RESUMEN
Using our in vitro and in vivo models of oxidative stress, the current study was designed to determine the neuroprotective potential of naringenin, alone or in combination with lipoic acid. In our mixed neuronal culture exposed to hypoxia and subsequent reoxygenation, naringenin was shown to provide significant neuroprotection against cell death at a concentration of 2.5 µmol/L. Lipoic acid (LA) did not produce neuroprotection at any concentration tested (0.25-100 µmol/L). In contrast, when naringenin was covalently combined with LA, producing a novel compound named "VANL-100", significant neuroprotection was observed at a concentration as low as 2×10-2 µmol/L (100-fold more potent). An ELISA for antioxidant capacity demonstrated that naringenin and VANL-100 likely resulted in neuroprotection by increasing the free radical scavenging capacity of the neuronal cells. Pretreatment of rats with the above compounds prior to middle cerebral artery occlusion (MCAO) followed by reperfusion, showed similar results. Naringenin significantly reduced infarct volume at a dose of 10 mg/kg while VANL-100 produced significant neuroprotection at a dose as low as 1×10-4 mg/kg (10 000-fold more potent). This VANL-100-induced neuroprotection persisted even when administered 1 and 3 hours into the reperfusion time course. Taken together, these results suggest that our novel compound, VANL-100 is neuroprotective, likely via a mechanism that involves increasing the antioxidant capacity of neuronal cells. Our results also show that VANL-100 is 100-10 000-fold more potent than the parent compounds, which adds to the growing evidence in support of combination therapy targeting oxidative stress in neurodegenerative diseases.
Asunto(s)
Flavanonas/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Ácido Tióctico/farmacología , Animales , Antioxidantes/metabolismo , Modelos Animales de Enfermedad , Femenino , Flavanonas/administración & dosificación , Flavanonas/uso terapéutico , Glucosa/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Oxígeno/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Ácido Tióctico/administración & dosificación , Ácido Tióctico/uso terapéuticoRESUMEN
In this study, we tested a novel synthetic pyrazole-containing compound, 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile (APPC), as an antioxidant in both in vitro and in vivo models of oxidative stress. In addition, the utility of covalently combining APPC with another well-established antioxidant, lipoic acid (LA), was also tested in both models. The in vitro results demonstrated that pretreatment with APPC in a mixed neuronal-glial culture exposed to oxygen-glucose deprivation (OGD) followed by reoxygenation-refeeding, resulted in significant neuroprotection at concentrations between 2.5 to 25 µmol/L. In contrast, LA was not neuroprotective following OGD alone or following reoxygenation-refeeding. However, the synthetic covalent combination of APPC with LA, named "UPEI-800", resulted in significant neuroprotection at concentrations between 0.027 and 2.7 µmol/L (100-fold more potent than APPC alone), an effect shown to be correlated with increased cellular antioxidant capacity. Further, in an in vivo model of ischaemia-reperfusion injury following transient occlusion of the middle cerebral artery (tMCAO), both APPC (0.1 and 1.0 mg/kg) and UPEI-800 (1×10-3 mg/kg) provided significant neuroprotection. Consistent with the in vitro findings, the in vivo results following tMCAO also demonstrated a 100-fold increase in the potency of the covalently linked compound UPEI-800 compared to APPC alone.
