Experience with allografts in the surgery of peripheral nerves (experimental study)

Peroneal nerve allografts four to seven cm in length were transplanted in 36 tissue typed beagle dogs, using a standard microsurgical technique. The influence of tissue typing on nerve regeneration through these grafts was studied with the help of electromyography and histology seven to nine months after nerve implantation. Better regeneration was found through the grafts with compatible than with noncompatible typing. The favourable effect of tissue compatibility became more evident when the length of the graft was longer than four cm. Tissue rejection reaction was much more marked and evident with longer grafts in noncompatible than in compatible groups. Radiation, with tissue typing, did not seem to confer any additional beneficial effect. Compatible tissue typed nerve allografts probably behave more like autografts.     

Quantitative study of muscle fibre atrophy and restitution after nerve grafts

Our comparative experimental studies on rabbits using clinical, electromyographical, and quantitative histological examinations of long autologous and homologous nerve grafts in 35 rabbits, paying special attention to quantitative histological changes in the gastrocnemius muscles, allow the statement that, compared to the short holografts, the long homografts showed worse results. Moreover, it became obvious that the regeneration rate of autografts was not influenced by increasing the lengths of the grafts. The answer to the question of how far even longer grafts may influence the quality of regeneration will need further investigations.     

Latent endothelial cell damage after experimental corneal cryopreservation

Ninety porcine corneas were evaluated by vital staining with alizarin red S and trypan blue in a three-step experiment. Central cell densities were counted (a) on freshly dissected corneas (n = 30), (b) on cryopreserved corneas directly after thawing (n = 30), and (c) after a postthawing organ culture interval of 24 h (n = 30). Two freezing methods were used: (a) minimum essential medium--containing 20% fetal calf serum and (b) the same but containing additionally 2% chondroitin sulfate. Directly after thawing neither method showed significant cell loss (3.9% and 3%) compared to fresh tissue. After postthawing organ culture, however, tissue that had been frozen without chondroitin sulfate displayed a cell loss of 73.5% compared to corneas of the same freezing protocol directly after thawing. Corneas in chondroitin sulfate containing medium showed a cell loss of only 33.2%. We conclude that reliable morphologic evaluation should not be obtained from cryopreserved corneas examined directly after thawing.     

Regeneration of cochlear efferent nerve terminals after gentamycin damage

Chickens recover auditory function after hair cell loss caused by ototoxic drug damage or acoustic overstimulation, indicating that mechanisms exist to reestablish appropriate neuronal connections to regenerated hair cells. However, despite similar hair cell regeneration times, hearing recovery takes substantially longer after aminoglycoside than after sound damage. We have therefore begun examining damage and regeneration of efferent nerve terminals by immunolabeling whole-mount cochleae for differentially localized synaptic proteins and by visualizing the distribution of label with confocal microscopy. In undamaged cochleae, the synaptic proteins synapsin and syntaxin show similar distribution patterns corresponding to the large cup-like terminals on short hair cells. After gentamycin administration, these terminals are disrupted as hair cells are lost, leaving smaller, more numerous synapsin-reactive structures in the sensory epithelium. Syntaxin reactivity remains associated with the extruded hair cells, indicating that the presynaptic membrane is still attached to the postsynaptic site. In contrast, after sound damage, both synapsin and syntaxin reactivity are lost from the epithelium with extruded hair cells. As regenerated hair cells differentiate after gentamycin treatment, the synapsin labeling associated with cup-like efferent endings reappears but is not completely restored even after 60 d of recovery. Thus, efferent terminals are reestablished much more slowly than after sound damage (), consistent with the prolonged loss of hearing function. This in vivo model system allows comparison of axonal reconnection after either complete loss (sound damage) or partial disruption (gentamycin treatment) of axon terminals. Elucidating the differences in recovery between these injuries can provide insights into reinnervation mechanisms.     

