External Counting and Scintiphotography in Rabbits with Arthritis By STEPHEN D. SHOLKOFF,M.D., MORTONG. GLICKMAN, M.D., JULIUSSCHACHTER, PH.D., AND MALCOLMROWLAND, PH.D. Bedsonia arthritis in rabbits was studied b y Anger camera scintiphotography and external counting, using pertechnetate 99m as the label. This method of evalu- A MEMBER of the bedsonia group of organisms (psittacosis, lymphogranuloma venereum, trachoma) has been identified as a possible etiologic factor in some cases of Reiter's syndrome.lS2 A long-term study concerning the bedsonia group is under way at this institution. Joints of rabbits and monkeys have been infected by intraarticular injection of bedsonia organisms isolated from patients with classical Reiter's syndrome? Neither clinical examination nor conventional roentgenography of these animals has satisfactorily demonstrated the varying intensity of arthritis. The clinical evaluation of arthritic activity is subject to observer error and cannot be recorded graphically. Roentgenography does not easily demonstrate rapidly changing i d a m matory manifestations, since structural changes of bone and cartilage are late in appearance and are slow to evolve,. In seeking an objective and graphic method for evaluating the arthritis, we From the Department of Radiology, the G. W . Hooper Foundation, and the Department of Pharmaceutical Chemistry, University of California, Schools of Medicine and Pharmacy, San Francisco. This investigation was supported b y NIH Training Grant GM 01272 frm the National Institutes of General Medical Sciences, and Research Grant Al 07698 from the National Institutes of Health. STEPHEN D. SHOLKOFF, M.D.: Research Fellow, Department o f Radiology, University of California School of Medicine, San Francisco, California. MORTONG. GLICKMAN,M.D.: Instructor in Residence, Department of Radiology, 220 ating the intensity of the arthritis and the changes in the disease proved more sensitive and more accurate than did clinical and roentgenographic examinations. studied external counting technics and gamma scintiphotography in rabbits infected with bedsonia. MATERIALSAND METHODS Nine rabbits were infected with bedsonia organisms by intraarticular injection into one knee joint. This injection consisted of 0.5 ml. of a 10 per cent yolk sac suspension of isolate 25 SM containing approximately 106 egg LD,,. The rabbits were evaluated by clinical, roentgenographic, and scintiphotographic examinations over a period of 3 weeks. The scintiphotographic studies were conducted with the Anger scintillation camera, equipped with a highspeed automatic digital printer.* A 4000 parallel hole, low energy collimator was used. The animals were immobilized with a recently devised apparatus to ensure identical positioning each time a rabbit was restudied.4 The knees were placed under the crystal, and the trunk and lower legs were shielded with lead (Fig. 1). Scintiphoto'Pho-Gamma I11 Scintillation Camera, Nuclear Chicago Corporation, 333 E. Howard Ave., Des Plaines, Ill. 60018. University of California School of Medicine, San Francisco, California. JULIUSSCWCHTF.R, Ph.D.: Assistant Professor of Epidemiology and Medicine, G. W . Hooper Foundution, University o f California School of Medicine, Sun Francisco, California MALCOLMROWLAND, Pn.D.: Assistant Professor, Department o f Pharmaceutical Chemistry, University of California School of Pharmucy, San Francisco, California. Reprint requests should be addressed to Dr. Sholkofl, Department of Radiology, Uniljersity of California, San Francisco Medical Center, San Francisco, California 94122. ARTHRITISAND RHEUMATISM, VOL. 12, No. 3 (JUNE 1969) 221 EXTERNAL COUNTING AND SCINTIPHOTOGRAPHY Fig. 1.