The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture.

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.

Early stages of facial nerve regeneration through silicone chambers in the rabbit.

The early stages of rabbit peripheral facial nerve regeneration [N = 16], over a 10-mm transectional gap, were analyzed at 1, 3, and 5 weeks of buccal nerve entubation in silicone chambers prefilled with saline. Normal nerve pooled data were obtained in nine nerves. Chronologic morphologic and morphometic light and electron microscopic computer-captured data reveal that the regeneration process can be subdivided into four stages: 1. The establishment of an acellular intergap matrix; 2. the ingrowth of mesodermally derived cells (macrophages, fibroblasts, endothelial cells, etc.) to form an intergap scaffolding; 3. the ingrowth of ectodermally derived cells (neurons, Schwann's cells, etc.) to reconstitute the transected peripheral nerve; and 4. neural maturation by progressive axonal enlargement, myelination, and compartmentalization. The first two stages occur during the first week, the third stage during the third week, and the fourth stage from the fifth week onwards of neural entubation. Morphometic analyses indicate that total neuroregenerates increase in number from 303 +/- 286 to 2693 +/- 1334 (+/- values are standard deviations) and myelination of axons increases from 12% to 48% during the third to fifth week of entubation. The thickness of the myelin (g-ratio) increases from 0.89 +/- .01 to 0.74 +/- .06 (SD) during the same period of time. At the end of the fifth week of entubation, the midchambers contain, on the average, 1279 +/- 980 myelinated axons (30% of normal counts) and 483 (+/- 11.2%) axons cross the chambers to innervate the distal transected neural stump (12.5%). Specific target-organ (quadratus labii superioris muscle) activity can be recaptured with a smaller number (12.5%) of regenerating myelinated axons from the buccal division of the facial nerve during the fifth week of entubation.

Rabbit facial nerve regeneration in NGF-containing silastic tubes.

Previous reports suggest that exogenous nerve growth factor (NGF) enhanced nerve regeneration in rabbit facial nerves. Rabbit facial nerve regeneration in 10-mm Silastic tubes prefilled with NGF was compared to cytochrome C (Cyt. C), bridging an 8-mm nerve gap. Three weeks following implantation, NGF-treated regenerates exhibited a more mature fascicular organization and more extensive neovascularization than cytochrome-C-treated controls. Morphometric analysis at the midtube of 3- and 5-week regenerates revealed no significant difference in the mean number of myelinated or unmyelinated axons between NGF- and cytochrome-C-treated implants. However, when the number of myelinated fibers in 5-week regenerates were compared to their respective preoperative controls, NGF-treated regenerates had recovered a significantly greater percentage of myelinated axons than cytochrome-C--treated implants (46% vs. 18%, respectively). In addition, NGF-containing chambers reinnervated a higher percentage of myelinated axons in the distal transected neural stumps (49% vs. 34%). Behavioral and electrophysiologic studies demonstrated spontaneous and induced activities in the target muscles when approximately one third of the myelinated axons were recovered in the midchamber (1280 axons). Horseradish peroxidase (HRP) studies demonstrated retrograde axonal transport to the midchamber and proximal transected neural stump. PC12 bioassay demonstrated persistent NGF activity in the intrachamber fluids at 3 (5:1 dilution) and 5 (2:1 dilution) weeks of entubation. Electrophysiologic tests demonstrated a slow conduction velocity of a propagated electrical impulse (43.5 m/s-1 vs. 67 m/s-1) and shallow wide compound action potential. In wider defects (15-mm chambers) and longer entubation periods (7 weeks), no regeneration or NGF activity was seen. Therefore, exogenous NGF provides an early but limited neurotrophic effect on the regeneration of the rabbit buccal division of the facial nerve and a limited behavioral and physiological improvement in the target muscles.

Facial nerve regeneration through semipermeable chambers in the rabbit.

