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Primary sciatic nerve repair using titanium staples
British Journal of Plastic Surgery (2002), 55, 330-334 9 2002 The British Associationof Plastic Surgeons doi: 10.1054/bjps.2002.3832
PLASTIC
SURGERY
Primary sciatic nerve repair using titanium staples C. E. Payne, S. P. Hunt* and B. G. H. Lamberty
Department of Plastic and Reconstructive Surgery, Addenbrooke's Hospital, Cambridge; and *Department of Anatomy and Developmental Biology, University College London, London, UK SUMMARY. The primary epineural repair of human peripheral nerves is most often achieved using non-absorbable microsutures, which can elicit a foreign-body reaction. We describe a new system for neural-tissue approximation, consisting of non-penetrating vascular closure staples (VCS) applied to the epineurium. These clips were initially developed for use in microvascular anastomosis, with no knowledge of their effectiveness in neural-tissue approximation. We compare the efficacies of VCS clips and monofilament nylon microsutures in the repair of transected sciatic nerves in 36 adult Sprague Dawley rats (18 treated with 9/0 sutures and 18 treated with VCS clips). In the rat, regeneration starts by day 5, and is well advanced by 4 weeks. To assess the overall repair success, the site of injury, after perfusion fixation, was harvested at 5, 7 and 30 days. The two methods were compared in terms of operative time, the degree of self-mutilation (autotomy), the macroscopic specimens in vivo and the microanatomical continuity through the repair site. Continuity was studied by using PGP-9.5-1abelled cryosections and fluorescent secondary antibodies to visualise axonal regeneration. Clip repair was significantly faster in the VCS group (mean_+ s.e.m. = 7 . 0 9 _ 0.36 min versus mean _ s.e.m. = 11.56 _+0.51 min in the sutured group) and an equal and minimal degree of autotomy was observed. Macroscopically, all 36 nerves were in continuity and free from neuroma. The use of VCS clips resulted in equivalent visualised regeneration across the repair site at each time point. We believe the use of VCS clips to be a faster and comparable alternative to non-absorbable sutures in primary nerve repair in this experimental model. 9 2002 The British Association of Plastic Surgeons
Keywords: primary nerve repair, epineural
sutures, VCS clips.
arrangement of sensory and motor fascicles is important. Where it is possible to define this arrangement, then fascicular or group-fascicular repair may give better results than epineural repair, 4 otherwise simple epineural coaptation will suffice. There is also increasing awareness of the negative effects of tension at the suture line, which has been seen experimentally to invite connective-tissue proliferation. 5 Nerve suture under non-physiological tension results in poor regeneration. Successful neuronal propagation across the repair site requires minimal escape of sprouting axons from the juncture site and a close approximation to the distal endoneural sheath. Standard monofilament nylon may elicit a foreignbody reaction, impair vascularity and potentially disrupt axonal regeneration. To prevent these complications, many different methods of nerve repair have been reported in the literature. Various sutureless techniques have been proposed as alternatives to the traditional suturing. These include the use of the fibrin-seal adhesive Tissucol 6'7 and the tissue adhesive cyanoacrylate, Histocryl, 8,9 for epineural repair, and the highly timeconsuming argon laser for fascicular repair, t~ Recently, a sutureless technique for microvascular anastomosis, using non-penetrating arcuate-legged titanium vascular closure staples (VCS clip applier system), has been approved for use. 11 The clips are quick to anastomose vessels as small as 1.3 m m and come in various sizes, from large (2.0 mm at the tip) to small (0.9 mm).
Lessons learnt and observations made by military surgeons during the First World War paved the way for the development of modern peripheral-nerve-surgery techniques. There was a nihilistic attitude to nerve repair during earlier conflicts, but in 1919 the neurosurgeon Charles Elsberg demonstrated that suturing the epineurium with fascicular apposition and tension-free repair improved the outcome. 1 The peripheral nervous system is capable of remarkable, although somewhat unpredictable, regeneration of severed axons. Nerve tissue has unique healing problems once transected. The ingrowth of connective tissue between the cut ends will impair or block regrowth and nerve conduction. Scar tissue and nerve tissue at the proximal end of the gap may then form a neuroma. The microenvironment at the injury site, involving complex neurochemical interactions, is the basis of neurone survival and outgrowth. 2 Disruption to the supply of neurotrophic factors can lead to increased cell death and aberrant regeneration. One important advancement in nerve surgery was the introduction of the operating microscope by Smith in 1964, 3 which made it possible to identify and manipulate nerve structures with improved accuracy. The topographic
Presented at the Summer Meeting of the British Association of Plastic Surgeons, 6 July 2001, Stifling, Scotland, UK.
330
Primary sciatic nerve repair using titanium staples The purpose of this study was to investigate the use of VCS clips for repairing transected adult rat sciatic nerves. In order to investigate the effects of clips on nerve regeneration, an animal study was conducted, in which clip repairs were performed on transected sciatic nerves and directly compared with repairs using interrupted 9/0 monofilament nylon sutures. To evaluate whether VCS repairs are superior or comparable to the standard technique, we recorded the repair time and the degree of self-mutilation (autotomy), together with the appearance of the macroscopic specimen in vivo and the microanatomical continuity across the repair site at set time points of 5, 7 and 30 days. Materials and methods The experiments were performed on 36 adult Sprague Dawley rats (UCL, London, UK, 250-280g body weight). Animal care and all procedures were authorised by the Home Office and were conducted in agreement with National Institute of Health guidelines. The small VCS clip applier was used (clip dimension at the tip 0.9 ram, s United States Surgical Corporation) (Fig. 1).
Operative procedure The animals were anaesthetised using halothane in oxygen (4 ml min-1) administered via a set anaesthetic apparatus. Using sterile techniques, the left sciatic nerve (approximate size: 2-2.18 mm) was isolated under an operating microscope (x 10) and then sharply transected 1 cm from the point of emergence from the greater sciatic foramen. Epineural nerve repair was immediately performed, using either four to five 9/0 monofilament nylon sutures or VCS clips, randomly assigned among the 36 animals so as to give 18 rats per group. Two epineural stay sutures were used in all animals to attain nerve approximation and alignment, before the repair was completed using sutures or clips. In two animals in each time period of the clip group, the stay sutures were removed prior to skin closure. The operative time was defined as the time necessary to complete the repair from the placement of the two stay sutures. After repair, the wounds were closed and topical postoperative antibiotics were applied. The left hind leg was not immobilised, and all animals were observed for self mutilation.
~1.4mm~
B
0.9 mm
Figure 1--(A) VCS small-clip applier, and (B) dimensions of the small clip.
331
Immunohistochemistry The animals were sacrificed with a lethal dose of intraperitoneal pentobarbital (1 ml) at intervals of 5, 7 and 30 days postoperatively (n = 12, six from each group, at each time point). The whole animals were fixed in cold 4% w/v paraformaldehyde (Merck) made up in 0.15M phosphate buffer (pH 7.4). The left sciatic nerve was then exposed, measured and photographed for macroscopic comparisons, before being harvested 1 cm proximal and distal to the repair site; the right sciatic nerve was taken as a control specimen. The specimens were placed in a cryoprotectant solution of 30% w/v sucrose (Merck) and 0.02% w/v sodium azide in 0.1 M phosphate buffer (pH 7.4), and stored at 4 ~ After embedding and freezing in CryoM-Bed embedding compound (Merck), longitudinal nerve cryosections (15 Ixm) were thaw mounted on Polysine slides, air dried and stored with desiccant at - 8 0 ~ For analysis, the sections were blocked at room temperature for 1 h to prevent non-specific binding, with 3% normal goat serum (Vector), 0.25% Triton X-100 (Sigma) and 0.02% sodium azide in 0.1M phosphate buffer. After rinsing, a blocking solution containing a 1:1000 dilution of monoclonal PGP 9.5 primary antibody (UltraClone) was applied overnight. Three 10 min washes in 0.1 M phosphate buffer were followed by blocking with 1:500 fluorescent goat anti-mouse IgG secondary blocking antibody (Molecular Probes) and staining with Texas Red for specimens obtained 5 days and 30 days postoperatively or Oregon Green for specimens obtained 7 days postoperatively. Two chromophores were used simply for ease of visualisation and identification of the specimens. Secondary incubations were carried out at room temperature for 2h in complete darkness. Cover slips and Citifluor mountant were applied to all slides after three 10min washes in 0.1 M phosphate buffer, and the slides were stored at 4 ~ Images of nerve sections were taken using a Leica microscope (10x, 20x and 40 • magnification) and Hamamatsu system in conjunction with Vision Explorer image collection software. Results
Operative time The mean_+ s.e.m, operative time for the sutured group was ll.56_+0.36min. The mean_+s.e.m, operative time for the VCS group was 7.09 _+0.51 rain. The times ranged from ll.01min to 12.40min in the sutured group, and from 6.03 min to 8.28 rain in the VCS group. The operative time decreased as the experience of the surgeon increased; hence the later animals took the shorter times in both groups. An unpaired two-tailed t-test showed a significant difference between the groups (P < 0.01, Table 1). The time required for clip repair was about two-thirds of Table 1 Operative times (all values are in minutes)
sutures VCS clips P<0.001.
Mean ( + s.e.m.)
Range
11.56 (• 0.36) 7.09 (• 0.51)
11.01-12.40 6.03-8.28
332
British Journal of Plastic Surgery
that required for the conventional suturing technique in this animal experiment.
Self mutilation Subjective evaluation of the degree of self mutilation at all time points showed no difference between the two groups. There was minimal autotomy, restricted primarily to the nibbling of nails (n = 7 in the sutured group and n = 5 in the VCS group) and toes (n = 3 in the sutured group and n = 2 in the VCS group). No animal needed to be destroyed because of self-amputation of the toes.
Macroscopic specimens All nerves were studied and photographed in vivo. All nerves maintained approximation at all time points. To demonstrate the ability of the clips alone to maintain approximation, the stay sutures had been removed prior to skin closure in some animals in the VCS group; on harvest the nerves in all these animals were still coapted. The nerve diameter at the time of the original operation was 2.0-2.18 mm. At 5 days and 7 days postoperatively the nerve diameter in the sutured group was m e a n + s.e.m. =2.26__0.08 mm, and at 30 days it was m e a n _ s.e.m. = 2.51 + 0.07 mm. The nerve diameter in the VCS group at 5 days and 7 days was mean+_s.e.m.= 2.22___0.08 mm, and at 30 days it was mean___ s.e.m. = 2.58 _ 0 . 0 7 m m (Table 2). At 5 days and 7 days normal nerve morphology was observed in all specimens in both groups (Fig. 2A). By 30 days no visible excessive swelling of the repair sites was seen, and all nerves had a normal surrounding inflammatory reaction. The clips were embedded into the epineurium; no clip was separated from the nerve in vivo (Fig. 2B). No significant differences in diameter were found between the experimental groups and the control specimens (P = 0.44-0.51).
