Authors would like to thank Brian Muntean, Shao Lo, Maki Takahashi and Blair Mell for their technical assistance. This work was supported by awards from the NIH (DK080640) and The University of Toledo &amp; ProMedica Translational Research Stimulation Award to Dr. Surya Nauli.

1. Cell pretreatment and metaphase preparation
<ol>
	<li>The cells are grown in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10-15% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37&deg;C with 5% CO2 incubator, until they reach 70-80% confluency.</li>
	<li>Treat the cells with colcemid solution at 0.05 &mu;g/ml for 30-60 min.</li>
	<li>Collect the medium containing any floating cells in 50-ml sterile falcon centrifuge tubes. Rinse the cells on the plate with sterile1X-PBS. After incubating with sterile trypsin for 1-2 min, harvest and collect the remaining cells into the same tube.</li>
	<li>Spin the tubes at 1000 r.p.m. for 5 min, aspirate the supernatant leaving 0.5 ml and loosen pellet by flicking with finger only.</li>
	<li>Depending on the pellet size, add 5-10 ml of hypotonic solution of 0.56% KCl in dH2O and incubate the suspension at 37&deg;C for 30-45 min.</li>
	<li>Add one drop of methanol/acetic acid (3:1 vol/vol) per ml of hypotonic cell suspension and invert the tube gently for mixing.</li>
	<li>Centrifuge at 1200 r.p.m. for 5 min and collect pellet as step 1.4, then add 5 ml of fresh methanol/acetic acid (3:1 vol/vol) fixative solution dropwise while flicking the pellet continously. This procedure is critical so that the metaphase spreads will not be trapped in the cells' clumps which would compromise the experiment.</li>
	<li>Centrifuge again at 1200 r.p.m. for 5 min and add 5-10 ml of ice-cold methanol/acetic acid fixative along the wall of the tube. At this stage, if needed, the cells can be stored in fixative solution in a tightened and sealed tube at -20&deg;C for short term or -80&deg;C for long term (years) for future use.</li>
	<li>Clean slides in absolute ethanol, then dip the slide in dH2O for approximately10X in order to form a sheath of water on the surface of the slide. Place the slide on a glass plate and drop 15-20&mu;l of cell suspension (from step 1.8) from 10" above the slide. Place the slide in a water bath set at 65-70&deg;C for 1-2 min and allow to dry.</li>
	<li>Check the slide under a light microscope using 10X and 40X dry objectives making sure there is metaphase chromosomes and the spreads are evenly spaced. Check for cytoplasm presence surrounding the chromosomes. If cytoplasm is present proceed with slide pretreatment (pepsin digestion), if no cytoplasm is present and the chromosomes have good morphology, then there is no need for slide pretreatment.</li>
</ol>
2. Slide pretreatment (pepsin treatment)
<ol>
	<li>Apply 120 &mu;l of 1:200 RNase solution (20 mg/ml) dissolved in 2X-SSC onto a 24 mm x 60 mm microscope coverglass and invert the metaphase slide face down on the coverglass then gently invert the metaphase slide face up and incubate at 37&deg;C for 45 min.</li>
	<li>Carefully remove the coverglass without scratching the slide and wash in 2X-SSC buffer in a coplin jar every 5 min for 15 min with shaking.</li>
	<li>Add 5-15 &mu;l of pepsin stock solution (100 mg/ml in dH2O) into a clean beaker and then add 100 ml of prewarmed (37&deg;C) 0.01M HCl. It is important that the pepsin is added into a clean beaker first and not directly into the HCl solution, otherwise it wouldn't dissolve in solution. Incubate the slide in a coplin jar containing the HCl/pepsin solution at 37&deg;C for 3-5 min. This step is very critical as too much digestion will cause the chromosomes to be overdigested and too little digestion will leave the cytoplasm undigested which might lead to non-specific binding of the probe and interfere with the hybridization signal.</li>
	<li>Wash the slide in a coplin jar containing 100 ml 1X-PBS for 5 min twice at room temperature.</li>
	<li>Wash the slide in a coplin jar containing 100 ml 1X-PBS/MgCl2 for 5 min at room temperature (50 ml of 1M MgCl2 in 950 ml of 1X-PBS).</li>
	<li>Place the slide in a coplin jar containing 100 ml 1% formaldehyde for 10 min at room temperature (1.7 ml of 37% formaldehyde in 100 ml of 1X-PBS/MgCl2).</li>
	<li>Wash the slide in a coplin jar containing 100 ml 1X-PBS for 5 min.</li>
	<li>Observe the slide under a light microscope using 40X dry lens to ensure that the slides are digested properly and no cytoplasm is present and the chromosome morphology is preserved. Select an area for hybridization using a diamond pen.</li>
</ol>
3. Chromosome and probe denaturation and hybridization
<ol>
	<li>Prepare fresh denaturing solution (70% formamide/2X SSC, pH 7.0) and prewarm to 70-80&deg;C in a coplin jar placed in a water bath Place the slide in the coplin jar containing the denaturing solution in a water bath at 70&deg;C for mouse chromosomes and 80&deg;C for human chromosomes for 30s-1.5 min.</li>
	<li>Immediately place the slide in ice-cold 70% ethanol for 3 min followed by 80% and 100% ethanol for 3 min each and air dry. Examine the slide for chromosome morphology. Good chromosome morphology is denoted by dark chromosomes and not "phase-light" or halo chromosomes.</li>
