Estrogen re-enhanced prenatal stress-related visceral sensitization and pain regulation

Background: Visceral pain is one of the most common sign of irritable bowel syndrome (IBS). Chronic stress during pregnancy may increase visceral pain sensitivity of offspring in a sexdependent way. Combining adult stress in offspring will increase this sensitivity. Based on the evidence implicating estrogen exacerbates visceral hypersensitivity in female rodents in pre-clinical models, we predicted that chronic prenatal stress (CPS) plus chronic adult stress (CAS) will maximize visceral pain sensitivity; and estrogen plays an important role in this hyperalgesia. Methods: The CPS plus CAS rodent model was established in which the balloon was used to distend colorectum. Meanwhile, the single ber recording in vivo and patch-clamp experiments in vitro were used to monitor neuronal activity. The RT-PCR, Western Blot, and Immunouorescence were used to study the effects of CPS and CAS on colon primary afferent sensitivity and molecular or transmission changes. We use Ovariectomy and Letrozole to treate female rats respectively in order to assess the role of estrogen in female-specic enhanced primary afferent sensitization. Letrozole mainly used to reduce estrogen levels. Results: As predicted, CPS signicantly increased single unit afferent ber activity in L6-S2 dorsal roots in response. Activity was further enhanced by CAS. And the activity in offspring females was signicantly greater than the males. Besides, the excitability of colon-projecting dorsal root ganglion (DRG) neurons increases in CPS + CAS rats that was associated with a decrease in transient A-type K+ current. Letrozole treatment decreases the colon DRG neuron excitability in females by decreasing the estrogen levels. Conclusions: This study adds to the growing evidence for the development of chronic stress induced visceral hypersensitivity in female, which involves estrogen-dependent sensitization of primary afferent colon neurons. Understanding this neurophysiological mechanisms will spur the development of female pain specic therapies.


Background
Visceral pain of colonic origin is the most prominent symptom in irritable bowel syndrome (IBS) patients [1]. Female IBS patients report more severe pain that occurs more frequently and with longer episodes than in male patients [1,2]. The ratio of female to male IBS is about 2:1 among patients seen in medical clinics [3]. Moreover, females show a higher prevalence of IBS co-morbidities such as anxiety and depression [4][5][6] and are more vulnerable to stress-induced exacerbation of IBS symptoms compared to males [3,7,8].
Clinical studies show that early life adverse experiences are risk factors for the development of IBS symptoms, including visceral pain and ongoing chronic stress, especially abdominal pain [9][10][11][12]. These factors contribute to the development of visceral hypersensitivity, a key component of the IBS symptom complex and one that may be responsible for symptoms of pain [13,14]. Our previous research found that the female offspring of mothers subjected to chronic prenatal stress (CPS) show a markedly greater visceral sensitivity than their male littermates following challenge by another chronic adult stress (CAS) protocol. A critical molecular event in the development of this female-enhanced visceral hypersensitivity is up-regulation of brain-derived neurotrophic factor (BDNF) expression in the lumbar-sacral spinal cord of female CPS + CAS rats [15]. However, the neurophysiological changes underlying this enhanced female-speci c visceral hypersensitivity and the role of hormone in the development of stress-induced visceral hypersensitivity are not well understood.
Visceral hypersensitivity in IBS involves abnormal changes in neurophysiology throughout the brain-gut axis. In IBS, there is evidence for sensitization of primary afferents to jejunal distention and electrical stimulation [16], and there is evidence for increased sensitivity of lumbar splanchnic afferents [17,18]. In animal models of either early life adverse events [19] or adult stress induced visceral hypersensitivity [20], there is evidence of colon primary afferent sensitization. However, these studies are performed in male rodents. Therefore, in this study, we established a CPS and CAS rodent model to analyse the impact on female colon afferent neuron function and the role of estrogen. Our hypothesis is that female CPS offspring subjected to chronic stress as adults exhibit greater colonic DRG neuron sensitization compared to their male littermates and that this female enhanced visceral sensitization and primary afferent sensitization are estrogen dependent.

