Sodium Valproate Improves Skin Flap Survival via Gamma-Aminobutyric Acid and Histone Deacetylase Inhibitory System

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Sodium Valproate Improves Skin Flap Survival via Gamma-Aminobutyric Acid and Histone Deacetylase Inhibitory System Moein Ala, MD,a,b Razieh Mohammad Jafari, PhD,a Hossein Nematian, MD,a,b Mohammad Reza Ganjedanesh, MD,a,b and Ahmad Reza Dehpour, PhDa,b,* a b

Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

article info

abstract

Article history:

Sodium valproate interacts with biological systems through different mechanisms such as

Received 2 January 2019

activation of gamma-aminobutyric acid (GABA)-sensitive chloride channels and inhibition

Received in revised form

of histone deacetylase. In this study, we examined the effect of sodium valproate in

23 July 2019

random-pattern skin flap of rats and investigated its mechanisms of action. Different types

Accepted 18 September 2019

of experiments were carried out. In acute treatment, different doses of sodium valproate

Available online xxx

(50, 100, 150, 300 mg/kg) were injected intraperitoneally 1 h before surgery. In chronic treatment, the substance was injected each day for 2 wk. The size of skin necrosis was

Keywords:

measured 1 wk after the surgery. The rate of secondary healing, amount of weight gain,

Valproate

hair growth, and wound regeneration were measured 2 wk after operation. In acute

Skin flap

treatment, sodium valproate (100 mg/kg) reduced significantly the length of skin necrosis

GABAergic

(P < 0.05). Administration of bicuculline (competitive antagonist of GABAA, 20 mg/kg)

HDAC

increased the length of skin necrosis (P < 0.05). In addition, administration of 100 mg/kg of sodium valproate and subeffective dose of bicuculline (10 mg/kg) prevented the protective effect of sodium valproate on skin flap necrosis (P < 0.05). In the chronically treated skin flap group, 100 mg/kg of sodium valproate reduced the length of necrosis (P < 0.01). Weight gain in the valproate group was more than that in the control group (P < 0.05). Skin also healed faster in the sodium valproate group than in the control group (P < 0.001). Combination therapy of sodium valproate and trichostatin A (330 nmol/kg) reversed the effect of valproate (P < 0.05). This study demonstrate that sodium valproate accelerates skin secondary healing in a rat model of skin flap probably through a GABA and histone deacetylaseedependent mechanism. ª 2019 Elsevier Inc. All rights reserved.

Introduction Skin interventions leading to disruption of perfusion finally brings focal necrosis. The scar formation after injury or skin

flap remains as a life-long disturbing outcome. Contrary to advanced surgical methods, skin lesions are unavoidable and may affect the patients’ appearance and self-confidence. Skin flap induces ischemic stress and production of inflammatory

* Corresponding author. Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, 13145-784, Iran. Tel.: þ98 21 88973652; fax: þ98 21 66402569. E-mail address: [email protected] (A.R. Dehpour). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.09.036

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mediators, which might affect the flap survival.1 Inflammatory cytokines such as interferon-gamma and tumor necrosis factor-alpha trigger apoptotic cell death in the ischemic area which potentially postpones tissue regeneration.2 The process of wound healing and angiogenesis involves production of a variety of growth factors that repairs the skin after injury.3e6 Sodium valproate is an anticonvulsant agent that enhances gamma-aminobutyric acid (GABA) receptors vastly.3,4,7 Also, valproate is increasingly used for the treatment of bipolar, schizoaffective disorders, neuropathic pain, and prophylaxis of migraine.8,9 It has been demonstrated that GABA receptors exist in antigen-presenting cells, and stimulation of these receptors contributes to suppression of inflammation.10 In the skin tissue, GABA modifies stimulation of mechanoreceptors.11 Furthermore, stimulation of GABA type A and B could present antipruritic effects in skin, which was mediated through modulating immune cells.12 GABA receptors, especially type A, are expressed in immune cells such as antigen-presenting cells and plays an obvious inhibitory role in immune system to ameliorate the inflammation.13 Also, valproate can modify meningeal neurogenic inflammation through a GABAdependent mechanism.14 Immune cells are scattered in skin tissue,15 so GABA pathway theoretically might counteract inflammation through immune cell inhibition. A recent study has shown that GABA-sensitive chloride channels are not the only known target for valproate. Histone deacetylase (HDAC), an enzyme which is expressed abundantly in the skin, is also inhibited by valproate.16 It appears that epigenetic modification by this enzyme modulates expression of specific proteins.17 Moreover, valproate increases bcl2 (antiapoptotic molecule) level and plays a neuroprotective effect in the central nervous system.18 Ximenes et al. revealed that administration of valproate controls rat paw inflammation by decreasing tumor necrosis factor-alpha level.19 Valproic acid enhances induced pluripotent stem cell generation,20 and previous researches proved valproic acid is one of the agents that can help the induction of induced pluripotent stem cell from somatic cells.21 It has been demonstrated that HDAC inhibition and epigenetic modulation are determinant factors in changing regeneration rate and collagen deposition after digital amputation.22 HDAC inhibitors regulate stem cell differentiation and self-renewal ability and affect tissue regeneration by modulation of cellular differentiation signaling pathways.23 The main purpose of this study was evaluation of sodium valproate on skin necrosis induced by skin flap. Here, we studied whether valproate affects the survival of skin flaps through GABA receptors and inhibiting HDAC signaling.

