Direct muscle neurotization after end-to-end and end-to-side neurorrhaphy An experimental study in the rat forelimb model

The need for the continuous research of new tools for improving motor function recovery after nerve injury is justified by the still often unsatisfactory clinical outcome in these patients. It has been previously shown that the combined use of two reconstructive techniques, namely end-to-side neurorrhaphy and direct muscle neurotization in the rat hindlimb model, can lead to good results in terms of skeletal muscle reinnervation. Here we show that, in the rat forelimb model, the combined use of direct muscle neurotization with either end-to-end or end-to-side neurorrhaphy to reinnervate the denervated flexor digitorum muscles, leads to muscle atrophy prevention over a long postoperative time lapse (10 months). By contrast, very little motor recovery (in case of end-to-end neurorrhaphy) and almost no motor recovery (in case of end-to-side neurorrhaphy) were observed in the grasping activity controlled by flexor digitorum muscles. It can thus be concluded that, at least in the rat, direct muscle neurotization after both end-to-end and end-to-side neurorrhaphy represents a good strategy for preventing denervation-related muscle atrophy but not for regaining the lost motor function.     

Interleukin-10 and regeneration in an end-to-side nerve repair model of the rat

End-to-side (ETS) neurorrhaphy is an option in peripheral nerve surgery. The aim of this study was to investigate whether the application of the anti-inflammatory cytokine interleukin-10 (IL-10) reduces scarring and thus enhances nerve regeneration in an ETS peroneal/tibial nerve lesion model of the rat. Twenty rats with a peroneal to tibial ETS neurorrhaphy were divided into two groups: (1) control group and (2) IL-10 group with intrafascicular application of 0.125 μg/100 μl IL-10. Survival time was 8 weeks. Nerve conduction velocities (NCVs) and motor function were analyzed and histomorphological evaluation with measurement of intraneural collagen level, axon count, total nerve area, and myelination index followed. Evaluation of motor function and nerve conduction did not show any statistical differences. Histological analyses revealed thicker myelin sheaths and higher myelination index in the IL-10 group (p < 0.001). Axon count showed no difference. The IL-10 group revealed lower collagen levels (p < 0.001). Comparison of total nerve area showed no statistical significance. At this dose, IL-10 evaluated at 8 weeks was not significantly different than placebo in functional, NCVs, and most morphological measures. However, there was a significant difference in thicker myelin sheaths and higher myelination index and lower collagen levels. This suggests that future experiments of IL-10 at different doses or longer periods of evaluation would be of interest.     

The glial reaction in the course of axon regeneration: a stereological study of the rat hypoglossal nucleus

Both hypoglossal nuclei were examined by electron microscope stereology after unilateral axotomy. The principal aim of this study was a quantitative assessment of the accompanying glial reaction. Volume densities (%) of neuronal and glial perikarya, as well as their processes, were evaluated in terms of volume plus surface densities (mm-1). In addition, specific surfaces (surface to volume ratio) of these neuronal and glial processes were assessed. First, a temporary decrease of dendritic volume density was detected on the ipsilateral side only. Further, the astrocytic reaction displayed differences between stem and lamellar processes. One day after axotomy, a bilateral decrease of volume density, as well as surface density of stem processes, was observed, yet their normal dimensions soon were reestablished. However, a more severe lamellar process reaction was evident. During the first 4 days, a significant increase of volume density and surface density was apparent. In the contralateral hypoglossal nucleus, this glial reaction also occurred but disappeared by day 14, whereas the ipsilateral nucleus continued to display a severe reaction of lamellar processes, only returning to normal status at day 77. In addition, a transient, severe reaction of presumptive microglia was established by employing the volume density and surface density quantitation procedure. Nevertheless, in comparison with the volume and surface contribution of astrocytic processes, the presumed microglial component was minimal. This study indicates a two-step involvement of astrocytes in regenerative repair. Namely, the first phase seems to result in an increase of lamellar processes through reshaping of the stem process.(ABSTRACT TRUNCATED AT 250 WORDS)     

End‐to‐side neurotization with different donor nerves for treating brachial plexus injury: An experimental study in a rat model

