The visceromotor response to noxious CRD
The VMR to 80 mmHg CRD was recorded in awake male (n = 33) and female (n = 23) rats under normal (e.g., non-irritated/non-inflamed) conditions, and following intracolonic mustard oil (). The magnitude of the VMR was significantly greater in normal and mustard oil-treated females compared to males (two-way RM ANOVA: sex p<0.0001; treatment p<0.0001). The mean increase in VMR magnitude was greater in females (18.7 ± 1.7) compared to males (14.8 ± 1.0; t-test, p<0.05). These data indicate the magnitude of the VMR in female rats is greater than males and intracolonic mustard oil has a greater effect in females.
The magnitude of VMR to 80 mmHg CRD in normal (CRD) and mustard oil-treated (MO+CRD) male and female rats. *** p<0.001 vs. male. # p<0.001 vs. same sex CRD.
The response of colonic primary afferent fibers to graded intensities of CRD
The CRD-evoked response of colonic afferents (LS and TL) was compared between normal and mustard oil-treated male and female rats to determine if sex differences in the VMR could be accounted for by differences in primary afferent responses to CRD. Surprisingly, there was no sex difference in the stimulus-response curves of LS colonic afferents in normal (male n=48 afferents, female n=42 afferents) and mustard oil-treated rats (male n=18, female n=14; two-way ANOVA; ), nor was there sensitization of the evoked response to CRD in males or females following intracolonic mustard-oil. Similarly, there was no difference in the response to distention between TL colonic afferents from normal or mustard oil-treated male and female rats (normal: male n=37, female n=35; mustard oil: male n=20, female n=23; two-way ANOVA; ). These data indicate that sex differences in the evoked response of colonic afferents to CRD in this model do not contribute to sex differences in the VMR.
Figure 2 A,B: The response of colonic afferent fibers in the LS and TL dorsal roots to graded intensities of colorectal distention. Legend in A is the same for B. C,D: the mean spontaneous activity in LS and TL colonic afferents recorded from normal rats and following (more ...)
In contrast to the absence of a sex difference in the evoked response in LS colonic afferents, there was a sex difference in the spontaneous activity (two-way ANOVA: sex p<0.01; treatment p<0.005; ). Prior to mustard oil injection, the mean spontaneous activity in male LS colonic afferents was greater than in females (p<0.01). Mustard oil increased the spontaneous activity of colonic afferents in males by 38% (t-test vs. normal males: p<0.05) and females by 58% (t-test: p<0.05) abolishing the sex difference. In TL colonic afferents there was a significant interaction between sex and treatment in spontaneous activity (two-way ANOVA: p<0.01; ). Spontaneous activity in normal males was greater than normal females (p<0.005). Mustard oil increased spontaneous activity in females by 44% (t-test: p<0.05), but not males (16% decrease, not significant) and the sex difference in spontaneous activity was lost.
Spinal cord Fos expression in response to CRD
In both the LS and TL spinal cord segments (n=8/group), colonic distention (80 mmHg) induced a comparable number of Fos-immunoreactive neurons per section in male (LS: 203 ± 19; TL: 55 ± 5) and female (LS: 224 ± 17; TL: 45 ± 4) rats (). There were a significantly greater number of Fos positive neurons in the LS compared to TL segments in both male and female rats (p< 0.001). Surprisingly, mustard oil plus CRD induced a slight, but non-significant increase in Fos expression in the LS segments (M: 252 ± 20; F: 242 ± 20) and no change in the TL segments (M: 57 ± 6; F: 51 ± 7).
The number of Fos labeled neurons per section in the LS and TL segments following CRD or mustard oil + CRD.
Response of spinal dorsal horn neurons to graded intensities of CRD
Although there was no sex difference in CRD-induced Fos expression, nor did mustard oil increase the number of visceroceptive neurons, single unit recording was used to determine if there was a sex difference in the magnitude of response of LS and TL dorsal horn neurons to CRD prior to and following treatment with mustard oil. Two phenotypes of dorsal horn neurons with excitatory responses to CRD were examined: Abrupt and Sustained.
