cortisol (C) in non ovariectomized, placebo treated ovariectomized (OVX + oil) and estradiol treated ovariectomized (OVX + E2) female rats.
in serum IL6and pituitary Il6 in non ovariectomized and ovariectomized female rats subjected to either CLP or LI in comparison to corresponding sham subgroups [CLP: non ovariectomized serum IL6 (ng/ml) 820.3 ± 92 vs. 517.7 ± 151.9, pituitary IL6 (ng/ml) 273.3 ± 29.1 vs. 125.2 ± 15.1; Ovariectomized serum IL6 (ng/ml) 865.1 ± 51.5 vs. 519.7 ± 151.9, pituitaryIL6 (ng/ml) 244.3 ± 27.2 vs. 127.2 ± 15.1] [LI: non ovariectomized serum IL6 (ng/ml) 864.8 ± 94.9 vs. 518.7 ± 151.9, pituitary IL6 (ng/ml) 128.6 ± 8.4 vs. 126.2 ± 15.1; Ovariectomized serum IL6 (ng/ml) 919.2 ± 297 vs. 513.2 ± 151.2, pituitary IL6 (ng/ml) 257.5 ± 26 vs. 126.3 ± 14.3] (Tables 1 and 2).
In addition, we found that E2 replacemt after ovariectomy was associated with significant decreases (p < 0.05) in the levels of serum IL6, brain IL6 and pituitary Il6 in comparison to placebo treated ovariectomized female rats after systemic or local inflammation. [Estradiol treated CLP: serum IL6 (ng/ml) 558 ± 103.4, brain IL6 (ng/ml) 195.6 ± 12.4, pituitary IL6 (ng/ml) 204.9 ± 10.8] [Estradiol treated LI: serum IL6 (ng/ml) 606.1 ± 105, brain IL6 (ng/ml) 200.7 ± 13.6, pituitary IL6 (ng/ml) 207.4 ± 8.3] (Tables 1 and 2).
When we compared different CLP subgroups to corresponding subgroups subjected to LI, we found that there were significant increases (p < 0.05) in the level of brain and pituitary IL6 with local inflammation in com parison to CLP. [Brain IL6 (ng/ml): ovariectomized 288.7 ± 7.7 vs. 252.7 ± 29.7, estradiol treated ovariectomized 200.7 ± 13.6 vs. 195.6 ± 12.4; pituitary IL6 (ng/ml): ovariectomized 257.5 ± 26 vs. 244.3 ± 27.2, estradiol treated ovariectomized 207.4 ± 8.3 vs. 204.9 ± 10.8] (Figures 3 (a)-(c)).
3.3. Nitrite (Brain, Pituitary)
We found that there was significant increase (p < 0.05) in the brain nitrite level in non ovariectomized and ovariectomized female rats subjected to either CLP or LI in comparison to corresponding sham subgroups [CLP: non ovariectomized brain nitrite (pg/ml) 271.3 ± 24.5 vs. 164 ± 8.5; Ovariectomized brain nitrite (pg/ml) 271.3 ± 24.5 vs 190.9 ± 12.4] [LI: non ovariectomized brain nitrite (pg/ml) 248.7 ± 43.7 vs. 165 ± 8.5; Ovariectomized brain nitrite (pg/ml) 258 vs. 24.7] (Tables 1 and 2).
In addition, we found that E2 replacemt after ovariectomy was associated with significant decreases (p < 0.05) in the levels of brain nitrite in comparison to placebo treated ovariectomized female rats after systemic or local inflammation. [Estradiol treated CLP: brain nitrite (pg/ml) 221.2 ± 15.4] [Estradiol treated LI: brain nitrite (pg/ml) 206 ± 36.5] (Tables 1 and 2).
When we compared different CLP subgroups to corresponding subgroups subjected to LI, we found that there was significant increase (p < 0.05) in the level of pituitary nitrite with local inflammation in comparison to
Table 1. Evaluation of the levels of E2, CRH, ACTH, cortisol, IL6 and nitrite in sham CLP and all CLP subgroups.
