Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

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II.RETROGRADE LABELING OF RETINAL GANGLION CELLS

In order to differentiate retinal ganglion cells from other retinal neurons in mixed culture, we take advantage of the unique anatomical connections between the brain and the cell bodies of RGCs. When an appropriate dye is injected into the superior colliculus of an anesthetized rat, the dye will flow via retrograde axonal transport in the axons of RGCs back to the cell bodies located in the inner retina [7]. Take care to select a fluorescent probe that will work in this manner. DiI-C18, DAPI, and several other dyes are available, but it is important that the dye chosen have an excitation/emission spectrum that will not interfere with the experimental probe. For example, if a red and green emitting dye such as JC-1 will be used for imaging ∆ΨM, then you should select a blue-violet dye (e.g., DAPI) for the retrograde labeling of RGCs. In the methods described below for RGC imaging of both ∆ΨM and superoxide anion, DAPI was used for retrograde labeling.

III. RETINAL GANGLION CELL CULTURE

Long-Evans rat pups, age P3, are injected with either DiI-C18 (Molecular Probes) or DAPI (Molecular Probes) into each superior colliculus for the retrograde labeling of retinal ganglion cells [8]. After waiting for at least 2 days, pups are sacrificed by CO2 asphyxiation and their retinas are dissected. The retinas are then dissociated according to previous methods [8], and plated on a poly-L-lysine- treated eight-well chambered cover glass for 2 h 37°C, 5% CO2, 80% humidity. After the incubation period is complete, follow the specific JC-1 or HEt staining protocols for imaging.

Cultures are incubated for 2 h for two reasons. First, the dissociated cell mix that is added to the well needs time to become attached to the poly-L-lysine– coated surface of the wells. Second, some of the cells in primary neuronal cultures such as these begin to undergo stressor axotomy-related apoptosis, which can be as much as 50% by 24 h [8,9]. Imaging soon after culture reduces the possibility that the cell being imaged will be in the process of dying from axotomy, although this obviously cannot be excluded. Longer-term cultures are more likely to have significant amounts of apoptosis.

IV. SETTING UP FOR IMAGING

A.Choice of Dyes

JC-1 (Molecular Probes) is a dual-emission fluorescent probe that associates with the mitochondrial inner membrane. The dye exists as two forms (monomer and

J-aggregates) depending on the membrane potential of the inner mitochondrial membrane with which the dye is associated. The monomer associates with the inner membrane regardless of membrane potential, but J-aggregates will only form if an adequate membrane potential is present. While both forms of JC-1 have identical excitation spectra, each form has its own unique emission spectrum. The monomer form emits at 535 nm and the J-aggregates have a peak emission at 580 nm. With the proper imaging equipment, these unique characteristics allow one to measure the emission intensity of each form of JC-1 and determine the membrane status of mitochondria in intact RGCs. Specifically, one can measure the ratio of 580/535 nm emission over time to detect changes in ∆ΨM brought on by specific pharmacologic treatments.

Dihydroethidium (HEt) is a dye that has a peak emission around 580–590 nm when converted to its active form by superoxide anion. When used in this method, it can be used to detect the superoxide burst occurring during apoptosis [10,11].

B. Equipment Needed

Listed below is the equipment necessary for imaging over short time intervals, and also the additional devices needed if one is to perform imaging experiments that will last several hours (microscope stage incubator, pH control, etc.). While each device can be purchased separately, there are some companies that will provide most, if not all, the necessary imaging equipment in a bundled package.

Equipment necessary for imaging:

1.Inverted fluorescent microscope (Zeiss Axiovert S100 or a similar model) fitted with appropriate filters (discussed below) and 100 oilimmersion objective

2.Sutter Instruments Lambda 10-2 filter wheel (or a similar model) with filters. To image with JC-1, a dual-emission probe, the filter wheel should be fitted along the emission light path (i.e., between the microscope and digital camera).

3.Dye-specific filter sets (obtain from Chroma or Omega Optical)

a. RGC identification

DAPI: Excitation: 330 nm Dichroic: 400 nm Emission: 450 nm

b.∆ΨM measurement

JC-1: Excitation: 480 nm Dichroic: 505 nm

Emission: 535 nm and 580 nm filters (Lambda 10-2 filter wheel)

c. Superoxide anion measurement

HEt: Excitation: 515 nm

Dichroic: 565 nm

Emission: 580 nm long pass

4.1.0 log (10%) neutral density filter. This should be fitted in the path of the microscope’s light source, and will cut down on the fading of JC-1.

5.Photometrics SenSys digital camera

6.UniBlitz shutter driver and shutter (important component for reducing the amount of dye fading)

7.Pentium III computer or better, loaded with MetaFluor 4.0 software (Universal Imaging Corporation), or later version

8.Lab-Tek II chambered cover glass for cell culture (0.2–0.5 mL working volume, 0.7 cm2 growth area) (Fisher Scientific)

Optional accessories for long-term studies (all but the CO2 tank and regulator are obtained from Harvard Apparatus):

CSMI microscope plate incubator (#65-0101)

TC-202A temperature controller w/ thermistor (#65-0045) pH controller (#70-2116)

Watson Marlow 205CA perfusion pump (#72-0501) Perfusion tubing (#72-0571)

CO2 tank with regulator

V.MEASURING MITOCHONDRIAL MEMBRANE POTENTIAL OR SUPEROXIDE ANION IN CULTURED RETINAL GANGLION CELLS

A.Dye Treatment

Due to the dyes’ extreme sensitivity to light, all procedures involving JC-1 or HEt should be carried out in the dark. Prepare a 4 mM (JC-1) or 3.2 mM (HEt) stock solution in dimethyl sulfoxide (DMSO). Aliquots should be stored at20°C and protected from light.

