Computer-assisted quantitative immunohistochemistry of the extracellular matrix in the dorsal and ventral spinal roots with the use of a lucia software
| Authors | |
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| Year of publication | 2001 |
| Type | Article in Periodical |
| Magazine / Source | The Histochemical Journal |
| MU Faculty or unit | |
| Citation | |
| Field | Neurology, neurosurgery, neurosciences |
| Keywords | quantitative immunohistochemistry; laminin; fibronectin |
| Description | The dorsal and ventral spinal roots contain different types of axons. The endoneurial extracellular matrix (ECM), produced by Schwann cells and fibroblasts under control of the axons, is important extrinsic factor of the nerve regeneration. A different content of the endoneurial ECM can be expected in the dorsal and ventral spinal roots in relation to their axon type differences, but no information is available to the present time. In the present paper we demonstrated a comparison of immunofluorescence intensity for chondroitin sulfate proteoglycan, fibronectin, tenascin and thrombospondin in the endoneurial ECM of the rat dorsal and ventral spinal roots. For this purpose the cryostat sections through the dorsal and ventral roots were cut and incubated simultaneously. We tested optimal incubation conditions with the primary and secondary antibodies to distinguish differences in immunostaining intensity of the endoneurial ECM for investigated molecules. The best results of distinct staining in the dorsal and ventral roots were obtained after incubation with the primary antibody for 4 hrs and after treatment with affinity purified secondary goat anti-mouse antibody conjugated with TRITC at room temperature (20-23oC) for 90 min. An intensity of the immunofluorescence staining was assessed by computer-assisted image analysis (Lucia-G v4.21) using interactive segmentation (HSI) to select the measured areas of digitized pictures. The connective tissue sheaths of both dorsal and ventral roots displayed a high intensity of immunofluorescence for all investigated molecules. The axoplasm of myelinated axons in both dorsal and ventral roots was slightly decorated by immunolabeling for chondroitin sulfate proteoglycan, tenascin and thrombospondin but not for fibronectin. The measurement of immunofluorescence brightness revealed that the endoneurial ECM of the dorsal roots is immunostained for investigated molecules in higher intensity than in the ventral roots. The results suggest quantitative differences of the endoneurial ECM content in the intact dorsal and ventral roots corresponding probably with presence of various types of axons. The different ECM contents of the intact spinal roots might be an initial stage related with different extrinsic conditions created alongside afferent and motor axons for promotion of their regeneration after the root injury. |
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