Co-Administered Vitamin E Isoforms d-α-tocopherol and d-δ-tocotrienol Rich Fraction Promote Regeneration of Skeletal Muscle in Diabetics

In diabetes the structural and functional recovery of skeletal muscle is impaired due to persistent hyperglycemiainduced oxidative stress. Vitamin E is known to be essential antioxidant for maintains the skeletal muscle homeostasis thus preventing oxidative damages. This study is designed to explore the effect of d-α-tocopherol and d-δ-tocotrienol rich fraction (d-δ-TRF) on crushed muscle regeneration in both healthy and diabetic rats. Diabetes was induced through single subcutaneous injection of aqueous alloxan at the dose of 100 mg/kg. Twenty four albino rats were divided into four groups; healthy control, diabetic control, healthy treated and diabetic treated. Treated groups received 100 mg/kg of d-α-tocopherol and d-δ-TRF each, orally, daily for three weeks. Through a horizontal mid-thigh skin incision and splitting of the fascia gluteus maximus was approached and crushed with Kocher’s forceps. Skin wound was closed with an absorbable suture. The crush-induced degenerative and regenerative changes in the muscle were studied by assessing the histological features, histomorphological measurements and biochemical analyses at the end of 3 rd weeks. Oneway ‘ANOVA’ and Student’s t-test were used for statistical analysis. All results revealed that the vitamin E isoforms have potency to maintain glycemic level, improve the antioxidant capacity and hasten the process of regeneration, revascularization, reinnervation and connective tissue remodeling in skeletal muscle after crush injury. It is therefore, concluded that the vitamin E isoforms are useful nutritional supplements for skeletal muscle functional and structural recovery in both healthy and diabetics.


Introduction
Skeletal muscle regenerative capacity involves the number, activation, proliferation and differentiation capacities of satellite cells as well as environment and oxidative stress [1,2].
Marked muscle atrophy is a characteristic feature of uncontrolled diabetes [3]. Oxidative stress severely impairs plasma membrane repair which is a fundamental cellular activity of the skeletal myocyte and failure to repair of the membrane may result into cell death by necrosis. Vitamin E is required for plasma membrane repair in skeletal myocytes [4,5] and also it protects the muscle from diabetes-induced oxidative damage in rats [3]. All above-mentioned studies are mainly based on the α-tocopherol isoform.
Another study [1] revealed that only low doses of tocotrienol rich fraction (TRF) promotes the proliferation capacity of the myoblasts but its high doses (> 200 g/ml) has negative effect on proliferation capacity and is cytotoxic to the myoblasts. Since TRF contain different vitamin E isoforms among which the -tocotrienol is the major constituent [6].
Our previous studies [7,8] revealed that the individual supplementation of d-α-tocopherol and d-δ-TRF helped to accelerate the skeletal muscle regeneration after crushed injuries. Therefore this present study is designed to assess the effect of co-administration of these compounds on muscle repair in both healthy and diabetic rats by using functional, histopathological, histomorphological and biochemical parameters.

Materials and Methods
After the approval by Institutional Animal Ethical Committee (No. 8937/2014), twenty four albino rats of either sex each weighing 230-320g were obtained from central animal house of JN medical college, AMU, Aligarh.
This present study followed the same method of animal care, induction of diabetes and monitoring of blood glucose level as described in our previous study [9].

Surgical Procedure
Surgical procedure (Figure 1), sample collection and fixation of tissues were followed the methods as described in our previous studies [7][8][9]. pointing the crushed parts of GM.

Histopathology and Histomorphology
Fixed tissue samples were processed for light microscopic studies. The 5µm thick paraffin sections were stained with Haematoxylin & Eosin (H & E), Aldehyde Fuchsin with Fast Green (AF with FG) and PicroSirus Red with Fast Green (PSR with FG).
Methods used in Histomorphological measurements and its calculations were same as described in our previous studies [7,8]

Lipid profile, Serum Creatinine and Total Protein
All lipid profiles, serum creatinine and serum total protein content were carried out by using Avantor Benesphera TM clinical chemistry Analyzer C61.

Enzymatic Antioxidant
Serum catalase was assayed by colorimetery as described [10]. The light absorbance of the sample was determined at 620 nm.

Non-invasive Biomarker (Oxidative Stress
Parameter) Serum total antioxidant capacity (TAC) was evaluated using ferric reducing antioxidant power (FRAP) assay [11]. The absorbance of sample was measured at 620 nm using photo colorimeter.

Statistical Analysis
All the data were statistically evaluated and the significance calculated using One-way 'ANOVA' followed by Tukey's test. Student's t-test were used for comparing the initial and final mean body weight of DC and blood glucose level in DXT group before and after treatment. All results were expressed as mean ± SD and P<0.05 and P< 0.0001 were considered as statistically significant.

