Ivermectin Increases Random-Pattern Skin Flap Survival in Rats: The Novel Role of GABAergic System
Mohammadreza Tabary, MD,a,b,1 Armin Aryannejad, MD,a,b,1 Nafise Noroozi, DVM,a,b Seyed Mohammad Tavangar, MD,c Razieh Mohammad Jafari, PhD,a Farnaz Araghi, MD,d Sahar Dadkhahfar, MD,d and Ahmad Reza Dehpour, PharmD, PhDa,b,*
a Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran
b Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
c Department of Pathology, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
d Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
a r t i c l e i n f o
Article history:
Received 5 May 2020 Received in revised form 1 September 2020
Accepted 22 September 2020 Available online xxx
Keywords: Ivermectin GABA a GABA B
Skin flap Rat
a b s t r a c t :
Background: Ivermectin (IVM) was first used as an antiparasitic agent; however, the role of this drug evolved into a broad spectrum. Many mechanisms have been proposed, including interaction with the GABAergic system. Considering the presence of GABA receptor in the skin tissue and its role in ischemia-reperfusion I/R injury, we aimed to evaluate the effect of IVM through GABA receptors on random-pattern skin flap survival.
Methods: Sixty Wistar male rats were used. Multiple doses of IVM (0.01, 0.05, 0.2, and 0.5 mg/ kg) were injected intraperitoneally before the surgery. Baclofen (selective GABAB agonist) and bicuculline (selective GABAA antagonist) were administered in combination with IVM to assess the role of the GABAergic system. Histopathological evaluations, immunohisto- chemical staining, quantitative assessment of IL-1b and TNFa, and the expression of GABAA a1 subunit and GABAB R1 receptors were evaluated in the skin tissue.
Results: IVM 0.05 mg/kg could significantly increase flap survival compared with the control group (P < 0.001). Subeffective dose of baclofen (0.1 mg/kg) had synergistic effect with the subeffective dose of IVM (0.01 mg/kg) (P < 0.001), whereas bicuculline 1 mg/kg reversed theeffect of IVM (0.05 mg/kg) (P < 0.001). IVM 0.05 mg/kg could also decrease the IL-1b andTNFa levels and increase the expression of GABAA a1 subunit and GABAB R1 receptors in the flap tissue compared with the control group.
Conclusions: IVM could improve skin flap survival, probably mediated by the GABAergic pathway. Both GABAA and GABAB receptors are involved in this process. This finding may repurpose the use of old drug, “Ivermectin.”
ª 2020 Elsevier Inc. All rights reserved.
Conflict of interest: None to declare.
* 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).
1 These two authors contributed equally to this work as first authors. 0022-4804/$ e see front matter ª 2020 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2020.09.010
Introduction
Random-pattern skin flap is a widely used approach in reconstructive surgeries for the closure of skin defects.1 After harvesting the flap, the blood supply depends greatly on dermal superficial and deep arterial plexuses.2 Necrosis, dehiscence, and flap failure are among the main complica- tions after random-pattern skin flap surgeries due to poor circulation.1 Ischemia-reperfusion (I/R) injury is one of the most studied biological processes involved in the progression of flap failure.3 Numerous techniques have been introduced to attenuate I/R injury, including ischemic and pharmacological preconditioning.4,5 Pharmacologic preconditioning refers to the administration of a drug before the global ischemia to improve the flap survival rate.6 Further, numerous drugs, including sumatriptan and sodium valproate, have been shown to increase flap survival through diverse mechanisms.7,8
Ivermectin (IVM) is a member of the avermectin’s family
introduced by Satoshi Omura in 1970. IVM is a derivative of naturally produced avermectin B1, composed of 80% 22,23- dihydro-avermectin B1a and 20% 22,23-dihydro-avermectin B1b.9,10 IVM was first used as an antiparasitic agent11; how- ever, the role of this drug evolved into a broad spectrum from regulating glucose and cholesterol level in diabetic mice12 to suppress malignant cell proliferation13 and inhibition of viral replication.14 Numerous mechanisms have been proposed to mediate these effects. Interaction with GABA type A (GABAA) receptor and farnesoid X receptor,15,16 acting via ligand-gated chloride channels, specifically those gated by glutamate (glutamate-gated chloride channels),17,18 and inhibiting WNT- TCF signaling in cancers19 are among the proposed mecha- nism of IVM action.
GABA, which is an inhibitory neurotransmitter, plays a roleboth in the nervous system and peripheral organs, including pancreas, thyroid, gastrointestinal tract, lung, testis, and liver.20,21 In addition, it has been shown that GABA plays an important protective role against the I/R injury in many or- gans, including the brain,22 spine,23 intestine,24 and kidney.25 Moreover, GABA receptors stimulation has been demon- strated to suppress inflammation,26 which is one of the main mechanisms involved in I/R injury.27 In the skin tissue, GABA interacts with mechanoreceptors.28 GABA receptors are also expressed in the skin tissue.29 In addition, it has been demonstrated that stimulation of GABAA and GABAB in the skin could have an antipruritic effect mediated through modulating immune cells.30Considering the presence of GABA receptor in the skin
tissue and its role in I/R injury, we aimed to evaluate the effect of IVM through mediating GABA receptors on random-pattern skin flap survival.
Pharmacology Department, Tehran University of Medical Sciences (Tehran, Iran). Animals were kept in the animal house, before and after surgeries, in transparent plastic cages at room temperature (23 2◦C), whereas they had free access to clean water and sufficient food. A 12/12-h light/dark cycle was also provided for the animals. Rats were generally anes- thetized before performing surgery and tissue sampling. They were kept warm during procedures using a lamp over the field,and artificial tear drops were used for the coverage of their eyes. All procedures and injections in each animal were per- formed far from the others to protect them against the stressful situation. This study was approved by the Ethics in Medical Research Committee of Tehran University of Medical Sciences (no. IR.TUMS.VCR.REC.46480). CO2 chambers were used to sacrifice the animals after performing the experiments.
Reagents
The combination of ketamine 10% (90 mg/kg) and xylazine 2% (9 mg/kg) was injected intraperitoneally (i.p.) to each rat to induce general anesthesia before any surgeries and tissue sampling; both were purchased from Alfasan (Woerden, Netherlands). IVM was received as a gift from (Gilaranco, Rasht, Iran). Bicuculline was purchased from Enzo Life Sci- ences (USA). Baclofen was purchased from Shimi Darou Kowsar (Tehran, Iran). IVM was dissolved in normal saline (0.9% NaCl [injectable]) by using propylene glycol as a solvent (with a total concentration of 10% [v/v]). Baclofen was dis- solved in dimethyl sulfoxide (DMSO) and was diluted with normal saline (DMSO concentration was 0.4% v/v). To dissolve bicuculline, chloroform with the final concentration of 2% v/v was used as a solvent. A weak acid (0.1 N HCl) was also added for better solubility (the final pH was approximately 4.5). Propylene glycol , DMSO, and HCl were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Solutions were injected (i.p.) before the surgeries based on the desired experiment.
Random-pattern skin flap model
A modified McFarlane skin flap model31 was performed in our study: after the induction of general anesthesia and shaving the dorsal hair of the rats, the dorsum of each rat was steril- ized, and a rectangular 8 × 2 cm area was marked with a
marker. The hip joints were used as the anatomical landmark
to place the caudal margin of the flap. The margins of the flap were incised gently using a sterilized surgical blade, except for the cephalad side, which remained attached in its original site. The main artery supplying the skin flap tissue from thecephalad side was carefully explored after elevating the flap
Materials and methods
Animals
Sixty male Wistar rats weighing 200-250 gr were used in this study. Animals were obtained from the animal house of the
tissue. The main artery was then cut to induce ischemia and establish a random-pattern skin flap. After that, the flap was left open in its site covered with a sterile gauze soaked with normal saline to ensure the ischemia. Then, the flap was su- tured back to the original site using simple stitches with 4/ 0 reverse cutting silk surgical sutures.
Study design
To investigate the most effective dose of IVM, multiple doses were administered before the surgeries in different groups. The most effective dose was initially injected based on the previous studies and then was adjusted as per the obtained results. In four different groups, 0.01, 0.05, 0.2, and 0.5 mg/kg IVM were injected (i.p.) 30 min before cutting the main artery, as mentioned previously. For the evaluation of the possible involvement of the GABAergic system on the skin flap sur- vival, baclofen was used as a selective GABAB receptor agonist. In three distinct groups, different doses of baclofen (0.1, 0.2, and 0.5 mg/kg) were administered 30 min before cutting the main artery. In a single group, the subeffective dose of IVM (which partially improved the skin flap survival) was coad- ministered with the subeffective dose of baclofen (a selective GABAB agonist) to examine the possible synergistic effect. In another experiment, the noneffective dose of bicuculline (a selective GABAA antagonist) was injected (i.p.) 30 min before the administration of the most effective dose of IVM (60 min before cutting the main artery), to evaluate the possible inhibitory role of bicuculline on the effect of the GABAergic system in skin flap survival. The control group just received
the vehicle (normal saline þ solvents) before the operation.
