Medication Ideas for Depression from Wenyang Jieyu Prescription / 中国实验方剂学杂志

Depression is a complex emotional and mental disorder. The traditional Chinese medicine （TCM） methods for treating depression mainly include soothing the liver and relieving depression. Our research team proposes that depression is caused by Yang Qi deficiency and obstructed Qi movement， which are closely related to neurological and psychological changes induced by early traumatic experiences. Therefore， we suggest that the treatment should focus on warming Yang， replenishing Qi， and promoting Qi movement and have formulated Wenyang Jieyu prescription based on Erxiantang for warming yang and Xiaoyaosan for relieving depression. The experiment with the mouse model of early trauma induced by maternal separation showed that Wenyang Jieyu prescription significantly improved the mouse activity and environmental exploration， reduced the immobility time in forced swimming and tail suspension tests， alleviated the behaviors such as aversion to darkness and fear of open space， enhanced social interaction and social cognitive abilities， altered decision-making biases， reduced depression-like behaviors， and improved the decision-making patterns. Additionally， the prescription lowered the serum level of cortisol， inhibited the cortisol surge in the dexamethasone/corticotropin-releasing hormone （Dex/CRH） test， up-regulated the expression of mineralocorticoid receptor （MR） and 11β-hydroxysteroid dehydrogenase 2 （11β-HSD2） in the hippocampus， down-regulated the expression of glucocorticoid receptor （GR） and corticotropin-releasing hormone receptor 1 （CRHR1）， inhibited the methylation of GR exon 1 and the expression of DNA methyltransferase 1 （DNMT1）， and restored the negative feedback of the hypothalamic-pituitary-adrenal （HPA） axis. Furthermore， Wenyang Jieyu prescription up-regulated the protein level of brain-derived neurotrophic factor （BDNF）， elevated the levels of postsynaptic density protein 95 （PSD95） and synaptophysin （Syn）， decreased the cell apoptosis index and B-cell lymphoma （Bcl-2）-associated X （Bax）/Bcl-2 ratio， suppressed the expression of Caspase-3， and enhanced the neuroplasticity and anti-apoptotic capacity in the hippocampus. Considering the research results， related articles， and clinical experience， we conclude that depression should be treated with liver-soothing and depression-relieving herbs， which can be supplemented with spleen-invigorating and Qi-regulating herbs to alleviate depressive symptoms. The Yang-warming and kidney-tonifying herbs can be used to eliminate the root cause and prevent relapse. Additionally， the wind-dispersing herbs can be supplemented to regulate the Qi movement throughout the body， thereby enhancing the efficacy of depression-relieving treatment.

Regulatory Effect of Wenyang Prescription， Jieyu Prescription， and Wenyang Jieyu Prescription on Pain Sensitivity and Depression-like Behaviors in Mice Induced by Maternal Separation and Chronic Neuropathic Pain / 中国实验方剂学杂志

ObjectiveTo observe the behavioral and pain threshold alterations， as well as the changes in indexes related to depression and pain in the serum and central system in mice stressed by maternal separation and chronic neuropathic pain， and explore the underlying mechanism of Wenyang prescription （WY）， Jieyu prescription （JY）， and Wenyang Jieyu prescription （WYJY） in improving depression and pain sensitivity. MethodThe birth date of mice was recorded as PD0. After birth， the mice were divided into a blank group and an experimental group. The neonatal mice in the experimental group underwent maternal separation in PD5-14 at 8 h·d-1. After ablactation， the mice were divided into a maternal separation group， a WY group （Erxian decoction， 5.84 g·kg-1）， a JY group （Xiaoyaosan， 12.00 g·kg-1）， a WYJY group （16.68 g·kg-1）， and a fluoxetine group （2.60 mg·kg-1）， with 15 mice in each group. Meanwhile， 15 male mice of the same age without maternal separation were assigned to the normal control group. Mice in the blank group and the maternal separation group were fed on a regular chow diet in PD21-PD90， while the remaining groups were fed on the corresponding drugs. In PD91， sciatic nerve ligation was performed to induce a model of maternal separation and chronic neuropathic pain. The open field test was used to observe the depression-like behaviors of mice in each group， and the mechanical and temperature pain thresholds were measured to detect the pain sensitivity of mice in each group. The serum levels of corticosterone （CORT）， substance P， and β-endorphin （β-EP） were determined by enzyme-linked immunosorbent assay （ELISA）， and the expression of the glucocorticoid receptor （GR） in the amygdala and β-EP protein in the hypothalamus was detected by immunohistochemistry. The mRNA expression levels of amygdala GR gene （Nr3c1）， FK506 binding protein 5 gene （FKBP5）， metabolic glutamate receptor 5 gene （GRM5）， and brain-derived neurotrophic factor （BDNF） were detected by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. ResultCompared with the blank group， the maternal separation group showed reduced stay time and total distance traveled in the 5-min open field test （P<0.01）， reduced mechanical pain threshold （P<0.01）， increased serum CORT and β-EP （P<0.01）， declining FKBP5 mRNA expression （P<0.01）， and increased hypothalamic β-EP expression （P<0.05）. Compared with the maternal separation group， the groups with drug intervention showed prolonged stay time （P<0.05， P<0.01） and up-regulated pain thresholds to different degrees. The total distance traveled in the 5-min open field test increased in the WY group， the WYJY group， and the fluoxetine group （P<0.05， P<0.01）. The JY group showed decreased serum CORT （P<0.01）， reduced β-EP ， and increased BDNF mRNA （P<0.01）. Nr3c1 and GRM5 mRNA decreased in the WY group （P<0.05， P<0.01）. The WYJY group showed decreased serum CORT （P<0.05）and decreased Nr3c1， GRM5， and BDNF mRNA （P<0.05， P<0.01）. The levels of β-EP expression were elevated to different degrees in the groups with drug intervention， but the differences were not significant. The levels of GR expression in the WY group， the JY group， and the WYJY group increased （P<0.05）. ConclusionWYJY can inhibit central pain sensitization and regulate hypothalamic-pituitary-adrenal gland （HPA） axis function by enhancing the expression of GR in the amygdala and inhibiting neuroplasticity and excitability in the amygdala to relieve depression-like behaviors and improve somatic hyperalgesia.

