JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
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Acknowledgements
Materials
Riferimenti
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Medicina

En Murine Model af iskæmisk Retinal Skade induceret af forbigående bilaterale fælles halspulsåren Okklusion

Published: November 12, 2020
doi:
10.3791/61865
, , , , , ,
1Laboratory of Photobiology,Keio University School of Medicine, 2Department of Ophthalmology,Keio University School of Medicine, 3Animal eye care,Tokyo Animal Eye Clinic, 4Tsubota Laboratory, Inc.

Summary

Her beskriver vi en musemodel af nethindeiskæmi ved forbigående bilateral fælles halspulsåre okklusion ved hjælp af enkle suturer og en klemme. Denne model kan være nyttig til at forstå de patologiske mekanismer i nethindeiskæmi forårsaget af hjerte-kar-abnormiteter.

Abstract

Forskellige vaskulære sygdomme såsom diabetisk retinopati, okklusion af nethinde vener eller arterier og okulær iskæmisk syndrom kan føre til nethinde iskæmi. For at undersøge patologiske mekanismer for nethindekemi skal der udvikles relevante eksperimentelle modeller. Anatomisk, en vigtigste retinale blod leverer fartøj er den oftalmiske arterie (OpA) og OpA stammer fra den indre halspulsåren af den fælles halspulsåren (CCA). Således kan afbrydelse af CCA effektivt forårsage nethindeisk iskæmi. Her etablerede vi en musemodel af nethindeiskæmi ved forbigående bilateral fælles halspulsåre okklusion (tBCCAO) for at binde den rigtige CCA med 6-0 silke suturer og for at okkludere venstre CCA forbigående i 2 sekunder via en klemme, og viste, at tBCCAO kunne fremkalde akut nethindeiskæmi, der fører til nethinde dysfunktion. Den nuværende metode reducerer afhængigheden af kirurgiske instrumenter ved kun at bruge kirurgiske nåle og en klemme, forkorter okklusion tid til at minimere uventede dyrs død, som ofte ses i mus modeller af midten cerebral arterie okklusion, og fastholder reproducerbarhed af fælles retinal iskæmiske fund. Modellen kan bruges til at undersøge patofysiologien af iskæmiske retinopatier hos mus og yderligere kan bruges til in vivo lægemiddelscreening.

Introduction

Nethinden er et neurosensorisk væv til visuel funktion. Da en betydelig mængde ilt er nødvendig for visuel funktion, nethinden er kendt som en af de højeste ilt krævende væv i kroppen1. Nethinden er modtagelig for vaskulære sygdomme som ilt leveres gennem blodkarrene. Forskellige typer af vaskulære sygdomme, såsom diabetisk retinopati og nethinde blodkar (vener eller arterier) okklusion, kan fremkalde nethinde iskæmi. For at undersøge patologiske mekanismer for nethindeiskæmi anses reproducerbare og klinisk relevante eksperimentelle modeller af nethindeisk iskæmi for nødvendig. Middle cerebral arterie okklusion (MCAO) ved indsættelse af en intraluminal filament er den mest almindeligt anvendte metode til udvikling af in vivo gnaver modeller af eksperimentel cerebral iskæmi2,3. På grund af nærheden af den oftalmiske arterie (OpA) til MCA bruges MCAO-modeller også samtidigt til at forstå patofysiologien af nethindeisk iskæmi4,5,6. For at fremkalde cerebral iskæmi sammen med nethinde iskæmi, lange filamenter er typisk indsættes gennem indsnit i den fælles halspulsåren (CCA) eller den eksterne halspulsåren (ECA). Disse metoder er vanskelige at udføre, kræver lang tid at afslutte operationen (over 60 minutter for en mus) og fører til høje variabiliteter i resultaterne efter operationen7. Det er fortsat vigtigt at udvikle en bedre model til at forbedre disse bekymringer.

