Documenta Ophthalmologia 108: 181–184, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Improved contact lens electrode for corneal ERG recordings in mice Botir T. Sagdullaev1, Paul J. DeMarco1,2,3 & Maureen A. McCall1,2 1
Department of Psychological and Brain Sciences, University of Louisville, Louisville, KY 40292, USA; Department of Ophthalmology and Visual Science, University of Louisville, Louisville, KY 40292, USA; 3 Louisville VA Medical Center, Louisville, KY 40208, USA 2
Accepted 4 February 2004
Key words: contact lens, electrode, electroretinogram (ERG), mouse
Abstract The electroretinogram (ERG) is routinely used to study retinal physiology in the clinic and in research. Due to their outstanding properties, contact lens electrodes (CLEs) are widely used for ERG recordings. Though the procedures for ERG recordings in mice are similar to those used in humans and larger vertebrates, use of CLEs in the mouse has been limited because of difficulties involved with the manufacturing of small contact lenses. We describe a simple instrument and method for manufacturing contact lenses and CLEs for stable ERG recordings in mice. The instrument operates like a hole-punch and is based on slip joint pliers incorporating a ball bearing on one jaw and forming plate on the other. These CLEs are simple to manufacture, inexpensive and provide stable, long-term recordings of corneal ERGs in mice. With minor modifications, these CLEs could be made for other small animals such as rats or fish.
Introduction The electroretinogram (ERG), a non-invasive tool for evaluating retinal function, is the metric of choice for screening mutants and genetically modified animals, [1–3]. It has become increasingly important to develop simple and standardized techniques and tools for recording the rodent ERG. Corneal drying however is a major problem when recording ERGs from an anesthetized animal. While this can be prevented by frequent irrigation of the corneal surface, it is not feasible since each application of fluid to the cornea results in swift changes in recording parameters due to the shunting effect of the fluid. Contact lenses provide a barrier to protect corneas from drying and to secure the placement of conducting material. Contact lens electrodes (CLEs) are widely used to record ERG in humans and large animals [4–9]. Nevertheless, due to technical difficulties in manufacturing small contact lenses, they have not been widely implemented in mice [5, 6, 10].
Here, we describe a simple and inexpensive instrument and method for manufacturing corneal contact lenses and CLEs for mice. These electrodes provide stable, long-term recordings of corneal ERG in mice and protect corneal surface. They have been extensively used in our lab to record ERGs from mice and rats. With minor modifications, CLEs could be made for other small animals including fish. Materials, methods and results Production of the contact lens Figure 1A shows the instrument designed in our laboratory for manufacturing the corneal contact lenses. This tool is made from standard 6-inch slip joint pliers whose jaws advanced in parallel to each other when squeezed. This feature is essential to yield an even curvature of the lens and smoothly cut edges. In addition, the loose joint allows the ball bearing to enter the hole in the forming plate in a self-guided manner, even if
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Figure 1. Instrument for manufacturing the contact lens and CLE for mouse. (A) Photograph and schematic drawing of the instrument for manufacturing a contact lens. See description in the text. Diameter of a ball bearing on the top jaw and a hole in the forming plate attached to the bottom jaw for manufacturing lenses for mouse is 3 mm. All measurements are in mm. (B) The photograph of a corneal lens electrode produced using instrument shown in A. Slits are cut through the edges of the contact lens and a conducting fiber is run across the inner surface of the contact lens. The fiber is held by a small alligator clip, which is then attached to a wire leading to a pre-amplifier.
