Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain

<p>Abstract</p> <p>Background</p> <p>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 <it>Wisteria floribunda </it>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.</p> <p>Results</p> <p>WFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA.</p> <p>Conclusion</p> <p>In general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.</p

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Sprague-Dawley rats were exposed to H-I at postnatal day 7 (P7), consisting of unilateral carotid artery ligation followed by 90 min exposure to 8% O₂. Minocycline, AG3340, or vehicle were administered once daily for 6 days, beginning 24 hours after insult. Animals were sacrificed at P14 for neurohistological assessments. Immunohistochemistry was performed to determine the degree of reactive astrogliosis and immune cell activation/recruitment. Neural injury was detected using the Fluoro-Jade stain, a marker that identifies degenerating cells.</p> <p>Results</p> <p>CD11b and glial fibrillary acidic protein (GFAP) immunopositive cells increased in ipsilateral cortex after treatment with vehicle alone, demonstrating microglia/macrophage recruitment and reactive astrogliosis, respectively. Fluoro-Jade staining was markedly increased throughout the fronto-parietal cortex, striatum and hippocampus. Treatment with minocycline or AG3340 inhibited microglia/macrophage recruitment, attenuated astrogliosis and reduced Fluoro-Jade staining when compared to vehicle alone.</p> <p>Conclusion</p> <p>The selective gelatinase inhibitor AG3340 showed equal efficacy in reducing neural injury and dampening neuroinflammation when compared to the anti-inflammatory compound minocycline. Thus, MMP-2 and MMP-9 may be viable therapeutic targets to treat neonatal brain injury.</p

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layers of hippocampus (B), indicating severe neural injury. OX-42 immunoreactivity showed CD11b – expressing cells at the cortical lesion site (C). Hippocampal OX-42 localized to the molecular layer of dentate gyrus and was evident in hippocampal regions seated ventral to CA1, but was not present in the pyramidal cell layers (D). Scale bars = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat"</p><p>http://www.jneuroinflammation.com/content/5/1/34</p><p>Journal of Neuroinflammation 2008;5():34-34.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527306.</p><p></p

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat-0

Cortex (B) compared to contralateral control (A). CD11b – expressing cells were abundant in corpus striatum of both hemispheres but showed distinct morphology (C,D). Cells in the ipsilateral striatum displayed the activated amoeboid phenotype (D) similar to those in ipsilateral cortex (E) when compared to the ramified morphology in the contralateral hemisphere (C) that is associated with a less activated state. Scale bars = 100 μm (A,B), 50 μm (C-E).<p><b>Copyright information:</b></p><p>Taken from "Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat"</p><p>http://www.jneuroinflammation.com/content/5/1/34</p><p>Journal of Neuroinflammation 2008;5():34-34.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527306.</p><p></p

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat-1

layers of hippocampus (B), indicating severe neural injury. OX-42 immunoreactivity showed CD11b – expressing cells at the cortical lesion site (C). Hippocampal OX-42 localized to the molecular layer of dentate gyrus and was evident in hippocampal regions seated ventral to CA1, but was not present in the pyramidal cell layers (D). Scale bars = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat"</p><p>http://www.jneuroinflammation.com/content/5/1/34</p><p>Journal of Neuroinflammation 2008;5():34-34.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527306.</p><p></p

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat-4

Ult. Intense OX-42 immunoreactivity was evident throughout the ipsilateral cerebral cortex of animals treated with vehicle alone (B). Microglia/macrophage labeling was nearly abolished after treatment with minocycline (D) and was markedly reduced after treatment with AG3340 (F). OX-42 immunoreactivity in contralateral control regions was faint and diffuse by comparison (A,C,E). Scale bars = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat"</p><p>http://www.jneuroinflammation.com/content/5/1/34</p><p>Journal of Neuroinflammation 2008;5():34-34.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527306.</p><p></p

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat-3

Ult. Animals treated with vehicle alone showed robust Fluoro-Jade labeling in ipsilateral cortex and corpus striatum (B). Fluoro-Jade staining was nearly abolished after treatment with minocycline (D) and greatly reduced after treatment with AG3340 (F) relative to vehicle alone (B). No labeling was detected in contralateral control regions (A,C,E). Quantification showed a significant reduction after treatment with either minocycline or AG3340 (G) compared to treatment with vehicle alone. N = 5, * = p < 0.05. Scale bars = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat"</p><p>http://www.jneuroinflammation.com/content/5/1/34</p><p>Journal of Neuroinflammation 2008;5():34-34.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527306.</p><p></p

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat-5

Cortex (B) compared to contralateral control (A). CD11b – expressing cells were abundant in corpus striatum of both hemispheres but showed distinct morphology (C,D). Cells in the ipsilateral striatum displayed the activated amoeboid phenotype (D) similar to those in ipsilateral cortex (E) when compared to the ramified morphology in the contralateral hemisphere (C) that is associated with a less activated state. Scale bars = 100 μm (A,B), 50 μm (C-E).<p><b>Copyright information:</b></p><p>Taken from "Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat"</p><p>http://www.jneuroinflammation.com/content/5/1/34</p><p>Journal of Neuroinflammation 2008;5():34-34.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527306.</p><p></p