Immunolocalization of membrane skeletal protein, 4.1G, in enteric glial cells in the mouse large intestine

4.1 family proteins are membrane skeletal proteins that interact with spectrin-actin networks and intramembraneous proteins. We reported that one of them, 4.1G, was immunolocalized in myelinated nerve fibers of the mouse peripheral nervous system, especially along cell membranes of paranodes and Schmidt-Lanterman incisures in Schwann cells. In this study, to examine 4.1G's appearance in unmyelinated peripheral nerve fibers, we focused on the enteric nervous system in mouse large intestines. In intestinal tissues prepared by an "in vivo cryotechnique" followed by freeze-substitution fixation, 4.1G was immunolocalized in Auerbach's myenteric plexus and connecting nerve fiber networks. Its immunostaining was mostly colocalized with glial fibrillar acidic protein, a marker of enteric glial cells, but not with c-Kit, a marker of interstitial cells of Cajal. Using whole-mount preparation after splitting inner and outer muscle layers, the nerve fiber networks including the plexus were clearly detected by the 4.1G immunostaining. By conventional pre-embedding immunoelectron microscopy, 4.1G was detected along cell membranes of enteric glial cells and their processes surrounding axons. These indicate that 4.1G may have some roles in adhesion and/or signal transduction in unmylinated PNS nerve fibers.     

The twitcher mouse. An alteration of the unmyelinated fibers in the PNS.

The twitcher is an authentic murine model of globoid cell leukodystrophy (GLD) in man. Extensive demyelination of the central and peripheral nervous systems (CNS and PNS) characterizes the neuropathologic features of GLD. In the common peroneal nerve of the twitcher, where demyelination was extensive, pronounced morphologic and quantitative alterations were noted in the unmyelinated fibers. They were 1) a large number of long and attenuated cellular processes of Schwann cells, which often enclosed only one or two axons; and 2) a threefold increase in the number of Schwann cell-axon units with reduced numbers of axons per unit. These results suggested increased branching of unmyelinated Schwann cells. Mild increase in unmyelinated fibers and mild decrease in myelinated fibers were additional features. In contrast, the sympathetic nerve trunk, which had only small numbers of myelinated and rare or no demyelinated fibers, showed much milder alterations in the unmyelinated fibers. Thus, the results of our study suggest that the alterations of the Schwann cells of the unmyelinated fibers in the twitcher are secondary to or in association with the chronic demyelinating process.     

Topographical significance of membrane skeletal component protein 4.1B in mammalian organs

The polarized architecture of epithelial cells is a fundamental determinant of cell structures and functions. Both formation and orientation of proper epithelial polarity are needed for cell-cell or cell-matrix adhesion, signal transduction and cytoskeletal interactions of multimolecular complexes at apical, lateral and basal cell membranes. These cell membrane domains are usually segregated by some junctional complexes. Recent molecular genetic studies on the anchor structure between myelin sheaths and axons have indicated the specific molecular organization for polarization of axolemma and the myelin sheaths at paranodes, termed 'septate-like junctions'. It was also speculated that other mammalian organs may use a similar junctional system. The protein 4.1 B was originally found to be localized in paranodes and juxtaparanodes of myelinated nerve fibers. Our recent immunohistochemical studies on protein 4.1B have indicated its significance for the cell-cell and/or cell-matrix adhesion in various rodent organs. The protein 4.1 family of proteins have been supposed to possess variable molecular domains relating to cell adhesion, ion balance, receptor responses and signal transduction. Therefore, more precise studies on the molecular structure and the functional domains of protein 4.1B, as well as on its changes under physiological and pathological conditions, may provide a clue for organogenesis in various mammalian organs.     

