Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

Neurogenic inflammation of gingivomucosal tissue in streptozotocin-diabetic rat

Previously it was assumed that nerve fibres are involved in the neurogenic inflammation induced by mechanical or chemical irriations. It has been also suggested that in diabetes mellitus the unmyelinated small diameter fibers are impaired as a result of diabetic neuropathy. Therefore, our aim was to study the alterations of the nerve processes in the gingivomucosal tissue in streptozotocin (STZ)-diabetic rats. Light- and electronmicroscopical examinations were made to analyze the changes in nerve fibres. After one week of steptozotocin treatment, the gingivomucosal tissue had inflammatory cell infiltration and some degenerated nerve fibres were also observed. Dense mitochondria, disorganization of cell organelles, and appearance of myelin-like dense bodies were found in the axons of degenerared nerve fibres. Semiquantitative analysis showed that 14 +/- 4% of the unmyelinated nerve fibres degenerated after one week of STZ treatment. However, degeneration of the myelinated nerve fibers was not observed. Two weeks after STZ treatment, most of the unmyelinated and myelinated nerve fibers showed degeneration (86 +/- 5%) and the placement of the ligature revealed a non-inflammatory connective tissue adjacent to a normal epithelium. The myelin sheath was disrupted and dark axoplasm with cytolysosomes became manifest. These findings demonstrated that both unmyelinated and myelinated nerve fibers are altered and inflammatory reaction exists in the gingivomucosal tissue only in the early stage of diabetes mellitus.     

SEDIMENTATION ANALYSIS OF RIBONUCLEIC ACID EXTRACTED FROM ISOLATED MAUTHNER NERVE FIBRE COMPONENTS

Abstract— Segments of spinal cords from goldfish or from carp were incubated in vitro in the presence of RNA precursors for varying periods of time. Mauthner nerve fibres were isolated from the fresh unfixed tissue, or, for the separate analyses of axon and myelin sheath, from the fixed spinal cord.
The myelinated Mauthner fibre isolated from the incubated spinal cord showed RNA synthesis. A considerable part of the material sedimented at 4 S , but part of the nucleic acid was recovered at higher sedimentation values, up to 30 S. Newly synthesized RNA was extracted from the isolated myelin sheath as well as from the axon. Isolated myelinated Mauthner fibres were also incubated in vitro with RNA precursors. In this case incorporation occurred exclusively in material sedimenting at 4 S or lower. The turnover rate for RNA from the fibre was of a higher order than that of the bulk of RNA from the spinal cord. The findings of RNA synthesis in these tissue components lacking nuclei could possibly be explained as owing to mitochondria.
Studies by electron microscopy demonstrated the extent of purity of the isolated components and it was found that contamination was so small as to make it unlikely that the RNA investigated originated in contaminating tissue.     

Histological Methods for Assessing Myelin Sheaths and Axons in Human Nerve Trunks

Although there are many histological techniques for assessing myelin sheaths and axons in paraffin embedded or frozen sections of the peripheral nervous system, modern approaches usually use plastic embedded material. Although plastic embedding is superior for small cutaneous branches, this method has limited value for histological assessment of nerve trunks. We report three methods which together yield a comprehensive approach for thorough and detailed investigation of human nerve trunks. The rapid osmication method permitted assessment of myelinated nerve fibers from frozen sections at operation, thus providing the surgeon with guidance on the extent of nerve resection. The modification presented here resulted in permanent slides, allowing comparison of results with those of the other two procedures. The new osmium-hematoxylin technique could be performed on paraffin embedded nerves. Paraffin, unlike plastic, permitted the study of the whole cross sectional area of the nerve in single sections. Moreover, the sharp image of the myelin permitted computerized morphometry. The significantly modified axonal silver impregnation technique was performed on frozen sections mounted on glass slides, as opposed to the time-consuming impregnation of free-floating sections. The latter technique had a high success rate and permitted semiquantitative assessment of axons in nerve trunks. These methods can be performed in any routine histology laboratory and resulted in greater accuracy compared to conventional methods.     