Materials and methods Animals Male Sprague-Dawley rats weighing 200-250 g were used and divided into 11 groups of 8 rats. The rats were kept in a temperature-controlled room (23  2 C) on a 12:12 h light/dark cycle. They had free access to food and water, and they were treated morally in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH US 86e23, eighth Ed.). The animals

were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg),24 and they were euthanized to harvest their skin tissue for staining and histopathologic measurements at the end of procedures. Each rat was tested only once.

Assessment of skin flap All rats underwent skin flap surgery after induction of general anesthesia. After deep anesthesia, dorsal skin was shaved to expose the surgery site. In each rat, after palpating the hip joints as an anatomical landmark of the caudal part of the flaps, dorsal skin was marked by constant size (measuring 2  8 cm) to determine the borders in the midline of rats’ dorsum and incision site. The skin flap was cranially based and centered in the midline then raised in a caudal to cranial manner.25 Meanwhile, perforator arteries beneath the flap were cut to ensure ischemia happens,26 and then the skin was placed on its first position. Flaps in the back of the animals were kept in moist and humid sterile gauzes during the procedure. To prevent hypothermia, a heat-emitting light was located next to rats. The animals were left in this condition for 20 min, and then the skin were sutured using 4.0 reversed cut nylon continuous stitches by the simple continuous method. Povidone-iodine was rubbed around the wound after suturing to prevent infection. The sterile wound dressing was applied to the area, and the rats were put back in clean cages. After 7 d, rats were anesthetized, their flaps were examined by the blinded investigator, and digital images and pathologic samples were obtained from the flaps. Survived area was measured using the Digimizer Image Analysis Software (MedCalc Software, Ostend, Belgium).

Drugs and procedure Sodium valproate (SCI-Taiwan, 1504P123) was dissolved in sterile normal saline. In single-dose treatment, we have investigated the increasing dose of sodium valproate (Alexis, 550-515-M250) on flap survival to evaluate dose response profile of the drug. This was achieved by intraperitoneal (i.p.) administration of 50, 100, 150, and 300 mg/kg of sodium valproate 1 h prior elevating the flap in different groups of rats. Bicuculline (10 mg/kg, GABA receptors antagonist) was administered solely and also alongside the most effective dose of sodium valproate (100 mg/kg) to clarify GABA pathway involvement in skin flap survival. In chronically treated animals, drugs were injected i.p. every day until 2 wk. To vivify the probable influence of HDAC pathway in this treatment, we coadministrated the effective dose of trichostatin A (TSA) (330 nmol/kg) (ab120850; Abcam, Cambridge, UK) with the effective dose of sodium valproate (100 mg/kg).25,27 A single dose of TSA as the HDAC inhibitor was administrated to compare drug effects. Control groups received pure sterile normal saline. TSA and bicuculline need a small amount of DMSO to be dissolved.28 All groups included eight rats.

Follow-up The size of necrosis after 7 d was measured as described.29 Furthermore, there are other determinants that can reflect the efficacy of sodium valproate in relieving skin flaps such as the speed of secondary healing and scar retraction, which was

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evaluated in this research after 2 wk of valproate injection. Further measurements were carried out which achieved interesting results such as comparing weight gain and hair growth speed in chronic sodium valproateetreated group.30,31 In addition, histopathology assessments were performed to investigate the effect of valproate on the skin flaps in cellular scale. Skin samples from the margin of necrotic area (1.5 cm  1.5 cm samples) were taken and kept in 10% formaldehyde for at least 24 h. Three specimens were sectioned from each block at a 5-mm diameter. These sections were prepared for hematoxylin and eosin staining. By H&E staining, the number of inflammatory cells, amount of edema, number of necrotic cells, and amount of blood vessels distribution were compared. All the samples were examined regarding different factors by the same pathologist who was also blinded to the groups. Each sample was investigated for capillary count under x400 magnification in different areas.

Statistical analysis Data are presented as mean  standard deviation and analyzed using the GraphPad Prism software (version 6.07) using one-way analysis of variance followed by post-hoc Tukey’s tests to analyze the data. Tests of homogeneity of variance were used to ensure normal distribution of the data. Probability (P) value less than 0.05 was considered significant.

Results Effect of acute sodium valproate administration on flap survival and necrosis length As shown in Figure 1 (A, B), on day 7, necrosis length decreased significantly in rats treated acutely with 100 mg/kg of sodium

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valproate (P < 0.05). The hair growth in control rats was better in shaving area than that in valproate-treated groups. Figure 1C illustrates the effect of acute administration of different doses of sodium valproate (50, 100, 150, or 300 mg/kg, i.p.) on necrosis length. Sodium valproate was administered 60 min before determination of skin ischemia induction. Acute sodium valproate (100 mg/kg, P < 0.05) significantly decreased the necrosis length compared with saline-treated control animals.

Effect of coadministration of TSA with chronic doses of sodium valproate on necrosis length Figure 2 shows the effect of chronic administration of sodium valproate (100 mg/kg, i.p.) on necrosis length. As shown in the figure, chronic administration decreased necrosis length better than acute (**P < 0.01). Simultaneous coadministration of TSA (330 nmol/kg, i.p.) with an effective dose of sodium valproate (100 mg/kg) reversed the potent protective effect of effective dose of sodium valproate (P < 0.05 versus sodium valproate), while the selective HDAC inhibitor TSA (330 nmol/ kg, i.p.) had insignificant changes in the skin flap survival compared with the control group (P > 0.05).

Effect of GABA antagonist on acute protective properties of effective dose of sodium valproate on necrosis length Administration of bicuculline, 20 mg/kg, as a potent antagonist of GABA receptors solely increased flap survival compared with the control group significantly (P < 0.05) (Fig. 3B). However, coadministration of bicuculline (10 mg/kg) and sodium valproate (100 mg/kg, i.p.) blocked the protective effect of sodium valproate, resulting in necrosis similar to that in the control group (P value > 0.05) (Fig. 3C).

Fig. 1 e (A) Control group and (B) treated group received 100 mg/kg of valproate and (C) comparative different doses of acute i.p. administration of sodium valproate on necrosis length in rat. Valproate decreased necrosis length and minimized skin flap 7 d after ischemia induction (mean ± SD in each group: 4.08 ± 0.51 cm in the control group and 3.22 ± 0.30 cm in the valproatetreated group). Control rats indicate better hair growth in shaving area, but alopecia is pronounced in valproate-treated groups. (C) Measuring the length of the necrotic area after passing of 1 wk after surgery revealed that single-dose injection of 100 mg/kg could diminish necrosis length and amplify flap survival (*P < 0.05). The effects were compared with those on a control group administered normal saline at the same time. Data are expressed as means ± SD of necrosis length in eight rats. *P < 0.05 compared with corresponding vehicle group. (Color version of figure is available online.) SD, standard deviation.

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Fig. 2 e (A) control; (B) coadministration of TSA and sodium valproate; (C) chronic treatment with sodium valproate for 2 wk, and (D) comparative necrosis length. Animals were operated at the same time, and after 1 wk, necrosis length decreased in treated groups (mean ± standard deviation of each group; 4.30 ± 0.057 cm in the control group, 3.14 ± 0.80 cm in sodium valproate-treated, 3.34 ± 0.29 cm in valproate and TSA combination therapy). As shown in D, coadministration of TSA (330 nmol/kg, i.p.) reversed the protective effects of sodium valproate effective dose (100 mg/kg) on skin flap. Each group consisted of at least 8 mice. **P < 0.01 and *P < 0.05 compared with vehicle/saline control group; #P < 0.05 compared with sodium valproate group. TSA alone did not lead to necrosis limitation. (Color version of figure is available online.)

