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

Figure 1 Schematic diagram outlining the steps used in preparing the isolated, living retina preparation. The posterior eyecup is stored and dissected in oxygenated artificial cerebrospinal fluid (aCSF) prior to being placed, ganglion cell layer up, into the recording/injection chamber. This chamber, which also receives oxygenated aCSF, fits onto the stage of an upright microscope. Single neurons in the isolated retina are viewed and targeted for intracellular analysis and injection by prestaining with the vital dye Acridine Orange. A high-resolution video monitor is used to provide visual stimulation of the retina, and an intracellular amplfier is used to obtain biophysical measurements.

result in mechanical damage to the retina. If vitreous remains attached to the posterior eyecup, it often can be removed prior to placing the retina into the recording and injection chamber using the reverse side of a pair of curved forceps and grasping the vitreous in an area of the retina that will not be examined. Treating the retina briefly with a low concentration (0.1%) of collagenase (Type II, C-6885, Sigma Chemical, St. Louis, MO) also has been used [20]. Again, it is important to try to peel the vitreous from the retina by detaching it from the margin of the eyecup and drawing it horizontally across the surface of the retina. A clean retinal surface is imperative for good oxygenation of the tissue, clean microelectrode penetrations and recordings of ganglion cells, and successful postinjection processing.

Once a posterior eyecup with a clean retinal surface is obtained, one can either separate the retina from the choroid and sclera using a fine (# 00) artist’s

brush, remove only the sclera using forceps and scissors, or leave the entire eyecup intact. Complete isolation of the retina is suitable for intracellular staining studies, but not those involving electrophysiological recordings, where dissec- tion-related damage to the photoreceptor and pigment epithelia layers could affect light responsiveness. For these studies it is best to either remove only the sclera or leave the eyecup intact. Final preparation of the retina for placement into the injection/recording chamber is performed in a petri dish containing aCSF. The eyecup is placed on a submersed, perforated, stainless steel platform, and a bubble stone is used to maintain oxygenation. Regardless of which type of isolated retina preparation will be used, three to four radial cuts, made away from the area of interest, are used to flatten the tissue; the optic disc, fovea, and retinal blood vessels serve as references for positioning these incisions. The retina then is placed, ganglion cell layer up, into the injection/recording chamber, which has been filled with oxygenated aCSF. While many different types of chambers are available commercially, we prefer one of our own design. The chamber consists of a rectangular piece of Plexiglas that has a circular well in the center. A pair of concentric Plexiglas rings fit into the well. The outer ring contains strands of nylon stocking that form a platform and hold the tissue above the chamber bottom. The inner ring also contains strands of nylon stocking, and when press fit into the outer ring, forms a “sandwich” that holds the tissue securely in place. The chamber then is mounted onto the stage of an upright microscope (Nikon Optiphot-2) equipped with epifluorescence. The tissue is perfused with warm (36°C), oxygenated aCSF using a gravity flow system. An aspirator bottle containing the aCSF is placed approximately 24 in. above the tissue preparation, and the solution is oxygenated with 95% O2/5% CO2 using a glass gas dispersion tube. The aCSF then passes through a flow meter and water jacket where the flow rate is adjusted to 4–6 cc/min and the solution is warmed just prior to delivery into the tissue chamber. The temperature of the water jacket, which consists of a series of stainless steel tubes traversing a water-filled compartment, is adjusted using a temperature-controlled circulating pump (Isotemp 2100, Fisher Scientific). The aCSF is warmed by making five passes through the Plexiglas water jacket before being dispensed to the tissue chamber. The solution flows over the tissue, and then is drawn off from a separate well using a vacuum pump and sidearm flask. Not drawing the aCSF directly from the injection/recording chamber prevents fluctuations in the chamber fluid level. The stainless steel needle used to draw off the fluid is moveable, thereby allowing one to adjust the depth of the aCSF in the chamber. This is of particular importance for the physiological recordings, where fluid depth affects the capacitance of the electrode, and therefore the quality of the electrical signals recorded.

