Clinical long-term in vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration

It was the purpose of this study to evaluate the clinical long-term effects of PLLA degradation in vivo on nerve regeneration in the rat sciatic nerve model. Thirty-one Sprague Dawley rats were utilized. Two groups of animals were selected. The control group of 10 animals received a 12 mm reversed isograft into the right sciatic nerve from 5 donor animals. The experimental group (n = 21) received a 12 mm empty PLLA conduits placed into a 12 mm defect in the right sciatic nerve. The left leg served as an internal control. Walking track analysis was performed monthly through 8 months. At the end of 4 and 8 months, animals in the control isograft and experimental group had the medial and lateral gastrocnemius muscles harvested and weighed for comparison. The midconduit/isograft and the distal nerve in these same animals were harvested and histomorphologically analyzed. Multiple samples were collected and expressed as means +/- standard error. A two-sample t-test and Wilcoxon rank sum test was used to compare the variables. Significance level was set at alpha = 0.05. After Bonferroni correction for multiple testing, a p value of < or = 0.01 was considered statistically significant. Throughout all time periods, the PLLA conduit remained structurally intact and demonstrated tissue incorporation and vascularization. There was no evidence of conduit collapse or breakage with limb ambulation. Moreover, there was no evidence of conduit elongation at 8 months as previously observed with the 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) conduits. The mean absolute value of the sciatic functional index (SFI) demonstrated no group differences from isograft controls measured over the 8 months except at 3 months where the isograft values were higher (p = 0.0379) and at 7 months were the isograft group was significantly lower (p = 0.0115). At 4 and 8 months, the weight of the gastrocnemius muscles of the experimental group was not significantly different from isografts. At 4 months the number of axons/mm2 and nerve fiber density was not significantly different between the isograft control and experimental groups in either the midconduit/isograft or distal nerve. At 8 months the number of axons/mm2 was significantly lower in the isograft compared to the midconduit experimental group (p = 0.006). The number of axons/mm2 in the distal nerve and the nerve fiber density in the midconduit and distal nerve were not significantly different between the two groups. The study confirmed our initial hypothesis that PLLA conduits are a viable scaffold for clinical long-term nerve gap replacement. We are critically aware however that longer evaluation of polymer degradation is warrented. Further studies on these individual nerve components are continuing, with the ultimate goal being the fabrication of a bioactive conduit that meets or exceeds the functional results of isografts.     

The effect of high outflow permeability in asymmetric poly(dl-lactic acid-co-glycolic acid) conduits for peripheral nerve regeneration

This study attempted to accelerate the peripheral nerve regeneration, using the high outflow rate of asymmetric poly(dl-lactic acid-co-glycolic acid) (PLGA) nerve conduits. Asymmetric PLGA nerve conduits of monomer ratio 85/15 were prepared by immersion-precipitation method to serve as possible materials. In this study, mandrels were immersed into a 20% (wt/wt) of PLGA/1,4-dioxane solution and precipitated in a non-solvent bath followed by freeze-drying. Different concentrations of isopropyl alcohol (95%, 40% and 20%) were used as precipitation baths where non-asymmetric (95%) and asymmetric (40% and 20%) conduits could easily form. The asymmetric nerve conduits that consisted of macrovoids on the outer layer, and interconnected micropores in the inner sublayer, possessed characters of larger outflow rate than inflow rate. The asymmetric conduits were implanted to 10mm right sciatic nerve defects in rats. Autografts, silicone and non-asymmetric PLGA conduits were performed as the control and the contrast groups. Implanted graft specimens of all groups were harvested for histological analysis at 4 and 6 weeks following surgery. The asymmetric PLGA conduits maintained a stable supporting structure and inhibited exogenous cells invasion during entire regeneration process. Asymmetric PLGA conduits were found to have statistically greater number of regenerated axons at the midconduit and distal nerve site of implanted grafts, as compared to the silicone and non-asymmetric groups at 4 and 6 weeks. Of interest was that the results of 4 weeks in asymmetric groups were better than the non-asymmetric groups at 6 weeks in number of axons. According to the results of permeability, the asymmetric structure in the conduit wall seemed to enhance the removal of the blockage of the waste drain from the inner inflamed wound in the early stage, which may have improved the efficacy of the peripheral nerve regeneration. The asymmetric structure could be adequately employed in the future as optimal nerve conduits in peripheral nerve regeneration.     

