92 4.1 INTRODUCTION Wound healing is a dynamic, interactive process involving soluble mediators, blood cells, extracellular matrix and parenchymal cells (Singer and Clark 1999). Acute and chronic wounds are at opposite ends of a spectrum of wound healing types that progress towards healing at different rates. In acute wounds, there is a precise balance between production and degradation of matrix proteins such as collagen; in chronic wounds this balance is lost and degradation plays too large a role. A burn is an injury that occurs as a result of exposure of the tissues to thermal, chemical, or electrical insults. Burns are classified based both on their depth and the surface area of the skin that is involved. First-degree burns that involve only the epidermal layer result in pain and erythema and usually heal within a few days without any scarring. Second-degree burns involve the entire epidermis and part of the underlying dermis. They are further classified as superficial partial-thickness or deep partial-thickness burns based on the depth of injury to the dermis. This distinction is important because many deep partial-thickness burns heal with significant scarring. Superficial partial- thickness burns are characterized by erythema, blister formation, and weeping. They are very painful, and the skin remains sensitive to touch and blanches when pressure is applied, indicating preservation of the dermal circulation. These superficial partial-thickness burns generally heal within 2 weeks with minimal scarring. In contrast, deep partial-thickness wounds involve the reticular as well as papillary layers of the dermis and are characterized by the presence of a nonelastic, red or white layer on top of the
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4.1 INTRODUCTION
Wound healing is a dynamic, interactive process involving soluble
mediators, blood cells, extracellular matrix and parenchymal cells (Singer and
Clark 1999). Acute and chronic wounds are at opposite ends of a spectrum of
wound healing types that progress towards healing at different rates. In acute
wounds, there is a precise balance between production and degradation of
matrix proteins such as collagen; in chronic wounds this balance is lost and
degradation plays too large a role.
A burn is an injury that occurs as a result of exposure of the tissues to
thermal, chemical, or electrical insults. Burns are classified based both on
their depth and the surface area of the skin that is involved. First-degree burns
that involve only the epidermal layer result in pain and erythema and usually
heal within a few days without any scarring. Second-degree burns involve the
entire epidermis and part of the underlying dermis. They are further classified
as superficial partial-thickness or deep partial-thickness burns based on the
depth of injury to the dermis. This distinction is important because many deep
partial-thickness burns heal with significant scarring. Superficial partial-
thickness burns are characterized by erythema, blister formation, and
weeping. They are very painful, and the skin remains sensitive to touch and
blanches when pressure is applied, indicating preservation of the dermal
circulation. These superficial partial-thickness burns generally heal within 2
weeks with minimal scarring. In contrast, deep partial-thickness wounds
involve the reticular as well as papillary layers of the dermis and are
characterized by the presence of a nonelastic, red or white layer on top of the
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burn that often does not blanch with pressure. Because many of the epithelial
appendages that give rise to restoration of the epidermis are destroyed, these
burns require up to 3 weeks healing and may be associated with significant
scarring. Full-thickness or third-degree burns involve the entire epidermis and
dermis and may appear as white, thick brown or tan and have a leathery
texture. The extent of the burn is expressed as the percentage of the total body
surface area (TBSA) that is involved and can be calculated using specialized
age-specific body charts, such as the Lund Browder chart (Lund and Browder
1944).
Significant advances have been made in understanding the cascade of
events (inflammation, tissue formation and remodeling) in normal healing
process. But, these phases are altered from their normal sequence in case of
infection. Especially, Pseudomonas aeruginosa, a gram-negative
opportunistic pathogen causes serious infection in burn wounds leading to
septicemia and if untreated results in mortality (Richard et al 1994).
Pathogenesis of Pseudomonas aeruginosa in burn wound is due to
various extracellular virulence factors such as elastase, exotoxins and
exozymes (Stieritz and Holder 1975), which are shown to influence directly
or indirectly the healing process. Since thermal injury induces an immuno
compromised state, infection due to Pseudomonas aeruginosa further
exacerbates the normally occurring array of events after burn injury.
Primarily, virulence factors may trigger serious weight loss (Steinstraesser et
al 2005). They contribute to delayed re-epithelialization, early dehiscence and
alter content of collagen (Smith and Enquist 1967, Robson 1997). Persistent
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infection after thermal injury impedes epidermal migration and maturation
under provisional matrix, resulting in increased scarring (Singer and McLain
2002). A burn wound infection is also known to spread systemically and
develop into sepsis with associated production of inflammatory cytokines,
mainly Intereukin-1β (IL-1β), Tumor Necrosis Factor-α (TNF- α) and
Intereukin -6 (IL-6). Bacterial endotoxins are the causative factors that induce
the production of these pro-inflammatory cytokines (Freudenberg et al 1993).
