Graefe's Archive Ophthalmology for Clinical and Experimental

Laboratory investigations

© Springer-Verlag 1992 Graefe's Arch Clin Exp Ophthalmol (1992) 230:380-384

Electrophysiological and morphological changes in rabbit retina after exposure to the light of the operating microscope Jorge Ramirez, Ursula Meyer, M. Stoppa, and Mareike Wenzel Augenklinik and Institut ffir Anatomic der Charit6, Humboldt-Universit/it, Schumannstrasse 20/21, O-1040 Berlin, Federal Republic of Germany Received December 27, 1989 / Accepted July 17, 199l

Abstract. H e a l t h y adult rabbit eyes were exposed to up to 4 h o f c o n t i n u o u s illumination with m o d e r a t e light intensity, as is p r o d u c e d by the l a m p o f an o p h t h a l m i c operating microscope. E l e c t r o r e t i n o g r a m s were recorded before a n d after the long period o f illumination. The depression o f the waves in the electroretinograms observed just following light exposure recovered within i h to n o r m a l values. Electron m i c r o s c o p y o f the retina revealed changes within the cells o f the p i g m e n t epithelium. These results are discussed in view o f their clinical implications in h u m a n patients.

Introduction The d a m a g i n g effects o f light on the h u m a n eye are well k n o w n in the f o r m o f sun or s n o w blindness. In experimental o p h t h a l m o l o g y , since the first description by Noell et al. [22], the disintegration o f retina p h o t o r e ceptors by long-term exposure to even m o d e r a t e light intensity has been well d o c u m e n t e d . In h u m a n patients, similar situations involving direct exposure o f the eye to light m a y occur, associated with diagnostic procedures such as indirect o p h t h a l m o s c o p y , intraocular fiber optic light as used in vitrectomy, and use o f the operating m i c r o s c o p e or slit l a m p [2, 10, 12, 17, 18]. Injury as a result o f light can be detected electrophysiologically with the electroretinogram ( E R G ) or a n a t o m i cally by light or electron microscopic tissue analysis. The degree o f the injury depends on the intensity, wavelength a n d d u r a t i o n o f light exposure, as well as the time when the observation is m a d e [7, 13, 15, 20, 24]. A detailed review o f earlier results has been given by L a n u m [14]. T h e aim o f the present animal investigation was to determine the potential d a m a g e that could be caused by o u r operating m i c r o s c o p e during routine eye surgery in h u m a n patients.

Correspondence to: U. Meyer, Anatomisches Institut, Fakult/it ffir Medizin (Charitb), Philippstrasse 12, O-1040 Berlin, Federal Republic of Germany

Materials and methods

Experiment The investigations were carried out in 12 healthy adult rabbits weighing 3.5-4.5 kg each. The animals were with 0.1 mg/kg body weight of atropine sulfate. After that, the animals were anesthetized with intravenous urethane (150 mg/kg of a 20% solution), and the pupils of both eyes were maximally dilated with 1.0% atropine and 10% phenylephrine. The animals were placed in a prone position, with their heads secured in an upright position, by using a harness. The ERGs recorded from the control and light-damaged eye were made by using a cornea ring electrode, and stimulation was done with a 20-W halogen lamp (1 s) in the operating microscope equipped with a camera shutter. During the exposure time when the operating microscope was being used, we stimulated the ERGs in the control eye by a green light-emitting diode (LED) for a duration of 1 s. Before the damage was inflicted, dark adaptation was reached. The ERG stimulation was repeated until dark adaptation had been reached (no further increase in the amplitude of the a-, b- and c-waves). Then we began with the light exposure. Only one eye was exposed to the light of the operating microscope (Carl Zeiss, Jena, 20-W halogen lamp) while the other served as control for 1-4 h. After the exposure time had ended, the ERG recordings were made until the initial amplitudes had been reached. During the experiment the corneas were kept moist by using physiological saline solution. After finishing the ERG recordings, the animals were killed and the bulbi enucleated for histological examination. All experiments were begun at the same time of day in order to exclude changes due to the circadian rhythm of cell activity.