Introduction: End‐to‐side neurotization is currently used to treat brachial plexus injury, but it is not clear which donor nerve yields the best outcome. We performed experiments to determine the optimal donor nerve. Methods: A total of 66 male Sprague‐Dawley rats were assigned to 1 of 3 groups. Group A was the control group. In Group B, the phrenic nerve was used as the donor, while the ipsilateral C7 nerve root served as the donor in Group C. The epineurial window was used in end‐to‐side neurorrhaphy. Behavioral observations, histology, electrophysiology, and fluorescence retrotracing were performed postoperatively. Results: Fluorescence retrotracing confirmed nerve regeneration in both Groups B and C upon end‐to‐side neurotization. The outcome of Group B was superior to that of Group C. Conclusions: Use of the phrenic nerve as the donor nerve yielded a better outcome than use of the ipsilateral C7 nerve root. Muscle Nerve 50 : 67–72, 2014     

The rat caudal nerves: a model for experimental neuropathies

This study provides a detailed investigation of the anatomy of the rat caudal nerve along its entire length, as well as correlated nerve conduction measures in both large and small diameter axons. It determines that rodent caudal nerves provide a simple, sensitive experimental model for evaluation of the pathophysiology of degeneration, recovery, and prevention of length‐dependent distal axonopathy. After first defining the normal anatomy and electrophysiology of the rat caudal nerves, acrylamide monomer, a reliable axonal toxin, was administered at different doses for escalating time periods. Serial electrophysiological recordings were obtained, during intoxication, from multiple sites along caudal and distal sciatic nerves. Multiple sections of the caudal and sciatic nerves were examined with light and electron microscopy. The normal distribution of conduction velocities was determined and acrylamide‐induced time‐ and dose‐related slowing of velocities at the vulnerable ultraterminal region was documented. Degenerative morphological changes in the distal regions of the caudal nerves appeared well before changes in the distal sciatic nerves. Our study has shown that (1) rat caudal nerves have a complex neural structure that varies along a distal‐to‐proximal gradient and (2) correlative assessment of both morphology and electrophysiology of rat caudal nerves is easily achieved and provides a highly sensitive index of the onset and progression of the length‐dependent distal axonopathy.     

Changes in extracellular space in optic nerves of regenerative and nonregenerative animals after hypertonic fixation: Implications for axon regeneration

The optic nerves of some regenerative and nonregenerative animals were compared using electron microscopy, after hypertonic perfusion. Optic axons and glial cell processes separated more readily in regenerative animals, creating large extracellular spaces. In mammals, cell processes remained in close proximity. These findings may indicate lesser adhesion between cell processes in optic nerves of regenerative animals, a characteristic that could allow “potential” space for axon regrowth after nerve injury.     

Persistent reduction of conduction velocity and myelinated axon damage in vibrated rat tail nerves

Prolonged hand‐transmitted vibration exposure in the workplace has been recognized for almost a century to cause neurodegenerative and vasospastic disease. Persistence of the diseased state for years after cessation of tool use is of grave concern. To understand persistence of vibration injury, the present study examined recovery of nerve conduction velocity and structural damage of myelinated axons in a rat tail vibration model. Both 7 and 14 days of vibration (4 h/day) decreased conduction velocity. The decrease correlated directly with the increased percentage of disrupted myelinated axons. The total number of myelinated axons was unchanged. During 2 months of recovery, conduction velocity returned to control level after 7‐day vibration but remained decreased after 14‐day vibration. The rat tail model provides insight into understanding the persistence of neural deficits in hand–arm vibration syndrome. Muscle Nerve, 2009     

Multiple measures of axonal excitability in peripheral sensory nerves: an in vivo rat model

Excitability measurements on human motor and sensory nerves have provided new insights into axonal membrane changes in peripheral nerve disorders. The aim of this study was to establish an in vivo rat preparation suitable for threshold tracking of sensory nerve action potentials (SNAPs) to model clinical sensory nerve excitability studies. In Sprague-Dawley rats anesthetized with ketamine and xylazine, current stimuli were applied to the base of the tail and SNAPs recorded from distal needle electrodes. Multiple excitability data were obtained as previously described for human nerves and compared to recordings from the motor tail axons and to sensory recordings from human median and ulnar nerves. The pattern of excitability changes in rats was broadly similar to that in humans, although some parameters differed significantly. Individual recordings were stable for at least 3 h. These data show that the rat tail enables excitability properties of sensory as well as motor axons to be studied experimentally, e.g., in models of nerve disease and during pharmacological interventions.     

