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Experimental study of neural repair of the transected spinal cord using peripheral nerve graft
J Orthop Sci (2004) 9:605–612 DOI 10.1007/s00776-004-0833-0
Experimental study of neural repair of the transected spinal cord using peripheral nerve graft Satoru Fukunaga1, Sadao Sasaki2, Tokuhide Fu1, Hiroshi Yokoyama1, Iluho Lee1, Ikuko Nakagaki2, Seiki Hori2, Hiroomi Tateishi1, and Souji Maruo1 1 2
Department of Orthopaedic Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan Department of Physiology, Hyogo College of Medicine Nishinomiya, Japan
Abstract It has been reported that transected spinal cord shows signs of axonal regeneration after peripheral nerve (PN) graft. We studied the membrane excitability and ion distribution in axons from transected rat spinal cord 3 weeks after PN graft using the spinal cord evoked potential, electron probe X-ray microanalysis, and the patch-clamp technique. Axonal structures were also observed using conventional electron microscopy. At the Th11 level, laminectomy was performed (⫽control) and the left thoracic segments of the spinal cord 2 mm in length were excised (⫽nongrafted group). PN sections from 8-week-old male Wistar rats were grafted into the spinal cord gap (⫽PN-grafted group). The spinal cord evoked potential in the PN-grafted group partly recovered in contrast to that in the nongrafted group, which showed no recovery. Higher Na, Cl, and Ca peaks and lower K peaks in the PN-grafted group were demonstrated compared with those in the nongrafted group. In the PN-grafted group, a higher current signal appeared in the axonal membrane of the spinal cord, suggesting a greater membrane activity compared with that in the nongrafted group. Unlike the nongrafted group, in which no myelinated axons were found, demyelinated axons that were myelinated by Schwann cells from the grafted peripheral nerve were observed in the PN-grafted group. These findings suggested that Schwann cells from the transplanted PN contributed to the repair of the transected spinal cord. Key words Spinal cord injury · Peripheral nerve graft · Patchclamp technique · Electron probe X-ray microanalysis
Introduction Injured spinal cord has been thought to be incapable of regeneration.11 However, recent studies have demonstrated that axonal regeneration after spinal cord injury
Offprint requests to: S. Fukunaga Received: December 22, 2003 / Accepted: August 9, 2004
occurs when provided with a suitable substratum, such as peripheral nerve,10,22,27 embryonic spinal cord,2,4–7 neural stem cells,21 Schwann cells,3,29,30 inhibitor of obstructive protein,26 or supplement of neurotrophic factor.13,25 It has been reported that the transected spinal cord with peripheral nerve (PN) graft proceeds to show signs of axonal regeneration across the peripheral nerve graft,22 although the PN-grafted animals continued to show functional deficits, ranging from complete paralysis to abnormal walking.2 Although neural stem cell graft has been considered useful,21 neural stem cells tend to develop into neuroglia and oligodendrocytes.12 For regeneration of injured spinal cord, it is important to induce axonal regeneration along with a new network system to achieve recovery of electrophysiological function and morphology.15 The present study examined the effects of PN graft on membrane excitability of axons using the spinal cord evoked potential (SCEP), electron probe X-ray microanalysis (XMA), and the patchclamp technique. The axonal structure was also observed using conventional electron microscopy.
Materials and methods Surgical procedure Animal experiments were carried out according to the ethical committee of Hyogo College of Medicine and the U.S. Department of Health and Human Services guide for the care and use of laboratory animals. Thirty male Wistar rats (8 weeks old and weighing 220–250 g at surgery) were used. Under anesthesia induced by intraperitoneal injection of Nembutal, laminectomy was performed at the Th11 level (control). A left thoracic segment, 2 mm in length, of the spinal cord (Th11– Th12) was excised by micro-scissors after the dura was incised longitudinally and retracted laterally (nongrafted group; Fig. 1A). A sciatic nerve segment
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cut sharply from another 8-week-old Wistar rat was grafted carefully into the spinal cord defect without suturing (PN-grafted group; Fig. 1B). The dura was closed with 10–0 nylon. Evaluations Three weeks postoperatively, neural connection was studied using the following techniques. Spinal cord evoked potential (SCEP) SCEP were recorded using an evoked response analyzer (NEC Synax ER 1100; Tokyo, Japan), from ten rats each for rats in the control, nongrafted, and PN-grafted groups. The electrodes for SCEP measurement (platinum electrodes, ON98-017; Unique Medical, Osaka, Japan) consisted of stimulating electrodes (platinum electrodes, ON98-017; Unique Medical) 2 cm proximal to the lesion, and an active recording electrode (platinum electrodes; ON98–017; Unique Medical) on the epidural space 2 cm distal to and the same side as the spinal cord lesion to minimize the SCEP recording from an intact nerve. The reference electrode (silver electrodes, 1.0 mm in diameter) was placed on the paravertebral muscle. Stimulation was performed at an intensity of 0.6–1.5 mA, self-sustaining for 0.2 ms, repeated 30–50 times using a short wave with a frequency of 5 Hz. Histopathological examination Spinal cords from the control, nongrafted, and PNgrafted groups were removed en bloc, fixed in 10% formalin, then decalcified. Histological sections were stained with hematoxylin and eosin (H&E.). Electron probe X-ray microanalysis For electron probe X-ray microanalysis (XMA), 30 rats were used, 10 each from the control, nongrafted, and PN-grafted groups. Segments of the spinal cord were quickly frozen using a metal block cooled in liquid ni-