Data collection and tissue sampling
On the seventh day after performing the skin flap operation, rats were generally anesthetized using ketamine and xylazine with the same protocol. Digital photographs of the flap area on the dorsum of each rat were captured using the same camera and environmental condition for all the study groups. Healthy skin was defined as soft and flexible pink skin with evidence of new hair growth, while hardened and thickened skin with a dark colored tissue without evidence of new hair growth was considered as necrotic skin. The necrotic area of each flap was eventually calculated using ImageJ software version 1.52 (National Institutes of Health, USA) as follows: necrotic area
(%) ¼ (area of necrotic skin/area of whole flap) × 100.32 Tissuesampling was performed by excising a full-thickness skin from the intermediate margin between the necrotic area and the grossly healthy area (1.5 cm in length). Then, this segment was halved in width into two 1.5 × 1 cm segments. Onesegment was frozen immediately after sampling using a liquidnitrogen tank, then was moved to a —80◦C freezer to be used for further evaluations, including western blot and enzyme- linked immunosorbent assay (ELISA) tests. The other segment was stored in 10% formaldehyde to be prepared for histopathological studies (at least 48 h).
Histopathological studies
A pathologist blinded to the treatment groups incised the samples in width into three equal segments, which then were embedded in paraffin. From each paraffin-embedded block, three specimens were cut with a diameter of 5 mm. Sections were stained with hematoxylin and eosin (H&E) to be used for histopathological scoring. Moreover, Masson’s trichrome staining was used to evaluate fibrin deposition in each sam- ple. The H&E histopathological scoring system included fourmain domains, namely inflammation, edema, degeneration, and fibrosis. It was defined as follows: 0 ¼ normal, 1 ¼ mild, 2 ¼ moderate, 3 ¼ severe. Neutrophil count was carried out under × 100 magnification in 10 different areas considering the mean count for further evaluations, and Masson’s tri- chrome studies were performed under × 20 magnification. The lower the histopathological scores are the more tissueecovery has been achieved, except for fibrosis, which a higher score shows a better recovery and higher regeneration.
Immunohistochemistry studies for CD31: evaluating the angiogenesis
Platelet endothelial cell adhesion molecule, known as cluster differentiation 31 (CD31), is a protein that is found in large amounts in endothelial cells. As it is involved in the angio- genesis process, it is used as an angiogenesis marker in immunohistochemistry (IHC) studies.33 IHC for CD31 was performed in this study to compare the angiogenesis rate among experimental groups. Specimens were obtained from formalin-fixed paraffin-embedded blocks and were placed on poly-L-lysineecoated slides. Citrate buffer (10 mM citric acid and 0.05% Tween 20, pH 6.0) was used for rehydration and retrieval of the slides. Slides were then warmed up by mi-
crowave (with the power of 600 W) for two ten-minute periods and then were blocked in 1% BSA in 1 × TBS at room tem- perature for 2 h 1 × TBS contained 50 mM Tris-Cl and 150 mM NaCl. After these process, endogenous peroxidases blocking
was performed by 0.3% H2O2 in methanol, followed by 50 mL amounts of primary CD31 antibodies (1:100), which then was stored overnight at 4◦C. The slides were washed again with
TBS, and 50 mL of goat antirat antibodies (1:100) was added, left
1 h at room temperature; At this stage,3,30-diaminobenzidine was added as the chromogen, and hematoxylin staining was performed (to stain nuclei). The slides then were assessed under × 200 and × 400 magnificence with Olympus light mi- croscope BH2 (Olympus Corporation, Tokyo, Japan) and the cell count was performed manually in three hotspots far from
the granulation tissues, as described previously,34 and the mean cell counts were used for further analyses.
Measurement of IL-1b and TNFa
Quantitative assessment of IL-1b and TNFa was performed using rat-specific ELISA kits (DuoSet ELISA development sys- tem) purchased from R&D Systems (Minneapolis, Minnesota, United States) as per the protocols provided by the manufac- turer. The optical density was determined at 450 nm. The final concentration was then adjusted for protein concentration, and the final result was reported as pg/mg protein.
Western blot analysis for GABAA a1 subunit and GABAB R1 receptors
Skin flap tissue samples were homogenized and incubated in lysis buffer comprising Tris-HCl, EDTA, NaCl, sodium deoxy- cholate, SDS, protease inhibitor cocktail, and NP40. Then, the homogenates were centrifuged at 12,000 × g at 4◦C for 10 min.
The final supernatants were then used for SDS-PAGE. Equal
amounts of supernatants (20 mL) were loaded to 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranepurchased from Sigma-Aldrich (St. Louis, Missouri, USA). Then, membranes were blocked and shaken for 75 min at room temperature with nonfat skimmed milk 2% and TBST buffer. The membranes were then incubated overnight at 4◦C with GAPDH (Santa Cruz Biotechnology, California, USA), Anti- GABA A receptor alpha 1 antibody (Abcam, UK), and GABAB R1 polyclonal antibody (Elabscience, USA). The mem-
branes were again washed with TBST buffer three times and incubated with mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology, CA, USA) at 1:1000 dilution for 75 min at room temperature. Samples were then visualized using an ECL Western Blotting Kit (Roche; Mannheim, Germany). The opti- cal density of each band was analyzed using ImageJ software (NIH; USA) and normalized to GAPDH band density. In addi- tion, data were recorded for at least three independent samples.
Statistical analysis
Data of the necrotic area of skin flaps are reported in per- centage as mean SD in each group. One-way analysis of variances followed by Tukey’s post hoc test was performed to compare the mean percentages among the groups. Nonpara- metric Kruskal-Wallis tests were used to analyze the results of ELISA tests, western blots, and histopathologic scores. Ana- lyses were performed and demonstrated utilizing Prism soft- ware version 8.2.1 for macOS (GraphPad Software, San Diego, California, USA) and SPSS version 24 (Chicago, Illinois, USA). The significance was considered as a P value of less than 0.05.
Results
The protective effect of IVM on skin flap survival: a dose- response effect
The mean percentage of the necrotic area in the control group (vehicle-treated operated rats) was 74 4. Based on our pilot studies, lower doses of IVM could exert more effective results than the therapeutic doses introduced by previous studies, which were 0.2 mg/kg.35 So, we initiated our experiments with a dosage of 0.01 mg/kg of IVM, and then it was increased to investigate the possible dose-response effect of IVM. In our first experiment, IVM 0.01 mg/kg was administered (i.p.) 30 min before cutting the main artery of the skin flap segment. The mean percentage of the necrotic area reached 34 4,which was significantly lower than the control group (P < 0.01). Then, the dosage was increased, and IVM 0.05 mg/kg was administered to another group of rats. The mean per-centage of the necrotic area in this group, 7 d after the surgery, decreased drastically to 10 2, which was significantly lower than the control group (P < 0.001). The necrotic area in thisgroup was also significantly lower than the IVM 0.01 mg/kg
group (P < 0.001). After that, the dosage was increased to the therapeutic dose of IVM, which was mentioned in the previ- ous studies (0.2 mg/kg).35 In a single group of rats, IVM 0.2 mg/ kg was administered (i.p.) before the surgery. After 7 d, the mean percentage of the necrotic area was 64 2 in this group, which was remarkably higher than the IVM 0.05 mg/kg groupand still lower than the control group (P < 0.05). As we
demonstrated, the protective effect of IVM was attenuated by increasing the dosage. So, we again increased the dosage IVM to examine the dose-response effect further. In another experiment, IVM 0.5 mg/kg was injected before the surgery. The average percentage of the necrotic area after 7 d was 73 3 in this group, which was not significantly different from the control group (P > 0.05). The mean percentage of the
necrotic area for the previously mentioned experiments, as well as the digital photos, is shown in Figure 1.
