Effects of flunixin meglumine, diclofenac sodium and metamizole

Effects of flunixin meglumine, diclofenac
sodium and metamizole sodium on experimental wound healing in rats
S. ERPEK1*, N. KILIC2, D. KOZACI3, E. DIKICIOGLU4 and T. KAVAK3
1
2
3
4
Department of Histology and Embryology, Faculty of Medicine, Adnan Menderes University, Aydin, Turkey.
Department of Surgery, Veterinary Faculty, Adnan Menderes University, Aydin, Turkey.
Department of Biochemisty, Faculty of Medicine, Adnan Menderes University, Aydin, Turkey.
Department of Pathology, Faculty of Medicine, Adnan Menderes University, Aydin, Turkey.
E-mail : [email protected]
SUMMARY
RÉSUMÉ
The aim of the study was to examine the effect of various nonsteroidal
anti-inflammatory drugs (NSAIDs) on the healing of excisional wounding
in the skin of rats. Twenty rats were equally divided among four groups.
After the excisional wounding, all animals received various NSAIDs for 14
days, except control group. Repair of full-thickness excisions was followed
up for 14 days and re-epithelialization, neovascularization, inflammatory
cell infiltration of the wound bed, collagen deposition and percent contraction of original wound size have been evaluated as the determinants of the
healing process. The percent wound closure of diclofenac and flunixin
meglumin treated animals was found to be significantly less compared to
control group animals. Histological findings demonstrated a relative delay
of healing tissue in NSAID treated animals except metamizole group.
Neovascularization was significantly less in the granulation tissue of diclofenac and flunixin meglumin treated groups on day 7 postwounding, than
that of the controls. The mean tissue hydroxyproline contents on days 0, 7
and 14 were not statistically significant among the study groups. NSAIDs
used in this animal model of wound healing caused a slight deficiency in the
healing process by decreasing wound contraction, and angiogenesis which
are two crucial factors in tissue integrity.
Effets de la flunixine méglumine, du diclofenac de sodium et du metamizole sodique sur la cicatrisation d’une blessure cutanée chez les rats.
Par S. ERPEK, N. KILIC, D. KOZACI, E. DIKICIOGLU et T. KAVAK.
Keywords : nonsteroidal anti-inflammatory drugs wound healing.
L’objectif de ce travail était d’évaluer l’effet des 3 anti-inflammatoires
non stéroïdiens (AINS) : la flunixine méglumine, le diclofénac de sodium et
le métamizole sodique, sur la cicatrisation de plaies cutanées chez le rat.
Quatre groupes de cinq rats ont été constitués. Les rats de trois groupes ont
été traités pendant 14 jours après l’excision cutanée avec un AINS, le quatrième groupe étant le groupe contrôle. La cicatrisation a été suivie pendant
14 jours et appréciée grâce aux marqueurs suivants : re-épithélisation, néovascularisation, infiltration cellulaire et diminution de la taille de la blessure. Par rapport au groupe contrôle, l’importance de la fermeture de la plaie
est significativement moins importante dans les groupes “diclofénac de
sodium” et “flunixine méglumine”. Les résultats histologiques ont mis en
évidence un retard dans le développement du tissu cicatriciel chez les animaux traités avec le diclofénac de sodium ou la flunixine méglumine mais
pas avec le métamizole sodique. A J7 après l’excision, la néovascularisation
du tissu granuleux est, par rapport au groupe contrôle, significativement
moins importante chez les rats traités avec le diclofénac de sodium et la flunixine méglumine. La quantité moyenne d’hydroxyproline tissulaire aux
jours 0, 7 et 14 n’était pas significativement différente dans les différents
groupes. Les AINS utilisés dans ce modèle animal ont entraîné une légère
perturbation de la cicatrisation en diminuant deux facteurs cruciaux : la
diminution de la taille de la blessure et l’angiogenèse.
Mots-clés : anti-inflammatoires non steroidiens - cicatrisation.
