The administration of intravenous fluid to critically ill patients is one of the most common, but also one of the most fiercely debated, interventions in intensive care medicine. Even though many thousands of patients have been enrolled in large trials of alternative fluid strategies, consensus remains elusive and practice is widely variable. Critically ill patients are significantly heterogeneous, making a one size fits all approach unlikely to be successful.New data from basic, animal, and clinical research suggest that fluid resuscitation could be associated with significant harm. There are several important limitations and concerns regarding fluid bolus therapy as it is currently being used in clinical practice. These include, but are not limited to: the lack of an agreed definition; limited and short-lived physiological effects; no evidence of an effect on relevant patient outcomes; and the potential to contribute to fluid overload, specifically when fluid responsiveness is not assessed and when targets and safety limits are not used.Fluid administration in critically ill patients requires clinicians to integrate abnormal physiological parameters into a clinical decision-making model that also incorporates the likely diagnosis and the likely risk or benefit in the specific patient’s context. Personalised fluid resuscitation requires careful attention to the mnemonic CIT TAIT: context, indication, targets, timing, amount of fluid, infusion strategy, and type of fluid.The research agenda should focus on experimental and clinical studies to: improve our understanding of the physiological effects of fluid infusion, e.g. on the glycocalyx; evaluate new types of fluids; evaluate novel fluid minimisation protocols; study the effects of a no-fluid strategy for selected patients and scenarios; and compare fluid therapy with other interventions. The adaptive platform trial design may provide us with the tools to evaluate these types of interventions in the intrinsically heterogeneous intensive care unit population, accounting for the explicit assumption that treatment effects may be heterogeneous. Although there is no universally accepted definition, personalised medicine has been described as “a medical model using characterization of individuals’ phenotypes and genotypes  for tailoring the right therapeutic strategy for the right person at the right time, and/or to determine the predisposition to disease and/or to deliver timely and targeted prevention”. This definition was used by EU Health Ministers in their Council conclusions on personalised medicine for patients, published in 2015 [Because of the way intensive care units (ICUs) have developed over the years, one could argue that they perhaps offer the ultimate environment for real-time personalised medicine. Modern ICUs provide a plethora of readily available patient-specific data, such as point of care blood tests, invasive and non-invasive haemodynamic and respiratory measurements, bedside ultrasound, and many other measures and variables, to help guide therapy. In addition, intensive care patients are significantly heterogeneous, underlining the need for personalised medicine principles.The administration of intravenous fluids is one of the most common interventions in the intensive care environment. There is on-going discussion regarding the benefits and risks of infusion therapy. While the application of fluid during shock may lead to circulatory stabilisation and can therefore be potentially lifesaving, fluid overload is associated with oedema development and worse outcomes . InteresThe paucity of evidence in this area of intensive care medicine may be related to the complexity of treatment in an intrinsically heterogeneous group of critically ill patients, as well as to considerable differences that exist in the clinical use of infusion therapy. Patient heterogeneity, even within groups of defined illnesses such as sepsis or acute respiratory distress syndrome (ARDS), remains one of the main challenges when it comes to developing robust evidence that therapeutic interventions such as fluid administration improve patient-centred outcomes, e.g. mortality and long-term function. The results of several large fluid studies have provided clinicians with some useful answers but also with many more questions and, as a result, a significant level of debate and controversy remains. These large randomised controlled trials, although considered the highest level of evidence, often fail to take into account patients’ heterogeneity, and consequently show negative results .Applying personalised medicine principles to fluid administration in critically ill patients requires careful attention to: context, indication, targets, timing, amount of fluid, infusion strategy, and type of fluid.It is important to differentiate between fluid substitution and volume substitution in intensive care patients. Different indications warrant different strategies and fluid choices, a distinction that has not always been appreciated sufficiently in the design of fluid studies. In addition, and again touching on patient heterogeneity, an intervention may be beneficial in one group of patients and harmful in another. This was elegantly demonstrated in a recent systematic review and meta-analysis of blood product transfusion strategy in the perioperative and acute care setting. In this study, the authors used a context-specific approach, based on patient characteristics and clinical settings, to estimate the risk or benefit of a restrictive transfusion strategy as compared with a liberal transfusion strategy. They found that a restrictive transfusion strategy was associated with an increased risk of composite cardiovascular events and death in patients with cardiovascular disease undergoing cardiac or vascular procedures, as well as in elderly patients undergoing orthopaedic surgery, but not in a mixed group of critically ill patients .As could be expected, the clinician’s diagnosis or working hypothesis of which underlying illness or process leads to abnormal physiology plays an important role in the decision-making process regarding fluid resuscitation. For example, in one Australian study, recognition and physician documentation of sepsis syndrome in patients presenting to the emergency department resulted in administration of significantly more volume of fluid . This ma2 pressure as measured by gastric tonometry, has been shown to be associated with worse patient outcomes [Abnormal physiology is often perceived to reflect a requirement for fluid resuscitation. A significant proportion of the evidence we generate in studies and translate into everyday practice is based on targeting intermediate end-points, such as improvement or correction of abnormal physiology. However, this approach may or may not result in improvement of relevant patient-centred outcomes. An excellent example of this dilemma can be found in studies in which tonometry-guided treatment was used in an attempt to improve outcomes . Gastroioutcomes . Howeveroutcomes . Anotheroutcomes . There aResuscitation of patients in shock needs to be timely and adequate. The Surviving Sepsis Guidelines suggest that a fluid bolus of 30 ml/kg crystalloid be administered to septic patients with hypotension or elevated lactate levels within 3 h of the time of presentation . Fluid bFirst, there is no universally accepted definition of what a fluid bolus is and how it should be administered, and what physiological effects clinicians expect as a result of a fluid bolus. This was illustrated in a recent survey amongst acute care physicians in Australia and New Zealand . This stSecond, the physiological effects of FBT in critically ill patients have not been well studied. This was highlighted in a systematic review of contemporary data of physiological changes after FBT in sepsis . No randThird, the haemodynamic response to a fluid bolus is usually small and short-lived, and the clinical relevance of this physiological effect is uncertain. In the previously mentioned systematic review of contemporary data of physiological changes after FBT, the mean arterial pressure (MAP) increased on average by 7.8 mmHg immediately after a fluid bolus, and returned close to baseline after 1 h, with no increase in urine output . These rFinally, FBT carries a significant potential for harm, especially if used indiscriminately. Rather alarmingly, clinicians do not appear to be particularly good at determining whether a patient will benefit from the administration of a fluid bolus, especially when basing this decision on clinical examination and static haemodynamic indices such as central venous pressure. In studies summarised in a review by Michard and Teboul in 2002 , around To date, the only randomised controlled trial of fluid resuscitation in sepsis is the FEAST trial  . The invCurrently, there is no evidence from randomised controlled trials that resuscitation with colloids, instead of crystalloids, reduces the risk of death in critically ill patients –39. InteBalanced fluids are crystalloid and colloid solutions with a more physiologically balanced electrolyte formulation, such as Hartmann's solution, PlasmaLyte, and Hextend. The use of these fluids for volume resuscitation can potentially prevent the development of hyperchloraemic acidosis, an electrolyte abnormality often encountered with the use of “normal” saline even after relatively low volume application –44. In aGoing forward, we need to focus on several important issues.First, we need to educate clinicians about the risks of fluid loading patients who are not fluid responsive. The potential of harm caused by fluid bolus therapy should more clearly feature in guidelines. Implementation of a physiologic, haemodynamically guided conservative approach to fluid therapy in patients with sepsis would possibly reduce the morbidity and improve the outcome . The safSecond, we urgently need to go back to the drawing board to design rigorous research to re-examine fluid therapy. The effects of fluid infusion on the immune system, on endothelial function, and on the integrity of the glycocalyx remain poorly understood. Degradation of the glycocalyx on the vascular luminal cell membrane has been identified to be an early step in septic vascular endothelial cell disorder . Fluid tThird, the concept of small-volume resuscitation using hypertonic fluids in sepsis deserves additional investigation. Hypertonic resuscitation may provide effective and rapid intravascular volume resuscitation. In addition, some preliminary data suggest that hypertonic fluid administration in sepsis may have beneficial effects on the global circulation and the cardiac function that exceed simple intravascular volume expansion . HypertoFinally, we should embrace new fluid trial designs that are capable of evaluating efficacy of multiple interventions in a heterogeneous ICU population, explicitly assuming treatment effects may be heterogeneous . AdaptivFluid resuscitation has long been one of the cornerstones of intensive care treatment, albeit with a limited evidence base in terms of its effects on outcome. An increasing body of literature suggests that fluid bolus therapy may contribute to fluid overload and cause harm, partly because clinicians do not routinely test for fluid responsiveness and rarely apply safety limits. The effects of fluid boluses on physiological parameters are not well studied, and seem small and short-lived at best.Personalised fluid administration in critically ill patients requires clinicians to integrate abnormal physiological parameters into a clinical decision-making model that also incorporates the likely diagnosis and the likely risk or benefit in the specific patient’s context. Personalised fluid resuscitation therefore requires careful attention to the mnemonic CIT TAIT: context, indication, targets, timing, amount of fluid, infusion strategy, and type of fluid.The research agenda should focus on basic research to improve our understanding of the physiological effects of fluid infusion, e.g. on the glycocalyx, and experimental and clinical studies to evaluate novel fluid minimisation protocols including no fluid strategies, and studies comparing fluid therapy with other interventions. The platform trial design may provide us with the tools to evaluate these types of interventions in the intrinsically heterogeneous ICU population, with the explicit assumption that treatment effects may be heterogeneous.Much of the progress that has been made in intensive care medicine is the result of identifying and abandoning potentially harmful interventions and treatments. Perhaps it is now time to add the indiscriminate use of fluid therapy to that list. Whenever tempted to give a fluid bolus to a patient, clinicians should remember “CIT TAIT” and should consider to just “sit tight”.


Colorectal cancer is one of the leading causes of cancer-related deaths. Although several therapeutic management strategies are available at the early colon cancer stages, such as endoscopic mucosal or submucosal dissection, associated complications often include bleeding or bowel perforations. As an alternative approach, we investigated endoscopic non-ablative fractional laser (eNAFL) irradiation as a minimally invasive therapeutic modality for the treatment of early-stage colorectal cancer. By implanting SL4-DsRed colon cancer cells into the colons of the C57BL/6 mice, we developed an orthotopic colon tumour mouse model and demonstrated the early-stage tumour growth delay following the eNAFL irradiation. Additionally, we evaluated the temperature changes in the eNAFL-irradiated area using numerical simulations, and induced inflammation using histological analysis. Our results indicate a minimal thermal damage confined to the irradiated spot, sparing the adjacent tissue and alteration in the tumour microenvironment. eNAFL irradiation may be clinically useful as a minimally invasive therapeutic intervention at the early stage of tumourigenesis. In future, an optimal eNAFL therapeutic dose should be determined, in order to increase the efficacy of this approach. Early-stage colon cancer is defined as cancer that is confined to the mucosa or submucosa and does not invade muscularis propria2. Currently, several therapeutic options for the early-stage colorectal cancer therapy using the endoscopy are available, such as snare polypectomy3, endoscopic mucosal resection4, and endoscopic submucosal dissection5. However, these treatment strategies are associated with complications such as bleeding or bowel perforations8. Additionally, despite the endoscopic tumour resection, residual tumour can be found, and approximately 13.1% of polyp recurrence rate was observed after endoscopic mucosal resection9. Other therapeutic options include radiation therapy, chemotherapy, immunotherapy, and targeted therapy, and a combination of these treatments may lead to the improvement in the progression-free or overall survival rates12. However, these approaches are primarily used for the treatment of the advance-stage colon cancer rather than the early-stage colon cancer13. Therefore, it would be beneficial for developing a treatment method that could easily be integrated with conventional endoscopic systems to suppress early tumour growth in a minimally invasive manner.Colorectal cancer is the second most frequent cause of cancer-related deaths in the US14. Currently, both ablative and NAFLs are widely used as a medical device. By replacing geriatric tissue with the newly-developed one, the texture and colour of the skin were shown to be significantly improved after irradiation15. Although the non-ablative laser treatment is less invasive, it was shown to have a limited therapeutic efficacy16, and fractional laser was developed to enhance the therapeutic efficacy, as it delivers high-energy light to the skin in a pixelated pattern, whereas non-fractionated lasers irradiate the entire projected area18.We investigated non-ablative fractional laser (NAFL) as an alternative therapeutic modality. NAFL delivers infrared-wavelength non-ablative laser light to the tissue with pixelated pattern, and this treatment selectively induces wound response in the dermal tissue, while minimizing the damage to the epidermis, in contrast with the ablative laser treatments, which remove the epidermis of the irradiated area19. These preventive effects may be useful for other epithelial tissues, such as colon and rectum, as well.Recently, it was reported that fractional lasers can be used for skin cancer prevention, showing that, following repeated irradiations of the mouse skin by the fractional laser, tumour occurrence rate and tumour progression in the skin of mice maintained under ultraviolet light, were significantly lower than those in the non-irradiated skin20. However, the ectopic model (colon cancer cells growing in skin) was shown to have limitations due to the differences in the tumour microenvironment22. Tumour response to therapy can be different depending on the localisation of the tumour23. Genetically engineered mouse models of cancer usually require a long time to develop tumours and the frequency, time, and location of primary tumours are less predictable22. Therefore, the use of the orthotopic tumour model with natural tumour environment is beneficial, as it is more predictable.We recently reported the therapeutic efficacy of NAFL irradiation in a subcutaneous tumour modelIn this study, we developed an endoscopic NAFL (eNAFL) irradiation system to determine its therapeutic effects on the early-stage tumours. Initially, we developed an orthotopic colon tumour model, matching the original anatomic location of the tumour by implanting SL4-DsRed cancer cells into the colon of C57BL/6 mouse. Following this, we examined the preclinical usefulness of fractional laser as a cancer therapeutic modality using the developed animal model. Additionally, using the numerical simulation and histological analysis, we examined the physiological processes induced by laser irradiation. Additional thermal imaging was performed to evaluate temperature changes in real time. To the best of our knowledge, this is the first study reporting the use of a NAFL as an early cancer therapeutic modality in an endoscopic setting.In Table We performed endoscopic imaging of the colonic walls in the control (n = 4) and eNAFL irradiated group (n = 5) every day for 1 week Fig. . Both whTo estimate the thermal effect of the eNAFL irradiation, we developed a computational numerical simulation model with a single irradiation spot on a tissue sample Fig. . In the Haematoxylin & Eosin (H&E) staining of the colon specimens from control and tumour-bearing mice was performed. In healthy mice, the colonic structure was shown to be preserved prior to the eNAFL irradiation Fig. . HoweverIn mice with the orthotopic colon tumours, similar histological changes were observed Fig. . Prior t20, the results of our approach indicate an improved clinical significance against orthotopic tumour growth in an endoscopic setting.We developed a novel eNAFL irradiation modality and examined its growth delay effects by using an orthotopic tumour model. To the best of our knowledge, this is the first endoscopic application of the thulium NAFL for the therapy of the gastrointestinal tract cancer. eNAFL irradiation resulted in statistically significant tumour growth delay. Although these results are consistent with those of a previous study that used the subcutaneous skin tumour modelet al.24 reported that, using this method, the tumour incidence in the surviving mice (95%) in over 200 implantation was 100%, always culminating with a tumour development at the injection site. Major complications associated with this procedure are bowel perforation and peritoneal seeding, which are primarily dependent on the required manual dexterity. Cancer cell leak into the abdominal cavity may lead to the peritoneal seeding without the tumour formation at the primary injection site.To create a reproducible orthotopic tumour model, we investigated different approaches, and showed that the cancer cell implantation mediated by the intra-colonic wall injection with laparotomy has a tumour-formation success rate comparable with that of the endoscopic implantation method. However, a dexterous procedure is required to inject cells into the submucosal layer of the thin colon, and the proper implantation of cancer cells is essential in this preclinical approach. In this study, the endoscopic implantation method resulted in a higher success rate. As a minimally invasive procedure, the advantage of this method is the lack of need for laparotomy, which inevitably induces a systemic inflammatory response. Zigmond 27, which can suppress tumour growth at the early tumour stages28. Similarly, our histological examinations at different time points after eNAFL treatment showed immune cell infiltration in the normal and tumour-harbouring colon tissue. Thermal injury and consequent tissue changes may induce the changes in the microenvironment of normal and tumour tissues. Tissue repairing processes after thermal damage require chemokines to recruit inflammatory cells in addition to vascular reactions. Here, we showed observed enhanced inflammatory responses in the colon tissue samples obtained from mice with and without tumour implantations. Therefore, the induced inflammation in the tumour microenvironment may have affected the observed tumour growth delay.The biological mechanisms underlying the eNAFL irradiation of tissue have not been completely understood. Previous studies, investigating fractional laser irradiation effects on skin remodelling reported that the irradiated tissue shows local inflammation29, are required to develop novel approaches that may increase therapeutic efficacy.To understand the effects of eNAFL irradiation on the colon wall, we devised an infrared thermal imaging experiment to directly monitor the thermal effect throughout this process (Supplementary Methods). Since no thermal endoscopic instruments are available yet, we used an alternative approach, the imaging of an exposed inner colonic wall after surgical incision of the colon . This result may be explained as follows. The advanced tumours tended to bleed, and therefore, the absorption of blood may interfere with the fluorescence signal. Additionally, with the increase in the tumour size, the attenuation of fluorescence signal due to the tissue thickness can affect the resultant signal. These factors are not easily controlled, particularly in the later stages of tumour growth. Therefore, we used fluorescence signals only to confirm the presence of the cancer cells in the early stage of tumour growth and not for the quantitative tumour growth analysis. However, as the fluorescence labelling allows the tracking of cells, our model could potentially be used to investigate the eNAFL treatment effects on metastases, when our orthotopic model is confirmed to produce consistent metastases to other organs.For the quantitative tumour evaluation, we used white light images instead of the fluorescence signals, since the fluorescence signal intensity does not correlate well with the visual grade of the tumour  agent; Avastin) was shown to increase clinical survival ratesIn conclusion, eNAFL therapy led to a tumour growth delay when applied for the treatment of the early-stage orthotopic colon tumour. However, both the treated and control groups showed persistent tumour growth in the later stages, showing that the application of this method alone may not lead to the desirable outcomes in clinic. Furthermore, a number