Neonatal Sepsis Alters the Excitability of Regular Spiking Cells in the Nucleus of the Solitary Tract in Rats.

OBJECTIVE: Sepsis is a leading cause of mortality and morbidity in infants. Although the measures of autonomic dysfunction (e.g., reduced heart rate variability) predict mortality in sepsis, the mechanism of sepsis-induced autonomic dysfunction has remained elusive. The nucleus of the solitary tract (NTS) is a vital structure for the integrated autonomic response to physiological challenges. In the present study we hypothesized that sepsis alters the excitability of NTS neurons in a rat model of neonatal sepsis (14-day-old rats). METHODS AND RESULTS: Sepsis was induced by intraperitoneal injection of cecal slurry (CS) in rat neonates. The presence of autonomic dysfunction was confirmed by observing a significant reduction in both short-term and long-term heart rate variably following CS injection. We investigated the effect of polymicrobial sepsis on the electrophysiological properties of the medial NTS neurons using a whole cell patch clamp recording. Our results showed that the resting membrane potential in regular spiking neurons was significantly less polarized in the septic group (-37.6 ±â€Š1.76 mv) when compared with the control group (-54.7 ±â€Š1.73 mv, P < 0.001). The number of spontaneous action potentials in the septic group was also significantly higher than the control group (P < 0.05). In addition, the frequency and amplitude of the spontaneous excitatory post synaptic potentials was significantly higher in neurons recorded in the septic group (P < 0.001). Interestingly, regular spiking cells in the CS group exhibited a rebound action potential following hyperpolarization. Injection of depolarizing currents was associated with lower first spike latency and changes in rise slope of action potential (P < 0.001). CONCLUSIONS: We showed that polymicrobial sepsis increases the excitability of regular spiking cells in the medial NTS. These alterations can potentially affect neural coding and thus may contribute to an abnormal homeostatic or allostatic physiological response to sepsis and systemic inflammation.

The effect of fractal-like mechanical ventilation on vital signs in a rat model of acute-on-chronic liver failure.

OBJECTIVE: The network of interactions between different organs is impaired in liver cirrhosis. Liver cirrhosis is associated with multi-system involvement, which eventually leads to multiple organ failure. This process is accelerated by a precipitating factor such as bacterial infection, which leads to respiratory distress, circulatory shock, neural dysfunction and very high mortality. Cirrhotic patients often have blunted respiratory sinus arrhythmia and impaired cardio-respiratory variability. Fractal-like mechanical ventilation is reported to enhance respiratory sinus arrhythmia and attenuate respiratory distress in experimental models. In the present study we hypothesise that fractal-like mechanical ventilation may improve the outcome of cirrhotic rats with multiple organ failure. APPROACH: Cirrhosis was induced by chronic biliary obstruction in rats. Acute multiple organ failure was induced by intraperitoneal injection of bacterial endotoxin in cirrhotic rats. The effect of conventional mechanical ventilation (with constant tidal volume and respiratory rate) or fractal-like ventilation (with the same average but variable tidal volume and respiratory rate) were assessed on vital signs, oxygen saturation and plasma alanine aminotransferase in anaesthetised cirrhotic rats. MAIN RESULTS: We demonstrated that fractal-like mechanical ventilation was accompanied by improved oxygen saturation, reduced heart rate and decreased liver injury following injection of bacterial endotoxin. Moreover, variable mechanical ventilation in cirrhotic rats reduced mortality and prevented a fall in short-term heart rate variability following endotoxin challenge in comparison with rats with constant mechanical ventilation. SIGNIFICANCE: We suggest further investigations into the beneficial effects of fractal-like ventilation strategy in critically ill patients with liver failure requiring organ support and mechanical ventilation.

Increased sample asymmetry and memory of cardiac time-series following endotoxin administration in cirrhotic rats.

