Victor Chaban

*Charles R. Drew University of Medicine and Science and University of California, Los Angeles, USA* 

#### **1. Introduction**

366 Endometriosis - Basic Concepts and Current Research Trends

[26] Thomas EJ, Campbell G. Molecular genetic defects in endometriosis. Gynecol Obstet

[27] Sendometriosisino C, Sendometriosisino A, Pietra G, et al. Role of major

[28] Xin Wang, Chunyu Liu, Endometriosis [J]. People leptin gene cloning and sequence

genetic control of endometriosis. Fertil Steril, 1995,64:909-916.

histocompatibility complex class I expression and natural killer-like T cells in the

Invest, 2000,50(supl 1)44-50.

determined , 2002,37(6):346- 348.

Patients with chronic pelvic pain frequently have pain from several pelvic organs. The most common diagnoses include endometriosis, interstitial cystitis, irritable bowel disease, pelvic floor tension myalgia, vulvar vestibulitis, and vulvodynia. Frequently, pain does not correlate with pathologic findings at the time of laparoscopy in the case of endometriosis, while vulvodynia, irritable bowel syndrome and pelvic floor tension myalgia and neuropathy may have no clearly demonstrable pathologic tissue changes. Most diagnoses associated with chronic pelvic pain have a high rate of recurrence and all are considered to be chronic conditions with a relapsing course. Endometriosis is a complex, poorly understood chronic illness of women in their reproductive age and pain is the major concern of women with this disease. Despite a successful reduction of pain using during the novel treatments pain returns in up to 75% of treated women. Pain is strongly associated with this disease and the lack of awareness to its pathology is further illustrated by the fact that the average time duration between the onset of pain and the diagnosis of endometriosis is 3 to 11 years despite the fact that 25-30% of women with chronic pelvic pain suffer from this disease. In women with endometriosis (mainly of reproductive age) alterations in the limbic and sympathetic nervous system and hypothalamic-pituitary-adrenal axis mediate a cycle of hypervigilance for pain sensations from pelvic organs, which can lead to descending induction of pathologic changes in pelvic organs. Chronic pelvic pain patients frequently have multiple diagnoses. Vicero-somatic and viscero-viseral hyperalgesia and allodynia result in the spread of a perception of pain from an initial site to adjacent areas. Chronic pelvic pain patients may initially have only one pain source in the pelvis, such as the uterus in dysmenorrhea or endometriosis implants, but a multitude of mechanisms involving the peripheral and central nervous system can lead to the development of painful sensations from other adjacent organs. Often the etiology of visceral pain is not clear, as there are many symptoms of the reproductive system, gastrointestinal and urinary tracts, musculoskeletal, neurological and psychological systems that often co-occur in the same patient. The variation of pain symptoms and pain perception and behavioral responses to pain in these patients is poorly understood. The treating clinician is often tempted to take a unidimensional approach and focus on one organ system and ignore the psychological and behavioral manifestations of the chronic pain.

The incidence of persistent, episodic, or chronic visceral pain are more prevalent in females thus defining the site(s) and mechanisms through which female steroid hormones modulate

Primary Afferent Nociceptors and Visceral Pain 369

poorly understood how endometriosis is associated with pain symptoms in different organs and how this nociceptive signaling is ameliorated by a hypoestrogenic state. One of the possible explanation can be that endometrial implants' sensory nerve supply and its