Body Weight and Blood Glucose Level
During the experimental period, typical clinic manifestations of the diabetes such as polyphagia, polydipsia and polyuria were observed in diabetic control rats while these clinical signs were reduced in diabetic treated groups after three weeks co-administration of d-α-tocopherol and dδ-TRF. Weight and blood glucose levels of all animals in each group were monitored at weekly intervals. Mean body weight in DC was significantly (P<0.0001) reduced whereas in all other groups it remained stable at the end of study period (Table 1). Mean blood glucose levels of healthy groups (HC & HXT) remained within normal limits. In DXT the mean blood glucose level was significantly (P<0.0001) reduced after three weeks treatment while in DC showed > 500 mg/dl throughout the experimental period ( Table 2).  Note that in DXT group the mean blood glucose level was significantly (P<0.0001) reduced after three weeks treatment while in DC showed > 500 mg/dl throughout the experimental period.

Changes Suggesting Degeneration
On 3 rd week the control groups showed more inflammatory cells, atrophic fibres with hypereosinophilic, hypertrophied and undulated sacrolemma and necrotic fibres with mineralization ( Figure 2 & 3). Other myopathic changes such as swollen, vacuolated, hyalinized, fragmented myofibers and hemorrhages in the myofibers and blood capillaries were noticed in DC ( Figure 3). Whereas in treated groups these degenerative and necrotic changes were hardly seen.

Changes Suggesting Regeneration
Only few activated myoblasts were noticed in control groups especially in DC as compared to treated groups ( Figure 4). More newly regenerated myofibers ( Figure 5), myofibers with central nuclei and split fibres were often noticed in treated groups as compared to control groups ( Figure 6).

Fibrosis and Fatty Connective Tissue
Quite frequent fatty depositions were found in the epimysium of control groups (Figure 2, 3 & 9) as compared to treated groups ( Figure 9) and in DC these features were also found in the in perimysium and interstitial connective tissues ( Figure 3). In control groups fibrosis was marked in all connective tissue coverings as compared to treated groups ( Figure 7).

Connective Tissue Remodeling and Neovascularization
Only few thin epimyseal elastin fibres were noticed in HC while these fibres were almost absent in the connective tissue coverings of DC. In treated groups these fibres were prominent were distributed in all connective tissue coverings ( Figure 8). In treated groups more proliferated blood capillaries have shown in the epimysium and at the site of newly regenerated myofibers ( Figure 5 & 9). But these features were less and observed only in the epimysium of control groups (Figure 9).  Note: Note-more collagen fibres (Red colour) in the all connective tissue layers of control groups but in treated groups these fibres were minimal, at initial magnification x100. PSR with FG stain.   Note: In treated groups myofibers' diameter/width were significantly (P<0.01) increased as compared to control groups.

Figure 11. Showing percentage of central nuclei in all groups in muscle TS (Mean ± SD).
Note: The percentages of central myonuclei in treated group were significantly (P<0.01) increased as compared to control groups.

Functional Repair by Reinnervation
In all groups epimyseal nerve bundles were observed ( Figure  9) and these features were also seen in relation to the recently regenerated myofibers of treated groups ( Figure 5 & 9).

Histomorphology
In control groups myofibers width and diameter were significantly (P<0.01) lesser as compared to treated groups ( Figure 10). Percentages of central myonuclei were significantly (P<0.01) high in treated groups as compared to control groups (Figure 11).

Serum Creatinine Level and Serum Total Protein Content
Serum creatinine level in DC were significantly higher ((P<0.01) as compared to all other groups. Serum total protein content in treated groups (HXT & DXT) was significantly higher (P<0.01) compared to control groups (HC & DC) (Table 4) [12].

