Complement Levels in Normal and Inflamed Aqueous Humor

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38 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 983 Vol. 24 iological responses of vertebrate eyes to the chemical irritant, nitrogen mustard. Invest Ophthalmol Vis Sci 24:84-9, 983. 8.

1 38 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 983 Vol. 24 iological responses of vertebrate eyes to the chemical irritant, nitrogen mustard. Invest Ophthalmol Vis Sci 24:84-9, Koester CJ, Roberts CW, Donn A, and Hoefle FB: Wide field specular microscopy: Clinical and research applications. Ophthalmology 87:849, DeRousseau CJ and Bito LZ: Intraocular pressure of rhesus monkeys (Macaca mulatto). II. Juvenile ocular hypertension and its apparent relationship to ocular growth. Exp Eye Res 32:47, 98.. Van Horn DL, Sendele DD, Seideman S, and Buco PJ: Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci 6:597, Laing RA, Sandstrom MM, and Leibowitz HM: Clinical specular microscopy. II. Qualitative evaluation of corneal endothelial photomicrographs. Arch Ophthalmol 97:72, 979. Complement Levels in Normal and Inflamed Humor Barrly J. Mondino and Haihwa Rao C2, C6, and C7 were measured by hemolytic assay and Factor B and IgG were measured by radial immunodiffusion in samples of normal and inflamed aqueous humor. Normal aqueous humor was found to contain functional C2, C6, and C7, but the small ratios of aqueous humor to serum measurements suggested that there was relatively little of these complement components in normal aqueous humor when compared to serum. The mean values of C2, C6, C7, and Factor B in aqueous humor and the median ratios of aqueous humor to serum measurements for each complement component were higher in patients with inflamed aqueous humor than in patients with normal aqueous humor. A comparison of the ratios of IgG to each complement component in normal and inflamed aqueous humor suggested that levels of IgG and complement increased proportionately in inflamed aqueous humor. Factor B, a component of the alternative pathway, was not detected in normal aqueous humor but measured in five of six samples of aqueous humor from eyes with anterior chamber inflammation. Invest Ophthalmol Vis Sci 24:38-384, 983 The complement system is an important mediator of inflammation, and little is known about this system in the aqueous humor of both normal and inflamed eyes. Using hemolytic assays, Chandler and associates demonstrated C4 in samples of aqueous humor from all seven patients that they studied, but C3 was found in only one of the samples and Cl in two of the samples from patients with anterior segment inflammation. We measured hemolytic levels of Cl, C4, C3, and C5 in aqueous humor samples obtained at the time of surgery from eyes without inflammation or previous surgery, eyes with a history of previous surgery and eyes with anterior uveitis. 2 We found that samples of normal aqueous humor contain functional Cl, C4, C3, and C5 and that the mean values of all complement components in aqueous humor and the ratios of aqueous humor to serum measurements were increased in patients with a history of previous surgery and were highest in patients with anterior uveitis. We extended our studies of complement in aqueous humor by measuring hemolytic levels of C2, C6, and C7 and levels of Factor B in normal and inflamed aqueous humor. We also measured levels of C2, C6, C7, and Factor B in each patient's serum to compare with levels found in aqueous humor. Finally we compared the ratios of IgG to complement in normal and inflamed aqueous humor as done by Chandler and associates to determine if levels of IgG and complement varied proportionately or disproportionately. Materials and methods. Samples of venous blood and aqueous humor were obtained from 4 patients. Group consisted of venous blood and aqueous humor samples from eight patients with cataracts and anterior chambers that showed no cells or flare (Tables A, B). Group 2 (Tables 2A, B) consisted of venous blood and aqueous humor samples from six patients with various degrees of anterior chamber inflammation (Tables 2A, B). Prior to collection of aqueous humor, patients received lid and retrobulbar injections of 2% mepivacaine hydrochloride mixed with an equal volume of.75% bupivacaine hydrochloride. Approximately.5 ml of aqueous humor was collected from all patients in a -cc tuberculin syringe with a 3-gauge needle. The anterior chamber was entered through the trephine groove in patients 2 and 4 undergoing corneal transplantation, through a superior limbal groove in patients -8 and undergoing cataract extraction and through the temporal limbus in patients 9,, and 3. The aqueous samples were placed in ice. Venous blood samples were also collected from all patients at the time of surgery. Both the aqueous humor and venous blood samples were picked up immediately by a technician. The venous blood was allowed to clot and then centrifuged at 2 rpm for min to separate the serum from the clot. The aqueous humor and serum were stored at -7 C. and aqueous humor from the same patient were analyzed on the same day with the same reagents /83/3/38/$.5 Association for Research in Vision and Ophthalmology Downloaded From: on 2/26/27

