Extracellular matrix proteins are time-dependent and regional-specific markers in experimental diffuse brain injury.
ABSTRACT: INTRODUCTION:The extracellular matrix (ECM) provides structural support for neuronal, glial, and vascular components of the brain, and regulates intercellular signaling required for cellular morphogenesis, differentiation and homeostasis. We hypothesize that the pathophysiology of diffuse brain injury impacts the ECM in a multi-dimensional way across brain regions and over time, which could facilitate damage and repair processes. METHODS:Experimental diffuse TBI was induced in male Sprague-Dawley rats (325-375 g) by midline fluid percussion injury (FPI); uninjured sham rats serve as controls. Tissue from the cortex, thalamus, and hippocampus was collected at 15 min, 1, 2, 6, and 18 hr postinjury as well as 1, 3, 7, and 14 days postinjury. All samples were quantified by Western blot for glycoproteins: fibronectin, laminin, reelin, and tenascin-C. Band intensities were normalized to sham and relative to ?-actin. RESULTS:In the cortex, fibronectin decreased significantly at 15 min, 1 hr, and 2 hr postinjury, while tenascin-C decreased significantly at 7 and 14 days postinjury. In the thalamus, reelin decreased significantly at 2 hr, 3 and 14 days postinjury. In the hippocampus, tenascin-C increased significantly at 15 min and 7 days postinjury. CONCLUSION:Acute changes in the levels of these glycoproteins suggest involvement in circuit dismantling, whereas postacute levels may indicate a restorative or regenerative response associated with recovery from TBI.
Project description:Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.
Project description:Traumatic brain injury (TBI) is a major public health concern affecting 2.8 million people per year in the United States, of whom about 1 million are children under 19 years old. Animal models of TBI have been developed and used in multiple ages of animals, but direct comparisons of adult and adolescent populations are rare. The current studies were undertaken to directly compare outcomes between adult and adolescent male mice, using a closed head, single-impact model of TBI. Six-week-old adolescent and 9-week-old adult male mice were subjected to mild-moderate TBI. Histological measures for neurodegeneration, gliosis, and microglial neuroinflammation, and behavioral tests of locomotion and memory were performed. Adolescent TBI mice have increased mortality (?2 = 20.72, p < 0.001) compared to adults. There is also evidence of hippocampal neurodegeneration in adolescents that is not present in adults. Hippocampal neurodegeneration correlates with histologic activation of microglia, but not with increased astrogliosis. Adults and adolescents have similar locomotion deficits after TBI that recover by 16 days postinjury. Adolescents have memory deficits as evidenced by impaired novel object recognition between 3-4 and 4-16 days postinjury (F1,26 = 5.23, p = 0.031) while adults do not. In conclusion, adults and adolescents within a close age range (6-9 weeks) respond to TBI differently. Adolescents are more severely affected by mortality, neurodegeneration, and inflammation in the hippocampus compared to adults. Adolescents, but not adults, have worse memory performance after TBI that lasts at least 16 days postinjury.
Project description:INTRODUCTION:Blast traumatic brain injury (B-TBI) affects military and civilian personnel. Presently, there are no approved drugs for blast brain injury. METHODS:Exendin-4 (Ex-4), administered subcutaneously, was evaluated as a pretreatment (48 hours) and postinjury treatment (2 hours) on neurodegeneration, behaviors, and gene expressions in a murine open field model of blast injury. RESULTS:B-TBI induced neurodegeneration, changes in cognition, and genes expressions linked to dementia disorders. Ex-4, administered preinjury or postinjury, ameliorated B-TBI-induced neurodegeneration at 72 hours, memory deficits from days 7-14, and attenuated genes regulated by blast at day 14 postinjury. DISCUSSION:The present data suggest shared pathologic processes between concussive and B-TBI, with end points amenable to beneficial therapeutic manipulation by Ex-4. B-TBI-induced dementia-related gene pathways and cognitive deficits in mice somewhat parallel epidemiologic studies of Barnes et al. who identified a greater risk in US military veterans who experienced diverse TBIs, for dementia in later life.
Project description:We inflicted TBI to wildetype (wt) mice in order to establish whether the anti-inflammatory agent cyclophosphamide can be used therapeutically. Cyclophosphamide was found to regulate distinct inflammatory cells such as activated microglia separate from invading phagocytes and dendritic cells. Cyclophosphamide postinjury selectively reduces antigen-presenting dendritic cells. Findings show feasibility of drug development to interfere with brain inflammation. TBI was carried out in injured wt B6 mice for postinjury treatment with cyclophospamide i.p. using saline as a control substance for comparison with injured but untreated mice. Total RNA was prepared from injured cerebral neocortex after three days. RNA samples were also from uninjured wt mice as reference for hybridization on Affymetrix microarrays.
