Biomedical subjects

G Allt

Publications and source records attributed to G Allt.

At least 19 recordsLinked to original sources

PubMed1992

Schwann cell endocytosis: a role in nerve regeneration?

Schwann cell plasma membrane vesicles have been shown to increase in numerical density after nerve injury but their function is unclear. In this study, ultrastructural tracers were micro-injected in vivo into crushed rat sciatic nerves after various time intervals to ascertain whether plasma membrane vesicles of Schwann cells are involved in the uptake and utilization of molecules from the endoneurium during axonal regeneration and remyelination. Horseradish peroxidase (HRP), a tracer of fluid-phase endocytosis, was taken up by macrophages and fibroblasts but remained external to Schwann cells throughout the study. After 14-16 days of crush injury, HRP was present within vessel lumina and in cytoplasmic vesicles of pericytes and vascular endothelia. Low-density lipoprotein-gold, which is primarily internalized by receptor-mediated endocytosis, and bovine serum albumin-gold, proposed as a tracer for fluid-phase endocytosis, were internalized by macrophages and fibroblasts but were not taken up by Schwann cells. Although Schwann cells formed pits in the plasma membrane and vesicles were evident in the cytoplasm, none of the tracers used were internalized by Schwann cells. It is suggested that Schwann cell plasmalemmal and cytoplasmic vesicles have a cellular role unrelated to endocytosis or alternatively the Schwann cell basal lamina may function as a diffusion barrier to the tracers employed.

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PubMed1992

Influence of fatty acid content of lysophosphatidyl choline on its myelinotoxic properties.

The efficacy of lysophosphatidyl choline (LPC) type I and type IV in producing demyelination was assessed in rat tibial and sural nerve. By light and electron microscopy, a greater myelinolytic activity was demonstrated with type I, and concomitantly electrophysiology showed a more severe conduction block. In teased nerve preparations and 1-microns thin sections, demyelinated fibres were more frequent with LPC type I. At 1 h after injection, electron microscopy showed much more extensive myelin lysis in the form of fine vesicular debris. By 6 days, completely demyelinated fibres were much more common and associated Schwann cells contained either small quantities or no myelin debris. With type IV LPC, cytopathological changes were more extensive at 1 h. A minority of Schwann cells showed swollen hydropic cytoplasm and degradation of organelles. Axonal retraction from the myelin sheath occurred in occasional fibres, and in a few unmyelinated fibres axoplasm showed organelle depletion and increased granularity. By 6 days, Schwann cells still contained large quantities of gross myelin debris and had often retracted to expose extensive areas of axolemma. The findings suggest that the two types of LPC have different myelinolytic actions, which may be related to their different fatty acid content. A possible role for the two types of LPC in 'bystander demyelination' is considered.

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PubMed1992

Distribution of anionic sites on the perineurium.

The distribution of anionic sites on the perineurial basal lamina (BL) and plasmalemma of dorsal root ganglia and sciatic nerves was determined using cationic ferritin (CF) and cationic gold (CCG). The probes were applied to the tissue before and after resin embedding and visualised by electron microscopy. There were no apparent differences in charge distribution between the 2 tissues. At physiological pH a strong anionic charge was distributed evenly over the BL as demonstrated by pre-embedding labelling with CF; the plasmalemma was only moderately anionic. A similar application of CCG at pH 2.0 revealed a quasi-regular distribution of anionic sites (presumably due to acidic carbohydrate moieties) on the BL, whilst CCG-labelling of L. R. White sections indicated a differential distribution of these moieties on the BL of the inner and outer perineurial lamellae. Cationic ferritin (12 nm diameter) crossed the BL and entered perineurial caveolae, but CCG (effective diameter of 15 nm) did not, suggesting that the BL is a size-restrictive filter. These results are discussed with regard to the ultrastructure and function of the BL of other tissues and the possible role of perineurial BL charge as a determinant of perineurial permeability.

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PubMed1991

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina of dorsal root ganglia.

Previous investigations of the blood-nerve barrier have correlated the greater permeability of ganglionic endoneurial vessels, compared to those of nerve trunks, with the presence of fenestrations and open intercellular junctions. Recent studies have demonstrated reduced endothelial cell surface charge in blood vessels showing greater permeability. To determine the distribution of anionic sites on the plasma membranes and basal laminae of endothelial cells in dorsal root ganglia, cationic colloidal gold and cationic ferritin were used. Electron microscopy revealed the existence of endothelial microdomains with differing labelling densities. Labelling indicated that caveolar and fenestral diaphragms and basal laminae are highly anionic at physiological pH, luminal plasma membranes and endothelial processes are moderately charged and abluminal plasma membranes are weakly anionic. Tracers did not occur in caveolae or cytoplasmic vesicles. In vitro tracer experiments at pH values of 7.3, 5.0, 3.5 and 2.0 indicated that the anionic charge on the various endothelial domains was contributed by chemical groups with differing pKa values. In summary, the labelling of ganglionic and sciatic nerve vessels was similar except for the heavy labelling of diaphragms in a minority of endoneurial vessels in ganglia. This difference is likely to account in part for the greater permeability of ganglionic endoneurial vessels. The results are discussed with regard to the blood-nerve and -brain barriers and vascular permeability in other tissues and a comparison made between the ultrastructure and anionic microdomains of epi-, peri- and endoneurial vessels of dorsal root ganglia and sciatic nerves.

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PubMed1990

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina.

The distribution of anionic sites on the cell membranes and basal laminae of vascular endothelial cells in the rat sciatic nerve was investigated using cationic ferritin (CF) and cationic colloidal gold (CCG). Nerves fixed by perfusion followed by immersion were chopped into 400 microns thick slices and incubated in CF or embedded in LR White resin for staining with CCG. Using electron microscopy, the distribution of these tracers was investigated. The results indicated that microdomains of various charge densities exist. Diaphragms of caveolae and transendothelial channels, and luminal endothelial processes are highly anionic, the basal laminae of endothelial cells and pericytes and luminal membranes are medium and abluminal membranes least anionic. Inter-endothelial tight junctions were unlabelled and not penetrated by CF. These structures are thought to represent charge and size filters that control permeability of the vasa nervorum. The distribution of these charge-size filters is discussed in terms of the blood-nerve barrier, a physiological property present in the endo- but absent in the peri- and epineurial vessels.

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PubMed1989

Diffusion barrier properties of the perineurium: an in vivo ionic lanthanum tracer study.

While the perineurium as a diffusion barrier has been extensively investigated by light and electron microscopy, such studies have been largely restricted to the use of protein tracers. In the present study the permeability of the perineurium to a physiologically more relevant ionic tracer has been assessed. In vivo the rat sural or tibial nerve was either microinjected with lanthanum nitrate solution for endoneurial application or bathed in the lanthanum solution for epineurial application. The findings generally demonstrated an effective barrier to the tracer which failed to penetrate the inner layers of the perineurium. Only at the highest lanthanum concentration and longest time intervals employed did trace quantities occasionally penetrate the barrier and then only in the presence of some cytopathological changes to the outermost perineurial cells. The usefulness of the microinjection method was limited by the slight but unavoidable trauma to the perineurium. The findings are related to those of other studies which have used electron dense tracers, also to studies using physiological including electrophysiological techniques and morphological including freeze-fracture methods.

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PubMed1989

Lysophosphatidylcholine-induced incipient demyelination: involvement of a new tubular structure.

Demyelination was induced in the rat sciatic and tibial nerves by microinjection with lysophosphatidylcholine (LPC). Accompanying early myelin lysis (1-24 h) was the formation of vesicles and tubular structures. The tubules which are novel structures have a diameter range of 24-27 nm, a centre-to-centre spacing 30-50 nm and may extend for 3 microns in length. In this form they are arranged as a monolayer in the periaxonal space. As demyelination progressed and the periaxonal space widened the tubules increased in number and became more irregularly arranged. The tubules are apparently derived from the myelin lamellae/Schwann cell plasma membrane, while the axolemma remains intact.

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PubMed1989

The vasa nervorum: microcorrosion casts for scanning electron microscopy.

Studies on the vasa nervorum have a long history, not least because of their beneficial application in surgical practice and in understanding the pathogenesis of some neuropathies. In the present study a method is described for the preparation of microcorrosion casts of the vasa nervorum suitable for examination by scanning electron microscopy. The results confirm the findings of earlier investigations but also demonstrate the advantages of an immediate three-dimensional representation of the vascular architecture together with the additional magnification and resolving power of electron microscopy.

