Establishment of anterior cruciate ligament reconstruction model in cynomolgus monkey with autogenous hamstring tendon transplantation / 中国修复重建外科杂志

OBJECTIVE@#To investigate the feasibility of establishing an anterior cruciate ligament (ACL) reconstruction model using hamstring tendon autograft in cynomolgus monkeys.@*METHODS@#Twelve healthy adult male cynomolgus monkeys, weighing 8-13 kg, were randomly divided into two groups ( n=6). In the experimental group, the ACL reconstruction model of the right lower limb was prepared by using a single bundle of hamstring tendon, and the ACL of the right lower limb was only cut off in the control group. The survival of animals in the two groups was observed after operation. Before operation and at 3, 6, and 12 months after operation, the knee range of motion, thigh circumference, and calf circumference of the two groups were measured; the anterior tibial translation D-value (ATTD) was measured by Ligs joint ligament digital body examination instrument under the loads of 13-20 N, respectively. At the same time, the experimental group underwent MRI examination to observe the graft morphology and the signal/ noise quotient (SNQ) was caculated.@*RESULTS@#All animals survived to the end of the experiment. In the experimental group, the knee range of motion, thigh circumference, and calf circumference decreased first and then gradually increased after operation; the above indexes were significantly lower at 3 and 6 months after operation than before operation ( P<0.05), and no significant difference was found between pre-operation and 12 months after operation ( P>0.05). In the control group, there was no significant change in knee range of motion after operation, showing no significant difference between pre- and post-operation ( P>0.05), but the thigh circumference and calf circumference gradually significantly decreased with time ( P<0.05), and the difference was significant when compared with those before operation ( P<0.05). At 6 and 12 months after operation, the thigh circumference and calf circumference were significantly larger in the experimental group than in the control group ( P<0.05). At 3 and 6 months after operation, the knee range of motion was significantly smaller in the experimental group than in the control group ( P<0.05). Under the loading condition of 13-20 N, the ATTD in the experimental group increased first and then decreased after operation; and the ATTD significantly increased at 3, 6 months after operation when compared with the value before operation ( P<0.05). But there was no significant difference between the pre-operation and 12 months after operation ( P>0.05). There was no significant change in ATTD in the control group at 3, 6, and 12 months after operation ( P>0.05), and which were significantly higher than those before operation ( P<0.05). At each time point after operation, the ATTD was significantly smaller in the experimental group than in the control group under the same load ( P<0.05). The MRI examination of the experimental group showed that the ACL boundary gradually became clear after reconstruction and was covered by the synovial membrane. The SNQ at each time point after operation was significantly higher than that before operation, but gradually decreased with time, and the differences between time points were significant ( P<0.05).@*CONCLUSION@#The ACL reconstruction model in cynomolgus monkey with autogenous hamstring tendon transplantation was successfully established.

Preliminary report of perioperative monitoring of six-gene-edited pig-to-cynomolgus monkey kidney xenotransplantation / 器官移植

