Name: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies (Video) | JoVE Journal
Uploaded: 2018-07-14T15:00:00.000+00:00
Duration: 12 min 42 s
Description: Learn to isolate retinal arterioles from rat retina for ex vivo cell physiology studies using patch clamp, calcium imaging, and pressure myography techniques. This method enables detailed investigation of vascular smooth muscle contractility and retinal blood flow regulation.

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Development of the protocols described in this paper was supported by grants from the following funding agencies: BBSRC (BB/I026359/1), Fight for Sight (1429 and 1822), The Juvenile Diabetes Research Foundation (2-2003-525), Wellcome Trust (074648/Z/04), British Heart Foundation (PG/11/94/29169), HSC R&#38;D Division (STL/4748/13) and MRC (MC_PC_15026).

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<li>McGahon, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPV2+Channels+Contribute+to+Stretch-Activated+Cation+Currents+and+Myogenic+Constriction+in+Retinal+Arterioles%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles.</a> Investigative Ophthalmology & Visual Science. 57 (13), 5637-5647 (2016).</li>
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<li>Fernández, J. A., Bankhead, P., Zhou, H., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+detection+and+measurement+of+isolated+retinal+arterioles+by+a+combination+of+edge+enhancement+and+cost+analysis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Automated detection and measurement of isolated retinal arterioles by a combination of edge enhancement and cost analysis.</a> PLoS One. 9, e91791(2014).</li>
<li>McGahon, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+downregulates+large-conductance+Ca2%2B-activated+potassium+beta+1+channel+subunit+in+retinal+arteriolar+smooth+muscle%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle.</a> Circulation Research. 100 (5), 703-711 (2007).</li>
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<li>McGahon, M. K., Dawicki, J. M., Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A-type+potassium+current+in+retinal+arteriolar+smooth+muscle+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">A-type potassium current in retinal arteriolar smooth muscle cells.</a> Investigative Ophthalmology & Visual Science. 46 (9), 3281-3287 (2005).</li>
<li>McGahon, M. K., Zhang, X., Scholfield, C. N., Curtis, T. M., McGeown, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+downregulation+of+the+BKbeta1+subunit+in+diabetic+arteriolar+myocytes%5BTitle%5D)+AND+%22Channels+%28Austin%29%22%5BJournal%5D)">Selective downregulation of the BKbeta1 subunit in diabetic arteriolar myocytes.</a> Channels (Austin). 1 (3), 141-143 (2007).</li>
<li>Ljubimov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basement+membrane+abnormalities+in+human+eyes+with+diabetic+retinopathy%5BTitle%5D)+AND+%22Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">Basement membrane abnormalities in human eyes with diabetic retinopathy.</a> Journal of Histochemistry and Cytochemistry. 44, 1469-1479 (1996).</li>
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<li>Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+physiology+of+retinal+and+choroidal+arteriolar+smooth+muscle+cells%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.</a> Microcirculation. 14 (1), 11-24 (2007).</li>
<li>Hinds, K., Monaghan, K. P., Frølund, B., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GABAergic+control+of+arteriolar+diameter+in+the+rat+retina%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">GABAergic control of arteriolar diameter in the rat retina.</a> Investigative Ophthalmology & Visual Science. 54 (10), 6798-6805 (2013).</li>
<li>Torring, M. S., Aalkjaer, C., Bek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constriction+of+porcine+retinal+arterioles+induced+by+endothelin-1+and+the+thromboxane+analogue+U46619+in+vitro+decreases+with+increasing+vascular+branching+level%5BTitle%5D)+AND+%22Acta+Ophthalmologica%22%5BJournal%5D)">Constriction of porcine retinal arterioles induced by endothelin-1 and the thromboxane analogue U46619 in vitro decreases with increasing vascular branching level.</a> Acta Ophthalmologica. 92 (3), 232-237 (2014).</li>
<li>P, S. kov J. ensen, S, M. etz M. ariendal P. edersen, Aalkjaer, C., Bek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+vasodilating+effects+of+insulin+and+lactate+are+increased+in+precapillary+arterioles+in+the+porcine+retina+ex+vivo%5BTitle%5D)+AND+%22Acta+Ophthalmologica%22%5BJournal%5D)">The vasodilating effects of insulin and lactate are increased in precapillary arterioles in the porcine retina ex vivo.</a> Acta Ophthalmologica. 94 (5), 454-462 (2016).</li>
</ol>

Dr. Joanna Kur is now an employee of Medtronic (Twin-Cities, Minnesota, USA), her present work does not conflict with that presented here.