Effects of nerve growth factor from genipin-crosslinked gelatin in polycaprolactone conduit on peripheral nerve regeneration--in vitro and in vivo

The gelatin solution crosslinked by genipin (0, 0.1, 0.5, 1.0, and 1.5% w/w) was studied as a nerve growth factor (NGF) carrier (GGp0, GGp0.1, GGp0.5, GGp1.0, and GGp1.5) in a polycaprolactone conduit in large-gap nerve regeneration. The GGp0 and GGp0.1 displayed the highest activity of PC12 cells and inhibited the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). No cytotoxicity was found in all groups by lactate dehydrogenase (LDH) release. The NGF-releasing characters were obtained by ELISA tests. A relatively fast release rate appeared during the first 10 days and then a subsequent slower release profile followed. NGF was higher in GGp0.1 than in GGp0 and GGp0.1 after 10 days. The bioactivity of the released NGF remains the same when measuring the neurite outgrowth of PC 12 cells. Finally, the controlled-release conduits were implanted into 12-mm long sciatic nerve gaps of rats. In addition, the best site of NGF carrier was determined either by filling carrier into the conduit lumen or by sucking carrier to the conduit wall. Four and 8 weeks after implantation, morphological analysis revealed that GGp0.1 conduits had markedly larger and more number of myelin axons in the midconduit and distal nerve. Further, sucking the carrier into the conduit wall was an efficient and convenient way to prevent the regeneration of axons and vessels from being impaired by the lumen's carrier. The genipin-crosslinked gelatin is a promising carrier in producing a high release concentration and a long release period of NGF to promote the regeneration over a large-gap nerve injury.     

Comparison between two different methods of immobilizing NGF in poly(DL-lactic acid-co-glycolic acid) conduit for peripheral nerve regeneration by EDC/NHS/MES and genipin

For surface modification and nerve regeneration, chitosan, followed by nerve growth factor (NGF), was immobilized onto the interior surface of poly (lactic acit-co-glycolic) conduits, using EDC/NHS/MES system (EDCs) and genipin (GP). Four new conduits were, therefore, obtained and named by immobilizing order-EDCs/EDCs, GP/EDCs, EDCs/GP, and GP/GP groups. The immobilized methods used were evaluated and compared, respectively. The researchers found that the EDCs- and GP-cross-linked chitosan displayed higher hydrophilic than pure poly (DL-lactic acid-co-glycolic acid) (PLGA) in water contact angle experiment, which meant the cell compatibility was improved by the modification. Scanning electron microscopic observations revealed that the GP-cross-linking of chitosan greatly improved cell compatibility while cultured rat PC12 cells were flatter and more spindle-shaped than EDCs-cross-linked chitosan. The results concerning the GP-cross-linked chitosan revealed significant proliferation of the seeded cells relative to pure PLGA films, as determined by counting cells and MTT assay. The NGF was released from the modified conduits in two separate periods--an initial burst in 5 days and then slow release from day 10 to day 40. The GP/EDCs group had the highest NGF value among all groups after the 5th day. Finally, the controlled-release conduits were used to bridge a 10 mm rat sciatic nerve defect. Six weeks following implantation, morphological analysis revealed the highest numbers of myelinated axons in the midconduit and distal regenerated nerve in GP/EDCs group. Therefore, the results confirm that GP/EDCs groups with good cell compatibility and effective release of NGF can considerably improve peripheral nerve regeneration.     

