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Dietary nitrate protects skin flap against ischemia injury in rats via enhancing blood perfusion
Biochemical and Biophysical Research Communications 515 (2019) 44e49
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dietary nitrate protects skin flap against ischemia injury in rats via enhancing blood perfusion Hao Cui a, b, Yaozhong Wang c, Yuanyong Feng a, b, Xiao Li a, b, Lingxue Bu a, b, Baoxing Pang a, b, *, Muyun Jia a, b, ** a b c
Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266003, PR China School of Stomatology of Qingdao University, Qingdao, 266003, PR China Department of Oral and Maxillofacial Surgery, Qingdao Stomatological Hospital, Qingdao, 266001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 May 2019 Accepted 7 May 2019 Available online 21 May 2019
Insufficient blood supply is associated with high levels of necrosis in reconstructive surgery. Restoring blood flow to undersupplied ischemic tissue is the most important impact factor determining skin flap viability. Dietary nitrate, a significant source of nitric oxide, has multiple physiological functions, including regulator of blood flow, angiogenesis, and vasodilatation. However, the effects of dietary nitrate on ischemic skin flap remain unknown. The present study evaluated whether dietary nitrate supplementation altered blood flow of ischemic skin flap in rats. Our results showed that nitrate treatment significantly enhanced ischemic tissue survival. Mechanistically, nitrate therapy significantly increased serum nitrate and nitrite levels, blood perfusion, and angiogenesis. In addition, the circulating levels of Inflammatory mediators were decreased by nitrate supplementation. In conclusion, we demonstrated that dietary nitrate supplementation protected ischemic skin flap by enhancing ischemia-induced revascularization. © 2019 Elsevier Inc. All rights reserved.
Keywords: Nitrate Nitric oxide Blood perfusion Skin flap Microvascular density
1. Introduction The transplantation of skin flaps is used as an important option to cover skin wounds and defect in reparative and reconstructive surgery [1,2]. The random skin flaps have been used more frequently because of its advantage of texture and color compatibility [3,4]. However, flap necrosis is the most common clinical complication in skin flap transplantation [5]. A series of pathological changes are involved in flap necrosis including inadequate blood flow, unpredictable vasospasm, thrombosis, as well as microvascular dysfunction [6,7]. In particular, restoring blood supply is a central role in vascularization supplied ischemic flap. Hence, to solve this problem, various pharmacological
* Corresponding author. Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266003, Jiangsu Road No.16, PR China. ** Corresponding author. Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266003, Jiangsu Road No.16, PR China. E-mail addresses: [email protected] (B. Pang), [email protected] (M. Jia). https://doi.org/10.1016/j.bbrc.2019.05.059 0006-291X/© 2019 Elsevier Inc. All rights reserved.
manipulations have been considered and performed for their efficacy in increasing blood flow to prevent tissue ischemia [8,9]. Nevertheless, no effective pharmacological therapy is currently available for flap necrosis. Nitric oxide (NO) is a bioactive gas that regulates various physiological responses. It plays a dominant role in the dilation of vessels and in the regulation of blood flow. NO thereby maximizes blood flow to ischemic tissues and minimizes tissue loss [10]. Dietary nitrate can be rapidly absorbed in the upper gastrointestinal tract, recycled in salivary glands, converted to nitrite by oral bacteria, and further metabolized to NO in the acidic stomach [11,12]. It has been reported that nitrate and nitrite serve as biological reservoirs for NO in hypoxia or acidic conditions [13]. Furthermore, many studies have suggested that dietary nitrates have the potential to prevent cardiovascular disease, ischaemic conditions, and high blood pressure in animal models and humans. Nitrate also improves hind limb blood flow during exercise in rats [14]. However, whether dietary nitrate might upregulate the skin flap blood flow to such an extent as to protect the skin flap from ischemic injury is still unclear. In this study, we investigated the effects of dietary nitrate on ischemia-induced skin flap injury in rats. Here, we report for the first time that nitrate may play an
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important role in protection against ischemia-induced skin flap injury via increasing blood perfusion.
perfusion change rate ¼ blood perfusion value measured day 1 or 3 or 7/blood perfusion value measured before operation 100).
2. Materials and methods
2.6. Determination of serum nitrate and nitrite content
2.1. Experimental animals and groupings
Blood was collected and centrifuged at 1500 g for 20 min at room temperature to obtain serum, then filtered and diluted prior to assaying. The concentrations of nitrate and nitrite in these samples were detected by Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit (PKGE001, R&D, USA) followed by standard experimental procedures [16].
