Audrey Jongen (Presenter)
Maastricht University Medical Centre
Bio: PhD-candidate on Colorectal Anastomotic Leakage at the department of General Surgery from the Maastricht University Medical Centre, the Netherlands.
Authorship: Audrey C.H.M. Jongen (1,2), Joanna W.A.M. Bosmans (1,2), Berta Cillero-Pastor (3), Gert B. Eijkel (3), Shane R. Ellis (3), Joep P.M. Derikx (4), Nicole D. Bouvy (1,2), Ron M. Heeren (3)
(1) Maastricht University Medical Centre, Department of General Surgery, Maastricht, the Netherlands (2) NUTRIM School of Nutrition and Translational Research in Metabolism, University of Maastricht, Maastricht, the Netherlands (3) Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht University, Maastricht, the Netherlands, (4) Pediatric Surgical Centre Amsterdam, Emma's Children's Hospital, Amsterdam Medical Centre/Vrije Universiteit Medisch Centrum, Amsterdam, the Netherlands
| Short Abstract Colorectal anastomotic leakage (CAL) remains the most dreaded complication after colorectal surgery despite extensive research, the implementation of fast track protocols and new surgical techniques. It occurs with an incidence between 7-12% and is associated with high rates of morbidity and mortality, decreased quality of life and a threefold increase in health care costs. In order to elucidate the pathophysiology of CAL, a discovery based longitudinal experiment was conducted to identify molecular pathways of anastomotic healing and anastomotic leakage using MALDI-IMS. Rat models for anastomotic healing and leakage were used, and clear differences in lipid expression could be observed between different time points and between control and anastomotic tissue. IMS was shown to be a valuable tool to identify molecules involved in the normal healing process and anastomotic leakage. |
Long Abstract
Introduction
Colorectal anastomotic leakage is the most dreaded complication after colorectal surgery because of its high morbidity and even – despite improved postoperative care – mortality[1]. The incidence of anastomotic leakage has remained stable over the last decades despite extensive research into risk factors, perioperative strategies and intra-operative interventions[2]. The pathophysiology of anastomotic leakage is largely unknown[3] and we strongly advocate that this should be elucidated prior to conducting intervention studies[4]. Despite some evidence regarding risk factors and some insight in the anastomotic healing process that involves the influx of inflammatory cells, interaction with intestinal microbiota and matrix metalloproteinases, it remains a challenge to study such a complex mechanistic pathways within biochemical and biological organisms.
Fortunately, rapid technological advances have been made in recent years to address these kinds of challenges. Among these, Mass Spectrometry Imaging (MSI) has emerged as an enabling technique to provide insight into the molecular entities within cells, tissues, and whole-body samples and to understand inherent complexities within biological metabolomes. With matrix-assisted laser desorption/ionization (MALDI) imaging, recent breakthroughs were achieved as it became possible to reveal disease-specific molecular features. This process is known as disease phenotyping and has already been done in atherosclerosis[5], renal cell carcinoma[6] and metastatic endometrial cancer[7].
Methods
Animals
A total of 28 Wistar rats with an average body weight of 250 gram were used. Animals were housed at the Central Animal Facilities of the Maastricht University. All animals were provided ad libitum access to food and water, and were cared for according to local standards. Postoperatively, welfare assessment was performed twice daily using a standardized method and animals were given pain medication in case of discomfort. The experimental protocol complied with the Dutch Animal Experimental Act and was approved by the Animal Experimental Committee of Maastricht University Medical Center (DEC2014-120).
Study design
The primary objective of this study was to investigate if the processes involved in anastomotic healing can be detected by means of MALDI MSI over time. Additionally, we explored if the components of this healing process change in case of anastomotic leakage, in order to identify factors that are essential for both anastomotic healing and anastomotic leakage. Anastomotic healing was investigated in a rat model with a sufficient anastomosis, whilst anastomotic leakage was studied in a non-steroidal anti-inflammatory (NSAID) induced model of anastomotic leakage. The colon was transected 2 cm distal from the cecum and an end-to-end anastomosis was created using 12 interrupted polypropylene 6/0 sutures (Prolene, Ethicon, Johnson & Johnson, Somerville, NJ). Time points of sacrifice: 6, 12 and 24 hours, 2, 3, 5, and 7 days after creation of the anastomosis, n=2 per time point. In case of the leakage model, rats were subjected to the oral administration of an NSAID (diclofenac) 12 hours before surgery, and during the first two days postoperatively.
