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EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF ACUTE INCISIONAL WOUND HEALING IN MALE ALBINO RAT By Saadia Ragab, Azza Hussein, Seham Abd El-Raof and Hanaa Hassanein Mohammed Department of Histology, Faculty of Medicine ABSTRACT: One of the most important functions of skin is tissue repair. This work was carried to study the process of healing of acute incisional rat wound. Fifty five adult male albino rats were used. Biopsies from 5 animals were used as control to study the normal skin. The rest of the animals were wounded. Histological techniques include hematoxylin and eosin stains and Masson trichrome stain for collagen. Immunohistochemical techniques include the expression of cyclooxygenase II and inducible nitric oxide synthase at different time points during wound healing. The study of the wound with hematoxylin and eosin showed inflammatory cell infiltration and creeping of new epidermal cells at 1 and 3 days postwounding. At 7, 14 and 30 days postwounding, a well established mature scar with thickening of the newly formed epithelium above it was formed. Histological study of the wound with Masson trichrome stain showed that a dense blue collagen network filled the wound gap at day three postwounding. Mature distinct scar formation with compact abnormally arranged collagen fibers was found at 7, 14 and 30 days postwounding. Immunohistochemical study showed that high COX-2 expression at the wound site started as early as 24 hours postwounding, continue to increase up to 7 days and markedly decreased 14 days postwounding. High iNOS expression was found 24 hours and 3 days postwounding. Seven days postwounding iNOS expression decreases and becomes faint at 14 and 30 days postwounding. It can be concluded that wound healing starts by inflammation, reepithelialisation and ends by formation of a scar of irregularly arranged collagen fibers. During the process of wound healing, there was also the expression of inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2). The two enzymes play important role in promotion of the wound healing process. KEY WORDS: Skin Healing Wound Histological Collagen COX2. NOS in inflammation, but also regulate other critical physiological responses. Currently three COX isoenzymes are known, COX-1, COX-2 and COX-3.(Kis et al., 2005).Cyclooxygenase-1 is considered a constitutive enzyme, being found in most mammalian cells. (Beiche et al., 1996 and Amin et al., 1997). On the other hand, Cyclooxygenase-2 is undetectable in most normal tissues. It is an inducible enzyme, becoming abundant in activated in cells at sites of inflammation (Chandrasekharan, 2002 and INTRODUCTION: Skin wound healing is a complex process in which there is repair of injuried epidermal and dermal tissues. During wound healing, many inflammatory mediators were found (Theoret 2004, Desmouliere et al., 2005 and Martin, 2005). Cyclooxygenase (COX) is the key enzyme required for the conversion of arachidonic acid to prostaglandins (PGs) which are compounds that present in a wide variety of human tissues. PGs not only play a central role 1 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Warner et al., 2002). Nitric oxide is a very important signaling molecule that acts in many tissues to regulate a wide range of physiological processes. (Witte, 2002). It was first discovered several years ago by a group that was attempting to identify the agent responsible for promoting blood vessel relaxation and the regulation of vascular tone. This particular agent was named endothelium-derived relaxing factor (EDRF), and was initially assumed to be a protein like most of the other signaling factors previously discovered. The discovery that EDRF was in fact nitric oxide, a small gaseous molecule that plays a key role in many biological proc-esses (Gryglewski, 1986). Nitric Oxide (NO) is produced by a group of enzymes called nitric oxide synthases (NOS). There are three isoforms of NOS which have been named according to their activity. They are neuronal NOS (nNOS), endothelial NOS (iNOS), and inducible NOS (iNOS) (Palmer, 1988). The aim of this study is to investigate the morphological changes in cells and tissues as a result of acute incisional skin wound and to evaluate the expression of some inflammatory mediators during acute incisional wound healing in male albino rat using histological and immunohistochemical techniques. were used as control to study the normal skin. The rest of the animals were wounded, briefly, the dorsal skin was shaved and swabbed with 70% alcohol, then one 1 cm full thickness incision was made on the back of each animal according to a template (Fig. 1, A). The wounds were unsutured and allowed to heal by secondary intention. The animals were housed in individual cages and allowed to