Aging-induced alterations in female rat colon smooth muscle: the protective effects of hormonal therapy

Aging is associated to oxidative damage and alterations in inflammatory and apoptotic pathways. Aging impairs secretion of several hormones, including melatonin and estrogens. However, the mechanisms involved in aging of smooth muscle are poorly known. We have studied the changes induced by aging in the colonic smooth muscle layer of female rats and the protective effect of hormonal therapy. We used young, aged, and ovariectomized aged female rats. Two groups of ovariectomized rats (22 months old) were treated either with melatonin or with estrogen for 10 weeks before sacrifice. Aging induced oxidative imbalance, evidenced by H(2)O(2) accumulation, lipid peroxidation, and decreased catalase activity. The oxidative damage was enhanced by ovariectomy. In addition, aged colonic muscle showed enhanced expression of the pro-inflammatory enzyme cyclooxygenase 2. Expression of the activated forms of caspases 3 and 9 was also enhanced in aged colon. Melatonin and estrogen treatment prevented the oxidative damage and the activation of caspases. In conclusion, aging of colonic smooth muscle induces oxidative imbalance and activation of apoptotic and pro-inflammatory pathways. Hormonal therapy has beneficial effects on the oxidative and apoptotic changes associated to aging in this model.     

Age-related effect of melatonin on permeability transition pore opening in rat brain mitochondria

Aging is accompanied by mitochondrial dysfunction related with lowering of the respiratory complex activity and decrease of ATP synthesis, as well as by an enhancement of oxidative stress and increased sensitivity to mitochondrial permeability transition pore (mPTP) opening in mitochondral triggering the programmed cell death. In the present work we studied the effect of natural antioxidant (melatonin) on parameters of mPTP detected in non-synaptic mitochondria isolated from the brain of young and old rats (3 and 18 months, resp.) with different melatonin treatments; namely, melatonin was either directly applied to the mitochondrial suspension or chronically administered to rats with drinking water. The data obtained have shown that mitochondria isolated from brain of old rats were more susceptive to induction of mPTP. Melatonin added directly to suspension of brain mitochondria isolated from young rats demonstrated a proapoptotic effect. A prolonged chronical treatment with melatonin of old rats produced an anti-apoptotic protective effect. Non-synaptic mitochondria isolated from the brain of old rats treated with melatonin were more resistant to the mPTP opening and demonstrated the activation of respiration of mitochondria as compared to the untreated rats.     

Lifelong caloric restriction prevents age-induced oxidative stress in the sympathoadrenal system of Fischer 344 x Brown Norway rats

Aging is associated with oxidative damage and an imbalance in redox signaling in a variety of tissues, yet little is known about the extent of age-induced oxidative stress in the sympathoadrenal system. Lifelong caloric restriction has been shown to lower levels of oxidative stress and slow the aging process. Therefore, the aims of this study were twofold: (1) to investigate the effect of aging on oxidative stress in the adrenal medulla and hypothalamus and (2) determine if lifelong 40% caloric restriction (CR) reverses the adverse effects of age-induced oxidative stress in the sympathetic adrenomedullary system. Adult (18 months) and very old (38 months) male Fischer 344 x Brown Norway rats were divided into ad libitum or 40% CR groups and parameters of oxidative stress were analyzed in the adrenal medulla and the hypothalamus. A significant age-dependent increase in lipid peroxidation (+20%, P < 0.05) and tyrosine nitration (+111%, P < 0.001) were observed in the adrenal medulla while age resulted in a reduction in the protein expression of key antioxidant enzymes, CuZnSOD (−27%, P < 0.01) and catalase (−27%, P < 0.05) in the hypothalamus. Lifelong CR completely prevented the age-induced increase in lipid peroxidation in the adrenal medulla and restored the age-related decline in antioxidant enzymes in the hypothalamus. These data indicate that aging results in a significant increase in oxidative stress in the sympathoadrenal system. Importantly, lifelong CR restored the age-related changes in oxidative stress in the adrenal medulla and hypothalamus. Caloric restriction could be a potential non-pharmacological intervention to prevent increased oxidative stress in the sympathetic adrenomedullary system with age.     

Light and melatonin schedule neuronal differentiation in the habenular nuclei

The formation of the embryonic brain requires the production, migration, and differentiation of neurons to be timely and coordinated. Coupling to the photoperiod could synchronize the development of neurons in the embryo. Here, we consider the effect of light and melatonin on the differentiation of embryonic neurons in zebrafish. We examine the formation of neurons in the habenular nuclei, a paired structure found near the dorsal surface of the brain adjacent to the pineal organ. Keeping embryos in constant darkness causes a temporary accumulation of habenular precursor cells, resulting in late differentiation and a long-lasting reduction in neuronal processes (neuropil). Because constant darkness delays the accumulation of the neurendocrine hormone melatonin in embryos, we looked for a link between melatonin signaling and habenular neurogenesis. A pharmacological block of melatonin receptors delays neurogenesis and reduces neuropil similarly to constant darkness, while addition of melatonin to embryos in constant darkness restores timely neurogenesis and neuropil. We conclude that light and melatonin schedule the differentiation of neurons and the formation of neural processes in the habenular nuclei.     

Changes in the Number of Primary Sensory Neurons in Normal and Vitamin-E-Deficient Rats during Aging

In the dorsal root ganglia (DRGs) of vitamin-E-deficient rats, we previously found an increase in the number of neurons during the first 5 months of life (Cecchini et al., 1993, 1994). This neurogenetic event seems to bring forward in time the increase in the number of primary sensory neurons that Devor et al. (1985) found in normal rats aged more than 1 year, but that other authors have not confirmed. The present study had two aims: first, to verify whether neurogenesis spontaneously occurs in DRGs of 14-month-old Sprague-Dawley rats; and, second, to determine whether the neurogenesis enhanced by vitamin E deficiency continues further in the long run, or whether it stops or reverses into neuron loss.

A quantitative and morphometric analysis was performed on neurons of L₃-L₆ DRGs in 14-month-old normal and vitamin-E-deficient rats: the results obtained were compared to those previously obtained in 1-month-old and 5-month-old animals of both dietetic treatment groups, in order to observe the effects of aging on these neuronal populations. The total number of DRG neurons in the control group was higher in older than in younger animals, whereas the value in the vitamin-E-deficient group was lower in older than in younger animals. The present data confirm that neurogenesis occurs in DRGs of normal rats during adult life. Moreover, they show that once the premature neurogenesis in the deficient rats is completed, no further increase in the number of neurons takes place.     

Cimetidine a promising radio-protective agent through modulating Bax/Bcl2 ratio: An in vivo study in male rats

Radiotherapy is one of the most effective modalities for treatment of neoplastic diseases. Radiation damage is to a large extent caused by overproduction of reactive oxygen species. To improve the therapeutic index, identifying effective substances for prevention or treatment of postirradiation intestinal and bone marrow injury should be prompted. This study was designed to evaluate the protective effects of cimetidine on the in rats exposed to γ-irradiation (5 Gy) and exploring the B-cell lymphoma 2 (Bcl2)/Bcl2 associated X (bax) pathway as a probable underlying mechanism. Eighteen adult male rats were randomly grouped into three: control, untreated irradiated rats, and irradiated rats pretreated with cimetidine. Seven days postirradiation the rats were culled, the bone marrow (BM) and jejunum tissue samples were collected for biochemical, histological, and immunohistological evaluation of BM cell count (BMCs), intestinal fibrosis, oxidative stress, tumor necrosis factor-α, Bcl2, and Bax. Cimetidine pretreatment significantly reversed the loss of BMCs, intestinal lining destruction, and fibrosis seen in the untreated irradiated rats and significantly decreased the underlying oxidative stress, inflammation, and Bax/Bcl2 ratio. There was a significant differential correlation between Bax/Bcl2 ratio, tissue oxidative stress level, and tissue injury. Cimetidine represents a very promising radioprotective agent with a potential differential beneficial effect on both cancer cells (inducing apoptosis) as previously proved through different studies and adjacent healthy cells (providing radioprotection via inhibiting apoptosis) as clearly demonstrated through this study, via its antioxidant effect and subsequent regulation of type 2 apoptotic pathway through modulation of Bax/Bcl2 ratio.     

Early melatonin supplementation alleviates oxidative stress in a transgenic mouse model of Alzheimer's disease

Multiple lines of evidence demonstrated that increased brain oxidative stress is a key feature of Alzheimer's disease (AD). Melatonin is a potent endogenous antioxidant and free radical scavenger. A transgenic mouse model for AD mimics the accumulation of senile plaques, neuronal loss, and memory impairment. Four-month-old transgenic mice were administrated melatonin at 10 mg/kg for 4 months. We investigated the long-term influence of melatonin on these mice before amyloid plaques were deposited. We found an increase in the levels of brain thiobarbituric acid-reactive substances (TBARS) and a decrease in glutathione (GSH) content, as well as accelerated upregulation of the apoptotic-related factors, such as Bax, caspase-3, and prostate apoptosis response-4 (Par-4) in transgenic mice, but not in wild-type (WT) littermates. Significantly, the increase in TBARS levels, reduction in superoxide dismutase activity, and GSH content were reinstated by melatonin. In addition, transgenic mice administered melatonin (10 mg/kg) showed a significant reduction in upregulated expression of Bax, caspase-3 and Par-4, indicating inhibited triggering of neuronal apoptosis. These results supported the hypothesis that oxidative stress was an early event in AD pathogenesis and that antioxidant therapy may be beneficial only if given at this stage of the disease process. In sharp contrast to conventional antioxidants, melatonin crosses the blood-brain barrier, is relatively devoid of toxicity, and constitutes a potential therapeutic candidate in AD treatment.     

Melatonin,a calpain inhibitor in the central nervous system: Current status and future perspectives

Dysregulation of neuronal Ca²⁺ and oxidative stress plays an important role in the activation of cysteine proteases including calpains and caspases that contribute to neuronal death. In neurodegenerative diseases, traumatic brain injury, stroke, and neuropathic pain calpain activities are markedly increased. Melatonin is a beneficial supplement in the treatment of central nervous system (CNS) disorders. Melatonin is a potent antioxidant and works as a free-radical scavenger to regulate a large number of molecular pathways, including oxidative stress, inflammation, apoptosis, and cell death under different pathological conditions. However, limited studies have evaluated the inhibitory effect of melatonin on calpains. This review summarizes the current knowledge related to the effects of melatonin on calpains in some of the common CNS disorders.     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin

Drug addiction is a chronic brain disease that is a serious social problem and causes enormous financial burden. Because mitochondrial abnormalities have been associated with opiate addiction, we examined the effect of morphine on mtDNA levels in rat and mouse models of addiction and in cultured cells. We found that mtDNA copy number was significantly reduced in the hippocampus and peripheral blood of morphine-addicted rats and mice compared with control animals. Concordantly, decreased mtDNA copy number and elevated mtDNA damage were observed in the peripheral blood from opiate-addicted patients, indicating detrimental effects of drug abuse and stress. In cultured rat pheochromocytoma (PC12) cells and mouse neurons, morphine treatment caused many mitochondrial defects, including a reduction in mtDNA copy number that was mediated by autophagy. Knockdown of the Atg7 gene was able to counteract the loss of mtDNA copy number induced by morphine. The mitochondria-targeted antioxidant melatonin restored mtDNA content and neuronal outgrowth and prevented the increase in autophagy upon morphine treatment. In mice, coadministration of melatonin with morphine ameliorated morphine-induced behavioral sensitization, analgesic tolerance and mtDNA content reduction. During drug withdrawal in opiate-addicted patients and improvement of protracted abstinence syndrome, we observed an increase of serum melatonin level. Taken together, our study indicates that opioid addiction is associated with mtDNA copy number reduction and neurostructural remodeling. These effects appear to be mediated by autophagy and can be salvaged by melatonin.     

Effect of isoflavone administration on age-related hepatocyte changes in old ovariectomized femal Wistar rats

Aging seems to be due to the accumulation of oxidative damage in cells and molecules. On the other hand, menopause and ovariectomy induce deleterious effects on different organs and systems that have been shown to be counteracted by estrogens and in a not so evident form also with phytoestrogens. The present study has investigated whether the administration of a commercial soy extract that contains approximately 10% isoflavones was able to modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female Wistar rats. Eighteen 22-month-old animals that had been previously ovariectomized at 12 months of age were divided into four groups: ovariectomized control rats, estradiol-treated ovariectomized females and ovariectomized rats treated with isoflavones. Six intact female rats of 2 months of age were used as reference group. Hepatocytes were isolated and cultured, and carbon monoxide (CO) and nitric oxide (NO) release, as well as adenosyl triphosphate (ATP), cyclic guanosyl monophosphate (cGMP), phosphatidylcholine (PC) and lipid peroxide (LPO) content of cells were evaluated. Uterus was also removed and weighed. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content as compared to young animals. Age also induced an increase in LPO cell content. NO, CO and cGMP were augmented with age, and PC synthesis showed a dramatic reduction. Treatment with either estradiol or isoflavones were able to improve all the mentioned parameters altered in hepatocytes isolated from old ovariectomized rats, and the magnitude of the improvement was similar for both treatments. Ovariectomy induced a significant reduction in uterine weight, which was significantly counteracted by estradiol treatment but not by isoflavone administration. In conclusion, the administration of a soy extract containing isoflavones seems to prevent oxidative changes in hepatocytes isolated from old ovariectomized female rats, without modifying uterus weight.     

Melatonin enhances hydrogen peroxide-induced apoptosis in human promyelocytic leukaemia HL-60 cells

Melatonin is an indoleamine secreted by the pineal gland that shows multiple tasks. This ubiquitously acting free radical scavenger has recently been shown to stimulate the production of reactive oxygen species (ROS) in tumour cells, making them undergo apoptosis, whilst it prevents apoptosis in healthy cells. The mechanisms by which melatonin exerts these dual actions are, however, not yet clearly understood. Thus, the aim of this study was to further investigate how melatonin can enhance oxidative stress-induced apoptosis in a leukaemia cell line. The results show that melatonin increased the apoptotic effects of H(2)O(2) in human myeloid HL-60 cells as assessed by cellular viability, mitochondrial permeability transition induction, mitochondrial membrane depolarization, ROS generation, caspases 3, 8 and 9 activity, phosphatidylserine externalization, and DNA fragmentation techniques. When healthy leucocytes were exposed to H(2)O(2), melatonin increased the viability of the cells. Taken together, the findings indicate that melatonin is a potential physiological tool capable of protecting healthy cells from chemotherapy-induced ROS production as well as inducing tumour cell death. Because cancer cells manifest increased oxidative stress as a result of their elevated metabolism, the use of melatonin may be useful in impairing their ROS buffering capacity.     

Melatonin Ameliorates Injury and Specific Responses of Ischemic Striatal Neurons in Rats

Studies have confirmed that middle cerebral artery occlusion (MCAO) causes striatal injury in which oxidative stress is involved in the pathological mechanism. Increasing evidence suggests that melatonin may have a neuroprotective effect on cerebral ischemic damage. This study aimed to examine the morphological changes of different striatal neuron types and the effect of melatonin on striatal injury by MCAO. The results showed that MCAO induced striatum-related dysfunctions of locomotion, coordination, and cognition, which were remarkably relieved with melatonin treatment. MCAO induced severe striatal neuronal apoptosis and loss, which was significantly decreased with melatonin treatment. Within the outer zone of the infarct, the number of Darpp-32+ projection neurons and the densities of dopamine-receptor-1 (D1)+ and dopamine-receptor-2 (D2)+ fibers were reduced; however, both parvalbumin (Parv)+ and choline acetyltransferase (ChAT)+ interneurons were not significantly decreased in number, and neuropeptide Y (NPY)+ and calretinin (Cr)+ interneurons were even increased. With melatonin treatment, the loss of projection neurons and characteristic responses of interneurons were notably attenuated. The present study demonstrates that the projection neurons are rather vulnerable to ischemic damage, whereas the interneurons display resistance and even hyperplasia against injury. In addition, melatonin alleviates striatal dysfunction, neuronal loss, and morphological transformation of interneurons resulting from cerebral ischemia.     

