This work was supported by grants from the Ernst und Berta Grimmke Stiftung and the Deutsche Ophthalmologische Gesellschaft (DOG).

The experimental procedures of this study were approved by the Animal Care Committee of Rhineland-Palatinate, Germany. Animal care conformed to the institutional guidelines and The Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Animals were treated according to the EU Directive 2010/63/EU for animal experiments. Male C57BL/6J mice (The Jackson Laboratory, Bar Harbour, ME, USA) aged 3-4 months were used for the experiments. Animals were housed under standard conditions (temperature 23 &#177; 2 &#176;C, humidity range 55 &#177; 10% and 12 h light/dark cycles), and had access to standard mouse chow and water ad libitum.
1. Isolation of Mouse Retina
<ol>
	<li>Place the dissection instruments on the preparation table and check if all instruments are complete to enable a quick preparation.</li>
	<li>Sacrifice the mouse by CO2 inhalation.</li>
	<li>Decapitate the mouse with steel scissors, cut the skull sagitally into two halves using the same pair of scissors and remove the skin and brain using eye scissors (Figure 1).</li>
	<li>Cut off the skull bone to leave only the orbital bone by using eye scissors.</li>
	<li>Transfer the orbit into a dissection dish containing ice-cold Krebs-Henseleit buffer.</li>
	<li>Keep the dissection dish on ice or change the buffer every 10 min.</li>
	<li>Cut the orbital bone using eye scissors and gently remove the eye globe together with the orbital tissue using type 4 precision tweezers and Student Vannas spring scissors (Figure 2).</li>
	<li>Gently remove the Harderian gland, connective tissue, and the extraocular muscles using type 5 precision tweezers and Vannas capsulotomy scissors, taking care not to damage the main branch of the ophthalmic artery.</li>
	<li>Ligate orbital branches of the ophthalmic artery and eventually the proximal end of the main branch of the ophthalmic artery using 10-0 Nylon sutures (Figure 3).</li>
	<li>Transfer the eye globe into a Petri dish containing 70% ethanol for 10 s using type 4 precision tweezers. Place the eye globe back into the dissection dish and replace the buffer with fresh Krebs solution. The cornea will appear whitish after ethanol exposure .</li>
	<li>Puncture the peripheral cornea with a 30 gauge needle and dissect the cornea using Vannas capsulotomy scissors. Gently cut off the iris tissue, place one sharp point of the Vannas capsulotomy scissors between the choroid and retina, and cut off the choroid and sclera. Leave some scleral tissue around the optic nerve (Figure 4).</li>
</ol>
2. Mounting the Retina in the Perfusion Chamber
NOTE: The perfusion chamber used for retinal arteriole experiments is home-made. It consists of a transparent reservoir with an in- and outflow tube (Figure 5).One end of a silicone tube is glued to the bottom of the chamber with histoacryl adhesive and the other end attached to a three-way stopcock. Connect a syringe containing fresh Krebs buffer to the three-way stopcock and fill the tube with buffer.
<ol>
	<li>Take a glass micropipette with a needle holder and push it into the end of the tube at the bottom of the perfusion chamber. Then break the tip of the micropipette with type 4 scissors to obtain a tip of 100 &#181;m diameter.</li>
	<li>Focus on the tip of the micropipette and flush it via the syringe connected to the tube. Take care that the capillary is not occluded by debris. In case of occlusion, remove it, flush the tubing and insert another micropipette.</li>
	<li>Fill the perfusion chamber with cold Krebs buffer and put the chamber under a stereo microscope.</li>
	<li>Place a transparent plastic platform into the perfusion chamber. This platform has an indentation of 1.8 mm width for the optic nerve and the ophthalmic artery and is placed on two steel wires of 1.6 mm diameter. Then, place a stainless-steel ring of 2.8 mm inner diameter and 4.0 mm outer diameter onto the platform.</li>
	<li>Transfer the retina with the optic nerve and attached ophthalmic artery from the dissection chamber into the perfusion chamber with extreme care using a spoon. This step is important to prevent potential stretching of the tissues as it may be detrimental to the experiment.</li>
	<li>Cut off the sutured part of the proximal end of the ophthalmic artery, place two preformed loops of 10-0 nylon suture onto the micropipette, and cannulate the ophthalmic artery with the micropipette. Tie the artery to the micropipette (Figure 6) and place the retina onto the transparent platform using fine-point-tweezers.</li>
	<li>Gently tear open the anterior lens capsula with two fine point tweezers, pull out the lens, and cut off the whole lens capsula using microscissors.</li>
	<li>Subsequently, make four radial incisions into the retina half the distance from the pars plana to the optic nerve and place a stainless-steel ring onto the retina to fix it to the bottom. The retina is now ready for the experiment (Figure 7).</li>
</ol>
3. Preparation of Retinal Arterioles for the Experiment
<ol>
	<li>Place the perfusion chamber under a light microscope. The objective is a 100X water-immersion objective lens.</li>
	<li>Connect the two tubings for in- and out-flow to a pericyclic pump and circulate the chamber with Krebs buffer oxygenated with 95% O2 and carbonated with 5% CO2 at 37 &#176;C. Use a flow rate between 100 and 120 mL/min.</li>
	<li>Connect the silicone tube, which is connected to the micropipette, to a reservoir silicone hose via a three-way stopcock. Then, fill the reservoir with Krebs buffer to a height corresponding to 50 mm Hg but&#160;do not open the three-way stopcock yet.</li>
	<li>Look through the objectives of the microscope and focus on the retinal surface. Search for blood vessels or red blood cells. It is possible that the blood vessels are completely collapsed, so sometimes only some red blood cells are visible.</li>
	<li>When a blood vessel is found, focus on it and open the three-way stopcock. The diameter of the vessel should increase immediately and the red blood cells move either centrifugally, if it is an arteriole, or centripetally in case of a venule. The arterioles are smaller in diameter compared to venules of the same order.</li>
	<li>Once a blood vessel with a well visible wall is found, it can be employed for experiments following an equilibration period of 30 min (Figure 8). During the equilibration period, the arterioles develop only weak intrinsic tone resulting in less than 10% of luminal diameter reduction.</li>
</ol>
4. Performing the Experiment
<ol>
	<li>Test the viability of vessels with the thromboxane mimetic 9,11-dideoxy-9&#945;,11&#945;-methanoepoxy prostaglandin F2&#945; (U46619) by adding it to the bath solution. This agent constricts mouse retinal arterioles by about 50% from resting diameter at concentrations of &#8805; 10-6 M.</li>
	<li>Once the vessel is confirmed viable, wash the agonist out of the bath by circulating the pump and supply fresh Krebs buffer into the bath.</li>
</ol>

<ol>
	<li>Toda, N., Nakanishi-Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+ocular+blood+flow,+glaucoma,+and+diabetic+retinopathy.">Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy.</a> Prog Retin Eye Res. 26 (3), 205-238 (2007).</li><li>Schuster, A. K., Fischer, J. E., Vossmerbaeumer, C., Vossmerbaeumer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography-based+retinal+vessel+analysis+for+the+evaluation+of+hypertensive+vasculopathy.">Optical coherence tomography-based retinal vessel analysis for the evaluation of hypertensive vasculopathy.</a> Acta Ophthalmol. 93 (2), e148-e153 (2015).</li><li>Cherecheanu, A. P., Garhofer, G., Schmidl, D., Werkmeister, R., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+perfusion+pressure+and+ocular+blood+flow+in+glaucoma.">Ocular perfusion pressure and ocular blood flow in glaucoma.</a> Curr Opin Pharmacol. 13 (1), 36-42 (2013).</li><li>Faraci, F. M., Sigmund, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+biology+in+genetically+altered+mice+:+smaller+vessels,+bigger+insight.">Vascular biology in genetically altered mice : smaller vessels, bigger insight.</a> Circ Res. 85 (12), 1214-1225 (1999).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Retinal+Blood+Flow+Using+Fluorescently+Labeled+Red+Blood+Cells.">Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells.</a> eNeuro. 2 (2), (2015).</li><li>Guevara-Torres, A., Joseph, A., Schallek, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label+free+measurement+of+retinal+blood+cell+flux,+velocity,+hematocrit+and+capillary+width+in+the+living+mouse+eye.">Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.</a> Biomed Opt Express. 7 (10), 4229-4249 (2016).</li><li>Schallek, J., Geng, Y., Nguyen, H., Williams, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+topography+of+retinal+pericytes+in+the+living+mouse+retina+using+in+vivo+adaptive+optics+imaging+and+ex+vivo+characterization.">Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.</a> Invest Ophthalmol Vis Sci. 54 (13), 8237-8250 (2013).</li><li>Gericke, 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=Identification+of+the+muscarinic+acetylcholine+receptor+subtype+mediating+cholinergic+vasodilation+in+murine+retinal+arterioles.">Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles.</a> Invest Ophthalmol Vis Sci. 52 (10), 7479-7484 (2011).</li><li>Gericke, 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=Functional+role+of+alpha1-adrenoceptor+subtypes+in+murine+ophthalmic+arteries.">Functional role of alpha1-adrenoceptor subtypes in murine ophthalmic arteries.</a> Invest Ophthalmol Vis Sci. 52 (7), 4795-4799 (2011).</li><li>Bohmer, 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=The+alpha(1)B+-adrenoceptor+subtype+mediates+adrenergic+vasoconstriction+in+mouse+retinal+arterioles+with+damaged+endothelium.">The alpha(1)B -adrenoceptor subtype mediates adrenergic vasoconstriction in mouse retinal arterioles with damaged endothelium.</a> Br J Pharmacol. 171 (16), 3858-3867 (2014).</li></ol>