Animals
Experiments were performed on pregnant Sprague-Dawley rats and their 8-16 week old male and female offspring.

CPS and CAS models
Pregnant dams were subjected to a CPS protocol that consisted of a random sequence of twice-daily applications of one of three stress sessions, one hour water avoidance stress, 45 min cold restraint stress or 20 min forced swim stress starting on 6th day and continuing until delivery (21st day). Male and female offspring from the stressed dams were designated CPS rats. Control dams received sham stress and their offspring were designated control rats. As adults (8-16 wks), control and prenatally stressed offspring were challenged by the same CAS protocol for nine days. Ovariectomy (OVX) or sham surgery was performed on female prenatal stress offspring in the 56th day. Daily Letrozole treatment was initiated on the 49th day, 2 weeks prior to initiation of adult stress. Treatment was continued through the stress protocol. Detail schematic diagram is present in Fig. 1A.

Rattreatment
Before the treatment of OVX or Letrozole, Vaginal smear test was used to identify the estrus cycle phases of female rats. The OVX or sham surgery was performed on female prenatal stress offspring in the 56th day. The aromatase inhibitor Letrozole (4,4'-(1H-1,2,4-triazol-l-yl-methylene)-bis-benzonitrile; 1.0 mg/kg, oral administration; Novartis) was used in experiment group; and Vehicle (hydroxypropyl cellulose 0.3% in water) was used in contral group once daily for 14 days. Direct transcutaneous intrathecal injections (i.t.) of estrogen and letrozole were performed respectively as described by Mestre et al. [21] In vivo single ber recording of L6-S2 DRG rootlets Multiunit afferent discharges were recorded from the distal ends of L6-S2 dorsal rootlets decentralized close to their entry into the spinal cord. A bundle of multiunit bers was distinguished into 2-6 single units off-line using wave mark template matching in Spike 2 software that differentiates spikes by shape and amplitude. Colonic afferent bers were identi ed by their response to graded colorectal distention (CRD). Iso urane, 2.5%, followed by 50 mg/kg, i.p. sodium pentobarbital induced general anesthesia that was maintained by infusing a mixture of pentobarbital sodium + pancuronium bromide + saline by intravenous infusion through the tail vein. Adequacy of anesthesia was con rmed by the absence of corneal and pupillary re exes and stability of end-tidal CO 2 level. A tracheotomy tube connected to a ventilator system provided a mixture of room air and oxygen. Expired CO 2 was monitored and maintained at 3.5%. Body temperature was monitored and maintained at 37 °C by a servo-controlled heating blanket. A laminectomy from T12 to S2 exposed the spinal cord. The head was stabilized in a stereotaxic frame.
The dura was gently opened and a warm mineral oil pool, contained by skin aps, covered the exposed spinal cord and roots as described previously [22].
Recording pipettes, pulled from borosilicate glass tubing, with resistance of 1-5 MΩ, were lled with solution containing (in mM): 100 KMeSO 3 , 40 KCl, and 10 HEPES, pH 7.25 adjusted with KOH (290 mOsm). DiI labeled neurons were identi ed under uorescent microscope. Whole-cell currents and voltage were recorded from DiI-labeled neurons using Dagan 3911 patch clamp ampli er. Data were acquired and analyzed by pCLAMP 9.2 (Molecular Devices, Sunnyvale, CA). The currents were ltered at 2-5 kHz and sampled at 50 or 100s per point. While still under voltage clamp, the Clampex Membrane Test program (Molecular Devices) was used to determine membrane capacitance, Cm and membrane resistance, Rm, during a 10 ms, 5 mV depolarizing pulse form a holding potential of -60 mV. The con guration was then switched to current clamp (0 pA) for determining other electrophysiological properties. After stabilizing for 2-3 min, resting membrane potential was measured. The minimum acceptable resting membrane potential was -40 mV. Spontaneous activity (SA) was then recorded over two 30 second periods separated by 60 s without recording as described by Bedi and Chen [24].

