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Background: Adjunctive pharmacological treatment may improve nerve regeneration. We investigated nerve regeneration processes of PXL01 - a lactoferrin-derived peptide - after repair of the sciatic nerve in healthy Wistar rats. Materials & methods: PXL01, sodium hyaluronate (carrier) or sodium chloride was administered around the repair. After 6 days axonal outgrowth, Schwann cell response, pan- (CD68) and pro-healing (CD206) macrophages in sciatic nerve, sensory neuronal response in dorsal root ganglia (DRG) and expression of heat shock protein 27 (HSP27) in sciatic nerves and DRGs were analyzed. Results: Despite a lower number of pan-macrophages, other investigated variables in sciatic nerves or DRGs did not differ between the treatment groups. Conclusion: PLX01 applied locally inhibits inflammation through pan-macrophages in repaired sciatic nerves without any impact on nerve regeneration or pro-healing macrophages.

Reconstruction of nerve defects is a clinical challenge. Autologous nerve grafts as the gold standard treatment may result in an incomplete restoration of extremity function. Biosynthetic nerve conduits are studied widely, but still have limitations. Here, we reconstructed a 10 mm sciatic nerve defect in healthy rats and analyzed nerve regeneration in poly (ε-caprolactone) (PCL) conduits longitudinally divided by gold (Au) and gold-cobalt oxide (AuCoO) nanoparticles embedded in poly-propylene poly-ethylene glycol (PPEG) membranes (AuPPEG or AuCoOPPEG) and compared it with unmodified PPEG-membrane and hollow PCL conduits. After 21 days, we detected significantly better axonal outgrowth, together with higher numbers of activated Schwann cells (ATF3-labelled) and higher HSP27 expression, in reconstructed sciatic nerve and in corresponding dorsal root ganglia (DRG) in the AuPPEG and AuCoOPPEG groups; whereas the number of apoptotic Schwann cells (cleaved caspase 3-labelled) was significantly lower. Furthermore, numbers of activated and apoptotic Schwann cells in the regenerative matrix correlated with axonal outgrowth, whereas HSP27 expression in the regenerative matrix and in DRGs did not show any correlation with axonal outgrowth. We conclude that gold and cobalt-oxide nanoparticle modified membranes in conduits improve axonal outgrowth and increase the regenerative performance of conduits after nerve reconstruction.

Objective: The aim of the study was to examine sensory behaviour and functional recovery in rats during nerve compression and after decompression. Compression injury is a far more common condition than nerve transection. The condition is characterised by numbness and a tingling/burning sensation, and some patients experience pain and allodynia during compression or after decompression treatment. The aetiology is in many cases unknown. Thus, further studies are of great importance for the understanding of this condition., Methods: In the present study, behavioural responses to tactile stimulation, thermal pain, as well as functional sensorimotor behaviour were investigated in rats before, during severe compression, and after decompression. The sciatic nerve of the rats was experimentally compressed for 3 or 28 days, whereafter surgical release, i.e. decompression, of the nerve was performed and the rats were examined up to ∼9 weeks., Results: An altered behaviour was found in response to compression injury, which is mitigated after early decompression treatment., Conclusions: These findings indicate that early intervention during severe compression injuries is of great importance for recovery and restoration of nerve function and, thus, should have an impact on clinical routines regarding treatment of compression injuries.

