2.1. Methylene Blue

Any interference with a tissue, like the introduction of a tissue tracer, will lead to inflammation. Proven by many studies, the anti-inflammatory action of MB protects against lesions such as pulmonary lesions induced by endotoxins, bacterial lipopolysaccharides that cause fevers, renal lesions made by ciclosporin, cardiac lesions given by the use of doxorubicin, pancreatic lesions made by streptozotocin, ischemic-reperfusion lesions, and lipid peroxidation suppression and increased inflammation by the time of reperfusion. It also accentuates the long-chain fatty acids oxidation [11,16].

Another important biological feature for the eventual use of the dye as a tissue tracer is its low molecular weight. MB is a partially liposoluble agent, which makes it a rapidly penetrating histological stain [49] that passes easily to the cellular membrane, accumulates in the mitochondrial matrix, enhances respiration which is realized via different processes (cytochrome C oxidase activity, O₂ consumption, ATP production), minimizes electrons leakage at the level of electron transport chain, and also decreases superoxide formation by the time of reperfusion [11,46].

At the cellular level MB modulates the function of different integral membrane proteins involved in solute transport like glucose and cations, such as Na⁺, K⁺, and H⁺. In addition, MB influences the function of voltage sensitive ions Na⁺, Ca²⁺, Ca²⁺ activated, and K⁺ channels, altering the excitability of neurons and making it suitable for use as a local anesthetic [11,50,51,52]. There are several factors that influence the molecular mechanisms which mediate MB effects, like routes of administration/application, light exposure, membrane potential, or the redox state of the cells [11].

When applied topically, MB improved fibroblast proliferation as well as collagen and elastin fibers production in tegument [34]. At the mitochondrial level, MB acts on the first, third, and fourth respiratory complex. The first complex, facing the cytosol, catalyzes the oxidation of hydrogen nicotinamide adenine dinucleotide (NADH), which gives two electrons to MB. In this case MB acts as an electron carrier, generating the LeucoMB form that is partially formed in the mitochondrial matrix [16]. Since MB has the redox potential of 11 mV, it suffers from the reduction process, thus allowing the continuous cycle between the two forms: MB and LeucoMB, as can be noticed in Figure 2 [53].

Diagram of MB as an alternative mitochondrial electron transporter. More details can be found in text. Reproduced with permission from [53].

The last compound presents redox indicator proprieties, which can be exploited in quantitative analysis of a large number of reductive agents, like glucose and ascorbic acid. Complex IV consumes more than 95% from O2 at the cellular level and the production of H2O2 and other oxidants at the level of complex I or III seems to be improved after blocking complex IV, thus reducing superoxide formation by the time of reperfusion [54]. Furthermore, complex IV assures that cytochrome c recycling is in its reduced form. The MB cycle takes place in mitochondrial electron transport chain (ETC) inhibiting the production of radical superoxide, competing with O₂ at the place of production of free radicals by NADH dehydrogenase of complex I. Cytochrome c is the electron transporter from complex III to complex IV, with complex I being the natural enzyme which reduces cytochrome c while complex II oxidizes it [53,55].

Decreasing NADH/NAD⁺ rapport, MB prevents cytoplasm acidification, the inhibition of glycogenosis and possibly oxidative metabolism including cyclooxygenase (COX) activity. In complex I and complex III there are two sites which are responsible for free radicals’ production by nonspecific transfer of electrons to O₂. Despite that one of the cell NAD(P)H-dependent dehydrogenase, the NADH-dehydrogenase of complex I is able to reduce artificial electron acceptors such as MB to MBH2 or O2 to superoxide radical. The cytochrome c, in the mitochondria, and methemoglobin are the only heme proteins reported to reoxidize MBH2 to MB [55]. This interaction between methemoglobin and MB leads to different effects of MB in a concentration-dependent manner as follows: high concentrations convert ferrous iron of reduced hemoglobin (Hmb) to the ferric form methemoglobin. Low concentrations have the opposite effect in drug-induced methemoglobinemia [56].

Furthermore, MB can stabilize the energetic metabolism and increase ATP synthesis after reoxygenation. When the reperfusion takes place the mitochondrial respiratory chain dysfunction leads to reactive oxygen species (ROS) accumulation. ROS affect mitochondrial membranes, targeting lipid peroxidation and mitochondrial lesions which lead to cytochrome c release in cytosol through mitochondrial permeability transition pore (MPTP), activating caspase 3 to induce apoptosis [54].

MB improves mitochondrial function by inducing peroxisome proliferator-activated receptor γ coactivator 1 PGC1α, a central mediator of mitochondrial biogenesis [56], and downregulates the expression of NACHT, Leucine-rich repeat (LRR), and Pyrin (PYD) dominions that contain protein 3 (NLRP3) an inflammasome produced by bone marrow derivatives from macrophages induced by ATP. In addition, MB might regulate inflammation, influencing the signaling way of pro-inflammatory nuclear factor-κB (NF-κB) and inhibiting caspase-1 activation to reduce apoptosis [57].

Post ischemic MB inhibits superoxide production competing with molecular oxygen at the iron-sulfur center of xanthine oxidase, acting as a willing electron acceptor for the last shunting of electron flow from the normal pathway. This switch in favor of an iron-sulfur center allows the anaerobic oxidation of purine substrates, short-circuiting O₂ generation at the flavin centers. Leucomethylene blue auto-oxidation produces predominantly hydrogen peroxide to the detriment of superoxide. Due to these facts, MB was proposed to attenuate the reperfusion pathophysiology after lesion in two ways. Thus, if MB is administrated before the ischemia, it causes attenuation of the hypoxanthine accumulation and permits anaerobic breakdown of hypoxanthine and xanthine to uric acid. The second option is related to the administration of MB immediately before injury or reperfusion to prevent superoxide formation [49].

All the effects produced by MB on the mitochondria, respiration, and antioxidant effects, are useful tools to reduce the tissue reaction at the invasiveness of a foreign substance on a living tissue.