URB447 Is Neuroprotective in Both Male and Female Rats after Neonatal Hypoxia-Ischemia and Enhances Neurogenesis in Females.

The need for new and effective treatments for neonates suffering from hypoxia-ischemia is urgent, as the only implemented therapy in clinics is therapeutic hypothermia, only effective in 50% of cases. Cannabinoids may modulate neuronal development and brain plasticity, but further investigation is needed to better describe their implication as a neurorestorative therapy after neonatal HI. The cannabinoid URB447, a CB1 antagonist/CB2 agonist, has previously been shown to reduce brain injury after HI, but it is not clear whether sex may affect its neuroprotective and/or neurorestorative effect. Here, URB447 strongly reduced brain infarct, improved neuropathological score, and augmented proliferative capacity and neurogenic response in the damaged hemisphere. When analyzing these effects by sex, URB447 ameliorated brain damage in both males and females, and enhanced cell proliferation and the number of neuroblasts only in females, thus suggesting a neuroprotective effect in males and a double neuroprotective/neurorestorative effect in females.

Prophylactic Treatment of Intestinal Ischemia-Reperfusion Injury Reduces Mucosal Damage and Improves Intestinal Absorption.

Purpose: Intestinal ischemia-reperfusion injury (i-IRI) involves a blood flow interruption in an intestinal segment followed by blood flow restoration. When blood flow is restored, oxidative and inflammatory molecules are distributed throughout the bloodstream, triggering both local and systemic damage. Our goal was to evaluate the potential of three antioxidant and/or anti-inflammatory compounds (curcumin, dexmedetomidine and α-tocopherol) to prevent or reverse local and systemic damage induced by i-IRI. Methods: i-IRI was induced by placing a microvascular clip in the superior mesenteric artery of female WAG/RijHsd rats; the clip was removed after 1h and reperfusion was allowed for 4h. Curcumin (200 mg/kg, orally), α-tocopherol (20 mg/kg, i.p.), and dexmedetomidine (5 or 20 µg/kg, s.c.; DEX5 and DEX20, respectively) were administered. Blood and terminal ileum specimens were collected for biochemical and histological determination. Furthermore, D-xylose absorption test was performed to evaluate intestinal absorption; after completing the 1-hour ischemia and 4-hour reperfusion period, 1 mL of aqueous D-xylose solution (0.615 mg/mL) was administered orally, and one hour later, plasma D-xylose levels were quantified. Results: The histological injury degree (HID) measured by the Chiu scale was significantly reduced when the treatments were applied (non-treated rats, 2.6 ± 0.75; curcumin, 1.54 ± 0.8; DEX5, 1.47 ± 0.7; DEX20 1.14 ± 0.5; and α-tocopherol, 1.01 ± 0.6); intestinal absorptive capacity also improved in all cases healthy rats (2.06 ± 0.07 µg/mL; non-treated, 1.18 ± 0.07 µg/mL; curcumin 1.76 ± 0.3 µg/mL; DEX5, 2.29 ± 0.2 µg/mL; DEX20, 2.25 ± 0.26 µg/mL; and α-tocopherol 1.66 ± 0.21 µg/mL). However, it failed to reduce liver enzyme levels. Finally, only dexmedetomidine significantly reduced urea and creatinine levels compared to non-treated animals. Conclusion: All drugs were effective in reducing HID, although α-tocopherol was effective to a greater extent. Only dexmedetomidine reverted intestinal absorption to normal values of healthy animals.

The Prevention of Ischemia-Reperfusion Injury in Elderly Rats after Lower Limb Tourniquet Use.

