Immobilization Research Articles

Remediation effect and mechanism of immobilized laccase on actual trichloromethane-contaminated soil samples

Trichloromethane (TCM) in soil causes adverse effects on the soil ecosystem and human health. Therefore, it is urgent to develop an efficient and low-cost soil TCM removal technology. In this study, we prepared sodium alginate-biochar-laccase immobilized pellets (SA-BC-LC) using composite immobilization technology, exploring the fitting impact of artificial neural network model (ANN) and general linear model (GLM) models on laccase loading and the correlation properties of SA-BC-LC. The SA-BC-LC exhibited a porous multi-layer network structure internally. Compared to free laccase, the relative activity of SA-BC-LC remained above 50 % after 5 reuses and still maintained at 48 % after 50 days of storage. Moreover, SA-BC-LC demonstrated significant capacity for adsorption and degradation of TCM in soil with ratios approximately at 31.3 % and 68.7 %, respectively. During the remediation process of TCM-contaminated soil from Taizhou Chemical plant, there was a rapid decrease in TCM concentration within the first hour. Under IM (Immobilization) treatment (added with 5 % SA-BC-LC), the removal rate of TCM reached up to 88.9 % in the first hour. The adsorption of TCM by SA-BC-LC was mainly chemical adsorption, which induced the improvement of the relative abundance of the co-metabolic dechlorinated microbial Pseudomonas. Our research presents a promising material for effectively remediating soils contaminated with chlorinated hydrocarbons (CHs), with excellent removal efficiency.

Role of gut-derived bacterial lipopolysaccharide and peripheral TLR4 in immobilization stress-induced itch aggravation in a mouse model of atopic dermatitis

Psychological stress and intestinal leakage are key factors in atopic dermatitis (AD) recurrence and exacerbation. Here, we demonstrate the mechanism underlying bacterial translocation across intestinal epithelial barrier damaged due to stress and further aggravation of trimellitic anhydride (TMA)–induced itch, which remain unclear, in AD mice. Immobilization (IMO) stress exacerbated scratching bouts and colon histological damage, and increased serum corticosterone and lipopolysaccharide (LPS). Orally administered fluorescein isothiocyanate (FITC)-dextran and surgically injected (into the colon) Cy5.5-conjugated LPS were detected in the serum and skin after IMO stress, respectively. The relative abundance of aerobic or facultative anaerobic bacteria was increased in the colon mucus layer, and Lactobacillus murinus, E. coli, Staphylococcus nepalensis, and several strains of Bacillus sp. were isolated from the spleens and mesenteric lymph nodes. Oral antibiotics or intestinal permeability blockers, such as lubiprostone (Lu), 2,4,6-triaminopyrimidine (TAP) and ML-7, inhibited IMO stress-associated itch; however, it was reinduced through intradermal or i.p. injection of LPS without IMO stress. I.p. injection of TAK-242 (resatorvid), a TLR4 inhibitor, abrogated IMO stress-associated itch, which was also confirmed in TLR4-KO mice. IMO stress alone did not cause itch in naïve mice. IMO stress-induced itch aggravation in TMA-treated AD mice might be attributed to the translocation of gut-derived bacterial cells and LPS, which activates peripheral TLR4 signaling.

Effects of two pharmaceuticals: Doxycycline and norfloxacin on plant and animal organisms living in the freshwater

The main reason for the appearance of pharmaceutical compounds in the aquatic environment is their regular excretion by humans and animals in an unchanged form or slightly metabolized. Pharmaceuticals limit the habitable living environment for aquatic organisms, because they can be toxic not only to bacteria but also to non-target organisms. Plants of the Lemnoideae subfamily and crustaceans Daphniindae family are widely used as bioindicators in freshwater environmental risk assessments. This study aimed to use biotests (Lemna test and Daphtoxkit) to determine the effect of two pharmaceuticals: antibiotic – Doxycycline (DOX) and semi-synthetic chemotherapeutic drug – Norfloxacin (NOR) on plants Lemna minor and crustaceans Daphnia magna. Standard Lemna test was extended to include pharmaceutical effects on plant chlorophyll content (LCC) and fluorescence (Fo, Fm, and Fv/Fm), and the confirmation of drug toxicity was the biotest Daphtoxkit assessing the immobilization (IM) of organisms. Studies have shown that DOX was more toxic than NOR on tested aquatic organisms: plants and crustaceans. The lowest observed effect concentration (EC20) of DOX and NOR reduced by 20 % LCC and Iy of L. minor was 2.14 and 8.11 mg × L−1, respectively. The LCC was an early and sensitive indicator of the phytotoxic effects of DOX in L. minor before morphological changes were observed. Confirmation of drug toxicity was the Daphtoxkit. The EC20 (180. min) of DOX and NOR, IM of daphnia by 20 % was 117.18 and 215.42 mg × L−1, respectively. In conclusion, DOX and NOR in aquatic environments may have significant implications for tested organisms and their ecosystems.

