The effects of nerve transection on the endoneurial collagen fibril sheaths

Summary The connective tissue changes during Wallerian degeneration and subsequent regeneration were studied in the distal stump of transected sciatic nerves of Wistar rats. In half of the animals regeneration was prevented by suturing the distal stump to muscle and in the rest spontaneous regeneration was allowed. Intact contralateral nerves served as controls. By 4 weeks after transection the Schwann cell columns became surrounded by a layer of thin collagen fibrils that were, on average, 25–30 nm in diameter. This was only half of the fibril diameter observed elsewhere in the endoneurium or in control nerves. The layer of thin fibrils diminished in thickness when axonal regeneration reached the distal stump, especially as the axons became myelinated. At all stages of the experiment the fibril diameter distribution in the surrounding normal endoneurial stroma was comparable with that observed in control nerves. Segments of Schwann cell basement membrane were observed to be closely associated with collagen fibrils both in freely regenerating, as well as in non-regenerating, nerves. The diameter of these fibrils corresponded to that observed in the zone of thin fibrils surrounding the Schwann cell columns. Such areas were not found in control nerves. The data obtained show that deposition of thin collagen fibrils occurs around the Schwann cell columns as a reaction to transection. Our observations on the regenerating nerves indicate that this connective tissue reaction does not prevent regeneration in the early phases following injury and that its progression is limited concomitantly with axonal regeneration.Supported by a grant (to V. S.) from the Emil Aaltonen Foundation and by institutional grants from the Turku University Foundation and the Sigrid Jusélius Foundation     

Pathology of local anesthetic-induced nerve injury

Summary Nerve fiber injury and endoneurial edema were induced by the injection of the local anesthetic 2-chloroprocaine, tetracaine, procaine, etidocaine or mepivacaine into the soft tissue and fascia surrounding the sciatic nerve of Sprague-Dawley rats. Light microscopy demonstrated that the perineurial barrier was not mechanically damaged by the surgical procedure but, at 48 h post-injection, perineurial permeability was increased. Previous observations of leakage of horseradish peroxidase and the present report of neutrophils and eosinophils in the endoneurium indicate a disruption of blood-nerve barrier systems. Endoneurial edema was observed in the subperineurial, interstitial and perivascular regions. Axonal degeneration and demyelination occurred; the latter associated with accumulation of large lipid droplets in Schwann cells. Degranulation of mast cells, proliferation of fibroblasts and macrophage activity were noteworthy in affected areas. The findings are remarkable in that this is the first model of endoneurial edema by a neurotoxin which penetrates the perineurium, disrupting barrier system and inducing nerve fiber injury.Supported in part by USPHS NS18715, NS14162 and NS07078 and the Veteran's Administration Research Service     

Wallerian degeneration of peripheral nerve

Summary The age-dependent loss of the major peripheral nerve lipids (cholesterol, phospholipid, and total galactolipid) was quantitated over a period of 9 weeks of Wallerian degeneration induced by surgical transection of rabbit sciatic nerves in animals of several ages. Proportionate losses of these lipids were determined by calculating the content of each lipid on a per nerve and on a per gram fresh weight basis remaining after a given period of Wallerian degeneration as a percent of original normal values at several times following surgery. The proportionate loss of each lipid from the distal stump was the most prompt and the most complete in nerves transected at 2 weeks of age, and the least in nerves transected at 20 weeks of age. The prompter clearance of these lipids from younger than older degenerating nerve gives convincing evidence that the suggestion from light-microscopic studies of faster clearance of neural debris in younger than in older animals is correct. A possible relationship between these biochemical findings and the phenomenon of greater functional recovery from peripheral nerve injury in younger than in older subjects is discussed.Supported by an N.I.H. grant (NS-10165)     

Peripheral nerve regeneration along collagen filaments

This paper describes the regeneration of severed peripheral nerve axons along collagen filaments without a tube. Two thousand collagen filaments were grafted to bridge 20 mm defects of rat sciatic nerve. The number of myelinated axons was approximately 4800 in the distal end of the nerve autograft at 8 weeks postoperatively; while in the collagen-filaments nerve guide it was 5500. The results suggested the collagen filaments guided regenerating axons effectively.     

