Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

神经生长因子与冻干异体神经桥接大鼠神经缺损的研究

实验采用冻干处理的异体神经与外源性神经生长因子（ＮＧＦ）结合来桥接大鼠的坐骨神经１．０ｃｍ的缺损。用雄性Ｗｉｓｔａｒ大鼠进行的四组实验结果表明：冻干处理的异体神经可降低其抗原性,但处理后并不损害雪旺氏细胞（ＳＣ）基底膜的完整性,在移植后可能成为轴突再生的通道和支架;外源性ＮＧＦ与冻干神经结合形成的复合体,可为神经的再生提供一个较好的微环境,具有成为理想桥接材料的可能性     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.     

乳鼠坐骨神经植块法的雪旺氏细胞培养

目的:改善并建立一种新的大鼠雪旺氏细胞(SCs)的培养方法,为研究外周神经损伤修复模型及其它外周神经相关实验提供高纯度、多数量的SCs。方法:麻醉后显微镜下解剖并分离新生3天内SD大鼠的坐骨神经,采取植块培养的方法,显微镜下尽量剥除坐骨神经纤维外膜,并梳理松解坐骨神经的神经纤维束。梳理后剪碎坐骨神经,每小块种植于培养皿中,使用纯血清培养4小时,再加入正常的DMEM/F12培养基,消化培养2-3代。最后用S-100及GFAP免疫荧光染色进行纯度鉴定。结果:本实验在总结前人实验的基础上,联合创新采用坐骨神经外膜剥除、神经内膜梳理、纯血清培养以及胰酶差速消化等方法,短时间内获得SCs的纯度可达99%以上,可用于进一步对雪旺氏细胞的功能进行研究。结论:这种选用乳鼠坐骨神经植块、血清培养的方法简单易操作,无需额外的生长因子及抑制因子,可在短期内获得大量高纯度的SCs。     

Exogenous Pleiotrophin Applied to Lesioned Nerve Impairs Muscle Reinnervation

Pleiotrophin (PTN) is a heparin-binding growth factor involved in nerve regeneration after peripheral nerve injury. After crush injury, PTN is found in distal nerve segments in several non-neural cell types, including Schwann cells, macrophages, and endothelial cells, but not in axons. To further clarify the role for PTN in nerve regeneration, we investigated the effects of PTN applied to lesioned peripheral nerve in vivo. PTN in a dose of 1 mg/kg impaired muscle reinnervation. Thus, gastrocnemius muscle failed to recover its contractile properties as assessed by in situ maximal isometric tetanic force. PTN also decreased non-neural cell densities and delayed macrophage recruitment in the distal crushed nerve. These results are discussed in the light of recent evidence that PTN is a multifunctional polypeptide.     

Effects of Axotomy on Distribution and Concentration of Elements in Rat Sciatic Nerve

X-ray microprobe analysis was used to determine the effects of axotomy on distribution and concentration (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in frozen, unfixed sections of rat sciatic nerve. Elemental concentrations were measured in axoplasm, mitochondria, and myelin at 8, 16, and 48 h after transection in small-, medium-, and large-diameter fibers. In addition, elemental composition was determined in extraaxonal space (EAS) and Schwann cell cytoplasm. During the initial 16 h following transection, axoplasm of small fibers exhibited a decrease in dry weight concentrations of K and Cl, whereas Na and P increased compared to control values. Similar changes were observed in mitochondria of small axons, except for an early, large increase in Ca content. In contrast, intraaxonal compartments of larger fibers showed increased dry weight levels of K and P, with no changes in Na or Ca concentrations. Both Schwann cell cytoplasm and EAS at 8 and 16 h after injury had significant increases in Na, K, and Cl dry weight concentrations, whereas no changes, other than an increase in Ca, were observed in myelin. Regardless of fiber size, 48 h after transection, axoplasm and mitochondria displayed marked increases in Na, Cl, and Ca concentrations associated with decreased K. Also at 48 h, both Schwann cell cytoplasm and EAS had increased dry weight concentrations of Na, Cl, and K. The results of this study indicate that, in response to nerve transection, elemental content and distribution are altered according to a specific temporal pattern. This sequence of change, which occurs first in small axons, precedes the onset of Wallerian degeneration in transected nerves.     

Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo

Abstract: Labeled palmitic acid ([16-¹⁴C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16-¹⁴C]palmitate to other long-chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2-diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16-¹⁴C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.     

Role of Non-coding RNAs in Axon Regeneration after Peripheral Nerve Injury

Peripheral nerve injury (PNI) may lead to disability and neuropathic pain, which constitutes a substantial economic burden to patients and society. It was found that the peripheral nervous system (PNS) has the ability to regenerate after injury due to a permissive microenvironment mainly provided by Schwann cells (SCs) and the intrinsic growth capacity of neurons; however, the results of injury repair are not always satisfactory. Effective, long-distance axon regeneration after PNI is achieved by precise regulation of gene expression. Numerous studies have shown that in the process of peripheral nerve damage and repair, differential expression of non-coding RNAs (ncRNAs) significantly affects axon regeneration, especially expression of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In the present article, we review the cellular and molecular mechanisms of axon regeneration after PNI, and analyze the roles of these ncRNAs in nerve repair. In addition, we discuss the characteristics and functions of these ncRNAs. Finally, we provide a thorough perspective on the functional mechanisms of ncRNAs in nervous injury repair, and explore the potential these ncRNAs offer as targets of nerve injury treatment.     

Dynamic Changes of p27<Superscript>kip1</Superscript> and Skp2 Expression in Injured Rat Sciatic Nerve

S phase kinase-associated protein 2 (Skp2), an F-box protein, is required for the ubiquitination and consequent degradation of p27^kip1. Previous reports have showed that p27^kip1 played important roles in cell cycle regulation and neurogenesis in the developing central nervous system. But the distribution and function of p27^kip1 and Skp2 in nervous system lesion and regeneration remains unclear. In this study, we observed that they were expressed mainly in both Schwann cells and axons in adult rat sciatic nerve. Sciatic nerve crush and transection resulted in a significant up-regulation of Skp2 and a down-regulation of p27^kip1. By immunochemistry, we found that in the distal stumps of transected nerve from the end to the edge, the appearance of Skp2 in the edge is coincided with the decrease in p27^kip1 levels. Changes of them were inversely correlated. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that p27^kip1 and Skp2 likely play an important role in peripheral nerve injury and regeneration. Ai-Guo Shen and Shu-Xian Shi contributed equally to this work.     

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me-^HC]choline and 15 μCi [2-³H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [¹⁴C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.     

Rapid Anterograde Spread of Premitotic Activity Along Degenerating Cat Sciatic Nerve

Peripheral nerve transection triggers a series of phenotypic alterations in Schwann cells distal to the site of injury. Mitosis is one of the earliest and best characterized of these responses, although the mechanism by which axonal damage triggers this critical event is unknown. This study examines the appearance and spatio-temporal spread of premitotic activity in distal stumps of transected cat tibial nerves. Premitotic activity was determined by measuring incorporation of [3H]thymidine (a marker of DNA synthesis during the S-phase of the cell cycle) into consecutive segments of desheathed tibial nerve. Incorporation of [3H]thymidine spread proximo-distally within distal nerve stumps between 3 and 4 days posttransection with an apparent velocity of at least 199 +/- 67 mm/day. This suggests that anterograde fast axonal transport may directly or indirectly be associated with the Schwann cell mitotic response to axon transection.     

Nerve Growth Factor of Red Nucleus Involvement in Pain Induced by Spared Nerve Injury of the Rat Sciatic Nerve

Nerve growth factor (NGF), a member of the neurotrophin family, is essential for the development and maintenance of sensory neurons and for the formation of central pain circuitry. The current study was designed to evaluate the expression of NGF in the brain of rats with spared nerve injury (SNI), using immunohistochemical technique. The results showed that the level of NGF in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the effect of NGF in the development of neuropathic pain, different doses of anti-NGF antibody (20, 2.0 and 0.2 μg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The data suggested that the higher doses of anti-NGF antibody (20 and 2.0 μg/ml) significantly attenuated the mechanical allodynia of neuropathic rats, while the 0.2 μg/ml antibody showed no analgesic effect. These results suggest that the NGF of RN is involved in the development of neuropathic allodynia in SNI rats.     

