Robert S. Garrett

Fine-structural localization of aldose reductase and ouabain-sensitive, K+-dependent p-nitro-phenylphosphatase in rat peripheral nerve

SummaryAldose reductase was visualized by light and electron microscopy using a goat anti-rat antibody with immunoperoxidase and immunogold, respectively. Ouabain-sensitive, K+-dependent, p-nitro-phenylphosphatase, a component of (Na+, K+)-ATPase, was localized at the electron microscopic level by enzyme histochemistry using p-nitro-phenylphosphate as substrate. In peripheral nerve, spinal ganglia and roots, the Schwann cell of myelinated fibers was the principal site of aldose reductase localization. Immunostaining was intense in the paranodal region and the Schmidt-Lanterman clefts as well as in cytoplasm of the terminal expansions of paranodal myelin lamellae and the nodal microvilli. Schwann cell cytoplasm of unmyelinated fibers were faintly labelled. Endoneurial vessel endothelia, pericytes and perineurium failed to bind appreciable amounts of aldose reductase antibody. However, mast cell granules bound antibody strongly. In contrast, p-nitro-phenylphosphatase reaction product was detected in the nodal axolemma, terminal loops of Schwann cell cytoplasm and the innermost layer of perineurial cells. In endothelial cells, reaction product was localized on either the luminal or abluminal, or on both luminal and abluminal plasmalemma. Endothelial vesicular profiles were often loaded with reaction product. Occasional staining of myelin and axonal organelles was noted. Mast cells lacked reaction product.

Demyelination in allergic and Marke's disease virus induced neuritis comparative electron microscopic studies

SummaryPatterns of demyelination were studied in sciatic nerves, spinal roots and ganglia of chickens afflicted with either Mareks disease (MD) or experimental allergic neuritis (EAN). MD was induced in susceptible chicks after hatching by inoculation of the JM strain of MD Herpes virus. Tissues from these chickens were examined 7–83 days after infection. EAN was studied 10–21 days after sensitization of 4 week old chickens to emulsions containing human peripheral nerve with complete Freunds adjuvant. In both conditions lesions were encountered which consisted of perivenular infiltrates of mononuclear cells that penetrated the basal lamina of the neurolemmal sheath, displaced Schwann cells, lysed and stripped myelin lamellae without damage to axons. Other lesions in MD were characterized by lymphomatous infiltrates that contained necrotic cells and disintegrating axons. The similarity of the demyelinating process in MD to that seen in EAN suggests that MD virus infection activates lymphocytes sensitized to peripheral nerve myelin. The findings are discussed with reference to acute idiopathic polyneuritis (Guillain-Barré syndrome) in patients with preceding or concurrent Herpes virus infections including those known to cause lymphoproliferative disorders.

Response of Glia, Mast Cells and the Blood Brain Barrier, in Transgenic Mice Expressing Interleukin-3 in Astrocytes, an Experimental Model for CNS Demyelination

Transgenic mice overexpressing cytokines facilitate analysis of the effects of these immunomodulators on indigenous cells of the central nervous system. This study examines morphological aspects of demyelination and permeability changes, in a recently described transgenic model (termed GFAP‐IL3). GFAP‐IL3 mice develop progressive motor disease at approximately 5 months. Lesions identified after disease onset, showed activation of microglia, astroglial proliferation with phagocytosis of lipids, and immigration of macrophages and mast cells into neural parenchyma. Lymphocytes failed to appear until the later stages of the disease. Later, cerebellar and brain stem white matter contained focal demyelinating lesions with intense macrophage infiltration and a proliferative astrocytosis. Dystrophic axonal changes were noted, in addition to demyelination in heavily infiltrated lesions. Mast cells, variably present in the thalamus and meninges of wild type mice, were greatly increased at these sites in GFAP‐IL3 mice. Blood‐brain barrier (BBB) defects were documented with leakage of intravenously injected horseradish peroxidase. Mast cell infiltration into the CNS and their degranulation at the site of injury, may represent initial events in a spontaneous process of macrophage mediated demyelination in which glial cells and macrophages are both involved in the phagocytic process.

Schwanm cell abnormalities in 2, 5-hexanedione neuropathy

SummaryDistinctive cytoplasmic alterations of Schwann cells were observed by electron microscopy in rats and mice with peripheral neuropathy induced by chronic exposure to 2,5-hexanedione. Pronounced enlargement of Schwann cells was due to accumulation of 100 Å cytoplasmic filaments and endoplasmic reticulum and was most often observed after 12–15 weeks exposure to 2,5-hexanedione. Examination of teased nerve fibres revealed segmental demyelination and remyelination involving axons of normal diameter as well as giant axons. The filament disorder induced by 2,5-hexanedione administration is not limited solely to axoplasmic contents. Possible mechanisms of demyelination are discussed and the changes are compared to those observed in human neuropathy for which 2,5-hexanedione appears to be the closest experimental model.

Tactile hyperesthesia, altered epidermal innervation and plantar nerve injury in the hindfeet of rats housed on wire grates

The effects of wire grates on nerve injury and recovery were examined in rats housed in cages with sawdust-covered solid flooring. For the first 3 weeks of the study, 20 rats were housed on sawdust alone and 20 rats were housed in cages with wire grates placed over the sawdust. For the remaining 9 weeks, 10 animals housed on sawdust had wire grates added to their cages, while grates were removed from the cages of 10 animals. The effects of tactile stimulation on hindpaw plantar skin was measured weekly using the Von Frey filament test. Intraepidermal innervation using PGP 9.5 immunostaining and plantar nerve histology were assessed at the end of the 12-week study. After just 1 week on grates, hindpaw withdrawal thresholds were already markedly decreased and remained low until the grates were removed at 3 weeks. Thresholds returned to normal by 4 weeks after removal of the grates. Wire grates also induced increases in PGP 9.5 immunoreactive intraepidermal fine nerve endings that were normalized after grate removal. Demyelination, Wallerian degeneration and Renaut bodies were induced in the medial plantar nerve in rats housed in cages with wire-grate flooring. Nerve injury was largely resolved after 9 weeks on sawdust flooring. These data demonstrate that wire grates rapidly induce hindpaw tactile hyperesthesia and plantar neuropathy in rats and emphasize a risk of using wire-grate cage flooring in studies assessing hindlimb function and structure.

Ferritin immune complex deposits in the choroid plexus

SummaryMice injected intrapertoneally with 20 mg of ferritin twice weekly for more than 4 weeks developed proteinuria due to the deposition of ferritin-antiferritin antibody complexes in renal glomeruli. Deposits of ferritin, immunoglobulin G (IgG) and third complement component (C3) also accumulated in the perivascular, extracellular space of the choroid plexus as demonstrated by immunofluorescence and electron microscopy. The findings confirm previous observations on immune complex deposits in the choroid plexus in spontaneous autoimmune disease and persistent viral infections. The occurrence of similar deposits in the human choroid plexus and the possibility of an associated disturbance of the blood-spinal fluid barrier are discussed.

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

Abstract: The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two‐ and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.

Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury

Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick‐embryo myoblasts showed similar prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose‐dependent fashion by addition of a neutralizing anti‐prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, prosaposin has myotrophic properties.

Canine GM1 GangliosidosisAn Ultrastructural and Biochemical Study

The ultrastructural and biochemical features of canine GM1 gangliosidosis were studied. β-Galactosidase activity assayed using both skin fibroblast tissue culture strains and fresh skin revealed enzyme activities in three groups (normals, heterozygotes, and homozygotes) corresponding to an autosomal recessive inheritance. The concentration of ganglioside GM1 was greatly increased in cerebral gray matter and kidney. A striking elevation of tissue oligosaccharides was found in liver, kidney, and spleen. Most neurons in the cerebral cortex and deep gray matter were filled by spherical lamellated inclusions. Hepatocytes contained vacuoles with an amorphous granular material which may correspond to the accumulation of galactoseoligosaccharides determined chemically. The disease in dogs has features similar to both the infantile and juvenile form of human GM, gangliosidosis.