Male adult Wistar rats (6e8 weeks of age; 200e250 g) were kept under standardized conditions at 21e22 C with 12-h light/ dark cycles. The rats were allowed to adapt to this environment in mesh-bottom cages with free access to distilled drinking water and regular pellet food for at least 7 days before the experiment. Twenty-four rats were randomly selected for three groups (n ¼ 8): the nitrate (5 mmol/L in distilled water), NaCl (5 mmol/L NaCl in distilled water), and control (distilled water) groups. All animal experiments were approved by the ethics committees of Labratory Animals of the Affiliated Hospital of the Qingdao University. 2.2. Nitrate supplementation Sodium nitrate (NaNO3) was dissolved in the distilled water (5 mmol/L) and was administered to the nitrate group for 14 days. The NaCl group was administered distilled water containing sodium chloride (NaCl) of the same dose. Animals underwent skin flap surgery on day 7. At day 14, all rats were subjected to blood sampling and histological analyses. 2.3. Flap ischemia model All rats were kept in a dark and quiet environment for 15 min before any intervention. Intramuscular administration of ketamine(100 mg/kg) and xylazine (10 mg/kg) was used for the anesthesia. After the removal of the hair and conventional disinfection, a caudal-based 3 cm 9 cm skin/panniculus carnosus flap was separated from the underlying fascia on the back of rats using the modified McFarlane Flap model (in the same position in all rats) [15]. Both sacral arteries supporting the blood supply of this flap model were sectioned completely. Immediately, skin flaps were sutured to the donor bed using a wedged-on cutting needle and continuous 4-0 silk sutures.
2.7. Histological staining The skin flaps in full thickness with subcutaneous tissues were dissected from sacrificed rats at postoperative day 7 for histological assessment. The samples (1 cm 1 cm) of central tissue from each flap were fixed in 4% for 24 h, embedded in paraffin and cut into 5mm sections for hematoxylin-eosin staining (H&E). 2.8. Immunohistochemistry For immunohistochemical staining, tissues were fixed in 4% paraformaldehyde, incubated in 3% H2O2 for 20 min, blocked in 5% BSA for 2 h, and stained with primary antibodies against CD34 (1:100; ab185732, Abcam, Cambridge, MA, USA) for 8 h at 4 C. After that, biotinylated goat anti-rabbit IgG serum (Beijing Zhongshan Golden Bridge Biotechnology Co., LTD, Beijing, China) were successively added to the sections for 3 h each at room temperature. Positive immune staining was detected using a DAB Substrate Kit (8059S, Cell Signaling Technology, Danvers, MA, USA) followed by standard experimental procedures. Microvascular density was calculated using the Image J software (NIH). 2.9. ELISA The levels of TNF-a and IL-6 in serum were measured according to the manufacturer's instructions. The ELISA kits were obtained from R&D Systems, Minneapolis, MN using the SpectraMax® M3 Microplate Reader (Molecular Devices, Sunnyvale, CA). Results were expressed as pg/ml.
2.4. Flap survival assessment 2.10. Statistical analysis The survival rate of each skin flap was observed on 1, 3, 7 days after the operation. Rats were anesthetized, and skin flap photos were taken with a digital camera. After the gross observation of the appearance of the skin flaps, zones of dark color and covered scabs were defined as necrotic area and the other zones of the skin flap were defined as the surviving area. To assess the percentage of the surviving area, the digital image was further processed with planimetric evaluation using Image-Pro Plus Software (Media Cybernetics, MD, USA). The results are expressed as a percentage of the surviving area relative to the total surface area of the skin flap (survival rate ¼ surviving area/total area 100%). 2.5. Measurement of blood perfusion The principle of laser Doppler flowmetry (LDF) for the assessment of skin flap blood flow was described previously [1]. A Moor VMS-LDF (Moor Instrument, UK) was used. Measurements were performed four times (before the operation, and 1, 3, 7 days after the operation) at two positions (1 and 5 cm distal from the baseline of the skin flaps) on the middle line of the skin flaps. The blood perfusion change rate was expressed as the percentage of the value obtained before the operation at the same position (blood
The data were expressed as the mean ± standard deviation. Differences between groups were analyzed by one-ANOVA followed by post-hoc Tukey's test, p < 0.05 was considered to be statistically significant. Statistical analyses were performed using SPSS version 23. 3. Results 3.1. Dietary nitrate improved the survival rate of skin flaps We observed the appearance of the skin flaps after the operation. On day 1 of postoperation, The distal regions became dark purple without obvious necrosis (Fig. 1A). On day 3, necrosis began to appear clear in the distal regions and the necrotic area was clearly distinguished from the survival area (Fig. 1B). On postoperative day 7, the circumscriptions between the necrotic and survival regions of the skin flap were more clearly identified. The necrotic parts became hard, shrink, and discolored (Fig. 1C). One day after the operation, skin flaps were treated nitrate exhibited larger survival areas than the other groups. However, the difference was not statistically significant at this time (Fig. 1D). The
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Fig. 1. Effect of dietary nitrate on survival rate of the ischemic skin flap. (AeC) Appearance of the ischemic skin flaps. (D) Flap survival rate at postoperative day 1. (E) Flap survival rate at postoperative day 3. (F) Flap survival rate at postoperative day 7. Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05; **p < 0.01 versus control group.
