Background and Purpose-Osteopontin (OPN) is an inducible, multifunctional, extracellular matrix protein that may be protective against blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH). However, the protective mechanisms remain unclear. Methods-We produced the endovascular perforation model of SAH in rats and studied the time course of OPN induction in brains by Western blotting and immunofluorescence (nϭ50). Then, 34 rats were randomly assigned to sham (nϭ3), shamϩOPN small interfering RNA (siRNA, nϭ3), SAHϩnegative control siRNA (nϭ14), and SAHϩOPN siRNA (nϭ14) groups, and 109 rats were allocated to shamϩvehicle (nϭ17), shamϩrecombinant OPN (nϭ17), SAHϩvehicle (nϭ33), SAHϩrecombinant OPN (nϭ31), and SAHϩrecombinant OPNϩL-arginyl-glycyl-L-aspartate motif-containing hexapeptide (nϭ11) groups. The effects of OPN siRNA or recombinant OPN on BBB disruption and related proteins were studied. Results-OPN was significantly induced in reactive astrocytes and capillary endothelial cells, peaking at 72 hours after SAH, during the recovery phase of BBB disruption. Blockage of endogenous OPN induction exacerbated BBB disruption and was associated with a reduction of angiopoietin-1 and mitogen-activated protein kinase (MAPK) phosphatase-1 (an endogenous MAPK inhibitor), activation of MAPKs, and induction of vascular endothelial growth factor-A at 72 hours after SAH, whereas recombinant OPN treatment improved it and was associated with MAPK phosphatase-1 induction, MAPK inactivation, and vascular endothelial growth factor-A reduction, which was blocked by L-arginyl-glycyl-L-aspartate motif-containing hexapeptide at 24 hours after SAH. Vascular endothelial growth factor-B and angiopoietin-2 levels were unchanged. Conclusions-OPN
Pigmented neurons in the substantia nigra pars compacta (SNc) and locus coeruleus (LC) show decreased numbers differentially in Parkinson's disease (PD) and multiple system atrophy (MSA). Recent reports have described that fast spin-echo T1-weighted magnetic resonance imaging (MRI) by a 3-tesla machine can visualize neuromelanin-related contrast of the noradrenergic and dopaminergic neurons respectively in the LC and the SNc. Using neuromelanin MRI at 3 T, we investigated possible alterations of these catecholaminergic neurons in 32 PD and 9 MSA patients, and compared the results with those of 23 normal volunteers. The contrast ratio of the LC and SNc was decreased in MSA and PD patients, most prominently in the LC in MSA patients. The contrast ratio of the SNc was correlated with the Hoehn-Yahr stage of PD and the severity of neuroradiological abnormalities in MSA. These results indicate a potential diagnostic value of neuromelanin MRI to distinguish MSA patients from normal and PD patients.
Objective-Accumulated evidence suggests that the primary cause of poor outcome after subarachnoid hemorrhage (SAH) is not only cerebral arterial narrowing, but also early brain injury (EBI). Our objective was to determine the effect of recombinant osteopontin (r-OPN), a pleiotropic extracellular matrix glycoprotein, on post-SAH EBI in rats. Design-Controlled in vivo laboratory study. Setting-Animal research laboratory.Subjects-One hundred seventy-seven male adult Sprague-Dawley rats, 300-370g.Interventions-The endovascular perforation model of SAH was produced. SAH or shamoperated rats were treated with an equal volume (1μL) of pre-SAH intracerebroventricular administration of two dosages (0.02 and 0.1μg) of r-OPN, albumin or vehicle. Body weight, neurological scores, brain edema and blood-brain barrier (BBB) disruption were evaluated, and Western blot analyses were performed to determine the effect of r-OPN on matrix metalloproteinase (MMP)-9, substrates of MMP-9 (zona occludens [ZO]-1, laminin), tissue inhibitor of MMP (TIMP)-1, inflammation (interleukin-1β), and nuclear factor (NF)-κ B signaling pathways. Measurements and MainResults-Treatment with r-OPN prevented a significant loss in body weight, neurological impairment, brain edema, and BBB disruption after SAH. These effects were associated with the deactivation of NF-κB activity, inhibition of MMP-9 induction, the maintenance of TIMP-1, and the consequent preservation of the cerebral microvessel basal lamina protein laminin, and the tight junction protein ZO-1.Conclusions-These results demonstrate that r-OPN treatment is effective for post-SAH EBI. Aneurysmal subarachnoid hemorrhage (SAH) is a common and devastating neurological disorder (1). Cerebral vasospasm has been believed to be a leading cause of mortality and morbidity (2). Recent randomized clinical trials, however, showed that clazosentan significantly reduced angiographic vasospasm but failed to improve long-term neurological outcome (3,4). This means that the treatment efforts targeting only angiographic vasospasm might not be enough to achieve a better outcome. Thus, new research efforts have focused on clarifying the pathophysiology of early brain injury (EBI) following SAH, and on developing protective strategies against it (5).Osteopontin (OPN) is a secreted extracellular matrix (ECM) glycoprotein that is involved in both physiological and pathological processes in a wide range of tissue (6). The biological functions of OPN are highly variable, and often seemingly contradictory depending on the biological scenario surrounding its induction (7). However, there is compelling evidence that OPN can, in a variety of situations, help cells survive an otherwise lethal insult (8). For example, endogenous OPN induction has consistently been found to have protective effects on ischemic injuries involving the brain and other organs (9,10). Moreover, the administration of recombinant OPN (r-OPN) markedly reduced the infarct size via anti-apoptotic actions in a transient focal cerebral ischemi...
A primate model was used to determine whether oxyhemoglobin (OxyHb), methemoglobin (MetHb), or bilirubin is likely to be responsible for cerebral vasospasm following subarachnoid hemorrhage (SAH). Forty cynomolgus monkeys were randomly assigned to one of five groups. On Day 0, each animal underwent angiography followed by right craniectomy and placement of an Ommaya reservoir with its catheter adjacent to the right middle cerebral artery (MCA). The animals received intrathecal injections twice a day for 6 days of one of the following solutions: mock cerebrospinal fluid (CSF); OxyHb; MetHb; bilirubin; or supernatant fluid from an incubated mixture of autologous blood and mock CSF. On Day 7, angiography was repeated and the animals were killed. Comparison of angiograms obtained on Day 0 and Day 7 of the experiment showed significant vasospasm of the right MCA and the right anterior cerebral and internal carotid arteries in the animal groups that had received OxyHb or supernatant fluid. There was a smaller reduction in diameter of the same vessels in the bilirubin group (not statistically significant), while no effects were observed in the groups receiving MetHb or mock CSF. Electron microscopy of the right MCA's gave results consistent with the angiographic findings. One monkey in the OxyHb group developed a delayed-onset right MCA infarction. These data suggest that OxyHb is the cause of cerebral vasospasm following SAH.
Objective-Osteopontin (OPN), a pleiotropic extracellular matrix glycoprotein, has been reported to be protective against ischemic lesions, but effects of OPN on vascular functions have not been investigated. The aim of this study was to assess whether recombinant OPN (r-OPN) could prevent cerebral vasospasm after subarachnoid hemorrhage (SAH) in rats.Methods-r-OPN was administered intraventricularly to rats undergoing SAH by the endovascular perforation, and its protective effects were evaluated by measuring the diameter of cerebral arteries and neurobehavioral testing. Western blotting and immunofluorescence were performed to explore the underlying mechanisms. An integrin receptor antagonist GRGDSP or mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 small interfering RNA (siRNA) was also administered to r-OPN-treated SAH rats, and those effects were evaluated.Results-Pre-SAH administration of r-OPN prevented vasospasm and neurological impairments at 24-72 hours post-SAH. r-OPN enhanced an endogenous MAPK inhibitor, MKP-1, and suppressed the phosphorylation of MAPKs, caldesmon and heat shock protein 27 in the spastic cerebral arteries at 24 hours post-SAH. Immunofluorescence revealed that MKP-1 was induced in the arterial smooth muscle layer. GRGDSP prevented r-OPN-induced MKP-1 upregulation, and MKP-1 siRNA abolished both MAPK inactivation and anti-vasospastic effects by r-OPN. Post-SAH r-OPN treatment also prevented vasospasm.Interpretation-r-OPN induced MKP-1 in the spastic cerebral arteries via binding to L-arginylglycyl-L-aspartate-dependent integrin receptors