Diabetic foot infection is one of the major complications of diabetes leading to lower limb amputations. Isolation and identification of bacteria causing diabetic foot infection, determination of antibiotic resistance, antimicrobial potential of protamine by electron microscopy and SDS-PAGE analysis, arethe aims of this study.Pseudomona aeruginosa was checked by minimum inhibitory concentration in 96 well micro-titer plates. The isolates were grown in bactericidal concentration of protamine on plates to isolate mutants. Effect of protamine on protein expression was checked by SDS- PAGE and ultra-structural morphological changes by transmission electron microscopy.285 pus samples from diabetic foot infection patients were collected from different hospitals of Karachi and Capital Health Hospital, Halifax, Canada. Clinical history of each patient was recorded. Bacterial isolates were cultured on appropriate media; identification was done by morphology, cultural and biochemical tests. Effect of protamine against multi drug resistant strains of Staphylococcus aureus 65 (23%), P. aeruginosa 80 (28.1%), Klebsiella spp. 37 (13%), Proteus mirabilis 79 (27.7%), and Escherichia coli 24 (12%). These isolates were highly resistant to different antibiotics. MIC value of protamine was 500 µg/ml against P. aeruginosa. SDS-PAGE analysis revealed that protamine can suppress expression of various virulence proteins and electron micrographs indicated condensation of cytoplasm and accumulation of protamine in cytoplasm without damaging the cell membrane. Results indicated prevalence of foot infection as 92% in diabetic patients. Major bacterial isolates were P. aeruginosa and S. aureus were the major isolates expressing multi-drug resistance and protamine sulfate represented good antimicrobial potential.  Pseudomona aeruginosa wound invasion. A major problem reported with P. aeruginosa infection is its high degree of resistance to some broad-spectrum antibiotics which make it to grow in different challenging environments  of clinical isolate of P. aeruginosa. Furthermore, SDS-PAGE analysis has also been done to investigate the changes produced by protamine sulphate in the protein profile of non-stressed and stressed P. aeruginosa isolated from diabetic foot infection. Protamine sulfate is a well-known cationic polypeptide (CAP) derived from salmon fish sperm. Bactericidal effect of protamine is well known . SeveralEthical certificateThe present study was approved by the Ethical Review Board, University of Karachi, Pakistan.Epidemiology of diabetic foot patientsA total of 350 diabetic patients with foot infection in the age groups of 30 to 80 years were included in the study. Both outdoor and hospitalized patients from different hospitals of Karachi, Pakistan were included. Clinical history was collected from all patients on a specially designed proforma along with the consent form to collect pus sample. Isolation, identification and antibiotic sensitivity of isolates°C in incubator  for 24 hr. The bacterial isolates were identified by standard biochemical methods .Pus samples were collected from ulcers in each case with Amies Transport medium swabs . All spe methods . AntibioDetermination of minimum inhibitory concentration (MIC) of protamineP. aeruginosa isolates, as reported previously . MIC was determined for protamine  against 20 MDR eviously . The culAntimicrobial stress effects on expression of outer membrane proteins in P. aeruginosaP. aeruginosa was performed as reported previously with some modifications . After 24 hr, optical density (OD) of cultures was measured at 480 nm by Spectrophotometer . Density of cultures was set to 1 OD. Broth was centrifuged at 6000 g  for 1 min to collect the cell pellet. Pellet was suspended in 1.0 ml cold PBS (pH 7.0). Cells were sonicated at setting of 10 for 1 min  with an interval of 4-5 min on ice (3 times). Unbroken cells were separated by centrifugation at 5000 g for 8 min at 4 °C. Supernatant was centrifuged at 100,000 g at 20 °C for 30 min. Membrane pellets were collected and stored at – 80 °C  till needed. Effect of antimicrobial potential of protamine sulfate against outer membrane proteins expressed in ications . Isolate°C for 50 min. Collected pellets were resuspended in 200 µl of 2x sample buffer  with 10% β-mercaptoethanol . Samples were heated for 5 min in boiling water bath and samples were loaded on to SDS-PAGE  and stained with silver as reported previously (Pellets were removed from freezer and were suspended in 500 µl of 2% (w/v) Na-lauryl-sarcosine (Sigma) till completely dissolved. Solution was again ultra-centrifuged at 100,000 g at 20 eviously .Standard linear regression curveP. aeruginosa were determined by standard linear regression curve . A standard curve of Rf values of standard proteins versus standard protein molecular weights was constructed to find out the best predicted values using the MS-Excel ver-2007 software. Relative molecular weights of unknown proteins of Characterization of protamine on P. aeruginosa by electron microscopyP. aeruginosa cells was assessed by visualizing through electron micrographs taken at a resolution of 121,000 X. Effect of protamine, with and without stress, was determined as previously reported by Garduno and coworkers , 9 ml of 100 mM sodium cocadylate  pH 7.3 and 0.5% Alcian blue) and negatively stained in aqueous uranyl acetate, dehydrated in acetone, and embedded in epoxy (TAAB 812) resin. Thin sections of 100 nm were cut with microtome  Thin sections were post stained with uranyl acetate-lead citrate and placed on 300 size mesh. The grids were air dried before they were electron micrographed with JEM 1230 Transmission Electron Microscope  at 80 kV. Images were captured using a Hamamatsu ORCA-HR digital camera . All materials and reagents for EM were obtained from Marivac Ltd .Antimicrobial potential of protamine sulphate on oworkers . Fresh cP. aeruginosa was one leading isolated pathogen exhibiting antibiotic resistance as presented in P. aeruginosa shared 80 isolates (28.1%) of the total isolated pathogens. These isolates were resistant to different groups of antibiotics, such as gentamicin, cefotaxime, coamoxiclav, tazobactum and meropenem  patients were having various bacterial and fungal infections as indicated in eropenem .P. aeruginosa from diabetic foot infections as well as revertant mutants of clinical P. aeruginosa 3194 and 3248. MIC values for P. aeruginosa 3194 and 3248 were also recorded, which was found to be 500 µg/ml of protamine. These isolates were subjected to maximum bactericidal concentration of protamine up to 2000 µg/ml.; these mutant isolates were reverted to normal when grown in protamine free medium.Antimicrobial activity of Protamine was assessed against 20 clinical MDR P. aeruginosa mutant 3248, two additional intense bands, band C (27 kDa) and band D (32 kDa) were identified in protamine induced expression  and G (32 kDa) were present in protamine stress condition, under non-stressed condition bands F (27 kDa) and G (32 kDa) were very weak i.e. corresponding proteins were found in low concentrations. We infer from these observations that different proteins were induced and/or suppressed after addition or in absence of Protamine , Staphylococcus epidermidis (3%) and other Streptococci (3%)  patients yielded positive culture for various bacterial and fungal pathogens. Among bacterial isolates, Gram-negative of cases , 24. In ng 18.9% . In anotcci (3%) .P. aeruginosa being the leading microbe. Most P. aeruginosa isolates were resistant to imipenam, meropenem, azotronam, ciprofloxacin, gentamycin, and piperacillin/tazobactam , is useful in treating P. aeruginosa infection, with 500 µg/ml. Action of protamine on different induced mutant strains revealed that they are effective in a range between 125 to 2000 mg/l, compared with 95 mg/l for the wild type strain. Protamine permeabilizes through cell membranes in both Gram-positive and Gram-negative bacteria  outer membrane proteins of P. aeruginosa isolates were absent without protamine as described by Schweizer  disappeared under protamine stress . We have observed the appearance of similar up-regulated results of mexAB- oprM proteins in clinical and mutant stains under protamine stress. This indicates the importance of protamine in lowering antibiotic resistance as documented by Broutin and workers  clinical isolate showed normal outer vesicular membrane structure without protamine. These cells show condensation of the cytoplasm, enlarged periplasmic space and accumulation of protamine within the cytoplasm. We infer from our repeated observations on electron micrographs that there was no cell membrane lyses or any damage in these bacterial cells ( et al. (Salmonella typhimurium cells where cells don’t lyse with the treatment of protamine. These results contradict the earlier studies where protamine stress was reported to disrupt and distend P. aeruginosa cells. These cells noticeably have damaged cytoplasmic membrane and granular cytoplasm (.In our experiments on ultra-structure, al cells  and 4. S( et al.  in Salmoytoplasm , 36.Listeria monocytogenes and Shewanella putrifaciens, as documented by Johansen et al. (Our data on protein profile and ultramicroscopic studies indicates otherwise  and 4. In et al. . Howevern et al. , 37, 38.n et al. . P. aeruginosa which represent it as a good antimicrobial agent. Protamine sulphate may have the potential to be used as an alternate to antimicrobial agent, may be in the form of wound washing agent to reduce the microbial load. This hypothesis is needed to be further investigated by in vivo diabetic animal infection model study to have an insight into the Protamine effect on tissues by histopathology studies and to check cell cytotoxicity before its applications. Results of SDS-PAGE indicate that Protamine can suppress various outer membranes proteins while results of Transmission Electron Microscopy revealed suppressed cellular activities within


