A Surgical Opinion on Hyperalgesia/Nociception

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Current Immunology Reviews, 2012, 8, 275-286 275 A Surgical Opinion on Hyperalgesia/Nociception, Inflammatory/Neurogenic Pain and Anti-inflammatory Responses and Drug Interventions Revisited: Current Breakthroughs and Views John J. Haddad*

Cellular and Molecular Physiology and Immunology Signaling Research Group, Biomedical Laboratory and Clinical Sciences Division, Department of Medical Laboratory Technology, Faculty of Health Sciences, Beirut Arab University, Beirut, Lebanon

Abstract: All sensory modalities are essentially important, but pain serves a protective function and is indispensable for survival, and, technically, pain is considered one of the most common symptoms of injuries and related diseases. Inflammatory cells and inflammatory mediators are crucially involved in the propensity, genesis, persistence and severity of pain, commonly known as nociception or hyperalgesia, following trauma, infection, or nerve injury. When it pins down to the essential understanding of pain/hyperalgesia pathways and their intricate interactions with myriad probabilities of milieu of inflammatory cytokines and related molecules, the amicable concept of specificity and complexity remains a major dilemma. Various hyperalgesic models have been established to investigate this intricate relationship between pain perception and inflammatory responses. Illness-induced hyperalgesia, for instance, is one of the most common aspects of pain related-inflammation and therapeutic approach to this pain should aim at interfering with various mediators of the inflammatory reactions, including neuropeptides, eicosanoids and cytokines. In this surgical synopsis, a trajectory of neurochemical events and cascades are delineated and unraveled in terms of the connection that has ostensibly evolved for hyperalgesia-inflammatory responses. The unprecedented intricacy of pain-inflammatory relationship and putative pathways bears surmountable clinical and physiological relevance. Keywords: Cytokines, endotoxin, hyperalgesia, inflammation, neurochemistry, neurogenic, nociception, pain.

1. INTRODUCTION AND SURGICAL BACKGROUND increases in intensity over a period of several seconds or minutes. Impulses for slow pain conduct along small- All of our sensory modalities are essentially important, diameter, unmyelinated C fibers. This type of pain may be but pain serves a protective function and is indispensable for excruciating and often has a burning, aching, or throbbing survival. Pain is technically the most common symptom of quality [1, 2]. Pain that arises from stimulation of receptors injuries and diseases [1]. The International Association for in the skin is called superficial somatic pain; stimulation of the Study of Pain (IASP) developed a consensus definition receptors in skeletal muscles, joints, tendons, and fascia of pain; according to the IASP, “pain is an unpleasant bodily causes deep somatic pain. Visceral pain results from sensory experience commonly produced by processes that stimulation of nociceptors in visceral organs. In many damage, or are capable of damaging [or inducing noxious instances of visceral pain, the pain is felt in or just deep to damage of], bodily tissue”. Nerve cell endings, or receptors, the skin that overlies the stimulated organ, or in a surface commonly known as nociceptors “(noci- is derived from the area far from the stimulated organ. This phenomenon is Latin for “hurt”)”, are central to pain sensation and called referred pain (Fig. 1) [1]. assessment. Nociceptors are chemoreceptive free nerve endings activated by tissue damage from intense thermal, A nociceptive stimulus at the nerve endings unleashes a mechanical, or chemical stimuli; they are found in virtually cascade of events throughout the nervous system, which can every tissue of the body except the brain. Nociceptors, ultimately lead (and loop) back to the site of injury. This thereby, have the ability to relay information to the central response prompts cells of a variety of types in the injured nervous system (CNS), particularly the brain, thus indicating area to release chemicals and neurochemicals that trigger an the location, nature and intensity of the ensuing pain [1]. immune response and thus influence the intensity and duration of pain [1, 2]. The inflammatory milieu that usually Technically, there are two types of pain: i) fast and ii) precedes, and accompanies, pain is, purportedly, slow. The perception of fast pain (acute, well localized) transcriptionally regulated, such as with the nuclear factor occurs rapidly because the nerve impulses propagate along (NF)-B, a major transcription factor, essentially involved in medium-diameter, myelinated A fibers. By contrast, slow mediating inflammatory processes and the release of pain begins after a stimulus is applied and gradually inflammatory mediators [1]. Current strategies aiming at controlling the origin, and propagation, of pain, as well as its manifestations, reveal the crucial role that this transcription *Address correspondence to this author at the Department of Medical Laboratory Technology, Faculty of Health Sciences, Beirut Arab University, factor has in modulating pain and nociception, following the Beirut, Lebanon; Tel: +961 1 300 110; Fax: +961 1 378 159; ensuing inflammatory cascade. In lieu with the E-mail: [email protected] aforementioned description, the author quotes a technical

Fig. (1). Schematics of nociceptors and common pain patterns/modules. (A) The nerve free ending of a nociceptor. Nociceptors are chemoreceptive free nerve endings activated by tissue damage from intense thermal, mechanical, or chemical stimuli. (B) Common patterns of referred visceral pain are shown in this graphic: (a) anterior and (b) posterior views. (Adapted, courtesy of Tortora et al., Principles of Anatomy and Physiology, 13th edition, 2012, John Wiley & Sons Publishers). note identifying the natural and current themes pertaining to pain, commonly known as nociception or hyperalgesia, inflammatory hyperalgesia and nociception pathways [1, 2]. following trauma, infection or nerve injury [1, 2]. It is essentially conspicuous that when studying hyperalgesia and “A natural assumption is that the sensation of pain arises inflammatory responses that animal, and recently human, from excessive stimulation of the same receptors that generate other somatic sensations. This is not [usually] the case. models can play an important role in the development, screening and evaluation of analgesic and anti-inflammatory Although similar in some [and probably in many] ways to the actions of clinical drugs [1, 2]. The availability of appropriate sensory processing of ordinary mechanical stimulation, the models, therefore, is of particular importance, if not crucial, for perception of pain (called nociception) depends on specifically such investigations [3, 4]. Different models have been utilized; dedicated receptors and pathways. Since alerting the brain to as an illustration, carrageenan, a polysaccharide extracted from the dangers implied by noxious stimuli differs substantially from informing it about innocuous somatic sensory stimuli, it makes the cell wall of red algae, has been used to induce peripheral inflammation and edema, restricted to the injected paw [5, 6]. good sense that a special subsystem be devoted to the This model has been, for example, used to evaluate the effects perception of potentially threatening circumstances. The of non-steroidal anti-inflammatory drugs (NSAIDs) [6, 7]. overriding importance of pain in clinical practice, as well as the Other studies have used the complete Freund’s adjuvant (CFA), many aspects of pain physiology and pharmacology that remain which contains attenuated heat-killed Mycobacteria in the imperfectly understood, continue to make nociception an extremely active [and burgeoning] area of research”. (Adapted, water-in-oil emulsion, which induces a severe inflammation of the injected hind paw characterized by erythema, edema and Scott T. Brady et al., In: Basic Neurochemistry: Principles of th hyperalgesia with a peak response lasting for several days post Molecular, Cellular, and Medical Neurobiology, 8 edition, induction [1, 2]. 2012, Academic Press Publishers). Localized and central endotoxin (ET) administration, Inflammatory cells and inflammatory mediators are crucially involved in the genesis, persistence and severity of another projected inflammatory model, has been shown to Hyperalgesia and Inflammatory Responses Current Immunology Reviews, 2012, Vol. 8, No. 4 277 induce, when injected intraperitoneally, systemic and, if set free, induce in mammalians potent hyperalgesia mediated by cytokines, which are released from pathophysiological effects (see Fig. 2). Chemically, they are the Kupffer cells and acting through the vagal nerve [8-10]. LPSs consisting of an O-specific chain, a core In addition, various models were utilized to induce, for oligosaccharide and a lipid component, termed lipid A, example, arthritic inflammation. CFA produced slow whose partial structures proved that the expression of arthritis when injected into joints of animals, and cytokines endotoxic activity depends on a unique primary structure and can induce similar effect [11]. Sodium urate has been also a peculiar endotoxic conformation. When bacteria multiply, used for the evaluation of analgesic and anti-inflammatory and also when they die and lyse, LPS is set free from the agents since its injection in the ankle joint of rats induced surface. LPS, orET, is recognized as a potent bacterial toxin, and in a model of gouty arthritis [12]. and acts as a major factor mediating toxic manifestations of On another front, it is well known that the inflammatory severe gram-negative infections and generalized sepsis [1-5]. On the other hand, LPS represents a highly active pain induced by various agents in animal models produces a immunomodulator that is capable of inducing nonspecific long lasting and, often, a generalized effect. However, it is resistance to viral and bacterial infections and holds great important that any model utilized should cause minimal promise as a potent immunological adjuvant [15-23]. distress to the animal, since it has been observed that in certain forms of acute pain, the induced behavioral changes like tweezing and scratching can influence nociceptive 2.2. The Pharmacological and Immunological Effects of assessment tests [13-15]. The lipopolysaccharide (LPS) Endotoxin layer, also known as ET, as mentioned above, isa major The complex effects of ET on mammalian systems constituent of the outer membrane of the cell wall of gram- includenon-specific activation of the immune system, negativebacteria (Fig. 2) [14]. In various mammalian activation of the complementcascades and induction of the species, ET has been shown to induce a number of pathophysiological changes associated with disease such as characteristic shock syndrome. Many effects of ET are secondary to the overproduction of cytokines, specifically fever, hypotension, disseminated intravascular coagulation, TNF- and IL-1 , which are proinflammatory cytokines peripheral blood cell changes, aphegia, adipsia, slow wave produced by stimulated macrophages and other non-immune sleep and decreased social interaction [14]. In addition, cells [24]. TNF- has been considered a central mediator of intraperitonealinjections of ET have been found to induce septic shock since it has been observed that a polyclonal long lasting hyperalgesia [13]. It is well known that ET interactswith various cell types among which neutralizing serum to TNF- was highly effective in preventing ET-mediated inflammatory responses [24]. macrophages/monocytes are ofparticular importance since Intravenous injection of IL-1 also produces shock and acute their interaction with ET results in an increased production lung injury with sequestration of neutrophils and of eicosanoids, reactive oxygen intermediates and cytokines permeability edema [21-24]. In addition, LPS-induced such as tumor necrosis factor- (TNF-), and interleukin activation of macrophages results in the production of (IL)-1, IL-6, IL-8 and IL-10 [10-15]. bioactive lipids, reactive oxygen species (ROS) and peptide Citing a critical example, intraperitoneal injection of sub- mediators of inflammation other than TNF- and IL-l, such physiologic, small doses of ET (e.g., 100-200μg) has alsobeen as IL-6, IL-8 and IL-10. Both IL-6 and IL-8 have been shown not to be able to cross the blood-brain barrier (BBB) but shown to be present in bacterial sepsis in primates. rather to stimulate the Kupffer cells of the liver to release both In its capacity to affect immune cells, endotoxin, eicosanoids and cytokines [16-18]. On the other hand, several studies reported that ET crosses the BBB since LPS injected recognized as a pleotropic macroamphiphile that interacts with several types of host cells, constitutes perhaps the most intravenously was detected in the cerebrospinal fluid (CSF) potent and multivalent molecule of bacterial origin [24]. [18]. It was suggested that an interaction between the Endotoxins also induce fever either by acting directly on the phospholipid bilayers of BBB and the amphipathic LPS pre-optic area of the anterior hypothalamic thermoregulatory molecules facilitate the entry of LPS through the BBB. It was centers in the CNS, or by sensitizing the monocyte lineage, also proposed that LPS can cross back to the blood by simple diffusion or lipid-mediated and plasma protein-mediated granulocytes and platelets resulting in the release of endogenous pyrogens, including IL-1 and prostaglandins transport mechanisms [19-21]. Furthermore, it was shown that [21-24]. A concise summary of substances, ions, chemicals the released IL-1and TNF-, rather than the prostaglandins, and neurochemicals following tissue damage and injury is mediate hyperalgesia by stimulating vagal (hepatic) afferents thereafter projected below: that induce the inflammatory pain. Inflammatory cytokines have been shown to induce hyperalgesia by a mechanism Inflammatory Substance Putative Source independent of both prostaglandins and the sympathetic nervous + Potassium (K ) Damaged cells system, whereas IL-8 has been shown to act via the sympathetic pathway [22, 23]. Serotonin Platelets Bradykinin Plasma 2. BACTERIAL ENDOTOXIN AT A GLANCE Histamine Mast cells 2.1. The Molecular Biochemistry of Endotoxin Prostaglandins Damaged cells Endotoxins of gram-negative microbes, which are Leukotrienes Damaged cells components of the outer membrane of the cell wall, as is Substance P (SP) Primary afferent fibers well known, perform a vital function for bacterial viability 278 Current Immunology Reviews, 2012, Vol. 8, No. 4 John J. Haddad

Fig. (2). The biochemical architectural structure of lipopolysaccharide, endotoxin (ET). (A, B) Drawings showing the prominent features comprising the cell envelope of a typical gram-negative cell. (C) Schematic of Salmonella serotype Typhimurium lipopolysaccharide layer showing the three characteristic features of an LPS layer from a gram-negative outer membrane. Lipid A, the endotoxin portion, is the most conserved, whereas the O-antigen, which is an antigenic determinant, is the least conserved. The O-antigen sugars as well as the branching structures are quite variable in different gram-negative species. Abbreviations: GlcN, N-acetylglucosamine; Core PS, polysaccharide core; Gal, galactose; Glc, glucose; Rha, rhamnose; Abe, abequose; Man, mannose. Hyperalgesia and Inflammatory Responses Current Immunology Reviews, 2012, Vol. 8, No. 4 279

3. PAIN PERCEPTION AND PATHWAYS This field is referred to as zone of ‘primary hyperalgesia.’ Another area extending beyond the injury site into normal 3.1. The Dogma of Pain – Current Themes skin is referred to as a zone of ‘secondary hyperalgesia’ [1, 2]. Within the primary zone, low-intensity thermal and A detailed molecular perspective on pain pathways and interactions has been emphasizedand well elaborated upon mechanical stimuli evoke pain response. Specific nociceptors, in addition, become sensitized to mechanical [1, 2, 25-29]. A stimulus that causes, or is on the verge of stimuli after the administration of inflammatory agents. causing, tissue damage usually elicits a sensation of pain. Secondary hyperalgesia is characterized mainly by Pain differs from other somatosensory modalities inthose sensitization to mechanical noxious stimuli. The touch- emotions such as fear and anxiety and feelings of evoked pain that characterizes secondary nociception unpleasantness are experienced along with the perceived sensation. Also, a painful stimulus can evoke a reflex escape appears to be mediated by an alteration in central processing such as the activation of low-threshold mechanoreceptors or withdrawal response as well as physiological changes [1]. In fact, there is an induced responsiveness of central similar to those elicited during fear, rage and aggression [1, neurons to input from mechanically-sensitive nociceptors 2]. The stimuli that typically induce pain result in a sensory (see Fig. 1). Studies of nociceptive neurons in the spinal cord experience plus a reaction, or a response to it [1]. of experimental animals have shown that the input properties The classification of pain types and syndromes is a and functional responses of these fibers can evidently change matter of debate among scientists and clinicians. Pain types following noxious stimulation of skin, muscles, joints and may be classified according to terminology based on viscera [1, 2]. anatomical origin, clinical characteristics or etiology. To this Peripheral tissue inflammation produced by purpose, some general concepts are frequently used when inflammatory agents induces an activation of the sensory discussing different types of pain [1]. Somatosensory or nocigenicpain originates from the skin, thus called fibers which form intermingled networks at the receptive fields in addition to sensitization of spinal cord sensory ‘superficial’pain, or from connective tissue, muscle, bone, neurons which show increased spontaneous and evoked articular pain or synovial membranes and thus called activity, decreased thresholds to noxious stimulation, and ‘deep’pain. This type is mediated by the fast-conducting, enlarged receptive fields, which may extend contralaterally. lightly myelinated A fibers. On the other hand, visceral These neurophysiological changes are induced by alterations painis derived from hollow organs in viscera or thorax and is often poorly localized. Visceral pain, which is frequently in sensory processing caused by an increased synaptic facilitation of afferent input and/or a reduction of the effect accompanied by autonomic and somatic reflexes, is of inhibitory pathways [1]. Tissue damage and inflammation commonly referred to a somatic region. Characteristics of involve pathophysiological signaling events that are visceral pain are like those of cutaneous pain evoked by C associated with the mechanisms inducing pain and fiber (Table 1) polymodal nociceptive afferents [1]. hyperalgesia (Fig. 3). These features implicate: (i) changes in Neurogenic pain, also known as neuropathic pain, refers to pain originating from injury or irritation of a nerve branch local blood flow and vascular permeability; (2) sensitization, activation and migration of immune cells; and (3) (peripheral neurogenic pain), or to pain derived from injury stimulation of peripheral nerve fibers (neurogenic aspects of or disease within the CNS. This type is called central inflammation) leading to hypersensitivity. Sensory neurons neurogenic pain [1, 2, 25-29]. detect and respond to a range of chemical stimuli and they are recognized as crucial elements in the integration of 3.2. Hyperalgesia and Nociception – The Magnitude inflammatory signals [1, 2].

Hyperalgesia is technically defined as a change in the 3.3. Sensory Circuits and Mechanisms – The Specialty stimulus response function that relates the magnitude of pain or its threshold to the stimulus intensity. Hypersensitivity Skin sensitivity is determined by several kinds of develops in normal subjects after a cutaneous injury, and receptors or, as they are called, sensitive spots, each may also develop in normal skin under certain neuropathic subserving a modality of cutaneous sensation: pain, touch, conditions. The areas of nociception induced by an injury to heat and cold. Histologists have defined a variety of receptor the skin include a zone that incorporates the site of injury.

Table 1. The Properties of Different Groups of Sensory Afferent Fibers Ostensibly Involved with Various Putative Inflammatory Pain Circuits and Intricate Pathways

Specificity Myelinated Unmyelinated

Fiber Type A A C Diameter 5 – 15 μm 1 – 5 μm 0.25 – 1.25 μm Conduction Velocity 3 – 100 m/s 10 – 30 m/s 1 – 2.5 m/s Receptor Type Specialized/Free Free Free

Light/Heavy Pressure; Light/Heavy Pressure; Light/Heavy Pressure; Response Heat 450C; Heat 450C; Heat 450C; Cooling Cooling Chemicals; Warmth 280 Current Immunology Reviews, 2012, Vol. 8, No. 4 John J. Haddad endings in skin and other tissues, the most common are the substantial gelatinosa at the level of laminae I and II. The free nerve endings. Anatomically discernible endings in cross section of the spinal cord shows the laminae or layers hairy skin and in smooth skin with overlapping receptive of cells in the dorsal horns which receive sensory neurons fields interconnect with sensory nerve fibers which, by virtue that project their axons toward the brain along ascending of their myelination, are subdivided into 3 major groups fiber tracts. Some of these fibers, which occupy the (Table 1): A, or large myelinated fibers, A, or small anterolateral divisions of the spinal cord, continue their myelinated, and C, or unmyelinated fibers. The majority of projections to the thalamus, forming the spino-thalamic tract, the sensory afferents are of the C type [1, 2]. The whereas the majority of the fibers penetrate tangles of short, specialization of every skin receptor would be defined in diffusely interconnected neurons that form the central core of terms of its position in a multidimensional space of the lower part of the brain, or the brain stem, also known as physiological variables. By definition the term ‘nociceptor’ the reticular formation. This area of the brain receives is an acronym of ‘nocireceptor’ and denotes a receptor for somatosensory input and projects information in a series of nociceptive stimuli [1]. These receptors, whose stimulation pathways and along complex routes to other regions of the produces pain, are located at the ends of small unmyelinated brain, including the hypothalamus. Such a highly specialized or lightly myelinated afferent fibers or neurons. Some of system is particularly involved in the process of pain them may respond preferentially to intense or noxious perception and integration, leading to the behavioral patterns mechanical stimulation, others to mechanical and thermal of stress, aggression, withdrawal and defense [30-35]. stimulation and still others to irritant chemicals as well. Nociceptors encode the intensity, duration and quality of 3.4. Current Themes in Pain Mechanisms and Pathways noxious stimuli and, by virtue of their topographically and the Inflammation Connection organized projections to the spinal cord, the location of the stimulus [30-35]. Nociceptive signaling can be considered as a correlate of motor reaction (reflex mainly) aiming at protecting the living

tissue. Nerve endings of thinly myelinated and/or Injury and Infection unmyelinated fibers are activated by excessive stimulation (e.g., mechanical, chemical and thermal) that can lead ultimately to a damage or injury. The beneficial aspect of this signaling and its crucial role for survival are strongly suggested from the major deformities and limited life-span of individuals with congenital analgesia (absence of pain)

Inflammation despite all measures made to compensate for this absence. From this aspect, acute nociception can be considered as a sensory modality like touch and taste, with the added survival value just alluded to. On the other hand, when nociceptive signaling persists for a long period of time Vascular Changes Immune Cells Activation Sympathetic Nerves exceeding its determining causes, it becomes a pathological entity by itself to be potentially labeled as chronic pain. The development of this entity can be considered as an end-result Increased Permeability Inflammatory Mediators Sensitization of a persistent cross-talk between environmental and

neurogenic factors leading, ultimately, to a vicious cycle. This condition usually occurs following tissue injury, inflammation and ischemic damage, which can result in Sensory Neurons transient or chronic changes in the environment of the nociceptors and in the function of the sensory nerve fibers [36-40]. In the case of inflammation or tissue damage, various inflammatory mediators and products of tissue breakdown are released within a time period ranging from minutes to Pain Perception hours. These include bradykinin, proteases, histamine, serotonin (5-hydroxytryptamine; 5-HT), nitric oxide (NO), prostanoids, neurotrophins, cytokines, ATP, protons and Fig. (3). The interactions of various elements during tissue injury other mediators that are released by injured and/or affected andinflammation inducing physiological, immunological and (including immune) cells. Most of these inflammatory neurological changes that lead to sensory neuron sensitization and products can irritate or sensitize the nerve terminals in the pain perception. affected area, which can lead to two main consequences: nociceptive signaling and neurogenic inflammation. The After entering the CNS, the primary afferents coming latter can contribute to the direct release of neuropeptides from nociceptors terminate in the dorsal horn of the spinal (SP, CGRP), or indirect (reflex) release of catecholamines cord, with the thinly myelinated Afibers ending in laminae I through the sympathetic efferents. Both neuropeptides and and V and the unmyelinated C fibers in lamina II [1]. Prior catecholamines are well known for their vasoactive to ascension to the brain, the nerve impulses diffuse through properties and their effects on various immune cells [1, 36- a region of short, densely packed nerve fibers known as the 40]. Hyperalgesia and Inflammatory Responses Current Immunology Reviews, 2012, Vol. 8, No. 4 281

The end result of the cross-talk between immunogenic potentiating mechanisms has been discussed systematically and neurogenic inflammation is chronic pain, on one side, in further details elsewhere [1-3]. and the various breakdown products in the inflammatory ‘soup’ or milieu, on the other hand, thus leading to the 4. MECHANISMS OF INFLAMMATION AND perpetuation of inflammation and to induction of critical NEUROGENIC PAIN changes in the properties of nerve terminals. Several important changes in the properties of afferent and efferent Tissue damage caused by a wound or by invasion of neurons are observed during chronic pain and inflammation. pathogenic microorganisms induces a complex sequence of These include, as illustration: i) activation of silent or events collectively known as the inflammatory response. dormant nociceptors; ii) peripheral and central sensitization, Although inflammation is an efficient means of protection leading to hyperalgesia (increased reactivity to nociceptive against invading pathogens and recovery from infection, the stimuli) and allodynia (nociceptive signaling and/or reaction cells and chemical mediators participating in the process of induced by innocuous stimuli under normal conditions); iii) inflammation are also capable of damaging tissues and changes in the ionic channels (Na+ channels) and in the interfering with the functioning of organs [1, 2, 40, 41]. properties of membrane receptors; and iv) significant alterations of the electrical signaling behavior of the injured 4.1. Cellular-Mediated Inflammation and the intact neighboring fibers. This constellation of events can occur totally, or in part, in various clinical and The inflammatory response, similar to the immune experimental situations leading to chronic pain, thus losing response, can be generated both from cells (such as its survival value as an alarm signal [1, 2, 35-40]. neutrophils, eosinophils, basophils, macrophages/monocytes, platelets, endothelium and nerve terminals) and from An important focus of pain research has been the study of circulating proteins (components of the complement cascade, chronic pain mechanisms, particularly the processes that lead to the abnormal sensitivity – spontaneous pain and coagulation factors, fibrinolysis or plasmin and kinin pathways). The cardinal signs of inflammation reflect three hyperalgesia – that is associated with these states. For some major events: (i) increased blood flow, resulting in the time it has been recognized that inflammatory mediators engorgement of the capillary network and, subsequently, released from immune cells can contribute to these persistent tissue redness or erythema and an increase in tissue pain states. However, it has only recently become clear that temperature occur; (ii) increased capillary permeability, immune cell products might have a crucial role not just in inflammatory pain, but also in neuropathic pain caused by which facilitates an influx of fluid and cells from capillaries into the surrounding tissue; the fluid that accumulates, or the damage to peripheral nerves or to the CNS. Because of the exudate, which has a high protein content, contributes to presence of a myriad of models alleged with pain and pain tissue swelling or edema; and (iii) an influx of phagocytic mechanisms, it is imperative, therefore, to dislodge leukocytes into the injured tissue. These phagocytes adhere (dissociate) the concepts applied with experimental models to the endothelial wall by margination; then, they move of pain from those associated with pain in humans, likely to be perceived, psychologically, physiologically and between the capillary endothelial cells into the inflamed tissue by diapedesis; and finally, they migrate through the mechanistically, at differential levels [1]. tissue to the site of the wound or infection by chemotaxis Recent reports include the inflammatory mediators in the [40-42]. special category of neuropathic pains, that are initially classified as resulting from discrete or obvious damage to the 4.2. The Chemokine Family of Inflammatory Cytokines central or peripheral nervous systems. These reports are based on animal experimentations showing an attenuation of Cytokines are peptide proteins that act as intercellular neuropathic manifestations by treatment with anti- signals that activate and recruit cells and, in some situations, inflammatory drugs and especially specific antagonists to control major physiological and pathophysiological processes pro-inflammatory cytokines. This implies that pro- [38-42]. Most of them can be produced by many cell types, inflammatory mediators (including cytokines) can constitute including macrophages/monocytes, keratinocytes, T lympho- an end product of neurogenic inflammation and can cytes, endothelial cells, Langerhans cells and neutrophils. The contribute further to the neuropathic pain by increasing the majority of these peptides have multiple activities with some inflammatory component of the vicious cycle [1, 2]. cytokines having extensively overlapping functions [40-42]. Purportedly, various strategies have been adopted to stop the The activities of cytokines on different target cells range from ‘snow ball’ phenomenon at the origin of chronic pain. These promoting cellular growth [e.g., IL-6 and platelet-derived include targeting of various mediators, considered to play a growth factor (PDGF)] or arresting growth [e.g., transforming key role in the inflammatory cascade, and based on the use growth factor- (TGF-) and TNF] to inducing viral resistance of specific antagonists to prostanoids, histamine, [e.g., interferon- (IFN-)], eliciting chemotaxis [e.g., IL-8 and neurotrophins and cytokines. A new strategy has recently macrophage chemotactic and activating factor (MCAF)] and emerged and is based on targeting the transcription factors at mediating inflammation (IL-1, IL-6, TNF-and TGF-). These the origin of expression of several pro-inflammatory peptides, or pro-inflammatory agents, stimulate the synthesis mediators and cytokines. The transcription factor of and release of other inflammatory mediators, such as nitric immense interest is, undoubtedly, NF-B. The focus on the oxide (NO), prostaglandins and substance P (SP), and thereby outcome of contemporary research as to how to understand participate in complex, interactive events that influence sensory the NF-B-pain nexus and the alleviation of chronic pain neurons to induce hyperalgesia (Fig. 4) [18, 23-27]. through specific targeting of this transcription factor 282 Current Immunology Reviews, 2012, Vol. 8, No. 4 John J. Haddad