Microscopy The eyes were immediately opened by coronal section; the cornea, lens, and vitreal body were removed and the remaining half fixed by immersion in formaline solution, embedding in paraffin, sectioning (10 gm thick), and staining with hematoxylin and eosin or Goldner's light green. For transmission electron microscopy (TEM) the posterior half of the bulb was fixed by immersion over night in cold 2.5% glutardialdehyde solution buffered with 0.1 M cacodylate buffer, pH 7.2, postfixed in buffered 1% osmium tetroxide solution and embedded in micropal. The ultrathin sections were taken from the retina and adjacent choroidea, stained with uranyl acetate and lead citrate solutions. For the observations and photographs a Tesla-B 500 electron microscope was used. Scanning electron microscopic (SEM) investigationss were performed at fracture

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30 and 60 min. The a- and b-waves were then comparable in size and shape to those before exposure. We noticed that recovery was first seen in the b-wave, but shortly afterward the a- and c-waves were also present. The only difference we found in the E R G recovery after 1-h exposure was that in this case the maximum amplitude response was reached faster than after 4-h exposure. Comparison between stimulation with higher intensity (20 W halogen lamp) and lower intensity (LED) stimulation did not show much difference in the recordings.

surfaces of the bulb. After fixation, the specimens were dehydrated in a graded series of ethanol and critical-point-dried with liquid carbon dioxide. They were then mounted on a specimen stub, sputtered with platinum, and viewed in a SEM-BS 300 (Tesla, Czechoslovakia).

Results

Electroretinography The recordings we present here were obtained from a rabbit that was exposed for 3 h (see legend to Fig. 1). In the first recording after damage (15.40) we can see no response to stimulation. After about 10 min (15.45) a small b-wave was registered. A growing amplitude from the a-, b-, and c-waves was recorded and after about 40 min the waves were comparable to those in the control eye. Similar recordings were made in the rabbits after exposure times 1-4 h. We noticed a difference in the time in which the first wave reappeared after damage. This recovery time varied between 5 and 12 rain. The restitution time of the full ERG-wave amplitudes comparable with the control eye varied between

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No significant light microscopic or SEM changes in the retinal structure or in the continuity of the pigment epithelium (PE) could be detected in eyes continuously exposed to light when compared to the controls even after 4-h exposure. With TEM the main changes occurred in the PE (Figs. 2 and 3). Parts of the outer segments of the photoreceptors frequently occurred as phagosomes deep in the PE cell cytoplasm. Moreover, in these cells large ovoid inclusion bodies were found to be filled mainly with homogeneous material. In some of these huge inclusions we recognized electron-dense areals of the size of melanin pigment granules, as well as electron-lucent fine strips or spots. No limiting membrane could be detected with certainty around the inclusion bodies. They were usually found to be positioned in the middle part of the PE cells, pushing away the region with fine tubules of the smooth endoplasmic reticulum (ER), and they were mainly situated between the basally situated mitochondria and the apically placed melanin pigment granules (melanosomes). At the surface of the PE cells the tips of the photoreceptor outer segments were seen close to the plasmalemma (Fig. 2). In addition, phagocytosis of the outer segments (Fig. 3) as well as myelin figures or vacuoles could be frequently observed in other PE cells. Lysosomes were found to be especially abundant in the apical cytoplasm. The photoreceptors and other retinal structures were not affected.

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From the literature it is known that continuous light appears to have an early effect on the outer segments of the photoreceptors or PE [4, 10, 14, 16, 19, 20, 24, 26, 29]. These changes can be followed in vivo with electroretinograms [15, 28]. In our investigations, the deep depression recorded just after continuous light exposure disappeared during the recovery time, thus indicating a minor injury to the photoreceptors or that the damaged area is small. The role of the PE in the sequence of light-induced impairment remains controversial. The damage it receives is believed to occur prior to the destruction of the receptors [8], or to take place simultaneously [22] or subsequently [14, 20]. O'Steen and Lytle [24] and O'Steen and Anderson [23] found the PE to be unaf-

382

Fig. 2. Electron micrograph of the pigment epithelium of a damaged eye. A large inclusion body (IB) is seen in the center of the cell; outer segments of photoreceptors are attached to the cell surface. Note the melanosomes (pigment granules) in the apical cell part, the mitochondria near the cell basis, and the mass of fine, tubulous, agranular endoplasmic reticulum (ER) in the cytoplasm (bar = 1 gin)

fected and degeneration to be limited to the photoreceptors. However, ultrastructural changes in the PE that occur after continuous light exposure have been described by Shear et al. [27] as the retraction of villous processes and increasing separation by intercellular spaces between the PE cells. Our investigations demonstrate ultrastructural signs of injury caused by the light of the ophthalmoscopic surgical microscope in pigmented cells despite the good recovery o f the E R G amplitudes. These changes consisted in unusually large ovoid paraplasmic inclusion bodies in the PE cells, which might be interpreted as large lipid drops. The diameter o f these large ovoid bodies (IB in Figs. 2 and 3) was several micrometers in size and occurred as single bodies in the middle of a PE cell. Therefore