	<li>Warm the SkyPaint probe (SKY paint kit; vial#1) at 37&deg;C with shaking for 20 min, vortex and centrifuge briefly at 1000 r.p.m. for a few seconds.</li>
	<li>Denature the probe in a thermocycler programmed for a two-step cycle at 85&deg;C for 5 min cycle followed by 37&deg;C for 60 min to allow labeled-probe DNA for preannealing.</li>
	<li>Apply 10&mu;l of the denatured probe onto the area of hybridization and cover with a 22 mm x 40 mm coverglass making sure not to trap air bubbles. Seal the edges of the coverglass with rubber cement and incubate in a humidified chamber at 37&deg;C for 48-72 hrs.</li>
</ol>
4. Fluorescent probe detection
<ol>
	<li>Remove the coverglass carefully and place the slide in a coplin jar containing prewarmed (45&deg;C) washing solution I (freshly prepared 50% formamide in 2X SSC). Wash for 5 min three times at 45&deg;C in a shaking water bath at 45 r.p.m.</li>
	<li>Wash the slide in washing solution II (1X SSC) at 45&deg;C for 5 min twice with shaking.</li>
	<li>Wash the slide in washing solution III (4X SSC/0.1% Tween 20) for 5 min at 45&deg;C with shaking.</li>
	<li>Apply 80 &mu;l of blocking reagent (SKY paint kit; vial#2), place a coverslip and incubate at 37&deg;C for 30 min.</li>
	<li>Remove the slide and allow the fluid to drain. Apply 80 &mu;l of Cy5 staining reagent (Concentrated Antibody Detection CAD kit; vial#3), apply a coverglass and incubate at 37&deg;C for 40 min.</li>
	<li>Wash the slide with washing solution III at 45&deg;C for 5 min three times with shaking.</li>
	<li>Apply 80 &mu;l of Cy5.5 staining reagent (Concentrated Antibody Detection CAD kit; vial#4), place a coverglass and incubate at 37&deg;C for 40 min.</li>
	<li>Wash the slide with washing solution III at 45&deg;C for 5 min three times with shaking.</li>
	<li>Tilt the slide and allow the fluid to drain. Apply 20 &mu;l of the anti-fade DAPI reagent (SKY paint kit; vial#5) and place a 24 mm x 60 mm microscope coverglass. Carefully remove air bubbles that might have formed. Slides can be imaged immediately or stored at 4&deg;C in the dark for no longer than 1 week.</li>
</ol>
5. Image acquisition and analysis
<ol>
	<li>Image acquisition is accomplished by viewing metaphase slides using an Olympus microscope equipped with a 60X oil immersion lens, a Spectral cube (custom designed triple band pass filter), a DAPI filter and a sagnac interferometer module with a CCD camera.</li>
	<li>Spectral-Karyotypes were carried out using SKY View software (Applied spectral imaging Version 1.62) following the user's manual.</li>
	<li>After analyzing the images, the chromosomes can be viewed as color images (with specific fluorescent colors), pseudo color images (with colors for classification) and inverted DAPI images (specific banding pattern).</li>
</ol>
6. Representative Results
A complete SKY procedure usually takes about one week time (Figure 1). This includes image acquisition and analysis provided that the metaphase cells are in adequate supply. Karyotyping analysis reveals normal mouse karyotype (40,XY) of cells from wild type mice (Figure 2a). In contrast, cells from Pkd1-/- mouse (PKD mouse model) shows a significant increase in chromosome number and structural abnormalities, such as chromosome deletions (Chromosome # 8) and translocations (chromosomes # 11 and 19) (Figure 2b). We also analyzed vascular tissues from the ADPKD patients. A simple study by counting the chromosomal numbers indicated that non-ADPKD and some ADPKD vascular samples had normal chromosomal numbers of 23 pairs (Figure 2c). In general, however, we observed failure of chromosomal segregation, resulting in 46 pairs of chromosomes in ADPKD samples instead of 23 pairs (Figure 2d).
<img alt="Figure 1" src="/files/ftp_upload/3887/3887fig1.jpg" /> 
Figure 1. SKY protocol flowchart. Flow chart of the SKY protocol illustrates steps to complete an experiment starting from cell pretreatment and metaphase preparations to image acquisition and analysis. An approximate one-week timeline is presented on the left with step-by-step procedures for each day.
<img alt="Figure 2" src="/files/ftp_upload/3887/3887fig2.jpg" /> 
Figure 2. Spectral karyotyping on immortalized mouse cell lines and freshly isolated human primary cells. Color and inverted DAPI images of individual chromosomes are shown before the chromosomal sorting. After sorting, chromosomes are presented in the "classification" table. a) SKY image of a metaphase spread from wild type mouse contains normal karyotype (40, XY). b) SKY image of a metaphase spread from Pkd1-/- mouse shows abnormal chromosome number (68 instead of 40) and chromosome abnormalities such as chromosome deletions (chromosome #8) and chromosome translocations (chromosomes #11 and 19). c) SKY image of a metaphase spread from the vascular tissue of a non-ADPKD patient has normal karyotype (46, XX). d) SKY image of a metaphase spread from the vascular tissue of an ADPKD patient contains abnormal karyotype (92, XXXX). Parts of the data have been previously reported and reused with permission 14.

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J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nature Protocols. 1, 3129-3142 (2006).</li><li>Schrock, E., Zschieschang, P., O'Brien, P., Helmrich, A., Hardt, T., Matthaei, A., Stout-Weider, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+of+human,+mouse,+rat+and+ape+chromosomes--applications+for+genetic+diagnostics+and+research.">Spectral karyotyping of human, mouse, rat and ape chromosomes--applications for genetic diagnostics and research.</a> Cytogenetic and Genome Research. 114, 199-221 (2006).</li><li>Padilla-Nash, H. M., Heselmeyer-Haddad, K., Wangsa, D., Zhang, H., Ghadimi, B. 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I., Allison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+genetic+complementation+controlling+the+chromosomal+abnormalities+and+loss+of+heterozygosity+formation+in+cancer.">A model for genetic complementation controlling the chromosomal abnormalities and loss of heterozygosity formation in cancer.</a> Cytogenetic and Genome Research. 116, 235-247 (2007).</li><li>Ried, T., Liyanage, M., du Manoir, S., Heselmeyer, K., Auer, K., Macville, M., Schrock, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cytogenetics+revisited:+comparative+genomic+hybridization+and+spectral+karyotyping.">Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping.</a> Journal of Molecular Medicine (Berlin, Germany). 75, 801-814 (1997).</li><li>AbouAlaiwi, W. A., Takahashi, M., Mell, B. R., Jones, T. J., Ratnam, S., Kolb, R. J., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+polycystin-2+is+a+mechanosensitive+calcium+channel+involved+in+nitric+oxide+signaling+cascades.">Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.</a> Circulation Research. 104, 860-869 (2009).</li><li>Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystins+1+and+2+mediate+mechanosensation+in+the+primary+cilium+of+kidney+cells.">Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.</a> Nature Genetics. 33, 129-137 (2003).</li><li>Nauli, S. M., Kawanabe, Y., Kaminski, J. J., Pearce, W. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cilia+are+fluid+shear+sensors+that+regulate+calcium+signaling+and+nitric+oxide+production+through+polycystin-1.">Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.</a> Circulation. 117, 1161-1171 (2008).</li><li>AbouAlaiwi, W. A., Ratnam, S., Booth, R. L., Shah, J. V., Nauli, S. 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No conflicts of interest declared.

