**2.7 Retrograde labeling**

DRG neurons innervating viscera were identified by retrograde labeling. Briefly, mice were anesthetized with isoflurane. For colonic afferents, the descending colon was exposed and Fluorogold (5% solution in PBS; Molecular Probes, Eugene, OR) was injected into the intestinal muscle wall (10 μl injections of into five to six different sites) using a Hamilton syringe (Hamilton Co., Reno, NV) with a 26-gauge needle. In another experiments we used uterus-specific DRG neurons in which tetramethylrhodamine (TMR) dye was injected in the uterus. Injection sites were carefully swabbed, the colon and uterus were extensively rinsed with 0.9% sodium chloride solution and sealed with New Skin to prevent dye leakage. The abdomen was sutured and the animals monitored for signs of pain or discomfort during the survival period. All animals were allowed to survive one week to allow for maximal transport of retrograde markers and housed in groups of two under 12/12 hours light cycle with food and water available *ad libitum.*

#### **2.8 Statistical analysis**

The amplitude of [Ca2+]*i* response represents the difference between baseline concentration and the transient peak response to drug stimulation. Significant differences in response to chemical stimulation will be obtained by comparing [Ca2+]*i* increases during the first stimulation with the second. A cell will be judged responsive if E2 inhibits the second [Ca2+]*<sup>i</sup>* transient by >30% of the first. This criterion was empirically derived in preliminary experiments. All of the data are expressed as the mean ± SEM. Statistical analysis was performed using Statistical Package for the Social Sciences 12.0 (SPSS, Chicago, IL, USA). To assess the significance among different groups, data were analyzed with one-way ANOVA followed by Schéffe post hoc test. A *P* <0.05 was considered statistically significant.

#### **3. Results**

#### **3.1 Role of P2X3 receptors in estrogen-induced nociceptive signaling in sensory neurons**

P2X3 and TRPV1 receptors expression were examined by western blot analysis of lysates from wild type, ERαKO, and ERβKO DRG tissues using a P2X3 specific primary antiserum (Fig.1 (a)). An intense band representing a ~64 kDa protein (P2X3) and a ~130 kDa (TRPV1) was seen

Primary Afferent Nociceptors and Visceral Pain 377

In our next experiments we evaluated P2X3 receptors modulation by ATP and E2 in sensory neurons. DRG neurons were loaded with fluorescent dye Fura-2 AM for one hour at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4. The coverslips were placed on a stage of Olympus IX51 inverted microscope. A fast superfusion system was used to perfuse the cells with HBSS and rapidly apply E2 and other chemicals. Fluorescence intensity was captured as digital images (sampling rates of 0.1- 2s). Regions of interest were identified within the soma from which quantitative measurements were made by re-analysis of stored image sequences using Slidebook® Digital Microscopy software. [Ca2+]i was determined by

**E2 E2**

**+ ICI 182.780**

Fig. 3. 17 β-Estradiol (E2) significantly reduced ATP-induced [Ca2+]i signaling *in vitro*. This

0 500 1000 1500 2000 2500 **Time, Sec**

**ATP ATP**

**3.2 Role of TRPV1 receptors in estrogen-induced nociceptive signaling in sensory** 

We found that nociceptive (small diameter) DRG neurons also express capsaicin-sensitive vanilloid (TRPV1) receptors. TRPV1positive neurons were categorized according to their labeling patterns and were expressed as a percentage of the total number of TRPV1 -positive cells. Immunohistochemical signal from ERαKO, ERβKO and WT mice was measured by

effect was blocked by ER antagonist ICI 182 780

**ATP**

computerized image analysis (Fig. 4)

0

100

200

300

400

500

600

**neurons**

ratiometric method.

in DRG lysates from wild type animals. There was a dramatic decrease in intensity of this band using lysates made from the both knock out DRG tissues when compared with wild type control animals (>4 fold decrease of control Fig.1). When the density in the control group was standardized to 1.0, the average densities were 0.172 ± 0.08 of ERαKO and 0.262 ± 0.10 of ERβKO in P2X3 receptors, and 0.59 ± 0.06 of ERαKO and 0.391 ± 0.04 of ERβKO in TRPV1 receptors, suggesting that both P2X3 and TRPV1 protein decreased in DRG, P<0.05, n=10.

Fig. 1. Western blot analysis of DRG lysates shows reduced expression of P2X3 and TRPV1 in both knock-out mice.

