Breast cancers are highly heterogeneous and their metastatic potential and response to therapeutic drugs is difficult to predict. A tool that could accurately gauge tumour invasiveness and drug response would provide a valuable addition to the oncologist’s arsenal. We have developed a 3-dimensional (3D) culture model that recapitulates the stromal environment of breast cancers by generating anisotropic  collagen scaffolds seeded with adipocytes and culturing tumour fragments therein. Analysis of tumour cell invasion in the presence of various therapeutic drugs, by immunofluorescence microscopy coupled with an optical clearing technique, demonstrated the utility of this approach in determining both the rate and capacity of tumour cells to migrate through the stroma while shedding light also on the mode of migration. Furthermore, the response of different murine mammary tumour types to chemotherapeutic drugs could be readily quantified. In addition, we aimed to develop techniques that permit assessment/visualization of this ‘metastatic’ potential and the response of invading cells to a panel of therapeutic drugs.Breast cancer mortality is a consequence of tumour metastasis to a variety of sites including lung, brain and bone. Distinguishing tumours that will metastasize from those that will not is challenging and often results in un-necessary or inappropriate treatment of women with primary breast cancer. As a step towards personalised medicine, it is essential to be able to predict the capacity of a tumour to metastasize and to respond to particular therapeutic regimes. A further confounding factor is the heterogeneous nature of many breast tumours where a subclone of tumour cells may behave differently to the bulk tumour. Thus, we sought to develop an in vitro culture models have been generated for studies of both the normal and malignant breast epithelium, all of which have defined utility13. The majority of these consist of cells traversing an isotropic  lattice. However, directional migration of tumour cells has been shown to be strongly influenced by chemical gradients and/or directional cues provided by the organisational structure of the scaffolding molecules that cells adhere to, known as the extracellular matrix (ECM). For example, the ECM protein collagen is frequently aligned in an anisotropic  manner in breast tumours with poor prognosis18. Therefore it is essential to recapitulate this collagen-rich anisotropic ECM structure in vitro in any study of breast cancer cell migration.A variety of 3D 20. Thus, it is important to incorporate this integral stromal component into any 3D model.Another crucial component of the tumour stroma is the fat pad, which provides an adipocyte-rich environment that the breast tumour cells must traverse/negotiate. Adipocytes are responsive to various hormones and secrete a variety of components including adipokines that influence migration21. However, breast tumours are heterogeneous in nature, and metastases arise from a minor yet critical subclone(s) of tumour cells that evolve within a specific tumour microenvironment. In this study, we sought to develop our model further and to utilise it to investigate the capacity of cells from primary tumours to migrate into a surrounding stroma. This is more relevant for breast tumour growth and metastasis in vivo as the invasive capacity of cells is analysed in the context of intact tumour architecture. Furthermore, this preserves the immediate tumour microenvironment comprising cancer-associated fibroblasts, immune cells, cytokines and ECM.In previous work, we developed 3D anisotropic engineered collagen scaffolds and demonstrated their value as a tool to measure the ability of individual cells from established breast cancer cell lines to invade the scaffoldWnt1 transgenic mouse model where overexpression of the Wnt1 proto-oncogene is driven by the MMTV promoter, resulting in adenocarcinoma development in FVB mice22. The second tumour model utilised was the TUBO cell line, derived from a mammary carcinoma that developed in a Balb/c-Her2/neu transgenic mouse, and injected into a syngeneic mouse mammary gland23. This model was chosen as overexpression of HER2 occurs in approximately 25% of human breast cancers and is related to a poorer prognosis than the more common oestrogen receptor positive disease24. Another advantage is that TUBO tumours allow faster experimental turnaround, as they arise approximately 5 weeks after cell inoculation. Once established, primary tumours were harvested and frozen for subsequent experiments to provide a biobank of near-identical tumour biopsies.Since there are multiple sub-types of breast cancer, and individual breast cancers are highly heterogeneous, we sought to compare the invasive behaviour of tumour cells derived from mouse mammary tumour models where carcinogenesis is initiated by different oncogenes. The first tumour model analysed was the well-established MMTV-in vitro model into a cancer therapeutic testing tool, a selection of available drugs were screened as a proof of principle. For this assessment, we selected three inhibitors of different pathways implicated in a variety of migratory mechanisms and processes. Firstly, we chose the Rho-associated protein kinase (ROCK) inhibitor, Y-27632 (denoted ROCKi hereafter), which affects a wide range of processes including proliferation, apoptosis, cell migration, adhesion, oncogenic transformation and the cytoskeleton26. Secondly, the pan-matrix metalloproteinase (MMP) inhibitor, GM6001, was selected as ECM remodelling and subsequent cancer cell migration is linked to MMP expression28. Lastly, the pan-ErbB inhibitor Canertinib was selected due to the role of the ErbB receptors EGFR29, HER230 and ErbB-431 in breast tumour growth and progression.To further develop our 32 followed by implanting primary mammary tumour fragments and measuring both the ability of tumour cells to invade the scaffold and observing their mode of migration. Our results show that this enhanced 3D model permits the distinct migratory behaviours of cells from different types of tumours to be observed and quantitatively analysed. Furthermore, this approach provides a rapid screen of the response of invading cells to therapeutic drugs. Thus, we can provide an in vitro platform for drug screening that will be useful in identifying efficacy and toxicity and may be utilised for personalised breast cancer medicine.Here we report significant enhancements to our original model by introducing pre-adipocytes into the scaffolds and differentiating these into lipid-filled adipocytes21. A synthetic fat pad was generated within the scaffolds by seeding preadipocytes (3T3-L1 cells), culturing for 7 days to fill the scaffold and then switching to differentiation media to induce these cells to differentiate into mature adipocytes32. The work flow to generate this model, that we name Engineered Tumour-Stroma Interaction Model (ET-SIM), is illustrated in Fig. To recapitulate the anisotropic collagen matrix surrounding mammary tumours, we synthesised 3D anisotropic collagen scaffolds using our previously described freeze drying protocol34. As shown in Fig. Previously, it has been reported that 3T3-L1 cells express the basement membrane proteins collagen IV and laminin upon adipogenesis36  and luminal  cells were seen invading collectively into the surrounding stroma  at both the free edge of the tumour as well as the edge in contact with the scaffold indicating that the tumour cells remain viable in ET-SIM cultures Fig. . The sca36 Fig. . In contoma Fig. . These toma Fig. . The resIHC observation of longitudinal sections of TUBO tumour fragment in ET-SIM cultures Fig. . This deWnt1 tumour fragments, the majority of migratory TUBO tumour cells migrated as small clusters (<10 cell)  Fig.  or rarerll) Fig. . Migratoll) Fig. . Thus thWnt1 tumour fragments were cultured with and without DMSO  or empty scaffolds to elucidate both therapeutic efficacy and adipocyte influence on migration. Migration distance was quantified through analysis of IHC sections of fixed samples using a modification of our previously published method21.Following on from this validation of ET-SIM as a method to distinguish different modes of tumour cell migration, we proceeded to test ET-SIM as a superior cancer therapeutic testing platform. MMTV-After 72 hours, treatment of tumour fragments in empty scaffolds with Canertinib resulted in a reduced migration distance, when compared to vehicle (DMSO) controls Fig. . This coIn addition to the migration distance of each cell, we measured the total number of cells that had migrated from the tumour fragment into the scaffold. After 72 hours, the total number of migratory cells was significantly decreased by treatment with Canertinib, when tumour fragments were seeded in either empty scaffolds Fig.  or ET-SIEach treatment condition was also compared individually for tumour fragments seeded in empty scaffolds versus ET-SIM cultures, at the 72 hour time point, to investigate the influence of adipocytes. During vehicle (DMSO) treatment, cells in ET-SIM cultures exhibited decreased migration distance and a diminished number of migratory cells compared to empty scaffolds . The MMP inhibitor, GM6001, showed anti-migratory effects in both empty scaffold and ET-SIM cultures, supporting the concept that MMP induced matrix remodelling assists tumour cell migration28 Fig. . ROCKi e28 Fig. . Indeed,28 Fig. . This suLastly, the total number of cancer cells that migrated at 10 days was analysed. Although not significant, these data suggest ROCKi treatment increases the total number of migratory cancer cells independent of adipocyte status when compared to DMSO vehicle controls Fig. . This is37, which increase tissue transparency by matching refractive indices, permit 3D immunolocalisation of protein and allows deeper laser penetration and z-sectioning by confocal microscopy of whole mounts. Our lab has shown TUBO tumours can be optically cleared and imaged using this approach38. Therefore we adapted this method to analyse scaffolds, tumours and their combination and were able to image these with stereoscopic imaging 14. Invasive MMTV-Wnt1 tumours remodel surrounding stromal ECM into a TACS-3 phenotype where collagen fibres run perpendicular to the tumour boundary creating localised anisotropy that provides a highway for metastatic cells. Importantly, a similar TACS-3 phenotype has been reported in human breast tumours that are associated with poor patient prognosis15. Our system mimics this in vivo microenvironment by seeding mammary tumour fragments into anisotropic collagen scaffolds. While other studies have reproduced collagen anisotropy in vitro using biaxial collagen gels43, our method uses a freeze drying approach21 to create collagen pores aligned perpendicular to the surface of a nucleation point in all directions. As the tumour fragments are seeded at the nucleation point, the scaffold mimics an anisotropic ECM at the periphery of the tumour in all directions. Additionally, these collagen scaffolds are not subject to the shrinkage issues associated with hydrogels mediated by cellular compaction44.Previous reports have shown breast tumours can be grouped according to three different tumour-associated collagen signatures (TACS)in vitro45. Interestingly, at 72 hours adipocytes had an anti-migratory effect in some conditions suggesting that adipocytes may secrete factors at earlier time points that inhibit specific drug activities. Indeed, consistent with this notion, exposing breast cancer cells to adipocyte conditioned medium has been shown to reduce the therapeutic efficacy of trastuzumab46. Collectively these observations highlight the need to incorporate adipocytes into assays of drug efficacy.To study adipocyte influences on tumour cell migration, we co-cultured mammary tumour fragments arising from either Wnt1 or Her2 overexpressing cells within ET-SIM cultures. In all culture conditions it was clear that the presence of adipocytes increased the migration distance of tumour cells after 10 days of culture. Adipocytes have been implicated in breast cancer progression through paracrine and endocrine signalling and can increase breast cancer cell line invasion Despite adipocytes positively influencing the distance that Wnt1 tumour cells migrate after 10 days in culture, adipocytes showed a tendency to reduce the overall number of migratory cells suggesting that they act as a physical selective barrier or filter – impeding some cells while facilitating penetration of more aggressive cancer cells. Importantly, ET-SIM thus reveals heterogeneous cellular behaviour and is therefore ideally suited to identify whether drugs target cellular subpopulations responsible for metastatic spread rather than generically de-bulking a tumour without effectively eliminating it.42 while others show that ROCK inhibition increases invasive potential49, suggesting that ROCKi exerts cell type specific and context dependent effects. Of note, ROCKi has been shown to promote cell survival and colony forming efficiency when used in organoid cultures of human breast, mouse mammary gland and MMTV-Wnt1 tumours53. Enhanced cell survival would explain the increased number of migratory cells but not necessarily the increased migration distance observed in ROCKi treated samples. Collectively, these data indicate that caution should be used when considering ROCK inhibitors as therapeutic agents. Migration distance was decreased although not entirely prevented, by the MMP inhibitor GM6001 suggesting that migration in our ET-SIM system is, at least partly, reliant on ECM remodelling and MMP function. Canertinib however, completely abrogated both TUBO and Wnt1 tumour cell migration in terms of both distance and number of cells after 10 days of treatment showing it to be the most effective therapy for these tumours.While examining the specific effects of three different inhibitors on Wnt1 tumour fragments we observed a striking increase in Wnt1 tumour cell migration during ROCKi treatment. Previous literature is divided on the effects of ROCKi with some studies showing that ROCK inhibition supresses or inhibits cancer cell migration in pre-aligned collagen gelsA limitation of our 3D cultures is the possibility that both inhibitory and stimulatory factors did not reach the core of the tumour fragment, due to the inefficient diffusion of nutrients in our static system. Tumour cells touching the scaffold may also be subject to diffusion issues, as they must obtain nutrients through the full scaffold thickness. Higher levels of cleaved caspase 3, a marker of apoptosis and some other forms of programmed cell death, were observed in this population, as compared to tumour cells at the free edge, and may have been a consequence of restricted diffusion. Nutrient restriction could also induce autophagy, although we have not investigated this directly. It is worth noting that the anisotropic structure of the scaffold should permit increased diffusion to the nucleation point. This is where the tumour fragments were seeded and where tumour cells touching the scaffold were located. Directional pores orientated from the edge of the scaffold - a nutrient rich area - may permit diffusion of essential nutrients to this location. Further investigation is required into the diffusion characteristics of the scaffold itself.Optical clearing techniques have vastly enhanced the depth of imaging achievable with confocal and 2pf microscopy. We have demonstrated that anisotropic collagen scaffolds, ET-SIM cultures, tumour fragments and their combination can be cleared using CUBIC successfully to enable fluorescent stereoscopic imaging and analysis of tumour cell migration. Additionally, CUBIC enabled deep (1000 µm) z-sectioning using immunofluorescence coupled with confocal microscopy, which facilitated a more detailed investigation into migratory cell phenotype and location. This simple technique provided novel insights into different forms of tumour cell migration. Furthermore, it allowed quantitative analysis of the diverse effects of pathway-specific inhibitory drugs and has significant potential as a tool for rapid ‘first pass’ investigation of drug efficacy.In summary, we have pioneered a new enhanced organotypic mammary tumour culture system. We demonstrate that this system provides both qualitative and quantitative insight into mammary tumour cell migration, the complex bivalent effects of adipocytes on this process and the effects of anti-metastatic drugs. We envisage that humanisation of this model will pave the way for improved personalised medicine strategies for patients with breast cancer. Patient derived tumour biopsy fragments containing both stromal and tumours cells, derived from different geographical regions and cultured in our ET-SIM system, would provide oncologists with another tool to use in concert with standard strategies to provide personalised medicine for breast cancers.3 TUBO cells into the abdominal mammary gland of BALB/c females. This cloned cell line was established from a mammary carcinoma that spontaneously arose in a BALB-neuT mouse and carries the Her-2/neu oncogene driven by the MMTV promoter54. Tumours were harvested before exceeding humane end points (4–5 weeks) and frozen for future use. MMTV-Wnt1 mice were crossed onto an FVB background for quicker tumour onset. Tumours were harvested at humane end points (2-12 months).Syngeneic TUBO mammary tumours were established by implanting 5 × 103 pieces for tumour fragment seeding.Tumours were placed in cryovials, covered with freezing medium , 40% FBS , 6% DMSO), placed immediately into a freezing container (ThermoFisher) followed by −80 °C overnight incubation. Cryovials were then stored in liquid nitrogen until required. For resuscitation cryovials were from hand warmed. Once thawing was first observed, room temperature medium was added. Tumours were decanted into tubes containing fresh tumour medium , 5 μg/ml Insulin , 10 ng/ml Epidermal Growth Factor ) making sure not to carry across any freezing medium. Decanting was repeated to wash away any remaining freezing medium. Tumours were mechanically fragmented using a scalpel into 1–3 mm21. Briefly, collagen from bovine Achilles tendon  was added to 0.05 M acetic acid overnight, homogenised and aspirated into engineered molds containing a copper pin. Using a freeze drier ice was then sublimated and the scaffolds cross-linked with 70% ethanol +33 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride +6 mM N-hydroxysuccinimide and sterilized in 70% ethanol overnight. Scaffolds were washed in phosphate buffered saline (PBS) and incubated in maintenance medium (MM) , 10% heat inactivated foetal bovine serum (FBS)  and 1x penicillin/streptomycin ) overnight to assure sterility.Anisotropic collagen scaffolds were synthesized using our freeze drying method32. Briefly, 1 × 106 3T3-L1 cells were seeded into scaffolds , left to proliferate for 7 days and differentiated using an adipogenic cocktail  and 3-isobutyl-1-methylxanthine ) for 7 days, changing media every other day. At this point scaffolds were moved into 6 well plates ready for tumour fragment seeding. All cell cultures were in 5% CO2 at 37 °C in a humid incubator.3T3-L1 cells were cultured in MM between passages 4–20. 3T3-L1 cells were seeded and differentiated in anisotropic collagen scaffolds using a modified previously published methodMechanically fragmented tumour pieces were seeded using sterile forceps into anisotropic collagen scaffold nucleation points with/without previously differentiated 3T3-L1 cells in 6 well plates. 1 ml of tumour media was carefully added so as not to dislodge the tumour fragment and left for 4 hours to attach. Following this an additional 4 ml of tumour media was added covering both the scaffold and tumour. A fresh 5 ml of tumour media was replaced each day and scaffolds were removed at different time points, fixed in 4% paraformaldehyde (PFA) and either paraffin embedded for immunohistochemistry or the CUBIC whole mount immunostaining protocol (see methods) was applied.After 24 hours of tumour fragment culture as mentioned above the media was replaced with 5 ml of tumour media supplemented with either 35.2 µM DMSO , 10 µM Y-27632 (ROCKi) , 10 µM GM6001  or 10 µM Canertinib . Media was replaced with fresh 5 ml tumour media plus therapeutic every 24 hours until fixation endpoint in 4% PFA.Paraffin embedded sections were deparaffinised, rehydrated in an ethanol series, permeabilised in 0.5% Triton-X/PBS and boiled in 10 mM sodium citrate for 11 mins in a pressure cooker for antigen retrieval. Samples were blocked in blocking buffer  in PBS-0.05% Triton-X . Primary antibodies were diluted in blocking buffer as follows: rabbit anti-β-catenin 1:1000 , mouse anti-αSMA 1:100 , mouse anti-E-cadherin 1:500 , mouse anti-Cytokeratin-18 pre-diluted (Progen Biotech 65028), rabbit anti-vimentin 1:100 , rabbit anti-Cytokeratin-14 , mouse anti-p63 1:100 , rabbit anti-cleaved caspase-3 1:800  and the nuclear dye Hoechst 33342 at 5 µg/ml . The following secondary antibodies were all purchased from life technologies and were all diluted in blocking buffer 1:500: AlexaFluor 488 goat anti-rabbit (A11008), AlexaFluor 647 goat anti-rabbit (A21245), AlexaFluor 488 goat anti-mouse (A11001), and AlexaFluor 647 goat anti-mouse (A21237).et al.37, that utilizes the CUBIC R1a method  as previously described by Lloyd-Lewis et al.38. Briefly, fixed samples were placed in R1a for 3 days in a dry incubator at 37 °C, changing to fresh R1a each day. Samples were blocked overnight in blocking buffer . Primary antibodies were diluted in blocking buffer for 4 days at 4 °C in the following concentrations: rabbit anti-perilipin 1:50 , rabbit anti-collagen IV 1:100 , rabbit anti-laminin 1:100 , rabbit anti-HER2 1:100  and the nuclear dye SYTO16 at 1 µM . Secondary antibody Alexa Fluor 647 goat anti-rabbit  was diluted 1:500 and incubated for 2 days at 4 °C.CUBIC clearing was based on the modified version of the protocol by Susaki 2pf and SHG was carried out on the LaVision BioTec TriM Scope II upright 2-photon scanning fluorescence microscope using a 25x water dipping objective. Filters used were: 645–695 nm , 495–560 nm  and 395–445 nm . A fixed 1040 nm laser excited Alexa Fluor 647 and a tuneable laser at 836 nm for SYTO16 and SHG excitation.55.Confocal imaging was carried out on the Leica TCS SP8 inverted confocal microscope and analysed on Fiji55.Standard fluorescence microscopy and tile scans were carried out on the Zeiss Imager H&E and fluorescence microscope, converted into TIFFs and analysed on Fiji21. Briefly, using tile scans generated from IHC, cell nuclei of migratory cells were marked using Fiji and their coordinates measured. The scaffold nucleation point was segmented and saved as coordinates. The closest distance from the nucleation point for each migratory nucleus was measured using formulae in Microsoft Excel and these results were then combined in GraphPad. Cancer cell nuclei were distinguished from stromal cells using the markers αSMA and β-catenin and were defined as migratory when found in or on any part of the scaffold away from the tumour fragment.Cancer cell migration was analysed using a modified version of our previously published methodMigration distances with multiple samples Figs  and 7a,bWork was carried out under UK Home Office Licence authority following ethical review of protocols by the local animal ethics committee of the University of Cambridge. All animal experimentation was carried out in accordance with the Animal (Scientific Procedures) Act 1986, and the European Union Directive 86/609. Mice were humanely sacrificed by dislocation of the neck.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Figures 1-6


Closed-loop resuscitation can improve personalization of care, decrease workload and bring expert knowledge in isolated areas. We have developed a new device to control the administration of fluid or simultaneous co-administration of fluid and norepinephrine using arterial pressure.We evaluated the performance of our prototype in a rodent model of haemorrhagic shock. After haemorrhagic shock, rats were randomized to five experimental groups: three were resuscitated with fluid and two with co-administration of fluid and norepinephrine. Among groups resuscitated with fluid, one was resuscitated by a physician and two were resuscitated according to two different closed-loop algorithms. Among groups resuscitated with fluid and norepinephrine, one was resuscitated by a physician and the other one by the closed-loop device. The precision of arterial pressure during the resuscitation period was assessed using rising time, time passed in the target area and performance error calculations.Groups resuscitated with fluid had similar performances and passed as much time in the target area of 80–90 mmHg as the manual group . Rats resuscitated with fluid and norepinephrine using closed-loop passed similar time in target area than manual group [closed-loop: 74.4% (58.4–84.5) vs. manual: 60.1% (46.1–72.4)] but had shorter rising time to reach target area [160 s (106–187) vs. 434 s (254–1081)] than those resuscitated by a physician. Rats resuscitated with co-administration of fluid and norepinephrine required less fluid and had less hemodilution than rats resuscitated with fluid alone. Lactate decrease was similar between groups resuscitated with fluid alone and fluid with norepinephrine.This study assessed extensively the performances of several algorithms for closed-loop resuscitation of haemorrhagic shock with fluid alone and with co-administration of fluid and norepinephrine. The performance of the closed-loop algorithms tested was similar to physician-guided treatment with considerable saving of work for the caregiver. Arterial pressure closed-loop guided algorithms can be extended to combined administration of fluid and norepinephrine.The online version of this article (10.1186/s13613-018-0436-0) contains supplementary material, which is available to authorized users. Haemorrhagic shock treatment requires immediate and appropriate resuscitation to preserve tissue perfusion while waiting for bleeding control. Fluid administration remains the cornerstone of the initial resuscitation of haemorrhagic shock. However, whereas fluid resuscitation is effective to restore arterial blood pressure, it may induce dilution of coagulation factors that subsequently increases blood loss . MoreoveTo fix these issues, closed-loop (CL) resuscitation systems using hemodynamic variables have been proposed to optimize and standardize fluid administration in burn and trauma patients –7. PreviBecause fluid therapy alone might not be sufficient to improve hemodynamic variables and excessive fluid might worsen traumatic coagulopathy, European guidelines also recommend the administration of vasopressors in case of life threatening hypotension . In clinThe first objective of this study was to compare the performances of CL resuscitation algorithms with physician manual resuscitation during early fluid resuscitation of haemorrhagic shock. The second objective was to compare the performances of CL resuscitation versus physician manual resuscitation with co-administration of fluid and norepinephrine.We focused the present study on rat model of controlled haemorrhagic shock. Rats are particularly adapted for exploratory studies . Rats have similar arterial pressure to humans and are frequently used for studies on arterial pressure-guided resuscitation . Rat mod−1 ketamine , 5 mg kg−1 xylazine hydrochloride  and 50 µg kg−1 buprenorphine . Anaesthesia was maintained throughout the experiment with additional injections of ketamine  using a peritoneal catheter. Anaesthesia efficacy was tested every 10 min using the pedal withdrawal reflex by pinching the foot pad. If the rat withdrew its leg in response to foot pad’s stimulation, then a complementary dose of anaesthesia was administered. After anaesthesia, rats were placed in the supine position on a heating blanket warmed at 38 °C. A tracheostomy was performed, and the rats breathed room air throughout the rest of the experiment. The right carotid artery was cannulated with a polyethylene catheter (PE ED 1.16 mm) which was used for exsanguination. The right femoral artery was cannulated (PE ED 0.69 mm), and this catheter was connected to a pressure transducer . The right jugular vein was cannulated (PE ED 0.92 mm) to administer fluid during resuscitation. Catheters were filled with NaCl 0.9%. The pressure transducer was linked to a data acquisition system . An additional pressure transducer  was necessary in the CL groups. It was connected to the carotid artery catheter and linked to the CL device.Animals were male Wistar rats aged between 12 and 16 weeks. Rats had free access to water and feeding until they were anesthetized. Inhalation anaesthesia was performed in an induction chamber using a concentration of 2–4% of isoflurane. Anaesthesia was relayed by an intraperitoneal injection of 90 mg kgHaemorrhagic shock was induced using a Wiggers’ model with fixed pressure . Rapid e−1 min−1). Infusion was started and stopped by an anaesthesiologist intensivist to target a SAP of 85 mmHg (M-F group). Two groups received automated treatment using CL based on different algorithms. The first one used a proportional–integral (PI) controller (CL-PI group) using continuous SAP acquisition to adapt infusion rate, while the second one used a fuzzy logic (FL) controller (CL-FL group) using mean SBP calculated every minute to adapt infusion rate . To achieve a progressive increase in fluid and norepinephrine, we used a protocol with alternating increase in fluid and norepinephrine. Three boluses of 10 mL kgThe algorithm used in the group treated with automated CL combining fluid and norepinephrine (CL-FNE group) used a combination of PI and FL. The CL-FNE combined a PI regulator for fluid and a FL regulator for NE. Several conditional rules were included to mimic the physician decisions. A schematic of the system set-up is presented in Fig. Arterial pressure was extracted every 200 ms. Analysis of performance during resuscitation was divided into two phases: the rising phase of arterial pressure until 80 mmHg of SAP and the stabilization phase into the target zone. During the stabilization phase, the performance was evaluated using the following variables as described elsewhere : the perIn all groups, fluid was administered using an infusion pump . Norepinephrine was infused using an appropriate infusion pump for small animals .Blood gas analyses was performed  and arterial lactate  and haematocrit were measured at the end of the haemorrhagic phase and immediately after the end of resuscitation using 100-µL blood gas capillary tubes.The CL system was based on a sbRIO 9626 EOM device  programmed with Labview connected to a WiFi module. The system was connected to a pressure transducer and to infusion pumps ]. Given the fact, on the one hand, that only large effects are sought and, on the other hand, that these experiments are complex, six–eight individuals per group are generally used. However, since the model of haemorrhagic shock is known in the literature to have significant variability, we decided to include 10 rats per group.Normality of distribution of variables was tested by the Shapiro–Wilk test. Since the studied variables were non-normally distributed, all data are presented as median—interquartile range.Among the three groups resuscitated with fluid, the effect of the resuscitation mode on the performance parameters and on the volume of fluid administered during resuscitation was analyzed globally using Kruskal–Wallis test. Comparisons of CL-PI or CL-FL with the M-F group were made using Dunn’s test. Among the two groups resuscitated with combined treatment, the effects of resuscitation mode were analyzed using Mann–Whitney test.In addition, we tested the hypotheses that systemic variables such as blood gas, lactate and haematocrit measured at the end of the exsanguination period and after resuscitation can be different between groups by nonparametric two-way ANOVA for repeated measures .To assess the effect of norepinephrine, we compared the groups with best performances in the presence or absence of norepinephrine by Mann–Whitney tests.http://cran.r-project.org/) using the nparLD package.Statistical analysis was performed with GraphPad Prism 6  and the R software (−1 (23.4–25.8) of blood, without any difference among groups, was withdrawn to achieve a target blood pressure of 30–35 mmHg during shock phase. Blood exsanguination induced a metabolic acidosis with a pH at 7.34 (7.31–7.37) and an increase in lactate concentration that peaked at 5.6 mmol L−1 (4.6–7.1) and a decrease in bicarbonate concentration to a bottom level of 22.3 mmol L−1 (21.2–23.7). Biological results are reported in Table A total of 53 rats were included in the protocol: 13 in the M-F group , 10 in the CL-PI group, 10 in the CL-FL group, 10 in the M-FNE group and 10 in the CL-FNE group. Rats weighted 348 g (318–373 g). Baseline MAP was 89 mmHg (85–99) without any difference among groups. 24.8 mL kgSAP during resuscitation is reported in Fig. −1 (22.2–44.1), 25.5 mL kg−1 (23.9–45.2) and 25.5 mL kg−1 (23.9–45.2) in the M-F group, the CL-PI group and the CL-FL group, respectively. Fluid resuscitation improved pH and decreased lactate and haematocrit in all groups (Table Rats received similar amount of fluid in the three groups: 33.6 mL kgp < 0.001 vs. M-FNE group). Note that the manual resuscitation required 20 (16–31.5) interventions to maintain SAP while CL-FNE performed 55 (30–112) adjustments (p = 0.002).SAP during resuscitation with fluid and norepinephrine is reported in Fig. −1 (13.9–21.6) of fluid and a mean dose of norepinephrine of 0.92 µg kg−1 min−1 (0.31–1.99) while those of CL-FNE group received 17.7 mL kg−1 (10–29.9) and a mean dose of norepinephrine of 0.61 µg kg−1 min−1 (0.21–1.39). In both groups, resuscitation improved pH and decreased lactate . This was associated to a lower dilution as attested by a more elevated haematocrit in the CL-FNE group than in the CL-PI group . However, pH improvement or lactate decrease was comparable to those observed in fluid resuscitation groups . FL is more adapted to correct coefficients when the response of the system is not known especially with discontinuous measurement of arterial pressure. In our algorithm, arterial pressure was acquired every minute in the CL-FL group. That is probably why the CL-FL appeared to be the worse. Performances of FL are appropriate when considering non-invasive blood pressure measurement and when the minimal measure of blood pressure is obtained every minute.Whereas norepinephrine has some advantages in haemorrhagic shock, it is never used alone because it might be deleterious , 21. NorCombined CL treatment with fluid and norepinephrine in the unstable situation of haemorrhagic shock is highly challenging. Here, the major difficulty was to guide fluid administration and NE administration simultaneously based only on SAP. Two parallel CL systems would induce important oscillations of arterial pressure.  There are several technical solutions to solve this problem such as using different targets for the two CL, using different time points to re-evaluate rate modifications or creating fusioned variables of both systems. Here, we have used a new parameter: the ratio of fluid volume/norepinephrine rate. In other words, a fluid volume is associated to a norepinephrine upper limit. We did not simply add two CL systems, but we integrated regulation to allow each treatment to influence the other as it is the case in clinical practice. We have used a combination of PI and fuzzy logic in this CL device. CL using fluid/norepinephrine ratio and conditional rules suppressed the highly oscillating response observed in preliminary experiments using parallel CL.Note that with CL-FNE we observed a small period of overshoot (4.9% of time > 90 mmHg) but that was limited at the initiation of the resuscitation and disappeared later in the maintenance phase. Further developments of the algorithms are likely to fix this issue.European guidelines recommend the early use of vasopressors in case of severity to reduce fluid volume and limit dilution . In addiHere, CL co-administration of fluid and norepinephrine reduced the volume of fluid required for resuscitation. This decrease in fluid reduced haemodilution, while pH improvement or lactate decrease was similar among groups resuscitated with fluid or a combination of fluid and norepinephrine. However, the optimal ratio between dose of vasopressor and volume of fluid that could improve hemodynamic with a dilution effect as low as possible, is not known.Performance of the resuscitation highly depends on the shock model. Here, we used pressure-controlled haemorrhagic shock severe enough to avoid spontaneous improvement in arterial pressure. Our algorithm used continuous arterial pressure measurement, and the performance using discontinuous measurements needs to be evaluated. We used a rodent model which has a different blood volume from the one of humans, and our results need to be confirmed in large animal model.This study explored extensively the performances of several algorithms for CL resuscitation of haemorrhagic shock with fluid alone and with co-administration of fluid and norepinephrine. The performance of the CL algorithms tested was similar to optimized manual treatment by physician with considerable saving of work for the caregiver. We also demonstrated that CL algorithms can be extended to co-administration of fluid and norepinephrine in the setting of haemorrhagic shock. Closed-loop resuscitation with fluid and norepinephrine can improve personalization of care, reduce the volume of fluid required for resuscitation and limit the dilution of coagulation factors, decrease workload and bring expert knowledge in isolated areas.Additional file 1. Description of closed-loop protocols and evaluation of performance parameters.


Tumour hypoxia has long presented a challenge for cancer therapy: Poor vascularisation in hypoxic regions hinders both the delivery of chemotherapeutic agents and the response to radiotherapy, and hypoxic cancer cells that survive treatment can trigger tumour regrowth after treatment has ended. Tumour-associated macrophages are attractive vehicles for drug delivery because they localise in hypoxic areas of the tumour. In this paper, we derive a mathematical model for the infiltration of an in vitro tumour spheroid by macrophages that have been engineered to release an oncolytic adenovirus under hypoxic conditions. We use this model to predict the efficacy of treatment schedules in which radiotherapy and the engineered macrophages are given in combination. Our work suggests that engineered macrophages should be introduced immediately after radiotherapy for maximum treatment efficacy. Our model provides a framework that may guide future experiments to determine how multiple rounds of radiotherapy and macrophage virotherapy should be coordinated to maximise therapeutic responses. Vehicles for targeted drug delivery are needed and macrophages have been shown to be promising candidates. Tumours release a number of chemoattractants, attracting monocytes that differentiate into tumour-associated macrophages (TAMs) once inside the tumour In addition to prodrugs, macrophages can also be used to deliver an oncolytic adenovirus to the hypoxic tumour cells Our model is specialised to describe a tumour spheroid’s growth and response to treatment. Grown in vitro, tumour spheroids are a useful experimental model system for drug penetration in an avascular tumour G0 cells Due to its widespread use as a frontline treatment for cancer, there are many mathematical models of radiotherapy. Of these, the linear-quadratic model is the most popular The remainder of this paper is organised as follows. In 22.1l, m, and n the volume fractions of the macrophages, tumour cells and extracellular material, respectively. The velocity, stress tensor, and pressure for each phase are denoted by vi, σi, iP for In this section we derive a model for the in vitro growth of an avascular tumour spheroid that is infiltrated by macrophages. We generally follow the approach used in l, m, and n, supplies:l, SmS, and nS represent the net rates of production for each phase. Equations for vl, vm, and vn are derived by applying conservation of momentum to each phase and neglecting inertial effects:Fijj by phase i, noting that Fa which is discussed below. Interfacial effects are modelled by the terms P∇l, P∇m, and P∇n where P is the interfacial pressure: these interfacial contributions arise by taking a continuum limit over discrete cells  of oxygen and the chemoattractant, respectively. As in cS and aS represent the net rates of production of oxygen and chemoattractant.Two additional phases, oxygen 2.2Fij, and the chemotactic force Fa. We begin by specifying functional forms for the net rates of production iS associated with each phase.lS and mS, it is convenient to introduce the “switch” function,α > 0, and B acts as a “threshold” such that β reaches half of its maximum value when ld(c) and md(c) model the rate at which macrophages and tumor cells die due to lack of oxygen:cc and pc are constant oxygen thresholds. Positive constants The system is assumed to be closed so that no mass is supplied to, or removed from, the system. Thus, at all points within the tumour,lS and mS that appear in nS follows naturally from The terms cS in aS in aλ are positive constants. The function aS accounts for chemoattractant production by macrophages and tumour cells under hypoxic conditions. It also accounts for the natural breakdown of chemoattractant at rate aλ.The net rates of production for oxygen and the chemoattractant are specified in a similar fashion. The oxygen consumption rate k in Fa drives macrophages up spatial gradients in the chemoattractant distribution and is assumed to be proportional to the volume fraction of macrophages l and spatial gradient of the chemoattractant concentration so thatχ > 0 governs the strength of chemoattraction.Following σi  instead of .Before specifying the stress tensors l, PmP, and nP. For simplicity, fix lD and mD that characterise the way in which the macrophages and tumour cells differ from inert bags of fluid. In what follows, we will show that lD and mD can be interpreted as diffusion coefficients for the two cell species  wherein the initial radius R(0) is also prescribed. Thus, we haver ≤ R(0) and that the tumour is initially devoid of macrophages and chemoattractant. We assume further that the initial tumour radius R0 is sufficiently small that the tumour is well-oxygenated with no hypoxic regions; oxygen has a spatially-uniform distribution that is consistent with the oxygen tension in the surrounding culture medium where We close the governing equations by prescribing the initial distributions of tumour cells, macrophages, oxygen, and chemoattractant for all 0 ≤ 2.4l, vmv, and nv that appear in the mass balance mσ in m supplies the following expression for mv:lv:P from the expressions for mv and lv provideslv and mv in the mass balance We reduce our model a system of diffusion-advection equations by using the momentum balances in 2.5In order to construct numerical solutions of the governing equations, it is convenient first to introduce a coordinate transformation in vitro tumour spheroids grown in the absence of macrophages and the time and radius at which these spheroids developed a necrotic core. Leek also performed a separate experiment where fluorescently labelled macrophages were co-culatured with tumour spheroids and the spatial distribution of macrophages within the tumour was measured at a fixed time point.The model is comprised of R(t) ≈ 17, which corresponds to approximately 200 μm; this is consistent with the experimental data from t ≤ 500 to reflect the limited lifespan of the spheroids and we refer interested readers to In l∞ increased from t < 50 the macrophages slowly migrate at low levels to the centre of the spheroid in response to the spatial gradient in chemoattractant which is produced by tumour cells under hypoxia. By pm), oxygen thresholds , the diffusion coefficient of oxygen (cD), and the rate of consumption of oxygen by tumour cells  where the position of the outer tumour radius R(t) satisfiesOn the tumour boundary, we define the velocity of tumour cells 4We solved Um, the uninfected tumour cells, caused by the radiotherapy which reduces the tumour size as the surviving cells concentrate into a smaller tumour mass. This reduction in tumour size allows oxygen to better penetrate into the tumour’s centre, and the cell death from radiotherapy causes an increase in chemoattractant. When macrophages are introduced into the culture medium surrounding the tumour at The model solution in For the numerical results presented in t ≤ 500 to reflect the limited lifespan of the tumour spheroid. t ≤ 3000. The tumour’s behaviour at later times is speculative, as the tumour is likely to begin shedding cells for t > 500 and this effect was not included in our model. However, these longer simulations allow for predictions of how treatment will affect a tumour in the long term.Tumour spheroids grown in vitro will begin to shed cells into the surrounding media after a certain period of time; Leek found this time to be approximately 16 days When simulating ϕ, cϕη, r) but no radiotherapy parameters. The full analysis is shown in The results of our model presented in 5We have developed a new continuum model that can be used to investigate how a novel, macrophage-based virotherapy acts and how, by suitable scheduling, its combination with radiotherapy can produce synergistic treatment responses. We can identify parameter values for which the model predictions are in good qualitative and quantiative agreement with independent results from in vitro and in vivo experiments Our model suggests that radiotherapy alone is not sufficient to prevent tumour regrowth, which is consistent with the experimental observations in While the long term simulations presented in this paper generate interesting predictions, our model is likely to be most accurate over shorter timescales. For the in vitro tumour spheroids considered in this paper, the tumour would begin to disintegrate by shedding cells into the surrounding media. For an in vivo tumour, the hypoxic tumour cells are likely to stimulate angiogenesis which would provide the tumour with the nutrients to fuel further growth A similar model by Jenner et al. used a system of three ODEs to model the changing levels of free virus particles, uninfected tumour cells, and infected tumour cells The theoretical studies presented in this paper illustrate how mathematical modelling can complement treatment development: The number of possible treatment schedules and combinations is too large to investigate exhaustively with experiments, but the modelling framework presented here can pinpoint promising treatment schedules that warrant further experimental investigation. In addition to the treatment schedules investigated in this paper, the model could be used to predict the effectiveness of additional strategies such as introducing a second dose of modified macrophages at a later time.A key contribution of this paper is illustrating how to decompose those tumour constituents or phases that contribute the most to the overall tumour volume into distinct “subphases” that share the same physical properties by using the theory of mixtures. While in this paper attention focused on the interplay between infected and uninfected tumour cells, natural extensions of the model include competition between native and engineered macrophages. Taken another step further, the model could be extended to include a cancer stem cell phase that is resistant to conventional therapy and/or a phase representing the vascular volume 6In this paper we have derived a tumour modelling framework based on the theory of mixtures. We have used this framework to investigate the effectiveness of radiotherapy and macrophage-delivered virotherapy when administered in combination to an in vitro tumour spheroid. While additional experimental work is needed to accurately parameterise the model, our results suggest that macrophages should be introduced immediately after radiotherapy in order to produce the maximum therapeutic effect. While validating these predictions requires further experimental investigation, our model represents a useful framework for identifying promising new treatment schedules and strategies.


Dexmedetomidine (DEX) has been reported to promote tumour metastases. However the underlying mechanisms remain unclear. The purpose of this study was to investigate whether DEX promotes tumour metastasis by inducing myeloid-derived suppressor cells (MDSC) in the context of surgery.DEX was given to lung cancer patients and its effects on expansion of monocytic MDSC (M-MDSC) were studied in the context of surgery. Spontaneous tumour metastasis was induced in C57BL/6 mice and the effects of DEX on M-MDSC expansion and metastasis formation were assessed.+CD33+HLA-DR–CD14+ M-MDSC in lung cancer patients after thoractomy. DEX-induced M-MDSC, in addition to have immunosuppressive activity, were more efficient in producing VEGF. Expansion of M-MDSC by DEX involved α2-adrenergic receptor. Using an experimental tumour metastasis mouse model, we demonstrated that the numbers of metastases on lung surface and CD11b+Ly6ChighLy6G− M-MDSC during postoperative period were enhanced in DEX-treated mice. Promotion of tumour metastasis by DEX-induced M-MDSC involved VEGF, a key factor for tumour angiogenesis.DEX increased CD11bDEX induces the proliferation of M-MDSC during postoperative period in lung cancer patients and this cell population is qualified with potent proangiogenic ability. Treatment of mice with DEX expands M-MDSC and promotes tumour metastasis through the increasing production of VEGF.The online version of this article (10.1186/s12967-018-1727-9) contains supplementary material, which is available to authorized users. The adice Fig. a, b. Tak+Ly6ChighLy6G− cells isolated from lungs of mice treated with DEX produced higher mRNA level of VEGF as compared with Ctrl and “DEX + YOH”-treated mice , (15.88 ± 3.37) and (8.63 ± 1.58) respectively  , 18, 19.immunity , and thiimmunity , DEX app+CD33+HLA-DR−-expressing MDSC significantly increased in lung cancer patients after thoracotomy and MDSC isolated after surgery from lung cancer patients were more efficient in promoting angiogenesis via VEGF [Another characteristic of tumor metastasis is that the formation of premetastatic niche correlates with immunosuppressive cells accumulation and subsequently formation of new blood vessels . Among tvia VEGF . Thus, DThe immunomodulatory properties of DEX could be beneficial in the context of inflammatory conditions that require sedation, such as sepsis, ischemia–reperfusion injury and ventilator-associated lung injury, among many others , 2. None2-AR, but there has been no report regarding the involvement of α2-AR in the expansion of MDSC. α2-Adrenoceptors are present in T lymphocytes, and agonists to this receptor decrease lymphocyte proliferation and both interferon-γ and IL-4 production [2-AR agonists [Previous studies have shown that a number of immune cells functions have been altered by αoduction . DEX couoduction . Naturalagonists . We may agonists –42. In sA better understanding of immune regulatory functions of DEX, which include the mechanisms by which DEX induces immunosuppressive ingredients and promotes tumour metastasis, has a potential to lead to new avenues for preventive intervention in cancer surgery by reducing their immunosuppressive effects and preventing tumour metastasis. Most nonsteroidal anti-inflammatory drugs (NSAIDs) function as cyclooxygenase-2 (COX-2) inhibitors that inhibit production of prostaglandin E2 (PGE2) . BecauseIn the context of surgery, the systemic administration of DEX effectively induces the proliferation of monocytic MDSC which possess proangiogenic and immunosuppressive activity and may promote tumour metastasis. This suggests that DEX administration may exert a deleterious effect for the prognosis of lung cancer patients via modulation of premetastatic niche. Prospective randomized controlled trials are needed to properly assess the effects of DEX on lung cancer patients and other cancers, and animal studies should further elucidate potential mediating mechanisms to be tested in cancer patients.Additional file 1: Figure S1. Expansion of M-MDSC by dexmedetomidine is inhibited by yohimbine.  CD11b+CD33+HLA-DR− cells (3 × 104 cells/well) isolated from lung cancer patients (n = 6) 24 h after surgery were cocultured with (A) dexmedetomidine (DEX) or (B) dexmedetomidine and yohimbine (DEX + YOH). Twenty-four hours after coculture, floating cells were collected and numerated using an automated cell counter. The percentage of CD11b+CD33+HLA-DR−CD14+ (M-MDSC) was analyzed by flow cytometry and the absolute number of M-MDSC was calculated.Additional file 2: Figure S2. DEX promotes tumour metastases in mice exposed to tumour excision. 1 × 106 LLC cells were injected s.c. into the dorsa of C57BL/6 mice. When tumors were 1500 mm3 in size, the mice were divided into 3 groups and treated with PBS (Ctrl group), dexmedetomidine (DEX group), or dexmedetomidine and yohimbine (DEX + YOH group). Then the mice immediately underwent surgical removal of the tumor. (A) When the mice showed dyspnea, they were euthanized and their lungs were removed. (B) On day 60 after tumor resection, the survived mice from Ctrl and DEX + YOH group were euthanized and their lungs were removed.Additional file 3: Figure S3. Expression of α2-AR on mouse MDSC. LLC cells were injected s.c. into the dorsa of C57BL/6 mice. When tumours were 1500 mm3 in size, the mice were divided into 3 groups and treated with PBS (Ctrl), DEX or DEX and YOH (DEX + YOH). Then the mice immediately underwent surgical removal of the tumor. Expression of α2-AR was analyzed on days 0, 1, 3 and 7  after surgery on the CD11b+Gr-1+ (MDSC), CD11b+Ly6ChighLy6G−cells (M-MDSC) and CD11b+Ly6ClowLy6G+ (G-MDSC) of lungs of mice (three mice per group at each time point) by flow cytometry. Mean fluorescent intensity (MFI) of α2-AR on (A) MDSC, (B) M-MDSC and (C) G-MDSC was assessed. *P < 0.001 as compared with T0 in Ctrl group; #P < 0.001 as compared with T0 in DEX group; †P < 0.001 as compared with T0 in DEX + YOH group.


The human brain is the organ with the highest metabolic activity but it lacks a traditional lymphatic system responsible for clearing waste products. We have demonstrated that the basement membranes of cerebral capillaries and arteries represent the lymphatic pathways of the brain along which intramural periarterial drainage (IPAD) of soluble metabolites occurs. Failure of IPAD could explain the vascular deposition of the amyloid-beta protein as cerebral amyloid angiopathy (CAA), which is a key pathological feature of Alzheimer's disease. The underlying mechanisms of IPAD, including its motive force, have not been clarified, delaying successful therapies for CAA. Although arterial pulsations from the heart were initially considered to be the motive force for IPAD, they are not strong enough for efficient IPAD. This study aims to unravel the driving force for IPAD, by shifting the perspective of a heart-driven clearance of soluble metabolites from the brain to an intrinsic mechanism of cerebral arteries . We test the hypothesis that the cerebrovascular smooth muscle cells, whose cycles of contraction and relaxation generate vasomotion, are the drivers of IPAD. A novel multiscale model of arteries, in which we treat the basement membrane as a fluid-filled poroelastic medium deformed by the contractile cerebrovascular smooth muscle cells, is used to test the hypothesis. The vasomotion-induced intramural flow rates suggest that vasomotion-driven IPAD is the only mechanism postulated to date capable of explaining the available experimental observations. The cerebrovascular smooth muscle cells could represent valuable drug targets for prevention and early interventions in CAA. The brain lacks a conventional lymphatic system, which in the rest of the body is responsible for removing waste products and excess fluid  Cipolla, . DeviatiProposed mechanism: vasomotion-driven IPAD. We propose the hypothesis of vasomotion-driven IPAD (denoted V-IPAD) and explain below the underlying mechanisms for this potential clearance process. The position of the contractile VSMCs within the artery wall appears to be ideal for allowing an immediate effect of their contractions on the conformation of the adjoining BM, which is a specialized sheet of extracellular matrix filled with interstitial fluid ; this model is denoted the V-IPAD model and presented in section 2.1  a porous and elastic solid matrix  and (ii) a fluid component  that occupies the connected pores, an example being a water filled bath sponge. Where the solid matrix deforms in response to an external load it transmits force (in the form of a pressure) to the fluid filling the pores; this subsequently leads to changes in the permeability of the solid matrix to fluid flow as, for example, the pores close up. As it will be shown, the elastic deformations of the poroelastic BM, induced by the contractile VSMCs, are critical in assuring high net fluid flow rates along the IPAD pathways, without the need of any intramural valves; such a behavior could not be obtained in a purely porous, underformable material. Models of poroelasticity have been widely applied to biological materials in applications ranging from fluid movement in bone Cowin, , to tumoThe mathematical details of the V-IPAD model are given in section 2. The simulation results are presented in section 3, followed by their discussion in section 4. We have written the paper such that the sections 3 and 4 can be read prior to section 2. In section 4, we also discuss suitable animal models  mouse models) for testing experimentally the V-IPAD hypothesis. In Supplementary Material In this section, we first describe the governing equations of the V-IPAD model and then explain their physiological significance. The V-IPAD model consists of two coupled models: (i) the BM model for the vasomotion-induced intramural periarterial flows through the deformable poroelastic BM, coupled to (ii) the arterial wall model for the elastic response of a rat MCA. We recall that the latter model is presented in detail in Supplementary Material h, running through the wall of a cylindrical vessel which is itself centered along the z-axis, undergoing axisymmetric deformation , as illustrated in Figure H is the undeformed thickness of the BM and 2h is the deformed thickness of the BM.The aim of this model is to quantify the amount of fluid eliminated from the brain tissue along the intramural vascular BM as a consequence of muscular contractions of cerebral arteries. The intramural vascular BM is modeled as a slowly varying sheet of width 2z, t); it depends on the contractile oscillations of the VSMCs and its value is previously determined from the elastic analysis of an active cerebral artery, rather than just being a prescribed function from the literature  affects the fluid flow through the BM by inducing deformations of its boundaries. Thus, the system depends on time only through the boundary conditions. Accounting for the symmetry of the system, as illustrated in Figure The poroelastic BM is a compressible elastic medium subjected to deformations in response to an external compressive stress and to changes in fluid pressure in the pores of the matrix. Specifically, the external compressive stress, denoted Σ, is a known input function of time and position, i.e., Σ = Σ, where the Cartesian y-coordinate is related to the cylindrical r-coordinated by the relationship rb = rm + 2y, where 2y ≪ rm and 2h ≪ rm; here, the expressions r = rb and r = rm describe the radial position of the outer and inner layer bounding the BM, respectively, as illustrated in Figure In general, solving a three-dimensional poroelastic model results in a highly complex system of equations. However, by exploiting the disparity between the length scales of the contracting arterial wall [BM thickness (0.4 μm) ≪ arterial radius (100 μm) ≪ wavelength of muscular contractions 2000 μm)], we derive a simplified version of the BM model by using a lubrication approximation  through the BM, as an effect of VSMC activity, we first need to solve the system described below in Equations (1–5) for the following five variables: the deformed thickness of the BM h, the volume fraction of fluid ϕf, the fluid velocity in the z-direction p and the principal effective Cauchy stress t is time and z is the position along the z-axis. The full derivation of this lubrication model of the poroelastic BM is given in Aldea (In order to quantitatively determine the fluid flow rate in Aldea . Howeverk is the deformation dependent permeability of the porous medium (details in Equation 6). H denotes the undeformed thickness of the upper half BM and Σ denotes the external constrictive stress; these two terms are the input of the BM model. The function where η is the fluid viscosity and The physiological significance of the BM model is outlined below Aldea, .s (solid) and ϕf (fluid) satisfy ϕs + ϕf = 1.Equations  represent conservation of fluid and solid mass, respectively, in the deformed configuration of the system. Assuming that the BM is comprised only of fluid and solid phases, the volume fractions ϕEquation (3) is the lubrication approximation of Darcy's law which relates the interstitial fluid velocity to the pore pressure gradient, the fluid viscosity and the deformation-dependent permeability. Various functions for deformation-dependent permeability have been discussed in the literature and, here, we choose the model described in Markert  which stk* is the permeability of the undeformed BM, κ is a positive parameter and where Applying an asymptotic analysis , where S acts as a parameter describing the local level of contractile activity of VSMCs. Here, we model the propagation of the muscular contractions of the VSMCs with the following wave form of the vascular toneEquation (4) represents the force-balance equation derived from the conservation of momentum. A close look at the elasticity of an arterial segment shows that Σ is the radial stress at the radial position of the BM  represents the vasomotion wave from the V-IPAD model, having the same pattern as the arterial vasomotion wave observed experimentally . The propagation velocities of the vasomotor response available in the literature describe the induced vasomotor response of arteries, rather than the (spontaneous) vasomotion wave  that describes the elastic behavior of the material under particular deforming conditions. Given the geometrical setup of the BM model, in which compressive forces are only significant in the y-direction, the relationship between the principal stress in the y-direction and the corresponding deformation is given (in terms of a strain energy function) byFinally, Equation (5) represents the constitutive equation, i.e., the stress-strain relationship for the hyperelastic BM. This means that the principal effective Cauchy stress y is the principal stretch ratio in the y-direction and is defined as WBM requires careful attention. Limited by the lack of experimental data on the elasticity of the cerebrovascular BM, a minimum number of material parameters able to describe the physiological response is desired. With this in mind, we choose the neo-Hookean model with only two material parameters from the study of Wirth et al.  is plotted in Figure J*). The Cauchy stress which is plotted in Figure The system of Equations (1–5) is simplified by replacing Equations  in Equation (1), which yields the non-linear equation for the deformed upper-half BMk(h) is given by Equation (6) and recalling that The BM is considered initially undeformed and uniform along the vessel, i.e.,z = 0 represents the proximal end of the BM, while z = Ls represents the distal end of the BM. Subsequently, both the deformation of the BM induced by the external compressive stress and the resulting flow through the BM, are investigated.where The ends of the BM are assumed to be at the same pressure h for a given Σ) and WBM. Once h is determined, all the other variables of the system can be calculated given their dependency on h. Finally, the fluid flow rate through the poroelastic BM is calculated as shown below.Equation (10) plays a paramount role in the dynamics of the system and is solved for S). The total flow rate of fluid through the infinitesimal volume of BM, generated by the vasomotion-induced deformation of the BM boundaries, is calculated by using the differential form of the Darcy's law and recalling that the cross sectional area of the BM is An infinitesimal element of the cerebral artery is considered, as illustrated in Figure QBM[μm3·s−1] is the volumetric flow rate through the entire thickness of the BM and rm[μm] denotes the radial position of the BM. Here, the extra factor of 2 appears because we have only been considering the upper half of the BM in our model from sections 2.1.1–2.1.3. It is emphasized that rm is a function of the wave of vascular tone, i.e., rm = rm), and its form is not prescribed, but is rather determined from the arterial wall model in Supplementary Material where  t)μm3·s− is the vThe net flow rate over one cycle of vasomotion isT represents the time period of the vasomotion wave. The parabolic Equation (10) is solved with the method of lines as described in Supplementary Material where We have considered the cerebral artery to be an idealized vessel  with uniform material properties determined experimentally by Bell et al. . For rear) at the middle of the arterial wall as the external constrictive stress (denoted Σ) acting on the BM, i.e., σr(r = rm) = Σ, where rm is the deformed radius r at the middle of the arterial wall  with units of stress, as shown in Figure S were determined elsewhere  and investigate the particular case in which the wavelength of the vasomotion wave is comparable with the characteristic length of a MCA. Moreover, for reasons of simplicity, we assume a uniform system and one that is sufficiently long in order to encompass several vasomotions waves simultaneously ), and the corresponding radial stress, Σ = Σ), depend on the vascular tone S through the arterial wall model ). The fluid movement through the BM is governed by Darcy's law. The change in the BM volume affects its porosity and, subsequently, its permeability to fluid flow, according to Equations . In this particular scenario, it has been allowed for the fluid volume fraction to drop to zero due to finite compressive forces that reduce the BM pores and, as a consequence, obstruct the path for fluid drainage; this behavior is similar to squeezing shut a fluid-filled sponge.The spatiotemporal variations in cerebral vascular tone influence the fluid flow through the BM by inducing deformations of its top and bottom boundaries via the radial constrictive stress Σ, which is derived using the strain energy function for neo-Hookean poroelastic materials from Wirth et al. . The abih and permeability k, as well as the resulting fluid flow rate QBM through the BM are shown in Figure 3·s−1 are obtained, the net flow rate during one cycle of vasomotion is only 360 μm3·s−1. The positive value indicates that the net intramural fluid flow rate during one cycle of vasomotion is always in the direction of the vasomotion wave . According to the V-IPAD hypothesis, the contractile VSMCs of cerebral arteries act as the drivers of IPAD in the brain by inducing BM deformations and, subsequently, net intramural fluid flows in the direction of the vasomotion wave. To our knowledge, this is the first quantitative study that explores the contribution of the cerebral VSMCs in the drainage of fluid from the brain. Although the exact physiological roles of vasomotion remain elusive, the most common view is that vasomotion serves as an auxiliary mechanism in tissue perfusion, especially under hypoperfusion conditions  and their anatomical position within the deep cortical layers make the investigation of brain clearance challenging to conduct in living humans and even in animal models. Physiologically-based mathematical and computational models represent an alternative tool for analysing potential clearance mechanisms of the brain, likely generating new mechanistic insights  in Arbel-Ornath et al. . Since t3·s−1 generated by the V-IPAD mechanism along only one BM layer is five orders of magnitude higher than the net periarterial flow rate induced by arterial pulsations , induced by the contracting and relaxing VSMCs, is in line with the experimental observations of vasomotion in large cerebral arteries  rat arterial strips elicited vasomotion with a velocity of 100 μm/s  or, equally, 0.04 μl·min−1·Despite the fact that the V-IPAD mechanism promotes significantly higher net intramural periarterial flow rates than the previously suggested mechanisms  of contractile cerebral vascular networks (Nishimura et al., We have shown that the oscillations in the contractile state of the cerebral VSMCs are sufficiently powerful to promote net intramural flow rates of notable magnitude for efficient drainage of fluid along the IPAD routes, but the predominant direction of vasomotion remains elusive. Within the context of IPAD of the brain, the vasomotion wave must propagate from penetrating arterioles toward leptomeningeal arteries on the surface of the cortex. Direct investigations of the cerebral cortex in humans when awake showed distinct regions of pial arterioles exhibiting slow sinusoidal vascular oscillations, with a frequency of ≈ 0.1 Hz, which propagated as spatial waves across the cortex (Rayshubskiy et al., It has recently been shown that the permeability of the brain interstitium is too low to allow development of any substantial bulk flow at physiological intracerebral pressure differences (Jin et al., While direct evidence is still missing, the aforementioned experiments (Beach et al., Various hypotheses have been advanced to explain CAA. It has been proposed that the Aβ deposited in the walls of arteries in CAA was derived from VSMCs Vinters, . HoweverThe impact of this study opens avenues for targeting vasomotion toward the facilitation of IPAD and prevention of CAA. This is key for the successful outcome of current immunization trials against Aβ in which plaques are solubilized, but Aβ becomes entrapped in the IPAD pathways, increasing the severity of CAA and most likely resulting in amyloid-related imaging abnormalities (Holmes et al., Using transgenic models with overproduction of mutant amyloid-beta peptides has many advantages in basic understanding of mechanism of disease (Klohs et al., Drugs such as cholinesterase inhibitors, commonly prescribed in the treatment of Alzheimer's disease, have enhanced high cognitive functions in demented patients who responded to treatment Birks, . Under tIn conclusion, this study has theoretically proven that the vasomotion wave initiated by the contractile VSMCs of cerebral arteries can represent the motive force for IPAD in the brain, shifting the view from a heart-driven clearance of fluid and solutes from the brain to an intrinsic mechanism of cerebral arteries. The V-IPAD model is the first one that quantitatively examines the contribution of the contractile cerebral VSMCs in the clearance of fluid from the brain, proposing a physically plausible driving force for IPAD. The model offers mechanistic insights into the behavior of the cerebrovascular BM and its interaction with the adjacent VSMCs. Once additional experimental data about the active response of cerebral arteries are provided, the V-IPAD model could be extended in order to improve its accuracy and simulate the effect of cerebral vascular aging on the IPAD of the brain. The hypothesis proposed and tested here stimulates further experimental investigation of the contribution of cerebral VSMCs in the clearance of fluid and solutes from the brain, particularly as these cells represent an accessible therapeutic target for CAA.The study was approved by the University of Kentucky Institutional Animal Care and Use Committee and conformed to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.RA, GR, and RC formulated the hypothesis. RA and GR formulated and solved the model. RC and DW designed the experimental pilot study, obtained, and analyzed the experimental data. All authors contributed to the draft of the manuscript, read, and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Varicella is normally a self-limited childhood disease caused by varicella-zoster virus infection. However, it sometimes causes severe diseases, especially in immunocompromised individuals. We report a case of severe varicella in a young woman.A 19-year-old woman presented to the emergency department with abdominal pain and a rash after taking methylprednisolone for 2 weeks for systemic lupus erythematosis. The laboratory data showed leukocytosis, thrombocytopenia, an elevated level of the liver transaminases and disseminated intravascular coagulation. Computed tomography of the abdomen revealed multiple air-fluid levels in the intestines. Hemorrhagic varicella was considered and antiviral therapy as well as immunoglobin were applied. Her condition deteriorated and she eventually died due to multi-organ failure and refractory shock. Next-generation sequencing performed on fluid from an unroofed vesicle confirmed the diagnosis of varicella.In its severe form, VZV infection can be fatal, especially in immunocompromised patients. Hemorrhagic varicella can be misdiagnosed by clinicians because of unfamiliar with the disease, although it is associated with a high mortality rate. In patients with suspected hemorrhagic varicella infection, antiviral therapies along with supportive treatment need to be initiated as soon as possible in order to minimize the case fatality rate. Varicella is normally a self-limited disease caused by varicella-zoster virus(VZV) infection. However, it sometimes causes severe diseases, especially in immunocompromised individuals such as those are taking chemotherapy or steroid therapy, and those who have had a renal transplant or a bone marrow transplant –4. Few iWe describe a case of atypical varicella in a woman who presented with abdominal pain and a rash after being treated with methylprednisolone.A 19-year-old Chinese woman was admitted to the emergency department of our hospital because of a three-day absence of defecation and a one-day history of abdominal pain. Two weeks previously, she had been discharged from the Department of Dermatology with a diagnosis of systemic lupus erythematosus(SLE). She was being treated with methylprednisolone at a dose of 24 mg per day.On examination, there was a diffuse brown pigmentation on the skin, with vesiculopapular rash on the forehead and over the trunk Fig. . There wIn its severe form, VZV infection can be fatal, especially in immunocompromised patients. Visceral disseminated disease is associated with a high mortality rate of 46–55% . In norm9/L [Intense abdominal pain is often an early symptom of dissemination, which reveals that multi-system organs are involved, such as the stomach, intestines and spleen  , 14, 21.9/L . Althoug9/L . In addi9/L .VZV is usually confirmed by polymerase chain reaction (PCR) amplified VZV DNA and enzyme-linked immunosorbent assay (ELISA) detection of anti-VZV antibody . In our The patient with SLE in our case was just discharged from the Department of Dermatology, where she had many opportunities for exposure to patients with herpes zoster or varicella. And the patient had never been vaccinated against varicella before. Vaccination of close contacts should be considered for immunocompetent individuals . However


There was an error in the Funding statement. The correct version is:This study was funded with grant UL-2010-4667 from the Dutch Cancer Society and with an unrestricted grant from Merck, Sharpe & Dohme (MSD). P.G.G., A.G.E. and S.M. are researchers employed by MSD. They have contributed to this study by critical appraisal of the data and the manuscript and by sharing expert opinion. Furthermore, MSD had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.