Abstract:Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank. sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C57BL/6 mice. After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile, real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice. Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas9. After microinjection, miRNA-29b1 gene-mutated mice were obtained. The sequencing results showed that there were two types of genotype for the mutated mice, one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion. Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly. Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR/Cas9 technology.

Abstract:Objective To study the feasibility of rat models of renal edema based on disease combined with traditional Chinese medicine syndrome yin-deficiency and yang-deficiency types. Methods Gastric gavage of thyroxine and tail vein injection of Adriamycin were performed to induce a rat model of kidney Yin deficiency edema, and intramuscular injection of hydrocortisone and tail vein injection of Adriamycin were used to establish a rat model of kidney Yang deficiency edema. The 24 h urine protein content, triiodothyronine (T3), thyroxine (T4), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), estradiol (E2) and testosterone (T) and other indicators were assayed to determine whether the rat models were successfully established. Result The rats of Yin deficiency edema group had clinical presentation such as hyperactivity, hair loss, dry stool, weight loss and temperature rise. Compared with the blank group, T3, T4, cAMP, E2, and 24 h urinary protein levels were significantly increased, cGMP and T content decreased, and cAMP/cGMP ratio was significantly increased, showing significant differences (P<0.05 for all). The Yang deficiency edema animals displayed reduced activity, diarrhea, decreased body weight and body temperature, and other signs of disease. Compared with the blank group, T3, T4, cAMP, and E2 contents were significantly decreased, cGMP, T, and 24 h urinary protein levels were significantly increased, and cAMP/cGMP ratio decreased, showing significant differences (P<0.05 for all). Conclusions The rat model of renal edema disease combined with TCM syndrome yin-deficiency was successfully induced by thyroxine in combination with Adriamycin, and the rat model of renal edema based on disease combined with TCM syndrome yang-deficiency is successfully established by administration of hydrocortisone plus Adriamycin. These two rat models demonstrate similar clinical manifestations of human renal edema based on disease combined with TCM syndrome yin-deficiency and yang-deficiency, respectively, therefore, may serve as useful tools for further research on this disease.

Abstract:Objective To investigate the effects of long-term consumption of Fallopia multiflora on mouse hematopoietic system. Methods Forty 10-month old female C57BL/6J mice were equally divided into two groups at random, the control group fed with normal food, and the experimental group, given food with added Fallopia multiflora. After 10 month, the mice were sacrificed, and the peripheral blood, spleen, thymus and bone marrow cells were examined by flow cytometry. Results In the mice fed with Fallopia multiflora, the percentage of B cells in the spleen and CD4⁺ cells in the thymus were increased, and CD8⁺ cells in the thymus and bone marrow hematopoietic stem cells were decreased, among the bone marrow cells, G0 cells were increased, but G1 and G2/S/M cells decreased. Conclusions Long-term proper consumption of Fallopia multiflora can delay the ageing of the hematopoietic system, and sustain its stability.

Abstract:Objective To explore the feasibility of transauricular arterial access for hepatic artery catheterization in rabbits. Methods Thirty healthy New Zealand White rabbits were randomly divided into 5 groups (n=6 in each group):transauricular vein injection group, transarterial infusion group, transarterial lipiodol group, transarterial gelfoam group and transhepatic puncture group. Every rabbit was prescribed elemene (20 mg/kg) via different access in 6 minutes. All the rabbits of hepatic artery catheterization were divided into two groups according to their serial number:transauricular arterial access group (odd, n=9) and transfemoral arterial access group (even, n=9). The arterial access could be changed each other due to the failure of one technique. The catheterization time, success rate and survival rate were compared between the two groups. Venous blood collection via auricular vein or jugular vein for pharmacokinetics was performed in each rabbit. Results Technical success rates of hepatic artery catheterization were 0% (0/9) and 88.9% (16/18) for transauricular and transfemoral arterial access, respectively. The time duration of transauricular and transfemoral access groups was 28.4±13.6 and 33.9±19.6 minutes, respectively (P>0.05). The survival rates of the transauricular and transfemoral access groups were 100% (9/9) and 88.9% (16/18), respectively. Blood samples were collected via auricular vein in 4 and jugular vein in 23 rabbits. Conclusions Hepatic artery catheterization via transauricular arterial access is technically not feasible, while transfemoral access is simple and suitable in rabbits. Blood collection via the jugular vein may be a more reliable and valuable method for pharmacokinetic studies in rabbits.

