Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated  Vascular endothelial growth factor (VEGF) is an essential mediator of vascularization under both physiological and tumour conditions. Due to the control function of tumour vascularization in development of both primary and secondary tumours + Gr1+ myeloid cells, which include neutrophils, macrophages and myeloid-derived suppressor cells, promote tumour progression independent of the adaptive immune system, and that these cells render tumours refractory to angiogenic blockade by VEGF antibodies in vivo kinetic studies of their actual roles in tumour progression remain challenging. Therefore, non-invasive visualization of the kinetic interaction between tumour cells and their microenvironment at high resolution will largely improve our understanding of basic cancer biology and will help to design new therapeutic strategies.Tumour progression is controlled by dynamic and reciprocal interactions between malignant tumour cells and non-malignant cells in their microenvironment Danio rerio, has been an important animal model for cancer, immune and stem cell research over the last decade in vivo analysis of tumour progression and the interactions between tumour cells and the host microenvironment The zebrafish, We have established a rapid, reproducible zebrafish embryonic xenograft model for simultaneous formation of a localized tumour and experimental micrometastasis by intravascular injection of tumour cells into the blood circulation of zebrafish embryos. With non-invasive high-resolution imaging we characterized the critical steps of tumour progression, including tumour vascularization and tissue invasion. Using this model, we found that myeloid cells are involved in these tumour processes, and especially that neutrophils condition the collagen matrix to facilitate metastatic niche formation and tumour invasion. Importantly, we show that VEGFR inhibitors suppress localized tumour growth but, in contrast, promote tumour invasion and micrometastasis formation by enhancing neutrophil migration.mN-phenylthiourea  was applied to prevent pigment formation from 1 day post-fertilization (dpf). For Pu.1 knockdown as previously published, Pu.1 MO  or Sunitinib , beclomethasone  and β-amino- proprionitrile  were applied directly to the egg water and refreshed every 2 days.Zebrafish and embryos were raised, staged and maintained according to standard procedures in compliance with the local animal welfare regulations. The transgenic lines Tg(fli1:GFP) and Tg(mpx:GFP)in vitro for > 10 passages. Zebrafish fibroblast cell lines ZF4 and PAC2 were cultured as previously described Murine aortic endothelial (MAE) cells and tumourigenic FGF2-over-expressing FGF2-T-MAE cells m above the ventral end of the duct of Cuvier where it opens into the heart. After implantation with mammalian cells, zebrafish embryos (including non-implanted controls) were maintained at 34 °C to compromise between the optimal temperature requirements for fish and mammalian cells Dechorionized 2dpf zebrafish embryos were anaesthetized with 0.003% tricaine (Sigma) and positioned on a 10 cm Petri dish coated with 1% agarose. Mammalian cells were trypsinized into single cell suspensions, resuspended in phosphate-buffered saline , kept at room temperature before implantation and implanted within 3 h. Non-fluorescent cells were labelled with the fluorescent cell tracker CM-DiI (Invitrogen) according to the manufacturer's instructions. The cell suspension was loaded into borosilicate glass capillary needles  and the injections were performed using a Pneumatic Pico pump and a manipulator (WPI). 50–400 cells, manually counted, were injected at approximately 60 µFor live imaging, embryos were anaesthetized using 0.016% tricaine (Sigma) and mounted in 0.6% low-melting agarose. Fixed embryos were imaged in PBST. Fluorescent image acquisition was performed using a Leica MZ16FA stereo microscope, a Leica TCS SPE confocal microscope or a Zeiss LSM exciter on an Axio Observer confocal microscope. Confocal stacks were processed for maximum intensity projections with Zeiss ZEN2009 software or ImageJ software. Images were adjusted for brightness and contrast using ImageJ. Overlays were created using Adobe Photoshop CS4 or ImageJ. 3D reconstructions and movies were assembled using ImageJ.Second-harmonic generation (SHG) was used to image the helical structures of collagen fibres, which are capable of combining two photons into one L-plastin was performed after the myeloperoxidase activity assay and the Mpx−/L-plastin+ cells were counted as macrophages in vivo+ cells from the posterior end of the tail fin in each embryo, which exhibited consistent intensity for at least 15 min, were tracked and analysed using the Manual Tracking plugin of ImageJ.In the Tg(fli1:GFP) line, the neutrophils in fixed embryos were detected using the Peroxidase/ Myeloperoxidase Leukocyte Kit (Sigma), as described previously L-plastin  and then wounded on the ventral side of the tail fin with the tip of a glass capillary needle, as described Tetramethylrhodamine dextran was injected into the sinus venosus of anaesthetized zebrafish embryos, as described t-tests with confidence intervals of 90% or 95%: ns, not significant (p > 0.05); *0.01 < p < 0.05; **0.0001 < p < 0.01; ***p < 0.0001.Statistical analysis was performed using Prism 4 software (GraphPad), two- or one-tailed unpaired In order to develop an experimental metastasis model, as frequently used in mice, we implanted up to 400 fluorescent tumour cells into the blood circulation of zebrafish embryos at 2 dpf. The tumour cells haematogenously disseminated in the embryo immediately after injection into the duct of Cuvier; a wide circulation channel on the embryonic yolk sac connecting the heart to the trunk vasculature A, B. WheTaking advantage of the transparency of zebrafish embryos, we performed high-resolution confocal imaging to characterize the localized tumour growth. Shortly after implantation, the non-disseminated tumour cells accumulated locally in close association with the primitive endothelial cells of the duct of Cuvier A, which de novo vascularization in a zebrafish xenograft system. This phenomenon is fundamentally different from tumour vascularization in a previously described angiogenesis assay, which monitors growth of new vessels from existing vessels towards the tumour implant Notably, only in the embryos where such functional tumour neovasculature had formed did the localized tumour cells proliferate and form a tumour mass of > 0.15 mm diameter by 6dpi. Phosphorylation of histone H3 (pH3), a proliferation marker, was significantly increased in both fluorescence-positive tumour cells and in fluorescence-negative stromal cells of the localized tumour mass after 4dpi H, when tThe occurrence of micrometastasis, independent of the localized tumour growth, was also characterized by high-resolution confocal imaging. Micrometastases originated from tumour cells disseminated haematogenously directly after implantation. Disseminated cells were arrested in the host vasculature in the first hour A, and exThe spatially specific invasion site was validated by other tested tumour cell lines . Such tu+ for neutrophils and Mpx−/L-plastin+ for macrophages Leukocytes are known to contribute to different steps of tumour progression, including the formation of tumour vasculature and metastasis m morpholino; m morpholino; To investigate the role of myeloid cells in tumour progression, we used morpholino oligonucleotides to knock down the transcription factor Pu.1, which controls the development and differentiation of myeloid cells in zebrafish embryos L-plastin+ myeloid cells To further decipher the impact of neutrophil CHT–TF transmigration on the spatially co-localized tumour cell invasion process, two-photon excited confocal microscopy and SHG was performed to visualize the extracellular fibrillar collagen matrix upon neutrophil migration m beclomethasone (m beclomethasone 4 h before implantation (Pharmacological approaches were taken to confirm the functional involvement of neutrophil migration in tumour cell invasion. A prototype glucocorticoid receptor agonist, beclomethasone, previously shown to inhibit neutrophil migration ethasone A, B, D. antation C. In addantation A–C. Notaantation D, indicaantation D and tumantation I. Taken Tumour vascularization is an important process in tumour progression. In our model, the localized tumour growth was determined by successful vascularization . AdminisWe then investigated the contribution of myeloid cells to the tumour cell invasion and micrometastasis promoted by VEGFR inhibition. We found that VEGFR inhibition led to a reduction of macrophage numbers, altered the typical elongated macrophage shape into a rounded morphology and suppressed the migratory activity of macrophages D, 9A–C. The recent discoveries in mice models and clinical studies showed that anti-angiogenic therapies by targeting the VEGF signalling inhibited primary tumour growth but promoted tumour invasion and metastasis. Unfortunately, the mechanism of this very relevant clinical problem is still elusive. Answering this question requires live imaging of tumour-microenvironment interactions at the cellular level—a process severely limited in current animal models.in vivo, including tumour vascularization, localized tumour growth, tumour invasion and micrometastasis formation, we discovered a new mechanism of the metastatic niche formation. In addition to the published roles of macrophages in this process Using a novel zebrafish xenograft model that allows simultaneous single-cell resolution monitoring of multi-step tumourigenesis Recently, the zebrafish myeloid cell population has been studied in detail and similarities with the human myeloid cell functions and embryonic differentiation have been demonstrated The involvement of myeloid cells in critical tumourigenesis, steps such as vascularization and invasion, was revealed by both genetic and pharmacological approaches. We demonstrated that myeloid cells, particularly macrophages, are essential for tumour vascularization. Importantly, we also obtained evidence that the micrometastasis site was determined by the physiological migration of neutrophils. Our results show that yet undefined environmental cues favour neutrophil transmigration at the posterior end of the caudal haematopoietic tissue, which remodels the fibrillar collagen matrix and in turn conditions the premetastatic niche for tumour cells to invade into the tail fin. These findings support the ‘seed and soil’ hypothesis, that tumour metastasis is controlled by both the tumourigenic property of disseminated cells and their microenvironment It is known that tumour-infiltrating immune cells can have both tumour-promoting and tumour-antagonizing roles Our data support the recent notion that inhibition of VEGF signalling may have opposite effects on localized tumour growth and on metastasis. It has been suggested that hypoxia-resistant cells may escape from a starving primary tumour treated with VEGFR inhibitors and then result in elevated malignancy in vivo cooperativeness between VEGF signalling and neutrophils in metastasis. In addition, we observed that macrophages were suppressed by the VEGFR inhibitors, which may indicate an indirect involvement of macrophages in this process. The remaining question is, what are the targets of the VEGFR inhibitors in the neutrophils that cause them to enhance their migration? The VEGFR inhibitors used in this study are sufficiently different in structure to ensure that the observations were not due to an off-target effect. However, we can not be certain that observed phenotypes are restricted to targeting of VEGF receptors, since sunitinib works also on PDGFR family members and affects pericyte coverage of endothelium, promoting extravasation of tumour cells and the formation of micrometastasis Our data provide evidence that the physiological migration of neutrophils is elevated upon inhibition of VEGF signalling, which in turn promotes tumour invasion. This is the first demonstration, to our knowledge, of the in vivo model. In conclusion, we exploited this model here to demonstrate that neutrophil migration affects metastasis and that this is enhanced by VEGFR inhibitors.Our findings implicate myeloid cells as critical mediators of tumour invasiveness elicited by VEGFR inhibitors, independent of inflammation and the adaptive immune system. It provides a new mechanism underlying the recent clinical observations of anti-VEGF therapies promoting tumour invasiveness. We propose that dual targeting of angiogenesis and myeloid cells can be beneficial in future treatments. Of course, it will only be relevant for medical studies if the neutrophil transmigration is not restricted to this model. Currently the role of macrophages in intra- and extravasation of cancer cells is well established


