Name: assessment and characterization of hyaloid vessels in mice
Uploaded: 2019-05-15T13:30:00
Description: Watch this Scientific Journal Video about Assessment and Characterization of Hyaloid Vessels in Mice at JoVE.com

Dette arbejde blev støttet af National Institutes of Health (NIH) tilskud (R01 EY024963 og EY028100) til J.C. Z.W. blev støttet af en Knights Templar Eye Foundation karriere starter Grant. Den hyaloide isolations procedure, der er beskrevet i dette studie, blev tilpasset med ændringer fra protokoller, der er generøst delt af DRs. Richard lang, Toshihide Kurihara og Lois Smith, som forfatterne er taknemmelige for.

Alle dyr blev behandlet i overensstemmelse med Association for forskning i vision og oftalmologi (ARVO) erklæring om anvendelse af dyr i oftalmisk og vision forskning for dyreforsøg, efterretnings linjerne for de nationale institutter for sundhed ( NIH) med hensyn til pasning og anvendelse af dyr til eksperimentelle procedurer og de regler, der er fastsat af den institutionelle dyrepleje og anvendelse Komité (IACUC) på Boston children's Hospital. Lrp5-/- mus (lager nr. 005823; Jackson Laboratory)...

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V., Adamek, E., Ohlmann, A., Lutjen-Drecoll, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norrie+gene+product+is+necessary+for+regression+of+hyaloid+vessels.">Norrie gene product is necessary for regression of hyaloid vessels.</a> Investigative Ophthalmology &amp; Visual Science. 45 (7), 2384-2390 (2004).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+expression+of+Wnt-pathway+mediated+genes+in+low-density+lipoprotein+receptor-related+protein+5+(Lrp5)+knockout+mice.">Retinal expression of Wnt-pathway mediated genes in low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice.</a> PLoS One. 7 (1), 30203 (2012).</li><li>Nayak, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+vascular+regression+is+regulated+by+a+Wnt/beta-catenin,+MYC+and+CDKN1A+pathway+that+controls+cell+proliferation+and+cell+death.">Developmental vascular regression is regulated by a Wnt/beta-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death.</a> Development. 145 (12), (2018).</li><li>Kurihara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+pVHL+and+HIF-alpha+isoforms+are+required+for+embryonic-to-adult+vascular+transition+in+the+eye.">Astrocyte pVHL and HIF-alpha isoforms are required for embryonic-to-adult vascular transition in the eye.</a> The Journal of Cell Biology. 195 (4), 689-701 (2011).</li><li>Huang, T. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+HIF-1alpha+partially+rescues+the+abnormal+hyaloid+vascular+system+in+Cited2+conditional+knockout+mouse+eyes.">Deletion of HIF-1alpha partially rescues the abnormal hyaloid vascular system in Cited2 conditional knockout mouse eyes.</a> Molecular Vision. 18, 1260-1270 (2012).</li><li>Yoshikawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regression+of+hyaloid+vasculature+is+triggered+by+neurons.">Developmental regression of hyaloid vasculature is triggered by neurons.</a> The Journal of Experimental Medicine. 213 (7), 1175-1183 (2016).</li><li>Garcia, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+VEGF-A+in+lens+development:+formation+of+the+hyaloid+capillary+network+and+protection+against+transient+nuclear+cataracts.">The function of VEGF-A in lens development: formation of the hyaloid capillary network and protection against transient nuclear cataracts.</a> Experimental Eye Research. 88 (2), 270-276 (2009).</li><li>Hurskainen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+maturation+of+the+retinal+vasculature+in+type+XVIII+collagen/endostatin+deficient+mice+and+changes+in+retinal+glial+cells+due+to+lack+of+collagen+types+XV+and+XVIII.">Abnormal maturation of the retinal vasculature in type XVIII collagen/endostatin deficient mice and changes in retinal glial cells due to lack of collagen types XV and XVIII.</a> FASEB journal. 19 (11), 1564-1566 (2005).</li><li>Fukai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+collagen+XVIII/endostatin+results+in+eye+abnormalities.">Lack of collagen XVIII/endostatin results in eye abnormalities.</a> The EMBO Journal. 21 (7), 1535-1544 (2002).</li><li>McKeller, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arf+tumor+suppressor+gene+promotes+hyaloid+vascular+regression+during+mouse+eye+development.">The Arf tumor suppressor gene promotes hyaloid vascular regression during mouse eye development.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (6), 3848-3853 (2002).</li><li>Hackett, S. F., Wiegand, S., Yancopoulos, G., Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiopoietin-2+plays+an+important+role+in+retinal+angiogenesis.">Angiopoietin-2 plays an important role in retinal angiogenesis.</a> Journal of Cellular Physiology. 192 (2), 182-187 (2002).</li><li>Chang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haploinsufficient+Bmp4+ocular+phenotypes+include+anterior+segment+dysgenesis+with+elevated+intraocular+pressure.">Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure.</a> BMC Genetics. 2, 18 (2001).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+Activation+of+Wnt+Signaling+by+Lithium+Normalizes+Retinal+Vasculature+in+a+Murine+Model+of+Familial+Exudative+Vitreoretinopathy.">Pharmacologic Activation of Wnt Signaling by Lithium Normalizes Retinal Vasculature in a Murine Model of Familial Exudative Vitreoretinopathy.</a> The American Journal of Pathology. 186 (10), 2588-2600 (2016).</li><li>Gong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PLoS One. 10 (7), 0132643 (2015).</li><li>Kishimoto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+characteristics+of+regressing+vessels+in+the+hyaloid+vascular+system+of+neonatal+mice:+Novel+implication+for+vascular+atrophy.">Histochemical characteristics of regressing vessels in the hyaloid vascular system of neonatal mice: Novel implication for vascular atrophy.</a> Experimental Eye Research. 172, 1-9 (2018).</li><li>Lang, R. 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Teknikker til at vurdere og karakterisere hyaloide fartøjer er intuitive og nødvendige procedurer for at observere hyaloid fartøjets regression i dyremodeller, for at tillade undersøgelser af de mekanismer, der underliggende den vaskulære regression under udvikling. Mens den in vivo retinale billeddannelse tillader langsgående observation af hyaloideregression i det samme dyr, kan adgang til et gnaver fundus Imaging system for OCT og FFA være en begrænsende faktor. Desuden er in vivo billeddannelse i levende mus ...

