Radiofrequency Combined with Intratumoral Immunotherapy: Preclinical Results and Safety in Metastatic Colorectal Carcinoma.

Radiofrequency ablation (RFA) of cancer induces an anti-tumor immunity, which is insufficient to prevent recurrences. In mice, RFA-intratumoral immunotherapy by granulocyte-macrophage colony-stimulating factor (GM-CSF) and Bacillus Calmette-Guerin resulted in complete metastases regression. Infectious risk in human needs replacement of live vaccines. Intratumoral purified protein derivatives (PPD) have never been tested in digestive cancers, and the safety of intratumoral immunotherapy after RFA has not yet been validated in human models. We investigated the therapeutic efficacy of combined radiofrequency ablation (RFA) and intratumoral immunotherapy (ITI) using an immune-muco-adherent thermogel (IMT) in a mouse model of metastatic colorectal cancer (CRC) and the safety of this approach in a pig model. Intratumoral stability of the immunogel was assessed using magnetic resonance imaging (MRI) and bioluminescent imaging. Seventy-four CT26 tumor-bearing female BALB/c mice were treated with RFA either alone or in combination with intratumoral IMT. Regression of distant metastasis and survival were monitored for 60 days. Six pigs that received liver radiofrequency and intralesional IMT injections were followed for 15 days. Experimental gel embolisms were treated using an intravascular approach. Pertinent rheology of IMT was confirmed in tumors, by the signal stability during 3 days in MRI and 7 days in bioluminescence imaging. In mice, the abscopal effect of RFA-intratumoral immunotherapy resulted in regression of distant lesions completed at day 16 vs. a volume of 350 ± 99.3 mm3 in the RFA group at day 25 and a 10-fold survival rate at 60 days. In pigs, injection of immunogel in the liver RFA area was safe after volume adjustment without clinical, hematological, and liver biology disorder. Flow cytometry showed an early increase in CD3 TCRγδ+T cells at D7 (p < 0.05) and a late decrease in CD29+-CD8 T cells at D15 (p < 0.05), reflecting the inflammation status changes. Systemic GM-CSF release was not detectable. Experimental caval and pulmonary thermogel embolisms were treated by percutaneous catheterism and cold serum infusion. RFA-intratumoral immunotherapy as efficient and safe mini-invasive interventional oncology is able to improve ablative treatment of colorectal liver metastases.

Nonliving versus Living Animal Models for Microvascular Surgery Training: A Randomized Comparative Study.

BACKGROUND: Ethical and financial considerations have encouraged the use of nonliving models for simulation-based training in microsurgery, such as commercially available chicken thighs. The purpose of this study was to compare the nonliving chicken thigh model to the one currently considered as the standard-namely, the living rat model-in the setting of an initiation microsurgery course. METHODS: Applicants to the 3-day basic microsurgery course of the Paris School of Surgery were assigned randomly to either one group that received the regular training of the school (RT group), including four hands-on sessions using only living rat models, or one group that received a modified curriculum in which a nonliving chicken thigh model was used for the first hands-on session (CT group). During the following session, all trainees were evaluated on living rat models, using a global rating scale and two task-specific scales (knot-tying and anastomosis); rates of anastomosis patency, animal survival, and technique completion were recorded. RESULTS: Ninety-three residents were enrolled. Global rating scale, knot-tying, and anastomosis task-specific scale scores were significantly higher in the CT group ( n = 51) than in the RT group, with mean differences of 2.6 points ( P = 0.0001), 1.3 points ( P < 0.0001), and 1.4 points ( P < 0.0001), respectively. Patency and survival rates were significantly higher in the CT group than in the RT group, with mean differences of 22% ( P = 0.0020) and 27% ( P < 0.0001), respectively; completion rates were not statistically different. CONCLUSION: Subject to the use of validated models, such as the chicken thigh, nonliving animal models are a suitable alternative to the living rat model in microsurgery initial training. CLINICAL RELEVANCE STATEMENT: The use of validated non-living models, such as the chicken thigh, is a suitable alternative to the living rat model in microsurgery initial training.

Training in microvascular anastomosis - A randomized comparative study between chicken thigh specimen and live rat.