2 No. 3 Reports 38 Table A. Hemolytic activities (CH 5 units/ml) in serum and aqueous humor from group (eyes without inflammation) C2 C6 C7 Patient/age (yr)/ sex, diagnosis /72/F Cataract 8,45 4, , ,82,628 57,958 2/7/M Cataract, , ,746 4,348 6,7 3/43/F Cataract 9, , ,889 73,28 4/74/F Cataract 8,955 7,279 9,9 59,849 87, _,48 5/46/F Cataract, ,3 87,599 3,579 25,263 6/8/F Cataract 6, , 89,79 64,865 39,663 7/72/F Cataract 9, ,689 64,23 9, /3/M Cataract 9, ,286 2,74 67,33 6 4,96 Median Ratio,57 ±,24 6 ± 3 4,785 ± 27,5 67 ±,657 83,79 ±9, ± 2,536 Hemolytic complement assays: Hemolytic activity of C2, C6, and C7 in serum and aqueous humor was determined by 5% hemolysis (CH 5 ) of sensitized sheep red blood cells. The cellular intermediates were prepared according to the method described by Borsos and Rapp. 3 The methods and controls that we used for assay of C2, C6, and C7 have been described in detail previously. 4 Radial immunodiffusion: Levels of Factor B (C3 activator) in serum and aqueous humor were determined by single radial immunodiffusion using plates from Calbiochem-Behring (San Diego, CA) that were Table IB. Levels of factor B and IgG (mg/ ml) in serum and aqueous humor from group (eyes without inflammation) Factor B IgG Patient Median Ratio ± 2., 83, ,59,2 8,3 ± ± Downloaded From: on 2/26/27

3 emolytic activities (CH 5 units/ml) in serum and aqueous humor from group 2 (eyes with anterior uveitis) C2 C6 C7 (yr)/sex sis Aqueo Seru sterior asional e flare), , , cells and 6, , , ens flare), , , topathy flare), , ,57,46 99 ous uveitis and flare) 2, ,264 3,268 4, atouveitis, cells and 9,756, ,74 5,98 5 8, 7,67 5 or Median,683 ±, ± ,998 ± 49,879,769 ± ,852 ± 29,94 3,389 ± 2, Downloaded From: on 2/26/27

4 No. 3 Reports 383 Table 2B. Levels of factor B and IgG (mg/ ml) in serum and aqueous humor from group 2 (eyes with anterior uveitis) Factor B IgG Patient 9 23., , , , , Median Ratio 2.2 ± ±.3 32,274 ± ± developed according to the technique of Mancini, Carbonara, and Heremans. 5 IgG levels were also measured by single radial immunodiffusion using regular and low level immunodiffusion plates (Meloy Laboratories, Springfield, VA). Statistical analysis: Analysis of variance on transformed (natural logarithm) data was used to determine if there were any statistically significant differences between Groups and 2 in levels of C2, C6, C7, Factor B, and IgG in serum and aqueous humor. The Student's t-test was then used to compare means between Groups and 2 where analysis of variance showed that there were statistically significant differences. The Mann-Whitney U test was used to determine if there were any statistically significant differences between Groups and 2 in the ratios of complement components in the patient's aqueous to serum and also in the ratios of IgG to each complement component in aqueous humor. Results. The values of C2, C6, C7, Factor B, and IgG in aqueous humor and serum and the ratios of aqueous to serum measurements are shown in Tables A and B (Group, eyes without inflammation) and Tables 2A and B (Group 2, eyes with anterior uveitis). There were no statistically significant differences in the two groups in serum values of C2, C6, C7, Factor B, and IgG (P =.79). The mean values of complement components in aqueous humor were higher in Group 2 than in Group, and the differences were statistically significant for C2 (P<.), C6 (P =.2), and C7 (/><.), Factor B (P =.4), and IgG (P =.5). We were unable to detect Factor B in aqueous humor from eyes without inflammation in Group, even when the wells were loaded multiple times with normal aqueous humor. In eyes with inflamed aqueous humor (Group 2), Factor B was not detected in aqueous humor from patient 9 who showed only an occasional cell and trace flare in the anterior chamber but was detected and measured in aqueous humor from the other five patients in this group with a greater anterior chamber response. The ratios of aqueous humor to serum measurements for all complement components were higher in Group 2 than in Group, and the differences were statistically significant for C2 (P =.), C6 (P =.2), C7 (P =.), Factor B (P =.) and IgG (P <.2). A comparison of the ratios of IgG to each complement component in aqueous humor showed no statistically significant differences between Groups and 2 (P>.2), and so these ratios are not included in the tables. Similar ratios of IgG to complement between Groups and 2 suggested that the levels of both complement and IgG increased proportionately in aqueous humor from Group 2. Discussion. In the present study, we measured C2, C6, and C7 by hemolytic assay and Factor B by radial immunodiffusion in aqueous humor. To the best of our knowledge, these complement components have never been identified before in aqueous humor. Hemolytic assays indicate that samples of aqueous Downloaded From: on 2/26/27