Histological and functional assessment of a Takotsubo cardiomyopathy model established by immobilization stress.

Takotsubo cardiomyopathy (TTC), also known as stress-induced cardiomyopathy, resembles acute heart failure syndrome but lacks disease-specific diagnosis and treatment strategies. TTC accounts for approximately 5-6% of all suspected cases of acute coronary syndrome in women. At present, animal models of TTC are often created by large amounts of exogenous catecholamines such as isoproterenol. However, isoproterenol injection cannot fully simulate the onset of stress-induced cardiomyopathy in humans since stress is not an instantaneous event. Rats were immobilized for 6h per day for 1-14 days. To examine whether the TTC model was successful, echocardiography was employed; Elisa detected serum sympathetic activation markers; and the Open-Field test (OFT) was used to analyze behavioral changes in rats after stress. Western blot and histology were used to assess sympathetic remodeling, inflammation levels, and fibrosis; qRT-PCR was used to explore the levels of fibrosis and myocardial hypertrophy. The electrical stability of ventricular was determined by electrophysiological testing. The rats showed severe stress behavior and local sympathetic remodeling of the heart after only 1 day of stress. After 3 days of stress, the induction of ventricular tachyarrhythmia increased prominently. The highest incidence of TTC in rats was at 5 days of immobilization stress. The pathological left ventricular remodeling caused by immobilization (IMO) stress includes inflammatory infiltration, fibrosis, and myocardial hypertrophy. Our study confirms the hypothesis that IMO stress can mimic Takotsubo cardiomyopathy, and the various effects on the heart depending on the duration of IMO stress. We observed the highest incidence of TTC occurred after 5 days of stress. Furthermore, there is a gradual occurrence of electrical and structural remodeling as the stress duration prolongs.

Combined orchiectomy and limb immobilization recapitulate early age‐related changes to skeletal muscle in mice

AbstractBackgroundMuscle mass and function decline in middle age, ultimately resulting in sarcopenia in the elderly and poor health outcomes, reducing quality of life. There is a lack of cost‐ and time‐effective murine models that recapitulate the physiological changes associated with muscle mass decline to study possible interventions to delay sarcopenia. We aimed to evaluate the effectiveness of combining orchiectomy (ORC) surgery to simulate age‐related androgen decline and hindlimb immobilization (IM) in inducing age‐related skeletal muscle changes.MethodsFour‐month‐old male C57BL/6J mice (n = 10) were subjected to ORC, followed by IM (right hindlimb casting) for 14 days. Upon completion of the casting period, ex vivo muscle contractile function, histology, and various mitochondrial markers were assessed, and results were compared with age‐matched controls (CON; n = 8) and middle‐aged (MA; 12 ± 1 months, n = 9) animals.ResultsIM combined with ORC induced a 30%–40% decrease in muscle mass across multiple hindlimb muscles (P < 0.0001), with the magnitude of muscle loss comparable with the MA group when corrected for body weight (P < 0.0001). In the IM limb of ORC mice, soleus muscle force significantly decreased when compared with the contralateral limb (P < 0.05) and aged‐matched CON group (P < 0.05). The decrements in muscle force and mass present in the IM limb of ORC mice were accompanied by a 70% reduction in the expression of the muscle structural protein dystrophin and various mitochondrial markers, including cytochrome C (−55%), peroxisome proliferator‐activated receptor gamma co‐activator 1‐beta (PGC1‐β) (−49%), and cytochrome oxidase IV (COX‐IV) (−73%) when compared with CON animals (P < 0.001). Lastly, our model also demonstrated specific fibre‐type shifts in fast‐ and slow‐twitch muscles, which mimicked changes in the MA group.ConclusionsApplying treatments during IM could target acute muscle atrophy in MA adults, while applying them following cast removal in a low‐testosterone environment could represent a window for rehabilitation therapeutics.

