Chapter 2 Endometriosis Associated Pain

 

2.1 Chapter Overview

2.2 Pain

Pain, as defined by the International Association for the Study of Pain (IASP), is “any unpleasant sensory or emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (Merskey et al., 1979). [Merskey, 1986, Merskey and Bogduk 1994] The IASP definition of pain amalgamated disparate concepts of pain, proposed down through the ages.

Historical theories of pain have generally focused on pain being conducted along specific pathways with man behaving like an automaton in response to a painful triggering event. This view does not fully support the potential complexities of the experience of pain.  More recent theories attempt to account for normal and pathological types of pain, generally proposing a reasonably complex matrix of communication orchestrated by central processes; both in response to, and influencing of, neuronal and non-neuronal cells within peripheral regions of the body.

2.2.1 Historical Theories of Pain

Aristotle (384 BC-322 BC) advanced Plato’s (424 BC-348 BC) philosophical teachings of the daily interplay between thoughts and emotions driving behavior to originate the idea that pain (and pleasure) is a ‘passion of the soul’. The emotion of pain did not belong to the five senses: vision, hearing, taste, touch and smell.

Hippocrates (460 BC-370 BC), widely acknowledged as the Father of Medicine, reasoned that disease had an organic origin rather than a mystical one. With detailed observation, he concluded pain was an imbalance of bodily fluids (Rey, 1993, Cervero, 2009, Redwine, 2012).

Figure 2.1 Descartes’ Theory of Pain (Basbaum AI and Bushnell C, 2009)

In 1664, René Decartes published Trait de l’homme (The Treatise of Man) positing that man is like a machine with defined pathways of cause and effect. When provoked by a stimulus, a fine thread opens a valve in the brain that allows the spirit to enter or leave body cavities resulting in protective behaviour (Figure 2.1). The Cartesian view of pain was revolutionary as it introduced the brain as a central component rather than the heart or soul, as proposed by Aristotle.

2.2.2 Modern Theories of Pain

The Cartesian model of pain was influential from its inception. It was advanced in 1811 when Sir Charles Bell (1774 – 1842) deduced, from animal studies, the existence of special sensory receptors configured to respond to one type of stimulus. His publication outlined the fundamental tenets of what became known as the Specificity Theory (Rey, 1995). Johannes Muller (1801 – 1858) furthered this notion in 1839 by divining that the sensory receptors, irrespective of the type of stimulation, sent a message to a particular part of the brain. It was the activation of a specific group of neurons, allocated to a particular sense organ, which gave rise to a specific sensation, including pain. Consistent with this idea of pain, Moritz Schiff (1823 – 1896) in 1858, demonstrated that touch and pain were transmitted along different spinal cord pathways. Schiff formulated the view that pain, as a sense, had its own neural conduits, independent of the other senses. Maximillan von Frey (1852 – 1932) advanced this theory in 1895 by outlining strict sensory pathways with a specific set of neuronal structures that transmit an incoming pain message along a pre-determined pathway from the periphery to the brain. This became the Specificity Theory (Melzack and Wall, 1965, Craig, 2002, Cervero, 2009, Moayedi, 2013), which continues to provide a framework for research today [Schmelz, Martin 2015]. By pressing fine filaments of increasing diameter to an area of skin (known as von Frey filaments and still in use), von Frey concluded that low threshold cutaneous receptors gave rise to a touch sensation, but when high threshold receptors were activated the result was pain.  His collaborator in these studies was Alfred Goldscheider (1858 – 1935) whose interpretation was framed by the Aristotelian view of pain as an emotion. Goldscheider argued that pain was a result of the intensity of the stimulus and summation occurring in the dorsal horns. The pain receptors were non-specific; it was how this incoming array of spatial and temporal inputs was processed by the brain that would determine the experience of pain. This became the Pattern Theory [Melzack and Wall, 1965, Rey, 1995, Craig, 2002, Cervero, 2009, Moayedi, 2013]. Whilst the Specificity Theory and the Pattern Theory have divergent philosophies; together, they have  provided a charter for investigation that persists to this day [Woolf 2007].

2.2.3 The Discovery of the Nociceptor

Looking at the problem in a new way, Sir Charles Sherrington (1857 – 1952), co-winner of the 1932 Nobel Prize for Physiology and Medicine, contended that the universal trigger for pain was tissue damage. Irrespective of the type of stimulus activating the sensory nervous system, it was the noxious quality of it that the body responded to (Sherrington, 1906). He observed that diverse stimuli applied to the receptive field of a sensory nerve elicited dissimilar responses, such as reflexive withdrawal or pain. Honing in on the sensory receptors associated with the thin, pain-conducting nerve fibres, he identified them as “nociceptors” from Latin noxa meaning “injurious”.  Sherrington introduced his concept of “the adequate stimulus”: the amount of physical phenomena required in order to initiate sensory transduction. Sherrington proposed that nociceptors exhibited selective excitability; yet, they did not play a role in subsequent behavior (Sherrington, 1906, Burke, 2007).

2.2.4 The Specificity and Pattern Theories of Pain 

The neural structures subserving pain perception could be specific, transmitting information through a labelled-line; or, pain transmission could be result of the pattern and intensity of the stimulus [Craig AD 2003]. Investigators attempted to match sensory nerve endings with specific sensations. They found that finely-arborized, free-nerve endings [Weddell 1945] encoded warmth, pain, touch [Hensel 1960] and even itch [Sinclair 1955]. Yet, bulbous, encapsulated nerve terminals [Hagan, 1953] were also associated with touch as we as well as other modalities [Weddell 1955b]. Whilst a linear relationship between modality and sensation could not be confirmed, certain neural structures are definitively associated with pain. Pain was conducted by small diameter, thinly- (Aδ) or unmyelinated (C) fibres [Adrian 1931], yet more than one type of sensory information could be carried by individual types of afferent fibres [Burgess PR and Perl ER 1967, Li, Jie, Kritzer, Elizabeth, 2015]. Afferent fibres were categorized on myelination [Bae, Jin Young 2015], diameter [Ramachandra R 2013], conductance velocity (Knibestol 1973] and mechanical or thermal sensitivity [Brown AG and Iggo A 1967, Abraira VE 2013].  Additionally, activation thresholds ranged from innocuous to noxious [Iggo A 1960] contributing to the idea that nerve terminals arbitrarily transduced the mechanical, thermal or chemical stimuli; yet, specific, pain-carrying fibres converged at the Central Nervous System (CNS).

2.2.5 Spinal Gate Theory

Attention turned to the role of the CNS in the transmission and perception of pain. The relationship between stimulus and pain perception was transformed by Melzack and Wall in 1965. They proposed the Spinal Gate Theory: there are spinal cord ‘gates’ which modulate sensory input by varied facilatory and inhibitory control [Melzack and Wall 1965]. The activity of the gate control system, located within the substantia gelatinosa of spinal dorsal horn, was dependent on the frequency and type of incoming information from small and large fibre afferents; thereby resulting in excitatory or inhibitory effects [Hagbarth KE and Kerr DIB 1954, Taub A 1964, Mendell LM and Wall PD 1965]. This introduced a ‘top-down’ approach to pain perception, with the spinal gate providing the first opportunity for incoming signal selection. The brain, therefore, could filter input and provide appropriate, body-wide responses to incoming sensory information [Melzack and Wall 1965]. Whilst this model has been shown not to be correct, it transformed the understanding of pain [Mendell LM 2014]

Myelinated and unmyelinated afferent fibres were known to transmit pain [Burgess PR and Perl ER, 1967, Bessou and Perl 1969]. Yet, the nature of the relationship between pain and afferent receptors remained elusive. The density of afferents was cofounded by the apparent paucity of afferent receptors [Melzack and Wall 1965, Mendell, LM 2014]. Furthermore, the adequate stimulus for nociceptive afferents was dynamic rather than fixed [Brown AG and Iggo, A 1967, Perl ER 1968]. A sub-population of afferent receptors responded exclusively to noxious mechanical stimulation – high-threshold (HT) mechanoreceptors [Burgess and Perl 1967, Boada, Danilo M et al 2014]. Moreover, these HT nociceptors were not stimulated with innocuous stimuli [Bessou and Perl 1969]. Additionally, the low-threshold (LT) mechanoreceptors could not signal potential or actual tissue damage [Bessou and Perl1969, Abraira VE and Ginty DD 2013]. Consequently, it was the variable sensitivities of the nociceptors that primed the CNS as to the quality of the incoming information.

2.2.6 Peripheral and Central Pain Mechanisms

Further functional studies of nociceptors, afferents and their relationship with pain yielded the concept of Peripheral Sensitization [Bessou and Perl 1969, Bessou 1971, Woolf 2007, Xu 2010]. Peripheral nociceptors become sensitized following injury: they exhibit a lowered threshold of activation and an enhanced magnitude of response following stimulation [Bessou and Perl 1969, Bessou 1971, Woolf and Ma 2007]. This phenomenon was only noted within the site of the injury and in the presence of inflammatory mediators [Perl ER 1976, Kocher, L 1987, Campbell, JN 1988, Binshtok AM 2008, Bishop T 2010]. Sensitized nociceptors due to an inflammatory state were activated, not only by the original thermal stimulus, but by other modes of stimulus as well [Bessou and Perl 1969, Hahn JF 1971].  The fact that other stimuli could contribute to post-injury pain sensitivity suggested that the afferent sense organs were polymodal receptors rather than specifically unimodal [Bessou and Perl 1969, Bessou and Perl 1971, Hahn JF 1971]. Moreover, in inflammatory peripheral sensitization, mechanically-insensitive afferents (MIA) become sensitized to mechanical stimuli [Wenk, Heather N., 2006]. This suggests diverse functionality of nociceptors, their associated afferent fibres and the transmission of pain.

Central sensitization is an amplified response to nociceptive stimulation that is mediated through the CNS [Woolf 1983]. The initial, conditioning stimulus in the periphery does not need to be noxious in quality in order to recruit central mechanisms that signal a threat. It is the increased excitability of nociceptive neurons and strengthened synaptic communication within the dorsal horn that contributes to the amplification of the signal [Woolf 2011]. Lloyd first described synaptic potentiation: a prolonged response in the CNS as evidenced by an enhanced response following repetitive stimulation through a mono-synaptic reflex arc pathway [Lloyd D.P.C. 1949]. It was the temporal aspect of pre-synaptic impulses that altered the spatial arrangement of pre- and post-synaptic communication [Eccles JC and Rall W. 1950, Eccles JC and McIntyre AK, 1951] giving rise to neuronal plasticity at the most basic level of neural communication.

2.2.7 Synaptic Adaptation

The adaptive capabilities of the CNS were demonstrated in the dorsal horn of the spinal column. Input from the thinly myelinated A-fibres, following repetitive low-frequency stimulation, resulted in inhibition at the dorsal horn, consistent with the Gate Theory [Melzack and Wall 1965, Mendell LM and Wall 1965, Janig W and Zimmermann M 1971]. Conversely the same stimulus applied to C-fibres gave rise to a cumulative increase in CNS neuronal firing [Mendell and Wall 1965, Wagman IH and Price DD 1969], termed ‘wind-up’. Wind-up is now thought to be homo-synaptic potentiation; or, an increased output following identical stimuli [Woolf 1988, Chen J 2000]. Both homo-synaptic potentiation and hetero-synaptic potentiation – which takes into account amplified responses to nociceptive and non-nociceptive neurons – are important contributors to neuronal plasticity and the perception of pain [Rottman, S 2008, Aziz Q 2009, Pfau DB 2011].