Objective To investigate the establishment of a six-gene-edited pig-to-non-human primate kidney xenotransplantation model. Methods The kidney of humanized genetically-edited pig (GTKO/β4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM) was transplanted into a cynomolgus monkey. The survival of the recipient and kidney condition after blood perfusion were observed. The parenchymal echo, blood flow changes, and size of the kidney were monitored on a regular basis. Routine blood test, kidney function test and electrolyte assessment were carried out. Dynamic changes of urine, feces and body mass were monitored. At the end of life, the transplant kidney, heart, liver, spleen, lung, and cecum were collected for pathological examination. Results The recipient died at postoperative 7 d. After blood flow was restored, the kidney was properly perfused, the organ was soft and the color was normal. At the end of the recipient's life, a slight amount of purulent secretion was attached to the ventral side of the kidney, with evident congestion and swelling, showing the appearance of "red kidney". Postoperatively, the echo of renal parenchyma was increased, blood flow was decreased, the cortex was gradually thickened, and a slight amount of effusion surrounded the kidney and abdominal cavity over time. In the recipient, the amount of peripheral red blood cells, hemoglobin, albumin, and platelets was progressively decreased, and serum creatinine level was increased to 308 μmol/L at postoperative 7 d, whereas the K+ concentration did not significantly change. Light yellow urine was discharged immediately after surgery, diet and drinking water were resumed within postoperative 3 h, and light yellow and normal-shape stool was discharged. The reddish urine was gradually restored to normal color within postoperative 1 d, which were consistent with the results of the routine urine test. A large amount of brown bloody stool was discharged twice in the morning of 2 d after surgery. Omeprazole was given for acid suppression, and the stool returned to normal at postoperative 4 d. The β2-microglobulin level was increased to 0.75 mg/L at postoperative 7 d. The body mass was increased by 1.7 kg. Autopsy pathological examination showed interstitial edema and bleeding of the transplant kidney, a large amount of infiltration of lymphocytes and macrophages, infiltration of lymphocytes in the arteriole wall and arterial cavity, accompanied by arteritis changes, lymphocyte infiltration in the cecal stroma and congestion in the spleen tissues. No significant abnormal changes were observed in other organs. Conclusions The humanized genetically-edited pig-to-non-human primate kidney xenotransplantation model is successfully established, and postoperative survival of the recipient is 1 week.

Comparison on the establishment of proprioceptive hypoesthesia model of anterior cruciate ligament by open surgery and minimally invasive surgery / 解剖学报

[Abstract] Objective To provide an optimized animal model for basic research by comparing the establishment of proprioceptive hypoesthesia model of anterior cruciate ligament (ACL) by open surgery and minimally invasive surgery. Methods Totally 30 normal cynomolgus monkeys were randomly divided into five groups: minimally invasive surgery group: unilateral ACL injury under arthroscope, n = 6; open surgery group: unilateral ACL injury through direct incision of knee joint, n = 6; minimally invasive sham operation group: unilateral ACL without injury through arthroscopic cleaning only, n = 6; open surgery group: unilateral ACL exploration without injury through direct incision of knee joint, n = 6; positive normal group: no surgical intervention, n = 6. Four weeks later, the neurophysiological examination [somatosensory evoked potentials (SEPs) and motor nerve conduction velocity (MCV)] was carried out. The macaque was killed and its unilateral ACL was stained with gold chloride. The number and variation of ACL proprioceptors were observed and recorded. Results In the aspect of neuroelectrophysiology, except the normal group, the latency of SEPs and MCV were prolonged, and the amplitude decreased. In terms of proprioceptors, the total number of minimally invasive surgery group: 578. 00± 12. 68, amplitude: 36. 33 ± 3. 72; total number of open surgery group: 367. 67 ± 9. 33, amplitude: 77. 00 ± 5. 55; total number of open sham operation group: 969. 00±18. 26, amplitude: 0±0; total number of normal group: 970. 46±16. 34, amplitude: 0± 0, the total number of proprioceptors decreased and the number of variation increased in all four groups except the normal group. At the same time, in the comparison of the two aspects, there were significant differences between the open operation group and the minimally invasive operation group, the open operation group and the open sham operation group, and the minimally invasive operation group and the minimally invasive sham operation group (P0. 05). Conclusion Both the open operation group and the minimally invasive operation group can build ACL proprioceptive hypoesthesia model, but the minimally invasive operation has less damage to the tissues around ACL, more scientific and single model, and less experimental error, which is of great significance in the basic research of ACL injury.

Effect of CRISPR/Cas9-mediated PD-1 gene knockout on proliferation, phenotype,IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation, phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNA of T cells was extracted for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3 antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands(243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly (both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.

