Date Published: June 20, 2013
Publisher: Public Library of Science
Author(s): Peng Kai Ong, Benoît Melchior, Yuri C. Martins, Anthony Hofer, Pamela Orjuela-Sánchez, Pedro Cabrales, Graziela M. Zanini, John A. Frangos, Leonardo J. M. Carvalho, Maria M. Mota.
Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.
Cerebral malaria (CM) is one of the most severe complications of malaria infection by Plasmodium falciparum that causes unacceptably high rates of mortality and morbidity, imposing devastating health and economic burdens especially in tropical countries . The murine model of CM using C57BL/6 mice infected by Plasmodium berghei ANKA (PbA) is a well accepted animal model for studying CM as it shares many common pathological features with human CM . The pros and cons of this model have been recently debated , , , . Murine or experimental CM (ECM) is associated with a vasculopathy  which is distinctively characterized by widespread cerebral arteriolar vasoconstriction, intense microvascular inflammation and markedly reduced cerebral perfusion , suggesting that cerebrovascular function is severely compromised in ECM. Cerebrovascular function is central to blood flow regulation which in turn is critical for maintaining metabolic homeostasis required for proper neurological functions . Since metabolic disturbances and neurological impairment are evident in ECM , , understanding the underlying mechanisms of cerebrovascular dysfunction will be imperative for providing mechanistic insights into pathogenesis of the disease.
The present study unveiled novel insights on changes in the status of NOS functions that contributes to cerebrovascular dysfunction in ECM. Substantial blunting of eNOS- and nNOS-elicited pial arteriolar dilatory responses during ECM development would point to a loss of enzymatic eNOS and nNOS activities involved in NO production, reinforcing the notion of low NO bioavailability in ECM that compromises cerebrovascular function. Several lines of evidence lent strong support to NOS uncoupling as a potential mechanism contributing to the NOS dysfunction which includes a state of BH4 deficiency, blunting of NO generation upon stimulation by agonists, decrease in total NOS activity, increase in lipid peroxidation levels, upregulation of eNOS protein expression and enhancement of eNOS and nNOS monomerization. In the case of eNOS, its dysfunction could be aggravated by a downregulation of phosphorylation activity at S1176 and this effect could be induced by the decrease in cerebrovascular wall shear stress during ECM development.