Axo-glial relations in the retina-optic nerve junction of the adult rat: electron-microscopic observations

Summary The retina-optic nerve junction (ROJ) was examined by electron microscopy in adult rats, with particular emphasis on the unmyelinated-myelinated nerve fibre transition. Both single sections and serial sections were used. The non-retinal part of the ROJ is covered by an extensively folded glia limitans, facing the choroidea, sciera and pia mater. The blood vessels within the ROJ follow a transverse course and are surrounded by unusually wide perivascular spaces with a glia limitans-like outer delimitation. The endothelial cells exhibit numerous pinocytotic vesicles on their abluminal aspect. In the unmyelinated part of the ROJ the axons are embedded in an extensive meshwork of fibrous astrocytic processes. Some unmyelinated axons exhibit patches of axolemmal undercoating with externally associated astrocytic processes. Typical oligodendrocytes are not found, but a few small dark glial cells of unknown identity can be observed. Atypical ensheathment and myelination of axons at this level by ectopic Schwann cells occurred in one case. In the transition segment of the ROJ a pattern similar to that along dysmyelinated axons is observed, including aberrant axo-glial contacts, unusually thin and short myelin sheaths, intercalated unmyelinated segments, distorted myelin termination regions, bizarre paranodes and myelin termination regions without associated nodally differentiated axolemma. Neither sheath length nor number of myelin lamellae is related to axon diameter in the transition region. Axon diameter tends to be somewhat larger at myelinated than unmyelinated levels of the same axon. We suggest that the unusual axo-glial relations in this region are due to a deficient proliferation and differentiation of oligodendroglial cells, and that the pattern of glial ensheathment in the ROJ might be a consequence of the locally deficient blood-brain barrier.     

Vagal nerve maturation in the fetal lamb: An ultrastructural and morphometric study

The maturation of the left vagal nerve was studied in the fetal lamb by transmission electron microscopy and by computer-assisted morphometry of sections of the entire nerve at seven gestational ages between 79 and 145 days (term is 147 days) and in the adult ewe. The number of unmyelinated axons per Schwann cell progressively decreased from 25 to 55 at 79 days to 1 to 5 at near-term. Unmyelinated axons of various sizes were enclosed within a single Schwann cell at all ages, but the mean axonal diameter increased in inverse relation to the number of unmyelinated axons. A few Schwann cells enclosed two myelinated axons, but in most instances myelination did not begin until a 1:1 ratio was achieved; some single axons with a Schwann cell remained unmyelinated in the adult. Myelinated fibers were rare at 79 days but myelination progressed rapidly thereafter until the adult ratio of myelinated: unmyelinated fibers was reached at about 100 days; myelinated axons were not uniformly distributed. The myelin sheaths and axons of small fibers progressively increased in diameter in late gestation, but new large fibers were not added. Early myelinating fibers and immature unmyelinated axons contained more microtubules than neurofilaments; neurofilaments predominated in mature axons with or without myelin. Cross-linkages between neurofilaments were already evident by 79 days. Maturation of the vagal nerve thus occurs first by an increase in number of myelinated fibers and then by an increase in the size of each fiber in this fixed population. The bimodal distribution in the size histogram of myelinated fibers is not achieved until 134 days gestation and correlates well with physiological maturation of respiratory patterns. © 1993 Wiley-Liss, Inc.     

Quantitative study of the apical nerve fibers of adult and juvenile rat molars

The rat molar has become an important model for studies of interactions between nerves and the pulp-dentin complex, yet there is only limited quantitative information on the number and size distribution of axons entering the roots of this tooth. This study was undertaken to provide such a detailed characterization of the apical innervation of the rat molar. An additional objective was to compare the apical nerve composition of young, recently erupted rat molars with that of mature teeth in order to determine whether there is ongoing maturation of the innervation after the teeth have attained functional occlusion. A complete census was made of the nerve fibers entering the roots of both mature and recently erupted juvenile mandibular first molars in Sprague-Dawley rats. Each of the four roots of the first molars was processed for electron microscopy of thin sections near the apex. The majority of intradental nerve fibers entered the molar via the two larger (mesial and distal) roots. Within the apical root pulp, most, but not all, axons occurred within well-defined fascicles associated with blood vessels. Molars from adult animals (age 4 months) had a mean total of 232 (S.D. = 49, N = 7 teeth) myelinated fibers and 806 (S.D. = 143) unmyelinated axons entering the four roots. Fibers exceeding the Aδ size range (circumference ≥ 19 μm) accounted for only 4% of the myelinated axons at the apex. Molars from juvenile animals (age 4 weeks) had fewer myelinated fibers (mean 176, S.D. 18, N = 8), but more unmyelinated axons (mean 1,174, S.D. 160) than adults. The mean ratio of unmyelinated axons to myelinated axons was 6.6:1 for juveniles compared to 3.5:1 for adults. Juvenile teeth contained no myelinated fibers that exceeded 19 μm in circumference. These results indicate that the innervation of the rat molar resembles that of teeth of non-rodent mammals in that (1) innervation density is high, (2) there is a high ratio of unmyelinated axons, and (3) most of the myelinated fibers are of thin caliber. Furthermore, it appears that after the molar erupts, maturation of the nerve fiber composition continues with processes that include both a marked decrease in the number of unmyelinated axons and an increase in the number and size heterogeneity of myelinated fibers. © 1994 Wiley-Liss, Inc.     