The effect of chronic stress on the immunogenicity and immunoprotection of the M6-TT vaccine in female mice

Active vaccination is an effective therapeutic option to reduce the reinforcing effects of opioids. Several studies showed that chronic stress affects the immune system decreasing the efficiency of some vaccines. Heroin withdrawal is a stressor and it is a stage in which the patient who abuses heroin is vulnerable to stress affects the immune response and consequently its immunoprotective capacity, then, the objective was to determine the effect of heroin-withdrawal and heroin-withdrawal plus immobilization, on the immune (immunogenicity) and protective response (behavioral response) of morphine-6-hemisuccinate-tetanus toxoid (M6-TT) vaccine in animals of two inbred mice strains with different sensitivity to drug-opioid and stress.Female BALB/c and C57Bl/6 inbred mice were immunized with the M6-TT. A solid-phase antibody-capture ELISA was used to monitor antibody titer responses after each booster dose in vaccinated animals. During the vaccination period, the animals were subjected to two different stress conditions: drug-withdrawal (DW) and immobilization (IMM). The study used tail-flick testing to evaluate the heroin-induced antinociceptive effects. Additionally, heroin-induced locomotor activity was evaluated. Stress decreased the heroin-specific antibody titer generated by the M6-TT vaccine in the two inbred mouse strains evaluated. In the two stress conditions, the antibody titer was not able to decrease the heroin-induced antinociceptive effects and locomotor activity. These findings suggest that stress decreases the production of antibodies and the immunoprotective capacity of the M6-TT vaccine. This observation is important to determine the efficacy of active vaccination as a potential therapy for patients with opioid drug use disorder, since these patients during drug-withdrawal present stress disorders, which could affect the efficacy of therapy such as active vaccination.

Atenolol Ameliorates Skeletal Muscle Atrophy and Oxidative Stress Induced by Cast Immobilization in Rats.

(1) Background: Skeletal muscle atrophy is a common and debilitating condition associated with disease, bed rest, and inactivity. We aimed to investigate the effect of atenolol (ATN) on cast immobilization (IM)-induced skeletal muscle loss. (2) Methods: Eighteen male albino Wistar rats were divided into three groups: a control group, an IM group (14 days), and an IM+ATN group (10 mg/kg, orally for 14 days). After the last dose of atenolol, forced swimming test, rotarod test, and footprint analysis were performed, and skeletal muscle loss was determined. Animals were then sacrificed. Serum and gastrocnemius (GN) muscles were then collected, serum creatinine, GN muscle antioxidant, and oxidative stress levels were determined, and histopathology and 1H NMR profiling of serum metabolites were performed. (3) Results: Atenolol significantly prevented immobilization-induced changes in creatinine, antioxidant, and oxidative stress levels. Furthermore, GN muscle histology results showed that atenolol significantly increased cross-sectional muscle area and Feret's diameter. Metabolomics profiling showed that glutamine-to-glucose ratio and pyruvate, succinate, valine, citrate, leucine, isoleucine, phenylalanine, acetone, serine, and 3-hydroxybutyrate levels were significantly higher, that alanine and proline levels were significantly lower in the IM group than in the control group, and that atenolol administration suppressed these metabolite changes. (4) Conclusions: Atenolol reduced immobilization-induced skeletal muscle wasting and might protect against the deleterious effects of prolonged bed rest.

Selenoprotein P deficiency protects against immobilization-induced muscle atrophy by suppressing atrophy-related E3 ubiquitin ligases.

The quality of skeletal muscle is maintained by a balance between protein biosynthesis and degradation. Disruption in this balance results in sarcopenia. However, its underlying mechanisms remain under-investigated. Selenoprotein P (SeP; encoded by Selenop in mice) is a hepatokine that is upregulated in type 2 diabetes and aging and causes signal resistances via reductive stress. We created immobilized muscle atrophy (IMM) model in Selenop knockout (KO) mice. IMM significantly reduced cross-sectional areas and the size of skeletal muscle fibers, which were ameliorated in KO mice. IMM upregulated the genes encoding E3 ubiquitin ligases and their upstream FoxO1, FoxO3, and KLF15 transcription factors in the skeletal muscle, which were suppressed in KO mice. These findings suggest a possible involvement of SeP-mediated reductive stress in physical inactivity-mediated sarcopenia, which may be a therapeutic target against sarcopenia.

