Wilson disease (WD), an autosomal recessive disease with abnormal copper metabolism, is caused by mutations in the ATP7B gene. The symptoms are characterized by liver, nervous system, and ocular manifestations related to the deposition of copper in the liver, lens nucleus, and cornea. The case reported in this article was a 30-year-old woman with high muscle tension, unable to stand or walk, low voice, and short-term memory loss. Brain MRI showed that patchy T1, long T2 signals, and high FLAIR presented signals in the basal ganglia thalamus area, subcortex, and brain stem. Low signal shadows were presented in magnetically sensitive bilateral basal ganglia region, callus, and cerebellar dentate nucleus. Ceruloplasmin was significantly reduced. With conventional treatments by western medicine, the symptoms were not alleviated. However, after the application of acupuncture-assisted treatment, the patient is capable with self-care ability, thus a desired effect was achieved. It is therefore suggested that acupuncture shall be applied to assist the treatment of WD to relieve symptoms and help patients regain self-care ability.

Heavy metal contamination in herbal medicines is a global threat to human beings especially at levels above known threshold concentrations. The concentrations of five heavy metals cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg) and copper (Cu) were investigated using Inductively Coupled Plasma Optical Mass Spectrometry (ICP-MS) with 1773 samples around the world. According to Chinese Pharmacopoeia, 30.51% (541) samples were detected with at least one over-limit metal. The over-limit ratio for Pb was 5.75% (102), Cd at 4.96% (88), As at 4.17% (74), Hg at 3.78% (67), and of Cu, 1.75% (31). For exposure assessment, Pb, Cd, As, and Hg have resulted in higher than acceptable risks in 25 kinds of herbs. The maximal Estimated Daily Intake of Pb in seven herbs, of Cd in five, of Hg in four, and As in three exceeded their corresponding Provisional Tolerable Daily Intakes. In total 25 kinds of herbs present an unacceptable risk as assessed with the Hazard Quotient or Hazard Index. Additionally, the carcinogenic risks were all under acceptable limits. Notably, As posed the highest risk in all indicators including Estimated Daily Intake, Hazard Index, and carcinogenic risks. Therefore further study on enrichment effect of different states of As and special attention to monitoring shall be placed on As related contamination.

We previously found that maternal cigarette smoke (CS) exposure resulted in impairment of central chemoreception and oxidative stress and mitochondrial dysfunction of parafacial respiratory group (pFRG, a critical site for mammalian central chemoreception) in neonatal rats. The present work was carried out to identify if maternal CS exposure could disturb the glutamate (GLU)-ergic and γ-aminobutyric acid (GABA)-ergic balance in pFRG of neonatal rats. We found that maternal CS exposure induced a decrease in GLU content and consequently in GLU/GABA ratio in pFRG of neonatal rats. Maternal CS exposure also decreased glutamine content and glutaminase and glutamine synthetase activity in offspring pFRG. In addition, expression of vesicular glutamate transporter 2 was depressed, and those of glutamate transporter 1 and GABA transporter 3 were elevated by maternal CS exposure. These results indicate that maternal CS exposure leads to a disturbance of GLU/GABA balance in pFRG of the neonatal rats, which might contribute to the suppression of central chemoreception in maternal CS-exposed offspring.

Prenatal cigarette smoke (CS) exposure causes numerous respiratory health problems in infants. This study aimed to investigate the effect of prenatal CS exposure on sevoflurane-induced respiratory suppression in neonatal rats and the protective role of H2S. We found that at baseline, minute ventilation (V’E), respiratory frequency (fR), and tidal volume (VT) were similar among tested groups, whereas sigh frequency (fS) was lower in CS group than in the Control group. During 3 % sevoflurane anesthesia, V’E was decreased, fR was slowed, VT was increased, and fS was reduced in all groups; however, the decline in fR and increase in VT was greater in CS group than in the Control group. During the recovery, fS remained lower in CS group. The above changes of respiratory response caused by prenatal CS exposure were alleviated by NaHS pretreatment (a donor of H2S, 56 μmol/kg/d, intraperitoneal injection). These results indicated that prenatal CS exposure alters the breathing into a much slower and deeper manner in neonatal rats during sevoflurane anesthesia, and H2S mitigates this respiratory change.

