Cytochemical demonstration of nucleoside phosphatase activity in myelinated nerve fibers of the rat

Summary The histochemical and cytochemical localization of end product from the Wachstein-Meisel reaction was examined in transverse frozen sections of rat ischiatic nerve and spinal nerve roots. A variety of fixatives, substrates, and inhibitors were used at varying pH. Following glutaraldehyde fixation, nucleoside phosphatase activity was noted in the axon-Schwann cell interface of both unmyelinated and myelinated nerve fibers. The presence of this enzymatic activity at this strategic location in the nerve fiber complex attests to the importance of this space as a metabolically active site.Additional deposits of end product occurred on neurofilaments of myelinated and unmyelinated axons and were observed as diffuse axonal stains by light microscopy. These precipitates had a predilection for tissue fixed in formalin or hydroxyadipaldehyde and were relatively more prominent following acidic incubations. The resemblance between these deposits and spurious axonal acid phosphatase precipitates was discussed.This study was supported by Grant NB 04161 of the National Institute of Neurological Disease and Blindness.     

Acetylcholinesterase: a histochemical identification of motor and sensory fascicles in human peripheral nerve and its use during operation

To evaluate the precision of acetylcholinesterase histochemical identification of motor and sensory fascicles, this study presents a systematic observation of human peripheral nerves by Karnovsky and Roots' histochemical method. The results indicate that either of the enzymatic activities of myelinated and unmyelinated fibers was different between motor and sensory fascicles. Fifty-seven percent of the myelinated fibers showed enzymatic activity in the motor fascicles, while none of the myelinated fibers in the sensory fascicles showed enzymatic activity. The unmyelinated fibers showing enzymatic activity in the sensory fascicles were far denser than those in the motor fascicles. Our study demonstrated that the unmyelinated fibers were sympathetic postganglionic unmyelinated fibers. From these results it is concluded that the motor and sensory fascicles may be identified not only according to the enzymatic activities of the myelinated fibers, but also according to the enzymatic activities of the sympathetic postganglionic unmyelinated fibers. An improved histochemical method was suggested for its applicability as a method of intraoperative nerve fascicle identification. Simulated experiments were done on the radial nerves and the median nerves in human cadavers. This improved histochemical process can be completed within 50 minutes and can be used in intraoperative nerve fascicle identification.     

Adenosine triphosphatase activity of cutaneous nerve fibers

Summary The histochemical study of Mg⁺⁺-activated adenosine triphosphatase (Mg⁺⁺-ATPase) activity was carried out on the peripheral nerves of mouse digital skin by light and electron microscopy. Under the light microscope, the ATPase activity was clearly demonstrated on the nerve fibers as a fine network in the subepidermal regions. Under the electron microscope, the reaction product of enzyme activity was located in the interspace between axolemma and the surrounding Schwann cells of the unmyelinated nerve fibers. No reaction product was observed in the space between the axolemma and the Schwann cells associated with myelinated nerve fibers. Demonstrable activity was absent at the nodes of Ranvier as well as on the para- and internodal regions of these myelinated axons. The part of the axolemma lacking a Schwann cell sheath failed to show a reaction product. The perineural epithelial cells surrounding the nerve fibers displayed reaction product in the caveolae. These results suggest a functional difference in the axon-Schwann interface of myelinated as compared to unmyelinated nerve fibers. The function of the perineural epithelial cell would be expected to be a regulatory one in transferring materials across the epithelium to keep the proper humoral environment around nerve fibers.     

Quantitative studies on the motor root of the mouse facial nerve. An electron-microscopic study.

Fiber count analysis was performed with the electron microscope on the motor root of the facial nerve in six mice. On an average, 3,433 (84.9%) of the total nerve fibers (4,046) were myelinated and 592 (14.6%) unmyelinated. The motor root consisted mostly of large myelinated fibers (large fiber group), but a part of the root consisted of small myelinated and unmyelinated fibers (small fiber group). The nerve fibers of the small fiber group appear to correspond with those of the intermediate nerve, and to pass through the motor root and enter the intermediate nerve near the geniculate ganglion.     

