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>Barraquer 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pericyclic pump&#160; (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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Drugs and Solutions</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 (CaCl2)</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 (KH2PO4)</td>
			<td>Carl Roth GmbH</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulphate (MgSO4)</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 (NaHCO3)</td>
			<td>Carl Roth GmbH</td>
			<td>0965.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-(D)-(+)- Glucose monohydrate</td>
			<td>Carl Roth GmbH</td>
			<td>6780.1</td>
			<td></td>
		</tr>
		<tr>
			<td>9,11-dideoxy-9&#945;,11&#945;-methanoepoxy prostaglandin F2&#945; (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. 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.

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

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...