Effectiveness of ozonated saline in the treatment of VX2 tumors in rabbits

Objective To investigate the efficacy, safety, and associated mechanisms of injected ozonated saline in the treatment of VX2 tumors. Methods A total of 90 rabbits bearing VX2 tumors on their left hind legs were randomly divided into three groups. The control group(A) received normal saline, while groups B and C received 20 μg/mL and 40 μg/mL O_3/O_2 ozonated saline, respectively. Rabbits were anesthetized and 2 mL of blood was drawn directly from the heart to measure serum concentrations of interleukin(IL-6) and tumor necrosis factor(TNF-α). The skin covering the VX2 tumor was cut in each rabbit and the maximum and vertical diameters of the tumors were measured under direct visualization. Several milliliters of saline, saline pre-treated with 20 μg/mL O_3/O_2, or saline pre-treated with 40 μg/mL O_3/O_2 were directly injected into the tumors of groups A, B, and C, respectively(injection volume(milliliter) =1/2 volume of the tumor, V = 1/2ab^2). On days 4, 8 and 12 following treatment, 10 rabbits were randomly selected from each group for blood sample collection, and serum IL-6 and TNF-α were measured. The tumor growth rate was calculated by measuring the maximum and vertical diameters of the VX2 tumors under direct visualization. All selected rabbits were euthanized and the tumors, livers, and lungs were removed for pathological examination. The tumor necrosis rate was calculated by cutting the tumors into half along the longitudinal axis and measuring the maximum diameters of the intratumoral necrotic regions. Results The average tumor volume in the three groups increased to different degrees at each time point; however, the average tumor growth rates in groups B and C were substantially lower than that in group A, exhibiting a statistically significant difference. The difference in the tumor growth rate between group B and group C was not statistically significant. The serum concentrations of IL-6 and TNF-α increased in the three groups at each time point, with larger increases occurring in groups B and C; however, the greater increases did not reach statistical significance. Although the diameters of the necrotic areas were larger in both groups B and C than that in group A, significant differences in necrotic area diameters were only found when comparing groups A and C on days 4 and 12 following treatment. Conclusion Direct injection of different concentrations of ozonated saline into VX2 tumors significantly increased intratumoral necrosis and reduced the tumor growth rate. The associated mechanism may be partially mediated by IL-6 and TNF-α, as the serum concentrations of these molecules increased after the treatment.

Establishment of Rabbit Liver VX2 Tumor Model Using Percutaneous Puncture Inoculation of Tumor Fragment Guided and Evaluated by Ultrasonography

The aim of the present study is to evaluate a method of establishing model of rabbit liver VX2 tumor using percutaneous puncture inoculation of tumor fragment guided by ultrasonography.VX2 tumor fragments were implanted into the liver of 13 New Zealand white rabbits flushed by 1 mL normal saline through percutaneous puncture needle guided by ultrasonography.Conventional ultrasonography and contrast-enhanced ultrasonography(CEUS)were performed 14 days after inoculation,and then the rabbits were sacrificed and pathologically examined.The success rate of inoculation was 100%.The average size of liver VX2 tumor was 1.7 cm×1.3 cm,CEUS of VX2 liver tumors showed the"rapid wash-in and wash-out"vascular pattern.There were significant differences between VX2 tumors and liver parenchyma in quantitative parameters of A,k and A×k(P<0.05),which meant that VX2 liver tumors were characterized by more blood flow volume and faster blood velocity than liver parenchyma.Tumor fragment flushed by normal saline into the liver through a needle may be a promising method for the induction of a hepatic tumor.And CEUS can be used for accurately assessing angiogenesis and blood perfusion of VX2 tumors.