I denne undersøgelse brugte vi simpelthen kort forbigående bilateral CCA okklusion (tBCCAO) med nåle og en klemme til at fremkalde nethindeisk iskæmi hos mus og analyserede typiske resultater af iskæmiske skader i nethinden. I denne video vil vi give en demonstration af tBCCAO-proceduren.

Protocol

Alle de metoder, der er beskrevet her, er blevet godkendt af Institutional Animal Care and Use Committee (IACUC) på Keio University School of Medicine. 1. Fremstilling af kirurgiske instrumenter og dyr Autoklave kirurgiske instrumenter og holde dem i 70% ethylalkohol. Forud for hver ny kirurgisk procedure, rene kirurgiske instrumenter omhyggeligt ved hjælp af 70% ethylalkohol. Forbered han-BALB/cAJc1-mus (6 uger gamle, 26-28 kg) i et specifikt patogenfrit (SPF) rum for at…

Representative Results

Efter systemisk cirkulation af FITC-dextran i 2 minutter blev nethindevakulaturer fra venstre og højre nethinde i de sham-opererede mus og tBCCAO-opererede mus undersøgt (Supplerende figur 1). FITC-dextran var fuldt synlig i begge nethinder i de sham-opererede mus og den venstre nethinde i de tBCCAO-opererede mus, mens den delvist kunne påvises i højre nethinde i de tBCCAO-opererede mus. Efter tBCCAO blev øjenlåg hængende undersøgt (Figur 2</strong…

Discussion

I undersøgelsen har vi vist, at tBCCAO ved hjælp af enkle suturer og en klemme kan fremkalde nethindeisk iskæmi og ledsagende nethindedysfunktion. Desuden har vi vist, at vores nuværende protokol for udvikling af en musemodel af nethindeisk iskæmi er lettere og hurtigere sammenlignet med andre tidligere protokoller til udvikling af nethindeisk iskæmisk skadesmodel2,3,7.

Anatomisk, venstre og hø…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af Grants-in-Aid for Scientific Research (KAKENHI) (18K09424 til Toshihide Kurihara og 20K18393 til Yukihiro Miwa) fra Ministeriet for Uddannelse, Kultur, Sport, Videnskab og Teknologi (MEXT).

Materials

Atipamezole hydrochloride Zenoaq Antisedan For anti-anesthesia
Applied Biosystems 7500 Fast Applied Biosystems – For qPCR
Butorphanol tartrate Meiji Seika Pharma Vetorphale For anesthesia
BZ-II Analyzer KEYENCE – For an image merge
BALB/cAJc1 CLEA – Mouse strain
β-Actin (8H10D10) Mouse mAb CST 3700 For western blot
Clamp Forcep World Precision Instruments WPI 500451 For surgery
Dumont forceps #5 Fine Science Tools 11251-10 For surgery
DAPI solution Dojindo 340-07971 For IHC
Envisu SD-OCT system Leica R4310 For OCT
FITC-dextran Merk FD2000S For retinal blood perfusion
Fluorescence microscope KEYENCE BZ-9000 For fluorescence detection
Gatifloxacin hydrate Senju Pharmaceutical Gachifuro For anti-bacterial infection
GFAP Monoclonal Antibody (2.2B10) Thermo 13-0300 For IHC
Heating pad Marukan RH-200 For surgery
HIF-1α (D1S7W) XP Rabbit mAb CST 36169 For western blot
ImageQuant LAS 4000 mini GE Healthcare – For chemiluminescence
Midazolam Sandoz K.K SANDOZ For anesthesia
Microtome Tissue-Tek TEC 6 Sakura – For sectioning
Medetomidine Orion Corporation Domitor For anesthesia
Needle holder Handaya HS-2307 For surgery
PuREC MAYO Corporation – For ERG
Scissor Fine Science Tools 91460-11 For surgery
Sodium hyaluronate Santen Pharmaceutical Hyalein For eye lubrication
Tropicamide/Penylephrine hydrochloride Santen Pharmaceutical Mydrin-P For mydriasis
6-0 silk suture Natsume E12-60N2 For surgery
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Riferimenti

  1. Anderson, B. Ocular effects of changes in oxygen and carbon dioxide tension. Transactions of the American Ophthalmological Society. 66, 423-474 (1968).
  2. Ingberg, E., Dock, H., Theodorsson, E., Theodorsson, A., Ström, J. O. Method parameters’ impact on mortality and variability in mouse stroke experiments: a meta-analysis. Scientific Reports. 6 (1), 21086 (2016).
  3. Atochin, D. N., Clark, J., Demchenko, I. T., Moskowitz, M. A., Huang, P. L. Rapid Cerebral Ischemic Preconditioning in Mice Deficient in Endothelial and Neuronal Nitric Oxide Synthases. Stroke. 34 (5), 1299-1303 (2003).
  4. Allen, R. S., et al. Severity of middle cerebral artery occlusion determines retinal deficits in rats. Experimental Neurology. 254, 206-215 (2014).
  5. Steele, E. C., Guo, Q., Namura, S. Filamentous Middle Cerebral Artery Occlusion Causes Ischemic Damage to the Retina in Mice. Stroke. 39 (7), 2099-2104 (2008).
  6. Minhas, G., Morishita, R., Anand, A. Preclinical models to investigate retinal ischemia: advances and drawbacks. Frontiers in Neurology. 3, 75 (2012).
  7. McColl, B. W., Carswell, H. V., McCulloch, J., Horsburgh, K. Extension of cerebral hypoperfusion and ischaemic pathology beyond MCA territory after intraluminal filament occlusion in C57Bl/6J mice. Brain Res. 997 (1), 15-23 (2004).
  8. Jiang, A. X., et al. Inducement and Evaluation of a Murine Model of Experimental Myopia. Journal of Visualized Experiments. (143), e58822 (2019).
  9. Miwa, Y., et al. Pharmacological HIF inhibition prevents retinal neovascularization with improved visual function in a murine oxygen-induced retinopathy model. Neurochemistry International. 128, 21-31 (2019).
  10. Adams, S., Pacharinsak, C. Mouse Anesthesia and Analgesia. Current Protocols in Mouse Biology. 5 (1), 51-63 (2015).
  11. Speetzen, L. J., Endres, M., Kunz, A. Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia. Journal of Visualized Experiments. (75), e4387 (2013).
  12. Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. Modeling stroke in mice – middle cerebral artery occlusion with the filament model. Journal of Visualized Experiments. (47), e2423 (2011).
  13. Lee, D., Kang, H., Yoon, K. Y., Chang, Y. Y., Song, H. B. A mouse model of retinal hypoperfusion injury induced by unilateral common carotid artery occlusion. Experimental Eye Research. 201, 108275 (2020).
  14. Li, S., et al. Retro-orbital injection of FITC-dextran is an effective and economical method for observing mouse retinal vessels. Molecular Vision. 17, 3566-3573 (2011).
  15. Tual-Chalot, S., Allinson, K. R., Fruttiger, M., Arthur, H. M. Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo. Journal of Visualized Experiments. (77), e50546 (2013).
  16. Lee, D., et al. A Fairy Chemical Suppresses Retinal Angiogenesis as a HIF Inhibitor. Biomolecules. 10 (10), (2020).
  17. Tomita, Y., et al. Pemafibrate Prevents Retinal Pathological Neovascularization by Increasing FGF21 Level in a Murine Oxygen-Induced Retinopathy Model. International Journal of Molecular Sciences. 20 (23), 5878 (2019).
  18. Yamamoto, H., Schmidt-Kastner, R., Hamasaki, D. I., Yamamoto, H., Parel, J. M. Complex neurodegeneration in retina following moderate ischemia induced by bilateral common carotid artery occlusion in Wistar rats. Experimental Eye Research. 82 (5), 767-779 (2006).
