Journal of Dermatology and Clinical Research

Cite this article: Elsy B, Maheshwari V, Khan AA (2016) Effects of d a-Tocopherol on Progression of Reepithelialization, Matrix Remodeling and Appearance of Epidermal Appendages in Secondary Skin Wounds of Diabetic Rats J Dermatolog Clin Res 4(4): 1081.

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*Corresponding authorAijaz Ahmed Khan, Department of Anatomy, JN Medical College, Aligarh Muslim University, Aligarh -202002 (U.P.) India. Tel: 91-9897216343; Email:

Submitted: 28 July 2016

Accepted: 10 September 2016

Published: 01 October 2016

Copyright© 2016 Khan et al.

OPEN ACCESS

Keywords•Diabetes•Reepithelialization•Remodeling•Tocopherol•Total antioxidant capacity

Research Article

Effects of d a-Tocopherol on Progression of Reepithelialization, Matrix Remodeling and Appearance of Epidermal Appendages in Secondary Skin Wounds of Diabetic RatsBijo Elsy1, Veena Maheshwari2, and Aijaz Ahmed Khan1*1Department of Anatomy, JNMC, India2Department of Pathology, JNMC, India

Abstract

In diabetes the delayed wound healing is believed to be due to many reasons such as hyperglycemia, infection, suppressed immunity and oxidative stress. Since vitamin E is an effective antioxidant and its active form is tocopherol, this study was designed to explore its role in wound healing process in both healthy and alloxan-induced diabetic rats. Twenty four albino rats were divided into four groups; healthy control, diabetic control, healthy treated and diabetic treated. Treated groups received d a-tocopherol (200 mg/kg body weight) orally daily for 3 weeks. Under general anesthesia, full-thickness excisional skin wounds were created on the dorsal surface of thoracic region. Progression of wound healing was assessed by macroscopic and microscopic features of wounds recorded at weekly intervals. Serum biochemical parameters were also estimated for each animal at the end of 3 weeks. It was observed that re-epithelialization, matrix remodeling and reappearance of epidermal appendages were earlier in treated groups as compared to control groups and this was also associated with significantly increased serum antioxidant status and total protein content. It is concluded that oral administration of d a-tocopherol promotes skin wound healing in both healthy and alloxan-induced diabetic rats, suggesting that its antioxidant potency is reasonably effective in the management of skin wounds.

ABBREVIATIONSAF with FG: Aldehyde Fuchsin with Fast Green; DC: Diabetic

Control; DPT: Diabetic d α-tocopherol treated; FRAP: Ferric Reducing Antioxidant Power; GHI: Global Healing Index; GRI: Global Remodeling Index; HC: Healthy Control; H&E: Haematoxylin & Eosin; HPT: Healthy d α-tocopherol treated; MT: Masson’s Trichrome; TAC: Total Antioxidant Capacity; VVG: Verhoeff Van Gieson

INTRODUCTIONIn many ways oxygen plays a pivotal role in the wound

healing process such as by oxidative bacterial killing, collagen synthesis and epithelialization. That why it is believed to be one of the reasons that the wound healing process is impaired under hypoxic conditions [1,2]. In the inflammatory phase of wound healing, neutrophils and macrophages arrive at the wound

site and secrete large amount of reactive oxygen species (ROS) along with pro-inflammatory cytokines [3]. Since, ROS being cytotoxic, redox balance must be strictly controlled in order to achieve normal wound healing. Interestingly, an optimal ROS level is distinctive for each step of wound healing. Hence, each antioxidative enzyme needs to be fine tuned as per requirement to maintain ROS levels suitable for each process of wound healing [4]. Both non-enzymatic antioxidants (e.g., glutathione, vitamin C, vitamin E) and enzymatic antioxidants (e.g., SOD, GPX, PRDX, and catalase) are involved in the fine tuning of ROS [5]. ROS are also involved in reepithelialization [4]. Superoxide a member of ROS has been shown to be linked with the activation of receptors for epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) [6,7] which also supports the migration and proliferation of epidermal cells.

In a 10 days study [8] it has been shown that topical tocopherol

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treatment enhances the rate of wound closure in streptozotocin-induced diabetic rat. Oral administration of both palm vitamin E and α-tocopherol (200 mg/kg) for 10 days significantly reduced oxidative stress markers and improved rates of wound closure in streptozotocin-induced diabetic rats when compared with control group [9,10].

