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Tubulization Increases Axonal Outgrowth of Rat Sciatic Nerve after Crush Injury
EXPERIMENTAL NEUROLOGY ARTICLE NO.
139, 238–243 (1996)
0097
Tubulization Increases Axonal Outgrowth of Rat Sciatic Nerve after Crush Injury PA¨ R DANIELSSON,*,1 LARS DAHLIN,†
AND
BO POVLSEN*,‡
*Department of Plastic Surgery, Hand Surgery & Burns, University of Linko¨ping, Linko¨ping, Sweden; †Department of HandSurgery, University Hospital, Lund University, Malmo¨, Sweden; and ‡Department of Orthopaedics, St Thomas’ Hospital, London, United Kingdom
It is important to develop methods which increase nerve regeneration since restoration of function following injury to peripheral nerves often requires outgrowth of the injured axons over long distances. In this study, axonal outgrowth after bilateral crush injury to the sciatic nerve of the rat was measured. One group with large-diameter nonpermeable silicone tubes and one group with large-diameter permeable silicone tubes applied around the crush site on one side had regeneration following nerve injury compared to controls on the other side. The length of regeneration of the regenerating axons were then measured 4, 5, and 6 days following the crush injury using the ‘‘pinch reflex test.’’ The presence of axons at the pinch level was confirmed by immunocytochemical staining for neurofilaments. The length of regeneration for rats with nonpermeable tubes was significantly greater than that of the contralateral control side and was so at all times ( p F 0.05). The effect was present but not that pronounced where permeable tubes were used. We conclude that the outgrowth of regenerating sensory axons after sciatic nerve crush injury in the rat can be increased by enclosing the regeneration site in a silicone tube. The observed effect may be due to local mechanisms such as macrophage invasion or prevention of rapid wash-out of fluid from the crush zone. r 1996 Academic Press, Inc.
distance is required. As degeneration and atrophy of the target organs and even the central structures occur (1, 2), the time required for reinnervation is of importance for the final outcome. It would therefore be advantageous to improve the regeneration process of the axons following an acute peripheral nerve injury. This has so far been beyond clinical therapeutic manipulation. In situations where the peripheral nerve trunk has previously been injured, i.e., ‘‘conditioning lesions’’ (31, 32), an increased axonal outgrowth has been found following a second injury to the nerve (3, 5–7, 36). For surgical reasons it is more appropriate to repair the injured nerve immediately and nerves cannot be preconditioned or predegenerated. For this reason the rate with which axons regenerate following an unplanned nerve injury is today beyond manipulation. Neurotrophic factors, which improve axonal outgrowth, are produced in an injured nerve trunk (24, 25, 27, 29). Theoretically, a silicone tube which is wrapped around the site of injury may reduce the wash-out of such factors and/or attract macrophages which can produce factors that may promote the production of neurotrophic factors (16, 22). Such a procedure may enhance the regenerating process and axonal outgrowth. The aim of the present study was to see whether enclosing a crush site in a silicone tube, permeable or nonpermeable, results in an increased axonal outgrowth of peripheral sensory nerves.
INTRODUCTION
MATERIAL AND METHODS
Disability following nerve injury represents a major clinical problem. The ultimate goal of peripheral nerve repair is to restore normal sensory and muscle function. Restoration of function following nerve injury often requires regrowth of the injured axons over relatively long distances and this situation may differ dramatically from the one encountered in animal experiments where nerve regeneration over a relatively short
Animals
1 To whom correspondence and reprint requests should be addressed at Department of Plastic Surgery, Hand Surgery & Burns, University Hospital, S-581 85, Linko¨ping, Sweden. Fax: 146 13 22 37 06.
Silicone Tubes (Nonpermeable/Permeable)
0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Seventy female adult Wistar rats weighing around 200 g were obtained from Mo¨llegaard AS, Denmark, and the study was approved by the Animal Ethical Committee of Lund University. The rats were kept on soft bedding in transparent cages at room temperature with a 1:1 light:dark cycle and free access to food and water.
