Microsatellite Instability (MSI) is a hallmark of colorectal cancer (CRC) and occurs in 15–16% of CRC. Molecular biological information of CRC in South Africa (SA) is largely unrecorded. This study was undertaken to determine the frequency of MSI, with particular reference to Lynch syndrome (LS) with a view to improve surveillance and prevention strategies. This was a retrospective study on CRC samples diagnosed between 2011–2015 at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). Samples diagnosed between 2011–2012 were screened for MSI by PCR and mismatch repair (MMR) immunohistochemistry (IHC), and additional BRAFV600E mutational analysis performed. T-tests, Fischer’s exact and Chi square statistical tests were applied. Twelve percent of patients displayed MSI, with increased frequency in black (15%) versus other ethnic group (OEG) (8%) patients. MSI patients were significantly younger than microsatellite stable (MSS) patients, however when stratified by ethnicity, black patients were predominantly younger (median age: 47), with increased MSH2/6 loss, and no BRAF mutations. These findings suggest a large proportion of young black SA CRC patients develop via the LS pathway due to earlier age onset and predominant MSH2/6 protein loss. SA patients of other ethnicities appear to follow the more well established sporadic MSI pathway. CIN or microsatellite stable (MSS) tumours account for 65–75% of CRC and mainly develop through mutant adenomatous polyposis coli (APC) gene with subsequent KRAS mutational activation, TP53 inactivation and somatic copy number alterations (SCNAs)5. MSI CRC occurs in approximately 15–16% of CRC due to inactivation of the DNA Mismatch Repair (MMR) system , with the majority (12%) presenting as sporadic MSI CRC as a result of epigenetic mutation (methylation silencing) of the promoter sequence of MLH1, and BRAF V600E oncogenic mutations10. The remaining subset (2–3%) is caused by germline mutations in either one of 4 MMR genes or the EpCAM gene and is a feature of Lynch syndrome (LS)14. CIMP CRC (10–20%) demonstrate hypermethylation of several promoter CpG island loci throughout the genome, leading to tumour suppressor and tumour-related gene inactivation. The CIMP and the MSI pathway overlaps in sporadic MSI CRCs, as these tumours display high levels of CIMP and MSI and are  characterized by a different type of precursor lesion in comparison to LS16. LS follows the classic adenoma-carcinoma sequence pathway as they present mainly with tubular adenomas (TAs) or tubulovillous adenomas (TVAs), whereas sporadic MSI CRCpremalignant lesions are sessile serrated adenomas (SSAs) developing through the serrated neoplasia pathway17. Clinicopathological features of sporadic MSI CRC include predominant occurrence in female patients, within the right colon and associated morphology includes signet ring cell and mucinous features with tumour infiltrating lymphocytes (TIL)20. LS patients are mainly associated with younger patients, with no gender preference and similar morphology as sporadic MSI tumours12.Colorectal cancer (CRC) develops through three major molecular pathways namely: Chromosomal Instability (CIN), Microsatellite Instability (MSI), and the epigenetic instability or CpG island methylator phenotype (CIMP) pathway23. These studies suggested a high frequency of LS, however additional validation studies and further molecular characterization of MSI CRC in black SA patients was recommended24.Previous CRC studies conducted in SA showed a higher frequency of MSI CRC in young black patients through MMR deficiency than white patients26. MSI/BRAF wild-type tumours are also more suggestive of LS and is crucial for improving cancer surveillance and prevention screening for patient family members, due to their increased risk of developing cancer. Identifying LS patients and treating with aspirin (600 mg per day) have also shown to reduce the risk of CRC27.MSI assessment has also shown important prognostic and predictive roles in CRC patient care, as MSI tumours are associated with a better prognosis than MSS tumours in early stage CRC, and treatment of MSI stage II tumours are not well responsive to 5-fluorouracil (5-FU) (standard treatment)This study assesses the frequency and features associated with MSI CRC over a 5-year period in a cohort of newly diagnosed CRC patients at the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) within SA. This data will provide insight into the CRC histopathological and molecular features associated with MSI CRC in black SA patients, with particular reference to MSI CRC frequency and the occurrence of suspected LS, a heretofore largely unassessed aspect of the disease.28. Immunophenotypic profiling of TILs was not performed. Family histories were not available from the histological reports. This research was conducted under the institution’s blanket ethics clearance (M10744) which allows for research to be carried out on all archived pathology specimens without informed consent from study participants. Additional study-specific ethics clearance was also obtained by the Ethics Committee of the University of the Witwatersrand (clearance number M120994), and all tests were performed according to the relevant guidelines and regulations.This was a retrospective study, comprising a 5-year cohort of 675 patients who had biopsy samples or colorectal resections fulfilling the histological criteria for adenocarcinoma of the colon or rectum from January 2011–December 2015, reported by the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) branch of the National Health Laboratory Service (NHLS)/Anatomical Pathology Division, Faculty of Health Sciences, University of the Witwatersrand. A total of 439 CRC cases with an MSI status by MMR immunohistochemistry (IHC) or MSI PCR result were included in this study. All cases were stratified by age, gender, ethnicity, tumour site, histological subtype, grade, stage (TNM classification by AJCC staging), presence and grade of precursor lesion, presence of tumour infiltrating lymphocytes (TIL) and Crohn’s-like inflammatory reaction (CIR). Tumour site was considered left-sided if distal from the splenic flexure and right-sided if proximal to the splenic flexure. This information was obtained from histology reports. A semi-quantitive H&E assessment using the Klintrup-Mäkinen score was used to determine the presence of TILsMolecular characterization of patient samples for frequency assessment was limited to the first 2 years 2011–2012) mainly due to the availability of funds . Samples with no allelic size variations in the 5 microsatellite markers were classified as microsatellite stable (MSS), allelic size variations found in 1 of the 5 microsatellites markers were classified as microsatellite instability-low (MSI-L) and variation in 2 or more microsatellite markers was classified as microsatellite instability-high (MSI-H)29. Previous studies have indicated black people show polymorphic variation frequencies within alleles NR21, BAT25 and BAT26 of approximately 10%32. For tumour samples that demonstrated instability in only 2 of these 3 markers, matched normal tissue was assessed to determine the instability status. In cases where no normal tissue could be assessed, samples were regarded as MSS.DNA was extracted from 4 × 10µm sections of tumour tissue from formalin fixed paraffin embedded (FFPE) blocks using the QIAamp DNA FFPE tissue kit . The extracted DNA was subjected to a pentaplex PCR panel of 5 markers comprising mononucleotide repeats amplified by 5 sets of primers, i.e.  as described in Haghighi MMR IHC was performed on MSI-H samples that were not previously assessed, utilizing the Dako EnVision™ FLEX detection system Kit and Autostainer Link 48 equipment  according to manufacturer’s instructions. The primary monoclonal antibodies used were FLEX monoclonal mouse antibodies against MLH1 , MSH2 , MSH6  and PMS2  . These were applied to 4-μm deparaffinized formalin fixed paraffin-embedded (FFPE) tissue sections. Blockage of endogenous peroxide activity was carried out with the EnVision™ FLEX Peroxidase-Blocking Reagent  and Antigen retrieval performed at 97 °C for 20 minutes (pH 9.0). Antigen-antibody reaction was visualized with the DAB (Diaminobenzidine) solution, and subsequently counter-stained with hematoxylin. Each sample was screened by an expert pathologist for the absence or presence of expression of each of the 4 MMR proteins. Negative protein expression was defined as complete absence of nuclear staining within tumor cells. Expression of MMR proteins in normal epithelium, lymphocytes and stromal cells was used as positive internal controls for each case. The four deficient (d) MMR patterns assessed were 1: dMLH1/PMS2; 2: dMSH2/MSH6; 3: dMSH6 and 4: dPMS2.® and Scorpions® technology, and includes a control assay to evaluate the adequacy of quality and quantity of DNA in a sample, before assessing the presence or absence of mutated DNA. Results were only deemed valid if the control run passed.All MSI-H samples were assessed for BRAF V600E mutations using the real-time Therascreen RGQ BRAF V600E mutation kit , according to the manufacturer’s instructions. The kit uses ARMSData obtained from the molecular characterization study (2011–2012) led to a more comprehensive study which included cases with an MSI status from histological reports for the total 5-year study period (2011–2015).All clinical data was collected in an excel sheet and statistical analysis was performed using Stata Intercooled 7.0  and Graphpad Prism version 7 . MSS and MSI-L cases were grouped together into one group (MSS), to establish unique associations with the counterpart MSI-H group. Patients were also analysed on the basis of ethnicity: black (African) versus other ethnic groups i.e., white (Caucasian), coloured (Mixed Ancestry), Indian (Asian); age and gender. The ability to draw statistically significant results on all ethnic groups was not possible due to small case numbers of certain groups. For this reason, White, Indian and Coloured patients were grouped into one “Other Ethnic Group” (OEG) to allow comparison with their black counterpart. Histopathological features were assessed as stated in the methodology section, and associations were evaluated using Fischer’s exact and Chi-square tests. A result with a P value less than 0.05 was considered statistically significant.A total 439 of 672 CRC patients diagnosed between January 2011-December 2015 had MSI CRC status assessment. Of these cases, 247 (56%) were male and 192 (44%) female, with an overall median age of 58 years. Black patients contributed to 60% (262/439) of cases.  (Column1 of Table ).MSI PCR performed on 267 cases 2011–2012) illustrated the majority were MSS , followed by MSI-L  and MSI-H . MMR IHC results demonstrated dMLH1 in most MSI CRC cases , followed by dMSH2/6 , dPMS2  and dMSH6 . BRAF mutational analysis was performed on all MSI-H cases with only 10% (3/31) harbouring a mutation, and all 3  occurred in dMLH1 tumours . A higher frequency of MSI tumours was seen in blacks , for the period 2011–2012. A significant relationship was seen between CRC subtype and tumour site (P < 0.0001), as MSS was associated with left sided tumours  and MSI with the right sided tumours [. A link between CRC molecular subtype and tumour subtype was also found (P < 0.0001), as mucinous adenocarcinoma occurred at a more frequent rate in MSI CRC;  than MSS CRC . Tumour infiltrating lymphocytes (TIL) response and subtype was found to have a correlation (P = 0.0045) as this occurred more often in the MSI group  than the MSS . Polyp subtypes tubular adenoma (TA) and tubulovillous adenoma (TVA) were associated with molecular subtypes (P = 0.0216) as TAs occurred more frequently in MSS CRC  and TVAs in MSI CRC .Male patients comprised 56% of the entire CRC cohort. Correlation between subtype and age was found (P = 0.0033) as median age was significantly lower in MSI CRC compared to the MSS subtype (51 vs 59 years respectively) (Table 1(Table ). A correlation with age was shown (P = 0.0006) as black patients were significantly younger compared to OEG patients . Tumour site and ethnicity showed an association for 2011–2012 data (P = 0.0105), as the majority of tumours were right sided within OEG patients , whereas black patients had left  and right-sided tumours  . Black patients with MSI tumours occurred in younger patients with left- (36%) and right-sided (59%) tumours, associated with dMSH2/6 (45%), and dMLH1/PMS2 (36%) tumours with a lack of BRAF mutations, indicating a possible LS cause37. Histopathological features were further stratified by ethnic groups (black versus OEG) to assess if any associations occurred with regard to gender, age, tumour site, MMR protein expression profile and BRAF mutation status. Within the 2011–2012 cohort, patients within the OEG were predominantly older (median age: 62), female (67%), possessed right sided tumours (89%), with deficient MLH1 (56%) expression, of which 60% harboured BRAF mutations. The 2011–2015 cohort however demonstrated an equal ratio of male to female patients, which could signify an under-diagnosis of female patients with MSI within the OEG. All other variables illustrated increased statistical associations within the 5-year cohort study. Collectively these features suggest the majority of OEG patients follow the more well established sporadic MSI CRC pathway20. Within the black patient group, all 2011–2012 data analysed with statistical significant associations revealed increased significance within the 5 year cohort, with the exception of tumour site where 2011–2012 data illustrated significance with an increased incidence of left-sided MSI tumours. This could possibly be due to pathologists generally screening more right than left-sided tumours for MSI CRC.In the SA cohort, MSI CRC was associated with younger age, right sided tumours, mucinous adenocarcinomas, TIL response, and TVA precursor lesions. This correlates well with findings in the literature38. This could possibly be indicative of aberrant non-functional proteins expressed or an alternative MMR protein involved in the development of MSI in this group of patients. This would suggest the 5 mononucleotide panel for MSI testing is the more sensitive tool for detecting instability in the South African setting. Perhaps this could be explored further for alternative causes of dMMR in these patients. For example, MSH3 loss of expression has recently been shown to be associated with MSI in African American patients40.MSI CRC also showed a high percentage of proficient (p) MMR expression (16%) for the universal 2-year screen, despite high concordance rates (95–96%) for MSI PCR versus IHC reportedMultiple logistic regression analysis was also conducted to control for the effect of confounding factors, young age (≤50 years), right-sided tumours, mucinous adenocarcinomas and TILS were associated with MSI CRC in SA patients.42. A study carried out by Cronje et al. in 2009 at CMJAH in Johannesburg demonstrated a large number of young black patients had increased loss of MMR protein expression, particularly MSH2, in comparison to their white counterpart, also suggesting a heritable cause21. Findings from this study confirm dMLH1 and dMSH2 associations within young OEG and black SA patients respectively.Limitations of the study included patient selection bias as patients from the 2013–2015 cohort were only selected if an MSI status was available. In addition, only a few patient samples with available data in certain subgroup analysis, such as TNM stage of disease, TILs, polyp subtype could be analysed, and this is mainly due to biopsy samples also included in the cohort. Only a subset of MSI samples  for the period 2011–2015 were included for BRAFV600E analysis, these largely from cases between period 2011–2012. This also represents a limitation of the study. The majority of patients diagnosed at CMJAH are of the black population group, therefore selection bias could play a role in the selection effect due to smaller sample numbers in the comparator OEG patient group. Previous MSI studies in SA however, have largely shown MLH1 mutations as the cause of hereditary and sporadic CRC in OEG patients 44. The Western and Northern Cape comprises mainly of the coloured population within SA, and LS have largely been identified in these 2 provinces  followed by white (15.6%) in this region44. LS genetic screening in Gauteng and remaining 6 provinces have been poor to non-existent, even though preliminary findings have suggested a possible hereditary cause, particularly in black patients from the 1990’s49.The Gauteng province, although the smallest, is the most densely populated and urbanized with nearly 700 people per square kilometre50. Genetic services such as testing and counselling are mostly offered in academic centres, and subsidies for these services are limited primarily due to the high medical costs associated with the HIV/AIDS epidemic46. Pathologists within the National Health Laboratory Services include a possible hereditary cause in histopathological reports of these patients with suspected LS, but genetic services are limited to counselling, particularly within the public sector of the Gauteng province. Colonoscopic surveillance of family members at risk for LS is difficult and expensive in a resource-poor country, however previous LS studies carried out within the Western Cape region of SA have shown the identification of the causative-mutation simplifies and reduces costs of surveillance considerably51.The healthcare financial system in SA is based on private and public funding, with the majority (57%) of the population utilizing public healthcare services, however 80% of healthcare expenditure in SA occurs in the private sectornd decade of life, to be repeated every 1–2 years52. LS patients treated with aspirin have a considerably reduced risk and reduced mortality rates associated with CRC53.If LS is the more prevalent MSI subtype in black SA patients, identifying the causative mutation together with regular colonoscopy screening is recommended for all affected individuals and families by the 2This study revealed MSI in black SA CRC patients is associated with earlier age onset, higher frequency of MSH2/6 loss and a lack of BRAFV600E mutations, suggesting Lynch or “Lynch-Like Syndrome” (LLS) as the main hereditary driver. Implementation of universal MSI screening is recommended as a routine diagnostic test within the SA setting to increase the detection rate of LS or LLS. This data warrants further molecular investigations in defining the underlying germline or somatic mutations. Appropriate early detection, surveillance and prevention strategies will reduce the risk and improve overall management of the disease in suspected LS patients and affected family members, particularly in the black population group in SA.This was a retrospective study on already available biological material and ethics clearance was obtained from the Human Ethics Research Committee, Faculty of Health Sciences: Ethics clearance reference number: M120994.Supplementary information


Cochlear implants provide an effective treatment option for those with severe hearing loss, including those with preserved low frequency hearing. However, certain issues can reduce implant efficacy including intracochlear tissue response and delayed loss of residual acoustic hearing. We describe a mouse model of cochlear implantation with chronic electric stimulation that can be used to study cochlear implant biology and related pathologies.Twelve normal hearing adult CBA/J mice underwent unilateral cochlear implantation and were evenly divided into one group receiving electric stimulation and one not. Serial impedance and neural response telemetry (NRT) measurements were made to assess implant functionality. Functionality was defined as having at least one electrode with an impedance ≤ 35 kOhms. Mouse cochleae were harvested for histology and 3D x-ray microscopy 21 days post-operatively, or, in case the implant was still functional, at a later time point when the implant failed. A separate experiment measured the hearing preservation rate in 7 adult CBA/J mice undergoing unilateral cochlear implantation with serial auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE).Implants maintained functionality for a mean of 35 days in the non-stimulated group and 19.8 days in the stimulated group. Reliable NRT and behavioral responses to electric stimulation were recorded. A robust intracochlear peri-implant tissue response with neo-ossification was seen in all cochleae. Six of seven mice maintained intact low frequency hearing up to 6 weeks following cochlear implantation.We demonstrate the feasibility of cochlear implantation and behaviorally significant electric stimulation in the mouse, with the potential for hearing preservation. This model may be combined with established mouse models of hearing loss and the large genetic and molecular research toolkit unique to the mouse for mechanistic and therapeutic investigations of cochlear implant biology. Cochlear implants (CI) have been established as a safe and effective strategy treating severe to profound sensorineural hearing loss in both adults and children. Advances in electrode design and ‘soft’ surgical techniques have enabled combined electrical and acoustic stimulation in many patients with functional low frequency hearing, which brings improved music perception , 2, speeAlthough advances in electrode design and speech processors have continued to improve CIs, there are still several pertinent issues that hamper their efficacy . The antMultiple animal models of cochlear implantation have been developed to study both intracochlear and auditory pathway responses to cochlear implantation and electric stimulation. Cat –28, guinMus musculus) has been extensively utilized for inner ear research. The ability to perform genetic manipulation and investigations in mice has yielded a variety of unique tools to aid inner ear research, including the development of transgenic reporter mice and models of genetic hearing loss [The mouse  was performed weekly. Starting 7 days post-operatively, all subjects were individually housed in custom stimulation cages. The stimulated group received electric stimulation for 5 hours, 5 days a week for 2 weeks. The non-stimulated group received no electric stimulation. We defined a functioning implant as having at least one intracochlear electrode with an impedance less than or equal to 35 kOhms, which is the upper limit permitted for stimulation programming. The primary endpoint involved two criteria. Firstly, after at least 21 post-operative days had passed and secondly, when no electrode channels (electrode) were programmable (corresponding to electrode impedances over 35 kOhms). When both criteria were met, animals were euthanized via decapitation under anesthesia with a mixture  of ketamine (87.5mg/kg body weight) and xylazine (12.5mg/kg body weight) and both cochleae harvested and fixed with 4% paraformaldehyde by perfusion through the oval window, followed by overnight submersion fixation. Arrays were left in the cochlea until 3D x-ray microscopy as described below.The surgical technique followed a modified version of the left post-auricular approach to the tympanic bulla for round window electrode array insertion described by Soken et al. . After eThe CI electrode array consisted of a silicone carrier with 3 half-banded platinum electrode contacts. The intracochlear portion measures 2.25mm in length and 0.15mm in diameter, tapering to 0.3mm in the extracochlear portion . The leaNRT and impedance measurements were made using clinical programming software . The electrodes were not preconditioned prior to measurement to accurately reflect the procedures used clinically with hearing preservation cases; impedance measurements were conducted in monopolar (MP) 1 + 2 mode, which measures the electrical impedance between one intracochlear electrode  and both monopolar extracochlear electrodes shorted together . NRT was measured using the Auto-NRT detect feature of the programming software using the Probe-Masker paradigm. The intracochlear electrode with the lowest impedance at the time was chosen as the active probe (stimulating) electrode and the intracochlear electrode with the next lowest impedance served as the active recording electrode; MP 1 and 2 served as the indifferent probe and recording electrodes, respectively. A charge balanced, biphasic square stimulus level series starting at an amplitude of 89 μA (90 Current Levels (CL); 2.22 nC/phase) and increasing to an amplitude of 377 μA  or until compliance limits are reached, was presented to determine an NRT threshold. The stimulus probe was presented with 25 μs pulse width and 7 μs interphase gap, at a probe rate of 80 Hz, with 50 sweeps. Recording was performed with a 98 μs recording delay, at a sampling rate of 20492 samples per second with 32 samples per stimulus sweep. The masking procedure was performed with the same probe electrode configuration as the active probes, at a masking current level 10 CL above the active current level with a 25 μs pulse width, 7 μs interphase gap, at a rate of 100 Hz with a 400 μs masker interval. The current level (CL) unit represents the amplitude of the current pulse on a log scale. Within the parameters of the 24RE CI emulator used for this study, 0 CL corresponds to 17.5 μA and 0.44 nC/phase and 255 CL to 1750 μA and 43.75 nC/phase.The CI processor was programmed with Custom Sound EP . All functioning intracochlear electrodes were shorted together via a software patch to effectively reduce impedance across the system and allow uniform stimulation amongst subjects. Volume sensitivity was set to maximum. The threshold and comfort (T&C) levels were matched at 30 CL below NRT threshold, ensuring a constant level of stimulation without dynamic range or amplitude modulation. This stimulus level was chosen to avoid any stimulation that may cause discomfort and adverse behavioral changes. The maximum sensitivity and maximum volume settings were selected within Custom Sound Programming Suite 4.2 , which sets the acoustic noise threshold to evoke a pulse train as 10 dB SPL. The programmed processors were connected to the implant emulator and connected to the electrode array assembly via the stimulation cage for 5 hours, 5 days a week. The stimulation CL was adjusted according to shifting NRT threshold. Stimulation history logs were checked to ensure the ambient animal housing noise levels drove the processors to deliver constant stimulation during the experimental hours.In-vivo intracochlear positioning of the electrode array was confirmed on post-operative day 7 via x-ray. X-ray was also used to investigate device integrity and positioning following sudden changes in electrode impedance or NRT threshold. Images were obtained on a Carestream MS FX Pro  with magnification stage at 35 KVP and 149 μA with 0.4 mm filtration and an exposure time of 40 seconds, producing pixel sizes of 42.6 microns. Animals were sedated with an anesthetic mixture  of ketamine (87.5mg/kg body weight) and xylazine (12.5mg/kg body weight) during in-vivo imaging.Fixed cochleae were prepared for non-destructive 3D x-ray microscopy with array in-situ. Images were obtained using a Zeiss Xradia Versa 3D x-ray Microscope  with a voxel size ranging 0.65–3μm. Three dimensional reconstructions and artifact rejection were implemented within Scout and Scan Control System and XM Reconstructor–Cone Beam 10 software . Image viewing and 3D reconstruction were conducted within Visual SI . An initial image was obtained with the array in-situ. The array was subsequently removed in a slow, controlled manner. Tissue was osmicated to enhance soft-tissue contrast. The cochlea was rinsed with 0.1M phosphate buffer. 1% osmium tetroxide with 1.5% potassium ferricyanide was gently perfused through the scalae and then immediately immersed in a 1% osmium tetroxide with 1.5% potassium ferricyanide solution and placed on rotator for 2 hours. Cochleae were then rinsed with 0.1M phosphate buffer solution and returned for imaging. The cochleae underwent repeat 3D x-ray microscopy and the resulting image series was merged with the previous array in-situ image series to obtain projections of the array within the cochlea.3D volume segmentation of the scala tympani was performed on 3D x-ray microscopy cochlea image stacks obtained after implant removal and specimen osmification. A gaussian filter was applied to image stacks prior to manual volume segmentation in Dragonfly software . This procedure was performed by a single laboratory staff blinded to group. Raw volumetric data for the scala tympani, implant tract, neo-ossification and soft tissue response was recorded. Fractional volumes of the neo-ossification and soft tissue response were calculated by dividing the volume of the respective region of interest by the total volume of the scala tympani.Following 3D x-ray Microscopy, osmicated cochleae are decalcified in 0.1M EDTA (pH 7.5). Cochleae are then dehydrated with graded alcohols. Plastic infiltration of cochlea is accomplished using a low viscosity embedding media Spurr’s kit (cat. 14300 Electron Microscopy Sciences). Before embedding in the epoxy resin, the cochlea is divided in the mid-modiolar plane by making a perpendicular cut through the round window and the apex. The halves are then placed in a block mold filled with Spurr’s resin and polymerization is achieved in an oven set at 75 degrees Celsius.1 μm thick sections are cut using a Reichert Ultramicrotome OmU3  and mounted on slides. Sections are then stained by using a Richardson’s staining method, covered slipped, examined and photographed with the 4X objective using a Nikon microscope with a Coolpix S10 digital camera  attached with a MM99 Martin Microscope Company adapter .A separate study was performed to determine the rate of hearing preservation in mouse cochlear implantation and observe for delayed hearing changes over a 6-week period in those with intact hearing following surgery. Successful hearing preservation was defined as less than or equal to 15 dB auditory brainstem response (ABR) threshold shift at 8 and 16 kHz. Seven (n = 7) 10–12 week old adult CBA/J mice underwent unilateral left cochlear implantation, 5 males and 2 females. The implanted array was identical to that described above except for the absence of extracochlear return electrodes and an alternative lead wire design. The surgical technique was similar to that described above with a 2.25mm depth of insertion, except with the additional use of cyanoacrylate glue and 9–0 nylon suture  to stabilize the implant near the bulla instead of dental cement. No electric stimulation was performed.Hearing was assessed by ABR and distortion product otoacoustic emissions (DPOAE) at pre-operative baseline and at 2, 4 and 6 weeks post-operatively in the left  ear. Right ear ABR and DPOAE was only performed at baseline and 6 weeks post-operatively to reduce total anesthetic burden. ABR and DPOAE was performed as previously described . BrieflyAll statistical analysis, where mentioned, was performed with GraphPad Prism software . Impedance data were analyzed via two-way ANOVA with repeated measures. Two-tailed t-test was used to analyze NRT and 3D x-ray volume segmentation data. ABR and DPOAE data were analyzed via one-way ANOVA with repeated measures with follow-up of significant effects with pairwise multiple comparisons via Holm-Sidak test. Statistical significance was set at an alpha level of 0.05 (p<0.05).Animals tolerated surgery well, with the exception of 2 cases of intra-operative mortality related to stapedial artery injury and 4 cases of early intra-operative complications related to anesthesia. Additional animals were used to reach study subject number goals. We found that low dose 1–3% isoflurane inhalational anesthesia reduced peri-operative mortality compared to injectable ketamine and xylazine. Animal feeding and grooming habits were not inhibited by the surgery and all animals maintained at least 90% of their pre-operative body weight; the animals did not show any alteration in their normal habits or ability to move within the cage. No signs of vestibular dysfunction, including abnormal gait or circling behavior were observed. Electric stimulation did not alter any of the aforementioned observations.At the onset of the study, a functioning electrode was defined as having an impedance less than or equal to 35kOhms based upon the parameters of Clinical CI programming software , which defines this as the impedance threshold below which an electrode can be programmed. NRT threshold was determined by visual confirmation of the lowest stimulus level at which a N1 and P2 peak response could be seen, which generally correlated with the threshold obtained from the software threshold auto-detect feature. Behavioral responses to electric stimulation at NRT threshold were consistently seen in non-sedated subjects. In response to a 30 second stimulus train at NRT threshold, animals were typically observed to have distinct changes in grooming habit corresponding with the offset / onset timing of the stimulus. The most common response was a temporary pause in grooming or feeding during the duration of the stimulus. No adverse signs of pain or discomfort were seen during these tests, including erratic behavior or circling. Chronic stimulation at 30 CL below NRT threshold did not produce any durable changes in normal behavior during the 5 hour stimulation blocks. Stimulation usage logs from the CI processor confirmed that the implant was stimulated for the entire 5 hour stimulation block.X-ray was used to assess intracochlear array position and to investigate the integrity of the electrode array lead or malposition following abrupt changes in electrode impedance. Fracture of the array lead wires were found in conjunction with abrupt impedance changes. Fractures were observed within the straight portion of the lead wire , adjacenA robust tissue response, including areas of neo-ossification was seen throughout all implanted cochleae in both groups, extending from the round window to an area immediately distal to the tip of the electrode array. This finding was confined to the scala tympani, with no tissue response seen within the scala media or scala vestibuli. An obvious implant tract could be seen in all subjects, with variable positioning of the electrode along the lateral wall to modiolus axis. No obvious signs of basilar membrane penetration or fracture of the osseous spiral lamina or modiolus were present.Segmentation of 3D x-ray microscopy image stacks allowed volumetric quantification of the post-implantation inflammatory response within the scala tympani . This anMerged images of 3D x-ray microscopy series were obtained with and without the array in-situ . ConsistIn a separate study examining hearing preservation in mouse cochlear implantation we applied inclusion criteria of successful hearing preservation as ≤ 15 dB ABR threshold shift at the 8 and 16 kHz frequencies at 2 weeks post-operatively. Within these criteria, 6/7 or 85.7% of subjects had successful hearing preservation after cochlear implantation. The one subject not meeting the hearing preservation criteria , post-op weeks 2 at 11314 Hz (p<0.05) and post-op weeks 2 and 4 at 32000 Hz (p<0.05); other pair-wise comparisons were non-significant (p>0.05).DPOAE SNR > 9 dB was considered a detectable response and is noted by a horizontal red dotted line in This study demonstrates the technical feasibility of mouse cochlear implantation with chronic electric stimulation, and the ability of hearing preservation surgery. Overall, 8/12 subjects maintained one or more functioning electrodes for at least 3 weeks, enabling electric stimulation over the same time period. Behavioral responses to electric stimulation and objective measures (NRT) evidenced successful stimulation of the mouse auditory system. Further, the surgery and experimental procedures were well tolerated by mice without any signs of vestibulopathy or implant related complications.An individual electrode impedance ≤ 35 kOhms is required for programming of electric stimulation within the Clinical CI programming software . This level represents the threshold for which electric stimulation can safely be administered without theoretical risk of tissue damage based on the Shannon equation . Impedanth cranial nerve [The NRT procedure measures the electrically evoked compound action potential (eCAP) response of the 8al nerve  and was al nerve  in Clinial nerve . Interesal nerve , 53, eleThere are several unique technical challenges to cochlear implant design and surgery in the mouse compared to other species. Scala tympani length and volume are less than that of other mammalian CI model species, which requires a smaller diameter and length electrode array. The CI The custom stimulation cage allowed continuous connectivity of the CI within the normal mouse housing environment, allowing repeated electric stimulation sessions and impedance and NRT recordings. This system improves ease of experimentation and may reduce animal stress associated with handling, change in housing environment and repeated anesthetic exposure . InclusiHearing preservation cochlear implantation has significantly expanded the CI candidate population and has shown the added benefit of combined electric and acoustic hearing –6, 8. HoA robust peri-implant tissue response confined to the scala tympani was seen in all subjects to a varying degree, composed of both soft tissue and neo-ossification. A similar tissue response has been seen 7 days after passive cochlear implantation of nylon monofilament in Balb/c mice  and follThe dual approach of non-destructive 3D x-ray microscopy followed by classic histologic sectioning shows complimentary utility. 3D x-ray microscopy provided reliable anatomic cochlear detail with delineation of the tissue response elements, facilitating easier quantification via 3D volume segmentation. The mean scala tympani volume measured in this study is similar to that found in other microCT studies , 57, addThe face validity  of this We describe a mouse model of cochlear implantation with chronic electric stimulation and the potential for hearing preservation implantation. The intracochlear tissue response seen in some human temporal bones is robustly reproduced in the CBA/J mouse, which may allow future mechanistic and therapeutic studies. The molecular and genetic research techniques available to the mouse, in addition to the radiologic, histologic and objective measures methods described here, provides unique advantages over other model species to CI biology investigations. Future studies with this model should employ established mouse models of hearing loss to more closely mirror human cochlear implantation recipients.


Maternal obesity and rapid infant weight gain have been associated with increased risk of obesity in childhood. Breastfeeding is suggested to be protective against childhood obesity, but no previous study has addressed the potential benefit of breastfeeding as a preventive method of childhood obesity amongst obese women. The primary aim of this study was to assess the relationship between mode of feeding and body composition, growth and eating behaviours in 6-month-old infants of obese women who participated in UPBEAT; a multi-centre randomised controlled trial comparing a lifestyle intervention of diet and physical activity to standard care during pregnancy.Three hundred and fifty-three mother and infant pairs attended a 6-months postpartum follow-up visit, during which they completed the Baby-Eating Behaviour Questionnaire, a parent-reported psychometric measure of appetite traits. Measures of infant body composition were also undertaken. As there was no effect of the antenatal intervention on infant feeding and appetite the study was treated as a cohort. Using regression analyses, we examined relationships between: 1) mode of feeding and body composition and growth; 2) mode of feeding and eating behaviour and 3) eating behaviour and body composition.p = 0.002) amongst formula fed infants, following adjustment for confounders. Independent of the mode of feeding, a measure of infant appetite was associated with sum of skinfold thicknesses , calculated body fat percentage , weight z-scores  and catch-up growth .Formula fed infants of obese women in comparison to those exclusively breastfed, demonstrated higher weight z-scores , higher rate of weight gain  and greater catch-up growth . There was also a lower enjoyment of food  contains supplementary material, which is available to authorized users. Childhood obesity is a global pandemic; in 2016 more than 41 million children < 5 years of age were overweight or obese , greaterThe World Health Organisation (WHO) recommend that all mothers exclusively breastfeed infants for the first 6 months of life and that breast feeding may protect against childhood obesity . HoweverSeveral studies have assessed eating styles and behaviours in infancy and their effect on weight development using the validated Baby Eating Behaviour Questionnaire (BEBQ) –19. Thesn = 1555), from UK inner-city settings of ethnic and social diversity. The intervention focused on reducing dietary glycaemic load and saturated fat intake whilst increasing physical activity and was delivered from 15+ 0–18+ 6 weeks’ gestation for 8 weeks. The results of the study indicated that the intervention had no effect on the primary maternal or neonatal outcomes, incidence of gestational diabetes (GDM) and large for gestational age infants respectively, between the groups. However, there was a difference in secondary maternal outcomes including a reduction in GWG , sum of skinfold thicknesses  and an increase in physical activity at 28 weeks’ gestation   [We previously undertook a randomised controlled trial (RCT), the UK Pregnancies Better Eating and Physical Activity Trial (UPBEAT). UPBEAT was a multi-centre RCT comparing the effect of a lifestyle intervention of diet and physical activity advice compared to standard antenatal care in a group of obese pregnant women ( 0.0015) .The primary aim of this study was to assess the role of mode of feeding, on measures of body composition, growth and eating behaviours in 6-month-old infants born to obese women. The investigation was a secondary analysis of the UPBEAT trial including 353 mother and infant dyads, all of whom had complete early feeding and body composition data as well as the parent-reported BEBQ. As no effect of the antenatal intervention was observed in any measures of infant feeding or appetite in this sub-group of participants, the data was treated as a cohort.+ 0 and 18+ 6 weeks’ gestation. The participants were from UK inner-city settings with high socioeconomic deprivation. Details of the trial intervention and inclusion and exclusion criteria have been published previously [n = 353) if they had: 1) attended the follow-up visit at 6-months of age; 2) completed the feeding questionnaires; and 3) infant anthropometric data were obtained. Infants were excluded if they were suffering from major ill health which could impact on growth and development or born ≤34 weeks’ gestation.Between July 2010 and May 2015, we conducted a planned follow-up at 6-months postpartum of infants and their mothers who had participated in the UPBEAT RCT. Women over the age of 16 years were recruited to the UPBEAT trial between 15eviously . Mother Data for mode, duration, type of milk and age of introduction of solids were collected using a validated feeding and growth questionnaire , adminisThe validated 18-item Baby Eating Behaviour Questionnaire was used to assess infant appetite and feeding behaviours [Maternal variables utilised in the present analysis included age, BMI, parity, ethnicity and socioeconomic status which were recorded at trial entry, and maternal GDM status, recorded following an oral glucose tolerance test at 24–28 week’s gestation. Neonatal data including gestational age at delivery and birthweight was collected at birth. In light of previously reported associations between cord blood leptin and infant adiposity , the lepThe outcomes of interest were measures of infant adiposity assessed by subscapular and triceps skinfold thicknesses z-scores derived using the WHO reference populations . Other op < 0.05. Analyses were performed using Stata version 14.0 .In this sub-group of participants, there was no effect of the antenatal intervention on any of the infant feeding or appetite variables . Further adjustment was made for early pregnancy maternal BMI, ethnicity, index of multiple deprivation, incidence of GDM and birthweight (Model 2). To assess the influence of infant appetite and satiety on measures of adiposity and growth at 