Anxiety and depression like behaviors in chronic Parkinsonism of cynomolgus monkey / 中华行为医学与脑科学杂志

Objective To establish and evaluate anxiety and depression like behaviors in MPTP-in-duced chronic Parkinsonism cynomolgus monkey. Methods Ten cynomolgus monkeys ( 15. 0 ± 1. 5 years old) were divided into normal group and MPTP-induced parkinsonism group(PD group),5 animals in each group. The behavioral methods for evaluating the anxiety and depression like behaviors in non-human prima-tes included Apathy Feeding Test (AFT),Human Intruder Test (HIT),Novel Fruit Test (NFT) and Preda-tor Confrontation Test (PCT). The changes in behavioral indicators between the two groups were analyzed. Results In AFT,PD group spent more time in observing food on both sides,and significantly higher than that in normal group (left:0. 73(0. 49) s,0. 50(0. 28) s,P<0. 01;right:0. 75(0. 60) s,0. 53(0. 16) s,P<0. 01),moreover PD group had limb disorder on the right side,and spent more time in taking food which sig-nificantly more than normal group (0. 83(0. 20) s,0. 57(0. 14) s,P<0. 01). In HIT,PD group moved and stood better than that in normal group,and occurred abnormal behaviors,such as climbed,grinded,scratched and shook cages (P>0. 05),while normal group spent more time in the back of cage during Stare and Back period( Stare:0(64. 70)s,111. 27(38. 94)s,P=0. 033; Back:0(25. 84) s,120(114. 69) s; H=17. 397). Finally,PD group had lower take-up rate and higher withdrawal rate than normal group in PCT(P>0. 05). Conclusion This study establishes a multi-angle evaluation of behavioral methods for anxiety and depres-sion in non-human primates. The analysis confirmed that chronic Parkinson's disease cynomolgus monkeys has different degrees of anxiety and depression,which provides a new method for preclinical intervention in Parkinson's disease.

Establishment of acute liver failure models induced by D-galactosamine in non-human primates / 器官移植

Objective To evaluate the effect of different doses of D-galactosamine (D-Gal) on non-human primate cynomolgus monkey and to establish a monkey model with different degree of acute liver failure (ALF). Methods Twelve cynomolgus monkeys were evenly divided into the low-, medium- and high-dose groups (n=4) with a dosage of 0.23, 0.25 and 0.27 g/kg, respectively. In each group, the corresponding dose of D-Gal solution was injected into the monkeys through the forearm vein at one time in a sober state (without anesthesia). The survival time of the cynomolgus monkeys was recorded. Digestive tract and hepatic encephalopathy symptoms were observed. Vital signs were measured at 0 h before and 12, 24, 36, 48, 60, 72, 96, 120 and 144 h after D-Gal administration. Alanine transaminase (ALT), total bilirubin (TB), prothrombin time (PT), blood ammonia and other parameters were detected from the blood samples. The liver tissues were prepared for hematoxylin-eosin (HE) staining to observe the pathological changes. Results All cynomolgus monkeys in the low-dose group survived and transient liver injury was noted without the hepatic encephalopathy symptoms. At 60 h after D-Gal administration, the liver function and coagulation indexes reached the peak, gradually recovered and then basically returned to the normal range at 120 h. In the medium-dose group, the course of disease was relatively slow and gradually recovered after the appearance of severe liver damage and hepatic encephalopathy symptoms and only one animal died. All cynomolgus monkeys in the high-dose group died after developing hepatic encephalopathy symptoms and severe liver damage with a mean survival time of (72±13) h. Pathological examination of liver tissue demonstrated that scattered liver cell necrosis and inflammatory cell infiltration were observed in the liver tissues of the low-dose group. In the medium- and high-dose groups, the hepatic lobule structure was not clear, and the liver cell necrosis in flakes accompanied by evident hemorrhage were documented. Conclusions The D-Gal dosage in the medium- and high-dose groups meet the standards of the ALF model. The degree of ALF in the medium-dose group is relatively slight, which is beneficial to the implementation of liver transplantation. ALF in the high-dose group is relatively severe, which is suitable for the evaluation of the clinical efficacy of therapeutic options.

Evaluation of fecal microbiomes associated with obesity in captive cynomolgus monkeys (Macaca fascicularis)

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.