recover. Animals were killed at 1, 3, 7, 14 and 30 days postwounding (10 at each time point) by chloroform overdose followed by cervical dislocation and animals were shaved if necessary. The cephalic and the lumber regions of the wound site were marked using non toxic marking pin and photographs of the wounds were taken (Fig. 1, B). The dorsal skin was then removed from the animals by using a sterile surgical blade down to and including the panniculus carnosus muscle; from each rat, one wound was dissected out with half a cm from the surrounding non-wounded surrounding skin and then bisected perpendicular to the plane of the wound, one half was used for histology and the other half was used for immunostaining. The specimens were fixed using 10% formal saline for 48 hours. After proper fixation, the specimens were dehydrated, cleared, impregnated with soft paraffin and then embeddd in hard paraffin and 6 microns sections were cut using rotatory microtome for histological staining. and the following methods were used: MATERIAL AND METHODS: This study was based on biopsies taken from the skin of 55 adult male Sprague Dawley rats. The animals aged approximately 12 weeks old and weight 225 to 250 gm. They were matched and singly housed in plastic cages and maintained in a light, humidity and temperature controlled environment for one week prior to the experiment. Standard rat diet and water were allowed. After wounding, animals were kept under the same conditions until the end of the experiment. Animals were briefly exposed to chloroform. Biopsies from 5 animals Histological techniques: Hematoxylin and eosin: stain and Massons trichrome stain. Immunohistochemical techniques: Cyclooxygenase-2 (COX2) antibody and inducible nitric oxide synthase (iNOS) antibody. Prior to immunolabelling, sections were fixed in acetone for 10 2 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ minutes. Endogenous peroxidases were quenched by treatment with 0.5% H2O2 in methanol followed by washing in tris buffer saline(TBS). Non specific binding of IgG was blocked using normal goat serum, diluted in 1:50 in 0.1% bovine serum albumin in TBS for 30 minutes. The sections were incubated with the diluted primary antibodies (iNOS 1: 1000 and COX2 1:500) at 4C overnight. Sections were then washed 3 times 5 minutes each and incubated for another 30 minutes with biotinylated secondary antibodies diluted 1:1000, followed by washing. Following a further 30 minutes incubation with Vectastin ABC kits (Avidin, biotinylated horse radish peroxidase complex) and washing for 10 minutes, the substrate, diaminobenzidine tetrahydrochloride (DAB) in distalled water was added for 5-10 minutes. The slides were dehydrated and mounted. This results in blue staining in positive sites. could be noticed at day three postwounding and the creeping new epidermal cells were also observed (Fig. 3). At 7 days postwounding, there was full reepithelialization with increase in the newly formed epithelial thickness and narrowing of the wound with matrix condensation leading to closure of the gap created by the injury and scar formation. Lack of hair follicles and so other skin appendages in the scar was also observed. More maturation in granulation tissue and decrease in the inflammatory cell infiltration were basic findings at this time point (Fig. 4). At 14 and 30 days wounds show a well established mature scar with thickening of the newly formed epithelium above it by comparison to the surrounding normal skin. The absence of hair follicles and other skin appendages at the scar site are still present at these time points (Figs. 5 & 6). New blood vessels beneath the scar were seen at the age of 30 days postwounding (Fig. 6). RESULTS: Haematoxylin and eosin: The epidermis of thin skin showed a single stratum germinativum as the thick skin however it contained a thin stratum spinosum and stratum corneum. Thin skin lacked a definite stratum lucidum and stratum granulosum, although individual cells of these layers were present in their proper locations. Thin skin contained a variety of appendages, mainly hair follicles, sweat glands, and sebaceous glands (Fig 1). Massons trichrome: Collagenous connective tissue of the dermis appears blue, parallel to the surface of the skin and arranged in honeycomb architecture. Elastic fibers appeared also in the dermis and stained red with Masson trichrome. They appeared especially abundant near sebaceous and sweat glands. Groups of smooth muscles were located in the deeper regions of the dermis and stained red also with Masson trichrome (Fig. 7). Wounds at early time points one day postwounding were covered by blood clot and the cut was through the whole thickness of the skin (Fig. 8).At day three postwounding, a dense collagen network stained blue by Masson trichrome filled the wound gap. Wounds showed abnormal collagen orientation at the wound site as compared to the normal orientation in At the earliest time point at day one postwounding there was blood clot over the wound and a provisional matrix with inflammatory cell infilt-ration. The start of creeping of new epidermal cells could be observed (Fig. 2). Marked increase in the inflammatory cell infiltration with degradation of the blood clot 3 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ the normal skin surrounding the wound. At 7 days postwounding: the fibrin-rich matrix was replaced by dense collagen staining. It showed condensation of the matrix and scar tissue formation which started to contract leading to narrowing of the wound (Fig. 9). Scars at 14 and 30 days postwounding showed increase in matrix condensation and mature distinct scar formation with compact abnormally arranged collagen fibers. At this stage the wound had remodelled to form a scar (Figs. 10 & 11). infiltrating the wound (Fig. 15). At 7 days postwounding, there was marked reduction in COX2 expression. It was found in the few inflammatory cells near the scar and in the endothelial cells of the blood vessels (Figs. 16 & 17). Scars at 14 and 30 days postwounding show faint COX2 expression. It could be observed in the thickened epithelium above the scar and in the endothelial cells of blood vessels deep in the scar. It was hardly seen in the scar and even less than in the surrounding normal skin where it was expressed mainly in hair follicles (Figs. 18 & 19). Wounds at early time points one day postwounding showed high iNOS expression at the wound site mainly localized to the creeping new epidermal cells at the wound edges and the inflammatory cells infiltrating the wound (Fig. 21). Marked increase in iNOS expression was observed three days postwounding localized mainly to the creeping new epidermal cells at the wound edges and the inflammatory cells infiltrating the wound (Fig. 22). There was little expression of iNOS in the keratinocytes as compared to the high expression in the inflammatory cells (Fig. 22,A). Expression of iNOS was also seen in the endothelial cells lining the blood vessels and the perivascular mast cells (Fig 22 B&C). At 7 days postwounding, there was marked reduction in iNOS expression. It was observed in the few inflammatory cells near the scar and in the endothelial cells of the blood vessels (Fig. 23).Scars at 14 and 30 days postwounding showed faint iNOS expression. It could be seen in the thickened epithelium above the scar. It was hardly seen in the scar and even less than in the surrounding normal skin where it was expressed mainly in hair follicles (Figs. 24 & 25). Immunocytochemistry: Cyclooxygenase-2 (COX2) expression appeared brown in positive staining. Weak expression of COX2 was found in the normal rat skin. It was mainly observed in hair follicles and in the epidermal cells (Fig. 12), inducible nitric oxide synthtase (iNOS) expression appeared brown in positive staining. Weak expression of iNOS is seen in the normal rat skin. It was mainly localized in hair follicles and in the epidermal cells (Fig. 20). Wounds at early time points one day postwounding showed high COX2 expression at the wound site mainly localized to the creeping new epidermal cells at the wound edges and the inflammatory cells infiltrating the wound (Fig. 13). There was little expression in the keratinocytes as compared to the high expression in the inflammatory cells (Fig. 14,A). COX2 expression was also observed in the endothelial cells of blood vessels (Fig. 14, B). The high COX2 expression in mast cells and fibroblasts was also noticed at this time point (Fig. 14,C). Marked increase in COX2 expression was noticed three days postwounding. It was mainly localized to the creeping new epidermal cells at the wound edges and the inflammatory cells 4 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Figure 1. photomicrograph showing normal thin skin with thin epidermal layers ( arrow), hair follicles (double arrows) with associated sebaceous glands (arrow with round head) and thin layer of keratin . (H&E) x40 Figure 2. photomicrograph of rat dermal wound 1day postwounding. showing the blood clot over the wound (star), inflammatory cells infiltrating the wound ( arrow) and the creeping new epidermal cells ( double arrows). (H&E x40. Figure 3. photomicrograph of skin 3 days postwounding showing the marked increase in the inflammatory cell infiltration at the wound site (arrow), the creeping new epidermal cells ( star) and the degradation of the blood clot .(H&E )x40. Figure 4. photomicrograph of skin 7 days postwounding showing closure of the wound, reepithelialisation with thicknening of the epithelium (arrow), condensation of the matrix and the scar tissue formation ( S). H&E x10 Figure 5. photomicrograph of skin 14 days postwounding showing the scar (S) with the thick epithelium over it (arrow). (H&E) x10. 5 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Figure 6. photomicrograph of skin 30 days postwounding. A) showing the scar (S), the loss of hair follicles , neovascularization (arrow) and absence of skin appendages in the H&E x40 Figure 7. photomicrograph of normal rat skin showing blue collagen fibres organised in a ‘honeycomb' formation in the normal dermis ( arrows) and the red colour of the muscle (double arrows). Masson trichrome x10 Figure 8. photomicrograph of 1 day dermal wound showing the junction between the full thickness wound with the blood clot over it (black arrow) and the normal skin (blue arrow). Masson trichrome x10 Figure 9. photomicrograph of 7 days dermal wound showing condensation of the matrix and scar formation (s). Masson trichrome x10. Figure 10. photomicrograph of 14 days dermal wound showing connective tissue fibres mainly collagen are densely packed to form a mature scar (S). The collagen contrasted sharply with the adjacent nomal dermal collagen. Masson trichrome x10. 6 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Figure 11. photomicrograph of 30 days dermal wound showing connective tissue fibres mainly collagen are densely packed to form a mature scar . Masson trichrome x10. Figure 12. Immunohistochemical evidence of COX2 expression in normal rat skin. There is faint COX2 staining mainly in the epidermal cells (arrow) and hair follicles (double arrows).x10. Figure 13. Immunohistochemical evidence of COX2 expression 1 day postwounding showing high COX2 expression at the wound site mainly localized to the creeping new epidermal cells at the wound edge ( arrow) and the inflammatory cells infiltrating the wound (double arrows). x10. Figure 14. Immunohistochemical evidence of COX2 expression 1 day postwounding. A) Showing COX2 expression in the skin at the wound edge. Notice the little expression in the keratinocyte (K) and the high expression in the inflammtory cells (arrow) in the dermis. B) COX2 is seen in the endothelial cells of blood vessels (arrow). C) COX2 is seen in mast cells (red arrows) and fibroblast like cells (black arrows) at the wound site. x40. 7 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Figure15. Immunohistochemical evidence of COX2 expression 3 day postwounding. High expression of COX2 in the growing newly formed epithelium at the edge of the wound ( arrow) and in the inflammatory cells infiltrating the wound (double arrows). x10. Figure 16. immunocytochemical evidence of COX2 expression 7 days postwounding showing marked reduction in COX2 immunoreactivity at the scar site (s). Few inflammatory cells expressing COX2 at the edges of the scar (arrows). x10. Figure 17. immunocytochemical evidence of COX2 expression 7 days postwounding showing COX2 expression in the endothelial cells of blood vessels (arrows). x10. Figure 18. Immunohistochemical evidence of COX2 expression in Faint COX2 14 days postwounding. expression in the thickened epithelium above the scar (arrow) and deep in the dermis (double arrows). x10. 8 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Figure 19. Immunohistochemical evidence of COX2 expression 30 days postwounding. COX2 is expressed in the endothelial cells of vessels deep in the scar ( arrow). It is hardly seen in the scar and even less than in the surrounding normal skin where it is expressed mainly in hair follicles (double arrows). x10. Figure 20. Immunohistochemical evidence of iNOS expression in normal rat skin. The ABC technique was used and it shows brown in positive staining. There is faint iNOS staining mainly in the epidermal cells ( arrow) and in the hair follicles (double arrows). x10. Figure 21. Immunohistochemical evidence of iNOS expression 1 day postwounding showing high iNOS expression at the wound site mainly localized to the growing epithelium at the wound edge (arrow) and the inflammatory cells infiltrating the wound (double arrows). x10. Figure 22. Immunohistochemical staining showing cellular localization of iNOS in rat dermal wound healing. A) iNOS is faintly expressed in keratinocytes (K) of the epidermis next to wound area, in inflammatory cells (arrow) infiltrating the dermis close to the wound edge. B) in the vascular endothelial lining (long arrow) and the perivascualr mast cells (short arrow) at the wound site. C) in the vascular endothelial cells (arrows). A x10, B and C x40. 