Endothelial dysfunction after repeated Chlamydia pneumoniae infection in apolipoprotein E-knockout mice.

BACKGROUND: Arterial relaxation is largely regulated by endothelial nitric oxide (NO). Its diminished activity has been associated with incipient atherosclerosis. We investigated the endothelium-dependent relaxation of aorta in apolipoprotein E-knockout (apoE-KO) mice exposed to single or repeated Chlamydia pneumoniae inoculation. METHODS AND RESULTS: Forty-eight apoE-KO mice, 8 weeks old, were inoculated intranasally with C pneumoniae (n=24) or saline (n=24) every 2 weeks over a 6-week period. Twenty mice (10 infected and 10 controls) were killed at 2 weeks and 6 weeks, respectively, after the first inoculation. The smooth muscle tone of aortic rings was measured in vitro at both time points. The norepinephrine-precontracted thoracic aortic rings were successively exposed to methacholine in the absence and presence of N:(G)-nitro-L-arginine methyl ester (L-NAME) and diclofenac. The methacholine-induced relaxation was attenuated in the infected mice at 6 weeks in both the absence and presence of L-NAME (P:<0.05 and P:<0.01, respectively). When administered together with L-NAME, diclofenac enhanced the relaxation of the L-NAME-pretreated aortas in infected mice at 2 weeks (P:<0.05) but not in noninfected mice. The relaxation response from infected mice tended to differ in the same manner at 6 weeks (P:<0.1). No intimal thickening was detected at either time point. CONCLUSIONS: C pneumoniae impairs arterial endothelial function, and the NO pathway is principally involved. Cyclooxygenase-dependent vasoconstricting products may also account for the infection-induced impaired relaxation. These findings further support the role of C pneumoniae infection in atherosclerosis development.

The effect of high post-mortem temperature on the development of pale, soft and exudative pork: Interaction with ultimate pH.

The effects of post-mortem temperature and ultimate pH (pH(u), 24 h post mortem) on the development of the pale, soft and exudative (PSE) characteristics in pig longissimus dorsi muscle were studied. Ten out of the 13 pigs used received pre-slaughter injections of adrenaline in order to deplete muscle glycogen stores. The two muscles from each pig were held at 12 or 35°C during rigor mortis development. Results from covariance analysis, using pH(u) as covariate, showed that a high temperature (35°C) resulted in a dramatic increase in internal light scattering (FOP) 24 h post mortem and a significant decrease in water- and salt-soluble proteins. Cooking loss, sarcomere length and drip loss did not vary significantly with rigor temperature. Interaction between temperature and pH(u) was estimated by assessing the relationship between pH(u) and the difference between the two rigor temperatures for selected traits. The best predictive model was a segmented quadratic model with a plateau which gave significant results for FOP, drip loss, water- and salt-soluble proteins. The effect of temperature decreased curvilinearly when pH increased until a constant value above which no noticeable difference was recorded (drip loss, water- and salt-soluble proteins) or a constant difference was reached (FOP). The pH values corresponding to the convergence points differed from one trait to another. They ranged from 5·72 to 6·22. These results illustrate the importance of muscle glycogen content at slaughter and subsequent pH(u) with regard to the development of temperature-induced PSE meat.

Effect of time between adrenaline injection and slaughter on the rate and extent of post-mortem metabolism in porcine skeletal muscle.

