The histochemical study of rat sciatic nerve cholinesterase in degeneration and regeneration

With a modified, less time-consuming, Karnovsky-Roots method, this study evaluated the reduction and recovery of rat sciatic nerve cholinesterase (ChE) in degeneration and regeneration. Characteristics of motor fascicles included not only partial staining of acetylcholinesterase (AchE) in the axon, but also a small amount of more intense staining of butyrylcholinesterase (BuchE) in the unmyelinated fibers, scattered between the myelinated fibers in a spot or fleck form. Characteristics of sensory fascicles included more intense staining of BuchE in the unmyelinated fibers, (appearing as irregular balls or pieces), and a scattering between unstained myelinated fibers. Mixed fascicles had characteristics of both motor and sensory fascicles. Unmyelinated fibers exhibited BuchE activity, but myelinated fibers exhibited AchE activity. Preparation time for a specimen to react to ChE-positive staining was about 1 to 2 hr; the method is very suitable for diagnosis during surgery. After nerve transection, AchE activity at the distal nerve end began to reduce gradually; no AchE could be tested after two weeks. But BuchE activity in unmyelinated fibers could be found until 30 days after transection. After epineurial suturing of the peripheral nerve transection, new AchE activity could be found at the anastomosis site until about two weeks and at 1 cm distal to the anastomosis site until about three weeks. It became more intense with the passage of time and, at about six weeks, regenerated AchE-positive myelinated fibers could be seen at the distal end of the nerve.     

Histochemical staining of nerve endings as an aid to free muscle transplantation.

Histochemical staining techniques that identify intact motor nerve fascicles are available to aid free muscle transplantation. Cholinesterase activity of myelinated axons can be identified by Karnovsky and Roots's technique. Axon viability can be assessed based on the presence of axoplasmic enzyme activity. By reacting serial sections for cholinesterase activity and carbonic anhydrase activity, which labels sensory axons, an accurate cross-sectional map of regenerating or functional sensory and motor nerve fibers can be constructed. Resolving the motor and sensory identities of fascicles in a mixed peripheral nerve should lead to more precise coaptation of recipient motor fibers to the motor nerve of the transferred muscle and enhance reinnervation.     

Histochemical study of retrograde degeneration in ligated peripheral nerves

The enzyme specificity of the Karnovsky staining was examined. Tetraisopropylpyrophosphoramide made the staining difference between anterior and posterior roots clearer. Rat sciatic nerve, gluteal muscle branch and sural nerve were ligated and retrograde changes were examined histologically and histochemically. In ligated sciatic nerve, axonal degeneration similar to Wallerian degeneration occurred within 5 mm proximal to ligation while acetylcholinesterase activity decreased in more proximal portions. Sections of ligated gluteal muscle branch showed marked increase of small myelinated fibers 6 weeks after ligation and the mean cross sectional area of myelinated fibers decreased due to the new small fibers. The mean cross sectional area of myelinated axons also decreased in ligated sural nerve but myelinated fibers did not so remarkably increase in number as ligated gluteal muscle branches. Histochemical funicular orientation could be reliable if performed soon after nerve injury before retrograde changes spread in the proximal stump.     

Both stump area and volume of distal sensory nerve segments influence the regeneration of sensory axons in rats.

We examined the influence of both stump area and volume of a distal sensory nerve segment on neurotropic induction of regenerating sensory axons in a rat saphenous nerve model. In group 1 (n = 10) the proximal stump of the severed saphenous nerve was inserted into the proximal channel, and a 2 cm free nerve segment and a double-barrelled 1 cm free nerve segment were inserted into the distal two channels of a silicone Y-chamber. In group 2 (n = 10), 2 cm and 1 cm free nerve segments were inserted into the distal two channels of a Y-chamber. The gap between the stumps was set at 4 mm. After six weeks, we counted and compared the number of regenerated myelinated sensory axons in the distal two channels. Significantly more axons regenerated in the wider stump area channel of group 1 and in the larger volume channel of group 2 than in the opposite channel in either group (p < 0.05 in each case).     

Diabetic autonomic neuropathy in BB rat. Ultrastructural and morphometric changes in parasympathetic nerves

Longitudinal electron-microscopic and morphometric studies of autonomic nerves containing predominantly parasympathetic fibers were undertaken in the spontaneously diabetic BB rat. Unmyelinated fibers of the diabetic vagus nerve and myelinated fibers of the penile nerve showed increased numbers of axonal glycogenosomes and axonal sequestration. Morphometric examination of myelinated and unmyelinated fibers of the vagus nerve revealed diminished fiber size compared with age-matched control animals. The distal myenteric nerve showed marked degenerative changes, whereas no structural changes could be demonstrated in intra-myenteric ganglion cells. These changes are similar to those described previously in somatic nerves of this model but different from those seen in sympathetic nerves of the diabetic BB rat.     

体神经-内脏神经吻合后神经纤维再生过程的光镜电镜观察

目的：观察大鼠体神经-内脏神经吻合后神经纤维的再生。方法：人工建立体神经-内脏神经反射弧大鼠模型，用电镜配合光镜观察12只大鼠术后1、4、8、24周神经变性与再生。结果：术后8、24周大体观察见神经吻合口位置稍许膨大，光镜观察发现术后8周吻合口位置可见新生的轴突，电镜观察见术后1周吻合口及其远近段神经纤维发生Waller变性，术后4周吻合口部位有再生的有髓和无髓纤维，术后8周新生的髓鞘进一步增厚，板层结构清晰可见，术后24周，髓鞘成熟，轴浆富含微管、微丝、线粒体。结论：体神经运动纤维能够再生长入并替代内脏神经节前纤维；再生的神经纤维具备基本正常的周围神经超微结构。     

Comparison of the neurotropic effects of motor and sensory Schwann cells during regeneration of peripheral nerves.

We examined the inductive ability of motor and sensory Schwann cells on regeneration of motor and sensory axons using a silastic Y chamber, and Lewis rats L5 ventral root (motor) and saphenous nerve (sensory). We developed four experimental models: motor-motor nerve group-proximal motor stump with distal fresh and frozen/thawed motor nerve segments (n = 7); sensory-sensory nerve group-proximal sensory stump with distal fresh and frozen/thawed sensory nerve segments (n = 7); motor-sensory nerve group-proximal motor stump with distal fresh and frozen/thawed sensory segments (n = 8); and sensory-motor nerve group-proximal sensory stump with distal fresh and frozen/thawed motor segments (n = 8). The gap was set at 4 mm. Six weeks postoperatively we compared the number of regenerated myelinated axons in the two distal channels, and found that sensory Schwann cells have a strong inductive ability for regeneration of both sensory and motor axons. Motor Schwann cells have weak inductive ability for regeneration of motor axons and no inductive ability for regeneration of sensory axons.     

Effects of long-term aldose reductase inhibition on development of experimental diabetic neuropathy. Ultrastructural and morphometric studies of sural nerve in streptozocin-induced diabetic rats

There is controversy over the efficacy of aldose reductase inhibitors in preventing the development of peripheral nerve lesions in experimental diabetes. This study was designed to show whether long-term (28-wk) inhibition of aldose reductase by ponalrestat influences structural changes in peripheral sensory nerve in rats with chronic streptozocin-induced diabetes. Sciatic nerve levels of sorbitol and fructose were significantly reduced but not completely normalized by ponalrestat treatment. myo-Inositol levels, which tended to decrease in diabetic rats, were significantly increased by ponalrestat treatment and exceeded the level in nondiabetic control rats (P less than 0.01). Ponalrestat treatment significantly increased nerve conduction velocity over the 28 wk of treatment (P less than 0.05), but levels remained well below those of control rats. Structural analysis of sural nerve of diabetic rats disclosed significant preventive effects of ponalrestat on the reduction in myelinated nerve fiber size and fiber occupancy. Axon-fiber size ratio was also preserved in the ponalrestat-treated group. However, diffuse deposition of glycogen and increased glycogenosomes within axons were not influenced by ponalrestat treatment. In contrast to the effect on myelinated nerve fibers, morphometry of unmyelinated nerve fibers did not reveal a significant effect of ponalrestat treatment. These results suggest that chronic treatment with an aldose reductase inhibitor has beneficial effects on the peripheral sensory nerve of experimentally diabetic rats. The effects were primarily on myelinated rather than unmyelinated nerve fibers.     

The influence of motor axon misdirection on muscle contraction force in early nerve repair in a rat sciatic nerve model.

We examined the influence of misdirection of regenerating motor axons toward the distal sensory Schwann tubes on the muscle contraction force in early nerve repair using a rat sciatic nerve model. At 0, 1, 2, 4 and 8 weeks after severing the tibial, peroneal and sural nerves, the proximal stump of the tibial nerve was anastomosed with the distal stumps of both the peroneal and sural nerves using tubulisation (n=10 in each of five groups). We intentionally used the distal stump of the sural nerve (a sensory nerve) to induce regeneration in motor axons from the proximal tibial nerve stump toward the distal sensory nerve stump. Twenty-four weeks after nerve repair, isometric contraction force and wet weight of the anterior tibial muscle were measured, and the numbers of regenerated myelinated axons (motor and sensory) in the distal sural and peroneal nerves were counted. The rates of sural nerve regeneration were significantly higher at weeks 0 and 1 than at the later repair times. However, muscle contraction force and muscle wet weight did not differ significantly between groups in which nerves were repaired within four weeks of severance. These results indicate that peripheral nerve repair within four weeks of severance does not influence the muscle contraction force of single muscle despite the misdirection of motor axons toward the distal sensory Schwann tubes.     

