3.2.1. Nitric Oxide
Since its recognition as the endothelium-derived relaxing factor [
61,
62], several studies have described NO as a strong vasodilator agent [
44]. After synthesizing in the endothelium, NO increases cyclic guanosine monophosphate (cGMP) in smooth muscles, by stimulating soluble guanylyl cyclase. cGMP finally inhibits proteins responsible for contraction, causing vasorelaxation [
44,
63]. In adjunct to this mechanism, NO induces the reduction of intracellular calcium, mediated by ATPases that actively transport calcium inside the sarcoplasmic/endoplasmic reticulum [
44,
63]. Other pathways toward vasodilation include the reduction in 20-hydroxyeicosatetraenoic acid [
64] and prostaglandins [
65]. NO may be key in regulating cerebral blood flow (CBF) [
66]. Indeed, in hyperoxia [
67,
68,
69], hypoxia [
67,
68], and hypercapnia [
70], it was found to be an essential mediator of CBF in animal models. Moreover, NO also has a leading role in coupling CBF and neuronal activity [
71].
Several studies explored the role of NO in critical neurologic illnesses. Indeed, early stages of severe brain injuries recognize a depletion of NO, probably contributing to secondary brain damage [
72]. By contrast, when present NO reduces injuries and has neuroprotective properties in ischemia/reperfusion animal models, both in stroke and cardiac arrest [
72]. For this reason, a prospective trial has been performed, proving the feasibility of iNO administration in cardiac arrest, and showing promising results in terms of survival [
73].
In subarachnoid hemorrhage, NOS inactivation is associated with alterations in microvascular density and hemostasis, leading to rebleeding, intracranial hypertension, and larger hemorrhage volume [
74]. Conversely, NO reduction is also correlated with a lower grade of neuroinflammation and better neurobehavioral function in rats [
75]. Studies in humans showed a correlation between elevated asymmetric dimethylarginine (which is an inhibitor of iNOS, and thus of the vasodilator molecule NO) with vasospasm and worse outcome [
76]; another small human study found elevated levels of NO metabolites, especially in subjects with poorer outcomes [
77]. Although results may be controversial, the literature suggests deleterious effects of NO depletion, supported by increased microthrombi formation and reduced cortical activity [
72]. A pilot human trial established the safety of iNO administration and showed promising results in treating delayed cerebral ischemia [
78].
Pathways in traumatic brain injury (TBI) appear more complex. Mechanical insults may induce up-regulation of iNOS and over-production of NO and ONOO
-. The RNS stimulate the production of glutamate, which triggers nNOS. Nevertheless, NO plays an uncertain role in this disease, and its implications are still unknown [
79]. Studies in newborn animal models are also contradictory. Some reports showed protective properties of NO by vasodilation, reducing ROS concentrations and scavenging radicals; others linked it to deleterious effects and greater brain injuries. These discrepancies might depend on the timing of production and concentrations of NO [
80].
The brain controls blood flow by interacting with vessel diameter. Autoregulation ensures constant blood supply; changes in systemic blood pressure -if they occur within certain limits- induce vasodilation or vasoconstriction. Additionally, the brain increases blood flow in areas where neurons are more active through a mechanism named neurovascular coupling. Although other pathways and elements participate, NO plays a significant role in both of them. NOS inhibition (and thus lowering NO production) increases the lower limit of mean arterial blood pressure where autoregulation acts, reducing its efficiency [
52,
53,
54]. Through neurovascular coupling, blood flow is directed to more active areas, where glutamate (the main excitatory neurotransmitter) induces calcium entry in neurons and activation of neuronal nNOS finally resulting in NO production and vasodilation [
65].
Interestingly, CO
2 may act as a regulator of NO. Indeed, the process of adjusting cerebral vascular tone in response to changes in arterial carbon dioxide partial pressure (PaCO
2) is known as chemoregulation, which could be the main trigger of endothelial NO-release [
81]. In humans, hypocapnia leads to vasoconstriction and reduced CBF, while hypercapnia and changes in perivascular pH result in vasodilation and increased CBF [
81]. Cerebral endothelial cells and astrocytes have been shown to release NO under normocapnic conditions, while NO production increases during hypercapnia and decreases during hypocapnia, regardless of pH levels [
82]. It has been proposed that these NO variations in response to PaCO
2 are specific to NOS regulation and that administering exogenous NO may influence the CO
2-dependent chemoregulation mechanism [
82].