Blodforsyningen i øjet er afgørende for at sikre den normale udvikling af nethinden og omgivende okulære væv og for at udstyre en ordentlig visuel funktion. Der er tre vaskulære senge i øjet: retinal vasculature, choroid, og en forbigående embryonale kredsløbssygdomme netværk af hyaloide fartøjer. Udviklingen af den okulære vaskulatur kræver rumlig og tidsmæssig koordination i hele embryogenese og vævs modning. Blandt de tre vaskulære senge, hyaloid Vaskulaturen er den første funktionelle blodforsyning system til at give næring og ilt til den nyligt dannede embryonale linse og den udviklende nethinden. Hyaloid fartøjer tilbagegang på samme tid, at nethinden vasculatures udvikle og modne1. Regression af hyaloid Vaskulaturen er afgørende for at give en klar visuel vej for udviklingen af visuel funktion; Derfor er denne vaskulære regressions proces så vigtig som væksten af retinale vasculature. Nedsat hyaloid regression kan føre til øjensygdomme. Desuden, regression af hyaloid fartøjer giver et model system til at undersøge de cellulære og molekylære mekanismer involveret i reguleringen af vaskulær regression, som kan have konsekvenser for angiogen regulering i andre organer samt.
Hyaloidevaskulaturen, afledt af hyaloidearterie (ha), består af Vasa hyaloidea propria (VHP), tunica vasculosa lentis (TVL) og pupil membran (PM). Det giver næring til den udviklende nethinden, den primære vitreous, og linsen under fosterudvikling2. Som følge af HA, VHP grene anteriorly gennem glaslegemet til linsen. TVL bægre den bageste overflade af linsen kapsel, og anastomoser til PM, som forbinder til de forreste ciliære arterier, dækker den forreste overflade af linsen2,3, hvilket resulterer i dannelsen af et netværk af fartøjer i PM 3 , 4 , 5. interessant, der er ingen vener i hyaloid Vaskulaturen, og systemet gør brug af koroidal vener til at udføre venøs dræning.
I det menneskelige embryon er hyaloid Vaskulaturen næsten færdig på omtrent den niende uge af drænings perioden og begynder at genvinde, når de første retinale fartøjer vises, i løbet af den fjerde måned af drænings perioden2. Begyndende med atrofi af VHP, regression af kapillar netværk af TVL, PM, og endelig, ha opstår efterfølgende2,3. I mellemtiden begynder de primære glasfiber trækker og den sekundære glasfiber at danne, sammensat af de ekstracellulære matrixkomponenter, herunder kollagen fibre. Den sjette måned af drænings måneden, den primære glaslegemet er reduceret til en lille gennemsigtig kanal, der strækker sig fra synsnerven disken til linsen, kaldet Cloquet kanalen eller hyaloid kanalen, og den sekundære glaslegemet bliver den vigtigste komponent i det bageste segment 2 , 3. den hyaloide cirkulation forsvinder for det meste på 35 til 36 ugers dræelse, lige før fødslen3.
I modsætning til mennesker, i hvem hyaloid Vaskulaturen er helt svandt ved fødslen, musen hyaloid vaskulære system begynder at genvinde efter fødslen. Da musen nethinden er født avaskulære og retinale fartøjer udvikle postnatalt, hyaloid fartøjer tilbagegang samtidig fra postnatal dag (P) 4 og er for det meste helt svandt af P216 (figur 1). PM forsvinder først mellem P10 og P12, og VHP forsvinder mellem P12 og P16, mens et lille antal TVL-og HA-celler forbliver selv på P16, og ved P21 er hyaloid vaskulære system regression næsten komplet6. I mellemtiden begynder retinal Vaskulaturen at udvikle sig efter fødslen. Det overfladiske lag af vaskulære plexus strækker sig fuldt ud til den perifere nethinden på P7 – P8, det dybe lag (placeret i det ydre plexiformlag) udvikler sig fra P7 – P12, og endelig udvikler det mellemliggende plexus i det inderste plexiformlag mellem P12 og P157 . Efterhånden som den retinale vaskulatur udvikler sig, erstatter den gradvist funktionen af samtidigt regresserende hyaloidekar, der giver næring og ilt til det udviklende øje. Den postnatale forekomst af hyaloid-fartøjs regression i mus giver en let tilgængelig eksperimentel model til at observere og studere hyaloidevaskulaturen samt det molekylære grundlag for vaskulære regressions processer under både fysiologiske og patologiske tilstande8.
