**6. The influence of endogenous NO on liver IRI**

Damage to the liver due to IRI is a culmination of inflammatory cross talk with the principal participants mentioned previously. Injury due to ischemia and reperfusion is the main cause

neutrophils adhere to endothelial cells at the initial stages of reperfusion, and subsequently transmigrate the endothelium where they continue to orchestrate tissue injury. The accumulation of activated neutrophils contributes to microcirculatory disturbances both locally and remotely. Activated neutrophils release reactive oxygen species, specifically

and macrophages are also activated shortly following reperfusion (Ysebaert et al., 2000). Recent studies propose an important role for lymphocytes, especially CD4+ T cells, in augmenting injury responses after IRI. However, lymphocytes may also play a protective role, but this is probably dependent on cell type and time course of injury (Ysebaert et al.,

During periods of ischemia, ROS and RNS are generated which can promote intracellular damage. Due to electron transport chain alterations, mitochondrial dysfunction ensues leading to reductions in ATP production and with subsequent loss of inner membrane stability resulting in mitochondrial swelling and rupture. With the reintroduction of oxygen during reperfusion, ROS are produced due to reactions of oxygen introduced during reperfusion and possible xanthine oxidase (or mitochondrial sources of ROS). ROS serve to stimulate other cell lines including Kupffer cells to produce proinflammatory cytokines (Diesen & Kuo, 2011). The major ROS are hydroxyl radical (OH•) and hydrogen peroxide.(H2O2). Reactions of ROS such as O2-• with NO yield products such as

Cytokines play a vital role in IRI, both by inducing and sustaining the inflammatory response, and by modulating IRI severity. Tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) are the two cytokines most commonly implicated in liver IRI. TNF-α is a pleiotropic cytokine generated by various different cell types in response to inflammatory and immunomodulatory stimuli. TNF-α modulates leukocyte chemotaxis and activation, and induces ROS production in Kupffer cells (Colletti et al., 1996). Additionally, IL-1 is known to promote production of ROS, induce TNF-α synthesis by Kupffer cells and induce

The complement system also contributes significantly to IRI and is composed of approximately thirty soluble and membrane-bound proteins. This system can be stimulated in three pathways: (1) the antibody-dependent classical pathway, (2) the alternative pathway, or (3) the mannose-binding lectin pathway (Qin & Gao, 2006). When activated, complement acts as a membrane-attacking complex that stimulates the production of proinflammatory cytokines and chemotactic agents. Furthermore, it can regulate adaptive

Damage to the liver due to IRI is a culmination of inflammatory cross talk with the principal participants mentioned previously. Injury due to ischemia and reperfusion is the main cause

**5.4 Reactive oxygen species (ROS) and reactive nitrogen species (RNS)** 

peroxynitrite (ONOO-), a RNS which can be an extremely aggressive oxidant.

neutrophil recruitment (Kato, Gabay, Okaya, & Lentsch, 2002).

**6. The influence of endogenous NO on liver IRI** 

•), proteases and various cytokines (Teoh & Farrell, 2003). Monocytes

superoxide radical (O2-

2000).

**5.5 Cytokines** 

**5.6 Complement** 

immunity (Boros & Bromberg, 2006).

of liver injury in response to vascular clamping during hepatic procedures such as hepatectomy and liver transplantation. This insult on the liver results in disturbances of the sinusoidal microcirculation and the generation of a variety of mediators such as reactive oxygen species, cytokines, activation of chemokines and other cell signaling molecules previously mentioned.

Hepatic IRI can cause severe hepatocellular injury that contributes to morbidity and mortality after liver surgery. As briefly mentioned previously, reductions of NO during liver IRI occur and are associated with increased liver injury (Köken & Inal, 1999). This is now appreciated to be due to decreases in NO steady-state production resulting from low concentrations of eNOS. This event coupled with NO inactivation due to reactions with abundant ROS, such as O2-•, results in reduced NO bioavailability. The consequences of this reduced bioavailability include but are not exclusive to increased oxidative stress, increased apoptosis, increased leukocyte adhesion, increased microcirculatory tone, and perturbed mitochondrial function. Interestingly, restoration with of NO to more "physiologic" concentrations serves to diminish the liver ischemia injury via countering the adverse actions mentioned previously. Other studies have demonstrated findings that are consistent with the premise that eNOS is crucial for minimizing injury during liver IRI. For example, liver injury was less in wild type mice compared to eNOS knockouts (eNOS -/-), in addition to the findings that agents given to increase eNOS expression or donate NO afford greater liver IRI protection (Duranski et al., 2006; Katsumi, Nishikawa, Yamashita, & Hashida, 2008). It is also well established that the NO concentrations during various inflammatory states are significantly increased by increased expression of inducible nitric oxide synthase or iNOS. However, the influence of iNOS and its true contribution in conferring liver protection deserves additional studies. In a rat model of liver IRI, iNOS expression was significantly increased as per increases in iNOS RNA at 1 and 5 hrs (Hur et al., 1999). This is consistent with other studies measuring iNOS expression of conditions of liver IRI. In a porcine model of IRI, intraportal injection of the selective iNOS inhibitor, aminoguanidine was demonstrated to decrease injury (M Isobe et al., 2000). In an intriguing study, NOS knockout mice (iNOS -/-) exposed to warm liver IRI demonstrated a much greater magnitude of injury compared to wild type mice. Interestingly, even though injury was greater in the iNOS knockout mice, little to no iNOS RNA was detectable in the wild type mice. It would appear for now, the true influence of iNOS's influence on liver injury during IR remains unclear.

A number of other endogenous NO-mediated mechanisms thought to confer protection have been published. For example, NO has been shown to inhibit caspase proteases via *S*nitrosylation, thereby inhibiting apoptosis (Maejima, Adachi, Morikawa, Ito, & Mitsuaki Isobe, 2005). This appears to be somewhat concentration dependent. Low physiological concentrations of NO may inhibit apoptosis. In contrast, higher concentrations may lead to the formation of toxic reactive nitrogen species such as ONOO- or reactive oxygen species which lead to cell necrosis and apoptosis (P. K. Kim, Zamora, Petrosko, & Billiar, 2001). Other published mechanisms of NO-mediated protection include inhibition of nuclear factor kappa B (Marshall, Hess, & Stamler, 2004), reversible inhibition of mitochondrial complex I, and decreased mitochondrial calcium accumulation (Burwell & Brookes, 2008). As to be expected, controversy exists concerning "if" and "how" NO exerts cellular protection. For instance, in a study by Jaeschke *et al* (Jaeschke et al., 1991), administration of a NO synthase inhibitor did not attenuate or accentuate liver injury during the initial reperfusion period. Inhibition of NO was observed not to influence neutrophil migration to the injured sites. While this contradicts a number of other studies, based on their findings, the authors concluded that NO availability was unlikely to be involved in the post-ischemic oxidant stress and reperfusion injury (Jaeschke, Schini, & Farhood, 1992). Nevertheless, the majority of published literature has demonstrated the beneficial effects of NO during liver IRI. These conflicting results might be explained by the fact that the mechanism of NO-mediated protection varies depending on cell type, quantities supplied, laboratory methods applied, timing and duration of NO exposure. Here, we summarize some key studies studying endogenous NO and NOS in hepatic IRI Table 1.


Table 1. Effect of endogenous NO and NOS on liver IRI
