iNOS is the one of the three isoforms that generates NO in a calcium-independent fashion and, despite its low abundance in the brain, it has been documented to generate the largest amount of NO and has been implicated to be involved with inflammatory conditions and mediation of oxidative stress [15]. Because of its dependence on calcium, once iNOS is expressed, it can produce copious amounts of NO for sustained periods and is only limited by cofactors and substrate availability [15]. Oxidative stress linked to iNOS is mediated largely via lipid peroxidation, DNA damage, inhibition, and damage of mitochondrial structures and enzymes [15]. INOS has been studied in relation to SAH in a number of animal models, and the exact role of iNOS in the pathogenesis of SAH is still unclear.

Some reports point to the possible deleterious role of iNOS after SAH, and how the enzyme may indeed drive or exacerbate pathogenesis and injury after hemorrhagic insult. Work by Suzuki et al. demonstrated that a blood by-product, Hemin, may indeed activate iNOS and subsequent pathways. Work by this team demonstrated that the overactivation of INOS resulted in the overdevelopment of NO, leading to nitrosative stress and peroxynitrite formation [32, 33]. This increased oxidative burden was linked to damaged smooth muscle cells in the vasculature and may cause a predisposition to a number of vascular complications observed after SAH. Furthermore, work by Sayama et al. demonstrated that iNOS activation and deleterious effects may be part of the EBI cascade of events [32, 33]. The team reported that there was an overexpression of iNOS mRNA in the basal pia 24 h after experimental SAH. iNOS expression was heightened in polymorphonuclear cells and mononuclear cells and in cells throughout the CNS 24 h after SAH. The group concluded that overactivation of iNOS resulted in the overproduction of free radicals and detectable lipid peroxidation, which correlated with the degree of MCA vasospastic burden.

Aminoguanidine (AG), an iNOS specific inhibitor, was used to help inhibit iNOS in an experimental SAH model. Fukada et al. demonstrated that AG inhibition of iNOS resulted in a significant reduction in endothelial and smooth muscle cell damage, which resulted in subsequent reduction in aneurysm formation in a model of aneurysm induction [9]. Reducing shear stress via Batroxobin (defibronyltic) reduced iNOS overexpression and vascular “overstimulation,” which was shown to be protective and reduced aneurysm rupture. Transgenic manipulation in experimental models of SAH also indicated a possible deleterious role of iNOS after SAH induction. Work by Saito demonstrated that mice that overexpress CuZn-SOD (an iNOS inhibitor) had significantly reduced vasospasm. The degree of vasospasm correlated directly with the amount of iNOS mRNA and protein expression. Additionally, the team reported that reactive oxygen species directly activate iNOS and may explain the trigger and exacerbation of complications after SAH [31].

Work by Lin et al. demonstrated that vasospasm after SAH may be driven at least partially by the observed upregulation of iNOS after the onset of hemorrhage. In the model reported by this group, animals demonstrated reductions in eNOS levels and significant upregulation of iNOS expression. The group then used adenosine A1 receptor agonists that partially prevented vasospasm and resulted in a significant reduction in eNOS expression. However, the agonists were not successful at reducing iNOS expression, which allowed the group to conclude that continued upregulation of iNOS may play a major role in vasospasm. Similarly, the use of 17beta-estrogen in male rats before experimental SAH reduced vasospasm significantly. 17beta-Estrogen was found to reduce the expression of iNOS mRNA and protein expression, and this reduction in iNOS bioavailability was linked to the reduction of iNOS, indicating a possible deleterious role of iNOS [16, 17].

Despite the many reports and studies that point toward a deleterious nature of iNOS after SAH, a few studies report a neutral and possibly beneficial role of iNOS and its oxidative properties after SAH. Inhibiting iNOS with P-toluenesulfonate after SAH conferred no attenuation of blood-brain barrier (BBB) damage, cerebral edema, or delayed neuronal apoptosis. Inhibiting iNOS did not improve outcome or neurological score after SAH, indicating that although it might be a pathological player, it may not be the biggest or most pertinent target [41].

