A profound understanding of how to tailor surface topographies of electrospun fibers is of great importance for surface sensitive applications including optical sensing, catalysis, drug delivery and tissue engineering. Hereby, a novel approach to comprehend the driving forces for fiber surface topography formation is introduced through inclusion of the dynamic solvent-polymer interaction during fiber formation. Thus, the interplay between polymer solubility as well as computed fiber jet surface temperature changes in function of time during solvent evaporation and the resultant phase separation behavior are studied. The correlation of experimental and theoretical results shows that the temperature difference between the polymer solution jet surface temperature and the dew point of the controlled electrospinning environment are the main influencing factors with respect to water condensation and thus phase separation leading to the final fiber surface topography. As polymer matrices with enhanced surface area are particularly appealing for sensing applications, we further functionalized our nanoporous fibrous membranes with a phosphorescent oxygen-sensitive dye. The hybrid membranes possess high brightness, stability in aqueous medium, linear response to oxygen and hence represent a promising scaffold for cell growth, contactless monitoring of oxygen and live fluorescence imaging in 3-D cell models. To alter the surface topography of e-spun fibers, two different principles have emerged: (1) post-processing procedures to add structural components or to selectively remove compounds from the fibers, (2) exploitation of phase separation processes during fiber formation aiming at selective and time dependent drying and crystallization processes. The first approach, controlled polymer crystallization on electrospun fibers either by incubation in polymer solution or by polymer solvent evaporation, creates lamellae on fibers with e.g. shish-kebab structures on fiber surfaces7. Alternatively, solvent extraction10, salt leaching11 or calcination of electrospun fibers made up of two components yields porous surfaces by selective removal of the sacrificial component12. The second approach, structuring by in-situ spinning procedures, has been demonstrated by electrospinning in a humid environment20, into liquid nitrogen21 or into non-solvent baths23 to result in porous fiber structures. Various attempts, mainly being inspired from porous polymer film formation techniques, have been made to explore underlying principles generating such surface structures of electrospun fibers. For instance, Srinivasarao et al.24 explain the ordered and monodispersed sized pores formed on polymer casted films to originate from breath figures. These represent imprints left by water droplets that condense on casted films due to evaporative cooling and have also been proposed to induce same characteristics on electrospun fibers18. On the other hand, the temperature drop caused by evaporative cooling of polymer solutions can result in thermally induced phase separation described as one of the porous membrane preparation techniques25. It is also argued as a possible reason behind the polymer jet in electrospinning separated into polymer rich and polymer lean regions26. Upon complete drying of the fibers, polymer lean regions are then expected to lead to the formation of inner pores or surface topographies. Further, thermodynamic instability can be introduced either by water from the humid environment acting as a non-solvent or by the addition of a non-solvent directly into the polymer solution mixture29. Thus, to create fibers with desired surface topographies, it is of crucial importance to properly choose a specific solvent and solvent mixture for each polymer. However, the most commonly used approach is to empirically select the solvents with defined volatility, water miscibility, dielectric constant and conductivity by trial and error. A more efficient approach is to thermodynamically predict the initial degree of solubility of a polymer in different solvents or solvent mixtures by use of solubility parameters32.The high surface to volume ratio of electrospun fibers and hence their high specific surface area allows their use in a great variety of applications. A further increase of the surface area by incorporating tailored surface topographies is therefore of major interest for many industrial and biomedical fields of applications including tissue engineering, drug delivery, catalysis, filtration and sensors33, Hansen Solubility Parameters are widely used to predict material properties such as the affinity of substances to each other, gelation behavior of organogels34, encapsulation efficiency of drugs35, barrier function of release systems36 as well as good and poor solvents for polymers37.Among the different solubility models38, has been extensively studied in the electrospinning field. Although it has been explored for solution, blend and emulsion electrospinning, in-situ structuring of PCL fibers has only recently been under investigation28. However, elaborated investigations are required to advance the understanding of the pore formation mechanism during fiber formation.Polycaprolactone (PCL), a biodegradable, FDA approved, semi-crystalline polyester which has been shown to be biocompatible for specific applications in the areas of drug delivery, medical sutures, dentistry, wound dressings as well as tissue engineeringHere, we demonstrate how electrospun fiber surface structure formation correlate with theoretically predicted polymer solubility alterations in different solvents during their evaporation, the corresponding solvent evaporation rates, subsequent polymer jet surface temperature changes and thus the inevitable influence on the solution thermodynamic equilibrium. As a model system PCL was used and spun at selected environmental humidity conditions. The extension of our findings to other polymer-solvent systems can establish a universal prediction tool for surface morphology evolution of electrospun fibers as shown for another hydrophobic polymer, poly L-lactic acid (PLLA) as well as for a hydrophilic polymer, polyvinylpyrrolidone (PVP).39. This composite was tested as 3-D scaffold for localized oxygen monitoring in culture of cancer cells.To highlight the application potential of developed porous morphologies in areas such as tissue engineering and biosensing, we have investigated the oxygen sensing ability of nanoporous structures in which a phosphorescent dye, Pt(II)-tetrakis (pentafluorophenyl) porphine (PtTFPP), was embedded in the PCL matrix after fabrication of the fibres−1), Polyvinylpyrrolidone (PVP) , dichloromethane (DCM) and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich (Switzerland), Polylactic acid (PLLA) (Ingeo™ Biopolymer 2500HP) from Natureworks, N,N- Dimethylformamide (DMF) from VWR Chemicals, Ethanol  and o-xylene from Fluka Analytical, chloroform  from Fischer Scientific and methanol  from Fluka. PtTFPP was from Frontier Scientific (USA). Calcein Green AM, cell growth medium, acetone, Na2SO3, KH2PO4, phosphate-buffered saline (PBS) and all the other reagents were from Sigma-Aldrich . All materials were used without any further purification.Polycaprolactone (PCL) . Additionally, electrical conductivity of the solutions was measured using a conductometer . PLLA solutions were prepared at a concentration of 10% (w/v) by using solvent mixtures of CHCl3:MeOH with volume ratios of 18:2 and 17:3 and CHCl3:DMF with volume ratios of 17:3, respectively. PVP solutions were prepared at a concentration of 10% (w/v) by using EtOH and a solvent mixture of EtOH:Xylene with the volume ratio of 14:6. PCL nanofibers were prepared according to the study of Guex et al.PCL solutions were prepared at a concentration of 15% (w/v) by using pure solvents DCM, CHCl40. Flow rate was held at 30 µL/min for all solutions and a potential of +8 kV on the needle and −3 kV on the counter electrode for PCL solutions with a fixed tip-to-collector distance of 25 cm was applied. The electrospinning process parameters were set as +12, −3 KV with 20 cm of distance for PLLA and as +7, −4 with 15 cm of distance for PVP solutions.All solutions were processed into fibers at three different relative humidity (RH) conditions, 12, 35 and 55%, respectively, by a custom-built electrospinning set-up as described previously2 with a surgical blade and the cut pieces were placed on a sample holder with a vertical sample fixation unit. Prior to loading, the samples were sputter coated with gold/palladium of 5 nm thickness to increase the electrical conductivity. The mean fiber diameters were calculated based on measurements of 90–100 fibers from the SEM micrographs using Image J software.Fiber diameters and their distribution as well as the inner and surface morphology of the electrospun fibers were investigated by Scanning Electron Microscopy (SEM)  using 2 kV accelerating voltage and 10 mA current flow. To examine the surface morphology, the sample patches were mounted on metal stubs with double-sided adhesive tape. In order to image the inner fiber morphology, cross sections were obtained by cutting fibers in liquid N41.Wide angle X-ray scattering (WAXS) experiments were performed on a STOE Imaging Plate Diffraction System IPDS II, using Mo Kα (λ = 0.71 Å) radiation in transmission geometry. The sample-to-detector distance was set to 200 mm allowing collecting data up to 2Θ = 38°. The diffraction patterns were acquired by means of an image plate detector system with exposure times of 15 min at room temperature. In order to calculate the crystallinity, the two dimensional scattering patterns were radially integrated into one-dimensional scattering functions, background subtracted, followed by a peak fitting to Pearson7 functions using Fityk 0.9.8 software42.PCL solubility in various solvents and solvent mixtures, relative solvent evaporation rates from these solutions, the consequent change in the Relative Energy Distance (RED) of solvent mixtures and the approximate solvent temperature change due to evaporation were calculated by use of the software Hansen Solubility Parameters in Practice (HSPiP) which has been developed by Steven Abbott, Charles M. Hansen and Hiroshi Yamamoto42. The RER calculations are implemented to electrospinning and fiber morphology correlation with the following assumptions: i) diffusive evaporation43 of the solvent, which is the main mechanism of solvent evaporation, takes place mainly from the straight region of the initiated jet from the tip and ii) the forced convective mass transfer of the solvent that is a result of the whipping motion is neglected to simplify the complex electrospinning process.As an approximation, the model which is introduced within this software accounts for the published Relative Evaporation Rates (RER) of the solvents, which are determined in comparison to the evaporation of standard solvent Butyl acetate (assuming that it evaporates in 100 time units) together with activity coefficients to simulate the evaporation rate of the solventsThe evaporation of solvents from a polymer solution prepared with a solvent mixture can alter the RED depending on the volatility differences of the individual solvents. These changes in RED values were simulated during the evaporation in function of time and the corresponding plots were generated.Twb) and calculated iteratively , washed once and proceeded to live cell imaging. Confocal phosphorescence lifetime imaging microscope (PLIM) was performed on an upright fluorescence microscope Axio Examiner Z1 (Carl Zeiss) with 20x/1.0 W-Plan Apochromat and Fluar 5x/0.25 objectives, heated incubator (37 °C), motorized Z-axis control, DCS-120 confocal TCSPC scanner (Becker & Hickl GmbH), R10467U-49 photon counting detector (Hamamatsu photonics) and dedicated SPCI software (Becker & Hickl GmbH) essentially as described previously44. The O2 calibration was performed using 390 nm LED excitation (650 nm emission) on inverted widefield PLIM microscope (Zeiss-LaVision), equipped with integrated CO2/O2/T control (Pecon)45. To achieve complete deoxygenation in PBS, a solution of 5 mg/ml KH2PO4; 5 mg/ml Na2SO3 was added to the membranes. Experiments were performed in triplicate.Human colon carcinoma HCT116 cells (ATCC) were handled as described previouslyp < 0.05 was considered statistically significant.All data were expressed as mean ± standard deviation of at least three samples. One-way analysis of variance (ANOVA) was performed to determine statistical significance of the data using Origin 9.1. A value of 46. The solubility parameter, δ, is defined as the square root of this cohesive energy, which reflects the degree of attractive forces holding the molecules together. Hansen subdivided this total solubility parameter into three fractions: dispersive interactions (δD), polar interactions (δP) and hydrogen bonding (δH) in units of MPa½. These three parameters can be visualized as coordinates in a 3-dimensional diagram termed 3-D Hansen space  is formulated as equation  of a liquid that is the energy of vaporization per volume unitace Fig.  in the H35. This ratio is called the RED46. A RED value lower than 1.0 indicates a high affinity or solubility (coordinate position within the sphere) whereas a RED value higher than 1.0, lower affinity to the polymer (coordinate position outside the sphere).In 3-D Hansen space, solvents are depicted as single points whereas the polymers are represented as spheres in which solvents with good solving or swelling properties for that polymer are located. The coordinate position of a substance with respect to a polymer solubility sphere is characterized by the ratio of the coordinates’ distance to the center of the sphere and the sphere’s interaction radiusD = 17.7 MPa½, δP = 5.0 MPa½ and δH = 8.4 MPa½) and interaction radius (R = 8) value taken from the HSPiP software42. In order to verify the HSP theory, experimentally known good solvents and non-solvents of PCL were selected47 and the RED values of these common electrospinning solvents such as CHCl3, DCM, DMSO, ethanol (EtOH), acetonitrile and methanol (MeOH) were calculated. Values for CHCl3 and DCM are located inside this sphere with RED values of 0.37 and 0.41, respectively. Values for DMSO, EtOH, Acetonitrile and MeOH were located outside of the PCL sphere due to RED values of 1.45, 1.53, 1.76 and 2.10, respectively  whereas CHCl3 evaporated with a slower rate and within 25 time units (a.u.).Electrospinning is a complex, multiphysics process involving electrohydrodynamics, mass and heat diffusion and transfer, and solidificationTwb)42. Besides, the temperature to which air can be cooled down reaching full saturation is the dew point (Tdp). Based on solution evaporation simulations, Twb can decrease to a theoretical minimum of −13.7 °C in the case of DCM but only to −4.6 °C in the case of CHCl3  > 0. Accordingly, ΔT of the DCM solution increased from 3.4 °C at 12% RH to 17.8 °C at 35% RH and to 24.4 °C at 55% RH  and it increased to 8.7 °C and 15.3 °C at 35% and 55% RH, respectively  rose above zero and the structures evolved with the increase in ΔT. The correlation between the calculated temperature difference and the resulting fiber surface structure formation is shown for the first time to the best of our knowledge. Additionally, ΔT values of DCM solutions were above zero at all RH conditions and this was also reflected as progressing structured fiber surfaces with increasing ΔT values following a RH increase.The fiber surface structure changed from smooth to textured topography in the case of CHCl3:MeOH (18:2) and CHCl3:DMSO (18:2), which have the same ratio of solvent to water miscible non-solvents, were compared. The initial RED values of the solvent mixtures with respect to PCL were calculated as 0.2 and 0.3 for CHCl3:MeOH and CHCl3:DMSO, respectively by use of the weighted volume average of individual solvents. Fibers spun from CHCl3:MeOH (18:2) mixture had 4.7 ± 0.2 µm, 4.6 ± 0.3 µm and 4.9 ± 0.5 µm of average fiber diameter at increasing RH conditions. On the other hand, fibers spun from CHCl3:DMSO (18:2) mixture had lower average fiber diameters of 2.5 ± 0.5 µm, 3.0 ± 0.4 µm and 3.4 ± 0.2 µm at increasing RH conditions  mixture or a CHCl3:DMSO (18:2) mixture was spun at 12% RH  mixture started to have surface limited pits whereas the ones electrospun from CHCl3:DMSO (18:2) mixture were obtained with irregular and more inter-connected surface topographies  mixture (26.7 a.u.) than from CHCl3:DMSO (18:2) mixture (33.3 a.u.)  Fig. . This leu.) Fig.  evaporatTwb of the CHCl3:MeOH (18:2) mixture was lower (−4.1 °C) compared to the CHCl3:DMSO (18:2) mixture (−3.5 °C), which was in agreement with the fact that the higher the volatility of a solvent, the lower the resulting Twb. The solvent evaporation resulted in increasing Twb of CHCl3:DMSO (18:2) while only a slight increase was obtained for CHCl3:MeOH (18:2) mixture. Additionally, since the Tdp of air at 12% RH is −10.3 °C, ΔT(Tdp − Twb) of both solutions were below zero from the beginning. On the other hand, Twb of CHCl3:MeOH (18:2) mixture was below the Tdp both at 35% RH (4.1 °C) and 55% RH (10.7 °C) which yielded positive values of ΔT and thus water condensation can be expected. In the case of CHCl3:DMSO (18:2) mixture however, Twb was rising above the Tdp both at 35% and 55% RH conditions after 26.7 (a.u.)  Fig. , which r3:MeOH (18:2) mixture was never above 1 which was defined as the solubility limit of a polymer in a given solvent. Therefore a RED value exceeding 1 causes a subsequent phase separation of the polymer solutions, which is composed of solvent:non-solvent mixtures, into polymer rich and polymer lean phases. On the other hand, CHCl3:DMSO (18:2) mixture RED value started at a higher value than the one of CHCl3:MeOH (18:2) and it raised up over 1.Solvent evaporation from a solution prepared in a mixture of solvent and non-solvent alters the solubility parameters of the mixture and thus, its compatibility with the polymer changes. This change can be examined in terms of RED change. The simulations revealed that RED value of CHCl48, as evidenced by the scattering corresponding to the crystalline orthorhombic phase50 and by the additional broad peak corresponding to the amorphous phase. The polymer crystallinity was found to be similar for all of the investigated samples revealing no significant influence of the used solvent systems and the applied RH and the respective surface structuring processes with respect to the ratio of amorphous to crystalline phases   and CHCl3:DMSO (16:4)  mixtures again at three different RH conditions. Varying the DMSO amount in the solution mixture altered the average fiber diameter  and CHCl3:DMSO (18:2) mixtures. At low RH, both PCL solutions prepared in CHCl3:DMSO (19:1) and CHCl3:DMSO (16:4) mixtures had smooth surfaces whereas fibers with structured surfaces were obtained at higher RHs  and it was delayed by the increase of DMSO amount in the solution to 33.3 time units (a.u.) for 18:2 mixture and to 40 time units (a.u.) for 16:4 mixture Figs  and 4c,dTwb of the solutions increased in value with increasing amounts of DMSO in the solution from −4.1 °C to −3.5 °C and finally to −2.4 °C  due to its inherent high photostability, excellent brightness (defined as the product of phosphorescent or fluorescent quantum yield and the molar extinction coefficient), as well as the possibility of oxygen calibration by luminescence lifetime reading in the microsecond range44. Upon evaporation, the solution is enriched in water, thus the dye diffuses through the membrane and precipitates within the mesh structure.The physical entrapment of PtTFPP has been obtained by choosing a mixture of acetone and water as non-solvents for PCL at room temperature with the former being a ‘good’ solvent and the latter being a non-solvent for the luminescent dye3 cps in solution, compared to >400 × 103 cps for the membrane . Compared to ‘blue light’ excitation (Soret band), 540 nm excitation mode is less destructive for cells and is more convenient for filtering out the biological autofluorescenceuts Fig. .2 sensing scaffold developed by use of commercial polystyrene-based AlvetexTM membranes stained with PtTFPP had phosphorescence lifetime of 56.5 μs at 0 kPa O2 and 20 μs at 21 kPa O244. PCL nanoporous membranes instead showed higher quenching effect with approximately five-fold change of phosphorescence lifetime.It was recently demonstrated that a hybrid O53. This is a unique finding for this type of systems and it is probably related to the higher accessibility of the dye to the quenching analyte due to the highly porous matrix morphology. This feature can be translated to steady sensitivity and accuracy of oxygen monitoring of cell grown in the 3-D structure.In addition, linear Stern-Volmer relationship was observed for PCL membranes Fig. 53. This 2-sensitivity  Stern-Volmer relationship.For comparison, we also stained smooth micro- and nanofiber PCL membranes with PtTFPP dye and evaluated them in solution. All tested types of membranes were sufficiently bright and photostable in PLIM microscopy, displaying comparable O2 were stabilized to values around 7–8 µs, indicating that membranes provided efficient O2 diffusion due to the highly porous fiber structure.Finally, the compatibility of the membranes impregnated with PtTFPP for biological experiments was investigated. Human colon carcinoma cells HCT116 were seeded and cultivated on membranes for 48 hours, stained with Calcein Green viability dye and live cells were imaged using confocal PLIM Fig. . Cells a2-sensitive indicator dye, PtTFPP. They additionally show cytocompatibility, allowing the investigation for growth and analysis of oxygenation of 3-D cell cultures. For O2-sensing applications, we found microfibers with porous surface morphology being the most suitable , compared with nanofiber and smooth microfiber materials. Hence, these electrospun fibrous membranes represent an interesting alternative to hybrid PS AlvetexTM and related 3-D scaffolds as their production is inexpensive, easy and up-scalable54.Thus, PCL membranes with different surface morphologies can be used for successful impregnation of the hydrophobic O55. Hence, it is a prerequisite to select appropriate solvents to obtain the desired surface morphology at a defined environmental condition. To reach a more universal theoretical approach for the complex electrospinning process, we utilized HSP to