5 384 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Morch 983 Vol. 24 humor from eyes without inflammation (Group ) contain functional C2, C6, and C7. The small ratios of aqueous humor to serum measurements of C2 (/ 72), C6 (/,657), and Cl (/,536) suggest that there is relatively little of these complement components in normal aqueous humor when compared to serum. We were unable to detect Factor B in normal aqueous humor. The single radial immunodiffusion technique that we used, however, may not be sensitive enough to detect small concentrations of Factor B that may be present in normal aqueous humor and fall below the working range of the plates. The mean values of all complement components and IgG were higher in inflamed aqueous humor (Group 2) than in normal aqueous humor (Group ). Complement and IgG levels in serum did not differ statistically between Groups and 2, so that increased serum levels could not be used to explain the increased aqueous levels in Group 2. Moreover, the ratios of aqueous humor to serum measurements for each complement component and IgG were higher in Group 2 than in Group. A comparison of the ratios of IgG to each complement component in normal and inflamed aqueous humor suggested that levels of IgG and complement increased proportionately in inflamed aqueous humor. The altered vascular permeability associated with anterior uveitis is probably responsible for the elevated levels of complement and IgG in aqueous humor samples from Group 2. The results of this and a previous study 2 indicate that normal aqueous humor contains components of the classical complement pathway: Cl, C4, C2, C3, C5, C6, and C7. Moreover, levels of these complement components are increased in inflamed aqueous humor. Factor B, a component of the alternative pathway, could not be demonstrated in normal aqueous humor but was found in five of six samples of inflamed aqueous humor. Key words: Complement, aqueous humor, anterior uveitis, classical pathway, alternative pathway Acknowledgments. Statistical analysis of the data was performed by Robert P. Hirsch, PhD, University of Pittsburgh School of Medicine. Margaret C. Sharrer, MD, provided aqueous humor from patient 3 and John C. Stuart, MD, from patient. From the Department of Ophthalmology, University of Pittsburgh School of Medicine and Eye and Ear Hospital of Pittsburgh. Supported in part by a grant from the National Eye Institute, RO EY 234 (Dr. Mondino). Submitted for publication June 28, 982. Reprint requests: Bartly J. Mondino, MD, Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 924. References. Chandler JW, Leder R, Kaufman HE, and Caldwell JR: Quantitative determinations of complement components and immunoglobulins in tears and aqueous humor. Invest Ophthalmol 3:5, Mondino BJ and Rao H: Hemolytic complement activity in aqueous humor. Arch Ophthalmol, in press. 3. Borsos T and Rapp HJ: Immune hemolysis: a simplified method for the preparation of EAC'4 with guinea pig or with human complement. J Immunol 99:263, Mondino BJ and Hoffman DB: Hemolytic complement activity in normal human donor corneas. Arch Ophthalmol 98:24, Mancini G, Carbonara AO, and Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Int J Immunochem 2:235, 965. Effect of Light on Dopamine Turnover and Metabolism in Rabbit Retina David Parkinson and Robert R. Rando Some neurochemical responses of dopaminergic neurons in the rabbit retina have been measured during prolonged light or dark adaptation. Light adaptation produced small increases (2%) in dopamine levels but larger increases (5- %) in the two metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Light also significantly increased tyrosine hydroxylase activity; the increase was more pronounced when activity was measured in vivo than in vitro. Dopamine turnover, was faster in the light than in the dark. All these data support the suggestion that light leads to the activation of dopaminergic neurons in the rabbit retina. Invest Ophthalmol Vis Sci 24: , 983 Dopamine has been identified as the neurotransmitter of a subset of amacrine cells ' 2 and work with the albino rat has suggested that light leads to the activation of these dopaminergic neurones. 3 We have observed light dependent activation of dopaminergic neurones in the chick retina also. 4 It would seem premature, however, to assume, a priori, that dopamine has the same function in all vertebrate retinae; especially since dopaminergic neurons in the Cebus monkey and teleost fish are interplexiform cells 5 rather than the amacrine cells of other vertebrate retinae /83/3/384/$.5 Association for Research in Vision and Ophthalmology Downloaded From: on 2/26/27

Effect of Light on Dopamine Turnover and Metabolism in Rabbit Retina

Effect of Light on Dopamine Turnover and Metabolism in Rabbit Retina 384 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Morch 1983 Vol. 24 humor from eyes without inflammation (Group 1) contain functional C2, C6, and C7. The small ratios of aqueous humor to serum measurements