Sodium butyrate (Na-Bu) is a potent histone deacetylase inhibitor (HDACi) and with therapeutic efficacy. Spleen tyrosine kinase (Syk) has been implicated in many immunological consequences and is a putative target for cancer treatment.Disrupted kinase and signaling pathways are found in many human cancers and they are implicated in carcinogenesis. Therefore, kinases have been important targets for the development of cancer therapeutics. Human vestibular schwannomas (VS) are the third most common intracranial tumours which occur in the vestibular branch of VIIIin vitro  and their possible effects on cell survival/death and levels of a few key proteins in the treated cells as compared to the untreated cells.The present study was undertaken in order to evaluate the effect Na-Bu, 2,4-Diamino-5-oxo-pyrimidine hydrochloride (CDKi), a broad spectrum kinase inhibitor and BAY 61-3606 (Syk inhibitor) on the survival of VS tumour tissues Fresh tumour tissues were collected randomly from 16 patients with sporadic, VS tumours, minced into pieces and maintained in primary cultures. Twenty four hours later these cells were exposed to Na-Bu, BAY 61-3606 or CDKi. Forty eight hours after exposure, the tissue lysates were analysed by western blotting for expression of pRb and other proteins involved in cell survival/death.cip1/waf1. The Untreated cells had uncleaved PARP-1 protein and the treated cells had cleaved PARP-1. The results show that the observed cell death in treated cells perhaps is mediated by modulation of the levels and processing of certain key proteins. The possible development of these components as therapeutics is discussed.The tissue samples used were positive for S100A protein, the maker for schwann cells confirming the VS tumour samples. The three individual treatments led to morphological change, DNA fragmentation and cell death and significantly reduced level of total and phosphorylated forms of pRb protein and drastically reduced EGF-R protein. These treatments also modulated levels of other proteins involved in cell survival/death such as PI3K, Caspase 3, TGF-β1, JNK, ASK1, Shh, NF-κB, p21 Human vestibular schwannomas (VS) arise from the vestibular branch of the 8th cranial nerve and are benign in nature. These tumours occur in both sporadic and familial forms. The sporadic VS tumours are generally unilateral, and the familial forms are bilateral and are also known as neurofibromatosis type 2 (NF2). Both the alleles of the NF2 gene are inactivated in all VS tumours and as a result these tumours lack the corresponding gene product, Merlin, or schwannomin. In familial cases, the NF2 tumours are known to occur at early ages as compared to sporadic cases. The symptoms associated with VS tumours include hearing loss, nausea, vertigo, tinnitus and facial paralysis, and hydrocephalus is found in severe cases. Due to its anatomical location, patients suffer great morbidity. Mortality rates are low due to this tumour; however, a higher rate of mortality is associated with secondary malignant tumours including glioma, meningiomas, etc. , 2.0-G1-S phase transition pyrimidin-5-yl] amino]pyridine-3-carboxamide hydrochloride) were obtained from Sigma-Aldrich, St. Louis, MO. The stock solutions were prepared in water. Twenty four hours after seeding the VS tumour tissues to the Petri plates, one set of plates was left untreated and used as control, and the others were treated only once with Na-Bu, CDKi, or BAY 61-3606 at a final concentration of 2 mM , 5 nM 1, and 10 Total DNA from the untreated and the treated VS cells were isolated using Tri-reagent  according to the protocol supplied by the manufacturer. Ten micrograms of each DNA sample were separated using a 1% agarose gel, stained with ethidium bromide, visualized under UV light, and photographed.Qualitative and quantitative analysis of the pRb protein in the untreated and treated tumour samples were done by Western blotting as previously described , 19. TheThe level of total pRb [110 kDa hypophosphorylated (pRb) + 112–114 kDa hyperphosphorylated bands (ppRb)] was measured using Bio-Rad  quantity 1 software for all the samples individually, and these values were normalized to the corresponding control 48 kDa γ-tubulin band from the same samples. The total pRb level for each sample was taken as 100%, and the percentage of phosphorylated form of ppRb in the untreated and the treated samples was estimated by calculating the corresponding (112–114 kDa) band intensities. We have also analysed the expression of TGF-β1, JNK1, ASK1, EGF-R, NF-κB, PI3K, Caspase-3, PARP-1, p21, SHH, and S-100A2 proteins in the lysates at 24, 48, and 72 h, and γ-tubulin was used as loading control in these experiments as well. The results were compared between the untreated and treated VS samples.t  test was performed for all the values. Generalized linear model Repeated Measures Analysis of Variants (RMANOVA) one-way classification was used. The overall levels of pRb protein between the untreated and CDKi and BAY 61-3606 treated samples were analysed for statistical significance. For statistical analysis, the percentages of phosphorylated pRb in the untreated and treated samples were transformed to angular transformation. The significance in the levels of phosphorylated form of pRb between the untreated and treated samples was also analysed. The significance in the levels of other proteins involved in the signalling pathways was also analysed between the treated and untreated samples. No statistical analysis was performed for the time course experiments as it was done with one tumour sample. However, statistical analysis was performed for the pooled data obtained from 16 VS tumour samples, 48 h post-treatments.Statistical analysis of the data was performed using Statistical Package for Social Sciences (SPSS) version 18.0. Paired The VS tumours were visualized by MRI/CT scans. Histological sections stained with hematoxylin and eosin (H&E) showed characteristic whirling pattern and the presence of Antony A and Antony B regions characteristic of VS tumours (data not shown). Among the 16 patients, 10 were males and 6 were females, and 7 patients belonged to the younger age group of 35 years or below and the remaining 9 were above 35 years of age. All the patients had very large tumours of approximately 3.5–3.8 cm in diameter or larger .in vitro  had the cell–cell contact and other biochemical properties similar to the tumour tissues as existed within the patients as the tissue integrity was not altered with drastic enzymatic digestions. The primary cultures used in this study therefore contained all the components of the VS tumour in the patient in vivo. The loose cells initially appeared round , 95% (NF-κB), and 60% (Shh) after 48 h of treatment as compared to the untreated control –c. The ueatments –c. No si control  and 5c.Expression of high-levels of 116 kDa PARP-1 and trace levels of 85 kDa cleaved PARP-1 protein was found in the untreated control cells. After treatments, there was an increase in 85 kDa band and a corresponding decrease in the 116-kDa band . HoweverThe levels of S100A2 remained higher in untreated control and this level decreased by 70% upon all three individual treatments .P  < 0.0001, and this was true for all three individual treatments.Statistical analysis of the data indicated that the increase or decrease in the levels of the various protein expression patterns in the treated samples as compared to the untreated controls was significant at 48 h post-treatment with a For the first time, we have analysed the effects of three components on the survival of VS tumour tissue samples from 16 patients in primary cultures. Our data indicates that a clear pathway could be involved in VS tumour cell death only in the tumour samples subjected to these treatments. The observed changes in the cellular morphology  19] and and19] aBased on the time course experiment, it was evident that optimum morphological and biochemical changes occurred at 48 h post-treatment. Therefore, this time point was selected for further experiments with 16 VS tumour samples , and 5c.in vitro  conditions. The pooled data from 16 patients were analysed for statistical significance.All the experiments in this study were performed with fresh VS tumour tissues. Molecular changes are known to happen during establishment of permanent cell lines, enzymatic treatments of patients’ tumour tissues and even in primary cultures when they are kept for a prolonged period. Often times these changes continue during serial propagation of these cell lines. The gene expression pattern in the normal cells, including normal Schwann cells, will be quite different than that of the VS tumour cells. It may not be appropriate to compare data from fresh VS tumour tissues obtained from patients to the data from established cell lines or other normal cells. Therefore, we have compared data between the treated and untreated VS samples from each patient that were maintained individually in identical The presence of increased levels of activated JNK  and b anThe carboxy terminal Ser 780 of the pRb protein has been shown to be efficiently phosphorylated in a cell cycle-dependent manner in T98G glioblastoma cell line . In our A recent study showed elevated expression of SYK in primary retinoblastoma tissue and retinoblastoma cell lines and BAY 61-3606 induced cell death in these samples with a concomitant increase in active Caspase-3 . Our datUndetectable or very low level of TGF-β1 protein by western blotting in all the untreated human VS samples observed by us –c is in cip1/waf1  protein  perhaps have similar characteristics, regardless of the age and gender of the patients. This is evident as only slight variations in gene expression (data in the present manuscript) were observed between the tumour samples. In this study, we have not used any tumour smaller than 3.5 cm in diameter and we have not used any bilateral NF2 tumour samples. The data presented here are preliminary, and the detailed study of the pathways leading to VS tumour cell death is underway. Detailed analysis of the proteins evaluated in this study is warranted in order to understand the actual mechanisms elicited by these proteins. Our preliminary data point towards the possibility to develop therapeutics to perturb the intracellular signalling to control the growth and progression of VS tumours.Data from the present study indicate that the levels of key cell survival proteins such as EGF-R and PI3K as well as phosphorylated pRb were drastically reduced only in the treated VS cells. The treatments also caused increased levels of proteins involved in cell death such as Shh, NF-κB, Caspase 3, and cleavage of PARP in the treated cells as compared to the untreated cells.Ethical clearance: This study was approved by NIMHANS Human Ethics Committee, clearance reference numbers: IEC No. RPA/105/07, dated 26 December, 2007 and No. NIMH/67th IEC/2009, dated 25 September 2009.Funding: This study was supported by financial aid from the Department of Science and Technology (DST), Government of India research project number SR/SO/HS-41/2007. RM is a Senior Research Fellow (SRF) supported by the DST project funds.Acknowledgement: The authors would like to thank Dr. B.A. Chandra Mouli, Dr. S. Sampath, Dr. D.P. Shukla, Dr. S. Dwarkanath, Dr. D.I. Bhat, Dr. N. Rao, Dr. M. Ranjan and others including the residents of the NIMHANS Neuro Surgery Department who provided the tumour tissues used in this study. We thank Dr. D.K. Subbakrishna and Ms. Vasuki Prathyusha for their help in statistical analysis. We thank the Department of Science and Technology (DST), Government of India for providing financial assistance for this study through a research project number SR/SO/HS-41/2007(PI-RG). RM is a Senior Research Fellow (SRF) supported by the DST project funds. The funding agency had no role in the study design, analysis and interpretation of data.Contributors: IDB provided the tumour tissues. RM, IB and RG designed the experiments and RM and RG analysed the data, performed the experiments and wrote the manuscript.