Different multiple regeneration capacities of motor and sensory axons in peripheral nerve

Abstract After peripheral nerve injury, axons often project sprouts from the node of Ranvier proximal to the damage site. It is well known that one parent axon can sprout and maintain several regenerating axons. If enough endoneurial tubes in the distal stump are present for the regenerating axons to grow along, then the number of mature myelinated nerve fibers in the distal stump will be greater than the number in the proximal stump. "Multiple regeneration" is used to describe this phenomenon in the peripheral nerve. According to previous studies, a prominent nerve containing many axons can be repaired by the multiple regenerating axons sprouting from another nerve that contains fewer axons. Most peripheral nerves contain a mixture of myelinated motor and sensory axons as well as unmyelinated sensory and autonomic axons. In this study, a multiple regeneration animal model was developed by bridging the proximal common peroneal nerve with the distal common peroneal nerve and the tibial nerve. Differences in the multiple regeneration ratio of motor and sensory nerves were evaluated using histomorphometry one month after ablating the dorsal root ganglion (DRGs) and ventral roots, respectively. The results suggest that the motor nerves have a significantly larger multiple regeneration ratio than the sensory nerves at two different time points.     

In vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration.

The present study provides in vivo trials of poly(L-lactic acid) (PLLA) as a porous biodegradable nerve conduit using a 10 mm sciatic nerve defect model in rats. The PLLA conduits, fabricated by an extrusion technique, had an inner diameter of 1.6 mm, an outer diameter of 3.2 mm, and a length of 12 mm. They were highly porous with an interconnected pore structure (of 83.5% porosity and 12.1 microm mean pore size). The conduits were interposed into the right sciatic nerve defect of Sprague Dawley rats using microsurgical techniques; nerve isografts served as controls. Walking track analysis was performed after conduit placement monthly through 16 weeks. At the conclusion of 6 and 16 weeks, sections from the isograft/conduit and distal nerve were harvested for histomorphometric analysis. The right gastrocnemius muscle was also harvested and its weight was determined. All conduits remained intact without breakage. Moreover, no conduit elongated during the 16 weeks of placement. Walking track analysis and gastrocnemius muscle weight demonstrated increasing regeneration over the 16 weeks in both the conduit and isograft control groups, with control values significantly greater. The nerve fiber density in the distal sciatic nerve for the PLLA conduits (0.16+/-0.07) was similar to that for the control isografts (0.19+/-0.05) at 16 weeks. The number of axons/mm2 in the distal sciatic nerve for the PLLA conduits was lower than that for the isografts (13 800+/-2500 vs. 10700+/-4700) at 16 weeks. The results for PLLA were significantly improved over those for 75:25 poly(DL-lactic-co-glycolic acid) of a previous study and suggest that PLLA porous conduits may serve as a scaffold for peripheral nerve regeneration.     

Bridging a 30-mm nerve defect using collagen filaments

This article describes a 30-mm regeneration of severed peripheral nerve axons along collagen filaments. Two thousand or 4000 31-mm-long collagen filaments were grafted to bridge a 30-mm defect of the rat sciatic nerve. A collagen tube was grafted as a control. The mean number and mean fiber diameter of regenerated myelinated axons were 330 +/- 227 and 2.7 +/- 0.9 microm in the distal end of the 2000 collagen-filaments nerve guide, and 564 +/- 275 and 2.5 +/- 1.1 microm in the distal end of the 4000 collagen-filaments nerve guide at 12 weeks postoperatively, whereas in the distal end of the collagen tube, no regenerated axon was found. These results suggest that the collagen filaments guide axons of the rat's sciatic nerve to regenerate for 30 mm and act as a scaffold for axonal regeneration. Thirty-millimeter nerve regeneration of a 1-mm-diameter rat sciatic nerve by an artificial nerve guarantees a clinical application of the implant which should be very important for patients and surgeons.     

Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration

The human amnion consists of the epithelial cell layer and underlying connective tissue. After removing the epithelial cells, the resulting acellular connective tissue matrix was manufactured into thin dry sheets called amnion matrix sheets. The sheets were further processed into tubes, amnion matrix tubes (AMTs), of varying diameters, with the walls of varying numbers of amnion matrix sheets with or without a gelatin coating. The AMTs were implanted into rat sciatic nerves. Regenerating nerves extended in bundles through tubes of 1-2 mm in diameter and further elongated into host distal nerves 1-3 weeks after implantation. Morphometrical analysis of the regenerated nerve cable at the middle of each amnion matrix tube 3 weeks after implantation was performed. The average numbers of myelinated axons were almost the same (ca. 80-112/10(4) microm(2)) in AMTs of 1-2 mm in diameter, as in the normal sciatic nerve (ca. 95/10(4) microm(2)). No myelinated fibers were found in AMTs composed of multiple thin tubes of 0.2 mm in diameter. The myelinated axons were thinner in implanted tubes than those in the normal sciatic nerve. The rate of occurrences of myelinated axons less than 4 microm in diameter was significantly higher in the AMTs, whereas axons in the normal sciatic nerve were diverse in distribution, with the highest population at 8-12 microm in diameter. Reinnervation to the gastrocnemius muscle was demonstrated electrophysiologically 9 months after implantation. It was concluded that the extracellular matrix sheet from the human amnion is an effective conduit material for peripheral nerve regeneration.     

Neurofilament elongation into regenerating facial nerve axons

Immunocytochemistry was used to show that neurofilaments advance into regenerating facial nerve axons at 2.5 mm/day, which is less than the rate of axonal elongation (4.3 mm/day), measured from the transport of radiolabeled protein into the axons. Thus, the distal region of the newly-regenerated axons is deficient in neurofilaments, and this was confirmed by electron microscopy. These neurofilament-free regenerating axons could also be detected by immunocytochemistry using antibody to protein B50 (GAP43), a component of growth-cones. Immunoblots of nerve segments, incubated with monoclonal antibodies against the three neurofilament proteins, showed that all three proteins were present in the neurofilaments elongating into the regenerating axons, and confirmed the more distal extensions of B50 immunoreactivity. These results show that neurofilament immunocytochemistry underestimates the extent of axonal regeneration, and it is suggested that this technique should be employed with caution in regeneration studies. When the facial nerve received a conditioning lesion 7 days prior to a test lesion, axonal regeneration rate increased to 6.0 mm/day, and there was a proportional increase in neurofilament elongation rate to 4.4 mm/day. This occurred in spite of the reduction in cell body neurofilament protein synthesis induced by the lesions. It is concluded that the rate of neurofilament extension into regenerating axons is not governed by cell body synthesis but by local interactions with other cytoskeletal materials which support the increased regeneration rate of conditioned axons.     

The response of regenerating peripheral neuntes to a grafted optic nerve

Summary Optic nerves, both viable (fresh or pre-degenerate) or non-viable (frozen-thawed) were grafted between the proximal and distal stumps of freshly transected sciatic nerves, using either 10/0 sutures or strips of nitrocellulose paper. The majority of regenerating peripheral neuntes, always in association with Schwann cells, avoided the viable optic nerve grafts, growing along the outside of the grafts in well vascularized minifascicles until they gained the distal stumps. A very small number of axons entered the grafts and grew, for distances typically less than 2mm, between layers of astrocyte processes. The number of axons entering was not increased by using predegenerate grafts or by blocking Schwann cell proliferation in the proximal stumps by pre-treating the latter with mitomycin C. There was no evidence of a continuous cellular-acellular partition between graft and host during the outgrowth phase of the neurites: it was concluded that axons failed to enter the grafts as a result of inhibitory interactions between Schwann cells and astrocytes. When grafts were rendered acellular, all structured debris, including recognizable components of the extracellular matrix, was rapidly removed and the space thus vacated was invaded by minifascicles of Schwann cells and regenerating neurites. Glial fibrillary acidic protein-positive astrocytes and carbonic anhydrase II-positive oligodendrocytes persisted within viable grafts for 17 months; they did not migrate into the surrounding nerve.     

Portion of a nerve trunk can be used as a donor nerve to reconstruct the injured nerve and donor site simultaneously

This study aims to estimate the effects of using a portion of a nerve trunk to repair itself and the injured nerve simultaneously. Proximal 1/2 median nerve served as donor nerve to repair the distal 1/2 median and whole ulnar nerve. Four months postoperation, the number of myelinated axons and nerve conduction velocities of the distal half median and ulnar nerve were (2033 ± 135 and 24.6 ± 5.3 m/s) and (1138 ± 228 and 30.3 ± 7.2 m/s). It suggests that using a portion of a nearby nerve truck to reconstruct itself and the injured nerve simultaneously is a practical method for severe peripheral nerve injury.     

Axonal uptake of horseradish peroxidase isoenzymes during Wallerian degeneration

Horseradish peroxidase isoenzymes were applied around crushed mouse hypoglossal nerves to study the influence of electrical charge on the uptake and ultrastructural distribution of macromolecules in axons distal to an injury. Both isoenzymes tested (Sigma type IX, cationic and type VII, anionic) were readily taken up into axons and moved in a distal direction along the nerve. Samples taken 1-3 mm below the crush showed that reaction products from both enzymes covered the inner surface of the axonal plasma membrane and were attached to organelles, particularly microtubules and neurofilaments. However, reaction product in the Schwann cell basement lamina and in the endoneurial collagen was much more dense with cationic peroxidase than with the anionic isoenzyme. Our study shows that both cationic and anionic macromolecules can move into axons distal to a nerve lesion. It can be assumed that also other agents can be taken up into axons and that 'wound substances' in this way may influence the processes by which axons are destroyed during Wallerian degeneration.     

Nerve injury, axonal degeneration and neural regeneration: basic insights

Axotomy or crush of a peripheral nerve leads to degeneration of the distal nerve stump referred to as Wallerian degeneration (WD). During WD a microenvironment is created that allows successful regrowth of nerve fibres from the proximal nerve segment. Schwann cells respond to loss of axons by extrusion of their myelin sheaths, downregulation of myelin genes, dedifferentiation and proliferation. They finally aline in tubes (Büngner bands) and express surface molecules that guide regenerating fibres. Hematogenous macrophages are rapidly recruited to the distal stump and remove the vast majority of myelin debris. Molecular changes in the distal stump include upregulation of neurotrophins, neural cell adhesion molecules, cytokines and other soluble factors and their corresponding receptors. Axonal injury not only induces muscle weakness and loss of sensation but also leads to adaptive responses and neuropathic pain. Regrowth of nerve fibres occurs with high specificity with formerly motor fibres preferentially reinnervating muscle. This involves recognition molecules of the L2/HNK-1 family. Nerve regeneration occurs at a rate of 3-4 mm/day after crush and 2-3 mm/day after sectioning a nerve. Nerve regeneration can be fostered pharmacologically. Upon reestablishment of axonal contact Schwann cells remyelinate nerve sprouts and downregulate surface molecules characteristic for precursor/premyelinating or nonmyelinating Schwann cells. At present it is unclear whether axonal regeneration after nerve injury is impeded in neuropathies.     

人工组织神经移植物引导大鼠再生坐骨神经通过10mm缺损早期的形态学观察

本实验采用壳聚糖导管和聚乙醇酸(PGA)纤维支架构成的人工组织神经移植物桥接大鼠10mm坐骨神经缺损,应用免疫荧光组织化学、激光扫描共聚焦显微镜和电镜技术对早期神经再生过程进行观察。结果显示,Schwann细胞和新生轴突沿PGA支架纤维有序地生长。术后4、7、10和14d,新生轴突和Schwann细胞沿支架由近及远长入的距离分别约150μm、500μm、1.3mm和3.5mm。术后4周,新生轴突前沿已经通过整段移植物长到远侧神经端。实验表明,该移植物有利于Schwann细胞和新生轴突的有序导向生长,能够有效地促进周围神经再生。     

Bioactive poly(L-lactic acid) conduits seeded with Schwann cells for peripheral nerve regeneration.