Sepsis, and other causes of acute systemic inflammation, can reduce heart rate variability (HRV) and increase cardiac cycle regularity in mammals. Thus, HRV monitoring has been used for early detection of sepsis in adults and neonates. Liver cirrhosis is associated with reduced basal HRV and the development of tolerance to the cardiac chronotropic effects of bacterial endotoxin. This may pose limitations on the use of heart rate monitoring in early detection of sepsis in this patient population. In a study to develop a physiomarker for the detection of sepsis in cirrhosis, we observed that endotoxin administration in adult cirrhotic rats leads to the development of transient heart rate decelerations, a phenomenon which has been reported in neonates with sepsis, and quantified using sample asymmetry analysis. In the present study, cirrhosis was induced by surgical ligation of the bile duct in rats. Cirrhotic rats were given intraperitoneal injections of either saline or endotoxin (1 mg kg-1). Changes in sample asymmetry and memory length of cardiac time-series were studied in conscious rats using implanted telemetric probes. Cirrhotic (but not control) rats exhibited increased sample asymmetry following endotoxin injection, which was consistent with the development of transient heart rate deceleration. Endotoxin administration in cirrhotic rats was associated with prolongation of memory length for observing decelerating perturbations in the cardiac rhythm. These findings may have application in the development of an HRV monitoring system for early detection of sepsis in cirrhosis.

Mesenteric artery responsiveness to acetylcholine and phenylephrine in cirrhotic rats challenged with endotoxin: the role of TLR4.

Cirrhosis is associated with vascular dysfunction and endotoxemia. These experiments were designed to investigate the hypothesis that the administration of a low-dose of lipopolysaccharide (LPS) worsens vascular dysfunction in rats subjected to bile-duct ligation (BDL), and to determine whether LPS initiates changes in vascular Toll-like receptor 4 (TLR4) expression. Four weeks after BDL, the animals were given an intraperitoneal injection of either saline or LPS (1.0 mg/kg body mass). Three hours later, the superior mesenteric artery was isolated, perfused, and then subjected to the vasoconstriction and vasodilatation effects of phenylephrine and acetylcholine, respectively. Our results show that phenylephrine-induced vasoconstriction decreased in the cirrhotic vascular bed (BDL rats) compared with the vascular bed of the sham-operated animals, and that the LPS injections in the cirrhotic (BDL) rats worsened this response. LPS injection administered to the sham-operated animals had no such effect. On the other hand, both the BDL procedure and the LPS injection increased acetylcholine-induced vasorelaxation, but LPS administration to the BDL rats had no effect on this response. The mRNA levels of TLR4 did not change, but immunohistochemical studies showed that TLR4 localization switched from the endothelium to vascular smooth muscle cells following chronic BDL. In conclusion, acute endotoxemia in cirrhotic rats is associated with hyporesponsiveness to phenylephrine and tolerance to the effects of acetylcholine. Altered localization of TLR4 may be responsible for these effects.

Prostaglandin F2α modulates atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats.

Endotoxemia induces various physiological adaptive responses such as tachycardia. There is evidence to show that inflammatory tachycardia might be linked to a direct action of prostanoids on the cardiac pacemaker cells. Recent reports have indicated that systemic inflammation may uncouple of cardiac pacemaker from cholinergic neural control in experimental animals; however, the exact mechanism of this phenomenon is uncertain. This study was aimed to explore the hypothesis that prostanoids modulate atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats. Male albino rats were given intraperitoneal injection of either saline or lipopolysaccharide (LPS, 1 mg/kg). 3 h after saline or LPS injection, the atria were isolated and chronotropic responsiveness to cholinergic stimulation was evaluated in an organ bath. The expression of atrial cyclooxygenases (COX)-1, COX-2 and COX-3 mRNA was assessed by quantitative real-time RT-PCR and cytosocalcium-dependent phospholipase A2 (cPLA2) activity was measured in the atria. The expression of atrial COX-2 mRNA and cPLA2 activity increased significantly in endotoxemic atria (P<0.05). Incubation with prostaglandin F2α (PGF2α, 100 pM) could significantly decrease chronotropic response to cholinergic stimulation in vitro. Likewise, LPS injection could induce a significant hyporesponsiveness to cholinergic stimulation, and incubation of isolated atria with either indomethacin (5 µM) or AL-8810 (a PGF2α antagonist, 10 µM) could reverse it (P<0.01, P<0.05, respectively), while SQ29548 (a thromboxane A2 antagonist, 10 nM) was failed (P>0.05). Our data showed that PGF2α may contribute to the atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats.

