Preferential motor reinnervation: a sequential double-labeling study

Previous experiments have shown that motor axons regenerating in mixed nerve will preferentially reinnervate a distal motor branch. The present experiments examine the mechanism through which this sensory-motor specificity is generated. An enclosed 0.5 mm gap was created in the proximal femoral nerves of juvenile rats. Two, three or eight weeks later the specificity of motor axon regeneration was evaluated by simultaneous application of horseradish peroxidase (HRP) to one distal femoral branch (sensory or motor) and Fluoro-Gold to the other. Motoneurons were then counted as projecting (i) correctly to the motor branch, (ii) incorrectly to the sensory branch, and (iii) simultaneously to both branches (double-labeled). Motor axon regeneration was random at 2 weeks, with equal numbers of motoneurons projecting to sensory and motor branches. However, the number of correct projections increased dramatically between 2 and 3 weeks. Twenty-six percent of neurons labeled at 2 weeks contained both tracers, indicating axon collateral projections to both sensory and motor branches. This number decreased significantly at each time period. Axon collaterals were thus 'pruned' from the sensory branch, increasing the number of correct projections at the expense of double-labeled neurons. These findings suggest random reinnervation of the distal stump, with specificity generated through trophic interaction between axons and the pathway and/or end organ.     

Chemical communication between regenerating motor axons and Schwann cells in the growth pathway

There are receptors on denervated Schwann cells that may respond to the neurotransmitters that are released from growth cones of regenerating motor axons. In order to ascertain whether the interaction of the transmitters and their receptors plays a role during axon regeneration, we investigated whether pharmacological block of the interaction would reduce the number of motoneurons that regenerate their axons after nerve section and surgical repair. Peripheral nerves in the hindlimbs of rats and mice were cut and repaired, and various drugs were applied to the peripheral nerve stump either directly or via mini-osmotic pumps over a 2–4-week period to block the binding of acetylcholine to nicotinic and muscarinic acetylcholine receptors (AChRs: α-bungarotoxin, tubocurarine, atropine and, gallamine) and binding of ATP to P2Y receptors (suramin). In rats, the nicotinic AChR antagonistic drugs and suramin reduced the number of motoneurons that regenerated their axons through the distal nerve stump. In mice, suramin significantly reduced the upregulation of the carbohydrate HNK-1 on the Schwann cells in the distal nerve stump that normally occurs during motor axon regeneration. These data indicate that chemical communication between regenerating axons and Schwann cells during axon regeneration via released neurotransmitters and their receptors may play an important role in axon regeneration.     

Chronic Schwann cell denervation and the presence of a sensory nerve reduce motor axonal regeneration

Motor axonal regeneration is compromised by chronic distal nerve stump denervation, induced by delayed repair or prolonged regeneration distance, suggesting that the pathway for regeneration is progressively impaired with time and/or distance. In the present experiments, we tested the impacts of (i) chronic distal sensory nerve stump denervation on axonal regeneration and (ii) sensory or motor innervation of a nerve graft on the ability of motoneurons to regenerate their axons from the opposite end of the graft. Using the motor and sensory branches of rat femoral nerve and application of neuroanatomical tracers, we evaluated the numbers of regenerated femoral motoneurons and nerve fibers when motoneurons regenerated (i) into freshly cut and 2-month chronically denervated distal sensory nerve stump, (ii) alone into a 4-cm-long distally ligated sensory autograft (MGL) and, (iii) concurrently as sensory (MGS) or motor (MGM) nerves regenerated into the same autograft from the opposite end. We found that all (315 +/- 24: mean +/- SE) the femoral motoneurons regenerated into a freshly cut distal sensory nerve stump as compared to 254 +/- 20 after 2 months of chronic denervation. Under the MGL condition, 151 +/- 5 motoneurons regenerated, which was not significantly different from the MGM group (134 +/- 13) but was significantly reduced to 99 +/- 2 in the MGS group (P < 0.05). The number of regenerated nerve fibers was 1522 +/- 81 in the MGL group, 888 +/- 18 in the MGM group, and 516 +/- 44 in the MGS group, although the high number of nerve fibers in the MGL group was due partly to the elaboration of multiple sprouts. Nerve fiber number and myelination were reduced in the MGS group and increased in the MGM group. These results demonstrate that both chronic denervation and the presence of sensory nerve axons reduced desired motor axonal regeneration into sensory pathways. A common mechanism may involve reduced responsiveness of sensory Schwann cells within the nerve graft or chronically denervated distal nerve stump to regenerating motor axons. The findings confirm that motor regeneration is optimized by avoiding even short-term denervation. They also imply that repairing pure motor nerves (without their cutaneous sensory components) to distal nerve stumps should be considered clinically when motor recovery is the main desired outcome.     

Guidance of regenerating motor axons in larval and juvenile bullfrogs

The segmental distribution of regenerating bullfrog motor axons was mapped in advanced tadpoles and juvenile frogs by stimulating selected muscle nerves and recording from the distal ends of the 3 lumbar ventral roots (VRs) that innervate the hindlimb. When motoneurons were axotomized by VR transection, they reestablished their original innervation fields, rarely, if ever, growing beyond the territory normally supplied by their spinal segment. However, when motoneurons were axotomized in the spinal nerves at the level of the hindlimb plexus, some of them regenerated into limb nerves that lay outside the axons' normal segmental boundaries, and many regenerated into the medial femoral cutaneous nerve, a pathway normally limited to sensory axons. These observations suggest that the ultimate destinations of regenerating axons are largely determined by structures the axons encounter as they penetrate the distal nerve stumps. Thus, axons regenerating from a severed VR grow into that root's own distal stump and reinnervate the hindlimb in a manner that is segmentally appropriate; axons transected near the plexus have access to the pathways of sensory, as well as motor, axons in all 3 lumbar segments, and establish innervation fields that are inappropriate for their segment of origin and their motor function.     

Preferential reinnervation of motor nerves by regenerating motor axons

Regeneration of axons into inappropriate distal nerve branches may adversely affect functional recovery after peripheral nerve suture. The degree to which motor axons reinnervate sensory nerves, and vice versa, has not been determined. In these experiments, HRP is used to quantify the sensory and motor neurons that reinnervate sensory and motor branches of the rat femoral nerve after proximal severance and repair. Motoneurons preferentially reinnervate the motor branch in juveniles and adults, even if the repair is intentionally misaligned or a gap is imposed between proximal and distal stumps. A specific interaction thus occurs between regenerating motor axons and the Schwann cell tubes that lead to the motor branch. This interaction is independent of mechanical axon alignment.     

Poor growth of Mammalian motor and sensory axons into intact proximal nerve stumps

Wallerian degeneration is very slow in the mouse strain now known as C57BL/Ola. Sensory axon regrowth following peripheral nerve lesions is very poor in these animals but motor axons succeed in reinnervating the distal nerve stump even while the majority of severed axons are still intact (Lunn et al., Eur. J. Neurosci., 1, 27 - 33, 1989). To see if motor axons could grow into a completely undegenerated portion of nerve, the proximal stumps of the peroneal and tibial nerves were sutured together in six BALB/c mice and the ability of large motor and sensory fibres from the tibial nerve to grow into the peroneal nerve was examined electrophysiologically in four of them. For the acute experiment the peroneal nerve was cut approximately 7 mm central to the point of suture to the tibial nerve. Both at 2 weeks and 7 weeks after surgery the size of the potential recorded in the ventral roots on stimulating the portion of peroneal nerve into which tibial axons were directed to grow was only approximately 8% of the potential recorded when the tibial nerve was itself stimulated. The potential recorded in the dorsal roots was only approximately 2%. Counts of axon numbers in electron micrographs showed a small but non-significant increase over normal in the number of unmyelinated axons in the peroneal nerves which had been connected to the tibial nerve in this way. It is concluded, in agreement with Langley and Anderson (J. Physiol., 31, 365 - 391, 1904), that axon growth into intact nerves is extremely limited in mammals and that the distal nerve stump of C57BL/Ola mice, although it degenerates very slowly, is not therefore equivalent to an intact peripheral nerve.     

