Sciatic Nerve Regeneration Through Alginate With Tubulation or ...

JOURNAL OF NEUROTRAUMA Volume 18, Number 3, 2001 Mary Ann Liebert, Inc.

Sciatic Nerve Regeneration Through Alginate With Tubulation or Nontubulation Repair in Cat WU SUFAN,1 YOSHIHISA SUZUKI,1 MASAO TANIHARA,4 KATSUNORI OHNISHI,2 KYOKO SUZUKI,1 KATSUAKI ENDO,3 and YOSHIHIKO NISHIMURA1

ABSTRACT A novel material for nerve regeneration, alginate, was employed in both tubulation and nontubulation repair of a long peripheral nerve defect injury. Twelve cats underwent severing of the right sciatic nerve to generate a 50-mm gap, which was treated by tubulation repair (n 5 6) or nontubulation repair (n 5 6). In the tubulation group, a nerve conduit consisting of polyglycolic acid mesh tube filled with alginate sponge was implanted into the gap and the tube was sutured to both nerve stumps. In the nontubulation group, the nerve defect was repaired by a simple interpolation of two pieces of alginate sponge without any suture. The animals in both groups exhibited similar recovery of locomotor function. Three months postoperatively, successful axonal elongation and reinnervation in both the afferent and efferent systems were detected by electrophysiological examinations. Intracellular electrical activity was also recorded, which is directly indicative of continuity of the regenerated nerve and restoration of the spinal reflex circuit. Eight months after operation, many regenerated myelinated axons with fascicular organization by perineurial cells were observed within the gap, peroneal and tibial branches were found in both groups, while no alginate residue was found within the regenerated nerves. In morphometric analysis of the axon density and diameter, there were no significant differences between the two groups. These results suggest that alginate is a potent material for promoting peripheral nerve regeneration. It can also be concluded that the nontubulation method is a possible repair approach for peripheral nerve defect injury. Key words: alginate; nerve defect injury; nerve regeneration; nontubulation; tubulation

INTRODUCTION

R

has been an intractable problem for several decades. In longer defects exceeding 20 mm, it is difficult to carry out repair by direct suturing, and bridging with a nerve autograft is usually required (Susan, 1997). However, using autografts has some unavoidable drawbacks, such as sacrifice of healthy functioning tissue. Preferred alternatives inEPAIR OF NERVE DEFECT INJURY

clude tissue grafts made from bone (Gluck, 1880), artery (Kosaka, 1990), vein (Pu et al., 1999), muscle (Norris et al., 1988; Santo-Neto et al., 1998), or combined tissues (Di-Benedetto et al., 1998), and artificial conduits made of silicone (Lundborg et al., 1982b; Francel et al., 1997), collagen (Tong et al., 1994; Kitahara et al., 1999), polyglactin (Molander et al., 1983), polyglycolic acid (PGA) (Mackinnon and Dellon, 1990), or a combination of them (Chamberlain et al., 1998a,b; Kiyotani et al.,

1 Departments of

Plastic and Reconstructive Surgery, 2Neurology, and 3Physiology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, and 4Graduate School of Materials Science, Nara Institute of Science and Technology, Nara, Japan.

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SUFAN ET AL. 1996; Suzuki, Y., et al., 1998). Although a variety of synthetic materials have been used as nerve conduits, most of them have been reported to support axon outgrowth in nerve defects of less than 30 mm. In clinical practice, however, considerably longer nerve defects must be repaired and, thus we sought to develop a more effective material which we have reported in a preliminary fashion (Suzuki, Y., et al., 1999b). Application of tubular devices is a common strategy for repairing nerve defects, which requires fixation to the nerve stumps by suturing. Regenerating axons are expected to elongate in the correct direction toward the distal stump through the tubular structure. However, it is believed that the orientation of nerve regeneration is guided by signaling factors released from the distal stump and target organ after nerve injury (Lundborg et al., 1982a; Seckel et al., 1984). On the other hand, no matter how slight, suturing itself is a form of trauma, which has the potential for scar and neuroma formation (Manders et al., 1987). It has been reported that nontubulation repair obtains better regeneration results than tubulation repair in the central nervous system (Guest et al., 1997), but no similar research has been performed in peripheral nerve regeneration. These factors led us to consider whether tubulation is always necessary in the repair of a nerve defect, or whether in the presence of an appropriate nontubulation medium, such as alginate, the regenerating nerve could still find and arrive at the distal stump of the transected nerve. Alginate, extracted from brown seaweed, is a copolymer of beta-D-mannuronic acid and alpha-L -guluronic acid. As a well-established biocompatible material, alginate has had wide medical applications for several decades. It has served as an inhibitor of strontium absorption from the diet of humans and animals (Hesp and Ramsbottom, 1965), a dental impression material (Leung et al., 1998), a carrier for chrondrocyte culture and transplantation (Paige et al., 1995; Gregory et al., 1999), and a microencapsulated drug delivery system (Zhou et al., 1997). It has been also used as a skin wound dressing (Gilchrist and Martin, 1983; Steenfos and Agren, 1998; Suzuki, Y., et al., 1998; Suzuki, Y., et al., 1999a). Recently, alginate was found to be quite effective for nerve regeneration in previous studies from our laboratory (Suzuki, Y., et al., 1999b; Suzuki, K., et al., 1999). Based on these lines of evidence, the present paper provides more complete data on alginate in nerve regeneration. Moreover, this study proposes “nontubulation repair using alginate” as a new and promising method for repairs of long nerve defect injury, requires no cumbersome microsurgical techniques which may offer great advantage in the clinical setting.

MATERIALS AND METHODS Experimental Animals A total of 12 adult American domestic short hair cats of both sexes, weighing between 3 and 4 kg, were used in the study. They were housed individually in cages with free access to food and water. All procedures were performed according to “Principles of Laboratory Animal Care” advocated by the Kyoto University Animal Experiment Committee. All animals survived the experiment until they were sacrificed. In each animal, the right sciatic nerve was subjected to ligation and repair, while the left was kept intact, serving as a control. No forced rehabilitation of walking was performed during the study.

Preparation of Alginate Sponge and Tubular Device Ethylenediamine and water-soluble carbodiimide were dissolved well in 1% sodium alginate (Wako Pure Chemical Industries Co. Ltd., Osaka, Japan) aqueous solution, to develop a transparent gel cross-linked with covalent bonds. The gel was washed with 2.5 mM calcium chloride and 143 mM sodium chloride to remove any residual contaminants and unreacted reagents. The gel was freeze-dried to become alginate sponge with a thickness of 3 mm, which was used for the nontubulation repair method. For the tubulation repair method, the gel was placed into a polyglycolic acid mesh tube measuring 4 mm in diameter, followed by freeze-drying treatment in a vacuum at 250°C for 12 h to become a tubular alginate conduit. The alginate conduit consisted of alginate sponge coated with polyglycolic acid mesh, which could be sutured to nerve stumps. These devices were sterilized by 25 kGy cobalt-60 g-irradiation. A scanning electron micrograph of alginate sponge is shown in Figure 1.

Operative Procedure All cats were anesthetized with an intramuscular injection of ketamine hydrochloride at 50 mg/kg. After shaving and preparing the skin with 10% povidone iodine, the right sciatic nerve was exposed through a posterior thigh incision, followed by nerve transection between the sciatic nerve notch and the distal bifurcation to create a 50-mm gap. The animals were divided into two groups for the tubulation (n 5 6) and nontubulation methods (n 5 6), respectively. In the tubulation group, the alginate tubular device with a length of 50 mm was implanted into the gap and fixed to both nerve stumps by suturing its PGA mesh to the epineurium with 7-0

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SCIATIC NERVE REGENERATION THROUGH ALGINATE electrodes (diameter, 0.5 mm) filled with 1M potassium citrate. Animals were immobilized with pancuronium bromide under artificial ventilation. The body temperature was kept within the normal range (36–37°C).

