The distribution of acetylcholine in normal and in regenerating nerves

1. The distribution of acetylcholine (ACh) in various nerves which had been regenerating for different periods after crushing has been compared with that in uncrushed nerves.2. Normal ventral roots from cats contained 78.1 +/- 22.7 (S.D.) mumole ACh/kg (blotted wet wt.); rabbit ventral roots contained 48.0 mumole/kg +/- 19.0 (S.D.) and rabbit sciatic nerves 16.6 +/- 7.3 (S.D.) mumole/kg. In the sciatic nerves the distal cm of 5 cm lengths taken from the thigh contained 30% more ACh than the most central cm portion. Possible explanations for this difference have been discussed.3. After both sciatic nerves and ventral roots had been crushed, there was an initial build-up (4 times control) of ACh central to the lesion and a decline ((1/4) control) distal to the lesion. These changes were maximum around 5 days after crushing. In sciatic nerves in which long periods of regeneration were investigated, the central build-up fell off to 1(1/2) times control by 25 days and the distal decline reversed to 2 times control in about 10 days. It then again decreased towards the control level by 25 days after crushing. These changes have been discussed in relation to the morphological changes which occur in a nerve following crushing.4. A peak of ACh content moved distally along the nerve from the crushed region at a rate of 1.0-1.5 mm/day. This was considered to represent an average rate of regeneration of the bulk of the axons. The amplitude of the peak declined progressively with time in the more distal parts of the nerve, probably because of dispersion as axons regenerated at different rates.     

Treatment of the neuromuscular junction with 4-aminopyridine results in improved reinnervation following nerve injury in neonatal rats

During early postnatal development, nerve injury results in the death of a large proportion of motoneurones and poor recovery of muscle function. Our previous results have shown that premature enhancement of transmitter release from nerve terminals prevents the death of motoneurones following neonatal nerve injury. Whether this increase in motoneurone survival is reflected in an improvement in the reinnervation of muscle was studied here. The muscles in one hindlimb of newborn rats were treated with 4-aminopyridine. Three days later, the sciatic nerve was crushed in the treated leg. When the animals were seven, 14 and 21days of age, the soleus and extensor digitorum longus muscles were removed and processed for GAP-43 (a 43-kDa growth-associated protein) and synaptophysin immunocytochemistry. Both GAP-43 and synaptophysin were expressed in normal soleus and extensor digitorum longus muscles at seven days. Synaptophysin was still expressed at 14 days, but GAP-43 expression had declined. Following nerve injury at three days of age, there was no GAP-43 or synaptophysin immunoreactivity in nerve terminals at seven days. By 21 days, there were 17.3+/-2.1 GAP-43-positive terminals per section in the soleus and 17.7+/-1.4 in the extensor digitorum longus, with mean terminal areas of 47.5+/-3.3 and 49.8+/-2.6 microm(2), respectively. In animals in which nerve crush was preceded by 4-aminopyridine treatment, at 21 days there were 32.9+/-2.6 GAP-43-immunoreactive terminals in the soleus and 44.9+/-2.3 in the extensor digitorum longus, with a mean area of 122.7+/-6.6 microm(2) in the soleus and 136.2+/-9.7 microm(2) in the extensor digitorum longus. These results indicate that in muscles pretreated with 4-aminopyridine, prior to nerve crush at three days, there are significantly more terminals, which occupy a larger area than in untreated muscles. Thus, increasing transmitter release prior to nerve injury significantly improved the ability of axons to reinnervate muscle.     

Dynamics of repair regeneration of rat cutaneous nerves after traumas of different severity

Studies on 163 mongrel with experimental crush trauma to cutaneous nerves addressed the dynamics of regeneration over a period of 10-50 days post-trauma. Two series of experiments were performed, in which a cutaneous nerve (the saphenous nerve) was crushed with a hemostatic clamp over lengths of 2 and 4 mm, respectively. Destructive processes in the L3 and L4 spinal ganglia, increases in the numbers of myelin fibers in the nerve distal to the trauma site and the rate of growth of the damaged nerve fibers towards the skin after nerve traumas of different lengths, recorded at 10-50 days, were identical in the two series. The rate of myelinization of regenerating fibers after 2-mm crush lesions was greater than that seen after 4-mm crush injuries only in the period up to 30 days post-trauma.     