Differences in incorporation of axonally transported protein in regenerating motor and sensory axons

Different patterns of labeling were obtained in regenerating rat sciatic nerve after incorporation of labeled, fast-transported protein by motor or sensory axons. In motor axons incorporation of label occurred principally at the distal end of the regenerating fibers, whereas in sensory axons incorporation occurred uniformly along the entire length of nerve between the original site of injury and the most rapidly growing tips. This difference is not a consequence of the different types of axonal trauma used to provoke regeneration but may reflect a less synchronous outgrowth of sensory axons after injury.     

Neurite-promoting influences of proliferating Schwann cells and target-tissues are not prerequisite for rapid axonal elongation after nerve crush

An important role in peripheral nerve regeneration has been ascribed to humoral trophic and tropic agents arising from the nonneuronal cells in the distal nerve stump and the denervated targets. In order to estimate their contribution to axonal elongation after crush injury to the rat sciatic nerve, an in vivo model was designed in which local cellular and target-derived influences were eliminated by 1) freeze-thawing of a long nerve segment distal to the crush site and 2) cutting the nerve far distally to the crush site, but within the frozen-thawed segment, and deflecting the frozen-thawed nerve stump in the opposite direction from its natural course. The sensory and motor axon elongation rate was estimated from the results of the nerve pinch test and choline acetyltransferase distribution along the nerve segment distal to the crush. The elongation rate of regenerating axons in deflected nerve segments, either non-treated or frozen-thawed, was close in magnitude to that obtained when target-derived influences were not eliminated. Neurotropism of axonal targets is therefore of little importance for axon elongation after nerve crush. In the absence of Schwann cells along the axonal path in frozen-thawed nerve segments, the elongation rate of both sensory and motor axons declined by about 40%. This implies that interactions between viable Schwann cells and growth cones of regenerating axons are not prerequisite for rapid axon elongation when Schwann cell basal lamina constitutes the growth substratum. Nevertheless, Schwann cells in Bungner bands possibly enhance the axon elongation rate by humoral or cell surface-mediated mechanisms.     

Local isoform‐specific NOS inhibition: A promising approach to promote motor function recovery after nerve injury

Physical injury to a nerve is the most frequent cause of acquired peripheral neuropathy, which is responsible for loss of motor, sensory and/or autonomic functions. Injured axons in the peripheral nervous system maintain the capacity to regenerate in adult mammals. However, after nerve transection, stumps of damaged nerves must be surgically joined to guide regenerating axons into the distal nerve stump. Even so, severe functional limitations persist after restorative surgery. Therefore, the identification of molecules that regulate degenerative and regenerative processes is indispensable in developing therapeutic tools to accelerate and improve functional recovery. Here, I consider the role of nitric oxide (NO) synthesized by the three major isoforms of NO synthases (NOS) in motor neuropathy. Neuronal NOS (nNOS) seems to be the primary source of NO that is detrimental to the survival of injured motoneurons. Endothelial NOS (eNOS) appears to be the major source of NO that interferes with axonal regrowth, at least soon after injury. Finally, NO derived from inducible NOS (iNOS) or nNOS is critical to the process of lipid breakdown for Wallerian degeneration and thereby benefits axonal regrowth. Specific inhibitors of these isoforms can be used to protect injured neurons from degeneration and promote axonal regeneration. A cautious proposal for the treatment of acquired motor neuropathy using therapeutic tools that locally interfere with eNOS/nNOS activities seems to merit consideration. © 2010 Wiley‐Liss, Inc.     

Identifying motor and sensory myelinated axons in rabbit peripheral nerves by histochemical staining for carbonic anhydrase and cholinesterase activities

Carbonic anhydrase (CA) and cholinesterase (CE) histochemical staining of rabbit spinal nerve roots and dorsal root ganglia demonstrated that among the reactive myelinated axons, with minor exceptions, sensory axons were CA positive and CE negative whereas motor axons were CA negative and CE positive. The high specificity was achieved by adjusting reaction conditions to stain subpopulations of myelinated axons selectively while leaving 50% or so unstained. Fixation with glutaraldehyde appeared necessary for achieving selectivity. Following sciatic nerve transection, the reciprocal staining pattern persisted in damaged axons and their regenerating processes which formed neuromas within the proximal nerve stump. Within the neuromas, CA-stained sensory processes were elaborated earlier and in greater numbers than CE-stained regenerating motor processes. The present results indicate that histochemical axon typing can be exploited to reveal heterogeneous responses of motor and sensory axons to injury.     

