Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48+/-3% (n = 5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94+/-4% (n = 5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48+/-3% (n = 5) to 35+/-4% (n = 5). Moreover, only 67+/-5% (n = 5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6+/-0.7 (n = 5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3+/-0.9 (n = 3) motor units were present. Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.     

Acetylcholine receptor and thymus in experimental autoimmune myasthenia gravis and experimental myositis.

This study was attempted to obtain information about biological properties of junctional acetylcholine receptor (AChR) and extrajunctional AChR, and about nerve influences on muscles AChRs under the pathological conditions of experimental myasthenia and myositis. Experimental autoimmune myasthenia gravis (EAMG) was induced in Wistar rats by immunizations with AChR purified from the electric organ of Narke Japonica without using Freund's complete adjuvant experimental myositis by immunization with rat muscle extract depleted of AChR. Thirty-five days after the initial immunization, unilateral dissection of the ischiadic nerve was performed in all immunized rats. Contents of AChR in both hind limb muscles were measured by double immunoprecipitation assay method 15 days after the experimental denervation. In the control animals the amount of AChR extractable from innervated muscles was 2.7 +/- 0.5 (mean +/- s.d.) pmole/g muscle and increased about 10-fold 15 days after the denervation (30 +/- 7.9). In rats with EAMG, AChR contents was reduced in both denervated (1.1 +/- 1.0) and innervated muscles (1.3 +/- 0.9). In experimental myositis, the increase of muscle AChR was impaired in denervated muscles (2.4 +/- 0.6), but AChR contents was not reduced in innervated muscles (2.7 +/- 0.9). These results suggest that nerves may influence AChR metabolism, keeping numbers of AChR constant even in inflammatory condition. In addition, germinal centre formation in thymic medulla was detected in EAMG rats.     

Long‐Term Effects of Neonatal Capsaicin Treatment on Intraepidermal Nerve Fibers and Keratinocyte Proliferation in Rat Glabrous Skin

Innervation is required to preserve several aspects of skin homeostasis. Previous studies in rodents have shown that sciatic nerve transection leads to epidermal thinning and reduced keratinocyte proliferation. As the sciatic nerve is composed of sensory and motor axons, it is not clear whether skin alterations reflect motor or sensory disturbances. In this study, we used neonatal capsaicin treatment to evaluate whether sensory chemical denervation affects keratinocyte proliferation at 1, 3, and 6 months of age. Using design‐based stereological methods, we estimated the total length of intraepidermal nerve fibers (IENF) that were of peptidergic type and the number of bromodeoxyuridine‐labeled (BrdU⁺) nuclei in the hind paw glabrous epidermis of control and capsaicin‐treated rats. We found that the treatment decreased the total fiber length of IENF immunoreactive for both protein gene product 9.5 (PGP⁺) and of IENF immunoreactive for calcitonin gene‐related peptide (CGRP⁺). The length of PGP⁺ fibers decreased by 83%, 81%, and 77% and that of CGRP⁺ fibers decreased by 48%, 58%, and 58% at 1, 3, and 6 months, respectively. Double‐immunofluorescence staining for neural beta III tubulin and CGRP revealed that the majority of the remaining fibers in the epidermis after capsaicin treatment were of peptidergic type. The number of BrdU⁺ nuclei was similar in both groups. Our findings suggest that IENF present after capsaicin treatment are sufficient to maintain epidermal replacement. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Reinnervation and recovery of mouse soleus muscle after long-term denervation