Introduction
The healing process in full-thickness skin wounds includes
coagulation, inflammation, tissue formation, and tissue
remodeling [20]. In such wounds there is an interruption of
tissue continuity with destruction of all the epidermal cell
layers and of mesenchymal tissue, including vascular elements. Initially, skin wounds are filled with blood and a
fibrin clot. The fibrin clot is the first protection against infection [21]. In addition to providing hemostasis, the fibrin clot
acts as a matrix for colonization by inflammatory cells which
are attracted to the wound site via chemotaxis. Inflammation,
the second phase of wound healing, occurs 1-5 days after
injury. Migrating inflammatory cells accumulate in the healing wound with neutrophils predominating in the early
hours of inflammation. Neutrophils help decontaminate the
wound through phagocytosis of bacteria and foreign bodies.
By the third day, macrophages outnumber neutrophils [20].
Macrophages engulf and phagocytose wound debris, cleaRevue Méd. Vét., 2006, 157, 4, 185-192
ring the way for the growth of new dermal matrix.
Subsequently, macrophages produce angiogenic and fibrogenic growth factors that promote new tissue formation at
the wound site [7].
Several studies implicate mast cells to as regulators of
wound healing. The capability of mast cells to release proinflammatory mediators, as well as factors that influence cell
proliferation and angiogenesis, and the accumulation of mast
cells in healing wounds, have led to the assumption that mast
cells are critical to the repair of injured tissue [11, 15, 43].
Re-epithelialization begins at the wound edges as early as 24
hours post injury, and granulation of the wound starts around
post-injury day 5 to re-establish the integrity of the epidermis and dermis at the wound site. Granulation tissue is formed as macrophages, fibroblasts, and endothelial cells move
into the wound space. Fibroblasts construct a new extracellular matrix to support cell growth while the newly formed
blood vessels meet metabolic needs for the formation and
maintenance of tissue. The phase of tissue remodeling
186
occurs as early as day 3 and may require months to be completed. Although new collagen continues to be deposited, net
resorption occurs due to increased degradation of old collagen by collagenases. As macrophages begin to disappear,
angiogenesis and fibroblast proliferation decrease [20].
As inflammation is central to the tissue repair process, a
relationship between wound healing and anti-inflammatory
drugs might be expected. Nonsteroidal anti-inflammatory
drugs (NSAIDs) are among the most widely used pharmaceuticals in humans and animals. The anti-inflammatory
effect of NSAIDs is thought to be mainly due to the
cyclooxygenases (COXs) [44]. COX proteins are the first in
a cascade of enzymes that convert arachidonic acid into
prostaglandins (PGs) and thromboxane (Tx), which are
involved in inflammation, cancer, and embryonic development [45]. NSAIDs such as aspirin, indomethacin, naproxen,
diclofenac, metamizole, and flunixin inhibit both COX-1
and COX-2 nonselectively and are widely used for the treatment of various inflammatory and noninflammatory conditions such as arthritis, cardiovascular diseases, and the management of postsurgical and post-traumatic pain in human and
animals [3, 4 , 23].
To investigate the role of various NSAIDs in wound repair,
we have used the established model of acute excisional
wounding in the skin of rats. Repair of full-thickness excisions was followed up for 14 day and re-epithelialization,
neovascularization, inflammatory cell infiltration of the
wound bed, collagen deposition and percent contraction of
original wound size have been evaluated as determinants of
the healing process, using macroscopical, biochemical, and
histological techniques.
Materials and Methods
ANIMALS
Twenty male Sprague-Dawley albino rats weighing 200250 g were used. All experimental protocols using animals
were approved by the Institutional Animal Care and Use
Committee at Adnan Menderes University Experimental
Research Laboratory. All rats were housed under standard
laboratory conditions (temperature : 23 ± 2 °C, 12 h light and
dark cycles). They had free access to standard laboratory
feed and water ad libitum.
EXPERIMENTAL DESIGN
The animals were randomly assigned into four equal
groups (5 rats/group). After the excisional wounding, all animals received various NSAIDs for 14 days, except the
control group. Group I served as controls, received physiologic saline (0.1 ml/100g body weight) per day subcutaneously. Group II received diclofenac sodium (Novartis,
Istanbul) 2.5 mg/kg [30], group III received metamizole
sodium (Aventis, Istanbul) 50 mg/kg [39], and group IV
received flunixin meglumine (Sanofi Dogu Ilac A.S.,
Istanbul) 2.5 mg/kg [42] per day subcutaneously on the back.