The effects of baclofen on skin flap survival: evaluating the role of the GABAergic system
Baclofen was administered alone to three different groups in three different doses, based on our pilot studies: 0.1, 0.2, and
0.5 mg/kg. It also showed a dose-response effect, the same as the protective effect of IVM. The mean percentage of the necrotic area 7 d after the administration of baclofen 0.1 mg/ kg was 42 3, which was lower than the control group
(P < 0.01). In the baclofen 0.2 mg/kg group, the mean per-
centage was 15 2 (P < 0.001 versus the control), which was also significantly lower than the baclofen 0.1 mg/kg group (P < 0.001). The baclofen 0.5 mg/kg group had a mean per- centage of 72 2, which was not significantly different from the control group (P > 0.05) e The appearance of skin flaps 7 d after treatment with different doses ofivermectin and the mean percentage of the necrotic area in each treatment group compared with the control group (vehicle treated and operated rats). The results are shown based on mean ± SD.
***P < 0.001, **P < 0.01 and *P < 0.05 versus control group.
(Color version of figure is available online.)
The coadministration of IVM and baclofen: evaluating the role of GABAA receptor
In a single group, IVM 0.01 mg/kg was injected (i.p.) concur- rently with baclofen 0.1 mg/kg. The mean percentage of the necrotic area in this group was 11 1, which exerted a remarkable protective effect against necrosis compared with
the control group (P < 0.001). The mean percentage in this
group was also statistically lower than IVM 0.01 mg/kg (mean:
34 4, P < 0.001) and baclofen 0.1 mg/kg (mean: 42 3,
P < 0.001) (Fig. 2).
The coadministration of IVM and bicuculline: evaluating the role of GABAB receptor
In a single group of rats, bicuculline 1 mg/kg (non-effective dose) was injected (i.p.) before the administration of IVM
0.05 mg/kg. Seven days after skin flap surgery, the mean percentage of the necrotic area was 52 3 in this group, which was significantly higher than the mean percentage of the necrotic area in the IVM 0.05 mg/kg group (mean: 10 2,
P < 0.001). Figure 2 shows digital photos of skin flaps and compares the mean percentages of the necrotic area between the study groups.
Histopathologic studies
Figure 3 shows the H&E and Masson’s trichrome-stained slides, and Figure 4 illustrates the mean histopathologic scores for each group, based on the defined scoring system described earlier. Severe inflammation and epithelial degen- eration were observed in the control group, whereas the fibrosis was mild. The mean scores for the control group were 2.75, 2.25, 2.75, and 0.50 in terms of inflammation, edema, epithelial degeneration, and fibrosis, respectively. These values for IVM 0.05 mg/kg were 0.75, 0.5, 0.75, and 2.25, respectively, which were significantly different from the control group. In this group, the mildest inflammation was observed as well as mildest edema and epithelial degenera- tion. The most severe fibrosis was also found in this group. In the coadministration of IVM 0.05 mg/kg and bicuculline 1 mg/ kg, histopathologic scores were reversed significantly The appearance of skin flaps 7 d after treatment with subeffective and effective doses of ivermectin, subeffective and effective doses of baclofen (GABAB agonist), and the coadministration of the subeffective IVM (0.01 mg/kg) with subeffective baclofen (0.1 mg/kg) and the effective IVM (0.05 mg/kg) with bicuculline 1 mg/kg (GABAA antagonist). The percentage of the necrotic area in each group is shown and compared with the control group based on mean ± SD. ***P < 0.001, and **P < 0.01 versus the control group, ###P < 0.001 versus the IVM 0.01 mg/kg group, and &&&P < 0.001 versus the IVM 0.05 mg/kg. Bac: baclofen, Bicu: bicuculline. (Color version of figure is available online.) Histopathologic studies: H & E staining ( 3 200 magnificence) and Masson’s trichrome staining ( 3 40 magnificence) of different treatment groups [1-5] and immunohistochemical staining for CD31 in the control group and the most effective dose of IVM (0.05 mg/kg) [6]. 1a, control group (H & E), which shows severe infiltration of inflammatory cells and moderate to severe epithelial degeneration ( 3 200). 1b, control group (Masson’s trichrome), which shows mild fibrin deposition and fibrotic bundles in the dermis and hypodermis ( 3 40). 2a, IVM 0.01 mg/kg (subeffective) group (H & E), shows moderate to severe infiltration of inflammatory cells and mild epithelial degeneration and edema within the tissue ( 3 200). 2b, IVM 0.01 (subeffective) group (Masson’s trichrome), which shows mild fibrin deposition within the tissue ( 3 40). 3a, IVM 0.01 mg/ kg D baclofen 0.1 mg/kg group (H & E), which shows mild infiltration of inflammatory cells and almost no epithelial degeneration ( 3 200). 3b, IVM 0.01 mg/kg D baclofen 0.1 mg/kg group (Masson’s trichrome), which reveals marked fibrin deposition and fibrotic bundles throughout the tissue ( 3 40). 4a, IVM 0.05 mg/kg (the most effective dose) group (H & E), which shows mild inflammatory cell infiltration and epithelial degeneration ( 3 200). 4b, IVM 0.05 mg/kg (the most effective dose) group (Masson’s trichrome), which reveals significant fibrin deposition and fibrotic bundles throughout the tissue ( 3 40). 5a, IVM 0.05 mg/kg D bicuculline 1 mg/kg group (H & E), which shows moderate to severe infiltration of inflammatory cells, and moderate epithelial degeneration can be seen in the slide ( 3 200). 5b, IVM 0.05 mg/kg D bicuculline 1 mg/kg group (Masson’s trichrome), which shows mild to moderate fibrin deposition within the tissue ( 3 40). 6a, control group (IHC for CD31), in which scattered stained cells with CD31 can be seen within the tissue in comparison with 6b, IVM 0.05 mg/kg (the most effective dose) group that shows countless cells stained with CD31 IHC staining throughout the tissue ( 3 200) [stained cells are pointed with arrows in both 6a and 6b slides]. (Color version of figure is available online.)2.25, 2.5, 0.5, respectively. The mean scores for the baclofen0.2 mg/kg group and also the coadministration of IVM 0.01 mg/ kg þ baclofen 0.1 mg/kg were significantly better than the control group (P < 0.001). Masson’s trichrome studies revealed remarkably higher fibrin deposition within the samples of IVM0.05 mg/kg and the combination of IVM 0.01 mg/kg þ baclofen0.1 mg/kg compared with the other groups. The amount of fibrin deposition was lowered after the administration of bicuculline.
ELISA assay for IL-1ß and TNF-a
The mean IL-1ß and TNF-a levels in the skin flap tissue of the control group were measured as 384 16 pg/mg protein and 139 5 pg/mg protein, respectively. Although the adminis- tration of IVM 0.01 reduced the levels, it was not statistically significant in comparison with the control group. A remark- able reduction in the IL-1ß and TNF-a levels in the skin flap tissues was detected after the administration of IVM 0.05 mg/
Histopathologic scores in different treatment groups. The scores are defined as: 0 [ normal, 1 [ mild, 2 [ moderate, and 3 [ severe. The results are shown as mean ± SD. *P < 0.05 versus the control group, ***P < 0.001 versus the control group, ###P < 0.001 versus the IVM 0.01 mg/kg group, and &&&P < 0.001 versus the IVM 0.05 mg/kg group. Lower scores in terms of inflammation, edema, and epithelial degeneration and higher scores in terms of fibrosis show a better recoveryfrom the I/R injury. (Color version of figure is available online.)g; the mean levels of IL-1ß and TNF-a were 123 17 pg/mg protein and 39 4 pg/mg protein, respectively (P < 0.001 versus the control group, and P < 0.05 versus IVM 0.01). After thecoadministration of IVM 0.01 mg/kg and baclofen 0.1 mg/kg, the mean levels were measured as 162 24 pg/mg protein for IL-1ß, and 64 17 pg/mg protein for TNF-a in the skin flap tissues, which were also significantly lower than the control
group (P < 0.05) (Fig. 5).
Immunohistochemical studies for CD31
IHC staining for CD31 was performed to evaluate the angio- genesis within the skin flap tissues 7 d following the surgery. These studies revealed that remarkably numberless
ImageFig. 5 e Quantitative measurement of IL-1ß and TNF-a levels in the skin flap samples of different treatment groups (pg/mg protein). The results are illustrated based on the mean ± SD for each group. *P < 0.05 versus the control group, ***P < 0.001 versus the control group, and &P < 0.05 versus the IVM 0.01 mg/kg group.endothelial cells were stained in the IVM 0.05 mg/kg group compared to the control group. The stained cells were counted in the epithelial layer and also in dermis and hypodermis tissue, far from any possible granulation tissues (Fig. 3: 6a and6b [ × 200]). After the administration of bicuculline with theIVM 0.05 mg/kg, the number of stained endothelial cells was lowered significantly than the IVM 0.05 mg/kg alone. In the IVM 0.01 mg/kg þ baclofen 0.1 mg/kg group, the ntained cells was also remarkably higher than the controgroup in the epithelial and dermal layer of the skin flap tissue.