9 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ Figure 23. Immunohistochemical evidence of iNOS expression 7 days postwounding showing marked reduction in iNOS immunoreactivity at the scar site (S). Few inflammatory cells expressing iNOS at the edges of the scar (black arrows). iNOS is also expressed in the endothelial cells of the blood vessels Figure 24. Immunohistochemical evidence of iNOS expression 14 days postwounding. Faint iNOS expression in the thickened epithelium above the scar (black arrow) and deep in the dermis (red arrows). x10. Figure 25. Immunohistochemical evidence of iNOS 30 days postwounding. It is hardly seen in the scar (s) and even less than in the surrounding normal skin where it is expressed mainly in hair follicles ( arrow). x10. in the wound area included inflamematory cell infiltration, blood clot formation, beginning of reepithelialisation and loss of the normal collagen orientation. These changes were in agreement with those of (Flanga, 2005). This was in favourite of the healing process since blood clotting stops excessive bleeding (Romo and Pearson, 2005). Reepithe-lialisation aimed at closing of the wound to enhance the healing process (Garg, 2000). The changes in the wound area DISCUSSION: This study aimed at the investigation of the histological changes that occur in rat skin during healing of acute incisional wounds at different time points 1, 3, 7, 14 and 30 days postwounding. The work investigated also the expression of inflammatory mediators cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS), which are known to contribute to the pathogenesis of wound healing and scar formation. The changes found 10 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ continued to occur throughout the time course; three days postwounding the wound showed degradation of the blood clot and marked increase in the inflammatory cell infiltrate, this was in agreement with (Shah et al., 1999). By day 7 postwounding the healing process was almost completed with complete reepithelialisation, scar formation and retraction of the cut muscle edges leading to narrowing of the wound. By day 14 the scar was maturated. These findings were in accordance with the results of other investigators ((Foreman et al., 1996). Although reepithelialisation started as early as one day postwounding, it increased with time and the wound showed complete reepithelialisation seven days postwounding, this was in agreement with findings of (Shah et al., 1999). Many studies (Ferguson et al., 2001 and Santoro, 2005) including the present one have shown that although the course of wound healing was controlled by three main phases inflammation, reepithelialisation and collagen deposition, there was overlapping between these three phases and there was predominance of each phase at certain time point; inflammation predominates in the first three days postwounding. Reepithelialisation predominates seven days postwounding. Collagen deposition predominates late in wound healing and this coincides with the results of (Sirsjo, 1996). It starts as early as 3 days postwounding and continued over the time course till it is fully mature by 30 days. The white distinct appearance of the scar is due to the loss of the normal collagen orientation in the wound area (Chamberlain et al., 1994). The induction of iNOS and COX-2 at the wound was during the early time points 1-7 days postwounding. Therefore, it was coincident with the infiltration of the wound by inflammatory cells and induction of cytokines. There was earlier induction and earlier downergulation of iNOS in the wounds by comparison to COX-2. While iNOS was highly expressed mainly during the first 2 days postwounding, COX-2 was highly expressed 2-7 days wound. This could be explained by (McCall & Vallance, 1992) who mentioned that iNOS is expressed by neutrophils, the first effector cells which migrate into the wound and predominate during the first 24 hours after injury. Following appropriate stimuli, neutrophils can express cytokines including interleukin -3, IL-1, IL-12, IL-8 and tumor necrosis factor (TNF), which are known to induce iNOS. However, COX-2 were expressed by fibroblasts and mast cells, which infiltrate wounds at late time point (sirsjo,1996). In addition, (Rosenberg, 2006) documented that transforming growth factor (TGF) and cytokines which is known to be upregulated in wounds at late time point can inhibit iNOS and induce COX-2. Other investigators (Salvemini et al., 1989) added that iNOS stimu-lates COX-2 and this could explain the lag in COX-2 activity. In summary, there are several factors, which could control the expression and the profile of iNOS and COX-2 in the wounds, including the wound cellularity, growth factors and the products of the enzymes.The pattern of expression of iNOS in the wound could be a physiological requirement for healing. Nitric oxide has anti-microbial and anti-proliferative activities which could overcome the microbial contamination in the early stages after injury (Nussler & Billiar, 1993). Moreover, nitric oxide produced by the vascular endothelial cells, maintains a high rate of blood flow in the wound. This increased blood flow facilitates both