Svigt af hyaloid regression kan ses i sygdomme som PHPV, som er en sjælden medfødt udviklingsmæssige anomali af øjet som følge af en mislykket eller ufuldstændig regression af den embryologiske, primære glasøse og hyaloid Vaskulaturen9. De mekanismer, der regulerer regressions processen af hyaloid vaskulatur er komplicerede og bredt undersøgt. En af de vigtigste molekylære veje, der er afgørende for den normale regression af hyaloide fartøjer, er WNT signalering pathway10, da genetiske mutationer i denne sti, der påvirker både WNT ligand og receptorer, er blevet forbundet med PHPV i mennesker9. Eksperimentelle undersøgelser identificerede en WNT ligand, Wnt7b, som produceres af makrofager omkring hyaloide fartøjer i det udviklende øje for at mægle denne regressions proces. Wnt7b aktiverer WNT signalering ved binding med receptorerne frizzled4 (FZD4)/LRP5 i tilstødende endotelceller til at initiere celle apoptose, hvilket fører til regression af hyaloid fartøjer10. Som et resultat, Wnt7b-mangelfulde mus viser en persistens af hyaloid fartøjer10. Tilsvarende binder en ikke-konventionel WNT ligand, Norrin (kodet af NDP -genet), også til FZD4/LRP5 for at inducere hyaloid-fartøjets regression under udviklingen. NDPy/-, Lrp5-/-, og Fzd4-/- mus alle display udskudt hyaloid fartøj regression, understøtter en kritisk regulerende rolle i WNT signalering11,12, 13,14,15,16. Desuden er en anden WNT coreceptor LRP6 overlapper med LRP5 i deres funktion på modulering af WNT signalering pathway i hyaloid vaskulære endotheliale celler17. Andre faktorer, der kan også bidrage til hyaloid regression omfatter hypoxia-inducerbarfaktor 18,19, vaskulær endotelial vækstfaktor20,21, kollagen-1822, 23, ARF24, angiopoietin-225, og knogle morfogenetisk protein-426. I dette papir bruger vi Lrp5-/- mus som en model af vedvarende hyaloide fartøjer til at demonstrere teknikker til vurdering og karakterisering af hyaloid Vaskulaturen gennem både in vivo og ex vivo metoder.
Visualisering af hyaloid Vaskulaturen in vivo og ex vivo er afgørende for at studere mekanismerne i hyaloid fartøjs regression. Nuværende metoder til at observere hyaloid Vaskulaturen primært fokusere på visualisering og analyse af VHP og ha, gennem Oct og FFA billeder, Eye Cross sektioner, og hyaloid Flat mount. OCT og FFA er kraftfulde in vivo Imaging værktøjer, så langsgående observation i levende dyr, efter at de har åbnet deres øjne. Desuden giver isoleret hyaloid Flat Mount visualisering af hele hyaloid Vaskulaturen og et middel til at opnå en nøjagtig kvantificering af fartøjs numrene. Men den skrøbelige og skrøbelige karakter af hyaloide fartøjer og de deraf følgende tekniske vanskeligheder ved dets isolation kan have begrænset dets anvendelse i øjen forskning noget10,17,27. I dette papir, vi giver en detaljeret protokol af visualisering af hyaloide fartøjer, der kombinerer både in vivo levende retinale Imaging og ex vivo isoleret hyaloid Flat mount for at øge gennemførligheden af disse teknikker. Denne protokol er blevet tilpasset med ændringer og udvidelser fra tidligere publikationer om in vivo-metoden med levende fundus og OCT Imaging28 og ex vivo - metoden for isoleret hyaloid Flat Mount11.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">AK-Fluor (fluorescein injection, USP)</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:156px;">17478-253-10</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Anti-CD31 antibody</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:156px;">ab28364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Antifade mounting medium</td>
			<td style="width:191px;">Thermo Fisher</td>
			<td style="width:156px;">S2828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifade Mounting Medium with DAPI</td>
			<td style="width:191px;">Vector Laboratories</td>
			<td style="width:156px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tear eyedrop</td>