Transient A-type K + current (I A ) recording method in Patch studies
To record voltage-gated K + current (Kv), Na + in control external solution was replaced with equimolar choline and the Ca 2+ concentration was reduced to 0.03 mM to suppress Ca 2+ currents and to prevent Ca 2+ channels becoming Na + conducting. The reduced external Ca 2+ would also be expected to suppress Ca 2+ -activated K + current. The current traces of Kv in DRG neurons were measured at different holding potentials. The membrane potential was held at -100 mV and voltage steps were from -40 to +30 mV to record the total Kv. The membrane potential was held at -50 mV to record the sustained Kv. The I A currents were calculated by subtracting the sustained current from the total current. The current density (in pA/pF) was calculated by dividing the current amplitude by cell membrane capacitance.

Real time RT-PCR
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA). One microgram of total RNA was reverse-transcribed using SuperScript TM III First-Strand Synthesis System. PCR was performed on a StepOnePlus thermal cycler with 18S as the normalizer using Applied Biosystems primer/probe set Rn02531967_s1 directed against the translated exon IX. Fold-change relative to control was calculated using the ΔΔCt method (Applied Biosystems).

Western Blot
Samples were lysed in RIPA buffer containing protease inhibitor cocktail and phenylmethanesulfonyl uoride. Lysates were incubated for 30 min on ice and then centrifuged at 10 000×g for 10 min at 4 °C.
The protein concentration in the supernatant was determined using BCA kits with bovine serum albumin as a standard. Equal amounts of protein (30 μg per lane) were separated with 10% SDS-PAGE and then transferred to nitrocellullose membranes (Bio-Rad, USA). The membrane was blocked in Li-Cor blocking buffer for 1 h at room temperature and then incubated with primary antibodies. BDNF antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) was used at 1:200 dilution; Nerve growth factor (NGF) antibody (Abcam, MA) was used at 1:1000 dilution; β-actin antibody (Sigma Aldrich, St Louis) was used at 1:5000 dilution. Secondary antibodies used were donkey anti-rabbit alexa uor 680 (Invitrogen) and goat antimouse IRDye 800 (Rockland). Images were acquired and band intensities measured using the Li-Cor Odyssey system (Li-Cor, Lincoln, Nebraska).

Immuno uorescence
Frozen sections of colon mounted on glass slides from control, CAS, CPS and CPS + CAS female rats were rehydrated in phosphate buffered saline at room temperature. All slides were treated for antigen retrieval and blocked with 10% normal goat serum (diluting in 0.3% phosphate buffered saline-Triton) for 1 h. Primary antibody NGF in antibody diluent (Renoir Red, Biocare Medical, Concord, CA) were incubated at 4 o C overnight. The slides were exposed to uorescent dye conjugated secondary antibody for 2 h at room temperature. Slides were counterstained with DAPI and coverslipped. Images were taken in uorescence mode on an Olympus laser scanning confocal microscope and the average signal intensity was calculated by the bundled software.

Data analyses
Single ber responses (impulses/second) to CRD were calculated by subtracting SA from the mean of 30 seconds of the maximal activity during distension. Fibers were considered responsive when CRD increased their activity 30% greater than the baseline value. Mechanosensitive single units were classi ed into high-threshold (>20 mmHg) and low-threshold (≤20 mmHg) on the basis of their response threshold and pro le during CRDs. Single ber activity data were analyzed using ANOVA with repeated measures; CRD intensity was the repeated factor and experimental group as the between group factor. If signi cant main effects were present, the individual means were compared using the Fisher post-hoc test.