Reinnervation and recovery of the mouse soleus muscle were studied 2-10 months after denervation periods of about 7 months. To maintain denervation the right sciatic nerve was frozen 14 times at 2-week intervals. Though initially intermittent muscle reinnervation occurred, contractile force of denervated muscles was reduced to less than 10% of the contralateral muscles by the fifth nerve freezing and further declined thereafter. Following reinnervation, recovery of soleus muscle force proceeded slowly to reach plateau values after 5-6 months. Tetanic muscle force reached on average 72% (range 58-86%, n = 12) of contralateral muscles after 5-10 months, (P less than 0.01, t-test for absolute values) and 87% of unoperated animals after 10 months (P less than 0.05, n = 5). Muscle fibre diameters were significantly reduced in reinnervated muscles, but frequency distributions were normal and similarly shaped in reinnervated and control muscles, suggesting complete muscle reinnervation and the absence of denervated fibres even at 2 months of reinnervation. Total numbers of muscle fibres were similar in reinnervated (842 +/- 73 S.D., n = 15), contralateral (854 +/- 104 S.D., n = 15) and control soleus muscles (853 +/- 77 S.D., n = 5). The number of myelinated axons in regenerating soleus nerves reached control values by 3 months after the last freezing, continued to increase till 6 months (150% of control), and declined thereafter (125% at 9-10 months). In the contralateral soleus nerves the number of myelinated axons remained constant during this period. Nerve fibre diameters remained abnormally small; even after 10 months of reinnervation fibre diameters were unimodally distributed with a mean diameter of 3.3 microns in contrast to the bimodal distribution in intact nerves (mean values 3.9 and 9.0 microns, respectively). Total fibre cross-section area per nerve increased with time but reached only 54% +/- 6 S.D., (n = 3) of contralateral nerves by 10 months. The relative thickness of the myelin sheath (g-ratio) returned to normal after 9-10 months. Anatomically, muscle reinnervation appeared to be complete by 7-8 weeks since unusually small muscle fibre profiles were absent.(ABSTRACT TRUNCATED AT 400 WORDS)     

The formation of synapses in reinnervated and cross-reinnervated striated muscle during development

1. A study has been made of the formation of synapses in reinnervated and cross-reinnervated developing striated muscles which normally receive either a focal or distributed innervation, using histological, ultrastructural and electrophysiological techniques.2. The focally innervated mammalian tibialis anterior muscle, denervated soon after birth, was reinnervated at both the original end-plates as well as on the new muscle added during the period of denervation; but not on the muscle present at the time of denervation. Nearly all the synapses which had formed, other than at the original end-plates, disappeared by 6 weeks post-natal.3. The avian anterior latissimus dorsi muscle (ALD), which receives a distributed innervation, was denervated during the first week post-hatched, and became reinnervated both at the original synaptic sites as well as on the new muscle added during the period of denervation; all these synapses were spaced approximately 200 mum apart along the length of individual muscle cells.4. The myofibres of the ALD muscle cross-reinnervated at hatching with the superior brachialis nerve, which contains fast motor axons that normally form a focal innervation, were each focally innervated by a single ;en plaque' terminal; these synapses had the same electrical properties as normal synapses formed by fast motor axons.5. Many of the myofibres of the avian posterior latissimus dorsi (PLD), which normally receive a focal innervation, received a distributed innervation from ;en grappe' terminals when cross-reinnervated with the ALD nerve at hatching.6. It is suggested that during development the nerve type determines the pattern of synapses over an effector; this is achieved by the nerve, after forming the initial synaptic contact, making the rest of the muscle cell membrane refractory to further synapse formation for some distance, this distance being determined by the nerve type.     