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PubMed1988

Lysophosphatidyl choline-induced demyelination. A freeze-fracture study.

Focal demyelination was produced in the rat sciatic nerve by microinjection of lysophosphatidyl choline (LPC). The demyelinating lesion was examined over the following 48 h using the freeze-fracture technique to examine myelin, Schwann cell and axonal membranes. Myelin lamellae were replaced by myriad spherical or oval membranous vesicles. The axonal and Schwann cell plasma membranes remained intact and the latter showed a large increase in caveolae-associated pores in some nerve fibres. The lysis of myelin lamellae and membranous vesicle formation are related to the known action of LPC on myelin and its membrane fusogenic properties. The importance of calcium ion influx and membrane protein aggregation and depletion in vesiculation are discussed.

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PubMed1988

Filipin-sterol complexes in disrupted myelin in the rat.

Using filipin as a cytochemical probe for cholesterol we have compared the distribution of filipin labelling in mildly disrupted myelin and normal myelin. The myelin lamellae in rat sciatic nerve were separated either by hypotonic saline (0.035-0.07 M) or nerve section (24-32 h) before aldehyde fixation and filipin treatment. Myelin separation was assessed in ultrathin sections and filipin distribution in freeze-fracture replicas. In separated myelin lamellae filipin labelling was similar throughout the myelin sheath while in normal control myelin filipin occurred most in the outer (abaxonal), least in the inner (adaxonal) and intermediate in the middle lamellae. It is concluded that this heterogeneous filipin labelling in normal myelin is a result of diffusion gradients to filipin within the myelin sheath and that in vivo cholesterol is uniformly distributed throughout all the lamellae of the myelin sheath. The site of the diffusion barrier to filipin within normal myelin is considered.

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PubMed1987

Distribution of filipin-sterol complexes in the unmyelinated nerve fibre.

The filipin-sterol technique was employed, together with freeze-fracture, to investigate the fine structure of the unmyelinated nerve fibre in the peripheral nerve. No heterogeneity was observed in the distribution of filipin labelling either in the Schwann cell plasma membrane or along the axolemma. The distribution of labelling is contrasted with that in the myelinated nerve fibre and related to the relative morphology and electrophysiology of the two types of fibre.

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PubMed1987

Filipin-sterol complexes at Schmidt-Lanterman incisures.

Employing the freeze-fracture technique, the distribution of filipin-sterol complexes was determined for membranes of peripheral nerve myelin. A heterogeneous distribution of complexes was observed with the greatest abundance on membranes associated with the cytoplasmic channels of Schmidt-Lanterman and longitudinal incisures. In addition there was an irregular network of well-labelled membrane bands in compact myelin. The results are related to a possible role for these channels and bands in the biochemical turnover of cholesterol in myelin.

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PubMed1987

The blood-nerve barrier: an in vivo lanthanum tracer study.

The permeability of the blood-nerve barrier was investigated using ionic lanthanum as an electron-dense tracer. The rat sural nerve was microinjected in vivo with lanthanum nitrate solution either into the endoneurial space or into the epineurium. Five to sixty minutes after injection the sural nerves were fixed by vascular perfusion or immersion. Using electron microscopy, lanthanum tracer was observed to be associated with endoneurial vessels in the perivascular spaces, in the inter-endothelial clefts and within the lumina. Furthermore, tracer was present in the spaces between adjacent endothelial cell layers and within vesicles and caveolae of endothelial cells. Epineurial vessels showed a similar distribution of tracer deposits but in greater quantities in inter-endothelial cell spaces and vessel lumina. The results are considered to demonstrate an absence of a blood-nerve barrier to ions as exemplified by lanthanum and are compatible with data from physiological experiments. The blood-nerve and blood-brain barriers are contrasted in their permeability to ions, their related fine structure and their physiological roles.

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PubMed1987

The development of Schmidt-Lanterman incisures: an electron microscope study.