they are not perfectly aligned during the machining of the separate parts. This simplifies the manufacture of the instrument using commonly available tools. Both jaws of the pliers are filed to remove the serrations. To manufacture a contact lens to fit a mouse eye, a 3 mm chrome steel ball bearing (Small Parts, Inc., Miami Lakes, FL) is soldered onto the top jaw in the guiding hole (3 mm wide by 1 mm deep). The curvature of the ball bearing approximates that of the adult (19–40 g) mouse cornea [11]. Abrading the ball bearing and applying an appropriate solder flux guarantees the bond between the ball bearing and the solder. A tapered hole aligned approximately to the placement of the ball bearing in the opposite jaw is drilled through the bottom jaw (see the Figure 1A). A hole of the same diameter as a ball
bearing is drilled through a rectangular piece of forged spring steel (available as a flat strip from Small Parts, Inc, or cut from a flat ACCO brand Banker’s clasp). The size of the hole on the forming plate should match precisely the diameter of the ball bearing, since it provides the cutting edge for the lens material. This plate is sanded to its final dimensions indicated in Figure 1A, polished on one side, abraded on another and glued with abraded side down to the bottom jaw of the pliers using an industrial strength twopart epoxy. The material used to produce the contact lenses is Aclar embedding film (Honeywell International, Inc.), 7.8 MIL (0.198 mm thick) (Ted Pella, Inc., Redding, CA). Aclar fluoropolymer film is optically clear (including the UVrange), biochemically inert, and it possesses a very low coefficient of friction (Honeywell datasheets found at www.aclar.com). The actual production of a lens is similar to the operation of a hole-punch. A sheet of film is inserted between the two jaws, and the jaws slowly squeezed to extrude a lens. The lens is formed and cut as the ball bearing fully enters the hole in the forming plate. A standard lens is shown in Figure 1B. Incorporating the conducting fiber into the contact lens The electrode material we use is a silver-coated 22/1-denier nylon fiber (Select Fabricators, Palmyra, NY, AgMedTex 22/1), modeled after the DTL fiber electrode [12, 13] (for more information www.ISCEV.org). In our initial experiments we used a multi-stranded fiber, however, a singlestranded fiber was preferred because it is easier to insert it through the slits in the contact lens. In addition, it forms a more secure contact with the alligator clip that we use to connect the electrode to the amplifier. To construct the CLE, small slits are cut into opposite edges of the contact lens. A piece of electrode fiber is then loosely inserted through the two slits. The thread is cut close to the lens at one end, and left approximately 1 cm long at the other end of the lens so that a small alligator clip can grasp it. The completed CLE is shown in Figure 1B. A drop of artificial tears containing 1% methylcellose is placed in the concavity of the
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Figure 2. Amplitude of the ERG b-wave during long-term recording. Variation in ERG b-wave amplitude over a 2-h period, measured from a mouse wearing a CLE. Each time point represents the average of of 30 ERG responses to a 10 ms white flash. Series of responses are presented at 5 min intervals. Silver needle electrodes, inserted under the skin of the bridge of the nose and tail, serve as reference and ground respectively. Bridged electrode impedance in this situation is typically below 15 K X. The mouse is light adapted to a 40 cd m)2 white background and the stimulus intensity is 220 cd m)2 s)1. The ERG is amplified (10 000·), band-pass filtered from 0.1–1000 Hz, and digitized at 5000 samples/s. The mouse is anesthetized with 25 mg/kg xylazine and 60 mg/ kg ketamine. Anesthesia is maintained by supplemental injections every 30–40 min. All animal protocols were approved by the Institution’s Animal Care and Use Committee and adhered to The Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research.
lens. This assists in adhering the CLE to the cornea, moisturizes the cornea and forms a salt bridge between the cornea and the electrode fiber. On occasion, a contact lens will spontaneously pull away from the eye if the electrode fiber is bent in a way to place tension on the lens. Thus attention must be paid to the bend in the fiber. In addition, applying a thin layer of petroleum jelly or other viscous ophthalmic lubricant around the edge of the lens assists in sealing the lens to the cornea and preventing evaporation of the artificial tear solution during long-term recordings. An example of data obtained during a longterm (2 h) ERG recording is shown in Figure 2. The b-wave amplitude did not change significantly over the recording session. Summary This instrument simplifies the manufacturing of contact lenses and CLEs to record the ERG in
mice. The instrument and contact lenses are easily fabricated from readily available materials and are inexpensive. In addition, the process we describe here has the advantage that heat is not used to form the Aclar, as has been mentioned in other ERG studies [14]. The CLEs constructed yield stable ERG recordings. During long-term recordings as well as multiple ERG screenings from the same animal, no damage to the cornea was evident if a sufficient amount of the artificial tear solution was applied to lubricate/moisturize the cornea. In agreement with earlier reports [5], we observed that CLEs once mounted required almost no adjustments or reinsertion, which provides stability of the recording and decreases the chances of damaging the corneal surface. Incorporating the conducting material into the contact lens facilitates mounting them on the corneal surface and securing a good electrical contact. We have found this feature useful when placing the electrodes onto small eyes under dim red light conditions needed for dark adaptation experiments. With some minor modifications of the ball bearing size and the forming plate, this technique could be used to produce contact lenses of various sizes to fit the eyes of other animals, such as fish or rats.
Acknowledgements This work was supported by NSF ISBN0079388, and the Department of Veterans Affairs.
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