  19. Cheng, L., Yu, H., Yan, N., Lai, K., Xiang, M. Hypoxia-Inducible Factor-1α Target Genes Contribute to Retinal Neuroprotection. Frontiers in Cellular Neuroscience. 11, 20 (2017).
  20. Mole, D. R., et al. Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts. The Journal of Biological Chemistry. 284 (25), 16767-16775 (2009).
  21. Majmundar, A. J., Wong, W. J., Simon, M. C. Hypoxia-Inducible Factors and the Response to Hypoxic Stress. Molecular Cell. 40 (2), 294-309 (2010).
  22. Newman, E. A. Glial cell regulation of neuronal activity and blood flow in the retina by release of gliotransmitters. Philosophical Transactions of the Royal Society B: Biological Sciences. 370 (1672), (2015).
  23. Vecino, E., Rodriguez, F. D., Ruzafa, N., Pereiro, X., Sharma, S. C. Glia-neuron interactions in the mammalian retina. Progress in Retinal and Eye Research. 51, 1-40 (2016).
  24. Symonds, C. The Circle of Willis. British Medical Journal. 1 (4906), 119 (1955).
  25. Lo, W. B., Ellis, H. The circle before willis: a historical account of the intracranial anastomosis. Neurosurgery. 66 (1), 7-18 (2010).
  26. Yang, G., et al. C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia. Brain Research. 752 (1), 209-218 (1997).
  27. Farkas, E., Luiten, P. G. M., Bari, F. Permanent, bilateral common carotid artery occlusion in the rat: A model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Research Reviews. 54 (1), 162-180 (2007).
  28. Morris, G. P., et al. A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice. PLOS ONE. 11 (2), 0148503 (2016).
  29. Tsuchiya, D., Hong, S., Kayama, T., Panter, S. S., Weinstein, P. R. Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice. Brain Research. 970 (1-2), 131-139 (2003).
  30. Kaelin, W. G., Ratcliffe, P. J. Oxygen Sensing by Metazoans: The Central Role of the HIF Hydroxylase Pathway. Molecular Cell. 30 (4), 393-402 (2008).
  31. Pauly, M., Sruthi, R. Ptosis: evaluation and management. Kerala Journal of Ophthalmolgy. 31 (1), 11-16 (2019).
  32. Averbuch-Heller, L., Leigh, R. J., Mermelstein, V., Zagalsky, L., Streifler, J. Y. Ptosis in patients with hemispheric strokes. Neurology. 58 (4), 620 (2002).
  33. Dutton, J. . Atlas of clinical and surgical orbital anatomy, second edition. 113, 1364 (2011).
  34. Ritzel, R. M., et al. Early retinal inflammatory biomarkers in the middle cerebral artery occlusion model of ischemic stroke. Molecular Vision. 22, 575-588 (2016).
  35. Crespo-Garcia, S., et al. Individual and temporal variability of the retina after chronic bilateral common carotid artery occlusion (BCCAO). PLOS ONE. 13 (3), 0193961 (2018).
  36. Qin, Y., et al. Functional and morphologic study of retinal hypoperfusion injury induced by bilateral common carotid artery occlusion in rats. Scientific Reports. 9 (1), 80 (2019).
  37. Block, F., Grommes, C., Kosinski, C., Schmidt, W., Schwarz, M. Retinal ischemia induced by the intraluminal suture method in rats. Neuroscience Letters. 232 (1), 45-48 (1997).
  38. Allen, R. S., et al. Progesterone Treatment in Two Rat Models of Ocular Ischemia. Investigative Ophthalmology & Visual Science. 56 (5), 2880-2891 (2015).
  39. Miller, R. F., Dowling, J. E. Intracellular responses of the Müller (glial) cells of mudpuppy retina: their relation to b-wave of the electroretinogram. Journal of Neurophysiology. 33 (3), 323-341 (1970).