Supraspinal contributions to central pain mechanisms were also examined with the use of repetitive stimulation [Bliss TVP and Lomo T 1973]. In the hippocampus, synaptic strength increased following persistent input; referred to as long-term potentiation (LTP) [Douglas RM and Goddard GV 1975, Lee KS 1980]. LTP was identified as a crucial underlying mechanism of learning and memory [Seligman ME 1970, Kupfermann, I. 1975, Bliss 1993]. Furthermore, LTP at synapses involving Aδ or C-afferents has been shown to participate in pain pathways [Randic M 1993]. Consequently, nociceptive neuronal input results in post-synaptic amplification of response [Sandkuhler J 2007]. This correlates with hyperalgesia suggesting LTP may be a contributing mechanism to this potentially pathological pain state [Kuner R 2010, Sandkuhler 2012].

2.2.8 Activity-Dependent Central Control of Pain

The Specificity and Convergent Pattern Theories of Pain have provided the framework for enquiry, particularly since Sherrington’s discovery of the nociceptor [Sherrington 1906]. Yet, it has been in the searching for a unified explanation of the relationship between neural structures and pain that the door has opened to a new model of pain [Craig AD ‘Bud’ 2003]. The contemporary model focusses on pain being an integrated, ‘centralized’ process: the CNS may alter pain signals dependent on peripheral, synaptic and central neuronal activity [Woolf 2011]. Accordingly, pain is not necessarily the result of a linear relationship between noxious input and perceived unpleasantness. Certainly a noxious stimulus will produce nociceptive pain. However, the ability of neural machinery to amplify input through peripheral sensitization [Ma, Weiya and Quirion, Remi 2014] and activity-dependent synaptic plasticity [Wang, Jun, Zhang, Xu, 2015]; distort input through increased neurotransmitter release and responsiveness [Galan A 2004, Schuh CD 2014] ; alter the degree and duration of input through changes in membrane excitability [Watson JL 2014], synaptic strength [Luo, Ceng and Kuner, Rohini 2014], receptive fields, long-term potentiation [Sandkuhler J 2007, Gruber-Schoffnegger D 2013, Halff, AW 2014] and descending modulation [Tao, Wenjuan et al 2014, Zhou, Lin 2014]; and recruit non-nociceptive neurons to produce supra-threshold action potentials [Woolf 1994, Galan A 2004, Kopach, O and Voitenko, Nana 2013] suggests a matrix of dynamic, centralized communication.

Each of these manifestations of central pain mechanisms will be discussed with respect to visceral pain pathophysiology and the chronic pelvic pain associated with endometriosis.

2.3 The Neurobiology of Pain

2.3.1 The Nervous System

The nervous system is broadly divided into the Central Nervous System (CNS) and the Peripheral Nervous System (PNS). The CNS, comprised of the brain and spinal column, is responsible for the processing of neural messages. All nerves located outside the dura mater (outer membrane) of the meninges of the CNS form the peripheral nervous system (Figure 2.2).

Figure 2.2 The organisation of the human nervous system. (www.highlands.edu)

The PNS subdivision includes the peripheral portions of cranial and spinal nerves, peripheral ganglia and their cell bodies, somatic and visceral sensory (afferent) nerve fibres, motor (efferent) nerve fibres and sensory receptors that respond to stimuli.

The role of the PNS is to transmit information to and from the CNS and the body. Functionally, the PNS has a sensory (afferent) division and a motor (efferent) division. The sensory component transmits information from the periphery to the CNS. It has somatic and visceral receptors. The somatic sensory branch conveys special sensory information including pain, temperature, touch, position, balance, smell, taste and vision. The visceral sensory receptors transmit messages about the environment and organs of the viscera.

The motor (efferent) component of the PNS transmits information from the CNS to the rest of the body. The motor component may be further divided into the somatic motor: the voluntary nervous system which innervates skeletal muscle; and, the autonomic motor: the involuntary nervous system that innervates cardiac muscle, smooth muscle and glands.

The Autonomic Nervous System (ANS) is comprised of the sympathetic and parasympathetic systems; working synergistically. The sympathetic system activates resources on demand whilst the parasympathetic system monitors and regulates internal bodily functions. Consequently, the ANS is responsible for cardiovascular function, respiration, digestion, blood pressure, temperature regulation and reproductive events. Control occurs through a reflex arc of visceral afferent transmission to the CNS with visceral motor output. Visceral afferents travel with efferent neurons. Together, with interneurons found in the spinal cord, they form a neural network; known as a plexus. A plexus can operate efficiently, somewhat independently of supra-spinal processing. This phenomenon is particularly noted in the plexus surrounding the gastrointestinal tract. Arbitrarily, this plexus is defined as the enteric division of the ANS. The ANS, therefore, utilizes two neurons between the CNS and the target organ or tissue. Consequently, the ANS, whilst functionally discrete, is comprised anatomically of CNS and PNS components.

The Female Reproductive Tract is innervated by autonomic, sympathetic and parasympathetic nerves, as well as contributions from the viscero and somato-sensory systems. The sensory system is the carrier of pain signals. Little is understood about the mechanisms of pain generation and pain sensitivity in the uterus and peritoneal regions of women with endometriosis who experience chronic pelvic pain. Emerging evidence of the sensory involvement in chronic pelvic pain in women with endometriosis highlights pelvic-CNS communication, the role of inflammation and neuroplasticity, neuro-endocrine effects and psychophysical influences as major contributors to endometriosis-associated pain. These will be discussed in the following sections.

2.3.2 Neurogenesis

Neurogenesis is the survival, development and repair of neuronal tissue; occurring under the control of neruotrophins and their receptors (Lindsay RM 1988). Neurotrophins and their receptors are differentially expressed throughout the central and peripheral nervous systems. Centrally, the major neurotrophin is brain-derived neurotrophic factor (BDNF), whilst peripherally, nerve growth factor (NGF) is dominant. Specific neurotrophin receptors are differentially expressed on subsets of neurons. This includes nociceptive (pain conducting) neurons, meaning that the function of a neuron may be characterized by its cytochemical constitution and its location (Keast JR and de Groat WC 1989). NGF and BDNF are known to have roles in pain mediation and modulation (Pezet 2005, Pezet and McMahon 2006).

NGF (Levi-Montalcini R 1952, 1986) is critical to the function of peripheral sensory neurons; particularly those involved in nociception.  Through exerting its action on axons, NGF promotes the proliferation, plasticity and sensitivity of sensory neurons (Twiss JL 2006). NGF binds to its high-affinity receptor, tyrosine kinase A (trkA) activating signaling pathways. The key signaling pathway is the phosphotidyl inositol 3’-phosphate-kinase (PI3K) (Cui 2006, Xu, Qinghao 2011) with downstream actions subsidizing neurite outgrowth and cellular differentiation in early development (Molliver DC 1995). In adulthood, the activation of these pathways facilitates pain processing (McMahon SB 2006, Xu, Qinghao, 2011) and enhanced pain behavior (Choi, Jeong 2012 Mol Pain). Consequently, NGF may be anti-apoptotic and pro-survival in early life, but later becomes a potent, activity-dependent mediator of pain (Malcangio M 1997, Wagner-Golbs 2012).

BDNF also protects adult autonomic DRG neurons whilst supporting the growth and differentiation of new cells and synaptogenesis (Acheson A Nature 1995, Musazzi L, 2014). It is particularly expressed in the hippocampus (Dayi, A 2015) as well as other CNS and PNS locations. Through binding to its key receptor, trkB, it influences learning, memory and higher thinking (Yamada K 2003, Vermehren-Schmaedick A 2014) and normal neural development (Emfors P 1995, Park, Jaewon 2014). Also implicated in potent pro-survival neuronal pathways with formation of reliable synapses is glial cell line-derived neurotrophic (GDNF) (Henderson CE 1994, Houssam D, 2014), neurotrophin-3 (NT-3) (Okragly AJ 1999) and neurotrophin-4 (NT-4) (Ochodnicky P 2012). Together, neurotrophins modulate neural plasticity, including pain processing, by exerting their influence through ligand-dependent signaling activity on sub-populations of neurons and synapses at all levels of the nervous system.

 

 

 

2.3.3 The Neuron

Figure 2.3 Structure of a typical neuron (www.enchantedlearning.com)

 

Humans interact with and respond to external and internal environments via the complex nervous system. At the core of this system lies the neuron (Fig. 2.3). It transforms intricate incoming messages, possibly from many other neurons, into a message that is transmissible by that particular neuron. Transmission is dependent on the physical location and morphology of that neuron. The “message” is an ionic trans-membrane change converting inputs to an electrical action potential. This voltage change across the membrane typically travels from the dendrites via the cell bodies (soma) and axon to the synapse. Contemporaries, Charles Sherrington and Santiago Ramon y Cajal in 1897, concluded a “synapse” is a specialized apposition, distinct from its neighbouring neuron, yet capable of mediating transmissions (Boron and Boulpaep, 2009). Neurotransmitters, released synaptically, are often simple molecules similar to those found in general metabolism; including extracellular adenosine triphosphate (ATP) and gaseous molecules, such as nitric oxide [the 50’s ref for ATP] (Burnett and Wesselmann, 1999, Keast, 2006). In some instances, a single neurotransmitter has divergent activity, binding with multiple receptors along a neuronal membrane causing a dynamic shift in the balance of incoming messages to post-synaptic cells. Conversely, a single ion channel may respond to multiple transmitters. Thus, complexity and efficiency of the nervous system is aided by synaptic amplification or inhibition.

 

2.3.4 The Structure and Function of the Neuron

A typical neuron consists of a cell body (soma), containing the nucleus and cellular machinery; dendrites arising from the soma, that branch to form a dendritic tree, capable of receiving messages from neighbouring neurons; and an axon (Figure 2.2). A neuron may have multiple dendrites but only one axon. The axon elongates from the soma at the axon hillock, varying in length, with a sensory neuron from the toes extending over a metre in humans. Both axons and dendrites may also be referred to as neurites; particularly if they are in an undifferentiated state.

Neurons may be categorized according to morphology and function. Broadly, they are afferent (sensory) neurons, responsible for the transmission of information from peripheral tissues and organs to the central nervous system (CNS); efferent (motor) neurons transmit signals to effector cells; and interneurons, which are contained within specific regions of the CNS.

Morphologically, the cell bodies (soma) of sensory neurons are found in the dorsal root ganglion (DRG). The DRG sit just outside the spinal cord and are mainly comprised of sensory fibres (Magendie 1822). These sensory (afferent) fibres come from the viscera, skin and other subcutaneous and deep tissues and synapse in the spinal cord. A few efferent fibres (which transmit messages away from the CNS) may be included in the DRG nerve bundles.  However, most motor (efferent) nerves and some autonomic nerves are found in the ventral roots of the spinal cord. This arrangement allows superfast processing of incoming sensory information: the motor response can be activated at the spinal level prior to the sensory input reaching the brain (Figure 2.4).