A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [³H]nisoxetine. However, traditional [³H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [³H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [³H]CFT binding to whole cells (at physiological [Na⁺] and 25 °C) were similar to potencies observed in the [³H]NE uptake assay. Inhibition constants (K_i) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R² = 0.99, p < 0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [³H]nisoxetine binding to membrane preparations (4 °C for 3 h, [Na⁺] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [³H]CFT binding became greater than that measured in a functional assay of [³H]NE uptake at physiological [Na⁺]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [³H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [³H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.     

Spinal neurogenesis and axon projection: A correlative study in the rat

The purpose of the present study was to determine the relationship between the duration of a spinal neuron's neurogenic period and the length of its axon or level of projection. Spinal segment L1 was chosen for examination and neurons were divided into four projection groups: (1) supraspinal projection (SSp), (2) long ascending propriospinal (LAPr), (3) short ascending propriospinal (SAPr), and (4) descending propriospinal (DPr). To determine the duration of the neurogenic period for each group, ³H-thymidine was administered to fetal rats during the proliferative period for spinal neuroblasts on one of embryonic (E) days E13 through E16. Between 50 and 100 days after birth neurons in each group were labeled with the retrograde fluorescent tracer Fluoro-Gold. To demonstrate nerve cells with SSp projections, spinal cords were hemisected at spinal segment C3 in one group of animals and Fluoro-Gold was applied to the sectioned surface of the cord. Three additional sets of animals were used to label nerve cells with LAPr, SAPr, and DPr projections by injecting Fluoro-Gold into the gray matter at spinal segments C6, T12, and L5, respectively. Neurons labeled with both Fluoro-Gold and ³H-thymidine and neurons labeled with Fluoro-Gold alone in each animal in each group were counted and the data statistically analyzed. Results showed that within each spinal lamina neurons with different projections were generated, i.e., completed cell division, at significantly different rates. Neurons with the longest axons, those with SSP projections, were generated first. These were followed by those with LAPr projections, and finally those with SAPr and DPr projections. In most laminae there was no significant difference between the neurogenic periods of rostrally projecting short propriospinal (SAPr) neurons versus caudally projecting short propriospinal (DPr) neurons. It was concluded that the duration of the neurogenic period for a given group of neurons within each spinal lamina is inversely related to the distance between the nerve cell and its projection site regardless of the direction of its projection. © 1993 Wiley-Liss, Inc.     

Regeneration of peripheral nerves after clenbuterol treatment in a rat model.

Clenbuterol is known to act as a neuroprotective substance in the central nervous system, and also reduces muscle atrophy after denervation. The aim of this study was to evaluate its influence on peripheral nerve regeneration. The rat sciatic nerve model was used in four groups (n = 8 per group). After complete nerve transection and microsurgical coaptation, two groups received a daily oral dose of 100 microg/kg clenbuterol and two served as controls. Regeneration was assessed clinically, histologically, and morphometrically after 4 and 6 weeks. The weight ratios of calf muscles were calculated. Histological examination showed significantly increased axon counts in the clenbuterol group and a better degree of myelination. Muscle weight ratios of the clenbuterol group were significantly increased after 6 weeks, and the animals showed improved function of the hindlimb. Thus, therapy with 100 microg/kg clenbuterol daily after coaptation of a sciatic nerve showed a positive influence on clinical, histological, and morphometrical parameters in the rat model. The underlying mechanism remains unclear.     

