 2012 52. Walwyn WM, Matsuka Y, Arai D, Bloom DC, Lam H, Tran C, et al: HSV-1-mediated NGF delivery delays nociceptive deficits in a genetic model of diabetic neuropathy. Exp Neurol 198:260–270, 2006 53. Wilson-Gerwing TD, Dmyterko MV, Zochodne DW, Johnston JM, Verge VM: Neurotrophin-3 suppresses thermal hyperalgesia associated with neuropathic pain and attenuates transient receptor potential vanilloid receptor-1 expression in adult sensory neurons. J Neurosci 25:758–767, 2005 54. Xu QG, Forden J, Walsh SK, Gordon T, Midha R: Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat. Laboratory investigation. J Neurosurg 112:890–899, 2010 55. Yang DY, Sheu ML, Su HL, Cheng FC, Chen YJ, Chen CJ,
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et al: Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid-derived mesenchymal stem cells regulated by stromal cell-derived factor-1a in a sciatic nerve injury model. Laboratory investigation. J Neurosurg 116:1357–1367, 2012
Manuscript submitted October 1, 2013. Accepted May 15, 2014. Please include this information when citing this paper: published online June 20, 2014; DOI: 10.3171/2014.5.JNS132132. Address correspondence to: Antos Shakhbazau, Ph.D., Department of Clinical Neuroscience, University of Calgary, HMRB 109-3330 Hospital Dr. NW, Calgary, AB T2N4N1, Canada. email: [email protected].
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Sensory recovery after cell therapy in peripheral nerve repair: effects of naïve and skin precursor-derived Schwann cells Laboratory investigation Antos Shakhbazau, Ph.D.,1,2 Chandan Mohanty, M.D.,1 Ranjan Kumar, M.Sc.,1–3 and Rajiv Midha, M.D., F.R.C.S.C.1,2 Department of Clinical Neuroscience, Faculty of Medicine, 2Hotchkiss Brain Institute, and 3Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada
1
Object. Cell therapy is a promising candidate among biological or technological innovations sought to augment microsurgical techniques in peripheral nerve repair. This report describes long-term functional regenerative effects of cell therapy in the rat injury model with a focus on sensory recovery. Methods. Schwann cells were derived from isogenic nerve or skin precursor cells and injected into the transected and immediately repaired sciatic nerve distal to the injury site. Sensory recovery was assessed at weeks 4, 7, and 10. Axonal regeneration was assessed at Week 11. Results. By Week 10, thermal sensitivity in cell therapy groups returned to a level indistinguishable from the baseline (p > 0.05). Immunohistochemistry at 11 weeks after injury showed improved regeneration of NF+ and IB4+ axons. Conclusions: The results of this study show that cell therapy significantly improves thermal sensation and the number of regenerated sensory neurons at 11 weeks after injury. These findings contribute to the view of skin-derived stem cells as a reliable source of Schwann cells with therapeutic potential for functional recovery in damaged peripheral nerve. (http://thejns.org/doi/abs/10.3171/2014.5.JNS132132)
Key Words • cell therapy • Schwann cells • skin precursors • stem cells • peripheral nerve • regeneration • thermal sensitivity
T
raumatic injury results in significant peripheral nerve damage in 2.8% of trauma patients, often leading to a long-term disability.32 Although the peripheral nervous system is commonly considered to possess a certain regenerative potential, existing microsurgical techniques have reached a plateau in terms of functional recovery.4 Further advances are expected from biological or technological innovations, such as improved nerve conduits, cell and gene therapy, electric stimulation, infusion of neurotrophic factors, and other techniques.3,11,24,39,41,43
Abbreviations used in this paper: CMAP = compound muscle action potential; IB4 = isolectin B4; NCV = nerve conduction velocity; nSC = nerve Schwann cell; PBS = phosphate-buffered saline; PFA = paraformaldehyde; SC medium = Schwann cell medium; SKP = skin precursor; SKP-SC = SKP-derived Schwann cell.
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In cell-based therapeutic approaches for peripheral nerve regeneration, Schwann cells are among the most appropriate candidates, being the myelinating glial cells in the peripheral nervous system and therefore key contributors to axonal regeneration and remyelination after injury.23,30 Guided by injury signals, Schwann cells divide and migrate to provide pathways for axonal regrowth by their alignment in the so-called bands of Büngner.15 Schwann cells also contribute to regeneration by secreting signaling molecules, cytokines, components of the extracellular matrix, cell adhesion molecules, and trophic factors.8,44 The therapeutic potential of autologous Schwann cells can be limited by lengthy expansion and the need This article contains some figures that are displayed in color online but in black-and-white in the print edition.
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A. Shakhbazau et al. to sacrifice a healthy nerve, resulting in neuropathic pain and donor site morbidity, and several groups have succeeded in deriving Schwann cells from bone marrow mesenchymal stem cells, adipose stem cells, and skin precursor (SKP) cells.2,9,10,17,21,26,42 Prior in vivo experiments from our group and others show some histological improvement after stem cell/Schwann cell therapies; however, the evidence for functional peripheral nerve recovery remains limited.16,47,48 In the present study, we aimed to evaluate the potential of SCs derived from the isogenic nerve and skin precursors for improved sensory recovery in a standard sciatic nerve transection model.
Methods Cell Isolation and Culture
Schwann cells were isolated from sciatic nerves of P2 Lewis rats according to established protocols22,47 with minor modifications. Briefly, sciatic nerves were excised, stripped of the epineurium, and cut into 1-mm2 pieces. Nerve segments were placed on poly-d-lysine–coated 35mm culture dishes in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.25 mg/ ml Fungizon for 3 days, allowing fibroblast migration out from the nerve. The medium was then changed to serumfree DMEM containing 1% N2, 10 ng/ml heregulin-1b, 4 mM forskolin (Schwann cell medium [SC medium]) for another 3 days to stimulate Schwann cell outgrowth. At Day 6, fragments were removed from the dish and explanted onto another 35-mm dish in SC medium with 2.5% FBS. The explant procedure was repeated until little outgrowth of fibroblasts was observed, then the explants were discarded and all dishes containing Schwann cells were purified in serum-free SC medium. Purity of nerve Schwann cell (nSC) cultures was assessed by p75 and GFAP immunolabeling and a heregulin exclusion test.35 Skin-derived precursor (SKP) cells were obtained from dermis of postnatal Day 2 Lewis rats and cultured according to published protocols.6,46,47 Briefly, skin from the dorsal torso was minced in HBSS (Gibco) on ice, incubated for 45 minutes in 0.1% collagenase XI at 37°C, dissociated mechanically, washed in cold DMEM, and passed through a 40-mm cell strainer. Filtrate was centrifuged at 1200 rpm, and the pellet was triturated and resuspended in culture medium (DMEM:F12 3:1, 100 U/ ml penicillin and 100 mg/ml streptomycin, 0.25 mg/ml Fungizon, 1× B27 supplement, 20 ng/ml EGF, and 40 ng/ ml bFGF [all from Gibco]). Skin-derived precursor cells were cultured and passaged as undifferentiated spherical aggregates at 37°C in a humidified atmosphere containing 5% CO2/95% air. The progenitor state of the cells within the aggregates was confirmed with nestin immunolabeling. To induce differentiation toward Schwann cells, aggregates were triturated and replated on poly-d-lysine/laminin– coated culture dishes (Corning) in SC medium (DMEM/ F12 supplemented with 100 U/ml penicillin and 100 mg/ ml streptomycin, 0.25 mg/ml Fungizon, 4 mM forskolin, 10 ng/ml heregulin-1b, and 1% N2 supplement [Gibco]), as described.6,40 Media were changed every 3–4 days. 424
