- Email: [email protected]
Novel method of chronically blocking retinal activity
Journal of Neuroscience Methods 87 (1999) 105 – 110
Novel method of chronically blocking retinal activity Glen T. Prusky a,*, Ary S. Ramoa b a
Department of Psychology and Neuroscience, The Uni6ersity of Lethbridge, 4401 Uni6ersity Dri6e, Lethbridge, Alberta, T1K 3M4 Canada b Department of Anatomy, Virginia Commonwealth Uni6ersity, Medical College of Vireirlia, Richmond, VA 23298 -0709, USA Received 12 October 1998; accepted 18 October 1998
Abstract The ethylene–vinyl acetate copolymer Elvax has been used as a vehicle to deliver bioactive substances to discrete areas of the nervous system. Here we report a novel use of Elvax to chronically block retinal activity. Small pieces of Elvax containing the sodium channel blocker tetrodotoxin (TTX) were surgically implanted into the vitreous humor of ferret eyes. Observations of the light-induced pupillary reflex combined with electrophysiological assays of vitreous humor confirmed that these implants completely blocked retinal activity for up to 25 days without apparent retinal damage. The advantages of this procedure over previous methods requiring multiple daily injections of TTX, and alternative experimental applications are discussed. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Elvax; LGN; Neural activity; Retina; Surgery; Sodium channel; Tetrodotoxin; TTX
1. Introduction Retinal activity has been shown to play an instructive role in regulating the form and function of the mammalian visual system (Shatz, 1990). The most striking demonstrations of the importance of retinal activity have blocked the generation of action potentials in retinal ganglion cells with the sodium channel blocker tetrodotoxin (TTX) and measured the resulting anatomical (Wong et al., 1991; Antonini and Stryker, 1993; Deyoe et al., 1995; Scheetz et al., 1995) and physiological (Chapman et al., 1986; Ramoa and Prusky, 1997) effects on the nervous system. Typically, TTX is injected through a steel needle into the vitreous humor of the eye where it dissipates and binds to sodium channels on cell membranes throughout the retina. While TTX delivered in this form is effective in blocking retinal ganglion cell activity, such intraocular injections have a number of disadvantages. First, the effective dose of TTX can be reduced within a few * Corresponding author. Tel.: +1-403-3295161; Fax: + 1-4033292555; e-mail: [email protected].
hours of an injection by the turnover of intraocular fluids (Barza, et al., 1983; Barza and McCue, 1983; Friedrich et al., 1997) and leakage of vitreous humor out of the hole created by the needle. Consequently, multiple daily injections may be required throughout an experiment to continuously block retinal activity. Since many experiments require activity blockade for many days, the likelihood of damaging the retina under these conditions is also considerable. Furthermore, in order to ensure constant retinal activity blockade in young animals. the concentration of injected TTX must be near a lethal dosage (unpublished observations). Second, the administration of anesthesia and manipulations that accompany intraocular injections can induce nonspecific physiological effects on the animals, including weight loss and the production of stress hormones that may affect the experimental outcome (Van Sluyters and Oberdorfer, 1991). Consequently, the development of techniques that reduce the number of surgical cycles are desirable. Third, the ability to monitor the effectiveness of TTX injections with a pupillary reflex can be compromised by retinal damage induced by the surgery. If unilateral injections are used, the consensual
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G.T. Prusky, A.S. Ramoa / Journal of Neuroscience Methods 87 (1999) 105–110
pupillary reflex in the contralateral eye can be examined to monitor activity blockade. however, this possibility is removed in experiments where bilateral retinal activity blockade is necessary. This paper describes an alternative to this methodology by using slow-release TTX implants. The ethylene vinyl co-polymer Elvax (DuPont) is normally used in the production of laminating adhesives, wood primers and road paints. However, it has also been utilized as an inert vector for the release of macromolecules (Langer and Folkman, 1976; Rhine et al., 1980) and delivery of low molecular weight drugs (Pasic and Rubel, 1989; Udin and Scherer, 1990; Chiaia et al., 1992; Berry et al., 1996; Scheetz, et al., 1996) to specific regions of the nervous system. A number of methodologies for the production of these implants have been reported (Rhine et al., 1980; Silberstein and Daniel, 1982; Smith et al., 1995), however, these vary widely in complexity and practicality. We have evaluated many different procedures for making slow-release Elvax and have adopted a simple method of producing Elvax containing an even distribution of TTX. We also report on a technique to implant small pieces of this Elvax into the vitreous humor of ferret eyes that are capable of continuously releasing TTX and blocking retinal activity for up to 25 days. Finally, we describe an electrophysiological assay of the vitreous humor that complements measures of pupillary reflex to monitor the effectiveness of the TTX.
2. Methods
2.1. El6ax washing Elvax 40-W (previously identified by DuPont as Elvax 40-P), was used as a slow release polymer in these experiments. Trace amounts of the antioxidant butylhydroxy-toluene (BHT) are added to Elvax during the manufacturing process to prevent discoloration over time. Although Elvax itself is biologically inert, BHT can be mildly inflammatory to some biological tissues and was removed with a washing protocol. Elvax beads were first placed in a beaker with 20 volumes of distilled water and a magnetic stirring bar. The beaker was then covered tightly with Parafilm and stirred at 40°C for at least 2 h/wash for five washes. In a similar manner, the beads were washed with 95% ethanol for 10 washes, followed by another five washes with 100% ethanol. The beads were then decanted through a Coors porcelain filter funnel without filter paper and 10 ml of each washing solution was used to confirm BHT removal by examining spectrophotometric absorption at 230 nm. Langer et al. (1987) reports that the initial absorbance may be 2 – 3 units and the final readings should be less than 0.02 units. Our washing resulted in
final BHT values below 0.1 units. After all washes were completed, the beads were placed in a Petri dish, dried overnight at 40°C, and stored in a sealed and labeled container at room temperature.
