Selected acute phase CSF factors in ischemic stroke: findings and prognostic value

<p>Abstract</p> <p>Background</p> <p>Study aimed at investigation of pathogenic role and prognostic value of several selected cerebrospinal fluid acute phase factors that can reflect the severity of ischemic brain damage.</p> <p>Methods</p> <p>Ninety five acute ischemic stroke patients were investigated. Ischemic region visualized at the twenty fourth hour by conventional Magnetic Resonance Imaging. Stroke severity evaluated by National Institute Health Stroke Scale. One month outcome of disease was assessed by Barthel Index. Cerebrospinal fluid was taken at the sixth hour of stroke onset. CSF pro- and anti-inflammatory cytokines were studied by Enzyme Linked Immunosorbent Assay. Nitric Oxide and Lipoperoxide radical were measured by Electron Paramagnetic Resonance. CSF Nitrate levels were detected using the Griess reagent. Statistics performed by SPSS-11.0.</p> <p><b>Results</b></p> <p>At the sixth hour of stroke onset, cerebrospinal fluid cytokine levels were elevated in patients against controls. Severe stroke patients had increased interleukin-6 content compared to less severe strokes (P < 0.05). Cerebrospinal fluid Electron Paramagnetic Resonance signal of nitric oxide was increased in patients against controls. Severe stroke group had an elevated Electron Paramagnetic Resonance signal of lipoperoxiradical compared to less severe stroke. Cerebrospinal fluid nitrate levels in less severe stroke patients were higher than those for severe stroke and control. Positive correlation was established between the initial interleukin-6 content and ischemic lesion size as well as with National Institute Health Stroke Scale score on the seventh day. Initial interleukin-6 and nitrate levels in cerebrospinal fluid found to be significant for functional outcome of stroke at one month.</p> <p><b>Conclusion</b></p> <p>According to present study the cerebrospinal fluid contents of interleukin-6 and nitrates seem to be the most reliable prognostic factors in acute phase of ischemic stroke.</p

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopolesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells

Protective effect of leptin against ischemia-reperfusion injury in the rat small intestine

BACKGROUND: The small intestine is extremely sensitive to ischemia-reperfusion (I/R) injury and a range of microcirculatory disturbances which contribute to tissue damage. Previous studies have shown that leptin plays an important physiological role in the microvasculature. The aim of this study was to evaluate the protective effects of leptin in I/R – induced mucosal injury in the small intestine. METHODS: Forty rats were divided into 5 groups (n = 8). Group I was subjected to a sham operation. Following mesenteric ischemia in group II (control); physiologic saline 1 cm(3), in group III; leptin 100 μg/kg, and physiologic saline 1 cm(3), in group IV; N(G)-L-arginine methyl ester (L-NAME) 20 mg/kg, and physiologic saline 1 cm(3), in group V; leptin 100 μg/kg, L-NAME 20 mg/kg, and physiologic saline 1 cm(3 )were given intra-peritoneally. In these groups, an I/R procedure was performed by occlusion of the superior mesenteric artery for 45 min followed by 120 min reperfusion. After reperfusion, the small intestines were resected for malondialdehyde (MDA) and nitric oxide (NO) concentration and histopathologic properties. Mucosal lesions were scored between 0 and 5. Tissue MDA and NO concentration and histopathologic grades were compared statistically. RESULTS: Tissue MDA level significantly increased (P < 0.05), tissue NO level significantly decreased in group V animals, compared to group III animals respectively (P < 0.001). Histopathologically, intestinal injury significantly decreased in the leptin treated ischemic group. CONCLUSION: Leptin can be used safely in mesenteric occlusive diseases, since it induces NO formation and release in mesenteric vessels

Collateral circulation: Past and present

Following an arterial occlusion outward remodeling of pre-existent inter-connecting arterioles occurs by proliferation of vascular smooth muscle and endothelial cells. This is initiated by deformation of the endothelial cells through increased pulsatile fluid shear stress (FSS) caused by the steep pressure gradient between the high pre-occlusive and the very low post-occlusive pressure regions that are interconnected by collateral vessels. Shear stress leads to the activation and expression of all NOS isoforms and NO production, followed by endothelial VEGF secretion, which induces MCP-1 synthesis in endothelium and in the smooth muscle of the media. This leads to attraction and activation of monocytes and T-cells into the adventitial space (peripheral collateral vessels) or attachment of these cells to the endothelium (coronary collaterals). Mononuclear cells produce proteases and growth factors to digest the extra-cellular scaffold and allow motility and provide space for the new cells. They also produce NO from iNOS, which is essential for arteriogenesis. The bulk of new tissue production is carried by the smooth muscles of the media, which transform their phenotype from a contractile into a synthetic and proliferative one. Important roles are played by actin binding proteins like ABRA, cofilin, and thymosin beta 4 which determine actin polymerization and maturation. Integrins and connexins are markedly up-regulated. A key role in this concerted action which leads to a 2-to-20 fold increase in vascular diameter, depending on species size (mouse versus human) are the transcription factors AP-1, egr-1, carp, ets, by the Rho pathway and by the Mitogen Activated Kinases ERK-1 and -2. In spite of the enormous increase in tissue mass (up to 50-fold) the degree of functional restoration of blood flow capacity is incomplete and ends at 30% of maximal conductance (coronary) and 40% in the vascular periphery. The process of arteriogenesis can be drastically stimulated by increases in FSS (arterio-venous fistulas) and can be completely blocked by inhibition of NO production, by pharmacological blockade of VEGF-A and by the inhibition of the Rho-pathway. Pharmacological stimulation of arteriogenesis, important for the treatment of arterial occlusive diseases, seems feasible with NO donors

Molecular control of nitric oxide synthesis through eNOS and caveolin-1 interaction regulates osteogenic differentiation of adipose-derived stem cells by modulation of Wnt/β-catenin signaling

BACKGROUND: Nitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation and osteogenesis. Endothelial nitric oxide synthase (eNOS), one of three NO-producing enzymes, is located in a close conformation with the caveolin-1 (CAV-1(WT)) membrane protein which is inhibitory to NO production. Modification of this interaction through mutation of the caveolin scaffold domain can increase NO release. In this study, we genetically modified equine adipose-derived stem cells (eASCs) with eNOS, CAV-1(WT), and a CAV-1(F92A) (CAV-1(WT) mutant) and assessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway. METHODS: NO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1(F92A) to eASCs, and osteogenesis and Wnt signaling was assessed by gene expression analysis and activity of a novel Runx2-GFP reporter. Cells were also exposed to a NO donor (NONOate) and the eNOS inhibitor, l-NAME. RESULTS: NO production as measured by nitrite was significantly increased in eNOS and CAV-1(F92A) transduced eASCs +(5.59 ± 0.22 μM) compared to eNOS alone (4.81 ± 0.59 μM) and un-transduced control cells (0.91 ± 0.23 μM) (p < 0.05). During osteogenic differentiation, higher NO correlated with increased calcium deposition, Runx2, and alkaline phosphatase (ALP) gene expression and the activity of a Runx2-eGFP reporter. Co-expression of eNOS and CAV-1(WT) transgenes resulted in lower NO production. Canonical Wnt signaling pathway-associated Wnt3a and Wnt8a gene expressions were increased in eNOS-CAV-1(F92A) cells undergoing osteogenesis whilst non-canonical Wnt5a was decreased and similar results were seen with NONOate treatment. Treatment of osteogenic cultures with 2 mM l-NAME resulted in reduced Runx2, ALP, and Wnt3a expressions, whilst Wnt5a expression was increased in eNOS-delivered cells. Co-transduction of eASCs with a Wnt pathway responsive lenti-TCF/LEF-dGFP reporter only showed activity in osteogenic cultures co-transduced with a doxycycline inducible eNOS. Lentiviral vector expression of canonical Wnt3a and non-canonical Wnt5a in eASCs was associated with induced and suppressed osteogenic differentiation, respectively, whilst treatment of eNOS-osteogenic cells with the Wnt inhibitor Dkk-1 significantly reduced expressions of Runx2 and ALP. CONCLUSIONS: This study identifies NO as a regulator of canonical Wnt/β-catenin signaling to promote osteogenesis in eASCs which may contribute to novel bone regeneration strategies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-016-0442-9) contains supplementary material, which is available to authorized users