Rat Cataract Induced by Suckling Mouse Cataract Agent

RAT CATARACT INDUCED BY SUCKLING MOUSE CATARACT AGENT H.

FRED CLARK, P H . D .

Philadelphia, Pennsylvania

The titer was 106'3 to 106·9 egg lethal doses so/ml. Rats. Pregnant Wistar-strain albino rats were provided by the animal care unit, State University of New York at Buffalo. New­ born rats (less than 48 hours old) were inoc­ ulated with 0.02 ml of suckling mouse cata­ ract agent into the left cerebral hemisphere. Eye examinations were initiated when the eyelids first opened at about 16 days of age and were continued at regular intervals thereafter. Eyes were examined by "naked eye," using oblique illumination with a mi­ croscope illuminating lamp (American Opti­ cal Company, model No. 651). Selected pairs of rats were killed for viral studies. Whole blood was diluted in phos­ phate-buffered saline, brain and liver-spleen tissues were triturated as 10% suspensions (w/v) in phosphate-buffered saline and each whole eye was triturated in 4.0 ml of phos­ phate-buffered saline. Each eye and blood sample was treated separately, while the brains and liver-spleen samples of each pair of animals were pooled. All specimens were assayed for suckling mouse cataract agent MATERIALS AND METHODS content by titration of the egg-lethal endSuckling mouse cataract agent. Propagation point in seven-day embryonated eggs. of the agent has been described in detail else­ RESULTS where.4 Briefly, seven-day embryonated EYE DISEASE IN RATS. eggs were inoculated into the yolk sac with The cataract incidence observed in rats in­ suckling mouse cataract agent. Allantoic fluid harvested when the embryos died, after four oculated as newborns with suckling mouse to six days of incubation, comprised the virus cataract agent is shown in Table 1 ; 97.5% stock. Virus used in this study had a history of the inoculated rats developed cataracts. of 11 or 12 passages in embryonated eggs. The majority were affected bilaterally, as re­ flected by a 94.0% incidence of cataracts in From the Department of Pediatrics, School of all eyes observed. No cataracts were ob­ Medicine, State University of New York, Buffalo. This study was supported by Research Grant Al- served in control animals inoculated with 6227 from the National Institute of Allergy and In­ normal allantoic fluid. fectious Disease. Cataracts appeared early, with 68% of Reprint requests to H. Fred Clark, Ph.D., Wistar Institute of Anatomy and Biology, Philadelphia, eyes showing gross changes by 17 days and Pennsylvania 19104. ψο of eyes affected by 19 days after inocu-

The suckling mouse cataract agent (SMCA) is an egg-lethal virus isolated from rabbit ticks in Georgia in 1961.* When inoc­ ulated by the intracerebral route into new­ born mice, this agent causes a high incidence of cataract development, preceded by retinitis and posterior uveitis. 2 ' 3 The eye disease is accompanied by high levels of virus infec­ tion in the eye and lifelong persistent infec­ tion of the brain. 4 ' 5 Suckling mouse cataract agent is not naturally transferred to the progeny of infected dams, but such progeny are protected against the cataractogenic ef­ fects of a challenge inoculation, apparently because of maternally acquired antibody.6"8 The highest SMCA-induced cataract rate obtained in albino strains of mice (eye ex­ amination without mydriasis) was approxi­ mately 45%. 4 The inefficiency of cataract in­ duction and the small size of the mouse eye limit the usefulness of this system as a model for studies of experimental cataract. This report describes superior results ob­ tained in newborn rats inoculated with suck­ ling mouse cataract agent.

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TABLE 1 INCIDENCE OF CATARACTS IN SMCA-INOCULATED NEWBORN RATS*

Inoculum Suckling mouse cataract agent Normal allantoic fluid

No. Litters

No. Rats

No. Rats with Cataract (s)

% Rats with Cataracts

% Total Eyes With Cataracts

22

118

115

97.5

94.0(218/

6

20

0

0.0

0.0(0/

232)

40)

* Incidence of cataracts observed at 25-35 days postinoculation in rats inoculated intracerebrally at age <48 hours with 1046 egg lethal doses5o of SMCA or, as a control, normal allantoic fluid (NAF).

lation. As in mice, the initial cataract was usually a peripheral opacity of the lens. The peripheral lesion progressed rapidly to a dense uniform opacity of the entire lens (fig. 1). This dense type of cataract appeared ear­ lier and much more consistently than in even the most susceptible mouse strain (pigmented C57/B1 mice). 3 The incidence of dense cataracts reached 50% by 21 days after inoculation, while 98% of diseased eyes progressed to this stage by 25 days after inoculation. In contrast, in hypersusceptible C57/B1 mice only 50% of cataracts became so severe and then only at 25 to 40 days postinoculation.3 In addition to cataracts, severe peribulbar inflammation frequently developed in the in­ oculated rats. Affected rats exhibited a gross swelling of the adnexa and eyelids that com­ monly completely prevented visualization of the bulbus oculi (fig. 2). This condition was

detected in 6.5% (16 of 246) of eyes at 25 to 30 days after inoculation. In nine litters of rats observed for several months, the inci­ dence of peribulbar disease increased from 3.1% (four of 139 eyes) at 30 days to 31.2% (38 of 122 eyes) at 60 to 90 days after inoculation. Similar lesions were not observed in control rats, and have been de­ tected only rarely in mice inoculated with suckling mouse cataract agent. Histopathologic studies of SMCA-induced eye disease in rats, performed by Drs. John Sheffer and Indira Kartha, will be re­ ported in detail elsewhere.2 Briefly, as in mice, an initial retinitis was followed rapidly by the development of posterior uveitis and cataract. At variance with the mouse disease was the occurrence of actual polymorphonuclear leukocytic infiltration of the lens, often progressing to abscess of the lens. Further­ more, the intensity of keratitis and anterior

Fig. 1 (Clark). Eye of rat inoculated with suckling mouse cataract agent, showing densi: cataract 52 days after inoculation.

Fig. 2 (Clark). Eye of rat inoculated with suckling mouse cataract agent, showing blepharitis and in­ flammation of adnexa 114 days postinoculation.

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TABLE 2 RECOVERY O F SMCA FROM THE TISSUES OF RATS INOCULATED INTRACEREBRALLY AS NEWBORNS WITH A DOSE OF APPROXIMATELY 10 4 ·' E L D 5 0

SMCA Titers*

Rat No.

Day Killed

175,176

2

6.6

4.7

n.d.f

2.4 2.1

2.1 2.3

163,164

4

7.3

5.5

n.d.

3.4 2.7

3.1 2.3

258,259

7

7.3

4.7

2.4 4.2

3.4 n.d.

4.3 n.d.

177,178

7

6.5

5.9

n.d.

7.1 6.4

7.3 6.9

264,265

11

8.1

5.7

<1.0 <1.0

6.7 6.2

6.7 7.4

165,166

15

7.5

6.5

<0.6 <0.6

6.3 6.1

5.4 6.5

246,247

25

5.0

1.8

<0.6 <0.6

5.3 3.7

4.1 3.5

36,37

90

3.9

<0.9

<0.6 <0.6

2.1 2.4

<0.5 1.7

72,73

180

3.8

<0.9

<0.6 <0.6

<0.5 <0.5

<0.5 <0.5

Brain

LiverSpleen

Blood

Left Eye

Right Eye

* Egg-lethal dosesso, logic per gm of tissue, ml of blood, or per whole eye. Brain and liver-spleen titers represent pooled tissues from two animals. t n.d.: Not determined.

uveitis in cataractous rat eyes was much higher than that previously observed in mice. Histologie observations of eyes with peribulbar inflammation revealed panophthalmitis with inflammation of the adnexa. Some eyes progressed to phthisis bulbi within a few months. Other eyes were apparently completely destroyed by the infection and subsequently resorbed. Rats inoculated with suckling mouse cata­ ract agent did not exhibit any excessive mor­ tality when compared to control animals. Minor symptoms of central nervous system disease, primarily circling and hyperexcitability, developed occasionally, as in mice. VIRAL STUDIES

The quantities of virus recovered from the eyes and viscera of SMCA-inoculated rats are listed in Table 2. Brain infection

was present in high titer by two days after inoculation, reached a peak level by 11-15 days and then diminished to a lower persis­ tent level. Liver-spleen tissues were consis­ tently infected from two through 15 days, but were clear of suckling mouse cataract agent by 25 days. Viremia was detected at seven days postinfection, but was absent at 11 days and thereafter. Suckling mouse cataract agent was recov­ ered from all eyes tested between two and 25 days after infection. Peak levels of eye in­ fection were detected from the seventh to the 15th day. During this period, a majority of eyes yielded from 10 60 to 10 7 " ELD 50 of virus each. Eye infection persisted for 90 days, but was not present at 180 days. This pattern of growth of suckling mouse cataract agent was similar to that previously observed in both highly susceptible4·5 and in

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relatively resistant6 strains of mice. The time of appearance of peak levels of infection in eyes and viscera was also essentially the same as in mice. However, while the titers of suckling mouse cataract agent in rat brains and viscera were similar to those found in susceptible strains of mice, the ti­ ters recovered from rat eyes were slightly higher than any detected in mouse eyes. Rat eyes yielded up to 107·4 ELD 50 . This is ap­ proximately 10 times the maximum level of infection detected in an infected mouse eye.6 DISCUSSION

It has been proposed that the SMCAsuckling mouse system may provide a useful model for the production of cataracts for ex­ perimental study. 1 ' 3 Such a system would be valuable for information which might be referrable to human virus-induced cataracts, such as may occur following infection with rubella virus,9 cytomegalovirus10'11 or the virus of Behçet's disease.12'13 A system capa­ ble of causing experimental cataracts may also be of use for basic studues on the bio­ chemistry and physiology of the cataractous lens in general. The possible usefulness of the suckling mouse cataract agent cataract-inducing sys­ tem is greatly enhanced by the potential for producing cataracts in newborn albino rats with an efficiency of greater than 95%, com­ pared to a maximum rate of about 45% in albino mice. An added feature of the rat dis­ ease is the frequent (about 30% at 60-90 days postinoculation) occurrence of a more severe syndrome, a panophthalmitis often leading to phthisis of the eye, following cata­ ract formation. As in mice, SMCA-inoculated rats devel­ oped an acute visceral infection, a chronic brain infection and eye infection persisting for about three months. The only striking difference between this pattern of infection and that seen in mice was a higher peak titer of suckling mouse cataract agent detected in eyes of rats. These higher levels of eye in­ fection in rats may explain the very high

cataract incidence obtained. A rough correla­ tion between maximum levels of SMCA eye infection and cataract rate has been noted in comparative studies of different strains of mice.1'2'8 SUMMARY

Inoculation of newborn albino rats with the suckling mouse cataract agent caused cataract(s) in 97.5% of test animals. This incidence of cataracts exceeds that observed in any strain of mice examined to date. SMCA-inoculated rats developed severe uni­ formly opaque cataracts consistently (98% of diseased eyes) and at an early age (by 25 days after inoculation). Subsequently, about 30% of rats developed severe inflammation of the eyelids and adnexa. Histopathologic studies of affected eyes revealed an initial posterior uveitis followed by retinitis and cataract development. The disease often progressed further to pan­ ophthalmitis and phthisis bulbi. Viral studies revealed an acute visceral infection and a chronic infection of the rat brain. Suckling mouse cataract agent was recovered from all eyes of rats killed between two and 25 days of age. A particularly high concentration of virus recovered from eyes between seven and 15 days after inoculation may explain the high incidence and severity of eye dis­ ease observed. REFERENCES

1. Clark, H. F. : Suckling mouse cataract agent. J. Infect. Dis. 114:476, 1964. 2. Kartha, I., Sheffer, J. and Clark, H. F. : His­ topathologic studies of SMCA-induced eye disease in rats. In preparation. 3. Olmsted, E., Prasad, S., Sheffer, J., Clark, H. F. and Karzon, D. T. : Ocular lesions induced in CS7 mice by the suckling mouse cataract agent (SMCA). Invest. Ophth. 5:413, 1966. 4. Clark, H. F. and Karzon, D. T.: Suckling mouse cataract agent (SMCA) in mice: 1. Factors affecting the incidence of cataracts and growth of virus in several strains of mice. J. Immunol. 101:776, 1968. 5. growth curve studies of the suckling mouse cataract agent in individual compartments of the eye. Proc. Soc. Exp. Biol. Med. In press. 6. Suckling mouse cataract agent (SMCA) in mice: II. Transfer of protection to progeny of

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SMCA-infected dams. J. Immunol. 101:782, 1968. 7. ■ Suckling mouse cataract agent (SMCA) in mice: III. The antigenic specificity of maternal protection against SMCA and the comparative abil­ ity of other viruses to replicate in the eye of the suckling mouse. J. Immunol. 101:890, 1968. 8. Demonstration of neutralizing antibody to the suckling mouse cataract agent (SMCA). Proc. Soc. Exp. Biol. Med. In press. 9. Gregg, N. M. : Congenital cataract following German measles in the mother. Tr. Ophth. Soc.

AUGUST, 1969

Australia 3 :35, 1941. 10. Christensen, L., Beeman, H. W. and Allen, A. : Cytomegalic inclusion disease. Arch. Ophth. 57 :90, 19S7. 11. Dvorak-Theobald, G. : Cytomegalic inclusion disease: Report of a case. Am. J. Ophth. 47:52, 1959. 12. Sezer, N. : Further investigations on the virus of Behçet's disease. Am. J. Ophth. 41:41, 1956. 13. Fenton, R. H. and Easom, H. A. : Behçet's syndrome. Arch. Ophth. 72.71, 1964.

ANTERIOR CHAMBER GLASS MEMBRANES LEWIS LAURING, M A J . (MC)

USA

San Francisco, California

New formation of glass membranes in the anterior chamber has been described as oc­ curring in a number of ocular diseases. There is little information, however, on their incidence. It is the purpose of this study to determine the diseases in which formation of glass membranes is most prevalent, and to correlate their pathologic characteristics with clinical features. Wangenmann 1 first recognized that the corneal endothelium could proliferate beyond Schwalbe's ring over the trabecular meshwork and iris. He described a blind, glaucomatous eye in which the endothelium had secreted a Descemetlike membrane covering a peripheral anterior synechia. Subsequently, Herbert 2 described similar new-formed glass membranes in eyes with iridocyclitis. He was impressed with the ability of the endo­ thelium to grow over "free surfaces" which had been covered with chronic inflammatory tissue. Reese3 studied pathologic material containing Descemetlike membranes and found that all the eyes were glaucomatous with deep chamber angles. He suggested that the "cuticular" membrane was a product of the trabecular endothelium and could be pro­ duced by primary trabecular disease, ante­ rior segment inflammation or trauma. ReinFrom the Eye Pathology Laboratory, Department of Ophthalmology, University of California. Reprint requests to Maj. Lewis Lauring, MC, Letterman General Hospital, San Francisco, Cali­ fornia 94129.

forcing Herbert's impression, Cogan4 wrote : The frequent finding of endothelium extending onto the surface of the iris in the presence of pe­ ripheral anterior synechiae may be looked upon as a special case of this propensity for the endothelium to attempt to surround and "enclose" anything in contact with it.

Wolter and Fechner 5 described similar glass membranes in five enucleated eyes and reviewed the literature. Since all their cases showed peripheral anterior synechiae, these authors concluded that this change was prereauisite for endothelial proliferation. Wolff and Zimmerman,6 in discussing monocular glaucoma in traumatic eyes with anteriorchamber angle recession, showed that a Des­ cemetlike membrane could be secreted by the endothelium in the absence of peripheral an­ terior synechiae and accompanied contusion angle deformity. MATERIALS AND METHODS

A total of 136 eyes with primary diag­ noses of contusion, angle-closure glaucoma or iridocyclitis were received at the Eye Pa­ thology Laboratory, University of Califor­ nia, from 1964-1968. These eyes were reexamined for the specific changes of endothelial proliferation and secretion of Descemetlike membranes posterior to Schwalbe's ring (fig. 1). Eyes with perforating injury or rupture of the globe were excluded, as endothelial proliferation is occasionally seen where there has been stromal overgrowth. A second se-