Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

Animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene functions.The field of genome modification in rabbits has progressed slowly.However,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic pathologies.These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice.This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

Diagnosis and Control of Rabbit Hemorrhagic Disease(RHD)

In this study, chronic-type rabbit hemorrhagic disease occurred in rabbit warrens of Shandong, Liaoning, Beijing and Hebei, which was mainly charac-terized by deaths of young and adult rabbits, longer durationand nerve symptoms, was surveyed by epidemiological investigation, bacterial isolation and incubation, artificial infection test and histopathologieal method to analyze the causes of chronic-type rabbit hemorrhagic disease and put forward the corresponding control measures.

Procyanidin B2 protects against diet-induced obesity and non-alcoholic fatty liver disease via the modulation of the gut microbiota in rabbits

BACKGROUND Procyanidins have beneficial effects on metabolic syndrome and antimicrobial activity,but the mechanisms underlying these effects are unclear.AIM To investigate the effects of procyanidin B2(PB2)on non-alcoholic fatty liver disease and to explore the possible mechanism.METHODS Thirty male New Zealand white rabbits were randomized into three groups.All of them were fed either a high-fat-cholesterol diet(HCD)or chow diet.HCD-fed rabbits were treated with vehicle or PB2 daily for 12 wk.Body weight and food intake were evaluated once a week.Serum biomarkers,such as total cholesterols,triglycerides,and aspartate transaminase,were detected.All rabbits were sacrificed and histological parameters of liver were assessed by hematoxylin and eosin-stained sections.Moreover,several lipogenic genes and gut microbiota(by 16S rRNA sequencing)were investigated to explore the possible mechanism.RESULTS The HCD group had higher body weight,liver index,serum lipid profile,insulin resistance,serum glucose,and hepatic steatosis compared to the CHOW group.PB2 treatment prevented HCD-induced increases in body weight and hypertriglyceridemia in association with triglyceride accumulation in the liver.PB2 also ameliorated low-grade inflammation,which was reflected by serum lipopolysaccharides and improved insulin resistance.In rabbit liver,PB2 prevented the upregulation of steroid response element binding protein 1c and fatty acid synthase and the downregulation of carnitine palmitoyltransferase,compared to the HCD group.Moreover,HCD led to a decrease of Bacteroidetes in gut microbiota.PB2 significantly improved the proportions of Bacteroidetes at the phylum level and Akkermansia at the genus level.CONCLUSION Our results indicate the possible mechanism of PB2 to improve HCD-induced features of metabolic syndrome and provide a new dietary supplement.

Enhancement of the Immune Response to Rabbit Hemorrhagic Disease Vaccine in Young Rabbits by Advanced Vaccination and Chinese Herbal Adjuvants

Experiments were conducted to determine the effects of advanced vaccination and Chinese herbal adjuvants （CHA）, containing astragalus polysaccharides （APS） and ginsenosides （GS） on the immune response to rabbit hemorrhagic disease （RHD） vaccine in young rabbits. In experiment 1, 5 New Zealand rabbits of each group at 30, 35, 40, or 45 days of age were injected with 2 mL of inactivated RHD vaccine, respectively. The dynamic changes of antibody titers were tested by the hemagglutination inhibition （HI） method. In experiment 2, 30 New Zealand rabbits at 35 days of age were randomly assigned to 5 treatment groups, representing inoculation with 3 mL of non-adjuvant RHD vaccine, CHA-RHD vaccine, CHA-HA vaccine （half dose antigen）, aluminium adjuvant-RHD vaccine, and PBS, respectively. The dynamic changes of peripheral lymphocyte proliferation and serum antibody titers were tested by the MTT method and the HI method. The results showed that the titer of maternal HI antibody in the 35-day-old rabbits was lower than the protective level of 3 log2, while on days 7 to 49 after the vaccination, the antibody titers were higher than 3 log2. The CHA promoted the lymphocyte proliferation and enhanced the serum antibody titer （P〈 0.05）. These findings from the two experiments suggested that advanced vaccination and Chinese herbal adjuvants significantly enhanced the immune response to vaccine against RHD, and effectively protected the young rabbits against RHD challenge.

Ultrastructural Pathologic Observation on the Gut-Associated Lymphoid Tissues of Sacculus Rotundus of Rabbits Infected with Rabbit Haemorrhagic Disease Virus

Ultrastructural pathological changes in the gut-associated lymphoid tissues of sacculus rotundus (SR) of rabbits infected with rabbit haemorrhagic disease virus (RHDV) were first observed. There were numerous holes at the luminal and basement membrane surfaces of the dome epithelium (DE), consistently accompanied by necrosis of lymphocytes and M-cells, and pronounced depletion of lymphocytes in the domes and follicles, decrease of DE complex with formation of pseudomembranous structure on the surface of the dome epithelium. A specific finding in lymphocytes and macrophages was that severe destruction detraction of the membrane of rough endoplasmic reticulum(RER) was accompanied by conspicious increase of solitary, ribo-some-like particles in the cytoplasm, with appearances of intranuclear particles and intranuclear inclusions. It was found that there were many round and dense virion-like particles, with 26 nm in diameter, in the nuclei and cytoplasm of lymphoctes, plasma cells, macrophages and fibroblasts, or in degenerated cells and cellular debris. At the same time, another round virion-like particles about 34 nm in diameter were also seen in the cytoplasm of some cells and interstitium. The results indicated that the appearances of the ribosome-like particles, virion-like particles and inclusion bodies were related to the replication and assembly of RHDV. The present observations suggested that DE of sacculus rotundus could be a open pathway and a transporting route for the entry of antigens into hosts. While the antigen is profoundly deleterious, DE may be as a closed portal or a barrier preventing the foreign antigenic materials from invading.

Prokaryotic Expression and Immunogenicity of Major Antigen Epitope of Rabbit Haemorrhagic Disease Virus VP60

[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was amplified by RT-PCR. It was cloned into pET-28b （ ＋ ） and expressed in E. coli Rosetta strain. The recombinant protein was detected by Western blot. The pudfied recombinant protein was used to immunize rabbits in order to observe its immunogenicity. [ Result] Western blot analysis revealed a clear band at approximately 24.0 kDa. The purified recom- binant protein reacted with the purified RHDV in ELISA. [ Conclusion] The prokaryotically expressed main antigen epitope of RHDV VP60 shows good immunogenicity.

Potential neuroprotection by Dendrobium nobile Lindl alkaloid in Alzheimer's disease models

At present,treatments for Alzheimer's disease can temporarily relieve symptoms but cannot prevent the decline of cognitive ability and other neurodegenerative changes.Dendrobium nobile Lindl alkaloid is the main active component of Dendrobium nobile Lindl.Dendrobium nobile Lindl alkaloid has been shown to resist aging,prolong life span,and exhibit immunomodulatory effects in animals.This review summarizes the mechanisms behind the neuroprotective effects reported in Alzheimer's disease animal models.The neuroprotective effects of Dendrobium nobile Lindl alkaloid have not been studied in patients.The mechanisms by which Dendrobium nobile Lindl alkaloid has been reported to improve cognitive dysfunction in Alzheimer's disease animal models may be associated with extracellular amyloid plaque production,regulation of tau protein hyperphosphorylation,inhibition of neuroinflammation and neuronal apoptosis,activation of autophagy,and enhanced synaptic connections.

Establishing a rabbit model of perianal fistulizing Crohn's disease

BACKGROUND Crohn’s disease(CD)is a chronic nonspecific intestinal inflammatory disease.The aetiology and pathogenesis of CD are still unclear.Anal fistula is the main complication of CD and is a difficult problem to solve at present.The main limitation of developing new therapies is bound up with the short of preclinical security and effectiveness data.Therefore,an ideal animal model is needed to establish persistent anal fistula and an inflamed rectal mucosa.AIM To improve the induction method of colitis and establish a reliable and reproducible perianal fistulizing Crohn’s disease animal model to evaluate new treatment strategies.METHODS Twenty male New Zealand rabbits underwent rectal enema with different doses of 2,4,6-trinitrobenzene sulfonic acid to induce proctitis.Group A was treated with an improved equal interval small dose increasing method.The dosage of group B was constant.Seven days later,the rabbits underwent surgical creation of a transsphincteric fistula.Then,three rabbits were randomly selected from each group every 7 d to remove the seton from the fistula.The rabbits were examined by endoscopy every 7 days,and biopsy forceps were used to obtain tissue samples from the obvious colon lesions for histological analysis.The disease activity index(DAI),colonoscopy and histological scores were recorded.Perianal endoscopic ultrasonography(EUS)was used to evaluate the healing of fistulas.RESULTS Except for the DAI score,the colonoscopy and histological scores in group A were significantly higher than those in group B(P<0.05).In the ideal model rabbit group,on the 7 th day after the removal of the seton,all animals had persistent lumens on EUS imaging,showing continuous fullthickness high signals.Histological inspection of the fistula showed acute and chronic inflammation,fibrosis,epithelialization and peripheral proctitis of the adjoining rectum.CONCLUSION The improved method of CD colitis induction successfully established a rabbit perianal fistula CD preclinical model,which was confirmed by endoscopy and pathology.

Awake rabbit model of ischemic spinal cord injury with delayed paraplegia:The role of ambient temperature

Background:Paraplegia after spinal cord ischemia is a devastating condition in the clinic.Here,we develop an awake rabbit model of spinal cord ischemia with delayed paraplegia and explore the influence of ambient temperature on the outcomes after injury.Methods:A total of 47 male rabbits were involved in the present study.Transient spinal cord ischemia was induced by occluding the infrarenal abdominal aorta of awake rabbits at different ambient temperatures.To find the optimal conditions for developing delayed paraplegia,hindlimb motor function after ischemia was evaluated between experiments.Results:The onset and magnitude of ischemic injury varied with the ambient temperature maintained during the peri-i schemia period.More serious spinal cord injury occurred when ischemia was induced at higher temperatures.At 18°C,25-minute ischemia resulted in 74%of rabbits developing delayed paraplegia.At a temperature of 28°C or higher,most of the animals developed acute paraplegia immediately.While at 13°C,rabbits usually regained normal motor function without paraplegia.Conclusion:This awake rabbit model is highly reproducible and will be helpful in future studies of delayed paraplegia after spinal cord ischemia.The ambient temperature must be considered while using this model during investigation of therapeutic interventions.

Methods to Reduce the Hypoglycemic Mortality of Alloxan in Diabetic Rabbit Model

Objective: To explore an intervention method to reduce the mortality of alloxan diabetes model, and to preliminarily analyze the mechanism of alloxan induced animal death. Methods: Healthy New Zealand rabbits were randomly divided into injection group, control group, experimental group and blank group. The single injection group was injected with 100 mg/kg alloxan once. The control group was given 5% glucose solution and 100 mg/kg alloxan was injected in two times. The experimental group was given 5% glucose solution orally, 100 mg/kg alloxan, 7 mL 0.9% NaCl intravenously and 5 mL 5% glucose intraperitoneally immediately, and blood glucose was continuously monitored, 10 mL 5% glucose intravenously and 10 mL 5% glucose intraperitoneally every 4 h in the hypoglycemic stage. The blank group does nothing. Liver and kidney tissues at different time periods were stained with HE and organ index was evaluated. Results: 1) A single injection of 100 mg/kg alloxan without any intervention resulted in 100% mortality. Before modeling, oral administration of 5% glucose solution, divided into two injections of 100 mg/kg alloxan, mortality reached 100%;A single injection of 100 mg/kg alloxan and continuous intervention of normal saline and glucose for 20 h can significantly reduce the mortality of alloxan induced diabetic rabbit model. 2) Liver and kidney tissues were damaged in different degrees at different time periods, and liver and kidney indexes were significantly increased after alloxan injection compared with the normal group, with statistical significance (P > 0.05). Conclusion: 1) Every 4 hours of hypoglycemia, 10 ml 5% glucose was injected intravenously 10 ml 5% glucose intraperitoneally. It can reduce the death rate of alloxan diabetic rabbit model and shorten the time of blood glucose measurement. 2) After the injection of alloxan, acute lesions of liver and kidney may occur in different degrees, or one of the causes of acute death of experimental animals.

Study on regulatory effect of acupuncture on rotation induced gastric dysrhythmia in rabbits *

AIM A model of experimental gastric dysrhythmia in rabbits was set up to evaluate the effect of different acupoints on regulating the gastric dysrhythmia in rabbits as to promoting the acupuncture treatment for this kind of disease.

Propofol Target-Controlled Infusion Modeling in Rabbits:Pharmacokinetic and Pharmacodynamic Analysis

This study aimed to establish a new propofol target-controlled infusion（TCI） model in animals so as to study the general anesthetic mechanism at multi-levels in vivo. Twenty Japanese white rabbits were enrolled and propofol（10 mg/kg） was administrated intravenously. Artery blood samples were collected at various time points after injection, and plasma concentrations of propofol were measured. Pharmacokinetic modeling was performed using Win Nonlin software. Propofol TCI within the acquired parameters integrated was conducted to achieve different anesthetic depths in rabbits, monitored by narcotrend. The pharmacodynamics was analyzed using a sigmoidal inhibitory maximal effect model for narcotrend index（NI） versus effect-site concentration. The results showed the pharmacokinetics of propofol in Japanese white rabbits was best described by a two-compartment model. The target plasma concentrations of propofol required at light anesthetic depth was 9.77±0.23 μg/m L, while 12.52±0.69 μg/m L at deep anesthetic depth. NI was 76.17±4.25 at light anesthetic depth, while 27.41±5.77 at deep anesthetic depth. The effect-site elimination rate constant（ke0） was 0.263/min, and the propofol dose required to achieve a 50% decrease in the NI value from baseline was 11.19 μg/m L（95% CI, 10.25–13.67）. Our results established a new propofol TCI animal model and proved the model controlled the anesthetic depth accurately and stably in rabbits. The study provides a powerful method for exploring general anesthetic mechanisms at different anesthetic depths in vivo.

Evaluation of the Effects of Cypermethrin on Female Reproductive Function by Using Rabbit Model and of the Protective Role of Chinese Propolis

The prophylactic effects of Chinese propolis against cypermethrin toxicity were evaluated by performing ovary and uterus histopathology, as well as by characterizing ovarian function, embryos, and litters. Cypermethrin induced atypia in the ovary and uterus, and decreased the ovulation sites and the number of embryos. Cypermethrin-induced oxidative stress during pregnancy, decreased the parturition rate as well as the number and weight of offspring and increased the incidence of morphological malformations in the offspring. Administration of propolis to cypermethrin-treated animals mitigated cypermethrin-induced reproductive toxicity.

Susceptibility-weighted imaging is suitable for evaluating signal strength in different brain regions of a rabbit model of acute hemorrhagic anemia

Acute hemorrhagic anemia can decrease blood flow and oxygen supply to brain, and affect its physiological function. While detecting changes in brain function in patients with acute hemorrhagic anemia is helpful for preventing neurological complications and evaluating therapeutic effects, clinical changes in the nervous systems of these patients have not received much attention. In part, this is because current techniques can only indirectly detect changes in brain function following onset of anemia, which leads to lags between real changes in brain function and their detection.

Pharmacokinetics of Native r-SAK in Rabbit's Femoral Artery Thrombosis Model

Objective: To study the pharmacokinetics of native r SAK in rabbit's femoral artery thrombosis model, the “lytic circle' method was used to determine plasma levels of r SAK. Methods: Thirty New Zealand rabbits were randomly assigned to the control (saline 10 ml, 30 min), r SAK low dose (0.25 mg/kg, 30 min), medial dose (0.50 mg/kg, 30 min), high dose (1.00 mg/kg, 30 min), single bolus (0.50 mg/kg, 2 min) and conjunctive therapy (initiated with heparin 200 U/kg, followed by infusion of r SAK 0.50 mg/kg for 30 min, and subsequently infused heparin 50 U/(kg·h) to endpoint) groups. The right femoral artery thrombosis model in rabbit was made by balloon injury, then the thrombolytic agents were infused through parallel ear vein and the blood samples were collected pre thrombolysis and at different time post thrombolysis to determine the plasma levels of r SAK by “lytic circle' method, the plasma levels of r SAK were processed by pharmacokinetic computing procedure to fit the model. Results: The plasma levels of r SAK and the diameters of lytic circles showed a pretty good linear correlation under the scope of 2.0×10 4 2.0×10 6 U/L, and the averaged recycle rate was (96.05±11.35)%(RSD =±11.82%).All peak concentration time in each infusion group was 30 min, and the peak concentrations positively correlated with the doses administrated in infusion groups(r=0.999 98, P <0.000 1). In single bolus group, Peak concentration time was 2 min, and the peak concentration reached (5.16±1.02) mg/L, which was significant higher than that in the same dose r SAK infusion group ( P <0.01). In conjunctive therapy group, the peak concentration showed no significant difference from that in the same dose r SAK infusion group ( P >0.05). The plasma levels of r SAK fit in two compartment model as processed by pharmacokinetic computing procedure in each group. Conclusion: The “lytic circle' method is a simple, practical and reliable method to determine the plasma level of r SAK, and the pharmacokinetics of native r SAK infusion fits in two compartment model in rabbit's femoral artery thrombosis model.

Comparison of two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma

AIM:To compare two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma.METHODS:Thirty New Zealand rabbits were randomly divided into two groups:A and B.Group A was assigned a traditional laparotomy method(embedding tumor fragments directly into the liver with tweezers).Group B was subjected to an improved laparotomy method(injection of tumor fragments into the liver through a 15 G syringe needle).The operation time, incision length, incision infection rate, and mortality rate were compared between the two groups after laparotomy.Magnetic resonance imaging(MRI) was performed to evaluate tumor formation rates and the characteristics of the tumors 2 wk after laparotomy.RESULTS:The mean operation times for the two groups(Group A vs Group B) were 23.2 ± 3.4 min vs 17.5 ± 2.9 min(P < 0.05); the incision length was 3.3 ± 0.5 cm vs 2.4 ± 0.6 cm(P < 0.05); and the mortality rate after 2 wk was 26.7% vs 0%(P < 0.05); all of these outcomes were significantly different between the two groups.The incision infection rates in the two groups were 6.7% vs 0%(P > 0.05), whichwere not significantly different.MRI performed after 2weeks showed that the tumor formation rates in the two groups were 90.9%vs 93.3%(P>0.05).These rates were not significantly different between the two groups.The celiac implantation rate and abdominal wall metastasis rate in the two groups were 36.4%vs 13.3%(P<0.05)and 27.2%vs 6.7%(P<0.05),respectively,which were significantly different between the two groups.CONCLUSION:The tumor formation rates were not significantly different between the two methods for modeling rabbit VX2 hepatocarcinoma.However,the improved method is recommended because it has certain advantages.

Experimental and finite element analysis of tibial stress fractures using a rabbit model

AIM: To determine if rabbit models can be used to quantify the mechanical behaviour involved in tibial stress fracture(TSF) development.METHODS: Fresh rabbit tibiae were loaded under compression using a specifically-designed test apparatus. Weights were incrementally added up to a load of 30 kg and the mechanical behaviour of the tibia was analysed using tests for buckling, bone strain and hysteresis. Structural mechanics equations were subsequently employed to verify that the results were within the range of values predicted by theory. A finite element(FE) model was developed using cross-sectional computer tomography(CT) images scanned from one of the rabbit bones, and a static load of 6 kg(1.5 times the rabbit's body weight) was applied to represent running. The model was validated using the experimental strain gauge data, then geometric and elemental convergence tests were performed in order to find the minimum number of cross-sectional scans and elements respectively required for convergence. The analysis was then performed using both the model and the experimental results to investigate the mechanical behaviour of the rabbit tibia under compressive load and to examine crack initiation.RESULTS: The experimental tests showed that un der a compressive load of up to 12 kg, the rabbit tibia demonstrates linear behaviour with little hysteresis Up to 30 kg, the bone does not fail by elastic buckling however, there are low levels of tensile stress which predominately occur at and adjacent to the anterio border of the tibial midshaft: this suggests that fatigue failure occurs in these regions, since bone under cycli loading initially fails in tension. The FE model predic tions were consistent with both mechanics theory and the strain gauge results. The model was highly sensi tive to small changes in the position of the applied load due to the high slenderness ratio of the rabbit s tibia. The modelling technique used in the curren study could have applications in the development o human FE models of bone, where, unlike rabbit tibia the model would be relatively insensitive to very sma changes in load position. However, the rabbit mode itself is less beneficial as a tool to understand the me chanical behaviour of TSFs in humans due to the sma size of the rabbit bone and the limitations of human scale CT scanning equipment.CONCLUSION: The current modelling technique could be used to develop human FE models. However, the rabbit model itself has significant limitations in under standing human TSF mechanics.

