A Morphologic Study of Deoxyribonucleic Acid Synthesis and ... · were prepared with Kodak AR-10...

A Morphologic Study of Deoxyribonucleic Acid Synthesisand Cell Proliferation in Regenerating Rat Liver;

Autoradiography with Thymidine-H3*

J. W. GRISHAMf

(Department of Pathology, Washington University School of Medicine, St. Louis, Missouri)

SUMMARY

A morphologic study of DNA synthesis and cellular proliferation in regeneratingrat liver has been made by means of incorporation of thymidine-H3 and autoradiog-

raphy. Appreciable numbers of hepatocytes began to synthesize DNA between 12and 18 hours after partial hepatectomy, the number increasing rapidly until a peakvalue of 29.4 Â±6.2 per cent was reached at 20 hours after hepatectomy, thereafterdecreasing more slowly toward normal. The peak incidence of hepatocytes synthesizingDNA preceded the peak incidence of such cells in mitosis by about 6 hours. At equivalent periods of time 6-8 times as many hepatocytes were labeled as were in mitosis.

At times when near maximal numbers of hepatocytes were synthesizing DNA, suchactive cells were predominantly located in zones 1 and 2 of the hepatic acinus. Somecells originally labeled in these areas at 20 hours after hepatectomy were apparentlyforced into zone 3 by further cellular proliferation. From 0 to 72 hours after hepatectomy about 80 per cent of all new hepatocytes were formed in zones 1 and 2. Synthesis of DNA in littoral and ductal cells began 8-12 hours later than it did in hepato

cytes, reaching a peak between 36 and 48 hours after hepatectomy. During the entireperiod of regeneration studied, about 75 per cent of total DNA synthesis could be accounted for by hepatocytes, and hepatocytes in zones 1 and 2 accounted for about 80per cent of this.

After two-thirds partial hepatectomy, the liver impossible to assess by conventional biochemicalof the rat quickly regains its lost mass. Many and histological technics.parameters of this restorative growth have been Autoradiographic localization of the deoxyribo-documented (8, 26) ; the predominant mechanism nucleic acid (DNA) tracer thymidine-H3 (TDR-is the proliferation of cells left in the hepatic rem- H3) represents a partial integration of these two

nant. Liver is composed of several kinds of cells in approaches. This method permits identification ofdistinct orientation, the most numerous of which specific cells undergoing DNA synthesis and theirare parenchymal, littoral, and bile ductal (3). The localization within the lobule, increases sensitivityrole of each kind of cell in restorative growth, as for detecting mitotic growth, and allows the sub-well as the role of hepatocytes in different spatial sequent course of labeled cells to be traced (19).locations, is largely unknown and is difficult or Because of these advantages we have utilized thy

midine-H3 and autoradiography to examine the re-S254Â°8kWaSSUpp0rtedby U'S' PublÃŒCHealth SerrÃ¬ee generating liver in young male rats after partial

Portions of this study were read at the 44th Annual Meeting hepatectomy.

of the American Society for Experimental Pathology, Atlantic TV^TJT t T c t X-T-Â»A TUT-nr^oCity, N.J., April 18-17, 1959, and at the llth Annual Meeting MA11SK1AÃ•O AJNJJ ALKIUUUS

of the Histochemical Society, New York, New York, April Male, Wistar albino rats, weighing 140-150 gm.9-10,1960. aj. ^he tjme of hepatectomy, were used. They were

t Life Insurance Medical Research Fund Fellow, 1959-1961. housed four to a cage in air-conditioned quarters

Receivedfor publication March 5, 1962. and were fed Purina chow and water ad libitum.

842

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GRISHAMâ€”CellRenewed in Regenerating Rat Liver 843

Animals were fasted for 12 hours prior to surgeryand were offered food and water ad libitum post-operatively. Partial hepatectomy was performedby the method of Higgins and Anderson (10). Onthe basis of expected liver weight (4.0 + 0.251percent of the body weight in ten rats of the samestrain and weight), 64.2 + 3.4 per cent of the liverwas removed. Operative mortality was 1.4 percent, and, after an initial postoperative weightloss, the animals grew at the same rate as thesham-operated controls or controls which had notbeen operated upon.

Thymidine-H3 (specific activity 360 me/mmole)2 was given intraperitoneally in doses of 0.2-0.4 Â¿Â¿e/ginbody weight (diluted in physiologicalsaline solution to a concentration of 50 Â¿ic/ml).This dose of TDR-H3 is well below that whichcauses inhibition of regeneration in liver of partially hepatectomized young rats (6).