Exogenous granulocyte-colony stimulating factor (G-CSF) has emerged as a drug candidate for improving the outcome after peripheral nerve injuries. We raised the question if exogenous G-CSF can improve nerve regeneration following a clinically relevant model - nerve transection and repair - in healthy and diabetic rats. In short-term experiments, distance of axonal regeneration and extent of injury-induced Schwann cell death was quantified by staining for neurofilaments and cleaved caspase 3, respectively, seven days after repair. There was no difference in axonal outgrowth between G-CSF-treated and non-treated rats, regardless if healthy Wistar or diabetic Goto-Kakizaki (GK) rats were examined. However, G-CSF treatment caused a significant 13% decrease of cleaved caspase 3-positive Schwann cells at the lesion site in healthy rats, but only a trend in diabetic rats. In the distal nerve segments of healthy rats a similar trend was observed. In long-term experiments of healthy rats, regeneration outcome was evaluated at 90days after repair by presence of neurofilaments, wet weight of gastrocnemius muscle, and perception of touch (von Frey monofilament testing weekly). The presence of neurofilaments distal to the suture line was similar in G-CSF-treated and non-treated rats. The weight ratio of ipsi-over contralateral gastrocnemius muscles, and perception of touch at any time point, were likewise not affected by G-CSF treatment. In addition, the inflammatory response in short- and long-term experiments was studied by analyzing ED1 stainable macrophages in healthy rats, but in neither case was any attenuation seen at the injury site or distal to it. G-CSF can prevent caspase 3 activation in Schwann cells in the short-term, but does not detectably affect the inflammatory response, nor improve early or late axonal outgrowth or functional recovery., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Knowledge about nerve regeneration after nerve injury and reconstruction in appropriate diabetic animal models is incomplete. Short-term nerve regeneration after reconstruction of a 10-mm sciatic nerve defect with either a hollow chitosan conduit or an autologous nerve graft was investigated in healthy Wistar and diabetic Goto-Kakizaki (GK) rats. After 21 days, axonal outgrowth, the presence of activated and apoptotic Schwann cells and the thickness of the formed matrix in the conduits were measured. In general, nerve regeneration was superior in autologous nerve grafts. In chitosan conduits, a matrix, which was thicker in diabetic rats, was formed and was positively correlated with length of axonal outgrowth. Axonal outgrowth in conduits and in nerve grafts extended further in diabetic rats than in healthy rats. There was a higher percentage of activating transcription factor 3 (ATF3)-immunostained cells in nerve segments from healthy rats than in diabetic rats after autologous nerve graft reconstruction. In chitosan conduits, more cleaved caspase 3-stained Schwann cells were generally observed in the matrix from the diabetic rats than in healthy rats. However, there were fewer apoptotic cells in the distal segment in diabetic rats reconstructed with a chitosan conduit. Preoperative glucose levels were positively correlated with axonal outgrowth after both reconstruction methods. Axonal regeneration was better in autologous nerve grafts than in hollow chitosan conduits and was enhanced in diabetic GK rats compared to healthy rats after reconstruction. This study provides insights into the nerve regeneration process in a clinically relevant diabetic animal model., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Multiple nerve branches were created during the regeneration procedure after a nerve injury and such multiple branches are suggested to be used to control, for example, prosthesis with many degrees of freedom. Transected rat sciatic nerve stumps were inserted into a nine mm long silicone tube, which contained four, five mm long, smaller tubes, thus leaving a five mm gap for regenerating nerve fibers. Six weeks later, several new nerve structures were formed not only in the four smaller tubes, but also in the spaces in-between. The 7-9 new continuous nerve structures, which were isolated as individual free nerves after removal of the tubes, were delineated by a perineurium and contained both myelinated and unmyelinated nerve fibers as well as blood vessels. Stimulation of the proximal nerve elicited contractions in distal muscles. Thin metal electrodes, inserted initially into the smaller tubes in some experiments, became embedded in the new nerve structures and when stimulated contractions of the distal muscles were observed. The "tubes within a tube" technique, creating multiple new nerves from a single "mother" nerve, can be used to record multiple signals for prosthetic device control or as sources for supply of multiple denervated targets.

Phosphorylation of extracellular-signal-regulated kinase 1/2 (p-ERK 1/2) was investigated by immunohistochemistry at 30 min, 1 h, and 48 h after nerve transection in the sciatic nerve of healthy and diabetic [streptozotocin (STZ)-induced diabetes mellitus and BioBreeding (BB; i.e. DR.lyp/lyp or BBDP)] rats. Transection injury increased the intensity of p-ERK 1/2 in nerve stumps at all time points. Staining was confined to Schwann cells with occasional faint staining in single axons. In diabetic rats, a lower intensity of p-ERK 1/2 was found at 1 and 48 h in the distal and proximal nerve stumps compared with healthy rats. STZ-induced diabetic rats were not different from BB rats. p-ERK 1/2 is activated differentially in Schwann cells after nerve injury in diabetic rats, whereas activation in STZ-induced diabetic rats did not differ from BB rats.

Background: A delay in surgical nerve repair results in impaired nerve function in humans, but mechanisms behind the weakened nerve regeneration are not known. Activating transcription factor 3 (ATF3) increases the intrinsic growth state of injured neurons early after injury, but the role of long-term changes and their relation to axonal outgrowth after a delayed nerve repair are not well understood. ATF3 expression was examined by immunohistochemistry in motor and sensory neurons and in Schwann cells in rat sciatic nerve and related to axonal outgrowth after transection and delayed nerve repair (repair 0, 30, 90 or 180 days post-injury). Expression of the neuronal cell adhesion molecule (NCAM), which is expressed in non-myelinating Schwann cells, was also examined., Results: The number of neurons and Schwann cells expressing ATF3 declined and the length of axonal outgrowth was impaired if the repair was delayed. The decline was more rapid in motor neurons than in sensory neurons and Schwann cells. Regeneration distances over time correlated to number of ATF3 stained neurons and Schwann cells. Many neurofilament stained axons grew along ATF3 stained Schwann cells. If nerve repair was delayed the majority of Schwann cells in the distal nerve segment stained for NCAM., Conclusion: Delayed nerve repair impairs nerve regeneration and length of axonal outgrowth correlates to ATF3 expression in both neurons and Schwann cells. Mainly non-myelinating Schwann cells (NCAM stained) are present in distal nerve segments after delayed nerve repair. These data provide a neurobiological basis for the poor outcomes associated with delayed nerve repair. Nerve trunks should, if possible, be promptly repaired.

