Validation of a Preclinical Model of Diethylnitrosamine-Induced Hepatic Neoplasia in Yucatan Miniature Pigs.

OBJECTIVE: The purpose of this study was to reduce the time to tumor onset in a diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) swine model via partial liver embolization (PLE) and to characterize the model for use in translational research. METHODS: Eight Yucatan miniature pigs were injected intraperitoneally with either saline (n = 2) or DEN (n = 6) solution weekly for 12 weeks. Three of the DEN-treated pigs underwent PLE. The animals underwent periodic radiological evaluation, liver biopsy, and blood sampling, and full necropsy was performed at study termination (∼29 months). RESULTS: All DEN-treated pigs developed hepatic adenoma and HCC. PLE accelerated the time to adenoma development but not to HCC development. Biomarker analysis results showed that IGF1 levels decreased in all DEN-treated pigs as functional liver capacity decreased with progression of HCC. VEGF and IL-6 levels were positively correlated with disease progression. Immunohistochemical probing of HCC tissues demonstrated the expression of several important survival-promoting proteins. CONCLUSION: To our knowledge, we are the first to demonstrate an accelerated development of hepatic neoplasia in Yucatan miniature pigs. Our HCC swine model closely mimics the human condition (i.e., progressive disease stages and expression of relevant molecular markers) and is a viable translational model.

An Improved Patient-Derived Xenograft Humanized Mouse Model for Evaluation of Lung Cancer Immune Responses.

Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord blood CD34+ stem cells (CD34+ HSC), and combined it with lung cancer cell line-derived human xenografts or patient-derived xenografts (Hu-PDX). Fresh CD34+ HSCs could reconstitute detectable mature human leukocytes (hCD45+) in mice at four weeks without the onset of graft-versus-host disease (GVHD). Repopulated human T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) increased in peripheral blood, spleen, and bone marrow over time. Although cultured CD34+ HSCs labeled with luciferase could be detected in mice, the cultured HSCs did not develop into mature human immune cells by four weeks, unlike fresh CD34+ HSCs. Ex vivo, reconstituted T cells, obtained from the tumor-bearing humanized mice, secreted IFNγ upon treatment with phorbol myristate acetate (PMA) or exposure to human A549 lung tumor cells and mediated antigen-specific CTL responses, indicating functional activity. Growth of engrafted PDXs and tumor xenografts was not dependent on the human leukocyte antigen status of the donor. Treatment with the anti-PD-1 checkpoint inhibitors pembrolizumab or nivolumab inhibited tumor growth in humanized mice significantly, and correlated with an increased number of CTLs and decreased MDSCs, regardless of the donor HLA type. In conclusion, fresh CD34+HSCs are more effective than their expanded counterparts in humanizing mice, and do so in a shorter time. The Hu-PDX model provides an improved platform for evaluation of immunotherapy.

A pilot trial of vascular targeted photodynamic therapy for renal tissue.

PURPOSE: Vascular targeted photodynamic therapy represents the newest generation of photodynamic therapy and a new paradigm for minimally invasive ablative therapy. We report a pilot trial of vascular targeted photodynamic therapy to evaluate the effect on porcine renal tissue. MATERIALS AND METHODS: Pigs underwent continuous infusion of WST-09 (Negma-Lerads, Toussous le Noble, France) and concurrent illumination with interstitial laser at a wavelength of 763 nm to the lower pole of the kidney. Drug doses were 0.5 to 1.0 mg/kg and light doses were 100 to 200 J. Nuclear renography was performed on postoperative day 5. On postoperative day 7 arteriography, pyelography, computerized tomography of the abdomen and necropsy were performed. RESULTS: Four of 7 animals completed therapy and all evaluations. Three animals died, including 1 of surgical complications and 2 of an anaphylactoid reaction to the Cremophor solvent in the compound. All kidneys in surviving animals functioned on nuclear renography. Renal function remained unchanged. No lesions or urine leakage was visible on imaging. On necropsy lesion size was 5 x 4 x 3 to 7 x 7 x 14 mm depending on the drug/light dose. Histology showed a distinct demarcation between the treated zone and the surrounding parenchyma at higher doses. Lesions were well demarcated with necrotic tubules, glomerular fibrinoid necrosis, capillary loop thrombosis, interstitial hemorrhage and lymphocytic infiltrates. CONCLUSIONS: Significant tissue effect with some necrosis was seen at these low drug/light combinations. This study provides the initial proof of principle that justifies further preclinical investigation of vascular targeted photodynamic therapy for renal tumors. A newer, water based formulation should decrease the incidence of reactions in swine. This newer formulation may allow further safe investigation of this novel treatment paradigm.

