Buffalo rats (Male, 8-10weeks, 350-400 g) were obtained from lab animal unit, The University of Hong Kong. Before operation, rats were anesthesia with pentobarbitone (40 mg/kg, ip) and after operation buprenorphine (0.05-0.1 mg/kg/12 hours) were given for attenuating the analgesics. Rats were housed in a standard animal laboratory with free activity and access to water and food. They were kept under constant environment conditions with a 12-hour light–dark cycle. All operations were performed under clean conditions. The study had been licensed according to Animal Ordinance Chapter 340 by the Department of Health, Hong Kong Special Administrative Region (ref.: (12–222) in DH/HA&P/8/2/3 Pt. 39). Hepatocellular carcinoma cell line McA-RH7777 (Purchased from the American Type Culture Collection, Number CRL1601, ATCC, Manassas, VA, USA) were used to establish the orthotopic liver cancer model in Buffalo rat . Two weeks after orthotopic liver tumor implantation, The branch of hepatic artery and portal vein to right and triangle lobes are clamped for 30 minutes (ischemia duration) following by reperfusion (release of the clamp). Major hepatectomy of tumor-bearing lobe (left lobe) is performed during the ischemia duration.
Treatment regimen and sample collection
YQ23 products were obtained from New B Innovation Limited. YQ23 product is the stabilized non-polymeric cross-linked tetrameric hemoglobin (65 kDa) with undetectable/low level of dimeric hemoglobin (32 kDa), phospholipid, DNA impurities and protein impurities. The concentration of YQ product is 10 g/dL and its pH range is 7.2-7.8. The osmolality and viscosity (at 37°C) are >250 mOsm/kg and 0.9 centipoise respectively. The p50 value is ~ 40 mmHg. The information for YQ product is shown in patent no. US7,932,356 B1, US 8,048,856 B1 and PCT/US12/46130. YQ23 (0.2 g/kg, treatment group) or the same volume of ringer’s acetate buffer (control group) were administrated intravenously at 1 hour before ischemia. An additional 0.2 g/kg YQ23 or the same volume of ringer’s acetate buffer was injected through hepatic portal vein immediately after reperfusion. Blood samples were taken at days 0, 1, 7, 14, 21 and 28 from tail vein for detecting the populations of circulating EPCs and Tregs. And then Buffalo rats were sacrificed at 4 weeks after major hepatectomy and partial hepatic I/R injury to monitor liver tumor metastasis. Liver and lung were sampled for further investigation.
Hematoxylin and Eosin (H & E) and Immunohistochemical (IHC) Staining
The histological changes were detected by H& E staining and the expression of CD34 (Santa Cruz Biotechnology) were detected by immunohistochemical staining. The details of H & E and IHC staining were described in our previous paper . In brief, after rehydrated in water, the paraffin sections placed in citric buffer (pH 6.0) and treated in a microwave. Afterwards, the sections underwent blocking with 10% FBS and then primary antibodies were applied (incubated at 4°C overnight). Then the sections underwent blocking with 3% peroxidase for 30 min and secondary antibodies from Dako EnVision System (DakoCytomation) were applied. Signals were developed with 3,3’-diaminobenzidine substrate solution (DakoCytomation).
Determination of microvessel density and tumor load analyses
Microvessel density (MVD) of intrahepatic metastatic tumor sections was evaluated . The mean number of tumor nodules in the lungs and liver, as well as tumor volume of intrahepatic metastatic nodules (L × W2/2) was calculated and expressed as average .
Detection of circulating EPCs and Tregs by flow cytometry
The number of circulating EPCs and Tregs were detected by flow cytometry. The details of flow cytometry were described in our previous paper . For analysis of EPC cell surface molecules, cells were stained with the following antibodies: unconjugated rabbit anti-CD133 (Abcam, Cambridge, UK), PE-conjugated anti-CD34 (Santa Cruz Biotech, Santa Cruz, CA), VEGFR2+ (BD Pharmingen, San Diego, CA) and goat anti-rabbit FITC secondary antibody (Abcam, Cambridge, UK). For analysis of Treg cells, cells were stained with PE-Cy5-conjugated anti CD4 (eBioscience, San Diego, CA) and PE-conjugated anti CD25 (BD Pharmingen, San Diego, CA), and then permeabilized with fixation/permeabiliation working solution and incubated with FITC-conjugated anti-Foxp3 (eBioscience, San Diego, CA).
Determination of liver oxygenation
Liver tissue oxygenation (Liver pO2) was directly monitored by the OxyLab® in vivo monitoring system (Oxford Optronix, UK) during the ischemia and reperfusion procedures in another group of Buffalo rats. Briefly, a large-area-surface (LAS) oxygen sensor (Oxford Optronix, UK) was placed between the right hepatic lobe and triangle lobe of the rat livers. The branch of hepatic artery and portal vein to right and triangle lobes were clamped for 30 minutes following by reperfusion. YQ23 (0.2 g/kg, treatment group) or ringer’s acetate buffer (control group) were administered intravenously at 1 hour before ischemia. An additional 0.2 g/kg YQ23 or ringer’s acetate buffer was injected through hepatic portal vein immediately after reperfusion. Liver oxygen tension was continuously measured during hepatic ischemia reperfusion injury: 1) baseline; 2) after infusion of YQ23 or ringer’s acetate buffer; 3) during ischemia; and 4) after onset of reperfusion.
Assessment of hepatic gene expression profiles
In order to investigate the effect of YQ23 on expressions of cytokines/chemokines, a new group of Buffalo rats were included for major hepatecotmy and partial hepatic I/R injury. YQ23 or ringer’s acetate buffer were administered at 1 hour before ischemia and immediately after reperfusion. Liver samples were collected at 6 hours after reperfusion and gene expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) . Gene expression levels were expressed as the folds relative to the normal liver. The sequences of the primers were listed as follows: CXCR3: Left AGCACAGACACCTTCCTGCT, Right CAGAGACCAGAGCCGAAAAC; TNF-α: Left GTCTGTGCCTCAGCCTCTTC, Right CCCATTTGGGAACTTCTCCT. IL6: Left GCCCTTCAGGAACAGCTATG, Right GTCTCCTCTCCGGACTTGTG; HO1: Left GAAGAAGATTGCGCAGAAGG, Right TTCATGCGAGCACGATAGAG.
Statistics and data analyses
Continuous variables were expressed as average. T-TEST was applied to delineate the difference between the treatment and control groups. Chi-Square (χ2) test was used to compare incidence of intrahepatic and lung metastasis after major hepatectomy and partial hepatic I/R injury. p < 0.05 was considered statistically significant. Calculations were performed by using the SPSS computer software version 16 (SPSS, Chicago, IL).