Development of astrocytes in the lamina cribrosa sclerae of the mouse optic nerve,with special reference to myelin formation

In the mouse optic nerve, the optic nerve fiber layer in the retina, the optic papilla and the lamina cribrosa sclerae (LCS) just after penetrating the eyeball failed to generate myelin, whereas the optic nerve proper in the orbit was occupied by myelinated nerve fibers. The present study investigated development of the architecture of LCS, where the axons develop from unmyelinated to myelinated type, to elucidate how the initial part of axons was unmyelinated. At the LCS of the adult optic nerve, well developed astrocytes densely formed a cytoplasmic mesh-like frame through which unmyelinated fibers passed. The astrocytes here contained numerous and densely packed intermediate glial filaments and cell organelles. This framework formed by astrocytes appeared to be completed between 7 and 14 postnatal days before oligodendrocyte progenitors, migrated from the chiasm side, reached the proximal end of LCS, and began myelin formation. Thus the failure in myelin formation at the intraocular part and LCS possibly depended upon unsuccessful migration of oligodendrocytes beyond LCS constructed by specialized astrocytes, although other inhibitory factors for myelin formation, such as adhesion molecules distributed around LCS, may be unsolved.     

Abnormalities of smooth muscle, basal laminae, and nerves in the aganglionic segments of the bowel of lethal spotted mutant mice

The terminal portion of the bowel of the lethal spotted mutant mouse (ls/ls) lacks an enteric nervous system due to the failure of neural crest precursors to colonize this region during embryonic life. As a result, the mouse develops congenital megacolon. We have postulated that the defect occurs because the microenvironment of the aganglionic segment is segmentally abnormal and does not permit the migration and/or survival of the enteric neural or glial precursors in the affected zone. We have examined the terminal segment of adult ls/ls and control mice by light and electron microscopy to determine if the defect is associated with identifiable structural abnormalities that persist to maturity. A striking abnormality is an overgrowth of the muscularis mucosa in the adult ls/ls mouse, particularly in the outer longitudinal layer. Electron microscopy also reveals an extensive thickening of the basal lamina around smooth muscle cells. In addition, nerves that are derived from fibers that are extrinsic to this area are abnormal. Large bundles of nerve fibers, some of which contain myelinated axons, large-caliber unmyelinated axons, and abundant collagen, are prominent in the intermuscular region of the aganglionic segments and often reach into the submucosa. The supporting cells of the unmyelinated and myelinated nerves in the aganglionic segment have voluminous perineural cytoplasm typical of immature Schwann cells. They also exhibit intermediate filaments in their cytoplasm. Otherwise they have the typical morphology of peripheral Schwann cells, rather than enteric glia, including individual ensheathment of axons and a surrounding basal lamina. We suggest that the extracellular matrix and/or cells of mesenchymal origin of the terminal bowel of the ls/ls mouse may prevent the ingrowth of the normal precursors of the glia as well as neurons of the enteric nervous system, but may permit or even encourage the ingrowth of abnormal numbers of extrinsic axons.     