Figure 2.4 Reflex arc (www.life.umd.edu)

2.3.5 The Sensory Neuron

Sensory neurons are considered to be pseudo-unipolar. True unipolar neurons only have one neurite extending from the soma. The soma of sensory neurons, on the other hand, extends a single process which branches into two: proximal, leading into the spinal column; and distal, leading to the periphery (Figure 2.5 a). The distal process may have free-nerve endings or be encapsulated (Figure 2.5 a & b). Additionally, sensory afferents have a receptive field, meaning that all receptors on an afferent may respond to an adequate stimulus (Figure 2.5 c).

Figure 2.5 Sensory Unit: afferent neuron and receptors (www.droualb.faculty.mjc.edu)

 

Afferent neurons may be myelinated or unmyelinated (Figure 2.3) and are involved in the transduction and transmission of pain signals. Myelin, produced by Schwann cells in the periphery and by neuroglia centrally, sheaths the axons to aid the velocity of transduction. The sheath is discontinuous at the Nodes of Ranvier. It is to these regions that the action potential propagates, effectively ‘jumping’ along the axon. Myelinated fibres are rapidly conducting: the key myelinated afferents are Aα, Aβ and Aδ.  Aα and Aβ carry touch and muscle information. Aδ fibres are more thinly myelinated than their A-fibre counterparts and are associated with cold sensation, pressure and nociceptive pain (Basbaum et al 2009). Aδ fibres terminate at Rexed laminae I and V of the spinal dorsal horn (Rexed B 1952).

Unmyelinated afferents are known as C-fibres. C-fibres are nociceptive, responding to thermal, mechanical or chemical stimuli and are associated with pain, warmth and light touch. Lacking myelin, C-fibres are slowly conducting with the action potential propagating in waves. C-fibres form Remak bundles. These bundles are organised with C-fibres, sometimes as few as 2 axons, being blanketed by protective Schwann cells (Murinson BB and Griffin JW 2004). Not only do the Schwann cells keep the bare C-fibres from the ions of the extracellular matrix (ECM), they promote neurotrophic actions (Gardiner NJ 2011). Consequently, non-myelinating Schwann cells may influence the electrochemical microenvironment of C-fibres and have a role in neural plasticity and neuropathic pain (Campana WM 2007). C-fibres terminate in the Rexed lamina II, or substantia gelatinosa of Rolando, of the spinal dorsal horn (Rexed B 1952)(Figure 2.7).

2.3.6 The Nociceptor

Nociceptive neurons are comprised of two main populations: small, thinly- or un-myelinated C-fibres and medium diameter, myelinated Aδ fibres.

2.3.7 Neural Connectivity

Sensory afferents transduce thermal, mechanical or chemical stimuli into an electrical signal (action potential) for propagation via the DRG to the synapse located within the superficial dorsal horn. The synapse is a specialized inter-neuronal structure that is critical for neural transmission and pain conductivity, perception and longevity (Perea G 2009).

2.4 The Central Nervous System

2.4.1 The Spinal Column

The CNS consists of the brain and spinal column. The spinal column transmits neural messages to and from the brain and the body; having a role in the voluntary movement of the trunk and limbs as well as conducting sensory input from these regions. The spinal cord is also responsible for messages involving most of the organs and vasculature of the viscera.

Protected by its bony vertebra, the spinal cord begins at the medulla oblongata in the brainstem and extends to the lumbar region. These regions are known as cervical, thoracic, lumbar, sacral and coccygeal, with 31 segments in total (Figure 2.6). The spinal cord is shorter than its protective vertebral column, meaning that the lower spinal nerves eg, lumbar, emerge from a higher segment (Barson AJ 1970, Barson AJ and Sands J 1975, Kameyama T etal 1996, Canbay S 2014). Spinal nerves are made up of both afferent and efferent fibres. The spinal nerves that innervate the female pelvis emerge from T10 to L2 (sympathetic fibres) and S2 to S4 (parasympathetic fibres).

Figure 2.6 The Spinal Column (www.biomed.brown.edu)

2.4.2 The Spinal Cord

In cross section, the spinal cord appears as white matter surrounding the H-shaped, grey matter (Figure 2.7). The grey matter consists of consists of neuronal cell bodies, with the functional layers determined by cell body size and distribution (Rexed 1952)( Bror Rexed (1914 – 2002) (Pearce JMS 2007). Proceeding from the apex of the dorsal horn to the ventral horn, these layers are Laminae I – IX with the tenth layer, Lamina X, surrounding the central canal that contains the cerebrospinal fluid. Large afferent fibres terminate in layers III, IV, VI, VII and IX. Large afferents are known to be involved in touch, pressure and proprioception. Of particular note, is Rexed Lamina II or substantia gelatinosa. Its gelatinous appearance is due to the predominance of thinly- or un- myelinated afferent fibres: Aδ and C, known to be involved in pain transmission.

Figure 2.7 The Spinal Cord (www.studyblue.com)

The white matter is formed by columns of axons. Axons grouped together in the same column are known as funiculus. White matter is comprised of dorsal, ventral, lateral and Lissauer’s funiculi. Smaller bundles of comparative axons within the column are called fasciculus. Functionally, the axons that start together, travel together and terminate together are known as a tract. A group of related tracts, such as the ascending tracts, are referred to as pathways.

 

 

2.4.3 Ascending Pain Pathways and Descending Modulation

The Dorsal Column Medial Lemniscus (DCSL) is a prominent, ascending pain pathway (Joshi SK and Gebhart GF 2000). It is comprised of large, myelinated, rapidly conducting fibres mostly concerned with discriminating fine touch, pressure, vibration and proprioception (position sense).

 

Fig 2.8 Ascending Pain Pathways  (www.droualb.faculty.mjc.edu)

The ventrolateral regions of white matter also have an important role to play in the transmission of pain and attendant consciousness. The ascending pathways of the Anterolateral System incorporate the spinothalamic, spinocerebellar and spinotectal tracts. The descending or efferent pathways include the corticospinal, vestibulospinal, tectospinal and reticulospinal tracts.

Normal pain: pain reduces with time. This is because noxious input stimulates the descending efferent pathways that inhibit the pain transmitting neurons. A negative feedback loop is established whereby the output from the pain-transmitting neurons is modulated due to the effect of the pain-inhibiting neurons. Over time, this reduces the perceived intensity of the pain. (Basbaum A and Fields H, 2004; Annals of Neurology 1978, vol 4, issue 5, pp 451-462) (See PAG)

2.4.4 Supra-spinal Processing

(on-going)

Supraspinal processing of TRPV1 modulates the emotional expression of abdominal pain: Jurik, Angela 2014, Pain, using TRPV1 KO mice: reduction in evoked abdominal constrictions in acute pancreatitis model of abdominal pain but no change in referred abdominal hyperalgesia or allodynia; reduction in nocifensive behaviour;  supraspinal TRPV1 mediates affective component of abdominal pain

Martins, D 2014: the role of TRPV1 in brain functions

2.5 Pain Perception

2.5.1 Pain Perception across the Menstrual Cycle

Pain perception reportedly differs throughout the menstrual cycle. Yet, the exact differences remain unclear. A meta-analysis examined studies of induced pain in healthy female volunteers across the menstrual cycle (Riley Iii et al., 1999). Thermal, mechanical and pressure stimuli produced greater sensitivity in luteal and menstrual phases compared with the follicular phase. However, electrical stimulation resulted in less sensitivity in the luteal phase over the other phases. The authors concluded that there was a menstrual phase effect on pain perception. More recently, thermal detection and thermal pain thresholds were psychophysically determined at the early follicular, peri-ovulatory and mid-luteal phases [Soderberg K 2006]. Serum estradiol and progesterone confirmed the phases. No major changes in pain sensitivity across the phases were noted. Similarly, in healthy women, with biochemically-confirmed phases, noxious stimuli produced minimal effect on invoked pain ratings across the menstrual cycle (Bartley et al., 2015). Additionally, quantitative sensory monitoring during the follicular and luteal phases indicated no changes of perception thresholds corresponding to the stimulation of A-beta, A-delta and C fibres across the menstrual cycle [Oshima, Masayuki 2002].

Alterations in pain perception across the menstrual cycle are often attributed to fluctuating hormonal levels. Earlier studies have emphasized higher estrogen

Another group sought to tightly restrict pain assessment in healthy women to just the mid-follicular and mid-luteal phase [Bartley E 2013]. This was because the

 

2.5.2 Pain perception in dysmenorrhagic women

2.5.3 In endometriosis

2.5.4 In chronic pelvic pain

2.5.5 With anxiety  and disposition

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.6 The Peripheral Nervous System

2.6.1 Innervation of the Female Reproductive Tract

Figure 2.9 Innervation of the Female Pelvis (www.netters-atlas-of-human-anatomy)

Innervation of the Female Reproductive Tract is mainly supplied by the hypogastric and pelvic nerves. These nerves are a combination of sympathetic and parasympathetic autonomics and sensory afferents (Cervero 1994, Wesselmann and Lai 1997). Sensory afferents may travel with parasympathetic nerves, thereby contributing to some types of pelvic sensory perception, but, most travel with sympathetic nerves.

Sympathetic nerve fibres travel through a chain of neural ganglia known as the sympathetic trunk, adjacent to the bony vertebra of the spine (Figure 2.9). The sympathetic trunk provides a conduit for sympathetic nerve fibres to join with spinal nerves that may be superior or inferior to the spinal nerves from which the sympathetic fibres originated (Rigaud J 2010, Kraima, Anne C, 2015). Spinal nerves communicate with the sympathetic trunk via the white and grey ramus communicans. These rami consist of myelinated (white) and unmyelinated (grey) visceral afferents that have their cell bodies in the dorsal root ganglion (DRG) and synapse within the spinal cord (Figure 2.7). These neural connections contribute to pain messaging in the ascending pain pathways (Giamberardino MA 1999, Winnard 2006, Wyndaele 2013).

The hypogastric nerve (HGN) innervates the uterus, ovaries, fallopian tubes and broad ligament with a contribution to the cervix, vagina and clitoris. Sensory afferents from these organs pass through the uterovaginal plexus to the inferior hypogastric plexus (IHG) (Jeyarajah S 2007, Rigaud J 2010). The IHG, also called the pelvic plexus, is located at the sides of the vagina and rectum (Figure 2.8). HGN afferents continue to the superior hypogastric plexus (SHG) and inferior mesenteric ganglion (IMG), onto the sympathetic chain and lumbar splanchnic nerves before projecting to the thoracolumbar region (T10 to L3) of the spinal cord (Berkley 1993, Winnard K 2006, Jobling 2014).

The pelvic nerve (PN), a mixture of visceral afferents and parasympathetic fibres, emerges from the sacral region of the spine (S2 – S4). Like the HGN, it passes through the pelvic plexus (or IHG) before contributing to the innervation of the uterus, cervix, vagina and clitoris. The vagina and external genitalia are also supplied by the pudendal nerve arising from the sacral plexus. Sacral afferents have a role in chronic pelvic pain (Desrosiers L 2015, Taghva A 2015).

