Measurement of Intraocular Pressure in Awake Mice

Abstract

purpose. To determine whether the Goldmann applanation tonometer can be modified to measure intraocular pressure (IOP) in the awake mouse.

methods. Tonometers with reduction of the biprism angles in the applanating tips and in the weight applied by the instrument were tested in anesthetized mice in calibration experiments. Then a tonometer with the appropriate configuration of tip and weight was used in conscious, unsedated mice.

results. Tonometry in mice required a biprism angle of 36° and weight applied of 25 mg per scale division (2 g full scale). This tonometer was calibrated in mice against manometrically measured IOP and showed good agreement across the range of IOP tested (0–50 mm Hg). In conscious mice the measured mean Goldmann value was 13.7 ± 3.2 mm Hg (mean ± SD; 95% confidence interval, 13.1, 14.2 mm Hg).

conclusions. The Goldmann tonometer, the standard for measuring the IOP in the human eye, was modified to measure this fundamental physiologic parameter in the awake mouse. This measurement is required to confirm success in genetically engineering a model in the powerful mouse system, which mimics elevated IOP in humans. The model will open new avenues for studying the causes of the optic neuropathy of glaucoma, the regulation of IOP, and new therapeutic approaches to prevent the irreversible loss of vision from this disease.

Primary open angle glaucoma (POAG), by far the most common form of glaucoma and the second leading cause of blindness, is characterized by a distinctive degeneration of the optic nerve by mechanisms that have been the focus of controversy since early in the twentieth century. The most important risk factor for this optic neuropathy is elevated intraocular pressure (IOP), also called ocular hypertension, and reducing IOP is the only available treatment. Goldmann applanation tonometry has long been the standard method for measuring IOP. ¹ The cause of the elevated IOP in POAG is a subtle defect in the fluid (aqueous humor) outflow pathway from the eye, and recently a causative gene for POAG in humans was identified. ² With this discovery it was anticipated that increased IOP would soon be induced by genetic engineering in the mouse, ³ which has an aqueous outflow pathway structure similar to the primate. ^{4 5} However, although there is no more fundamental tool for investigating a disease than an animal model, one is still not available for POAG because it depends crucially for phenotypic assessment on IOP measurement in the mouse, noninvasively, at multiple time points and without the confounding effects on IOP of general anesthesia. We have adapted the Goldmann tonometer to the eyes of awake mice. 

Applanation tonometry is based on the Imbert-Fick hypothesis. ¹ Briefly, it holds that when a flat surface is pressed against a closed, thin-walled sphere with a given internal pressure, 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. The Goldmann tonometer has a transparent plastic applanating (flattening) tip in the shape of a truncated cone. A biprism (two prisms touching at their apices) within the tip optically doubles the image of the flattened surface as the tip contacts the cornea, with the two components separated by a fixed amount, dependent on the apex angles of the prisms. The area of contact is viewed through the tonometer tip with a slit lamp biomicroscope as force is applied to the cornea through the tip, which is connected by a lever arm to the tonometer body containing a variable weight, until the diameter of the applanated area reaches that fixed separation. 

Methods

Modified Applanation Tonometer

The eyes of mice are naturally protuberant, and so they are accessible to the applanating tip of this tonometer without reduction of its external dimensions and without having to retract the eyelids. Tonometer tips for empiric testing in the mouse (corneal diameter, 3.4 mm; radius of curvature, 1.75 mm; thickness, 0.1 mm) were fabricated with biprism angles less than the 48° required for the rat. ⁶ The body of the tonometer was modified to apply the same weight as used for the rat: 25 mg per Goldmann scale division (2 g full scale). 

Calibration

Calibration experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the tenets of the Declaration of Helsinki, and the guidelines of the Unit for Laboratory Animal Medicine (ULAM) of the University of Michigan. The testing was carried out in adult CS7 Black mice (ULAM; n = 5) under general anesthesia with intraperitoneal ketamine (100 mg/kg, Ketaset; Fort Dodge, Fort Dodge, IA) and xylazine (10 mg/kg, Rompun; Bayer, Shawnee Mission, KA). A 30-gauge cannula was placed through the peripheral cornea into the anterior chamber. An IOP pressure-regulating device ⁷ was interposed between the cannula and a pressure transducer (P23; Statham Instrument Co., Hato Rey, PR). IOP was recorded on a Grass Polygraph (Grass Instrument Co., Quincy, MA), which had been calibrated with a mercury manometer. IOP was adjusted to a series of pressure settings, both in random and in step fashion, always masked from the person who made an applanation reading at each setting from 0 to 50 mm Hg. 

Restraint of Awake Mice

Awake adult CS7 Black mice were restrained for applanation tonometry in a polyethylene cone (DecapiCone; Braintree Scientific, Inc., Braintree, MA) with its apex trimmed to allow exposure of the head. This method restrained mice securely, and they responded to it docilely. 

Applanation Tonometry in Awake Mice

Restrained, awake mice (n = 10) were positioned on a small platform at the slit-lamp biomicroscope. Whiskers were trimmed because if caught between the tonometer tip and the cornea, artifactually high readings can result, a version of which has been reported with human hair. ⁸ Applanation tonometry was performed just as it is in humans: after topical anesthesia with an eye drop of proparacaine hydrochloride 0.5% and another, minute drop of fluorescein sodium 2% dye to outline the applanated area. Repeated tonometer readings were made by one person as another person maintained the animal’s position; a series of applanation readings, to the nearest half scale division, was obtained in each eye, the right eye first, at intervals of 15 to 45 seconds. 

