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Objective To establish and evaluate a method for rapid and sensitive S.xylosus detection using qPCR(real-time quantitative PCR).Methods A gehM gene fragment was selected as the target for S.xylosus.A set of specific primers was synthesized and a qPCR method was established to detect S.xylosus.A S.xylosus standard strain and other non-target strains were chosen for analysis.DNA of S.xylosus was diluted 10-fold to determine its sensitivity.Clinical samples were tested,and positive products were sequenced.The result were compared with those of bacterial culture.Results S.xylosus had a specific amplification curve,whereas other non-S.xylosus species did not,indicating that the primers were specific for S.xylosus.Sensitivity was 100 fg/μL DNA.Repeatability within and between groups was less than 3％.A total of 60 clinical samples were analyzed,of which five samples had a typical S curve.qPCR products were sequenced and BLAST searched.The similarity of the gene sequences was 99.63％,indicating that the sample was positive for the S.xylosus gehM gene with a positivity rate of 8.3％.However,the positivity rate of bacterial culture was 6.7％.The positivity rate of qPCR was slightly higher than that of the culture.Conclusions The established qPCR method is rapid with high sensitivity and specificity,and can be used to detect S.xylosus.
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The microbiological quality of laboratory animals is crucial for the validity and reproducibility of scientific research data, as well as human health and animal welfare. Currently, individual ventilation cages (IVC) have become the mainstream feeding system for rodent laboratory animals. The most commonly used pathogen monitoring method for this feeding system is soiled bedding sentinels (SBS). This method monitors the microbial carrying status of mouse colony through indirect contact and delayed feedback. It can effectively monitor pathogens transmitted via the fecal-oral route, such as mouse hepatitis virus and reovirus. However, this method has difficulty detecting pathogens mainly transmitted through aerosols or direct contact, such as Sendai virus and Pasteurella pneumotropica. The exhaust air dust (EAD)-PCR monitoring method involves swab sampling in the IVC exhaust ducts to monitor the corresponding racks of the ducts; swab sampling before the prefiltration of the host to monitor the entire IVC rack; and EAD collection device sampling to monitor all racks connected to the same host. Different IVC manufacturers have developed corresponding EAD collection devices for their respective IVC systems, making operations convenient and standardization easy. Compared with the SBS method, the EAD-PCR method significantly improves detection rate and timeliness, with the fastest detection possible after one week of exposure. It can serve as a supplement or replacement for the SBS method. Currently, increasing evidence supports that EAD-PCR testing is a more reliable, sensitive, and cost-effective monitoring method, and is more beneficial to animal welfare. This article reviews the application progress of these two methods for monitoring pathogens, analyzes the existing limitations of the EAD-PCR method, and proposes solutions based on its implementation in our laboratory and examination units. The EAD-PCR method helps reduce the number of live sentinel animals used in pathogen monitoring, in order to better maintain the "3Rs" principle of laboratory animal welfare.
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ObjectiveTo establish a method for rapid and sensitive detection of Staphylococcus aureus. MethodsThe specific gene nuc of Staphylococcus aureus was selected as the target gene. A pair of specific primers and a TaqMan probe were designed and synthesized according to the published sequence of the nuc gene. Establish a nucleic acid detection method for nuc gene using fluorescence quantitative PCR technology, and apply it clinically in the detection of fecal samples from rats and mice. ResultsThe DNA extracted from Staphylococcus aureus and other non-Staphylococcus aureus strains was detected by qPCR. The results showed that Staphylococcus aureus had a specific amplification curve, while other non-Staphylococcus aureus did not, indicating that the designed primers and probes were specific for Staphylococcus aureus. The sensitivity of this method was determined by diluting the DNA of Staphylococcus aureus by 10 times. The results showed that the detection limit of this method was 10 fg DNA, which was 2 orders of magnitude higher than that of ordinary PCR method. A total of 91 clinical samples were detected in this study, of which 4 rat samples from the same facility had a typical S-curve. The PCR products were sequenced and BLAST compared. The gene sequence of this sample was 100% similar to that of Staphylococcus aureus, indicating that the sample was positive for the nucleic acid of Staphylococcus aureus nuc gene, with a positive rate of 4.40%. The result was consistent with that obtained by bacterial culture method. The nucleic acid extraction adopted a full-automatic nucleic acid purification instrument, and the time required from nucleic acid extraction to detection result determination was less than 1.5 h. ConclusionThe qPCR method established in this study to identify Staphylococcus aureus with nuc gene as the target gene has the advantages of fast, high sensitivity and specificity, and can be used for the detection of Staphylococcus aureus in feces of rats and mice.
