OR I G I N A L A R T I C L E

Histopathological, immunohistochemical, and molecular studiesfor determination of wound age and vitality in rats

Azem A. Khalaf1 | Eman I. Hassanen2 | Amr R. Zaki3 | Adel F. Tohamy1 |Marwa A. Ibrahim4

1Department of Forensic Medicine andToxicology, Faculty of Veterinary Medicine,Cairo University, Cairo, Egypt2Department of Pathology, Faculty ofVeterinary Medicine, Cairo University,Cairo, Egypt3Department of Forensic Medicine andClinical Toxicology, Faculty of Medicine,Beni-Suef University, Beni-Suef, Egypt4Department of Biochemistry and Chemistryof Nutrition, Faculty of VeterinaryMedicine, Cairo University, Cairo, Egypt

CorrespondenceMarwa Ibrahim. Biochemistry Department,Faculty of Veterinary Medicine, CairoUniversity, Egypt.Email: marwaibrahim@cu.edu.eg

AbstractIn forensic medicine, it is vital to verify with the best attainable accuracy once inju-

ries occurred during vital or post-mortem conditions. An immunohistochemical

study was carried out to examine the time-dependent expression of macrophage-

specific gene CD68 (cluster of differentiation 68), alpha-smooth muscle actin

(α-SMA), and vascular endothelial growth factor (VEGF) in different skin woundtimings (0, 1, 3, 5, 7, and 14 days) in rats. Histopathological studies were per-

formed to assess the wound age and vitality. Eighteen male albino Wister rats

(weighing 170-200 g) were used for wound induction. Rats (n = 3) were

euthanised at 0, 1, 3, 5, 7, and 14 days from the starting point of wound induction.

Histopathological examination showed that the epidermal re-epithelialisation was

completed 14 days after skin incision. The inflammatory phase was recorded dur-

ing the first 3 days of healing and reached the maximum levels at 5 days, then

declined after 7 days, and completely removed at 14 days. The beginning of the

proliferative phase was dated to day 3 and the peak at days 5 and 7. The initiation

of the granulation tissue formation and remodelling phase of the healing process

was observed 5 days after wounding. By immunohistochemical staining, negative

VEGF gene expressions at early stages (0-3 days) were observed, as well as neither

CD68+ macrophages nor α-SMA+ myofibroblast cells were detected. By increas-ing the wound ages (5-7 days), granulation tissue and angiogenesis were observed,

with the migration of macrophages and fibroblast, which expressed VEGF, CD68,

and α-SMA positive reaction. Time-dependent expression of the above markerssuggested that they would be useful indicators for the determination of wound age.

Both VEGF and transforming growth factor-beta 1 (TGFb1) mRNA levels were

determined in different skin wound ages. The transcription of TGFb1 and VEGF

increased shortly after wounding, until post-wounding day 7. It then declined con-

stantly, reaching minimal values on day 14.

KEYWORD S

gene expression, immunohistochemistry, TGFb1, VEGF, wound aging

Received: 16 July 2019 Revised: 5 August 2019 Accepted: 11 August 2019

DOI: 10.1111/iwj.13206

© 2019 Medicalhelplines.com Inc and John Wiley & Sons Ltd

Int Wound J. 2019;1–10. wileyonlinelibrary.com/journal/iwj 1

https://orcid.org/0000-0001-7935-8379mailto:marwaibrahim@cu.edu.eghttp://wileyonlinelibrary.com/journal/iwj

1 | INTRODUCTION

Until now, it has been difficult to determine wound timingin forensic medicine; however, it will contribute to thereconstruction of crime scenes and cause the arrest of a sus-pect.1 Forensic pathologists should establish the temporalorder of injuries in cases involving multiple traumas by dif-ferent offenders. In cases of killing, the major focus is onwhether an injury was caused while the individual was aliveor during the post-mortem period and how long the victimsurvived after the wound was inflicted.2