We previously reported that maternal cigarette smoke (CS) exposure resulted in impairment of central chemoreception and induced mitochondrial dysfunction in offspring parafacial respiratory group (pFRG), the kernel for mammalian central chemoreception. We also found that hydrogen sulfide (H2S) could attenuate maternal CS exposure-induced impairment of central chemoreception in the rat offspring in vivo. Mitochondrial ATP sensitive potassium (mitoKATP) channel has been reported to play a significant role in mitochondrial functions and protect against apoptosis in neurons. Thus, we hypothesize here that mitoKATP channel plays a role in the protective effects of H2S on neonatal central chemoreception in maternal CS-exposed rats. Our findings revealed that pretreatment with NaHS (donor of H2S, 22.4mM) reversed the central chemosensitivity decreased by maternal CS exposure, and also inhibited cell apoptosis in offspring pFRG, however, 5-HD (blocker of mitoKATP channels, 19mM) attenuated the protective effects of NaHS. In addition, NaHS declined pro-apoptotic proteins related to mitochondrial pathway apoptosis in CS rat offspring pFRG, such as Bax, Cytochrome C, caspase9 and caspase3. NaHS or 5-HD alone had no significant effect on above indexes. These results suggest that mitoKATP channels play an important role in the protective effect of H2S against impairment of central chemoreception via anti-apoptosis in pFRG of rat offspring exposed to maternal CS.

We have reported that smoking during pregnancy is associated with deficit in neonatal central chemoreception. However, the underlying mechanism is not well clarified. In this study, we developed a rat model of maternal cigarette smoke (CS) exposure. Pregnant rats were exposed to CS during gestational day 1-20. Offspring were studied on postnatal day 2. Reactive oxygen species (ROS) content and expressions of antioxidant proteins in retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) were examined by fluorogenic dye MitoSOX™ Red and Western blotting, respectively. The response of hypoglossal rootlets discharge to acidification was also detected with micro-injection of H2O2 into RTN/pFRG of offspring brainstem slices in vitro. Results showed that maternal CS exposure led to an increase in ROS production, and brought about decreases in mitochondrial superoxide dismutase and Kelch-like ECH-associated protein-1, and an increase in NF-E2-related factor 2 in offspring RTN/pFRG. Catalase and glutathione reductase expressions were not significantly changed. Moreover, oxidative stress induced by micro-injection of H2O2 into RTN/pFRG in vitro inhibited the discharge response of hypoglossal rootlets to acidification. These findings suggest that maternal CS exposure results in oxidative stress in RTN/pFRG of rat offspring, which might play a role in the impairment of central chemoreception.

We previously found that hydrogen sulfide (H2S) ameliorated the dysfunction of central chemoreception caused by prenatal cigarette smoke exposure (CSE). In the present study, we further explored whether the parafacial respiratory group (pFRG) is involved in the protection of central chemoreception by H2S against prenatal CSE-induced injury. We found that NaHS, a donor of H2S, restored the expression of Phox2b, which was downregulated by prenatal CSE, in the pFRG region of neonatal rats. NaHS also relieved the prenatal CSE-induced excitatory synapse disturbance in the pFRG region of neonatal rats. Additionally, NaHS prevented the increase in the malondialdehyde level and suppression of antioxidase activity in the pFRG region of neonatal rats induced by prenatal CSE. Furthermore, NaHS prevented the downregulation of the expression of antioxidases and Nrf2 in the pFRG region of neonatal rats with prenatal CSE. These results suggest that H2S can protect the pFRG of neonatal rats against prenatal CSE-induced injury via an antioxidative effect.

Background/Aims: Cigarette smoke exposure (CSE) during pregnancy is a well-recognized health hazard that causes placental damage. Hydrogen sulfide (H2S) has been reported to protect multiple organs from injury. However, the protective effects of H2S have not been tested in the placenta. This study aimed to explore the potential of H2S in protecting placenta against oxidative injury induced by CSE during pregnancy and the possible underlying mechanisms. Methods: Pregnant SD rats were randomly divided into 4 groups: NaCl, NaHS (a donor of H2S), CSE and CSE+NaHS. Placental oxidative damage was detected by 8-hydroxy-2-deoxyguanosine (8-OHdG) stain and malondialdehyde (MDA) assay. Placental redox status was assessed by measuring reactive oxygen species (ROS), total antioxidant capacity (T-AOC) and glutathione (GSH) levels, as well as copper/zinc SOD (SOD1), manganese SOD (SOD2), catalase (CAT) and glutathione peroxidase (GPx) activities and expressions. Meanwhile, nuclear factor erythroid 2-related factor 2 (Nrf2) was analyzed by immunohistochemistry, real-time PCR and Western blot. Results: We found that NaHS markedly reduced the elevated levels of 8-OHdG and MDA induced by CSE. Further, NaHS treatment effectively mitigated CSE-induced placental redox imbalance by inhibiting ROS production, restoring T-AOC level, increasing GSH/GSSG ratio, and augmenting SOD1 SOD2, CAT and GPx activities and expressions. More notably, NaHS administration also reversed the aberrant decrease of Nrf2 due to CSE in rat placentas. Conclusion: Our data demonstrate that H2S can protect against CSE-induced placental oxidative damage probably by alleviating redox imbalance via Nrf2 pathway.

Cigarette smoke (CS) exposure negatively affects neurodevelopment. We established a CS exposure rat model to determine how maternal CS exposure induces oxidative stress and mitochondrial dysfunction in parafacial respiratory group (pFRG) essential to central chemoreceptive regulation of normal breathing. Pregnant rats were exposed to CS during gestational days 1-20, and the offspring were studied on postnatal day 2. Our data showed that maternal CS exposure resulted in elevated accumulation of ROS, which left a footprint on DNA and lipid with increases in 8-hydroxy-2'-deoxyguanosine and malondialdehyde contents. Furthermore, maternal CS exposure induced decreases in manganese superoxide dismutase, catalase and glutathione reductase activities as well as reduction in glutathione content in pFRG in the offspring. Moreover, maternal exposure to CS led to mitochondrial ultrastructure changes, mitochondrial swelling, reduction in ATP generation, loss of mitochondrial membrane potential and increase in mitochondrial DNA copy number. These findings suggest that maternal exposure to CS alters normal development of pFRG that is critical for normal respiratory control.

Maternal cigarette smoke exposure (CS) during pregnancy is a well recognized causative factor for placental injury. Hydrogen sulfide (H2S), a gaseous signal molecule, has been shown to exert a protective effect against tissue injury in many organs. This study aimed to investigate whether H2S could alleviate CS-induced placental injury in rats. Pregnant SD rats were randomly divided into 4 groups: NaCl, CS, CS + NaHS (a donor of H2S) and NaHS. On gestational day 21, placental and fetal weights were measured. Placental H2S content was assessed by methylene blue method. Placental histological changes were examined by light microscopy and transmission electron microscopy. The results showed that administration of NaHS significantly attenuated CS-induced decrease in placental and fetal weights. Moreover, NaHS injection markedly alleviated CS-induced reduction in placental H2S content. Meanwhile, NaHS treatment obviously improved CS-induced morphological changes of placental junctional and labyrinthine zones. More notably, NaHS administration in CS rats was also found to ameliorate placental ultrastructural alterations, as indicated by decrease in the thickness of placental barrier, prevention of apical microvilli loss, alleviation of endoplasmic reticulum swelling and reduction in the number of vacuoles in trophoblast cells. Taken together, these findings demonstrate that administration of NaHS can alleviate CS-induced placental injuries in rats, suggesting that H2S may have therapeutic potential for treatment of placental injury caused by CS.

The present study was designed to investigate whether H2S could improve the respiratory responses to hypercapnia blunted by prenatal CSE in neonatal rats in vivo. Respiratory activities were recorded with head-out body plethysmography. The results showed that during baseline, respiratory frequency (FR), tidal volume (VT) and minute ventilation (VE) were similar among tested groups; frequency of spontaneous apnea (FSA), not post-sigh apnea (FPA), was significantly elevated by prenatal CSE. During hypercapnia, the increases in FR and VE were significantly reduced, but VT was not markedly different, in CSE group; both FSA and FPA were decreased, although FSA remained higher in CSE group. All the aforementioned effects induced by CSE on respiratory activities were relieved by NaHS (donor of H2S, 56 μmol/kg by intraperitoneal injection). These data indicate that H2S could ameliorate the disruption of respiratory responses to hypercapnia induced by prenatal CSE in neonatal rats.

We previously demonstrated that hydrogen sulfide (H2S) protected neonatal rat medulla oblongata from prenatal cigarette smoke exposure (CSE) via anti-apoptotic effect. The present work further investigated the involvement of anti-oxidative and anti-inflammatory effects of H2S in the protection. Pregnant Sprague-Dawley rats were randomly divided into NaCl, CSE, CSE + NaHS (a donor of H2S) and NaHS groups. All the tests were performed with corresponding neonatal rats. Nissl staining revealed that NaHS treatment ameliorated neuronal chromatolysis in the hypoglossal nucleus and nucleus ambiguus resulted from prenatal CSE. Moreover, NaHS eliminated decrease of glutathione level, increase of malondialdehyde content and inhibition of superoxide dismutase activity within neonatal rat medulla oblongata caused by prenatal CSE. NaHS also relieved the up regulation of tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6 in the medulla oblongata of the neonatal CSE rats. These results suggest that H2S can alleviate prenatal CSE-induced injuries of neonatal rat medulla oblongata through anti-oxidative and anti-inflammatory effects.

Normal breathing in rodents requires activity of glutamatergic Dbx1-derived (Dbx1(+)) preBötzinger Complex (preBötC) neurons expressing somatostatin (SST). We combined in vivo optogenetic and pharmacological perturbations to elucidate the functional roles of these neurons in breathing. In transgenic adult mice expressing channelrhodopsin (ChR2) in Dbx1(+) neurons, photoresponsive preBötC neurons had preinspiratory or inspiratory firing patterns associated with excitatory effects on burst timing and pattern. In transgenic adult mice expressing ChR2 in SST(+) neurons, photoresponsive preBötC neurons had inspiratory or postinspiratory firing patterns associated with excitatory responses on pattern or inhibitory responses that were largely eliminated by blocking synaptic inhibition within preBötC or by local viral infection limiting ChR2 expression to preBötC SST(+) neurons. We conclude that: (1) preinspiratory preBötC Dbx1(+) neurons are rhythmogenic, (2) inspiratory preBötC Dbx1(+) and SST(+) neurons primarily act to pattern respiratory motor output, and (3) SST(+)-neuron-mediated pathways and postsynaptic inhibition within preBötC modulate breathing pattern.

Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii (Chuan Xiong) has been proved to protect the medulla oblongata from chronic hypoxia injury. However, the underlying mechanism remains unclear. The purpose of this study was to determine whether the protective effects of TMP are associated with the heme oxygenase-1 (HO-1) dependant pathway in adult rats. The morphological changes of neurons in the hypoglossal nucleus (12N), the nucleus ambiguus (Amb), the nucleus tractus solitarius (NTS), and the pre-Bötzinger complex (pre-BötC) were investigated by Nissl staining; the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured to evaluate the anti-oxidant effect; some apoptosis parameters, Bax mRNA and Bcl-2 mRNA, were tested; and the double immunochemistry staining of active caspase-3/NeuN was performed. Meanwhile, the HO-1 protein expression and heme oxygenase (HO) activity were examined. Tin-protoporphyrin (SnPP), a potent inhibitor of HO, was used to further confirm the effect of HO-1. We found that TMP ameliorated the neuron loss in 12N, Amb and NTS, the decrease in SOD activity and the increase in MDA content, the decrease in Bcl-2 mRNA of medulla oblongata (P<0.05), and the increase in percentage of apoptotic neurons in Amb (P<0.05) induced by chronic hypoxia. Co-administration with SnPP abolished the beneficial effects above of TMP to some extent (P<0.05). Moreover, TMP significantly increased HO activity and HO-1 protein expression, which was most likely enhanced in the neurons (P<0.05), and co-administration of SnPP reduced these up-regulated effects (P<0.05). This study demonstrated that HO-1 dependant pathway may be involved in the protective action of TMP against chronic hypoxic damage on medulla oblongata in the rats.

