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Estimating anatomical Уfunctional position coordinates in liver tissue.

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THE ANATOMICAL RECORD 228:267-276 (1990)
Estimating Anatom ical-F unctional Position
Coordinates in Liver Tissue
E. BARBERA-GUILLEM, A. ALONSO-VARONA, M.D. BOYANO, AND
F. VIDAL-VANACLOCHA
Department of Cell Biology and Morphological Sciences, School of Medicine and Dentistry,
University of the Basque Country, Leioa, 48940 Vizcaya, Spain
ABSTRACT
Hepatocyte enzyme activity was demonstrated by examining
adult C57BL/6 mouse liver cryostat sections under a succinate dehydrogenase
(SDH) histochemical reaction, and quantified by microspectrophotometry and microdensitometry. The hepatocyte SDH activity gradient along the path between
the portal veins (PV) and efferent terminal hepatic venules (THV) was analyzed by
measuring the concefitration of the chromophore precipitated in 10 consecutive
hepatic parenchymal domains located along imaginary lines drawn across the
entire PV-to-THV distance. The profiles of intensity or of normalized relative
optical density obtained on a high number of lines were correlated with distance
values along the PV-to-THV pathway, enabling us to establish a general mathematical function relating SDH activity (chromophore concentration) to position
values on a scale of 0 to 10 corresponding to the theoretical PV-to-THV distance.
The equation can be used to interpolate the SDH activity surrounding any intrahepatic object located between the PV and the THV, thus making it possible to
calculate the object’s anatomical-functional position coordinates in the liver acinus. To demonstrate how this method is used, we have calibrated the intrahepatic
position of hemopoietic foci induced in the liver tissue of adult mice treated with
phenylhydrazine (PHZ), and show that these foci are located on coordinate 3.31
(maximum range 1.25-4.86) of the sinusoidal domain-that is, on the borderline
between Rappaport’s acinar zones 1 and 2.
The biological variability of the hepatocytes and sinusoidal cells found along the hepatic sinusoidal pathway results in a functional zonation of adult liver tissue (Jungermann and Katz, 1982; Barbera-Guillem
and Vidal-Vanaclocha, 1988; Gumucio and Chianale,
1988). Consequently, a regionalized distribution of normal functional processes and of pathological behaviour
(Rappaport, 1976) is recognized in each zone. Given
this functional heterogeneity, numerous studies have
tried to correlate certain functional phenomena with
their location within the hepatic lobule. Examples are
the studies made of the intralobular distribution of
Kupffer cells (Sleyster and Knook, 1982; Bouwens e t
al., 19861, Pit cells (Kaneda and Wake, 1983) and fat
storing cells (Wake, 1989), or studies on the possible
existence of specific places within the hepatic lobule
where certain functional activities take place
(Thurman and Kauffman, 1985; Lamers et al., 1988;
Teutsch, 1988; Barbera-Guillem et al., 1989a). However, despite the need to pinpoint the intralobular location of the element or process under study in order to
interpret it at a more general functional level, the intralobular topographical coordinates used are poorly
defined, due to the biological irregularity of the structural and functional organization of the liver tissue
itself (Rappaport et al., 1954; Lamers et al., 1989;
Teutsch, 1988). Certain studies, therefore, indicate the
place where the phenomenon in question occurs by
measuring in microns its distance from the portal or
0 1990 WILEY-LISS, INC.
centrilobular veins (Hildebrand and Schleicher, 1986;
Ebert et al., 1987; Arber et al., 1988). In other cases,
the object is related to the different zones of microcirculatory influence (Rappaport, 1973; Sleyster and
Knook, 1982). What are never given, however, are precise topographical references that would make it possible to establish a correlation with the functional
characteristics of the location concerned.
In this study, we have used cytophotometry and densitometry to quantify the SDH activity of the hepatocytes along the sinusoidal pathway from the portal
venules (PV) to the terminal hepatic venules (THV).
The data obtained make it possible to establish standard correlation curves between hepatocyte SDH activity and a microvascular reference position. In addition,
the mathematical expression of these functions enables
us to calculate through interpolation the position of
any object with regard to its microcirculatory reference
position, given the SDH activity of its environment.
These coordinates of relative position arrived at
through the enzyme activity of the neighboring hepa-
Received November 15, 1989; accepted March 20, 1990.
Address reprint requests to F. Vidal-Vanaclocha, Department of
Cell Biology and Morphological Sciences, School of Medicine and Dentistry, University of the Basque Country, Leioa, 48940 Vizcaya,
Spain.
268
E. BARBERA-GUILLEM E T AL.
tocytes guarantee a n empirical standardization of results that was lacking in other procedures.
To demonstrate how this procedure is used to calculate position according to hepatocyte SDH activity, this
study calibrates the exact location of the hemopoietic
foci induced in the livers of adult mice subjected to
acute hemolytic treatment using phenylhydrazine. Our
results show that these hemopoietic foci occupy a specific, well-defined band within the sinusoidal structure.
MATERIALS AND METHODS
Animals
Adult (age: 7 weeks, weight: 18-20 g) C57BL/6 mice
were purchased from Iffa Credo Laboratories (France)
and kept on a 12 h day-night rhythm with free access
to food and water (standard diet). All animals were
fasted 6 h before the experiments, and were anesthetized by intraperitoneal injection of pentobarbital (50
mgtkg body wt.) prior to sacrifice.
Histochemistry
An in situ histochemical reaction to reveal succinate
dehydrogenase (SDH) activity was carried out on 10
pm cryostat sections as follows: tissue slides were incubated 3 min a t 37°C with 2-3 ml of a substrate solution made of sodium succinate (Merk, WG), nitrobluetetrazolium I11 (NBT) (Sigma Chem. Co., St. Louis,
MO), and phosphate-buffered salts prepared as described previously (Barbera-Guillem et al., 1989b). After incubation, the slides were rinsed in cold phosphate
buffer, fixed in 10% formaldehyde for 3 min, rinsed in
distilled water, and mounted on glycerol gel. For SDH
activity to show up more clearly in micrographs, the
reaction was prolonged to 20 min in the photographed
liver tissue sections. Section thickness homogeneity
was evaluated by analytical interferential microscopy
(POL optics, Zeiss).
In Situ Cytophotometric Measurement of
Cromophore Content
Cytophotometric measurement of chromophore concentration was taken by a n integrating method with a
0.25 pm step size (0.06 pm2) to reduce distribution error, given the normally heterogeneous histochemical
reaction with NBT. Four hundred points (24 pm2)were
measured on a field of cytoplasm conforming to the
shape of each hepatocyte. The wave length selected was
585 nm with a 5 nm band width (Butcher, 1978). The
measurements were made with a Zeiss microspectrophotometer (Zeiss, Oberkochen, WG) connected on line
with a p.c. microcomputer which was programmed to
record the movements of the object and the 0.25 pm
step scanning of irregularly shaped areas previously
defined by the operator according to the form of the
cytoplasm of each hepatocyte.
Hepatocyte samples were obtained by selecting 10
lines connecting the portal venules (PV) and nearby
terminal hepatic venules (THV) from each of 10 tissue
slides selected from five different animals. There was
at least 1 mm between each tissue slide. Next, the cytoplasmic chromophore content ( E ) of each of the hepatocytes around each line (Fig. 15 was measured. E ,
corresponded to integrated extinction of the 400 points
included in the cytoplasmic field defined for each hepatocyte. The data were recorded in a file (“A”), with x
being the relative chromophore concentration for each
0.06 pm2 point in the hepatocyte (E,/400), and y the
position of the hepatocyte on the line a s measured on a
scale of 0 to 10, beginning at the PV and going on up to
10 a t the THV.
Densitometric Measurements of Histochemically
Demonstrated Hepatic SDH Activity
Quantification of liver SDH activity and further processing of the image was run with a n equipment setup
of a n integrated automatic image analysis system
(Southern Microcomp. Instruments, Inc., Atlanta), and
a VANOX AHBT optical microscope (Olympus Optical
Co., Ltd., Japan) equipped with a SIT66 TV camera
(Dage-MTI, Inc., Michigan) used under the following
conditions: 585 nm wavelength monochromatic lighting; recording of light intensity at each pixel on a scale
of 0 to 255; projection of the object using a 20 x objective lens so that each pixel represents a n object area
measuring 0.12 pm2 (imaginary circle of 0.39 pm in
diameter). Once this was done, 100 lines were again
drawn between the PV and the THV on the same 10
slides used previously and the intensity values of each
of the pixels (Z,) on the lines were recorded in a file,
together with a number indicating order of placement
on the line (taking the PV as the starting point). The
same operation as before was again carried out, but
this time using a negative image so that the greater
intensity now corresponded directly to the cells with
highest chromophore content. Later, the data for each
hepatocytic pixel were transformed, giving us two different files: File “B,” containing for each pixel a n x
value equivalent to the Z, of the negative image and a
y value equivalent to the position of the pixel on the
line mapped onto a 0 to 10 scale. File “C” contained for
each pixel a n x value equivalent to the normalized relative optical density (NROD) obtained by transformation according to the Z, values of the positive image:
(a)E,
=
log(255/1,)
(b) NROD
=
E p - Emin
Emax - Emin
where Em,, is the value of maximum E , found on a
tissue slide and Eminis the value of minimum E found
on the tissue slide. They value is the same as i n h l e B.
Using the data in Files A, B, and C, adjustments
were made for each, calculating the correlation between the values found and functions of n degrees expressed as
y = ko
+ k l x + kzxz + . . . + k,x”
from which we can extrapolate any position value,
given the relative chromophore concentration, the negative image intensity, or the normalized relative optical density.
Image Analysis of Hepatic Parenchymal Cells
Using the integrated image analysis system, the hepatocytes stained with NBT due to the SDH reaction
were digitalized and represented in only four gray levels using the integrated image analysis system. On
these images, the relative surface area occupied by
each gray level was calculated morphometrically.
ESTIMATING POSITION COORDINATES IN LIVER TISSUE
269
Fig. 1. Liver cryostat section following an SDH histochemical reaction showing the procedure used for cytophotometric measurement of
chromophore concentration in the cytoplasm of hepatocytes prior to
correlation with position on the sinusoidal pathway from the portal
vein (PV) to the terminal hepatic venule (THV). The imaginary line
that runs from the PV to the THV is divided into 10 segments of equal
length, with each of these measurements made in well-defined areas
within the cytoplasm of the hepatocytes.
Phenylhydrazine-InducedHepatic Hemopoiesis in
Adult Mice
value was calculated for each focus by solving the corresponding function:
Animal treatment with phenylhydrazine has been
described in a previous work (Barbera-Guillem et al.,
1989b). Briefly, adult mice were administered with one
daily intraperitoneal injection of 0.1 ml phosphatebuffered saline (PBS) containing 1mg of phenylhydrazine (PHZ) hydrochloride (Sigma Chem. Co., St. Louis,
MO) for 7 days.
Calibrating the Position of Hemopoietic Foci Within the
Liver Tissue
The cytoplasmic SDH activity of hepatocytes surrounding both portal and central vessels and hemopoietic foci was recorded by means of a computer-assisted
Zeiss microspectrophotometer as described above. Five
SDH activity measurements were taken around each
type of liver structure on the basis of the maximum
number of vessels and of hemopoietic foci found in 15
tissue sections from five different mice. Spectrophotometric values were expressed in arbitrary units normalized with respect to average periportal hepatocyte
SDH activity. These values were then used to calculate
the average for each focus, which was recorded in a file
a s the x descriptor of each focus. Later, the y position
y = ko
+ klx, + kzx; + . . . + k,&
Statistics
The Wilcoxon rank sum test was used to determine
the significance of SDH activity differences in the hepatocyte populations measured (periportal,perivenous,
and perihemopoietic focus).
RESULTS
Identification of Hepatic Parenchymal Cell Phenotypes
According to Their Succinate Dehydrogenase Activity
The SDH histochemical reaction enables us to obtain
a clearly defined view of the hepatic tissue and its functional zones on cryostat sections. The hepatic areas of
greatest enzyme activity are found around the portal
veins as revealed by their dark color, while the areas of
lesser SDH activity, found around the terminal hepatic
venules, stand out in contrast due to their much lighter
color (Fig. 1).
Cytophotometric measurement of the hepatocyte
chromophore content in hepatic cryostat sections subjected to the SDH histochemical reaction reveals a
clear difference between the cells in the area closest to
270
E. BARBERA-GUILLEM ET AL.
TABLE 1. Chromophore concentration in hepatic
parenchymal cells of liver cryostat sections under a
succinate dehydrogenase histochemical reaction'
Parameters
Total chromophore
content
Ranee
AveFage chromophore
Concentration
Intraacinar heDatic areas
Peri-THV
Peri-PV
244.5
2
103.27
121-327
0.71 k 0.31
435.17 t 230.64
205-665
1.35 k 0.71
'THV, terminal hepatic venule; PV, portal venule Data are expressed
as mean values f SD from measurements of integrated extintion of
the 400 points included in the cytoplasmic field defined for each hepatocyte. The number of measurements per hepatic area was higher
than 2000, being obtained from five mice (two tissue sections per
mouse).
the portal venules (PV) and those around the terminal
hepatic venules (THV) (Table 1).In each of these areas,
there is a high degree of variation, however, and a
considerable percentage of hepatocytes could be erroneously classified if chromophore content were the only
criterion taken into account. In fact, when we examined the distribution of the hepatocytes in the periportal and perivenous regions (Fig. 21, we found a small
proportion of hepatocytes from the two regions with a
clear overlap in their chromophore concentration (between 0.6 and 0.9%).
Moreover, analysis of hepatocyte images pertaining
to the three acinar zones shows a heterogeneous chromophore distribution inside the cells themselves. In
general, the dye produces heavy precipitations in certain cytoplasmic zones, which contrast with zones
where the coloring is relatively weak (Fig. 3). After
grouping the entire range of optical densities into four
levels (represented by four gray levels) with the image
analyzer, it became clear that the majority of the pixels
corresponding to hepatocyte images belong either to
the densest class (level 4),or else to the low-intermediate category (level 2). By contrast, those corresponding to levels 1 and 3 are very scarce. Examination of
these hepatocyte images also shows that in periportal
cells, level-4 pixels occupy up to 80% of the total surface area of the hepatocyte, while in the THV region
the hepatocytes offer the inverse image, since over 80%
of these pixels are of the level-2 type. Between these
two extremes there is a large number of hepatocytes
whose cytoplasmic surface is partially occupied by pixels of all shades in varying proportions. The intermediate zone is where this type of hepatocyte is the most
frequent.
lntraacinar Cytophotometric Mapping of Hepatocyte SDH
Activity Phenotypes
To study the profile of the decline in hepatocyte SDH
activity, cytophotometry was used to determine the average chromophore concentration in the cytoplasm of
hepatocytes located along the sinusoidal PV or septumto-THV pathway. The results show the existence of hepatocytes with highly variable average concentration
values, although in general they tend to decrease from
the periportal to the perivenous area. Therefore, if we
use these concentrations to determine a possible association between relative optical density value and lo-
0
0.5
1
1.5
2
CHROMOPHORE CONCENTRATION
(ARBITRARY UNITS,
Fig. 2. Histograms of frequency distribution of hepatocyte SDH activity around portal venules (dotted area) and terminal hepatic
venules (open area). The average chromophore concentration, expressed in normalized arbitrary units, was 1.00 0.14 for periportal
hepatocytes and 0.43 i 0.13 for peri-THV hepatocytes.
*
cation on the 0 to 10 scale corresponding to the points
of measurement on the line between the portal and
terminal hepatic veins, we find that the higher correlation coefficient responds to the adjustment of a third
degree polynomic equation (correlation coefficient =
0.989) (Fig. 4). This standard curve makes i t possible,
therefore, to correlate the different hepatocyte SDH activity values with precise locations on the microcirculatory network. Moreover, the mathematical expression of this curve enables us to calculate through
interpolation the position of any object on the sinusoidal pathway if we know the SDH activity of the surrounding hepatocytes.
lntraacinar Densitometric Mapping of Hepatocyte SDH
Activity Phenotypes
Densitometric measurement of chromophore concentration in the cytoplasm of hepatic parenchymal cells
subjected to a reaction in order to reveal SDH activity
coincides with the data obtained in the cytophotometric
measurement. Quantifying the intensity of light in the
pixels located on lines drawn from the PV to the THV
on negative images of microscopic hepatic fields (Fig. 5)
reveals a drop in the SDH activity gradient that becomes especially evident when we eliminate the pixels
corresponding to the noncytoplasmic structures such as
the sinusoidal lumen or cell nuclei (Fig. 6). Also, when
we correlate the values of normalized relative optical
intensity or density with those of the distance from the
PV on the lines between the PV and the THV (Fig. 7),
we once again obtain a standard curve that graphically
represents a mathematical function. This equation relates the SDH activity (measured indirectly a s chromophore concentration) or independent variable, with
the distance from the PV, or dependent variable.
Calibrating the Position of PHZ-Induced Hemopoietic Foci
in Adult Liver Tissue
As previously described (Ploemacher and van Soest,
19771, daily administration of phenylhydrazine to
adult C57BLi6 mice produced a n acute hemolytic anemia, which was immediately accompanied by -a strong
ESTIMATING POSITION COORDINATES IN LIVER TISSUE
Fig. 3.Digitalized image of hepatic tissue subjected to a histochemical reaction for SDH. On the left is zone 1, and on the right, the zone
closest to the THV (zone 3). The image is formed by pixels in four gray
levels. The majority of zone 1hepatocyte pixels are of the highest gray
271
level (41. By contrast, most zone 3 hepatocytes have gray-level 2 pixels. There are also hepatocytes with intermediate characteristics in
which there is a balanced combination of both gray-level 2 and 4
pixels.
hemopoietic reaction involving several organs includ- paport, 1973), numerous studies were done in search of
ing liver tissue. On the seventh day of PHZ treatment, topographical correlations between different metabolic
hepatic sinusoids had expanded and showed a n in- activities (Jungermann and Katz, 1982) or between
creased infiltration of hemopoietic cells. These cells
formed small focal aggregates not associated with portal and central veins, which occluded sinusoidal lumen
(Fig. 8). The average SDH activity of hepatocytes surrounding hemopoietic foci was similar to that of periportal hepatocytes and significantly different from that
of peri-THV hepatocytes (P<O.Ol) (Table 2). Hemopoietic foci seemed to be located, therefore, in the high
SDH activity domain of liver acini. Thus, to verify their
exact location, the SDH activity values of the hepatocytes surrounding each focus were interpolated on the
curve of the intraacinar SDH activity gradient in order
to calculate the position of these values on the scale of
0 to 10 t ha t goes from the PV to the THV of any hepatic
acinus. The result of these interpolations can be seen in
Figure 9, which shows that the hemopoietic foci implant in the adult liver tissue of PHZ-treated mice in
the sinusoidal segment extending from point 1.25 to
4.86 on the relative scale superimposed on the PV-to0
0.5
1
THV pathway, with 3.31 being the most probable point
of location.
-
HEPATOCYTE S D H A C T I V I T Y
[ A S CHROMOPHORE CONCENTRATION
ARBITRARY UNITS 1
DISCUSSION
Once the existence of well-differentiated functional
zones had been demonstrated in the liver tissue (Rap-
Fig. 4. Acinar gradient curve of hepatocyte SDH activity calculated
from step-by-step recording for this enzyme activity at 10 consecutive
equidistant points on an imaginary line drawn on the portalto-central vein distance.
272
E.
BARBERA-GUILLEM ET AL.
Fig. 5. Digitalized image of a liver tissue field taken from positive (a)and negative (b) images in 255
gray levels (a)and 255 intensity levels (b).The line is made up of all the lined-up pixels used to calculate
the relation between the optic density (a)or the intensity of (b) and the correlation of either with the
portal-to-central vein distance.
certain cell types and the functional zone where they
are found (Loud, 1968; Sleyster and Knook, 1982;
Vidal-Vanaclocha and Barbera-Guillem, 1985; Wake,
1989). However, the delineation of the classical lobules
and of the hepatic acini is often imprecise, and their
form and function do not correspond to the geometric
models normally used to describe them (Matsumoto
and Kawakami, 1982; Teutsch, 1988; Lamers et al.,
TABLE 2. Hepatocyte succinate dehydrogenase
1989). Consequently, most of these studies have run up
activity around portal venules, terminal hepatic
against difficulties in calculating exact locations
venules, and phenylhydrazine-induced hemopoietic
within the lobular or acinar structure of the hepatic
foci and relative position of foci within the liver
acini of C57BL/6 mice
tissue. The only clearly identifiable points of reference
are the portal vessels (PV) and the terminal hepatic
Average SDH
Distance from the venules (THV), but these two points are separated by
Hepatocyte
activity
portal venule
variable distances and delimit theoretical areas of dif(RODU)'
(DFPV)~
populations
ferent sizes and shapes. The sinusoidal pathway itself,
1.00 2 0.20*
0
Periportal venules
although theoretically radial in form and leading toPeriterminal hepatic
0.44 i 0.11
10
ward the THV, is also erratic when seen in histological
venule
section, due to a tridimensional superstructure that
0.83 2 0.24*
3.31
Perihemopoietic foci
(range 1.25-4.86) is difficult to express on a plane (Matsumoto and
Kawakami, 1982).
'Succinate dehydrogenase activity was expresssed as relative optical
All these features of biological irregularity make i t
density units (RODU) ? SD. The number of measurements was at
least of 10,000, being obtained from five mice (15 liver tissue sections difficult to use distance to the hepatic vessels measured
in microns a s a precise measurement of position correg%
e:;'tT
'::
DFPV of hemopoietic foci was obtained by interpolating latable to the data derived from the functional zonation
perihemopoietic hepatocyte SDH activity values in the corresponding
of the hepatic tissue. For these reasons, we feel that it
curve of the intraacinar SDH activity gradient.
*Significant differences from the peri-terminal hepatic venule SDH is important to transform these intrahepatic distance
coordinates into others directly related to some wellactivity by Wilcoxon rank sum test, P<O.Ol.
273
ESTIMATING POSITION COORDINATES IN LIVER TISSUE
250
200
>
k
150
v)
2
W
zI-
100
50
O
1J
,
,
,
50
75
,
,
,
,
I
,
I
250
200
>
C
150
v)
z
W
z_
+
100
50
0
0
25
100 125 150 175 200 225 250
DISTANCE FROM THE PORTAL VEIN
(pm)
Fig. 6. Representation of all the intensity values of the pixels on a
line joining the portal vein with the central vein (top) in relation with
their position in microns (at the 10 intervals used to calculate their
relative position). Bottom, the pixels that actually are found on hepatocyte cytoplasm have been shaded. The fall in the intensity gradient is evident when the background pixels are eliminated (curve).
known functional activity in order for the position coordinates to have a real and uniform value.
Highly technical, detailed studies have demonstrated that hepatocytes have an activity gradient for
some specific enzymes down the length of the sinusoidal pathway connecting the PV with the THV (Wimmer and Pette, 1979; Hildebrand and Schleicher, 1986;
Ebert et al., 1987; Teutsch, 1988). Thus, relative position on these gradients (between maximums and minimums) could constitute a distance value closely linked
to the functional activity of each hepatic topographical
zone and, therefore, would represent a high information-content measurement.
Studies that examine the form of the gradients
mention the difficulty involved in measuring them
with precision due to the difficulty of drawing lines of
development of this gradient between zones (Hildebrand and Schleicher, 1986). Normally, straight lines
are drawn between the portal and central vessels, and
microbiochemical (Ebert et al., 1987) or microdensitometric (Hildebrand and Schleicher, 1986) measurements are taken down the length of the lines.
However, there will inevitably be disparity in these
measurements due to the very nature of the tissue, the
histochemical reaction, and the methods of measurement.
What we have done is take SDH activity as the reference enzyme activity-although any other hepatic
enzyme recognized as zonal and with an intraacinar
distribution along an activity gradient (Lamers et al.,
1988)could be used as well. The histochemical reaction
of SDH has a close correlation between the enzyme
activity and quantity of chromophore precipitated
(Nolte and Pette, 1972; Lippold, 1982). Measurements
of the hepatocyte cytoplasmic chromophore content
serve to establish a correlation between functional activity and positions in lobules or acini, as we confirmed.
a
b
U
0
50
100
150
200
250
I N T E N S I T Y (ARBITRARY UNITS)
0
0.5
NORMALIZED
R.O.D. U N I T S
Fig. 7. Intensity (a)or normalized relative optical density (b) and distance values of the pixels located
on 100 lines drawn between the PV and the THVs. The curves are the graphic representations of the
functions adapted to the intensityidistance correlation (a)or to the NRODidistance correlation (b).
1
274
E. BARBERA-GUILLEM ET AL.
Fig. 8. Light micrograph of a liver cryostat section under an SDH
histochemical reaction from phenylhydrazine-treated mice. Hemopoietic colonies were identified as small unstained foci (arrows) selectively located within the high SDH activity parenchymal regions
(dark areas surrounding portal veins defined a s Rappaport’s zone 1.
Low SDH activity parenchymal cells were defined as Rappaport’s zone
3, where hemopoietic foci were never found. PV, portal vessels; THV,
terminal hepatic venules. x 200
An ordering of the hepatocytes along the gradient was
demonstrated, with a maximum found near the PV and
septum and the minimum around the THV. Also, the
standardization of geometric distance values between
the PV and THV makes i t possible to establish a general function t h a t provides us with a position scale in
the hepatic acinus correlatable with this functional zonation.
The difficulty in measuring by microspectrophotometry is that what must be measured is the integration
of the chromophore content in each cell. In order to
avoid the problems posed by the irregular shape of each
hepatocyte, we propose measuring a constant polygon
within each hepatocyte cytoplasm, rather than the hepatocyte itself. This makes it possible to avoid measuring other neighboring types of cell, ensures identical
adjustment, and reduces enormously the time taken in
carrying out the measurement.
We also tested the viability of the method with TV
microdensitometry. The main difficulty in using this
instrument is that the unit of measurement used is the
intensity value of each pixel, the size of which depends
on the optics used. After a detailed analysis of the densitometric image of the hepatocytes, we verified that
the chromophore content or concentration is a very pre-
cise amount that is difficult to calculate with the intensity values of the pixels, since the latter fall within
very few gray levels. The concentration or quantity
values, on the other hand, are arrived a t by adding the
different proportions of pixels.
According to our results, it is possible to find pixels of
all values in any zone of the hepatic acinus, and what
varies is the probability of finding a given value in
each zone. These circumstances have already been
described in image analysis studies of the liver and
its functional zones (Hildebrand and Schleicher,
1986). Our examination of a large number of pixels
showed that, although there is enormous disparity on a
line drawn from a PV to a THV, i t is possible to discover the gradient by programming out all the pixels
that do not belong to hepatocyte images. With this
approach a gradient is established with characteristics
similar to the gradient obtained through microspectrophotometric measurement. In this regard, it might be
a s useful to use direct intensity measurements obtained from a n inverted image (a negative) of a section
of liver subjected to the SDH reaction, a s to use values
programmed into standard units of optical density. In
this case, however, it would be necessary to first introduce a function transforming the intensity of the
ESTIMATING POSITION COORDINATES IN LIVER TISSUE
____-
L I V E R ACINUS
--
/
i-/
L- J
/
Z0,NE 1
53187 from the University of the Basque Country
awarded to F.V.-V.
Q
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1
DOMAIN OF
HEMOPOIETIC
LITERATURE CITED
\
‘
PV
\SINUSOIDAL
275
PATHWAY
HEMOPOIETIC FOCI
& - - -%
L I V E R LOBULE
Fig. 9. Diagrammatic representation of acinar and lobular models of
liver tissue organization and the distributions of liver hemopoietic
foci from phenylhydrazine-treated adult mice. Liver sinusoids run
from the terminal portal venules (PV) to the terminal hepatic venules
(CV) and define a functional pathway that we have scaled 0 (at the PV
end) to 10 (at the CV end). The dotted area defines the specific domain
of hemopoietic foci implantation. The average position of hemopoietic
foci on the sinusoidal pathway was 3.31 (range 1.25-4.86).
pixels into their corresponding normalized optic density value.
To summarize the procedure, we calculate the anatomical-functional position of a n object in a liver section subjected to SDH histochemical reaction using the
following procedure: (1) Verification that the experiment in question does not alter the pattern of functional activity of the hepatocytes used a s our reference
for determining the gradients. (2) Calculation of maximum and minimum activity by the procedure preferred for the histological section as a whole. (3) Calculation of the function relating the enzyme activity
gradient with its relative position. (4) Calculation of
hepatocyte activity around the object. (5) Interpolation
of the anatomical-functional position using the value
t h a t represents the average activity of the hepatocytes
surrounding the object in the mathematical function
relating both variables.
From earlier studies, we knew that the position of
PHZ-induced hemopoietic foci in the liver is intrasinusoidal (Ploemacher and van Soest, 1977), but it was not
known in what functional area they occurred. The
present results show that hemopoietic foci are not disturbed a t random, but occupy positions in Rappaport
zone 1. The exact estimate of anatomical-functional
coordinates demonstrates foci within a narrow functional band. This border area (Barbera-Guillem et al.,
1989b) is between Rappaport’s acinar zone 1 and zone
2. We feel that the value of this procedure is in pinpointing the precise spot where implantation occurs,
and demonstrating the regularity with which this position is selected. Possible functional and cellular implications associated with this selective implantation
process in hepatic tissue need to be addressed.
ACKNOWLEDGMENTS
Supported by C.I.C.Y.T. Grant PR-0910184 awarded
to E.B.-G. and by Grants 075.327-52/87 and 075.327-
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