Results
Effects of CPS plus CAS on primary afferent responses to CRD in male and female rats The basal activity of a spinal afferent ber was de ned as the average number of action potentials per second (impulses/sec) in the 60 second period before the onset of a distention stimulus. 66% of the afferent bers under study displayed SA in male controls. In the female controls, SA was signi cantly greater than control males, 0.71 ± 0.21 vs 1.24 ± 0.20 imp/sec (Fig. 1B). The average single ber activity in response to CRD was signi cantly higher in female control rats compare to male controls (Fig. 1C). We found that the enhanced sensitization in female rats mainly came from the low threshold bers (Fig. 1D,   E).
To assess the effects of CPS + CAS on colon afferent ber activities, we compared average single colon afferent ber activities projecting from dorsal roots S1-L6 in response to CRD in male and female control, CPS, control + CAS and CPS + CAS rats recorded approximately 24 hours after the last stressor. Within females, CPS signi cantly increased single unit afferent activities in response to CRD vs control female rats (Fig. 1F). CAS alone enhanced single unit activities compared to control. The average afferent responses after CAS in female prenatally stressed rats were signi cantly greater (44.0% increase rate compare to female control and 39.3% increase rate compare to CAS only female rats). Within males, CPS had no signi cant effect on primary afferent responses (Fig. 1G). When we compared males to females within each experimental group, we found that average single ber activities were signi cantly higher in female compared to male CPS + CAS rats (Fig. 1F, G). This greater activity may contribute to the enhanced female visceral hypersensitivity previously reported in this model. Average single ber activity of control, CPS and CAS females were signi cantly greater than their respective male experimental groups (Fig. 1F, G). Both CAS and CPS + CAS signi cantly increased primary afferent responses compared to control and to CPS. Thus, our CPS and CAS protocols generally produce sensitization of colon projecting primary afferent bers with greatest effects produced by the combination of CPS + CAS in both males and females.

Increase in Excitability of Colon-projecting Lumbosacral DRG Neurons from female CPS + CAS rats
To elucidate the electrophysiological basis for enhanced stress-induced primary afferent activity in female rats, we performed patch clamp studies on acutely dissociated retrograde labelled colon projecting neurons from the L6-S2 DRG from control, prenatal stress, adult stress only and CPS + CAS female rats isolated 24 hours after the last adult stressor (Fig. 2A). Input resistance (Fig. 2C) and rheobase (Fig. 2D) were signi cantly decreased in neurons from CPS + CAS rats compared to the other three groups. The number of action potentials elicited at either 2X or 3X the rheobase were signi cantly greater in adult stress and CPS + CAS neurons compared to control and to CPS neurons (Fig. 2B, E). CAS signi cantly increased action potential overshoot with or without CPS (Fig. 2F), but it did not signi cantly alter the other electrophysiological characteristics, such as spontaneous spike number, membrane capacitance (pF), resting membrane potential, cell diameter, time constant, action potential amplitude, and duration of DRG neurons (Table 1). The value equal means ± SE. Statistical signi cance is indicated by *(P < 0.05), **(P < 0.01) and ***(P < 0.001) compared with the control group. n = number of observations.
There were a signi cantly greater percentage of neurons displaying SA in CPS + CAS rats vs. control or CPS only rats (Fig. 2G). Under voltage-clamp conditions, neurons from female CPS + CAS, CAS, CPS and control groups exhibited I A and sustained outward recti er K + currents (I K ) (Fig. 2H). Compared with the other three groups, DRG neurons from CPS + CAS rats demonstrated a signi cantly reduced average I A (p < 0.05) (Fig. 2H). However, the average I K density although decreased was not altered signi cantly.

Effects of CPS and/or CAS on plasma estrogen concentrations
We did the vaginal smear test to identify the estrus cycle phases by identifying the Vaginal Cytological cell types. Estrogen concentration was signi cantly higher in CPS proestrus/estrus phase compared to control diestrus, control proestrus/estrus and CPS diestrus porestrus (p < 0.05; Fig. 3A). Comparison of the plasma estrogen concentrations in control, CAS, CPS, CPS + CAS showed that CPS signi cantly increased plasma estrogen levels compared to the control rats and that CAS increased plasma estrogen level compared to the control and CPS rats (Fig. 3B).
To determine whether estrogen contributed to stress-induced visceral hypersensitivity in prenatal stressed females, we reduced plasma estrogen levels with either OVX or Letrozole treatment. OVX signi cantly lowered serum estradiol levels before and after CAS (Fig. 3C). Treatment was continued throughout CAS.
After treatment with Letrozole, serum estradiol levels were signi cantly reduced (Fig. 3D). To study the effects of gender and stress on norepinephrine and ACTH levels, we measured plasma norepinephrine levels in female rats from all four experimental groups. CAS alone signi cantly increased plasma norepinephrine levels compared to control and to CPS alone (Fig. 3E) Plasma norepinephrine levels were signi cantly increased in CPS + CAS rats compared to CAS alone as well as to control and CPS. Plasma ACTH levels were signi cantly increased in CPS + CAS rats compared to control. (Fig. 3F).

Effects of Letrozole treatment on colon DRG neuron excitability
We performed patch clamp experiments on acutely isolated retrograde labelled DRG neurons from CPS + CAS females with or without letrozole treatment 24 hours after the last adult stressor. Letrozole treatment signi cantly increased rheobase (Fig. 4A), and signi cantly reduced input resistance (Fig. 4B). Action potential overshoot (Fig. 4C) and the number of action potentials elicited by a current injection at either 2X or 3X rheobase were signi cantly reduced by letrozole treatment (Fig. 4D). Other electrophysiological properties were not signi cantly altered ( Table 2). We also recorded Electromyographic activity to determine whether the reduction in visceral sensitivity in female CPS + CAS rats caused by OVX or systemic Letrozole treatment can reduced visceromotor response. We found that the Electromyographic of CRD was signi cantly reduced in OVX rats after 24 hours after the last adult stressor ( Supplementary  Fig. 1A) or in systemic Letrozole treated rats compared to vehicle both before and after CAS ( Supplementary Fig. 1B). These ndings demonstrated a signi cant decrease in excitability of colon projecting L6-S2 neurons. The value equal means ± SE. Statistical signi cance is indicated by *(P < 0.05), **(P < 0.01) and ***(P < 0.001) compared with the control group. n = number of observations.

Spinal cord BDNF levels regulated by estrogen
To investigate the effect of estrogen on BDNF expression, we measured BDNF mRNA and protein levels in lumbar sacral spinal cord of OVX and Sham CPS + CAS female rats. Systemic estradiol administration to naïve cycling females produced signi cant increases in plasma estrogen (Fig. 5A), lumbar sacral spinal cord BDNF mRNA (Fig. 5B) and protein (Fig. 5C). We also measured BDNF mRNA and protein levels in lumbar sacral spinal cord of OVX and Sham CPS + CAS female rats. BDNF mRNA and protein expression were signi cantly suppressed by OVX compared to Sham rats ( Supplementary Fig. 1C). Another experiment showed that intrathecal infusion of estrogen into naïve female rats signi cantly increased BNDF protein levels, which proved that estrogen can reverse the experimental results and contribute to the response to visceral pain ( Supplementary Fig. 1D).
Peripheral NGF level increased in CPS + CAS female rats We examined NGF expression in the colon in females from all four experimental groups by immunohistochemistry (Fig. 6A). Morphometric analysis showed that CAS and CPS + CAS signi cantly increased NGF levels in the colon wall with the increase in CPS + CAS signi cantly greater that of CAS alone (Fig. 6B). Western blotting showed that NGF protein was signi cantly up-regulated in CPS + CAS compared to controls (Fig. 6C).