Low contrast medium and radiation dose for hepatic computed tomography perfusion of rabbit VX2 tumor

AIM:To evaluate the feasibility of low contrast medium and radiation dose for hepatic computed tomography(CT) perfusion of rabbit VX2 tumor.METHODS:Eleven rabbits with hepatic VX2 tumor underwent perfusion CT scanning with a 24-h interval between a conventional tube potential(120 k Vp) protocol with 350 mg I/m L contrast medium and filtered back projection,and a low tube potential(80 k Vp) protocol with 270 mg I/m L contrast medium with iterative reconstruction.Correlation and agreement among perfusion parameters acquired by the conventional and low dose protocols were assessed for the viable tumor component as well as whole tumor.Image noise and tumor-to-liver contrast to noise ratio during arterial and portal venous phases were evaluated.RESULTS:A 38% reduction in contrast medium dose(360.1 ± 13.3 mg I/kg vs 583.5 ± 21.5 mg I/kg,P < 0.001) and a 73% decrease in radiation dose(1898.5 m Gy·cm vs 6951.8 m Gy·cm) were observed.Interestingly,there was a strong positive correlation in hepatic arterial perfusion(r = 0.907,P < 0.001;r = 0.879,P < 0.001),hepatic portal perfusion(r = 0.819,P = 0.002;r = 0.831,P = 0.002),and hepatic blood flow(r = 0.945,P < 0.001;r = 0.930,P < 0.001) as well as a moderate correlation in hepatic perfusion index(r = 0.736,P = 0.01;r = 0.636,P = 0.035) between the low dose protocol with iterative reconstruction and the conventional protocol for the viable tumor component and the whole tumor.These two imaging protocols provided a moderate but acceptable agreement for perfusion parameters and similar tumorto-liver CNR during arterial and portal venous phases(5.63 ± 2.38 vs 6.16 ± 2.60,P = 0.814;4.60 ± 1.27 vs 5.11 ± 1.74,P = 0.587).CONCLUSION:Compared with the conventional protocol,low contrast medium and radiation dose with iterative reconstruction has no significant influence on hepatic perfusion parameters for rabbits VX2 tumor.

Feasibility of hyperspectral analysis for discrimination of rabbit liver VX2 tumor

BACKGROUND Hepatocellular carcinoma is one of the most common malignant tumors worldwide. Currently, the most accurate diagnosis imaging modality for hepatocellular carcinoma is enhanced magnetic resonance imaging. However, it is still difficult to distinguish cirrhosis lesions, and novel diagnosis modalities are still needed.AIM To investigate the feasibility of hyperspectral analysis for discrimination of rabbit liver VX2 tumor.METHODS In this study, a rabbit liver VX2 tumor model was established. After laparotomy,under direct view, VX2 tumor tissue and normal liver tissue were subjected to hyperspectral analysis.RESULTS The spectral signature of the liver tumor was clearly distinguishable from that of the normal tissue, simply from the original spectral curves. Specifically, two absorption peaks at 600-900 nm wavelength in normal tissue disappeared but a new reflection peak appeared in the tumor. The average optical reflection at the whole waveband of 400-1800 nm in liver tumor was higher than that of the normal tissue.CONCLUSION Hyperspectral analysis can differentiate rabbit VX2 tumors. Further research will continue to perform hyperspectral imaging to obtain more information for differentiation of liver cancer from normal tissue.

Role of MR-DWI and MR-PWI in the radiotherapy of implanted pulmonary VX-2 carcinoma in rabbits

Objective： To detect the activity of tumor cells and tumor blood flow before and after the radiotherapy of implanted pulmonary VX-2 carcinoma in rabbit models by using magnetic resonance diffusion-weighted imaging（MR-DWI） and magnetic resonance perfusion weighted imaging（MR-PWI）, and to evaluate the effectiveness and safety of the radiotherapy based on the changes in the MR-DWI and MR-PWI parameters at different treatment stages.Methods： A total of 56 rabbit models with implanted pulmonary VX-2 carcinoma were established, and then equally divided into treatment group and control group. MR-DWI and MR-PWI were separately performed using a Philips Acheiva 1.5T MRI machine（Philips, Netherland）. MRI image processing was performed using special perfusion software and the WORKSPACE advanced workstation for MRI. MRDWI was applied for the observation of tumor signals and the measurement of apparent diffusion coefficient（ADC） values; whereas MR-PWI was used for the measurement of wash in rate（WIR）, wash out rate（WOR）, and maximum enhancement rate（MER）. The radiation treatment was performed using Siemens PRIMUS linear accelerator. In the treatment group, the radiotherapy was performed 21 days later on a once weekly dosage of 1,000 c Gy to yield a total dosage of 5,000 c Gy.Results： The ADC parameters in the region of interest on DWI were as follows： on the treatment day for the implanted pulmonary VX-2 carcinoma, the t values at the center and the edge of the lesions were 1.352 and 1.461 in the treatment group and control group（P〉0.05）. During weeks 0-1 after treatment, the t values at the center and the edge of the lesions were 1.336 and 1.137（P〉0.05）. During weeks 1-2, the t values were 1.731 and 1.736（P〈0.05）. During weeks 2-3, the t values were 1.742 and 1.749（P〈0.05）. During weeks 3-4, the t values were 2.050 and 2.127（P〈0.05）. During weeks 4-5, the t values were 2.764 and 2.985（P〈0.05）. The ADC values in the treatment group were significantly higher than in the control group. After the radiotherapy（5,000 c Gy）, the tumors remarkably shrank, along with low signal on DWI, decreased signal on ADC map, and remarkably increased ADC values. As shown on PWI, on the treatment day for the implanted pulmonary VX-2 carcinoma, the t values of the WIR, WOR, and MER at the center of the lesions were 1.05, 1.31, and 1.33 in the treatment group and control group（P〉0.05）; in addition, the t values of the WIR, WOR, and MER at the edge of the lesions were 1.35, 1.07, and 1.51（P〉0.05）. During weeks 0-1 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 1.821, 1.856, and 1.931（P〈0.05）; in addition, the t values of the WIR, WOR, and MER at the edge of the lesions were 1.799, 2.016, and 2.137（P〈0.05）. During weeks 1-1 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.574, 2.156, and 2.059（P〈0.05） and the t values of the WIR, WOR, and MER at the edge of the lesions were 1.869, 2.058, and 2.057（P〈0.05）. During weeks 2-3 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.461, 2.098, and 2.739（P〈0.05） and the t values of the WIR, WOR, and MER at the edge of the lesions were 2.951, 2.625, and 2.154（P〈0.05）. During weeks 3-4 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.584, 2.107, and 2.869（P〈0.05） and the t values of the WIR, WOR, and MER at the edge of the lesions were 2.057, 2.637, and 2.951（P〈0.05）. During weeks 4-5 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.894, 2.827, and 3.285（P〈0.05） and the t values of the WIR, WOR, andMER at the edge of the lesions were 3.45, 3.246, and 3.614（P〈0.05）. After the radiotherapy（500 c Gy）, the tumors shrank on the T1 WI, WIR, WOR, and MER; meanwhile, the PWI parameter gradually decreased and reached its minimum value.Conclusions： MR-DWI and MR-PWI can accurately and directly reflect the inactivation of tumor cells and the tumor hemodynamics in rabbit models with implanted pulmonary VX-2 carcinoma, and thus provide theoretical evidences for judging the clinical effectiveness of radiotherapy for the squamous cell carcinoma of the lung.

Expression of MMP-2 in residual VX2 liver tumor after transcatheter arterial embolization combined with portal venous embolization in an animal model

Objective:This study aimed to analyze the effects of transcatheter arterial embolization(TAE)combined with portal venous embolization(PVE)on the expression of MMP-2 in residual VX2 liver tumor tissues,liver function and non-embolic lobe regeneration.Methods:A total of 72 rabbits were randomly divided into Sham,TAE,PVE and TAE+PVE groups(n=18/group).The tissue samples from each group were taken at 6 h,3 days and 7 days after interventional operation,respectively.MMP-2 expression was detected by immunohistochemistry,Real-time PCR,and Western-blotting.The main indicators(such as AST,ATL,and TBIL)of liver function and the volume of non-embolized hepatic lobes were measured in each group after operation.One-way ANOVA and Kruskal-wallis method were used for statistical analysis.Results:The expression of MMP-2 mRNA and protein remained the highest in the Sham group,and the expression of MMP-2 mRNA and protein in TAE,PVE and TAE+PVE groups were successively increased,and the expression of MMP-2 in TAE+PVE group was always significantly higher than TAE group.The AST and ALT levels in each group on day 7 after operation showed a significant declination,and all groups have recovered to the preoperative baseline level and TBIL has a slight fluctuation in each group after operation with no statistical difference.On day 7 after operation,the increasing volume of non-embolized liver lobes in TAE+PVE group showed a more significant effect than those in PVE group,but there was no statistical significance(37.62±1.54 ml VS 36.18±1.15 ml,P=0.881),and its volume was significantly higher than those in the sham group(27.03±1.11 ml).Conclusion:TAE+PVE is considered to be an efficient and safe approach for treating rabbit VX2 liver transplantation tumor,but the expression of MMP-2 increased fastest after TAE+PVE,which might promote tumor cell invasion and metastasis.

A Correlative Study between CT Perfusion Parameters and Angiogenesis in Rabbit VX2 Liver Tumors

Objective: The purpose of this study was to evaluate the correlation between CT perfusion parameters and the hypoxia-inducible factor-1 alpha (HIF-1α), vascular en-dothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and microvessel density (MVD) marked by CD34 molecular of rabbit VX2 liver tumors and to investigate the value of CT perfusion imaging in evaluating tumor angiogenesis. Material and methods: Twenty-four cases of rabbit VX2 liver tumor were performed by CT perfusion scanning. Hepatic artery perfusion (HAP), portal vein perfusion (PVP), total hepatic blood flow (THBF) and hepatic perfusion index (HPI) were measured by perfusion software. HIF-1α, VEGF and MMP-2 expression and MVD were detected in the 24 rabbit VX2 liver tumor tissue samples using immunohistochemical method. The correlation between the HIF-1α, VEGF, MMP-2 expression and MVD and CT perfusion parameters were analyzed. Results: Correlation analysis revealed that the expression of HIF-1α, MMP-2, MVD were positively related to the HAP, THBF, HPI (p < 0.01), but no relations with PVP (p > 0.05);and correlation analysis revealed that the expression of VEGF was positively related to the HAP, HPI (p 0.05). There was a positive relationship between the expression of HIF-1α, VEGF, MMP-2 and MVD (p < 0.01). Conclusions: CT perfusion imaging can reflect the blood perfusion of the rabbit VX2 liver tumors and evaluate the information of angiogenesis about tumors.

兔肝脏、肌肉、皮下VX2肿瘤模型的建立和对比研究

目的通过对肝脏、肌肉及皮下不同部位兔VX2肿瘤模型的建立和比较,寻求更适合于肿瘤经皮介入治疗的荷瘤动物模型。方法将54只新西兰白兔按VX2肿瘤接种部位不同随机分为肝脏组、肌肉组和皮下组,每组18只。三组动物术后12、16、20 d均进行CT灌注扫描,测量肿瘤大小,每次各组分别处死6只兔进行病理观察,并对三组模型的建模手术时间、肿瘤体积及并发症等情况进行比较。结果皮下组肿瘤生长最慢,肌肉组其次,肝脏组生长最快,组间差异有统计学意义(P<0.05)。接种第12天,肝脏组肿块长径已约1 cm,而皮下组则需20 d肿块长径才达1 cm左右,肌肉组肿块生长速度介于二者之间。三组建模所用时间分别为肝脏组(5.54±0.51)min,肌肉组(1.02±0.20)min,皮下组(0.98±0.21)min,肝脏组明显较长。除了肝脏组出现腹水、肝内转移及腹壁种植外,皮下组、肌肉组均无明显影响模型的情况。结论与肝脏和皮下建模相比,兔肌肉内VX2肿瘤模型具有操作简单,成瘤稳定,肿瘤生长速度适中的优点,更适合作为兔VX2瘤的经皮介入治疗模型。