differences in the survival area became more robust on 3 and 7 days after the operation. The skin flaps were treated with nitrate showed significantly less tissue necrosis versus skin flaps treated with NaCl or untreated skin flaps (Fig. 1E and F). 3.2. Dietary nitrate increased nitrate and nitrite levels and decreased histological lesions Intake of nitrate by drinking water showed an increase in serum nitrate and nitrite levels compared with the control and NaCl groups.(p < 0.05) (Fig. 2A). To further investigate the effects of nitrate on skin flap, the ischemia skin flaps treated or not with nitrate for 14 days were analyzed by histochemistry. H&E staining revealed that the nitrate-treated tissues showed more neovascularization and less inflammatory cell infiltration (Fig. 2B). 3.3. Dietary nitrate increased blood perfusion and vessel density The rate of blood perfusion change at 1 cm distal flap from the baseline of the skin flaps showed a tendency to increase in a timedependent manner without a significant difference among the groups (Fig. 3A). At 5 cm, the results also showed a tendency to increase in a time-dependent manner. The blood perfusion change rate dropped severely after operation regardless of nitrate administration. On a postoperative day 3 and 7, the blood perfusion change was significantly improved in nitrate treated rats compared with those in NaCl and control rats (Fig. 3B). To investigate whether the improvement in tissue perfusion was mediated in part by increased revascularization, blood density was determined 7 days after the operation. Thus, we examined the expression of endothelial cell marker CD34 by immunohistochemistry. The vessel density was directly reflected in the number of CD34-positive vessels/mm2. The vessel density was higher in the nitrate-treated group compared with controls. (p < 0.05) (Fig. 3C).
3.4. Dietary nitrate attenuated inflammatory mediators The inflammatory response plays an important role in the wound healing process. To demonstrate whether dietary nitrate can decrease the inflammatory response, serum TNF-a and IL-6 levels were detected using ELISA kits. The results revealed that levels of TNF-a and IL-6 were significantly decreased by nitrate treatment (Fig. 4A and B).
4. Discussion Skin flap ischemia injury, a common complication of repairing soft tissue injuries, requires remodeling of the blood flow to sustain its viability [17]. The physiological repair response, however, is often insufficient, and therapeutic blood reperfusion remains an unmet medical need. In the present study, we demonstrate that dietary nitrate supplementation significantly enhances the blood perfusion change rate as well as the survival rate following skin flaps ischemia injury. These salutary effects are coupled to an altered inflammatory response with the raised generation of NO and changes in microvascular. This is the first description of the beneficial effects of nitrate-enriched diet in protecting the flaps against ischemic insult. Inorganic nitrate has emerged as a dietary component that can regulate diverse bodily functions in physiological activity. Numerous experimental studies have demonstrated the dietary nitrate supplementation defense of the gastric mucosa against injury and increase the gastric mucosal blood flow [18e20]. Another research demonstrated that dietary nitrate supplementation strongly augments blood perfusion recovery in the ischemic hind-limb [21]. Bier, S.C. et al. also declared that nitrite therapy effectively stimulates ischemic tissue vascular remodeling in the setting of metabolic dysfunction [22]. Moreover, Other studies demonstrating the anti-inflammatory effects of dietary nitrate and nitrite are emerging in different experimental inflammatory
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Fig. 2. Dietary nitrate increased nitrate and nitrite levels and decreased histological lesions. (A, B) Nitrate and nitrite levels after 7-day nitrate feed. (C) H&E staining of the skin flap. Scale bar ¼ 100 mm, magnification:40 (upper); Scale bar ¼ 200 mm, Original magnification:100 (lower). Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05; **p < 0.01 versus control group.
