Nociception and conditioned fear in rats: strains matter.

When using rats in pain research, strain-related differences in outcomes of tests for pain and nociception are acknowledged. However, very little is known about the specific characteristics of these strain differences. In this study four phylogenetically distant inbred rat strains, i.e. Wistar Kyoto (WKY), Fawn Hooded (FH), Brown Norway (BN) and Lewis (LE), were investigated in different tests related to pain and nociception. During Pavlovian fear conditioning, the LE and WKY showed a significantly longer duration of freezing behaviour than the FH and BN. Additionally, differences in c-Fos expression in subregions of the prefrontal cortex and amygdala between rat strains during retrieval and expression of conditioned fear were found. For example, the BN did not show recruitment of the basolateral amygdala, whereas the WKY, FH and LE did. During the hot plate test, the WKY and LE showed a lower thermal threshold compared to the BN and FH. In a follow-up experiment, the two most contrasting strains regarding behaviour during the hot plate test and Pavlovian fear conditioning (i.e. FH and WKY) were selected and the hot plate test, Von Frey test and somatosensory-evoked potential (SEP) were investigated. During the Von Frey test, the WKY showed a lower mechanical threshold compared to the FH. When measuring the SEP, the FH appeared to be less reactive to increasing stimulus intensities when considering both peak amplitudes and latencies. Altogether, the combined results indicate various differences between rat strains in Pavlovian fear conditioning, nociception related behaviours and nociceptive processing. These findings demonstrate the necessity of using multiple rat strains when using tests including noxious stimuli and suggest that the choice of rat strains should be considered. When selecting a strain for a particular study it should be considered how this strain behaves during the tests used in that study.

Predictability of painful stimulation modulates the somatosensory-evoked potential in the rat.

Somatosensory-evoked potentials (SEPs) are used in humans and animals to increase knowledge about nociception and pain. Since the SEP in humans increases when noxious stimuli are administered unpredictably, predictability potentially influences the SEP in animals as well. To assess the effect of predictability on the SEP in animals, classical fear conditioning was applied to compare SEPs between rats receiving SEP-evoking electrical stimuli either predictably or unpredictably. As in humans, the rat's SEP increased when SEP-evoking stimuli were administered unpredictably. These data support the hypothesis that the predictability of noxious stimuli plays a distinctive role in the processing of these stimuli in animals. The influence of predictability should be considered when studying nociception and pain in animals. Additionally, this finding suggests that animals confronted with (un)predictable noxious stimuli can be used to investigate the mechanisms underlying the influence of predictability on central processing of noxious stimuli.

Antinociceptive effects of low dose lumbosacral epidural ropivacaine in healthy ponies.

The objective of this study was to evaluate the safety and efficacy of low dose lumbosacral epidural ropivacaine in ponies. Antinociceptive effects of epidural ropivacaine were evaluated by means of mechanical nociceptive thresholds (MNTs) at several spinal levels in conscious ponies. The effects of ropivacaine on nociceptive afferent transmission to the spinal cord were also assessed by measuring spinal cord somatosensory evoked potentials (SSEPs) in anaesthetised ponies. Ataxia scores were determined in conscious ponies to assess the effects on motor function. A randomised, placebo controlled, double blind cross-over design was used. Low dose lumbosacral epidural ropivacaine led to increases in MNTs at various anatomical locations with a maximum effect at the lumbosacral and sacrococcygeal regions, both with respect to increase in threshold and duration of effect. Analysis of SSEPs showed that epidural ropivacaine influenced both Aß- and Aδ-mediated afferent transmission to the spinal cord at the level of the lumbosacral junction. Ponies showed mild ataxia after low dose lumbosacral epidural ropivacaine, but all ponies remained standing. Application of low dose lumbosacral epidural ropivacaine provided safe and efficacious antinociceptive effects in conscious and anaesthetised ponies, and could therefore be a valuable addition to multimodal analgesic protocols in Equidae.

Bispectral index and the clinically evaluated anaesthetic depth in dogs.