壳聚糖-抗坏血酸盐胶体用于牙周炎模型大鼠牙周组织的修复与再生：效果验证

背景：随着对牙周病的病因和发病机制的深入认识,牙周结缔组织破坏的直接原因在于宿主的免疫反应,而非细菌本身,学者们进行了大量具有宿主调节功能的牙周局部用药的研究。壳聚糖是目前较受关注的一种材料,用其附带抗坏血酸盐来处理牙周疾病值得探索。 目的：评估壳聚糖-抗坏血酸盐促进牙周炎模型大鼠牙周组织修复与再生的作用。 方法：采用56只Wistar大鼠建立牙周炎模型,随机分成两组,实验组用壳聚糖-抗坏血酸盐胶体,对照组用生理盐水,隔天牙周袋底注射治疗。治疗后不同时间进行临床检查、形态学测量和组织学观察。 结果与结论：实验组菌斑指数、牙龈指数、牙周袋深度、附着丧失和牙齿松动度均低于对照组,最大差值分别为1.18(28 d)、0.92 (2 d)、0.33 mm(4 d)、0.49 mm(28 d)、1.69(28 d)。实验组牙槽骨丧失量在0,2,4周分别是(1.18±0.08),(1.10±0.12), (1.03±0.08) mm;而对照组分别为 (1.17±0.11),(1.31±0.09),(1.42±0.14) mm。组织学观察实验组和对照组都没有明显的新附着形成,但实验组炎症浸润程度明显低于对照组,且有再生修复。说明壳聚糖-抗坏血酸盐能够促进牙周炎模型大鼠牙周组织修复与再生,是一种很有治疗意义和开发价值的牙周局部应用药物。 关键词：壳聚糖;抗坏血酸盐;牙周炎;动物模型;口腔材料 doi:10.3969/j.issn.1673-8225.2010.12.045     

TC10 as an essential molecule in axon regeneration through membrane supply and microtubule stabilization

Mammalian central nervous system(CNS)neurons lose axon regenerative ability as they mature.This failure to regenerate shows a clear contrast to a remarkable potential of axon growth during embryonic development and after an injury in the peripheral nervous system(PNS)(Hilton and Bradke,2017).The absence of regeneration in the mature CNS neurons is caused by an inhibitory influence of the environment of the injured axons and the deficit of intrinsic factors that enable regeneration in the PNS(He and Jin,2016).     

Increase in inflammatory cytokines in median nerves in a rat model of repetitive motion injury

We examined cytokines in rat median nerves following performance of a high repetition reaching and grasping task at a rate of 8 reaches/min for up to 8 weeks. IL-1alpha, IL-1beta, TNF-alpha, IL-6 and IL-10 were analyzed by immunohistochemistry. Double-labeling immunohistochemistry for ED1, a marker of phagocytic macrophages, was also performed. We found increased immunoexpression of IL-6 by week 3, increases in all 5 cytokines by week 5. This response was transient as all cytokines returned to control levels by 8 weeks of performance of a high repetition negligible force task. Cytokine sources included Schwann cells, fibroblasts and phagocytic macrophages (ED1-immunopositive). These findings suggest that cytokines are involved in the pathophysiology of repetitive motion injuries in peripheral nerves.     

右美托咪啶对创伤性脑损伤大鼠模型的神经保护作用实验研究

目的探讨右美托咪啶对创伤性脑损伤组织炎症与细胞凋亡影响。方法建立创伤性脑损伤大鼠模型,设置实验组和对照组,其中实验组予以右美托咪啶6μg/kg腹腔注射干预,对照组予以等量生理盐水腹腔注射,损伤后第3天和第7天,采用ELISA检测损伤组织内肿瘤坏死因子(TNF-α)和白介素-1β(IL-1β)浓度水平,Western-bolt分析损伤组织内凋亡信号关键蛋白caspase-3表达,TUNEL法观察损伤组织细胞凋亡情况。结果右美托咪啶处理可明显降低创伤性脑损伤组织内炎性因子TNF-α和IL-1β浓度(P0.05),减少凋亡信号关键蛋白caspase-3表达(P0.05),显著抑制创伤性脑损伤组织中神经细胞凋亡(P0.05)。结论右美托咪啶处理具有明显神经保护作用,其机制可能与减轻创伤性脑损伤组织炎性反应和抑制神经细胞凋亡有关。     

Changes in myelinated and unmyelinated axon numbers in the proximal parts of rat sural nerves after two types of injury

