I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
|
Protocol no. 60
EYE LENS ORGAN CULTURE
Long-term cultures of bovine whole lenses are
used to assess the effect of drugs and chemicals on the refractive index
(focal length) and transparency of the lens tissue. These endpoints are
measured simultaneously by a computer-driven scanning laser system.
CONTACT
Professor Jacob Sivak
Faculty of Science School of Optometry University
of Waterloo
Waterloo Ontario N2L 3G1 Canada
Tel: 519-885-1211 ext 3174 Fax: 519-746-7937
RATIONALE
The lens has been somewhat neglected in terms
of investigations into toxic effects on the eye. This does not, however,
mean that damage to the lens caused by external agents is not an important
factor worthy of study. The lens plays an important role in focusing light
onto the retina and impairment of this function can have dire consequences
to vision. Certain features of the lens, i.e. its high protein content
and the retention of older tissue as new lens fibres develop, make it liable
to damage by chemicals and some forms of electromagnetic radiation. Since
the principal function of the lens is to help the cornea focus light onto
the retina, the most appropriate means of investigating the effects of
chemicals on lens integrity, is to examine the lens focal properties. A
computer-driven scanning laser system can be used to measure the focal
length of various points on the cultured lens. A graphic profile of the
lens focal variation is then plotted from such measurements and the effect
that treatments have on this profile can be assessed, quantitatively. Bovine
lens cultures are suitable for this type of test system, as the lens tissue
is readily available from local abattoirs. The fact that long term cultures
of lenses can be maintained, permits the study of reversible damage over
a period in this system, as well as for studying early lens damage. PROCEDURE
SUMMARY Lenses are removed from 2-3 year-old bovine eyes. A preliminary
range finding experiment is performed in which lenses are cultured at 35øC
in beakers containing a wide range of concentrations of the test compound.
Three concentrations are selected for testing. Each lens is cultured in
a specially prepared two-chambered cell to ensure that each side of the
lens is bathed in medium. The cultures are incubated at 35øC and the medium
is changed every 48 hours. Treatment and control lenses are cultured for
24 hours before experimental use to ensure that they have not been damaged
during dissection. Treated and untreated lenses are cultured for a further
1-10 days in an incubator. The lenses are transferred once or twice a day
to the laser scanner for measurement of focal properties and scatter. The
lenses are scanned at a frequency that is determined from the preliminary
range finding experiments. In the intervening period between the scans,
the cultures are maintained at 35øC with routine media changes. The optical
properties of the lens are monitored by a laser scanning system, which
comprises a scanning helium-neon laser beam, video camera and video frame
digitizer. The system first locates the optical centre of the lens (the
position of no refractive deviation of the beam). The laser then scans
the lens in steps, the size of which is dependent on the diameter of the
lens as well as the focal length. For toxicological work using bovine lenses
a step size of 0.5mm (22 steps) is ideal in terms of the visibility and
quantity of the data. The focal length for each beam position is measured
by a digitizer and a graphic profile of the lens focal variation is plotted.
The effects of treatments on this profile can then be assessed.
CRITICAL ASSESSMENT
Previously, in vitro tests were performed on
short-term lens cultures, and involved qualitative assessments of lens
opacity from measurements taken from the refraction of laser beams by the
lens culture (Saar, Neumann & Gershon, 1989). In this protocol, an
improved method permits the monitoring of early lens damage that occurs
prior to opacification. The use of long term cultures makes it possible
to study transient lens damage and lens repair mechanisms. The automated
scanning system makes data collection a relatively simple procedure and
produces quantitative results; the automation of the procedure makes it
more suited than the method of Saar, Neumann & Gershon, 1989 to the
large-scale screening of compounds. The bovine lens culture conditions
aim to represent as closely as possible the in vivo conditions. The culture
medium osmolarity is approximately 310 mOsm and varies within 5% of that
of fresh bovine aqueous and vitreous humours (Sivak, Yoshimura & Weerheim,
1990b). Using the initial cell chambers and laser scanning system the lenses
have been maintained in culture for periods ranging from 325 to 900 hours,
during which time control lenses have shown no significant change in focal
length. Due to the transfer of lens cultures to and from the laser scanner,
it is not possible to maintain the incubation temperature of the cells
at precisely 35øC throughout the culture period. For example, the lenses
may be exposed to a temperature of 21øC for periods of 5-15 minutes/day
during transfer. Experimental controls suggest that this amount of time
out of the incubator does not affect the results. The temperatures to which
cells are exposed when maintained for long periods of time in the incubator
may vary from 36 to 37øC. The design of the initial scanning laser system
has been improved to produce a simpler, more accurate and more compact
instrument. Various changes in hardware and software have led to an increase
in scanning speeds such that it is now possible to scan the lens, i.e.
locate the optical centre and measure focal length for 20 lens positions
along the X and Y directions, in about 60 seconds. Due to the development
of an effective culture technique, and improvements to the design of the
2-chambered cells, it is possible to perform culture experiments of at
least 1000 hours duration. The prototype scanners are built in the workshops
of the School of Optometry, University of Waterloo, Canada, at a cost of
approximately $25000 for each instrument, mass production would reduce
this cost somewhat. A number of graduate and undergraduate students have
learned to use the system without difficulty. The results of preliminary
tests using the system described in this protocol, suggest that it is suitable
for the evaluation of lens damage. Sivak et al, (1990a) used this system
to investigate the effect of three chemical agents, hydrogen peroxide,
DL-propranolol and prednisone, on lens refractive index and transparency.