The protocols described above require practice but should be achievable with minimal troubleshooting. On an average day we would obtain 6 - 8 usable arterioles from the isolation and achieve 3 - 4 successful experiments. If problems are encountered, however, there are some steps that can be taken to help improve success rates. Occasionally we have found, particularly when using younger rats (&#60; 8 weeks), that the yield of arterioles can be low. To circumvent this problem, we would suggest centrifuging the retinal tissue (10 - 30 s at 500 x g) between each of the trituration steps in steps 1.12 - 1.14. This often helps to improve the yield of vessels but will also increase the amount of cell debris within the preparation.
When cannulating vessels for pressure myography studies it is important to check the arterioles carefully for any side-branches that may have been cleaved close to the bifurcation site. This represents a common cause of leakage and loss of pressure during experimentation. The main issue that can arise with the [Ca2+]i protocols is the level of dye loading. Poor dye loading leads to low signal-to-noise ratios, while overloading results in disruption of normal Ca2+ homeostasis. To reduce the likelihood of either of these issues, a small aliquot of retinal homogenate can be removed, and isolated vessels checked periodically during the loading protocol. When undertaking patch-clamp experiments, the success rate of gigaseal formation is highly dependent on how well the basal lamina has been digested. When initially testing this protocol or using new lots of enzymes it may be necessary to adjust the concentration/duration of enzyme digestion. Carefully monitoring the separation of the endothelial and smooth muscle layers will ensure sufficient digestion to remove enough basal laminal to gain access. Careful monitoring is also necessary to avoid over-digestion, which can manifest as the vessel begins to constrict. If this occurs, the smooth muscle cells are often too fragile for patch clamp recording. When applying enzymes, it is important to include DNAse I to ensure removal of strands of DNA liberated from damaged cells during the isolation process. DNA fragments are sticky and cause the forceps to adhere to the arteriole during the final stages of the cleaning process (step 4.4), often resulting in vessel loss. Cleaning of vessels is technically difficult and is best performed at high magnification (20X) with gentle sweeping motions of closed forceps of the finest possible tip diameter. Between sweeps, clean the forceps with lab roll.
As highlighted earlier, a key motivation behind the development of the protocols outlined in this manuscript was to better understand why blood flow is disrupted during retinal vascular diseases. Most of our work to date has focused on diabetic eye disease28,33. Arterioles can be isolated from the retinas of experimental rodent models of diabetes using the methods described in section 1. When experimenting on isolated retinal arterioles from diabetic animals, it is important to try to closely replicate the hyperglycemic conditions experienced by the vessels in vivo. For this reason, we would normally raise the D-glucose levels in our isolation and experimental solutions to 25 mM. Thickening of vascular basement membranes is a well-recognized phenomenon in retinal vessels during diabetes34,35. When using animals with prolonged diabetes (&#62; 1 months disease duration), increased enzyme concentrations or digestion times are often needed to enable the application of patch-clamp recording methods.
An important limitation of using ex vivo isolated retinal arterioles to study retinal vascular physiology and pathophysiology is the loss of the surrounding retinal neuropile. Although removal of the retinal glial and neuronal cells enables easy access to the retinal vascular smooth muscle cells for cell physiology studies, the response of the vessels to vasoactive mediators can change dramatically in the absence and presence of retinal tissue. The actions of adenosine tri-phosphate (ATP), for example, provide a good illustration of this point. In isolated rat retinal arterioles, addition of ATP triggers a robust constriction of the vessels36, while in the presence of an intact neuropile, the vessels dilate37. Therefore, wherever possible, we usually try to validate key findings from our isolated arteriole preparations using ex vivo retinal whole-mounts and in vivo measurements of vessel diameter and blood flow16,37. Of note, new methods have recently emerged for studying small arterioles and capillaries in whole perfused porcine retinas ex vivo38,39. Such preparations are likely to greatly improve our understanding of how the retinal neuropile regulates retinal arteriolar and capillary tone and how changes in retinal haemodynamics modulate neuronal activity in the retina.
Although the procedures described in this paper are focused on the use of isolated rat retinal arterioles for understanding arteriolar smooth muscle cell physiology, we are currently developing protocols to also enable the study of endothelial cell function in these vessels. In preliminary work, we have been successful in modifying the enzymatic digestion of retinal arteriolar segments to yield viable endothelial cell tubes that are amenable to Ca2+ imaging and patchclamp recording studies. Cannulation of the isolated arterioles at both ends, enabling intraluminal delivery of drugs, could in the future also enable endothelium-dependent vasodilatory responses to be investigated in these vessels.