Following 1–2 weeks incubation, cells appearing under phase contrast to have bipolar Schwann cell morphology were isolated with cloning cylinders and their fate specification was confirmed with GFAP and p75-NTR antibody staining. Cultures were further enriched for p75-NTR– positive cells by means of fluorescence activated cell sorting (FACS) using flow cytometer FACSAria II (Beckton Dickinson) and expanded in the SC medium until ~ 95% purity was achieved. These cells were subsequently referred to as SKP-derived Schwann cells, or SKP-SCs. SKP-SCs grow as adherent monolayers in static cultures. Both nSCs and SKP-SCs were then routinely maintained on plastic tissue culture flasks and plates (Falcon) at 37°C in a humidified atmosphere containing 5% CO2/95% air. In Vitro Myelination
To confirm the myelinating properties of cultured cells, SKP-SCs and nSCs were seeded onto the neurite network formed by 1-week culture of dorsal root ganglia from postnatal (P10) Lewis rats in Neurobasal medium containing B27 supplement, 10% FBS, 50 ng/ml NGF, 1% penicillin/streptomycin and 0.25 mg/ml Fungizon. Assay was performed in 8–well chamber slides (Nunc) coated with Matrigel. Host Schwann cells from dorsal root ganglia were killed by the addition of 7 mM cytosine arabinoside. Nerve Schwann cells (nSCs) and SKP-SCs were labeled with CellTracker CM-DiI (Invitrogen) prior to seeding. Co-cultures were maintained in SC medium containing 50 ng/ml NGF, and ascorbic acid (50 mg/ ml, Sigma) was added to induce myelination.42 The coculture was maintained for 2 weeks, then fixed with 2% paraformaldehyde (PFA) in PBS, blocked/permeabilized with 2% BSA/ 0.3% Triton-X100 in PBS and stained for myelin basic protein (MBP) with secondary antibodies with an emission wavelength of 488 nm. Animals
A total of 27 male Lewis rats weighing 225–250 g (Charles River) were used in this study (as detailed below). Animals were maintained in a temperature- and humidity-controlled environment with a 12-hour light/dark cycle. Food (Purina) and water were available ad libitum. Surgical interventions were carried out under inhalation anesthetic (Isoflurane, 99.9%, Halocarbon Laboratories), and pain control was provided by means of intraperitoneal or oral administration of buprenorphine (30 mg/kg). Surgical procedures were carried out aseptically, and standard microsurgical techniques were used with an operating microscope (Wild M651; Wild Leitz). Animals were killed at endpoint under deep anesthesia using an overdose of intracardiac Euthanol (Bimeda-MTC). All efforts were made to minimize suffering and animal numbers by using appropriate protocols. The protocol was approved and monitored by the University of Calgary animal care committee and adhered strictly to guidelines set by the Canadian Council on Animal Care.
Surgical Procedures
Surgery was performed unilaterally, in the right hindlimb, in all animals. The sciatic nerve was exposed at J Neurosurg / Volume 121 / August 2014
Sensory recovery after cell therapy in peripheral nerve repair mid-thigh level after splitting the biceps femoris muscle and transected ~ 1 cm proximal to the trifurcation with a small segment removed to create a 5-mm gap. All animals received an immediate entubulation repair within a silicon tube, inner diameter 1.5 mm, secured with 9-0 Prolene microsutures at each end, leaving a 5-mm gap between the proximal and distal stumps, using previously reported techniques.19,38 Immediately following repair, 10 ml of nSCs (n = 6) or SKP-SCs (n = 5) or carrier medium (n = 6) was injected using a 10-ml Hamilton syringe with a 30-gauge needle into the segment of transected sciatic nerve immediately distal to the graft, giving a total of 1 × 106 cells. Schwann cells were labeled 20 minutes prior to injection with CellTracker CM-DiI (Invitrogen) according to the manufacturer’s guidelines. Briefly, cells were trypsinized from culture vessels and resuspended to 1 × 106 cells/ml in medium containing 1 mM DiI staining solution. Following incubation for 20 minutes in the dark, cells were washed 3 times in DMEM and stored on ice until injection. To monitor cell/medium delivery, 0.1% FastGreen was added to the cells and medium immediately before injection. In a non-repair group (n = 5), severed nerve was not repaired and no injections were made. In a sham-surgery group (n = 5), nerve was exposed but not operated. Wounds were closed in layers, and the animals were housed as described above with appropriate analgesic administration (4 days), until they were killed at the 11-week time point. Electrophysiology
Electrophysiological recovery was assessed by measuring compound muscle action potential (CMAP) amplitude and latency. Electrical conductivity across the injury site was evaluated by stimulating the sciatic nerve proximally (at the sciatic notch) and measuring the CMAP amplitude evoked in the gastrocnemius muscle 5 and 8 weeks following the surgery. Animals were anesthetized with isoflurane as described above, stimulating bipolar electrodes were inserted percutaneously into the sciatic notch proximal to the nerve repair site, and a supramaximal stimulus was delivered to the sciatic nerve. Recording electrodes were placed longitudinally into the gastrocnemius muscle, and the ground electrode was placed subcutaneously in the base of the tail. Recordings were obtained using a VikingQuest electrodiagnostic system (Nicolet). The distance between the electrodes was measured, and latencies were used to calculate nerve conduction velocity (NCV).
Thermal Sensitivity Testing
Sensory recovery of the experimental animals was evaluated by analyzing the responses of injured and contralateral intact hindlimbs to thermal stimulus by the Hargreaves plantar test according to published methods.13,52,53 The animals were placed in a Plexiglas chamber atop a transparent glass surface that housed a radiant heat source and acclimatized for 5 minutes. The heat source was placed directly under the plantar surface of the hind paw and activated, starting a digital timer. The time count stopped when the paw was withdrawn from the heat source, indicating the paw withdrawal latency (seconds). J Neurosurg / Volume 121 / August 2014
Three or four evaluations for each paw were performed by an observer blinded to the nature of the groups. Intervals of 5 minutes were allowed between the readings to prevent paw sensitization, and withdrawal latency of the injured (right) limb was normalized to the uninjured side to generate latency ratio. Baseline levels were established 1 week before the injury/cell injection. Sensitivity testing resumed 4 weeks after surgery and was also performed at weeks 7 and 10 postinjury. Target Muscle Weight
As the animals were killed at the 11-week time point as described above, the gastrocnemius muscles were immediately exposed and dissected bilaterally. Muscles were explanted accurately to prevent excessive bleeding, and the wet muscle weights were recorded. Muscle weights were normalized to the contralateral side to account for individual differences between animals.
Immunohistochemistry
At 11 weeks following surgery and cell implantation, nerve segments immediately distal to the injury site were excised, removed carefully from silicon tubes, and fixed overnight in 2% PFA in PBS. Samples were washed three times in PBS, cryoprotected in 30% sucrose, and embedded in optimal cutting temperature (OCT) compound (Sakura Fine technical Co.). Longitudinal sections 12 mm in length were cut with a cryostat (Leica Microsystems Inc.) at −23°C and mounted on Superfrost slides (Fisher Scientific). Sections were blocked/permeabilized with 2% BSA/0.3% Triton-X100 in PBS and incubated overnight in primary antibody. Antibodies used were antineurofilament NF200 (1:500, from Sigma) and FITC-IB4 (1:100, from Vector Laboratories). Following 3 washes with PBS, NF200-labeled slides were incubated with secondary antibody (Alexa Fluor anti–mouse IgG 488) for 3 hours. Slides were then washed and cover-slipped using Fluorosave reagent (Calbiochem) and viewed under a fluorescence microscope (Olympus BX51). Omission of primary or secondary antibody was used as negative control for the staining process. On average, 5 or 6 sections were stained for each regenerating nerve, and in each of these sections, a line was drawn perpendicular to the center of the section and antibody-positive profiles crossing this line were counted and summed, similar to previously described methods.18
Statistical Analysis
Differences between groups were analyzed using a 1-way ANOVA with a post hoc t-test. Statistical significance was accepted at p < 0.05 for pairwise comparisons. All results are presented as the mean ± SEM