diseases [23,24]. Studies in animal models of ischemia and reperfusion have now revealed a central role of nitrate in hypoxia signaling [25]. The fact that the doses of nitrate used in these studies are easily achieved by the diet brings an interesting nutritional aspect to this research. In our study, we chose to use random skin flap in order to ischemic injury and be able to study the blood reperfusion effect of dietary supplementation with sodium nitrate. Nitric oxide (NO) is not only a vasodilator but also is known to mediate angiogenesis [26]. NO plays a key role in the regulation of cardiovascular function, cellular energetics, neurotransmission, and immune function [27]. Increased nitrate and nitrite levels in the blood may serve as NO reservoir for maintaining the systemic NO 3NO-2-NO homeostatic balance in the body. The L-arginine-NOS pathway is oxygen dependent, whereas the nitrate-nitrite-NO pathway is gradually activated as oxygen tensions fall. In this sense, NOS-independent NO formation when oxygen and L-arginine supply is limited [28]. Nitrite appears to signal protection through pathways distinct from those classicly ascribed to NO [29]. Thus, dietary nitrate may protect against skin flap ischemia injury associated with blood flow deficiencies by regulating NO homeostasis in the body. Our present study also demonstrated that dietary nitrate exerts skin flap protective effects through increased NO-like effects of nitrate and nitrite. The mechanism by which nitrate enhances the blood reperfusion of ischemic skin flaps seems to involve its angiogenesis effect. Pharmacological treatment with nitrate, an oxidation product of NO, may offer an alternative therapeutic opportunity. Evidence suggests that dietary nitrate supplementation increases the
regenerative capacity of ischemic tissue and that this effect may offer a strategy to improve ischemia-induced revascularization [30]. Further investigations in ischemic tissues highlighted the potential of dietary nitrate in angiogenesis [21,31]. Improving angiogenesis and increasing blood supply to ischemic areas was considered to promote the survival of random skin flap [32]. Here, we found that vessel density from immunohistochemistry results for CD34 showed more neovascularization in skin flaps by nitrate treatment. Thus, we thought that dietary nitrate supplementation augments blood perfusion recovery in ischemic skin flap may have resulted from a significant increase in blood vessel density. Alleviated ischemia injury of skin flap was accompanied by decreased inflammatory cytokine levels. Inflammation is a major contributor to cell death after ischemia of skin flaps since it potentiates cellular apoptosis. The greater the extent of inflammation, the more pronounced the necrosis, ultimately compromising flap success [33]. The inflammation reaction due to tissue ischemia induces damage to the blood vessels and exacerbate ischemia, which creates a vicious circle [34]. Interleukins facilitate cellular infiltration, leading to increased neutrophil counts that eventually cause tissue damage by releasing various cytokines and proteolytic enzymes. Therefore, the examination of pro-inflammatory cytokine regulatory factors can prove instrumental for effective treatment of ischemia. The current study examined that dietary nitrate decreased the levels of TNF-a and IL-6. Thus, the role of nitrate alleviating skin flap ischemia injury is related to lessening the levels of proinflammatory mediators. Nitrate and nitrite were previously considered to be harmful
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Fig. 3. Dietary nitrate improved blood perfusion and microvascular density. (A) Blood perfusion change rates at 1 cm distal from the baseline of the skin flaps. (B) Blood perfusion change rates at 5 cm distal from the baseline of the skin flaps. (C) CD34-positive vessels as assessed by immunohistochemistry. The arrows showing CD34 positive cells. (Scale bar ¼ 50 mm, Original magnification 400 ). Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05 versus control group.