Immunohistochemistry The monoclonal PGP 9.5 antibody showed, in all nerve sections at all time points, specific strong pan-neuronal immunoreactivity with low background staining. Proximal to the repair site there was intense immunolocalisation to the reactive neuronal cytoplasmic polypeptides at 5, 7 and 30 days. Distally, Wallerian degeneration had removed these reactive antigens at days 5 and 7. At day 5, early regeneration across the repair site was typically seen more peripherally in the sutured nerves and more centrally in the clip-repaired nerves (Fig. 3A). No excessive reaction at the repair site was seen. In one Table 2
Macroscopic specimen: diameter o f the nerve (mm),
values are given as mean + s.e.m. Nerve diameter (mm) 5 days and 7 days a
sutures VCS clips aP = 0.444; bP=0.51.
2.26 _+0.08 2.22 + 0.08
30 days b
2.51 _ 0.07 2.58 + 0.07
Figure 2--(A) In vivo clipped nerve specimen at day 5, prior to harvest, showing minimal disruption to the nerve morphology. (B) In vivo clipped nerve specimen at day 30, prior to harvest. Note that there are no loose clips in the tissue, and the clips have embedded into the epineurium. The specimen shows a normal surrounding inflammatory reaction.
Primary sciatic nerve repair using titanium staples
Figure 3 ~ ( A ) At 5 days regeneration has commenced across the cliprepair site, shown centrally by Texas Red in this specimen. (B) At 7 days the axonal advancement (shown by Oregon Green) has progressed as far as the division into the two main terminal branches, the common peroneal and tibial nerves.
suture-repaired specimen there was no regeneration across the site. Regeneration was more advanced by 7 days (shown by Oregon Green). Generally, there was little variation between the operative groups in terms of PGP 9.5 staining. Axonal regeneration had advanced (563 Ixm) to the division into the two main terminal components, the common peroneal and tibial nerves (Fig. 3B). Complete specimen PGP 9.5 immunoreactivity was visualised by Texas Red at 30 days. The neuronal architecture at the repair site had become more organised in both groups. Under fluorescent microscopy, the repair site was easily identified, partly by the cellular reaction around the suture in the sutured group and the stay suture in the clipped group. No reaction to the clips could be identified. The strong pan-neuronal immunoreactivity continued to the end of the mounted specimen and the separation into the common peroneal and tibial nerves.
Discussion It is generally agreed that primary repair by end-to-end approximation of the stumps is the best surgical choice after severance of a peripheral nerve. Digital nerve injuries can comprise 68% of the nerve injuries presenting to a department. 12 The development of a painful neuroma, particularly in a digit, can be very disabling; the search
333 for non-penetrating sutureless microsurgical repair techniques stems from the desire to improve nerve coaptation and clinical results. Previous experimental sutureless techniques have been proposed, but have led to unpredictable outcomes. The cyanoacrylates s are known cytotoxics, 9 and fibrin seals 6'7 are unable to hold the nerve together against its natural retraction. VCS clips can quickly and successfully anastomose large and small arteries 11,13 and veins, 14 and aid the insertion of vascular prostheses. VCS clips have been used effectively to close other non-vascular tubular structures, and have been used quickly and easily to close linear incisions in the gallbladder 15 and longitudinal ureterotomies. 16 The comparable and minimal inflammatory reaction elicited by the titanium is well documented, tl We have shown that VCS clips can be used as a sutureless method of repairing peripheral nerves. The major advantage of using the clip applier rather than conventional suturing to repair small peripheral nerves is the reduction in the repair time. During the course of these experiments, the clip repair time decreased as experience with the applier increased. Speed does not compromise the quality of the nerve repair. In this study, we found clip repair to be about one-third faster than suture repair, and the difference was statistically significant (P<0.01). Although the speed of application is not crucial when repairing a single nerve, the savings in time during the repair of multiple nerves and arteries (for example in palmar lacerations) could compensate for the additional cost of the clips. The rat sciatic nerve has a poor epineural and perineural structural framework, and the fascicles bulge freely when the nerve is transected. ~7 This intrinsic property of the rat nerve lengthened the operative time compared with that normally expected when repairing a human peripheral nerve. This difficulty was encountered in both experimental groups; hence the times are comparable. Autotomy is an unknown but observable quantity. In all the animals there was a minimal degree of autotomy, and we suggest that the presence of titanium near neural tissue does not cause ectopic firing or hinder the propagation of axonal growth cones. Neuronal hyperplasia forming a neuroma is an important cause of failure of primary nerve repairs. Subjective simple observation of the gross intact specimen in vivo prior to harvest gives an indication of the activity at the repair site. If the clips produced inadequate coaptation, a macroscopic deformity would result at the repair site, but this was not seen in any harvested specimen, and the nerve diameters were within normal limits. The clips are non-penetrating, which may suggest a potential to become lost in the tissues and to fail to maintain approximation of the two ends. No clips were found free from the nerve, and it is also worth noting that the clips are easy to remove and atraumatic to the epineurium if a mistake is made with the applier. We subjectively analysed the data from the immunohistochemistry to determine, at the microscopic level, whether the clips impede the progression of the growth cones across the repair site. PGP 9.5 is an excellent and specific pan-neuronal marker, immunolocalising to neuronal cytoplasmic polypeptides, of as yet unknown
334
function. Monoclonal primary antibody produces enhanced specific staining, ts compared with polyclonal antibodies. At all time points in both operative groups there was unhindered progression of the growth cones across the repair site. Secondary visualisation with both chromophores picked up the strong PGP immunoreactivity in the regenerating axons. The use of two chromophores allowed specific separation and identification of the early time points from the later experiments. Overall, we demonstrated that the titanium had little detrimental effect on axon growth and did not cause excessive buildup of neuronal tissue at the repair site. It would be of supplementary benefit to study the repair site by quantitative assessment at all time points for each operative technique. Analysis of transverse sections, to determine the number of axons crossing the repair site and calculate their diameters, may further demonstrate that titanium clips are an equal repair mechanism to sutures. It can be assumed that non-penetrating clips maintain nerve approximation, have no detrimental effect on nerve regeneration, and are a fast accurate way of achieving peripheral nerve repair. We suggest that this method is comparable to non-absorbable microsuture in the repair of small-diameter nerves.