Changes of Microglia in Hippocampus of Mice Induced by Maternal Separation with Restraint Stress and Regulatory Effect of Wenyang Jieyu Prescription / 中国实验方剂学杂志

Objective:To observe the activation of microglia in hippocampus of depressed and anxious mice induced by maternal separation with acute restraint stress and the expression of interleukin-1<italic>β</italic>（IL-1<italic>β</italic>），interleukin-6（IL-6），tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）， investigating the mechanism of Wenyang Jieyu prescription in treating anxiety and depression. Method:Eighty four male C57BL offspring were randomly divided into control group， acute restraint stress group and model group on postnatal day 0（PD0）. Maternal separation combined with acute restraint stress was used to prepare anxious and depressed model mice， dividing the model mice into model group， Wenyang， Jieyu， Wenyang Jieyu and fluoxetine group according to random number table method. During the period of PD21-PD90， the control， acute restraint stress and model mice were fed with normal diet， with the other groups fed with corresponding medicine mixed diet. The Wenyang， Jieyu and Wenyang Jieyu groups were given 5.85， 12.03 and 16.71 g·kg^-1·d^-1 respectively. The fluoxetine group was given 2.60 mg·kg^-1·d^-1. Open field， zero maze test and social interaction tests were used to evaluate the anxiety and depression of model mice. The expression of Iba-1 in hippocampal microglia was detected by immunohistochemistry（IHC）. The mRNA expression of IL-1<italic>β</italic>， IL-6， TNF-<italic>α</italic>， Iba-1 and glucocorticoid receptor（GR）were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with the control group， total movement distance and time spent in central zone in 5 min of the model mice significantly decreased（<italic>P</italic><0.01）， time spent in opened arm and total movement distance decreased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）， investigation time during testing and training increased significantly（<italic>P</italic><0.01）. The expression of Iba-1 protein and mRNA，IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> mRNA significantly increased（<italic>P</italic><0.01）， the expression levels of GR mRNA significantly decreased（<italic>P</italic><0.01）. The result of IHC staining showed that microglia were over activated. Compared with the model group， total movement distance and time spent in central zone in 5 min of mice in the Wenyang Jieyu and fluoxetine group significantly increased（<italic>P</italic><0.05，<italic>P</italic><0.01）.Time spent in opened arm significantly increased（<italic>P</italic><0.01）. Investigation time during testing and training significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of Iba-1 protein and mRNA，IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> mRNA significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of GR mRNA increased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）. IHC staining showed the microglia recovered. Time spent in opened arm of mice in the Wenyang group and Jieyu group significantly increased （<italic>P</italic><0.01）， time spent investigating during testing decreased significantly （<italic>P</italic><0.05）， the expression levels of Iba-1 protein and mRNA，IL-6 mRNA significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of GR mRNA of mice in the Wenyang group significantly increased （<italic>P</italic><0.05）， the expression of TNF-<italic>α </italic>mRNA significantly decreased （<italic>P</italic><0.05）. Total movement distance of mice in the Jieyu group increased significantly （<italic>P</italic><0.01）， time spent investigating during training decreased significantly （<italic>P</italic><0.05），the expression level of IL-1<italic>β </italic>mRNA significantly decreased （<italic>P</italic><0.05）. IHC staining showed that microglia recovered partly in both groups. Conclusion:The comprehensive curative effect and pharmacological action of Wenyang Jieyu prescription were better than Wenyang prescription and Jieyu prescription. Wenyang Jieyu prescription can treat anxiety and depression in maternal separation and acute restraint stress mice， its possible mechanism may be related to the decreased activation of microglia， down-regulation of IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> expression and up-regulation of GR expression.