Melatonin and tryptophan as therapeutic agents against the impairment of the sleep-wake cycle and immunosenescence due to aging in Streptopelia risoria

All organisms present circadian rhythm in most of their physiological functions, and among them there stand out sleep, motor activity, immune function, the secretion of melatonin, and the production and release of numerous neurotransmitters, in particular of serotonin because of its relationship with the aforementioned factors. Aging changes these rhythms, altering sleep quality and contributing to immunosenescence. Treatment with exogenously administered melatonin or tryptophan may restore these impaired functions due to aging. In our animal model (Streptopelia risoria), both the hormone and the amino acid acted on the activity-rest rhythms, modulating the circulating levels of melatonin and serotonin, and increased the cell viability and resistance to induced oxidative stress of blood heterophils, at the same time as enhancing the phagocytic function and neutralizing the superoxide anions deriving from this immune function. Also, in the old individuals, the treatments with melatonin and tryptophan at the concentrations and times of administration considered suitable improved nocturnal rest besides reverting the immunosuppressory and oxidative effects accompanying phagocytosis at these advanced ages.     

LncRNA TUG1 inhibits neuronal apoptosis in status epilepticus rats via targeting the miR-421/mTOR axis

Status epilepticus (SE) induces apoptosis of hippocampal neurons. However, the underlying mechanism in SE is not fully understood. Recently, lncRNA TUG1 is reported as a significant mediator in neuronal development. In present study, we aimed to investigate whether lncRNA TUG1 induces apoptosis of hippocampal neurons in SE rat models. TUG1 expression in serum of normal volunteers and SE patients, SE rats and neurons with epileptiform discharge was detected. SE rat model was established and intervened with TUG1 to evaluate hippocampal neuronal apoptosis. The experiments in vitro were further performed in neurons with epileptiform discharge to verify the effects of TUG1 on neuronal apoptosis of SE rats. The downstream mechanism of TUG1 was predicted and verified. miR-421 was intervened to perform the rescue experiments. Levels of oxidative stress and inflammation-related factors and mTOR pathway-related proteins in SE rats and hippocampal neurons were detected. TUG1 was highly expressed in serum of SE patients, SE rats and neurons with epileptiform discharge. Inhibition of TUG1 relieved pathological injury, oxidative stress and inflammation and reduced neuronal apoptosis in SE rats, which were further verified in hippocampal neurons. TUG1 upregulated TIMP2 expression by targeting miR-421. Overexpressed miR-421 inhibited hippocampal neuronal apoptosis. TUG1 knockout inactivated the mTOR pathway via the miR-421/TIMP2 axis to relieve neuronal apoptosis, oxidative stress and inflammation in SE rats and hippocampal neurons. Taken together, these findings showed that downregulation of lncRNA TUG1 inhibited apoptosis of hippocampal neurons in SE rats, and attenuated oxidative stress and inflammation damage through regulating the miR-421/mTOR axis.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.     

Selective alteration at the growth-hormone- releasing-hormone nerve terminals during aging in GHRH-green fluorescent protein mice

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.     

Critical function of endogenous XIAP in regulating caspase activation during sympathetic neuronal apoptosis

In sympathetic neurons, unlike most nonneuronal cells, growth factor withdrawal-induced apoptosis requires the development of competence in addition to cytochrome c release to activate caspases. Thus, although most nonneuronal cells die rapidly with cytosolic cytochrome c alone, sympathetic neurons are remarkably resistant unless they develop competence. We have identified endogenous X-linked inhibitor of apoptosis protein (XIAP) as the essential postcytochrome c regulator of caspase activation in these neurons. In contrast to wild-type neurons that are resistant to injection of cytochrome c, XIAP-deficient neurons died rapidly with cytosolic cytochrome c alone. Surprisingly, the release of endogenous Smac was not sufficient to overcome the XIAP resistance in sympathetic neurons. In contrast, the neuronal competence pathway permitted cytochrome c to activate caspases by inducing a marked reduction in XIAP levels in these neurons. Thus, the removal of XIAP inhibition appears both necessary and sufficient for cytochrome c to activate caspases in sympathetic neurons. These data identify a critical function of endogenous XIAP in regulating apoptosis in mammalian cells.