NMDA receptor within nucleus accumbens shell regulates propofol self-administration through D1R/ERK/CREB signalling pathway.

Addictive properties of propofol have been demonstrated in both humans and animals. The nucleus accumbens (NAc) shell (NAsh) in the brain, along with the interactions between N-methyl-D-aspartate receptor (NMDAR) and the dopamine D1 receptor (D1R), as well as their downstream ERK/CREB signalling pathway in the NAc, are integral in regulating reward-seeking behaviour. Nevertheless, it remains unclear whether NMDARs and the NMDAR-D1R/ERK/CREB signalling pathway in the NAsh are involved in mediating propofol addiction. To investigate it, we conducted experiments with adult male Sprague-Dawley rats to establish a model of propofol self-administration behaviour. Subsequently, we microinjected D-AP5 (a competitive antagonist of NMDARs, 1.0-4.0 µg/0.3 µL/site) or vehicle into bilateral NAsh in rats that had previously self-administered propofol to examine the impact of NMDARs within the NAsh on propofol self-administration behaviour. Additionally, we examined the protein expressions of NR2A and NR2B subunits, and the D1R/ERK/CREB signalling pathways within the NAc. The results revealed that propofol administration behaviour was enhanced by D-AP5 pretreatment in NAsh, accompanied by elevated expressions of phosphorylation of NR2A (Tyr1246) and NR2B (Tyr1472) subunits. There were statistically significant increases in the expressions of D1Rs, as well as in the phosphorylated ERK1/2 (p-ERK1/2) and CREB (p-CREB). This evidence substantiates a pivotal role of NMDARs in the NAsh, with a particular emphasis on the NR2A and NR2B subunits, in mediating propofol self-administration behaviour. Furthermore, it suggests that this central reward processing mechanism may operate through the NMDAR-D1R/ERK/CREB signal transduction pathway.

Sevoflurane but not propofol enhances ovarian cancer cell biology through regulating cellular metabolic and signaling mechanisms.

Perioperative risk factors, including the choice of anesthetics, may influence ovarian cancer recurrence after surgery. Inhalational anesthetic sevoflurane and intravenous agent propofol might affect cancer cell metabolism and signaling, which, in turn, may influence the malignancy of ovarian cancer cells. The different effects between sevoflurane and propofol on ovarian cancer cell biology and underlying mechanisms were studied. Cultured ovarian cancer cells were exposed to 2.5% sevoflurane, 4 µg/mL propofol, or sham condition as the control for 2 h followed by 24-h recovery. Glucose transporter 1 (GLUT1), mitochondrial pyruvate carrier 1 (MPC1), glutamate dehydrogenase 1 (GLUD1), pigment epithelium-derived factor (PEDF), p-Erk1/2, and hypoxia-inducible factor 1-alpha (HIF-1α) expressions were determined with immunostaining and/or Western blot. Cultured media were collected for 1H-NMR spectroscopy-based metabolomics analysis. Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to analyze metabolomics data. Sevoflurane increased the GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α expressions but decreased the PEDF expression relative to the controls. In contrast to sevoflurane, propofol decreased GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α but increased PEDF expression. Sevoflurane increased metabolite isopropanol and decreased glucose and glutamine energy substrates in the media, but the opposite changes were found after propofol treatment. Our data indicated that, unlike the pro-tumor property of sevoflurane, propofol negatively modulated PEDF/Erk/HIF-1α cellular signaling pathway and inhibited ovarian cancer metabolic efficiency and survival, and hence decreased malignancy. The translational value of this work warrants further study. • Sevoflurane promoted but propofol inhibited ovarian cancer cell biology. • Sevoflurane upregulated but propofol downregulated the GLUT1, MPC1, and GLUD1 expressions of ovarian cancer cells. • Sevoflurane enhanced but propofol inhibited ovarian cancer cellular glucose. metabolism and glutaminolysis. • Sevoflurane downregulated PEDF but upregulated the Erk pathway and HIF-1α, while propofol had the adverse effects on ovarian cancer cells.

The excitatory transmission from basolateral nuclues of amygdala to nucleus accumbens shell regulates propofol self-administration through AMPA receptors.

Propofol addictive properties have been demonstrated in humans and rats. The glutamatergic transmission from basolateral nucleus of amygdala (BLA) to the nucleus accumbens (NAc) modulates reward-seeking behaviour; especially, NAc shell (NAsh) is implicated in reward-seeking response. Previous studies indicated the interactions between AMPA receptors (AMPARs) and dopamine D1 receptor (D1R) in NAc mediated drug addiction, but whether the circuit of BLA-to-NAsh and AMPARs regulate propofol addiction remains unclear. We trained adult male Sprague-Dawley rats for propofol self-administration to examine the changes of action potentials (APs) and spontaneous excitatory postsynaptic currents (sEPSCs) in the NAsh. Thereafter, optogenetic stimulation with adeno-associated viral vectors microinjections in BLA was used to explore the effect of BLA-to-NAsh on propofol self-administration behaviour (1.7 mg/kg/injection). The pretreatment effects with NBQX (0.25-1.0 µg/0.3 µl/site) or vehicle in the NAsh on propofol self-administration behaviour, the expressions of AMPARs subunits and D1R/ERK/CREB signalling pathway in the NAc were detected. The results showed that the number of APs, amplitude and frequency of sEPSCs were enhanced in propofol self-administrated rats. Propofol self-administration was inhibited in the NpHR3.0-EYFP group, but in the ChR2-EYFP group, there was a promoting effect, which could be weakened by NBQX pretreatment. NBQX pretreatment also significantly decreased the expressions of GluA2 subunit and D1R in the NAc but did not change the expressions of GluA1 and ERK/CREB signalling pathway. The evidence supports a vital role of BLA-to-NAsh circuit in regulating propofol self-administration and suggests this central reward processing may function through the interaction between AMPARs and D1R in the NAsh.

Effect of Regional vs General Anesthesia on Incidence of Postoperative Delirium in Older Patients Undergoing Hip Fracture Surgery: The RAGA Randomized Trial.

Importance: In adults undergoing hip fracture surgery, regional anesthesia may reduce postoperative delirium, but there is uncertainty about its effectiveness. Objective: To investigate, in older adults undergoing surgical repair for hip fracture, the effects of regional anesthesia on the incidence of postoperative delirium compared with general anesthesia. Design, Setting, and Participants: A randomized, allocation-concealed, open-label, multicenter clinical trial of 950 patients, aged 65 years and older, with or without preexisting dementia, and a fragility hip fracture requiring surgical repair from 9 university teaching hospitals in Southeastern China. Participants were enrolled between October 2014 and September 2018; 30-day follow-up ended November 2018. Interventions: Patients were randomized to receive either regional anesthesia (spinal, epidural, or both techniques combined with no sedation; n = 476) or general anesthesia (intravenous, inhalational, or combined anesthetic agents; n = 474). Main Outcomes and Measures: Primary outcome was incidence of delirium during the first 7 postoperative days. Secondary outcomes analyzed in this article include delirium severity, duration, and subtype; postoperative pain score; length of hospitalization; 30-day all-cause mortality; and complications. Results: Among 950 randomized patients (mean age, 76.5 years; 247 [26.8%] male), 941 were evaluable for the primary outcome (6 canceled surgery and 3 withdrew consent). Postoperative delirium occurred in 29 (6.2%) in the regional anesthesia group vs 24 (5.1%) in the general anesthesia group (unadjusted risk difference [RD], 1.1%; 95% CI, -1.7% to 3.8%; P = .48; unadjusted relative risk [RR], 1.2 [95% CI, 0.7 to 2.0]; P = .57]). Mean severity score of delirium was 23.0 vs 24.1, respectively (unadjusted difference, -1.1; 95% CI, -4.6 to 3.1). A single delirium episode occurred in 16 (3.4%) vs 10 (2.1%) (unadjusted RD, 1.1%; 95% CI, -1.7% to 3.9%; RR, 1.6 [95% CI, 0.7 to 3.5]). Hypoactive subtype in 11 (37.9%) vs 5 (20.8%) (RD, 11.5; 95% CI, -11.0% to 35.7%; RR, 2.2 [95% CI, 0.8 to 6.3]). Median worst pain score was 0 (IQR, 0 to 20) vs 0 (IQR, 0 to 10) (difference 0; 95% CI, 0 to 0). Median length of hospitalization was 7 days (IQR, 5 to 10) vs 7 days (IQR, 6 to 10) (difference 0; 95% CI, 0 to 0). Death occurred in 8 (1.7%) vs 4 (0.9%) (unadjusted RD, -0.8%; 95% CI, -2.2% to 0.7%; RR, 2.0 [95% CI, 0.6 to 6.5]). Adverse events were reported in 106 episodes in the regional anesthesia group and 102 in the general anesthesia group; the most frequently reported adverse events were nausea and vomiting (47 [44.3%] vs 34 [33.3%]) and postoperative hypotension (13 [12.3%] vs 10 [9.8%]). Conclusions and Relevance: In patients aged 65 years and older undergoing hip fracture surgery, regional anesthesia without sedation did not significantly reduce the incidence of postoperative delirium compared with general anesthesia. Trial Registration: ClinicalTrials.gov Identifier: NCT02213380.