of parameters should be considered, and some of them have not been optimised in this study, such as laser energy, the number of irradiation spots, irradiation duration, and others. Further optimisation and potential combination with other therapeutic modalities are warranted to improve treatment efficacy. This newly developed endoscopic approach has a great potential as a minimally invasive therapeutic interventional technology that can be applied at the early stage of tumourigenesis.A thulium fibre-based fractional laser system  was combined with a multi-channel endoscopy system Fig. . After dThis study was approved by the Institutional Animal Care and Use Committee of the Gwangju Institute of Science and Technology (GIST-2016-32) and all experiments were performed in accordance with relevant guidelines and regulations. We attempted three different approaches in order to find an optimal orthotopic colon tumour model.6 SL4-DsRed cells in 50 µL PBS suspensions were injected via the PE10 tube into the colonic lumen. After 30-min incubation, the suture and PE10 tips were removed. Mouse peritoneum and the abdominal wall were sutured with polyglactin suture materials . Ketoprofen (5 mg/kg) was subcutaneously injected for pain control.The first approach was the intraluminal seeding with laparotomy of colon cancer cells. Six-week-old female C57BL/6 mice were placed in the supine position anaesthetised with zoletil/xylazine mixture in a saline solution (60/10 mg/kg body weight). A polyethylene tube  was applied to the tip of the 30-gauge needle. After mouse laparotomy, distal colon was found with intestinal forceps. The upper portion of the colon was lightly ligated with polyamide monofilament, non-absorbable suture materials . PE10 tube tip was inserted via anus to the ligated site of the colon. Trypsin-EDTA solution  was introduced into the lumen via PE10 tip and incubated for 10 min to irritate mucosal barrier, which was followed by washing with PBS. The distal portion of the colon was temporarily ligated using the same suture. Afterwards, 1 × 105 SL4-DsRed cells in 10 µL PBS were injected into the colonic wall using 28-gauge needle attached to the 10-µL Hamilton syringe . After the injection, mouse peritoneum and the abdominal wall were sutured as described.The second approach was the intra-colonic wall injection with laparotomy of colon cancer cells. Laparotomies were performed under anaesthesia, and 1 × 1037. Endoscopic needle injection was described previously24. Under anaesthesia, the endoscopic tip was inserted into mouse anus. Eight-inch needle  was inserted via the instrument channel of an endoscope, and 1 × 105 SL4-DsRed cells in 30 µL PBS were injected.The final approach was the endoscopic implantation of colon cancer cells. A home-built multi-channel mouse endoscopic system was used for the endoscopic investigationAfter the orthotopic cancer cell implantation, we performed endoscopic imaging to compare tumour formation rate and the follow-up period. Successful implantation was determined by the presence of red fluorescence in the colonic wall during the follow-up endoscopic examinations, performed twice weekly. Overall tumour production success rate and the last follow-up days to tumour formation were recorded. After comparing these three orthotopic tumour implantation methods, we selected the best approach for eNAFL irradiation. Fluorescence images were obtained using a scientific colour CCD camera  and the RGB-based image was converted into a 16-bit grayscale image using the ImageJ software .20. To imitate the fractionated laser treatment, we used a simple irradiation at four spots. The control group mice did not receive any treatment. Follow-up endoscopic examinations were performed using the fluorescence and white light imaging.Nine mice with orthotopic tumours were randomly separated into the irradiation (n = 5) and control (n = 4) groups Fig. . All ortMurine colon cancer cells (SL4-DsRed) were cultured for the use in syngeneic xenograft tumour models. This cell line was kindly provided by the Edwin L. Steel Laboratory . SL4-DsRed cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F12) 1:1 medium supplemented with L-glutamine and 2.438 g/L NaHCO3, 10% foetal bovine serum (FBS), and 1% penicillin/streptomycin solution. All media and reagents were purchased from Gibco .p, k, t, Qbio, and Qlaser are the temperature of tissue, density, specific heat, thermal conductivity, time, non-directional heat exchange by blood perfusion, and heat source by laser absorption into the tissue, respectively. Qbio is based on the Pennes bioheat equation, presented as Eq.  of 500 μm. The laser beam has been modelled as a spatially distributed heat source on the surface by using a built-in Gaussian pulse function, where the beam size  with a standard deviation of three has been considered, which accounts for 99.7% of the total laser power (35 mW), in order to closely match the real experimental conditions. The laser irradiation lasted for 2 s on the tissue surface. The surrounding colon tissue was modelled using the extra-fine tetrahedral swept mesh method, which facilities only a single thin element through the thickness, while maintaining reasonable size in the plane. The numerical computation of the photothermal analysis of the colon model was performed using a commercially available software . Eq. .Tumour growth in the colonic lumen was examined using a custom-built endoscope capable of both white light and fluorescence imaging. Endoscopic scoring of tumour development was done according to the methods described in the previous reportsTo evaluate the tissue responses to the eNAFL irradiation, histological analyses of both control and tumour-bearing mice were performed. Control and tumour-bearing mouse colons were extracted before, 6 h after, and 48 h after eNAFL irradiation. Tissue changes and immune cell infiltration rates were evaluated using the H&E staining.The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.Supplementary Video S1Supplementary information


AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis‐dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF‐1α, caspase‐3 (CASP3) and metalloproteinase‐2 (MMP2) protein levels. We found that AQP1 knock‐down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA‐treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1‐dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF‐1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.Aquaporin‐1 ( Aquaporins (AQPs) are transmembrane channel proteins responsible for water flux across the plasma membrane driven by osmotic and hydrostatic gradient Tumour angiogenesis is the formation of new blood vessels necessary to provide nutrients and oxygen for tumour to growth. The progression of the migrating endothelial cells requires the degradation of the extracellular matrix mediated by matrix metalloproteinase (MMPs), a family of zinc‐dependent endopeptidases. MMPs are secreted by cells resident in the tumour, such as tumour cells, fibroblasts and activated endothelial cells, and mainly by immigrating immune cells The relationship between AQP1 expression and metastatic potential and survival has been previously shown in invading lung cancer, particularly in adenocarcinomas, where AQP1 overexpression correlated with metastases and unfavourable survival rates We report here the proof of concept that AQP1 inhibition is also able to strongly reduce cell's ability to metastasize by crossing endothelial barrier and further support AQP1 as proangiogenic protein and potential target for cancer therapy.Procedures involving animals were carried out in compliance with the European directive on animal use for research and Italian law on animal care. This project has been approved by the Italian Health Department (Approved Project no. 100/2014‐B). All experiments were designed to minimize the number of animals used and animal suffering.www.lgcstandards-atcc.org) were grown in DMEM medium supplemented with 10% heat‐inactivated foetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin and maintained at 37°C in a 5% CO2 incubator. Cell culture reagents were purchased from Thermofisher (www.thermofisher.com). In experiments using staurosporine , cells were cultured for 24 hrs, treated with drugs for 48 hrs and then collected.The murine melanoma B16F10 cells CRL 6475 (www.thermofisher.com).AQP1 siRNA (5′‐ACUCCAGGGUGGAGAUGAAGCCCAA‐3′) and a scrambled CTRL siRNA (5′‐CGAUGGAGAAGGCCAACUAGGGACU‐3′) www.envigo.com). Animals were kept in a regulated environment  with a 12 hrs light/dark cycle with food and water ad libitum. 106 B16F10 cells in 200 μl PBS were injected subcutaneously into the back of the mice. RNAi experiments were performed 10 days after the injection of the tumour cells (day 0) as previously described www.thermofisher.com) in a total volume of 200 μl of 5% glucose in water. After 20 min of incubation, the siRNA‐oligofectamine complexes were injected into the tumour using an insulin syringe with a permanently attached needle. At day 6, 24 μg of siRNA was used, and the amount of oligofectamine was scaled up accordingly. Tumour length (L) and width (W) were measured every 3 days after implantation using a caliper, and tumour volume was calculated as L × W2 × 0.5. Mouse survival was closely monitored (at least three times per day) throughout the entire experimental period. After death, tumour tissues were divided to be kept at −80°C and fixed in 4% paraformaldehyde for immunofluorescence analysis. Lungs were harvested and fixed in freshly prepared 4% paraformaldehyde for 4 hrs. Tumour foci on the surfaces of the lungs were counted under a stereomicroscope. Each organ was observed for metastasis analysis.Male C57BL6/J mice (8 weeks of age) were obtained from Harlan Laboratories (www.qiagen.com) following the manufacturer's guidelines. The extracted gDNA was quantified using NanoDrop. For analysis, 3 μg of each sample was analysed by agarose gel electrophoresis.Genomic DNA (gDNA) was extracted from frozen tumour tissue using the Qiagen DNeasy Blood and Tissue kit (www.scbt.it), rabbit polyclonal anti‐HIF‐1α‐CHIP grade (ab2185) 1:500 (www.abcam.com); rabbit polyclonal anti‐CASP3 (3015‐100) dilution 1:100 (www.biovison.com). The incubation time was O/N at 4°C for Western blot and 1 hr at RT for immunofluorescence analysis. The following secondary antibodies were used for Western blot: horseradish peroxidase (HRP) conjugated goat anti‐rabbit IgG (sc‐2004), and goat antimouse IgG (sc‐2005), donkey anti‐goat IgG (sc‐2020) dilution 1:5000 (www.scbt.it). The secondary antibody used for immunofluorescence analysis was a donkey anti‐rabbit Alexa 488‐conjugated (A21206) dilution 1:1000 (www.thermofisher.com).The following primary antibodies were used goat polyclonal anti‐actin (I‐19) (sc‐1616) dilution 1:500 and mouse monoclonal anti‐MMP2 (8B4) (sc‐13595) dilution 1:200 (www.lifescience.roche.com). The lysis was performed on ice for 1 hr, and the samples were then centrifuged at 22,000 × g for 1 hr. The protein content of the supernatant was measured with a bicinchoninic acid (BCA) Protein Assay Kit (www.Thermoscientific.com/Pierce).Excised tumours were solubilized in at least seven volumes of RIPA buffer  added with a cocktail of protease inhibitors (www.Thermoscientific.com/NuPage) and transferred to polyvinylidene difluoride membranes (www.merckmillipore.com). Membranes with blotted proteins were incubated with primary antibodies, washed, and incubated with peroxidase‐conjugated secondary antibodies. Reactive proteins were revealed with an enhanced chemiluminescent detection system (www.Thermoscientific.com) and visualized on a Chemidoc imaging system (www.bio-rad.com). Densitometric analysis was performed with ImageJ software (www.imagej.nih.gov).Western blot analysis was performed as previously described www.leica-microsystems.com/) and stored on positively charged glass slides (www.Thermoscientific.com). The sections were fixed in 4% paraformaldehyde, washed with PBS, blocked using 0.1% gelatin diluted in PBS for 30 min at room temperature (RT), incubated for 1 hr with primary antibodies, washed with PBS‐gelatin, incubated with Alexa Fluor488 conjugated secondary antibody and mounted with a medium containing 50% Glycerol and 1% n‐propylgallate in PBS. Sections were examined with a Leica DMRXA photomicroscope equipped for epifluorescence and PL Fluotar 16× and 100× objectives (www.leica-microsystems.com/). Digital images were obtained with a DX M1200 digital camera (www.nikoninstruments.com) and ACT‐1 version 2.20 software (www.nikoninstruments.com). Once captured, the auto contrast function was applied to the whole images using Adobe Photoshop CC 2014 (www.adobe.com). Mean fluorescence intensity analysis was performed with ImageJ (www.imagej.nih.gov): 10 fields for each condition were converted in grey scale, and the mean grey value was obtained for each whole image.Eight micrometre transverse sections were prepared using a cryostat . Data are expressed as mean ± S.E.M.A Kaplan–Meier plot was generated to determine mouse survival. Significance of differences in survival was calculated using Log‐rank (Mantel‐Cox) test. Statistically significant differences in number of lung nodules and densitometric analysis were computed using the Student's We have previously demonstrated that AQP1 silencing by RNA interference (RNAi) reduced primary tumour growth in the mouse model of melanoma Starting from day 0, tumours were measured every 3 days to monitor tumour growth that, in line with previous experiments, was found significantly reduced in AQP1 siRNA compared with CTRL siRNA and untreated mice  digestion and VEGF proteolytic release from the tumour matrix This generates a persistent hypoxic condition, which is in turn responsible for the apoptosis of tumour cells and the reduced size of the tumours.The increased expression of both VEGF and MMP2 is frequently synchronized by the common regulator HIF‐1α In conclusion, this study shows that AQP1 is not only able to promote tumour angiogenesis but it can also facilitate tumour cell intravasation and metastatic formation, probably promoting an increase in endothelial permeability. We believe this study adds knowledge on the intricate role of AQP1 at the vascular level and in tumour biology, which is key in view of potential alternatives to the current antiangiogenic therapy mainly based on VEGF pathways.L.S., C.D.G., F.P., A.C. and M.G.M. performed the research, L.S., C.D.C. and G.P.N. analysed the data, G.P.N., A.F. and M.S. designed the research. G.P.N. wrote the manuscript with substantial critical revision from L.S., C.D.G., A.F. and M.S.The authors declare no conflict of interest.


Ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS) is caused by tumours releasing ACTH. Ectopic ACTH-producing tumour regression is rarely induced using steroidogenesis inhibitors. We presented a case of EAS in which ACTH production by a lung tumour was reduced by metyrapone (MTP) and also reviewed previous cases of ectopic ACTH production suppressed via steroidogenesis inhibition.A 71-year-old female with general fatigue, central obesity and impaired glucose tolerance was diagnosed with Cushing’s syndrome due to elevated ACTH , cortisol  and 24-h urinary free cortisol (UFC)  levels. Chest computed tomography identified a solid 26.6 × 22.9 × 30.0 mm tumour with a cavity in the upper lobe of the left lung. There was no adrenal gland enlargement. Tumour markers were not significantly elevated; ACTH levels were not suppressed by 8-mg dexamethasone. A corticotropin-releasing hormone stimulation test revealed blunted ACTH response . She was diagnosed with EAS due to a lung lesion. MTP treatment was started to reduce cortisol production. ACTH levels and cortisol and UFC levels were normalised and the ACTH-producing lung tumour was ablated after MTP treatment. In several reported cases, plasma ACTH levels reduced during steroidogenesis inhibitor treatment for EAS. Among the 10 patients, three cases of pheochromocytoma, one of thymic carcinoid and one of islet cell carcinoma were reported. In four cases, the tumour was not detected. In our case, the pathology of the lung tumour was unknown because of lack of tumour cells in biopsy. The patients were treated with ketoconazole (KTZ) and/or MTP and exhibited ACTH and cortisol/UFC suppression, but tumour regression was observed only in our case.MTP and/or KTZ may reduce ACTH and cortisol production. The tumour spontaneously regressed after MTP treatment, indicating that MTP may reduce the tumour size without surgery. The mechanisms of therapeutic effects of steroidogenesis inhibitors and prognosis of spontaneous remission should be elucidated further via molecular biology studies.The online version of this article (10.1186/s12902-018-0246-2) contains supplementary material, which is available to authorized users. Cushing’s syndrome (CS) is associated with excess mortality and morbidity due to the long-term sequela of excessive cortisol levels . SteroidEctopic adrenocorticotropic hormone (ACTH) syndrome (EAS) is caused by tumours that release ACTH . EAS accIn this study, we report a rare case of EAS in which ACTH levels, as well as those of cortisol and urinary free cortisol (UFC), are normalised by treatment with MTP. In addition, the lung tumour, which was considered the ACTH-producing tumour, was ablated after the administration of MTP. This is the first reported case of the regression of an ectopic ACTH-producing tumour upon treatment with a steroidogenesis inhibitor using functional imaging. Moreover, we reviewed reported cases of ectopic ACTH production that was suppressed by steroidogenesis inhibition.A 71-year-old female presented with general fatigue, leg oedema and impaired glucose tolerance. Her medical history included Graves’ disease, colon polyp, sarcoidosis and uveitis following glucocorticoid treatment. Upon hospitalisation, she exhibited weight gain, central obesity, moon facies, proximal muscle weakness and elevated ACTH , cortisol  and 24-h UFC levels . Normal diurnal variation of cortisol was lost. Brain magnetic resonance imaging revealed an empty sella with no pituitary adenoma. Chest computed tomography (CT) identified a solid tumour of 26.6 mm × 22.9 mm × 30.0 mm in size with a cavity in the upper lobe of the left lung Fig. . There wTo differentiate between Cushing disease and EAS, an overnight 8-mg dexamethasone suppression test was performed. ACTH and cortisol levels were 184.8 pg/mL and 76.5 μg/dL, respectively, before the administration of dexamethasone. ACTH and cortisol levels 12 h after suppression with 8-mg dexamethasone were 176.7 pg/mL and 67.2 μg/dL, respectively. Lack of suppression of ACTH and cortisol by 8-mg dexamethasone is consistent with that with EAS. A corticotropin-releasing hormone (CRH) stimulation test revealed a blunted response of ACTH . According to these findings, the patient was diagnosed with EAS due to a lung lesion. MTP treatment was started to reduce cortisol production on the first day of hospitalisation (day 1).d-glucan levels were elevated to 23.85 and 69.6 mg/dL , respectively, whereas the patient’s white blood cell count was normal (8300/μL). Imipenem/cilastatin  and caspofungin acetate  were administered as antimicrobial and antifungal treatments, respectively.On day 3, because cortisol levels fell below the normal range (2.8 μg/dL), hydrocortisone treatment was started for supplementation. On the same day, the patient developed pneumonia and pleural effusion. C-reactive protein and beta-On day 14, CT detected enlargement of the lung tumour to a 5-cm-diameter mass  together with suppressed cortisol, regression of the lung nodule and disappearance of the pleural effusion. No tumour cells were revealed by bronchoscopy and CT-guided lung biopsy. Hypokalaemia and hyperglycaemia were also improved. Therefore, surgery was cancelled. She was discharged 6 weeks after admission. Six months after admission, CT revealed a small residual lesion in the lungs Fig.  and somaWe have presented a unique case of ectopic ACTH production with ACTH normalisation upon shrinkage of a pulmonary lesion. It is rare that steroidogenesis inhibitors suppress ectopic ACTH production and induce tumour regression. The therapeutic effect of MTP or cyclic CS may explain the regression of ACTH-producing cells. This is the first reported case of the regression of an ectopic ACTH-producing tumour following treatment with a steroidogenesis inhibitor as confirmed using functional imaging.In several reported cases, plasma ACTH levels were reduced during steroidogenesis inhibitor treatment for EAS Table  3–9]. A–9. A3–9]The mechanisms by which steroidogenesis inhibitors suppress ectopic ACTH production are unclear. The primary mechanism by which KTZ inhibits steroidogenesis is the inhibition of 17-hydroxylase, 11β-hydroxylase and the cholesterol side-chain cleavage enzyme , versus Direct impairment of ACTH biosynthesis by steroidogenesis inhibitors is also considered a cause of ACTH reduction. An in vitro study found that KTZ inhibited ACTH secretion at therapeutic doses by impairing adenylate cyclase activation in corticotrophs , which iTumour haemorrhage or infarction has been postulated as the cause of spontaneous remission of pituitary-dependent CS . AlthougAnother potential differential diagnosis of this patient was cyclic CS, which involves the fluctuating over-production of cortisol over months or years. In this patient, aberrant ACTH production occurred 1 month after the initiation of MTP, although it has since been suppressed for 1 year and 6 months. She has been observed carefully to determine whether hypercortisolemia will reappear. She is very pleased with the condition resolving itself without surgical intervention.We presented a rare case of EAS in which ACTH production by a lung tumour was reduced by MTP. The tumour itself regressed spontaneously after the initiation of MTP, indicating that this drug may reduce tumour size without surgery. The mechanisms of the therapeutic effect of steroidogenesis inhibitors and the prognosis of spontaneous remission should be elucidated further via molecular biology studies.Additional file 1:Figure S1. Enlargement of the lung tumour on day 14. (TIFF 48 kb)


