Perit Dial Int
29(Supplement_2):
40-44
2009
© 2009 International Society for Peritoneal Dialysis
Part 2: Cellular and Molecular Biology of the Peritoneum
and Peritoneal Dialysis |
TECHNICAL ASPECTS IN STUDYING PERITONEAL MORPHOLOGY IN ANIMAL MODELS OF PERITONEAL DIALYSIS
Soner Duman1 and
Sait 
en2
Departments of Nephrology1 and
Pathology,2 Ege University Medical School, Izmir,
Turkey
Correspondence to: S. Duman, Nefroloji Bilim Dali, Ege Üniversitesi Tip
Fakültesi, Bornova 35100, Izmir, Turkey.
dumans{at}med.ege.edu.tr
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ABSTRACT
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Objective: Peritoneal biopsies are considered useful
for gaining a better understanding of the pathophysiology of the peritoneum
during experimental peritoneal dialysis (PD). Different peritoneal tissue
samples (i.e., abdominal wall, liver, diaphragm, intestine, and
omentum) may be used, but there can be artifacts due to peritoneal tissue
processing.
Aim: To investigate differences in peritoneal membranes
from different parts of the peritoneum, and also 2 different fixatives, in
experimental PD and a peritonitis model in rats.
Methods: Peritoneal tissues from the anterior abdominal
wall, liver, omentum, and intestine were taken from each of 3 groups of
animals: sham, experimental PD, and peritonitis model. Tissue samples were
immediately fixed with 4% formaldehyde and routinely processed for
histological examination. Two parietal peritoneal tissue samples according to
longitudinal and horizontal sections of anterior wall inner abdominal muscle
were also taken. All samples were immediately fixed with 4% formaldehyde and
B5 fixative (B5), and then routinely processed for histological
examination.
Results: In all groups, histopathological findings were
more commonly seen in the abdominal wall samples. There were no changes
observed in peritoneal membranes other than those of anterior abdominal wall
samples from both sham and PD model rats. However, there was a significant
difference between anterior and posterior facets of liver in the peritonitis
model. Furthermore, the antimesenteric site of intestinal peritoneum was less
affected than the mesenteric site. There were no significant histopathological
differences between B5 and 4% formaldehyde fixation (p >
0.05).
Conclusion: Our results suggest that peritoneum
obtained from the anterior abdominal wall is the most affected area and
therefore the most suitable site to investigate peritoneal changes in the
experimental rat PD model. There were no significant differences between
fixation with 4% formaldehyde and B5 solution. Abdominal wall samples should
be of the same direction of inner abdominal muscle, that is, horizontal
sectioning should be used for measurements of the submesothelial area.
KEY WORDS: Peritoneal histology; experimental studies; fixatives; standardization.
Peritoneal dialysis (PD) has become an acceptable alternative to
hemodialysis. Today, 4% – 70% (average 15%) of all end-stage renal
disease patients are on PD (1).
During long-term PD, various changes in peritoneal morphology take place,
including mesothelial denudation, interstitial fibrosis, neovascularization,
and vascular alterations
(2,3).
Among the suggested causes of those histological and functional alterations
are recurrent infections and the nature and products of PD solutions
(4,5).
Peritoneal biopsies have been considered useful for gaining a better
understanding of the pathophysiology of PD
(6). The growing interest in
the study of the morphology, physiology, and pathophysiology of the peritoneum
during PD is therefore not surprising. Different peritoneal tissue samples,
including anterior abdominal wall, liver, diaphragm, intestine, and omentum,
may be used for this purpose
(7–12).
Loss of mesothelial cells and changes in peritoneal interstitium are found
during PD (13). However, it
should be noted that, in studying the peritoneum, artifacts are a constant
threat. The peritoneum is a delicate tissue. Owing to a differential in
shrinkage between tissue components during the processing protocol, the
mesothelium may be stretched or contorted. In fact, most biopsy specimens of
mesothelium show alterations often erroneously attributed to pathology when
they are, in fact, due to technical problems of sampling
(14).
Histological distortion, cell shrinkage, and shedding of mesothelium are
not uncommon during tissue processing based on 10% formaldehyde
(7,10,11).
The parietal peritoneum is more difficult to fix and section than other
peritoneal tissues due to thick abdominal muscle fibers. Therefore, in the
present study, we compared various peritoneal tissues (visceral and parietal
peritoneum) and methods of fixation (B5 and 4% formalin) with the aim of
establishing a better tissue and fixative for morphological examination of
biopsy specimens of experimental PD models.
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MATERIALS AND METHODS
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Parietal peritoneal samples were obtained from nonuremic male albino Wistar
rats weighing 200 – 220 g. The rats were housed in polycarbonate cages,
fed a standard laboratory diet, and given free access to water. The rats
received daily intraperitoneal injections (sham group; n = 10) or
daily intraperitoneal infusions of 10 mL 3.86% dextrose (PD group; n
= 11), or intraperitoneal colchicine plus 3.86% dextrose (experimental
peritonitis group; n = 7). Duration of the experiment was 4
weeks.
In step 1, peritoneal tissues from the anterior abdominal wall, liver,
omentum, and intestine were taken from all groups. Tissue samples were
immediately fixed with 4% formaldehyde and routinely processed for
histological examination. In step 2, two parietal peritoneal tissue samples
(longitudinal and horizontal sections, according to inner abdominal muscle
direction) from the anterior abdominal wall were obtained and immediately
fixed with either 4% formaldehyde or B5 solution. After fixation, the samples
were processed for histological examination. Paraffin sections (4 – 5
µm) were taken from each tissue sample and stained with hematoxylin and
eosin. Sections were evaluated under light microscopy.
Histopathological changes, the quality of tissue preservation, and
artifacts of parietal peritoneum or abdominal muscles were evaluated by light
microscopy. The same pathologist (SS) performed semiquantitative evaluations
of the number and reactivity of mesothelial cells, the homogeneity of
peritoneal thickness, and the presence of inflammation, submesothelial edema,
fibroblastic activity and fibrosis, and vascularization. Details of the
histopathological evaluations were described in a previous study of ours
(12). An image-analysis
microscope linked to an IBM-compatible computer captured one histopathological
image including mesothelium, submesothelial area, and inner abdominal muscle
for each section. Mean thicknesses of the peritoneum and submesothelial area
were measured (AxioVision 2.0, demo version; Carl Zeiss AB, Hamburg, Germany).
We compared two measurements in each specimen.
Data were analyzed using the Pearson correlation, paired t-test, and
Mann–Whitney test. A p value of less than 0.05 was considered
significant.
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RESULTS
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In all groups, histopathological findings were more commonly found in the
abdominal wall samples. No changes were observed in peritoneal membrane
samples from liver, antimesenteric, or omental sites in groups 1 and 2 (sham
and PD models), but there were significant changes in the abdominal wall
samples. In group 3 (peritonitis model), however, there was a significant
difference in peritoneal membrane from the posterior facet of the liver
compared to the anterior facet (p < 0.05)
(Figure 1). Furthermore, also
in the peritonitis model, antimesenteric sites of intestinal peritoneum were
less affected than mesenteric sites (p < 0.05).
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Figure 1 — Peritoneal samples of liver [posterior (A) and anterior (B)
facets], intestinal wall [mesenteric (C) and antimesenteric (D) sides], and
anterior abdominal wall [horizontal (E) and longitudinal (F) sections of inner
abdominal muscle] in the rat peritonitis model. Abdominal wall is more
affected than the other samples.
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We observed small differences in histopathological parameters but there
were no significant differences (p > 0.05) between 4% formalin and
B5 fixation (Figure 2). Values
of submesothelial area and mean thickness in horizontally sectioned samples
were more stable than in longitudinal sections, but there were no significant
differences (p > 0.05) (Figure
3).
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Figure 2 — Fixation with B5 (A,C) and 4% formaldehyde (B,D) of sections of the
anterior abdominal wall in the dextrose group: inner abdominal muscle fibers,
horizontal (A,B) and longitudinal (C,D). Measurement of the submesothelial
area in the horizontal sections is more reliable than in longitudinal
sections.
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Figure 3 — Histological images and measurements from computer images of
horizontal (A) and longitudinal (B) samples.
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DISCUSSION
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The present study compares various peritoneal samples and fixation methods
for peritoneal tissues. The results show that anterior abdominal wall samples
were more affected than other peritoneal areas. There were no significant
differences between 4% formaldehyde and B5 fixatives. We believe that 4%
formaldehyde is better than conventional 10% formaldehyde. Based on our
experience from our previous study
(12), we suggest that inner
abdominal muscle fibers should be taken from the same direction during
parietal peritoneal sampling. Horizontal sections are safer than longitudinal
for measuring submesothelial area and peritoneal thickness.
Peritoneal tissue can easily be damaged. It has been suggested that studies
of peritoneal histology and ultrastructure may have encountered some artifacts
(14). Mesothelium dries
quickly, it reacts topically to blood and plasma exudates, and even mild
abrasion from the surgeon's gloves and tools may induce histopathological
damage. In the present study, the same pathologist (SS) carefully dissected
and collected all peritoneal tissues and found that mesothelial cell artifacts
may still appear during tissue processing.
There is no standard method to adequately assess peritoneal morphology. It
is important to know which peritoneal tissue should be sampled. Samples from
the abdominal wall, liver, and mesentery of the small intestine have been used
in previous studies
(7–12).
We compared abdominal wall, liver, and intestine samples. Significant anterior
abdominal wall changes were found in all groups; however, there were no
changes in the liver and intestine samples in the sham and PD model groups. In
the peritonitis model, anterior abdominal wall changes were more prominent
than in samples from the other sites. Some studies use visceral peritoneum
taken from the liver
(8,15,16).
Morphological findings of the peritoneum in these studies might be affected by
injections (higher dosage and twice daily) and the presence of the catheter
itself. We also found morphological changes in the anterior facet of the liver
in a chronic PD model using a catheter
(16).
Measurements of peritoneal thickness and submesothelial area may vary from
section to section within the same tissue sample. We have also observed that
the direction of the inner abdominal muscle affects the submesothelial area
and thickness. If the inner abdominal muscle fibers are horizontal, peritoneal
thickness varies up to tenfold in the same section, whereas peritoneal
thickness generally does not vary in longitudinal muscle sections
(homogeneity) (12). However,
submesothelial area measurements within the longitudinal sections might show
more differences between different histopathological sections in the same
sample. Neither other's nor our older studies mention these differences
(7,10,11).
In our previous study (12), we
suggested that both types of sections can be used, but that the horizontal and
longitudinal sections showed systematic differences. All samples in a study
should be taken using the same section pattern, either longitudinal or
horizontal.
In the present study, we found that horizontally sectioning the inner
muscle fibers is more reliable than longitudinal sectioning. Horizontal
sectioning minimizes variation in measurements of submesothelial
thickness.
Preservation of the original volume of the cells and tissues during the
processing period is a well-known prerequisite. Formaldehyde and alcohol both
may seriously shrink tissue
(17). On the other hand, B5
and Bouin's solution cause little tissue shrinkage; these solutions have been
suggested as better fixatives. We compared B5 and 4% formaldehyde in the
routine processing protocol and found no significant differences between them.
When we used 10% formaldehyde in our previous studies, we observed occasional
detachment of basal lamina and mesothelium from the muscle, leading to
submesothelial artifacts similar to those seen in other studies
(7,10,11).
In the present study, there were no such artifacts.
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CONCLUSION
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Our results suggest that peritoneum obtained from the anterior abdominal
wall is the most affected area, and therefore is the best site to investigate
peritoneal changes in an experimental rat PD model. There were no significant
differences between fixation with 4% formaldehyde and B5 solution.
Furthermore, fixation with 4% formaldehyde may be superior to conventional 10%
formaldehyde in the quality of the preparations. For measuring submesothelial
area or peritoneal thickness, all abdominal wall samples in a study should be
taken using the same sectioning pattern, either longitudinal or
horizontal.
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ACKNOWLEDGMENTS
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The authors thank Professor Dimitrios G. Oreopoulos for reviewing the final
draft and making useful suggestions.
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ABSTRACT
MATERIALS AND METHODS
