Recovery of cutaneous pain sensitivity after end-to-side nerve repair in the rat.

OBJECT: The hypothesis that collaterally sprouting axons from an uninjured donor nerve may provide recovery of pain sensitivity in the skin after end-to-side nerve repair was investigated in rats. In addition, the effect of this technique on the donor nerve was examined. METHODS: The distal stump of the transected peroneal nerve was sutured end to side to the intact sural nerve. No epineurial window or perineurial slit was made in the sural nerve at the site of coaptation. Other nerves in the leg were transected and ligated. Eighteen weeks later, the sural nerve was transected at a site distal from the coaptation site. The residual pain sensitivity in the peroneal innervation field in the instep was documented using the skin pinch test in three of 11 animals. The area of sensitivity encompassed 19 to 40% of the maximum nociceptive innervation area of the normal peroneal nerve. The nerve pinch test revealed functional sensory axons in all communicating peroneal nerves, in which 277+/-119 myelinated axons (mean +/- standard deviation) were found by histological investigation. CONCLUSIONS: The authors conclude that at least partial recovery of sensory function due to collateral sprouting of axons after end-to-side nerve repair is possible in principle. However, the presence of functional sensory axons in the peroneal nerve stumps did not guarantee the recovery of skin sensitivity to pain in all animals. No functional or morphological evidence of an untoward effect of collateral sprouting into the end-to-side communicating nerve was detected in the axons of the donor nerve itself.     

Specificity in peripheral nerve regeneration: a discussion of the issues and the research

 Epineurial and epiperineurial nerve suturing with orientation of the intraneurial funicular pattern are the only useful nerve repair techniques. The research on axonal regeneration was reviewed to determine whether basic research findings may support a new clinical approach to nerve repair. The research indicates that Schwann cell migration from the distal nerve stump is important in tissue specificity; sensory regeneration, but not motor regeneration, shows selectivity; sensory Schwann cells in the distal nerve segment induce not only sensory axons but also motor axons, which are the strongest 1 week after denervation and are influenced by the stump area and the volume of the distal nerve segment; and evidence of topographic specificity is weak. The strong inductive ability of sensory Schwann cells to misdirect motor nerve regeneration to the distal sensory Schwann tubes may not support the use of tube techniques for nerve repair. Received: February 1, 2002     

Reinnervation of muscles in rats after repair of transsected sciatic nerves with Y-shaped and X-shaped silicone tubes. Muscle reinnervation after nerve repair.

Reinnervation of the gastrocnemius and anterior tibial muscles was assessed by measurements of tetanic force after repair of sciatic nerves with Y-shaped or X-shaped silicone tubes in rats. The transsected proximal stump of either the tibial or the peroneal fascicle was introduced into the opening of a Y-shaped silicone tube, or both fascicles were introduced into an X-shaped tube. The distal tibial and peroneal fascicles were inserted into the distal outlets of the tubes leaving a gap of 4 mm between proximal and distal stumps. In the X-shaped tubes the proximal inserts were placed opposite or adjacent to their respective distal parts. Sixteen weeks later reinnervation was evaluated by measurements of tetanic force of the gastrocnemius and anterior tibial muscles after electrical stimulation of the fascicles. There was preferential reinnervation in both types of tubes. In Y-shaped tubes about 90% of the tetanic force could be recorded from both muscles after stimulation of the peroneal and tibial fascicles, respectively. Recovery was lower in the X-shaped tubes, amounting to about 75%. Contractions evoked by misrouted fibres were similar (roughly 40%) in both models. We conclude that motor axons preferentially, but not exclusively, selected a path to reinnervate their original target muscle.     

Lack of topographical specificity in peripheral nerve regeneration in rats.

In a previous study we found that sensory regeneration was neurotropically selective regardless of the end organ, but motor regeneration was not, which made us doubt the existence of topographic specificity. The purpose of the present study was to confirm the existence of topographic specificity in rats. The proximal stump of either the peroneal or tibial nerve was inserted into the proximal limb of a silicone Y-chamber. Both distal stumps of peroneal and tibial nerve were inserted into the distal limbs. The gap between the stumps was set at either 4 mm (n = 8, on each subgroup) or 8 mm (n = 8, on each subgroup). Six weeks later the number of regenerated axons in the distal two limbs were counted and compared. The number of regenerated axons towards the distal tibial nerve side was significantly larger in every model. Regenerated axons from the proximal peroneal stump did not preferentially choose the distal peroneal stump. The existence of topographic specificity is unlikely.     

