RESUMEN
Hypoxic preconditioning of stem cells and neural progenitor cells has been tested for promoting cell survival after transplantation. The present investigation examined the hypothesis that hypoxic preconditioning of bone marrow mesenchymal stem cells (BMSCs) could not only enhance their survival but also reinforce regenerative properties of these cells. BMSCs from eGFP engineered rats or pre-labeled with BrdU were pre-treated with normoxia (20% O(2), N-BMSCs) or sub-lethal hypoxia (0.5% O(2). H-BMSCs). The hypoxia exposure up-regulated HIF-1α and trophic/growth factors in BMSCs, including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF) and its receptor FIK-1, erythropoietin (EPO) and its receptor EPOR, stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). Meanwhile, many pro-inflammatory cytokines/chemokines were down-regulated in H-BMSCs. N-BMSCs or H-BMSCs were intravenously injected into adult rats 24h after 90-min middle cerebral artery occlusion. Comparing to N-BMSCs, transplantation of H-BMSCs showed greater effect of suppressing microglia activity in the brain. Significantly more NeuN-positive and Glut1-positive cells were seen in the ischemic core and peri-infarct regions of the animals received H-BMSC transplantation than that received N-BMSCs. Some NeuN-positive and Glut-1-positive cells showed eGFP or BrdU immunoflourescent reactivity, suggesting differentiation from exogenous BMSCs into neuronal and vascular endothelial cells. In Rotarod test performed 15days after stroke, animals received H-BMSCs showed better locomotion recovery compared with stroke control and N-BMSC groups. We suggest that hypoxic preconditioning of transplanted cells is an effective means of promoting their regenerative capability and therapeutic potential for the treatment of ischemic stroke.
Asunto(s)
Trasplante de Médula Ósea/fisiología , Isquemia Encefálica/patología , Isquemia Encefálica/terapia , Precondicionamiento Isquémico/métodos , Trasplante de Células Madre Mesenquimatosas , Neovascularización Fisiológica/fisiología , Neurogénesis/fisiología , Animales , Western Blotting , Recuento de Células , Diferenciación Celular/fisiología , Células Cultivadas , Inmunohistoquímica , Inflamación/patología , Inflamación/prevención & control , Ligadura , Masculino , Arteria Cerebral Media/fisiología , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Recuperación de la Función/fisiología , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/terapiaRESUMEN
Focal adhesion kinase (FAK) plays key roles in cell adhesion and migration. We now report that the delayed rectifier Kv2.1 potassium channel, through its LD-like motif in N-terminus, may interact with FAK and enhance phosphorylation of FAK(397) and FAK(576/577). Overlapping distribution of Kv2.1 and FAK was observed on soma and proximal dendrites of cortical neurons. FAK expression promotes a polarized membrane distribution of the Kv2.1 channel. In Kv2.1-transfected CHO cells, formation of the Kv2.1-FAK complex was stimulated by fibronectin/integrin and inhibited by the K(+) channel blocker tetraethylammonium (TEA). FAK phosphorylation was minimized by shRNA knockdown of the Kv2.1 channel, point mutations of the N-terminus, and TEA, respectively. Cell migration morphology was altered by Kv2.1 knockdown or TEA, hindering cell migration activity. In wound healing tests in vitro and a traumatic injury animal model, Kv2.1 expression and co-localization of Kv2.1 and FAK significantly enhanced directional cell migration and wound closure. It is suggested that the Kv2.1 channel may function as a promoting signal for FAK activation and cell motility.
Asunto(s)
Movimiento Celular , Polaridad Celular , Corteza Cerebral/enzimología , Quinasa 1 de Adhesión Focal/metabolismo , Neuronas/enzimología , Canales de Potasio Shab/metabolismo , Animales , Células CHO , Adhesión Celular , Membrana Celular/enzimología , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Forma de la Célula , Corteza Cerebral/efectos de los fármacos , Córnea/fisiopatología , Lesiones de la Cornea , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Activación Enzimática , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Ratones , Complejos Multiproteicos , Neuronas/efectos de los fármacos , Fosforilación , Mutación Puntual , Bloqueadores de los Canales de Potasio/farmacología , Mapeo de Interacción de Proteínas , Seudópodos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potasio Shab/antagonistas & inhibidores , Canales de Potasio Shab/genética , Tetraetilamonio/farmacología , Transfección , Cicatrización de HeridasRESUMEN
Recombinant human erythropoietin (rhEPO), a neurovascular protective agent, therapeutically supports angiogenesis after stroke by enhancing endogenous up-regulation of vascular endothelial growth factor (VEGF). Increased VEGF expression has been characterized to negatively impact the integrity of the blood brain barrier (BBB), causing brain edema and secondary injury. The present study investigated the rhEPO-induced BBB protection after stroke and how it might be achieved by affecting VEGF pathway. rhEPO treatment (5,000 U/kg, i.p., 30 min before stroke and once a day for three days after stroke) reduced Evans blue leakage and brain edema after ischemia. The expression of the BBB integrity markers, occludin, alpha-catenin and beta-catenin, in the brain was preserved in animals received rhEPO. rhEPO up-regulated VEGF expression; however, the expression of VEGF receptor-2 (fetal liver kinase receptor, Flk-1) was significantly reduced in rhEPO-treated animals three days after stroke. We propose that, disregarding increased VEGF levels, rhEPO protects against ischemia-induced BBB damage at least partly by down-regulating Flk-1 expression and the response to VEGF signaling in the acute phase after stroke.
Asunto(s)
Barrera Hematoencefálica/patología , Eritropoyetina/uso terapéutico , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/patología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Edema Encefálico/prevención & control , Circulación Cerebrovascular/efectos de los fármacos , Circulación Cerebrovascular/fisiología , Regulación hacia Abajo/efectos de los fármacos , Azul de Evans , Técnica del Anticuerpo Fluorescente , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/prevención & control , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Microscopía Confocal , Ocludina , Proteínas Recombinantes , Accidente Cerebrovascular/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
K+ and Cl- homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K+ and Cl- efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl- current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C2-ceramide, or serum deprivation. Cl- channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl- channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K+ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl- and K+ homeostasis in regulating different apoptotic events.
Asunto(s)
Apoptosis/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Canales de Cloruro/metabolismo , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Canales de Cloruro/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Microscopía Electrónica de Transmisión , Neuronas/metabolismo , Neuronas/ultraestructura , Nitrobenzoatos/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacosRESUMEN
PURPOSE: To study retinal changes in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a spontaneously diabetic rat, and evaluate it as a possible new diabetic retinopathy model. METHODS: We examined the retinas of OLETF rats and Long-Evans Tokushima Otsuka (LETO) rats as controls under both a transmission electron microscope (TEM) and a scanning electron microscope (SEM). RESULTS: We detected higher blood sugar level in the OLETF rats than in the LETO rats beginning at 5 months of age. The inner nuclear layers of the retina decreased from 3-4 rows to 2 rows, whereas the photoreceptor cell nuclei decreased from 8 rows to 3-6 rows. Retinal pigment epithelial cells decreased in height, and basal infoldings were poorly developed. Retinal capillary basement membranes were significantly thicker in the OLETF rats than in the LETO rats, and endothelial cell damage was observed. The SEM of vascular corrosion casts of OLETF rats showed tortuosity, microaneurysms, and loop formations. CONCLUSIONS: The development of spontaneous hyperglycemia in OLETF rats was associated with alterations in retina ultrastructure. Changes were similar to those seen in diabetic retinopathy, but did not include either hemorrhages or exudates. The OLETF rat may be a useful animal model for the study of ocular complications in humans.