Histological Analysis

FIG. 1.

Scanning electron micrograph of the alginate sponge.

nylon suture (Proline®; Ethicon Inc., Somerville, NJ) (Fig. 2A). In the nontubulation group, the nerve gap was simply filled with two pieces of sponge with the same size of 60 3 10 3 3 mm, which were sandwiched between the proximal and distal nerve stumps (Fig. 2B). Muscle and skin were then closed with 5-0 sutures. The left sciatic nerve was kept intact as a control in all animals.

Electrophysiological Evaluation

The animals were sacrificed at an average of 10 months after operation. Under deep anesthesia with intramuscular injection of pentobarbital sodium at a dose of 500 mg/kg, the animals were perfused transcardially with 0.5 L phosphate-buffered solution (PB), pH 7.4, followed by 0.5 L of 1% glutaraldehyde (GA) in 0.1 M phosphate-buffered saline (PBS), pH 7.4. After sacrifice, the operated position was reexposed, and the regenerated nerve was observed and photographed. Specimens were taken from the middle position of the regenerated sciatic nerve, and from the peroneal and tibial nerves 5 mm distal to the bifurcation. They were fixed in 2% GA for 2 h and rinsed with 0.1 M PBS, and then postfixed in 1% osmium tetroxide for 2 h, and dehydrated in a series of ethanol and propylene oxide. The specimens were finally embedded in epon. For light microscopy, 1-mm-thick sections were stained with 1% toluidine blue solution, while 80-nm-thick sections were double-stained with uranyl acetate and lead nitrate for transmission electron microscopy (H-7000® ; Hitachi, Tokyo, Japan).

Morphometric Analysis

Electrophysiological evaluations were performed monthly using a Nicolet Viking Quest® device as described previously (Nicolet Biomedical Inc., Madison, WI) (Suzuki, Y., et al., 1999b). Animals were anesthetized by intramuscular injection of ketamine hydrochloride at a dose of 50–80 mg/kg. Electrical activity was recorded with percutaneously inserted stainless steel bipolar needle electrodes in both sides of the hindlimb. Compound muscle action potentials (CMAP) were recorded from the gastrocnemius muscle following single stimulation in the proximal sciatic nerve at the level of the sciatic notch, whereas somatosensory evoked potentials (SEP) were recorded as an average of 50 responses from the skull over the somatosensory cortex following stimulation of tibial nerve. The recovery index was calculated by the following formula: Recovery index 5 peak latency of regenerated side/ peak latency of contralateral normal side Intracellular recordings were performed from the lumbosacral motoneurons and interneurons using glass micro-

The myelinated fibers were analyzed in nerve crosssections stained with toluidine-blue from the mid-portion of the regenerated sciatic nerve, peroneal nerve, and tibial nerve. The images of nerve sections were captured to a computer (Apple Macintosh®) via a digital video cam-

FIG. 2. Illustration of tubulation and nontubulation repair methods. In the tubulation method, an alginate conduit was implanted into nerve gap and fixed to both nerve stumps by suturing (A). In the nontubulation method, the nerve gap was simply filled with two pieces of sponge, which were sandwiched between the proximal and distal nerve stumps (B).

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SUFAN ET AL. paired cat indicated that activation of regenerated nerves had evoked antidromic spike potentials and excitatory postsynaptic potentials (EPSP) in the hindlimb motoneurons (Fig. 4), which is direct evidence of functional restoration of the spinal reflex arcs.