Activating mGluR8 Attenuates Visceral Hypersensitivity in Neonatal Maternally Separated Rats / 胃肠病学

Activating metabolite glutamate receptor 8 (mGluR8) has anti-hyperpathia effect in central nervous system, however, studies of effects in gastrointestinal tract are rare. Visceral hypersensitivity is one of the pathogenesis factors of irritable bowel syndrome (IBS). Aims: To investigate the effect and potential mechanism of activating mGluR8 on visceral hypersensitivity in neonatal maternally separated (NMS) rats. Methods: Twenty-four male newborn SD rats were randomly divided into normal control (NC) group, NMS group and mGluR8 agonist (S)-3, 4-DCPG group (3, 10 mg/kg). Newborn rats were subjected to 3 hours daily maternal separation on postnatal day 2-14 to establish the NMS model; in (S)-3, 4-DCPG group, (S)-3, 4-DCPG (3 or 10 mg/kg) were administered 1 hour prior to the visceral sensitivity test in NMS rats. Abdominal withdrawal reflex (AWR) score and abdominal electromyography (EMG) activity were used to measure visceral sensitivity. mGluR8 mRNA and protein expressions in colon mucosa were measured by RT-PCR and Western blotting, respectively; TNF-α, IL-1β and IL-6 mRNA expressions in colon mucosa were measured by RT-PCR. The protein expression of myeloperoxidase (MPO) was measured by immunohistochemistry. Results: AWR score and EMG activity in NMS group were significantly higher than those in NC group under different colorectal distension (CRD) pressure. AWR score and EMG activity were significantly decreased in (S)-3, 4-DCPG group. mGluR8 mRNA and protein expressions in NMS group were significantly higher than those in NC group (P<0.05). Compared with NMS group, TNF-α mRNA expression was significantly decreased in 3 mg/kg (S)-3, 4-DCPG group (P<0.05), and MPO protein expression was significantly decreased in 10 mg/kg (S)-3, 4-DCPG group (P<0.05). Conclusions: Activating mGluR8 attenuates visceral hypersensitivity in NMS rats, the mechanism may be related to decrease of pro-inflammatory cytokine TNF-α.

Proteome profiling of different rat brain regions reveals the modulatory effect of prolonged maternal separation on proteins involved in cell death-related processes

BACKGROUND: Early-life stress in the form of maternal separation can be associated with alterations in offspring neurodevelopment and brain functioning. Here, we aimed to investigate the potential impact of prolonged maternal separation on proteomic profiling of prefrontal cortex, hippocampus and cerebellum of juvenile and young adult rats. A special attention was devoted to proteins involved in the process of cell death and redox state maintenance. METHODS: Long-Evans pups were separated from their mothers for 3 h daily over the first 3 weeks of life (during days 2-21 of age). Brain tissue samples collected from juvenile (22-day-old) and young adult (90-day-old) rats were used for label-free quantitative (LFQ) proteomic analysis. In parallel, selected oxidative stress markers and apoptosis-related proteins were assessed biochemically and by Western blot, respectively. RESULTS: In total, 5526 proteins were detected in our proteomic analysis of rat brain tissue. Approximately one tenth of them (586 proteins) represented those involved in cell death processes or regulation of oxidative stress balance. Prolonged maternal separation caused changes in less than half of these proteins (271). The observed alterations in protein expression levels were age-, sex- and brain region-dependent. Interestingly, the proteins detected by mass spectrometry that are known to be involved in the maintenance of redox state were not markedly altered. Accordingly, we did not observe any significant differences between selected oxidative stress markers, such as the levels of hydrogen peroxide, reduced glutathione, protein carbonylation and lipid peroxidation in brain samples from rats that underwent maternal separation and from the corresponding controls. On the other hand, a number of changes were found in cell death-associated proteins, mainly in those involved in the apoptotic and autophagic pathways. However, there were no detectable alterations in the levels of cleaved products of caspases or Bcl-2 family members. Taken together, these data indicate that the apoptotic and autophagic cell death pathways were not activated by maternal separation either in adolescent or young adult rats. CONCLUSIONS: Prolonged maternal separation can distinctly modulate expression profiles of proteins associated with cell death pathways in prefrontal cortex, hippocampus and cerebellum of juvenile rats and the consequences of early-life stress may last into adulthood and likely participate in variations in stress reactivity.