Establishment of a method for culturing cynomolgus T lymphocytes induced by human CD3Ab / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To establish a method for in vitro isolation and culture of T lymphocytes from peripheral blood of cynomolgus monkeys that induced by human CD3 antibody based on the foundation of protein homology of CD3 from human, cynomolgus monkey and porcine. Methods: The amino acid sequences of human, cynomolgus monkeys and porcine CD3 proteins were obtained from NCBI, and the sequence, homology and phylogenetic tree were analyzed by DNAMAN software. Western blotting was used to detect the expression of CD3 protein on T cell membranes from the three species. PBMCs of healthy cynomolgus were isolated and divided into three groups: group A was stimulated with anti-human CD3Ab alone, group B was stimulated with IL-2 alone, and group C was costimulated with human CD3Ab and IL-2. Cell morphology and growth status were observed under inverted microscope and the cell growth curve was plotted. Cell viability was detected by trypan blue staining and the expressions of CD3, CD4 and CD8 on T cell surface were detected by flow cytometry. Results: The homology of the amino acid sequence of human CD3 protein to cynomolgus monkey and porcine were 86.9% and 65.6% respectively. The expression levels of CD3 protein on cynomolgus and porcine T cell membrane were 79% and 17% contrast to human, respectively. Cells of group A did not proliferate. Proliferation, viability and CD3 expression [(93.8±3.6)% vs (70.3±4.7)%, P<0.01] in T cells of group C were significantly higher than those in group B. Growth curve of T cells in group C showed an S-shape, which is consistent with Logistic growth curve. T cells in group C exhibited high purity and expressed high level CD3; moreover, the CD8+T cell took a high proportion. Conclusion: The membrane of T lymphocytes from peripheral blood of cynomolgus can express CD3 protein that highly homological to human. Co-stimulation of human CD3Ab, IL-2 and 1% PHAcan induce the proliferation and differentiation of T lymphocytes of cynomolgus, and obtain T lymphocytes with good growth status, high proliferation ability and high purity.

Analysis of the ammonia level in air and heavy metals in drinking water in a cynomolgus monkey feedlot / 中国比较医学杂志

Objective The ammonia level in air and heavy metals in drinking water were explored in a cynomolgus monkey feedlot. Methods Air ammonia from different communities and feeding patterns of cynomolgus monkeys were collected at three time-points per day and determined by Nessler's reagent spectrophotometry. Inductively coupled plasma mass spectrometry was applied to detect the level of heavy metals in drinking water from 5 different sampling sites in the feedlot,incluing the outlet of underground water,the water tank,both monkey cages equipped with PVC or iron pipes and sewage lagoon,respectively. Results Air ammonia levels in quarantine inspection flock cages(0.59 ± 0.03 mg/m3)were significantly higher than in the cages of both reproductive flock(0.34 ± 0.03 mg/m3)and sale flock(0.27 ± 0.04 mg/m3). The ammonia level in air in different feeding patterns ranks as following:cage rearing of quarantine inspection flock(0.59 ± 0.03 mg/m3)>cage rearing of reproductive flock(0.48 ± 0.02 mg/m3)>captive bleeding of sale flock(0.30 ± 0.02 mg/m3)>cage rearing of sale flock(0.25 ± 0.01 mg/m3)> captive bleeding of reproductive flock(0.22 ± 0.02 mg/m3). The air ammonia concentrations of both former flocks were statistically higher than the latter three flocks. The highest air ammonia level among different flocks and feeding patterns occurred in the morning, before waste discharge clean-up. The iron concentration in drinking water in the cages equipped with iron pipes was higher than Chinese drinking water standard. Conclusions The air ammonia level was lower than the Chinese air quality standard. The iron concentration in drinking water in the cages equipped with iron pipes was higher than the Chinese drinking water standard.