Spectral Karyotyping (SKY) is a cytogenetics technique used in studying genomic and chromosomal compositions. This technique takes advantage of chromosome painting probes, and the detection of these probes are acquired through a sagnac interferometer. The complete SKY process usually takes about one week, and it involves several key steps (Figure 1). The SKY uses a standard protocol, which was first described by Padilla-Nash et al 3. The protocol has since been adapted by various cyto...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30088-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penecillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>SV30010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Colcemid</td>
			<td>Roche Diagnostics</td>
			<td>10 295 892 001</td>
			<td>10 &mu;g/ml</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>S77375-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Scientific</td>
			<td>SH30256-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SKY paint probe kit (Human)</td>
			<td>Applied Spectral Imaging</td>
			<td>SKY000028</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SKY paint probe kit (Mouse)</td>
			<td>Applied Spectral Imaging</td>
			<td>SKY000030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Concentrated antibody detection kit</td>
			<td>Applied Spectral Imaging</td>
			<td>SKY000033</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Scientific</td>
			<td>SH30236.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A433P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A38-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Roche Diagnostics</td>
			<td>10 109 169 001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fluka</td>
			<td>63069-500ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Mallinckrodt Baker</td>
			<td>2106-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td>Promega</td>
			<td>V4261</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
			<td>prepare just before use</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-549</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass 24x60mm</td>
			<td>VWR Vista Vision</td>
			<td>16004-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rubber cement</td>
			<td>Elmer's (local retail store)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hybridization/ humidifiedchamber/Tray</td>
			<td>Simport</td>
			<td>M920-2</td>
			<td>put wet paper towels at the bottom</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Eppendorf</td>
			<td>Epgradient S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaking platform/Orbital shaker</td>
			<td>Bellco Biotechnology</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaking/water bath</td>
			<td>Precision Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI filter cube</td>
			<td>Chroma Technology</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SKY filter cube</td>
			<td>Chroma Technology</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectraCube</td>
			<td>Applied Spectral Imaging</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inverted cell culture microscope</td>
			<td>Nikon</td>
			<td>Nikon Eclipse TS100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX70</td>
			<td>60X oil</td>
		</tr>
	</tbody>
</table>

spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

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Research

JoVE Journal

Medicine

38.2K visualizações.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Cariotipagem espectral (SKY) é uma técnica avançada citogenética para identificar as aberrações cromossômicas e cromossômicas. Essa técnica leva vantagem das sondas cromossômicas de pintura, que permitem a classificação de todos os cromossomos. SKY também pode identificar aberrações cromossômicas e defeitos complexos de segregação em camundongos e humanos, com várias doenças, incluindo doença renal policística.

Vídeo: Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...