Mesenchymal stromal cells ameliorate diabetes-induced muscle atrophy through exosomes by enhancing AMPK/ULK1-mediated autophagy.

Diabetes and obesity are associated with muscle atrophy that reduces life quality and lacks effective treatment. Mesenchymal stromal cell (MSC)-based therapy can ameliorate high fat-diet (HFD) and immobilization (IM)-induced muscle atrophy in mice. However, the effect of MSCs on muscle atrophy in type 2 diabetes mellitus (T2DM) and the potential mechanism is unclear. Here, we evaluated the efficacy and explored molecular mechanisms of human umbilical cord MSCs (hucMSCs) and hucMSC-derived exosomes (MSC-EXO) on diabetes- and obesity-induced muscle atrophy. Diabetic db/db mice, mice fed with high-fat diet (HFD), mice with hindlimb immobilization (IM), and C2C12 myotubes were used to explore the effect of hucMSCs or MSC-EXO in alleviating muscle atrophy. Grip strength test and treadmill running were used to measure skeletal muscle strength and performance. Body composition, muscle weight, and muscle fibre cross-sectional area (CSA) was used to evaluate muscle mass. RNA-seq analysis of tibialis anterior (TA) muscle and Western blot analysis of muscle atrophy signalling, including MuRF1 and Atrogin 1, were performed to investigate the underlying mechanisms. hucMSCs increased grip strength (P=0.0256 in db/db mice, P=0.012 in HFD mice, P=0.0097 in IM mice), running endurance (P=0.0154 in HFD mice, P=0.0006 in IM mice), and muscle mass (P=0.0004 in db/db mice, P=0.0076 in HFD mice, P=0.0144 in IM mice) in all models tested, with elevated CSA of muscle fibres (P<0.0001 in db/db mice and HFD mice, P=0.0088 in IM mice) and reduced Atrogin1 (P=0.0459 in db/db mice, P=0.0088 in HFD mice, P=0.0016 in IM mice) and MuRF1 expression (P=0.0004 in db/db mice, P=0.0077 in HFD mice, P=0.0451 in IM mice). MSC-EXO replicated all these hucMSC-mediated changes (P=0.0103 for grip strength, P=0.013 for muscle mass, P<0.0001 for CSA of muscle fibres, P=0.0171 for Atrogin1 expression, and P=0.006 for MuRF1 expression). RNA-seq revealed that hucMSCs activated the AMPK/ULK1 signalling and enhanced autophagy. Knockdown of AMPK or inhibition of autophagy with 3-methyladenine (3-MA) diminished the beneficial anti-atrophy effects of hucMSCs or MSC-EXO. Our results suggest that human umbilical cord mesenchymal stromal cells mitigate diabetes- and obesity-induced muscle atrophy via enhancing AMPK/ULK1-mediated autophagy through exosomes, with implications of applying hucMSCs or hucMSC-derived exosomes to treat muscle atrophy.

Effect of Electrical Stimulation on Disuse Muscular Atrophy Induced by Immobilization: Correlation With Upregulation of PERK Signal and Parkin-Mediated Mitophagy.

The aims of the study are to investigate the effect of electrical stimulation on disuse muscular atrophy induced by immobilization (IM) and to explore the role of PERK signal and Parkin-dependent mitophagy in this process. In the first subexperiment, 24 rabbits were divided into four groups, which underwent different periods of IM. In the second subexperiment, 24 rabbits were divided into four groups on average in accordance with different kinds of interventions. To test the time-dependent changes of rectus femoris after IM, and to evaluate the effect of electrical stimulation, the wet weights, cross-sectional area and fat deposition of rectus femoris were assessed in this study, along with the protein levels of atrogin-1, p-PERK, Parkin, and COXIV. The wet weights and cross-sectional area decreased, and the fat deposition increased in rectus femoris after IM, along with the elevated protein levels of atrogin-1, p-PERK, Parkin, and decreased protein levels of COXIV. The above histomorphological and molecular changes can be partially ameliorated by electrical stimulation. Immobilization of unilateral lower limb could induce rectus femoris atrophy, which can be partially rectified by electrical stimulation. PERK signal and Parkin-mediated mitophagy may be the mechanisms by which electrical stimulation can play a significant role.