Two experiments were performed. In the first,TDR-H3 was injected at 6-hour intervals from 0 to72 hours after hepatectomy. Animals were killed 2hours after injection, in groups of five to ten, todetermine the number and kind of cells undergoingDNA synthesis and the number and kind of cellsin mitosis. To more precisely define maximumpoints for these values, other animals were giveninjections at 20 and 26 hours after hepatectomy.Zero-time animals included controls, which hadnot been operated upon, sham-operated animals,and animals given injections and killed immediately after hepatectomy. Since neither the number ofcells synthesizing DNA nor the number of cells inmitosis differed in these various animals, theywere grouped together for control purposes. Timesof operation were adjusted so that animals weregiven injections between 5 and 7 A.M. and werekilled between 7 and 9 A.M.,when both DNA synthesis (19) and mitosis (11) are at near-maximaldiurnal rates.

In the second experiment all animals were giveninjections of TDR-H3 at one timeâ€”that at whichDNA synthesis is maximal in hepatocytes (20hours after hepatectomy in these rats). The animals in this experiment were killed in groups offive to eight at 6-hour intervals up to 60 hours afterhepatectomy. Again all animals were given injections between 5 and 7 A.M.This experiment wasdesigned specifically to determine whether any migration of hepatocytes occurred after initial labeling.

Liver was fixed in neutral buffered formalin andwas routinely embedded in paraffin. Sections forautoradiograms were cut at 3â€”4microns. These

1Mean + the standard deviation.2Schwarz Bioresearch, Inc., Mt. Vernon, N.Y.

were prepared with Kodak AR-10 Stripping Film3by the method of Doniach and Pele (4) or withKodak Type NTB-3 Nuclear Track Emulsion4 bythe method (slightly modified) of Messier andLeblond (18). Sections were deparaffinized andstained by the Feulgen technic or were left unstained. Only a few preparations were coated with1 per cent celloidin. This film interposed betweensection and emulsion interferes with detection ofweakly labeled cells and thus reduces sensitivity(25) ; also, it considerably impedes staining and dehydration of sections through the emulsion. Inthe absence of this protective covering, formalin(which can reduce silver in photographic emulsion)was removed from deparaffinized sections by washing them in rapidly running water overnight.Emulsion-coated slides were exposed (for 4-32weeks) and developed according to the conditionsoutlined by Messier and Leblond (18). Feulgen-stained sections and some unstained sections werethen dehydrated, cleared, and mounted by routinehistological methods. After washing and prior todehydrating and mounting, some sections werestained through the emulsion with hematoxylin.

Artifactitious lines of silver grains arrayed alongtissue spaces were occasionally encountered indipped autoradiograms (12). These caused no difficulty in interpretation, since they occurred onlyover empty spaces. Low magnifications were usedto determine the localization of labeled cells in relation to hepatic landmarks. Exposure periods upto 8 months were necessary to achieve sufficientcontrast for photography, and these long exposuresresulted in some elevation of background. Forcounting grains, smears of dispersed cells were exposed for 4 weeks resulting in 10-80 grains overeach labeled nucleus. Cells were separated essentially by the method of Harrison (9). Suspensionsof separated cells were smeared on albumin-coatedslides, fixed in alcohol-ether, and then treated inthe same manner as sectioned material. Hepatocytes were easily identified by their abundant cytoplasm. Naked nuclei were excluded from theestimation.

Types of cells studied were hepatocytes (paren-chymal cells), ductal cells, and littoral cells. Hepatocytes included only large polygonal cells forminghepatic plates. Ductal cells included cells formingall intrahepatic bile ducts from the largest to thesmallest encountered and thus consisted of cholan-gioles or ductules as well as ducts. Littoral cellsconsisted of sinusoidal endothelial cells. In considering the spatial localization of labeled cells, portalareas and hepatic veins were used as reference

8Kodak Limited, London, England.4Eastman Kodak Co., Rochester, N.Y.



844 Cancer Research Vol. 22, August 1962

points. The distance between adjacent landmarkswas divided into three equal distances, corresponding to periportal area, midlobular area, and peri-hepatic area. By limiting observations to regionsdirectly connecting adjacent terminal portal spacesand terminal hepatic veins, the above areas correspond to zone 1, zone 2, and zone 3, respectively,of the acinar concept of hepatic structure (21). Thelatter terminology will be used, although the results can be viewed from either the hexagonal lobular or the acinar concepts of hepatic structure.The relationship of large portal spaces or large central veins to areas of parenchyma containing labeled hepatocytes was inconstant.

Labeled cells of each type were determined asthe per cent of all cells (labeled and unlabeled) ofthat specific type present in random, nonrepeti-tive, oil-immersion fields, as well as in selected oil-immersion fields located (by prior scanning atlower magnification) in zone 1, zone 2, or zone 3.Obviously, only portal spaces were examined incounting'bile ductal cells. At least 3,000 to 5,000

cells of each type were counted for each animal.To further determine the distribution of labeled

cells in different areas, such cells were marked ongraph paper in relation to terminal portal areasand terminal hepatic veins with the use of an ocular reticule made in the pattern of the graph paper.Circles, the diameter of which equaled one-thirdthe distance between portal spaces and adjacenthepatic veins, were drawn on the map so that threecircles just filled the distance along a straight lineconnecting these landmarks. The first circle (zone1) had one edge tangential to the edge of a terminalportal space and the opposite edge tangential tothe second circle (zone 2), whereas the third circle(zone 3) was drawn tangential to the edge of theterminal hepatic vein and to the second circle directly opposite. Labeled cells in all three areaswere totaled, and the numbers in each area wereexpressed as per cent of this total number.