Results Cells
Our first aim was to confirm phenotypic stability of nSCs and SKP-SCs after isolation from nerves or differentiation from dermis stem cells. Both cell types retained 425
A. Shakhbazau et al. characteristic Schwann cell morphology and were positive for p75 neurotrophin receptor, a canonical Schwann cell marker,30,47 after purification and expansion in culture (Fig. 1 and B). An in vitro myelination assay further demonstrated that both nSCs and SKP-SCs formed robust and abundant myelination profiles when seeded on dorsal root ganglion neurite network, evidence that purification and in vitro culture did not impair Schwann cells’ ability to myelinate (Fig. 1C and D). In the case of SKP-SCs, in vitro myelination testing also further confirmed successful differentiation of SKP cells into functional Schwann cell analogs. Expanded cells were next used for injections into transected rat sciatic nerves. Surgeries
All operated animals survived the surgeries, the operations were well tolerated, and wounds healed without signs of infection, pain, or discomfort. A total of 1 × 106 cells or 10 μl of medium alone was used for injection into transected sciatic nerve, as described in Methods. The intraoperative spread of cell/medium injection was monitored with Fast Green and was typically observed from the injection site to 1–1.2 cm distal, confined within the nerve with no significant leakage outside. Graft repair remained intact and regenerating cables formed in all repair groups across the prior injury and repair site,
characterized by areas of irregular fibers of connective tissue, invading macrophages, Schwann cells, debris, and regenerating axons. Interestingly, DiI-positive cells were observed 11 weeks postsurgery in both the nSC and the SKP-SC injection groups. This may not necessarily be a characteristic of cell survival (since the lipophilic dye may dilute when cells divide or myelinate37), but the finding definitely suggests that DiI CellTracker has a potential for effective long-term labeling (Fig. 1E and F). Electrophysiology
After the surgery the electrophysiological parameters (CMAP amplitude and NCV) were decreased at Weeks 5 and 8 in all injury groups in comparison with findings in the sham-surgery group, with the non-repair group predictably showing the lowest values. The cell therapy groups demonstrated a trend toward improved electrophysiological recovery, with the SKP-SC group being the only repair group to reach significant difference in NCV from non-repair by Week 8 after injury (p < 0.05). In agreement with our previous data,47 peak CMAP amplitude measurements also revealed a trend for improved recovery in the SKP-SC group (Fig. 2). However, the advantage of this group’s amplitude versus other operated groups did not reach statistical significance in this study.
Fig. 1. Representative images of nSCs (A, C, and E) and SKP-SCs (B, D, and F). A and B: p75 immunoprofiling of nSCs and SKP-SCs in culture: co-localization with nuclear stain Hoechst confirms population purity. C and D: In vitro myelination of dorsal root ganglion neurites by DiI-labeled nSCs and SKP-SCs. E and F: In vivo survival of DiI-labeled nSCs and SKP-SCs in the regenerating cable at Week 11 after injection. Original magnification ×100. Bar = 50 µm.
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Fig. 2. Electrophysiological parameters of nerve regeneration at Week 8 time point: NCV and normalized CMAP amplitude. *p < 0.05 vs non-repair group.
Thermal Sensitivity
In our sciatic nerve transection model, all injury groups developed, by Week 4, a significant decrease in sensitivity to thermal stimulation in the injured hind paw (p < 0.01) compared with baseline levels. This hypoalgesic state remained significantly higher compared with baseline in non-repair and medium groups at all time points analyzed and until the end of experiment (p < 0.01). However, cell therapy with 1 × 106 of either nSCs or SKP-SCs markedly reversed thermal responses to a normalgesic level, which was no longer significantly different from the baseline (p > 0.05) by Week 10 (Fig. 3).