脐带间充质干细胞移植可延缓大鼠失神经肌肉的萎缩

背景：周围神经断伤后生长缓慢,失神经支配的肌肉萎缩及运动终板纤维化,导致肢体功能不可逆障碍。脐带间充质干细胞已经广泛应用于多学科研究,但应用于周围神经损伤中延缓大鼠失神经肌肉萎缩鲜有报道。 目的：观察异种异基因脐带间充干细胞移植于大鼠离断坐骨神经断端,延缓失神经肌肉萎缩的效果。 方法：新鲜脐带采集于健康足月产妇,分离鉴定脐带间充质干细胞。制备大鼠坐骨神经SunderlandⅣ度损伤模型,去神经束5 mm,神经外膜修复,5 mm小间隙移植脐带间充质干细胞模型,对照组仅在小间隙内注入同体积生理盐水。测定大鼠坐骨神经功能指数,小腿三头肌湿质量,坐骨神经干潜伏期、动作电位传导速度、波幅,以及骨骼肌纤维横截面积维持率。 结果与结论：造模后4,8及12周,脐带间充质干细胞组大鼠坐骨神经功能指数、右侧小腿三头肌湿质量及骨骼肌纤维横截面积维持率均显著高于对照组(P < 0.05)。造模后12周肌电图显示,脐带间充质干细胞组大鼠坐骨神经干潜伏期显著低于对照组,动作电位传导速度及波幅显著高于生理盐水对照组(P < 0.001)。提示脐带间充质干细胞移植于大鼠离断的坐骨神经断端,可促进神经生长,延缓失神经肌肉萎缩,维持失神经肌肉形态及功能。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Effects of chronic stimulation on the size and speed of long-term denervated and innervated rat fast and slow skeletal muscles

This study seeks to identify the mechanisms which motoneurones use to control the contractile force and speed of skeletal muscles. We have stimulated directly slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of adult rats intermittently at 100 Hz for 1-9 months. The muscles were either chronically denervated, denervated and reinnervated, or normally innervated. The stimulation started either immediately, or more commonly, after 1-9 months of denervation. Stimulation starting several months after denervation increased the mean maximum tetanic tension 37 times in SOL and eight times in EDL. These values represented 40 and 12% of the increases obtained by reinnervation after comparable periods of time. In denervated SOL and EDL muscles stimulated directly for more than 2 months, the mean isometric twitch contraction times were 13 and 12.7 ms, as in normal EDL muscles (13 ms). In innervated SOL muscles stimulated directly for 1-4 months, the mean twitch contraction times were 23.6 ms (normally innervated) and 19.2 ms (reinnervated), which were considerably shorter than in normal control SOL muscles (39.2 ms). Single motor unit recordings revealed that the natural (background) nerve impulse activity was essentially unaffected by the stimulation. Twitch contraction time and percentage of type II fibres in SOL muscles were related. The fastest muscles (denervated and stimulated) consisted of 100% type II fibres (with one exception), the second fastest (reinnervated and stimulated) of 70-50%, the third fastest (normally innervated and stimulated) of 45-0%, the second slowest (reinnervated) of 15-0%, and the slowest muscles (innervated controls) of 5-0% type II fibres.     

Contribution of Endothelial Nitric Oxide Synthase in the Blunted Renal Responses to Volume Expansion in Diabetic Rats

The renal excretory responses to volume expansion (VE), by 10 % body wt, were determined in groups of anaesthetised streptozotocin-induced diabetic rats with one denervated and one innervated kidney in the presence and absence of nitric oxide synthase (NOS) inhibitors. VE in diabetic rats increased (P < 0.001) cumulative urine sodium excretion (CuU(Na)V) to 104 +/- 9 and 69 +/- 6 micromol min(-1) (g kidney wt)(-1) in the denervated and in the innervated kidneys, respectively, which were both less (P < 0.001) than in the non-diabetic rats, at 225 +/- 14 and 148 +/- 14 micromol min(-1) (g kidney wt)(-1), respectively, in the denervated and the innervated kidney. The non-selective NOS inhibitor, N(G)-nitro-L-arginine-methyl-ester (L-NAME) given to the diabetic rats with intact renal innervation enhanced CuU(Na)V after VE by 43 % (P < 0.001), while the combination of L-NAME and renal denervation restored CuU(Na)V to a value comparable to that of non-diabetic rats. In diabetic rats treated with either a relatively selective inhibitor for the neuronal isoform of NOS, 7-nitroindazole, or a relatively selective inhibitor for the inducible isoform of NOS, aminoguanidine, CuU(Na)V after VE was similar to the untreated diabetic rats irrespective of whether or not the renal nerves were present. This investigation demonstrated that NO production contributed, at least partly, to the depressed ability to excrete a saline load in diabetes mellitus. The endothelial isoform of NOS was most probably responsible for generating NO which caused the blunted excretory responses. The ability of NO to attenuate the excretory responses to volume expansion was an action independent of the renal innervation status. Experimental Physiology (2001) 86.4, 481-488.     

The adductor part of the adductor magnus is innervated by both obturator and sciatic nerves

The hip adductor group, innervated predominantly by the obturator nerve, occupies a large volume of the lower limb. However, case reports of patients with obturator nerve palsy or denervation have described no more than minimal gait disturbance. Those facts are surprising, given the architectural characteristics of the hip adductors. Our aim was to investigate which regions of the adductor magnus are innervated by the obturator nerve and by which sciatic nerve and to consider the clinical implications. Twenty‐one lower limbs were examined from 21 formalin‐fixed cadavers, 18 males and 3 females. The adductor magnus was dissected and was divided into four parts (AM1‐AM4) based on the locations of the perforating arteries and the adductor hiatus. AM1 was supplied solely by the obturator nerve. AM2, AM3, and AM4 received innervation from both the posterior branch of the obturator nerve and the tibial nerve portion of the sciatic nerve in 2 (9.5%), 20 (95.2%), and 6 (28.6%) of the cadavers, respectively. The double innervation in more than 90% of the AM3s is especially noteworthy. Generally, AM1–AM3 corresponds to the adductor part, traditionally characterized as innervated by the obturator nerve, and AM4 corresponds to the hamstrings part, innervated by the sciatic nerve. Here, we showed that the sciatic nerve supplies not only the hamstrings part but also the adductor part. These two nerves spread more widely than has generally been believed, which could have practical implications for the assessment and treatment of motor disability. Clin. Anat. 27:778–782, 2014. © 2013 Wiley Periodicals, Inc.     

Renal nerve blunts natriuretic and diuretic response to atrial natriuretic peptide in conscious rabbits

The contribution of the renal nerve to the natriuretic and diuretic responses to rat atrial natriuretic peptide (rAMP) was investigated in conscious rabbits with unilateral renal denervation. Renal nerve activity (RNA) was measured at the contralateral innervated kidney. Catheters were bilaterally implanted into the ureters. Urine samples were collected from each kidney by gravity drainage at 10-min clearance intervals. In rabbits with all baroreflexes intact, infusion of rANP at 0.3 micrograms/(kg.min) for 30 min decreased mean arterial pressure by 8 +/- 4 mmHg and increased RNA by 53 +/- 13%. After sinoaortic baroreceptor denervation (SAD), hypotensive response to infusion of rANP was greater than that in intact rabbits, while RNA did not change. After SAD plus vagotomy, infusion of rANP lowered mean arterial pressure by 21 +/- 4 mmHg and RNA by 19 +/- 6%. In the denervated kidney, infusion of rANP increased Na+ excretion by 16.1 +/- 4.5 from 3.5 +/- 1.0 muEq/min and water excretion by 0.17 +/- 0.05 from 0.08 +/- 0.02 ml/min. In the contralateral innervated kidney, infusion of rANP increased the amount of Na+ and water excretion by 4.5 +/- 3.2 muEq/min and 0.07 +/- 0.04 ml/min, which were significantly less than those in the denervated kidney. These results indicate that infusion of rANP increases RNA, due to baroreceptor reflexes, and that this increase in RNA blunts natriuretic and diuretic action of rANP.     

Collateral reinnervation and expansive regenerative reinnervation by sensory axons into “foreign” denervated skin: an immunohistochemical study in the rat

Summary Immunohistochemistry has been used to study, the capacity of different types of sensory axons in the saphenous nerve to extend into denervated glabrous skin territory after a chronic sciatic nerve lesion. In this study, the extension of the intact or regenerating thin peptidergic and coarse saphenous nerve fibres in adult and neonatal rats was determined. Substance P (SP) and calcitonin gene-related peptide (CGRP) antibodies were used as markers for thin axons and neurofilament (NF) antibodies for coarse axons. In addition, S-100 protein (S-100) antibodies, which primarily stain Schwann cells associated with myelinated axons, as well as innervated lamellated cells of Meissner corpuscles, were used. After a chronic sciatic nerve lesion in adult rats, thin dermal and epidermal SP-immunoreactive (IR) and CGRP-IR saphenous nerve fibres were present in an area lateral to that normally innervated by the saphenous nerve in the foot sole. In neonatally lesioned animals, thin dermal and epidermal SP-IR and CGRP-IR, as well as coarse dermal NF-IR fibres and S-100-IR cells, all of which derived from the saphenous nerve, were found in the sciatic nerve territory. In addition, some dermal SP-IR and CGRP-IR fibres were transiently present in the lateral part of the foot sole. After chronic sciatic nerve lesion and a concomitant crush injury of the saphenous nerve in adults or neonatals, thin dermal and epidermal SP-IR and CGRP-IR fibres, as well as coarse dermal NF-IR fibres and S-100-IR cells, were found in the innervation area normally occupied by the sciatic nerve. After a sciatic nerve cut and a concomitant crush injury of the saphenous nerve in adult rats, the SP-IR and CGRP-IR fibres, as well as the NF-IR fibres and S-100-IR cells were restricted to the medial part of this area. After a sciatic nerve cut and a concomitant crush injury of the saphenous nerve in neonatal rats, a few thin dermal SP-IR and CGRP-IR fibres were found in the lateral part of the foot sole as well. The findings of the present study together with those of previous morphological studies indicate that intact thin axons from the saphenous nerve, including those exhibiting peptide immunoreactivity, but not coarse saphenous axons, are capable of extending into foreign denervated glabrous skin after chronic sciatic nerve injuries. In neonatally sciatic-nerve-injured animals, both groups of axons spread from the intact saphenous nerve into the sciatic nerve territory. This was also the case when the saphenous nerve had been crushed and allowed to regenerate in rats injured neonatally, or as adults. However, judging from previous physiological data, the regenerating axons do not develop into functional low-threshold mechanoreceptors.     

Effect of sciatic denervation on cell-mediated immunity

Local semiallogenic graft-versus-host (GvH) and host-versus-graft (HvG) reactions were induced in mice whose right hind limbs had been partially denervated surgically. The denervation was performed by removing a 2 mm segment of the sciatic nerve, while sham-operated and non-operated rats served as controls. Eight days after denervation the mice were injected with spleen cells from 3-month-old C57BL female, or from (C57xCBA)F1 male mice, and the GvH and HvG reactions respectively were evaluated, 7 or 10 days later, by quantitatively estimating the weight (enlargement) of the popliteal lymph-nodes. Significant weight increases of 51% (for the GvH reaction) and 63% (for the HvG reaction) were observed in the denervated mice, when compared to the sham-operated ones. This increased cell-type reactivity in a denervated limbs seems to be related to the immunostimulatory activity of cytokines which are released by cells engaged in repair processes of damaged nerves, as well as related to the reduction of the immunosuppressive effect of noradrenergic innervation in the denervated animals.     

Effects of short-term denervation and subsequent reinnervation on motor endplates and the soleus muscle in the rat

The rat sciatic nerve was locally frozen, and changes in the nerve, motor endplates, and the soleus muscle were examined for up to 6 weeks by light and electron microscopy. The wet weights of denervated soleus muscles compared with contralateral values progressively declined to a minimum at 2 weeks after injury (60.7 +/- 2.5%) and began to reverse following 3 weeks. The sciatic nerve thoroughly degenerated after freezing. However, numerous regenerated myelinated and thin nerve fibers were observed at 3 weeks. They were considerably enlarged but still smaller than normal counterparts at 6 weeks postoperatively. Nerve terminals containing synaptic vesicles of endplates disappeared at day 1 and mostly reappeared at 3 weeks (about 70% of the endplates). All endplates examined were reinnervated at 4, 5, and 6 weeks. On the other hand, postsynaptic folds of muscle fibers seemed to be only slightly influenced by denervation or reinnervation. Ultrastructural alterations of myofibrils, in particular the loss of register, immediately appeared after denervation, spread progressively, peaked at 2 weeks, ameliorated following reinnervation, and became significantly normalized at 6 weeks after freezing. The proportion of type II fibers in the soleus muscle similary showed an increase and a decrease with a short delay in response to denervation and reinnervation, respectively. This study clearly demonstrated that the nerve supply affects the ultrastructural integrity of skeletal muscles. In addition, changes in the endplates and the soleus muscle evaluated in this study after short-term denervation are largely reversible following reinnervation.     

Systemic arterial baroreceptors in ducks and the consequences of their denervation on some cardiovascular responses to diving

1. In the duck systemic arterial baroreceptors which cause bradycardia in response to induced hypertension are located in the walls of the ascending aorta, innervated by the depressor nerves.2. The location of the baroreceptors was confirmed both histologically and by recording activity from the depressor nerve. Stimulation of the central cut end of a depressor nerve caused transient bradycardia and a fall in blood pressure which was maintained throughout the period of stimulation.3. Cardiovascular adjustments to submergence of 2 min duration were monitored in intact, sham-operated and denervated ducks. The sham-operated and denervated ducks were used in the experiments some 20-50 days post-operation. The denervations were checked at post-mortem.4. In the first series of experiments on young ducks mean arterial pressure during a 2 min dive fell by 30% in intact, 17.5% in sham-operated, and 48% in denervated ducks. In all ducks heart rate was reduced by 84-85%.5. In a second series of experiments on older ducks sciatic artery blood flow was also recorded and mean arterial blood pressure fell by 9.2% in intact and by 53% in denervated animals, although there were no significant differences in heart rate during the 2 min dives. In normal animals sciatic vascular resistance increased after 2 min submergence by 7.86 +/- 1.7 times, whereas in denervated ducks it increased by only 2.32 +/- 0.5 times.6. The role of systemic arterial baroreceptors in generation of the cardiovascular responses to submergence in ducks is discussed in terms of the input supplied by the baroreceptors to the central nervous system.     

Response of the granulation tissue to acetylcholine and noradrenaline during denervation

Granulation tissue from the wounds of back of both paws was studied in rats on 7th day of healing following crushing of right sciatic nerve and after repeated applications of 0.2 g/l of acetylcholine (Ach) and noradrenaline (NA). In control group (rats with intact nerves) applications of Ach intensified inflammatory reaction and negatively affected the state of regenerating skin tissues: the effect of NA consists in promoting fibroblastic and leukocytic reactions. Simultaneous crushing of right sciatic nerve and infliction of the wound amplified the pathologic reaction in the wounds of denervated rats as compared to that in animals who did not undergo denervation, leading to rapid ulceration. NA administration caused negative consequences in wounds both in right and left sides. Crushing of the nerve was performed 7 days prior to wound infliction and on day 7 of the study of neurodystrophic processes has shown that Ach influence on denervated and non denervated wounds was positive, while NA influence was negative. After reinnervation adreno- and cholinoreactivity of granulation tissue was partially normalized both on the right and left sides.     