Fracture healing and callus innervation after peripheral nerve resection in rats

The effects of femoral and sciatic nerve resection on fracture healing and innervation of the fracture callus were studied using a stable fracture model. In 34 rats the right tibia was subjected to a standardized closed fracture and stabilized with a modular intramedullary nail. In half of the animals, resection of 1 cm of the femoral and sciatic nerves was performed (nerve resection group), whereas the other animals had sham operations (sham group). To avoid unequal load-bearing between the two groups, all fractured hindlimbs were immobilized in a plaster of Paris cast. The trial was terminated after 5 weeks of fracture healing. Callus size was scored radiographically, and bone mineralization was measured by 85-strontium incorporation. Seven rats from each group had immunohistochemical examination for neural regeneration and ingrowth. Antisera for protein gene product 9.5, neurofilaments, neural growth associated protein 43/B-50, calcitonin gene related peptide, and substance P were used. The mechanical properties of the healing fractures were recorded in a three-point cantilever bending test. After 5 weeks, the normally innervated, fractured tibias had regained approximately 50% strength compared with the unfractured side, in comparison with only 20% in the animals that had nerve resection. Although the fracture calluses were mechanically weaker, they were significantly larger in the nerve resection group, indicating defects in tissue composition or organization rendered by the nerve injury. The mineralization rate, as measured by 85-strontium incorporation, was the same in the two groups. However, the nerve resection did not provide complete denervation but changed the innervation pattern of the healing fracture, as the density of sensory nerve fibers immunostaining for substance P and neurofilaments was less in the group with femoral and sciatic nerve resection. The results suggest that intact innervation is essential for normal fracture healing because nerve injury induced a large, but mechanically insufficient, fracture callus.     

Antioxidants in peripheral nerve

Oxidative stress and antioxidants have been related in a wide variety of ways with nervous tissue. This review attempts to gather the most relevant information related to a) the antioxidant status in non pathologic nervous tissue; b) the hypothesis and evidence for oxidative stress (considered as the disequilibrium between prooxidants and antioxidants in the cell) as the responsible mechanism of diverse neurological diseases; and c) the correlation between antioxidant alterations and neural function, in different experimental neuropathies. Decreased antioxidant availability has been observed in different neurological disorders in the central nervous system, for example, Parkinson's disease, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, cerebral ischaemia, etc. Moreover, the experimental manipulation of the antioxidant defense has led in some cases to interesting experimental models in which electrophysiological alterations are associated with the metabolic modifications induced. In view of the electrophysiological and biochemical effects of some protein kinase C inhibitors on different neural experimental models, special attention is dedicated to the role of this kinase in peripheral nervous tissue. The nervous tissue, central as well as peripheral, has two main special features that are certainly related to its antioxidant metabolism: the lipid-enriched membrane and myelin sheaths, and cellular excitability. The former explains the importance of the glutathione (GSH)-conjugating activity towards 4-hydroxy-nonenal, a biologically active product of lipid peroxidation, present in nervous tissue and in charge of its inactivation. The impairment of the latter by oxidative damage or experimental manipulation of antioxidant metabolism is discussed. Work on different experimental neuropathies from author's laboratory has been primarily used to provide information about the involvement of free radical damage and antioxidants in peripheral nerve metabolic and functional impairment.     

Growth hormone enhances regeneration of nitric oxide synthase-containing penile nerves after cavernous nerve neurotomy in rats

PURPOSE: As growth hormone has been reported to improve nerve regeneration, we studied the effect of rat growth hormone (GH) on the regeneration of nitric oxide synthase (NOS)-containing penile nerves and the neurons in the pelvic ganglia after unilateral cavernous nerve neurotomy in rats. MATERIALS AND METHODS: Male rats were divided into three groups: sham operation (n = 14); unilateral neurotomy of a 5 mm. segment of the cavernous nerve (n = 14) with subsequent injection of buffer solution only; and unilateral neurotomy with GH injection (n = 14). Electrostimulation of the intact cavernous nerve was performed at 1 and 3 months. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in penile nerve fibers of the mid-shaft segment and in neurons of the pelvic ganglia. RESULTS: One month after unilateral neurotomy, both the buffer alone and GH-treated groups showed a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves on the side of neurotomy. At 3 months, the number of NOS-containing nerve fibers in the buffer alone group did not increase, while the GH-treated group showed a significant increase. In the GH-treated group at 3 months, more NOS-positive neurons in the pelvic ganglia were found on the intact side than on the side of neurotomy (p <0.034), indicating that the regeneration derives from pelvic ganglion neurons on the intact side. Furthermore, electrostimulation in the GH-treated group revealed a greater maximal intracavernosal pressure and a shorter latency period at 3 months than in those given buffer alone. CONCLUSIONS: Our results show that GH injection significantly enhances the regeneration of NOS-containing fibers in the dorsal and intracavernosal nerves after unilateral cavernous nerve injury. We believe that GH administration may present a new and more physiologic approach to the treatment of erectile dysfunction after radical pelvic surgery.     

The effect of aprotinin on extraneural scarring in peripheral nerve surgery: an experimental study

BACKGROUND: Although laser resurfacing has become increasingly popular in the treatment of Caucasian skin, concerns about healing and postinflammatory pigmentary changes have limited its use in Asian skin. OBJECTIVE: The purpose of this study was to determine the efficacy and safety of the Erbium(ER):YAG laser used in the treatment of Asian skin. METHODS: Fifty Asian individuals with Rhytids, scars, pigmentary alteration, and a variety of cutaneous growth were treated with the Erbium:YAG laser. Patients were evaluated for clinical efficacy, wound healing, post-operative erythema, and post-inflammatory pigmentary changes. RESULTS: Significant improvement was noted in all individuals. Shorter periods for re-epithelization and erythema duration were noted when compared to previously reported results following carbon dioxide laser resurfacing. CONCLUSION: The Erbium:YAG laser is safe and effective in the treatment of Asian skin.     

Alteration to the levels of galanin in uninjured contralateral rat saphenous nerves after unilateral saphenous nerve section

Previous work in this laboratory has demonstrated that unilateral injury to the rat saphenous nerve decreases levels of substance P in the uninjured contralateral saphenous nerve during the subsequent regenerative period. Furthermore this contralateral nerve also has a decreased ability to elicit a neurogenic inflammatory response. In this present study the levels of galanin were investigated under similar experimental conditions. Levels of this peptide were found to be significantly decreased in the uninjured contralateral nerve. No significant changes in the total protein level of these nerves was found as compared to the control uninjured animals. The implications of this, in the context of the other contralateral changes found after peripheral nerve injury, are discussed.     

Nerve fibre velocity and refractory period distributions in nerve trunks

With the use of a double stimulus technique, nerve fibre velocity range measurements were performed over a single conduction distance in 13 normal subjects and over two conduction distances in another 12 normal subjects. The velocity ranges were found to be dependent upon the conduction distance, owing to unknown refractory period delays. Refractory period values were calculated for the 12 subjects and also direct refractory period distribution measurements were made on 15 normal subjects using a twin stimulus and automatic subtraction technique. Corrections to the velocity range measurements were made upon differing assumptions as to the correlation between refractory period and fibre conduction velocity. It was concluded that a single median value refractory period obtained from the distribution was the best correction to use, based upon the hypothesis that for group A fibres the random scatter of refractory period values is far greater than any variation due to a correlation between refractory period and fibre conduction velocity. It was found important to recognize that calculated values of velocity range are a function not only of the spread of fibre conduction velocities but also of refractory periods.     

The nerve fibre layer in ocular hypertension. Preliminary results

A group of healthy subjects (IOP < 22 mmHg) and a simple ocular hypertension group (IOP > or = 22 mmHg) were examined with a view to checking if there were any differences between their nerve fibre layers. Results indicate thinner fibres in the ocular hypertensives significantly correlative with pressure increase. Among the two groups, some corresponding fibre thicknesses were found.     

Heat shock protein 27: developmental regulation and expression after peripheral nerve injury

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.     

Changes in peripheral nerves of rats four months after induction of streptozotocin diabetes. A qualitative and quantitative study

Eight streptozotocin-injected Wistar rats and eight controls were fixed by whole-body perfusion 4 months after beginning of the experiment, the nervus radialis was dissected and processed for light and electron microscopy. After light-microscopic study standard photographs of nerve cross sections were measured by means of a semiautomatic image analyzer. The following measurements were obtained: (1) surface of fibers, axons, and myelin sheaths, (2) ratio of myelin to axon surface, and (3) percent of endoneural space. Group means and standard deviations were calculated, and cumulated size class distributions were made. Representative nerve specimens from all animals were also studied by electron microscopy. The quantitative study revealed in the diabetics a severe reduction of the average myelin surface, a mild increase of axonal cross section and of endoneural space, a reduction of myelin/axon ratio and a mild reduction in cross section of the nerve. Ultrastructural lesions of minor degree were found in the cytoplasm of Schwann and mesenchymal cells, no lesion was observed in axons. These findings demonstrate the presence of neuropathy 4 months after induction of diabetes and support the pathogenetic role of the Schwann cell in our experimental model.     

The effect of age on peripheral motor nerve function after crush injury in the rat

OBJECTIVE: To determine the ontogeny of functional recovery after peripheral nerve crush injury. DESIGN: Comparative study in rats of varying ages. MATERIAL AND METHODS: Sixty-second crush injury was performed on the left posterior tibial nerve. Control animals underwent either nerve transection or sham procedure. Nerve function was evaluated 2, 4, and 8 weeks following injury by walking track analysis. Print length ratio (PLR), (ratio of normal right-sided print length to experimental left-sided print length), was used to evaluate functional recovery. MEASUREMENTS AND MAIN RESULTS: Two weeks after crush injury, adult rats experienced significantly greater functional impairment than both 4-day-old and 3-week-old animals (p < 0.05). Four weeks after injury, the difference in function between 4-day-old and adult rats and between 3-week-old and adult rats became insignificant. Complete recovery had been achieved by 8 weeks in all groups. CONCLUSIONS: These results demonstrate faster functional recovery after nerve injury in immature rats than in adults.