Silicone tubes were made of medical-grade silicone tubing (Mentor Co., U.S.A.; internal diameter, 1.6 mm;
238
INCREASED AXONAL REGENERATION IN RAT SCIATIC NERVE
external diameter, 2.7 mm; and length, 10 mm). The internal diameter of the silicone tubes was chosen so as not to create any compression due to swelling of the enclosed nerve segment (6). The sciatic nerve diameter at the crush site prior to injury was <0.9–1.1 mm and at pinch time 1.4–1.5 mm. The permeable tubes were produced by punching holes with a diameter of <0.35 mm spread uniformly over the tube and equivalent to <10% of the surface area. The tubes were cut open with a longitudinal incision and then wet sterilized. Surgery The rats were anesthetized ip with 0.8 ml/100 g rat of sodium pentobarbital (60 mg/ml) in 0.9% saline in a 1:10 volume proportion. The sciatic nerves were exposed bilaterally at the midthigh and mobilized over approximately 14 mm and then subjected to a crush injury bilaterally using a pair of smooth microforceps (2 3 20 s). The crush sites were marked with a 9/O epineurial Ethilon suture. On one side a silicone tube was applied around the nerve with the crush site at the midpoint of the tube and then closed using a 9/O suture. In 36 rats regular nonpermeable silicone tubes were used and in 34 rats the permeable tubes were used. The wounds were closed in layers and the rats were allowed unrestricted movement directly after the anesthesia. Evaluation of Regeneration Pinch reflex test. Four, five, and six days after the nerve injury, different rats were anesthetized each day with 0.7 ml/100 g rat ip of the solution mentioned above. The sciatic nerves were reexposed bilaterally and the silicone tube was removed. The sciatic nerve was carefully dissected free distally, including the peroneal and tibial nerve, and cut at the level of the ankle. The leading axons were localized by pinching the nerve in a proximal direction with a pair of forceps until a withdrawal reflex was elicited. In some nerves this point was marked with a ligature. The pinch reflex test is a reliable method for studying outgrowth of sensory axons (15, 18, 23, 39). The distance between this point and the marked crush site was measured with a caliper and regarded as the length of regeneration of sensory axons. Neurofilament staining. To confirm the pinch test results, some of the sciatic nerves were processed for immunocytochemical localization of neurofilaments. Following the pinch test, the nerve was rinsed in cold Ringer’s solution (pH 7.2, 4°C), embedded in Tissue Tek, and frozen. Longitudinal sections at a thickness of 8 µm were cut on a cryostat and mounted on poly-L-lysintreated objective slides. The sections were washed in phosphate-buffered saline (PBS), fixed in ice-cold acetone (5 min), rinsed in PBS (3 3 5 min), and incubated for 20 min in 5% dry milk diluted in PBS to prevent
239
nonspecific antibody absorption. The sections then were exposed to 68-kDa rabbit antibovine neurofilament antibody at a dilution of 1:40 in PBS at room temperature overnight. Following washing in PBS for 3 3 5 min, the sections were treated with fluoresceinconjugated swine to rabbit polyclonal antibodies at a dilution of 1:40 for 30 min at room temperature. They were washed 3 3 5 min in PBS, mounted in buffered paraphenylendiamine-containing glycerol, coded, and examined under a fluorescence microscope. Control sections were incubated without either the primary or the secondary antibody. Statistics The paired nonparametric Wilcoxon signed rank test (two-tailed; In STAT, version 1.12) was used to compare the regeneration lengths of the experimental sides. A p value # 0.05 was considered statistically significant. Results are presented as means 6 SD. The regeneration rate (mm/day) and the initial delay, i.e., on which day after injury the axons began to grow, were calculated by simple regression analysis and then extrapolated to the X-axis. RESULTS
Macroscopic Examination The mobilized regions on both sides of the sciatic nerves were examined under an operation microscope. The control side showed an irregular fibrin layer covering the nerve while the nonpermeable tube side showed a smooth epineurial cover with no signs of compression. The permeable tube side showed signs of a moderate fibrin covering with some spins corresponding to the tube holes, most pronounced at Day 6. Regeneration The length of axonal outgrowth was measured at Days 4, 5, and 6 postsurgery and the results are shown in Table 1. On the nonpermeable tube side the regeneration lengths were statistically significantly longer than those on the control side (Table 1). The regeneration on the control side of all animals was linear (Fig. 1; rate, 3.6 mm/day), with an initial delay of 1.4 days. The nerves surrounded by permeable tubes showed a statistically significant greater increase in length compared to controls at Days 4 and 6, but the increase was not as pronounced as that for the rats with nonpermeable tubes. At Day 4 the axonal outgrowths for both types of tubes were longer compared to controls and the regeneration at Days 4 and 5 was almost linear to that of the controls. The effect was somewhat more pronounced at Day 6, the rats with the nonpermeable tubes having an average axonal outgrowth distance 21% longer than those with the permeable tubes. However, when a
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DANIELSSON ET AL.
TABLE 1 Regeneration Length (mm) and p Values for Each Animal Number (No.) after 4, 5, and 6 Days on the Control (Ctrl) and the Tube Sides for Nonpermeable (Npt) and Permeable (Pt) Tubes Day 4 No. 1 2 3 4 5 6 7 8 9 10
Mean: SD: p-values: All controls Mean: SD:
Day 5
Day 6
Ctrl
Npt
No.