It is now clear that medulloblastoma (MB), one of the most clinically challenging paediatric brain tumours, is not a single disease entity. Rather, it comprises four distinct molecular subgroups (Wnt pathway activated (WNT), Sonic hedgehog pathway activated (SHH), and the less well-characterised Group 3 and Group 4) , 15, whik-means consensus clustering on all CpG probes with a standard deviation >0.25  clearly indicated the presence of four subgroups  showed a higher correlation than the maximum correlation between different donors, indicating the robustness of this assay for archival tissue . This signature allows for simple classification of single clinical samples without the need for comparison against a larger reference dataset. The data for these signature probes for the Heidelberg frozen tumour cohort are given in Supplementary Table 1.The 450k array is also suitable for analysis of DNA from FFPE material. Profiling of the same tumour from both frozen and FFPE material . For both fresh-frozen and FFPE DNA, there was a very high correlation between profiles at all input quantities down to 50 ng . SNP genotyping from the array indicated that four samples were also part of the Heidelberg frozen cohort. As with the frozen versus FFPE comparison, the correlation between these paired samples run in different facilities was higher than any other pairwise comparison . The derived methylation subgroups of the remaining 56 unique samples again gave a very close overlap with the expression-defined subgroups , showing the robustness of this platform for the classification of MB, independent of where the data are generated .To further validate the broad applicability of this technique, we examined an additional independent tumour cohort with matching expression subgrouping data (derived as previously described ), for whMYC/MYCN/GLI2 gene amplifications, from the FFPE as well as the frozen tumour samples  in Group 3/Group 4; Fig. TP53 mutations  in SHH-MB [A further major benefit of using this comprehensive array platform rather than a targeted gene panel is the ability to generate genome-wide copy-number profiles using the intensity measures of the methylation probes, with a good concordance to other copy-number platforms such as CGH or SNP arrays, as we have recently described . This alles Fig. f, h. Steroup 3, i7q in Groroup 3, i7q in Groroup 3, i7q in Gron SHH-MB . This toIn summary, we demonstrate here a method for reliable classification of medulloblastoma into molecular subgroups, and tumour copy-number profiling, using a commercially available DNA methylation array platform that performs well on either frozen or FFPE tumour material. We also show that this technology can be reproducibly applied with low amounts of starting material, at different institutes, and with the benefit of easier handling compared with FFPE-derived RNA. We therefore believe that this platform holds great potential for refining the information obtainable from large, archival tumour series. Most importantly, we also expect that this will become one of the key technologies for risk stratification and patient cohort selection in the next generation of large, biology-led, multi-centre clinical trials.Supplementary material 1 Supplementary material 2 (XLS 123 kb)Below is the link to the electronic supplementary material.


Dynamic contrast-enhanced MRI revealed anti-vascular effects of MLN0518 following 3 days treatment. Hypoxia confers chemo- and radio-resistance, and alongside PDGF, is implicated in evasive resistance to agents targeted against VEGF signalling. PDGFR antagonists may improve potency and efficacy of other therapeutics in combination. This study highlights the challenges of identifying appropriate quantitative imaging response biomarkers in heterogeneous models, particularly considering the multifaceted roles of angiogenic growth factors.Angiogenesis, the development of new blood vessels, is essential for tumour growth; this process is stimulated by the secretion of numerous growth factors including platelet derived growth factor (PDGF). PDGF signalling, through its receptor platelet derived growth factor receptor (PDGFR), is involved in vessel maturation, stimulation of angiogenesis and upregulation of other angiogenic factors, including vascular endothelial growth factor (VEGF). PDGFR is a promising target for anti-cancer therapy because it is expressed on both tumour cells and stromal cells associated with the vasculature. MLN0518 (tandutinib) is a potent inhibitor of type III receptor tyrosine kinases that demonstrates activity against PDGFRα/β, FLT3 and c-KIT. In this study a multi-parametric MRI and histopathological approach was used to interrogate changes in vascular haemodynamics, structural response and hypoxia in C6 glioma xenografts in response to treatment with MLN0518. The doubling time of tumours in mice treated with MLN0518 was significantly longer than tumours in vehicle treated mice. The perfused vessel area, number of alpha smooth muscle actin positive vessels and hypoxic area in MLN0518 treated tumours were also significantly lower after 10 days treatment. These changes were not accompanied by alterations in vessel calibre or fractional blood volume as assessed using susceptibility contrast MRI. Histological assessment of vessel size and total perfused area did not demonstrate any change with treatment. Intrinsic susceptibility MRI did not reveal any difference in baseline R Tumour growth is dependent upon angiogenesis, the development of new blood vessels, to provide a nutritive blood supply The platelet derived growth factor (PDGF) family of growth factors exert their cellular effects by binding to, and activating, the structurally related tyrosine kinase receptors PDGFRα and PDGFRβ in vitroMLN0518  is a potent, ATP-competitive and reversible inhibitor of type III receptor tyrosine kinases that crosses the blood brain barrier and demonstrates activity against PDGFRα/β, FLT3 and c-KIT in the submicromolar range The identification and investigation of biomarkers of treatment response is imperative in the development of molecularly targeted anti-cancer drugs In this study, a multi-parametric MRI and histopathological approach was used to interrogate the vascular structural and haemodynamic response, and the hypoxic changes in C6 glioma xenografts in response to MLN0518.C6 rat glioma cells  were maintained in Ham's F-10 medium supplemented with 10% (v/v) foetal bovine serum and 5 mM L-glutamine .All procedures undertaken at the Institute of Cancer Research were approved by the Institute of Cancer Research Ethical Review Committee and with the authority of Personal and Project Licences issued by the UK Home Office under the Animals (Scientific Procedures) Act 1986, and in accordance with the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research 6) were injected subcutaneously into the flanks of female NCr nude mice under isoflurane anaesthesia. Tumour size was monitored using callipers and growth curves were obtained for individual tumours using the formula for ellipsoid volume (L×W2)/2, where L and W were the two largest dimensions of the ellipsoid. Once tumours reached 200±100 mm3 they were randomised to receive either 20 mg/kg MLN0518 or vehicle (5% dextrose) subcutaneously twice daily for either three or ten days.C6 cells  using a 3 cm birdcage coil or a Varian Inova 7T horizontal bore system  using a 63 mm quadrature coil. For susceptibility contrast and intrinsic susceptibility MRI, anaesthesia was induced with a 10 ml/kg intraperitoneal injection of fentanyl citrate (0.315 mg/ml) plus fluanisone ), midazolam ) and sterile water (1∶1∶2). For dynamic contrast-enhanced MRI, anaesthesia was induced with 3–4% isoflurane in air (2L/min) and maintained at 1–2%. For the contrast-enhanced protocols a lateral tail vein was cannulated with a 27G butterfly catheter  to enable the remote administration of either USPIO particles  or Gd-DTPA . Core body temperature was maintained by warm air blown through the magnet bore.Following either three or ten days treatment with 20 mg/kg MLN0518 or vehicle, mice underwent 2-weighted sequence  = 36ms, repetition time (TR) = 5000 ms) was first used for localisation of the tumour and measurement of tumour volume. Multi-gradient echo  ranging from TE = 6.2 to 28.2 ms, flip angle = 45°, 16 averages), spin echo  and diffusion-weighted spin echo  images were acquired for quantification of R2*, R2 and ADC, respectively, from 3 contiguous 1mm thick axial slices through the largest extent of the tumour, with a 64×64 matrix over a 3×3 cm field of view A rapid acquisition with relaxation enhancement (RARE) TR = 200 ms, 8 echo times ranging from TE = 6.2 to 28.2 ms, flip angle = 45°, 16 averages) were acquired for quantification of R2* from three contiguous axial slices through the largest extent of the tumour. Carbogen (5% CO2/95% O2) was then continuously administered via a nose piece at 1L/min; following a transition period of at least 10 minutes a second set of MGE images were acquired. This protocol was performed on vehicle treated (n = 5) and MLN0518 treated (n = 6) animals.A morphological RARE sequence was used for tumour localisation, following which MGE images  imaging sequence , using a 64×128 matrix (subsequently zero-filled to 128×128) over a 6×6 cm field of view). Gd-DTPA (0.1mmol/kg) was manually injected i.v. over 5 seconds after 10 baseline scans were acquired, and image acquisition continued for a further 70 scans Fast spin echo Ta posteriori algorithm, which took into account the Rician distribution of noise in magnitude MR data in order to provide unbiased parameter estimates 2* and R2 were determined from the susceptibility contrast MRI data. Changes in each measured relaxation rate following delivery of USPIO (ΔR2* and ΔR2) were also evaluated, from which fractional blood volume  and vessel size index  were estimated 2* and ΔR2* from the intrinsic susceptibility MRI data were calculated in the same manner, to reflect changes induced by carbogen breathing. All data were fitted on a pixel-by-pixel basis using in-house software . The median value of each parameter in each tumour was determined. For the DCE MRI data whole tumour ROIs were drawn and no regional segmentation performed. The concentration of Gd-DTPA was determined from the basal T1 and the previously measured relaxivity at 7T of 3.2/mM/s. Parametric IAUGC  maps were subsequently generated for the whole image Parameter estimation was undertaken using a Bayesian maximum Tumour bearing mice were administered 60 mg/kg hypoxia marker pimonidazole hydrochloride  in phosphate-buffered saline (PBS) i.p. After at least 45 minutes 15 mg/kg of the perfusion marker Hoechst 33342  in PBS was injected intravenously through a lateral tail vein Hoechst 33342 fluorescence signals from whole frozen tumour sections  were recorded at 365 nm using a motorised scanning stage  attached to a BX51 microscope  driven by CellP . In addition, individual images from localised regions of each section were acquired. From these images the vascular morphology was visualised and the largest vessel diameter measured using CellP The same sections were then processed for the simultaneous detection of pimonidazole adducts and vascular endothelial marker CD31 Alpha smooth muscle actin (α-SMA) immunohistochemistry was performed on 5 µm formalin-fixed paraffin-embedded (FFPE) sections (n = 12 per treatment group) using mouse monoclonal anti-α-SMA antibodies  and EnVision Dual link HRP-labelled polymer . Composite images of whole tumour sections were acquired using the same microscope system under light. Whole tumour images were acquired to determine areas of α-SMA staining; positively stained vessels were counted from 6 fields at 200× magnification per tumour section FFPE sections were also