RESUMEN
Background Preeclampsia is a prominent risk factor for long-term development of cardiovascular disease. Although existing studies report a strong correlation between preeclampsia and heart failure, the underlying mechanisms are poorly understood. One possibility is the glycoprotein growth factor activin A. During pregnancy, elevated activin A levels are associated with impaired cardiac global longitudinal strain at 1 year, but whether these changes persist beyond 1 year is not known. We hypothesized that activin A levels would remain increased more than 1 year after a preeclamptic pregnancy and correlate with impaired cardiac function. Methods and Results To test our hypothesis, we performed echocardiograms and measured activin A levels in women approximately 10 years after an uncomplicated pregnancy (n=25) or a pregnancy complicated by preeclampsia (n=21). Compared with women with a previously normal pregnancy, women with preeclampsia had worse global longitudinal strain (-18.3% versus -21.3%, P=0.001), left ventricular posterior wall thickness (0.91 mm versus 0.80 mm, P=0.003), and interventricular septal thickness (0.96 mm versus 0.81 mm, P=0.0002). Women with preeclampsia also had higher levels of activin A (0.52 versus 0.37 ng/mL, P=0.02) and activin/follistatin-like 3 ratio (0.03 versus 0.02, P=0.04). In a multivariable model, the relationship between activin A levels and worsening global longitudinal strain persisted after adjusting for age at enrollment, mean arterial pressure, race, and body mass index (P=0.003). Conclusions Our findings suggest that both activin A levels and global longitudinal strain are elevated 10 years after a pregnancy complicated by preeclampsia. Future studies are needed to better understand the relationship between preeclampsia, activin A, and long-term cardiac function.
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Cardiopatías/etiología , Ventrículos Cardíacos/fisiopatología , Contracción Miocárdica/fisiología , Periodo Posparto/fisiología , Preeclampsia/fisiopatología , Función Ventricular Izquierda/fisiología , Activinas/sangre , Adulto , Biomarcadores/sangre , Ecocardiografía , Femenino , Estudios de Seguimiento , Cardiopatías/diagnóstico , Cardiopatías/fisiopatología , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Preeclampsia/sangre , Preeclampsia/diagnóstico , Embarazo , Pronóstico , Estudios Retrospectivos , Factores de TiempoRESUMEN
Background Approximately 60% of women have Stage B heart failure 1 year after a preeclamptic delivery. Emerging evidence suggests that the profibrotic growth factor activin A, which has been shown to induce cardiac fibrosis and hypertrophy, is elevated in preeclampsia and may be inhibited by aspirin therapy. We hypothesized that preeclamptic women receiving aspirin would have lower activin A levels and reduced global longitudinal strain (GLS), a sensitive measure of cardiac dysfunction, than women who do not receive aspirin. To test our hypothesis, we performed a cohort study of women with preeclampsia or superimposed preeclampsia and compared activin A levels and GLS in parturients who did or did not receive aspirin. Methods and Results Ninety-two parturients were enrolled, of whom 25 (27%) received aspirin (81 mg/day) therapy. GLS, plasma activin A, and follistatin, which inactivates activin A, were measured. Women receiving aspirin therapy had lower median (interquartile range) levels of activin A (8.17 [3.70, 10.36] versus 12.77 [8.37, 31.25] ng/mL; P=0.001) and lower activin/follistatin ratio (0.59 [0.31, 0.93] versus 1.01 [0.64, 2.60] P=0.002) than women who did not receive aspirin, which also remained significant after multivariable analysis. Furthermore, GLS was worse in patients who did not receive aspirin (-19.84±2.50 versus -17.77±2.60%; P=0.03) despite no differences in blood pressure between groups. Conclusions Our study suggests that antepartum aspirin therapy reduced serum activin A levels and improved GLS in preeclamptic patients, suggesting that aspirin may mitigate the postpartum cardiac dysfunction seen in women with preeclampsia.
