Immunohistochemical study of skin nerve regeneration after toe-to-finger transplantation: correlations with clinical, quantitative sensory, and electrophysiological evaluations

Cutaneous nerve regeneration following toe-to-finger transplantation was studied by immunohistochemical technique using antibody to protein gene product 9.5 (PGP 9.5) which is a specific neuronal marker. By this technique, epidermal and dermal nerves were semi-quantified and the Meissner's corpuscles were quantified. There were also quantitative sensory tests (QST) including pinprick, pressure and temperature, as well as electrophysiological studies including digital nerve sensory conduction, digital nerve somatosensory evoked potentials and sympathetic skin response at the pulp of the transplanted toes. The opposite corresponding normal finger and normal toe served as controls. Study subjects were 20 adult patients with toe-to-finger transplantation for at least one year. A score system was used to quantify the results of histochemical, psychophysiological and electrophysiological studies. Clinically 7 patients had good recovery and 13 patients had poor recovery. Cutaneous nerve regeneration in the transplanted toes was incomplete with epidermal nerve, dermal nerve and the Meissner's corpuscle significantly reduced. The nerve regeneration was correlated with clinical recovery, QST and electrophysiological data. These findings indicate that immunohischemical technique is useful to evaluate skin nerve regeneration following toe-to-finger transplantation, and that although nerve regeneration did occur, it was incomplete and correlated with the severity of hand injury.     

Peripheral nerve regeneration and intraneural revascularization

Peripheral nerves are particularly vulnerable to injuries and are involved in numerous pathologies for which specific treatments are lacking. This review summarizes the pathophysiological features of the most common traumatic nerve injury in humans and the different animal models used in nerve regeneration studies. The current knowledge concerning Wallerian degeneration and nerve regrowth is then described. Finally, the involvement of intraneural vascularization in these processes is addressed. As intraneural vascularization has been poorly studied, histological experiments were carried out from rat sciatic nerves damaged by a glycerol injection. The results, taken together with the data from literature, suggest that revascularization plays an important role in peripheral nerve regeneration and must therefore be studied more carefully.     

Sarcoplasmic masses in equine skeletal muscle

Sarcoplasmic masses in humans have been associated with various myopathies, although the significance remains elusive. Similar structures have also been observed in equine muscle. The aim of this study was to determine the frequency of such structures in normal and abnormal equine muscle, and to characterize these structures using histological, histochemical, immunohistochemical, ultrastructural, and morphometric analyses. The histological and histochemical appearance was similar to that of human sarcoplasmic masses with a central or subsarcolemmal distribution. Of interest was a predilection for the gluteus medius muscle in younger horses and type 2B fibers. Ultrastructurally they contained disorganized myofibrils and other cellular components that were not membrane bound and were present in both normal and abnormal equine muscle without a specific disease association, suggesting they are a non-pathological degenerative structure. The relatively frequent occurrence of sarcoplasmic masses in horses may make this species a good model for studying the pathogenesis of these structures.     

Skin biopsy as a diagnostic tool in peripheral neuropathy

Skin biopsy is a safe, minimally invasive, painless and cheap tool for providing diagnostic information on small nerve fibers, which are invisible to routine neurophysiological tests. Biopsy can be performed in hairy skin to investigate unmyelinated and thinly myelinated fibers and in glabrous skin to examine large myelinated fibers. Morphometric analysis of skin nerves is readily accomplished through the use of immunohistochemical techniques, and has proved to be reliable, reproducible and unaffected by the severity of neuropathy. One further advantage of skin biopsy over conventional nerve biopsy is that it allows somatic nerve fibers to be distinguished from autonomic nerve fibers. Morphological changes, axonal degeneration and abnormal regeneration occur in cutaneous nerves very early in the course of peripheral neuropathies, making skin biopsy a promising tool for investigating the progression of neuropathy and the effect of neuroprotective treatments in clinical practice and trials. This article reviews the techniques that are used to investigate the innervation of human skin, the possible uses of skin biopsy in diagnosing and monitoring peripheral neuropathies, and correlations between skin biopsy findings and those of other diagnostic methods.     

Immuno-lectin histochemistry and ultrastructure in two cases of globoid cell leukodystrophy (Krabbe's disease).