Uterine pain is transmitted through different levels of the spinal cord as uterine afferents arise from different spinal segments and track to different regions of the uterus. The upper, intra-peritoneal fundus and body is innervated by the HGN and the sub-peritoneal, cervical region is innervated by the PN (Peters 1987, Berkley 1988). The PN was found to be more sensitive to mechanical and chemical stimuli (Berkley 1990) than the HGN (Robbins A 1990, Cunningham ST 1991). Using electrophysiological techniques in a female rat model, Berkley deduced that innocuous conditions produced single organ responses whilst noxious stimulation resulted in an expanded receptive field and multi-organ response. Additionally, neurophysiological observations were matched with behaviours, finding that the PN messaging was more likely to be employed in with various sensory, pain, mating and conception behaviours (Clark Ann S 2011) whilst the HGN was activated in times of threat, pain and during parturition (Berkley 1990).

Innervations for a number of pelvic organs pass through the inferior hypogastric plexus (IHG). In addition to those of the female reproductive tract, they include the bladder (Deffieux, X 2007, Gomez-Amaya, SM 2015) and urethra (Giamberardino, MA 2002, Kanao, H 2015), distal ureter ( Ditting T , Linz P 2012) and terminal portions of the colon (Capek, S 2015). The convergent neural pathways for pelvic organs have implications in pelvic pain transmission (Giamberardino MA 2010).

 

2.7 Innervation of the Eutopic Endometrium and Peritoneal Region and Pain in Endometriosis

2.7.1 Neurogenesis, Pain and Endometriosis

Neurotrophins and their receptors are expressed in endometriotic lesions: peritoneal, ovarian and DIE with strongest expression in DIE (Anaf 2002, Tok 2007, Odagiri 2009, Wanf 2009, 2009)

BDNF, NT-3 and NT4 in endometriotic glands and stroma in ovarian endometriomas (Tran 2009)

TNFa may increase NGF [Woolf CJ 1997]

NGF effect on endo-associated symptoms: [Barcena de Arellano ML 2011]

2.7.2 Innervation of the Eutopic Endometrium and Pain in Endometriosis

2.7.3 Neurogenesis and Innervation of the Peritoneal Lesions, Peritoneal Fluid and Pain in Endometriosis

DIE: readily activates nociceptive system due to highest density of NF, mast cells and various inflammatory cells and mediators [Anaf]

Peritoneal fluid: [Eisermann J 1988] increased levels of TNFa in PF; have been correlated with pain [Scholl B 2009] but not with lesion type [D’hooge TM 2001]; TNFa produce mechanical hyperalgesia in rodents [ Parada CA 2003]; inflammatory response in humans [Groves RW]; clinical trials using anti-TNFa antidbodies have not brought relief from endo pain [Koninckx PR 2008, Lu D, Song H Shi G, Cochrane database CD008088 2013]

Il-6 in PF:  [D’Hooghe TM 2001, Drosdzol-Cop A 2012], [Punnonen J 1996], [Wickiewicz D 2013]

NGF in PF: [Barcena de Arellano ML 2011] may promote neurite outgrowth in lesions;

Neurotrophic over-expression in PL: [Barcena de Arellano ML 2013]

Prediction of endometriosis with PF markers: [Bedaiwy MA 2002]

Rat model: innervation of ectopic endo [Berkley KJ 2004]

 

2.8 Convergent Pelvic Pain Pathways

2.8.1 Referred Pain

Chronic pelvic pain is often diffuse, poorly localized and referred to regions other than the affected organ. This phenomenon is known as referred pain, first labelled by Henry Head in 1893 (Head H , Brain 16 1893, pp 1 – 333). Visceral referred pain is usually perceived in a neurally-associated somatic region. Accordingly, uterine pain may be experienced in the lower back or radiating through the trunk and down the back of the legs. These areas are consistent with dermatomes T10 – L2. Dermatomes are cutaneous areas that correspond to innervation by a particular spinal nerve (Figure x dermatomes). Neural information from the uterus enters the spinal cord in the same segment as these particular spinal nerves. Due to the common spinal projection, pain transmission is then referred to, in this case, cutaneous regions. Consequently, neural convergence underlies a number of pelvic pain pathways (Giamberardino MA 1999).Visceral and somatic afferents converge onto the same sensory neurons in the spinal cord (MacKenzie,  J 1906, Symptoms and their interpretation, Shaw & sons London) resulting in viscerovisceral, viscerosomatic and somatovisceral convergence (Giamberardino MA 2010) with enhanced pain sensitivity ( Cervero F 2000) .

2.8.2 Viscerovisceral convergence

Transduction of a noxious stimulus proceeds from the primary afferent innervating a pelvic organ to synapse within the dorsal horn. Plastic alterations in synaptic communication (Lloyd 1949, Woolf 2000) and an expansion in the neuronal receptive field (Cook AJ 1987, Treede RD 1992) mean that neighbouring afferents transmit the message antidromically – from the DRG to the periphery. Notably, this output is from afferents localized to lamine VI-VII (Malykhina 2007), not general effector axons.

In a rat model, the DRG that innervates the uterus, colon and bladder via the HGN (Chaban V 2007) and converge at the same DRG (Winnard K 2006). Inflammation in one of these organs means inflammation in others (Berkley KJ 2005, Winnard K 2006)

Animal studies Berkley KJ, Giamberardino MA

Visceral pain is accompanied by emotional distress and symptoms associated with the autonomic nervous system eg, nausea, raised temp, sweating, pallor

2.8.3 Viscerosomatic convergence

Animal studies of Berkley KJ, Giamberardino MA

 

 

 

 

 

 2.9 Pathophysiology of Chronic Pelvic Pain

2.9.1 Visceral Pain

Visceral pain is diffuse, changing, poorly localized, not evoked from all viscera and is associated with exaggerated behavioral modification and emotional distress. The pain may be referred to unrelated areas of skin or muscles or may overlap with other organs of the viscera making the origin difficult to determine. Adding to the difficulty of pinpointing the source is that visceral pain is often not concomitant with injury and is initiated by a set of stimuli that differ from those responsible for somatic pain. The neurobiology of somatic pain cannot be extrapolated to account for visceral pain. Visceral pain is induced by distention of hollow organs, ischemia and inflammation. Visceral pain hypersensitivity is the outcome of a combination of nociceptive, inflammatory or neuropathic pain. Each of these pain types display considerable neural plasticity and each are prospective pathways to endometriosis-associated pain.

2.9.2 Chronic Pain

Chronic pain is pain that persists for greater than 6 months duration or pain that extends beyond the expected time for healing (Turk DC in Bonica JJ et al Eds: Boncia’s management of pain, 2001).

Chronic pelvic pain in females of reproductive age is often gynaecological in origin. It is experienced in the abdomino-pelvic region, lower back and may radiate up through the trunk to the shoulders or down the back of the legs to the knees. CPP is characterized as vague, diffuse, deep, sharp, aching, dull and dragging.

Chronic pain may be representative of the plasticity of the nervous system. A feature of neuroplasticity is that sensory neurons can become sensitized; both innocuous and noxious stimuli are perceived as painful. Firstly, nociceptors in the periphery can be sensitized to the local milieu of hormones and inflammatory mediators, exhibiting responsiveness at lower thresholds of stimulus.

The next step on this sensitization-to-chronic pain pathway incorporates the plasticity of the spinal cord neurons. A sub-set of spinal nociceptive neurons, found in the outer layer of the dorsal horn (Mantyh P 1995, 1997, 1999), sensitize almost certainly by low frequency input from sensitized peripheral neurons (Mantyh P late 90’s, Sandkuhler J Mol pain 2007). This results in strengthened synaptic transmission between these neurons. This is known as long-term potentiation (LTP).

LTP, first identified in the rabbit hippocampus (Terje Lomo 1966, Lomo 2003) and now thought to be possible at every excitatory synapse in the brain (Malenka R, Bear M, 2004, Neuron),  is considered to be a key underlying mechanism of learning and memory (Bliss T and Lomo T, 1973, Douglas R and Goddard G, 1975, Bliss TV et al Nature 1993) and is implicated in neuropathic pain, anxiety and depression.

 

 

2.9.3 Nociceptive Pain

 

2.9.4. Inflammatory Pain

 

2.9.5 Neuropathic Pain (NP)

Neuropathic Pain is defined by the IASP as “pain initiated or caused by a lesion or disease of the somatosensory nervous system” (www.iasp-pain.org/taxonomy; accessed 17/02/2015). This definition has been revised since the most recent Classification of Chronic Pain Taxonomy was published (Merskey H. and Bogduk N., 1994). The revision came about in order to distinguish neuropathic pain as just involving the somatosensory system rather than implying the involvement of other types of neural pathways as was indicated in the previous definition (Treede, R.-D. et al,  2008)

Consequently, neuropathic pain may be thought of as pain arising in the nociceptive system despite apparently inadequate activation of the nociceptive system.

Neuropathic pain is an abnormal pain state. It is generated by stimuli that do not normally cause pain eg. light touch. Neuropathic pain is a representation of the plasticity of the nervous system and is a dysfunction of peripheral nerves.

 

2.10 Peripheral and Central Pain Mechanisms in Chronic Pelvic Pain

2.10.1 Peripheral Sensitization or Primary Hyperalgesia

Peripheral sensitization is when high threshold (HT) nociceptors, usually only responsive to noxious stimuli, become sensitized after injury [Iggo A, 1960, Perl, 1968, Bessou P 1969]. These HT nociceptors display a reduced threshold, particularly for heat pain [ ], only within the site of the injury [ ]. As inflammatory modulators are recruited to the injury site, peripheral sensitization or primary hyperalgesia, explains pain hypersensitivity associated with inflammation. See refs list.

Increased responsiveness to noxious stimuli that includes an increased duration of response yet no expansion of the site of response is best shown by sunburn.

2.10.2 Secondary hyperalgesia

Secondary hyperalgesia occurs when there is expansion of the nociceptive receptive field. Adjacent, healthy tissue now contributes to the sensation of pain. Pain sensitivity is increased in both duration and area affected.

2.10.3 Wind-up or Temporal Summation

2.10.4 Long-term potentiation

2.10.5 Recruitment of non-neural tissue to produce supra-threshold action potentials

2.10.6 Descending modulation

 

Notes: Central sensitization is defined as “an amplification of neural signaling within the CNS that elicits pain hypersensitivity” [Woolf CJ 2011].

Assess the contribution of CS to inflammatory, neuropathic and other dysfunctional pain disorders

Local levels of NGF affect nociceptor function

Regulation by NGF: increased NGF in inflammation; experimentally increased NGF = thermal hyperalgesis [ Refs and Woolf, Mendell, McMahon]

NGF sesntive fibres express BDNF at low levels; NGf can upregulate BDNF; changes in NGF alter the excitability of the dorsal horn through the release of substance P = central sensitization

 

 

 

2.11 Nociceptors

Nociceptive neurons are comprised of two main populations: small, thinly- or un-myelinated C-fibres and medium diameter, myelinated Aδ fibres.