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Activinas/sangre , Aspirina/administración & dosificación , Preeclampsia/sangre , Preeclampsia/fisiopatología , Atención Prenatal , Función Ventricular Izquierda/efectos de los fármacos , Adulto , Aspirina/efectos adversos , Biomarcadores/sangre , Regulación hacia Abajo , Esquema de Medicación , Femenino , Folistatina/sangre , Proteínas Relacionadas con la Folistatina/sangre , Humanos , Preeclampsia/diagnóstico , Embarazo , Estudios Prospectivos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Mechanisms of peri-operative ischaemic optic neuropathy remain poorly understood. Both specific pre-operative and intra-operative factors have been examined by retrospective studies, but no animal model currently exists. OBJECTIVES: To develop a rodent model of peri-operative ischaemic optic neuropathy. In rats, we performed head-down tilt and/or haemodilution, theorising that the combination damages the optic nerve. DESIGN: Animal study. SETTING: Laboratory. ANIMALS: A total of 36 rats, in four groups, completed the functional examination of retina and optic nerve after the interventions. INTERVENTIONS: Anaesthetised groups (n>8) were supine (SUP) for 5âh, head-down tilted 70° for 5âh, head-down tilted/haemodiluted for 5âh or SUP/haemodiluted for 5âh. We measured blood pressure, heart rate, intra-ocular pressure and maintained constant temperature. MAIN OUTCOME MEASUREMENTS: Retinal function (electroretinography), scotopic threshold response (STR) (for retinal ganglion cells) and visual evoked potentials (VEP) (for transmission through the optic nerve). We imaged the optic nerve in vivo and evaluated retinal histology, apoptotic cells and glial activation in the optic nerve. Retinal and optic nerve function were followed to 14 and 28 days after experiments. RESULTS: At 28 days in head down tilted/haemodiluted rats, negative STR decreased (about 50% amplitude reduction, Pâ=â0.006), VEP wave N2-P3 decreased (70% amplitude reduction, Pâ=â0.01) and P2 latency increased (35%, Pâ=â0.003), optic discs were swollen and glial activation was present in the optic nerve. SUP/haemodiluted rats had decreases in negative STR and increased VEP latency, but no glial activation. CONCLUSION: An injury partly resembling human ischaemic optic neuropathy can be produced in rats by combining haemodilution and head-down tilt. Significant functional changes were also present with haemodilution alone. Future studies with this partial optic nerve injury may enable understanding of mechanisms of peri-operative ischaemic optic neuropathy and could help discover preventive or treatment strategies.
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Modelos Animales de Enfermedad , Inclinación de Cabeza/efectos adversos , Hemodilución/efectos adversos , Traumatismos del Nervio Óptico/diagnóstico por imagen , Neuropatía Óptica Isquémica/diagnóstico por imagen , Animales , Electrorretinografía/métodos , Hemodilución/métodos , Masculino , Traumatismos del Nervio Óptico/etiología , Traumatismos del Nervio Óptico/fisiopatología , Neuropatía Óptica Isquémica/etiología , Neuropatía Óptica Isquémica/fisiopatología , Atención Perioperativa , Ratas , Ratas Sprague-DawleyRESUMEN
Women with hypertensive disorders of pregnancy have an increased risk of subsequent heart failure and cardiovascular disease when compared with women with normotensive pregnancies. Although the mechanisms underlying these findings are unclear, elevated levels of the biomarker activin A are associated with myocardial dysfunction and may have predictive value. We hypothesized that elevated levels of antepartum activin A levels would correlate with postpartum cardiac dysfunction in women with hypertensive disorders of pregnancy. We prospectively studied 85 women to determine whether increased antepartum activin A levels were associated with cardiac dysfunction at 1 year postpartum as measured by global longitudinal strain. Thirty-two patients were diagnosed with preeclampsia, 28 were diagnosed with gestational or chronic hypertension, and the remainder were nonhypertensive controls. Activin A levels were measured with ELISA both in the third antepartum trimester and at 1 year postpartum. Comprehensive echocardiograms including measurement of global longitudinal strain were also performed at enrollment and at 1 year postpartum. Antepartum activin A levels correlated with worsening antepartum global longitudinal strain (r=0.70; P=0.0001). Across the entire cohort, elevated antepartum activin A levels were associated with the development of abnormal global longitudinal strain at 1 year (C statistic 0.74; P=0.004). This association remained significant after multivariable adjustment for clinically relevant confounders (C statistic 0.93; P=0.01). Postpartum activin A levels also correlated with increasing left ventricular mass index (P=0.02), increasing mean arterial pressures (P=0.02), and decreasing E' values (P=0.01). Activin A may be a useful tool for identifying and monitoring patients at risk for postpartum development of cardiovascular disease.