Two cases of globoid cell leukodystrophy (Krabbe's disease) have been studied by means of conventional histological, immunohistochemical and ultrastructural methods. Lectins specific for several different carbohydrates, Ricinus communis (RCA-1), peanut agglutinin (PNA), wheat germ agglutinin (WGA) and Concanavalin A (Con A) have been utilized both at optical and ultrastructural level. In both cases a strong positive labeling of the globoid cells could be observed light-microscopically. Ultrastructurally immunogold deposits only of RCA-I and WGA in the typical Krabbe's inclusions have been clearly demonstrated.     

High- and low-frequency transcutaneous electrical nerve stimulation delay sciatic nerve regeneration after crush lesion in the mouse

Abstract   The stimulation of peripheral nerve regeneration has been studied in different ways, including the use of electrical fields. The capacity of this modality to enhance nerve regeneration is influenced by the parameters used, including current type, frequency, intensity, and means of administration. Transcutaneous electrical nerve stimulation (TENS) is a frequently used form of administering electrical current to the body, but its effects on peripheral nerve regeneration are not known. This study assessed the influence of TENS on sciatic nerve regeneration, using a model of crush lesion in the mouse. Mice were stimulated 30 min a day, 5 days a week, for 5 weeks with both high- (100 Hz) and low- (4 Hz) frequency TENS. Control animals had the sciatic nerve crushed but were not stimulated. Assessment was performed weekly by functional analysis using the Static Sciatic Index for the mouse and at the end of the experiment by light and electron microscopy. The results showed that although there were no differences between the groups regarding the Static Sciatic Index values, TENS led to nerves with morphological signs of impaired regeneration. At light microscopy level, TENS nerves presented more axons with dark axoplasm, signs of edema, and a less organized cytoarchitecture. Electronmicrographs showed fewer and thinner thick myelinated fibers and increased number of Schwann cell nuclei. Myelinated axon diameters and density and diameter of nonmyelinated fibers were not affected by TENS, leading to the conclusion that this regimen of electrical stimulation leads to a delayed regeneration after a crush lesion of the sciatic nerve in the mouse. All these effects were more pronounced on high-frequency TENS nerves.     

Synthetic nerve graft containing collagen and synthetic Schwann cells inproves functional, electrophysiological, and histological parameters of peripheral nerve regeneration

Current methods of peripheral nerve repair are to directly suture cut nerve stumps, or to bridge large gaps with an autograft repair. Autograft-associated problems include donor site morbidity and limited supply. Many of the present limitations of nerve repair might be overcome by expanding the patients own Schwann cells in vitro, then combining the cells with other neuro-tropic and -trophic materials into an Artificial Nerve Graft (ANG) for bridging a nerve gap. In this 4.5 month experiment, a rat peroneal nerve model with a 10 mm gap was used to evaluate the effect of live Schwann cells on peripheral nerve regeneration. Nerve gaps were repaired with cellular ANGs containing live Schwann cell, dead Schwann cell, or mixed fibroblast/Schwann cell populations suspended in a collagen I matrix, and with sutured autografts or ANGs containing just collagen or medium. Regenerated nerves were evaluated by walking track analysis, qualitative and quantitative histology, and electrophysiology. Overall, the autograft was the best repair method, while the ANG containing live Schwann cells was statistically superior to other ANG repair methods. This study demonstrates that an ANG containing cultured syngeneic Schwann cells improves functional, histological, and electrophysiological parameters of peripheral nerve regeneration.     

Etifoxine provides benefits in nerve repair with acellular nerve grafts

Introduction: Acellular nerve grafts are good candidates for nerve repair, but the clinical outcome of grafting is not always satisfactory. We investigated whether etifoxine could enhance nerve regeneration. Methods: Seventy‐two Sprague‐Dawley rats were divided into 3 groups: (1) autograft; (2) acellular nerve graft; and (3) acellular nerve graft plus etifoxine. Histological and electrophysiological examinations were performed to evaluate the efficacy of nerve regeneration. Walking‐track analysis was used to examine functional recovery. Quantitative polymerase chain reaction was used to evaluate changes in mRNA level. Results: Etifoxine: (i) increased expression of neurofilaments in regenerated axons; (ii) improved sciatic nerve regeneration measured by histological examination; (iii) increased nerve conduction velocity; (iv) improved walking behavior as measured by footprint analysis; and (v) boosted expression of neurotrophins. Conclusions: These results show that etifoxine can enhance peripheral nerve regeneration across large nerve gaps repaired by acellular nerve grafts by increasing expression of neurotrophins. Muscle Nerve 50:235–243, 2014     