A quantitative electron microscope analysis of peripheral nerve in the urodele amphibian in relation to limb regenerative capacity

An approximate 1:1 ratio of myelinated to unmyelinated fibers was established in counts from electron micrograph montages in nerves of the newt, Triturus (Notophthalmus) viridescens. The number of myelinated fibers correspond to the number counted with the light microscope after osmium fixation. Light microscope counts of silver impregnated sections yielded a value slightly higher suggesting that, except for bundles of unmyelinated fibers, the silver technique revealed mainly myelinated fibers. The results were used to reassess previous quantitative studies on the relation between number of nerve fibers and the control which nerves exert on regeneration. For a truer estimate of the number of axons affecting regeneration, fiber values previously reported should now be doubled to include the large number of unmyelinated fibers. However, calculations show that the unmyelinated fibers contribute less than 3% of the total neuroplasm in the peripheral nerve. Finally, counts made of Schwann cells and fibroblasts show that the latter are few in number.     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 +/- 28 nerve fibers consisting of 63 +/- 24 unmyelinated and 12 +/- 6 myelinated fibers. The PAN was composed of 195 +/- 50 nerve fibers, namely 129 +/- 28 unmyelinated and 66 +/- 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 microm with a maximum between 0.3 and 0.6 microm. The myelinated nerve fibers in the MAN ranged from 1 to 8 microm with a peak between 2 and 5 microm. In the PAN, their diameters ranged from 1 to 12 microm with a clearly visible peak at 4-5 microm and a plateau at 8-9 microm that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 &#45 28 nerve fibers consisting of 63 &#45 24 unmyelinated and 12 &#45 6 myelinated fibers. The PAN was composed of 195 &#45 50 nerve fibers, namely 129 &#45 28 unmyelinated and 66 &#45 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 &#119 m with a maximum between 0.3 and 0.6 &#119 m. The myelinated nerve fibers in the MAN ranged from 1 to 8 &#119 m with a peak between 2 and 5 &#119 m. In the PAN, their diameters ranged from 1 to 12 &#119 m with a clearly visible peak at 4-5 &#119 m and a plateau at 8-9 &#119 m that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Distribution, origin and sensitivity to capsaicin of primary afferent substance P-immunoreactive nerves in the heart

This report is intended as an overview of the distribution, origin and sensitivity to capsaicin of substance P-immunoreactive (SP-I) primary afferent cardiac nerves. Immunohistochemical and physiological methods were employed to compare the presence and density of these nerve fibers in the guinea pig and rat hearts. SP-I fibers are numerous in the guinea pig heart including the parietal pericardium, atria, ventricles, valves, coronary arteries and around intrinsic cardiac ganglion cells. The rat heart contains few SP-I fibers. Vagotomy does not influence the number of intensity of immunoreactive fibers in the guinea pig heart. By stimulating the atrium or ventricle and recording from the second or third thoracic dorsal roots Ad1, Ad2 and C fibers were demonstrated in the atria, but only Ad fibers in the guinea pig ventricle; in addition, only Ad fibers were recorded from the vagus nerves. Only Ad1 fibers were demonstrated in the rat heart. Treatment with capsaicin depletes the SP-I and decreases the conduction velocity of C-fibers and some Ad2 fibers in the guinea pig heart. We suggest that SP-I primary afferent nerve fibers are unmyelinated (C-type) or small myelinated (Ad2-type) nerves in the guinea pig heart and that their cell bodies of origin are predominantly in dorsal root ganglia.     

Pathological changes of human unmyelinated nerve fibers: a review

In the cutaneous nerves, unmyelinated nerve fibers outnumber the myelinated ones but are scarcely analyzed, especially at autopsy. This indifference toward the pathology of unmyelinated nerve fibers may be due to the necessity of electron microscopic analyses and, more importantly, the obscurity of pathological alteration of unmyelinated nerve fibers in aging as well as in peripheral nerve disorders. The aim of this article is to review (1) the normal appearance including postmortem changes, (2) the age-related changes, and (3) the pathological alteration in various neuropathic and non-neuropathic conditions, of unmyelinated nerve fibers in the sural nerve. For the complete analyses of sural nerve, qualitative and quantitative estimation of unmyelinated nerve fibers in all specimens should be recommended and it sometimes has an important diagnostic value.     

