Biomedical subjects

C H Berthold

Publications and source records attributed to C H Berthold.

At least 19 recordsLinked to original sources

PubMed1997

Removal of retrogradely transported material from rat lumbosacral alpha-motor axons by paranodal axon-Schwann cell networks.

The aim of this study was to investigate the potential ability of Schwann cells to sequester axonally transported material via so called axon-Schwann cell networks (ASNs). These are entities consisting of sheets of Schwann cell adaxonal plasma membrane that invade the axon and segregate portions of axoplasm in paranodes of large myelinated mammalian nerve fibres. Rat hindlimb alpha-motor axons were examined in the L4-S1 ventral roots using light/fluorescence, confocal laser, and electron microscopy for detection of retrogradely transported red-fluorescent latex nanospheres taken up at a sciatic nerve crush, and intramuscularly injected horseradish peroxidase endocytosed by intact synaptic terminals. Survival times after tracer administration ranged from 27 hours to 4 weeks. During their retrograde transport toward the motor neuron perikarya, organelles carrying nanospheres/peroxidase accumulated at nodes of Ranvier, where they often appeared in close association with the paranodal myelin sheath. Serial section electron microscopy showed that many of the tracer-containing bodies were situated within ASN complexes, thereby being segregated from the main axon. Four weeks after nanosphere administration, several node-paranode regions still showed ASN-associated aggregations of spheres, some of which were situated in the adaxonal Schwann cell cytoplasm. The data establish the ability of Schwann cells to segregate material from motor axons with intact myelin sheaths, using the ASN as mediator. Taken together with our earlier observations that ASNs in alpha-motor axons are also rich in lysosomes, this process would allow a local elimination and secluded degradation of retrogradely transported foreign substances and degenerate organelles before reaching the motor neuron perikarya. In addition, ASNs may serve as sites for disposal of indigestable material.

Animals↗

PubMed1997

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody.

Gangliosides are characteristic glycolipid components of plasma cell membranes, especially enriched in the CNS and PNS. In some diseases involving the PNS, in particular motor neuropathies associated with conduction block, IgM autoantibodies against ganglioside GM1 have been implicated as a pathogenic factor. In order to study the GM1 distribution in peripheral nerves we have investigated its in situ localization using a new anti-GM1 monoclonal antibody, GM1:1. Immunization and production of the monoclonal antibody was made by common protocols and binding specificity was investigated by using structurally related glycolipids and modified GM1-molecules. The result showed that an alpha 2-3 bound sialic acid together with a terminal galactose moiety were essential for GM1:1 binding. In situ localization of GM1 in rat dorsal and ventral spinal roots was investigated by conventional immunomicroscopy. GM1 immunoreactivity was the same in both roots and appeared like a finely granular, in places confluent, material confined to Schmidt-Lanterman's incisures, to myelin sheath paranodal end segments and to some extent to the abaxonal Schwann cell cytoplasm; all of these structures are likely to be the target for GM1 antibodies in peripheral neuropathies. Nodal gaps and fibre contours showed a weak non-specific fluorescence. The localization of GM1 to the incisures of Schmidt-Lanterman and the paranodal end segments of the myelin sheaths might indicate a role of gangliosides as adhesion molecules.

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PubMed1997

The existence of a layer IV in the rat motor cortex.

We have reconstructed the laminar pattern of rat primary motor cortex (Fr1) using a computerized analysis system based on the so-called 'optical dissector'. Data were visualized on a graphics terminal. In contrast to current views, which state that there is no prominent layer IV in the motor cortex of the rat, our method of analysis revealed a genuine layer IV consisting of densely packed small neurons.

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PubMed1996

Aspects of the quantitative analysis of neurons in the cerebral cortex.

We address three problems concerning the quantitative analysis of nerve cell distribution in the cerebral cortex: (i) preparatory tissue deformation (shrinkage); (ii) difficulties in differentiating between small neurons and astroglia; and (iii) the bias introduced by the counting method. We found that staining with Richardson's solution led to no shrinkage in Vibratome-cut sections of aldehyde-fixed rat brains, but did result in staining of the neurons and left the glial cells unstained. This was in striking contrast to Nissl staining which introduced a linear shrinkage of 20-30% and stained all kinds of cortical cells indiscriminately. A computer-based unbiased counting method was implemented by taking advantage of the stereological procedure referred to as the 'optical disector' (Gundersen, H.J.G. (1986) Stereology of arbitrary particles, J. Microsc., 143: 3-45).

