The rabbit brachial plexus as a model for nerve repair surgery--histomorphometric analysis.

One of the most devastating injuries to the upper limb is trauma caused by the avulsion. The anatomical structure of the rabbit's brachial plexus is similar to the human brachial plexus. The aim of our study was to analyze the microanatomy and provide a detailed investigation of the rabbit's brachial plexus. The purpose of our research project was to evaluate the possibility of utilizing rabbit's plexus as a research model in studying brachial plexus injury. Studies included histomorphometric analysis of sampled ventral branches of spinal nerves C5, C6, C7, C8, and Th1, the cranial trunk, the medial part of the caudal trunk, the lateral part of the caudal trunk and peripheral nerve. Horizontal and vertical analysis was done considering following features: the axon diameter, fiber diameter and myelin sheath. The number of axons, nerve area, myelin fiber density and minimal diameter of myelin fiber, minimal axon diameter and myelin area was marked for each element. The changes between ventral branches of spinal nerves C5-Th1, trunks and peripheral nerve in which the myelin sheath, axon diameter and fiber diameter was assessed were statistically significant. It was found that the g-ratio has close value in the brachial plexus as in the peripheral nerve. The peak of these parameters was found in nerve trunks, and then decreased coherently with the nerves travelling peripherally.

Induction of phosphorylated c-Jun in neonatal spinal motoneurons after axonal injury is coincident with both motoneuron death and regeneration.

c-Jun activation has been implicated not only in neuronal degeneration, but also in survival and regeneration. Here, we investigated c-Jun activation in injured motoneurons by using a nerve crush model in neonatal rats. We identified two distinct subpopulations of motoneurons: about 60% underwent degeneration following injury whereas the remaining 40% survived and induced a regeneration response at 3 weeks post injury. However, all motoneurons examined expressed phosphorylated-c-Jun-immunoreactivity (p-c-Jun-IR) at the early stage of 3 days following injury. These results suggest that active c-Jun was induced in all neonatal motoneurons following nerve crush injury, regardless of whether they were destined to degenerate or undergo successful regeneration at a later stage. Our findings therefore support the hypothesis that active c-Jun is involved in both neuronal degeneration and regeneration.

Regulation of Schwann cell differentiation and proliferation by the Pax-3 transcription factor.

Pax-3 is a paired domain transcription factor that plays many roles during vertebrate development. In the Schwann cell lineage, Pax-3 is expressed at an early stage in Schwann cells precursors of the embryonic nerve, is maintained in the nonmyelinating cells of the adult nerve, and is upregulated in Schwann cells after peripheral nerve injury. Consistent with this expression pattern, Pax-3 has previously been shown to play a role in repressing the expression of the myelin basic protein gene in Schwann cells. We have studied the role of Pax-3 in Schwann cells and have found that it controls not only the regulation of cell differentiation but also the survival and proliferation of Schwann cells. Pax-3 expression blocks both the induction of Oct-6 and Krox-20 (K20) by cyclic AMP and completely inhibits the ability of K20, the physiological regulator of myelination in the peripheral nervous system, to induce myelin gene expression in Schwann cells. In contrast to other inhibitors of myelination, we find that Pax-3 represses myelin gene expression in a c-Jun-independent manner. In addition to this, we find that Pax-3 expression alone is sufficient to inhibit the induction of apoptosis by TGFß1 in Schwann cells. Expression of Pax-3 is also sufficient to induce the proliferation of Schwann cells in the absence of added growth factors and to reverse K20-induced exit from the cell cycle. These findings indicate new roles for the Pax-3 transcription factor in controlling the differentiation and proliferation of Schwann cells during development and after peripheral nerve injury.

[Preparation of three-dimensional porous scaffold of PLGA-silk fibroin-collagen nanofiber and its cytocompatibility study].