Discussion
Our enhanced chronic stress-induced visceral hypersensitivity in female prenatally rats is associated with an increase in the responses of lumbosacral nerve bers to CRD in both male and female rats. These ndings are further supported by our data showing increased excitability of colon projecting DRG neurons from females in patch clamp studies. The magnitude of this sensitization is greatest in female CPS + CAS rats suggesting that this sensitization makes a major contribution to the observed enhanced female visceral hypersensitivity in our model. We focused on lumbar sacral afferent bers and dissociated neurons in patch clamp studies. It is possible TL neurons also contribute (CAS in males sensitizes TL neurons).
Chronic stress is known to increase the excitability of colon projecting DRG neurons in rats and mice. In adult male Sprague Dawley rats, colon DRG neuron sensitization was driven by increases in NGF expression in the colon muscularis externa [20]. In our model we observe a signi cant increase in colon NGF, but its potential role in primary afferent sensitization and visceral hypersensitivity was not investigated in this study. Other studies in male mice show that stress in the form of water avoidance stress signi cantly increases excitability of colon projecting DRG neurons and that stress mediators corticosterone and norepinephrine working together can increase DRG neuron excitability in vitro [25,26]. Neonatal maternal deprivation sensitizes colon projecting neurons in adult males [27]. In our study, we nd signi cant increases in serum levels of norepinephrine in CPS + CAS females. However, daily systemic treatment with adrenergic antagonists during the adult stress protocol failed to reduce visceral hypersensitivity in female newnatal + adult stress rats [20] suggesting that norepinephrine did not play a major role in the acquisition of enhanced female visceral hypersensitivity or primary afferent sensitization in our model. We nd signi cant decreases in the transient potassium I A current in neurons isolated from CPS + CAS females compared to the other three experimental groups. Declines in A type Kv currents in DRG neurons are associated with persistent pain in multiple chronic pain models [28]. Whether this decline is caused by changes in channel properties or expression was not investigated in this study. However, another study demonstrates that estrogen signi cantly shifted the activation curve for I A current in the hyperpolarizing direction and that estrogen inhibited Kv (+) channels in mouse DRG neurons through a membrane ERactivated non-genomic pathway [29].
Our results show that excitability of colon projecting neurons in CPS + CAS females is signi cantly reduced by systemic letrozole treatment suggesting that estrogen contributes to this sensitization process. Previous studies show that estrogen receptors expressed on primary afferent neurons contribute to enhanced sensitivity in various pain models [30][31][32][33]. One study nds no decline in the responses of colon projecting nerve bers to CRD following OVX and nds no detectable estrogen receptor alpha immunoreactivity in colon projecting DRG neurons [34]. The reasons for these differing results are not clear although local production of estrogen in DRG neurons could be su cient to sustain sensitization.
NGF and its receptors play important roles in the mechanism of visceral pain and hyperalgesia in woman.
For example, the endometriosis is an estrogen-dependent and commonly encountered disease in women. The main symptoms are various types of pelvic pain which have a serious effect on women's physical and mental health, but the mechanisms of pain are still unclear. Recent studies showed that NGF can promote neural cell proliferation and differentiation, induce the expression and release of neuropeptides and increase the number of sensory neurons through binding nerve cell receptor.

Conclusion
In this study, we examine the difference of sex and effects estrogen on the acquisition of enhanced visceral hypersensitivity in femal CPS + CAS rats. Our study shows that estrogens act in the spinal cord and the primary afferent neurons to enhance visceral nociception. Acute blockade of the endogenous synthesis of estrogens in the rat spinal cord may signi cantly reduce visceral hypersensitivity, suggesting that locally produced estrogen can regulate nociceptive neurons to modulate visceral hypersensitivity. Chronic stress-estrogen-BDNF axis sensitizes visceral hypersensitivity in female offspring subjected to CPS. The development of chronic stress induced visceral hypersensitivity in female rats is estrogen dependent. A key component of this hypersensitivity is estrogen dependent sensitization of primary afferent colon neurons. Our ndings provide key scienti c evidence in a preclinical model in support of developing gender-based treatment for pain in IBS.
We investigated herein whether estrogen re-enhanced visceral pain sensitivity in chronic prenatal stress (CPS) plus chronic adult stress (CAS) rodent models. After using physical Ovariectomy (OVX) or chemical inhibitor Letrozole treatment to reduce estrogen levels, we nd that the visceral hyperalgesia, colonic afferant neuronal excitability, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and Estrogen all were increased. The ndings indicate that the chronic stress induced visceral hypersensitivity is estrogen dependent and the hypersensitivity is estrogen dependent sensitization of primary afferent colon neurons, which provide key scienti c evidence in a preclinical model in support of developing gender-based pain management.
Abbreviations IBS: irritable bowel syndrome; CPS: chronic prenatal stress; CAS: chronic adult stress; DRG: dorsal root ganglion; BDNF: brain-derived neurotrophic factor; OVX: ovariectomy; CRD: colorectal distention; SA: Spontaneous activity; I A : transient A-type K + current; Kv: voltage-gated K + current; NGF: nerve growth factor; ACTH: adrenocorticotropic hormone Declarations Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional supplementary les.