Morphometry and ultrastructure of the squirrel monkey (Saimiri sciureus) vestibular nerve

Vestibular nerves of squirrel monkeys (Saimiri sciureus) embedded in plastics and epoxies were examined with light microscopy (LM) and transmission electron microscopy (TEM), and computerized measures were obtained and analyzed statistically. An average of 12,412 perikarya and 12,005 myelinated nerve fibers was obtained. Approximately 0.7% of the perikarya appeared unmyelinated under LM. About 500 unmyelinated fibers were counted. The cross-sectional area of 1,864 perikarya was 200-650 micron 2. The cross-sectional area of 1,346 nerve fibers was 3-11 micron 2 for the axoplasm and 11-12 micron 2 for the myelin sheath of the same fiber. Myelin thickness was directly proportional to the axoplasm cross-sectional area of the nerve fibers. The cross-sectional area of central axons and peripheral dendrites differed significantly (p less than 0.001). The initial segments of peripheral dendrites were usually smaller, but longer than the initial segments of the central axons. Both initial segments increased in diameter after the first node of Ranvier. Schmidt-Lantermann incisures were more abundant in thick and heavily myelinated fibers than in thin and lightly myelinated fibers. Larger perikarya usually had larger fibers and vice versa, within the first 100-200 micron from the first node of Ranvier. No major ultrastructural differences were found between myelinated and unmyelinated perikarya, except at the hillock region. The Nissl substance was preferentially located in the peripheral cytoplasm.     

The morphological consequences of neonatal treatment with capsaicin on primary afferent neurones in adult rats

A comparison was made using our work and that reported in the literature of the losses of myelinated and unmyelinated fibres in a variety of nerves and also of losses of nerve cells in dorsal root ganglia, after treatment of neonatal rats with capsaicin. In L3 and L4 dorsal roots 85-93% of unmyelinated fibres and 9-33% of myelinated fibres were lost after 50-100 mg/kg capsaicin neonatally. In rats treated with 85 mg/kg capsaicin, percentage losses of unmyelinated (89%) and myelinated (36%) fibres of L4 dorsal roots were remarkably similar to the calculated losses of small dark (92%) and large light (34%) neurones respectively in these ganglia. Studies with monoclonal antibody RT97 which labels the large light neurones only, confirmed that some RT97 negative cells (i.e. small dark neurones) remain after capsaicin treatment. At present no evidence exists to suggest that the cell death of small dark neurones or C fibres after neonatal capsaicin treatment is completely selective for subgroups of these neurones, either in relation to sensory modality, or in relation to immunocytochemical cell markers and peptide content. However much more data is required to establish whether this cell death is really nonselective as regards immunocytochemical markers.     

Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman microspectroscopy

Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm^?1, and 2,945 cm^?1, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.     

Effect of oculomotor and trigeminal nerve section on the ultrastructure of different myoneural junctions in the rat extraocular muscles

Summary The intramuscular nerves and myoneural junctions in the rat rectus superior, medialis and inferior muscles from 10 hours to about 10 days after section of the trigeminal and oculomotor nerves were studied with the electron microscope. Two different kinds of myoneural junctions are to be observed; one type derives from myelinated nerves and is similar to the ordinary myoneural junctions (motor end plates) of other striated skeletal muscles, while the other type derives from unmyelinated nerves, is smaller in size and has many myoneural synapses distributed along a single extrafusal muscle fibre.Section of the trigeminal nerve caused no changes in the myoneural synapses. After section of the oculomotor nerve degenerative changes occur in both the myelinated and unmyelinated nerves and in both types of myoneural junctions. In the axon terminals of both the myelinated and unmyelinated nerves the earliest changes are to be observed 10 to 15 hours after section of the nerve. First, swelling of the axoplasm, fragmentation of microtubules and microfilaments and swelling of mitochondria takes place, somewhat later agglutination of the axonal vesicles and mitochondria. The axon terminals are separated from the postsynaptic muscle membrane by hypertrophied teloglial cells about 24 hours after section of the nerve. The debris of the axon terminals is usually digested by the teloglial cells within 42 to 48 hours in both types of myoneural junction.Changes in the postsynaptic membrane are observed in the myoneural junctions of the unmyelinated nerves as disappearance of the already earlier irregular infoldings, whereas no changes take place in the infoldings of the motor end plates. The postsynaptic sarcoplasm and its ribosomal content increase somewhat.The earliest changes occur along unmyelinated axons 10 to 15 hours and along myelinated axons 15 to 24 hours after nerve section. The unmyelinated axons are usually totally digested within 48 hours, whereas the myelinated axons took between 48 hours and 4 days to disappear. The degeneration, fragmentation and digestion of the myelin sheath begin between 24 and 42 hours and still continues 10 days after the operation.The results demonstrate that in the three muscles studied structures underlying the physiologically well known double innervation of the extraoccular muscles are all part of the oculomotor system.We are grateful to Professor Antti Telkkä, M. D. Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.     

The stomatogastric nervous system of the house cricket Acheta domesticus L. I. The anatomy of the system and the innervation of the gut

The distribution of the ganglia and nerves of the stomatogastric nervous system and the innervation of the extrinsic and intrinsic muscles are described. Median unpaired frontal and hypocerebral ganglia and paired ingluvial ganglia are present. The anterior pharynx is innervated by branches of the frontal nerve and by the anterior and posterior pharyngeal nerves, originating from the frontal ganglion. The posterior pharyngeal nerves are linked to nerves innervating the posterior part of the pharynx which have their origin in the hypocerebral ganglion, the anterior portion of which has previously been regarded as part of the recurrent nerve. Paired esophageal nerves run the length of the esophagus and crop between the hypocerebral and and ingluvial ganglia, innervating the muscularis by serial side branches. From each ingluvial ganglion runs an ingluvial nerve which innervates the gizzard and a cecal nerve which innervates the midgut and its ceca. At the posterior end of the midgut there is a poorly developed nerve ring. Nerves running posteriorly from this nerve ring link the stomatogastric nervous system with the proctodeal innervation from the terminal abdominal ganglion. Multipolar peripheral neurons are present on the muscularis of the whole of the foregut, rather randomly distributed on the crop and gizzard but forming fairly definite groupings at some points on the pharynx. Though of varied appearance, these cells could not be divided into discrete morphological categories. Peripheral neurons on the midgut are of different and characteristic morphology, though a few cells of the same appearance as those of the foregut occur at the midgut-hindgut boundary. Nerve fibers on the gut almost invariably terminate on the fibers of the muscularis.     

Isolation and characterization of axolemma-enriched fractions from rabbit and bovine peripheral nerve

Axolemma-enriched fractions were isolated from bovine spinal accessory nerves, bovine intradural dorsal roots, and rabbit sciatic nerve by differential centrifugation and separation on a linear 10–40% sucrose (w/w) gradient. The fractions were enriched 4 to 10 fold in acetylcholinesterase, a biochemical marker for axolemma. Axolemma-enriched fractions isolated from uniformly well-myelinated fibers (bovine spinal accessory nerve) contained lower CNPase activity and higher acetylcholinesterase activity than comparable fractions isolated from variably myelinated fibers (rabbit sciatic nerve and bovine intradural roots). Separation by polyacrylamide electrophoresis showed that the molecular weight distribution of all peripheral nerve axolemma-enriched fractions was similar and ranged from 20 to over 150 kilodaltons. All axolemma-enriched fractions appeared to contain a small but variable amount of myelin-specific proteins. Based on biochemical properties, peripheral nerves containing uniformly well-myelinated fibers yield an axolemma-enriched fraction which is least contaminated with myelin-related membranes.     

