Functional recovery after peripheral nerve injury via sustained growth factor delivery from mineral-coated microparticles

The gold standard for treating peripheral nerve injuries that have large nerve gaps where the nerves cannot be directly sutured back together because it creates tension on the nerve, is to incorporate an autologous nerve graft. However, even with the incorporation of a nerve graft, generally patients only regain a small portion of function in limbs affected by the injury. Although, there has been some promising results using growth factors to induce more axon growth through the nerve graft, many of these previous therapies are limited in their ability to release growth factors in a sustained manner and tailor them to a desired time frame. The ideal drug delivery platform would deliver growth factors at therapeutic levels for enough time to grow axons the entire length of the nerve graft. We hypothesized that mineral coated microparticles(MCMs) would bind, stabilize and release biologically active glial cell-derived neurotrophic factor(GDNF) and nerve growth factor(NGF) in a sustained manner. Therefore, the objective of this study was to test the ability of MCMs releasing growth factors at the distal end of a 10 mm sciatic nerve graft, to induce axon growth through the nerve graft and restore hind limb function. After sciatic nerve grafting in Lewis rats, the hind limb function was tested weekly by measuring the angle of the ankle at toe lift-off while walking down a track. Twelve weeks after grafting, the grafts were harvested and myelinated axons were analyzed proximal to the graft, in the center of the graft, and distal to the graft. Under physiological conditions in vitro, the MCMs delivered a burst release of NGF and GDNF for 3 days followed by a sustained release for at least 22 days. In vivo, MCMs releasing NGF and GDNF at the distal end of sciatic nerve grafts resulted in significantly more myelinated axons extending distal to the graft when compared to rats that received nerve grafts without growth factor treatment. The rats with nerve grafts incorporated with MCMs releasing NGF and GDNF also showed significant improvement in hind limb function starting at 7 weeks postoperatively and continuing through 12 weeks postoperatively when compared to rats that received nerve grafts without growth factor treatment. In conclusion, MCMs released biologically active NGF and GDNF in a sustained manner, which significantly enhanced axon growth resulting in a significant improvement of hind limb function in rats. The animal experiments were approved by University of Wisconsin-Madison Animal Care and Use Committee(ACUC, protocol# M5958) on January 3, 2018.     

Hyperbaric oxygen treatment has different effects on nerve regeneration in acellular nerve and muscle grafts

Effects of hyperbaric oxygen treatment (HBO) on nerve regeneration in acellular nerve and muscle grafts were investigated in rats. Nerve and muscle grafts were made acellular by freeze-thawing and the obtained grafts were used to bridge a 10-mm gap in the sciatic nerve on the left and right sides, respectively. Rats were treated with HBO (100% oxygen for 90 minutes at 2.5 atmospheres absolute pressure ATA) twice a day for 7 days. Axonal outgrowth, Schwann cell migration and invasion of macrophages were examined 10 days after the graft procedure by staining neurofilaments, S-100 proteins and the macrophage antibodies ED1 and ED2, respectively. Axonal outgrowth and Schwann cell migration in acellular nerve grafts were superior to that found in the acellular muscle grafts. However, there was no difference between HBO-treated and nontreated rats in acellular nerve grafts. Such a difference was found in acellular muscle grafts concerning both axonal outgrowth and Schwann cell migration from the proximal nerve end. No differences in the content of macrophages or neovascularization (alkaline phosphatase staining) in either of the grafts and treatments were seen. It is concluded that there is a differential effect of HBO-treatment in acellular nerve and muscle grafts and that HBO-treatment has no effect on the regeneration process in acellular nerve grafts, in contrast to fresh cellular nerve grafts where a beneficial effect has previously been reported.     

The role of vascularization in nerve regeneration of nerve graft

Vascularization is an important factor in nerve graft survival and function. The specific molecular regulations and patterns of angiogenesis following peripheral nerve injury are in a broad complex of pathways. This review aims to summarize current knowledge on the role of vascularization in nerve regeneration, including the key regulation molecules, and mechanisms and patterns of revascularization after nerve injury. Angiogenesis, the maturation of pre-existing vessels into new areas, is stimulated through angiogenic factors such as vascular endothelial growth factor and precedes the repair of damaged nerves. Vascular endothelial growth factor administration to nerves has demonstrated to increase revascularization after injury in basic science research. In the clinical setting, vascularized nerve grafts could be used in the reconstruction of large segmental peripheral nerve injuries. Vascularized nerve grafts are postulated to accelerate revascularization and enhance nerve regeneration by providing an optimal nutritional environment, especially in scarred beds, and decrease fibroblast infiltration. This could improve functional recovery after nerve grafting, however, conclusive evidence of the superiority of vascularized nerve grafts is lacking in human studies. A well-designed randomized controlled trial comparing vascularized nerve grafts to non-vascularized nerve grafts involving patients with similar injuries, nerve graft repair and follow-up times is necessary to demonstrate the efficacy of vascularized nerve grafts. Due to technical challenges, composite transfer of a nerve graft along with its adipose tissue has been proposed to provide a healthy tissue bed. Basic science research has shown that a vascularized fascial flap containing adipose tissue and a vascular bundle improves revascularization through excreted angiogenic factors, provided by the stem cells in the adipose tissue as well as by the blood supply and environmental support. While it was previously believed that revascularization occurred from both nerve ends, recent studies propose that revascularization occurs primarily from the proximal nerve coaptation. Fascial flaps or vascularized nerve grafts have limited applicability and future directions could lead towards off-the-shelf alternatives to autografting, such as biodegradable nerve scaffolds which include capillary-like networks to enable vascularization and avoid graft necrosis and ischemia.     

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.     

Spatiotemporal progress of nerve regeneration in a tendon autograft used for bridging a peripheral nerve defect

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.     

Variation in nerve autograft length increases fibre misdirection and decreases pruning effectiveness: an experimental study in the rat median nerve

OBJECTIVES: In the clinical set, autologus nerve grafts are the current option for reconstruction of nerve tissue losses. The length of the nerve graft has been suggested to affect outcomes. Experiments were performed in the rat in order to test this assumption and to detect a possible mechanism to explain differences in recovery.METHODS: The rat median nerve was repaired by ulnar nerve grafts of different lengths. Rats were evaluated for 12 months by behavioural assessment and histological studies, including ATPase myofibrillary histochemistry and retrograde neuronal labelling.RESULTS: It was demonstrated that graft length interferes in behavioural functional recovery that here correlates to muscle weight recovery. Short nerve grafts recovered faster and better. Reinnervation was not specific either at the trunk level or in the muscle itself. The normal mosaic pattern of Type I muscle fibres was never restored and their number remained largely augmented. An increment in the number of motor fibres was observed after the nerve grafting in a predominantly sensory branch in all groups. This increment was more pronounced in the long graft group. In the postoperative period, about a 20% reduction in the number of misdirected motor fibres occurred in the short nerve graft group only.CONCLUSION: Variation in the length of nerve grafts interferes in behavioural recovery and increases motor fibres misdirection. Early recovery onset was related to a better outcome, which occurs in the short graft group.     

The impact of a muscle target organ on nerve grafts with different lengths—A histomorphological analysis

The present study was done in order to evaluate the influence of a target muscle on the regenerative processes in long nerve grafts. In 21 rabbits the saphenous nerve was used as a nerve graft and coapted to the cut motor nerve of vastus medialis. The animals were separated into three groups with different graft lengths, namely 3, 5, and 7 cm. In a second stage the distal end of the graft (Graft.dist.) was coapted to the motor branch of rectus femoris. Cross sections of the normal vastus nerve and the Graft.dist. before and 7 months after the connection to rectus femoris were analyzed histomorphometrically. Before coaptation to the target organ mean fiber number in the Graft.dist. of the 3-cm-long grafts was 3380 and decreased to 2413 in the 7-cm-long grafts. Seven months after coaptation the results showed a statistically significant decrease of fibers in the Graft.dist. of group two and three and a distinct decrease of the fibers in group one. Summarizing, in a two-stage nerve grafting procedure the reinnervation of the muscle target organ leads to a down-regulation of fibers in the distal end of short and long nerve grafts. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:618–627, 1998.     

The impact of motor and sensory nerve architecture on nerve regeneration

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve's Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p < 0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p < 0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.     

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.     

Functional characterization of optimized acellular peripheral nerve graft in a rat sciatic nerve injury model

Abstract

Objectives: Acellular grafts are a viable option for use in nerve reconstruction surgeries. Recently, our lab created a novel optimized decellularization procedure that removes immunological material while leaving the majority of the extracellular matrix structure intact. The optimized acellular (OA) graft has been shown to elicit an immune response equal to or less than that elicited by the isograft, the analog of the autograft in the rat model. We investigated the performance of the OA graft to provide functional recovery in a long-term study.

Methods: We performed a long-term functional regeneration evaluation study using the sciatic functional index to quantify recovery of Lewis rats at regular time intervals for up to 52 weeks after graft implantation following 1 cm sciatic nerve resection. OA grafts were compared against other decellularized methods (Sondell treatment and thermal decellularization), as well as the isograft and primary neurorrhaphy.

Results: The OA graft supported comparable functional recovery to the isograft and superior regeneration to thermal and Sondell decellularization methods. Furthermore, the OA graft promoted early recovery to a greater degree compared to acellular grafts obtained using either the thermal or the Sondell methods.

Discussion: Equivalent functional recovery to the isograft suggests that the OA nerve graft may be a future clinical alternative to the current autologous tissue graft.     

Adipose derived stem cells and nerve regeneration

Injuries to peripheral nerves are common and cause life-changing problems for patients alongside high social and health care costs for society. Current clinical treatment of peripheral nerve injuries predominantly relies on sacrificing a section of nerve from elsewhere in the body to provide a graft at the injury site. Much work has been done to develop a bioengineered nerve graft, precluding sacrifice of a functional nerve. Stem cells are prime candidates as accelerators of regeneration in these nerve grafts. This review examines the potential of adipose-derived stem cells to improve nerve repair assisted by bioengineered nerve grafts.     

Immunohistochemical assessment of rat nerve isografts and immunosuppressed allografts

Objective: Autologous peripheral nerve grafts are commonly used clinically as a treatment for peripheral nerve injuries. However, in research using an autologous graft is not always feasible due to loss of function, which in many cases is assessed to determine the efficacy of the peripheral nerve graft. In addition, using allografts for research require the use of an immunosuppressant, which creates unwanted side effects and another variable within the experiment that can affect regeneration. The objective of this study was to analyze graft rejection in peripheral nerve grafts and the effects of cyclosporine A (CSA) on axonal regeneration.

Methods: Peripheral nerve grafts in inbred Lewis rats were compared with Sprague-Dawley (SD) rats to assess graft rejection, CSA side effects, immune responses, and regenerative capability. Macrophages and CD8+ cells were labeled to determine graft rejection, and neurofilaments were labeled to determine axonal regeneration.

Results: SD rats without CSA had significantly more macrophages and CD8+ cells compared to Lewis autografts, Lewis isografts, and SD allografts treated with CSA. Lewis autografts, Lewis isografts, and SD autografts had significantly more regenerated axons than SD rat allografts. Moreover, allografts in immunosuppressed SD rats had significantly less axons than Lewis rat autograft and isografts.

Discussion: Autografts have long been the gold standard for treating major nerve injuries and these data suggest that even though CSA is effective at reducing graft rejection, axon regeneration is still superior in autografts versus immunosuppressed allografts.     

Regeneration across preserved peripheral nerve grafts

The potential to store nerve grafts for a prolonged period of time was assessed in a rat sciatic nerve model. Three-centimeter syngeneic nerve grafts were stored in Belzer/University of Wisconsin cold storage solution at different temperatures (5°C, 22°C, or 37°C) for varying time periods (6 h, 24 h, or 3 weeks) prior to transplantation. Functional assessment using serial walking track analyses revealed no difference between storage times and temperatures. At 14 months postengraftment, the conduction velocities and the number of myelinated fibers that had regenerated across all grafts stored at 5°C for all time periods tested were superior to grafts stored at either 22°C or 37°C. Nerve grafts stored for up to 3 weeks at 5°C acted as effective conduits for proximal regenerating fibers and resulted in histologic, electrophysiologic, and functional results equivalent to fresh nerve grafts. Nerve graft storage may be applicable to nerve allografts and potentially provide allograft material that requires reduced or no associated host immunosuppression.© 1995 John Wiley & Sons, Inc.     

