Autosomal dominant polycystic kidney disease (ADPKD) is typified by the accumulation of fluid-filled cysts and abnormalities in renal epithelial cell function. The disease is principally caused by mutations in the gene encoding polycystin-1, a large basolateral plasma membrane protein expressed in kidney epithelial cells. Our studies reveal that, in normal kidney cells, polycystin-1 forms a complex with the adherens junction protein E-cadherin and its associated catenins, suggesting a role in cell adhesion or polarity. In primary cells from ADPKD patients, the polycystin-1/polycystin-2/E-cadherin/beta-catenin complex was disrupted and both polycystin-1 and E-cadherin were depleted from the plasma membrane as a result of the increased phosphorylation of polycystin-1. The loss of E-cadherin was compensated by the transcriptional upregulation of the normally mesenchymal N-cadherin. Increased cell surface N-cadherin in the disease cells in turn stabilized the continued plasma membrane localization of beta-catenin in the absence of E-cadherin. The results suggest that enhanced phosphorylation of polycystin-1 in ADPKD cells precipitates changes in its localization and its ability to form protein complexes that are critical for the stabilization of adherens junctions and the maintenance of a fully differentiated polarized renal epithelium.

Changes in extracellular space (ECS) diffusion parameters in astrogliotic tissue around a unilateral cortical stab wound were determined from concentrationtime profiles of tetramethylammonium (TMA ϩ ) using TMA ϩ -selective microelectrodes. Three diffusion parameters-ECS volume fraction ␣ (␣ ϭ ECS volume/ total tissue volume), tortuosity ( 2 ϭ D/ADC; where D is the free and ADC is the apparent diffusion coefficient of TMA ϩ in the brain), and nonspecific TMA ϩ uptake kЈ-were determined at 3, 7, 21, and 35 days postwounding (dpw), in the hemispheres ipsilateral and contralateral to the lesion. Following diffusion experiments, tissue sections were immunostained for glial fibrillary acidic protein (GFAP) and chondroitin-sulphate proteoglycans (CSPG). In the area 300-1000 µm around the wound, ␣ was increased at 3, 7, and 21 dpw by about 20% but returned to control values at 35 dpw; was increased at all four intervals, reaching a maximum at 7 dpw. kЈ was lower than in the contralateral hemisphere at 7, 21, and 35 dpw. Measurements 1,500-2,000 µm from the wound revealed only an increase in at 7 dpw. The time course of changes in ECS diffusion parameters closely correlated with increased staining for GFAP and CSPG. Our results show that astrogliosis significantly changes the diffusion properties of nervous tissue, making it less permissive. Both hypertrophied astrocytic processes and an enhanced formation of some extracellular matrix molecules could affect, through changes in the diffusion of molecules in the ECS, neuron-glia communication, ''cross-talk'' between synapses, extrasynaptic transmission, and regenerative processes.

A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery.

Vascular cells provide a neural stem/progenitor cell (NSPC) niche that regulates expansion and differentiation of NSPCs within the germinal zones of the embryonic and adult brain under both physiologic and pathologic conditions. Here, we examined the NSPC-endothelial cell (NSPC/EC) interaction under conditions of ischemia, both in vitro and after intracerebral transplantation. In culture, embryonic mouse NSPCs supported capillary morphogenesis and protected ECs from cell death induced by serum starvation or by transient oxygen and glucose deprivation (OGD). Neural stem/progenitor cells constitutively expressed hypoxia-inducible factor 1alpha (HIF-1alpha) transcription factor and vascular endothelial growth factor (VEGF), both of which were increased approximately twofold after the exposure of NSPCs to OGD. The protective effects of NSPCs on ECs under conditions of serum starvation and hypoxia were blocked by pharmacological inhibitors of VEGF signaling, SU1498 and Flt-1-Fc. After intracerebral transplantation, NSPCs continued to express HIF-1alpha and VEGF, and promoted microvascular density after focal ischemia. These studies support a role for NSPCs in stabilization of vasculature during ischemia, mediated via HIF-1alpha-VEGF signaling pathways, and suggest therapeutic application of NSPCs to promote revascularization and repair after brain injury.

Hypertensive small vessel disease is a major cause of vascular cognitive impairment (VCI). Spontaneously hypertensive/stroke prone rats (SHR/SP) with unilateral carotid artery occlusion (UCAO) and a Japanese permissive diet (JPD) have white-matter (WM) damage similar to that seen in VCI. We hypothesized that WM injury was due to hypoxia-mediated, blood-brain barrier (BBB) disruption. Twelve-week-old SHR/SP had UCAO/JPD and were studied with immunohistochemistry, biochemistry, multimodal magnetic resonance imaging (MRI), and Morris water maze (MWM) testing. One week after UCAO/JPD, WM showed a significant increase in hypoxia inducible factor-1α (HIF-1α), which increased further by 3 weeks. Prolyl hydroxylase-2 (PHD2) expression decreased at 1 and 3 weeks. Infiltrating T cells and neutrophils appeared around endothelial cells from 1 to 3 weeks after UCAO/JPD, and matrix metalloproteinase-9 (MMP-9) colocalized with inflammatory cells. At 3 weeks, WM immunostained for IgG, indicating BBB leakage. Minocycline (50 mg/kg intraperitoeally) was given every other day from weeks 12 to 20. Multimodal MRI showed that treatment with minocycline significantly reduced lesion size and improved cerebral blood flow. Minocycline improved performance in the MWM and prolonged survival. We propose that BBB disruption occurred secondary to hypoxia, which induced an MMP-9-mediated infiltration of leukocytes. Minocycline significantly reduced WM damage, improved behavior, and prolonged life.

A biocompatible heterogeneous hydrogel of poly [N-(2-hydroxypropyl) methacrylamide] (PHPMA), was evaluated for its ability to promote tissue repair and enhance axonal regrowth across lesion cavities in the brain and spinal cord in adult and juvenile (P17 P21) rats. Incorporation of PHPMA hydrogels into surrounding host tissue was examined at the ultrastructural level and using immunohistochemical techniques. In addition, and in parallel to these studies, diffusion parameters (volume fraction and tortuosity of the gel network) of the PHPMA hydrogels were evaluated pre- to postimplantation using an in vivo real-time iontophoretic method. The polymer hydrogels were able to bridge tissue defects created in the brain or spinal cord, and supported cellular ingrowth, angiogenesis, and axonogenesis within the structure of the polymer network. As a result, a reparative tissue grew within the porous structure of the gel, composed of glial cells, blood vessels, axons and dendrites, and extracellular biological matrices, such as laminin and/or collagen. Consistent with matrix deposition and tissue formation within the porous structure of the PHPMA hydrogels, there were measurable changes in the diffusion characteristics of the polymers. Extracellular space volume decreased and tortuosity increased within implanted hydrogels, attaining values similar to that seen in developing neural tissue. PHPMA polymer hydrogel matrices thus show neuroinductive and neuroconductive properties. They have the potential to repair tissue defects in the central nervous system by replacing lost tissue and by promoting the formation of a histotypic tissue matrix that facilitates and supports regenerative axonal growth. () ()

BackgroundMicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response.MethodsIntravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO.ResultsIn the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1α, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-β and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls.ConclusionsBased on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0753-x) contains supplementary material, which is available to authorized users.

Pulsed electromagnetic fields (PEMF) have been demonstrated to have anti-inflammatory and pro-regenerative effects in animals and humans. We used the FDA-approved Sofpulse (Ivivi Health Sciences, LLC) to study effect of PEMF on infarct size and poststroke inflammation following distal middle cerebral artery occlusion (dMCAO) in mice. Electromagnetic field was applied within 30-45 min after ischemic brain damage and utilized twice a day for 21 consecutive days. Ischemic infarct size was assessed using MRI and histological analysis. At 21 days after dMCAO, the infarct size was significantly (by 26%) smaller in PEMF-treated animals as compared to controls. Neuroinflammation in these animals was evaluated using specialized cytokine/chemokine PCR array. We demonstrate that PEMF significantly influenced expression profile of pro- and anti-inflammatory factors in the hemisphere ipsilateral to ischemic damage. Importantly, expression of gene encoding major pro-inflammatory cytokine IL-1α was significantly reduced, while expression of major anti-inflammatory IL-10 was significantly increased. PEMF application significantly downregulated genes encoding members of the major pro-apoptotic tumor necrosis factor (TNF) superfamily indicating that the treatment could have both anti-inflammatory and anti-apoptotic effects. Both reduction of infarct size and influence on neuroinflammation could have a potentially important positive impact on the poststroke recovery process, implicating PEMF as a possible adjunctive therapy for stroke patients.