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

II. METHODS

A.Animal Care and Anesthesia

We have mainly used adult female Sprague-Dawley rats (200–225 g) for our experiments. The rats were obtained from the breeding colony of Murcia University or from Harlan Interfauna lbe´rica (Barcelona, Spain). Animal care and experimental procedures were done according to institutional guidelines, European Union regulations, and policies on the use of animals of the association for research and vision in ophthalmology (ARVO). All surgical manipulations were carried out using general anesthesia.

Different types of anesthesia were used. For retrograde labeling, animals were administered a mixture of ketamine (75 mg/kg) (Ketolar , Parke-Davis, S.L. Barcelona, Spain) and xylazine (10 mg/kg) (Rompu´n , Bayer, S.A. Barcelona, Spain) in sterile saline. For neuroprotection studies, animals were anesthetized with intraperitoneal injections of 7% chloral hydrate in saline (0.42 mg/g body weight), because both ketamine and xylazine have been reported to have neuroprotective effects on ischemia-induced neuronal damage [1,2].

After experimental manipulations, animals were placed in their cages to recover from anesthesia and a steroid-antibiotic ointment containing neomycin and dexamethasone (Fludronef , Iquinosa, Madrid, Spain) was applied over the ocular surface to prevent corneal desiccation. Rats were fed ad libitum and kept in cages in temperature-controlled rooms with a 12-h light/dark cycle (light period from 8 a.m. to 8 p.m.). Light intensity in the cages ranged from 8 to 24 lux. An operating microscope and standard microsurgical instruments were used for all experimental procedures.

B.Is the Retina Suitable for Neuroprotection Studies?

The retina is a highly specialized part of the central nervous system that takes its position in the front of the head during development. From this location, the retina looks at the outside world and captures electromagnetic waves within the visible spectrum; these are transduced into electrical signals that are in turn processed through the different retinal layers to produce a retinal output. The retinal ganglion cell (RGC) axons convey this output along the optic nerve toward several relay stations in the midbrain, where it is further analyzed and sent over to the visual cortex where visual perception occurs. Several characteristics of the retinocollicular system of the adult rodent make it suitable for in vivo studies of neuronal survival after injury and administration of neuroprotective substances.

1. Location

The retina is encapsulated within the eye globe, which is located in the orbit, and is easily accessible for experimental manipulations. Moreover, the retina is somewhat isolated from the rest of the CNS, and thus experimental manipulations of one retina do not usually involve damage to the other retina or other parts of the brain, and this allows animal survival for shortand long-term studies.

2. Natural Reservoir

The eye globe may act as a reservoir for substances injected into the vitreous. These substances may be used to treat the retinal cells, or to label selectively the entire axonal population of RGCs, thus allowing identification of retinal projections into the brain [3].

3. Vascular Supply

In the rat, the ophthalmic artery trifurcates into two posterior ciliary arteries (nasal and temporal) and one central retinal artery (CRA). The CRA penetrates into the eye through the inferior and nasal aspect of the optic nerve [4–7]. Seven or eight terminal radial branches of the CRA are the main vascular supply to the inner layers of the retina. The choroidal blood vessels provide nutrients to the outermost one-third of the retina, and originate from the two posterior ciliary arteries. Arterioles arising from the posterior ciliary arteries, as well as from the CRA, are the main source of blood flow to the optic nerve head [6]. Because the retina is encapsulated within the sclera, which is a semi-rigid structure, increments of the intraocular pressure directly affect retinal blood flow. In addition, the ophthalmic artery may be identified intraorbitally on its course along the ON sheath before it trifurcates and is dissected and ligated, thus interrupting retinal and choroidal blood supply.

4. Optic Nerve Manipulations

The intra-orbital segment of the optic nerve can be readily identified, dissected, and injured. The ophthalmic artery does not enter the ON, like in most mammals, but courses within the ON sheath, and this allows experimental manipulations of the ON without compromising retinal blood supply [8,9].