Asunto(s)
Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Pirazoles/farmacología , Animales , Antioxidantes/metabolismo , Muerte Celular/efectos de los fármacos , Técnicas de Química Sintética , Glucosa/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Oxígeno/metabolismo , Pirazoles/síntesis química , Pirazoles/química , Ratas , Daño por Reperfusión/patologíaRESUMEN
Previously, our laboratory provided evidence that lipoic acid (LA) covalently bonded to various antioxidants, resulted in enhanced neuroprotection compared to LA on its own. The naturally occurring compound scopoletin, a coumarin derivative, has been shown in various in vitro studies to have both antioxidant and anti-inflammatory mechanism of actions. The present investigation was designed to determine if scopoletin on its own, or a co-drug consisting of LA and scopoletin covalently bonded together, named UPEI-400, would be capable of demonstrating a similar neuroprotective efficacy. Using a rat stroke model, male rats were anesthetized (Inactin®; 100 mg/kg, iv), the middle cerebral artery was permanently occluded for 6 h (pMCAO), or in separate animals, occluded for 30 min followed by 5.5 h of reperfusion (ischemia/reperfusion; I/R). Pre-administration of either scopoletin or UPEI-400 significantly decreased infarct volume in the I/R model (p < 0.05), but not in the pMCAO model of stroke. UPEI-400 was â¼1000 times more potent compared to scopoletin alone. Since UPEI-400 was only effective in a model of I/R, it is possible that it may act to enhance neuronal antioxidant capacity and/or upregulate anti-inflammatory pathways to prevent the neuronal cell death.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/farmacología , Daño por Reperfusión/prevención & control , Escopoletina/análogos & derivados , Escopoletina/farmacología , Accidente Cerebrovascular/prevención & control , Ácido Tióctico/análogos & derivados , Ácido Tióctico/farmacología , Animales , Masculino , Fármacos Neuroprotectores/administración & dosificación , Ratas , Ratas Sprague-Dawley , Escopoletina/administración & dosificación , Ácido Tióctico/administración & dosificaciónRESUMEN
Inflammation-related cerebral damage mediated by infiltrating neutrophils following reperfusion plays a role in reperfusion-induced brain damage subsequent to a stroke event. The ELR-CXC family of chemokines are CXCR1 and CXCR2 agonists that are known to drive neutrophil migration and activation. The present study demonstrated the benefit of anti-inflammatory therapy in the treatment of ischemic stroke with the administration of the competitive ELR-CXC chemokine antagonist, CXCL8(3-72)K11R/G31P (G31P). Male Sprague-Dawley rats were anaesthetized, and the middle cerebral artery (MCA) was occluded for 30 min followed by 5.5 h of reperfusion. Pretreatment with G31P resulted in a significant, dose-dependent (approximately 61-72%) decrease in infarct volumes compared to vehicle-treated animals, but neuroprotection was also observed when G31P (0.5 mg/kg) was administered 1 or 3 h following the start of reperfusion. The neuroprotection observed following the administration of this competitive CXCR1/CXCR2 antagonist may present therapeutic opportunities for addressing reperfusion-induced inflammatory damage in patients presenting with transient ischemic episodes.
Asunto(s)
Antiinflamatorios/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Quimiocinas CXC/antagonistas & inhibidores , Interleucina-8/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/patología , Infarto Encefálico/tratamiento farmacológico , Infarto Encefálico/patología , Isquemia Encefálica/etiología , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Quimiocinas CXC/inmunología , Infarto de la Arteria Cerebral Media/complicaciones , Interleucina-8/farmacología , Masculino , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Ratas Sprague-Dawley , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/patologíaRESUMEN
Previous work in our laboratory demonstrated the utility of synthetic combinations of two naturally occurring, biologically active compounds. In particular, we combined two known anti-oxidant compounds, lipoic acid and apocynin, covalently linked via an ester bond (named UPEI-100). In an animal model of ischemia-reperfusion injury (tMCAO), UPEI-100 was shown to produce equivalent neuroprotection compared to each parent compound, but at a 100-fold lower dose. However, it was determined that UPEI-100 was undetectable in any tissue samples almost immediately following intravenous injection. Therefore, the present investigation was done to determine if biological stability of UPEI-100 could be improved by replacing the ester bond with a more bio cleavage-resistant bond, an ether bond (named UPEI-104). We then compared the stability of UPEI-104 to the original parent compound UPEI-100 in human plasma as well as liver microsomes. Our results demonstrated that both UPEI-100 and UPEI-104 could be detected in human plasma for over 120 min; however, only UPEI-104 was detectable for an average of 7 min following incubation with human liver microsomes. This increased stability did not affect the biological activity of UPEI-104 as measured using our tMCAO model. Our results suggest that combining compounds using an ether bond can improve stability while maintaining biological activity.