OBJECTIVE: This study investigated whether the bispectral index (BIS monitor) corresponded with the clinical assessment of anaesthetic depth in dogs. STUDY DESIGN: Prospective clinical study. ANIMALS: Sixty-five dogs undergoing anaesthesia for surgery. METHODS: Dogs were assigned to one of three different anaesthetic techniques. A three point scale was devised to determine the clinical depth of anaesthesia (CDA); CDA 1 represented light, CDA 2 surgical and CDA 3 excessive depth of anaesthesia. BIS values were recorded and CDA assessed at specific times and points throughout surgery. Data were statistically analysed using mixed model regression. RESULTS: Clinical depth of anaesthesia was assessed as CDA 1 on 68, 2 on 748 and 3 on four occasions. The BIS recorded for CDA 1 differed significantly from that for CDA 2 (p<0.001). However, individual BIS values measured at light and surgical levels of anaesthesia overlapped considerably. The sensitivities and specificities calculated for BIS to diagnose CDA 1 compared to CDA 2 in the three anaesthetic protocols were 28-86% and 55-85%. The accompanying positive predictive value was 0.08-0.29 and the negative predictive value was 0.95-0.97. End-tidal isoflurane concentrations (anaesthetic techniques 1 and 3) and propofol infusion (technique 2) at CDA 1 was significantly lower than those at CDA 2 (p=0.001). CONCLUSIONS: Although BIS values overall distinguished between CDA scores, the calculated specificities, sensitivities and predictive values were low, and there were anomalous results in individual cases. CLINICAL RELEVANCE: The use of the BIS as the sole method to determine anaesthetic depth in dogs is imprudent.

Neurophysiological assessment of the sedative and analgesic effects of a constant rate infusion of dexmedetomidine in the dog.

The sedative and analgesic effects of continuous rate infusion (CRI) of dexmedetomidine (DEX) were investigated in Beagle dogs (n=8) using auditory and somatosensory evoked potentials (AEPs and SEPs) recorded before, during and after a CRI of saline or DEX (1.0, 3.0, 5.0 µg/kg bolus, followed by 1.0, 3.0, 5.0 µg/kg/h CRI, respectively). The results showed a significant reduction in AEP at doses of 1.0 µg/kg/h and above and a significant reduction of the SEP at doses of 3.0 and 5.0 µg/kg/h. Neither the AEP nor the SEP was further reduced at 5.0 µg/kg/h when compared to 3.0 µg/kg/h, although a slower return towards baseline values was observed at 5.0 µg/kg/h. The mean plasma levels (±SEM) of DEX during infusion were 0.533±0.053 ng/mL for the 1.0 µg/kg/h dose, 1.869±0.063 ng/mL for the 3.0 µg/kg/h dose and 4.017±0.385 for the 5.0 µg/kg/dose. It was concluded that in adult dogs, a CRI of DEX had a sedative and analgesic effect that could be described quantitatively using neurophysiological parameters. Sedation was achieved at lower plasma levels than required for analgesia, and DEX had a longer (but not larger) effect with infusion rates above 3.0 µg/kg/h.

The alpha(2)-adrenoceptor agonist dexmedetomidine suppresses memory formation only at doses attenuating the perception of sensory input.

It was investigated whether continuous rate infusion of the alpha(2)-adrenoceptor agonist dexmedetomidine can suppress memory formation by mechanisms other than reducing perception of sensory input in a fear-conditioning paradigm. Different groups of rats infused with either saline or dexmedetomidine (2.0, 4.0 or 10.0microg/kg bolus, followed by 2.0, 4.0 or 10.0microg/kg/h continuous rate infusion respectively), were subjected to a somatosensory-evoked potential (SEP) fear-conditioning paradigm. This paradigm combined the pairing of an innoxious conditioned stimulus (CS) and a noxious unconditioned stimulus (US), of which the latter was used to generate the SEPs (training phase).The following day, the perception of the US during the training phase was assessed by presenting the CS only and subsequently scoring the resulting duration of freezing behaviour (testing phase). Freezing behaviour was reduced only in those groups which demonstrated reduced SEPs. Based on these findings, it is concluded that dexmedetomidine suppresses memory formation only at doses reducing central nervous system activity in response to sensory input.

Use of epidurally derived evoked potentials for quantification of caudal nociception in ponies.