Ctrl
Pt
No.
Ctrl
Npt
No.
Ctrl
Pt
No.
Ctrl
Npt
No.
Ctrl
Pt
11,4 7,6 6,4 9,1 9,4 9,8 9,4 9,5 9,0 9,9
10,9 9,1 8,7 11,0 9,9 9,4 9,5 11,3 11,6 11,7
11 12 13 14 15 16 17 18 19 20
9,8 9,7 9,5 10,0 9,1 8,7 9,0 10,0 9,9 9,1
10,2 8,6 10,1 12,9 9,3 10,2 9,7 11,7 12,2 9,1
21 22 23 24 25 26 27 28 29 30
15,6 11,9 14,0 11,6 14,9 13,5 13,4 12,9 13,5 12,9
13,8 15,6 16,3 14,0 15,0 15,5 14,8 14,3 14,4 13,2
31 32 33 34 35 36 37 38 39 40
13,2 13,3 12,8 12,5 13,0 13,3 12,8 12,8 13,5 13,1
14,6 13,9 13,3 12,6 17,7 12,3 12,6 13,8 13,7 16,1
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
24,3 13,6 17,1 17,6 19,4 15,1 14,2 13,6 15,7 16,7 16,9 16,1 17,5 16,0 16,1 18,0
35,9 33,3 29,5 33,8 17,4 19,4 38,2 23,7 14,9 28,0 18,2 18,9 18,6 17,2 17,3 20,0
57 58 59 60 61 62 63 64 65 66 67 68 69 70
16,7 16,5 16,7 16,9 14,1 16,2 17,3 16,9 14,5 16,2 16,8 16,9 15,5 17,1
17,0 17,9 16,0 29,8 18,6 28,0 16,5 20,5 17,7 25,6 19,6 17,4 19,0 15,8
9,2 10,3 1,3 1,1 0.0195
9,5 10,4 0,5 1,4 0.0391
13,4 14,7 1,2 0,9 0.0273
9,3 1,0
13,0 14,1 0,3 1,7 0.0645
16,7 24,0 2,6 7,8 0.0019
13,2 0,9
16,3 19,9 1,0 4,5 0.0085 16,5 2,0
Mann–Whitney U test is applied to the data, the difference between permeable and nonpermeable tubes at Day 6 is not statistically different. Neurofilament Staining The immunocytochemical localization of neurofilaments in all investigated specimens confirmed the presence of axons at the pinch level. A longitudinal section of the distal portion of the sciatic nerve at the marked pinch level stained for neurofilaments is shown in Fig. 2. DISCUSSION
FIG. 1. Mean values and SD of the regeneration length with controls, nonpermeable, and permeable tubes at Days 4, 5, and 6.
This study indicates that the outgrowth of axons can be increased following a crush lesion if the crush site is enclosed in a nonpermeable or permeable silicone tube. Such an effect was not so pronounced when the silicone tube was perforated with holes (permeable tubes). The control nerve injury without tubes showed a linear outgrowth with a regeneration rate of 3.6 mm per day and with an initial delay of 1.4 days. These values are similar to previously published studies using a crush lesion (4, 10, 14, 19, 20, 37). The method used to evaluate regeneration—the pinch reflex test—is a reliable and consistent method for studying the axonal outgrowth of sensory axons comparable to measure-
INCREASED AXONAL REGENERATION IN RAT SCIATIC NERVE
241
FIG. 2. Staining of neurofilaments of a crushed sciatic nerve enclosed in a nonpermeable tube 6 days postcrush. The longitudinal section shows positive staining for neurofilament at the distal level of positive pinch marked with a suture (arrow), indicating the distal tip of outgrowing axons. Length of bar, 100 µm.
ment of axonal transport and immunohistochemical detection of neurofilaments (3, 23, 33, 34). The pinch reflex test does not overestimate axonal outgrowth, which is confirmed by the results of the neurofilament staining in this study. The silicone tube model has been used for many years to study the regeneration process. Following transection and introduction of the proximal and distal nerve stumps into the tube a fibrin matrix develops between the nerve ends. The fibrin matrix is invaded by macrophages and later by ingrowth of Schwann cells and axons (8, 27, 28, 40). It has also been observed that the fibrin matrix contains interleukin-1b and this substance also accumulates in the silicone tube with a peak at the seventh day (12). At that time the majority of the mononuclear cells are monocytes/macrophages (12). Application of a silicone tube around an uninjured nerve also induces accumulation of ED1- and ED2positive macrophages around the nerve and it has been suggested that such an accumulation of macrophages leads to a release of factors that stimulate the proliferation of nonneuronal cells in the endoneurial space, without any sign of injury to the axons (6). The stimulation of an inflammatory reaction and the application of tissue containing numerous macrophages to a peripheral nerve increase the outgrowth of sensory axons (5, 8, 9). Furthermore, application of inflammatory cells close to the nerve cell bodies in the dorsal root ganglia increases nerve regeneration (26).