Our study show that nociceptive capsaicin-sensitive TRPV1 receptors and ATP-sensitive P2X3 receptors express in DRG neurons. DRGs section were immunostained with primary antibodies against P2X3 and TRPV1. Neuronal profiles from each four mouse with ERαKO, ERβKO as well as wild type mice were quantified for each fluorescent probe. Both P2X3 and TRPV1 receptors present in DRGs (Fig. 2).

Fig. 2. Expression of P2X3 receptors in DRG neurons from wild type, ERαKO, and ERβKO *in vivo*

in DRG lysates from wild type animals. There was a dramatic decrease in intensity of this band using lysates made from the both knock out DRG tissues when compared with wild type control animals (>4 fold decrease of control Fig.1). When the density in the control group was standardized to 1.0, the average densities were 0.172 ± 0.08 of ERαKO and 0.262 ± 0.10 of ERβKO in P2X3 receptors, and 0.59 ± 0.06 of ERαKO and 0.391 ± 0.04 of ERβKO in TRPV1 receptors, suggesting that both P2X3 and TRPV1 protein decreased in DRG, P<0.05, n=10.

Fig. 1. Western blot analysis of DRG lysates shows reduced expression of P2X3 and TRPV1

Our study show that nociceptive capsaicin-sensitive TRPV1 receptors and ATP-sensitive P2X3 receptors express in DRG neurons. DRGs section were immunostained with primary antibodies against P2X3 and TRPV1. Neuronal profiles from each four mouse with ERαKO, ERβKO as well as wild type mice were quantified for each fluorescent probe. Both P2X3 and

Fig. 2. Expression of P2X3 receptors in DRG neurons from wild type, ERαKO, and ERβKO

in both knock-out mice.

*in vivo*

TRPV1 receptors present in DRGs (Fig. 2).

In our next experiments we evaluated P2X3 receptors modulation by ATP and E2 in sensory neurons. DRG neurons were loaded with fluorescent dye Fura-2 AM for one hour at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4. The coverslips were placed on a stage of Olympus IX51 inverted microscope. A fast superfusion system was used to perfuse the cells with HBSS and rapidly apply E2 and other chemicals. Fluorescence intensity was captured as digital images (sampling rates of 0.1- 2s). Regions of interest were identified within the soma from which quantitative measurements were made by re-analysis of stored image sequences using Slidebook® Digital Microscopy software. [Ca2+]i was determined by ratiometric method.

Fig. 3. 17 β-Estradiol (E2) significantly reduced ATP-induced [Ca2+]i signaling *in vitro*. This effect was blocked by ER antagonist ICI 182 780

#### **3.2 Role of TRPV1 receptors in estrogen-induced nociceptive signaling in sensory neurons**

We found that nociceptive (small diameter) DRG neurons also express capsaicin-sensitive vanilloid (TRPV1) receptors. TRPV1positive neurons were categorized according to their labeling patterns and were expressed as a percentage of the total number of TRPV1 -positive cells. Immunohistochemical signal from ERαKO, ERβKO and WT mice was measured by computerized image analysis (Fig. 4)

Primary Afferent Nociceptors and Visceral Pain 379

tracing we identified cutaneous and visceral afferent neurons *in vivo and in vitro*. We also found a subset of DRG neurons which innervate both visceral organs: uterus and

Endometriosis is one of the most common benign gynecological diseases, characterized by the presence of endometrial tissue outside the uterine cavity, most commonly implanted over visceral and peritoneal surfaces within the female pelvis. Clinical studies have shown that it may occur in up to 10-15% of women of reproductive age. Symptoms of endometriosis are usually associated with pelvic pain, including recurrent painful periods, painful intercourse, and painful defecation during menstruation, chronic lower abdominal pain and hypersensitivity. Unfortunately, understanding of the mechanisms of endometriosis-associated pain and its management in women is currently insufficient. Studies have shown possible mechanisms of chronic pelvic pain associated with endometriosis could be due to persistent nociceptive input from endometrial tissues that lead to peripheral and central sensitisation resulting in increased responsiveness of dorsal root ganglion and dorsal horn neurons. Indeed, recent studies in human and animals have shown that peritoneal endometrial lesions are richly innervated by Aδ and C sensory nerve fibers, which positively stained by substance P or calcitonin gene-related

Several lines of evidence indicated that there is a close relationship between nerve fiber density and endometriosis-associated pain. There is a significant increase in nerve fiber density in women with endometriosis who reported pelvic pain, suggesting these nerve fibers may play an important role in the mechanisms of pain generation. Accumulating literatures described that SP presents in the myometrium and is involved in the inflammatory and pain responses, suggesting a possible role of SP nerve fibers in the generation of pain related to endometriosis. SP, which is synthesized and contained in 20– 30% of DRG neurons, is involved in the transmission of nociceptive information to the central nerve system. SP is contained primarily in, and co-released from, small-diameter primary afferent fibers on noxious stimulation. Activation of nociceptive C and Aδ primary afferent fibers by electrical, chemical, or mechanical stimulation has been reported to release SP. Visceral nociceptive C-fibers can be activated by SP, representing an endogenous system regulating inflammatory, immune responses, and visceral hypersensitivity. SP afferent fibers play an important role in the pathogenesis of visceral hyperalgesia, suggesting critical role of SP in regulation of pelvic nociception associated with endometriosis. ATP is a peripheral mediator of pain which contributes to the activation of sensory afferents by activating ATP receptors following inflammation or nerve injury. It may correlate with SP release and play an important role in modulating nociception in primary sensory neurons. Local injections of ATP and ATP analogs to the rat hindpaw elicit spontaneous pain behaviors, hyperalgesia and allodynia which can be augmented by inflammation, indicating ATP might be involved in visceral hyperalgesia associated with endometriosis. Although these findings reveal the greater abundance of primary sensory nerve fibers clearly present within the peritoneal endometrial lesions in patients diagnosed with endometriosis, and these nerve fibers may play an important role

colon.

peptide.

**4. Conclusion** 

Fig. 4. Expression of TRPV1 receptors in dorsal root ganglion neurons from Wt, ERαKO, and ERβKO *in vivo*.

Capsaicin-induced TPRV1 receptor-mediated changes in [Ca2+]i may represent a level of DRG activation to noxious cutaneous stimulation while ATP-induced changes in [Ca2+]i may reflect the level of DRG neuron sensitization to noxious visceral stimuli since ATP is released by noxious stimuli and tissue damage near the primary afferent nerve terminals . In the view of this fact, TRPV1 receptor expression and activity might be considered as markers for a specific subtype of sensory neurons, and their activation by exogenous stimuli (e.g. capsaicin) could be a useful tool to exam the possible modulatory effects of pain-related substances.

#### **3.3 Primary afferent sensory neurons receive input from different visceral organs**

An important test of our hypothesis will be to establish that E2 modulates visceral afferents. A corollary of that hypothesis was that cutaneous pain may be differently modulated compared with visceral pain. We have proposed that E2 preferentially acts on visceral afferents to modulate the nociception. In a series of experiments using retrograde tract

Fig. 5. Identification of colon-specific (with FG), uterus-specific (with TMR) and DRG receiving input from both organs *in vitro* 

tracing we identified cutaneous and visceral afferent neurons *in vivo and in vitro*. We also found a subset of DRG neurons which innervate both visceral organs: uterus and colon.

## **4. Conclusion**

378 Endometriosis - Basic Concepts and Current Research Trends

Fig. 4. Expression of TRPV1 receptors in dorsal root ganglion neurons from Wt, ERαKO, and

Capsaicin-induced TPRV1 receptor-mediated changes in [Ca2+]i may represent a level of DRG activation to noxious cutaneous stimulation while ATP-induced changes in [Ca2+]i may reflect the level of DRG neuron sensitization to noxious visceral stimuli since ATP is released by noxious stimuli and tissue damage near the primary afferent nerve terminals . In the view of this fact, TRPV1 receptor expression and activity might be considered as markers for a specific subtype of sensory neurons, and their activation by exogenous stimuli (e.g. capsaicin) could be a useful tool to exam the possible modulatory effects of pain-related

**3.3 Primary afferent sensory neurons receive input from different visceral organs** 

FG staining TMR staining Double (FG/TMR) sta*i*ning

Fig. 5. Identification of colon-specific (with FG), uterus-specific (with TMR) and DRG

receiving input from both organs *in vitro* 

An important test of our hypothesis will be to establish that E2 modulates visceral afferents. A corollary of that hypothesis was that cutaneous pain may be differently modulated compared with visceral pain. We have proposed that E2 preferentially acts on visceral afferents to modulate the nociception. In a series of experiments using retrograde tract

ERβKO *in vivo*.

substances.

Endometriosis is one of the most common benign gynecological diseases, characterized by the presence of endometrial tissue outside the uterine cavity, most commonly implanted over visceral and peritoneal surfaces within the female pelvis. Clinical studies have shown that it may occur in up to 10-15% of women of reproductive age. Symptoms of endometriosis are usually associated with pelvic pain, including recurrent painful periods, painful intercourse, and painful defecation during menstruation, chronic lower abdominal pain and hypersensitivity. Unfortunately, understanding of the mechanisms of endometriosis-associated pain and its management in women is currently insufficient. Studies have shown possible mechanisms of chronic pelvic pain associated with endometriosis could be due to persistent nociceptive input from endometrial tissues that lead to peripheral and central sensitisation resulting in increased responsiveness of dorsal root ganglion and dorsal horn neurons. Indeed, recent studies in human and animals have shown that peritoneal endometrial lesions are richly innervated by Aδ and C sensory nerve fibers, which positively stained by substance P or calcitonin gene-related peptide.

Several lines of evidence indicated that there is a close relationship between nerve fiber density and endometriosis-associated pain. There is a significant increase in nerve fiber density in women with endometriosis who reported pelvic pain, suggesting these nerve fibers may play an important role in the mechanisms of pain generation. Accumulating literatures described that SP presents in the myometrium and is involved in the inflammatory and pain responses, suggesting a possible role of SP nerve fibers in the generation of pain related to endometriosis. SP, which is synthesized and contained in 20– 30% of DRG neurons, is involved in the transmission of nociceptive information to the central nerve system. SP is contained primarily in, and co-released from, small-diameter primary afferent fibers on noxious stimulation. Activation of nociceptive C and Aδ primary afferent fibers by electrical, chemical, or mechanical stimulation has been reported to release SP. Visceral nociceptive C-fibers can be activated by SP, representing an endogenous system regulating inflammatory, immune responses, and visceral hypersensitivity. SP afferent fibers play an important role in the pathogenesis of visceral hyperalgesia, suggesting critical role of SP in regulation of pelvic nociception associated with endometriosis. ATP is a peripheral mediator of pain which contributes to the activation of sensory afferents by activating ATP receptors following inflammation or nerve injury. It may correlate with SP release and play an important role in modulating nociception in primary sensory neurons. Local injections of ATP and ATP analogs to the rat hindpaw elicit spontaneous pain behaviors, hyperalgesia and allodynia which can be augmented by inflammation, indicating ATP might be involved in visceral hyperalgesia associated with endometriosis. Although these findings reveal the greater abundance of primary sensory nerve fibers clearly present within the peritoneal endometrial lesions in patients diagnosed with endometriosis, and these nerve fibers may play an important role

Primary Afferent Nociceptors and Visceral Pain 381

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#### **5. References**


in pain generation associated with endometriosis, pain mechanisms associated with endometriosis are still not well known, and the role of these primary sensory nerve fibers

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**5. References** 


**21** 

*Slovenia* 

**Embryo Quality and Pregnancy Outcome in** 

Investigation of one complex pathological condition is definitively a challenging task, but trying to find the connection(s) between two is even more difficult. This is very true in the case of infertility and endometriosis. Both conditions have numerous symptoms, very diverse clinical pictures and multifactorial etiologies. The first step toward understanding the connection between the two is to prove the correlation between them. The next task is to try to understand the mechanisms by which they affect each other, which involves examination and comparison of numerous variables specific for each condition. The results

At present, there is little debate that endometriosis and infertility are actually associated. For example, early retrospective studies (Hasson, 1976; Drake & Grunet, 1980; Strathy et al., 1982) and one more recent prospective study (Mahmood & Templeton, 1991) performed in women who underwent laparoscopy (for various reasons) showed that endometriotic lesions were significantly more frequent in women who were treated for infertility than in those who requested laparoscopy for tubal sterilization. Prevalence of endometriosis in infertile women ranged from 21 - 48% which was in clear contrast to the prevalence of 1.3 – 5% in fertile women (Hasson, 1976; Drake & Grunet, 1980; Strathy et al., 1982). Another line of evidence of the existence of a link between endometriosis and infertility came from studies in women who underwent donor insemination because of severe male infertility. In these studies, women with endometriosis had significantly fewer conceptions per procedure than women without this condition (Hammond et al., 1986; Yeh & Seibel, 1987). In studies where peritoneal endometriosis was induced in rabbits (Schenken & Asch, 1980), primates (Schenken et al., 1984) and rodents (Vernon & Wilson 1985; Barragan et al., 1992) it was clearly demonstrated that endometriosis was strongly associated with infertility regardless

While it is relatively easy to document the link between the two conditions, defining precise pathophysiologic mechanisms and proving a causal relationship between endometriosis and

**1. Introduction** 

are still the subject of many controversies.

of localization and/or extension of the lesions.

infertility is much more difficult.

**1.1 Endometriosis and infertility** 

**Infertile Patients with Endometriosis** 

Veljko Vlaisavljević, Marko Došen and Borut Kovačič *Department of Reproductive Medicine and Gynecologic Endocrinology, Clinics for Gynecology and Perinatology, University Clinical Center Maribor,* 