大鼠坐骨神经压榨损伤后早期降钙素基因相关肽的变化

目的：研究大鼠坐骨神经压榨损伤后早期降钙素基因相关肽(CGRP)的动态变化及与神经再生的关系。方法：SD大鼠坐骨神经压榨损伤后分别存活1d到21d,免疫组化技术观察CGRP分布和含量的变化。结果：(1)1d组神经CGRP大量堆积,压榨近端明显多于远端,随即下降,21d组基本消失。(2)1d组背根节、脊髓后角和前角CGRP开始增高,并分别在3～5d、5～7d和7d组达峰值,随后渐降,21d组脊髓前角CGRP阳性运动神经元仍明显高于假手术组和对照侧。结论：神经压榨损伤后CGRP表达变化呈明显的时空模式,可能参与了神经元保护并介导了损伤信号的传导。     

Influence of peripheral inflammation on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia and on nerve recovery after crush injury

An experimental study was performed to investigate the influence of the inflammation in peripheral target tissue on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia (DRG) and on recovery of crushed nerve. Fifty-four male Wistar rats were used for this study. The sciatic nerve was operatively crushed unilaterally with an aneurysm clip. Inflammation in peripheral target tissue was induced by injection of complete Freund's adjuvant (CFA) at 1 week before crush. In crushed or crushed with arthritis rats DRGs were examined in immunohistochemistry for GAP-43 and the sciatic nerves were observed in Epon embedded sections with toluidine blue stain. In addition, electrophysiological studies of the nerves were performed to evaluate the recovery of function. Immunohistochemical studies showed the ratio of GAP-43 immunopositive cells in crushed with arthritis rats was significantly lower than that in crushed rats at 1 week after crush (P<0.01). Electrophysiological studies at 4 weeks after crush showed functional nerve recovery in crushed with arthritis rats was significantly suppressed compared with that in crushed rats (P<0.01). Histological studies showed the mean diameter of the axons in crushed with arthritis rats was significantly smaller than that in crushed rats (P<0.01). All these findings indicate that inflammation in peripheral target tissue suppresses GAP-43 expression in DRG and eventually suppresses functional and morphological recovery of the crushed nerve.     

Changes in slow axonal transport of tubulin induced by local application of colchicine to rabbit vagus nerve

Archer , D. R., Dahlin , L. B. & MCLEAN, W. G. 1994. Changes in slow axonal transport of tubulin induced by local application of colchicine to rabbit vagus nerve. Acta Physiol Scand 150, 57–65. Received 15 April 1993, accepted 30 July 1993. ISSN 0001–6772. Department of Pharmacology and Therapeutics, University of Liverpool, UK and Department of Hand Surgery, Lund University, Malmo, Sweden. The biochemical and morphological responses of the rabbit vagus nerve to local application of colchicine and to nerve crush were investigated. Fourteen days after the cervical vagus nerve had been crushed or subjected to local application of colchicine for 2 h, nodose ganglia of anaesthetized rabbits were either injected with [³⁵S]methionine or [³H]leucine for studies of slow and fast axonal transport, respectively, or prepared for light microscopical examination. The radio-labelled proteins of the faster of the two slow transport groups (SCb; 25–30 mm day¹) were separated by one- or two-dimensional polyacrylamide gel electrophoresis and both radio-labelled tubulin and actin were quantified by densitometry from resulting fluorographs of gels. A relative increase in radio-labelled tubulin was found in SCb in the crushed and colchicine-treated nerves; this increase persisted for up to 50 days after nerve crush. Morphological changes in nerve cell bodies induced by colchicine were similar, but smaller in magnitude than those in crushed nerves. It is concluded that a temporary arrest of axonal transport produced by colchicine can lead to a redistribution of tubulin transport comparable with that found in regenerating nerve.     

Recovery of functional response in the nucleus of the solitary tract after peripheral gustatory nerve crush and regeneration.