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1 045 myelinated axons and 4 160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2 476 in the proximal stump to one of 2 219.

The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones.

These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.     

The effect of hyperbaric oxygen treatment on early regeneration of sensory axons after nerve crush in the rat

Abstract The effect of hyperbaric oxygen treatment (HBO) on sensory axon regeneration was examined in the rat. The sciatic nerve was crushed in both legs. In addition, the distal stump of the sural nerve on one side was made acellular and its blood perfusion was compromised by freezing and thawing. Two experimental groups received hyperbaric exposures (2.5 ATA) to either compressed air (pO2 = 0.5 ATA) or 100% oxygen (pO2 = 2.5 ATA) 90 minutes per day for 6 days. Sensory axon regeneration in the sural nerve was thereafter assessed by the nerve pinch test and immunohistochemical reaction to neurofilament. HBO treatment increased the distances reached by the fastest regenerating sensory axons by about 15% in the distal nerve segments with preserved and with compromised blood perfusion. There was no significant difference between the rats treated with different oxygen tensions. The total number of regenerated axons in the distal sural nerve segments after a simple crush injury was not affected, whereas in the nerve segments with compromised blood perfusion treated by the higher pO2, the axon number was about 30% lower than that in the control group. It is concluded that the beneficial effect of HBO on sensory axon regeneration is not dose-dependent between 0.5 and 2.5 ATA pO2. Although the exposure to 2.5 ATA of pO2 moderately enhanced early regeneration of the fastest sensory axons, it decreased the number of regenerating axons in the injured nerves with compromised blood perfusion of the distal nerve stump.     

An in vivo model to quantify motor and sensory peripheral nerve regeneration using bioresorbable nerve guide tubes

An in vivo preparation is presented to study the rate and time course of motor and sensory axonal regeneration. The cut ends of a transected sciatic nerve were inserted into each end of a 5-6 mm non-toxic and bioresorbable nerve guide tube to create a 4 mm nerve gap in adult mice. Subsequently, cell bodies in the ventral spinal cord and L3-L5 dorsal root ganglia that had regenerated axons across the gap were retrogradely labeled with horseradish peroxidase (HRP). The HRP was applied 3 mm distal to the nerve guide and was accessible only to axons that had regenerated through the nerve guide. Labeled cells were counted in 40 micron serial sections at 2, 4 and 6 weeks after initial nerve transection. The results indicate a significant increase in the number of labeled motor and sensory cell bodies over time. By 6 weeks after transection, approximately two thirds as many ventral horn motor cells and one third as many dorsal root ganglion sensory cells were labeled as in control non-transected animals. These data serve as a baseline to compare differential effects of additives to the nerve guide lumen in terms of sensory and motor neuron response.     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

Regeneration of taste buds by nongustatory nerve fibers

Previous cross-reinnervation studies in situ by other investigators have demonstrated that cutaneous sensory and motor axons are incapable of trophically supporting mammalian taste buds. The present experiments examined the gustatory trophic potency of chemosensory and barosensory axons of the carotid sinus nerve. We report here that morphologically normal taste buds appeared on cat circumvallate papillae at 2 to 19 months after cross-anastomosis of the carotid sinus and lingual nerves, branches of the IXth cranial (glossopharyngeal) nerve. However, neurophysiologic and histologic data also indicated that, despite microsurgical procedures designed to direct regenerating lingual nerve fibers toward the carotid body and carotid sinus, some lingual axons escaped the anastomosis and subsequently grew within their native distal stump. The principal objective of this study was thus to determine whether foreign innervation of taste buds did indeed occur, or regenerated lingual nerve fibers were instead responsible for the newly formed buds. Our results showed that stray lingual fibers were not responsible for the reappearance of taste buds because transection of the original proximal lingual nerve stump (cross-anastomosed to the distal carotid sinus nerve stump) did not reduce the incidence of taste buds or the accumulation of radiolabeled material axoplasmically transported from the petrosal (sensory) ganglion. Autoradiography of labeled tissue samples showed that more than 90% of the taste buds were labeled at 8 and 9 days after lingual nerve transection. These data support the hypothesis that sensory axons in the carotid sinus nerve share an important trophic chemistry with gustatory neurons.     