放射性物质辐射后的异种神经移植

将经~(60)Co辐射、长8mm的成年狗的胫神经移植于纯种成年新西兰兔的胫神经。术后4、8、10、12周可见再生神经纤维通过近端吻合部长入移植神经,继而越过远端吻合部向远段胫神经延伸,移植神经和远段胫神经内的再生有髓和无髓纤维成束集聚或散在分布,再生轴突被Schwann细胞包绕。8周后的腓肠肌AChE阳性,AChE结合镀银染色的切片上可见再生神经终末与银颗粒覆盖的AChE阳性部位——运动终板相连。各存活期的移植神经和远段胫神经均未见淋巴细胞和浆细胞。     

Morphofunctional Interactions of Peripheral Nerve Fibers of the Iris with Neurons Developing in the Anterior Chamber of the Eye in Rats

Electron microscopic studies were performed on intraocular transplants of embryonic septal and hippocampal tissue developing in the anterior chamber of the eye in rats for 3–4 months. The aim of the study was to seek ultrastructural identification of peripheral nerve fibers entering transplants from the iris, and to assess their ability to establish true synaptic contacts with transplanted CNS neurons. Bundles of myelinated and unmyelinated axons surrounded by Schwann cell cytoplasm were seen within the perivascular spaces of ingrowing blood vessels. Both types of peripheral fiber were also identified in the neuropil areas of transplants. At the ultrastructural level, unmyelinated axons were found to be free of glial Schwann cell sheaths and to form typical asymmetrical synapses with the dendrites and dendritic spines of transplant neurons. These results provide evidence of the high morphofunctional plasticity of both parts (central, peripheral) of the nervous system.     

The ultrastrcture of wallerian degeneration in the severed optic nerve of the newt (Triturus viridescens)

Wallerian degeneration in the severed newt's (Triturus viridescens) optic nerve is complete between the 10–14th post operative day (p.o.d.). Consequently, the newt optic nerve displays one of the most rapid degenerative responses yet reported for the central nervous system of vertebrates. In most cases it also exhibits the speed of degenerative phenomena in the vertebrate peripheral nervous system. The degeneration of unmyelinated axons is most rapid and is completed by 2–3 p.o.d., compared to myelinated axons, most of which degenerate between 2–10 p.o.d. Myelin ring formation (vesicular transformation) is the principal form of lamellar breakdown and occurs in a highly organized manner which can be clearly staged. The glial cell response to Wallerian degeneration is two-fold: cytoplasmic hypertrophy and myelin-lytic. Glial hypertrophy subsides by the 10–14 p.o.d. with the ingrowth of numerous regenerating nerve fibers. The myelin-lytic response accounts for most of the myelin destruction. Leukocyte-like and microglialike cells also participate in myelin breakdown but to a lesser degree.     

In vivo proliferation,migration and phenotypic changes of Schwann cells in the presence of myelinated fibers

Following injury to a peripheral nerve, changes in the behavior of Schwann cells help to define the subsequent microenvironment for regeneration. Such changes, however, have almost exclusively been considered in the context of Wallerian degeneration distal to an injury, where loss of axonal contact or input is thought to be critical to the changes that occur. This supposition, however, may be incorrect in the proximal stumps where axons are still in contact with their cell bodies. In this work, we studied aspects of in vivo Schwann cell behavior after injury within the microenvironment of proximal stumps of transected rat sciatic nerves, where axons are preserved. In particular we studied this microenvironment proximal to the outgrowth zone, in an area containing intact myelinated fibers and a perineurial layer, by using double immunolabelling of Schwann cell markers and 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells.In normal sciatic nerve, Schwann cells were differentiated, in an orderly fashion, into those associated with unmyelinated fibers that labeled with glial fibrillary acidic protein (GFAP) and those associated with myelinated fibers that could be identified by individual axons and myelin sheaths. After sciatic nerve transection, there was rapid and early expansion in the population of GFAP-labeled cells in proximal stumps that was generated in part, by de novo expression of GFAP in Schwann cells of myelinated fibers. Schwann cells from this population also underwent proliferation, indicated by progressive rises in BrdU and GFAP double labeling. Finally, this Schwann cell pool also developed the property of migration, traveling to the distal outgrowth zone, but also with lateral penetration into the perineurium and epineurium, while in intimate contact with new axons.The findings suggest that other signals, in the injured proximal nerve stumps, beyond actual loss of axons, induce 'mature' Schwann cells of myelinated axons to dedifferentiate into those that up-regulated their GFAP expression, proliferate and migrate with axons.     

