Viscous properties of teleost aqueous humor

Comp. Biochem. Physiol., 1969, Vol. 28, pp. 1411 to 1417. Pergamon Press. Printed in Great Britain

VISCOUS PROPERTIES OF TELEOST AQUEOUS HUMOR J. R U S S E L L H O F F E R T and PAUL O. F R O M M Department of Physiology, Michigan State University, East Lansing, Michigan 48823

(Received 30 aYuly 1968) Abstract--1. The viscosity of rainbow trout (Salmo gairdneri) aqueous humor was found to be 37.44 centistokes measured at 20°C. 2. The viscosity of teleost aqueous humor is about ten times greater than that of canine aqueous humor. 3. Treatment of samples of teleost aqueous humor with hyaluronidase for 30 min and u.v. light for 1 hr caused respective 89 and 84 per cent decreases in the viscosity below that of controls. INTRODUCTION IN OUR EARLIER studies we observed that the aqueous humor of trout eyes appeared to be much more viscous than that from eyes of other vertebrates. It was noted by Balazs (Balazs & Jeanloz, 1965a) that aqueous humor from tuna and carp was likewise quite viscous. The substance responsible for this viscosity in tuna and carp has been isolated and identified as a polysaccharide made up of equimolar amounts of hexosamine and hexuronic acid; both glucosamine and galactosamine were found to be present. The glycosaminoglycan present in fish aqueous humor was found to be identical to that isolated from fish vitreous body. Although the glycosaminoglycan present in tuna and carp ocular fluids differs from hyaluronic acid this polysaccharide is rapidly degraded by testicular, bacterial and leach hyaluronidase. A possible relationship between the occurrence of corneal and lenticular lesions in trout and exposure of animals to sunlight has been suggested (Hoffert & Fromm, 1965). At that time we indicated that there was a probable decrease in the viscosity of the aqueous humor accompanying the development of keratectasia, keratoglobus and cataract formation. McCandless et al. (1969) have established that fish in hatchery race-ways are exposed to some u.v. radiation and much of the radiant energy of wavelengths between 300 and 400 m/z is absorbed by the aqueous humor. Radiation in the visible range is not absorbed significantly by the transparent ocular structures and/or fluids and the i.r. radiation (above 800 m/z) is almost entirely absorbed by the environmental water, consequently insufficient amounts reach the ocular structures to cause any thermal damage. A method was devised for the determination of absolute viscosity of aqueous humor with samples as small as 0.1 ml and having kinematic viscosities as high as 150 centistokes (cs). This report is concerned with determinations of the absolute viscosity of aqueous humor from trout and dogs and with the effects of hyaluronidase and u.v. light on the viscosity of fish aqueous humor. 1411

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J. RUSSELLHOFFERTAND PAUL O. FROMM

MATERIALS AND METHODS Rainbow trout (Salmo gairdneri) from the Michigan Conservation Department were held in fiberglass-lined tanks at 13°C under a daily regimen of 16 hr light and 8 hr darkness. With a 1-ml disposable syringe, aqueous humor samples were obtained by inserting a 26-gage needle into the anterior chamber at the nasal aspect of the scleral--corneal junction. About 0"1 ml of aqueous humor could be withdrawn from each eye of fish weighing 100150 g and in order to have a sufficient volume for analyses, samples from both eyes of a single fish were pooled. Adequate volumes of aqueous humor were obtained in a sLmilar manner from mongrel dogs which were to be used in classroom experiments. After obtaining samples they were Lmmediately placed in a microviscosimeter similar to the one described by Somrner & Wear (1949) and Lillard (1952). T h e viscosimeter consists basically of a capillary tube 44 cm long with an internal radius of 0"595 m m mounted in a water jacket. A temperature of 20 + 0'25°C was maintained by circulating water from a constant-temperature bath through the water jacket. T h e whole assembly was mounted on a platform which could be accurately positioned to within 0.5 ° from - 9 0 ° to + 90 ° with the tube being horizontal at an angle of 0 °. A 11 "95-cm segment of the capillary tube was marked off and the time necessary for the front meniscus of a sample to pass through the segment could be measured to the nearest 0.01 sec. Flow times ranging between 2 and 30 sec were obtained by adjusting the angle of inclination. Each sample was run at three or more angles and at each angle the average of four flows to the right and four flows to the left was determined. Prior to each measurement the capillary tube was washed by aspirating through it a 1% detergent solution followed by distilled water, acetone and dry air in that order. Calibration of the viscosimeter was done using distilled water and standard sucrose solutions (Handbook of Chemistry and Physics, 1957) and with American Society of Testing Materials viscosity standards obtained from the Cannon Instrument Co., State College, Pa. Flow times for these standards were obtained as noted above and the data were treated in a similar manner but without some of the assumptions described by Sommer & Wear (1949). This was possible because of the fact that all of our measurements were made at a single temperature and the range of viscosity of the biological fluids tested was much less than that investigated by the aforesaid authors. I n the mathematical treatment of the experimental data use is made of the fact that the log-log plot of angle of inclination vs. efflux time results in a straight-line relationship (Fig. 1). The constant (T~) is equivalent to the efflux time at a 1° inclination and this value may be determined graphically by extrapolating a fitted line to the time axis or calculated from the summation of the individual points: log T M = log (a) (s)/N = log a l s l + l o g a~s2+log artsn where N is the n u m b e r of angles at which measurements were made; a, the angle of inclination of the tube in degrees; and s, efflux time in seconds at angle (a). Viscosities for each of the standard solutions are plotted on the centistoke axis (Fig. 1) and straight lines (conversion lines) are drawn from these points to the TM value for that solution. The experimental standardization lines and conversion lines meet at the time axis forming, within experimental error, a constant angle. Since the conversion lines are parallel it follows that efflux time is directly proportional to the kinematic viscosity in centistokes.

v=k.T~ where v is the viscosity of sample in centistokes, T~/, mean effiux time at 1 ° angle; and k, proportionality constant. T h e proportionality constant (k) was determined by substituting in the above equation the data from standard solutions. Twenty-seven determinations on eight different viscosity standards ranging from 1"002 to 47"52 cs were made and the average value for k + S.E. was 0-0511 + 0.001. For the hyaluronidase studies aqueous humor samples were diluted with an equal volume of a solution containing 0-1 M acetate buffer (pH 6.0) and 0"15 M NaC1. After dilution, the

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VISCOSITY OF TROUT AQUEOUS H U M O R

viscosity of one aliquot was measured immediately; 1 mg powdered hyaluronidase was added to the second aliquot. T h e viscosity of the latter was determined after 30 rnin incubation at 20°C. T h e hyaluronidase used had an activity of 400 units per rag, was of bovine testes origin and was obtained from M a n n Research Lab. Inc., New York.

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FIo. 1. Log-log plot of angle of inclination and flow time for four representative viscosity standards. Flow times at 1° inclination (T~) are connected by conversion lines to centistoke values for the respective standards.

Investigations of the effect of u.v. light on aqueous humor were made as follows. Samples were diluted 1 : 1 with distilled water and the viscosity of the first aliquot determined immediately. T h e second aliquot was placed in a quartz cuvettc which had a surface area of 4 cm s and was positioned 5 cm from a Blak Ray XX-15 High Intensity Lamp containing two G.E. FST8-BLB 15 W long wave u.v. bulbs (Ultra Violet Products Inc., San Gabriel, Calif.); exposure time was 1 hr. This lamp produces 97 per cent of its radiation between 300 and 400 m p with maximum intensity at a wavelength of 355 nap. Calculations indicated that the aqueous humor samples received an irradiance of approximately 860 p W per crnI"

J. RUSSELL HOFFERT AND PAUL0. FROMM

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RESULTS All measurements of viscosity were made at velocities substantiallyless than those which would result in turbulent flow, i.e. if one exceeds a Reynoldsnumber of 1400. Trout aqueous humor samples showed constantflowrates when flowtime was 2-30 sec for the 11.95-cm segment. Uneven flow was observed when flow times were above or below these values and data for these observations were discarded. Sinceno correlationwas found betweenthe angleof inclination(shear rate) and calculatedviscosity,flowappeared to be essentiallythat of a Newtonian liquid with flow times ranging between 2 and 30 sec. To test the effectof sample size viscositymeasurements were made on sixteen samples varying in size from 3.8 to 13.8 cm as measured on the capillary tube. The Kendall rank correlation test (Siegel, 1956) was applied and it was found that the correlation coefficientbetween sample size and viscositywas not significantly different from zero at the 5 per cent level. It was further established that the viscosityof untreated aqueous humor samples remainedconstantover a l-hr period. Data presented in Table l show that the viscosityof aqueous humor of trout is some ten times greater than that from the dog. T h e coefficient of skewness (gl) indicates that all distributions are significantly different from a normal distribution TABLE 1--KINEMATIC

VISCOSITY I N CENTISTOKES OF VARIOUS TELEOST BODY FLUIDS AND CANINE AQUEOUS HUMOR MEASURED AT 2 0 ° C

Sample

Number

Mean

Standard error

g 1*

Trout aqueous humor Canine aqueous humor Trout plasma Trout blood (Hct = 28%)

59 9 17 13

37.44 3"35 3"62 7'70

3"809 0"522 0"217 0'298

457"3 59"4 1"4 24"1

* Coefficient of skewness. Hct = hematocrit. at the 5 per cent level. T h e trout aqueous h u m o r data show the greatest departure from normality and its frequency distribution is depicted in Fig. 2. T h e nonparametric M a n n - W h i t n e y U test was used to verify that the teleost aqueous h u m o r was significantly more viscous (P = 0.01) than canine aqueous humor. A 30-min hydrolysis of diluted aqueous h u m o r samples with hyaluronidase caused an 89 per cent decrease in the average viscosity (Table 2). T h e nonparametric, one-tailed Walsh test (Siegel, 1956) indicated that the viscosity of the treated samples was significantly below that of controls at the P = 0.031 level. DISCUSSION Earlier qualitative observations in our laboratory and by others have indicated that the aqueous h u m o r of fishes is m u c h more viscous than that from other vertebrate eyes. Quantitative data presented in this report show that the viscosity of

VISCOSITY OF TROUT AQUEOUS HUMOR

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rainbow trout aqueous h u m o r is some ten times more viscous than canine aqueous humor. Beswick & McCulloch (1956) reported that fresh bovine aqueous h u m o r

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FIO. 2. Frequency distribution of viscosity of rainbow trout aqueous humor. Data from fifty-nine samples are plotted in 5-centistoke increments. T A B L E 2 - - E F F E C T OF TREATMENT OF DILUTED TROUT AQUEOUS HUMOR SAMPLES W I T H HYALURONIDASE AND ULTRAVIOLET L I G H T

Experiment: hyaluronidase Control Hyaluronidase treated Difference: % change in viscosity = 88"7 Experiment: u.v. Control u.v.-treated Difference: % change in viscosity = 83"7

No. of data

Viscosity (centistokes)

5 5

20"76 2.33 18"43

6 6

33"11 5"40 27.71

has a low relative viscosity similar to canine aqueous humor. It is also known that, in general, the hyaluronic acid concentration in bovine aqueous humors is quite low and for this reason they are not greatly affected by treatment with hyaluronidase.

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J. RUSSELL HOFFERT AND PAUL O. FROMM

It has been established (Balazs & Jeanloz, 1965a) that hyaluronidase can cause liquefication of vitreous bodies and that the action of this enzyme is on the hyaluronic acid present therein. Our data clearly show that treatment with hyaluronidase significantly decreased the viscosity of trout aqueous humor; thus we have concluded that the substance responsible for the high viscosity of this humor is hyaluronic acid or a glycosaminoglycan which is susceptible to enzymatic degradation with hyaluronidase. Irradiation of pure hyaluronic acid solutions and vitreous bodies in vitro has been shown (Balazs & Jeanloz, 1965b) to result in a decrease in viscosity. This decreased viscosity was found to be the result of a depolymerization of the hyaluronic acid. Data presented herein show that irradiation with near u.v. light causes a very significant decrease in the viscosity of trout aqueous humor samples. The wavelengths with which the samples were irradiated are those which have been shown to penetrate the teleost cornea (McCandless et aL, 1969) and are also wavelenths present in the solar radiation that reaches the earth's surface. In summary, evidence is presented in support of our hypothesis that irradiation of trout eyes in vivo with near u.v. light can cause changes in the viscosity of the aqueous humor. Decreased viscosity appears to be due to a breakdown of hyaluronic acid or a glycosaminoglycan present in this structure which is sensitive to the action of hyaluronidase. Changes in viscosity of aqueous humor of this nature may result in the establishment of new diffusion and hydrodynamic steady states which are not compatible with maintenance of normal ocular physiology but conducive to the development of ocular lesions similar to those we have observed in hatchery trout. Acknowledgements--This research was supported in part by Grant NB-04125 from the National Institutes of Neurological Diseases and Blindness, United States Public Health Service. The authors wish to express appreciation for technical assistance to Mr. Michael Fairbanks and Mrs. Esther Brenke. REFERENCES

BALMSE. A. & JF.ANLOZR. W. (Editors) (1965a) The Amino Sugars, Vol. IIA. Academic Press, New York. BALAZSE. A. & JEANLOZR. W. (Editors) (1965b) The Amino Sugars, Vol. IIB. Academic Press, New York. BESWlCKJ. A. & McCuLLOCHC. (1956) Effect of hyaluronidase on the viscosity of the aqueous humor. Br. ~. Ophthal. 40, 545-546. Handbook of Chemistry and Physics (1957), 39th edn. The Chemical Rubber Co., Cleveland, Ohio. HOF~RT J. R. & FROMMP. O. (1965) Biomicroscopic, gross and microscopic observations of corneal lesions in the lake trout, Salvelinus namaycush. •. Fish. Res. Bd Can. 22, 761-766. LILLAROJ. G. (1952) Modified method for viscosity measurement of small samples. Analyt. Chem. 24, 1042-1043. McCANDI2~ R. L., HOFrXRTJ. R. & FROMMP. O. (1969) Light transmission by corneas, aqueous humor and crystalline lenses of fishes. Vision Res. 9, 223-232.

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SmGEL S. (1956) Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. SOMlVmRJ. V. & WEAR G. E. C. (1949) Some microphysical apparatus and methods for inspection of petroleum products. Proc. 14th Mid-year Meetg, Am. Petrol. Inst., Div. of Ref. Vol. 29M (111), pp. 12-24. Key Word Index---Aqueous humor; teleosts; u.v. light; viscocity; hyaluronic acid; Salmo gairdneri.