Evaluation of a rabbit rectal VX2 carcinoma model using computed tomography and magnetic resonance imaging

AIM： To establish a rabbit rectal VX2 carcinoma model for the study of rectal carcinoma.METHODS： A suspension of VX2 cells was injected into the rectum wall under the guidance of X-ray fluoroscopy. Computed tomography （CT） and magnetic resonance imaging （MRI） were used to observe tumorgrowth and metastasis at different phases. Pathological changes and spontaneous survival time of the rabbits were recorded.RESULTS： Two weeks after VX2 cell implantation, the tumor diameter ranged 4.1-5.8 mm and the success implantation rate was 81.8%. CT scanning showed low-density loci of the tumor in the rectum wail, while enhanced CT scanning demonstrated a symmetrical intensification in tumor loci. MRI scanning showed alow signal of the tumor on T1-weighted imaging anda high signal of the tumor on T2-weighted imaging.Both types of signals were intensified with enhanced MRI. Metastases to the liver and lung could beobserved 6 wk after VX2 cell implantation, and a largearea of necrosis appeared in the primary tumor. The spontaneous survival time of rabbits with cachexia and multiple organ failure was about 7 wk after VX2 cell implantation.CONCLUSION： The rabbit rectal VX2 carcinoma model we established has a high stability, and can be used in the study of rectal carcinoma.

Establishment of a chronic left ventricular aneurysm model in rabbit

Objectives To establish a cost-effective and reproducible procedure for induction of chronic left ventricular aneurysm （LVA） in rabbits. Methods Acute myocardial infarction （AMI） was induced in 35 rabbits via concomitant ligation of the left anterior descending （LAD） coronary artery and the circumflex （Cx） branch at the middle portion. Development of AMI was co n-firmed by ST segment elevation and akinesis of the occluded area. Echocardiography, pathological evaluation, and agar i n-tra-chamber casting were utilized to validate the formation of LVA four weeks after the surgery. Left ventricular end systolic pressure （LVESP） and diastolic pressure （LVEDP） were measured before, immediately after and four weeks after ligation. D i-mensions of the ventricular chamber, thickness of the interventricular septum （IVS） and the left ventricular posterior wall （LVPW） left ventricular end diastolic volume （LVEDV） and systolic volume （LVESV）, and ejection fraction （EF） were recorded by echo-cardiography. Results Thirty one （88.6%） rabbits survived myocardial infarction and 26 of them developed aneurysm （83.9%）. The mean area of aneurysm was 33.4% ＆#177; 2.4% of the left ventricle. LVEF markedly decreased after LVA formation, whereas LVEDV, LVESV and the thickness of IVS as well as the dimension of ventricular chamber from apex to mitral valve annulus significantly increased. LVESP immediately dropped after ligation and recovered to a small extent after LVA formation. LVEDP progressively increased after ligation till LVA formation. Areas in the left ventricle （LV） that underwent fibrosis included the apex, anterior wall and lateral wall but not IVS. Agar intra-chamber cast showed that the bulging of LV wall was prominent in the area of aneurysm. Conclusions Ligation of LAD and Cx at the middle portion could induce develo pment of LVA at a mean area ratio of 33.4%＆#177;2.4%which involves the apex, anterior wall and lateral wall of the LV.

Establishment of VX2 breast carcinoma model in rabbit by injecting tumor mass suspension

Objective: To establish a stable model of VX2 breast carcinoma in rabbit and select the optimal way. Methods: Thirty female New Zealand rabbits were randomly divided into 3 groups with 10 in each. Tumor cell suspensions or tumor mass suspensions were injected into breast tissues of rabbits of group A and B, respectively. Tumor blocks were surgically implanted in rabbit breasts of group C. Tumor formation rate, tumor growth rate, and tumor-bearing survival time was compared, and the histological feature of tumor was observed. Results: Models were established conveniently and successfully in rabbits received injection of tumor mass suspensions. Tumor proliferated rapidly with the biological feature of squamous cell carcinoma. Conclusion: VX2 breast carcinoma model in rabbit was established successfully. Intramammary injection of tumor mass suspension is the best method.