6-months of age, further adjustment was made for cord blood leptin concentration as leptin has been implicated in appetite regulation in offspring of obese mothers , and modn = 116) and confounder data  . Assessment was undertaken to determine known predictors of missingness and data was subsequently imputed to create 50 datasets using 10-burn in iterations.To assess for potential selection bias, comparisons were made between mother-offspring pairs included and excluded within this study. At the 6-month follow-up visit 698 infants had complete anthropometric data. Infants were excluded from the analysis due to missing mode of feeding and/or BEBQ data n = 16 and coFor the 698 infants who attended the 6-month visit, 353 with complete data were included in this analysis Fig. . Of the p = 0.02). After adjustment for maternal and infant confounding factors detailed in Additional file p = 0.002) in comparison to children who were breastfed , sum of skinfolds (SSFT), total body fat (%), weight z-scores and catch-up growth following adjustment for maternal and offspring confounders Model 2)  were significantly older, were more likely to be nulliparous and more likely to be white, in comparison to those excluded from the analysis  . Centre, maternal age, number of years in full time education and gestation at delivery were identified to be significant predictors of missingness, suggesting missing data was ‘missing at random’  using the same questionnaire [Exclusively breastfed infants were more likely to demonstrate enjoyment of food as assessed at 6-months of age compared to formula or mixed fed infants. This observation has also been made recently in an Australian cohort of weight heterogeneous women  and ‘my baby enjoys feeding time’. Rather than concluding that enjoyment of food is associated with greater food intake, an alternative explanation could be that the infants of obese women enjoyed human milk more than formula milk possibly due to a preferred odour of human milk . FurtherEarly eating patterns and behaviours can determine later life eating habits and food preferences and have been associated with the development of childhood overweight and obesity , 41. In Strengths of the study include our sample of mothers and their infants are from a prospective cohort, recruited from UK inner-city populations. As 80% of the study population were in the highest quintile of socioeconomic deprivation and compromised entirely of obese women, this study is well placed to assess the mode of early feeding on infant anthropometric outcomes within this high-risk group amongst whom associations between maternal obesity and childhood obesity have frequently been described , 44. FurLimitations of the study include loss of follow-up of the study population which may result in selection bias and this limits transferability of findings to the general population. However, there were no differences in BMI, incidence of GDM or infant characteristics between those included and excluded within the analyses. Furthermore, complete outcome data was only available for 50% of the follow-up participants, however, sensitivity analyses including the use of multiple imputation did not result in differences in the observed relationships. The BEBQ is a parent-reported measure and subject to recall bias, it was used to assess appetite and satiety of the infant but did not distinguish between delivery of breastmilk via breast or bottle or volume of milk consumed; this may confound interpretation of results as previous studies have suggested that mode of delivery may influence appetite  and voluIn summary, exclusive breastfeeding in obese pregnant women modified early life childhood growth trajectories in infants at 6-months of age, in comparison to formula or mixed fed infants. Measures of general appetite in early infancy were also associated with measures of adiposity, weight and catch up growth in infants born to obese mothers from deprived inner-city UK populations. Given the association between maternal obesity and obesity in later life of the child these findings strongly support provision of lactation support for obese women, recognised to have difficulties in breastfeeding . A novelAdditional file 1:Table S1. Univariate analysis of infant body composition at 6 months of age stratified by mode of early feeding in offspring born to obese women (n = 353). (DOCX 14 kb)Additional file 2:Table S2. Postnatal characteristics previously implicated with infant adiposity, by UPBEAT randomisation allocation. (DOCX 15 kb)Additional file 3:Table S3. Description and reasoning behind potential confounders associated with mode of early feeding and infant anthropometry at 6 months of age [s of age , 54–59. Additional file 4:Figure S1. Distributional assessment of infant anthropometric measurements at 6 months of age by stratified by offspring sex. (DOCX 33 kb)Additional file 5:Figure S2. Associations between measures of general appetite with infant obesity and growth at 6 months of age, in offspring born to obese women (n = 353). (DOCX 22 kb)Additional file 6:Table S4. Associations between measures of infant appetite including measures of enjoyment of food, food responsiveness and slowness in eating with infant adiposity and anthropometry at 6 months of age, in offspring born to obese women (n = 353). (DOCX 15 kb)Additional file 7:Table S5. The association between mode of early feeding, timing of introduction of solid foods and infant anthropometry at 6 months of age. (DOCX 15 kb)Additional file 8:Table S6. Comparison of baseline maternal and neonatal demographic, clinical, and anthropometric characteristics between those included (n = 353) and excluded (n = 1167) from the analysis. (DOCX 15 kb)Additional file 9:Table S7. Sensitivity analysis of removal of infants born > 34 weeks’ gestation and ≤ 37 weeks’ gestation (n = 8 excluded). (DOCX 14 kb)Additional file 10:Table S8. Predictors of missing exposure (mode of infant feeding) and maternal covariate data in infants with detailed anthropometric data at 6 months of age. (DOCX 17 kb)Additional file 11:Table S9. Sensitivity analyses assessing the role of mode of early feeding on measures of infant anthropometry at 6 months of age., in offspring born to obese women (n = 353) using multiple imputation. (DOCX 15 kb)


Preterm newborns typically require supplemental oxygen but hyperoxic conditions also damage the premature lung. Oxygen-induced lung damages are mainly studied in newborn mouse models using oxygen concentrations above 75% and looking at short-term effects. Therefore, we aimed at the investigation of long-term effects and their dependency on different oxygen concentrations.2) for 14 days followed by a longer period of normoxic conditions. Lung-related parameters were collected at an age of 60 or 120 days.Newborn mice were exposed to moderate vs. severe hyperoxic air conditions  or altered expressions of antioxidant enzymes  and lung elasticity-relevant proteins  in adulthood. In contrast to severe hyperoxia, moderate hyperoxia was less lung damaging but also not free of long-term effects .Severe but not moderate neonatal hyperoxia causes emphysematous lungs without persisting oxidative stress and inflammation in adulthood. As the existing fragmentation of the elastic fibers seems to play a pivotal role, it indicates the usefulness of elastin-protecting compounds in the reduction of long-term oxygen-related lung damages. The respiratory management of preterm newborns includes supplemental oxygen because the immature lung is unable to maintain sufficient gas exchange . However2•-) molecules through the mitochondrial oxidative phosphorylation system in a higher amount than they can be simultaneously detoxified by dismutation to hydrogen peroxide [2•- is increasingly generated by members of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme family at the outer cell membrane [2•- excess then causes the generation of other types of ROS molecules with subsequent cell damage through the oxidation of lipids and proteins, which again leads to secondary ROS generation by attracted immune cells [An increase in intra- and extra-cellular ROS due to the increased supply of oxygen and, therefore, increased alveolar oxygen concentration plays a pivotal role in mediating lung cellular damages . Initialne cells .2) from birth until PNDs 4–14 as they focused on the investigation of BPD development [Newborn mice are commonly used as animal model to investigate cellular and molecular processes that contribute to the hyperoxia-mediated lung injury in preterm infants. In contrast to human alveolar development beginning prior to birth, murine alveolarization begins after birth on postnatal day (PND) 3, and the saccular phase of the lung development is already completed by PND15 . Most moelopment . Howeverelopment –13, not 1) and/or forced expiratory flow rates at 25–75% vital capacity (FEF25–75%) in young-adult survivors of BPD [Another disadvantage of existing mouse experimental studies is the lack of lung functional analyses recording especially the peak expiratory flows, in addition to compliance and airway resistance, in order to better assess the elastic recoil of the lung tissues. This would be of high importance as clinical investigations showed lower forced expiratory volumes in 1 s .Surprisingly low is also the number of mouse experimental studies assessing long-term oxidative damages of lung proteins , 19, and2), moderate hyperoxic  and severe hyperoxic  normobaric air conditions. This study was approved by the local Commission for Animal Protection. Hyperoxic air was created in a polycarbonate chamber equipped with computerized gas flow control units  and O2 gas detector , which was large enough for three standard cages. Maximum six newborns of each sex per lactating dam were exposed to either N or mH conditions in one experimental cycle or they were exposed to either N or sH conditions in another experimental cycle. Lactating dams were rotated between hyperoxic and normoxic conditions every day to minimize maternal effects. Because of this experimental procedure, our study generated more mice of the N than mH or sH group. Analyses were performed at the PNDs 14, 60 and/or 120. At PND30, 17 female mice each treatment group were individually placed in a standard cage equipped with a running wheel until PND60. Wheel-running activity (PNDs 53–60) was recorded by use of a data acquisition unit connected to the wheel-running unit as described [Newborn mice of the C57BL/6 N strain  were treated from birth until PND14 with different concentrations of oxygen to reach normoxic . Newborns were marked by foot tattoos for their identification. General health and body weights of the experimental mice were checked every day till PND15, every 2 days between PNDs 16–30, and every week after PND31. At the PND of analysis, mice were killed by cervical dislocation, and lung and blood were prepared for further analysis.A bronchoalveolar lavage (BAL) was performed three times with 0.3 ml phosphate-buffered saline and then pooled. After counting the cell number, BAL cells were centrifuged on a slide and stained with the HemaDiff Quick Staining kit  for cytological investigations. Cell-free BAL fluid was obtained