u-0126 and Edema

Aquaporin-4 (AQP4) regulates retinal water homeostasis and participates in retinal oedema pathophysiology. β-dystroglycan (β-DG) is responsible for AQP4 polarization and can be cleaved by matrix metalloproteinase-9 (MMP9). Retinal oedema induced by ischemia-reperfusion (I/R) injury is an early complication. Bumetanide (BU) has potential efficacy against cytotoxic oedema. Our study investigated the effects of β-DG cleavage on AQP4 and the roles of BU in a rat retinal I/R injury model. The model was induced by applying 110 mm Hg intraocular pressure to the anterior eye chamber. BU and U0126 (a selective ERK inhibitor) were intraperitoneally administered 15 and 30 min, respectively, before I/R induction. Rhodamine isothiocyanate extravasation detection, quantitative real-time PCR, transmission electron microscopy, hematoxylin-eosin staining, immunofluorescence staining, western blotting, and TUNEL staining were performed. AQP4 lost its polarization in the retinal perivascular domain as a result of β-DG cleavage. BU rescued AQP4 depolarization, suppressed AQP4 protein expression, attenuated retinal cytotoxic oedema, and downregulated β-DG and AQP4 mRNA expression. BU suppressed glial responses and mitochondria-mediated apoptotic protein expression, including that of Caspase-3 and Cyto C, raised the Bcl-2/Bax ratio, and lowered the number of apoptotic cells in the retina. Both BU and U0126 downregulated p-ERK and MMP9 expression. Thus, BU treatment suppressed β-DG cleavage, recovered AQP4 polarization partially via inhibiting ERK/MMP9 signaling pathway, and possess potential neuroprotective efficacy in the rat retinal ischemia-reperfusion injury model.

Topics: Animals; Aquaporin 4; Bumetanide; Dystroglycans; Edema; Matrix Metalloproteinase 9; Neuroprotection; Papilledema; Rats; Reperfusion Injury; Retina

Peripheral modulation of wind-up enhancement induced by peripheral tissue injury is investigated in rat spinal wide-dynamic-range (WDR) neurons. After subcutaneous (s.c.) injection of melittin, a pain-related peptidergic component separated from bee venom, the responsiveness of spinal cord WDR neuron to repeated suprathreshold (1.5T, the intensity threshold) electrical stimuli is enhanced. Comparing with the less effects on early response (0-100 ms), melittin significantly increases late response (100 ms to the next stimulus artifact) and after-discharge (starting from 2s after the last stimulus artifact) with 189% and 546%, respectively. Peripheral administration of a specific MEK inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis-[o-aminophenylmercapto] butadiene (U0126, 1 microg) gradually suppresses, but not completely blocks melittin-enhanced wind-up to the similar level of baseline. The inhibitions of U0126 are mainly on late response and after-discharge with 49% and 65%, respectively. Peripheral administration of three doses of U0126 (0.1, 1, 10 microg) has no effects on melittin-induced local paw edema regardless of either pre- or post-treatment of the drug. We conclude that peripheral ERKs pathway in the primary injury site is required to maintain melittin-enhanced wind-up of rat spinal cord wide-dynamic-range neurons.

Topics: Action Potentials; Analysis of Variance; Animals; Butadienes; Edema; Electric Stimulation; Enzyme Inhibitors; Foot; Male; MAP Kinase Signaling System; Melitten; Microelectrodes; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Nitriles; Plethysmography; Rats; Rats, Sprague-Dawley; Spinal Cord; Time Factors

Emerging data suggest that matrix metalloproteinase-9 (MMP-9) plays a critical role in the pathophysiology of brain injury. However, the regulatory mechanisms involved in vivo remain unclear. In this study, we focus on a mitogen activated protein kinase (MAPK) pathway that may trigger MMP-9 after traumatic brain injury. We aim to show that inhibition of the extracellular signal regulated kinase (ERK) would attenuate MMP-9 levels, reduce blood-brain barrier damage, and attenuate edema after trauma induced by controlled cortical impact in mouse brain. Western blots showed that phospho-ERK was rapidly upregulated after trauma. Treatment with U0126, which inhibits MEK, the kinase upstream of ERK, effectively prevented the activation of ERK. After trauma, gelatin zymography showed an increase in MMP-9. U0126 significantly reduced trauma-induced MMP-9 levels. Correspondingly, U0126 ameliorated the degradation of the tight junction protein ZO-1, which is an MMP-9 substrate, and significantly attenuated tissue edema. At 7 days after trauma, traumatic lesion volumes were significantly reduced by U0126 compared with saline-treated controls. These data indicate that the ERK MAPK pathway triggers the upregulation in MMP-9 after trauma, and further suggest that targeting the upstream signaling mechanisms that regulate deleterious MMP-9 activity may reveal new therapeutic opportunities for traumatic brain injury.

Topics: Animals; Brain Injuries; Butadienes; Down-Regulation; Edema; Enzyme Inhibitors; Male; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Nitriles

The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.

Topics: Animals; Anti-Inflammatory Agents; Butadienes; Edema; Enzyme Inhibitors; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Protein Serine-Threonine Kinases