Hung Ming Chang

Melatonin preserves longevity protein (sirtuin 1) expression in the hippocampus of total sleep-deprived rats.

Abstract:  Sleep disorders cause cognitive dysfunction in which impaired neuronal plasticity in the hippocampus may underline the molecular mechanisms of this deficiency. As sirtuin 1 (SIRT1) plays an important role in maintaining metabolic homeostasis and neuronal plasticity, this study is aimed to determine whether melatonin exerts beneficial effects on preserving SIRT1 activation following total sleep deprivation (TSD). TSD was performed by disc on water method for five consecutive days. During this period, animals daily received melatonin at doses of 5, 25, 50 or 100 mg/kg. The cytochrome oxidase (COX) histochemistry, SIRT1 immunohistochemistry together with Morris water maze learning test were performed to examine the metabolic, neurochemical, as well as the behavioral changes in neuronal plasticity, respectively. The results indicate that in normal rats, numerous COX and SIRT1 positive‐labeled neurons with strong staining intensities were found in hippocampal pyramidal and granular cell layers. Following TSD, both COX and SIRT1 reactivities were drastically decreased as revealed by reduced staining pattern and labeling frequency. Behavioral data corresponded well with morphological findings in which spatial memory test in water maze was significantly impaired after TSD. However, in rats receiving different doses of melatonin, both COX and SIRT1 expressions were successfully preserved. Considerably better performance on behavioral testing further strengthened the beneficial effects of melatonin. These findings suggest that melatonin may serve as a novel therapeutic strategy directed for preventing the memory deficits resulting from TSD, possibly by effectively preserving the metabolic function and neuronal plasticity engaged in maintaining cognitive activity.

Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.

Melatonin attenuates the neuronal NADPH‐d/NOS expression in the nodose ganglion of acute hypoxic rats

Excessive production of nitric oxide (NO) may play a detrimental role in the process of hypoxia‐related neuropathology. This study explored whether treatment with melatonin would attenuate the neuropathological changes in the vagal ganglia following a severe hypoxic insult. Thirty minutes prior to hypoxia treatment, young adult rats were pre‐treated with melatonin at 5, 25 or 100 mg/kg injected intraperitoneally. Hypoxia was achieved by subjecting the rats to a barometric pressure of 0.2 atm (PO2=43 Torr) for 4 hr in an altitude chamber. Nicotinamine adenine dinucleotide phosphate‐diaphorase (NADPH‐d) histochemistry combined with the neuronal nitric oxide synthase (nNOS) immunohistochemistry were used to detect the NADPH‐d/nNOS reactivity in the nodose ganglion (NG) at various time points following the hypoxic exposure. In normal untreated rats, about 43% of the neurons in the NG displayed NADPH‐d/nNOS reactivity. Following hypoxic exposure, both the percentage and the staining intensity of NADPH‐d/nNOS positive neurons in the NG were markedly increased, but these were reduced in longer surviving animals. Quantitative analysis of cell counts revealed that about 17% of the neurons died at 14 days after hypoxia treatment. However, in hypoxic rats given different doses of melatonin pre‐treatment, neuronal death as well as the frequency and staining intensity of NADPH‐d/nNOS reactivity of the nodose neurons were significantly decreased. The effect of melatonin on neuronal survival and NADPH‐d/nNOS expression was dose‐dependent. It is therefore suggested that melatonin exerts a neuroprotective effect and may serve as a potential therapeutic strategy for prevention and/or reducing the susceptibility of nodose neurons to NO‐mediated hypoxic neuropathy.

Melatonin preserves superoxide dismutase activity in hypoglossal motoneurons of adult rats following peripheral nerve injury.

Abstract:  Peripheral nerve injury (PNI) produces functional changes in lesioned neurons in which oxidative stress is considered to be the main cause of neuronal damage. As superoxide dismutase (SOD) is an important antioxidative enzyme involved in redox regulation of oxidative stress, the present study determined whether melatonin would exert its beneficial effects by preserving the SOD reactivity following PNI. Adult rats subjected to hypoglossal nerve transection were intraperitoneally injected with melatonin at ones for 3, 7, 14, 30 and 60 days successively. The potential neuroprotective effects of melatonin were quantitatively demonstrated by neuronal nitric oxide synthase (nNOS), mitochondrial manganese SOD (Mn‐SOD), and cytosolic copper‐zinc SOD (Cu/Zn‐SOD) immunohistochemistry. The functional recovery of the lesioned neurons was evaluated by choline acetyltransferase (ChAT) immunohistochemistry along with the electromyographic (EMG) recordings of denervation‐induced fibrillation activity. The results indicate that following PNI, the nNOS immunoreactivity was significantly increased in lesioned neurons peaking at 14 days. The up‐regulation of nNOS temporally coincided with the reduction of ChAT and SOD in which the Cu/Zn‐SOD showed a greater diminution than Mn‐SOD. However, following melatonin administration, the nNOS augmentation was successfully suppressed and the activities of Mn‐SOD, Cu/Zn‐SOD, and ChAT were effectively preserved at all postaxotomy periods. EMG data also showed a decreased fibrillation in melatonin‐treated groups, suggesting a potential effect of melatonin in promoting functional recovery. In association with its significant capacity in preserving SOD reactivity, melatonin is suggested to serve as a powerful therapeutic agent for treating PNI‐relevant oxidative damage.

Sleep deprivation predisposes liver to oxidative stress and phospholipid damage: a quantitative molecular imaging study

Sleep disorders are associated with an increased rate of various metabolic disturbances, which may be related to oxidative stress and consequent lipid peroxidation. Since hepatic phosphatidylcholine plays an important role in metabolic regulation, the aim of the present study was to determine phosphatidylcholine expression in the liver following total sleep deprivation. To determine the effects of total sleep deprivation, we used adult rats implanted for polygraphic recording. Phosphatidylcholine expression was examined molecularly by the use of time‐of‐flight secondary ion mass spectrometry, along with biochemical solid‐phase extraction. The parameters of oxidative stress were investigated by evaluating the hepatic malondialdehyde levels as well as heat shock protein 25 immunoblotting and immunohistochemistry. In normal rats, the time‐of‐flight secondary ion mass spectrometry spectra revealed specific peaks (m/z 184 and 224) that could be identified as molecular ions for phosphatidylcholine. However, following total sleep deprivation, the signals for phosphatidylcholine were significantly reduced to nearly one‐third of the normal values. The results of solid‐phase extraction also revealed that the phosphatidylcholine concentration was noticeably decreased, from 15.7 µmol g–1 to 9.4 µmol g–1, after total sleep deprivation. By contrast, the biomarkers for oxidative stress were drastically up‐regulated in the total sleep deprivation‐treated rats as compared with the normal ones (4.03 vs. 1.58 nmol mg–1 for malondialdehyde levels, and 17.1 vs. 6.7 as well as 1.8 vs. 0.7 for heat shock protein 25 immunoblotting and immunoreactivity, respectively). Given that phosphatidylcholine is the most prominent component of all plasma lipoproteins, decreased expression of hepatic phosphatidylcholine following total sleep deprivation may be attributed to the enhanced oxidative stress and the subsequent lipid peroxidation, which would play an important role in the formation or progression of total sleep deprivation‐induced metabolic diseases.

The Role of Procalcitonin for Acute Pyelonephritis and Subsequent Renal Scarring in Infants and Young Children

PURPOSE We assessed the usefulness of procalcitonin as a biological marker in diagnosing acute pyelonephritis and for predicting subsequent renal scarring in young children with a first febrile urinary tract infection. MATERIALS AND METHODS Children 2 years old or younger with a first febrile urinary tract infection were prospectively studied. Renal parenchymal involvement was assessed by (99m)Tc-dimercaptosuccinic acid scan within 5 days of admission and after 6 months. Serum samples from all patients were tested for procalcitonin, C-reactive protein and white blood cell count measurements. RESULTS The 112 enrolled patients (age range 24 days to 24 months old) were divided into acute pyelonephritis (76) and lower urinary tract infection (36) groups according to the results of (99m)Tc-dimercaptosuccinic acid scans. Median values of procalcitonin, C-reactive protein and white blood cell count at hospitalization were significantly higher in patients with acute pyelonephritis than in those with lower urinary tract infection. The area under receiver operating characteristic curves showed that procalcitonin was superior to C-reactive protein and white blood cell count as a marker for diagnosing acute pyelonephritis. Initial and post-antibiotic treatment procalcitonin values were significantly higher in children with renal scarring than in those without scarring (p <0.001). Procalcitonin values at hospitalization and after treatment were independent predictors of later renal scarring on logistic regression analysis. CONCLUSIONS Our results indicate the superior diagnostic accuracy of procalcitonin for predicting acute pyelonephritis in children 2 years old or younger. Higher initial and posttreatment procalcitonin values are independent risk factors for later renal scarring.

Neuregulin Facilitates Nerve Regeneration by Speeding Schwann Cell Migration via ErbB2/3-Dependent FAK Pathway

Background Adequate migration of Schwann cells (Sc) is crucial for axon-guidance in the regenerative process after peripheral nerve injury (PNI). Considering neuregulin-erbB-FAK signaling is an essential pathway participating in the regulation of Sc migration during development, the present study is aimed to examine whether neuregulin would exert its beneficial effects on adult following PNI and further determine the potential changes of downstream pathway engaged in neuro-regeneration by both in vitro and in vivo approaches. Methodology and Principal Findings Cultured RSC96 cells treated with neuregulin were processed for erbB2/3 immunofluorescence and FAK immunoblotings. The potential effects of neuregulin on Sc were assessed by cell adherence, spreading, and migration assays. In order to evaluate the functional significance of neuregulin on neuro-regeneration, the in vivo model of PNI was performed by chronic end-to-side neurorrhaphy (ESN). In vitro studies indicated that after neuregulin incubation, erbB2/3 were not only expressed in cell membranes, but also distributed throughout the cytoplasm and nucleus of RSC96 cells. Activation of erbB2/3 was positively correlated with FAK phosphorylation. Neuregulin also increases Sc adherence, spreading, and migration by 127.2±5.0%, 336.8±3.0%, and 80.0±5.7%, respectively. As for in vivo study, neuregulin significantly accelerates the speed of Sc migration and increases Sc expression in the distal stump of injured nerves. Retrograde labeling and compound muscle action potential recordings (CMAP) also showed that neuregulin successfully facilitates nerve regeneration by eliciting noticeably larger CMAP and promoting quick re-innervation of target muscles. Conclusions As neuregulin successfully improves axo-glial interaction by speeding Sc migration via the erbB2/3-FAK pathway, therapeutic use of neuregulin may thus serve as a promising strategy to facilitate the progress of nerve regeneration after PNI.

Melatonin restores the cytochrome oxidase reactivity in the nodose ganglia of acute hypoxic rats

Abstract:  This study aimed to elucidate whether melatonin would exert beneficial effects on the neuronal functions of the nodose ganglion (NG) following acute hypoxic insult. The cytochrome oxidase (COX) and the nicotinamine adenine dinucleotide phosphate diaphorase (NADPH‐d) histochemistry along with the nitric oxide synthase (NOS) immunofluorescence were used to examine the metabolic stage and nitric oxide production in nodose neurons respectively. Adult rats were injected intraperitoneally with melatonin at 5 or 100 mg/kg. Hypoxia was achieved by placing the rats into an altitude chamber (PO2 = 43 torr) for 4 hr. The results show that in normal untreated rats, nearly all and about 43% of the NG neurons displayed COX and NOS/NADPH‐d reactivities with various staining intensities respectively. However, COX reactivity was drastically decreased while NOS/NADPH‐d reactivity was significantly upregulated following hypoxia treatment. In melatonin pretreated rats, the hypoxia‐induced reduction of COX reactivity was obviously prevented and the augmentation of NOS/NADPH‐d reactivity was successfully suppressed. The deficit in the metabolic stage and the over‐activation of NOS would contribute to the generation of oxidative stress. By effectively preventing the metabolic disruption, melatonin may have potential utility in therapeutic treatment of neuronal dysfunctions where oxidative stress is a participant.

Axotomy along with hypoxia enhances the neuronal NADPH-d/NOS expression in lower brain stem motor neurons of adult rats

This study was aimed to determine whether axotomy coupled with hypoxia would exert a more profound effect on injury-induced neuronal nitric oxide synthase (NOS) expression. In this connection, the vagus and the hypoglossal nerves of adult rats were transected unilaterally in the same animal, and half of the operated animals were subjected to hypoxia treatment. Both the neuronal NOS immunohistochemistry and the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry were used to assess the neuronal NOS expression. The present results have shown that the number of NADPH-d/NOS-positive [NADPH-d/NOS(1)] neurons in the hypoglossal nucleus (HN) peaked at 14 days after axotomy, while that in dorsal motor nucleus of vagus (DMN) and nucleus ambiguus (NA) was progressively increased up to 60 days. The up-regulation of NADPH-d/NOS in HN and DMN was more pronounced in hypoxic than in normoxic animals, a feature that was not evident in the NA. Quantitative analysis showed that the number of surviving motoneurons in normoxic animals was significantly higher than those subjected to hypoxia at 14 days postaxotomy in HN and at all postaxotomy time points in DMN. The difference may be attributed to their different functional components. Since O2 deprivation leads to poor cellular function, the stronger expression of NADPH-d/NOS and the more drastic neuronal loss following nerve transection in the hypoxic animals compared with the controls suggest that hypoxia plays an important role in peripheral neuropathies in which NO is implicated.

Sleep deprivation impairs Ca2+ expression in the hippocampus: ionic imaging analysis for cognitive deficiency with TOF-SIMS.

Sleep deprivation causes cognitive dysfunction in which impaired neuronal plasticity in hippocampus may underlie the molecular mechanisms of this deficiency. Considering calcium-mediated NMDA receptor subunit 1 (NMDAR1) and neuronal nitric oxide synthase (nNOS) activation plays an important role in the regulation of neuronal plasticity, the present study is aimed to determine whether total sleep deprivation (TSD) would impair calcium expression, together with injury of the neuronal plasticity in hippocampus. Adult rats subjected to TSD were processed for time-of-flight secondary ion mass spectrometry, NMDAR1 immunohistochemistry, nNOS biochemical assay, cytochrome oxidase histochemistry, and the Morris water maze learning test to detect ionic, neurochemical, bioenergetic as well as behavioral changes of neuronal plasticity, respectively. Results indicated that in normal rats, strong calcium signaling along with intense NMDAR1/nNOS expression were observed in hippocampal regions. Enhanced calcium imaging and neurochemical expressions corresponded well with strong bioenergetic activity and good performance of behavioral testing. However, following TSD, both calcium intensity and NMDAR1/nNOS expressions were significantly decreased. Behavioral testing also showed poor responses after TSD. As proper calcium expression is essential for maintaining hippocampal neuronal plasticity, impaired calcium expression would depress downstream NMDAR1-mediated nNOS activation, which might contribute to the initiation or development of TSD-related cognitive deficiency.