they appeared to differ quite a lot in number, structure, shape, and size from the usual phagosomes seen as a result of photoreceptor disk shedding within the PE cytoplasm. Retinal disk shedding and phagocytic activity of the PE are regularly occurring phenomena [5, 6, 9, 11, 25, 30]. D o M i n g and Gibbons [5] have described phagotized, detached outer segments in the PE as "lamellar inclusion bodies." Light with increasing intensity stimulates the phagocytosis of detached outer segments that, in the PE cytoplasm, degrade via the lysosomal system [6, 16] to become finally small vacuoles, phagosomes, small inclusion particles, and lipofuscin. A multitude of such small phagosomes and small autophagic vacuoles or lipofuscin granules have been fre-

383

Fig. 3. A pigment epithelial cell from a damaged eye in detail. Next to the large inclusion body (IB) several pigment granules (PG) and lipid droplets, secondary lysosomes, or lipofuscin granules (L) are seen in the endoplasmic reticulum (ER)-rich cytoplasm. Phagocytosis of an outer segment of a photoreceptor (0S) (bar= 1 ~m)

quently described even in normal PE of various species. Quantitative estimations have been made by Rem6 et al. [25], demonstrating a sharp increase in the number of phagosomes after illumination, especially when following a dark period. Even a circadian rhythm in the phagocytic activity of the PE has been found. Likewise, Feeny [6] described a multitude of small (about I I.tmdiameter) lipofuscin and melanin granules within the human PE. To our knowledge, large individual inclusion bodies such as we found have not been described in EM investigations in experimental eye research. So far, we have not done any quantitative estimations. The question of regional differences in the metabolic reaction of the retina has been raised by Johnson [11] who in the rabbit found degeneration and edematous swelling of retinal neurons after acute ischemia - mainly in the periphery of the retina, but also to a lesser degree in the inner region of the visual streak. Our tissue pieces for TEM were taken only from the latter, so we cannot decide if there are regional differences in the intracellular deposit of paraplasmic substances in the PE. Larger amounts of material accumulated by the PE cells have also been described by Mc Kechnie et al. [19], but only when light exposure was combined with ischemia. The material accumulated was supposed to be proteinaceous [19], containing lysosomal enzymes and also lipids [6]. Our large IB might have developed from the rapid confluence of a multitude of lipid droplets and melanolipofuscin granules within the cell. These lipids are presumed to originate mainly from the phospholipids of phagocytized outer segment disk membranes, which are rich in polyunsaturated fatty acids [1]. After longterm exposure, in the photoreceptors the light may activate oxidizing enzymes that generate free-oxygen radicals by their reactions [21], followed by rapid outer segment disk shedding and phagocytosis by the PE. Phos-

pholipids are the major constituents of highly specialized biomembranes. These phospholipids are most sensitive to free-radical reactions, and a sudden liberation of free fatty acids from membranes of the phagocytized parts of outer segments takes place within the PE cell cytoplasm. Other recent investigations also reinforce the oxidative hypothesis of light injury, with the formation of oxygen radicals initiating the sudden release of lipids freed by membrane degradation [10]. These findings support our view that the production of such large inclusion bodies is the consequence of enhanced phagocytosis of the outer segments, followed by the rapidly enhanced membrane degradation activity of the PE during the long-term light exposure. If such large amounts of paraplasmic compounds can be metabolized, it seems unlikely that this formation is then a reversible process, especially in the elderly, in whom lipofuscin deposits increase in PE cells [6]. Based on our results, it could be concluded that in some clinical situations light impairment of the PE can also occur in human patients. Therefore, the time of exposure to indirect ophthalmoscopy should be minimized to avoid potential light-induced retinal damage [18]. The same may be the case when using the stereo ophthalmoscope, which employs a similar intense light source. The danger of accelerating retinal damage with any kind of intense light is too great to be ignored. Both surgeons and anesthesiologists should be aware of the potential effect of high-oxygen levels on retinal phototoxicity [10]. Ophthalmologic devices and instruments for diagnosis and therapy should be equipped with light sources that are not dangerous for the patient. In contrast to this demand, however, our modern surgical microscopes may cause light-induced maculopathy or a "pseudophakic cystoid macular edema" [3, 12, 17, 18].

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