This study attempted to enhance the efficacy of peripheral nerve regeneration using our previously tested poly(L-lactic acid) (PLLA) conduits by incorporating them with allogeneic Schwann cells (SCs). The SCs were harvested, cultured to obtain confluent monolayers and two concentrations (1 x 10(4) and 1 x 10(6) SC/ml) were combined with a collagen matrix (Vitrogen) and injected into the PLLA conduits. The conduits were then implanted into a 12 mm right sciatic nerve defect in rats. Three control groups were used: isografts, PLLA conduits filled with collagen alone and empty silicone tubes. The sciatic functional index (SFI) was calculated monthly through four months. At the end of second and fourth months, the gastrocnemius muscle was harvested and weighed for comparison and the graft conduit and distal nerve were harvested for histomorphologic analysis. The mean SFI demonstrated no group differences from isograft control. By four months, there was no significant difference in gastrocnemius muscle weight between the experimental groups compared to isograft controls. At four months, the distal nerve demonstrated a statistically lower number of axons mm2 for the high and low SC density groups and collagen control. The nerve fiber density was significantly lower in all of the groups compared to isograft controls by four months. The development of a "bioactive" nerve conduit using tissue engineering to replace autogenous nerve grafts offers a potential approach to improved patient care. Although equivalent nerve regeneration to autografts was not achieved, this study provides promising results for further investigation.     

Collagen filaments as a scaffold for nerve regeneration

This article describes repair of peripheral nerve defect using collagen filaments instead of tubes. Many tube-shaped nerve guides induce regeneration of severed peripheral nerve axons within a limited distance. Substantial regeneration of nerve axons has not been reported without a tubular conduit. Here we show the regeneration of peripheral nerve axons along filaments of collagen without a tube. Cables of collagen filaments were grafted to repair 20-mm defects of rat sciatic nerves. Nerve autografts and collagen tubes were grafted as controls. The mean number and the mean fiber diameter of regenerated myelinated axons were approximately 4800 and 3.3 microm in the distal end of the nerve autograft at 8 weeks postoperatively while in the distal end of the collagen-filaments nerve guide, they were approximately 5500 and 2.3 microm. Collagen tubes failed to bridge the nerve defect. Histologic studies suggest that nerve axons regenerated substantially along the collagen filaments.     

Stimulation and inhibition mechanisms in peripheral nerve regeneration]

To better analyse the early growth of peripheral nerve regeneration, we recently developed a film model. Following transection of a peripheral nerve, e.g. the common peroneal nerve in mice, both proximal and distal stumps of the transected nerve are sandwiched between two sheets of film, and kept in vivo for various timed intervals after axotomy. The regenerating neurites sprout not only from the nodes of Ranvier close to the transected nerve end but also from the terminal bulbs which are formed at the transected nerve end. All of the regenerating neurites consist of naked axons for at least 2 days after axotomy, and elongate on the film with a growth rate of 77 microns/day. On migrating from a parent nerve to the regenerating axons, Schwann cells promote the axons to grow with a 4 times higher speed. Thereafter, a distal nerve stump of the transected nerve release some stimulating factors toward the regenerating nerves, and the axonal growth rate is increased by approximately 1.5 fold. Some inhibitory factors, one of which is myelin-associated glycoprotein, are at the same time released from the distal nerve segment for a week from the 7th post-operative day, and keep new axons from sprouting and inhibit outgrowth of young naked axons in order to optimum regeneration and maturation of outgrowing pioneer axons. It means a so-called pruning phenomenon.     

