Preliminary reference intervals and the impact of citrate storage time for thrombelastography in cats including delta and the velocity curve.

BACKGROUND: Thrombelastography is a useful tool in assessment of hemostasis. Beside the traditional variables, the velocity curve and the variable delta have lately earned attention. The velocity curve provides knowledge about the speed of clot formation including information about thrombin generation. Delta, which only reflects enzymatic coagulation, allows the determination of the origin of hypercoagulability when compared to clot rigidity, a variable that reflects both platelet and enzymatic activity. The aim was to establish preliminary reference intervals for feline thrombelastography including the velocity curve variables and delta obtained after 60 min of storage including the assessment of coefficients of variation. Furthermore, the effect of citrate storage time (30 versus 60 min) on feline thrombelastography will be determined. RESULTS: Prolonged storage times significantly reduced reaction (R) (P = 0.019) and clotting (K) (P = 0.008) times, split point (SP) (P = 0.019) and time to maximum rate of thrombus generation (TMRTG) (P = 0.023) values whereas maximum rate of thrombus generation (MRTG) significantly increased (P = 0.040). Preliminary reference intervals: R (min): 2.7-18.1; K (min): 0.8-3.9; alpha (°): 27.6-75.2; maximum amplitude (mm): 18.5-62.5; clot rigidity (dyn/cm2): 1.2-8.2; coagulation index: -4.6 - 2.6; SP (min): 2.4-15.4; delta (min): 0.3-3.1; thrombus generation (mm/min): 255.3-751.2; MRTG (mm/min): 4.0-19.3; TMRTG (min): 3.5-22.0; maximum rate of lysis (mm/min): 0.0-4.7 and time to maximum rate of lysis (min): 0.4-55.8. CONCLUSION: Storage for 60 versus 30 min induces hypercoagulable tracings including the velocity curve, some of which variables (MRTG, TMRTG) might function as sensitive markers for changes in the coagulation activity. Because of the impact of citrate storage time on thrombelastography, reference intervals have to be established using a specific and constant storage time in each laboratory.

Vasopressin V1A receptors mediate the stabilization of intestinal mucosal oxygenation during hypercapnia in septic rats.

BACKGROUND: Microvascular oxygen saturation (µHBO2) plays an essential role in the development and outcome of sepsis. Hypercapnia (HC) improves the microvascular oxygenation of the mucosa in both healthy and septic animals. Vasopressin V1A receptor blockade prevents this positive effect under otherwise physiological conditions. The aim of this study was to investigate the effects and mechanisms of the vasopressin system during hypercapnia under septic conditions. METHODS: 80 rats were randomized into 8 groups (N=10). Colon ascendens stent peritonitis (CASP) or sham surgery was performed on 40 animals each to establish a moderate polymicrobial sepsis or sham control, respectively. 24h after sepsis induction the animals were subjected to 120min of volume-controlled and pressure-limited ventilation with either normocapnic (pCO2 35-45mmHg) or moderate hypercapnic (pCO2 of 65-75mmHg) ventilation targets. Animals received either vasopressin V1A receptor blockade (SR 49059, 1mgkg(-1) i.v.) or vehicle solution (dimethyl sulfoxide, 1%). Blood pressure, heart rate, pO2 and pCO2 were measured and microcirculatory oxygenation (µHBO2) and microcirculatory flow (µflow) were recorded using tissue reflectance spectrophotometry. Oxygen supply (µDO2) and consumption (µVO2) were calculated from intermittent blood gas analysis. RESULTS: In septic animals, µHBO2 declined during normocapnia (-11±10.3) but remained unchanged during hypercapnia. µHBO2 declined with vasopressin V1A receptor blockade both during normocapnia (-7.4±10.6) and hypercapnia (-9.2±9.8). Microcirculatory oxygen consumption was significantly reduced by hypercapnia in septic animals (-2.4·10(5) [AU]±2.4·10(5) [AU]). In sham animals, µHBO2 and µVO2 did not change. CONCLUSION: Vasopressin V1A receptors mediate the beneficial effects of hypercapnia on microcirculatory oxygenation during sepsis. The effects of vasopressin on µHBO2 might be related to decreased oxygen consumption during hypercapnia.

Glycine transporter GlyT1, but not GlyT2, is expressed in rat dorsal root ganglion--Possible implications for neuropathic pain.

Glycinergic inhibitory neurotransmission plays a pivotal role in the development of neuropathic pain. The glycine concentration in the synaptic cleft is controlled by the glycine transporters GlyT1 and GlyT2. GlyT1 is expressed throughout the central nervous system, while GlyT2 is exclusively located in glycinergic neurons. Aim of the present study was to investigate whether GlyTs are also expressed in the peripheral sensory nervous system and whether their expression is modulated in experimental neuropathic pain. Neuropathic pain was induced in male Wistar rats by Chronic Constriction Injury (CCI) and verified by assessment of mechanical allodynia (von Frey method). Expression patterns of GlyTs and the glycine binding subunit NR1 of the N-methyl-d-aspartate (NMDA) receptor in the spinal cord and dorsal root ganglia (DRG) were analyzed by Western blot analysis, PCR and immunohistochemistry. While both GlyT1 and GlyT2 were detected in the spinal cord, only GlyT1, but not GlyT2, was detected in DRG. Immunofluorescence revealed a strictly neuronal localization of GlyT1 and a co-localization of GlyT1 and NR1 in DRG. Compared to sham procedure, spinal cord and DRG expression of GlyT1 was not altered and NR1 was unchanged in DRG 12 days after CCI. GlyT1, but not GlyT2, is expressed in the peripheral sensory nervous system. The co-expression of GlyT1 and NMDA receptors in DRG suggests that GlyT1 regulates glycine concentration at the glycine binding site of the NMDA receptor. Differential regulation of GlyT1 expression in the spinal cord or DRG, however, does not seem to be associated with the development of neuropathic pain.

Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats.

BACKGROUND: MicroRNAs (miRNAs) are involved in the neuroplastic changes which induce and maintain neuropathic pain. However, it is unknown whether nerve injury leads to altered miRNA expression and modulation of pain relevant target gene expression within peripheral nerves. In the present study, expression profiles of miR-1 and the pain-relevant targets, brain derived neurotrophic factor (BDNF) and Connexin 43 (Cx43), were studied in peripheral neuropathic pain, which was induced by chronic constriction injury (CCI) of the sciatic nerve in rats. The expression of miR-1 was investigated in the sciatic nerve, dorsal root ganglion (DRG) and the ipsilateral spinal cord by qPCR. Changes of BDNF and Cx43 expression patterns were studied using qPCR, Western blot analysis, ELISA and immunohistochemistry. RESULTS: In sciatic nerves of naïve rats, expression levels of miR-1 were more than twice as high as in DRG and spinal cord. In neuropathic rats, CCI lead to a time-dependent downregulation of miR-1 in the sciatic nerve but not in DRG and spinal cord. Likewise, protein expression of the miR-1 targets BDNF and Cx43 was upregulated in the sciatic nerve and DRG after CCI. Immunohistochemical staining revealed an endoneural abundancy of Cx43 in injured sciatic nerves which was absent after Sham operation. CONCLUSIONS: This study demonstrates that CCI leads to a regulation of miRNAs (miR-1) in the peripheral nervous system. This regulation is associated with alterations in the expression and localization of the miR-1 dependent pain-relevant proteins BDNF and Cx43. Further studies will have to explore the function of miRNAs in the context of neuropathic pain in the peripheral nervous system.

The beneficial effects of acute hypercapnia on microcirculatory oxygenation in an animal model of sepsis are independent of K(+)ATP channels.

BACKGROUND: Acute hypercapnia maintains the microcirculatory oxygenation of the splanchnic region during sepsis. The first aim of this study was to characterize the role of K(+)ATP channels on the microcirculatory flow and oxygenation during acute moderate hypercapnia. The second aim was to investigate whether a short period of hypercapnia induces detrimental effects in an otherwise undamaged rodent lung. METHODS: Experiments were performed on 60 male Wistar rats. A moderate polymicrobial sepsis was induced by colon ascendens stent peritonitis (CASP) surgery. 24h after induction of sepsis volume-controlled and pressure-limited ventilation was established for 120 min, with either normocapnic (pCO2 35-45 mmHg) or moderate hypercapnic ventilation targets (pCO2 65-75 mmHg) and with or without non-selective K(+)ATP channel blockade with glibenclamide. Microcirculatory blood flow of the colonic wall as well as oxygen delivery and consumption were assessed with tissue laser Doppler and reflectance spectrophotometry. Hemodynamic variables were recorded and plasma cytokine levels and myeloperoxidase levels of the lungs were analyzed. RESULTS: In septic animals microcirculatory oxygenation deteriorated progressively with normocapnia (-11.7 ± 11.8%) but was maintained (-2.9 ± 5.6%) with hypercapnia. This effect was associated with an increased microcirculatory oxygen consumption in septic animals with normocapnia (+25.7 ± 37.1%) that was decreased in the hypercapnia groups (-7.2 ± 28.1%). The effect of hypercapnia in septic animals was not altered by additional K(+)ATP channel blockade (-5.7 ± 32.7%). Hypercapnia neither induced an inflammatory response in lungs nor altered the systemic cytokine response. CONCLUSIONS: The observed beneficial effect of hypercapnia on microvascular oxygenation of the colon in sepsis does not seem to be mediated via K(+)ATP channels.