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Improving the reproducibility of biomedical research results remains a major challenge. Transparent and accurate reporting of progress can help readers evaluate the reliability of research results and further explore an experiment by repeating or building upon its findings. The ARRIVE 2.0 guidelines, released in 2019 by the UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs), provide a checklist applicable to any in vivo animal research report. These guidelines aim to improve the standardization of experimental design, implementation, and reporting, as well as the reliability, repeatability, and clinical translatability of animal experimental results. The use of the ARRIVE 2.0 guidelines not only enriches the details of animal experimental research reports, ensuring that information on animal experimental results is fully evaluated and utilized, but also enables readers to understand the content expressed by the author accurately and clearly, promoting the transparency and integrity of the fundamental research review process. At present, the ARRIVE 2.0 guidelines have been widely adopted by international biomedical journals. This article is the second part of the Chinese translation of the complete interpretation of the ARRIVE 2.0 guidelines published in PLoS Biology in 2020 (original text can be found at https://arriveguidelines.org) and based on the best practices for following the ARRIVE 2.0 guidelines in international journals. This part includes Items 4-7 of "ARRIVE Essential 10" in the ARRIVE 2.0 guidelines: "Randomization", "Blinding", "Outcome Measurement", and "Statistical Methods". Our Chinese translated version aims to promote the full understanding and use of the ARRIVE 2.0 guidelines by domestic researchers, enhancing the standardization of experimental animal research and reporting, and promoting the high-quality development of experimental animal technology and comparative medicine research in China.
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Neuropathic pain (NPP) is the main culprit among chronic pains affecting the normal life of patients. Procaine is a frequently-used local anesthesia with multiple efficacies in various diseases. However, its role in modulating NPP has not been reported yet. This study aims at uncovering the role of procaine in NPP. Rats were pretreated with procaine by intrathecal injection. Then NPP rat model was induced by sciatic nerve chronic compression injury (CCI) and behavior tests were performed to analyze the pain behaviors upon mechanical, thermal and cold stimulations. Spinal expression of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) was detected by qRT-PCR and western blot. JAK2 was also overexpressed in procaine treated model rats for behavior tests. Results showed that procaine pretreatment improved the pain behaviors of model rats upon mechanical, thermal and cold stimulations, with the best effect occurring on the 15th day post model construction (p<0.05). Procaine also inhibited JAK2 and STAT3 expression in both mRNA (p<0.05) and protein levels. Overexpression of JAK2 increased STAT3 level and reversed the improvement effects of procaine in pain behaviors (p<0.01). These findings indicate that procaine is capable of attenuating NPP, suggesting procaine is a potential therapeutic strategy for treating NPP. Its role may be associated with the inhibition on JAK2/STAT3 signaling.
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Animals , Humans , Rats , Anesthesia, Local , Behavior Rating Scale , Blotting, Western , Chronic Pain , Injections, Spinal , Janus Kinase 2 , Models, Animal , Neuralgia , Procaine , RNA, Messenger , Sciatic Nerve , STAT3 Transcription FactorABSTRACT
BACKGROUND:Osteoporosis caused by diabetes melitus as common secondary osteoporosis has been paid more and more attention recently. Zoledronic acid serves as a novel drug for osteoporosis, and its effect on osteoblasts in vivo remains unclear. OBJECTIVE:To investigate the changes of the expression of bone morphogenetic protein 2 andNoggin in the femur of type 1 diabetes melitus rats and the effect of zoledronic acid on them. METHODS:Models of type 1 diabetes melitus were established by intraperitoneal injection of streptozotocin in 130 Wistar rats. 3 days later, rats with blood sugar > 16.7 mmol/L for three consecutive times were considered as successful models, 120 in total. These models were randomly divided into model, prevention and treatment groups. Rats in the prevention and treatment groups were intravenously administered zoledronic acid (0.1 mg/kg) on the day of modeling and 2 weeks after model establishment. An additional 40 rats were injected with citrate buffer solution as control group. RESULTS AND CONCLUSION: Compared with the control group, femur bone mineral density, serum alkaline phosphatase levels, and femur bone morphogenetic protein 2 mRNA expression levels were significantly lower in the model group (P < 0.05), butNoggin mRNA expression significantly increased (P < 0.05). Compared with the model group, bone mineral density and bone morphogenetic protein 2 mRNA expression levels were significantly higher in the prevention and treatment groups (P < 0.05), butNoggin mRNA expression significantly lower (P < 0.05), and serum alkaline phosphatase levels gradualy restored. These results indicated that the bone metabolic disturbance occurs in early stage in rats with type 1 diabetes melitus. Zoledronic acid can promote bone formation, increase bone density, and improve bone metabolism.