Long-term results of peripheral nerve repair: a comparison of nerve anastomosis with ethyl-cyanoacrylate and epineural sutures.

The morphological and functional recovery after repair of lesions to the sciatic nerve was studied in adult rats. We compared conventional microsuturing with a synthetic ethyl-cyanoacrylate adhesive. Six months after a unilateral lesion and subsequent repair the tibial branch to the lateral gastrocnemius muscle and the caudal sural cutaneous nerve were examined with electrophysiological measurements of motor and sensory conduction velocity, motor nerve action potentials, and quantitative histological examinations. There was functional reinnervation of motor and sensory nerves in both groups, as shown by equivalent recovery of motor and sensory conduction velocities, and motor nerve action potentials. Histological examination showed no significant difference in the mean diameter, fibre density or the number of regenerated myelinated motor and sensory axons distal to the repair site between the two groups. We conclude that anastomosis of the nerve with ethyl-cyanoacrylate adhesive supports morphological and functional recovery comparable to that of conventional epineural sutures after a unilateral lesion of the sciatic nerve in adult rats.     

Overcoming peripheral nerve gap defects using an intact nerve bridge in a rabbit model

This study investigated the intact nerve bridge technique for overcoming peripheral nerve gap defects in a rabbit model. To create the intact nerve bridge, a 1-cm segment of the peroneal nerve is resected leaving a gap defect. The proximal and distal peroneal nerve stumps are sutured 1 cm apart, in an end-to-side fashion, to the intact tibial nerve epineurium. Four experimental groups were used (n = 10): primary repair of resected segment; intact nerve bridge; nerve autograft; and gap in situ control. Evaluation after 12 weeks included measurement of isometric muscle contraction force, axonal counting, wet muscle weights, and histologic examination. The results of this study support two main conclusions, in a rabbit model: (a) regenerating axons can use the epineurium of an intact nerve to bridge a gap defect; (b) there is no significant difference in the functional recovery between standard nerve autografts and the intact nerve bridge technique.     

Comparison of the neurotropic effects of motor and sensory Schwann cells during regeneration of peripheral nerves.

We examined the inductive ability of motor and sensory Schwann cells on regeneration of motor and sensory axons using a silastic Y chamber, and Lewis rats L5 ventral root (motor) and saphenous nerve (sensory). We developed four experimental models: motor-motor nerve group-proximal motor stump with distal fresh and frozen/thawed motor nerve segments (n = 7); sensory-sensory nerve group-proximal sensory stump with distal fresh and frozen/thawed sensory nerve segments (n = 7); motor-sensory nerve group-proximal motor stump with distal fresh and frozen/thawed sensory segments (n = 8); and sensory-motor nerve group-proximal sensory stump with distal fresh and frozen/thawed motor segments (n = 8). The gap was set at 4 mm. Six weeks postoperatively we compared the number of regenerated myelinated axons in the two distal channels, and found that sensory Schwann cells have a strong inductive ability for regeneration of both sensory and motor axons. Motor Schwann cells have weak inductive ability for regeneration of motor axons and no inductive ability for regeneration of sensory axons.     

Differential response of sensory and motor axons in nerve allografts after withdrawal of immunosuppressive therapy