Training in microsurgical techniques on live rats is the gold standard, but raises ethical issues related to animal welfare and cost. The aim of this study was to compare acquisition of microsurgical techniques with primary training on chicken thigh specimens. Seventy six students were randomly assigned: 23 to exclusive rat training and 53 to primary chicken-leg training. Both groups were then evaluated on aortic suture and jugular aortic bypass surgery in live rats. The primary criterion for successful anastomosis was the patency test. The survival of the rat, the number of severe vascular wounds and the quality of the dissection were also assessed. Aortic anastomoses were of significantly better quality in the chicken group (p = 0.041). There was no significant difference in the number of serious injuries, rat mortality, or quality of dissection (p > 0.05). For jugular aortic bypass surgery, dissection quality (p = 0.02) and patency test (p = 0.05) were better in the chicken-leg group. There was no significant difference in number of severe wounds or rat mortality (p > 0.05). Students who started their microsurgical training on a chicken leg did not perform worse than those with exclusive live rat training. Initial training on chicken thigh specimens seems to be a reliable alternative to training on live models. LEVEL OF EVIDENCE: Level II - Randomized controlled trial.

Relevance of a Simulation Model to Microvascular Surgery for Military Surgical Residents.

Introduction: Microsurgical training is an asset for deployed military orthopaedic surgeons who frequently treat hand or nerve injuries in the field. The objective of this study was to evaluate a microvascular surgery simulation model intended to prepare residents prior to their enrolment in conventional microsurgery degree training. Methods: An experimental study was conducted to evaluate technical progress and satisfaction of military surgical residents using a model based on Japanese noodles with four tests of increasing difficulty. Objective endpoints included instruments handling, distribution, and quality of stitches, as well as anastomoses duration. Responses to the Structured Assessment of Microsurgery Skill self-assessment questionnaire were also analyzed. Results: Nine residents from different specialties participated in the study. Their anastomoses quality and average satisfaction significantly increased between the first and the last session (p < 0.05). Conversely, the average operating time decreased significantly over the sessions (p < 0.001). Conclusion: This simulation model seems to constitute a satisfactory initiation to microsurgery and could limit the use of animal models. It could also be included in the continuing education of military surgeons who have an occasional microsurgical practice during deployments.

International microsurgery simulation society (IMSS) consensus statement on the minimum standards for a basic microsurgery course, requirements for a microsurgical anastomosis global rating scale and minimum thresholds for training.

INTRODUCTION: Microsurgery is a surgical technique that uses optical magnification as well as specific instruments to address necessary reconstructive procedures in different medical specialties. The apprenticeship of this technique requires overcoming a steep learning curve. There is a need for standardization of the training criteria in microsurgery. The International Microsurgery Simulation Society (IMSS) was born in 2011, since then its main objective has been to connect the main international specialists and educators of this sub-specialty to share and discuss the ethical and scientific basis of preclinical microsurgery teaching. METHODS: In order to achieve a consensus on the minimum standards for the organization of basic microsurgery training courses, the requirements for a microsurgical anastomosis global rating scale and minimum thresholds for training, a total of nineteen independent global experts participated in a formal consultative consensus development program. The agreement criteria for each statement was established when consensus of 65-100% was reached. RESULTS: There have been established six recommendations concerning minimum standards for a basic microsurgery course, one recommendation in relation to minimum thresholds for training and four recommendations regarding the global rating scale as gold standard for a microsurgical anastomosis assessment. The eleven defined recommendations reached the agreement threshold of 65-100%. CONCLUSIONS: The development of this consensus sets the minimum recommended requirements for conducting basic microsurgery training courses, as well as suggestions for objective assessment of the learning curve and skills of trainees.

Interfacial fluid transport is a key to hydrogel bioadhesion.

Attaching hydrogels to soft internal tissues is a key to the development of a number of biomedical devices. Nevertheless, the wet nature of hydrogels and tissues renders this adhesion most difficult to achieve and control. Here, we show that the transport of fluids across hydrogel-tissue interfaces plays a central role in adhesion. Using ex vivo peeling experiments on porcine liver, we characterized the adhesion between model hydrogel membranes and the liver capsule and parenchyma. By varying the contact time, the tissue hydration, and the swelling ratio of the hydrogel membrane, a transition between two peeling regimes is found: a lubricated regime where a liquid layer wets the interface, yielding low adhesion energies (0.1 J/m2 to 1 J/m2), and an adhesive regime with a solid binding between hydrogel and tissues and higher adhesion energies (1 J/m2 to 10 J/m2). We show that this transition corresponds to a draining of the interface inducing a local dehydration of the tissues, which become intrinsically adhesive. A simple model taking into account the microanatomy of tissues captures the transition for both the liver capsule and parenchyma. In vivo experiments demonstrate that this effect still holds on actively hydrated tissues like the liver capsule and show that adhesion can be strongly enhanced when using superabsorbent hydrogel meshes. These results shed light on the design of predictive bioadhesion tests as well as on the development of improved bioadhesive strategies exploiting interfacial fluid transport.

A New Synthetic Model for Microvascular Anastomosis Training? A Randomized Comparative Study Between Silicone and Polyvinyl Alcohol Gelatin Tubes.

INTRODUCTION: Assessment of a resident's microsurgical competency with the rodent model remains the current gold standard. However, cost and ethical issues related to animal welfare may limit training opportunities. Therefore, synthetic alternatives such as silicone tubes have been developed to provide easy access to training, shorten the learning curve, and have been incorporated into microsurgical courses as a low-fidelity model for basic skills acquisition. This study compares the use of polyvinyl alcohol (PVA) gelatin tubes with silicone for resident microsurgical training. MATERIAL AND METHODS: Residents were randomized into silicone (S) or PVA (P) groups and underwent the same training. Following basic instruction, microsurgical anastomoses were performed with the rat's aorta or carotid artery or both. Performance was assessed using the Objective Structured Assessment of Technical Skills (OSATS) score and 5 different items to assess the quality of the anastomosis. Posttest questionnaires were also conducted for qualitative assessment of both students' and trainers' experience with silicone and PVA in comparison with rat vessels. RESULTS: OSATS score in Group P was higher than Group S (18.2 ± 2.6 vs 16.6 ± 2.5, p = 0.015). Results of anastomoses were similarly better in Group P based on OSATS score (19.3 ± 1.2 vs 17.7 ± 0.7, p = 0.027). Subjectively, both students and trainers found that PVA tubes resembled the rat aorta more closely than silicone. The number of rats used was also significantly lower in Group P than Group S (65 vs 75 total, p = 0.023). CONCLUSION: PVA gelatin tubes may be a viable alternative to silicone for microsurgical training because this synthetic model mirrors better rat vessels and can improve training performance based on objective assessment while using less animals overall.

Determination of ketamine and its main metabolites by liquid chromatography coupled to tandem mass spectrometry in pig plasma: Comparison of extraction methods.

A rapid, sensitive and specific liquid chromatography coupled to tandem mass spectrometry method was developed for the simultaneous quantification pig plasma of ketamine and its two principal metabolites, norketamine and dehydronorketamine. Three extraction procoles were assessed including acetonitrile precipitation, Oase™ microplate extraction, and liquid-liquid extraction. Oase™ microplate extraction induced no significant matrix effect, important signal/noise ratio and good recoveries, ranging from 82 to 87% for the considered compounds. Using this extraction procedure, the assay was linear in the dynamic range 10-3000ng/mL (R2>0.99) regardless of the analytes. Intra- and inter-day accuracies were less than 12% for all compounds and intra- and inter-day precisions expressed as RSD were within <9.9%. Samples were stable in different storage conditions. High ketamine, norketamine and dehydronorketamine concentrations up to 15,000ng/mL can be determined with good precision using appropriate sample dilution. The assay was successfully applied to pig plasma samples to determine the pharmacokinetics of ketamine and the consecutive metabolites after buccal administration of a 4mg/kg ketamine base solutions.

Dual-acting biofunctionalised scaffolds for applications in regenerative medicine.

Off the shelf scaffolds for replacing ultra-small diameter vascular grafts are valuable for reconstruction of diseased or damaged vessels. The limitations for such grafts include optimal handling with ready availability of varied lengths of grafts, graft patency with the ability to replace the function of active cellular mechanisms and adequate mechanical properties to maintain physicochemical function. We used a well-established, solvent casting method for potential tissue replacement scaffold fabrication with incorporated bioactive molecules, which we have previously explored to confer haemocompatibility. These grafts were tested in-vivo within the abdominal aorta of 10 Wistar rats and the patency was clinically and echographically evaluated. Haemocompatibility and endothelialisation were assessed on explants. Biofunctionalised scaffolds were also grafted subcutaneously and intraperitoneally to evaluate integration, inflammation and angiogenesis reactions. The potential wider applications of this dual acting scaffold were evaluated for its interactions with human dermal fibroblasts as well as bronchial epithelial cells. Physicochemical property evaluation of the functionalised grafts has clarified the mechanical strength and permeability. This study confirmed the microsurgical suturability of tubular grafts and graft patency of functionalized scaffolds. The study demonstrated the potential of a dual acting biofunctionalised scaffold's use for a wide range of tissue engineering applications where micro-porous, yet impermeable scaffolds are needed.

Hepatic proliferation and angiogenesis markers are increased after portal deprivation in rats: a study of molecular, histological and radiological changes.

BACKGROUND & AIMS: To determine the pathogenesis of liver nodules, and lesions similar to obliterative portal venopathy, observed after portosystemic shunts or portal vein thrombosis in humans. METHODS: We conducted an experimental study comparing portacaval shunt (PCS), total portal vein ligation (PVL), and sham (S) operated rats. Each group were either sacrificed at 6 weeks (early) or 6 months (late). Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa), proliferation (Il6st, Mki67, Hgf, Hnf4a), angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf), oxidative stress (Nos2, and 3, Hif1a), and fibrosis (Tgfb). PCS and PVL were compared to the S group. RESULTS: Periportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups. CONCLUSIONS: Portal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.

The rat model in microsurgery education: classical exercises and new horizons.

Microsurgery is a precise surgical skill that requires an extensive training period and the supervision of expert instructors. The classical training schemes in microsurgery have started with multiday experimental courses on the rat model. These courses have offered a low threat supervised high fidelity laboratory setting in which students can steadily and rapidly progress. This simulated environment allows students to make and recognise mistakes in microsurgery techniques and thus shifts any related risks of the early training period from the operating room to the lab. To achieve a high level of skill acquisition before beginning clinical practice, students are trained on a comprehensive set of exercises the rat model can uniquely provide, with progressive complexity as competency improves. This paper presents the utility of the classical rat model in three of the earliest microsurgery training centres and the new prospects that this versatile and expansive training model offers.

A new murine model of endovascular aortic aneurysm repair.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 1990(1) in a rat, then described in mice(2). Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate.

BACKGROUND: Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29. METHODS: Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. RESULTS: DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. CONCLUSIONS: Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy.

Chapter 4: Methods and protocols in peripheral nerve regeneration experimental research: part I-experimental models.

This paper addresses several basic issues that are important for the experimental model design to investigate peripheral nerve regeneration. First, the importance of carrying out adequate preliminary in vitro investigation is emphasized in light of the ethical issues and with particular emphasis on the concept of the Three Rs (Replacement, Reduction, and Refinement) for limiting in vivo animal studies. Second, the various options for the selection of the animal species for nerve regeneration research are reviewed. Third, the two main experimental paradigms of nerve lesion (axonotmesis vs. neurotmesis followed by microsurgical reconstruction) are critically outlined and compared. Fourth, the various nerve models that have most commonly been employed are overviewed focusing in particular on forearm mixed nerves and on behavioural tests for assessing their function: the ulnar test and the grasping test which is useful for assessing both median and radial nerves in the rat. Finally, the importance of considering the influence of various factors and diseases which could interfere with the nerve regeneration process is emphasized in the perspective of a wider adoption of experimental models which more closely mimic the environmental and clinical conditions found in patients.

[25 years review of vascular microsurgery evaluation--perspectives]. / Les transplants libres: 25 ans de microchirurgie vasculaire--bilan--perspectives.

Free-tissue transfer has become an accepted method for reconstructing complex surgical defects. We review 25 years' experience of this approach. In France, microsurgery was first performed in 1974. I myself constructed 839 consecutive free flaps in 821 patients during a 25-year period. Here we distinguish between two different indications, namely malignant and benign lesions. In oncology, the patient recovers good quality of life even if the prognosis is poor. In contrast, the transplant offers permanent cure for patients with benign lesions. Microvascular anastomoses were constructed with separated stitches (90% of anastomoses); end-to-end anastomoses were preferred (85%). Manual suture with thread is the best technique. Post-operative flap monitoring included clinical observation, Doppler sonography, thermic probing, and endoscopy. The overall success rate of free flap reconstruction was 95.5%. In cancer patients, surviving flaps resulted in wound healing and did not delay post-operative irradiation or chemotherapy. The incidence of major complications (death 0.36%, necrosis 4.5%) and minor post operative complications (27.1%) was acceptable. The mean hospital stay was 20 days. Careful selection of the transplants yielded good reliability. Six donor sites were sufficient to reconstruct the majority of defects. Teaching of microsurgery is one of our main preoccupations. This is an indispensable technique in all fields of reconstruction. Rigorous training is necessary and much time must be spent before reaching a high level of reliability.