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ObjectiveTo investigate the effects of different time administration of propofol on cytochrome c (Cyt c) in the cytoplasm in rat hippocampal neurons with hypoxia-reoxygenation (H/R) injury.MethodsPrimary cultured hippocampal neurons were randomly divided into 5 groups ( n =5 each):control group (group C),model of H/R injury group (group M),and different time administration of propofol groups (group Ⅰ,Ⅱ,Ⅲ ).In groups M,Ⅰ,Ⅱ and Ⅲ,the neurons were exposed to 95％ N2 + 5％ CO2 for6 h followed by 12 h reoxygenation.In groups Ⅰ, Ⅱ,Ⅲ,propofol was added to the culture medium before hypoxia,immediately after reoxygenation and at 2 h of reoxygenation (T0-2) respectively,with the final concentration of 20 μmol/L.The cell apoptosis was observed at T1,2 and at 24 h of reoxygenation ( T3 ) and the concentration of Cyt c in the cytoplasm was detected at T1-3.ResultsCompared with group C,the concentration of Cyt c in the cytoplasm was significantly increased at T1-3 in group M and at T1,2 in groups Ⅰ,Ⅱ,Ⅲ (P ＜ 0.05).Compared with group M,the concentration of Cyt c in the cytoplasm was significantly decreased at T1-3 in group I and at T1,2 in groups Ⅱ and Ⅲ ( P ＜0.05).The concentration of Cyt c in the cytoplasm was significantly higher at T1,2 in groups Ⅱ and Ⅲ than in group Ⅰ,and at T2 in group Ⅲ than in group Ⅱ ( P ＜ 0.05).The neuronal apoptosis was significantly decreased in groups Ⅰ,Ⅱ and Ⅲ as compared with group M.ConclusionDifferent time administration of propofol can reduce the mitochondrial Cyt c release to the cytoplasm,inhibit apoptosis in hippocampal neurons,and reduce H/R injury in rats,with better effect when given before hypoxia.
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Objective To investigate the effects of a pulsed electromagnetic field (PEMF) on cell proliferation,differentiation and the genetic expression of osteogenesis specific transcription factor-Osterix (OSX) in the calvaria-derived osteoblasts of SD rats.MethodsPrimary osteoblasts were separated from the calvarias of Sprague-Dawley rats 24 hours after birth by trypsinazation and collagenase digestion.The osteoblasts at passage 4 were randomly divided into a control group and a PEMF group,Cells in the PEMF group were placed onto PEMF therapeutic apparatus and exposed to 11 mT radiation at 12 Hz for 1 h per day for 3 days.The osteoblasts' proliferation ability was then detected via methylthiazdyl tetrazolium assay.Alkaline phosphatase (ALP) activity in the cell lysate and the supernatant fluid was assayed through disodium phenyl phosphate colorimetric determination to determine the cells'differentiation ability.OSX mRNA expression was determined using real time reverse transcription polymerase chain reaction (RT-PCR).ResultsThe proliferation of osteoblasts in the PEMF group was significantly greater than in the control group.PEMF exposure suppressed ALP activity in both the lysate and the supernatant of the osteoblasts.The OSX mRNA expression in the PEMF group was significantly down-regulated compared with the control group.ConclusionPEMF at 12 Hz and 11 mT can promote the proliferation of osteoblasts,but it inhibits cellular differentiation and down-regulates the mRNA expression of OSX.
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Objective To investigate the alteration of nuclear factor kappa B( NF-κB) and tumor necrosis factor-a (TNF-α) mRNA and protein in hippocampus in chronic constrictive injury (CCI) model of rats. Methods Seventy-six male Wistar rats were randomly divided into 2 groups ( n = 38): the CCI group which received the chronic constriction injury and the sham group which received the sham operation as control. The mechanical and thermal nociceptive thresholds were assessed with paw withdrawal latency (PWL) to von Frey filaments and radiant heat at 1d before and ld,4d,7d,14d and 28d after CCI operation. Five animals were sacrificed at each time point for real-time polymerase chain reaction (real-time PCR) and another three animals sacrificed at 7d postoperation for immunofluorescence histochemical staining. Results The thresholds to mechanical and thermal stimuli decreased obviously after operation in CCI group. The expressions of TNF-α and NF-κB mRNA began to increase at ld( (2.079 ±0. 104)times and 4d( ( 1.640 ± 0.064) times) after operation and reached the peak at 7d ((2.748 ±0.147)times, (2.010 ±0.096)times) ,then the expressions of TNF-a mRNA began to decrease,while the expressions of NF-kB mRNA maintained at a high level throughout the experiment. The result of immunofluorescence histochemical staining revealed that NF-kB and TNF-α protein expressions at 7 day increased significantly on the hippocampus,which was consisted with NF-κB and TNF-a mRNA levels. Conclusion The activation NF-κB and TNF-α in hippocampus may be involved in the procession of neuropathic pain.
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Objective To investigate the effects of propofol on mitochondrial membrane permeability during hypoxia/reoxygenation (H/R) in rat hippocampal neurons. Methods Primary cultured hippocampal neurons of fetal rats obtained from Wistar (17-18 days of gestation) were randomly divided into 3 groups: Ⅰ control group (group C), Ⅱ H/R group and Ⅲ propofol + H/R group. Neurons were cultured in the culture medium with combined oxygen glucose deprivation for 2 h followed by reoxygenation in group Ⅱ and Ⅲ. In group Ⅲ propofol was added to the culture medium with the final concentration of 20 μ mol/L before combined oxygen glucose deprivation.Neuronal viability was detected by MTT assay and mitochondrial membrane potential (MMP) with flow cytometry at 0, 4, 8, 12 and 24 h after reoxygenation (T1-5) and the permeability of cell membrane and mitochondrial membrane was monitored at T5 using laser confocal scanning microscope. Results The neuronal viability and MMP were significantly decreased (P ＜ 0.05), while the permeability of cell membrane and mitochondrial membrane was increased at T5 in group Ⅱ as compared to group Ⅰ . The neuronal viability at T1-4 and MMP at T1-5 were significantly increased (P ＜ 0.05), while the permeability of cell membrane and mitochondrial membrane was decreased at T5 in group Ⅲ compared to group Ⅱ . Conclusion Propofol can protect rat hippocampal neurons against H/R injury through increasing MMP, improving the cell and mitochondrial membrane permeability, and increasing the neuronal viability.
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Objective The present study was designed to attempt a continuous assessment of cerebral autoregulation by continuously monitoring jugular venous bulb oxygen saturation (SjO 2) in patient undergoing cardiac operation. Methods Twenty-four patients undergoing cardiac surgery under cardiopulmonary bypass (CPB) were included. Among them 16 were male, 8 female. No neuropsychological deficit was found in any of them before or after operation. Patients had neither diabetes mellitus nor immunological diseases. Normocapnia was maintained during operation. SjO 2 and other routine parameters, such as blood pressure, temperature, heart rate, pulmonary atery pressure and cardiac output etc were monitored and recorded continuously via a computerized anesthetic recording system. The numerical data of SjO 2 and mean arterial pressure (MAP) were sampled every 30s. Correlation between SjO 2 and MAP was evaluated before, during and after CPB. According to the slope "a" of the regression line "SjO 2= b + a MAP", cerebral autoregulation was estimated. The cerebral autoregulation was taken as deteriorated if the two parameters were highly correlated with a ≥0.4, showed as dysautoregulation (+). Those with a
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AIM: To establish the experimental acute pulmonary embolism(APE) model and observe the left and right ventricular pressure-volume relationship in different overload situations. METHODS: The present study consisted of seven anesthetized mongrel dogs that were divided into the control group, moderate APE group and severe APE group according to the various phase and different pressure load during the experiment. APE model was induced by suture piece injection through right cardiac catheterization. The hemodynamic indexes were measured by the simultaneous cardiac catheterization and echocardiography.RESULTS: (1) In the group with moderate APE, the pressure-volume relationship of right ventricle tended to right-upward shift, the area of chart increased, the shape of chart transformed form triangle to rectangle. The mild parallel leftward shift, the area of chart decreased mildly and no change of chart shape was observed in the pressure-volume relationship of left ventricle. (2) In the group with severe APE, the chart of right ventricular pressure-volume relationship tended to right-upward shift continuously, the area of chart decreased. The chart of the left ventricle tended to left-downward shift and no change of chart shape was observed in the pressure-volume relationship of left ventricle, the area of chart decreased. The erose shape of charts was also found.CONCLUSION: The chart of ventricular pressure-volume relationships is a practical and reliable method to evaluate left and right ventricular hemodynamic in APE.