Effects of varying periods of immobilization of a limb on the morphology of a peripheral nerve

Immobilization of a hind limb of the cat in the resting position was found to result in morphological changes in the nerve supplying the medial head of the gastrocnemius muscle. A reduction in the diameter of the larger myelinated fibres (greater than 12 micrometers) concomitant with a reduction in the mean fibre diameter of the nerve were observed. This reduction in the mean fibre diameter of the nerve was found to be directly proportional to the period of immobilization. Prolonged immobilization beyond 8 weeks, resulted in the splitting of the myelin lamellae, formation of myelin rings and figures in some of the larger axons in addition to an abundant increase in collagen. These structural changes indicate a definite neural involvement in the disuse atrophy of the muscle as a result of immobilization extending for a period of 8 weeks and above.     

RADIOAUTOGRAPHIC STUDIES OF CHOLINE INCORPORATION INTO PERIPHERAL NERVE MYELIN

This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.     

A comparative study of oculomotor,trochlear and abducens nerves in Arabian foals

We investigated the microscopic structure of transverse sections of the oculomotor, trochlear and abducens nerves of Arabian foals using stereological methods. Bilateral nerve pairs from 2-month-old female Arabian foals were analyzed. The tissues were embedded in plastic blocks, then 1 µm thick sections were cut and stained with osmium tetroxide and methylene blue-azure II. Stereology was performed using light microscopy. Morphometry showed that the right and left pairs of nerves were similar. The transverse sectional areas of the oculomotor, trochlear and abducens nerves were 1.93 ± 0.19 mm², 0.32 ± 0.06 mm² and 0.70 ± 0.08 mm², respectively. The oculomotor nerve exhibited a significantly greater number of myelinated axons (16755 ± 1279) and trochlear (2656 ± 494) and the abducens nerves (4468 ± 447). The ratio of the axon diameter to myelinated nerve fiber diameter was 0.58, 0.55 and 0.55 for the oculomotor, trochlear and abducens nerves, respectively. Of the three nerves studied, the abducens nerve exhibited the greatest nerve fiber area, myelin area, nerve and axon diameters, and myelin thickness. The ratio of small myelinated nerve fibers was greatest in the oculomotor nerve.     

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.     

Evidence for receptor nerve endings in tendons and related tissues of an electric teleost, Gnathonemus petersii

Tendons and muscle aponeurosis associated with muscle lodged in the mental appendage of a weakly electric mormyrid fish, Gnathonemus petersii, have been examined for nerve terminations in them, using methods of silver impregnation, histology and electron microscopy. Large myelinated afferent nerve fibres innervate the tendinous tissues and form nerve terminals in the perimysium, the muscle-tendinous junctions and the tendons, the last recalling the tendon organs of higher vertebrates. Nerve terminals are characterized by loss of myelin, Schwann sheath and basal lamina, the neurolemma being freely exposed to either fibroblasts and their laminar processes or the collagen fibres and the tendon intercellular matrix. Nerve terminals lack synaptic specialisation of their membrane. This study is the first demonstration of sensory nerve terminals (tension receptors) associated with tendons and related tissue in a teleost fish.     

Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria

Cultures of whole fetal rat sensory ganglia which had matured and myelinated in culture were treated for 1-3 h with a pulse of 0.2% trypsin. The tissue was observed during the period of treatment and during subsequent weeks using both light and electron microscopy. Within minutes after trypsin addition the matrix of the culture was altered and the nerve fascicles loosened. Progressive changes included the retraction of Schwann cell processes from the nodal region the detachment of the myelin-related paranodal Schwann cell loops from the axon, and lengthening of the nodal region as the axon was bared. The retraction of myelin from nodal stabilized several hours after trypsin withdrawal. Breakdown of the altered myelin segments was rare. There were no discernable changes in neurons or their processes after this exposure to trypsin. The partial repair which occured over a period of several weeks included the reattachment of paranodal Schwann cell loops to the axolemma and the insertion of new myelin segments where a substantial length of axolemma had been bared. The significance of these observations to the characterization of the Schwann cell-axolemmal junctions on myelinated nerve fibers is discussed. The dramatic degree of myelin change that can occur without concomitant myelin breakdown is particularly noted, as is the observation that these altered myelin segments are, in part, repaired.     

