Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model.

BACKGROUND: The process of angiogenesis is a key element for tumor growth and proliferation and therefore one of the determining factors for aggressiveness and malignancy. A better understanding of the underlying processes of tumor induced angiogenesis is crucial for superior cancer treatment. Furthermore, the PeriCam perfusion speckle imager (PSI) system high resolution (HR) model by PERIMED presents a noninvasive method for semi-quantitative measurement of blood perfusion, based on laser speckle contrast analysis (LASCA). Aim of the present study was to utilize the chick chorioallantoic membrane (CAM) model as an in-ovo-tumor-model which enables rapid neovascularization of tumors while allowing real-time observation of the microcirculation via LASCA. METHODS: Fertilized chicken eggs were grafted with embryonal/alveolar rhabdomyosarcoma cells or primary sarcoma tumors. The blood perfusion was measured before and after tumor growth using LASCA. The procedure is accelerated and simplified through the integrated PIMSoft software which provides real-time graphs and color-coded images during the measurement. RESULTS: Sarcoma cells and primary sarcoma tumors exhibited satisfactory growth processes on the CAM. LASCA visualized microcirculation accurately and enabled an extensive investigation of the angiogenic potential of sarcoma cells on the CAM. We were able to show that sarcoma cells and primary sarcoma tumors induced larger quantities of neovasculature on the CAM than the controls. CONCLUSIONS: The utilization of LASCA for the investigation of tumor angiogenesis within the CAM model appears to be a highly beneficial, cost-efficient and easily practicable procedure. The proposed model can be used as a drug-screening model for individualized cancer therapy, especially with regards to anti-angiogenic agents.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

Background: The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Objectives: Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Methods: Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. Results: IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Conclusions: Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

New, Innovative, Three-Dimensional In Vivo Model for High-Level Microsurgical and Supermicrosurgical Training: A Replacement for Animal Models.

SUMMARY: Microsurgery and supermicrosurgery are surgical subdomains necessary for a large variety of surgical disciplines. So far, there is no training model for lymphatic surgery or perforator flap surgery, and the most commonly used microsurgical training models are living animals. However, the ethical principles of replacement, refinement, and reduction (the three Rs) of living animals for training purposes were implemented, highlighting the necessity of an animal-sparing microsurgical training model. Formed during embryogenesis, the chick chorioallantoic membrane resembles a highly vascularized, noninnervated membrane within fertilized chicken eggs. The aim of this study was to utilize the chorioallantoic membrane model as an innovative and versatile training model for supermicrosurgery and microsurgery that can reduce the number of animals used for these purposes. The variety of different sized vessels for the implementation of an anastomosis proved the chorioallantoic membrane model as a well-functioning supermicrosurgical and microsurgical training model. The circulatory system is resilient enough to withstand the mechanical stress applied to the tissue, and the patency of the implemented anastomosis can be tested for the verification of the procedures. In summary, the integration of the chorioallantoic membrane model into a surgical training program can benefit its quality by representing a realistic anatomical and physiological model with a high variety of vascular structures. Moreover, the chorioallantoic membrane model satisfies the principles of replacement, refinement, and reduction as an animal-sparing model, indicating the potential of this model as an innovative microsurgical training model for the improvement of surgical skills.

An Innovative Simulation Model for Microvascular Training.

SUMMARY: Preclinical/clinical microsurgical training is essential for clinical practice. Therefore, various training models have been established, such as synthetic and cadaveric models. The most common limitation of these models is the lack of circulation, which limits the simulation of real intraoperative circumstances. Thus, the authors aimed to create a novel model that provides blood circulation with an extracorporeal perfusion device that they attached to rat cadavers for the reestablishment of a circulatory system. Patent blue and heparin were added to the perfusion fluid to visualize circulation and to dissolve thrombosis, and indocyanine green fluorescent imaging was applied to show the perfusion of the entire body. The femoral and brachial vessels were dissected, and an end-to-end anastomosis was performed on the femoral artery. The patency of the operated vessel was visualized with indocyanine green fluorescent imaging. Indocyanine green fluorescent imaging showed appropriate vessel patency and extremity perfusion through the anastomosis. The use of this novel rat model enables a solution for ethical problems encountered when using rats for surgical training courses. By practicing on these animal-sparing models with intact circulation, microsurgical skills can be improved. Future studies on further microsurgical techniques and vascular perfusion of organs or tumors may benefit from our model.

Extended analysis of intratumoral heterogeneity of primary osteosarcoma tissue using 3D-in-vivo-tumor-model.

BACKGROUND: Osteosarcomas are a rare, heterogeneous and malignant group of bone tumors that have a high potential for metastasis and aggressive growth patterns. Treatment of metastasized osteosarcoma is often insufficient and research is compromised by problems encountered when culturing cells or analyzing genetic alterations due to the high level of intratumoral and intertumoral heterogeneity. The chick chorioallantoic membrane (CAM) model, a 3D-in-vivo-tumor-model, could potentially facilitate the investigation of osteosarcoma heterogeneity at an individual and highly specified level. OBJECTIVE: Objective was to establish the grafting and transplantation of different primary osteosarcoma tissue parts onto several consecutive CAMs for tumor profiling and investigation of osteosarcoma heterogeneity. METHODS: Various parts of primary osteosarcoma tissue were grafted onto CAMs and were transplanted onto another CAM for five to seven consecutive times, enabling further experimental analyzes. RESULTS: Primary osteosarcoma tissue parts exhibited satisfactory growth patterns and displayed angiogenic development on the CAM. It was possible to graft and transplant different tumor parts several times while the tissue viability was still high and tumor profiling was performed. CONCLUSIONS: Primary osteosarcoma tissue grew on several different CAMs for an extended time period and neovascularization of serial transplanted tumor parts was observed, improving the versatility of the 3D-in-vivo-tumor-model.