The Adenosine A2A Receptor Activation in Nucleus Accumbens Suppress Cue-Induced Reinstatement of Propofol Self-administration in Rats.

Propofol has shown strong addictive properties in rats and humans. Adenosine A2A receptors (A2AR) in the nucleus accumbens (NAc) modulate dopamine signal and addictive behaviors such as cocaine- and amphetamine-induced self-administration. However, whether A2AR can modulate propofol addiction remains unknown. AAV-shA2AR was intra-NAc injected 3 weeks before the propofol self-administration training to test the impacts of NAc A2AR on establishing the self-administration model with fixed ratio 1 (FR1) schedule. Thereafter, the rats were withdrawal from propofol for 14 days and tested cue-induced reinstatement of propofol seeking behavior on day 15. The propofol withdrawal rats received one of the doses of CGS21680 (A2AR agonist, 2.5-10.0 ng/site), MSX-3 (A2AR antagonist, 5.0-20.0 µg/site) or eticlopride (D2 receptor (D2R) antagonist, 0.75-3.0 µg/site) or vehicle via intra-NAc injection before relapse behavior test. The numbers of active and inactive nose-poke response were recorded. Focal knockdown A2AR by shA2AR did not affect the acquisition of propofol self-administration behavior, but enhance cue-induced reinstatement of propofol self-administration compared with the AAV-shCTRLgroup. Pharmacological activation of the A2AR by CGS21680 (≥ 5.0 ng/site) attenuated cue-induced reinstatement of propofol self-administration behavior. Similarly, pharmacological blockade of D2R by eticlopride (0.75-3.0 µg/site) attenuated propofol seeking behavior. These effects were reversed by the administration of MSX-3 (5.0-20.0 µg/site). The A2AR- and D2R-mediated effects on propofol relapse were not confounded by the learning process, and motor activity as the sucrose self-administration and locomotor activity were not affected by all the treatments. This study provides genetic and pharmacological evidence that NAc A2AR activation suppresses cue-induced propofol relapse in rats, possibly by interacting with D2R.

Short-term perfluorooctane sulfonate exposure impairs Leydig cell regeneration in the adult rat testis via targeting hedgehog signaling.

Perfluorooctane sulfonate is related to male reproductive dysfunction in rats and humans. However, the underlying mechanism remains unknown. Here, we reported the effects of short-term exposure to perfluorooctane sulfonate on the regeneration of Leydig cells in vivo and investigated possible mechanisms in vitro. After adult male Sprague-Dawley rats were gavaged perfluorooctane sulfonate (0, 5 or 10 mg/kg/day) for 7 days and then injected intraperitoneally ethane dimethane sulfonate next day to eliminate Leydig cells, the Leydig cell regeneration process was monitored. Perfluorooctane sulfonate significantly lowered serum testosterone levels, reduced the number of regenerated Leydig cells, down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Dhh) and their proteins at doses of 5 and 10 mg/kg 35 and 56 days after ethane dimethane sulfonate. Using a 3D seminiferous tubule culture system to study the development of stem Leydig cells, we found that perfluorooctane sulfonate inhibited stem Leydig cell proliferation and differentiation and hedgehog signaling pathway. In conclusion, a short-term exposure to perfluorooctane sulfonate can inhibit the development of stem Leydig cells into the Leydig cell lineage via direct suppression of hedgehog signaling pathway and indirect inhibition of desert hedgehog section by Sertoli cells.

Apoptosis and necroptosis occur in the different brain regions of hippocampus in a rat model of hypoxia asphyxia.