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PubMed1996

Development of nodes of Ranvier in feline nerves: an ultrastructural presentation.

The ultrastructure of developing nodes of Ranvier and adjacent paranodes of future large myelinated fibers in feline lumbar spinal roots is described. The development starts before birth concurrent with myelination and is finished at the end of the first postnatal month when the nodal regions of future large fibers, now 4-5 microns of diameter, for the first time appear like miniatures of those of their 4 times thicker and fully mature counterparts. At this stage the fibers also begin to show mature functional properties. The latent maturation process is denoted "nodalization" and includes two major events: (1) the formation of a narrow node gap bordered by compact myelin segments and filled with Schwann cell microvilli that interconnect an undercoated nodal axolemma with rapidly increasing accumulations of mitochondria lodging in the longitudinal cords of Schwann cell cytoplasm that is distributed outside a more and more crenated paranodal myelin sheath; (2) the setting of a fixed number of nodes along the axons; an event that includes segmental axonal and myelin sheath degeneration and is concluded by the elimination of supernumerary Schwann cells.

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PubMed1996

Heterogeneity in the columnar number of neurons in different neocortical areas in the rat.

We have investigated the number of neurons in three neocortical areas of the rat brain. Our results challenge the uniformity concept proposed by Rockel et al. [Brain, 103 (1980) 221-244]. Area Fr1, HL and Oc2 (primary motor, primary somatosensory and secondary visual cortex) from Sprague-Dawley rats were examined. The brains were glutaraldehyde fixed, sectioned in 50 mu m thick sagittal slices and stained in Richardson's solution. The counting was carried out using a computerized system based on the optical disector. The cortical thickness was measured to be 1.9 mm, 1.9 mm, and 1.4 mm in area Fr1, HL, and Oc2, respectively. The number of neurons under 1 mm2 cortical surface was calculated to be 91 100 in Fr1, 133 500 in HL and 106 100 in Oc2. The number of neurons in a volume of tissue 30 x 25 mu m through the depth of the cortex was calculated to be 68 in Fr1, 100 in HL and 80 in Oc2. The density of neurons was calculated to be 48 500 neurons/mm3 in Fr1, 69 400 neurons/mm3 in HL and 76,900 neurons/mm3 in Oc2. There were significant (P < 0.01) differences between all areas regarding both the number of neurons under a certain area of surface as well as the neuron density. The results indicate that there is no basic uniformity in the number of neurons under a certain area of cortical surface.

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PubMed1996

Horseradish peroxidase in axons of the dorsal funiculi of the cat: distribution after an injection of the enzyme into the dorsal column nuclei.

Horseradish peroxidase (HRP) was injected into the left dorsal column nuclei of adult cats. Large dorsal funiculi axons of the C3, C5, C8 and L7 segments were searched for HRP-activity after 12, 24, 36 and 48h using light and electron microscopy. Accumulations of intra-axonal HRP-positive bodies occurred at nodes of Ranvier in the C3-C8 segments at 12, 24 and 36h and in the L7 segments at 24, 36, and 48h. The accumulations of HRP in three spatio-temporally different consecutive patterns, noted earlier at nodes of Ranvier in the peripheral nervous system (PNS) portion of feline alpha motor axons for more than 70h after an intramuscular injection of the enzyme, were not observed in the present material. We suggest that the differences in the modes in which large PNS and CNS axons interact with retrogradely transported HRP are due to differences in the organization of the respective nodal regions. We also emphasize that endocytosis via axon terminals in the CNS normally represents uptake of material from an extracellular space which is controlled and protected by the blood-brain barrier. This is in contrast to endocytosis via axon terminals in a muscle, which represents uptake of material from an extracellular space openly exposed to influx of different substances from the blood stream.

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PubMed1995

Axonal constriction at Ranvier's node increases during development.

We have studied the ratio between the nodal and the internodal diameter (the dn/d(in) ratio) of large myelinated axons in the L7 ventral spinal root of the cat during pre- and postnatal development using light and electron microscopy. A substantial nodal constriction, dn/d(in) = 0.6, was found at the beginning of myelination, about 2 weeks before birth. The ratio decreased during the subsequent 10 weeks and approached the adult value of 0.47 (SE 0.01, N = 45) in the 8 weeks old kitten. The observations are discussed with respect to the maturation of the nodal region and to our earlier idea that the constricted nodal axon segments of large peripheral myelinated nerve fibres of adult cats and kittens 2 months and more of age are sites capable of interacting with and perhaps even controlling the passage of axonally transported materials.

Age Factors↗

PubMed1995

Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood-brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S-100 immunohistochemical staining patterns in the hippocampus of wild-type and mutant mice suggested a normal abundance of astrocytes in GFAP-negative mice, however, in contrast to wild-types, GFAP-negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up-regulation of other intermediate filament proteins, such as vimentin. The GFAP-negative mice displayed post-traumatic reactive gliosis, which suggests that GFAP up-regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

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PubMed1995

Automated correction of linear deformation due to sectioning in serial micrographs.

This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi-thin and ultra-thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi-linear, i.e. two first-order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two-dimensional sections and is primarily aimed at three-dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.

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PubMed1994

A method for 2D reconstruction of intracellularly labeled neurons from sequential sections.

A technique for 2D reconstruction of intracellularly labeled neurons from sequential sections is described. The system consists of a Charged Coupled Device-camera mounted on a microscope, a videomixer and a IBM-compatible PC with a framegrabber. The neurons (interneurons from the spinal cord of the cat) were labeled iontophoretically by horshradish peroxidase and subsequently cut in 60 microns sections. The sections were aligned using the video mixer by fitting the cut dendrites and axon from one section with their counterparts in the following section. The images were then digitized in the PC where they were fused to create a superimposed picture of the aligned parts of the neuron; a 2D reconstruction was created.

Animals↗

PubMed1994

Dimensions of individual alpha and gamma motor fibres in the ventral funiculus of the cat spinal cord.

Using light and electron microscopy, axon diameter, myelin sheath thickness (measured as number of myelin lamellae) and internodal length of alpha and gamma motor axons of the L7 ventral root and spinal cord segment were investigated in serial cross-sections. The CNS internodes of the alpha motor fibres had, on average, an axon diameter of 8.6 microns, 105 myelin lamellae and a length of about 560 microns. The CNS internodes of the gamma motor fibres had, on average, an axon diameter of 3.4 microns, 66 myelin lamellae and a length of about 440 microns. Axon diameter at the nodes of Ranvier was 30-40% of the internodal axon diameter. Axon diameter, number of myelin lamellae and internodal length varied considerably between consecutive internodes. Statistical analysis showed no systematic increases or decreases. Regression analyses of the scatter plots of the number of myelin lamellae and internodal length against axon diameter showed large variations and correlation coefficients of r < 0.50. In conformity with ventral root (PNS) internodes (Nilsson & Berthold, 1988) the plotting of intrafunicular (CNS) internodal myelin volume against internodal axon mantle area showed linear correlations with correlation coefficients of r > 0.90. The mean axon diameter of the investigated CNS internodes was similar to, the mean number of myelin lamellae somewhat lower than, and the mean internodal length considerably shorter than that of internodes of axons of the L7 ventral root (Nilsson & Berthold, 1988). In contrast to the ventral root, the intrafunicular alpha motor fibres had higher g values (axon diameter/fibre diameter value) and lower il/d ratios (internodal length/axon diameter ratio) than is considered optimal for conduction. We consider that these deviations from the theoretical optimum are not large enough to impair the conduction properties of the CNS parts of the motor axons in a significant way.

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PubMed1993

Axoplasmic organelles at nodes of Ranvier. I. Occurrence and distribution in large myelinated spinal root axons of the adult cat.

Using light microscopy (LM) and electron microscopy (EM) we have examined the occurrence and distribution of axoplasmic organelles in large myelinated nerve fibres of the L7 ventral and dorsal spinal roots of the cat with special reference to the paranode-node-paranode (pnp)-regions. Ninety-eight percent of the 550 Toluidine Blue-stained paranode-node-paranode-regions examined in the light microscope contained dark-blue bodies accumulated distal to the midlevel of the paranode-node-paranode-region. Further, a veil of Toluidine Blue positive material was observed in about 50% of the paranode-node paranode-regions. In about 25% of these paranode-node-paranode-regions the veil lay distal to the midlevel of the paranode-node-paranode-region and in the remainder it lay proximally. Electron microscopy suggested that the ultrastructural equivalents of the dark-blue bodies and of the veil were dense lamellar bodies and a diffuse granular material, respectively. Our calculations indicate that from 70% to more than 90% of some organelles (dense lamellar bodies, multivesicular bodies and vesiculo-tubular membranous organelles) present in an axon are accumulated in the paranode-node-paranode-regions. The occurrence of these organelles in the individual paranode-node-paranode-regions varied within wide limits also in adjacent fibres. The dense lamellar and multivesicular bodies dominated the distal part of the paranode-node-paranode-regions while the vesiculo-tubular membranous organelles dominated the proximal part, i.e. the organelles showed a mutual proximo-distal segregation with reference to the midlevel of the paranode-node-paranode-region. Of seventeen paranode-node-paranode-regions analyzed ultrastructurally, seven were classified as 'fully segregated', that is 67% or more of the lamellar and multivescular bodies, present in the whole paranode-node-paranode-region, lay distal to the mid-level, and 67% or more of the vesiculo-tubular membranous organelles lay proximal to it.

Animals↗

PubMed1993

Axoplasmic organelles at nodes of Ranvier. II. Occurrence and distribution in large myelinated spinal cord axons of the adult cat.

The occurrence and distribution of axoplasmic organelles in large myelinated axons of the ventral, the lateral and the dorsal funiculi of L7 spinal cord segments of the cat have been studied using electron microscopy (EM). Most organelles were found to be concentrated to the paranode-node-paranode (pnp)-regions and they showed their highest relative concentration in the constricted part of these regions, i.e. at the nodes of Ranvier. In the paranode-node-paranode-regions of the lateral and dorsal funiculi, large dense bodies predominated distal to the nodal mid-level and vesiculo-tubular membranous organelles proximal to it. This pattern of organelle distribution, a proximo-distal (with reference to the neuron soma) segregation of the organelles, was only faintly indicated in the paranode-node-paranode-regions of the alpha motor axons of the ventral funiculus. These paranode-node-paranode-regions were, apart from a weak proximo-distal segregation of a few organelles, characterized by deposits of electron dense granules and clusters of large round mitochondria. We conclude that there are two types of organelle accumulation and distribution in the paranode-node-paranode-regions of large spinal cord nerve fibres of the cat. One type is found in the lateral and dorsal funiculi, i.e. in axons with terminal (synaptic) fields inside the blood-brain-barrier. The other type is found in the alpha motor axons of the ventral funiculus, i.e. in axons with their terminal field in the PNS and thus outside the blood-brain barrier. It should be noted that retrogradely transported material in the alpha motor axons has passed through a long sequence of paranode-node-paranode-regions equipped with Schwann cells before it reaches the CNS, while material transported retrogradely in the axons of the dorsal and lateral funiculi has not. The following discussion includes a comparison of the organelle accumulation and distribution in these two types of CNS paranode-node-paranode-regions with the organelle accumulation and distribution observed in the paranode-node-paranode-regions of PNS axons.

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PubMed1992

Metabolic relationships between proteins of myelin and paranodally shedded, partially degraded myelin fragments in the rabbit CNS.

The "close-to-node" regions of myelinated nerve fibres, i.e., the paranodal end segments, are generally thought to be sites of high metabolic activity and myelin sheath turnover. Data on turnover rates of individual myelin constituents are conflicting but there exists a common belief that myelin is metabolized as independent molecules rather than as a unit. The occurrence of paranodal Marchi-positive bodies, with morphological and biochemical properties consistent with partially degraded myelin, prompted us to examine the temporal dynamics of the incorporation of radioactive precursor label in the major proteins of myelin and the Marchi-positive bodies. 3H-leucine was administered intrathecally in adult rabbits. After various survival times, the spinal cord was subfractionated by ultracentrifugation in a discontinuous two-step 0.32 M/0.85 M sucrose gradient. Myelin was collected from the interface and a floating fraction, heavily enriched in Marchi-positive bodies, was recovered on top of the 0.32 M sucrose. By scintillation counting and by gel fluorography combined with immunoblotting, a gradual appearance with time of partially degraded peptides of myelin-associated protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase was seen in the floating fraction but not in myelin. The temporal dynamics of the specific activities of these two proteins and myelin-basic protein and proteolipid protein were consistent with a typical source-product relationship between myelin and the material in the floating fraction. In conjunction with earlier morphological and biochemical findings, these data may suggest that Marchi-positive bodies appear as a consequence of myelin catabolism.

Animals↗

PubMed1992

Computer-assisted realignment of light micrograph images from consecutive section series of cat cerebral cortex.

Two computer- and image-analysis-based procedures for realignment of images of consecutive light micrographs of nerve tissue (cat motor cortex) have been developed. One procedure (CENT) was interactive, employing the subjective overall 'best fit' of two images to each other and the other (AUTOCENT) was based on an automatic comparison of two images (image thresholding and binary comparison). Images of light micrographs of several hundred consecutive sections were realigned using the interactive and the automatic methods. The interactive procedure was easy to use; realignment of an image took only a few minutes in the hands of an experienced operator and should be easy to implement on commercially available smaller computers like PCs and workstations, but the realignment was possibly disturbed by the operator's subjective expectancy of regular forms. The automatic procedure realigned 1-4 images per hour, might be implemented on smaller computers if the programming is made more efficient, was probably objective and restored the 'true position of images'.

Animals↗

PubMed1991

Lysosomal activity at nodes of Ranvier in dorsal column and dorsal root axons of the cat after injection of horseradish peroxidase in the dorsal column nuclei.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e. lysosomes, in dorsal column and dorsal root axons of the spinal cord segments C8 and L7 in adult cats was analyzed by light and electron cytochemical methods after injection of horseradish peroxidase (HRP) in the dorsal column nuclei. Axonal lysosomes were, with few exceptions, concentrated at the nodes of Ranvier. We found no changes in nodal occurrence and distribution of lysosomes in axons of the HRP-injected sides, as compared to axons of the uninjected sides or of animals not exposed to HRP. Axonal lysosomes were very rare in the dorsal columns, where the frequency of nodes containing light microscopically detectable AcPase-positive bodies was 0-5% at the HRP-injected sides, 0-6% at the contralateral sides, and 0-3% in control animals. The corresponding values in the cervical and lumbar dorsal roots were 6-23%, 9-20%, 10-12% and 19-37%, 21-40%, 26-43%, respectively. In view of our recent observations in alpha-motor neurons, the results point at a noteworthy difference in local degradative ability between dorsal column axons and alpha-motor axons, the latter being able to accumulate intramuscularly injected and retrogradely transported HRP at their PNS nodes of Ranvier for 48-60 h, during which period the axoplasmic AcPase activity/concentration increases at some nodes. Such a degradative activity, which could protect the motor neurons by restricting axoplasmic transport of exogenous materials imbibed by their axon terminals outside the CNS, may not be of the same significance for neurons, e.g. dorsal root ganglion neurons, the axon terminals of which are located within the CNS.

Acid Phosphatase↗

PubMed1991

Lysosomal activity in developing cat alpha-motor axons under normal conditions and during retrograde axonal transport of horseradish peroxidase.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e., lysosomes, in lumbosacral alpha-motor axons of kittens, 0-16 weeks of age, was analyzed by light and electron cytochemical methods under normal conditions and after intramuscular injection of horseradish peroxidase (HRP). Axonal lysosomes were rare early postnatally. In 3-week-old animals, a few AcPase-positive bodies appeared in the axoplasm at some nodes of Ranvier in the peripheral nervous system (PNS) and internodally in the intrafunicular motor axon parts within the central nervous system (CNS). From 6 weeks postnatally, a nodal concentration of AcPase-positive bodies was also noted in the CNS. The number of AcPase-positive bodies continued to increase gradually in the course of neuronal maturation. In 16-week-old animals, axonal AcPase activity was still at considerably lower levels than at adult stages. At all ages, acid hydrolase-containing organelles were most commonly found at ventral root nodes. After injection of HRP in the medial gastrocnemius muscle, accumulations of AcPase-positive bodies were seen in the axoplasm at some PNS nodes of the HRP-injected sides of kittens aged 8, 12, and 16 weeks. Incubation for demonstration of both HRP and AcPase activity showed that some organelles at HRP-transporting nodes contained both types of reaction product. The nodal AcPase activity in the intrafunicular, CNS parts of alpha-motor axons of the HRP-exposed sides did not differ from that of the contralateral, uninjected sides. In view of our previous observations in alpha-motor neurons of adult cats in which a lysosome-mediated degradation of axonally transported materials may take place at PNS nodes of Ranvier, the present study illuminates possible differences in the ability to interfere with axonal transport between developing and mature neurons. The infrequent presence of lysosomes in developing alpha-motor axons and the implied disability of their nodal regions to interfere with axonally transported constituents in a way similar to that seen in adult animals may be of significance in that trophic and chemical signals can pass unhindered between the periphery and perikaryon. However, this could also have negative consequences for the vulnerable immature neuron in that various materials retrieved at the axon terminals outside the CNS are permitted a more-or-less free access to the perikaryon.

Aging↗