Table 2. Evaluation of the levels of E2, CRH, ACTH, cortisol, IL6 and nitrite in sham LI and all LI subgroups.
CLP. [Pituitary nitrite (pg/ml): non ovariectomized 244.8 ± 39.1 vs. 159.7 ± 30.7, estradiol treated ovariectomized 250.2 ± 22.5 vs. 226.9 ± 20.2] (Figure 4 (a)-(b)).
In our study, we found that both the level of estradiol hormone and the type of inflammation (stress) could play an important role in the degree of HPA response. Regarding the level of estradiol hormone, we found that in both CLP and LI the reduced level of estradiol induced by stress (inflammation) or surgical ovariectomy was associated with increases in the plasma levels of CRH, ACTH and Cortisol. Meanwhile, estradiol replacement after ovariectomy induced significant increase in estradiol to certain physiological level, which was associated with significant decreases in the plasma levels of CRH, ACTH and Cortisol.
Previous studies demonstrated that the stress released hormones such as CRH, vasopressin and glucocorticoids could inhibit the gonadal steroid production . With low level of estradiol the other inflammatory mediators take the upper hand over sex hormones on the HPA response resulting in stimulatory effects at the level of hypothalamic CRF release or directly on the pituitary itself to stimulate ACTH secretion [26,27]. In addition, previous studies demonstrated that castration enhances the ACTH and corticosterone response to IL-1ß and lipopolysaccharide (LPS) and this was reversed by E2 replacement [28-31]. However, our study differ in that we used not only CLP model, which appears to be the best existing alternative for emulating human polymicrobial sepsis to date and could mimic the pathophysiologic
All values are expressed in means ± SD. $P < 0.05 vs. corresponding CLP groups.
Figure 3. Evaluation of the effects of cecal ligation and puncture (CLP) and local inflammation(LI) on the plasma level of interleukin 6 (IL6) (A), the brain level of IL6 (B) and the pituitary level of IL6 (C) in non ovariectomized, placebo treated ovariectomized (OVX+oil) and estradiol treated ovariectomized (OVX+E2) female rats.
consequences of sepsis [32,33], but also local inflammation as many studies have been largely restricted to the HPA response to LPS. However, it is doubtful that LPS activation of the HPA axis can be viewed as a general model by which infectious/inflammatory insults influence neuroendocrine activity . Another difference inour studies is that we evaluated the HPA response by the measurement of the plasma levels of CRH, ACTH
All values are expressed in means ± SD. $P < 0.05 vs. corresponding CLP groups.
Figure 4. Evaluation of the effects of cecal ligation and puncture (CLP) and local inflammation (LI) on the brain level of nitrite (A) and the pituitary level of nitrite (B) in non ovariectomized, placebo treated ovariectomized (OVX + oil) and estradiol treated ovariectomized (OVX + E2) female rats.
and cortisol instead of the measurement of the tissue content of CRH and ACTH, which may not well reflect the rapidly releasable pool of these hormones, which is the more important parameter in acute hormonal responses. This is because tissue contents of hormones, in general, represent a difference between an increase by synthesis and a decrease by release, which thus raises the possibility that similar hormonal contents in different experimental groups could result if both the synthesis and release are altered to a similar degree but in an opposite direction [28,30,35,36].
In contrast to our study, Watanobe and Yoneda  found that OVX significantly diminished the LPS-induced ACTH release, and that this effect was completely abolished by supplementing E2 suggesting that estrogen actually promotes inflammatory induced stress response. In addition, Nappi et al.  reported that CRH gene was highly expressed in the parvocellular subdivision of the paraventricular nucleus during the morning of proestrus (higher sex hormones) compared with diestrous females (lower sex hormones) subjected to systemic injection of LPS. Lunga and Herbert  showed also that basal and stress-activated corticosterone levels have been shown to vary across the oestrous cycle, being at their maximal during the proestrous phase when circulating E2 levels are high. Those discrepancies between our results and previous results could be due to differences in experimental circumstances, species and the concentration of estradiol reached after ovariectomy and achieved by treatment.
