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.

Crystalloid inclusions in brain macrophages and hemopoietic tissue in GFAP-IL3 mice resemble inclusions identified in multiple sclerosis.

Crystalloid inclusions or "pole bodies" observed in brain macrophages in human demyelinating disease represent a morphological enigma. Similar inclusions were detected in brain macrophages from the GFAP-IL3 mouse, a transgenic murine model for macrophage mediated demyelination. Mice also showed inclusions in hematopoietic tissue. They appear to be related to phagocytosis and secretion, respectively, as evidenced by the fact that in phagocytosing cells they often merged with lysozomes and that affected cells showed empty channels open to the interstitium. Based on ultrastructural and immunolocalization studies using chaperonin-10, lysozyme, and cathepsin the authors suggest that these inclusions are consistent with phagocytosis-related secretory products. This study may provide insight into the nature and significance of similar macrophage inclusions recently identified in multiple sclerosis.

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.

Necrotizing myopathy induced by overexpression of interferon-gamma in transgenic mice.

A transgenic mouse model has been established in which the cytokine interferon-gamma (IFN-gamma) is overexpressed through the action of the acetylcholine receptor epsilon promoter acting at the neuromuscular junction. While originally developed as a model for the study of the pathogenesis of myasthenia gravis, there are important differences from both human myasthenia gravis and its animal model, experimental autoimmune myasthenia gravis. By 4 months of age there was a well-established inflammatory, predominantly necrotizing myopathy, with marked dystrophic calcification. Dystrophic and degenerative changes in terminal axons and adjacent Schwann cells were also apparent. The acetylcholine receptor was not the primary target of the inflammatory response, since at 10 weeks of age the receptor content was not decreased and antibodies were not detected bound to the receptor. The IFNgamma transgenic mouse model may provide a clinically relevant model of necrotizing myopathy for investigation of the pathological changes associated with, and presumably precipitated by, overexpression of the pro-inflammatory cytokine interferon-gamma on the neuromuscular junction, intramuscular nerves and myofibers.

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.

Abnormal iron deposition associated with lipid peroxidation in transgenic mice expressing interleukin-6 in the brain.

Transgenic mice, named GFAP-IL6, that express interleukin-6 in astrocytes in the central nervous system (CNS) have a constitutive blood-brain barrier (BBB) defect and develop a progressive neurodegenerative disease. Based on ultrastructural observations showing electron-dense pigment in the brain of the GFAP-IL6 mice, we hypothesized that iron metabolism was altered in the brains of these animals. Enhanced histochemical methods revealed abnormal iron deposition in the cerebellum from 1 month of age that worsened with progression of the disease. Immunohistochemical analysis of iron-binding proteins (IBP) showed increased ferritin immunoreactivity and a decreased signal from the transferrin receptor in symptomatic animals. Atomic absorption spectroscopy revealed a 40% increase of total iron concentration in the cerebellum at the symptomatic stage. In order to obtain evidence that accumulation of this oxidizing metal was toxic, we looked for the presence of oxidative damage. Using the MAL-2 antibody, extensive lipid peroxidation (LP) was detected in the neocortex and the cerebellum in symptomatic animals. Ultrastructural analysis indicated lipofuscin deposition at the sites of neuro-axonal degeneration and abnormal iron deposition. These results suggest that the IL6-induced BBB defect precipitates iron accumulation in the GFAP-IL6 mouse brain and that subsequent IBP regulation mediates protective responses. As these defenses become overwhelmed, the iron overload seems to promote LP, which may contribute to the neurodegeneration that ensues. This transgenic mouse model of IL6-mediated neurodegeneration provides a unique opportunity to examine several aspects of iron metabolism in the brain, including its entry at the site of the BBB, its distribution through the IBP, and its mechanisms of toxicity.

Changes in aldose reductase after crush injury of normal rat sciatic nerve.

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.

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

Aldose 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.

Selective vulnerability of unmyelinated fiber Schwann cells in nerves exposed to local anesthetics.