Evaluating the effectiveness of training strategies: performance goals and testing.

The Public Health Service policy, Animal Welfare Act regulations, and the Guide for the Care and Use of Laboratory Animals all require that institutions provide training for personnel engaged in animal research. Most research facilities have developed training programs to meet these requirements but may not have developed ways of assessing the effectiveness of these programs. Omission of this critical activity often leads to training that is ineffective, inefficient, or unnecessary. Evaluating the effectiveness of biomedical research and animal care training should involve a combination of assessments of performance, competence and knowledge, and appropriate tests for each type of knowledge, used at appropriate time intervals. In this article, the hierarchical relationship between performance, competence, and knowledge is described. The discussion of cognitive and psychomotor knowledge includes the important distinction between declarative and procedural knowledge. Measurement of performance is described and can include a variety of indirect and direct measurement techniques. Each measurement option has its own profile of strengths and weaknesses in terms of measurement validity, reliability, and costs of development and delivery. It is important to understand the tradeoffs associated with each measurement option, and to make appropriate choices of measurement strategy based on these tradeoffs arrayed against considerations of frequency, criticality, difficulty of learning, logistics, and budget. The article concludes with an example of how these measurement strategies can be combined into a cost-effective assessment plan for a biomedical research facility.

Type I insulin-like growth factor as a liver reserve assessment tool in hepatocellular carcinoma.

Chronic liver diseases (CLDs) encompass a wide range of illnesses, including nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and viral hepatitis. Deterioration of liver capacity, with subsequent progression into cirrhosis and hepatocellular carcinoma (HCC), ultimately leads to a further decrease in the hepatic reserve. The Child-Turcotte-Pugh scoring system is the standard tool for assessing underlying liver reserve capacity in routine practice and in clinical trials of CLD and HCC. In this review, we highlight the clinical significance of insulin-like growth factor-I (IGF-I) and the growth hormone (GH) signaling pathway in HCC. IGF-I could be a marker for liver reserve capacity in CLDs and HCC in clinical practice. This approach could improve the risk assessment and stratifications of patients on the basis of their underlying liver reserve, either before active treatment in routine practice or before they are enrolled in clinical trials.

Urinary catheterization of male rabbits: a new technique and a review of urogenital anatomy.

Rabbits are widely used as an animal model for urologic research studies in which urinary bladder catheterization is required. However, standard manual retrograde urinary catheterization proved to be difficult to perform on anesthetized male rabbits in a research study, with frequent misplacement of the catheter into the vesicular gland. Attempts to reposition the catheter into the bladder after initial entry into the vesicular gland frequently failed and resulted in exclusion of the animal from the study. We assessed the normal anatomy of the lower urinary tract of male rabbits to determine the cause of catheterization misdirection into the vesicular gland and to develop a more reliable technique for urinary bladder catheterization. A modified 'digital (finger) pressure' catheterization technique was developed for successful urinary catheterization of male rabbits. Retrospective statistical analysis of 45 rabbits used for urinary catheterization studies showed improvement in the success rate of catheterization by using the digital pressure technique over the standard method of retrograde urinary catheter insertion. In addition, we here review the relevant gross and histologic anatomy of the urogenital tract of male rabbits.

Training veterinary care technicians and husbandry staff improves animal care.

Our animal care facility has always relied on an animal health team consisting of veterinarians, veterinary care technicians, and husbandry staff to provide a high level of animal care. As our rodent population increased, it became necessary to modify the roles and responsibilities of these staff members to accommodate the program's expansion. To accomplish that modification, we developed a training program that focused primarily on technicians by using a case-management algorithm. To support our technicians, we provided additional training to animal husbandry staff as they assumed the primary role in the initial assessment of the animals' health. After completing the training, technicians made the transition from simply identifying health issues to actually making decisions for treating and euthanizing rodents. This training program empowered all team members and resulted in a staff that could provide consistent, high-quality veterinary care more efficiently.

