Experimental traumatic cataract

Transcription

1 Experimental traumatic cataract II. A transmission electron microscopy and extracellular tracer study P. P. Fagerholm and B. T. Philipson Lens changes caused by injury to the anterior part of the lens were studied toith Procion yellow as an extracellular tracer and by transmission electron microscopy at different time intervals after trauma. Both rats and rabbits were used. The findings were related to the slit-lamp appearance of the wounded lenses. In the rat lens a posterior subcapsular cataract developed within the first hour after trauma. Within 1 hr after injury the fluorescent tracer was seen at the wound but was also conspicuous at the posterior pole. Swelling of lens fiber cells and the formation of large syncytical aggregates were found as the posterior opacity enlarged. These changes reached the anterior subcapsular cortex via the equatorial cortex after about 1 month. In the rabbit lens a slight cellular swelling was seen in the subcapsular cortex. Only in one of 15 lenses a posterior subcapsular opacity developed after about 1 week in spite of a large wound. The uptake of Procion yellow was most prominent in the wound area and was never observed at the posterior pole. In both species, no further penetration of the dye occurred through the wound after the epithelium, by regeneration, had sealed the wound. The importance of epithelial wound sealing and that of a restored cellular barrier at the posterior pole are discussed as well as the significance of these factors in the cataract progression. Key words: experimental traumatic cataract, transmission electron microscopy, extracellular tracer, Procion yellow, wound healing, progression of subcapsular cataract, posterior lens barrier, species difference I erforating trauma to the anterior part of the lens always induces a local opacification at the wound. In the human lens, a subsequent opacification in the posterior subcapsular region is frequent. Earlier experimental studies have been focused on the repair of the wound From the Departments of Medical Biophysics and Ophthalmology, Karolinska Institutet, Stockholm, Sweden. This investigation was supported by grants from the Swedish Medical Research Council (Project No. 4204), Karolinska Institutet, H. Hiertas Foundation, Carmen och Bertil Regners Foundation, and The Swedish Work Environment Fund. Submitted for publication May 24, Reprint requests: Per Fagerholm, M.D., Dept. of Medical Biophysics, Karolinska Institutet, S Stockholm 60, Sweden. except for Fisher and Wakely 1 who studied late morphological changes in the rabbit lens. Wound healing after minor lens injuries has been studied by light and electron microscopy in mice 2 ' 3 and in rabbit. 4 The progressive subcapsular opacification after lens injury was studied by us in rats and rabbits with a quantitative microradiographic technique. The progressive changes in the rat lens following injury have many similarities to those found in the human traumatic cataract. 5 In the present investigation the aim was to further study the morphological and physiological characteristics of such progressive opacities in lens of the rat and the rabbit. The progression of a subcapsular cataract was followed biomicroscopically and was also studied by transmission electron microscopy /79/ $01.20/ Assoc. for Res. in Vis. and Ophthal., Inc

2 Volume 18 Number 11 Experimental traumatic cataract. II Fig. 1. Light micrograph of the wound area in a rat lens 5 min after trauma. Lens fibers protrude through the capsular defect. The capsular edge is rolled up, and epithelial cells (e) are flattened or missing adjacent to the wound. (Bar = 100 /am.) and with an extracellular tracer, Procion yellow. Materials and methods Two species, rats and rabbits, were chosen because it is known from previous studies that they react differently to lens trauma. 6 Thirty-six male Sprague-Dawley rats, initially weighing 250 gm, were used. The right lens in each animal was subjected to trauma, the left served as control. After intraperitoneal anesthesia with 50 mg/kg sodium pentobarbital and iris dilation with 0.5% tropicamide, the rats were wounded in their right eye through the cornea with a spring-suspended needle. This needle was 0.4 mm in diameter, penetrating about 0.5 mm into the lens close to the anterior pole. Thirty-six female New Zealand rabbits, initially weighing 1.5 kg, were also used. The rabbits were anesthetized with intravenously injected sodium pentobarbital at a dosage of about 40 mg/kg. The iris was dilated with 0.5% tropicamide. The right lens was injured with a knife-needle, and the left lens was used as control. Two superficial incisions were made into the lens. Each incision was about 4 mm long and about 0.5 mm deep. These cuts formed a cross with its center at the anterior pole. The eyes were examined biomicroscopieally in a Zeiss slit lamp before lens injury, during the first hour after injury, daily during the first week, and then weekly during the following month. Six animals of each species were killed by an overdose of sodium pentobarbital at each of the following intervals after injury: 5 min, 1 hr, 24 hr, 3 days, 1 week, and 1 month. The eyes were enucleated, and the lenses were carefully extracted by a posterior route under a stereomicroscope. Three pairs of lenses were immediately prepared for histological and electron microscopical examination. Fig. 2. Schematic drawing of the wound area (inner circle). The size of the area with defect or missing epithelial cells is indicated by the large circle, c, Capsule; e, epithelium. After 2 hr fixation in 4% glutaraldehyde in 0.1M phosphate buffer (ph 7.4), small specimens were cut from the wound area, the equatorial cortex, and the posterior cortex. These were postfixed in 1% osmium tetroxide in 0.1M phosphate buffer. After dehydration the specimens were embedded in Epon. Sectioning was performed on a LKB Ultrotome. Three pairs of lenses were used for Procion yellow tracer studies. Procion yellow (ICI) has a molecular weight of about 500. Its fluorescence is activated at 460 nm, and the emittance is at 550 nm. The dye was dissolved in Dulbecco's buffer, ph adjusted to 7.3. In the fluorescence microscope (Leitz Orthoplan, filter system G) the dye was seen yellow, clearly distinguishable from the light green autofluorescent lens material. Intact rat and rabbit lenses showed no uptake of Procion yellow at dye concentrations of 0.1% or less, and the most suitable concentration of Procion dye was found to be 0.01%. 7