C-fibres, sensitive to nerve growth factor (NGF), synthesize calcitonin-gene related peptide (CGRP) and substance P, projecting to laminae I and outer laminae II of the spinal dorsal horn [Christensen BN and Perl ER 1970, Light AR and Perl ER 1977, Kumazawa T and Perl ER 1978]. Non-peptidergic C-fibres, lacking a trkA receptor, are sensitive to glial cell-derived neurotrophic factor (GDNF), projecting to inner laminae II. This population of nociceptive neurons can be labelled by isolectin B4 (IB4) and are known to express both the purinergic receptor (P2X) and transient receptor potential (TRP). TRP is also expressed in the NGF-sensitive population.

[Poetntial refs: Brodie and Gelfand 1994, Mitsuma 2004, Henderson 1994, review Snider WD and McMahon SB 1998]

Figure 2.1 Central termination patterns of 2 classes of nociceptive neurons. [Snider WD and McMahon SB 1998]

The central termination pattern of these two groups of nociceptive neurons provides evidence of their respective roles in pain generation (Figure 2.1). Knocking out the substance P receptor, NK1, in laminae I had no effect on the nociceptive response; yet, the introduction of the TRP-specific stimulus, capsaicin, induced hyperalgesia [Mantyh 1997], suggesting a role for NGF-sensitive C-fibres in maladaptive pain. Likewise, in mice that lacked protein kinase C (PKC), found in the laminae II target of IB4 neurons, there was no change in acute nociceptive behavior, yet they failed to develop neuropathic pain following nerve ablation [Malmberg 1997]. This suggests a role for IB4 neruons expressing P2X and TRP in the generation of neuropathic pain [Snider WD and McMahon SB 1998 – core review, Millan MJ 1999 –core review, Polgar, E, et al 1999, Miletic V eral 2000, Piers C etal 2014 J of Comparative Neurology – review].

Neuropathic pain is characterized by an amplified response within spinal cord neurons [Woolf 1994]. It is the dorsal horn that is the crucial processing region for nociceptive input. Nociceptive input arrives via the transduction of the stimulus into an electrical impulse [Woolf 1991, Dubna and Ruda 1992]. In neuropathic or chronic pain, excitation in the dorsal horn occurs with minimal primary afferent input due to reduction in thresholds (allodynia) and increased responsiveness to noxious inputs (hyepralgesia) [Woolfe 1983, Laird and Cervaro 1989, Neugebour 1990, Malmberg and Vaksh 1992, Woolf 2011]. Secondary hyperalgesia occurs through expansion of the nociceptive receptive field, allowing inputs from healthy cells to produce pain [Larid and cervaro 1989, Woolf 2011]. Within the dorsal horn, this induces large, myelinated fibres to synapse with small, nociceptive fibres in the dorsal horn (Woolfe, 1983, Qing-Ping Ma and Woolfe, 1995, Cohen Steven P. 2014]. Expansion in nociceptive receptive fields correlates with pain sensitivity [Neziri 2010]. Through this mechanism, pro-nociceptive molecules within the periphery, such as inflammatory mediators, ATP, protons, cytokines, neurotrophins and nitric oxide, exert influence within the CNS [Cohen Steven P. 2014] and contribute to maladaptive pain. Neurotrophins, non-neuronal and immune-competent cells also play a role in nociceptor sensitization [ ], modulation [ ] and central processing [ ] .

2.11.1 Responsiveness of Nociceptive neurons

Thermal Sensitivity, Mechanical Sensitivity, Chemical Sensitivity

 

 

 

 

 

 

 

2.12 Purinergic Receptor

Purinergic receptors (P2X) form a superfamily of membrane ion channels responding to extracellular adenosine 5’-triphosphate (ATP) (Holton, 1959, Valera et al., 1994, North and Barnard, 1997). These ATP-gated receptors are widely distributed throughout the central and peripheral nervous systems and in non-excitable tissue (Burnstock, 1972), with an ionotropic sub-class: P2X; and a metabotropic sub-class: P2Y, P2U and P2T. Of particular interest is the P2X receptor; of which, there are seven subunits: P2X(1-7) (North, 2002). P2X is implicated in primary afferent transmission (Jiang et al., 2003), nociceptive pain (North, 2004), inflammatory pain (Khakh and North, 2006), central modulation (North and Verkhratsky, 2006, Browne and North, 2013)and neuropathic pain (North, 2004, North and Verkhratsky, 2006, Khakh and North, 2006, Surprenant and North, 2009).

2.12.1 Structure

Structurally, each P2X subunit consists of two membrane-spanning domains (TM-1 and TM-2) (Werner et al., 1996, North and Barnard, 1997) separated by a large extracellular loop (Figure 2.6.xx). The extracellular domain has a consensus glycosylation sequence (North, 2002), an ATP binding motif (Jiang et al., 2000) and  a conserved sequence of 10 cysteine residues forming disulfide bonds (Khakh and North, 2006). Both the amino and carboxy termini are intracellular. There is no reported homology between P2X and other proteins.

 

 

Figure 2.6.xx Purinergic Receptor

1. A. Membrane topology (See text above for detail). B. Schematic of P2X receptor. The trimer forms an inverted pyramidal structure with a crown-shaped outer surface (Mio et al., 2005)

 

Cloned DNA studies show P2X receptors form trimers as their functional unit (Brake et al., 1994, Valera et al., 1994, Surprenant et al., 1995, Mio et al., 2005, Kawate et al., 2009).P2X channel formation occurs with a maximum of three subunits (Stoop et al., 1999, Aschrafi et al., 2004), which is a stable arrangement (Nicke et al., 1998, Gonzales et al., 2009). Trimer subunits may be identical: homo-mulitmeric (North, 2002); or, exist as hetero-mulitmers (Lewis et al., 1995, Collo et al., 1996b, Jiang et al., 2003). Homo-multimeric channels are formed by P2X1, P2X2, P2X3, P2X4, P2X5 and P2X7 (North, 2002, Dubyak, 2007), whilst the homomer of P2X6 is poorly expressed (MacKenzie et al., 1999, Burnstock, 2013a). Hetero-multimers of  P2X1/2 (Brown et al., 2002) , P2X1/4 (Nicke et al., 2005), P2X1/5 (Torres et al., 1998, Haines et al., 1999), P2X2/3 (Lewis et al., 1995, Radford et al., 1997, Newbolt et al., 1998), P2X2/6 (King et al., 2000), P2X4/6 (Le et al., 1998) and P2X4/7 (Dubyak, 2007, Guo et al., 2007) have been identified. These structural differences confer functional variations (North, 2002).

2.12.2 Function

Purinergic receptors function as cation-selective channels, gated by ATP. These channels open in the presence of extracellular ATP or its analogue, αβmethylene-ATP (αβ-meATP). P2X1, P2X3 and P2X6 respond to ATP or αβ-meATP. P2X2, P2X4 and P2X5 form an open channel with bound ATP only, not its analogue (MacKenzie et al., 1999, Burnstock, 2013a). Continuous exposure to ATP results in pore dilation (Dubyak, 2007)allowing permeability to larger cations (North, 2002). As progressively larger ions flow across the membrane (MacKenzie et al., 1999, North, 2002, Dubyak, 2007) the transmembrane potential is altered(Mio et al., 2005, Khakh and North, 2006), influencing the action potential threshold. Altered thresholds are a hallmark of chronic pain states.  As ATP is known to evoke pain in humans (Bleehen et al., 1976, North, 2002), the role of purinergic receptors and extracellular ATP has implications in chronic pain states.

2.12.3 Distribution

The P2X family of ligand-gated, cationic-channel receptors is widely distributed throughout neuronal and non-neuronal tissue (Table 2.6.1). Purinergic receptors are involved in a number of physiological processes. These include contractility of smooth muscle, maintenance of vascular tone, cardiac rhythm and sensory transmission of pain.

Purinergic receptors are associated with sensory neurons (Holton and Holton, 1954, Burnstock, 1972), both centrally (Abbracchio et al., 1995) and peripherally (Surprenant et al., 1995), with P2X2 and P2X3  being the predominantly expressed sub-types (Vulchanova et al., 1997). P2X2 is expressed on small-diameter  primary afferents (Krishtal et al., 1983), the superficial dorsal horn (Tsuzuki et al., 2003), spinal (Jo and Schlichter, 1999, Studeny et al., 2005)and supra-spinal neurons (Stoeckel et al., 2003, Pougnet et al., 2014). P2X3 is confined to primary afferents (North, 2004), sub-populations of small-diameter cell-bodies within the DRG (Bradbury et al., 1998) and DRG projections into lamina II of the spinal dorsal horn (Jahr and Jessell, 1983, Vulchanova et al., 1998, North, 2002). P2X2 and P2X3 are implicated in nociceptive signaling (Tsuda et al., 2000, Dirajlal et al., 2003), inflammatory and chronic pain (Vulchanova et al., 2001) as well as the development of neuropathic pain (Krames, 2014).

Table 2.6.1 Purinergic Receptor Characterisation

P2X Receptor Subtypes	Distribution	Role	Refs
P2X1	CNS and PNS: Sensory neurons; Smooth muscle: ovary, uterus and blood vessels; Endocrine secretory cells: pituitary; Immune cells*	Sympathetic vasoconstriction in small arteries and arterioles; promotion of apoptosis	(Valera et al., 1995, Scase et al., 1998, Sun et al., 1998, Bardini et al., 2000, Mulryan et al., 2000, Valdecantos et al., 2003, Jiang et al., 2005, Vial and Evans, 2005, Lecut et al., 2009, Burnstock, 2013a)
P2X2	CNS and PNS: predominant sub-type found on sensory neurons in periphery and spinal column; Smooth muscle: blood vessels; Endocrine secretory cells: pituitary; Immune cells*	Sensory transduction and transmission and modulation of synaptic function; Nociceptive signaling; chronic, inflammatory pain; taste; hearing; bladder volume	(Rassendren et al., 1997, Lynch et al., 1999, Jiang et al., 2003, Illes and Ribeiro, 2004, Shieh et al., 2006, Chaumont et al., 2008, Pannek et al., 2009, Hausmann et al., 2012, Burnstock, 2013a)
P2X3	CNS and PNS: primary sensory afferents; DRG (only, not spinal column); Smooth muscle: blood vessels; Endocrine secretory cells: pituitary	Sensory transduction and transmission; Nociceptive signaling; Acute inflammatory pain;	(Collo et al., 1996a, Shieh et al., 2006, Lewis et al., 1995, Burnstock, 2013a)
P2X4	CNS and PNS: Sensory neurons, Smooth muscle: blood vessels; predominant subtype expressed in brain Human endometrial epithelial cells; Endocrine secretory cells: pituitary; Immune cells* (See below)	Brain ischaemia, muscular dystrophy, upregulation following nerve injury	(Valdecantos et al., 2003, Cavaliere et al., 2003, Shieh et al., 2006, Burnstock, 2013a)
P2X5	CNS and PNS: Sensory neurons; Smooth muscle; vasculature; Human endometrial epithelial cells; Endocrine secretory cells: pituitary; Immune cells*		(Collo et al., 1996a, Burnstock, 2013a, Burnstock, 2014)
P2X6	CNS and PNS: Sensory neurons, Smooth muscle, vasculature, predominant subtype expressed in brain (less than P2X4); Human endometrial epithelial cells Endocrine secretory cells: pituitary		(Collo et al., 1996a, Burnstock, 2013a)
P2X7	CNS: astrocytes; Immune cells: macrophages, Human endometrial epithelial cells; Endocrine secretory cells: pituitary; Immune cells*	Induction of apoptosis in epithelial cells; Neuropathic and inflammatory pain	(Valdecantos et al., 2003, Illes and Ribeiro, 2004, Shieh et al., 2006, Dubyak, 2007, Li et al., 2007, Burnstock, 2013a)
P2X2/3	CNS and PNS: Sensory neurons: primary afferents, Smooth muscle, vasculature	Longer-lasting nociceptive sensitivity; Chronic inflammatory pain; Neuropathic pain; Possibly a greater physiological role than P2X3 in primary afferents	(Radford et al., 1997, Vulchanova et al., 1997, Virginio et al., 1998, North, 2004, Wilkinson et al., 2006, Burnstock, 2013b)
P2X1/2; P2X1/4; P2X1/5; P2X2/6; P2X4/6; P2X4/7	CNS and PNS: Sensory neurons, Smooth muscle, vasculature	Roles have not been fully determined	(Burnstock, 2013a, Dubyak, 2007)

