Free iron, total F2-isoprostanes and total F4-neuroprostanes in a model of neonatal hypoxic–ischemic encephalopathy: neuroprotective effect of melatonin

Abstract:   Oxidative stress due to free radical formation and initiation of abnormal oxidative reactions is involved in several diseases of newborns, such as hypoxic–ischemic encephalopathy. Melatonin, an endogenously produced indoleamine primarily formed in the pineal gland, is a potent free radical scavenger as well as an indirect antioxidant. The present study was conducted to evaluate the formation of oxidative damage mediators and the possible effect of melatonin treatment in a model of hypoxic–ischemic encephalopathy in 7-day-old rats. Pups were subjected to permanent ligation of the right common carotid artery and exposed for 2.5 hr to a nitrogen–oxygen mixture (92% and 8%, respectively) (hypoxia–ischemia, HI). Melatonin was injected intraperitoneally to a group of rats at the dose of 15 mg/kg 30 min before starting the ischemic procedure (HI–Melatonin). After 24 hr of treatment, in homogenized cerebral cortex, desferoxamine (DFO)-chelatable free iron, total F₂-isoprostanes and total F₄-neuroprostanes, originating from the free radical-catalyzed peroxidation of arachidonic and docosahexaenoic acids, respectively, were determined. HI induced a significant increase in DFO-chelatable iron, total F₂-isoprostanes and F₄-neuroprostanes in both right and left side of the cerebral cortex. In HI–Melatonin-treated animals the levels of free iron, F₂-isoprostanes, and F₄-neuroprostanes were significantly lower than that in HI rats and the values were similar to controls. These data show the important neuroprotective role of melatonin in reducing oxidative damage resulting from HI. Melatonin could represent a potential safe approach to perinatal brain damage in humans.     

Melatonin decreases mortality following severe subarachnoid hemorrhage

Abstract:  Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease that is associated with significant morbidity and mortality. There is substantial evidence to suggest that oxidative stress is significant in the development of acute brain injury following SAH. Melatonin is a strong antioxidant that has low toxicity and easily passes through the blood–brain barrier. Previous studies have shown that melatonin provides neuroprotection in animal models of ischemic stroke. This study hypothesizes that melatonin will provide neuroprotection when administered 2 hr after SAH. The filament perforation model of SAH was performed in male Sprague–Dawley rats weighing between 300 and 380 g. Melatonin (15 or 150 mg/kg), or vehicle was given via intraperitoneal injection 2 hr after SAH. Mortality and neurologic deficits were assessed 24 hr after SAH. A significant reduction in 24-hr mortality was seen following treatment with high dose melatonin. There was no improvement in neurologic scores with treatment. Brain water content and lipid peroxidation were measured following the administration of high dose melatonin to identify a mechanism for the increased survival. High dose melatonin tended to reduce brain water content following SAH, but had no effect on the lipid peroxidation of brain samples. Large doses of melatonin significantly reduces mortality and brain water content in rats following SAH through a mechanism unrelated to oxidative stress.     

Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia–ischemia

Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia–ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP‐1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT‐1 induced by HI, and this effect was observed in the same neurons that over‐express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT‐1 expression, suggesting that SIRT‐1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.     

Antenatal antioxidant treatment with melatonin to decrease newborn neurodevelopmental deficits and brain injury caused by fetal growth restriction

Fetal intrauterine growth restriction (IUGR) is a serious pregnancy complication associated with increased rates of perinatal morbidity and mortality, and ultimately with long‐term neurodevelopmental impairments. No intervention currently exists that can improve the structure and function of the IUGR brain before birth. Here, we investigated whether maternal antenatal melatonin administration reduced brain injury in ovine IUGR. IUGR was induced in pregnant sheep at 0.7 gestation and a subset of ewes received melatonin via intravenous infusion until term. IUGR, IUGR + melatonin (IUGR + MLT) and control lambs were born naturally, neonatal behavioral assessment was used to examine neurological function and at 24 hr after birth the brain was collected for the examination of neuropathology. Compared to control lambs, IUGR lambs took significantly longer to achieve normal neonatal lamb behaviors, such as standing and suckling. IUGR brains showed widespread cellular and axonal lipid peroxidation, and white matter hypomyelination and axonal damage. Maternal melatonin administration ameliorated oxidative stress, normalized myelination and rescued axonopathy within IUGR lamb brains, and IUGR + MLT lambs demonstrated significant functional improvements including a reduced time taken to attach to and suckle at the udder after birth. Based on these observations, we began a pilot clinical trial of oral melatonin administration to women with an IUGR fetus. Maternal melatonin was not associated with adverse maternal or fetal effects and it significantly reduced oxidative stress, as evidenced by reduced malondialdehyde levels, in the IUGR + MLT placenta compared to IUGR alone. Melatonin should be considered for antenatal neuroprotective therapy in human IUGR.     

Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain

Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical-induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or 3 hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 538.2+/-160.7 pM/mL; brain homogenates: 13.8+/-2.8 pM/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, SOD activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.     

Melatonin regulates the calcium‐buffering proteins,parvalbumin and hippocalcin,in ischemic brain injury

Abstract: Melatonin has anti‐oxidant activity and it exerts a neuroprotective effects during ischemic brain injury. Calcium‐buffering proteins including parvalbumin and hippocalcin are involved in neuronal differentiation and maturation through calcium signaling. This study investigated whether melatonin moderates parvalbumin and hippocalcin expression in cerebral ischemia and glutamate toxicity‐induced neuronal cell death. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Male Sprague‐Dawley rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortical tissues were collected 24 hr after MCAO. Parvalbumin and hippocalcin levels were decreased in vehicle‐treated animal with MCAO, whereas melatonin prevented the ischemic injury‐induced reduction in these proteins. In cultured hippocampal cells, glutamate toxicity decreased parvalbumin and hippocalcin levels, while melatonin treatment prevented the glutamate exposure‐induced diminished in these proteins levels. Melatonin also attenuated the glutamate toxicity‐induced increase in intracellular Ca²⁺ levels. These results suggest that the maintenance of parvalbumin and hippocalcin levels by melatonin in ischemic injury contributes to the neuroprotective effect of melatonin against neuronal cell damage.     

Melatonin reduces apoptosis and necrosis induced by ischemia/reperfusion injury of the pancreas

The pancreas is highly susceptible to the oxidative stress induced by ischemia/reperfusion (IR) injury leading to the generation of acute pancreatitis. Melatonin has been shown to be useful in the prevention of the damage by ischemia-reperfusion in liver, brain, myocardium, gut and kidney. The aim of the study was to evaluate the cytoprotective properties of melatonin against injury induced by IR in pancreas. The obstruction of gastro-duodenal and inferior splenic arteries induced pancreatic IR in male Wistar rats. Melatonin was intraperitoneally administered before or/and after IR injury. The animals were killed at 24 and 48 hr after reperfusion and there were evaluated parameters of oxidative stress (lipoperoxides, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione), glandular endocrine and exocrine function (lipase, amylase, insulin) and cell injury (apoptosis and necrosis). The IR induced a marked enhancement of oxidative stress and impaired pancreatic function. The histological analysis showed that IR induced acute pancreatitis with the accumulation of inflammatory infiltrate, disruption of tissue structure, cell necrosis and hemorrhage. Melatonin administration before or after pancreatic IR prevented all tissue markers of oxidative stress, biochemical and histological signs of apoptosis and necrosis, and restored glandular function. No histological signs of pancreatitis were observed 48 hr after reperfusion in 80% of the animals treated with melatonin, with only a mild edematous pancreatitis being observed in the remaining rats. Preventive or therapeutic administration of melatonin protected against the induction of oxidative stress and tissue injury, and restored cell function in experimental pancreatic IR in rats.     

Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Abstract:  Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.     

Melatonin prevents down-regulation of astrocytic phosphoprotein PEA-15 in ischemic brain injury

Melatonin functions as a free-radical scavenger and has a neuroprotective effect against ischemic brain damage. PEA-15 (phosphoprotein enriched in astrocytes 15) regulates various cellular processes including cell proliferation and apoptosis. In this study, we investigated whether melatonin regulates the levels of PEA-15 and the two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in a middle cerebral artery occlusion (MCAO)-induced injury model and neuronal cells exposed to glutamate. Adult male rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortex tissues were collected 24 h after MCAO. PEA-15 levels after ischemic brain injury were monitored using a proteomic approach. Melatonin pretreatment prevented the ischemic injury-induced reduction in PEA-15 levels. Moreover, Western blot analysis demonstrated that melatonin attenuated the ischemic injury-induced reduction in PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116) levels. Neuronal cells exposed to glutamate showed decreased expression of PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116), while melatonin pretreatment prevented the glutamate toxicity-induced decreases in the levels of these proteins. The reduction in the levels of phospho-PEA-15 proteins indicates the inhibition of anti-apoptotic function of PEA-15. Together, in vivo and in vitro results suggest that melatonin protects neurons against ischemic injury by maintaining levels of phospho-PEA-15 proteins.     

Melatonin prevents the injury-induced decline of Akt/forkhead transcription factors phosphorylation

Abstract:  Melatonin plays a neuroprotective role against brain injury through the activation of Akt and the inhibition of apoptotic cell death. This study investigated whether melatonin modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, FKHR, AFX, and 14-3-3. Adult male rats were treated with melatonin (5 mg/kg) prior to middle cerebral artery occlusion (MCAO) and brain tissues were collected at 24 hr after MCAO. This study confirmed that melatonin significantly reduces infarct volume and decreases the number of TUNEL-positive cells in the cerebral cortex. Potential activation was measured by phosphorylation of PDK1 at Ser²⁴¹, Akt at Ser⁴⁷³, FKHR at Ser²⁵⁶, and AFX at Ser¹⁹³ using Western blot analysis. Melatonin prevented the injury-induced reduction of pPDK1, pAkt, pFKHR, and pAFX. However, melatonin did not affect the level of 14-3-3, which acts as an anti-apoptotic factor through interaction of pFKHR. Further, in the presence of melatonin, the interaction of pFKHR and 14-3-3 increased, compared with that of control animals. This study suggests that melatonin plays a potent protective role against brain injury and that Akt activation and FKHR phosphorylation by melatonin mediated these protective effects.     

Chronic and acute melatonin effects in gerbil global forebrain ischemia: long-term neural and behavioral outcome

Abstract:  Melatonin attenuates the short-term consequences of brain ischemia in several animal models. However, there is scant information regarding its efficacy for improving the long-term outcome. To further address that issue, we subjected gerbils to 5-min bilateral carotid occlusion. Some gerbils received acute peri-surgical administration of melatonin while others received continuous melatonin in their water. The gerbils' brains were histologically assessed at 20 wk postsurgery. Chronic but not acute melatonin attenuated ischemia-induced hyperactivity at 3 days postsurgery. Twenty weeks postsurgery, the ischemic gerbils showed varying degrees of bilateral loss of hippocampal CA1 pyramidal cells and elevation of glial fibrillary acidic protein immunoreactivity there. Both the cell loss and the immunoreactivity were markedly asymmetrical for some gerbils. Neither acute nor chronic melatonin altered this pattern of CA1 cell loss and glial immunoreactivity increase. Ischemia increased the number of CA1 cells that were immunoreactive for doublecortin (DCX), a marker for newborn neurons. This increase in CA1 DCX expression was not affected by either melatonin treatment. However, both acute and chronic melatonin reduced the number of DCX immunoreactive neurons in the dentate gyrus. Thus, neither acute nor chronic melatonin altered the long-term neural outcome of forebrain ischemia, although chronic administration seemed to attenuate the short-term behavioral effect. It is suggested that persistently high brain levels of melatonin may be essential for long-term neuroprotection against ischemia. The possibility that melatonin may modulate hippocampal neurogenesis merits further exploration both in normal animals and in models of brain insult.     

Effects of progesterone on the neonatal brain following hypoxia-ischemia

Progesterone displays a strong potential for the treatment of neonatal hypoxic-ischemic encephalopathy since it has been shown to be beneficial in the treatment of the central nervous system injuries in adult animals. Here, we evaluated the effects of the administration of progesterone (10 mg/kg) in seven-days-old male Wistar rats submitted to neonatal hypoxia-ischemia (HI). Progesterone was administered immediately before ischemia and/or 6 and 24 h after the onset of hypoxia. The body weight of the animals, the volume of brain lesion and the expression of p-Akt and procaspase-3 in the hippocampus were evaluated. All animals submitted to HI showed a reduction in the body weight. However, this reduction was more remarkable in those animals which received progesterone before surgery. Administration of progesterone was unable to reduce the volume of brain damage caused by HI. Moreover, no significant differences were observed in the expression of p-Akt and procaspase-3 in animals submitted to HI and treated with either progesterone or vehicle. In summary, progesterone did not show a neuroprotective effect on the volume of brain lesion in neonatal rats submitted to hypoxia-ischemia. Furthermore, progesterone was unable to modulate p-Akt and procaspase-3 signaling pathways, which may explain the absence of neuroprotection. On the other hand, it seems that administration of progesterone before ischemia exerts some systemic effect, leading to a remarkable reduction in the body weight.     

Melatonin attenuates decrease of protein phosphatase 2A subunit B in ischemic brain injury

Melatonin is an antioxidant that has neuroprotective functions in ischemic brain injury. Protein phosphatase 2A (PP2A) is a serine and threonine phosphatase that modulates cell metabolism and cell survival. This study investigated whether melatonin modulates PP2A subunit B in focal cerebral ischemia and glutamate toxicity-induced neuronal cell death in a rat model. Middle cerebral artery occlusion (MCAO) was performed to induce permanent cerebral ischemic injury. Adult male rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortex tissues were collected 24 hr after MCAO. A proteomic approach elucidated the decrease in PP2A subunit B in MCAO-operated animals. Melatonin treatment attenuated injury-induced reductions in PP2A subunit B levels. Western blot analyses indicated that melatonin prevents injury-induced decrease in PP2A subunit B levels. In neuronal cells, glutamate toxicity induced a lowering of PP2A subunit B, while melatonin treatment attenuated the glutamate exposure-induced decreases in PP2A subunit B. These results suggest that the maintenance of PP2A subunit B by melatonin in ischemic injury is critical to the neuroprotective function of melatonin during neuronal cell damage.     

An age-related decline in melatonin secretion is not altered by food restriction

Melatonin has been found to exhibit youth-maintaining and disease-preventing properties. The current study examined whether the age-retarding regimen of chronic food restriction (FR) slowed the decline in melatonin secretion reported to occur with age. Total nocturnal melatonin secretion was assessed by radioimmunoassay of the primary metabolite, 6-sulphatoxymelatonin (6-S-OH-MLT), in urine. Measurements were made through adulthood (70 to 765 days) on male Wistar rats maintained on the FR regimen (60% of the normal intake) with the control animals fed ad libitum (AL). The data of animals exhibiting gross pathology were excluded. Analyses of covariance found the FR regimen had no effect on either the levels or pattern of decline observed in 6-S-OH-MLT excretion through adulthood. However, the FR body-weight-indexed metabolite measures were approximately double those of the AL (p = .06). The possibility that this result may reflect unusually high melatonin peaks in the FR tissues is discussed.     

Melatonin improves sleep and reduces nitrite in the exhaled breath condensate in cystic fibrosis – a randomized, double-blind placebo-controlled study

Abstract:  Cystic fibrosis (CF) is a chronic progressive disorder characterized by repeated episodes of respiratory infection. Impaired sleep is common in CF leading to reduced quality of life. Melatonin, a secretory product of the pineal gland, has an important function in the synchronization of circadian rhythms, including the sleep–wake cycle, and has been shown to possess significant anti-oxidant properties. To evaluate the effects of exogenous melatonin on sleep and inflammation and oxidative stress markers in CF, a randomized double-blind, placebo-controlled study initially involving 20 patients with CF was conducted. One individual failed to conclude the study. All subjects were clinically stable when studied and without recent infectious exacerbation or hospitalization in the last 30 days. Groups were randomized for placebo ( n  = 10; mean age 12.1 ± 6.0) or 3 mg melatonin ( n  = 9; mean age 16.6 ± 8.26) for 21 days. Actigraphy was performed for 6 days before the start of medication and in the third week (days 14–20) of treatment. Isoprostane and nitrite levels were determined in exhaled breath condensate (EBC) at baseline (day 0) and after treatment (day 21). Melatonin improved sleep efficiency ( P  = 0.01) and tended to improve sleep latency ( P  = 0.08). Melatonin reduced EBC nitrite ( P  = 0.01) but not isoprostane. In summary, melatonin administration reduces nitrite levels in EBC and improves sleep measures in clinically stable CF patients. The failure of melatonin to reduce isoprostane levels may have been a result of the low dose of melatonin used as a treatment.     

Melatonin induces apoptotic death in LNCaP cells via p38 and JNK pathways: therapeutic implications for prostate cancer

Abstract:  Reactive oxygen species (ROS) are involved in pathophysiology of ischemia/reperfusion injury. Melatonin is a potent scavenger of ROS. Thus, this study was designed to elucidate its effects in a combined hepatic warm ischemia and resection model. The right lateral and caudate lobes (32% of liver volume) of Sprague–Dawley rats underwent warm ischemia for 30 min followed by reperfusion and subsequent resection of the nonischemic liver tissue. Some rats were gavaged with 50 mg/kg melatonin 2 hr before the onset of experiments. Controls received the same volume of microcrystalline cellulose. Survival, transaminases, histology, flow cytometry, inducible nitric oxide synthase (iNOS) expression, and activation of signal transduction pathways [c-Jun N-terminal kinase (JNK), cJUN, IκB kinase α (IKKα), proliferating cell nuclear antigen (PCNA), and Ki67] were assessed for hepatic injury, oxidative stress, and cell proliferation. Melatonin significantly improved animal survival and decreased transaminase levels, the indices for necrosis, liver damage, leukocyte infiltration, and iNOS expression. In parallel, the expression of IKKα, JNK1, and cJUN decreased by 35–50% after melatonin ( P  <   0.05). At the same time, melatonin reduced the expression of both PCNA and Ki67 in liver ( P  <   0.05). Melatonin is hepatoprotective most likely via mechanisms including inhibition of IKK and JNK pathways and regulation of cell proliferation.     

Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats

Abstract:  Oxidative stress is believed to contribute to functional and histopathologic disturbances associated with chronic cerebral hypoperfusion (CCH) in rats. Melatonin has protective effects against cerebral ischemia/reperfusion injury. This effect has mainly been attributed to its antioxidant properties. In the present study, we evaluate the effects of melatonin on chronic cerebral hypoperfused rats and examined its possible influence on oxidative stress, superoxide dismutase (SOD) activity, reduced glutathione (GSH) levels, and heat shock protein (HSP) 70 induction. CCH was induced by permanent bilateral common carotid artery occlusion in ovariectomized female rats. Extensive neuronal loss in the hippocampus at day 14 following CCH was observed. The ischemic changes were preceded by increases in malondialdehyde (MDA) concentration and HSP70 induction as well as reductions in GSH and SOD. Melatonin treatment restored the levels of MDA, SOD, GSH, and HSP70 induction as compared to the ischemic group. Histopathologic analysis confirmed the protective effect of melatonin against CCH-induced morphologic alterations. Taken together, our results document that melatonin provides neuroprotective effects in CCH by attenuating oxidative stress and stress protein expression in neurons. This suggests melatonin may be helpful for the treatment of vascular dementia and cerebrovascular insufficiency.     

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 protects kidney grafts from ischemia/reperfusion injury through inhibition of NF-kB and apoptosis after experimental kidney transplantation

Abstract:  Free radicals are involved in pathophysiology of ischemia/reperfusion injury (IRI). Melatonin is a potent scavenger of reactive oxygen and nitrogen species. Thus, this study was designed to elucidate its effects in a model of rat kidney transplantation. Twenty Lewis rats were randomly divided into 2 groups (n = 10 animals each). Melatonin (50 mg/kg BW) dissolved in 5 mL milk was given to one group via gavage 2 hr before left donor nephrectomy. Controls were given the same volume of milk only. Kidney grafts were then transplanted into bilaterally nephrectomized syngeneic recipients after 24 hr of cold storage in Histidine–Tryptophan–Ketoglutarate solution. Both graft function and injury were assessed after transplantation through serum levels of blood urea nitrogen (BUN), creatinine, transaminases, and lactate dehydrogenase (LDH). Biopsies were taken to evaluate tubular damage, the enzymatic activity of superoxide dismutase (SOD) and lipid hydroperoxide (LPO), and the expression of NF-kBp65, inducible nitric oxide synthase (iNOS), caspase-3 as indices of oxidative stress, necrosis, and apoptosis, respectively. Melatonin improved survival ( P  < 0.01) while decreasing BUN, creatinine, transaminases, and LDH values up to 39–71% ( P  < 0.05). Melatonin significantly reduced the histological index for tubular damage, induced tissue enzymatic activity of SOD while reducing LPO. At the same time, melatonin down-regulated the expression of NF-kBp65, iNOS, and caspase-3. In conclusion, donor preconditioning with melatonin protected kidney donor grafts from IRI-induced renal dysfunction and tubular injury most likely through its anti-oxidative, anti-apoptotic and NF-kB inhibitory capacity.