Role of haeme oxygenase-1 in resolution of oxidative stress-related pathologies: focus on cardiovascular, lung, neurological and kidney disorders.

The present review examines the role of the cytoprotective enzyme haeme oxygenase-1 (HO-1) in adaptive responses to inflammatory disease and explores strategies for its clinical use, with particular emphasis on use of therapeutic use of the enzyme using phytochemical inducers of HO-1 such as extracts of Ginkgo biloba, curcumin, and flavonoids extracted from seeds of the sour cherry (Prunus cerasus). This laboratory has identified strategies by which combinations of dietary phytochemicals may be configured to synergistically strengthen immunoregulatory mechanisms that normally prevent inflammation from leading to disease. A major focus of this research initiative has been HO-1, which is capable of substantially reducing oxidative stress by several mechanisms. HO-1 metabolizes haeme that accumulates in tissues because of red blood cell turnover. Two products of this degradation - carbon monoxide (CO) and bilirubin - have potent capacity for reducing oxidative stress and for counteracting its effects. A description will be provided of how HO-1 products maintain healthy tissue function and remediate oxidative tissue damage. This will be explored in four major organ systems, including the cardiovascular system, the lungs, the central nervous system and the kidneys. Particular focus will be given to the physiological coordination of cardiovascular functions mediated by CO produced by HO-1 and to nitric oxide (NO), a gaseous second messenger expressed by nitric oxide synthetase. A major unifying theme of the present review is an exploration of the potential use of dietary phytochemical formulations as tools for the clinical application of HO-1 in therapeutic reduction of oxidative stressors, with resultant improved treatment of inflammatory pathologies.

Inhibition of cardiac leptin expression after infarction reduces subsequent dysfunction.

Leptin is known to exert cardiodepressive effects and to induce left ventricular (LV) remodelling. Nevertheless, the autocrine and/or paracrine activities of this adipokine in the context of post-infarct dysfunction and remodelling have not yet been elucidated. Therefore, we have investigated the evolution of myocardial leptin expression following myocardial infarction (MI) and evaluated the consequences of specific cardiac leptin inhibition on subsequent LV dysfunction. Anaesthetized rats were subjected to temporary coronary occlusion. An antisense oligodesoxynucleotide (AS ODN) directed against leptin mRNA was injected intramyocardially along the border of the infarct 5 days after surgery. Cardiac morphometry and function were monitored by echocardiography over 11 weeks following MI. Production of myocardial leptin and pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 were assessed by ELISA. Our results show that (1) cardiac leptin level peaks 7 days after reperfused MI; (2) intramyocardial injection of leptin-AS ODN reduces early IL-1ß and IL-6 overexpression and markedly protects contractile function. In conclusion, our findings demonstrate that cardiac leptin expression after MI could contribute to the evolution towards heart failure through autocrine and/or paracrine actions. The detrimental effect of leptin could be mediated by pro-inflammatory cytokines such as IL-1ß and IL-6. Our data could constitute the basis of new therapeutic approaches aimed to improve post-MI outcome.

The role of exogenous carbon monoxide in the recovery of post-ischemic cardiac function in buffer perfused isolated rat hearts.

Isolated rat hearts were perfused for 10 min with oxygenated buffer and equilibrated with carbon monoxide (CO) of 0.001% and 0.01% before the induction of 30 min global ischemia followed by 120 min of reperfusion. These concentrations of CO significantly improved the post-ischemic recovery of coronary flow (CF), aortic flow (AF), and left ventricular developed pressure (LVDP). The improvement in recovery reflected in the reduction of infarct size and the incidence of reperfusion-induced ventricular fibrillation (VF). Thus, hearts subjected to 0.001% and 0.01% of CO exposure via the perfusion buffer, infarct size was reduced from the CO-free control value of 39% +/- 5% to 21% +/- 3% (*p<0.05) and 18% +/- 4% (*p<0.05), respectively. In the presence of 0.001% and 0.01% CO, the incidence of VF was also reduced from its control value of 92% to 17% (*p<0.05) and 17% (*p<0.05), respectively. Increasing the CO exposure to 0.1% in the buffer, all hearts showed VF combined with ventricular tachycardia or bradycardia and various rhythm disturbances indicating the direct toxic effects of CO on the myocardium. The results show that cardioprotective concentrations (0.01% and 0.001%) of exogenous CO related to an increase in cGMP levels and guanylate cyclase activities.