AIM OF THE STUDY: Hypoxic-ischemic encephalopathy (HIE) is a major cause of newborn brain injury. Apoptosis and necroptosis are two forms of cell death which may occur in HIE but reported data are yet limited. This study investigates the expression of receptor interacting protein kinase (RIPK) 1 and 3, and caspase3, the key modulators of necroptosis and apoptosis, respectively, in a model of HIE to determine whether both forms of cell death occur in the corresponding brain regions. MATERIALS AND METHODS: Postneonatal day 7 Sprague-Dawley rats were subjected to right carotid artery ligation followed by hypoxia or subjected to skin incision under surgical anesthesia without ligation and hypoxia. Neuroglioma (H4) cell was cultured and subjected to 24 h hypoxic insults. Necrostatin-1, a RIPK1 inhibitor, was administered in both in vivo and in vitro settings before insult. RESULTS: After hypoxic-ischemic insults, both RIPK1 and RIPK3 expression were significantly increased in the region of hippocampal dentate gyrus in the injurious hemisphere. However, cleaved caspase3 was significantly increased in the hippocampal cornu ammonis 1 region in the injurious hemisphere. After hypoxic insults, RIPK1 and RIPK3 expression was also found in H4 cells. In addition, it was identified that the increased RIPK1 and RIPK3 can be inhibited by necrostatin-1 in both in vivo and in vitro. CONCLUSIONS: These data indicated that apoptosis and necroptosis occur in different brain regions of hippocampus in a model of HIE which may suggest that strategies to prevent each form of neuronal death is valuable to be developed.

Regulation of spermatid polarity by the actin- and microtubule (MT)-based cytoskeletons.

It is conceivable that spermatid apico-basal polarity and spermatid planar cell polarity (PCP) are utmost important to support spermatogenesis. The orderly arrangement of developing germ cells in particular spermatids during spermiogenesis are essential to obtain structural and nutrient supports from the fixed number of Sertoli cells across the limited space of seminiferous epithelium in the tubules following Sertoli cell differentiation by ∼17 day postpartum (dpp) in rodents and ∼12 years of age at puberty in humans. Yet few studies are found in the literature to investigate the role of these proteins to support spermatogenesis. Herein, we briefly summarize recent findings in the field, in particular emerging evidence that supports the concept that apico-basal polarity and PCP are conferred by the corresponding polarity proteins through their effects on the actin- and microtubule (MT)-based cytoskeletons. While much research is needed to bridge our gaps of understanding cell polarity, cytoskeletal function, and signaling proteins, a critical evaluation of some latest findings as summarized herein provides some important and also thought-provoking concepts to design better functional experiments to address this important, yet largely expored, research topic.

The association of the optimal bolus of dexmedetomidine with its favourable haemodynamic outcomes in adult surgical patients under general anaesthesia.

AIMS: Dexmedetomidine is highly specific α2-adrenoceptor agonist. A single bolus of dexmedetomidine can achieve clinical therapeutic effect. Therefore, it is essential to know the safety margin between the clinical effectiveness dosages of dexmedetomidine and its side effect. METHODS: A total of 42 patients who underwent elective thyroidectomy were enrolled in this study. Dexmedetomidine was given as a single bolus injection 30 min towards the end of surgery. The up-and-down sequential schedule was used in this study. The starting dose of dexmedetomidine was set at 0.1 µg/kg in the first patient and the next patient would then receive a dose of dexmedetomidine decremented by 0.05 µg/kg if the prior patient's baseline heart rate (HR) had a decrease of ≥20% and/or mean arterial blood pressure (MAP) increase or decrease of ≥20%, otherwise, the following patient would receive an incremental 0.05 µg/kg dose of dexmedetomidine. The analytic techniques of linear, linear-logarithmic, exponential regressions and centred isotonic regression were used to determine the ED50 of dexmedetomidine and the residual standard errors were calculated for the comparison of goodness of fit among the different models. RESULTS: The median (interquartile range [range]) lowest HR was 57 beats/min (53-63.3[46-76]) with an average HR decrease of 8.0 beats/min (5-13 [4 to 23]). The median (interquartile range [range]) highest MAP was 98 mmHg (91.8-105 [83-126]) with a MAP increase of 10.0 mmHg (6.8-18.0 [2-24]). The ED50 (95% confidence interval) from 4 different statistical approaches (linear, linear-logarithmic, exponential regressions and centred isotonic regression) were 0.262 µg/kg (0.243, 0.306), 0.252 µg/kg (0.238, 0.307), 0.283 µg/kg (0.238, 0.307), and 0.278 µg/kg, respectively. Among the 4 models, the exponential regression had the least residual standard error (0.03618). CONCLUSION: The ED50 derived from 4 statistical models for an intravenous bolus of dexmedetomidine without significant haemodynamic effects was distributed in a narrow range of 0.252-0.283 µg/kg, and the exponential regression was the model to best match the study data.