Several researchers have investigated the presence of nerve fibers in endometriotic lesions in both human and animal study. Using different types of specific immunohistochemical neuronal markers such as substance P (SP) and calcitonin gene related peptide (CGRP) sensory nerve fibers markers) in human peritoneal endometriotic lesions from women with visually and biopsy proven endometriosis, investigators have demonstrated multiple, small unmyelinated nerve fibers are present in peritoneal endometriotic lesions, and these peritoneal endometriotic lesions contain both Aδ and C nerve fibers. Accumulating evidence has shown these nerve fibers may play a critical role in pain production in patients with endometriosis, and a close histological relationship has been identified between these nerve fibers and endometriosis associated pain. Tulandi *et al*. (2001) reported that the distance between endometriotic glands and nerve fibers in endometriotic lesions from women with pain was closer than in women with no pain. The density of nerve fibers in peritoneal endometriotic lesions was much greater than in normal peritoneum in women with no endometriosis. The nerve fiber density in endometriotic lesions can be markedly reduced by hormonal drugs such as gonadotropin releasing hormone (GnRH) analogues and combined oral contraceptives, which have been used efficaciously to treat endometriosis-associated pain, indicating that modulation of these nerve fibers might alter pelvic nociception. The fact that peritoneal endometriotic lesions are innervated by sensory Aδ, sensory C nerve fibers raises the intriguing questions, what kind of role do these nerve fibers play in the mechanisms by which endometriotic lesions produce pain and hyperalgesia, and how do

The demonstration of Aδ and C sensory fiber innervations to peritoneal endometriotic lesions, suggesting these innervations contributes to both visceral hyperalgesia and pelvic pain that occur in patients with endometriosis brings up the interesting questions, how do these sensory fibers transmit and modulate visceral nociception in endometriosis? Immunohistochemical staining of these nerve fibers in endometriosis showed co-localization of SP, CGRP, implicating SP and CGRP might be involved in modulation of visceral nociception. Endometriosis is an inflammatory disease, which is known to contain proinflammatory cytokines, prostaglandins, and other neuroactive agents that could readily activate the CGRP- and SP-positive C-fiber nociceptive afferents found in the endometriotic

When these sensory nerve fibers are stimulated by inflammatory substances, neurotransmitters such as SP, CGRP can be secreted from sensory nerve endings. SP and CGRP can contribute to the inflammatory response by causing vasodilation, plasma extravasation and cellular infiltration by interacting with endothelial cells, arterioles, mast cells, neutrophils and immune cells. SP can also act on mast cells in the vicinity of sensory nerve endings to evoke de-granulation and the release of TNF-α, histamine, prostaglandin D2 (PGD2) and leukotriene, providing a positive feedback. CGRP has a wide range of biological activities, including sensory transmission, regulation of glandular secretion, and inhibiting SP degradation by a specific endopeptidase to enhance SP release, thereby

potentially estradiol-modulated influence on the nociception.

they modulate pain perception in these condition?

lesions.

amplifying the effects.

visceral nociception is an important step in understanding the gender differences in pain perception and in designing appropriate therapies for females. One such mechanism may be the convergence of nociceptive stimuli and estrogen input on the primary afferent neurons which innervate viscera. Based on our results, it is likely that estrogen receptors (ERs) expressed in primary afferent neurons modulate nociceptive signaling. Our recent data suggest that estrogen acting on primary afferent nociceptors modulates the response to proand anti-nociceptive signals associated with the clinical presentation of functional disorders such as endometriosis.

#### **1.2 The nociception of endometriosis**

Endometrial tissues outside the uterus can cause severe pain and this pain can be diminished with therapies that suppress estrogen production (Berkley *et al*. 2005). The mechanism of endometriosis-induced nociceptive signaling is poorly understood and in some cases pain can be exacerbated by co-morbidity with other chronic pelvic pain syndromes such as irritable bowel syndrome, painful bladder syndrome, vulvodynia and fibromyalgia. It has also been shown that ectopic implants develop sensory nerve supply both in women and in animal models of endometriosis. Sensory input arriving from the visceral organ to the spinal cord divergences at the level of primary sensory neurons which further transmit considerable information from periphery to the central nervous system. Visceral pain may be manifestation from a single organ such as uterus or may arise from algogenic conditions affecting more than one organ (Malykhina 2007). This type of pain is important not only because it is difficult to diagnose its clinical conditions but also for its therapeutic implications. It is quite possible to modulate pain from one viscus to another. Recent study by Giamberardino and others showed that the treatment of the endometriotic lesions results in the improvement of spontaneous and referred urinary symptoms (Giamberardino *et al*. 2010).