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Activinas/sangre , Presión Sanguínea/fisiología , Ventrículos Cardíacos/diagnóstico por imagen , Hipertensión Inducida en el Embarazo/fisiopatología , Periodo Posparto , Disfunción Ventricular Izquierda/sangre , Función Ventricular Izquierda/fisiología , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Ecocardiografía , Femenino , Ventrículos Cardíacos/fisiopatología , Humanos , Embarazo , Tercer Trimestre del Embarazo , Estudios Prospectivos , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
PURPOSE: Ischemia-associated retinal degeneration is one of the leading causes of vision loss, and to date, there are no effective treatment options. We hypothesized that delayed injection of bone-marrow stem cells (BMSCs) 24 h after the onset of ischemia could effectively rescue ischemic retina from its consequences, including apoptosis, inflammation, and increased vascular permeability, thereby preventing retinal cell loss. METHODS: Retinal ischemia was induced in adult Wistar rats by increasing intraocular pressure (IOP) to 130-135 mmHg for 55 min. BMSCs harvested from rat femur were injected into the vitreous 24 h post-ischemia. Functional recovery was assessed 7 days later using electroretinography (ERG) measurements of the a-wave, b-wave, P2, scotopic threshold response (STR), and oscillatory potentials (OP). The retinal injury and anti-ischemic effects of BMSCs were quantitated by measuring apoptosis, autophagy, inflammatory markers, and retinal-blood barrier permeability. The distribution and fate of BMSC were qualitatively examined using real-time fundus imaging, and retinal flat mounts. RESULTS: Intravitreal delivery of BMSCs significantly improved recovery of the ERG a- and b-waves, OP, negative STR, and P2, and attenuated apoptosis as evidenced by decreased TUNEL and caspase-3 protein levels. BMSCs significantly increased autophagy, decreased inflammatory mediators (TNF-α, IL-1ß, IL-6), and diminished retinal vascular permeability. BMSCs persisted in the vitreous and were also found within ischemic retina. CONCLUSIONS: Taken together, our results indicate that intravitreal injection of BMSCs rescued the retina from ischemic damage in a rat model. The mechanisms include suppression of apoptosis, attenuation of inflammation and vascular permeability, and preservation of autophagy.
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Células de la Médula Ósea/citología , Isquemia/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Degeneración Retiniana/terapia , Vasos Retinianos/patología , Animales , Apoptosis , Barrera Hematorretinal , Permeabilidad Capilar , Modelos Animales de Enfermedad , Electrorretinografía , Etiquetado Corte-Fin in Situ , Inyecciones Intravítreas , Isquemia/diagnóstico , Isquemia/metabolismo , Masculino , Ratas , Ratas Wistar , Degeneración Retiniana/diagnóstico , Degeneración Retiniana/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/fisiopatologíaRESUMEN
PURPOSE: We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. METHODS: Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. RESULTS: Eyes injected with hypoxic BMSC-conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. CONCLUSIONS: The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.
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Células de la Médula Ósea/citología , Medios de Cultivo Condicionados/farmacología , Hipoxia/fisiopatología , Isquemia/terapia , Precondicionamiento Isquémico , Trasplante de Células Madre Mesenquimatosas/métodos , Retina/patología , Enfermedades de la Retina/terapia , Animales , Apoptosis , Células Cultivadas , Modelos Animales de Enfermedad , Electrorretinografía , Etiquetado Corte-Fin in Situ , Inyecciones Intraoculares , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Retina/fisiopatología , Enfermedades de la Retina/patología , Enfermedades de la Retina/fisiopatología , Células Ganglionares de la Retina/patologíaRESUMEN
PURPOSE: Delayed treatment after ischemia is often unsatisfactory. We hypothesized that injection of bone marrow stem cell (BMSC) conditioned medium after ischemia could rescue ischemic retina, and in this study we characterized the functional and histological outcomes and mechanisms of this neuroprotection. METHODS: Retinal ischemia was produced in adult Wistar rats by increasing intraocular pressure for 55 minutes. Conditioned medium (CM) from rat BMSCs or unconditioned medium (uCM) was injected into the vitreous 24 hours after the end of ischemia. Recovery was assessed 7 days after ischemia using electroretinography, at which time we euthanized the animals and then prepared 4-µm-thick paraffin-embedded retinal sections. TUNEL and Western blot were used to identify apoptotic cells and apoptosis-related gene expression 24 hours after injections; that is, 48 hours after ischemia. Protein content in CM versus uCM was studied using tandem mass spectrometry, and bioinformatics methods were used to model protein interactions. RESULTS: Intravitreal injection of CM 24 hours after ischemia significantly improved retinal function and attenuated cell loss in the retinal ganglion cell layer. CM attenuated postischemic apoptosis and apoptosis-related gene expression. By spectral counting, 19 proteins that met stringent identification criteria were increased in the CM compared to uCM; the majority were extracellular matrix proteins that mapped into an interactional network together with other proteins involved in cell growth and adhesion. CONCLUSIONS: By restoring retinal function, attenuating apoptosis, and preventing retinal cell loss after ischemia, CM is a robust means of delayed postischemic intervention. We identified some potential candidate proteins for this effect.
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Células de la Médula Ósea/citología , Isquemia/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Retina/patología , Enfermedades de la Retina/terapia , Animales , Apoptosis , Western Blotting , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Modelos Animales de Enfermedad , Electrorretinografía , Citometría de Flujo , Etiquetado Corte-Fin in Situ , Inyecciones Intravítreas , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratas , Ratas Wistar , Retina/fisiopatología , Enfermedades de la Retina/patología , Enfermedades de la Retina/fisiopatología , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
In previous studies, it was shown that post-conditioning, a transient period of brief ischemia following prolonged severe ischemia in the retina, could provide significant improvement in post-ischemic recovery, attenuation of cell loss, and decreased apoptosis. However, the mechanisms of post-conditioning in the retina have not been elucidated. We hypothesized that two kinases, mitogen-activated protein kinase p38α and protein kinase B (Akt), were involved in the mechanism of post-conditioning. Ischemia was induced in rat retina in vivo. Recovery after ischemia followed by 8 min of post-conditioning early in the reperfusion period after prolonged ischemia was assessed functionally (electroretinography) and histologically at 7 days after ischemia. We examined the role of p38α and Akt subtypes 1-3 in post-conditioning by intravitreal injection of interfering RNA 6 h prior to ischemia and post-conditioning and compared the results to injection of non-silencing interfering RNA sequence. The blockade of p38α significantly decreased the recovery after ischemia and post-conditioning, and enhanced cell loss and disorganization of the retina. Blockade of Akt1, and to a lesser degree, Akt2, significantly decreased the recovery after ischemia and enhanced cell loss and disorganization. These differences in the effects of blockade of Akt subtypes were not explainable by distribution of Akt subtypes in the retina, which were similar. In conclusion, both p38 and Akt are essential components of the neuroprotection induced by post-ischemic conditioning in the retina.
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Poscondicionamiento Isquémico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Retina/metabolismo , Retina/fisiopatología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Biomarcadores/metabolismo , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Electrorretinografía , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Retina/citología , Retina/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
In previous studies, it was shown that post-conditioning, a transient period of brief ischemia following prolonged severe ischemia in the retina, could provide significant improvement in post-ischemic recovery, attenuation of cell loss, and decreased apoptosis. These studies showed that post-conditioning effectively prevented damage after retinal ischemia when it was instituted early (within 1 h) in the post-ischemic period. While post-ischemic conditioning holds high promise of clinical translation, patients often present late after the onset of retinal ischemia and therefore immediate application of this anti-ischemic maneuver is generally not feasible. In this study, we examined the hypothesis that application of a post-conditioning stimulus at 24 h or greater following the end of prolonged ischemia would decrease the extent of ischemic injury. Ischemia was induced in rat retina in vivo. Recovery after ischemia followed by 5 min of post-conditioning brief ischemia 24 or 48 h after prolonged ischemia was assessed functionally (electroretinography) and histologically at 7 days after ischemia and post-conditioning or sham post-conditioning. We found that the brief ischemic stimulus applied 24, but not 48 h after prolonged ischemia significantly improved functional recovery and decreased histological damage induced by prolonged ischemia. We conclude that within a defined time window, delayed post-ischemic conditioning ameliorated post-ischemic injury in rats. Compared to earlier studies, the present work demonstrates for the first time the novel ability of a significantly delayed ischemic stimulus to provide robust neuroprotection in the retina following ischemia.