Peripheral neural regeneration, over a 10-mm transectional gap, was determined in 70 rabbit buccal divisions of the facial nerve using two entubational systems (semipermeable and impermeable silicone chambers) prefilled with three natural occurring media (serum, blood, and saline) during a 5-week period. The number of myelinated axonal regenerates at the midchamber and at 2 mm in the distal transected neural stump were counted in each group and compared to pooled myelinated axonal counts in 9 normal rabbit buccal divisions of the facial nerve. Semipermeable porous chambers had an overall greater regeneration success rate (75% vs. 42.8%) and regained, on the average, a higher number of myelinated axons (51.4% vs. 26.1%) than silicone chamber regenerates. Semipermeable chambers prefilled with serum or blood had significantly higher regeneration success rates, myelinated axonal counts, and percentages of neural innervation of the distal transected neural stump. Both entubational systems produced similar axonal counts with intraluminal saline. The highest overall success rate (93.7%) and average number of myelinated axons per chamber (3072) were achieved in semipermeable chambers prefilled with serum. The greatest variability in myelinated axonal counts (0 to 3266 axons) and percentage of distal stump innervation (5.5% to 98.1%) was seen in silicone chambers filled with saline. The percentage of myelinated axons from the midchamber that innervated the distal stump was greater in semipermeable chambers with blood (73%) and serum (54%) than in silicone saline chambers (43%). On the average, the distal stumps from semipermeable chambers filled with serum (47%) and blood (33.5%) regained a higher percentage of normal myelinated axonal counts than silicone-saline chambers (12.5%). These results suggest that both the construction of entubational chamber and the intraluminal medium can have significant influence on neurite regeneration. Semipermeable chambers prefilled with serum have a strong neurite-promoting potential in peripheral neural regeneration of rabbit facial nerves.

Comparison of rabbit facial nerve regeneration in nerve growth factor-containing silicone tubes to that in autologous neural grafts.

Previous reports suggest that nerve growth factor (NGF) enhanced nerve regeneration in rabbit facial nerves. We compared rabbit facial nerve regeneration in 10-mm silicone tubes prefilled with NGF or cytochrome C (Cyt C), bridging an 8-mm nerve gap, to regeneration of 8-mm autologous nerve grafts. Three weeks following implantation, NGF-treated regenerates exhibited a more mature fascicular organization and more extensive neovascularization than Cyt C-treated controls. Morphometric analysis at the middle of the tube of 3- and 5-week regenerates revealed no significant difference in the mean number of myelinated or unmyelinated axons between NGF- and Cyt C-treated implants. However, when the numbers of myelinated fibers in 5-week regenerates were compared to those in their respective preoperative controls, NGF-treated regenerates had recovered a significantly greater percentage of myelinated axons than Cyt C-treated implants (46% versus 18%, respectively). The number of regenerating myelinated axons in the autologous nerve grafts at 5 weeks was significantly greater than the number of myelinated axons in the silicone tubes. However, in the nerve grafts the majority of the axons were found in the extrafascicular connective tissue (66%). The majority of these myelinated fibers did not find their way into the distal nerve stump. Thus, although the number of regenerating myelinated axons within the nerve grafts is greater than that of axons within silicone tube implants, functional recovery of autologous nerve graft repairs may not be superior to that of intubational repairs.

Effects of thrombin and protease nexin-1 on peripheral nerve regeneration.

Thrombin and serine protease inhibitors such as protease nexin-1 (PN-1) have been implicated in neurite outgrowth activity. We compared rat sciatic nerve regeneration in 10-mm silicone tubes, bridging an 8-mm nerve gap, that were prefilled with thrombin (1.5 IU) and PN-1 (50 micrograms/mL or 1 mg/mL) to those filled with saline solution (control). Neural regeneration and fibrinoid matrix progression (deposition of extracellular matrix) were analyzed at 1, 4, 17, and 21 days after silicone tube implantation. At 1 and 4 days after implantation, thrombin reduced fibrinoid matrix length propagation from both the proximal and distal transected nerve stumps, but PN-1 and saline did not interfere with matrix progression (p < .05). Seventeen days after implantation, the number of silicone tubes containing myelinated neuronal regenerates at the mid-tube region was 1 of 6 for thrombin, 6 of 9 for PN-1, and 6 of 10 for saline solution. Twenty-one days after implantation, 11 of 11 tubes with saline solution, 9 of 11 with PN-1 at 1 mg/mL, and 7 of 9 with PN-1 at 50 micrograms/mL had myelinated neural regenerates in the mid-silicone tube region, while only 2 of 9 thrombin-containing silicone tubes contained myelinated axons. There was no statistically significant difference in myelinated neurite regenerates at 17 and 21 days after implantation among silicone tubes prefilled with saline solution and PN-1 (50 micrograms/mL or 1 mg/mL). Thrombin interfered with matrix progression and significantly reduced the number of myelinated neurite regenerates (p = .01). The PN-1 and saline solutions did not inhibit matrix progression or affect the number of myelinated axonal regenerates (p = .92).

Influence of ganglion age, nonneuronal cells and substratum on neurite outgrowth in culture.

