PERIODIC AND RHYTHMIC XITOTIC ACTIV~TY m THE KIDNEY O F THE ALBINO RAT CHARLES M. BLUMESFELD Uepnrtmenl of Anatomy, Unkie?-sity of Utah. Salt L t r L c C i t y The morphology of mitosis lias been stndi ed extensively. In unicellular and some of the simpler multicellular organisms the factors regulating it have been investigated. Experiments hare been conducted iii tvliicli stimulation and inhibition of this form of cell division have been attempted. Mitogenetic radiation is the object of much attention. Few studies have been made concerning the physiology of this process in higher forms. lllitosis is probably the commoner form of cell dirision in most tissues and organs. As such, it i s thc essential feature of growth by Iiyperplasia, and knowledge of the manner in which it is controlled ~.r-ouldbe of importaiice in both normal and abnormal increase. Great difficulties surround tlie problem. Although removal of an organ, part of an organ, o r a few cells to an artificial meciium makes direct obscrvation feasible, it eliminates the influence of the vest of tlie organism, wliich is of obvious importance in normal mitotic activity. Direct microscopic obserration of parts of the living organisms is reduced in value by the Iimited areas which can bc studied, and by the abnormal conditions wliidi are sometimes created. K k T E H I A I i S AND METIIODS As an approach to the problem it was decided to determine whether mitotic activity was rhythmic or periodic. Such studies have been made in mammals by F o r t q n - v a n Leyden 433 THE 4 N A T O 3 I I r 4 T ~RTCONI), \ U J 7 2 , Xi0 4 A N D W P P T P \ l F N l 436 CHARLES &I. BLUMENFELD ('17, '26), Ortiz PicGn ('33), and Carletoii ('34). Their observations will be discussed later. F o r this purpose 240 male albino rats of the TJTistarstrain, higlily inbred, mere employed. When 28 days old they were killed with chloroform, the executions having been timed so that twenty rats were killed sometime within each 2-hour interval of the day and night. TVhe11 litter mates were used they mere killed at equidistant points in the 24-hour period so that inherited differences in rate of growth would not by chance be concentrated at certain limes. The rats wcre autopsied as rapidly as possible, and usually all parts were removed and in the fixatioe within 5 minutes after death. With the exception of the adrenal and thyroid glands, which were fixed whole, thin slices, to insure rapid fixation, were taken of tlie following structures : brain, liver, kidney, pancreas, skin, small intestine, submandibular gland, testis, thymus and tongue. Bouin's fluid was employed as the fixative. Dchydrstion, clearing, infiltration with and enibedding in paraffin were done in the nsnal manner. Thus far the cortex of the kidneys from the first 96 rats has been studied. Each 2-honr interval u7as represented by eight rats. From the left kidney o l each rat a central segment, approximately 5 mm. thick, was sectioned serially a t 8 p . Every twentieth section was mounted. Heideiihaiii's iron hematoq-lin was uscd first, but trial showed that careful staining with Harris' hematoxplin arid eosiii P,much less time-consuming, gave preparations as good for counting mitotic figures. As an index of the mitotic activity of a specimen the number of mitoses in 1000 fields was counted. A field was demarcated by two pairs of hairs arranged t o form a square and cemented in a cardboard I-ing set in the eyepiece of a microscope. Tlie couriting was done at a magnification of 1000 diameters. To make the findings more representative of a kidney, instead of counting 1000 consecutive fields in a single section, 250 consecutive fields in each of four sections were examined. The four sections studied u7ere usually the first, fifth, tenth and fiiteenth, and since each of these sections was every twentieth of the serial ribbon, there was 437 PERIODIC AND R H Y T H N l C MITOTIC ACTIVITY an interval of a t least 480 ~1 between them. Thus there was no danger of counting the same mitosis twice. Counting was always begun at the lateral border of the kidney so that approximately the same portion of each kidney was inspected. The findings were recorded as number of mitoses per 1000 fields. I n this way values of mitoses per I000 fields of renal cortex were obtained from each of eight rats for each 2-hour intcrval. The values for individual rats are listed in table 1 according to the time thc rats were killed. The means of the values f o r each 2-hour interval were calculated (table 1) and from these a curve if-as constructecl. The points on tlic curve TABLE 1 Individ?cal and mean values of mitoses per 1000 fields, based on eight rats per &hoar interval for a period of 24 hours ~ 6-8 8-10 10-12 12-2 2-4 lo-la P.X. P.X. P.M. A.M. A.M. AX. ~ 36 63 44 72 62 17 47 103 55.5 I I 66 47 41 35 36 68 67 122 59.8 38 I I 68 45 51 73 26 42 71 2.5 44 59 54 35 74 40 36 ~ 46.0 12 16 54 21 21 22 61 31 0 2 59 27 12 37 46 20 34 33.6 28.5 ~ 50.4 40 25 32 39 6s 47 37 33 ~ 41 .o 33 25 30 3R 42 83 63 53 27 40 10% 42 33 25 31 13 ~ ~ Ai.3 41.4 representing mean values were placed midway i n the corresponding time intervals; i.e., the average value for the period 8 P.M. to 10 r m . was placed at 9 P.M. The c u r w is presented in figure 1. OBSERVATIONS The data show that in the cortcx of the kidneys studied there was periodicity of mitotic activity. The lowest rate of activity occurred during the period from 10 P.M. to 1 2 night, the mean value being 28.5 mitoses per 1000 fields. The highest rate of activity occurred during the period from 2 P.M. to -1 P,M., thc mean value being 59.8 mitoses per 1000 fields. From the point of lowest to the point of highest activity there mas an interval of 16 hours; from high to low point, 8 hours. 438 CHARLES M. BLUMENFELU The irregular curve seems to be composed of 6-hour cycles. Starting from the lowest point of mitotic activity a t 11 P.M., there is a rise to 1 A.M., another rise to 3 A.M., then a drop to 5 A.M. I n the next 6-hour interval there is a rise in mitotic activity from 5 A.M. to 7 A.M., another rise from 7 A.M. to 9 A.M., then a drop to 11A.M. Tn the third 6-hour interval there is a rise from 11A.M. to 1P.M., another rise t o 3 P.M., then a drop to 5 P.M. But in the last 6-hour interral there was a rise from A CURVE OF M I T O T I C A C T I V I T Y IN T H E RENAL CORTEX OF THE A L B I N O RAT F O R A P E R I O D O F 2 4 H O U R S 8PM IZPM 4AM 8AM 12 M 4 PM 8 PM TIME IN HOURS Figure I 5 P.M. to 7 P.M., then a fall t o 9 P.M., and a second fall to 11P.M., the lowest point of activity during the 24-hour period. The similarity in character of curvcs representing three of the 6-hour intervals, and the striking departure from these of the fourth is graphically portrayed in figure 2. Table 1, in which individual values are listed, shows that f o r any period there is wide variation of these values. Presentation of means, alone, gives too little consideration to the PERIODIC AND R H Y T H M I C MJTOTIC ACTIVITY 439 variability. I n making a statistical study of the data, using standard biometric methods, comparisons were drawn between mitotic activity during the day and night, aud betwecn the first and second halves of each of these intervals. The period from 8 A.M. t o 8 P.M. was designated as day; that from 8 P.M. to 8 A.M., as night. These periods, divided in half, vere desigr , first half of day, second half of day, nated, r e s p e c t i ~ ~ las: first half of night and second half of night. &lean values and SIX-HOUR SEGMENTS O F THE 2 4 - H O U R C U R V E O F M I T OTIC A C T I V I T Y I N T H E R E N A L CORTEX O F T H E A L B I N O RAT 60 Lr, a 0 h f 30 T I M E l N HOURS Figure 2 statistical data f o r comparisoiis made between various periods are giren in table 2. From these data it was learned that the mean lcrel of mitotic activity was significantly greater during the day than at night. Mitotic activity was a t its lowest level during the first half of the night, and rose, by gradually decreasing steps, to the highest level during the second half of the day. Comparisons drawn between successive halves of the night and 440 CHARLES M. GIAUMEKFELD day revealed no sigmificant differences. Mitotic activity was significantly lower during the first half of the night and significantly higher during the second half of the day than during the rest of the 24-hour period. TABLE 2 A slntistzral coinpartson of average Eewls of mitotic acfivity dtwing onrtous porlzons of the 24 hour period I I No. Time T I X E INTERVALS POMPARFT) iiIcankP.E.1 Tlme I I DIFF. S.R. *r.x. KO. 5LeankP.E. ~ I US. 8 P.Y. to 8 A.M. 48 38.16L1.34 8 A.M. to 8 P.M. 48 5 0 . 2 0 i . O F 8 P.N. t o 2 24 34.37%f?.l(i 2 A.M. to 8 A.M. 24 41.9Si2.06 s A.M. t o 2 P.M. 24 48.3713.03 -6.42 04 52.04&2.$9 -1-3.67 -3.67 1.7 8 P.N. 14.12 0.9 8 P.X. 72 47.4.521.57 S P.N. t o 2 P.N. SO, 4 1 3 6 f l . K A.N. 2 A.M. t o 8 A.M. 24 41.95&2.06 8 A.M. to 3 P.N. 24 48.37-1-3.03 2 8 P.M. t o 2 24 34.37rt?.l(i 2 A . % . A.Y. 2 P.M. to 8 P.M. 24 52.04*2.79 P.X. t o to -12.01 22.58 -7.58 -1-2.99 -13.08 *2.67 10.48 t3.16 4.6 2.5 4.9 3.3 S o . , number o i renal cortices examined. Mean, mean number of mitoses per 1000 fields. P.E., probablc error. Diff., differcncc. S.R., signifiranre ratio, a n expression of the probability t h a t a n observed difference is due to random sampling. The greater the significance ratio the less likely it i s t h a t the observed difference is accidental. I n this report a signjficance ratio of 3 or more i s assumed to indicate significance because there is less than 1 chance in 20 t h a t such a differencc could occur by chance. IIIXCUSAION It has been established that mitotic activity exhibits periodicity in rate in various plants. From such studies Fortuynvan Leyden ('l7), was led to investigate mitotic activity in animal tissues. She nsed six kittens, killed on the second day after birth, and counted the number of mitoses seen in sections of the cornea, small intestine, liver, lymph node, thymus, spleen and bone marrow, and in spreads of the mesentery. H e r method of obtaining an index of mitotic activity was to count from 2500 to 10,000 nuclei, and note the nnmber that PERIODIC A N D R I I Y T H M I C M I T O T I C AUTITJTL' 441 mere in mitosis. Mitoses were so few aiicl clifficult t o recognize in the liver that she omitted it from further consideration. I n the cornea, activity was greatest at 10.30 P.M., least at 10.30 A.M. ; in the crypts of Lieberkuhn of the small intestine, greatest at 10.30 r.M. and least at 10.30 li.nI. ; in the mesentery, greatest at 2.30 AX. and least at 10.30 A.M. The findings in the lymph node, thymus, spleen ancl bone marrow u7ere essentially similar. The same investigator reported ( '26) studies of mitotic activity in the crypts of Lieberkuhii and cornea of mice 2 weeks old. No mitoses mere seen in the cornea, but in the crypts of Lieberkulin mitotic activity was greatest at 10.30 A.M. and least a t 2.30 AX. H e r results are open t o tlie criticism of paucity of samples. Ortiz Pic6n ('33) in the course of an investigation of mitotic activity in the epidermis of mice of various ages, made some observations concerning daily variations in rate of activity. He utilized skin from the lumbar region of six mice, grouped in three pairs. One mouse from each pair was killed a t 1 2 M., the others at 7 P.M., 8 P.M. and 1 2 night. An index of mitotic actirity was obtained by counting all the mitoses in 1 sq.mm. of skin. Mitoses in the epidermis were more than three times as numerous in the mice killed at noon than in those killed at night. IIis results, also, are subject to the criticism of too few samples. Carleton ( '34) reported studies of mitotic activity in tlie epidermis and epithelial cells of the hair follicles of forty-eight mice killed from 8 hours t o 7 days after birth. She employed eight litters of six mice each, and killed the members of each litter a t 4hour intervals, beginning a t 1 2 M. Five thousand cells were counted in each specimen. The per cent of tliem in mitosis was considered an index of mitotic activity. The results indicated that mitotic activity mas greatest at 8 P.M. and least at 12 M. The differeiices in the results obtained by these investigators may well be due to the small size of the samples used by two of tliem, t o differences in organs examined, ancl to differences iii the ages and orders of mammals employed (cat, mouse). I n cats, F'oriuyn-van Leyden observed the greatest 442 C H A R L E S M. BLUMENPELD mitotic activity a t night, the least during the day; in mice she found activity lowest at night and greatest during the day. Ortiz Pic6n found mitotic activity in the epidermis of mice greatest at noon and lowest at night, but Carlcton, who used a far larger number of animals, found the opposite situation in the same organ of the same animal. My observations, which showed greatest activity during the day and least at night, agree with the findings of Fortuyn-van Leyden and Ortiz Pic6n for the mouse. Horn-ever, comparison of results obtained by various investigators will be of value orily when such factors as size of samples, animals used, physiologic age of animals, organs studied, arid external conditions have been made more uniform. I n one respect the previous and present findings were alike : daily periodicity of mitotic activity was found in various organs of cats, micc and rats. I n the present investigation it is not possible to explain the findings from data thus far available. The sudden drop in mitotic activity which occurred during the transition from day t o night is suggestive of a causal relationship, but besides being a matter of the possible direct effects of light, changes are produced in the environment and activities of the rats in a n experimental colony. The 6-hour cycles pointed out are indicative of rhythmic mitotic activity, but these, too, cannot as pet be explained. It is felt that the chief d u e of such studies at the present lies in furnishing a tool for worlring on the problem of growth by hyperplasia under normal circumstances. I f it be established that the mean level o f mitotic activity jn the renal cortex of the rat is twice as great at 3 P.M. as it is a t 11 P.M., then a consideration of every measurable difference between kidneys obtained at thesc two times may help unravel the problem of the regulation of growth by mitosis. PERIODIC AND RHYTHMIC MITOTIC ACTIVITY 443 CONCLUSIONS 1. The renal cortex of ninety-six male albino rats, 28 days old, was examined for mitotic activity, an index of which was established by counting the mitoses seen in 1000 fields. Eight specimens were obtained during each 2-hour interval of a 24-hour period. 2. Mitotic activity was greatest from 2 P.M. to 4 P.M., and least from 10 P.M. to 12 night. 3. The curve plotted from the mean values appeared to be composed of four 6-hour segments, three of which closely resembled each other in character. This was interpreted as indicating rhythmic waves of mitotic activity. 4. Biometric studies revealed that the mean level of mitotic activity was significantly greater during the day (8 A.M. to 8 P.M.) than during the night (8 P.M. to 8 A.M.) ; that it was significantly less during the first half of the night (8 P.M. to 2 A.M.) and significantly greater during the second half the day (2 P.M. to 8 P.M.) than during the rest of the 24' period. 5. Application of these findings may prove useful in f studies of the factors regulating growth by mitosis. LITERATURE CITED CARLETON, A. 1934 A rhythmical periodicity in the mitotic division of cells. J. Anat., vol. 68, pp. 251-263. FORTUYN-VAN LEYDEN,C. E. DROUGLEEVER 1917 Some observations on perio nuclear division in the cat. Proc. Akad. wet. Amsterdam, Bd. 19, S. 3 8 4 4 . 1926 Day and night period in nuclear divisions. Proc. Akad. wet. Amsterdam, Bd. 29, S. 979-988. ORnz PIC6N, J. M. 1933 Uber Zellteilungsfrequcnz und Zellteilungsrhythmus in der Epidermis der Maus. Ztschr. f . Zellforsli. u. mikr. Anat., Bd. 23, 5. 779-789.