2.2. El6ax preparation A sample of 100 mg of washed Elvax beads were placed in a capped, 12 × 45 mm, flat-bottomed, glass scintillation tube containing 1 ml methylene chloride (Sigma). After 2–3 h, the Elvax was completely depolymerized and the solution was mixed on a Vortex. Two milligrams of TTX (Sankyo, supplied through Calbiochem) was dissolved in 20 m1 of citrate buffer and the solution was sonicated until clear. This was then added to the Elvax/methylene chloride mixture, the vial was recapped, and the entire solution was vortexed (at least 5 min at high speed) until the drug was completely disbursed throughout the fluid in very small droplets. Mixing was best accomplished by repeatedly holding the vial against the edge of the Vortex cap at a 45° angle for a few seconds, and then allowing it to spin in a circumferential fashion around the inside perimeter of the cap for a few seconds. The former disbursed the water droplets in the Elvax solution and the latter spun the air bubbles out. Ten microlitres of a 1% Fast Green (Sigma) solution (DMSO; Sigma) was then added to color the solution and mixed as above on the Vortex. On the flnal mix, special care was taken to ensure that all air bubbles were spun out of the mixture so they did not become a permanent fixture in the final suspension. Once mixing was completed, the tube was uncapped and immediately placed upright on dry ice in order to freeze the solution into a cylindrical shape. The frozen mixture was then moved into −70°C where it remained for 4 days, followed by another 4 days at − 20°C. This procedure slowly evaporated the methylene chloride and reduced the frozen Elvax into a cylindrical-shaped pellet the original volume of the raw beads. The TTX impregnated Elvax was cut (200–400 mm) in round crossections on a freezing microtome, after gluing the pellet to the stage with cyanoacrylate and embedding it in frozen Tissue-Tek (Sakura). Individual sections were mounted on a gelatinized slide and stored at −20°C until they were used. Before surgery, small strips were cut from the Elvax and washed for a few seconds in 70% EtOH. The strips were then washed in sterile saline for 30 min to remove TTX from the surface of the polymer. Control Elvax containing only citrate buffer was produced and treated in an otherwise identical fashion.
2.3. Surgery Male and female ferrets ranging in age from postnatal day (P)0 to adult were used as experimental subjects
G.T. Prusky, A.S. Ramoa / Journal of Neuroscience Methods 87 (1999) 105–110
in this study. Animals were anesthetized by trifluoroethane (to effect in a vapor enclosure mixed with room air) or sodium pentobarbital (35 mg/kg, i.p.). A topical antiinflammatory agent (prednisolone acetate, Pred Forte, Allergan) was applied to the eye and a small incision was made in the sclera near the limbus with a micro unitome knife (Beaver) to gain access to the vitreous chamber. Elvax strips were inserted through the hole and placed in the vitreous humor adjacent to the lens, taking care to avoid direct contact with the retina and lens. The number and size of the implanted strips was determined by the age of the animal and the size of the eye. In small neonatal eyes, usually two strips (approx. 2 ×0.2 × 0.2 mm) were implanted, and by adulthood, up to four larger pieces (approx. 4×2×0.2 mm) were used. Animals were recovered and monitored for behavioral signs of surgery-induced abnormalities. We observed no postoperative inflammation or infection in the vast majority of the animals in this study. The few animals that experienced post-operative complications were removed as subjects. Both monocular and binocular surgeries were undertaken, however, binocular implants were most successful in animals older than P14. Following eye opening, light-induced pupillary reflexes were examined in the stimulated and the non-stimulated eyes daily to monitor retinal activity. The lack of a pupillary reflex in the stimulated eye could, in some circumstances, be due to a blockade of neural input or damage to the iris. However, we also tested the consensual reflex in the non-stimulated eye in animals with unilateral implants, which is only blocked if retinal activity in the stimulated eye is blocked.
2.4. Electrophysiology In order to obtain direct evidence of the effectiveness of the TTX implants in animals before eye opening, electrophysiological assays of the vitreous hurnor were undertaken. Samples of vitreous fluid were obtained from the eyes of animals implanted with Elvax at different time intervals following the surgery. Approximately 1 ml of vitreous was collected by placing the broken tip of a micropipette, pulled from borosilicate glass capillary (TW100F-4, WPI), into a small incision made near the limbus. Removing the vitreous by capillary action had the advantage of causing minimal eye disturbance. As a result, animals could be used for additional testing, including extraction of vitreous fluid, tract tracing or electrophysiological recordings. The effect of the vitreous fluid on the generation of action potentials in the lateral geniculate nucleus (LGN) of ferrets was tested. Vibratome-cut slices (400 mm) containing the LGN were obtained from deeply anesthetized (100 mg/kg sodium pentobarbital, i.p.) ferrets as described previously (Ramoa and Mc-
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Cormick, 1994). Slices were placed in an interface-type chamber (FST) where they rested on lens paper. To increase mechanical stability, a piece of nylon mesh was placed over the slices. In this way the flow rate could be changed from null (during the local application of the vitreous), to a high flow rate within seconds that lead to superfusion of the slices (for washing-out vitreous). Approximately 2 h following placement of slices in the chamber, extracellular recordings of action potential activity was conducted using a tungsten-in-glass electrode (Levick, 1972) lowered into one of the geniculate layers. Retinal afferents in the LGN activated with constant current stimuli (100 ms duration, 30–300 mA intensity, 10–20 s intervals) were delivered through a bipolar stimulating electrode positioned in the optic tract. The tip of the pipette containing the vitreous fluid was positioned near the recording site and vitreous was pressure ejected while action potentials were recorded. Supplementary evidence of the efficacy of the TTX treatments was derived from experiments in which the pupillary reflex was examined in the eyes of rats. A 2–5 ml sample of vitreous fluid was removed from the eyes of ferrets implanted with Elvax. This sample was then injected into one eye of a P25 rat under anesthesia (30 mg/kg sodium pentobarbital). After recovery from the surgery, the pupillary reflex in the eye was tested.
3. Results
3.1. El6ax preparation The Elvax preparation protocol produced cylindricalshaped pellets of Elvax (approx. 7 mm dia.× 4 mm h; Fig. 1C) from an initial 1 ml volume of solution.
Fig. 1. Prepared Elvax mounted on a glass slide. (A). Series (top right to bottom left) of 200 mm thick sections of Elvax impregnated with TTX. The annular shape of the first four sections is the result of cutting through the concave top of the Elvax block. (B). Small strips cut from an Elvax section suitable for intraocular implantation. (C). Top (left) and side (right) views of the cylinder-shaped blocks of prepared Elvax before sectioning. The width of the blocks is 7 mm.
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Fig. 2. Effects of vitreous fluid from TTX-treated and control eyes on action potential activity in the slice preparation. Examples of extracellular recordings show that neuronal action potential activity evoked by optic tract stimulation was reversibly blocked (A,B,C) following local application of vitreous fluid obtained from a P12 ferret injected at P9 implanted with Elvax containing TTX. In contrast, action potential activity in a different slice was not affected (D,E) by local application of vitreous fluid collected from an eye treated with Elvax containing only citrate buffer. Each panel shows ten superimposed recordings.
Sections of Elvax (200 – 400 mm) (Fig. 1A) contained an even distribution of spheres formed by droplets containing dissolved TTX. Small strips cut from the sections were used as implants (Fig. 1B).
3.2. Pupillary reflex Elvax implants containing TTX displayed chronic and consistent sustained release properties as measured with a light-induced pupillary reflex in the stimulated eyes and the consensual reflex in non-stimulated eyes. The pupillary reflex was absent in all animals implanted with TTX-Elvax by the time they recovered from anesthesia. This reflex was absent for at least 7 days in all animals and up to 25 days in some cases. After 25 days, the pupillary reflex recovered in all animals. Since a normal pupillary reflex is dependent upon an intact retina, its recovery following sustained TTX blockade indicates that the implantation surgery did not substantially damage the retina. Animals implanted with control Elvax exhibited a robust pupillary reflex immediately following the surgery and for the duration of the experiments.
3.3. Electrophysiology When vitreous humor obtained from the eyes of animals implanted with TTX-Elvax for 3 days was pressure ejected near LGN neurons, it induced a complete and reversible cessation of action potential activity (Fig. 2). Activity resumed within 1 – 2 min of terminating the puffing, confirming that the vitreous
humor contained sufficient TTX to effectively block neuronal sodium channels. Similar effects were observed in all samples of vitreous fluid collected from TTX-treated eyes following 1 day (n= 4), 3 days (n=4) and 4 days (n=3) of intraocular implantation. There was no noticeable difference in the ability of the vitreous fluid to block action potentials whether it was drawn from a location near the implant or not, suggesting that TTX diffused throughout the entire eye. In contrast, the vitreous fluid collected from eyes (n=7) treated with control Elvax did not affect action potential activity of LGN neurons as illustrated in Fig. 2. Additional confirmation of TTX-Elvax effectiveness was obtained in rats (n= 2) injected intraocularly with the vitreous fluid of ferrets (n= 2) treated for 3 days. These animals displayed a reversible loss of their pupillary reflex, while animals (n= 2) injected with vitreous fluid extracted from control eyes showed no effect.