Remote ischemic preconditioning preserves Connexin 43 phosphorylation in the rat heart in vivo.

BACKGROUND: Remote ischemic preconditioning (RIPC) protects the heart from ischemia and reperfusion (I/R) injury. The underlying molecular mechanisms are unclear. It has been demonstrated that Connexin 43 (Cx43) is critically involved in cardioprotective interventions including classical ischemic preconditioning. In the present study we investigated the influence of RIPC on the expression patterns of Cx43 after I/R in the rat heart in vivo. METHODS: Male Wistar rats were subjected to 35 min regional myocardial ischemia followed by 2 h reperfusion with or without 4 cycles of 5 minutes bilateral hind limb ischemia and reperfusion (RIPC), to RIPC without ischemia or underwent no intervention (Sham). Infarct size was measured by TTC staining. The myocardium was divided into area at risk (AAR) and area not at risk (non AAR). Expression of Cx43-mRNA and protein was analyzed by qPCR and Western Blot analysis, respectively. Localization of Cx43 was visualized by confocal immunofluorescence staining. RESULTS: RIPC reduced the infarct size (I/R: 73 ± 5% vs. RIPC I/R: 34 ± 14%, p < 0.05). Expression of Cx43 mRNA did not differ between groups. I/R caused a strong decrease of relative Cx43 protein expression in the AAR that was partly abolished by RIPC. Furthermore, RIPC decreased the level of ischemia-induced dephosphorylation of Cx43. Confocal immunofluorescence staining showed that I/R caused a loss of the Cx43 signal at the intercalated discs, while the Cx43 signal at the intercalated discs was partly sustained after RIPC. CONCLUSION: Preservation of Cx43 protein expression and phosphorylation after RIPC might protect the rat heart in vivo.

Effects of remote ischemic preconditioning and myocardial ischemia on microRNA-1 expression in the rat heart in vivo.

Remote ischemic preconditioning (RIPC) is an easily applicable method for protecting the heart against a subsequent ischemia and reperfusion (I/R) injury. However, the exact molecular mechanisms underlying RIPC are unknown. We examined the involvement of microRNAs (miRNAs) and in particular the expression of miRNA-1 (miR-1) in RIPC and myocardial ischemia. Remote ischemic preconditioning was conducted by four cycles of 5-min bilateral hind-limb ischemia in male Wistar rats. Cardiac ischemia was induced by ligation of the left anterior descending coronary artery for 35 min followed by 2 or 6 h of reperfusion. MicroRNA expression was analyzed by Taqman miRNA arrays and quantitative polymerase chain reaction assays. Luciferase assays were performed to validate the miR-1 target gene brain-derived neurotrophic factor (BDNF). Brain-derived neurotrophic factor mRNA and protein levels were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Remote ischemic preconditioning led to a differential expression of miRNAs. The most abundant cardiac miRNA, miR-1, was downregulated by RIPC without following ischemia as well as after I/R and RIPC followed by I/R after 2 h of reperfusion. After 6 h of reperfusion, RIPC led to an upregulation of miR-1, whereas ischemia had no effect on miR-1 expression. Luciferase assays confirmed the interaction of miR-1 with BDNF, a protein that has been shown to exert cardioprotective effects. Brain-derived neurotrophic factor protein levels in rat hearts measured by enzyme-linked immunosorbent assay were not significantly altered after 2 or 6 h of reperfusion in all intervention groups. Remote ischemic preconditioning leads to changes in the expression levels of the most abundant cardiac miRNA, miR-1. MicroRNA 1 levels did not correlate with protein levels of BDNF, a known miR-1 target, in vivo. Further studies are needed to explore the biological significance of changes in miR-1 expression levels and the potential interaction with BDNF in RIPC-induced cardioprotection.

Long-term application of glycine transporter inhibitors acts antineuropathic and modulates spinal N-methyl-D-aspartate receptor subunit NR-1 expression in rats.