大鼠坐骨神经超微结构的老年性变化

为了研究老年大鼠坐骨神经超微结构特点,随机取3月龄(成年组)和24月(老年组)龄正常SD大鼠各10只,用电镜观察两组间坐骨神经超微结构的差异。结果显示:老年组大鼠坐骨神经内有髓纤维的百分比、轴突间胶原纤维密度以及Schwann细胞胞质中脂褐质沉积密度均多于成年组(P<0.05);但无髓纤维之百分比少于成年组(P<0.05)。上述结果提示坐骨神经内的有髓纤维与无髓纤维百分比、轴突间胶原纤维密度以及Schwann细胞胞质中脂褐质沉积密度是衡量大鼠坐骨神经老化的形态标志之一。     

Myelination of regenerated optic fibers in peripheral nerve graft of adult cats

Retinal ganglion cells of adult cats have the potential to regenerate their axons into autografted peripheral nerve. Two months after transplantation of the sciatic nerve to the axotomized optic stump, regenerated axons were labeled anterogradely with biocytin, and myelin formation by Schwann cells was examined electron microscopically. Both myelinated and unmyelinated fibers were labeled with biocytin. Among 511 axons labeled in three grafts, 96 fibers (18.8%) were myelinated and 415 (81.2%) were unmyelinated. Mean diameter with SD of myelinated fibers was 1.28 ± 0.39 m (range 0.71–2.47) and that of unmyelinated fibers was 0.76± 0.38 m (range 0.18–2.46). The ratio of inner to outer diameters of the myelin sheath (g value) was 0.82, which is close to the value (0.8) for the optic fibers of intact adult cats.     

Unmyelinated nerve fibers

The paper presents a critical review of various current concepts of the structure and kinetics of unmyelinated nerve fiber. A classification of nerve fibers, different from the earlier ones, is proposed, that demonstrates not only the morphological fiber types, but also the kinetics of their reversible transition stages from non-glial to myelinated fiber. Evidence is presented to show the erroneousness of conceptions, still appearing in many publications, that consider the unmyelinated nerve fiber as "the Remak's cable type fiber". According to the current data, "Remak's fiber" is a glial-neurite complex, i.e. a bundle of unmyelinated nerve fibers covered with a single glial cell. Using the electron microscope, it was demonstrated that comparable glial-neurite complexes of myelinated nerve fibers, formed in CNS in a similar way by a single oligodendrocyte, cannot be named a single fiber. Cutting the nerves makes visible that the single fibers forming "the Remak's fiber" stem from different cells, therefore they cannot be a single "fiber". It has been shown for the first time experimentally, that in extreme situations, as a result of contraction of gliocyte processes, unmyelinated fibers may "leave" the glial-neurite complexes and become the nonglial fibers. Some data are presented that may serve as criteria for differentiation of unmyelinated fiber with a stratified sheath from developing myelinated fiber.     

Nerve fiber caliber analysis in the mouse facial trunk distal to the stylomastoid foramen with the electron microscope

Nerve fiber counts and caliber analysis were performed with the electron microscope on 5 facial trunks distal to the stylomastoid foramen from 3 mice. On an average, about 89% of the total nerve fibers (3,205) were myelinated and about 11% unmyelinated. Majority (80.5%) of the myelin sheaths of the myelinated fibers in the nerves were 2.0-4.5 microns and majority (86.4%) of their axons were 1.0-3.0 micron in minor diameter. Their frequency distributions were similar in pattern to that of the large fiber zone of the motor root of the mouse facial nerve. Majority (86%) of the unmyelinated fibers in the facial trunk distal to the stylomastoid foramen were 0.1-0.5 micron in minor diameter. Its frequency distribution showed that the unmyelinated fibers of the facial trunk tend to be rather smaller than that of the other nerves examined in the previous studies. Ratios of the circumferential length of the myelinated fibers to that of the circles with the same areas ranged from 1.00 to 1.98, with 1.02-1.10 being most frequent (72%). Ratios of the axons of the myelinated fibers ranged from 1.00 to 2.54, with 1.04-1.14 being most frequent (51.8%). Ratios of the unmyelinated fibers ranged from 1.00-1.18 (84%), and those which over 1.20 were rare.     

A light and electron microscopic study on pulpal nerve fibers in the lower incisor of the mouse.