Simplified technique for percutaneous transrenal arteriovenous dialysis graft creation in a swine model.

PURPOSE: To investigate the experimental creation of a percutaneous arteriovenous graft via the renal vessels using a simplified technique and to report on its safety, complications, and 1-month follow-up. MATERIALS AND METHODS: Transrenal arteriovenous grafts were created from the renal artery to the renal vein in six swine. Using a combined transfemoral and percutaneous approach, a 7 mm x 150 mm stent graft was deployed to create a renal vein limb, and a 6 mm x 150 mm stent graft was deployed to form the renal artery limb. The external portion of the arterial limb was telescoped into the venous limb to form one continuous loop and was placed into a subcutaneous pocket. The duration and technical success of the procedure were recorded. Shunt patency was assessed by auscultation and angiography, as well as by necropsy and histopathology. RESULTS: Arteriovenous graft creation was technically successful in all six animals with rapid arteriovenous shunting documented with angiography at completion. The mean procedure duration was 84 minutes (range, 70-130 minutes). Auscultation and angiography revealed that four of the six shunts were occluded at 4 weeks. Moderate (50% to 75%) diffuse in-stent angiographic stenosis was present in the two remaining animals. No technical complications occurred. Midterm complications of localized and gradual concentric stenosis due to mural thrombosis occurred in three animals. Graft infection resulting in late abrupt thrombosis occurred in four animals. CONCLUSIONS: Transrenal arteriovenous graft creation using the simplified technique can be performed safely in swine. Delayed complications including graft infection and in-stent stenosis must be addressed prior to use of the technique in clinical settings.

Evaluation of cerebral microemboli during radiofrequency ablation of lung tumors in a canine model with use of impedance-controlled devices.

PURPOSE: To evaluate the incidence of cerebral microemboli during radiofrequency (RF) ablation of lung tumors in a canine model and evaluate the adverse effects of these microemboli on the brain parenchyma with use of magnetic resonance (MR) imaging and histopathologic examination. MATERIALS AND METHODS: Percutaneous RF ablation of 12 lung tumors in 12 dogs was performed under computed tomography (CT) guidance with use of impedance-controlled devices. The common carotid artery was continuously monitored in each animal during RF ablation with duplex Doppler ultrasonography. All animals underwent brain MR imaging shortly after RF ablation. Delayed brain MR imaging (5-8 days after RF ablation) was performed in eight animals. The MR examinations included diffusion-weighted echo-planar imaging. The animals were euthanized 3-11 days after RF ablation. The brain was harvested from each animal and examined by an experienced veterinary pathologist for evidence of ischemia. RESULTS: RF ablation was technically successful in all animals. Microbubbles were detected in the carotid artery in two animals (17%). Acute and delayed MR studies demonstrated no evidence of ischemic brain injury in any of the animals. Gross and histopathologic assessment of brain tissue also demonstrated no ischemic changes. CONCLUSIONS: During RF ablation of lung tumors, microbubbles are detected in the carotid arteries in a small number of cases. These microbubbles are too few and too small to be detected by CT imaging of the brain and do not cause ischemic brain injury.

Transarterial versus transhepatic portal vein embolization to induce selective hepatic hypertrophy: a comparative study in swine.

