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Clinical Science ( 1 989) 76, 269-276
269
Multi-organ damage resulting from experimental faecal
peritonitis
D. TIGHE, R. MOSS, S. BOGHOSSMN, M. F. HEATH, B. CHESSUM AND E. D. BENNETT
Departments of Medicine 1, Electron Microscopy, Haematology and Microbiology,St Georges Hospital Medical School, London, and
Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, U.K.
(Received 25 January/29 April 1988; accepted 11 July 1988)
SUMMARY
1. Using specific-pathogen-free New Zealand White
rabbits, we have compared the effects of faecal peritonitis
over a period of 5 h in eight test animals with eight controls in which a sham operation was performed.
2. There was morphological damage to lungs, liver and
spleen of test animals. Lung capillaries and sinusoids of
the liver showed occlusion by cell debris and leucocytes,
with endothelial damage. The lungs also showed alveolar
epithelial disruption, basement membrane exposure and
type I1 pneumocytes lacking lamellar bodies. In the liver
there was fibrin deposition and swollen Kupffer cells. The
spleen showed degranulating neutrophils, fibrin deposits,
p!atelet aggregates and activated macrophages, with no
damage to the endothelium.
3. There was no morphological damage to the kidney
or heart of test animals or to any organs of sham-operated
animals.
4. There were mixed anaerobes and aerobes in faecal
material used to induce peritonitis. Cultures of liver,
spleen and kidney isolated four different types of
micro-organisms. Blood cultures showed two types
of micro-organisms. Cultures of lung and heart showed
one type of micro-organism.
5. The presence of micro-organisms in an organ C O U ! ~
not be correlated with the degree of histological damage
to that organ.
6. In test animals an early significant reduction in
circulating leucocytes and platelets was sustained for the
duration of the experiment with significant diffuse intravascular coagulation.
7. There was no change in test animal neutrophil
adhesiveness until 120 min, when significant reduction
was observed.
8. Serum phospholipase A, (EC 3.1.1.4) activity in the
test group showed a threefold increase at 300 min.
Correspondence: Dr D. Tighe, Department of Medicine 1, St
Georges Hospital Medical School, Cranmer Terrace, London
SW17 O R E .
Key words: histological change, multi-organ damage,
neutrophil adhesiveness, peritonitis, phospholipase A,.
Abbreviations: ARDS, adult respiratory distress syndrome; LM, light microscopy; MABP, mean arterial
blood pressure; PLA,, phospholipase A, (EC 3.1.1.4);
SEM, scanning electron microscopy; TEM, transmission
electron microscopy.
INTRODUCTION
Multi-organ failure associated with septicaemia is a
common clinical problem with a mortality rate approaching 100%. Anatomical and physiological changes occurring in the lung after sepsis [l,21 and endotoxic shock [3]
have demonstrated leucostasis, endothelial damage and
pulmonary hypertension. Scant attention has been paid,
however, to damage that occurs in other organs.
There is little documentation of the relationship
between the presence of micro-organisms and tissue
damage affecting various organs in bacteraemia. In
addition, it is unclear whether the degree of organ damage
is proportional to the number of micro-organisms
present.
Leucocytopaenia has been shown in animal models of
endotoxic shock and may be related to leucostasis occurring in various organs. Such leucostasis may be due to
increased neutrophil adhesiveness to the endothelium,
although little work has been done to establish whether
there is any such increase in septic shock in humans.
Phospholipase A, (PLA,; E C 3.1.1.4) activates the
arachidonic acid cascade, the products of which may be
responsible for some of the changes seen in septic shock.
Serum PLA, has been demonstrated to be elevated in
patients with septicaemia [4] and in a rabbit model of
endotoxic shock [5].We have shown previously that
administration of mepacrine, a PLA, antagonist,
markedly attenuates histological changes occurring in the
rabbit lung after faecal peritonitis [l].
We undertook the present study using a rabbit peritonitis model described previously [l], to examine tissue
270
D. Tighe et al.
changes in lungs, liver, kidney, heart and spleen by
electron microscopy. Bacterial cultures of these tissue
were performed to establish any correlation between
tissue damage and the presence of micro-organisms. In
view of the leucostasis that occurs in this animal model,
we determined whether any increase in neutrophil adhesiveness took place. We also wished to confirm that serum
PLA, activity would increase in this animal model.
METHOD
Faecal peritonitis was induced in eight specific-pathogenfree New Zealand White rabbits (2-2.5 kg) [l]. In brief,
after anaesthesia a limited laparotomy was performed, the
caecum opened, 5 ml of caecal contents removed and the
intestinal incision closed. The caecal aspirate was then
spread evenly around the peritoneal cavity and the laparotomy closed. A similar laparotomy was performed in
the eight controls, but the caecum was not opened.
Animals were killed after 300 min when samples of
lung, kidney, liver, heart and spleen were taken for light
microscopy (LM) and for transmission (TEM) and scanning electron microscopy (SEM). Tissue for TEM was
fixed in 3% (v/v) glutaraldehyde in cacodylate buffer,
post-fixed in 1% (w/v) osmium tetroxide in buffer, dehydrated in ascending concentrations of ethanol and
embedded in Spurs' resin. Tissue for SEM was similarly
fixed and dehydrated by alcohol and then taken through a
freon alcohol series and critical point dried.
Samples of lung, liver, heart, spleen, kidney, blood and
caecal content were taken for bacteriological examination.
The organ samples were dipped into boiling water for a
few seconds to sterilize external surfaces and then the
sample was cut in half. The size of the surface was noted
and was then smeared over the surface of the plates of
media. Cultures were incubated for 48 h in 10% CO, in
air and anaerobically with 10% CO, for 48 h. Blood
samples were taken and cultured using a poured plate
technique with 0.1,0.5 and 1.0 ml of blood per plate. The
plates were incubated both anaerobically and aerobically
with 10% CO,. Three millilitres were also cultured in a
Casteneda and a thioglycollate blood culture broth bottle.
These two bottles were subcultured at 2 days and 5 days
after inoculation on both aerobic and anaerobic media.
Blood samples were taken at baseline, 30,60, 180 and
300 min for total and differential leucocyte counts, basic
clotting parameters and neutrophil adhesion to nylon
fibre in a Pasteur pipette by the method of MacGregor el
aI. [6]. Fibrinogen was assayed by the method of Clauss
[71.
Serum PLAz levels were estimated on clotted plasma at
baseline and 300 min. The method was a modification of
those of Heath & Jacobson [8,9].The substrate used was
1-palmitoyl 2-[ 1-14C]oleoylphosphatidylcholine (Applied
Science, Oud-Beijerland, Netherlands) diluted in egg
phosphatidylcholine (Sigma, Poole, U.K.) to give 50 000
d.p.m. per assay tube, dispersed by brief ultrasonication at
a concentration of 0.8 mmol/l in the assay buffer: 50
mmol/l 4-( 2-hydroxyethy1)-1-piperazine-ethanesulphonic
acid/HCl (pH 7.5 at 37"C), 10 mmol/l CaCI,, 0.1% (w/v)
bovine serum albumin. To 20 pl of rabbit serum were
added 80 pl of sodium deoxycholate (5 mmol/l, in 150
mmol/l NaCI), and 100 pl of substrate. The assay tubes
were incubated for 1 h at 3 7 T , then acidified with 25 pl
of 0.5 mol/l H,SO,, and extracted with chloroform/methanol (containing 25 p g of oleic acid) by the procedure of
Bligh & Dyer [lo]. Fifty microlitres of the chloroform
layer was analysed by t.1.c. on LK6D silica gel plates
(Whatman, Maidstone, U.K.),developed first with chloroform/methanol/water (65:25:4, v/v), then after drying,
with hexaneldiethy1 ether/acetic acid (69:29:2, v/v).
Iodine vapour was used to locate the oleic acid and phosphatidylcholine spots, which were scraped off for scintillation counting. PLA, activity was calculated from the
proportion of radioactivity recorded in the fatty acid spot,
and adjusted for the activity found with reagent blanks
(20 p1 of NaCl instead of serum).
Results are expressed as means fSEM. Statistical significance was evaluated by Student's r-test.