Disorder of intestinal amino acid pathway in depression-like offspring rats induced by maternal separation / 浙江大学学报·医学版

To investigate the intestinal amino acids pathway in depression-like offspring rats induced by maternal separation. Sprague-Dawley (SD) female rats were randomly divided into a control group (=8) and a maternal separation group (=8). After normal delivery, the maternal rats were separated from offsprings for 14 consecutive days and 3 h per day in maternal separation group; while rats in the control group was received no interventions in postpartum. Depression-like behaviors of offspring rats were evaluated using the sucrose preference test, novelty suppressed feeding test, and forced swimming test. Amino acid analyzer was used to detect the changes of amino acid contents in the small intestine, and the expressions of alanine-serine-cysteine transporter 2 (ASCT2), solute carrier superfamily 6 member 19 (BAT1) and L-type amino acid transporter 1(LAT1) were detected by Western blot. The weight of the offspring rats in the maternal separation group was significantly lower than that of the control group at 21 and 28 d (=4.925 and 5.766, all <0.01). Compared with the control group, the percentage of sucrose preference of the offspring rats in the maternal separation group was significantly reduced (=2.709, <0.05), and the feeding latency was significantly prolonged (=-13.431, <0.01). The immobility time in FST of maternal separation group was significantly longer (=-3.616, <0.01).Increased concentration of aspartic acid (=-6.672, <0.01) and down-regulation of glutamine (=3.107, <0.01) and glycine (=9.781, <0.01) were observed in maternal separation group. Western blot analysis revealed that the protein expressions of ASCT2 (=6.734, <0.01) and BAT1 (=9.015, <0.01) in maternal separation group were reduced, while the expression of LAT1 was increased (=-8.942, <0.01). Maternal separation can induce the depression-like behavior in offspring rats; the amino acid contents and the amino acid transporter expression in the small intestine are reduced, which may be related to depression-like behavior induced by maternal separation.

Activation of corticotropin-releasing hormone system in the locus ceruleus induces the visceral sensitivity of irritable bowel syndrome rats / 中华消化杂志

Objective@#To study the role of corticotropin-releasing hormone (CRH) system in locus ceruleus (LC) in irritable bowel syndrome (IBS) and to explore its molecular mechanism.@*Methods@#The IBS rat was established by maternal separation following with postnatal stress. The tissues sample of LC was obtained by micropunched nuclei. The expression of c-Fos, CRH and its receptors including corticotropin-releasing hormone receptor (CRHR) 1 and CRHR2 of rats’ LC tissues of control group and IBS group was detected by quantitative polymerase chain reaction(PCR). The expression of DNA methyltransferase (DNTM) 1, DNMT3a and DNMT3b at the mRNA level were also measured. In addition, the expression of histone methyltransferase ASH2-like protein (ASH2L) and SET and MYND domain containing 2 (SMYD2) was determined by Western blotting. T test was used for statistical analysis.@*Results@#The rectal pneumatic pressure of IBS group was lower than that of control group ((69.82±5.47) mmHg vs. (86.86±5.98) mmHg; 1 mmHg=0.133 kPa), however compared with that of control group, the expression of c-Fos at the mRNA level increased (2.11±0.44 vs.1.00±0.19), and the differences were statistically significant (t=6.215 and 2.321, P<0.01 and 0.05). In addition, compared with that of control group, the expression of CRH at the mRNA level increased (1.99±0.35 vs.1.00±0.13), and the difference was statistically significant (t= 2.797, P<0.05). Compared with that of control group, the expression of SMYD2 at the protein level up-regulated (1.04±0.21 vs. 0.61±0.12), and the difference was statistically significant (t=4.451, P<0.01). However, there were no statistically significant differences in the expression of CRHR-1, CRHR2, DNMT1, DNMT3a, DNMT3b at the mRNA level, and the expression of ASH2L between IBS group and control group (0.96±0.13 vs. 1.00±0.26, 1.35±0.63 vs. 1.00±0.43, 1.40±0.61 vs.1.00±0.19, 1.39±0.58 vs. 1.00±0.21, 1.45±0.71vs.1.00±0.39 and 0.80±0.19 vs. 1.05±0.26, respectively; all P>0.05).@*Conclusions@#Maternal separation combined with postnatal stress affect the transcription of Crh gene in LC and cause the activation of the stress regulation network CRH and norepinephrine system, resulting in the increase of the visceral sensitivity of rats. The abnormal transcription of Crh gene may be related with SMYD2-mediated histone H3K36 methylation, but not related with the modification of DNA methylation.