Histologic Observation

FIG. 3. Electrophysiologic recording of compound muscle action potentials (CMAP) and somatosensory evoked potentials (SEP, average of 50 responses, arrows indicate peak latencies) 3 months after operation. CMAP and SEP showed remarkable restoration in both the tubulation and nontubulation groups. Compared with normal nerves, the alginate-repaired nerves showed satisfactory recovery.

era (Fuji, Tokyo, Japan) connected to a light microscope. In each animal, three images (an approximate area of 12 3 104 mm 2 ) of one respective cross-section of sciatic, tibial, and peroneal nerves were randomly selected and retouched with Adobe Photoshop® to suit the image analysis. The density and diameter of the myelinated axons was determined using public domain NIH computer software. The standardized diameter of each nerve fiber was calculated as the diameter of a circle of the same perimeter so as to minimize the bias caused by tissue shrinkage during the fixation procedure (Karnes et al., 1977). For controls, three normal cat sciatic, tibial and peroneal nerves were randomly selected and subjected to the same analysis. The number and diameter distribution of myelinated axons were calculated, and the mean is shown (see Fig. 7 below).

Macroscopically, the regenerated nerve cable was observed bridging the 50-mm gap in all animals in both groups, which appeared thinner and wider than the normal sciatic nerve (Fig. 5). In both groups 8 months after operation, a large number of well-myelinated axons were observed both in mid-portion of the gap and in the tibial and peroneal nerves (Fig. 6). The regenerated myelinated fibers were smaller in diameter and thinner in myelination compared to normal nerve, and were organized as small fascicular structures by surrounding perineurial cells. Numerous unmyelinated fibers were also observed by electron microscopy. Neither residual fragments of alginate nor inflammatory cells were found in the specimens.

Morphometric Analysis The results of morphometric analysis are shown in Table 2. The axonal density (axon number/mm2 ) of myelinated fibers was similar in the two groups, which was higher than that of normal nerves. In the distribution of axon diameter, the normal nerve had two peaks at 4–6 mm and 14–16 mm, whereas the regenerated nerve had only one peak at 4 mm (Fig. 7). Compared with normal axons, the regenerated axons showed a smaller diameter and higher density.

DISCUSSION The major finding of the present study was that the non-tubulation repair method using alginate also pro-

RESULTS

TABLE 1. RECOVERY INDEX OF REGENERATED NERVES 3 MONTHS A FTER SURGERY

Electrophysiological Evaluation The earliest electrophysiologic restoration was found 2 months after operation in both groups. At 3 months after surgery, CMAP and SEP showed remarkable restoration in both the tubulation and nontubulation groups. Regenerated nerves showed almost normal amplitude and latency compared with normal nerves (Fig. 3). The recovery index of the respective potentials are shown in Table 1. The intracellular recordings of the non–tubulation-re-

Nontubulation (n 5 6) Tubulation (n 5 6)

CMAP

SEP

0.866 6 0.21 0.922 6 0.12

1.02 6 0.21 1.06 6 0.37

Recovery index 5 peak latency of regenerated nerve/peak latency of normal nerve. CMAP, compound muscle action potentials; SEP, somatosensory evoked potentials.

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SCIATIC NERVE REGENERATION THROUGH ALGINATE

FIG. 4. Intracellular potentials and evoked potentials recorded from a nontubulation-repaired cat 3 months postoperatively. (A) Antidromic spike potentials (M-spikes) followed by slow excitatory postsynaptic potentials (EPSP) recorded intracellularly from a hindlimb motoneuron with stimulation of the tibial nerve. (B) Repetitive spike discharges of a presumed Renshow cell in the ventral horn following stimulation of the tibial nerve. (C,D) Somatosensory evoked potentials (SEP) recorded in the medullary dorsal column nucleus (C) and the cerebral cortex (D) with stimulation of the tibial nerve. (E) Electromyograms evoked in the triceps sural muscle with stimulation of the sciatic notch. (F) Compound nerve action potentials (CNAP) recorded at the sciatic notch following stimulation of the tibial nerve. Upward deflections show positivity in all recordings.