Secondary healing Measuring the duration of wound healing in chronic valproate-treated group and control group revealed a significant (P < 0.01) result in secondary healing as represented in Figure 4D. Sodium valproate accelerates secondary healing. When scar length after 14 d was measured, it showed a significant (P < 0.01) decrease after treating rats with a chronic (100 mg/kg) valproate injection. As it is obvious in Figure 4C, both animals underwent simultaneous surgery, but valproatetreated rats recovered significantly after 14 d. While as Figure 4E comparison of scar length between the control group

and chronic combination therapy of TSA (330 nmol/kg) and sodium valproate (100 mg/kg) showed a significant (P < 0.05) remission, the effect of single therapy of TSA in scar length reduction was less than that of the combination therapy.

Weight gain and hair growth Fourteen days of injection of 100 mg/kg of sodium valproate decreased hair growth notably, and just 1 of 8 rats had full pattern of hair growth, but in the control group, 5 of 8 rats revealed a full pattern of hair growth. Also, weight gain was more in the valproate group (P < 0.05).

Fig. 3 e (A) control; (B) bicuculline (20 mg/kg); (C) bicuculline (10 mg/kg) D sodium valproate (100 mg/kg); and (D) comparative these groups necrosis length. Bicuculline (20 mg/kg) itself increased necrosis length (*P < 0.05) and combination therapy of subeffective dose of bicuculline (10 mg/kg) and valproate (100 mg/kg) could prevent the curative effect of valproate on skin flap model (##P < 0.01) (mean ± standard deviation of each group; 4.08 ± 0.51 cm in control group, 5.02 ± 0.70 cm in 20 mg/kg bicuculline, and 4.66 ± 0.076 cm in combination therapy of sodium valproate plus subeffective dose of bicuculline). Bicuculline was administered before i.p. injection of sodium valproate. Each group consisted of 8 mice. *P < 0.05; compared with saline control group. ##P < 0.01; compared with sodium valproate 100 mg/kg. (Color version of figure is available online.)

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Fig. 4 e Secondary healing and scar formation 2 wk after surgery. (A) control; (B) sodium valproate-treated; (C) (Left) treated with valproate; and (right) received normal saline. Valproate accelerated wound healing, decreased scar length, and suppressed hair growth (mean ± SD of scar length in the control group was 3.78 ± 0.92 cm and 2.42 ± 0.69 cm in valproatetreated group). Rats were operated at the same time. (D) Secondary healing progress was accelerated after chronic injection of 100 mg/kg of sodium valproate (***P < 0.001) and valproate-treated groups skin healed before control groups. (E) Scar length assessment 14 d after flap induction showed that treating rats with sodium valproate (100 mg/kg) decreased the scar length (**P < 0.01), although coadministration of valproate (100 mg/kg) and TSA (330 nmol/kg) decreased the effect of sodium valproate (*P < 0.05). The data represent the mean ± standard deviation of eight independent experiments. The statistical analysis was carried out using the one-way analysis of variance followed by Tukey’s test. *P < 0.05 and **P < 0.01 versus control. (Color version of figure is available online.)

Histopathology assay Comparing the microstructure of the skin showed that the epithelial layer had better structure after treating with valproate, considering the number of inflammatory cells, necrotic cells, regenerative cells, and vessels distribution.

Discussion After the vast number of surgeries and skin manipulation that can affect wound healing, different drugs with various mechanisms have been examined to minimize scar formation. In the present study, we demonstrated that sodium valproate can affect secondary healing through GABA stimulatory and HDAC inhibitory mechanisms.

As reported previously, valproate can relieve skin manifestations of Nelson syndrome.32e37 In this study, it has been demonstrated that sodium valproate administration, either acute or chronic, prevents skin degeneration and accelerates its regeneration. There are different possible mechanisms for these effects. Considering the most important known targets of sodium valproate led us to investigate the role of GABA and HDAC32e34 in a rat model of skin flap survival. After testing different dosages of sodium valproate as shown in Figure 1B, 100 mg/kg exhibited the best outcome. Furthermore, as shown in Figure 3, injection of a single-dose of 20 mg/kg of bicuculline (a GABA receptor antagonist35) significantly increased the length of necrosis (Fig. 3B). Coadministration of a subeffective dose of bicuculline with sodium valproate presented a fully different outcome in comparison with valproate alone, demonstrating that a GABA-dependent

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pathway might be involved in relieving skin injury induced by ischemia. Increasing neurons or other cells’ resting potential in skin tissue and preventing their stimulation36 might have been involved in this process. Different studies revealed that stimulating GABA receptors can modulate and suppress the inflammatory responses.10 GABA receptors express in both neurons and immune cells (which exist in skin tissue15), and they can suppress cytokine secretion and affect the migration of immune cells.37 Also, GABA and GABA type A receptor (GABA-A-R) agonists decrease cytotoxic immune responses and cutaneous delayed-type hypersensitivity reactions.38,39

So the GABA-ergic system is a considerable mechanism that valproate can act through to minimize skin inflammation and damage (Fig. 5). On the same note, chronic injection of TSA, as an HDAC inhibitor, could decrease the necrosis length and scar size, but its effect was less than that of a combination therapy of TSA and sodium valproate. Besides, the effect of combination therapy was less than that of a single therapy of sodium valproate (Fig. 2D). However, both valproate and TSA are HDAC inhibitors, and it seems they have different effects on the gene expression which may lead to expression of different proteins.

Fig. 5 e Cell survival by 2 wk, 100 mg/kg doses of sodium valproate was determined 2 wk after addition of ischemia using the H&E assay. (A) Control and (B) sodium valproate treated in different magnification. As shown in (A), epithelial cells disrupt, and there are more inflammatory cells. Interestingly, despite the control, there are not necrotic cells in the epithelial layer of valproatetreated animals. The presence of inflammatory cells in the control group is clearer. (Color version of figure is available online.)

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valproate impedes hair growth and amplifies weight gain. This study illustrated that sodium valproate may act through two mechanisms to achieve these effects: GABA-ergic and HDAC inhibitory system.

Acknowledgment This study was financially supported by Experimental Medicine Research Center, Tehran University of Medical Sciences and Health Services, Tehran, Iran (grant no. 96002757, 96-0330-35223) from the Iran National Science Foundation (INSF) (grant no. 96002757).

Fig. 6 e Effects of sodium valproate on the weight gain of animals. Comparing weight gain in the control group with the chronic injection of sodium valproate demonstrated that chronic administration of sodium valproate leads to excessive weight gain (P < 0.05). Results are shown as the mean ± SD.

Therefore, a consequent effect of simultaneous usage of valproate and TSA is that TSA might interact with the epigenetic pathways. It seems that inhibition of HDAC plays an important role in changing the outcome of skin flap. HDAC enzyme manipulates genes structure and modulates their expression,40,41 so HDAC inhibitors such as valproate and TSA modulate gene expression, and through this change, sodium valproate might reinforce expression of growth factors and structural proteins42 which are required for skin regeneration. In addition, HDAC inhibitors may decrease the expression of inflammatory factors and prevent their destruction. Fibroblasts, keratinocytes, vascular pericytes,43 and endothelial cells make the major part of the skin tissue, so the effect of sodium valproate might be associated with upregulation of growth factor such as fibroblast growth factor, vascular endothelial growth factor, transforming growth factor-b, keratinocyte growth factor, and epidermal growth factor.44e46 Understanding the effects of drugs such as sodium valproate and other gene expression modulators in skin regeneration process is important and may lead to development of new therapeutic pathways in future.47 As said in the results section, combination therapy of TSA and sodium valproate had a better outcome, so it shows that valproate acts through different mechanisms to reduce skin necrosis and accelerate its remission. In this study, treating rats with sodium valproate accelerated secondary healing, and we demonstrated that GABA-ergic system and HDAC inhibitory system might be involved in flap survival and regeneration of skin flaps. However, the mechanism of hair loss and weight gain by valproate (Fig. 6) might be different as these effects were not affected by bicuculline or TSA.

Conclusion These findings demonstrate that sodium valproate increases flap survival and accelerates wound healing. Also, sodium

Disclosure The authors have no conflict of interest to declare.

references

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