BACKGROUND: Dysfunction of spinal glycinergic neurotransmission is a major pathogenetic factor in neuropathic pain. The synaptic glycine concentration is controlled by the two glycine transporters (GlyT) 1 and 2. GlyT inhibitors act antinociceptive in various animal pain models when applied as bolus. Yet, in some studies, severe neuromotor side effects were reported. The aim of the current study was to elucidate whether continuous inhibition of GlyT ameliorates neuropathic pain without side effects and whether protein expression of GlyT1, GlyT2, or N-methyl-D-aspartate receptor subunit NR-1 in the spinal cord is affected. METHODS: In the chronic constriction injury model of neuropathic pain, male Wistar rats received specific GlyT1 and GlyT2 inhibitors (ALX5407 and ALX1393; Sigma-Aldrich, St. Louis, MO) or vehicle for 14 days via subcutaneous osmotic infusion pumps (n = 6). Mechanical allodynia and thermal hyperalgesia were assessed before, after chronic constriction injury, and every 2 days during substance application. At the end of behavioral assessment, the expression of GlyT1, GlyT2, and NR-1 in the spinal cord was determined by Western blot analysis. RESULTS: Both ALX5407 and ALX1393 ameliorated thermal hyperalgesia and mechanical allodynia in a time- and dose-dependent manner. Respiratory or neuromotor side effects were not observed. NR-1 expression in the ipsilateral spinal cord was significantly reduced by ALX5407, but not by ALX1393. The expression of GlyT1 and GlyT2 remained unchanged. CONCLUSIONS: Continuous systemic inhibition of GlyT significantly ameliorates neuropathic pain in rats. Thus, GlyT represent promising targets in pain research. Modulation of N-methyl-D-aspartate receptor expression might represent a novel mechanism for the antinociceptive action of GyT1 inhibitors.

Evaluation of a new side-stream, low dead space, end-tidal carbon dioxide monitoring system in rats.

The aim of this study was to evaluate a newly developed infrared side-stream capnograph with minimal sample volume for the continuous measurement of end-tidal carbon dioxide (CO2) concentrations in small rodents. Thirty-four male Wistar rats (weight 345 ± 70 g) were treated in accordance with the National Institutes of Health (NIH) guidelines for animal care. All experiments were performed with approval of the local animal care and use committee. Sepsis was induced by implanting an 18 gauge stent into the colon 24 h prior to the experiments, allowing a constant fecal leakage into the peritoneal cavity (25 septic and nine control animals). Hemodynamic variables and end-tidal CO2 were recorded continuously and arterial blood (5 × 120 µL) was sampled periodically for arterial blood gas analysis. After baseline controlled mechanical ventilation was randomized and titrated to either normocapnia (35-45 mmHg) or hypercapnia (65-75 mmHg) with exogenous application of CO2. A total of 155 paired CO2 measurements comparing end-tidal and arterial partial pressure were conducted. Side-stream capnography underestimated the CO2 partial pressure with a bias of -6.1 mmHg and a 95% limit of agreement from 6.7 to -19.1 mmHg. Our results suggest that side-stream end-tidal CO2 monitoring with a low dead space could be utilized in rats as a surrogate for the arterial CO2 measurement over a wide range of partial pressures in normal and septic animals.

Acute, short-term hypercapnia improves microvascular oxygenation of the colon in an animal model of sepsis.

INTRODUCTION: The deterioration of microcirculatory oxygenation of the gut plays a vital role in the development of sepsis. Acute hypercapnia enhances the microcirculatory oxygenation of the splanchnic region under physiological conditions, while the effect of hypercapnia under sepsis is unknown. The aim of this study was to investigate the effects of acute hypercapnia and hypercapnic acidosis on the colonic microcirculation and early cytokine response in polymicrobial sepsis. METHODS: Experiments were performed on 103 male Wistar rats. Colon ascendens stent peritonitis (CASP) surgery with varying stent diameters was conducted to establish a moderate polymicrobial sepsis model. In a second series, 24h of sepsis development induced by CASP surgery was followed by 120min of volume-controlled and pressure-limited ventilation with either normocapnic (pCO2 45±5mmHg) or moderate hypercapnic ventilation targets (pCO2 75±5mmHg) via exogenous carbon dioxide application. The effect of acidosis was investigated by metabolically buffering the hypercapnic acidosis with tromethamine. Microcirculatory oxygenation of the colon wall (tissue reflectance spectrophotometry) and hemodynamic variables were recorded continuously and arterial blood gas and cytokine (TNF-α, IL-6, IL-10) levels were analyzed intermittently. RESULTS: In septic animals the microcirculatory oxygenation of the colon deteriorated under normocapnia (-7.0±7.6% at 90min) but was maintained under hypercapnic acidosis (+3.6±7.6%) and buffered hypercapnia (+1.5±4.4%). Cytokine levels were significantly higher in septic animals as opposed to sham animals but did not differ between normocapnic and hypercapnic groups. CONCLUSIONS: Acute hypercapnic acidosis and buffered hypercapnia both improve splanchnic microcirculatory oxygenation in a septic animal model, thereby counteracting the adverse effect induced by sepsis. The circulating pro- and anti-inflammatory cytokine levels are not modulated after 120min of hypercapnia.