Bridging sciatic nerve gap using tissue-engineered nerves constructed with neural tissue-committed stem cells derived from bone marrow

BACKGROUND: Schwann cells are the most commonly used cells for tissue-engineered nerves. However, autologous Schwann cells are of limited use in a clinical context, and allogeneic Schwann cells induce immunological rejections. Cells that do not induce immunological rejections and that are relatively easy to acquire are urgently needed for transplantation.OBJECTIVE: To bridge sciatic nerve defects using tissue engineered nerves constructed with neural tissue-committed stem cells (NTCSCs) derived from bone marrow; to observe morphology and function of rat nerves following bridging; to determine the effect of autologous nerve transplantation, which serves as the gold standard for evaluating efficacy of tissue-engineered nerves.DESIGN, TIME AND SETTING: This randomized, controlled, animal experiment was performed in the Anatomical laboratory and Biomedical Institute of the Second Military Medical University of Chinese PLA between September 2004 and April 2006.MATERIALS: Five Sprague Dawley rats, aged 1 month and weighing 100-150 g, were used for cell culture. Sixty Sprague Dawiey rats aged 3 months and weighing 220-250 g, were used to establish neurological defect models. Nestin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 antibodies were provided by Santa Cruz Biotechnology, Inc., USA. Acellular nerve grafts were derived from dogs.METHODS: All rats, each with 1-cm gap created in the right sciatic nerve, were randomly assigned to three groups. Each group comprised 20 rats. Autograft nerve transplantation group: the severed 1-cm length nerve segment was reverted, but with the two ends exchanged; the proximal segment was sutured to the distal sciatic nerve stump and the distal segment to the proximal stump. Blank nerve scaffold transplantation group: a 1-cm length acellular nerve graft was used to bridge the sciatic nerve gap. NTCSC engineered nerve transplantation group: a 1-cm length acellular nerve graft, in which NTCSCs were inoculated, was used to bridge the sciatic nerve gap.MAIN OUTCOME MEASURES: Following surgery, sciatic nerve functional index and electrophysiology functions were evaluated for nerve conduction function, including conduction latency, conduction velocity, and action potential peak. Horseradish peroxidase (HRP, 20%) was injected into the gastrocnemius muscle to retrogradely label the L4 and L5 nerve ganglions, as well as neurons in the anterior horn of the spinal cord, in the three groups. Positive expression of nestin, NSE, GFAP, and S-100 were determined using an immunofluorescence double-labeling method.RESULTS: NTCSCs differentiated into neuronal-like cells and glial-like cells within 12 weeks after NTCSC engineered nerve transplantation. HRP retrograde tracing displayed a large amount of HRP-labeted neurons in L4-5 nerve ganglions, as well as the anterior horn of the spinal cord, in both the autograft nerve transplantation and the NTCSC engineered nerve transplantation groups. However, few HRP-labeled neurons were detected in the blank nerve scaffold transplantation group. Nerve bridges in the autograft nerve transplantation and NTCSC engineered nerve transplantation groups exhibited similar morphology to normal nerves. Neither fractures or broken nerve bridges nor neuromas were found after bridging the sciatic nerve gap with NTCSCs-inoculated acellular nerve graft, indicating repair. Conduction latency, action potential, and conduction velocity in the NTCSC engineered nerve transplantation group were identical to the autograft nerve transplantation group (P>0.05), but significantly different from the blank nerve scaffold transplantation group (P<0.05). CONCLUSION: NTCSC tissue-engineered nerves were able to repair injured nerves and facilitated restoration of nerve conduction function, similar to autograff nerve transplantation.     

Surgical repair of peripheral nerve injury

Magnification, use of fine interfascicular grafts for repair, and development of intraoperative electrophysiologic measurements of function have had a substantial impact on this field in the last 10 to 20 years. Basic surgical principles established during and since World War II remain the foundation for surgical repair of peripheral nerve injury but have been complemented nicely by these more recent advances. Selection of patients for surgery, as well as the timing of such, has been reviewed with emphasis on the differences between suspected transections and lesions in continuity, as well as comments on serious peripheral entrapments and tumors affecting nerve. The importance of not only preoperative electromyographic studies but also the intraoperative use of stimulation and stimulation and recording of nerve action potentials (NAPs) for lesions in continuity has been stressed. Operative techniques such as neurolysis, NAP recordings, suture, split repair, and interfascicular graft repair have been reviewed and some commentary on results provided. There has been a gradual evolution of centers in this country and abroad for care of the more serious surgical nerve problems. It is anticipated that in the future, such centers will be able to provide improved data concerning results with civilian nerve injuries.     