Peripheral nerve regeneration in RGD peptide incorporated collagen tubes

This paper describes the regeneration of lesioned sciatic nerve with collagen tubes incorporated with RGD cell-adhesive peptide. Collagen implants of 14 mm were grafted to bridge a gap length of 10 mm nerve defect in a rat model. The regenerated tissues were analyzed histomorphologically. The number of myelinated axons in the regenerated mid-graft of the RGD peptide incorporated groups was statistically significant (p<0.05) than control collagen tube and autograft control after 30 days postoperatively. After 90 days of implantation, the mean counts were still statistically significant in the case of RGD peptide group than control collagen and autograft groups. Immunofluorescence studies demonstrated the staining of S100 proteins in the peripherally located cells indicating the proliferation of Schwann cells in the early days of regeneration. The staining pattern of integrin-alphaV was observed mostly in the perineurial regions in close proximity to the RGD peptide incorporated collagen tubes. Other studies like sciatic functional index, conduction velocity at 90 days postoperatively suggest complete regeneration of lesioned nerves with RGD incorporated collagen implants.     

Decreased L5 spinal nerve ligation nociceptive behavior following L4 spinal nerve transection

This study used the escape/avoidance paradigm to explore the role of the L4 spinal nerve in L5 ligation nociception. Unlike L5-ligated controls, L5-ligated/L4-transected animals had normal mechanical withdrawal threshold and did not escape/avoid mechanical stimulation of the afflicted paw. The result from the escape/avoidance paradigm, which does not rely on a reflexive withdrawal response, directly supports the hypothesis that the L4 spinal nerve contributes to L5 ligation neuropathic pain.     

Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection

The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC) from cell death after axotomy. Here, we assessed EPO's neuroprotective properties in vivo by examining RGC survival and axon regeneration at 4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection (5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye and transection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. In addition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitreal EPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our in vivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability to cross the blood-brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair.     

Tremor in the human hand following peripheral nerve transection and reinnervation

Slow movements and position holding by the digits are both characterised by 8-10 Hz tremor which appears to be centrally generated. Denervation and subsequent reinnervation lead to significant alterations in peripheral connectivity and reflex organisation. We have tested the hypothesis that 8-10 Hz tremor is present in the digits of subjects following a complete nerve lesion. The frequency content of abduction and adduction movements was recorded in 12 index fingers and nine little fingers reinnervated subsequent to a complete ulnar nerve transection. An optical position laser transducer was used to measure digital movements, minimising mechanical interference to the system. Concurrently, surface electromyograms (EMG) were also recorded from first dorsal interosseus muscles (1DI) and abductor digiti minimi brevis (ADMB) muscles for index and little fingers, respectively. The maximal voluntary contraction (MVC) of the reinnervated muscles varied from 5.9% to 100% of those of the unimpaired, contralateral hands. The subjects performed abduction-adduction movements of the index and little fingers and a position holding task. Significant peaks in PSD curves of acceleration and rectified integrated EMG traces were identified. Tremor in the 8-10 Hz range was evident in both the acceleration and EMG signals for the majority of digits during both the slow movement and position holding tasks. These findings demonstrate the robust nature of these 8-10 Hz oscillations, even following the significant changes in peripheral connectivity of muscle and nerve resulting from nerve transection and reinnervation.     

Peripheral nerve abnormalities in pediatric patients with spinal muscular atrophy

We examined the specific nerve conduction deficits distinguishing spinal muscular atrophy (SMA) subtypes I and II. Five SMA I patients (age, 0.2–1.1 years) and 10 SMA II patients (age, 1.0–2.8 years) were examined. Patients were compared to age-matched controls for motor and sensory conduction velocity (MCV and SCV) changes, compound muscle and sensory nerve action potential amplitudes (CMAP and SNAP), and F-wave occurrence (FO). Slower MCVs were found in three of five SMA I patients; all five exhibited markedly decreased CMAP amplitudes. Tibial nerve CMAP amplitudes significantly reduced in SMA II patients (p < 0.01). Slower SCVs and decreased SNAP amplitudes were observed in three of five SMA I patients but not in SMA II patients. Although FOs were reduced in both extremities of SMA I patients, the reduction was prominent in the tibial nerve of SMA II patients (p = 0.031). Loss of motor units may be widespread in the early stage of SMA I, while specific to the legs in young SMA II patients. SMA I showed sensory nerve degeneration, especially of large myelinated fibers. SMA II showed no sensory nerve abnormalities.     