constituents in the development of gastric cancer and other malignancies [35,36]. However, recent reports have indicated no harm to human health of normal dietary nitrate [37e39]. Moreover, green vegetables are the major source of inorganic nitrate. Thus, dietary nitrate could be a promising preventive and therapeutic strategy in clinical trials. In conclusion, dietary nitrate can effectively protect the skin flap against ischemia-induced injury. This protection by nitrate is related to the acceleration of blood flow recovery, stimulation angiogenesis, and inhibition of the inflammatory response. However, the specific mechanism of the protective effect by nitrate requires further exploration. Conflicts of interest There are no competing interests. Acknowledgments This work was supported by Shandong Provincial Natural Science Foundation (ZR2017BH034). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.059 References
Fig. 4. Dietary nitrate attenuated Inflammatory mediators in serum. (A) The levels of the TNF- a in serum. (B) The levels of the IL-6 in serum. Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05; **p < 0.01 versus control group.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dietary nitrate protects skin flap against ischemia injury in rats via enhancing blood perfusion Hao Cui a, b, Yaozhong Wang c, Yuanyong Feng a, b, Xiao Li a, b, Lingxue Bu a, b, Baoxing Pang a, b, *, Muyun Jia a, b, ** a b c
Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266003, PR China School of Stomatology of Qingdao University, Qingdao, 266003, PR China Department of Oral and Maxillofacial Surgery, Qingdao Stomatological Hospital, Qingdao, 266001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 May 2019 Accepted 7 May 2019 Available online 21 May 2019
Insufficient blood supply is associated with high levels of necrosis in reconstructive surgery. Restoring blood flow to undersupplied ischemic tissue is the most important impact factor determining skin flap viability. Dietary nitrate, a significant source of nitric oxide, has multiple physiological functions, including regulator of blood flow, angiogenesis, and vasodilatation. However, the effects of dietary nitrate on ischemic skin flap remain unknown. The present study evaluated whether dietary nitrate supplementation altered blood flow of ischemic skin flap in rats. Our results showed that nitrate treatment significantly enhanced ischemic tissue survival. Mechanistically, nitrate therapy significantly increased serum nitrate and nitrite levels, blood perfusion, and angiogenesis. In addition, the circulating levels of Inflammatory mediators were decreased by nitrate supplementation. In conclusion, we demonstrated that dietary nitrate supplementation protected ischemic skin flap by enhancing ischemia-induced revascularization. © 2019 Elsevier Inc. All rights reserved.
Keywords: Nitrate Nitric oxide Blood perfusion Skin flap Microvascular density
1. Introduction The transplantation of skin flaps is used as an important option to cover skin wounds and defect in reparative and reconstructive surgery [1,2]. The random skin flaps have been used more frequently because of its advantage of texture and color compatibility [3,4]. However, flap necrosis is the most common clinical complication in skin flap transplantation [5]. A series of pathological changes are involved in flap necrosis including inadequate blood flow, unpredictable vasospasm, thrombosis, as well as microvascular dysfunction [6,7]. In particular, restoring blood supply is a central role in vascularization supplied ischemic flap. Hence, to solve this problem, various pharmacological
* Corresponding author. Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266003, Jiangsu Road No.16, PR China. ** Corresponding author. Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266003, Jiangsu Road No.16, PR China. E-mail addresses: [email protected] (B. Pang), [email protected] (M. Jia). https://doi.org/10.1016/j.bbrc.2019.05.059 0006-291X/© 2019 Elsevier Inc. All rights reserved.
manipulations have been considered and performed for their efficacy in increasing blood flow to prevent tissue ischemia [8,9]. Nevertheless, no effective pharmacological therapy is currently available for flap necrosis. Nitric oxide (NO) is a bioactive gas that regulates various physiological responses. It plays a dominant role in the dilation of vessels and in the regulation of blood flow. NO thereby maximizes blood flow to ischemic tissues and minimizes tissue loss [10]. Dietary nitrate can be rapidly absorbed in the upper gastrointestinal tract, recycled in salivary glands, converted to nitrite by oral bacteria, and further metabolized to NO in the acidic stomach [11,12]. It has been reported that nitrate and nitrite serve as biological reservoirs for NO in hypoxia or acidic conditions [13]. Furthermore, many studies have suggested that dietary nitrates have the potential to prevent cardiovascular disease, ischaemic conditions, and high blood pressure in animal models and humans. Nitrate also improves hind limb blood flow during exercise in rats [14]. However, whether dietary nitrate might upregulate the skin flap blood flow to such an extent as to protect the skin flap from ischemic injury is still unclear. In this study, we investigated the effects of dietary nitrate on ischemia-induced skin flap injury in rats. Here, we report for the first time that nitrate may play an
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important role in protection against ischemia-induced skin flap injury via increasing blood perfusion.
perfusion change rate ¼ blood perfusion value measured day 1 or 3 or 7/blood perfusion value measured before operation 100).
2. Materials and methods
2.6. Determination of serum nitrate and nitrite content
2.1. Experimental animals and groupings
Blood was collected and centrifuged at 1500 g for 20 min at room temperature to obtain serum, then filtered and diluted prior to assaying. The concentrations of nitrate and nitrite in these samples were detected by Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit (PKGE001, R&D, USA) followed by standard experimental procedures [16].
Male adult Wistar rats (6e8 weeks of age; 200e250 g) were kept under standardized conditions at 21e22 C with 12-h light/ dark cycles. The rats were allowed to adapt to this environment in mesh-bottom cages with free access to distilled drinking water and regular pellet food for at least 7 days before the experiment. Twenty-four rats were randomly selected for three groups (n ¼ 8): the nitrate (5 mmol/L in distilled water), NaCl (5 mmol/L NaCl in distilled water), and control (distilled water) groups. All animal experiments were approved by the ethics committees of Labratory Animals of the Affiliated Hospital of the Qingdao University. 2.2. Nitrate supplementation Sodium nitrate (NaNO3) was dissolved in the distilled water (5 mmol/L) and was administered to the nitrate group for 14 days. The NaCl group was administered distilled water containing sodium chloride (NaCl) of the same dose. Animals underwent skin flap surgery on day 7. At day 14, all rats were subjected to blood sampling and histological analyses. 2.3. Flap ischemia model All rats were kept in a dark and quiet environment for 15 min before any intervention. Intramuscular administration of ketamine(100 mg/kg) and xylazine (10 mg/kg) was used for the anesthesia. After the removal of the hair and conventional disinfection, a caudal-based 3 cm 9 cm skin/panniculus carnosus flap was separated from the underlying fascia on the back of rats using the modified McFarlane Flap model (in the same position in all rats) [15]. Both sacral arteries supporting the blood supply of this flap model were sectioned completely. Immediately, skin flaps were sutured to the donor bed using a wedged-on cutting needle and continuous 4-0 silk sutures.
2.7. Histological staining The skin flaps in full thickness with subcutaneous tissues were dissected from sacrificed rats at postoperative day 7 for histological assessment. The samples (1 cm 1 cm) of central tissue from each flap were fixed in 4% for 24 h, embedded in paraffin and cut into 5mm sections for hematoxylin-eosin staining (H&E). 2.8. Immunohistochemistry For immunohistochemical staining, tissues were fixed in 4% paraformaldehyde, incubated in 3% H2O2 for 20 min, blocked in 5% BSA for 2 h, and stained with primary antibodies against CD34 (1:100; ab185732, Abcam, Cambridge, MA, USA) for 8 h at 4 C. After that, biotinylated goat anti-rabbit IgG serum (Beijing Zhongshan Golden Bridge Biotechnology Co., LTD, Beijing, China) were successively added to the sections for 3 h each at room temperature. Positive immune staining was detected using a DAB Substrate Kit (8059S, Cell Signaling Technology, Danvers, MA, USA) followed by standard experimental procedures. Microvascular density was calculated using the Image J software (NIH). 2.9. ELISA The levels of TNF-a and IL-6 in serum were measured according to the manufacturer's instructions. The ELISA kits were obtained from R&D Systems, Minneapolis, MN using the SpectraMax® M3 Microplate Reader (Molecular Devices, Sunnyvale, CA). Results were expressed as pg/ml.
2.4. Flap survival assessment 2.10. Statistical analysis The survival rate of each skin flap was observed on 1, 3, 7 days after the operation. Rats were anesthetized, and skin flap photos were taken with a digital camera. After the gross observation of the appearance of the skin flaps, zones of dark color and covered scabs were defined as necrotic area and the other zones of the skin flap were defined as the surviving area. To assess the percentage of the surviving area, the digital image was further processed with planimetric evaluation using Image-Pro Plus Software (Media Cybernetics, MD, USA). The results are expressed as a percentage of the surviving area relative to the total surface area of the skin flap (survival rate ¼ surviving area/total area 100%). 2.5. Measurement of blood perfusion The principle of laser Doppler flowmetry (LDF) for the assessment of skin flap blood flow was described previously [1]. A Moor VMS-LDF (Moor Instrument, UK) was used. Measurements were performed four times (before the operation, and 1, 3, 7 days after the operation) at two positions (1 and 5 cm distal from the baseline of the skin flaps) on the middle line of the skin flaps. The blood perfusion change rate was expressed as the percentage of the value obtained before the operation at the same position (blood
The data were expressed as the mean ± standard deviation. Differences between groups were analyzed by one-ANOVA followed by post-hoc Tukey's test, p < 0.05 was considered to be statistically significant. Statistical analyses were performed using SPSS version 23. 3. Results 3.1. Dietary nitrate improved the survival rate of skin flaps We observed the appearance of the skin flaps after the operation. On day 1 of postoperation, The distal regions became dark purple without obvious necrosis (Fig. 1A). On day 3, necrosis began to appear clear in the distal regions and the necrotic area was clearly distinguished from the survival area (Fig. 1B). On postoperative day 7, the circumscriptions between the necrotic and survival regions of the skin flap were more clearly identified. The necrotic parts became hard, shrink, and discolored (Fig. 1C). One day after the operation, skin flaps were treated nitrate exhibited larger survival areas than the other groups. However, the difference was not statistically significant at this time (Fig. 1D). The
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Fig. 1. Effect of dietary nitrate on survival rate of the ischemic skin flap. (AeC) Appearance of the ischemic skin flaps. (D) Flap survival rate at postoperative day 1. (E) Flap survival rate at postoperative day 3. (F) Flap survival rate at postoperative day 7. Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05; **p < 0.01 versus control group.