The rats were treated daily for up to14 days with drugs, and
euthanized at the end of the experiment.
ERPEK (S.) AND COLLABORATORS
EXCISIONAL SKIN WOUNDING
Rats anesthetized by ether inhalation, the hair of the dorsal
skin was shaved and cleaned with povidone-iodine and dried
with sterile towels. In the shaved area two circular, fullthickness excisional dermal wounds, 2 cm in diameter, was
made extending through epidermis and dermis to the level of
subcutaneous fat. Skin samples were collected for biochemical analysis. Skin wounds were placed on the opposite sides
of midline at the scapula level. No postoperative antibiotic
was used. The wounds and general health of the animals
were monitored daily for up to 2 week postsurgery. No complications were encountered. The percent wound closure was
recorded on day 4, 7, 11 and 14 and the wounds were photographed to measure the wound surface area. The actual value
was converted into percent value taking the size of the
wound at the time of wounding as 100% [37]. One biopsy
comprising the total wound area and 3 mm of non wounded
skin on each side was collected from each animal, bisected in
the center, and prepared for histochemistry and biochemistry
at days 7, and 14 after injury.
HISTOCHEMISTRY
For histological examination the samples were fixed in
10% neutral-buffered formalin and embedded in paraffin
blocks. Serial sections 5 µm in thickness were taken from
each block. Two adjacent paraffin sections were stained with
haematoxylin-eosin for routine histological examination.
Another set of two consecutive sections was stained by
Masson’s trichrome to identify collagen fibers. The last set
of two consecutive sections was stained by Toluidin blue for
mast cell identification. Specimens were examined by an
Olimpus BH2 microscope. Light microscopic examination
was performed in blinded fashion, in terms of healing parameters and inflammatory changes in the granulation tissue.
Epithelial regeneration, fibrin layer, edema, inflammatory
cell infiltration, fibroblast proliferation, collagen deposition
and angiogenesis were scored semiquantitatively using the
modified Ehrlich / Hunt numerical scale [12]. The samples
were graded on a score of 0 to 4 for epithelial regeneration.
The scores were given as (0) no epithelium, (1) minimal epithelial regeneration, (2) single layer epithelium with partial
closure, (3) moderate epithelial regeneration, and (4) multilayer epithelium with complete closure. Fibrin layer was
scored as being present or absent. For the other histological
parameters, five fields chosen randomly were scored for
each tissue specimen at 400x magnification. A total of 10
fields were sampled per animal. The density of newly formed vessels (angiogenesis density), edema, inflammatory
cell infiltration (granulocytic cell infiltration, mononuclear
cell infiltration), mast cell infiltration, fibroblast proliferation in granulation tissue which was classified into four
grades semi quantitatively, i.e. Grade 0=absent (no finding is
present in the fields), Grade 1=slight (<25% of the fields
contained evidence of any finding), Grade 2=moderate (from
25- 50% of the fields contained evidence of any finding),
Grade 3=severe (>50% of the fields contained with abovementioned histological parameters). All the specimens were
examined by two researchers separately and means of the
scores were calculated.
Revue Méd. Vét., 2006, 157, 4, 185-192
EFFECTS OF FLUNIXIN DICLOFENAC AND METAMIZOL ON WOUND HEALING IN RATS
QUANTITATION OF HYDROXYPROLINE/COLLAGEN
CONTENT
Tissue samples were collected on day 0, 7 and 14 and were
stored in -85°C until assayed. Hydroxyproline contents in
skin tissue were determined by using the modified method
developed by Reddy and Enwemeka [38]. Samples were
homogenized in 2N NaOH by sonication. Homogenates
were hydrolysed by autoclaving at 120°C for 20 min.
Chloramin-T was added to hydrolyzed samples, mixed
gently and kept at room temperature for 25 min. for oxidation. Erlich`s aldehyde reagent was then added to each
sample to generate a colored product which was then meaused at 550 nm spectrophotometrically. BCA protein assay kit
from Pierce was used to determine protein levels in tissues
according to the manufacturer’s instructions. All hydroxyproline values are expressed as pg/mg protein (Mean ±
SEM).