Specificity in regenerative outgrowth and target reinnervation by mammalian peripheral axons

There are indications that specific factors are present in the distal stump of transected nerves which preferentially attract axons of the corresponding proximal stump into the distal nerve stumps. However, the impact of these factors is unclear, since there is abundant evidence that numerous regenerating motor and sensory axons are topographically misdirected after nerve transection and repair. Topographic reinnervation is improved after fascicular repair of fasciculated nerves, and quite precise after nerve crush. The latter may not be true, however, for non-myelinated axons, which show a high degree of aberrant growth even after crush. In contrast, regenerative outgrowth appears to be topographically specific after neonatal nerve transection. Reinnervation of muscle fibers appears to be unspecific in adult mammals, but specific after neonatal injury under certain circumstances. Some preference for reinnervation of the appropriate sensory receptors seems to exist although this preference does not preclude reinnervation of receptors by 'foreign' sensory fibers. In conclusion, incorrect topographic and target reinnervation commonly occurs after peripheral regeneration in adult mammals, and most certainly explains some of the functional disturbances after peripheral nerve lesions. Topographic regeneration appears to be better after nerve injury in developing mammals indicating that mechanisms from the developmental period may persist and aid in accurate regenerative outgrowth.     

Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model

Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (± 4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (± 4.2%) after direct coaptation, and 25% (± 6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.     

Treadmill training enhances axon regeneration in injured mouse peripheral nerves without increased loss of topographic specificity

We investigated the extent of misdirection of regenerating axons when that regeneration was enhanced by using treadmill training. Retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial and common fibular nerves 2 or 4 weeks after transection and surgical repair of the mouse sciatic nerve. The spatial locations of retrogradely labeled motoneurons were studied in untreated control mice and in mice receiving 2 weeks of treadmill training, according to either a continuous protocol (10 m/minute, 1 hour/day, 5 days/week) or an interval protocol (20 m/minute for 2 minutes, followed by a 5‐minute rest, repeated four times, 5 days/week). More retrogradely labeled motoneurons were found in both treadmill‐trained groups. The magnitude of this increase was as great as or greater than that found after using other enhancement strategies. In both treadmill‐trained groups, the proportions of motoneurons labeled from tracer applied to the common fibular nerve that were found in spinal cord locations reserved for tibial motoneurons in intact mice were no greater than in untreated control mice and significantly less than those found after electrical stimulation or chondroitinase treatment. Treadmill training in the first 2 weeks following peripheral nerve injury produces a marked enhancement of motor axon regeneration without increasing the propensity of those axons to choose pathways leading to functionally inappropriate targets. J. Comp. Neurol. 517:245–255, 2009. © 2009 Wiley‐Liss, Inc.     

Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.     

Neurotropism revisited

The purpose of this study is to re-examine the probable directive effect of the distal stump of a severed peripheral nerve on regenerating axons. Forssman postulated the existence of such a directive influence and Cajal interpreted it as chemotactic in nature. This view was subsequently refuted by Weiss and Taylor. In our study the proximal stumps of transected rodent sciatic nerve were inserted into the single inlet end of a Y-shaped autogenous inferior vena cava graft. Into one limb of the double outlet end, namely the common iliac nerve bifurcation, the distal stump of the same sciatic nerve was inserted, while the counter limb was ligated in one group, left open in the second group, inserted with a segment of autogenous tendon in the third, and grafted with a segment of autogenous nerve in the fourth group. Both outlets were left unoccupied in yet another group as the control. The vena cava conduit was prepared so that a 1.5 cm gap existed between the proximal stumps of the sciatic nerve and the distal sciatic nerve stumps and the tendon grafts respectively. The grafted sciatic nerves were explored and biopsied after 12 weeks. The direction of nerve tissue regeneration in each group was analyzed histologically. Predilection of the regenerating nerve fibers toward the distal stumps was observed in each of the test groups. These results indicate the existence of a guiding influence at the distal stump toward the regeneration nerve fibers.