Hypoxia-ischemia (H-I) can produce widespread neurodegeneration and deep cerebral white matter injury in the neonate. Resident microglia and invading leukocytes promote lesion progression by releasing reactive oxygen species, proteases and other pro-inflammatory mediators. After injury, expression of the gelatin-degrading matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are thought to result in the proteolysis of extracellular matrix (ECM), activation of cytokines/chemokines, and the loss of vascular integrity. Thus, therapies targeting ECM degradation and progressive neuroinflammation may be beneficial in reducing H-I - induced neuropathy. Minocycline has MMP-inhibitory properties and is both anti-inflammatory and neuroprotective. AG3340 (prinomastat) is an MMP inhibitor with high selectivity for the gelatinases. The purpose of this study was to determine whether these compounds could limit H-I--induced injury when administered at a delayed time point.

The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), post-synaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 microg protein for PSD-95 to 0.23 microg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.

Proteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability.