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Correlation Between Peritonitis and Incisional Infections in Horses
Journal Pre-proof Correlation between peritonitis and incisional infections in horses Renata G.S. Dória, PhD, Silvio H. Freitas, PhD, Luciane M. Laskoski, PhD, Laura P. Arruda, PhD, Antônio C. Shimano, PhD PII:
S0737-0806(19)30652-5
DOI:
https://doi.org/10.1016/j.jevs.2019.102903
Reference:
YJEVS 102903
To appear in:
Journal of Equine Veterinary Science
Received Date: 15 March 2019 Revised Date:
18 September 2019
Accepted Date: 21 December 2019
Please cite this article as: Dória RGS, Freitas SH, Laskoski LM, Arruda LP, Shimano AC, Correlation between peritonitis and incisional infections in horses, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/j.jevs.2019.102903. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.
1
Correlation between peritonitis and incisional infections in horses
2
Renata G.S. Dória1* PhD, Silvio H. Freitas1 PhD, Luciane M. Laskoski2 PhD, Laura P.
3
Arruda3 PhD, Antônio C. Shimano4 PhD
4
1
5
University of São Paulo, Rua Duque de Caxias Norte, 225, Jardim Elite, 13.635-900,
6
Pirassununga, São Paulo, Brazil.
7
*Correspondence email: [email protected]
8
2
9
Brazil.
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering,
Center of Biological and Nature Sciences, Federal University of Acre, Rio Branco, Acre,
10
3
11
Grosso, Brazil.
12
4
13
Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Mato Grosso Company of Research, Assistance and Rural Extension, Cáceres, Mato
Department of Biomechanics, Medicine and Rehabilitation of the Musculoskeletal System,
14 15
Abstract
16
Surgical site infection of abdominal incisions is an important complication
17
following laparotomy with increased risk of incisional hernia formation in horses. This
18
study aims to evaluate the healing process of abdominal incisions and correlate peritonitis
19
with the occurrence of surgical site infection and incisional hernias. Nine horses underwent
20
standardized laparotomy, intestinal exploration, and induced septic peritonitis. Standardized
21
relaparotomy was performed two (n=3), four (n=3) and six (n=3) months later to evaluate
22
the abdominal cavity for adhesions and to collect the sutured ventral abdominal wall to
23
evaluate and prepare it for histopathological and tensile strength study. All horses presented
24
endotoxemia, controllable peritonitis, heat and touch-sensitive ventral abdominal edema 1
25
and surgical wound infection with presence of purulent discharge. Adhesion of the cecum
26
or colon to the internal portion of the surgical wound was observed. Healing of the infected
27
surgical wounds occurred by second intention and a space between the rectus abdominis
28
muscles developed due to the presence of a scar, which was related to incisional hernia. In
29
the histopathological evaluation, the collagen content increased, and the inflammation
30
decreased over time. The tensile strength increased over time and was highest after 6
31
months. After the second surgical intervention, there was no infection of the surgical
32
wound in any of the animals and healing by first intention occurred. Surgical site infection
33
may be a symptom of peritonitis in horses recovering from abdominal surgery. Infected
34
surgical wounds heal by second intention, which favors the spacing of rectus abdominis
35
muscle and the formation of incisional hernia.
36
Keywords: abdominal infection, abdominal surgery complications, horses, second
37
intention healing, wound infection.
38 39
Introduction
40
Abdominal disorders are frequently found in equine clinics and may be associated with
41
several complications. Post-operative septic peritonitis can be considered a challenge for
42
veterinary clinics. It is diagnosed based on a combination of clinical signs in association
43
with abnormal peritoneal fluid evaluation and can reach up to 20% of horses undergoing
44
diagnostic and therapeutic laparotomies [1,2]. Among the possible etiologies of peritonitis,
45
fecal contamination is a frequent and serious cause [3-5]. Currently, there is a high rate of
46
survival of horses after colic surgeries; therefore, postoperative complications, such as
47
suppuration of laparotomy wounds and incisional hernia, have become relevant [6]. Despite
48
scientific advances, the prevalence of incisional complications following laparotomies, as 2
49
incisional drainage, dehiscence and hernia formation, may reach 40%, or even 87.5% when
50
reintervention is needed [7,8,9]. Wound infections were recorded in 29 to 36% of horses
51
after intestinal surgery and the rates of wound complications were significantly different in
52
relation to intraperitoneal contamination at surgery (63.6%) compared with no
53
contamination (27.4%) and development of post-operative peritonitis (85.7%) compared
54
with no peritonitis (27.3%) [1,9]. The risk of developing an incisional hernia is
55
significantly associated with the occurrence of incisional suppuration [1,10-11]. Incisional
56
complications associated with median laparotomy in horses are caused by several
57
predisposing factors, including factors inherent to the patient, surgical/anesthesia
58
procedures, and the postoperative period [6-10].
59
Incisional complications may result in extended postoperative care, increased
60
convalescence time, surgical reinterventions for hernia repair, or death. Complications
61
associated with ventral midline incisions include hematoma in the rectus abdominis muscle,
62
edema, fistulae, local infection, dehiscence, and herniation [7,9,12]. Herniation is related to
63
failure of the suture material, aggressive postoperative recovery, surgical wound infection,
64
and uncontrolled exercise in the early postoperative period [8-9,12-13].
65
Most studies on ventral median incisions in horses have focused on healthy horses
66
[6,12-15]. Notably, the surgeon should minimize factors that promote surgical wound
67
infection. However, there have been few reports regarding follow-up of the healing of
68
equine abdominal wounds with peritonitis and the correlation of such healing with the onset
69
of incisional hernias. The objective of this study was to evaluate the healing process of
70
abdominal incisions and correlate peritonitis with the occurrence of surgical site infection
71
and incisional hernias.
72 3
73
Material and methods
74
Twelve healthy, no-defined-breed horses (6-12 years of age; nine females and three males;
75
mean body weight of 350 ± 50 kg), which had no history of previous abdominal surgeries,
76
were included in this study. The horses were divided into two experimental groups:
77
peritonitis group (PG), consisting of nine animals, which underwent laparotomy, intestinal
78
exploration, and induction of septic peritonitis; and the control group (CG), which included
79
three animals that died due to causes not related with septic peritonitis. All horses of the PG
80
were dewormed and kept in a stall for a minimum of 2 weeks before the initiation of the
81
study. They were fed Tifton hay (2% body weight, daily), commercial equine feed (1%
82
body weight, daily), and water ad libitum.
83 84
First experimental phase
85
After 12 hours of fasting, the PG animals received 2% xylazine (0.5 mg·kg-1 IV) as
86
pre-anesthetic medication; after 10 minutes, the animals received guaiacol glyceryl ether
87
(100 mg·kg-1 IV) for myorelaxation. Anesthetic induction was conducted with 10%
88
ketamine (1 mg·kg-1 IV) and midazolam (0.1 mg·kg-1 IV) mixed in the same syringe;
89
anesthesia was maintained with halothane and spontaneous ventilation. The same surgeon
90
performed all surgical procedures. The animals were positioned in dorsal decubitus, and the
91
abdominal ventral region was clipped from the sternum to the groin. The surgical site was
92
prepared for surgery by scrubbing the area with povidone-iodine scrub for 3 minutes,
93
followed by povidone-iodine solution and 70% isopropyl alcohol application. A 20-cm
94
incision was made in the skin, subcutaneous tissue, and linea alba. This was followed by
95
opening of the peritoneum on the falciform ligament, and exploration of the abdominal
96
cavity. Initially, the cecum and pelvic flexure were exposed, followed by the sternal and 4
97
diaphragmatic flexures. Subsequently, the small intestine was exposed; starting from the
98
ileum toward the jejunum; the duodenum and stomach were palpated. The small intestine
99
contents were conducted to drainage into the cecum. Thereafter, the small intestine was
100
returned to the abdominal cavity and the small colon was exposed, enabling palpation of
101
the rectum and transverse colon. Additionally, the nephro-splenic ligament and the ventral
102
and right dorsal colon were palpated. After this procedure, the pelvic flexure was
103
repositioned within the abdominal cavity. At this stage, septic peritonitis was induced in
104
accordance with a modified surgical model standardized by Alves [5] (1997): 1 mL cecal
105
intestinal contents (cecal fluid with diluted vegetable matter, obtained via puncture at the
106
cecal apex) were aspirated with a syringe and needle (40 × 16) and 1 mL blood was
107
collected with syringe and needle (40 × 12) from the jugular vein. Both aspirates were
108
diluted in 1 L of lactated Ringer’s solution (1 L solution comprising lactated Ringer’s
109
solution, blood, and cecal contents). After repositioning the cecum, the abdominal cavity
110
was washed with this prepared solution (Figure 1A,B). The linea alba was sutured,
111
involving the peritoneum, with the cruciate suture pattern; this ensured standardized
112
spacing of 1.5 cm from the edge of the incision and 1.5 cm between each point, using a
113
synthetic non-absorbable thread (nylon, 0.60 cm). The subcutaneous tissue was
114
approximated with continuous horizontal mattress pattern and synthetic absorbable thread
115
(polyglycolic acid, number 1); the skin was approximated with interrupted horizontal
116
mattress suture and synthetic non-absorbable thread (nylon, 0.60 cm). The duration of the
117
complete surgical procedure was standardized to less than 1 hour. All animals recovered
118
from anesthesia and received benzathine penicillin (40,000 IU/kg, IM, every 48 h, three
119
applications), gentamicin (6.6 mg/kg, IV, once per day, for 5 days), and flunixin
5
120
meglumine (1.1 mg/kg, once per day, for 3 days). Every 12 hours, the animals underwent a
121
general physical examination and were monitored for signs of abdominal pain, peritonitis,
122
laminitis, and ileus. The animals were kept in stalls, without changes in food or water
123
management. Cleaning sutures were performed twice a day with povidone-iodine solution
124
during 10 days.