瘤内注射臭氧致兔VX2瘤组织超微结构改变的研究

目的 研究瘤体内注射不同浓度臭氧（O3）后兔VX2瘤组织超微结构的改变，以探讨臭氧用于实验研究/临床应用的安全有效浓度，并根据超微结构改变持续时间，推断重复治疗可能的间隔时间。方法 20只荷瘤大白兔分为4组，对照组2只，T20（20%臭氧）、T40（40%臭氧）和T60组（60%臭氧）治疗组各6只，直视下按瘤体积2倍量不同浓度的O2/O3混合气体用注射器直接注入瘤体内，对照组（仅模拟穿刺）及各治疗组取2只兔在实验完成后即刻取材，剩余兔分别于术后第4、8天各处死2只取材、制片，用透射电镜观察。结果 T20组注射臭氧后即刻可见肿瘤细胞有轻微的损伤改变，线粒体轻微嵴溶解，少许线粒体呈球状肿胀，内质网扩张，部分核周间隙增宽，细胞间隙正常。T40组和T60组注射臭氧后即刻均可见肿瘤细胞有明显损伤，表现为线粒体明显肿胀，内质网扩张，细胞核肿胀，核周间隙增宽，部分细胞膜崩裂，组织结构崩解，术后第4天，可见大片坏死，炎细胞浸润，肿瘤细胞膜不完整，溶酶体线粒体形成（呈新月形黑颗粒）或线粒体溶解，内质网扩张，部分胞质溶解，细胞核不规则，核周间隙扩张，T60组更明显。血管内皮细胞也有损伤改变。3组的细胞损伤在术后第8天仍可观察到，但呈逐渐修复状态。结论 兔VX2瘤内注射20% ～ 60%臭氧后，都会对肿瘤细胞、瘤内血管内皮细胞造成损伤甚至死亡，20%的损伤较轻微，40% ～ 60%的比较明显，但综合安全性及治疗效果， 40%臭氧可能更为明显。虽然有部分修复，但术后第8天仍可见较明确的细胞损伤改变，故治疗间隔以4 ～ 8 d为宜。

光敏剂-磁性纳米粒子螯合剂对磁性纳米粒子在VX2肝转移癌细胞内靶向性分布的影响

目的:研究光敏剂-磁性纳米粒子螯合物促进磁性纳米粒子在VX2肝转移癌细胞内的靶向性分布.方法:取健康新西兰大白兔40只,体质量2.5-3.0kg.建立VX2肝转移癌模型,随机分成空白对照组、光敏剂组、磁性纳米粒子组和光敏剂-磁性纳米粒子螯合物组,在成瘤后的第16、18和20d,经兔耳缘静脉分别注射生理盐水、光敏剂、磁性纳米粒子和螯合物.于第22d处死试验兔,剖腹观察肿瘤生长情况,取正常肝组织和癌结节,作普鲁氏蓝铁染色、透射电镜检查和原子光谱吸收半定量检测.结果:(1)普鲁氏蓝染色后,在螯合剂组癌结节内见到大量蓝染铁颗粒,明显多于其他各组,而肝细胞内少见蓝染铁颗粒;(2)电镜检查,螯合剂组癌细胞内有大量散在的黑色细小颗粒,主要分布在溶酶体、内质网、线粒体和细胞核内;而肝细胞内只有少量黑色细小颗粒;(3)原子光谱吸收半定量检测显示,在癌结节内,螯合剂组铁含量(9.09mg/L)明显高于空白对照组、光敏剂组和磁性纳米粒子组(P<0.01),癌组织内铁含量也明显高于正常肝组织的铁含量(P<0.01).结论:光敏剂-磁性纳米粒子螯合剂能够促进磁性纳米粒子在肝转移癌结节内的聚集.

双歧杆菌对兔VX2瘤组织的靶向性及其对肝癌细胞抑制作用的研究

目的探讨青春型双歧杆菌对兔VX2瘤肿瘤组织的靶向性及其对人肝癌细胞的体外抑制作用。方法①建立荷VX2瘤兔模型,实验组经耳缘静脉注射青春型双歧杆菌;对照组经耳缘静脉注射生理盐水。处死动物,取出脏器和肿瘤组织粉碎后进行厌氧培养和革兰氏染色,观察双歧杆菌对肿瘤的靶向性。②采用超声波细胞粉碎法提取双歧杆菌细胞壁(BCW),应用MTT法和镜下观察来证明BCW体外对人肝癌细胞HepG-2的抑制作用。结果①实验组瘤组织中可见革兰氏阳性菌落,对照组及实验组各正常组织培养未查见。②不同浓度BCW均能抑制HepG-2细胞生长,且BCW浓度越大,抑制作用越强(P<0.05),且镜下观察到HepG-2细胞的生长状态逐渐减弱。结论青春型双歧杆菌能够在兔VX2瘤组织中靶向定植,且其细胞壁能够在体外抑制肝癌细胞的生长。