Sample preparation
Tissue samples (the anastomosis with a 5mm margin on both sides and a control sample 2 cm distal from the anastomosis) were embedded in a 10% glycerol solution, snap frozen in liquid nitrogen and stored at -80°C. The samples were then cut into 10 μm sections for MS analysis, and into 4 μm sections for conventional staining using a cryomicrotome (CM 1860 UV, Leica Microsystems GmbH, Wetzlar, Germany) set at -20°C. 10 μm serial sections for MALDI imaging were thaw mounted on Indium-Tin-Oxide (ITO) coated slides (Delta Electronics (Loveland, CO, USA, 4–8 Ω sq−1), whilst the 4 μm slides were thaw mounted on non-conducting tissue slides. The ITO slides coated with 10 μm tissue sections for MS analyses were then placed in a desiccator for 15 minutes. Tissue sections were sprayed with norharmane matrix (7 mg/ml in 1 2 methanol chloroform) using an automated sprayer (TM-Sprayer, HTX Technologies, Carrboro, NC, USA). The samples were dried and stored in the desiccator until MS analysis.
Mass-spectometry acquisition
Biomolecules present in the anastomotic and control tissue were desorbed, ionised and separated according to their mass-to-charge (m/z) ratios using a Waters Synapt G2-Si mass spectrometer (Waters, Manchester, UK) in positive ion mode at a raster size of 100 µm, and an m/z range of 100-2000. Calibration was performed prior to each measurement with red phosphorous. Regions of interest (ROI) were created by using the HDI v1.4 software (Waters). ROI were based on the histological layers of the gut and in specific the mucosal layer in interaction with gut microbiota, both in control and anastomotic tissue. Commensal microbes and pathogenic bacteria colonizing the intestine have the potential to acquire an aggressive and virulent tissue degrading phenotype, resulting in anastomotic breakdown as was shown by both animal and human research[8].
Each m/z value present in this spectral collection was converted to an image by using dedicated imaging software (see also data analysis). The chemical structure of each ion detected from the tissue surface was identified after its isolation and fragmentation inside the mass spectrometer (tandem mass spectrometry, MS/MS).
Data analysis
Comparisons between the different time points and between the anastomotic and control tissue of the same animals were conducted using Biomap (Novartis) and principal component analysis (PCA). Total ion count (TIC) was used for spectral normalization in all cases. In short, PCA is an unsupervised statistical method that aims at pooling a maximum amount of variance in a minimum number of independent variables. Data pre-treatment and PCA were performed using our in-house built ChemomeTricks toolbox for MATLAB version 2014a (The MathWorks, Natick, MA, USA). The peak assignments were performed according to the bibliography and LIPID MAPS software (http://www.lipidmaps.org/tools/ index.html).
Hematoxylin-Eosin staining
After analyses, the MSI slides were rinsed in 70% Ethanol for 10 minutes to remove matrix. Tissue specimens were then rehydrated with graded alcohol to H2O. The slides were submerged into haematoxylin for 3 minutes and rinsed with running tap water for 3 minutes. Subsequently, the slides were counterstained in eosin for 30 seconds and rinsed with running tap water for one minute. Finally, the slides were washed in 100% Ethanol for 2 minutes and dehydrated in xylene for 30 seconds. Optical images were acquired using a MIRAX Desk Scanner (Zeiss, Gottingen, Germany).
Results
We focused on differences in lipid profiles (lipidomics) between colonic tissues. Changes in the distribution and density of cellular constituents were visible as a result of changed distribution of membrane lipids, possibly resulting from altered metabolism or cell-response in the process of anastomotic healing.
Clear differences were observed between different time points and between control and anastomotic tissue. The signal of m/z 824.50 (most likely a phosphatidylcholine (16:0/20:212) showed high correlation with the mucosa and showed an increased expression and signal spreading over time after construction of an anastomosis. The signal of m/z 725.40, which is likely to be sphingomyelin (d18:1/16:013) shows exclusive presence in damaged tissue, i.e. after construction of the anastomosis and an increased expression over time. The control tissue on day 7 shows an expression profile that is more similar to the anastomotic tissue compared to the control tissue on day 3, which shows relatively low expression of most mass signals. Control tissue that has been exposed to a longer follow-up (7 days), shows expression profiles that are more similar to the anastomotic tissue when compared to control tissue after 3 days.