125 126
Second experimental phase
127
At 2 (2-month subgroup, n = 3), 4 (4-month subgroup, n = 3), and 6 months (6-
128
month subgroup, n = 3) after the surgical procedure, with the animals under general
129
inhalation anesthesia (using the anesthetic protocol described above) and positioned in
130
dorsal decubitus, the skin scar was opened; a blunt dissection of the subcutaneous tissue
131
was performed to expose the fascial-muscle plane and the previously constructed suture
132
line. At this stage, the sutured ventral abdominal wall (20 cm long × 8 cm wide) was
133
removed in preparation for the study. Two-centimeter samples were collected from the
134
cranial, middle, and caudal portions of the scar, and then stored in 10% formaldehyde for
135
histopathological evaluation; the remaining tissue was immediately frozen at -70ºC for a
136
subsequent tensile strength test. Abdominal cavity synthesis was conducted by repeating
137
exactly the previously described technique; the animals recovered from anesthesia,
138
underwent the same post-surgical therapy and cleaning sutures described above, and
139
returned to their routine activities when fully recovered.
140 141
Control Group
6
142
Three horses that did not undergo surgical interventions and died of other causes not
143
related to septic peritonitis were used in the CG; the ventral abdominal musculature (20 cm
144
× 8 cm, with the linea alba in the center) of these horses was collected and subjected to
145
histopathological evaluation and tensile strength analysis.
146 147
Laboratorial evaluations
148
For each horse in the PG group, blood and peritoneal fluid samples were collected
149
immediately before surgery, at 24 hours and 48 hours post-surgery, and at every 48 hours
150
on subsequent days until the 10th postoperative day. Hematologic and peritoneal fluid
151
evaluation was conducted; this comprised blood counts, total plasma protein and plasma
152
fibrinogen measurement for blood samples and visual examination, and evaluation of
153
density, total protein content, red blood cell count, leukocyte global count, and differential
154
leukocyte count for abdominal fluid samples.
155
Histological sections were prepared and evaluated with respect to collagen content
156
and maturity by using hematoxylin-eosin (H&E) staining. Tensile strength was evaluated
157
with a breaking strength test for the abdominal wall by using an electromechanical drive
158
tensiometer connected to a microcomputer. The speed of the break test calibrated in the
159
apparatus was 30 mm/min. Segments of the abdominal wall were attached to aluminum
160
claws parallel to the suture line (the threads were not removed); those claws were
161
connected to the apparatus, which exerted tensile force perpendicular to the suture. Values
162
were presented in Newtons (N).
163
The experimental data underwent analysis of the normality of residues by using the
164
Shapiro-Wilk test, followed by analysis of variance and Tukey's mean test. The non-
7
165
parametric Kruskal-Wallis test was used for data that did not exhibit a normal distribution,
166
followed by Dunn's mean rank test. A significance level of 5% was used in all analyses.
167 168
Results
169
All horses recovered from anesthesia without complications. In the immediate
170
postoperative period, the animals exhibited controllable peritonitis and endotoxemia, with
171
apathy, hyperthermia (T > 38.5ºC), increased heart and respiratory rates, reduction or
172
absence of intestinal motility, reddened mucous membranes with a thin purple line above
173
the teeth (toxic line), reluctance to move, splinting of the abdominal wall and sensitivity to
174
external abdominal pressure. Horses maintained this state for approximately 72 hours, after
175
which hyperthermia, tachycardia and tachypnea ended, intestinal motility returned, and the
176
other endotoxemia signs disappeared. Furthermore, surgical site infection was successfully
177
induced in all PG animals: heat and touch-sensitive ventral abdominal edema around the
178
skin incision and wound infection were observed between the 5th and 7th day post-surgery,
179
with the presence of purulent discharge (Figure 1E,F).
180
Two animals showed abdominal discomfort: one animal at 7 days post-surgery (2-
181
month subgroup); and another animal at 10 days post-surgery (6-month subgroup). Both
182
animals demonstrated an accumulation of gas and compaction of contents in the digestive
183
tract. After symptomatic clinical treatment, both animals exhibited symptom reversal and
184
remained in their respective experimental subgroups. Two other horses exhibited intense
185
pain and entero-gastric reflux at 8 to 10 days post-surgery, such that they underwent a
186
separate exploratory celiotomy; on observation of jejunal adhesion to the surgical wound,
187
these animals were excluded from the experimental groups.
8
188
Peritoneal fluid collection was possible only in the pre-surgical period, as well as at
189
24 and 48 hours after surgery. At other timepoints, it was not possible to collect peritoneal
190
fluid because a high concentration of fibrin was observed in the samples, beginning at 48
191
hours. Important changes were observed in the post-surgical fluid collections. These
192
became more pronounced after 24 hours: changes in color from light yellow to amber, total
193
protein content increased from 1.72 g/dL ± 1.18 g/dL to 4.41 g/dL ± 1.89 g/d, red blood cell
194
count increased from 592.14/µL ± 437.58/µL to 61,843.88/µL ± 64,668.85/µL, and the
195
leukocyte count increased from 1,088.80/µL ± 1,257.75/µL to 50,773/µL ± 76,615.79/µL,
196
and polymorphonuclear cells predominated, with degenerate neutrophils. These changes
197
indicated the presence of postoperative inflammatory reaction in all PG horses [16].
198
In the hematological analysis, a progressive elevation was observed in the evaluated
199
parameters over the time. These became more pronounced at 10 days post-surgery: total
200
leukocyte count increased from 9,140/µL ± 1,554.35/µL to 19,966.67/µL ± 5,153.51/µL,
201
neutrophil count increased from 5,721.90/µL ± 1,064.79/µL to 15,055.17/µL ±
202
5,678.76/µL, with toxic neutrophils and fibrinogen level increased from 366.67 mg/dL ±
203
150.55 mg/dL to 600 mg/dL ± 141.42 mg/dL. These changes were indicative of the
204
hematological profile of horses with peritonitis and surgical wound infection in the post-
205
surgical period [17].
206
All experimental animals in the PG recovered from septic peritonitis and wound
207
infection; skin sutures had healed but some points for drainage of purulent secretions
208
persisted during the first phase of the experimental period.
209
After 2 (2-month subgroup), 4 (4-month subgroup), and 6 (6-month subgroup)
210
months, the second experimental phase was undertaken: the collection of surgical scars for
211
histological evaluation and tensile strength analysis. In this second experimental phase, it 9
212
was possible to observe the events inside the abdominal cavity after exploratory laparotomy
213
in cases of septic peritonitis. In all horses, adhesion of the cecum or colon to the internal
214
portion of the surgical wound effectively sealed the abdominal cavity opening and
215
vascularized the surgical wound (Figure 1C). Neovascularization was observed from the
216
intestinal serosa, which extended through the fibrous scar tissue of adhesion, between the
217
intestinal loop and the surgical wound; this presumably supported the wound healing.
218
Anatomical preservation was noted in all cases without interference with the positioning of
219
other intestinal loops in the abdominal cavity.
220
At 2 months post-surgery, nylon threads that had been positioned in the linea alba
221
were no longer in place; these had been expelled into the subcutaneous tissue and the linea
222
alba was completely healed. Surgical wounds exhibited purulent secretions drained by
223
small sinuses in the skin. These drainage points on the skin led directly to the subcutaneous
224
tissue, where "abscesses" had formed; notably, these abscesses encapsulated the nylon
225
threads that had been expelled from the linea alba (Figure 1D).
226
Infected surgical wounds healing occurred by second intention and a space between
227
the rectus abdominis muscles developed due to the presence of a scar (Figure 2). The linea
228
alba in CG animals was 0.3 cm wide, without variation; in PG animals, the distance
229
between the rectus abdominis muscles, filled with scar tissue, ranged from 2 to 8 cm
230
(median, 4 cm; interquartile range, 3.5 to 5.75 cm), independent of the experimental
231
subgroup. This spacing between the rectus abdominis muscles was likely responsible for
232
the bulging of the abdomen observed in all PG animals, which was related to the presence
233
of an incisional hernia (Figure 1G,H).
234
At 2 months post-surgery, the scars of surgical wounds were thicker (median, 2.85
235
cm; interquartile range, 2.1 to 3 cm) than the linea alba of CG animals (median, 0.8 cm; 10
236
interquartile range, 0.7 to 0.8 cm) and reduced in thickness over time (figure 2); thus, the
237
scars of animals in the 4-month subgroup (median, 1.75 cm; interquartile range, 1.5 to 1.8
238
cm) were thicker than those of animals in the 6-month subgroup (median, 1.05 cm;
239
interquartile range, 0.9 to 1.4 cm) and thinner than those of animals in the 2-month
240
subgroup (Table 1).
241
The tensile strength increased with time with the highest tensile strength observed at
242
6 months post-surgery (median, 415.4 N), compared with 2 months post-surgery (median,
243
341.1 N); this was the sole statistically significant difference observed in this measurement
244
(Table 2 and Figure 3). Notably, when there was a rupture during this test, the rupture sites
245
were lateral to the scars at the point of insertion, next to the rectus abdominis muscle.
246
In the histopathological evaluation, the collagen content, visualized using H&E
247
staining, showed differences between horses in the CG and horses evaluated at 2, 4, and 6
248
months post-surgery. It was possible to observe a lower amount of collagen fibers in horses
249
evaluated at 2 months, compared with controls and with animals evaluated at 4 and 6
250
months; notably, these fibers were disorganized. This variation was also observed in the
251
same animal (in the same individual), with differences among analyzed regions (cranial,
252
medial, or caudal portion of the surgical wound). Some sites exhibited more orderly
253
arrangement of the collagen fibers; in other areas, there was a larger quantity of
254
extracellular matrix and smaller quantity of collagen. This was related to a larger quantity
255
of fibroblasts, as well as increased inflammation and vascularization. With the increased
256
length of time, such as in animals evaluated at 4 and 6 months, there was a progressive
257
reduction in the number of fibroblasts, blood vessels, and inflammatory infiltrates;
258
moreover, there was an increase in the quantity of collagen and the extent of remodeling. In
259
the 6-month animals, there were areas with larger quantities of collagen fibers, which were 11
260
denser and exhibited greater reinforcement, compared with those same areas in control
261
animals. Overall inflammatory infiltration ranged from mixed to mononuclear,
262
predominantly multifocal, and perivascular, and was present around the suture. This
263
inflammation was intense-to-moderate in the 2-month subgroup, moderate-to-light in the 4-
264
month subgroup, and discreet in the 6-month subgroup.
265
The second surgical intervention involved substantial divulsion of the subcutaneous
266
tissue, muscle bleeding and extensive tissue removal; subsequently, there was no infection
267
of the surgical wound in any of the experimental animals, and healing occurred by first
268
intention.
269 270
After the two experimental phases were completed, all animals showed full postoperative recovery and were donated to rural owners.
271 272
Discussion
273
Septic peritonitis in horses is one of the most serious complications of colic, especially in
274
cases requiring surgical intervention. Incisional complications observed following
275
laparotomy results in the delay of surgical wound healing and increases convalescence
276
period, duration of hospitalization, costs of treatment, and it may be fatal [6,9,12,18-20].
277
In this study, the induction of septic peritonitis was performed in accordance with a
278
modified version of the surgical model standardized by Alves [5] (1997). This protocol,
279
which involved the aspiration of 1 mL of blood and 1 mL of cecal contents, both diluted in
280
1 L of lactated Ringer’s solution placed inside the abdominal cavity of healthy horses,
281
proved informative, because there were changes in clinical, hematological, and peritoneal
282
fluid levels; these were consistent with the onset of septic peritonitis [16,19-22], in nine
283
experimental horses but did not result in fatality. Although the abdominal fluid parameters, 12
284
specially leukocyte counts, shown by the model of peritonitis induction in this study should
285
be compared to the leukocyte counts described in ponies after abdominal exploratory
286
surgery, the association of serial sampling of both peritoneal fluid and peripheral blood, and
287
physical examinations were mandatory in order to distinguish the presence of septic
288
peritonitis [2,23]. In this study, the animals showed consistent clinical signs of post-
289
operative endotoxemia and peritonitis, cytologic examination of peritoneal fluid
290
demonstrating degenerative cell changes and peripheral blood with neutrophilia and toxic
291
neutrophils, characterizing systemic and local responses to the inflammatory peritoneal
292
reaction caused by the presence of fecal content.
293
Importantly, using this protocol for peritonitis induction, it was possible to promote
294
the infection of surgical wounds in all experimental horses. Conversely, the removal of a
295
20-cm × 8-cm segment of the abdominal scar from the same horses resulted in healing by
296
primary intention, without any complications. Numerous factors are associated with
297
surgical wound infection in horses. These include the patient, surgical technique, surgical
298
time, incision size, and postoperative management [6-7,9,12,24]. In contrast, in cases of
299
equine colic, the most common causes of surgical wound contamination are endogenous
300
sources. Enterotomy and enterectomy are significantly associated with wound infection,
301
increasing the incidence of incisional hernia by up to 16% [7,12-13,24-26]. The results of
302
this study indicate that peritonitis may be an important factor to be considered after
303
abdominal surgery in horses with surgical site infection of the abdominal incision. It was
304
possible to observe, in this study, a negative correlation between incisional infection and
305
pre-, intra-, and postoperative procedures, once they were completely standardized in both
306
experimental phases; the most relevant difference between the experimental phases was the
13
307
first phase peritonitis induction and only after this phase the horses presented surgical site
308
infection.
309
Interestingly, to our knowledge, no previous study has evaluated the interior of the
310
abdomen at time points after laparotomies in horses with septic peritonitis. Subsequent
311
surgical interventions in this study, after 2, 4, and 6 months of peritonitis, allowed
312
evaluation of the interior of the animals’ abdominal cavities, in addition to the scars. In all
313
cases, the presence of adhesions was observed in surgical wounds in contact organs, such as
314
the cecum and colon. All other intestinal segments were anatomically and visually normal.
315
Abdominal infection was confirmed to aid in the formation of adhesions in the
316
intestinal loops adjacent to the surgical wound. This is detrimental to horses because of the
317
complications that may be caused by the adhesions [6,12,27]. However, the adhesions aim
318
to defend the body against external hazard or infection, because intestinal loops adhere to
319
the surgical wound; they act as a mechanical barrier to seal the cavity and prevent
320
evisceration, while favoring scarring of the surgical wound. Moreover, the scar tissue strip
321
that arises from the serosa of the adhered organ provides vascularization for the surgical
322
wound [27]. Importantly, the possibility of small bowel adhesion to the surgical wound,
323
which causes severe abdominal discomfort, was observed in two horses (18%); these were
324
then excluded from the study.
325
Many factors related to the mid-ventral incision can affect healing and the period of
326
convalescence in each horse [1,9,11,14]. During this study, it was possible to confirm that
327
surgical wound healing in horses with peritonitis had occurred by second intention (i.e.,
328
there was spacing between the rectus abdominis muscles, which was filled with scar tissue).
329
When evaluating abdominal scars at 2, 4, and 6 months post-surgical, it was possible to
14
330
observe the formation of thick scars, which gradually became thinner and longer, allowing
331
the formation of incisional hernias in 100% of the experimental animals.
332
It is important to note that edema and tissue granulation contribute to the thickness
333
of the “linea alba” during healing, but do not provide resistance [14]. Peritoneal
334
inflammation, resulting from abdominal infection, promotes the release of inflammatory
335
mediators, which have deleterious effects on the wound environment, delaying healing [8-
336
9,20]. Bacterial activity and local inflammation result in significant tissue weakening,
337
which is associated with endotoxemia—a factor that delays the healing of surgical wounds
338
[10,20]. In addition, because of its fibrous nature and vascular shortage [13,28], healing of
339
the linea alba is relatively slower than healing after paramedian laparotomy or procedures
340
conducted through the flank [10,15,29]. Hence, there might have been a delay in the
341
healing of the linea alba due to abdominal infection; because the weight of the intestinal
342
loops on the surgical wound promoted separation of the sutured edges, this space was filled
343
by elongated scar tissue that was responsible for bulging of the ventral abdominal region.
344
In support of this hypothesis, histopathological evaluation revealed that scarring
345
occurred by second intention [30], and that the rate of formation differed according to the
346
degree of inflammation; this demonstrates that the process of infection directly affected the
347
delay in the healing of surgical wounds. Notably, more intense inflammatory events were
348
associated with shorter healing time, in a manner similar to that of non-homogeneous
349
healing between different regions of the surgical wound (cranial, medial, or caudal); this
350
was observed, depending on the local inflammatory process, in the same animal. Thinner
351
scars and reduction of the ventral abdominal volume were observed proportionally
352
throughout the 2, 4, and 6-month time periods, due to the reduction in edema and tissue
353
granulation, as well as the maturation and remodeling of scar tissue, with residual ventral 15
354
bulging caused by scar elongation. Notably, although it was possible to histologically
355
observe collagen maturation over time, in this study, abdominal scars exhibited an active
356
inflammatory process up to 6 months postoperatively; this demonstrated an important delay
357
in the healing process, in a region where the action of forces and weight support can make
358
such healing challenging [13,28].
359
The evaluation of the tensile strength of scars, although performed in a small
360
number of animals, suggested that equine owners should wait for more than 2 months
361
before returning their animals to athletic activities (i.e., until this resistance equals or
362
exceeds that of the control group). This differs from the report by Chism et al. [14] (2000),
363
in which uninfected ventral abdominal wounds were evaluated in horses. It is known that
364
incised fascia sutured with non-absorbable thread, such as nylon, reach up to 50% of their
365
resistance on the 50th day and 80% on the 100th day [7,15,29]. However, there is no precise
366
information in literature regarding this duration for the healing of infected tissue; only the
367
occurrence of delayed healing is clear. Studies related to tensile strength and temporal
368
morphological changes during healing of the incised linea alba in healthy horses were
369
conducted by Chism et al [14]. (2000). Retrospective studies are the basis for
370
recommendations regarding return to exercise after ventral median celiotomy in horses with
371
peritonitis [9]. In this study, peritonitis induced infection of the surgical wound, and led to
372
healing delay; this resulted in healing by second intention in all experimental animals, with
373
spacing of the rectus abdominis muscles and filling of the space by scar tissue. Moreover,
374
100% of the horses showed incisional hernia, despite remaining at rest in their stalls. It is
375
possible that early athletic return might promote the formation of a more pronounced
376
ventral bulge. In the intervening time, the use of abdominal bandages in the postoperative
16
377
period could reduce the formation of incisional hernias, because they promote better
378
distribution of the weight of the abdominal loops over the surgical wound.
379
This study demonstrated that surgical wound infection may be a symptom of
380
peritonitis in horses recovering from abdominal surgery. Surgical site infection of the
381
abdominal incision results in a second intention healing, along with spacing of the rectus
382
abdominis muscles and filling of the space with scar tissue. We hypothesize that with
383
greater spacing of the rectus abdominis muscles during healing, the incisional hernia will be
384
more pronounced. We suggest remaining 6 months out of work as the recovery time for
385
horses that have identified surgical incisional infection of the abdominal incision.
386
In addition, when scars were evaluated 2 months after surgery, the peritoneal
387
surface was fully healed. Nylon threads were present, encapsulated in the subcutaneous
388
tissue, without proper support of the surgical wound. The drainage of the secretions through
389
the skin revealed continuity in these subcutaneous pockets, demonstrating the need for
390
removal of the nylon threads in order to resolve infection in the surgical wound.
391
However, some limitations should be noted in this study. First, there is not a true
392
control group, but the horses served as their own controls during the experimental second
393
phase after the tissue samples had been harvested for further study. At that point, the
394
incisions were closed but peritonitis was not induced. Second, the use of a nonabsorbable
395
suture, as nylon, and cruciate pattern were not the standardized closure technique utilized
396
most frequently in horses undergoing abdominal surgery, but they might be recommended
397
for surgical incisions classified as potentially contaminated. Third, the small number of
398
horses used in this study makes it difficult to find a significant relationship between tensile
399
strength of scars and the length of time to remain out of work after abdominal surgery for
400
horses that have identified incisional infection. 17
401
We concluded that infection of surgical wounds may be a result of infection of the
402
abdominal cavity in the postoperative period in horses with gastrointestinal disorders.
403
Infected surgical wounds heal by second intention, which favors the spacing of rectus
404
abdominis muscles and the formation of incisional hernia.
405 406
Author’s declaration of interests: No conflict of interest has been declared.
407 408
References
409
[1] Mair TS, Smith LJ. Survival and complication rates in 300 horses undergoing surgical
410
treatment of colic. Part 2: Short-term complications. Equine Vet J 2005; 37:303-309.
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[2] Sapper C; Gerhards H. Examination of peritoneal fluid after diagnostic and therapeutic
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laparotomies in horses. Pferdeheilkunde 2005; 1:20-28.
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[3] Tulleners EP. Complications of abdominocentesis in the horse. J Am Vet Med Assoc
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1983; 182: 232-234.
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[4] White II NA, Desrochers A, McKenzie HC. Diagnosis of gastrointestinal disease, in
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Blinkslager AT, White II NA, Moore J, Mair TS (eds). The Equine Acute Abdomen (ed 4),
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USA, Wiley-Blackwell, 2017, pp 221-310.
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[5] Alves GES. Treatment of experimental peritonitis in horses with the association of
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DMSO, heparin and enrofloxacin: clinical, surgical and pathological study. Doctoral
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Thesis. UFMG Veterinary School, 1997, pp 180.
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[6] Freeman DE. Fifty years of colic surgery. Equine Vet J 2018; 0: 1-13.
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[7] Kobluk CN, Ducharme NG, Lumsden JH, Pascoe PJ, Livesey MA, Hurtig M, Horney,
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FD, Arighi M. Factors affecting incisional complication rates associated with colic surgery
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in horses: 78 cases (1983-1985). J American Vet Med Assoc 1989; 195: 639-642. 18
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[8] Honnas CM, Cohen ND. Risk factors for wound infection following celiotomy in
426
horses. J Am Vet Med Assoc 1997; 210: 78-81.
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[9] Freeman DE, Rötting AK, Inoue OJ. Abdominal closure and complications. Clin Tech
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Equine Pract 2002; 1: 174-187.
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[10] French NP, Smith J, Edwards GB, Proudman CJ. Equine surgical colic: risk factors for
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postoperative complications. Equine Vet J 2002; 34: 444-449.
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[11] Isgren CM, Salem SE, Archer DC, Worsman FCF, Townsend NB. Risk factors for
432
surgical site infection following laparotomy: Effect of season and perioperative variables
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and reporting of bacterial isolates in 287 horses. Equine Vet J 2017; 49:39-44.
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[12] Mair TS, Smith LJ, Sherlock CE. Evidence-Based Gastrointestinal Surgery in Horses.
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Vet Clin North Am Equine Pract 2007; 23: 267-292.
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[13] Trostle SS, Wilson DG, Stone WC, Markel MD. A study of the biochemical properties
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of the adult equine linea alba: relationship of the tissue bite size and suture material to
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breaking strength. Vet Surg 1994; 23: 435-441.
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[14] Chism PN, Latimer FG, Patton CS, Rohrbach BW, Blackford JT. Tissue strength and
440
wound morphology of the equine linea alba after ventral midline celiotomy. Vet Surg 2000;
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29: 145-151.
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[15] Korenkov M, Beckers A, Koebke J, Lefering R, Tiling T, Troidl H. Biomechanical and
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morphological types of the linea alba and its possible role in the pathogenesis of midline
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incisinal hernia. Eur J Surg 2001; 167: 909-914.
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[16] Mendes LCN, Marques LC, Bechara GH, Peiró JR. Experimental peritonitis in horse:
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peritoneal fluid composition. Braz J Vet Anim Sci 1999, 51: 217-221.
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[17] Mendes LCN, Marques LC, Schocken-Iturrino RP, Ávila FA, Malheiros EB.
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Experimenal peritonitis in horses. Hematologicas and biochemistry aspects. Braz J Vet 19
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Anim Sci 2000, 37: 146-152.
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[18] Mair TS, Hillyer MH, Taylor FGR. Peritonitis in adult horses: a review of 21 cases.
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Vet Rec 1990; 126: 567-570.
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[19] Faria EP, Marques JR AP, Alves GES. Cellular and biochemical characteristics of
453
peritoneal fluid of equines submitted to experimental peritonitis. Braz J Vet Anim Sci 1999;
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51: 1-15.
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[20] Werners AH. Treatment of endotoxaemia and septicaemia in the equine patient. J Vet
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Pharmacol Ther 2017; 40: 1-15.
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[21] Trent AM. The peritoneum and peritoneal cavity, in Kobluk CN, Ames TR, Geor RJ
458
(eds). The Horse: Diseases & Clinical Management, Philadelphia, Saunders, 1995, pp. 373-
459
401.
460
[22] Lopes MAF, Dearo ACO, Biondo AW, Godin LFP, Iamaguti P, Thomassian A,
461
Kohayagawa A. Peritoneal fluid exam and hemogram of horses submitted to laparotomy
462
and carboxymethylcellulose intraperitoneal infusion. Ciência Rural 1999; 29: 79-85.
463
[23] Santschi EM, Grindem CB, Tate LP, Corbett WT. Peritoneal fluid analysis in ponies
464
after abdominal surgey. Vet Surg 1988; 17:6-9.
465
[24] Ingle-Fehr JE, Baxter G, Howard R, Trotter G, Stashak T. Bacterial culturing of
466
ventral median celiotomies for prediction of incisional complications in horses. Vet Surg
467
1997; 26: 7-13.
468
[25] Phillips TJ, Walmsley JP. Retrospective analysis of the results of 151 exploratory
469
laparotomies in horses with gastrointestinal disease. Equine Vet J 1993; 25: 427-431.
470
[26] Wilson DA, Baker GJ, Boero MJ. Complications of celiotomy incisions in horses. Vet
471
Surg 1995; 24: 506-514.
472
[27] Maciver AH, McCall M, James Shapiro AM. Intra-abdominal adhesions: cellular 20
473
mechanisms and strategies for prevention. Int J Surg 2011; 9: 589-594.
474
[28] Edwards GB. Cirurgia abdominal, in Hickman J. (ed). Cirurgia y Medicina Equinas.
475
Buenos Aires, Argentina, Hemisferio Sur, 1988, pp 123-216.
476
[29] Chism PN, Latimer FG, Blackford JT, Patton CS, Rohrbach BW. Tissue strength and
477
collagen content of the equine linea alba following ventral midline celiotomy. AAEP
478
Proceedings 1998; 44: 258-259.
479
[30] Theoret CL. Wound repair in the horse: problems and proposed innovative solutions.
480
Clin Tech Equine Pract 2004; 3: 134-140.
481 482
21
483
Figures legends
484
Figure 1. Illustration of the puncture at the cecal apex and cecal contents aspirated with a
485
syringe and needle (A); Cecal contents (1 mL) and blood (1 mL) diluted in 1 L of lactated
486
Ringer’s solution (B); Adhesion of the cecum to the internal portion of the surgical wound
487
(C); subcutaneous surface presenting "bag" formed in the subcutaneous tissue with
488
encapsulated nylon threads (D; arrow); surgical wound infection, with drainage of purulent
489
secretions (E- lateral view and F- ventral view); Bulging of the abdomen, which was related
490
to the presence of an incisional hernia (G- lateral view and H- ventral view).
491
Figure 2. Illustration of the secondary intention healing of the surgical wound. Observe the
492
space between the rectus abdominis muscles developed due to the presence of a scar
493
(arrows). Note that the scars of surgical wound reduced in thickness over time. A and B –
494
control group; C and D – 2-month subgroup (fresh and formalized samples); E and F – 4-
495
month subgroup (fresh and formalized samples); G and H – 6-month subgroup (fresh and
496
formalized samples).
497
Figure 3. Box plot of tensile strength in the control group and in the 2-, 4-, and 6-month
498
subgroups.
499
22
500
Tables
501
Table 1. Medians and interquartile ranges of the thickness of the linea alba (surgical
502
wound) of animals in the control group (CG) and in the 2-, 4-, and 6-month subgroups. CG
503
2-month subgroup 2.85 (2.1 – 3) b
4-month subgroup 1.75 (1.5-1.8) b
0.8 (0.7-0.8) a Thickness (cm) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 1.05 (0.9-1.4) c
504 505
Table 2. Medians and interquartile ranges of the tensile strength (N) in the control group
506
(CG) and in the 2-, 4-, and 6-month subgroups. CG
507
2-month subgroup 341.1 (154-360.3) a
4-month subgroup 421.5 (354-463) ab
331.4 Tensile strength (301.3-405.8) ab (N) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 415.4 (364.2-483) b
508
23
TABLES Table 1. Medians and interquartile ranges of the thickness of the linea alba (surgical wound) of animals in the control group (CG) and in the 2-, 4-, and 6-month subgroups. CG
2-month subgroup 2.85 (2.1 – 3) b
4-month subgroup 1.75 (1.5-1.8) b
0.8 (0.7-0.8) a Thickness (cm) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 1.05 (0.9-1.4) c
Table 2. Medians and interquartile ranges of the tensile strength (N) in the control group (CG) and in the 2-, 4-, and 6-month subgroups. CG
2-month subgroup 341.1 (154-360.3) a
4-month subgroup 421.5 (354-463) ab
331.4 Tensile strength (301.3-405.8) ab (N) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 415.4 (364.2-483) b
•
Surgical site infection may be a symptom of peritonitis.
•
Peritonitis favors the development of wound infection and incisional hernia.
•
Second intention healing of abdominal incision favors incisional hernia formation.
S0737-0806(19)30652-5
DOI:
https://doi.org/10.1016/j.jevs.2019.102903
Reference:
YJEVS 102903
To appear in:
Journal of Equine Veterinary Science
Received Date: 15 March 2019 Revised Date:
18 September 2019
Accepted Date: 21 December 2019
Please cite this article as: Dória RGS, Freitas SH, Laskoski LM, Arruda LP, Shimano AC, Correlation between peritonitis and incisional infections in horses, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/j.jevs.2019.102903. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.
1
Correlation between peritonitis and incisional infections in horses
2
Renata G.S. Dória1* PhD, Silvio H. Freitas1 PhD, Luciane M. Laskoski2 PhD, Laura P.
3
Arruda3 PhD, Antônio C. Shimano4 PhD
4
1
5
University of São Paulo, Rua Duque de Caxias Norte, 225, Jardim Elite, 13.635-900,
6
Pirassununga, São Paulo, Brazil.
7
*Correspondence email: [email protected]
8
2
9
Brazil.
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering,
Center of Biological and Nature Sciences, Federal University of Acre, Rio Branco, Acre,
10
3
11
Grosso, Brazil.
12
4
13
Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Mato Grosso Company of Research, Assistance and Rural Extension, Cáceres, Mato
Department of Biomechanics, Medicine and Rehabilitation of the Musculoskeletal System,
14 15
Abstract
16
Surgical site infection of abdominal incisions is an important complication
17
following laparotomy with increased risk of incisional hernia formation in horses. This
18
study aims to evaluate the healing process of abdominal incisions and correlate peritonitis
19
with the occurrence of surgical site infection and incisional hernias. Nine horses underwent
20
standardized laparotomy, intestinal exploration, and induced septic peritonitis. Standardized
21
relaparotomy was performed two (n=3), four (n=3) and six (n=3) months later to evaluate
22
the abdominal cavity for adhesions and to collect the sutured ventral abdominal wall to
23
evaluate and prepare it for histopathological and tensile strength study. All horses presented
24
endotoxemia, controllable peritonitis, heat and touch-sensitive ventral abdominal edema 1
25
and surgical wound infection with presence of purulent discharge. Adhesion of the cecum
26
or colon to the internal portion of the surgical wound was observed. Healing of the infected
27
surgical wounds occurred by second intention and a space between the rectus abdominis
28
muscles developed due to the presence of a scar, which was related to incisional hernia. In
29
the histopathological evaluation, the collagen content increased, and the inflammation
30
decreased over time. The tensile strength increased over time and was highest after 6
31
months. After the second surgical intervention, there was no infection of the surgical
32
wound in any of the animals and healing by first intention occurred. Surgical site infection
33
may be a symptom of peritonitis in horses recovering from abdominal surgery. Infected
34
surgical wounds heal by second intention, which favors the spacing of rectus abdominis
35
muscle and the formation of incisional hernia.
36
Keywords: abdominal infection, abdominal surgery complications, horses, second
37
intention healing, wound infection.
38 39
Introduction
40
Abdominal disorders are frequently found in equine clinics and may be associated with
41
several complications. Post-operative septic peritonitis can be considered a challenge for
42
veterinary clinics. It is diagnosed based on a combination of clinical signs in association
43
with abnormal peritoneal fluid evaluation and can reach up to 20% of horses undergoing
44
diagnostic and therapeutic laparotomies [1,2]. Among the possible etiologies of peritonitis,
45
fecal contamination is a frequent and serious cause [3-5]. Currently, there is a high rate of
46
survival of horses after colic surgeries; therefore, postoperative complications, such as
47
suppuration of laparotomy wounds and incisional hernia, have become relevant [6]. Despite
48
scientific advances, the prevalence of incisional complications following laparotomies, as 2
49
incisional drainage, dehiscence and hernia formation, may reach 40%, or even 87.5% when
50
reintervention is needed [7,8,9]. Wound infections were recorded in 29 to 36% of horses
51
after intestinal surgery and the rates of wound complications were significantly different in
52
relation to intraperitoneal contamination at surgery (63.6%) compared with no
53
contamination (27.4%) and development of post-operative peritonitis (85.7%) compared
54
with no peritonitis (27.3%) [1,9]. The risk of developing an incisional hernia is
55
significantly associated with the occurrence of incisional suppuration [1,10-11]. Incisional
56
complications associated with median laparotomy in horses are caused by several
57
predisposing factors, including factors inherent to the patient, surgical/anesthesia
58
procedures, and the postoperative period [6-10].
59
Incisional complications may result in extended postoperative care, increased
60
convalescence time, surgical reinterventions for hernia repair, or death. Complications
61
associated with ventral midline incisions include hematoma in the rectus abdominis muscle,
62
edema, fistulae, local infection, dehiscence, and herniation [7,9,12]. Herniation is related to
63
failure of the suture material, aggressive postoperative recovery, surgical wound infection,
64
and uncontrolled exercise in the early postoperative period [8-9,12-13].
65
Most studies on ventral median incisions in horses have focused on healthy horses
66
[6,12-15]. Notably, the surgeon should minimize factors that promote surgical wound
67
infection. However, there have been few reports regarding follow-up of the healing of
68
equine abdominal wounds with peritonitis and the correlation of such healing with the onset
69
of incisional hernias. The objective of this study was to evaluate the healing process of
70
abdominal incisions and correlate peritonitis with the occurrence of surgical site infection
71
and incisional hernias.
72 3
73
Material and methods
74
Twelve healthy, no-defined-breed horses (6-12 years of age; nine females and three males;
75
mean body weight of 350 ± 50 kg), which had no history of previous abdominal surgeries,
76
were included in this study. The horses were divided into two experimental groups:
77
peritonitis group (PG), consisting of nine animals, which underwent laparotomy, intestinal
78
exploration, and induction of septic peritonitis; and the control group (CG), which included
79
three animals that died due to causes not related with septic peritonitis. All horses of the PG
80
were dewormed and kept in a stall for a minimum of 2 weeks before the initiation of the
81
study. They were fed Tifton hay (2% body weight, daily), commercial equine feed (1%
82
body weight, daily), and water ad libitum.
83 84
First experimental phase
85
After 12 hours of fasting, the PG animals received 2% xylazine (0.5 mg·kg-1 IV) as
86
pre-anesthetic medication; after 10 minutes, the animals received guaiacol glyceryl ether
87
(100 mg·kg-1 IV) for myorelaxation. Anesthetic induction was conducted with 10%
88
ketamine (1 mg·kg-1 IV) and midazolam (0.1 mg·kg-1 IV) mixed in the same syringe;
89
anesthesia was maintained with halothane and spontaneous ventilation. The same surgeon
90
performed all surgical procedures. The animals were positioned in dorsal decubitus, and the
91
abdominal ventral region was clipped from the sternum to the groin. The surgical site was
92
prepared for surgery by scrubbing the area with povidone-iodine scrub for 3 minutes,
93
followed by povidone-iodine solution and 70% isopropyl alcohol application. A 20-cm
94
incision was made in the skin, subcutaneous tissue, and linea alba. This was followed by
95
opening of the peritoneum on the falciform ligament, and exploration of the abdominal
96
cavity. Initially, the cecum and pelvic flexure were exposed, followed by the sternal and 4
97
diaphragmatic flexures. Subsequently, the small intestine was exposed; starting from the
98
ileum toward the jejunum; the duodenum and stomach were palpated. The small intestine
99
contents were conducted to drainage into the cecum. Thereafter, the small intestine was
100
returned to the abdominal cavity and the small colon was exposed, enabling palpation of
101
the rectum and transverse colon. Additionally, the nephro-splenic ligament and the ventral
102
and right dorsal colon were palpated. After this procedure, the pelvic flexure was
103
repositioned within the abdominal cavity. At this stage, septic peritonitis was induced in
104
accordance with a modified surgical model standardized by Alves [5] (1997): 1 mL cecal
105
intestinal contents (cecal fluid with diluted vegetable matter, obtained via puncture at the
106
cecal apex) were aspirated with a syringe and needle (40 × 16) and 1 mL blood was
107
collected with syringe and needle (40 × 12) from the jugular vein. Both aspirates were
108
diluted in 1 L of lactated Ringer’s solution (1 L solution comprising lactated Ringer’s
109
solution, blood, and cecal contents). After repositioning the cecum, the abdominal cavity
110
was washed with this prepared solution (Figure 1A,B). The linea alba was sutured,
111
involving the peritoneum, with the cruciate suture pattern; this ensured standardized
112
spacing of 1.5 cm from the edge of the incision and 1.5 cm between each point, using a
113
synthetic non-absorbable thread (nylon, 0.60 cm). The subcutaneous tissue was
114
approximated with continuous horizontal mattress pattern and synthetic absorbable thread
115
(polyglycolic acid, number 1); the skin was approximated with interrupted horizontal
116
mattress suture and synthetic non-absorbable thread (nylon, 0.60 cm). The duration of the
117
complete surgical procedure was standardized to less than 1 hour. All animals recovered
118
from anesthesia and received benzathine penicillin (40,000 IU/kg, IM, every 48 h, three
119
applications), gentamicin (6.6 mg/kg, IV, once per day, for 5 days), and flunixin
5
120
meglumine (1.1 mg/kg, once per day, for 3 days). Every 12 hours, the animals underwent a
121
general physical examination and were monitored for signs of abdominal pain, peritonitis,
122
laminitis, and ileus. The animals were kept in stalls, without changes in food or water
123
management. Cleaning sutures were performed twice a day with povidone-iodine solution
124
during 10 days.
125 126
Second experimental phase
127
At 2 (2-month subgroup, n = 3), 4 (4-month subgroup, n = 3), and 6 months (6-
128
month subgroup, n = 3) after the surgical procedure, with the animals under general
129
inhalation anesthesia (using the anesthetic protocol described above) and positioned in
130
dorsal decubitus, the skin scar was opened; a blunt dissection of the subcutaneous tissue
131
was performed to expose the fascial-muscle plane and the previously constructed suture
132
line. At this stage, the sutured ventral abdominal wall (20 cm long × 8 cm wide) was
133
removed in preparation for the study. Two-centimeter samples were collected from the
134
cranial, middle, and caudal portions of the scar, and then stored in 10% formaldehyde for
135
histopathological evaluation; the remaining tissue was immediately frozen at -70ºC for a
136
subsequent tensile strength test. Abdominal cavity synthesis was conducted by repeating
137
exactly the previously described technique; the animals recovered from anesthesia,
138
underwent the same post-surgical therapy and cleaning sutures described above, and
139
returned to their routine activities when fully recovered.
140 141
Control Group
6
142
Three horses that did not undergo surgical interventions and died of other causes not
143
related to septic peritonitis were used in the CG; the ventral abdominal musculature (20 cm
144
× 8 cm, with the linea alba in the center) of these horses was collected and subjected to
145
histopathological evaluation and tensile strength analysis.
146 147
Laboratorial evaluations
148
For each horse in the PG group, blood and peritoneal fluid samples were collected
149
immediately before surgery, at 24 hours and 48 hours post-surgery, and at every 48 hours
150
on subsequent days until the 10th postoperative day. Hematologic and peritoneal fluid
151
evaluation was conducted; this comprised blood counts, total plasma protein and plasma
152
fibrinogen measurement for blood samples and visual examination, and evaluation of
153
density, total protein content, red blood cell count, leukocyte global count, and differential
154
leukocyte count for abdominal fluid samples.
155
Histological sections were prepared and evaluated with respect to collagen content
156
and maturity by using hematoxylin-eosin (H&E) staining. Tensile strength was evaluated
157
with a breaking strength test for the abdominal wall by using an electromechanical drive
158
tensiometer connected to a microcomputer. The speed of the break test calibrated in the
159
apparatus was 30 mm/min. Segments of the abdominal wall were attached to aluminum
160
claws parallel to the suture line (the threads were not removed); those claws were
161
connected to the apparatus, which exerted tensile force perpendicular to the suture. Values
162
were presented in Newtons (N).
163
The experimental data underwent analysis of the normality of residues by using the
164
Shapiro-Wilk test, followed by analysis of variance and Tukey's mean test. The non-
7
165
parametric Kruskal-Wallis test was used for data that did not exhibit a normal distribution,
166
followed by Dunn's mean rank test. A significance level of 5% was used in all analyses.
167 168
Results
169
All horses recovered from anesthesia without complications. In the immediate
170
postoperative period, the animals exhibited controllable peritonitis and endotoxemia, with
171
apathy, hyperthermia (T > 38.5ºC), increased heart and respiratory rates, reduction or
172
absence of intestinal motility, reddened mucous membranes with a thin purple line above
173
the teeth (toxic line), reluctance to move, splinting of the abdominal wall and sensitivity to
174
external abdominal pressure. Horses maintained this state for approximately 72 hours, after
175
which hyperthermia, tachycardia and tachypnea ended, intestinal motility returned, and the
176
other endotoxemia signs disappeared. Furthermore, surgical site infection was successfully
177
induced in all PG animals: heat and touch-sensitive ventral abdominal edema around the
178
skin incision and wound infection were observed between the 5th and 7th day post-surgery,
179
with the presence of purulent discharge (Figure 1E,F).
180
Two animals showed abdominal discomfort: one animal at 7 days post-surgery (2-
181
month subgroup); and another animal at 10 days post-surgery (6-month subgroup). Both
182
animals demonstrated an accumulation of gas and compaction of contents in the digestive
183
tract. After symptomatic clinical treatment, both animals exhibited symptom reversal and
184
remained in their respective experimental subgroups. Two other horses exhibited intense
185
pain and entero-gastric reflux at 8 to 10 days post-surgery, such that they underwent a
186
separate exploratory celiotomy; on observation of jejunal adhesion to the surgical wound,
187
these animals were excluded from the experimental groups.
8
188
Peritoneal fluid collection was possible only in the pre-surgical period, as well as at
189
24 and 48 hours after surgery. At other timepoints, it was not possible to collect peritoneal
190
fluid because a high concentration of fibrin was observed in the samples, beginning at 48
191
hours. Important changes were observed in the post-surgical fluid collections. These
192
became more pronounced after 24 hours: changes in color from light yellow to amber, total
193
protein content increased from 1.72 g/dL ± 1.18 g/dL to 4.41 g/dL ± 1.89 g/d, red blood cell
194
count increased from 592.14/µL ± 437.58/µL to 61,843.88/µL ± 64,668.85/µL, and the
195
leukocyte count increased from 1,088.80/µL ± 1,257.75/µL to 50,773/µL ± 76,615.79/µL,
196
and polymorphonuclear cells predominated, with degenerate neutrophils. These changes
197
indicated the presence of postoperative inflammatory reaction in all PG horses [16].
198
In the hematological analysis, a progressive elevation was observed in the evaluated
199
parameters over the time. These became more pronounced at 10 days post-surgery: total
200
leukocyte count increased from 9,140/µL ± 1,554.35/µL to 19,966.67/µL ± 5,153.51/µL,
201
neutrophil count increased from 5,721.90/µL ± 1,064.79/µL to 15,055.17/µL ±
202
5,678.76/µL, with toxic neutrophils and fibrinogen level increased from 366.67 mg/dL ±
203
150.55 mg/dL to 600 mg/dL ± 141.42 mg/dL. These changes were indicative of the
204
hematological profile of horses with peritonitis and surgical wound infection in the post-
205
surgical period [17].