Characteristics of liver on magnetic resonance diffusion-weighted imaging:Dynamic and image pathological investigation in rabbit liver VX-2 tumor model

AIM: To investigate dynamical and image pathological characteristics of the liver on magnetic resonance (MR) diffusion-weighted imaging (DWI) in the rabbit VX-2 tumor model. METHODS: Forty New Zealand rabbits were included in the study and VX-2 tumor piece was implanted intrahepatically. Fifteen animals received two intrahepatic implantations while 25 had one intrahepatical implantation. DWI, T1- and T2-weighted of magnetic resonance imaging (MRI) were carried out on the 7th and the 14th d after implantation and DWI was conducted, respectively on the 21th d. Ten VX-2 tumor samples were studied pathologically. RESULTS: The rate of lump detected by DWI, T1WI and T2WI was 78.7%, 10.7% and 53.5% (χ2 = 32.61,P < 0.001) on the 7th d after implantation and 95.8%, 54.3% and 82.9% (χ2 = 21.50, P < 0.001) on the 14th d. The signal of most VX-2 tumors on DWI was uniform and it was equal on the map of apparent diffusion coefficient (ADC). The signal of VX tumors did not decrease on the 7th d after implantation, most of them slowly growing during the week following implantation without significant cell dying within the tumor. VX-2 tumors grew increasingly within 14 d after implantation but the signal of most VX-2 tumors on DWI or on the map of ADC was uniform or uneven and ADC of VX tumors decreased obscurely or slightly because tumor necrosis was still not obvious. On the 21th d after implantation, the signal of most VX-2 tumors on DWI or on the map of ADC was uneven because tumor necrosis was evident and ADC of VX-2 tumor necrotic areas decreased. The areas of viable cells in VX-2 tumors manifested a high signal on DWI and a low signal on the map of ADC. The areas of dead cells or necrosis in VX-2 tumors manifested low signals on DWI and low, equal or high signals on the map of ADC but they manifested high signals on DWI and on the map of ADC at the same time when the areas of necrotic tumor became liquefi ed or cystic. The border of tumors on DWI appeared gradually distinct and internal signals of tumor became progressively uneven. CONCLUSION: The manifestations of viable, necrotic and liquefi ed or cystic areas in VX-2 tumors on DWI are typical and DWI is of significant and potential values in clinical application in both the early detection and diagnosis of liver tumors.

Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and Strept Avidin-Biotin Complex (SABC) immunohistochemical staining. RESULTS: The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P < 0.001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P < 0.001). PCNAand nm23 expression in the VX-2 tumor periphery first increased and then decreased (P < 0.001 and P = 0.03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm2, there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P < 0.001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

Characteristics and pathological mechanism on magnetic resonance diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

AIM: To investigate dynamic characteristics and pathological mechanism of signal in rabbit VX-2 tumor model on diffusion-weighted imaging (DWI) after chemoembolization. METHODS: Forty New Zealand rabbits were included in the study and forty-seven rabbit VX-2 tumor models were raised by implanting directly and intrahepatically after abdominal cavity opened. Forty VX-2 tumor models from them were divided into four groups. DWI was performed periodically and respectively for each group after chemoembolization. All VX-2 tumor samples of each group were studied by pathology. The distinction of VX-2 tumors on DWI was assessed by their apparent diffusion coefficient (ADC) values. The statistical significance between different time groups, different area groups or different b-value groups was calculated by using SPSS12.0 software. RESULTS: Under b-value of 100 s/mm2, ADC values were lowest at 16 h after chemoembolization in area of VX-2 tumor periphery, central, and normal liver parenchyma around tumor, but turned to increase with further elongation of chemoembolization treatment. The distinction of ADC between different time groups was significant respectively (F = 7.325, P < 0.001; F = 2.496, P < 0.048; F = 6.856, P < 0.001). Cellular edema in the area of VX-2 tumor periphery or normal liver parenchyma around tumor, increased quickly in sixteen h after chemoembolization but, from the 16th h to the 48th h, cellular edema in the area of normal liver parenchyma around tumor decreased gradually and that in the area of VX-2 tumor periphery decreased lightly at, and then increased continually. After chemoembolization, Cellular necrosis in the area of VX-2 tumor periphery was more significantly high than that before chemoembolization. The areas of dead cells in VX-2 tumors manifested low signal and high ADC value, while the areas of viable cells manifested high signal and low ADC value. CONCLUSION: DWI is able to detect and differentiate tumor necrotic areas from viable cellular areas before and after chemoembolization. ADC of normal liver parenchyma and VX-2 tumor are influenced by intracellular edema, tissue cellular death and microcirculation disturbance after chemoembolization.