Fig. 1A,B. Micrographs illustrating the procedures for peripheral nerve (PN) graft. A Left thoracic segments (Th11–Th12: 2 mm) of the spinal cord are transected by micro-scissors (nongrafted group). B PN sections from 8-week-old Wistar rat are grafted into the spinal cord cavity (PN-grafted group). Bars 5 mm
trogen. Frozen sections (0.1 µm) were then cut on an ultramicrotome (MT-7000, Cryosectioning System CR21; RMC, Tucson, AZ, USA) maintained at ⫺130°C. The sections were mounted on 100-square Au grids, transferred to a freeze-drying apparatus (Freeze Dryer FL-100S; Nihon Freezer, Tokyo, Japan), and dried at ⫺120°C and 10⫺4 Torr overnight. After carbon coating (Vacuum Evaporator HUS-4; Hitachi, Tokyo, Japan), the specimens were analyzed using a transmission electron microscope with an EMAX-3770 X-ray microanalysis system (Horiba, Kyoto, Japan) operating at 75 kV. A probe current of 10⫺9–10⫺10 A was employed. The diameter of the probe was 0.1–1 µm and the detection time was 200 s. Scanning transmission electron micrographs and energy dispersive X-ray spectra of the axon and myelin of the spinal cord nerve fibers were obtained. The X-ray microanalyzer was also interfaced to an EMAX-3770 quantianalysis system. Special utility programs included those for statistical analysis, and details of the procedures have been published previously.1,17,19,23,24 Peak counts of various elements and the characteristic peak/continuum ratios were computed. The values were converted to concentrations using a program that we designed from data obtained by X-ray microanalysis of standard albumin frozen sections. Patch-clamp technique A segment of spinal cord 2 mm distal to the grafted lesion was harvested and placed on a petri dish in external solution (NaCl, 140 mM; KCl, 5 mM; CaCl2, 1 mM; MgCl2, 1.2 mM; HEPES-NaOH: pH 7.2, 5 mM). Patch pipettes were prepared in a two-stage process: pulling a pipette using a puller (PP-83; Narishige, Tokyo, Japan) and heat polishing the pipette tip using the microforge (MF-83; Narishige). About 1–2 µm of the pipette tip was used. The patch pipette was filled with external solution, then placed in the pipette holder, and finally stuck into the axon of that spinal cord forming a cellattached patch14 under a stereoscope (Fluovert; Leica,
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Heidelberg, Germany). Suction was applied, then the giga-seal formation was monitored. After stimulation with 20, 35, and 50 mV was applied by the stimulators (Axopatch 200A; Axon Instruments, Union City, CA, USA, and DigiData 1200 INTERFACE, Axon), the ion current of the myelin membrane was measured using the patch-clamp analyzer (Compaq Desk PRO GGM; Houston, TX, USA) under voltage clamp. Conventional electron microscopy Tissue blocks were fixed with 2.5% glutaraldehyde followed by 1% osmium tetroxide for approximately 3 h at 4°C. After dehydration through a graded series of ethanol, specimens were embedded in Epon. Semithin sections (1–0.5 µm) cut on an ultramicrotome (MT-7000, Cryosectioning System CR-21; RMC) were stained with toluidine blue, then examined with a light microscope. Ultrathin sections were obtained with a glass knife and double-stained with uranium acetate and lead citrate for transmission electron microscopy.
Fig. 2. Change in spinal cord evoked potential (SCEP). In the PN-grafted group (GG), the amplitude of the potential is restored to the 70% level of the normal counterpart. C, control group; NG, nongrafted group
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Results Spinal cord evoked potential (SCEP) Figure 2 shows the change in SCEP. In the nongrafted group (NG), recovery of the evoked potential was hardly noted. In the PN-grafted group (GG), however, the amplitude of the potential recovered to about 70% of the control value (25 ⫾ 5.6 µV vs. 36 ⫾ 3.1 µV, n ⫽ 10; P ⬍ 0.05) with longer latency than the control group (15 ⫾ 3.8 ms vs. 8.3 ⫾ 1.4 ms, n ⫽ 10; P ⬍ 0.05). Light microscopy Figure 3 shows light micrographs of sagittal sections of the resected spinal cord stained with H&E. In the nongrafted group (Fig. 3A), the spinal cord defect was covered by scar. In the PN-grafted group (Fig. 3B), the transplanted nerve was united with the spinal cord. Electron probe X-ray microanalysis (XMA) Figure 4 shows a scanning transmission electron micrograph of unfixed frozen section from a control rat. Spectra of axons and myelin membranes of spinal cord from a control rat are presented in Fig. 5. A typical X-ray spectra over the axons and myelin membranes from a control rat showed high peak K and peaks for Na, P, S, Cl, and Ca (Fig. 5, top). The spectrum over the myelin showed high peaks for P and K (Fig. 5, bottom). The calculated electrolyte concentrations of axons and myelin membranes in the three groups are summarized in Figs. 6 and 7. In the PN-grafted group, concentrations of Na, Cl, and Ca in the axons were significantly higher than those in the nongrafted group (Na: 20 ⫾ 2.51 vs. 16 ⫾ 1.26 mmol/kg wet weight; Cl: 37 ⫾ 10.1 vs. 28 ⫾ 8.23 mmol/kg wet weight; Ca: 0.44 ⫾ 0.12 vs. 0.32 ⫾ 0.25 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05), whereas K in the PN-grafted group was lower than that in the nongrafted group (K: 72 ⫾ 19.2 vs. 83 ⫾ 22.2 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05) (Fig. 6). Concentrations of Na, Cl, and Ca in the myelin membranes in the PNgrafted group were significantly higher than those in the nongrafted group (Na: 19 ⫾ 5.83 vs. 12 ⫾ 3.24 mmol/kg wet weight; Cl: 75 ⫾ 22.3 vs. 72 ⫾ 6.0 mmol/kg wet