In the present study the axonal outgrowth was already longer at Days 4 and 5 in nerves that were enclosed in a nonpermeable silicone tube. The effect was somewhat pronounced at Day 6 and the nonpermeable tubes had longer axonal outgrowth than the permeable tubes at all times. We therefore suggest that the application of a silicone tube may attract macrophages with a subsequent inflammatory response, leading to improved outgrowth due to factors released by the macrophages. The somewhat increased average axonal outgrowth in rats with nonpermeable tubes at Day 6 may indicate that the inflammatory response is not immediate but somewhat delayed. It is also possible that there may be a less pronounced accumulation of factors such as interleukin-1b and neuronotrophic factors close to the crush site when the permeable tubes were used. A decrease in concentration of these factors due to wash-out may explain the effect observed. Neurotrophic activity, probably explained by the presence of CNTF and NGF, has recently been reported to occur in silicone tubes where transected nerve ends were introduced (13). Treatment with CNTF can also potentiate the regeneration of axons as shown by an increased regeneration rate compared to that of controls (35). Permeable silicone tubes have been used in previous studies. Jenq and Coggeshall (17) observed that the newly formed nervelike structure between the transected nerve ends enclosed in ‘‘holey’’ tubes had a different organization with a large number of minifascicles
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DANIELSSON ET AL.
similar to that found in a study where Gor-Tex chambers were used (11). It was found that Gor-Tex chambers with pores showed a somewhat disorganized pattern compared to an innercoated chamber. Jenq and Coggeshall suggested a better regeneration in permeable tubes based on the number of regenerating fibres. We suggest that our findings together with those of previously published articles using nonpermeable silicone tubes indicate that the outgrowth of sensory axons as well as the organization of the growth is better in nonpermeable tubes than in permeable tubes. The present study raises the question of whether the regeneration rate is always fixed from the start or can be altered after some days (6 days) since the nonpermeable tubes seem to show a pronounced effect at Day 6. Due to technical reasons it was not possible to measure the length of outgrowth after 6 days but this will be studied in future studies. Other methods to improve the outgrowth of axons have been described. Treatment with pulsed electromagnetic fields following a crush injury can stimulate outgrowth of sensory axons by increasing the regeneration rate by up to 21% (34). Conditioning treatments such as pretreatment with pulsed electromagnetic fields or compression of a peripheral nerve have also been shown to increase axonal outgrowth (6, 7, 21). In clinical cases where the silicone tube model has been used to repair human digital median and ulnar nerves it seems that regeneration proceeds at a higher initial rate in nerves treated with silicone tubes even at points far distal to the site of the lesion (30, 38). We conclude that the axonal outgrowth of sensory axons following a crush injury to rat sciatic nerve can be increased by enclosing the crush site in a silicone tube. Such a phenomenon may be based on local changes, such as invasion of macrophages, in the zone of regeneration and that the factors released from these cells are prevented from leaking out from the crush site.
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ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (5188), the Swedish Society for Medical Research, Riksfo¨rbundet fo¨r Trafik-och Polioskadade, Neurologiskt Handikappades Riksfo¨rbund, Trygg-Hansa SPP Research Foundation, and ¨ stergo¨tland. Lions of O
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MCQUARRIE, I. G., AND B. GRAFSTEIN. 1973. Axon outgrowth enhanced by a previous nerve injury. Arch. Neurol. 29: 53–55. MCQUARRIE, I. G., B. GRAFSTEIN, AND M. D. GERSHON. 1977. Axonal regeneration in the rat sciatic nerve: Effect of a conditioning lesion and of dbcAMP. Brain Res. 132: 443–453. REDSHAW, J. D., AND M. A. BISBY. 1987. Proteins of fast axonal transport in regenerating rat sciatic sensory axons: A conditioning lesion does not amplify the characteristic response to axotomy. Exp. Neurol. 98: 212–221. RUSOVAN-JOHNSSON, A., AND M. KANJE. 1991. Stimulation of regeneration of the rat sciatic nerve by 50 Hz sinusoidal magnetic fields. Exp. Neurol. 112: 312–316. SAHENK, Z., J. SEHARASEYON, AND J. R. MENDELL. 1994. CNTF potentiates peripheral nerve regeneration. Brain Res. 655: 246–250. SISKEN, B. F., M. KANJE, G. LUNDBORG, E. HERBST, AND W. KURTZ. 1989. Stimulation of rat sciatic nerve regeneration with pulsed electromagnetic fields. Brain Res. 485: 309–316. SJO¨ BERG, J., M. KANJE, AND A. EDSTRO¨ M. 1988. Influence of nonneuronal cells on regeneration of the rat sciatic nerve. Brain Res. 453: 221–226. VILJANEN, V. 1995. Nerve reconstruction with a silicone tube, a case report. In Proceedings of the 6th Congress of International Federation and Societies for Surgery of the Hand, Helsinki, Finland July 3–7, O151. YOUNG, J. Z., AND P. B. MEDAWAR. 1940. Fibrin suture of peripheral nerves: Measurement of the rate of regeneration. Lancet 2: 126–128. ZHAO, Q., L. B. DAHLIN, M. KANJE, AND G. LUNDBORG. 1993. Repair of the transected rat sciatic nerve: Matrix formation within silicone tubes. Restor. Neurol. Neurosci. 5: 197–204.