The wound is defined as the morphologic functional dis-ruption of the continuity of a tissue structure. Skin woundhealing is considered a complicated and well-organised bio-logical response composed of three phases including inflam-mation, granulation tissue formation, and remodelling,which involve large numbers of regulatory molecules, likecytokines and growth factors.3 Dysregulation in cytokine orgrowth factor expression alters the normal wound healingprocess.4

These biological responses are collectively termed“vitality,” which is related to whether the victim was alive atthe time of the trauma and how long before the death of thevictim the trauma was inflicted.5 Wound vitality can bedetermined through morphological, cytological, and molecu-lar biological techniques. A variety of biomarkers concernedin vital reactions reportedly increase the accuracy of wound-age estimation.6

Currently, the wound age is a principal parameter inforensic investigations. It is essential to determine the timeof wounds whether it occurred before or after death. It canbe determined using immunohistochemical techniques,growth factors and cytokines.7 A variety of methods for esti-mation of the wound age have been established, such as rou-tine histopathological examination, immunohistochemicalstaining, and reverse transcription polymerase chain reaction(RT-PCR).6

In forensic pathology, immunohistochemistry is themethod of choice because of its reliability and simple appli-cation in formalin-fixed paraffin-embedded tissue. Contraryto most techniques, this morphological technique permits thelocation of the substance of interest inside the tissue or cellsubstructures. Generally, throughout the first few minutesafter the wound occurrence, the microscopic examinationscannot verify whether a wound was sustained before or afterdeath. However, the mRNA levels of cytokines and enzymestypically change sooner than the protein levels and thehistomorphology after wounding.8 So that, assays based onmRNA are appropriate for estimating the age of early-stagewounds. Although RNA is less stable than protein, it hasbeen detected in a long-preserved sample.9 Total RNA ofsufficient quality and quantity can be obtained from samples

that are many months, even years, old.10 Thus, the mRNAlevels of inflammatory cytokines and wound-healing factorsare measured using the real-time PCR to determine thewound age. Because quantitative PCR (qPCR) is a highlysensitive technique to notice even slight changes in geneexpression, it is important to be careful at every step includ-ing data analysis.11 Data normalisation by using referencegenes may be a crucial step for accurate analysis to noticeinevitable experimental variations, particularly disparitieswithin the step of sample loading. The expression of somehousekeeping genes is upregulated after an injury, and thisis considered a critical problem.12 Thus, it is important tospot a stably expressed internal control for effectivenormalisation.

In the past, it was suggested that histological characteris-tics could classify a vital wound, but this method isunreliable and showed a high rate of negative cases.13

Recently, molecular pathology techniques14 and biologicalsubstances, such as vascular endothelial growth factor(VEGF) and transforming growth factor (TGF) and pro-inflammatory cytokines such as interleukin (IL)1, IL6, andtumor necrosis factor have become useful markers forwound age determination.15

VEGF is an essential angiogenic factor in the formationof new granulation tissue in the case of wound healing, so itis used as a marker for wound age determination.16

TGFα and TGFb1 are the foremost promising markers inthis cluster of molecules. Indeed, Grellner and Madea inves-tigated their relevance for skin wound age estimation andobserved upregulation of TGFb1 within several minutesafter wounding, with a peak between 30 and 60 minutes.3

However, simultaneous detection of other markers, as acluster of differentiation 68 (CD68), which is a transmem-brane protein expressed on inflammatory cells, may beeffective in the estimation of wound age.17 Closure of the

Key Messages• aim of work is to determine wound age at differ-

ent times as well as to differentiate between vitaland non-vital wound

• immunohistochemical examinations used to eval-uate CD68 and alpha-smooth muscle actin, anddiscuss the practical availability of the abovegenes as a marker for wound age determination

• the vascular endothelial growth factor and TGFb1gene expressions in rat skin wounds at differentages were performed for identification of suchmarkers used for estimating wound age

2 KHALAF ET AL.

wound is an essential process in healing of the wound. Myo-fibroblasts are recognised to play a central role in closing thewound tissue through alpha-smooth muscle actin (α-SMA).The α-SMA plays important roles in many cellular pro-cesses, including cell division, cell motility, and the genera-tion of contractile force.18

It is believed that there are many established parametersthat yield this knowledge due to the non-specificity, poorrepeatability, and inadequate diagnostic performance of bio-markers besides the limitations of the techniques used. There-fore, systematic and specific criteria for distinguishing usefulmarkers are required, and many advanced techniques ought tobe applied to generate data with more accuracy and objectiv-ity. Because the wound-age estimation is intricate and elabo-rate downside, the use of a combination of several parameterscould minimise the faults in the wound-age estimation.