Thus, there are only few short-term (10 days) studies [8-10] conducted on streptozotocin-induced diabetic rats by the time when wound is not fully closed. And therefore, the present study has been undertaken to evaluate the antioxidant effects of d α-tocopherol on skin wound healing for an extended period of 21 days by using gross, histological, histomorphological and biochemical parameters.

MATERIALS AND METHODSAfter clearance from Institutional Animal Ethical Committee

(No. 8937/2014), 24 albino rats of either sex each weighing 230- 320g were obtained from central animal house of JN medical college, AMU, Aligarh. Prior to commencement of the experiments, animals were acclimatized to the new environmental condition for a period of one week. They were kept in a well ventilated room and were supplied standard pellet diet.

Induction of Diabetes

After deprivation of food for 4 hours, single dose of alloxan (100 mg/kg of body weight; Alloxan monohydrate from Sigma-Aldrich) was injected subcutaneously at hip region. Food and water were provided after one hour of injection. Blood sugar level was monitored by using Glucometer (Dr Morepen gluco one) on the 4th day of alloxan injection. Animals with blood sugar level at 250 mg/dl and above were selected as diabetic for this study. Weight and blood glucose levels of all animals in each group were monitored at weekly intervals.

Experimental Groups

Animals were divided into four groups having 6 rats in each group: (1) Healthy Control- HC; (2) Diabetic Control- DC; (3) Healthy d α-Tocopherol treated- HPT and (4) Diabetic d α-Tocopherol treated- DPT (200mg/kg body weight, orally, daily for 3 weeks. d α-Tocopherol Myra e capsule [Vitamin E] manufactured by PT Daya- Baria laboratoria Tbk, Indonesia; Imported and packed by United laboratories, Inc, 66 United St, Philippines). Dosage of d α-tocopherol (200mg/kg body weight) was based on previous studies carried out [9,10].

Creation of Skin Wound; Collection and Fixation of Tissue and Blood Samples

Under ether general anesthesia, dorsal surface of thoracic region was shaved and from the pinched skin fold, full-thickness of 8.5 ± 0.48 mm diameter (an area equivalent to 46.74 ± 0.32 mm2) excisional wounds were made. Type and size of wound model were very akin to the murine excisional wound model described earlier [11]. Povidone-iodine solution was applied on the wound and 0.5 ml Voveran (analgesic) and 2 mg single shot of Gentamycin (antibiotic) were also injected simultaneously. On completion of 3 weeks animals were sacrificed under deep ether anesthesia and skin bearing healing wound were excised in a manner to include some adjacent normal skin also. The

excised tissues were immersion-fixed in 10% neutral buffered formalin. Blood samples were collected into sterilized vials by direct puncture of heart at the time of sacrifice. Samples were allowed to clot, centrifuged at 2500 rpm for 30 min, the serum was separated and stored in vials and subsequently assayed for serum catalase activity, total antioxidant capacity and total protein content.

Macroscopic Examination

The macroscopic changes in the wounds undergoing healing were observed and recorded photographically on 1st, 7th, 14th & 21st day of creation of wounds.

Histopathology and Histomorphometry

Fixed tissue samples were processed for light microscopical studies. The 5µm thick sections were stained with Haematoxylin & Eosin (H & E), Masson’s Trichrome (MT), Verhoeff Van Gieson (VVG) and Aldehyde Fuchsin with Fast Green (AF with FG).

Histomorphometry was performed on both H & E and MT stained sections. While H & E sections were used for measuring the Global Healing Index (GHI), MT stained sections were used for estimation of Global Remodeling Index (GRI). Histological features under x 4 objective lens of trinocular microscope (Olympus, BX40, Japan) were recorded by digital camera (Sony 18.2 MP, Japan) and measurements were made by using software Motic image version 2.0. Measurements related to epidermal thickness and calculation of healing indices were based on the mathematical model for healing and remodeling matrix [12].

Biochemical Estimation and Analysis

a. Serum total protein content was carried out by using Avantor BenespheraTM clinical chemistry Analyzer C61.

b. Enzymatic antioxidant: Serum catalase was assayed by colorimetery as described [13]. The light absorbance of the sample was determined at 620 nm.

c. Non-invasive biomarker (oxidative stress parameter):

Serum total antioxidant capacity (TAC) was evaluated using ferric reducing antioxidant power (FRAP) assay [14]. The absorbance of sample was measured at 620 nm using photocolorimeter.

Statistical Analysis

All the data were statistically evaluated and the significance calculated using one way ‘ANOVA’ followed by Tukeys test. All the results were expressed as Mean ± SD and P <0.05 was considered as statistically significant.

RESULTSWeight and blood glucose levels of all animals in each group

were monitored at weekly intervals. Mean body weight in healthy control (HC) and treated groups (HPT& DPT) remained stable while diabetic control group (DC) showed slight decrement at the end of study period. Mean blood glucose levels of healthy groups (HC & HPT) remained within normal limits while diabetic groups (DC & DPT) showed > 450 mg/dl throughout the experimental period (Table 1,2).

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Table 1: Body weights (g) of the animals of all groups during the period of study.Groups Day 0 Day 7 Day 14 Day 21HC 270 ± 35.59 266.67 ± 15.28 283.33 ± 20.82 290 ± 21.60DC 277.5 ± 25 247.5 ± 17.08 235 ± 23.80 227.5 ± 22.17HPT 260 ± 33.17 250 ± 21.60 286.25 ± 18.87 293.33 ± 20.82DPT 267.5 ± 29.86 240 ± 20.5 257.5 ± 17.08 272.5 ± 22.17Note the mean body weight in healthy control (HC) and treated groups (HPT& DPT) remained stable while diabetic control group (DC) showed slight decrement at the end of study period.

Table 2: Blood sugar (mg/dl) of the animals of all groups during the period of study.Groups Day 0 Day 7 Day 14 Day 21HC 146± 28.21 124 ± 19.98 160.67 ± 18.01 167 ± 17.06DC 540.25 ± 47.12 553 ± 39.42 574.25 ± 30.20 578 ± 34.73HPT 124 ± 14.23 126.5 ± 17.52 136 ± 18.70 147.12 ± 18.12DPT 546.5 ± 35.80 555.2 ± 29.95 507.4 ± 36.14 478.4 ± 36.64Note that the mean blood sugar levels of healthy groups (HC & HPT) remained within normal limits while the diabetic groups (DC & DPT) showed hyperglycemic state throughout the period of study.

Macroscopic Observations

Progressive wound healing was observed in both control and treated groups which lead to gradual decrease in the actual wound areas. However, by the end of 14th day those in treated groups were remarkably smaller than diabetic control (Figure 1).

Microscopic Observations

Histomorphometry: Neoepidermis in general develops in all groups by the end of 2nd week. However, on completion of 3 weeks, in both the treated groups (HPT & DPT) the neoepidermis was found to be significantly thicker (P <0.01) than the thickness of the epidermis on their wound borders (Table 3). During the study period GHI and GRI in HPT were significantly higher (P <0.01) as compared to all other groups. And even the said values in DPT remained significantly high (P <0.01) as compared to DC (Figure 2,3) suggestive of beneficial effect of treatment in both healthy and diabetics.

Reepithelialization: At the end of study period, complete reepithelialization were noticed in all the groups and interdigitations at dermoepidermal junction appeared in major portion of the wound in treated groups whereas in control groups these features were restricted at the margins of wounds (Figure 4,5).

Matrix remodeling and Skin appendages: The collagen fibres in the regenerated dermis were mostly horizontally arranged and interwoven compactly in treated groups (HPT & DPT) but they were found to be obliquely placed in HC. Poorly interlaced collagen fibres in the suprahypodermal area were observed in DC (Figure 5,6). Elastin fibres in control groups were found in the wound margins while in the treated groups they were noticed one step beyond the border towards the central part of the wound (Figure 7). In treated groups hair follicles and sebaceous glands were in advance stage into the regenerating dermis whereas in control groups they remained at wound margins (Figure 4,5).

Biochemical Analysis

Effects of d α-tocopherol supplementation on serum catalase

Figure 1 Photographs showing the condition of the healing skin wounds of different groups at weekly intervals. Note that even at 14 days the healing is at much advanced stage in treated groups.

Table 3: Border & neoepidermal thickness (mm; Mean ± SD) at the end of 2nd & 3rd week.

Groups

2 weeks 3 weeksBorder

epidermis Neoepidermis Border epidermis Neoepidermis

HC 0.078 ± 0.018 0.109 ± 0.013 0.075 ± 0.014 0.115 ± 0.014DC 0.058 ± 0.017 0.075 ± 0.025 0.061 ± 0.013 0.092 ± 0.043HPT 0.089 ± 0.023 0.123 ± 0.014 0.070 ± 0.015 0.143 ± 0.022DPT 0.075 ± 0.019 0.118 ± 0.022 0.072 ± 0.018 0.136 ± 0.028Note that the neoepidermal thickness in treated groups (HPT & DPT) is almost double to their respective border epidermal thickness at the end of 3rd week.

activity, total antioxidant capacity and total protein content exhibited significant (P <0.05) reduction in DC as compared to HC. All serum analyses values improved significantly after supplementation of d α-tocopherol in HPT as compared to all

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Figure 2 Weekly mean values (in mm) of Global Healing Index (GHI).

Figure 3 Weekly mean values of Global Remodeling Index (GRI).

Figure 4 Representative images from H & E stained sections of different groups at initial magnification x100.

Figure 5 Representative images from MT stained sections of different groups showing the thickness of neoepidermis and presence of hair follicles in the regenerating dermis of HPT and DPT at initial magnification x200.

Figure 6 Representative images from VVG stained sections of different groups showing arrangement of collagen fibres at initial magnification x400.

Figure 7 Representative images from AF with FG stained sections of different groups at initial magnification x400. Arrows (→) pointing the presence of elastin fibres (violet colour) WB: wound borders; RD: regenerating dermis.

other study groups (P <0.01) and in DPT as compared to DC (P <0.05), (Table 4).

DISCUSSIONThere are many studies which suggest that hyperglycemia

promotes generation of highly reactive free radical and leads to the development of oxidative stress [15] which in turn accelerates the development of diabetes and its associated complications [16]. α-Tocopherol is known to be the most abundant and active form of vitamin E in humans [17,18] and it is also known to have scavenging effect on reactive oxygen species and also has an stabilizing effect on damaged cell membrane [19,20].

Since simple murine excisional wounds provide a valid and reproducible wound model that heals by both contraction and reepithelialization [11], in the present study full thickness

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excisional wounds of 8.5 ± 0.48 mm diameter were made on dorsal surface of thoracic region. Macroscopic observation of healing wounds revealed relatively smaller wound size in treated groups even on14th day, suggestive of faster recovery in the treated groups. Skin consists of two major components-epidermis(epithelium) and dermis (connective tissue).The reepithelialization is widely accepted to be one of the major processes in wound healing that ensures successful repair [21-23]. Basal keratinocytes from both the wound edge and epidermal appendages such as hair follicles, sweat glands and sebaceous glands constitute the main sources for cells responsible for the reepithelialization [24].

Thickness of the epidermis is a good indicator for the superficial changes in the wound [12]. The mean values of histomorphological measurement in the present study showed that the neoepidermis was regenerated during the second week. In treated groups (HPT & DPT) on completion of 3 weeks it was almost twice thicker than the epidermis on the wound borders. The GHI is believed to be a key parameter during initial healing and constitute a relevant marker for direct comparison between different treatment groups. In cases of stronger wound remodeling the GRI can go up to 1 [12]. The mean values of GHI and GRI in the present study were significantly (P <0.01) high during the entire experimental period in HPT as compared to all other study groups. In addition, these values were significantly high in DPT as compared to DC.

A 10 days study [8] showed complete epithelialization in all diabetic rats irrespective of topical application of tocopherol cream. All layers of epithelium were observed in tocopherol treated group however these epithelial layers were immature and thin in untreated diabetic rats. Presence of interdigitation between the epithelium and dermis were observed in diabetic rats by topical application of 0.29% tocopherol cream. In the present study complete reepithelialization were noticed in all the groups and interdigitations appeared in major area of neoepidermis in treated groups whereas these were found only at the margins of neoepidermis in control groups at the end of 3 weeks. These interdigitations are known to provide both physical and tropic support.

