(Investigative Ophthalmology and Visual Science. 2001;42:340-342.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Goldmann Applanation Tonometry in the Conscious Rat
Bruce E. Cohan1 and
David F. Bohr2
1 From the Eye Research Fund Laboratory and the
2 Department of Physiology, Medical School, University of Michigan, Ann Arbor.
 |
Abstract
|
|---|
PURPOSE. To determine whether the Goldmann applanation tonometer can be modified to
measure intraocular pressure (IOP) in the conscious rat.
METHODS. In anesthetized rats Goldmann tonometers were tested that had reduced
biprism angles in the applanating tips and reduced weights in the
tonometer body from those used in humans and species with similar size
eyes. Tonometers with tips with biprism angles of 48° and an applied
weight of 25 mg per Goldmann scale division (2 g full scale) were
calibrated for the rat against manometrically measured IOP. Tonometers,
thus modified, were then used in conscious, unsedated rats.
RESULTS. In conscious rats the measured mean Goldmann value was 15.5 ± 0.6
mm Hg (confidence interval = 14.1, 16.6 mm Hg). This was the
plateau level reached after the repeated applanations (approximately
10) required to eliminate an artifactual decline in initial Goldmann
readings, which was larger than that in humans.
CONCLUSIONS. The Goldmann applanation tonometer was modified to measure IOP in the
conscious, unsedated rat. This instrument, the standard for measuring
this key physiological parameter in the human eye, can now be applied
to the laboratory rat. This may advance the use of this important
animal as a model in IOP and glaucoma research.
|
Introduction
|
|---|
The animal model, one of the most important tools in
biomedical research, was introduced to studies of glaucoma by
Gaasterland and Kupfer1
and Quigley and
Hohman,2
who convincingly demonstrated that the condition
resembling ocular hypertension in humans could be experimentally
produced in the monkey. The most productive laboratory investigations
of the effects of elevated intraocular pressure (IOP) in the past
quarter century have been conducted with this model. Recently, in part
because of the expense of acquiring and maintaining monkeys in sizable
numbers and the ease and familiarity of work with the laboratory rat,
this animal has begun to share the role of model for IOP and glaucoma
studies. The anatomy of the aqueous humor outflow pathway in this
rodent eye is similar to the primate,3
unlike rabbit, dog,
and cat eyes, which therefore are rarely used in glaucoma-related
studies. Morrison’s group4
5
and Sharma’s
group6
have described methods for producing ocular
hypertension in the rat.
Noninvasive IOP measurement is accomplished by tonometry, and two
tonometers, that were designed for the human eye and cannot be modified
for the rat eye, have been used to detect qualitative IOP elevation in
this animal. The pneumotonometer, which lacks calibration studies in
the rat, has been used in these animals under general
anesthesia.7
The Tono-Pen, which has been subjected to
calibration studies in the rat,8
9
has been applied in the
conscious animal,4
10
but with
difficulty.11
A third tonometric method is Goldmann applanation
tonometry.12
It depends on the Imbert-Fick hypothesis,
which holds that when a flat surface is pressed against a closed sphere
with a given internal pressure, an equilibrium will be attained when
the force exerted against the spherical surface is balanced by the
internal pressure of the sphere exerted over the area of contact.
Ideally, the sphere applanated by the flat surface is thin, perfectly
elastic, and perfectly flexible, and the only force acting against it
is the pressure of the applanating surface. In any case the applanated
area and the subsequently displaced volume must be small in relation to
the total area and volume of the sphere.
The Goldmann tonometer has a transparent plastic applanating tip in the
shape of a truncated cone through which corneal contact is observed
with the slit-lamp biomicroscope. The tip contains a biprism (two
prisms touching at their apices), which produces optical doubling of
the image of the flattened surface and separates the two components by
a fixed amount, dependent on the apex angles of the prisms. The
tonometer tip is connected by a lever arm to the tonometer body, which
contains a variable weight. Force is applied to the cornea through the
tip until the diameter of the applanated area reaches that fixed
separation.
Goldmann empirically tested eyes while measuring IOP with a manometer.
He found that for the human eye biprism angles of 60° gave the
appropriate diameter of the area of corneal applanation, relating the
force of a variable weight applied to it (100 mg per scale division of
his instrument; 8 g full scale) to the manometrically measured
IOP. He reported that this diameter in the human eye (and in monkey) is
3.06 mm and in rabbit, dog, and cat 4 mm.
|
Materials and Methods
|
|---|
Modification of the Goldmann Applanation Tonometer
Because the eyes of rats are naturally protuberant, they are
readily accessible to the applanating tip of the Goldmann tonometer
without reduction of its external dimensions and without having to
touch the eye lids. In anesthetized rats a range of reductions of the
biprism angles of the Goldmann tonometer tip were tested empirically in
calibration experiments. The results of these tests determined the
values of the angles of the biprism in the tonometer tip (48°; Fig. 1
) and of the weight applied by the instrument to the cornea (25 mg per
scale division; 2 g full scale). The resulting changes yielded a
smaller area, approximately 2 mm in diameter, of corneal applanation
than in the larger eyes of the species in Goldmann’s experiments.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Schematic of Goldmann applanation tonometer tip showing the biprism
angle for the human (60°) and for the rat (48°) eye.
|
|
Calibration of the Modified Goldmann Applanation Tonometer
The experiments adhered to the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and the guidelines of the
Unit for Laboratory Animal Medicine of the University of Michigan
Medical School. The modified tonometer was calibrated in the eyes of
brown Norway retired breeder male rats (Harlan Sprague–Dawley,
Indianapolis, IN; n = 5). With these animals under
general anesthesia with sodium pentobarbital (50 mg/kg
intraperitoneal), a 27-gauge cannula was positioned in the anterior
chamber. An IOP pressure–regulating device was interposed between the
cannula and a pressure transducer (P23; Statham Instrument Co., Hato
Rey, PR) from which IOP was recorded on a Grass Polygraph (Grass
Instrument Co., Quincy, MA), which had been calibrated by a mercury
manometer. IOP was then adjusted to a series of pressure
settings8
both in random and in step fashion, always
masked from the person who made an applanation reading at each setting,
from 0 to 60 mm Hg (Fig. 2) .
View larger version (18K):
[in this window]
[in a new window]
|
Figure 2. A tracing of manometrically measured IOP from a cannula in the anterior
chamber of a rat eye to calibrate the Goldmann applanation tonometer.
(A) IOP was adjusted with a pressure-regulating device, here
demonstrated in a step tracing, from 0 to 50 mm Hg, and readings made
with a modified Goldmann tonometer at each IOP step as noted.
(B) Comparison of the manometer measurement and the Goldmann
reading data of this tracing by linear regression analysis.
|
|
Application of the Goldmann Applanation Tonometer in Conscious Rats
The modified tonometer was used in conscious, unsedated rats
(n = 10) that were wrapped in a small towel and held
gently on a small platform at the slit-lamp biomicroscope, with one
person holding the animal and another making the applanation readings.
Brown Norway rats are especially docile and require no training or
frequent handling before an applanation session. Goldmann tonometry was
performed as it is in humans, with topical anesthesia obtained with an
eye drop of proparacaine hydrochloride 0.5% and the edge of the
applanated area accentuated by staining the tears with a minute drop of
sodium fluorescein 2% dye. A series of applanation readings of both
eyes was obtained in 194 animal-sessions. Readings were generally made
at intervals of from 15 to 45 seconds and in a few sessions at 1- or at
2-minute intervals, over an 8-minute test period for each eye, the
right eye first. Tonometer tip-corneal contact time was usually 10 to15
seconds.
|
Results
|
|---|
Calibration
The results, compared by linear regression analysis of the
Goldmann readings with the manometer measurements, obtained both in
random and step fashion, showed good agreement across the range of IOP
tested (Fig. 2)
. And one Goldmann scale unit on the tonometer dial
corresponded to 1.1 mm Hg IOP.
Applanation Tonometry in the Conscious Rat
Because the means of the Goldmann readings in the conscious rats
did not differ significantly by ANOVA between eyes of an animal, among
animals, or at different times of the day, all readings of all test
sessions of the 10 animals were pooled. It was found that repeated
applanations resulted in a decline in Goldmann readings to a plateau or
steady state level. A nonlinear model was applied to this pooled data
to determine the number of tonometer contacts (±SE) required to reach
the asymptote: "mathematical" stability was reached at 11.3 ±
1.2 (95% confidence interval [CI] = 9.3, 13.3 applanations). The
means of Goldmann readings were close to each other at 9, 10, 11, 12,
and 13 applanations, and their confidence intervals were narrow (Table 1)
. At 11.3 applanations, the interpolated mean Goldmann reading
in this series of conscious, unsedated brown Norway rats was 15.5 ± 0.6 (95% CI = 14.1, 16.6 mm Hg), the same mean that Goldmann
reported for the normal human eye.12
The decline was from
a mean initial reading of 23.5 ± 0.4 (95% CI = 22.7, 24.4
mm Hg).
