Validity, practical utility and reliability of Actigraph accelerometry for the measurement of habitual physical activity in dogs.

OBJECTIVES: To test the validity, practical utility, and reliability of the Actigraph GT3-X accelerometer for measurement of habitual physical activity in pet dogs. METHODS: In the validation study, 30 dogs wore the accelerometer for 1 day while being filmed. Accelerometer and film were synchronised and 10-minute periods of the filmed records were extracted with dogs in continuous periods of sedentary behaviour, light intensity physical activity indoors, light to moderate intensity physical activity outdoors and vigorous physical activity outdoors. For the practical utility and reliability studies, 20 dogs wore the GT3-X accelerometers for 1 week: practical utility was quantified as data loss and was also assessed by owner questionnaire; reliability was determined by 2 to 7 days of monitoring using the Spearman-Brown prophecy formula. RESULTS: In the validation study, accelerometry output differed significantly between activity intensities (Friedman test, P<0·01). In the practical utility study, no data were lost from any dogs and dog owners reported that accelerometry was well tolerated. Reliability of accelerometry output was high: for 3 days of wear, it was 91% [95% confidence interval (CI) 82 to 96] and for 7 days of wear, it was 94% (CI 88 to 97). CLINICAL SIGNIFICANCE: The GT3-X accelerometer is valid, practical and reliable for the measurement of habitual physical activity in dogs.

Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells.

Schwann cells synthesize two heparan sulfate proteoglycans, one that is a component of the Schwann cell basement membrane and a smaller one that is an integral component of the Schwann cell plasma membrane. To determine the functions of these molecules, Schwann cell-nerve cell cultures were grown in medium containing a specific inhibitor of proteoglycan biosynthesis, 4-methylumbelliferyl-beta-D-xyloside. Treatment with 1 mM beta-D-xyloside caused a 90% reduction in the accumulation of 35SO4-labeled proteoglycans in the cell layer of the cultures. Gel filtration analysis revealed that both the basement membrane and plasma membrane proteoglycans were affected. Inhibition of proteoglycan biosynthesis was accompanied by an inhibition of laminin deposition into extracellular matrix as determined by immunostaining of cultures and by immunoblotting of cell-associated proteins. This occurred even though there was no decrease in the amount of laminin detected in the medium of beta-D-xyloside-treated cultures. Deposition of collagen type IV was similarly affected. In addition, there was no myelin produced in beta-D-xyloside treated cultures. However, when beta-xyloside-treated cultures were supplied with exogenous basement membrane, Schwann cells produced numerous myelin segments. These results indicate that Schwann cell proteoglycans play an essential role in basement membrane assembly, and that the integral plasma membrane proteoglycan is not required for the basement membrane to exert its effects on Schwann cell differentiation.

Schwann cell myelination in a chemically defined medium: demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation.

Primary cultures of embryonic rat Schwann cells and sensory nerve cells can be grown in the serum-free defined medium N2, but the Schwann cells fail to deposit extracellular matrix and do not ensheath or myelinate axons. Previously these functions could be induced only in media supplemented with serum and chick embryo extract. Here we show that supplementing N2 medium with ascorbic acid and a commercial preparation of the glycoprotein fetuin or purified bovine serum albumin in the absence of serum or other undefined media components leads to increased production of Schwann cell extracellular matrix and extensive myelin formation by Schwann cells. Ascorbic acid is required for production of collagen type IV. Both ascorbic acid and one of the proteins are required for optimal extracellular matrix formation and myelination. These results lend support to the hypothesis that production of extracellular matrix by Schwann cells is necessary for myelination of nerve fibers.

A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells.