This study characterizes the outgrowth patterns of superior cervical ganglia (SCG) obtained from embryonic (E15), perinatal (E20-21), and adult (P35) rats when placed in culture on various substrata. Outgrowth morphology, degree of fasciculation, and outgrowth length were examined on collagen (COL), polyornithine (PO), polylysine (PL), fibronectin (FN), and nonneuronal cells (NNCs) from the ganglion. COL and FN supported extensive neuritic outgrowth; PO and PL provided poor support. Outgrowth pattern, degree of fasciculation, neurite growth rate, and the number of NNCs in the outgrowth varied considerably depending upon the COL configuration. When undiluted COL (approximately 5 mg/ml) was air dried, a three-dimensional loose fibrillar network was formed. Upon COL dilution or gelling undiluted COL by ammoniation, an essentially two-dimensional layer was formed. On two-dimensional COL, NNCs were able to proliferate and migrate extensively from ganglia of all ages; their presence influenced the form and extent of neurite growth. E15, E20, and P35 neurites responded differently to their endogenous NNCs. E15 neurites extended in relation to NNC surfaces and were predominantly nonfasciculated. E20 neurites became more fasciculated in the presence of NNCs that exhibited morphological and behavioral differences from those migrating from E15 ganglia. E20 neurite bundles became defasciculated when they extended into E15 outgrowth. Far fewer neurites grew from P35 explants in the presence of their NNCs. Three-dimensional COL greatly slowed NNC migration and thus allowed investigation of neurite outgrowth from ganglia of differing age in the absence of NNCs. We conclude that neuritic outgrowth patterns on varying substrata reflect not only neurite differences depending upon ganglion age but also variation in the behavior of accompanying NNCs.

Efficacy of nerve growth factor in regeneration of the mandibular nerve: a preliminary report.

An experimental study evaluating the potential utility of nerve growth factor (NGF) on sensory nerve regeneration was conducted in a rabbit mandibular nerve model. In 10 animals, bilateral 7-mm nerve gaps were created and repaired with the placement of 10-mm Silastic (American Scientific Products, McGaw Park, IL) conduits into which NGF (left side) and a control solution (cytochrome C, right side) was instilled. After 90 days, the nerve repairs were removed and the two sides compared by clinical appearance, and histologic and electrophysiologic assessment. The conduits instilled with control solution failed to result in nerve regeneration in any of the animals. The conduits with NGF solution, however, consistently displayed neural connections between the proximal and distal ends with the presence of slowed, but recordable, conduction velocities. The axonal numbers in the NGF repairs were significantly less than those of the normal nerve, but when adjusted for fascicular size, the axonal densities were comparable. In addition, osteoid tissue was observed around many of the NGF-induced nerve regenerates, but not on the control solution side, suggesting a possible influence of this protein on bone formation.

Nerve growth factor facilitates regeneration across nerve gaps: morphological and behavioral studies in rat sciatic nerve.

The rat sciatic nerve does not possess a high potential for regeneration through silastic tubes when the interstump nerve gap is greater than 10 mm. In this study, the effect of NGF treatment on regeneration of the rat sciatic nerve in 10- and 15-mm silastic chambers was compared. In addition, regeneration in 15-mm silastic chambers was compared to regeneration in 15-mm semipermeable chambers. Sections of tubing were implanted and filled with NGF or a control solution of cytochrome C (Cyt. C). Tube implants were removed at various postoperative times and regeneration was assessed histologically and behaviorally. NGF treatment promoted regeneration success rate. It enhanced the initial outgrowth of nonneuronal cells and neuronal fibers into the chamber producing more cellular, organized regenerates. At 2 weeks, in 10-mm chambers, NGF-treated regenerates had fourfold more unmyelinated fibers than controls. At 3 weeks, NGF-treated regenerates possessed threefold more myelinated fibers than controls. After 4 weeks all regenerates had similar numbers of myelinated nerves at the chamber's midpoint. This initial "head start" was sustained peripherally as indicated by the earlier return of sensory function (response to a noxious temperature stimulus) in NGF-treated animals. Finally, regeneration success rate in 15-mm semipermeable tubes is greater than that in 15-mm silastic chambers (NGF and Cyt. C). However, regenerates in silastic chambers possessed twofold more myelinated fibers than regenerates in semipermeable chambers. The positive effects of NGF on neural regeneration and recovery of sensory function provide support for the potential use of NGF in treating peripheral nerve injuries.