In all cases, the retinal dissections are performed in dim light, and the retinas are allowed 30 min in compete darkness in the injection/recording chamber before being studied. Single ganglion cells are viewed under epifluorescence us-

ing a 40 water immersion objective (Nikon, N.A. 0.55) with a working distance of about 1.6 mm. In order to aid visualization, the retinas are treated periodically with two to three drops of the vital dye Acridine Orange (A-4912, Sigma, 1mM in aCSF, see Fig. 1). This often is not necessary when working with a completely isolated cat retina, where individual ganglion cells can be viewed under standard illumination using either differential contrast optics, or by simply misadjusting the microscope condenser. However, because of the increased number of ganglion cell axons, it is difficult to view individual cells in the primate retina without Acridine prestaining. When ganglion cells are viewed using epifluorescence, a neutral density filter (ND4) is used to reduce the intensity of the mercury vapor light reaching the tissue, and cells are viewed only long enough to either align the electrode with the selected cell (physiology studies), or quickly penetrate the cell (anatomical studies).

V.ELECTRODE PREPARATION AND INTRACELLULAR STAINING

Intracellular injections and/or recordings are made using glass microelectrodes and a four-axis hydraulic micromanipulator (Narishighe, Japan). The glass micropipettes are pulled on a Flaming-Brown P-87 horizontal micropipette puller (Sutter Instruments, Novato, CA). For anatomical studies, we have found that a 3% solution of the fluorescent dye Lucifer Yellow CH (Sigma, L-0259) in 0.1 M LiCl (pH 7.6) works well for labeling both cat and primate ganglion cells. For combined electrophysiological and anatomical studies, we fill the pipettes with a solution containing 2% Neurobiotin (Vector Labs, Burlingame, CA) and 0.05% pyranine (Aldrich, Milwaukee, WI) in 1 M potassium acetate buffer (pH 7.6); Lucifer Yellow CH precipitates in 1 M potassium acetate, and therefore we do not use it with this buffer. However, it may remain soluble in 200 mM acetate buffer. Because both fluorescent dyes have excitation and emission wavelengths similar to Acridine Orange, it is possible to view the electrode tip and the prestained ganglion cells using the same epifluorescence filter combination (B- 3A/DM505, Nikon). This makes it easier to target single ganglion cells and results in a more stable preparation, because filter cubes do not need to be exchanged during cell penetrations. The electrodes are beveled from an initial resistance of 80–100 MΩ to a final resistance of about 35–45 MΩ using a K.T. Brown type beveler (BV-10, Sutter Instruments). For intracellular injection with Lucifer Yellow CH, the electrode is positioned adjacent to the cell soma and advanced slowly along its axis until it is seen to penetrate the cell soma. Successful penetration of the cell membrane is recognized by the sudden filling of the soma by the small amount of dye that leaks from the electrode tip. Complete filling of the soma, dendritic tree, and intraretinal axon then is achieved by passing 1–5 nA of

negative current through the electrode. Typically-2 min is required to fill parasol, midget, and beta ganglion cells, whereas 2–3 min is needed for the larger alpha cells. Intracellular penetration and recording using the potassium acetate-Neuro- biotin electrodes is accomplished using a similar strategy; however, here the negative capacitance circuitry of the intracellular amplifier (AxoClamp 2B, Axon Instruments, Union City, CA) is used to gently “ring” the electrode tip and aid with penetration of the cell membrane. Successful penetration is indicated by a negative deflection of about 50–60 mV in the recorded electrode potential, and large intracellular spikes. Injection of Neurobiotin is achieved using positive current pulses (1–2 nA) of 100–200 ms duration, delivered for 3–5 min.

Regardless of the injection procedure, it is imperative that optimal electrode configuration and injection parameters be established for the intracellular dyes and buffers being used, and the cells being studied. Nonoptimal conditions can result in morphological artifacts that could be misinterpreted as degenerative changes. In addition, because it is not uncommon for a bond to develop between the cell membrane and electrode, quick withdrawl of the electrode is needed in order to prevent the cell soma from being damaged mechanically or excised from the tissue.