206
All experimental animals in the PG recovered from septic peritonitis and wound
207
infection; skin sutures had healed but some points for drainage of purulent secretions
208
persisted during the first phase of the experimental period.
209
After 2 (2-month subgroup), 4 (4-month subgroup), and 6 (6-month subgroup)
210
months, the second experimental phase was undertaken: the collection of surgical scars for
211
histological evaluation and tensile strength analysis. In this second experimental phase, it 9
212
was possible to observe the events inside the abdominal cavity after exploratory laparotomy
213
in cases of septic peritonitis. In all horses, adhesion of the cecum or colon to the internal
214
portion of the surgical wound effectively sealed the abdominal cavity opening and
215
vascularized the surgical wound (Figure 1C). Neovascularization was observed from the
216
intestinal serosa, which extended through the fibrous scar tissue of adhesion, between the
217
intestinal loop and the surgical wound; this presumably supported the wound healing.
218
Anatomical preservation was noted in all cases without interference with the positioning of
219
other intestinal loops in the abdominal cavity.
220
At 2 months post-surgery, nylon threads that had been positioned in the linea alba
221
were no longer in place; these had been expelled into the subcutaneous tissue and the linea
222
alba was completely healed. Surgical wounds exhibited purulent secretions drained by
223
small sinuses in the skin. These drainage points on the skin led directly to the subcutaneous
224
tissue, where "abscesses" had formed; notably, these abscesses encapsulated the nylon
225
threads that had been expelled from the linea alba (Figure 1D).
226
Infected surgical wounds healing occurred by second intention and a space between
227
the rectus abdominis muscles developed due to the presence of a scar (Figure 2). The linea
228
alba in CG animals was 0.3 cm wide, without variation; in PG animals, the distance
229
between the rectus abdominis muscles, filled with scar tissue, ranged from 2 to 8 cm
230
(median, 4 cm; interquartile range, 3.5 to 5.75 cm), independent of the experimental
231
subgroup. This spacing between the rectus abdominis muscles was likely responsible for
232
the bulging of the abdomen observed in all PG animals, which was related to the presence
233
of an incisional hernia (Figure 1G,H).
234
At 2 months post-surgery, the scars of surgical wounds were thicker (median, 2.85
235
cm; interquartile range, 2.1 to 3 cm) than the linea alba of CG animals (median, 0.8 cm; 10
236
interquartile range, 0.7 to 0.8 cm) and reduced in thickness over time (figure 2); thus, the
237
scars of animals in the 4-month subgroup (median, 1.75 cm; interquartile range, 1.5 to 1.8
238
cm) were thicker than those of animals in the 6-month subgroup (median, 1.05 cm;
239
interquartile range, 0.9 to 1.4 cm) and thinner than those of animals in the 2-month
240
subgroup (Table 1).
241
The tensile strength increased with time with the highest tensile strength observed at
242
6 months post-surgery (median, 415.4 N), compared with 2 months post-surgery (median,
243
341.1 N); this was the sole statistically significant difference observed in this measurement
244
(Table 2 and Figure 3). Notably, when there was a rupture during this test, the rupture sites
245
were lateral to the scars at the point of insertion, next to the rectus abdominis muscle.
246
In the histopathological evaluation, the collagen content, visualized using H&E
247
staining, showed differences between horses in the CG and horses evaluated at 2, 4, and 6
248
months post-surgery. It was possible to observe a lower amount of collagen fibers in horses
249
evaluated at 2 months, compared with controls and with animals evaluated at 4 and 6
250
months; notably, these fibers were disorganized. This variation was also observed in the
251
same animal (in the same individual), with differences among analyzed regions (cranial,
252
medial, or caudal portion of the surgical wound). Some sites exhibited more orderly
253
arrangement of the collagen fibers; in other areas, there was a larger quantity of
254
extracellular matrix and smaller quantity of collagen. This was related to a larger quantity
255
of fibroblasts, as well as increased inflammation and vascularization. With the increased
256
length of time, such as in animals evaluated at 4 and 6 months, there was a progressive
257
reduction in the number of fibroblasts, blood vessels, and inflammatory infiltrates;
258
moreover, there was an increase in the quantity of collagen and the extent of remodeling. In
259
the 6-month animals, there were areas with larger quantities of collagen fibers, which were 11
260
denser and exhibited greater reinforcement, compared with those same areas in control
261
animals. Overall inflammatory infiltration ranged from mixed to mononuclear,
262
predominantly multifocal, and perivascular, and was present around the suture. This
263
inflammation was intense-to-moderate in the 2-month subgroup, moderate-to-light in the 4-
264
month subgroup, and discreet in the 6-month subgroup.
265
The second surgical intervention involved substantial divulsion of the subcutaneous
266
tissue, muscle bleeding and extensive tissue removal; subsequently, there was no infection
267
of the surgical wound in any of the experimental animals, and healing occurred by first
268
intention.
269 270
After the two experimental phases were completed, all animals showed full postoperative recovery and were donated to rural owners.
271 272
Discussion
273
Septic peritonitis in horses is one of the most serious complications of colic, especially in
274
cases requiring surgical intervention. Incisional complications observed following
275
laparotomy results in the delay of surgical wound healing and increases convalescence
276
period, duration of hospitalization, costs of treatment, and it may be fatal [6,9,12,18-20].
277
In this study, the induction of septic peritonitis was performed in accordance with a
278
modified version of the surgical model standardized by Alves [5] (1997). This protocol,
279
which involved the aspiration of 1 mL of blood and 1 mL of cecal contents, both diluted in
280
1 L of lactated Ringer’s solution placed inside the abdominal cavity of healthy horses,
281
proved informative, because there were changes in clinical, hematological, and peritoneal
282
fluid levels; these were consistent with the onset of septic peritonitis [16,19-22], in nine
283
experimental horses but did not result in fatality. Although the abdominal fluid parameters, 12
284
specially leukocyte counts, shown by the model of peritonitis induction in this study should
285
be compared to the leukocyte counts described in ponies after abdominal exploratory
286
surgery, the association of serial sampling of both peritoneal fluid and peripheral blood, and
287
physical examinations were mandatory in order to distinguish the presence of septic
288
peritonitis [2,23]. In this study, the animals showed consistent clinical signs of post-
289
operative endotoxemia and peritonitis, cytologic examination of peritoneal fluid
290
demonstrating degenerative cell changes and peripheral blood with neutrophilia and toxic
291
neutrophils, characterizing systemic and local responses to the inflammatory peritoneal
292
reaction caused by the presence of fecal content.
293
Importantly, using this protocol for peritonitis induction, it was possible to promote
294
the infection of surgical wounds in all experimental horses. Conversely, the removal of a
295
20-cm × 8-cm segment of the abdominal scar from the same horses resulted in healing by
296
primary intention, without any complications. Numerous factors are associated with
297
surgical wound infection in horses. These include the patient, surgical technique, surgical
298
time, incision size, and postoperative management [6-7,9,12,24]. In contrast, in cases of
299
equine colic, the most common causes of surgical wound contamination are endogenous
300
sources. Enterotomy and enterectomy are significantly associated with wound infection,
301
increasing the incidence of incisional hernia by up to 16% [7,12-13,24-26]. The results of
302
this study indicate that peritonitis may be an important factor to be considered after
303
abdominal surgery in horses with surgical site infection of the abdominal incision. It was
304
possible to observe, in this study, a negative correlation between incisional infection and
305
pre-, intra-, and postoperative procedures, once they were completely standardized in both
306
experimental phases; the most relevant difference between the experimental phases was the
13
307
first phase peritonitis induction and only after this phase the horses presented surgical site
308
infection.
309
Interestingly, to our knowledge, no previous study has evaluated the interior of the
310
abdomen at time points after laparotomies in horses with septic peritonitis. Subsequent
311
surgical interventions in this study, after 2, 4, and 6 months of peritonitis, allowed
312
evaluation of the interior of the animals’ abdominal cavities, in addition to the scars. In all
313
cases, the presence of adhesions was observed in surgical wounds in contact organs, such as
314
the cecum and colon. All other intestinal segments were anatomically and visually normal.
315
Abdominal infection was confirmed to aid in the formation of adhesions in the
316
intestinal loops adjacent to the surgical wound. This is detrimental to horses because of the
317
complications that may be caused by the adhesions [6,12,27]. However, the adhesions aim
318
to defend the body against external hazard or infection, because intestinal loops adhere to
319
the surgical wound; they act as a mechanical barrier to seal the cavity and prevent
320
evisceration, while favoring scarring of the surgical wound. Moreover, the scar tissue strip
321
that arises from the serosa of the adhered organ provides vascularization for the surgical
322
wound [27]. Importantly, the possibility of small bowel adhesion to the surgical wound,
323
which causes severe abdominal discomfort, was observed in two horses (18%); these were
324
then excluded from the study.
325
Many factors related to the mid-ventral incision can affect healing and the period of
326
convalescence in each horse [1,9,11,14]. During this study, it was possible to confirm that
327
surgical wound healing in horses with peritonitis had occurred by second intention (i.e.,
328
there was spacing between the rectus abdominis muscles, which was filled with scar tissue).
329
When evaluating abdominal scars at 2, 4, and 6 months post-surgical, it was possible to
14
330
observe the formation of thick scars, which gradually became thinner and longer, allowing
331
the formation of incisional hernias in 100% of the experimental animals.