In the LS segments of normal rats, the response of Abrupt neurons was significantly more robust in females (n=11 neurons) compared to males (n=14 neurons; two-way ANOVA: sex, p<0.01; ), paralleling the sex difference in the VMR of normal rats. Intracolonic mustard oil had no effect on the magnitude of response in males (two-way RM ANOVA: p>0.05; ), but increased the response to CRD in females (two-way RM ANOVA: treatment, p<0.001; ). Thus, following colonic mustard oil the response in females was significantly greater than males (two-way ANOVA: sex, p<0.001; ) and the magnitude of the sex difference increased from 49% in normal rats to 72% following mustard oil (comparing the difference in the area under the curve in 4Ai and 4Aiv).
Figure 4 The response of LS Abrupt (A), LS Sustained (B) and TL Abrupt (C) dorsal horn neurons in normal rats and following intracolonic mustard oil. i) The response of neurons in normal male and female rats. ii) The response of neurons in males before and after (more ...)
LS Sustained neurons were qualitatively different from LS Abrupt neurons. In normal rats, similar to Abrupt neurons, the response of LS Sustained neurons in female rats (n = 9) was greater than males (n = 11; two-way ANOVA: sex, p<0.001; ). However, contrary to Abrupt neurons, colonic mustard oil increased the response of Sustained neurons in male rats (two-way RM ANOVA: treatment, p<0.001; ) and decreased the response in female rats (two-way RM ANOVA: treatment, p<0.05; treatment x pressure, p<0.05; ). This resulted in the loss of the sex difference in Sustained neurons following intracolonic mustard oil (two-way ANOVA: p>0.05; ) from 120% in normal rats to 49% following mustard oil.
The TL spinal cord is unlikely to contribute to the VMR in normal rats [48
]. Nevertheless, Abrupt neurons were recorded in these segments in order to determine the effect of mustard oil. Prior to mustard oil the response of TL Abrupt neurons was significantly greater in females (n=7) compared to males (n=11; two-way ANOVA: sex, p
< 0.01; ).
Following intracolonic mustard oil the response of Abrupt neurons to CRD in both male (two-way RM ANOVA: treatment, p
<0.001; ) and female rats (two-way RM ANOVA: treatment, p
<0.001; ) increased, maintaining the direction (two-way ANOVA: sex, p
<0.005; ) and magnitude (40% in normal rats, 43% following mustard oil) of the sex difference. Sustained neurons were rarely encountered in the TL segments of male rats [66
] and therefore were not studied.
Sex difference in NMDA receptor activity
NMDA receptors contribute to spinal processing of acute nociceptive visceral stimuli and estradiol increases NMDA receptor expression in the spinal cord of ovariectomized rats increasing the magnitude of the VMR [59
]. This prompted us to determine if sex differences in NMDA receptor activity contribute to the sex difference in the VMR.
The NMDA receptor antagonist APV dose-dependently attenuated the VMR in normal males and females (). 100 nmol APV produced 92% attenuation of the VMR (%MPE) in males compared to 55% in females (t-test, p<0.001). 200 nmol APV further attenuated the VMR to 74% MPE in females equalizing the %MPE to 100 nmol in males (t-test, p=0.1303). Overall, APV had a greater effect in males (%MPE: two-way ANOVA: sex p<0.0001; APV p<0.0001). Following intracolonic mustard oil there was still a dose-dependent attenuation of the VMR in rats of both sexes, but the sex difference was lost (two-way ANOVA: dose p<0.0001; ) as 100 nmol APV attenuated the VMR by 86% in females.
Figure 5 A, B: dose-response curves to i.t. APV from normal (A) and mustard oil-treated (B) rats. § p<0.0001 vs. male. * p<0.05 in that pressure. C: Western blot and quantification showing total GluN1 from the LS spinal cord of normal males (more ...)
Western blots were used to measure the expression of the obligatory component of the NMDA receptors, GluN1 (formerly NR1), in the LS and TL spinal segments in normal and mustard oil-treated rats. Normal males had more total GluN1 protein in the LS spinal cord compared to females (t-test: p<0.01; ), but females had greater GluN1 expression in the membrane fraction (t-test: p<0.05; ). In the TL spinal cord, there was no sex difference in the membrane expression of GluN1 (data not shown). Taken together, these data suggest greater NMDA receptor activity in the LS spinal cord in females might underlie the sex difference in the magnitude of the VMR under normal conditions.
Following colonic mustard oil there was no longer a sex difference in membrane expression of GluN1 in the LS spinal cord (t-test: p>0.05; ). This was due to an increase in GluN1 in males (t-test, p<0.001; ), but not females (). In contrast, there was significantly more GluN1 expression in the membrane fraction in the TL segments of female rats (t-test: p<0.05; ).