Isometric contractile function following nerve grafting: A study of graft storage

In order to assess the effects of storage on nerve grafts, the isometric contractile function of the gastrocnemius muscle was assessed 14 months following sciatic nerve autografting in the rat. Three-centimeter sciatic nerve grafts were stored at either 5°C or 22°C for 6 h, 24 h, or 3 weeks in an organ transplant solution. Muscle mass and maximal force in the fresh control graft group returned to 47% and 36% of normal levels, respectively, which was similar to stored grafts. Storage at 5°C was superior to 22°C and there was no decrement in contractile function in grafts stored up to 3 weeks at 5°C. These findings suggest that the storage of nerve grafts is a feasible technique that might be applied to nerve allografts, thus permitting elective reconstruction of large peripheral nerve gaps. © 1994 John Wiley & Sons, Inc.     

改良法脱细胞后坐骨神经的生物力学特性

Nerve grafts are able to adapt to surrounding biomechanical environments if the nerve graft itself exhibits appropriate biomechanical properties (load, elastic modulus, etc.). The present study was designed to determine the differences in biomechanical properties between fresh and chemically acellularized sciatic nerve grafts. Two different chemical methods were used to establish acellular nerve grafts. The nerve was chemically extracted in the Sondell method with a combination of Triton X-100 (nonionic detergent) and sodium deoxycholate (anionic detergent), and in the modified method with a combination of Triton X-200 (anionic detergent), sulfobetaine-10 (SB-10, amphoteric detergents), and sulfobetaine-16 (SB-16, amphoteric detergents). Following acellularization, hematoxylin-eosin staining and scanning electron microscopy demonstrated that the effect of acellularization via the modified method was similar to the traditional Sondell method. However, effects of demyelination and nerve fiber tube integrity were superior to the traditional Sondell method. Biomechanical testing showed that peripheral nerve graft treated using the chemical method resulted in decreased biomechanical properties (ultimate load, ultimate stress, ultimate strain, and mechanical work to fracture) compared with fresh nerves, but the differences had no statistical significance (P > 0.05). These results demonstrated no significant effect on biomechanical properties of nerves treated using the chemical method. In conclusion, nerve grafts treated via the modified method removed Schwann cells, preserved neural structures, and ensured biomechanical properties of the nerve graft, which could be more appropriate for implantation studies.     

Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts

The aim of this study was to investigate the effects of vascular endothelial growth factor (VEGF) on regeneration of the rat sciatic nerve in vivo. To that end we used 10-mm long cell-free nerve grafts to bridge a gap in the sciatic nerve. The grafts were pretreated with either VEGF (50, 100 or 250 ng/ml), nerve growth factor (NGF, 100 ng/ml) or laminin (100 ng/ml) before implantation. Outgrowth of axons, Schwann cells, blood vessels and macrophages were studied 10 days post-implantation by the use of immunocytochemistry and histochemistry. Grafts pretreated with VEGF stimulated the outgrowth of Schwann cells and blood vessels but not axons. In such grafts, the Schwann cells also exhibited a dramatic change in morphology and became filled with large lipid-containing vacuoles. These cells also showed an intense immunoreactivity for the VEGF receptor flk-1. Neither pretreatment with laminin nor NGF affected the outgrowth of Schwann cells. However, NGF treatment increased the number of axons in the graft but was not able to counteract injury-induced downregulation of substance P in the dorsal root ganglia. The results show that local application of VEGF promotes at least two events, invasion of Schwann cells and neovascularization, which are important during nerve regeneration. The findings suggest that the effects of the pretreatment by the growth factors is local and limited to the graft, whereas central events like neuropeptide synthesis is not affected.     

Near-nerve needle sensory conduction study of the medial calcaneal nerve: New method and report of four cases of medial calcaneal neuropathy

There has been one previously published antidromic method for studying medial calcaneal nerve (MCN) conduction. However, the origin of the compound nerve action potentials (CNAPs) with this technique is uncertain because of the antidromic nature of stimulation. We report a new orthodromic method for MCN conduction study using the near-nerve needle technique. In 35 normal controls, maximum nerve conduction velocity (NCV) and negative-peak NCV of MCN were 42.4 +/- 3.9 m/s and 33.6 +/- 3.0 m/s, respectively. The amplitude of the CNAP was 4.1 +/- 2.2 micro V. We also report four cases of medial calcaneal neuropathy, three of which were confirmed by this technique. We conclude that the present technique is capable of recording the sensory nerve action potentials of MCN in isolation and confirming the diagnosis of medial calcaneal neuropathy electrophysiologically.