IntroductionInappropriate fluid prescriptions result in excess morbidity and mortality in surgical patients. The majority of prescriptions are done by foundation year one doctors (FY1s) despite repeated evidence of poor knowledge and prescription habits among them when it comes to prescribing fluids.Materials and methodsThis was a retrospective observational study conducted at a 798-bed district general teaching hospital. Data for one year from an out-of-hours (OOHs) electronic task record system was extracted. An analysis was performed on all surgical ‘Fluid Reviews’ jobs recorded in the period from August 1, 2018, to August 7, 2019.ResultsDuring the 371-day study period, 1,283 requests for fluid reviews were made. Of these, 1,228 (95.7%) were assigned to the FY1 and 1,185 (92.3%) were requested by nurses. There was a mean of 3.5 ±2.1 requests per day. A bimodal distribution of requests was noted with peaks at 1900 and 2400. There was no discernible variation between different days of the week.ConclusionFluid reviews were most frequently requested by nursing staff at times that coincide with their handover and the commencement of a new fluid chart at midnight. Reducing the number of inappropriate requests for fluid reviews may reduce the opportunity for inappropriate fluid prescribing. Improvements could be achieved through interventions in the ward rounds and by encouraging a multidisciplinary approach to education on fluid prescribing. Reducing the number of fluid prescriptions OOHs promotes continuity of care and education through patient follow-ups. Inappropriate fluid prescribing has been well documented as a source of morbidity and mortality in surgical wards. Complications usually arise from regimes that are either too restrictive or too liberal -3, whileConsiderable efforts have been made to understand the importance of appropriate fluid prescribing in the pre and intraoperative periods. However, fluid prescribing in the postoperative period is a topic that is less well researched . The genDespite the complexities involved in fluid prescribing, the majority of prescriptions are performed by foundation year one doctors FY1s) . . 7]. A sPrevious studies have focused on the type and quantity of fluids prescribed and the implications they have on patient morbidity ,15. TheyThis was a retrospective observational study conducted at a 798-bed district general teaching hospital in southwest England. General surgery and urology, including acute surgical admissions, manage 119 beds in the hospital. OOHs surgical ward cover and admissions, defined as 1700 to 0800 from Monday to Sunday, are covered by one FY1, one senior house officer (SHO), and one registrar. Handover for doctors is at 2030 and 0800 while handover for nurses is at 1930 and 0730.Data were derived from an electronic task record system known as the ‘Doctor’s Taskboard’ . This software is used to request and assign jobs to doctors during the OOHs period. All requests must be set to one of 12 predefined job types. All jobs falling within one year of foundation training were extracted . Duplicate and blank entries were removed. Tasks assigned to ‘Other’ were reassigned to ‘Fluid Reviews’ if appropriate. The resultant dataset was filtered to include all surgical ‘Fluid Reviews’ during the OOHs period prior to analysis.This project was approved as a service evaluation study by the Clinical Audit Department at the Royal Devon and Exeter NHS Foundation Trust.Over the 371-day (53-week) study period, 1,283 requests were made for fluid reviews in the OOHs period. The FY1 was assigned 1,228 (95.7%) of those, and the SHO was assigned 55 (4.3%). The colorectal, upper gastrointestinal, and urology wards made 518 (40.4%), 390 (30.4%), and 245 (19.1%) requests respectively. The remaining 130 (10.1%) reviews were requested in other wards. The majority of requests were made by nurses  with a small proportion requested by doctors . It is not known who requested the remaining 51 (4%).Fluid reviews were uniformly requested OOHs Monday to Sunday with an average of 3.5 ±2.1 reviews per night , with no discernible variation between different days of the week. Fluid reviews were commonly requested and peaked at 1900 and 2400, coinciding with nursing handover and the commencement of a new fluid chart at midnight. While a causal relationship could not be determined in this study, it suggests that the change of nursing staff and completion of the fluid balance chart may prompt the need for a fluid review.Some fluid reviews may be unnecessary and result in the prescription of unwarranted fluids, particularly given fluid reviews are poorly performed by FY1 doctors -12,14. IFluid reviews OOHs were predominantly requested by nursing staff with the majority completed by the FY1; this trend has not changed over the last decade . FurtherThis study has several limitations. Primarily, we used data from a single centre. However, this study identifies data for one year from a large district general hospital and may be generalised to other hospitals adopting a similar OOHs system. The study did not consider requests made during normal working hours, in-person or via the bleep system, and may have therefore underestimated the volume during this period. Nor did it assess how many reviews resulted in prescriptions and whether the prescriptions were appropriate or not. This study captures data representing 70% of the year by including times that fall outside of the normal working week .Our study found that fluid reviews were commonly requested by nursing staff at times that coincide with their evening handover and the commencement of a new fluid chart at midnight. Foundation doctors need to be even more vigilant at these times, as fluids could be overprescribed in this time period. Interventions in the ward rounds and those involving nurses may reduce the number of referrals OOHs and subsequent opportunity for inappropriate prescription of fluids. Future studies could explore the appropriateness of fluid reviews OOHs and whether fluids are appropriately prescribed.


This prospective, randomized, blinded, interventional cross-over study investigated the distribution, elimination, plasma volume expansion, half-life, comparative potency, and ideal fluid prescription of three commonly prescribed intravenous (IV) fluids in 10 healthy conscious cats using volume kinetic analysis that is novel to veterinary medicine. Each cat received 20 mL/kg of balanced isotonic crystalloid (PLA), 3.3 mL/kg of 5% hypertonic saline (HS), and 5 mL/kg of 6% tetrastarch 130/0.4 (HES) over 15 min on separate occasions. Hemoglobin concentration, red blood cell count, hematocrit, heart rate, and blood pressure were measured at baseline, 5, 10, 15, 20, 30, 40, 50, 60, and every 15 min until 180 min. Urine output was estimated every 30 min using point-of-care bladder ultrasonography. Plasma dilution derived from serial hemoglobin concentration and red blood cell count served as input variables for group and individual fluid volume kinetic analyses using a non-linear mixed effects model. In general, the distribution of all IV fluids was rapid, while elimination was slow. The half-lives of PLA, HS, and HES were 49, 319, and 104 min, respectively. The prescribed fluid doses for PLA, HS, and HES resulted in similar peak plasma volume expansion of 27–30%. The potency of HS was 6 times higher than PLA and 1.7 times greater than HES, while HES was 3.5 times more potent than PLA. Simulation of ideal fluid prescriptions to achieve and maintain 15 or 30% plasma volume expansion revealed the importance of a substantial reduction in infusion rates following initial IV fluid bolus. In conclusion, volume kinetic analysis is a feasible research tool that can provide data on IV fluid kinetics and body water physiology in cats. The rapid distribution but slow elimination of IV fluids in healthy conscious cats is consistent with anecdotal reports of fluid overload susceptibility in cats and warrants further investigation. Intravenous fluid alters the volume of expanded body fluid space (v) over time as IV fluid is distributed and eliminated from the body fluid space. Elimination occurs in proportion to volume expansion (v − V) governed by a first-order elimination rate constant (k10). Distribution of IV fluids can be described using kinetic models analogous to the compartmental models in PK analysis. Intravenous fluid can distribute within a single body fluid space, or be distributed between two body fluid spaces, the central (vc) and peripheral (vp) compartment, in proportion to volume expansion of each respective body fluid space governed by first-order bi-directional intercompartmental rate constants (k12 and k21). The schematic diagrams of the one-volume fluid space (1-VOFS) and two-volume fluid space (2-VOFS) kinetic models are shown in The principles of VK are beyond the scope of this manuscript and readers are directed to current reviews , 18, 20.Although VK has been studied extensively in humans and animal research models , 18, 20,ad-libitum access to fresh water.This prospective, randomized, blinded, three-treatment and three-period (3 × 3) cross-over experimental study was performed at the Ontario Veterinary College, University of Guelph. This study, which required the sampling of ~24 mL of blood per cat over a duration of 3 weeks, was approved by and conducted in accordance with the guidelines of the University of Guelph's Animal Care Committee. Ten male intact purpose-bred domestic shorthair cats were enrolled into this study between June and July 2017. Each cat was certified healthy based on physical examination, complete blood count, serum biochemistry profile, total thyroxine level, urinalysis, symmetric dimethylarginine level, and N-terminal pro B-type natriuretic peptide level. All cats underwent a 7-days acclimatization period and were conditioned to tolerate general handling, simple restraint techniques, and wearing an anti-anxiety shirtTwo days prior to the experiment, each cat was instrumented with a central venous jugular catheterFood and water were withheld during each 3-h experimental period. Each cat received 50 mg (≤5 kg body weight) or 100 mg (>5 kg body weight) trazodoneEach cat received three randomly assigned IV fluid boluses on three separate days with a minimum 72-h washout period between treatments. Intravenous fluid treatment consisted of 20 mL/kg of balanced isotonic crystalloid solutionDuplicate blood samples (0.4 mL × 2) were collected at baseline prior to each fluid administration. Subsequently during IV fluid infusion, single blood samples were collected every 5 min for the first 20 min, then every 10 min until the 60-min time point, and every 15 min thereafter until completion of the 3-h observation period . At each blood sampling occasion, 0.4 mL of whole blood was collected into a 500 μL ethylenediaminetetraacetic acid (EDTA) microtainer tube and a total of 0.4 mL of saline flush was returned to the cat. Blood samples were collected from the central venous catheter using a three-syringe technique as follows: instillation of 0.2 mL of saline flush followed by aspiration of at least 0.5 mL of diluted blood sample, collection of desired blood sample, then return of initial diluted blood sample followed by an additional 0.2 mL of saline flush. Hemoglobin (Hb) concentration, red blood cell (RBC) count, and hematocrit (HCT) were analyzed by an automated hematology analyzerAt similar time points for blood sampling, systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), and heart rate (HR) were recorded using an oscillometric blood pressure deviceThe average coefficients of variation (CV) for Hb concentration and RBC count were determined using the respective duplicate baseline measurements . The meaThese calculations were performed using a computer softwareAll data sets, regardless