The authors have no competing financial interests.

The measurement of vascular responses in the mouse retina is challenging due to the small size of retinal blood vessels. With the presented technique, retinal arterioles are visualized by transmitted light microscopy. This is possible, because the isolated retina is translucent. The advantage of the technique is the high optical resolution. The calculated spatial resolution is 11 px/&#181;m. However, the real resolution for this optical system that uses white light is between 200 and 300 nm, which is explained by the Abbe diffraction limit. Since the first branch of mouse retinal arterioles has an internal diameter of 20 to 30 &#181;m, diameter changes of approximately 1% are detectable with this system. There is no need of additional technical devices or fluorescent dyes as reported in other studies to visualize small retinal arterioles5,6,7. A disadvantage of the technique is the long preparation time, which is between 90 to 120 min by trained investigators. If the preparation time exceeds 180 min, the endothelial function starts to be attenuated.
There are several major critical steps in this technique. First, it is important to thoroughly ligate all retrobulbar arterial branches. If ligation is incomplete, retinal arterioles will not be pressurized due to leakage. Second, the immersion in ethanol for 10 s before opening the eye globe is important to deactivate the smooth muscle reactivity of the ophthalmic artery. We previously demonstrated that immersing for 10 s in 70% ethanol completely deactivates the ophthalmic artery smooth muscle8. In contrast, retinal arterioles are unlikely to be directly affected by ethanol, since they are protected by the bulbar wall. If this step is omitted, diameter changes in the ophthalmic artery may influence the perfusion of the retina. Of note, reactivity of the ophthalmic artery may markedly differ from that of retinal arterioles in response to the same agonist9,10. Third, avoid torsion of the ophthalmic artery. Especially while mounting the retinal preparation onto the platform, the ophthalmic artery may become twisted, resulting in occlusion of the lumen. Also, check carefully that the tip of the glass capillary is not occluded to enable pressurization of retinal arterioles. We suggest not to perform more than three consecutive concentration-response curves in the same retinal preparation, because we observed that responses to acetylcholine became weaker when repeating the concentration-response curves for more than three times. However, there may be differences regarding the repeatability of concentration-response curves depending on the agent applied and, thus, should be tested individually.
We applied pharmacological agents extraluminally in our studies, although intraluminal perfusion using a servo control pump is also possible. Since the technique allows to measure for local mechanisms of vascular reactivity in the retina of small laboratory animals including mice and rats, the advantages of gene-targeting technology by using genetically modified animals can be accessed with this method.