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Poscondicionamiento Isquémico , Daño por Reperfusión/prevención & control , Enfermedades de la Retina/prevención & control , Vasos Retinianos/fisiología , Animales , Apoptosis/fisiología , Presión Sanguínea/fisiología , Recuento de Células , Citoprotección , Electrorretinografía , Presión Intraocular/fisiología , Ratas , Ratas Wistar , Recuperación de la Función , Daño por Reperfusión/fisiopatología , Enfermedades de la Retina/fisiopatología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Tonometría OcularRESUMEN
We previously described the phenomenon of retinal ischemic pre-conditioning (IPC) and we have shown the role of various signaling proteins in the protective pathways, including the mitogen-activated protein kinase p38. In this study we examined the role in IPC of mitogen-activated protein kinase phosphatase-1 (MKP-1), which inactivates p38. Ischemia was produced by elevation of intraocular pressure above systolic arterial blood pressure in adult Wistar rats. Preconditioning was produced by transient retinal ischemia for 5 min, 24 h prior to ischemia. Small interfering RNA (siRNA) to MKP-1 or a control non-silencing siRNA, was injected into the vitreous 6 h prior to IPC. Recovery was assessed by electroretinography (ERG) and histology. The a-and b-waves, and oscillatory potentials (OPs), measured before and 1 week after ischemia, were then normalized relative to pre-ischemic baseline, and corrected for diurnal variation in the normal non-ischemic eye. The P2, or post-photoreceptor component of the ERG (which reflects function of the rod bipolar cells in the inner retina), was derived using the Hood-Birch model. MKP-1 was localized in specific retinal cells using immunohistochemistry; levels of mitogen-activated protein kinases were measured using Western blotting. Injection of siRNA to MKP-1 significantly attenuated the protective effect of IPC as reflected by decreased recovery of the electroretinogram a and b-waves and the P2 after ischemia. The injection of siRNA to MKP-1 reduced the number of cells in the retinal ganglion cell and outer nuclear layers after IPC and ischemia. Blockade of MKP-1 by siRNA also increased the activation of p38 at 24 h following IPC. MKP-1 siRNA did not alter the levels of phosphorylated jun N-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK) after IPC. The results suggest the involvement of dual-specificity phosphatase MKP-1 in IPC and that MKP-1 is involved in IPC by regulating levels of activated MAPK p38.
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Fosfatasa 1 de Especificidad Dual/fisiología , Precondicionamiento Isquémico , Daño por Reperfusión/prevención & control , Enfermedades de la Retina/prevención & control , Vasos Retinianos/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Técnicas de Cultivo de Célula , Electrorretinografía , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , MAP Quinasa Quinasa 4/metabolismo , Fosforilación , Interferencia de ARN , Ratas , Ratas Wistar , Daño por Reperfusión/enzimología , Daño por Reperfusión/fisiopatología , Retina/enzimología , Retina/fisiopatología , Células Bipolares de la Retina/fisiología , Enfermedades de la Retina/enzimología , Enfermedades de la Retina/fisiopatología , Células Ganglionares de la Retina/metabolismoRESUMEN
Ischemic pre-conditioning (IPC) provides neuroprotection in the rat retina from the damaging effects of severe ischemia. Recently, neuroprotection by retinal ischemic post-conditioning (Post-C), i.e., transient ischemia after more lengthy, damaging ischemia, was described, but its mechanisms are not yet known. One possible explanation of the effectiveness of Post-C is that it augments intrinsic neuroprotective mechanisms initiated during ischemia. Increasing duration of the damaging ischemic insult may therefore impact the effectiveness of Post-C. IPC, in contrast, sets in motion a series of neuroprotective events prior to the onset of ischemia. Thus, IPC and Post-C may operate by differing mechanisms. Accordingly, we examined the effect of retinal ischemic duration on post-ischemic outcome in vivo in rats after adding Post-C, and the impact of combining pre- and post-conditioning. Recovery after ischemia performed 24 h after IPC, or after Post-C performed 5 min after ischemia ended, was assessed functionally (electroretinography) and histologically at 7 days after ischemia. Durations of ischemia of 45 and 55 min were studied. Since recovery with IPC or Post-C alone, with 55 min of ischemia, did not achieve the same degree of effect (i.e., not complete recovery) exhibited in our previous studies of IPC using a different ischemia model, we also combined IPC and Post-C to test the hypothesis of the possible additive effects of the IPC and Post-C. We found that the recovery after Post-C was enhanced to a greater degree when ischemia was of longer duration. Post-C led to greater post-ischemic recovery compared to IPC. Both IPC and Post-C also attenuated structural damage to the retina. Contrary to our hypothesis, IPC and Post-C did not combine to enhance recovery after ischemia. In earlier studies, IPC attenuated post-ischemic apoptosis. To begin to examine the mechanism of Post-C, we studied its impact on apoptosis following ischemia. We examined apoptosis by determining the percentage of TUNEL-positive cells at 24 h after ischemia. Post-C attenuated apoptosis, but when combined with IPC, TUNEL was similar in the combined group to that of ischemia alone. We also examined the role of the recruitment of an inflammatory response in ischemia and Post-C. We found that inflammatory markers increased by ischemia were not altered by Post-C. We conclude that Post-C effectiveness depends upon the duration of ischemia; Post-C is not additive with IPC, and Post-C functions, in part, by preventing apoptotic damage to the inner retina. Post-C has considerable promise for clinical translation to eye diseases that cause blindness by ischemia.
