February 2001
Volume 42, Issue 2
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Glaucoma  |   February 2001
Goldmann Applanation Tonometry in the Conscious Rat
Author Affiliations
  • Bruce E. Cohan
    From the Eye Research Fund Laboratory and the
  • David F. Bohr
    Department of Physiology, Medical School, University of Michigan, Ann Arbor.
Investigative Ophthalmology & Visual Science February 2001, Vol.42, 340-342. doi:
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      Bruce E. Cohan, David F. Bohr; Goldmann Applanation Tonometry in the Conscious Rat. Invest. Ophthalmol. Vis. Sci. 2001;42(2):340-342.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

The animal model, one of the most important tools in biomedical research, was introduced to studies of glaucoma by Gaasterland and Kupfer 1 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 group 4 5 and Sharma’s group 6 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. 
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 settings 8 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)
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). 
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. 
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 Moses 14 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. 
 
Figure 1.
 
Schematic of Goldmann applanation tonometer tip showing the biprism angle for the human (60°) and for the rat (48°) eye.
Figure 1.
 
Schematic of Goldmann applanation tonometer tip showing the biprism angle for the human (60°) and for the rat (48°) eye.
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.
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.
Table 1.
 
Calculated Goldmann Readings from a Nonlinear Model of the Experimental Data in the Conscious Rat for a Range of Repeated Applanations
Table 1.
 
Calculated Goldmann Readings from a Nonlinear Model of the Experimental Data in the Conscious Rat for a Range of Repeated Applanations
No. of Applanations Expected Goldmann Reading* Confidence Interval, †
9 16.6 ± 0.5 15.6, 17.7
10 15.6 ± 0.6 14.5, 16.8
11 15.7 ± 0.6 14.5, 16.8
12 15.3 ± 0.6 14.1, 16.4
13 15.7 ± 0.6 14.4, 17.0
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 ( Image not available Image not available ∗) 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.)
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 ( Image not available Image not available ∗) 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.)
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. 
Gaasterland D, Kupfer C. Experimental glaucoma in the rhesus monkey. Invest Ophthalmol. 1974;13:455–457. [PubMed]
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Morrison JC, Fraunfelder FW, Milne ST, et al. Limbal microvasculature of the rat eye. Invest Ophthalmol Vis Sci. 1995;36:751–756. [PubMed]
Moore CG, Morrison JC, Johnson EC, et al. Effect of glaucoma drops on unilateral expermentally elevated IOP in rats [ARVO Abstract]. Invest Ophthalmol Vis Sci. 1995;36(4)S738.Abstract nr 3412.
Morrison JC, Moore CG, Deppmeier LMH, et al. A rat model of chronic pressure-induced optic nerve damage. Exp Eye Res. 1997;64:85–96. [CrossRef] [PubMed]
Shareef SR, Garcia-Valenzuela E, Salierno A, et al. Chronic ocular hypertension following episcleral venous occlusion in rats (Letter). Exp Eye Res. 1995;61:379–382. [CrossRef] [PubMed]
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Moore CG, Milne ST, Morrison JC. Noninvasive measurement of rat intraocular pressure with the Tono-Pen. Invest Ophthalmol Vis Sci. 1993;34:363–369. [PubMed]
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Cabrera CL, Wagner LA, Schork MA, et al. Intraocular pressure measurement in the conscious rat. Acta Ophthalmol Scand. 1999;77:33–36. [PubMed]
Goldmann H. Applanation tonometry. Newell FW eds. Glaucoma. Transactions of the Second Conference. 1957;167–220. Josiah Macy, Jr. Foundation New York, NY.
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Bynke H, Krakau CET, Wilke K. Repeated applanation tonometry in optic atrophy. Acta Ophthalmol Scand. 1972;50:240–246.
Wilke K. Effects of repeated tonometry: genuine and sham measurements. Acta Ophthalmol Scand. 1972;50:574–581.
Krakau CET, Wilke K. Effects of loading of the eye on the intraocular pressure and on the episcleral venous pressure. Acta Ophthalmol Scand. 1974;52:107–124.
Figure 1.
 
Schematic of Goldmann applanation tonometer tip showing the biprism angle for the human (60°) and for the rat (48°) eye.
Figure 1.
 
Schematic of Goldmann applanation tonometer tip showing the biprism angle for the human (60°) and for the rat (48°) eye.
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.
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.
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 ( Image not available Image not available ∗) 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.)
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 ( Image not available Image not available ∗) 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.)
Table 1.
 
Calculated Goldmann Readings from a Nonlinear Model of the Experimental Data in the Conscious Rat for a Range of Repeated Applanations
Table 1.
 
Calculated Goldmann Readings from a Nonlinear Model of the Experimental Data in the Conscious Rat for a Range of Repeated Applanations
No. of Applanations Expected Goldmann Reading* Confidence Interval, †
9 16.6 ± 0.5 15.6, 17.7
10 15.6 ± 0.6 14.5, 16.8
11 15.7 ± 0.6 14.5, 16.8
12 15.3 ± 0.6 14.1, 16.4
13 15.7 ± 0.6 14.4, 17.0
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