5. Identification of Retinal Neurons

The great majority of RGCs in the albino rat send their axon to the superficial layers of the superior colliculi (SCi) [10] and a proportion (35%) of these also send a collateral to the dorsal lateral geniculate nuclei (dLGNi) [11]. Application

of retrogradely transported tracers to the main retinorecipient territories (e.g., the SCi), or to their cut axons in the ON results in the selective labeling of the RGC population. Because the retina may be examined as a flat mount, the entire RGC population can be observed in one preparation, and this facilitates quantitative estimations of the RGC population in the normal or experimental retina. In addition to RGCs, the retina comprises five other different classes of neurons, whose cell somata and processes are all confined to the retina. The cell somata of these are organized into three nuclear layers (outer, inner, and RGC layer) with two intermingled synaptic layers (outer and inner plexiform layers). This layered structure may be readily examined in retinal cross-sections. Furthermore, specific classes and subclasses of retinal neurons and cells may be identified using retrograde labeling techniques combined with immunocytochemical techniques [12].

C.Retrograde Labeling of the RGC Population

It is difficult to distinguish RGCs from the many displaced amacrine cells in the RGC layer [13] on the basis only of classical anatomical methods [12]. Furthermore, identification of injured RGCs is hindered because these may suffer phenotypic changes, including morphological [14], immunocytochemical [8,12], and molecular [15,16]. To identify the RGC population we have used retrogradely transported neuronal tracers applied to the terminals or cut axons of the RGCs. Identifying and counting RGCs is important when evaluating whether a substance may have neuroprotective effects.

1. Use of FluoroGold as Retograde RGC Tracer

The RGC population was retrogradely labeled from the superior colliculi (SCi) with the fluorescent tracer FluoroGold (FG) (Fluorochrome Inc., Engelwood, CO) following protocols that were originally described in 1988 [17]. In brief: The Midbrain was exposed, the pia mater overlying both SCi was gently removed, and a small piece of gelatin sponge (Spongostan Film, Ferrosan, Denmark) soaked in a solution of 3% FG and 10% dimethyl sulfoxide in saline was laid over the SCi. Because most RGCs in the rat project to the SCi [10], this procedure results in the labeling of almost the entire RGC population in both retinas [17].

Previous studies from our laboratory [18,19] have shown that following FG application to the SCi, some RGCs appear labeled as early as 3 days after dye application, and most RGCs appear labeled by 7 days after dye application. At this time, the densities of FG-labeled RGCs are similar to those obtained when other fluorescent and nonfluorescent retrogradely transported tracers are applied to the main retinorecipient target regions in the brain [12,17,20]. Furthermore, FG application to the SCi [19] or the lateral rectus muscle [21] results in the labeling of the entire population of RGCs or abducens motoneurons, respectively,

for up to 4 weeks after tracer application without leakage or apparent fading. Longer survival periods, however, were associated with a decrease in the numbers of labeled cells [19,21], and by 3 months only one-third of the population of abducens motoneurons was still labeled with FG [21]. At this time point, the entire population of abducens motoneurons could be labeled again if FG was reinjected into the ipsilateral rectus muscle, suggesting that FG is not toxic to neurons but disappears slowly from the neurons with increasing survival periods [21].

2. Use of di-ASP as Retrograde RGC Tracer

To identify RGCs in long-term studies we have used two different methods. In a first method, we labeled the RGC population with dil, a lipophilic carbocyanine which persists within the RGC somata for periods of up to 21 months after tracer application without leakage or fading [17,20]. A second method consists of applying retrograde tracers to the axons [12,22] or terminals [23] of RGCs, shortly before animal processing.

Here we describe the use of DiAsp applied intraorbitally to the ocular stump of the transected ON. The lipophilic dye 4Di-10Asp [DiAsp, D29, N-4-4-4-dide- cylaminostyryl-N-methylpyridinium iodide, Molecular Probes, Eugene, OR] can be applied to the RGC target territories or their cut axons to retrogradely label RGCs, both in control and experimental animals [22,24,25]. In brief: Three days before sacrifice, the left ON is exposed in the orbit, dissected from its surrounding sheaths, and cut close to its origin in the optic disc [8]. DiAsp is then applied to the ocular stump of the intraorbitally transected ON. To facilitate dye uptake by cut axons, crystals of DiAsp were previously dissolved in dimethylformamide and the solvent allowed to evaporate. Intraorbital transection of the ON induces RGC death [12,20], but this first appears between day 4 and 5 after axotomy [18,26]. Therefore, it is likely that the densities of DiAsp-labeled RGCs 3 days after dye application would not be affected by axotomy-induced RGC death. However, we cannot exclude the possibility that transient ischemia of the retina prior to DiAsp application shortens this 4–5 day period before the onset of cell death.

D. Induction of Retinal Ischemia

A variety of procedures have been described to induce retinal ischemia in laboratory animals. These may be divided into two main groups. One consists of increasing the intraocular pressure (IOP) above systolic levels to interrupt blood flow within the eye, and the other consists of the ligature of the blood vessels supplying the retina. Retinal ischemia may be permanent or transient if reperfusion is allowed (for review, see Table 3, Ref. 27). In the following section we describe the methods used in our laboratory to induce transient ischemia of the retina by elevation of the IOP [19] or by selective ligature of the ophthalmic vessels [28–32].

1. General Considerations

Ischemia-induced CNS damage is influenced by body temperature, which should be kept constant throughout the duration of the experiment. This is also the case for experiments aimed at studying ischemia-induced RGC death [33]. In a previous study, in which the room temperature was maintained constant at 23°C, the body temperature of a group of rats anesthetized and subjected to transient retinal ischemia was monitored with a rectal probe. The body temperature fluctuations in these animals ranged from 35.4°C right after initiation of retinal ischemia to 33.8°C 2 h later [19]. Because in a large series of similarly prepared experiments the densities of RGCs surviving different periods of ischemia were very consistent, it is possible that small body temperature fluctuations did not interfere with RGC survival [19]. Furthermore, the temperature within the eye was not monitored; thus, we ignore whether these small changes in body temperature also influenced retinal temperature and if this in turn affected RGC survival in those experiments.

The retinal blood flow may be monitored through the operating microscope. Inspection of the eye fundus is facilitated by pupil dilation with a topical drop of 1% tropicamide (Cusi Laboratories, El Masnou, Barcelona, Spain). Corneal desiccation throughout the experiment is avoided by applying on the cornea a solution of 2% hydroxypropylmethylcellulose (Gonioftal 4000, Cusi Laboratories, El Masnou, Barcelona, Spain). The placement of a coverslip over the cornea facilitates direct visualization of the eye fundus through the microscope. The eye fundus of the albino rat shows the central retinal artery dividing into six or seven radial vessels as well as six or seven radial veins that converge toward the disc into the central retinal vein.

2.Increase of the Intraocular Pressure Above Systolic Levels

We induced retinal ischemia in the left eye by increasing the intraocular pressure (IOP) above systolic arterial levels (see below). In these animals, as well as in those in which ischemia was induced by selective ligature of the ophthalmic vessels, the right intact eye served as control with the animals under deep anesthesia, two nylon monofilament 6/0 sutures were placed on the superior and inferior bulbar conjunctiva of the left eye close to the corneo-scleral limbus. These sutures were pulled tangentially in opposite directions until retinal blood flow was interrupted completely; this was assessed by examination of the eye fundus. The sutures were then tied to a metal frame, specially devised for these studies, to maintain the IOP above systolic levels. Inspection of the eye fundus was required throughout the duration of the ischemic period to ascertain blood flow arrest (Fig. 1a). Retinal ischemia is characterized by pallor of the iris and intense pallor of the eye fundus, as well as by blood flow arrest within radial retinal vessels. These were either empty or showing fragmented columns of arrested red blood cells.

At the end of ischemia, the sutures were released slowly and this allowed restoration of the retinal blood flow. Reestablishment of blood flow was characterized by increasing movement of the fragmented columns of red cells, the progressive filling of the radial vessels to their normal pattern, and the pink appearance of the fundus. Reestablishment of blood flow within the retina, if not spontaneous, may be facilitated with gentle eye massage. Animals in which restoration of the retinal blood flow did not occur, or was associated with retinal or vitreous hemorrhages, were discarded from the study. Regular findings at the termination of the period of ischemia were a moderate edema of conjunctiva and cornea [19].

3. Selective Ligature of the Ophthalmic Vessels

Under deep anesthesia, the left optic nerve was exposed in the orbit and the ON sheath was opened longitudinally [8] (Figs. 1b, 2a). A fine 10/0 monofilament suture was carefully introduced between the ON and the sheath, and tied around the sheath, avoiding damage to the ON (Fig. 2b). Because the ON sheath contains the ophthalmic artery [6], this procedure interrupts retinal and choroidal blood flow. The ligature may be released after different ischemic intervals to allow retinal reperfusion. The eye fundus may be examined directly through the operating microscope to assess retinal blood flow before, during, and after retinal ischemia (Fig.3) [30–32].