STATISTICAL ANALYSIS
Histopathologic scores of the groups were expressed as
means, while percent wound closure and tissue hydroxyproline concentrations were expressed as mean ± SEM.
Comparisons between experimental and control groups were
performed with Kruskal-Wallis test and paired comparisons
of the groups were done by Mann-Whitney U test. As we
employed multiple comparisons between each group of the
study (namely six), we adjusted the α using Bonferroni’s formula (α = 0.05/6=0.008) in order to avoid infected type I
error [26].
Results
MACROSCOPIC ASSESSMENT
The percentage of the wound closure on days 4, 7, 11 and
14 was less in the NSAID treated animals compared to
controls (Table I). The percentage of the wound closure of
diclofenac treated animals on days 4, 7, 11 and 14 was found
to be significantly less compared to control group animals
(p=0.004). For the flunixin meglumine treated group, the
wound closure was significantly less only for days 4, 7 and
11 (p=0.004). The wound closure for flunixin and diclofenac
groups on days 4, 7, and 11 was also significantly less compared to the metamizole group (p= 0.008). The wounds healed fastest in the control group followed by the metamizole
group. Diclofenac and flunixin groups had significant delays
¥
in wound healing. The treatment of rats by these two drugs
slowed the healing process which is especially evident on
days 4, 7 and 11 (p=0.004).
HISTOPATHOLOGICAL ASSESSMENT
The skin structure of the rat is similar to human skin. Both
are formed by dermis (connective tissue) and epidermis (epithelium), and the tissues have the same general characteristics.
Specimens harvested from the animals underwent histological assessment for epithelial regeneration, fibrin layer,
edema, inflammatory cell infiltration, fibroblast proliferation, collagen content and angiogenesis (Table II).
POSTWOUNDING ON DAY 7
Control groups
The wounds were covered with a scab with severe infiltrating polymorphonuclear cells and phagocytic macrophages.
In this inflammatory stage, the tissue was edematous and
characterized by multiple empty spaces. The effusion was
formed a thick fibrin layer, and inflammatory cells advance
along lines of fibrin within a clot and along capillaries that
are growing into the wound. The granulation tissue was rich
with mixed inflammatory cells and neo-vascularization (Fig.
1-a). Around the neo-vascularization area, fibroplasia and
collagen synthesis had begun. Epithelialization was also
started at the edge of the wound (Fig. 2-a).
Treated groups
Neovascularization was significantly less in the granulation tissue of the treated groups on day 7 postwounding, than
that of the controls (p=0.008) (Fig. 1-b). Fluxin and diclofenac treated animals demonstrated a relative paucity of healing tissue on seventh day. Although there were some new
blood vessels present and a small number of inflammatory
cells, there was minimal collagen and epithelial regeneration
(Fig. 2-b). Though it was not statistically significant, edema
was more pronounced in flunixin and diclofenac groups
compared with controls and the metamizole group (Fig. 1-b).
In contrast, metamizole-treated animals showed increased
epithelial regeneration, fibroblast proliferation and collagen
production compared to the other study groups. However,
this finding was also statistically not significant (p>0.008).
Furthermore, infiltrations of inflammatory and mast cells
were less in the granulation tissue of the treated groups on
day 7 postwounding, than that of the controls. The differences were statistically significant for eosinophils between
: p= 0.004, ¢ : p= 0.004, * : p=0.008, # : p= 0.008
TABLE I. — Effect of metamizole, diclofenac and flunixin meglumine on % wound closure of excision wound
(Values are mean±SE).
Revue Méd. Vét., 2006, 157, 4, 185-192
187
188
ERPEK (S.) AND COLLABORATORS
MNC : mononuclear cell
C : Control group
F : Flunixin group
M : Metamizole group
D : Diclofenac group
TABLE II. — Histologic evaluation of wound healing in skin tissue. (The samples were graded on a score of 0 to 4 for epithelial regeneration. The scores were
given as (0) no epithelium, (1) minimal epithelial regeneration, (2) single layer epithelium with partial closure, (3) moderate epithelial regeneration, and (4)
multi-layer epithelium with complete closure. Fibrin layer was scored as being present or absent. The density of newly formed vessels (angiogenetic density), edema, inflammatory cell infiltration (granulocytic cell infiltration, mononuclear cell infiltration), mast cell infiltration, fibroblast proliferation in granulation tissue which was classified into four grades semi quantitatively, i.e. Grade 0=absent (no finding is present in the fields), Grade 1=slight (<25% of
the fields contained evidence of any finding), Grade 2=moderate (from 25- 50% of the fields contained evidence of any finding), Grade 3=severe (>50% of
the fields contained with abovementioned histological parameters).
FIGURE 1. — The granulation tissue on postwounding day 7. (a) The granulation tissue was rich with mixed inflammatory cells and neo-vascularization in
control group (Masson’s trichrome, x10). (b) Neovascularization and inflammatory cells were less in the granulation tissue of the flunixin treated group than
those of the controls (Masson’s trichrome, x10).
FIGURE 2. — Epithelial regeneration on postwounding day 7. (a) Epithelialization is visible at the edge of the wound in control group (Haematoxylin and eosin,
x4). (b) There were minimal epithelial regeneration in flunixin treated group (Haematoxylin and eosin, x4). (arrow : end of the epithelialization ; asterisks :
border between granulation and normal tissues).
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EFFECTS OF FLUNIXIN DICLOFENAC AND METAMIZOL ON WOUND HEALING IN RATS
189
FIGURE 3. — Accumulation of mast cells in healing wound on postwounding day 7. The severity of mast cell infiltration granulation tissues are graded as “three”
in the control group (a) and “two” in the diclofenac treated group (b) for abundance of mast cells in these microphotographs (Toluidin blue, x20).
FIGURE 4. — Fibroplasia and collagen deposition on postwounding day 14. On day 14, neovascularization and the infiltration of inflammatory cells were milder, while fibroplasia and collagen deposition become more remarkable in control group (a) compared to the diclofenac treated group (b) (Masson’s trichrome, x10).
the flunixin group and controls (p=0.008), and for mast cells
between the diclofenac group and controls (p=0.008) (Fig. 3a., b).
POSTWOUNDING ON DAY 14
Control groups
The scab became thin and regenerated epithelium with
partial closure was observed on the surface of the healing tissue. There was no edema in the granulation tissue except for
one specimen. Neovascularization and the infiltration of
inflammatory cells became milder. Fibroplasia and collagen
deposition was more remarkable than that of day 7 (Fig. 4-a).
Collagen densities had increased, but were still different
from dermal collagen bundles in unwounded dermis.
Treated groups
Histological findings showed that epithelial regeneration
was more prominent in the metamizole group compared to
the other groups on day 14 post wounding. However, this
finding was not statistically significant (p>0.008). There was
Revue Méd. Vét., 2006, 157, 4, 185-192
minimal edema in the granulation tissue of all treated animals. On postwounding day 14, neutrophil and mononuclear
cell infiltrations were higher. Only flunixin group had statistically higher levels of mononuclear cells compared to the
controls (p=0.008). Eosinophil infiltration was lower in treated animals compared to the controls. The presence of mast
cells was higher in diclofenac group than the others
(p>0.008). Fibroblast proliferation and collagen content was
less in the granulation tissue of the treated groups on day 14
postwounding, than that of the controls (p>0.008) (Fig. 4-a,
b). These histological findings demonstrated a relative delay
of healing tissue in NSAID-treated animals except the metamizole group.
HYDROXYPROLINE CONTENT
Hydroxyproline as a measure for collagen was quantitated
in tissue samples from the animals on day 0, 7 and 14 (Table
III). The mean tissue hydroxyproline contents on day 0, 7
and 14 were not different among the study groups (p>0.008).
190
ERPEK (S.) AND COLLABORATORS
TABLE III. — Effect of metamizole, diclofenac and flunixin meglumine on hydroxyproline
content (pg/mg protein) of excision wound (Values are mean±SE).
Discussion
The healing process following injury is one of the most
fundamental defence mechanisms of an organism against its
environment. Wound healing involves a series of overlapping phases, including hemostasis, inflammation, proliferation, and remodeling which together ensure an efficient closure of the wound and restore the integrity of the lost tissue.
