Salvatore Guarini

Efferent Vagal Fibre Stimulation Blunts Nuclear Factor-κB Activation and Protects Against Hypovolemic Hemorrhagic Shock

Background—We investigated whether electrical stimulation (STIM) of efferent vagus nerves may suppress nuclear factor (NF)-&kgr;B activation and the inflammatory cascade in hemorrhagic (Hem) shock. Methods and Results—Rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Hem shock was induced by intermittent withdrawing of blood until mean arterial pressure stabilized within the range of 35 to 40 mm Hg. Application of constant voltage pulses to the caudal vagus ends (STIM; 5 V, 2 ms, 1 Hz for 12 minutes, 5 minutes after mean arterial pressure stabilization) increased survival time (VGX+Hem+Sham STIM=38±3 minutes; VGX+Hem+STIM >180 minutes), reverted the marked hypotension (VGX+Hem+Sham STIM=33±3 mm Hg; VGX+Hem+STIM=66±5 mm Hg), inhibited I&kgr;B&agr; liver loss, and blunted the augmented NF-&kgr;B activity, decreased hepatic tumor necrosis factor (TNF)-&agr; mRNA (VGX+Hem+Sham STIM=1.42±0.5 amount of TNF-&agr; m-RNA; VGX+Hem+STIM=0.51±0.2 amount of TNF-&agr; mRNA), and reduced plasma TNF-&agr; (VGX+Hem+Sham STIM=190±24 pg/mL; VGX+Hem+STIM=87±15 pg/mL). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Conclusions—Our results show a parasympathetic inhibition of NF-&kgr;B by which the brain opposes NF-&kgr;B activation in the liver and modulates the inflammatory response during acute hypovolemic hemorrhagic shock.

α-MSH and other ACTH fragments improve cardiovascular function and survival in experimental hemorrhagic shock

Hypovolemic shock was produced in rats by withdrawing about 50% of the estimated total blood volume. Following mean arterial pressure stabilization in the range of 15–25 mm Hg, with a pulse pressure of 7–12 mm Hg, the rats were given intravenous bolus injections either of ACTH fragments or of saline. The following ACTH fragments or analogs were used: ACTH-(4–10), α-MSH, ACTH-(1–16), ACTH-(1–17), ACTH(1–18), [Nle4, D-Phe7]α-MSH, [β-Ala1,Lys17]ACTH-(1–17)-4-amino-n-butilamide (alsactide). ACTH-(1–24) and human synthetic ACTH-(1–39) were used for comparison. All animals treated with saline died in 22.51 ± 3.62 min. Treatment with ACTH fragments (160 μg/kg i.v.) increased blood pressure and pulse amplitude, the effect starting within a few minutes, gradually increasing, and reaching a maximum in 15–30 min. The blood and pulse pressure increases were sustained, remaining almost stable until the end of the 2 h recording. Two out of nine rats treated with alsactide, which was the least active, died within 2 h after treatment, while all rats treated with the other ACTH fragments or analogs were still surviving at that time. Both on a weight and on a molar basis, the most active was ACTH-(1–24), followed by ACTH-(1–16), by the α-MSH analog [Nle4,D-Phe7]ACTH-(1–13), by ACTH-(1–18) and by ACTH-(1–17). The present results show that melanocortins reverse otherwise fatal hypovolemic shock, and suggest a new therapeutic approach for shock treatment. Since endogenous opioids play a key role in the pathophysiology of shock, these data provide further experimental support to the hypothesis of a melanocortin-opioid homeostatic system, involved in many important body functions.

Hydrogen sulfide slows down progression of experimental Alzheimer’s disease by targeting multiple pathophysiological mechanisms

It has been previously reported that brain hydrogen sulfide (H2S) synthesis is severely decreased in Alzheimers disease (AD) patients, and plasma H2S levels are negatively correlated with the severity of AD. Here we extensively investigated whether treatment with a H2S donor and spa-waters rich in H2S induces neuroprotection and slows down progression of AD. Studies with sodium hydrosulfide (a H2S donor) and Tabianos spa-water were carried out in three experimental models of AD. Short-term and long-term treatments with sodium hydrosulfide and/or Tabianos spa-water significantly protected against impairment in learning and memory in rat models of AD induced by brain injection of β-amyloid1-40 (Aβ) or streptozotocin, and in an AD mouse model harboring human transgenes APPSwe, PS1M146V and tauP301L (3xTg-AD mice). The improvement in behavioral performance was associated with hippocampus was size of Aβ plaques and preservation of the morphological picture, as found in AD rats. Further, lowered concentration/phosphorylation levels of proteins thought to be the central events in AD pathophysiology, namely amyloid precursor protein, presenilin-1, Aβ1-42 and tau phosphorylated at Thr181, Ser396 and Ser202, were detected in 3xTg-AD mice treated with spa-water. The excitotoxicity-triggered oxidative and nitrosative stress was counteracted in 3xTg-AD mice, as indicated by the decreased levels of malondialdehyde and nitrites in the cerebral cortex. Hippocampus reduced activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in phosphorylation of tau protein but also in inflammation and apoptosis, was also found. Consistently, decrease in tumor necrosis factor-α level, up-regulation of Bcl-2, and down-regulation of BAX and the downstream executioner caspase-3, also occurred in the hippocampus of 3xTg-AD mice after treatment with Tabianos spa-water, thus suggesting that it is also able to modulate inflammation and apoptosis. Our findings indicate that appropriate treatments with H2S donors and Tabianos spa-waters, and may be other spa-waters rich in H2S content, might represent an innovative approach to slow down AD progression in humans by targeting multiple pathophysiological mechanisms.

Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.

ABSTRACT The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited I&kgr;B&agr; liver loss, blunted the augmented nuclear factor-&kgr;B activity, decreased hepatic tumor necrosis factor (TNF)-&agr; mRNA (VGX + SAO + Sham STIM = 1.0 ± 1.9 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 ± 0.2 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-&agr; (VGX + SAO + Sham STIM = 118 ± 19 pg/mL; VGX + SAO + STIM = 39 ± 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 ± 1 U/g tissue; VGX + SAO + STIM = 3.1 ± 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 ± 1.0 U/g tissue; VGX + SAO + STIM = 3.2 ± 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-&kgr;B and TNF-&agr; in SAO shock.

Oxidative stress causes nuclear factor-κB activation in acute hypovolemic hemorrhagic shock

Nuclear Factor kappaB (NFkappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA; 6 +/- 2.2 microm), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 microm), both studied to evaluate OH(*) production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of IkappaBalpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

Vagus nerve mediates the protective effects of melanocortins against cerebral and systemic damage after ischemic stroke.

A vagus nerve-mediated, efferent cholinergic protective pathway activated by melanocortins is operative in circulatory shock and myocardial ischemia. Moreover, melanocortins have neuroprotective effects against brain damage after ischemic stroke. Here we investigated cerebral and systemic pathophysiologic reactions to focal cerebral ischemia in rats induced by intrastriatal microinjection of endothelin-1, and the possible protective role of the melanocortin-activated vagal cholinergic pathway. In the striatum and liver of saline-treated control rats, the activation of extracellular signal-regulated kinases, c-jun N-terminal kinases, and caspase-3, the increase in tumor necrosis factor-α (TNF-α) concentration and DNA fragmentation, as well as the increase in TNF-α plasma levels, occurred 10 and 20 h after the ischemic insult suggesting an activation of inflammatory and apoptotic responses. Treatment with [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH; 3 or 9 h after stroke) suppressed the inflammatory and apoptotic cascades at central and peripheral level. Bilateral vagotomy and pharmacologic blockade of peripheral nicotinic acetylcholine receptors blunted the protective effect of NDP-α-MSH. The present results show that focal brain ischemia in rats causes significant effects not only in the brain, but also in the liver. Moreover, our data support the hypothesis that a protective, melanocortin-activated, vagal cholinergic pathway is likely operative in conditions of ischemic stroke.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

In circulatory shock, melanocortins have life‐saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists.

Adrenocorticotropin reversal of experimental hemorrhagic shock is antagonized by morphine

ACTH-(1-24) dose-dependently improved cardiovascular function in rats and dogs subjected to experimental hemorrhagic shock, and intravenous dose of 160 and 100/microgram/kg, respectively, completely restoring arterial blood pressure and pulse amplitude. All saline-treated animals died within 30 min of bleeding, while all ACTH-treated animals were still alive at the end of the observation period (2 hr). The injection of ACTH-(1-24) also dramatically improved the respiratory function. Morphine, i.v. injected into rats at the dose of 2.5 mg/kg, antagonised the effect of ACTH-(1-24) to a greater or lesser degree, depending on the dose of peptide employed: at 160/microgram/kg, antagonism was complete, at 320/microgram/kg antagonism was only partial, while at 480/microgram/kg antagonism was almost completely overcome. These data further support the idea that melanocortins are physiological antagonists of opioids, and suggest that melanocortin peptides may prove to be rational and effective drugs in the treatment of hypovolemic shock.

A highly reproducible model of arterial thrombosis in rats

The objective of this investigation was to develop a reproducible and reliable method of arterial thrombosis in a small laboratory animal. Rats were anesthetized with urethane, and a common carotid artery was exposed. A completely occlusive thrombus was produced by applying an electric current to the arterial wall (2 mA for 5 min) while simultaneously constricting the artery with a hemostatic clamp placed immediately downstream from the electrodes. A complete and persistent cessation of blood flow was obtained in all the control rats starting 10-15 min after the thrombogenic lesion. Histological examination revealed a picture of mixed white and red thrombus, stratified and rich in platelets aggregates and fibrin, with piles of red cells trapped in the fibrin network. On the other hand, stasis alone (clamping) was ineffective at all, whereas electric current application alone caused non-occlusive thrombosis only in 30% of animals. An antithrombotic dose of heparin (3 mg/kg i.v., 30 min before thrombus induction) prevented the formation of a persistent thrombus, blood flow being progressively restored (up to 59% of basal value within 45 min). Similarly, a thrombolytic dose of urokinase-type plasminogen activator (2 mg/kg, for 120 min, starting 15 min after thrombus induction) caused a rapid and progressive resumption of blood flow (up to 80% of basal value). This method gives highly consistent and reproducible results and may be suitable for the study or the screening of antithrombotic as well as thrombolytic agents.

Reversal of haemorrhagic shock in rats by cholinomimetic drugs.

1 In an experimental model of haemorrhagic shock resulting in the death of all rats within 20–30 min, the intravenous (i.v.) injection of the tertiary amine cholinesterase inhibitor physostigmine (17–70 μgkg−1) induced a prompt, sustained and dose‐dependent improvement of cardiovascular and respiratory function, with marked increase in the volume of circulating blood and survival of all treated animals, at least for the 2 h of observation. 2 Similar results were obtained with the i.v. injection of the cholinoceptor agonist oxotremorine (5–25 μg kg−1), while neostigmine (54 or 70 μgkg−1), a quaternary cholinesterase inhibitor which cannot cross the blood‐brain barrier, had negligible effects. 3 The anti‐shock activities of oxotremorine and physostigmine were blocked by the intracerebroventricular injection of either of the combined nicotinic and M2‐muscarinic receptor antagonists gallamine and pancuronium, or of the nicotinic antagonist mecamylamine. They were also blocked by intraperitoneal injection of the adrenergic neurone blocking agent guanethidine, but they were not antagonized by either the combined M1‐ and M2‐muscarinic receptor antagonist atropine, the M1‐muscarinic receptor antagonist pirenzepine, or the M2‐muscarinic receptor antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methobromide. 4 It is concluded that cholinomimetic drugs can reverse hypovolaemic shock through central activation (seemingly mediated by nicotinic receptors) of sympathetic tone, with mobilization and redistribution of the residual blood.