and prevented vasospasm after SAH. Therapeutic induction of MKP-1 may be a novel approach for the prevention and treatment of cerebral vasospasm.Aneurysmal subarachnoid hemorrhage (SAH)is a common and devastating neurological disorder.1 Cerebral vasospasm, the delayed narrowing of large capacitance arteries at the base of the brain, has been believed to be a leading cause of mortality and morbidity and to be the most preventable complication secondary to SAH.2 Recent randomized clinical trials, however, showed that currently available anti-vasospastic drugs are not enough to achieve a better outcome.3 , 4 Thus, further research efforts are needed to develop the new therapy against vasospasm. Osteopontin (OPN) is a multifunctional extracellular matrix glycoprotein that is known to be protective against ischemic injuries involving the brain and other organs.5 , 6 However, no studies have investigated the effect of OPN on vascular functions that might contribute to the protective effect. Considering pleiotropic effects of OPN,6 it would be interesting to examine if OPN modulates the vessel diameter in a pathological condition. In fact, OPN is reported to affect some signaling pathways such as protein kinase C (PKC) that is potentially involved in vascular smooth muscle contraction.7 , 8 We recently reported that recombinant OPN (r-OPN) suppressed interleukin (IL)-1β-induced nuclear factor (NF)-κB activation and matrix metalloproteinase...
Neonatal hypoxia-ischemia (HI) is an important clinical problem with few effective treatments. Granulocyte-colony stimulating factor (G-CSF) is an endogenous peptide hormone of the hematopoietic system that has been shown to be neuroprotective in focal ischemia in vivo, and is currently in phase I/II clinical trials for ischemic stroke in humans. We tested G-CSF in a rat model of neonatal hypoxia-ischemia in postnatal day 7 unsexed rat pups. Three groups of animals were used: hypoxia ischemia (HI, n=67), hypoxia-ischemia with G-CSF treatment (HI+G, n=65), and healthy control (C, n=53). G-CSF (50 μg/kg, subcutaneous) was administered 1 hour after HI, and given on four subsequent days (five total injections). Animals were euthanized 24 hours, 1, 2, and 3 weeks after HI. Assessment included brain weight, histology, immunohistochemistry, and Western blotting. G-CSF treatment was associated with improved quantitative brain weight and qualitative Nissl histology after hypoxia-ischemia. TUNEL demonstrated reduced apoptosis in group HI+G. Western blot demonstrated decreased expression of Bax and cleaved caspase 3 in group HI+G. G-CSF treatment was also associated with increased expression of STAT3, Bcl-2, and Pim-1, all of which may have participated in the anti-apoptotic effect of the drug. We conclude that G-CSF ameliorates hypoxic-ischemic brain injury, and that this may occur in part by an inhibition of apoptotic cell death.
A right-sided subarachnoid hemorrhage (SAH) was created in 12 monkeys. Only the right (clot-side) cerebral arteries developed angiographic vasospasm (VSP), which was maximal 7 days after SAH. Eight animals were killed at this time and the remainder at 14 days. At the time of killing the middle cerebral arteries (MCAs) were harvested, and four normal, left (non-clot-side) MCAs were vasoconstricted in vitro with prostaglandin F2… All MCAs were studied with scanning and transmission electron microscopy. Right MCAs in maximal VSP 7 days from SAH were undistinguishable on scanning electron microscopy from normal arteries vasoconstricted in vitro: both groups demonstrated a mean 57% reduction in vessel caliber and a 5-fold increase in vessel wall thickness compared to normal, nonvasoconstricted left MCAs. On transmission electron microscopy, however, arteries in SAH-induced VSP showed degenerative changes in the tunica intima and media. These changes were still evident at 14 days. despite considerable resolution of VSP. These findings, as well as those from other pathological studies of animal and human cerebral arteries in VSP, suggest that the arterial narrowing and vessel wall thickening seen within several weeks of SAH is due primarily to medial contraction, but unlike simple vasoconstruction, is associated with degenerative ultrastructural changes in the endothelium and vascular smooth muscle cells which may denote a temporarily irreversible state.
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