The aim of this work was to study the effect of time between adrenaline injection and slaughter on the rate and extent of post-mortem metabolism in pig muscle. Five pigs were subcutaneously injected with adrenaline (0·3 mg/kg) or with a saline solution 4 h, 1 h or 15 min prior to slaughter. pH(1), pH(u) and FOP(u) were measured in Longissimus dorsi (LD) and Biceps femoris (BF) muscles. m.LD samples were taken 20 days before slaughter using biopsy sampling, immediately after bleeding and 45 min post mortem for biochemical analysis. m.LD glycolytic potential (very close to glycogen content) was decreased by the injection of adrenaline 4 h and 1 h prior to slaughter, when comparing values at rest (20 days before slaughter) and values determined immediately after bleeding. The depletion was greater fro the injection performed 4 h before slaughter. In this group pH(u) was higher foe adrenaline-injected pigs (5·69 versus 5·47 for pigs injected with saline solution) but the difference was not significant. Pigs injected with adrenaline 1 h prior to slaughter exhibited higher glucose, glucose-6-phosphate and lactate levels immediately after slaughter, lower pH(1) and higher FOP values in m.LD than control pigs. Since the differences in pH(1) were not explained by differences in the rate of build-up of lactate, it was hypothesized that pigs injected with adrenaline 1 h prior to slaughter had lower muscle pH at slaughter. It was concluded that the rate or the extent of post mortem metabolism in pig muscle may be affected independently, by manipulating the time between adrenaline injection and slaughter.

CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine.

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.

Middle-latency auditory evoked potentials during induction of thiopentone anaesthesia in pigs.

A method is described for measuring middle-latency auditory evoked potentials (MLAEP) in consciously awake, non-sedated pigs during the induction of thiopentone anaesthesia (0.6 ml/kg, 2.5% thiopentone solution). It was done by using autoregressive modelling with an exogenous input (ARX). The ability to perceive pain during the induction was compared with (1) the changes in latencies and amplitudes of the MLAEP, (2) the change in a depth of anaesthesia index based on the ARX-model and (3) the change in the 95% spectral edge frequency. The pre-induction MLAEP was easily recordable and looked much like the one in man, dogs and rats. The temporal resolution in the ARX method was sufficiently high to describe the fast changes occurring during induction of thiopentone anaesthesia. As previously reported from studies in man, dogs and rats, induction of thiopentone anaesthesia resulted in significantly increased latencies and decreased amplitudes of the MLAEP trace as well as in a significantly reduced depth of anaesthesia index and spectral edge frequency. None of the changes, however, related well to the ability to react to a painful stimulus. Whether an ARX-based depth of anaesthesia index designed especially for pigs might be better than the present index (designed for man) for assessing depth of anaesthesia must await the results of further studies.

Effects of CO2 anaesthesia on central nervous system activity in swine.

The objective of the study was to examine the changes in central nervous system (CNS) activity and physical behaviour during induction and awakening from CO2 anaesthesia. Two studies, each using pigs immersed into 90% CO2 gas for a period of 60 s were performed. In study 1, we monitored middle latency auditory evoked potentials (changes in latencies, amplitudes and a depth of anaesthesia index), electroencephalographic parameters (delta, theta, alpha and beta electroencephalographic power and 95% spectral edge frequency) and heart rate; and in study 2, we monitored body movements and arterial and venous partial pressure of CO2 and O2. No behavioural signs of distress were observed during the early part of the induction. The swine exhibited muscular activity from 13-30 s after induction-start as well as during awakening from anaesthesia, possibly because of a transitory weaker suppression of the brain stem than of the cortex. The CNS and blood gas parameters started to change from the very start of induction. The CNS suppression lasted only approximately one minute after the end of the induction period. The two studies indicated a good temporal relationship between changes in amplitude, depth of anaesthesia index, spectral edge frequency, and arterial PCO2 during the induction period.

Transient neocortical, hippocampal and amygdaloid EEG silence induced by one minute inhalation of high concentration CO2 in swine.