神经退变和再生的构筑变化

将夹伤的大鼠坐骨神经分离成单根纤维,观察98d内轴突和许旺细胞的构筑变化过程发现,损伤既使轴浆转运阻断、积累的细胞器退变,也使髓鞘板层,特别是斯兰氏切迹撕裂、变形或侵入轴突。轴突或髓鞘虽可各呈单一的退变,但以两者并存多见。伤后1d即出现富含微管的再生芽,它被增殖的许旺细胞突起及其基底膜包绕,并逐步发育成熟。根据再生的特征性构筑变化,提出了再生芽、无髓和有髓纤维、斯兰氏切迹、朗氏结与神经小束的初见、发育和成熟高峰期的时间顺序。无髓纤维的发育成熟早于有髓纤维。     

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.     

Sex Differences in Sensory and Motor Branches of the Pudendal Nerve of the Rat

The morphology of the pudendal nerve was quantified in adult male and female rats. The sensory branch of the pudendal nerve was about three times as large in cross section in males as in females, and the motor branch was about five times as large. Electron microscopy was used to determine the ultrastructural bases of these gross size differences. Differences that were found included greater packing density of both myelinated and unmyelinated axons in females, larger myelinated and unmyelinated axons in males, larger myelin sheaths of sensory axons in males, more numerous myelinated axons in both branches of males, and more numerous unmyelinated axons in the sensory branch of males. There was also some indication that myelinated sensory axons were more likely to branch in the dorsal clitoral nerve of females than in the homologous nerve of males. Morphological differences in the structure of pudendal axons, their associated Schwann cells, and the extracellular matrix as well as differences in sensory and motor axonal number all have potential implications for the sexual differentiation of the central nervous system and behavior.     

Quantitative electron-microscopy analysis of cranial nerves III, IV, and VI and of the extrinsic eye muscles in fog.

A comparative study of the quantitative data of the frog extraocular muscles and the cranial nerves that innervate them was performed. Oculorotatory muscles contain muscle fibres of at least 4 types which are arranged in heterogeneous layers. The zonal arrangement of the muscles does not occur on the cross-sections in the vicinity of muscle insertions. In these regions only two muscle fibre types are present and the total number of fibres is smaller by 70% than in the central region of the muscle. Most numerous are muscle fibres in the rectus inferior muscle, while the smallest number of fibres is found in rectus interior muscle. Three distinct types of nerve fibres are distinguished according to the following criteria: occurrence and thickness of myelin sheath, fibre diameter and ratio "g". The fibres with thin myelin sheaths indicate small diameters (1-5--6- mum) and their ratio "g" equals 0-82 +/- 0-08. They constitute about 30% of the myelinated fibres in the nerve supply of the oculorotatory muscles and about 14% in the supply of the retractor bulbi muscle. Both the value of the ratio "g" and a greater number of these fibres in the nerve supply of the muscles that contain slow contracting muscle fibres indicate that they are rather slow conducting nerve fibres. The range of the diameters of the fibres with thick myelin sheaths is greater (3-5--13-5 mum) and their "g" equals 0-66 +/- 0-06. These fibres constitute about 70% of the myelinated ones in the nerve supply of the oculorotatory muscles and 86% in the supply of the retractor bulbi muscles. The value of the ratio "g" in these fibres indicates that they are fast contracting ones. The smallest diameters are found in the myelinated fibres (0-5--1-7 mum). These fibres occur frequently in all the examined nerves; they constitute 36--47% of the total number of all the nerve fibres. The frog extraocular muscles are characterized by an abundal nerve supply which is reflected in the low innervation ratio (1:4--1:5). On the distal cross-section of nerves the number of nerve fibres is greater than on the proximal ones. Ganglionic neurons occur sporadically around the nerves; in the nerve III synaptic contacts between two neurons were observed.     