Discussion & Conclusion
This is a pilot report of a discovery based longitudinal experiment of lipidomic molecular activities in healing and leaking intestinal anastomoses in a rat model. For the first time, localised lipid differences between control and anastomotic tissue has been detected through MALDI-MSI. These differences will be expanded on in the future. The fact that control tissue after 7 days showed similar expression profiles to the anastomotic tissue suggests that the processes involved in healing are not limited to the damaged tissue at the anastomotic site, but instead spread over a larger segment over time. Some mass signals show specific expression in damaged tissue, such as m/z 725.40, sphingomyelin[8]. Sphingomyelins have several structural and functional roles in the cell; they are found in the cell membrane and play a role in apoptosis. Recent studies have linked marked alterations in sphingolipid biology to several diseases[9,10]. Particularly detected lipid profiles are specific for pathophysiological processes and can aid in finding new targets for therapy[11]. This has already led to the identification of a mitochondrial key factors in ischemia/reperfusion[12,13].
This experiment shows that mass spectrometry imaging is a valuable tool to identify molecules that are involved in the normal healing process or in anastomotic leakage. When combined with other modalities (IHC, conventional staining, proteomics, peptidomics, metabolomics), this technique can be used to map the molecular pathways involved in the normal anastomotic healing process, as well pathways involved in leaking which are currently still largely unknown. Current studies from our research group are focusing on unravelling the biochemical processes involved in healing and leaking. Subsequent, specific analyses can be run on the same tissues in order to discover biomarkers for leakage and possibly to develop novel preventive techniques.
References & Acknowledgements:
1. Crombe T, Bot J, Messager M, Roger V, Mariette C, Piessen G. Malignancy is a risk factor for postoperative infectious complications after elective colorectal resection. Int J Colorectal Dis 2016;31:885-94.
2. McDermott FD, Heeney A, Kelly ME, Steele RJ, Carlson GL, Winter DC. Systematic review of preoperative, intraoperative and postoperative risk factors for colorectal anastomotic leaks. The British journal of surgery 2015;102:462-79.
3. Shogan BD, Carlisle EM, Alverdy JC, Umanskiy K. Do we really know why colorectal anastomoses leak? Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract 2013;17:1698-707.
4. Bosmans JW, Jongen AC, Bouvy ND, Derikx JP. Colorectal anastomotic healing: why the biological processes that lead to anastomotic leakage should be revealed prior to conducting intervention studies. BMC gastroenterology 2015;15:180.
5. Martin-Lorenzo M, Alvarez-Llamas G, McDonnell LA, Vivanco F. Molecular histology of arteries: mass spectrometry imaging as a novel ex vivo tool to investigate atherosclerosis. Expert review of proteomics 2016;13:69-81.
6. Alfaro CM, Jarmusch AK, Pirro V, et al. Ambient ionization mass spectrometric analysis of human surgical specimens to distinguish renal cell carcinoma from healthy renal tissue. Analytical and bioanalytical chemistry 2016;408:5407-14.
7. Mittal P, Klingler-Hoffmann M, Arentz G, et al. Proteomics of endometrial cancer diagnosis, treatment, and prognosis. Proteomics Clinical applications 2016;10:217-29.
8. Krezalek MA, Alverdy JC. The role of the microbiota in surgical recovery. Current opinion in clinical nutrition and metabolic care 2016.
9. Miyamoto S, Hsu CC, Hamm G, et al. Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator. EBioMedicine 2016;7:121-34.
10. Melland-Smith M, Ermini L, Chauvin S, et al. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia. Autophagy 2015;11:653-69.
11. Rocha B, Cillero-Pastor B, Eijkel G, et al. Characterization of lipidic markers of chondrogenic differentiation using mass spectrometry imaging. Proteomics 2015;15:702-13.
12. Martens JC, Keilhoff G, Halangk W, et al. Lipidomic analysis of molecular cardiolipin species in livers exposed to ischemia/reperfusion. Molecular and cellular biochemistry 2015;400:253-63.
13. Ji J, Baart S, Vikulina AS, et al. Deciphering of mitochondrial cardiolipin oxidative signaling in cerebral ischemia-reperfusion. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 2015;35:319-28.
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