Experimental skin flaps and nitric oxide

Surgical flaps are used in plastic surgery to reconstruct tissue defects due to trauma or cancer removal. Occasionally flaps are subjected to ischemia and reperfusion injury leading to flap failure. Nitric oxide (NO), a small gaseous molecule, has vast physiological importance as it participates in the regulation of blood pressure, blood flow, neurotransmission and immune response. NO is synthesized by the enzyme NO synthase (NOS), which exists in both constitutive and inducible forms. Constitutive NOS in endothelial cells (eNOS) continuously synthesizes NO in small amounts causing vasodilation and inhibition of platelet and leukocyte activity. Inducible NOS (iNOS) in leukocytes and inflamed tissue synthesizes NO in large amounts, which under certain circumstances leads to tissue destruction. Ischemia and reperfusion injury has great clinical impact and affects tissues such as the brain (stroke), heart (myocardial infarction) and surgical flaps (necrosis). The mechanisms underlying this tissue damage are not fully understood and methods to prevent and treat flap necrosis would be of great clinical value.

In the present thesis experimental flaps in the rat were studied with special reference to the role of NO. Different experimental skin flap models in the rat were used. NOS activity, flap ultrastructure, flap blood flow and flap survival after modulation of NOS and administration of NO were studied. Constitutive NOS activity was demonstrated in intact skin by citrulline assay. In an ischemic dorsal random flap model this constitutive NOS gradually decreased after flap surgery. Concurrently, increasing signs of endothelial damage and accumulation of leukocytes and platelets was observed by transmission electron microscopy. Inhibition of the constitutive NOS led to a decreased flap blood flow, as measured by laser Doppler technique, and also to a decrease in flap survival. Intact skin did not display any iNOS activity, whereas in the dorsal flaps iNOS activity was seen to gradually appear after surgery. At the same time an accumulation and extravasation of leukocytes was seen. Treatment with dexamethasone was found to prevent the induction of iNOS and also to increase flap survival.

Besides enzymatic formation of NO, non-enzymatic formation through the reduction of nitrite (N02) under acidic and reducing conditions has been described. With this knowledge at hand, a cream containing increasing concentrations of N02- and vitamin C was mixed and applied to the surface of an epigastric flap model. The cream generated NO in a concentration dependent manner, as measured by chemi luminescence, and increased the supplying and superficial blood flow in the flaps, as measured by transit-time ultrasound technique and laser Doppler technique respectively. Furthermore, the gas nitrogen dioxide was generated by the cream.

Taken together, the results show that constitutive NO, probably mainly derived from eNOS, is important for flap survival as it maintains blood flow and possibly also inhibits accumulation, aggregation and activation of leukocytes and platelets. Furthermore, the results indicate that induction of iNOS, which is capable of producing large concentrations of NO, could be negative for flap tissue. NO at high concentrations has previously been demonstrated to be tissue destructive, both in itself and also through the formation of different free radicals. Inhibition of the negative effects of NO and administration of NO to counteract a decrease in endogenous, constitutive NO synthesis could prove beneficial to flap tissue and might become useful in a clinical setting. Local administration, for example through the application of a cream to the flap surface, is an interesting and attractive way of treatment.

List of scientific papers

I. Gribbe O, Lundeberg T, Samuelson UE, Wiklund NP (1997). Nitric oxide synthase activity and endothelial ultrastructure in ischaemic skin-flaps. Br J Plast Surg. 50(7): 483-90.
https://pubmed.ncbi.nlm.nih.gov/9422945

II. Gribbe O, Lundeberg T, Samuelson UE, Wiklund NP (1999). Dexamethasone increases survival and attenuates induction of inducible nitric oxide synthase in experimental skin flaps. Ann Plast Surg. 42(2): 180-4.
https://pubmed.ncbi.nlm.nih.gov/10029484

III. Gribbe O, Samuelson UE, Wiklund NP (2007). Effects of nitric oxide synthase inhibition on blood flow and survival in experimental skin flaps. J Plast Reconstr Aesthet Surg. 60(3): 287-93. Epub 2006 Nov 22
https://pubmed.ncbi.nlm.nih.gov/17293287

IV. Gribbe O, Gustafsson LE, Wiklund NP (2007). Transdermally administered nitric oxide by application and acidified nitrite increases blood flow in rat island flaps. European Journal of Pharmacology. Oct 3: Epub ahead of print.
https://pubmed.ncbi.nlm.nih.gov/17976572