BEHAVIOURAL STUDY

Teflon coated stainless steel wire electrodes (Cooner Wires, Chatworth, California, USA) were stitched into the external oblique musculature immediately superior to the inguinal ligament for electromyographic (EMG) recording. Arterial and venous catheters and EMG electrodes were tunnelled subcutaneously and externalised at the back of the neck. In some experiments, an intrathecal catheter (PE10, 8.5 cm) was extended to the lumbar enlargement through a slit in the atlanto-occipital membrane. The intrathecal catheter was externalised at the back of the neck together with the arterial catheter and EMG electrodes in these experiments. After recovery from anaesthesia, rats were housed separately for at least three days prior to testing.

For colorectal distension (CRD), a 6–7 cm long, 2–3 cm diameter balloon was inserted intra-anally into the descending colon and rectum. The balloon catheter was connected to a distension control device (Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA) via a low volume pressure transducer and intracolonic pressure was continuously monitored (see Gebhart and Sengupta17 for details). CRD in awake rats results in two readily quantifiable pseudaffective (that is, spinobulbospinal) responses18: an increase in mean arterial pressure (MAP) and contraction of the abdominal and hindlimb musculature, termed the visceromotor response (VMR). VMR was quantified by measuring EMG activity in the external oblique musculature. The arterial catheter was connected to a low volume pressure transducer (Cobe Labs, Linkwood, Colorado, USA) and the EMG signal was amplified (×10 000, 300–1000 Hz) and filtered (200 Hz high pass, 4 pole Butterworth; graphic equaliser, Yamaha). Distending pressure, arterial pressure, and EMG were digitised at 1000 Hz (DT280, Data Translation, Marlboro, Massachusetts, USA) and processed using programs written in ASYST. The EMG signal was rectified and averaged over 500 ms, reducing the effective sampling to 2 Hz. All signals were viewed on line and also recorded for subsequent analysis.

Staircase distension was used for CRD.19 Briefly, starting at 0 mm Hg, intracolonic pressure was incremented in steps (10–20 mm Hg) over about 80 seconds to a final pressure of 80 mm Hg. Changes in MAP and EMG were quantified during the initial five seconds of each distension step. Three to five staircase distensions, at four minute intervals, were given six hours after intracolonic instillation of either saline or 1 ml of 5% acetic acid (HAc)20 to establish predrug baseline response magnitude and threshold. Octreotide was then administered intravenously or intrathecally using a single dose or cumulative dosing paradigm; two staircase distensions were given four and eight minutes after drug administration, followed by the next dose of drug (intrathecal study).

SINGLE FIBRE RECORDING

The trachea was cannulated to allow artificial ventilation with room air. For intra-arterial drug administration, a catheter was passed into the descending aorta via the left common carotid artery. Mean arterial blood pressure was monitored continuously and maintained >80 mm Hg with intravenous 5% dextrose in saline given in a bolus of 1–1.5 ml as required. The lower abdomen was exposed by a 3–4 cm long incision laterally at the left flank. The pelvic nerve was isolated from surrounding tissues and wrapped with a pair of Teflon coated stainless steel wires stripped at the tips and sealed with non-reactive Wacker gel (Wacker Silicone Corp., Adrian, MI, USA). The spinal cord was exposed by laminectomy (T13–S2) and covered with warm (37°C) mineral oil. Recordings were made from the distal cut end of pelvic nerve afferent fibres in the S1 dorsal root. The dorsal rootlet was split into fine filaments to obtain a single unit. Electrical activity of the single unit was recorded by a monopolar silver-silver chloride electrode. Action potentials were monitored continuously by analogue delay and displayed on a storage oscilloscope after initial amplification through a low noise AC differential amplifier. Action potentials were processed through a window discriminator and counted (one second binwidth) using the spike2/CED 1401 data acquisition program. Peristimulus time histograms, intracolonic pressure, and blood pressure were displayed on line continuously. All parameters were also recorded for off line analysis.