To our knowledge, our study is the first that compare the effects of CLP and LI on HPA response with different estradiol levels. We found that LI is associated with enhancement of HPA response in comparison to CLP. This could suggest that mechanism involved in HPA enhancement differ between LI and CLP. This could support both Mannino et al. and Turnbull and Rivier [27-39], who showed that the immediate response to intra-plantar formalin (phase 1) results from the stimulation of primary afferent nociceptors, whereas the latter response, phase 2, is generated by a reduced but continuing stimulation of peripheral nociceptors, and activation of inflammatory mediators, and that both phases have an effect on the HPA response during local inflammation. For this reason, our second aim was to measure central and peripheral formed IL6, which plays a major role in endotoxin-induced stimulation of the HPA axis, as indicated by the fact that antibodies against IL6 almost completely block the HPA response to endotoxin [40-42], and central formed nitrite (as an indicator of NO production). We wanted to see if there are differences in IL6 and NO production with different estradiol levels and different type of inflammation that could explain the previous observed differences in HPA responses. We found that low physiological level of estradiol induced by stress and surgical ovariectomy associated with significant increases in serum and pituitary IL6 both in LI and CLP. However, estradiol replacement after ovariectomy succeded to increase estradiol to certain physiological level associated with significant lowering of serum, pituitary and brain IL6.
Our finding support previous studies, which showed relation between estradiol and IL6, such as Chiu et al. , who observed a negative correlation between circulating IL6 and E2 concentrations during the normal menstrual cycle. Moreover, Puder et al.  demonstrated that E2 attenuates the endotoxin-induced stimulation of IL6, and TNF-alpha release in the postmenopausal women.
The exact mechanisms by which estrogen interferes with cytokine activity are still incompletely known but may potentially include interactions of the estrdiol receptor (ER) with other transcription factors, modulation of IL6 promoter and nitric oxide activity, an anti-oxidative effect, a plasma membrane action, and changes in immune cell function[45,46].
In addition, we found that low physiological level of estradiol associated with significant increases in the brain nitrite, and significant lowering of the brain nitrite was achieved after estradiol replacement. These findings could suggest the enhancement of NO induced stimulation of HPA axis during inflammation, as indicated by Puder et al. , who suggested that the endogenous NO seems to restrain the HPA response to inflammatory stimuli. Uribe et al.  found also a temporal correlation between endotoxin-induced activation of the HPA axis and stimulation of neuronal NO synthase in the paraventricular nucleus of the hypothalamus. Although, previous studies demonstrated the inhibitory effect of estrogen on NO productions [45,48] our study is the first to our knowledge that showed the inhibitory effect of high physiological level of estradiol on NO production during local and systemic inflammation. The mechanism by which estradiol has differential effects on the different NO synthase isoforms is not well understood. Both nuclear and extra-nuclear pathways are used by estradiol to affect the NOS isoforms. However, the inhibitory effect of estradiol on iNOS may be due to the inhibition of cytokines production .
We found also significant differences between CLP and LI in central produced nitrite and IL6 (brain and pituitary), with higher production associated with LI. This finding could partial explain the previous noted LI induced higher HPA response and support the important role of central formed NO and Il6 on HPA response during local inflammation. However, we could not exclude the involvement of other factors such as the nociceptive afferent fiber [8,34] in the differences between local inflammation and systemic inflammation induced HPA axis responses. However, we were surprised that central rather than peripheral formed IL6 showed significant difference during local inflammation, this observation raise an important question that need further investigation: whether the peripheral inflammatory mediators or the neural afferent stimulation is responsible for the observed enhanced central inflammation during local inflammation?
From our study, we suggest that both estradiol level and the type of stress are important determinant factors for HPA response, and their effects on HPA could be mediated through IL6 and NO. However, further molecular study will provide additional mechanistic information and further support for the conclusions derived in this study.
The skillful technical assistance of Afaf, Aza and Tarek is appreciated.