British Journal o f Plastic Surgery
8. 9. 10. 11.
12.
13.
microsuture in peripheral nerve repair. J Hand Surg 1988; 13A: 273-8. Siedentop KH, Loewy A. Facial nerve repair with tissue adhesive. Arch Otolaryngol 1979; 105: 423--6. Lehman RA, Hayes GJ, Leonard E Toxicity of alkyl 2-cyanoacrylates. I. Peripheral nerve. Arch Surg 1966; 93: 441-6. Almquist EE, Nachemson A, Auth D, Almquist B, Hall S. Evaluation of the use of the argon laser in repairing rat and primate nerves. J Hand Surg 1984; 9A: 792-9. Zhu YH, Kirsch WM, Cushman R, et al. Comparison of suture and clip for rnicrovascular anastomosis. Surg Forum 1985; 36: 492-5. de Medinaceli L, Prayon M, Merle M. Percentage of nerve injuries in which primary repair can be achieved by end-to-end approximation: review of 2,181 nerve lesions. Microsurgery 1993; 14: 244-6. Zeebregts CJ, van den Dungen JJ, Kalicharan D, Cromheecke M, van der Want J, van Schilfgaarde R. Nonpenetrating vascular clips for small-caliber anastomosis. Microsurgery 2000; 20: 131-8.
We thank Dr Michelle Robinson PhD for all her help at University College London in the setting up of the immunohistochemistry experiments.
14. Leppaniemi AK, Wherry DC, Pikoulis E, et al. Arterial and venous repair with vascular clips: comparison with suture closure. J Vasc Surg 1997; 26: 24-8. 15. Leppaniemi AK, Wherry DC, Soltero RG, et al. A quick and simple method to close vascular, biliary and urinary tract incisions using the new Vascular Closure Staples: a preliminary report. Surg Endosc 1996; 10: 771-4. 16. Leppaniemi AK, Wherry DC, Pikoulis E, Hufnagel HV, Fishback N, Rich NM. Ureteral repair with titanium staples: comparison with suture closure. Urology 1998; 51: 553-7. 17. Greene EC. Anatomy of the Rat. New York: Hafner Publishing Company, 1963; XXVII: 131-4. 18. Wilson POG, Barber PC, Hamid QA, et al. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol 1998; 69: 91-104.
References
The Authors
1. Elsberg CA. Technique of nerve suture and nerve grafting. JAMA 1919; 73: 14224. 2. Yin Q, Kemp GJ, Frostick SP. Neurotrophins, neurones and peripheral nerve regeneration. J Hand Surg 1998; 23B: 433-7. 3. Smith JW. Microsurgery of peripheral nerves. Plast Reconstr Surg 1964; 33: 317-29. 4. Lundborg G. A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg 2000; 25A: 391--414. 5. Terzis J, Faibisoff B, Williams B. The nerve gap: suture under tension vs. graft. Plast Reconstr Surg 1975; 56: 166-70. 6. Becker CM, Guening CO, Graft GL. Sutures or fibrin glue for divided rat nerves: Schwann cell and muscle metabolism. Microsurgery 1985; 6: 1-10. 7. Moy O J, Peimer CA, Koniuch MP, Howard C, Zielezny M, Katikaneni PR. Fibrin seal adhesive versus nonabsorbable
Caroline E. Payne MSc, FRCS, Senior House Officer in Plastic Surgery B. George H. Lamberty MA, FRCS, Consultant Plastic Surgeon
Acknowledgements
Department of Plastic and Reconstructive Surgery, 4th Floor, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK.
Steven P, Hunt PhD, Professor Department of Anatomy and Developmental Biology, Medawar Building, University College London, Gower Street, London WC1E 6BT, UK. Correspondence to Miss C. E. Payne. Paper received 1 October 2001. Accepted 11 Febmary 2002, after revision.
