Sex and oestrous cycle differences in visceromotor responses and vasopressin release in response to colonic distension in male and female rats anaesthetized with halothane{dagger}

A. Holdcroft1, S. Sapsed-Byrne1, D. Ma1, D. Hammal2 and M. L. Forsling3

1Departments of Anaesthesia and Intensive Care and 2Medical Statistics and Evaluation, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0HS, UK. 3Department of Physiology, St Thomas’ Hospital, King’s College School of Medicine, Lambeth Palace Road, London SE1 7EH, UK*Corresponding author

{dagger}Presented in part to the Anaesthetic Research Society 1998 and published as an abstract in Br J Anaesth 1999; 82: 469–470P.

Accepted for publication: June 12, 2000


    Abstract
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 Abstract
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 Methods and Results
 Comment
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Visceromotor responses and vasopressin release before and after colonic visceral distension were compared between male (n=5 (n=4 for vasopressin)) and female rats and between females during the oestrous cycle (proestrus n=6, oestrus n=5, metestrus n=5, diestrus n=6) at a controlled depth of anaesthesia. Pre-stimulation vasopressin and blood pressures demonstrated oestrous cycle variability. The mean (SEM) colonic balloon pressure triggering visceromotor responses was significantly higher in males (64 (4) mm Hg) than females (41 (1) mm Hg), P=0.002 and within females, proestrus rats had the lowest thresholds, (29 (1) mm Hg, P<0.01). Post-stimulation, vasopressin concentrations increased significantly in all groups (males 1.34 (0.39) to 2.24 (0.74) pmol litre–1; females 1.54 (0.24) to 2.88 (0.58) pmol litre–1; P=0.002). Within groups statistically significant differences were measured in proestrus 2.06 (0.56) to 3.42 (1.12) and oestrus 1.16 (0.38) to 2.76 (0.60) pmol litre–1 (P<0.05). High vasopressin concentrations coupled with low-pressure stimulation during proestrus shows sex-hormone dependent integration of the neuroendocrine response to noxious visceral stimulation.

Br J Anaesth 2000; 85: 907–10

Keywords: hormones, vasopressin; rat; sex; anaesthetics volatile, halothane


    Introduction
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 Abstract
 Introduction
 Methods and Results
 Comment
 References
 
Aversive visceromotor and cardiovascular responses to visceral distension in animals can be measured repetitively using colonic balloon inflation1 and are influenced in female rats by the oestrous cycle.2 Confirmation of the relationship between vasopressin (AVP) release and noxious visceral stimuli is considered important in the understanding of stress responses as well as the fundamental differences between sexes and normal cyclical sex-hormone variations. The sensitivity and rapidity of AVP release coupled with a short half-life necessitates standardized experimental conditions that include gentle handling, specific time-related activities,3 cardiovascular stability and controlled anaesthetic depth.4

It was hypothesized that AVP release could be stimulated by noxious visceral distension in anaesthetised rats and the magnitude of this response would vary with sex and oestrous cycle phase.


    Methods and Results
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 Abstract
 Introduction
 Methods and Results
 Comment
 References
 
Studies on 5 male and 4-day cycling female (6, 5, 5, and 6 in proestrus, oestrus, metestrus and diestrus respectively) adult Wistar rats were completed during the morning at the end of a 12 h dark cycle (Home Office PPL70/4211) using the method we previously described.2 A saline vaginal smear was taken for cellular identification (a high proportion of cornified cells being present on the day of oestrus) to determine the oestrous phase.5 This was read at the end of the experiment. Anaesthesia was induced with 5% halothane in oxygen then maintained with 2% halothane by a funnel closely applied to the airway. A calibrated Datex Capnomac II measured inspired halothane continuously at the nose. An 8 cm long double-lumen colonic balloon was fully inserted through the rectum into the colon and secured with tape, then a rectal thermometer to maintain normothermia. After securing the catheters the halothane concentration was reduced to 1%. The arterial catheter was connected via a strain gauge transducer to an Apple Macintosh Performa 475. Arterial blood pressures, with mean arterial pressures (MAP) and heart rates derived from it, and colonic balloon pressures were recorded as shown in Figure 1. Bilateral electromyography electrodes were connected to leg flexor muscles and the raw signal was used to confirm the onset of the visual visceromotor response.



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Fig 1 Measurements of the electromyogram (EMG), arterial blood pressure (BP), heart rate (HR), mean arterial blood pressure (MAP) and intracolonic pressure (COLP) showing the changes from baseline in response to a single colonic balloon distension (BALP) in a rat in metestrus. The arrow on the EMG trace corresponds with the balloon pressure that triggered a visual visceromotor response.

 
After 20 min stabilization, volume replacement allowed 1 ml blood to be removed for AVP immunoassay. These sampling techniques do not lead to AVP release.6 Vasopressin was determined by immunoassay in extracts of plasma prepared using C18 Sep-Pak columns (Waters Associates Ltd., Northwick, Essex, UK). Determinations were performed as described by Forsling and Peysner3 using the First International Standard for AVP (77/501). The lower limit of detection for the assay was 0.12 (0.02) pmol litre–1 with intra and interassay variations of 7.7 and 11.9% respectively for 2.5 pmol litre–1.

Colonic distension was achieved by slow manual balloon inflation (15–30 s), up to the threshold triggering the visceromotor response, with a 100 mm Hg maximum to prevent rupture, followed by deflation. Every 2 min threshold pressure and the associated MAP was measured. After six intermittent inflations, post-stimulation AVP and arterial blood gas analysis was taken and the rat killed by anaesthetic overdose. All rats had normal blood gases.

Mixed model ANOVA (Genstat, Numerical Algorithms Group, Oxford, UK) was used for three outcome variables: colonic pressure, MAP and AVP concentrations. Five groups were analysed, the four oestrous cycle phases within the female group and one male group. Cycle phase and time were taken as factors for inclusion in the model. The main results are presented as ratios of geometric means because log transformations were performed to comply with the assumptions of ANOVA. There were no statistically significant changes with time.

Baseline AVP concentrations, which lie in the normal physiological range for conscious rats,3 and MAP are shown in Table 1. In all rats AVP increased post-stimulation. There were no significant differences in AVP concentrations (pmol litre–1) between male and female groups (pre-stimulation mean (SEM): 1.34 (0.38) and 1.54 (0.24) respectively) with pre-stimulation male to female ratio of 1.167 (95%CI 0.539, 2.527; P=0.683). However, males have concentrations 17% greater than females. Similarly, post-stimulation the male: female ratio was 0.860 (95%CI 0.397, 1.863; P=0.689) with post-stimulation mean (SEM) values of 2.24 (0.74) and 2.88 (0.58) pmol litre–1 respectively. Although non-significant, males have concentrations approximately 14% smaller than females. Thus, over time, the magnitude of the change was smaller for males than females. The highly significant differences pre- and post-stimulation, taking males and females as one group, can be expressed as the ratio 1.941 (95%CI 1.309, 2.879; P=0.002). Thus the mean AVP concentration post-stimulation was 94% greater than the mean AVP concentration pre-stimulation. In addition, within the female group, after the colonic stimulation a statistically significant increase in AVP concentrations was detected in proestrus and oestrus.


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Table 1 AVP concentrations (pmol 1itre–1), MAP and threshold balloon pressures (mm Hg) before and after stimulation by colonic balloon distension. All values are expressed as mean (SEM) (range). a Within groups: P<0.05; b between female groups P<0.01 (comparing each group to the diestrus group); between male and female groups: c P<0.05, d P<0.01
 
Visceromotor responses were triggered at mean (SEM) balloon pressures for males and females of 63.8 (4) and 41.0 (1) mm Hg respectively. Proestrus rats have the lowest mean balloon pressure and males the largest. The ratio of males to females is 1.574 (95%CI 1.198, 2.068; P=0.002), so that the pressure to trigger a visceromotor response in males is approximately 1.6 times larger than females.

