Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve

The expression of the neural cell adhesion molecules L1 and N-CAM and of their shared carbohydrate epitope L2/HNK-1 was studied during the development and after the transection of mouse sciatic nerves. During development, L1 and N-CAM were detectable on most, if not all, Schwann cells at embryonic day 17, the earliest stage tested. With increasing age, the immunoreactivity was reduced being confined to non-myelinating Schwann cells by post-natal day 10, at which stage the staining pattern resembled that seen in adult sciatic nerves. Double-immunolabelling experiments revealed a complete overlap between L1 and N-CAM antibodies. The L2/HNK-1 epitope was not detectable in developing sciatic nerves until the end of the 2nd post-natal week, when it appeared to be associated with the outer profiles of thick myelin sheets, as also seen in adult sciatic nerves. Three days after the transection of adult sciatic nerves, L1 antigen and N-CAM was detectable in more Schwann cells in the distal nerve end than in untreated control nerves. The peak level of the reappearance of L1 antigen and N-CAM in Schwann cells occurred between 2 and 4 weeks after transection. The reduction of L1-antigen expression to its normal adult level took more than a year, thus recapitulating normal development, but on a more protracted time scale. Similarly, the L2/HNK-1 epitope remained undetectable until the transected nerve had returned to its normal state of myelination, i.e. approximately 1 year after transection.     

Collateral sprouting occurs following end-to-side neurorrhaphy

Recent evidence supports the use of end-to-side neurorrhaphy for the treatment of certain peripheral nerve disorders. However, the mechanism by which nerves regenerate following this procedure is still unclear. To address this question, the authors designed a new end-to-side coaptation model in rats in which the donor nerves were uninjured. The regenerated axons at the coaptation site were observed directly using fluorescent dye as the neural tracer. The sciatic nerve from adult Wistar rats was transplanted between the left and right median nerves. Fifteen rats were divided into three groups. In group I, the donor (right median) nerve was sutured end to side to the divided grafted nerve using a noninjury technique. In group II, the aponeurosis of the spinal muscles was harvested and the sciatic and right median nerves were coapted end to side noninjuriously by wrapping them in the excised aponeurosis. In group III, a perineurial window was created and a partial neurectomy was carried out at the suture site, after which the sciatic and right median nerves were sutured end to side. Sixty days after the operation, nerve regeneration was evaluated by recording action potentials in the grafted nerve, by performing electromyography in the flexor muscles in the forearm, and by histological examination. The grafted nerves were fixed and sectioned, the number of regenerated nerve fibers was counted, and axonal diameters were measured. Fluorescent dye crystal was used, in conjunction with confocal microscopy, to observe the regenerated axons at the co-aptation site. The results showed that nerve regeneration had occurred in the animals, as determined electrophysiologically and histologically. Both the right and left flexor muscles of the forearm contracted simultaneously as a result of indirect electric stimulation of the grafted nerve, which suggests that the regenerated nerve was physiologically connected with the donor nerve. Nerve fiber counts did not show any differences among groups (p > 0.05), but axonal diameters were significantly greater in group III than in the other two groups. Fluorescent dye staining revealed the presence of regenerated nerve fibers beyond the coaptation site. In group III, the regenerating nerves were observed within the whole section of the coaptation site and collateral sprouting was found to occur even at a site distal to the suture. From these results, the authors conclude that in end-to-side neurorrhaphy, nerve regeneration occurs by collateral sprouting from the donor nerve.     

Functional studies on sciatic nerve blood flow in respect to its vascular supply and tonic neural activity.

An introduction of laser flow meters for a continuous measurements of a tissue blood flow has opened new avenues for an accurate assessment blood flow in peripheral nerves. The aim of our study was: 1) to carry out a functional verification of anatomical sources of a sciatic nerve blood supply in the rat; 2) develop a measurement technique to facilitate standardisation of results; 3) to determine the role of nerve fibres tonic activity in the maintenance of a resting blood flow in the sciatic nerve. Based on results of the present study the following conclusions have been drawn out: 1) in order to obtain a real values of the blood flow through the sciatic nerve it is necessary to remove its muscular fascia; 2) an uninjured epineurium plays a crucial role in maintaining the resting blood flow; 3) major blood supply of sciatic nerve comes from inferior gluteal and popliteal arteries; 4) the tonic neural activity plays a role in the maintenance of the resting sciatic nerve blood flow in anaesthetised rats.     

Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I

In this report, we have identified two apolipoproteins (apo), apoD and apoA-IV, that, together with the previously identified apoA-I and apoE, accumulate in the regenerating peripheral nerve. These four apolipoproteins were identified in regenerating rat sciatic nerves by their molecular weights, their isoelectric points, and their recognition by specific antibodies. Antibodies were also used to document the changing concentrations of these apolipoproteins in homogenates of regenerating sciatic nerves collected 1 day to 6 weeks after a denervating crush injury. By 3 weeks after injury, at their peak accumulation, apoA-IV and apoA-I had increased 14- and 26-fold, respectively, relative to their concentrations in the normal nerve. Apolipoproteins D and E, in contrast, increased over 500- and 250-fold, respectively, by 3 weeks. These same apolipoproteins also accumulated in the regenerating sciatic nerves of two other species, the rabbit and the marmoset monkey. Immunocytochemistry showed that apoD was produced by astrocytes and oligodendrocytes in the normal central nervous system, and by neurolemmal or fibroblastic cells in the normal peripheral nervous system. Metabolic labeling of both apoD and apoE by [35S]methionine during an in vitro incubation of regenerating rat sciatic nerve segments confirmed that these apolipoproteins are synthesized by the nerve. Neither apoA-IV nor apoA-I was metabolically labeled, however, suggesting that they enter the nerve from the plasma. The results from this study provide evidence that several different apolipoproteins from various sources may play a role in lipid transport within neural tissues.     

Age-related variations of elements as compared among optic, radial, and sciatic nerves

To elucidate changes of peripheral nerves with aging, the authors studied age-related changes of element contents in the optic, radial, and sciatic nerves by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The contents of phosphorus and sulfur remained constant through ages 61 to 97 yr in three nerves, the optic, radial, and sciatic nerves. It was found that there were age-related differences in calcium content among the optic, radial, and sciatic nerves: The calcium content of the optic nerve increased progressively with aging; in the radial nerve, it was hardly changed with aging; in contrast, the calcium content of the sciatic nerve decreased gradually with aging. In addition, it was found that in the radial nerve there were moderate correlations between age and zinc or sodium content, whereas significant correlations between age and the content of silicon or iron were found in the sciatic nerve. Furthermore, there was a correlation between the silicon and iron contents in the sciatic nerves.     

The effects of ultrasonic energy on peripheral nerves: implications for ultrasound-assisted liposuction

The integration of ultrasound-assisted liposuction with traditional suction-assisted lipoplasty has extended the role of liposuction in body contouring. Although there are ample data regarding the effects of ultrasound on peripheral nerves from studies with the Cavitron ultrasound surgical aspirator, there is little information concerning the effects of modern ultrasound body contouring equipment on neural tissue. This study was designed to evaluate the functional and histologic effects of ultrasound energy on rat peripheral nerves (sciatic nerves) using a commonly-used ultrasound-assisted liposuction generator. After the application of ultrasound to exposed rat sciatic nerves, operative magnification was used to assess any visible injury. The sciatic function index was serially measured to quantify immediate and long-term functional effects on the nerves. Our results showed immediate visible injury using low amplitude settings (level 6), but no functional evidence of injury until much higher settings were used (level 9). All animals in the groups with initial functional impairment had returned to normal or near-normal function at completion of the study (51 days). Histologic examination revealed no evidence of damage in the low amplitude groups. Histologic analysis of the high amplitude groups displayed diffuse infiltration of the nerve, with foamy histiocytes and an increased number of mast cells, consistent with remote neural injury followed by myelin breakdown and repair.     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Moderately Different NADPH-Diaphorase Positivity in the Selected Peripheral Nerves after Ischemia/ Reperfusion Injury of the Spinal Cord in Rabbit

1. To vicariously investigate the nitric oxide synthase (NOS) production after spinal cord injury, NADPH-d histochemistry was performed on the selected peripheral nerves of adult rabbits 7 days after ischemia. The effect of transient spinal cord ischemia (15 min) on possible degenerative changes in the motor and mixed peripheral nerves of Chinchilla rabbits was evaluated.2. The NADPH-diaphorase histochemistry was used to determine NADPH-diaphorase activity after ischemia/reperfusion injury in radial nerve and mediane nerve isolated from the fore-limb and femoral nerve, saphenous nerve and sciatic nerve separated from the hind-limb of rabbits. The qualitative analysis of the optical density of NADPH-diaphorase in selected peripheral nerves demonstrated different frequency of staining intensity (attained by UTHSCSA Image Tool 2 analysis for each determined nerve).3. On the seventh postsurgery day, the ischemic spinal cord injury resulted in an extensive increase of NADPH-d positivity in isolated nerves. The transient ischemia caused neurological disorders related to the neurological injury—a partial paraplegia. The sciatic, femoral, and saphenous nerves of paraplegic animals presented the noticeable increase of NADPH-d activity. The mean of NADPH-diaphorase intensity staining per unit area ranged from 134.87 (±32.81) pixels to 141.65 (±35.06) pixels (using a 256-unit gray scale where 0 denotes black, 256 denotes white) depending on the determined nerve as the consequence of spinal cord ischemia. The obtained data were compared to the mean values of staining intensity in the same nerves in the limbs of control animals (163.69 (±25.66) pixels/unit area in the femoral nerve, 173.00 (±32.93) pixels/unit area in saphenous nerve, 186.01 (±29.65) pixels/unit area in sciatic nerve). Based on the statistical analysis of the data (two-way unpaired Mann–Whitney test), a significant increase (p≤0.05) of NADPH-d activity in femoral and saphenous nerve, and also in sciatic nerve (p≤0.001) has been found. On the other hand, there was no significant difference between the histochemically stained nerves of fore-limbs after ischemia/reperfusion injury and the same histochemically stained nerves of fore-limbs in control animals.4. The neurodegenerative changes of the hind-limbs, characterized by damage of their motor function exhibiting a partial paraplegia after 15 min spinal cord ischemia and subsequent 7 days of reperfusions resulted in the different sensitivity of peripheral nerves to transient ischemia. Finally, we suppose that activation of NOS indirectly demonstrable through the NADPH-d study may contribute to the explanation of neurodegenerative processes and the production of nitric oxide could be involved in the pathophysiology of spinal cord injury by transient ischemia.     

Investigation of the mechanisms for wireless nerve stimulation without active electrodes

Electric-field stimulation of neuronal activity can be used to improve the speed of regeneration for severed and damaged nerves. Most techniques, however, require invasive electronic circuitry which can be uncomfortable for the patient and can damage surrounding tissue. A recently suggested technique uses a graft-antenna—a metal ring wrapped around the damaged nerve—powered by an external magnetic stimulation device. This technique requires no electrodes and internal circuitry with leads across the skin boundary or internal power, since all power is provided wirelessly. This paper examines the microscopic basic mechanisms that allow the magnetic stimulation device to cause neural activation via the graft-antenna. A computational model of the system was created and used to find that under magnetic stimulation, diverging electric fields appear at the metal ring's edges. If the magnetic stimulation is sufficient, the gradients of these fields can trigger neural activation in the nerve. In-vivo measurements were also performed on rat sciatic nerves to support the modeling finding that direct contact between the antenna and the nerve ensures neural activation given sufficient magnetic stimulation. Simulations also showed that the presence of a thin gap between the graft-antenna and the nerve does not preclude neural activation but does reduce its efficacy.     

七叶莲对蟾蜍坐骨神经动作电位的影响

目的：观察七叶莲水煎液对蟾蜍离体坐骨神经复合动作电位的幅度及传导速度的影响,研究其对坐骨神经电生理特性的作用。方法：将制备的蟾蜍坐骨神经干分为4组,分别在任氏液和浓度为10％,20％,40％的七叶莲水煎液中浸泡,用MedLab生物信号采集处理系统引导神经干复合双相动作电位,并分别测定各组不同浸泡时间的坐骨神经干动作电位的幅度和传导速度两项电生理指标。记录不同浓度的七叶莲水煎液对蟾蜍离体坐骨神经复合动作电位的幅度和传导速度的影响。结果：10％,20％,40％3种浓度的七叶莲水煎液均使坐骨神经复合动作电位的幅度变小（P〈0．01）,传导速度变慢（P〈0．01）并最终使坐骨神经动作电位消失,且经过一段时间后动作电位的幅度和传导速度均能恢复。结论：七叶莲能可逆地阻滞神经动作电位的传导。     