of fluid type, were first analyzed together, and both 1-VOFS and 2-VOFS kinetic models were fitted to the data. Following that, data sets were analyzed separately according to the different fluid types, and both kinetic models were fitted to the data. Sub-analysis of HS infusion was also performed using a three-volume fluid space kinetic model taking into account the effects of osmotic fluid . The varThe differential equations used for the 2-VOFS kinetic model were:R0) for PLA, HS, and HES were obtained directly from the prescribed fluid dosages. The average estimated urine outputs measured throughout the study were used to estimate k10 as follows:Serial plasma dilution and estimated urine output were used as input variables for VK analysis. Estimated urine output was included to stabilize the kinetic models. Intravenous infusion rates  and elimination (k10), were generated using a specialized PK modeling and simulation softwareThe fixed parameters in the VK models, i.e., p < 0.01), the 95% confidence interval (CI) was statistically significant (excludes 1), and the inter-individual variability or coefficient of variation (CV) was <50%.Fluid type, SAP, DAP, MAP, HR, age, and body weight were evaluated as potential covariates in the VK model. The continuous variables SAP, DAP, MAP, and HR were evaluated at every time point, while age and body weight were examined once for each individual data set. The first-order conditional estimation with extended least squares (FOCE-ELS) search routine was applied to provide maximum likelihood estimates for fixed and random effects. A search for trends in plots of random effects was also used to identify potentially significant covariates. A covariate was accepted if it significantly improved the curve-fit through reduction of -2 log likelihood (-2 LL) greater than the critical value of 6.64 . Forward and backward stepwise covariate search was used to identify statistically significant covariates through the reduction of -2 LL > 6.64 (p < 0.01).Volume kinetic and covariate analyses were performed by an independent individual (RH) using the specialized PK modeling and simulation softwaren = 1), catheter site infection (n = 1), and non-salvageable catheter occlusion (n = 2). Throughout the study periods, all cats were normotensive with a median SAP of 116 mmHg (IQR 108–124) and median MAP of 86 mmHg . The median HR was 157 beats per minute (IQR 139–187). The mean baseline Hb concentration and HCT at the start of the study were 99.5 ± 11.0 g/L and 30.3 ± 3.4%, respectively. The final Hb concentration and HCT recorded at the end of the study were 76.9 ± 9.5 g/L and 23.8 ± 2.8%, respectively.Ten cats with a median age of 33 weeks (range 27–33) and mean body weight of 4.79 ± 0.69 kg completed all phases of the study with no adverse effects. All cats maintained normal activity levels and good appetite. No vomiting, diarrhea, abnormal respiratory patterns, or other concerning constitutional signs were recorded. Central venous catheters were replaced in four cats over a duration of 3 weeks due to inadvertent catheter dislodgment (R0) for PLA, HS, and HES were 6.5, 1.1, and 1.6 mL/min, respectively.Thirty experimental data sets  were acquired from 10 cats on 3 separate days. One set of PLA data was determined to be unsuitable for inclusion in the analysis based on serial plasma dilution that exceeded three standard deviations from the mean, likely due to spurious baseline measurement. Of the total 540 paired serial Hb concentrations and RBC counts, 37 (6.8%) measurements were removed due to incomplete data and 18 (3.3%) measurements from the unsuitable PLA data set were excluded from further analysis. The average CV for baseline Hb concentration and RBC count were 3.53 and 3.38%, respectively. The estimated total urine outputs for cats receiving PLA, HS, and HES were 37.3 ± 26.5, 16.2 ± 8.4, and 19.5 ± 14.8 mL, respectively. The mean infusion rates (n = 3) resulted in lower Vc and higher k10 with the differences within an acceptable range of 2–10%.A total of 18 blood samples (~8 mL of whole blood) were collected from each cat for every individual experiment. The plasma dilution-time curve with and without correction for Hb loss from serial blood sampling of the three randomly selected experimental data sets diverged very little over time . In compp < 0.01), and thus is considered the better model for group fluid analysis.Twenty-nine data sets, regardless of fluid type, were analyzed together, and both 1-VOFS and 2-VOFS kinetic models were fitted to the data. The VK parameter estimates of both kinetic models are summarized in p < 0.01) as supported by the improved residual plots  for HS and −1.298 (95% CI −1.621 to −0.975) for HES. Therefore, the Vc of individual fluid types can be expressed as the population Vc to the base of the natural logarithm of the covariance between a specific fluid type and Vc:In the 2-VOFS kinetic model, covariate analysis identified fluid type and MAP as significant covariates . The individual k12 can be expressed by the equation:The covariance between MAP and n = 9), HS data set (n = 10), and HES data set (n = 10) were analyzed separately according to fluid type, and both 1-VOFS and 2-VOFS kinetic models were fitted to the data. Sub-analysis of HS infusion was also performed using a three-volume fluid space kinetic model, taking into account the effects of osmotic fluid shift. The 2-VOFS kinetic model was statistically justified (p < 0.01) for PLA and HS, while the 1-VOFS kinetic model was statistically justified (p < 0.01) for HES based on the reduction of -2 LL > 6.64. The VK parameter estimates of statistically justified kinetic models for PLA, HS, and HES are summarized in The PLA data set (n = 9) demonstrated trends in the plots of random effects, however, none of the tested covariates was statistically significant. The t1/2 for PLA was 49 min (95% CI 29–147). On the other hand, MAP was identified as a significant covariate (p < 0.01) to k10 for both HS and HES with a covariance of −2.865 (95% CI −5.350 to −0.380) and 2.904 (95% CI 0.017–5.790), respectively. The individual k10 can be expressed by the equation:Covariate analysis for PLA  and 104 min (95% CI 69–213), respectively.In consideration of MAP as a significant covariate to The plasma dilution-time curves of PLA, HS, and HES administered at the presumed equipotent doses demonstrated an almost identical peak plasma volume expansion of ~27–30% . ComputeThe ideal PLA, HS, and HES fluid prescriptions to achieve and maintain 15 or 30% plasma volume expansion in a 5 kg healthy conscious cat simulated using VK parameter estimates generated from the respective statistically justified models are summarized in This exploratory study demonstrated the feasibility and potential of VK modeling and analysis in evaluating the distribution, elimination, plasma volume expansion, half-life, potency, and ideal fluid prescriptions of PLA, HS, and HES in healthy conscious cats using serial Hb- and RBC- derived plasma dilution supported by estimated urine output. All cats successfully completed each phase of the experiment with only minor indwelling central venous catheter complications and tolerated the frequent blood sampling well. The frequency of measurement time points was sufficient to complete the VK analysis, although the 3-h observation period did not capture the entire return of plasma dilution to baseline. Kinetic models were successfully fitted to all experimental data sets and a novel approach of VK analysis, where all fluids were analyzed in a unified model, produced similar findings as the conventional individual fluid analysis approach. Overall, the results generated from this study were logical and physiologically sound. The demonstration of similar peak plasma volume expansion following administration of approximately equipotent fluid doses, as well as half-life and fluid potency that conform to expectations suggest that VK modeling and analysis has the potential to investigate IV fluid kinetics and body water physiology in cats.Population kinetics is a PK analysis technique that identifies and quantifies sources of variability in substance concentrations between individuals , which is useful for therapy customization . In thisWhen individual fluid analyses were performed, the 2-VOFS kinetic model was better fitted to PLA, while the 1-VOFS kinetic model was appropriate for HES in our population of healthy conscious cats. This is consistent with VK studies of healthy conscious human volunteers , 27, 30,post-hoc without improvement of these models, suggesting that the estimated urine output truly represents fluid elimination. Regardless, urine output is not a crucial input variable in VK analysis although it serves to stabilize the VK model, if available  and vice versa (k21), were found to be very fast in our study subjects. The distribution phase of PLA and HS in our healthy cats were between 5 and 10 min  in the group fluid analysis, and fluid elimination (k10) for HS and HES. As MAP increases, transcapillary fluid filtration contributes to the increased fluid distribution while stimulation of baroreceptors and activation of renal blood pressure regulation feedback system promotes fluid elimination. The only unexpected finding was a negative covariance between MAP and k10 for HS, in which an increase in MAP decreases fluid elimination. The reason for this phenomenon is uncertain at this time.Although cats remained normotensive throughout the experiments, MAP was found to be a significant covariate for fluid distribution out of the central compartment  but considered acceptable for a preliminary study. For this type of experiment, erroneous baseline measurements had considerable consequences as evidenced by the exclusion of an entire data set even though subsequent serial measurements were appropriate, which further reduced our study sample size. In addition, the average CV of baseline Hb concentration of 3.53% and RBC of 3.38% obtained in our study were higher than the recommended 1% for VK studies  and adhered to the University of Guelph Animal Care Policy and Procedures, the provincial legislation and regulations of the Animals for Research Act, and the national guidelines and policies of the Canadian Council on Animal Care. All individuals with direct animal involvement in this study completed the University of Guelph's Animal User Training Program.XY, SB, and AB contributed to the conception and design of the study. XY and SB conducted the research. XY acquired and organized the data and wrote the first draft of the manuscript. RH performed the data analysis. XY, SB, and RH contributed to data interpretation. SB, AB, and RH provided critical revision of the manuscript. All authors read and approved the final manuscript.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. The handling editor WM declared a past supervisory role with one of the authors.


The current paper is aimed to investigate the effects of waviness, random orientation, and agglomeration factor of nanoreinforcements on wave propagation in fluid-conveying multi-walled carbon nanotubes (MWCNTs)-reinforced nanocomposite cylindrical shell based on first-order shear deformable theory (FSDT). The effective mechanical properties of the nanocomposite cylindrical shell are estimated employing a combination of a novel form of Halpin-Tsai homogenization model and rule of mixture. Utilized fluid flow obeys Newtonian fluid law and it is axially symmetric and laminar flow and it is considered to be fully developed. The effect of flow velocity is explored by implementing Navier-Stokes equation. The kinetic relations of nanocomposite shell are calculated via FSDT. Moreover, the governing equations are derived using the Hamiltonian approach. Afterward, a method which solves problems analytically is applied to solve the obtained governing equations. Effects of a wide range of variants such as volume fraction of MWCNTs, radius to thickness ratio, flow velocity, waviness factor, random orientation factor, and agglomeration factor on the phase velocity and wave frequency of a fluid conveying MWCNTs-reinforced nanocomposite cylindrical shell were comparatively illustrated and the results were discussed in detail.  One substantial concern of engineers in designing structures and researchers in analyzing structures is the durability and permanence of the structure while it is subjected to various kinds of static and dynamic loadings. This issue has become an interesting topic on manufacturing, designing and analyzing structures made of various materials. Composite materials owing to their superb chemical and physical properties are one of suitable candidate to be selected as constituent materials of structures . ComposiBesides, the reinforcements can be in various scales and shapes including fiber as macro-reinforcement and particle as nano-reinforcement. The materials reinforced with the nanoreinforcements are called nanocomposite. Carbon-base materials such as carbon fiber, graphene, graphene oxide (GO), graphene platelets (GPLs), and carbon nanotubes (CNTs) are among the best candidates for being used as reinforcement. CNTs can be divided to Single-walled CNT (SWCNT) and Double-walled CNT (DWCNT) and Multi-walled CNT (MWCNT). They have similar properties and application but, among them, MWCNTs have slightly better properties. The existence of multi-walled nanotube makes MWCNTs more appropriate to utilize as reinforcements. Several investigations have addressed the mechanical responses of reinforced nanocomposite structures ,19,20,21Several problems still challenge the development of the reinforced composite structures. Orientation, waviness, and agglomeration of reinforcements are some of these problems which can substantially affect the mechanical behavior of composite structures. In this context, the analysis and design of composite structures concerning the mentioned factors are of crucial significance. Therefore, the influences of reinforcements’ orientation, waviness, and aggregation on the mechanical response of composite structures have been surveyed by several researchers. Jam, et al.  exploredAnalysis of propagation of waves in structures is one of significant analyses that study mechanical behavior of structures. Besides, this analysis can be helpful in designing, modeling and analyzing structure which are utilized in different systems. Also, in non-destructive tests and structural health monitoring, this analysis can be advantageous ,46,47. IIn the present work, the effects of MWCNTs’ orientation, waviness, and agglomeration on wave dispersion behavior of a fluid conveying MWCNT-reinforced polymeric nanocomposite were investigated in the framework of a novel form of Halpin-Tsai homogenization model. In the new form of Halpin-Tsai micromechanical model, the noted effects can be covered and studied implicitly. Plus, FSDT was implemented to attain kinetic relations of nanocomposite cylindrical shells. In the next step, the obtained governing equations were analytically solved to probe the effect of various variables on the variation of phase velocity and wave frequency of fluid conveying MWCNTs-reinforced nanocomposite cylindrical shells.L and h is depicted in A schematic view of a fluid-conveying MWCNTs-strengthened cylindrical shell with respective length and height of E represent Young’s modulus, ρ represent mass density, and υ represent Poisson’s ratio. Furthermore, subscripts of r and m stand for MWCNT and polymer matrix, respectively. rL is MWCNTs’ length and rd shows its diameter. mV and rV denote volume fraction of matrix and MWCNTs, respectively which have the following relationship: In the current section, a new form of Halpin-Tsai homogenization model and rule of the mixture was applied to determine the material properties of the nanocomposite shell. The effective mechanical properties of nanocomposite shell will be calculated as :(1)E(z)=Rf is the random orientation factor. MWCNTs were assumed to be oriented randomly in three directions in this research and the value of random orientation factor equals 1/6 [Rf=1/3. Wf is waviness factor which can be defined as [A stands for amplitude a wavy MWCNT and W stands for the amplitude and half-wavelength of a wavy MWCNT, as indicated in Waviness, random orientation, and agglomeration factors can significantly influence the value of elastic modulus. The mentioned factors can be considered through the following equation :(5) ζ=fRuals 1/6 . If MWCNfined as :(6)fW=1−Af is agglomeration factor which has the following definition [finition :(7)fA=e−u is axial displacement, v is circumferential displacement, w is lateral displacementsx, θ is rotation about axial direction, and ψθ is rotation about circumferential direction respectively. Hence, the strains of a cylindrical shell that are not zero can be written as:R denotes radius of cylindrical shell. Then, to achieve Euler-Lagrange equations of MWCNTs-reinforced cylindrical shell, Hamiltonian approach was applied and it can be stated as:U indicates strain energy, K indicates kinetic energy, and ncW indicates work done by external loadings. The strain energy’s variation for an elastic structure is written as follows:Pursuant to the first-order shear deformable shell theory, the displacement fields at every point of a fluid-conveying MWCNTs-reinforced nanocomposite cylindrical shell can be defined as : (8)u1(xThe resultant forces and momentum can be stated as follow:By using Equations (18)–(20), Equation (17) can be rewritten in the following form:Moreover, the variation of kinetic energy can be described by:Dot-subscript is related to differentiation with respect to time. fρ and P stand for density of the fluid and pressure of the fluid. Due to the reciprocating identity in the contact points between the speed and acceleration of the structure and fluid, the following equations are developed:vx stands for the mean velocity of fluid flow; shear stress (τ) and viscosity (fμ) relations are formulated as:Herein, the viscous fluid flow in MWCNTs-reinforced cylindrical shells is supposed to be, Newtonian, laminar, fully developed and axially symmetric . Therefofμ and RV are the viscosity and fluid flow of the fluid, respectively.In the above equation the parameters The variation of work done by the exterior loading can be stated as:sκ is a factor for shear correction which is taken as 5/6.Therefore, to attain the motion equations of the cylindrical shell, Equations (21), (22) and (29) will be replaced into Equation (16) leading to the following relations:Integrating the aforementioned equations over the thickness of shell, the following relations will be obtained:At last, by mixing Equations (21)–(27) with Equation (31), the governing ss of fluid conveying MWCNT-reinforced nanocomposite cylindrical shells can be obtained as written below:mU, m,V and mW, while rotation amplitudes are representing by Θxm and Θψm. Moreover, xk is longitudinal wave number and nk is circumferential wave number. Finally, mω shows the circular natural frequency. By inserting displacement fields from Equation (51) in Equations (46)–(50), the following relation can be achieved:A method that consist of an exponential function was considered to analytically solve the obtained governing equations of fluid-conveying MWCNT-reinforced nanocomposite cylindrical shells. Therefore, the displacement fields will be:K, C and M illustrates stiffness, damping and mass matrices, respectively. The elements of every matrix are provided in the By solving the following relation, natural frequency can be calculated:By dividing circular frequency to 2π, the wave frequency can be computed. Furthermore, by setting In present section, several plots are provided to clarify the influence of different parameters on propagation of wave in fluid-conveying MWCNTs-reinforced nanocomposite cylindrical shells. In this research, the thickness of the shell was supposed to be 5 cm; also, both the length and radius of the shell were taken 30 times greater than its thickness. The material properties of MWCNTs and polystyrene are given in a) and phase velocity (b) against wave number for various MWCNTs’ volume fractions is illustrated in xv = 2000. It can be said that both phase velocity and wave frequency increased by an increment in the volume fraction of MWCNTs but the curve of rV = 12% had lower values than rV = 9% which can be assigned to the agglomeration factor. Moreover, because of the existence of fluid in the cylindrical shell, there is a damping effect which caused a reduction in the phase velocity and wave frequency at a certain wave number after which, the phase velocity, and wave frequency rose. The wave number at which the damping effect was observed increased by selecting lower volume fractions of MWCNTs. Generally, by rising the wave number, the wave frequency first increases followed by a decline at damping wave number; after that, it again rises. For the phase velocity, a reduction is first observed until a damping wave number which is then followed by an increase.Variation of wave frequency (a) and phase velocity (b) against wave number is surveyed in rV = 5%. To clarify the effect of waviness factor on wave frequency and phase velocity, agglomeration factor, and fluid flow velocity were not taken into account. The waviness factor descended the variation of phase velocity and wave frequency. Moreover, by rising the wave number, phase velocity first declined and then grew after damping wave number (β = 6).The effect of waviness factor on the changes of wave frequency (a) and phase velocity (b) by wave number alternation regardless of waviness factor and fluid flow velocity at rV = 5%. As mentioned before, similar to waviness, the agglomeration had a negative and decreasing effect. Hence, the trend of diagrams  wave frequency and (b) phase velocity versus the wave number for different fluid flow velocities at rV = 5% considering all factors. According to diagrams, the cylindrical shell without fluid flow exhibited greater phase velocity and wave frequency as compared to its peers possessing various fluid flow. This means that fluid flow had a reducing influence on changes of phase velocity and wave frequency. The damping wave number also varied by changing the fluid flow velocity. On the other hand, the critical flow velocity can be defined for wave numbers whose natural frequency reached its minimum values. Finally, The phase velocity and wave frequency of the fluid-conveying MWCNT-reinforced nanocomposite cylindrical shell decreased with enhancing the fluid flow velocity.An increment in the radius to thickness ratio declined the phase velocity and wave frequency of the fluid-conveying MWCNT-reinforced nanocomposite cylindrical shells.Agglomeration and waviness factor affected the mechanical behavior of nanocomposite shells in a decreasing manner.Regardless of the agglomeration factor, an enhancement in the MWCNT content augmented the elastic modulus, wave frequency, and phase velocity.The increment of parameter α plays a decreasing role in the variation of phase velocity.Resolving the MWCNTs agglomeration is vital to enhance the mechanical behavior of nanocomposite cylindrical shells.The present investigation was aimed to assess the wave dispersion in a fluid-conveying MWCNTs-strengthened nanocomposite cylindrical shell considering the influences of the nanofillers’ waviness, agglomeration, and orientation. The effective mechanical properties of nanocomposite cylindrical shells were estimated using a combination of a new form of Halpin-Tsai homogenization model and rule of mixture. Based on this model, the noted effects were investigated implicitly. Viscous fluid flow was considered Newtonian, axially symmetric, laminar, and fully developed. Navier-Stokes equation was applied to study the effect of flow velocity. To this end, FSDT and Hamilton’s principle were used to derive the governing equations of MWCNTs-reinforced nanocomposite cylindrical shell. Eventually, the obtained governing equations were analytically solved and wave frequency and phase velocity values were calculated. To verify present method, the obtained results were compared to other investigations. The most remarkable highlights can be expressed as:


Subcutaneous mouse tumour models are widely used for the screening of novel antitumour treatments, although these models are poor surrogate models of human cancers.We compared the antitumour efficacy of the combination of ionising radiation (IR) with two DNA damage response inhibitors, the PARP inhibitor olaparib and the ATR inhibitor AZD6738 , in subcutaneous versus orthotopic cancer models.Olaparib delayed the growth of irradiated Lewis lung carcinoma (LL2) subcutaneous tumours, in agreement with previous reports in human cell lines. However, the olaparib plus IR combination showed a very narrow therapeutic window against LL2 lung orthotopic tumours, with nearly no additional antitumour effect compared with that of IR alone, and tolerability issues emerged at high doses. The addition of AZD6738 greatly enhanced the efficacy of the olaparib plus IR combination treatment against subcutaneous but not orthotopic LL2 tumours. Moreover, olaparib plus AZD6738 administration concomitant with IR even worsened the response to radiation of head and neck orthotopic tumours and induced mucositis.These major differences in the responses to treatments between subcutaneous and orthotopic models highlight the importance of using more pathologically relevant models, such as syngeneic orthotopic models, to determine the most appropriate therapeutic approaches for translation to the clinic. For years, subcutaneous tumour models implanted in immunocompromised mice have been a standard for the preclinical evaluation of novel drugs and therapeutic combinations without filling this requirement, and possibly leading to incorrect assessments for translation into the clinic. Preclinical data in tumours grafted orthotopically in immunocompetent hosts are sparse.Radiotherapy (RT) is a mainstay of current anticancer strategies. One of its limitations lies in its toxicity to normal tissues that are close to the radiation field.5 Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear protein involved in base-excision repair (BER), and is critical for the repair of single-strand breaks (SSBs) and single-strand intermediates.7 During genome duplication, the collision of replication forks with unrepaired SSBs and/or trapped PARP–DNA complexes, resulting from PARP inhibition, leads to the formation of potentially lethal DNA double-strand breaks (DSBs), the repair of which is highly dependent on homologous recombination (HR).6 Thus, HR-deficient cells are extremely sensitive to PARP inhibition.8 This synthetic lethality has been extensively investigated in BRCA-mutated ovarian cancer, leading to the approval of the PARP inhibitor olaparib in over 60 countries. However, the efficacy of PARP inhibitors (PARPi) in the clinic may be limited by the emergence of resistance. The restoration of HR competency and replication fork stabilisation (fork protection) has been described as two critical compensatory PARPi- resistance mechanisms.10 As the damage induced by PARP inhibition is generated during the S phase, PARPi-treated cells rely heavily on the DDR protein ATR (ataxia telangiectasia and Rad3-related), which plays a major role in survival during DNA replication stress.10 ATR inhibition significantly enhanced the cytotoxicity of PARPi, not only in BRCA-mutated but also in BRCA-proficient and PARPi-resistant human cancer cells.13Radiotherapy (RT) exerts its cytotoxic effect by inducing DNA damage. Cells have evolved to respond to extensive DNA damage through sophisticated cell-cycle checkpoints and DNA repair pathways. Inhibiting the DNA damage response (DDR) in tumour cells is a rational strategy to augment the cytotoxicity of RT.31 In this study, LL2-luc Lewis lung carcinoma murine tumour cells were implanted either subcutaneously or orthotopically. Established tumours were treated with combination treatments of ionising radiation (IR), PARP inhibitor (olaparib) and ATR inhibitor . As expected, olaparib or AZD6738 radiosensitised LL2-luc cells in vitro, and improved the efficacy of radiotherapy against LL2-luc subcutaneous tumours in vivo. The triple-combination treatment  delayed tumour growth even further. However, the olaparib plus IR combination treatment showed limited efficacy against LL2-luc orthotopic tumours, and signs of toxicity were revealed. The addition of AZD6738 to this therapeutic combination did not augment the antitumour efficacy. In addition, mucositis was observed in a head and neck orthotopic model treated with this triple combination. Thus, our study highlights different responses to antitumour treatments and different therapeutic windows between subcutaneous and orthotopic tumour model settings, warranting the need to define and systematically use clinically relevant preclinical tumour models.Numerous preclinical studies have previously demonstrated that PARP or ATR inhibition radiosensitises human cancer cells in vitro, and improves RT efficacy in vivo in human tumour models xenografted subcutaneously into immunodeficient mice.2O2 was purchased from Sigma-Aldrich. Cells were irradiated with an X‐ray XRAD320 tube .LL2-luc cells were purchased from Caliper Life Sciences . TC1-luc cells generated by the HPV16 E6/E7 and c-H-ras retroviral transduction of lung epithelial cells of C57BL/6 origin were kindly provided by T.C. Wu . The following antibodies were used: anti-Poly-ADP Ribose , anti-PARP-1 , anti-GAPDH , anti-p-Chk1 , anti-Chk1 , anti-actin  and secondary antibodies . For protein extraction, cells were harvested in SDS–urea buffer for PAR staining, or otherwise harvested in RIPA buffer containing protease and phosphatase inhibitors ). Olaparib and AZD6738 were provided by AstraZeneca. HFor proliferation assays, cells were treated with various doses of drug(s), and a WST-1 assay was conducted 72 h later, according to the manufacturer’s instructions . Cells were incubated with the WST-1 reagent, and the absorbance, which is correlated to the number of viable cells, was measured with a spectrophotometer. For clonogenic assays, cells were plated at a single-cell density, treated with the drug(s), and then irradiated or not 1 h later. The colonies were stained with crystal violet. The surviving fraction (SF) was calculated as follows: SF = number of colonies formed/number of cells seeded × plating efficiency of the nonirradiated group.31Animal procedures were performed according to protocols approved by the Ethics Committee CEEA26 . Female C57BL/6 mice  were purchased from Janvier (France) and housed in the Gustave Roussy animal facility . Mice were used after an acclimation time of 7 days. They had free access to food  and water. They were housed on a 12-h light/dark cycle at a room temperature of 22 °C ± 2 °C and a relative humidity of 55% ± 15% . Irradiation was performed locally to the subcutaneous tumour, the whole thorax or the snout with an X‐ray XRAD320 tube , the rest of the body being protected by a lead shield. Olaparib and AZD6738 were administered intraperitoneally 1 h before irradiation in the morning. Olaparib and AZD6738 were formulated in 10% DMSO + 10% kleptose in purified sterile water, and in 10% DMSO + 40% propylene glycol + 50% sterile water, respectively. The control groups were injected with these vehicles and were sham-irradiated. We chose to administer olaparib and AZD6738 at doses previously used in combination with radiation in preclinical studies.3, the mice were allocated to different treatment groups (day 1). The tumour size was measured with an electronic calliper. The tumour volume was estimated from two-dimensional tumour measurements (volume = length × width2/2). The endpoint for survival was a tumour exceeding 500 mm3.For subcutaneous grafts, 600,000 LL2-luc cells  were injected into the right flank. When the tumours reached ~80 mm32 For head and neck tumours, TC1-luc cells were injected at a submucosal site in the right inner lip  under anaesthesia (2% isoflurane).33 Tumour growth was monitored using bioluminescence imaging. For both orthotopic models, the ethical endpoint for survival was loss of more than 20% of the initial weight or severe clinical symptoms.For orthotopic lung tumours, under anaesthesia (2% isoflurane), the skin was incised, 600,000 LL2-luc cells (in PBS + Matrigel (BD#356231), 10 µl) were injected directly into the lung through the pleura and then the wound was closed by suture clips. Tumour growth was monitored by bioluminescence imaging using the Xenogen In Vivo Imaging System 50  under anaesthesia (2% isoflurane).All animals were killed by cervical dislocation. All the experimental procedures were conducted in a laboratory.P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001). Survival data were analysed using the Kaplan–Meier method, and compared statistically using the log-rank test (the Bonferroni correction was used to adjust for multiple comparisons). Tumour growth  was analysed using a two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons .Clonogenic survival is depicted as the mean ± SEM. Statistical analyses were performed using GraphPad Prism software    against LL2-luc subcutaneous tumours implanted in syngeneic immunocompetent mice  Fig. , while o/L) Fig. . Olapariice Fig. .Fig. 1Th32 and tumour growth was followed by bioluminescence imaging. Surprisingly, the antitumour efficacy of IR was not enhanced by concomitant treatment with 50 mg/kg olaparib , concomitant treatment with 50 mg/kg olaparib augmented the antitumour efficacy of RT against subcutaneous LL2-luc tumours Fig. , in agreons Fig. . Thus, t36 Chk1 phosphorylation was also augmented by IR alone   Figs. . Surprisy) Figs. . We incry) Figs. . At highy) Figs. . Tolerabdel Fig. . Notably43 Differences in the responses to antitumour treatment between subcutaneous and orthotopic tumours generated with the same tumour cell line have seldom been reported. In a lung tumour model, cisplatin but not mitomycin C exerted a significant antitumour effect against orthotopic tumours, reflecting the clinical situation, while subcutaneous tumours were sensitive to mitomycin C and less sensitive to cisplatin.44 Sensitivity to doxorubicin has also been described to depend on tumour location, with high sensitivity in subcutaneous sites and low sensitivity in the liver and in the lung, whereas the highest sensitivity to 5-FU was observed in the lung for the same tumour cell  line.45 Immunotherapy was shown to exert differential effects against murine orthotopic and subcutaneous tumours.48 Here, we showed that tumour location affects the efficacy of DDR inhibitors combined with IR in a murine lung tumour model and in a murine head and neck tumour model. Our results suggest that the limited efficacy of the olaparib + IR or olaparib + AZD6738 + IR combination treatments against LL2-luc and TC1-luc orthotopic tumours might be accompanied by tolerability issues. Experiments with the subcutaneous model failed to reveal tolerability issues and the narrowness of the therapeutic window, while such information might be essential for the potential transfer of these therapeutic strategies into the clinic.By including the stromal and immune environment, syngeneic orthotopic tumour models are thought to more closely resemble the complex process of human tumour progression and healthy tissue reaction. Orthotopic models generally grow faster, are more vascularised, are less hypoxic and are able to metastasise.43 Indeed, PARP inhibitors generally increase tumour vessel perfusion, and olaparib radiosensitises hypoxic rather than well-oxygenated tumours in vivo.28 Furthermore, lung orthotopic and subcutaneous implantations may not provide the same immune microenvironment, as previously demonstrated for other tumour sites, such as the colon or the kidney.48 Through the induction of extensive DNA damage, DDR inhibitors can modulate tumour inflammation, immune priming and immunosuppression.51 Thus, the differences in the response to the antitumour treatments in our study might also derive in part from differences in the reaction of the immune system across different tumour settings.The differential responses to the antitumour treatment observed between orthotopically and subcutaneously implanted tumours might be due in part not only to decreased tolerability but also to differences in vascularisation and tumour perfusion, as subcutaneous tumours are known to be less oxygenated than orthotopic tumours.Our study reinforces the critical importance of using clinically relevant preclinical tumour models instead of the subcutaneous implantation model that is too often used. We think that syngeneic orthotopic tumours grafted in immunocompetent mice are far more informative to appropriately select drugs and design clinical trials, and we recommend the extensive use of these models in preclinical studies.Supplementary information


Therefore, the ‘Funding’ section of the Declarations for this paper should have been written as follows:Following the publication of this article, the authors have realized that one of the grant numbers published in the ‘Funding’ section of their paper had been written incorrectly: The grant no. for the granted received from the Medical Science and Technology Program of Guangzhou of China should have been written as “20171A011Funding329 and 20171A010325), Youth Scientific Research Project of Guangzhou Medical University of China (grant no. 2015A14) and the Medical Research Foundation of Guangdong Province of China (grant no. A2018093).The current work was supported by funding from Science and Technology Plan Project of Guangzhou of China (grant no. 201510010177), Medical Science and Technology Program of Guangzhou of China (grant no. 20171A011The authors apologize to the funders of their research project, and to the readership of the Journal for any inconvenience caused.