Fibroblast growth factor homologous factor 1 stimulates Leydig cell regeneration from stem cells in male rats.

Fibroblast growth factor homologous factor 1 (FHF1) is an intracellular protein that does not bind to cell surface fibroblast growth factor receptor. Here, we report that FHF1 is abundantly present in Leydig cells with up-regulation during its development. Adult male Sprague Dawley rats were intraperitoneally injected with 75 mg/kg ethane dimethane sulphonate (EDS) to ablate Leydig cells to initiate their regeneration. Then, rats daily received intratesticular injection of FHF1 (0, 10 and 100 ng/testis) from post-EDS day 14 for 14 days. FHF1 increased serum testosterone levels without affecting the levels of luteinizing hormone and follicle-stimulating hormone. FHF1 increased the cell number staining with HSD11B1, a biomarker for Leydig cells at the advanced stage, without affecting the cell number staining with CYP11A1, a biomarker for all Leydig cells. FHF1 did not affect PCNA-labelling index in Leydig cells. FHF1 increased Leydig cell mRNA (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, Nr5a1 and Hsd11b1) and their protein levels in vivo. FHF1 increased preadipocyte biomarker Dlk1 mRNA level and decreased fully differentiated adipocyte biomarker (Fabp4 and Lpl) mRNA and their protein levels. In conclusion, FHF1 promotes Leydig cell regeneration from stem cells while inhibiting the differentiation of preadipocyte/stem cells into adipocytes in EDS-treated testis.

mTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

Lipoxin A4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial-mesenchymal transition.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by alveolar epithelial disruption. Lipoxins (LXs), as so-called "braking signals" of inflammation, are the first mediators identified to have dual anti-inflammatory and inflammatory pro-resolving properties. METHODS: In vivo, lipoxinA4 was administrated intraperitoneally with 1 µg/per mouse after intra-tracheal LPS administration (10 mg/kg). Apoptosis, proliferation and epithelial-mesenchymal transition of AT II cells were measured by immunofluorescence. In vitro, primary human alveolar type II cells were used to model the effects of lipoxin A4 upon proliferation, apoptosis and epithelial-mesenchymal transition. RESULTS: In vivo, lipoxin A4 markedly promoted alveolar epithelial type II cells (AT II cells) proliferation, inhibited AT II cells apoptosis, reduced cleaved caspase-3 expression and epithelial-mesenchymal transition, with the outcome of attenuated LPS-induced lung injury. In vitro, lipoxin A4 increased primary human alveolar epithelial type II cells (AT II cells) proliferation and reduced LPS induced AT II cells apoptosis. LipoxinA4 also inhibited epithelial mesenchymal transition in response to TGF-ß1, which was lipoxin receptor dependent. In addition, Smad3 inhibitor (Sis3) and PI3K inhibitor (LY294002) treatment abolished the inhibitory effects of lipoxinA4 on the epithelial mesenchymal transition of primary human AT II cells. Lipoxin A4 significantly downregulated the expressions of p-AKT and p-Smad stimulated by TGF-ß1 in primary human AT II cells. CONCLUSION: LipoxinA4 attenuates lung injury via stimulating epithelial cell proliferation, reducing epithelial cell apoptosis and inhibits epithelial-mesenchymal transition.

Flurbiprofen Inhibits Androgen Productions in Rat Immature Leydig Cells.

Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague-Dawley rats were cultured with 0-100 µM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 µM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 µM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3ß-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 µM for rat enzyme and 6.31 µM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 µM. Flurbiprofen also down-regulated Lhcgr expression at 100 µM. Flurbiprofen at 10 and 100 µM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.

Statistical Analysis Plan for "An international multicenter study of isoelectric electroencephalography events in infants and young children during anesthesia for surgery".

