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Brain Research 681 (1995) 105-108

Research report

Predegeneration enhances regeneration into acellular nerve grafts Nils Danielsen a,b,., James M. Kerns c, BjiSrn Holmquist d, Qing Zhao a, G&an Lundborg a, Martin Kanje e a Department of Hand Surgery, University Hospital, University ofLund, Malrai~, Sweden b Department of ExperimentalResearch, University Hospital University ofLuna~ Malm6, Sweden c Department of Anatomy, Rush Presbyterian St Luke's Medical Center, Chicago, USA d Department of Mathematical Statistics, University ofLund, Lund, Sweden e Department of Animal Physiology, Uniuersity ofLund, Lund, Sweden Accepted 22 February 1995

Abstract

In the present study, we determined the regeneration rate and the initial delay in rat sciatic nerve grafts first made hypercellular by predegeneration then acellular by freeze-thawing. 7-day predegenerated nerve pieces from the distal nerve stump on the fight side were made acellular by repeated freeze-thawing and inserted as grafts into a 10-ram long freshly created defect on the left contralateral side. Freshly made (no predegeneration period) acellular nerve grafts were used as controls. Both types of grafts supported outgrowth of regenerating axons as demonstrated by the sensory pinch test. However, the predegenerated acellular nerve grafts had a significantly shorter initial delay period (2.7 days) as compared with freshly made acellular nerve grafts (9.5 days). The initial delay period for predegenerated acellular nerve grafts was similar to that for fresh cellular nerve grafts but significantly longer than that for predegenerated cellular nerve grafts [24]. The rate of regeneration appeared independent of the type of grafts used. We suggest that modifications of the basal lamina and/or factors produced during the predegeneration period by non-neuronal cells survive the freeze-thawing cycle and account for the decrease in the initial delay period.

Keywords: Rat sciatic nerve; Nerve regeneration; Nerve degeneration; Nerve grafting; Nerve repair; Schwann cell

1. Introduction

We and others have shown that predegenerated nerve grafts (PNG; taken from the distal nerve stump of a previously injured donor nerve) are superior to fresh nerve grafts (FNG) for repair of peripheral nerve defects in the rat [9,24,37,40]. PNG have a reduced initial delay period, i.e., the time interval before the axons enter the graft while the rate of regeneration is unaffected [9,24]. The mechanism behind this effect is not known. However, it is reasonable to assume that it is related to non-neuronal cells (e.g., Schwann cells) since they are more numerous in predegenerated grafts [1,6,33]. These cells produce a variety of factors which stimulate axonal growth, including neurotrophic factors, like N G F [20,27,38], but also basal

* Corresponding author. Department of Experimental Research, University Hospital, 205 02 MaimS, Sweden. Fax: (46) (40) 336207. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved

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lamina components, like laminin, which has a strong neurite-promoting effect in vitro [4,10-12,28]. A number of studies have demonstrated the importance of living non-neuronal cells in the graft or the distal nerve segment [2,18,19,36]. If these cells are killed, regeneration is severely impaired. However, both axons and Schwann cells can grow into acellular nerve grafts (ANG) [3,17,23]. It appears that it is sufficient to provide the axons with a substratum, like the basal lamina, to initiate regeneration since acellular pieces of muscles support outgrowth of axons [13-15]. Interestingly, frozen sections from predegenerated nerves, but not from freshly frozen nerves, support axonal outgrowth from dorsal root ganglion neurons in tissue culture [5]. The questions raised in the present study were whether predegenerated A N G (PANG) are superior to A N G (frozen and thawed without a predegeneration period) in vivo and how such grafts compare with cell-containing nerve grafts [24].

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N. Danielsen et al./ BrainResearch 681 (1995)105-108

2. Materials and methods

2.1. Animals The young adult Wistar rats (female, 180-200 g) used in this study were kept in soft bedding cages on an ad libitum food/water diet with a 12-h light/dark cycle. The experimental protocol was reviewed and approved by the Swedish Animal Ethics Committee at the University of Lund. The animals were anaesthetized for aseptic surgery with a 1.8-ml i.p. injection of sodium pentobarbital (60 m g / m l ) and saline in a 1:10 vol. proportion.