stained with haematoxylin and eosin (H&E) for the assessment and quantification of necrosis (n = 14 per treatment group). Composite images of whole tumour sections were recorded, and the degree of tumour necrosis was quantified and expressed as a percentage of the whole tumour section area Results are presented as the mean ±1 standard error of the mean (s.e.m.). Significance testing used a Student's unpaired t-test or Mann-Whitney test with a 5% confidence level to compare vehicle and MLN0518 treated tumours at the study endpoint.Mean growth curves for tumours grown in mice receiving 20 mg/kg MLN0518 or vehicle, expressed as a percentage of the tumour volume immediately prior to the first dose, are shown in v) are shown in v and ADC and between vehicle and MLN0518 treated tumours. Quantification of total Hoechst 33342 uptake and vessel size measurements taken from Hoechst 33342 stained tumour sections also showed no significant differences in total perfusion and vessel size, respectively, with MLN0518 treatment (At the end of the ten day treatment period tumours were investigated by MRI. Representative parametric maps of apparent diffusion coefficient (ADC), a measure of the magnitude of Brownian water diffusion within tissue and an imaging biomarker of cellularity, fractional blood volume (fBV) and vessel size index . Dynamic contrast-enhanced MRI performed following three days treatment with MLN0518 or vehicle revealed that the initial area under the gadolinium concentration curve (IAUGC) was significantly lower in tumours in MLN0518 treated mice than in control tumours  or by histological vessel size measurement. Considering the role of PDGF in vascular maturation and developing a vascular hierarchy, coupled with evidence that the number of tumour blood vessels possessing a smooth muscle element to their wall was lower in treated tumours than controls, an increase in vessel calibre following MLN0518 treatment was anticipated No difference in vessel calibre was observed between control and MLN0518 treated tumours using susceptibility contrast MRI  and magnetic resonance elastography (MRE).


Pancreatic cancer is the fourth leading cause of tumour death in the western world. However, appropriate tumour models are scarce. Here we present a syngeneic murine pancreatic cancer model using 7 Tesla MRI and evaluate its clinical relevance and applicability.6606PDA murine pancreatic cancer cells were orthotopically injected into the pancreatic head. Liver metastases were induced through splenic injection. Animals were analyzed by MRI three and five weeks following injection. Tumours were detected using T2-weighted high resolution sequences. Tumour volumes were determined by callipers and MRI. Liver metastases were analyzed using gadolinium-EOB-DTPA and T1-weighted 3D-Flash sequences. Tumour blood flow was measured using low molecular gadobutrol and high molecular gadolinium-DTPA.3+/-243 mm3) with MRI (mean 918 mm3+/-193 mm3) with MRI being more precise. Histology (n = 5) confirmed MRI tumour measurements (mean size MRI 38.5 mm2+/-22.8 mm2 versus 32.6 mm2+/-22.6 mm2 (histology), p < 0,0004) with differences due to fixation and processing of specimens. After splenic injection all mice developed liver metastases with a mean of 8 metastases and a mean volume of 173.8 mm3+/-56.7 mm3 after 5 weeks. Lymphnodes were also easily identified. Tumour accumulation of gadobutrol was significantly (p < 0.05) higher than gadolinium-DTPA. All imaging experiments could be done repeatedly to comply with the 3R-principle thus reducing the number of experimental animals.MRI handling and applicability was similar to human systems, resolution as low as 0.1 mm. After 5 weeks tumour volumes differed significantly (p < 0.01) when comparing calliper measurments (n = 5, mean 1065 mmThis model permits monitoring of tumour growth and metastasis formation in longitudinal non-invasive high-resolution MR studies including using contrast agents comparable to human pancreatic cancer. This multidisciplinary environment enables radiologists, surgeons and physicians to further improve translational research and therapies of pancreatic cancer. Pancreatic cancer is the third most common cause of gastrointestinal malignancies and the fourth leading cause of cancer death in the northern hemisphere. Pancreatic cancer accounts for approximately 33,000 cancer deaths in the United States and almost 13,000 in Germany ,5 the alCurrently, several mouse models of pancreatic cancer have been established . These iThe aim of this study was the evaluation of clinically relevant murine pancreatic cancer models for longitudinal diagnostic and therapeutic studies. An orthotopic pancreatic cancer model as well as a liver metastases model using the cell line 6606PDA  were stuSix to eight weeks old male C57/BL6 mice with a body weight of 20 to 23 g were obtained from Charles River Laboratories  and allowed to adapt to the new surrounding for about seven to fourteen days. They were maintained in a specific pathogen-free environment receiving sterilized food  and tap water ad libitum. Stress levels were kept to a minimum before starting experiments. Animal rooms had a twelve to twelve hour light-dark/day-night cycle and were maintained at constant temperature and humidity. Before starting experiments all animal studies had been approved by the ethics committee for animal care of Mecklenburg-Vorpommern, Germany.D12G allele. Cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin (referred to as "complete medium"). Tissue culture reagents were obtained from Gibco . Cell cultures were kept in a humidified incubator at 37°C with 5% CO2. Cell cultures were kept pathogen-free and were regularly tested for Mycoplasma species. They were consistently negative for Mycoplasma contamination.The murine pancreatic adenocarcinoma cell line 6606PDA was a kind gift from Prof. David Tuveson, Cambridge, UK . 6606PDASubconfluent cultures of 6606PDA cells were harvested using a 0.05% Trypsin-solution, washed twice in PBS, and re-suspended as single-cell suspensions in serum-free RPMI-1640. Cell viability was tested using trypan blue exclusion. Viability was always greater than 95%.® Pfizer Pharma, Berlin, Germany) and xylometazoline hydrochloride  at concentrations of 87 mg/kg and 13 mg/kg injected intraperitoneally. The abdominal cavity was opened by a 1.5 cm wide transverse laparotomy pointing slightly to the right. The head of the pancreas was identified and lifted up by a cotton wool tip. 2.5 × 105 tumour cells in a total volume of 20 μl consisting of equal volumes of PBS and matrigel  were slowly injected into the head of the pancreas using a 27-gauge needle. Matrigel is a substance with a more watery consistency at 4°C but turns into a gel-like substance when reaching temperatures above 22°C. It was used to prevent local spillage and thereby the early development of local peritoneal tumour growth. To further prevent leakage a cotton wool tip was pressed onto the injection site for 30 seconds. To ensure the same injection technique for all experiments the injection was always carried out by the same person (M.S.). The pancreas was then replaced into the abdominal cavity. The abdominal cavity was closed by a running single layer 4/0 polyester suture . To decrease post surgical pain and the effects of surgery we subcutaneously injected buprenorphine hydrochloride at a dose of 0,1 mg/kg. So far, we have not had any fatalities related to surgery.General anaesthesia in mice was induced using a combination of ketamine hydrochloride  for at least 4 hours before starting MRI scans. For MRI scans all mice were kept in a supine position . The depth of anaesthesia was monitored by the breathing rate. MRI sequences were triggered by the breathing rate. An anaesthesia adjusted breathing rate of 40 per minute was found to be optimal. To reduce the influence of bowel motility in all MRI examinations mice were always kept % - 1.5%.Tumour bearing mice were scanned in a high field 7.0 Tesla MRI scanner for small animals ). MRI analyses were performed in a whole mouse body coil  applied to all mice. However, difficult findings or images were always discussed with a senior radiologist. The investigator had been thoroughly trained by analyzing numerous MR scans and optimized the MR analyses for the presented murine models in cooperation with the involved radiologists. Satellite tumours were considered as part of the original tumour if there was true and continuous tumour between original tumour and satellite tumour. Metastases and secondary tumours without continuous tumour growth connecting to the original tumour were excluded when measuring the primary tumour. The tumour borders/edges could easily be identified even without contrast media due to their characteristic shape and appearance. MR images were compared with corresponding macroscopic tumour growth on exploration and with corresponding microscopic tumour growth using histological specimens.3 water of defined volume  in a standardized 1.5 ml Eppendorf tube as an MRI phantom.To analyze the acquired MRI data we used CIT (Center for Information Technology) and NIH  developed algorithms in MIPAV-software . To val®, Bayer Schering Pharma, Berlin, Germany) were injected intravenously 10 minutes before starting MRI scans. Primovist® was used at a concentration of 0.025 mmol/ml and was injected via the tail vein. Also for these experiments mice were kept in a supine position using the whole mouse body coil. For the detection of liver metastases a high resolution T1 flash3d-vibe („volumetric interpolated breathhold examination") weighted sequence was used. Scans were carried out in the frontal as well as the horizontal plane . This was followed by the generation of high resolution T2-weighted images in frontal and horizontal planes as above. The volumes of liver metastases as well as the volume of the liver itself were calculated using MIPAV using the above described algorithm.For the generation of images of liver metastases 200 μl of the contrast medium Gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid  and the high molecular weight contrast medium gadolinium-DTPA . So far, neither of them has induced any adverse reactions.For imaging studies of the blood flow within the tumour two different contrast media were used: the low molecular weight contrast medium gadobutrol  was inserted into the tail vein of the mouse. The catheter was connected with an injection line and linked to the injection pump . Again, for these experiments mice were kept in a supine position using the whole mouse body coil. For the tumour assessment two fast high resolution T2-weighted scans were done: in the frontal as well as the horizontal plane .Volume Interpolated Breathhold Examination, horizontal plane: TR: ca. 3.98 ms; TE: 1.89 ms; FA: 12°; FoV: 50 mm × 50 mm; matrix: 192 × 256; 8 slices at 1.5 mm each slice, acquisition time: 637 sec, total number of pictures: 130, acquisition time: ca. 13:00 min). The contrast medium was injected via the tail vein. The optimal concentration and amount of the contrast medium was calculated using the weight of the mouse. The standard concentration of the contrast medium was 0.1 mmol/kg body weight, i.e. 0.0025 mmol/ml for a 25 g mouse. For a standardized application of the contrast medium (bolus of 200 μl within 3 seconds) an injection pump with bolus function was used .Thereafter, the FoV was determined. It was always essential to have the centre of the FoV within the largest diameter of the tumour. This was followed by T1-weighted pre-contrast scans . Several pre-contrast scans were taken for an acquisition of a statistically stable baseline After completing 9 pre-contrast scans the dynamic contrast medium enhanced scans were performed using the VIBE-recording technique  reflecting the tumour blood flow the base line SI0 was subtracted from SIt divided by the base line value: ΔSIt = (SIt - SI0)/SI0The basis for measuring tumour blood flow was a model in which the accumulation of contrast medium within the tumour over time was used as the marker. The accumulation was analyzed through the change of signal intensity within a defined ROI. This ROI was placed around the whole tumour. Before injecting the contrast agent/medium the base line of the signal intensity was measured. For this altogether 10 analyses were carried through. The mean of analyses 4 through 9 was taken as the base line SIt was measured and calculated for each animal. Finally, the mean SI-index of each group was determined for comparisons between the groups. This method using the above radiological and mathematical means is feasible and reproducible for the exact measurement of tumour blood flow. Furthermore, this method could be used for analyzing different factors that might influence tumour blood flow. The functional distribution of the contrast medium within the tumour tissue was analyzed using the two compartments model of pharmacokinetics [This SI-index ΔSIkinetics .Tumour-bearing animals were sacrificed 5 weeks after tumour injection following the final MRI scan. Pancreatic tumours and/or livers were resected. The volume of tumours was estimated by electronic calliper measurements. Tumour volume was calculated using the formula for ellipsoid tumours V = L × W × H × (π/6). L was the longest distance from right to left, W was the largest dorsal/ventral diameter and H was the largest rostral/caudal diameter . FurtherAll specimens were analyzed by histology, 5 sections were cut per specimen for routine analysis. Specimens were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Tumour rotation during the embedding process was prevented by marking the tumour/pancreatic specimen in situ e.g. by stitches or markers. Then, after fixation, the specimens were transferred into the embedding chambers holding the specimens in position until the paraffin was solid to prevent further rotation. After cutting 4 μm serial sections they were stained using haematoxylin and eosin. Sections were evaluated for various pathologic parameters using a light microscope . Also, the serially cut specimens  were compared with their corresponding MR images. The appropriate matches were found when slide and image showed the same landmarks.In vitro and in vivo results were statistically analyzed using the program GraphPad InStat version 3.06 . The unpaired or the paired t-test with Welch's correction or the unpaired Mann-Whitney test (for non-Gaussian distributions) or the Wilcoxon signed rank test were taken to compare the means of values of the experiments. A p-value below 0.05 was considered to be statistically significant.3 (SD +/- 27.2 mm3). The one sample t-test (p = 0.36) and the Wilcoxon Signed Rank Test (p = 0.5) revealed no significant  difference to the hypothetical value of 1000 mm3.The validation of the used volumetric-algorithms for the compatibility with our MR-system using Eppendorf tubes showed a mean discrepancy to the expected volume of 1.8% with a calculated mean of all coronal and transverse planes of 982 mmAll mice orthotopically injected with tumour cells developed tumours within the head of the pancreas (i.e. 100%). Survival analyses have shown a median survival of about 50 days. To study the size of tumours high resolution T2-weighted images in the frontal as well as the horizontal plane were analyzed. The tumour within the head of the pancreas could be safely identified in all tumour bearing animals. The tumours showed strong signal intensities. With increasing tumour size a typical central zone of tumour necrosis could be identified Figure  and 4.3 . 35 days following tumour injection tumours had increased in volume to 918 mm3 . In addition, tumour size was assessed by using electronic callipers using the formula LxWxHxΠ/6 (see above). Following the last MRI scan after 35 days of tumour growth mice were sacrificed and organs were harvested for further analyses. Tumours measured by callipers were always slightly larger compared to tumours measured by MRI with a mean of 1065 mm3  versus 498.1 mm3 +/-252.8 mm3 , p = 0.069; and 316.5 mm3 +/- 318 mm3 (MRI) versus 463.1 mm3 +/-331.9 mm3  p < 0.007, paired t-test).With the help of breathing rate triggered sequences and the reduction of bowel motility by keeping mice NPO the margins of the tumour could be focused sharply. This was essential for the exact determination of the tumour volume. Tumour growth could be closely monitored over time n = 5): 21 days following tumour injection the mean tumour volume could be measured at 540 mm: 21 days2 +/-22.8 mm2 versus 32.6 mm2 +/- 22.6 mm2 histological tumours, p < 0,0004, paired t-test). This difference was due to tumour shrinking following fixation and possibly alterations after cutting, staining and coverslipping procedures of tumours and was a systematic effect. Therefore, these differences were constantly present and did not differ significantly between animals. Also, the standard deviation was almost exactly the same, pointing out the systematic effect. Furthermore, the very high spatial resolution of the 7 Tesla MRI enabled the investigator to detect changes as small as 0.1 mm. Therefore, tumour infiltration e.g. of the bowel could be seen on MR images or MR changes were highly suggestive of tumour infiltration. This was confirmed by histological analyses  would have been easily detected by MRI. Yet, MRI immediately after injection would have caused too much stress to the mice. The earliest time point of MR imaging was 10 days following injection showing mean tumour volumes of 320 mm3 . Matrigel has not shown any effects on tumour growth or MR imaging. Previous in vitro experiments using matrigel and without matrigel had shown almost similar growth and migration of tumour cells. This was confirmed in in vivo experiments (data not shown). Lymphnode metastases were easily identified ranging from 0 to 8  after 5 weeks in this tumour model (data not shown). They presented as round hyper intense lesions measuring up to 5.0 mm in diameter  there were only small though significant but expected differences in tumour sizes albeit standard deviations were the same. Tumour sizes were compared by using similar sections of MR images and specimen sections the size being measured and compared after electronic scanning  was detected. After five weeks these had significantly (p < 0.03) increased to an average of 8 metastases (range 5 - 12) with a mean volume of 173.8 mm3 (range 123.6 - 235.3 mm3 +/-56.7 mm3) After measuring the total volume of the liver the percentage of the liver volume that was taken up by the metastases could be determined  developed liver metastases. First, small tumours developed within the spleen before metastasizing via the splenic and the portal vein into the liver (data not shown). After three weeks liver metastases could be detected by MRI. The T1 weighted 3D-Flash sequences proved to be an excellent technique for a three dimensional picture of all anatomical structures. This was accomplished within a short overall scanning time. Primovistr Figure  and 7. Td Figure .® as well as the high molecular weight contrast medium Gadomer® resulted in an equivalent accumulation within the vascular system  a quantitative comparison of the different signal intensities was carried out. These tests showed that an analysis of the tumour blood flow was possible and reproducible using either contrast medium. However, using the same concentrations of the contrast medium the two substances showed different signal intensities within the vascular system. Analysing the signal intensities within the aorta (ROI/mean) GadomerFor tumour blood flow analyses we found a concentration of the contrast medium of 0.0025 mmol/ml to be ideal. Using this concentration the analysis of the enhancement of the contrast medium employing VIBE sequences was optimal. At the same time the otherwise increasing possibility of biochemical interferences using higher concentrations of contrast media could be avoided.In vitro and in vivo experiments are necessary to better understand the development and nature of malignant tumours. Furthermore, models that resemble the human situation are critical for developing and testing new therapeutic strategies. Here we used a 7 Tesla MRI for monitoring tumour growth and metastasis formation within liver and lymphnodes in a murine pancreatic cancer model. In addition, the blood flow within tumours could be analyzed. This model reliably mimics the clinical situation of diagnosing and monitoring pancreatic cancer patients by employing MRI [ying MRI ,21.The here utilized 7.0 Tesla small animal MRI scanner by Bruker  is part of a multidisciplinary core facility at Greifswald University. The basic work flow and work charts are similar to the already installed human MRI system which is controlled by Siemens software as is the small animal MRI. One new feature of the system, the possibility of generating breathing rate triggered sequences, has proved indispensable. The depth of anaesthesia controls nicely the breathing rate, enabling the investigator to keep the rate fairly stable. This is essential for breathing rate triggered sequences. Employing this technique, volumes of parenchymatous organs like the liver or the volume of solid tumours could be measured accurately down to differences in size as small as 0.1 mm. In the past, tumour growth and metastasis formation had been studied by direct measurement after sacrificing the animal. The MRI technique enables the researcher to monitor tumour development over time, if necessary several times, in the same animal. This has significantly reduced the number of required animals for longitudinal studies. At the same time, each mouse can serve as its own control comparing images at different time points. Also, for treatment experiments, tumours and their response to treatment can be easily monitored over time. The presented murine model in combination with the 7 Tesla MRI scanner is an excellent example of multidisciplinary cooperation. In this case radiologists, surgeons and physicians have shared their expertise to implement new and valuable tumour models to further improve the research of pancreatic cancer.in vivo models have combined human cells and immunodeficient mice [Many ent mice -11. Thesent mice . Therefoent mice -15. Modeent mice -25.in vivo imaging systems are required.Other tumour models have been introduced working simply with subcutaneously injected tumour cells ,26. The The first reports on the use of MRI in small animals were published some 30 years ago . The tecThe determination of sizes or volumes of implanted tumours without imaging techniques has employed the measurement using callipers or histology. In subcutaneous models calliper measurement is reasonable, since the mouse can be monitored over time without sacrificing it. In orthotopic tumour models this is not possible. Only after sacrificing the animal tumour sizes/volumes can be determined. Using callipers only approximations of the actual size or volumes of in situ tumours are possible. Tomayko et al.  have pubUsing histology we have found a very close correlation between histological size and size determined by MR. In fact, there was no difference in standard deviations. However, due to shrinking during fixation, changes during the processing of specimens including embedding, cutting and staining histological measurements were always somewhat smaller than MR measurements. The correctness of MR measurements were validated by algorithms developed by CIT and NIH specialists employing MIPAV software . FurtherThe 7 Tesla small animal MRI scanner employed here has an excellent spatial resolution. Tumours were always readily detected. Using breathing controlled T2-weighted sequences tumour volumes could be measured very accurately. We could find a plane image resolution of 0.17 mm × 0.13 mm when using slices of 0.7 mm. Due to this high resolution MRI was highly suggestive of tumour infiltration of surrounding organs if invasion was present. When MRI was inconclusive histological examination could differentiate tumour infiltration from inflammation . In factin vivo imaging systems other than MRI, such as CT, CT-PET, ultrasound, bioluminescence and fluorescence imaging techniques, have been developed for determining tumour size and volume in orthotopic experimental models [Several l models . These tl models : each anl models . Also, Ml models . Also, tl models .At the beginning of small animal MRI scanning many problems had been caused by moving artefacts due to difficulties in anaesthesia, the breathing of animals and bowel motility. Modern techniques and the use of the 7 Tesla MRI scanner have greatly reduced these problems . The inhFor MRI scans of the abdomen the whole body mouse coil generated the best images. In contrast to conventional surface coils it is a volume coil and has a very high spatial resolution although a slightly reduced signal to noise ratio. Conventional surface coils like the rat brain coil (2 × 2), which could also be used, showed decreased signal intensities with increased coil distance. The whole body mouse coil avoided inhomogeneities of the magnetic fields. This guarantees the continuous good quality of images of the whole FoV including a good signal to noise ratio.