This Statistical Analysis Plan details the statistical procedures to be applied for the analysis of data for the multicenter electroencephalography study. It consists of a basic description of the study in broad terms and separate sections that detail the methods of different aspects of the statistical analysis, summarized under the following headings (a) Background; (b) Definitions of protocol violations; (c) Definitions of objectives and other terms; (d) Variables for analyses; (e) Handling of missing data and study bias; (f) Statistical analysis of the primary and secondary study outcomes; (g) Reporting of study results; and (h) References. It serves as a template for researchers interested in writing a Statistical Analysis Plan.

Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes.

Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor ß (TGF-ß). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-ß, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry.

Current Status and Challenges of NRF2 as a Potential Therapeutic Target for Diabetic Cardiomyopathy.

Diabetic cardiomyopathy is one of the main causes of heart failure and death in patients with diabetes mellitus. Reactive oxygen species produced excessively in diabetes mellitus cause necrosis, apoptosis, ferroptosis, inflammation, and fibrosis of the myocardium as well as impair the cardiac structure and function. It is increasingly clear that oxidative stress is a principal cause of diabetic cardiomyopathy. The transcription factor nuclear factor-erythroid 2 p45-related factor 2 (NRF2) activates the transcription of more than 200 genes in the human genome. Most of the proteins translated from these genes possess anti-oxidant, anti-inflammatory, anti-apoptotic, anti-ferroptotic, and anti-fibrotic actions. There is a growing body of evidence indicating that NRF2 and its target genes are crucial in preventing high glucose-induced oxidative damage in diabetic cardiomyopathy. Recently, many natural and synthetic activators of NRF2 are shown to possess promising therapeutic effects on diabetic cardiomyopathy in animal models of diabetic cardiomyopathy. Targeting NRF2 signaling by pharmacological entities is a potential approach to ameliorating diabetic cardiomyopathy. However, the persistent high expression of NRF2 in cancer tissues also protects the growth of cancer cells. This "dark side" of NRF2 increases the challenges of using NRF2 activators to treat diabetic cardiomyopathy. In addition, some NRF2 activators were found to have off-target effects. In this review, we summarize the current status and challenges of NRF2 as a potential therapeutic target for diabetic cardiomyopathy.

ResolvinD1 stimulates epithelial wound repair and inhibits TGF-ß-induced EMT whilst reducing fibroproliferation and collagen production.

Acute and chronic inflammatory lung diseases are often associated with epithelial cell injury/loss and fibroproliferative responses. ResolvinD1 (RvD1) is biosynthesized during the resolution phase of inflammatory response and exerts potent anti-inflammatory and promotes resolution of inflammatory lung diseases. The aim of this study was to investigate whether RvD1 exerts protective effects on alveolar epithelial cell function/differentiation and protects against fibroproliferative stimuli. Primary human alveolar type II cells were used to model the effects of RvD1 in vitro upon wound repair, proliferation, apoptosis, transdifferentiation, and epithelial-mesenchymal transition (EMT). Effects of RvD1 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also examined. RvD1 promoted alveolar type II (ATII) cell wound repair and proliferation. RvD1 protected ATII cells against sFas-ligand/TNF-α-induced apoptosis and inhibition on cell proliferation and viability. RvD1 promoted ATII cells transdifferentiation. Moreover, we demonstrate that RvD1 inhibited EMT in response to TGF-ß. Furthermore RvD1 inhibited human lung fibroblast proliferation, collagen production, and myofibroblast differentiation induced by both TGF-ß and bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients. The effects of RvD1 were PI3-kinase dependent and mediated via the resolvin receptor. RvD1 seems to promote alveolar epithelial repair by stimulating ATII cells wound repair, proliferation, reducing apoptosis, and inhibiting TGF-ß-induced EMT. While RvD1 reduced fibroproliferation, collagen production, and myofibroblast differentiation. Together, these results suggest a potential new therapeutic strategy for preventing and treating chronic diseases (such as idiopathic pulmonary fibrosis) as well as the fibroproliferative phase of ARDS by targeting RvD1 actions that emphasizes natural resolution signaling pathways.

Translation and validation of Simplified Chinese version of the Pain Catastrophizing Scale in chronic pain patients: Education may matter.