Work by Vellimana et al. focused on studying the differential expression of all three NOSes after experimental SAH under preconditioning or non-preconditioning situations. The study reported that preconditioning resulted in a more favorable outcome after SAH, and only eNOS demonstrated an increase in expression and was considered likely be protective when preconditioning was provided. Both nNOS and iNOS were deemed to not play a major protective role after SAH [40].

Work by our group focused on using simvastatin as an acute treatment and as prophylaxis in experimental mice in SAH. Simvastatin was observed to reduce the expression of iNOS when given prophylactically, but when administered after the induction of SAH, iNOS expression remained elevated after 48 h of induction. Despite the sustained increased expression in the post-SAH treatment group, animals still demonstrated reduced vasospasm, apoptosis, and microthromboembolism, indicating that iNOS may be a minor player in the pathogenesis of SAH in this particular model [30].

INOS has been reported to possibly confer protective effects, solely based on its proximity and expression in the vascular walls after SAH. Work by Pluta et al. postulated that because Hemin (a blood by-product) is readily available after SAH and is also an activator of iNOS, there should be an increased amount of iNOS expression and activation around the vicinity of hemorrhage. Pluta et al. postulated that iNOS may be a source of NO after SAH because of the involvement of macrophages after SAH, specifically in and around the endothelium of large- and middle-sized vessels. This expression of NO and overabundance of iNOS expression may help alleviate SAH-induced vasospasm [22].

In a primate model of SAH, nNOS expression was found to be markedly reduced and correlated greatly with the degree of vasospastic burden. SAH induction was associated with reduced staining of nNOS in the vascular adventitia in animals with significant vasospasm. These findings point toward the possible protective role of nNOS, and indicate that NO synthesized by nNOS may indeed be important in the homeostatic modulation of vascular tone in large- and middle-sized vessels [25].

Work by Sehba et al. demonstrated that following experimental SAH there was a heightened measurable expression of nNOS in neurons and the microvasculature. nNOS expression and concentration was significantly increased in the SAH group alone. The heightened expression of nNOS was deemed to be a protective response caused by acute stress and decreased cerebral blood flow, and likely driven to introduce increased expression of NO to reverse any pathological consequences of the hemorrhage [36].

nNOS involvement and protective role could also be a major player in the formation and rupture of aneurysms. Experimental aneurysms were induced in mice and the role of eNOS and nNOS was assessed. eNOS knock-out (KO) mice demonstrated a compensatory upregulation of nNOS expression in the walls of cerebral aneurysms. Interestingly, introducing aneurysms in eNOS and nNOS KO mice resulted in an increase in the amount of aneurysms present, with evidence of late-stage aneurysm formation. Double KO animals demonstrated a significantly increased amount of macrophage (the main cell mediators of inflammation) infiltration, vascular degradation, and progression to aneurysmal rupture. These findings point to the possible protective role of nNOS in maintaining the integrity and structural stability of cerebral aneurysms [2].

Recent work by our lab focused on understanding the role of NOSes after SAH. We reported that nNOS was upregulated in eNOS KO SAH mice, and likely to have been upregulated for compensatory purposes and possible protective expression of NO under pathological conditions such as SAH, where oxidative stress is a major player of pathogenesis [27].

The other well-studied, perhaps most-studied, constitutive NOS isoform is eNOS. It is usually found in endothelial cells, and often responsible for generating NO that is used for protective homeostatic functions such as endogenous anti-microthrombosis, vasomotor regulation, and smooth muscle growth modulation. eNOS has been the target of several research studies pertaining to SAH because of its involvement in vascular regulation, possible role in middle/large vessel vasospasm, and physiological expression in the brain. Several studies focused on the measurement of and detecting levels of eNOS after SAH, and debated its possible protective, deleterious, or even neutral role.