OBJECTIVE: To determine whether epidurally derived evoked potentials (EPs) can be used to reliably assess nociception and antinociception in ponies. ANIMALS: 7 ponies. PROCEDURES: EPs and electromyograms (EMGs) from the quadriceps femoris muscles were recorded simultaneously, following electrical stimulation applied to the distal portion of the hind limb. The effect of increasing stimulus intensity, conduction velocities of the stimulated nerves, effect of epidurally applied methadone, and effect of systemically administered propofol were evaluated. RESULTS: In the EP and EMG waveforms, 2 distinct complexes, the EP N25 and P50 and the EMG P27 and N62, respectively, were identified. On the basis of their latency and calculated conduction velocities, the EP P50 and EMG N62 were considered to be related to nociception (AD-mediated). All complexes increased significantly in amplitude with increasing stimulus intensity and decreased significantly following epidural administration of methadone or systemic administration of propofol. CONCLUSIONS AND CLINICAL RELEVANCE: Although the experimental setup allowed successful discrimination between tactile- and nociceptive-associated responses, the identified EPs, considered to reflect activity in the spinal cord, could not be definitively differentiated from activity in the lumbosacral epaxial musculature. Further research is required to refine measurement techniques to allow for discrimination between these 2 signals. Similar to other species, neurophysiologic variables such as EPs could potentially become a useful additional tool in quantifying nociception in equidae.

Nociception-related somatosensory evoked potentials in awake dogs recorded after intra epidermal electrical stimulation.

At present, the specific neurophysiologic methodology of recording pain-related evoked potentials is considered a most promising approach to objectively quantify pain in man. This study was designed to characterise and evaluate the use of somatosensory evoked potentials to study nociception in a canine model. To this aim, somatosensory evoked potentials were evoked by intra-epidermal electrical stimulation and recorded from the scalp in 8 beagle dogs. Characteristics determined were: (1) the conduction velocities of the peripheral nerve fibres involved, (2) the stimulus intensity response characteristics and (3) the evaluation of possible disturbance of the signals by muscular activity from the hind paw withdrawal reflex (EMG artefact). The results showed (1) the conduction velocities to be in the A-delta fibre range (i.e. fibres involved in nociception), (2) an increase in amplitude and a decrease in latency of the evoked potential following increasing stimulus intensities and (3) the absence of EMG artefact in the signals. These data indicate that the evoked potentials recorded, are related to nociception and thus are suited to quantitatively characterise the perception of noxious stimuli making this model useful for pain- and analgesia-related research.

The cardiorespiratory effects of a fentanyl infusion following acepromazine and glycopyrrolate in dogs.

We investigated whether the analgesic mu-opioid fentanyl can be used safely in dogs in everyday clinical veterinary practice, with limited and non-invasive monitoring. To this end, the cardiorespiratory effects of fentanyl, administered in doses reported to be adequate for inducing opiate analgesia in spontaneously breathing canine patients, were evaluated by measuring the respiration rate, oxygen saturation (SpO2), heart rate, respiratory sinus arrhythmia (RSA), and rectal body temperature. Ten Beagle dogs, all spontaneously breathing room air, underwent three separate sessions in which they received in random order either saline, fentanyl 5 microg/kg/h or fentanyl 10 microg/kg/h. Each session started with a non-medication period, followed by acepromazine with glycopyrrolate, followed by a loading dose and infusion of saline or fentanyl, and ended with the administration of the antagonist naloxone. At the doses studied, fentanyl did not significantly change the respiration rate or have a clinically relevant effect on SpO2 or RSA, whereas it significantly decreased the heart rate and core body temperature. In the dose range tested and under the conditions described in this protocol, we conclude that fentanyl can be safely administered to healthy dogs spontaneously breathing room air.

Evaluation of analgesic and sedative effects of continuous infusion of dexmedetomidine by measuring somatosensory- and auditory-evoked potentials in the rat.

OBJECTIVE: To study, the analgesic and sedative effects of different constant rate infusions (CRI) of dexmedetomidine, in the rat, by measurement of specific electroencephalographic parameters. The recorded parameters were somatosensory-evoked potentials (SEPs) and auditory-evoked potentials (AEPs), which have been shown to be related to analgesia and sedation respectively. ANIMALS: Nine male Wistar rats (HsdCpb:Wu, Harlan Netherlands BV, body weight 300-350 g). METHODS: Somatosensory-evoked potentials were recorded from the primary somatosensory cortex and the vertex location (SI/Vx-SEPs). Auditory-evoked potentials were recorded from the primary auditory cortex and vertex location (AI/Vx-AEPs). Primary somatosensory cortex and vertex location recorded SEPs and AI/Vx-AEPs were recorded alternately, during CRI of dexmedetomidine (4.0, 10.0, 20.0 microg kg(-1) hour(-1)) and a control (saline). RESULTS: The primary somatosensory cortex-evoked potentials were not affected by the dexmedetomidine CRI, but the other three parameters were significantly affected; although the AI-SEP to a lesser extent than the Vx-SEP and Vx-AEP. A maximum effect on the Vx-AEP was reached at lower doses than on the Vx-SEP. CONCLUSIONS: Based on the present findings, it is suggested that CRI of dexmedetomidine provided profound sedation at low doses, whereas higher doses are needed to provide concurrent analgesia. CLINICAL RELEVANCE: A constant rate infusion of dexmedetomidine can be a valuable adjunct in the provision of sedation and/or analgesia. However, analgesia cannot be produced without sedation, and sedation is not necessarily accompanied by comparative degrees of analgesia.

EMG activity of the muscular and stromal layer of the cervix in relation to EMG activity of the myometrium and cervical dilatation in PGF2alpha induced parturition in the cow.

The goal of this study was to quantify and characterize the electromyographic (EMG) activities in the cervical outer muscular layer and in the cervical stromal layer, and to characterize their relationship with myometrial EMG activity and cervical dilatation during PGF2alpha-induced parturition in term pregnant cows. We continuously measured the EMG activity of the uterine myometrium and cervical outer muscular layer as well as the cervical stromal layer in five cows using bipolar electrodes while at the same time measuring changes in the cervical diameter with ultrasound cervimetry. This we did from the moment a prostaglandin analogue was injected until the expulsion of the calf. In contrast to the cervical stromal layer, the cervical outer muscular layer showed distinct EMG activity, which began to increase at about the same time as the EMG activity of the myometrium, i.e. some 12 h before the start of cervical dilatation. However, the rate of this increase was lower than in the myometrium and it was not characterized, like in the myometrium, by an increase in maximum EMG amplitude. Although the cervical outer muscular layer showed contracture and contraction like EMG activity in unison with in the myometrium, it was also characterized by a more irregular EMG activity, which occurred independently from the myometrium. These data suggest that while the outer muscular layer of the cervix may be considered to be a caudal continuation of the myometrium, it also displays activity independently from the myometrium. The physiological relevance of this activity remains to be explored.

The effect of a single oxytocin or carbetocin treatment on uterine contractility in early postpartum dairy cows.

The uterotonic characteristics and effectiveness of a single treatment with either oxytocin or carbetocin were quantified in early postpartum dairy cows after normal, uncomplicated calvings. Both the short-term (within 4 h), and the long-term effects (between 12 and 36 h) of the two treatments were compared. Between 14 and 16 h after parturition, 27 multiparous Holstein-Friesian dairy cows, without fetal membrane retention, were selected and divided into three groups. The first group (n = 9) was administered 50 IU oxytocin intramuscularly, the second group (n = 10) received 0.35 mg carbetocin, while animals of the third group (n = 8), serving as a control, were administered 5 mL saline solution. A transcervically introduced open tip catheter system was used for the non-invasive acquisition of the intrauterine pressure (IUP) recording. After digitalization, the signals were analyzed, using a specially adapted graphical software program. A significant short-term effect was found both in the oxytocin and carbetocin treated groups from the analysis of the contraction frequencies (FREQ) and of the total area under the curve (TAUC). After significant peaking during the first post-treatment hour, the values of the parameters for these two groups remained higher during the second hour, returning to the initial levels again during the third hour and reaching the level of the control group by the 12th hour. Mean amplitude (AMP), duration (DUR) and area under the curve (AUC) of pressure cycles were not significantly affected by any of the treatments. Although mean FREQ and TAUC significantly declined from the initial values to 12, 24 and 36 h in all groups, mean AMP and AUC in the oxytocin and carbetocin treated groups, and mean DUR only in the carbetocin treated group to 12 and 36 h, the long-term analysis revealed no significant treatment differences for any IUP parameters. Because treatment with either oxytocin, or carbetocin elicited similar uterotonic effects in healthy, early postpartum cows, it cannot be expected, that using carbetocin in preference to oxytocin, will result in a more beneficial clinical effect on uterine involution during this period.

Validation of pressure measurements and electromyographic results for the uterus of cattle during the early postpartum period.