Understanding how blood flow is controlled in the retina is an important goal, since abnormal blood flow has been implicated in the pathogenesis of a variety of sight-threatening retinal diseases1,2,3,4. The retinal circulation, which supplies the inner retinal neurons and glial cells, has an end artery arrangement, with all of the blood from the retinal arteries and arterioles passing through the capillaries to the retinal venules and finally veins5. Blood flow in the retina is regulated by the tone of the retinal arteries and arterioles as well as the contractile activity of the pericytes located on the walls of the retinal capillaries and post-capillary venules6,7,8. The control of retinal vascular tone is complex and is modulated by a range of inputs from the circulatory system and surrounding retinal tissue, including blood gases, circulating molecules and hormones, and vasoactive substances released from the retinal vascular endothelium and macroglia9,10,11. The retinal arterioles are the small arterial branches of the retina and are composed of a single layer of vascular smooth muscle cells and an inner lining of longitudinally arranged endothelial cells12,13,14. These vessels form the main site of vascular resistance within the retinal circulation and therefore play an important role in the local control of retinal blood flow. Retinal arterioles regulate capillary blood flow in the retina by dilating or constricting their luminal diameter, mediated by changes in vascular smooth muscle contractility10,15,16. Understanding the molecular mechanisms through which retinal arterioles regulate retinal perfusion therefore requires preparations where the arteriolar smooth muscle cells can be accessed and studied in conditions as close to physiological as possible.
Ex vivo preparations of isolated retinal blood vessels provide access to the vascular smooth muscle cells, whilst still retaining their functionality and interconnectivity with the underlying endothelium. Most studies to date using isolated vessels have focused on large bovine or porcine arterial vessels (60 - 150 &#181;m). These can be mounted in commercially available wire or pressure myograph systems to enable pharmacological interrogation of vascular smooth muscle cell contractile mechanisms17,18. Such preparations have greatly contributed to our knowledge of retinal vascular physiology under normal conditions. Few studies have used retinal arterioles isolated from small laboratory animals as their smaller diameter (~ 8 - 45 &#181;m) prevents their use in conventional myography systems19,20,21,22. An important advantage, however, of using vessels from small laboratory animals is the wide availability of genetically modified, transgenic and retinal disease models. Small laboratory animals are also more amenable for in vivo intervention studies.
Here we describe straightforward protocols for isolating and cannulating rat retinal arterioles for pressure myography experiments. Ca2+ imaging and electrophysiology protocols using these vessels are also detailed. These can provide further insights into the regulation of vascular smooth muscle contractility and blood flow in the retina.