Costimulation blockade can prevent rejection of nerve allografts in short-term studies. We tested if costimulation blockade also prevented rejection of nerve allografts in long-term experiments, thereby improving functional recovery. A 7-mm sciatic nerve defect in C57/BL6 mice was bridged either by nerve allografts from Balb/C mice or by isogenic nerve grafts (isografts) from C57/BL6 mice. Costimulation blockade in the form of a triple treatment with anti-LFA-1, anti-CD40L, and CTLA4Ig was given at post-operative days 0, 2, 4, and 6 (intraperitoneal). Control mice (placebo; allografts) with nerve grafts were treated with isotype antibodies during the same time period. After 49 days, tetanic muscle force, wet weight of gastrocnemius muscle, histology, and morphometry in the tibial nerve were evaluated. Costimulation blockade diminished rejection of the nerve allografts. Axons bridged the graft. Treatment increased wet weight of the gastrocnemius muscle and resulted in a higher mean myelin area/nerve fiber in the tibial nerve distal to the nerve grafts. Tetanic muscle force and number of axons in tibial nerve showed no differences between groups. We conclude that rejection is suppressed by costimulation blockade. Treatment improves recovery of target muscle and myelination after nerve allografting.

Cultured Schwann cells in tendon autografts for nerve repair improve the early phase of nerve regeneration in rat sciatic nerves as judged by the rate of axonal outgrowth. We tested the long-term effects on functional recovery using measurements of muscle force, the number of axons and myelination, using morphometry. In addition, we recorded wet weight of the gastrocnemius muscle. Schwann cell cultures were prepared from predegenerated nerves. Ten and 15 mm defects in rat sciatic nerves were bridged using bilateral tendon autografts with Schwann cell-seeded tendon autografts on one side, and untreated tendon autografts on the other. Animals were evaluated at six and 12 weeks, respectively. At six weeks, myelination, as judged by G-ratio (ratio of axonal diameter to diameter of nerve fibres), was significantly increased in tendon autografts pretreated with Schwann cells in 10mm defects. No such difference was seen in the 15 mm defects. We found no difference in functional recovery, other morphometric variables, or muscle weight between the two grafts. We conclude that early effects on nerve regeneration using transplantation of cultured Schwann cells in rat sciatic nerves are temporary. Other strategies are necessary to obtain lasting effects on functional recovery.

The aim of this study was to investigate if costimulation blockade could be used to modulate the immune response, to prevent rejection, and to stimulate regeneration into nerve allografts. Nerve allografts from Balb/C mice, and isogenic nerve grafts (isografts) from C57/BL6 mice, were used to bridge a 7-mm gap of the sciatic nerve in C57/BL6 mice. Allograft recipients were treated with either a triple treatment with anti-lymphocyte function antigen-1 (anti-LFA), anti-CD40 ligand (anti-CD40L), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (anti-CTLA4Ig) or isotype antibodies (placebo) at postoperative days 0, 2, 4, and 6 (intraperitoneal). After 5 or 9 days, the nerve grafts, together with the proximal and the distal nerve segments, were evaluated by histology and immunocytochemistry for inflammatory cells [CD4-positive (CD4+) and CD8-positive (CD8+) staining cells] and axonal outgrowth (neurofilaments). The immune response was inhibited by costimulation blockade with less extensive inflammation and a lower number of CD4+ staining cells in triple-treated allografts at 9 days. The regeneration rate was significantly faster in isografts (0.75 mm/day) compared with allografts with placebo treatment (0.39 mm/day), but not when compared with triple-treated allografts (0.49 mm/day). At 9 days, the axons were significantly longer in nerve isografts than in nerve allografts, irrespective of treatment. Hence, costimulation blockade neither increased the regeneration rate nor the outgrowth length in triple-treated allografts. We conclude that costimulation blockade inhibits the immune response in nerve allografts without deterring early axonal outgrowth.

Staining by activating transcription factor 3 (ATF3), a neuronal marker of nerve injury, was examined by immunocytochemistry in neurons and Schwann cells after crush or transection (regeneration inhibited) of rat sciatic nerve. ATF3 immunoreactivity peaked in neurons after three days and then gradually subsided to normal within 12 weeks after the crush. The response lasted somewhat longer and declined over time in spinal cord neurons but not in those of dorsal root ganglia (DRG) after transection, indicating a differential regulation of sensory and motor neurons. ATF3 expression was more pronounced in Schwann cells, and remained longer after transection, implying that to some extent regenerating axons produce signals that reduce ATF3 expression in Schwann cells. However, even after transection without repair (no contact with regenerating axons), ATF3 expression in Schwann cells in the distal segment decreased over time suggesting that regenerating axons are not entirely responsible for the down-regulation. These findings have clinical implications on when it is worthwhile to reconstruct nerve injuries.

A new method of acute dissociation of Schwann cells was used to study the effect of addition of such cells to a tendon autograft--a recently-described graft material--on peripheral nerve regeneration in rats. Autologous Schwann cells were obtained from enzymatic dissociation of predegenerated nerves. The tendon autografts were supplied with Schwann cells through brief in vitro coincubation. Schwann cell-free tendon autografts were used as controls. Axonal outgrowth was measured immunohistochemically after four, seven, and 10 days. At seven days, outgrowth was significantly longer in the pretreated autografts. The use of acutely-dissociated Schwann cells is a new approach to tissue engineering in nerve reconstruction, and may abolish the need for time-consuming culture of Schwann cells.