determine not only the initial polymer-solvent compatibility but more importantly the solubility changes for the model polymer PCL during solvent evaporation throughout the electrospinning process. The findings were then also tested for PLLA and PVP to gain deeper insights on the applicability of the tool for other polymers.Electrospun fibers with tailored surface topographies can be generated by adapting solution parameters such as solvent system and polymer molecular weight and/or environmental parameters such as temperature and relative humidity56. To date, the hypotheses of evaporative cooling and phase separation yielding surface topographies on electrospun fibers have never been correlated in a theory-driven approach to understand, predict and control the surface texturing of e-spun fibers in relation with Tdp of the electrospinning environment and simulated Twb on the polymer jet surface. By investigating the electrospun fibers produced from solutions of PCL in two different good solvents (CHCl3 and DCM) and respective calculation of the Twb’s, it was concluded that the higher the solvent volatility, the lower the Twb  mixture at 12% RH can be explained with the negative value of ΔT corresponding to lack of water condensation as well as a RED value below 1  mixture at 12% RH had also negative ΔT referring to no water condensation and thus smooth surface morphologies were obtained. However, its RED value increased to higher values than that of CHCl3:MeOH (18:2) mixture, although this increase was not high enough to induce phase separation at low RH condition by itself. The irregular and inter-connected surface pores obtained at 35% and 55% RH can be the synergistic result of positive ΔT values till increasing Twb reached to Tdp with an increasing RED value in the meantime .Additionally, the comparison of DMSO amounts in the solution mixture revealed that at the highest RH condition, a more pronounced structuring of the fibers was obtained due to an increase of phase separation deriving from higher DMSO concentrations Figs  and 4b. Overall, we propose the pore formation mechanism as depicted in Fig. In summary, the surface structure formation on electrospun PCL fibers originates from the interplay between polymer solubility changes and jet surface temperature decrease deriving from differences in solvent volatility. The synergistic effect of these parameters together with the relative humidity defines the extent of surface structuring on electrospun PCL fibers as well as PLLA and PVP fibers.2 sensing in 3-D cell culture in this study.These findings hold great potential to extend the tool-box for tailoring the topographies of electrospun fibers with better efficiency, control and reproducibility for surface sensitive applications such as drug delivery, advanced tissue engineering, catalysis, filtration and biosensing technology as demonstrated with OSupplementary PDF File


There is an ongoing debate on the therapeutic potential of vaso-modulatory interventions against glioma invasion. Prominent vasculature-targeting therapies involve tumour blood vessel deterioration and normalisation. The former aims at tumour infarction and nutrient deprivation induced by blood vessel occlusion/collapse. In contrast, the therapeutic intention of normalising the abnormal tumour vasculature is to improve the efficacy of conventional treatment modalities. Although these strategies have shown therapeutic potential, it remains unclear why they both often fail to control glioma growth. To shed some light on this issue, we propose a mathematical model based on the migration/proliferation dichotomy of glioma cells in order to investigate why vaso-modulatory interventions have shown limited success in terms of tumour clearance. We found the existence of a critical cell proliferation/diffusion ratio that separates glioma responses to vaso-modulatory interventions into two distinct regimes. While for tumours, belonging to one regime, vascular modulations reduce the front speed and increase the infiltration width, for those in the other regime, the invasion speed increases and infiltration width decreases. We discuss how these  Gliomas are aggressive brain tumours typically associated with poor prognosis, sharp deterioration in the patients’ quality of life and low survival rates, making this disease challenging to treat. According to the World Health Organization (WHO)245The influence of the microenvironment on glioma cell behaviour plays a crucial role in the resulting diffusive tumour growth and its invasive capacity. Hypoxia, the presence of abnormal and sustained low oxygen levels in the tumour tissue, strongly correlates with glioma invasiveness and malignancy89104101213A particularly important component of the tumour microenvironment is the vascular network. Accumulating evidence suggests the existence of various positive and negative feedback mechanisms between glioma cells and the vasculature. Indeed, gliomas are reported as highly vascularised neoplasias15171921192021The high degree of angiogenesis and vascular pathologies observed in gliomas has been the target of several therapeutic vaso-modulatory strategies24262724303128303324Mathematical modelling has the potential to improve our understanding of the complex biology of gliomas and their interactions with the microenvironment, as well as it may help in the design of more effective and personalised treatment strategies353637383940414216384445464748495051525455444546471638ρ and functional tumour vasculature v, as well as the concentrations of oxygen σ and pro-angiogenic factors a in the tumour microenvironment, where d is the dimension of the system. We develop a mathematical model that describes the growth of vascularised gliomas focusing on the interplay between the migration/proliferation dichotomy and vaso-occlusion at the margin of viable tumour tissue. The system variables are the density of glioma cells [A1] Glioma cells switch phenotypes between proliferative (normoxic) and migratory (hypoxic) depending on the oxygen concentration in the tumour microenvironment4101213[A2] Under hypoxia conditions glioma cells secrete pro-angiogenic factors151718[A3] Pro-angiogenic factors trigger the formation of blood vessels and regulate vasculature remodelling18[A4] Endothelial cells forming the vascular network uptake pro-angiogenic factors18[A5] Functional tumour blood vessels releases oxygen1517[A6] Oxygen is essential for glioma growth and progression1718[A7] Glioma cells consume oxygen provided by the functional vasculature17[A8] Prothrombotic factors and increased mechanical pressure in regions of high glioma cell density induce blood vessel occlusion and collapse192358ρ1 and proliferative (normoxic) ρ2, depending on the concentration of oxygen in the tumour microenvironment σ. More precisely, we consider two linear switching functions, f21(σ) = λ1 − σ and f12(σ) = λ2σ, that represent the rate at which glioma cells change from migratory to proliferative and vice versa, respectively. Although there is experimental evidence of a positive correlation between oxygen availability and cell proliferation, the exact functional form of the oxygen-dependent phenotypic switching remains unknown. Accordingly, we consider the simplest case, i.e. a linear switching between proliferative and migratory phenotypes, in line with previous studies61λ1 and λ2 are positive constants, see the Based on the migration/proliferation dichotomy4101213Cell motility is modelled as a diffusive process mimicking the net infiltration of glioma cells into the surrounding brain tissue, while a logistic growth term is considered for tumour cell proliferation. The system of equations for the migratory and proliferative glioma cells is given byt and spatial x coordinates in the arguments of variables have been omitted for notational simplicity. Dρ and bρ are the diffusion and proliferation coefficients of migratory and proliferative glioma cells, respectively. N represents the brain tissue carrying capacity, i.e. the maximum number of cells that can be supported by the environment. The parameters Dρ, bρ and N are positive constants.where the temporal ρ = ρ1 + ρ2 by assuming that f12(σ)ρ1 = f21(σ)ρ2. This is a plausible assumption since intracellular processes, such as signalling pathways regulating the phenotypic switch, operate at much shorter time scales than cell migration and proliferation. Thus, we assume that phenotype switching is a mechanism faster compared to cell division and motility, which allows to express ρ1 and ρ2 as a function of ρ in the following formThe system (1)-(2) can be reduced to a single equation for the total density of glioma cells where we have thatandρ1 and ρ2, the equation for the total density of (migratory and proliferative) glioma cells ρ is given bySumming equations  and 2),,2), and α(σ) and β(σ) are defined as followswhere the oxygen-dependent functions andα(σ) + β(σ) = 1, we can rewrite Then, taking into account that α(σ) and β(σ) in σ0 > 0 be the physiological oxygen concentration in the normal brain tissue. Then, by normalising Dρ = D/α(σ0) and bρ = b/β(σ0) the classical Fisher-Kolmogorov equation is recovered under the assumption of a constant oxygen concentration in the tumour microenvironment, given byNotice that 55D and b are positive parameters that represents the intrinsic diffusion and proliferation rates of glioma cells, respectively. We remark that, 164955where v. We further consider that pro-angiogenic factors are exclusively produced by glioma cells under hypoxic conditions at a rate proportional to the tumour cell density, and therefore neglecting hypoxia-independent pathways. In addition, endothelial cells forming the vascular network uptake pro-angiogenic factors which also undergo natural decay. The equation for the effective pro-angiogenic factor concentration a is given byNeovascularisation in tumours takes place when pro-angiogenic factors overcome anti-angiogenic stimuli. However, in gliomas there is evidence of a wide range of pro- and anti-angiogenic factors involved, each of them acting through different vascularisation mechanisms1524t and spatial x coordinates in the arguments of variables have been omitted for notational simplicity. Da is the diffusion coefficient of pro-angiogenic factors. Assuming the quasi-steady state approximation of where the temporal k1, k2 and k3 represent the production, consumption and natural decay rates of pro-angiogenic factors, respectively, where H(ξ), defined as H(ξ) = 1 if ξ ≤ 0 and H(ξ) = 0 if ξ > 0, given byThe positive parameters θ is a positive parameter that controls the steepness of σ is lower than the hypoxic oxygen threshold where v = 1/2. Thus, the limit case v = 0 represents an avascular tissue, while on the contrary v = 1 describes a hypothetical scenario characterised by excessive vascularisation.Histopathological studies have shown that the vascular structure and function in brain tumours is markedly abnormal1718a > 0, leading to the formation of new functional vasculature according to a logistic growth term. The rate at which functional tumour vasculature is generated follows the Michaelis-Menten kinetics depending on the pro-angiogenic factor concentration, where a constant dispersal rate of endothelial