Background: Lower limb ischemia-reperfusion injury (IRI-LL) is a common major complication of orthopedic surgery, especially in elderly patients. It has previously been demonstrated that folinic acid (FA) reduced IRI-LL damage in 3−4-month-old rats. This current work analyses the effect of FA in the prevention of IRI-LL in elderly animals. Methods: Forty-two 18-month-old male WAG/RijHsd rats were subjected to 3 h of ischemia. Eighteen animals received FA (2.5 mg/kg, ip) 20 min before the end of the ischemia period, while the other half received the same volume of saline solution. The animals were sacrificed after 3 h, 24 h, and 14 days of reperfusion for biochemical (tissue damage markers and electrolytes), histopathological studies of the gastrocnemius muscle and the daily assessment of the limb function by the Rota Rod test, respectively. Results: The administration of FA prior to the end of the ischemia period reduced the increase in LDH and CK observed in non-treated animals by 30−40% (p < 0.0001). When the histological sections were analyzed, FA was found to have reduced the number of damaged muscle fibers per field by 20% (60 ± 17.1 vs. 80.7 ± 16.4, p < 0.0001). The functional test revealed that FA also led to an improvement in the muscle function, assessed by the length of time that the animals kept running on the rod, compared to untreated animals. Conclusions: The administration of FA, prior to the end of the ischemic period, decreases the damage induced by IRI-LL, also achieving a faster recovery of mobility.

Conditioned Medium from H2O2-Preconditioned Human Adipose-Derived Stem Cells Ameliorates UVB-Induced Damage to Human Dermal Fibroblasts.

Human skin exposure to ultraviolet B (UVB) radiation can result in acute photodamage through oxidative modifications of cellular components and biomolecules involved in the metabolism of dermal cells. Recently, the therapeutic potential of human adipose-derived stem cells (hASCs) has been investigated as a novel strategy for photoprotection due to their pro-angiogenic properties, protective activity against oxidative stress and paracrine effect on dermal cells. To enhance these therapeutic properties, hASCs can be preconditioned by exposing them to sublethal cellular stressors. In this study, we first analyzed response capacity against UVB-induced oxidative stress in H2O2-preconditioned hASCs (called HC016 cells); and second, we evaluated the photoprotective effect of HC016-conditioned medium (CM) in an in vitro UVB irradiation model in cultured human foreskin fibroblasts (hFFs). The results demonstrated that HC016 cells have a greater capacity to respond efficiently to UVB-induced oxidative stress, evidenced by higher Nrf2 antioxidant system activity and enhanced viability and migration capacity. Further, HC016-CM treatment increased viability, migratory capacity and collagen type I synthesis in hFFs exposed to UVB radiation, as well as reducing their cytotoxicity, apoptosis, senescence and IL-6 secretion. Collectively, these findings support the view that HC016 cells could protect against UVB-induced photodamage via paracrine mechanisms.

Proposal of New Safety Limits for In Vivo Experiments of Magnetic Hyperthermia Antitumor Therapy.

BACKGROUND: Lately, major advances in crucial aspects of magnetic hyperthermia (MH) therapy have been made (nanoparticle synthesis, biosafety, etc.). However, there is one key point still lacking improvement: the magnetic field-frequency product (H × f = 4.85 × 108 Am-1s-1) proposed by Atkinson-Brezovich as a limit for MH therapies. Herein, we analyze both local and systemic physiological effects of overpassing this limit. METHODS: Different combinations of field frequency and intensity exceeding the Atkinson-Brezovich limit (591-920 kHz, and 10.3-18 kA/m) have been applied for 21 min to WAG/RijHsd male rats, randomly distributed to groups of 12 animals; half of them were sacrificed after 12 h, and the others 10 days later. Biochemical serum analyses were performed to assess the general, hepatic, renal and/or pancreatic function. RESULTS: MH raised liver temperature to 42.8 ± 0.4 °C. Although in five of the groups the exposure was relatively well tolerated, in the two of highest frequency (928 kHz) and intensity (18 kA/m), more than 50% of the animals died. A striking elevation in liver and systemic markers was observed after 12 h in the surviving animals, independently of the frequency and intensity used. Ten days later, liver markers were almost recovered in all of the animals. However, in those groups exposed to 591 kHz and 16 kA/m, and 700 kHz and 13.7 kA/m systemic markers remained altered. CONCLUSIONS: Exceeding the Atkinson-Brezovich limit up to 9.59 × 109 Am-1s-1 seems to be safe, though further research is needed to understand the impact of intensity and/or frequency on physiological conditions following MH.