There exists a balance of inflammatory cytokines related to severity and
mortality (Walley et al 1996); hence control is of paramount importance.
Especially IL-1β and TNF- α are commonly reported, exhibiting synergism
and hence the net effect should be considered when correlating cytokines
levels and severity of disease (Dinarello 2000). These cytokines also play a
major role in regulation of immune response, hematopoiesis and inflammation
(Reddy et al 2001). Amongst these cytokines IL-6 has short peak time in
normal healing process, while IL-1β and TNF- α are shown to persist for
longer time (Moulin 1995, Neely et al 1996, Grellner 2002). Major cellular
infiltrates that secrete these cytokines are neutrophils and macrophages and
their localization during granulation is detrimental to healing process
(Appleton et al 1993). Impeded epidermal migration mentioned above is due
to altered expression pattern of major matrix remodeling component, Matrix
metalloproteinases (MMPs) (Armstrong and Jude 2002). These MMPs
constitute large family of Zn2+ and Ca2+ dependent endopeptidases, implicated
in tissue remodeling and chronic inflammation. They possess broad and
overlapping specificities and collectively have the capacity to degrade all
components of extracellular matrix (ECM) (Werb 1997, Shapiro 1998).
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Despite the systemic antibiotic therapy, prevention of infection at the
wound site greatly influences the inflammatory and remodeling events. Hence
the use of topical antibiotics are shown to be more effective in reducing the
incidence of wound infection (Halasz 1977, Scher and Peoples 1991). Topical
treatment offers the advantage of immediate effect, lower systemic levels and
higher local tissue bioavailability.
The present in vivo experiment is aimed at investigating qualitatively
the difference between Doxycycline loaded microspheres treated and control
rats, with deep second degree burn wounds challenged with Pseudomonas
aeruginosa, through histological, immunohistochemical localization of
proinflammatory cytokines. In addition quantitative assessment of collagen
turn over, tissue level expression of MMP-1, MMP-2 and MMP-9 to assess
the efficacy of Doxycycline loaded microspheres treated rats to combat
bacterial challenge and subsequently to exhibit faster healing in comparison to
controls. The influence of early infection on various cascading phases during
healing process has been analyzed with special reference to efficient
granulation tissue formation and effective remodeling.
4.2 MATERIALS AND METHODS
4.2.1 Materials
Reagents for biochemical estimation of Collagen, Hexosamine and
zymogram analysis of MMPs, Hydroxyproline, Glucosamine HCl, Para
900 mL), paraffin embedded and 4-5 μm thick sections were cut using a
microtome and mounted on glass slides. Histological sections were then
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de-paraffinzed and stained with H&E to detect the dermal and connective
tissue changes. The staining was performed according to the standard protocol
(Culling 1974).
4.2.2.11.b Immunohistochemical analysis
The early and late course of inflammatory response during healing
was observed by the proinflammatory cytokines IL-1β, IL-6 and TNF-α
expression. Immunohistochemistry was performed according to previously
mentioned procedure (Moulin 1995). The sections were de-paraffinized using
xylene, air-dried and serially hydrated using aqueous ethanol (gradient of
70%) and finally hydrated completely. The hydrated sections were then
incubated for 1 hour with 2 N HCl at 370C (for antigen exposure) and kept
immersed in PBS (containing 0.5% Tween 20) overnight. The sections of
different post burn days (till day 12) were incubated for 1-2 hours at 370C,
individually with polyclonal rabbit antibody for IL-1β, IL-6 and TNF-α to
detect the inflammatory status, while that of control rats taken on day 1 and
day 9 were probed with collagen IV to observe the burn depth (as mentioned
in 4.2.2.3). All the primary antibodies were used at a dilution of 1:100 in 2%
BSA. Then the sections were incubated with alkaline phosphatase tagged anti
rabbit IgG (secondary antibody, diluted to 1:200 dilutions) for 45 mins–1 hr at
370C. Sections were then detected with fast red substrate (prepared with
buffer provided in the kit by manufacturers), washed overnight in water (in
dark), counterstained with Mayer’s Haematoxylin for 15 minutes and finally
mounted using crystal mountant.
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4.2.2.12 Statistical analysis
All graphical illustrations in this study are represented as mean ± S.D
and analyzed with Man Whitney test using SPSS software. Test for
significance was performed with confidence limit of 95%, i.e., p<0.05 was
considered statistically significant.
4.3 RESULTS
The present investigation deals with several responses in magnitude
and temporal pattern of wound repair in Pseudomonas aeruginosa (ATCC
25619) challenged standard deep second-degree rat burn model. It was
observed that there exists a strong relationship between severity of early
infection and subsequent healing events.
4.3.1 Burn Depth Observation
The depth of burn wound created using the thermal device designed
by us resulted in deep second-degree burn wounds. The tissue section excised
along with surrounding healthy skin stained with H&E shows (Figure 4.4) the
complete loss of epidermis, destruction of dermis spreading just above the
muscle layer (thin line of adnexial dermis can be visualized) with complete
loss of hair follicles and vessel walls. To show the comparison of the depth, a
section of unwounded tissue with healthy dermis, intact suprabasal layer with
partial dissociation of the epidermis due to spreading of sub-epidermal
blistering is shown in inset (marked as b). The depth was confirmed by the
presence of Type IV collagen in hair follicle by immuno staining. Tissue
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biopsy on day 1 (Figure 4.4) showed scattered spots of collagen IV in deep
dermis. To reaffirm the observations, section on day 9, when immunostained,
shows (Figure 4.5) presence of positive Type IV collagen just above the
subcutaneous-paraspinal region, over which thin layer of granulation can be
observed.
Figure 4.4 Determination of Wound Depth by Hematoxylin and Eosin Staining (i) Day 1 tissue section (inset) shows the wound depth along
with the adnexial intact epidermis (ep). Serrated arrow shows sub dermal blistering (b). Curved line shows the borderline of wound surface along the dermal-cutaneous layer (c).
(ii) Partially adherent dermis (d) above the muscle layer (m)
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Figure 4.5(i) Immunohistochemical Localization of Collagen IV on Day 1
Day 1 section showing Collagen IV spotted along borderline of adherent tissue layer (arrow), above the muscle layer (m).
Figure 4.5(ii) Immunohistochemical Localization of Collagen IV on Day 9 Immunoreaction of Collagen IV, on day 9, between the granulation (gt)-cutaneous layer junction (arrow heads)
4.3.2 Body Weight Assessment
The average body weight of the rats (n=60) was 184 grams. The
control group showed a constant decrease (5%) from their initial weight till
day 15, after which only a marginal increase is seen but they did not attain the
initial weight until complete healing. In treated group there was marginal
weight loss (1%) till day 9 but they regained their initial weight and showed
an increase of 3% from their initial body weight as shown in Figure 4.6.
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Figure 4.6 Determination of Body Weight at Various Time Intervals
4.3.3 Wound Closure Examination
The control group showed a significant increase in wound size
till day 15 (20% from initial size), followed by a positive healing, which was
slow when compared to the treated groups. The treated group showed an
increase of wound size till day 9, after which it exhibited positive healing
response achieving complete healing by day 24, which was significant when
compared with the control rats (complete healing observed only by 37 days).
From this investigation it is noteworthy that, control group rats exhibited
significantly larger wound size against treated groups at all time points
(p<0.05) (Figures 4.7 and 4.8).
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Figure 4.7 Efficiency of Doxy-MS-CS on Burn wounds in comparison
to control group
Figure 4.8 Percentage Wound Closure
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4.3.4 Influence of Infection on Healing
The control and treated rats infected with Pseudomonas aeruginosa
(107 cfu) exhibited differential response with regard to their microbial load
over the course of healing. As it can be observed from Figure 4.9, that on day
3 the number of cfu significantly increased by 100 fold in control group
(2x105 cfu –107 cfu, microbial count determined in the slough of the wound
since granulation was not obtained), while the treated rats did not show a
significant increase or decrease (2 fold increase, from 2x105 cfu – 4x105cfu).
However on day 6, in treated group a significant decrease (3 fold) in
microbial load was observed (4x105 cfu to 2x102 cfu), which constantly
reduced on post burn days. Treated rats showed 99.9% decrease by day 9
whilst, in control group, the number of cfu did not decrease significantly till
day 15. Almost 75% of control group showed mild to severe purulent
discharge, another sign indicating the onset of positive infection. The severity
of infection was obvious in control group, leading to mortality of 3 animals
indicating local infection has detrimental effect on healing.
Figure 4.9 Quantitative Determination of Microbial Burden in Control and Treated Rats
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4.3.5 Collagen Turn Over
A common pattern of change was observed in both the groups that a
steep increase in collagen content was observed followed by equally a steep
decrease of the collagen content from day 9 for treated and day 15 for control
group. In control group, collagen content (63.8 mg / mg) attained the peak on
day 15, whereas peak levels are found to be on day 9 for treated group. The
treated group exhibited a significant difference in collagen content till day 12
when compared with control (Figure 4.10).
Figure 4.10 Collagen Content in Granulation Tissue