A significant sex difference in MAP exists post- but not pre-stimulation. The ratio of the geometric means in males to females, for pre-stimulation is 1.017 (95%CI 0.996, 1.038; P=0.113) and for post-stimulation 1.023 (95%CI 1.002, 1.044; P=0.031) with an average increase of 4%. When comparing MAP in females the most significant difference was between diestrus and the other groups. Pre- and post-stimulation diestrus rats had values 16–18% greater than either the proestrus or oestrus groups and approximately 5% greater than the metestrus group.


    Comment
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 Abstract
 Introduction
 Methods and Results
 Comment
 References
 
Pre-stimulation AVP concentrations confirmed the oestrous cycle variations originally described by Forsling and Peysner.3 Although there were no statistical differences between males and females in baseline or post-stimulation AVP release despite the marked differences in balloon pressures required to elicit a visceromotor response, the magnitude of increase was smaller in males. The larger pressure for a visceromotor response and less AVP release may indicate that the hypothalamo-pituitary axis is more reactive in females than males. Metestrus showed least changes, which might be associated with a lack of hormonal changes during this phase. An increase in MAP with visceral stimulation is most likely to be the result of central nervous system stimulation in response to visceral afferent nerve fibre activity.1 Noxious stimuli are associated with increased sympathetic nervous system activity causing hypertension. When MAP is elevated by non-hypothalamo-pituitary mediated mechanisms the response is to decrease AVP release. The increase in AVP release can be considered to relate to noxious stimulation rather than as a response to cardiovascular changes and possibly could be expected to be of greater magnitude if MAP was maintained at baseline values and did not increase with stimulation.

The higher balloon pressures observed in this study in males supports previous studies of sex differences in pain thresholds.7 Autonomic nerves can transmit noxious stimuli from peripheral visceral structures to the hypothalamus. If afferent impulses can be traced to the hypothalamus, then what factors determine the variations in visceromotor and stress responses during the oestrous cycle? Evidence from double labelling immunocytochemistry demonstrates colocalization of oestrogen receptor beta with vasopressin in the supraoptic nucleus and paraventricular nucleus of the hypothalamus.8 Electrophysiological changes in the frequency and intensity of peripheral nerve impulses from reproductive organs have also been shown during the oestrous cycle7 particularly during proestrus and oestrus. These variations in peripheral nerve activity may modulate the central release of AVP, which is a stress hormone. In addition, central nervous system sensitisation may further modulate not only neural but also endocrine responses. Interactions between the central and peripheral neuroendocrine activity during noxious stimulation require further investigation.


    References
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 Abstract
 Introduction
 Methods and Results
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 References
 
1 Ness TJ, Gebhart GF. Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characteristics of pseudoaffective reflexes in the rat. Brain Res 1988; 450: 153–69[ISI][Medline]

2 Sapsed-Byrne S, Ma D, Ridout D, Holdcroft A. Estrus cycle phase variations in visceromotor and cardiovascular responses to colonic distension in the anaesthetised rat. Brain Res 1996; 742: 10–16[ISI][Medline]

3 Forsling ML, Peysner K. Pituitary and plasma vasopressin concentrations and fluid balance throughout the oestrous cycle of the rat. J Endocrinol 1988; 117: 397–402[Abstract]

4 Holdcroft A, Bose D, Sapsed-Byrne SM, Ma D, Lockwood GG. Arterial to inspired partial pressure ratio of halothane, isoflurane, sevoflurane and desflurane in rats. Br J Anaesth 1999; 83: 618–21[Abstract/Free Full Text]

5 Montes GS, Luque EH. Effects of ovarian steroids on vaginal smears in the rat. Acta Anat Basel 1988; 133: 192–9[Medline]

6 Wells T, Forsling ML, Windle RJ. The vasopressin response to centrally administered hypertonic solutions in the conscious rat. J Physiol 1990; 427: 483–93[Abstract]

7 Berkley KJ. Sex differences in pain. Behav Brain Sci 1997; 20: 371–80[ISI][Medline]

8 Alves SE, Lopez V, McEwen BS, Weiland NG. Differential colocalisation of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study. Proc Natl Acad Sci USA 1998; 95: 3281–6[Abstract/Free Full Text]