Folinic Acid Potentiates the Liver Regeneration Process after Selective Portal Vein Ligation in Rats.

Liver resection remains the gold standard for hepatic metastases. The future liver remnant (FLR) and its functional status are two key points to consider before performing major liver resections, since patients with less than 25% FLR or a Child-Pugh B or C grade are not eligible for this procedure. Folinic acid (FA) is an essential agent in cell replication processes. Herein, we analyze the effect of FA as an enhancer of liver regeneration after selective portal vein ligation (PVL). Sixty-four male WAG/RijHsd rats were randomly distributed into eight groups: a control group and seven subjected to 50% PVL, by ligation of left portal branch. The treated animals received FA (2.5 m/kg), while the rest were given saline. After 36 h, 3 days or 7 days, liver tissue and blood samples were obtained. FA slightly but significantly increased FLR percentage (FLR%) on the 7th day (91.88 ± 0.61%) compared to control or saline-treated groups (86.72 ± 2.5 vs. 87 ± 3.33%; p < 0.01). The hepatocyte nuclear area was also increased both at 36 h and 7days with FA (61.55 ± 16.09 µm2, and 49.91 ± 15.38 µm2; p < 0.001). Finally, FA also improved liver function. In conclusion, FA has boosted liver regeneration assessed by FLR%, nuclear area size and restoration of liver function after PVL.

Effects of Folinic Acid Administration on Lower Limb Ischemia/Reperfusion Injury in Rats.

Surgery under ischemic conditions, lasting up to 3 h, is routinely performed in orthopedic surgery, causing undesirable injury due to ischemia-reperfusion syndrome, with short and medium-term functional repercussions. To date, there is no established prophylactic treatment. In this work we evaluated folinic acid (FA) in a rodent model of lower limb ischemia-reperfusion (IRI-LL). 36 male WAG rats underwent 3 h of lower limb ischemia. In the saline group, rats received intraperitoneal administration of saline (used as vehicle for treatment). In the experimental group, rats were pretreated with FA (2.5 mg/kg) before the end of ischemia. After ischemia, animals were sacrificed at 3 h, 24 h or 14 days (for biochemical determination (Na+, K+, Cl-, urea, creatinine, CK, LDH, ALP, ALT, and AST), pathological assessment, or functional study using the rotarod test; respectively). Another six animals were used to establish the reference values. The prophylactic administration of FA significantly reduced the elevation of biochemical markers, especially those that most directly indicate muscle damage (CK and LDH). In addition, it also improved direct tissue damage, both in terms of edema, weight, PMN infiltrate and percentage of damaged fibers. Finally, the administration of FA allowed the animals to equal baseline values in the rotarod test; what did not occur in the saline group, where pre-ischemia levels were not recovered. Following 3 h of lower limb ischemia, FA minimizes the increase of CK and LDH, as well as local edema and leukocyte infiltration, allowing a faster recovery of limb functionality. Therefore, it could be considered as a prophylactic treatment when tourniquet is used in clinics.

A reproducible method for biochemical, histological and functional assessment of the effects of ischaemia-reperfusion syndrome in the lower limbs.