			<td style="width:191px;">Systane</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bovine serum albumin (BSA)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">C57BL/6J mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO: 000664</td>
			<td></td>
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			<td height="21" style="height:21px;width:355px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">C1016</td>
			<td></td>
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			<td height="21" style="height:21px;width:355px;">Cryostat</td>
			<td style="width:191px;">Leica</td>
			<td style="width:156px;">CM3050S</td>
			<td></td>
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			<td style="width:191px;">Leica</td>
			<td style="width:156px;">CM3050 S</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Cyclopentolate hydrochloride and phenylephrine hydrochloride eyedrop</td>
			<td>Cyclomydril</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">G9382</td>
			<td></td>
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			<td height="42" style="height:42px;width:355px;">Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:156px;">A-11008</td>
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			<td height="21" style="height:21px;">Heating board</td>
			<td style="width:191px;">Lab-Line Instruments Inc.</td>
			<td style="width:156px;">N/A</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:355px;">Isolectin GS-IB4, 594 conjugate</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:156px;">I21413</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ketamine hydrochloride injection</td>
			<td style="width:191px;">KetaVed</td>
			<td style="width:156px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Lrp5-/- mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO. 005823</td>
			<td>Developed by Deltagen Inc., San Mateo, CA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Micron IV and OCT</td>
			<td style="width:191px;">Phoenix Research Labs</td>
			<td style="width:156px;">N/A</td>
			<td>Imaging software: InSight</td>
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			<td height="21" style="height:21px;width:355px;">Microscope</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:156px;">discovery v8</td>
			<td></td>
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			<td height="21" style="height:21px;width:355px;">Microsurgery forceps</td>
			<td style="width:191px;">Scanlan International</td>
			<td style="width:156px;">4004-05</td>
			<td></td>
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			<td height="21" style="height:21px;width:355px;">Microsurgery scissors</td>
			<td style="width:191px;">Scanlan International</td>
			<td style="width:156px;">6006-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
			<td style="width:191px;">Tissue-Tek</td>
			<td style="width:156px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
			<td style="width:191px;">Agar Scientific</td>
			<td style="width:156px;">AGR1180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (16%)</td>
			<td>Electron&#160;Microscopy&#160;Sciences</td>
			<td style="width:156px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Peel-A-Way disposable embedding molds (tissue molds)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:156px;">12-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS) buffer (10X)</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:156px;">P0496</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Slide cover glass</td>
			<td style="width:191px;">Premiere</td>
			<td style="width:156px;">94-2222-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost microscope slides&#160;</td>
			<td style="width:191px;">Fisherbrand</td>
			<td style="width:156px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Triton X-100</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Xylazine sterile solution</td>
			<td style="width:191px;">Akorn: AnaSed</td>
			<td style="width:156px;">NDC: 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment and characterization of hyaloid vessels in mice

