Cardiovascular effects of levosimendan during rewarming from hypothermia in rat

BackgroundPrevious research aimed at ameliorating hypothermia-induced cardiac dysfunction has shown that inotropic drugs, that stimulate the cAMP, – PKA pathway via the sarcolemmal β-receptor, have a decreased inotropic effect during hypothermia. We therefore wanted to test whether levosimendan, a calcium sensitizer and dose-dependent phosphodiesterase 3 (PDE3) inhibitor, is able to elevate stroke volume during rewarming from experimental hypothermia.MethodsA rat model designed for circulatory studies during experimental hypothermia (4 h at 15 °C) and rewarming was used. The following three groups were included: (1) A normothermic group receiving levosimendan, (2) a hypothermic group receiving levosimendan the last hour of stable hypothermia and during rewarming, and (3) a hypothermic placebo control group. Hemodynamic variables were monitored using a Millar conductance catheter in the left ventricle (LV), and a pressure transducer connected to the left femoral artery. In order to investigate the level of PKA stimulation by PDE3 inhibition, myocardial Ser23/24-cTnI phosphorylation was measured using Western-blot.ResultsAfter rewarming, stroke volume (SV), cardiac output (CO) and preload recruitable stroke work (PRSW) were restored to within pre-hypothermic values in the levosimendan-treated animals. Compared to the placebo group after rewarming, SV, CO, PRSW, as well as levels of Ser23/24-cTnI phosphorylation, were significantly higher in the levosimendan-treated animals.ConclusionThe present data shows that levosimendan ameliorates hypothermia-induced systolic dysfunction by elevating SV during rewarming from 15 °C. Inotropic treatment during rewarming from hypothermia in the present rat model is therefore better achieved through calcium sensitizing and PDE3 inhibition, than β-receptor stimulation.     

Plasma catecholamines in rats during rewarming from induced hypothermia

The present study sought to quantitate the levels of plasma catecholamines [norepinephrine (NE), epinephrine (E), and dopamine (DA)] during induction and rewarming from hypothermia. Male rats (317 +/- 8 g) were made hypothermic by exposure to 0.9% halothane at -10 to -15 degrees C while blood pressure (carotid artery), heart rate, and colonic temperature (Tc) were monitored. Anesthesia was discontinued when Tc reached 28 degrees C. Tc continued to fall but was held at 20-20.5 degrees C for 30 min. Rewarming was then initiated by raising ambient temperature to 22 degrees C. Arterial blood samples were taken 1) before cooling, 2) just before rewarming, 3) when Tc reached 22 degrees C during rewarming, and 4) when Tc reached 27 degrees C during rewarming. Plasma was assayed radioenzymatically for catecholamines using both phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase procedures, and hypothermic induction resulted in significant increases in NE, E, and DA above control levels (P less than 0.01). With rewarming to Tc = 22 degrees C, all catecholamines increased above the level observed during hypothermia (P less than 0.01), and NE and DA increased still further (P less than 0.01) when Tc reached 27 degrees C. The levels of plasma catecholamines observed during hypothermia and during the rewarming phase indicate a role of the sympathoadrenal medullary system in the metabolic adjustments associated with hypothermia and recovery. During rewarming, the levels of E and NE attained exceed those at which both substances may be expected to act as circulating hormones.     

Peripheral blood flow during rewarming from mild hypothermia in humans

During the initial stages of rewarming from hypothermia, there is a continued cooling of the core, or after-drop in temperature, that has been attributed to the return of cold blood due to peripheral vasodilatation, thus causing a further decrease of deep body temperature. To examine this possibility more carefully, subjects were immersed in cold water (17 degrees C), and then rewarmed from a mildly hypothermic state in a warm bath (40 degrees C). Measurements of hand blood flow were made by calorimetry and of forearm, calf, and foot blood flows by straingauge venous occlusion plethysmography at rest (Ta = 22 degrees C) and during rewarming. There was a small increase in skin blood flow during the falling phase of core temperature upon rewarming in the warm bath, but none in foot blood flow upon rewarming at room air, suggesting that skin blood flow seems to contribute to the after-drop, but only minimally. Limb blood flow changes during this phase suggest that a small muscle blood flow could also have contributed to the after-drop. It was concluded that the after-drop of core temperature during rewarming from mild hypothermia does not result from a large vasodilatation in the superficial parts of the periphery, as postulated. The possible contribution of mechanisms of heat conduction, heat convection, and cessation of shivering thermogenesis were discussed.     

Cardiac arrhythmias during rewarming of patients with accidental hypothermia.

Accidental hypothermia has a high mortality and is associated with cardiac arrhythmias. To determine the incidence of arrhythmias and their importance 22 patients with accidental hypothermia (core temperature less than 35 degrees C) were studied by 12 lead electrocardiography and continuous recording of cardiac rhythm. Although 14 of the patients died (64%), only six died while hypothermic. Prolongation of the Q-T interval and the presence of J waves were related to the severity of the hypothermia. Supraventricular arrhythmias, including atrial fibrillation, were common (nine cases) and benign. Ventricular extrasystoles were also common (10 cases), but ventricular tachycardia or fibrillation did not occur during rewarming. In eight patients who died while being monitored the terminal rhythm was asystole. There was no correlation between the severity of hypothermia or the rate of rewarming and the clinical outcome. In the absence of malignant arrhythmias there is no indication for using prophylactic antiarrhythmic treatment in patients with accidental hypothermia. The presence or absence of severe underlying disease is the main determinant of prognosis.     

Effects of milrinone on left ventricular cardiac function during cooling in an intact animal model

A significant reduction in immunogenicity has been observed in some frozen-thawed tissues after cryopreservation. The objective of this study was to evaluate the effects of programmed cryopreservation on immunogenicity of rabbit bladder mucosa and on the extent of immunological rejection caused by the allograft. This study would provide theoretical support for the application of allogenic frozen-thawed bladder mucosa in the treatment of urethral stricture. Forty-two adult male New Zealand rabbits were used in this study. The immunogenicity was detected by mixed lymphocyte reaction using the allograft of bladder mucosa (fresh and frozen-thawed) and spleen lymphocytes. Twelve urethral stricture models were established in New Zealand rabbits for substitution urethroplasty using the allograft of bladder mucosa, which were divided into fresh and frozen-thawed group. Two weeks after operation, lymphocyte proliferation was detected in both blood and spleen of recipient rabbits. At the same time, immunohistochemical staining of urethral allograft was performed and the expression of CD3, CD4 and CD8 were observed. The mRNA of bladder mucosa (fresh and frozen-thawed) was extracted and the expressions of RLA-I, RLA-II and RLA-III gene were detected by real-time PCR. By mixed lymphocyte reaction, we found that allogenic lymphocyte proliferation stimulated by frozen-thawed bladder epithelial cells was significantly weaker than that of the fresh cells. The blood and spleen lymphocytes from fresh bladder mucosa group showed significantly higher proliferation rate than frozen-thawed group. Compared with the fresh group, the expression of CD3+ and CD8+ T cells infiltrated in the operation locus of bladder mucosa urethroplasty was significantly decreased in the frozen-thawed group. However, the expressions of RLA genes did not change significantly after the freeze-thaw procedure. This study demonstrates for the first time that a programmed freeze-thaw procedure of rabbit bladder mucosa could reduce its immunogenicity in allogenic bladder mucosa urethroplasty and thus restrict the extent of immunological rejection, therefore, provides theoretical support for the application of frozen-thawed bladder mucosa in the treatment of urethral stricture.     

Respiration during hypothermia: effect of rewarming intermediate areas of ventral medulla

We studied respiration (phrenic nerve activity) during progressive hypothermia to as low as 30.5 degrees C in five anesthetized, paralyzed, glomectomized, and vagotomized cats. PCO2 was maintained at a constant level throughout the experiments. We confirmed the results of a previous study (J. P. Kiley, F. L. Eldridge, and D. E. Millhorn, J. Appl. Physiol. 58: 295-312, 1985) in which respiratory minute output decreased progressively with cooling and respiratory frequency decreased markedly. In addition we show that focal rewarming to normal temperature (37.5 degrees C) of the structures in the intermediate areas on the ventral surface of the medulla resulted in a significant reversal of the depressed respiratory minute activity observed with hypothermia. Respiratory frequency, however, was unaffected by intermediate area rewarming. We conclude that the decreased respiratory activity during hypothermia is due to a generalized interference with neural function. A major portion of these effects is due to cooling of the intermediate areas, but the slowing of respiratory frequency appears to be an independent effect.     

Myocardial gene expression profiling of rewarming shock in a rodent model of accidental hypothermia

Background

Severe accidental hypothermia represents a cardiovascular emergency associated with high mortality and poor recovery of cardiac function. The biochemical changes occurring within the heart during the development of hypothermia and subsequent resuscitation are not known.

Methods

By mRNA expression profiling, we have characterized gene expression changes occurring within the myocardium in an intact rat model of accidental hypothermia during cooling to a core temperature of 15 °C and subsequent rewarming to 37 °C. During the rewarming phase, these animals develop a profound low-output cardiac failure.

Results

Hypothermia induces expression of known mediators of thermotolerance, including heat-shock protein 70 and several factors involved in protection against apoptotic cell death. Upregulation of genes involved in autophagy and increased abundance of autophagosomal vesicles suggest involvement of autophagic degeneration in the development of myocardial dysfunction occurring during rewarming from hypothermia. Rewarming from hypothermia also induces expression of several pro-inflammatory genes involved in the nuclear factor kappa B (NFκB) signaling cascade.

Conclusions

Our data demonstrate that rewarming from hypothermia is associated with the induction of a cellular stress–response, including upregulation of autophagy and activation of pro-inflammatory signaling cascades. These data provide a framework for understanding the molecular changes that occur during induction of and rewarming from severe hypothermia, and identifies potential targets for cardioprotective interventions in resuscitation of victims of hypothermia.     

Genetic effects on an animal model of anxiety

Genetic effects on behavioural measures thought to model anxiety have been reported on 15 mouse chromosomes. In general the individual effect from each locus is small, contributing to 10% or less of the total variation, but through use of crosses between inbred rodents the power to detect such effects is high: 39 loci have been reported at stringent levels of significance. Novel multivariate analyses of these data go some way to characterizing the genetic architecture of anxiety and also to validating the tests that are used for its measurement. However, we are still some way from finding the molecular variants that explain the heritability of the trait.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.     

Efficacy of forced-air and inhalation rewarming by using a human model for severe hypothermia

Goheen, M. S. L., M. B. Ducharme, G. P. Kenny, C. E. Johnston, John Frim, Gerald K. Bristow, and Gordon G. Giesbrecht. Efficacy of forced-air and inhalation rewarming by using a humanmodel for severe hypothermia. J. Appl.Physiol. 83(5): 1635-1640, 1997.We recentlydeveloped a nonshivering human model for severe hypothermia by usingmeperidine to inhibit shivering in mildly hypothermic subjects. Thisthermal model was used to evaluate warming techniques. On threeoccasions, eight subjects were immersed for ~25 min in 9°C water.Meperidine (1.5 mg/kg) was injected before the subjects exited thewater. Subjects were then removed, insulated, and rewarmed in anambient temperature of 20°C with either1) spontaneous rewarming (control),2) inhalation rewarming withsaturated air at ~43°C, or 3)forced-air warming. Additional meperidine (to a maximumcumulative dose of 2.5 mg/kg) was given to maintain shiveringinhibition. The core temperature afterdrop was 30-40% less duringforced-air warming (0.9°C) than during control (1.4°C) andinhalation rewarming (1.2°C) (P < 0.05). Rewarming rate was 6- to 10-fold greater during forced-airwarming (2.40°C/h) than during control (0.41°C/h) andinhalation rewarming (0.23°C/h) (P < 0.05). In nonshivering hypothermic subjects, forced-air warming provided a rewarming advantage, but inhalation rewarming did not.

     

Heart rate variability and electrocardiogram waveform as predictors of morbidity during hypothermia and rewarming in rats

This study examined electrocardiogram (ECG) waveform, heart rate (HR), mean blood pressure (BP), and HR variability as potential autonomic signatures of hypothermia and rewarming. Adult male Sprague-Dawley rats had telemetry transmitters surgically implanted, and 2 weeks were allowed for recovery prior to induction of hypothermia. Rats were lightly anesthetized (sodium pentobarbital, 35 mg/kg i.p.) and placed in a coil of copper tubing through which temperature-controlled water was circulated. Animals were cooled to a core temperature (Tc) of 20 degrees C, maintained there for 30 min, and then rewarmed. Data (Tc, BP, HR from ECG, and 10-s strips of ECG waveforms) were collected every 5 min throughout hypothermia and rewarming. Both HR and BP declined after initial increases with the drop in HR starting at a higher Tc than the drop in BP (29.6 +/- 2.4 degrees C vs. 27.1 +/- 3.3 degrees C, p < 0.05). Animals that were not successfully rewarmed exhibited a significant (p < 0.05) increase in the normalized standard deviation of interbeat intervals (IBI) throughout cooling compared with animals that were successfully rewarmed. The T wave of the ECG increased in amplitude and area with decreasing Tc. T-wave amplitude and IBI variability show potential as predictors of survival in hypothermic victims.     

Cardiovascular effects of atrial natriuretic extract in the whole animal

Atrial tissue extract (AE) and ventricular tissue extract cause identical decreases in total peripheral resistance when they are injected i.v. into anesthetized rats. However, only AE causes significant hypotension because of cardiac inhibition. This involves both bradycardia and failure of stroke volume to increase appropriately. The observations cannot be explained by direct action of AE on myocytes, but are more likely to be the result of interactions with cardiovascular reflex mechanisms. Excitation of chemosensitive cardiac receptors with vagal afferents appears to be an important afferent mechanism. The efferent limb for the negative chronotropic response resides partly in the vagus nerves and partly in cardiac sympathetic nerves. The negative inotropic response of AE was not altered by vagotomy, spinal section, atropine, or propranolol. These results suggest that atrial peptides may cause the release of a negatively inotropic substance from a site that is not yet identified.     

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.     

The prevention of inadvertent hypothermia during fetal surgery: an ovine model

The effect of application of a reflective thermoblanket was compared with varying degrees of partial removal of the ovine fetus from the uterus. The thermoblanket reduced heat loss by a 53.3% slower fall in rectal temperature when compared to exposed fetuses during the same surgery (Welch t statistic = 3.68, d.f. = 26.08, P less than 0.001). This was further reduced by limiting the area of fetal exposure without thermoblanket application.