(Utterbach, 1962) haemorrhages, fevers were sometimes observed even under conditions. (Received 28 August 1986)

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1 J. Physiol. (1987), 390, pp With 6 text-figures Printed in Great Britain FEVER INDUCED IN RABBITS BY INTRAVENTRICULAR INJECTION OF RABBIT AND HUMAN SERUM ALBUMIN BY AKIO MORIMOTO, NAOTOSHI MURAKAMI, TOMOKI NAKAMORI AND TATSUO WATANABE From the Department of Physiology, Yamaguchi University School of Medicine, Ube Yamaguchi 755, Japan (Received 28 August 1986) SUMMARY 1. Intraventricular injection of rabbit and human serum albumin, and rabbit endogenous pyrogen produced dose-dependent fevers in rabbits. The pyrogenicity of albumin was less than one-twentieth of the pyrogenicity of endogenous pyrogen. 2. Fevers induced by ventricular albumin were significantly suppressed by intraventricular injection of indomethacin which is a cyclooxygenase inhibitor. In contrast, subcutaneous injection had no effect. 3. Ventricular endogenous pyrogen induced several of the acute phase responses, i.e. decreases in the plasma concentration of iron and zinc, and increases in both the plasma concentration of copper and the white blood cell count. Albumin induced none of these responses. 4. It is concluded that fever induced by ventricular albumin is processed by prostaglandins synthesized within the central nervous system. However, ventricular albumin does not activate the central mechanism to induce acute phase responses. INTRODUCTION Fever is one of the major symptoms of various infections. According to the current theory (Atkins, 1960), a bacterial endotoxin causes fever by inducing circulating and/or reticuloendothelial macrophages to produce endogenous pyrogens, for example interleukin 1 (Dinarello, 1984). Subsequently endogenous pyrogen raises body temperature by its actions on the central nervous system (c.n.s.). However, in the cases of intraventricular (Rudy, Westergaard & Yaksh, 1978) or subarachnoid (Utterbach, 1962) haemorrhages, fevers were sometimes observed even under conditions in which there were no infections. Such fevers might be induced by constituents of circulating blood. In 1979, Hattingh, Laburn & Mitchell reported fevers associated with intravenous injections of bovine serum albumin. They concluded that fever following bovine albumin injection had characteristics similar to fever caused by endogenous pyrogen. Therefore, it is inferred that serum albumin has intrinsically pyrogenic action when it enters the C.N.S. In the present study, we investigated the effect of intravenous and intraventricular injections of rabbit and human serum albumin on the body temperature of rabbits.

2 138 A. MORIMOTO AND OTHERS In the results, both albumins showed strong pyrogenicity when ventricularly injected, but intravenous injections did not produce fever. Subsequently, in order to clarify the characteristics of serum albumin and endogenous pyrogen, which mediate febrile responses, we investigated pyrogenicity and some of the acute phase responses (i.e. plasma trace metals and blood cell counts) during fever induced by ventricular injection of serum albumin and endogenous pyrogen. METHODS New Zealand White male rabbits, weighing kg, were used. At least 10 days prior to the start of the experiment, all animals (n = 7) were implanted with a stainless-steel cannula (1-0 mm o.d.) in the third cerebral ventricle, by standard stereotaxic techniques under general anaesthesia (pentobarbitone sodium, 20 mg/kg). Intraventricular injections were made through the cannula with a microsyringe pump for a period of 5 min. The volume infused into the cerebral ventricle was always 20,ul, while the doses of injected substances varied. Intravenous injections were made into the marginal ear vein through a sterile needle (25 gauge). Rabbit endogenous pyrogen (REP) was prepared from white blood cells of male rabbit (New Zealand White strain). The white blood cells were stimulated by lipopolysaccharide of Salmonella typhosa endotoxin (Difco Laboratories). The general procedures have been described in detail elsewhere (Morimoto, Watanabe, Ono, Sakata & Murakami, 1986). Partial purification of REP was achieved by ultrafiltration using two types of membranes (loym1o, 1OXM50, Amicon), which removed all substances outside the range of to molecular weight. Consequently, this partially purified REP contained a protein concentration of 1 mg/ml. Rabbit (Sigma) and human (Kyowa) serum albumin were dissolved in sterile saline, and prostaglandin E2 was also dissolved in saline mixed with ethanol (2 %). The indomethacin as cyclooxygenase inhibitor was dissolved in sesame oil (20 mg/ml) for subcutaneous injection and in ethanol (99 %) for ventricular injection. On the day of the experiment, animals were minimally restrained in conventional stocks. For each rabbit, the experiment was performed at a room temperature of C between and h under the same lighting conditions. Throughout the experiment, the rectal temp'erature was measured with a copper-constantan thermocouple. The rectal temperature in each animal was allowed to stabilize for at least 90 min before any injections were made. Furthermore, for measuring the blood cell counts and the plasma concentration of iron, zinc and copper, about 5 ml of blood was withdrawn through the marginal ear vein. Blood samplings were made three times:1 h before, and 8 and 24 h after intraventricular injections of saline (201I), rabbit serum albumin (RSA) (400,ug) and REP (10 sag). Immediately after collecting the blood, the number of white blood cells and the number of red blood cells were measured with an automatic counter (Coulter, model S plus II). The remaining blood was centrifuged at 2000 rev/min for 15 min at 4 C, and plasma was separated. The plasma concentration of iron was measured by an autoanalyser (Olympus Super-Multi AS8,000), while the plasma concentrations of zinc and copper were measured by an atomic absorption spectrophotometer (Hitachi model ). Data were analysed for statistical significance by Student's t test for unpaired data. RESULTS Fig. 1 shows the effects of intravenous and intracerebroventricular injections of RSA and REP on the rectal temperature. About 30 min after intraventricular injection of RSA (200 4ag), the rectal temperature gradually started to rise and a prolonged fever was observed. Intravenous injection of RSA (80 mg/kg) did not induce any fever (Fig. 1 A). In contrast, an intravenous injection of REP (0-1 mg/kg) produced a typically monophasic fever with short latency. Intraventricular injection of REP ltg) (10 also induced fever but the latency to fever onset and the febrile pattern were similar to those induced by that of RSA. As shown in Fig. 2, the results obtained from human serum albumin (HSA) were almost the same as those from

3 ALBUMIN AND FEVER * B 1*0. I. I. I. I. I Fig. 1. Mean changes (mean + S.E. of mean) in rectal temperature (AT,) in the same group of seven rabbits after intravenous (I.v.) or intracerebroventricular (i.c.v.) injections (arrows) of RSA or REP. A: 0, i.v. injection of RSA (80 mg/kg); *, I.c.v. injection of RSA (200 jug). B: A, i.v. injection of REP (0 1 mg/kg; A, i.c.v. injection of REP (10 jug) *0 Fig. 2. Mean changes (mean + S.E. of mean) in rectal temperature (AT,) in the same group of seven rabbits after intravenous (I.v., El, 80 mg/kg) or intracerebroventricular (I.c.V., *, 200,ug) injections (arrow) of HSA. RSA. The mean maximum rise in the rectal temperature for 4 h after ventricular injection of several doses of RSA, HSA and REP are presented in Fig. 3. The Figure shows that the pyrogenicities of RSA and HSA were almost identical but less than one-twentieth of the pyrogenicity of REP.

4 140 A. MORIMOTO AND OTHERS 20 F IL -t E E x F AL T 0*5 1 AL 0 I I l Injection dose (,g) Fig. 3. Mean maximum rise (mean + S.E. of mean) in rectal temperature (ATr) in the same group of seven rabbits after intracerebroventricular injections of several doses of RSA (O), HSA (O) and REP (-) I~ a I 0 20 m a n. B 251- io0- -&- a 1*0 j; *t I Fig. 4. Mean changes (mean + S.E. of mean) in rectal temperature (ATr) in the same group of seven rabbits after intracerebroventricular (i.c.v.) injections of RSA and prostaglandin E2 (PGE2). A, effects of indomethacin (INDO) by i.c.v. injection (@, 400,ug) and subcutaneous injection (s.c., A, 20 mg/kg) 15 min before RSA injection. An i.c.v. injection of ethanol vehicle (EtOH, 0, 20,tl) was used as the control. B, effect of i.c.v. indomethacin 400 gtg) and ethanol vehicle (EtOH, 0, 20,1u) on fever induced by i.c.v. PGE2 (10 ug).

5 ALBUMIN AND FEVER To investigate whether fever induced by ventricular albumin is processed by inducing prostaglandin production in the C.N.S., the effects of ventricular and subcutaneous injections of indomethacin on febrile responses were investigated. When indomethacin (400,ug) was administrated intraventricularly before injection of RSA, RSA-induced fever was significantly suppressed (Fig. 4A). In contrast, subcutaneous indomethacin (20 mg/kg) did not affect the rise in body temperature induced. Moreover, as shown in Fig. 4B, intraventricular indomethacin did not affect the fever induced by prostaglandin E2 (10,g) injected into the ventricle, indicating that F- TI E 30 < L 1II \M 1 [: m I L:: CU LX li I1X'N 200 TK -: T SE 100k E ~ ~ 1 h F[p cle L T CU) E ~-50 l h 8 h 24 h pre-injection post-injection post-injection Fig. 5. Mean changes (mean+s.e. of mean) in plasma concentration of iron, zinc and copper in the same group of seven rabbits after intracerebroventricular injections of saline (open bars, 20 #s1), RSA (stippled bars, 200 jug) and REP (hatched bars, 10 psg). * P < ventricular indomethacin (400,ug) did not suppress the C.N.S. mechanisms involved in the development of fever. These results indicate that fever produced by albumin is accomplished by prostaglandins produced in the C.N.s. Fig. 5 shows the changes in the concentration of the plasma iron, zinc and copper levels 8 and 24 h after intraventricular injection of saline (20 #1), RSA (200,ug) and REP (10 jug). Intraventricular REP decreased the concentrations of plasma iron and zinc but increased the concentration of copper. Intraventricular RSA had no effect on these concentrations. Fig. 6 shows the changes in the white and red blood cell counts. Intra-

6 142 ~0 x ~8 6EL A. MORIMOTO AND OTHERS I *~~~~~~~~~~ -o4 _E T 0 1 h 8 h 24 h pre-inj ection post-injection post-injection Fig. 6. Mean changes (mean + S.E. of mean) in white and red blood cell counts in the same group of seven rabbits after intracerebroventricular injections of saline (open bars, 20 ul), RSA (stippled bars, 200,ug) and REP (hatched bars, 10 jug). * P < ventricular REP caused an increase in the white blood cell count but did not induce changes in the red blood cell count. Intraventricular RSA did not induce any changes in the blood cell counts. DISCUSSION Hattingh, Laburn & Mitchell (1979) showed that a small dose (4 mg) of bovine serum albumin induced consistent fever in rabbits when intravenously injected. In the present results, intraventricular injections of rabbit and human albumin produced fever, but intravenous injections did not cause fever even in high doses (80 mg/kg). This may indicate that serum albumin is intrinsically pyrogenic in the C.N.S. In clinical cases, we sometimes come across patients with pyrexia who exhibit no signs of infection in either physical or biochemical examinations. Doctors have treated such cases as 'fever of unknown origin' (Wolff & Dinarello, 1980), but no significant therapies have been explored. The present results show that serum albumin has strong pyrogenicity in the cerebral ventricle. This indicates that fever is developed even under non-infectious conditions, when blood or serum albumin pathologically flows into the cerebral ventricle. Circulating blood normally contains serum albumin within the range of mg/ml. Thus, less than 0 1 ml blood in the cerebral ventricle could induce a significant fever. Since Milton & Wendlandt (1971) observed the strong pyrogenic action of ventricular prostaglandin E, prostaglandins have been believed to be the final mediators in the pathogenesis of fever in the c.n.s. The fevers induced by ventricular albumin, as well as endogenous pyrogen (Morimoto, Murakami, Nakamori & Watanabe, 1987), are believed to be processed by prostaglandins synthesized within the C.N.S., because these fevers were significantly suppressed by ventricular, but not by subcutaneous, indomethacin which is well known as the most potent of cyclooxygenase inhibitors (Krupp & Ziel, 1979). Since indomethacin resists diffusion into the blood-brain

7 ALBUMIN AND FEVER barrier (Hucker, Zacchei, Cox, Brodie & Cantwell, 1966), it is considered that subcutaneous indomethacin had no effect on the c.n.s. However the pyrogenicity of albumin was less than one-twentieth, on a weight basis of protein content, as compared with the pyrogenicity of endogenous pyrogen. It is now generally recognized that the acute phase response represents a primary host defence response to infection and other acute inflammatory stimuli (Gordon & Koj, 1985). The acute phase response includes fever and changes in the plasma concentration of certain trace metals and glycoproteins. It is currently believed that endogenous pyrogen induces peripherally and/or centrally several of these acute phase responses (Kampschmidt, 1980), in addition to fever (Morimoto et al. 1987). In the present results, intraventricular endogenous pyrogen caused decreases in the plasma concentration of iron and zinc and increases in the plasma concentration of copper and the white blood cell count. These results further indicate that the central action of endogenous pyrogen is involved in the development of some of the acute phase responses. However, ventricular albumin did not cause changes in the plasma trace metals or the white blood cell count. Nevertheless, the magnitudes of febrile responses induced by REP (10 jug) and RSA (200 jig) were almost identical. Previous reports show that acute phase responses were not observed during fever induced by intraventricular (Merriman, Upchurch & Kampschmidt, 1974) or intrahypothalamic (Blatteis, Hunter, Llanos-Q, Ahokas & Mashburn, 1984) injections of prostaglandins. Therefore, it is considered that albumin produces fever by inducing prostaglandin synthesis in the c.n.s. just as endogenous pyrogen does, but that albumin does not activate the central mechanism to induce the acute phase responses. From the view of the acute phase response, fever induced by albumin is quite different from fever usually occurring under infectious conditions. REFERENCES ATKINS, E. (1960). Pathogenesis of fever. Physiological Reviews 40, BLATTEIS, C. M., HUNTER, W. S., LLANOS-Q, J., AHOKAS, R. A. & MASHBURN JR, T. A. (1984). Activation of acute-phase responses by intrapreoptic injections of endogenous pyrogen in guinea pigs. Brain Research Bulletin 12, DINARELLO, C. A. (1984). Interleukin- 1. Review of Infectious Diseases 6, GORDON, A. H. & KoJ, A. (1985). The Acute Phase Response to Injury and Infection. Amsterdam: Elsevier. HATTINGH, J., LABURN, H. & MITCHELL D. (1979). Fever induced in rabbits by intravenous injection of bovine serum albumin. Journal of Physiology 290, HUCKER, H. B., ZACCHEI, A. G., Cox, S. V., BRODIE, D. A. & CANTWELL, N. H. R. (1966). Studies on the absorption, distribution and excretion of indomethacin in various species. Journal of Pharmacology and Experimental Therapeutics 153, KAMPSCHMIDT, R. F. (1980). Metabolic alterations elicited by endogenous pyrogen. In Fever, ed. LIPTON, J. M., pp New York: Raven Press. KRUPP, P. J. & ZIEL, R. (1979). Antipyretics. In The Body Temperature, ed. LOMAX, P. & SCH6NBAUM, E., pp New York: Dekker. MERRIMAN, C. R., UPCHURCH, H. F. & KAMPSCHMIDT, R. F. (1974). Prostaglandin E1, aspirin and the action of leukocytic endogenous mediator. Journal of Pharmacology and Experimental Therapeutics 188, MILTON, A. S. & WENDLANDT, S. (1971). Effect on body temperature of prostaglandins of A, E and F series on injection into the third ventricle of unanaesthetized cats and rabbits. Journal of Physiology 218,

8 144 A. MORIMOTO AND OTHERS MORIMOTO, A., MURAKAMI, N., NAKAMORI, T. & WATANABE, T. (1987). Evidence for separate mechanisms of induction of biphasic fever inside and outside the blood-brain barrier in rabbits. Journal of Physiology 383, MORIMOTO, A., WATANABE, T., ONo, T., SAKATA, Y. & MURAKAMI, N. (1986). Rat endogenous pyrogen and fever. American Journal of Physiology 250, R RUDY, T. A., WESTERGAARD, J. L. & YAKSH, T. L. (1978). Hyperthermia produced by stimulated intraventricular hemorrhage in the cat. Experimental Neurology 58, UTTERBACH, R. A. (1962). Extracranial vascular disease. In Baker Clinical Neurology, pp New York: Hoeber-Harper. WOLFF, S. M. & DINARELLO, C. A. (1980). Fever of unknown origin. In Fever, ed. LIPTON, J. M., pp New York: Raven Press.