Current methodology described to mimic lower limb ischaemia-reperfusion injury (LL-IRI) does not accurately define the procedures and pressures exerted to induce and maintain ischaemia. In this piece of work, we propose a well-defined and detailed rat model that simulates the conditions established in clinical practice guidelines for tourniquet application and allows us to test treatments that aim to prevent/reduce LL-IRI. Eighty-six male WAG/RijHsd rats were subjected to hind limb IRI (LL-IRI), using a mechanical system applying a 1 kg tension to induce and maintain ischemia for 2 or 3 h, and assessed the damage caused by reperfusion at biochemical and muscular levels at different time points. At the biochemical level, both 2 and 3 h of ischemia induced changes (except for electrolyte levels); 3 h of ischemia induced greater changes in specific markers of muscular damage: creatine kinase (CK) and lactate dehydrogenase (LDH). At the histopathological level, 3 h of ischemia and 24 h of reperfusion was associated with an increase in hind limb girth, cross-sectional area, and weight and presence of neutrophils, as well as histological damage in more than 60% of muscle fibres. Our model allows to reliably reproduce the damage associated with the use of a pneumatic tourniquet. CK and LDH, as well as measures of tissue damage, allow to define and characterize the response to LL-IRI-related damage. A period of 3 h of ischemia followed by 3 h of reperfusion caused only local damage but showed greater sensitivity to detect differences in future studies on prophylactic treatments against LL-IRI.

Curcumin Reduces Colorectal Cancer Cell Proliferation and Migration and Slows In Vivo Growth of Liver Metastases in Rats.

BACKGROUND: New therapeutic approaches are an essential need for patients suffering from colorectal cancer liver metastases. Curcumin, a well-known plant-derived polyphenol, has been shown to play a role in the modulation of multiple signaling pathways involved in the development and progression of certain cancer cells in vitro. This study aims to assess the anti-tumor effect of curcumin on CC531 colorectal cancer cells, both in vitro and in vivo. METHODS: On CC531 cultures, the cell viability and cell migration capacity were analyzed (wound healing test) 24, 48, and 72 h after treatment with curcumin (15, 20, 25, or 30 µM). Additionally, in WAG/RijHsd tumor-bearing rats, the total and individual liver lobe tumor volume was quantified in untreated and curcumin-treated animals (200 mg/kg/day, oral). Furthermore, serum enzyme measurements (GOT, GPT, glucose, bilirubin, etc.) were carried out to assess the possible effects on the liver function. RESULTS: In vitro studies showed curcumin's greatest effects 48h after application, when all of the tested doses reduced cell proliferation by more than 30%. At 72 h, the highest doses of curcumin (25 and 30 µM) reduced cell viability to less than 50%. The wound healing test also showed that curcumin inhibits migration capacity. In vivo, curcumin slowed down the tumor volume of liver implants by 5.6-fold (7.98 ± 1.45 vs. 1.41 ± 1.33; p > 0.0001). CONCLUSIONS: Curcumin has shown an anti-tumor effect against liver implants from colorectal cancer, both in vitro and in vivo, in this experimental model.

Evolution of Culture on Patient Safety in the Clinical Setting of a Spanish Mutual Insurance Company: Observational Study between 2009 and 2017 Based on AHRQ Survey.

BACKGROUND: Patient safety (PS) is a key factor in reducing or even eradicating adverse incidents and events. Many health organizations promote strategies to improve PS, while also pointing out the importance of measuring it. For more than eight years, our institution has developed strategies focused on improving PS-culture among our personnel. The goal of this paper is to analyze the PS-culture between the years 2009 and 2017. METHODS: A cross-sectional survey focused on PS, and developed by the American Agency for Healthcare Research and Quality (AHRQ), was conducted in 2009 and in 2017 among all healthcare workers at Mutualia, anonymously and voluntarily. RESULTS: The overall response rate was similar in both 2009 and 2017 (37.2% and 38.5%, respectively). The average rating obtained showed a significant improvement over the period (7.7 vs. 8.1; p < 0.05). Itemizing by question, the main strengths were found in management support, organizational learning and continuous improvement, and, especially, in teamwork. Regarding weaknesses, the two lowest scores were those which refer to the balance between clinical safety and workload and the freedom to question the decisions made by superiors. CONCLUSIONS: The results obtained from the PS-surveys show that the overall PS-culture in our institution has increased, suggesting that the strategies focused on the improvement of PS-culture were well adopted among our personnel. The overall score places Mutualia at similar levels to those reached by the AHRQ and Spanish National Health System.

Biochemical and Metabolomic Changes after Electromagnetic Hyperthermia Exposure to Treat Colorectal Cancer Liver Implants in Rats.

