Researchers have studied nitric oxide (NO) extensively for the past 40 years. However, there has been an increased interest within the past 15 years. In 1998, the Nobel Prize in Physiology and Medicine was awarded to scientists who worked out the signaling mechanisms for NO in the human body.
Nitric oxide is an endogenous gas produced by cells with many diverse physiological effects. The substrate arginine is converted by the enzyme nitric oxide synthase (NOS) to citrulline with the liberation of NO (see “A Closer Look At Nitric Oxide Production” below).1 After NO is released, it has a half-life of seconds with subsequent binding to receptors on or within the cell that causes a second messenger cascade. The result of this cascade depends on the type of NOS. There are many subtypes of NOS including: nNOS, which are found in neurons; iNOS, which are inducible forms found throughout the body; and eNOS, which are found in blood vessel cells. The iNOS and eNOS subtypes are particularly interesting with their implications in the lower extremity, especially when it comes to wound healing.
There are two relevant components of NO on wound healing. The first is the vascular component. Endogenous NO acts upon endothelial cells to cause dilatation of vessels (both arterial and venous). Contrarily, inhibition of NO synthesis causes vasoconstriction and subsequent hypertension.1 Evidence also suggests there is decreased NO activity with an increase in activity levels by vasoconstrictors in the diabetic rat model.2 Studies of humans have also demonstrated decreased NO production in diabetic patients with peripheral neuropathy.3 Further, the vessels of insulin dependent diabetic patients demonstrate less responsiveness to NO and potentially a decrease in availability of NO.4,5 Researchers have also shown that NO increases blood flow to the microcirculation adjacent to wound sites.6 
The second component of NO on wound healing is in the inflammatory process. Researchers have measured NO at higher levels in proximity to wound sites.7 Cytokines stimulate macrophages and fibroblasts to produce NO.8,9 Nitric oxide appears to have cytotoxic properties and this suggests some level of antimicrobial activity.10 Also keep in mind that inhibition of NO synthesis retards collagen synthesis and deposition which we know are key components in providing principal strength characteristics of wounds.11
There is growing interest in the clinical use of NO in the lower extremity. Clinically, we know that NO can play a role in two key pathologic components in the diabetic foot: vascular compromise and peripheral neuropathy.
There have been promising results for the use of NO in treating painful peripheral neuropathy. Researchers have demonstrated that monochromatic near-infrared photoenergy (MIRE®, Anodyne® Therapy System) increases sensation and decreases pain.12-14 The proposed principle behind MIRE is that photon energy induces the release of NO, thereby increasing blood flow to vascularly compromised peripheral nerves. Clinicians have also utilized topical NO, in the form of a spray, for the treatment of painful peripheral neuopathy with good success.15
However, the direct application of NO for the treatment of diabetic wounds has not been well researched. While it is possible to prevent wound occurrence by reversing or retarding the neuropathic process that often leads to wounds, the question remains on whether NO can expedite the healing of existing wounds.
There is anecdotal evidence to support this theory. In regard to isosorbide dinitrate (nitroglycerin), a liberator of NO, podiatric physicians have been using topical nitroglycerin to treat ischemic wounds. There are two published studies demonstrating that topical application of nitroglycerin in the form of a paste may increase perfusion to the foot.16,17 Further, a study conducted by Wheeland, et. al., demonstrated that the application of nitroglycerin led to healing of ischemic digital ulcerations.18 However, these studies were small with non-stringent methodology. No systematic, large-scale study has evaluated the efficacy of topical nitroglycerin application in wound healing in the lower extremity. We have submitted for publication a placebo-controlled, randomized, double-blind study evaluating the ability of topical nitroglycerin to measurably increase perfusion to the foot.19 We examined the use of a nitroglycerin patch on healthy subjects and measured perfusion levels using transcutaneous thermometry and photoplethysmography. Our study revealed that nitroglycerin at a low dose does not measurably increase local perfusion. We did not examine higher doses due to potential side effects including headache and dizziness.
A follow-up study is needed to examine the use of topical nitroglycerin in the lower extremity. Specifically, a placebo-controlled study is required to examine the ability of nitroglycerin to expedite wound healing in the diabetic foot.
Some fundamental questions remain regarding the clinical application of NO in wound healing. In our study, we were unable to demonstrate that topical application of nitroglycerin is able to increase perfusion to the foot. However, it is possible that topical application of nitroglycerin can increase local perfusion at higher doses. Assuming that topical nitroglycerin is able to increase local perfusion in the foot, would this increase be able to overcome large vessel compromise upstream?
There are other concerns regarding the application of higher doses of topical nitroglycerin. There is a possibility that they increase the risk for systemic effects. Patients who suffer from chronic wounds often suffer from other disease processes including cardiac disease. There may be a potential interaction with other cardiac drugs and nitroglycerin at higher doses. There may also be unwanted direct effects on the heart. Accordingly, do these risks outweigh the potential benefits of the topical application of nitroglycerin?
Another important question is whether NO has positive effects on wounds in the non-ischemic foot. If a patient has adequate blood flow, can augmentation of NO facilitate wound healing? We know that NO plays a role in inflammation. However, the specifics of this role in the chronic wound environment of a diabetic foot are not clear. Researchers have demonstrated that supplementation with exogenous nitric oxide donors such as molsidomine or supplementation of the substrate L-Arginine promotes wound healing in diabetic rats.20,21 However, there is some evidence to suggest that high levels of NO can be destructive to tissues.7 Some also argue that with the potential harmful effects of NO, future research efforts should develop therapies to inhibit NO production.22 Direct application in a controlled study would better delineate the ability of NO to facilitate healing.
Nitric oxide plays an important role in the vascular system and the inflammatory process. However, we need more data in order to better understand the place of NO in the armamentarium for the treatment of pathologies in the diabetic foot.
Dr. Kim is an Assistant Professor at the Arizona Podiatric Medicine Program, Midwestern University College of Health Sciences. He is the author of multiple clinical and basic science publications and has lectured throughout the country.
Dr. Steinberg (pictured) is an Assistant Professor in the Department of Surgery at the Georgetown University School of Medicine in Washington, D.C. He is a Fellow of the American College of Foot and Ankle Surgeons.
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