In this study, we developed a near-physiological non-spontaneously precipitating fluid for an accelerated in vitro calcification assessment, and validated it by analyzing the calcification potential of two prosthetic materials within two reference-tests. The first test focused on the comparison of four calcification fluids under dynamic contact with n=12 commercial bovine pericardium patches. The second one focused on the validation of the most appropriate fluid by analyzing the calcification potential of pericardium vs. polyurethane. The patches were mounted in separate test compartments and treated simultaneously with the respective fluids at an accelerated test frequency. Calcification propensity and progression were detected macroscopically and microscopically. Structural analyses of all deposits indicated hydroxyapatite by X-ray powder diffraction, which is also most commonly observed in vivo. Histological examination by von Kossa staining showed matrix internal and superficial calcifications, depending on the fluid composition. The present study reveals promising results towards the development of a meaningful, cost and time saving in vitro analysis of the calcification potential of bioprosthetic heart valves.Calcification is a major failure mode of bioprosthetic heart valves. So far, cost and time saving  The heterogeneous nucleation represents the material-induced calcification, a relevant parameter for the comparison of different valve materials, which can occur intrinsically within the materials’ matrix and on its surface. Homogeneous nucleation, on the other hand, occurs independently from the presence of any test substrate, and will thus lead to diffuse and primarily superficial calcification, which does not permit an objective comparison of different materials. To avoid this form of calcification in the development of a suitable in-vitro test method, we deliberately focused our previous fluid study on the development of non-spontaneous precipitating fluids. In the present study, we now investigated the heterogeneous calcification tendency of the non-spontaneous precipitating fluids in contact with potentially calcifying materials under dynamic conditions. Additionally, we now investigated two spontaneously precipitating fluids of our preliminary fluid studyin vivo. The most common calcium phosphate phase, which occurs in vivo, is hydroxyapatite (HAP). According to Elliot,via OCP as a metastable precursor during the recrystallization process. 18Calcification is a major reason for the failure of bioprosthetic heart valves.via pores and penetration of the calcification to the subsurface is considered controversial. In addition, the role of mechanical stress as a contributor to calcification is seen controversially. The bond strength between the calcified layer and the polyurethane surface is described as very weak, approximately in the range of Van-der-Waals forces.2+ trap mechanism by phospholipids or due to residual cell fragments that have been devitalized by glutaraldehyde treatment is most probably responsible for the calcification.29In the present study, we conducted two reference tests. The first one focused on the comparison of the precipitation behavior of four different fluids All tests were accompanied by the structural analysis of the resulting deposits and histological localization of the calcification within the tested material or onto its surface.The calcification propensity of four differently composed fluids was tested under accelerated frequency and physiological pressure loads on bovine pericardial patches.All chemicals used were of analytical grade. The water used for the calcification solutions was sterile double-distilled water.n = 3). All patches were of the same order number, of the same pretreatment by the manufacturer and of similar geometry .Twelve commercial glutaraldehyde crosslinked bovine pericardium patches (Edwards bovine pericard 4700) were tested using four different fluids  without contact to a potentially calcifying substrate, a part of each fluid was kept as a reference in a separate polyethylene (PE) bottle within the durability tester at 37 °C. Thus, the reference fluids were exposed to slight vibrations comparable to the ones within the compartments.The compositions of the investigated calcification solutions based on the preliminary fluid studyT concentrations, and fluid C additionally in the PT concentration. For the same ionic strength, this resulted in different activities of free calcium and HPO42-, which allowed the different ion products and saturation levels to be adjusted, especially with regard to the solubility product of DCPD. As the saturation degree concerning the solubility product of DCPD is considered as an initial factor of in vivo calcification and spontaneous precipitation,T] was increased in order to increase the ionic product towards the Ksp of DCPD and thus decrease the degree of undersaturation towards saturation.All selected fluids had an ionic strength of ~ 0.16 M,The fluid preparation was performed at room temperature due to a better laboratory handling. Thus the pH adjustment of the barbital buffer by addition of 1N hydrochloric acid solution also took place at room temperature, considering the temperature dependency of the barbital buffer.13Preservation of the calcification fluids during the test period was done by addition of 0.05 % sodium azide to each fluid. Due to the photosensitivity of the barbital buffer, the fluid preparation as well as the calcification testing were performed under light-protected conditions.The initial concentrations of total calcium and total phosphate in the fluids were determined by colorimetry . The calcification propensity and progression during the test were detected macroscopically and microscopically on the patches´ surfaces. Additionally, the course of the test was controlled by colorimetric measurement of the calcium and phosphate decrease within the fluid of each test compartment. The sodium and potassium concentrations of the fluids were determined potentiometrically, both with the Vitros Chemistry System 350 .θ at a sweep rate of 0.1° 2θ/min or with a D8 Advance Bruker AXS powder diffractometer between 10° and 80° 2θ at a sweep rate of 0.1° 2θ/min. When using the D8 device, the sample material was fixated on the sample carrier with silicone paste. The noise component of the signal caused by the silicone paste was determined by taking a reference spectrum of silicone paste, under the same conditions as the actual sample measurements. For XRD measurement, the deposits were mostly separated from the pericardial material, dehydrated with ethanol 98%, suction filtered and dried in a desiccator.Structural analysis of the deposits was carried out by X-ray powder diffraction (XRD) according to the availability with a Philips X’Pert Pro (MILIDI) powder diffractometer between 10° and 80° 2Histological examinations of the calcified pericardium patches were made from 5 µm thin slices of the cross section areas by HE-, EvG-, Alizarin red- and von Kossa staining. The histological examinations were conducted at the immunohistochemistry facility of the IZKF of the medical faculty at RWTH Aachen University.The calcification propensity of glutaraldehyde crosslinked bovine pericardium patches was tested against polyurethane patches under accelerated frequency and physiological pressure loads using the most appropriate calcification fluid of the Fluid-Reference-Test, fluid L.For the chemicals used here, the same specifications apply as described for the Fluid-Reference-Test.Three commercial glutaraldehyde crosslinked bovine pericardium patches  were tested against three polyurethane  patches produced in-house. All patches were of a similar geometry .The used test system, the mounting of the test patches and the adjusted test conditions were the same as described in the Fluid-Reference-Test. But for the Fluid-Material-Differentiation-Test the same fluid composition was used for all test patches and the test duration of 9 weeks.The composition of the calcifying solution used was similar to fluid L of the Fluid-Reference-Test Table .Fluid preparation, preservation and testing were performed according to the same protocol as described for the Fluid-Reference-Test.Fluid concentration measurements and the detection of propensity and progression of the calcifications on the patch surface were performed as described for the Fluid-Reference-Test.θ at a sweep rate of 0.1° 2θ/min. The sample material was fixated on the sample carrier with vaseline. The noise component of the signal caused by the vaseline was determined by taking a reference spectrum of vaseline, under the same conditions as the actual sample measurements. For XRD measurement, the deposits were mostly separated from the patch material, dehydrated with ethanol 98 %, suction filtered and dried in a desiccator.Structural analysis of the deposits was carried out by X-ray powder diffraction (XRD) with a D8 Advance Bruker AXS powder diffractometer between 10° and 80° 2The histological examination of the calcified patches was carried out in the same way as described for the Fluid-Reference-Test.The reference samples of the fluids C and E showed a slight precipitation within the PE bottles during each week. The reference samples of the fluids F and G showed no precipitation.In contact with potentially calcifying pericardium patches the fluids C and E revealed a progressing calcification over 4 weeks Fig. , which sThe patches tested in the fluids F and G showed no calcification within a testing period of 4 weeks. Thus, the test period with fluid G was prolonged up to 9 weeks. Fluid F, as the most undersaturated fluid, was replaced by its modification, renamed fluid L Table  up from All calcified pericardium patches showed the beginning of calcification right next to the circular line of fixation, independent of the fluid used. Radial inwards growth of the deposits was observed during the following weeks Figs.  and 2.After four (fluids C and E) or nine (Fluid L) weeks of testing, respectively, the resulting deposits from the patches were analyzed by XRD. The patches P10 to P12, tested in fluid G, did not reveal sufficient deposits for analysis. Additionally to the diffractograms of the deposits of the patches P2 and P6 Figs. a to deteThe principal tissue stains HE and EvG, exemplarily depicted for cross sections of P2 mainly show collagen fibers of the pericardium Figs. a.1–4b.2 The special stains used to identify mineralization in tissue sections, Alizarin Red and von Kossa, are also depicted in Figure The calcification behavior differed distinctly between the two test groups, pericardium and polyurethane test patches. The reference part of fluid L exhibited no precipitation. The pericardium patches showed a recognizable start of calcification at 6 weeks right next to the circular line of fixation. Radial inwards growth of the deposits was observed during the following 3 weeks Fig. . HoweverThe structural analysis was conducted from the patches with the most amount of deposits, Peri1 and Peri2. X-ray powder diffractograms of the patch-deposits are depicted in Figure Cross sections of the calcified area of Peri3 indicated collagen fibers at the principal stains HE and EvG Figs. a.1–7b.2,To provide a cost- and time-saving method for the assessment of the calcification potential of bioprosthetic heart valves, several attempts towards an accelerated in-vitro model were undertaken. Due to the problem of superficial calcification or spontaneous precipitation, which occurred in the fluids applied,In  bioprosthetic valve replacement, which is often preferred to mechanical valve replacement because of the lack of need for lifelong anticoagulation, failure of the valve due to calcification-related stiffening of the leaflets after about 10 to 12 years is a serious problem.5As expected from the preliminary fluid study,Besides the calcification potential and the macroscopic localization of the occurring calcification, the structural characterization and microscopic or histological localization of the deposits played a decisive role in the assessment of the fluids.According to the comparison with the reference maps for DCPD, OCP and HAPvia OCP. The conversion from ACP or OCP to HAP depends on the Ca2+-availability and takes place within 24–72 h as stated in literature.2+ concentration of the considered fluids, so that it can be assumed that in the pure fluid study  the conversion of OCP to HAP was delayed or prevented as a function of the lower Ca2+-availability, which was exceeded by the material effect in the patch study. Fluid E, which had a distinctly higher Ca2+-content, already showed HAP as the predominant phase of the spontaneous precipitates in the fluid study2+-availability as a controlling factor in OCP/HAP transformation.Even Fluid C, which in the previous fluid studyAll von Kossa stains Figs. d.1–4f.6 As expected for fluid L from the Fluid-Reference-Test, the pericardial patches showed the macroscopic beginning of calcification after week 6 of testing. Comparable to the Fluid-Reference-Test, the calcification of the pericardial patches commenced at the area where the mechanical bending stress due to the clamping was highestBased on the results for the patches PU1 and PU3, it was found that the tested polyurethane in the shape of foils did not represent a heterogeneous calcification nucleator.As the comparison with the deposits of the Fluid-Reference-Test and the reference maps for DCPD, OCP, HAPThe result of von Kossa staining of the patch cross-section of Peri3 underlines the pronounced occurrence of intrinsic calcification Figs. d.2–7d.4 via fetal calf serum and addition of suitable cells. The in vitro use of these fluids is a challenge, especially because of the need to avoid long-term contamination, and is part of our ongoing studies. For further application in standard in vitro tests, the use of such fluids would also represent a cost issue.Since we have deliberately kept the fluid composition simple and have limited it to the essential physiological calcification factors, i.e. we have refrained from adding fluid-side inducers or inhibitors, a possible effect of non-collagenous proteins, as discussed in the literature,Another limitation arose from the fact that we wanted to develop an accelerated test to get results within a reasonable time. The increased mechanical load applied for this purpose led to expansions of the firmly clamped patches, especially the pericardial patches, which are certainly not comparable with deformations under physiological conditions. In order to achieve a more realistic performance under accelerated conditions, further systematic calcification studies with heart valve prostheses that react differently to stress due to their opening and closing behavior need to be conducted. The influence of patch deformation on calcification behavior was not investigated in this study, as this study only focused on evaluating the fluid used, based on its ability to differentiate between different calcification propensities of the tested materials, regardless of what caused these different tendencies.in vitro calcification tests from four resp. five different fluid compositions. The comparison of the XRD analyses of the in vitro calcifications with literature diagrams of synthetic hydroxyapatite and human aortic valve calcifications, revealed HAP as the main phase formed in pericardial contact. The Fluid-Material-Differentiation-Test confirmed the calcification potential of Fluid L and showed its ability to differentiate between materials with different calcification propensities by means of pericardial patches and polyurethane foils. Also in this test, HAP could be confirmed as the main mineral phase of the deposits, as well as the distinct matrix internal intrinsic localization of the deposits, besides a smaller amount near the substrates’ surface.The fluid reference study allowed us to select the most suitable non-spontaneously precipitating fluid for dynamic In further tests, it would be interesting to examine differently pretreated bioprosthetic heart valves in Fluid L instead of patches. It may even be necessary to switch to Fluid G with even weaker calcification potential, since bioprosthetic heart valves may have a higher calcification potential in the dynamic test setting than the patches, due to different load distributions.in vitro calcification method will be the adaptation of the fluid to systemic physiological conditions in order to include protein action and possibly cell participation in the calcification model and to create a suitable environment for the assessment of systemically active anti-calcification treatments.A further step in the development of a suitable