BACKGROUND: Hyperthermia (HT) therapy still remains relatively unknown, in terms of both its biological and therapeutic effects. This work aims to analyze the effects of exposure to HT, such as that required in anti-tumor magnetic hyperthermia therapies, using metabolomic and serum parameters routinely analyzed in clinical practice. METHODS: WAG/RigHsd rats were assigned to the different experimental groups needed to emulate all of the procedures involved in the treatment of liver metastases by HT. Twelve hours or ten days after the electromagnetic HT (606 kHz and 14 kA/m during 21 min), blood samples were retrieved and liver samples were obtained. 1H-nuclear-magnetic-resonance spectroscopy (1H-NMR) was used to search for possible diagnostic biomarkers of HT effects on the rat liver tissue. All of the data obtained from the hydrophilic fraction of the tissues were analyzed and modeled using chemometric tools. RESULTS: Hepatic enzyme levels were significantly increased in animals that underwent hyperthermia after 12 h, but 10 d later they could not be detected anymore. The metabolomic profile (main metabolic differences were found in phosphatidylcholine, taurine, glucose, lactate and pyruvate, among others) also showed that the therapy significantly altered metabolism in the liver within 12 h (with two different patterns); however, those changes reverted to a control-profile pattern after 10 days. CONCLUSIONS: Magnetic hyperthermia could be considered as a safe therapy to treat liver metastases, since it does not induce irreversible physiological changes after application.

Antioxidant-Based Therapy Reduces Early-Stage Intestinal Ischemia-Reperfusion Injury in Rats.

Intestinal ischemia-reperfusion injury (i-IRI) is a rare disorder with a high mortality rate, resulting from the loss of blood flow to an intestinal segment. Most of the damage is triggered by the restoration of flow and the arrival of cytokines and reactive oxygen species (ROS), among others. Inactivation of these molecules before tissue reperfusion could reduce intestinal damage. The aim of this work was to analyze the preventive effect of allopurinol and nitroindazole on intestinal mucosal damage after i-IRI. Wag/RijHsd rats were subjected to i-IRI by clamping the superior mesenteric artery (for 1 or 2 h) followed by a 30 min period of reperfusion. Histopathological intestinal damage (HID) was assessed by microscopic examination of histological sections obtained from injured intestine. HID was increased by almost 20% by doubling the ischemia time (from 1 to 2 h). Nitroindazole reduced HID in both the 1 and 2 h period of ischemia by approximately 30% and 60%, respectively (p < 0.001). Our preliminary results demonstrate that nitroindazole has a preventive/protective effect against tissue damage in the early stages of i-IRI. However, to better understand the molecular mechanisms underlying this phenomenon, further studies are needed.

Histological Assessment of Rat Retinas with Ischemia-Reperfusion Injury.

INTRODUCTION: Retinal ischemia-reperfusion (IR) injury occurs in pathological situations that interrupt the blood flow to the retina, such as is the case during central retinal artery occlusion (CRAO). The animal models described in the literature are based on the pressure produced by the weight of a given quantity of saline elevated to a certain height; however, to establish these parameters it is necessary to perform mathematical calculations that cannot be easily redone in the case of punctual variations of intraocular pressure (IOP). The aim of this study was to present a new system that allows us to reproduce the conditions of retinal IR and thereby properly assess the level of injury in retinal histological samples. METHODS: We developed a retinal IR model in WAG/RijHsd rats based on CRAO through increasing IOP. To develop this model, we produced ischemia for 1 h using a hydrostatic pressure system that maintained a constant high IOP and then allowed reperfusion for 1 h. The injury attributable to IR was assessed by histological examination of retinal samples, determining whether there was histological damage and/or dendritic swelling and counting the outer nuclear layer cells showing cytoplasmic swelling. RESULTS: The increase in IOP to 150 mm Hg produced CRAO, in turn causing observable histological damage and dendritic swelling in all retinas subjected to IR. Counting the number of cells showing cytoplasmic swelling yielded a mean of 102.5 ± 35 cells/field. The contralateral retinas were healthy, showing no significant changes. CONCLUSION: The retinal IR model proposed is simple, reproducible, and allows variable durations of ischemia and reperfusion, and most importantly, it allows easy correction by adjusting the pressure of the sphygmomanometer, of any change in IOP to keep the ischemia stable, without having to recalculate the elevation height of the ischemia induction system. Moreover, the damage caused by IR can be effectively assessed by the type of histopathological assessment performed. For these reasons, it can be considered a reliable method for studying drugs that may prevent retinal IR injury.