2.2. Surgical procedures The sciatic nerve on the right side was isolated proximally at the sciatic notch region and transected. The proximal stump was ligated with a 3 - 0 silk ligature to prevent regenerating axons to re-innervate the distal nerve stump during the predegeneration period. After 7 days of predegeneration, the nerve on the right side was re-exposed and a 12-13-mm long nerve piece was removed from the distal nerve stump. This nerve piece was first frozen in liquid nitrogen for 30 s and then thawed for 30 s in sterile water. This procedure was repeated 5 × . The ends of the frozen nerve piece were trimmed to make a 10-mm long PANG. The PANG was transposed to a 10-mm long freshly made defect in the left sciatic nerve of the same rat. Three perineurial sutures each ( 9 - 0 Ethilon) were used for the proximal and distal anastomosis, respectively. Fresh ANG were used as controls. In these experiments, resected pieces (10 mm of length) from both sciatic nerves were frozen and thawed according to the protocol described above. The nerve piece from the right side was used as an ANG on the left side and vice versa. In both the PANG and ANG groups, the muscle incision was closed with 6 - 0 sutures and the skin with 4 - 0 sutures or wound clips. After recovery from the anaesthetic, the animals were returned to the animal quarters.

intervals in a proximal direction until the typical muscle response was observed. The nerve was marked with a ligature at this point and the entire length of the sciatic nerve was then removed for measurements of the regeneration distances with a caliper. Several of our previous studies have demonstrated the accuracy of this test as compared with neurofilament staining for measuring the regeneration distance [7,24,30-32].

2.4. Calculation of regeneration rate and initial delay period The individual regeneneration distances were plotted vs. the postoperative intervals and the regression line was calculated according to Holmquist et al. (1993) [22]. The slope of the regression line represents the regeneration rate ( m m / d a y ) and the interception of the regression line with the x axis represents the initial delay period. The mathematical details of this model are presented elsewhere [22]. The model accounts for the 'regeneration failures' (i.e., those nerves showing 0-mm regeneration distances within the evaluation period) and also for those nerves where the axons have reached beyond the most distal available pinch site. For comparison, the regression lines for PNG and FNG from a previous study [24] were included in the statistical calculations. The statistical differences between various regeneration rates and initial delay periods were calculated using the t test with Bonferroni-Dunn correction for multiple comparisons ( P < 0.05).

3. Results The individual regeneration distances with the calculated regression lines for the PANG and ANG groups are shown in Fig. 1 together with the regression lines for their 30A a

2.3. Evaluation of regeneration distance The sensory pinch test was used to evaluate regeneration distances [16,35,39] on postoperative days 2, 4, 6, 8 and 10 (n = 8 at each evaluation interval) for the PANG group and at postoperative days 6, 10, 12, 14, 16, 18, 21 and 28 (n = 6 - 8 at each evaluation interval) for the ANG group. The rats were lightly anaesthetized with a 0.2-ml i.p. injection of a solution containing sodium pentobarbital (60 m g / m l ) , saline and diazepam (5 m g / m l in 1:1:2 vol. proportions). The sciatic nerve was re-exposed and an intact proximal branch to the biceps femoris muscle was pinched to determine that the level of anesthesia allowed the animal response. The most distal branches of the sciatic nerve were pinched with a forceps before cutting and then the nerve was carefully pinched at ~ 0.5-mm

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Fig. 1. Diagram showing individual regeneration distances from sensory pinch test and corresponding calculated regression lines [22] for PANG and ANG groups (for clarity of diagram 28-day postoperative data for ANG group are not shown but they were included in calculations). Included in diagram are also regression lines (dotted lines) for FNG and PNG groups (from a previousstudy [24]).