In the present study, expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in rat dorsal root ganglion (DRG) neurons and sciatic nerve following experimental sciatic nerve compression was studied with the use of quantitative immunohistochemistry and in situ hybridization. Previously, we have investigated changes in PACAP expression after nerve transection and, here, the far more frequently encountered condition of nerve compression injury is examined. Nerve compression was performed unilaterally on the rat sciatic nerve, at mid-thigh level, by application of a narrow silicone tube around the nerve for 3, 7, 14 or 28 days, respectively. We detect a statistically significant upregulation in the number and density of PACAP mRNA expression in both small and large DRG neurons in response to nerve compression. An increased number of PACAP-immunoreactive neurons is also found in the ipsilateral DRG. In addition, PACAP immunoreactivity is observed in the compressed sciatic nerve segment and adjacent nerve tissue after nerve compression. The present findings can be compared with previous studies where we have shown that PACAP expression is upregulated in DRG; in response to peripheral inflammation (primarily in small-medium neurons), and after axotomy (dramatic upregulation in medium-large neurons). In view of the recent findings of an increased PACAP expression in DRG after nerve compression, as well as the previous findings of a modulation of PACAP expression in response to axotomy and inflammation, it is likely that PACAP is also involved in the modulation of the response to peripheral nerve compression.

Purpose: To compare the longitudinal suture model for bridging nerve defects with direct approximation under tension or with autologuos nerve grafting., Methods: Seven mm nerve defects in the rat sciatic nerve were repaired by either of these three methods. Evaluation was performed at twelve weeks by morphometry of the tibial nerve distal to the repair site and by weight of the gastrocnemius muscle, an indicator of target reinnervation., Results: The number of nerve fibers and myelin areas in the tibial nerve were similar for all repair methods as were the weight of the gastrocnemius muscle., Conclusions: Longitudinal sutures can be used to bridge short nerve defects and could be an alternative to nerve grafting., (Copyright 2004 IOS Press)

Galanin is probably involved in nociceptive sensory processing in spinal cord. We investigated whether a common injury, peripheral nerve compression, induced up-regulation of galanin (immunocytochemistry) in sensory neurons in rats 6 or 14 days post-injury and compared the response with other nerve injuries. Sciatic nerve compression increased the number of galanin positive sensory neurons as compared to uninjured and contralateral dorsal root ganglia. Complete transection was more efficient than a partial transection and a slight compression injury as an inducer of galanin. Mainly small diameter sensory neurons became positive but also some large diameter neurons. We conclude that nerve compression up-regulates galanin in sensory neurons. The extent of the induction could be related to the severity of nerve injury.

Previous experimental studies have shown positive effects of hyperbaric oxygen treatment in the early regeneration phase in the first few days following a nerve injury. In this study, functional effects of hyperbaric oxygen treatment were studied in 2 series of rats after an injury to the sciatic nerve - a standardized crush injury and nerve transection and repair, respectively. Postoperatively the animals were treated with 100% oxygen at 2.5 atmospheres absolute pressure for 90 minutes and the treatment was employed twice daily for 7 days. The animals were evaluated with walking track analysis up to twice weekly. The experiments were terminated after 90 days when the tetanic force was measured in the tibial anterior and gastrocnemius muscles. No statistically significant differences were found in either of these tests. It is concluded that hyperbaric oxygen treatment, given in accordance with clinical protocols used in limb crush injuries and other peripheral conditions, was not effective in the restoration of gait or the muscular strength after 90 days in rats after these nerve injuries. This study does not support nerve crush injury or nerve transection and repair as indications for hyperbaric oxygen treatment.

One hindlimb of a rat was subjected to tourniquet compression (150, 200 and 300 mmHg; 2 h). After 6 days a 10 mm sciatic or tibial nerve graft from the compressed limb was sutured to bridge a 3-4 mm gap in the sciatic nerve of the non-compressed limb. The distances of regenerating sensory axons were measured 6 days post surgery (tibial grafts, 8 days). Compression at 200 and 300 mmHg led to significantly longer regeneration distances than those seen in controls. Incorporation of BrdU and expression of p75 receptor by non-neuronal cells (Schwann cells) in sciatic nerves 6 days after compression (150 and 300 mmHg; 2 h) was also increased as a sign of Schwann cell activation. Tourniquet compression may be used as a non-invasive method to enhance nerve regeneration in nerve grafts.