-The rabbit is in immobolized position for the study with neck extended and drawn through a notch in the boards. The camera will be positioned over the unshielded knees. The lead extends beyond the margins of the crystal so that only the knees are monitored. graphic examinations were performed after intravenous injection of 500 pc of pertechnetate 99m (99mTc0,-) at varying intervals after infection, as shown in Table 1. The crystal was split electrically so that each knee was counted separately. The counts were collected continuously for 3 to 13 hours after each injection, and the number collected was printed automatically at 1min. intervals during each study period. Photographs were exposed by collecting 2000 counts. Control studies were performed prior to the bedsonia infection. Three animals had intravenous 99mTc04- studies beginning at 8 and 48 hours after injection of 1.0 ml. saline into a knee joint. This was done to ensure that needle introduction did not produce early detectable change in the labeling characteristics of the joints. Subsequent intravenous studies 1 to 12 weeks later were per- formed on these rabbits to c o & m that the previous intraarticular injections had been innocuous. RESULTS Neither scintiphotography, external counting, roentgenography, nor physical examination demonstrated any abnormalities in the control studies. At frequent intervals after infection, all the joints were examined for heat, swelling, and passive range of motion. During the first 12 hours after infection, no abnormality was discernible. At 48 hours, severe inflammatory response was demonstrated in all the inoculated joints. By 3 weeks, the range of motion in all rabbits had de- SHOLKOFF ET AL. Fig. 2.-Roentgenogram 3 days after infection of the left knee. Although not clearly reproduced, minimal soft tissue swelling is present over the left knee on the original film. No other abnormalities are seen. creased and in 5 of these soft tissue thickening was detectable. By that time, however, neither warmth over the joint nor effusion was evident. The roentgenograms demonstrated minimal soft tissue swelling as early as 24 hours after inoculation (Fig. 2). This finding remained constant, but neither bone changes nor evidence of cartilage destruction was present at any time during the 3 weeks of study. As early as 4 hours after infection, the intravenous pertechnetate studies showed increased radioactivity in the affected knee relative both to the contralateral knee and to the knees as they had appeared prior to infection (Figs. 3A and B, 4A and B). This relationship persisted throughout the study, as shown by the scintiphotographs in Figs. 5 and 6. The results can be expressed numerically as the ratio, Ci/C,, where C , = maximum counts per minute from the infected knee, and C, = counts at that time from the contralateral (normal) knee (Table 1).For those animals studied prior to infection, the counts over the knees were maximum between 10 and 50 min. (mean = 33) after injection of pertechnetate. In the infected animals, the maximum count rate was reached at 20 to 58 min. (mean = 42). The range of counts collected per minute at the maximum for each study was 3200 to 10,000. The room background was 200 counts per minute. It was measured before and after each study, using the same detector. The varying intensity of the arthritis is easily appreciated by comparing the ratios at different times. The use of ratios rather than absolute numbers eliminated inaccuracies caused by differences in animal weight, symmetry of the extremities, response to infection, rate of excretion of pertechnetate, and other natural variables. Histologic examination of the infected joints of 2 rabbits at the end of the study confirmed the presence of chronic inflam- EXTERNAL COUNTING AND SCINTIPHOTOGRAPHY 223 Fig. 3.-(A) Rabbit 1: Intravenous 9SmTcOp- study performed one week after intraarticular injection of 1.0 ml. saline into the right knee, and prior to infection shows no detectable abnormality. (B) Rabbit 1: Increased labeling of the left knee is present as early as 6 hours after infection with bedsonia. SHOLKOFP ET AL. Fig. 4.-(A) Rabbit 2: Normal intravenous 9grnTcO4- study before infection. (B) Rabbit 2: Twenty-fourhours after infection of the left knee. .TERNAL COUNTING AND SCINTIPHOTOGRAPHY Fig. 5.-Rabbit 1: Intravenous study at 3 weeks. Fig. 6 . R a b b i t 2: Intravenous study at 3 weeks. 226 SHOLKOFF ET AL. Table 1.