Abstract:Objective To observe the 12-lead ECG (electrocardiogram) characteristics of Japanese rabbits, and provide basic ECG data for cardiovascular disease research. Methods The 12-lead ECG and X-rays of 55 male Japanese rabbits were recorded in supine position after intraperitoneal injection of 20% urethane. Results ECG:① The 12-lead ECG characteristics of male Japanese rabbits were similar to humans. The rabbit heart rate was 265.5±36.8 beats/min, faster than that of humans. No arrhythmia was found in all the 55 rabbits. ② The supine position average ECG axis was between 19° to 250°, varying a lot. ③ P wave:The shape of P wave was blunt round or a little bit sharp. P waves were all in accordance with the sinus P wave rules, which were more obvious in lead II, aVF and all chest leads. ④ The PR interval was 0.063±0.007 s. ⑤ The QRS duration was 0.040±0.005 s. The main waves were mostly upward in leads Ⅱ, Ⅲ, and aVF. The same as humans, the R/S ratios were increased by degrees in chest leads. ⑥ The ST segment was short, and was located in the equipotential line. ⑦ The shapes of T wave were mostly round, partly had twin peaks. T waves were more obvious in leads Ⅱ, Ⅲ, aVR, and AVF and chest leads. ⑧ The QT interval was 0.142±0.015 s, and QTc was 0.306±0.034 s. In the X-rays, most heart shadows were in the center and right chest. Conclusions The normal values of 12-lead ECG characteristics of Japanese rabbits are obtained in this study, which are of certain application value in experimental studies of cardiovascular diseases.

Abstract:Objective To explore the effect of Bushencupailuan decoction, a Chinese medicine formula, on polycystic ovary syndrome (PCOS) and its mechanism through observing the sex hormones, anti-Müllerian hormone (AMH) and the morphology of ovaries of PCOS rat models. Methods Bushencupailuan decoction was administered in DHEA-induced PCOS model rats. The pathology of ovaries was examined, and the serum sex hormones and AMH were detected. Results Compared with the normal group, the ovaries of model group rats had severe diffuse cystic dilatation and cystic cavities of different sizes. The granular layer cells were compressed into flat or cubical shapes. Corpora lutea and follicles in various stages of development were rare. Bushencupailuan decoction obviously improved the numbers of mature follicles and corpora lutea, levels of sex hormones and AMH of PCOS model rats compared with those of model rats (P<0.05). Conclusions Bushencupailuan decoction can treat PCOS by improving the serum sex hormone levels in PCOS model rats, reduce serum AMH and help to restore ovulation.

Abstract:Objective To preliminarily explore the effects and brain protective mechanism of intermittent hypoxia preconditioning (IHP) on rats with seizures induced by lithium-pilocarpine (Li-pilo). Methods A total of 96 8-week old male Sprague Dawley rats (clean grade) were randomly divided into control group,seizure group and four IHP-seizure groups. The animal model of epilepsy was established by intraperitoneal injection of Li-pilo in the seizure group and four IHP-seizure groups (Li-pilo was injected at 1, 3, 7, or 14 days after a 5-day regimen of IHP). Subsequent seizure behavior, the latency period and percentage of generalized seizures were quantitatively evaluated for 240 min and the cognitive function was tested by Morris water maze task, and followed by the detection of hippocampus neuron apoptosis and related protein (BCL-2, Bax, and cleaved-caspase-3) by TUNEL labeling and Western blot, respectively. Results The induced seizure peaked on an average between 50-150 min after Li-pilo administration, scored using a modified Racine scale. The average scores of modified Racine scale in the IHP-3d seizure group was significantly lower than that in the other groups. The latency period and percentage of generalized seizures in the IHP-3d seizure group rats were significantly different from the parameters in the seizure group rats (P<0.05). IHP-3d seizure rats showed lower escape latency, neuronal apoptosis counts and higher percentage of time in the probe quadrant compare with the seizure group and the other three IHP-seizure groups (P<0.05). Compared with the control group, the parameters of water maze and apoptosis detection in the IHP-3d seizure group showed no significant changes (P>0.05). Conclusions The results indicate that IHP treatment may help to decrease the susceptibility to epilepsy by reducing abnormal apoptosis, and has a brain protective effect on the seizure rats.