I.Staining protocol for JC-1

1.Thaw out the 4 mM stock solution of JC-1 in the 37°C water bath.

2.Add 7 mL of Neurobasal medium to a 15 mL Falcon tube, and place in a 37°C water bath.

3.Once thawed, add 5.37 L of the 4 mM stock to 7 mL of Neurobasal

medium in a sterile incubator. Important: Do not add the JC-1 directly to the media. Tilt the tube containing the media at a 45° angle, then

vary substantially. Therefore, one cannot simply measure the average fluorescence from a well if the purpose is to measure the timing of acute changes in ∆ΨM. Instead, individual cells should be observed for these types of experiments, and this can be achieved by selecting specific regions for measurement using the MetaFluor software. An advantage of measuring ∆ΨM in this way is that small changes in ∆ΨM due to pharmacological treatments can accurately be detected. However, the number of RGCs in a single field of a mixed retinal culture under a 100 objective is low, and does not allow for the measurement of many cells simultaneously. A section below describing data analysis discusses how to combine experiments to obtain significance.

During imaging, the digital camera, filter wheel, and microscope shutter are all under the control of the imaging software program (MetaFluor), which integrates all of their functions. This allows the experimenter to take a relatively passive role during the actual acquisitions of images. Before beginning any experiment, the program must be set up properly. Variables such as acquisition number, time between acquisitions, camera settings, optical filters to use, and so on must be decided on beforehand. The advantage is that MetaFluor does the rest after a simple mouse click starts the imaging process. Remember that for measuring ∆ΨM, two separate emission filters are needed (535 nm areas of low ∆ΨM, 580 nm areas of high ∆ΨM). Thus, a typical imaging cycle for JC-1 will involve taking one picture at 535 nm, followed by a picture at 580 nm. MetaFluor can then automatically calculate a ratio of the 580 nm and 535 nm fluorescent intensities (580/535 nm). The data can be transferred while imaging to Microsoft Excel, for further analysis after the experiment. A representative from the software company is an excellent resource for describing the complete capabilities of the software package.

The experiments measuring ∆ΨM start by obtaining images once per minute for 5 min and calculating the ratio of 580/535 nm. After this initial baseline fluorescence has been determined, a chemical treatment is added to the well. Then, additional images are acquired once per minute for 20 min, up to 12 h (long-term imaging equipment needed, see above.) Chemical treatments are selected for their ability (proven or proposed) to alter ∆ΨM in some fashion. An excellent control is 10 µM valinomycin, a K ionophore for the mitochondrial inner membrane, which causes collapse of the ∆ΨM within minutes.

1.Imaging Protocol

1.Using an appropriate filter set for DAPI, locate an RGC in the well.

2.Switch to the 100 oil-immersion lens, and refocus. The nucleus should be stained a bright, vivid blue-violet color. Check that the cell appears intact and living.

Note: Other light purple–colored cells may be found in the well, but

these are not retinal ganglion cells. Some DAPI-stained RGCs die and lyse during the incubation period, releasing small amounts of DAPI that may be taken up by nearby cells (bipolar cells, macrophages, etc.). These cells that have secondarily taken up DAPI appear as very faint purple cells scattered throughout the plate. If one concentrates on locating only cells that have their nucleus stained (not the entire cytoplasm), and that staining is an intense purple (not faint), then there should be no confusing the RGCs from other retinal cells in this system.

3.Change the filter block to the position for JC-1 excitation. The cell should have bright orange spots or streaks (these are the stained mitochondria) against a bright blue background. If the cell appears entirely green, it is probably dead as a result of the dissociation procedure, and should not be studied. Focus on the cell, and again check that it is centered in the field of view. Manually close the shutter and divert the microscope light to the filter wheel/digital camera apparatus

4.Using the imaging software, complete one cycle of imaging. The digital camera will acquire one image of the fluorescent intensity at both 535 nm and 580 nm.

5.Using the imaging software, encircle a region of the just-imaged cell for measurement.

6.Start the experiment, which will continue to image the same cell to the specifications of the program setting while making fluorescent measurement.

2.Imaging Protocol for Dihydroethidium (HEt)

This procedure is essentially identical to that used for imaging ∆ΨM with JC-1.

3.Common Problems with Obtaining an Image

•The light path in the microscope was not properly diverted to the filter wheel/digital camera apparatus.

•The shutter did not open. Light should pass through the plate during the image acquisition—if it did not, then the shutter did not open properly. This usually can be fixed by turning the power for the shutter controller off, then back on after a few seconds. Or, try hitting the Reset button on the shutter controller a few times, then try imaging again.

•The proper filters are not in the filter block and/or filter wheel.

•The cell moved out of the camera’s field of view. Oil microscopy using a 100 objective can be tricky. Any slight jarring of the microscope apparatus or vibration of the table it is placed on may cause the cell to move. Addition of chemical treatments by pipetting into the well while

imaging may also cause problems. Make sure the microscope is on a very stable, heavy table. The poly-L-lysine solution used to adhere the cells should be in good condition. If necessary, addition of agents by perfusion pump may be necessary.

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