(From the Division of Preventable Diseases, Minnesota Department of Health, and the University of Minnesota, Minneapolis)

SPECIFICITY IN THE EFFECTS ON BRAIN METABOLISM OF TWO DIFFERING NEUROTROPIC VIRUSES* BY MARGARET NICKLE AND HERMAN KABAT, M.D. (From the Division of Preventable Diseases, Minnesota Department of Health, and the University of Minnesota, Minneapolis) (Received for publication, May 26, 1944) Brain tissue infected with the virus of poliomyelitis has been shown to have a decreased ability to metabolize glucose under anaerobic conditions while oxidation of glucose is unimpaired (1). Preliminary studies also suggested that oxidation of lactate, succinate, and pyruvate by poliomyelitic brain tissue did not differ from normal. Dehydrogenase activity of poliomyelitis-infected brain was unaffected with glucose, lactate, or succinate as substrates but was greater than normal in the absence of added substrate. Sodium fluoride had a greater inhibitory effect on anaerobic glycolysis of normal than of poliomyelitic mouse brain suspensions. Preliminary studies of the chemical changes in the brain, resulting from infection with the virus of poliomyelitis indicate that the content of lactic acid is decreased (2), that of adenosine triphosphate is increased while that of phosphocreatine and residual organic phosphate is decreased (3). A considerable decrease in anaerobic glycolysis of chick embryonic tissue has been observed 2 to 4 days following inoculation of tissue cultures with the Western strain of equine encephalomyelitis virus (4). This effect on tissue metabolism was prevented by inoculation of the tissue with virus mixed with specific Western equine encephalomyelitis antiserum but was still observed after tissue inoculation with virus plus anfimeningococcus serum. Using mouse embryonic brain tissue, it was possible to demonstrate a decrease in anaerobic glycolysis following inoculation with St. Louis encephalitis virus and Jungeblut-Sanders murine poliomyelitis virus (5). In the present report, the effects on brain metabolism of infection with two distinct neurotropic viruses are compared. While both Western equine encephalomyelitis and poliomyelitis viruses decrease anaerobic glycolysis of mouse brain tissue, specific differences become apparent when a more extended survey of metabolic activities is carried out. Methods The poliomyelitis virus suspension was prepared from spinal cords of paralyzed mice which had been infected with the Lansing strain from successive passages made during the course of * Aided by a grant from The National Foundation for Infantile Paralysis, Inc. 247

248 BRAIN METABOLISM AND TWO NEUROTROPIC VIRUSES the experiment. A i0 per cent suspension was centrifuged and Swiss albino mice about 3 to 4 weeks of age were inoculated intracerebrally with 0.03 cc. of the supernatant. In most cases, the brain was removed for metabolic study as soon as paralysis appeared but some determinations were carried out with mice showing more severe signs of illness. A few mice showed no paralysis but had definite symptoms of encephalitis. Brains of mice infected with Western equine encephalomyelitis virus (W.E.E.) were obtained from animals used in routine neutralization tests. Mice 5 to 6 weeks of age were inoculated with 0.03 cc. of a mixture of infected mouse brain suspension and patient's serum, the final dilution of virus being from 104 to 10"% Those showing definite encephalitic signs (hence with no protection from the serum) were used in the experiments. A few mice inoculated only with ]0 per cent Western equine encephalomyelitis virus suspension and showing signs of infection with it were also studied. Aerobic and anaerobic metabolic activities of normal, W.E.E.-infected and poliomyelitisinfected mouse brain suspensions were measured in the Barcroft-Warburg apparatus atj38 C. Determination s were made simultaneously on three samples of each brain suspension and two brains were studied in each experiment. The freshly removed brain was crushed in a mortar and a I0 per cent suspension in Locke's solution was prepared. Unfortunately, normal brains were not always studied at the same time as infected ones. This was a matter of economy, which involved the use of infected mice when they were available and normal ones when infected mice were not available. A. For aerobic metabolism, each Warburg flask contained the following: i0 per cent brain suspension... 0.5 cc. Locke's solution... 1.0 cc. IV/ Phosphate buffe r ~-~, ph 7.4... 0.3 cc. Substrate 0.16 ~r... 0.i cc. Sodium chloride 0.9 per cent... to total 3.0 cc. The apparatus was filled with air. The vessel containing this mixture was attached to the manometer and equilibrated at 38 C. for 10 minutes. Then the stopcocks were dosed and readings of oxygen consumption taken at 15 minute intervals. B, Foranaerobic metabolism: I0 per cent brain suspension... 0.5 cc. Sodium bicarbonate 0.16 M... 0.5 cc. Substrate 0.16 M... 0.2 cc. Sodium chloride 0.9 per cent... to total 3.0 cc. In preparing this mixture, CO2 was flushed through the bicarbonate solution immediately before pipetting into the flasks and the glucose, when used, was added last. The apparatus when then flushed with a mixture of 95 per cent N~ and 5 per cent CO2 for 12 to 15 minutes, placed in water bath at 38 C., and equilibrated for I0 minutes before dosing the stopcocks. Readings of CO2 evolution were taken at 15 minute intervals. The Locke's solution contained 0.25 per cent glucose. Thus some glucose was added to the brain in every experiment, even when other substrates were being studied. While significant and specific differences in metabolism of the brain infected with the two viruses were observed under these conditions, the differences might be expected to be even more striking if Ringer's solution were substituted for the Locke's solution, thereby omitting the additional glucose. I~SU-LTS Normal brain suspensions as well as suspensions prepared from brains infected with the viruses of poliomyelitis or Western equine encephalomyelitis,

MARGARET NICKLE AND HERMAN KABAT 249 I

250 BRAIN METABOLISM AND TWO NEUROTROPIC VIRUSES show a wide variation in oxygen consumption and in anaerobic glycolysis. The factors responsible for this variation in tissue metabolism in vitro are poorly understood. Because of this variation, it is necessary to apply statistical techniques to determine whether a significant difference exists. The essential data on anaerobic metabolism are presented in Table I. While the number of brains of each type studied was small (9 to 14), the differences in the findings with infected brains and normal ones are significant by both the TA Comparison of Effects on Oxygen Utilization of Brain Tissue Inj ( and lactate-g Acrobic-glucose 121 rag. per cent 1st hr. --L Mean... Number of brains:~................................. 14 ] 14 Standard deviation................................. 24.7 ] 23.4 Probable error of mean............................. I 4.45 4.21 Probable error of difference......................... 6.13 Actual difference................................... 1.8 Actual difference Probable d i f f error e r eof n c ~ O. 29 t... 0.134 n... 26 Change from normal, per cent....................... i P 12.5 2.36 5.04 13.1 2.6 1;? N 02.4 14 19.7 3.54 2nd hr. Total 2 hrs. E P N E 940 70 30;0 205 19.8 ] 11.1, 42.8 41.~ 3.56 2.01 7.7 7.52 5.02 4.1 10.75 7.8 23.4 9.5 1.55 5.7 0.88 1.01 3.6 0.57 26 1-22.825 26 _!_ 22.8 4.2 8.8 36.5 4.1, 2~6: --1.5.9 N 133.9* 15 18.6 3.26 N = Normal E = Equine encephalomyelitis (Western strain) P = Poliomyelitis (Lansing strain) * Oxygen consumption in cubic millimeters per.50 mg. wet weight per hour ~t Three determinations run simultaneously on each brain and averaged "t" value (6) and the "critical ratio." It is of interest that anaerobic C02 output is significantly below normal in both poliomyelitis and W.E.E. brain tissue when the added glucose concentration is high, while only W.E.E. anaerobic metabolism is depressed significantly when the glucose concentration is low. With high glucose concentration, anaerobic metabolism of the encephalitic brain is 28 per cent below normal while that of the poliomyelitic brain is only 16.6 per cent below normal. It is worth noting that the mean CO2 output for the poliomyelitic brain is higher when the glucose concentration is low than when it is high. The values for anaerobic metabolism of the W.E.E. brain are the same whether the glucose concentration is low or high. Anaerobic metabolism is increased only slightly in normal brain by a large increase in glucose concentration. Data on oxygen utilization with various concentrations of added glucose or

MARGARET NICKLE AND HERMAN KA.BAT 251 Err lactate are presented in Table II. With glucose added to make a concentration of 121 rag. per cent, the poliomyelific brain shows a significant decrease from normal in 2 hours while the small decrease shown by the encephalitic brain is not significant. On the other hand, when the glucose concentration is increased or lactate is added to the glucose, the oxygen utilization of the encephalitic brain is significantly below normal whereas that of the poliomyelitic brain is not.,d with the Viruses of Poliomyelitis and Equine Encephalomyelitis ose used as substrates) 5.0 20.9 5.08 4.46 6.03 5.54 9.5 6.6 3.23 1.19 2.2 0.79 24 23 Aerobic-glucose 217 rag. per cent Aerobic-lactate 48 rag. per cent glucose 121 rag. per cent L hr. I 2nd hr. Total 2 hrs.,, 1st hr. 2nd hr. Total 2 hrs. E P E P NI, P P N E P N E P 1 4.411 L271"3 101.8 80.39 8716 111995 ~8 8 86.7 154.5 178.3 12 9 I 3.12 14.5, 23.9 3.1t 5.09 4.4 5]97 21..514.2 4.8! 2.38 3.0~ 1.51 17 18 --21.1 %o 184.8,049 9 10 32.6 27.52 43.9 6.96 6.11 9.36 9.3 11.66 46.1 26.0 4.9( 2.23 3.1', 1.43 17 18-20.2 103.2 12 19.77 t 7.74 369, 13:5, 19.4 4.8'/ [ 3.0. ~ 9.9 2.22 4.3 3.1 0.72 0.41 20 64.1 5113 13 17.8 9.64 3.33 1.87 3.81 12.8 3.36 2.12 23 L4.2 32.8 14.4 20.0 3.18 6.13 2.8 4.49 4.6 6.7 7.6 5.3 32.2 8.4 1.15 4.7 0.71 3.0 20 23-17.2 1.1 0.65 20 The normal brain tissue shows practically no increase in oxygen consumption from a considerable increase in glucose concentration. The encephalific brain has a higher oxygen uptake with low glucose than with high glucose. On the other hand, the poliomyelitic brain shows a greater aerobic metabolism at the higher glucose concentration. Addition of lactate to the glucose decreases greatly in the 2nd hour the oxygen utilization of the normal and of the two infected brains and markedly decreases oxygen uptake of the encephalitic brain in the 1st hour. The aerobic metabolism of the normal and infected brains, on addition of pyruvate-glucose and succinate-glucose as substrates, is presented in Table III. With pyruvate-glucose added, the mean oxygen utilization of the infected brains is below normal but the difference is not statistically significant. The addition of pyruvate to the glucose decreases oxygen consumption of the

252 BRAIN 3~[ETABOLIS3~ AND TWO NEUEOTROPIC VIRUSES,,, + i ~N N " " 2 ~:.~ "r, ~g : :~ :~ ~.o II II II <DI~

MARGARET NICKLE AND HERMAN KABAT 253 normal brain, particularly in the 2nd hour. Addition of succinate to the glucose greatly accelerated oxygen utilization by both the normal and poliomyelitic brains. In the Ist hour, aerobic metabolism of the poliomyelitic brain is higher than the normal by 13.5 per cent and this difference is statistically significant. The comparison of the effects on brain metabolism of infection with the viruses of poliomyelitis and Western equine encephalomyelitis is summarized in Table IV. The percentage change from normal is calculated from the total 2 hour metabolism. It is evident from this table that the metabolic effects of these two neurotropic virus infections on the brain are decidedly different Specificity of Effects on Added substr~te TABLE IV Brain Metabolism of Two Different Virus Infections Concentration rag. $er cent 121 Western equine encephalomyelitis Change from ] Statistically normal [ significant per cent --4.1 No Poliomyelitis ange norma_...~l from [ Statistically significant per cent [ --15.9 ] Yes 217 -- 20 Yes --11.2 [ No Aerobic Lactate 48 121 --17.2 Yes --4.5 No Pyruvate 47 121 --9.5 No --8 No Anaerobic 37.5 229.5 --23.8 Yes --28 Yes --7 No - 16.6 Yes and quite specific. Under four of the six substrate conditions studied, there is a statistically significant decrease in brain metabolism from one virus and no significant change from normal in the case of the other virus. DISCUSSION One may conclude in a preliminary way that the metabolic effects on brain tissue of infection with the viruses of poliomyelitis and Western equine encephalomyelitis are different and quite specific. Because of the small number of brains investigated and the wide variation in metabolic activity in vitro of both normal and infected brains, it would be hazardous to consider these observations conclusive. Nevertheless, the marked specificity of the metabolic effects of the two differing neurotropic virus infections, and the fact that the

254 BRAIN METABOLISM AND TWO NEI..YROTROPIC VIRUSES differences from normal are clearly significant statistically by two different tests suggest that more extended investigation should confirm these observations. It has been possible in this study to confim the results of Racker and Kabat (1) who demonstrated that with a relatively high concentration of glucose present, the poliomyelitie brain shows a decreased anaerobic glucolysis but the oxygen consumption is unaffected. On the other hand, we have shown that at lower glucose concentrations, the oxygen consumption of poliomyelitic brain is significantly decreased while anaerobic glycolysis is not significantly below normal. Furthermore, there is suggestive evidence that poliomyelitic brain may show a greater oxygen utilization than normal, with succinate-glucose as the substrate. Western equine encephalomyelitic brain shows a significant decrease in anaerobic metabolism at both high and low glucose concentrations. On the other hand, oxygen utilization is unaffected at a moderate glucose concentration and is significantly decreased at a high glucose concentration. Aerobic metabolism of W.E.E. brain is also below normal with lactate-glucose as the substrate. It is noteworthy that the decrease in anaerobic glycolysis of mouse brain infected with W.E.E. virus is only 28 per cent while the decrease in anaerobic glycolysis in embryonic chick tissue infected with this virus is approximately 50 per cent (4). An interesting example of specificity in the action of viruses on tissue cells has been reported by Andrewes (7). He observed that cultivation of influenza A virus in simple culture of cells rendered the latter unsuited to the increase of a biologically distinct strain of influenza A virus when this was added 24 hours later. Such an altered tissue culture was still favorable to the multiplication of an unrelated virus such as that of lymphogranuloma venereum. Whichever strain of influenza A virus was added to the culture first could bring about conditions preventing the increase of the other. The reason for this phenomenon is obscure. SIJ'N31ARY 1. Brain tissue infected with the virus of Western equine encephalomyelitis shows specific differences in metabolism from brain tissue infected with the virus of poliomyelitis. 2. With added glucose concentration of 121 nag. per cent, oxygen utilization of pouomyelitic brain is significantly below normal and that of encephalitic brain is not. 3. With added glucose concentration of 217 rag. per cent, oxygen utilization of encephalitic brain is significantly below normal and that of poliomyelitic brain is not. 4. With lactate-glucose as the substrate, oxygen utilization of encephalitic brain is significantly below normal and that of poliomyelitic brain is not.

MARGARET NICKLE AND HERMAN KABAT 255 5. With pyruvate-glucose as the substrate, neither encephalitic brain nor poliomyelitic brain differs significantly from normal in oxygen consumption. 6. With succinate-glucose as the substrate, oxygen utilization of poliomyelific brain is significantly above that of the normal control in the 1st hour. 7. With added glucose concentration of 37.5 mg. per cent, anaerobic metabolism of encephalitic brain is significantly below the normal whereas that of poliomyelitic brain is not. 8. With added glucose concentration of 229.5 rag. per cent, anaerobic metabolism of both encephalitic brain and poliomyelitic brain is significantly below the normal. BIBLIOGRAPHY 1. Racker, E., and Kabat, H., J. Exp. Med., 1942, 76, 579. 2. Kabat, H., Erickson, D., Eklund, C., and Nickle, M., Science, 1943, 98, 589. 3. Kabat, H., Science, 1944, 99, 63. 4. Victor, J., and Huang, C. H., J. Exp. Med., 1944, 79, 129. 5. Huang, C. H., Proc. Soc. Exp. Biol. and Med., 1943, 54, 160. 6. Croxton, F. E., and Cowden, D. J., Applied general statistics, New York, Prentice- Hall, Inc., 1940. 7. Andrewes, C. H., Brit. J. Exp. Path., 1942, 23, 214.