Abstract:Objective To explore different methods for establishing animal models of Ureaplasma urealyticum (UU) infection and to select the best method of establishment of this disease. Methods SPF female BALB/c mice and Wistar rats were multiple-infected with small dose of UU liquid or single-infected with high-dose UU liquid, pretreated with estradiol benzoate, to establish models of Ureaplasma urealyticum infection. Cervical secretion was taken for UU culture at 14, 28, 42 and 56 days after the first time UU treatment, and the reproductive organs were taken for histopathological examination. Results Gross examination of the UU-infected rats showed generally less serious lesions than the mice. The low-dose estrogen group was worst, showing hyperemia and edema, cervical hypertrophy, hydrosalpinx, enlargement, and more rigid with poor elasticity. Significant differences were observed in the vaginal UU colonization rates between the low-dose estrogen and low-dose non-estrogen groups, high-dose estrogen and non-estrogen groups, low-dose estrogen and high-dose estrogen groups, low-dose estrogen and non-estrogen groups, in both the mice and rats (P<0.05 for all). The same dose estrogen treatment caused a higher UU colonization rate in the mice than rats (P<0.05). Histological examination showed that the low-dose estrogen treated rats had no obvious pathological changes, except loose tissue edema in some rats, but apparent pathological changes were observed in the low-dose estrogen treated mice. Conclusions The vulvar lesions in the mice are worse than in the rats. Estrogen pre-treatment can increase the Ureaplasma urealyticum colonization rate in the animals. The UU colonization rate in the vagina of mice is higher than in the rats, and multiple low-dose UU treatment produces a higher UU colonization rate than a single high-dose UU treatment. Therefore, BALB/c mice are more susceptible to Ureaplasma urealyticum infection, with more serious pathological changes, and are more suitable for experimental studies of related diseases.

Abstract:Objective To evaluate the rat model of dry eye induced by topical administration of benzalkonium chloride and compare the morphological characteristics of corneal and conjunctival tissues of the normal and model rats. Methods Topical administration of benzalkonium chloride (0.3%) twice per day for seven days to induce the rat model of dry eye. Pathology with HE and periodic acid-Schiff staining and transmission electron microscopy were performed to evaluate the corneal and conjunctival changes. Results The corneal and conjunctival epithelial cells were sloughed off, the number of goblet cells was decreased in the dry eye model. Microvilli were obviously lost in the surface epithelial cells of the dry eye rat cornea and conjunctiva compared with the normal rats, as indicated by the amount and size of microvilli. Conclusions The changes in the corneal and conjunctival epithelia of BAC-induced rat dry eye are similar to the changes in human dry eye. This rat model may play an essential role for dry eye studies in the future.