Chondrosarcoma is the third most common primary tumour of the bone, after myeloma and osteosarcoma. Most of the chondrosarcoma grow slowly and rarely metastasize, and they have an excellent prognosis after adequate surgery. However most of them are chemo or radio-resistant. We report a case of primary chondrosarcoma of proximal humerus in a 36-year-old female who presented with a six years history of left shoulder swelling and restricted range of motion. Trucut biopsy showed a well-differentiated chondrosarcoma. The patient underwent forequarter amputation of left upper limb and was started on chemotherapy following operation. Chondrosarcoma constitutes a heterogeneous group of neoplasms that have the production of cartilage matrix by tumour cells in common2. This primary sarcoma of bone in adults has a male predominance and is well demonstrated between the 3rd and 7th decades of life. Low grade chondrosarcoma has similar appearance to enchondroma and osteochondroma and has occasional binucleated cells. High grade chondrosarcoma has increased cellularity, atypia, and mitoses3. We report a case of a well-differentiated giant chondrosarcoma of the proximal left humerus in a 36-year-old female patient who underwent forequarter amputation with good recovery.Primary bone tumours are uncommon and this has certainly contributed to the scarcity of data about their relative frequency, and to the limited understanding of the risk factors. Overall, bone sarcomas account for 0.2% of all malignancies, and the adjusted incidence rate for all bone and joint malignancies is 0.9 per 100,000 persons per year, while the 5-year overall survival rate is 67.9%A 36-year-old female patient was admitted to our department, presenting progressive enlarging mass and restricted range of motion of the left shoulder  for 6 yeAn informed consent was obtained from the patient prior to the operation. A multi-disciplinary intervention by the orthopaedic, radiological, anaesthetist and oncology team was adopted. She underwent forequarter amputation of her left upper limb. The tumour tissues were excised with maximum possible surgical safety margins. Intraoperatively there was neither excessive bleeding nor development of major complications. The resected mass measured 450 × 415 × 250mm and weighed 31kg  and was Microscopically, the sections showed lobulated tumour composed of chondrocytes with hyperchromatic nuclei. There was minimal cellularity which exhibited rare small, dark nuclei and multinucleated forms. Mitotic activity was rare. Minimal tumour necrosis was present. Lymph vascular permeation -noted. There was no osteoid formation. The tumour cells involved part of the adjacent bone and its marrow, adjacent soft tissue and adjacent muscle. No joint or neurovascular bundle at margin were involved by tumour cells. Two lymph nodes were positive for metastatic chondrosarcoma.Chondrosarcoma of the proximal humerus is an uncommon malignant bone tumour, and limited information is available about treatment. Preservation of the functional capacities of the involved limb along with a complete removal of the tumour is considered as the most important criterion in the surgical management of tumour of the proximal humerus. Although literatures support wide local excision for Grade I conventional chondrosarcoma rather than amputation, decision of forequarter amputation was made after taking into consideration of the tumour size which was too extensive and it was fungating and ulcerated. Furthermore the neurovascular structures of left upper extremity were severely affected. Therefore forequarter amputation with adequate surgical margin remained the best option procedure.Forequarter amputation of her left upper extremity posed challenges to the surgeon due to the enormous size of the tumour. Pre-operative planning with MRI and CT scan were not possible to be performed. It made the resection more challenging as we could not assess the extent of the chondrosarcoma and delineate the extent of soft tissue involvement for clear resection of the tumour. With the help of angiography of left upper limb the risk of bleeding during operation was minimized as anatomical position of major vessels were identified and no other anomalies such as vessel aneurysm was detected pre-operatively.4. The prognosis of this patient is good in view of the histopathological finding of the tumour which supports grade I chondrosarcoma. Although patient defaulted treatment for 6 years, there was no evidence of distant metastasis in solid organs based on CT scan done postoperatively.Chondrosarcoma of bone generally has a good prognosis when optimally diagnosed at an early stage. Histopathological grading, at present, is the best predictor of clinical behaviour. A series from the 1980s reported a 5-9% risk of metastasis with low grade conventional chondrosarcoma, whereas recent series reported 3% or no risk of metastasisIt is generally believed that chondrosarcoma is relatively chemo- and radiotherapy resistant due to the extracellular matrix, poor vascularity and low percentage of dividing cells. However the patient was initiated on adjunct chemotherapy in view of her young age, high tumour load and evidence of regional lymph node- metastasis with adjacent bone marrow involvement.Currently the patient is on six monthly follow up and she is satisfied with her current condition. She is looking forward to finding a new job after completing her chemotherapy.


Resistance to radiotherapy is frequently encountered in patients with glioblastoma multiforme. It is caused at least partially by the high glutathione content in the tumour tissue. Therefore, the administration of the glutathione synthesis inhibitor Buthionine-SR-Sulfoximine (BSO) should increase survival time.BSO was tested in combination with an experimental synchrotron-based treatment, microbeam radiation therapy (MRT), characterized by spatially and periodically alternating microscopic dose distribution. One hundred thousand F98 glioma cells were injected into the right cerebral hemisphere of adult male Fischer rats to generate an orthotopic small animal model of a highly malignant brain tumour in a very advanced stage. Therapy was scheduled for day 13 after tumour cell implantation. At this time, 12.5% of the animals had already died from their disease.The surviving 24 tumour-bearing animals were randomly distributed in three experimental groups: subjected to MRT alone (Group A), to MRT plus BSO (Group B) and tumour-bearing untreated controls (Group C). Thus, half of the irradiated animals received an injection of 100 μM BSO into the tumour two hours before radiotherapy.Additional tumour-free animals, mirroring the treatment of the tumour-bearing animals, were included in the experiment. MRT was administered in bi-directional mode with arrays of quasi-parallel beams crossing at the tumour location. The width of the microbeams was ≈28 μm with a center-to-center distance of ≈400 μm, a peak dose of 350 Gy, and a valley dose of 9 Gy in the normal tissue and 18 Gy at the tumour location; thus, the peak to valley dose ratio (PVDR) was 31.After tumour-cell implantation, otherwise untreated rats had a mean survival time of 15 days. Twenty days after implantation, 62.5% of the animals receiving MRT alone (group A) and 75% of the rats given MRT + BSO (group B) were still alive. Thirty days after implantation, survival was 12.5% in Group A and 62.5% in Group B. There were no survivors on or beyond day 35 in Group A, but 25% were still alive in Group B. Thus, rats which underwent MRT with adjuvant BSO injection experienced the largest survival gain.In this pilot project using an orthotopic small animal model of advanced malignant brain tumour, the injection of the glutathione inhibitor BSO with MRT significantly increased mean survival time. Glioblastoma multiforme (WHO IV) is a highly malignant brain tumour listed as a rare disease , with a Metastudies show that radiotherapy is the only independent factor determining outcome in glioblastoma multiforme , 5. One We have now combined the administration of the glutathione synthesis inhibitor BSO with a new experimental radiotherapeutic paradigm, in a small animal model of a very aggressive malignant brain tumour in an advanced stage of development. This paradigm, proposed for the treatment of malignant neoplasms, is a unique micro-radiosurgical method based on the principles of grid radiotherapy using synchrotrons X-rays , 17. If In the clinical radiotherapy of the brain, dose limits are dictated by the risks of cerebral edema, brain tissue necrosis and longterm changes in the white matter which can lead to cognitive deficits. MRT might be a good approach to overcome those limitations.Tumour cell implantation and irradiation with the aim to study the potential of the glutathione inhibitor BSO in combination with MRT in vivo were conducted at ID 17, the biomedical beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.F98 glioma cells from a commercially available cell line CRL-2397, ATCC) were used to generate orthotopic brain tumours in 27 male young adult Fischer rats 275–305 g). F98 gliomas share many characteristics with the cells of the highly malignant human brain tumour glioblastoma multiforme, such as fast aggressive growth with infiltration of normal brain structures Fig.  and deve397, ATCC5 g. F98 The animals were housed and cared for in a temperature-regulated animal facility exposed to a 12-h light/dark cycle.R in air inhalation), the scalp of the animals was shaved and disinfected. A sagittal midline incision was made in the scalp. The periosteum was retracted. A burr hole was placed 3 mm to the right of the sagittal suture and 3 mm posterior to the coronal suture. Using a small animal stereotactic frame, one hundred thousand F98 glioma cells were implanted into the anterior aspect of the right cerebral hemisphere of 27 rats. The cell suspension was injected through a 27 G needle attached to the Hamilton syringe, the tip of which was carefully lowered through the burr hole and inserted 3 mm below the cortical surface.Ten million F98 glioma cells were harvested from cultures, suspended in 1 ml of Hanks serum and stored on ice. For each animal, 10 μl of this cell suspension were aspirated in a Hamilton syringe which was mounted on the vertical arm of a small animal stereotactic frame. Under general anaesthesia (1.5–2% Isoflurane®/kg s.c. before surgery and a second dose at 12 h after surgery.The cell suspension was injected over a period of 4 min (2.5 μl / min) using an automated injector pump  to limit injury to the brain from the injection process. After completion of the tumour cell injection, the needle was left in place for an additional minute to allow the cell suspension to evenly distribute within the tissue. The needle was then withdrawn, the burr hole sealed with bone wax and the scalp sutured. The animals were allowed to recover. For analgesia, each animal received one dose of 0.05 mg BuprenorphineOut of our 27 tumour-bearing animals, three animals (12.5%) died before the day of the scheduled radiotherapy from their progressive disease Fig. . These aR collimator with slits spaced 400 μm in the instrument. Because of the minimal divergence of the beam, the spacing was 411 μm at the level of the goniometer [The white X-ray beam generated by the synchrotron source and the wiggler was filtered , resulting in a spectrum that extends from about 50 to well above 350 keV, with a mean energy of 105 keV . The micniometer .The tumours were expected to have diameters of 4.5–6.0 mm at irradiation on day 13 after tumour cell implantation. In the absence of an option for pre-therapeutic imaging, we irradiated a large tissue volume surrounding the tumour using two crossfired arrays centered on the tumour, each array ≈10 mm wide and 14 mm high, comprising 50 microplanar parallel microbeams of ≈28.3 μm FWHM at the level of the goniometer, with a center-to-center distance of ≈205.5 μm between adjacent microbeams  [The dose rate was measured for broad beam conditions in solid water plates   using a 3) . With thAccording to our Monte Carlo calculations, the value of the peak-to-valley dose ratio (PVDR) for MRT with the parameters used in our study was around 31. Therefore, the MRT valley dose would have been about 9 Gy per port for the 350 Gy peak entrance dose. Thus, in the normal tissue, the valley dose in our experimental design would have agreed with the recommendations resulting from the QUANTEC study, which was aimed at determining threshold values for normal tissue tolerance [n = 8 per group): Animals in Group A were submitted to MRT alone. Animals in Group B also underwent MRT but additionally received one injection of BSO into their tumours two hours before MRT. Using the small animal stereotactic frame again, four microliter of a 100 μM BSO solution were injected through the burr hole which had been used previously for tumour cell implantation, with the tip of the needle 3 mm below the cortical surface. Thus, BSO was injected in the same location where the tumour cells had been deployed 13 days earlier and the BSO was assumed to be deposited in the centre of the tumour that had developed from those F98 glioma cells. The dose of BSO was chosen based on the results of experiments described by Ataelmannan [The surviving 24 tumour-bearing animals were randomly distributed into three experimental groups .MRT was administered in a single session, with a peak skin entry dose of 350 Gy in each direction. The rats were positioned in a prone position on the goniometer, orthogonally to the direction of beam propagation, with the top of the skull horizontal  was used to evaluate the Cognitive dysfunction is frequently reported in patients after cerebral irradiation with conventional sources, especially in patients who were irradiated at a young age , 37. In Rodents show a strong tendency to approach and explore novel rather than familiar objects. This feature of cognitive behaviour, related to the animals’ ability to form new memory contents, is exploited in the Object Recognition Test (ORT) developed by Ennaceur and Delacour .For memory assessment, each animal was habituated to the empty test environment (a 40 cm × 40 cm × 60 cm open black Perspex box with a light grey floor) during a single session of 4 min duration. The following day, the animals were returned to the test environment where two identical objects had been placed on the floor of the cage (test part T1). Each animal was allowed to explore the environment including the two equal objects for 4 min before returning to its home cage. Confronted with this scenario, animals will typically spend equal times exploring each of the objects. The time spent exploring each of the objects was recorded for each animal. After the end of the test period, animals were returned to their home cages. Retrieved again from their home cages after a 4 h inter-trial interval, animals were inserted in the test environment for the second part of the ORT (test part T2). For this second session, one of the already familiar objects was replaced by a new object with similar salience. As before, the time spent exploring each of the objects was recorded for each animal. This test is based on the hypothesis that, in the second part of the test (T2), rats with normal memory function will spend more time examining the new rather than re-exploring the previously encountered object. Animals with memory encoding or retrieving impairments, however, will again spend equal times exploring both objects.There is an ongoing debate regarding the relation between hippocampal function and performance in the new object recognition test. Results of a metaanalysis published in 2015 support the idea that the validity of the ORT depends on the length of interval between the two test sessions: the hippocampus appears to be necessary for object recognition memory only if the recall interval is longer than 10 min . Thus, wp = 0.010) and MRT + BSO (P = 0.003).The increase of survival times within the irradiated groups, compared to untreated tumour-bearing animals, is illustrated in Fig. All untreated tumour-bearing animals had died by day 20 after tumour cell implantation, with a mean survival time of 15 days (SD ± 1.85). Median survival was also 15 days, equal to the median survival seen in tumour-bearing animals in a previous experiment with identical tumour parameters who received BSO only. Median survival was 20 days for animals undergoing MRT only and 27 days for animals undergoing MRT + BSO injection prior to radiotherapy. The confidence interval for both the mean and the median was 95%.Out of all other experimental groups, survival at day 20 after tumour cell implantation was 62.5%  in Group A (MRT only) and 75%  in Group B (MRT + BSO). On day 30 after tumour cell implantation, survival was 12.5% in Group A and 62.5% in Group B. There were no survivors on or beyond day 35 in Group A, but 25%  were still alive in Group B. The last animal in Group B died 41 days after tumour cell implantation (Table No significant memory deficit was detected either early or late after MRT alone Fig. . This isSurvival times for ≥90% of untreated intracerebral tumour-bearing control animals were about 20 days or more in most small animal models previously used for radiotherapy studies at the synchrotron , 40–43. Biston and colleagues, using an orthotopic F98 brain tumour model generated from 1000 implanted cells, reported a median survival time of 25 days for untreated tumour-bearing animals and a 31 day median survival after 5 Gy irradiation with synchrotron X-rays at 78.8 keV . In our Biston et al.  showed tDose escalation by spatial hypofractionation for better tumour control is not entirely new in the history of radiotherapy for patients with glioblastoma multiforme. Stereotactic radiosurgery has been used successfully to boost tumour target doses beyond 60 Gy and increase overall survival , 45. FolFew studies have investigated potential adverse effects of MRT on brain function. Earlier work has suggested that the structure of normal tissues in the path of the beam is greatly preserved after MRT , 50. We F98, like the human glioblastoma multiforme that it is supposed to model, is characterized by extensive invasion of normal brain structures with tumour cell clusters located at varying distances from the primary tumour bulk, also along the Virchow-Robin spaces , 51–53. Interestingly, it has been found that after intravenous injection the rate of BSO entry into gliomas is higher than the entry into tumour-free brain, by about one order of magnitude . FurtherThe adjuvant administration of BSO significantly increases survival times in several types of malignant brain tumour in vivo , 56. TheData obtained in a recent pre-clinical study support the concept of an intravenous administration of BSO as radioenhancer . Those dThe results of at least two Phase I clinical trials in which BSO was administered intravenously as adjuvant therapy in patients with solid malignant tumours have been published , 56. An Furthermore it was shown in vitro and in vivo that BSO increased the sensitivity to temozolomide, the standard chemotherapy agent in the treatment of patients with high grade glioma, by modifying ROS production , 60. BasThe preliminary results obtained in our pilot experiment should be confirmed in an expanded study with pre-therapeutic imaging to ensure that all tumours were of similar size at the time of treatment and to include a group of tumour-bearing animals that just receives an injection of BSO and no radiotherapy. Based on all data, the integration of MRT into a clinical schedule of whole brain irradiation could then be tested, possibly in combination with an intravenous administration of the glutathione synthesis inhibitor BSO. This might contribute to a significantly better tumour control even in a brain tumour at a very advanced stage of the disease.Furthermore, it would also be worthwhile to conduct a well-designed experiment using BSO as adjuvant with broad beam irradiation, with and without MRT as integrated boost. Such an experiment might answer the question whether even with conventional radiotherapy, patients might benefit from adjuvant BSO.The results of this pilot study suggest that MRT in combination with the glutathione synthesis inhibitor BSO results in a significant increase of mean survival time in an orthotopic small animal model of highly malignant brain tumour in a very advanced stage of development. Survival time was doubled compared to untreated animals. Two thirds of the time gained can be attributed to the glutathione synthesis inhibitor. A follow-up study in a larger number of animals is required to increase statistical power and confirm these promising results.We have also shown that MRT alone does not impair new memory formation. These preliminary results should be confirmed in an expanded study.