Epidemiological and experimental evidence have indicated that these chemicals
are potential cataract-forming agents. Using the scanning laser system,
it was found that hydrogen peroxide caused an increase in average focal
length and an increase in the variability of focal length. Propranolol
increased the variability of focal length but did not cause an increase
in overall focal length. At the concentration tested (0.03mM), and over
the 21 day duration of the study, prednisone was not found to affect lens
optical properties. The fact that hydrogen peroxide had a different effect
on focal length to DL-propranolol is believed to indicate that different
effects on refractive index are induced by the different modes of action
of chemicals, and, therefore, it is possible that specific refractive index
changes can be related to specific physiological and anatomical damage.
It may also prove possible to study local lens damage, since the laser
scanner can be used to examine any site on the lens. Evidence of the sensitivity
of the laser scanning system comes from the study by Sivak et al, (1990b)
in which a focal change was detected 18 days after the treatment of bovine
lens cultures with 0.5mg/ml gentamicin. Previously, other in vitro studies
had used higher dose concentration of 5mg/ml before lens damage was observed.
In vivo studies involving the injection of 0.4mg/ml of gentamicin into
the eyes of cynomolgus monkeys caused no observable damage when examined
by ophthalmoscopy and electroretinography. Besides testing chemicals, it
is also possible to assess the impact of UV radiation on the optical properties
of the lens, (Stuart et al, 1991). Experiments into UV-induced lens damage
using animals usually involve acute exposure which is not truly representative
of chronic human exposure to environmental levels of UV. The laser scanning
system represents a sensitive and suitable means of assessing the risk
of cataract from environmental UV. It also makes it possible to monitor
lens damage and repair over a longer period than is achieved in in vivo
studies. There are several features of this system that should be considered
when analysing the results. The refractive, anatomical and physiological
properties of lenses vary from species to species, therefore this should
be taken into account when interspecies comparisons are made. It also is
important to note that the control of spherical aberration of the lens
is determined both by its shape and its refractive index distribution.
Therefore, it is possible for a change in lens shape to be counteracted
by an accompanying change in lens refractive index. For example, the intake
of water may reduce the radius of curvature of the lens surface, whilst
at the same time reducing overall refractive index. Since these two changes
have opposite refractive effects, the end result may be little or no change
in lens focal length. However, it is unlikely that a counteraction will
be perfect at all points from lens centre to periphery. Transient increases
in focal length have been noted when lenses are exposed to UV light (see
Figure 5). Such effects may be due to the disruption of the water balance
within the lens. Such a disruption of water balance has been implicated
in the opacification and swelling observed in in vivo experiments involving
UV exposure. Future work may entail isolating the two lens surfaces since
the anterior lens surface is the metabolically active lens component. In
summary, the scanning laser system is a sensitive and effective means of
quantifying early and subtle changes in lens optical function.
TEST STATUS
Undergoing in-house development. One validation
study for alcohols has been completed (Sivak, et al, 1992), and a second
study on surfactants is underway. The eye lens organ culture system will
be included in the MEIC programme.
CHEMICALS TESTED
Chloramphenicol
DL-Propranolol
Gentamicin
Hydrogen peroxide
Prednisone
REFERENCES
- Saar, I., Neumann, E. & Gershon, D. (1989)
Effects of gentamicin and chloramphenicol on the transparency of cultured
rat lenses. Opthalmic Res, 21, 118.
- Sivak, J.G., Stuart, D.D., Herbert, K.L., Van
Oostrom, J.A. & Segal, L. (1992) Optical properties of the cultured
bovine ocular lens as an in vitro alternative to the Draize eye toxicity
test: Preliminary validation for alcohols. Toxicology Methods, in press.
Sivak, J.G., Yoshimuro, M. and Dovrat, A. (1990a) Effect of hydrogen peroxide,
DL-propranolol, and prednisone on bovine lens optical function in culture.
Investigative Ophthalmology & Visual Science 31(5) 954-963.
- Sivak, J.G., Yoshimura, M. & Weerheim, J.A.
(1990b) Effect of gentamicin and chloramphenicol on bovine lens optical
function during culture. J.Toxicol.- Cut. & Ocular Toxicol. 9(4) 265-275.
- Stuart, D.D., Sivak, J.G., Cullen, A.P, Weerheim,
J.A. & Monteith, C.A. (1991) UV-B radiation and the optical properties
of cultured bovine lenses. Current Eye Research 10(2) 177-184.
- Weerheim, J.A. & Sivak, J.G. (1991) Scanning
laser measure of optical quality of the cultured crystalline lens. Opthal.
Physiol. Opt., 12, 72-79.
IP-60 C October 1992
|
PLEASE NOTE THAT TO OBTAIN DETAILS OF THE PROTOCOL YOU SHOULD REGISTER 
