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A quantitative method of measuring liver regeneration in the dog
A
QUANTITATIVE
R.
METHOD REGENERATION
ROBINSON
BAKER, HENRY
M.D.,* N.
WAGNER,
THE ability of the liver to regenerate following subtotal hepatectomy has been the subject of extensive experimental study [ 11. It is known that different types of hepatic cells regenerate at different rates. Increased desoxyribonucleic acid synthesis begins in the parenchymal cell, followed in 24 hours by similar changes in the Kupffer’s cells. Regeneration of connective tissue occurs at a considerably slower rate. These experiments are designed to develop a quantitative method of measuring the rate of regeneration of liver mass following a subtotal hepatectomy in the dog. The basic method is a modification of that described by Stern [6] for measuring liver regeneration in the rat.
MATERIALS
AND
METHODS
The method of measuring the rate of regeneration of liver cell mass is based on the following: (1) 80 to 95% of an intravenous dose of radioactive colloidal gold is phagocytized by the macrophages of the liver and From the Department of Surgery and the Division of Nuclear Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md. * Advanced Clinical Fellow, American Cancer Society. This work was aided by U.S. Public Health Service Grant GM10548. Submitted for publication Jan. 13, 1967. 578
OF MEASURING IN THE DOG TOHRU
MIGITA, JR.,
LIVER
M.D.,
AND
M.D.
remains within these cells for at least several weeks; (2) at varying intervals following the subtotal hepatectomy the extent of hepatectomy can be accurately determined by comparing the sum of the radioactivity in the operative specimen and the sacrificed specimen to that in the operative specimen; and (3) the weight of the liver remnant following subtotal hepatectomy can be determined by multiplying the weight of the operative specimen by a fraction based on the calculated extent of hepatectomy. To verify the basic assumption of the method, the uniformity of distribution of intravenously injected radioactive colloidal gold was measured in a series of 13 dogs. Eighteen hours after the intravenous injection of 2 millicuries of Aulgs (0.3 to 0.8 mg./ml. ),* a 70% hepatectomy was performed. In 7 dogs the liver remnant was removed immediately following the operative procedure. The hepatectomy specimen and the remaining liver were divided into respective lobes and homogenized. Four representative samples of each lobe weighing 5 gm. were placed in a well scintillation counter and the counts per minute per gram determined. The total radioactivity in both the operative specimens (OL) and the liver remnant (RL) was determined by multiplying the actual weight in grams of these specimens times the counts * Aurogold,
E.R.
Squibb
and
Sons.
BAKER
ET
AL.:
MEASURING
LIVER
REGENERATION
IN
THE
DOG
per minute per gram of each specimen. The extent of hepatectomy could then be accurately determined by dividing the total counts per minute in the operative specimen (OL) by the total counts per minute in the operative specimen (OL) plus the total counts per minute in the liver remnant (RL). The weight of the liver remnant was then determined by solving the equation: 100% -
% hepatectomy
% hepatectomy
RL = -OL
For example, the operative specimen of Dog No. 1 weighed 186 gm. and on radioassay contained 205,313 counts per minute per gram or a total of 38,188,218 counts per minute. The liver remnant weighed 86 gm. and contained 205,432 counts per minute per gram or a total of 17,667,152 counts per minute. The extent of hepatectomy was calculated in the following manner : Total counts per minute of both specimens ZI 17,667,152 + 38,188,218 = 55,855,370 The percent
of hepatectomy was equal to 38,188,218 or 68.470 the ratio of -55,855,370 Therefore, was equal to
the weight of the liver 100 - 68.4% X 68.4%
remnant
= -186 X = 85.6 gm.
In the remaining 6 dogs, the distribution of Aulgs was determined at 2- to lo-day intervals following hepatic resection. Sixty-seven random samples of liver tissue weighing from 5.2 to 7.2 gm. were analyzed from both the opera-
y0 regeneration =
Fig.
1. The right lateral lobe and a portion of the caudate lobe remain after an estimated 70% hepatectomy.
was performed (Fig. l), and after removal of the gallbladder the operative specimen was weighed and stored at 5°C. At 2- to 18-day intervals following the hepatectomy the dogs were autopsied. To correct for radioactive decay the radioactivity of the operative specimen of the liver (OL) and that of the sacrificed specimen (SL) were determined at the same time. The extent of hepatectomy was determined by the previously described method. On the basis of these determinations, an average of 70.5 f 4.85% (1 SD.) hepatectomy was performed in the 36 dogs. Following determination of the weight of the liver remnant the percentage regeneration following a 70% hepatectomy was calculated according to the equation:
weight of liver at autopsy (SL) - weight of liver remnant ( RL) weight of liver removed at surgery (OL) x 100
tive and regenerative specimens of liver and the counts per minute per gram measured. Following these initial studies the rate of liver regeneration was studied in a series of 36 dogs. Two millicuries of Aulgs were injected intravenously 18 hours prior to the operative procedure. An estimated 70% hepatectomy
RESULTS The ratio of the percent dose per gram in the operative specimen (OL) compared with that in the liver remnant (RL) removed immediately following operation in 7 dogs was 579
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
7
NO.
12,
1.005 f 0.0041 (1 SD.), as indicated in Table 1. On the basis of these determinations it was also possible to verify the accuracy of the method utilized in determining the weight of the liver remnant. The actual weight of the Table
1.
Relative
Specific
Activity
CPM”
2.
The Actual to the Weight
per
Specimen
and Liver CPM
gm.
in Sacrifice Liver 205,432 161,840 91,096 87,490 100,995 174,185 151,202
1 205,313 2 155,707 3 90,158 4 93,929 5 98,027 6 173,334 7 158,348 Mean S.D. * CPM = countsper minute. Table
liver remnant compared to the weight of the liver remnant calculated by the isotope dilution technique is presented in Table 2. Radioactive colloidal gold was also uniformly distributed within the specimens of liver removed at operation and within the
of Au198 in the Operative
CPMa per gm. in Operative Liver
Dog No.
1967
DECEMBER
Remnant
per gm. of OL
CPM per gm. of RL 0.999 0.962 0.990 1.074 0.971 0.995 1.047 1.005 0.041
Weight of the Liver Remnant Immediately Following Hepatectomy Compared of the Liver Remnant Calculated by the Isotope Dilution Technique
RL” RLb Dog No. (P.) 1 186 86 38,188,218 2 272 42,352,304 108 3 183 62 16,498;914 4 61 15,780,072 168 5 215 108 21,075,805 6 146 55 25,306,764 25,177,332 7 159 85 a SL = amountof liver presentafter surgery. b RL = liver remnant. c OL = operative specimen. Table
3.
Uniformity
of Au IQ8 Distribution Operative
Case No.
Dose/gm.
8 9 10
0.175 0.158 0.126
11
0.149
12 13 Average 580
0.146 0.165
in the Operative
SD.
0.0118 0.0090 0.0049 0.0056 0.0037 0.0100
and Regenerated
Hepatectomy (%)
68.4 70.8 74.4 74.7 65.9 72.5 66.2
Liver Removed
S.D.
Regenerating Liver Mean y0
S.D.
(%)
Dose/gin
(%I
Liver
Mean y0
RLb (cm. 1 17,667,152 17,478,720 5,647,952 5,336,890 10,907,460 9,580,175 12.852,170
6.7 5.7 3.9 3.6 2.5 6.1 4.8
0.113 0.136 0.070 0.079 0.070 0.085
SD.
0.0049 0.0048 0.0036 0.0040 0.0118
4.3 3.6 6.9 4.6 ( 1::97, 6.5
Determined by Isotope Dilution Technique
85.6 111.5
62.2 57.1 111.8 55.5 81.1
at Autopsy Sacrifice Time (days)
; 10
10 10 10
BAKER
ET
AL.:
MEASURING
LIVER
REGENERATION
IN
THE
DOG
. :. :
l .
-
.
.
.
I !.
.
2
4
I
I
!
6
8
IO
DAYS AFTER
Fig. 2.
.
Percent regeneration
I
I
1
1
12
14
16
I8
OPERATION
SL-RL OL
X 100 as
a linear function of time. regenerated liver removed at intervals from 2 to 10 days after hepatectomy. The result of these studies in 6 dogs is presented in Table 3. The relative standard deviation of the percent dose per gram of the specimens removed at operation was 4.8%, while that of the regenerating was 6.5%. The rate of liver regeneration in the 36 dogs proved to be extremely variable. In Figure 2 we have plotted the percent regeneration as a function of time, t (days), after surgery. This was calculated by means of the equation: SL-RL OL
x 100%
where SL = the amount of liver present at time, t, after surgery RL = the remnant of liver left behind at operation OL = the amount of liver removed by hepatectomy Thus, if the liver had completely regenerated, SL would equal loo%,, RL = 30%, and OL = 70y0,, and the percentage regenerated would equal 100%. It can be seen from Figure 2 that there was considerable variation in the degree of liver regeneration. When we fitted a straight line by the method of least squares, we obtained the equation: % regeneration = 3% per day + 25% and a correlation coefficient of 0.60. It is clear that this fit is inadequate, since the points originate at zero. Therefore, we graphed the
/
1
1
(
I
1
/
2
4
6
8
IO
12
14
DAYS
Fig. 3.
AFTER
Percent regeneration
I
I
16
I8
OPERATION
SL - RL
X 100 as
OL an exponential function of time after operation,
percent regeneration on semilog paper as a function of time (Fig. 3). The following equation fit the experimental results: SL=RL+
(SL,-RL)
(l-e-bt)
or SL-RL -OL
SLI - RL (1 - e-bt) =-
OL
where SL = the amount of liver present at the time, t SLr = the amount of SL as “t” approaches infinity. When this was done, the correlation coefficient was 0.80. Thus, we concluded that the rate of regeneration was more nearly exponential than linear. The fractional regeneration was 0.06 per day or 6% per day, based on the assumption that the liver regenerated at 100% of the original weight, a reasonable assumption in view of the correlation coefficient of 0.80.
DISCUSSION Quantitative measurements of the rate of liver regeneration following subtotal hepatectomy are based upon: ( 1) accurate determina581
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
7
NO.
tions of the fraction of liver removed and the size of the liver remnant which remains following resection; and (2) the weight of the regenerated specimen at varying intervals following operation, Since the operative and regenerative specimens can be weighed, the crucial factor in calculating the degree of regeneration is the estimation of the weight of the liver remnant. In previous experiments on liver regeneration in the dog by Child [2], Fisher [3], and Grindlay [4] the weight of the liver remnant was estimated by multiplying the weight of the operative specimen by a fraction based on the estimated degree of hepatectomy (3/7 in the case of a 70% hepatectomy). While a relatively standard subtotal hepatic resection can be performed in the dog, this method is dependent upon a constant relationship between the various lobes of the liver. MacDonald [5] has demonstrated that a socalled standard 66% hepatectomy in the rat actually is subject to an error of from I2 to 14%. This same error is undoubtedly present or even greater in view of the larger size of the dog. These experiments demonstrate that radioactive colloidal gold is evenly distributed within the normal and regenerating liver of the dog. The accuracy of the method is further verified in 7 dogs sacrificed immediately following the operative procedure (Table 2). The technique thus provides a means of accurately determining rather than estimating the size of the liver remnant following a subtotal hepatectomy. The results of the second series of experiments in 36 dogs indicate that the liver of mongrel dogs regenerates at an
582
12,
DECEMBER
1967
extremely variable rate. In view of the extreme variation in the rate of regeneration mongrel dogs seem to be unsatisfactory animals in which to study the effects of cytotoxic agents or variations in blood flow, such as the effect of a portacaval shunt on the course of regeneration following subtotal hepatic resection. SUMMARY A quantitative method of measuring liver regeneration in the dog is described. Following development of this experimental method a 7070 hepatectomy was performed in 36 dogs. Regeneration from 2 to 18 days was found to be extremely variable. It appeared to proceed in an exponential rather than a linear fashion. REFERENCES 1. 2.
3.
Bucher, N. L. R. Regeneration of mammalian liver. Znt. Rev. Cytol. 15:245, 1963. Child, C. G., Barr, D., Holswade, T. R., and Harrison, C. S. Liver regeneration following portocaval transposition. Ann. Surg. 138:600, 1953. Fisher, B., Russ, C., Updegraff, H., and Fisher, E. R. Effect of increased hepatic blood flow upon liver regeneration. A.M.A. Arch. Surg. 69:263,
1954. 4. Grindlay, 5.
6.
J. H., and Ballman, J. L. Regeneration of the liver in the dog after partial hepatectomy. Surg. Gynec. Obstet. 94:491, 1952. MacDonald, R. A., Rogers, A. E., and Pechet, G. Regeneration of the liver: Relation of regenerative response to size of partial hepatectomy. Lab. Znuest. 11:544, 1962. Stern, R. Studies on reticuloendothelial function in relation to growth processes. Ann. N.Y. Acad. Sci. 88:252, 1960.
QUANTITATIVE
R.
METHOD REGENERATION
ROBINSON
BAKER, HENRY
M.D.,* N.
WAGNER,
THE ability of the liver to regenerate following subtotal hepatectomy has been the subject of extensive experimental study [ 11. It is known that different types of hepatic cells regenerate at different rates. Increased desoxyribonucleic acid synthesis begins in the parenchymal cell, followed in 24 hours by similar changes in the Kupffer’s cells. Regeneration of connective tissue occurs at a considerably slower rate. These experiments are designed to develop a quantitative method of measuring the rate of regeneration of liver mass following a subtotal hepatectomy in the dog. The basic method is a modification of that described by Stern [6] for measuring liver regeneration in the rat.
MATERIALS
AND
METHODS
The method of measuring the rate of regeneration of liver cell mass is based on the following: (1) 80 to 95% of an intravenous dose of radioactive colloidal gold is phagocytized by the macrophages of the liver and From the Department of Surgery and the Division of Nuclear Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md. * Advanced Clinical Fellow, American Cancer Society. This work was aided by U.S. Public Health Service Grant GM10548. Submitted for publication Jan. 13, 1967. 578
OF MEASURING IN THE DOG TOHRU
MIGITA, JR.,
LIVER
M.D.,
AND
M.D.
remains within these cells for at least several weeks; (2) at varying intervals following the subtotal hepatectomy the extent of hepatectomy can be accurately determined by comparing the sum of the radioactivity in the operative specimen and the sacrificed specimen to that in the operative specimen; and (3) the weight of the liver remnant following subtotal hepatectomy can be determined by multiplying the weight of the operative specimen by a fraction based on the calculated extent of hepatectomy. To verify the basic assumption of the method, the uniformity of distribution of intravenously injected radioactive colloidal gold was measured in a series of 13 dogs. Eighteen hours after the intravenous injection of 2 millicuries of Aulgs (0.3 to 0.8 mg./ml. ),* a 70% hepatectomy was performed. In 7 dogs the liver remnant was removed immediately following the operative procedure. The hepatectomy specimen and the remaining liver were divided into respective lobes and homogenized. Four representative samples of each lobe weighing 5 gm. were placed in a well scintillation counter and the counts per minute per gram determined. The total radioactivity in both the operative specimens (OL) and the liver remnant (RL) was determined by multiplying the actual weight in grams of these specimens times the counts * Aurogold,
E.R.
Squibb
and
Sons.
BAKER
ET
AL.:
MEASURING
LIVER
REGENERATION
IN
THE
DOG
per minute per gram of each specimen. The extent of hepatectomy could then be accurately determined by dividing the total counts per minute in the operative specimen (OL) by the total counts per minute in the operative specimen (OL) plus the total counts per minute in the liver remnant (RL). The weight of the liver remnant was then determined by solving the equation: 100% -
% hepatectomy
% hepatectomy
RL = -OL
For example, the operative specimen of Dog No. 1 weighed 186 gm. and on radioassay contained 205,313 counts per minute per gram or a total of 38,188,218 counts per minute. The liver remnant weighed 86 gm. and contained 205,432 counts per minute per gram or a total of 17,667,152 counts per minute. The extent of hepatectomy was calculated in the following manner : Total counts per minute of both specimens ZI 17,667,152 + 38,188,218 = 55,855,370 The percent
of hepatectomy was equal to 38,188,218 or 68.470 the ratio of -55,855,370 Therefore, was equal to
the weight of the liver 100 - 68.4% X 68.4%
remnant
= -186 X = 85.6 gm.
In the remaining 6 dogs, the distribution of Aulgs was determined at 2- to lo-day intervals following hepatic resection. Sixty-seven random samples of liver tissue weighing from 5.2 to 7.2 gm. were analyzed from both the opera-
y0 regeneration =
Fig.
1. The right lateral lobe and a portion of the caudate lobe remain after an estimated 70% hepatectomy.
was performed (Fig. l), and after removal of the gallbladder the operative specimen was weighed and stored at 5°C. At 2- to 18-day intervals following the hepatectomy the dogs were autopsied. To correct for radioactive decay the radioactivity of the operative specimen of the liver (OL) and that of the sacrificed specimen (SL) were determined at the same time. The extent of hepatectomy was determined by the previously described method. On the basis of these determinations, an average of 70.5 f 4.85% (1 SD.) hepatectomy was performed in the 36 dogs. Following determination of the weight of the liver remnant the percentage regeneration following a 70% hepatectomy was calculated according to the equation:
weight of liver at autopsy (SL) - weight of liver remnant ( RL) weight of liver removed at surgery (OL) x 100
tive and regenerative specimens of liver and the counts per minute per gram measured. Following these initial studies the rate of liver regeneration was studied in a series of 36 dogs. Two millicuries of Aulgs were injected intravenously 18 hours prior to the operative procedure. An estimated 70% hepatectomy
RESULTS The ratio of the percent dose per gram in the operative specimen (OL) compared with that in the liver remnant (RL) removed immediately following operation in 7 dogs was 579
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
7
NO.
12,
1.005 f 0.0041 (1 SD.), as indicated in Table 1. On the basis of these determinations it was also possible to verify the accuracy of the method utilized in determining the weight of the liver remnant. The actual weight of the Table
1.
Relative
Specific
Activity
CPM”
2.
The Actual to the Weight
per
Specimen
and Liver CPM
gm.
in Sacrifice Liver 205,432 161,840 91,096 87,490 100,995 174,185 151,202
1 205,313 2 155,707 3 90,158 4 93,929 5 98,027 6 173,334 7 158,348 Mean S.D. * CPM = countsper minute. Table
liver remnant compared to the weight of the liver remnant calculated by the isotope dilution technique is presented in Table 2. Radioactive colloidal gold was also uniformly distributed within the specimens of liver removed at operation and within the
of Au198 in the Operative
CPMa per gm. in Operative Liver
Dog No.
1967
DECEMBER
Remnant
per gm. of OL
CPM per gm. of RL 0.999 0.962 0.990 1.074 0.971 0.995 1.047 1.005 0.041
Weight of the Liver Remnant Immediately Following Hepatectomy Compared of the Liver Remnant Calculated by the Isotope Dilution Technique
RL” RLb Dog No. (P.) 1 186 86 38,188,218 2 272 42,352,304 108 3 183 62 16,498;914 4 61 15,780,072 168 5 215 108 21,075,805 6 146 55 25,306,764 25,177,332 7 159 85 a SL = amountof liver presentafter surgery. b RL = liver remnant. c OL = operative specimen. Table
3.
Uniformity
of Au IQ8 Distribution Operative
Case No.
Dose/gm.
8 9 10
0.175 0.158 0.126
11
0.149
12 13 Average 580
0.146 0.165
in the Operative
SD.
0.0118 0.0090 0.0049 0.0056 0.0037 0.0100
and Regenerated
Hepatectomy (%)
68.4 70.8 74.4 74.7 65.9 72.5 66.2
Liver Removed
S.D.
Regenerating Liver Mean y0
S.D.
(%)
Dose/gin
(%I
Liver
Mean y0
RLb (cm. 1 17,667,152 17,478,720 5,647,952 5,336,890 10,907,460 9,580,175 12.852,170
6.7 5.7 3.9 3.6 2.5 6.1 4.8
0.113 0.136 0.070 0.079 0.070 0.085
SD.
0.0049 0.0048 0.0036 0.0040 0.0118
4.3 3.6 6.9 4.6 ( 1::97, 6.5
Determined by Isotope Dilution Technique
85.6 111.5
62.2 57.1 111.8 55.5 81.1
at Autopsy Sacrifice Time (days)
; 10
10 10 10
BAKER
ET
AL.:
MEASURING
LIVER
REGENERATION
IN
THE
DOG
. :. :
l .
-
.
.
.
I !.
.
2
4
I
I
!
6
8
IO
DAYS AFTER
Fig. 2.
.
Percent regeneration
I
I
1
1
12
14
16
I8
OPERATION
SL-RL OL
X 100 as
a linear function of time. regenerated liver removed at intervals from 2 to 10 days after hepatectomy. The result of these studies in 6 dogs is presented in Table 3. The relative standard deviation of the percent dose per gram of the specimens removed at operation was 4.8%, while that of the regenerating was 6.5%. The rate of liver regeneration in the 36 dogs proved to be extremely variable. In Figure 2 we have plotted the percent regeneration as a function of time, t (days), after surgery. This was calculated by means of the equation: SL-RL OL
x 100%
where SL = the amount of liver present at time, t, after surgery RL = the remnant of liver left behind at operation OL = the amount of liver removed by hepatectomy Thus, if the liver had completely regenerated, SL would equal loo%,, RL = 30%, and OL = 70y0,, and the percentage regenerated would equal 100%. It can be seen from Figure 2 that there was considerable variation in the degree of liver regeneration. When we fitted a straight line by the method of least squares, we obtained the equation: % regeneration = 3% per day + 25% and a correlation coefficient of 0.60. It is clear that this fit is inadequate, since the points originate at zero. Therefore, we graphed the
/
1
1
(
I
1
/
2
4
6
8
IO
12
14
DAYS
Fig. 3.
AFTER
Percent regeneration
I
I
16
I8
OPERATION
SL - RL
X 100 as
OL an exponential function of time after operation,
percent regeneration on semilog paper as a function of time (Fig. 3). The following equation fit the experimental results: SL=RL+
(SL,-RL)
(l-e-bt)
or SL-RL -OL
SLI - RL (1 - e-bt) =-
OL
where SL = the amount of liver present at the time, t SLr = the amount of SL as “t” approaches infinity. When this was done, the correlation coefficient was 0.80. Thus, we concluded that the rate of regeneration was more nearly exponential than linear. The fractional regeneration was 0.06 per day or 6% per day, based on the assumption that the liver regenerated at 100% of the original weight, a reasonable assumption in view of the correlation coefficient of 0.80.
DISCUSSION Quantitative measurements of the rate of liver regeneration following subtotal hepatectomy are based upon: ( 1) accurate determina581
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
7
NO.
tions of the fraction of liver removed and the size of the liver remnant which remains following resection; and (2) the weight of the regenerated specimen at varying intervals following operation, Since the operative and regenerative specimens can be weighed, the crucial factor in calculating the degree of regeneration is the estimation of the weight of the liver remnant. In previous experiments on liver regeneration in the dog by Child [2], Fisher [3], and Grindlay [4] the weight of the liver remnant was estimated by multiplying the weight of the operative specimen by a fraction based on the estimated degree of hepatectomy (3/7 in the case of a 70% hepatectomy). While a relatively standard subtotal hepatic resection can be performed in the dog, this method is dependent upon a constant relationship between the various lobes of the liver. MacDonald [5] has demonstrated that a socalled standard 66% hepatectomy in the rat actually is subject to an error of from I2 to 14%. This same error is undoubtedly present or even greater in view of the larger size of the dog. These experiments demonstrate that radioactive colloidal gold is evenly distributed within the normal and regenerating liver of the dog. The accuracy of the method is further verified in 7 dogs sacrificed immediately following the operative procedure (Table 2). The technique thus provides a means of accurately determining rather than estimating the size of the liver remnant following a subtotal hepatectomy. The results of the second series of experiments in 36 dogs indicate that the liver of mongrel dogs regenerates at an
582
12,
DECEMBER
1967
extremely variable rate. In view of the extreme variation in the rate of regeneration mongrel dogs seem to be unsatisfactory animals in which to study the effects of cytotoxic agents or variations in blood flow, such as the effect of a portacaval shunt on the course of regeneration following subtotal hepatic resection. SUMMARY A quantitative method of measuring liver regeneration in the dog is described. Following development of this experimental method a 7070 hepatectomy was performed in 36 dogs. Regeneration from 2 to 18 days was found to be extremely variable. It appeared to proceed in an exponential rather than a linear fashion. REFERENCES 1. 2.
3.
Bucher, N. L. R. Regeneration of mammalian liver. Znt. Rev. Cytol. 15:245, 1963. Child, C. G., Barr, D., Holswade, T. R., and Harrison, C. S. Liver regeneration following portocaval transposition. Ann. Surg. 138:600, 1953. Fisher, B., Russ, C., Updegraff, H., and Fisher, E. R. Effect of increased hepatic blood flow upon liver regeneration. A.M.A. Arch. Surg. 69:263,
1954. 4. Grindlay, 5.
6.
J. H., and Ballman, J. L. Regeneration of the liver in the dog after partial hepatectomy. Surg. Gynec. Obstet. 94:491, 1952. MacDonald, R. A., Rogers, A. E., and Pechet, G. Regeneration of the liver: Relation of regenerative response to size of partial hepatectomy. Lab. Znuest. 11:544, 1962. Stern, R. Studies on reticuloendothelial function in relation to growth processes. Ann. N.Y. Acad. Sci. 88:252, 1960.