Br. J. Surg. Vol. 67 (1980) 851-855 Printed in Great Britain
The effect of local infection upon wound healing:
an experimental study
T. E. BUCKNALL*
SUMMARY
Local infection was introduced into rat abdominal
wounds using a loa bacteriallml inoculum. Three
groups of infection were used: Staphylococcus aureus,
Pseudomonas aeruginosa and a combination group of
Escherichia coli and Proteus mirabilis. Infection was
shown to delay healing as judged by bursting tests.
Fibroblast proliferation was depressed at the wound edges
but there was an increase in the total amount of hydroxy-
proline present. Small vessel angiogenesis was increased
in areas of abscess formation but larger vessels were
commonly blocked by thrombus or distorted by surround-
ing inflamed tissue. The possible causes of these effects are
discussed.
WHEN one considers that in a healing wound cell and
tissue production is proceeding at a rate which exceeds
that seen in most malignant tumours, it is humiliating to
admit how little we know of the mechanisms involved.
Nearly half of all postoperative complications are
wound complications. Among these, infection and
dehiscence are the most common (1 -3).
The present study investigates the effects of local
infection on the healing of abdominal wounds and the
interplay of cellular and vascular aspects of the healing
process in response to bacteria.
Materials and methods
The series comprised 120 adult female Sprague-Dawley rats
whose weights ranged from 200 to 300 g. In all animals a 5.0 cm
vertical midline abdominal incision was made. Bacteriological
swabs were taken from the skin wound in order to assess the
bacterial growth under the experimental conditions. The
incision was then closed aseptically using one layer of 2/0 (3
metric) sterile braided black silk on a cutting needle. A
standard continuous through-and-through suture technique
was used. Each suture would admit a curved forceps beneath it,
ensuring that it was not tight.
In the infection y p s , I . O m l of a 10' bacteria y m!
inoculum, prepared y the technique described by Edlic et a1
(4), was introduced gently into the wound using a tuberculin
syringe and fine needle. There were three infection groups
based on the organisms used: Staphylococcus aureus.
Pseudomonas aeruginosa and a combination group of
Escherichia coli and Proteus mirabilis. In the control wounds
1 .O ml of sterile normal saline was introduced using the same
technique. Swabs were taken on day 5 to confirm the presence
of the expected organisms in the pus, this being the only
criterion accepted for infection.
Bursting srrength of rhe abdominal wall
Forty rats (10 in each group) were killed on day 5 and 36 on day
14 (4 having died). The bursting strength was measured by the
technique previously described from this laboratory by De
Haan et al. ( 5 ) as a modification of the method devised by
Prudden et al. (6). This comprised the insertion of a rubber
condom into the peritoneal cavity through a defect made in the
apex of the vagina. The balloon was connected to a cylinder of
oxygen and slowly inflated at a constant rate. The bursting
pressure was measured on a connecting mercury manometer at
the time of wound disruption. The abdominal wall sutures were
removed before this procedure was carried out.
Hydroxyproline estimates
The hydroxyproline content of tissues was estimated by a
modification of the method of Woessner (7). Using this method
the hydroxyproline content of standard segments of the
midline incision on the control and infected rats on day 7 were
estimated.
Histology and autoradiography
Twenty rats, 5 in each group, were used. Seven days after a
midline abdominal incision had been sutured, and either
infected or acting as control, the rats were given an intra-
peritoneal injection of tritiated thymidine 2 pCi/g body weight,
and were killed with ether after 60 min. Two segments of the
skin wound from the centre of the incision were removed, fixed
in 10 per cent formalin and sections were cut and stained with
haematoxylin and eosin. One set of sections was examined
histologically by light microscopy. Autoradiographs were
prepared with the second set by the dip ing technique using
Ilford Nuclear Research Emulsion KS. d e s e were exposed for
3 weeks and subsequently examined under the light micro-
scope. Using a graticule, all the cells in 3'fields on either side of
the incision at the wound edge were counted, that is 6 fields
were counted in each wound. The labelling index is the
percentage of cells that are labelled in each specimen. The
percentage of labelled fibroblasts and capillary endothelial cells
in the granulation tissue under the epidermis provided an
estimate of cellular proliferation. The criterion for labelling
was 8 grains/nucleus, which was well above the background
grain density.