To provide a simple numerical formulation based on fixed grids, a wavelet element method for fluid–solid modelling is introduced in this work. Compared with the classical wavelet finite element method, the presented method can potentially handle more complex shapes. Considering the differences between the solid and fluid regions, a damping-like interface based on wavelet elements is designed, in order to ensure consistency between the two parts. The inner regions are constructed with the same wavelet function in space. In the time and spatial domains, a partitioned approach based on Jacobi iteration is combined with the pseudo-parallel calculation method. Numerical convergence analyses show that the method can serve as an alternative choice for fluid–solid coupling modelling. In the context of structural health monitoring, as well as seismic and acoustic exploration, the numerical modelling and analyses of fluid–solid interfaces and coupling have been considered crucial issues, due to the complexity of the related physical phenomena. For waves propagating in a single medium , nearly all types of numerical methods can be used, such as the finite difference method ,2 and itIn fluid–solid interaction problems, both the fluid domain and the solid domain are deformed. For elastic and incompressible problems, the solid region is usually modelled by a fixed grid technique, due to its small deformation, while the grid of the fluid part can be altered between moving and fixed grids . The arbBesides grid techniques , simulation techniques and solving techniques are other aspects to be considered. The fluid–solid interaction problem can be implemented in a monolithic or partitioned formulation . The monIn this work, we developed an isoparametric wavelet element formulation with Jacobian matrix, which allows the method to potentially handle more complex shapes. With the consideration of the balance between physical and geometric interpolation, a B-spline-based wavelet is used as the interpolating/shape function. Different with the classical interpolating/shape function, the transform matrix is introduced to avoid the use of wavelet coefficient and controlling point for B-spline-based wavelets. The superiorities of B-spline in structural analysis and geometric approximation make the corresponding wavelet element a promising selection for the inviscid fluid–solid coupling problem. Considering the difference between solid and fluid regions, in terms of their degrees of freedom, a damping-like interface based on wavelet elements is designed. The inner regions, both for the fluid and the solid, are constructed using the same wavelet function in the spatial domain. A partitioned approach based on Jacobi iteration  is used,u denotes the displacement of the solid. For the two-dimensional case, u is expressed by T. The symbols σ and ε denote the stress and strain tensors, respectively. The symbols λ and μ are the Lame parameters, and sρ is the mass density of the solid waveguide, referring to the inertial force of the inspected region. The matrix I is the identity tensor. The vector sf defines the general external force in the solid region. The function tr(·) is employed to obtain the trace of the bracketed term. The fluid region, where only the acoustic wave propagates, is governed by the conservation and dynamics equations:v denotes the velocity of the fluid, the constant k is the bulk modulus of fluid. The mass density of fluid is defined by fρ.In order to simplify the analysis and to highlight the performance of the presented numerical process, in this paper, the fluid part is considered inviscid and incompressible, while the solid part is considered elastic and isotropic. The strong form  of the wave motion equation in the solid region can be written, in vector form, as Equations (1)–(3) based on Lame parameters:v is replaced by the gradient of the potential function As the fluid is irrotational, the variable u and v. To address this problem, Komatitsch et al. T for the solid region, and x, y) for the fluid region, and integrating by parts over the field of interest, we obtain Equation (10) for the solid region:s and Ωf, respectively, and the interface between them is defined by the symbol fs, in the solid region, which can be implemented into the fluid region. Based on the interface components in Equations (10) and (11), the solid and the fluid system are coupled. In numerical formulation, the Dirichlet–Neumann decomposition method is used to connect the coupled regions:In finite element methods, problems are solved using their corresponding weak forms. By dotting the partial differential equation with the trial function (or interpolating function) mth-order j scale B-spline wavelets on the interval (BSWI) interpolating function mth-order 0 scale B-spline functions and the corresponding wavelets given by Goswami , respectively, depicting the normalized x and y co-ordinates. The two-dimensional interpolating function is formulated based on the Kronecker product between the two vectors in Equation (15); namely, u is approximated by the interpolating function N and node vectors ue as:Based on Equation (14), the interpolating functions, in horizontal and vertical directions, are defined as:N is replaced by T is required to transform the wavelet coefficients into the physical domain, in which the interpolating function N yields:e depicts “elemental”. Equilibrium is also satisfied in each element. Based on the Hamilton principle, we further obtain the wavelet element method in matrix form:Note that the wavelet coefficients can be obtained when the interpolating function M is the mass matrix and K is the stiffness matrix. The pseudo-damping C is the coupling matrix of the interface, which acts on the first order temporal derivatives of u and J is the Jacobian matrix which transforms the normal region to a curved region, and the constants iw and jw are the weights referring to the Gaussian integral. As an isoparametric method, the shape function related to J is also defined by the same BSWI used for interpolation. Here, we give the explicit expression of the Jacobian matrix based on BSWI43  element.Assembling the elementary matrices together, we further obtain:j and order m should yield:The even order spline is usually used in structural analysis; thus, the fourth-order spline is selected. In order to ensure the existence of inner wavelet, scale j is fixed at 3.Then scale 0 is given by [3 function The BSWI4given by , then tha like:F depicts the unknow function to be interpolated. Thus, T matrix is employed to connect wavelet coefficient a with physical region u as:T matrix. The Jacobian matrix:Similarly, we can obtain d = T to distinguish with deflection u. The main entries of J matrix are calculated by:Here the location is denoted by The main components of J matrix can be calculated according to Equation (37).3 element is presented in The interpolating results of a quarter circle idealized by one BSWI4The central difference time integration scheme for temporal discretization is Equation (45):t and time step Δt define the varying times in the numerical calculation. Two difficulties are implicated in solving the above equations directly: (1) the large dimension of the stiffness matrix and (2) the iteration between two mediums in different time steps.The subscripts related to the time t  in the central difference time integration scheme, in order to ensure convergence. In each time step, the matrix calculation and the inverse computation should be iteratively solved. Therefore, Equation (45) is difficult to solve on the level of the global matrix. We chose the pseudo-parallel method : (a) in the solid region, in order to estimate the displacement field teration , as showK, a semi-full matrix, is not used in the calculation; instead, it is established on an elementary level by the equations M and interface matrix C are assembled in our process, the mass matrix M is a diagonal matrix, which can be treated as a vector, and its inverse can be easily calculated . The interface matrix C is similar to M but sparser. The presented calculations are all accomplished in only one grid system, where the absence of a staggered grid guarantees the simplicity of the structure of the developed algorithm.Based on the definition in Equation (45), the algorithms in (1)Cases A–C were designed for different regions  and different interfaces .(2)Case A-1 and Case A-2 for Case A). These two sub-cases were used to simulate wave propagation from solid to fluid and fluid to solid, respectively.Two sub-cases were set in each case Cases A–C only present some qualitative comparisons with the theoretical wavefront in snapshots. Quantitative analyses are given at the end of this section, in terms of convergence analysis.(4)Cases A–C, convergence analysis was only conducted for Case C, the most complex case among them.Considering the similarity of Numerical validations were organized in the following manner:f1 = 75 kHz and the modulation frequency f2 = 75 kHz:In order to demonstrate the performance of the presented method, a simple fluid–solid coupling example is conducted in this section. The inspected regions are treated as the plane strain problem and the material parameters are given in The wave propagation is illustrated by contours in the following parts. In the solid region, the contours represent the displacement defined by The solid part, with dimensions 0.125 m × 0.25 m, was divided by 10 × 20 BSWI elements—20,301 nodes and 40,602 dofs (degrees of freedom)—and the fluid part, with dimensions 0.125 m × 0.25 m, was also meshed by 10 × 20 BSWI elements—20,301 nodes and 20,301 dofs. It should be noted that the BSWI elements used in this paper had 121 nodes per element, thus having 121 dofs and 242 dofs for fluid and solid, respectively. The number of dofs seemed to be not large enough, compared with some commercial software; however, it becomes further multiplied when time iteration . The wavefronts of these two secondary waves also qualitatively agreed with the theoretical wavefronts. The above results validate the effectiveness of the present method in normalized regions.To bilaterally validate the effectiveness of the interface, the source was then moved to the fluid region for verification. The grid was kept the same. In Case B, a rectangular region divided by a curve, is shown in Case B-1 Grid-1, the densest mesh composed of 1920 elements  for the fluid part and 800 solid elements  for the solid part.(2)Grid-2, the grid used in (3)Grid-3, a denser mesh composed of 648 elements  for the fluid part and 324 solid elements  for the solid part.(4)Grid-4, a sparse mesh composed of 392 elements  for the fluid part and 196 solid elements  for the solid part.Convergence analyses were conducted on It should be mentioned that the grids were similar in structure, but had different densities.Case C-1, in which the wave travelled from the solid part to the fluid part. The wave source was located at −0.0360 m and 0 m, and assigned vibrations along X-direction. The receiver was located at −0.0625 m and 0 m in the solid region. The fluctuation of the wave source determined that the X-direction responses at the receiver were dominated by the P-wave  can achieve a satisfying iteration speed, while under-relaxations converge slowly.It is clear that Aitken relaxation performed best among the inspected methods. From the convergence behavior of Aitken relaxation shown in In this work, the inviscid fluid–solid coupling problem was solved by an iterative model using the wavelet element method and a partitioned approach. Based on the use of the Jacobian matrix in the wavelet element method, problems relating to curved boundaries can be solved. The interface was integrated into the model as a pseudo-damping layer, which can conduct transformations between force vectors in the solid region and pressure in the inviscid fluid region. As validated by our results, the presented method can serve as an alternative choice for the issue under consideration, both for normal regions and the curved region.This work focused on inviscid fluid, but the problems relating to the influence of viscosity on the interface and numerical format  can be further considered and investigated. To achieve efficient computation, fixed grid technology was used for the fluid region, by use of a Cartesian grid. As a result, the flow solver may be simple and fast; however, it sacrifices accuracy near the interface, due to the interpolations used. The use of a finite element method in both solid and fluid regions makes the formulation simple; however, the accuracy depends on the ratio between the fluid and solid grid size. Due to the effects of temporal iteration and numerical oscillation on convergence behavior, the sizes of elements are determined by the highest frequency domain considered, which may necessitate a memory- and time-related computational burden.