RESUMEN
The pregnancy hormone relaxin protects tissue from ischemic damage. The ability of relaxin-3, a relaxin paralog, to do so has not been explored. The cerebral expression levels of these peptides and their receptors make them logical targets for study in the ischemic brain. We assessed relaxin peptide-mediated protection, relative relaxin family peptide receptor (RXFP) involvement, and protective mechanisms. Sprague-Dawley rats receiving permanent (pMCAO) or transient middle cerebral artery occlusions (tMCAO) were treated with relaxin peptides, and brains were collected for infarct analysis. Activation of the endothelial nitric oxide synthase pathway was evaluated as a potential protective mechanism. Primary cortical rat astrocytes were exposed to oxygen glucose deprivation and treated with relaxin peptides, and viability was examined. Receptor involvement was explored using RXFP3 antagonist or agonist treatment and real-time PCR. Relaxin and relaxin-3 reduced infarct size after pMCAO. Both peptides activated endothelial nitric oxide synthase. Because relaxin-3 has not previously been associated with this pathway and displays promiscuous RXFP binding, we explored the receptor contribution. Expression of rxfp1 was greater than that of rxfp3 in rat brain, although peptide binding at either receptor resulted in similar overall protection after pMCAO. Only RXFP3 activation reduced infarct size after tMCAO. In astrocytes, rxfp3 gene expression was greater than that of rxfp1. Selective activation of RXFP3 maintained astrocyte viability after oxygen glucose deprivation. Relaxin peptides are protective during the early stages of ischemic stroke. Differential responses among treatments and models suggest that RXFP1 and RXFP3 initiate different protective mechanisms. This preliminary work is a pivotal first step in identifying the clinical implications of relaxin peptides in ischemic stroke.
Asunto(s)
Infarto de la Arteria Cerebral Media/prevención & control , Relaxina/uso terapéutico , Animales , Astrocitos/efectos de los fármacos , Encéfalo/patología , Células Cultivadas , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Infarto de la Arteria Cerebral Media/patología , Masculino , Distribución Aleatoria , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Péptidos/agonistas , Receptores de Péptidos/antagonistas & inhibidores , Proteínas Recombinantes/uso terapéutico , Relaxina/farmacologíaRESUMEN
The present study demonstrates the benefits of combinatorial antioxidant therapy in the treatment of ischemic stroke. Male Sprague-Dawley rats were anaesthetised and the middle cerebral artery (MCA) was occluded for 30 minutes followed by 5.5 hours of reperfusion. Pretreatment with resveratrol 30 minutes prior to MCA occlusion resulted in a significant, dose-dependent decrease in infarct volume (p<0.05) compared to vehicle-treated animals. Neuroprotection was also observed when resveratrol (2 × 10(-3) mg/kg; iv) was administered within 60 minutes following the return of blood flow (reperfusion). Pretreatment with non-neuroprotective doses of resveratrol (2 × 10(-6) mg/kg) and lipoic acid (LA; 0.005 mg/kg) in combination produced significant neuroprotection as well. This neuroprotection was also observed when resveratrol and LA were administered 15 minutes following the onset of MCA occlusion. Subsequently, we synthetically combined resveratrol and LA in both a 1 ⶠ3 (UPEI-200) and 1 ⶠ1 (UPEI-201) ratio, and screened these new chemical entities in both permanent and transient ischemia models. UPEI-200 was ineffective, while UPEI-201 demonstrated significant, dose-dependent neuroprotection. These results demonstrate that combining subthreshold doses of resveratrol and LA prior to ischemia-reperfusion can provide significant neuroprotection likely resulting from concurrent effects on multiple pathways. The additional protection observed in the novel compound UPEI 201 may present opportunities for addressing ischemia-induced damage in patients presenting with transient ischemic episodes.
Asunto(s)
Antioxidantes/farmacología , Isquemia Encefálica/tratamiento farmacológico , Daño por Reperfusión/tratamiento farmacológico , Estilbenos/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Ácido Tióctico/farmacología , Animales , Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/fisiopatología , Resveratrol , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Edaravone, an electron spin trapper with radical scavenging activity, has been shown to be effective in reducing infarct volume in humans following ischemic stroke. However, concerns of edaravone-induced renal toxicity have limited its clinical adoption. Previous work has demonstrated that edaravone produced significant neuroprotection when injected prior to a period of ischemia and/or reperfusion. The current investigation was designed to determine if a newly synthesized co-drug consisting of lipoic acid and edaravone, named UPEI-300, could produce neuroprotection in in vitro and/or an in vivo rodent model of stroke. UPEI-300 produced dose-dependent neuroprotection in vitro and was subsequently tested in vivo. Male rats were anaesthetized and the middle cerebral artery was occluded for 30 min followed by 5.5 h of reperfusion (ischemia/reperfusion; I/R). Pre-administration of UPEI-300 dose-dependently decreased infarct volume. Significant neuroprotection was also observed when UPEI-300 (1.0 mg/kg) was injected during the 30 min period of ischemia as well as up to 60 min following the start of reperfusion. These results indicate that a co-drug consisting of edaravone and lipoic acid is a potent neuroprotectant, and clinically, the use of such a novel co-drug following an ischemic stroke might maintain neuroprotection while potentially decreasing edaravone associated renal toxicity.
Asunto(s)
Antipirina/análogos & derivados , Isquemia Encefálica/prevención & control , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Daño por Reperfusión/prevención & control , Accidente Cerebrovascular/prevención & control , Ácido Tióctico/análogos & derivados , Animales , Antipirina/farmacología , Antipirina/uso terapéutico , Isquemia Encefálica/patología , Hipoxia de la Célula , Células Cultivadas , Combinación de Medicamentos , Masculino , Neocórtex/citología , Neuroglía/patología , Neuronas/patología , Fármacos Neuroprotectores/uso terapéutico , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Accidente Cerebrovascular/patología , Ácido Tióctico/farmacología , Ácido Tióctico/uso terapéuticoRESUMEN
Resveratrol, a dietary polyphenol with antioxidant and anti-inflammatory activity, has been shown to provide neuroprotection in models of ischemia. However, the mechanism of action of resveratrol-induced neuroprotection remains unclear. Previous work in our laboratory has provided evidence that acute, systemic administration of resveratrol is neuroprotective in a permanent model of cerebral ischemia, an effect that was blocked when animals received the non-selective estrogen receptor antagonist, ICI, 182,780. The present study was designed to investigate whether the source of neuroprotection afforded by resveratrol action within the cerebral cortex itself is mediated preferentially via selective activation of either α or ß estrogen receptor subtype. Intracortical injection of resveratrol (0.1 and 1.0 µM) 10 min prior to 30 min of ischemia followed by 5.5h of reperfusion significantly reduced infarct volume in the prefrontal cortex. This neuroprotective effect was significantly attenuated when resveratrol injection (1.0 µM) was preceded by injection of a selective estrogen receptor α antagonist, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1N-pyrozole dihydrochloride (MPP) or a selective estrogen receptor beta (ERß) antagonist, 4-[2-phenyo-5,7-bis(trifluoromrthyl)pyrazolo(1,5-a)pyrimidin-3-yl]phenol (PHTPP). These results provide evidence for rapidly induced neuroprotection mediated by resveratrol activation of either estrogen receptor subtype within the ischemic cortex of rats.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Estilbenos/administración & dosificación , Animales , Isquemia Encefálica/diagnóstico , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Masculino , Fármacos Neuroprotectores/administración & dosificación , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/diagnóstico , Resveratrol , Resultado del TratamientoRESUMEN
After ischemic stroke, early thrombolytic therapy to reestablish tissue perfusion improves outcome but triggers a cascade of deleterious cellular and molecular events. Using a collaborative approach, our groups examined the effects of guanosine (Guo) in response to ischemic reperfusion injury in vitro and in vivo. In a transient middle cerebral artery occlusion (MCAO) in rats, Guo significantly reduced infarct volume in a dose-dependent manner when given systemically either immediately before or 30 min, but not 60 min, after the onset of the 5.5-hr reperfusion period. In a separate experiment, Guo significantly reduced infarct volume after 24 hr of reperfusion when administered 5 min before reperfusion. Western blot analysis did not reveal any significant changes either in endoplasmic reticulum (ER) stress proteins (GRP 78 and 94) or HSP 70 or in levels of m-calpain. In vitro oxygen and glucose deprivation (OGD) significantly increased production of both reactive oxygen species (ROS) and interleukin-8 (IL-8) in the primary astrocytes. Guo did not alter ROS or IL-8 production when given to the astrocytes before OGD. However, Guo when added to the cells prior to or 30 min after reperfusion significantly reduced IL-8 release but not ROS formation. Our study revealed a dose- and time-dependent protective effect of Guo on reperfusion injury in vitro and vivo. The mechanisms by which Guo exerts its effect are independent of unfolded proteins in ER or the level of intracellular calcium or ROS formation. However, the effect may be induced, at least partially, by inhibiting IL-8, a marker of reperfusion-triggered proinflammatory events.
Asunto(s)
Infarto Encefálico/prevención & control , Guanosina/administración & dosificación , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Daño por Reperfusión/prevención & control , Análisis de Varianza , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Infarto Encefálico/etiología , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/deficiencia , Proteínas de Choque Térmico/metabolismo , Hipoxia , Interleucina-8/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reperfusión/efectos adversos , Daño por Reperfusión/complicaciones , Factores de TiempoRESUMEN
Background. Lipoic acid (LA), which has significant antioxidant properties, may also function as a potent neuroprotectant. The synthetic compounds INV-155, INV-157, INV-159, and INV-161 are physiochemical combinations of lipoic acid and captopril. We sought to determine if these compounds have neuroprotective potential following middle cerebral artery occlusion (MCAO) in rats. Methods. Male Sprague-Dawley rats were injected intravenously with captopril (1-50 mg/kg) 30 minutes prior to MCAO. Blood pressure, heart rate, baroreceptor reflex sensitivity, and infarct size were measured. In addition, dose response effect on infarct size and cardiovascular parameters was determined using INV-155, INV-157, INV-159, and INV-161 and compared to captopril and LA. Results. Pretreatment with captopril and LA at all doses tested was neuroprotective. The compounds INV-159 (0.5-10 mg/kg) and INV-161 (1-10 mg/kg) produced a significant,dose-dependent decrease in infarct size. In contrast, INV-155 and INV-157 had no effect on infarct size. Conclusions. Combined pretreatment with captopril potentiated the neuroprotective benefit observed following LA alone. Both INV-159 and INV-161 were also neuroprotective. These results suggest that patients taking combinations of captopril and LA, either as combination therapy or in the form of INV-159 or INV-161, may also benefit from significant protection against cerebral infarction.
RESUMEN
Previous work in our laboratory has provided evidence that preadministration of apocynin and lipoic acid at subthreshold levels for neuroprotection enhanced the neuroprotective capacity when injected in combination. Therefore, the present investigation was designed to determine whether a co-drug consisting of lipoic acid and apocynin functional groups bound by a covalent bond, named UPEI-100, is capable of similar efficacy using a rodent model of stroke. Male rats were anesthetized with Inactin (100 mg/kg iv), and the middle cerebral artery was occluded for 6 h or allowed to reperfuse for 5.5 h following a 30-min occlusion (ischemia/reperfusion, I/R). Preadministration of UPEI-100 dose-dependently decreased infarct volume in the I/R model (P < 0.05), but not in the middle cerebral artery occlusion model of stroke. Using the optimal dose, we then injected UPEI-100 during the stroke or at several time points during reperfusion, and significant neuroprotection was observed when UPEI-100 was administered up to 90 min following the start of reperfusion (P < 0.05). A time course for this neuroprotective effect showed that UPEI-100 resulted in a decrease in infarct volume following 2 h of reperfusion compared with vehicle. The time course of this neuroprotective effect was also used to study several mediators along the antioxidant pathway and showed that UPEI-100 increased the level of mitochondrial superoxide dismutase and oxidized glutathione and decreased a marker of lipid peroxidation due to oxidative stress (HNE-His adduct formation). Taken together, the data suggest that UPEI-100 may utilize similar pathways to those observed for the two parent compounds; however, it may also act through a different mechanism of action.
Asunto(s)
Acetofenonas/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Accidente Cerebrovascular/tratamiento farmacológico , Ácido Tióctico/análogos & derivados , Ácido Tióctico/uso terapéutico , Acetofenonas/síntesis química , Acetofenonas/química , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Disulfuro de Glutatión/biosíntesis , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Fármacos Neuroprotectores/síntesis química , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/prevención & control , Superóxido Dismutasa/biosíntesis , Ácido Tióctico/síntesis química , Ácido Tióctico/químicaRESUMEN
Apocynin is a well-known NADPH-oxidase inhibitor currently being investigated for its potential therapeutic use in patients with cardiovascular disease, such as occlusive stroke. However, the use of apocynin as a potential neuroprotective agent has come under criticism due to a narrow experimental therapeutic dose range and possible pro-oxidant effects at high doses. Lipoic acid is a powerful antioxidant due to its ability to scavenge free radicals at very low doses and has been demonstrated to enhance the therapeutic value of several other classes of drugs. Therefore, the present study was designed to determine if co-administration of previously determined non-neuroprotective doses of lipoic acid and apocynin in combination could enhance their neuroprotective ability thus extending the therapeutic dose range. We tested the hypothesis in a rat model of stroke and reperfusion injury. The middle cerebral artery (MCA) in male Sprague-Dawley rats was occluded for 30 min followed by 5.5h of reperfusion. Pre-treatment with several doses of apocynin (0.05, 0.1 and 1.0 mg/kg) in combination with a single dose of lipoic acid (0.005 mg/kg) resulted in a dose-dependent reduction in infarct volume up to ~50%. These results demonstrate that a non-effective dose of lipoic acid can enhance the neuroprotective ability of apocynin at doses which were previously demonstrated to be non-neuroprotective. Co-administration of apocynin with lipoic acid may overcome the criticisms of the use of apocynin as a neuroprotectant and provide an effective therapy in the prevention of cell death following stroke.
Asunto(s)
Acetofenonas/administración & dosificación , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Encéfalo/fisiopatología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/fisiopatología , Ácido Tióctico/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Combinación de Medicamentos , Humanos , Masculino , Fármacos Neuroprotectores , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
The present study was designed to determine a dose-response relationship between apocynin and infarct volume as well as to provide a possible molecular mechanism mediating this effect. We tested the hypothesis that apocynin protects against cell death following stroke and reperfusion injury. Apocynin was administered 30 min prior to, or immediately following removal of sutures used to occlude the middle cerebral artery (MCA) in male Sprague-Dawley rats. Following removal of the sutures, the MCA was allowed to undergo 5.5h of reperfusion. Pretreatment with apocynin 30 min prior to occlusion resulted in a dose-dependent reduction in infarct volume by â¼50 %. Analysis of tissue from the ischemic cortex of apocynin-treated rats showed an increase in the level of glutathione (GSH), protein adducts (HNE-His), hydrogen peroxide (H(2)O(2)) and DNA fragmentation (apoptotic cell death) was also observed. This suggests that apocynin may increase antioxidant defense systems (GSH) to limit the degree of ischemia-induced cellular stress. In addition, this moderate cell stress results in more apoptotic vs necrotic cell death, and thus may limit the spreading depression and total cell death that occurs following ischemia/reperfusion. These effects may serve as a potential novel mechanism of action contributing to the apocynin-induced neuroprotection observed.
Asunto(s)
Acetofenonas/farmacología , Apoptosis/efectos de los fármacos , Isquemia Encefálica/patología , Fármacos Neuroprotectores/farmacología , Reperfusión , Animales , Antioxidantes/farmacología , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glutatión/análisis , Peróxido de Hidrógeno/análisis , Masculino , Arteria Cerebral Media/efectos de los fármacos , Arteria Cerebral Media/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/prevención & controlRESUMEN
In the present study, we tested the hypothesis that selective activation of estrogen receptor subtypes (ERα and ERß) would be neuroprotective following ischemia and/or ischemia-reperfusion, as well as prevent the associated autonomic dysfunction. The selective ERα agonist, PPT, when administered 30 min prior to occlusion of the middle cerebral artery (pMCAO), resulted in a dose-dependent neuroprotection as measured 6 hours postpermanent MCAO, but not following 30 mins of MCAO followed by 5.5 hrs of reperfusion (I/R). In contrast, 30 min pretreatment with the selective ERß agonist, DPN, resulted in a dose-dependent neuroprotection following I/R, but was not protective following pMCAO. Both drugs prevented the ischemia-induced autonomic dysfunction as measured by a decrease in the baroreceptor reflex sensitivity (BRS). The data presented here suggest a differential role of each ER subtype in targeting the mechanisms of cell death that occur in ischemia versus reperfusion injury.
RESUMEN
Previously we have found that extracellular guanosine (Guo) has neuroprotective properties in in vitro and in vivo. Moreover, extracellular Guo significantly increased in the ipsilateral hemisphere within 2h following focal stroke in rats, and remained elevated for one week. Therefore, we hypothesized that Guo could be a potential candidate for a non-toxic neuroprotective agent. In the present study, we examined the effects of Guo on rats following permanent middle cerebral artery occlusion (MCAO). We also determined whether Guo can precondition neurons by modulating endoplasmic reticulum (ER) stress proteins. As most therapies employ a combination treatment regimen, we optimized the neuroprotection by combining pre- and post-MCAO treatments with Guo, attempting to reduce both ischemic cell death and improve functional recovery. A combination of 4mg/kg Guo given 30min pre-stroke and 8mg/kg Guo given 3, 24 and 48h post-stroke exerted the most significant decrease in infarct volume and sustainable improvement in neurological function. Moreover, these effects are not attributable to Guo metabolites. Measurements taken 6h post-MCAO from animals pre-treated with Guo did not reveal any significant changes in ER stress proteins (GRP 78 and 94) or HSP 70, but did reveal significantly increased levels of m-calpain. Thus, our data indicate that there is a treatment regimen for Guo as a neuroprotectant following ischemic stroke. The mechanism by which Guo confers neuroprotection may involve an increase in m-calpain, possibly resulting from a mild increase in intracellular calcium. M-calpain may be involved in the preconditioning response to ischemia by upregulating endogenous pro-survival mechanisms in neurons.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Guanosina/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Conducta Animal/efectos de los fármacos , Western Blotting , Peso Corporal/efectos de los fármacos , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Guanosina/administración & dosificación , Proteínas de Choque Térmico/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Masculino , Glicoproteínas de Membrana/metabolismo , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/patologíaRESUMEN
Lipoic acid (LA) is a naturally occurring compound and dietary supplement with powerful antioxidant properties. Although LA is neuroprotective in models of stroke, little is known about the cellular mechanisms by which it confers protection during the early stages of ischemia. Here, using a rat model of permanent middle cerebral artery occlusion (MCAO), we demonstrated that administration of LA 30 min prior to stroke, reduces infarct volume in a dose dependent manner. Whole-cell patch clamp techniques in rat brain slices were used to determine if LA causes any electrophysiological alterations in either healthy neurons or neurons exposed to oxygen and glucose deprivation (OGD). In healthy neurons, LA (0.005 mg/ml and 0.05 mg/ml) did not significantly change resting membrane potential, threshold or frequency of action potentials or synaptic transmission, as determined by amplitude of excitatory post synaptic currents (EPSCs). Similarly, in neurons exposed to OGD, LA did not alter the time course to loss of EPSCs. However, there was a significant delay the onset of anoxic depolarization as well as in the time course of the depolarization. Next, intracellular calcium (Ca(2+)) levels were monitored in isolated neurons using fura-2. Pretreatment with 0.005 mg/ml and 0.05 mg/ml LA for 30 min and 6 h did not significantly alter resting Ca(2+) levels or Ca(2+) response to glutamate (250 µM). However, pretreatment with 0.5 mg/ml LA for 6 h significantly increased resting Ca(2+) levels and significantly decreased the Ca(2+) response to glutamate. In summary, these findings suggest that LA does not affect neuronal physiology under normal conditions, but can protect cells from an ischemic event.
Asunto(s)
Antioxidantes/farmacología , Calcio/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Neuronas/efectos de los fármacos , Ácido Tióctico/farmacología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
Lipoic acid (LA) is a known antioxidant currently used as a therapy in patients with vascular and metabolic disorders. We tested the hypothesis that lipoic acid is protective against the cell death observed following stroke. Lipoic acid was administered 30minutes prior to, or immediately following removal of sutures used to occlude the middle cerebral artery (MCA) in male Sprague-Dawley rats. Following removal of the sutures, the MCA territory was allowed to undergo 5.5hrs of reperfusion. This ischemia/reperfusion (I/R) resulted in a focal infarct restricted to the prefrontal cortex (24±3mm(3)). Pretreatment with LA 30minutes prior to occlusion resulted in a dose-dependent reduction in infarct volume. This reduction in infarct volume was not observed when the LA was administered immediately prior to reperfusion (30minutes post-occlusion). To investigate a potential hemodynamic mechanism for this LA-induced neuroprotection, blood pressure, heart rate and baroreceptor reflex sensitivity (BRS) were measured. Intravenous administration of LA did not result in any significant changes in any of these parameters compared to saline-treated rats. Similarly, there was no significant contribution of systemic nitric oxide or alteration in cerebral perfusion measured following pretreatment with lipoic acid or during the course of occlusion and reperfusion compared with saline-treated rats. Western blot analysis of tissue from the ischemic cortex showed an increase in protein expression of superoxide dismutase (SOD2), but not SOD1, in LA pretreated rats. This suggests a potential mechanism of action contributing to the LA-induced neuroprotection observed. Furthermore, the data in the present investigation suggest the potential use of LA pretreatment as a neuroprotectant in stroke patients.