Chronic administration of ketamine ameliorates the anxiety- and aggressive-like behavior in adolescent mice induced by neonatal maternal separation

Ketamine has long been used as an anesthetic agent. However, ketamine use is associated with numerous side effects, including flashbacks, amnesia, delirium, and aggressive or violent behavior. Ketamine has also been abused as a cocktail with ecstasy, cocaine, and methamphetamine. Several studies have investigated therapeutic applications of ketamine, demonstrating its antidepressant and anxiolytic effects in both humans and rodents. We recently reported that neonatal maternal separation causes enhanced anxiety- and aggressive-like behaviors in adolescent. In the present study, we evaluated how acute and chronic ketamine administration affected the behavioral consequences of neonatal maternal separation in adolescent mice. Litters were separated from dams for 4 hours per day for 19 days beginning after weaning. Upon reaching adolescence (post-natal day 35–49), mice were acutely (single injection) or chronically (7 daily injections) treated with a sub-anesthetic dose (15 mg/kg) of ketamine. At least 1 h after administration of ketamine, mice were subjected to open-field, elevated-plus maze, and resident-intruder tests. We found that acute ketamine treatment reduced locomotor activity. In contrast, chronic ketamine treatment decreased anxiety, as evidenced by increased time spent on open arms in the elevated-plus maze, and remarkably reduced the number and duration of attacks. In conclusion, the present study suggests that ketamine has potential for the treatment of anxiety and aggressive or violent behaviors.

Early maternal separation induces cognitive deficits in adult rats via the dysregulation of hippocampal neuronal nitric oxide synthase expression / 中国神经精神疾病杂志

Objective To investigate the effect of maternal separation (MS) on cognitive function in adult male rats through the expression of neuronal nitric oxide synthase (nNOS) in hippocampus, and to reveal the roles of early life stress (ELS) on neural development in rats. Methods Healthy SD pregnant rats (n=12) were randomly divided into maternal separation group (MS group) and control group (NMS group) (n=6 for each group). The newborn rats in the MS group were separated from the mother rats for 3 h every day from postnatal day 3 to 22 whereas no intervention was taken in the NMS group. At the age of 10 weeks, Morris water maze was used to test the learning and memory abilities of two groups of offspring male rats. Neuron immunofluorescence staining was used to examine the number and distribution of neurons in dentate gyrus (DG) of two groups of offspring male rats. Western Blot method was used to detect nNOS, eNOS, Bax/BCL2, Caspase-3 and P53 levels in the hippocampus of the two groups. Ki67/DCX immunofluorescence staining were used to examine the proliferation and differentiation of neurons in the DG area of the hippocampus. TUNEL staining was used to detect the neuronal degeneration and death in the DG area of the hippocampus. Results Behavioral tests showed that the escape latency of male rats in MS group was prolonged, the target quadrant residence time and the number of platform crossing decreased (P<0.05) compared with NMS group. Compared with NMS group, the number of normal and degenerated neurons in hippocampal DG area of MS group had no significant change (P＞0.05). However, the expression of nNOS and eNOS in hippocampus was decreased (P<0.05) and the expression of Bax/BCL2 was increased (P<0.05), but the expression of caspase-3 and P53 remained unchanged (P＞0.05). In addition, Neuronal proliferation and differentiation were decreased and apoptosis was increased in MS group (P<0.05). Conclusion Repeated MS reduces the expression levels of nNOS in the hippocampus, affects the neuronal function in the DG area, and has a long-term influence on the neurodevelopment, which results in cognitive deficits related to learning and memory abilities in adult rats.

Effect of maternal separation stress on behavior of neonatal rd mice / 中国比较医学杂志

Objective To evaluate the effect of maternal separation stress on the behavior of neonatal rd mice.Methods Neonatal rd mice were divided into maternal separation (MS) group (n=9) and control group (n=9).MS-stress was induced in the MS group by 4-hour-separation per day for 28 days.Open field test,elevated plus maze test,forced swim test and tail suspension test were used to evaluate the anxiety-like and depression-like behavior of the neonatal rd mice.Results The stay time and distance travelled of MS group in the central zone were 0.88％ and 28.17±5.65 cm,respectively,significantly shorter than that of the control group (2.61％,109.9±9.79 cm.P =0.04,P =0.001).Compared with the control group,the stay time in open arms of the MS group was significantly decreased (P<0.01),while the immobility time in forced swim test and tail suspension test of the MS group were 126.5±10.22 s and 21.56±6.83 s,significantly longer than that of the control group (77.75±16.83 s,P =0.02,7.37±3.22 s,P =0.03).Conclutions The 28-day maternal separation stress can significantly increase the anxiety-like and depression-like behavior in neonatal rd mice.

Maternal Separation Does Not Produce a Significant Behavioral Change in Mice

Early life adversities together with genetic predispositions have been associated with elevated risks of neuropsychiatric disorders during later life. In order to investigate the underlying mechanisms, many chronic, early-life stress paradigms in multiple animal models have been developed. Previously, studies reported that maternal separation (MS) in the early postnatal stages triggers depression-and/or anxiety-like behaviors in rats. However, similar studies using mice have reported inconsistent behavioral outcomes. In this study, we sought to assess behavioral outcomes from two different early-life stress paradigms; a conventional 3-hour MS and a maternal separation with early weaning (MSEW) paradigm using C57BL/6J male mice with independent cohorts. Our data demonstrated that both MS and MSEW paradigms did not produce reported behavioral anomalies. Therefore, MS paradigms in mice require further validation and modification.