Establishment of a cynomolgus monkey model of gastrocnemius muscle atrophy / 中国实验动物学报

Objective To establish a monkey model of muscle atrophy by fixing the right lower limb and set up e-valuation criteria for that model. Methods Twelve healthy,6-7 year old male cynomolgus monkeys(body weight 6-7 kg)were used in this study. All animals were normal and had no malformed lower limb bones. Right lower limbs of the an-imals were fixed with fiberglass bandage(from knee joint to anklebone)for 15 weeks. Body weight,crus perimeter and crus volume were recorded every two weeks. MRI examinations of the gastrocnemius muscle were conducted at weeks 11,13 and 15. Muscle biopsy was taken for pathological examination at week 15. Results There were no obvious changes of body weight during the experiment. At 15 weeks after modeling,the right crus perimeter and crus volume of the experimental an-imals were significantly decreased(P <0.05),and some biochemical indexes such as serum creatinine,gloucose and al-kaline phosphatase were significantly decreased(P <0.05), while cholesterol was significantly increased(P <0.05).MRI showed atrophy of the right gastrocnemius muscle. Muscle biopsy also showed muscle atrophy and muscle fibers be-came thinner in the right gastrocnemius,and the cross-section area of the muscle fibers was significantly decreased. Con?clusions It is easy to operate and establish a muscle atrophy model in Cynomolgus monkeys by fixing the right lower limb. This model is suitable for objective and easy evaluation of muscle atrophy by measurement of crus perimeter and crus volume of the limb,blood biochemical parameters,MRI examination,and histopathological examination in combination.

Establishment of immune liver fibrosis models in cynomolgus monkeys / 中国免疫学杂志

Objective:To establish liver fibrosis models in cynomolgus monkey using calf serum.Methods:Healthy cynomolgus monkeys were randomly divided into control group and model group,and there were 6 animals in each group.Model group were treated with calf serum subcutaneously at the dose of 0.4 ml/kg,and control group were received the same dose of normal saline.Calf serum will be administrated twice a week.Results:After 8 weeks of administration,the serum HA,PC Ⅲ,Ⅳ C,TGF-β1 and α-SMA in model group were increased differently when compared with modeling before(P＜0.05,P＜0.01),and the serum HA and PC Ⅲ in model group were also increased significantly when compared with control group.After 12 weeks of administration,the serum Ⅳ C,TGF-β1 and α-SMA in model group were increased significantly compared with control group(P＜0.05),and the serum LN and TCH in model group were increased obviously when compared with modeling before(P＜0.05),the levels of serum AST and ALT in model group were also increased distinctly as compared with control group and modeling before(P＜0.05),while the serum ALB in model group was decreased significantly when compared with modeling before(P＜0.05).At week 16,the serum LN,TCH and TG in model group were increased significantly when compared with control group(P＜0.05);while the serum ALB in model group was decreased distinctly when compared with control group(P＜0.05);the strong echo were showed in liver of model group by using ultrasonography;pathological examination showed that there were necrosis and monocyte infiltrating in liver of model group,and collagen fiber proliferation were observed in portal area of model group.Conclusion:Liver fibrosis models can be established successfully in cynomolgus monkeys by subcutaneously injecting calf serum for 16 weeks.

Effect of Eylea on recombinant AAV2-VEGF-induced choroidal neovascularization / 中华实验眼科杂志