OBJECTIVE: To develop three-dimensional (3D) porous nanofiber scaffold of PLGA-silk fibroin-collagen and to investigate its cytocompatibility in vitro. METHODS: Method of electrostatic spinning was used to prepare 3D porous nanofiber scaffold of PLGA-silk fibroin-collagen (the experimental group) and 3D porous nanofiber scaffold of PLGA (the control group). The scaffold in each group was observed by scanning electron microscope (SEM). The parameters of scaffold fiber diameter, porosity, water absorption rate, and tensile strength were detected. SC harvested from the bilateral brachial plexus and sciatic nerve of 8 SD suckling rats of inbred strains were cultured. SC purity was detected by S-100 immunohistochemistry staining. The SCs at passage 4 (5 x 10(4) cells/mL) were treated with the scaffold extract of each group at a concentration of 25%, 50%, and 100%, respectively; the cells treated with DMEM served as blank control group. MTT method was used to detect absorbance (A) value 1, 3, 5, and 7 days after culture. The SC at passage 4 were seeded on the scaffold of the experimental and the control group, respectively. SEM observation was conducted 2, 4, and 6 days after co-culture, and laser scanning confocal microscope (LSCM) observation was performed 4 days after co-culture for the growth condition of SC on the scaffold. RESULTS: SEM observation: the scaffold in two groups had interconnected porous network structure; the fiber diameter in the experimental and the control group was (141 +/- 9) nm and (205 +/- 11) nm, respectively; the pores in the scaffold were interconnected; the porosity was 87.4% +/- 1.1% and 85.3% +/- 1.3%, respectively; the water absorption rate was 2 647% +/- 172% and 2 593% +/- 161%, respectively; the tensile strength was (0.32 +/- 0.03) MPa and (0.28 +/- 0.04) MPa, respectively. S-100 immunohistochemistry staining showed that the SC purity was 96.5% +/- 1.3%. MTT detection: SC grew well in the different concentration groups and the control group, the absorbance (A) value increased over time, significant differences were noted among different time points in the same group (P < 0.05), and there was no significant difference between the different concentration groups and the blank control group at different time points (P > 0.05). SEM observation: in the experimental group, SC grew well on the scaffold, axon connection occurred 4 days after co-culture, the cells proliferated massively and secreted matrix 6 days after co-culture, and the growth condition of the cells was better than the control group. The condition observed by LSCM 4 days after co-culture was the same as that of SEM. CONCLUSION: The 3D porous nanofiber scaffold PLGA-silk fibroin-collagen prepared by the method of electrostatic spinning is safe, free of toxicity, and suitable for SC growth, and has good cytocompatibility and proper aperture and porosity. It is a potential scaffold carrier for tissue engineered nerve.

An ultrasonographic and histological study of intraneural injection and electrical stimulation in pigs.

BACKGROUND: In this study we evaluated the minimum stimulating current associated with intraneural needle placement and sonographic appearance of intraneural injection. METHODS: We inserted a needle 2 cm inside 28 pig nerves (brachial plexus in vivo), recorded the minimum current to elicit a motor response, and injected dye (5 mL) under ultrasound (US) imaging. RESULTS: The minimum current to elicit a motor response was 0.43 mA (range: 0.12-1.8 mA). Nerve expansion was visualized by US in 24 of 28 nerves. Histology revealed penetration of the epineurium in these same 24 nerves. There was no evidence of dysplasia within the fascicle of any nerve. CONCLUSIONS: US may prove useful to detect intraneural injection, whereas a motor response above 0.5 mA may not exclude intraneural needle placement. The correlation between intraneural injection and neurological dysfunction remains unclear.

Subnuclear localization of FOS-like immunoreactivity in the rat parabrachial nucleus after nociceptive stimulation.

The effect of noxious stimulation on the expression of FOS-like immunoreactivity (FOS-LI) in neurons of the parabrachial nucleus (PB) was studied in awake, freely moving rats. In one series of experiments, the rats were subjected to noxious mechanical stimulation (pinch) of either the nape of the neck or the base of the tail for 20 seconds every 5 minutes for 90 minutes, and then they were killed by transcardial perfusion after 45-210 minutes. Control animals received innocuous mechanical stimulation (brush) of the tail. Noxious stimuli resulted in FOS-LI in neurons in the dorsal part of the lateral PB, with heavy labeling in the superior lateral (PBsl) and the dorsal lateral (PBdl) subnuclei. FOS-LI was also elicited in the central lateral subnucleus (PBcl) and, although much more sparsely, in the external lateral subnucleus and the Kölliker-Fuse nucleus. Tail and neck stimulation resulted in similar labeling patterns, but more neurons, particularly in PBsl, expressed FOS-LI after pinch of the tail than of the neck. In another series of experiments, rats received injection of 5% formalin into one hindpaw. After 75-90 minutes, FOS-LI was seen in the same parts of PB as after noxious mechanical stimulation. The heaviest labeling was seen on the side contralateral to the injection side, with statistically significant (P < 0.05) side differences present in PBsl and PBdl. In a third series of experiments, rats were hemisected at low cervical-upper thoracic segments, allowed 2 weeks to recover, and then given formalin injections in both hindpaws. Significantly more neurons were FOS-labeled in PBdl, PBsl, and PBcl on the side contralateral to the hemisection than on the ipsilateral side. These observations are discussed in relation to the organization of the spinal afferent input and the efferent connections of PB. It is concluded that the FOS-LI expression in PBdl and PBsl and probably also in PBcl, to a large extent, is evoked by the ascending spinal nociceptive input to PB. Because these subnuclei project to several hypothalamic regions, it is suggested that neurons in PB that express FOS after noxious mechanical and chemical stimulation primarily are involved in autonomic and homeostatic responses to behavioral situations that involve tissue-damaging stimuli.

Androgen regulation of neuromuscular junction structure and function in a sexually dimorphic muscle of the frog Xenopus laevis.