Regulation of ventricular fibrillation by heme oxygenase in ischemic/reperfused hearts.

We have assessed the relationship between reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO) mRNA expression using northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme activity in isolated working ischemic/reperfused rat hearts. Isolated hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. Upon reperfusion with VF, cardiac function was registered (n = 6 in each group), and HO mRNAs and enzyme activities were measured at the end of reperfusion in hearts that showed VF or did not develop VF. The expression of HO-1 mRNA (about fourfold) was observed in ischemic/reperfused nonfibrillated myocardium in comparison with the nonischemic control hearts. In those hearts when VF was developed, the expression of HO-1 mRNA was not observed in comparison with the nonischemic control myocardium. The results measured by RT-PCR and enzyme analysis support the data obtained by northern blotting. In additional studies, we decided to approach the question from a different angle. Thus, the purpose of our work was also to study the role of HO expression and enzyme activity in electrically fibrillated hearts without the ischemic/reperfused protocol. To simulate the period of 10 min of reperfusion-induced VF, hearts were electrically fibrillated, then defibrillated, and perfused for an additional 110 min, and HO-1 mRNA expression and enzyme activities were determined. Thus, electrically induced VF resulted in about 60%, 60%, and 70% reduction in HO-1 mRNA expression, RT-PCR signal intensity, and enzyme activity, respectively, compared with the nonfibrillated ischemic/reperfused group. In conclusion, our data provide evidence that the development of reperfusion-induced VF inhibits HO-1 mRNA expression and enzyme activity in both electrically fibrillated myocardium and ischemic/reperfused fibrillated hearts. The results clearly show that HO-1 mRNA expression and enzyme activity were increased in ischemic/reperfused nonfibrillated myocardium, suggesting that interventions that are able to increase HO-1 mRNA expression and enzyme activity may prevent the development of VF.

Non-specific caspase inhibition reduces infarct size and improves post-ischaemic recovery in isolated ischaemic/reperfused rat hearts.

Myocardial ischaemia and reperfusion lead to myocardial cell death due, at least in part, to apoptotic mechanisms. Although cysteinyl aspartate-specific proteinase (caspase) activation is a major event and the most-cited culprit in the development of apoptosis, its potential contribution to ischaemic myocardial cell death is largely unknown. To study the role of caspase activation, isolated rat hearts (n=6 per group) were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion. A non-selective [0.1 or 0.5 microM acetyl-Tyr-Val-Ala-Asp chloromethylketone (YVAD-cmk)] or selective caspase inhibitors [0.07 or 0.2 microM acetyl-Asp-Glu-Val-Asp-cmk (Ac-DEVD-cmk, caspase-3 inhibitor); 0.07 or 0.2 microM benzoxycarbonyl-Leu-Glu-OMe-His-Asp(OMe)-fluoromethylketone (z-LEHD-fmk, caspase-9 inhibitor)] were added to the perfusate at the start of reperfusion. Non-selective caspase inhibition with 0.1 or 0.5 microM YVAD-cmk limited infarct size: (21 +/- 4%, P<0.05; 17 +/- 3%, P<0.05, respectively) compared with the ischaemic/reperfused control (32 +/- 5%). In hearts treated with 0.1 or 0.5 microM caspase II non-selective inhibitor, the fraction of terminal-deoxynucleotidyl-transferase deoxyuridine nick end labelling (TUNEL)-positive myocyte nuclei in the infarcted zone was reduced from the ischaemic/reperfused non-treated control of 11.2 +/- 2.1% to 6.2 +/- 1.6% (P<0.05) and 1.2 +/- 0.2% (P<0.05), respectively. The recovery of post-ischaemic cardiac function (coronary flow, aortic flow and left-ventricular developed pressure) improved significantly with the application of the non-selective caspase inhibitor as well. In hearts perfused with specific caspase inhibitors (caspase-3 and caspase-9) there was no significant reduction in the infarct size, no improvement in post-ischaemic cardiac function and no reduction of apoptotic cell death. We conclude that non-specific inhibition of caspases may be therapeutically beneficial in myocardial ischaemia/reperfusion-induced damage, while selective caspase inhibitors may fail to prevent such reperfusion-induced injury in our model system.

Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts.