Two factors responsible for the development of denervation hypersensitivity

1. Innervated adult skeletal muscle is sensitive to acetylcholine at the end-plate region only. After denervation the entire muscle membrane becomes chemosensitive. The period of greatest increase in sensitivity in rat soleus muscles following section of the sciatic nerve in the thigh is between 48 and 72 hr post-operatively.2. Direct electrical stimulation was found to prevent the onset of the development of denervation hypersensitivity during the first 2-3 days after nerve section. Thereafter, electrical stimulation only reduced the sensitivity of denervated muscles to acetylcholine (ACh).3. The period of greatest increase in sensitivity follows loss of transmission and degeneration of the nerve terminals. Once this degeneration is under way, electrical stimulation is no longer as effective in preventing the development of denervation hypersensitivity.4. Hypersensitivity is also seen in muscles on which a small piece of thread or degenerating nerve has been placed. Hypersensitivity following these procedures declines within a few days, unlike denervation hypersensitivity which persists until innervation is restored.5. The present results suggest that activity alone cannot prevent the development of hypersensitivity in the presence of degenerating nerve fibres, or muscle damage. Activity does however counteract increased sensitivity. It is suggested that two factors interact to produce denervation hypersensitivity; the presence of degenerating nerve tissue and concomitant cellular changes bring about changes in the muscle fibre membrane causing it to become hypersensitive; and the loss of muscle activity, resulting in the persistence of hypersensitivity until innervation is restored.     

Muscle spindle reinnervation following phenol block

Infusion of phenol into peripheral nerves is used clinically to manage spasticity. It produces relief of symptoms by chemical denervation. We simulated the clinical procedure by bathing the lateral plantar nerve of rats in 7% phenol solution for 20 min. We studied the innervation of muscle spindles in the plantar lumbrical muscles of untreated rats and in rats 4 and 6 weeks after a single phenol block. Spindles were identified by the immunoreactivity of nuclear bag(1) fibers to slow tonic myosin (antibody ALD 19). The integrity of the sensory and motor reinnervation of spindles was evaluated using a monoclonal antibody specific for a high molecular weight neurofilament protein. Four weeks after phenol block, muscle spindles were difficult to find, as their immunoreactivity to antibody ALD 19 was reduced. In those spindles studied, most (>80%) were completely denervated. The remainder of which were innervated by afferents only. None received efferent (gamma) innervation. After 6 weeks, spindles were readily identified and nearly all (>90%) received recognizable afferent innervation. A much smaller number (38%) received gamma innervation. Phenol block thus results in a complete denervation of muscle spindles, followed by a fairly rapid sensory reinnervation. Reinnervation by gamma motor neurons is either incomplete or significantly delayed.     

The sensitivity to glutamate of denervated muscles of the crayfish

The effect of denervation on the sensitivity of muscle fibres to glutamate was studied in leg muscles of crayfish.1. When the motor nerve was cut close to the proximal accessory flexor muscle the distal end of the nerve degenerated. Neuromuscular transmission failed and spontaneous miniature potentials disappeared after two months. Several stages of nerve terminal degeneration were seen in muscles denervated between 1 and 3 months, and after 4 months no remains of synapses could be found.2. Following denervation for periods up to 8 months there was no significant change in sensitivity to glutamate, a substance that mimics the action of the neural transmitter. Depolarizations produced by various concentrations of glutamate in the bathing solution were the same in denervated and control muscle fibres. Moreover, the sensitivity to iontophoretically applied glutamate was localized to discrete patches as in innervated muscles.3. Supersensitivity of muscles apparently does not occur after denervation in the crayfish.     

Non-equivalence of impulse blockade and denervation in the production of membrane changes in rat skeletal muscle.

1. A complete and long lasting blockade of nerve impulses was established in the sciatic nerve of rats, by implanting silastic cuffs of critical internal diameters. Either marcaine-impregnated or plain cuffs were used. The contralateral sciatic nerve was sectioned. 2. At various days after the initial procedures, the extensor digitorum longus muscles of the two sides were examined with intracellular electrodes. 3. Decrease in resting membrane potential, fibrillatory activity and resistance of the action potential to tetrodotoxin developed not only in the denervated but also in the impulse-blocked muscles. In the latter, the fibres were normally innervated since they displayed miniature end-plate potentials and were excitable by nerve stimulation distal to the blocking cuff. 4. However, all of the above mentioned denervation-like changes were significantly less pronounced in the blocked muscles than in the denervated ones. 5. It is concluded that in addition to loss of nerve impulses, some other neural factor must be taken into account to explain the origin of muscle changes induced by denervation. The possible relation of this additional factor with nerve degeneration is discussed.     

Remodeling of the vascular bed and progressive loss of capillaries in denervated skeletal muscle

Very little is known regarding structural and functional responses of the vascular bed of skeletal muscle to denervation and about the role of microcirculatory changes in the pathogenesis of post-denervation muscle atrophy. The purpose of the present study was to investigate the changes of the anatomical pattern of vascularization of the extensor digitorum longus muscle in WI/HicksCar rats 1, 2, 4, 7, 12, and 18 months following denervation of the limb. We found that the number of capillaries related to the number of muscle fibers, i.e. the capillary-to-fiber ratio (CFR), decreased by 88%, from 1.55 +/- 0.35 to 0.19 +/- 0.04, during the first 7 months after denervation and then slightly declined at a much lower rate during the next 11 months of observation to 10% of the CFR in normal muscle. Between months 2 and 4 after denervation, the CRF decreased by 2.4 times, from 58% to 24% of the control value. The loss of capillaries during the first 4 months following nerve transection was nearly linear and progressed with an average decrement of 4.16% per week. Electron microscopy demonstrated progressive degeneration of capillaries following nerve transection. In muscle cells close to degenerating capillaries, the loss of subsarcolemmal and intermyofibrillar mitochondria, local disassembly of myofibrils and other manifestations of progressive atrophy were frequently observed. The levels of devascularization and the degree of degenerative changes varied greatly within different topographical areas, resulting in significant heterogeneity of intercapillary distances and local capillary densities within each sample of denervated muscle. Perivascular and interstitial fibrosis that rapidly developed after denervation resulted in the spatial separation of blood vessels from muscle cells and their embedment in a dense lattice of collagen. As a result of this process, diffusion distances between capillaries and the surfaces of muscle fibers increased 10-400 times. Eighteen months after denervation most of the capillaries were heavily cushioned with collagen, and on the average 40% of the muscle cells were completely avascular. Devascularization of the tissue was accompanied by degeneration and death of muscle cells that had become embedded in a dense lattice of collagen. Immunofluorescent staining for the vascular isoform of alpha-actin revealed preservation of major blood vessels and a greater variability in thickness of their medial layer. Hyperplastic growth of the medial layer in some blood vessels resulted in narrowing of their lumens. By the end of month 7 after denervation, large deposits of collagen around arterioles often exceeded their diameters. Identification of oxidative muscle fibers after immunostaining for slow-twitch myosin, as well as using ultrastructural criteria, has shown that after 2 months of denervation oxidative muscle fibers were less susceptible to atrophy than glycolytic fibers. The lower rate of atrophy of type I muscle fibers at early stages of denervation may be explained by their initially better vascularization in normal muscle and their higher capacity to retain capillaries shortly after denervation. Thus, degeneration and loss of capillaries after denervation occurs more rapidly than the loss of muscle fibers, which results in progressive decrease of the CFR in denervated muscle. The change of capillary number in denervated muscle is biphasic: the phase of a rapid decrease of the CFR during the first 7 months after nerve transection is followed by the phase of stabilization. The presence of areas completely devoid of capillaries in denervated muscle and the virtual absence of such areas in normal muscle indicate the development of foci of regional hypoxia during long-term denervation. The anatomical pattern of muscle microvascularization changes dramatically after nerve transection. Each muscle fiber in normal muscle directly contacts on average 3-5 capillaries. (ABSTRACT TRUNCATED)