In the current study, histopathological as well ashistomorphometric studies were performed to determine thewound age at different times as well as to differentiatebetween vital and non-vital wound. Immunohistochemicalexaminations were used to evaluate CD68, α-SMA, andVEGF gene expressions in rat skin wounds at different ages,and discuss the practical availability of the above genes asmarkers for wound age determination. Also, the m-RNAlevels of VEGF and TGFb1 genes were performed for iden-tification of such markers used for estimating wound age.From our point of view, this is the first study focused on thedetermination of wound vitality in addition to the timing ofboth vital and non-vital wound at a long period of time usinghistopathological and immunohistochemical studies as wellas mRNA gene analysis.

2 | MATERIALS AND METHODS

2.1 | Ethical considerations

All procedures were conducted according to the protocolapproved by the Institutional Animal Care and Use Committeeat Cairo University (IACUC, CU-II-F-18-19), Cairo, Egypt.

2.2 | Animals

Eighteen male Wister rats (weighing 170-200 g) wereobtained from Holding Company for Biological Productsand Vaccines (VACSERA), Helwan, Egypt. All animalswere housed in plastic cages (three rats per cage) in a well-ventilated environment and received a daily illumination of16 hours of light. They were fed on dry commercial standardpellets and gained access to tap water ad libitum throughoutthe experimental period. They were acclimatised to the envi-ronment for 2 weeks prior to the onset of the experiment useto ensure their healthy state.

2.3 | Induction of wound

Prior to wound induction, rats were anaesthetised with intra-muscular injections of xylazine 10 mg/kg and ketamine90 mg/kg. The area was marked with a pencil to determinewound margins and then shaved with an electric clipper atthe dorsal back of the animal.19 Full-thickness (extending upto adipose tissue) circular wounds (2 cm2 × 2 cm2) was cre-ated using a sterile biopsy punch. Wounds were left openwithout treatment for 14 days. Rats (n = 3) were euthanisedat 0, 1, 3, 5, 7, and 14 days from the starting point of woundinduction. To evaluate the impact of post-mortem degenera-tion, skin wound excision was performed at 1, 3, 5, and7 days after the rats were euthanised to compare betweenvital and post-mortem wound.

2.4 | Sampling

At the euthanisation time, skin wound specimen was col-lected and preserved in 10% neutral buffer formalin (pH 7.0)for further histopathological and immunohistochemical stud-ies, while others were preserved in liquid nitrogen for RNAextraction and RT-PCR gene expression.

2.5 | RNA isolation and qRT-PCR

Each sample was homogenised using liquid nitrogen, andthe total RNA was extracted using the Qiagen RNeasy MiniKit according to the manufacturer's instructions. Both theconcentration and purity of the isolated RNA were tested byNanoDrop.20 The RT-PCR was performed using theRevertAid First Strand cDNA Synthesis Kit according to theguidelines. Quantitative real-time PCR (qRT-PCR) was per-formed using the Luminaries Color HiGreen Low ROXqPCR Master kit (Thermo Scientific, K0371) 21 according tothe manufacturer's protocol. All values were normalised tothe level of GAPDH22 mRNA. The primers for VEGF1were designed using the Primer3 software; sense primer 50-GCAATGATGAAGCCCTGGAG-30 and antisense 50-GCTTGTCACATACGCTCCAG-30 and the transforminggrowth factor-beta 1 (TGF-β1) primer sequences were as fol-lows: 50-CACTCCCGTGGCTTCTAGTG-30 and 50-GGACTGGCGAGCCTTAGTTT-30. The cDNA was amplified by40 cycles of denaturation at 95�C for 45 seconds, annealingat 57�C for 45 seconds and extending at 72�C for45 seconds. Duplicate plates were tested, and the cyclethreshold (Ct) values were used to calculate the gen-e/GAPDH ratio, with a value of 1.0 used as the calibra-tor. The normalised expression ratio was calculatedusing ΔΔCt.23