Specific forelimb muscles in anurans are sexually dimorphic and underlie the androgen-dependent clasping response of males during amplexus. Previous studies have reported that androgen treatment slows the contractile properties of these sexually dimorphic forelimb muscles. In amphibians, the expression of functionally distinct acetylcholine (ACh) receptors, the levels of acetylcholinesterase (AChE), the extent of multiple innervation, and the structure of individual end plates vary with the contractile properties of the muscle fibers. In higher vertebrates, androgens have been reported to alter the expression of ACh receptors, AChE, and the neuromodulator, calcitonin gene-related peptide (CGRP). To determine whether the known androgen-dependent changes in contraction of androgen-sensitive forelimb muscles are accompanied by concomitant changes in synaptic structure or function, we have compared functional neuromuscular transmission, the pattern of innervation, and CGRP immunoreactivity in nerve or muscle preparations from castrated (C) and castrated and testosterone-treated (CT) adult male Xenopus laevis. CGRP expression in androgen receptor (AR)-immunopositive neurons was increased in CT animals. However, no significant differences were found in ACh-mediated single channel or macroscopic currents, the extent of multiple end plates, or end plate morphology for forelimb fibers isolated from C and CT Xenopus. In contrast, analysis of forelimb fibers from gonadally intact adult females and juvenile animals of both sexes revealed that macroscopic synaptic currents were significantly shorter in these animals than in either C or CT adult males. Our data suggest that forelimb fibers in sexually dimorphic muscles of Xenopus do show significant differences in synaptic transmission; however, neither end-plate organization nor functional neuromuscular transmission are subject to activational effects of androgens in adult male frogs.

An experimental study of contralateral C7 root transfer with vascularized nerve grafting to treat brachial plexus root avulsion.

Experimental rat models of simulated brachial plexus injuries were devised to compare the effect of contralateral C7 root transfer with phrenic neurotization. The effect of vascularized nerve grafting (VNG) was also compared with the use of conventional nerve grafts (CNG) in the treatment of root avulsion of the brachial plexus. 160 rats were randomly divided into four groups of 40 each; contralateral C7 root transfer with a vascularized ulnar nerve graft (C7-VNG), contralateral C7 root transfer with conventional ulnar nerve grafting (C7-CNG), ipsilateral phrenic nerve transfer with a vascularized ulnar nerve graft (P-VNG) and ipsilateral phrenic nerve transfer with conventional ulnar nerve grafting (P-CNG). Electrophysiological and histological examinations and functional evaluation were performed at different post-operative intervals. C7 root transfer was found to be superior to phrenic nerve transfer and VNG superior to CNG. Severance of the C7 nerve root was not found to affect limb function on the healthy side.

Lesion-induced changes in the central terminal distribution of galanin-immunoreactive axons in the dorsal column nuclei.

Rats that sustained forelimb removal on either embryonic day (E) 16, on the day of birth (P-0), or transection of the brachial plexus in adulthood had brainstem sections stained for galanin, calcitonin gene-related peptide (CGRP), or substance P (SP) at various intervals after these lesions were made. In normal adult rats, only a few galanin-immunoreactive fibers are present in the cuneate nucleus and most are located in its caudal portion. CGRP-positive axons are also sparse in the cuneate and are distributed mainly in the periphery of the nucleus. SP-positive axons are seen throughout the cuneate nucleus. In rats that sustained forelimb removals at birth or transection of the brachial plexus in adulthood, dense galanin immunoreactivity was present throughout the cuneate nucleus at all rostrocaudal levels on the side of the brainstem ipsilateral to the lesion. The changes after lesions that were made in the adult animals were apparent within 1 week, the earliest time analyzed. Increases in galanin immunoreactivity in the cuneate of animals that sustained forelimb removals on P-0 were first visible on P-2. Neither forelimb removal at birth nor brachial plexus lesions in adulthood had any qualitative effect upon the distribution or density of CGRP- or SP-immunoreactivity in the cuneate nucleus. Removal of a forelimb on E-16 did not increase the density of galanin-immunoreactive fibers in the cuneate nucleus. Such lesions also failed to produce any appreciable change in the density of either CGRP- or SP-positive fibers in the cuneate nucleus. The present data raise the possibility that large caliber, non-peptidergic primary afferent axons which innervate the cuneate nucleus may express galanin after damage at birth or in adulthood.

Changes in microglial cell numbers in the spinal cord dorsal horn following brachial plexus transection in the adult rat.

The effect of peripheral nerve transection on the size of the microglial cell population in cytoarchitecturally distinct regions of the spinal cord dorsal horn of rats was evaluated at selected intervals 2 through 35 days after unilateral brachial plexotomy. The identification of cells was verified by electron microscopic examination of a representative random sample of cells included in the counts. Microglial cell numbers were increased in laminae I, II as well as the arbitrarily defined deeper laminae 3.5 days after surgery. Although microglial cell numbers in laminae I were within normal range 35 days after axotomy, those of the more ventrally located laminae remained significantly greater than control values for the duration of the experimental period. These findings demonstrate that: 1) microglial cell proliferation in the dorsal horn is an early event in the central changes that are attendant to peripheral nerve injury 2) the time course of the response varies in cytoarchitecturally different regions.