I øjet nærer de embryonale hyaloide fartøjer den udviklende linse og nethinden og genstigning, når nethinde skibene udvikler sig. Persisterende eller mislykket regression af hyaloide fartøjer kan ses i sygdomme som vedvarende hyperplastisk primær glaslegemet (PHPV), hvilket fører til en blokeret lysvej og nedsat visuel funktion. Forståelse af mekanismerne bag hyaloid-fartøjets regression kan føre til ny Molekylær indsigt i den vaskulære regressions proces og mulige nye måder at håndtere sygdomme med persistente hyaloide fartøjer på. Her beskriver vi procedurerne for billeddannelse hyaloid i levende mus med optisk kohærens tomografi (OCT) og fundus fluorescein angiografi (FFA) og en detaljeret teknisk protokol om isolering og flad montering hyaloid ex vivo til kvantitativ analyse. Lavdensitets lipoprotein receptor-relaterede protein 5 (LRP5) Knockout-mus blev brugt som en eksperimentel model af vedvarende hyaloide fartøjer for at illustrere teknikkerne. Tilsammen kan disse teknikker lette en grundig vurdering af hyaloide fartøjer som en eksperimentel model af vaskulær regression og undersøgelser af mekanismen af persistente hyaloide fartøjer.

In vivo billeddannelse af hyaloide fartøjer i levende mus Figur 3 A afslører tværsnits udsigt over OLT billeder til nethinden og hyaloid væv i 3-måneders-gamle WT og Lrp5-/-mus, en dyremodel med vedvarende hyaloid. WT Eye viser fraværet af hyaloid væv, mens Lrp5-/-Eye viser to vedvarende hyaloide fartøjer afledt af synsnerven hoved. Figur 3 B viser FFA billeder ...