Abstract:Objective To compare three rat models of acute stress response and to explore an ideal experimental rat model for research of acute stress response. Methods A total of 40 clean grade male SD rats were randomly (by random number) divided into 4 groups (n=10 each):Normal group (group I), caudal trunk transection group (group II), burn group (group III), and amputation group (group IV). The group I received no special treatment, the group II received inferior caudal trunk transection at the level between sacral spinal nerve 3 and 4 (S3, S4), the group III was inflicted with 30% total body surface area (TBSA) grade 3 burn on the back, and the group IV had an amputation of the left lower limb as severe traumatic stress. Rats in each group were killed at 30 minutes after treatment and blood samples were collected for measuring serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels by ELISA. The c-fos protein expression in hypothalamic tissue was examined by immunohistochemistry. Results Compared with the group I, serum ACTH and CORT levels, and hypothalamic expression of c-fos protein in the group II, III, IV were significantly increased (P<0.05).Compared with the group III, serum ACTH and CORT levels, and hypothalamic expression of c-fos protein were significantly higher in the group II and IV (P<0.05), while there was no significant difference between the group II and IV (P>0.05). Conclusions The preparation of acute stress response model induced by inferior caudal trunk transection has simple operation steps and produces a traumatic injury to a similar degree, and quite well reflects the acute stress response in humans caused by sudden accident. Therefore it is a quite good method to establish acute stress response and deserves further investigation.

Abstract:Objective To study the liver pathomorphological and serum biochemical changes in Beagle dog with spontaneous hepatocirrhosis and establish the backgroud information of experimental animals for GLP. Methods The ALT,AST,TP,ALB,ALP,TBIL,TC,TG and GGT were detected using an automatic biochemical analyzer,and compared the differences of above index between blank control and diseased animal.The histological changes of the liver were observed by optical microscopy. Results Compared with the blank control,the ALT,AST,ALP,TBIL and GGT of diseased dogs were increased significantly,and the ALB decreased significantly. Compared with normal dogs, the liver cells had nodular regeneration, arranged irregularly and pseudolobule formation.The pseudolobules were packaged with collagen fibers.Conclusion It is suggested that spontaneous lesions in Beagle dogs should be monitored so as to provide appropriate experimental animal histopathologicalbackgroud information for drug safety evaluation.

Abstract:Objective To develop a new method to expose the stellate ganglion to increase the success rate of establishing a dog model of atrial fibrillation indinced by sympathetic stimulation. Methods A total of 28 adult dogs were randomly divided into traditional group and improvement group, 14 dogs in each group. The stellate ganglions were separated by the two different methods, respectively, to establish a sympathetic stimulation induced atrial fibrillation model in all the dogs. Changes of vital signs, survival rate of the dogs and the voltage required to stimulate the stellate ganglion were recorded intraoperatively. Changes of cardiac electrophysiology were recorded before and after electric stimulation. The levels of released neurotransmitters were detected by immunohistochemistry. Results The survival rate of the improvement group was 100% (14/14), significantly higher than the 64.3% (9/14) of the traditional group (P<0.05). The operation time of the improvement group was 122.71±3.62 min, significantly shorter than the 269.44±8.79 min of the traditional group (P<0.05). The threshold voltage of the improvement group was significantly lower than that of the traditional group (P<0.05). Conclusions Our modified surgical procedure can effectively reduce the mortality of dogs, significantly shorten the operation time, and reduce the intraoperative blood loss, keeping a more intact stellate ganglion, and maintains a more stable voltage of electric stimulation, Therefore, it is a new method more suitable for establishment of a sympathetic stimulation induced atrial fibrillation model in dogs.

Abstract:Objective To investigate the method to prevent the displacement of tissue expander implanted between the panniculus carnosus and deep fasia in rabbits. Method 40 rabbits were divided into two groups randomly:the experimental group and the control group. Through an incision on the back, a pocket was made by dissecting between the rabbit panniculus carnosus and deep fasia. The panniculus carnosus at the pocket boundary were sutured with the deep fascia inside the pocket by interrupted 3-0 sutures. A 50 mL kidney-shaped tissue expander was implanted. The expander was inflated with 10 mL saline after the wound was sutured. 7 days after implantation, black tattoos were made on the skin around the expander to mark the site of expander. Two months after implantation, the expanders in the experimental group were inflated with 10 mL saline weekly to a total of 140 mL. 7 days after implantation the expanders of control group were inflated with 10 mL saline weekly to a total of 140 mL. The incidence of expander displacement, dehiscence of incision, reversal of injection pot and death were recorded.Results There was no expander displacement in the 19 rabbits of experimental group. All the expanders were located within the tattoo area. There was no expander displacement in 11 rabbits of the control group. 4 expanders of the control group moved from the back to belly. The difference between them was statistically significant (P<0.05). The incidence of dehiscence of incision in the experimental group was not significantly lower than that in the control group (P>0.05). Meanwhile there was no significant difference in the incidences of reversal of injection pot, infections and deaths (P>0.05 for all). Conclusions Sutures between the panniculus carnosus and the deep fascia at the pocket boundary asisted by delayed inflating can prevent the displacement of tissue expander implanted between the panniculus carnosus and deep fasia in rabbits, thus to obtain effective expansion.