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Fig. 3A,B. Sagittal sections of the spinal cord of the nongrafted group (left) and PN-grafted group (right) are counterstained with hematoxylin and eosin (H&E) methods. A The spinal cord cavity is covered by scar (S). B The transplanted nerve unites with the spinal cord. Bars 500 µm
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Fig. 4. Transmission electron micrograph of a freeze-dried 0.1-µm section of the spinal cord (control group). A, axon; M, myelin. Bar 1 µm
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Fig. 5. Energy dispersion X-ray spectra of axon (top) and myelin (bottom) in membranes of the spinal cord in the control group
Fig. 6. Calculated axonal electrolyte concentration. Na, Cl, and Ca peaks in the PN-grafted group are higher than those in the nongrafted group. The K peak in the PN-grafted group is lower than that in the nongrafted group
weight; Ca: 0.39 ⫾ 0.37 vs. 0.32 ⫾ 0.29 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05), while K in the PN-grafted group was lower than that in the non-grafted group (K: 89 ⫾ 23.9 vs. 93 ⫾ 25.1 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05) (Fig. 7). Patch-clamp technique Figure 8A shows change in the ion current on the axon membranes. There was no change in current in the
nongrafted group (NG), but a higher current signal was noted in the PN-grafted group (GG). Figure 8B shows an amplitude histogram of patch-clamp records from the control group, indicating a binomial distribution. Figure 8C shows an amplitude histogram in the PN-grafted group. Its peak was not clear, although it had dissociated. Figure 8D shows I–V curves of both groups. Although conductance in the PN-grafted group remained lower than that in the control group, the conductance had recovered to about 70%.
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Fig. 7. Calculated myelin electrolyte concentration. The findings are similar to those for the axons shown in Fig. 6
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D Fig. 8. A The trace shows a continuous current record. The top figure (C) shows an example of typical current seen in the control group. Although no change in the current is recognized in the nongrafted group (NG) (center), a higher current signal appears in the PN-grafted group (bottom). B Histogram
of the control group shows binomial distribution. C Histogram of the PN-grafted group. Its peak is not clear, although it had dissociated. D I–V curves of both groups. The conductance in the PN-grafted group recovered to about 70%
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myelinated by Schwann cells were found in the PNgrafted group (Fig. 10C).
Discussion
Fig. 9. Electron micrograph shows demyelinated axons in the nongrafted group. No myelinated axons are found. Arrows indicate demyelinated axons. Bar 1 µm
Regeneration after spinal cord injury has been the focus of various studies including grafting of embryonic spinal cord,2,4–7 neural stem cells,21 peripheral nerve,10,22,27 and other investigations. Although its usefulness has been proven and various experiments involving neural stem cell graft have been performed,21 it is not yet an established technique. Cheng et al. reported axonal regeneration and functional recovery using peripheral nerve graft.10 However, their findings have not been retested, because their experimental technique was complicated, and functional recovery was limited. In addition, changes in the elemental ion concentration and membranous current detected by XMA and patch-clamp technique after experimental PN graft have not previously been reported. Therefore, we further investigated the effect of peripheral nerve graft after spinal cord injury on membrane excitability using XMA and the patch-clamp technique. The axonal structure was also observed using conventional electron microscopy. Spinal cord evoked potential (SCEP)
A,B
C Fig. 10A–C. Electron micrographs show myelinated and demyelinated axons in the PN-grafted group. A A typical Schwann cell myelinating an axon (arrowheads). B Demyelinated axons (arrows). C Fascicles of axons, myelinated by Schwann cells (arrowheads), or demyelinated (arrows). Nu, nucleus. Bars 1 µm
Conventional electron microscopy Figure 9 shows an electron micrograph of a cross section of nerve fibers from the non-grafted group. Many demyelinated axons were found, but there was no myelinated axon. Figure 10 shows electron micrographs of a cross section of nerve fibers from the PN-grafted group. Figure 10A shows an axon myelinated by a Schwann cell, and Fig. 10B shows demyelinated axons. Many axons
Imaizumi et al. reported that electrophysiological examination of transected dorsal columns supports the conclusion that transplantation of Schwann cells is capable of facilitating long-distance regeneration of functionally remyelinated axons within the spinal cord.15 In the PN-grafted group, the amplitude of the potential recovered, and we thought that axons of the grafted nerve proceeded across the grafted lesion. However, the potential recovery was only 70%. Therefore, we believe that only limited numbers of axons proceeded across the grafted lesion. Electron probe X-ray microanalysis XMA allows observation of an organism using an electron microscope and enables X-ray detection analysis using an equipment attached to the electron microscope. The elemental ion concentration in a minute part can then be determined based on the X-ray energy spectrum.1,17,19,23,24 Moreover, observation of fresh specimens is possible by freezing the organism in liquid nitrogen. In the grafted group, Na, Cl, and Ca concentrations rose compared with those in the nongrafted group. Therefore, we speculated that in the grafted group, when membranous activity increased and continuous depolarization occurred, Na, Cl, and Ca flowed into the