<table><tbody><tr><td>Beakers</td><td>Fisherbrand</td><td>15409083</td><td>Or any equilavent product</td></tr><tr><td>Curved Scissors</td><td>Fisher Scientific</td><td>50-109-3542</td><td>Or any equilavent product</td></tr><tr><td>Disposable plastic pipette/ transfer</td><td>Sarstedt</td><td>86.1174</td><td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td></tr><tr><td>Dissecting microscope</td><td>Brunel Microscopes LTD</td><td></td><td>Or any equilavent product</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #5 14095</td><td>Any equivalent fine forceps</td></tr><tr><td>Pasteur pipette</td><td>Fisherbrand</td><td>11546963</td><td>Fire polished to reduce friction but not enough to narrow the tip</td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>P7741</td><td>Or any equilavent 10cm product</td></tr><tr><td>Pipette teat</td><td>Fisherbrand</td><td>12426180</td><td>Or any equilavent product</td></tr><tr><td>Purified water supply</td><td>Merek</td><td>Milli-Q Integral Water Purification System</td><td>Or any equilavent system</td></tr><tr><td>Round bottomed test tube</td><td>Fisherbrand</td><td>14-958-10 B</td><td>5 mL; any equilavent product</td></tr><tr><td>Seratted forceps</td><td>Fisher Scientific</td><td>17-467-230</td><td>Or any equilavent product</td></tr><tr><td>Single edge blades</td><td>Agar Scientific</td><td>T585</td><td>Or any equilavent product</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>184</td><td>Or any pliable surface&amp;nbsp;</td></tr><tr><td>Testtube rack</td><td>Fisherbrand Derlin</td><td>10257963</td><td>Or any equilavent product</td></tr><tr><td>Pressure myography</td><td></td><td></td><td></td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>x2 (or any equilavent product)</td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software&amp;nbsp;</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Analysis plugin</td><td>Myotraker&amp;nbsp;</td><td>https://doi.org/10.1371/journal.pone.0091791.s002</td><td>Custom plugin for imageJ Freely available for download</td></tr><tr><td>Cannulation pipette glass</td><td>World Percision instruments</td><td>TW150F-4</td><td>Or any equilavent product</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fluo-4-AM</td><td>Thermo Fisher Scientific</td><td>F14201</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #7 14097</td><td>Any equivalent fine forceps</td></tr><tr><td>Fura-2-AM</td><td>Thermo Fisher Scientific</td><td>F1221</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Helper pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Manometer</td><td>Riester</td><td>LF1459</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W558818</td><td>75&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Or any equilavent product</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Video capture software</td><td>Hypercam</td><td>version 2.28.01</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Patch clamping</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2411</td><td>3 mg disolved daily in 50 &amp;mu;L of DMSO (sonicate to dissolve)</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200a</td><td>Or any equilavent product</td></tr><tr><td>Analogue digital converter</td><td>Axon</td><td>Digidata 1440A</td><td>Or any equilavent product</td></tr><tr><td>Collagenase Type 1A</td><td>Sigma-Aldrich</td><td>C9891&amp;nbsp;</td><td>0.1mg/mL in LCH</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>DNAse I</td><td>Millipore</td><td>260913</td><td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td>Custom made</td><td></td><td>Any equilavent commerical product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Patching software</td><td>Molecular Devices</td><td>Pclamp v10.2</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Protease Type XIV</td><td>Sigma-Aldrich</td><td>P5147&amp;nbsp;</td><td>0.01mg/mL in LCH</td></tr><tr><td>Single channel pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>Whole cell pipette glass</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>x40 lens</td><td>Nikon</td><td>40x/0.55, WD 2.1, &amp;infin;/1.2</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; imaging</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Acquisition software&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Acquisition Engine V1.1.5</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software for confocal imaging</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Confocal microscope</td><td>Leica Geosystems</td><td>SP5&amp;nbsp;</td><td>Or any equilavent product; confocal</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE2000</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Monochromator&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Optoscan</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Software for confocal microscope</td><td>Leica Geosystems</td><td>LAS-AF version 3.3.</td><td>Or any equilavent product; confocal</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x100 lens</td><td>Nikon</td><td>x100 N.A. 1.3 oil</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Leica Geosystems</td><td>HCX PL FLUOTAR, 20x/0.50, &amp;infin;/0.17/D</td><td>Or any equilavent product; confocal</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x63 lens</td><td>Leica Geosystems</td><td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &amp;infin;/0.17/E</td><td>Or any equilavent product; confocal</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>&lt;strong&gt;Pipettes&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Specifications and settings for fabrication of pipettes for experimentation&lt;/strong&gt;</td></tr><tr><td>Helper pipette</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;10 M&amp;Omega;</td></tr><tr><td>Cannulation pipette</td><td>World Percision instruments</td><td>TW150F-4</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 290&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 58&lt;br/&gt; Time: 150&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 3-10 &amp;mu;m&lt;br/&gt; Polishing: no&lt;br/&gt; Final Resistance: &amp;lt;1 M&amp;Omega;</td></tr><tr><td>On-cell patching</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;5 M&amp;Omega;</td></tr><tr><td>Whole-cell patching</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 296&lt;br/&gt; Pressure: 200&lt;br/&gt; Heat: 287&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 50&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 2-3 &amp;mu;m&lt;br/&gt; Polishing: helpful but not necessary&lt;br/&gt; Final Resistance: 1-2 M&amp;Omega;</td></tr></tbody></table>

isolation of retinal arterioles for ex vivo cell physiology studies

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

isolation-of-retinal-arterioles-for-ex-vivo-cell-physiology-studies

Research

JoVE Journal

Biology

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Intensive preparation of intact mouse cerebral endothelial "tubes" from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Here, we provide a detailed methodology for isolating the retina from the mouse eye for extended ex vivo experimentation. This protocol emphasizes making this technically demanding approach accessible for researchers who would like to take advantage of the research avenues afforded by keeping retinal glia in situ in live tissue.

An Ex Vivo Explant Model for Studying Glial Interactions in the Mouse Retina

Learn to isolate retinal arterioles from rat retina for ex vivo cell physiology studies using patch clamp, calcium imaging, and pressure myography techniques. This method enables detailed investigation of vascular smooth muscle contractility and retinal blood flow regulation.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

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