Our previous study in vitro showed that hydrogen sulfide (H2S) could protect the medullary respiratory centers from injury induced by acute hypoxia in brainstem slices of neonatal rats. The present study was carried out to determine if H2S could exhibit similar protective effects in adult rats and to explore the underlying mechanisms of its protection. It was observed that hypoxia induced a diphasic respiratory response, an excitatory phase followed by an inhibitory one, as indicated by an increase followed by a decrease in frequency of rhythmic discharge of the diaphragm. Nissl staining revealed that some of the neurons in the medullary respiratory related nuclei were impaired in hypoxia rats. Hypoxia led to increases in the content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as a decrease in the level of Bcl-2 mRNA of the medulla oblongata. Intracerebroventricular injection of 2.5mM NaHS (a donor of H2S) or L-cysteine (L-Cys, a substrate for H2S) could prevent inhibitory respiratory effect occurred in the rats with hypoxia. Exogenous application of NaHS and L-Cys could also reduce the content of MDA and the activities of SOD and GSH-Px, and increase the level of Bcl-2 mRNA expression of medulla oblongata caused by hypoxia. These results indicate that H2S could protect the medullary respiratory centers against injury induced by acute hypoxia in adult rats partly due to its anti-oxidant and anti-apoptotic effects.

It has been postulated that prenatal cigarette smoke exposure (CSE) increases the risk for sudden infant death syndrome. The victims of infant death syndrome suffer from respiratory abnormalities, such as central apnea, diminished chemoreflex and alteration in respiratory pattern during sleep. However, no experimental evidence on CSE model exists to confirm whether prenatal CSE gives rise to reduction of neonatal central chemoreception in in vitro preparations in absence of peripheral sensory feedback. The aim of the present study was to test the hypothesis that maternal CSE during pregnancy depresses central chemoreception of the neonatal rats. The pregnant rats were divided into two groups, control (n = 8) and CSE (n = 8). Experiments were performed on neonatal (0-3days) rat pups. Fictive respiratory activity was monitored by recording the rhythmic discharge from the hypoglossal rootlets of the medullary slices obtained from the neonatal rats. The burst frequency (BF) and integrated amplitude (IA) of the discharge were analyzed. Their responses to acidified artificial cerebrospinal fluid (aCSF) were tested to indicate the change of the central chemosensitivity. Under condition of perfusing with standard aCSF (pH 7.4), no significant difference was detected between the two groups in either BF or IA (P>0.05). Under condition of perfusing with acidified aCSF (pH 7.0), BF was increased and IA was decreased in both groups (P<0.01). However, their change rates in the CSE group were obviously smaller than that in the control group, 66.98 ± 10.11% vs. 143.75 ± 15.41% for BF and -22.38 ± 2.51% vs. -44.90 ± 3.92% for IA (P<0.01). In conclusion, these observations, in a prenatal CSE model, provide important evidence that maternal smoking during pregnancy exerts adverse effects on central chemoreception of neonates.

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, microtubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the supernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

H2S has been found to act as a neuroprotectant. Cystathionine-beta-synthase (CBS) is one of three enzymes for production of H2S in mammals. The present study was conducted to investigate whether CBS expression can be affected by in utero cigarette smoke exposure (CS) in the neonatal rats. SD pregnant rats were randomly divided into control and CS groups. The neonatal offspring rats were used in experimental observation. The methylene blue assay method was used to examine H2S production rate by medulla oblongata. CBS mRNA and protein expression in medulla oblongata were analysed by RT-PCR and Western blot, respectively, and the levels of CBS expression in neurons and glia of medullary nuclei were examined with immunohistochemical technique. In medulla oblongata of CS group, compared to control group, H2S production rate was increased (P

We previously revealed that cystathionine-beta-synthase (CBS) for production of H 2 S is expressed in neonatal rat medulla oblongata and CBS-H 2 S pathway contributes to protection of medullary respiratory centers against injury induced by acute hypoxia in both neonatal and adult rats. The present study was carried out to explore if expression of CBS/H 2 S in medulla oblongata is upregulated by chronic intermittent hypoxia (CIH) in adult rats. Adult SD rats were randomly divided into control and CIH groups. The levels of mRNA and protein of CBS in medulla oblongata were analyzed by RT-PCR and Western blot, respectively. The expression of CBS protein in medullary respiration-related nuclei was examined with immunohistochemistry. H 2 S production rate by medulla oblongata was measured by methylene blue assay. It was indicated that the levels of both mRNA and protein of CBS in medulla oblongata were higher in CIH group ( P P 2 S by medulla oblongata was elevated in CIH group ( P 2 S in medulla oblongata can be upregulated by CIH, suggesting that CBS-H 2 S pathway may be involved in protection of medullary respiratory centers against CIH injury in adult rats.