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Periodic and rhythmic mitotic activity in the kidney of the albino rat.

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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.
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