Asunto(s)
Antioxidantes/farmacología , Isquemia Encefálica/complicaciones , Fármacos Neuroprotectores , Daño por Reperfusión/prevención & control , Ácido Tióctico/farmacología , Animales , Barorreflejo/efectos de los fármacos , Barorreflejo/fisiología , Western Blotting , Isquemia Encefálica/patología , Infarto Cerebral/patología , Circulación Cerebrovascular , Citosol/enzimología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Hemodinámica/efectos de los fármacos , Masculino , Arteria Cerebral Media/efectos de los fármacos , Arteria Cerebral Media/fisiología , Mitocondrias/enzimología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/fisiología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
We have developed a novel rodent animal model of reperfusion injury following stroke. In this model, blood flow through the middle cerebral artery (MCA) is temporarily occluded by placing gentle pressure on sutures behind the artery along three separate branches. The sutures remain in place for a period of time (occlusion), and are then removed for an additional amount of time (reperfusion) to study the effects of drug treatment on the ischemic core and/or reperfusion injury. This approach resulted in a highly reproducible focal infarct restricted to the prefrontal cerebral cortex with an intra-operative mortality rate of less than 1%. To validate this new model of reperfusion injury, we used two well characterized neuroprotectants, estrogen and edaravone. Estrogen and edaravone have been studied extensively in many animal models, and our lab as well as others have consistently demonstrated significant reductions in infarct size following edaravone or estrogen pretreatment. In this novel model, intravenous pretreatment of animals with either estrogen or edaravone resulted in significant, dose-dependent, reduction in infarct size following reperfusion. In conclusion, our results demonstrate the validity of using this novel model to study the mechanism of neuroprotection following stroke. Based on the low mortality rate and reproducibility of the focal infarct volume, this novel rodent model is ideal for preclinical studies to screen drugs for potential therapy against reperfusion injury following stroke.
Asunto(s)
Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/complicaciones , Daño por Reperfusión/etiología , Animales , Antipirina/administración & dosificación , Antipirina/análogos & derivados , Antipirina/farmacología , Circulación Cerebrovascular , Relación Dosis-Respuesta a Droga , Edaravona , Estrógenos/administración & dosificación , Estrógenos/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Masculino , Arteria Cerebral Media/patología , Arteria Cerebral Media/fisiopatología , Arteria Cerebral Media/cirugía , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Procedimientos Neuroquirúrgicos/efectos adversos , Procedimientos Neuroquirúrgicos/métodos , Corteza Prefrontal/irrigación sanguínea , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/patología , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/patología , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Ischemic tolerance describes a phenomenon whereby subcritical stimuli evoke cellular protective mechanisms resulting in increased tolerance to subsequent ischemia. In the present study we propose that the cytoprotective effects attributed to 17beta-estradiol and tunicamycin in an in vivo rodent model of ischemia are reflected by changes in neuronal tissue levels of m-calpain, HSP70, GRP94 and GRP78. Rats pretreated with 17beta-estradiol, tunicamycin or both demonstrated dose-dependent reductions in infarct area following 4 h of permanent middle cerebral artery occlusion (MCAO). Western blot analysis revealed that 4 h of MCAO was associated with decreased cortical expression of HSP70 and m-calpain and increased expression of GRP78. Pretreatment with 12.5 microg/kg 17beta-estradiol did not change this pattern of protein expression following MCAO. While GRP94 expression was elevated in sham-operated rats pretreated with 17beta-estradiol, the ensuing ischemic tolerance did not appear to be mediated by changes in cellular stress proteins. Pretreatment with 50 microg/kg tunicamycin significantly reduced HSP70 in cortical tissue samples taken from sham-operated rats and appeared to attenuate the threshold for activation of m-calpain in rats undergoing 4 h of MCAO. Lastly, a combined treatment in which rats undergoing MCAO were pretreated with both tunicamycin (24 h prior) and 17beta-estradiol (30 min prior) was associated with an attenuated stress response as indicated by reduced expression of GRP78 and GRP94 when compared to saline-treated controls. The results of this study suggest that the ischemic tolerance observed following MCAO in rats pretreated with either 17beta-estradiol or tunicamycin is likely mediated in part through differential effects on cellular stress proteins.