Nephrol Dial Transplant (2001) 16: 664±665
The rabbit model in evaluating the biocompatibility
in peritoneal dialysis
Guido Garosi and Nicola Di Paolo
Nephrology and Dialysis Division, Policlinico Le Scotte, Siena, Italy
Abstract Rat and rabbit are the most common animal
models for peritoneal dialysis. Rats are cheap and easy
to keep. Rabbits allow dialysis to be performed for
longer periods and in a way very similar to that used in
human patients. Recent progress in histomorphometry
enables accurate comparison of the biocompatibility
of different peritoneal dialysis solutions. Preliminary
data in the rabbit indicate that peritoneal dialysis is
associated with a change in both the number and size of
milky spots, which are peritoneal corpuscles involved
in peritoneal defence.
Keywords: biocompatibility; histomorphometry;
peritoneal dialysis; peritoneum; rabbit; rat
Peritoneal structure is similar in all mammals; there-
fore, in order to reproduce the changes caused by
peritoneal dialysis (PD) in humans as closely as
possible, the limiting factor is the practical possibility
of dialysing a mammal in the same way as a human.
Small mammals, especially rats, are often preferred
w1,2x because they are easy to obtain and keep, and
are economical. However, their small size is already
a source of difference with respect to humans in so far
as the way in which PD is performed. The main
problem with rats is that it is dif®cult to dialyse them
for a long period. The duration of dialysis is crucial for
reproducing histological alterations associated with
PD. In rats, it is dif®cult to use a catheter and often
the PD solution is injected blindly into the abdomen
with a needle: there is a probability of piercing the
viscera, the quantity of PD solution is small and the
dialysate is dif®cult to recover. The catheter itself does
not induce peritoneal alterations w3x. If we look at the
ratio of peritoneal surface area to exchange volume,
Correspondence and offprint requests to: Guido Garosi, Nephrology
and Dialysis Division, Policlinico Le Scotte, Viale Bracci 16, I-53100
Siena, Italy.
# 2001 European Renal Association±European Dialysis and Transplant Association
we see quite similar values for humans and rabbits,
but a much higher value for rats.
Larger mammals, such as the rabbit, have fewer
such disadvantages w4x. Rabbits survive much longer
than rats: we lose ;40% in 6 months, mainly due to
peritonitis. In rabbits, a catheter can be inserted quite
easily with the exit site on the dorsal part of the neck.
After 2 weeks to allow cicatrization, PD is commenced,
usually with two or three exchanges of 30 mlukguday
for several months.
Irrespective of the animal model chosen, a major
problem in studying peritoneal membrane structure is
sampling. The mesothelium dries very quickly when
exposed to air w5x. In order to prevent damage due
to artefacts, the specimen is usually taken and ®xed
as soon as the peritoneal cavity is opened. In our
experience w4x, in vivo ®xation is the best way to protect
specimens from damage. When the animal has to be
sacri®ced, 2% glutaraldehyde solution buffered with
0.2 M sodium cacodylate is infused simultaneously in
a peripheral vein (usually 20 ml for a rabbit) and into
the abdominal cavity via the catheter (30 mlukg for
rabbits). The animal dies immediately and the whole
peritoneal cavity and surrounding tissues are ®xed
prior to surgical dissection and exposure to air.
To evaluate biocompatibility, it is essential to
quantify as precisely as possible the morphological
alterations induced by PD. Wide use has been made
of semiquantitative methods w4x. One or two expert
observers judge the presence or absence of alterations
in a subjective manner, and if necessary their entity.
The limits of semiquantitative methods are obvious:
any single alteration, such as cubic transformation of
the mesothelium or submesothelial oedema, can only
be de®ned as present or absent, or at most quanti®ed
with a series of pluses and minuses. Observer-based
variations are also signi®cant.
Semiautomatic histomorphometric systems used with
the light microscope have recently become available.
They are image analysers that provide accurate measures
of distance and area on digital images obtained with
a videocamera connected to a light microscope. These
systems enable quantitative morphometric analysis of
peritoneal changes induced by PD. We found this
The rabbit model in evaluating the biocompatibility in peritoneal dialysis
method useful for comparing the biocompatibility of
PD solutions: in particular, we found that solutions
with amino acids are more biocompatible than
solutions with glucose w6x.
The method can quantify several PD-related altera-
tions. The cubic transformation of the mesothelium
can be quanti®ed by determining the percentage
of cubic mesothelial cells: we measure the thickness of
the cytoplasm of 100 mesothelial cells at high magni-
®cation; each cell is de®ned as normal if its cytoplasmic
thickness is -16 mm, and cubic if P16 mm. The con-
tinuity of the mesothelial layer can be quanti®ed by
determining the percentage of peritoneum without
mesothelium in many different sections observed at
moderate magni®cation, up to a linear extension of
3000 mm. Submesothelial oedema is analysed over
10 microscope ®elds of submesothelial connective
tissue: the area occupied by tissue and that free between
the tissue components due to oedema are de®ned by
the system semiautomatically, according to colour
differences: this provides an estimate of the percentage
of oedematous tissue. Morphometric analysis of the
arteriole walls is carried out by determining lumen
perimeter, intimal thickness, intimal area, external
intimal perimeter, muscle layer thickness, muscle layer
area and external perimeter of the muscle layer on clear
transverse sections of arterioles.
We currently are studying some morphological
aspects in the rabbit that could be important in
peritoneal defence mechanisms: little is known about
the milky spot system. Our preliminary data from
two rabbits dialysed for 30 days (1.36% glucose, two
exchanges of 30 mlukguday) and two non-dialysed
rabbits show a change in both the number and
size of milky spots after PD. These semiquantitative
data need to be veri®ed by quantitative analysis in
a larger number of animals; however, they suggest
a possible mechanism for the PD-associated decrease
in peritoneal defence.
Milky spots w7,8x are corpuscles found mainly on the
great omentum. They are 0.1±2 mm in size, hardly
visible to the naked eye, and under low magni®cation
look like tufts of cotton wool. They consist mainly
of macrophages, B and T lymphocytes and mast
cells, immersed in a network of connective tissue,
vascularized by fenestrated capillaries and, in places,
by evident lymphatic vessels. Studies with monoclonal
antibodies have shown that the macrophages in the
centre of milky spots are precursors of activated
macrophages, which are situated in the peripheral
665
areas. Similarly, B and T lymphocytes show various
stages of maturation in the milky spots.
An interesting feature w7,8x is that close to milky
spots, the mesothelium is practically devoid of base-
ment membrane; macrophages and lymphocytes do
not encounter any obstacles in the peritoneal lumen.
Milky spots may also be associated with stomata,
i.e. with microscopic lacunae between mesothelial cells.
These spaces presumably facilitate communication
with lymphatic vessels.
Thus we can say that milky spots are deposits of
white cells that are completing their maturation and
are ready to enter the intraperitoneal space if needed.
In mice, peritonitis induced by bacterial toxin and
adjuvant causes a large increase in the activity of the
milky spots.
These features suggest an important role for milky
spots in peritoneal defence mechanisms. The prelimin-
ary observation of a change in these corpuscles
during PD if con®rmed by quantitative studies,
constitutes real morphological evidence of a decrease
in peritoneal defence. It should also be interesting
to observe whether milky spots increase during
peritonitis associated with PD.
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