PURPOSE: Portal vein embolization (PVE) is used to induce liver hypertrophy for surgical candidates with marginal future liver remnant (FLR) volumes. We compared the feasibility, safety, and effectiveness of a transarterial approach for PVE (TA-PVE) with those of a transhepatic approach for PVE (TH-PVE) in a swine model. MATERIALS AND METHODS: Ten experimental pigs (TA-PVE, n = 5; TH-PVE, n = 5) and six controls (TA, n = 3; TH, n = 3) were studied. For TA-PVE, a microcatheter was advanced into arteries supplying the left and left middle hepatic lobes. A 3 to 1 Ethiodol-ethanol mixture was infused into selected arteries to cross the arterioportal peribiliary plexus and remain within the portal veins (PVs). For TH-PVE, PVs in the same lobar distribution were embolized with 355- to 500-micro m polyvinyl alcohol particles and coils. Controls were similarly catheterized for saline infusion. Computed tomography with volumetry was performed before and 7, 14, 21, and 28 days after PVE to assess FLR hypertrophy (absolute FLR volume change and FLR/total liver volume [TLV]). Computed tomographic volumetry, laboratory data, and histopathology were compared between groups. RESULTS: All procedures were technically successful. The increases in mean absolute FLR volume (TA-PVE, 148 +/- 84 cm(3); TH-PVE, 62 +/- 19 cm(3); P = .082), mean FLR hypertrophy (TA-PVE, 93.2%; TH-PVE, 48.4%; P = .178), and mean FLR/TLV (TA-PVE, 31.0%; TH-PVE, 16.2%; P = .130) from day 0 to day 28 between experimental groups were better for TA-PVE. Changes in laboratory data among all groups were minimal. Two complications occurred from TA-PVE (right gastric artery embolization [n = 2] without sequela) and two from TH-PVE (acute segmental right PV thrombosis [n = 1]; death 3 weeks after PVE of unknown cause [n = 1]). CONCLUSIONS: Transarterial portal vein embolization is feasible, safe, and effective for inducing future liver remnant hypertrophy in swine and may represent an improvement over previously reported transhepatic portal vein embolization methods.

An in vivo orthotopic canine model to evaluate distribution of intraprostatic injectate: implications for gene therapy and drug delivery for prostate cancer.

OBJECTIVES: Complete and reliable infiltration of the entire prostate is important to improve the efficacy of intraprostatic gene therapy, as well as drug delivery. We sought to evaluate the optimal injection scheme and feasibility of magnetic resonance imaging (MRI) using a surrogate solution to assess the distribution of injectate in an orthotopic canine model. METHODS: Twelve dogs were anesthetized, and laparotomy was performed. Four dogs in each group were injected with a 1:10 dilution of 1% methylene blue and gadolinium-diethylenetriamine pentaacetic acid using 3, 10, and 20-core injection schemes. The dogs subsequently underwent MRI and then were killed. The prostates were harvested, sectioned, and photographed for methylene blue distribution. The cross-sectional area of the stained pathology and MRI slides was calculated, and correlation studies were performed. Statistical analysis was performed using the paired t test and Pearson's correlation test. RESULTS: The gadolinium distribution reflected the methylene blue distribution, with a Pearson's correlation coefficient of 0.97 (P <0.001). The fractional volume distribution in the 3, 10, and 20-core injection schemes was 10.2%, 28.5%, and 30.1%, respectively. The volume of distribution was significantly greater for the 10-core (P = 0.0024) and 20-core (P = 0.0025) injection schemes than for the 3-core scheme. However, no significant difference was found between the 10 and 20-core schemes (P = 0.52). CONCLUSIONS: MRI using gadolinium-diethylenetriamine pentaacetic acid is an excellent modality to evaluate the effective volume distribution of injectate in an in vivo orthotopic prostate model. The 10-core injection scheme seemed to be as good as the 20-core scheme in achieving adequate distribution of injectate within the prostate.

Magnetic resonance imaging assessment of a convective therapy delivery paradigm in a canine prostate model.

PURPOSE: To quantitatively investigate the feasibility of MRI as a tool for assessing the spatial distribution of a convectively delivered agent using a canine prostate model. MATERIALS AND METHODS: Canine prostates (ex vivo, n = 3; in vivo, n = 12) were injected under several injection paradigms with a solution of gadolinium-DTPA for MR contrast and methylene blue as a grossly visible surrogate drug marker. Ex vivo and in vivo distributions were assessed at 1.5T and quantitatively compared. RESULTS: Measured distributions using MRI and methylene blue pathology photographs were analyzed using a Bland-Altman method. The fractional percentage volume covered (V frac) compared the measurements grossly: Pearson's correlation coefficients were R = 0.99 for ex vivo and R = 0.77 for in vivo (P < 0.05). The fractional percentage of area covered (A frac) demonstrated the high degree of spatial correlation between individual slices: R = 0.93 for ex vivo and R = 0.98 for in vivo (P < 0.05). There was no statistically observable bias in scale or offset between the measurements. CONCLUSION: Measured distributions using MRI and pathology were highly correlated and unbiased, indicating the potential of MRI as a tool for quantitative assessment of interstitial delivery of injected therapies in vivo.

Impaired angiogenesis in aminopeptidase N-null mice.

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.