Asunto(s)
Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Retina/ultraestructura , Vasos Retinianos/ultraestructura , Animales , Glucemia/análisis , Peso Corporal , Molde por Corrosión , Hiperglucemia/patología , Masculino , Microscopía Electrónica de Rastreo , Ratas , Ratas Endogámicas OLETF , Ratas Long-EvansRESUMEN
The long-term effects of the angiotensin-converting enzyme (ACE) inhibitor cilazapril on retinal and choroidal circulation in rats with spontaneous diabetes type 2 were assessed by corrosion casts, scanning electron microscopy (SEM) and transmission electron microscopy. One group of 20 male Otsuka Long-Evans Tokushima Fatty (OLETF) rats was treated with 10 mg/kg/day of cilazapril from 4 to 64 weeks of age, and 20 other OLETF rats received no treatment. A third group, 20 male Long-Evans Tokushima Otsuka (LETO) rats, served as age-matched controls. At regular intervals, the rats were weighed, and their blood glucose was measured. Before the experiment, their systolic blood pressure and total cholesterol level were determined. At 64 weeks of age, the OLETF rats weighed significantly less than the cilazapril-treated OLETF and the LETO rats (p < 0.0001). At the same age, 100% of the untreated OLETF rats had bilateral cataracts, while the lens was clear and no fundus abnormality was detected in the cilazapril-treated OLETF rats and the LETO rats. Cilazapril lowered systolic blood pressure to a nearly normal level, significantly prevented the increase in blood sugar and inhibited the increase in serum cholesterol in the OLETF rats throughout the treatment. In the 64-week-old OLETF rats without treatment, corrosion cast and SEM revealed diabetic retinal and choroidal vascular changes: tortuosity of the vessels, variations in caliber, narrowing of arteries, arterio-arteriolar anastomoses and hairpin loop formation in precapillary arterioles, sparse collecting venules in the choroid and marked capillary changes such as caliber irregularity, narrowing, tortuosity, loop formation and decreased capillaries, outpouching and microaneurysms. In the cilazapril-treated OLETF rats, these changes were markedly decreased to the level seen in the LETO rats, in which the retinal and choroidal blood vessels had a definite and fairly constant pattern and the capillaries were more regularly and densely arranged and had a remarkably uniform caliber. Our results show that the long-term administration of cilazapril before or from the initial onset significantly prevented the increase in blood sugar and inhibited the increases in serum cholesterol in OLETF rats throughout the treatment, lowered systolic arterial pressure to a nearly normal level and prevented diabetic ocular complications. The effects of cilazapril on the diabetic retinal and choroidal vasculature are described for the first time. SEM of corrosion casts is a valuable and easy technique for showing precisely and three-dimensionally the effects of some drugs on the vasculature.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Coroides/irrigación sanguínea , Cilazapril/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/patología , Vasos Retinianos/patología , Animales , Glucemia/análisis , Presión Sanguínea , Vasos Sanguíneos/patología , Peso Corporal , Colesterol/sangre , Molde por Corrosión , Fondo de Ojo , Masculino , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Ratas , Ratas Endogámicas OLETF , Ratas Long-Evans , SístoleRESUMEN
We studied rat retinal changes due to aluminum (Al) toxicosis with a transmission electron microscope (TEM) and an energy dispersive X-ray analyzer (EDXA). Normal 4-week-old Wistar Kyoto rats were divided randomly into Al toxicosis and control groups. The Al toxicosis group was injected ip with 0.3 ml of 4% aluminum chloride (AlCl3) per day every day for 16 weeks. The retina was examined with a TEM and EDXA at 8, 12, and 16 weeks after starting injections with AlCl3. There was a statistically significant increase in the serum Al concentration in the Al toxicosis group (p < 0.001). We observed prominent pathologic changes at 16 weeks after the first injections. Thin retinal pigment epithelium (RPE), and disappearance of the photoreceptor outer and inner segments and nuclei were observed. There were high-density irregular granules in the outer and inner plexiform layers and in the inner nuclear layer. We found dense granules in the cells, which remained between the RPE and the inner nuclear layer. EDXA detected Al in the high-density irregular granules in these areas. Al injected ip caused accumulation of Al in the rat retina and the destruction of photoreceptor cells. These findings indicate that Al is toxic to the retina.