View this table:
[in this window]
[in a new window]
|
Table 1. Calculated Goldmann Readings from a Nonlinear Model of the
Experimental Data in the Conscious Rat for a Range of Repeated
Applanations
|
|
Decline in Goldmann Readings with Repeated Applanations
That in the rat the decline in Goldmann readings with repeated
applanations was not the result of a response of the conscious animal
to the measurement process was suggested by its presence in both the
first-measured right eye and the left eye. Also, it is not due to a
reflex because it persisted after both retrobulbar anesthesia and
under general anesthesia (with lower initial and plateau levels). When
this decline in the rat was first observed, the impulse was to
attribute it to the expression of aqueous humor during applanation.
Experiments to test this possible explanation were subsequently
performed on rats under general anesthesia with a cannula in the
anterior chamber to measure IOP manometrically; the pressure regulator
was not used, and IOP was allowed to reach a steady state, generally
approximately 12 mm Hg. It was noteworthy then that with repeated
applanations, the manometrically measured IOP baseline remained
unchanged (Fig. 3)
. This showed that the decline in Goldmann tonometer readings could not
be attributed to expression of fluid from the eye during applanation
and confirmed the validity for the rat eye of the Imbert-Fick
requirement that displaced volume is small in relation to its total
area and volume. Further, when initial Goldmann readings were compared
simultaneously with manometrically measured IOP, the Goldmann readings
were higher and with repeated applanations declined to match the
manometrically measured level. This showed that the higher initial
readings were artifactual.
View larger version (10K):
[in this window]
[in a new window]
|
Figure 3. A representative tracing of manometrically measured IOP from a cannula
in the anterior chamber of a rat eye, during multiple readings with a
modified Goldmann applanation tonometer. The baseline remains
stable during five applanations
(||*)
showing that the initial decrease in Goldmann readings is not the
result of expression of fluid from the eye during applanation. (The
rhythmic IOP spikes are from microscopic nystagmoid movements.)
|
|
|
Discussion
|
|---|
The phenomenon in the rat of a decline in Goldmann tonometer
readings with repeated applanations has a close parallel in the human
eye in which a similar, usually smaller, decline has long been
recognized and clinically either ignored or taken into account by
repeating applanations until the readings stabilize and discarding
initial readings.13
Initially observed by
Goldmann,12
this effect was characterized by
Moses14
and by Bechrakis,15
whose name is
sometimes attached to it. The phenomenon has been most studied by
Krakau’s group,16
17
18
19
but even now its mechanism(s)
remains obscure.
We speculated that in the rat this decline phenomenon is of corneal
origin but could not demonstrate, in images of projected patterns,
corneal flattening after the 8-minute test sessions of multiple
applanations, perhaps because our corneal topography was not
sufficiently sensitive. It still seems plausible that repeated
applanations may subtly alter the central rat cornea, making it
approach more closely a perfect membrane. Goldmann did describe in
human eyes a decline in readings when the tonometer tip was allowed to
remain in contact with the cornea, which he attributed to creep or
"flowing" of the tissue.12
To try to reduce the
decline phenomenon in the rat by using tonometer tips with smaller
biprism angles would require reducing further the weight applied to the
cornea, which is beyond the practical limit of the instrument (Pfister
R, personal communication, 2000).
The rat is by a factor of 10 the most widely used experimental animal
in biomedical research, and its physiology and pathology have been
extensively studied. Inbred rat strains provide models of many
diseases, among them systemic arterial hypertension, heart failure,
diabetes, and obesity. A reliable method for measurement of IOP in the
conscious, unsedated rat is now available using the world standard
instrument, the Goldmann applanation tonometer, modified as we have
described. The availability of this fundamental measurement of this key
physiological parameter in this important experimental animal opens new
avenues for IOP and glaucoma research and the possibility of
development of an inbred rat strain with spontaneous elevated IOP
(ocular hypertension) by transgenic and inbreeding strategies.
|
Acknowledgements
|
|---|
The authors thank Haag-Streit International where Jürg
Schnetzer (Köniz/Bern, Switzerland) provided the modified
tonometer tips and Rolf Pfister (Mason, OH) reduced the tonometer
weight. Statistical analysis was by Niko Kaciroti and M. Anthony
Schork.
|
Footnotes
|
|---|
Supported in part by a research grant from the Lloyd and Mabel Johnson Foundation.
Submitted for publication June 15, 2000; revised October 25, 2000; accepted November 2, 2000.
Commercial relationships policy: N.
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1998.
Corresponding author: Bruce E. Cohan, Eye Research Fund Laboratory, 2350 Washtenaw Avenue, Ann Arbor, MI 48104. becohan{at}umich.edu
|
References
|
|---|
-
Gaasterland, D, Kupfer, C. (1974) Experimental glaucoma in the rhesus monkey Invest Ophthalmol 13,455-457[Abstract/Free Full Text]
-
Quigley, HA, Hohman, RM (1983) Laser energy levels for trabecular meshwork damage in the primate eye Invest Ophthalmol Vis Sci 24,1305-1306[Abstract/Free Full Text]
-
Morrison, JC, Fraunfelder, FW, Milne, ST, et al (1995) Limbal microvasculature of the rat eye Invest Ophthalmol Vis Sci 36,751-756[Abstract/Free Full Text]
-
Moore, CG, Morrison, JC, Johnson, EC, et al (1995) Effect of glaucoma drops on unilateral expermentally elevated IOP in rats [ARVO Abstract] Invest Ophthalmol Vis Sci 36(4),S738Abstract nr 3412.