Western blot tests
To assess the expression of GABAA a1 subunit and GABAB R1 receptors, western blotting tests were performed. The data showed that the expression of both GABAA a1 subunit and GABA B R1 receptors increased significantly in the skin flap
tissue after the administration of IVM 0.05 mg/kg compared with the control group (P < 0.01). The coadministration of IVM
0.01 mg/kg and baclofen 0.1 mg/kg also exerted similar effects.
After the administration of bicuculline 1 mg/kg concurrently with IVM 0.05 mg/kg, the expression of both GABAA a1 subunit and GABA B R1 receptors raised significantly compared with the control group (P < 0.001 for both tests), whereas the expression of the GABAB R1 receptor was more prominent in this group (Fig. 6).
Discussion
Highlighting the main results, the present study indicated that IVM improved skin flap survival, probably mediated by the GABAergic system. In addition, a biphasic dose-response pattern was found between the IVM dosage and the necrotic Western blot analysis of GABAA a1 subunit and GABAB R1 in different skin flapesampled tissues from different treatment groups. Densities were normalized toGAPDH band density. **P < 0.01 versus the control group and ***P < 0.001 versus the control group.area. The most effective dose of IVM was 0.05 mg/kg in our study. We also found a higher expression of GABAA a1 subunit and GABAB R1 receptor after administration of the effective dose of IVM. Further, CD31 staining showed increased angio- genesis in the group treated with IVM (0.05 mg/kg) compared to the control group.
IVM, a derivative of naturally produced avermectin B1,9 is believed to act through many ligand-gated channels, including GABA receptors.15 GABA, which is primarily syn- thesized from glutamate, possesses three main types of re- ceptors, including two ionotropic receptors (GABAA and GABAc receptors) and a metabotropic receptor (GABAB recep- tor).36 It has been shown that GABA may play a protective role in I/R injuries, mainly in the cerebral tissue.37 This protective effect was described by the potential inhibitory role of enhanced GABA pathway from glutamate release.22 Some studies also showed the protective effect of GABA agonists against cerebral ischemia. For instance, baclofen has been shown to improve functional independence after stroke.38 In addition, GABA enhancers (rac-hopantenic acid and adaptol) remarkably improved psychopathologic, psychometric, and detailed somatic functions in patients with chronic cerebral ischemia39,40; however, these effects are highly dose related.41 The protective effect of the GABAergic system has also been studied in other tissues rather than the brain.
TheGABAergic system has been shown to play a crucial role in I/R injury of the spinal cord,42 gastric,43 intestinal,24 renal,44 he- patic,45 and myocardial46 tissue.
Recently, a study by Ala et al. showed that the GABAergic system could mediate the protective effect of sodium val- proate in skin flap survival.8 They found that GABAA antago- nist could reverse this protective effect, which is consistent with our findings. Many mechanisms have been proposed for this protective effect. First, increasing neurons or other cells’ resting potential in the skin tissue and preventing them from stimulation may play a role in this process.47 Our data also showed that IL-1ß and TNF-a concentrations in the skin tissue were lowered after the administration of IVM, indicating the suppression of inflammatory process, which is consistent with previous findings about the GABA role in the modulation of inflammatory responses.26 GABA receptors are expressed in immune cells, and they can suppress cytokine secretion.48,49
We found that both GABAA and GABAB receptors are
involved in this process. Baclofen, a selective GABAB agonist, had a synergistic effect with IVM. Further, bicuculline, a se- lective GABAA antagonist, could reverse the protective effect of IVM against skin flap survival. Previous studies have confirmed the role of both GABA receptors in I/R injury. One study showed that the coadministration of GABAA and GABAB receptors had a better result than given alone in the in vitro ischemia models.50 In addition, fewer adverse effects were observed when using both receptors agonist concurrently (e.g., coadministrations of etomidate and propofol; and co- administrations of zolpidem and diazepam).51 It can be claimed, with some justification, that to achieve better anti- ischemic effect, GABAA and GABAB receptors in the skin tis- sue should be stimulated before skin flap surgeries.