Partial hepatectomy enhances the growth of CC531 rat colorectal cancer cells both in vitro and in vivo.

Partial hepatectomy (PHx) is the gold standard for the treatment of colorectal cancer liver metastases. However, after removing a substantial amount of hepatic tissue, growth factors are released to induce liver regeneration, which may promote the proliferation of liver micrometastases or circulating tumour cells still present in the patient. The aim of this study is to assess the effect of PHx on the growth of liver metastases induced by intrasplenic cell inoculation as well as on in vitro proliferation of the same cancer cell line. Liver tumours were induced in 18 WAG/RijHsd male rats, by seeding 250,000 syngeneic colorectal cancer cells (CC531) into the spleen. The left lateral lobe of the liver was mobilized and in half of the animals it was removed to achieve a 40% hepatectomy. Twenty-eight days after tumour induction, the animals were sacrificed and the liver was removed and sliced to assess the relative tumour surface area (RTSA%). CC531 cells were cultured in presence of foetal calf serum, non-hepatectomised (NRS) or hepatectomized rat serum (HRS), and their proliferation rate at 24, 48, and 72 h was measured. RTSA% was significantly higher in animals which had undergone PHx than in the controls (non-hepatectomised) (46.98 ± 8.76% vs. 18.73 ± 5.65%; p < 0.05). Analysing each lobe separately, this difference in favour of hepatectomized animals was relevant and statistically significant in the paramedian and caudate lobes. But in the right lobe the difference was scarce and not significant. In vitro, 2.5% HRS achieved stronger proliferative rates than the control cultures (10% FCS) or their equivalent of NRS. In this experimental model, a parallelism has been shown between the effect of PHx on the growth of colorectal cancer cells in the liver and the effect of the serum on those cells in vitro.

Desarrollo de un plan integral para la adquisición de la competencia de comunicación en el grado en Odontología de la Universidad del País Vasco: un reto institucional / Development of a comprehensive plan for the acquisition of communication skills in the degree in dentistry at the University of the Basque Country: an institutional challenge

INTRODUCCIÓN: La praxis de la odontología requiere el desarrollo adecuado de las habilidades de comunicación, competencia transversal que ha de estar garantizada durante los estudios de grado. Esto supone un reto institucional, por cuanto las facultades han de poner los mecanismos académicos para evaluar el nivel de desarrollo de dichas habilidades. MÉTODOS: Se plantea un plan integral de carácter colaborativo para el desarrollo de las habilidades comunicativas en el grado en Odontología de la Universidad del País Vasco/Euskal Herriko Unibertsitatea, que contempla cuatro fases: a) recogida y socialización de la información sobre el desarrollo de la competencia; b) diseño de desarrollo trasversal y vertical de la competencia; c) implementación y análisis de resultados; y d) recogida de evidencias del proceso y divulgación de los resultados. RESULTADOS: Tras la detección de las fortalezas y debilidades, se definieron 40 habilidades comunicacionales, estructuradas en cinco bloques (comunicación oral con pacientes/familiares, comunicación oral con otros profesionales, comunicación escrita con pacientes/familiares, comunicación escrita con otros profesionales sanitarios y técnicas de comunicación odontólogo-paciente) y tres niveles de desempeño (identificación de la información, desempeño con alta supervisión y desempeño autónomo con moderada supervisión). Para su desarrollo, se diseñaron 19 actividades formativas y de evaluación, actualmente en implementación. CONCLUSIÓN: El plan se está desarrollando con éxito, y las evidencias del proceso, recogidas en un portafolio, servirán para el seguimiento de la titulación

