Neurofilament M and calbindin D28k are present in mutually exclusive subpopulations of enteric neurons in the rat submucous plexus

Immunocytochemical methods have been used to examine the localisation of 3 neurofilament proteins and the calcium binding protein, calbindin D_28k, in whole mount preparations of the submucous plexus in the Wistar rat. Neurofilament-M (160 kDA protein) was present in 40% of the submucosal neurons, staining fine filaments in the soma and the axonal processes. Calbindin D_28k was present in 40% of the submucosal neurons staining both the soma and nerves within the plexus. The neurofilament proteins and calbindin D_28k were never observed within the same neurons. Neurofilament-M was co-localised with substance P and calcitonin gene-related peptide but not somatostatin or the other neuropeptides investigated. Calbindib D_28k was co-localised with vasoactive intestinal polypeptide and neuropeptide Y. Galanin- and somatostatin-immunoreactive neurons did not contain either the neurofilament proteins or calbindin D_28k. The results demonstrate the presence of subsets of submucosal neurons that can be distinguished by the presence of neurofilament-M or calbinsin D_28k.     

Intrathecal vincristine: a fatal chemotherapeutic error with devastating central nervous system effects

Summary The devastating neuropathological changes wrought by the intrathecal administration of vincristine are reported, with a detailed account of the widespread lesions in the brain and spinal cord, found in post-mortem light- and electron-microscope studies.     

Developmental patterns of intermediate filament gene expression in the normal hamster brain

We have examined the patterns of expression of the major intermediate filament (IF) protein mRNAs during development of the hamster brain. Quantitative northern blotting was used to examine changes in the levels of mRNAs for the low, middle and high molecular weight neurofilament proteins (NF-L, NF-M, NF-H) as well as peripherin, vimentin and glial fibrillary acidic protein (GFAP). Total RNA was isolated from hamster brains at embryonic (E) days 12 and 14 and postnatal (P) days 1, 3, 5, 7, 9, 11, 13, 15, 20, 28 and 60-90 (adult), and probed with specific IF cDNAs. Northern blotting revealed that NF-L and NF-M mRNAs were present at very low levels in embryonic brain and that significant expression of these genes only occurred postnatally when the levels increased dramatically until P28 and then declined again in the adult. Increases in NF-H mRNA levels were somewhat delayed relative to those of NF-L and NF-M. NF-H mRNA was not seen at embryonic stages and was expressed at very low levels prior to P9; after that time the levels increased rapidly until P28 and then declined in the adult. Two of the type III IF genes, peripherin and vimentin, followed a pattern of expression opposite that of the NF genes. Both peripherin and vimentin mRNAs were present in embryonic brain and were expressed at higher levels during early postnatal stages than at later times. The magnitude and rate of reduction in vimentin gene expression in the postnatal interval was much greater than that of peripherin. GFAP mRNA levels were extremely low prior to P9 after which a robust increase occurred, followed by a decline in the adult. We discuss the implication of the dramatic changes in IF isotype expression in brain to the pathways of both neuronal and glial development in vivo.     

Immunohistological study of grumose degeneration of the dentate nucleus in progressive supranuclear palsy

The grumose degeneration observed in the dentate nuclei of 7 cases of progressive supranuclear palsy (PSP) was studied with a panel of antibodies which included 2 neurofilaments, Tau and ubiquitin. Dentate nucleus neurons were negative with all antibodies except ubiquitin which showed a slightly positive homogeneous pattern of staining. The amorphous material surrounding swollen or normal neurons was strongly positive for neurofilament and subunits and numerous torpedoes were observed in the granular layer of the cerebellar cortex. Our results confirm that grumose degeneration consists in degeneration of terminal axons of Purkinje cells in the dentate nucleus. The positivity of dentate nucleus neurons for ubiquitin may support the concept of synaptic dysfunction between Purkinje cells and dentate nucleus neurons.     

Morphogenesis of the primary arterial trunks of the forelimb in the rat embryos: the trunks originate from the lateral surface of the dorsal aorta independently of the intersegmental arteries

It has been believed that the primary arterial trunk of the mammalian forelimb is derived from the 7th intersegmental artery. Here we examined the early morphogenesis of the arteries and nerves in the forelimb region by adopting a method that combined intravascular dye-injection with nerve staining to whole mounted rat embryos. The study was carried out on greater numbers of specimens at smaller intervals of embryonic stages and from earlier stages than those in previous reports. We report that: (1) The multiple primary arterial trunks in the forelimb region (primary subclavians) originate directly from the lateral surface of the dorsal aorta independently of the intersegmental arteries, previous to the formation of limb buds. (2) The tips of the 8th (and the 9th) primary subclavians that originate from the aorta near the origin of the 8th (or the 9th) intersegmental artery bend cranially and/or caudally. With the formation of limb bud, they extend to form the longitudinal trunks in the presumptive axillary region. The primary arteries in the free arm region branch off from this longitudinal trunk, and one of them develops into the axial artery. (3) The origins of the primary subclavians shift their positions on the surface of the dorsal aorta and approach the origins of the neighboring intersegmental arteries to join them, and then replace the latter. Consequently, the primary subclavians appear to be ”the lateral branches of the in tersegmental arteries.” (4) The 8th primary subclavian is dominant at first, but is replaced by the 7th primary subclavian, which develops into the definitive subclavian artery. (5) With the brachial nerve plexus formation, the axillary arterial plexus derived from the longitudinal trunk develops to form two stems of the axillary artery. Accepted: 15 April 1999     

Ultrastructural observations on the vincristine-induced neuronal crystalloid inclusion in young rats

Summary Vincristine-induced crystalloid inclusions were examined in the neurons and neuronal processes of young rats by the electron microscope (EM) equipped with a tilting stage. Using a computer system that reproduces three-dimensional organization, an optical transformation method was applied to the microtubules and neurofilaments in an attempt to clarify the morphological appearance and internal pattern of crystalloid inclusions. The dimensional models obtained were compared with actual EM photographs, and the characteristic ultrastructural component and morphology were drawn out.Basically, a crystalloid inclusion is composed of four strands of intermediate 10 nm neurofilaments connected to one another by four side-arms producing a circular profile on a transverse section. These four side-arms seemed to arise from nodules within the filaments at regular intervals simulating a bead-like appearance. These data did not significantly differ from those obtained from EM images. Characteristically, these crystalloid inclusions began to appear 6 h after the administration of 10^–3 M vincristine sulfate and persisted up to a period of 6 days. Beyond that, however, these inclusions were no longer demonstrable suggesting a transient state. This was in contrast to neurofibrillary tangles which appeared to be permanent changes.     

Characterization of a shared epitope in cortical Lewy body fibrils and Alzheimer paired helical filaments

The straight fibrils of the Lewy body contain an epitope related to phosphorylation of the KSPV motif common to the C termini of the 200- and 170-kDa neurofilament subunits and . To further characterize this phosphorylated neurofilament/ epitope in Lewy bodies and to analyze the constituents of isolated Lewy bodies we used a combined biochemical and immunochemical approach. In formalin-fixed paraffin-embedded tissue cortical Lewy bodies were labelled by monoclonal antibodies directed to phosphorylation-dependent KSPV epitopes in the sequences of neurofilament and phosphorylation-independent epitopes. Immunoblotting of solubilized Lewy body fibrils with the same antibodies which stained Lewy bodies in tissue sections revealed that the immunoreactive Lewy body proteins were phosphorylated neurofilament subunits. An antibody to the 68-kDa neurofilament subunit labelled Lewy bodies and Lewy body protein at 50–68 kDa. We conclude that the shared phosphorylated epitope in Lewy body fibrils and paired helical filaments is related to the common KSPV sequence in neurofilament and , and that all three neurofilament subunits are present in the Lewy body. This result indicates that although Lewy bodies and neurofibrillary tangles share epitopes they are comprised of distinct structural subunits.Supported by grants from the Parkinson Foundation of Canada and the Alzheimer Society of Canada to C.B. M.S.P. is a recipient of a Medical Research Council of Canada Studentship     

Reversibility of neurofilamentous inclusion formation following repeated sublethal intracisternal inoculums of AlCl3 in New Zealand white rabbits