  40. Block, F., Grommes, C., Kosinski, C., Schmidt, W., Schwarz, M. Retinal ischemia induced by the intraluminal suture method in rats. Neuroscience Letters. 232 (1), 45-48 (1997).
  41. Lee, J. H., Shin, J. M., Shin, Y. J., Chun, M. H., Oh, S. J. Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion. Anatomy & Cell Biology. 44 (1), 25-34 (2011).
  42. Li, S. Y., et al. Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury. PLOS ONE. 6 (1), 16380 (2011).
  43. Furashova, O., Matthé, E. Retinal Changes in Different Grades of Retinal Artery Occlusion: An Optical Coherence Tomography Study. Investigative Ophthalmology & Visual Science. 58 (12), 5209-5216 (2017).
  44. Zadeh, J. K., et al. Short-Time Ocular Ischemia Induces Vascular Endothelial Dysfunction and Ganglion Cell Loss in the Pig Retina. International Journal of Molecular Sciences. 20 (19), (2019).
  45. Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. Rodent stroke model guidelines for preclinical stroke trials (1st edition). Journal of Experimental Stroke & Translational Medicine. 2 (2), 2-27 (2009).
  46. Tang, Y., et al. Hypothermia-induced ischemic tolerance is associated with Drp1 inhibition in cerebral ischemia-reperfusion injury of mice. Brain Research. 1646, 73-83 (2016).
  47. Barone, F. C., Knudsen, D. J., Nelson, A. H., Feuerstein, G. Z., Willette, R. N. Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. Journal of Cerebral Blood Flow & Metabolism. 13 (4), 683-692 (1993).
  48. Pula, J. H., Yuen, C. A. Eyes and stroke: the visual aspects of cerebrovascular disease. Stroke and Vascular Neurology. 2 (4), 210 (2017).
  49. Steele, E. C., Guo, Q., Namura, S. Filamentous middle cerebral artery occlusion causes ischemic damage to the retina in mice. Stroke. 39 (7), 2099-2104 (2008).
  50. Sim, D. A., et al. The Effects of Macular Ischemia on Visual Acuity in Diabetic Retinopathy. Investigative Ophthalmology & Visual Science. 54 (3), 2353-2360 (2013).
  51. Wu, K. K., Huan, Y. Streptozotocin-induced diabetic models in mice and rats. Current Protocols in Pharmacology. , (2008).
  52. Mubarak, A., Hodgson, J. M., Considine, M. J., Croft, K. D., Matthews, V. B. Supplementation of a high-fat diet with chlorogenic acid is associated with insulin resistance and hepatic lipid accumulation in mice. Journal of Agricultural and Food Chemistry. 61 (18), 4371-4378 (2013).
  53. Ansari, S., Azari, H., McConnell, D. J., Afzal, A., Mocco, J. Intraluminal middle cerebral artery occlusion (MCAO) model for ischemic stroke with laser doppler flowmetry guidance in mice. Journal of Visualized Experiments. (51), e2879 (2011).
  54. Hedna, V. S., et al. Validity of Laser Doppler Flowmetry in Predicting Outcome in Murine Intraluminal Middle Cerebral Artery Occlusion Stroke. Journal of Vascular and Interventional Neurology. 8 (3), 74-82 (2015).

Tags

Ischemic Retinal InjuryTransient Bilateral Common Carotid Artery OcclusionMurine ModelRetinal IschemiaPathophysiologyIschemic RetinopathiesIn Vivo Drug ScreeningVascular DiseasesDiabetic RetinopathyOcclusion Of Retinal Veins Or ArteriesOcular Ischemic SyndromeBALB/c MiceEyelid Drooping
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Citazione di questo articolo
Lee, D., Miwa, Y., Jeong, H., Ikeda, S., Katada, Y., Tsubota, K., Kurihara, T. A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion. J. Vis. Exp. (165), e61865, doi:10.3791/61865 (2020).
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