Ultrastructural and morphometric analysis of long-term peripheral nerve regeneration through silicone tubes

Summary Light and electron microscopy were used to investigate long-term regeneration in peripheral nerves regenerating across a 10 mm gap through silicone tubes. Schwann cells and axons co-migrated behind an advancing front of fibroblasts, bridging the 10 mm gap between 28 and 35 days following nerve transection. Myelination of regenerated fibres started between 14 and 21 days after transection and occurred in a manner similar to that reported during development. Although these early events were successful in producing morphologically normal-appearing regenerated fibres, complete maturation of many of these fibres was never achieved. Axonal distortion by neurofilaments, axonal degeneration and secondary demyelination were seen at 56 days following nerve transection. These changes progressed in severity with time as more axons advanced through the distal stump towards their peripheral target. Since regeneration occurs in the absence of endoneurial tubes, and because constrictive forces act on the nerve during regeneration, we suggest that these extrinsic factors limit the successful advancement of axons through the distal stump to their target organ.     

The axoplasmic reticulum within myelinated axons is not transported rapidly

The axoplasmic reticulum in myelinated axons is an extensive system of branched smooth membranous tubules which is found throughout the length of large axons. To investigate its motility and possible role in fast axonal transport, a focal chilling method was used to arrest transport at two sites separated by a 3 mm wide warm region along the saphenous nerve of mice. The experiments ran for 3-4 h since axoplasmic material travelling faster than 25 mm/day would clear from the central warm region. The nerve was subsequently fixed and processed by a technique that enhances the electron density of the axoplasmic reticulum. Thin and thick sections from several regions along the nerve were then systematically studied using conventional and high voltage electron microscopy. In these studies we found that: 1. the axoplasmic reticulum does not accumulate against the proximal sides of the cold blocks; 2. although often closely associated, there is no evidence of continuity between the axoplasmic reticulum and the discrete membranous compartments that do accumulate proximal to the chilled regions; 3. the axoplasmic reticulum remains in the central 3 mm wide warm region; 4. the axoplasmic reticulum does not accumulate against the distal sides of the cold blocks; 5. retrogradely moving elements that do accumulate distal to the cold blocks do not fuse with the axoplasmic reticulum and are not contained in it; and 6. both retrograde and anterograde vector types are often closely associated with elements of axoplasmic reticulum. These results were supported by quantitative morphometric analysis. We conclude that the axoplasmic reticulum represents a discrete membrane system, separate from either anterogradely or retrogradely moving rapid transport vectors, and that this interconnected cisternal system itself is not rapidly transported.     

Regrowth of PNS axons through grafts of the optic nerve of the Browman-Wyse (BW) mutant rat

Summary We have examined the behaviourin vivo of regenerating PNS axons in the presence of grafts of optic nerve taken from the Browman-Wyse mutant rat. Browman-Wyse optic nerves are unusual because a 2–4 mm length of the proximal (retinal) end of the nerve lacks oligodendrocytes and CNS myelin and therefore retinal ganglion cell axons lying within the proximal segment are unmyelinated and ensheathed by processes of astrocyte cytoplasm. Schwann cells may also be present within some proximal segments. Distally, Browman-Wyse optic nerves are morphologically and immunohistochemically indistinguishable from control optic nerves.When we grafted intact Browman-Wyse optic nerves or triplets consisting of proximal, junctional and distal segments of Browman-Wyse optic nerve between the stumps of freshly transected sciatic nerves, we found that regenerating axons avoided all the grafts which did not contain Schwann cells, i.e., proximal segments which contained only astrocytes; regions of Schwann cell-bearing proximal segments which did not contain Schwann cells; junctional and distal segments (which contained astrocytes, oligodendrocytes and CNS myelin debris). However, axons did enter and grow through proximal segments which contained Schwann cells in addition to astrocytes. Schwann cells were seen within grafts even after mitomycin C pretreatment of sciatic proximal nerve stumps had delayed outgrowth of Schwann cells from the host nerves; we therefore conclude that the Schwann cells which became associated with regenerating axons within the grafts of Browman-Wyse optic nerve were derived from an endogenous population. Our findings indicate that astrocytes may be capable of supporting axonal regeneration in the presence of Schwann cells.