INTRODUCTION: The practice of dentistry requires the proper development of communication skills, which must be guaranteed during undergraduate studies. This is an institutional challenge, because the schools have to put the academic mechanisms to evaluate the level of development of these skills. METHODS: The University of the Basque Country (UPV/EHU) is developing a comprehensive collaborative plan that includes four phases: a) collection and socialization of information on the current situation of the development of communication skills; b) design of a training plan to improve these skills, c) training plan implementation and results assessment, and d) gathering evidence of the process and dissemination of the results. RESULTS: After the detection of strengths and weaknesses in communication, we defined a total of 40 communication skills, structured in five blocks (oral communication with patients/family members; oral communication with other professionals; written communication with patients/family members; written communication with other health professionals, and dentist-patient communication techniques) and three levels of performance (identifying information, performance with high supervision, autonomous with moderate supervision). For its development, 19 training and evaluation activities were designed, currently being implemented. CONCLUSION: The plan is being developed successfully and the evidence of the process, collected in a portfolio, will be used to monitor the degree

Intra-Arterial Infusion of Magnetic Nanoparticle-Based Theragnostic Agent to Treat Colorectal Cancer Liver Implants in Rats.

INTRODUCTION: Nowadays, surgical excision remains the gold standard to treat liver metastases of colorectal cancer (CRCLM). However, as more than 50% of patients are not eligible for surgery, other alternatives such as percutaneous or intravascular interventional therapies (thermal ablation, chemoembolization, or radioembolization), are quite relevant. Recently, the use of magnetic nanoparticles (MNPs) has been suggested as an adjuvant for these therapies, as they could increase their necrotising effect on the tumour while reducing doses and exposure times of thermal therapies. To investigate the potential curative effect of these compounds, animal models are needed, both for the development of experimental interventional procedures and for MNPs toxicity and distribution assessment. Herein, we describe both an experimental infusion procedure in CRCLM-bearing rats and analytical and histological methods to evaluate MNPs deposits in the tissue. METHODS: Eighteen male WAG/RijHsd rats were subjected to intrahepatic injection of 250,000 colorectal cancer cells. Twenty-eight days later, half of the tumour-positive animals (n = 6) were administered with MNPs while the other half (n = 6) did not receive any injection and were used as control. Under microscope magnification, the splenic artery was carefully and completely dissected, and a catheter was inserted through the splenic artery to the common hepatic artery where 1 mL MNPs suspension was administered in 5 min; then STIR, DP*, and T2 MRI sequences were obtained (and signal intensity measured) and both tumour and liver tissue samples were collected for elemental and histological analyses. CONCLUSION: Our method for selective administration of MNPs is reproducible and well-tolerated and it fairly mimics the approach used in clinical practice when intravascular interventional therapies are applied.

Ultrasound Tumor Size Assessment, Histology and Serum Enzyme Analysis in a Rat Model of Colorectal Liver Cancer.

During tumor development, tissue necrosis appears as a natural phenomenon directly associated with an increase in tumor size. The aim of this study was to assess the use of ultrasound (US) for predicting natural tumor necrosis in a rat liver implant model of colorectal cancer. To achieve this goal, we sought to establish a correlation between US-measured tumor volume, serum enzyme levels and histopathological findings, particularly those regarding necrosis phenomena in liver implants. Under US guidance, CC531 colorectal cancer cells were injected into the left liver lobe of WAG/RijHsd rats. Twenty-eight days after cell inoculation, tumor volume was measured by US, and rats were sacrificed to obtain samples of tumor tissue as well as blood serum. In hematoxylin and eosin-stained tumor samples, the percentage of tumor that was necrotic was estimated. The association between percentage tumor necrosis and US-measured tumor volume was assessed by univariate logistic regression analysis, and a linear regression equation was obtained. Serum enzyme levels did not differ significantly between tumor-bearing and tumor-free rats. Tumor implants appeared as well-defined hyper-echoic regions with a mean volume of 0.61 ± 0.39 mL and tumor necrosis percentage of 8.6 ± 7.7%. Linear regression analysis revealed a very strong relationship (Pearson correlation coefficient r = 0.911) between US-measured tumor volume and tumor necrosis percentage; the regression equation was tumor necrosis percentage = 21 × US-measured tumor volume (in mL) - 3.1. The study found US to be a useful tool in animal-based trials. Tumors inside the liver (ranging in volume from 0.24-1.37 mL) can be observed by US, and moreover, US-measured tumor volume on day 28 can be used to estimate tumor necrosis occurring as the natural evolution of tumor implants.