This study aimed to determine the efficacy and complications of intravitreal chemotherapy-assisted endoresection for refractory International Classification of Retinoblastoma (ICRB) group D retinoblastoma in monocular patients.In this retrospective case series, intravitreal chemotherapy-assisted endoresection by pars plana vitrectomy was performed in 11 eyes with refractory ICRB group D retinoblastoma unresponsive to standard therapies in monocular patients.Across a mean follow-up period of 42.7 months, globe salvage was attained in all 11 eyes (100%). There were no cases of extra-ocular tumour seeding or remote metastasis. In 9 eyes (81.8%), tumour control was achieved with one pars plana vitrectomy; in 2 cases (18.2%), repeated treatment, such as laser therapy, intravitreal chemotherapy or a second pars plana vitrectomy, was needed. Retinal reattachment was achieved in all 4 eyes (100%) with previous retinal detachment. Four eyes (36.4%) required subsequent cataract surgery due to secondary cataract. Ten eyes (90.9%) had improvement in best-corrected visual acuity at the last follow-up.Intravitreal chemotherapy-assisted endoresection appears to be a safe and effective globe-salvaging method for refractory group D retinoblastoma. It is a promising alternative to enucleation and a supplementary therapeutic strategy for those unresponsive to standard therapies, especially for the monocular retinoblastoma patients. Over the past two decades, the management of retinoblastoma has dramatically changed from eye-sacrificing methods to eye-preserving alternatives . Eye preIViC was first introduced in 2003 , and it Theoretically, endoresection by pars plana vitrectomy (PPV) is the ideal way to eradicate a tumour and solve vitreoretinal complications, but its use in the treatment of retinoblastoma has been controversial due to the high risk of metastasis, orbital seeding and extraocular extension . HoweverThis retrospective cohort study was conducted between May 2013 and March 2019. The children were referred for this study when refractory retinoblastoma was unresponsive with uncontrolled intravitreal seeding or tumour recurrence after receiving six cycles of systemic chemotherapy with a standard vincristine/etoposide/carboplatin (VEC) regimen in all cases and different strategies of local chemotherapy  was performed once, 1 week before surgery. While performing 25-gauge PPV, intraoperative IViC of 5 μg/ml topotecan in irrigation fluid  was applied. The visible intravitreal tumour cells were removed through vitrectomy; the subretinal tumour lesions were removed with extended resection accompanied by electrocoagulation at least 3 mm from the margin of the mass. To avoid retinal detachment, laser coagulation was applied to the boundaries after resection  were applied to all three of the sutured incision sites.After surgery, all patients underwent routine eye examinations as well as neuroimaging (CT and MRI) to evaluate if there was any tumour recurrence, metastasis or orbital/extraocular extension. IViC and laser treatment were applied when there was any sign of recurrence. Additional PPV was performed when recurrence could not be controlled by IViC and/or laser treatment. Cataract surgery was performed for secondary cataract at least 6 months after the first PPV if there was no sign of recurrence or involvement of the anterior segment. Silicon oil removal was considered when any oil-related complications occurred in the eye, such as oil emulsification, oil migration, secondary glaucoma, etc.This study was performed after obtaining informed consent from the patients and their family, and it was approved by the Board of Ethics at Sun Yat-Sen University Zhongshan Ophthalmological Center.A total of 11 eyes were included in this retrospective cohort study conducted between May 2013 and February 2019. The basic information, ocular features and treatment history of the patients are presented in Table The follow-up time, tumour recurrence and metastasis, post-operative complications and subsequent treatments are presented in Table Two cases (8 and 9) showed tumour recurrence after primary PPV without intravitreal seeding or anterior chamber involvement. In case 9, tumour recurrence was found 2 months after PPV, and it was observed as a focal solid tumour at the edge of previous endoresection. After repeated IViC  and retinal laser photocoagulation, the tumour regressed and no recurrence was observed during the follow-up period Fig. . In caseThree of the 4 cases with pre-surgical retinal detachment achieved anatomical restoration of the retina after undergoing PPV once. One case (case 9) had recurrent retinal detachment 12 months after the first surgery, and retinal reattachment was achieved after a second PPV with no recurrent retinal detachment during subsequent follow-up. Secondary cataract was observed in 4 cases  and cataract surgery (phacoemulsification and intraocular lens implantation) was performed. Silicone oil removal was performed in 2 cases  when silicone oil emulsification was seen. Silicon oil was not removed when focal recurrence was found (case 9); in this situation, further IViC and laser treatment were done in the silicon oil-filled eye.The changes in best corrected visual acuity (BCVA) are shown in Table For many years, the risk of dissemination, seeding and extraocular spread has been the biggest obstacle for surgical intervention in patients with retinoblastoma. Honavar et al. reported an unfavourable outcome in 75% of patients with retinoblastoma who underwent PPV in 2001 . With thIViC-assisted endoresection should be performed with strict control of the indications and contraindications. We suggest that only previously treated (IVC/IAC/IViC) refractory ICRB group D cases with obvious vitreous and/or subretinal seeding should be considered for this form of treatment. The direct and definite removal of tumour is the significant advantage of endoresection, so it is especially helpful for cases with a high burden of vitreous and/or subretinal seeding. The IViC-assisted endoresection by PPV can reduce the tumour size and its resulting burden while facilitating extensive and uniformed distribution of the chemotherapeutic drug in the vitreous cavity, which further enhances the efficiency of the drug and reduces the amount of treatment required and the retinal toxicity of repeated IViC .The contraindication of endoresection for retinoblastoma is any sign of tumour metastasis in the anterior segment or extraocular metastasis, which suggests that ICRB group E cases should be excluded. In the study by Zhao et al., the retinoblastoma was not controlled by one-time use of PPV in 4 patients, all of whom demonstrated tumour metastases in the anterior chamber of the eyes, and only one eye was preserved . This reAlthough many preventive measures have been used, the tumour can still recur. In our study, two cases had single or multiple focal subretinal tumour recurrence. Fortunately, the recurrent tumours could be eradicated by repeated IViC, laser coagulation or a second PPV. The relatively good prognosis was mostly attributed to the absence of recurrent vitreous seeding. We assumed the silicone oil tamponade helped confine the tumour as a focal lesion of the retina, thus limiting intravitreal tumour seeding. Therefore, intravitreal silicone oil tamponade is recommended during PPV to prevent tumour dissemination, especially vitreous seeding. Since the tumour may recur a long time after surgery , we suggest that the silicone oil tamponade time should be prolonged appropriately, unless any oil-related complications occur.In addition to eradicating intraocular tumours, IViC-assisted PPV can also help address the sight-threatening vitreoretinal complications of retinoblastoma, such as vitreous haemorrhage and persistent retinal detachment. In our study, 4 of the11 eyes had pre-surgical retinal detachment, and all of them achieved successful anatomical retinal reattachment after PPV. This is especially crucial for monocular patients who have no other alternative for retaining visual function and who require eye preservation. In our study,10 of the 11 monocular patient had improved vision after PPV; in 4 cases, the patient’s vision had improved to ≥20/200. The favourable visual function outcomes also proved the value and good prospect of this treatment strategy.However, this study has some limitations. Like any new technique, it is difficult to conduct a randomised controlled trial for treating retinoblastoma, as it is a relatively rare disease. The follow-up period in this study was also relatively short, with a mean of 42.7 months (ranging from 12 months to 83 months). Since retinoblastoma could recur years later, further follow-up is required to evaluate the long-term effects of this treatment method. We also used topotecan instead of melphalan because topotecan was the only chemotherapy drug currently approved in mainland China for the treatment of retinoblastoma.This study showed encouraging results for using IViC-assisted endoresection to control ICRB group D retinoblastoma. This suggests that it might be considered to be a supplementary therapeutic strategy for some refractory cases that are unresponsive to standard therapies, especially for monocular patients for whom the other affected eye has been previously enucleated.