Chi Gu

Melatonin-enhanced autophagy protects against neural apoptosis via a mitochondrial pathway in early brain injury following a subarachnoid hemorrhage.

Melatonin is a strong antioxidant that has beneficial effects against early brain injury (EBI) following a subarachnoid hemorrhage (SAH) in rats; protection includes reduced mortality and brain water content. The molecular mechanisms underlying these clinical effects in the SAH model, however, have not been clearly identified. This study was undertaken to determine the influence of melatonin on neural apoptosis and the potential mechanism of these effects in EBI following SAH using the filament perforation model of SAH in male Sprague Dawley rats. Melatonin (150 mg/kg) or vehicle was given via an intraperitoneal injection 2 hr after SAH induction. Brain samples were extracted 24 hr after SAH. The results show that melatonin treatment markedly reduced caspase‐3 activity and the number of TUNEL‐positive cells, while the treatment increased the LC3‐II/LC3‐I, an autophagy marker, which indicated that melatonin‐enhanced autophagy ameliorated apoptotic cell death in rats subjected to SAH. To further identify the mechanism of autophagy protection, we demonstrated that melatonin administration reduced Bax translocation to the mitochondria and the release of cytochrome c into the cytosol. Taken together, this report demonstrates that melatonin improved the neurological outcome in rats by protecting against neural apoptosis after the induction of filament perforation SAH; moreover, the mechanism of these antiapoptosis effects was related to the enhancement of autophagy, which ameliorated cell apoptosis via a mitochondrial pathway.

Melatonin attenuates inflammatory response‐induced brain edema in early brain injury following a subarachnoid hemorrhage: a possible role for the regulation of pro‐inflammatory cytokines

Melatonin is a strong anti‐oxidant that has beneficial effects against early brain injury (EBI) following a subarachnoid hemorrhage (SAH) in rats; protection includes the reduction of both mortality and neurological deficits. The molecular mechanisms underlying these clinical effects in the SAH model have not been clearly identified. This study examined the influence of melatonin on brain edema secondary to disruption of the blood–brain barrier (BBB) and the relationship between these effects and pro‐inflammatory cytokines in EBI following SAH using the filament perforation model of SAH in male Sprague–Dawley rats. Melatonin (150 mg/kg) or vehicle was given via an intraperitoneal injection 2 hr after SAH induction. Brain samples were extracted 24 hr after SAH. Melatonin treatment markedly attenuated brain edema secondary to BBB dysfunctions by preventing the disruption of tight junction protein expression (ZO‐1, occludin, and claudin‐5). Melatonin treatment also repressed cortical levels of pro‐inflammatory cytokines (IL‐1β, IL‐6, and TNF‐α), which were increased in EBI 24 hr after SAH. To further identify the mechanism of this protection, we demonstrated that administration of melatonin attenuated matrix metallopeptidase 9 expression/activity and vascular endothelial growth factor expression, which are related to the inflammatory response and BBB disruption in EBI after SAH. Taken together, this report shows that melatonin prevents disruption of tight junction proteins which might play a role in attenuating brain edema secondary to BBB dysfunctions by repressing the inflammatory response in EBI after SAH, possibly associated with regulation of pro‐inflammatory cytokines.

Progesterone attenuates early brain injury after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE Although the neuroprotective effects of progesterone against early brain injury (EBI) after trauma have been demonstrated in several studies, whether progesterone reduces EBI after subarachnoid hemorrhage (SAH) remains unknown. In this study, we explored the effect of progesterone on cell apoptosis, stability of the blood-brain barrier (BBB), brain edema, and mortality in male Sprague-Dawley rats subjected to subarachnoid hemorrhage-induced EBI by endovascular perforation. METHOD Rats (n=66) were randomly assigned to sham, SAH+vehicle, and SAH+progesterone groups. Progesterone (16 mg/kg) or an equal volume of vehicle was administered at 1h, 6h and 12h after SAH. Mortality within 24h, neurological scores, brain edema, Evans blue dye extravasation, cell apoptosis, and the expression of caspase-3 and matrix metalloproteinase (MMP)-9 were assayed after 24h of SAH. RESULT Progesterone treatment significantly reduced mortality, brain edema, Evans blue dye extravasation, cell apoptosis, expression of caspase-3 and MMP-9, and improved neurological scores compared with the vehicle group. CONCLUSION Progesterone may reduce EBI after SAH by inhibiting cell apoptosis and stabilizing the BBB.

Endoplasmic reticulum stress is associated with neuroprotection against apoptosis via autophagy activation in a rat model of subarachnoid hemorrhage

Endoplasmic reticulum (ER) stress might play an important role in a range of neurological diseases; however, this phenomenons role in subarachnoid hemorrhage (SAH) remains unclear. In this study, we explored the potential role of endoplasmic reticulum stress in early brain injury following SAH.84 rats were used for an endovascular perforation-induced subarachnoid hemorrhage model. The rats were intraperitoneally pretreated with the ER stress inducer tunicamycin (Tm) or with the inhibitor tauroursodeoxycholic acid (TUDCA) before SAH onset. An intracerebral ventricular infusion of autophagy inhibitor 3-methyladenine (3-MA) was also used to determine the relation between autophagy and ER stress in early brain injury following SAH. At 24h, rats were neurologically evaluated, and their brains were extracted for molecular biological and histological studies. ER stress was activated in rats after 24h of SAH. Enhanced ER stress via Tm pretreatment significantly improved neurological deficits, attenuated the expression of pro-apoptotic molecules of caspase-3 and reduced the number of TUNEL-positive cells. In contrast, the ER stress inhibitor TUDCA aggravated neurological deficits and apoptotic cell death. Western blot analysis revealed that levels of the autophagic protein Beclin 1 and the ratio of LC3-II to LC3-I were both increased by Tm infusion and reduced by TUDCA administration. The suppression of autophagic activity with 3-MA attenuated Tm-induced anti-apoptotic effects. Our study indicates that ER stress alleviates early brain injury following SAH via inhibiting apoptosis. This neuroprotective effect is most likely exerted by autophagy activation.

Melatonin-mediated mitophagy protects against early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation

The NLRP3 inflammasome is activated in the early period following subarachnoid hemorrhage(SAH), resulting in inflammatory responses. Recent studies have shown that activation of NLRP3 inflammasome is suppressed by autophagy, but the potential mechanism is unclear. In this study, we examined whether mitophagy was involved in the beneficial effect of melatonin and its relationship with NLRP3 inflammasome activation after SAH. In total, 130 adult-male SD rats were randomly divided into four groups: sham group, SAH + vehicle group, SAH + melatonin group, and SAH + 3-methyladenine (3-MA) + melatonin group. Brain samples were used for brain water content analysis, ROS assay, Western blot, immunohistochemistry and transmission electron microscopy. The results showed that melatonin treatment markedly increased the expression of both autophagy markers(LC3-II/LC3-I and Atg 5), and mitophagy markers(Parkin and PINK-1) following SAH induction. Additionally, melatonin treatment attenuated pathological changes in mitochondria and reduced ROS generation, which are closely related to NLRP3 inflammasome activation. Consequently, melatonin-mediated upregulation of proteins associated with mitophagy inhibited NLRP3 inflammasome activation and significantly reduced pro-inflammatory cytokine levels after SAH. Conversely, 3-MA, an autophagy inhibitor, reversed these beneficial effects of melatonin on mitophagy and the NLRP3 inflammasome. These results suggest that mitophagy-associated NLRP3 inflammasome inhibition by melatonin is neuroprotective against early brain injury post-SAH in rats.

Inhibiting HIF-1α by 2ME2 ameliorates early brain injury after experimental subarachnoid hemorrhage in rats.

Although hypoxia-inducible factor-1α (HIF-1α) has been extensively studied in brain injury following hypoxia-ischemia, the role of HIF-1α in early brain injury (EBI) after subarachnoid hemorrhage (SAH) remains unclear. The present study was under taken to investigate a potential role of HIF-1α in EBI after SAH. Rats (n=60) were randomly divided into sham+vehicle, SAH+2-methoxyestradiol (2ME2), and SAH+vehicle groups. The SAH model was induced by endovascular perforation and all the rats were subsequently sacrificed at 24h after SAH. We found that treatment with 2ME2 suppressed the expression of HIF-1α, BNIP3 and VEGF and reduced cell apoptosis, blood-brain barrier (BBB) permeability, brain edema, and neurologic scores. Double fluorescence labeling revealed that HIF-1α was expressed predominantly in the nuclei of neurons and TUNEL-positive cells. Our work demonstrated that HIF-1α may play a role in EBI after SAH, causing cell apoptosis, BBB disruption, and brain edema by up-regulating its downstream targets, BNIP3 and VEGF. These effects were blocked by the HIF-1α inhibitor, 2ME2.

The Polarization States of Microglia in TBI: A New Paradigm for Pharmacological Intervention

Traumatic brain injury (TBI) is a serious medical and social problem worldwide. Because of the complex pathophysiological mechanisms of TBI, effective pharmacotherapy is still lacking. The microglial cells are resident tissue macrophages located in the brain and have two major polarization states, M1 phenotype and M2 phenotype, when activated. The M1 phenotype is related to the release of proinflammatory cytokines and secondary brain injury, while the M2 phenotype has been proved to be responsible for the release of anti-inflammation cytokines and for central nervous system (CNS) repair. In animal models, pharmacological strategies inhibiting the M1 phenotype and promoting the M2 phenotype of microglial cells could alleviate cerebral damage and improve neurological function recovery after TBI. In this review, we aimed to summarize the current knowledge about the pathological significance of microglial M1/M2 polarization in the pathophysiology of TBI. In addition, we reviewed several drugs that have provided neuroprotective effects against brain injury following TBI by altering the polarization states of the microglia. We emphasized that future investigation of the regulation mechanisms of microglial M1/M2 polarization in TBI is anticipated, which could contribute to the development of new targets of pharmacological intervention in TBI.

Melatonin attenuates neurogenic pulmonary edema via the regulation of inflammation and apoptosis after subarachnoid hemorrhage in rats.

Neurogenic pulmonary edema (NPE) is a serious non‐neurological complication that can occur after a subarachnoid hemorrhage (SAH) and is associated with decreased survival and a poor neurological outcome. Melatonin is a strong antioxidant that has beneficial effects against SAH in rats, including reduced mortality and reduced neurological deficits. The molecular mechanisms underlying these clinical effects in the SAH model, however, have not been clearly identified. This study was undertaken to determine the influence of melatonin on SAH‐induced NPE and the potential mechanism of these effects using the filament perforation model of SAH in male Sprague Dawley rats. Either melatonin (150 mg/kg) or a vehicle was given via an intraperitoneal injection 2 hr after an SAH induction. Lung samples were extracted 24 hr after SAH. The results show that the melatonin treatment attenuated SAH‐induced NPE by preventing alveolar–capillary barrier dysfunctions via inhibiting the disruption of tight junction proteins (ZO‐1 and occludin). Moreover, the treatment downregulated the levels of mature interleukin (IL) ‐1β, myeloperoxidase (MPO), and matrix metallopeptidase (MMP) 9 expression/activation, which were increased in the lung; also, melatonin treatment improved neurological deficits. Furthermore, the melatonin treatment markedly reduced caspase‐3 activity and the number of TUNEL‐positive cells in the lung. Taken together, these findings show that administration of melatonin attenuates NPE by preventing alveolar–capillary barrier dysfunctions via repressing the inflammatory response and by anti‐apoptosis effects after SAH.

Methylene blue attenuates neuroinflammation after subarachnoid hemorrhage in rats through the Akt/GSK-3β/MEF2D signaling pathway

Subarachnoid hemorrhage (SAH) is a serious medical problem with few effective pharmacotherapies available, and neuroinflammation has been identified as an important pathological process in early brain injury (EBI) after SAH. Methylene blue (MB) is an older drug that has been recently proven to exert extraordinary neuroprotective effects in several brain insults. However, no study has reported the beneficial effects of MB in SAH. In the current investigation, we studied the neuroprotective effects of MB in EBI after SAH and focused on its anti-inflammatory role. A total of 303 rats were subjected to an endovascular perforation process to produce an SAH model. We found that MB could significantly ameliorate brain edema secondary to BBB disruption and alleviate neurological dysfunction after SAH. MB administration also promoted the phosphorylation of Akt and GSK-3β, leading to an increased concentration of MEF2D in the nucleus. The cytokine IL-10 was up-regulated, and IL-1β, IL-6 and TNF-α were down-regulated after MB administration. MB administration could also alleviate neutrophil infiltration and microglia activation after SAH. MK2206, a selective inhibitor of Akt, abolished the neuroprotective effects of MB, inhibited the phosphorylation of Akt and prevented the nuclear localization of MEF2D. MK2206 also reduced the expression of IL-10 and increased the expression of pro-inflammatory cytokines. In conclusion, these data suggested that MB could ameliorate neuroinflammatory responses after SAH, and its anti-inflammatory effects might be exerted via activation of the Akt/GSK-3β/MEF2D pathway.

Rosiglitazone attenuates early brain injury after experimental subarachnoid hemorrhage in rats

Early brain injury (EBI) plays a crucial role in the pathological progress of subarachnoid hemorrhage (SAH). This study was designed to determine whether rosiglitazone protects the brain against EBI in rats, and discuss the role of the anti-apoptotic mechanism mediated by Bcl-2 family proteins in this neuroprotection. 86 male Sprague-Dawley rats were divided into the sham group, the SAH+ vehicle group and the SAH+ rosiglitazone group. SAH was induced via an endovascular perforation technique and rosiglitazone (3mg/kg) or vehicle was administered. Mortality, neurological scores, brain water content, Evans blue dye assay, TUNEL stain assay, Gelatin zymography, and western blot analysis were performed. Rosiglitazone significantly improved mortality, neurological scores, brain water content, blood brain barrier (BBB) and apoptosis compared with the vehicle group within 24h after SAH. The TUNEL staining assay demonstrated that apoptosis was ameliorated. Cleaved Caspase-3 and MMP-9 expression was reduced, whereas Bcl-2 and p-Bad was markedly preserved by rosiglitazone. A significant elevation of p-Akt was detected after rosiglitazone treatment. Our study demonstrated that rosiglitazone plays a neuroprotective role in EBI after SAH via attenuation of BBB disruption, brain edema and apoptosis.