The inflammatory reaction is characterized by local edema,
pain, redness, fever, and discomfort and involves the sequential infiltration of neutrophils, macrophages, and lymphocytes [20]. The basic mechanism of action of NSAIDs, prescribed to deter inflammatory discomfort, possess analgesic,
antipyretic, and anti-inflammatory properties at a varying
degree depending on the particular drug and the clinical
condition [17]. Drugs that suppress inflammation could therefore be suspected to adversely affect wound healing.
This comparative study gives a better understanding on
effects of NSAIDs during wound healing. The major categories of non-steroidal anti-inflammatory drugs include salicylates, propionic acid derivatives (ibuprofen, naproxen), phenylacetic acid derivatives (diclofenac sodium), pyrazolon
derivatives (metamizole sodium), anthranilic acids (meclofenamic acid), and the aminonicotinic acid derivative flunixin meglumine [14]. Previous studies have demonstrated
that NSAIDs do not behave alike in their influence on wound
healing [8]. Some researchers have stated that NSAIDs
delayed the wound healing, whereas others claimed that the
same substances had a positive effect or had no effect on the
healing process. NSAIDs may affect wound healing by assisting or interfering with different phases of the processes.
Re-epithelialization, the migration of epithelial cells from
the wound margin to restore epithelial continuity, is an
essential process for cutaneous wound healing [5]. For healthy wound healing, there is a need for restoration of a continuous epithelial “barrier” crucial for protection of granulation tissue against mechanical injuries and infections. In our
study, we observed that diclofenac and flunixin caused a
slight decrease in re-epithelialization, whereas metamizole
seemed to hasten epithelialization. However, this finding
was not statistically significant. Similar findings have been
reported by Kampfer et al. [19] showing that diclofenac markedly reduced epithelization in excision wounds in mice. On
the contrary, BLOMME et al. [2] demonstrated that diclofenac did not delay keratinocyte proliferation and differentiation in sutured skin wounds in mice, following full-thickness
incisions. Similar to our observations on skin there have
been studies reporting that indomethacin significantly inhibited gastric wound re-epithelialization both in vivo and in
vitro conditions [24, 32, 34].
Angiogenesis, the formation of new capillary blood vessels, is a critical component of the healing process that is
essential for wound healing since blood flow is necessary for
oxygen and nutrients to be delivered to the healing site.
NSAIDs have been shown to inhibit angiogenesis through
direct effects on endothelial cells. Traditional NSAIDs may
delay wound healing via decreasing prostaglandin synthesis
by non-selectively inhibiting both COX-1 and COX-2,
which are important in the regulation of angiogenesis [18].
Indeed, in our study, we observed that angiogenesis was
less pronounced in the granulation tissue of the NSAID-treated groups, compared to controls, on day 7 postwounding
(p=0.008). This difference become less visible at the later
stages of the healing process. Our results are consistent with
those of KAMPFER et al. [19] who reported there was no
formation of vascular structures from endothelial cells
within the granulation tissues of diclofenac-treated mice 5
day after excisional wounding. Furthermore, NSAIDs such
as diclofenac, metamizole and indomethacin, have been
shown to block angiogenesis in the ulcerated gastric tissue of
rats [1, 16, 40, 41]. This inhibition of angiogenesis by
NSAIDs is a causal factor in the delay of ulcer healing. On
the other hand, other investigators reported no effect of
diclofenac or indomethacin administration on neovascularization following full-thickness incisional, sutured skin
wounding in mice [2, 29]. The difference between our study
and the latter studies might be that we applied excisional
wound whereas they applied an incisional one.
An adequate inflammatory response is characterized by
the sequential infiltration of neutrophils, macrophages, and
lymphocytes. Studies have shown that defects in the inflammatory phase of healing directly cause failure in the subsequent fibroblast growth and collagen synthesis [25, 28].