When peripheral nerves of experimental rats are exposed to local anesthetics, distinctive and reproducible pathologic changes occur involving the perineurial sheath and endoneurial contents. Application of intermediate strength concentrations of the local anesthetics, 2-chloroprocaine, lidocaine, etidocaine, and intermediate or high concentrations of procaine to the surface of rat sciatic nerves resulted in the following changes. By 48 hours, the perineurial sheath exposed to the drug was disrupted and became permeable to granulocytes which infiltrated the subjacent endoneurium in conjunction with edema formation in the endoneurial interstitium. Application of 10% procaine to exposed nerve resulted in extensive demyelination. The most striking pathologic change occurring with either intermediate or high doses was accumulation of lipid droplets in Schwann cells, a phenomenon that occurred often in myelin-producing Schwann cells but much less frequently in unmyelinated fiber Schwann Cells. Lipid accumulation appears to be one of several reactive changes that affect Schwann cells of myelinated fibers and is dose-dependent. On the other hand, while reactive changes were infrequently seen in unmyelinated fiber Schwann cells, these cells appeared more susceptible to injury as shown by electron microscopy. Injury to Schwann cells by local anesthetics is temporary because these cells can replicate quickly. Autoradiographic studies of thymidine incorporation 1 week after procaine administration to the sciatic nerve showed intense proliferation of Schwann cells, but no such activity in controls. These findings support the view that their neurotoxic properties may account in some part for the function of local anesthetics, that Schwann cells of small unmyelinated fibers are more vulnerable to these agents than those of myelinated fibers, and that destruction of their supporting cells is followed by vigorous mitotic activity in the endoneurium.

Virus persists in beta cells of islets of Langerhans and infection is associated with chemical manifestations of diabetes. II. Morphologic observations.

Persistence of lymphocytic choriomeningitis (LCM) virus in the islets of Langerhans was associated with mild hyperglycemia and abnormal glucose tolerance test results. Early histopathologic events consisted of occasional perivascular inflammatory mononuclear cells around both islet and acinar cells. Morphometric studies showed an increase in the size of islets from virus-infected mice. By electron microscopy, LCM virions were found within infected beta cells. Cytolytic injury of beta cells was minimal and did not account for the abnormalities of glucose metabolism. In contrast to the findings in islets, ultrastructural studies of acinar cells revealed LCM virions in abundance, vacuolar degeneration, and intracytoplasmic inclusions. This study extends the previous observation that LCM virus infection may persist in beta cells of the islets of Langerhans without causing structural injury but be associated with abnormalities resembling the chemical and histopathologic features of the early stage of Type II (adult-onset) human diabetes mellitus.

Effect of octanoate injection on rat blood-brain barrier.

Serum concentrations of short and medium chain fatty acids, including octanoate, are elevated in hepatic encephalopathy and Reye syndrome. Injection of octanoate into animals produces features reminiscent of Reye syndrome, but the mechanisms are unknown. To evaluate the effect of octanoate on blood-brain barrier permeability, three techniques were used. Entry of horseradish peroxidase and trypan blue into brain was not observed after octanoate injection. Brain uptake of tryptamine, tyrosine and methionine was increased significantly by octanoate, while uptake of insulin was unchanged. This study suggests that octanoate may produce central nervous system alterations by facilitating entry of certain low molecular weight compounds into brain. This may represent one mechanism for the development of encephalopathy in liver disease and Reye syndrome.

Pituitary dwarfism in mice persistently infected with lymphocytic choriomeningitis virus.

Most strains of mice injected intracerebrally with lymphocytic choriomeningitis virus grow to adulthood maintaining a persistent virus infection associated with chronic virus-induced immune complex disease. However, mice on a k background are highly susceptible to neonatal infection and develop the clinical syndrome of pituitary dwarfism and hypoglycemia. Examination of pituitary tissue fails to reveal morphologic alteration by light and electron microscopy. Within the pituitary, viral antigens are exclusively distributed within the cells of the adenohypophysis. Using ultrastructural colloidal gold-labeling techniques, we demonstrate the presence of mature virus particles budding from the surface of growth hormone containing cells from the pituitary. This study indicates that persistent lymphocytic choriomeningitis virus infection of the growth hormone cells in susceptible mice is associated with pituitary dwarfism without producing visible structural damage.

Canine GM1 gangliosidosis. An ultrastructural and biochemical study.

The ultrastructural and biochemical features of canine GM1 gangliosidosis were studied. beta-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 galactose-oligosaccharides determined chemically. The disease in dogs has features similar to both the infantile and juvenile form of human GM1 gangliosidosis.

Cycloleucine encephalopathy.

Cycloleucine, a non-metabolizable amino acid analogue produces status spongiosus in cerebral white matter of rats and mice as well as a distinctive lesion of astrocytes. Its mechanisms of action include competition with natural amino acids from transport across the blood-brain barrier leading to inhibition of entry of circulating amino acids into brain, interference with ribosomal RNA maturation, and blockage of transmethylation reactions, including the conversion of homocystine to methionine. Cycloleucine also affects the kidney, producing aminoaciduria. Electron microscopy of cerebral white matter reveals spongiform changes of myelin sheaths caused by separation of myelin lamellae along intraperiod lines and accumulation of whorls of filaments in astrocytes. The myelinopathy is dose related and its toxicity is cumulative due to its long half-life in animals. The findings are discussed with reference to other spongiform myelinopathies, including status spongiosus observed in homocystinuria and other aminoacidurias.