Watch this Scientific Journal Video about Assessment and Characterization of Hyaloid Vessels in Mice at JoVE.com

Assessment and Characterization of Hyaloid Vessels in Mice

Denne protokol beskriver både in vivo og ex vivo metoder til fuldt ud at visualisere og karakterisere hyaloide fartøjer, en model af vaskulær regression i muse øjne, ved hjælp af optisk kohærens tomografi og fundus fluorescein angiografi for levende billeddannelse og ex vivo isolering og efterfølgende flad mount af hyaloid for kvantitativ analyse.

Vurdering og karakterisering af Hyaloide fartøjer i mus

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

Vores protokol giver både in vivo og ex vivo foranstaltninger til visualisering og karakterisering af hyaloid vaskulatur i øjet som en nyttig eksperimentel model for at studere vaskulær regression. Denne teknik kombinerer både in vivo langsgående observation af hyaloid fartøjer i levende mus og ex vivo visualisering og nøjagtig kvantificering af dissekeret hele hyaloid fartøjer. Vores teknik giver en praktisk og gennemførlig metode til undersøgelse af patogenese og grundlæggende mekanismer i vedvarende føtal vaskulatur.Denne metode giver også indsigt i cellulære og molekylære mekanismer, der er involveret i reguleringen af okulær vaskulær regression, som kan være relevante for angiogenese forskning i andre organer. Den hyaloid isolation trin kan være teknisk udfordrende for begyndere. Masser af praksis og tålmodighed vil bidrage til at sikre en vellykket dissektion.Visualisering af denne metode vil give detaljerede praktiske oplysninger og tips om den teknik, der kan hjælpe seerne med at mestre færdighederne mere effektivt. For optisk sammenhæng tomografi eller OLT billeddannelse, justere musen og sonden, så den optiske nerve hovedet er i midten af det visuelle felt i fundus billedet og justere fokus og vinklen på den angivne linje i OCT imaging software til at afsløre den vedvarende hyaloid fartøjer, før du får billeder. For fundus fluorescein angiografi billeddannelse af hyaloid fartøjer, justere tilpasningen lidt for at placere synsnerven hovedet i midten af synsfeltet og ændre mikroskop filter til en grøn fluorescerende kanal.Fokuser derefter på de vedvarende hyaloidbeholdere og få flere billeder på et, tre, fem og 10 minutter efterindtøbning for at bestemme det bedste tidspunkt for signal-til-baggrund-forholdet til observation af fartøjerne. For ex vivo hyaloid fartøj dissektion og visualisering bruge mikrokirurgi pincet til at åbne øjenlågene i en postnatal dag-otte mus og placere buede mikrokirurgi pincet under kloden i kredsløb om det ene øje til at forstå synsnerven uden at klemme øjeæblet. Træk forsigtigt og fjern øjeæblet med scer og fastgør øjet i 4% paraformaldehyd ved stuetemperatur.Efter 30 minutter overføre de faste øjne til iskold PBS under en dissektion mikroskop og injicere 50 mikroliter af gelatine opløsning i glaslegeme af øjeæblet på fire forskellige jævnt fordelte steder omkring limbus. Derefter inkubere øjeæblet på is i 30 minutter for at størkne den injicerede gelatine i glaslegeme rummet. Når gelatine har hærdet overføre øjeæblet til en petriskål af PBS under dissekere mikroskop og bruge mikrokirurgi saks til at foretage et snit på limbus at fjerne hornhinden.Klip og fjern synsnerven under det dissekerende mikroskop. Ved hjælp af to par saftler skalle af og kassere sclera, choroid og retinale pigment epitellag, efterfulgt af fjernelse af iris. Med synsnerven side af nethinden vender opad og linserne vender nedad, injicere 50 mikroliter af PBS lige under nethinden kop til at tillade ophobning af PBS mellem gelatinerede glaslegeme og nethinden.Brug mikrokirurgi kraftpind til forsigtigt at skrælle nethinden kop og ciliaere krop fra glaslegeme. Ved hjælp af en overførsel pipet overføre gelatine kop indeholder hyaloid væv nedsænket i PBS til et mikroskop dias. Og drej de resterende væv over, så linsen vender opad.Løft derefter linsen og forsigtigt løsne forbindelsen mellem linsen tunica vasculosa lentis og vasa hyaloidea propria før du skærer hyaloid arterie med mikrokirurgi saks til at fjerne linsen tunica vasculosa lentis. Hold vasa hyaloidea propria regionen hyaloid kop til den flade mount. Til flad montering af hyaloid beholderen prøver forsigtigt skylle