nutrition and cell infiltration. Further-more, this low level of nitric oxide could neutralize the oxygen free radicals released by 11 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ infiltrating leukocytes, thus protecting cells from damage by oxygen and hydroxyl radicals (Romo and Pearson, 2005). On the other hand, cytokines released by infiltrating macrophages and associated with bacterial invasion several hours postwounding, could stimulate poly-morphnuclear neutronphils (PMNs) and macrophages to produce large amounts of nitric oxide, which in turn might inhibit cytokine and nitric oxide produ-ction by infiltrated cells (Griscavage et al., 1993). This could provide a negative regulation of the inflammatory response of the wound, since severe inflame-mation and high levels of nitric oxide can cause massive tissue damage. Transforming growth factor (TGF), which is known to be a potent fibro-genic cytokine that enhances wound healing, has been reported to inhibit nitric oxide production by macrophages (Eichler and Carlson, 2005).The PGs are known to have anti-inflammatory effects and Recently, (Kis et al., 2005) found that PGs enhance wound repair by enhancing blood flow in the neovascularisation of repairing wounds. Thus the course of wound healing is normally controlled by both iNOS and COX-2 and it is likely that, interference with these physiological responses to injury either by inhibitors or inducers for these enzymes could alter the healing process. However delayed healing which occurs under conditions associated with low expression of the enzymes e.g. in diabetes and malnutrition (Schaffer et al., 1997) or excessive upregulation of these enzymes such as chronic wounds might be treated using inhibitors or inducers (Flanga, 2004 and Scholar,2006). (Koh et al., 2003) showed that COX-2 activity was increased during early time points of wound healing. Using immunostaing they showed that COX2 staining was particularly prominent in the inflammatory cells, fibroblast and newly formed vascular endothelial cells. These data suggest that COX-2 is constitutively expressed in epidermis and is associated with keratinocyte differentiation. Pervious studies (Etscheid et al., 2005)) observed that the expression of COX2 was mainly within the basal layer of the epidermis, peripheral cells in the outer root sheath of hair follicles and fibroblast-like cells and capillaries near epidermal wound edges. 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The American Journal of Surgery, 183(4): 406-412. 14 EL-MINIA MED., BULL., VOL. 19, NO. 2, JUNE, 2008 Ragab et al ___________________________________________________________________________________ الدراسه الهستولوجيه والمناعه الكيميائيه الخلويه اللتئام الجرح الحاد القاطع لذكر الفأراالبيض سعديه رجب -عزه حسين على -سهام عبد الرءوف – هناء حسانين محمد قسم الهستولوجى -كلية طب المنيا ان أحد اهم وظائف الجلد اصالح النسيج .وقد تم هذا العمل لدراسة عملية التئاام الجارح الحاد القاطع للفئران وقد استخدم لذلك خمسة وخمسون من ذكورالفئران البيضاء البالغة.وتم اخد عيناا مان خمساة ائاران كمجموعاة ضاابطة وتام قتال بقياة الفئاران بعاد جرحاااا اليااوم ا ول والثالث والسابع والرابع عشار والثالثاين .وقاد اوضاح دراساة الجارح بصابغة الايماتوكسايلين وا يوسين ان نشع خاليا ا لتاا وححف خاليا البشارا الجديادا يكاون اا الاثالث اياام ا ولا بعد الجرح .وا ا يام السابع والرابع عشر والثالثين تم تكوين ندبا ناضاجا حسان التاسايع ماع حياادا ساامك النساايج الطالئااا حااديث التكااوين اوقااه .امااا الدراسااه الاسااتولوجيه للجاارح باسااتخدام صبغة الماسون تراى كروم اقد بينا ان شابكه كثيفاه مان الكاو جين المصابوزة باا حر تماال اجاوا الجاارح عناد اليااوم الثالا ث .اماا اااا ا ياام السااابع والرابااع عشار والثالثااين اقاد وجااد النااد الناضج المكون من الياف الكو جين الملتحمه المترتباه زيار طبيعياا .وقاد بينا دراساة المناعاة الكيميائية الخلوية ان التعبير المرتفع نحيم السيكلواوكسجينيح اثنين عند مكان الجرح يبدا مبكرا بعد اربع وعشرين ساعة من الجرح ويستمر ا ا رتفاع حتاا سابعة اياام ويقال جادا عناد الياوم الرابع عشر والثالثينن ويكاون هاذا ا رتفااع علاا ا خاك ااا كال مان النسايج الطالئاا حاديث التكوين عند حااتا الجرح ،والنسيج المتحب اا وسط الجرح ،وا وعية الدموية حديثة التكوين وخاليا ا لتاا التا تنش ع اا الجرح .ويكون التعبير قليل للغاية ااا البشارا وا دماة بعيادا عان مكان الجرح. وقااد كااان التعبياار مرتفعااا نااحيم النيتريااك اوكساايد سااينثيح المسااتحث اااا اليااومين ا ول والثالااث بعااد الجاارح ووجااد علااا ا خااك اا كاال ماان الخاليااا المبطنااه لالوعيااة الدمويااه حديثااة التكااوين والنساايج الطال ئااا .ولكنااه يقاال جاادا اااا نساايج النااد عنااد ا يااام السااابع والرابااع عشاار والثالثين .ويمكن من ذلك استنتاج ان عملية التئام الجروح تشتمل علا ا لتااا واعاادا النسايج الطالئا وتكوين الند من اليااف الكاو جين المترتباه ااا صاور زيار طبيعياه .وتشاتمل ايضاا علا تعبير كل من انحيم السيكلواوكسجينح اثنين وانحيم النيتريك اوكسيد سينثيح المستحث حيث ان لاما دورا ماما ا تحفيح عملية ا لتئام. 15