Western blotting was chosen to evaluate the level ofexpression of GABAA a1 subunit and GABAB R1 receptors as previous studies have shown that avermectin family might alter the expression of these receptors.52 Interestingly, we found a higher expression of GABAA a1 subunit and GABAB R1 receptors after the administration of IVM in random-pattern skin flap surgery. Previous studies also revealed a higher expression of GABA receptors after the administration of the avermectin family. A study by Chen et al. showed a higher expression of GABAA and GABAB receptors in pigeon after exposure to avermectin.52 Zhao et al. also found increased expression of the GABAA a1 subunit after exposure to aver- mectin in Carassius auratus gibelio.53 The expression rate of GABA receptors during I/R injury depends on many factors, including the target tissue and the time passed after injury. One study showed a higher expression of the GABAA receptor in caudate putamen, and at the same time, no changes were found in cerebral cortexes in the rat cerebral ischemia model.54 In terms of time, a study by Neumann-Haefelin showed that the protein content of the a1 subunit decreased 7 d after I/R injury in rats with brain ischemia,55 although the increased expression of GABAA receptor subunits was confirmed.56 Thus, we can conclude that the expression of GABA receptors depends strongly on the target tissue and the time after I/R injury. In the skin, we observed higher expres- sion of GABAA a1 subunit and GABAB R1 7 d after surgery; however, more studies are needed to detect these levels more religiously before and after this time point.
On the other hand, expression of GABAA a1 subunit and GABAB R1 receptors has increased after the administration of bicuculline. This phenomenon is called antagonist-induced increase in receptor expression and was shown in other re- ceptors such as serotonin 1A (5-HT1A) receptor after the administration of its specific antagonist.57
Studies have shown that angiogenesis in flap tissue can be assessed using CD31 antibody staining.58 In this case, we found increased angiogenesis after the administration of IVM. This effect can also be justified by the role of the GABA signaling pathway in angiogenesis. A study by et al. showed important angiogenesis signaling pathways might be either under the direct control of or actively interacting with the endothelial GABA signaling pathway.59 This shows that the GABAergic pathway could increase flap survival probably by increasing angiogenesis.
Authors’ contribution: A.D. contributed to study concep- tion and design. M.T., A.A., N.N., and S.T. contributed to acquisition of data. M.T., A.A., and R.M. contributed to anal- ysis and interpretation of data. M.T., A.A., F.A., R.M., A.D., and
S.D. contributed to drafting of manuscript. R.M., A.D., S.T., and
S.D. contributed to critical revision.
Disclosure
This work was supported by a grant from the Experimental Medicine Research Center, Tehran University of Medical Sci- ences (Grant No. 99-1-101- 46480).
r e f e r e n c e s
As inflammation is another factor which contribute to the Bicuculline
survival of skin flap tissue,60 measuring its level may help to better understand the response to anti-ischemic drugs. Pre- vious studies used IL-1ß and TNF-a in flap tissue to show the level of inflammatory response.61 We also observed decreased levels of both IL-1ß and TNF-a in treatment groups with the maximal effect (the IVM 0.05 mg/kg group and the IVM þ baclofen group) compared with controls. This can un- derscore the role of inflammation in skin flap survival; in other words, anti-ischemic drugs which possess an anti- inflammation property may be a better choice for pharmaco- logic preconditioning.
In this study, we showed that IVM improved skin flap survival, probably mediated by the GABAergic system. We also indicated that both GABAA and GABAB receptors are involved in this process. Indeed, this study has some limitations. Many other mechanisms have been proposed for IVM, including chloride channels,62 WNT-TCF signaling pathway,19 and the farnesoid X receptor.16 Evaluating these mechanisms were out of the scope of this study. In addition, our study was an experimental study, and all the limitations of the experi- mental studies are also applicable to ours.
Conclusion
Ivermectin could improve skin flap survival, probably medi- ated by the GABAergic system. Both GABAA and GABAB re- ceptors are involved in this process. This finding may repurpose the use of the old drug, “IVM.”
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Acknowledgment
Funding: This work was supported by a grant from Experi- mental Medicine Research Center, Tehran University of Medical Sciences (Grant No. 99-1-101- 46480). Special thanks to Iran National Science Foundation (INSF) for their support.
Conflict of interest: None to declare.
Availability of data: Data will be shared by request. Ethical approval: This study was approved by the Ethics in
Medical Research Committee of Tehran University of Medical Sciences (no. IR.TUMS.VCR.REC.46480).
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