Acupuncture alters pro-inflammatory cytokines in the plasma of maternally separated rat pups / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the potential alleviating effects of acupuncture on maternal separation (MS)-induced changes in plasma pro-inflammatory cytokine levels of rat pups.</p><p><b>METHODS</b>On postnatal day 15, rat pups were randomly assigned to 4 groups (n=6 per group) using a random number table: normal, MS, MS with acupuncture stimulation at Shenmen (HT 7) acupoint (MS+HT 7), and MS with acupuncture stimulation at Chengshan (BL 57) acupoint (MS+BL 57) groups. Rat pups in the normal group were housed with their mothers under standard conditions; those in the MS, MS+HT 7 and MS+BL 57 groups were maternally separated and individually maintained. Acupuncture stimulation was performed at HT 7 or BL 57 acupoints once a day for 7 consecutive days. A tail suspension test was performed to measure immobility time of rats and the plasma was collected on postnatal day 21, then levels of corticosterone (CORT), interleukin (IL)-1β, IL-6 and glial cell-derived neurotrophic factor (GDNF) in plasma were measured.</p><p><b>RESULTS</b>Compared with the normal group, the immobility time and the plasma levels of CORT, IL-1β, IL-6 and GDNF in the MS group were significantly increased (P<0.05 or P<0.01). Compared with the MS group, the immobility time and the plasma levels of CORT, IL-1β, IL-6 and GDNF were significantly reduced in the MS+HT 7 group (P<0.05 or P<0.01). Moreover, the immobility time and plasma levels of IL-1β and IL-6 in the MS+HT 7 group were significantly lower than those in the MS+BL 57 group (P<0.05).</p><p><b>CONCLUSION</b>Acupuncture stimulation at HT 7 can alleviate the behavioral impairment and changes of the cytokines by MS, indicating that acupuncture can help to relieve MS-induced depression.</p>

Review of the application of neonatal maternal separation model in irritable bowel syndrome (IBS) studies / 中国实验动物学报

The neonatal maternal separation ( NMS) model has been ubiquitously used in studies of irritable bowel syndrome ( IBS) ,which mimics the disease characterized by visceral hypersensitivity, and GI dismotility and mental disor-der.This minireview focuses on the features and certain pathological mechanism of the animal models.

Efecto de la alopregnanolona sobre la ansiedad en ratas con separación materna durante la lactancia / The effect of allopregnanolone on anxiety induced in rats separated from their young during breastfeeding

Antecedentes. La relación madre-hijo y el cuidado parental al recién nacido son fundamentales en el desarrollo fisiológico y emocional de los individuos. Evidencias asocian el estrés temprano con el desarrollo de enfermedades mentales. El modelo de separación materna durante la lactancia (SMDL) se ha utilizado para inducir estrés temprano en ratas y estudiar efectos a largo plazo. Diversos estudios han encontrado que, en ratas separadas de sus madres, disminuyen los niveles del receptor GABA-A y esos bajos niveles están asociados a comportamientos ansiosos. Objetivo. Evaluar el efecto de la alopregnanolona, un neuroesteroide agonista del receptor GABA-A, sobre la ansiedad inducida por SMDL. Materiales y métodos. Se utilizaron 30 ratas Wistar dividas en dos grupos, uno control y uno experimental (SMDL). La SMDL se realizó desde el día postnatal 1 hasta el día postnatal 21, durante 180 minutos en la mañana y 180 minutos en la tarde. Desde el día 22 los sujetos se alojaron en cajas por sexo y tratamiento y continuaron su desarrollo normal hasta el día 60, en el que se hizo la inyección con alopregnanolona y la prueba comportamental en el laberinto en cruz elevado. Resultados. El estrés crónico causado por la SMDL afecta el comportamiento de los individuos, perfil comportamental que varía dependiendo del sexo. Se encontró que los machos presentan comportamientos más ansiosos que las hembras, las cuales a su vez muestran más actividad locomotora y exploración Conclusiones. Al aplicar alopregnanolona el repertorio comportamental varía en los animales con SMDL; estos resultados sugieren que la alopregnanolona, a través de su unión al receptor GABA-A, puede llegar a revertir los efectos de la separación materna, sobre los comportamientos relacionados con ansiedad.