by centrifugation and analyzed for protein with the Pierce™ bicinchoninic acid (BCA) Protein Assay kit , for IgM with the mouse IgM ELISA kit  and for soluble receptor for advanced glycation end-products (sRAGE) with the mouse RAGE DuoSet kit . Heparinized blood was taken after transection of the vena cava and subjected to automated blood cell count using the scil Vet ABC™ Hematology Analyzer .2O. After embedding in paraffin, 4-μm-thick sections were cut, dewaxed, rehydrated and incubated in hematoxylin-eosin solutions  to stain nuclei and tissue proteins. Lung elastic fibers were visualized by staining the sections with Weigert resorcin-fuchsin  followed by counterstaining with tartrazine . Images of the stained sections were taken with the Axiovert microscope equipped with Spot Camera . Lung histological parameters were analyzed by use of the ImageJ 1.47v software [Lung histology was assessed by use of the left lung, which was fixed with 4% phosphate-buffered formaldehyde for 15 min at a filling pressure of 20 cmHsoftware . The measoftware . The radsoftware .Total RNA and protein was isolated from the right lung by use of the TRIzol™ Reagent (Thermo Fisher Scientific). In addition, we prepared tissue lysates in 1% phosphate-buffered sodium dodecyl sulfate solution supplemented with protease inhibitors. Equal protein amounts were separated by gel electrophoresis with subsequent standard immuno-blot procedure using polyclonal rabbit antibodies against SP-B , SP-C (Santa Cruz), Cu/ZnSOD , catalase (Abcam), MnSOD , PEX14  or GAPDH (Santa Cruz). The expression of the antioxidant enzymes was determined by slot blot procedure using the Minifold slot blot system  with subsequent antibody detection. The LAS 3000 computer-based imaging system  equipped with AIDA 2.0 software  was used for signal detection and quantification of the signal intensities. All data were normalized per total protein stain of the blotted proteins, which was performed with 0.5% amido black solution.SFTPB , SFTPC  or ELN . Relative mRNA amounts were quantified by use of an external cDNA standard because the normalization per another gene  appeared to be inappropriate.Equal amounts of total RNA were reverse transcribed into cDNA with the M-MLV RT Rnase H Minus . Then, the cDNA was subjected to quantitative PCR using the iCycler iQ™ Real-Time PCR Detection System . PCR was performed with mouse-specific primers for − 1 wet tissue) containing 1 μg∙ml− 1 saponine  as mild detergent and 50 μM deferoxamine mesylate (Sigma) as Fe3+ chelator. Nuclei were then removed by centrifugation (600 g for 10 min). 10 μl nuclei-free cytoplasmic fraction were mixed after 90 min (Fe3+ chelate effect completed) with 90 μl 1 mM 1-hydroxy-3-carboxy-2,2,5,5,-tetramethyl-pyrrolidine  as a spin trap for O2•-. A computer-operated electron paramagnetic resonance spectrophotometer  equipped with MiniScope Control v2.7.3 analysis software  measured the time-dependent formation of 3-carboxy-2,2,5,5-tetra-methyl-pyrrolin-1-oxyl (•CP) at room temperature every 10 min. O2•- data were expressed as •CP formation rate∙min− 1.ROS are formed in mitochondria and to high extent also in other cellular compartments of lung samples . Therefo− 1) via tracheal cannulation. Through cannulation of pulmonary artery and left atrium of the heart we ensured a constant perfusion flow of the lung vascular bed (1 ml∙min− 1 at 37 °C). RPMI 1640 medium (Thermo Fisher Scientific) was used as perfusion buffer and constantly purged with 5% CO2/95% N2 to yield pH 7.4 and O2-reduced conditions. Thereafter, the ventilation was switched to negative pressure ventilation with constant minimal pleural pressure (− 2 cmH2O) and specific maximal pleural pressures. Since the isolated lung is not surrounded by the pleurae, repeating augmented breaths every 3 min at − 20 cmH2O maximal pleural pressure stabilize the tidal volume (TV) that would otherwise decline steadily. The PULMODYN® Pulmonary Mechanics Data Acquisition software recorded the lung physiological parameters.The buffer-perfused mouse lung system from Hugo Sachs Elektronik-Harvard Apparatus  analyzed the lung respiratory mechanics. We described this ex vivo method before in detail , 26. In P values ≤0.05 indicate a significant difference between the groups.The software SigmaStat 3.5 and SigmaPlot 10  were used for statistical calculations and data presentations. The ANOVA test followed by Holm-Sidak method (parametric data) or ANOVA on Ranks followed by Dunn’s method (non-parametric data) was used for multiple comparisons. The Student’s t-test (parametric data) was used for comparing two groups. The survival of mice was tested for significance by log-rank test. All mice of our study were treated with normoxia (N), moderate hyperoxia (mH) or severe hyperoxia (sH) until PND14. Expect for lung functional analysis in fully grown mice at PND120, most other parameters were analyzed earlier at PND60. Oxidative stress-related parameters were additionally studied in newborns and directly after treatment (PND14).At PND60, the treatment of newborn mice with neonatal hyperoxia slightly reduced their survival rate which was significant for mice of the mH group and only a trend for those of the sH group Table . This re2•- in mouse lung tissues [2•--scavenging enzymes, as well as catalase, which detoxifies resultant H2O2, in lung samples depending on development (age) and treatment. Development-dependent analyses showed increasing protein amounts of all three enzymes with early changes in catalase and late changes in MnSOD and Cu/ZnSOD  mice in an initial experiment. This comparison revealed an age-related increase of the lung compliance as indicated by changes in the pleural pressure-TV relations Fig. a accompaOur mouse experimental study demonstrated that neonatal hyperoxia at severe condition causes structural and functional changes of the adult lung indicating towards an emphysematous lung phenotype. This lung phenotype is not accompanied by oxidative stress and inflammation in adulthood. Also, it is not severe enough to restrict the relatively high running activity of mice. Assessments of the elastic fiber lengths and comparative analyses of the mRNA and protein level of elastin and selected surfactant proteins indicate a pivotal role of severe neonatal hyperoxia on long-lived extra-cellular matrix structures in adult lungs. In contrast to neonatal hyperoxia at severe condition, moderate hyperoxia does not cause such significant changes in lung histology and function in adulthood but, surprisingly, somewhat more lung oxidative stress. Moreover, direct comparisons of the respiratory mechanics of our young-adult mice treated with moderate neonatal hyperoxia with the respiratory mechanics of older untreated mice suggest potential functional impairment at advanced ages.2) for a comparable time period (10–14 d). The increased thickness of the alveolar walls and, therefore, lower cell number per total lung tissue area identified in our study might result from an aberrant expression of elastin and other extracellular matrix molecules [Computed tomography-based investigations of survivors of the severe BPD often show structural changes of the lung indicating lung emphysema , 17. Thiolecules . In cont1 and FEF25–75% [Clinical investigations of young-adult survivors of severe BPD also identified limitations in the expiration as indicated by lower values for FEVEF25–75% –16, whicEF25–75% –13, 33, The higher sensitivity of the lung tissue to ventilation-mediated damages might be caused by the increased lung compliance together with lower expiratory flows because the volume of inspired air is critically high at higher pleural pressures. As isolated lungs are not restricted by the thoracic cage, this is also of particular case in our ex vivo lung analysis. In addition to differences in peak expiratory flow and lung compliance, severe neonatal hyperoxia causes reduced peak inspiratory flows without alterations in the airway resistance which suggests a general impairment of the lung elasticity in adulthood. Important reason for the lower lung elasticity might be the fragmentation of elastic fibers, which can be caused by an increase in neutrophile elastase during administered hyperoxia  and by aIn contrast to the reduced length of elastic fibers, the total amounts of elastin as marker of fibroblasts , selecteOur study also included analyses of selected antioxidant enzymes important for the removal of intracellular ROS. In accordance with most findings of Berkelhamer et al. , their a2 for 14 d) showed restrictions in their physical capability [The data of study also showed that experimental hyperoxia using oxygen concentration of 75% until the completion of the saccular phase in mice  cause changes of the respiratory mechanics in adulthood. In this regard, we identified higher lung compliance but without increase in the peak expiratory flow as expected from an older comparison group. This observation suggests that neonatal hyperoxia at moderate conditions may induce an age-related lung emphysema [In clinical situations, the surfactant replacement may mediate significant antioxidant activity due to enzymatic and non-enzymatic compounds naturally present in animal surfactants . This ismphysema  earlier mphysema –17.Lung tissues of adult mice exposed as newborns to moderate hyperoxia also showed a slight increase in free ROS associated with somewhat more immune cells located in the BAL fluid. It is well conceivable that the NADPH-dependent generation of ROS in BAL cells contributes to an increased lung level in free ROS . AlthougAs already discussed above, our animal experimental study using newborn mice identified some similarities to the hyperoxia-mediated lung pathologies described in human survivors of the preterm birth. The existence of such similarities indicates the usefulness of this mouse model to study the effect of neonatal hyperoxia on lung structure and function in adulthood. However, the lung of newborn mice is not that premature (late saccular stage) as the lung of most premature human infants requiring the treatment with high concentrations of oxygen. Also, newborn mice are adapted to survive with a lung in the late saccular stage without supplemental oxygen. Therefore, lung tissues of human preterm infants may be more susceptible to the same oxygen concentrations used in our animal study.Moderate and severe hyperoxic conditions in newborn mice cause differential lung phenotypes in adulthood. Emphysematous lungs without persisting oxidative stress and inflammation are the result of severe neonatal hyperoxia. As the existing fragmentation of the elastic fibers seems to play a pivotal role in the development of this lung phenotype, more effort should be made to identify useful elastin-protecting compounds which can be applied in addition to surfactant therapy in the future. Non-emphysematous lungs with slight increase in ROS formation and some other changes are the result of moderate neonatal hyperoxia. However, detailed experimental studies are still needed in order to understand their possible importance in the compensation of hyperoxic lung damages.Additional file 1: Figure S1. Altered lung structure in response to neonatal hyperoxia.