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Poscondicionamiento Isquémico , Daño por Reperfusión/prevención & control , Enfermedades de la Retina/prevención & control , Vasos Retinianos/fisiopatología , Animales , Apoptosis/fisiología , Electrorretinografía , Técnica del Anticuerpo Fluorescente Indirecta , Etiquetado Corte-Fin in Situ , Mediadores de Inflamación/metabolismo , Presión Intraocular/fisiología , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/fisiopatología , Factores de Tiempo , Isquemia TibiaRESUMEN
In previous studies, inhibition of mitogen-activated protein kinase (MAP) p38 significantly improved recovery and attenuated apoptosis after retinal ischemia in rats. Yet, ischemic preconditioning (IPC) attenuated the ischemia-induced increase in p38 expression. We hypothesized that p38 was required for induction of ischemic tolerance by IPC. We examined the mechanisms of involvement of p38 in IPC neuroprotection. IPC or ischemia was induced in rat retina in vivo. Recovery after ischemia performed 24h after IPC was assessed functionally (electroretinography) and histologically at 7d after ischemia in the presence or absence of inhibition of p38. We examined the role of p38alpha in the mimicking of IPC produced by opening mitochondrial KATP channels using diazoxide, or stimulation of p38 activation by anisomycin. The importance of adenosine receptors in p38 activation after IPC was assessed using specific blockers of adenosine A1 and A2a receptors. Interfering RNA (siRNA) or SB203580 was used to block p38alpha. Phosphorylated p38 levels were measured. Phosphorylated p38 protein increased with IPC. Interfering RNA (siRNA) to p38alpha prior to IPC, or inhibiting p38 activation with SB203580, with ischemia following 24h later, significantly attenuated the neuroprotective effect of IPC. Anisomycin administered to increase p38 mimicked IPC, an effect blocked by SB203580. IPC-mimicking with diazoxide, an opener of mitochondrial KATP channels, was diminished with p38alpha siRNA. Adenosine receptor blockade did not decrease the elevated levels of phosphorylated p38 after IPC. Specific inhibition of p38alpha suggests that this MAPK is involved in the protective effects of IPC, and that p38 is downstream of mitochondrial KATP channels, but not adenosine receptors, in this neuroprotection.
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Isquemia/terapia , Precondicionamiento Isquémico , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Daño por Reperfusión/prevención & control , Neuronas Retinianas/enzimología , Vasos Retinianos/fisiopatología , Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Animales , Anisomicina/farmacología , Diazóxido/farmacología , Modelos Animales de Enfermedad , Electrorretinografía , Activadores de Enzimas/farmacología , Imidazoles/farmacología , Presión Intraocular , Isquemia/complicaciones , Isquemia/enzimología , Isquemia/patología , Isquemia/fisiopatología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/genética , Fosforilación , Canales de Potasio/agonistas , Canales de Potasio/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Daño por Reperfusión/enzimología , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Neuronas Retinianas/patología , Transducción de Señal , Factores de TiempoRESUMEN
BACKGROUND: The purpose of this study was to examine the role of erythropoietin in retinal ischemic preconditioning (IPC). METHODS: Rats were subjected to retinal ischemia after IPC. Electroretinography assessed functional recovery after ischemia; retinal sections were examined to determine loss of retinal ganglion cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to assess apoptosis. Levels of downstream mediators were measured in retinal homogenates by Western blotting. To assess the involvement of erythropoietin in IPC, Western blotting was used to measure levels of erythropoietin and its receptor (EPO-R) in retinal homogenates after IPC. To examine erythropoietin's role in IPC, the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC was studied. RESULTS: Erythropoietin levels did not change after IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histologic neuroprotection produced by IPC in comparison to control injection of denatured sEPO-R. Apoptotic damage after ischemia was enhanced in the sEPO-R-treated retinas as indicated by fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Phosphorylated extracellular-signal-regulated kinase and heat shock protein 27, but not protein kinase B, upregulated in denatured sEPO-R-treated retinae, were attenuated in eyes injected with sEPO-R. CONCLUSIONS: These results indicate that EPO-R upregulation is a critical component of the functional, histologic, and antiapoptotic protective effect of IPC on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.