cells (vasculature) is assumed. Notice that the Michaelis-Menten term is commonly used to model a saturating response at high doses in biological systems22631923v is given byBlood vessels in gliomas are not stable, being continuously formed, occluded and destroyed. Neovascularisation takes place by different angiogenic and vasculogenic processes induced by complex signalling mechanisms that are not well understood1166t and spatial x coordinates in the arguments of variables have been omitted for notational simplicity. Dv is the diffusion coefficient representing the net dispersal of tumour vasculature, g1 is the formation rate of functional blood vessels, μ is the pro-angiogenic factor concentration at which g1 is half-maximal, g2 is the vaso-occlusion rate and n is a parameter that regulates the degree of blood vessel occlusion depending on the density of glioma cells. The vaso-occlusion term, g2vρn, models the mechanical pressure exerted on blood vessels in regions of high glioma cell density, see the 32N/2, where N is the brain tissue carrying capacityDv, g1, μ, g2 and n are positive constants.where again the temporal a is much smaller than the uptake/internalisation rate by endothelial cells, i.e. k3 ≪ k270Plugging K = μk2/k1 represents the concentration of pro-angiogenic factors at which the formation rate of functional tumour vasculature is half-maximal, see the where σ0 and that in the tumour interstitium. These assumptions result in the equation for the oxygen concentration σ given byOxygen is delivered to the brain tissue via functional blood vessels, spreads into the tumour bulk and is consumed by glioma cells. Transport of oxygen within tissues occurs by diffusion and convectiont and spatial x coordinates in the arguments of variables have been omitted for notational simplicity. Dσ is the oxygen diffusion coefficient, h1 is the permeability coefficient of functional vasculature and h2 is the oxygen consumption rate by glioma cells. The parameters Dσ, h1, σ0 and h2 are positive constants. Notice that similar assumptions have been previously considered to model oxygen dynamics in vascular tumour growthwhere the temporal The proposed glioma-vasculature interplay model comprises a system of coupled partial differential equations given byα(σ) and β(σ) are given by equations (where the oxygen-dependent functions quations –5), res, resα(σ)ρ0, σ0 and v0 are the initial density of glioma cells spatially distributed in a segment of length ε, the density of functional tumour vasculature and the oxygen concentration, respectively. The positive parameter γ controls the steepness of x − ε) with ε > 0, and L > 0 is the length of the one-dimensional computational domain. In addition, we consider an isolated host tissue in which all system behaviours arise solely due to the interaction terms in where the positive parameters Tf > 0 is an arbitrary simulation time, i.e. the end of simulations. The conditions above also imply that no cell or molecule leaves the system through the domain boundaries.where Model III, is a generalisation of two simpler models which are also of interest for the study of glioma growth and invasion. As shown in v = v0 (Model II), and also a constant oxygen concentration σ = σ0 (Model I). More precisely, Model II is obtained from Model III by setting g1 = g2 = 0 in Model I is obtained from Model II by setting h2 = 0 in The glioma-vasculature interplay model (14)–(16) referred to as Model I corresponds to the classical Fisher-Kolmogorov 55Model II given by Model II is assumed constant, the blood perfusion can be considered stable and we therefore neglect tumour-induced vascular pathologies. The latter is a reasonable assumption, particularly for low-grade gliomas, where an abnormal vascular structure is not prominentModel II is to consider tumour-associated vascularisation dynamics. This is precisely what defines Model III, which is used to investigate the effects of vaso-modulatory interventions on glioma invasion. Taking into account that Model I has been extensively studied, we begin with the analysis of Model II as an intermediate step towards analysing Model III, see ρ at the end of simulations Tf. In turn, the infiltration width is defined by the difference between the points where glioma cell density is 80% and 2% of the maximum cellular density at simulation time Tf. These specific features of tumour invasion have been reported crucial to determine glioma malignancy and predict therapeutic failure1650We characterise glioma invasion by the tumour front speed and infiltration width, see D and proliferation b rates from the effective rates that depend on the oxygen concentration in the tumour microenvironment. The effective diffusion Deff and proliferation beff rates of glioma cells are defined as followsUnlike the classical Fisher-Kolmogorov andL is the length of the one-dimensional domain of simulation. Notice that Dρ = D/α(σ0) and bρ = b/β(σ0), where D and b are the intrinsic glioma cell diffusion and proliferation rates, respectively. We then investigate the dependence of Deff and beff, as well as the tumour front speed and infiltration width, at simulation time Tf on different values of parameters h2 (glioma cell oxygen consumption) and g2 (vaso-occlusion).where Parameter values considered in the model simulations are taken from published data wherever possible or estimated to approximate physiologic conditions based on appropriate physical and biological arguments, see Deff and proliferation beff rates defined in equations (D and b. Model simulations in The proposed glioma-vasculature interplay model 14)–(16) is first considered to investigate the effects of variations in the tumour cell oxygen consumption and vaso-occlusion rates on the effective invasive behaviour of gliomas. –(16) is quations  and 18)Deff and h2 or vaso-occlusion g2 results in more diffusive and less proliferative tumours. The model supports that at high h2 and g2 values, the oxygen concentration in the tumour microenvironment significantly decreases, which may result in hypoxia and necrosis. The lack of oxygen limits the proliferative capacity of glioma cells, and in turn enhances the hypoxia-induced cell migration to better-oxygenated brain tissue areas. In particular, variations in the cell oxygen consumption and vaso-occlusion rates are predicted to have a major impact on highly infiltrative and/or rapidly growing gliomas. Thus, the precise way in which such cellular and microenviromental changes affect the overall invasive potential of tumours can be expected to depend on the specific intrinsic glioma cell features.Comparative simulation maps in D and b. In particular, these properties of tumour invasion are determined by a non-linear relationship between the effective diffusion Deff and proliferation beff rates of glioma cells. For instance, in the simplest model corresponding to the classical Fisher-Kolmogorov equation (Model I), the tumour front speed is proportional to Model II, i.e. under the assumption of a constant density of functional tumour vasculature, reveals that variations in the rate at which glioma cells consume oxygen h2 produce opposing effects on the tumour front speed. More precisely, b* for which the front speed in tumours characterised by b > b* decreases at higher values of h2, while on the contrary tumours with b < b* invade faster displaying diffusely infiltrative growth patterns. Assuming that the tumour front speed is proportional to the product of effective diffusion and proliferation rates, we can readily explain the aforementioned simulation results for variations of h2. On one hand, in tumours with glioma cell proliferation rates b above the critical threshold b*, the effective migration and proliferation mechanisms compensate each other, leaving almost-invariant the speed of the invading front. On the other hand, in the cases of tumours with b < b*, while the effective proliferation rate is not significantly affected, the migratory activity of glioma cells is higher for increasing values of h2, which results in faster tumour front propagation speeds.Analysis of the The flatness/steepness of the tumour front is proportional to a ratio of effective glioma cell diffusion and proliferation rates. When oxygen in the microenvironment is not limited, highly diffusive tumours evolve with large and flat fronts, whereas increased glioma cell proliferation results in short and steep fronts. However, under oxygen-limiting conditions the shape of the evolving tumour front is markedly influenced by the specific rate at which glioma cells consume oxygen. Model III reveal that for increasing vaso-occlusion rates g2, the tumour front speed is differently affected depending on the intrinsic diffusion and proliferation rates of glioma cells. In addition to the modulatory effects of oxygen availability on glioma growth and invasion, these processes are also influenced by vascularisation mechanisms. Comparative simulation maps in D and b inside a region delimited by a critical rate b+ and an approximate ratio between diffusion and proliferation rates Λ+ = b/D, the invading front moves faster as g2 increases. Besides, the front speed slightly decreases or remains almost constant in the rest of tumours, i.e. with parameter values of D and b outside of such region. In particular, tumours characterised by b < b+ evolve at low cellular density and thus vascular occlusive events due to increased mechanical pressure by glioma cells hardly occur. On the other hand, increasing vaso-occlusion rates in tumours with b > b+ enhances the effective cell migration towards better vascularised brain tissue regions. Although vascular occlusion limits the proliferative activity of glioma cells, faster tumour front speeds are predicted as long as the triggered migratory activity dominates over cell proliferation.Simulations of the b < b+ is almost unaffected for increasing vaso-occlusion rates as shown in b > b+ are also separated by an approximated linear relation between D and b with respect to variations in the infiltration width. In particular, more occlusion of the blood vessel results in larger flat fronts in tumours with cell proliferation/diffusion ratios above the critical value Λ+ for b > b+, while the infiltration width is reduced in the rest of tumours.The infiltration width in tumours with In this work, we proposed a deterministic mathematical model of glioma growth and invasion that is formulated as a system of reaction-diffusion partial differential equations. Our glioma-vasculature interplay model accounts for the dynamics of normoxic and hypoxic glioma cells based on the Go-or-Grow mechanism which is in turn influenced by the functional tumour vasculature and the concentration of oxygen in the microenvironment. In particular, we focused on the effect of variations in the glioma cell oxygen consumption and vascular occlusion on prognostically-relevant characteristics of tumour invasion, i.e. the front speed and infiltration width. The main model results are summarised in The model analysis revealed that increasing glioma cell oxygen consumption and vaso-occlusion rates results in more diffusive and less proliferative tumours. In both scenarios, the average oxygen concentration in the tumour microenvironment decreases, which limits glioma cell proliferation and enhances hypoxia-induced migration. This is in line with previous clinical and histopathological observations that hypoxia strongly correlates with glioma malignancy192021Model I) critical behaviours are not observed. Assuming or not tumour vascularisation dynamics, the Go-or-Grow induced criticality is expressed either in the form of a proliferation/diffusion ratio Λ+ = b/D for b > b+ or a critical proliferation rate b* of glioma cells, respectively. More precisely, the critical thresholds b* and Λ+ for b > b+ separate tumour behaviours in regimes where the front speed and infiltration width are differently affected by changes in the glioma cell oxygen consumption and vaso-occlusion rates. These findings highlight the importance of further investigating the therapeutic potential of targeting the Go-or-Grow phenomenon as a strategy to reduce glioma cell migration. Based on our model results, we can argue that one-size-fits-all vaso-modulatory interventions should be expected to fail to control glioma invasion due to the complexity of the mechanisms involved and inter-patient heterogeneity. This study supports the value of personalised medicine and provides a simplified, but useful modelling framework with predictive potential based on a precise tumour profiling from possible biopsy measurements and medical imaging. In particular, patient-based estimation of tumour cell proliferation and diffusion rates would be crucial components of such future tailored approaches to individualise treatment selection for glioma patients.Recently, it has been shown that the migration/proliferation dichotomy can introduce a critical threshold on the glioma cell density that separates tumour growth and extinction dynamics, a phenomenon called Allee effect+ = b/D for b > b+, a pro-thrombotic or an anti-vasogenic therapeutic technique may reduce the tumour front speed but at the same time leads to highly infiltrative behaviours, which makes this treatment strategy rather inappropriate. However, normalisation of the tumour blood vessels may result in faster growing gliomas with compact, less invasive morphologies. Thus, surgical resection could be considered to remove such compact tumours. In turn, the benefits of conventional treatment modalities such as chemo-, radio- and immunotherapy might significantly increase in well-vascularised and therefore normally oxygenated tumours243031We believe that this work substantially expands the theoretical concepts of the invasive behavior of gliomas, suggesting that any vasculature-targeting therapeutic intervention will inevitably lead to a trade-off between the tumour front speed and infiltration width. This result suggests that vaso-modulatory interventions should be embedded in a personalised combination of different treatment protocols, in which anti-angiogenesis might be integrated with individually adjusted strategies targeting cell proliferation, metabolic transformation or immune responses. For instance, in the case of gliomas characterised by a cell proliferation/diffusion ratio above Λin silico findings provide new insights into the impact of functional vascular changes on glioma invasion. Furthermore, the migration/proliferation dichotomy of glioma cells has been modelled in the simplest possible way and more informed formulations depending on other tumour-related factors should be considered. In turn, intratumoral genetic diversity is not directly considered, but instead we take into account phenotypic diversity depending on oxygen availability, which has long been recognized as an important therapeutic factor. The latter is supported by evidence that genetic diversity is tumour-subtype specific and not significantly affected during treatment, while phenotypic heterogeneity is significantly different before and after therapy42We conclude by pointing out a number of related future research directions, as well as discussing some limitations of this work. Although in our model the vaso-occlusion term in How to cite this article: Alfonso, J. C. L. et al. Why one-size-fits-all vaso-modulatory interventions fail to control glioma invasion: in silico insights. Sci. Rep.6, 37283; doi: 10.1038/srep37283 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Both proteins were found to be overexpressed e.g. in tumor cells, mediating cell proliferation and migration as well as tumor angiogenesis and neuroinflammation. The extracellular domain of Axl (sAxl) and Gas6 were found in the peri-tumoral edema and blood of animals as well as in human glioma tissue. Therefore, we monitored the level of sAxl and Gas6 in human blood samples. To increase the sensitivity of protein detection beyond commonly used standard methods we preliminary tested the innovative Proseek Single-Plex Protein Assay Kit System from Olink Bioscience together with new antibodies against the soluble RTK sAxl and its ligand Gas6. We conclude that the Proseek method is a highly sensitive and fast procedure that can be used as a possible powerful tool compared to routinely used ELISA-methods. Within this group, glioblastome multiforme (GBM) WHO IV is the most aggressive subtype and represents the biggest group therein. Anaplastic glioma WHO grade III imply astrocytoma and oligodendroglioma . The curSince the receptor tyrosine kinase (RTK) Axl and its ligand growth arrest specific gene 6 (Gas6) are known to be co-expressed in HGG tissue correlating with a poor prognosis , we wereThe receptor tyrosine kinase Axl, beside Tyro3/Sky and Mer (syn. TAM family), is characterized by an extracellular domain (ECD) of two immunoglobuline-like domains abreast of two fibronection-type III domains ,8.After ligand binding , the tyrosine kinase domain of the receptors of the TAM family activates the intracellular signal transduction downstream by receptor dimerisation and initiation of autophosphorylation ,10. Axl/22.1To generate the new method to measure the detection of sAxl and Gas6, 3 patients of ≥18 years and ≤80 years of age with the diagnosis of a first or second recurrent or progressed high-grade glioma measured by standard MRI were included. For comparison, the serum of a healthy 25-year old woman was added (control). Sera of patients with recurrent or progressive HGG receiving anti-angiogenic bevacizumab therapy  were used. After baseline and treatment start with Avastin, clinical follow ups started with serum sample collections at week 1 ± 1 day, followed by clinical follow up visits at week 2. Afterwards every second week until clinical progression , serum samples were collected and used for measurements. Ethical approval for this study was granted by the local research ethics committee (AM3752_LEK).2.2g. Aliquots of the supernatants were frozen to −80 °C. Serial dilutions for the proteins sAxl and Gas6 were prepared as a positive control and to establish a calibration curve. To this end, the recombinant and affinity purified Axl  and Gas6  have been diluted in Calibrator Diluent as follows: Axl 139 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 100 pg/ml, 10 pg/ml, 1 pg/ml, 0.1 pg/ml, 0.01 pg/ml, 0 ; Gas6 230 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 100 pg/ml, 10 pg/ml, 1 pg/ml, 0.1 pg/ml, 0.01 pg/ml, 1 fg/ml, 0 .For serum sample preparation, standard laboratory tests including chemistry , coagulation, biochemistry) and haematology panels have been performed. At baseline evaluation a serum pregnancy test  and a test for infections  have been carried out. All patients have been consented for the collection and storage of blood , and markers have been evaluated using above-mentioned method. At baseline and during periodic follow-up visits in the course of an antiangiogenic treatment with Avastin (Bevacimzumab 10 mg/m2 body surface every two weeks), serum samples have been collected at baseline, in the first and second week and afterwards every 2 weeks  until tumor progression for precise Proseek analysis. Therefore one serum tube derived from vein puncture for testing the combination of the new markers associated with tumor neovascularization  by Proseek-method have been used. Therefore blood samples were let for 10 min at room temperature and centrifuged for 10 min at 1408 × 2.3Proseek is a reagent Kit system from Olink Bioscience  to detect and quantify proteins in sample material like serum and plasma, based on the Proximity Extension Assay (PEA) technology. The PEA is a method for single protein detection based on a Proximity-dependent DNA polymerization event. It can be performed using 2 Proximity probes. For this project, we took two polyclonal antibodies already established in ELISAs, but not for the Proseek reagent Kit system, to detect sAxl  and Gas6 . The Proseek Assay Development kit has been performed as described in the user manual version 3.0. First, the antibodies were conjugated either to the oligonucleotides (Oligonucleotide-labeled antibodies) A or B by using Proseek Probemaker A (Art.no. 93001-0010) or B (Art. No. 93002-0010) to create Proseek probes A and B. A dilution series of recombinant human sAxl  and recombinant human Gas6  as antigen standard in Calibrator diluent  for standard curve was used. For negative control, Calibrator diluent without antigen was prepared for method application. The incubation of the dilution series and serum samples with Proseek probes A and B leads to the binding to the target protein. To dilute the Proseek probes and lower their real concentration, the Pre-Extension solution has been adducted before the Extension master mix has been added. Thereafter, the addition of a DNA polymerase will enable the extension of the hybridized oligonucleotides and the resulted sequence is detected and quantified by qPCR .2.4® Protein Assays Probe Development Protocol . Proseek reagents have been prepared according to the Proseek user manual . Samples, buffer (recommended background control by the TaqMan® Protein Expression Assay Protocol) or recombinant antibody for standard curve were incubated according to manual instruction. The PCR was performed using the TaqMan® Protein Expression Assay reagent kit according to the protocol . Data analysis and sample calculation were carried out using Microsoft Excel 2007. For the calibration curve of each test of sAxl and Gas6, the calibrators were measured in duplicates. Afterwards the cycle of quantification (Cq) was calculated as the mean value of 3 independent experiments and the background values of the control were subtracted. To assess the concentration of the proteins the formula y = kx + d has been used.For this study, a two-step real-time PCR (qPCR) was performed by using the fluorophore FAM based on the TaqMan2.52SO4 and determination of the optical density of each well has immediately been started at 450 nm.Calibration curves using human sAxl  and Gas6  were also analyzed using the DuoSet ELISA Development Kit for sandwich ELISA. Both Kit systems contain components required to measure natural and recombinant human Gas6 and sAxl according to the manufactures protocols. Detection was performed using a Siemens BEP III ELISA Reader and results have been processed using Microsoft Excel 2007. In detail, the capture antibody has been diluted in PBS and immediately coated on a 96-well