Lipid Metabolism in Peripheral Nerve Cell Culture (Rich in Schwann Cells) from Normal and Trembler Mice

Abstract: A culture of peripheral nerve cells, very rich in Schwann cells, was developed from sciatic nerve. In both normal and Trembler, typical spindle-shaped cells were seen; most of the cells were surrounded by basement membrane-like material (predominantly in-between adjacent cells). In Trembler cells, cultivated in the presence of labelled acetate, the fatty acids were slightly altered; phosphatidylcholine was slightly reduced and phosphatidyl-ethanolamine increased. Sulfatides were increased four times.     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.     

碱性成纤维细胞生长因子对大鼠晚期周围神经再生作用的实验研究

目的探讨外源性碱性成纤维细胞生长因子(bFGF)对晚期周围神经再生的作用.方法50只SD大鼠随机分治疗组、对照组各25只,切断右侧坐骨神经,12周后予以修复,修复术后每日分别给予bFGF和生理盐水,行神经电生理和组织学检查.结果治疗组和对照组修复处远段神经均有不同程度再生,4周时已可见到再生轴突,且治疗组多见.计量分析治疗组运动神经传导速度、神经肌肉动作电位幅值、髓鞘厚度、再生轴突直径和截面积明显优于对照组.治疗组与对照组相比,差异有显著性.结论bFGF能促进晚期周围神经再生.     

c-fos Expression in Gracilothalamic Tract Neurons after Electrical Stimulation of the Injured Sciatic Nerve in the Adult Rat

The number of c-fos protein-like immunoreactive (Fos-LI) cells in the gracile nucleus was determined after electrical stimulation at Aα/Aβ-fiber strength of the normal and of the previously injured sciatic nerve in adult rats. No Fos-LI cells were seen after electrical stimulation of the noninjured sciatic nerve, or after sciatic nerve injury without electrical stimulation. However, stimulation 21 days after sciatic nerve transection resulted in numerous Fos-LI cells in the ipsilateral gracile nucleus. Combined Fos immunocytochemistry and retrograde labeling from the thalamus showed that the majority (76%; range = 70–80%) of the cells in the gracile nucleus that expressed Fos-LI after nerve injury projected to the thalamus. The results indicate that morphological, biochemical, and physiological alterations in primary sensory central endings and second-order neurons, which have earlier been demonstrated in the dorsal column nuclei after peripheral nerve injury, are accompanied by changes in the c-fos gene activation pattern after stimulation of the injured sciatic nerve. A substantial number of the c-fos-expressing neurons project to the thalamus.     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Acrylamide-Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve

The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of approximately 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.     

Regulation of Sulfoglucuronyl Glycolipid Synthesis in the Developing Rat Sciatic Nerve

Sulfoglucuronyl glycolipids (SGGLs) have been considered as target antigens in demyelinating peripheral neuropathies associated with IgM monoclonal gammopathy. The regulation of expression of SGGLs in the rat sciatic nerve during development was studied by assaying the levels of SGGLs and activities of four glycosyltransferases sequentially involved in their synthesis from lactosylceramide. The levels of SGGLs in the sciatic nerve increased with development and reached a maximum at sixty days after birth. The rate of increase in the level of SGGLs between day 5 to 20 was similar to rate of deposition of myelin in the nerve. Analysis of the activities of the glycosyltransferases showed that only lactotriosylceramide galactosyltransferase (LcOse₃Cer-GalTr) increased in parallel with the levels of SGGLs during development. The other three enzymes were not co-relative with the synthesis of SGGLs. The product of LcOse₃Cer-GalTr reaction, nLcOse₄Cer is the key intermediate for all neolactoglycolipids, particularly NeuAc2-3nLcOse₄Cer or nLM1, which is the major ganglioside (60%) of myelin in rat sciatic nerve. The results suggest that in the sciatic nerve SGGLs are mostly associated with Schwann cell myelin and their synthesis is regulated by LcOse₃Cer-GalTr, unlike in the cerebral cortex and cerebellum where SGGLs are associated with the neuronal membranes and their synthesis is regulated by lactosylceramide N-acetylglucosaminyltransferase (LcOse₂Cer-GlcNAcTr).