MR diffusion-weighted imaging of rabbit liver VX-2 tumor

AIM: To investigate the implanting method of rabbit liver VX-2 tumor and its MR diffusion-weighted imaging (DWI) characteristics.METHODS: Thirty-five New Zealand rabbits were included in the study. VX-2 tumor was implanted subcutaneously in 14 rabbits and intrahepatically in 6 for pre-experiments. VX-2 tumor was implanted intrahepatically in 12 rabbits for experiment and three were used as the control group. DWI, T1- and T2-weighted of MR1 were performed periodically in 15 rabbits for experiment before and after implantation. The distinction of VX-2 tumors on DWI was assessed by their apparent diffusion coefficient (ADC) values. The statistical significance was calculated byanalysis of variance (ANOVA) of the randomized block design using SPSS10.0 software. RESULTS: The successful rate of subcutaneous implantation of VX-2 tumor was 29% (4/14) while that of intrahepatic implantation of it was 33% (2/6) in the preexperiment. The successful rate of intrahepatic implantation of VX-2 tumor in the experiment was 83% (10/12) and 15 tumors grew in 10 successfully implanted rabbits. The DWT signal of VX-2 tumor was high and became lower when the b value increased step by step. The signal of VX-2 tumor on the map of ADC was low. When the b value was 100 or 300 s/mm2, the ADC value of normal group and VX-2 tumor group was respectively 2.57±0.26, 1.73±0.31, 1.87±0.25 and 1.57±0.23 mm2/s. Their distinction was significant (F= 43.26, P＜0.01), the tumor ADC value between b values 100 and 300 s/mm2 wassignificant (Tukey HSP, P＜0.05) and the ADC value between VX-2 tumor and normal liver was also significant (Tukey HSP, P＜0.01). VX-2 tumor developed quickly and metastasized early to all body, especially to the lung, liver, lymph nodes of mediastinum, etc.CONCLUSION: The DWI signal of rabbit VX-2 tumor has its characteristics on MR DWI and DWI plays an important role in diagnosing and discovering VX-2 tumor.