moted transected nerves to regenerate across a 50-mm gap, and yielded results comparable to those with the tubulation repair method. Electrophysiological examination showed that the nontubulation repair method produced remarkable restoration of function. Stimulation of the regenerated nerve evoked sensory responses in the cerebral cortex and dorsal column nuclei, which were comparable to those in normal preparations. Furthermore, with the intracellular recording method, stimulation of the regenerated tibial nerve evoked antidromic and orthodromic responses in motoneurons, indicating functional restoration of the spinal reflex, which suggested that the transected nerve had undergone adequate regeneration. In addition, the histologic findings indicated that the nontubulation group obtained similar regeneration to the tubulation group. These results suggest that under appropriate conditions, tubulation and suturing are not necessary for repair of a nerve defect injury. The correct orientation of nerve regeneration toward the distal stump is probably due to some inherent mechanisms of the body, such as guidance by

FIG. 5. Anatomic appearance of tubulation (A) and nontubulation (B) repaired nerves 8 months after operation. In both groups, the regenerated nerves are observed bridging the gap. In the middle position, regenerated nerves appear thinner and wider than the normal sciatic nerve (C). The peroneal and tibial nerves show similar appearances to normal nerves (arrows toward distal position).

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SUFAN ET AL.

FIG. 6. Histologic appearances 8 months after operation. A large number of well-myelinated axons were observed both in the mid-portion of the gap (B, non-tubulation; C, tubulation) and in the tibial (D, nontubulation; E, tubulation) and peroneal nerves (F, nontubulation; G, tubulation). The regenerated myelinated fibers were smaller in diameter and thinner in myelination compared to the normal nerve (A), and were organized as small fascicular structures by surrounding perineurial cells. Numerous unmyelinated fibers were also observed by electron microscopy (H, nontubulation; I, tubulation). No obvious difference was found between the two groups. Neither residual fragments of alginate nor inflammatory cells were found in the specimens. Bar 5 50 mm, toluidine blue stain (A–G); 2 mm, double-stained with uranyl acetate and lead nitrate (H,I).

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FIG. 6.

Continued.

factors released from the distal stump, rather than the use of tubulation or suturing. Although there are some reports of sutureless techniques in nerve repair, other fixation procedures have been employed instead of suturing, such as the use of a nerve coupler (Marshall et al., 1989), laser (Trickett et al., 1997), or fibrin glue (Hamm et al., 1988; Maragh et al., 1990). To date, there have been few reports of nontubulation and nonfixation repair of nerve defect injury. It was reported that the maximal length of nerve defect for regeneration is about 20 mm in rodents (Susan, 1997) and that in rats axons usually fail to regenerate over a 15-mm gap and in mice axons usually fail to regenerate over a gap of more than 6 mm (Labrador et al., 1998). Although there are some reports of successful regeneration beyond this limit, such as 25 mm in the rat (Foidart et al., 1997), little success was achieved over a gap longer than 50 mm, even using muscle autografts in humans (Calder and Norris, 1993). For a cat, 50-mm gap in sciatic nerve is also too long to obtain a recovery without treatment. In our preliminary experiments, several cats were subjected to an empty 50-mm gap in their sciatic

nerves, and neither histologic nor electrophysiologic evidence of regenerated nerve across the gap was found. The results of the present study suggest that alginate is an excellent potential material for peripheral nerve regeneration. Bioabsorbability is the first advantage of using alginate in nerve regeneration. It is now widely accepted that absorbable materials are more suitable for nerve regeneration (Brunelli et al., 1994), and that nonabsorbable conduits have little clinical application (Terris and Fee, 1993). The alginate we developed is a bioabsorbable substance, which disappears within a few months after implantation into tissues. In our previous studies, alginate resulted in less residual debris when used for skin wound dressing (Suzuki, Y., et al., 1998; Suzuki, Y., et al., 1999a) and nerve regeneration (Suzuki, Y., et al., 1999b; Suzuki, K., et al., 1999). In the present study also, as seen in Figure 6, no alginate debris could be detected. The second merit is that alginate was demonstrated to have little inhibitory effect on cells in vitro and to cause little foreign body reaction in tissues in vivo (Suzuki, Y., et al., 1998; Suzuki

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SUFAN ET AL. TABLE 2. MYELINATED AXONS DENSITY 8 MONTHS AFTER SURGERY (COUNTS /MM 2) Nontubulation

Frequency distribution (%)

Normal

35

Tubulation

Sciatic nerve 6,224 6 453 4,649 6 800 3,875 6 494 Peroneal nerve 5,384 6 260 10,189 6 1068 9,708 6 237 Tibial nerve 5,536 6 475 9,729 6 943 9,624 6 288 Data are mean 6 SD.