139, 238–243 (1996)
0097
Tubulization Increases Axonal Outgrowth of Rat Sciatic Nerve after Crush Injury PA¨ R DANIELSSON,*,1 LARS DAHLIN,†
AND
BO POVLSEN*,‡
*Department of Plastic Surgery, Hand Surgery & Burns, University of Linko¨ping, Linko¨ping, Sweden; †Department of HandSurgery, University Hospital, Lund University, Malmo¨, Sweden; and ‡Department of Orthopaedics, St Thomas’ Hospital, London, United Kingdom
It is important to develop methods which increase nerve regeneration since restoration of function following injury to peripheral nerves often requires outgrowth of the injured axons over long distances. In this study, axonal outgrowth after bilateral crush injury to the sciatic nerve of the rat was measured. One group with large-diameter nonpermeable silicone tubes and one group with large-diameter permeable silicone tubes applied around the crush site on one side had regeneration following nerve injury compared to controls on the other side. The length of regeneration of the regenerating axons were then measured 4, 5, and 6 days following the crush injury using the ‘‘pinch reflex test.’’ The presence of axons at the pinch level was confirmed by immunocytochemical staining for neurofilaments. The length of regeneration for rats with nonpermeable tubes was significantly greater than that of the contralateral control side and was so at all times ( p F 0.05). The effect was present but not that pronounced where permeable tubes were used. We conclude that the outgrowth of regenerating sensory axons after sciatic nerve crush injury in the rat can be increased by enclosing the regeneration site in a silicone tube. The observed effect may be due to local mechanisms such as macrophage invasion or prevention of rapid wash-out of fluid from the crush zone. r 1996 Academic Press, Inc.
distance is required. As degeneration and atrophy of the target organs and even the central structures occur (1, 2), the time required for reinnervation is of importance for the final outcome. It would therefore be advantageous to improve the regeneration process of the axons following an acute peripheral nerve injury. This has so far been beyond clinical therapeutic manipulation. In situations where the peripheral nerve trunk has previously been injured, i.e., ‘‘conditioning lesions’’ (31, 32), an increased axonal outgrowth has been found following a second injury to the nerve (3, 5–7, 36). For surgical reasons it is more appropriate to repair the injured nerve immediately and nerves cannot be preconditioned or predegenerated. For this reason the rate with which axons regenerate following an unplanned nerve injury is today beyond manipulation. Neurotrophic factors, which improve axonal outgrowth, are produced in an injured nerve trunk (24, 25, 27, 29). Theoretically, a silicone tube which is wrapped around the site of injury may reduce the wash-out of such factors and/or attract macrophages which can produce factors that may promote the production of neurotrophic factors (16, 22). Such a procedure may enhance the regenerating process and axonal outgrowth. The aim of the present study was to see whether enclosing a crush site in a silicone tube, permeable or nonpermeable, results in an increased axonal outgrowth of peripheral sensory nerves.
INTRODUCTION
MATERIAL AND METHODS
Disability following nerve injury represents a major clinical problem. The ultimate goal of peripheral nerve repair is to restore normal sensory and muscle function. Restoration of function following nerve injury often requires regrowth of the injured axons over relatively long distances and this situation may differ dramatically from the one encountered in animal experiments where nerve regeneration over a relatively short
Animals
1 To whom correspondence and reprint requests should be addressed at Department of Plastic Surgery, Hand Surgery & Burns, University Hospital, S-581 85, Linko¨ping, Sweden. Fax: 146 13 22 37 06.
