Objective A novel compound based on near-infrared fluorescence(NIRF)probe was prepared to achieve dynamic monitoring of an inflammatory brain edema model in mice and real-time evaluation of therapeutic effects through in vivo imaging.Methods The NIRF probe IR-783 was chemically linked with clinical brain edema therapeutic drug furosemide(FSM)to obtain the new compound,IR-783-FSM.The ultraviolet fluorescence properties of the compound were evaluated using an ultraviolet spectrophotometer.The uptake of the compound by mouse macrophage cells RAW 264.7 was detected with in vitro cellular experiments.Its cytotoxicity was evaluated through CCK8 assays.A brain edema model was established in BALB/c mice via intraperitoneal injection of lipopolysaccharide(LPS),confirmed by HE staining and dry-wet weight methods for brain tissues.The mice in the brain edema model were divided into control group,IR-783,and IR-783-FSM treatment groups,receiving intraperitoneal injections of PBS,IR-783,and IR-783-FSM,respectively.Real-time in vivo fluorescence imaging was then performed.The mice in each group were euthanized after 10 hours.Ex vivo brain imaging and dry-wet weight measurements were performed to observe the NIRF imaging characteristics and therapeutic effects of IR-783-FSM on brain edema model.Results The newly synthesized compound,IR-783-FSM,retained the excellent near-infrared fluorescence characteristics of IR-783.It could target mouse macrophages with an IC50 of 48.82 μmol/L.A brain edema model could be successfully constructed with intraperitoneal injection of LPS,with significantly higher brain tissue water content compared to the control group(P＜0.01).In vivo imaging showed that IR-783-FSM had a significantly stronger fluorescence signal in the brain edema model than IR-783.Compared to the control group,the brain water content was significantly reduced in the 2,5,and 8 mmol/L IR-783-FSM treatment groups(P＜0.01).Conclusion The newly synthesized NIRF probe IR-783-FSM facilitates dynamic monitoring of brain edema and real-time evaluation of therapeutic effects.

Objective In this study,inbred BALB/c mice infected with the pneumonia virus of mice(PVM)were used to establish an animal model of viral pneumonia,and the changes in the pro-inflammatory alarmin molecule,high mobility group box 1 protein(HMGB1),during PVM infection were observed,as well as the in vivo intervention effects of the HMGB1 inhibitor,glycyrrhizic acid(GA),on PVM-induced lung injury.Methods Three-week-old female BALB/c mice were randomly divided into three groups,each consisting of 6 mice.One group,uninfected by PVM,served as the control group(Control).The other two groups were inoculated intranasally with PVM at a dose of 1×104 50％tissue culture infective dose(TCID50)/25 μL,and subsequently treated with GA saline solution(GA group)or plain saline solution(normal saline,NS group)via gavage for 15 consecutive days.During this period,changes in body weight and appearance were monitored in each group.At the end of the experiment,lung tissue samples were collected from all groups.The distribution of PVM and HMGB1 proteins in the lung tissues was analyzed using hematoxylin-eosin staining and immunohistochemistry.The expression levels of HMGB1 and its Toll-like receptor 4(TLR-4),advanced glycosylation end-product-specific receptor(AGER),and inflammatory cytokines such as interleukin(IL)-1β,IL-2,and tumor necrosis factor-α(TNF-α)in lung tissues of mice were measured using real time fluorescence quantitative PCR.Results Compared with the Control group,the NS group showed a significant weight loss after 6 days(P＜0.05).Histopathological tests revealed pronounced inflammatory lesions in their lungs.Immunohistochemistry results showed that HMGB1 was released from the nucleus to the cytoplasm,and real time fluorescence quantitative PCR results indicated that the expression levels of HMGB1,IL-1β,and IL-2 were significantly upregulated(P＜0.05).In the GA group,there was no significant change in the clinical symptoms or body weight.However,compared with the NS group,the pathological damages of lung tissues in the GA group were significantly reduced,and the expression levels of HMGB1,IL-1 β,IL-2,and interferon-γ(IFN-γ)in lung tissues were also significantly decreased(P＜0.05),although the expression level of AGER was significantly increased(P＜0.05).Conclusion PVM infection can cause significant inflammatory pathological lung damages in mice,and GA can effectively alleviate the damages.Its therapeutic effect may be related to the activation of HMGB1 signaling pathway.