3 1162 Fagerholm and Philipson Invest. Ophthalmol, Visual Sci, November 1979 Fig. 3. Fluorescence micrograph from the wound area of a rat lens incubated in Procion yellow, taken 5 min after trauma. Lens fibers (f) are protruding. The capsule (c) is separated from the lens fibers, and the space (s) in between contains the bright fluorescent dye. The fluorescent tracer is also seen between lens fibers and in a space filled with lens fiber debris (d). (Bar = 100 fim.) Fig. 4. Procion yellow fluorescence in a rat lens section taken adjacent to the wound. The lens was taken 5 min after trauma. The dye can be seen penetrating between the epithelial cells (e). (Bar = 100 ^m.) The lenses were incubated for 1 hr in 0.01% Procion yellow and were then prepared for fluorescence microscopy by fixation in 10% formalin for at least 2 hr. Following dehydration and embedding in paraffin the specimens were sectioned to a thickness of about 10 /xm. Unstained, deparaffinated sections were examined in the fluorescence microscope. Results Rat lens Wound area. The lens was first studied 5 min after trauma. The capsule had loosened from the lens fibers, and its edges were often rolled up. Swollen lens fibers protruded through the wound (Fig. 1). The epithelial cells were often missing up to a distance of 0.5 mm from the wound (Figs. 1 and 2). In the remaining epithelial cells the nuclei were often pyknotic, and the cytoplasm contained swollen mitochondria, myelin-like figures, and abundant ribosomes. Incubation of the lens in Procion yellow resulted in a penetration of the stain in between the detached epithelium and the lens fibers. Procion yellow fluorescence was also seen in the extracellular space along the lens

4 Volume 18 Number 11 Experimental traumatic cataract. II mcapsule Fig. 5A. Electron micrograph of epithelium close to the wound in a rat lens one hour after trauma. The remaining epithelial cells contain pyknotic nuclei (n), and the lens fiber cells are disrupted to spherical bodies (sb). (Bar = 1 ju-m.) fibers surrounding the wound (Fig. 3). Here, only the 15 to 20 most superficial fibers were surrounded with the tracer. The uptake was most conspicuous between the lens fibers and the epithelial cells (Fig. 4). One hour after trauma the wound area basically had the same microscopical appearance as immediately after trauma, but the protruding lens fibers were more swollen and membrane-enclosed spheres were seen at the free fiber ends. The epithelial cells left on the detached part of the capsule showed the same cellular changes as at 5 min after trauma (Fig. 5A). Several layers of flattened lens fiber cells were found in the subcapsular region adjacent to the wound (Fig. 5B). Twenty-four hours after trauma further breakdown of the protruding lens fibers was seen. Procion yellow leaked into all the wounded rat lenses during the first 3 days after trauma. One week after trauma epithelial regeneration had sealed the wound in all the lenses studied (Fig. 6). Outside the epithelium elongated cells were seen embedded in an extracellular substance rich in fibrous material, The regenerated epithelial cells were in close contact with each other. These cells were rounder and less regularly arranged than normal epithelial cells. Procion yellow did not penetrate through regenerated epithelium 1 week after trauma (Fig. 7). Subcapsular cortex. In lenses taken 5 min after injury, the equatorial region lens fibers and lens epithelium appeared normal. At the posterior pole the only abnormality observed was swelling of the most superficial lens fibers (Fig. 8). Still after 1 hr no pathological signs could be seen in the equatorial region, but at the posterior pole swollen lens fibers with extracellular vacuoles were observed underneath the capsule in all lenses. An opacification was then visible in the posterior subcapsular region. In the injured rat lenses, Procion yellow filled the extracellular vacuoles at the posterior pole after 1 hr, in some lenses even earlier (Fig. 9). Furthermore, the lens fibers were outlined by the tracer in the entire subcapsular region. After 24 hr, lens fiber swelling was observed at the posterior pole in the three