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intracellular space from the extracellular space while K flowed out. Patch-clamp technique The patch-clamp technique is a method of recording the ion current passing along the cell membrane. Neher and Sakmann14 first demonstrated the activity of an ion channel molecule in 1976. Thereafter, this technique was applied to many cell systems following establishment of the giga-seal method, along with the development of some additional variations. In the present study, we succeeded in measuring the minute current in myelin by producing a patch pipette using a two-stage process and establishing a giga-seal. In the PN-grafted group, the membranous activity increased and some ion channels opened, then higher membranous current signals appeared. Conventional electron microscopy Generally, in the peripheral nerve, a Schwann cell wraps around one axon and the nucleus and cytoplasm of the Schwann cell exist along the axon. However, in the central nervous system, the cell body containing the nucleus of the oligodendrocyte separates from the nerve fiber to form myelin, and it is supposed that the nucleus does not absolutely appear in the outer tongue of myelin and dendrite, which combines with myelin.8,9,16,18,20,28 In the PN-grafted group, regenerating axons were found across the neural connection. Therefore, the myelin surrounding the demyelinated axons could be regarded as a grafted Schwann cell. Conclusion The effects of PN graft on membrane excitability were studied using the spinal cord evoked potential, electron probe X-ray microanalysis, and the patch-clamp technique. Spinal cords from the PN-grafted group were more active than those from the non-grafted group. When PN is grafted to the transected spinal cord, Schwann cells in the PN seem to facilitate repair of the transected spinal cord. Acknowledgments. We thank Drs. Y. Aoki, A. Arakawa, A. Miyawaki, and H. Itohara of Hyogo College of Medicine for technical advice and encouragement.
References 1. Aoki Y, Maruo S, Arakawa A, et al. Experimental study of spinal cord compression by epidural tumors using electron probe X-ray microanalysis and confocal laser scanning microscopy. J Orthop Sci 1997;2:434–41.
611 2. Asada Y, Kawaguchi S, Hayashi H, et al. Neural repair of the injured spinal cord by grafting: comparison between peripheral nerve segments and embryonic homologous structures as a conduit of CNS axons. Neurosci Res 1998;31:241–9. 3. Bamber NI, Li H, Lu X, et al. Neutrophins BDNF and NT-3 promote axonal re-entry into the distal host spinal cord through Schwann cell-seeded mini-channels. Eur J Neurosci 2001;13:257– 68. 4. Bernstein-Goral H, Bregman BS. Spinal cord transplants support the regeneration of axotomized neurons after spinal cord lesions at birth: a quantitative double-labeling study. Exp Neurol 1993; 123:118–32. 5. Bregman BS. Spinal cord transplants permit the growth of serotonergic axons across the site of neonatal spinal cord transection. Dev Brain Res 1986;34:265–79. 6. Bregman BS, Kunkel-Bagden E, McAtee M, et al. Extension of the critical period for developmental plasticity of the corticospinal pathway. J Comp Neurol 1989;282:355–70. 7. Bregman BS, Bernstein-Goral H. Both regenerating and latedeveloping pathways contribute to transplant-induced anatomical plasticity after spinal cord lesions at birth. Exp Neurol 1991;112: 49–63. 8. Bunge RP, Glass PM. Some observations on myelin-glial relationships and on the etiology of the cerebrospinal fluid exchange lesion. Ann NY Acad Sci 1965;122:15–28. 9. Bunge RP. Glial cells and the central myelin sheath. Phys Rev 1968;48:197–251. 10. Cheng H, Cao Y, Olson L. Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function. Science 1996;273: 510–3. 11. Feigin I, Geller EH, Wolf A. Absence of regeneration in the spinal cord of the young rat. J Neuropathol Exp Neurol 1951; 10:420–5. 12. Giovanini MA, Reier PJ, Eskin TA, et al. Characteristics of human fetal spinal cord grafts in the adult rat spinal cord: influences of lesion and grafting conditions. Exp Neurol 1997;148:525– 43. 13. Grill R, Murai K, Blesch A, et al. Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J Neurosci 1997;17: 5560–72. 14. Hamill OP, Marty A, Neher E, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cellfree membrane patches. Pflugers Arch 1981;391:85–100. 15. Imaizumi T, Lankford KL, Kocsis JD. Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord. Brain Res 2000; 854:70–8. 16. Kruger L, Maxwell DS. Electron microscopy of oligodendrocytes in normal rat cerebrum. Am J Anat 1966;118:411–35. 17. Maeda T, Hori S, Sasaki S, et al. Effects of tension at the site of coaptation on recovery of sciatic nerve function after neurorrhaphy: evaluation by walking-track measurement, electrophysiology, histomorphometry, and electron probe X-ray microanalysis. Microsurgery 1999;19:200–7. 18. Meinecke DL, de F Webster H. Fine structure of dividing astroglia and oligodendroglia during myelin formation in the developing mouse spinal cord. J Comp Neurol 1984;222:47–55. 19. Nakagaki I, Sasaki S, Shiguma M, et al. Distribution of elements in pancreatic exocrine cells determined by electron probe X-ray microanalysis. Pflugers Arch 1984;401:340–5. 20. Peters A. Further observations on the structure of myelin sheaths in the central nervous system. J Cell Biol 1964;20:281–96. 21. Pincus DW, Goodman RR, Fraser RAR, et al. Neural stem and progenitor cells; a strategy for gene therapy and brain repair. Neurosurgery 1998;42:858–68. 22. Richardson PM, McGuinness UM, Aguayo AJ. Peripheral nerve autografts to the rat spinal cord: studies with axonal tracing methods. Brain Res 1982;237:147–62.