Sacral anterior root stimulation and cryotechnique: A new option for selective urethral sphincter block and reversible deafferentation in the future?

A possible application of cryotechnique might be a selective block of nerve fiber activity during sacral anterior root stimulation to achieve selective block of urethral sphincter and reversible deafferentation. In 13 foxhounds, electrical stimulation of sacral anterior roots S2 was performed while the accompanying spinal nerves were simultaneously cooled down from +25 degrees C in a stepwise fashion until a block of urethral sphincter activity was observed. The effects of cold block on the urethral sphincter and bladder were monitored by urodynamic investigation. In 2 additional dogs sacral posterior roots S2 were cooled down to +3 degrees C while accompanying anterior and posterior roots were stimulated distal to the cryothermode. Compound action potentials (APs) were registered proximal to the cryothermode before, during and after cooling and recovery time of cold blocked nerves was evaluated. Complete cold block of the urethral sphincter during spinal nerve cooling was achieved in all cases. Block temperature averaged +12 degrees C. Detrusor pressure was a mean 5,2 cm water. Recovery time was on average 5 min. The cold block was always reversible. In both dogs of the second series the compound action potentials disappeared nearly completely at +3 degrees C. Three min after the end of the cooling period the appearance of the compound action potentials was back to normal. In this study, cryotechnique proved to be effective for selective and reversible block of nerve fibers during sacral anterior root stimulation. In functional electrical stimulation this technique may lead to an improvement of quality of life in para- or tetraplegic patients resulting in optimization of voiding, standing, walking and grasping and does so without the necessity of surgical dorsal root rhizotomy.     

Quantitative studies on the intermediate nerve of the mouse with the electron microscope.

Quantitative counts of the intermediate nerve fibers of the mouse with electron microscope have shown that, on an average, 1,267 (79.3%) and 301 (18.9%) of the total nerve fibers (1,597) were myelinated and unmyelinated fibers, respectively. The number of unmyelinated fibers in the intermediate nerve was less than one-half the number of unmyelinated fibers in the greater petrosal nerve found in previous studies. Therefore, it is postulated that some of the unmyelinated fibers in the greater petrosal nerve may come from the intermediate nerve, while the rest may be derived from another source.     

Fiber analysis of human ventral and dorsal roots on the basis of different acetylcholinesterase activity]

Marked differences in the AChE activity of myelinated nerve fibers of ventral and dorsal roots could be established in human post mortem material. After a fixation time of 3 h and a critical incubation period of 24 h, in the mean 96% of the myelinated ventral root but only 4% of dorsal root fibers showed reaction product, detectable by the light microscope. The percentage of stained fibres varies, to some extent, in the different segments. Groups of very thin myelinated fibres within the ventral roots between the segments C-8 and L-3, showing a conspicuous high enzyme activity, are interpreted as pre-ganglionic sympathetic fibres; similar elements in the sacral ventral roots may represent parasympathetic fibres. The method of Karnovsky, applied under conditions established in this study, can be used for analysis of fibre types in a given human peripheral nerve.     

Substance P-immunoreactive nerve fibers in tracheobronchial lymph nodes of the guinea pig: origin, ultrastructure and coexistence with other peptides.

Double-labeling immunofluorescence of guinea pig tracheobronchial lymph nodes revealed complete coincidence of SP and CGRP immunoreactivities in perivascular nerves and axons of the medullary lymphatic tissue. Additional dynorphin A or cholecystokinin immunoreactivity was seen only in some of the medullary fibers. Ultrastructurally, all SP-immunoreactive axons were unmyelinated and displayed vesicle-containing varicosities. Retrograde neuronal tracing combined with immunohistochemistry revealed a sensory origin from dorsal root ganglia of SP/CGRP-immunoreactive fibers ramifying within paratracheal lymph nodes, and an additional neuronal population being devoid of SP/CGRP immunoreactivity. The findings provide evidence for several types of sensory nerve fibers innervating lymph nodes.     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。