Microglial cell responses in the rabbit retina following transection of the optic nerve

The response of retinal microglial cells, which accompanies retrograde degeneration of ganglion cell axons and perikarya (induced by transection of the optic nerve), was studied in whole-mounted rabbit retinae labeled enzyme-histochemically for nucleoside diphosphatase (NDPase), which is a microglial cell marker. A few days after transection, the number of microglial cells/mm2, as well as their staining intensity, began to increase in the inner plexiform layer. The mosaic-like distribution of the star-shaped microglial cells present in the inner plexiform layer of a normal rabbit retina was preserved during ganglion cell degeneration. As in the normal retina, processes of individual cells never overlapped with those of neighboring cells in the inner plexiform layer because individual cells in the "degenerating" retina acquired shorter processes, i.e., the cells occupied a smaller territory compared to the normal retina. In the nerve fiber layer the number and staining intensity of NDPase-labeled microglial cell processes (most of which are aligned in parallel with degenerating ganglion cell axons) transiently increased and returned to normal values by 5 months post-transection. Microglial cells that are not detectably NDPase labeled in the outer plexiform layer of a normal rabbit retina acquire intense staining a few days after the nerve is cut. The functional significance of the increased NDPase activity in the plasma membrane of microglial cells during degeneration remains to be determined.     

壳聚糖导管桥接周围神经缺损的实验研究

目的　应用壳聚糖神经导引管作为神经再生室桥接大鼠坐骨神经缺损 ,观察对神经再生的作用。方法　选用体重 2 0 0± 2 0g的Wistar大鼠 30只 ,手术造成右侧坐骨神经长约 12mm的缺损 ,以壳聚糖导管桥接神经缺损 ,以左侧正常坐骨神经作为对照 ,分别于术后 4、 12、 2 4周进行大体及显微解剖观察、组织学检查、免疫组化检查、电镜观察和神经电生理测定。结果　术后大鼠右后肢感觉、运动功能有不同程度的恢复 ;光镜和电镜组织学检查发现术后 12周再生轴突已长过神经缺损间隙 ,2 4周再生完全 ,髓鞘化良好 ,神经纤维排列整齐规则 ,导管大部分被降解吸收 ;神经电生理检查在术后 2 4周记录到再生坐骨神经的复合动作电位。结论　壳聚糖神经导引管为大鼠坐骨神经再生提供一个良好的再生微环境 ,再生坐骨神经功能恢复良好     

The dynamics of macrophage recruitment after nerve transection

In the present study rat sciatic nerves (n = 60) were transected; in half of the animals the nerve was allowed to regenerate freely, in the other half the regeneration was prevented by suturing beside the point of transection. Macrophages were stained with ED-1 antibody and counted (number/mm²) in both the epi- and endoneurium 3, 7, 14, 48 and 56 days post transection. Macrophages were observed first in the epineurium; the local density of macrophages was considerably higher in the epineurium than in the endoneurium during the first few days. The number of macrophages in the epineurium was maximal at 3 days (1000–2000/mm²), and thereafter it declined sharply. In the endoneurium macrophages were most abundant after 2 weeks (1000/mm²), after which their number declined steadily. A migration of epineurial macrophages appeared to take place through the perineurium from epineurial areas containing a high concentration of macrophages. Initially an endoneurial accumulation of macrophages was noted in the subperineurial area. These findings suggest an alternative route for macrophages into the endoneurial space. No statistical difference was observed between the regenerating and non-regenerating experimental groups. The present study indicates that regenerating axons do not have an effect on the number of macrophages in either the epineurium or the endoneurium. Received: 21 March 1996 / Revised, accepted: 25 September 1996     

Abnormal sensory and motor reinnervation of rat muscle spindles following nerve transection and suture

Summary Muscle spindles from the lower lumbrical muscles of rats were studied by transmission and scanning electron microscopy following reinnervation (i) after a single sciatic nerve crush lesion and (ii) after transection and immediate epineurial suture of the sciatic nerve. In all muscle spindles, regenerated sensory or motor nerve endings were encountered 3 months after making the lesions although in the nerve-transection group, regenerated nerve endings were seen less frequently before 6 months of recovery. Abnormalities in reinnervated spindles following neurotomy comprised: (1) multiplicity of axonal sprouts (hyperinnervation), sometimes irregularly related to, although never in direct contact with, regenerated sensory nerve endings; (2) altered contact relationships between sensory nerve endings and intrafusal muscle fibers; (3) abnormal structure of nerve endings; and (4) irregular association of Schwann cell processes to regenerated sensory nerve endings. These findings indicate that reinnervation of muscle spindles following transection and suture of a peripheral nerve, i.e., after complete interruption of its continuity, in fact, occurs although the fine structural abnormalities observed are supposed to interfere with adequate functional restoration.Supported by a grant from the Deutsche Forschungsgemeinschaft: Di 386/1-1     

Peripheral nerve ultrasonography in patients with transthyretin amyloidosis

Objective

To systematically study peripheral nerve morphology in patients with transthyretin (TTR) amyloidosis and TTR gene mutation carriers using high-resolution ultrasonography (US).

