Oxygen-glucose deprivation promotes gliogenesis and microglia activation in organotypic hippocampal slice culture: involvement of metalloproteinases.

Organotypic hippocampal cultures are used as an alternative model for studying molecular mechanism(s) of neurogenesis after combined oxygen-glucose deprivation (OGD) mimicking ischemic conditions. The aim of the present work was to investigate the effect of OGD on stem/progenitor cells proliferation and/or differentiation in the hippocampus. Our attention was primarily focused on the relationship between neurogenesis-associated processes and activity of matrix metalloproteinases (MMPs). Cell proliferation was detected by using BrdU incorporation. Newly generated BrdU (+) cells were identified by labeling with specific cell markers. In order to check the activity and localization of MMPs we conducted in situ zymography in conjunction with immunohistochemistry. In our experimental conditions OGD-insult followed by 24 h of recovery caused the damage of neuronal cells in CA1. At 1 week cell death appears all over the hippocampus. We found that expected stimulation of endogenous neurogenesis fails as a source of compensation for the lost neurons in OGD-treated cultures. The modulation of culture microenvironment after ischemia favors the dominant proliferation of glial cells expressed by the enhancement of newly-generated oligodendrocyte progenitors. In addition, during our study we also detected some BrdU labeled nuclei encapsulated by GFAP positive processes. However, the majority of BrdU positive cells expressed microglial specific stain, particularly pronounced in CA1area. The OGD-promoted responses involved activation of metalloproteinases, which matches the progression of gliogenesis. On the other hand, the high activity of MMPs associated with microglial cells implicate their involvement in the mechanism participating in OGD-induced cell damage.

Pivotal role for beta-1 integrin in neurovascular remodelling after ischemic stroke.

beta1 integrin is a cell surface molecule that is critical for endothelial cell adhesion, migration and survival during angiogenesis. In the present study we employed in vivo and in vitro models to elucidate the role of beta1 integrin in vascular remodelling and stroke outcomes. At 24 h after cerebral ischemia and reperfusion (I/R), the ischemic cortex (ipsilateral area) exhibited modest beta1 integrin immunoreactivity and a robust increase was observed at 72 h. Double-label immunohistochemical analysis for beta1 integrin with neuronal (NeuN), microglial (Iba-1), astrocyte (GFAP), progenitor cell (Ng2) and blood vessel (collagen 4) markers showed that beta1 integrin expression only localized to blood vessels. In vitro studies using cultured endothelial cells and a beta1 integrin blocking antibody confirmed that beta1 integrin is required for endothelial cell migration, proliferation and blood vessel formation. In vivo studies in the cerebral I/R model using the beta1 integrin blocking antibody further confirmed that beta1 integrin signaling is involved in vascular formation and recovery following ischemic stroke. Finally, we found that beta1 integrin is critically involved in functional deficits and survival after a stroke. These results suggest that beta1 integrin plays important roles in neurovascular remodelling and functional outcomes following stroke, and that targeting the beta1 integrin signalling may provide a novel strategy for modulating angiogenesis in ischemic stroke and other pathological conditions.

Long-term stable fibrin gels for cartilage engineering.

It is essential that hydrogel scaffold systems maintain long-term shape stability and mechanical integrity for applications in cartilage tissue engineering. Within this study, we aimed at the improvement of a commercially available fibrin gel in order to develop a long-term stable fibrin gel and, subsequently, investigated the suitability of the optimized gel for in vitro cartilage engineering. Only fibrin gels with a final fibrinogen concentration of 25mg/ml or higher, a Ca(2+) concentration of 20mm and a pH between 6.8 and 9 were transparent and stable for three weeks, the duration of the experiment. In contrast, when preparing fibrin gels with concentrations out of these ranges, turbid gels were obtained that shrank and completely dissolved within a few weeks. In rheological characterization experiments, the optimized gels showed a broad linear viscoelastic region and withstood mechanical loadings of up to 10,000 Pa. Bovine chondrocytes suspended in the optimized fibrin gels proliferated well and produced the extracellular matrix (ECM) components glycosaminoglycans and collagen type II. When initially seeding 3 million cells or more per construct (5mm diameter, 2mm thick), after 5 weeks of culture, a coherent cartilaginous ECM was obtained that was homogenously distributed throughout the whole construct. The developed fibrin gels are suggested also for other tissue engineering applications in which long-term stable hydrogels appear desirable.

Roles of mineralocorticoid and glucocorticoid receptors in the regulation of progenitor proliferation in the adult hippocampus.

New neurons are produced continually in the dentate gyrus of the hippocampus. Numerous factors modulate the rate of neuron production. One of the most important is the adrenal-derived corticoids. Raised levels of corticoids suppress proliferation of progenitor cells, while removal of corticoids by adrenalectomy reverses this. The exact mechanisms by which corticoids mediate such regulation are unknown, but corticoids are believed to act through the receptors for mineralocorticoids (MR) and glucocorticoids (GR). Previous reports regarding the roles of these receptors in regulating cell proliferation came to contrasting conclusions. Here we use both agonists and antagonists to these receptors in adult male rats to investigate and clarify their roles. Blockade of MR with spironolactone in adrenalectomised male rats implanted with a corticosterone pellet to reproduce basal levels enhanced proliferation, whereas treatment with the GR antagonist mifepristone had no effect. However, mifepristone reversed the suppressive effect of additional corticosterone in intact rats. Both aldosterone and RU362, agonists of MR and GR, respectively, reduced proliferation in adrenalectomised rats, and combined treatment with both agonists had an additional suppressive action. These results clearly show that occupancies of both receptors act in the same direction on progenitor proliferation. The existence of two receptors with different affinities for corticoids may ensure that proliferation of progenitor cells in the adult dentate gyrus is regulated across the range of adrenal corticoid activity, including both basal and stressful contexts. Although a small proportion of newly formed cells may express GR and MR, corticosterone probably regulates proliferation indirectly through other local cells.