No abstract

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.

The neuropeptide galanin (Gal) and its receptors (GalR1, GalR2, and GalR3) are expressed in spinal cord. We have characterized the pharmacology of the antinociceptive effects of intrathecally (i.t.) administered galanin and its analogs in the formalin test in rats, using an automated flinch detection system. Intrathecal injection of rat galanin (Gal 1-29 ) or human galanin (Gal 1-30 ) produced a dose-dependent inhibition of formalinevoked flinching in phase 2, but not in phase 1. Relative potency of galanin homologs is Gal 1-29 Ն Gal 1-30 Ͼ galanin-like peptide 1-24 Ն Gal 2-11 ϭ Gal 3-29 (an inactive analog). Galanin 1-29 and Gal 1-30 are both high-affinity agonists to GalR1/R2, whereas Gal 2-11 is a GalR2 receptor agonist. Our data suggest that i.t. galanin-produced antinociception is mediated by activation of GalR1 receptors. When comparing antinociceptive effects of i.t. Gal 1-29 to morphine and to 2-amino-5-phosphonopentanoic acid (AP-5, an N-methyl-D-aspartate antagonist), Gal 1-29 is of intermediate potency between these two analgesic agents based on the ED 50 values. An isobolographic analysis showed synergy between Gal 1-29 and morphine and between Gal 1-29 and AP-5 on the second phase. Fixed ratio dose combinations of morphine and Gal 1-29 , or AP-5 and Gal 1-29 produced significantly greater antinociception than predicted from simple additivity. In summary, the present findings reveal that 1) spinal galanin produces a reliable inhibition of formalin-induced facilitated nociceptive processing, an effect possibly mediated by GalR1 receptors; and 2) galanin potentiates i.t. morphine and AP-5-induced antinociception.

Outflow facility is reduced in transgenic Col1a1(r/r) mice with IOP elevation. The inverse correlation of IOP elevation to facility reduction indicates that increased resistance in the aqueous outflow pathway contributes to ocular hypertension in Col1a1(r/r) mice. These mice may be useful as a model for open-angle glaucoma, as well as for assessing the relationship between collagen type I metabolism and aqueous outflow.

Purpose To determine the effect of molecular size on the drainage route of dextrans injected into the rat anterior chamber (AC). Methods Anesthetized adult rats received monocular AC injections of a mixture of 3-kDa dextran-cascade blue, 40-kDa dextran-Texas red, and 500-kDa dextran-FITC. After exsanguination of the rats 2, 4, 6, 12, 24, or 72 hours later, the eyes, facial lymph nodes, and cervical lymph nodes were isolated, and the total content of each dextran type was determined by spectrofluorometry. Also, lymph nodes were evaluated histologically 4 and 24 hours after AC injection of 40-kDa dextran-FITC. Results The speed of tracer exit from the eye varied with 3-kDa dextran > 40-kDa dextran > 500-kDa dextran. No 3-kDa dextran was detected in either facial lymph nodes or cervical lymph nodes at any time point. The average recovery of 40-kDa dextran in the facial and cervical lymph nodes peaked at 52.6% of the amount injected. In contrast, average recovery of 500-kDa dextran in the facial and cervical lymph nodes peaked at 1.8% of amount the injected. Histology showed 40-kDa dextran was mostly contained within lymph node cells at both 4 and 24 hours after injection. Conclusions Transport of 40-kDa dextran from the AC to the facial lymph nodes and cervical lymph nodes is markedly more efficient than that of 500-kDa dextran. In contrast, there is negligible transport of 3-kDa dextran. These results demonstrate that different sized aqueous macromolecules can exit the eye by different routes.