N. Danielsen et al. / Brain Research 681 (1995) 105-108

Table 1 Calculated regeneration rates and initial delay periods for cellular and acellular nerve grafts Type of graft Regeneration Initial delay Observations rate (ram/day) period(days) (n) PNG 1.7 0.0 40 APNG 1.2 2.7 a,b 40 FNG 1.5 3.6 a,b 44 ANG 2.1 9.5 a 62 Regeneration rate (ram/day; slope of regression line) and initial delay period (days; x-axis intercep0were calculatedaccordingto Holmqnistet al. (1993) [22]. a Statisticaldifferencesas comparodwith PNG. b Statistical differencesas comparedwith ANG. Calculatedregeneration rates and initial delay periods for PNG and FNG groups are from a previous study [24].

corresponding cell-containing grafts, PNG and FNG, from a previous study [24]. The ANG group showed a drastic shift of the regression line to the right as compared with the other three groups, indicating a much slower onset of regeneration. In this group, 7 / 1 0 nerves showed 0-mm regeneration distance at postoperative day 6. 0 values were also recorded at days 10 and 12. The regression line for the PANG group was similar to that of the FNG group (Fig. 1). The PANG group also showed some 0-mm regeneration distance at early evaluation periods. 3 / 8 nerves had 0 values at postoperative day 2. For postoperative days 4 and 6, the numbers were 4 / 8 and 1/8, respectively. The calculated regeneration rates and initial delay periods and the statistical comparisons between the four groups are shown in Table 1. The initial delay period for the ANG group was 9.5 days which was significantly longer than for the PANG (2.7 days), FNG and PNG groups. The regeneration rates varied less among the four groups, from 1.2-2.1 mm/day, as compared with the differences in initial delay periods (Table 1). Despite this range, the differences between various regeneration rates were not statistically significant.

4. Discussion The main finding in the present study was that the PANG group had a significantly shorter initial delay period as compared with the ANG group. Thus, the main effect of predegeneration, at decrease in the initial delay period [9,24], survived the freeze-thawing procedure. Furthermore, this study confirms previous observations by other methods that regeneration is impaired in nerve grafts devoid of non-neuronal cell,,; [2,18,19]. What then makes a predegenerated nerve graft superior to a fresh graft? We favour a multifactorial hypothesis which includes that both modifications of the basal lamina, which serves as a substratum for regenerating nerve fibers [3,10,14], and factors synthetized and released from non-

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neuronal cells are of importance. During the predegeneration period, the Schwann ceils proliferate and the distal nerve stump, which is later to be used as the source for the PNG, is invaded by inflammatory cells. During Wallerian degeneration, these cells together with the Schwann cells interact to produce and release a variety of factors which influence regeneration [8,20,21,26,27,34]. Furthermore, Schwann cells also produce basal lamina components, such as laminin and collagen IV [29]. Together, all these products could profoundly change the composition and structure of the basal lamina and the surrounding microenvironment in a way that favours nerve regeneration. At the time of grafting, many of these changes have already taken place in the PNG while they are just beginning in the FNG. Hence, the superiority of predegenerated grafts. The freezing and thawing procedure kills the cells in the graft [18,23]. Therefore, the observed effect of predegeneration as compared with an ANG should mainly reflect alterations of the extracellular matrix which occurred during the predegeneration period. We cannot presently determine if these changes are structural, compositional or both. Compositional changes could include binding of trophic factors to the basal lamina [29]. Interestingly, there is a suggestive temporal correlation between NGF accumulation in a distal nerve stump after nerve injury and the effects of predegeneration on nerve regeneration [9,20,25]. For example, a shortening of the initial delay can be demonstrated in grafts predegenerated for only 1 day [9], a time period which coincides with the retrograde accumulation of NGF in the part of the PNG facing the regenerating nerve fibers [20,25]. This implies that the availability of trophic factors may play an important role for the predegeneration effect. It is conceivable that some of the compositional and structural alterations in the microenvironment induced by predegeneration survive the repeated freeze-thawing procedure.

Acknowledgements This study was supported by grants from the Swedish Medical Research Council (5188); the Swedish Natural Science Research Council; the Faculty of Medicine, University of Lund; Research Foundations Administered by University Hospital, Malm6; Zoega's Fund for Medical Research; and by a Senior International Fellowship to J.M. Kerns (1 F06 TWO1697-01A1) from the Fogarty International Center NIH, Bethesda, Maryland, MD.

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