In vivo assessment of intratumoral aspirin injection to treat hepatic tumors

AIM: To study the antineoplastic efficacy of 10% aspirin intralesional injection on VX2 hepatic tumors in a rabbit model. METHODS: Thirty-two male rabbits (age: 6-9 wk; body weight: 1700-2500 g) were inoculated with VX2 hepatic tumor cells (104 cells/rabbit) via supraumbilical median laparotomy. On day 4 post-implantation, when the tumors were about 1 cm in diameter, the rabbits were randomly divided into the following groups (n = 8 each group) to assess early (24 h) and late (7 d) antineoplastic effects of intratumoral injection of 10% bicarbonate aspirin solution (experimental groups) in comparison to intratumoral injection of physiological saline solution (control groups): group 1, 24 h control; group 2, 24 h experimental; group 3, 7 d control; group 4, 7 d experimental. The serum biochemistry profile (measurements of glycemia, alkaline phosphatase, gamma-glutamyl transferase, aspartateaminotransferase, and alanine aminotransferase) and body weight measurements were obtained for all animals at the following time points: D0, before tumor implant; D4, day of treatment; D5, day of sacrifice for groups 1 and 2; D11, day of sacrifice for groups 3 and 4. Gross assessments of the abdominal and thoracic cavities were carried out upon sacrifice. The resected liver tissues, including hepatic tumors, were qualitatively (general morphology, signs of necrosis) and quantitatively (tumor area) assessed by histopathological analysis. RESULTS: Gross examination showed no alterations, besides the left hepatic lobe tumors, had occurred in the thoracic and abdominal cavities of any animal at any time point evaluated. However, the features of the tumor foci were distinctive between the groups. Compared to the control groups, which showed normal unabated tumor progression, the aspirin-treated groups showed imprecise but limited tumor boundaries and a general red-white coloration (indicating hemorrhaging) at 24 h post-treatment, and development of yellow-white areas of a cicatricial aspect at 7 d after treatment. At all time points evaluated, all except one biochemical parameters tested within the reference range (P > 0.05); a significant increase was detected in the alkaline phosphatase level of the control group 3 on D11 (P < 0.05). At 24 h post-treatment, the aspirintreated groups showed extensive coagulation necrosis accompanied by a remarkable absence of viable tumor foci; at 7 d after treatment, the tumors had completely disappeared in these animals and fibrous necrotic nod- ules had developed. In contrast, throughout the study course, the tumors of the control groups remained unchanged, showing tumor nodules without necrosis at the time point corresponding to 24 h post-treatment and increased amounts of tumor nodules at the time point corresponding to 7 d post-treatment. Quantitative analysis of the remaining tumor area revealed that the aspirin-treated groups had significantly smaller tumor foci at 24 h post-treatment (8.5% ± 0.7%) andat 7 d after treatment (11.0% ± 4.2%), compared to those in the control groups (24 h: 98.5% ± 1.5% and 7 d: 94.0% ± 2.7%; both,P < 0.005). CONCLUSION: Intralesional injection of a 10% aspirin solution causes destruction of VX2 hepatic tumors in rabbits without evidence of relapse at 7 d after treat- ment administration.

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.

Sphere-forming-like cells(squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma

Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell（CSC） traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas（SCCs） and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation（CD） 44, CD133, acetaldehyde dehydrogenase 1（ALDH1）, B cell-specific Moloney murine leukemia virus integration site 1（Bmi-1）, Nestin, octamer-binding transcription factor 4（Oct4）and reduced expression protein-1（Rex-1） expression with reverse transcription-polymerase chain reaction（RT-PCR）; chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers（CD44, Bmi-1, Nestin, Oct4 and Rex-1）, capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts（with histological features resembling those of the original VX2 rabbit buccal SCC） from the transplantation of 103 undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.

经肝动脉应用重组人白细胞介素-2联合碘化油乳剂治疗兔VX2肝肿瘤的实验研究

目的观察重组人白细胞介素-2联合碘化油乳剂经肝动脉灌注对兔VX2肝肿瘤的治疗作用。方法建立30只兔VX2肝肿瘤模型,随机分为对照组、常规组和实验组,经胃十二指肠动脉至肝动脉分别灌注生理盐水、MMC(0.2mg/kg)+适量超液化碘油以及重组人白细胞介素-2(20万IU/m2)+适量超液化碘油。结果术后实验组兔在生存率上优于常规组,对照组、常规组和实验组介入治疗后4周的生存率分别为70.0%、80.0%和90.0%,且实验组血清肝功能指标及病理检查显示肝功能损害轻。结论经肝动脉灌注重组人白细胞介素-2联合碘化油乳剂对兔VX2肝肿瘤具有较好的治疗作用。

Radiosensitivity of β-elemene on rabbit VX2 renal transplant carcinoma model

Objective To investigate the effects of low dosage of β-elemene on the radiosensitivity of rabbit VX2 renal transplant carcinoma model. Methods We took the rabbit VX2 renal transplant carcinoma as the model. Experimental rabbits were divided into three groups: the control group, the radiation group, and the radiation +β-elemene (radiosensitivity) group. The change of tumor was observed by Spiral CT and B ultrasound to compare its regrowth period. The tumor was measured by light microscopy and electron microscopy. Results The tumor in radiosensitivity group was restrained obviously and the sensitization enhancement ratio (SER) of β-elemene was 1.89. Different apoptosis was observed under transmission electron microscopy. Conclusion Low dosage β-elemene can enhance the radiosensitivity of rabbit VX2 renal transplant carcinoma model and induce the apoptosis of tumor cells, but the mechanism needs further study. It promotes apoptosis in mechanisms in vitro.