Herman Kwansa

Resveratrol protects against experimental stroke: Putative neuroprotective role of heme oxygenase 1

Epidemiological and experimental reports have linked mild-to-moderate wine and/or grape consumption to a lowered incidence of cardiovascular, cerebrovascular, and peripheral vascular risk. This study revealed that resveratrol, an enriched bioactive polyphenol in red wine, selectively induces heme oxygenase 1 (HO1) in a dose- and time-dependent manner in cultured mouse cortical neuronal cells and provides neuroprotection from free-radical or excitotoxicity damage. This protection was lost when cells were treated with a protein synthesis or heme oxygenase inhibitor, suggesting that HO1 induction is at least partially required for resveratrols prophylactic properties. Furthermore, resveratrol pretreatment dose-dependently protected mice subjected to an optimized ischemic-reperfusion stroke model. Mice in which HO1 was selectively deleted lost most, if not all, of the beneficial effects. Together, the data suggest a potential intracellular pathway by which resveratrol can provide cell/organ resistance against neuropathological conditions.

Decreased Damage From Transient Focal Cerebral Ischemia by Transfusion of Zero-Link Hemoglobin Polymers in Mouse

Background and Purpose— Transfusion of large polymers of hemoglobin avoids the peripheral extravasation and hypertension associated with crosslinked tetrameric hemoglobin transfusion and may be more effective in rescuing brain from focal ischemia. Effects of transfusion of high-oxygen-affinity hemoglobin polymers of different weight ranges were determined. Methods— Hypervolemic exchange transfusion was performed during 2 hours of middle cerebral artery occlusion in mice. Results— Compared to transfusion with a 5% albumin solution or no transfusion, infarct volume was reduced 40% by transfusion of a 6% solution containing hemoglobin polymers in the nominal range 500 to 14 000 kDa. Infarct volume was not significantly reduced by transfusion of a lower concentration of 2% to 3% of this size range of polymers, 6% hemoglobin solutions without removal of polymers <500 kDa or >14000 kDa, or crosslinked hemoglobin tetramers with normal oxygen affinity. Exchange transfusion with the 6% solution of the 500 to 14 000 kDa hemoglobin polymers did not improve the distribution of cerebral blood flow during focal ischemia and, in mice without ischemia, did not affect flow to brain or other major organs. Conclusion— An intermediate size range of polymerized bovine hemoglobin possessing high oxygen affinity appears optimal for rescuing mouse brain from transient focal cerebral ischemia. A minimum concentration of a 6% solution is required, the rescue is superior to that obtained with crosslinked tetrameric hemoglobin possessing normal oxygen affinity, and tissue salvage is not associated with increased blood flow. This polymer solution avoids the adverse effects of severe renal and splanchnic vasoconstriction seen with crosslinked tetrameric hemoglobin.

Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor

J. Neurochem. (2012) 121, 168–179.

Transfusion of hemoglobin-based oxygen carriers in the carboxy state is beneficial during transient focal cerebral ischemia

Exchange transfusion of large volumes of hemoglobin (Hb)-based oxygen carriers can protect the brain from middle cerebral artery occlusion (MCAO). Hb in the carboxy state (COHb) may provide protection at relatively low volumes by enhancing vasodilation. We determined whether transfusion of rats with 10 ml/kg PEGylated COHb [polyethylene glycol (PEG)-COHb] at 20 min of 2-h MCAO was more effective in reducing infarct volume compared with non-carbon monoxide (CO) PEG-Hb. After PEG-COHb transfusion, whole blood and plasma COHb was <3%, indicating rapid release of CO. PEG-COHb transfusion significantly reduced infarct volume (15 ± 5% of hemisphere; mean ± SE) compared with that in the control group (35 ± 6%), but non-CO PEG-Hb did not (24 ± 5%). Chemically dissimilar COHb polymers were also effective. Induction of MCAO initially produced 34 ± 2% dilation of pial arterioles in the border region that subsided to 10 ± 1% at 2 h. Transfusion of PEG-COHb at 20 min of MCAO maintained pial arterioles in a dilated state (40 ± 5%) at 2 h, whereas transfusion of non-CO PEG-Hb had an intermediate effect (22 ± 3%). When transfusion of PEG-COHb was delayed by 90 min, laser-Doppler flow in the border region increased from 57 ± 9 to 82 ± 13% of preischemic baseline. These data demonstrate that PEG-COHb is more effective than non-CO PEG-Hb at reducing infarct volume, sustaining cerebral vasodilation, and improving collateral perfusion in a model of transient focal cerebral ischemia when given at a relatively low dose (plasma Hb concentration < 1 g/dl). Use of acellular Hb as a CO donor that is rapidly converted to an oxygen carrier in vivo may permit potent protection at low transfusion volumes.

Development of Zero-Link Polymers of Hemoglobin, Which do not Extravasate and do not Induce Pressure Increases upon Infusion

Intramolecular crosslink of hemoglobin tetramers solved the problem of urine elimination and short intravascular retention time of cell free hemoglobin infusion. It also produced a family of crosslinked hemoglobins with P50 between 18 and 30 mmHg. However, it did not solve the problem of MAP increases in infused animals. It was proven that extravasation of hemoglobin into interstitial fluid was responsible for MAP increases. Extravasation and the MAP increase was avoided using a hemoglobin polymer with average size near 25 MDa. In spite of a very high oxygen affinity, this polymer delivered oxygen to tissues, producing either vasodilation or vasoconstriction according to oxygen needs. It was also proven that cell free hemoglobins are more efficient than red cells in delivering oxygen to tissues.

Adenosine A2A receptor contributes to ischemic brain damage in newborn piglet

Pharmacologic inactivation or genetic deletion of adenosine A2A receptors protects ischemic neurons in adult animals, but studies in neonatal hypoxia-ischemia (H-I) are inconclusive. The present study in neonatal piglets examined the hypothesis that A2A receptor signaling after reoxygenation from global H-I contributes to injury in highly vulnerable striatal neurons where A2A receptors are enriched. A2A receptor immunoreactivity was detected in striatopallidal neurons. In nonischemic piglets, direct infusion of the selective A2A receptor agonist CGS 21680 through microdialysis probes into putamen increased phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor NR1 subunit and Na+, K+-ATPase selectively at protein kinase A (PKA)-sensitive sites. In ischemic piglets, posttreatment with SCH 58261, a selective A2A receptor antagonist, improved early neurologic recovery and preferentially protected striatopallidal neurons. SCH 58261 selectively inhibited the ischemia-induced phosphorylation of NR1, Na+, K+-ATPase, and cAMP-regulated phosphoprotein 32 KDa (DARPP32) at PKA-sensitive sites at 3 hours of recovery and improved Na+, K+-ATPase activity. SCH 58261 also suppressed ischemia-induced protein nitration and oxidation. Thus, A2A receptor activation during reoxygenation contributes to the loss of a subpopulation of neonatal putamen neurons after H-I. Its toxic signaling may be related to DARPP32-dependent phosphorylation of PKA-sensitive sites on NR1 and Na+, K+-ATPase, thereby augmenting excitotoxicity-induced oxidative stress after reoxygenation.

Role of Nitric Oxide Scavenging in Peripheral Vasoconstrictor Response to ββ Cross-Linked Hemoglobin

Transfusion with many crosslinked hemoglobin solutions causes an increase in arterial pressure that may be mediated by scavenging of nitric oxide (NO). If so, we postulated that inhibiting synthesis of NO after hemoglobin transfusion would fail to cause vasoconstriction ordinarily seen with such inhibition. In pentobarbital anesthetized cats, we tested whether administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), produced peripheral vasoconstriction after isovolemic exchange transfusion with hemoglobin to the same extent as occurs with L-NAME infusion in time controls and in controls matched for reduced hematocrit (17%) with albumin transfusion. Bovine hemoglobin was treated aerobically with bis-(3,5-dibromosalicyl) fumarate to produce βbeta;-81 lysine crosslinks. Hemoglobin exchange transfusion increased mean arterial blood pressure and there was no further increase after L-NAME. In contrast, L-NAME increased pressure in the time controls and albumin controls. Hemoglobin...

Endothelin rather than 20-HETE contributes to loss of pial arteriolar dilation during focal cerebral ischemia with and without polymeric hemoglobin transfusion

Partial exchange transfusion with a cell-free hemoglobin (Hb) polymer during transient middle cerebral artery occlusion (MCAO) reduces infarct volume but fails to increase blood flow, as might be expected with the induced decrease in hematocrit. In ischemic brain, endothelin antagonists are known to produce vasodilation. In nonischemic brain, pial arterioles constrict after Hb exchange transfusion, and the constriction is blocked by an inhibitor of 20-HETE synthesis. We tested the hypothesis that a 20-HETE synthesis inhibitor and an endothelin A receptor antagonist increase pial arteriolar dilation after Hb exchange transfusion during MCAO. Pial arteriolar diameter was measured in the ischemic border region of the distal MCA border region through closed cranial windows in anesthetized rats subjected to the filament model of MCAO. During 2 h of MCAO, pial arteriolar dilation gradually subsided from 37 +/- 3 to 7 +/- 5% (+/-SE). Compared with residual dilation at 2 h of MCAO with vehicle superfusion (14 +/- 3%), loss of dilation was not prevented by superfusion of a 20-HETE synthesis inhibitor (21 +/- 5%), partial Hb exchange transfusion (7 +/- 5%) that decreased hematocrit to 23%, or a combination of the two (5 +/- 5%). However, loss of dilation was prevented by superfusion of an endothelin A receptor antagonist with (35 +/- 4%) or without (32 +/- 5%) Hb transfusion. Pial artery constriction during reperfusion was attenuated by HET0016 alone and by BQ610 with or without Hb transfusion. Systemic administration of the endothelin antagonist during prolonged MCAO increased blood flow in the border region. Thus loss of pial arteriolar dilation in the ischemic border region during prolonged MCAO depends on endothelin A receptor activation, and this effect was independent of the presence of cell-free Hb polymers in the plasma. In contrast to previous work in nonischemic brain, inhibition of oxygen-dependent 20-HETE synthesis does not significantly influence the pial arteriolar response to polymeric Hb exchange transfusion during focal ischemia.

Adipyl crosslinked bovine hemoglobins as new models of allosteric systems.

As indicated by peptide analyses and mass spectrometry estimations, intramolecular crosslink with bis(3,5‐dibromosalicyl)adipate of bovine hemoglobin results in the formation of two main components covalently bridged across the β‐cleft. In one component the crosslink joins the β1V1‐β2K81 residues (XL‐Peak‐1), in the other the bridge is between the β1K81‐β2K81 residues (XL‐Peak‐2). Both components are tetrameric with a mass near MW = 67 kDa as estimated by gel filtration, and a hydrodynamic radius near 3.20 nm, estimated by dynamic light scattering. They have very low oxygen affinity with Pm near100 mmHg (XL‐Peak‐1) and near 70 mmHg (XL‐Peak‐2) respectively at 37° C, at neutral pH. The Bohr effect is almost absent in XL‐Peak‐1, while in XL‐Peak‐2 it is very near normal. Both systems show oxygen binding cooperativity with an index near n = 2.0. Flash photolysis kinetics of the recombination with CO could be resolved into a fast and a slow component. The amplitude of the fast rates were not concentration‐dependent. The stopped‐flow kinetics were autoaccelerating, consistent with their ligand‐binding cooperativity. All rates were very similar to those of normal hemoglobin, suggesting that the oxy‐ rather than the deoxy‐forms of the systems were affected by the crosslink. Proteins 2000;39:166–169.

Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.