A comparison of electric and magnetic compound action signals as quantitative assays of peripheralo nerve regeneration

The evaluation of peripheral nerve regeneration is of great interest in clinical as well as in experimental situations. However, there are few techniques that give early and quantitative information on the states of the regeneration process. If quantitative assays would be available, different surgical techniques and medications could be evaluated more accurately in relation to axonal ingrowth and functional recovery. The purpose of this study was to investigate the merits of nerve compound action signals (NCASs) recorded electrically and signals recorded with a novel magnetic recording technique. We compared the two techniques in the rabbit peroneal nerve, 2, 4, 6, and 8 weeks after a nerve reconstruction. Our conclusions are that the signals recorded with the magnetic sensor are far more reproducible and less prone to stimulus artifact than the electrically recorded signals. Furthermore, the magnetic recording shows that the number of axons that have regenerated increases with time. Previously, this could only be determined with histological studies. Other ingrowth parameters that can be quantified are the average ingrowth distance, and the variation between axons in ingrowth velocity. © 1993 John Wiley & Sons, Inc.     

PDLLA／NGF复合导管促进周围神经缺损再生的形态学研究

目的探究聚d,L-乳酸,神经生长因子(PDLLA/NGF)可吸收性复合导管桥接对大鼠坐骨神经缺损再生修复的促进作用,为临床上周围神经损伤再生修复提供实验依据。方法60只SD大鼠随机均分4组:自体神经移植(A组),单纯PDLLA导管(B组),PDLLA加一次性给NGF(C组),复合导管(D组)。制作坐骨神经10 mm缺损,按上述分组桥接,手术后1、2、3月每组各取5只修复处组织。行大体观察、三头肌湿重、光镜观察与图像处理、电镜超微结构观察。结果同时间段组间比较,三头肌恢复率A组高,D组次之,C组较差,B组最差(P<0.05)。组织学观察及图像分析显示,D组再生神经的数、质量都显著优于B、C组(P<0.05),接近A组。电镜观察显示A、D组中段神经纤维密度大、直径大、髓鞘厚,内有发育良好的神经丝和微管。B、C组神经稀、直径小、髓鞘薄、无髓神经相对多。结论PDLLA/NGF复合导管具有良好的组织相容性。用该管桥接修复大鼠受损的坐骨神经,能有效地促进其再生。     

Centronuclear myopathy.

Centronuclear myopathy occurring sporadically in two African female children is reported, with details of clinical history and histological, histochemical, and ultrastructural findings, and a review of 58 previously reported cases. In spite of distinctive histological features, the clinical presentation of this condition is variable, there are different modes of inheritance, and the pathogenesis remains unclear.     

Differentiating astroglia in nervous tissue histogenesis/regeneration: studies in a model system of regenerating peripheral nerve

The role of astroglia in nervous system histogenesis/regeneration (morphogenesis) was studied as a function of cell age. The effect of inoculated astroglia at different cell maturation stages on axonal growth was examined in a peripheral nerve regenerating model system. This model system consists of rat sciatic nerve stumps that regenerate through an empty silicone chamber (Lundborg et al.: Journal of Neuropathology and Experimental Neurology 41:412-422, 1982). Rat astroglial cell populations grown in cell culture were suspended either in a liquid (physiological solution) or in a solid (isotonic collagen gel) medium and inoculated within the silicone chamber at the time of surgery. Immature and mature cell populations, at ages corresponding to 9-69 postnatal days (P9-P69), were inoculated, and their effect on neural growth was analyzed by histological, immunocytochemical, and ultrastructural techniques, 2-6 weeks later. Astroglial cells differentially modulated the process of nerve regeneration, an effect that is a function of the cells' developmental stage. P19 astroglia and older cells inhibited the regeneration process, encapsulating the axons in a glia-limitans-like structure. Immature astrocytes (P9) did not seem to interfere with the regeneration process; nerve outgrowth in their presence resembled and was comparable to the ones obtained in the presence of inoculated Schwann cells. The differential effects of the developing astroglia were not significantly changed by the inoculation media, e.g., liquid or solid, and were already pronounced 2 weeks after neural transection. It is suggested by the results of the study that the role and function of astroglia in nervous system morphogenesis are changing with cell differentiation. Adult astrocytes seem to downregulate axonal growth; presumably, their function is to confine the neurites within designated structural and functional boundaries.     