®, comparable to the human situation, metastatic lesions could be accurately detected with a significantly improved focus of the lesions. Using the MRI software MIPAV this is an ideal model for comparative and therapeutic studies involving either liver metastases alone or liver metastases and their primary tumour. The amount and size of liver lesions and, if present, the primary tumour, can be exactly monitored over time without sacrificing animals, again complying with the 3R principles [For the induction of liver metastases several models including the portal vein injection technique  and the inciples .® for the detection of liver metastases and the intravenous contrast media Gadovist® and Gadomer® for tumour blood flow studies employing an orthotopic murine pancreatic cancer model. These models very closely resemble the human situation. Interestingly, He et al. [In this study we have routinely used the intravenous contrast medium Primoviste et al.  describee et al. .® as well as the high molecular weight contrast medium Gadomer® can be used for dynamic contrast enhanced imaging of the tumour blood flow and for analyzing different factors that might influence tumour blood flow.The low molecular weight contrast medium Gadovist® shows a much higher accumulation of contrast medium within the parenchyma of the tumour than the high molecular weight Gadomer®. However, high molecular weight contrast media could be used to study tumour neoangiogenesis [® could show an increased accumulation in areas of increased VEGF activity and a decreased accumulation in areas of decreased or blocked activity. In those studies the dynamic contrast enhanced MRI would demonstrate direct therapeutic effects. Also, they could be monitored over time, again significantly reducing the number of research animals as asked for by Russel and Burch [Gadovistogenesis . VEGF asnd Burch .The analysis of murine pancreatic cancer models can be done comparably to the human situation. Employing a 7 Tesla small animal MRI in a multidisciplinary environment involving radiologists, physicians and surgeons, allows the exact clinical detection and monitoring of tumour growth, tumour volumes, lymphnode and liver metastasis formation as well as the tumour blood flow. The number of research animals can be significantly reduced in longitudinal studies according to the 3R principles. The presented models can be used for imaging studies as well as non-surgical and surgical treatment purposes.The authors declare that they have no competing interests.LIP worked on the study conception and its design and participated in all experiments, statistics and drafting and revision of the manuscript. He read and approved the final manuscript. AK also worked on the study conception and its design and participated in all MR and mouse experiments including surgery. He was involved with statistics, the revision of the manuscript and read and approved the final manuscript. MS performed MR and mouse experiments including surgery. He was involved with interpretation of data, revision of the manuscript as well as reading and approval of the final manuscript. NA carried out MR, histology and mouse experiments including surgery. She took part in the interpretation of data, the revision of the manuscript and read and approved its final version. JPK worked on the study design and MR experiments as well as the interpretation of data and the revision of the manuscript. He read and approved the final manuscript. SS took part in the study design and MR and histology experiments. He interpreted the data and helped with statistics and revision of the manuscript. He read and approved the final manuscript. SR participated in the study design, histology experiments, statistics, the interpretation of data as well as revision of the manuscript. He read and approved its final version. FS also participated in the study design, histology experiments, statistics, the interpretation of data and the revision of the manuscript. He read and approved the final manuscript. SD was involved in MR experiments, the revision of the manuscript and read and approved the final manuscript. SK collected MR and calliper data, performed mouse experiments including surgery and was involved with statistics, the interpretation of data and the revision of the manuscript. She read and approved the final manuscript. FUW participated in the study conception, the interpretation of data and revision of the manuscript. He read and approved the final manuscript. JM worked on the interpretation of data, the revision of the manuscript and read and approved its final version. MML participated in the interpretation of data, the revision of the manuscript and read and approved the final manuscript. SH worked on MR experiments, the interpretation of data and the revision of the manuscript. He read and approved the final manuscript. NH was involved with the interpretation of data, the revision of the manuscript and read and approved its final version. CDH interpreted the data, revised the manuscript and read and approved the final manuscript. RP was also involved with the interpretation of data and the revision of the manuscript. He read and approved the final manuscript. WvB also worked on the study conception and its design. He performed statistics, interpreted the data and was involved with drafting and revising the manuscript. He read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/40/prepub


The unsteady natural convection flow of an incompressible viscous fluid near a vertical plate that applies an arbitrary shear stress to the fluid is studied using the Laplace transform technique. The fluid flow is due to both the shear and the heating of the plate. Closed-form expressions for velocity and temperature are established under the usual Boussinesq approximation. For illustration purposes, two special cases are considered and the influence of pertinent parameters on the fluid motion is graphically underlined. The required time to reach the steady state in the case of oscillating shear stresses on the boundary is also determined. It is encountered, for instance, in the cooling of nuclear reactors or in the study of environmental heat transfer processes. Soundalgekar and Gupta et al.It is also important to specify that the “no slip” boundary condition may not be necessarily applicable to flows of some polymeric fluids that can slip or slide on the boundary. Thus, the shear stress boundary condition is particularly meaningful –(12) we use the Laplace transform technique to eliminate the variable q is the transform parameter.Applying the Laplace transform to (9) and using the corresponding initial and boundary conditions for temperature, we find the next problem in the transform domainThe solution of the problem (13) and (14) is given by2 and the natural condition (12)2 at infinity. However, in this form, the boundary condition (11)2 seems not to be satisfied. In order to eliminate this drawback, we present for Now, applying the inverse Laplace transform to this last equality, we find for the dimensionless temperature see also (16)whichsee also (17)in teThe Nusselt number Nu, which measures the heat transfer rate at the plate, can be obtained using any of the above expressions for temperature. However, we must be very careful because the immediate results can be misleading. If we use (16), for instance, the correct resultFor velocity, we apply the Laplace transform to (8) and use both the corresponding initial and boundary conditions and (15) for Inverting this result, we get for see also  in order1, for instance, it is sufficient to show that the derivative with respect to y of the first term from (22) can be written in the equivalent formDirect computations show that The temperature distribution corresponding to this case can be directly obtained by making For velocity, by making In the absence of thermal effects, as expected, in all cases the velocity is reduced to its mechanical componentThe previous solutions can be used to obtain the dimensionless temperature, Nusselt number and velocity distributions corresponding to any motion problem with physical relevance. For illustration purposes, two special cases are considered, and the corresponding solutions are graphically discussed.Suppose that the vertical plate suddenly applies a constant shear stress Let us now assume that the vertical plate applies an oscillatory shear The temperature distribution is the same as before, but the corresponding velocities are obtained by introducing (35) in (22) or (29). The starting solutions (35) are important for those who want to eliminate the transients from their experiments. They can be written as a sum of steady-state and transient solutions. In order to determine the required time to reach the steady-state, namely the time after which the fluid flows according to the steady-state solutions, we present here the steady-state solutions only, namely see also (36)(37)The steady-state components (36) and (37), unlike the starting solutions (35), differ by a phase shift. This is the reason why we presented the solutions separately, corresponding to the sine or cosine shear stresses on the boundary.The unsteady natural convection flow of an incompressible viscous fluid near an infinite vertical plate with exponential heating is analytically studied. Initially, the plate is at rest and then it suddenly applies an arbitrary time-dependent shear stress to the fluid. Exact solutions are established for dimensionless temperature, Nusselt number and velocity without any restriction. Equivalent forms are presented for the temperature, while the fluid velocity y. Its values at any distance y are always higher for In order to get some physical insight of the results corresponding to a constant shear on the boundary, some numerical calculations have been carried out for different values of pertinent parameters that describe the flow characteristics. y are shown in t. It is clearly seen that the free convection effects are significant and the boundary layer thickness increases in both cases.The effects of Pr and Gr numbers on the fluid motion are presented in Finally, the required time to reach the steady-state for motions due to a plate that applies oscillatory shear stresses to the fluid is determined by The purpose of this work is to provide exact solutions for the unsteady natural convection flow of an incompressible viscous fluid near an infinite plate with shear stress boundary conditions and exponential heating. More exactly, after time Finally, in order to emphasize the influence of pertinent parameters on the temperature, Nusselt number and velocity distributions, and to get some physical insight of the obtained results, two special cases are considered and some numerical calculations and graphs have been carried out. The main findings are:Exact solutions for temperature and velocity are obtained in terms of the complementary error function. They satisfy all imposed initial and boundary conditions.The velocity of the fluid The thermal boundary layer, as well as the temperature of the fluid, increases in time and decreases with respect to the Prandtl number Pr.Nusselt number profiles decrease with respect to Pr near the surface of the plate and then increase. Nusselt number values tend to a steady value as the time increases.The effects of Pr and Gr numbers on the fluid motion are significant and fluids with higher Prandtl number or smaller positive Grashof number move slowly.The fluid velocity, as expected, is an increasing function with respect to Gr in the case of the cooling of the plate. A reverse effect is observed in case of the heating of the plate.The required time to reach the steady-state is much smaller in the case of cosine oscillations in comparison to sine oscillations of the shear stress on the boundary. This is obvious, because at time