The development of Schmidt-Lanterman incisures was investigated in the rat sural nerve during an active phase of postnatal myelination (5-21 days post partum). Two distinct populations of incisures were recognised and the following nomenclature for their developmental stages is proposed. Primary incisures which appear ab initio in myelination and always extend across the whole radial thickness of the myelin sheath but initially around only part of its circumference. Consequently they appear in transverse section as sectoral incisures (occupying less than half the circumference) and in longitudinal section as asymmetric incisures (involving one side only of the myelin sheath). Secondary incisures appear later, in regions of a compact myelin sheath, initially traversing only part of its radial thickness but commonly occupying its whole circumference. Thus they usually appear in transverse section as circumferential incisures and in longitudinal section as symmetric incisures (involving both sides of the myelin sheath). Less commonly secondary incisures may form in a sector of the myelin sheath but still in regions of compact myelin and thus appear asymmetric in longitudinal section and sectoral in transverse section. Secondary incisures appear mainly adaxonally in the earlier stages examined and mainly abaxonally in the later stages. The maturation of primary and secondary incisures into the radially and circumferentially complete incisure characteristic of the mature myelinated nerve fibre is described. The above mechanisms of incisural formation are contrasted with mechanisms previously suggested to occur during normal development and remyelination and related to the plasticity and ultrastructure of the myelin sheath.

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PubMed1985

Axonal microtubules: a computer-linked quantitative analysis.

Employing current computer-aided morphometric techniques, axonal microtubule density was determined for the rat sural nerve. Analysis of extensive data showed that while microtubule number increases with axon size, the increase is not directly proportional. Thus the relationship between microtubule density and axonal size is inversely related, so that microtubule density is greater in smaller axons than in larger axons. When a proximal and distal site, separated by 2 cm, were compared for microtubule density there was no significant difference, using pooled data for all fibre diameters. The results are interpreted in terms of our present knowledge of axonal-microtubule quantitative relationships, which is reviewed.

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PubMed1985

Filipin-sterol complexes at nodes of Ranvier.

Using the filipin-sterol technique, regional heterogeneity in the axonal and Schwann cell plasma membranes was investigated at the node of Ranvier and paranodes. Filipin-sterol complexes were abundant at the nodal axolemma but infrequent throughout the paranodal axolemma. The paranodal Schwann cell plasma membrane was rich in complexes which extended over the nodal Schwann cell microvilli. There were no regional differences in filipin labelling of the nodal-paranodal Schwann cell plasma membrane in relation to features such as paranodal cytoplasmic columns or mesaxonal furrows. However, the paranodes of adjacent Schwann cells were sometimes markedly different from each other in the amount of filipin labelling. The extent to which filipin labelling is indicative of cholesterol membrane content is discussed and the findings are related to current concepts of distribution, mobility and interaction of protein and lipid in biomembranes, with particular reference to the nodal axolemma.

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PubMed1985

Axonal degeneration in large and small nerve fibres. An electron-microscopic and morphometric study.

Using computer-aided morphometric methods, axonal degeneration following nerve crush was analysed to reassess whether small fibres degenerate before large fibres or vice versa, or simultaneously. Axonal microtubule density was used as the criterion for determining the extent of fibre degeneration. Axonal areas and axonal microtubule numbers were recorded from a large sample of myelinated fibres in the right unoperated rat sural nerve and distal to crush in the left sural nerve. Both samples were divided into small and large fibre groups, according to axonal areas. Statistical analysis of the data confirmed a significant loss of microtubules from the left crushed nerve fibres but no significant difference in the relative loss of microtubules from small and large fibres. It is concluded, therefore, that in Wallerian degeneration, axonal breakdown, as assessed by microtubule loss, occurs simultaneously in small and large fibres. The findings are related to the electrophysiological changes which occur in Wallerian degeneration.

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PubMed1985

Distribution of filipin-sterol complexes in the myelinated nerve fiber.

Using filipin as a cytochemical probe to reveal the distribution of cholesterol, myelinated peripheral nerve fibers were examined in freeze-fracture replicas. Filipin-sterol complexes were most abundant in the Schwann cell and axonal plasma membranes. In the Schwann cell plasma membrane there was no heterogeneity in complex distribution in relation to the subjacent cytoplasmic network. In myelin lamellae there was a decrease in complexes from outer to inner lamellae and some aggregation of complexes in individual lamellae. The density of complexes in cytoplasmic organelles varied from absent in mitochondria to high in lysosome-like bodies. The results are interpreted in terms of the related biochemical composition and biophysical properties of cell membranes, with particular reference to the myelinated nerve fiber. The influence of diffusion barriers and gradients on the formation of complexes by filipin is considered.

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