Yewei Liu

Mammalian models of chemically induced primary malignancies exploitable for imaging-based preclinical theragnostic research.

Compared with transplanted tumor models or genetically engineered cancer models, chemically induced primary malignancies in experimental animals can mimic the clinical cancer progress from the early stage on. Cancer caused by chemical carcinogens generally develops through three phases namely initiation, promotion and progression. Based on different mechanisms, chemical carcinogens can be divided into genotoxic and non-genotoxic ones, or complete and incomplete ones, usually with an organ-specific property. Chemical carcinogens can be classified upon their origins such as environmental pollutants, cooked meat derived carcinogens, N-nitroso compounds, food additives, antineoplastic agents, naturally occurring substances and synthetic carcinogens, etc. Carcinogen-induced models of primary cancers can be used to evaluate the diagnostic/therapeutic effects of candidate drugs, investigate the biological influential factors, explore preventive measures for carcinogenicity, and better understand molecular mechanisms involved in tumor initiation, promotion and progression. Among commonly adopted cancer models, chemically induced primary malignancies in mammals have several advantages including the easy procedures, fruitful tumor generation and high analogy to clinical human primary cancers. However, in addition to the time-consuming process, the major drawback of chemical carcinogenesis for translational research is the difficulty in noninvasive tumor burden assessment in small animals. Like human cancers, tumors occur unpredictably also among animals in terms of timing, location and the number of lesions. Thanks to the availability of magnetic resonance imaging (MRI) with various advantages such as ionizing-free scanning, superb soft tissue contrast, multi-parametric information, and utility of diverse contrast agents, now a workable solution to this bottleneck problem is to apply MRI for noninvasive detection, diagnosis and therapeutic monitoring on those otherwise uncontrollable animal models with primary cancers. Moreover, it is foreseeable that the combined use of chemically induced primary cancer models and molecular imaging techniques may help to develop new anticancer diagnostics and therapeutics.

Pharmacologic effects of cannabidiol on acute reperfused myocardial infarction in rabbits: evaluated with 3.0T cardiac magnetic resonance imaging and histopathology

Abstract: Cannabidiol (CBD) has anti-inflammatory effects. We explored its therapeutic effects on cardiac ischemia–reperfusion injury with an experimental imaging platform. Reperfused acute myocardial infarction (AMI) was induced in rabbits with a 90-minute coronary artery occlusion followed by 24-hour reperfusion. Before reperfusion, rabbits received 2 intravenous doses of 100 &mgr;g/kg CBD (n = 10) or vehicle (control, n = 10). Evans blue was intravenously injected for later detection of the AMI core. Cardiac magnetic resonance imaging was performed to evaluate cardiac morphology and function. After euthanasia, blood troponin I (cTnI) was assessed, and the heart was excised and infused with multifunctional red iodized oil dye. The heart was sliced for digital radiography to quantify the perfusion density rate, area at risk (AAR), and myocardial salvage index, followed by histomorphologic staining. Compared with controls, CBD treatment improved systolic wall thickening (P < 0.05), significantly increased blood flow in the AAR (P < 0.05), significantly decreased microvascular obstruction (P < 0.05), increased the perfusion density rate by 1.7-fold, lowered the AMI core/AAR ratio (P < 0.05), and increased the myocardial salvage index (P < 0.05). These improvements were associated with reductions in serum cTnI, cardiac leukocyte infiltration, and myocellular apoptosis (P < 0.05). Thus, CBD therapy reduced AMI size and facilitated restoration of left ventricular function. We demonstrated that this experimental platform has potential theragnostic utility.

Visualization, Quantification and Characterization of Caerulein-Induced Acute Pancreatitis in Rats by 3.0T Clinical MRI, Biochemistry and Histomorphology.

Purpose: To investigate whether Caerulein-induced acute pancreatitis (AP) in rats could be noninvasively studied by clinical magnetic resonance imaging (MRI) techniques and validated by enzymatic biochemistry and histomorphology. Materials and Methods: The study was approved by the institutional animal ethical committee. The AP was induced in 26 rats by intraperitoneal injections of Caerulein, as compared to 6 normal rats. T2-weighted 3D MRI, T2 relaxation measurement and contrast enhanced T1-weighted MRI were performed at 3 Tesla. Pancreatic volume and contrast ratio of pancreas against surrounding tissues were measured by MRI. Animals were scarified at 3, 8, 24 and 48-hr respectively for analyses of serum lipase and amylase levels, and biliopancreatic perfusion-assisted histomorphology. Results: The AP could be observed on MRI 3-hr onwards after Caerulein-administration. T2 relaxation within the pancreas was prolonged due to high water content or edema. Increase of vascular permeability was indicated by T1 contrast enhancement. Both edema and vascular permeability gradually recovered afterwards (p<0.05/0.01), paralleled by declining serum enzyme levels (p<0.05). Microscopy revealed cell vacuolization and edema for early stage, and increased inflammatory cell infiltration and acinar cell loss after 24 and 48-hr. Conclusion: Multiparametric MRI techniques at 3.0T could facilitate noninvasive diagnosis and characterization of Caerulein induced AP in rats, as validated by a novel ex vivo method.

Characterization of a rat orthotopic pancreatic head tumor model using three-dimensional and quantitative multi-parametric MRI.

The purpose of this study was to investigate the reliability of 3D isotropic MRI and quantitative multi‐parametric MRI characterization on an orthotopic pancreatic head tumor model in rats. 3D isotropic T2‐weighted MRI was performed as a routine for tumor longitudinal follow‐up and volume estimation. Common bile duct diameter was measured from 3D multiplanar reconstruction. Quantitative multi‐parametric measurements including pixel‐wise T2, T1 relaxivity, apparent diffusion coefficient (ADC) and apparent diffusion kurtosis mapping were performed twice throughout tumor growth. Semi‐quantitative and quantitative analyses based on an extended Tofts model were applied to region‐of‐interest‐based dynamic contrast‐enhanced imaging, followed by contrast ratio measurement on standard contrast‐enhanced imaging. Moreover, low‐level texture‐based analysis was inspected for T2, T1, ADC and contrast ratio measurements. Results indicated that multi‐parametric MRI showed good reproducibility for tumor characterization; the measurements were not affected by tumor growth. Tumor growth was further confirmed with histology examinations. To conclude, state‐of‐the‐art clinical MRI techniques were translated to this preclinical tumor model with high reliability, and have paved the way for translational oncology studies on this tumor model.

A methodology for constraining power in finite element modeling of radiofrequency ablation.

Radiofrequency ablation (RFA) is a minimally invasive thermal therapy for the treatment of cancer, hyperopia, and cardiac tachyarrhythmia. In RFA, the power delivered to the tissue is a key parameter. The objective of this study was to establish a methodology for the finite element modeling of RFA with constant power. Because of changes in the electric conductivity of tissue with temperature, a nonconventional boundary value problem arises in the mathematic modeling of RFA: neither the voltage (Dirichlet condition) nor the current (Neumann condition), but the power, that is, the product of voltage and current was prescribed on part of boundary. We solved the problem using Lagrange multiplier: the product of the voltage and current on the electrode surface is constrained to be equal to the Joule heating. We theoretically proved the equality between the product of the voltage and current on the surface of the electrode and the Joule heating in the domain. We also proved the well-posedness of the problem of solving the Laplace equation for the electric potential under a constant power constraint prescribed on the electrode surface. The Pennes bioheat transfer equation and the Laplace equation for electric potential augmented with the constraint of constant power were solved simultaneously using the Newton-Raphson algorithm. Three problems for validation were solved. Numerical results were compared either with an analytical solution deduced in this study or with results obtained by ANSYS or experiments. This work provides the finite element modeling of constant power RFA with a firm mathematical basis and opens pathway for achieving the optimal RFA power.

The first study on therapeutic efficacies of a vascular disrupting agent CA4P among primary hepatocellular carcinomas with a full spectrum of differentiation and vascularity: Correlation of MRI-microangiography-histopathology in rats: Vascular disrupting agent CA4P in rats with HCCs

To better inform the next clinical trials of vascular disrupting agent combretastatin‐A4‐phosphate (CA4P) in patients with hepatic malignancies, this preclinical study aimed at evaluating CA4P therapeutic efficacy in rats with primary hepatocellular carcinomas (HCCs) of a full spectrum of differentiation and vascularity by magnetic resonance imaging (MRI), microangiography and histopathology. Ninety‐six HCCs were raised in 25 rats by diethylnitrosamine gavage. Tumor growth was monitored by T2‐/T1‐weighted‐MRI (T2WI, T1WI) using a 3.0 T scanner. Early vascular response and later intratumoral necrosis were detected by dynamic‐contrast‐enhanced (DCE) MRI and diffusion‐weighted‐imaging (DWI) before, 1 and 12 hr after CA4P iv‐administration. In vivo MRI‐findings were validated by postmortem‐techniques. Multi‐parametric MRI revealed rapid CA4P‐induced tumor vascular shutdown within 1 hr, followed by variable intratumoral necrosis at 12 hr. Tumor volumes decreased by 10% at 1 hr (p < 0.05), but resumed at 12 hr. Correlations of semi‐quantitative DCE parameter initial‐area‐under‐the‐gadolinium‐curve (IAUGC30) with histopathology proved partial vascular closure and compensational reopening (p < 0.05). The higher grades of vascularity prevented those residual tumor tissues from CA4P‐caused ischemic necrosis. By histopathology using a 4‐scale cellular‐differentiation criteria and a 4‐grade tumor‐vascularity classification, percentage of CA4P‐induced necrosis negatively correlated with HCC differentiation (r = −0.404, p < 0.001) and tumor vascularity (r = −0.370, p < 0.001). Ordinal‐logistic‐regression helped to predict early tumor responses to CA4P in terms of tumoral differentiation and vascularity. Our study demonstrated that CA4P could induce vascular shutdown in primary HCCs within 1 hr, resulting in various degrees of tumor necrosis at 12 hr. MRI as a real‐time imaging biomarker may help to define tumor vascularity and differentiation and further to predict CA4P therapeutic outcomes.

Micro-HCCs in rats with liver cirrhosis: paradoxical targeting effects with vascular disrupting agent CA4P