DVIVEDI et al. [10] reported that ibuprofen and diclofenac
sodium noticeably reduced the cardinal histologic features of
inflammation; namely macrophages, plasma cells, histiocytes and neutrophils. Likewise, DONG et al. [9] have
demonstrated that ibuprofen treatment decreased neutrophil
function and lymphocyte infiltration into sponges implanted
in rats at the time of burn injury. Our results pointed a slight
decrease in inflammatory and mast cells infiltration in the
wounds of the NSAIDs treated groups on day 7 postwounding. Our findings are in agreement with BLOMME et al. [2]
reporting that diclofenac caused a minimal subjective
decrease in the recruitment of inflammatory cells on days 1
and 3 in full thickness incisional wounding in mice.
Diclofenac sodium was also able to reduce inflammatory
reactions to cat-gut [13] and cotton [35] sutures in rat subcutaneous tissues. Diclofenac sodium greatly reduced both leuRevue Méd. Vét., 2006, 157, 4, 185-192
EFFECTS OF FLUNIXIN DICLOFENAC AND METAMIZOL ON WOUND HEALING IN RATS
kocyte migration and lesion formation [36]. KAMPFER et
al. [19], on the other hand, showed that moderate dose of
diclofenac almost completely diminished infiltration of neutrophils and macrophages into the wound site in mice with
excisional wounding. Diclofenac was also reported to reduce
both edema and the accumulation of inflammatory cells
within the paratenon in the Achilles tendon [27].
Wound healing is mainly achieved by synthesis of the
connective tissue matrix. Breakdown of collagen, a major
protein of the extracellular matrix liberates free hydroxyproline and its peptides. Measurement of hydroxyproline, therefore, has been used as an index of collagen turnover. The
increased hydroxyproline content of the excision wounds
indicates faster collagen turnover leading to rapid healing
[37]. It is generally assumed that hydroxyproline content
parallels the healing process. While some studies have
shown that defects in the inflammatory phase of healing
directly cause failure in the subsequent fibroblast growth and
collagen synthesis [25, 28], some others reported that collagen deposition and cell density within the granulation tissue
were not affected by NSAIDs [29]. In agreement with
MÜLLER-DECKER et al. [29], we could not show difference in hydroxyproline contents from granulation tissue
between treatment and control groups, in rats. Indeed, some
NSAIDs have been shown to have a positive effect on softtissue healing where they stimulate collagen synthesis in the
early phases of repair during skin and ligament healing [6].
On the contrary, MUSCARA et al. [31] showed that
naproxen significantly decreased collagen deposition at the
wound site in rats. They evaluated the effects of naproxen in
an incisional wound model using implanted sponges and
showed a decrease in hydroxyproline content, suggesting a
role for naproxen in delayed wound healing via inhibition of
COX-1 and COX-2 which are necessary for tissue granulation formation and remodeling. One explanation for the differences between our findings and the study of MUSCARA
et al. [31] could be the models used for wound formation
(excisional vs incisional). Furthermore, the NSAIDs we
investigated in our study, although quite similar in mechanism of action, differ from the ones used by MUSCARA et
al.
Wound contraction is another key feature of wound healing which contributes to minimization of infection and promotion of rapid wound closure. In this study diclofenac and
flunixin caused significant delay in wound contraction on
day 4, 7, 11, (p=0.004). However, treatment with metamizole
resulted in a slight decrease in contraction when compared
with the controls. Other researchers have found similar
results using different drugs ibuprofen, phenybutazone, aspirin, indomethacin, in similar rat excisional wound models
during first or second week [8, 22]. DIWAN and
KULKARNI [8] also showed that enfenamic acid, another
NSAID did not retard wound contraction at any stage if anything, it appeared to marginally promote the process. This
compound also hastened epithelization. OGIHARA and
OKABE [33] reported that the repeated administration of
indomethacin delayed the healing in acetic acid-induced gastric ulcers in rats via inhibiting the contraction of the ulcer
base.
Revue Méd. Vét., 2006, 157, 4, 185-192
191
In summary, our findings indicate that NSAIDs used in
this animal model of wound healing caused a slight deficiency in the healing process by decreasing wound contraction, and angiogenesis which are two crucial factors in tissue
integrity. Microscopic examination showed no significant
difference in collagen production and cell infiltration in the
granulation tissue. These data suggest that NSAIDs should
be used judiciously to minimize their adverse effects on
wound healing in humans.
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