differences in the survival area became more robust on 3 and 7 days after the operation. The skin flaps were treated with nitrate showed significantly less tissue necrosis versus skin flaps treated with NaCl or untreated skin flaps (Fig. 1E and F). 3.2. Dietary nitrate increased nitrate and nitrite levels and decreased histological lesions Intake of nitrate by drinking water showed an increase in serum nitrate and nitrite levels compared with the control and NaCl groups.(p < 0.05) (Fig. 2A). To further investigate the effects of nitrate on skin flap, the ischemia skin flaps treated or not with nitrate for 14 days were analyzed by histochemistry. H&E staining revealed that the nitrate-treated tissues showed more neovascularization and less inflammatory cell infiltration (Fig. 2B). 3.3. Dietary nitrate increased blood perfusion and vessel density The rate of blood perfusion change at 1 cm distal flap from the baseline of the skin flaps showed a tendency to increase in a timedependent manner without a significant difference among the groups (Fig. 3A). At 5 cm, the results also showed a tendency to increase in a time-dependent manner. The blood perfusion change rate dropped severely after operation regardless of nitrate administration. On a postoperative day 3 and 7, the blood perfusion change was significantly improved in nitrate treated rats compared with those in NaCl and control rats (Fig. 3B). To investigate whether the improvement in tissue perfusion was mediated in part by increased revascularization, blood density was determined 7 days after the operation. Thus, we examined the expression of endothelial cell marker CD34 by immunohistochemistry. The vessel density was directly reflected in the number of CD34-positive vessels/mm2. The vessel density was higher in the nitrate-treated group compared with controls. (p < 0.05) (Fig. 3C).
3.4. Dietary nitrate attenuated inflammatory mediators The inflammatory response plays an important role in the wound healing process. To demonstrate whether dietary nitrate can decrease the inflammatory response, serum TNF-a and IL-6 levels were detected using ELISA kits. The results revealed that levels of TNF-a and IL-6 were significantly decreased by nitrate treatment (Fig. 4A and B).
4. Discussion Skin flap ischemia injury, a common complication of repairing soft tissue injuries, requires remodeling of the blood flow to sustain its viability [17]. The physiological repair response, however, is often insufficient, and therapeutic blood reperfusion remains an unmet medical need. In the present study, we demonstrate that dietary nitrate supplementation significantly enhances the blood perfusion change rate as well as the survival rate following skin flaps ischemia injury. These salutary effects are coupled to an altered inflammatory response with the raised generation of NO and changes in microvascular. This is the first description of the beneficial effects of nitrate-enriched diet in protecting the flaps against ischemic insult. Inorganic nitrate has emerged as a dietary component that can regulate diverse bodily functions in physiological activity. Numerous experimental studies have demonstrated the dietary nitrate supplementation defense of the gastric mucosa against injury and increase the gastric mucosal blood flow [18e20]. Another research demonstrated that dietary nitrate supplementation strongly augments blood perfusion recovery in the ischemic hind-limb [21]. Bier, S.C. et al. also declared that nitrite therapy effectively stimulates ischemic tissue vascular remodeling in the setting of metabolic dysfunction [22]. Moreover, Other studies demonstrating the anti-inflammatory effects of dietary nitrate and nitrite are emerging in different experimental inflammatory
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Fig. 2. Dietary nitrate increased nitrate and nitrite levels and decreased histological lesions. (A, B) Nitrate and nitrite levels after 7-day nitrate feed. (C) H&E staining of the skin flap. Scale bar ¼ 100 mm, magnification:40 (upper); Scale bar ¼ 200 mm, Original magnification:100 (lower). Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05; **p < 0.01 versus control group.