Long-term endoneurial changes after nerve transection

Summary Long-term endoneurial changes in the distal stump of transected rat sciatic nerve were examined from 8 to 50 weeks after nerve transection. The morphological alterations were followed both in nerves which were allowed to regenerate and in nerves in which regeneration was prevented by suturing. The nerves prevented from regenerating showed markedly atrophied Schwann cell columns after 20 weeks and a disappearance of some Schwann cell columns after 30 weeks. The surrounding endoneurial fibroblast-like cells gradually lost their delicate cytoplasmic extensions and formed rough fascicles around numerous shrunken Schwann cell columns or around areas from which Schwann cells had apparently disappeared. Inside the fascicles, the Schwann cell loss was replaced by collagen fibrils or occasionally, by a dense accumulation of microfibrils. The loss of endoneurial cytoplasmic processes continued up to 50 weeks, leaving behind patches of thin fibrils around numerous shrunken Schwann cell columns or around collagenous areas where Schwann cells were lost. The endoneurial matrix showed presence of thin 25- to 30-nm collagen fibrils close to shrunken Schwann cell columns up to 50 weeks but in areas with advanced degeneration a shift towards regular 50- to 60-nm collagen fibrils occurred. The degenerated areas resembled those described in Renaut bodies and neurofibromas. Despite suturing of transected nerves to prevent sprouting, occasional regenerating sprouts were noted in the Schwann cell columns. These axons were surrounded in a sheath-like fashion by pre-existing endoneurial cell fascicles covered by a basal lamina. In the reinnervating nerves the endoneurial space gradually lost its compartmentized structures consisting of collagen fibrils and endoneurial fibroblast-like cells. After 20 weeks the endoneurial cells were inconspicuous and the extracelluar matrix consisted mainly of 50- to 60-nm collagen fibrils. During axonal growth and maturation, Schwann cells containing unmyelinated axons surrounded large, myelinated axons in a collar-like fashion. Close to these collars of Schwann cells, thin 25- to 30-nm collagen fibrils were noted in focal areas, even after 50 weeks. Occasionally, numerous clusters of regenerating axonal sprouts were noted in the perineurium. These were surrounded by multiple layers of cells possessing basal lamina. The present results show that after nerve transection the distal stump of the severed nerve shows dynamic changes in the endoneurial space, especially in nerves where reinnervation is prevented. The endoneurial fascicles around occasional axonal sprouts in sutured nerves, representing possibly a delayed type of regeneration, show that axons have a strong ability to grow but on the other hand endoneurial structures are unable to respond normally to axonal growth after advanced degeneration.     

Degeneration in the cochlear nerve of the rat following cochlear lesions

Left unilateral cochlear lesions were performed on 26 albino rats at 1.5 months of age. After survival times ranging from 1 h to 6 months, the animals were perfused via the aorta with mixed aldehydes. Blocks including the cochlear nerves were removed, embedded in Araldite, sectioned in a plane transverse to the longitudinal axis of the nerve, and analyzed in the light microscope. Degenerating fiber profiles were grouped into 4 categories, and their relative frequencies were counted, as were numbers of normal fibers and glial cell nuclei. The cross-sectional areas of the nerves were measured. Lesion extent was evaluated by means of sections through operated cochleas from short and long survival times, and right cochlear nerves from 11 of the animals were used as controls. In the left nerves, segmental swelling of fibers occurred as early as 16 h survival, followed by collapse of fibers and breakdown of myelin sheaths. Starting at 36 h survival, increased numbers of glial cells were seen in the nerve. At longer survival times there were decreases in the cross-sectional area of the nerve and in the packing density of degenerating fiber profiles. At the longest survival times, a substantial amount of debris remained which resembled that seen in early stages. Finally, there was evidence of continued loss of nerve fibers occurring over a period of weeks to months.     