PLASTIC
SURGERY
Primary sciatic nerve repair using titanium staples C. E. Payne, S. P. Hunt* and B. G. H. Lamberty
Department of Plastic and Reconstructive Surgery, Addenbrooke's Hospital, Cambridge; and *Department of Anatomy and Developmental Biology, University College London, London, UK SUMMARY. The primary epineural repair of human peripheral nerves is most often achieved using non-absorbable microsutures, which can elicit a foreign-body reaction. We describe a new system for neural-tissue approximation, consisting of non-penetrating vascular closure staples (VCS) applied to the epineurium. These clips were initially developed for use in microvascular anastomosis, with no knowledge of their effectiveness in neural-tissue approximation. We compare the efficacies of VCS clips and monofilament nylon microsutures in the repair of transected sciatic nerves in 36 adult Sprague Dawley rats (18 treated with 9/0 sutures and 18 treated with VCS clips). In the rat, regeneration starts by day 5, and is well advanced by 4 weeks. To assess the overall repair success, the site of injury, after perfusion fixation, was harvested at 5, 7 and 30 days. The two methods were compared in terms of operative time, the degree of self-mutilation (autotomy), the macroscopic specimens in vivo and the microanatomical continuity through the repair site. Continuity was studied by using PGP-9.5-1abelled cryosections and fluorescent secondary antibodies to visualise axonal regeneration. Clip repair was significantly faster in the VCS group (mean_+ s.e.m. = 7 . 0 9 _ 0.36 min versus mean _ s.e.m. = 11.56 _+0.51 min in the sutured group) and an equal and minimal degree of autotomy was observed. Macroscopically, all 36 nerves were in continuity and free from neuroma. The use of VCS clips resulted in equivalent visualised regeneration across the repair site at each time point. We believe the use of VCS clips to be a faster and comparable alternative to non-absorbable sutures in primary nerve repair in this experimental model. 9 2002 The British Association of Plastic Surgeons
Keywords: primary nerve repair, epineural
sutures, VCS clips.
arrangement of sensory and motor fascicles is important. Where it is possible to define this arrangement, then fascicular or group-fascicular repair may give better results than epineural repair, 4 otherwise simple epineural coaptation will suffice. There is also increasing awareness of the negative effects of tension at the suture line, which has been seen experimentally to invite connective-tissue proliferation. 5 Nerve suture under non-physiological tension results in poor regeneration. Successful neuronal propagation across the repair site requires minimal escape of sprouting axons from the juncture site and a close approximation to the distal endoneural sheath. Standard monofilament nylon may elicit a foreignbody reaction, impair vascularity and potentially disrupt axonal regeneration. To prevent these complications, many different methods of nerve repair have been reported in the literature. Various sutureless techniques have been proposed as alternatives to the traditional suturing. These include the use of the fibrin-seal adhesive Tissucol 6'7 and the tissue adhesive cyanoacrylate, Histocryl, 8,9 for epineural repair, and the highly timeconsuming argon laser for fascicular repair, t~ Recently, a sutureless technique for microvascular anastomosis, using non-penetrating arcuate-legged titanium vascular closure staples (VCS clip applier system), has been approved for use. 11 The clips are quick to anastomose vessels as small as 1.3 m m and come in various sizes, from large (2.0 mm at the tip) to small (0.9 mm).
Lessons learnt and observations made by military surgeons during the First World War paved the way for the development of modern peripheral-nerve-surgery techniques. There was a nihilistic attitude to nerve repair during earlier conflicts, but in 1919 the neurosurgeon Charles Elsberg demonstrated that suturing the epineurium with fascicular apposition and tension-free repair improved the outcome. 1 The peripheral nervous system is capable of remarkable, although somewhat unpredictable, regeneration of severed axons. Nerve tissue has unique healing problems once transected. The ingrowth of connective tissue between the cut ends will impair or block regrowth and nerve conduction. Scar tissue and nerve tissue at the proximal end of the gap may then form a neuroma. The microenvironment at the injury site, involving complex neurochemical interactions, is the basis of neurone survival and outgrowth. 2 Disruption to the supply of neurotrophic factors can lead to increased cell death and aberrant regeneration. One important advancement in nerve surgery was the introduction of the operating microscope by Smith in 1964, 3 which made it possible to identify and manipulate nerve structures with improved accuracy. The topographic
Presented at the Summer Meeting of the British Association of Plastic Surgeons, 6 July 2001, Stifling, Scotland, UK.
330
Primary sciatic nerve repair using titanium staples The purpose of this study was to investigate the use of VCS clips for repairing transected adult rat sciatic nerves. In order to investigate the effects of clips on nerve regeneration, an animal study was conducted, in which clip repairs were performed on transected sciatic nerves and directly compared with repairs using interrupted 9/0 monofilament nylon sutures. To evaluate whether VCS repairs are superior or comparable to the standard technique, we recorded the repair time and the degree of self-mutilation (autotomy), together with the appearance of the macroscopic specimen in vivo and the microanatomical continuity across the repair site at set time points of 5, 7 and 30 days. Materials and methods The experiments were performed on 36 adult Sprague Dawley rats (UCL, London, UK, 250-280g body weight). Animal care and all procedures were authorised by the Home Office and were conducted in agreement with National Institute of Health guidelines. The small VCS clip applier was used (clip dimension at the tip 0.9 ram, s United States Surgical Corporation) (Fig. 1).