We sought to investigate anticancer efficacy of a vascular disrupting agent (VDA) combretastatin A-4 phosphate (CA4P) in relation to tumor size among hepatocellular carcinomas (HCCs) in rats using magnetic resonance imaging (MRI) and postmortem techniques. Nineteen rats with 43 chemically-induced HCCs of 2.8–20.9 mm in size on liver cirrhosis received CA4P intravenously at 10 mg/kg. Tumor-diameter was measured by T2-weighted imaging (T2WI) to define microcancers (< 5 mm) versus larger HCCs. Vascular responses and tissue necrosis were detected by diffusion-weighted imaging (DWI), contrast-enhanced T1-weighted imaging (CE-T1WI) and dynamic contrast enhanced (DCE-) MRI, which were validated by microangiography and histopathology. MRI revealed nearly complete necrosis in 5 out of 7 micro-HCCs, but diverse therapeutic necrosis in larger HCCs with a positive correlation with tumor size. Necrosis in micro-HCCs was 36.9% more than that in larger HCCs. While increased diffusion coefficient (ADCdiff) suggested tumor necrosis, perfusion coefficient (ADCperf) indicated sharply decreased blood perfusion in cirrhotic liver together with a reduction in micro-HCCs. DCE revealed lowered tumor blood flow from intravascular into extravascular extracellular space (EES). Microangiography and histopathology revealed hypo- and hypervascularity in 4 and 3 micro-HCCs, massive, partial and minor degrees of tumoral necrosis in 5, 1 and 1 micro-HCCs respectively, and patchy necrotic foci in cirrhotic liver. CD34-PAS staining implicated that poorly vascularized micro-HCCs growing on liver cirrhosis tended to respond better to CA4P treatment. In this study, more complete CA4P-response occurred unexpectedly in micro-HCCs in rats, along with CA4P-induced necrotic foci in cirrhotic liver. These may help to plan clinical applications of VDAs in patients with HCCs and liver cirrhosis.We sought to investigate anticancer efficacy of a vascular disrupting agent (VDA) combretastatin A-4 phosphate (CA4P) in relation to tumor size among hepatocellular carcinomas (HCCs) in rats using magnetic resonance imaging (MRI) and postmortem techniques. Nineteen rats with 43 chemically-induced HCCs of 2.8-20.9 mm in size on liver cirrhosis received CA4P intravenously at 10 mg/kg. Tumor-diameter was measured by T2-weighted imaging (T2WI) to define microcancers (< 5 mm) versus larger HCCs. Vascular responses and tissue necrosis were detected by diffusion-weighted imaging (DWI), contrast-enhanced T1-weighted imaging (CE-T1WI) and dynamic contrast enhanced (DCE-) MRI, which were validated by microangiography and histopathology. MRI revealed nearly complete necrosis in 5 out of 7 micro-HCCs, but diverse therapeutic necrosis in larger HCCs with a positive correlation with tumor size. Necrosis in micro-HCCs was 36.9% more than that in larger HCCs. While increased diffusion coefficient (ADCdiff) suggested tumor necrosis, perfusion coefficient (ADCperf) indicated sharply decreased blood perfusion in cirrhotic liver together with a reduction in micro-HCCs. DCE revealed lowered tumor blood flow from intravascular into extravascular extracellular space (EES). Microangiography and histopathology revealed hypo- and hypervascularity in 4 and 3 micro-HCCs, massive, partial and minor degrees of tumoral necrosis in 5, 1 and 1 micro-HCCs respectively, and patchy necrotic foci in cirrhotic liver. CD34-PAS staining implicated that poorly vascularized micro-HCCs growing on liver cirrhosis tended to respond better to CA4P treatment. In this study, more complete CA4P-response occurred unexpectedly in micro-HCCs in rats, along with CA4P-induced necrotic foci in cirrhotic liver. These may help to plan clinical applications of VDAs in patients with HCCs and liver cirrhosis.

Vascular disrupting agent in pancreatic and hepatic tumour allografts: observations of location-dependent efficacy by MRI, microangiography and histomorphology

Background:Tumours growing in organs of different vascular environment could exhibit diverse responses to vascular disrupting agent (VDA). This study was aimed to identify in vivo imaging biomarkers for evaluation of pancreatic and hepatic tumours and comparison of their responses to a VDA Combretastatin A4 Phosphate (CA4P) using multiparametric MRI.Methods:Male WAG/Rij rats were used for orthotopic pancreatic head tumour and hepatic tumour implantation; tumour growth was monitored by 3D isotropic MRI using a 3.0-T clinic scanner. Therapeutic intervention using CA4P was investigated by in vivo quantitative MRI measurements including T2/T1 relaxation mapping, diffusion kurtosis imaging and dynamic contrast-enhancement (DCE) imaging. Animals were scarified 10 h after CA4P treatment for ex vivo validation using microangiography and histomorphology.Results:State-of-the-art clinical MRI protocols were successfully adapted for imaging small animal tumour with high reliability. One hour after CA4P injection, marked vascular shutdown was detected with DCE MRI in both pancreatic and hepatic tumours. However, 10 h later, therapeutic necrosis was limited in pancreatic tumours compared with that in hepatic tumours (P<0.01). Heterogeneous therapeutic changes were depicted in tumour lesions using pixel-wise Tofts model, which was generated from dynamic T1 mapping. In addition, tumour responses including haemorrhage, oedema and necrosis were detected using quantitative T2/T1 relaxation maps and diffusion kurtosis images, and were validated using histomorphology.Conclusions:Using multiparametric imaging biomarkers, hepatic tumours were found to be significantly more responsive to CA4P than pancreatic tumours, which could be of reference for designing future clinical trials on this agent.