周围神经再生早期过程中内源性和外源性巨噬细胞数量变化的实验研究

目的：周围神经再生过程中巨噬细胞发挥了重要的作用,然而目前对于神经内内源性和外源性巨噬细胞的具体作用了解的却很少,因此本实验研究了小鼠坐骨神经损伤后早期再生过程中内源性和外源性巨噬细胞数量比例变化的情况,探索周围神经再生的规律。方法：移植CAG-EGFP转基因小鼠的全骨髓有核细胞到骨髓灭活野生型C5781／6小鼠体内建立嵌合体小鼠模型。待移植成功3个月后夹伤小鼠一侧坐骨神经,并在损伤后第2、7、14和28天取材、切片,使用巨噬细胞特异性抗体cD68进行免疫荧光染色,分析损伤神经段中内源性巨噬细胞（CD68＋／EGFP-）、外源性巨噬细胞（CD68＋／EGFP＋）的数量及其比例变化情况。结果：①夹伤骨髓移植模型小鼠坐骨神经后,参与坐骨神经损伤修复的巨噬细胞可分为两类,即内源性巨噬细胞（CD68＋／EGFP-）和外源性巨噬细胞（CD68＋／EGFP＋）;②夹伤坐骨神经后,浸润的总巨噬细胞数量从第2天开始逐渐增加,到第14天达到高峰,约为正常情况下的60倍,随后逐渐减少;③起初外、内源性巨噬细胞间的比例是1：1,差值最大出现在损伤后第14天为4：l。结论：小鼠坐骨神经夹伤后,内外源性巨噬细胞共同参与了受损神经组织远心段的修复和再生过程,损伤初期发挥作用的主要是内源性巨噬细胞,随后大量浸润的外源性巨噬细胞占主导作用。本实验首次连续观察并定量分析了神经损伤后早期内源性和外源性巨噬细胞的数量改变,证实了瓦勒氏变性过程中内源性和外源性巨噬细胞在不同阶段对巨噬细胞总量的贡献作用。     

Peripheral nerve lesion-induced uptake and transport of choleragenoid by capsaicin-sensitive c-fibre spinal ganglion neurons

In the present experiments the effect of systemic capsaicin treatment on the retrograde labelling of sensory ganglion cells was studied following the injection of choleratoxin B subunit-horseradish peroxidase conjugate (CTX-HRP) into intact and chronically transected peripheral nerves. In the control rats CTX-HRP injected into intact sciatic nerves labelled medium and large neurons with a mean cross-sectional area of 1,041 +/- 39 gm2. However, after injection of the conjugate into chronically transected sciatic nerves of the control rats, many small cells were also labelled, shifting the mean cross-sectional area of the labelled cells to 632 +/- 118 microm2. Capsaicin pretreatment per se induced a moderate but significant decrease in the mean cross-sectional area of the labelled neurons (879 +/- 79 microm2). More importantly, systemic pretreatment with capsaicin prevented the peripheral nerve lesion-induced labelling of small cells. Thus, the mean cross-sectional areas of labelled neurons relating to the intact and transected sciatic nerves, respectively, did not differ significantly. These findings provide direct evidence for a phenotypic switch of capsaicin-sensitive nociceptive neurons after peripheral nerve injury, and suggest that lesion-induced morphological changes in the spinal cord may be related to specific alterations in the chemistry of C-fibre afferent neurons rather than to a sprouting response of A-fibre afferents.     

Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.     

Temporal progression and extent of the return of sensation in the foot provided by the saphenous nerve after sciatic nerve transection and repair in the rat - implications for nociceptive assessments

Sensory testing, by providing stimuli for nociceptors of the foot, is a popular method of evaluating sensory regeneration after damage to the sciatic nerve in the rat. In the following study, 20 rats were submitted to double transection of the sciatic nerve. The subsequent 14 mm gap was repaired through guidance interponation. In order to evaluate nerve regeneration, sensory testing was performed additionally to other methods, which included motor testing, morphometry, and electron microscopic assessments of nerves. Somatosensory testing revealed that all animals exhibited next to the same amount of sensory reinnervation on their foot regardless of their experimental group. In motor tests, however, two out of the three experimental groups did not improve at all. These groups also failed to show neural regrowth in morphometric and electron microscopic assessments of the associated nerve. Retrograde tracing was able to prove the saphenous nerve as an alternative source of sensory reinnervation in animals with failed sciatic regeneration. This means that results of sensory testing in the rat should be treated with caution, taking into account the areas tested and the likelihood that in these areas saphenous sprouting could have taken place. Furthermore, it is strongly advised that somatosensory testing should be conducted only on toe 5.     

THE EFFECTS OF UNDERNUTRITION ON THE PROTEINS OF OPTIC AND SCIATIC NERVES DURING DEVELOPMENT

Abstract— Polyacrylamide gel electrophoresis has been used to assess the appearance of some optic and sciatic nerve proteins in normal developing rats and in undernourished rats. Of the myelin proteins, the'Wolfgram'proteolipid is already present about the time myelination begins. The basic myelin proteins appear later, first in sciatic and then in optic nerve. A non-myelin basic protein, assumed to be a histone, is present at high levels in both nerves before myelination begins. There is no apparent effect of undernutrition on the appearance and amount of myelin proteins at 12, 16 and 22 days of age. The'histone'protein is reduced in optic and sciatic nerves at times corresponding roughly to the transition periods from cellular proliferation to myelin formation. The possibilities are discussed that myelin basic proteins are synthesized as compact myelin formation occurs, and that there may be retarded cellular proliferation in nerves of undernourished rats.     

Bioelectric properties of frog sciatic nerves during exposure to stationary magnetic fields

The bioelectric properties of frog sciatic nerves have been measured during exposure to homogeneous, stationary magnetic fields. The action potential amplitude, conduction velocity, absolute refractory period and relative refractory period were found to be unaffected by a continuous 4-h exposure to perpendicular or parallel 2.0 T (1 T equal 10(4) G) magnetic fields. These parameters also remained unchanged during a 1-h post-exposure period. The conduction velocity was similarly found to be unchanged when the field was applied continuously for 17 h. Exposure of sciatic nerves to a 1.0-T field led to no alteration in the threshold for neural excitation. The absence of magnetic field effects on nerve electrical activity observed in the present experiments contrasts with the positive findings reported previously by other investigators. These discrepancies may be attributable to an inadequate control of ambient temperature in the earlier studies.     

Chemical and structural changes of neurofilaments in transected rat sciatic nerve

The sequence of changes occurring in transected rat sciatic nerve was examined by electron microscopy and by sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. Representative segments of transected nerves were processed for ultrastructural examinations between 0 and 34 days after the transection of sciatic nerves immediately below the sacro-sciatic notch. The remainder of the transected nerves and the intact portions of sciatic nerves were desheathed and immediately homogenized in 1 percent SDS containing 8 M urea and 50 mM dithioerythritol. Solubilized proteins were analyzed on 12 percent gels at pH 8.3 in a discontinuous electrophoretic system. Initial changes were limited to the axons of transected nerve fibers and were characterized by the loss of microtubules and neurofilaments and their replacement by an amorphous floccular material. These changes became widespread between 24 and 48 h after transection. The disruption of neurofilaments during this interval occurred in parallel with a selective loss of 69,000, 150,000 and 200,000 mol wt proteins from nerve homogenates, thus corroborating the view that these proteins represent component subunits of mammalian neurofilaments. Furthermore, the selective changes of neurofilament proteins in transected nerves indicate their inherent lability and suggest their susceptibility to calcium-mediated alterations. Electrophoretic profiles of nerve proteins during the 4-34-day interval after nerve transection reflected the breakdown and removal of myelin, the proliferation of Schwann cells and the deposition of endoneurial collagen. A marked increase of intermediate-sized filaments within proliferating Schwann cell processes was not accompanied by the appearance of neurofilamentlike proteins in gels of nerve homogenates.