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Eritropoyetina/análisis , Precondicionamiento Isquémico , Receptores de Eritropoyetina/análisis , Daño por Reperfusión/prevención & control , Retina/efectos de los fármacos , Vasos Retinianos , Animales , Apoptosis , Western Blotting , ADN Nucleotidilexotransferasa , Electrorretinografía , Eritropoyetina/farmacología , Eritropoyetina/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de Choque Térmico HSP27 , Etiquetado Corte-Fin in Situ , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/enzimología , Retina/citología , SolubilidadRESUMEN
Potent endogenous protection from ischemia can be induced in the retina by ischemic preconditioning (IPC). Protein kinase B/Akt is a cellular survival factor. We hypothesized that Akt was integral to IPC based upon differential effects of Akt subtypes. Rats were subjected to retinal ischemia after IPC or IPC-mimicking by the opening of mitochondrial KATP (mKATP) channels. The effects of blocking Akt using wortmannin, API-2, or small interfering RNA (siRNA) were examined. Electroretinography assessed functional recovery after ischemia, and TUNEL examined retinal ganglion cell apoptosis. We studied the relationship between Akt activation and known initiators of IPC, including adenosine receptor stimulation and the opening of mKATP channels. The PI-3 kinase inhibitor wortmannin 1 or 4 mg/kg (i.p.), the specific Akt inhibitor API-2, 5-500 microM in the vitreous, or intravitreal siRNA directed against Akt2 or -3, but not Akt1, significantly attenuated the neuroprotective effect of IPC. Interfering RNA against any of the three Akt subtypes significantly but time-dependently attenuated mKATP channel opening to mimic IPC. Adenosine A1 receptor blockade (DPCPX), A2a blockade (CSC), or the mKATP channel blocker 5-hydroxydecanoic acid significantly attenuated Akt activation after IPC. Interfering RNA directed against Akt subtypes prevented the ameliorative effect of IPC on post-ischemic apoptosis. All three Akt subtypes are involved in functional retinal neuroprotection by IPC or IPC-mimicking. Akt is downstream of adenosine A1 and A2a receptors and mKATP channel opening. The results indicate the presence in the retina of robust and redundant endogenous neuroprotection based upon subtypes of Akt.
Asunto(s)
Isquemia/enzimología , Precondicionamiento Isquémico/métodos , Proteínas Proto-Oncogénicas c-akt/fisiología , Vasos Retinianos/enzimología , Androstadienos/farmacología , Animales , Apoptosis , Western Blotting/métodos , Clorpropamida/análogos & derivados , Clorpropamida/farmacología , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Electrorretinografía/efectos de los fármacos , Electrorretinografía/métodos , Isquemia/prevención & control , Isoenzimas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Vasos Retinianos/patología , WortmaninaRESUMEN
The purpose of our study was to determine the specific subtypes of protein kinase C involved in the neuroprotection afforded by retinal ischemic preconditioning (IPC), their relationship to the opening of mitochondrial KATP (mKATP) channels, and their role in apoptosis after preconditioning and ischemia. Rats were subjected to retinal ischemia after IPC, or retinas were rendered ischemic after pharmacological opening of mKATP channels. Using immunohistochemistry and image analysis, we determined cellular localization of PKC subtypes. We blocked PKC-delta and -epsilon to study the effect on protection with IPC or with IPC-mimicking by the opening of mKATP channels. PKC subtypes were inhibited pharmacologically or with interfering RNA. Electroretinography assessed functional recovery after ischemia. IPC was effectively mimicked by injection of diazoxide to open the mKATP channel. IPC and/or its mimicking were attenuated by the PKC-delta inhibitor rottlerin and by interfering RNA targeting PKC-delta or -epsilon. Using TUNEL staining and Western blotting for caspase-3 and fodrin breakdown we assessed apoptosis. The injection of interfering RNA to PKC-delta and -epsilon before preconditioning significantly enhanced TUNEL staining as well as the cleavage of caspase-3 and fodrin after ischemia. In summary, our experiments have shown that both PKC-delta and -epsilon subtypes are involved in the cellular signaling that results in neuroprotection from IPC and that both are downstream of the opening of mKATP channels.