Effects of the calcineurin inhibitor FK506, the platelet-activating factor (PAF) antagonist, and free radical scavenger Ginkgo biloba extract, EGb 761, and their combination on reperfusion-induced ventricular fibrillation (VF), ventricular tachycardia (VT), and recovery of cardiac function were studied after 30 min of global ischemia followed by 2 h of reperfusion in isolated rat hearts. In the first series of studies, rats received a daily (oral) dose of 0, 1, 5, 10, 20, or 40 mg/kg/day FK506 for 10 days. FK506 dose-dependently reduced the incidence of reperfusion-induced total (irreversible plus reversible) VF from a value of 92% for untreated animals to 92% (NS), 83% (NS), 67% (NS), 33% (p<0.05), and 25% (p<0.05), for doses of 1-40 mg/kg/day, respectively, with effects on incidence of VT showing the same pattern. FK506, between 20 and 40 mg/kg/day, also resulted in significant recovery of postischemic cardiac function. In the second series of studies, rats were treated with EGb 761 alone or in combination with FK506. Whereas no significant reduction in arrhythmias or improvement in cardiac function resulted from a single intervention of EGb 761 at 25 mg/kg/day, combined treatment of rats with 25 mg/kg/day of EGb 761 and 1 or 5 mg/kg/day of FK506 resulted in a reduction in total and irreversible VF of 92% and 92% to 42% (p<0.05) and 33% (p<0.05), 25% (p<0.05) and 8% (p<0.05), respectively, versus untreated control animals, paralleled by similar effects on the incidence of VT and accompanied by significant improvements in postischemic cardiac function. Our results demonstrate a novel cardioprotective characteristic of FK506 and suggest that combination therapy by using FK506 plus EGb 761 synergistically improves postischemic cardiac function, while reducing the incidence of reperfusion-induced VF and VT, which may expand the clinical utility of FK506 and allow therapy with FK506 at lower doses than are currently useful.

Synaptic connections of horizontal canal mediated ascending Deiters tract axons on medial rectus motoneurons in cat.

This study demonstrates the termination of ascending tract of Deiters' (ATD) axons on ipsilateral medial rectus (MR) motoneurons. Horseradish peroxidase (HRP) was iontophoretically injected into ATD axons which were recorded in the MR motoneuron pool of the oculomotor nucleus. MR motoneuron cell bodies were identified by retrograde transport of HRP injected into MR muscles in the orbit. ATD axons were identified by Type I responses to horizontal rotation, monosynaptic responses on stimulation of the ipsilateral labyrinth, and no response on contralateral labyrinth or contralateral abducens nucleus or on ipsilateral MR nerve stimulation. Light microscopic examination showed the main stem axons to be lateral to the medial longitudinal fasciculus, and terminal boutons were in contact with ipsilateral identified MR motoneurons (Furuya and Markham: Exp. Brain Res. 43: 289-303, 1981). Light microscopy and semi-thin sections showed boutons of ATD in contact with identified MR motoneuron cell bodies and proximal dendrites. The electron micrographs (EM) showed the HRP-injected ATD axons have synapses on MR motoneurons. ATD boutons made axosomatic and axodendritic synapses on MR motoneurons. The boutons contained numerous spheroidal synaptic vesicles. Several examples showed clear asymmetrical post-synaptic membrane specialization. This confirms the synaptic connection between horizontal canal activated elements in the ATD and MR motoneurons.

Choline and acetylcholine metabolism in rat neostriatal slices.

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).

The preservation of nerve cells in rat neostriatal slices maintained in vitro: a morphological study.

The in vitro preservation of neurons in 300 micron thin neostriatal slices, which are routinely used for electrophysiological studies, was examined by light and electron microscopy and was compared to 700 micron thick neostriatal slices. The thin slices displayed well-preserved cells after up to 5 h of incubation. This finding correlated well with whether electrical activity could be recorded. In cross-section, the thin slices consisted of three layers: the inner layer contained many intact cells (80%) and was sandwiched between the outer layers where deteriorating cells predominated. In contrast to the thin slices, the thick slices (700 micron) displayed no layering of intact cells in cross-section. Instead, the majority of cells throughout these thick slices was swollen (98%), with only small patches of intact cells. Two types of deteriorating cells were apparent: swollen cells and dark (pycnotic) cells. The proportion of swollen cells increased with incubation time. In the thin slices this swelling occurred in the outer layers with the middle layer of intact cells remaining relatively unchanged over long incubation periods, whereas all cells in the thick slices were swollen after 2 h of incubation. Dark cells were localized to the outer portion of both slices and the number of such dark cells did not change with incubation time.