4. Discussion The Elvax preparation method described in this paper is a simple and effective way to create TTX impregnated Elvax with reproducible release properties. It is important when curing the Elvax that the frozen methylene chloride/Elvax solution evaporates slowly so that a homogeneous matrix is maintained. Frost-free freezers should be avoided when using this method, since they increase their temperature to near 0°C daily to reduce the amount of condensation in the chamber. This defrosting cycle allows the methylene chloride to evaporate rapidly, Causing air bubbles to form inside the matrix. It is also crucial that the scintillation tube is uncapped in the freezer to allow the evaporating fumes to escape. Methylene chloride is toxic and precautions should be taken to avoid inhaling the fumes or having them contaminate something else in the freezer. We have tried making larger Elvax blocks by dissolving 200 mg of Elvax in 2 ml methylene chloride, however, there is a high probability of air bubbles forming in the solution as a result of the polymerizing surface of the cylinder preventing the escape of evaporated methylene chloride at the core. Unless there is good reason to make different-shaped pellets to generate very large slabs of Elvax, a 1 ml solution in scintillation tubes will work reliably and generate relatively large sections. Inferior quality Elvax is also produced if more than 20 ml of a drug solution is added to the 1 ml Elvax/Methylene chloride mixture. The increased volume of fluid in the polymer results in a lower density matrix that is difficult to cut and has less reliable release properties. The Elvax implantation technique used in this study undoubtedly produces less experimental trauma than previous intraocular injection protocols. Because an effective dose of TTX is released
G.T. Prusky, A.S. Ramoa / Journal of Neuroscience Methods 87 (1999) 105–110
from the Elvax in a sustained manner for 7 – 25 days, this eliminates the need to make multiple daily injections of TTX to continuously block retinal activity. In addition, the cumulative detrimental effects of multiple surgical procedures are reduced. We have found that implanting multiple small strips of Elvax is more effective than implanting one large piece. Small strips allow for a smaller incision in the eye, thereby reducing the overall trauma and leakage of intraocular fluid. In addition, the release of TTX from multiple polymer strips may provide a more even distribution of TTX throughout the vitreous than release from a single implant. If longer duration release than that described in this paper is desired, we suggest that surgical implants of new TTX/Elvax be made every 7 – 25 days. We are currently investigating whether thicker sections of Elvax implanted in the eye release TTX for longer periods of time. In one animal, we have implanted two strips of TTX-Elvax approximately 4×1 ×0.4 mm, and achieved reversible blockade of retinal activity for 25 days. We expect in some experiments, however, that any added benefit in TTX release time will have to be balanced against the detrimental effects of making a larger incision in the sclera to accommodate the larger strip of Elvax. We have not tried to increase the concentration of TTX in the Elvax beyond 2 mg/100 mg to produce longer duration implants, since it is very difficult to dissolve more than 2 mg of TTX/sodium citrate in 20 ml of solution. In addition, the duration of release is more likely a property of the polymer matrix than the concentration of the TTX. The methodology described in this paper could be used to investigate the effect of many different drugs on specific aspects of visual function. TTX has the property of nonspecifically blocking all action potential activity in retinal ganglion cells, thus enabling the use of a pupillary reflex to monitor the sustained release properties of TTX impregnated Elvax. The pupillary reflex can not always be used as a measure of a drug’s effectiveness in the retina, since most drugs target a subset of receptors on cells not necessarily linked to the function of the pupillary reflex. Under these circumstances, an electrophysiological assay like that described here could be developed to test the efficacy of a particular drug. In addition, a high performance liquid chromatography (HPLC) analysis of the vitreous humor may be able to establish the precise concentration of a drug in the intraocular fluid. Collectively, the results of this paper indicate that the sustained release of pharmacological agents from Elvax implanted in the eye can be used to examine the role of retinal activity and retinal neurotransmis-
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sion in visual system development, even before patterned visual function is possible.
Acknowledgements This work was supported by a NSERC research grant to G.T.P., and by NSF (IBN-9431983) and Whitehall Foundation grants to A.S. Ramoa.
References Antonini A, Stryker MP. Development of individual geniculocortical arbors in cat striate cortex and effects of binocular impulse blockade. J Neurosci 1993;13:3549 – 73. Barza M, Kane A, Baum J. Pharmacokinetics of intravitreal carbenicillin, cefazolin, and gentamicin in rhesus monkeys. Invest Ophthalmol Vis Sci 1983;24:1602 – 1606. Barza M, McCue M. Pharmacokinetics of aztreonam in rabbit eyes. Antimicrob Agents Chemother 1983;24:468 – 73. Berry FB, Prusky GT, Brown IR. Alteration of CamI mRNA expression in the developing rat superior colliculus following chronic treatment with an NMDA receptor antagonist. Dev Brain Res 1996;91:171 – 80. Chapman B, Jacobson MD, Reiter HO, Stryker MP. Ocular dominance shift in kitten visual cortex caused by imbalance in retinal electrical activity. Nature 1986;324:154 – 6. Chiaia NL, Fish SE, Bauer WR, Bennett-Clarke CA, Rhoades RW. Postnatal blockade of cortical activity by tetrodotoxin does not disrupt the formation of vibrissa-related patterns in the rat’s somatosensory cortex. Dev Brain Res 1992;66:244 – 50. Deyoe EA, Trusk TC, Wong-Riley MT. Activity correlates of cytochrome oxidase-defined compartments in granular and supragranular layers of primary visual cortex of the macaque monkey. Vis Neurosci 1995;12:629 – 39. Friedrich S, Saville B, Cheng YL. Drug distribution in the vitreous humor of the human eye: the effects of aphakia and changes in retinal permeability and vitreous diffusivity. J Ocul Pharmacol Ther 1997;13:445 – 59. Langer R, Folkman J. Polymers for the sustained release of proteins and other macromolecules. Nature 1976;963:797–800. Langer R, Brown L, Edelman E. Controlled release and magnetically modulated release systems for macromolecules, in: Methods in Enzymology, vol. 142, 1987, pp. 399 – 422. (Method 4 p. 404). Levick WR. Another tungsten rnicroelectrode. Med Biol Eng 1972;10:510 – 5. Pasic TR, Rubel EW. Rapid changes in cochlear nucleus cell size following blockade of auditory nerve electrical activity in gerbils. J Comp Neurol 1989;283:474 – 80. Ramoa AS, McCormick DA. Developmental changes in electrophysiological properties of LGNd neurons during reorganization of retinogeniculate connections. J Neurosci 1994;14:2089–97. Ramoa AS, Prusky GT. Retinal activity regulates developmental switches in functional properties and ifeprodil sensitivity of NMDA receptors in the lateral geniculate nucleus. Dev Brain Res 1997;101:165 – 75. Rhine WD, Hsieh DS, Langer R. Polymers for sustained macromolecule release: procedures to fabricate reproducible delivery systems and control release kinetics. J Pharm Sci 1980;69:265– 70. Scheetz AJ, Prusky GT, Constantine-Paton M. Chronic receptor antagonism during retinotopic map formation depresses CaM
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kinase II differentiation in rat superior colliculus. Eur J Neurosci 1996;8:1322 – 8. Scheetz AJ, Williams RW, Dubin MW. Severity of ganglion cell death during early postnatal development is modulated by both neuronal activity and binocular competition. Vis Neurosci 1995;12:605 – 10. Shatz CJ. Impulse activity and the patterning of connections during CNS development. Neuron 1990;5:745–56. Silberstein GB, Daniel CW. Elvax 40P implants: sustained, local release of bioactive molecules influencing mammary ductal development. Dev Biol 1982;93:272–8. Smith AL, Cordery PM, Thompson ID. Manufacture and release
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characteristics of Elvax polymers containing glutamate receptor antagonists. J Neurosci Methods 1995;60:211 – 7. Udin SB, Scherer WJ. Restoration of the plasticity of binocular maps by NMDA after the critical period in Xenopus. Science 1990;249:669 – 72. Van Sluyters RC, Oberdorfer MD. Preparation and maintenance of higher mammals during neuroscience experiments. Report of a National Institutes of Health Workshop. NIH publication 1991; c 91-3207:37 – 9. Wong RO, Herrmann K, Shatz CJ. Remodeling of retinal ganglion cell dendrites in the absence of action potential activity. J Neurobiol 1991;22:685 – 97.
Novel method of chronically blocking retinal activity Glen T. Prusky a,*, Ary S. Ramoa b a
Department of Psychology and Neuroscience, The Uni6ersity of Lethbridge, 4401 Uni6ersity Dri6e, Lethbridge, Alberta, T1K 3M4 Canada b Department of Anatomy, Virginia Commonwealth Uni6ersity, Medical College of Vireirlia, Richmond, VA 23298 -0709, USA Received 12 October 1998; accepted 18 October 1998
Abstract The ethylene–vinyl acetate copolymer Elvax has been used as a vehicle to deliver bioactive substances to discrete areas of the nervous system. Here we report a novel use of Elvax to chronically block retinal activity. Small pieces of Elvax containing the sodium channel blocker tetrodotoxin (TTX) were surgically implanted into the vitreous humor of ferret eyes. Observations of the light-induced pupillary reflex combined with electrophysiological assays of vitreous humor confirmed that these implants completely blocked retinal activity for up to 25 days without apparent retinal damage. The advantages of this procedure over previous methods requiring multiple daily injections of TTX, and alternative experimental applications are discussed. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Elvax; LGN; Neural activity; Retina; Surgery; Sodium channel; Tetrodotoxin; TTX
1. Introduction Retinal activity has been shown to play an instructive role in regulating the form and function of the mammalian visual system (Shatz, 1990). The most striking demonstrations of the importance of retinal activity have blocked the generation of action potentials in retinal ganglion cells with the sodium channel blocker tetrodotoxin (TTX) and measured the resulting anatomical (Wong et al., 1991; Antonini and Stryker, 1993; Deyoe et al., 1995; Scheetz et al., 1995) and physiological (Chapman et al., 1986; Ramoa and Prusky, 1997) effects on the nervous system. Typically, TTX is injected through a steel needle into the vitreous humor of the eye where it dissipates and binds to sodium channels on cell membranes throughout the retina. While TTX delivered in this form is effective in blocking retinal ganglion cell activity, such intraocular injections have a number of disadvantages. First, the effective dose of TTX can be reduced within a few * Corresponding author. Tel.: +1-403-3295161; Fax: + 1-4033292555; e-mail: [email protected].
hours of an injection by the turnover of intraocular fluids (Barza, et al., 1983; Barza and McCue, 1983; Friedrich et al., 1997) and leakage of vitreous humor out of the hole created by the needle. Consequently, multiple daily injections may be required throughout an experiment to continuously block retinal activity. Since many experiments require activity blockade for many days, the likelihood of damaging the retina under these conditions is also considerable. Furthermore, in order to ensure constant retinal activity blockade in young animals. the concentration of injected TTX must be near a lethal dosage (unpublished observations). Second, the administration of anesthesia and manipulations that accompany intraocular injections can induce nonspecific physiological effects on the animals, including weight loss and the production of stress hormones that may affect the experimental outcome (Van Sluyters and Oberdorfer, 1991). Consequently, the development of techniques that reduce the number of surgical cycles are desirable. Third, the ability to monitor the effectiveness of TTX injections with a pupillary reflex can be compromised by retinal damage induced by the surgery. If unilateral injections are used, the consensual
0165-0270/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 7 0 ( 9 8 ) 0 0 1 7 2 - 1
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pupillary reflex in the contralateral eye can be examined to monitor activity blockade. however, this possibility is removed in experiments where bilateral retinal activity blockade is necessary. This paper describes an alternative to this methodology by using slow-release TTX implants. The ethylene vinyl co-polymer Elvax (DuPont) is normally used in the production of laminating adhesives, wood primers and road paints. However, it has also been utilized as an inert vector for the release of macromolecules (Langer and Folkman, 1976; Rhine et al., 1980) and delivery of low molecular weight drugs (Pasic and Rubel, 1989; Udin and Scherer, 1990; Chiaia et al., 1992; Berry et al., 1996; Scheetz, et al., 1996) to specific regions of the nervous system. A number of methodologies for the production of these implants have been reported (Rhine et al., 1980; Silberstein and Daniel, 1982; Smith et al., 1995), however, these vary widely in complexity and practicality. We have evaluated many different procedures for making slow-release Elvax and have adopted a simple method of producing Elvax containing an even distribution of TTX. We also report on a technique to implant small pieces of this Elvax into the vitreous humor of ferret eyes that are capable of continuously releasing TTX and blocking retinal activity for up to 25 days. Finally, we describe an electrophysiological assay of the vitreous humor that complements measures of pupillary reflex to monitor the effectiveness of the TTX.
2. Methods
2.1. El6ax washing Elvax 40-W (previously identified by DuPont as Elvax 40-P), was used as a slow release polymer in these experiments. Trace amounts of the antioxidant butylhydroxy-toluene (BHT) are added to Elvax during the manufacturing process to prevent discoloration over time. Although Elvax itself is biologically inert, BHT can be mildly inflammatory to some biological tissues and was removed with a washing protocol. Elvax beads were first placed in a beaker with 20 volumes of distilled water and a magnetic stirring bar. The beaker was then covered tightly with Parafilm and stirred at 40°C for at least 2 h/wash for five washes. In a similar manner, the beads were washed with 95% ethanol for 10 washes, followed by another five washes with 100% ethanol. The beads were then decanted through a Coors porcelain filter funnel without filter paper and 10 ml of each washing solution was used to confirm BHT removal by examining spectrophotometric absorption at 230 nm. Langer et al. (1987) reports that the initial absorbance may be 2 – 3 units and the final readings should be less than 0.02 units. Our washing resulted in
final BHT values below 0.1 units. After all washes were completed, the beads were placed in a Petri dish, dried overnight at 40°C, and stored in a sealed and labeled container at room temperature.
2.2. El6ax preparation A sample of 100 mg of washed Elvax beads were placed in a capped, 12 × 45 mm, flat-bottomed, glass scintillation tube containing 1 ml methylene chloride (Sigma). After 2–3 h, the Elvax was completely depolymerized and the solution was mixed on a Vortex. Two milligrams of TTX (Sankyo, supplied through Calbiochem) was dissolved in 20 m1 of citrate buffer and the solution was sonicated until clear. This was then added to the Elvax/methylene chloride mixture, the vial was recapped, and the entire solution was vortexed (at least 5 min at high speed) until the drug was completely disbursed throughout the fluid in very small droplets. Mixing was best accomplished by repeatedly holding the vial against the edge of the Vortex cap at a 45° angle for a few seconds, and then allowing it to spin in a circumferential fashion around the inside perimeter of the cap for a few seconds. The former disbursed the water droplets in the Elvax solution and the latter spun the air bubbles out. Ten microlitres of a 1% Fast Green (Sigma) solution (DMSO; Sigma) was then added to color the solution and mixed as above on the Vortex. On the flnal mix, special care was taken to ensure that all air bubbles were spun out of the mixture so they did not become a permanent fixture in the final suspension. Once mixing was completed, the tube was uncapped and immediately placed upright on dry ice in order to freeze the solution into a cylindrical shape. The frozen mixture was then moved into −70°C where it remained for 4 days, followed by another 4 days at − 20°C. This procedure slowly evaporated the methylene chloride and reduced the frozen Elvax into a cylindrical-shaped pellet the original volume of the raw beads. The TTX impregnated Elvax was cut (200–400 mm) in round crossections on a freezing microtome, after gluing the pellet to the stage with cyanoacrylate and embedding it in frozen Tissue-Tek (Sakura). Individual sections were mounted on a gelatinized slide and stored at −20°C until they were used. Before surgery, small strips were cut from the Elvax and washed for a few seconds in 70% EtOH. The strips were then washed in sterile saline for 30 min to remove TTX from the surface of the polymer. Control Elvax containing only citrate buffer was produced and treated in an otherwise identical fashion.
2.3. Surgery Male and female ferrets ranging in age from postnatal day (P)0 to adult were used as experimental subjects
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in this study. Animals were anesthetized by trifluoroethane (to effect in a vapor enclosure mixed with room air) or sodium pentobarbital (35 mg/kg, i.p.). A topical antiinflammatory agent (prednisolone acetate, Pred Forte, Allergan) was applied to the eye and a small incision was made in the sclera near the limbus with a micro unitome knife (Beaver) to gain access to the vitreous chamber. Elvax strips were inserted through the hole and placed in the vitreous humor adjacent to the lens, taking care to avoid direct contact with the retina and lens. The number and size of the implanted strips was determined by the age of the animal and the size of the eye. In small neonatal eyes, usually two strips (approx. 2 ×0.2 × 0.2 mm) were implanted, and by adulthood, up to four larger pieces (approx. 4×2×0.2 mm) were used. Animals were recovered and monitored for behavioral signs of surgery-induced abnormalities. We observed no postoperative inflammation or infection in the vast majority of the animals in this study. The few animals that experienced post-operative complications were removed as subjects. Both monocular and binocular surgeries were undertaken, however, binocular implants were most successful in animals older than P14. Following eye opening, light-induced pupillary reflexes were examined in the stimulated and the non-stimulated eyes daily to monitor retinal activity. The lack of a pupillary reflex in the stimulated eye could, in some circumstances, be due to a blockade of neural input or damage to the iris. However, we also tested the consensual reflex in the non-stimulated eye in animals with unilateral implants, which is only blocked if retinal activity in the stimulated eye is blocked.
2.4. Electrophysiology In order to obtain direct evidence of the effectiveness of the TTX implants in animals before eye opening, electrophysiological assays of the vitreous hurnor were undertaken. Samples of vitreous fluid were obtained from the eyes of animals implanted with Elvax at different time intervals following the surgery. Approximately 1 ml of vitreous was collected by placing the broken tip of a micropipette, pulled from borosilicate glass capillary (TW100F-4, WPI), into a small incision made near the limbus. Removing the vitreous by capillary action had the advantage of causing minimal eye disturbance. As a result, animals could be used for additional testing, including extraction of vitreous fluid, tract tracing or electrophysiological recordings. The effect of the vitreous fluid on the generation of action potentials in the lateral geniculate nucleus (LGN) of ferrets was tested. Vibratome-cut slices (400 mm) containing the LGN were obtained from deeply anesthetized (100 mg/kg sodium pentobarbital, i.p.) ferrets as described previously (Ramoa and Mc-
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Cormick, 1994). Slices were placed in an interface-type chamber (FST) where they rested on lens paper. To increase mechanical stability, a piece of nylon mesh was placed over the slices. In this way the flow rate could be changed from null (during the local application of the vitreous), to a high flow rate within seconds that lead to superfusion of the slices (for washing-out vitreous). Approximately 2 h following placement of slices in the chamber, extracellular recordings of action potential activity was conducted using a tungsten-in-glass electrode (Levick, 1972) lowered into one of the geniculate layers. Retinal afferents in the LGN activated with constant current stimuli (100 ms duration, 30–300 mA intensity, 10–20 s intervals) were delivered through a bipolar stimulating electrode positioned in the optic tract. The tip of the pipette containing the vitreous fluid was positioned near the recording site and vitreous was pressure ejected while action potentials were recorded. Supplementary evidence of the efficacy of the TTX treatments was derived from experiments in which the pupillary reflex was examined in the eyes of rats. A 2–5 ml sample of vitreous fluid was removed from the eyes of ferrets implanted with Elvax. This sample was then injected into one eye of a P25 rat under anesthesia (30 mg/kg sodium pentobarbital). After recovery from the surgery, the pupillary reflex in the eye was tested.
3. Results
3.1. El6ax preparation The Elvax preparation protocol produced cylindricalshaped pellets of Elvax (approx. 7 mm dia.× 4 mm h; Fig. 1C) from an initial 1 ml volume of solution.
Fig. 1. Prepared Elvax mounted on a glass slide. (A). Series (top right to bottom left) of 200 mm thick sections of Elvax impregnated with TTX. The annular shape of the first four sections is the result of cutting through the concave top of the Elvax block. (B). Small strips cut from an Elvax section suitable for intraocular implantation. (C). Top (left) and side (right) views of the cylinder-shaped blocks of prepared Elvax before sectioning. The width of the blocks is 7 mm.
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Fig. 2. Effects of vitreous fluid from TTX-treated and control eyes on action potential activity in the slice preparation. Examples of extracellular recordings show that neuronal action potential activity evoked by optic tract stimulation was reversibly blocked (A,B,C) following local application of vitreous fluid obtained from a P12 ferret injected at P9 implanted with Elvax containing TTX. In contrast, action potential activity in a different slice was not affected (D,E) by local application of vitreous fluid collected from an eye treated with Elvax containing only citrate buffer. Each panel shows ten superimposed recordings.
Sections of Elvax (200 – 400 mm) (Fig. 1A) contained an even distribution of spheres formed by droplets containing dissolved TTX. Small strips cut from the sections were used as implants (Fig. 1B).
3.2. Pupillary reflex Elvax implants containing TTX displayed chronic and consistent sustained release properties as measured with a light-induced pupillary reflex in the stimulated eyes and the consensual reflex in non-stimulated eyes. The pupillary reflex was absent in all animals implanted with TTX-Elvax by the time they recovered from anesthesia. This reflex was absent for at least 7 days in all animals and up to 25 days in some cases. After 25 days, the pupillary reflex recovered in all animals. Since a normal pupillary reflex is dependent upon an intact retina, its recovery following sustained TTX blockade indicates that the implantation surgery did not substantially damage the retina. Animals implanted with control Elvax exhibited a robust pupillary reflex immediately following the surgery and for the duration of the experiments.