OBJECT: Rejection of nerve allografts and loss of regenerated host axons after withdrawal of immunosuppressive therapy poses an ongoing challenge in peripheral nerve repair. The present report is of a blinded prospective controlled study in which an established rat model of nerve allotransplantation is used to examine the effect of fiber type on survival and degeneration of nerve allografts after discontinuation of immunosuppression. The authors hypothesized that sensory axons will selectively resist a rejection response, whereas motor axons will degenerate. METHODS: Four-centimeter nerve segments from ACI rats were grafted into peroneal and sural (mixed) or saphenous (sensory) nerve gaps in Lewis rats. In some rats, L4-6 dorsal root ganglia were ablated before grafting, creating pure motor sural and peroneal nerves. All rats received 12 weeks of immunosuppressive therapy to support nerve regeneration into allografts. Immunosuppression with cyclosporin was then withdrawn. At planned death (12-18 weeks postsurgery), graft tissue was subjected to histomorphometric analysis for evaluation of axon survival and loss. Graft rejection led to loss of all axons in approximately 60% of the allograft segments. The mixed nerve group was most prone to complete rejection, with significantly lowered axon counts at Weeks 16 and 18 compared with the Week 12 baseline. Axons from the sensory nerve were least likely to degenerate. The pure motor nerve group axons demonstrated intermediate sensitivity, with a selective loss of larger axons at Week 16 and a significant decrease in axon counts from the Week 12 baseline at Week 18. CONCLUSIONS: Whereas the majority of axons are lost after withdrawal of immunosuppressive therapy from nerve allografts, there is a selective survival of axons from cutaneous sensory nerves and smaller-diameter motor fibers. The biological and molecular mechanisms that make some axons impervious to injury remain to be determined.     

Reverse end-to-side neurotization in a regenerating nerve

Bypass grafting around a neuroma-in-continuity entails coapting a nerve graft above and below the injured segment using two sequential end-to-side repairs. The proximal repair is analogous to what has been classically described as an end-to-side repair; the axons from the intact nerve sprout into the end of a recipient nerve and travel distally. At the distal connection, however, axons in the graft must enter the side of the intact nerve and find their way to appropriate end organs. This process has not been well investigated. To examine this, a reverse end-to-side repair, suturing the distal end of the peroneal nerve to the side of a transected and repaired tibial nerve, was performed in 20 rats. A primary end-to-end repair of the tibial nerve was performed in 10 additional rats. Twelve weeks later, contraction forces of the gastrocnemius muscle were measured following proximal stimulation. Measurements were repeated following elimination of potential axonal pathways to identify which axons (peroneal or tibial) had achieved greater reinnervation. The results indicated that both groups of axons had achieved significant reinnervation. This study supports the idea that a reverse end-to-side repair can result in axonal invasion of an intact but regenerating nerve and achieve functional recovery.     

Effect of conduit repair on aberrant motor axon growth within the nerve graft in rats

After peripheral nerve injury, minimizing axonal misdirection has been a matter of importance to obtain good functional outcomes. In general, it becomes more challenging as the nerve defect length is longer. As previous works suggested that a conduit repair leaving a short gap could induce some target-specific reinnervation, we expected that a distally placed conduit combined with nerve graft would enhance the specificity of reinnervation, especially in dealing with a long gap. To test this, a 14-mm-long gap was created in the rat sciatic nerves and repaired with either 1) whole nerve graft (WG), 2) interfascicular nerve grafts (FG), or 3) whole nerve graft combined with distally placed silicone tube leaving a 5-mm gap (TUBG). At the end of follow up, the extent of target specific reinnervation (measurement of the compound muscle action potentials evoked by stimulation of the sciatic nerve and its tibial and common peroneal fascicles) and the accuracy of motoneuronal projection (sequential retrograde labeling of the common peroneal motor pool) were assessed. Both assessments revealed that groups FG and TUBG had a similar selectivity, which was significantly higher than in group WG. Consistent with these results, the functional recovery as assessed by walking track analysis showed no significant difference between groups FG and TUBG, whereas those were significantly superior to group WG. In contrast, histomorphometric assessment of the regenerating axons and wet muscle weight showed no significant difference among the three groups. In conclusion, conduit repair would have some effects on reducing motor axonal misdirection, and it might be more effective when used in the management of a large defect in combination with nerve graft.     