Silicone Tubes (Nonpermeable/Permeable)
0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Seventy female adult Wistar rats weighing around 200 g were obtained from Mo¨llegaard AS, Denmark, and the study was approved by the Animal Ethical Committee of Lund University. The rats were kept on soft bedding in transparent cages at room temperature with a 1:1 light:dark cycle and free access to food and water.
Silicone tubes were made of medical-grade silicone tubing (Mentor Co., U.S.A.; internal diameter, 1.6 mm;
238
INCREASED AXONAL REGENERATION IN RAT SCIATIC NERVE
external diameter, 2.7 mm; and length, 10 mm). The internal diameter of the silicone tubes was chosen so as not to create any compression due to swelling of the enclosed nerve segment (6). The sciatic nerve diameter at the crush site prior to injury was <0.9–1.1 mm and at pinch time 1.4–1.5 mm. The permeable tubes were produced by punching holes with a diameter of <0.35 mm spread uniformly over the tube and equivalent to <10% of the surface area. The tubes were cut open with a longitudinal incision and then wet sterilized. Surgery The rats were anesthetized ip with 0.8 ml/100 g rat of sodium pentobarbital (60 mg/ml) in 0.9% saline in a 1:10 volume proportion. The sciatic nerves were exposed bilaterally at the midthigh and mobilized over approximately 14 mm and then subjected to a crush injury bilaterally using a pair of smooth microforceps (2 3 20 s). The crush sites were marked with a 9/O epineurial Ethilon suture. On one side a silicone tube was applied around the nerve with the crush site at the midpoint of the tube and then closed using a 9/O suture. In 36 rats regular nonpermeable silicone tubes were used and in 34 rats the permeable tubes were used. The wounds were closed in layers and the rats were allowed unrestricted movement directly after the anesthesia. Evaluation of Regeneration Pinch reflex test. Four, five, and six days after the nerve injury, different rats were anesthetized each day with 0.7 ml/100 g rat ip of the solution mentioned above. The sciatic nerves were reexposed bilaterally and the silicone tube was removed. The sciatic nerve was carefully dissected free distally, including the peroneal and tibial nerve, and cut at the level of the ankle. The leading axons were localized by pinching the nerve in a proximal direction with a pair of forceps until a withdrawal reflex was elicited. In some nerves this point was marked with a ligature. The pinch reflex test is a reliable method for studying outgrowth of sensory axons (15, 18, 23, 39). The distance between this point and the marked crush site was measured with a caliper and regarded as the length of regeneration of sensory axons. Neurofilament staining. To confirm the pinch test results, some of the sciatic nerves were processed for immunocytochemical localization of neurofilaments. Following the pinch test, the nerve was rinsed in cold Ringer’s solution (pH 7.2, 4°C), embedded in Tissue Tek, and frozen. Longitudinal sections at a thickness of 8 µm were cut on a cryostat and mounted on poly-L-lysintreated objective slides. The sections were washed in phosphate-buffered saline (PBS), fixed in ice-cold acetone (5 min), rinsed in PBS (3 3 5 min), and incubated for 20 min in 5% dry milk diluted in PBS to prevent
239
nonspecific antibody absorption. The sections then were exposed to 68-kDa rabbit antibovine neurofilament antibody at a dilution of 1:40 in PBS at room temperature overnight. Following washing in PBS for 3 3 5 min, the sections were treated with fluoresceinconjugated swine to rabbit polyclonal antibodies at a dilution of 1:40 for 30 min at room temperature. They were washed 3 3 5 min in PBS, mounted in buffered paraphenylendiamine-containing glycerol, coded, and examined under a fluorescence microscope. Control sections were incubated without either the primary or the secondary antibody. Statistics The paired nonparametric Wilcoxon signed rank test (two-tailed; In STAT, version 1.12) was used to compare the regeneration lengths of the experimental sides. A p value # 0.05 was considered statistically significant. Results are presented as means 6 SD. The regeneration rate (mm/day) and the initial delay, i.e., on which day after injury the axons began to grow, were calculated by simple regression analysis and then extrapolated to the X-axis. RESULTS
Macroscopic Examination The mobilized regions on both sides of the sciatic nerves were examined under an operation microscope. The control side showed an irregular fibrin layer covering the nerve while the nonpermeable tube side showed a smooth epineurial cover with no signs of compression. The permeable tube side showed signs of a moderate fibrin covering with some spins corresponding to the tube holes, most pronounced at Day 6. Regeneration The length of axonal outgrowth was measured at Days 4, 5, and 6 postsurgery and the results are shown in Table 1. On the nonpermeable tube side the regeneration lengths were statistically significantly longer than those on the control side (Table 1). The regeneration on the control side of all animals was linear (Fig. 1; rate, 3.6 mm/day), with an initial delay of 1.4 days. The nerves surrounded by permeable tubes showed a statistically significant greater increase in length compared to controls at Days 4 and 6, but the increase was not as pronounced as that for the rats with nonpermeable tubes. At Day 4 the axonal outgrowths for both types of tubes were longer compared to controls and the regeneration at Days 4 and 5 was almost linear to that of the controls. The effect was somewhat more pronounced at Day 6, the rats with the nonpermeable tubes having an average axonal outgrowth distance 21% longer than those with the permeable tubes. However, when a
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TABLE 1 Regeneration Length (mm) and p Values for Each Animal Number (No.) after 4, 5, and 6 Days on the Control (Ctrl) and the Tube Sides for Nonpermeable (Npt) and Permeable (Pt) Tubes Day 4 No. 1 2 3 4 5 6 7 8 9 10
Mean: SD: p-values: All controls Mean: SD:
Day 5
Day 6
Ctrl
Npt
No.
