Degree Of Axonal Regeneration Research Articles

Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

Neural Stem Cell– and Schwann Cell–Loaded Biodegradable Polymer Scaffolds Support Axonal Regeneration in the Transected Spinal Cord

Biodegradable polymer scaffolds provide an excellent approach to quantifying critical factors necessary for restoration of function after a transection spinal cord injury. Neural stem cells (NSCs) and Schwann cells (SCs) support axonal regeneration. This study examines the compatibility of NSCs and SCs with the poly-lactic-co-glycolic acid polymer scaffold and quantitatively assesses their potential to promote regeneration after a spinal cord transection injury in rats. NSCs were cultured as neurospheres and characterized by immunostaining for nestin (NSCs), glial fibrillary acidic protein (GFAP) (astrocytes), betaIII-tubulin (immature neurons), oligodendrocyte-4 (immature oligodendrocytes), and myelin oligodendrocyte (mature oligodendrocytes), while SCs were characterized by immunostaining for S-100. Rats with transection injuries received scaffold implants containing NSCs (n=17), SCs (n=17), and no cells (control) (n=8). The degree of axonal regeneration was determined by counting neurofilament-stained axons through the scaffold channels 1 month after transplantation. Serial sectioning through the scaffold channels in NSC- and SC-treated groups revealed the presence of nestin, neurofilament, S-100, and betaIII tubulin-positive cells. GFAP-positive cells were only seen at the spinal cord-scaffold border. There were significantly more axons in the NSC- and SC- treated groups compared to the control group. In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord. Further, these multichannel biodegradable polymer scaffolds effectively serve as platforms for quantitative analysis of axonal regeneration.

The Effects of Trapidil on Fibroblastic Activity, Schwann Cell Proliferation, and Platelet-derived Growth Factor Receptor Levels in the Experimental Sciatic Nerve Injury

Background Trapidil, an antianginal drug with inhibitory effects on platelet-derived growth factor (PDGF), has been shown to reduce reactive fibrosis in models of central nervous system injury. Therefore, we evaluated trapidil's effects on fibroblast activity, Schwann cell proliferation, and PDGF receptor levels in rats after transection and anastomosis of the sciatic nerve. We also evaluated the effect of Schwann cell proliferation on regeneration of anastomosed sciatic nerves. Methods Wistar rats were randomly divided into 3 groups. Group 1 (N=10) was used to determine normal peripheral nerve morphology (via light and electron microscopy) and PDGF receptor levels. Group 2 (N=20) underwent sciatic nerve anastomosis after nerve transection to examine the influence of the surgical procedure on PDGF receptor levels and the microscopic findings. Group 3 (20 rats) received a single intraperitoneal dose of trapidil (40 mg/kg) immediately after the surgical procedure to investigate its effects on fibroblast activity, Schwann cell proliferation, and PDGF levels. Results PDGF-A and PDGF-B receptor levels were lower in group 3, the trapidil-treated group, than group 2 on all posttransection days examined. Ultrastructural analysis revealed that group 3 also had lower levels of fibroblast activity and Schwann cell proliferation than group 2. However, the peripheral nerve ultrastructure and degree of axonal regeneration were similar between groups 2 and 3 at 14 days posttransection. Conclusions Trapidil treatment significantly decreased reactive fibrosis and PDGF receptor expression after peripheral nerve injury, and thus, may be useful therapeutically. These results also suggest that Schwann cells alone are not effective in promoting neurite formation.

Long term assessment of axonal regeneration through polyimide regenerative electrodes to interface the peripheral nerve

Polyimide sieve electrodes were implanted between the severed ends of the sciatic nerve in rats. The degree of axonal regeneration through the electrode was examined by physiological and histological methods from 2 to 12 months postimplantation. Regeneration was successful in the 30 animals implanted. Functional reinnervation of hindlimb targets progressed to reach maximal levels at 6 months. Comparatively, the reinnervation of distal plantar muscles was lower than that of proximal muscles and of digital nerves. The number of regenerated myelinated fibers increased from 2 to 6 months, when it was similar to control values. The majority of myelinated fibers crossing the via holes and regenerated through the distal nerve had a normal appearance. However, in a few cases decline of target reinnervation and loss of regenerated nerve fibers was found from 6 to 12 months postimplantation. Motor axons labeled by ChAT immunoreactivity regenerated scattered within minifascicles, although they were found at higher density at the periphery of the regenerated nerve. The number of ChAT-positive axons was markedly lower distally than proximally to the sieve electrode.

Morphologic and functional evaluation of peripheral nerve fibers regenerated through polyimide sieve electrodes over long-term implantation.

We evaluated by morphologic and functional analysis the regeneration of peripheral nerve fibers through polyimide regenerative-type electrodes over long-term implantation. Polyimide sieve electrodes were placed in silicone chambers and implanted between the severed ends of the sciatic nerve in rats. The sieve part had 281 round via holes of 40 microm in diameter, with nine integrated recording-stimulating electrodes arranged around the via holes. The degree of axonal regeneration was examined at 2, 7, and 12 months postimplantation (mpi). Regeneration was successful in 12 of the 13 animals implanted. Reinnervation of distal muscle and nerves increased with time, reaching a plateau about 7 mpi. The number of myelinated fibers increased from 2 to 7 months, at which time it was similar to control values. With time the myelinated fibers matured, with significant increases in axon diameter and myelin thickness. Only 0.6% of the regenerated axons showed evidence of compression near the implanted electrode. The majority of the myelinated fibers that crossed the via holes and had been regenerated through the distal nerve had a normal appearance. Sieve electrodes were useful for nerve stimulation at postimplantation. Stimulation through different active electrodes excited nerve bundles, evoking compound muscle action potentials of varying shape and amplitude, indicative of selective axonal stimulation.

Enhanced Axonal Regeneration Following Combined Demyelination plus Schwann Cell Transplantation Therapy in the Injured Adult Spinal Cord

We have treated spinal cord injured rats with demyelination plus Schwann cell transplantation and assessed neurite outgrowth in a quantifiable model of axonal regeneration. Axonal injuries of differing severity were induced in the dorsal funiculus of adult rats using a micromanipulator-controlled Scouten knife. Demyelinated regions were produced so as to overlap with the injury site by the injection of galactocerebroside antibodies plus complement one segment cranial to the axonal injury site. Schwann cells were isolated from the sciatic nerve, expanded in vitro, and transplanted into the injury site 1 day later. Animals were killed after an additional 7 days. Schwann cells were evenly distributed throughout the region of demyelination, which extended 6–7 mm cranial to the axonal injury site. The severity of axonal injury was quantified by counting degenerate axons in transverse resin sections. The degree of axonal regeneration was assessed by an electron microscopic analysis of growth cone frequency and distribution relative to the site of axonal injury. Quantification of growth cones at a distance from the site of axonal injury indicated a strong linear relationship (P < 0.001) between the number of growth cones and the number of severed axons; the ratio of growth cones to severed axons was increased by 26.5% in demyelinated plus transplanted animals compared to demyelinated animals without a transplant. Furthermore, only the demyelinated plus transplanted animals contained growth cones associated with myelin in white matter immediately outside of the region of complete demyelination. Growth cones were absent in transplanted-only animals at a distance from the site of axonal injury. These findings indicate that combined demyelination plus Schwann cell transplantation therapy enhances axonal regeneration following injury and suggests that growth cones are able to overcome myelin-associated inhibitors of neurite outgrowth in the presence of trophic support.