Muscle damage induced by maximal eccentric exercise of the elbow flexors after 3-week immobilization.

The present study investigated the effects of a 3-week immobilization (IM) on muscle damage induced by maximal eccentric exercise (MaxEC) to test the hypothesis that the IM would make muscles prone to muscle damage. Young healthy sedentary men were pseudo-randomly assigned to IM or control group (n=12/group). Non-dominant arms of the IM group participants were immobilized at 90° elbow flexion by a cast for 21 days. All participants performed MaxEC consisting of five sets of six elbow flexor contractions by lowering a dumbbell set at 100% of pre-exercise maximal voluntary isometric contraction (MVC) strength of the non-dominant arm. This was performed at 2 days after the cast removal for the IM group. MVC torque, range of motion (ROM), muscle thickness (MT), muscle hardness, position sense (PS), and joint reaction angle (JRA) of the elbow flexors were measured at baseline, post-immobilization, and before, immediately after, and one to 5 days after MaxEC. The IM decreased MVC torque (-17 ± 2%), ROM (-2 ± 1%), MT (-7 ± 3%), and JRA (-12 ± 6%), and increased in muscle hardness (20 ± 6%) and PS (11 ± 2%) (p < 0.05). Changes in MVC (e.g., 2 days: -40± 5 vs. -30 ± 9%), ROM (2 days: -11 ± 2 vs. -9 ± 3%), muscle soreness (peak: 63 ± 22 vs. 48 ± 14 mm), plasma CK activity (peak: 7820 ± 4011 vs. 4980 ± 1363 IU/L), PS (maximal change: -23 ± 2 vs. -18 ± 3%), and JRA (maximal change: -37 ± 4 vs. -26 ± 3%) after MaxEC were greater (p < 0.05) for the IM than control group. These results supported the hypothesis and showed that the IM made the muscles more vulnerable to muscle damage induced by eccentric exercise.

Interaction between the hypothalamo-pituitary-adrenal and thyroid axes during immobilization stress.

The location of corticotropin-releasing hormone receptor 2 (CRH-R2) on thyrotropes within the avian anterior pituitary (APit) and its activation by different stressors indicate a possible communication between hypothalamo-pituitary-adrenal (HPA) and thyroid (HPT) axes. Therefore, an experiment was designed to 1) compare the timing of major components of the HPT axis to those of the HPA axis; 2) address whether stressors activating the HPA axis may simultaneously upregulate components of the HPT axis. Blood, brain, and APit were sampled from chicks prior to stress (control) and 15, 30, 60, 90, and 120min following immobilization (IM) stress. The nucleus of the hippocampal commissure (NHpC) and paraventricular nucleus (PVN) were cryo-dissected from brains for RT-qPCR. Gene expression of thyrotropin-releasing hormone (TRH) and its receptors (TRH-R1 and TRH-R3), urocortin3 (UCN3), deiodinase 2 (D2), and the second type of corticotropin-releasing hormone (CRH2) within the NHpC and PVN was measured. Additionally, gene expression of TRH receptors, thyroid stimulating hormone subunit beta (TSHβ), and D2 was determined in the APit and corticosterone assayed in blood. In brains, a significant upregulation in examined genes occurred at different times of IM. Specifically, UCN3 and CRH2 which have a high affinity to CRH-R2 showed a rapid increase in their mRNA levels that were accompanied by an early upregulation of TRHR1 in the NHpC. In the APit, a significant increase in gene expression of TSHβ and TRH receptors was observed. Therefore, results supported concurrent activation of major brain and APit genes associated with the HPA and HPT axes following IM. The initial neural gene expression originating within the NHpC resulted in the increase of TSHβ mRNA in the APit. Specifically, the rapid upregulation of UCN3 in the NHpC appeared responsible for the early activation of TSHβ in the APit. While sustaining TSHβ activation appeared to be due to both CRH2 and TRH. Therefore, data indicate that CRH-producing neurons and corticotropes as well as CRH- and TRH-producing neurons and thyrotropes are activated to produce the necessary energy required to maintain homeostasis in birds undergoing stress. Overall, data support the inclusion of the NHpC in the classical avian HPA axis and for the first time show the concurrent activation of the HPA axis and components of the HPT axis following a psychogenic stressor.