Object: The authors have previously shown that longitudinal sutures without artificial tube support regeneration across a 7-mm gap in the rat sciatic nerve. In the present study, the authors compared this new approach with the use of autologous nerve grafts across short defects and examined whether the approach could be used to support regeneration across extended gaps and whether the interposition of a short nerve segment (the stepping-stone procedure) was applicable in this model., Methods: Longitudinal sutures were used to bridge 7- and 15-mm gaps in the rat sciatic nerve. Contralateral comparisons were made to nerve autografts in the 7-mm group and to sutures plus a short interposed nerve segment in the 15-mm group. Regeneration was evaluated at 2, 4, and 12 weeks by using immunocytochemical analysis for Schwann cells, neurofilament protein, and macrophages and at 12 weeks also by using histological examination, including morphometry in the distal tibial trunk and tetanic force measurements in the gastrocnemius muscle., Conclusions: The authors found that the results of regeneration after repair with longitudinal polyglactin sutures across short defects were not significantly different from those produced by the use of autologous nerve grafts. Regeneration, although poor, occurred along sutures across extended gaps and was significantly enhanced by an interposed nerve segment acting as a Schwann cell resource in this model.

We studied the effect of hyperbaric oxygen treatment on axonal outgrowth in grafts of sciatic nerves in 40 rats. The sciatic nerve was transsected and a 10 mm long segment from the opposite side was immediately sutured in as a nerve graft. Postoperatively 17 animals were treated with 100% oxygen at 3.2 atmospheres absolute pressure for 45 minutes and the treatment was repeated at four and eight hours postoperatively and then every eight hours until evaluation. At seven days the axonal outgrowth was evaluated by immunohistochemical staining of neurofilaments in the nerve grafts. The axonal outgrowth was significantly longer in animals treated with hyperbaric oxygen. We conclude that hyperbaric oxygen can improve nerve regeneration in sciatic nerve grafts in rats.

To determine whether drying of nerve grafts can affect axonal outgrowth and proliferation of non-neuronal cells, nerve segments dried for 0-60 min were used as nerve grafts to bridge a gap in transected rat sciatic nerves. Axonal outgrowth was measured by pinch reflex test and confirmed by immunocytochemical staining of neurofilaments. The proliferation of non-neuronal cells was measured by incorporation of BrdU in the dried nerve segments. Drying of the nerve segment for 60 min reduced the length of axonal outgrowth to 66 and 76% 3 and 6 days, respectively, after the grafting procedure. At that time point the number of proliferating cells was reduced by 51%. It is concluded that the number of proliferating non-neuronal cells is reduced in dried nerve segments which only partly impairs axonal outgrowth. Factors other than Schwann cells are probably important for an optimal mileue for regeneration in nerve grafts.

A long defect (15 mm) in rat sciatic nerve was repaired with a bioartificial nerve graft composed of a silicone tube and seven synthetic filaments of five types (polyamide, catgut, polydioxanone, and two types of polyglactin, normal and quickly-absorbed) inserted longitudinally into the tube. In all cases in which filaments were used a regenerating bridge was obtained in the tube after three months in contrast to empty silicone tubes, in which no structure was observed. There was a 6%-46% recovery of isometric muscle contractility of the anterior tibial and gastrocnemius muscles with positive pinch reflex test in most cases. Myelinated axons were seen in the regenerating tissue between the filaments but not directly in contact with them, and there were varying numbers of macrophages close to the filaments. Silicone tubes with filaments, regardless of type of filament, induced nerve tissue to regenerate and resulted in functional recovery through a 15 mm nerve gap not achieved with empty tubes. Nerve promoting factors may be applied to the filaments and the model is a valuable tool for further development of artificial nerve grafts.

We have proposed that it is sufficient to provide a simple substratum on which regenerating axons may traverse a gap in a peripheral nerve. To test this hypothesis we set up a new experimental model in which sutures were used to bridge a 10 mm long defect in a peripheral nerve. A defect was created bilaterally in 25 rat sciatic nerves. The cut ends of the nerve were joined by three laps of a continuous suture, on one side with 8/0 polyamide (non-absorbable) and on the other with 8/0 polyglactin (absorbable), leaving a 7 mm gap. At two weeks a matrix that contained capillaries, fibroblast-like cells, and mononuclear cells had formed between the nerve endings, and the sutures were surrounded by foreign-body-like tissue reactions. At four weeks axons had grown into the distal nerve segment on both sides in 65%-90% of the cases as indicated by a response to the pinch reflex test and immunocytochemistry for presence of neurofilament protein. Axons were organised in minifascicles and these tended to grow larger as the demarcation of the entire regenerated segment by a perineurial-like structure improved with time. At 12 weeks axonal counts of cross-sections of the distal tibial trunk showed many myelinated nerve fibres but no significant difference in axonal counts or degree of myelination between the polyamide and polyglactin sides. The results show that conventional sutures alone are sufficient to support regeneration across a short gap in a peripheral nerve, a method that may be of potential clinical value.

We have previously shown that a tendon autograft from the rat tail can support regeneration across a gap in the continuity of the rat sciatic nerve. In this study, we characterized the spatiotemporal progress of regeneration in such a graft bridging a 10-mm defect in the sciatic nerve of the rat. Regeneration was assessed 7, 10, 14, or 18 days postoperatively, by immunocytochemistry for axons, Schwann cells, and macrophages and histochemistry for blood vessels. Axonal regrowth into the grafts showed an initial delay period of 6.8 days, whereafter axons grew at a rate of 1.0 mm/day. Schwann cells grew into the grafts from both the proximal and distal nerve segments, proximally just ahead of the axonal front. Macrophages were initially preferentially located at the periphery of the grafts, but gradually increased inside the grafts. Blood vessels entered the grafts from both the proximal and distal aspects of the severed nerve. The onset of vascularization appeared to coincide with axonal regeneration into the grafts.