Upon completion of the injections and/or recordings, the retina is removed from the chamber and drop-fixed in a solution containing 4% paraformaldehyde in 0.1 M phosphate buffer. Retinas containing Lucifer Yellow CH-filled ganglion cells then are washed with buffer, mounted onto subbed slides, and coverslipped using DePeX (BDH Laboratory, Poole, England), a non-autofluorescing mounting medium. Neurobiotin-labeled retinas are washed with 0.01 M phosphate buffered saline, dissected from the sclera and pigment epithelium using a fine artist’s brush (# 00), and processed using Vector Lab’s Vectastain ABC Elite (PK-6100) and DAB substrate (SK-4100) kits. These retinas also are then mounted and coverslipped using DePeX.

VI. RETINAL SAMPLING, MAPPING,

AND ANALYSIS

Although the intracellular technique provides detailed filling of single ganglion cells, it does not permit large numbers of neurons to be sampled across the entire retina. Therefore, one typically needs to select a specific area of the retina for study. This also is necessary because ganglion cell size is not constant, but increases with increased retinal eccentricity. Thus, for accurate morphological comparisons to be made, it is necessary to match ganglion cell samples for retinal location. Fortunately, the ability to visualize specific retinal landmarks, such as the optic disc, fovea, area centralis, and retinal blood vessel pattern, in the isolated retina make it possible to coordinate cell sampling across retinae.

cells, and one that is not readily available using standard video image capture and analysis systems. Aside from the ease of capture and the ability to freely manipulate cell images, a third advantage of being able to apply confocal microscopy is that most quantitative measurements can be made directly from the digital images using the system’s own morphological analysis software. This enhances the efficiency of the analysis and reduces the potential for magnification-based errors. However, since most confocal image formats now are compatible with current image analysis packages, cell images can be downloaded and analyzed offline to reduce cost and increase flexibility. For our purposes, we combined intracellular staining and both video-enhanced and confocal microscopy to compare ganglion cell morphologies following either neonatal damage to visual cortex in the cat [19] or experimental glaucoma in primates [25]. Qualitative and quantitative ganglion cell comparisons included the assessment of differences in dendritic field organization, cell body size, dendritic field area, and axon diameter for ganglion cells of specific classes. In the cat studies, we found that a selective loss of beta ganglion cells due to neonatal damage to visual cortex affects only the development of surviving beta, and not alpha, ganglion cells. This suggests that the dendritic fields of ganglion cells in the vertebrate retina achieve their final morphologies based on competitive interactions among ganglion cells of the same, and not different, classes. With respect to the primate studies, application of the intracellular staining technique allowed us to demonstrate for the first time that the earliest signs of ganglion cell degeneration in glaucoma occur at the level of the distal dendritic tree, and that the pattern of degeneration is similar for both midget and parasol type cells. In brief, the degenerative changes include a thinning of the proximal and distal dendrites, abrupt reductions in dendritic process diameter at branch points, and a general decrease in the complexity of the dendritic tree (Fig. 2). These findings have at least three significant implications. First, they indicate that the onset of glaucoma-related ganglion cell death occurs earlier than previously thought based on Nissl-stained estimates of ganglion cell loss alone. Second, because ganglion cells receive all of their input from more distal retinal elements via their dendrites, it is reasonable to assume that early deficits in ganglion cell function also occur; this is the focus of our structurefunction studies. And finally, since changes at the level of the cell soma occur later, they suggest a window of opportunity for effective neuroprotection intervention.

Because our structure-function studies employ the use of a nonfluorescent dye, the ganglion cells for this work are analyzed using the Neurolucida/ NeuroExplorer cell reconstruction and analysis system (MicroBrightField, Inc., Colchester, VT). This system incorporates a motorized stage and video-based (Hamamatsu C-5985 chilled CCD camera) image analysis software. Individual cells that have been filled with Neurobiotin are reconstructed three-dimensionally

VII. USE OF THE ISOLATED RETINA

PREPARATION FOR BIOPHYSICAL

AND VISUAL STIMULATION STUDIES

As noted previously, one of the main advantages of working with the isolated retina preparation is that its neural circuitry is preserved. Thus one can analyze not only the morphology, but also the intrinsic and visual response properties of single neurons (Fig. 3). Biophysical analyses, which evaluate membrane integrity, include measurements of resting membrane potential, membrane time constants, threshold levels for activation, and rate of firing in response to intracellularly applied steps of depolarizing current. In our setup, the stimuli used for these tests are presented, and the cellular responses collected and analyzed, using a commercial stimulus presentation and data acquisition and analysis package (pClamp6, Axon Instruments). Visual stimulation of the isolated retina can be achieved by presenting light stimuli (flashing or patterned) via either the camera port or the condenser of the microscope. Because we do not remove the sclera from the retina preparation, our system involves positioning a high-resolution XYZ video monitor (Tektronix 608) over the camera port of the microscope (Fig. 1). The video monitor is aligned with the center of the camera port, and the cell to be studied is positioned in the center of the microscope field. Visual patterns consisting of drifting and counter-phased light and dark bars are projected onto the isolated retina through the 40 objective. The various patterns are generated and presented in random order by computer control of a Picasso CRT Image Synthesizer (Innisfree, Fenstanton, Cambridgeshire, England). Sinusoidal-modu- lated color diodes also are used to examine the response properties of primate ganglion cells.

VIII. USE OF THE WHOLE-MOUNTED RETINA FOR STUDYING NEUROPROTECTION

As a first assessment of neuronal degeneration or survival following a particular manipulation or treatment strategy, it often is useful to start by examining a large population of neurons over a wide region of the tissue being studied. Since this is not possible using the isolated retina-intracellular technique, we used Nisslstained retinal whole mounts in our initial studies of retinal ganglion cell survival following optic nerve injury and brain-derived neurotrophic factor (BDNF: Regeneron Pharmaceuticals, Tarrytown, NY) treatment in the cat [13]. The retinal whole mount is a unique tissue preparation, and it is ideally suited for studying changes in ganglion cell size, number, and density. Cats too, offer several advantages for use in neuroprotection studies. First, as noted previously, the morpholo-

A central issue in any study that involves the measurement of ganglion cell numbers is cell identification. In the cat, as with most vertebrate retinas the ganglion cell layer also contains a significant number of amacrine cells [52–54]. While most ganglion cells in the rat can be identified using the sponge-based retrograde labeling method described previously, this approach is not readily applicable in the cat due to the greater depth and spatial separation of the colliculus and visual thalamus. Nevertheless, in the cat one can distinguish most ganglion cells and “displaced” amacrine cells based on soma size and cytoplasmic differences; the amacrine cells are among the smallest neurons in the ganglion cell layer, they show a low ratio of cytoplasmic-to-nuclear volume, and they often contain a prominent basophilic nuclear fold [52].

In our analysis of the neuroprotective effects of BDNF in cats following optic nerve injury, we selected for quantitative analysis a region of the retina that occupied 1.74 mm2 and was located 3.0 mm above and 1.5 mm temporal to the area centralis. This region was chosen because of the relatively constant size and density of ganglion cells in this area of the cat retina (Fig. 4)[26]. Welldefined retinal landmarks such as the optic disc, area centralis, and retinal blood vessel pattern were used, along with the microscope stage digitizer, to properly orient each retina on the microscope stage, and to standardize the starting point and stage movements for cell sampling. From the starting point, 42 digital images (41,000 m2/image) then were obtained systematically using the Hamamatsu high-resolution video camera and a 40 objective. The retinal images were collected as three dorsal-ventral passes composed of 14 images each. Double counting was avoided by separating each sample column horizontally by 500 m (using the Digitizer readout), and vertically by using each previous image as a reference for the next. Cell size, density, and number were determined directly from the digital images using an image analysis and measurement software package (Image Pro Plus, Media Cybernetics).

By standardizing the measurements and using a region of the retina that could be identified reliably and sampled systematically across retinas, we were able to demonstrate the neuroprotective ability of BDNF in an eye comparable in size to that of the primate. Further, by testing different amounts of the drug, we were able to demonstrate not only that 30 g of BDNF is needed to obtain maximum ganglion cell survival in the cat, but also that increasing the drug dose results in a decrease, rather than increase, in the level of neuroprotection (Fig. 5). Because the sample areas were standardized for each retina, changes in cell density mirrored the changes in cell number. Cell size measurements suggested a complex response among the different classes of ganglion cells. While 30 g of BDNF retained the largest number of ganglion cells, 90 g minimized the loss of medium-sized neurons and retained normal proportions of large, medium, and small ganglion cells.