Circulating angiogenic cells (CAC) have been identified as important regulators of vascular biology. However, there is still considerable debate about the genotype and function of CAC. iv). Genes and pathways of interest were further evaluated using qPCR comparing CACiv versus CD14+ monocytic cells. The CACiv transcriptome strongly related to tissue macrophages, and more specifically to regulatory M2c macrophages. The cytokine expression profile of CACiv was predominantly immune modulatory and resembled the cytokine expression of tumor-associated macrophages (TAM). Pathway analysis revealed previously unrecognized biological processes in CACiv, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Analysis of endothelial-specific genes did not show evidence for endothelial transdifferentiation.  Data from publicly available gene expression data sets were used to analyse the transcriptome of in vitro cultured CAC (CACiv are genotypically similar to regulatory M2c macrophages and lack signs of endothelial differentiation. CAC These EPCs were able to differentiate in vitro into an endothelial phenotype and induce neovascularization in vivo. After more than a decade of vigorous research, during which the early findings of Asahara et al. have been extended, the dogma that postnatal neovascularization relies solely on the proliferation, migration and remodeling of fully differentiated endothelial cells, has been largely revised. Nowadays, neovascularization is considered to be a dynamic process in which local endothelial cell proliferation and circulating progenitor cells join forces to engage in the restoration of tissue perfusion.Endothelial progenitor cell (EPC) therapy is an appealing strategy for the treatment of cardiovascular diseases. The concept of EPCs dates back to a landmark study published in 1997 by Asahara et al. [However, there is still considerable debate about the phenotype and function of EPCs and much of the uncertainty is caused by a high degree of confusion about the definition of EPCs. First of all, over the years, different culture protocols have emerged, all claiming to produce EPCs from peripheral blood mononuclear cells. Furthermore, a variety of molecular marker combinations have been advocated for the characterization of circulating EPCs. Obviously, the ambiguity that surrounds the term ‘EPC' has not facilitated the understanding and advancement of EPC biology.iv for these in vitro cultured blood-derived mononuclear cells. The potential use of these cells to aid in the restoration of impaired neovascularization has been investigated [iv-specific gene signature, to determine the CACiv cytokine-cytokine receptor fingerprint and to investigate the biological processes that are important in CACiv biology. This approach is not unprecedented, since, for instance in oncology, genetic profiling has revolutionized tumor characterization and yielded new insights into tumor biology [In the present article, we have investigated an EPC subtype that has been renamed as circulating angiogenic cell (CAC) , early Estigated . We used biology . Using t biology  could be biology .iv strongly relate to tissue macrophages, and more specifically to regulatory M2 macrophages, without evidence for endothelial transdifferentiation.In the present paper, we provide evidence that CACiv, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Together, our findings provide novel insights into the field of CAC biology.The cytokine expression profile is predominantly immune modulatory and resembles the cytokine expression of tumor-associated macrophages (TAMs). Pathway analysis has revealed previously unrecognized biological processes in CACiv, we collected blood of healthy volunteers. The CACiv culture protocols were reviewed and approved by the local ethics committee of the Antwerp University Hospital (EC number 12/10/101). Written informed consent was obtained from all participants.The data that is reported in this manuscript used publicly available published data sets from other studies. The data collected from GSE2040 involved cell cultures of human volunteers and the data obtained from GSE5099 involved cell material from blood donor buffy coats. Both studies were in compliance with the Helsinki Declaration on research involving human subjects, human material or human data and under the approval of an appropriate local ethics committee. For a qPCR study of CACiv gene signature, a publicly available gene expression data set (GSE2040) (HG-U95Av2 microarray (Affymetrix Inc.), see supplementary data file (https://www.ncbi.nlm.nih.gov). This gene expression data set contained 3 gene expression profiles of CACiv and 3 gene expression profiles of CD14+ monocytes, all of which were included in the analysis. Raw expression data were normalized using GCRMA and probe sets with a fluorescence intensity above 100 in at least 25% of the arrays were filtered for further analysis. The gene signature was generated using the nearest shrunken centroid method implemented in the R-package Prediction Analysis of Microarrays (PAM). Using a leave-one-out cross-validation procedure, a ∂-value was selected in such a way that the misclassification error rate was minimal. The global clustering pattern of the CACiv signature genes was evaluated using unsupervised hierarchical clustering (UHC) with the Euclidean distance as distance measure and complete linkage as the dendrogram drawing method. Using the global test [iv and CD14+ monocytes.To develop the CACata file ), targethttp://www.ingenuity.com) software was used to identify biological networks, functions and canonical pathways important to CACiv biology. To appreciate the genetic resemblance of the CACiv gene expression profile in relation to other cell types of interest , the expression of CACiv signature-related genes was analyzed using the Reference database for gene Expression Analysis , together with an extensive review of the literature.The Ingenuity Pathways knowledge base Analysis (IPA) , see supplementary data file) including a total of 44928 entries representing more than 33,000 human genes containing expression data on the differentiation of monocytes into macrophages, and of macrophages into an M1 or M2 macrophage subtype was downloaded from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo). Raw expression data were normalized using GCRMA and probe sets with a fluorescence intensity above 100 in at least 25% of the arrays were filtered for further analysis. Using PAM, we generated gene centroids for the monocyte, macrophage, M1 macrophage and M2 macrophage cell fractions. The leave-one-out cross-validation procedure was used to select a ∂-value in such a way that the misclassification error rate was minimal. The centroids were applied onto the GSE2040 data set using the nearest centroid classification routine. Samples were classified by correlating the centroid-specific gene expression profile of each sample in the data set with the shrunken centroids generated by the PAM algorithm. Positive correlation coefficients indicate resemblance of the tested sample to the cell fraction represented by the centroid. Mann–Whitney U tests were used to compare the resulting correlation coefficients between CACiv and monocytes.The following strategy was adopted to evaluate the possible macrophage genotype of cultured CACiv and endothelial cells, we retrieved an endothelial-specific gene list reported by Bhasin et al. [iv and endothelial cells with respect to the endothelial-specific gene list. In addition, we calculated the average gene expression of the endothelial-specific genes and compared this level between CACiv and CD14+ monocytes to evaluate whether a difference in expression existed between both cell types with respect to the set of endothelial-specific genes.To evaluate the degree of resemblance between CACn et al. . We usedThe data that is reported in this manuscript used publicly available published data sets from other studies. The data collected from GSE2040 involved cell cultures of human volunteers and the data obtained from GSE5099 involved cell material from blood donor buffy coats. Both studies were in compliance with the Helsinki Declaration on research involving human subjects, human material or human data and under the approval of an appropriate local ethics committee.n = 4) by density gradient centrifugation using lymphocyte separation medium (Lonza). The CD14+ cell fraction was isolated by using CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. CACiv were cultured out of the mononuclear cell fraction of blood specimens of healthy volunteers (n = 4) according the method first described by Dimmeler et al. [6 mononuclear cells were plated on human fibronectin-coated 24-well culture dishes and maintained in EBM-2 basal medium with EGM-2-MV SingleQuots and 20% fetal bovine serum (FBS) (Lonza). After 3 days in culture, nonadherent cells were removed by washing with PBS and adherent cells were further incubated in fresh medium until day 7. Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and cultured in EBM-2 basal medium with EGM-2 SingleQuots for 14 days . Cells from passage 6 and 7 were used.Mononuclear cells were extracted out of blood specimens of healthy volunteers n =  by densiRNA was isolated using the RNeasy mini kit (Qiagen) following the manufacturer's instructions. On-column DNAse treatment (Qiagen) was used to remove contaminating DNA leftovers. RNA concentration and purity were analyzed using Nanodrop spectrophotometer (Nanodrop technologies) readings at 260 and 280 nm. Complementary DNA (cDNA) was synthesized using Invitrogen superscript kit according to the manufacturer's instructions and using random hexamer primers for reverse transcription. Reverse transcription was performed at 50°C for 55 minutes, followed by 5 minutes of incubation at 85°C to inactivate the reverse transcriptase enzyme. cDNA samples were placed on ice and stored at -20°C until further use.