5 1164 Fagerholm and Philipson Invest. Ophthalmol. Visual Sri. November 1979 Fig. 5B. Electron micrograph of the same lens as in Fig. 5A, but a little more distant from the wound. Underneath the apparently intact epithelium (e) a layer of swollen lens fibers (f) are seen. Deeper into the cortex the lens fibers are extremely flattened. (Bar = 1 jam.) 5B Fig. 6. Light micrograph of the wound area in a rat lens 1 week after trauma. Regenerating epithelial cells cover the wound. (Bar = 100 /nm.) Fig. 7. Fluorescence micrograph from the wound area of a lens, taken 1 week after trauma. Procion yellow is seen in the extracellular space of the scar tissue (t). The lens epithelium (e) has regenerated underneath the scar tissue. The epithelium is not penetrated by Procion yellow. (Bar = 1 fj-m.)

6 Volume Numbe, Experimental traumatic cataract. II Fig. 8. Electron micrograph from the posterior pole of a rat lens 5 min after trauma. Lens fibers immediately inside the capsule (c) appear irregular and swollen. (Bar = 1 /u.m.) lenses studied and was very extensive in two of them. The equatorial region remained unaffected. Large extracellular spaces filled with fluorescent dye were seen underneath the capsule of the posterior pole after 3 days. After 1 week the opacification had reached the equatorial subcapsular cortex. The lenses appeared different morphologically. In one of the three lenses large, swollen lens fiber cells filled the entire posterior subcapsular region (Fig. 10). In another lens a large space, free of cells, was formed at the posterior subcapsular region (Fig. 11). In the third lens a multilayered formation of regenerated lens cells covered the posterior capsule (Fig. 12). In these three lenses extracellular vacuoles were observed in between the superficial lens fibers, being most prominent in the equatorial region. In the posterior cortex and in the equatorial cortex from the three lenses, immense lens fiber cells were seen. Discontinuities of varying size in the membranes between adjacent lens fibers were frequently found here (Fig. 13). Through these membrane discontinuities the cytoplasm from adjacent lens fibers was continuous, and consequently large syncytical cells were formed. The discontinuities appeared as holes when serial sections were examined. They were usually found between already enlarged cells. At the equator these syncyticallike aggregates were always separated from the capsule by nuclei-containing lens fibers, apparently newly formed. The uptake of Procion yellow 1 week after injury was still prominent at the posterior pole in the large

7 1166 Fagerholm and Philip son Incest. Ophthalmol. Visual Sri. November 1979 Fig. 9. Fluorescence micrograph from the posterior subcapsular region of a rat lens, taken 5 inin after trauma. Minor changes in the form of slightly swollen cells and an increased extracellular fluorescence are seen in the most superficial layers. (Bar = 100 /u.m.) Fig. 10. Light micrograph from the posterior pole of a rat lens 1 week after trauma. Swollen lens fibers are seen in the outermost 0.5 mm of the cortex. (Bar = 100 yum) extracellular spaces underneath the capsule in two of the four lenses studied. One month after trauma the lenses had basically the same appearance as after 1 week. Lens fiber changes such as swelling and fusion of cells were seen in the entire subcapsular cortex (Fig. 14). Furthermore, in one of the three lenses examined, regenerated nuclei-containing lens cells were observed. Rabbit lens Wound region. The morphological appearance in the wound region was similar to that observed in the rat lens. The wound was sealed 1 month after trauma by a single layer of almost normal-appearing epithelial cells. Fig. 11. Light micrograph from the posterior pole of a rat lens 1 week after trauma. In a space (s) inside the capsule (c), cells are missing. The subcapsular lens fibers are extensively swollen. (Bar = 100 /xm.) On the inside of regenerated epithelium normal-appearing lens fibers were seen. Further into the lens about 0.2 mm deep, swollen and irregular fibers were found. Procion yellow leaked into all the wounded lenses during the first week, but no uptake could be detected 1 month after injury or after histological wound sealing. Subcapsular cortex. In the posterior subcapsular cortex signs of slight swelling of the two to three outermost lens fiber layers were observed after 24 hr in all lenses (Fig. 15). The equatorial and the anterior cortex, except for the region around the injury, appeared identical to the corresponding regions in the control lenses.

8 Volume 18 Number 11 Experimental traumatic cataract. II <Y:<,?>- Fig. 12. Light (A)'and electron (B) micrograph from the posterior pole of a rat lens 1 week after trauma. A, Multilayers of nuclei containing cells are seen on the inside of posterior lens capsule (c). Deeper into the lens cortex a more or less structureless space (s) is observed. (Bar = 10 /urn.) B, In the nuclei-containing cells very few cell organelles are seen. (Bar = 1 /xm.) 13C X Fig. 13. Electron micrographs from the equatorial region in a rat lens 1 week after trauma. A, Aggregates of lensfibersprobablyformedby swelling and cellular fusion are seen. (Bar = 1 /xm.) B, and C, Cell membranes between a cell aggregate and the adjacent lensfibersin higher magnification. Discontinuities in the membranes are seen. (Bar = 1 fim in B and 0.1 /xm in C.) 13C No uptake of Procion yellow could be detected at the posterior pole even after 24 hr and in spite of the large trauma (4 by 4 mm). Here, the tracer penetrated at most just beyond the equatorial region where slightly swollen cells could be seen. In the superficial subcapsular cortex 1 week after trauma, vacuoles were observed in all the lenses and in all lens regions but were most numerous in the equatorial region (Fig. 16). A slight swelling of the equatorial and the anterior subcapsular lens fibers was also observed. The posterior cortex appeared then almost normal, but a slight swelling could be observed in the two to four outermost lens fiber layers. However, in one lens, more extensive fiber changes were seen (Figs. 17A and 17B). In the anterior cortex about 0.1 mm inside the capsule, swollen lens fibers with abnormal membrane formations could be seen at various distances from the wound in all three lenses. Generally similar cellular changes were observed 1 month after injury. Discussion The injury of the lens had to be of a certain optimal size in order to induce a progressive pacification, similar to the posterior subcap-

9 1168 Fagerholm and Philipson Invest. Ophthalmol. Visual Sri. November 1979 Fig. 14. Light micrograph from the anterior subcapsular region of a rat lens 1 month after trauma. Large cells irregularly arranged and vacuoles between superficial lens fibers are seen. The lens fiber cells frequently contain nuclei. (Bar = Fig. 15. Light micrograph from the posterior region of a rabbit lens 1 hr after trauma. A swelling of the outermost lens fiber layers is noted. It is seen that the fibers in the outermost one to two cell layers are enlarged and irregularly arranged compared to the hexagonal cortical cells. (Bar = 100 (im.) sular cataract seen in the human lens after trauma. 5 In the rabbit the injured area of the anterior lens surface had to have a diameter of about 4 mm, exceeding that in the rat lens by 10 times. Even so, most of the rabbits did not develop typical posterior subcapsular cataracts. The area with defect or missing epithelial cells was found to be much larger than the area actually injured (Fig. 2). In the rat lens this meant a doubling of the diameter and, consequently, an increase by a factor of four of the initial epithelial wound area. Through this enlarged epithelial wound Procion yellow was able to penetrate into the lens. Furthermore, the biomicroscopically visible opacity associated with the wound had approximately the same size as the region exhibiting morphological changes, the area showing the functional defect, and the region with reduced concentration of dry mass. 6 In the rat lens a subcapsular opacification developed in the posterior region within 1 hr after trauma. The appearance of this subcapsular cataract was biomicroscopically similar to that of the human traumatic cataract. The subcapsular changes consisted of swollen lens fiber cells, formation of large syncytical aggregates, large spaces free of cells, or regenerated lens cells. These changes, with the exception of the regenerated lens cells, corresponded to a marked reduction of dry mass found microradiographically 6 in the entire subcapsular cortex. In the rabbits, followed 1 week or more, only one posterior subcapsular cataract developed in the 12 lenses examined. However, the subcapsular lens fibers showed moderate swelling, which was in agreement with the microradiographically observed slight hydration. 6 The zonular opacity visible about 0.1 mm into the cortex consisted of swollen and irregular lens fibers cor-

10 Volume 18 Number 11 Experimental traumatic cataract. II capsule 16A Fig. 16. Light (A) and electron (B) micrographs from the equatorial region of a rabbit lens 1 week after trauma. A, Numerous vacuoles are seen in the subcapsular layers. (Bar = 100 pm.) B, Epithelial cells showing that large vacuoles to be extracellular. (Bar = 1 fim.) responding to marked reduction of dry mass determined microradiographically. This zone of cellular damage probably corresponds to the zonular opacification examined in the electron microscope by Fisher and Wakely 1 in rabbit lenses 3 years after trauma. Biomicroscopically visible opacities in injured lenses from both rats and rabbits always corresponded to marked cellular changes and a reduction in dry mass concentration. This is probably caused by an increased cellular water uptake resulting in lens fiber damage. In the wounded rat and rabbit lenses the extracellular tracer dye was confined to the outermost 10 to 15 layers of lens fibers, except in the region adjacent to the injury. The reason for this limit to dye penetration is not known. A combination of the following two morphological explanations seems most likely: (1) a gradually diminished extracellular space towards the lens nucleus, 8 and (2) very slow penetration of the dye due to abundant cell junctions. The diffusion of the dye within the extracellular space along the subcapsular lens fibers was rapid in both rat and rabbit lenses. This is consistent with an earlier study by Paterson, 9 using radioactive tracers. The uptake of Procion yellow in both rat and rabbit lenses was most prominent in areas with morphological changes, reflecting an enlargement of the extracellular space. After healing of the traumatized area by regenerating epithelium, Procion yellow could not penetrate into the lens from the anterior side. Procion yellow uptake could be seen in some of the rat lenses even after epithelial wound sealing. However, this was due to leakage through the posterior side of the lens. In two of six rat lenses cell regeneration was observed covering the posterior capsule. This probably is an attempt by the epithelium to heal the posterior lens barrier. A similar cell migration has been seen in human lenses with posterior subcapsular cataract 10 and in rat lenses after x-ray irradiation. 11 This indicates that in the injured rat lens the posterior barrier which offers resis-

11 **%*«. : * ' 1170 Fagerholm and Philipson Invest. Ophthalmol. Visual Sci. November 1979 capsule Figs. 17A and 17B. Electron micrographs from the posterior pole of a rabbit lens 1 week after trauma. Fig. 17A. Superficial lens fiber cells contain intracellular vacuoles of varying size. (Bar = 1 /im.) 17A We gratefully acknowledge the expert technical assistance of Christina Lindqvist. REFERENCES 17B Fig. 17B. Formation containing ribosomes and mitochondria is shown in detail. (Bar =0. tance to ionic movement 9 is often damaged during the first week. Leakage through this defect barrier explains the progression of the subcapsular cataract in spite of the anterior wound healing. 1. Fisher, R.F., and Wakely, J.: Changes in lens fibers after damage to the lens capsule. Trans. Ophthalmol. Soc. U.K. 96:278, Rafferty, N.S., and Gossens, W.: Ultrastructural studies of traumatic cataractogenesis: observations of a repair process in mouse lens. Am. J. Anat. 142:177, Nelson, K.J., and Rafferty, N.S.: A scanning and electron microscopic study of lens fibers in healing mouse lens. Exp. Eye Res. 22:335, Unakar, N.J., Harding, C.V., Reddan, J.R., and Shapiro, R.A.: Characterization of wound healing in the rabbit lens. I. Light and electron microscope observations. J. Microsc. 16:309, Fagerholm, P.P., and Philipson, B.T.: Human traumatic cataract. A quantitative microradiographic

12 Volume Numbe Experimental traumatic cataract. II and electronmicroscopic study. Acta Ophthalmol. 57:20, Fagerholm, P.P., and Philipson, B.T.: Experimental traumatic cataract. I. A quantitative microradiographic study. INVEST. OPHTHALMOL. VISUAL SCI. 18:1151, Fagerholm, P.P.: Procion yellow as an extracellular tracer in cataract research. Manuscript. 8. Paterson, C. A.: Extracellular space of the crystalline lens. Am. J. Physiol. 218:797, Paterson, C.A.: Anteroposterior cation gradients in bovine lenses. INVEST. OPHTHALMOL. 12:861, Streeten, B.W., and Eshaghian, J.: Human posterior subcapsular cataract. A gross and flat preparation study. Arch. Ophthalmol. 96:1653, Worgul, B.V., Merriam, G.R., Jr., Szechter, A., and Shrinivasan, B.D.: Lens epithelium and radiation cataract. I. Preliminary studies. Arch. Ophthalmol. 94:996, Copyright information The appearance of a code at the bottom of the first page of an original article in this journal indicates the copyright owner's consent that copies of the article may be made for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., P.O. Box 765, Schenectady, N.Y , /518/ , for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale.