|Year : 1981 | Volume
| Issue : 2 | Page : 91-96
Effect of endotoxins on rabbit lens
SK Angra, S Kunnatur, RL Mathur, M Mohan
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, A.I.I.M.S., New Delhi, India
S K Angra
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, A.I.I.M.S., New Delhi-110029
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Angra S K, Kunnatur S, Mathur R L, Mohan M. Effect of endotoxins on rabbit lens. Indian J Ophthalmol 1981;29:91-6
| Introduction|| |
Cataracta complicata, was a concept introduced by Otto Becker (1879) to embrace the lenticular changes which frequently appear in various ocular diseases and, in general are characterized by punctate, striate or diffuse opacities often accompanied with a polychromatic luster. It has been believed that in majority of cases, such cataracts are presumably due to the effect of toxins either front the inflammatory focus or from the products of degeneration caused by diseases, and owing to the thinness of the posterior capsule and its lack of supporting epithelial barrier, the earliest clinical changes are typically seen in the region of the posterior pole. Posterior subcapsular changes in rabbit lens after intravitreal injection of horse serum, and Shigella flexneri endotoxin have been observed., As far as we know the effect of other autotoxins on crystalline lens of rabbits have not been reported earlier. In the present investigation we have studied the effect of eadotoxins released from E. coli, C. albicans and E. histolytica on the transparency of rabbit crystalline lens. We have determined the contents of sodium, potassium, and reducing sugars in the lenses of those rabbits which showed lenticular opacities
| Materials and methods|| |
Pigmented as well as albino rabbits weighing between 2-2.5 Kg were used for this study. Pure cultures of E. coli and C. albicans were isolated from human material. Suspensions in saline were prepared corresponding to No. 10 tube of turbidometer. The suspensions were kept in boiling water bath for 30 minutes, cooled and centrifuged at 20,000 'g' for 30 minutes. The supernatant was used for intravitreal and suprachoroidal injections. Ultrasonicated antigen of E. histolytica was obtained from Microbiology Department of our Institute. The preparation had a protein content of 1.366 mg/ml.
Intravitreal and suprachoroidal injections of the different extracts were given by anaesthetizing the animals with intravenous injection of sodium barbitone in a doze of 25mg/kg body weight. Xylocaine 4% was used for topical anaesthesia. Three such injections of different toxins used, were given at weekly intervals to the respective animal.
| Clinical examination|| |
All the animals were subjected to external, biomicroscopic and ophthalmoscopic examination by dilating the pupils with homatropine 2% drops. The animals were examined twice a week for a period of 60 days.
At the end of the period of observation, the animals were sacrificed by air embolism and aqueous was withdrawn by a tuberculin syringe with 26 gauge needle. The eyes were then enucleated and leas and vitreous were dissected out for further analysis.
Estimation of biochemical parameters :
Lenses were homogenized in 5% trichloroacetic acid and the homogenate centrifuged for 30 minutes at 20,000 'g'. The supernatant was used for estimation of sodium and potassium and the pellet was dried at 60° C for 8 hrs and weighed. Lens water was calculated by deducting the dry weight of pellet from the wet weight of lens as described by Barber, 1958. Serum, aqueous humour and vitreous body were used as such for sodium and potassium estimations. The sodium and potassium contents were determined by flame photometery.
Reducing sugars were estimated in separate lenses by the method of Fplin and Wu as cited by Varley (1963).
| Observations|| |
Examination of the animals after intravitreal injections of 0.1 ml of saline, E. coli, C. albicans and E. histolytica extracts revealed no positive abnormalities in cornea, anterior chamber, iris and pupillary reflex. A mild to moderate conjunctival congestion was observed in all the animals at the site of injection which disappeared within 1-2 weeks. A mild to moderate vitreous flare was noticed in all the animals except the control group. The vitreous flare disappeared by 5th week in the C. albicans group but persisted in 50% animals in which E. colt and E. histolytica extracts were injected. The lenses remained clear in all the groups till the 5th week of first injection but at the end of 6th week, haziness of the posterior subcapsular region, in the eyes of rabbits given E. coli extracts, were seen. By the end of 8th week, granular opacities in the posterior sub-capsular region were seen [Figure - 1][Figure - 2] in eight out of ten eyes. No haziness or opacities were seen in the lenses of those eyes which were given intravitreal injections of C. albicans and E. histolytica extracts. Similarly, suprachoroidal injections of different extracts did not show any change in cornea or signs of anterior uveitis. Conjunctival congestion subsided in 1-2 weeks and mild to moderate vitreous flare was present up to 8 weeks. However, in the E. histolytica group. the vitreous flare was seen in the initial stages. At the 6th week of observation a definite irregular opacity around the lens suture, granular in appearance with ill defined margins was seen in 4 out of 10 eyes in the E. coli group. By the end of 8th week two lenses showed haziness in the posterior subcapsular region and four lenses showed a granular opacity.
| BIOCHEMICAL OBSERVATIONS:|| |
Our data on the contents of sodium in the lens, aqueous, vitreous and serum of control group shows a selective distribution of this cation in these tissues and body fluids [Table - 1] Similarly, potassium is not distributed in equal concentrations but does not follow the same distribution pattern as sodium. This becomes more evident when we compare the sodium: potassium ratio which is 39.74; 0.21; 33.82 and 26.72 fot the crystalline lens, aqueous humour, vitreous body and serum respectively. The contents of reducing sugars are very low in the vitreous followed by crystalline lens, aqueous and serum in the increasing order [Table - 1].
The contents of sodium in the crystalline lens are increased by about 2.6 and 3.2 times after intravitreal and suprachoroidal injections of E. coli extracts respectively. On the other hand, a decrease of about 23% in the sodium contents of vitreous is observed [Table - 1]. The potassium contents remain unaltered in all the tissues studied by us.
There is a slight increase in the contents of reducing sugars of aqueous humour and vitreous body but no statistically significant change is observed in the crystalline lens and serum of rabbits given either intravitreal or suprachoroidal E. coli extract [Table - 1].
| Discussion|| |
In the present study, we have undertaken the work to see the effect of various toxins from different organisms, each representing a class of bacteria, fungus and protozoae. This appeared important in view of the fact that among the patients with cataract seen in our lens clinic, positive stool examinations (parasites) were present in 48.7% of children with cataract where no other definite etiology could be found. However, in the group of children with cataract of known etiology, positive stool examinations were present in only 23.3% subjects. In cases of cataract having typical features of complicated cataract, it is not always possible to demonstrate a causative ocular pathology. Such cases may have a subclinical disease. However, it is also possible that in such cases the toxins liberated by organism present in the body act on the lens directly or indirectly through structures like the uvea thereby leading to a cats-act.
In our experiments, we observed conjunctival congestion in all rabbits. The congestion was more after suprachoroidal injection than after intravitreal injection. The congestion in any group was comparable with the route of injection. So it can be surmised that the trauma of incising the conjunctiva prior to suprachoroidal injection was the factor responsible for more congestion after this route.
The cornea remained essentially clear and anterior uveitis was not present as shown by the absence of iritis, the pupil being round, briskly reacting to light and fully dilating on applying homatropine 2% eye drops.
Lenticular changes in the posterior subcapsular area were present in 60% of the eyes receiving a suprachoroidal injection of E.coli extract and in 80% of the eyes receiving an intravitreal injection of E. coli extract by the end of the period of observation. It is interesting to note that no lenticular changes were observed in any of the other eyes which received saline, C. albicans or E histolytica extract either suprachoroidally or intravitreally.
The vitreous showed a mild to moderate flare in all the eyes injected with the extracts of E. coli, C. albicans or E. histolytica. Although it has been reported that intravitreal injection of horse serum produced subcapsular changes in the posterior region of rabbit lens, but the opacities produced were not well defined. Intravitreal injection of Shigella flexneri endotoxin has been shown to produce lenticular opacities in mice, initially in the posterior subcapsular region, but the eyes showed severe endophthalmitis and progressed to pthisis bulbi 4. It is suggested that the lenticular changes produced by E. coli extract were not solely due to the uveitis induced, as C. albicans and E. histolytica extract produced comparable uveitis but no lenticular changes whatsoever. The toxins present seem to have acted directly in addition through the mechanism of uveitis.
Our observations on the contents of sodium, potassium and reducing sugars in the crystalline lens and aqueous humour of the rabbits in control group are in good agreement with the earlier reported values. A marked increase of sodium contents in the crystalline lens after intravitreal and suprachoroidal injection of E. coli extract is accompanied with a decrease of this cation in the vitreous body [Table - 1]. The contents of potassium on the other hand remain unchanged. The sodium ions penetrate the posterior surface of the crystalline lens more rapidly than potassium. Therefore, in our study, the gain of sodium in the lens is reflected by a simultaneous loss in the vitreous body whereas aqueous humour does not show such alteration. The loss of lens potassium is suggested to be a late manifestation of cataractogenesis.
The contents of reducing sugars are increased by about 32% and 39% in aqueous humour and vitreous body respectively after suprachoroidal injections of E, coli extract ([Table - 1], p < 0.01). In vitro studies have shown the formation of cataracts when lensvitreous preparations were incubated in medium containing glucose. It seems reasonable to conclude that the injections of E. coli extract bring about alterations in the lens environment which may have harmful effects on the posterior capsule of the lens resulting in an influx of sodium ions from the vitreous body. An increase in the contents of sodium ions in the crystalline lens appears to be a typical manifestation of cataractogenesis and is known to occur in different type of cataracts and is known to occur in different types of cataracts and in the aging lens,.
| Summary|| |
Lens opacities have been produced after intravitreal and suprachoroidal injection of E. coli extract in rabbits. These opacities are present in the posterior subcapsular region of the lens, which is accompanied with mild uveitis, and are similar to a picture of complicated cataract. The contents of sodium, potassium and reducing sugars were measured in the aqueous, lens, and vitreous about three fold in the cataractous lenses, accompanied with a decrease in the vitreous while the sodium contents of aqueous remain unaltered. The contents of potassium on the other hand, remain unchanged. An increase of 33.6% and 39.3% in the reducing sugars is observed in the aqueous and vitreous after suprachoroidal injection of E. coli extract. Suprachoroidal and intravitreal injections of C. albicans and E. histolytica extracts produced uveitis but no lenticular changes, even after a follow up of eight weeks. E. coli endotoxins seem to act directly or in combination with uveitis on the lens transparency.
| References|| |
Becker, 0, 1876. cited in "Systems of Ophthalmology". Vol. XI Diseases of the lens and vitreous; Glaucoma and Hypotony. Duke Elder, S. Editor. Henry Kimpton. London. 1969. p.p. 210.
Pau, H., 1950, cited in "Systems of Ophthalmology". Vol. XI Diseases of the lens and vitreous, Gloucoma and Hypotony. Duke Elder, S. Editor. Henry Kimpton"London. pp 210.
Testa, M., Bocci, N. Fiori, C., Calbro, S., and Aurichio, G., 1968, Exptl. Eye. Res. 7 : 473.
Shimizu, M., 1978, Folia. Ophthalmol. Jap. 29 : 562.
Barber, W., 1968, Invest. Ophthalmol. 7: 564.
Varley, H., 1963, "Practical biochemistry'. New. York., 1963., William-Heinmann Medical books Ltd. and Interscience books Inc. pp 33.
Reddy, D.V.N. 1969, "The rabbit in eye research". J.H. Prince. Editor Spring field, U.S.A. 1964. Charles. C. Thomas. pp 362.
Kinsey, V.E, and Reddy, D.V.N. 1965, Invest. Ophthalmol. 4 : 104.
Kinoshita, J.H. and Merola, L,O. 1964, Invest. Ophthalmol 3 : 577.
Chylack, L.T. Jr and Kinoshita, J.H. 1973, Exptl. Eye. Res. 15:
Adler, F.H. 1953, "Physiology of the eye". The C.V. Mosby Co, St. Luis. pp 215.
Kuck, J.F.R. 1970, "Cataract formation" in Biochemistry of the Eye. C.N. Graymore. Editor. New York. 1970. Academic Press, pp 324-325.
Duncan, G. and Bushell, A.R. 1975, Exptl. Eye. Res. 20 : 223.
Harris, J.E. and Becker, B, 1965 Invest. Ophthalmol 4 :709.
[Figure - 1], [Figure - 2]
[Table - 1]