COMPARISON OF THE NEUROTROPIC EFFECTS OF MOTOR AND SENSORY SCHWANN CELLS DURING REGENERATION OF PERIPHERAL NERVES

We examined the inductive ability of motor and sensory Schwann cells on regeneration of motor and sensory axons using a silastic Y chamber, and Lewis rats L5 ventral root (motor) and saphenous nerve (sensory). We developed four experimental models: motor-motor nerve group-proximal motor stump with distal fresh and frozen/thawed motor nerve segments (n = 7); sensory-sensory nerve group-proximal sensory stump with distal fresh and frozen/thawed sensory nerve segments (n = 7); motor-sensory nerve group-proximal motor stump with distal fresh and frozen/thawed sensory segments (n = 8); and sensory-motor nerve group-proximal sensory stump with distal fresh and frozen/thawed motor segments (n = 8). The gap was set at 4 mm. Six weeks postoperatively we compared the number of regenerated myelinated axons in the two distal channels, and found that sensory Schwann cells have a strong inductive ability for regeneration of both sensory and motor axons. Motor Schwann cells have weak inductive ability for regeneration of motor axons and no inductive ability for regeneration of sensory axons.     

人体膈神经与副膈神经的组织化学研究

研究人体膈神经与副膈神经的运动纤维数量及有髓纤维截面积。方法：运用Ｋａｍｏｖｓｋｙ－Ｒｏｏｔｓ［１］乙酰胆碱酯酶组织化学方法，研究１２例成人膈神经及并存的５例副膈神经。对轴索内酶活性反应呈阳性的纤维，进行计数及纤维截面积图象分析。结果：膈神经与副膈神经绝大部分有髓纤维酶反应呈阳性，所含阳性有膈纤维分别为２６８６根、６３４根。膈神经与副膈神经并存时有髓纤维总数达３２１８根。有髓纤维截面积分别为１０３．８μｍ２、９４．７μｍ２。结论：膈神经含２６８６根运动纤维，有髓纤维截面积为１０３．８μｍ２，是良好的移位动力神经；副膈神经与其并存，有重要的临床意义。     

Diffusional delay in local anesthetic block in vitro

The diffusion of lidocaine to myelinated and unmyelinated axons was compared on individual afferent fibers of rabbit vagus nerve. The criterion consisted of the time required for more than 95% completion of the asymptotic increase in impulse conduction time produced by a weak, nonblocking concentration of lidocaine. Measurements on sheathed and desheathed nerves for both myelinated and unmyelinated axons detected an apparent but statistically not significant diffusional lag at the perineurial sheath, averaging four minutes in this model; there was no significant difference in the mean time for attainment of criterion in myelinated and unmyelinated axons, which averaged an additional 13 min in both types of fiber. From these observations the authors conclude that lidocaine diffused as readily through the nodal gap to the excitable membrane of the myelinated fiber as through the Schwann cell mesaxon to the unmyelinated fiber. Thus differential diffusion within a nerve seems unlikely to be a contributing factor to clinical differential block.     

Identification of myelinated motor and sensory axons in a regenerating mixed nerve

The common peroneal nerves of Wistar rats were transected and repaired to compare the sequential changes in the numbers of regenerating motor and sensory myelinated axons in a single mixed nerve. At sequential intervals (2, 4, and 12 weeks) after nerve repair, 3 kinds of staining were performed: cholinesterase staining (Karnovsky's staining) for motor axons, carbonic anhydrase staining for sensory axons, and antineurofilament immunohistochemical staining for all axons. At 2 weeks there was a large number of carbonic anhydrase-positive axons (600 +/- 98; mean +/- SD) and cholinesterase-positive axons were occasionally seen. Subsequently, there was a striking increase of cholinesterase-positive myelinated axons, reaching to 302 +/- 50 at 12 weeks. The results suggest that the myelinated sensory axons regenerate faster in the early stage of nerve regeneration and that regeneration of the myelinated motor axons is prominent in the subsequent stage. (J Hand Surg 2000; 25A:104-111.     