Angiography
Twenty rats, 5 in each group, were used. Ten days after a
midline abdominal incision had been sutured, and either
infected or acting as a control, the rats were anaesthetized with
ether. The method of angiography used was a modification of
the technique described by Myers and Cherry (8); the descend-
ing thoracic aorta, approached through a left thoracotomy
wound, was cannulated with a h e r fitting cannula. The
vascular system was flushed through ntl with warm normal
saline. An equal parts solution ( 0 . g mr/g body weight) of
barium sulphate and methylene blue was then introduced
slowly at a rate of 1.0 d / 1 5 s in order to minimize trauma to
the vessels by keeping within physiological pressure. The
abdominal wall was then excised and the skin carefully
removed. The specimens were laced on cardboard to revent
shrinkage and into 10 per cent Formalin for 48 h. After &ation
they were radiographed on Kodak X-ray film. Serial sections
were then taken at 0.5 cm intervals for histological
examination.
Results
Bacteriological studies revealed insignificant growth in
rat skin wounds under normal conditions. The method
used to produce infection caused 100 per cent of the
wounds to become infected. The mortality was 3.3 per
cent.
The observed levels of bursting strength, hydroxy-
proline levels and labelling indices were not normally
distributed. For this reason the Mann-Whitney U test
was used to analyse the data. As there was not an apriori
assumption that the bursting strength in infected rats
was less than in controls, two-tailed probability values
* Westminster Hospital, London SWlP 2AP.

852 T. E. BucknPll
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O' Control Staph. Pseudo. E.coli&Proteus.
Fig. 1. Hydroxyproline levels in infected and control
wounds on the seventh postoperative day. (For details see
Table II.)
are given in the results. The null hypothesis was that
there was no significant difference between controls and
the infection groups.
The pus forming dose of bacteria
A pilot study was performed with serial concentrations
of bacteria with P . aeruginosa as the trial organism. It
was found that at 10" cells/ml all the test animals died
with an overwhelming infection. At lo4 cells/ml ap-
proximately 50 per cent of the wounds showed no sign
of infection, but at 10" cells/ml 100 per cent of the
wounds produced pus without mortality and therefore
this concentration was used throughout.
Bursting strength of the abdominal wall
At 5 days the bursting pressure was greater in control
rats than in any of the infected groups. This difference is
statistically significant between controls and staphylo-
coccal wounds. and between control and pseudomonad
wounds (P<O.OOl) but not between control and
wounds infected with E. colilP. mirabilis. At 14 days
the difference in the bursting pressure is significantly
greater in control rats than in any of the infected
groups (P<0401) (Table I). There was a significant
ranking order of bursting strength from strongest to
weakest, i.e. control, E. coli/P. mirabilis, S . aureus, P .
aeruginosa. From day 5 to day 14 there was a constant
increase in strength in each group. During this 9-day
period there was a mean increase in strength of 360 per
cent.
Hydroxyprohe estimations
There was a larger amount of hydroxyproline present in
the infected wounds than the control wounds on
postoperative day 7. This reached significance with the
E. coli/P. mirabilis infected group (P<O.O5) (Table II,
Fig. 1).
Histological and autoradiographic studies
In the control group (Fig. 2) the cellular component on
day 7 was seen to consist mainly of fibroblasts, many of
Fig. 2 Non-infected healing wound at 7 days HE x 17.
Fig. 3. Disor anized appearance of s~phy locd- in fec ted
wound at 7 fays. Note haemorrhage into vessels and
abscess formation (arrowed). HE x 17.
Table I: BURSTING STRENGTH (BS) OF
ABDOMINAL WOUNDS IN RATS*
BSmmHg Median
Group (sd.) BS Day
5 Control 93.2 (18.8) 103
14 Control 341.2 (22.6) 345
55 5 S. aureus
5 P. aeruginosa 28.7 (13)t
5 E. colilP. mirabilis 77.2 (29) 84
14 S. aureus 199.5 (25.3)t 202
14 P. aeruginosa 105.3 (26.9)t 1 14
14 E. colilP. mirabilis 277.7 (29.0)t 267
54 4 4.2)t 33
* n = 10 in all groups except P. aeruginosa on day 14,
where n = 6.
tPC0401.
Table 11: HYDROXYPROLINE (OHP) LEVELS IN
SKIN WOUNDS OF RATS ON DAY 7*
Mean OHP Median OHP
Group (mdg s.d.) ( m d d
~
Controls 27.97 (13.56) 2640
S. aureus 35.42 (15.35) 33.10
P . aeruginosa 32.37 (13.60) 29.65
E. colilP. mirabilh 35.78 (13.65)t 3540
* n = 5 .
t P<0.05.