Применение тонкослойной хроматографии для индентификации дифенгидрамина гидрохлорида и прокаина гидрохлорида при их совместном присутствии

ТЕХНОЛОГИЯ ФАРМАЦЕВТИЧЕСКАЯДИМЕДРОЛНОВОКАИНХРОМАТОГРАФИЯ ТОНКОСЛОЙНАЯ /ИС

Enhanced expressions of microvascular smooth muscle receptors after focal cerebral ischemia occur via the MAPK MEK/ERK pathway

<p>Abstract</p> <p>Background</p> <p>MEK1/2 is a serine/threonine protein that phosphorylates extracellular signal-regulated kinase (ERK1/2). Cerebral ischemia results in enhanced expression of cerebrovascular contractile receptors in the middle cerebral artery (MCA) leading to the ischemic region. Here we explored the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126.</p> <p>Methods and result</p> <p>Rats were subjected to a 2-h middle cerebral artery occlusion (MCAO) followed by reperfusion for 48-h and the ischemic area was calculated. The expression of phosphorylated ERK1/2 and Elk-1, and of endothelin ET_Aand ET_B, angiotensin AT₁, and 5-hydroxytryptamine 5-HT_1Breceptors were analyzed with immunohistochemistry using confocal microscopy in cerebral arteries, microvessels and in brain tissue. The expression of endothelin ET_Breceptor was analyzed by quantitative Western blot. We demonstrate that there is an increase in the number of contractile smooth muscle receptors in the MCA and in micro- vessels within the ischemic region. The enhanced expression occurs in the smooth muscle cells as verified by co-localization studies. This receptor upregulation is furthermore associated with enhanced expression of pERK1/2 and of transcription factor pElk-1 in the vascular smooth muscle cells. Blockade of transcription with the MEK1 inhibitor U0126, given at the onset of reperfusion or as late as 6 hours after the insult, reduced transcription (pERK1/2 and pElk-1), the enhanced vascular receptor expression, and attenuated the cerebral infarct and improved neurology score.</p> <p>Conclusion</p> <p>Our results show that MCAO results in upregulation of cerebrovascular ET_B, AT₁and 5-HT_1Breceptors. Blockade of this event with a MEK1 inhibitor as late as 6 h after the insult reduced the enhanced vascular receptor expression and the associated cerebral infarction.</p

Contractility Dominates Adhesive Ligand Density in Regulating Cellular De-adhesion and Retraction Kinetics

Cells that are enzymatically detached from a solid substrate rapidly round up as the tensile prestress in the cytoskeleton is suddenly unopposed by cell–ECM adhesions. We recently showed that this retraction follows sigmoidal kinetics with time constants that correlate closely with cortical stiffness values. This raises the promising prospect that these de-adhesion measurements may be used for high-throughput screening of cell mechanical properties; however, an important limitation to doing so is the possibility that the retraction kinetics may also be influenced and potentially rate-limited by the time needed to sever matrix adhesions. In this study, we address this open question by separating contributions of contractility and adhesion to cellular de-adhesion and retraction kinetics. We first develop serum-free conditions under which U373 MG glioma cells can be cultured on substrates of fixed fibronectin density without direct matrix contributions from the medium. We show that while spreading area increases with ECM protein density, cortical stiffness and the time constants of retraction do not. Conversely, addition of lysophosphatidic acid (LPA) to stimulate cell contractility strongly speeds retraction, independent of the initial matrix protein density and LPA’s contributions to spreading area. All of these trends hold in serum-rich medium commonly used in tissue culture, with the time constants of retraction much more closely tracking cortical stiffness than adhesive ligand density or cell spreading. These results support the use of cellular de-adhesion measurements to track cellular mechanical properties

Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

<p>Abstract</p> <p>Background</p> <p>Late cerebral ischemia carries high morbidity and mortality after subarachnoid hemorrhage (SAH) due to reduced cerebral blood flow (CBF) and the subsequent cerebral ischemia which is associated with upregulation of contractile receptors in the vascular smooth muscle cells (SMC) via activation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide a therapeutic window for treatment of cerebral ischemia after SAH.</p> <p>Results</p> <p>Here we demonstrate that SAH increases the phosphorylation level of ERK1/2 in cerebral vessels and reduces the neurology score in rats in additional with the CBF measured by an autoradiographic method. The intracisternal administration of SB-386023-b, a specific inhibitor of raf, given 6 h after SAH, aborts the receptor changes and protects the brain from the development of late cerebral ischemia at 48 h. This is accompanied by reduced phosphorylation of ERK1/2 in cerebrovascular SMC. SAH per se enhances contractile responses to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and angiotensin II (Ang II), upregulates ET_B, 5-HT_1Band AT₁receptor mRNA and protein levels. Treatment with SB-386023-b given as late as at 6 h but not at 12 h after the SAH significantly decreased the receptor upregulation, the reduction in CBF and the neurology score.</p> <p>Conclusion</p> <p>These results provide evidence for a role of the ERK1/2 pathway in regulation of expression of cerebrovascular SMC receptors. It is suggested that raf inhibition may reduce late cerebral ischemia after SAH and provides a realistic time window for therapy.</p

Comparison of neuropathic pain and neuronal apoptosis following nerve root or spinal nerve compression

Altered dorsal root ganglion (DRG) function is associated with neuropathic pain following spinal nerve injury. However, compression of the cauda equina and dorsal rhizotomy proximal to the DRG do not induce significant pain, whereas in the spinal nerve and peripheral nerve, injury distal to the DRG does induce neuropathic pain. Caspase signaling induces apoptosis, and caspase inhibitors prevent pain-related behavior. The degree of DRG neuronal apoptosis is thought to play a role in pain behavior. We suggest that differences in pain behavior according to the injury sites within the DRG may be related to imbalances in apoptotic injuries. The aim of this study was to determine which compression injury was more painful and to compare behavior with expression of tumor necrosis factor (TNF)-alpha in DRG and apoptosis in the DRG following crush injury to the L5 nerve root or L5 spinal nerve. Sprague–Dawley rats received a crush injury to the L5 spinal nerve (distal to the DRG), crush injury to the L5 nerve root (proximal to the DRG), or no crush injury (sham). Mechanical allodynia was determined by the von Frey test. Expression of TNF-alpha was compared among three groups using immunoblot findings. Furthermore, we compared the percentage of neurons injured in the DRG using immunostaining for apoptotic cells and localization of activated caspase 3. Mechanical allodynia was observed in both crush injury groups. The duration of mechanical allodynia in the distal crush group was significantly longer than in the proximal crush group (P < 0.05). TNF-alpha expression was increased in DRG neurons following injury. DRG apoptosis in the distal crush group was significantly higher than in the proximal group at each time point (P < 0.05). This study suggests that spinal nerve crush injuries produce a greater degree of DRG apoptosis than do corresponding nerve root crush injuries, and that the former injuries are associated with longer lasting mechanical allodynia. Thus, differences in the time course of mechanical allodynia might be associated with an imbalance in DRG apoptosis

Enhancement of endogenous neurogenesis in ephrin-B3 deficient mice after transient focal cerebral ischemia

Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3−/− (knockout) and ephrin-B3+/+ (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3−/− compared to ephrin-B3+/+ mice. However, prominent post-ischemic neurogenesis in ephrin-B3−/− mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4 weeks. Ischemic injury in ephrin-B3−/− mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3+/+ animals, infarct size in ephrin-B3−/− mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3−/− mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke

Hepcidin Is Involved in Iron Regulation in the Ischemic Brain

Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain