636 Reports Invest. Ophthalmol. Visual Sci.

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1 636 Reports Invest. Ophthalmol. Visual Sci. three optically determined volumes. Furthermore, no statistically significant difference existed between the optically determined volumes and the fluorescein distribution volumes. The root mean square difference between the optical and the fluorescein volumes was 17 jul. Therefore 95% of paired measurements by these two techniques should agree within 34 /*1. Discussion. The photographic technique of measuring the volume of the anterior chamber described here has the advantage that it can be performed with unmodified, commercially available equipment. The anterior chamber scale is easily reproduced. Others could be made for use with other slit-lamp camera systems. We have found that a pocket calculator is helpful for multiplying each scale reading by the appropriate weighting factor and for adding up the totals. Once a photograph has been made, we found that it took approximately 2 min. to determine the anterior chamber volume using the anterior chamber scale and the pocket calculator. The sources of error of optical reconstruction of the anterior chamber have been discussed by Jones and Maurice 1 and by Brown. 5 The use of a vertical slit is probably less reliable than the use of a horizontal slit, which allows observation of a greater portion of the peripheral anterior chamber, but a vertical slit is more convenient. The most important step to avoid errors with the vertical slit technique is to take care that the angle setting and the camera position (either left or right) are the same for the experimental eye as for the model eye. Small errors in alignment of the center of the slit with the optic axis of the experimental eye are bound to occur. To study the effect of such alignment errors, we photographed an artificial anterior chamber intentionally misaligning the center of the slit plane with respect to the optic axis of the artificial cornea. Less than 5% error was introduced by moving the slit 1 mm. left or right of center, moving the slit 3 mm. up or down from center, or having the eye "look" up or down 1. Less than 1% error was introduced by having the eye "look" left or right 1. Misalignments greater than these introduced significantly larger errors. In practice, alignment errors as large as these are easily observed by the photographer, particularly if care is taken to note whether the catoptric images of the slit light lie within the slit plane. If the center of the pupil does not coincide with the geometric center of the eye, the volume might be overestimated or underestimated. In our artificial eye, lateral or medial displacement of 1 mm. caused less than 5% error. Upward or downward displacement of the pupil may result in erroneous application of the scale to the photograph. In our artificial eye, a.5 mm. upward displacement of the pupil introduced a +21% error, and a.5 mm. downward displacement introduced a -18% error. Therefore, if the pupil appears eccentric, other landmarks obtained from a straight-on photograph must be used for proper centering of the scale. The fluorescein distribution volume of the rhesus monkey eye appears to be nearly the same as the optically determined volume. A similar relationship probably exists in normal human eyes, making this simplified photographic method applicable for calibrating fluorescein techniques for measuring aqueous humor flow. From the Department of Ophthalmology, Mayo Clinic, Rochester, Minn. Submitted for publication Feb. 7, Requests for reprint (or transparencies ): Dr. Richard F. Brubaker, Department of Ophthalmology, Mayo Clinic, Rochester, Minn Key words: fluorescein, anterior chamber, volume, anterior chamber scale, anterior chamber depth, photogrammetry. REFERENCES 1. Jones, R. F., and Maurice, D. M.: New methods of measuring the rate of aqueous flow in man with fluorescein, Exp. Eye Res. 5:28, Nagataki, S.: Aqueous humor dynamics of human eyes as studied using fluorescein, Jpn. J. Ophthalmol. 19:235, Heim, M.: Photographische Bestimmung der Tiefe und des Volumens der menschlichen Vorderkammer, Ophthalmologica 12:193, Jones, R. F., and Maurice, D. M.: A simple photographic method of measuring the volume of the anterior chamber, Exp. Eye Res. 2:233, Brown, N.: Quantitative slit-image photography of the anterior chamber, Trans. Ophthalmol. Soc. U.K. 93:277, The effect of theophylline on the breakdown of the blood-aqueous barrier in the rabbit eye. ELISABETH BENGTSSON. A disruption of the blood-aqueous barrier in rabbit eyes was elicited by topical prostaglandin E!y infrared irradiation of the iris, or subcutaneous a-melanocyte stimulating hormone (a-msh). The course of the inflammatory reaction was followed by photoelectrical measurements of the aqueous flare in the anterior chamber. Pretreatment with intravenous theophylline, a phosphodiesterase inhibitor, significantly increased the protein leakage caused by prostaglandin E t and a-msh, but the response to infrared irradiation was slightly but not significantly enhanced. Intravenous theophylline given in higher doses caused per se an aqueous flare increase, which could not be inhibited by

2 Volume 16 Number 7 Reports 637 pretreatment with topical indomethacin. Our results indirectly indicate that accumulation of intraocular camp promotes a barrier damage and that camp might be the common effector of the barrier breakdown caused by prostaglandin as well as by nonprostaglandin agents. Various authors have suggested that cyclic adenosine 3',5'-monophosphate (camp) is the ultimate effector of the breakdown of the bloodaqueous barrier induced by different traumatic stimuli to the eye (see literature cited in ref. 1). Imidazole, which increases camp phosphodiesterase activity, 2 antagonizes when given systemically the barrier damage induced by prostaglandin 1 as well as by the nonprostaglandin stimulus a-melanocyte stimulating hormone (a-msh). 3 Topical imidazole, however, has no effect on the response to prostaglandin-mediated traumata 1-3 but strongly potentiates the a-msh response, 3 conceivably by promoting the prostaglandin uptake in the iris and ciliary body. 4 Further experiments were carried out in an attempt to determine the role of camp in mediating the a-msh response. Agents which inhibit camp phosphodiesterase activity should increase the a-msh effects. Theophylline can inhibit the camp phosphodiesterase activity 2 and has been used to enhance the accumulation of labelled camp for assay of adenyl cyclase activity in ciliary process tissue. 5 We 3 have studied the capability of theophylline to affect the damage of the blood aqueous barrier induced by a-msh as compared to prostaglandin and infrared irradiation of the iris (a prostaglandin-mediated trauma) (see references in ref. 3). Experiments. Disruption of the blood aqueous barrier in eyes of pigmented rabbits was elicited using the following three irritative stimuli: (1) 1.5 or 3 fig of prostaglandin E 2 (PGE 2 ) locally applied to the cornea, (2) infrared irradiation of the iris for 2 min., and (3) 2 i"g/kg. body weight a-msh given subcutaneously. The barrier damage was quantified by measuring photoelectrically the course of the aqueous flare response seen in the anterior chamber, reflecting the protein leakage across the blood-aqueous barrier. In some series the pupillary diameter was also recorded. Chemical preparations, 3 infrared irradiation, aqueous flare, (see ref. 3) and pupillary measurements 6 were performed as described in previous papers. The flare was measured in arbitrary units. The effect of theophylline (Theon; Draco, Lund, Sweden) in a dose of 75 mg./kg. body weight given intravenously 3 min. before stimulation of one eye with PGE 2 or infrared irradiation or simultaneously with a-msh was tested. For control of each experiment the three irritative stimuli were randomly administered to the same groups of rabbits at least 14 days before or after the actual test. For PGE = and infrared irradiation the contralateral eye was used for control. For a-msh the mean of the aqueous flare in the two eyes was used. The effect of A*1 of theophylline (1 mg./ ml.) given topically to one eye every 1 min. during the hour previous to administration of the three traumatic agents to both eyes was also tested. The effect of intravenous ( to 1 mg./kg. body weight) and topical theophylline on the physiological aqueous flare was also checked. Statistics. Wilcoxon's test for paired differences was used for calculating the significance of differences; p refers to the null hypothesis (H o ). Results. Intravenous theophylline slightly increased (Ho: p <.5) the flare response to 1.5 ng of PGE 2 (eight rabbits) but did not change either the pupillary reaction or the time relationships (Fig. 1). However, when 3 /ug of PGE 2 was applied topically to five rabbits (mean of the maximal aqueous flare response, 148. ± 12.1 arbitrary units), the barrier damage induced was not significantly (H o : p >.1) affected by intravenous theophylline (mean of the maximal flare response, 152. ± 2.2 arbitrary units). The barrier damage or pupillary response caused by infrared irradiation (nine rabbits) was not significantly (H o : p >.5) changed by intravenous theophylline although a slight promotion of the flare increase was seen. The maximal flare response to irradiation occurred about 3 min. earlier when pretreatment with intravenous theophylline had been given (Fig. 1). The flare response to a-msh, however, was significantly (H o : p <.1) increased by intravenous theophylline (Fig. 1). In seven out of 1 unselected rabbits tested, there was no aqueous flare response when merely a-msh had been given. In the other three there was a faint to modest flare response. When the same group of rabbits were given intravenous theophylline previous to the injection of a-msh, only one rabbit did not get a significant aqueous flare increase (> % increase of the physiological aqueous flare). In all the others a modest maximum flare value was recorded. Theophylline given intravenously in a dose of mg./kg. body weight (six rabbits) or 75 mg./kg. body weight (15 rabbits) did not change the physiological aqueous flare in any of the cases (Table I). In five out of 1 rabbits 1 mg./kg. body weight caused no barrier damage at all (nonresponders), but in five rabbits a moderate or dense aqueous flare was induced (responders) (Table I). The mean of the flare values after 2 hr. in these five rabbits was , and a typical time course of an aqueous flare response is shown in Fig. 2.

3 638 Reports Invest. Ophthalmol. Visual Sci. The effect of intravenous theophylline (Th) on the aqueous flare response () and the pupillary diameter ((/)) Prostaglandin Ej Infrared irradiation L-Melanocyte stimulating hormone o- - - o eyes pretreated with theophylline (Th) t T control eyes - no pretreatment 75 t t Th PGE2 3 hours t t 1 t Th«tt-MSH Fig. 1. Effect of 75 mg./kg. body weight intravenous theophylline (Th) on the pupillary diameter () (in appropriate series) and aqueous flare response () to 1.5 /tg of prostaglandin E 2 (PGE 2 ) given topically (8 rabbits), infrared irradiation of the iris (9 rabbits) and 2 Mg/kg. body weight a-melanocyte stimulating hormone (a-msh) given subcutaneously (1 rabbits). Ordinate: flare values ± S.E.M. in arbitrary units and pupillary diameter - S.E.M. in millimeters. Table I. The effect of intravenous theophylline on the aqueous flare Theophylline dose (mg./kg. body weight) 75 1 (nonresponders) 1 (responders) No. of rabbits Mean aqueous flare ± S.E. (arbitrary units) Initial value Value after 2 hr ± ± ± ± ± 11.8 =fc 14.1 ± 74.2 ± In order to test whether the barrier disruption due to theophylline was mediated via enhanced prostaglandin synthesis, we repeated the intravenous injection of theophylline (1 mg./kg. body weight) about 2 weeks later in the same group of rabbits. However, one eye was now pretreated topically with.1 ml. of indomethacin (1 mg./ml. distilled water) (water-soluble preparation from Dumex, Copenhagen) four times every 15 min., which is a dose known to inhibit even strong prostaglandin-mediated effects. 3 The mean of the maximal flare increase in the indomethacin treated eyes was 61. ± 23.3 as compared to 68.5 ± 22. in the contralateral control eyes. Thus the flare response to theophylline was not significantly (H o : p >.1) affected in any direction by indomethacin. Topical theophylline had no effect on the breakdown of the barrier induced by any of the three irritative stimuli tested; nor as used in