COMPARISON OF THE NEUROTROPIC EFFECTS OF MOTOR AND SENSORY SCHWANN CELLS DURING REGENERATION OF PERIPHERAL NERVES

We examined the inductive ability of motor and sensory Schwann cells on regeneration of motor and sensory axons using a silastic Y chamber, and Lewis rats L5 ventral root (motor) and saphenous nerve (sensory). We developed four experimental models: motor-motor nerve group-proximal motor stump with distal fresh and frozen/thawed motor nerve segments (n = 7); sensory-sensory nerve group-proximal sensory stump with distal fresh and frozen/thawed sensory nerve segments (n = 7); motor-sensory nerve group-proximal motor stump with distal fresh and frozen/thawed sensory segments (n = 8); and sensory-motor nerve group-proximal sensory stump with distal fresh and frozen/thawed motor segments (n = 8). The gap was set at 4 mm. Six weeks postoperatively we compared the number of regenerated myelinated axons in the two distal channels, and found that sensory Schwann cells have a strong inductive ability for regeneration of both sensory and motor axons. Motor Schwann cells have weak inductive ability for regeneration of motor axons and no inductive ability for regeneration of sensory axons.     

Choline acetyltransferase activity in collateral sprouting of peripheral nerve after surgical intervention: experimental study in rats.

The purpose of this study was to establish an assay of choline acetyltransferase (ChAT) activity to investigate the regeneration of injured peripheral nerve, repaired by end-to-end or end-to-side neurorrhaphy. Murine sciatic and peroneal nerves were exposed, and the peroneal nerve was transected at a site 5 mm from its ramification. For end-to-side neurorrhaphy, an epineurotomy producing a 5-x5-mm window was carried out on the tibial nerve, just above the level of gastrocnemius muscle ramification. The peroneal nerve stump was then sutured end-to-side to the tibial nerve window. For end-to-end neurorrhaphy, the peroneal stump was directly sutured end-to-end. ChAT activity was measured at a site distal to the peroneal stump at 1 to 3 months postoperatively, and the results were compared among four groups: 1) end-to-end neurorrhaphy group; 2) end-to-side neurorrhaphy group; 3) unrepaired group; and 4) positive controls. ChAT activity in the end-to-side neurorrhaphy yielded approximately two-thirds the value of the end-to-end neurorrhaphy, and more than half the value of positive controls at 3 months postoperatively. Histologic sections of the end-to-side and end-to-end sutured peroneal nerve demonstrated large numbers of myelinated axons and Schwann cells at the third postoperative month. All the results demonstrated that end-to-side neurorrhaphy is comparable to well-performed end-to-end neurorrhaphy, thus providing another option for surgical treatment of avulsion nerve injury and massive nerve defect.     

远端神经变性对神经再生放大规律的影响

目的 探讨应用正常供体神经修复预变性受体神经时,再生神经纤维放大的效果.方法 将雄性SD大鼠12只,随机分为变性组、对照组,所有动物均实行手术.变性组应用部分正常腓总神经修复变性8周胫神经远端;对照组应用部分正常腓总神经修复即刻损伤后胫神经远端.术后3个月进行神经电生理测量、肌力测量、组织学观察、神经锇酸染色及有髓神经纤维计数,并计算两组神经再生放大率.结果 变性组和对照组神经传导速度分别为(16.992±3.737)m/s和(23.092±2.788)m/s;肌肉强直收缩力恢复率为(39.642±5.865)%和(71.098±6.778)%;再生有髓神经纤维数分别为1718.2±282.0和3340.0±506.5;神经再生放大率1.581±0.329和3.098± 0.642;以上指标变性组均低于对照组(P＜0.05).结论 应用正常神经修复8周变性神经远端时,神经纤维再生放大率降低,说明损伤远端神经较长时间变性,接受再生神经纤维长入的能力降低.     

Structural and functional regeneration of muscle-related axons after transection and repair of the rat sciatic nerve using nonvascularized autologous fascia as a barrier between tibial and peroneal nerve fascicles

Aberrant reinnervation of target organs caused by misdirected axonal growth at the repair site is a major reason for the poor functional outcome usually seen after peripheral nerve transection and repair. This study investigates whether the criss-crossing of regenerating rat sciatic nerve axons between tibial and peroneal nerve fascicles can be reduced by using non-vascularized autologous fascia as a barrier. The left sciatic nerve was transected and repaired at midthigh as follows: epineurialy sutures (Group A); fascicular repair of tibial and peroneal nerve fascicles (Group B); fascicular repair of tibial and peroneal nerve fascicles separating the two fascicles by non-vascularized autologous fascia (Group C). In the control Group D, only the left tibial fascicle was transected and repaired. Five months postoperatively, the outcome of regeneration was evaluated by histology, by retrograde tracing, and by assessment of the contraction force of the gastrocnemius and tibial anterior muscles. The tracing experiments showed that muscle reinnervation was less abnormal in Group C than in Groups A and B. However, muscle contraction force was not better in Group C than in Groups A and B. With respect to the peroneal nerve innervated muscle, the contraction force in Group C was significantly lower than in Group B. The histologic picture indicated that this inferior result in Group C was due to nerve compression caused by fibrotic scar tissue at the site of the fascia graft. Results of this study show that a non-vascularized autologous fascial graft used as a barrier between two sutured nerve fascicles in adjacency reduces criss-crossing of regenerating axons between the fascicles, but causes significant nerve compression.     

Selectivity of distal reinnervation of regenerating mixed motor and sensory nerve fibres across muscle grafts in rats

This study investigated target specificity during axonal regeneration of a mixed motor and sensory nerve towards respective targets. The femoral nerves in rats were divided and allowed to grow across a 6 mm gap interposed with frozen and thawed muscle grafts towards their distal motor and sensory nerve stumps. Fourteen weeks later the number of motoneurons projecting axons into the motor and sensory branches were determined by retrograde axonal tracing using horse-radish peroxidase. There were significantly higher numbers of motoneurons (p = 0.0034) projecting into the motor nerve than the sensory nerve. Efferent axons of a mixed nerve selectivity grew into motor branches when allowed to regenerate across a 6 mm gap interposed with muscle grafts. It is possible that a deliberately created 'structured gap' during repair of mixed nerves could improve axonal matching by allowing expression of neurotropism.     

Specificity of Reinnervation and Motor Recovery after Interposition of an Artificial Barrier Between Transected and Repaired Nerves in Adjacency – An Experimental Study in the Rat

Summary  Non-specific re-innervation of target organs caused by misdirected axonal growth at the repair site is regarded as one reason for a poor functional outcome after peripheral nerve transsection and repair. This study investigates the rate of aberrant re-innervation and its influence on motor recovery in the rat sciatic nerve using artificial sheets as barrier between tibial and peroneal nerves.  The sciatic nerve was transsected and repaired as follows: epineural sutures (A×6), fascicular repair of tibial and peroneal nerves respectively (B×8), and the same as in group B, but separating both nerves using an Integra?-sheet with silicone (C×8), or Integra? without silicone (D×8). As control, solely the tibial nerve was transsected and repaired (E×5).  Final investigations after 4 months revealed that in group C, 50% of the Integra?-silicone sheets were dislocated. No dislocation was found in group D. Muscle contraction force of the gastrocnemius muscle was significantly higher in group E as compared to all other groups. However although not significant, group D showed a consistently higher muscle contraction force than groups A, B, and C. Histology in groups A, B, and C with dislocated sheets demonstrated multiple axons growing from the tibial to the peroneal nerve and vice versa. In groups D and E, no such axonal growth was visible. These findings were confirmed by a significantly higher rate of specific re-innervation of the soleus muscle using sequential retrograde double labelling technique.  Results of this study suggest that an artificial sheet such as Integra? bears the potential of preventing aberrant re-innervation between repaired adjacent nerves resulting in improved motor recovery. Clinically, this technique may be of importance for brachial plexus, sciatic nerve, and facial nerve repair.     

Lack of topographical specificity in sensory nerve regeneration through muscle grafts in rats.

Regenerating sensory axons of each receptor class make new connections with similar denervated receptors. This study investigates to what extent these axons return to their original receptive field. The lateral cutaneous nerves of the thigh in rats were divided and allowed to regenerate across a 6 mm. gap interposed with frozen and thawed muscle graft towards their original distal nerve stump and a "foreign" sensory nerve, the saphenous nerve. 16 weeks later, myelinated axon counts of 26 pairs of distal nerves showed no preferential growth towards the original receptive field. Lack of topographic specificity during sensory nerve regeneration may explain the faulty localisation of sensation after nerve repair in clinical practice. Following sensory nerve regeneration, the somatosensory cortex receives accurate afferent information but from disparate skin sites; this probably alters the relationship of overlapping sensory fields and may be the cause of distorted pattern recognition.     