Activation of central muscarinic receptor type 1 prevents development of endotoxin tolerance in rat liver.

Endotoxin tolerance is a mechanism in which cells receiving low doses of endotoxin, enter a transient phase with less inflammatory response to the next endotoxin challenges. Central nervous system is known to modulate systemic inflammation through activation of the cholinergic system; however, the role of central anti-inflammatory pathway in pathophysiology of hepatic endotoxin tolerance is unknown. Our study was designed to assess the effect central muscarinic type 1 receptor (M1) activation on development of endotoxin tolerance in rat liver. Endotoxin tolerance was induced by daily intraperitoneal injection of endotoxin (1 mg/kg) for 5 days. Animals were randomly divided into two groups which received intracerebroventricular injection of either MCNA-343 (an M1 agonist, 5 ng/kg) or saline 1h after intraperitoneal injection of saline or endotoxin. The responsiveness to endotoxin was assessed by measuring hepatic MCP-1 (monocyte chemotactic protein-1), iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis-α) mRNA expression 3h after intraperitoneal administration of endotoxin using quantitative RT-PCR. A significant reduction in hepatic expression of MCP-1, iNOS and TNF-α was observed in rats with 5 days endotoxin challenge in comparison with rats given a single dose of endotoxin. There was no significant difference in hepatic expression of MCP-1, iNOS or TNF-α between acute and chronic LPS-treated groups in rats given MCNA-343. Central MCNA-343 stimulation could prevent the induction of hepatic endotoxin tolerance in animals receiving repeated doses of endotoxin. This indicates that M1 cholinergic receptor activation in the central nervous system can modulate endotoxin tolerance in rat liver.

The role of α7 nicotinic acetylcholine receptor in modulation of heart rate dynamics in endotoxemic rats.

Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in modulation of heart rate dynamics during systemic inflammation.

Effect of endotoxin on heart rate dynamics in rats with cirrhosis.

Reduced heart rate variability (HRV) is a hallmark of systemic inflammation which carries negative prognostic information in sepsis. Decreased HRV is associated with partial uncoupling of cardiac pacemaker from cholinergic neural control during systemic inflammation. Sepsis is a common complication in liver cirrhosis with high mortality. The present study was aimed to explore the hypothesis that endotoxin uncouples cardiac pacemaker from autonomic neural control and reduces HRV in an experimental model of cirrhosis. Cirrhosis was induced by surgical ligation of the bile duct in rats. Cirrhotic rats were given intraperitoneal injection of either saline or lipopolysaccharide (endotoxin, 1mg/kg). Changes in HRV indices were studied in conscious rats using implanted telemetric probes. The atria were isolated and chronotropic responsiveness to cholinergic stimulation was assessed in vitro. Endotoxin injection induced a significant tachycardia and decreased short-term and long-term HRV indices in control rats. However, endotoxin was unable to increase heart rate in cirrhotic animals. In contrast with control rats, endotoxin induced biphasic changes in short-term HRV in cirrhotic rats. Acute endotoxin challenge reduced long-term HRV with 60-min delay in comparison with control animals. Endotoxin injection was associated with a significant hypo-responsiveness to cholinergic stimulation in control rats in vitro. Endotoxin did not change atrial chronotropic responsiveness to cholinergic stimulation in cirrhotic rats. Our data shows that cirrhosis is associated with development of tolerance to cardiac chronotropic effect of endotoxin in rats.