Abstract:Objective To establish a real-time fluorescent quantitative PCR (FQ-PCR) method for detection of murine norovirus (MNV) in laboratory mouse and provide the basis for establishment of a standard detection method for MNV. Methods Specific primers were designed and MNV DNA standards were prepared according to the MNV genome sequences published on NCBI. The specificity, sensitivity, repeatability and stability of the established Q-PCR method were tested. The established Q-PCR method was applied to detect 766 mouse caecum content samples to explore preliminarily the infection status of laboratory mice in Beijing. Results No cross reaction showed in human norovirus and feline calicivirus with the established Q-PCR method.The sensitivity was up to 10 copies/μL. The coefficient of variation (CV) of intra-assay and inter-assay was less than 2%. There were 301 positive cases detected in the 766 samples of laboratory mice. Conclusions The established FQ-PCR method is accurate and effective with high specificity, sensitivity and repeatabiliy in the quantitative detetion of nucleic acid, and can be applied to rapidly and quantitatively screen MNV in laboratory mice.

Abstract:Through analyzing the characteristics of the experimental animal industry, supply and demand status and application prospect of mobile internet industry, as the "mobile Internet + experimental animals" combination to study the function and effect and operation mode of the experimental animals mall platform. The platform will be provide open e-commerce services for experimental animal industry, forming an innovative pattern of "resource sharing, mutual benefit and win-win", promote the balanced allocation of the experimental animal industry chain resources, build "effectiveness, efficiency, effect" good operation management environment.

Abstract:Objective To introduce the operation skills of tail vein injection in conscious rats, and improve the success rate of ingection. Methods The rat was fixed by the operator with a self-made binding clothes, one person assists to fix the rat tail, one person performs puncture, and one person performs injection. During the injection process, we should minimize the injury to the rat tail and the stress caused by operation, strictly limit the needle point, the number of puncture, the maximum dose and injection speed, and to make the needle position away from the injection site to avoid contamination and waste of the drug solution. Results The operation method was successfully established and it was fast, stable, with good repeatability and high degree of coordination. Conclusion This operation is rapid, reliable and stable, worthy of recommendation, especially for the intravenous injection of expensive drugs.

Abstract:Skin sensitization (allergic contact dermatitis, ACD), is a serious condition caused by small reactive molecules and is characterized by a delayed-type hypersensitivity. Animal tests were usually used in the evaluation of sensitizing potential of chemical substances in the past. However, there is an increasing interest from the public for reducing and ultimately replacing animal tests. The European Union (EU) has posed a ban on animal testing of cosmetic ingredients that includes skin sensitization since 2013. Therefore, alternative in vitro tests are the main tendency in chemical substances and cosmetic sensitizing potential research in the future. In this study, different kinds of in vitro test methods that were adopted by OECD or on research(LLNA, DPRA, KeratinoSens ^TM, h-CLAT)were reviewed through recent years literature, comprehensive introduction and evaluation were made to obtain reliable hazard and potency information on potential skin sensitizers.

Abstract:Laboratory animal welfare is an important guarantee for the accuracy and reliability of experimental results, so it has been paid attention in scientific research. But the situation is not optimistic in the teaching practice. This paper has specially analyzed laboratory animal welfare and ethics in experiment teaching and provided specific measures involved in legislation, spread of animal welfare and implementation of 3R principle to ensure animal welfare.