332
It is important to note that edema and tissue granulation contribute to the thickness
333
of the “linea alba” during healing, but do not provide resistance [14]. Peritoneal
334
inflammation, resulting from abdominal infection, promotes the release of inflammatory
335
mediators, which have deleterious effects on the wound environment, delaying healing [8-
336
9,20]. Bacterial activity and local inflammation result in significant tissue weakening,
337
which is associated with endotoxemia—a factor that delays the healing of surgical wounds
338
[10,20]. In addition, because of its fibrous nature and vascular shortage [13,28], healing of
339
the linea alba is relatively slower than healing after paramedian laparotomy or procedures
340
conducted through the flank [10,15,29]. Hence, there might have been a delay in the
341
healing of the linea alba due to abdominal infection; because the weight of the intestinal
342
loops on the surgical wound promoted separation of the sutured edges, this space was filled
343
by elongated scar tissue that was responsible for bulging of the ventral abdominal region.
344
In support of this hypothesis, histopathological evaluation revealed that scarring
345
occurred by second intention [30], and that the rate of formation differed according to the
346
degree of inflammation; this demonstrates that the process of infection directly affected the
347
delay in the healing of surgical wounds. Notably, more intense inflammatory events were
348
associated with shorter healing time, in a manner similar to that of non-homogeneous
349
healing between different regions of the surgical wound (cranial, medial, or caudal); this
350
was observed, depending on the local inflammatory process, in the same animal. Thinner
351
scars and reduction of the ventral abdominal volume were observed proportionally
352
throughout the 2, 4, and 6-month time periods, due to the reduction in edema and tissue
353
granulation, as well as the maturation and remodeling of scar tissue, with residual ventral 15
354
bulging caused by scar elongation. Notably, although it was possible to histologically
355
observe collagen maturation over time, in this study, abdominal scars exhibited an active
356
inflammatory process up to 6 months postoperatively; this demonstrated an important delay
357
in the healing process, in a region where the action of forces and weight support can make
358
such healing challenging [13,28].
359
The evaluation of the tensile strength of scars, although performed in a small
360
number of animals, suggested that equine owners should wait for more than 2 months
361
before returning their animals to athletic activities (i.e., until this resistance equals or
362
exceeds that of the control group). This differs from the report by Chism et al. [14] (2000),
363
in which uninfected ventral abdominal wounds were evaluated in horses. It is known that
364
incised fascia sutured with non-absorbable thread, such as nylon, reach up to 50% of their
365
resistance on the 50th day and 80% on the 100th day [7,15,29]. However, there is no precise
366
information in literature regarding this duration for the healing of infected tissue; only the
367
occurrence of delayed healing is clear. Studies related to tensile strength and temporal
368
morphological changes during healing of the incised linea alba in healthy horses were
369
conducted by Chism et al [14]. (2000). Retrospective studies are the basis for
370
recommendations regarding return to exercise after ventral median celiotomy in horses with
371
peritonitis [9]. In this study, peritonitis induced infection of the surgical wound, and led to
372
healing delay; this resulted in healing by second intention in all experimental animals, with
373
spacing of the rectus abdominis muscles and filling of the space by scar tissue. Moreover,
374
100% of the horses showed incisional hernia, despite remaining at rest in their stalls. It is
375
possible that early athletic return might promote the formation of a more pronounced
376
ventral bulge. In the intervening time, the use of abdominal bandages in the postoperative
16
377
period could reduce the formation of incisional hernias, because they promote better
378
distribution of the weight of the abdominal loops over the surgical wound.
379
This study demonstrated that surgical wound infection may be a symptom of
380
peritonitis in horses recovering from abdominal surgery. Surgical site infection of the
381
abdominal incision results in a second intention healing, along with spacing of the rectus
382
abdominis muscles and filling of the space with scar tissue. We hypothesize that with
383
greater spacing of the rectus abdominis muscles during healing, the incisional hernia will be
384
more pronounced. We suggest remaining 6 months out of work as the recovery time for
385
horses that have identified surgical incisional infection of the abdominal incision.
386
In addition, when scars were evaluated 2 months after surgery, the peritoneal
387
surface was fully healed. Nylon threads were present, encapsulated in the subcutaneous
388
tissue, without proper support of the surgical wound. The drainage of the secretions through
389
the skin revealed continuity in these subcutaneous pockets, demonstrating the need for
390
removal of the nylon threads in order to resolve infection in the surgical wound.
391
However, some limitations should be noted in this study. First, there is not a true
392
control group, but the horses served as their own controls during the experimental second
393
phase after the tissue samples had been harvested for further study. At that point, the
394
incisions were closed but peritonitis was not induced. Second, the use of a nonabsorbable
395
suture, as nylon, and cruciate pattern were not the standardized closure technique utilized
396
most frequently in horses undergoing abdominal surgery, but they might be recommended
397
for surgical incisions classified as potentially contaminated. Third, the small number of
398
horses used in this study makes it difficult to find a significant relationship between tensile
399
strength of scars and the length of time to remain out of work after abdominal surgery for
400
horses that have identified incisional infection. 17
401
We concluded that infection of surgical wounds may be a result of infection of the
402
abdominal cavity in the postoperative period in horses with gastrointestinal disorders.
403
Infected surgical wounds heal by second intention, which favors the spacing of rectus
404
abdominis muscles and the formation of incisional hernia.
405 406
Author’s declaration of interests: No conflict of interest has been declared.
407 408
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409
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410
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DMSO, heparin and enrofloxacin: clinical, surgical and pathological study. Doctoral
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wound morphology of the equine linea alba after ventral midline celiotomy. Vet Surg 2000;
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29: 145-151.
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[15] Korenkov M, Beckers A, Koebke J, Lefering R, Tiling T, Troidl H. Biomechanical and
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incisinal hernia. Eur J Surg 2001; 167: 909-914.
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Experimenal peritonitis in horses. Hematologicas and biochemistry aspects. Braz J Vet 19
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Kohayagawa A. Peritoneal fluid exam and hemogram of horses submitted to laparotomy
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[29] Chism PN, Latimer FG, Blackford JT, Patton CS, Rohrbach BW. Tissue strength and
477
collagen content of the equine linea alba following ventral midline celiotomy. AAEP
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479
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480
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481 482
21
483
Figures legends
484
Figure 1. Illustration of the puncture at the cecal apex and cecal contents aspirated with a
485
syringe and needle (A); Cecal contents (1 mL) and blood (1 mL) diluted in 1 L of lactated
486
Ringer’s solution (B); Adhesion of the cecum to the internal portion of the surgical wound
487
(C); subcutaneous surface presenting "bag" formed in the subcutaneous tissue with
488
encapsulated nylon threads (D; arrow); surgical wound infection, with drainage of purulent
489
secretions (E- lateral view and F- ventral view); Bulging of the abdomen, which was related
490
to the presence of an incisional hernia (G- lateral view and H- ventral view).
491
Figure 2. Illustration of the secondary intention healing of the surgical wound. Observe the
492
space between the rectus abdominis muscles developed due to the presence of a scar
493
(arrows). Note that the scars of surgical wound reduced in thickness over time. A and B –
494
control group; C and D – 2-month subgroup (fresh and formalized samples); E and F – 4-
495
month subgroup (fresh and formalized samples); G and H – 6-month subgroup (fresh and
496
formalized samples).
497
Figure 3. Box plot of tensile strength in the control group and in the 2-, 4-, and 6-month
498
subgroups.
499
22
500
Tables
501
Table 1. Medians and interquartile ranges of the thickness of the linea alba (surgical
502
wound) of animals in the control group (CG) and in the 2-, 4-, and 6-month subgroups. CG
503
2-month subgroup 2.85 (2.1 – 3) b
4-month subgroup 1.75 (1.5-1.8) b
0.8 (0.7-0.8) a Thickness (cm) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 1.05 (0.9-1.4) c
504 505
Table 2. Medians and interquartile ranges of the tensile strength (N) in the control group
506
(CG) and in the 2-, 4-, and 6-month subgroups. CG
507
2-month subgroup 341.1 (154-360.3) a
4-month subgroup 421.5 (354-463) ab
331.4 Tensile strength (301.3-405.8) ab (N) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 415.4 (364.2-483) b
508
23
TABLES Table 1. Medians and interquartile ranges of the thickness of the linea alba (surgical wound) of animals in the control group (CG) and in the 2-, 4-, and 6-month subgroups. CG
2-month subgroup 2.85 (2.1 – 3) b
4-month subgroup 1.75 (1.5-1.8) b
0.8 (0.7-0.8) a Thickness (cm) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 1.05 (0.9-1.4) c
Table 2. Medians and interquartile ranges of the tensile strength (N) in the control group (CG) and in the 2-, 4-, and 6-month subgroups. CG
2-month subgroup 341.1 (154-360.3) a
4-month subgroup 421.5 (354-463) ab
331.4 Tensile strength (301.3-405.8) ab (N) Different letters represent a statistically significant difference; p<0.05.
6-month subgroup 415.4 (364.2-483) b
•
Surgical site infection may be a symptom of peritonitis.
•
Peritonitis favors the development of wound infection and incisional hernia.
•
Second intention healing of abdominal incision favors incisional hernia formation.