Schwann cells cocultured with sensory neurons in a serum-free medium accumulate a single species of radiolabeled heparan sulfate proteoglycan (HS-PG) during incubation in medium containing 35SO4. This HS-PG was poorly extracted from cultures by solutions containing 1% Triton X-100 in low salt buffer or by solutions containing 1 M KCl, 4 M urea plus dithiothreitol, 1 mM Tris-HCl, 5 mM EDTA, or 100 micrograms/ml of heparin. The HS-PG was efficiently extracted, however, by 1% Triton X-100 in the presence of 1 M KCl or by 1% deoxycholate. These treatments solubilize both cell membranes and the Schwann cell cytoskeleton. In intact cells the HS-PG was digested by trypsin, indicating it was at least partially exposed on the cell surface. When solubilized HS-PG was applied to a column of octyl-sepharose CL-4B, more than 90% was retained by the column, but was quantitatively eluted by a solution containing 1% Triton X-100. In addition, the solubilized HS-PG could be incorporated into artificial phospholipid vesicles. These results indicate the HS-PG is an integral plasma membrane protein. The inability of low ionic strength solutions containing Triton X-100 to solubilize the HS-PG suggested it was bound to an additional structure. To determine whether the HS-PG was associated with the cytoskeleton we isolated cytoskeletons by detergent lysis of cells and centrifugation. The major protein components of isolated cytoskeletons were spectrin (Mr 225,000), vimentin (Mr 58,000), and actin (Mr 45,000). When 35SO4-labeled cells were used to prepare cytoskeletons approximately 80% of the total HS-PG was recovered in the cytoskeleton fraction. These results suggest the HS-PG is an externally exposed integral plasma membrane protein that is anchored to the Schwann cell cytoskeleton.

Schwann cell myelination: induction by exogenous basement membrane-like extracellular matrix.

Exposing rat Schwann cells co-cultured with nerve cells to a reconstituted basement membrane induced the formation of myelin segments by Schwann cells. This occurred in a serum-free culture medium in which, in the absence of this matrix, Schwann cells proliferate but fail to differentiate. This reconstituted basement membrane was prepared from solubilized extracellular matrix proteins synthesized by a basement membrane-producing murine tumor. The major constituents of this reconstituted matrix are collagen type IV, laminin, heparan sulfate proteoglycan, entactin, and nidogen. The matrix also elicited striking morphological changes in Schwann cells, inducing them to spread longitudinally along the nerve fibers (a necessary early step in the process of ensheathment of nerve fibers). Several observations indicated that the effect of the matrix was exerted directly on Schwann cells and not indirectly through an effect on nerve cells. First, the matrix-induced cell spreading occurred only in areas in which Schwann cells directly contacted the matrix; Schwann cells that were associated with the same nerve fibers but that did not themselves directly contact the matrix did not exhibit spreading. Second, the matrix-induced alteration in Schwann cell morphology was observed in cultures in which the nerve cells were removed. These results provide direct evidence that basement membrane contact induces normal Schwann cell differentiation, and support the idea that Schwann cell differentiation in vivo may be regulated by the appearance of the basement membrane, which normally envelops terminally differentiating Schwann cells.

Sulfated proteoglycans produced by rat dorsal root ganglion cells.

Primary organotypic cultures of embryonic rat dorsal root ganglia, which contain sensory neurons, Schwann cells, and fibroblasts, produce an extensive extracellular matrix. These cultures actively incorporated 35SO4 into glycosaminoglycans, of which 30% were heparan sulfate, 65% chondroitin sulfate, and 5% dermatan sulfate. Sulfate-labeled proteoglycans made by these cells were extracted with 4 M guanidine-hydrochloride and purified by CsCl density gradient centrifugation, gel filtration chromatography, and DEAE-cellulose chromatography. Eight individual species were resolved, of which five were subjected to further analysis. One low-density (less than 1.35 g/ml) proteoglycan, with a Kav on Sepharose Cl-4B = 0.34 contained heparan sulfate chains of Mr = 20,000. The other four proteoglycans, with Kav on Sepharose Cl-4B of 0.04, 0.35 (two), and 0.44, contained predominantly chondroitin sulfate chains with Mr ranging from 30,000 to 40,000. To determine possible functions of these proteoglycans, cultures were grown in medium containing 1 mM 4-methyl-umbelliferyl-beta-D-xyloside. The drug inhibited 35SO4 proteoglycan accumulation in the cell layer by approximately 90%. Drug-treated cultures exhibited several developmental abnormalities, including decreased migration of fibroblast-like cells, abnormal morphology of these cells, and decreased myelination of axons by Schwann cells.

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans.

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.