diseases [23,24]. Studies in animal models of ischemia and reperfusion have now revealed a central role of nitrate in hypoxia signaling [25]. The fact that the doses of nitrate used in these studies are easily achieved by the diet brings an interesting nutritional aspect to this research. In our study, we chose to use random skin flap in order to ischemic injury and be able to study the blood reperfusion effect of dietary supplementation with sodium nitrate. Nitric oxide (NO) is not only a vasodilator but also is known to mediate angiogenesis [26]. NO plays a key role in the regulation of cardiovascular function, cellular energetics, neurotransmission, and immune function [27]. Increased nitrate and nitrite levels in the blood may serve as NO reservoir for maintaining the systemic NO 3NO-2-NO homeostatic balance in the body. The L-arginine-NOS pathway is oxygen dependent, whereas the nitrate-nitrite-NO pathway is gradually activated as oxygen tensions fall. In this sense, NOS-independent NO formation when oxygen and L-arginine supply is limited [28]. Nitrite appears to signal protection through pathways distinct from those classicly ascribed to NO [29]. Thus, dietary nitrate may protect against skin flap ischemia injury associated with blood flow deficiencies by regulating NO homeostasis in the body. Our present study also demonstrated that dietary nitrate exerts skin flap protective effects through increased NO-like effects of nitrate and nitrite. The mechanism by which nitrate enhances the blood reperfusion of ischemic skin flaps seems to involve its angiogenesis effect. Pharmacological treatment with nitrate, an oxidation product of NO, may offer an alternative therapeutic opportunity. Evidence suggests that dietary nitrate supplementation increases the
regenerative capacity of ischemic tissue and that this effect may offer a strategy to improve ischemia-induced revascularization [30]. Further investigations in ischemic tissues highlighted the potential of dietary nitrate in angiogenesis [21,31]. Improving angiogenesis and increasing blood supply to ischemic areas was considered to promote the survival of random skin flap [32]. Here, we found that vessel density from immunohistochemistry results for CD34 showed more neovascularization in skin flaps by nitrate treatment. Thus, we thought that dietary nitrate supplementation augments blood perfusion recovery in ischemic skin flap may have resulted from a significant increase in blood vessel density. Alleviated ischemia injury of skin flap was accompanied by decreased inflammatory cytokine levels. Inflammation is a major contributor to cell death after ischemia of skin flaps since it potentiates cellular apoptosis. The greater the extent of inflammation, the more pronounced the necrosis, ultimately compromising flap success [33]. The inflammation reaction due to tissue ischemia induces damage to the blood vessels and exacerbate ischemia, which creates a vicious circle [34]. Interleukins facilitate cellular infiltration, leading to increased neutrophil counts that eventually cause tissue damage by releasing various cytokines and proteolytic enzymes. Therefore, the examination of pro-inflammatory cytokine regulatory factors can prove instrumental for effective treatment of ischemia. The current study examined that dietary nitrate decreased the levels of TNF-a and IL-6. Thus, the role of nitrate alleviating skin flap ischemia injury is related to lessening the levels of proinflammatory mediators. Nitrate and nitrite were previously considered to be harmful
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Fig. 3. Dietary nitrate improved blood perfusion and microvascular density. (A) Blood perfusion change rates at 1 cm distal from the baseline of the skin flaps. (B) Blood perfusion change rates at 5 cm distal from the baseline of the skin flaps. (C) CD34-positive vessels as assessed by immunohistochemistry. The arrows showing CD34 positive cells. (Scale bar ¼ 50 mm, Original magnification 400 ). Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05 versus control group.
constituents in the development of gastric cancer and other malignancies [35,36]. However, recent reports have indicated no harm to human health of normal dietary nitrate [37e39]. Moreover, green vegetables are the major source of inorganic nitrate. Thus, dietary nitrate could be a promising preventive and therapeutic strategy in clinical trials. In conclusion, dietary nitrate can effectively protect the skin flap against ischemia-induced injury. This protection by nitrate is related to the acceleration of blood flow recovery, stimulation angiogenesis, and inhibition of the inflammatory response. However, the specific mechanism of the protective effect by nitrate requires further exploration. Conflicts of interest There are no competing interests. Acknowledgments This work was supported by Shandong Provincial Natural Science Foundation (ZR2017BH034). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.059 References
Fig. 4. Dietary nitrate attenuated Inflammatory mediators in serum. (A) The levels of the TNF- a in serum. (B) The levels of the IL-6 in serum. Date are expressed as the mean ± SD, n ¼ 8, *p < 0.05; **p < 0.01 versus control group.
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