Rat model of cholelithiasis with human gallstones implanted in cholestasis-induced virtual gallbladder.

AIM To facilitate translational research on cholelithiasis, we have developed a rat model of human gallstones by exploiting the unique biliopancreatic features of this species. METHODS Under anesthesia, 16 adult rats of equal genders underwent two times of abdominal surgery. First, their common bile duct (CBD) was ligated to cause cholestasis by total biliary obstruction (TBO). On day 0, 1, 3, 7, 14, 21 and 28 after TBO, magnetic resonance imaging (MRI) was conducted to monitor the dilatation of the CBD, and blood was sampled to analyze total serum bilirubin (TSB). Secondly, on day 30, the abdomen was re-opened and gallstone(s) collected from human patients were implanted in the dilated CBD as a virtual gallbladder (VGB), which was closed by suture ligation. This rat cholelithiasis model was examined by MRI, clinical observation, microcholangiography and histology. RESULTS All rats survived two laparotomies. After ligation, the CBD was dilated to a stable size of 4 to 30 mm in diameter on day 21-28, which became a VGB. The rats initially showed signs of jaundice that diminished over time, which paralleled with the evolving TSB levels from 0.6 ± 0.3 mg/dL before ligation, through a peak of 10.9 ± 1.9 mg/dL on day 14, until a nearly normalized value after day 28. The dilated CBD with thickened wall allowed an incision for implantation of human gallstones of 1-10 mm in diameter. The rat cholelithiasis was proven by in vivo MRI and postmortem microcholangiography and histomorphology. CONCLUSION A rat model cholelithiasis with human gallstones has been established, which proves feasible, safe, reliable, nontoxic and cost-effective. Given the gallstones of human origin, applications of this model may be of help in translational research such as optical detection and lysis of gallstones by systemic drug administration.

Differential diagnosis of gallstones by using hypericin as a fluorescent optical imaging agent

AIM To explore the feasibility of using hypericin as an optical imaging probe with affinity for cholesterol for differential fluorescent detection of human gallstones. METHODS Cholesterol, mixed and pigment stones from cholecystectomy patients were incubated with hypericin or solvent. After 72 h, the stones were analysed for fluorescence (365 nm) and treated with 2-propanol/dimethyl sulfoxide for high performance liquid chromatography (HPLC) analysis. Rats with virtual gallbladder containing human cholesterol, mixed or pigment gallstones (VGHG) received 5 mg/kg hypericin or solvent and VGHG rats with cholesterol stones were given different hypericin doses (5-15 mg/kg). Twelve hours later, the stones were analysed at 365 nm. Biliary excretion and metabolites of hypericin were assessed in common bile duct (CBD) cannulated rats for 9 h using fluorospectrometry, HPLC and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS Homogeneous high fluorescence was seen on cholesterol stones either pre-incubated with hypericin or extracted from VGHG rats receiving hypericin. Mixed stones showed a dotted fluorescent pattern, whereas pigment and solvent-treated ones lacked fluorescence. HPLC showed 7.68, 6.65 and 0.08 × 10(-3) M of cholesterol in extracts from cholesterol, mixed, and pigment gallstones, respectively. Hypericin accounted for 2.0, 0.5 and 0.2 × 10(-6) M in that order. On cholesterol stones from VGHG rats receiving different hypericin doses, a positive correlation was observed between dose and fluorescence. In the bile from CBD-cannulated rats, fluorescence represented 20% of the injected dose with two peaks in 9 h. HPLC analysis revealed that hypericin conjugates reached 60% of the peak area. By MALDI-TOF MS, hypericin-glucuronide was detected. CONCLUSION This study proves the potential use of hypericin for differential fluorescent detection of human gallstones regarding their chemical composition.