Rehabilitation following operative treatment of acute Achilles tendon ruptures: a systematic review and meta-analysis.

The aim of this systematic review and meta-analysis was to compare re-rupture rates, complication rates, functional outcomes, as well as return to work (RTW)/sport (RTS) among different rehabilitation protocols following operative treatment of acute Achilles tendon ruptures. Systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Five databases were searched for randomized controlled trials (RCTs) comparing at least two rehabilitation protocols following surgical repair of acute Achilles tendon ruptures. Rehabilitation protocols were classified as a variation of either non-weightbearing (NWB) or weightbearing (WB) and immobilization (IM) or mobilization (M). The data collection consisted of re-ruptures, complications, and RTW/RTS. Out of 2760 studies screened, 20 RCTs with 1007 patients were eligible. Fourteen studies included a group consisting of WB + M (Group 1), 11 of WB + IM (Group 2), 3 of NWB + M (Group 3), and 13 of NWB + IM (Group 4). Outcome parameters available for a meta-analysis were re-ruptures, complications, RTW, and RTS. Re-ruptures overall occurred in 2.7%, with prevalences ranging between 0.04 and 0.08. Major complications occurred in 2.6%, with prevalences ranging between 0.02 and 0.03. Minor complications occurred in 11.8% with prevalances ranging between 0.04 to 0.17. Comparing the odds-ratios between the four different groups revealed no significant differences with overall favourable results for group 1 (WB+M). Early functional rehabilitation protocols with early ankle M and WB following surgical repair of acute Achilles tendon ruptures are safe and they apparently allow for a quicker RTW and RTS and seem to lead to favourable results.

PACAP-PAC1R modulates fear extinction via the ventromedial hypothalamus

Exposure to traumatic stress can lead to fear dysregulation, which has been associated with posttraumatic stress disorder (PTSD). Previous work showed that a polymorphism in the PACAP-PAC1R (pituitary adenylate cyclase-activating polypeptide) system is associated with PTSD risk in women, and PACAP (ADCYAP1)-PAC1R (ADCYAP1R1) are highly expressed in the hypothalamus. Here, we show that female mice subjected to acute stress immobilization (IMO) have fear extinction impairments related to Adcyap1 and Adcyap1r1 mRNA upregulation in the hypothalamus, PACAP-c-Fos downregulation in the Medial Amygdala (MeA), and PACAP-FosB/ΔFosB upregulation in the Ventromedial Hypothalamus dorsomedial part (VMHdm). DREADD-mediated inhibition of MeA neurons projecting to the VMHdm during IMO rescues both PACAP upregulation in VMHdm and the fear extinction impairment. We also found that women with the risk genotype of ADCYAP1R1 rs2267735 polymorphism have impaired fear extinction.

Stress alters the transcriptional activity of Leydig cells dependently on the diurnal time.

The increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored. Here, the stress-response of the testosterone-producing rat Leydig cells depending on the time of stressful events was studied. The study analyzes the effects of 3-h immobilization (IMO) applied at different periods during the day. The IMO performed once [1 time immobilization stress (1×IMO)] or repeated in 10 consecutive days [10 time repeated immobilization stress (10×IMO)]. Both types of IMO increased corticosterone and decreased testosterone blood level. However, the effect of 10×IMO occurring in the active phase on blood testosterone was less pronounced. This is related to different sensitivity to IMO-events depending on the diurnal time. Most steroidogenesis-related genes [gene encoding luteinizing hormone/choriogonadotropin receptor (Lhcgr), gene encoding cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), gene encoding hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 1 (Hsd3b1/2), and gene encoding cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1)] were downregulated in the inactive phase but unchanged or even upregulated in the active phase of the day. Both types of IMO stimulated the expression of clock elements gene encoding aryl hydrocarbon receptor nuclear translocator-like (Bmal1)/aryl hydrocarbon receptor nuclear translocator-like (BMAL1), gene encoding period circadian regulator 1 (Per1)/period circadian regulator 1 (PER1) regardless of the day's stage and reduced gene encoding nuclear receptor subfamily 1, group D, member 1 (Rev-erba) in the inactive phase. The principal component analysis (PCA) confirmed a major shift, for both IMO-types, in the transcription of the genes across the passive/active stage. Further, 10×IMO changed a diurnal pattern of the glucocorticoid receptor [gene encoding nuclear receptor subfamily 3, group C, member 1 (Nr3c1)/nuclear receptor subfamily 3, group C, member 1 (GR)] expression, whereas the observed time-dependent IMO-response of the Leydig cells correlated with different corticosterone engagements. Altogether, the Leydig cell's stress response depends on the daytime of the stressful event, emphasizing the importance of the circadian system in supporting androgen homeostasis and male fertility.