Objective To evaluate the effect of Eylea (aflibercept) on recombinant adeno-associated virus 2 (AAV2)-vascular endothelial growth factor (VEGF)-induced choroidal neovascularization (CNV).Methods Two normal cynomolgus monkeys were used in this study.Recombinant AAV2-VEGF was delivered by subretinal injection (60 μl/eye,7.0×1011 IU/ml) into both eyes for each cynomolgus monkey.Both eyes of one animal were treated with single intravitreal injection of Eylea (50 μl/eye,40 mg/ml) 3 weeks after AAV2-VEGF injection,and the two eyes of the other animal were untreated.Optical coherence tomography (OCT),electroretinography (ERG),fluorescein angiography (FFA) and ocular photography were conducted.The experiment was approved by the Institutional Animal Care and Use Committee (IACUC) at JOINN Laboratories (Suzhou) (IACUC serial number:ACU15-1112).Results OCT showed that subretinal hyperreflective material,retinal edema,choroid edema and local pigment epithelial detachment were found after injection of recombinant AAV2-VEGF.All of those symptoms were relieved after treated with Eylea,but aggravated after 4 weeks treated with Eylea.FFA showed that the area of fluorescein leakage expanded obviously after 2 weeks treated with AAV2-VEGF,and it was observed at the whole of fundus posterior pole at 5 weeks after injection.After 1 week treated with Eylea,the area of fluorescein leakage decreased obviously,but increased gradually after 4 weeks treated with Eylea.ERG results showed that the amplitude of ERG decreased gradually after injection of recombinant AAV2-VEGF.After intravitreal injection of Eylea,the amplitude of ERG increased gradually,but it decreased again after 4 weeks treated with Eylea.Conclusions Injection of recombinant AAV2-VEGF is capable of inducing CNV and pathological retinal in cynomolgus monkey.Eylea can prevent the development of clinically relevant CNV,but the effect just lasts for a period.

Population reproductive regularity and reproductive performance of rhesus monkeys and cynomolgus monkeys in Kunming area / 中国比较医学杂志

Objective To explore the population reproductive regularity and reproductive performance of rhesus and cynomolgus monkeys in Kunming area, and to provide reference data for the construction of rhesus and cynomolgus monkey breeding base,reproductive biology research and biological resources protection.Methods The reproductive regularity and reproductive performance in a rhesus monkey population of 20 males and 130 females, and a cynomolgus monkey population of 120 males and 780 females in a large-scale experimental monkey breeding base in Kunming were observed for a whole year and statistically analyzed.Results There was a distinct seasonal variation in the reproduction of rhesus monkeys in Kunming area, while no obvious seasonal changes was observed in cynomolgus monkeys.The pregnancy rate, reproductive rate and neonatal survival rate of rhesus monkeys were 76.15%, 69.23% and 90.70%, respectively.The pregnancy rate, neonatal reproduction rate and survival rate of cynomolgus monkeys were 78.98%, 74.87% and 94.81%, respectively.The menstrual cycle and pregnancy period of rhesus monkeys were (28.80 ± 2.33) d and (165.87 ± 7.52) d, respectively.The menstrual cycle and pregnancy period of cynomolgus monkeys were (29.35 ± 3.05) d and (157.93 ± 5.42) d, respectively.The average birth weight and weaning weight of rhesus monkey pups were (425.00 ± 100.50) g and (1491.67 ± 172.35) g, and those of cynomolgus monkeys were (314.33 ± 61.18) g and (1013.50 ± 115.50) g, respectively.Conclusions The reproduction regularity of rhesus and cynomolgus monkeys in Kunming area are defined.The reproductive performance of rhesus and cynomolgus monkeys are reported in detail.It provides basic data for the breeding of rhesus and cynomolgus monkeys in Kunming area and the development of research of experimental monkey reproductive biology.

Effects of anti CD3 monoclonal antibody on immunity and gene expression profiles of cynomolgus monkeys / 中国药学杂志

OBJECTIVE: To investigate the effects of anti CD3 monoclonal antibody (Ant-CD3 McAb) on immune function in cynomolgus monkeys, using the traditional immunotoxicity assessment methods and T cell dependent antibody response (TDAR) test, microarray, etc, and further explore the mechanism of Ant-CD3 McAb. METHODS: Eighteen Cynomolgus monkeys were individually dosed with saline, 0.5 and 2.5 mg·kg-1Ant-CD3 McAb for 7 d. The clinical observation, body weight, hematological test, antibody detection, TDAR test, lymphocyte subsets, bone marrow, histopathological examination and whole blood gene expression profile analysis were evaluated. RESULTS: Ant-CD3 McAb significantly inhibited the immune response to keyhole limpet hemocyanin (KLH) in cynomolgus monkey, and caused the decrease of peripheral white blood cell counts and CD3+ cells, the decrease of lymphocytes in thymus cortex. Microarray test showed that the gene function of differently expressed genes mainly involved in cell proliferation, immune response, cytokine secretion, apoptosis etc. CONCLUSION: Ant-CD3 McAb could induce obvious immunosuppression effects in cynomolgus monkeys. Detection of gene expression profile of whole peripheral blood is feasible in immunotoxicity evaluation. The apoptosis induction and proliferation inhibition of T lymphocytes might be the main cause of the decreased T lymphocytes. IL-8 might play an important role in the mechanism of Ant-CD3 McAb.

EXPERIMENTAL STUDY ON LENTIVIRUS-MEDIATED MULTI-GENES CO-TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF KNEE OSTEOARTHRITIS IN CYNOMOLGUS MONKEY / 中国修复重建外科杂志

OBJECTIVE: To observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA). METHODS: BMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×107 cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR. RESULTS: No significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P<0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P<0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P<0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P<0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P<0.05). CONCLUSIONS: Lentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.

PCR test of Helicobacter pylori and“Helicobacter macacae”infections in rhesus and cynomolgus monkey breading colonies / 中国比较医学杂志

Objective To investigate the status of Helicobacter pylori and “Helicobacter macacae” infection in rhesus and cynomolgus monkeys in China.Methods With the use of 16S rRNA specific primers for Helicobacter spp and Helicobacter pylori ( HP ) from published literatures, and new 16S rRNA specific primers designed for “Helicobacter macacae” ( HM ) , we investigated the infection status of these two Helicobacter spps in both of 45 rhesus and 90 cynomolgus monkeys by qPCR or conventional PCR on stool samples.Results All three primer sets for 16S rRNA exhibited excellently sensitivity and specificity.Both the infection rates of HP and HM were 100% among 45 young adult rhesus monkeys.The infection rate of HP and HM in 90 young adult cynomolgus monkeys were 100% and 97.8%, respectively.Conclusions Helicobacter pylori and “Helicobacter macacae” are present in almost every artificially bred adult rhesus and cynomolgus individuals which may adversely affect the health of laboratory monkeys and the accuracy of related animal experiments.

Establishment of delay eyeblink conditioned reflex and the acquisition rule in cynomolgus monkey / 中华行为医学与脑科学杂志

Objective To investigate the method of establishing a model of delay eyeblink condi-tioning ( DEC) in no surgical cynomolgus monkey and analyze the related acquisition rule.Methods Using the special monkey chairs to fix 6 adult male cynomolgus monkeys,they were sitting on the chairs and keep-ing awake,and heads could rotate freely during the whole training session.In the experiment,the 6 monkeys were trained in DEC with a tone (500 ms,85 dB) as the conditioned stimulus (CS),paired with a corneal oxygen-puff (100 ms,5 psi) as the unconditioned stimulus (US).There were 120 trials per-session,and 2 sessions per-day.During the experiments,an infrared emitter/detector attached to spectacles for eyeblink re-cording,the data of conditioned response ( CR) and startle response ( SR) were analyzed offline.Result-s Among the 6 cynomolgus monkeys,4 of them completed all of the training process of DEC and 3 monkeys had successful acquisition of DEC.The average CR rate in the last training session reached ( 64.67 ± 2.00)%,(P<0.01);1 monkey only showed a high acquisition rate( 85.00%) at the first training session, and the low CR rate in the next training sessions with the average rate of 18.16%,suggesting that the DEC model was failed to established.The SR rates in 4 cynomolgus monkeys were low with average rate of 5.47%. Conclusion Although the DEC behavior training of the cynomolgus monkey is easy to be influenced by many inside or outside factors,DEC can be obtained under the condition of non surgery and proper braking.

Reference values of hematology, biochemistry, and blood type in cynomolgus monkeys from cambodia origin / 한국실험동물학회지

Cynomolgus monkeys as nonhuman primates are valuable animal models because they have a high level of human gene homology. There are many reference values for hematology and biochemistry of Cynomolgus monkeys that are needed for proper clinical diagnosis and biomedical research conduct. The body weight information and blood type are also key success factors in allogeneic or xenogeneic models. Moreover, the biological parameters could be different according to the origin of the Cynomolgus monkey. However, there are limited references provided, especially of Cambodia origin. In this study, we measured average body weight of 2,518 Cynomolgus monkeys and analyzed hematology and serum biochemistry using 119 males, and determined blood types in 642 monkeys with Cambodia origin. The average body weight of male Cynomolgus monkeys were 2.56±0.345 kg and female group was 2.43±0.330 kg at the age from 2 to 3 years. The male group showed relatively sharp increased average body weight from the 3 to 4 age period compared to the female group. In hematology and biochemistry, it was found that most of the data was similar when compared to other references even though some results showed differences. The ABO blood type result showed that type A, B, AB, and O was approximately 15.6, 33.3, 44.2, and 6.9%, respectively. The main blood type in this facility was B and AB. These biological background references of Cambodia origin could be used to provide important information to researchers who are using them in their biomedical research.

Reference values of clinical pathology parameters in cynomolgus monkeys (Macaca fascicularis) used in preclinical studies / 한국실험동물학회지

Nonhuman primates are increasingly used in biomedical research since they are highly homologous to humans compared to other rodent animals. However, there is limited reliable reference data of the clinical pathology parameters in cynomolgus monkeys, and in particular, only some coagulation and urinalysis parameters have been reported. Here, we reported the reference data of clinical chemical, hematological, blood coagulation, and urinalysis parameters in cynomolgus monkeys. The role of sex differences was analyzed and several parameters (including hematocrit, hemoglobin, red blood cell, blood urea nitrogen, total bilirubin, alkaline phosphatase, creatinine kinase, gamma-glutamyl tranferase, and lactate dehydrogenase) significantly differed between male and female subjects. In addition, compared to previous study results, lactate dehydrogenase, creatinine kinase, and aspartate aminotransferase showed significant variation. Interstudy differences could be affected by several factors, including age, sex, geographic origin, presence/absence of anesthetics, fasting state, and the analytical methods used. Therefore, it is important to deliberate with the overall reference indices. In conclusion, the current study provides a comprehensive and updated reference data of the clinical pathology parameters in cynomolgus monkeys and provides improved assessment criteria for evaluating preclinical studies or biomedical research.

Dynamic monitoring of virus antibody during the establishment of SPF cynomolgus monkey population / 中国实验动物学报

Objective To investigate the positive and suspicious rates of BV, SRV, SIV and STLV-1 antibody of the cynomolgus monkeys introduced from Laos, dynamic monitoring of virus antibody during the establishment of SPF cyno-molgus monkey population, and then comparing the positive rate of virus antibody in the baby monkeys between the common with SPF populations.Methods Continuous monitoring four kinds of virus in the monkeys was carried out using special reagent kits and analyzed the data.Results In the introduced cynomolgus monkeys (n=1998), the positive rate of BV antibody was 52.35%, suspicious rate of BV antibody was 8.31%, and negative rate of BV antibody was only 39.34%, the positive rates of SRV and STLV-1 antibody were 7.45%and 8.56%, respectively, and positive or suspicious SIV anti-body in the cynomolgus monkeys was not detected.After screening and establishment of the SPF population, the positive rates of BV, SRV and STLV-1 antibody were 5.24%, 1.01%and 0.4%, respectively, in the year 2010.Then after five years of screening and elimination, by the end of 2014, the positive rates of BV, SRV and STLV-1 antibodies were 0.82%, 0.27%and 0.27%, respectively, and positive or suspicious SIV antibody in the cynomolgus monkeys was not de-tected.The BV antibody positive rate of the baby monkeys was 9.71%and the suspicious rate was 1.85%in the common population, but only 0.22%of the baby monkeys in the SPF population.Conclusions Continuous monitoring virus anti-bodies and eliminating positive and suspicious animals have important significance in the establishment of a SPF cynomolgus monkey population.