FIG. 2.

Expression of gelatinases MMP-2 and MMP-9 in microdissected brain structures. (a) Gelatin zymography. To increase their concentration, the gelatinases were purified from brain structure lysates on gelatin-Sepharose beads, using the method of Zhang and Gottschall (67), and loaded on a 9% polyacrylamide gel containing gelatin (0.07%) and activated (20 h at 37°C), and then the gels were stained. Constitutive expression of active MMP-2 (65 kDa) was detected in all sham-inoculated structures, while faint MMP-9 proteolytic activity (92 kDa) was seen only in the mesencephalon, brain stem, cerebellum, and spinal cord. MMP-2 and pro-MMP-9 were markedly upregulated in infected brain structures, in particular in the rostral part of the brain. (b) Densitometric analysis. Data expressed as the ratio of infected/sham-inoculated normalized values (relative units) showed upregulation of MMP-2 and MMP-9 mainly in the hippocampus and, to a lesser extent, in the cortex and hypothalamus of infected mice. cx, cortex; mes, mesencephalon; hip, hippocampus; hyp, hypothalamus; bs, brain stem; cb, cerebellum; sc, spinal cord. (c) APMA treatment of hippocampal lysates. To determine if gelatinases are expressed as prozymogens or active enzymes, hippocampal lysates from sham-inoculated and infected mice at 14 dpi were treated with APMA and then electrophoresed as above. The zymograms for the untreated samples (lanes 1 and 2) showed two clear bands of respective apparent molecular masses of 65 and 92 kDa, presumably corresponding to active MMP-2 and pro-MMP-9, which were strongly upregulated in infected hippocampus (lane 2). In the APMA-treated samples (lanes 3 and 4), the 92-kDa product disappeared while a 75-kDa apparent molecular mass product became visible; the 65-kDa product corresponds to active MMP-2 that remained unchanged. Culture supernatant from PMA-treated BHK21 cells (containing the MMP-2 active forms) and TNF-α-treated DEV cells (containing the MMP-9 active form) were used as positive controls. Lanes 1 and 3, hippocampus from a sham-inoculated mouse; lanes 2 and 4, hippocampus from an infected mouse