hyaloid kop med frisk PBS at fjerne eventuelle dissektion vragrester.Med vævet flyder i PBS bruge mikrokirurgi kraftaffræt til forsigtigt at arrangere og justere placeringen af gelatineret hyaloid kop, således at kanten af koppen vender nedad. Placer dias med hyaloid kop nedsænket i PBS på et dias varmere ved 37 grader Celsius. Efter gelatine smelter og hyaloid flader fjerne dias, når det er knap tør og tilføje en dråbe anti-fade montering medium med DAPI at plette kerner i hyaloid fartøjer.Derefter forsigtigt placere en coverslip på hyaloid at fuldføre den flade mount. For at afbilde DAPI farvning af hyaloid fartøjer overføre den monterede prøve på scenen af en fluorescerende mikroskop og bekræfte, at den centrale hyaloid arterie er synlig. Derefter identificeres de kargrene, der er direkte afledt af hyaloid arterien, og antallet af kargrene tælles manuelt til kvantificering.Her, tværsnitsvisninger af OCT billeder til nethinden og hyaloid væv i tre måneder gamle vilde type og lav densitet lipoprotein receptor-relaterede protein fem eller LRP5 knockout mus, en dyremodel med vedvarende hyaloid fartøjer, er vist. I disse fundus fluorescein angiografi billeder af vedvarende hyaloid fartøjer otte grene af hyaloid fartøjer er observeret i glaslegeme af seks uger gamle knockout mus, mens ingen rest af hyaloid fartøjer er observeret i vilde type dyr af samme alder. I disse hyaloid fartøj flade mounts de vaskulære celler og tilhørende makrofager kan identificeres ved deres nukleare DAPI farvning.Og hver linje af celler forgrening fra den centrale hyaloid arterie repræsenterer et fartøj af vasa hyaloidea propria. Vilde type mus har et gennemsnit på 12 grene af hyaloid fartøjer på postnatal dag otte, mens alder-matchede LRP5 knockout hvalpe udviser omkring 25 grene af hyaloid fartøjer viser en signifikant nedsat regression af hyaloid vaskulatur. Desuden, forsinket og ufuldstændig nethinden vaskulær udvikling er også observeret i LRP5 knockout hvalpe.Faktisk kan hyaloid fartøjer stadig identificeres i tværsnit af LRP5 knockout øjenvæv sektioner på postnatal dag otte. Mens de vilde type øjne ikke længere vise disse fartøjer. Det vigtigste at huske er at være blid og tålmodig, når isolere hyaloid systemet fra retinal og andre okulære væv.Efter isolering af hyaloid fartøjer immunstaining med forskellige celle markører kan udføres for at afdække de cellulære interaktioner regulerer hyaloid regression. Denne teknik udforsker de underliggende cellulære og molekylære mekanismer vedvarende føtal vaskulatur i okulær angiogenese forskning. Paraformaldehyd, ketamin og xylazin er farlige.Opberedning af disse reagenser i en kemisk røghætte ved hjælp af det korrekte personlige værneudstyr.

assessment-and-characterization-of-hyaloid-vessels-in-mice

Research

JoVE Journal

Medicine

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

Vurdering af gastrisk tømning hos ikke-overvægtige diabetiske mus ved anvendelse af en [<sup&gt; 13</sup&gt; C]-octansyre Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Ekkokardiografisk Vurdering af højre hjerte i mus

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Halspulsåren infusioner farmakokinetisk og farmakodynamisk analyse af Taxaner i mus

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Ultralyd funderet vurdering af koronararterie Flow og koronar Flow Reserve Brug af trykoverbelastning Model i mus

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Ekkokardiografiske Strategier og Protokoller for omfattende fænotypisk karakterisering af Valvular hjertesygdom i mus

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Vurdering af respiratoriske funktion i bevidst mus af dobbelt-kammer Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Non-invasiv vurdering af Dorsiflexor muskelfunktion i mus

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Induktion og karakterisering af pulmonal hypertension hos mus ved hjælp af Hypoxi/SU5416-modellen

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Invasiv Hæodynamisk vurdering for det højre ventrikel system og hypoxia-induceret pulmonal arteriel hypertension i mus

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Brug af en kemisk biopsi til graft kvalitetsvurdering

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...