Objective Pain catastrophizing is linked to many aspects of pain perception and defines a unique dimension in predicting pain intensity and physical disability. Pain Catastrophizing Scale (PCS) is an effective, validated,self-report measure, commonly used in clinical trials. Here, we present a Simplified Chinese PCS (SC-PCS) version developed in Chinese patients suffering from chronic pain. Methods The SC-PCS was generated in five steps and tested on an initial patient cohort (N = 30). A convenience sample (N = 200) of in-hospital patients with non-malignant pain lasting for more than 12 weeks were recruited for the study, of which 81 completed 5 additional pain questionnaires. A subset (N = 24) of the patients completed an additional SC-PCS, 10 days after the initial query to assess test-retest validation. Results Intra-class correlations coefficient indicated high reproducibility and temporal consistency, (0.97), for the total score. Cronbach's alpha determined high internal consistency across the SC-PCS total score and its three subscales (0.87, 0.85, 0.62, and 0.65). The SC-PCS total score moderately or weakly (R = -0.2 to 0.49), but significantly, correlated with other measurements, such as pain Visual Analog Scale, Beck Depression Inventory, Pain Anxiety Symptoms Scales, Positive and Negative Affect Schedule, and education. We used exploratory factor analysis to examine the dimensionality of the SC-PCS, which indicated instability of the current three-factor model. However, a confirmatory factor analysis indicated that the three-factor model had the best goodness-fitting. Conclusions We demonstrate the successful translational adaptation from English to Simplified Chinese as well as the reliability and validity of SC-PCS. An important discovery was education level significantly correlated with SC-PCS, identifying a future consideration for other cross-cultural development of self-reported measures.

VEGF mitigates histone-induced pyroptosis in the remote liver injury associated with renal allograft ischemia-reperfusion injury in rats.

Clinical evidence has indicated a possible link between renal injury and remote liver injury. We investigated whether extracellular histone mediates remote hepatic damage after renal graft ischemia-reperfusion injury, while vascular endothelial growth factor (VEGF) is protective against remote hepatic injury. In vitro, hepatocyte HepG2 cultures were treated with histone. In vivo, the Brown-Norway renal graft was stored in 4°C preservation solution for 24 hours and then transplanted into a Lewis rat recipient; blood samples and livers from recipients were harvested 24 hours after surgery. Prolonged cold ischemia in renal grafts enhanced liver injury 24 hours after engraftment. Caspase-1, ASC, NLRP3, and AIM2 expressions in hepatocyte, CD68+ -infiltrating macrophages, tissue, and serum interleukin-1ß and -18 were greatly elevated, indicating that pyroptosis occurred in the liver and resulted in acute liver functional impairment. Blocking the caspase-1 pathway decreased the number of necrotic hepatocytes. VEGF treatment suppressed the hepatocyte pyroptosis and liver function was partially restored. Our data suggested that renal allograft ischemia-reperfusion injury is likely associated with acute liver damage due to hepatocyte pyroptosis induced by histone and such injury may be protected by VEGF administration. VEGF, therefore, may serve as a new strategy against other remote organ injuries related to renal transplantation.

Dopamine D1 Receptor Within Basolateral Amygdala Is Involved in Propofol Relapse Behavior Induced by Cues.

Propofol has been proven to be potentially abused by humans and laboratory animals; however, studies that have examined propofol relapse behavior are limited, and its underlying mechanism remains unclear. In this study, we examined whether basolateral amygdala-specific or systematic administration of the dopamine receptor antagonist alters cue-induced propofol-seeking behaviors in a rat model. Male Sprague-Dawley rats first received 14 days of propofol self-administration training, where active nose poke resulted in the delivery of propofol infusion paired with a tone and light cues. After 1-30 days of forced abstinence, the cue-induced propofol-seeking behaviors were tested in the operant chamber. We demonstrated, for the first time, after a few days of withdrawal from intravenous bolus administration of propofol, propofol-related cues could induce robust reinstatement of drug-seeking behavior. Systematic administration of dopamine D1 receptor antagonist (SCH-23390) or dopamine D2 receptor antagonist (spiperone) inhibited propofol relapse behavior induced by drug-related cues. Furthermore, we show that microinfusion of SCH-23390 into basolateral amygdala dose-dependently attenuated cue-induced propofol drug-seeking behavior, whereas infusion of spiperone had no effect on the propofol relapse behavior. Our results reveal the involvement of dopamine receptors within the basolateral amygdala in the cue-induced propofol relapse behavior in rats.