Results

Calibration

In mice under general anesthesia, calibration tests of the tonometer tips showed that a tip with biprism angles of 36° was appropriate for these eyes. 

The calibration results of a tonometer with this tip, compared by linear regression analysis of the applanation readings with the manometer measurements, obtained both in random and step fashion, showed good agreement across the range (0–50 mm Hg) of IOP tested (Fig. 1) . One applanation scale division on the tonometer dial corresponded to 1.19 ± 0.033 mm Hg (mean ± SE; 95% confidence interval [CI], 1.13, 1.26 mm Hg) IOP. 

Applanation Tonometry in Awake Mice

Because the means of the applanation readings in the conscious mice 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. In the conscious mouse the mean Goldmann value was 13.7 ± 3.78 mm Hg (mean ± SD; 95% CI, 13.1, 14.2 mm Hg). Because repeated applanations resulted in a decline in Goldmann readings to a plateau, this was taken into account by applying a nonlinear model to the pooled data to determine the number of tonometer contacts required to reach the asymptote:“ mathematical” stability in the mouse eye was reached at 5.4 ± 3.79 (mean ± SD; 95% CI, 4.1, 6.6) applanations. The mouse Goldmann mean value reported here is interpolated at the calculated point the plateau was reached. 

Discussion

The present study parallels those of Goldmann, ¹ who found empirically the combination of biprism angles in the applanation tonometer tip and weight applied from the body of the tonometer that flattened the appropriate area of the cornea to relate the tonometer readings to manometrically measured IOP. In the eyes of dogs, cats, and rabbits the required diameter of the area of corneal applanation was 4 mm. In human eyes the appropriate diameter of the applanated area was 3.06 mm, obtained with a tonometer tip with 60° biprism angles and an applanating weight of 100 mg per scale division (8 g full scale); the same combination was used in monkey eyes. In the rat ⁶ the Goldmann method required applanation of an area 2 mm in diameter, achieved with a tonometer tip with biprism angles of 48°, and the weight applied was 25 mg per Goldmann scale division (2 g full scale). In the present study of the mouse eye, the area of applanation was 1.5 mm in diameter, obtained with 36° biprism angles and the same applied weight as in the rat. 

Mean IOP in the awake mouse by Goldmann applanation tonometry is 13.7 mm Hg. It is 15.5 mm Hg in both conscious rats ⁶ and humans. ¹ In several mouse strains under general anesthesia, John et al. ⁹ reported IOP means from 7.7 to 13.7 mm Hg, measured via a cannula in the anterior chamber. In awake mice the deviation of applanation readings from a one-to-one relationship with manometric IOP can be adjusted for by using either a nomogram or slightly larger biprism angles in the tonometer tip. 

In the mouse the decline in Goldmann applanation readings to a plateau with repeated corneal touches is especially puzzling. Having speculated that this artifact of this tonometric method may be corneal in origin, ⁶ we had expected that it would be most marked in the mouse, because the ratios of the corneal to applanated diameters are 25% in human, 36.3% in rat, and 44.1% in mouse. However, the mouse mean decline (3.5 mm Hg) is closer to the human (∼2 to 4 mm Hg) ⁸ than is the rat (8.0 mm Hg). ⁶ So the source of this phenomenon is elusive in rodents as it has been in humans, the subject a generation ago of numerous studies with inconclusive results by among others the pioneer investigators of Goldmann applanation tonometry. ⁸  

The cause of the optic neuropathy of POAG continues to provoke controversy in large part because research in humans has had essentially fixed limitations: clinically, the optic nerve is studied descriptively, based on observation of its surface at low magnification; and human POAG tissue is scarce, usually from eyes harvested hours after death. In response to these limitations the optic nerves of monkeys have been studied after IOP was elevated by laser, damaging the pathway of aqueous humor outflow from the eye. ^{10 11} More recent studies in the rat, which has an aqueous humor outflow pathway structure similar to the primate, ¹² impeded ocular venous drainage by sclerosing ^{13 14} or by ligating ¹⁵ veins, and then elevation of IOP was detected under general anesthesia ^{16 17} and in conscious animals. ^{13 17 18} The results were qualitative because the measuring instruments were designed for the human and could not be modified for these small eyes. ¹⁹ Although the eye of the conscious rat is accessible to Goldmann tonometry, ⁶ the potential for obtaining IOP elevation in this species, spontaneously by breeding strategies or induced by genetic engineering, currently seems problematical. By contrast, the mouse system, especially powerful in investigating mammalian genetics, is the most practical candidate for inducing elevated IOP levels that mimic ocular hypertension in humans, whether by knockout or by overexpression techniques. Among the goals of such a mouse model will be contributions to a fuller understanding of mechanisms of IOP regulation, the role of IOP in the optic neuropathy of POAG, and evaluation of new pharmacological, laser, and surgical approaches to glaucoma therapy. 

 Supported in part by research grants from the Lloyd and Mabel Johnson Foundation and from Keith A. Kobet, MD.

 Submitted for publication March 16, 2001; revised May 23, 2001; accepted June 19, 2001.

 Commercial relationships policy: N.

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2001.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Figure 1.

 

Calibration of the modified Goldmann applanation tonometer in the eyes of two mice. Comparison was made by linear regression analysis of IOP measured manometrically from a cannula in the anterior chamber and readings made with the tonometer as IOP was adjusted with a pressure-regulating device to settings from 0 to 50 mm Hg. In one mouse, pressure settings were adjusted in random fashion (○); in another mouse, pressure settings were adjusted in sequences of ascending and descending steps (•).

 

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. Melissa S. Aniol provided technical assistance in mouse restraint.