神经型布氏杆菌病周围神经损害的临床与电生理研究

目的研究神经型布氏杆菌病周围神经损害的临床特征,探讨电生理对其的诊断价值。方法对32例神经型布氏杆菌病周围神经损害患者(病例组)和32名性别及年龄与病例组匹配的正常对照组进行神经电生理检查,并对所得检查结果进行统计学分析。结果病例组与对照组在运动末梢潜伏期(distal motor latency,DML)、复合肌肉动作电位(compound motor active potentials,CMAP)波幅、运动神经传导速度(motor nerve conduction velocity,MCV)、感觉神经动作电位潜伏期(sensory nerve action potential latency,SL)、感觉神经动作电位(sensory nerve action potential,SNAP)波幅及感觉神经传导速度(sensory nerve conduction velocity,SCV)方面的比较,差异均有统计学意义(P0.05)。电生理检查提示上下肢周围神经损害,感觉神经及运动神经均受累,其中感觉神经占55.47%,运动神经占16.80%,上肢以正中神经(64条)最多见,下肢以腓肠神经(16条)最多见。四肢运动神经256条中43条传导速度减慢,占16.80%,四肢感觉神经256条中142条传导速度减慢,占55.47%,SCV较MCV改变明显,上肢病变重于下肢。结论神经电生理检查为神经型布氏杆菌病周围神经损害的临床诊断提供了客观依据。     

Novel purinergic sensitivity develops in injured sensory axons following sciatic nerve transection in rat

Teased fibers were made from 153 spontaneous A afferents ending in sciatic nerve end neuromas of 3-14 days standing, 21 A afferents from intact sensory endings in the contralateral sciatic nerve, and 50 intact A afferents from the sciatic nerve in intact rats. Ninety-two percent of the injured fibers responded to adenosine 5'-triphosphate (ATP) (i.v.). However, few fibers from the contralateral nerve or nerves from intact animals responded to ATP. P2 receptor antagonist suramin or reactive blue 2 blocked the ATP-induced response in 76% of the fibers tested, whereas the P1 receptor antagonist aminophylline blocked the ATP-evoked effect in only 18% of the fibers tested. Sympathectomy did not affect the ATP-induced effects in injured axons. Close-arterial injection of ATP caused similar results as i.v. injection of ATP. The present study suggests that a novel purinergic sensitivity is developed at the injury site after sciatic nerve transection in rats, which may play a role in neuropathic pain under some conditions such as sympathetic activation.     

Value of nerve biopsy in the diagnosis and follow-up of leprosy: the role of vascular lesions and usefulness of nerve studies in the detection of persistent bacilli

Nerve biopsy specimens from 53 patients with leprosy and neuropathy were taken from the sural, the dorsal branch of the ulnar, or the superficial radial nerves and processed for light and electron microscopy. There was inflammation in 40 cases (75%), 7 with a granulomatous reaction, various stages of fibrosis in 35 (66%), and endoneurial vascular neoformation in 7. In two cases, small focal infarcts were associated with marked endoneurial inflammation compressing the vessels, in addition to endoneurial lymphocytic vasculitis. Most had an axonal neuropathy of varying degree, some with total fibre loss, others with predominant small myelinated and unmyelinated fibre loss. Signs of demyelination and remyelination were the main findings in 9 cases (17%). Bacilli were present in endothelial, perineurial, Schwann cells and in macrophages. On two occasions, they lost their alcohol acid resistance, were suspected in semithin sections, and confirmed ultrastructurally. The biopsy was decisive for the diagnosis of leprosy in 15 cases (28%), most without skin lesions. We evaluated the effectiveness of the treatment in 20 (37.7%), 12 without and 8 with bacilli, despite negativity in the skin. The diagnosis of leprosy based on skin lesions was confirmed with the nerve biopsy in 9 cases, 6 had an inflammatory neuropathy suggestive of leprosy in the absence of bacilli, and 3 had nonspecific changes in the sural nerve since the neuropathy was in the upper limbs. We conclude that nerve biopsy is indicated for the diagnosis of leprosy in cases without clinically visible skin lesions and to evaluate the effectiveness of the treatment. In these cases the ultrastructural studies are important for recognition of the bacilli. Vascular lesions may play an important role in the progression of the nerve damage, including the occurrence of focal nerve infarcts which, to our knowledge, have not been previously reported in association with leprosy. Received: 8 December 1995 Received in revised form: 8 December 1996 Accepted: 13 January 1997