For counting mitoses, sections were cut at 10-15

p and were stained with iron hematoxylin or by theFeulgen method. The number of mitotic and inter-phase nuclei in random, nonrepetitive, oil-immersion fields were counted. For each animal and eachkind of cell at least 50 mitotic figures were enumerated, and the results expressed as per cent of allcells.

Poor penetration of tritium beta rays is a disadvantage as well as an advantage in autoradio-graphie localization of this isotope. Labeled nucleimust be virtually in contact with emulsion to bedetected, and with sections of appreciable thickness some nuclei will appear falsely unlabeled.Thus, values given in this paper are doubtless

somewhat lower than the true values. Since uniformly thin sections were used, comparisons between different treatment groups within this studyare valid, but close comparison with other studiesmay be only qualitatively valid.

RESULTSCONTROLLIVEK

In control rats which had not been operatedupon, sham-operated rats, and rats given injections immediately after hepatectomy, 0.26 + 0.10per cent of hepatocytes, 0.08 Â±0.12 per cent ofductal cells, and 0.76 + 0.53 per cent of littoralcells were labeled. At the same time 0.06 Â±0.06per cent of hepatocytes were in mitosis. Mitoticfigures were so few in ductal and littoral cells as topreclude reliable enumeration. Labeled hepatocytes and littoral cells were randomly distributedthroughout the distance between terminal portalspaces and terminal hepatic veins. Thus, there wasno zonation in intact livers.

REGENERATINGLIVERTemporal relationship of DNA synthesis in hepa

tocytes, in ductal cells, and in littoral cells.â€”As

judged by appearance of labeled nuclei, DNA synthesis began suddenly in hepatocytes between 12and 18 hours after hepatectomy (Chart 1; Figs. 1-10). From a control value of 0.26 + 0.10 per centand a value at 12 hours of 1.27 Â±0.62 per cent,the incidence of labeled hepatocytes reached amaximum at 20 hours after hepatectomy, when29.4 Â±6.2 per cent were labeled. This value differed significantly from values at 18 hours (P <0.01) and at 24 hours (P < 0.05 > 0.02) afterhepatectomy. The number of hepatocytes synthesizing DNA decreased somewhat more slowlythan it had increased. At 48 hours after hepatectomy the incidence of labeled hepatocytes was only4.8 Â±1.0 per cent.

A significant increase in the number of labeledductal or littoral cells did not occur until between20 and 24 hours after hepatectomy (Chart 1; Figs.4, 8, 9, and 11-14). For both groups of cells theincrease was gradual, and a peak value was notreached until 42 hours. For ductal cells the peakvalue was 20.0 + 6.0 per cent, and for littoral cellsit was 14.3 + 3.4 per cent. Although mean valuesfor both ductal and littoral cells were highest atthis time, values for 36 and 48 hours were not significantly different (P > 0.1 in both instances).Thus, the point of maximal DNA synthesis inthese two kinds of cells was not sharply defined.However, peak synthesis in them occurred at least16 hours later than peak synthesis in hepatocytesand perhaps as much as 28 hours afterward. For



GRISHAMâ€”CellRenewal in Regenerating Rat Liver 845

the 72 hours of regeneration studied, addition ofpercentages of cells labeled at 6-hour intervalsgave total values of 84.0 per cent of hepatocytes,78.7 per cent of littoral cells, and 88.5 per cent ofductal cells.

Temporal relationship of DNA synthesis and mitosis.â€”The curve for the portion of hepatocytes

undergoing DNA synthesis at different times afterhepatectomy has been described above. The curvefor the temporal incidence of mitotic cells wasqualitatively similar but was displaced in time andwas less in magnitude (Chart 2). Initial elevationand peak incidence of mitotic rate followed corresponding points on the curve of per cent labeledcells by about 6-8 hours. The mitotic rate began toincrease sharply by about 20 hours after hepatec-

PARENCHYMAL CELLS

12 24 36 48 60 72HOURS AFTER HEPATECTOMY

CHART1.â€”Percent of hepatocytes, ductal cells, and littoralcells labeled at different intervals after hepatectomy. TDR-H3was injected at intervals indicated by points, and animals werekilled 2 hours later. Vertical lines represent two standard deviations around the mean. Each point represents results fromfive to ten animals.

tomy and reached a peak of 3.6 + 0.80 per cent at26 hours. By 54 hours only 0.64 + 0.34 per centof hepatocytes were in mitosis. For equivalentpoints on each curve the ratio of the per cent ofhepatocytes synthesizing DNA over the per cent inmitosis was 6-8.