4 Volume 16 Number 7 Reports 639 our experiments, did it alter the physiological flare. Discussion. Intravenous theophylline promotes the damaging effect of a-msh on the bloodaqueous barrier. The aqueous flare response to prostaglandin and infrared irradiation was also somewhat enhanced, although the difference in the case of these two stimuli was weak or not significant, respectively. As concerns the three traumatic agents it was observed that within the groups the percentage increase of the flare caused by theophylline was smaller in proportion as the damage from merely the traumatic agents was greater. In those rabbits in which the stimuli per se caused a fairly dense flare response, intravenous theophylline caused no potentiation or only a minor one. This condition is in accordance with the observation that theophylline caused about % increase of the flare response to 1.5 /*g of PGE 2 whereas in no case did it significantly affect the flare increase due to 3 /*g of PGE 2, which caused a much more severe barrier damage. Perhaps this is also the reason why Zink et al. 7 could not demonstrate any effect of intraperitoneal aminophylline (1 mg./kg. body weight) on the intraocular pressure response to prostaglandin, since they tested a high dose of topical prostaglandin such as 5 Mg- Zink et al. 7 also reported that doses of aminophylline higher than 1 mg./kg. were lethal to their albino rabbits. Not even drowsiness was observed by us in any of the 1 pigmented rabbits given 1 mg./kg. theophylline intravenously. Theophylline has been reported to depress the systemic blood pressure, 8 but the principal part of this pressure fall is rapidly reversed within 3 min. after the injection of theophylline. Any considerable pressure fall caused by theophylline can therefore hardly remain at the point of time when our traumatic stimuli exert their effects. The observation that theophylline per se is capable of causing a barrier damage and that this effect is not inhibited by pretreatment with indomethacin indicates that an enhanced concentration of intraocular camp, without any coexisting increase of the intraocular prostaglandin synthesis, is able to elicit a barrier disruption. The present results also support the hypothesis that camp is the ultimate effector of the breakdown of the blood-aqueous barrier caused by prostaglandin. 1 In the literature, it has been suggested that the a-msh effect on biological systems is due to its ability to increase the amount of intracellular camp by either stimulating the adenyl cyclase system 9 or by inhibiting the enzyme camp phosphodiesterase. 1 Our present results are in accordance with this hypothesis and also support pur earlier assumption 6 that the primary damaging o- -o right eye «left eye 3 hours Fig. 2. Typical time course of the aqueous flare response () in one rabbit caused by 1 mg./kg. body weight intravenous theophylline (Th). Ordinate: flare values in arbitrary units. effect of a-msh on the barrier might be caused by a direct enhancement of the intraocular camp concentration. From the Department of Experimental Ophthalmology, University Eye Clinic, Lund, Sweden. Submitted for publication Feb. 14, Reprint requests: Elisabeth Bengtsson, M.D., Department of Experimental Ophthalmology, University Eye Clinic, S Lund, Sweden. Key words: blood-aqueous barrier, aqueous flare, prostaglandin, infrared irradiation, a-msh, theophylline. REFERENCES 1. Zink, H. A., Podos, S. M., and Becker, B.: Modification by imidazoles of ocular inflammatory and pressure responses, INVEST. OPH- THALMOL. 14:28, Butcher, R. W., and Sutherland, E. W.: Adenosine 3',5'-phosphate in biological materials. I. Purification and properties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3',5'-

5 64 Repents Invest. Ophthalmol. Visual Sci. phosphate in human urine, J. Biol. Chem. 237:1244, Bengtsson, E.: The effect of imidazole on the disruption of the blood-aqueous barrier in the rabbit eye, INVEST. OPHTHALMOL. 15: 315, Bengtsson, E., and Ehinger, B.: The effect of experimental uveitis on the uptake of prostaglandin Ei in the rabbit iris-ciliary body, Acta Ophthalmol. (accepted for publication ). 5. Waitzman, M. B., and Woods, W. D.: Some characteristics of an adenyl cyclase preparation from rabbit ciliary process tissue, Exp. Eye Res. 12:99, Bengtsson, E.: Interaction of adrenergic agents with a-melanocyte stimulating hormone and infrared irradiation of the iris in the rabbit eye, INVEST. OPHTHALMOL. VISUAL SCI. (accepted for publication). 7. Zink, H. A., Podos, S. M., and Becker, B.: Inhibition by imidazole of the increase in intraocular pressure induced by topical prostaglandin E, Nature New Biol. 245:21, Genee, E., and Geissendbrfer, T.: Blutdruckandernde Medikamente und Augeninnendruck im Tierversuch, Albrecht v. Graefes Arch. Klin. Exp. Ophthalmol. 195:187, van de Veerdonk, F. C. G., and Brouwer, E.: Role of calcium and prostaglandin (PGEi) in the MSH-induced activation of adenylate cyclase in Xenopus laevis, Biochem. Biophys. Res. Commun. 52:13, Goldman, M., and Hadley, M. E.: Evidence for separate receptors for melanophore stimulating hormone and catecholamine regulation of cyclic AMP in the control of melanophore responses. Br. J. Pharmacol. 39:16, 197. Distribution of axonal transport blockade by acute intraocular pressure elevation in the primate optic nerve head. HARRY A. QUIGLEY AND DOUGLAS R. ANDERSON. We studied the degree of axonal transport blockade in various areas of the optic nerve head with acute intraocular pressure (IOP) elevation in 19 squirrel monkey eyes. When IOP was raised to 2 to mm. Hg for 7 hr., mild axonal transport blockade occurred in each area of the disk, most prominently in nerve fiber bundles of the superior pole. With 7 hr. IOP elevations between and 9 mm. Hg, a somewhat greater degree of transport blockade occurred throughout the nerve head, although again the superior and inferior poles were somewhat more affected. The distribution of short-term transport blockade over the entire nerve head corresponds to the diffuse damage of acute glaucoma, but the pattern hints at the preference for damage near the poles of the disk seen in chronic glaucoma. However, before these results can be fully evaluated, further information is needed on axonal pathways through the optic nerve head and on the relationship between transport obstruction and ganglion cell death. In recent experiments, acute elevation of intraocular pressure (IOP) in primates produced a blockade of the normal axonal transport of material within nerve fibers at the level of the scleral lamina cribrosa. 1 ' 2 The elevated IOP blocks both orthograde (eye to brain) and retrograde (brain to eye) axonal transport at the same site. 3 It is not yet known whether abnormal IOP interrupts axonal transport by mechanical compression of axons or by vascular insufficiency. Nor is it definite that the pathogenetic pathway from elevated IOP to cell damage in chronic glaucoma involves the process of axonal transport block. Chronic glaucoma damages axons of retinal ganglion cells, leading to a characteristic sequence of visual field loss. Most commonly, early field loss appears in the arcuate or Bjerrum area and involves the nasal periphery, whereas the central and temporal fields are typically affected late in the process. 4 This progression suggests a differential sensitivity of axons or axonal bundles to pressure-induced damage. The explanation of this selective effect may be quite important in the understanding of pathogenetic mechanisms in glaucomatous visual field loss. We attempted to determine whether the distribution of acute axonal transport block by elevated IOP was uniform over the whole disk or if it showed a selective sensitivity of axons in certain regions, particularly with moderate IOP elevations. Rapid orthograde axonal transport was studied as in previous experiments, but with serial section autoradiographs through each nerve head to compare transport block in macular, arcuate, and nasal nerve bundles. Methods. Squirrel monkeys (Saimiri sciurea) were selected for study, since their retina has a foveal pit and their optic nerve head anatomy closely resembles that of the human optic nerve head. After anesthesia was induced by intramuscular phencyclidine and intraperitoneal pentobarbital, systemic blood pressure was monitored by means of femoral artery catheters, and IOP was controlled by means of an anterior chamber needle connected to a variable-height reservoir. Axonal transport was studied by intravitreal injection of.1 ml. (1 /uci) of 3 H-leucine just prior to IOP elevation (L-leucine-4,5-3 H[N], 3 to Ci/mmol; New England Nuclear Corp.; Boston, Mass.). The perfusion pressure was defined as the mean blood pressure minus IOP, where mean blood pressure = diastolic + % (systolic - diastolic). In each eye, the IOP was maintained for 7 hr. at a perfusion pressure of either 3, 6, or 9 mm. Hg. At the end of the experiment, 4% paraformaldehyde was infused