Diabetic autonomic neuropathy in BB rats and effect of ARI treatment on heart-rate variability and vagus nerve structure

The preventive effect of the aldose reductase inhibitor (ARI) ponalrestat on heart-rate variability and the development of autonomic neuropathy in the vagus nerve was investigated in the spontaneously diabetic BB rat. ARI treatment completely prevented the characteristic decrease in heart-rate variability and axonal atrophy of the vagus nerve for 4 mo in hyperglycemic BB rats. After 6 mo of treatment, the preventive effect on heart-rate variability was partial, and the vagus nerve demonstrated an increase in regenerating myelinated and unmyelinated fibers. These data suggest that autonomic neuropathy involving the vagus nerve is metabolically induced by demonstrating that inhibition of the polyol pathway significantly delays the occurrence of functional and structural autonomic neuropathy despite the presence of hyperglycemia.     

Selective regeneration of motor and sensory axons in an experimental peripheral nerve model without endorgans.

We assessed the selectivity of motor and sensory axon regeneration towards the distal motor and sensory nerve segments that were disconnected from endorgans in a rat silicone Y chamber model. The L5 ventral root was used as a pure motor nerve, and the saphenous nerve was used as a sensory nerve. In experiment 1 (n=11), the proximal stump of the L5 ventral root, a 1-cm-long L5 ventral root segment and a saphenous nerve segment were inserted into a silicone Y chamber. In experiment 2 (n=11), the proximal stump of the saphenous nerve, a L5 ventral root segment and a saphenous nerve segment were inserted into a Y chamber. The distance between the nerve stumps was 5 mm. Six weeks later, the number of regenerated myelinated motor and sensory axons was measured and compared in the distal two channels. Motor axons showed no selective regeneration, but sensory axons did.     

感觉神经植入兔带蒂皮瓣转移再造阴茎后的再生神经形态学观察

目的 观察兔隐神经与阴茎背神经吻接植入腹壁浅血管蒂岛状皮瓣阴茎再造术后，再造阴茎感觉神经的再生过程和机制，探索再造阴茎感觉功能重建的有效方法。方法 雄性新西兰兔40只随机均分为实验（神经植入）组和对照（未植神经）组。建立隐神经与阴茎背神经吻接植入腹壁浅血管蒂岛状皮瓣阴茎再造术的动物模型，术后1、2周及1、3、6个月，应用组织学、免疫组化及电镜等方法对再造阴茎感觉神经的再生情况进行形态学观察。结果 组织学观察发现实验组皮瓣内神经束数量不断增多，至术后6个月时尚可见神经长人脂肪层，对照组皮瓣内残存神经呈萎缩改变；免疫组化结果显示：术后实验组神经植入后再生感觉神经纤维、触觉小体、表皮内游离神经末梢的密度和数量明显高于对照组；电镜结果表明：术后3个月以内早期，皮肤感觉神经的再生以无髓神经纤维为主，之后有髓纤维和无髓纤维均有出现。结论 隐神经移植与阴茎背神经端端吻合植入兔腹壁浅血管蒂岛状皮瓣阴茎再造术，再造阴茎至少在6个月时可以获得良好的感觉神经再生和末梢感受器的神经再支配。     

Preferential block of small myelinated sensory and motor fibers by lidocaine: in vivo electrophysiology in the rat sciatic nerve.

BACKGROUND: Controversy still surrounds the differential susceptibility of nerve fibers to local anesthetics and its relation to selective functional deficits. In the current study we report features of conduction blockade in different classes of rat sciatic nerve fibers after injection of lidocaine by a percutaneous procedure that closely resembles clinical applications. METHODS: In 30 adult male Sprague-Dawley rats (weight, 300-400 g) during general anesthesia, impulses were recorded in different classes of sensory axons (large, Aalpha and beta fibers; small, Adelta myelinated fibers and unmyelinated C fibers) and motor axons (large, Aalpha fibers; small, Agamma myelinated fibers) classified by conduction velocity. The sciatic nerve was stimulated distally, and impulses were recorded from small filaments teased from L4-L5 dorsal (sensory) and ventral (motor) roots sectioned acutely from the spinal cord. Lidocaine at concentration of 0.05-1% was injected percutaneously in 0.1-ml solutions at the sciatic notch. Both tonic (stimulated at 0.5 Hz) and use-dependent (stimulated at 40 Hz for Adelta and Agamma fibers and at 5 Hz for C fibers) impulse inhibitions by lidocaine were assayed. RESULTS: Minimal effective (threshold) lidocaine concentrations (i.e., to block conduction in 10% of fibers) were, for sensory, 0.03% for Adelta, 0.07% for Aalphabeta, and 0.09-0.1% for C fibers, and for motor, 0.03% for Agamma and 0.05% for Aalpha fibers. The order of fiber susceptibility, ranked by concentrations that gave peak tonic fiber blockade of 50% (IC50s), was Agamma > Adelta = Aalpha > Aalphabeta > C. Faster-conducting C fibers (conduction velocity > 1 m/s) were more susceptible (IC50 = 0.13%) than slower ones (conduction velocity < 1 m/s; IC50 = 0.30%). At 1% lidocaine, all fibers were tonically blocked. Use-dependent effects accounted for only a modest potentiation of block (at a lidocaine concentration of 0.25%) in Adelta and Agamma fibers, and in C fibers phasic stimulation had even smaller effects and sometimes relieved tonic block. CONCLUSIONS: Susceptibility to lidocaine does not strictly follow the "size principle" that smaller (slower) axons are always blocked first. This order of fiber blockade is qualitatively consistent with previous reports of the order of functional deficits in the rat after percutaneous lidocaine, that is, motor = proprioception > nociception, if we assume that motor deficits first arise from conduction failure in Agamma fibers and that nociception relies on C fiber conduction.     