Target Muscle Weights
To analyze potential myotrophic effects of cell therapy in our injury model, we weighed the gastrocnemius muscles 11 weeks after surgery and normalized them to their respective contralateral (left) sides to compare with the denervated controls (non-repair group) and shamoperated animals. Weights of the muscles in all repair groups were approximately 41%–44% of the uninjured side, significantly (p < 0.001) higher than in the non-repair group (12% of normal). The differences between the repair groups were, however, not significant (Fig. 4).
Fig. 3. Thermal perception recovery in the experimental groups (injured right limb normalized to contralateral limb). The nSC and SKP-SC groups demonstrate steady return to the normalgesic state by Week 10 (no significant difference from baseline, p > 0.05), while the medium and non-repair groups values remain significantly hyposensitive. **p < 0.01 versus baseline.
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Fig. 4. Gastrocnemius muscle weights at Week 11 (normalized to uninjured side).
Axonal Regeneration
To anatomically confirm our findings on sensory recovery, regenerated nerves were excised at the termination time point (Week 11), fixed in PFA, and sectioned longitudinally, as described above. Axonal outgrowth was analyzed distal to the injury site by immunohistochemistry for isolectin B4 (IB4) as a robust and specific marker of the sensory neuron subpopulation and for neurofilament NF200 as a more general marker. Both NF200 and IB4 immunohistochemistry revealed prominent regenerative improvement in both cell therapy groups (30%–70% increase in NF+ profiles and 70%–110% in sensory-specific IB4 staining in cell therapy groups compared with the medium alone group, Fig. 5).
Discussion
Cell therapies with Schwann cells and other cell types have gained increased attention in recent years as a potent tool to improve the outcomes of nerve recovery beyond those achieved with the current gold standard— direct microsurgical repair.25,29,33,34,49,55 It has been found that pre-differentiation of stem cells into the Schwann cell phenotype in vitro prior to injection results in remarkably better outcomes compared with the use of undifferentiated SKPs or mesenchymal stem cells.16,47,48 In previous studies, we and others showed that SKPs, predifferentiated in vitro into Schwann cells (SKP-SCs), survived in vivo and promoted robust axonal regrowth both in vitro and in acute and chronic models of rodent nerve injury.5,47,51 In the present study, we therefore moved on to assess the effects of nSC and SKP-SC therapy on the functional sensory recovery of an injured limb. Transection (Grade V) nerve injury results in a dramatic deficit in both motor and sensory functions of the injured limb.1,29,54 Observed changes in thermal responses vary from hyperalgesia in less severe to hypoalgesia in more severe injuries.14 In our study, non-repair and medium-treated groups predictably became hypoalgesic after injury and remained so until the end of the experiment (as late as Week 10 postsurgery). Present time points for the regeneration analysis were based on our earlier observations of peripheral nerve regeneration after transection injury.19,20,47 In sharp contrast to the medium injection group, animals that received cell therapy showed by 427
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Fig. 5. A: Quantitative analysis of mean (± SEM) numbers of axonal profiles stained for NF200 and IB4 at the Week 11 time point. The values obtained in the cell therapy groups are significantly different from those obtained in the medium group. *p < 0.05, **p < 0.01. B and C: Representative images of IB4 staining after nSC (B) and medium (C) injections. D and E: Representative images of NF200 staining after SKP-SC (D) and medium (E) injections. Original magnification ×100. Bar = 50 µm.