25 20 15 10 5 0

normal

non-tubulation

tubulation

Axon diameter of sciatic nerve

Frequency distribution (%)

35 30

0-2u 2-4u 4-6u 6-8u 8-10u 10-12u 12-14u 14-16u 16-18u

25 20 15 10 5 0

normal

non-tubulation

tubulation

Axon diameter of tibial nerve 35 Frequency distribution (%)

Y., et al., 1999a), and it thus can be employed in tissue regeneration. Moreover, as an extract from seaweed, compared with those from animals, it has a lower chance of carrying transferable diseases and is therefore safer for use in humans. One mechanism by which alginate sponge promotes nerve regeneration is that alginate may be a suitable environment for regenerating axons and endogenous neurotrophic factors. The orientation of nerve regeneration is guided by signaling factors released from the distal stump and target organ after nerve injury (Lundborg et al., 1982a; Seckel et al., 1984). The multiple pore structure of alginate (Fig. 1) is probably a suitable pathway for the passage of neurotrophic factors, as well as for axon elongation. At the early stage, alginate sponge becomes a gel-like substance, within which regenerated axons were observed (data not shown). In addition, alginate may provide a scar-free space for elongating axons during its process of biodegradation. In the present study, few fibroblasts were found within the regenerated nerve. The tubulation method, usually suturing a tube-like nerve conduit to the nerve stumps, is a traditional procedure that is mainly used for the study of peripheral nerve regeneration. Alginate could probably play the same roles as a tubulation nerve conduit, providing a pathway for regenerating axons and neurotrophic factors, and preventing surrounding tissue invasion. Our successful results imply that suturing and a tubular structure are not always necessary for peripheral nerve regeneration. In summary, the present study suggests that alginate is a potent material for promoting peripheral nerve regeneration, and that the nontubulation method is a promising approach for repair of peripheral nerve defects. Clinically, this finding will provide another possible choice of treatment of nerve defects, especially in certain anatomic positions where it is difficult to perform suturing. Although alginate showed beneficial effects on nerve regeneration in the present study, its exact mechanisms are still unclear. Further research will be needed to disclose the precise mechanisms.

0-2u 2-4u 4-6u 6-8u 8-10u 10-12u 12-14u 14-16u 16-18u

30

30

0-2u 2-4u 4-6u 6-8u 8-10u 10-12u 12-14u 14-16u 16-18u

25 20 15 10 5 0

normal

non-tubulation

tubulation

Axon diameter of peroneal nerve

FIG. 7. Distribution of myelinated axon diameter of sciatic, tibial, and peroneal nerves (normal nerve group, n 5 3; tubulation group, n 5 6; and nontubulation group, n 5 5). The normal nerve has two peaks located at 4–6 mm and 14–16 mm, whereas the regenerated nerves have only one peak at around 4 mm. Regenerated nerves show similar distribution of myelinated axon diameter in sciatic, tibial, and peroneal nerves.

ACKNOWLEDGMENTS We thank Dr. Fujioka for his assistance in producing electron microscopic photographs. This study was supported in part by a grant for “Research for the Future” (JSPS-RFTF 96100203) from the Japan Society for the Promotion of Science, by grants for scientific research (no. 11307037, 11877240) from the Japanese Ministry of Education and by a grant from Mitsui Life Social Welfare Foundation.

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Address reprint requests to: Yoshihisa Suzuki Department of Plastic and Reconstructive Surgery Kyoto University Graduate School of Medicine Sakyo-ku Kyoto 606-8507, Japan E-mail: [email protected]

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