Isolamento delle arteriole retiniche per studi di fisiologia delle cellule Ex Vivo

Understanding how blood flow is controlled in the retina is an important goal, as abnormal blood flow has been implicated in the development of a variety of sight-threatening retinal diseases, such as diabetic retinopathy, glaucoma, and branch vein occlusions. Retinal arterioles play a key role in the regulation of blood flow in the retina by dilating and constricting their luminal diameter, mediated by changes in the contractility of the smooth muscle cells that encircle these vessels. Understanding the molecular mechanisms underlying the regulation of retinal perfusion therefore requires preparations where retinal arteriolar smooth muscle cells can be accessed and studied in conditions as close to physiological as possible.

In this video, we demonstrate a straightforward protocol for the isolation of arterioles from the rat retina that can be used in patch clamp, calcium imaging, and pressure myography studies. Over the last number of years, this preparation has been used to provide further insights into the regulation of vascular smooth muscle contractility and blood flow in the retina in both health and disease. Make up a one-liter solution of low calcium containing Hanks'or LCH, as shown in the materials table.

Assemble the isolation equipment prior to the collection of tissue. The glass Pasteur pipette must be fire polished to smooth, but not narrow the tip. Trim a plastic pipette to an aperture of approximately five to seven millimeters.

Place the eyes on the dissection dish, immersed in LCH solution. Use one set of forceps to anchor the eye to the dish by holding the orbital muscle attachments or optic nerve. Ensure the anchoring point is as close as possible to the sclera to stabilize the eye.

Using the blade, cut through the cornea along the ora serrata, and remove the lens by pressing gently on the sclera with forceps. The small, circular region that we see at the back of the eye in this image is the optic nerve. Using the blade, cut the eyecup in half symmetrically through the optic disc.

Using closed forceps, gently brush out the retinas from the two halves of the eyecup, taking care to remove any remaining attachments at the optic disc. Repeat the process with the second eye, and transfer the dissected retinas into the test tube using the plastic pipette and a small drop of LCH medium. Using the plastic pipette, fill the test tube with LCH to approximately five milliliters, and allow the retinas to settle to the bottom of the tube.

Wash the tissue approximately three times by removing approximately four milliliters of solution from the tube and adding fresh LCH using the plastic pipette. Where necessary, extraneous tissue should be removed. Wash the inside of the glass Pasteur pipette with polished tip with LCH to prevent the tissue from sticking to the pipette.

Using the same pipette, remove approximately four milliliters of solution from the test tube, and add approximately two milliliters of fresh LCH. Gently dissociate the retinas by drawing the tissue through the tip of the glass pipette slowly, and expel the contents into the test tube. Try not to introduce bubbles at this stage, and repeat the process until the retinas are broken up to the size of approximately two to three millimeters squared.

Wash the inside of the pipette with approximately two milliliters of LCH, and expel this into the test tube. Allow the contents to settle to the bottom over five to 10 minutes. Repeat the trituration process as described previously with a little more force until the tissue pieces are approximately one millimeter squared in size.

Allow the tissue to settle, and repeat once more with even more force until the contents are fully homogenized and no pieces of retina remain. The solution at this stage should appear milky in appearance. This technique will yield up to eight arteriole segments per isolation, measuring from 200 to 2, 500 micrometers in length.

Cannulation of one end of the arteriole with occlusion of the opposite end allows for the measurement of pressure-induced vasoconstriction, also known as the myogenic response. Arteriolar pressure myography is carried out as follows. An aliquot of retinal homogenate is transferred to a physiological recording chamber mounted on the stage of an inverted microscope.

Leave the homogenate to settle on the bottom of the chamber for at least five minutes. Visually scan across the recording chamber to identify arterioles greater than 200 micrometers in length and possessing an open end through which the vessel can be cannulated. Anchor down one end of the vessel using fine forceps and a small tungsten wire slip placed over the vessel.

Using the overlapping ends of the tungsten wire, maneuver the vessel to run horizontally across the bath, such that the open end is in line with the pressurization cannula. Perfuse the chamber with zero calcium Hanks'at 37 degrees Celsius. Cannulations are performed with glass pipettes.

The cannula is positioned at the open end of the vessel using a fine micromanipulator. The tip is positioned immediately adjacent to the opening, as assessed by adjusting the plane of focus on the microscope, such that both the end of the vessel and the cannula tip are in focus at the same time and the pressurization cannula advanced into the vessel aperture. A helper pipette is required to assist with the cannulation process and is used to gently restrain the arteriole and guide the arteriolar wall over the pressurization cannula at the same time as the cannula is advanced into the vessel lumen.