Ctrl
Pt
No.
Ctrl
Npt
No.
Ctrl
Pt
No.
Ctrl
Npt
No.
Ctrl
Pt
11,4 7,6 6,4 9,1 9,4 9,8 9,4 9,5 9,0 9,9
10,9 9,1 8,7 11,0 9,9 9,4 9,5 11,3 11,6 11,7
11 12 13 14 15 16 17 18 19 20
9,8 9,7 9,5 10,0 9,1 8,7 9,0 10,0 9,9 9,1
10,2 8,6 10,1 12,9 9,3 10,2 9,7 11,7 12,2 9,1
21 22 23 24 25 26 27 28 29 30
15,6 11,9 14,0 11,6 14,9 13,5 13,4 12,9 13,5 12,9
13,8 15,6 16,3 14,0 15,0 15,5 14,8 14,3 14,4 13,2
31 32 33 34 35 36 37 38 39 40
13,2 13,3 12,8 12,5 13,0 13,3 12,8 12,8 13,5 13,1
14,6 13,9 13,3 12,6 17,7 12,3 12,6 13,8 13,7 16,1
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
24,3 13,6 17,1 17,6 19,4 15,1 14,2 13,6 15,7 16,7 16,9 16,1 17,5 16,0 16,1 18,0
35,9 33,3 29,5 33,8 17,4 19,4 38,2 23,7 14,9 28,0 18,2 18,9 18,6 17,2 17,3 20,0
57 58 59 60 61 62 63 64 65 66 67 68 69 70
16,7 16,5 16,7 16,9 14,1 16,2 17,3 16,9 14,5 16,2 16,8 16,9 15,5 17,1
17,0 17,9 16,0 29,8 18,6 28,0 16,5 20,5 17,7 25,6 19,6 17,4 19,0 15,8
9,2 10,3 1,3 1,1 0.0195
9,5 10,4 0,5 1,4 0.0391
13,4 14,7 1,2 0,9 0.0273
9,3 1,0
13,0 14,1 0,3 1,7 0.0645
16,7 24,0 2,6 7,8 0.0019
13,2 0,9
16,3 19,9 1,0 4,5 0.0085 16,5 2,0
Mann–Whitney U test is applied to the data, the difference between permeable and nonpermeable tubes at Day 6 is not statistically different. Neurofilament Staining The immunocytochemical localization of neurofilaments in all investigated specimens confirmed the presence of axons at the pinch level. A longitudinal section of the distal portion of the sciatic nerve at the marked pinch level stained for neurofilaments is shown in Fig. 2. DISCUSSION
FIG. 1. Mean values and SD of the regeneration length with controls, nonpermeable, and permeable tubes at Days 4, 5, and 6.
This study indicates that the outgrowth of axons can be increased following a crush lesion if the crush site is enclosed in a nonpermeable or permeable silicone tube. Such an effect was not so pronounced when the silicone tube was perforated with holes (permeable tubes). The control nerve injury without tubes showed a linear outgrowth with a regeneration rate of 3.6 mm per day and with an initial delay of 1.4 days. These values are similar to previously published studies using a crush lesion (4, 10, 14, 19, 20, 37). The method used to evaluate regeneration—the pinch reflex test—is a reliable and consistent method for studying the axonal outgrowth of sensory axons comparable to measure-
INCREASED AXONAL REGENERATION IN RAT SCIATIC NERVE
241
FIG. 2. Staining of neurofilaments of a crushed sciatic nerve enclosed in a nonpermeable tube 6 days postcrush. The longitudinal section shows positive staining for neurofilament at the distal level of positive pinch marked with a suture (arrow), indicating the distal tip of outgrowing axons. Length of bar, 100 µm.