Structural and functional investigations of the murine cavernosal nerve: a model system for serial spatio-temporal study of autonomic neuropathy

OBJECTIVE: To illustrate the ultrastructural fibre composition of the rat cavernosal nerve at serial levels, from its origin in the main pelvic ganglion to its termination in the corpus cavernosum of the distal penile shaft, and to develop a technique that permits repeated electrophysiological recording from the fibres that form the cavernosal nerve distinct from the axons of the dorsal nerve of the penis (DNP). MATERIALS AND METHODS: For the light microscope and ultrastructural studies, Sprague-Dawley rats were anaesthetized and the pelvic organs and lower limbs were perfused with glutaraldehyde through the distal aorta. Tissue samples were embedded in epoxy resin and prepared for light and electron microscopy. Frozen tissue was used for the immunohistochemical studies and sections were stained with rabbit anti-nitric oxide synthetase 1 (NOS1). For the electrophysiology, anaesthetized rats were used in sterile conditions. Nerve conduction velocity for the cavernosal nerve was assessed from a point 2 mm below the main (major) pelvic ganglion after stimulating the nerve at the crus penis; multi-unit averaging techniques were used to enhance the recording of slow-conduction activity. Recordings from the DNP were obtained over the proximal shaft after stimulation at the base of the penis. RESULTS: Step-serial sections of the cavernosal nerve revealed numerous ganglion cells in the initial segments and gradually fewer myelinated fibres at distal levels. At the point of crural entry, the nerve contained almost exclusively unmyelinated axons. As it descended the penile shaft, the nerve separated into small fascicles containing only one to four axons at the level of the distal shaft. In the corpus cavernosum, vesicle-filled presynaptic axon preterminals were close to smooth muscle fibres, but did not seem to be in direct contact. Immunohistochemical evaluation of NOS1 activity showed intense staining of the fibres of the DNP and most of the neurones in the main pelvic ganglion. There was also scattered NOS1 activity in the nerve bundles of the corpus cavernosum. Electrophysiology identified activity in C fibres on the cavernosal nerve and in Aalpha-Adelta fibres in the DNP. CONCLUSION: These results show that it is possible to perform integrated cavernosal pressure monitoring and ultrastructural and electrophysiological studies in this model. These yielded accurate data about the erectile status of the penis, and the state of unmyelinated and myelinated fibres in the DNP and cavernosal nerves of the same animal. This study provides a useful template for future studies of experimental diabetic autonomic neuropathy.     

Carbonic anhydrase histochemistry. A potential diagnostic method for peripheral nerve repair

Many large-diameter myelinated axons in spinal dorsal roots contain carbonic anhydrase activity, whereas few small-diameter ventral root axons stain for this enzyme. This differential localization of carbonic anhydrase in sensory and motor nerve fibers is indicative of the potential of carbonic anhydrase histochemistry to provide a convenient method for identifying predominantly motor or sensory fascicles in cut ends of peripheral nerves, thereby facilitating coaptation of fascicles in peripheral nerve repair.     

Lidocaine (without epinephrine) does not affect the fine structure or microtubules of the trigeminal nerve in vivo.

The authors examined the fine structure and microtubules of unmyelinated axons and Schwann cells in the infraorbital branch of rat trigeminal nerve 1, 3.5 and 24 hours after intraneural injection of lidocaine HC1, 1--4 per cent, 0.2 ml, or saline solution, 0.2 ml; untreated control nerves were also examined. These concentrations of lidocaine are more than sufficient to block impulse conduction and rapid axonal transport in rat infraorbital nerve, but contrary to previous reports, significant structural change was not found as compared with control nerves.