Single-unit recording and transganglionic tracing techniques were used to assess the properties of, and inputs to, neurons within the rostral nucleus of the solitary tract (NST) after peripheral gustatory nerve injury and regeneration in adult hamsters (Mesocricetus auratus). Tastant-evoked responses were recorded from 43 neurons in animals in which the ipsilateral chorda tympani (CT) nerve was crushed 8 wk earlier (experimental animals) and from 46 neurons in unlesioned control animals. The 89 neurons were separated into three functional clusters named according to the best stimulus for neurons in the cluster: S, sucrose; N, sodium acetate; and H, HCl or KCl. Stimulus-evoked spike rates across all stimuli were 35.4 +/- 4.4% lower in the experimental hamsters. The largest difference in evoked spike rates occurred for neurons in the H cluster, in which the response to KCl also was delayed relative to normal responses. The response of S-cluster units to sucrose and saccharin was also lower in the experimental animals. The mean response rate and the time course of response of neurons in the N cluster did not differ between the two groups. For each cluster, the spontaneous rates and mean response profiles across eight stimuli were very similar in the experimental and control animals, and the breadth of tuning hardly differed. In both groups, Na+ responses in the N cluster were amiloride sensitive, and responses to the water rinse after stimulation with HCl were common in the S cluster. At 8-20 wk after nerve crush, biotinylated dextran tracing of the CT nerve revealed that the regenerated CT fibers did not sprout outside the normal terminal zone in the NST, but the density of the central terminal fibers was 36.9 +/- 6.35% lower than normal. After CT nerve crush and regeneration, the overall reduction in taste-evoked spike rates in NST neurons is likely a consequence of this change in terminal fibers; this in turn likely results from the known reduction in CT fibers regenerating past the crush site. In the face of this reduction, the normal taste-evoked spike rate in N-cluster neurons requires explanation. The observed recovery of normal specificity could be mediated by a restoration of specific connections by primary afferent fibers peripherally and centrally or by central compensatory mechanisms.     

Environmental factors contribute to the enhanced regeneration of frog sciatic sensory axons by a conditioning lesion

The outgrowth of regenerating frog sciatic sensory axons after a crush lesion was measured from the distribution of axonally transported radioactive proteins. Five days after a single test crush the regeneration distance was 3.6 +/- 0.3 mm. If the nerve was subjected to a conditioning lesion 15 mm distal to and 5, 10 or 17 days prior to the test crush, there was an increased outgrowth distance (5.1 +/- 0.2 mm) 5 days after the test crush when the conditioning interval was 10 days. Prolongation of the time interval between the two lesions to 17 days did not significantly change the enhanced outgrowth. In contrast, if the conditioning and test lesions were applied at the same site, the regeneration distance 5 days after the test crush was already significantly increased after a conditioning interval of 5 days (5.9 +/- 0.5 mm). It was further enhanced after an interval of 10 days (7.8 +/- 0.5 mm) and yet further after 17 days (9.2 +/- 0.5 mm). When the conditioning and test lesions were superimposed, the regeneration occurred through a region of the nerve that was pre-degenerated due to the conditioning lesion. The positive correlation between the degree of degeneration and the outgrowth distance supports the theory that environmental factors contribute to the regenerating ability of peripheral nerves.     

Problems with the use of the toe-spreading reflex in rats as an assay in nerve regeneration studies

Stages in the return of the toe-spreading reflex after sciatic nerve injury were examined using the rat. It was found that the earliest stages in the return of the reflex do not indicate nerve regeneration, but rather reflect the development of adrenalin sensitivity in denervated muscles. Probably systemic adrenalin released during the reflex-testing procedure causes muscle contractions which imitate a nerve-induced toe-spread reflex.     

Re-innervation and recovery of rat soleus muscle and motor unit function after nerve crush

In this study we have investigated the effects of peripheral nerve crush on the contractile properties of the adult rat soleus muscle. The soleus nerve was crushed close to the muscle and functional re-innervation was assessed by the measurement of the force produced by contraction induced by electrical nerve stimulation. Whole soleus muscle and single motor unit (MU) properties were studied at increasing re-innervation times 7-56 days after crush. Results showed progressive re-innervation as 50% of the axotomized motoneurones had re-innervated their muscle 7 days after crush, 72% at 14 days and re-innervation was complete at 28 days. The force parameters recovered more slowly. Tetanic contractions faded at high frequency stimulation, which did not occur in the control muscle. This disruption in the tetanic response was more pronounced in single MUs. Our results demonstrate for the first time a process of progressive axonal re-innervation by the axotomized motoneurones and provide a functional picture of the effective restoration of the neuromuscular function.     

Origin of sympathetic innervation of the knee joint in the cat: a retrograde tracing study with horseradish peroxidase

In the cat the origin of sympathetic nerve fibers in the medial and posterior articular nerve (MAN and PAN) of the knee joint was studied using retrograde labeling with horseradish peroxidase (HRP). Three to 5 days after uptake of HRP by MAN per cat 404 +/- 136 labeled somata (mean +/- S.D., n = 4 cats) were found mainly in the paravertebral ganglia L4 and L5 of the ipsilateral sympathetic trunk, and uptake of HRP by PAN labeled per animal 532 +/- 155 cell bodies (mean +/- S.D., n = 4 cats) mainly in the paravertebral ganglia L5 and L6. The cross-sectional areas of the labeled somata ranged from 200 to 1300 microns 2 with a mean of about 620 microns 2.     

Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve

Adhesion molecules are important in supporting axonal regeneration. Qualitative studies have described increased expression of neural cell adhesion molecule (NCAM) and N-cadherin in models of nerve injury allowing active regeneration. In this study we have used quantitative immunohistochemistry to compare expression of NCAM and N-cadherin after nerve injury either with active regeneration (crush) into the distal stump or without (axotomy and capping). Quantification was performed 15 days after axotomy in proximal and distal stumps. Quantification after crush either proximal, distal or within the crushed area was performed at 2, 7, 15 and 30 days after injury. Axotomy induced increases in expression in proximal stumps between two and three times those in uninjured nerves for both molecules. In distal stumps, N-cadherin levels increased seven-fold, yet NCAM levels did not exceed control values. After crush, NCAM immunoreactivity increased in the crushed area and distal stump in contrast to axotomy and NCAM-positive axons co-localized with PGP9.5. N-cadherin levels in the distal stump increased above control levels, but the magnitude of the increase seen after crush was different to those seen after axotomy. In conclusion, increased expression of adhesion molecules, particularly NCAM, in the distal stump of injured nerves is dependent upon the presence of regenerating axons.     

Effects of ageing on spinal motor and autonomic pain responses

The course of ageing leads to various changes in the nervous system, which can affect pain processing in the elderly. However, the affection of different components of the nociceptive system remains unclear. To investigate basic nocifensive responses, we compared age-related changes of autonomic and motor reflex responses to noxious electrical stimulation. In 39 healthy young subjects (mean +/- S.D.; 24.1 +/- 3.3 years) and 52 healthy elderly subjects (mean +/- S.D.; 71.9 +/- 5.3 years) the nociceptive flexion reflex (NFR) and the sympathetic skin response (SSR) were determined using noxious electrical stimulation of the sural nerve. Verbal pain ratings were assessed in addition. No ageing effects on the NFR and on verbal pain ratings were found, whereas the SSR amplitude declined significantly with ageing. Since both SSR and NFR share comparable primary afferent pathways and the motor as well as the subjective responses to noxious stimulation were preserved, our data seem to suggest that central or peripheral efferent sympathetic functions are altered by age.     

Myelination of regenerated axons in goldfish optic nerve by Schwann cells

Summary This study uses immunohistochemistry and EM to examine the site of injury in goldfish optic nerve during axonal regeneration. Within seven days of nerve crush axons begin to regrow and a network of GFAP⁺ reactive astrocytes appears in the nerve on either side of the injury. However, the damaged area remains GFAP^–. By 42 days after nerve crush, the sheaths of new axons acquire myelin marker 6D2, and the crush area becomes populated by a mass of longitudinally-orientated S-100⁺ cells. Ultrastructurally, the predominant cells in the crush area bear a strong resemblance to peripheral nerve Schwann cells; they display a one-to-one association with myelinated axons, have a basal lamina and are surrounded by collagen fibres. It is proposed that these cells are Schwann cells which enter the optic nerve as a result of crush, where they become confined to the astrocyte-free crush area.     

Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve

Basic and acidic fibroblast growth factors (FGF) were implanted next to the proximal stump of the transected optic nerve of adult rats, in order to assess whether these molecules have neurotrophic activity in vivo. Of the 119,973 +/- 2484 (S.E.M.) retinal ganglion cells present in retinae of unoperated control rats, 11,375 +/- 2413 (S.E.M.) remained at 30 days after transection of the optic nerve in control operated rats. After implantation of gel foam soaked in basic FGF, the number of retinal ganglion cells surviving at 30 days after axotomy tripled (36,387 +/- 3270 (S.E.M.], after acidic FGF, it increased almost 4-fold (40,916 +/- 5405 (S.E.M.]. These results indicate that FGF has neurotrophic activity in the adult central nervous system, and that this molecule is able to rescue adult retinal ganglion cells from axotomy induced cell death. It remains to be shown whether FGF acts directly on retinal ganglion cells or indirectly via glial cells or other cells.     

Uptake and retrograde axonal transport of horseradish peroxidase in regenerating facial motor neurons of the mouse

Summary Uptake and retrograde axonal transport of intravenously injected horseradish peroxidase (HRP) was studied during regeneration after a crush injury of the facial nerve of the mouse. The circulation time of HRP was 12 to 24 h. HRP injected immediately after the crush diffused into injured axons in the crushed region and accumulated subsequently in perikarya of facial neurons in the brain stem. After a time interval of 1 h or 5 days between the crush and the injection only a faint HRP accumulation occurred in a few facial neurons. After an interval of 7 days a moderate number of neurons had incorporated the tracer, while after more than 9 days the HRP activity in the regenerating neurons was more pronounced than in the contralateral neurons. Ultrastructurally, muscles of the vibrissae showed denervated subneural apparatuses 6 days after the crush. 8 days after the crush regenerating axon terminals containing small clusters of synaptic vesicles, dense cored vesicles and some HRP-labelled vesicles, were found over some gutters and after 10 to 13 days all examined gutters contained axon terminals with large numbers of synaptic vesicles and some HRP-containing vesicles. More than one axon terminal profile was seen in the same synaptic gutter. 32 and 64 days after the crush the neuromuscular junctions had regained a more mature appearance. The calibre spectra of the crushed facial nerves still showed a shift towards smaller diameters 134 days after the crush, at a time when a slight increase in HRP activity in the facial neurons persisted.     

大鼠骶神经钳夹伤致神经源性膀胱模型的建立及评估

目的建立并评估大鼠骶神经钳夹伤后神经源性膀胱模型。方法将25只健康雌性大鼠随机分为对照组(n=10)和钳夹伤组(n=15)。钳夹伤组对大鼠双侧骶2神经进行钳夹伤,对照组仅暴露双侧骶2神经。造模后观察大鼠自主排尿情况,4周后检测尿流动力学指标,并对膀胱和骶神经进行组织形态学观察。结果造模48 h后钳夹伤组大鼠下腹部膀胱充盈,自主排尿受限,对照组大鼠可自主排尿。造模4周后钳夹伤组大鼠的最大膀胱容量大于对照组,差异具有统计学意义(P<0.05);钳夹伤组大鼠漏尿点压及膀胱内压高于对照组,差异具有统计学意义(P<0.05)。造模4周后钳夹伤组大鼠的膀胱明显胀大,HE染色膀胱壁肌层厚度小于对照组,差异具有统计学意义(P<0.05)。结论大鼠双侧骶2神经钳夹伤可建立神经源性膀胱模型,造模方法简单易行且模型稳定。     

Comparison of blood-nerve barrier disruption and matrix metalloprotease-9 expression in injured central and peripheral nerves in mice

To investigate the involvement of blood-born factors and extracellular proteases in axonal degeneration and regeneration in both PNS and CNS, we directly compared the differences of blood-nerve barrier (BNB) disruption and matrix metalloprotease-9 (MMP-9) induction between the sciatic nerve and optic nerve after crush injury in the same animal. In sciatic nerve, BNB disruption, fibrin(ogen) deposition and MMP-9 expression were observed only in the first week following injury. Neurofilament (NF) immunoreactivity dramatically decreased in the first 2 days, gradually recovered to the normal levels by day 28. In contrast, the immunoglobulin G deposits spanned from 4 h to 28 days in crushed optic nerves. Fibrin(ogen) deposition was only observed in the first 2 days, while MMP-9 induction did not occur until a week after injury but lasted for 3 weeks in the crushed optic nerves. The NF immunoreactivity did not change much until day 7 and almost completely disappeared on day 28. The decrease of NF immunoreactivity coincided with the induction of MMP-9 after optic nerve crush. These results show that BNB disruption and MMP-9 induction are differentially regulated in the PNS and CNS after injuries, and they may contribute to the different regeneration capacities of the two systems.     