The role of PACAP/PAC1R in PTSD: effects on fear extinction via the ventromedial hypothalamus

Introduction The incidence and severity of posttraumatic stress disorder (PTSD) is higher in women than men because of environmental and biological factors. Specific mechanisms in the PACAP-PAC1R (pituitary adenylate cyclase-activating polypeptide and its type I receptor) system may confer PTSD risk in women. Interestingly, while the PACAP (ADCYAP1) - PAC1R (ADCYAP1R1) system is expressed highly in the hypothalamus, no relationship has been described for this pathway in the hypothalamus with fear processing or in PTSD.ObjectivesWe studied whether the estrous/menstrual cycle at the moment of trauma predicts PTSD and the involvement of the PACAP neurons in the amygdala and hypothalamus during traumatic stress.MethodsMice: DREADDs, immunohistochemistry and behavior. Humans: fear-potentiated startle and questionnaires.Results Here, we show that acute stress immobilization (IMO) produces fear extinction impairments in female mice. Also, IMO elicits Adcyap1 and Adcyap1r1 mRNA upregulation in the hypothalamus, PACAP/c-Fos downregulation in the medial amygdala (MeA), and PACAP/FosB/ΔFosB upregulation in the ventromedial hypothalamus dorsomedial part (VMHdm) after fear extinction. We also found that women with the risk genotype of ADCYAP1R1 rs2267735 SNP show impaired fear extinction. In mice, DREADD-mediated inhibition of the MeA neurons projecting to the VMHdm during IMO rescues both PACAP upregulation in VMHdm and the fear extinction impairment. We ruled out contributions from inherent hormonal states showing that the menstrual or estrous cycle phase at the moment of trauma does not result in a vulnerable phenotype.ConclusionsOur data suggest that the PACAP-PAC1R hypothalamic system may be a novel candidate to treat and prevent PTSD symptoms including fear dysregulations.DisclosureNo significant relationships.

P613. Study of PPM1F as a Translational Marker of Traumatic Stress

Our recent findings showed that the expression of PPM1F phosphatase, member of CaMKIIg pathway, is downregulated in PTSD patients, and differentially regulated in the mPFC and the amygdala of male and female mice previously exposed to acute immobilization (IMO). Chronic unpredictable mild stress (CUMS) is a reliable model of depression in rodents that could have also impact on PPM1F expression supporting a role of PPM1F on chronic stress. On the other hand, IMO impairs cued-conditioned fear extinction, but little is known about sex differences in the processing of fear memory after exposure to acute stress.

H2S Protects Against Immobilization-Induced Muscle Atrophy via Reducing Oxidative Stress and Inflammation.

Chronic inflammation and oxidative stress are major triggers of the imbalance between protein synthesis and degradation during the pathogenesis of immobilization-induced muscle atrophy. This study aimed to elucidate the effects of hydrogen sulfide (H2S), a gas transmitter with potent anti-inflammatory and antioxidant properties, on immobilization-induced muscle atrophy. Mice were allocated to control and immobilization (IM) groups, which were treated with slow (GYY4137) or rapid (NaHS) H2S releasing donors for 14 days. The results showed that both GYY4137 and NaHS treatment reduced the IM-induced muscle loss, and increased muscle mass. The IM-induced expressions of Muscle RING finger 1 (MuRF1) and atrogin-1, two muscle-specific E3 ubiquitin ligases, were decreased by administration of GYY4137 or NaHS. Both GYY4137 and NaHS treatments alleviated the IM-induced muscle fibrosis, as evidenced by decreases in collagen deposition and levels of tissue fibrosis biomarkers. Moreover, administration of GYY4137 or NaHS alleviated the IM-induced infiltration of CD45 + leukocytes, meanwhile inhibited the expressions of the pro-inflammatory biomarkers in skeletal muscles. It was found that administration of either GYY4137 or NaHS significantly attenuated immobilization-induced oxidative stress as indicated by decreased H2O2 levels and 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunoreactivity, as well as increased total antioxidant capacity (T-AOC), nuclear factor erythroid-2-related factor 2 (NRF2) and NRF2 downstream anti-oxidant targets levels in skeletal muscles. Collectively, the present study demonstrated that treatment with either slow or rapid H2S releasing donors protected mice against immobilization-induced muscle fibrosis and atrophy. The beneficial effects of H2S on immobilization-induced skeletal muscle atrophy might be due to both the anti-inflammatory and anti-oxidant properties of H2S.