Light and electron microscopic studies were made on pulpal nerve fibers in mouse lower incisors, typical continuously growing teeth. Serial sections, from the apex of the odontogenic sheath to the incisal edge of the apical foramen, were examined by light microscopy to identify myelinated fibers passing through the apical foramen. The fine structure of the pulpal nerves was examined by electron microscopy at three sites: 1) the level at the incisal edge of the apical foramen; 2) a level 5 mm incisal from the apex of the odontogenic sheath; and 3) the level where the incisor comes out of the alveolar bone. No myelinated fibers were found passing through the apical foramen; they were also lacking at the three levels of the pulp. At level 2, unmyelinated axons were seen in close contact with smooth muscle fibers of arterioles. At level 3, nerve fibers were difficult to distinguish from processes of fibroblasts and odontoblasts. Degenerating axons were present in Schwann cells, and fine unmyelinated axons running through the odontoblast cell layer were seen. Various types of unmyelinated axons were observed in the apical region (level 1). These axons were classified into 6 types on the basis of their fine structures: Type I, bundles of unmyelinated axons completely or partly ensheathed by Schwann cell cytoplasm (mature type); Type II, bundles of unmyelinated axons in a space formed by a Schwann cell membrane (regenerating type); Type III, bundles of unmyelinated axons ensheathed not by a Schwann cell, but merely by a basal lamina (regenerating type); Type IV, single axons in direct contact with the basal lamina (regenerating or terminal type); Type V, naked, electron-dense axons with many vesicles and mitochondria (growth cone-like type); and Type VI, electron opaque axons, due to loss of axonal organellae (degenerating type). The significance of these structures is discussed in relation to the continuous growth of the rodent incisor.     

Nerve fiber composition of the intracranial portion of the oculomotor, trochlear, and abducens nerves in the sheep

In the present investigation, the fiber content and the diameter spectra of the intracranial portion of the three oculomotor nerves (oculomotor, trochlear, and abducens nerves) were analysed in sheep by light and electron microscopy. It was determined that up to 14.98% of fibers in the oculomotor nerve, 17.01% in the trochlear nerve, and 11.87% in the abducens nerve were unmyelinated. The myelinated fibers showed a bimodal distribution in their size spectrum in all three nerves, with a majority of large myelinated axons, but a considerable proportion of small myelinated fibers, as well. The sensory function of the unmyelinated fibers present in the three oculomotor nerves is discussed also on the basis of our previous morphofunctional investigations.     

Nerve fiber analysis and age-related changes of the human mandibular nerve

We morphometrically analyzed nerve fibers of the human mandibular nerve with a discriminative staining method that makes it possible to separate nerve fibers into myelinated and unmyelinated fibers. We counted numbers and transverse areas of myelinated axons under the microscope using an on-line image-analyzer. This study revealed the morphometric changes which affect the human mandibular nerve during the aging process.     

Role of transthyretin in posttraumatic regeneration of the sciatic nerve in mouse

Although thyroid hormones are known to have a significant influence on the development of nervous system, the absence of changes in the brain of mice deficient in transthyretin--a protein providing thyroid hormone transport across the blood-brain and blood-nerve barrier--remains unexplained. Therefore, the aim of this study was to determine the effect of transthyretin on the formation of myelinated and unmyelinated nerve fibers in sciatic nerve of mice. The myelinated and unmyelinated nerve fibers were counted in sciatic nerve of 3-months-old normal and transthyretin-knockout (transthyretin(-/-)) mice 15 and 30 days after nerve crushing. No differences were detected in the number of myelinated and unmyelinated nerve fibers in intact control (wild-type) animals group vs. transthyretin(-/-) mice. By days 15 and 30 after nerve crushing the number of myelinated nerve fibers was diminished by 54.7 and 71.8%, respectively, in transthyretin(-/-) mice, as compared to that in control animals. The number of unmyelinated nerve fibers at day 15 after the injury was not different in transthyretin(-/-) and control mice, however, by day 30 the number of these fibers in control group was found to increase significantly, exceeding that one in transthyretin(-/-) mice by 27.9%. These results indicate the important contribution of transthyretin, as a thyroxin carrier protein, to the process of posttraumatic regeneration of sciatic nerve. The absence of changes in nerve fiber numbers in transthyretin-knockout mice in postembryonic period suggests the presence of transthyretin-independent mechanism of thyroxin transport into the peripheral nervous system.