Horizontal alignment of collagen fibres in the wound matrix indicates better tissue remodeling [25]. Interestingly, during early stage of healing the collagen fibres in dermis revealed different orientation and packing density. They were horizontally placed and densely packed in the treated groups whereas in HC more fibres were obliquely placed. In DC poorly interlaced collagen fibres in the suprahypodermal area were observed. Presence of elastin fibres in the healing wound indicates final stages of matrix remodeling [26]. In the present study the elastin

fibres were found at the wound margins of control groups. In treated groups their presence was more evident towards the regenerating dermis. The growth of hair follicles and sebaceous glands in the regenerating dermis were also observed in treated groups which indicate faster healing and quicker remodeling of the wound matrix [25].

Antioxidant capacity of plasma is the primary measure and marker to evaluate the status and potential of oxidative stress in the body [27]. Total antioxidant capacity showed significant reduction (P <0.001) in plasma and liver homogenate FRAP of diabetic rats when compared with control animals [28-30]. The observation of present work with significant reduction (P <0.05) in serum FRAP of diabetic control when compared with healthy control is in agreement with the findings of above mentioned workers. Antioxidant power in diabetic treated group has been found significantly (P <0.05) improved with d α-tocopherol treatment as compared to diabetic control group similar to one observed earlier [29].

Catalase is a preventive antioxidant which inhibits the initial production of free radicals. When H2O2 is generated in large quantities, the enzyme catalase is also used for its removal [31]. It has been shown that the catalase activity in plasma, liver and kidney of diabetic rats are significantly decreased when compared with those of control rats [32,33]. The results of the present study also demonstrated that in diabetic control group serum catalase activity decreased significantly (P <0.05) as compared to healthy control which is in agreement with the observations of above studies [32,33]. It has been suggested that decreased catalase activity in plasma and tissues of streptozotocin diabetic rats may be due to its increased utilization for scavenging the toxic products of lipid peroxidation or due to decreased availability of H2O2 [32]. Vitamin E treatment has been shown to normal ize the catalase activity in the control group [33]. The findings of present study showing significant (P <0.05) improvement in the catalase activity by supplementation of d α-tocopherol in diabetic treated rats for 3 weeks as compared to diabetic control is in agreement with those of previous study [33].

The total protein content is also known to be an indicator for the protein level and cellular proliferation of the wound tissue [34]. Diabetic rats commonly show marked reduction in serum total protein level and when treated with vitamin E its level improves significantly [35].The result of present study indicates that the tocopherol treatment enhances protein synthesis in treated groups (HPT & DPT) as compared to control groups (P <0.05, P <0.01).This finding is also in agreement with other previous studies [34,8].Thus the enhancement of wound healing in the present study is supported by the increased protein level-dependent collagen synthesis. From afore-

Table 4: Effects of d α-tocopherol supplementation on biochemical parameters (Mean ± SD).

Serum Analyses HC DC HPT DPT

Catalase (u/ml)* 0.0672 ± 0.004 0.0438 ± 0.005 0.088 ± 0.004 0.066 ± 0.006

TAC (µmol/L) 1285.5 ± 67.18 1000 ± 67.88 1481.3 ± 78.02 1309.5 ± 101.12

Total protein (g/dl) 5.05 ± 0.07 4.5 ± 0.14 5.4 ± 0.15 5.15 ± 0.08Note that all biochemical parameters reveal significant reduction in diabetic control group (DC) as compared to all other groups (P <0.05). Catalase (u/ml)*u-µmols of H2O2 utilised/mt.

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mentioned observations it appears that supplementation of the d α-tocopherol leads to increased serum catalase activity, total antioxidant capacity, total protein content, regeneration of epithelium, matrix remodeling and reappearance of epidermal appendages in both treated groups as contrast to control groups.

CONCLUSIONFrom the present experimental study it is concluded that oral

administration of d α-tocopherol promotes skin wound healing in both healthy and alloxan-induced diabetic rats. Therefore, d α-tocopherol seems to hold strong therapeutic potential in the management of skin wounds in future.

ACKNOWLEDGEMENTSAll kinds of support availed from the Department of Anatomy,

JN Medical College, Aligarh Muslim University is gratefully acknowledged.

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Elsy B, Maheshwari V, Khan AA (2016) Effects of d a-Tocopherol on Progression of Reepithelialization, Matrix Remodeling and Appearance of Epidermal Append-ages in Secondary Skin Wounds of Diabetic Rats J Dermatolog Clin Res 4(4): 1081.

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