KHALAF ET AL. 3

2.6 | Histopathological studies

Formalin-fixed paraffin-embedded wound tissue sampleswere cut into 4-μm tissue sections. The sections were stainedwith haematoxylin and eosin (H&E) and Masson's trichromefor histopathological examination.24

The four-point scoring system was used to evaluate the dif-ferent stages of wound healing at a different time related to thewound age. Wound tissue sections were graded according tothe following parameters: congestion, oedema, haemorrhage,inflammatory cell infiltration, fibroblast proliferation, re-epithelialisation, angiogenesis, and collagen deposition.25 Theabove parameters were categorised as follows: (−) = normalhistology (no alterations), (+) = 50% (severe).

2.7 | Immunohistochemical studies

Formalin-fixed paraffin-embedded tissues were cut into 4-μmtissue sections. The deparaffinised and dehydrated slides werequenched in 3% hydrogen peroxide to neutralise endogenousperoxidase activity, then washed in PBS-T, and blocked in

1% bovine serum albumin. Slides were incubated with pri-mary antibody against vascular endothelial growth factorVEGF or CD68 or α-SMA (Abcam) at 1:400 dilutions over-night at 4�C. The slides were washed and incubatedwith biotinylated-conjugated goat antimouse IgG antibody(Abcam) at 1:400 dilutions for 1 hour at room temperature.Slides were washed and immediately treated with diaminobenzidine (DAB) chromogenic substrate for 5 minutes, thencounterstained in haematoxylin, and rinsed in deionised water.Slides were then rehydrated in alcohol and xylene, dried, andmounted on a distyrene plasticizer xylene (DPX) mountingmedium and then examined under a light microscope to eval-uate the severity of gene expressions.

2.8 | Statistical analysis

The results are expressed as the mean ± SD. Continuousvariables were analysed with one-way analysis of variance(ANOVA), followed by the Bonferroni post hoc test. A P-value of

3 | RESULTS

3.1 | Histopathological examination for thevital wound

The histopathological examination of skin tissue sections atday 0 did not exhibit any inflammatory reaction (Figure 1A),only there was ulceration of the epidermis associated withmild dermal oedema in the wound margins (Figure 1B).Masson trichrome stain contains normal dense thick collagenirregularly arranged at different directions (Figure 1C).

Skin tissue sections at day 1 showed ulceration of theepidermis with coagulated necrosis to the full thickness ofthe dermis (Figure 1D) and the subcutaneous fat. Conges-tion, oedema, mild haemorrhage, and minimal inflamma-tory cells' infiltrations were noticed in the dermal layer(Figure 1E). Subcutaneous fat showed mild haemorrhagein addition to polymorph nuclear inflammatory cell infil-tration. Masson's trichrome stain contains thin slightly

stained collagen irregularly arranged at different direc-tions (Figure 1F).

Skin tissue sections at day 3 showed severe necrotic der-matitis associated with loss of hair follicles, sweat, and seba-ceous gland (Figure 1G). There were severe congestion,oedema, haemorrhage, inflammatory cells' infiltration mainlyneutrophils, eosinophils, and macrophages (Figure 1H).Masson trichrome staining noticed the wound bed filled withprominent deposition of very thin collagen bundles arrangedin a different direction in a meshwork pattern (Figure 1I).Subcutaneous fat showed severe congestion, haemorrhage,and inflammatory cells' infiltration.