An error was introduced in the funding information. Correction text below:An error was introduced in the funding information during the preparation of this article for publication. The correct funding information is as follows: This work was supported by a grant from the Hertie Foundation to RS and TMR, by grants from the German Competence Network 'Degenerative Dementias' (01GI1005B) and the Fritz Thyssen Foundation (grant 10.12.1.192) to TMR and a grant from the Center for Clinical Research (IKFZ) Tübingen (grant 170-0-0) to RS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


The first two authors, Alexander Zoufaly and Jakob P. Cramer, should be noted as having contributed equally to the article.In addition, a funding organization and grant are incorrectly omitted from the Funding statement. The following sentence should be included with the Funding statement: “This research was funded by a grant from the German Ministry of Health (BMG).”


Neural probe technologies have already had a significant positive effect on our understanding of the brain by revealing the functioning of networks of biological neurons. Probes are implanted in different areas of the brain to record and/or stimulate specific sites in the brain. Neural probes are currently used in many clinical settings for diagnosis of brain diseases such as seizers, epilepsy, migraine, Alzheimer's, and dementia. We find these devices assisting paralyzed patients by allowing them to operate computers or robots using their neural activity. In recent years, probe technologies were assisted by rapid advancements in microfabrication and microelectronic technologies and thus are enabling highly functional and robust neural probes which are opening new and exciting avenues in neural sciences and brain machine interfaces. With a wide variety of probes that have been designed, fabricated, and tested to date, this review aims to provide an overview of the advances and recent progress in the microfabrication techniques of neural probes. In addition, we aim to highlight the challenges faced in developing and implementing ultra-long multi-site recording probes that are needed to monitor neural activity from deeper regions in the brain. Finally, we review techniques that can improve the biocompatibility of the neural probes to minimize the immune response and encourage neural growth around the electrodes for long term implantation studies. In the To date, a wide range of neural electrodes have been used in basic neuroscience and neural prosthetic research (brain machine interfaces) starting with the early electrolyte-filled micropipettes , 34-37 aA number of reviews have been previously published for neural probes and neuroMEMS by Najafi , Banks , Heidusc2.Neural probes are microstructures that form the connection between the biological neural tissue with physical devices and electronics. Implantable neural probes for neuroscience and brain machine interfaces are generally preferred to have a minimum footprint as possible to minimize neural damage and incorporate structural features that facilitate easy entry and movement through the brain tissue. The smaller footprint for the neural probe will allow the probe to approach the target neurons as closely as possible and thus improving the signal-to-noise ratio of the recorded activity or target a very specific region to be stimulated. Further, there is growing need to insert large numbers of accurately spaced recording sites into small volumes which is promoting the application of thin-film and micromachining techniques to the microelectrode production.Typically, a MEMS-based neural probe consists of a single or multiple long protruding structures or shafts (of lengths ranging from 200 μm to 15 mm) preferably with thin thickness (thicknesses ranging from 10 μm to 200 μm). Each shaft includes the recording sites, interconnect traces and back carrier area carrying the bonding pads to connect the probes to electronics. Ideally, shafts with minimum thickness are preferred to reduce damage to the tissue when the probe is inserted into the brain. Moreover, miniaturization of the neural probes is very crucial to approach and efficiently interface the neural tissue at the scales of neural cells whose size typically range between 10 and 50 μm in diameter and spaced 20 nm apart . In contMany research groups investigating neural probe technologies are faced with different challenges including non-standard and unconventional fabrication processes leading to low yield and high cost, lack of on-site and monolithic Integrated Circuit (IC) integration leading to high noise and reduced sensitivity, shorter sized probes mainly limited by fabrication technology, low design flexibility, and limited selection of materials having the mechanical properties that fulfill both the implantation application requirements and being compatible with standard microfabrication processing. In addition, biocompatibility of the neural probes to minimize the foreign-body immune response plays an important role in determining long-term function of neural probes during implantation. These parameters in combination ultimately determine the microfabrication process needed to manufacture the neural probes.3.Metal microelectrodes, which enabled the initiation of many pioneering studies in neural sciences, represent the most widely used neural probes . The eleet al. [Typically used electrode materials include platinum, iridium, platinum-iridium, gold, stainless steel, tungsten, and molybdenum. Quartz glass, Teflon, polyimide, and Parylene materials are used for the insulation of these electrodes. The insulating materials ideally should have a high dielectric constant in order to minimize stray capacitances affecting the electrode during recording. A multiple-electrode array of these insulated wires can be made by gluing individual metal wire electrodes together or by using cutoff wire bundles. For instance, Vassiliy Tsytsarev et al.  have devThe exposed or non-insulated area of metal wire electrodes which forms the recording site is further processed for manipulating the impedance of the site. For instance, Skrzypek and Keller covered a tungsten rod with polymethyl methacrylate (PMMA) and placed it in a Scanning Electron Microscope (SEM) such that the desired area on the probe tip was exposed by manual control of the SEM electron beam. The sample probes are shown in et al. has reported a floating array of metal probe by employing laser machining of a ceramic substrate to form recording holes [et al. as shown in More recently, Musallam ng holes . The lasAlthough metal based electrodes are still employed successfully in both acute and chronic recording from the brain tissue, these kinds of electrodes can record activity only at their exposed tip. Increasing the number of recording sites requires increasing the number of electrodes which results in a linear increase in overall probe size and causes undesirable neural tissue damage. Further, it is difficult to insert a large number of probes accurately into a small volume of brain tissue. The main advantage of metal electrodes is the simplicity in their manufacturing. This simplicity also leads to its disadvantage which is the lack of common standards and automation in electrode manufacturing from one institution to other. Due to the lack of automation, the electrical characteristics of the recording tip vary from one microelectrode to the next within a batch, from batch to batch, and from one laboratory to other.et al. [The micromachining of metal wire based neural probe arrays has been demonstrated using electroplating technique as it provides high aspect ratio structures for various metals. This process consists of fabricating multiple metal shafts on the same silicon substrate and then releasing them by back etching of the silicon. The electrodes with an insulation layer, consisting of Parylene-C as shown in et al.  employedet al. , 68. Theet al. .The fabrication starts with patterning multiple regions of seed layer with all except one layer being electrically isolated from the electroplating. The electroplated nickel which was used in the process will grow upward and outward from the edges of the patterned seed layer and results in a structure with rounded edges both in- and out- of plane as shown in During electrodeposition as the height of the electroplated metal equals the height of the neighboring electrically isolated region of the seed layer, plating will occur even on the insulated regions. The electroplating technique is mainly limited by anisotropic deposition rate due to the current crowding effect. Specifically, the electric field lines leave the cathode uniformly and crowd on the exposed metal regions on the wafer.The result is a higher deposition rate laterally than vertically. Moreover, wire-bonding cannot be used to connect the probes to external circuitry so wires have to be manually soldered to the probe bond pads. The silicon based probes discussed in the next section appear to be more attractive option than metal probes as each can carry many recording sites that are placed at relatively known positions on the probe. Thus, the increase in the number of the recording sites does not increase the overall probe size.4.The introduction of surface and bulk micromachining techniques played a significant role in standardizing the production of microprobes with very well defined shanks and precise placement of recording sites , 71. Thepia arachnoid membrane covering the brain. Silicon has well-recognized mechanical advantages suitable for neural probes and it allows the use of the well-established microfabrication technologies and equipment infrastructure developed for the integrated circuits industry leading to high precision and well-defined structural features. The use of silicon photolithography process allow excellent control over the recording site size, shape and spacing enabling multiple recording sites to be placed at variable heights on a single electrode shank. Such ability allows insertion of a large number of recording sites in a small volume which is not possible with metal wire arrays or