Objective To establish a mouse model of pregnancy pain-depression comorbidity induced by chronic unpredictable stress(CUS),complete Freund's adjuvant(CFA),and formalin,and to systematically evaluate the associated phenotypes and preliminarily explore the pathological basis of the comorbidity.Methods Eight-week-old C57BL/6J female mice were randomly strarified divided into a control group(no intervention before pregnancy)and a CUS model group(CUS intervention before pregnancy)based on sucrose preference test(SPT)data.After completing the CUS treatment,female and male mice were paired and mated.Pain was induced by injecting 50％CFA and 5％formalin in the right hind foot during pregnancy to create a model of pregnancy pain-depression comorbidity.The experiment was divided into 8 subgroups:control-blank group,CUS-blank group,control-CFA group,CUS-CFA group,control-formalin group,CUS-formalin group,control-CFA+formalin group,and CUS-CFA+formalin group,with 10 mice in each group.The mice in each group were subject to behavioral tests,including the SPT,forced swimming test,tail suspension test,and open field test before and after CUS intervention,during pregnancy,and after delivery.Pain sensitivity changes were measured using mechanical allodynia and thermal hyperalgesia tests.Mice were then euthanized.Levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in hippocampus,as well as cortisol and adrenocorticotropic hormone(ACTH)in serum,were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with the control-blank group,the CUS-blank group showed a significant depression-like behavior with reduced pain threshold(P＜0.001).The control-CFA+formalin group showed a decrease in pain threshold after both CFA injection and formalin injection(P＜0.01).Compared with the control-blank and control-formalin groups,the pain threshold was significantly lower in the CUS-formalin group(P＜0.01),with a sequential decrease among the three.Compared with the control-blank and control-CFA groups,the pain threshold was significantly lower in the CUS-CFA group(P＜0.001),with a sequential decrease among the three.Compared with the control-blank and control-CFA+formalin groups,the mechanical pain threshold of mice in the CUS-CFA+formalin group was significantly lower(P＜0.001)and the thermal radiation tolerance time was shorter(P＜0.01),both with sequential decreases among the three.Compared with the control-CFA+formalin and the CUS-blank groups,the CUS-CFA+formalin group had a significantly lower percentage of sucrose preference(P＜0.001),longer immobility time during the forced swimming test(P＜0.001)and tail suspension test(P＜0.001),reduced central exploration time in the open field test(P＜0.001),reduced total exploration distance(P＜0.001),and reduced percentage of distance traveled for central exploration(P＜0.001).Compared with the control-CFA+formalin and CUS-blank groups,the serum cortisol and ACTH levels of the CUS-CFA+formalin group were significantly higher(P＜0.01),and the levels of IL-6 and TNF-α in the hippocampus were higher(P＜0.05).Conclusion The combination of CUS+CFA+formalin injections is an ideal method for establishing a C57BL/6J mouse model of pregnancy pain-depression comorbidity.The behavioral changes in model mice may be attributed to the regulation of inflammatory response in hippocampus and hormone levels in the hypothalamic-pituitary-adrenal(HPA)axis.

Objective To investigate the effects of microRNA(miRNA,miR)-887-3p on the proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells and its underlying molecular mechanism.Methods Annulus fibrosus tissues were obtained from 8-week-old SPF-grade SD male rats,centrifuged to prepare and identify annulus fibrosus cells.Rats in the experiment were randomly divided into four groups:a Normal group consisting of primary annulus fibrosus cells without any treatment;a Control group treated with 10 ng/mL interleukin-1β(IL-1β)for 24 hours to establish a degenerative cell model;an interference group(miR-887-3p inhibitor)transfected with miR-887-3p inhibitor using Lipo3000 based on the Control group;and an overexpression group(miR-887-3p mimics)transfected with miR-887-3p mimics using Lipo3000 based on the Control group.CCK-8 assay was used to assess cell viability;flow cytometry was used to measure cell apoptosis rates;real-time fluorescence quantitative PCR(qPCR)was used to detect the expression levels of miR-887-3p and murine double minute 4(MDM4)mRNA;Western blotting was used to measure the protein expression levels of MDM4,Bcl-2,and Caspase-3.Results Immunofluorescence staining of isolated and cultured cells revealed a Collagen I positive rate of over 90％in rat intervertebral disc annulus fibrosus cells,indicating a cell purity level greater than 90％.Real-time fluorescence qPCR results showed that after establishing an annulus fibrosus degenerative cell model using IL-1β,the expression level of miR-887-3p significantly increased compared to the Normal group(P＜0.001).Compared to the Control group,transfection with miR-887-3p inhibitor resulted in a significant decrease in its expression level(P＜0.001).The CCK-8 assay showed that compared to the Normal group,cell viability significantly decreased in the Control group(P＜0.001).Compared to the Control group,cell proliferation ability significantly increased after miR-887-3p inhibition,and significantly decreased after overexpression of miR-887-3p.Flow cytometry results revealed that compared to the Normal group,the apoptosis rate in the Control group significantly increased(P＜0.001).Compared to the Control group,the cell apoptosis rate significantly decreased in the miR-887-3p interference group(P＜0.001)and increased in the overexpression group(P＜0.001).Western blotting analysis showed that compared to the Normal group,Bcl-2 expression level significantly decreased(P＜0.001)and Caspase-3 expression level significantly increased(P＜0.001)in the Control group.Compared to the Control group,Bcl-2 and MDM4 expression levels significantly increased(P＜0.01),and Caspase-3 expression level significantly decreased(P＜0.01)in the miR-887-3p interference group;whereas in the overexpression group,Bcl-2 and MDM4 expression levels significantly decreased(P＜0.05),and Caspase-3 levels significantly increased(P＜0.05).Real-time fluorescence qPCR and protein immunoblotting results showed that after interfering with miR-887-3p,the expression of MDM4 protein and mRNA increased(P＜0.001);after overexpressing miR-887-3p,their expression decreased(protein,P＜0.01;mRNA,P＜0.001).Conclusion MiR-887-3p may modulate the cell proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells by regulating MDM4 expression,thereby influencing the development and progression of disc degeneration.

Objective To observe the effects of a 1 470 nm semiconductor laser on vaporization cutting,coagulation,and thermal injury of ex vivo animal tissues,aiming to explore the feasibility of its application in the treatment of benign prostatic hyperplasia.Methods The experimental group and control group were treated with HANS-D1 and ML-DD01FI 1 470 nm semiconductor laser therapy equipment,respectively.Fresh ex vivo pig bladder tissue was exposed to lasers with the optical fiber placed at distances of 0.5 cm and 1 cm from the tissue for 5 s.The effects of layers at powers of 60,90,120,150,and 160 W on tissue injury were observed.Ex vivo dog prostate and pig kidney tissues were used for vaporization ablation and cutting to observe the effects of lasers at the same power levels on tissue vaporization and cutting thermal injury.Additionally,in coagulation mode,the effects of 30,40,and 50 W semiconductor lasers on tissue coagulation were observed after irradiating ex vivo pig kidney tissue for 5,10,and 15 seconds.Results When the optical fiber was placed 1 cm away from the tissue,the 1 470 nm semiconductor lasers did not cause accidental damage to adjacent normal bladder tissue.However,at a distance of 0.5 cm,the 120 W,150 W,or 160 W lasers caused slight damage to the bladder tissue.In addition,with the increase in output power,the vaporization ablation efficiency of 60-160 W lasers on dog prostate tissue gradually increased,showing a good linear correlation between vaporization volume and total energy consumption(P＜0.001).Histopathological HE staining results indicated that the coagulation layer thickness in the experimental group was 292.20-309.98 μm,and the vaporization layer depth was 1.49-4.52 mm.In the control group,the coagulation layer thickness was 289.91-303.53 μm,and the vaporization layer depth was 1.88-4.43 mm.There was no significant difference between the two groups(P＞0.05).Moreover,when performing vaporization cutting on ex vivo pig kidney tissue with a cross-sectional area of 1 cm2,the efficiency of vaporization cutting by the 60-160 W 1 470 nm semiconductor lasers increased with the increase in output power(P＜0.05).The coagulated layer thickness in the experimental group was 496.04-514.47 μm,while that in the control group was 489.39-518.53 μm.Additionally,in coagulation mode,when ex vivo pig kidney tissue was irradiated for 5,10,and 15 s with 30,40,and 50 W semiconductor lasers,the coagulation diameter,groove depth,and coagulation efficiency gradually increased with the increase in laser output power(P＜0.05).The coagulation layer thickness in the experimental group and control group was 399.10-449.98 μm and 392.97-447.65 μm,respectively,and the vaporization layer depth was 3.05-7.09 mm and 2.70-7.14 mm,respectively.There was no significant difference between the two groups(P＞0.05).Conclusion The 1 470 nm semiconductor laser shows good vaporization ablation,cutting,and coagulation effect on ex vivo tissues,with a good linear correlation between the effect and the output energy.

Traumatic fractures and stress fractures are common orthopedic diseases,and there is great potential in researching bone turnover,repair,and promotion of fracture healing.Basic medical experiments often use animal models of long bone fractures in limbs to study the mechanisms of various interventions on fracture healing.Fracture healing is a complex process influenced by multiple factors and involves multiple molecules and pathways.Therefore,to explore the mechanisms more deeply,accelerate the translation of results,and improve the clinical efficacy,it is particularly important to choose the appropriate animal fracture modeling methods in experimental research.Based on this,this paper conducts a literature review of animal species and modeling methods commonly used for long bone fracture models in experimental research.It summarizes five methods:bone defect method,physical impact method,mechanical bending method,open osteotomy method,and drilling method.A side-by-side comparison of their advantages,disadvantages,and scope of application is made,aiming to provide suitable fracture models for studyingthe mechanisms of fracture healing interventions.

Objective To analyze the composition of microbial community in gut of elderly male Nycticebus bengalensis,aiming to explore the health impact factors associated with artificial captivity.Methods Fecal samples of 4 adult male Nycticebus bengalensis were collected and the V4 region was amplified using bacterial 16S rDNA universal primers.Illumina NovaSeq sequencing platform was used for microbial sequencing analysis.The complexity and similarity of the samples were analyzed using the QIIME software analysis tool.The species structure and abundance of the intestinal flora were analyzed at the phylum and genus levels based on validated data.The PICRUSt software was applied to predict the metabolic function of the flora.Results The results showed that the diversity index(Shannon index)of the flora in the youngest Nycticebus bengalensis was higher than that of the others.PCoA analysis showed that the bacterial community compositions of the four samples had a certain degree of similarity.Bacteria identified in the Nycticebus bengalensis feces included 12 phyla,18 classes,28 orders,49 families,93 genera,and 59 species.Among them,the dominant phyla were Bacteroidetes and Firmicutes with average relative abundances of 46％and 28％,respectively;The genera Bacteroides,Bifidobacterium,and Fusobacterium had higher abundances,with relative abundances of 33％,6％,and 6％,respectively;The beneficial genus Bifidobacterium was found in all samples,with the highest relative abundance found in the younger Nycticebus bengalensis.PICRUSt function prediction analysis showed that the abundance of the functional genes related to amino acid transport and metabolism,carbohydrate transport and metabolism,was relatively high.Conclusion The use of Illumina NovaSeq high-throughput sequencing technology can comprehensively detect the fecal microbial community of the Nycticebus bengalensis.The bacterial composition of Nycticebus bengalensis has rich diversity.Many bacteria are not identified and have higher relative abundance,which need further study.

Specific pathogen-free(SPF)chickens are widely used in the research of avian diseases and vaccines.Vertically transmissible diseases are transmitted to chickens through vertical transmission,seriously affecting their survival rate,increasing production costs,and causing significant economic losses to the poultry industry,while severely impacting the breeding and use of SPF chickens.Therefore,it is crucial for researchers and managers to enhance their understanding of vertically transmissible pathogens in chickens and to develop effective monitoring measures.Quality monitoring is an important part of ensuring the quality of SPF chickens,with pathogen detection being the primary step.Based on this,it is necessary to cultivate qualified SPF chickens through purification methods and biosecurity measures.This paper reviews the major vertically transmissible pathogens in chickens,including viral pathogens,bacterial pathogens and mycoplasmas,as well as their detection methods.This study compares the differences in microbiological testing items and methods for SPF chickens between the U.S.corporate standard and the Chinese national standard.Analysis of the results shows that in both standards,vertically transmissible pathogens such as Escherichia coli,Proteus mirabilis,Salmonella,and avian leukosis are not included in the microbiological testing items for SPF chickens.Instead,these pathogens are characterized by mixed infections,and outbreaks can seriously affect flock health.To produce higher-quality SPF chickens,it is necessary to include these pathogens in the mandatory testing items.The aim of this paper is to help readers understand the relevant standards for microbiological monitoring of SPF chickens,the hazards of vertically transmissible pathogens,and prevention and control strategies,so as to provide a reference for the detection and purification of pathogens in SPF chickens.

Objective To study the effects of different pellet feed hardness on the growth and reproduction,feed utilization rate,and environmental dust in laboratory mice.Methods One hundred of fifty 50 3-week-old SPF-grade C57BL/6JGpt and 150 ICR laboratory mice were randomly divided into three groups,with an equal number of males and females.They were fed diets with different hardness of 18.62 kg,23.15 kg,and 27.89 kg.Body weight,feed utilization rate,and dust levels in cages were recorded and calculated for mice aged 3-10 weeks.Forty-five 6-week-old male mice and ninety 4-week-old female mice from each strain were randomly divided into three groups and fed pellet feeds with three different hardness levels.After 2 weeks of adaptation to the same hardness feed,the mice were paired at a 1:2 male-to-female ratio and monitored for reproductive data for 3 months.Results At the age of 4 weeks,the body weight of male C57BL/6JGpt mice in 23.15 kg group was significantly higher than that in the 18.62 kg and 27.89 kg groups(P＜0.01),and the body weight of females in the 18.62 kg group was significantly higher than that in the 27.89 kg group(P＜0.05).There was no significant difference in body weight among ICR mice aged 3-10 weeks across different feed hardness groups(P＞0.05).For both strains,feed utilization rate for males was higher than that for females across different feed hardness groups at all weeks of age(P＜0.01).Compared to the 27.89 kg group,both the 18.62 kg and 23.15 kg groups showed a significant increase in the 50-mesh dust levels in cages for both strains aged 4-8 weeks(except for 7-week-old C57BL/6JGpt mice)(P＜0.05).For both C57BL/6JGpt and ICR mice,there was no significant difference in basic reproductive performance such as interval between the first litter and the monthly production index among the three feed hardness groups during the experimental period(P＞0.05).However,the monthly production index of C57BL/6JGpt mice first increased and then decreased with the increase of feed hardness,while that of ICR mice increased with increasing feed hardness,though these differences were not statistically significant(P＞0.05).Conclusion Different strains and genders had different tolerance to feed hardness.C57BL/6JGpt mice are more adapted to lower hardness feeds,while ICR mice are better suited to slightly higher hardness feeds.

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.