Mitoses were found less readily in ductal andlittoral cells. Compared with the ratio for hepatocytes of 6-8, the ratio of the per cent of ductal or

littoral cells synthesizing DNA over the per centin mitosis was about 15-20. For example, between

36 and 48 hours after hepatectomy when values

12 24 36 48 60 72HOURS AFTER HEPATECTOMY

CHAKT2.â€”Percent of parenchymal cells undergoing DNAsynthesis (upper graph) and mitosis (lower graph) at differentintervals after hepatectomy. Arrows indicate the time of maximal incidence on the opposite curve. Vertical lines representtwo standard deviations around the mean. Each point represents results from five to ten animals. Points at 20 and 24 hourson the curve for labeled cells differ significantly (P < 0.05 >0.02), as do points at 26 and 30 hours on the curve for mitosis(P < 0.01).

for percentage of labeled cells were 17.2 + 3.6 percent, 20.0 + 6.0 per cent, and 14.5 Â±4.3 per centin ductal cells, the values for mitoses were 1.0+ 0.9 per cent, 1.2 + 0.7 per cent, and0.9 + 0.8 per cent. Likewise, when maximum values for labeled littoral cells ranged from 11.7 + 4.6per cent to 14.3 Â±3.4 per cent, values for mitosesranged from 0.5 Â±0.4 per cent to 0.7 Â±0.4 percent. For both ductal and littoral cells, temporalcurves for mitoses were so flat that maximumpoints could not be discerned.

Lobar and lobular localization of cells synthesizingDNA at different times after hepatectomy.â€”At alltimes studied following hepatectomy, all lobules inall parts of residual lobes were similarly labeled.Neither lobes nor lobules free of labeled cells were




noted at intervals when substantial DNA synthesiswas occurring.

When TDR-H3 was injected at any time between 18 and 30 hours after hepatectomy and animals killed 2 hours later, there was a sharp localization of labeled hepatocytes to zones 1 and 2. Atthese times 18-43 per cent of all hepatocytes inthese two areas combined were labeled, and 85 to95 per cent of all labeled hepatocytes were locatedthere (Table 1; Chart 3; Figs. 1, 2, 5, and 6). Atthese intervals the incidence of hepatocytes syn-

l"I5 60

245

^30

fe

thesizing DNA was greatest (Chart 1). With thewaning of DNA synthesis in hepatocytes after36 hours, zonal distinction was progressively obscured, and by 42 hours there was no difference inthe relative number of labeled cells in any area(Table 1; Chart 3; Figs. 3, 4, and 7-10).

Labeled littoral cells were randomly distributedthroughout lobules at all times (Figs. 4, 9, and 10).Labeled ductal cells were, of course, confined toportal spaces (Figs. 4, 9, and 11-13).

Temporal change in lobular location and in grain

â€¢ â€¢Zone Ioâ€”o Zone 2â€¢â€”*Zone3

18 30 42 54 66HOURS AFTER HEPATECTOMY

CHART8.â€”Labeled hepatocytes in the three zones for thehepatic acinus or lobule at different times after hepatectomyand expressed as a per cent of total labeled hepatocytes in allzones. Animals were given injections of TDR-H3 at the intervals indicated by points and killed 2 hours later. Each point

TABLE 1

PERCENTOFALLHEPATOCYTESIN DIFFERENTAREASOFTHEACINUSORLOBULETHATARELABELEDATDIFFER

ENTTIMESAFTERHEPATECTOMY

TIME(HOCUS)01218202480864248AREA

or Aerara OBLOnl'i.cZone

1(Periportnl)0.24+

0.09*1.6Â±0.786.5

Â±10.4(Â¡7.8+12.249.3Â±10.224.8

Â±7.212.3Â±5.56.2+2.74.5Â±1.1Zone

Ã•(Midlobule)0.29+0.061.0

Â±0.67.8+4.218.7Â±6.415

5+5.310.8+4.011.4+4.87.0+2.94.8

Â±1.0Zone

3(Perihepatici0.28+0.151.3

Â±0.51.4+0.82.4+0.82.6Â±0.73.1Â±1.14.8Â±2.05.7+2.45.0

+1.8

* Each value represents the mean Â±the standard deviationfor 6ve to ten animals.

represents results from five to ten animals. Standard deviationsare not shown to avoid cluttering the chart. There is no significant difference in localization of labeled cells at 42 hours afterpartial hepatectomy or thereafter.

count of initially labeled hepatocytes.â€”At20 hoursafter hepatectomy, when the number of labeledhepatocytes was maximal, such cells were sharplylocalized to zones 1 and 2 (Figs. 16 and 19). WhenTDR-H3 was injected at this time and animalskilled at 6-hour intervals thereafter, labeled cellsultimately became distributed throughout the lobules (Chart 4; Figs. 16-20). As the mitotic wavebegan at 20-24 hours after hepatectomy many ofthe originally labeled cells divided (Figs. 16 and17), and, as they did so, some were apparentlyforced nearer to the hepatic veins.