Counts of myelinated and unmyelinated axon profiles have been made from normal, uninjured rat sural nerves and from nerves injured 6 months earlier in one of two ways. In one group of rats the nerve was simply cut and left to regenerate, leading to the development of a neuroma in continuity, while in the second group the nerve was cut but then ligated as well to prevent regeneration; this led to stump neuroma formation. After nerve transection and regeneration, with subsequent formation of a neuroma in continuity, there was no change in the number of myelinated axon profiles found 25 mm proximal to the old injury site when compared with control, but there was an 18% reduction (P < 0.05) in the number of unmyelinated axon profiles. Immediately proximal to the injury site the picture was similar, with there still being the same number of myelinated axon profiles as in control material but here the reduction in unmyelinated axon numbers was slightly greater at 24% (P < 0.05). In the proximal part of nerves that had been cut and stump neuroma formation induced there was a large increase (33%) in myelinated axon profiles over and above control values (P < 0.001) but the number of unmyelinated profiles was the same as in controls. Closer to the stump neuroma the number of myelinated axon profiles had increased yet further to be 88% (P < 0.001) above control while the number of unmyelinated ones remained no different from control. Our interpretation of these results is that after nerve transection and regeneration there is no loss of peripheral neurons supporting myelinated axons but some loss of those supporting unmyelinated ones. If a cut nerve is prevented from regenerating and a stump neuroma forms, however, a vigorous sprouting response is triggered in neurons with myelinated axons while those supporting unmyelinated axons are possibly prevented from dying. The reaction of peripheral neurons to injury is such that the number of axons they support varies along the nerve as one goes disto-proximally away from the injury site. Thus discrepancies in results from different laboratories have come about because material for axon counting has been taken from different points along the nerve relative to the injury site and also because the material has been taken from nerves injured in different ways.     

Peripheral nerve grafts to the frog optic tectum: a morphological study of foreign axon regeneration in the central nervous system

The proximal stump of a transected mandibular nerve was grafted onto the rostrodorsal surface of the optic tectum in adult Rana pipiens to investigate the morphologic characteristics of nonspecific axonal regeneration in a highly organized region of central nervous system (CNS). Within the first 3 weeks postgraft surgery (WPS), the nerve-tectum interface became firmly established. Concomitant with this was an invasion of the host tectum by a small number of fine "pioneerlike" axons from the nerve. By 6 WPS there developed a concerted instreaming of a large number of peripheral fibers. Once within the CNS, the foreign axons distributed themselves throughout the rostrocaudal extent of the tectum, but primarily its dorsal aspect within superficial layers 8 and 9. Presence of intact optic fibers at the time of mandibular fiber invasion served somewhat to restrict the regenerating aberrant axons in their course through layer 9. This restriction could be avoided by removal of the optic input either before or during peripheral ingrowth. However, once peripheral fibers had entered and established themselves in the host environment, no subsequent manipulation of the retinotectal projection had any effect. The aberrant growth pattern, which appeared remarkably stable after 6 WPS, consisted of a plexus of medium- and fine-caliber peripheral axons. Many of these fibers had numerous branches and "en passant" varicosities, the latter encompassing a variety of shapes and sizes. Terminal swellings and arborizations were also found. When comparing the regeneration of optic and mandibular nerve fibers in the tectum, two distinctions were made. Whereas optic axons revealed a fascicular and layered organization, mandibular axons showed a highly segregated and disordered growth pattern. These characteristic differences were maintained even when the two fiber systems were allowed to coregenerate into the same target tectum. Thus, each of the two groups of axons interacts with the tectal substrate in a distinct manner, apparently independent of the other.     

Relation between the number of myelin lamellae and axon circumference in fibers of vagus and sciatic nerves of mice

Nerve fiber populations of the vagus and sciatic nerves of mice were classified according to the number of myelin lamellae present in the sheaths. This method for classifying fiber populations was superior to others used previously since it provided a more sensitive procedure for the analysis of individual fibers and better control over the technical factors involved in tissue processing. The relationship of the number of myelin lamellae in the sheath to axon circumference was found to be linear. In fresh tissue there was one myelin lamella for every 0.24 μ increase in axon circumference above da value of 2.32 μ (the mean circumference of an average-sized nonmyelinated fiber). A formula was proposed which may be useful for understanding how axons control myelin development and interpreting developmental stages, as well as for evaluating pathologic conditions affecting the peripheral nervous system. The critical diameter above which fibers were found to be myelinated was about 0.8 μ for fixed nerve and 1.1 μ for fresh nerve. The ratio of axon diameter to fiber diameter ranged between 0.5 and 0.9 and was not related to fiber size.