*Immune cells: macrophages, neutrophils, lymphocytes, mast cells, dendritic cells (Burnstock, 2013a)

2.12.4 Purinergic Signaling

The purinergic signaling hypothesis, proposed by Geoffrey Burnstock (Burnstock, 1972), identifies extracellular ATP as the neurotransmitter released by non-adrenergic, non-cholinergic nerves. ATP, as a neurotransmitter (Eccles, 1964) had been identified in sensory neurons of the cat (Holton and Holton, 1954). In fact, every nerve in the CNS (Franke and Illes, 2014) and PNS (Furuya et al., 2014) uses ATP as a co-transmitter (Burnstock, 1976, Abbracchio et al., 2009).

Extracellular ATP, when released from damaged tissue in humans, causes pain [Bleehen T 1976 and check 1977]. ATP is a potent excitor of P2X and, due to heteromization of P2X and the potential for more than one P2X subtype per cell (Petruska et al., 2000) , there is diversity in purinergic signaling. Variation in signaling is dependent on the time required for channel desensitization (temporary inactivation) and recovery (Rettinger and Schmalzing, 2003, Bhargava et al., 2012), agonist or antagonist binding (Sokolova et al., 2004) and regulation by, for example, calcium in the extracellular environment (Fabbretti et al., 2004). The predominant purinergic subtypes expressed on sensory neurons are P2X2 and P2X3. Homomeric P2X2 can only be activated by ATP and is slowly desensitizing. Conversely, P2X3 is responsive to ATP and its analogue, αβmethylene-ATP (αβ-meATP) with rapid desensitization; clearly contributing to the multiplicity of purinergic signaling. See Table 2.6.2 for a summary of the properties of the P2X receptor family.

Table 2.6.2 Properties of P2X receptors

P2X Receptor Subtypes	Agonists: ATP or αβ-meATP	Permeation properties	Desensitisation	Refs
P2X1	ATP; αβ-meATP	Cation-selective: Na+, Ca2+, K+	Fast	(Evans et al., 1996, North, 2002, Roberts et al., 2006a)
P2X2	αβ-meATP	Cation-selective; larger cations	Slow	(Evans et al., 1996, North, 2002, Roberts et al., 2006a)
P2X3	ATP; αβ-meATP	Cation-selective	Fast	(North, 2002, Roberts et al., 2006a)
P2X4	αβ-meATP	Cation-selective; larger cations	Slow	(North, 2002, Roberts et al., 2006a)
P2X5	αβ-meATP	Cation-selective	Slow	(North, 2002, Roberts et al., 2006a)
P2X6	ATP; αβ-meATP	Cation-selective	Slow	(North, 2002, Roberts et al., 2006a)
P2X7	ATP: high concentration; αβ-meATP	Cation-selective; larger cations	Slow	(North, 2002, Dubyak, 2007)
P2X2/3	ATP; αβ-meATP	Cation-selective	Slow	(North, 2002, Roberts et al., 2006a)
P2X1/2	ATP; αβ-meATP	Cation-selective	Fast/slow	(Roberts et al., 2006a)
P2X1/4	Unknown (for ATP); αβ-meATP	Cation-selective	Slow	(Roberts et al., 2006a)
P2X1/5	ATP; αβ-meATP	Cation-selective	Fast/slow	(North, 2002, Roberts et al., 2006a)
P2X2/6	ATP	Cation-selective	Slow	(North, 2002, Roberts et al., 2006a)
P2X4/6	ATP; αβ-meATP	Cation-selective	Slow	(North, 2002, Roberts et al., 2006a)
P2X4/7	Unknown	Cation-selective	unknown	(Dubyak, 2007)

 

The P2X channel opens rapidly in response to the binding of fewer than 3 molecules of ATP (Bean, 1990, Stelmashenko et al., 2012), allowing the ions, particularly calcium, to flow. Pore permeability is then affected by the time-course of response of that particular P2X channel to the presence of the agonist. P2X2, P2X4 and, most notably, P2X7 undergo a change in pore size with the continued presence of ATP, resulting in permeability to larger cations (Surprenant et al., 1995, Dubyak, 2007, Stelmashenko et al., 2014), altering the transmembrane potential (Mio et al., 2005, Khakh and North, 2006). The remaining P2X subtypes respond to the ongoing presence of an agonist by either rapidly or slowly desensitizing. Desensitization is the reduced response to an agonist in the continued presence of the agonist (Burnstock and Kennedy, 2011), leading to the closure of the pore. Agonist unbinding is required for receptor recovery from the desensitized state. P2X1 and P2X3 are rapidly desensitizing receptors and P2X2, P2X4, P2X5 and P2X7 are slow (Rettinger and Schmalzing, 2003, Roberts et al., 2006a, Kaczmarek-Hajek et al., 2012). Additionally, more than one P2X subtype may assemble in one cell (Cook et al., 1997, Thomas et al., 1998, Grubb and Evans, 1998). This means the P2X receptor family substantially influences the presence of extracellular ATP, the membrane potential and intracellular Ca²⁺. This, in turn, underpins a number of physiological functions including sensory transduction and pain (Fabbretti, 2013).

2.12.5 Purinergic Signaling in CNS and PNS

P2X receptors mediate fast synaptic communication in a number of ways. Firstly, the potent activity of ATP (or αβ-meATP) allows the channel to open within milliseconds of binding. Fast or slow desensitization of the channel regulates the on-going neuronal response to ATP (Roberts et al., 2006a). If there is increased ATP there will be an increased number of membrane depolarisations, affecting channel recovery time, subsequent P2X expression and rapidity of successive cycles of extracellular ATP binding (Chen et al., 2005). Combined with the constant ATP binding and unbinding, is the trafficking of P2X receptors to the cell surface. The efficiency of trafficking, therefore, plays a role in signaling.

P2X receptors are synthesized, with some glycosylation, in the rough endoplasmic reticulum. Further glycosylation occurs in the Golgi apparatus, with plasma membrane trafficking occurring via vesicle exocytosis. Recycling of these vesicle proteins occurs through endocytosis (Heuser and Reese, 1973), ‘kiss-and-run’ retrieval of proteins prior to full vesicle collapse (Fesce et al., 1994), rapid reversal of vesicle-surface connection (Royle and Lagnado, 2003) and through the intrinsic pore permeability to Ca²⁺  (Hollins and Ikeda, 1997). Combined, these factors mediate fast intra-cellular communication and contribute to neuronal plasticity in pain states (Wu et al., 2014).

2.12.6 Purinergic Signaling in Pain States

Visceral pain appears to arise due to nociception, local inflammatory influences and neuropathic factors (Giamberardino MA 1999, Mayer EA 1994) and is different from cutaneous pain (Chang 2002). Emerging evidence implicates purinergic receptor subtypes P2X2 and P2X3 in visceral pain pathways, contributing to nociceptive, inflammatory and persistent pain states. P2X2 and P2X3 are expressed on primary afferents and in sub-populations of small, sensory neurons in the DRG [North RA 2002] innervating the viscera [Bradbury EJ 1998] and projecting to the dorsal horn [Rexed B 1952]. P2X2 is widely expressed, including post-synaptically [Jahr and Jessel 1983], whilst P2X3 expression is confined to the peripheral sensory neurons {Burnstock 2013a]. A loss of P2X2 and P2X3 DRG sub-populations in knockout mice decreases the response to noxious stimuli (Bradbury 1998). Additionally, in P2X3 null mice, there is a loss of ATP-induced depolarization in the DRG with a corresponding reduction in pain behaviour [Cockayne 2000, Zhong Y , Cockayne DA 2001].

The purinergic receptor contributes to nociceptive pain transmission through mediation by extracellular ATP [Tsuzuki K 2003, Pannek J 2009, Gao, Yun 2010, Benarroch, EE 2015]. ATP is released peripherally from sensory neurons [Holton and Holton 1954] and non-neuronal cells [Chambers 2007, Lambert 2008, Balse 2012] in response to a nociceptive event. Nociceptive triggering may occur in the presence of actual or potential tissue damage. Therefore, ATP may be released from healthy cells  (Lazarowski, 2012) to participate in neuronal signal transduction (Armstrong and Hille, 1998). Consequently, both damaged or apoptotic and healthy cells may release ATP in response to chemical and mechanical stimuli. These stimuli are present in certain conditions such as inflammation, vasoconstriction or dilation and maintenance of the local hormonal mileau. As a result, local influences on extracellular ATP binding, calcium influx, membrane depolarization and action potential generation are increasingly important in detailing nociceptive, inflammatory and neuropathic pain states (Snyder, 1992) [Fabbretti 2013].

For instance, extracellular ATP modulates vascular tone, angiogenesis and regeneration of damaged vessels via the puringergic receptor (Burnstock and Ralevic, 2014)[Yegutkin G 2015]. The purinergic hypothesis suggests that ATP released from endothelial cells, possibly due to smooth muscle constriction and shear-stress from turbulent blood flow, may bind to co-localised primary afferents [Burnsotck 1981], amplifying acute nociceptive signals (Birder, 2005) resulting in hyperalgesia  [Joseph E 2013].

Likewise, ATP, released from visceral organ endothelial cells in response to excessive distention, initiates a pain pathway (Burnstock, 1996). Specifically, ATP released from the over-filled bladder wall activates mechanically-insensitive (MIA) nociceptors, signaling pain. Reduced pain behaviour was noted in P2X2 and P2X3 knockout mice (Cockayne et al., 2000), despite increased bladder volume (Wang et al., 2005)and decreased voiding frequency (Cockayne et al., 2000). This suggests a pro-nociceptive role for the P2X receptor (Burnstock, 1999, Joseph et al., 2014, de Groat et al., 2015).