Project description:Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins ?1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase ?/? isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.
Project description:The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate-to-severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro- (IL-1? and TNF?) and anti-inflammatory (Arg1 and TGF?) cytokines with a single-phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro-inflammatory response peaks at 4 h and 7 days, and a delayed anti-inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post-TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.
Project description:The accumulation of extracellular matrix (ECM) and glial scar formation are considered important factors for the failure of regeneration in central nervous system (CNS) injury and multiple sclerosis. Theiler's murine encephalomyelitis (TME) as a model of multiple sclerosis served to evaluate the spatio-temporal course of ECM alterations in demyelinating conditions. Microarray analysis revealed only mildly upregulated gene expression of ECM molecules, their biosynthesis pathways and pro-fibrotic factors, while upregulation of matrix remodeling enzymes was more prominent. Immunohistochemistry demonstrated progressive accumulation of chondroitin sulfate proteoglycans, glycoproteins and collagens within demyelinated TME lesions, paralleling the development of astrogliosis. Deposition of collagen IV, laminin, perlecan and tenascin-C started 28 days postinfection (dpi), collagen I, decorin, entactin and neurocan accumulated from 56 dpi on, and fibronectin from 98 dpi on. The basement membrane (BM) molecules collagen IV, entactin, fibronectin, laminin and perlecan showed perivascular and parenchymal deposition, while the non-BM components collagen I, decorin, neurocan and tenascin-C only accumulated in a nonvascular pattern in demyelinated areas. Contrary, phosphacan expression progressively decreased during TME. The immunoreactivity of aggrecan and brevican remained unchanged. The spatio-temporal association of matrix accumulation with astrogliosis suggests a mainly astrocytic origin of ECM deposits, which in turn may contribute to remyelination failure in TME.
Project description:Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.
Project description:We inflicted TBI to wildetype (wt) mice in order to establish whether the anti-inflammatory agent cyclophosphamide can be used therapeutically. Cyclophosphamide was found to regulate distinct inflammatory cells such as activated microglia separate from invading phagocytes and dendritic cells. Cyclophosphamide postinjury selectively reduces antigen-presenting dendritic cells. Findings show feasibility of drug development to interfere with brain inflammation. Overall design: TBI was carried out in injured wt B6 mice for postinjury treatment with cyclophospamide i.p. using saline as a control substance for comparison with injured but untreated mice. Total RNA was prepared from injured cerebral neocortex after three days. RNA samples were also from uninjured wt mice as reference for hybridization on Affymetrix microarrays.
Project description:As populations age, the number of patients sustaining traumatic brain injury (TBI) and concomitantly receiving preinjury antiplatelet therapy such as aspirin (ASA) and clopidogrel (CLOP) is rising. These drugs have been linked with unfavorable clinical outcomes following TBI, where the exact mechanism(s) involved are still unknown. In this novel work, we aimed to identify and compare the altered proteome profile imposed by ASA and CLOP when administered alone or in combination, prior to experimental TBI. Furthermore, we assessed differential glycosylation PTM patterns following experimental controlled cortical impact model of TBI, ASA, CLOP, and ASA + CLOP. Ipsilateral cortical brain tissues were harvested 48 h postinjury and were analyzed using an advanced neuroproteomics LC-MS/MS platform to assess proteomic and glycoproteins alterations. Of interest, differential proteins pertaining to each group (22 in TBI, 41 in TBI + ASA, 44 in TBI + CLOP, and 34 in TBI + ASA + CLOP) were revealed. Advanced bioinformatics/systems biology and clustering analyses were performed to evaluate biological networks and protein interaction maps illustrating molecular pathways involved in the experimental conditions. Results have indicated that proteins involved in neuroprotective cellular pathways were upregulated in the ASA and CLOP groups when given separately. However, ASA + CLOP administration revealed enrichment in biological pathways relevant to inflammation and proinjury mechanisms. Moreover, results showed differential upregulation of glycoproteins levels in the sialylated N-glycans PTMs that can be implicated in pathological changes. Omics data obtained have provided molecular insights of the underlying mechanisms that can be translated into clinical bedside settings.