-Ratio Rabbit Number Initial Ratio 1.08 033 0.91 1.03 0.96 1.04 0.93 N.D. $ 1 2 3 4 5 6 7 8 9 N.D.$ Ci/C, for Each Intravenous Pertechnetate Study 4 to 6 hr.. ratio 24 hr., ratio Time After Infection* 48 hr.. ratio 98 hr., rPti0 1.18 2.34t 2.18 0.93 ** 1.15 3 wk. IUtiO’ 1.21 1.40 1.36 1.20 1.24 1.26 1.44 1.72 1.90 * The observations at the above times were compared to the initial values and found to be signifi0.01.The ratios obtained in the studies after infection have each been divided by their cant, P corresponding initial value so that they will be comparable with one another. t Clinically the most severely involved, the only animal in which severe decreased range of motion was evident during the h s t week after infection. $ N.D. =Not done. The mean of the other studies was 0.98. The theoretically expected value was 1.00. ** Confirmed negative study by scintiphotography. All the others were obviously positive by sdntiphotography. < gressed. Structural bone abnormalities and osteoporosis were not manifest during the study and would not be expected to respond to changes in the disease. Scintiphotographic technics demonDISCUSSION strated evidence of inflammation before Organisms of the bedsonia group of bac- such inflammation was seen clinically. No teria produce a chronic inflammatory ar- false positive or false negative results were thritis in rabbits. Regardless of whether this obtained. The intensity of local involveagent is related to Reiter’s syndrome, acute ment by inflammation could be graded oband chronic arthritis develops in rabbits in- jectively by external counting and scintifected with bedsonia. These animals there- photographs. On serial studies, scintifore are ideal models for the study of meth- photographs and ratios of counts collected ods to evaluate arthritis. from the knees correlated well with each In our experimental animals, clinical ex- other and with the changing clinical state. amination failed to detect the disease in its We believe that this method of examinaearly stages and was consistently accurate tion is the most valid, simple method of only in the most florid stage. Because the evaluating early stages of arthritis and its results were not quantifiable, an accurate changing state. It is useful in animal reestimate of improvement, progression, or search, where objectivity is essential and a stability of the disease was impossible on clinical history is not possible. Its potential usefulness in clinical practice will be the subsequent examinations. The roentgenograms demonstrated soft subject of a later report. Pertechnetate scintiphotography is not tissue swelling early, but the degree of swelling did not change as the disease pro- specific for detection of arthritis. Any local- matory changes. Bedsonia organisms were reisolated from the synovial membranes. The noninjected knee of each rabbit was normal. 227 EXTJ3RNAL COUNTING AND SCINTIPHOTOGRAPHY ized inflammatory process might produce similar abnormal scintiphotos and count ratios and could therefore be studied by this method. SUMMARIO IN INTERLINGUA Arthritis a Bedsonia in conilios esseva studiate per scintiphotographia a camera Anger e contation externe, con le us0 de pertechnetato-99m como marca. Iste methodo d e evalutar le intensitate del arthritis e le alterationes occurrente in le curso del tempore in le morbo se provava plus sensibile e plus accurate que examines clinic e roentgenographic. REFERENCES 1. Schachter, J., Barnes, M. G., Jones, J. P., Jr., Engleman, E. P., and Meyer, K. F.: Isolation of bedsoniae from the joints of patients with Reiter's syndrome. Proc. SOC. Exp. Biol. Med. 122:283, 1966. 2. Amor, B., Coste, F., and Delbarre, F.: Mise en Lvidence, par culture d'agents du genre BedSonia dans le liquide articulaire en cas de rheu- matisme de Fiessinger-Leroy-Reiter (F. L. R. ). C. R. Acad. Sci. [D] 264:1365, 1967. 3. Smith, D. E., James, P., Schachter, J., EngIeman, E. P., and Meyer, K. F.: Experimental bedSonia arthritis in the rabbit. In preparation. 4. Sholkoff, S. D., Glickman, M. G., and Powell, M. R.: Restraint of small animals for radiopharmaceutical studies. In preparation.