OBJECTIVE: To evaluate methods for on-farm measurements of uterine contractility in postpartum dairy cows by comparing data simultaneously recorded by use of 2 intrauterine pressure (IUP) devices and quantified electromyographic (EMG) signals. ANIMALS: 5 cows during the first 48 hours after parturition. PROCEDURE: 2 EMG electrodes were implanted on the surface of the gravid uterine horn. Parturition was induced by injection of a prostaglandin F2alpha analogue at day 274 of gestation. An open-tip catheter and pressure microtransducer were transcervically inserted and affixed to a caruncle immediately after calving. Changes in IUP were recorded concurrent with EMG recordings during 2-hour periods at 2, 6, 12, 18, 24, 36, and 48 hours after parturition. Novel acquisition and analysis software programs were used with a digital data-filtering capability for evaluation of IUP and EMG signals. RESULTS: The method for intrauterine fixation of the 2 pressure measurement instruments was effective and allowed easy, externally guided removal of the devices 48 hours after parturition. There was a high correlation between the data obtained by the 2 pressure measuring systems. Good correlation was also found between pressure data obtained by the open-tip catheter system and EMG signals. Although the quantified IUP and EMG signals were highly comparable, synchronization was not always evident during visual inspection of these signals. CONCLUSIONS AND CLINICAL RELEVANCE: The open-tip IUP catheter system with a special fixation method is suitable for use in on-farm studies. It will enable investigators to record natural and pharmacologically influenced uterine contractility in early postpartum dairy cows.

Characteristics of bovine early puerperal uterine contractility recorded under farm conditions.

A non-invasive, digital technique was used to measure and quantify intrauterine pressure (IUP) changes in early postpartum dairy cows kept under farm conditions in order to document physiological changes in uterine contractility after uncomplicated calvings. In addition, possible relationships between characteristics of uterine contractility and blood ionized calcium (Ca(2+))-concentrations were investigated. Recordings of uterine contractility were made by using a transcervically inserted open tip catheter in 12 healthy cows during their first 48h after calving. The IUP recording technique appeared easily applicable under farm conditions. Although mean frequency (FREQ), amplitude (AMP) and area under the curve (AUC) of the myometrial contractions significantly decreased due to time, untreated early postpartum cows showed a high variability in characteristics of uterine contractility. There was no correlation between blood Ca2+ -concentrations and any of the contractility parameters.

Reducing chloride conductance prevents hyperkalaemia-induced loss of twitch force in rat slow-twitch muscle.

Exercise-induced loss of skeletal muscle K(+) can seriously impede muscle performance through membrane depolarization. Thus far, it has been assumed that the negative equilibrium potential and large membrane conductance of Cl(-) attenuate the loss of force during hyperkalaemia. We questioned this idea because there is some evidence that Cl(-) itself can exert a depolarizing influence on membrane potential (V(m)). With this study we tried to identify the possible roles played by Cl(-) during hyperkalaemia. Isolated rat soleus muscles were kept at 25 degrees C and twitch contractions were evoked by current pulses. Reducing [Cl(-)](o) to 5 mM, prior to introducing 12.5 mM K(o), prevented the otherwise occurring loss of force. Reversing the order of introducing these two solutions revealed an additional effect, i.e. the ongoing hyperkalaemia-related loss of force was sped up tenfold after reducing [Cl(-)](o). However, hereafter twitch force recovered completely. The recovery of force was absent at [K(+)](o) exceeding 14 mM. In addition, reducing [Cl(-)](o) increased membrane excitability by 24%, as shown by a shift in the relationship between force and current level. Measurements of V(m) indicated that the antagonistic effect of reducing [Cl(-)](o) on hyperkalaemia-induced loss of force was due to low-Cl(-)-induced membrane hyperpolarization. The involvement of specific Cl(-) conductance was established with 9-anthracene carboxylic acid (9-AC). At 100 microm, 9-AC reduced the loss of force due to hyperkalaemia, while at 200 microm, 9-AC completely prevented loss of force. To study the role of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in this matter, we added 400 microm of the NKCC inhibitor bumetanide to the incubation medium. This did not affect the hyperkalaemia-induced loss of force. We conclude that Cl(-) exerts a permanent depolarizing influence on V(m). This influence of Cl(-) on V(m), in combination with a large membrane conductance, can apparently have two different effects on hyperkalaemia-induced loss of force. It might exert a stabilizing influence on force production during short periods of hyperkalaemia, but it can add to the loss of force during prolonged periods of hyperkalaemia.