Operative procedure The animals were anaesthetised using halothane in oxygen (4 ml min-1) administered via a set anaesthetic apparatus. Using sterile techniques, the left sciatic nerve (approximate size: 2-2.18 mm) was isolated under an operating microscope (x 10) and then sharply transected 1 cm from the point of emergence from the greater sciatic foramen. Epineural nerve repair was immediately performed, using either four to five 9/0 monofilament nylon sutures or VCS clips, randomly assigned among the 36 animals so as to give 18 rats per group. Two epineural stay sutures were used in all animals to attain nerve approximation and alignment, before the repair was completed using sutures or clips. In two animals in each time period of the clip group, the stay sutures were removed prior to skin closure. The operative time was defined as the time necessary to complete the repair from the placement of the two stay sutures. After repair, the wounds were closed and topical postoperative antibiotics were applied. The left hind leg was not immobilised, and all animals were observed for self mutilation.
~1.4mm~
B
0.9 mm
Figure 1--(A) VCS small-clip applier, and (B) dimensions of the small clip.
331
Immunohistochemistry The animals were sacrificed with a lethal dose of intraperitoneal pentobarbital (1 ml) at intervals of 5, 7 and 30 days postoperatively (n = 12, six from each group, at each time point). The whole animals were fixed in cold 4% w/v paraformaldehyde (Merck) made up in 0.15M phosphate buffer (pH 7.4). The left sciatic nerve was then exposed, measured and photographed for macroscopic comparisons, before being harvested 1 cm proximal and distal to the repair site; the right sciatic nerve was taken as a control specimen. The specimens were placed in a cryoprotectant solution of 30% w/v sucrose (Merck) and 0.02% w/v sodium azide in 0.1 M phosphate buffer (pH 7.4), and stored at 4 ~ After embedding and freezing in CryoM-Bed embedding compound (Merck), longitudinal nerve cryosections (15 Ixm) were thaw mounted on Polysine slides, air dried and stored with desiccant at - 8 0 ~ For analysis, the sections were blocked at room temperature for 1 h to prevent non-specific binding, with 3% normal goat serum (Vector), 0.25% Triton X-100 (Sigma) and 0.02% sodium azide in 0.1M phosphate buffer. After rinsing, a blocking solution containing a 1:1000 dilution of monoclonal PGP 9.5 primary antibody (UltraClone) was applied overnight. Three 10 min washes in 0.1 M phosphate buffer were followed by blocking with 1:500 fluorescent goat anti-mouse IgG secondary blocking antibody (Molecular Probes) and staining with Texas Red for specimens obtained 5 days and 30 days postoperatively or Oregon Green for specimens obtained 7 days postoperatively. Two chromophores were used simply for ease of visualisation and identification of the specimens. Secondary incubations were carried out at room temperature for 2h in complete darkness. Cover slips and Citifluor mountant were applied to all slides after three 10min washes in 0.1 M phosphate buffer, and the slides were stored at 4 ~ Images of nerve sections were taken using a Leica microscope (10x, 20x and 40 • magnification) and Hamamatsu system in conjunction with Vision Explorer image collection software. Results
Operative time The mean_+ s.e.m, operative time for the sutured group was ll.56_+0.36min. The mean_+s.e.m, operative time for the VCS group was 7.09 _+0.51 rain. The times ranged from ll.01min to 12.40min in the sutured group, and from 6.03 min to 8.28 rain in the VCS group. The operative time decreased as the experience of the surgeon increased; hence the later animals took the shorter times in both groups. An unpaired two-tailed t-test showed a significant difference between the groups (P < 0.01, Table 1). The time required for clip repair was about two-thirds of Table 1 Operative times (all values are in minutes)
sutures VCS clips P<0.001.
Mean ( + s.e.m.)
Range
11.56 (• 0.36) 7.09 (• 0.51)
11.01-12.40 6.03-8.28
332
British Journal of Plastic Surgery
that required for the conventional suturing technique in this animal experiment.
Self mutilation Subjective evaluation of the degree of self mutilation at all time points showed no difference between the two groups. There was minimal autotomy, restricted primarily to the nibbling of nails (n = 7 in the sutured group and n = 5 in the VCS group) and toes (n = 3 in the sutured group and n = 2 in the VCS group). No animal needed to be destroyed because of self-amputation of the toes.
Macroscopic specimens All nerves were studied and photographed in vivo. All nerves maintained approximation at all time points. To demonstrate the ability of the clips alone to maintain approximation, the stay sutures had been removed prior to skin closure in some animals in the VCS group; on harvest the nerves in all these animals were still coapted. The nerve diameter at the time of the original operation was 2.0-2.18 mm. At 5 days and 7 days postoperatively the nerve diameter in the sutured group was m e a n + s.e.m. =2.26__0.08 mm, and at 30 days it was m e a n _ s.e.m. = 2.51 + 0.07 mm. The nerve diameter in the VCS group at 5 days and 7 days was mean+_s.e.m.= 2.22___0.08 mm, and at 30 days it was mean___ s.e.m. = 2.58 _ 0 . 0 7 m m (Table 2). At 5 days and 7 days normal nerve morphology was observed in all specimens in both groups (Fig. 2A). By 30 days no visible excessive swelling of the repair sites was seen, and all nerves had a normal surrounding inflammatory reaction. The clips were embedded into the epineurium; no clip was separated from the nerve in vivo (Fig. 2B). No significant differences in diameter were found between the experimental groups and the control specimens (P = 0.44-0.51).
Immunohistochemistry The monoclonal PGP 9.5 antibody showed, in all nerve sections at all time points, specific strong pan-neuronal immunoreactivity with low background staining. Proximal to the repair site there was intense immunolocalisation to the reactive neuronal cytoplasmic polypeptides at 5, 7 and 30 days. Distally, Wallerian degeneration had removed these reactive antigens at days 5 and 7. At day 5, early regeneration across the repair site was typically seen more peripherally in the sutured nerves and more centrally in the clip-repaired nerves (Fig. 3A). No excessive reaction at the repair site was seen. In one Table 2
Macroscopic specimen: diameter o f the nerve (mm),
values are given as mean + s.e.m. Nerve diameter (mm) 5 days and 7 days a
sutures VCS clips aP = 0.444; bP=0.51.
2.26 _+0.08 2.22 + 0.08
30 days b
2.51 _ 0.07 2.58 + 0.07
Figure 2--(A) In vivo clipped nerve specimen at day 5, prior to harvest, showing minimal disruption to the nerve morphology. (B) In vivo clipped nerve specimen at day 30, prior to harvest. Note that there are no loose clips in the tissue, and the clips have embedded into the epineurium. The specimen shows a normal surrounding inflammatory reaction.
Primary sciatic nerve repair using titanium staples
Figure 3 ~ ( A ) At 5 days regeneration has commenced across the cliprepair site, shown centrally by Texas Red in this specimen. (B) At 7 days the axonal advancement (shown by Oregon Green) has progressed as far as the division into the two main terminal branches, the common peroneal and tibial nerves.
suture-repaired specimen there was no regeneration across the site. Regeneration was more advanced by 7 days (shown by Oregon Green). Generally, there was little variation between the operative groups in terms of PGP 9.5 staining. Axonal regeneration had advanced (563 Ixm) to the division into the two main terminal components, the common peroneal and tibial nerves (Fig. 3B). Complete specimen PGP 9.5 immunoreactivity was visualised by Texas Red at 30 days. The neuronal architecture at the repair site had become more organised in both groups. Under fluorescent microscopy, the repair site was easily identified, partly by the cellular reaction around the suture in the sutured group and the stay suture in the clipped group. No reaction to the clips could be identified. The strong pan-neuronal immunoreactivity continued to the end of the mounted specimen and the separation into the common peroneal and tibial nerves.
Discussion It is generally agreed that primary repair by end-to-end approximation of the stumps is the best surgical choice after severance of a peripheral nerve. Digital nerve injuries can comprise 68% of the nerve injuries presenting to a department. 12 The development of a painful neuroma, particularly in a digit, can be very disabling; the search
333 for non-penetrating sutureless microsurgical repair techniques stems from the desire to improve nerve coaptation and clinical results. Previous experimental sutureless techniques have been proposed, but have led to unpredictable outcomes. The cyanoacrylates s are known cytotoxics, 9 and fibrin seals 6'7 are unable to hold the nerve together against its natural retraction. VCS clips can quickly and successfully anastomose large and small arteries 11,13 and veins, 14 and aid the insertion of vascular prostheses. VCS clips have been used effectively to close other non-vascular tubular structures, and have been used quickly and easily to close linear incisions in the gallbladder 15 and longitudinal ureterotomies. 16 The comparable and minimal inflammatory reaction elicited by the titanium is well documented, tl We have shown that VCS clips can be used as a sutureless method of repairing peripheral nerves. The major advantage of using the clip applier rather than conventional suturing to repair small peripheral nerves is the reduction in the repair time. During the course of these experiments, the clip repair time decreased as experience with the applier increased. Speed does not compromise the quality of the nerve repair. In this study, we found clip repair to be about one-third faster than suture repair, and the difference was statistically significant (P<0.01). Although the speed of application is not crucial when repairing a single nerve, the savings in time during the repair of multiple nerves and arteries (for example in palmar lacerations) could compensate for the additional cost of the clips. The rat sciatic nerve has a poor epineural and perineural structural framework, and the fascicles bulge freely when the nerve is transected. ~7 This intrinsic property of the rat nerve lengthened the operative time compared with that normally expected when repairing a human peripheral nerve. This difficulty was encountered in both experimental groups; hence the times are comparable. Autotomy is an unknown but observable quantity. In all the animals there was a minimal degree of autotomy, and we suggest that the presence of titanium near neural tissue does not cause ectopic firing or hinder the propagation of axonal growth cones. Neuronal hyperplasia forming a neuroma is an important cause of failure of primary nerve repairs. Subjective simple observation of the gross intact specimen in vivo prior to harvest gives an indication of the activity at the repair site. If the clips produced inadequate coaptation, a macroscopic deformity would result at the repair site, but this was not seen in any harvested specimen, and the nerve diameters were within normal limits. The clips are non-penetrating, which may suggest a potential to become lost in the tissues and to fail to maintain approximation of the two ends. No clips were found free from the nerve, and it is also worth noting that the clips are easy to remove and atraumatic to the epineurium if a mistake is made with the applier. We subjectively analysed the data from the immunohistochemistry to determine, at the microscopic level, whether the clips impede the progression of the growth cones across the repair site. PGP 9.5 is an excellent and specific pan-neuronal marker, immunolocalising to neuronal cytoplasmic polypeptides, of as yet unknown
334
function. Monoclonal primary antibody produces enhanced specific staining, ts compared with polyclonal antibodies. At all time points in both operative groups there was unhindered progression of the growth cones across the repair site. Secondary visualisation with both chromophores picked up the strong PGP immunoreactivity in the regenerating axons. The use of two chromophores allowed specific separation and identification of the early time points from the later experiments. Overall, we demonstrated that the titanium had little detrimental effect on axon growth and did not cause excessive buildup of neuronal tissue at the repair site. It would be of supplementary benefit to study the repair site by quantitative assessment at all time points for each operative technique. Analysis of transverse sections, to determine the number of axons crossing the repair site and calculate their diameters, may further demonstrate that titanium clips are an equal repair mechanism to sutures. It can be assumed that non-penetrating clips maintain nerve approximation, have no detrimental effect on nerve regeneration, and are a fast accurate way of achieving peripheral nerve repair. We suggest that this method is comparable to non-absorbable microsuture in the repair of small-diameter nerves.
British Journal o f Plastic Surgery
8. 9. 10. 11.
12.
13.
microsuture in peripheral nerve repair. J Hand Surg 1988; 13A: 273-8. Siedentop KH, Loewy A. Facial nerve repair with tissue adhesive. Arch Otolaryngol 1979; 105: 423--6. Lehman RA, Hayes GJ, Leonard E Toxicity of alkyl 2-cyanoacrylates. I. Peripheral nerve. Arch Surg 1966; 93: 441-6. Almquist EE, Nachemson A, Auth D, Almquist B, Hall S. Evaluation of the use of the argon laser in repairing rat and primate nerves. J Hand Surg 1984; 9A: 792-9. Zhu YH, Kirsch WM, Cushman R, et al. Comparison of suture and clip for rnicrovascular anastomosis. Surg Forum 1985; 36: 492-5. de Medinaceli L, Prayon M, Merle M. Percentage of nerve injuries in which primary repair can be achieved by end-to-end approximation: review of 2,181 nerve lesions. Microsurgery 1993; 14: 244-6. Zeebregts CJ, van den Dungen JJ, Kalicharan D, Cromheecke M, van der Want J, van Schilfgaarde R. Nonpenetrating vascular clips for small-caliber anastomosis. Microsurgery 2000; 20: 131-8.
We thank Dr Michelle Robinson PhD for all her help at University College London in the setting up of the immunohistochemistry experiments.
14. Leppaniemi AK, Wherry DC, Pikoulis E, et al. Arterial and venous repair with vascular clips: comparison with suture closure. J Vasc Surg 1997; 26: 24-8. 15. Leppaniemi AK, Wherry DC, Soltero RG, et al. A quick and simple method to close vascular, biliary and urinary tract incisions using the new Vascular Closure Staples: a preliminary report. Surg Endosc 1996; 10: 771-4. 16. Leppaniemi AK, Wherry DC, Pikoulis E, Hufnagel HV, Fishback N, Rich NM. Ureteral repair with titanium staples: comparison with suture closure. Urology 1998; 51: 553-7. 17. Greene EC. Anatomy of the Rat. New York: Hafner Publishing Company, 1963; XXVII: 131-4. 18. Wilson POG, Barber PC, Hamid QA, et al. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol 1998; 69: 91-104.
References
The Authors
1. Elsberg CA. Technique of nerve suture and nerve grafting. JAMA 1919; 73: 14224. 2. Yin Q, Kemp GJ, Frostick SP. Neurotrophins, neurones and peripheral nerve regeneration. J Hand Surg 1998; 23B: 433-7. 3. Smith JW. Microsurgery of peripheral nerves. Plast Reconstr Surg 1964; 33: 317-29. 4. Lundborg G. A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg 2000; 25A: 391--414. 5. Terzis J, Faibisoff B, Williams B. The nerve gap: suture under tension vs. graft. Plast Reconstr Surg 1975; 56: 166-70. 6. Becker CM, Guening CO, Graft GL. Sutures or fibrin glue for divided rat nerves: Schwann cell and muscle metabolism. Microsurgery 1985; 6: 1-10. 7. Moy O J, Peimer CA, Koniuch MP, Howard C, Zielezny M, Katikaneni PR. Fibrin seal adhesive versus nonabsorbable
Caroline E. Payne MSc, FRCS, Senior House Officer in Plastic Surgery B. George H. Lamberty MA, FRCS, Consultant Plastic Surgeon
Acknowledgements
Department of Plastic and Reconstructive Surgery, 4th Floor, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK.
Steven P, Hunt PhD, Professor Department of Anatomy and Developmental Biology, Medawar Building, University College London, Gower Street, London WC1E 6BT, UK. Correspondence to Miss C. E. Payne. Paper received 1 October 2001. Accepted 11 Febmary 2002, after revision.