Untersuchungen über die Rolle von Matrix-Metalloproteinasen und deren Inhibitoren bei der demyelinisierenden murinen Theilervirus-Enzephalomyelitits mittels RT-qPCR ; Investigations on the role of matrix-metalloproteinases and their inhibitors in demyelinating Theiler´s murine encephalomyelitis employing RT-qPCR

In the literature section a short review of the present knowledge of the virology, epidemiology, disease signs, pathology and pathogenesis of chronic demyelinating Theiler`s murine encephalomyelitis (TME) is given. The investigations concerning the constitutive expression of matrix-metalloproteinases (MMPs) and their endogenous inhibitors ("tissue inhibitors of matrix-metalloproteinases", TIMPs) in the spinal cord are based on the results obtained from 12 adult, female SJL/JHanHsd control mice. For the investigations upon the role of MMPs and TIMPs in demyelinating TME, 76 female SJL/JHanHsd mice, infected with the BeAn-strain of TMEV at the age of five weeks and 77 placebo (sham)-infected female SJL/JHanHsd mice were killed in groups of six, 0, 1, 4, 7, 14, 28, 42, 56, 70, 98, 147, and 196 days post infection (d.p.i.). For the description of the course of chronic demyelinating TME a clinical evaluation was performed. A 60-second-hangtest, a rotarod assay and an evaluation of stride length were performed as special tests for the evaluation of motor coordination and strength. For the histological examination hematoxylin-eosin and luxol-fast-blue-cresylechtviolet stained sections of formalin-fixed, paraffin-embedded spinal cord were examinated. The latter consisted of a semiquantitative scoring of the degree of meningitis, poliomyelitis, leukomyelitis, hypercellularity of the gray and white matter, demyelination and malacia in defined cervical, thoracal and lumbal segments of the spinal cord For the quantification of the mRNA expression of the MMPs (-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, -24), TIMPs (-1, -2, -3, -4), the four housekeeping genes (GAPDH, ß-actin, HPRT, SDHA) and the TMEV-RNA in the spinal cords, RT-qPCR using the Brilliant® SYBR® Green QPCR Core Reagent Kit (Stratagene®) was performed employing the MX4000TM Multiplex Quantitative PCR System (Stratagene®) with an absolute external cDNA standard. No clinical signs were observed during the early infection phase from 0 to 21 d.p.i. except for a ...