[Analysis of Biological Properties of Human Adult Mesenchymal Stem Cells and Their Effect on Mouse Hind Limb Ischemia].

BACKGROUND: Due to their self-renewal, proliferation, differentiation, and angiogenesis-inducing capacity, human adipose mesenchymal stem cells (AMSC) have potential clinical applications in the treatment of limb ischemia. AMSC from healthy donors have been shown to induce neovascularization in animal models. However, when cells were obtained from donors suffering from any pathology, their autologous application showed limited effectiveness. We studied whether liposuction niche and obesity could determine the regenerative properties of cells meaning that not all cell batches are suitable for clinical practice. METHODS: AMSC obtained from 10 donors, obese and healthy, were expanded in vitro following a good manufacturing practice-like production protocol. Cell viability, proliferation kinetics, morphological analysis, phenotype characterization, and stemness potency were assessed over the course of the expansion process. AMSC selected for having the most suitable biological properties were used as an experimental treatment in a preclinical mouse model of hind limb ischemia. RESULT: All cell batches were positively characterized as mesenchymal stem cells, but not all of them showed the same properties or were successfully expanded in vitro, depending on the characteristics of the donor and the extraction area. Notably, AMSC from the abdomen of obese donors showed undesirable biological properties. AMSC with low duplication times and multilineage differentiation potential and forming large densely packed colonies, were able, following expansion in vitro, to increase neovascularization and repair when implanted in the ischemic tissue of mice. CONCLUSION: An extensive AMSC biological properties study could be useful to predict the potential clinical efficacy of cells before in vivo transplantation. Thus, peripheral ischemia and possibly other pathologies could benefit from stem cell treatments as shown in our preclinical model in terms of tissue damage repair and regeneration after ischemic injury.

Antitumor magnetic hyperthermia induced by RGD-functionalized Fe3O4 nanoparticles, in an experimental model of colorectal liver metastases.

This work reports important advances in the study of magnetic nanoparticles (MNPs) related to their application in different research fields such as magnetic hyperthermia. Nanotherapy based on targeted nanoparticles could become an attractive alternative to conventional oncologic treatments as it allows a local heating in tumoral surroundings without damage to healthy tissue. RGD-peptide-conjugated MNPs have been designed to specifically target αVß3 receptor-expressing cancer cells, being bound the RGD peptides by "click chemistry" due to its selectivity and applicability. The thermal decomposition of iron metallo-organic precursors yield homogeneous Fe3O4 nanoparticles that have been properly functionalized with RGD peptides, and the preparation of magnetic fluids has been achieved. The nanoparticles were characterized by transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), electron magnetic resonance (EMR) spectroscopy and magnetic hyperthermia. The nanoparticles present superparamagnetic behavior with very high magnetization values, which yield hyperthermia values above 500 W/g for magnetic fluids. These fluids have been administrated to rats, but instead of injecting MNP fluid directly into liver tumors, intravascular administration of MNPs in animals with induced colorectal tumors has been performed. Afterwards the animals were exposed to an alternating magnetic field in order to achieve hyperthermia. The evolution of an in vivo model has been described, resulting in a significant reduction in tumor viability.