Intrinsic cholinergic excitation in the rat neostriatum: nicotinic and muscarinic receptors.

An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 muM), d-tubocurarine (3 muM) or hexamethonium (100 muM), but not by the antimuscarinic agent atropine (100 muM). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1--20 muM, physostigmine 0.1--0.5 muM), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1--3 Hz to 10--30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1--100 muM). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.

Intrinsic excitation in the rat neostriatum mediated by acetylcholine.

Synaptic excitation elicited by local stimulation in neostriatal slices was found to be mediated by acetylcholine (ACh). The synapses generating this excitation belong to intrinsic neurons. Thus, for the first time, direct evidence for the existence of intrinsic excitatory cholinergic neurons in the neostriatum is provided.

Intraaxonal transport of Herpes simplex virus in the rat central nervous system.

Light and electron microscopic observation 3--4 days after microinjection of Herpes simplex virus (HSV) into the left neostriatum of rat demonstrated the following results. (1) Virus labeled nerve cells were found in the ipsilateral substantia nigra; a large number of infected neurons were in the zona compacta and some were in the zona reticulata. No virus infection was evident in the contralateral side. (2) Virus labeled neurons were found in the cortex, a greater number ipsilaterally than contralaterally, and in the dorsal raphé nuclei. Cortical microinjection of HSV led to infection of some cortical cells but no neostriatal cells. We conclude, therefore, that spread of the virus to the cortex, the substantia nigra and the dorsal raphé following neostriatal injection was by retrograde axonal transport. (3) The left neostriatum, where HSV was injected, showed a surprisingly small number of virus infected neurons. The infected neurons were mostly the large neurons; the majority of medium sized neurons were well preserved. There was massive degeneration of nerve terminals throughout the neuropil. Most of these degenerating nerve terminals are considered to be afferent fibers.

Electron microscopic investigation of the vestibular projection to the cat trochlear nuclei.

Ultrastructural degeneration studies were carried out on the cat trochlear nucleus following lesion of the vestibulo-trochlear pathway in order to characterize the location and type of presynaptic endings involved in this pathway. Four types of boutons are found in the normal trochlear nucleus. Types I and II are large and demonstrate typical en passant profiles with small diameter synaptic vesicles (35 and 40 nm). These terminals are characterized by the absence of neurofilaments in the Type II endings. Types III and IV are smaller boutons, located more axondendritically, and contain larger diameter synaptic vesicles (45 nm). Type V terminals contain large, granulated vesicles and occur only rarely. Following the interruption of the ascending projection from the ipsilateral superior and medial vestibular nuclei by parasagittal medullary lesions, degeneration of Type II boutons was commonly encountered in the ipsilateral trochlear nucleus. Predominantly Type III degeneration was found in the contralateral trochlear nucleus. Electrical stimulation of the vestibular nerve showed that these lesions resulted in (1) a complete loss of inhibition in the ipsilateral trochlear nucleus and (2) a significant (75-90%) reduction in the contralateral excitatory pathway to the trochlear nucleus. Midline sagittal lesions in the floor of the fourth ventricle interrupting the decussating fiber projection from the bilateral medial vestibular nuclei resulted in selective degeneration of only Type III boutons in both trochlear nuclei. We conclude that inhibitory vestibular neurons eminating from the superior vestibular nucleus terminate on trochlear motoneurons with Type II boutons and excitatory vestibular neurons from the contralateral medial vestibular nucleus end on trochlear motoneurons with Type III boutons.

Regional distribution of choline acetyltransferase and acetylcholinesterase activity in baboon brain.

(1) The activities of choline acetyltransferase (ChAc) (EC 2.3.1.6) and acetylcholinesterase (AChE) (EC 3.1.1.7) were determined in about one hundred regions and subregions of baboon brain. The activities and distributions of these enzymes were in comparable to those found previously in the brains of other species. (2) ChAc activity was highest in the interpeduncular nucleus, where it was about twice that in the putamen, the region previously thought to be the richest in this enzyme. The caudate nucleus, the substantia perforata, the nucleus basalis, the central part of the amygdala and the oculomotor nucleus also had high activities. The activities in the cerebral and cerebellar cortex were less than one twentieth of that in the interpeduncular nucleus. (3) The distribution of AChE activity was not entirely in parallel with that of ChAc.