Cross-sensitization in the pelvis implies the transmission of noxious stimuli from one organ to another through an adjacent normal structure resulting in functional (rarely organic) changes. Pelvic organ cross-sensitization is considered as one of the factors contributing to chronic pelvic pain (Pezzone *et al.* 2005). Chronic pelvic pain (CPP) syndrome affects up to 25% of reproductive age women and results in dysmenorrhea, menstrual irregularities, back pain and reduced fecundity. One of the most common causes of CPP is endometriosis. Chronic pain adversely affects mood, social and professional life and general well being. Thus, assessing the impact of the pain on various domains of a patient's existence has become an important focus in the clinical management. Most women with complaints of pelvic pain will undergo laparoscopy to both diagnose and treat these diseases, but laparoscopy is often is unsuccessful due to lack of intraperitoneal pathology or altered pain processing. Pain out of proportion to identifiable pathology is the most immediate and dramatic consequence of disorders associated with CPP and is responsible for a highly negative impact on quality of life and substantial workforce loss. Results of a national survey determined that 15% of women in the United States have experienced CPP and only 10% of these consulted a gynecologist and 75% did not consult a health care provider of any type. Due to the alarming situation and unmet need, the USA and other countries have launched a call for more focused research on improving the diagnosis and treatment of CPP syndrome.

There is often no clear relationship between the severity of the chronic pelvic pain and pathology in the pelvic viscera, including reproductive tract (ovaries and uterus). It is still

visceral nociception is an important step in understanding the gender differences in pain perception and in designing appropriate therapies for females. One such mechanism may be the convergence of nociceptive stimuli and estrogen input on the primary afferent neurons which innervate viscera. Based on our results, it is likely that estrogen receptors (ERs) expressed in primary afferent neurons modulate nociceptive signaling. Our recent data suggest that estrogen acting on primary afferent nociceptors modulates the response to proand anti-nociceptive signals associated with the clinical presentation of functional disorders

Endometrial tissues outside the uterus can cause severe pain and this pain can be diminished with therapies that suppress estrogen production (Berkley *et al*. 2005). The mechanism of endometriosis-induced nociceptive signaling is poorly understood and in some cases pain can be exacerbated by co-morbidity with other chronic pelvic pain syndromes such as irritable bowel syndrome, painful bladder syndrome, vulvodynia and fibromyalgia. It has also been shown that ectopic implants develop sensory nerve supply both in women and in animal models of endometriosis. Sensory input arriving from the visceral organ to the spinal cord divergences at the level of primary sensory neurons which further transmit considerable information from periphery to the central nervous system. Visceral pain may be manifestation from a single organ such as uterus or may arise from algogenic conditions affecting more than one organ (Malykhina 2007). This type of pain is important not only because it is difficult to diagnose its clinical conditions but also for its therapeutic implications. It is quite possible to modulate pain from one viscus to another. Recent study by Giamberardino and others showed that the treatment of the endometriotic lesions results in the improvement of spontaneous and referred urinary symptoms

Cross-sensitization in the pelvis implies the transmission of noxious stimuli from one organ to another through an adjacent normal structure resulting in functional (rarely organic) changes. Pelvic organ cross-sensitization is considered as one of the factors contributing to chronic pelvic pain (Pezzone *et al.* 2005). Chronic pelvic pain (CPP) syndrome affects up to 25% of reproductive age women and results in dysmenorrhea, menstrual irregularities, back pain and reduced fecundity. One of the most common causes of CPP is endometriosis. Chronic pain adversely affects mood, social and professional life and general well being. Thus, assessing the impact of the pain on various domains of a patient's existence has become an important focus in the clinical management. Most women with complaints of pelvic pain will undergo laparoscopy to both diagnose and treat these diseases, but laparoscopy is often is unsuccessful due to lack of intraperitoneal pathology or altered pain processing. Pain out of proportion to identifiable pathology is the most immediate and dramatic consequence of disorders associated with CPP and is responsible for a highly negative impact on quality of life and substantial workforce loss. Results of a national survey determined that 15% of women in the United States have experienced CPP and only 10% of these consulted a gynecologist and 75% did not consult a health care provider of any type. Due to the alarming situation and unmet need, the USA and other countries have launched a call for more focused research on

There is often no clear relationship between the severity of the chronic pelvic pain and pathology in the pelvic viscera, including reproductive tract (ovaries and uterus). It is still

such as endometriosis.