Codonopsis lanceolata and its active component Tangshenoside I ameliorate skeletal muscle atrophy via regulating the PI3K/Akt and SIRT1/PGC-1α pathways

Background: Skeletal muscle atrophy is caused by aging, disuse, malnutrition, and several diseases. However, there are still no effective drugs or treatments for muscle atrophy. Codonopsis lanceolata (CL), a traditional medicinal plant and food, has been reported to have anti-oxidative, anti-inflammatory, anti-tumor, and anti-obesity effects.Purpose: This study aimed to investigate the efficacy and active component of CL on muscle atrophy in vitro and to confirm the effect of CL and its active component on muscle atrophy and the underlying molecular mechanisms in vivo.Study: design/Methods This study used the dexamethasone (Dex)-induced muscle atrophy C2C12 myotube model and immobilization (IM)-induced muscle atrophy C57BL/6 mice model. In vitro study, the myotube diameter was measured. In vivo study, the grip strength, muscle mass (quadriceps, gastrocnemius, and soleus) and muscle fiber cross-sectional area (CSA) was measured. Western blot analysis and qRT-PCR were performed to confirm the underlying molecular mechanismsResults:In vitro study, CL and its main component, Tangshenoside I (TSI), effectively restored C2C12 myotube diameters decreased by Dex. Surprisingly, TSI was identified as the active component responsible for the overall efficacy of CL on muscle atrophy. In vivo study, CL and TSI, dose-dependently increased grip strength, mass muscle, and muscle fiber CSA reduced by IM. In the molecular mechanism studies, CL and TSI increased muscle protein synthesis via activating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin complex 1 (mTORC1) pathway and decreased muscle protein degradation via inhibiting the muscle ring finger-1 (MuRF1) and muscle atrophy F-box protein (Atrogin-1) expressions. It also upregulated mitochondrial biogenesis via the silent information regulator 1 (SIRT1)/ peroxisome proliferator-activated receptor gamma and coactivator-1 alpha (PGC-1α) pathway.Conclusion: This study suggests that CL and its active component, TSI, can be potential drug candidates for the prevention and treatment of muscle atrophy.

The Effects of Ethyl Pyruvate on Behavioral and Biochemical Parameters in Immobilization Stress-Induced Mice

Objective: Ethyl pyruvate (EP) is a molecule with antioxidant, anti-inflammatory, and anti-apoptotic features. The present study investigates EP's effects on behavioral and biochemical alterations induced by immobilization (IM) stress. &#x0D; Methods: In this outcome, 45 male BALB/c mice were separated into five groups [CONT (control), IM, IM+EP, EP, IM+F (fluoxetine)]. IM applied groups had 6 hours of immobilization stress procedure a day for a week. EP (50 mg/day) and fluoxetine (5 mg/day) were applied to the mice intraperitoneally. After the stress induction protocol, passive avoidance, open field (OF), and forced swimming tests (FST) were executed. After behavioral tests, brain tissues were obtained for biochemical investigations. &#x0D; Results: Immobilization application increased immobility times in the FST and decreased the total distance moved and the center time in the OF test. Immobile times were reduced with fluoxetine and EP applied groups compared with the IM group due to decreased learned helplessness. The IM group had decreased brain-derived neurotrophic factor (BDNF), superoxide dismutase, and glutathione peroxidase levels, which were increased with EP applications.&#x0D; Conclusion: These results show that EP generates antidepressant-like effects with decreasing oxidation and increasing BDNF levels.