Disturbances in retinal perfusion have been implicated in the pathogenesis of various ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and glaucoma1,2,3. Thus, studies aimed at measuring vascular reactivity in the retina are important to understand the pathophysiology of these diseases and to develop new treatment approaches.
Due to the possibility of gene manipulation in the murine genome, the mouse has become a widely used animal model for studies of the cardiovascular system4. However, because of the small size of retinal blood vessels (&#8804; 30 &#181;m), measurement of vascular reactivity in the mouse retina is challenging. For example, stereomicroscopic techniques for in vivo measurement are limited in their optical resolution and therefore only allow to exactly detect changes in diameter or blood flow in small blood of less than &#8804; 30 &#181;m diameter when equipped with additional sophisticated devices, such as a confocal microscope using fluorescent dyes or the Adaptive Optics Scanning Light Ophthalmoscope5,6. Moreover, the interpretation of in vivo measurements aimed at identifying local signaling mechanisms in retinal blood vessels can be confounded by anaesthetics, changes in systemic blood pressure and the influence of retrobulbar blood vessels.
Therefore, we developed a method to measure responses of mouse retinal blood vessels with high optic resolution ex vivo. The technique presented herein allows visualization of retinal arterioles via transmitted light microscopy. This method, which can also be used in rats, provides access to the advantages of gene targeting technology in ocular vascular research.

<table><tbody><tr><td>Steel Scissors</td><td>Carl Roth GmbH</td><td>3576.1</td><td>1x 140 mm</td></tr><tr><td>Eye Scissors</td><td>Geuder</td><td>G-19390</td><td>1x straight, 10.5 cm</td></tr><tr><td>Precision tweezers, straight with fine tips</td><td>Carl Roth GmbH</td><td>LH68.1</td><td>2x type 4</td></tr><tr><td>Precision tweezers, straight with extra fine tips</td><td>Carl Roth GmbH</td><td>LH53.1</td><td>2x type 5</td></tr><tr><td>Vannas capsulotomy scissors</td><td>Geuder</td><td>19760</td><td>1x straight, 77 mm</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91501-09</td><td>1x curved,</td></tr><tr><td>&lt;em&gt;Barraquer&lt;/em&gt; Needle Holder</td><td>Geuder</td><td>G-17500</td><td>1x curved, 120 mm</td></tr><tr><td>Needle</td><td>Becton, Dickinson and Company</td><td>305128</td><td>1x 30 G</td></tr><tr><td>Glass Capillaries (for producing micropipettes)</td><td>Drummond Scientific Company</td><td>9-000-1211</td><td>1x (1.2 x 0.8 mm; outer/inner diameter)</td></tr><tr><td>Nylon Suture</td><td>Alcon</td><td>198001</td><td>1x 10-0</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>153066</td><td>1x 35 mm diameter</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>172931</td><td>1x 100 mm diameter</td></tr><tr><td>Discofix C</td><td>Braun</td><td>16500C</td><td>10 cm</td></tr><tr><td>Histoacryl adhesive</td><td>B. Braun Surgical, S.A.</td><td>1050052</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pericyclic pump&amp;nbsp; (CYCLO II)</td><td>Carl Roth GmbH</td><td>EP76.1</td><td>1x</td></tr><tr><td>Vertical Pipette Puller Model 700C</td><td>David Kopf Instruments</td><td></td><td>1x</td></tr><tr><td>Microscope (Vanox-T AH-2)</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Water immersion objective LUMPlanFL, 1.0 NA</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Digital camera (TK-C1381)</td><td>JVC</td><td></td><td>1x</td></tr><tr><td>Perfusion chamber</td><td>self-made</td><td></td><td>1x</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Drugs and Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth GmbH</td><td>K928.4</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>5239.1</td><td></td></tr><tr><td>Kalium chloride (KCl)</td><td>Carl Roth GmbH</td><td>6781.1</td><td></td></tr><tr><td>Kalium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>3904.2</td><td></td></tr><tr><td>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>261.2</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Carl Roth GmbH</td><td>9265.2</td><td></td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>0965.3</td><td></td></tr><tr><td>&amp;alpha;-(D)-(+)- Glucose monohydrate</td><td>Carl Roth GmbH</td><td>6780.1</td><td></td></tr><tr><td>9,11-dideoxy-9&amp;alpha;,11&amp;alpha;-methanoepoxy prostaglandin F2&amp;alpha; (U-46619)</td><td>Cayman Chemical</td><td>16450</td><td></td></tr><tr><td>Acetylcholine chloride</td><td>Sigma-Aldrich</td><td>A6625-25G</td><td></td></tr></tbody></table>

preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

U-46619 produced concentration-dependent vasoconstrictor responses in retinal arterioles from wild-type mice of the C57Bl/6J background. At a concentration of 10-6 M, reduction in luminal diameter was &#8776;50% from resting diameter. Figure 9A shows a representative concentration-response curve of one retinal arteriole. In arterioles preconstricted with U46619, cumulative administration of acetylcholine evoked concentration-dependent increases in luminal diameter to &#8776;25% from the preconstricted diameter at 10-5 M, indicative of an intact vascular endothelium (Figure 9B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56199/56199fig1.jpg" /> 
Figure 1: Dissection of the mouse skull. Skin on the decapitated mouse head is removed to expose the eyes and orbital cavity. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56199/56199fig2.jpg" /> 
Figure 2: Isolation of eye globe and orbital tissue. The orbital bone was cut with eye scissors and the eye globe together with the retrobulbar tissues and optic nerve were carefully isolated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56199/56199fig3.jpg" /> 
Figure 3: The eye globe and preparation of the ophthalmic artery. Once the surrounding orbital tissues were dissected with fine microscissors, the ophthalmic artery was carefully exposed and their small branches ligated with 10-0 nylon monofilament sutures. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56199/56199fig4.jpg" /> 
Figure 4: Dissection of ocular structures. To visualize the retina, the sclera and uvea were dissected with Vannas capsulotomy scissors. Some scleral tissue around the optic nerve was left to avoid damage of the ophthalmic artery. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56199/56199fig5.jpg" /> 
Figure 5: Organ chamber for real-time video microscopy. The chamber was home made. It consisted of a Petri dish of 100 mm diameter with an in- and out-flow tube. The tubes were glued with histoacryl adhesive. Externally oxygenated and carbonated Krebs-Henseleit buffer was circulated by a peristaltic pump via these tubes. One end of a silicone tube was glued to the bottom of the chamber and the other end attached to a three-way stopcock. At the end of the tube, a glass micropipette with a tip of 100 &#181;m was inserted, which served for cannulation of the ophthalmic artery. Via the silicone tube, the ophthalmic circulation was pressurized by filling a silicone tube with Krebs buffer to a level corresponding to 50 mm Hg. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56199/56199fig6.jpg" /> 
Figure 6: Cannulation of the ophthalmic artery. The ophthalmic artery was cannulated with glass micropipette and sutured with a 10-0 nylon suture. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56199/56199fig7.jpg" /> 
Figure 7: Cannulation of the ophthalmic artery. After placing the retina onto the transparent platform, the lens with the capsular bag was removed, four radial incisions were made into the retina, and a stainless steel of ring of 2.8 mm inner diameter and 4.0 mm outer diameter was placed onto the retina to fix it to the bottom. The retina was then ready for the experiment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56199/56199fig8.jpg" /> 
Figure 8: Visualization of retinal blood vessels. An exemplary retinal arteriole with red blood cells inside.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56199/56199fig9.jpg" /> 
Figure 9: Functional studies using video microscopy. Representative concentration response curves from a single retinal arteriole. (A) Concentration-response curve for the vasoconstrictor, U46619 (10-9 to 10-6 M), and (B) for the endothelium-dependent vasodilator, acetylcholine (10-8 to 10-5 M). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56199/56199fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

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

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

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

preparation-steps-for-measurement-reactivity-mouse-retinal-arterioles

Nanyang Technological University

Research

JoVE Journal

Medicine

7.1K Views.  University Medical Center of the Johannes Gutenberg University Mainz. Veel visie-bedreigende oogbeschadigingen en/of ziekten worden geassocieerd met disfunctionele retinale microvessels. De meting van de retinale arteriole reacties is daarom belangrijk om te onderzoeken van de onderliggende pathofysiologische mechanismen. Dit artikel beschrijft een gedetailleerd protocol voor muis retinale arteriole isolatie en voorbereiding te beoordelen van de effecten van vasoactieve stoffen op vasculaire diameter.

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

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Neuroscience

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 ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

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.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology 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 ...

Biology

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

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

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