E.Administration of Neuroprotective Substances

Substances to be tested for their neuroprotective effects may be administered before, during, or after the insult. These may be given systemically or topically or delivered directly into the vitreous [34]. Topical instillation is a simple procedure that may be done in the alert animal.

For direct administration into the vitreous, the animal is sedated with anesthesia, the pupil is dilated, and topical anesthetic eyedrops are also instilled on the eye. A 30-G needle is used to produce a penetrating puncture through the conjunctiva, sclera, choroid, and retina at approximately 1–2 mm from the cor- neo-scleral limbus. The needle of 5 L Hamilton microsyringe is then introduced through this puncture into the vitreous and directed toward the posterior pole to avoid injury to the lens [34]. Direct injury to the lens results in lens opacity and may have neuroprotective effects on RGC survival [35,36]. Injection volumes were not greater than 5 L, and saline was used in most instances as vehicle.

F.Tissue Processing

After various survival times, animals were given an overdose of anesthetics (ketamine and xylazine) and perfused through the heart first and briefly with 0.9%

Figure 3 Micrographs illustrating the eye fundus of the retina before ischemia (a), during ischemia (b), and shortly after reperfusion (c). These micrographs were taken through a video camera connected to the operating microscope. (a) A normal eye fundus shows typical radial retinal vessels. (b) During the period of transient ischemia induced by selective ligature of the ophthalmic vessels, these appear empty or with fragmented columns of red cells that do not move. (c) Shortly after release of the ligature around the ophthalmic vessels, blood flow is restored within radial retinal vessels.

NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Both eyes were enucleated and an 8/0 suture was placed in the superior aspect of the conjunctiva near the limbus for orientation purposes. The retina may be examined in cross-sections or in whole-mounts. In the following section we describe the methodology employed to obtain flattened whole-mounts.

1. Retinal Whole-Mounts

The retinas were dissected out and prepared as flattened whole-mounts by making four radial cuts, the deepest one indicating the superior pole of the retina and the others indicating the nasal, temporal, and inferior pole of the retina, respectively. Flattened whole-mounts were transferred to a piece of filter paper and this was immersed in the same fixative for 1 h. The retinas were released from the filter paper, washed in the buffer solution, and mounted vitreal side up on subbed slides.

For experiments in which DiAsp was used to retrogradely label RGCs, 0.1 M sodium carbonate buffer (pH 9) was used as mounting media. In animals in which FG was used as retrograde tracer, mounting media consisted of 50% glycerol in 0.1 M sodium carbonate buffer (pH 9) containing 0.04% of p-phenylenedi- amine [37].

Retinas were examined under fluorescence microscopy (Axiophot, Zeiss, Oberkochen, Germany) with fluorescein (BP 450-490, LP520) and ultraviolet (BP 365/12, LP 397) filters that allow the observation of the green-yellowish fluorescence of DiAsp and of the white-gold fluorescence of FG, respectively. It is our experience that examination of DiAsp should be done within the first 24 h after processing the animal because the dye tends to dissipate and fade away, thus losing its fluorescence quality soon after mounting.

2. Photographing and Counting Labeled RGCs

Densities of surviving RGCs in the flat mounted retinas were estimated following previously described methods to determine densities of RGCs within the central regions of the retina [12,17–20,30], where RGCs are the most prevalent [38,39]. In brief, FG-labeled RGCs were counted in a masked fashion from printed micrographs ( 400) taken from 12 standard rectangular areas of 0.0864 mm2 of each retina situated, three in every retinal quadrant, at approximately 0.875, 1.925, and 2.975 mm, respectively, from the optic disc. A labeled RGC was counted if the whole cell or its nucleus was visible within the micrograph. Counts in the 12 regions were averaged and divided by the area of the picture to obtain a mean RGC density (cells/mm2) per retina. For groups of similarly treated retinas, results were reported as mean RGC densities SD (standard deviation). Cell counts were done in a masked fashion from printed micrographs; the identity of the retinas that led to the micrographs was not known until cell counts