G.F. Machado1; N.R. Marangoni2; G.D. Melo3; O.C. Moraes3
Supported by FAPESP (Process #05/60132-1).
Canine visceral leishmaniasis (CVL) frequently causes a chronic systemic disease, characterized by loss of weight, skin lesions, lymphadenopathy, hepatosplenomegaly, renal failure and also neurologic symptoms, like paraparesis and paraplegia. Inflammation in choroid plexus and leptomeninges, and hyaline deposits in blood vessel walls are related as the major histopathologic alteration in central nervous system (CNS). To facilitate their entry in CNS, leukocytes produce matrix metalloproteinases (MMPs), resulting in disruption of the blood vessels basal membrane, promoting the breakdown of blood-brain-barrier (BBB). MMPs are zinc-dependent endopeptidases acting on extracellular matrix remodeling, and in CNS, high concentrations of MMPs perpetuate inflammatory response. The aim of this study was to investigate the involvement of MMP-2 and MMP-9 in CNS damage in naturally infected dogs by Leishmania chagasi. Samples of nervous tissue of dogs with positive diagnosis for CVL, symptomatic (n = 5) or asymptomatic (n = 9), and dogs with negative diagnosis (control group, n = 8) were evaluated by gelatin zymography. Gels were analyzed by ImageJ software 1.40x (NIH) and data were evaluated by means of one-way ANOVA followed by Tukey post test with significance level of 5%.
We detected only inactive forms of both MMP-2 and MMP-9. ProMMP-2 was detected only in dogs with positive diagnosis for CVL, but its density was increased in symptomatic animals (p < 0.0001). ProMMP-9 was found in all groups, but a significant difference (p = 0.0096) was observed only in asymptomatic and control dogs. MMPs exist in very low concentrations in normal CNS, but they are found in cases of severe brain injury and neuronal disorders. MMPs are secreted as proenzymes and their extracellular activity is regulated by activators and inhibitors, and TIMPs (tissue inhibitor of metalloproteinases) are considerate the major regulator of MMPs activity in tissues. The considerable different levels in studied MMPs observed among controls and infected asymptomatic or symptomatic dogs suggested a growing production of MMP-2 and -9 during the disease progression. This is in agreement with the increasing lymphocyte number we have observed in CNS of dogs with CVL (data not shown). Apparently, there are also powerful control mechanisms of MMPs activation in tissue nervous of dogs during CVL. Our results demonstrate a kind of nervous compartment preservation, at least in the stages of infection studied. Further studies in brain tissue of dogs with CVL and nervous symptoms, and also the involvement of TIMPS are necessary to confirm our hypothesis.