Histopathological examinations of wound tissue sectionsat days 5 to 7 were similar and noticed evident inflammatoryreactions including macrophages and lymphocytes associatedwith fibroblast proliferation and neovascularisation. Moderateto complete epidermal regeneration (re-epithelialisation) wasobserved at the wound tips and margin (Figure 2A,D). Wound

FIGURE 2 Histopathological examinations of skin wound in rats at the late phase of healing (proliferation and remodelling). A-C, Rat skinwound at 5 day: A, moderate epidermal regeneration (arrow) associated with fibroblast proliferation and neovascularisation filling the wounded area(star); B, dermal fibrogenesis (arrow) and neovascularisation (arrowhead) irregularly arranged to form granulation tissue associated with severeinflammation and haemorrhage (star); C, slightly stained thin immature collagen arranged irregularly. D-F, Rat skin wound at 7 day: D, completeepidermal re-epithelialisation (arrow) associated with dermal granulation tissue formation (star); E, dermal fibroblast proliferation regularly arrangedparallel to the regenerated epidermis and perpendicular to the angioblast (arrow head) forming organised tissue, noticed haemorrhage (star); F,mature collagen fibre (star) arranged parallel to the regenerated epidermis. G-I, Rat skin wound at 14 day: G, full epidermal re-epithelialisation(arrow) in addition to organised tissue formation in the underline dermis; H, mature organised tissue formation (arrow) and neither inflammation norneovascularisation was observed; I, dense thick mature collagen regularly arranged parallel to the regenerated epidermis (star)

KHALAF ET AL. 5

area was filled with granulation tissue ormed from macro-phages, fibroblast, and new blood capillaries (Figure 2B,E).Immature collagen fibre detected by Masson trichrome stainexhibits a meshwork pattern at day 5 (Figure 2C). Whileprominent thin collagen fibres arranged in one direction paral-lel to the regenerated epidermis were detected at 7 days andboth associated with the severe inflammatory reaction as wellas haemorrhage (Figure 2F).

At 14 days, skin wound sections showing full re-epithelialisation and some of the mature collagen fibres stillremained at the centre without inflammatory reactions(Figure 2G,H). Mature collagen fibres at the wound bed sta-ined blue by Masson's trichrome without any inflammatoryreactions (Figure 2I).

3.2 | Histopathological examinations for thenon-vital wound

Microscopic picture of non-vital skin wound tissue sectionsat day 0 showed autolysis, oedema, and congestion(Figure 3A). At 1 and 3 days old, non-vital wound tissuesections showed moderate to severe autolysis, oedema,

bleeding, and fibrin deposition in the dermal layer(Figure 3C,D). Blood vessels were filled with haemolysisblood (Figure 3B) without any inflammatory cells' infiltra-tion. At 5 and 7 days old, non-vital wound tissue sectionsshowed extensive autolysis (Figure 3E) with mild-to-moderate loss (Figure 3F) of the tissue in the wound areas.The data are summarised in Table 1.

3.3 | Immunohistochemical studies

Immunohistochemical examination of the vital woundsummarised in Table 2 showed that there were negative tomild expression of α-SMA and VEGF at 0, 1, and 3 days inskin tissue sections. Infiltration of a high number of CD68+macrophages was observed from day 3 at the wound surfaceand extended to the superficial dermal layers, but negativelyexpressed at days 0 and 1. All of the abovementioned immu-nohistochemical markers positively expressed from day5 and reach to the peak at day 7 and then declined and notexpressed at day 14 except for α-SMA still stronglyexpressed at day 14 in some sections (Figure 4).

FIGURE 3 Microscopicexamination of the non-vitalwound in rats. A, At 0 dayshowing complete epidermal lossassociated with post-mortemautolysis, congestion (arrows) ofthe dermal layer (star). B,C, At1 day old showing B, dermalcongestion (arrows) and C, post-mortem bleeding (arrows). D, At3 days old showing moderatedermal autolysis and oedema(star). E, At 5 days old showingsevere dermal autolysis. F, At7 days old showing severe dermalautolysis with loss of some partswithout healing (star)

6 KHALAF ET AL.

3.4 | Relative mRNA expression of VEGF andTGFb2 genes

A significant upregulation in both VEGF and TGFb1 mRNAlevels was observed in all of the vital skin wounds at theearly stages (0, 1, 3, and 5 days) and reached to the peak atday 7. However, a sharp downregulation was detected in themRNA level of both the VEGF and TGFb1 at day14 (Figure 5).

4 | DISCUSSION

The competence to resolve whether the skin wound occurredduring life or after death is considered a crucial issue inforensic medicine. The determination of wound age andvitality is always required to elucidate the relationshipbetween wounds and the cause of death. Skin-wound healingstarts immediately after wounding and consists of threephases: inflammation, proliferation, and maturation.6

In our study, histopathological examination of skinwound tissue section showed mild oedema and/or conges-tion of vessels at 1 day old. Margination of polymorphs wasobserved at 1 and 3 days after wounding. Our finding is inagreement with the previous study where it was noticed thatearly polymorph infiltration was found at 4 to 7 hours andreach the maximum levels after 24 hours.26

Mononuclear cell infiltration was observed at 1, 3, and7 days, as well as the earliest re-epithelialisation and

fibroplasia were observed at 5 days. Similar observationsreported that the earliest mononuclear infiltration wasnoticed at 24 hours.27 Another study reported that the epithe-lial regeneration starts as early as 30 hours and is clearly vis-ible by 72 hours in most cases. The granulation tissuedeposition was noticed in our study at 5 and 7 days. Thisobservation in accordance with a previous study confirmedthat the granulation tissue formation is seen by 5 to 8 days.28

The collagen tissue was noticed at 7 and 14 days old. Theprevious study confirmed that collagen formation begins at3 to 6 days and later increases in density until 14 days.26

Our histopathological results of the non-vital skin woundtissue sections at 0, 1, 3, 5, or 7 days old showed neitherinflammatory reactions nor healing process (re-epithe-lialisation, angiogenesis and fibrogenesis). Our findings werein agreement with other several studies which prove that cer-tain changes cannot be inflicted in the post-mortem, such asinflammation, resorption, and wound repair processes thatwere considered as active responses of the body.16 Our resultsconfirmed the pivotal role of histopathological examinationsin deciding both the vitality and age of the skin wound.

With advances in the biochemical and immunohistochemi-cal techniques, several markers have been applied to thewound age determination. The immunohistochemical analysisis now exclusively utilised for wound age determination.29

In the current study, immunohistochemical stainingtogether with the mRNA level showed that the VEGF wasoverexpressed at the late stages of healing (5 and 7 days)and not expressed at the early stages (0, 1, and 3 days).VEGF is a signal protein produced by cells that stimulate theformation of blood vessels and have an important role dur-ing angiogenesis.30 VEGF expression in normal skin isabsent. However, cutaneous damage prompted a sharpupregulation of VEGF expression. Excessive transcription ofVEGF is associated with the proliferation of new blood ves-sels at the site of injury.31 Expression of VEGF isupregulated by several proinflammatory cytokines such asIL1 and IL6, which are enhanced during the early stage of

TABLE 1 Semiquantitative analysisof microscopic picture of wound tissuesections of different ages for assessmentof wound aging

0 1 3 5 7 14

Congestion − ++ +++ +++ ++ −

Oedema + ++ +++ + + −

Haemorrhage − + +++ +++ ++ −

Inflammatory cells' infiltration − + +++ +++ ++ −

Fibroblast proliferations (fibrogenesis) − − − +++ +++ ++

Angiogenesis − − − +++ +++ +

Epithelialisation − − − ++ +++ +++

Mature collagen fibres (fibrosis) − − − _ ++ +++

Note: −, none; +, mild; ++, moderate; +++, severe.

TABLE 2 Semiquantitative analysis of immunohistochemicalexaminations of wound tissue sections of different ages

0 1 3 5 7 14

CD68 − − ++ +++ ++ −

α-SMA − − − +++ +++ +++

VEGF − − − +++ ++ +

Note: −, none; +, mild; ++, moderate; +++, severe.