Background. The mother-child relationship and parental care for the newborn are fundamental in individuals' physiological and emotional development. Evidence-based research associates early stress with the development of mental illnesses. Maternal separation during lactation (MSDL) models have been used to induce early stress in rats and for studying the long-term effects of such intervention. Several studies have found decreased GABA-A receptor levels in separated rats from their mothers and such low levels have been associated with anxious behaviour. Objective. Assessing the effect of allopregnanolone (a GABA-A receptor neurosteroid agonist) on MSDL-induced anxiety. Materials and methods. Thirty Wistar rats were divided into two groups: control and experimental (MSDL). SMDL occurred for 180 minutes in the morning and 180 minutes in the afternoon. Subjects were housed in boxes by gender and treatment following day 22 and their normal development was allowed to continue until day 60 when they were injected with allopregnanolone and underwent a behavioural test in an elevated plus maze (EPM). Results. Chronic stress induced by MSDL affected individuals' behaviour, their behavioural profile varying according to their gender. Males exhibited more anxious behaviour than females who engaged in more locomotive and exploratory activity. Conclusions. MSDL animals' behavioural repertoire varied due to the allopregnanolone injection, suggesting that the effect of allopregnanolone due to GABA-A receptor interaction could reverse the effects of maternal separation on anxiety-related behaviour.

Establishment and assessment of the diarrhea rat model of liver-QI stagnation with spleen deficiency / 中国实验动物学报

Objective To establish a diarrhea rat model using multiple-stimulating factors and choosing the best indexes to assess whether the model is consistent with the disease characteristics of liver -QI stagnation with spleen deficien-cy in traditional Chinese medicine .Methods Newborn SD rats were randomly divided into model group ( n=20 ) and control group (n=10).The rats of model group were stimulated by maternal separation , restraint stress and rectum acetic acid irritation, while the rats in control group were fed as normal .Weight changes, rectal sensitivity, Bristol scores and wa-ter content of feces and histology of the colon tissues were used as evaluation indexes to assess whether the model meets the demands for further studies .Results The rats in the model group showed loss of appetite , increase of water intake and u-rine reduction .Some rats showed increased activity , and even mania .Bristol scores and water content of feces were signifi-cantly higher than that of the control group , and the rectal sensitivity was significantly increased .The colon mucosa showed slightly thickened submucosal layer and mild inflammatory cell infiltration in the model rats .Conclusions The rat model established in this study is better to simulate the clinical manifestation of liver -QI stagnation with spleen deficiency in Chi-nese medicine , and may meet the demands of related researches of this disease .

Early repeated maternal separation induces alterations of hippocampus reelin expression in rats.

The long-term effects of repeated maternal separation (MS) during early postnatal life on reelin expression in the hippocampus of developing rats were investigated in the present study. MS was carried out by separating Wistar rat pups singly from their mothers for 3 h a day during postnatal days (PND) 2–14. Reelin mRNA and protein levels in the hippocampus were determined using qRT-PCR and Western blotting, at PND 22, PND 60 and PND 90. MS resulted in the loss of body weight in the developing rats, and reelin mRNA and protein levels in the hippocampus generally were down-regulated over the developing period, but the reelin mRNA and protein levels in the hippocampus of 90-day-old male rats were up-regulated. These findings suggest that the long-term effects of MS on the expression levels of hippocampal reelin mRNA and protein depends on the age at which the stressed rats’ brains were collected; reelin had important implications for the maternal-neonate interaction needed for normal brain development. In conclusion, repeated MS occurring during early postnatal life may cause the alterations of hippocampal reelin expression with the increasing age of developing rats.

Down-regulation of Caveolin-l, GFAP, BDNF expression in hippocampus of neonatal rats induced by maternal sepa-ration / 临床儿科杂志

Objective To explore the potential mechanism of abnormal behavior resulted from maternal separation in neo-natal period in rat. Methods Neonatal rats were equally and randomly divided into maternal separation group and control group. The rats in maternal separation group were separated from the dam for 3h per day on postnatal days (PND) 2 to 21, nothing was done to the rats in the control group. The brain tissues were taken out after being killed on PND 7, 14, and 21. The expressions of Caveolin-l, BDNF and GFAP in hippocampal formation were detected by immunohistochemistry. Semiquantitative assessment of immunohistochemical images was performed by Image-Pro Plus software. Results Compared with control groups, the expres-sion of Caveolin-l on PND 7 had no signiifcant change, while BDNF and GFAP were signiifcantly increased in maternal separa-tion group (P<0.05). On PND 14 and 21, the expressions of Caveolin-l, BDNF and GFAP were signiifcantly decreased in maternal separation group (P<0.05). Conclusions Decreased expressions of Caveolin-l, BDNF and GFAP caused by maternal separation in neonatal period may be associated with abnormal behaviors in adulthood in rat.