Comparative study of the neuronal plasticity along the neuraxis of the vibrissal sensory system of adult rat following unilateral infraorbital nerve damage and subsequent regeneration

 The aim of the present study was to examine the physiological consequences of a unilateral infraorbital nerve lesion and its regeneration at different levels of the somatosensory neuraxis. In animals whose right infraorbital nerve had been crushed, a large unresponsive area was found in the main brainstem trigeminal nucleus (Pr5). Responses evoked by ipsilateral vibrissal deflection in the middle of Pr5 reappeared only on days 22–35 after the nerve had been transected, whereas recovery from the nerve crush took only 7–9 days. However, no sign of short-term neuronal plasticity was observed in Pr5 after peripheral nerve injury. An enlargement of the receptive fields in two-thirds of the units and a lengthening in the delay of the evoked responses were observed as long-term plastic changes in Pr5 neurons after peripheral-nerve regeneration. In the ventral posteromedial nucleus of the thalamus (VPM) of partly denervated animals, however, only minutes or hours after the nerve crush, certain units were found to respond in some cases not only to the vibrissae, but also to mechanical stimulation of the face over the eye (two units), the nose (one unit), and the midline (one unit). Apart from the experiments involving incomplete denervation, the vibrissal representation areas of the VPM were unresponsive to stimulation of both the vibrissae and other parts of the face until nerve regeneration had occurred. In the somatosensory cortex, an infraorbital nerve crush immediately resulted in a large cortical area being unresponsive to vibrissal deflection. It was noteworthy, however, that shortly after the nerve crush, this large unresponsive whisker representation cortical area was invaded from the rostromedial direction by responses evoked by stimulation of the forepaw digits. In spite of the reappearance of vibrissa-evoked responses 7–10 days after the nerve crush, an expanded digital representation could still be observed 3 weeks after the nerve crush, resulting in an overlapping area of digital and vibrissal representations. The withdrawal of the expanded representation of forepaw digits was completed by 60 days after the nerve crush. The results obtained in Pr5, the VPM, and the cortex strongly suggest that the higher the station in the neuraxis, the greater the degree of plasticity after infraorbital nerve injury. Received: 28 May 1998 / Accepted: 5 January 1999     

The reflex bradycardia during brain ischemia in the rabbit.

We examined the responses of whole aortic nerve activity, aortic baroreceptor activity, heart rate (HR), and arterial pressure (AP) to brain ischemia sustained for approximately 30 s in anesthetized spontaneously breathing rabbits. The minimum values of HR observed during brain ischemia were 76 +/- 11 beats/min (mean +/- S.E., n = 14) before sectioning the bilateral aortic nerve (BAN), and 161 +/- 12 beats/min after sectioning the left aortic nerve (LAN), and 225 +/- 11 beats/min after sectioning the LAN and right aortic nerve (RAN). Averages for reflex fall in HR during BAN, LAN, and RAN activation were 140 +/- 9, 78 +/- 7, and 62 +/- 7 beats/min, respectively, by subtracting the HR fall responses to brain ischemia in the absence of aortic baroreceptor afferents from their control values. The heights of the integrated whole left and right aortic nerve activities in systole slightly increased during brain ischemia, whereas the brain ischemia remarkably increased those activities during the diastolic phases. The brain ischemia induced a hysteresis in the mean AP-aortic barorecept or activity relationship. These results suggest that the total activity of aortic nerve fibers would determine the bradycardia evoked by brain ischemia and that the difference between the relative contributions of LAN and RAN on the brain ischemia-induced reflex bradycardia would reflect the total impulse frequency of aortic myelinated and non-myelinated fibers.