microplate, á 100 μl and incubated at RT for ∼24 h. Each well has been washed 3-fold with 400 μl wash buffer by an autowasher and remained buffer has been removed by blotting the plate against paper towels. Plates were blocked in 300 μl adequate blocking buffer for 1 h at RT. In the meantime, samples and standards were diluted in the required concentrations. Plates were aspirated like before and samples and standards diluted in RD buffer were added (100 μl/well) and incubated for 2 h at RT. Afterwards, the aspiration step was repeated and 100 μl of the detection antibody per well was added for 2 h. Washing procedure followed, before the working dilution of streptavidin-HRP, 100 μl, for 20 min in the dark, starts. Plate has been washed as before and 100 μl substrate solution was added in the dark. 20 min later, the reaction has been stopped by adding 50 μl 2 N H33.1A linear serial dilution of a human Gas6 standard AF885 from R&D systems was generated between 1 pg/ml and 10 ng/ml and used for the amplification. The mean Cq value for each concentration standards was calculated based on measurements from three independent experiments performed in duplicates n = 3; A, B. The3.2A linear serial dilution of a human sAxl standard  was generated between 10 pg/ml and 10 ng/ml and used for the amplification. The mean Cq value for each concentration standards was calculated based on measurements from three independent experiments performed in duplicates n = 3; A, B. TheResults revealed that the Proseek method was more sensitive to detect low concentrations of sAxl (10 pg/ml–10 ng/ml) as compared to ELISA detection (125 pg/ml–2 ng/ml) C.3.3After establishing the standard curves for Gas6  and sAxl4The aim of the study was the establishment of alternative methods beside routinely used sandwich enzyme-linked immunosorbent assay (ELISA) to measure levels of sAxl and Gas6 with the intention of developing a sensitive diagnostic instrument capable of detecting the growth and recurrence of HGG before the tumor reaches larger sizes required for direct visualization. Taken that sAxl and Gas6 plasma concentration are interesting parameters to identify different types of diseases such as HGGs, ELISA methods have been recently developed, allowing henceforth accurate analysis of sAxl in human serum for routine diagnostics . TherefoThe combination of DNA ligation and real-time PCR and the combination with PEA-established methods can be performed with standard laboratory equipment. The advantages of a PEA-based technique are that it is an elegant format and does not required time-consuming washing procedures leading to material loss. Moreover, measurements can be performed in sample volume as small as 1 μl, which is crucial for cases of precious biobanked material or paraffin-embedded tissues. Additionally, measurements were performed using commercially available polyclonal or monoclonal antibodies from R&D systems within 1 day. The advantage of using antibodies is that it is a powerful tool to combine protein detection with PCR amplification. The antibodies established for the Proseek Assay Development Kit are affinity-purified and have no BSA or other carrier proteins included that could interfere with conjugation . After preparing the dilution series of the antigen standards , the antibody will bind to the target protein, the antigen. By extending the bonded probes with Oligos using Extension master mixes from the Kit through a DNA polymerization event, the real-time PCR amplicon has been created for PCR to increase sensitivity. The limitations of the assay are that the primary antibody has to be compatible and well selected which suits to the assay, e.g. R & D systems. Additionally the method needs some time for establishment to ensure precise detections . Also the needs of time has to be hold, especially for the time of binding to reduce unspecific clustering of proteins or due to low target antigen levels in body fluids (2 h room temperature or at +4 °C over night).Further investigation is still needed to correlate the progression of sAxl and Gas6 concentrations in serum of HGG patients with disease progression, but Proseek Multi-Plex Protein Assay Kit system seemed to be a powerful tool to combine those 2 proteins for further analysis.


Kllsm4Δ1 mutant, which accumulates mRNAs, ages much faster that the wild type strain and undergoes regulated-cell-death. We also found that in Kllsm4Δ1 cells the mutation rate increases during chronological life span indicating that the capacity to handle oxidized RNAs in yeast declines with aging. Lowering intracellular ROS levels by antioxidants recovers the wild-type phenotype of mutant cells, including reduced amount of oxidized RNAs and lower mutation rate. Since mRNA oxidation was reported to occur in different neurodegenerative diseases, decapping-deficient cells may represent a useful tool for deciphering molecular mechanisms of cell response to such conditions, providing new insights into RNA modification-based pathogenesis.Despite recent advances in understanding the complexity of RNA processes, regulation of the metabolism of oxidized cellular RNAs and the mechanisms through which oxidized ribonucleotides affect mRNA translation, and consequently cell viability, are not well characterized. We show here that the level of oxidized RNAs is markedly increased in a yeast decapping  The lower fidelity of RNA synthesis and even lower fidelity of translation (~1 per 104 amino acids) are tolerated because they cause no mutation. Damaged RNAs and proteins have thus been considered dispensable. Actually, considering the fact that the pool size of ribonucleotides is hundreds of times larger than that of 2′-deoxyribonucleotides [DNA damage can lead to permanent sequence changes and many studies dealt with DNA damage checkpoint complexes and how cells handle damaged DNA. On the contrary, the possibility and the mechanisms of repairing damaged RNA and proteins has been considered unlikely to even exist for long time. The error rate of transcription is one mistake per 10leotides , 4. Receleotides . Uncoverleotides –12. ThesMoreover, since the discovery of yeast apoptosis  multipleKllsm4Δ1 cells, expressing a truncated form of KlLSM4, the subunit of the decapping activator Lsm1-7 complex [lsm mutants can also be prevented by the addition to the growth medium of antioxidant compounds, such as Acetyl-l-carnitine (ALC) or apple extracts [We also reported that cell death in decapping mutants, affected in early steps of mRNA degradation, is accompanied by elevated histone mRNA levels, persisting throughout the cell cycle, and is associated with defects in the S-phase progression . The pre complex , 18, can complex , 19. Preextracts , 21, sugKllsm4Δ1 mutant and its effect on cellular aging and cell death.Here we present data concerning the accumulation of oxidized RNAs in the S. cerevisiae strains MCY4/Kllsm4Δ1 , MCY4/313Kllsm4Δ1/LSM4 , CML39-11A  [We used the rp1-289) , 19. CelTotal RNA from yeast cells was isolated using a hot phenol procedure  and sepa600 ~ 0.7) following the addition of cycloheximide to a final concentration of 100 µg/mL for 10 min at room temperature. Cells were washed twice with LB buffer  and cell extract was prepared by vortexing cell suspension with 1 ml glass beads  followed by centrifugation at 13.200 rpm for 10 min at 4 °C. RNA concentration was measured  and extract aliquots corresponding to 30 OD260 units in 400 µl were loaded onto a 7 and 47 % sucrose gradient prepared in buffer SG  using ACTA Prime chromatography system . Samples were centrifuged in the SW41 rotor  at 39,000 rpm for 90 min at 4 °C, gradients were fractionated by upward displacement with Fluorinert (3 M), using a gradient fractionator  and ACTA Prime with fraction collector Frac-950.Polysomes were prepared from 100 ml of yeast culture grown to the late exponential phase  was carried out exactly as described [In vivo protein labelling with escribed .Kllsm4Δ1cell cultures. Total RNAs were extracted after 1 day of growth and analysed by Northwestern as described. Trp + reversion frequency was determined in the absence or in the presence of antioxidants.For reversion frequency of Trp-phenotype and the occurrence of Canavanine resistant clones three independent cultures of the considered strains were cultivated at 28 °C in SD medium and auxotrophic requirement as needed. For each culture, three plates of selective medium lacking tryptophan or containing L-canavanine  were inoculated and incubated at 28 °C for 3–5 days. The frequency of the Trp + reversion and of Canavanine resistant mutants was normalized to the number of viable cells in each experiment. Viability at day 1 was determined with the microcolonies test , while fFigures Kllsm4Δ1 compared to the wild type strain CML39-11A, with ribosomal RNAs being the most evident  and we observed a very similar profile in both strains . Similarly, no mutations were detected in the SUP70 gene that codes for tRNAGlnCUG, whose mutations are known to suppress the UAG stop codon present in the Trp1-289 allele [Kllsm4Δ1 [+ revertants were greatly reduced in the presence of apple extracts and ALC during cell growth  phenotype originating from mutations in CAN1, the arginine permease gene involved in the uptake of arginine as well of its cytotoxic analogue canavanine [CAN1 gene amplified from about 10 Canr clones of each strain after 1 day of growth. Six out of ten clones showed the wild type CAN1 sequence in the Kllsm4Δ1 mutant, while base substitutions/insertions occurred in the remaining four clones, whereas the wild type base substitutions/insertions were observed in six out of seven clones in, with only one clone showing the wild type sequence (Table Kllsm4Δ1 mutant is similar to, or even lower, than that observed in the wild type. Sequencing of additional Kllsm4Δ1-derived Canr clones after six days of growth revealed that 5 out of 11 clones had no sequence changes, a result very close to that observed after 1 day of growth. The remaining clones showed base substitution/insertion/deletion, three of which were already present on day 1, suggesting that their appearance occurred in the early stages of growth (Table + revertants reported above, suggesting the involvement of other mechanisms enhancing the reversion frequency (Fig. A common way to measure the mutation frequency in yeast is based on the canavanine resistance (Cannavanine . To verith Table . The obsDespite the evidence that oxidative damage to RNA is associated with numerous disease states, this phenomenon has received relatively little attention . It has Kllsm4Δ1 mutant cells accumulate oxidized RNAs, starting from the early stage of growth, leading to genome instability, as indicated by the exceptional high reversion frequency of the Trp− phenotype. Nevertheless, this was not reflected in an increase in DNA mutation frequency, suggesting that the abnormally high reversion observed in the mutant might result from post-transcriptional/translational defects.We previously reported a link between mRNA decapping defects and the onset of premature aging and apoptosis in yeast cells , 12. AltKllsm4Δ1 mutant. Although preliminary, these results open an interesting scenario that correlates RNA oxidation and the onset of cellular response mechanisms during cell aging. In this respect, yeast decapping mutants might represent useful tools for understanding the effects of RNA and protein damage on the cellular lifespan.We hypothesize that the mechanism underlying the suppression of the auxotrophic mutations under selective pressure may be due to translational errors induced by oxidized mRNAs. Consistently, we found that reducing the intracellular ROS level by the addition of antioxidants can recover the wild type situation with respect to the amount of oxidized RNAs as well the mutation rate in the


Dysbiosis is linked to the cause of several human diseases, many of which having an immunity related component. This work investigated whether mice genetically selected for low or high antibody production display differences in intestinal bacterial communities, and consisted in the comparison of fecal 16SV6–V8 rDNA PCR amplicons resolved by temperature gradient gel electrophoresis (TGGE) of five each of low (LIII) and high (HIII) antibody producing mice. 16SV6 rDNA amplicons of 2 mice from each line were sequenced.LIII mice were grouped in a single TGGE cluster, displayed a low α-diversity, and were distinguished by low Firmicutes/Bacteroidetes ratio.The results suggest that genetically driven low antibody production in mice is associated with gut dysbiosis. Salmonella typhimurium infection [Mycobacterium leprae heat shock protein Hsp65 [Trypanosoma cruzi infection than animals of the LIII lineage, a feature which was shown to be gender-specific  and correlated with interferon-γ and nitric oxide production by peritoneal lymph node cells [Proper functioning of the immune system (IS) depends on its modulation by body associated microorganisms, in a process starting early after birth, lasting through all life time and which contributes for the shaping of microbial ecosystem itself, ultimately influencing body homeostasis. The importance of a fine-tuned microbiota-immunity balance can be illustrated by multiple human disorders having as an underlying basis defects in immune response and accompanying gut dysbiosis, such as autoimmune and inflammatory bowel diseases . The usenfection , higher nfection  and highin Hsp65 . On the de cells . The polde cells . Given sde cells , we inveS. typhimurium flagellar antigen, and their corresponding Ab title determined by agglutinin assays.Comprised 8–12 weeks-old female mice of Selection III, derived from the original outbred population , and raiFive each of low (LIII) and high (HIII) antibody producer lines were randomly taken for fecal bacteriome determination.The stools were submitted to DNA purification, using the QiaAMP DNA stool mini kit  and the fecal DNA used as templates for PCRs carried out to amplify the sequences encoding the 16SV6–V8 rRNA (16SV6–V8 rDNA). The resulting 433 bp amplicons were resolved by temperature gradient gel electrophoresis (TGGE). PCR primers were U968 (GAACGCGAAGAACCTTAC) attached to a 40 bases GC clamp and L1401 (GCGTGTGTACAAGACCC). PCR conditions and components are described elsewhere . Fecal D16SV6–V8 rDNA amplicons were purified as described above and run in a 0.8 mm polyacrylamide  gel in a TGGE mini system  for 2 h at 130 V, under a 40–44 °C temperature gradient, with 1×TAE as the running buffer. Following electrophoresis, the gel was fixed, silver stained according to the TGGE mini system manufacturer’s protocol, dried at room temperature and submitted to image capturing at the Gel doc EZ Imager . Similarities or divergence among bacterial communities of distinct fecal samples were determined by pair-wise comparison of band profiles from the 16SV6–V8 rDNA amplicons TGGE. The relatedness among mice fecal bacteriome were represented in a dendrogram constructed with the help of DendroUPGMA [8 DNA molecules per microliter and then a pool was formed by mixing 5 µL of each of distinct amplicons. The pooled DNA was attached to the surface of ion sphere particles (ISPs) using the IonPGM™ Template OT2 400 kit  and the corresponding protocol . The emulsion PCR was carried out in the Ion OneTouch™ 2 System (Life Technologies), the quality of amplification was checked and ISP enrichment was performed in the Ion OneTouch™ Enrichment System (Life Technologies). Sequencing primers were then annealed to the ISPs’ single stranded DNA, following Ion PGM sequencing 400 kit protocol (Life Technologies Publication No. MAN0007242). Sequencing proceeded in the Ion torrent personal genome machine  in a ISP loaded 314™ chip. Low quality and polyclonal sequence reads, as well as primers and barcodes were filtered out of the data and the 16SV6 rDNA sequences were available as a FastQ file.Sequencing was preceded by clonal amplification, by an emulsion PCR, of the 16SV6 rDNA amplicons. To accomplish this, each of the four amplicons was diluted to a concentration of 1.56 × 10http://metagenomics.anl.gov/).Quality filtered FastQ files were uploaded to MG-RAST server , where rComparative analysis of fingerprints generated by TGGE of 16SV6–V8 amplicons of the fecal DNA enabled the grouping of all LIII mice, but not of HIII, in a single cluster Fig. , a resulAverage alfa diversity for HIII and LIII was 8.2 ± 2.1 and 6.2 ± 1.9, respectively, with at least 97% of the 16SV6 rDNA reads in both HIII and LIII mice corresponding to Firmicutes (F) and Bacteroidetes (B) Fig. . F/B ratElizabethkingia meningoseptica  and Clostridium hathewayi (Clostridiaceae) in HIII . Although 43 and 54 distinct species were found exclusively in LIII and HIII lines respectively, only the particular association of Ruminococcus torques (41 ± 14 reads) with HIII mice was statistically significant (p = 0.03).Comparative analysis, at the species level, of the sequencing data from the two mice of each lineage, taking in account only the most abundant Bacteria (arbitrarily considered as those found with a minimum of 100 reads in at least one of the mice’s bacteriomes), significant interline variation was seen only at the higher prevalence of S. typhimurium antigen (selection III) [The present study aimed at investigating whether genetically accumulated differences in the ability to produce antibody could influence the structure of mice’s intestinal bacteriome. This was performed by the analysis of stools from Biozzi mice, derived from Swiss lineage by successive inbreeding to display differential quantitative response to ion III) . TGGE ofion III) . TherefoRuminococcus torques (Ruminococcaceae), shown here as an eventual microbiota signature of the HIII line, is less abundant in CD patients, particularly those displaying a C-reactive protein positive reaction [Clostridium hathewayi, along with other butyrate producing Clostridia have been demonstrated to induce the proliferation of T-regulatory (Treg) cells and to attenuate symptoms in experimental colitis and allergic diarrhea when administrated orally to mice [Lachnospiraceae and Ruminococcaceae, primary butyrate producers in human gut, have been negatively associated with human diarreal disease . In the reaction . Clostri to mice . All of LIII and HIII mice are respectively susceptible and resistant to PIA, and susceptible animals show a higher number of pro-inflammatory cytokines IL-1β, IL-12 and TNF-α producing splenocytes . In cyclAlthough the high inter-individual variation among HIII mice, deduced from TGGE fingerprints, could mean lack of a particular feature in this line’s bacteriome, two of them (H3 and its similar fingerprint displaying H4 line’s match) were shown to possess a higher diversity and displaying a taxa composition which, in the light of available literature information, could approximate to a non-disease favoring environment in the gut. Opposing to this normobiosis picture, the less heterogeneous LIII mice’s bacteriome included bacterial taxa associated with a pro-inflammatory dysbiosis condition. These observations should nonetheless be validated by future studies, with the investigation of higher number of animals as well as taking in account other variables such as cage effect , to mini


Myocardial hemorrhage is a frequent complication following reperfusion in acute myocardial infarction and is predictive of adverse outcomes. However, it remains unsettled whether hemorrhage is simply a marker of a severe initial ischemic insult or directly contributes to downstream myocardial damage. Our objective was to evaluate the contribution of hemorrhage towards inflammation, microvascular obstruction and infarct size in a novel porcine model of hemorrhagic myocardial infarction using cardiovascular magnetic resonance (CMR).N = 27) were subjected to coronary balloon occlusion followed by reperfusion and divided into three groups (N = 9/group): 8 min ischemia with collagenase (+HEM); 45 min infarction with saline (I-HEM); and 45 min infarction with collagenase (I+HEM). Comprehensive CMR was performed on a 3 T scanner at baseline and 24 h post-intervention. Cardiac function was quantified by cine imaging, edema/inflammation by T2 mapping, hemorrhage by T2* mapping and infarct/microvascular obstruction size by gadolinium enhancement. Animals were subsequently sacrificed and explanted hearts underwent histopathological assessment for ischemic damage and inflammation.Myocardial hemorrhage was induced via direct intracoronary injection of collagenase in a novel porcine model of ischemic injury. Animals  also resulted in an observable inflammatory response, similar to that arising from a mild ischemic insult (I-HEM). CMR findings were in good agreement with histological staining patterns.Hemorrhage is not simply a bystander, but an active modulator of tissue response, including inflammation and microvascular and myocardial damage beyond the initial ischemic insult. A mechanistic understanding of the pathophysiology of reperfusion hemorrhage will potentially aid better management of high-risk patients who are prone to adverse long-term outcomes. Acute Myocardial Infarction (AMI) occurs due to cessation of coronary blood flow to the heart muscle and the extent of injury follows a wavefront pattern of necrosis in proportion to the duration of occlusion . While pThe presentation of myocardial hemorrhage as a consequence of IRI has been well documented in both humans  and animAlthough some of these observational clinical studies have reported the prognostic implications of myocardial hemorrhage utilizing the predictive power of CMR, the findings have not been consistent in the literature. Several other investigators have found hemorrhage to only be associated with infarct size and presence of MVO, but not offering prognostic significance beyond these parameters , 16. GivA critical question is whether hemorrhage is simply a bystander and a marker of severe microvascular injury or does it actively contribute to downstream myocardial and microvascular damage beyond the initial ischemic insult. Furthermore, hemorrhage has always been associated with MVO , 17; howN = 27) subjected to a coronary occlusion: 1) 8 min ischemia with collagenase resulting in only hemorrhage without infarction  [N = 9) [N = 9).In order to evaluate the contribution of hemorrhage alone in ischemia-reperfusion injury, hemorrhage was artificially induced in porcine hearts by direct intracoronary injection of collagenase. A recent study has demonstrated that intracoronary collagenase is capable of inducing myocardial bleeding in a dose-dependent manner without any mortality attributed to collagenase . This mo, N = 9) ; 2) 45 m [N = 9) ; and 3) Female Yorkshire pigs  were employed in the study and all procedures were conducted in accordance with protocols approved by the Animal Care Committee of Sunnybrook Research Institute. Before catheterization, animals were sedated with an anesthetic cocktail comprising of atropine (0.05 mg/kg) and ketamine (30 mg/kg). Animals were then intubated, mechanically ventilated and maintained under anesthesia using isoflurane (1-5%). Complete coronary occlusion was achieved distal to the second diagonal branch of the left anterior descending (LAD) artery using an over-the-wire angioplasty balloon dilatation catheter ; note that the same level of occlusion was maintained across all animals to ensure consistency. Blood flow was occluded for 8 min (+HEM group) or 45 min (I-HEM and I+HEM groups) and was followed by reperfusion; this was verified by X-ray fluoroscopy . One minute prior to balloon deflation (reperfusion), either 3.2 ml PBS (I-HEM group) or PBS containing collagenase (+HEM and I+HEM groups)  was injected through the guide wire port. Cardiac events during ischemia-reperfusion were managed by antiarrhythmic drugs (75 mg over 5 min amiodarone bolus and lidocaine drip as needed) or defibrillation whenever necessary. Animals were then recovered and examined by CMR at 24 h post-intervention and subsequently sacrificed.CMR was performed on a 3 T whole body scanner  at baseline  and at 24 h post-intervention. Cardiac function was evaluated using a steady-state-free-precession (SSFP) sequence in cine mode: 12-15 short-axis slices, TR/TE = 4/1.7 ms, flip angle = 45°, FOV = 24x21.6 cm, matrix = 224x160, slice thickness = 5 mm, 8 views/segment, 20 phases/slice. Edema was quantified using a previously validated T2 mapping sequence ; spiral Cine SSFP images were analyzed using CVI42 software  to quantify cardiac function parameters: ejection fraction (EF), end diastolic and end systolic volumes  and LV mass. Infarct volume was quantified by segmenting the LGE images using the full-width-half-maximum technique  and regiAnimals were euthanized at 24 h following the CMR examination, hearts were explanted and the LV was sectioned into short-axis slices. Prior to sacrifice, a 4% solution of fluorescent dye thioflavin S  was intravenously injected as a bolus to examine endothelial cell distribution and arterial patency in the heart [p value <0.05 was considered statistically significant.All data  were represented as continuous variables and expressed as mean ± SD. For data involving multiple time points i.e. baseline and 24 h, animal groups were compared using a repeated measures 2-way analysis of variance (ANOVA) statistical analysis with multiple comparisons carried out using the Sidak correction factor ; both within group and across group comparisons were performed. For comparisons across groups only at a single time point (24 h), a one way ANOVA statistical test was performed. Linear regression with goodness of fit analysis was performed between infarct zone T2 and T2* values to determine relationship between inflammation and hemorrhage. A N = 7 animals in each group for further CMR and histological analysis. There were no adverse effects noted or any mortality attributed as a result of the collagenase administration.In the +HEM group, one animal died during the interventional procedure and one animal died post procedure and could not undergo a CMR examination. Two animals died during the infarct procedure in both the I-HEM and I+HEM groups, overall representing a 20% mortality rate associated with the ischemic event. The mortality and exclusions resulted in p < 0.01) and I + HEM  groups, indicative of edematous swelling .Cardiac function parameters by CMR  were not significantly different across the three groups at 24 h post intervention See Fig. . Howevering Fig. . In the p < 0.0001); see bar plot in Fig. CMR revealed distinct patterns of injury across the three groups of animals at 24 h. Figure p < 0.0001) and I + HEM  groups compared to baseline values, indicating the presence of hemorrhage . Hemorrhage volume based on T2* mapping demonstrated that the extent of hemorrhage was significantly greater in the I+HEM group compared with the +HEM group   Fig. .Fig. 3Hep < 0.0001). In the I-HEM group, edema in the infarcted tissue was also mild  and not significantly different than that in the +HEM group (p = NS). The severity of edema in the infarct zone was the greatest in the I+HEM group  and was significantly greater than the other two groups (p < 0.001)  and was the greatest in I+HEM (15.8 ± 8.3%) compared to + HEM (1.7 ± 1.7%) and I-HEM (4.4 ± 2.4%) groups ; see plot in Fig. Figure 01) Fig. . Percentups Fig. . In the Figure This is the first study to systematically and mechanistically demonstrate that hemorrhage is a critical component of ischemia-reperfusion injury. The novel preclinical model developed in this study was able to create three distinct patterns of ischemic injury – hemorrhage only, infarction without hemorrhage and infarction with hemorrhage. This offered a means of probing the contribution of myocardial hemorrhage in AMI in a progressive manner. Key findings of our study were as follows: 1) hemorrhage alone can induce a mild inflammatory response in the heart; 2) hemorrhage when combined with a mild ischemic insult, can further aggravate inflammation and result in greater myocardial injury beyond the initial ischemic insult; 3) hemorrhage can contribute towards the occurrence of an MVO; and 4) edema is correlated with the degree of hemorrhage and the hemorrhagic sites are targeted by infiltrating macrophages. Overall, these observations suggest that a significant interaction occurs between hemorrhage, the inflammatory response and ischemic injury. Thus, hemorrhage is not simply a bystander, but an active modulator of cardiac injury and the reparative processes that follow mechanical reperfusion in AMI.The pathophysiological mechanisms leading to reperfusion hemorrhage in humans are currently unclear. A comprehensive review on intramyocardial hemorrhage in AMI has been recently published by Betgem et al. . In as eClinical studies have shown that patients with durations of ischemia >4 h are at significantly high risk of experiencing major adverse cardiovascular events, such as re-infarction, repeat revascularization, heart failure and death . These aTo understand the consequences of hemorrhage, animal models have been utilized to show differences between hemorrhagic and non-hemorrhagic infarcts using either fixed or variable occlusion times , 19. ForCollagenases are a group of metalloproteinases that can disrupt the interstitial and basal membrane collagen matrix ; when inAlthough the occurrence of myocardial hemorrhage following AMI has been known from human autopsy studies from the 1980s and preclinical tissue specimens , 34, thiThe sole effect of hemorrhage in tissue has been extensively studied in the brain for understanding the implication of intracerebral hemorrhage –41. CMR Following successful reperfusion, the ‘no-reflow’ phenomenon is often encountered, characterized by ischemia-induced microvascular obstruction (MVO) and has been correlated with adverse left ventricular (LV) remodeling and poor patient outcome . Recent Hence, mechanistic studies are still lacking, particularly related to complex interaction of hemorrhage with the ischemic tissue bed and vasculature. Specifically, once hemorrhage is present, does it contribute to worsening the ischemic injury and aggravating the MVO ? Our study was designed to address this aspect and we speculate that vascular compression by hemorrhage and the associated inflammatory response (edema) could potentially worsen an MVO. In the + HEM group, we did not observe the classic MVO, however histology confirmed the presence of a perfusion deficit with the Thioflavin S stain. A possible explanation for this could that the regions of perfusion abnormality were too small and subtle to be detected on CMR given that this group did not present with a classical infarct. However, our findings indicate that ischemia in association with hemorrhage (I+HEM group) leads to a greater incidence of MVO suggesting a potential causal relationship between the two. Furthermore, a secondary inflammatory response arising from hemorrhage could also contribute to the no-reflow phenomenon as suggested in the literature . We woulWe did not note any significant difference in cardiac function parameters, particularly EF at 24 h post-intervention across the three groups. This could potentially be attributed to the relatively small infarct sizes observed in the I-HEM and I+HEM groups with mean infarct size of 2% and 12% (of LV mass) respectively; +HEM group did not show an infarction on CMR. Based on our previous study utilizing a 90 min occlusion model , an infaWe acknowledge several limitations. Firstly, the collagenase-based model for artificially induced hemorrhage is not a true representation of reperfusion hemorrhage resulting from injured/leaky microvasculature. Nevertheless, the model was able to achieve a similar microvascular response. Histological findings clearly identified the extravasation of blood in the interstitial spaces, creating a blood distribution pattern identical to that seen in a 90 min LAD occlusion hemorrhagic model of porcine AMI , 19. ThuOur study also cannot address or distinguish the effects (eg. inflammatory response) of collagenase itself from that arising from hemorrhage, which occurs as a result of its administration; this is an inherent limitation of the model. Nevertheless, the collagenase model does offer an elegant methodology for creating hemorrhagic tissue, similar to that utilized in brain hemorrhage studies , 48. OurDespite a fixed dose of intracoronary collagenase, the amount of myocardial hemorrhage induced was variable across animals; in some animals hemorrhage was predominantly epicardial with some right ventricular involvement. This would be expected in a cohort of animals since the degree of microvascular response to enzymatic action and ischemia is not predictable. Nevertheless, the selection of a high dose collagenase regimen ensured a consistent induction of myocardial hemorrhage within a given group.Lastly, T2 measurements can be influenced by both edema and hemorrhage; T2 is elevated in edematous conditions whereas it is lowered by hemorrhage and its by products , 33, 42.Hemorrhage is not simply a bystander but an active contributor to cellular and microvascular damage and inflammation beyond the initial ischemic insult. CMR is a powerful investigational tool that was able to reveal the underlying interactions of hemorrhage and thus demonstrate its critical role in ischemia-reperfusion injury. Future studies can be designed to address the chronic effects of hemorrhage using this novel animal model. A mechanistic understanding of the pathophysiology of reperfusion hemorrhage in the setting of AMI will potentially aid better management of the high-risk patients who are prone to adverse long-term outcomes.