Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.     

The role of extracellular matrix in peripheral nerve regeneration: a wound chamber study

Summary A wound chamber model was used for the study of the interaction between axon, Schwann cell and extracellular matrix during peripheral nerve regeneration. Impermeable silicone tubes, 8 mm long and 1.4 mm in internal diameter were sutured to transected rat sciatic nerve and the contents of the tubes were removed at intervals for chemical, histological, immunocytochemical and electron microscopic studies. There was an initial phase of fluid accumulation and the formation of a fibrin/fibronectin clot or cable which connected the cut ends of the nerve. The chamber fluid was shown to have a protein profile similar to that of rat serum. Schwann cells, endothelial cells and fibroblasts migrated first into the cable, apparently mediated by cell-fibrin interaction. Axons buried within the Schwann cell cytoplasm were led into the cable but an axon-fibrin interaction was not observed. After 1 week, the fibrin matrix underwent dissolution, with replacement by collagen. This marked the onset of myelination and the organization of nerve fibers into fascicles. The findings from the present study suggest that the interactions between axon and Schwann cell and between Schwann cell and a changing extracellular matrix are the essential driving force in nerve growth and differentiation during peripheral nerve regeneration.Supported by a grant from the National Science Council of R. O. C. (NSC 80-0412-B075-67)     

Insulin-like growth factor-I promotes nerve regeneration through a nerve graft in an experimental model of facial paralysis

Among the pathological sequelae of facial paralysis is a paralytic eye. Apart from the psychological and aesthetic deficits, facial paralysis if left untreated can lead to dryness, ulceration and eventual blindness. Although numerous restorative microsurgical approaches have been introduced to address the sequelae of this problem, complete restoration of function to denervated facial muscles remains elusive.Utilizing the rat model of facial paralysis the present research has as an objective to examine a dual treatment approach. Specifically, this study combined the current microsurgical treatment of the cross-facial nerve graft with local administration of insulin-like growth factor I (IGF-I).The efficacy of this combined approach (cross-facial nerve graft + IGF-I) was assessed in the following ways: (a) behavior measurement of the blink response and (b) histomorphometry light and electron microscopy of the entire nerve graft. These data will help provide insight into the restoration of facial muscle function after trauma and assist in the future development of more potent treatment strategies.7he local adnünistration of IGF-I (50 micro g/ml) to the cross-facial nerve graft was found to restore the blink response faster and to strengthen the degree of eye closure. Light microscopy examination revealed that IGF-I significantly enhanced axonal regeneration within a nerve graft (a 22% increase in the mean number of axons), and increased the mean nerve fiber diameter and myelin thickness. Electron microscopy assessment of the nerve grafts demonstrated that the IGF-I treated grafts possessed a greater density of microtubules, which were evenly distributed within the axoplasm.     

Role of extracellular matrix in the regeneration of a pacinian corpuscle

The pacinian corpuscle is composed of an inner and an outer core or bulb. The former is formed by tightly packed and multi-layered thin cellular processes (lamellae) of lamellar cells which surround a centrally located axon terminal, and the latter, also called the capsule, is made up of very loosely piled layers of thin cells which encircle the inner core. Lamellar cells of the inner core are considered to be specialized Schwann cells, and the outer core cells are modified perineurial cells. In the present study, the matrix filling the extracellular spaces of the inner core consisted of basal lamina-like amorphous materials, sparce fine collagen fibrils, and the ground substance embedding these structural components. No definite basal laminae were found on the inner core lamellae except on the peripherally located ones which had distinct basal laminae. Outer-core cells were invested along the entire contour by distinct basal laminae. The interspace between the inner and outer cores was a continuation of the nerve endoneurium. The purpose of this investigation was to determine whether the extracellular matrix of the pacinian corpuscle, especially that of the inner core, has the ability to cause corpuscle regeneration, i.e. to make the regenerating axons and Schwann cells differentiate into corpuscular axon terminals and inner core cells, respectively. Pacinian corpuscles in the periosteum at the distal end of the fibula of mice were repeatedly frozen (3-5 times) in situ with forceps cooled with liquid nitrogen. Within 2-3 days, all the cellular constituents of the corpuscle had degraded, while the extracellular matrices of the inner and outer cores apparently remained undamaged. After 5-7 days, regenerating axons and accompanying immature Schwann cells entered these extracellular matrices of the inner cores. A remarkable finding was that these immature Schwann cells were detached from the axon, and sent thin cellular processes around the axon in a characteristic fashion, basically forming the same pattern as lamellae in a normal corpuscle. The regeneration of the inner core was completed by about 40 days after the freezing treatment. In the outer core, perineurial cells proliferated and extended through the basal lamina tubes of the old cells, becoming new outer core cells. These findings indicate that the extracellular matrix of the pacinian corpuscle has a specific property to cause the regeneration of the corpuscle.     

Role of the extracellular matrix in myelination of peripheral nerve.

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination.     

Altered synaptic organization in facial nucleus following facial nerve regeneration: An electrophysiological study in man

Seven patients with postfacial palsy contracture and mass contractions were investigated electrophysiologically. In 3 patients the early blink reflex showed an unusually high amplitude, which can be attributed to enhanced excitability of facial motor neurons. In 5 patients the early blink reflex had acquired a crossed character. It is assumed that changes in organization of the facial nucleus contribute to the altered function of reinnervated facial muscles.     

Effect of hyaluronidase on brain extracellular matrix in vivo and optic nerve regeneration

In the rat, intracerebral injection of bacterial hyaluronidase resulted in the almost complete disappearance of hyaluronic acid (HA) and glial hyaluronate-binding protein (GHAP) from cerebral hemispheres, brain stem, and cerebellum (but not from optic nerves and chiasm) starting 2–3 hr after the injection. HA and GHAP reappeared throughout the brain in characteristic patches 2–3 days after the injection. The patches gradually became confluent and after 12 days the brain appeared virtually normal. In normal rat optic nerve, staining for HA and GHAP ceased abruptly in the region of the lamina cribrosa. The retina was completely negative. HA and GHAP disappeared from hyaluronidase-injected optic nerve, chiasm, and contralateral optic nerve. In hyaluronidase-injected crushed optic nerves, regenerated axons were able to grow for short distances (about 500μm) into the distal stump undergoing Wallerian degeneration. No such growth was observed in saline-injected controls. © 1993 Wiley-Liss, Inc.     

Axonal and extracellular matrix responses to experimental chronic nerve entrapment

We have analyzed the ultrastructural and histopathological changes that occur during experimental chronic nerve entrapment, as well as the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG). Adult hamsters (n = 30) were anesthetized and received a cuff around the right sciatic nerve. Animals survived for varying times (5 to 15 weeks) being thereafter perfused transcardially with fixative solutions either for immunohistochemical or electron microscopic procedures. Experimental nerves were dissected based upon the site of compression (proximal, entrapment and distal). CSPG overexpression was detected in the compressed nerve segment and associated with an increase in perineurial and endoneurial cells. Ultrastructural changes and data from semithin sections were analyzed both in control and compressed nerves. We have observed endoneurial edema, perineurial and endoneurial thickening, and whorled cell-sparse pathological structures (Renaut bodies) in the compressed nerves. Morphometrical analyses of myelinated axons at the compression sites revealed: (a) a reduction both in axon sectional area (up to 30%) and in myelin sectional area (up to 80%); (b) an increase in number of small axons (up to 60%) comparatively to the control group. Distal segment of compressed nerves presented: (a) a reduction in axon sectional area (up to 60%) and in myelin sectional area (up to 90%); (b) a decrease in axon number (up to 40%) comparatively to the control data. In conclusion, we have shown that nerve entrapment is associated with a local intraneural increase in CSPG expression, segmental demyelination, perineurial and endoneurial fibrosis, and other histopathological findings.     

Vascularized nerve grafts: an experimental study

Objective: The aim of this study is to define an experimental model in order to promote the functional recovery of the nerves using grafts with vascular support (Vascular Nerve Grafts – VNG). The aim of this study is to define, on an experimental model in normal recipient bed, whether the functional recovery with VNG is superior to that obtained non-vascularized graft (NNG).

Methods: Twenty male rabbits, which underwent dissection of sciatic nerve, were later treated by reinnervation through an autograft. In 10 animals the reconstruction of sciatic nerve was realized with VNG; in 10 control animals the reconstruction of sciatic nerve was realized with NNG.

Results: The VNG group showed a better axonal organization and a significantly higher number of regenerated axons in the early phases (after 30 days) than the NNG group, whereas the difference in the axonal number at day 90 was less significant; besides, the axon diameter and the myelin thickness were not significantly improved by VNG group.

Discussion: Our data suggests that the use of VNG leads to a faster regeneration process and a better functional recovery, although the final results are comparable to those of the NNG. VNG improve the quality of the axonal regeneration (axonal diameter and Schwann cells), although the increase in the axonal number is not significant and does not improve the long-term functional outcome.     

Analysis of axonal regeneration through the silicone regeneration chamber: A retrograde tracing study in the rabbit facial nerve

Transected facial nerve buccal branches in the adult rabbit were sutured to a silicone growth chamber and regeneration was observed at 3, 5, and 7 weeks postoperation. Using the retrograde axonal transport of horseradish peroxidase technique the soma in the facial motor nucleus were counted and the number was correlated with the number of axons found in the mid-cross section of the regenerating buccal branch and with the number of axons in the original nerve. The somatotopic reorganization in the facial motor nucleus was examined. The mean number of facial motoneuron soma labeled with HRP in the control was 68.0% (+/- 18.6, SE) of the axons counted at periphery. In the regenerating nerves, the labeled soma represented 2.4% of the preoperative controls after 3 weeks and rose to 8.6 and 43.9% at 5 and 7 weeks, respectively. At 3 weeks postoperative time, four of six regenerating nerves did not contain any myelinated axons at the center cross section. The ratios of labeled soma to the regenerating myelinated axons counted at the center cross section at 5- and 7-week time points were 42.2 and 61.1%, respectively. The location of the soma found in the early regeneration phase was similar to the normal distribution except in the dorsal subnucleus. After 7 weeks the proportion of labeled soma in the intermediate subnucleus declined but the general pattern replicated the distribution found in normal control preparations.     

脱细胞细胞外基质及其预构建桥接物修复坐骨神经缺损的实验研究

目的 比较脱细胞细胞外基质(acellular extracellular matris，AECM)同周围神经并行端侧吻合预构建桥接物修复坐骨神经缺损并与自体神经移植后神经再生的效果比较，探讨其修复长段周围神经缺损的可行性。方法 将组织工程化技术制备的AECM 10mm、15mm、20mm预构建，形成一种新的神经桥接物并对其进行了观察和动物移植试验。预构建即：将AECM与坐骨神经并行端侧吻合2周。结果 发现组织工程化技术制备的AECM虽然细胞被完全消蚀，但还具备原有三维结构及生物活性，经预构建后用其修复SD大鼠15mm坐骨神经缺损时，取得了接近自体神经移植的结果，并且没有缺血或炎性表现，无瘢痕形成。结论 这种新的AECM预构建桥接物有可能成为自体神经移植的替代材料。     

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.     

Complement components of nerve regeneration conditioned fluid influence the microenvironment of nerve regeneration

Nerve regeneration conditioned fluid is secreted by nerve stumps inside a nerve regeneration chamber.A better understanding of the proteinogram of nerve regeneration conditioned fluid can provide evidence for studying the role of the microenvironment in peripheral nerve regeneration.In this study,we used cylindrical silicone tubes as the nerve regeneration chamber model for the repair of injured rat sciatic nerve.Isobaric tags for relative and absolute quantitation proteomics technology and western blot analysis confirmed that there were more than 10 complement components(complement factor I,C1q-A,C1q-B,C2,C3,C4,C5,C7,C8β and complement factor D) in the nerve regeneration conditioned fluid and each varied at different time points.These findings suggest that all these complement components have a functional role in nerve regeneration.     

Complement components of nerve regeneration conditioned lfuid inlfuence the microenvironment of nerve regeneration

Nerve regeneration conditioned lfuid is secreted by nerve stumps inside a nerve regeneration chamber. A better understanding of the pro-teinogram of nerve regeneration conditioned lfuid can provide evidence for studying the role of the microenvironment in peripheral nerve regeneration. In this study, we used cylindrical silicone tubes as the nerve regeneration chamber model for the repair of injured rat sciatic nerve. Isobaric tags for relative and absolute quantitation proteomics technology and western blot analysis conifrmed that there were more than 10 complement components (complement factor I, C1q-A, C1q-B, C2, C3, C4, C5, C7, C8β and complement factor D) in the nerve regeneration conditioned lfuid and each varied at different time points. These ifndings suggest that all these complement components have a functional role in nerve regeneration.     

Postoperative facial and vestibular nerve palsy: experimental study of its pathophysiological mechanisms

The 7th and 8th cranial nerves were shifted in the cerebellopontine (CP) angle of dogs by cerebellar retractions that were similar to those performed in humans with monitoring of auditory evoked brainstem responses (ABR). Postoperatively, the vestibular, facial nerves, and brainstem were histologically examined. Caudal-to-rostral shifts of the nerves could induce vestibular and/or facial nerve damages. The most vulnerable portion of the vestibular nerve was located between the vestibular ganglions and the area vestibularis-the most lateral end of the internal auditory canal. This indicated that due to traction force derived from surgical interventions, the nerves and vessels were avulsed at the fundus of the internal auditory canal. The vestibular nerve may be potentially injured more easily and frequently than the cochlear and facial nerves in retromastoid craniectomies with lateral decubitus position in humans. Direct injuries of the facial nerves in the CP angles were not observed in this study. It was elucidated that the facial nerve was usually injured in the facial canal proximal to the geniculate ganglion due to traction force derived from manipulations in the CP angle. It is likely that as facial nerve edema progresses postoperatively, the facial nerve is gradually compressed within the narrow labyrinthine portion of the facial canal. This may be the cause of delayed postoperative facial nerve palsy. The importance to recognize how not only cochlear but also vestibular and facial nerve are injured by the usual manipulations in the CP angle is stressed.