KHALAF ET AL. 7

wound healing.32 The role of VEGF in wound healingincludes synchronisation of vascular permeability, the prolif-eration of endothelial cells, and the influx of inflammatorycells into the site of injury.33 During the skin repair,upregulation of VEGF in dermal capillaries was reported.34

In our study, CD68+ macrophages expressed at 3, 5, and7 days after wounding while α-SMA + myofibroblastsappeared at 5 and 7 days. This finding is in agreement withthe previous study, thus demonstrating that macrophageswere observed at day 3 or later after wounding in humanskin wounds.35 During the process of skin wound healing,myofibroblasts and macrophages are presumed to play animportant role in angiogenesis due to the secretion of VEGFin skin wound healing.

The transcription of TGFb1 and VEGF increased shortlyafter wounding, until post-wounding day 7. It then declined

constantly, reaching minimal values on day 14. Among thewound repair factors, TGFb1 has the widest range of actions,influencing various cell types involved in all stages ofwound repair.36 It is a potent cytokine that has a rolein wound healing. Following cutaneous injury, TGFb1is promptly secreted by macrophages, platelets, andkeratinocytes.37 Upregulation in the gene expression wasrecorded straight away after skin damage.38 TGF-b1 is criti-cal for inflammation initiating and granulation tissue forma-tion. It is essential to facilitate cell migration and promotewound healing.39 The inhibitory effect of TGFb1 on woundhealing might be attributed to the increased inflammatorycytokines, which directly suppress the expression of genesthat regulate keratinocyte migration.36 The overexpressionof this cytokine was suggested to be associated with protrac-tion of the wound healing process.38

FIGURE 4 Immunohistochemical examination of the wound tissue sections at 3, 7, and 14 days. A, Immunostaining of CD68 positivelyexpressed in the phagocytic macrophage in the superficial dermal layers. B, C, Negative alpha-smooth muscle actin (α-SMA) and VEGF geneexpression in the dermal layer. D, Severe CD68+ macrophages' infiltration throughout the granulation tissue in the underline dermis. E, Severeα-SMA+ spindle-shaped fibroblast cells' proliferation in the wound area. F, Strong VEGF expression among the phagocytic macrophages andaround the neovessels. G, Mild CD68+ macrophages' infiltration. H, Spindle-shaped fibroblast cells positively immunostained by α-SMA. I,Negative VEGF gene expression among the dermis

8 KHALAF ET AL.

5 | CONCLUSION

The determinations of wound vitality apart from the timingof both vital and non-vital wound at a long time period arevery crucial subjects for the field of forensic medicine. Thecurrent study evaluated the potential capacity of CD68,α-SMA, VEGF, and TGFb1 to be used as biomarkers forwound age determination.

CONFLICT OF INTEREST

The authors declare no potential conflict of interest.

ORCID

Marwa A. Ibrahim https://orcid.org/0000-0001-7935-8379

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https://orcid.org/0000-0001-7935-8379https://orcid.org/0000-0001-7935-8379https://orcid.org/0000-0001-7935-8379

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How to cite this article: Khalaf AA, Hassanen EI,Zaki AR, Tohamy AF, Ibrahim MA.Histopathological, immunohistochemical, andmolecular studies for determination of wound age andvitality in rats. Int Wound J. 2019;1–10. https://doi.org/10.1111/iwj.13206

10 KHALAF ET AL.

https://doi.org/10.1111/iwj.13206https://doi.org/10.1111/iwj.13206

Histopathological, immunohistochemical, and molecular studies for determination of wound age and vitality in rats1 INTRODUCTION2 MATERIALS AND METHODS2.1 Ethical considerations2.2 Animals2.3 Induction of wound2.4 Sampling2.5 RNA isolation and qRT-PCR2.6 Histopathological studies2.7 Immunohistochemical studies2.8 Statistical analysis

3 RESULTS3.1 Histopathological examination for the vital wound3.2 Histopathological examinations for the non-vital wound3.3 Immunohistochemical studies3.4 Relative mRNA expression of VEGF and TGFb2 genes

4 DISCUSSION5 CONCLUSION CONFLICT OF INTERESTREFERENCES