The Fine Anatomy of the Optic Nerve of Anurans—An Electron Microscope Study

In the optic nerve of Anurans numerous myelinated and unmyelinated axons appear under the electron microscope as compact bundles that are closely bounded by one or several glial cells. In these bundles the unmyelinated fibers (0.15 to 0.6 µ in diameter) are many times more numerous than the myelinated fibers, and are separated from each other, from the bounding glial cells, or from adjacent myelin sheaths, by an extracellular gap that is 90 to 250 A wide. This intercellular space is continuous with the extracellular space in the periphery of the nerve through the numerous mesaxons and cell boundaries which reach the surface. Numerous desmosomes reinforce the attachments of adjacent glial membranes. The myelinated axons do not follow any preferential course and, like the unmyelinated ones, have a sinuous path, continuously shifting their relative position and passing from one bundle to another. At the nodes of Ranvier they behave entirely like unmyelinated axons in their relations to the surrounding cells. At the internodes they lie between the unmyelinated axons without showing an obvious myelogenic connection with the surrounding glial cells. In the absence of connective tissue separating individual myelinated fibers and with each glial cell simultaneously related to many axons, this myelogenic connection is highly distorted by other passing fibers and is very difficult to demonstrate. However, the mode of ending of the myelin layers at the nodes of Ranvier and the spiral disposition of the myelin layers indicate that myelination of these fibers occurs by a process similar to that of peripheral nerves. There are no incisures of Schmidt-Lantermann in the optic myelinated fibers.     

Histological analysis of the aortic nerve in the rat raised in space (Rapid communication on Neurolab Project).

To study development of the aortic nerve baroreflex under conditions of microgravity, we examined the cross section of the left aortic nerve (LAN), which is the afferent of the baroreflex, in the neonate rats aged 25 days raised in microgravity on the space shuttle Columbia (flight:FLT group) for 16 days. In this paper, we report a part of the result obtained from the data of the myelinated fibers of LAN analyzed with an electron microscope. Two kind of ground control groups were compared to the FLT group; one was asynchronous ground control (AGC) group where the rats were housed in the same cage as that on the shuttle, and the other was vivarium(VIV) group where the rats were housed in a commercial cage. The LANs in each group were extirpated the from rats perfused with a fixative and embedded for histological analysis. We observed the transverse sections of LAN and took pictures of several areas (magnified to x 2K to x 200K). No irregular myelination was found in all fibers of FLT group when they were compared with two control groups. The thickness of myelin of the maximally myelinated fibers were 0.55 +/- 0.17 micrometer in FLT(n=5), 0.45 +/- 0.10 micrometer in AGC(n=5), and O.47 +/- 0.06 micrometer meter in VIV(n=5). There was no significant difference among three groups (unpared t-test). The results suggest that there is no effect of space environment on the myelin formation of each nerve fiber in the aortic nerve.     

THE ULTRASTRUCTURE OF ADULT VERTEBRATE PERIPHERAL MYELINATED NERVE FIBERS IN RELATION TO MYELINOGENESIS

Adult chameleon myelinated peripheral nerve fibers have been studied with the electron microscope in thin sections. The outer lamella of the myelin sheath has been found to be connected as a double membrane to the surface of the Schwann cell. The inner lamella is connected as a similar double membrane with the double axon-Schwann membrane. The relations of these double connecting membranes suggest that the layered myelin structure is composed of a double membrane which is closely wound about the axon as a helix. These findings support the new theory of myelinogenesis proposed recently by Geren. The possible significance of these results with respect to cell surface membranes and cytoplasmic double membranes is discussed.     

Conduction velocities and fiber diameters in a cutaneous nerve of the pigeon

Summary The characteristics of fibers of a cutaneous nerve supplying the wing skin of the pigeon have been investigated with electrophysiological and electron microscopic techniques.Recordings of the compound action potential showed four distinct peaks with conduction velocities of about 30 m/s, 12 m/s, 4 m/s and 0.5 m/s.From electron micrographs both fiber diameters and thickness of myelin sheath were assessed and used as criteria for segregating various fiber populations. Altogether four groups could be discerned: large thickly myelinated fibers, small thickly myelinated fibers, small thinly myelinated fibers, and unmyelinated or C-fibers. The subdivision of the thickly myelinated fibers into two populations is evidenced mainly by corresponding peaks in the compound action potential. The thinly myelinated fibers with a mean diameter of 2 m contributed about 90% of all myelinated fibers in this nerve.When comparing fiber dimensions and conduction velocities of this avian nerve with those of mammalian cutaneous nerves, the lower CV's of avian nerve fibers can be explained by smaller diameters and thinner myelin sheaths.The results of this investigation are a prerequisite for latency considerations in central somatosensory pathways in birds.Abbreviations CAP compound action potential - CV conduction velocity - D fiber diameter - d axon diameter - g ratio d/D - m thickness of myelin sheath     

Morphometric Analysis of Normal, Mutant, and Transgenic CNS: Correlation of Myelin Basic Protein Expression to Myelinogenesis

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)