Labeled mitotic figures were frequent in animalsgiven injections of TDR-H3 and killed 4-6 hourslater. The average grain count above labeled pa-renchymal nuclei (in smears) decreased from 52.7+ 11.2 per nucleus in animals killed 2 hours afterinjection at 20 hours after hepatectomy to 28.3 +9.2 per nucleus in animals killed 24 hours after in-




jection at this same time (P = 0.02). Concomi-tantly the number of labeled hepatocy tes in similarly treated animals increased from 29.4 Â±6.2 percent of all cells to 49.8 Â±10.3 per cent of all cells(P < 0.01).

DISCUSSIONThis study demonstrates that different kinds of

cells in liver and hepatocytes in different areas contribute to total hepatic cellular proliferation andDNA synthesis in qualitatively and quantitativelydifferent ways. Temporal differentials between periods of proliferation in hepatocytes and in ductal

to

90

Â¡75

60

gÂ«

fe

15

ent in the liver and on the mean degreeof ploidy of each (3) (Table 2). It is evidentthat hepatocytes contribute the preponderance ofcells proliferated and of DNA synthesized between0 and 72 hours after hepatectomy, accounting forabout 62 per cent of the former and 75 per cent ofthe latter. However, the extent to which hepatocytes predominate in total proliferation and inDNA synthesis varies at different times after hepatectomy. Between 0 and 36 hours about 76 per centof cells proliferated, and about 84 per cent of DNAsynthesized are accounted for by hepatocytes,

â€¢â€”â€¢Zone Ioâ€”o Zone2â€¢â€”â€¢ZoneS

20 26 32 38 44 50 56HOURS AFTER HEPATECTOMY

62

CHART4.â€”Labeled liepatocytes in the three zones of thehepatic acinus or lobule Â¡isa per cent of total labeled hepatocytes in all zones. All animals were given injections of TDR-H3at 20 hours after hepatectomy and killed at intervals thereafter as indicated by the points. Each point represents results

and littoral cells are clear-cut (1, 5). However, during the entire course of regeneration almost identical total percentages of all three types were labeled. These total percentages are the result ofaddition of values from "flash" labeling at 6-hour

intervals and are almost certainly not true percentages of residual cells proliferated. Their degreeof error depends on the relation of this 6-hour interval to the length of the DNA synthetic periodas well as on the extent of the inefficiency of theautoradiographic method. Nevertheless, they doindicate the relative percentages of the three typesof cells that have proliferated.

These values allow a rough estimate of the portion of total hepatic cellular proliferation and oftotal hepatic DNA synthesis contributed by eachtype of cell to be computed on the basis ofknown percentages of each type of cell pres-

from five to eight animals. Standard deviations are not shownto avoid cluttering the chart. There is no significant differencein localization of labeled cells at 18 hours after injection orthereafter.

whereas between 36 and 72 hours correspondingfigures are 43 per cent and 55 per cent, respectively(Table 2). Furthermore, it is apparent that non-homogeneity of DNA synthesis exists within thepopulation of hepatocytes as it does between themand other kinds of cells. For instance, zones 1 and2 account for 80 per cent of all DNA synthesis occurring in hepatocytes between 0 and 72 hoursafter hepatectomy (and thus for about 60 per centof total hepatic DNA synthesis). Certain experimental manipulations may cause these nonhomo-geneities to be even more pronounced or to havea different quality (13, 22). TDR-H3 incorporationwith autoradiography allows assessment of the extent of such unevenness.

The ratio of the per cent of cells labeled to theper cent in mitosis at a given time has been saidto be a function of the relative time taken for DNA




synthesis as compared with mitosis, and the ratio of6:1-8:1 for parenchymal cells found in this studycompares well with that in previous reports (19,23). This suggests that the time needed for synthesis is roughly 6-8 times greater than that neededfor mitosis. Whether the ratio of 15:1-20:1 forlittoral and ductal cells indicates a longer time forDNA synthesis or a shorter time for mitosis ascompared with similar values in hepatocytes is unknown, although the former possibility has beensuggested (5).

The numbers of hepatocytes synthesizing DNAin the normal rat liver are few, and such cells arerandomly distributed in the hepatic lobule (19, 23).Removal of two-thirds of the liver causes a strikingalteration in this pattern. Between 18 and 36 hoursafter hepatectomy, when large numbers of hepatocytes are synthesizing DNA, proliferating cells aresharply localized to zones 1 and 2. Statements thatproliferation is random in regenerating liver (2,15, 20) may be explained by the fact that manyinvestigators have considered intervals at or after48 hours when lobular segregation no longer exists.