612 23. Sasaki S, Nakagaki I, Mori H, et al. Intracellular carcium store and transport of elements in acinar cells of the salivary gland determined by electron probe X-ray microanalysis. Jpn J Physiol 1983;33:69–83. 24. Sasaki S, Sawada Y, Morita M, et al. Effects of benzothiazepine calcium blocker on electrolyte alteration in human ischemic and reperfused myocardium. J Mol Cell Cardiol 1994;26:1037–44. 25. Sayers ST, Khan N, Ahmed Y, et al. Preparation of brain-derived neurotrophic factor and neurotrophin-3-secreting Schwann cells by infection with a retroviral vector. J Mol Neurosci 1998;10:143– 60. 26. Schnell L, Schwab ME. Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature (Lond) 1990;343:269–72.
S. Fukunaga et al.: Neural repair by PN graft 27. Senoo E, Tamaki N, Fujimoto E, et al. Effects of prelesioned peripheral nerve graft on nerve regeneration in the rat spinal cord. Neurosurgery 1998;42:1347–56. 28. Waxman SG, Sims TJ. Specificity in central myelination: evidence for local regulation of myelin thickness. Brain Res 1984;292:179– 85. 29. Xu XM, Guenard V, Kleitman N, et al. Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord. J Comp Neurol 1995;351:145–60. 30. Xu XM, Zhang SX, Li H, et al. Regrowth of axons into the distal spinal cord through a Schwann-cell-seeded mini-channel implanted into hemisected adult rat spinal cord. Eur J Neurosci 1999;11:1723–40.
Experimental study of neural repair of the transected spinal cord using peripheral nerve graft Satoru Fukunaga1, Sadao Sasaki2, Tokuhide Fu1, Hiroshi Yokoyama1, Iluho Lee1, Ikuko Nakagaki2, Seiki Hori2, Hiroomi Tateishi1, and Souji Maruo1 1 2
Department of Orthopaedic Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan Department of Physiology, Hyogo College of Medicine Nishinomiya, Japan
Abstract It has been reported that transected spinal cord shows signs of axonal regeneration after peripheral nerve (PN) graft. We studied the membrane excitability and ion distribution in axons from transected rat spinal cord 3 weeks after PN graft using the spinal cord evoked potential, electron probe X-ray microanalysis, and the patch-clamp technique. Axonal structures were also observed using conventional electron microscopy. At the Th11 level, laminectomy was performed (⫽control) and the left thoracic segments of the spinal cord 2 mm in length were excised (⫽nongrafted group). PN sections from 8-week-old male Wistar rats were grafted into the spinal cord gap (⫽PN-grafted group). The spinal cord evoked potential in the PN-grafted group partly recovered in contrast to that in the nongrafted group, which showed no recovery. Higher Na, Cl, and Ca peaks and lower K peaks in the PN-grafted group were demonstrated compared with those in the nongrafted group. In the PN-grafted group, a higher current signal appeared in the axonal membrane of the spinal cord, suggesting a greater membrane activity compared with that in the nongrafted group. Unlike the nongrafted group, in which no myelinated axons were found, demyelinated axons that were myelinated by Schwann cells from the grafted peripheral nerve were observed in the PN-grafted group. These findings suggested that Schwann cells from the transplanted PN contributed to the repair of the transected spinal cord. Key words Spinal cord injury · Peripheral nerve graft · Patchclamp technique · Electron probe X-ray microanalysis
Introduction Injured spinal cord has been thought to be incapable of regeneration.11 However, recent studies have demonstrated that axonal regeneration after spinal cord injury
Offprint requests to: S. Fukunaga Received: December 22, 2003 / Accepted: August 9, 2004
occurs when provided with a suitable substratum, such as peripheral nerve,10,22,27 embryonic spinal cord,2,4–7 neural stem cells,21 Schwann cells,3,29,30 inhibitor of obstructive protein,26 or supplement of neurotrophic factor.13,25 It has been reported that the transected spinal cord with peripheral nerve (PN) graft proceeds to show signs of axonal regeneration across the peripheral nerve graft,22 although the PN-grafted animals continued to show functional deficits, ranging from complete paralysis to abnormal walking.2 Although neural stem cell graft has been considered useful,21 neural stem cells tend to develop into neuroglia and oligodendrocytes.12 For regeneration of injured spinal cord, it is important to induce axonal regeneration along with a new network system to achieve recovery of electrophysiological function and morphology.15 The present study examined the effects of PN graft on membrane excitability of axons using the spinal cord evoked potential (SCEP), electron probe X-ray microanalysis (XMA), and the patchclamp technique. The axonal structure was also observed using conventional electron microscopy.
Materials and methods Surgical procedure Animal experiments were carried out according to the ethical committee of Hyogo College of Medicine and the U.S. Department of Health and Human Services guide for the care and use of laboratory animals. Thirty male Wistar rats (8 weeks old and weighing 220–250 g at surgery) were used. Under anesthesia induced by intraperitoneal injection of Nembutal, laminectomy was performed at the Th11 level (control). A left thoracic segment, 2 mm in length, of the spinal cord (Th11– Th12) was excised by micro-scissors after the dura was incised longitudinally and retracted laterally (nongrafted group; Fig. 1A). A sciatic nerve segment
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cut sharply from another 8-week-old Wistar rat was grafted carefully into the spinal cord defect without suturing (PN-grafted group; Fig. 1B). The dura was closed with 10–0 nylon. Evaluations Three weeks postoperatively, neural connection was studied using the following techniques. Spinal cord evoked potential (SCEP) SCEP were recorded using an evoked response analyzer (NEC Synax ER 1100; Tokyo, Japan), from ten rats each for rats in the control, nongrafted, and PN-grafted groups. The electrodes for SCEP measurement (platinum electrodes, ON98-017; Unique Medical, Osaka, Japan) consisted of stimulating electrodes (platinum electrodes, ON98-017; Unique Medical) 2 cm proximal to the lesion, and an active recording electrode (platinum electrodes; ON98–017; Unique Medical) on the epidural space 2 cm distal to and the same side as the spinal cord lesion to minimize the SCEP recording from an intact nerve. The reference electrode (silver electrodes, 1.0 mm in diameter) was placed on the paravertebral muscle. Stimulation was performed at an intensity of 0.6–1.5 mA, self-sustaining for 0.2 ms, repeated 30–50 times using a short wave with a frequency of 5 Hz. Histopathological examination Spinal cords from the control, nongrafted, and PNgrafted groups were removed en bloc, fixed in 10% formalin, then decalcified. Histological sections were stained with hematoxylin and eosin (H&E.). Electron probe X-ray microanalysis For electron probe X-ray microanalysis (XMA), 30 rats were used, 10 each from the control, nongrafted, and PN-grafted groups. Segments of the spinal cord were quickly frozen using a metal block cooled in liquid ni-
Fig. 1A,B. Micrographs illustrating the procedures for peripheral nerve (PN) graft. A Left thoracic segments (Th11–Th12: 2 mm) of the spinal cord are transected by micro-scissors (nongrafted group). B PN sections from 8-week-old Wistar rat are grafted into the spinal cord cavity (PN-grafted group). Bars 5 mm
trogen. Frozen sections (0.1 µm) were then cut on an ultramicrotome (MT-7000, Cryosectioning System CR21; RMC, Tucson, AZ, USA) maintained at ⫺130°C. The sections were mounted on 100-square Au grids, transferred to a freeze-drying apparatus (Freeze Dryer FL-100S; Nihon Freezer, Tokyo, Japan), and dried at ⫺120°C and 10⫺4 Torr overnight. After carbon coating (Vacuum Evaporator HUS-4; Hitachi, Tokyo, Japan), the specimens were analyzed using a transmission electron microscope with an EMAX-3770 X-ray microanalysis system (Horiba, Kyoto, Japan) operating at 75 kV. A probe current of 10⫺9–10⫺10 A was employed. The diameter of the probe was 0.1–1 µm and the detection time was 200 s. Scanning transmission electron micrographs and energy dispersive X-ray spectra of the axon and myelin of the spinal cord nerve fibers were obtained. The X-ray microanalyzer was also interfaced to an EMAX-3770 quantianalysis system. Special utility programs included those for statistical analysis, and details of the procedures have been published previously.1,17,19,23,24 Peak counts of various elements and the characteristic peak/continuum ratios were computed. The values were converted to concentrations using a program that we designed from data obtained by X-ray microanalysis of standard albumin frozen sections. Patch-clamp technique A segment of spinal cord 2 mm distal to the grafted lesion was harvested and placed on a petri dish in external solution (NaCl, 140 mM; KCl, 5 mM; CaCl2, 1 mM; MgCl2, 1.2 mM; HEPES-NaOH: pH 7.2, 5 mM). Patch pipettes were prepared in a two-stage process: pulling a pipette using a puller (PP-83; Narishige, Tokyo, Japan) and heat polishing the pipette tip using the microforge (MF-83; Narishige). About 1–2 µm of the pipette tip was used. The patch pipette was filled with external solution, then placed in the pipette holder, and finally stuck into the axon of that spinal cord forming a cellattached patch14 under a stereoscope (Fluovert; Leica,
S. Fukunaga et al.: Neural repair by PN graft
Heidelberg, Germany). Suction was applied, then the giga-seal formation was monitored. After stimulation with 20, 35, and 50 mV was applied by the stimulators (Axopatch 200A; Axon Instruments, Union City, CA, USA, and DigiData 1200 INTERFACE, Axon), the ion current of the myelin membrane was measured using the patch-clamp analyzer (Compaq Desk PRO GGM; Houston, TX, USA) under voltage clamp. Conventional electron microscopy Tissue blocks were fixed with 2.5% glutaraldehyde followed by 1% osmium tetroxide for approximately 3 h at 4°C. After dehydration through a graded series of ethanol, specimens were embedded in Epon. Semithin sections (1–0.5 µm) cut on an ultramicrotome (MT-7000, Cryosectioning System CR-21; RMC) were stained with toluidine blue, then examined with a light microscope. Ultrathin sections were obtained with a glass knife and double-stained with uranium acetate and lead citrate for transmission electron microscopy.
Fig. 2. Change in spinal cord evoked potential (SCEP). In the PN-grafted group (GG), the amplitude of the potential is restored to the 70% level of the normal counterpart. C, control group; NG, nongrafted group
A
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Results Spinal cord evoked potential (SCEP) Figure 2 shows the change in SCEP. In the nongrafted group (NG), recovery of the evoked potential was hardly noted. In the PN-grafted group (GG), however, the amplitude of the potential recovered to about 70% of the control value (25 ⫾ 5.6 µV vs. 36 ⫾ 3.1 µV, n ⫽ 10; P ⬍ 0.05) with longer latency than the control group (15 ⫾ 3.8 ms vs. 8.3 ⫾ 1.4 ms, n ⫽ 10; P ⬍ 0.05). Light microscopy Figure 3 shows light micrographs of sagittal sections of the resected spinal cord stained with H&E. In the nongrafted group (Fig. 3A), the spinal cord defect was covered by scar. In the PN-grafted group (Fig. 3B), the transplanted nerve was united with the spinal cord. Electron probe X-ray microanalysis (XMA) Figure 4 shows a scanning transmission electron micrograph of unfixed frozen section from a control rat. Spectra of axons and myelin membranes of spinal cord from a control rat are presented in Fig. 5. A typical X-ray spectra over the axons and myelin membranes from a control rat showed high peak K and peaks for Na, P, S, Cl, and Ca (Fig. 5, top). The spectrum over the myelin showed high peaks for P and K (Fig. 5, bottom). The calculated electrolyte concentrations of axons and myelin membranes in the three groups are summarized in Figs. 6 and 7. In the PN-grafted group, concentrations of Na, Cl, and Ca in the axons were significantly higher than those in the nongrafted group (Na: 20 ⫾ 2.51 vs. 16 ⫾ 1.26 mmol/kg wet weight; Cl: 37 ⫾ 10.1 vs. 28 ⫾ 8.23 mmol/kg wet weight; Ca: 0.44 ⫾ 0.12 vs. 0.32 ⫾ 0.25 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05), whereas K in the PN-grafted group was lower than that in the nongrafted group (K: 72 ⫾ 19.2 vs. 83 ⫾ 22.2 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05) (Fig. 6). Concentrations of Na, Cl, and Ca in the myelin membranes in the PNgrafted group were significantly higher than those in the nongrafted group (Na: 19 ⫾ 5.83 vs. 12 ⫾ 3.24 mmol/kg wet weight; Cl: 75 ⫾ 22.3 vs. 72 ⫾ 6.0 mmol/kg wet
B
Fig. 3A,B. Sagittal sections of the spinal cord of the nongrafted group (left) and PN-grafted group (right) are counterstained with hematoxylin and eosin (H&E) methods. A The spinal cord cavity is covered by scar (S). B The transplanted nerve unites with the spinal cord. Bars 500 µm
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Fig. 4. Transmission electron micrograph of a freeze-dried 0.1-µm section of the spinal cord (control group). A, axon; M, myelin. Bar 1 µm
S. Fukunaga et al.: Neural repair by PN graft
Fig. 5. Energy dispersion X-ray spectra of axon (top) and myelin (bottom) in membranes of the spinal cord in the control group
Fig. 6. Calculated axonal electrolyte concentration. Na, Cl, and Ca peaks in the PN-grafted group are higher than those in the nongrafted group. The K peak in the PN-grafted group is lower than that in the nongrafted group
weight; Ca: 0.39 ⫾ 0.37 vs. 0.32 ⫾ 0.29 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05), while K in the PN-grafted group was lower than that in the non-grafted group (K: 89 ⫾ 23.9 vs. 93 ⫾ 25.1 mmol/kg wet weight; n ⫽ 50; P ⬍ 0.05) (Fig. 7). Patch-clamp technique Figure 8A shows change in the ion current on the axon membranes. There was no change in current in the
nongrafted group (NG), but a higher current signal was noted in the PN-grafted group (GG). Figure 8B shows an amplitude histogram of patch-clamp records from the control group, indicating a binomial distribution. Figure 8C shows an amplitude histogram in the PN-grafted group. Its peak was not clear, although it had dissociated. Figure 8D shows I–V curves of both groups. Although conductance in the PN-grafted group remained lower than that in the control group, the conductance had recovered to about 70%.
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Fig. 7. Calculated myelin electrolyte concentration. The findings are similar to those for the axons shown in Fig. 6
A
B,C
D Fig. 8. A The trace shows a continuous current record. The top figure (C) shows an example of typical current seen in the control group. Although no change in the current is recognized in the nongrafted group (NG) (center), a higher current signal appears in the PN-grafted group (bottom). B Histogram
of the control group shows binomial distribution. C Histogram of the PN-grafted group. Its peak is not clear, although it had dissociated. D I–V curves of both groups. The conductance in the PN-grafted group recovered to about 70%
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myelinated by Schwann cells were found in the PNgrafted group (Fig. 10C).
Discussion
Fig. 9. Electron micrograph shows demyelinated axons in the nongrafted group. No myelinated axons are found. Arrows indicate demyelinated axons. Bar 1 µm
Regeneration after spinal cord injury has been the focus of various studies including grafting of embryonic spinal cord,2,4–7 neural stem cells,21 peripheral nerve,10,22,27 and other investigations. Although its usefulness has been proven and various experiments involving neural stem cell graft have been performed,21 it is not yet an established technique. Cheng et al. reported axonal regeneration and functional recovery using peripheral nerve graft.10 However, their findings have not been retested, because their experimental technique was complicated, and functional recovery was limited. In addition, changes in the elemental ion concentration and membranous current detected by XMA and patch-clamp technique after experimental PN graft have not previously been reported. Therefore, we further investigated the effect of peripheral nerve graft after spinal cord injury on membrane excitability using XMA and the patch-clamp technique. The axonal structure was also observed using conventional electron microscopy. Spinal cord evoked potential (SCEP)
A,B
C Fig. 10A–C. Electron micrographs show myelinated and demyelinated axons in the PN-grafted group. A A typical Schwann cell myelinating an axon (arrowheads). B Demyelinated axons (arrows). C Fascicles of axons, myelinated by Schwann cells (arrowheads), or demyelinated (arrows). Nu, nucleus. Bars 1 µm
Conventional electron microscopy Figure 9 shows an electron micrograph of a cross section of nerve fibers from the non-grafted group. Many demyelinated axons were found, but there was no myelinated axon. Figure 10 shows electron micrographs of a cross section of nerve fibers from the PN-grafted group. Figure 10A shows an axon myelinated by a Schwann cell, and Fig. 10B shows demyelinated axons. Many axons
Imaizumi et al. reported that electrophysiological examination of transected dorsal columns supports the conclusion that transplantation of Schwann cells is capable of facilitating long-distance regeneration of functionally remyelinated axons within the spinal cord.15 In the PN-grafted group, the amplitude of the potential recovered, and we thought that axons of the grafted nerve proceeded across the grafted lesion. However, the potential recovery was only 70%. Therefore, we believe that only limited numbers of axons proceeded across the grafted lesion. Electron probe X-ray microanalysis XMA allows observation of an organism using an electron microscope and enables X-ray detection analysis using an equipment attached to the electron microscope. The elemental ion concentration in a minute part can then be determined based on the X-ray energy spectrum.1,17,19,23,24 Moreover, observation of fresh specimens is possible by freezing the organism in liquid nitrogen. In the grafted group, Na, Cl, and Ca concentrations rose compared with those in the nongrafted group. Therefore, we speculated that in the grafted group, when membranous activity increased and continuous depolarization occurred, Na, Cl, and Ca flowed into the
S. Fukunaga et al.: Neural repair by PN graft
intracellular space from the extracellular space while K flowed out. Patch-clamp technique The patch-clamp technique is a method of recording the ion current passing along the cell membrane. Neher and Sakmann14 first demonstrated the activity of an ion channel molecule in 1976. Thereafter, this technique was applied to many cell systems following establishment of the giga-seal method, along with the development of some additional variations. In the present study, we succeeded in measuring the minute current in myelin by producing a patch pipette using a two-stage process and establishing a giga-seal. In the PN-grafted group, the membranous activity increased and some ion channels opened, then higher membranous current signals appeared. Conventional electron microscopy Generally, in the peripheral nerve, a Schwann cell wraps around one axon and the nucleus and cytoplasm of the Schwann cell exist along the axon. However, in the central nervous system, the cell body containing the nucleus of the oligodendrocyte separates from the nerve fiber to form myelin, and it is supposed that the nucleus does not absolutely appear in the outer tongue of myelin and dendrite, which combines with myelin.8,9,16,18,20,28 In the PN-grafted group, regenerating axons were found across the neural connection. Therefore, the myelin surrounding the demyelinated axons could be regarded as a grafted Schwann cell. Conclusion The effects of PN graft on membrane excitability were studied using the spinal cord evoked potential, electron probe X-ray microanalysis, and the patch-clamp technique. Spinal cords from the PN-grafted group were more active than those from the non-grafted group. When PN is grafted to the transected spinal cord, Schwann cells in the PN seem to facilitate repair of the transected spinal cord. Acknowledgments. We thank Drs. Y. Aoki, A. Arakawa, A. Miyawaki, and H. Itohara of Hyogo College of Medicine for technical advice and encouragement.
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