Week 10 clear signs of return to the normalgesic state according to a standard Hargreaves plantar test. The SKP-SC group showed maximum recovery potential, which was, however, closely followed by the nSC group. Reduced response latencies in the cell therapy groups were paralleled by increased signal conduction velocity, as showed by standard electrophysiological tests.19,47 Normalized thermal response latencies for the cell therapy groups were restored to levels indistinguishable from the pre-surgery baseline, but did not extend beyond normal thermal response to hyperalgesic state. 428
To complement our sensory response data histologically, immunohistochemical analysis was performed on nerve longitudinal sections using IB4 from Griffonia (Bandeiraea) simplicifolia, a long-known marker for sensory neurons in the peripheral nervous system.31,45 IB4 binds Ret tyrosine kinase7 which controls a number of ion channels (Nav1.8, Nav1.9, Trp family) and receptors (delta opioid, Mrg family) critical for the detection and transduction of sensory signals.12 IB4 was ranked the most specific and predictive marker of nociceptive C fibers, while NF200 is expressed in both A and C fibers.36 J Neurosurg / Volume 121 / August 2014
Sensory recovery after cell therapy in peripheral nerve repair Our immunohistochemical data suggest that cell therapy is highly beneficial for the successful regrowth of sensory IB4+ neurons, being (especially in the case of SKP-SCs) a part of a more general regenerative improvement (Fig. 5). This histoanatomical finding is well in line with our observations on electrophysiological and thermal sensation recovery. Our data on target muscle recovery/preservation suggest that cell therapies with either nSCs or SKP-SCs in the present injury model are apparently not additive to microsurgical repair. This is in contrast to our earlier findings in a chronic denervation model50 and the work of others,16 but the difference can be explained by the more severe degree of injury in the chronic model and in the work of Keilhoff et al.,16 leading to a more pronounced deficit in the no-cell group. Ipsilateral/contralateral ratios in Keilhoff’s cell therapy groups remained around 40%, consistent with our study (Fig. 4). Of note, cell therapy did not show any improvement in mechanical sensitivity (von Frey filament test) in our injury model (our unpublished observations; interestingly, thermal but not mechanical sensory recovery was also shown in a neuropathic pain model53). Assessment of skilled locomotion using the ladder-rung technique19,27,28 also did not reveal a significant additive effect of cell therapy after sciatic nerve transection, but we found a noticeable improvement with SKPSC treatment in a tibial nerve repair model.20 Observed differences in recovery between small and large peripheral nerves may lead to a better understanding of regenerative potential span for stem cell therapies. Contribution of nSCs and SKP-SCs to the regenerative process may result from a variety of effects, being very likely a convergence of many. The most obvious effects would be a competent myelination of the regenerating axons, together with the enhancement of Wallerian degeneration and accelerated clearance of inhibitory debris.30 Injected nSCs and SKP-SCs may also release a variety of growth factors and other molecules supportive for axonal regeneration and the remodeling of the microenvironment. SKP-SCs were previously shown by our lab to produce increased amounts of NGF, NT-3 and BDNF.47 This may also imply the cooperation of exogenous nSCs and SKP-SCs with the host cells. Schwann cells and their stem cell–derived counterparts may recruit into the injury site cells of other types that participate in debris clearance and tissue reconstruction and revascularization.48,49 The major novel findings in our study are: 1) Cell therapy with the use of nSCs and SKP-SCs is highly additive to (enhances) the regrowth of IB4+ sensory neurons as well as general regeneration of NF+ axons. 2) SKP-SCs and naïve Schwann cells facilitate paw sensory recovery in the regenerating sciatic nerve in an 11-week time frame. 3) SKP-SCs support functional recovery in peripheral nerve to an extent equal to or higher than achieved with nSCs. Our data therefore add to the evidence for significant potential of SKP-SCs as a tool for cell therapies in reconstructive neurosurgery. Acknowledgments We thank Joey Grochmal, Jeff Biernaskie, Bhagat Singh, and Laura Craig for their kind assistance.
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Manuscript submitted October 1, 2013. Accepted May 15, 2014. Please include this information when citing this paper: published online June 20, 2014; DOI: 10.3171/2014.5.JNS132132. Address correspondence to: Antos Shakhbazau, Ph.D., Department of Clinical Neuroscience, University of Calgary, HMRB 109-3330 Hospital Dr. NW, Calgary, AB T2N4N1, Canada. email: [email protected].
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