In this report, we describe the clinical, topographical and immunohistochemical characteristics of neurofilament (NF) inclusion formation induced by the intracisternal inoculation of young adult New Zealand white rabbits at 28-day intervals with 100 g AlCl₃ over the course of 267 days. The ability to recover following cessation of aluminum exposure has also been assessed. The extent of neurofilamentous inclusion formation was proportionate to the cumulative amount of AlCl₃ inoculated and initially consisted of fusiform axonal distention in the ventral spinal cord at day 51 following the initial inoculum. Spinal motor neuron perikaryal inclusions and discrete axonal spheroids were observed at day 107 and supraspinal neurofilamentous pathology by day 156. Perikaryal inclusions were immunoreactive to antibodies recognizing both poorly phosphorylated (SMI 32) and more highly phosphorylated high molecular weight NF (NFH). In contrast, axonal spheroids were intensely immunoreactive at all stages with antibodies recognizing highly phosphorylated NFH and an age-dependent NFH phosphorylation state (SMI 34) with only faint SMI 32 immunoreactivity. Immunoreactivity to an antibody recognizing ubiquitin-protein conjugates did not appear until day 156, whereas inclusions were not immunoreactive to antibodies recognizing either phosphatase-dependent or-independent microtubule-associated protein tau at any stage. Upon withdrawal from further AlCl₃ exposure after intervals of 51, 107 or 156 days following the initial inoculum, clinical recovery ensued in all rabbits. In all but the most severely affected rabbits, perikaryal neurofilamentous inclusions resolved. However, axonal spheroids continued to be prominent. However, axonal spheroids continued to be prominent. These studies demonstrate that the repetitive intracisternal inoculation of AlCl₃ in New Zealand white rabbits induces a reversible process of neurofilamentous inclusion formation that preferentially affects motor neurons, and in which recovery will occur in those inclusions containing an admixture of both poorly and highly phosphorylated NFH.     

Axonal cytoskeleton changes in experimental optic neuritis

Axonal loss and degeneration in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE) have been suggested by brain imaging, pathological and axonal transport studies. Further elucidation of the processes and mechanisms of axonal degeneration in demyelinating diseases is therefore of potential importance in order to alleviate the permanent disabilities of MS patients. However, detailed studies in this area are impeded by the small number of reliable models in which the onset and location of demyelination can be well-controlled. In this study, microinjection of polyclonal rabbit anti-galactocerebroside (anti-Gal C) antibody and guinea pig complement was used to induce local demyelination in the rat optic nerve. We found that treatment with appropriate volumes of the antibody and complement could induce local demyelination with minimal pressure- or trauma-induced damage. Local changes in neurofilaments (NFs) and microtubules (MTs) were examined with both immunohistochemistry (IHC) and electron microscopy (EM). On day 1 after microinjection, we observed moderate NF and MT disassembly in the local demyelinated area, although in most cases, no apparent inflammatory cell infiltration was seen. The NF and MT changes became more apparent on days 3, 5, 7 after microinjection, along with gradually increased inflammatory cell infiltration. These results suggested that acute demyelination itself may induce local cytoskeleton changes in the demyelinated axons, and that the ensuing local inflammation may further enhance the axonal damage. When the lesions were stained with specific antibodies for T lymphocytes, macrophages, and astrocytes, we found that most of the cells were macrophages, suggesting that macrophages may play a greater role in inflammation-related axonal degeneration and axonal loss. These results were confirmed and further characterized on the ultrastructural level.     

β,β′-Iminodipropionitrile toxicity in normal and congenitally neurofilament-deficient Japanese quails

Morphological effects of a neurotoxin, ,-iminodipropionitrile (IDPN) were analyzed in normal and cogenitally neurofilament (NF)-deficient Japanese quails. These quails (6 weeks old) were injected intraperitoneally with IDPN (0.2 g/kg body weight) three times every 3 days. They were necropsied at 10 to 12 days after the first injection. In normal quails, axonal swellings were observed histologically in the ventral motoneurons, ventral root, commissura grisea and spinal ganglion in the cervical and synsacral spinal cord. Electron microscopically, the changes consisted of increased NFs, with scattered mitochondria, smooth endoplasmic reticulum and microtubules. The myelin sheaths of the involved nerves were thinner than those of the normal axons. These lesions were similar to those induced by IDPN intoxication in mammalian experimental animals. In NF-deficient quails injected with IDPN, no axonal changes were detected. These findings suggested that IDPN selectively attacked the NFs.