The study is part of a series of investigations performed with the ultimate goal of obtaining an objective evaluation of the ethical aspects and the narcotic efficiency of CO2 inhalation used as pre-slaughter anaesthesia for swine. Six Yorkshire swine were exposed twice to 80% CO2 for 1 min during simultaneous recording of the EEGs from the frontal neocortex, the dorsal hippocampus, and the amygdaloid region via permanently implanted electrodes. In five of the animals myoclonic jerks started at 28 +/- 1 s of CO2 exposure and lasted for 6 +/- 2 s. Neocortical slow wave (delta) activity and increased amplitude of the hippocampal theta (5-7 Hz) waves (i.e. EEG changes seen during the second stage of barbiturate anaesthesia) had developed before the brief period of myoclonic jerks. After this period the EEG activity gradually declined, resulting in neocortical EEG silence at the end of the exposure. This apparent isoelectricity lasted for on average 1 min. The return of the neocortical EEG activity exhibited a pattern reverse to its disappearance, but was much prolonged in comparison to the EEG extinction. Pre-exposure neocortical EEG pattern was not regained until 3-5 min post-exposure. In eight out of 11 experiments the CO2 inhalation also induced hippocampal EEG silence lasting for on average 30 s. EEG flattening was further obtained when recording from the amygdaloid nuclear complex and the adjacent pyriform cortex. The observed changes in the neocortical and hippocampal EEGs suggest that the present swine were unconscious already when they exhibited motor reactions. This does not exclude the possibility that CO2-independent stress/arousal factors present in a slaughterhouse environment may facilitate the development of motor phenomena similar to seizures, with the result that such reactions become manifest before the neocortical EEG exhibits an anaesthesia pattern. The duration of the observed EEG silence implies that, from the ethical point of view, exsanguination might safely be performed within 1 min after the moment when the animal is removed from the high concentration CO2. However, the slow return to a pre-exposure neocortical EEG pattern suggests that the swine remains unconscious for at least another minute.

Inefficiency of bilateral amygdaloid lesions to reduce the transient motor reactions exhibited by swine during exposure to CO2.

The aim of the study was to evaluate from the ethical point of view the importance of transient muscular jerks commonly exhibited by swine at an early stage of pre-slaughter CO2-anaesthesia. The influence of 5 min restraint upon plasma concentrations of adrenaline (A) and noradrenaline (NA) was studied in swine (n = 6) before and after bilateral lesioning of the amygdaloid region of the brain, as were the motor reactions of the same and three other animals during 1 min exposure to 80% CO2. The A and NA responses to the restraint became almost extinguished after amygdaloid lesioning in three of the animals, were reduced by about 50% in one swine, whereas no reduction was seen in the other two animals investigated. The amygdaloid lesioning did not visibly influence the latency for, and the duration and intensity of the muscular jerks manifested by the swine during the CO2-exposure. The results do not favour the possibility that transient motor reactions exhibited by swine during pre-slaughter CO2-exposure are manifestations of emotional stress. Instead, the study indirectly supports the idea that the cause of the muscular jerks may be disinhibition of subcortical motor centres being inactivated by the CO2-anaesthesia somewhat later than neocortical cells normally exerting the inhibition.

Acidosis, hypoxia and stress hormone release in response to one-minute inhalation of 80% CO2 in swine.

The study pertains to a series of investigations on the effects of CO2 inhalation as used for pre-slaughter anaesthesia in swine. Acid/base parameters, blood oxygen tension, plasma Na, K, Ca and stress hormone concentrations were monitored in Yorkshire swine before, during, and for 10 min after the animals were descended for 1 min into 80% CO2 in air. Severe respiratory acidosis (PaCO2 approximately 50 kPa, arterial pH approximately 6.6) and hypoxia (PaO2 approximately 4kPa) had developed after 45 s of the CO2 inhalation. The corresponding changes in venous blood were less drastic (PvCO2 approximately 17 kPa, pH 7.1, PvO2 approximately 4 kPa). Readjustment to PaCO2 approximately II kPa, arterial pH 7.2, and PaO2 approximately 13 kPa had occurred at 1 min post CO2. Four minutes later the respiratory acidosis had become converted into metabolic acidosis subjected to partial respiratory compensation (arterial pH 7.3 in the presence of moderate hypocapnia and hyperoxaemia). The cause of this metabolic acidosis (present also at 10 min post CO2) was apparently hypoxia-induced anaerobic metabolism (= lactic acid production). Apparently due to hydrogen ion transport into the cells in exchange for other cations, hyperkalaemia (K approximately 6.6 mmol l-1), and a 7 mmol l-1 increase in plasma Na had developed at 1.5 min later. The CO2 inhalation did not change the total plasma Ca significantly. The transport of the swine from the stable to the immediate pre-experimental situation induced a 3-fold increase in plasma cortisol concentration (PC, to approximately 130 mmol l-1). No further increase in PC occurred in response to the CO2 inhalation. It indicates that no additional emotional strain was imposed upon the animals during the CO2 exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone secretion during acute respiratory acidosis and NH4Cl-induced metabolic acidosis in the goat.

Acute respiratory acidosis was induced in goats by inhalation of 6% or 8% CO2 in air for 30 min. The lower CO2 concentration caused a significant rise in plasma cortisol (PC), but had no appreciable influence upon plasma aldosterone (PA), and did not affect the arterial blood pressure (aBP). A more pronounced PC response was observed in association with the inhalation of 8% CO2, but also here without concomitant increase in PA. However, the aBP became elevated by about 30% during the CO2 exposure with a simultaneous increase in glomerular filtration rate and a water diuresis, suggesting that the release of arginine vasopressin temporarily became inhibited. It was confirmed that metabolic acidosis induced by duodenal NH4Cl administration is preceded by a transient rise in PA. Dexamethasone-induced feedback inhibition of the ACTH secretion blocked the PA response, which possibly reflects NH4 ion stimulation of the ACTH release. The combined results of the CO2 and NH4Cl experiments seem to justify the conclusion that increases in PA seen in conjunction with acidosis do not reflect a direct hydrogen ion stimulation of the adrenal glomerulosa cells.

Observations on normal EEG activity in different brain regions of the unrestrained swine.

This methodological and electrophysiological study was undertaken with the aim of obtaining possibilities for evaluating objectively the ethical value of using high concentration CO2 inhalation as pre-slaughter anaesthesia for swine. A technique for implantation of recording electrodes into the porcine neocortex and limbic system is described, as are results of EEG recording from these brain regions in the unrestrained animal. In addition to expected desynchronized activity in the awake animal, bilateral recordings from the frontal neocortex revealed the irregular occurrence of 'sleep spindle' during apparent non-REM sleep. The 'spindles' were sometimes unilateral, and when bilaterally simultaneous, the amplitude was often larger in one of the hemispheres. The EEG recorded from the dorsal hippocampus was characterized by theta-wave activity. The offering of food to the hungry animal, and subsequent brief feeding periods, were associated with marked accentuation of this theta activity. Recording via electrodes placed within the amygdaloid nuclear complex showed high-frequency activity irregularly interrupted by slow waves. However, if one of the 'amygdaloid' twin electrodes hit the ventral pole of the hippocampus, theta activity was dominant. Electrodes placed adjacent to the amygdaloid nuclei in the pyriform cortex exhibited a basic awake activity similar to that recorded from these nuclei. During apparent non-REM sleep, however, trains of slow waves were seen simultaneously with the appearance of 'sleep spindles' in the ipsilateral neocortex.

Carbon dioxide narcosis: influence of short-term high concentration carbon dioxide inhalation on EEG and cortical evoked responses in the rat.

The effects of 1 min exposure to 80% CO2 inhalation was studied in rats under light general anaesthesia with N2O. It was found that 80% CO2 gave a rapid slowing of the EEG as well as a rapid decrease of the amplitude of cortical somatosensory evoked potentials. Only minor influences upon the blood pressure and heart rate were seen. The changes were found to be transient and most of the EEG effects had disappeared about 2-3 min following the end of the CO2 exposure. The findings are related to current procedures for preslaughter anaesthesia in swine using 80% CO2. The present experiments show that 80% CO2 rapidly induces an interruption of afferent sensory transmission to the cortex as well as slowing of the EEG. Both findings indicate that 80% CO2 is an effective agent for inducing adequate anaesthesia for slaughter procedures. However, the transient nature of the CO2 effects upon the central nervous system revealed here has to be taken into account to optimize the slaughter house procedures.