-
Morrison, JC, Moore, CG, Deppmeier, LMH, et al (1997) A rat model of chronic pressure-induced optic nerve damage Exp Eye Res 64,85-96[Medline][Order article via Infotrieve]
-
Shareef, SR, Garcia-Valenzuela, E, Salierno, A, et al (1995) Chronic ocular hypertension following episcleral venous occlusion in rats (Letter) Exp Eye Res 61,379-382[Medline][Order article via Infotrieve]
-
Garcia-Valenzuela, E, Shareef, SR, Walsh, JB, et al (1995) Programmed cell death of retinal ganglion cells during experimental glaucoma Exp Eye Res 61,33-44[Medline][Order article via Infotrieve]
-
Mermoud, A, Baerveldt, G, Minckler, DS, et al (1994) Intraocular pressure in Lewis rats Invest Ophthalmol Vis Sci 35,2455-2460[Abstract/Free Full Text]
-
Moore, CG, Milne, ST, Morrison, JC (1993) Noninvasive measurement of rat intraocular pressure with the Tono-Pen Invest Ophthalmol Vis Sci 34,363-369[Abstract/Free Full Text]
-
Moore, CG, Johnson, EC, Morrison, JC (1996) Circadian rhythm of intraocular pressure in the rat Curr Eye Res 15,185-191[Medline][Order article via Infotrieve]
-
Cabrera, CL, Wagner, LA, Schork, MA, et al (1999) Intraocular pressure measurement in the conscious rat Acta Ophthalmol Scand 77,33-36[Medline][Order article via Infotrieve]
-
Goldmann, H. (1957) Applanation tonometry Newell, FW eds. Glaucoma. Transactions of the Second Conference ,167-220 Josiah Macy, Jr. Foundation New York, NY.
-
Whitacre, MM, Stein, R. (1993) Sources of error with use of Goldmann-type tonometers Surv Ophthalmol 38,1-30[Medline][Order article via Infotrieve]
-
Moses, RA (1961) Repeated applanation tonometry Ophthalmologica 142,663-668[Medline][Order article via Infotrieve]
-
Bechrakis, VE (1966) Über den spontanen Druckabfall bei Applanationstonometrie Ophthalmologica 151,604-614[Medline][Order article via Infotrieve]
-
Krakau, CET, Wilke, K. (1971) On repeated tonometry Acta Ophthalmol Scand 49,611-614
-
Bynke, H, Krakau, CET, Wilke, K. (1972) Repeated applanation tonometry in optic atrophy Acta Ophthalmol Scand 50,240-246
-
Wilke, K. (1972) Effects of repeated tonometry: genuine and sham measurements Acta Ophthalmol Scand 50,574-581
-
Krakau, CET, Wilke, K. (1974) Effects of loading of the eye on the intraocular pressure and on the episcleral venous pressure Acta Ophthalmol Scand 52,107-124
This article has been cited by other articles:
|
|
|
|
 
Z. He, B. V. Bui, and A. J. Vingrys
Effect of Repeated IOP Challenge on Rat Retinal Function
Invest. Ophthalmol. Vis. Sci.,
July 1, 2008;
49(7):
3026 - 3034.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-H. Wang, J. C. Millar, I.-H. Pang, M. B. Wax, and A. F. Clark
Noninvasive Measurement of Rodent Intraocular Pressure with a Rebound Tonometer
Invest. Ophthalmol. Vis. Sci.,
December 1, 2005;
46(12):
4617 - 4621.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Danias, A. I. Kontiola, T. Filippopoulos, and T. Mittag
Method for the Noninvasive Measurement of Intraocular Pressure in Mice
Invest. Ophthalmol. Vis. Sci.,
March 1, 2003;
44(3):
1138 - 1141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. E. Cohan, A. C. Pearch, P. T. Jokelainen, and D. F. Bohr
Optic Disc Imaging in Conscious Rats and Mice
Invest. Ophthalmol. Vis. Sci.,
January 1, 2003;
44(1):
160 - 163.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. E. Cohan and D. F. Bohr
Measurement of Intraocular Pressure in Awake Mice
Invest. Ophthalmol. Vis. Sci.,
October 1, 2001;
42(11):
2560 - 2562.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