RESULTS
Histological analysis
Samples of lung tissue (Fig. 1) showed reduction in
capillary lumen area, with marked leucostasis and capillary loop occlusion by neutrophils, lymphocytes and cell
debris. Although platelets were seen, aggregates never
formed. Neutrophils were adherent to the capillary endothelium which showed hypertrophy with occasional disruption and exposure of basement membrane. Alveolar
epithelial cells showed swelling and some bleb formation.
Type I1 pneumocytes appeared deranged, having a
reduced number of lamellar bodies.
Neutrophils showed progressive degranulation of
primary granules with the development of translucent
vacuoles, many of which had lost their membranes. Areas
of pooled glycogen and phagosomes containing bacteria
were also seen. There was no interstitial oedema or fibrin
deposition.
Liver samples (Fig. 2) revealed dense occlusion of the
sinusoids by degranulating neutrophils, lymphocytes, cell
debris and fibrin deposits. Rod-like bacteria were present
in the lumen of the sinusoids, some being phagocytosed
by neutrophils. Kupffer and endothelial cells showed
hypertrophy with consequent narrowing of the sinusoidal
lumina and the space of Disse, with a reduction in sinusoidal luminal area. SEM revealed the sieve plates of the
sinusoidal endothelium to be disrupted by areas of larger
fenestrations, especially when adjacent to activated
Kupffer cells.
Most of the occlusion by leucocytes, Kupffer cells,
endothelial cells and fibrin deposition occurred in sinusoids of zone 1 of the Rappaport acinus system. Sinusoids
of zone 2 appeared constricted but with little damage,
whilst the sinusoids of zone 3 appeared more patent
where they entered the hepatic vein. Each acinus defines
an area of liver tissue organized around the terminal
branch of the portal vein in such a way that cells at the
centre of the acinus, zone 1, are the first to receive blood,
Multi-organ damage in rabbit peritonitis
27 1
Fig. 1. Lung. (a) Section of lung from a sham-operated animal showing erythrocytes (RBC)in
patent thin-walled capillaries and thin interstitiurn. LM:original magnification X 550. ( b )Section
from test animal showing capillary occlusion by polymorphonuclear leucocytes (PMN) and
lymphocytes, with thickened interstitium. LM:original magmfication x 550. ( c )Septal wall from
test animal showing degranulating PMN occluding capillaries, with endothelial and epithelial
damage (arrows). T E M original magnification x 1700. ( d )Degranulating PMN with phagosome
containing a bacterium, occluding a capillary within a thickened septa1 wall. TEM: original magnification X 6000. ( e )Fractured capillary loop from a sham-operated animal. SEM: original magnification X3400. (f)Fractured capillary loop from a test animal showing obstruction by an
adherent PMN and RBC. S E M original magnification x 3400.
followed by zone 2, and cells located toward the
periphery and collecting vein, zone 3, being the last to
receive blood.
The spleen (Fig. 3) showed degranulating neutrophils
in the red pulp, arterioles and venous sinuses. Many
aggregates of platelets were seen in the red pulp, although
there was no capillary occlusion. Macrophages showed
evidence of activation and contained large amounts of
phagocytosed cell debris. Fibrin was also found adjacent
to the endothelium of the venous sinuses and red pulp.
There was no damage to the endothelium.
Heart and kidney histology showed no adherent leucocytes, endothelial disruption or fibrin deposition. There
were no histological changes seen in any of the organs
taken from the sham-operated animals.
Microbiology
Within the test group a number of different species of
organisms were isolated.
The caecal aspirate contained mixed aerobic and
anaerobic bacteria to a count of at least 1.4 x loy per ml
of 'emulsion'. The predominant species found were Bacteroides,Enterobacteriaceae and Streptococcusfaecalis.
All blood cultures taken at baseline were sterile but
those taken at 120 and 300 min grew Enterobacter
agglomerans together with a Bacteroides species. Shamoperated animals did not have cultures taken.
Cultures of heart and lung rarely produced more than a
few colonies of E. agglomerans (Table 1). Cultures of
liver, spleen and kidneys produced mixed growths of
272
Fig. 2. Liver. ( a ) Section from a portal area from a sham-operated animal showing patent
sinusoids, several with erythrocytes (REX).LM: original magnification x 550. ( b )Section from a
portal area from a test animal showing open sinusoids (*) followed by capillary obstruction from
polymorphonuclear leucocytes (PMN), cell debris and fibrin (arrow) leading to more narrowed
and obstructed sinusoid. LM: original magnification x 550. (c)Section from a test animal showing
total obstruction of a sinusoid by a Kupffer cell (KC)and degranulating PMN (PMN).Fibrin ( F ) is
present between obstructive cells. Endothelial cells and space of Disse cannot be clearly seen.
TEM: original magnification X 1600. ( d )Section from a test animal showing fibrin ( F )deposition
and cell debris occluding a sinusoid between enlarged endothelial cells (EC). The space of Disse
can be seen at (D), but is mainly absent where endothelial cells flatten the microvilli on to the
hepatocyte surfaces (arrow). TEM: original magnification x 1850. (e) Fractured surface from test
animal showing sinusoidal lumen (SL), Kupffer cells (KC) and sinusoidal obstruction (Ob) by
PMN, RI3C and cell debris. SEM: original magnification X 600. (f)Enlarged Kupffer cell (KC)
obstructing sinusoidal lumina (SL). The endothelium close to Kupffer cells shows fenestrations
larger than in other areas (arrows). Part of a hepatic chord (HC). SEM. original magnification
X 2200. ( g ) Sinusoidal lumen from a sham-operated animal showing small endothelial fenestrations (FE). Hepatic microvillus surface (large arrow) of a hepatic chord (HC). Small arrow shows
the bile canaliculi. SEM: original magnification x 3750. ( h )Sinusoidal lumen from a test animal
showing large endothelial fenestrations through which the microvillus surface of the hepatocytes
can be seen. SEM: original magnification X 3750.
Multi-organ damage in rabbit peritonitis
273
Fig. 3. Spleen. ( a ) Section from a test animal showing a venous sinus (VS), red pulp area (Rp)
with erythrocytes. Also present are many polymorphonuclear leucocytes (PMN) (P) and
aggregates of platelets (PL).TEM: original magnification X 1100. ( 6 )Section from a test animal
showing platelet aggregation, a macrophage (M) the remains of which surround a degranulating
PMN (P).TEM: original magnification X 1700. ( c ) Fibrin deposit (F),reticular fibres (arrow) and
macrophage (M) adjacent to venous sinus (VS). TEM: original magnification x 3150. ( d )Fibrin
deposit showing characteristic banding. TEM: original magnification x 3500.
Table 1. Microbiology and histology in test animals
Micro-organisms: - ,no colonies; + ,colonies present. Tissue changes: + ,present, - ,absent.
Lung
Micro-organisms
E. agglornerans
S. faecalis
Bacteroides
Gram-positive anaerobic rods
+
Tissue changes
Degranulating neutrophils
Endothelial damage
Fibrin deposits
Disseminated intravascular
coagulation
Heart
+
Blood
Liver
Spleen
Kidney
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+-
++-
-
-
-
-
-
-
+
+-
-
-
-
+
-
-
Table 2. Differential blood cell count
Neutrophils, lymphocytes and platelets at time 0 and 300 min in test and sham-operated animals.
Results are expressed as meant- SEM. Statistical significance: *P= 0.02, **P= 0.001.
x Cell count (no. of cells/l)
Time
(min)
Test animals
Sham-operated animals
0
300
0
300
Neutrophils
Lymphocytes
2.9 f 0.9
0.6+-0.1*
2.4 f 0.5
5.6 f 1.0*
2.4 f0.3
0.9 f 0.1 **
3.4 f 1.2
3.3 f0.7
Platelets
*
660 89
290 f 51**
700 f 82
710+55
D. Tighe et al.
274
Table 3. Blood clotting and PLA,
Prothrombin time (PT), thrombin time (TT), kaolin partial thromboplastin time (KPTT), fibrinogen and PLA, at time 0 and 30 min in test and sham-operated animals. Results are expressed as
mean k SEM. Statistical significance: *P= 0.05, **P= 0.01.
Clotting time (s)
Time
(min)
PT
TT
KPTT
Test animals
0
300
5.2k0.3
6.5 k 0.5*
12.5k0.8
20.6 k 3.7
Sham-operated animals
0
300
5.5k0.3
5.3k0.4
12.1k1.6
10.9k1.8
E. agglomerans, S. faecalis, two types of Bacteroides
species and a Gram-positive anaerobic rod.
Haematology
Peripheral neutrophil count in the test animals (Table
2 ) fell significantly to 19% of the baseline at 300 min. In
the sham-operated rabbits there was a significant increase
to 133% of baseline at 300 min.
Peripheral lymphocyte count in the test animals fell
significantly to 37.5% of baseline at 300 min. There was
no significant change in the sham-operated group.
Peripheral platelet count in the test animals fell significantly to 41% of baseline at 300 min. There was no significant change in the sham-operated group.
Prothrombin time in the test group (Table 3) at 300 min
increased significantly to 125% of baseline. The thrombin
time increases to 165% and kaolin partial thromboplastin
time to 145% were not significant. Plasma fibrinogen fell
significantly to 69%.
Plasma fibrinogen in the sham-operated animals fell
significantly to 93%; the other parameters showed no
significant change.
Neutrophil adhesion in the test animals 30 min after
peritonitis induction fell to 64% of baseline, 68% after 60
min and significantly to 74% after 120 min (Fig. 4). The
sham-operated animals showed no significant changes.
Serum PLA, activity in the test group showed a significant increase to 383% of baseline after 300 min (Table 3).
In the sham-operated group there was a fall to 56% of
baseline.
DISCUSSION
We have developed a rabbit model of faecal peritonitis
simulating clinical peritonitis, a condition often associated
with the development of multi-organ failure and the adult
respiratory distress syndrome (ARDS). We have demonstrated an early and maintained reduction in peripheral
blood leucocyte and platelet counts. There was evidence
of progressive disseminated intravascular coagulation
with significant reduction of plasma fibrinogen, although
fibrin degradation products were not measured. These
results are similar to those obtained by other groups using
a variety of techniques [2,3].
120
8
g
.-
Fibrinogen
(dl)
PLAz
(i.u./l)
44.5k4.7
64.0 f 12
3.4k0.5
2.3 k 0.3**
0.9 k 0.5
3.6 rt 1 .O*
40.5k4.6
36.0k5.1
2.9k0.1
2.7kO.l"
1.8 k 0.7
1.Ok0.1
-
100-
ao-
M
c
2
6040
20
-
1
I
I
I
We have also demonstrated the expected histological
abnormalities in the lungs showing capillary leucostasis,
endothelial disruption and type I1 pneumocyte damage [2,
31. The changes shown in the liver and spleen have been
reported less frequently but are similar to the findings of
Balk et al. [2] and Coalson et al. [3]. In the liver there was
fibrin deposition with evidence of severe damage to the
sinusoids of zone 1 of the hepatic acinus but none to the
hepatocytes or bile canaliculi. The spleen showed evidence of fibrin deposition, activation of tissue macrophages and aggregates of degranulating neutrophils but
with no endothelial disruption. In marked contrast, the
heart and kidney did not show any of these changes.
Coalson et al. [3]were also unable to show leucostasis in
these organs of their septic baboon model.
There was heavy polymicrobial growth from the caecal
aspirant and micro-organisms were cultured from blood.
Polymicrobal growths of similar micro-organisms were
found in the liver, spleen and kidneys, although the lungs
and heart showed only a few colonies of one species.
These findings strongly suggest that there is no direct relationship between the presence of micro-organisms in an
Multi-organ damage in rabbit peritonitis
organ and the degree of damage to that organ (Table 1).
Although liver and spleen had polymicrobal growth with
severe histological damage, this relationship was not true
of the lungs where only one species was isolated but again
with severe histological damage. The kidney with the
presence of polymicrobial growth and heart where only
one species was isolated showed no evidence of histological change. We should emphasize that there could have
been functional changes in the organs not reflected by
histological damage. These findings are compatible with
those of Goris et al. [ll], who produced multi-organ
damage in rats by inducing sterile peritonitis with
zymosan. These data show that organ damage is likely to
be produced by mediators released as a result of the
peritoneal inflammation.
This poses the question as to why leucocytes aggregate
in the liver, lungs and spleen but not in other organs. We
were unable to show any increase in neutrophil adhesiveness to account for leucostasis; indeed, we demonstrated
a decrease that became significant at 120 min in those
neutrophils still circulating in the peripheral blood. It may
be postulated that those cells aggregating in the organs do,
however, show an increase in adhesiveness which we were
unable to measure. It is also possible that changes occur in
endothelial adhesiveness modifying the relationship
between the activated leucocytes and the endothelium.
Fowler et al. [12] showed that there was an absence of any
humoral factor in the blood of ARDS patients capable of
causing chemotaxis or enzyme secretion in normal
neutrophils. Zimmerman et al. [13] showed that plasma
from ARDS patients was unable to promote a respiratory
burst from healthy neutrophils.
We have shown a fourfold increase in serum PLA, in
response to experimental faecal peritonitis, this change in
circulating activity being concurrent with the haematological and multi-organ histological damage.
Infusion of endotoxin has been found to produce an
11-fold increase in PLA, [5]. Infusions of a rabbit plasma
fraction containing PLA, caused a small effect on mean
arterial blood pressure (MABP) which could be prevented by pretreatment of the material with an inhibitor
of PLA, [5]. Vadas & Hay [5] suggested that circulating
PLA, releases fatty acids and lysophosphatides from
phospholipids and that these, and their metabolites, are
responsible for both hypotension and margination of
leucocytes.
The data showing that MABP starts to fall before any
significant change occurs in circulating PLA, [5] offers an
alternative hypothesis for the role of elevated serum
PLA,. It suggests that PLA, is secreted and has a local
action while still at a high concentration, before some or
all of the secreted enzyme escapes into the circulation. To
attain high tissue concentrations secondarily, by diffusion
out of the circulation, seems less likely. The serum level of
PLA, would thus indicate escape of PLA, from the tissues, and would depend on (i)the rate of secretion within
the tissues, and (ii)the rate of clearance into the circulation. Increased mediator production (e.g. prostaglandins)
would result from an increase in PLA, secretion, or a decrease in clearance of PIA,. On the other hand, an
275
increased serum PLA, level would arise with an increase
in secretion or an increase in clearance into the circulation. This may not therefore be a reliable index of mediator production, and so of potential damage and clinical
prognosis.
Elevation of serum PLA, has been found in patients at
risk for multi-organ failure [l],the levels showing some
correlation with prognosis. Absolute levels in humans are
very much lower than those in rabbits [8],the peak in
severe human cases after several days of illness being only
15% of the level measured in rabbits 5 h after endotoxin
infusion. Preliminary results from another group [ 14, 151
show similar clinical correlations, but much greater absolute values. Assay methods used by this group of investigators were very different, thus comparisons may not be
meaningful.
Although the eicosanoids are important in the production of inflammation, cytokine polypeptides also play
a role in this process. Thus micro-organisms, endotoxin,
C5a and other agents induce inflammation by the release
of the mediators interleukin-1 [16] and tumour necrosis
factor [ 171. These cytokines, produced by mononuclear
leucocytes, can stimulate neutrophil activation and adherence to capillary endothelium and can stimulate endothelium to produce more interleukin-1 and platelet-activating factor [ 181that can also stimulate neutrophils.
This study has therefore shown that the production of
faecal peritonitis in rabbits results in severe damage to
lungs, liver and spleen with marked leucostasis within
these organs. In contrast, the heart and kidney showed no
evidence of damage. Furthermore, the presence of microorganisms in an organ is not a prerequisite for histological
damage. There was the expected reduction in peripheral
leucocytes and platelets with evidence of diffuse intravascular coagulation. There was a significant reduction in
neutrophil adhesiveness. Although we have demonstrated
a marked rise in serum PLA, activity, it is uncertain
whether this is a cause or effect of the organ damage that
we found.
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