Maternal separation during breastfeeding induces changes in the number of cells immunolabeled to GFAP

The mother-child relationship is fundamental to the establishment and maintenance of synaptic networks and physiological and emotional development. Animal models including maternal separation have been used to study changes at behavioral and neurobiochemical levels. Due to the relevance of glial cells during development, our aim was to determine if short periods of maternal separation during breastfeeding induce permanent changes in a number of astrocytes labeled with the glial fibrillary acidic protein in different brain areas. Wistar rats were housed under standard laboratory conditions with reversed light/dark cycle; food and water ad libitum. Pups were separated from their mothers for 6 h daily during breastfeeding period. On day 22, pups were separately housed according to gender and treatment. At day 60, subjects were evaluated in the elevated plus maze and, after processing for immunohistochemistry, 20-μm sections were made. Prefrontal cortex, paraventricular nucleus, preoptic area, hippocampus and amygdala were localized. Labeled cells were quantified using Image-J program. Results showed that separated females had more entries into open arms and spend more time as compared with the control groups. In the prefrontal cortex we identified a decrease in staining cells in separated females, whereas there was an increase in staining cells in separated males. In the hippocampus and preoptic area, we observed a decrease only in separated males. We did not find any differences in the paraventricular nucleus or amygdala. Our results indicate that maternal separation during breastfeeding induces permanent changes in the number of astrocytes in different brain areas of both males and females.

Efectos de la spparación materna temprana sobreel desempeño en el laberinto enn cruz elevado en ratas adultas / Effects of Early Maternal Separation on The Performance in the Elevated Plus Maze in Adult Rats

Se ha demostrado que alteraciones en la interacción madre-cría produce efectos duraderos sobre el desarrollo cerebral y comportamental; de esta forma sujetos expuestos a estrés por separación materna temprana (SMT) presentan variaciones en los comportamientos indicadores de ansiedad. El objetivo del presente estudio fue investigar los efectos específicos del estrés por SMT sobre los comportamientos indicadores de ansiedad en ratas Wistar adultas machos y hembras. Las ratas fueron anidadas con ciclo luz/oscuridad invertido (7 p.m./7 a.m.), agua y comida ad libitum. El procedimiento de separación se realizó dos veces diarias durante los días postnatales 1 al 21 (7:00 a 10:00 y 13:00 a 16:00 p.m.). Los comportamientos indicadores de ansiedad fueron evaluados por medio del laberinto en cruz elevado (LCE) cuando las crías alcanzaron un peso de 230 g. Se encontró que el estrés por SMT tiene efectos específicos para cada sexo sobre los comportamientos relacionados con la ansiedad, las hembras separadas maternalmente presentaron un perfil ansioso menor que las no separadas y los machos separados mostraron mayor conflicto exploración/evitación. Estos resultados corroboran hallazgos preliminares de nuestro laboratorio, en los que se evidencia interacción entre la vulnerabilidad ante desafíos ambientales tempranos y los mecanismos compensatorios del cuidado materno.

It has been demonstrated that disruption of mother-pup interaction during early life exerts long-lasting effects on the brain and behavioral development. Therefore subjects exposed to early maternal separation stress (MS) show variations in anxiety-like behaviors. The aim of this study was to investigate the specific effects of SMT stress on anxiety-like behaviors in adult male and female Wistar rats. Rats were housed with reversed light dark cycle (light on at 7 p.m., off at 7 a.m.), water and food ad libitum. Separation was carried out in postnatal days 1 to 21, twice daily in dark cycle (7:00 a 10:00 y 13:00 a 16:00 p.m.). The anxiety-like behaviors were tested through the elevated plus maze (EPM) when the pups reached 230 g of weigh. We found that the MS stress has sex specific effects on anxiety-like behaviors: the maternal separated females displayed a lesser anxious outline than the not separated ones and the separated males showed a large exploration/avoidance conflict. These results confirm previous effects of our labs, which may be related to an interaction between vulnerability to environmental challenge and maternal care compensatory behaviors.

Effect of maternal deprivation on emotion and expression of dopamine transporter in adult rats / 中南大学学报(医学版)

Objective To investigate the change of emotion and striatum dopamine transporter(DAT) expression in adult male rats experiencing maternal deprivation, and to explore whether DNA methylation is involved in the regulatory mechanism of DAT expression. Methods Newborn rats were randomly divided into 2 groups: a maternal separation group (n=16) and a control group (n=14). The maternal deprivation group were separated from their mother for 6 hours (09∶00-15∶00) per day from postnatal day 1 to 14, while the controls (n=14) without the deprivation. When the rats in the 2 groups were 12 week, their spontaneous anxiety levels and exploratory ability in novel environments were assessed by an elevated plus maze and an open field test. DAT mRNA expression in the striatum was detected by reverse transcription-PCR, and its DNA methylation level was measured by bisulfated DNA sequencing. Results Maternally-deprived rats showed lower ability of exploring in a new environment and lower levels of anxiety than the controls. The expression of DAT mRNA in the striatum of the maternal separation group (0.236±0.043) was significantly lower than that in the control group (0.480±0.107) (P＜0.05). However the DNA methylation level in the promoter region of DAT was not significantly different between the 2 groups. Conclusion Maternal deprivation influenced the emotion and expression of dopamine transporter in adult rats and DNA methylation may not be involved.