There is emerging importance of specific features of P2X2 in pain signaling [Burnstock 2014].  As noted above, the P2X2 pore dilates during sustained ATP-analogue application [Virginio C 1999, Compan 2012] allowing permeability to larger ions [Khakh 1999] potentially altering neuronal function [Virginio c 1999]. In its open-state, P2X is particularly sensitive to extracellular ATP [Yan 2010]; with animal studies indicating increased extracellular ATP in inflamed tissues in vivo [Bours M 2011] suggesting amplified P2X2 activity on sensory neurons. The open and dilated P2X2 pore has greater ion conductance [Rokic and Stojilkovic 2013]; yet, desensitises, or closes, in the presence of extracellular calcium [Khadra,   2012]. It is then the level of intracellular calcium that potentiates ATP, or its analogue, via downstream signaling cascades [Fabbretti 2013, Hagenston A 2014], modulating neurotransmitter release {Gu and MacDermott 1997, Dolphin 2013] and increasing nociceptive signaling [Fabbretti E 2013] and inflammation [Bours 2011].

P2X3, also, appears to contribute to inflammatory and chronic pain (Barclay et al., 2002, Fabbretti, 2013); specifically, via its properties of fast-onset desensitisation and slow recovery (Table 2.6.2). Desensitisation is the loss of receptor responsiveness in the continued presence of an agonist (Giniatullin and Nistri, 2013). Repetitive agonist applications in animals studies resulted in reduced nociceptive behaviour linked to a P2X3 nociceptive pathway (BlandWard and Humphrey, 1997, Barclay et al., 2002, Ren et al., 2006). This behaviour also potentially arises due to another property of P2X3 known as high-affinity desensitisation (HAD)(Sokolova et al., 2006, Giniatullin and Nistri, 2013). HAD means that the receptor can desensitize in the presence of very low concentrations of an agonist, yet signal transduction does not occur (Karoly et al., 2008). ATP, the predominant P2X3 agonist, is also a potent inhibitor of P2X3 (Sokolova et al., 2004, Helms et al., 2013) with as few as two ATP molecules (Stelmashenko et al., 2012) inactivating P2X3 (Grote et al., 2008). Given the ambient level of extracellular ATP present at any moment, this suggests that a proportion of P2X3 will always be inactive, possibly to reduce inappropriate nociceptive signaling (Illes et al., 2008, Giniatullin and Nistri, 2013).

Recovery from desensitisation is generally slow in P2X3 (Cook et al., 1998); yet, ‘resensitisation’ occurs more rapidly with increased temperature (Khmyz et al., 2008) higher levels of extracellular calcium (Cook et al., 1998, Fabbretti et al., 2004), high levels of neurotrophins (Fabbretti et al., 2006, D’Arco et al., 2007, Russell et al., 2014)and extracellular acidification which occurs in inflammation (Gerevich et al., 2007, Mo et al., 2013).  It is thought that in inflammatory and chronic pain states conditions alter to support pro-nociceptive effects of P2X3; namely, the recruitment of inflammatory mediators (Viatchenko-Karpinski et al., 2013), release of neuropeptides (Fabbretti, 2013),increased neurotrophins (Osikowicz et al., 2013),removal of ambient ATP (Grote et al., 2008), extracellular calcium fluctuations due to other P2X activity (Ding and Sachs, 2000), reduced HAD (Pratt et al., 2005), improved recovery times (Pratt et al., 2005)and, even heterodimerisation with P2X2 (North, 2002, North, 2004). Together, these factors shift the balance from steady-state P2X3 to open-channeled, active pain transmission.

 

2.12.7 Purinergic Receptors in Female Reproductive Tissue

P2X receptors are identified on various female reproductive tissues. P2X1 and P2X2 mediate smooth muscle contraction and are found in the myometrium of the mouse (Ninomiya and Suzuki, 1983), cat (Bulbring et al., 1968), guinea pigs (Piper and Hollingsworth, 1996), rats (Bardini et al., 2000) and humans (Ziganshin et al., 2006, Hutchings et al., 2009). P2X2 is found in the vascular endothelial cells of the fallopian tubes (Bardini et al., 2000) and mediates sperm capacitation (a necessary step for successful fertilization of the oocyte) (Banks et al., 2010). P2X2 also mediates non-pregnant uterine spasms (Piper and Hollingsworth, 1996) and pain (North, 2002). P2X3 is on sensory nerves and has a role in the sensory-motor reflex of contraction and pain (Chaban, 2008). The heterodimer, P2X2/3 is expressed by rat uterine smooth muscle (Bardini et al., 2000) increasing during pregnancy (Ziganshin et al., 2002) and, with P2X2, mediates co-ordinated contractions at term (Ziganshin et al., 2006).

In the female reproductive tract, P2X4 mediates the regulation of epithelial transport, necessary for the sperm and egg to meet. Additionally, P2X4 possibly has a role in luminal acidification of the cervix and vagina (Belleannee et al., 2010). Luminal acidification assists spermatozoa in reaching full fertilizing ability. P2X5 is involved in cellular differentiation and is anti-proliferative (Bardini et al., 2000).  P2X7 is expressed in the myometrium of pregnant rats (Urabe et al., 2009, Miyoshi et al., 2010, Miyoshi et al., 2012). Likewise, endometrial cells express P2X7; noted in the rat uterine epithelium (Bardini et al., 2000), rat luminal epithelium and stromal cells (Tassell et al., 2000) and human epithelial cells (Li et al., 2007, Gorodeski, 2009). P2X receptors are found in all reproductive tissues with differential expression throughout the menstrual cycle and pregnancy (Burnstock, 2014).

2.12.8 Purinergic Signaling in Female Reproductive Tissue

Extracellular ATP mediates steroidogenesis (Park et al., 2003, Peluso et al., 2007), endocrine modulation (Del Canto et al., 2007), trans-epithelial transport through the fallopian tubes (Keating and Quinlan, 2008), maintenance of uterine and peritoneal fluid environment throughout the different phases of the menstrual cycle  (Keating and Quinlan, 2012, Aliagas et al., 2013), angiogenesis (Haynes et al., 2003, Burnstock, 2014, Burnstock and Ralevic, 2014), neurogenesis (Tsai et al., 1996), limits the local inflammatory processes (Zanin et al., 2012), implantation (Slater et al., 2002), syncytiotrophoblastic ion transportation (Roberts et al., 2006b), myometrial (Ziganshin et al., 2006) and smooth muscle contractility (Ziganshin et al., 2002, Burnstock and Knight, 2004) within the female reproductive tract.

Extracellular ATP is implicated in the temporal and spatial transformation of the cellular architecture of the endometrium throughout the menstrual cycle. These changes include cell proliferation (Villavicencio et al., 2009), programmed cell death (apoptosis) (Thompson et al., 1999, Haughian et al., 2006, Insel et al., 2012), autocrine and paracrine immune cell recruitment (Ho et al., 2006, Berbic et al., 2009), lymph-angiogenesis and neurogenesis (Hill et al., 2010, Hey-Cunningham et al., 2013), endocrine-induced proliferation (Chan et al., 2013), inflammation and apoptosis (Li et al., 2006, Kizilay et al., 2008, Vazquez-Cuevas et al., 2013), tissue regeneration (Makarenkova and Shestopalov, 2014), synthesis and secretion of endometrial fluid (Chan et al., 1997), differential protein expression during the window of implantation (Li et al., 2011) and endometrial receptivity (Ruan et al., 2012, Ruan et al., 2014).  Endometrium is a highly dynamic tissue with extracellular ATP and its purinergic receptor playing a crucial role in endometrial cyclic remodeling.

2.12.9 Purinergic signaling in Endometriosis Associated Pain

Direct evidence for the role of the purinergic receptor in endometriosis is limited; yet, emerging evidence suggests activation of the purinergic receptor is a major contributor to pro-nociceptive, neuroendocrine, angiogenic and neurogenic signaling in endometriosis-associated pain.

Activation of the purinergic receptor can be highly influenced by the presence of estrogen. Estrogen receptors α and β (ERα and ERβ) are expressed in the DRG of small diameter, sensory neurons [Papka 2002] as are P2X receptors [Cho T 2012]. In ERα and ERβ knockout mice, ATP induced measureable calcium influx in L1- S1 DRG [Chaban 2003] via P2X [Cho T 2012] suggesting a pro-nociceptive role for estrogen. More specifically, activation of ERα appears to be anti-nociceptive whilst ERβ is pro-nociceptive [Coulombe MA 2011]; although, this is disputed [Ji y, Tang B, Traub RJ 2011] with a rat model indicating that ERα mediates visceral pain via a local effect at the DRG [Chaban 2011, Traub 2013]]. It is thought that high local estrogen attenuates ATP-induced depolarization [Chaban 2011] increasing pain sensitivity. Estrogen, therefore, is exerting a local, non-reproductive effect, most likely through a rapid onset, non-genomic, alternative pathway [ Micevych and Dominguez 2009], in the DRG [Chaban and Micevych 2005], with estrogenic effects taking seconds [Kelly 1976] rather than hours [Cornil C 2006] and occurring irrespective of systemic levels of estrogen [Amandusson A and Blomqvist, A 2013].

Estrogen is also neuroprotective [Wang 2003], exerts regulatory effects on neurotransmitters [McEwan 2012], stimulates synaptic remodeling [Sellers K 2015] and neurite sprouting [Barth C 2015]. Sprouting, sensory innervations [McAllister SL, Pyatok SA 2012] in a rat model of endometriosis contributes to hyperalgesic pain behaviours [McAllister SL 2012], with pro-nociceptive, inflammatory molecules [Scholl B 2009] released by endometriotic lesions [Alvarez P 2012] sensitizing local afferents [Gold and Gebhart 2010] through increased levels of extracellular ATP successfully gating the purinergic receptor.

The purinergic receptor plays a significant role in the maintenance of vascular tone and remodeling [Burnstock 2014]. P2X is expressed in the endothelial cells of the vasculature as well as the surrounding smooth muscle [Burnstock 2014]. The endothelial cells release ATP due to shear stress [Burnstock 1999] or in the presence of inflammatory cells [Burnstock 2014b]. This promotes non-neuronal, neuronal cross-talk with neuronal P2X activity [Franceschini A 2013], modulated by released ATP [Inoue and Tsuda 2012] triggering nociceptive signaling [Magni G 2014] and contributing to inflammatory aspects of neuropathic pain [Fabbretti 2013].  Extracellular ATP also has an angiogenic effect promoting re-endothelialization [Shen and Di Corleto 2008], proliferation [Hoeben A 2004] and neovascularization [Kaczmarek  M 2005].