Enhanced recovery may be viewed as a comprehensive approach to improving meaningful outcomes in patients undergoing major surgery. Evidence to support enhanced recovery pathways (ERPs) is strong in patients undergoing colorectal surgery. There is some controversy about the adoption of specific elements in enhanced recovery “bundles” because the relative importance of different components of ERPs is hard to discern . There is evidence that specific approaches to fluid management are better than alternatives in patients undergoing colorectal surgery; however, several specific questions remain.In the “Perioperative Quality Initiative (POQI) Fluids” workgroup, we developed a framework broadly applicable to the perioperative management of intravenous fluid therapy in patients undergoing elective colorectal surgery within an ERP.We discussed aspects of ERPs that impact fluid management and made recommendations or suggestions on topics such as bowel preparation; preoperative oral hydration; intraoperative fluid therapy with and without devices for goal-directed fluid therapy; and type of fluid. We recommend unrestricted access to clear fluids for oral intake up to 2 h before the induction of anesthesia to maintain hydration while minimizing the risk of aspiration.We recommend that the clear fluid used to maintain oral hydration contain at least 45 g of carbohydrate to improve insulin sensitivity (except in type I diabetics due to their insulin deficiency state). We suggest that complex carbohydrate  be used when available.We recommend that clinicians avoid administration of intravenous fluids to replace preoperative “fluid losses” in patients who received iso-osmotic bowel preparation provided there was unrestricted intake of clear fluids for up to 2 h before the induction of anesthesia. There is no evidence that iso-osmotic mechanical bowel preparation leads to adverse effects on preoperative volume status.We recommend against the use of hyper-osmotic or hypo-osmotic bowel preparations prior to surgery since there is no benefit relative to iso-osmotic bowel preparation and there may be adverse effects on preoperative volume status.5.We recommend the application of a hemodynamic framework to guide clinical decision-making during surgery. We have developed such a framework and suggest that the use of intraoperative goal-directed fluid therapy (GDFT) is likely to be safe in the majority of patients undergoing major colorectal surgery. GDFT has little risk, and the use of advanced hemodynamic monitoring equipment may enhance clinical decision-making when compared with the use of conventional monitors.6.We suggest that the advanced hemodynamic monitoring equipment used to guide clinical decision-making intraoperatively be selected based on a combination of surgical patient and institutional factors since such monitoring can minimize both hypovolemia (by promoting therapy in volume responders) and hypervolemia (by restricting therapy in non-responders).7.We recommend that in isolation, intraoperative oliguria should not trigger fluid therapy, as low urine output is a normal physiologic response during surgery and anesthesia. We also recommend that intraoperative oliguria be investigated and that absolute (as opposed to relative) hypovolemia be ruled out.8.We recommend that intraoperative and postoperative anuria warrant immediate attention since anuria is pathological.9.We recommend that fluid management strategies focus on the following: first, identifying if there is a clinical problem that can be solved by fluid therapy and then identifying what fluid and how much is appropriate. Rather than treating every instance of abnormal hemodynamic values , clinicians must establish causation based on available information about the patient and clinical context.10.We recommend that therapy attempt to reverse the most likely cause of a hemodynamic derangement. Absolute hypovolemia may or may not be responsible for observed hemodynamic abnormalities. For instance, stroke volume variation above 13 % soon after the induction of anesthesia and with the institution of mechanical ventilation should prompt consideration of vasodilation (relative hypovolemia) rather than as the cause of fluid responsiveness. The patient may hence require vasoconstrictors rather than bolus fluid therapy provided clear fluids have been consumed preoperatively and iso-osmotic bowel preparation has been used.11.We recommend the use of buffered isotonic crystalloids for the treatment of hypovolemia in patients undergoing colorectal surgical procedures. We acknowledge that the restrictions on the use of starch solutions are based on extrapolations from the critical care literature.12.We suggest that patients tolerating fluids orally after surgery be given unrestricted access to such fluids as this increases patient satisfaction and as it is likely that intravenous fluid administration offer no added benefit.13.We suggest that the hemodynamic framework utilized intraoperatively be extended into the postoperative period to the extent possible, in situations where patients might benefit from such postoperative monitoring (high-risk patients or those with significant blood loss or complications during surgery).Outcomes such as complication rates, readmissions, and length of stay may be highly variable across different centers conducting colorectal surgery , was invited to participate. In total, 32 experts from around North America and Europe met in Durham, NC, on March 4–5, 2016, to iteratively discuss the evidence supporting enhanced recovery paradigms and develop consensus statements with practical recommendations for clinicians.all the experts present at the first POQI meeting.A list of relevant questions was collectively developed and circulated electronically prior to the meeting. Based on literature searches performed by members, questions were formulated. In the first plenary session, the POQI perioperative fluid management subgroup presented these questions to the entire POQI workgroup, to receive feedback and assistance in refining the questions. The subgroup then worked together to formulate answers to these questions, supported by evidence when available and by expert opinion when no clear evidence was available. These were presented in the second plenary session. After receiving feedback, the subgroup refined a series of consensus statements, which was then reviewed with and modified by the entire POQI group in the final plenary session. This manuscript is based on these multiple rounds of feedback from Based on both discussions (held prior to the conference) and the literature (identified by the participants), the following questions were considered most relevant to perioperative fluid management before, during, and after colorectal surgery within an ERP:(i)What are the effects of preoperative oral intake of clear solutions (containing complex versus simple carbohydrates) up to 2 h prior to the induction of anesthesia?(ii)Does mechanical bowel preparation contribute to preoperative hypovolemia?(iii)Is urine output a valid indicator of perioperative fluid needs?(iv)Is there a rational approach to intraoperative fluid management based on the current evidence?(v)Which types of fluids should be used intraoperatively?(vi)How do variations in surgical and anesthesia technique affect intraoperative fluid management?(vii)How should fluid therapy be managed postoperatively?It has known that both simple  and complex  carbohydrate-containing solutions prevent protein catabolism following exercise  , the oral intake of clear liquids should occur more than 2 h prior to the induction of anesthesia. ASA guidelines recommend modification of preoperative fasting on an individual basis in the presence of “gastroesophageal reflux disease, dysphagia symptoms, or other gastrointestinal motility disorders.”  may lead to hypovolemia due to gastrointestinal losses prior to colorectal surgery, was supported by a study comparing ten subjects randomized to Picolax (magnesium citrate (a hyper-osmotic laxative) and sodium picosulfate (a stimulant)) versus not. There was significantly more orthostasis and tachycardia in the group that received the hyper-osmotic bowel preparation  based on transpulmonary thermodilution, after induction of anesthesia. The authors concluded that hypovolemia was likely to be present , the mean cardiac index was 2.66 L/mModern MBP techniques typically utilize iso-osmotic agents, which in theory do not produce dehydration (no osmotic shift in fluids toward the bowel lumen). When combined with the emphasis on intake of clear fluids up to 2 h before surgery (see question i) in compliance with the ASA Fasting Guidelines, concerns related to the impact of MBP on volume status are minimal. There is no need for fluid therapy to treat presumed fluid losses from iso-osmotic MBP and starvation. The shift in practice away from empiric administration of fluid therapy toward therapy based on the detection of “fluid responsiveness”  has further diminished arbitrary preoperative intravenous hydration. If clinicians are able to rapidly identify hypovolemia intraoperatively, excessive preoperative fluid losses can be detected and managed objectively. Since it has not been established that iso-osmotic MBP predisposes patients to hypovolemia, and since clinicians can identify patients in whom MBP might have produced excessive fluid losses promptly  group, is more challenging to interpret as abnormal, especially when other indicators of overall tissue perfusion are normal  is a natural response to anesthesia and surgery. The resorptive actions of vasopressin on the collecting duct in nephrons lead to the retention of water with accompanying oliguria—this may not indicate organ dysfunction . Minimally invasive cardiac output monitoring-guided fluid management has been studied in multiple randomized controlled trials in patients undergoing diverse procedures  . Several investigators have examined device-guided GDFT in the modern era of ERPs. Three such groups independently tested a “zero balance” or “restrictive” strategy against conventional minimally invasive cardiac output monitoring -guided GDFT within the context of colorectal ERPs, and all found no difference in the length of stay or incident complications   to crystalloid in critically ill patients—found no difference in the primary outcome of 28-day mortality . Several surgical maneuvers  as well as by anesthetic interventions , utilization of thoracic epidural analgesia with local anesthetics) may impact measures of “fluid responsiveness.” Understanding the physiologic implications of these maneuvers can help clinicians contextualize changes in device-based measures and avoid the use of fluid therapy based only on the presence of “fluid responsiveness” without attendant absolute hypovolemia.While Trendelenberg (“head down”) positioning has been used for over a century in an effort to improve hemodynamics by augmenting venous return, its intraoperative use is primarily for better visualization of the operative site. Immediately after placing a patient in the head down position, there is a transient increase in right ventricular preload and stroke volume from increased venous blood flow (from the lower extremities and unstressed compartments). This subsequently leads to increased left ventricular output and cardiac output measurements taken 3–5 min after initiating the head down position show an increase in cardiac index    respiratory variation as guides to “fluid responsiveness.”Intraoperative mechanical ventilation may now incorporate PEEP  most commonly used for open abdominal surgical procedures. TEA with local anesthetics can leads to hypotension from reduction in venous return (sympathectomy with venodilation) and decreases in cardiac index  and calories (through fluids or solid food)). In such fasting patients, replacement fluids must be provided intravenously. With ERPs a change in philosophy has occurred with surgeons often allowing oral intake immediately after surgery—provided there is no active nausea/vomiting. Given unrestricted access to oral fluids, patients can regulate their intake to preserve intravascular volume (as long as their thirst mechanisms are intact). Isolating the impact of this paradigm shift toward early postoperative oral intake is a challenge primarily because of the heterogeneity among various published colorectal ERPs. For instance, in a recent meta-analysis examining the characteristics of 13 colorectal ERPs, 9 of 13 centers allowed MBP, and only 8 of 13 centers protocolized perioperative fluid administration  after abdominal surgery is directly related to both length of stay and the incidence of complications ?Is there a clinical outcome difference between simple versus complex carbohydrate loading?https://clinicaltrials.gov/ct2/show/NCT01424150), the focus of which is liberal versus restrictive fluid administration but which plans to examine the effect of GDFT using a statistical test of interaction. However, this study does not specifically focus on GDFT in the context of ERPs.Is a protocolized “restrictive” or “zero balance” technique equivalent to GDFT? This may be answered to some extent by the ongoing RELIEF trial (What risk stratification tool(s) best predict outcomes in patients undergoing colorectal surgery and what are the clinical and financial implications of using risk stratification to influence monitoring decisions and hemodynamic management in patients undergoing colorectal surgery?Do colloids offer any benefits over crystalloid for intraoperative GDFT in non-septic patients undergoing colorectal surgery?Are potential benefits of chloride-restrictive electrolyte solutions  demonstrable in prospective studies and are there differences within available choices of such solutions ?Iso-osmolar bowel preparation is unlikely to lead to preoperative hypovolemia requiring intravenous fluid therapy provided patients are given unrestricted access to clear fluids orally. In patients that present to the operating room in a hypovolemic state, rapid detection is feasible by dynamic indicators of fluid responsiveness such as arterial (or plethysmographic) respiratory variation. Inclusion of carbohydrates in preoperative oral fluids is likely to improve insulin sensitivity (particular when complex carbohydrates are used) and may reduce protein catabolism. Anuria is abnormal and requires immediate attention. In general, oliguria is common during and after anesthesia and surgery and should trigger diagnostic efforts but not fluid therapy until hypovolemia is established as the cause. Intraoperative fluid therapy should be based on a framework where all available information is integrated to determine if there is a physiologic problem requiring reversal. Low tidal volumes and PEEP alter the sensitivity and specificity of dynamic indicators of fluid responsiveness but have a minimal impact on cardiac index at levels commonly utilized in the operating room. To the extent possible, the approach to intraoperative fluid management should continue postoperatively.


Allrecipes.com are compared with those of 100 main meal recipes from five bestselling cookery books from popular celebrity chefs and 100 ready meals from the three leading UK supermarkets. The comparison is made using nutritional guidelines published by the World Health Organization and the UK Food Standards Agency. The main conclusions drawn from our analyses are that Internet recipes sourced from Allrecipes.com are less healthy than TV chef recipes and ready meals from leading UK supermarkets. Only 6 out of 5,237 Internet recipes fully complied with the WHO recommendations. Internet recipes were more likely to meet the WHO guidelines for protein than other classes of meal (10.88 v 7% (TV), p < 0.01; 10.86 v 9% (ready), p < 0.01). However, the Internet recipes were less likely to meet the criteria for fat (14.28 v 24 (TV) v 37% (ready); p < 0.01), saturated fat (25.05 v 33 (TV) v 34% (ready); p < 0.01), and fiber . More Internet recipes met the criteria for sodium density than ready meals , but fewer than the TV chef meals . For sugar, no differences between Internet recipes and TV chef recipes were observed (81.1 v 81% (TV); p = 0.86), although Internet recipes were less likely to meet the sugar criteria than ready meals (81.1 v 83% (ready); p < 0.01). Repeating the analyses for each year of available data shows that the results are very stable over time.A government’s response to increasing incidence of lifestyle-related illnesses, such as obesity, has been to encourage people to cook for themselves. The healthiness of home cooking will, nevertheless, depend on what people cook and how they cook it. In this article, one common source of cooking inspiration—Internet-sourced recipes—is investigated in depth. The energy and macronutrient content of 5,237 main meal recipes from the food website  Globally, more than two-thirds (68.8%) of adults and almost 3 in 4 men (74%) are considered to be overweight or obese . In 2008One of the major contributing factors to such illnesses is poor dietary habits, in particular diets high in sugar, carbohydrates, and fat and diets low in fiber –6. ConsiHoward et al.  investigAllrecipes.com. The site has a community of 40 million home cooks accessing 3 billion pages annually across 19 sites in 24 countries with recipes available in 13 languages .Rather than sampling from the dataset , we used all of the recipes available, which meet the criteria applied in the previously published analysis . This waTable 2.1The following nutritional information is available in our dataset about each recipe: year of publication; the recommended number of servings; and total energy (kJ), protein (g), carbohydrate (g), sugar (g), sodium (mg), fat (g), saturated fat (g), and fiber (mg) content.viaAllrecipes.com and collected during the main crawl. Allrecipes.com estimates the nutritional content for an uploaded recipe by matching the contained ingredients with those of the ESHA research database  collected have no nutritional information available. These recipes were excluded from our analyses. How these exclusion criteria influenced the number of recipes analyzed is shown in Table 2.2Throughout our analyses, we make use of two internationally recognized standards for measuring the healthiness of meals: the World Health Organization (WHO) guidelines  and the The WHO has defined 15 ranges of macronutrients, which should be considered in a daily meal plan. We follow the approach of Howard et al.  who chosA similar approach is taken to derive an FSA traffic light labeling system score. The FSA score relates only to 4 macronutrients . The scale is green , amber, and red .Following the procedure described in Howard et al.’s paper , for eacχ2 tests, we compared the percentage of energy derived from macronutrients in the meals of different types with the nutrient intake goals for preventing diet-related chronic diseases recommended by WHO  according to a modified version of the 2007 FSA guidance on its recommended labeling scheme . The traAllrecipes.com. This allowed us to determine how stable the results are over time.In addition to the statistical analyses performed by Howard et al., we calculated the same values for recipes published each year on 3Allrecipes.com compared to TV chef recipes and supermarket ready meals. The TV chef recipes contain more energy  v 2,066 kJ (ready)) and more protein (37.46 v 29.50 g (Internet) v 27.85 g (ready)) than both Internet recipes and ready meals . Internet recipes are significantly lower in terms of carbohydrate (35.20 v 49.48 g (TV) v 51.05 g (ready), p < 0.01) and sugar (5.50 v 8.25 g (TV) v 6.80 g (ready), p < 0.01, p = 0.02) content, but significantly higher than TV chef recipes in terms of sodium density . There is no significant difference between the sodium content of Internet recipes and ready meals, which are known for unhealthy high levels of salt  . By inve4.1This study is the first to comprehensively evaluate the nutritional content of Internet-sourced main meals. To our knowledge, it is not only the largest study investigating the healthiness of online sourced recipes but also the first to systematically compare the nutritional properties of Internet recipes with those of other sources of cooking inspiration. Moreover, it is also the first investigation to provide insights on the nutritional content of Internet recipes over a long period of time . Although we examined the food website with the largest traffic volumes on the Internet, it is possible (and perhaps likely) that recipes from different Internet sources may have provided different results.Allrecipes.com collection as a whole. The collection is not subject to the same editorial or space constraints as a book or supermarket shelf, and thus no restrictions are placed on the recipes published on the site. It could be, therefore, that certain classes of recipes  are over or underrepresented in the collection, which would bias the findings. Moreover, it could be that recipes, popular with users of the food portal, have different nutritional properties to unpopular recipes. Further analyses we performed confirm this  has since overtaken the international version in terms of numbers of visits from UK users.A further limitation of the study is that The datasets describing the supermarket ready meals and cookery books were created sometime ago and published in Howard et al.’s work  in 2012.Allrecipes.com data was taken; the TV chef recipes were analyzed using the WinDiets software  sourced od blogs . The dis4.3Allrecipes.com. Schneider et al. also discovered differences in sodium levels across seasons. A further line of research could similarly investigate temporal patterns in the nutritional properties of uploaded recipes.When investigating the nutritional content of recipes from food blogs, Schneider et al.  found thvia online recipe databases  and declare: no support from any companies for the submitted work; no relationships with any companies that might have an interest in the submitted work in the previous three years; and no non-financial interests that may be relevant to the submitted work.All authors have completed the ICMJE uniform disclosure form at