Objectives: Exposing a hind leg of a rat to vibration induces an injury to the sciatic nerve--a so called conditioning lesion. After such injury induced by vibration the regenerative capacity of the nerve is improved and can be detected as an increased axonal outgrowth from a test crush lesion to the same nerve. The purpose was to study whether the effect of a conditioning lesion induced by vibration can be prevented by local treatment with a Ca2+ channel blocker D600., Methods: D600 (methoxyverapamil) or Ringer's solution was locally applied to the sciatic nerve on one side through a silicone tube connected to a miniosmotic pump, which was implanted subcutaneously. During the same period the hind leg was exposed to vibration (80 Hz; 32 m/s2 root mean squared) for five hours daily for five consecutive days. The other hind leg was not vibrated. After the end of exposure to vibration the sciatic nerves were crushed bilaterally (test crush lesions) and three or six days later the regeneration distances of sensory axons were measured by the pinch reflex test., Results: Nerves in the control animals (without implanted miniosmotic pumps and nerves on to which Ringer's solution was locally applied) that were exposed to vibration showed a significantly increased outgrowth length of sensory axons from the test crush lesion compared with the non-vibrated side. Such an effect of a conditioning lesion from the exposure to vibration was suppressed by local application of D600., Conclusions: Local administration of a Ca2+ channel blocker D600 can prevent the effect of a conditioning lesion-that is, the nerve injury induced by vibration can be inhibited by D600. This may have implications for the treatment of patients with neuropathy of the hand induced by vibration.

Application of granulation tissue, which is rich in macrophages, to a peripheral nerve induces a conditioning effect, in that it enhances the regeneration capability of peripheral nerves after a test crush lesion. The temporal aspects of this response and the role of macrophages and interleukin-1 beta (IL-1 beta) were studied in the sciatic nerves of 71 rats. Granulation tissue was implanted close to the sciatic nerve and test crush lesions were applied after various periods of time (0-21 days). Regeneration was evaluated after an additional two, three, four, or six days. Regeneration distances were longer in granulation-treated nerves than in nerves treated with subcutaneous tissue. Furthermore, in animals in which the test crush lesion was made at the same time as the granulation tissue was implanted (n = 6), regeneration distances were longer, 8.1 (0.8) mm compared with 7.2 (0.6), than those in which the crush was made after conditioning intervals of 3 (n = 6, 7.6 (0.4) compared with 6.9 (0.4), p = 0.03); 7 (n = 6, 7.4 (0.4) compared with 6.6 (0.1), p = 0.03); and 21 days [(n = 8, 7.2 (0.6) compared with 6.4 (0.5)]. Inactivation of the granulation tissue by freezing suppressed the conditioning effect. There were numerous ED1 and ED2 positive macrophages as well as positive staining for IL-1 beta in the granulation tissue on day 0. Positive staining for IL-1 beta was also seen in nerve fibres as well as in non-neuronal cells after a conditioning interval. The results suggest that regeneration is stimulated by factors released from the cells of the granulation tissue, and that the amount of factors released or the responsiveness of the regenerating nerve change during the conditioning interval.

We have studied the effects of vibration on the regeneration capacity of the peripheral nerve. A rat model was used where one hind limb was subjected to vibration of defined magnitude and duration while the contralateral hind limb was not exposed to vibration. Seven days later, the sciatic nerves were transected bilaterally and cross-joined giving the following groups: group A, a proximal vibrated nerve end sutured to a non-vibrated distal nerve end; group B, a non-vibrated proximal nerve end sutured to a distal vibrated nerve end, and group C, non-vibrated proximal nerve end sutured to a non-vibrated distal nerve end. The regeneration distances were measured 3, 6 and 8 days after surgery. The control group showed a normal linear outgrowth. The outgrowth in the two experimental groups was initially not different to controls but later became significantly different, indicating a retardation of outgrowth in these groups. It is concluded that short-term exposure to vibration can impair nerve regeneration after transection and nerve repair.

Effects of acute compression for 2 hours around the sciatic nerve trunk at 30 or 80 mmHg on the regeneration potential in rat sciatic nerves were studied. Sham compression or mobilization was performed contralaterally. A week later a crush injury was inflicted proximal to the compressed segment. After another 3 or 6 days the length of axonal outgrowth was measured, using the pinch test technique. We found that compression at either level caused an increased length of axonal outgrowth compared to the mobilized or sham-compressed nerves. The results show that an acute compression at low pressures does have a conditioning lesion effect on peripheral nerves.

The formation of a regenerate between sciatic nerve segments or stumps inserted into Y-tunnelled silicone chambers was studied under conditions where regenerating axons were prevented from entering the chamber. This was accomplished by using an isolated segment of the nerve as a proximal insert. After one week, a cellular regenerate spanned the proximal and distal inserts. The size of the regenerate increased if circulation was preserved in the distal inserts. At four weeks, a perineurium-like sheath surrounded the regenerate and longitudinally oriented Schwann cell columns could be observed throughout the regenerate. A similar 'pseudo-nerve' formed towards a piece of distally inserted tendon. Thus, the information required for the formation of a nerve-like structure is inherent to the non-neuronal cells entering the chamber. Schwann cells, in contrast to regenerating axons, do not exhibit preferential growth towards nervous tissue.

Specificity of muscle reinnervation and the recovery of muscle contractility were studied after repair of the transected rat sciatic nerve. Six different techniques were compared: epineurial suture, perineural suture, whole nerve graft, interfascicular grafts, skeletal muscle bridge and tubulization. Muscle tetanic force and specificity of reinnervation were evaluated 12 weeks after nerve repair. Recovery of tetanic force was superior after repair with epineurial sutures. There was no statistical significance between the other methods in respect of tetanic force. The specificity of muscle reinnervation was best after tubulization, repair with interfascicular grafts and perineurial suture.

The effects of application of a chromic catgut suture (conditioning lesion) placed close to the sciatic or tibial nerves on regeneration of the sciatic nerve after a crush lesion (test lesion), that had been induced after an appropriate conditioning interval (two or four weeks) were assessed. The catgut suture induced an inflammatory reaction around the nerve during the four weeks after application of the catgut suture (conditioning interval) but no signs of degeneration of nerve fibres were seen. There was a significant increase in length of outgrowth of sensory nerve fibres as measured by the pinch reflex test when the test lesion was applied after two and four weeks' exposure to the catgut suture. The rate of regeneration was increased by about 11% when the catgut suture had been applied for four weeks before the test lesion was made. The findings indicate that an inflammatory reaction around a peripheral nerve may act as a conditioning lesion, thereby stimulating regeneration of the nerve.

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.

To study the effects of drying on regeneration, crush lesions were made on the sciatic nerve of 38 rats and the injured area was then air-dried for various periods of time (0-60 min). The regeneration distance of sensory nerve fibres was measured by the pinch reflex test three or six days later. Regeneration was significantly impaired by drying the nerve for 30 or 60 min, but shorter periods (10 min) also seemed to affect axonal outgrowth. Regeneration distances were increased in nerves that were subjected to drying and then allowed to recover for a week (conditioning interval) before the nerve was crushed. The results show that drying of a peripheral nerve is detrimental to nerve regeneration, and we suggest that nerves should not be exposed to air for more than a few minutes during operation without irrigation.

The hind leg of adult rats was exposed to vibrations (82 Hz; amplitude peak-to-peak 0.21 mm) for 4 hours during 5 consecutive days. Light and electron microscopic examination of the plantar and sciatic nerves were done immediately after the exposure period or after a 2- or 4-week recovery period. Light microscopic examination did not reveal any distinct signs of injury. However, ultrastructurally unmyelinated fibers in the plantar nerves showed distinct changes, with deranged axoplasmic structure and/or accumulation of smooth endoplasmatic reticulum. These changes were to a large extent reversible in 2 weeks and appeared normalized after a 4-week recovery period. No ultrastructural changes could be observed in the sciatic nerve. However, when the sciatic nerve was crushed after 5 days of vibration exposure, axonal outgrowth was increased 23% as compared with controls. These findings confirm that vibration induces nerve fiber damage, in this experimental model expressed as a "conditioning effect" contributing to increased regeneration potential of the corresponding neurones.

Regeneration of severed peripheral nerves is unfortunately often incomplete, due to loss of nerve fibers and neuroma formation. A new approach is presented with the intention of improving the conditions for nerve repair. In the first of the two stages, a pseudosynovial tube is formed around a silicone rubber rod, surrounded by a stainless steel spiral, which was placed in the backs of rats. This tube, in the second stage, is used as a free "tube graft" to bridge gaps of about 10-12 mm lengths in the severed sciatic nerve. The tube was kept open by the metal spiral. Regenerating nerve fibers with their sprouts grew into the initially open space in the tube. A new nerve trunk was formed, comprised of closely packed myelinated and unmyelinated axons, organized into fascicles. Demonstration by electron microscopy and by EMG recording of reinnervation of foot muscles supported successful long-term results. The fascicles were delimited by perineurial and epineurial sheaths and, furthermore, showed signs of maturation. It was also demonstrated that the nerve-fiber regeneration ceased after a few weeks if there was no distal nerve inserted into the tube. The importance of optimizing the interaction between local factors and regenerating nerve fibers for reestablishment of functionally valuable motor units is discussed.

In 1944 Weiss & Taylor presented experimental evidence against "neurotropism" in nerve regeneration. We used a silicone Y-chamber system to repeat some of those experiments. The proximal stump of transected rat sciatic nerve was introduced into the proximal inlet of the Y. One of the distal outlets was left empty, plugged or occupied by a tendon graft, the other outlet being occupied by a nerve graft. Analysis after 4 and 12 weeks showed in all cases a preferential or exclusive axonal growth towards the nerve piece. The results, indicating the existence of "tissue specificity", are contradictory to the results reported by Weiss & Taylor (30). The findings are discussed with respect to possible influence of humoral, cellular and molecular factors associated with the distal nerve stump as well as the matrix, formed between both nerve segments.