This procedure requires simultaneous, controlled movement of both manipulators and extensive practice to achieve a high success rate. After one minute of recording at zero millimeters of mercury, the intraluminal pressure is increased to 40 millimeters of mercury, and the vessel should rapidly dilate, confirming successful cannulation. Pressure-induced vasoconstriction, that is, the myogenic response, will subsequently develop over a period of approximately 15 minutes.

Patch clamp recording from retinal vascular smooth muscle cells enables investigation of the plasma membrane ionic currents that regulate intracellular calcium and hence cellular contractility. Whole-cell and single-channel recording is possible from individual smooth muscle cells still embedded within their parent arterioles as follows. Retinal arterioles are isolated and anchored down in the recording chamber with tungsten wire slips.

Basal lamina needs to be digested away to enable a high resistance seal to be formed between the patch pipette and the arteriolar smooth muscle cell membrane. The arterioles are superfused with a sequential series of enzyme solutions at 37 degrees Celsius, which also results in electrical uncoupling of adjacent cells, as indicated by the arrows on the image. The level of digestion is initially evaluated on visual separation of endothelial and smooth muscle layers during the collagenase step.

Removal of any remaining strands of basal lamina and/or peripheral neuropile is achieved by carefully sweeping the closed tips of fine forceps along the surface of the vessel. For patch clamping, the tip of the patch pipette is positioned vertically over the cell of interest and lowered gradually using the fine, slow movement of the micromanipulator to make contact with the arteriolar smooth muscle membrane. This is judged by cell movement and a change in the pipette resistance, measured using a cell seal test protocol in the acquisition software.

As the pipette is lowered onto the cell, the seal resistance will increase approximately fivefold. Negative pressure is applied transiently to the back of the pipette, and a gigaseal is gradually formed. This requires repeated applications of negative pressure over the course of one to five minutes.

For whole-cell recording, the perforated patch-clamp method is predominantly used. A gigaseal is formed, as previously described, with a pipette containing intracellular-like solution and amphotericin B.The access resistance is monitored using the membrane test protocol in the acquisition software. Once the access resistance falls to less than 15 megaohms, series resistance compensation is performed.

Typically it is possible to compensate the series resistance by approximately 75%in perforated patch mode. Voltage steps or ramp protocols may then be used to measure whole-cell currents. Following dissociation of the retina, primary, secondary, and pre-capillary arterioles can be identified based on their caliber and the arrangement of the vascular smooth muscle cells.

The first-and second-order arterioles appear visually similar under bright-field microscopy, but can be distinguished on the basis of their size. The pre-capillary arterioles are the smallest arterial vessels in the preparation and are easily recognizable due to the intermittent arrangement of vascular smooth muscle cells. The isolated arterioles can be clearly differentiated from capillaries and venules within the isolation.

Capillaries are apparent as a meshwork of small-caliber vessels approximately four to 10 micrometers in diameter, while venules are thin-walled and lack smooth muscle cell coverage. Primary, secondary, and pre-capillary arterioles are suitable for pressure myography, calcium imaging, and patch-clamp studies. Using pressurized arterioles, we have investigated the molecular mechanisms involved in the generation of the myogenic response.

The photomicrographs in this image show a rat retinal arteriole at various time points during the course of a pressure myography experiment. The time-course plot beneath shows the changes in vessel diameter during the full course of the experiment. Immediately upon pressurization, the vessel dilates, which is then followed by an active myogenic constriction that reaches a steady-state level after 15 minutes.

Addition of wortmannin in a zero calcium Hanks'solution at the end of the experiment dilates the vessel to its passive diameter for normalization purposes. This slide shows an example of a single-channel patch-clamp recording from a retinal arteriole smooth muscle cell prior to and following membrane stretch. This on-cell patch contains two stretch-activated TRPV2 channels, the activity of which increases with application of negative pressure to the patch pipette.

Protocols described here require practice, but should be achievable with minimal troubleshooting. We would recommend using the isolated arterioles on the same day as isolation. The method is optimized for rat retinal arterioles, but can be used on mice retinas.

Now we have used this preparation extensively, but where possible we also try to validate our key findings using ex vivo retinal whole-mounts and in vivo measurements of blood vessel diameter and blood flow.