bundles. Inclusion of integrated circuits can be performed directly on the probes and offers benefits in terms of better signal acquisition, reducing power line interference, and ultimately reducing the overall probe size by reducing the electrical cable sizes [The substrate forms an important part of the probe structure providing the mechanical support. Ideally, the substrate must be biocompatible, small enough to obviate damage to the tissue and strong enough to penetrate the le sizes -83.A well-known example of silicon based neural probes are the Michigan probes  and an epia layer of the brain needed special guide tools for insertion. Mechanical weakness of the probes causes the probes to crack and shatter and may cause severe damage and disturbance to the brain tissue during insertion. Another well-known silicon based-probes incorporating multiple-electrode arrays is the Utah probe and an example of which is shown in Michigan probes have been successfully used in a number of neuroscience applications, but they also suffer from some disadvantages related to probe thickness and durability. Typically, wet etching can be employed for limited probe thickness and thus we find that the typical thickness of the Michigan probes is 15 μm. These probes when pierced through the The Utah electrode arrays have a unique structure with the arrays pointing upwards  as opposed to all other silicon neural probes which are built lying down . As a result, the probe length of the Utah electrode arrays is limited by the silicon wafer thickness. The longest reported probe length is only 1.5 mm. Further, only one recording site can be made on each probe shaft and the fabrication process is not a typical batch process and therefore suffers from low production rates. The interconnection of the Utah electrode arrays is made of a set of polyimide insulated gold wires and ultrasonically bonded on the back of the array to a set of aluminum read-out pads. The stiffness of the metal wire bundle makes these unsuitable for chronic implantation in human brain and especially for the high-density electrode arrays.4.1.2) or glass sandwiched between a thin layer of silicon (device layer) and the silicon substrate (handle wafer). SOI based neural probes employ the SiO2 layer as an etch-stop layer. Several etching techniques are available where the etch rates for SiO2 are much lower than for silicon. The handle wafer is back-etched to release the probe shaft which means that the probe shaft thickness is defined by the thickness of the device layer. Different techniques have been used to develop neural probes using SOI wafers, starting with the use of backside wet-etching using potassium hydroxide (KOH) [SOI wafers are produced by placing a thin, insulating layer such as silicon oxide (SiOde (KOH)  solutionde (KOH) , 102.et al. [et al. and these fabricated probes are shown in et al. used double-sided DRIE of a SOI substrate with the buried SiO2 layer acting as an etch stop to pattern forklike probe shafts as shown in A combination of plasma etching (to shape the probe shafts) with KOH wet etching of the handle wafer (to release the shafts) was used by Kewley et al. . Anotheret al. . An impret al. used DRIE in combination with direct write laser lithography (DWL) for neural probe fabrication [More recently, Kindlundh rication . DWL is 4.2.There are many emerging MEMS and CMOS-MEMS processes that can be used to fabricate neural probes with specific features such as ultra-long reinforced structures and integrated signal processing capability. Typically, we find micromachined electrode arrays are constructed using silicon as the structural support layer with gold as the recording sites material. These materials are available in the standard MEMS processes such as SOIMUMPS from MEMSCAP and MicraGEM from Micralyne. Further, the use of standard fabrication processes yields mass-producible and well-defined probe structures. More recently, CMOS-MEMS process from DALSA 0.8 μm process with bulk micromachining can be employed for development of active and intelligent neural electrodes. However, post-fabrication processing is required for the CMOS-MEMS process to deposit gold or iridium on the electrode recording sites.The authors recently developed prototype neural electrodes using the MicraGEM process. The prototype includes high-aspect ratio silicon electrodes which have 10 μm thickness and up to 6.5 mm in length, although longer probes are possible. Ultra-long probes having lengths longer than 5 mm can be used for recording in deeper regions of the brain. Many probes developed previously for this purpose by several research groups failed (cracked or shattered) during implantation as they were unable to withstand the insertion axial forces, retraction forces, and tension forces of the brain tissue. To overcome the problem, the developed prototype investigates a novel principle of reinforcing the probes in their more susceptible areas to improve their strength during implantation without increasing the dimensions of the probe.Numerical studies are performed to determine the reliability of the ultra-long neural probes when subjected to significant shear stresses due to imperfect insertion into the brain and/or due to movement of the brain relative to the skull. The simulations are used to predict the behaviour of the probes when subjected to a buckling force under axial compression. The horizontal component of the force is very important for neural electrodes as it determines the force at which the probe tip penetrates the brain tissue. As seen from the simulation results in 5.The use of polymer materials for neural probes has been widely studied to replace the normal silicon nitride or silicon dioxide insulation layer deposited during the fabrication of silicon neural electrodes , 106-108et al. [et al. [et al. [et al. [Rousche et al.  demonstr [et al.  as shown [et al.  and is s [et al. .The flexible polyimide electrodes suffer from disadvantages due to the lack of rigidity which causes buckling during insertion into the brain. Given this reason, long length polyimide probes cannot be used as they bend due to lack of rigidity and reduces the accuracy in targeting a selected region in the brain. Moreover, polyimide-based probes are prone to failure due to possible moisture absorption by polyimide. Given this, silicon-based neural probes appear to be more successful owing to the mechanical properties of the silicon and the flexibility of patterning using standard microfabrication technologies.6.Perhaps the greatest challenge facing the use of neural implants is the resultant tissue response to the neural injury which effects the long term functioning of the implanted neural probe. Signal deterioration soon after implantation of arrays has been reported by many groups. For these devices to be useful, they should ideally remain functional indefinitely for enabling self-contained and self-governing brain machine interfaces. Signal deterioration has been attributed to the general immune activation of the brain in response to the presence of a foreign device. Insertion of the electrode ruptures the vasculature and destroys all neurons in its path. Immediately following the insertion of an array, an acute immune response is activated which facilitates the recruitment of glial cells . Activatet al. [Using immunohistology, Biran et al.  showed tet al. . The braet al.  and headet al. . This moet al. . Local det al. . Anti-inet al. . This meet al. recently showed that using nanostructured porous silicon (PSi) surface for implants improved biocompatibility [In contrast to directly battling the immune response, some groups have proposed using neural growth factors to encourage neural growth around the electrode. Kennedy used a glass cone filled with growth factor releasing tissue and showed that using neural growth factors (NGF) improves recording over many months . In additibility . They retibility , 126-1287.The paper aimed to give an overview of the commonly used microfabrication technologies to develop neural probes for prosthesis and brain machine interfaces. Technological advancements in the areas of microelectronics along with surface and bulk micromachining have led to the development of a great number of passive and active probes. In the review, we described metal wire, silicon, and polymer based neural electrodes. Silicon based probes appear to be the most versatile and widely accepted technology for neural microelectrodes. We notice that there are three main considerations for the development of neural electrodes, namely, (i) probe material selection, (ii) microfabrication process, and (iii) biocompatibility interface. The combination of these three factors ultimately determines the functionality, usability, and long-term implantation reliability of the neural electrodes.Implantable neural probes are generally preferred to have a minimum footprint as possible to minimize neural damage and to facilitate easy entry and movement through the brain tissue. The above requirement combined with the growing interest to undertake neuroscience studies from deeper regions of the brain has necessitated the need to develop ultra-long probes (lengths longer than 5mm) with thicknesses less than 50μm. Such high aspect ratio structures pose a great design challenge as the probes have to be able to withstand the insertion axial forces, retraction forces, and tension forces of the brain tissue. New techniques such as probe reinforcement as described in this review have to be developed for these emerging applications. In near future, the standardization of commercial MEMS processes and emerging CMOS-MEMS processes could lead to the development of new neural probes that are cost-effective and mass-produced with ultra-long probe shafts and on-site/on-probe signal processing circuitry.


The Infection Prevention Society (IPS) is a charity that supports members to ensure no person is harmed by a preventable infection. In 2009, IPS began a strategy planning and evaluation exercise that has acted as the catalyst for transformation, achieved through the use of innovative change techniques and a social media campaign.To establish a new strategy and social media campaign.Qualitative and quantitative approacheswere used; a needs assessment of members, a creative thinking workshop using 3 novel techniques drafted a vision, mission, strategic aims and objectives, feedback exercises followed at IPS' annual conferences. This included a Strategy Decision Tree and Strategy Wall to gather free text that was analysed. In parallel, a baseline survey of members’ use of social media was issued and a follow up process for twitter analysis established.In 2011, IPS issued strategic objectives: a) to lead, shape and inform the infection prevention agenda; b) to generate and promote the evidence base for infection prevention, and c) to be the organisation of choice to sustain improvement.The member survey found that equal proportions of members (39%) thought that use of Twitter would enhance and not enhance IPS. Implementation and on-going evaluation exercises were designed from this intelligence. In 2011, 1% of members stated that Twitter would serve a purpose. In 2013, 37% of IPS members were signed up to Twitter. Within 1 month the impact of the @IPS_infection Twitter account increased 2 and a half fold, .This approach engaged members in strategy development and allowed for articulation of achievable but stretching objectives. The use of social media and Twitter in particular is being used to reinvigorate networking and influence, and is already yielding results. A range of measures are being employed to support on-going strategy implementation, to track progress and to role model to other societies.None declared.


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