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Precondicionamiento Isquémico , Proteína Quinasa C/fisiología , Daño por Reperfusión/prevención & control , Enfermedades de la Retina/prevención & control , Vasos Retinianos/enzimología , Acetofenonas/farmacología , Animales , Apoptosis , Benzopiranos/farmacología , Western Blotting , Células Cultivadas , Diazóxido/farmacología , Electrorretinografía , Inhibidores Enzimáticos/farmacología , Etiquetado Corte-Fin in Situ , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Canales de Potasio/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/farmacocinética , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/enzimología , Enfermedades de la Retina/enzimología , Enfermedades de la Retina/patología , Vasos Retinianos/patología , Vasodilatadores/farmacologíaRESUMEN
PURPOSE: To examine the mechanisms of ischemic preconditioning (IPC) related to the opening of mitochondrial KATP (mKATP) channels in the retina. METHODS: Rats were subjected to retinal ischemia after IPC, or retinas were rendered ischemic after pharmacological opening of mKATP channels. The effects of blocking mKATP channel opening, nitric oxide synthase (NOS) subtypes, or protein kinase C (PKC) on the protective effect of IPC or on the opening of mKATP channels were studied. Electroretinography assessed functional recovery after ischemia. Immunohistochemistry and image analysis were used to measure changes in levels of reactive oxygen species (ROS) and NOS subtypes and to determine their cellular localization. RESULTS: IPC was effectively mimicked by injection of the mKATP channel opener diazoxide. Both IPC and its mimicking by diazoxide were completely attenuated by the mKATP channel blocker 5-hydroxydecanoic acid (5-HD). Nonspecific blockade of NOS by N(omega)-nitro-L-arginine (L-NNA), but not by specific inducible (i)NOS or neuronal (n)NOS inhibitors, blunted IPC and IPC-mimicking, as did blockade of PKC. IPC and diazoxide IPC-mimicking significantly enhanced mitochondrial ROS production in the inner retina, an effect blocked by 5-HD. Mitochondrial ROS colocalized with e- and nNOS in retinal cells after stimulation with diazoxide. CONCLUSIONS: The results showed that IPC in the retina requires opening of the mKATP channel, and that IPC could be effectively mimicked using the mKATP channel opener diazoxide. eNOS-generated nitric oxide, PKC, and ROS are activated by opening of the mKATP channel.
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Precondicionamiento Isquémico , Canales de Potasio/metabolismo , Daño por Reperfusión/prevención & control , Retina/metabolismo , Vasos Retinianos/fisiología , Animales , Ácidos Decanoicos/farmacología , Diazóxido/farmacología , Electrorretinografía , Técnica del Anticuerpo Fluorescente Indirecta , Hidroxiácidos/farmacología , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitroarginina/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The giant excised patch variant of patch clamp recording combines microsecond time resolution of macroscopic currents with rapid exchange of the experimental solutions at the intracellular membrane surface. This technique has been applied to a limited number of cell types, including Xenopus oocytes, muscle cells, and photoreceptors. We have applied this technique to recording recombinant ion channel currents expressed in membrane patches excised from HEK293 cell lines. Giant inside-out patch recordings of Na(+) channels and SK(Ca) type calcium-activated potassium channels show high temporal resolution and excellent signal to noise characteristics. This technique will facilitate the study of recombinant ion channels expressed in mammalian cells.
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Canales Iónicos/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodos , Animales , Artefactos , Línea Celular , Humanos , Magnesio/farmacología , Mamíferos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Fibras Musculares Esqueléticas/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Canales de Potasio/fisiología , Canales de Potasio Calcio-Activados/fisiología , Proteínas Recombinantes , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Canales de Sodio/fisiología , XenopusRESUMEN
Small conductance, Ca(2+)-activated K(+) channels (SK channels) regulate neuronal excitability. We used patch clamp to study the actions of the neuroprotective drug riluzole on recombinant SK2 channels expressed in HEK293 cells and native SK channels underlying the afterhyperpolarization current (I(AHP)) in cultured hippocampal neurons. External riluzole activated whole-cell SK2 channel currents in HEK293 cells dialyzed with a Ca(2+)-free intracellular solution. When applied to the intracellular aspect of the membrane of giant inside-out patches, riluzole enhanced the membrane current activated by 100 nM Ca(2+) in a reversible and concentration-dependent manner; 30 microM riluzole applied to the intracellular aspect of the patches sensitized the channels to activation by Ca(2+), resulting in a leftward shift of the Ca(2+) activation curve. Riluzole also enhanced the I(AHP) and reduced the spontaneous action potential frequency in chemically stimulated neurons. Modulation of SK channel activity by riluzole may contribute to its cellular, behavioral, and clinical effects.