Discussion
In diabetes increased reactive oxygen species (ROS) generation leads to myopathy due to failure of myocyte plasma membrane repair. Vitamin E promotes the myocyte plasma membrane repair and thus maintains the skeletal muscle homeostasis [4]. Myonecrosis is a complication of longstanding diabetes which most commonly affects the lower limb muscles [13].
In muscle injury the necrotic fibers are enlarged with altered internal architecture, influx of calcium ions, loss of the plasmalemma and an increased number of mononucleated cells [14,15]. In this current study the control groups showed more inflammatory cells, atrophic fibres with hypereosinophilic, hypertrophied and undulated sacrolemma and necrotic fibres with mineralization. Other myopathic changes such as swollen, vacuolated, hyalinized, fragmented and hemorrhagic myofibers and plethora of blood capillaries were noticed in diabetic control group. Three weeks coadministration of d-α-tocopherol and d-δ-TRF seems to hasten the replacement of degenerated myofibers in treated groups.
Muscle regeneration and physiological activity of stem cells are inhibited by chronic inflammatory response [14]. On 3 rd week, more cellular infiltrations were seen in the epimysium and between myofibres of control groups. In treated groups the decreased inflammations points out towards the helpful effect of d-α-tocopherol and d-δ-TRF in creating the favorable environment for muscle regrowth after injury.
Presence of split myofibers during muscle regeneration suggests incomplete fusion of fibers regenerating within the same basal lamina [16,17]. On 3 rd week in control groups the recently regenerated myofibers were minimal. In treated groups presence of more newly regenerated myofibers along with the split fibres revealed the efficiency of vitamin E isoforms in structural recovery of injured skeletal muscle.
Diameter of myofibers and the number of central myonuclei are two useful markers for fusion-based muscle regeneration process [18]. Reduction in the diameter of hind limb myofibres were observed in STZ-diabetic rats [19] and in the present study similar histomorphological results were observed in diabetic control group. The recently regenerated fibres are larger in diameter and have central nuclei. These myonuclei moves to the periphery at the end of muscle regeneration [20]. More number of central nuclei and increased size of muscle fibres were found in treated groups than control groups. These explain the combined effect of dα-tocopherol and d-δ-TRF on fast repair of myofibers after crushed injury.
The repair of damaged myofibers occurred by the activation, proliferation and differentiation of satellite cells into myoblasts [15]. In treated groups more myoblasts were seen as compared to control groups, suggesting thereby that both d-α-tocopherol and d-δ-TRF have potency to promote the proliferation and differentiation process of satellite cells into myoblasts.
The initial fibrosis which occurs during healing process has important role in support, strength and protection of the injury site. However, the overproduction of collagens within the injured area often leads to heavy scarring and the loss of muscular function [14]. In the present study, even on 3 rd week more fibrosis and fatty depositions in the epimyseal and perimyseal connective tissue layers were observed in control groups. Treated groups had lesser fibrosis and thus providing suitable environment for muscles regeneration evidenced as superior effects of co-administration of d-αtocopherol and d-δ-TRF.
Extra cellular remodeling is a one of the fundamental requirements for myoblast migration and fusion during development and regeneration [21]. On 3 rd week in HC the reappearance of thin elastin fibres were observed only in epimysium while even these were absent in DC connective tissue coverings. Treated groups showed marked elastin fibres in all connective tissue coverings which is in agreement with our previous study [12] that the d-αtocopherol and d-δ-TRF have beneficial role in connective tissue remodeling. Regeneration process is completed only after the penetration of new blood vessels into the injured area [22]. In the present study control groups showed lesser proliferated blood capillaries in the epimysium as compared to treated groups. In addition to this treated groups had neovascularization at the site of regenerated myofibers as well. This result correlates with our many previous studies [23,24] that both d-α-tocopherol and d-δ-TRF supplementation supports revascularization after crushinjuries.
Reinnervation after injury is helpful for the maturation and functional recovery of regenerating myofibres. The nerve activities can directly influence protein turnover and gene expression in multinucleated regenerating myotubes and indirectly influence the proliferation and differentiation of satellite cells [25][26][27]. At the end of 3 rd week the reinnervation were observed in epimysium of all groups. In treated groups the newly formed myofibers were more mature and attained functional recovery by presence of regenerated nerve bundles in relation to repaired myofibers. This result is in agreement with previous studies [23,24] that both d-α-tocopherol and d-δ-TRF have potency in nerve regeneration after crush injuries.
Increased levels of cholesterol, triglycerides and phospholipids are the common features in hyperlipidemia [28]. Maintaining the optimal level of lipids is a necessary requirement in diabetes to prevent the diabetic microangiopathy, macroangiopathy, cerebral vascular disease and arteriosclerosis [29]. The data of the present study indicates that the mean values of total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels were higher and high density lipoprotein (HDL) level was lower in diabetic control group, indicating a state of significant dyslipidemia in untreated diabetic rats [30]. Lower mean values of TC, TG, LDL and VLDL levels and high HDL level were recorded in diabetic co-administered group (DXT) after three weeks treatment, this result is in agreement with other related study [31].
Catalase is a preventive antioxidant which inhibits the initial production of free radicals and removes the excess H 2 O 2 [32]. The present study showed serum catalase activity value was lower in DC this is in agreement with other studies [33,34]. This activity was normalized in control group after vitamin E treatment [34]. Three weeks co-administration of d-α-tocopherol and d-δ-TRF also helped to increase the serum catalase activity in treated groups [12].
Antioxidant capacity of plasma is the primary measure and marker to evaluate the status and potential of oxidative stress in the body [35]. The present work observed that serum total antioxidant level in diabetic control was significantly lower (P<0.05) compared to healthy control which is in agreement with the findings of other study [36]. Improved serum antioxidant capacity was observed in treated groups after coadministration of d-α-tocopherol and d-δ-TRF for three weeks [12].

Conclusion
Based on all findings it is concluded that co-administration of d-α-tocopherol and d-δ-TRF have potency to maintain glycemic level, improve the antioxidant capacity and promote functional and structural recovery of myofibers after crush injuries by regeneration, revascularization, reinnervation and connective tissue remodeling. Therefore it is suggested that these vitamin E isoforms are hopeful option for the treatment of skeletal muscle crush-injuries in both healthy and diabetics.