Skeletal muscle and peripheral nerve changes caused by chronic arterial insufficiency--significance and clinical correlations--histological, histochemical and ultrastructural study

A histological, histochemical and ultrastructural study on muscle changes in chronic arterial insufficiency has been carried out in 40 patients. Muscle biopsy probes were taken in each patient either from the gastrocnemius or rectus femoris. In 7 patients submitted for amputation of a lower limb due to a more severe vascular disease, specimens were also obtained from both the sciatic and sural nerve for light and electron microscopic study. Our findings confirm the previous histological, histochemical and ultrastructural data on chronic ischemia of the skeletal muscle, suggesting that muscle damage is mainly based on a neurogenic noxa initiated by an ischemic peripheral neuropathy. In addition, many morphologic features also show the presence of a primary myopathic noxa, possibly depending on the direct damage of the mitochondrial respiratory chain by the deficit of the muscle blood supply.     

Exenatide promotes regeneration of injured rat sciatic nerve

Damage to peripheral nerves results in partial or complete dysfunction. After peripheral nerve injuries, a full functional recovery usually cannot be achieved despite the standard surgical repairs. Neurotrophic factors and growth factors stimulate axonal growth and support the viability of nerve cells. The objective of this study is to investigate the neurotrophic effect of exenatide(glucagon like peptide-1 analog) in a rat sciatic nerve neurotmesis model. We injected 10 μg/d exenatide for 12 weeks in the experimental group(n = 12) and 0.1 m L/d saline for 12 weeks in the control group(n = 12). We evaluated nerve regeneration by conducting electrophysiological and motor functional tests. Histological changes were evaluated at weeks 1, 3, 6, and 9. Nerve regeneration was monitored using stereomicroscopy. The electrophysiological and motor functions in rats treated with exenatide were improved at 12 weeks after surgery. Histological examination revealed a significant increase in the number of axons in injured sciatic nerve following exenatide treatment confirmed by stereomicroscopy. In an experimentally induced neurotmesis model in rats, exenatide had a positive effect on nerve regeneration evidenced by electromyography, functional motor tests, histological and stereomicroscopic findings.     

野百合碱诱导大鼠肺动脉高压模型的建立

背景：目前尚缺乏简单易行、实用、操作性强的肺动脉高压动物模型。 目的：建立一种实用的注射野百合碱诱导的肺动脉高压动物模型。 方法：采用一次性皮下注射野百合碱60 mg/kg的方法制备SD大鼠肺动脉高压模型。 结果与结论：野百合碱注射后第1,2,3,4周,大鼠平均肺动脉压明显升高,右心室肥厚明显。光镜下可见肺小血管肌化程度增强,相对中膜厚度增加,肺血管密度减少,以上症状均随野百合碱注射时间的延长逐渐加重。证实此方法建立的大鼠肺动脉高压模型造模成功。     

Traumatic brain injury in piglets of different ages: techniques for lesion analysis using histology and magnetic resonance imaging

Quantitation of lesions in large gyrencephalic brains presents a variety of technical challenges. Specific techniques are required when comparing lesions in subjects of different ages in order to assess maturational effects. We have modified existing techniques to attain reliable, consistent and reproducible paraffin-embedded histological sections for volumetric lesion analysis and correlation with magnetic resonance imaging (MRI) in piglets of different ages following focal traumatic brain injury. Twenty-four Yorkshire domestic piglets at three different ages (5 days, 1 month, and 4 months old) underwent scaled cortical impact injury to the fronto-parietal cortex. This contusion model utilizes a rapid volume of indentation scaled proportionally to the growth of the brain, allowing for examination of maturational influences on the brain's response to focal mechanical trauma. To overcome problems with differential processing and embedding of brains ranging from 43 to 107 g, we developed a piglet parallel brain slicing apparatus. Along with specific methods for processing, embedding, mounting, and slide preparation, these techniques enabled excellent quality 10-microm serial coronal sections to be obtained for histology and immunohistochemical analysis. Accurate co-registration of histologic, immunohistochemical and radiologic images at different ages was possible, which may enhance understanding of developmental aspects of brain injury pathophysiology.     

EOSINOPHILIC FASCIITIS: AN ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL STUDY OF THE INTERMEDIATE FILAMENT PROTEIN SKELETIN IN REGENERATING MUSCLE FIBRES

Thornell L.-E. & Bjelle A. (1981) Neuropathology and Applied Neurobiology 7, 435–449
Eosinophilic fasciitis: an ultrastructural and immunohistochemical study of the intermediate filament protein skeletin in regenerating muscle fibres
In a case of eosinophilic fasciitis with pronounced muscle involvement non-specific ultrastructural signs of degeneration were observed in areas of perifascicular atrophy. By combined immunohistochemical and electron microscopical techniques intermediate skeletin filaments were demonstrated. These filaments, when found in combination with nuclei with large nucleoli and an abundance of polyribosomes are a sign of regeneration of muscle fibres. This finding is a useful indicator in assessing the prognosis and therapy of muscle involvement in eosinophilic fasciitis.     

Mechanism research of cryoanalgesia

Abstract

The sciatic nerves of rabbits were frozen at different temperatures (-20°C/ - 60°C, - 700°C, - 140°C, and - 180°C). The morphology and function of the frozen nerves were examined with light microscopy (hematoxylin and eosinophilin stain and a histochemical thiocholine method) and electron microscopy. The function of the nerve after freezing was assessed using short latency somatosensory evoked potentials, sensory conduction velocity, and electromyogram at various intervals after freezing. There were no changes in morphology or function of nerves cryolesioned at - 20°C. The nerve fibers cryolesioned at -60°C showed signs of freezing degeneration and lost their conductive function although, these nerves all recovered. Approximately half of nerve fibers cryolesioned at -100°C showed Wallerian degeneration, and although the time to remyelination was delayed, nerve regeneration was still complete. At -140°C and -180°C the nerve fibers showed immediate necrosis, with destruction of basal membranes and proliferation of collagen fibers. The results explained the mechanism of cryoanalgesia. Our study demonstrates that cryo-temperatures lower than - 140°C will cause permanent alterations in nerve morphology and function, whereas warmer temperatures do not result in permanent nerve damage and are therefore not likely to provide long-term analgesia to patients. [Neurol Res 1995; 17: 307-311]     

Digital electron microscopic examination of human sural nerve biopsies

Diabetic peripheral polyneuropathy is characterized by axonal degeneration and regeneration as well as by Schwann cell and microvascular changes. These changes have been described at both the light (LM) and the electron microscopic (EM) levels; however, EM has not been applied to large clinical trials. Our goal was to adapt the rigorous techniques used for quantifying human biopsies with LM image analysis to accommodate ultrastructural analyses. We applied digital image capture and analysis to the ultrastructural examination of axons in sural nerve biopsies from diabetic patients enrolled in a multicenter clinical trial. The selection of sural nerve biopsies was based on the quality of specimen fixation, absence of physical distortion, and nerve fascicle size (> or =100,000; < or =425,000 microm2). Thin sections were collected on formvar-coated slot grids, stabilized with carbon and scanned on a Phillips CM100 transmission electron microscope. Digital images were captured with a Kodak Megaplus 1.6 camera. A montage was constructed using software derived from aerial mapping applications, and this virtual image was viewed by EM readers. Computer-assisted analyses included identification and labeling of individual axons and axons within regenerating clusters. The average density of regenerating myelinated axon clusters per mm2 was 65.8 +/- 5.1, range of 0-412 (n = 193). These techniques increase the number of samples that may be analyzed by EM and extend the use of this technique to clinical trials using tissue biopsies as a primary endpoint.