ment of axonal transport and immunohistochemical detection of neurofilaments (3, 23, 33, 34). The pinch reflex test does not overestimate axonal outgrowth, which is confirmed by the results of the neurofilament staining in this study. The silicone tube model has been used for many years to study the regeneration process. Following transection and introduction of the proximal and distal nerve stumps into the tube a fibrin matrix develops between the nerve ends. The fibrin matrix is invaded by macrophages and later by ingrowth of Schwann cells and axons (8, 27, 28, 40). It has also been observed that the fibrin matrix contains interleukin-1b and this substance also accumulates in the silicone tube with a peak at the seventh day (12). At that time the majority of the mononuclear cells are monocytes/macrophages (12). Application of a silicone tube around an uninjured nerve also induces accumulation of ED1- and ED2positive macrophages around the nerve and it has been suggested that such an accumulation of macrophages leads to a release of factors that stimulate the proliferation of nonneuronal cells in the endoneurial space, without any sign of injury to the axons (6). The stimulation of an inflammatory reaction and the application of tissue containing numerous macrophages to a peripheral nerve increase the outgrowth of sensory axons (5, 8, 9). Furthermore, application of inflammatory cells close to the nerve cell bodies in the dorsal root ganglia increases nerve regeneration (26).
In the present study the axonal outgrowth was already longer at Days 4 and 5 in nerves that were enclosed in a nonpermeable silicone tube. The effect was somewhat pronounced at Day 6 and the nonpermeable tubes had longer axonal outgrowth than the permeable tubes at all times. We therefore suggest that the application of a silicone tube may attract macrophages with a subsequent inflammatory response, leading to improved outgrowth due to factors released by the macrophages. The somewhat increased average axonal outgrowth in rats with nonpermeable tubes at Day 6 may indicate that the inflammatory response is not immediate but somewhat delayed. It is also possible that there may be a less pronounced accumulation of factors such as interleukin-1b and neuronotrophic factors close to the crush site when the permeable tubes were used. A decrease in concentration of these factors due to wash-out may explain the effect observed. Neurotrophic activity, probably explained by the presence of CNTF and NGF, has recently been reported to occur in silicone tubes where transected nerve ends were introduced (13). Treatment with CNTF can also potentiate the regeneration of axons as shown by an increased regeneration rate compared to that of controls (35). Permeable silicone tubes have been used in previous studies. Jenq and Coggeshall (17) observed that the newly formed nervelike structure between the transected nerve ends enclosed in ‘‘holey’’ tubes had a different organization with a large number of minifascicles
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similar to that found in a study where Gor-Tex chambers were used (11). It was found that Gor-Tex chambers with pores showed a somewhat disorganized pattern compared to an innercoated chamber. Jenq and Coggeshall suggested a better regeneration in permeable tubes based on the number of regenerating fibres. We suggest that our findings together with those of previously published articles using nonpermeable silicone tubes indicate that the outgrowth of sensory axons as well as the organization of the growth is better in nonpermeable tubes than in permeable tubes. The present study raises the question of whether the regeneration rate is always fixed from the start or can be altered after some days (6 days) since the nonpermeable tubes seem to show a pronounced effect at Day 6. Due to technical reasons it was not possible to measure the length of outgrowth after 6 days but this will be studied in future studies. Other methods to improve the outgrowth of axons have been described. Treatment with pulsed electromagnetic fields following a crush injury can stimulate outgrowth of sensory axons by increasing the regeneration rate by up to 21% (34). Conditioning treatments such as pretreatment with pulsed electromagnetic fields or compression of a peripheral nerve have also been shown to increase axonal outgrowth (6, 7, 21). In clinical cases where the silicone tube model has been used to repair human digital median and ulnar nerves it seems that regeneration proceeds at a higher initial rate in nerves treated with silicone tubes even at points far distal to the site of the lesion (30, 38). We conclude that the axonal outgrowth of sensory axons following a crush injury to rat sciatic nerve can be increased by enclosing the crush site in a silicone tube. Such a phenomenon may be based on local changes, such as invasion of macrophages, in the zone of regeneration and that the factors released from these cells are prevented from leaking out from the crush site.
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15. 16. 17.
ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (5188), the Swedish Society for Medical Research, Riksfo¨rbundet fo¨r Trafik-och Polioskadade, Neurologiskt Handikappades Riksfo¨rbund, Trygg-Hansa SPP Research Foundation, and ¨ stergo¨tland. Lions of O
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