Finally, as an NO reservoir, the vasodilating effects of nitrites have long been investigated. Although some results failed to find any vasoactive activity [
83], most studies confirmed that nitrite has vasorelaxant effects through the activation of guanylate cyclase [
46,
84,
85,
86,
87]. >
In summary, NO stands as a potent vasodilator, playing diverse roles in the regulation of blood flow and brain protection. Its mechanism is intricate and involves numerous interactions. When it comes to brain injuries, a deficiency in NO could contribute to secondary brain damage, while NO possesses neuroprotective qualities in animal models of ischemia/reperfusion, including stroke. Interestingly, NO inhalation has been suggested as a neuroprotective intervention during cardiopulmonary resuscitation [
88]. Consequently, extensive research is needed to gain a deeper understanding of its mechanisms and potential therapeutic effects.
3.2.2. Peroxynitrite
Peroxynitrite (ONOO
-) has shown vasoactive properties, producing vasorelaxation of arterioles in vitro [
89] and in vivo [
20], although its potency seems very low, up to 50-fold less than that of NO [
90]. The effect doesn't appear to involve the endothelium [
91], and the vasoactive mechanism is still unclear. Some authors proposed that the effect is linked to the opening of ATP-sensitive potassium channels [
20,
92], by directly activating the channels or reducing ATP concentration by interfering with cellular metabolism [
20,
92,
93]. Other mechanisms proposed to explain this vasodilating activity include elevation of cGMP levels, membrane hyperpolarization via K
+ channel activation, activation of myosin phosphatase activity, and interference with cytosolic calcium movement and cellular membrane Ca
2+ entry [
89].
However, in contrast with all the above results, Daneva et al. induced an increase in ONOO
- by upregulating iNOS in mice, reporting an increase in pulmonary arterial pressure by impeding calcium entry in smooth muscle cells of pulmonary arteries [
94]. Similarly, Ottolini et al. investigated the same pathway in diet-obese mice, finding a link between obese hypertension and high ONOO
- levels [
95]. Finally, in vitro and animal studies [
96,
97] have observed that peroxynitrite may induce vasoconstriction in cerebral arteries. This effect is likely due to inhibiting the cerebral K
+-dependent calcium channel. Interestingly, the addition of glutathione inhibited this cerebral vasoconstrictive effect.
Therefore, the net effects of peroxynitrite appear conflicting, and are not completely understood, probably being dose and time-dependent [
98].
3.2.3. Nitroxyl
HNO has been proven to act as an endothelium-derived vasorelaxant [
99,
100,
101,
102] not susceptible to tolerance [
100]. Moreover, HNO has multiple effects, including inhibiting platelet aggregation, limiting vascular smooth muscle proliferation, and interacting with metallo and thiol-containing proteins.
HNO promotes vasodilation by activating several molecular pathways [
50,
101,
102,
103]. These pathways include the stimulation of soluble guanylyl cyclase (sGC), leading to an increase in cGMP. From animal studies, it has been suggested that the sGC-cGMP pathway may be necessary and sufficient for HNO-induced vasodilation in vivo [
104]. Others proposed that HNO may activate ATP-sensitive potassium (K
ATP) channels and voltage-dependent potassium (K
v) channels, resulting in the outflow of potassium ions. This causes the cell membrane to become hyperpolarized and leads to a reduction in intracellular calcium levels. Finally, from an elegant animal study, Eberhardt et al. suggested a vasodilation HNO mediated vasodilation via the transient receptor potential channel A1 (TRPA1) and calcitonin gene-related peptide (CGRP) [
105]. This HNO-TRPA1-CGRP signaling pathway could be a crucial component in the neuroendocrine regulation of vascular tone, mediated by HNO.