Angiogenesis is critical for repair, regeneration, growth and differentiation within the endometrium, being primarily augmented by vascular endothelial growth factor (VEGF) and under the control of estrogen and progesterone [Girling Jane E 2005]. Hormonal regulation of endometrial angiogenesis is complex and appears to be dysregulated in endometriosis [Hey-Cunningham 2013]. The eutopic endometrium and peritoneal lesions of women with endometriosis appear to have greater angiogenic potential [Li, Y. Z. 2013, VEGF meta-analysis, da Silva C M 2014]. These trophic actions are mediated by P2X [Burnstock 2014] affecting cytoskeletal changes, cellular adhesion and motility of newly-formed blood vessels [Kaczmarek M 2005]. Additionally, the inflammatory environment of endometriosis stimulates the activity of VEGF [Veikkola T 2000] further promoting angiogenesis [Rumjahn 2009]. Moreover, inflammatory conditions also increase extracellular ATP[Scodelaro B 2012], which forms a complex with VEGF, giving rise to added proliferation of endothelial cells [Gast 2011]. Neo-vascularisation associated with endometriosis, co-localised with P2X expressing sensory neurites, appears to contribute to endometriosis-associated pain [Zhang G 2008].

 

2.13 Vanilloid Receptor

The Transient Receptor Potential Vanilloid sub-types 1 – 6 (TRPV1 – 6) forms a branch of a super-family of non-voltage-gated, non-selective cation channels [Montell C 2002], with TRPV1, the first mammalian member of this part of the family, expressed on small-diameter sensory neurons [Caterina MJ 1997] as well as in the brain [Toth A 2005, Puente, Nagore 2015] and non-neuronal tissue [Birder LA 2002]; playing an important [Caterina MJ 1997] and ever-increasing role in the pain pathway [Fischer Michael JM 2013].  These multi-modal receptors transduce the principle types of pain stimuli: thermal and chemical [Caterina MJ 1997, Tominaga M 1998]. TRPV1 is gated by heat (>42°C) [Caterina MJ 1997], exogenous vanilloids [Gavva NR 2004] and protons, with acidic conditions (pH </= 5.9) activating TRP at room temperature [Tominaga M 1998]. Activation of thermo sensitive TRP channels allows calcium influx, membrane depolarization, action potential generation and nociceptive transmission. Together, the thermo-sensitive TRP family detects signals ranging from noxious heat to noxious cold [Romanovsky 2007].

 

 

 

 

2.13.1 Structure

Figure 2.6. Structural details of a single TRPV1 subunit [Liao M 2013]

TRPV1 topology (Figure 2.6. ) consists of four symmetrical subunits and 6 transmembrane  helices (S1 – S6) forming a pore between S5 and S6, with the C and N termini located intracellularly, and a glycosylation site available in the pore region[Vannier B 1998]. S5 faces the lipid membrane and S6 lines the pore [Clapham DE 2005]. The pore is also lined by S1 – S4 subunits, creating a large extracellular vestibule and a short ion selectivity filter [Liao M 2013]. S1 – S4 contains a conserved region of 5 amino acids [Clapham DE 2005]. These aromatic amino acids seem to stabilize TRPV1 with S1 to S4 anchoring the channel and the S4-S5 linker being the fulcrum around which the pore opens [Liao M 2013] in response to ligand binding [Cao 2103]. Experimental ligands included capsaicin, plant-based resiniferatoxin (RTX) and double-knot toxin (DkTx ) from tarantula venom in complementary studies utilizing cryo-electron microscopy to detect three conformational states of TRPV1 [Liao M 2013, Cao E 2013]. The studies revealed a dual gate with RTX/DkTx binding in the wide-mouthed funnel, allowing ion conduction, and capsaicin nestling in a hydrophobic region beyond the ion selectivity filter and closer to the S1-S4 subunit (Figure 2.6. ). Protons were found to bind at the outer pore; and the team plan future studies to determine how temperature gates TRPV1 [Cao E 2013, Liao M 2013].

Figure 2.6.2.x Channel conformation of TRPV1 (Henderson R 2013)

Whilst the architecture of TRPV1 may be similar to other ion channels [van Geest Marleen 2000]; its high permeability to calcium and other functional attributes define its physiological role [Jordt SE 2000, Clapham D E 2005].

 

 

2.13.2 Function

TRPV1 is key sensor of heat pain and its activation contributes to persistent pain sensitivity. TRPV1 is activated by temperatures above 43°C [Caterina 1997], acid [Tominaga 1998], anandamide [Zygmunt PM 1999], N-arachidonayl-dopamine (NADA) and N-oleoyldopamine [Hwang PNAS 2000 97; 6155-6160, Hwang PNAS 2002 99;8400-8405], 12-hydroxyperoxyeicosatetranoic acid (HPETE), 15-HPETE and Leukotriene B4 [Shin PNAS 2002, 99; 10150-10155].

TRPV1 is indirectly activated or sensitized by NGF [Chuang Nature 2001 411;957-962], bradykinin [Chuang 2001], prostaglandins [Moriyama , Molecular Pain 2005, 17;1-13], histamine [Shim, J. Neurosci, 2007; 28;2331-2337], serotonin [Sugiura J. Neurosci, 2004; 24;9521-9530], prokineticin  [Negri, J. Neurosci, 2006, 26;6716-6727] and proteases [Amadesi, J. Neurosci, 2004, 24;4300-4312].

 

 

involved in the transduction of noxious stimuli and the transmission of pain.

2 open states with plant-based resiniferatoxin (RTX) and double-knot toxin (DkTx ), from tarantula venom; gates can communicate with one another; underpins TRPV1 modulation and contribution to pain hypersensitivity

TRPV1 is the first of the 27 member TRP family to have the structure observed in its entirerity

 

2.13.3 Distribution

TRPV1 is synthesized in DRG and transported peripherally along small Aδ and C fibres and, centrally, to the dorsal horn {Szallasi 1995, Caterina 1997, Tominaga 1998].

Expressed in non-sensory cells

TRPV1-4: regulated by nicotinic acid (NA aka vit B3 or niacin); NA causing cutaneous flushing; lowers activation threshold of TRPV1, inducing action at body temp [Ma, Linlin 2015]

Properties of TRP family are detailed in Clapham D et al 2005 including TRPV 1– 6 etc

Table 2.6.x.x. Properties of TRPV1 [Clapham D 2005]

TRPV1	Details	References
Ion Selectivity	Ca2+ >  Mg2+ >  Na+  ~ K+  ~  Cs+	Caterina and Julius 2001
Activation	Increasing temperature shifts activation to within physiological range; acid	Caterina MJ 1997;  Jordt SE 2000; Brauchi 2004
Inactivation	Intracellular Ca2+: cAMP-dependent protein kinase directly phosphorylates TRPV1	Premkumar LS and Ahern GP 2000; Vellani V 2001; Bhave G 2002;
Activators	Capsaicin, RTX, DxTx, heat >42°C, extracellular protons, anandanmide; Indirect: NGF, bradykinin, prostaglandins, serotonin etc	Caterina MJ 1999; Jordt SE 2000; Chuang HH 2001;
Blockers	Capsazepine, ruthenium red, iodo-resiniferatoxin, BCTC, PIP2	Chuang HH 2001; Zhuang ZY 2004
Inhibitors	PIP2; calmodulin	Prescott and Julius 2003; Chaudury 2011
Distribution	Primary afferents, DRG, CNS and non-excitable, non-neuronal tissue	Birder LA 2002;
Physiological functions	Depolarization and calcium influx due to heat, exogenous or endogenous ligands; pain sensation; vasodilation	Caterina MJ 2000; Woodbury CJ 2004;
Pathophysiology	Pain; Pain hypersensitivity; Inflammatory hyperalgesia	Caterina MJ 1997; Caterina MJ 2000; Davies JB 2000;  Zhuang ZY 2004; Fischer MJM 2013
Pharmacological significance	Potential analgesic site; blocking causes hyperthermia and decreased response to painful heat in humans	Gavva, NR 2004; Fischer MJM 2013
Comments	TRPV1 is not activated by Ca2+ store depletion	Clapham D 2005

 

2.13.4 Vanilloid Signaling

Pore dilation: time and agonist-dependent manner [Munns, Clare 2015]; larger cation permeability

2.13.5 Vanilloid Signaling in CNS and PNS

Zhang X….McNaughton PA 2005: NGF rapidly increases membrane expression of TRPV1

2.13.6 Vanilloid Signaling in Pain States

VR1 activity ameliorated by inflammatory mediators such as NGF, somatostation, bradykinin, prostaglandins, serotonin

VR1 is integrated in multiple signaling pathways in nociceptor sensitization

TRPV1 potentiates TRPA1 activity and vice versa by forming a complex and interacting with Tmem100 in DRG to mediate persistent pain [Weng, Hao-Jui 2015] Tmem KO mice show reduced inflammatory mechanical hyperalgesia; context dependent modulation of TRPV1

2.13.7 Vanilloid Receptor in Female Reproductive Tissue

2.13.8 Vanilloid Signaling in Female Reproductive Tissue

2.13.9 Vanilloid Signaling in Endometriosis associated Pain

VR1 elevated at PL and correlated with CPP (Rocha MG et al Reprod Sci 2011)

 

 

 

 

 

2.14 Psychological Processing of Visceral Pain

Notes: Nociception and the interaction with the neural matrix: complex and involing those areas associated with emotion, thought, behavior and sexual response. PAG is potentially involved in neuromodulation and this may be one mechanism by which nociception is influenced at the spinal level.

At the spinal level, normal sensations and nociception utilize the same small afferent fibres from the viscera. Whether the message is normal or noxious depends on the intensity of the message ie the number of afferent signals transmitted to the dorsal horn.

This is in contrast to messaging in somatic tissue where the Adelta / C fibres code for nociception and the Abeta fibre transmits light touch.

Because of  the relationship between the neruomatrix and neuromodulation at the spinal level, it is thought that the intensity coding of visceral stimuli contributes to the greater influence of psychological neuromodulation / aspects of perception of visceral pain (as compared with the psychological influences in somatic pain).

 

Notes: Psychological contributors to the experience of pain

Psychology plays a role in nociception/pain neuromodulation at the higher level. The network of interaction may facilitate or reduce the nociceptive signal reaching consciousness impacting pain perception and the pain experience/behaviors.

Functional MR imaging has shown that the psychological neuromodulation of visceral pain occurs in a number of ways. Eg. Reducing pain through distraction probably engages a different area of the brain than the area of the brain influencing how mood impacts pain reduction. Quick response psychological neuromodulation of nociception (in the above examples) is a handy survival tool however there is a flipside to psychological neurmodulation. The neuroplasticity of the neuromatrix means there is the opportunity for long-term potentiation of visceral nociception ie. Learning. Intimate involvement of higher centres or networks may be significant in neuroprocessing of nociception and pain.

Neuroplasticity is a feature of the central and peripheral nervous system. Activation by a particular stimulus modality or modalities may establish neural pathways that leave  an individual vulnerable to perceiving sensations as painful or more painful than a non-affected individual.

Stress should not be overlooked. It may be defined as a threat to homeostasis, be intrinsic or extrinsic and physical or psychological. The response to stress may involve a number of corporal systems including the nervous, endocrine and immune systems. The potential for long term systemic abnormalities from stress is a mechanism by which life events can leave a physiological imprint and should be considered in persistent pain states.

 

 

2.15 Peripheral and Central Mechanisms in Endometriosis Associated Pain

(schematic)

 

 

 

 

 

 

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