During this interval of rapid growth the patternof cellular proliferation is that of a renewing population as described by Messier and Leblond (19).These authors have classified all tissues on thebasis of their normal rates of proliferation. Normaladult liver is considered to be an expanding population, since there is a small but constant proliferation of cells within it. By comparison, there is alarge turnover of cells in renewing populations andlittle or no apparent proliferation in static populations. Proliferation characteristically occurs diffusely throughout tissue in the case of expandingpopulations, whereas in rapidly proliferating, renewing populations, formation of cells often occursin circumscribed zones. This situation obtains inthe liver at early stages of regeneration after partial hepatectomy.

Cells formed in restricted zones of proliferationcharacteristically migrate from these sites intoother areas (19). In regenerating liver there is anapparent migration of cells from zones 1 and 2 tozone 3, since many of the cells initially labeled inthe former areas may ultimately be found in the

TABLE2CALCULATIONOFTHEPORTIONOFHEPATICCELLULARPROLIFERATIONANDDNA

SYNTHESISCARRIEDOUTBYHEPATOCYTES,LITTORALCELLS,ANDDUCTALCELLSDURINGREGENERATIONAFTERPARTIALHEPATECTOMY

Based on a normal liver composition of 60.6 per cent hepatocytes, 83.4 per cent littoral cells, and 2.0 per cent ductal cells with mean degrees of ploidy of 8.5 for hepatocytes and 2.0 for littoral and ductal cells (8).

Total per cent ofcellslabeledRelative

number ofcellsproliferated*Per

cent of total hepatic cells prolifera tedfRelative

amount ofhepatic DNA

synthesized}Per

cent of total hepatic DNA synthesized!TYPE

OFCELI.Hepatocytes

LittoralcellsDuctalcellsHepatocytes

Littoral cellsDuctalcellsHepatocytes



Littoral cellsDuctal cellsLENGTH

OF PERIODOFREGENERATION0-36

Hours61.8

28.080.537.4

940.675.9

18.91.1130.918.8

1.283.2

11.91.036-7Ã•

Hours22.2

50.758.013

516.9

1.241.0

51.73.647.3

83.82.454.340.52.80-7Â«

Hours84.0

78.788.550.9

26.31.861.8

32.02.2178.2

52.63.674.1

21.61.4

* Total per cent of cells of specific type labeled X percentage of that type of cell inliver X 100.

. Relative number of cells of specific type proliferatedSum of relative numbers of all types of cells proliferated

Ã•Relative number of cells of specific type proliferated X mean degree of ploidy ofthat type of cell.

. Relative amount of DNA synthesized by specific type of cellSum of relative amount of DXA synthesized by all types of cells



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FIGS. 1-Ã¡Ã¼.â€”Allthe figures are atltoradiographs of sectionsof liver from |)arti;ill,v hepatectomized rats given injeetions ofTDK-II3 at indicatili times after hepatectomy and killed atthe stated intervals after injection. In all figures in which theyappear, solid arrows indicate hepatic veins and open arrowsindicate portal spaces.

Fio. 1.â€”Ratgiven injection at 18 hours and killet! a hourslater. Note that labeled nuclei were located only in zones 1 andâ€¢iwith none in zone 3. Hematoxylin stain. Mag. X150.

FIG. a.â€”liÃ¢tgiven injection at 30 hours and killed -2hourslater, /.one 3 still contained comparatively fewer labeled cellsthan did zones 1 and i. Hematoxylin stain. Mag. XIÃ±u.

FIG. 3.â€”Rat given injection at 36 hours and killed '2hourslater. Labeled cells were present throughout the lobule, but inthis animal there was still a periportal predominance. Hematoxylin stain. Mag. X15Ãœ.

FIG. 4.â€”Rat given injection at 48 hours and killed '2hourslater. Labeled cells were scant and had no lobular orientation.Many labeled littoral and ductal cells were seen. Hematoxylinstain. Mag. X150.

FIG. 5.â€”Periportal area of rat given injection at at) hoursand killed '2 hours later. This illustrates that the majority ofhepatocytes in the immediate vicinity of terminal portal spaceswere labeled at this time. Hematoxylin stain. Mag. X5(KI.

FIG. 6.â€”Aperihepaticarea from the same animal as in Fig.;>.There were only three labeled nuclei in the area illustrated.Hematoxylin stain. Mag. X5(K).

FIG. 7.â€”Periportal area from a rat given injection at 3(ihours and killed ^ hours later. A lesser portion of hepatocyteswere labeled than at Â¿0hours, and several littoral and ductalcells were labeled. Hematoxylin stain. Mag. X45U.

FIG.8.â€”Perihepatic area from the same animal as in Fig. 7.Frequent labeled hepatocytes were present, as well as a fewlabeled littoral cells. Hematoxylin stain. Mag. X450.



'â€¢>..rxtÃ ffÃ¯rzÃ¯; yÂ«â€¢Â¿t*v* TÃŽ*&:.Â¿Ã®;**">'â€¢ V-V-** ?..*o-.Â¿<;-'Â»V--"S, ^ ,â€¢'v'*..v' ;*^.^->.*>!^i?-s/^s-* Â¿Ã•* Â».' â€¢.%â€¢ A^^ <-i>?* -*



FIG. 9.â€”Periportal area from a rat given injection at 48hours and killed i hours later. Note the paucity of labeledhepatocytes. Numerous labeled ductal cells, littoral cells, andportal macrophages were seen. Heinatoxylin stain. Mag.X600.

FIG. 10.â€”Perihepatic area from the same animal as in Fig.Ã•).No labeled hepatocytes were seen. Many littoral cells werelabeled. Hernatoxylin stain. Mag. XÂ«><>.

FIG. 11.â€”Labeled cells in a small bile duct at 48 hours.Heinatoxylin stain. Mag. X4(M).

FIG. H. â€”[libeled cells in a terminal bileductuleat 48 hours.Hematoxylin stain. Mag. X400.

FIG. 13.â€”Labeled cells in a moderate-sized intrahepaticbile duct at 48 hours. Heinatoxylin stain. Mag. X700.

FIG. 14.â€”Labeled littoral cells at 48 hours. Hepatocyteswere not labeled. Feulgen stain. Mag. X8IMI.

FIG. 15.â€”I-abeledmitotic figure at '.!<>hours after hepa-tectomy and (i hours after injection. One interphase nucleuswas also labeled. Feulgen stain. Mag. XH.50.



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Fio. 16.â€”Ratgiven injection at Ã<)hours and killed 4 hourslater. labeled cells were locateci predominantly in zones 1 and*>.Ileinatoxylin stain. Mag. X480.

FIG. 17.â€”Ratgiven injection at "Â¿0hours and killed 6 hourslater. labeled cells were still located predominantly in zones1 and a. Note several labeled mitotic figures. Hematoxylinstain. Mag. X480.

FIG. 18.â€”Rat given injection at Â¿0hours and killed Â¿4hours later. Labeled cells now were located along the entiredistance between portal space and hepatic vein. J,al>el wasmuch lighter (lower grain count) over individual nuclei than inFigs. 16 and 17. Hematoxylin stain. Mag. XÃ•60.

FIG. 19.â€”Unstained autoradiograph from a rat given injection at 20 hours and killed a hours later. This preparationshows the periportal localization of labeled cells clearly. Theopen Â¡irrowindicates the site of a portal space and the closedarrow the site of a hepatic vein in underlying tissue as revealedby phase microscopy. Unstained. Mag. X75.

FIG. <0.â€”Asimilar preparation from a rat given injectionat ~i(\hours and killed Â¿4hours later. Labeled cells now occupythe entire distance between portal space and hepatic vein.Silver grains over individual nuclei were much fewei than inFig. 1!). Figs. 19 and Â¿0were exposed, developed, and printedunder as nearly identical conditions as possible. Unstained.Mag. X75.



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latter. The presence of labeled mitotic figures andthe temporally related decrease in average graincount over nuclei originally labeled at 20 hours,with concomitant increase in the total number oflabeled cells, demonstrates that division of theoriginally labeled cells has occurred.

Since the majority of new hepatocytes areformed in zones 1 and 2 after partial hepatectomy,it may be valid to refer to these areas as the hepaticgrowth zone. More correctly, it might be termeda reserve growth zone, since cellular replacementoccurs uniformly from all parts of the lobule inintact liver, in liver after comparatively smalllosses of tissue, and in liver after two-thirds removal at times later than 36 hours after hepatectomy(2, 7, 15, 20). Preliminary evidence indicates thatappearance of a restricted type of proliferationmay be related to the amount of liver removed ordestroyed and that at least 20-30 per cent mustbe removed before this occurs.5 This observationmay explain reports that, after hepatic damage resulting from carbon tetrachloride administration(14), fat accumulation (17), and biliary obstruction (16), hepatocytes synthesizing DNA are uniformly distributed within the lobule. It is possiblethat in the usual instances these stimuli result inless than 20-30 per cent destruction of liver at anygiven time. We have observed that sufficientlylarge doses of carbon tetrachloride can produceproliferation in a restricted zone.6

Since hepatocytes in all lobules remaining afterhepatectomy are similarly labeled, it is concludedthat new lobules do not form primarily but thatgrowth of residual lobules occurs. The inevitableconsequence is increase in lobular size. Evidence ofthis increase has been previously obtained (24),and we have confirmed this for early intervals afterhepatectomy. Whether enlarged lobules are secondarily divided by subdivision and extension of terminal hepatic veins is not known.

REFERENCES1. ABEKCROMBIB,M., and HARKNESS,R. D. The Growth of

Cell Populations and the Properties in Tissue Culture ofRegenerating Liver in the Rat. Proc. Roy. Soc., s. B, 138:544-61, 1951.

2. BRUES,A. M., and MARBLE,B. B. An Analysis of Mitosisin Liver Restoration. J. Exp. Med., 65:15-27, 1937.

8. DAOUST,R. The Cell Population of Liver Tissue and theCytological Reference Bases. In: R. W. BRAUER(ed.),Liver Function, A Symposium on Approaches to the Quantitative Description of Liver Function, pp. 8-10. Washington: American Institute of Biological Sciences, 1958.

4. DONIACH,I., and PELC,S. R. Autoradiographic Technique.Brit. J. Radio!., 23:184-92, 1950.

6J. W. Grisham, unpublished observations.eA. Mikata and J. W. Grisham, unpublished observations.

5. EDWARDS,J. L.: SMITH,S. W.; WESTMARK,E. R.; andYoucis, P. M. Interrelations of DNA Synthesis and CellDivision in Normal and Regenerating Liver. Fed. Proc.,18:475, 1959.

6. GRISHAM,J. W. Inhibitory Effect of Tritiated Thymidineon Regeneration of the Liver in the Young Rat. Proc. Soc.Exp. Biol. & Med., 106:555-58, 1960.

7. HARKNESS,R. D. The Spatial Distribution of DividingCells in the Liver of the Rat after Partial Hepatectomy.J. Physiol., 116:878-79, 1952.

8. . Regeneration of Liver. Brit. Med. Bull., 13:87-93,1957.

9. HARRISON,M. F. Percentage of Binucleate Cells in Liversof Adult Rats. Nature, 171:611, 1958.

10. HIGGINS,G. M., and ANDERSON,R. M. ExperimentalPathology of the Liver; Restoration of the Liver of theWhite Rat Following Partial Surgical Removal. Arch.Pathol., 12:186-202, 1931.

11. JAFFE, J. J. Diurnal Periodicity in Regenerating Liver.Anat. Ree., 120:985-54, 1954.

12. JOFTES, D. L. Liquid Emulsion Autoradiography withTritium. Lab. Investigation, 8:131-86, 1959.

13. KELLY,L. S.; DOBSON,E. L.; FINNEY,C. R.; and HIRSCH,J. D. Proliferation of the Reticuloendothelial System in theLiver. Am. J. Physiol., 198:1134-38, 1960.

14. LEEVY,C. M.; HOLLISTEH,R. M.; SCHMID,R.; MAC.DON-ALD,R. A.; and DAVIDSON,C. S. Liver Regeneration inExperimental Carbon Tetrachloride Intoxication. Proc.Soc. Exp. Biol. & Med., 102:672-75, 1959.

15. MACÃœONALD,R. A. "Lifespan" of Liver Cells. Autoradiographic Study Using Tritiated Thymidine in Normal, Cir-rhotic, and Partially Hepatectomized Rats. Arch. Int.Med., 107:385-43, 1961.

16. M.tcDoNALD,R. A., and PECHET,G. Liver Cell Regeneration due to Biliary Obstruction. Arch. Pathol., 72:138-41,1961.

17. MACÃœONALD,R. A.; SCHMID,R.; and MALLORY,G. K.Regeneration in Fatty Liver and Cirrhosis. Autoradiographic Study Using Tritiated Thymidine. Arch. Pathol.,69:175-80, 1960.

18. MESSIER,B., and LEBLOND,C. P. Preparation of CoatedRadioautographs by Dipping Sections in Fluid Emulsion.Proc. Soc. Exp. Biol. & Med., 96:7-10, 1957.

19. . Cell Proliferation and Migration as Revealed byRadioautography after Injection of Thymidine-H3 intoMale Rats and Mice. Am. J. Anat., 106:247-85, 1960.

20. PECHET,G., and MACÃœONALD,R. A. Repair of NutritionalCirrhosis. Autoradiographic and Histological Study afterPartial Hepatectomy. Cancer, 14:963-70, 1961.

al. RAPPAPORT,A. M.; BOROWY,A. J.; LOUGHEED,W. M.;and LOTTO,W. N. Subdivision of Hexagonal Liver I/>bulesinto a Structural and Functional Unit. Anat. Ree., 119:11-88, 1954.

22. RUBIN,E.; HUTTERER,F.; GALL,E. C.; and POPPFR,H.Nature of Increased Protein and DNA in Chronic HepaticInjury. Nature, 192:886-87, 1961.

28. SCHULTZE,B., and OEHLKIIT,W. Autorndiographic Investigation of the Incorporation of H3-Thymidine into Cellsof Rat and Mouse. Science, 131:737-88, I960.

24. SIDOROVA,V. F. The Structure of Regenerating Liver inRats. Bull. Exp. Biol. & MetÃ.,48:1020-24, I960.

25. TONNA,E. A., and CRONKITE,E. P. Factors Which Influence the Latent Image in Autoradiography. Stain Technol.,33:255-60, 1958.

26. WEINBREN,K. Regeneration of Liver. Gastroenterology,37:657-68, 1959.



1962;22:842-849. Cancer Res J. W. Grisham

3with Thymidine-HCell Proliferation in Regenerating Rat Liver; Autoradiography A Morphologic Study of Deoxyribonucleic Acid Synthesis and

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