Peripheral neuropathy represents a well-known complication from long-term exposure to vibration. In the present study an experimental model is presented with the purpose of analyzing the formation of intraneural edema following vibration exposure. Vibration (82 Hz, peak-to-peak amplitude 0.21 mm) was induced in the hind limb of rats by the use of vibrating electric motors during 4 h/d for 5 d. Tracer techniques (with albumin Evans blue and horseradish peroxidase) were used to study the permeability of intraneural microvessels after the vibration exposure on day 5. It was found that the vibration trauma in this model induced epineurial edema in the sciatic nerve. It is hypothesized that the formation of intraneural edema may be an important pathophysiological factor in the occurrence of vibration-induced neuropathy.

Preformed, autologous mesothelial chambers were utilized to study axonal growth following selective predegeneration of the distal nerve stump and/or preconditioning of the proximal nerve stump. The left and/or right sciatic nerve of rats was exposed and transected in the thigh. Two weeks after transection, the left proximal nerve stump was cross-anastomosed with the right distal nerve stump by using a mesothelial chamber leaving a 15-mm gap between the two nerve stumps. Previous studies have shown that axonal overgrowth normally does not occur over this gap distance to the distal stump. Three months after cross-anastomosing, regeneration across the 15-mm gap was evaluated by muscle action potential recordings and light microscopical examination. In experiments in which a distal nerve stump was selectively degenerated and the proximal segment was freshly cut, axons had bridged the 15-mm gap in six of seven rats. When a proximal preconditioned nerve stump was matched with a freshly cut distal stump, axonal overgrowth occurred in only 4 of 10 experiments. In experiments including a proximal preconditioned nerve stump and a distal predegenerated stump, axons bridged the gap in 6 of 8 experiments. We concluded that a priming lesion, including manipulation with proximal and/or distal stump, enhances axonal growth in mesothelial chambers.

An experimental model is presented for studying nerve regeneration over gaps of various lengths between the both ends of a severed nerve. After transferring left and right sciatic nerves of rat to the back, the gap between the two nerve ends could be bridged by a preformed, tube-shaped mesothelial chamber of a desired length. When the gap length was 10 mm or less, a well developed nerve structure was generated in the chamber between the nerve ends, and axons from the left sciatic nerve reinnervated muscles in the right limb via the right sciatic nerve. When the gap length was extended to 15 mm or more no such regeneration occurred. When no distal nerve end was introduced into the chamber, there was a limited growth into this chamber over only 5-6 mm. This "limited growth phenomenon" is discussed with respect to a lack of "trophic" or cellular support from a distal nerve segment. It is also proposed that the termination of growth, seen under these circumstances, may suggest a new principle for avoiding the development of neuromas after nerve transections.

An experimental model is presented for studying axonal growth after experimental hyperthyroidism and hypothyroidism. The left sciatic nerve of the rat was transected and transposed to the back. The proximal nerve stump was inserted into a 50-mm-long mesothelial chamber leaving the distal end of the chamber open. Different groups of young adult rats were given daily injections of thyroxine (10 micrograms/100 g body weight) or the goitrogen, thiamazol, in the drinking water (0.125 g/liter) for 12 weeks. Thyroxine treatment increased significantly the extent of axonal outgrowth from the proximal nerve stump compared with untreated rats. Experimental hypothyroidism (thiamazol treatment), evidenced by a retarded body growth, did not affect the extent of axonal outgrowth. In other experiments the left proximal nerve stump was cross-anastomosed with the right distal nerve stump. The two nerve stumps were bridged with a mesothelial chamber leaving a 15-mm gap. This gap distance is known from our previous studies to inhibit axonal overgrowth to the distal nerve stump. As evidenced by histological evaluation, in three of six thyroxine-treated rats, axons had bridged the 15-mm gap. We conclude that experimentally induced hyperthyroidism enhances axonal growth in mesothelial chambers.

We have compared the anatomic and functional regeneration of a transected sciatic nerve following regrowth from its proximal stump through either preformed empty mesothelial chambers or autologous nerve grafts bridging a 10 mm gap. Within the mesothelial chambers an organized multifascicular nerve trunk forms between the proximal and distal stumps. After 3 months, distal segment cross sections from the mesothelial chamber and nerve graft groups did not differ with respect to axonal density or distribution of axonal diameters. Mean conduction velocities across the gaps were also similar, although the nerve graft group had a wider distribution of velocities. Little or no regeneration was evident when the gap between the nerve stumps was left empty. These results suggest that if the regrowing proximal stump is in an appropriate environment, it can form a well organized and oriented nerve trunk. In the mesothelial chambers, the regenerating nerve is surrounded by a loose cellular stroma and a small amount of interstitial fluid, which was found to contain trophic activity for cultured rodent sensory neurons. Such factors may also support nerve regeneration in vivo.