(Giamberardino *et al*. 2010).

improving the diagnosis and treatment of CPP syndrome.

**1.2 The nociception of endometriosis** 

poorly understood how endometriosis is associated with pain symptoms in different organs and how this nociceptive signaling is ameliorated by a hypoestrogenic state. One of the possible explanation can be that endometrial implants' sensory nerve supply and its potentially estradiol-modulated influence on the nociception.

Several researchers have investigated the presence of nerve fibers in endometriotic lesions in both human and animal study. Using different types of specific immunohistochemical neuronal markers such as substance P (SP) and calcitonin gene related peptide (CGRP) sensory nerve fibers markers) in human peritoneal endometriotic lesions from women with visually and biopsy proven endometriosis, investigators have demonstrated multiple, small unmyelinated nerve fibers are present in peritoneal endometriotic lesions, and these peritoneal endometriotic lesions contain both Aδ and C nerve fibers. Accumulating evidence has shown these nerve fibers may play a critical role in pain production in patients with endometriosis, and a close histological relationship has been identified between these nerve fibers and endometriosis associated pain. Tulandi *et al*. (2001) reported that the distance between endometriotic glands and nerve fibers in endometriotic lesions from women with pain was closer than in women with no pain. The density of nerve fibers in peritoneal endometriotic lesions was much greater than in normal peritoneum in women with no endometriosis. The nerve fiber density in endometriotic lesions can be markedly reduced by hormonal drugs such as gonadotropin releasing hormone (GnRH) analogues and combined oral contraceptives, which have been used efficaciously to treat endometriosis-associated pain, indicating that modulation of these nerve fibers might alter pelvic nociception. The fact that peritoneal endometriotic lesions are innervated by sensory Aδ, sensory C nerve fibers raises the intriguing questions, what kind of role do these nerve fibers play in the mechanisms by which endometriotic lesions produce pain and hyperalgesia, and how do they modulate pain perception in these condition?

The demonstration of Aδ and C sensory fiber innervations to peritoneal endometriotic lesions, suggesting these innervations contributes to both visceral hyperalgesia and pelvic pain that occur in patients with endometriosis brings up the interesting questions, how do these sensory fibers transmit and modulate visceral nociception in endometriosis? Immunohistochemical staining of these nerve fibers in endometriosis showed co-localization of SP, CGRP, implicating SP and CGRP might be involved in modulation of visceral nociception. Endometriosis is an inflammatory disease, which is known to contain proinflammatory cytokines, prostaglandins, and other neuroactive agents that could readily activate the CGRP- and SP-positive C-fiber nociceptive afferents found in the endometriotic lesions.

When these sensory nerve fibers are stimulated by inflammatory substances, neurotransmitters such as SP, CGRP can be secreted from sensory nerve endings. SP and CGRP can contribute to the inflammatory response by causing vasodilation, plasma extravasation and cellular infiltration by interacting with endothelial cells, arterioles, mast cells, neutrophils and immune cells. SP can also act on mast cells in the vicinity of sensory nerve endings to evoke de-granulation and the release of TNF-α, histamine, prostaglandin D2 (PGD2) and leukotriene, providing a positive feedback. CGRP has a wide range of biological activities, including sensory transmission, regulation of glandular secretion, and inhibiting SP degradation by a specific endopeptidase to enhance SP release, thereby amplifying the effects.

Primary Afferent Nociceptors and Visceral Pain 371

neurophysiological and pharmacological characteristics (Chang and Heitkemper 2002). The pathophysiology of visceral hyperalgesia is less well-known than its cutaneous counterpart, and our understanding of visceral hyperalgesia is colored by comparison to cutaneous hyperalgesia, which is believed to arise as a consequence of the sensitization of peripheral nociceptors due to long-lasting changes in the excitability of spinal neurons. Endometriosis is currently defined as a chronic functional syndrome characterized by recurring symptoms of abdominal discomfort or pain. In the context of visceral pain, the TRPV1 receptor is a sensory neuron-specific cation channel which plays an important role in transporting thermal and inflammatory pain signals. Evidence for TRPV1's role is that mice lacking TRP1 receptor gene have deficits in thermal- or inflammatory-induced hyperalgesia (Davis *et al*. 2000). Activation of both TRPV1 and P2X receptors induce mobilization of [Ca2+]i in cultured

Sex hormones and 17β-estradiol (E2) in particular may directly influence the functions of primary afferent neurons since both ERs are present on small-diameter DRG neurons (Papka and Storey-Workley 2002). Despite the broad spectrum of E2 effects in the nervous system, the mechanisms of hormonal pain modulation remain unclear. There are two subforms: estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) which were traditionally thought of as ligandactivated transcription factors. However, recent work has demonstrated multiplicity of E2 actions (membrane, cytoplasmic and nuclear) (Nadal *et al*. 2001). ER distributed through CNS and PNS including regions that mediate nociception. For example, ERs are expressed in dorsal horn neurons of the spinal cord and DRG neurons. DRG neurons express both ERα and ERβ *in vivo* (Papka and Storey-Workley 2002) and *in vitro* (Chaban 2010). These findings suggest that E2 may modulate sensory input at the primary afferent level. E2 can alter gene transcription, resulting in pro-nociceptive (reducing β-endorphin expression) or anti-nociceptive (increasing enkephalin expression) changes of endogenous opioid peptides , opioid receptors (Micevych and Sinchak 2001) and, by increasing levels of CCK, an anti-nociceptive and anti-opioid

E2 can modulate cellular activity by altering ion channel opening and second messenger signaling by stimulating G-proteins (Chaban *et al*. 2003) , the signal transduction pathways traditionally associated with membrane receptor activation. Many of these effects have been ascribed to membrane-associated receptors. The results from other laboratories (Lee *et al.* 2002) and our data (Chaban *et al*. 2003) indicate that E2 is acting to modulate L-type VGCC. The cloned TRPV1 receptor is a nonselective cation channel with a high permeability for Ca2+. TRPV1's are distributed in peripheral sensory nerve endings and are involved in the transduction of different stimuli in sensory neurons. TRPV1 functions as molecular integrator of painful chemical and physical stimuli (noxious heat (>43º C) and low pH). Various inflammatory mediators such as prostaglandin E2 (PGE2) and bradykinin potentiate TRPV1. The potentiation of TRPV1 activity can be quantified by measuring the differences of capsaicin-induced Ca2+ concentration changes before and after receptor activation (Petruska *et al*. 2000). Significantly, a subset of DRG neurons respond to both capsaicin and ATP indicating that there may be cross-activation of these receptors that may underlie the sensitization of visceral nociceptors. 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

DRG neurons (Gschossmann *et al.* 2000).

molecule (Micevych *et al.* 2002).

primary afferent nerve terminals (Burnstock 2001).

Dorsal root ganglion (DRG) neurons can be activated or modulated by the activation of chemosensitive receptors on peripheral terminals and ATP has been implicated in sensory transduction of noxious stimuli by activating purinergic P2X receptors (Dunn *et al.* 2001). Once released into the intercellular areas, the action of ATP is mediated by primarily P2X3 receptors which are expressed on primary afferent fibers and cell bodies within DRG (Burnstock 2001). The capsaicin-sensitive primary afferent neurons of small- and mediumdiameter neurons mediate nociceptive-like behaviors suggesting that TRPV1 expressing neurons are nociceptors. Activation of purinergic (P2X3) and transient potential receptors family vcanilloid-1 (TRPV1) receptors results in the depolarization and opening of voltagegated Ca2+channels (VGCC) (Koshimizu *et al.* 2000). A sensation of pain is produced by depolarization of the peripheral nerve terminals.