Using intact nerve to bridge peripheral nerve defects: an alternative to the use of nerve grafts

This preliminary study was conducted to determine whether a regenerating peripheral nerve in a rat model can use the epineurium of an intact nerve to bridge a nerve gap defect. To create the intact nerve bridge a 1-cm segment of the peroneal nerve is resected leaving a gap defect. The proximal and distal peroneal nerve stumps are sutured 1-cm apart, in an end-to-side fashion, to the epineurium of the intact tibial nerve. The following experimental groups were used (n = 12): group A, immediate primary repair of resected segment; group B, intact nerve bridge technique; group C, nerve autograft; and group D, gap in situ control. Evaluation 12 weeks after surgery included measurement of the tibialis anterior muscle contraction force, axonal counting, wet weight of the tibialis anterior muscle, and histologic examination. The results of this animal study support 3 main conclusions: regenerating axons can use the epineurium of an intact nerve to bridge a gap in nerve continuity; when using functional recovery to assess regeneration, there is no significant difference between standard nerve autografts and the intact nerve bridge technique; and based on histologic examination, the intact nerve bridge technique does not injure the intact tibial nerve used to bridge the gap defect. Taken together, the results of this preliminary animal study suggest that the intact nerve bridge technique may be a potential alternative to standard nerve autografts in appropriate circumstances. Further investigation in a higher animal model is warranted before considering clinical application of the intact nerve bridge technique.     

Motor functional and morphological findings following end-to-side neurorrhaphy in the rat model.

Nerve repair cannot always be achieved by the conventional end-to-end technique. This study evaluated the functional recovery of nerves repaired with end-to-side neurorrhaphy in a rat model. The right peroneal nerves of 80 female rats were transected and divided into four groups. In group A, the nerve ends were separated and remained unrepaired; in group B, the distal peroneal ends were directly sutured to the epineurium of the tibial nerves in end-to-side fashion; in group C, the distal ends were sutured through an epineurial window at the repair site in end-to-side fashion; and in group D, the nerve ends were reconnected by the traditional end-to-end technique. Evaluation included gait analysis by calculation of a peroneal functional index, measurement of contractile function of the extensor digitorum longus muscle, wet weight of the extensor digitorum longus, and histological examination. The findings of this study suggested the following: (a) end-to-side neurorrhaphy allows effective motor functional recovery, demonstrated by earlier improvement of the peroneal functional index, stronger muscle contractile function, greater muscle weight, and higher density of regenerated axons compared with unrepaired nerves; (b) removal of the epineurium of the donor nerve at the nerve coaptation site increases the effectiveness of end-to-side neurorrhaphy, but the epineurium appears to be a partial barrier to axonal regeneration; (c) removal of the epineurium does not affect the structure and function of the donor nerve; and (d) end-to-end repair achieved the best functional recovery among the four groups; therefore, end-to-side repair should be considered as a potential alternative only when no proximal nerve is available.     

Morphological and functional evaluation of leg-muscle reinnervation after coupler coaptation of the divided rat sciatic nerve

Mechanical couplers are successfully used for microvascular venous anastomoses. The advantages include a simple and fast technique and a high patency rate. Couplers offer a secluded coaptation site, and might also be of use in peripheral nerve repair. The present study was designed to investigate coupler coaptation of the rat sciatic nerve, evaluating the number and locations of motor and sensory neurons projecting to the selected muscles as well as stimulation-induced muscle contraction force. Adult rats underwent either suture or coupler repair after left sciatic nerve transection. In all rats, the experimental side was compared to the healthy right side. Evaluation after 20 weeks included retrograde labeling of motoneurons and dorsal root ganglion neurons projecting to the tibial anterior muscle and to the tibial posterior muscle, histology, muscle contraction force (tibial anterior muscle and gastrocnemius muscle), and a pinch reflex test. The results show that the suture and the coupler groups did not differ significantly regarding the examined parameters, except for discrete signs of nerve compression at the coaptation site after coupler repair due to fibrous tissue ingrowth. However, this did not impair axonal regeneration. Importantly, axonal outgrowth from the repair site to the surrounding tissue was not observed after coupler coaptation, but it was observed after suture repair. These results suggest that couplers may be of value for repair of nerves in adjacency to avoid axonal crisscrossing between nerves during regeneration.