3.3. Electrophysiology When vitreous humor obtained from the eyes of animals implanted with TTX-Elvax for 3 days was pressure ejected near LGN neurons, it induced a complete and reversible cessation of action potential activity (Fig. 2). Activity resumed within 1 – 2 min of terminating the puffing, confirming that the vitreous
humor contained sufficient TTX to effectively block neuronal sodium channels. Similar effects were observed in all samples of vitreous fluid collected from TTX-treated eyes following 1 day (n= 4), 3 days (n=4) and 4 days (n=3) of intraocular implantation. There was no noticeable difference in the ability of the vitreous fluid to block action potentials whether it was drawn from a location near the implant or not, suggesting that TTX diffused throughout the entire eye. In contrast, the vitreous fluid collected from eyes (n=7) treated with control Elvax did not affect action potential activity of LGN neurons as illustrated in Fig. 2. Additional confirmation of TTX-Elvax effectiveness was obtained in rats (n= 2) injected intraocularly with the vitreous fluid of ferrets (n= 2) treated for 3 days. These animals displayed a reversible loss of their pupillary reflex, while animals (n= 2) injected with vitreous fluid extracted from control eyes showed no effect.
4. Discussion The Elvax preparation method described in this paper is a simple and effective way to create TTX impregnated Elvax with reproducible release properties. It is important when curing the Elvax that the frozen methylene chloride/Elvax solution evaporates slowly so that a homogeneous matrix is maintained. Frost-free freezers should be avoided when using this method, since they increase their temperature to near 0°C daily to reduce the amount of condensation in the chamber. This defrosting cycle allows the methylene chloride to evaporate rapidly, Causing air bubbles to form inside the matrix. It is also crucial that the scintillation tube is uncapped in the freezer to allow the evaporating fumes to escape. Methylene chloride is toxic and precautions should be taken to avoid inhaling the fumes or having them contaminate something else in the freezer. We have tried making larger Elvax blocks by dissolving 200 mg of Elvax in 2 ml methylene chloride, however, there is a high probability of air bubbles forming in the solution as a result of the polymerizing surface of the cylinder preventing the escape of evaporated methylene chloride at the core. Unless there is good reason to make different-shaped pellets to generate very large slabs of Elvax, a 1 ml solution in scintillation tubes will work reliably and generate relatively large sections. Inferior quality Elvax is also produced if more than 20 ml of a drug solution is added to the 1 ml Elvax/Methylene chloride mixture. The increased volume of fluid in the polymer results in a lower density matrix that is difficult to cut and has less reliable release properties. The Elvax implantation technique used in this study undoubtedly produces less experimental trauma than previous intraocular injection protocols. Because an effective dose of TTX is released
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from the Elvax in a sustained manner for 7 – 25 days, this eliminates the need to make multiple daily injections of TTX to continuously block retinal activity. In addition, the cumulative detrimental effects of multiple surgical procedures are reduced. We have found that implanting multiple small strips of Elvax is more effective than implanting one large piece. Small strips allow for a smaller incision in the eye, thereby reducing the overall trauma and leakage of intraocular fluid. In addition, the release of TTX from multiple polymer strips may provide a more even distribution of TTX throughout the vitreous than release from a single implant. If longer duration release than that described in this paper is desired, we suggest that surgical implants of new TTX/Elvax be made every 7 – 25 days. We are currently investigating whether thicker sections of Elvax implanted in the eye release TTX for longer periods of time. In one animal, we have implanted two strips of TTX-Elvax approximately 4×1 ×0.4 mm, and achieved reversible blockade of retinal activity for 25 days. We expect in some experiments, however, that any added benefit in TTX release time will have to be balanced against the detrimental effects of making a larger incision in the sclera to accommodate the larger strip of Elvax. We have not tried to increase the concentration of TTX in the Elvax beyond 2 mg/100 mg to produce longer duration implants, since it is very difficult to dissolve more than 2 mg of TTX/sodium citrate in 20 ml of solution. In addition, the duration of release is more likely a property of the polymer matrix than the concentration of the TTX. The methodology described in this paper could be used to investigate the effect of many different drugs on specific aspects of visual function. TTX has the property of nonspecifically blocking all action potential activity in retinal ganglion cells, thus enabling the use of a pupillary reflex to monitor the sustained release properties of TTX impregnated Elvax. The pupillary reflex can not always be used as a measure of a drug’s effectiveness in the retina, since most drugs target a subset of receptors on cells not necessarily linked to the function of the pupillary reflex. Under these circumstances, an electrophysiological assay like that described here could be developed to test the efficacy of a particular drug. In addition, a high performance liquid chromatography (HPLC) analysis of the vitreous humor may be able to establish the precise concentration of a drug in the intraocular fluid. Collectively, the results of this paper indicate that the sustained release of pharmacological agents from Elvax implanted in the eye can be used to examine the role of retinal activity and retinal neurotransmis-
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sion in visual system development, even before patterned visual function is possible.
Acknowledgements This work was supported by a NSERC research grant to G.T.P., and by NSF (IBN-9431983) and Whitehall Foundation grants to A.S. Ramoa.
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