μl. We used the geNorm algorithm to determine an optimal combination of reference genes for internal normalization . All qPCR reactions were carried out as follows: after an initial denaturation-activation step at 95°C for 10 min, amplifications consisted of 45 cycles of denaturation at 95°C for 10s, annealing at 60°C for 15 s and measurement of fluorescence at 72°C for 1 s. Cycle number (Cq) was measured using the baseline-independent second derivative maximum method. Normalized relative gene expression was determined by the E-∆∆Cq method. Assay efficiency (E) was measured by serial dilution of cDNA of pooled samples based on the slope of the standard dilution curve (E = 10(1/-slope)-1).Taqman® gene expression assays (Applied Biosystems) were used for qPCR analysis on a LightCycler® 480 instrument (Roche). All primers were designed to be intron spanning. qPCR was performed using the LightCycler® Taqman® Master Mix (Roche) in a final reaction volume of 20 p-value of <0.05 indicated statistical significance.Statistical analysis was performed in PASW® statistics 18 (IBM Corp.). Graphs were created in GraphPad Prizm®. Data are expressed as mean ± SEM. Student's t-test was used for statistical analysis of relative expression data after logarithmic transformation because of non-normality of data subsets. A two-sided iv and CD14+ monocytes using a publicly available gene expression data set (GSE2040) that has previously been used to investigate the neovascularization capacity [iv [iv [iv versus CD14+ monocytic cells  (We composed a gene signature that discriminates between CACcapacity  and prodcity [iv  and to vate = 0) .iv signature with the cell specific expression data in the RefExA database yielded abundant evidence for macrophage-associated gene expression or genes that were related to monocyte/macrophage differentiation, such as GPNMB, APOE, APOC1, ACP2 and CYP27A1. In total, 43 of the 70 CACiv signature genes showed to be highly expressed and/or specific for macrophages. Considering the downregulated genes, repression of the monocytic lineage transcriptome was evident . Furthermore, the CACiv signature revealed the upregulation of several genes associated with the differentiation and function of DCs  and osteoclasts  (Comparison of the CACnd MITF) .iv evidenced a high resemblance to that of ‘alternatively activated' M2 macrophages, and more specifically to the M2c subtype that is induced by the anti-inflammatory cytokine IL10 and is characterized by the upregulation of CD163 and CCL18 [CD163 and CCL18, together with other markers specific for M2 differentiation of macrophages, such as SLCO2B1, were identified in the CAC gene signature. The upregulation of the M2c trait was further substantiated with qPCR gene expression analysis of CD163, CCL18 and SCLO2B1 in CACiv from healthy volunteers compared to CD14+ monocytes and HUVEC  and plaque remodeling by atherosclerotic plaque-associated macrophages , and additionally observed repression of several genes regulating transformation of macrophages into foam cells .Additionally, the expression of M1 inflammatory macrophage differentiation markers, such as ATF3, IL1B and CCL3, was found to be significantly downregulated in the microarray data. Interestingly, we identified several genes in the CACiv, we used a microarray data set (GSE5099) on the differentiation of monocytes into macrophages, and of macrophages into the M1 or M2 macrophage subtype . We found that the CACiv expression profile was discordant with the monocytic cell signature (p = 0.001) but significantly correlated with the signature of macrophages (p = 0.011). Concerning M1 versus M2 differentation, CACiv exhibited a predominant M2 expression profile  differentiation. We compared the CACiv signature to the RefExA database and two published data sets of endothelial restricted genes [RNASE1, we found no evidence for induction of endothelial-specific gene expression in the CACiv. Furthermore, comparison of the CACiv signature to a published gene signature of tumor-derived endothelium (ovarian carcinoma) yielded only one tumoral vascular marker, GPNMB, which was upregulated in both profiles [iv from healthy volunteers confirmed RNASE1 and VEGFB upregulation in CACiv versus CD14+ monocytes and HUVEC. However TIE-2 (TEK) expression pertained exclusively to HUVEC whereas both CACiv and CD14+ monocytes failed to show any TIE-2 expression  to clusonocytes . RNASE1,iv give rise to cells with an endothelial genotype, but would rather suggest that CACiv closely resemble M2c macrophages.Together, these findings do not seem to support the hypothesis that CACiv biology, we investigated their cytokine and cytokine receptor expression. Using a global test, we evaluated differences in cytokine-cytokine receptor expression for genes annotated to the KEGG pathway ‘Cytokine-cytokine receptor interaction' and were able to demonstrate clear differences in interleukin, chemokine and chemokine receptor expression between CACiv and monocytes. Monocytes exhibited a classical inflammatory genotype, with expression of inflammatory cytokines, such as IL1ß, (IL6), IL8, (IL12α), IL12ß and TNF , such as MMP2 and MMP9, and inhibits the expression of ADAMTS1, an angiogenesis inhibitor [α has also been shown to upregulate MMP9 activity and enhance angiogenesis [iv with important tissue remodeling and angiogenic properties in the setting of MI.Interestingly, CACnhibitor . IL23α hogenesis . Togetheiv, suggesting that differentiated CACiv, do not display increased hypoxia-directed tissue homing capacity compared to CD14+ monocytic cells. On qPCR analysis, the expression of IL10 and CCR7 was comfirmed to be significantly upregulated in CACiv  and FXR/RXR  signaling pathways and riboflavin metabolism  were significantly upregulated in CACiv compared to CD14+ monocytes , was found to be associated with the monocyte-like profile and appeared to be repressed during differentiation towards CACiv. This finding is in accordance with the cytokine expression profile of CACiv and is indicative of a shift towards immune-modulatory IL10 signaling pathways. The upregulation of the FXR/RXR and LXR/RXR associated genes APOE and NR1H3 in CACiv compared to CD14+ monocytes was confirmed by qPCR analysis  outgrowth endothelial cells (OEC), also known as endothelial colony-forming cells (ECFC). ECFCs were closely related to endothelial cells, whereas the CACiv genotype clustered with monocytic cells and evidenced an alternative activated M2 macrophage genotype [iv and ECFC with various other cell types of hematopoietic and mesenchymal origin and concluded that CACiv display a genotype that is restricted to the hematopoietic lineage, whereas ECFC, together with endothelial cell subtypes, belong to a large mesenchymal cell cluster.Several studies have dealt with the lineage commitment of EPC. A first study questioning the true progenitor cell nature of EPC was published by Rehman et al. , who reva et al. , revealegenotype . Finallygenotype  providediv signature is evident and, together with the evidence from previous studies, almost excludes direct transdifferentiation of CACiv into EC. Most likely, as demonstrated by Prokopi et al. [The absence of endothelial markers in the CACi et al. , this hyß and TNF, play a role in Th1 responses and in the killing of pathogens and tumor cells. M2 macrophages, on the other hand, display anti-inflammatory properties, facilitate Th2 responses and engage in active tissue remodeling and tumor promotion. Recently, this dichotomized view on macrophage differentiation was challenged by a new paradigm [iv profile probably reflects a macrophage subset with M2 predominance, closely resembling regulatory M2c macrophages. Still, CACiv express some M1-associated pro-inflammatory markers, such as IL1α and IL23α, which could aid CACiv to engage in important biological processes, such as tissue remodeling and angiogenesis. Additional studies, comparing the CACiv transcriptome with these and other macrophage-related cell types, are warranted.Macrophages show a remarkable degree of plasticity in response to specific environmental stimuli and many distinct macrophage subsets have been described . Broadlyparadigm  accordiniv transcriptome.Using IPA analysis, we identified riboflavin metabolism and the LXR/RXR and FXR/RXR pathways as the most significantly upregulated biological processes in the CAC+CD14− macrophages in normal intima devoid of atherosclerotic disease [iv expression profile resembled that described by Trogan et al. [iv biology, could hint a possible role for CAC in reverse plaque remodeling.LXRs are a family of cholesterol-sensing nuclear receptors regulating lipid homeostasis and cholesterol transport . In macr disease . The CACn et al. , who shoiv profile. Activation of FXR pathways in mouse models of atherosclerosis could almost completely inhibit aortic atherosclerotic lesion formation [ß, IL6 and TNF [κB-mediated inflammation [in vivo [We also demonstrated upregulation of FXR/RXR cholesterol-sensing nuclear receptors in the CACormation , attenua and TNF  and negaammation . Further[in vivo .iv biology. Riboflavin or vitamin B2 is the central element of the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors are essential for mitochondrial oxidoreductase reactions, cellular oxidative stress resistance and endothelial nitric oxide synthase (eNOS) coupling and eNOS activity [Finally, IPA analysis indicated riboflavin metabolism as significantly upregulated in CACactivity .iv profile, seem to relate to reverse cholesterol transport, immunomodulation, energy metabolism, oxidative stress resistance and NO bioavailability. These findings hint at a possible beneficial effect of CACiv therapy in the treatment of atherosclerotic and cardiovascular disease. Alternatively, pathophysiological conditions leading to impaired in vivo CAC function might induce endothelial dysfunction and atherosclerotic plaque formation and progression. However, future experimental studies are required to confirm these hypotheses.Together, the biological pathways observed in our CACiv are genotypically related to regulatory M2c macrophages. CACiv, however, show little evidence of endothelial cell transdifferentiation. We propose new mechanisms by which CACiv could be efficiently applied in the broad field of cardiovascular pathophysiology, more specifically by immunomodulation, tissue remodeling, enhancement of cholesterol efflux and vasculoprotection.In conclusion, our data indicate that CAC