Pelvic nerve input to the S1 dorsal root was identified first by electrical stimulation of the pelvic nerve (single 0.5 ms square wave pulse at 3–8 mV). Fibres were classified on the basis of their conduction velocities; those with conduction velocities <2.5 m/s were considered unmyelinated C fibres and those with conduction velocities >2.5 m/s were considered thinly myelinated Aδ fibres. If a fibre responded to CRD, responses to phasic distending pressures of 5, 10, 20, 30, 40, 60, 80, and 100 mm Hg, 30 seconds at four minute intervals, were determined; a noxious intensity of CRD (80 mm Hg, 30 seconds) was used thereafter in these experiments.

For acute inflammation of the colon, the balloon was removed and 1.5–2 ml of 5% HAc in saline was instilled intra-anally into the descending colon. The irritant was held in the colon for 15 minutes, the colon was flushed with warm saline, and the balloon reinserted. Other rats received identical treatment with vehicle (saline). In three rats, the balloon was removed and zymosan (25 mg/ml, 2 ml in 30% ethanol) was instilled into the colon as a second model of sensitisation of responses to CRD.21 The balloon was reinserted and responses to 80 mm Hg CRD were studied 30 minutes after intracolonic treatment.

The effects of octreotide were tested on responses of mechanosensitive afferent fibres to 80 mm Hg CRD. Octreotide was administered intra-arterially or intravenously using a cumulative dose paradigm. Each dose of drug was given 90 seconds before the onset of distension. Drugs were tested on responses of fibres recorded from untreated and HAc or zymosan treated colons. As a positive control, the kappa opioid receptor agonist U50,488, which attenuates responses of pelvic nerve afferent fibres to noxious distension,22 ,23 was tested in two experiments after octreotide had been tested.

DATA ANALYSIS

Differences between resting EMG activity and MAP before and after drug administration were compared using a non-parametric Wilcoxon test to determine whether there were changes in these parameters attributable to drug action. Dose-response relationships were constructed to show dose dependent changes in response magnitudes at innocuous (20 mm Hg) and noxious (60 mm Hg) intensities of distension.

The resting activity of an afferent fibre was counted for 60 seconds before CRD and the response to distension was determined as the increase in discharge during distension above its resting activity. Results were analysed using analysis of variance (ANOVA) for repeated measures.

All data are expressed as mean (SEM). A value of p<0.05 was considered statistically significant.

DRUGS

Commercially available octreotide acetate (Sandostation; Sandoz Pharmaceutical Co., East Hanover, New Jersey, USA) was used throughout. Octreotide is a cyclic octapeptide; the acetate salt has a molecular weight of 1019.3. U50,488 (molecular weight 465.4) was purchased from Research Biochemicals Inc. (Natick, Massachusetts, USA). Zymosan was purchased from Sigma Chemical Co., (St Louis, Missouri, USA).

BEHAVIOURAL STUDY

All rats exhibited monotonic increases in visceromotor and pressor responses to graded intensities of CRD (10–80 mm Hg, 20 seconds) (figs1, 2). The mean maximal pressor responses to 80 mm Hg CRD did not differ between rats treated intracolonically six hours earlier with saline (21 (3) mm Hg) or 5% HAc (18 (2) mm Hg), consistent with previous results in this model.20 Response magnitudes for VMR at 80 mm Hg CRD were 150 (10.6)% and 249 (21)% of control in saline and HAc treated rats (p<0.05), respectively, revealing an enhanced response to a noxious intensity of distension (that is, hyperalgesia).20 There were no changes in response threshold.

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Figure 1

Stimulus-response functions (SRFs) for visceromotor (A) and pressor (B) responses to colorectal distension before (vehicle) and after intravenous administration of octreotide (100 μg) in saline and acetic acid treated rats. The broken line represents the control response to 80 mm Hg distension as 100%. All data are expressed as percentage of maximal control response (mean (SEM)). The basis for n is the number of rats used in constructing each SRF. *Significantly different from control group (ANOVA).

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Figure 2

Stimulus response functions (SRFs) for visceromotor (A) and pressor (B) responses to colorectal distension (CRD) before (vehicle) and after cumulative doses of intrathecal administration of octreotide (5–15 μg) in saline and acetic acid treated rats. The broken line represents the control response to 80 mm Hg distension as 100%. All data are expressed as percentage of control response (mean (SEM)). The basis for n is the number of rats used in constructing each SRF. Intrathecal octreotide dose dependently attenuated the visceromotor (A) and pressor (B) responses to CRD in saline treated rats.

When given intravenously, the single dose of octreotide tested (100 μg/kg) had no effect on VMR in saline or HAc treated rats (fig 1). However, octreotide significantly attenuated the increase in MAP in response to CRD but only in HAc treated rats (fig 1). The mean maximal pressor response to 80 mm Hg CRD was reduced from 20.5 (3.8) mm Hg to 10.6 (3.6) mm Hg by octreotide in HAc treated rats. Response magnitude at all intensities tested was less without apparent effect on response threshold. There was no effect of octreotide on resting MAP in either group (saline treated 135.8 (3.3) mm Hg; HAc treated 134.2 (4.5) mm Hg).

When administered intrathecally, octreotide dose dependently attenuated both the visceromotor and pressor responses to CRD in saline treated rats (fig 2). Octreotide (10 and 15 μg) significantly decreased response magnitude and increased the VMR threshold. In HAc treated rats, octreotide evidenced little effect on the VMR and differences were not statistically significant. This likely reflects the visceral hyperalgesia produced by intracolonic instillation of HAc (see above and Burton and Gebhart20). Incomplete data were collected for the pressor response in HAc treated rats.

SINGLE FIBRE STUDY

A total of 15 afferent fibres in the decentralised S1 dorsal root, identified by electrical stimulation of the pelvic nerve and response to CRD, were studied. Ten fibres were myelinated Aδ fibres (mean conduction velocity 13.0 (1.9) m/s) and five fibres were unmyelinated C fibres (mean conduction velocity 2.0 (0.2) m/s). In the presence of the balloon in the descending colon, 11 fibres exhibited an ongoing discharge (mean 1.0 (0.3) imp/s; range 0.01–3.2). All 15 fibres were characterised for responses to graded intensities of CRD (5–100 mm Hg) and all gave monotonic increases in response to increasing pressures of CRD which were temporally linked to stimulation. None gave on-off, rapidly adapting responses suggestive of mucosal afferent fibres. Cumulative intravascular administration of octreotide did not affect the responses to noxious CRD of any of 13 mechanosensitive pelvic nerve afferent fibres tested. Figure 3 illustrates examples of lack of effect of octreotide on responses of two different Aδ afferent fibres to CRD in uninflamed colon and zymosan inflamed colon. Note the enhanced response to CRD (that is, sensitisation) after intracolonic instillation of zymosan, which was unaffected by octreotide. Two fibres, in which octreotide did not attenuate responses to CRD, were tested subsequently with U50,488, a kappa opioid receptor agonist. U50,488 attenuated responses to CRD (fig 3), as we have documented previously.22 ,23

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Figure 3

Effects of cumulative systemic administration of octreotide on responses of pelvic nerve afferent fibres to noxious colorectal distension (CRD) (80 mm Hg, 30 seconds). Examples of lack of effect of octreotide on responses of different Aδ afferent fibres to CRD in uninflamed colon (A) and zymosan inflamed colon (B). Thirty minutes after intracolonic injection of zymosan (25 mg/ml, 2 ml in 30% ethanol), the fibre in (B) was sensitised (response magnitude increased from 679 imp/30s to 897 imp/30s, an increase to 132% of the pre-zymosan control (“C”) response). Octreotide was injected intra-arterially in a cumulative dose as indicated by the arrows. Responses of the fibres are illustrated as peristimulus time histograms (one second binwidth); distending pressure (80 mm Hg, 30 seconds every four minutes) is presented below. In (C), the effect of U50,488 (16 mg/kg) is shown for a fibre that had previously received cumulative doses of octreotide (2.4 mg/kg).

Figure 4 summarises individual responses of fibres from untreated (n=8) and HAc (n=2) or zymosan (n=3) treated colons in the presence of a broad range of cumulative doses of octreotide. There was no inhibition of responses of two afferent fibres to CRD when octreotide was given in doses of 50 and 100 μg/kg (data not shown).

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Figure 4

Absence of dose dependent effects of octreotide on responses of individual pelvic nerve afferent fibres to noxious colorectal distension (80 mm Hg, 30 seconds, as percentage of control). Octreotide failed to attenuate responses of pelvic nerve afferent fibres recorded from either untreated or zymosan treated colon.