4.3. Inflammatory Mediators Other than Cytokines Bradykinins When influenced by inflammatory mediators, or following the administration of noxious irritants, sensory fibers are sensitized and exhibit a spontaneous responsiveness and evoked activity [1]. Inflammatory mediators induce changes in the cellular neurochemistry of IL-1 TNF nociceptors enhancing neurogenic interactions with surrounding central and peripheral tissues that control injury and facilitate tissue repair, but which may also boost signal transmission by releasing neurotran-smitters in the spinal iNOS; COX; B1 IL-6/IL-8 cord, a process referred to as central sensitization [40-42]. A variety of substances are released or synthesized following injury by trauma or disease. These factors have a profound impact on the generation of inflammation and the consequent events [18, 19]. Cytostatic/cytotoxic mediators are produced Prostaglandins such as nitric oxide, and reactive oxygen intermediates or free radicals. Moreover, modulators and chemical agents of

NGF inflammation including a variety of proinflammatory Sympathetic Neurons Opioids cytokines, adhesion molecules, cascades of enzymes, receptors and hematopoietic growth are produced. Finally, an integration of a system of peripheral and central tissue responses facilitate neural (afferent and sympathetic) circuit Hyperalgesia signaling [1-3].

Neurogenic factors also contribute to inflammatory pain Fig. (4). Cytokines and inflammatory hyperalgesia. Bradykinin and hyperalgesia. In one study, the sequestration of stimulates IL-1 and TNF production. TNF induces hyperalgesia by endogenous nerve growth factor (NGF) in the adult rat using initiating a cascade of other cytokines including IL-1, IL-6 and IL- a trkA-IgGfusion protein has been shown to abolish the 8. IL-1 induces prostanoid formation and a number of secondary 2+ hyperalgesia associated with carrageenan inflammation. A effects, including NGF production as well as the induction of Ca - population of nociceptive sensory neurons expresses the high independent form of nitric oxide synthase (iNOS) and affinity NGF receptor, trkA. Blocking the receptor for NGF, cyclooxygenase (COX), and expression of B1 receptors. The therefore, on sensory afferents has been shown to effectively expression and release of opioids from immune cells act on sensory control hyperalgesia [15-23]. Furthermore, the injection of and sympathetic neurons to reduce neurogenic inflammation and NGF mediates increased sensitivity to noxious stimulation hyperalgesia. since animals exposed to anti-NGF antiserum have shown reduced responses to painful and inflammatory stimuli [24-

Sensory Neurons – Neurokinin Release

Sympathetic Blood Vessels Immune Cells Mast Cells Spinal Cord Neurons

Vasodilation Proliferation Histamine Neurotransmitters Transmission SP and CGRP Plasma Chemotaxis 5-HT Reflexes Excitability Extravasation Cytokines Proteinases Changes

Fig. (5). Neurokinins induce inflammatory hyperalgesia via actions on the vasculature, stimulation of immune cells and sympathetic fibers, mast cell degranulation and as neurotransmitters in the spinal cord facilitating central sensitization. Hyperalgesia and Inflammatory Responses Current Immunology Reviews, 2012, Vol. 8, No. 4 283

30]. NGF-induced expression of specific transcription In reiteration, it is noted that “a strategy to block NGF activators induce the release of mRNAs that encode either by sequestering the factor itself or blocking its binding neurokinin precursors, the pre-proneurokinins, including SP, to TrkA has proven to be efficacious in ameliorating pain in neurokinin A (NKAl and a genomically unrelated peptide, several animal models. Treatment with anti-NGF agents has the CGRP, or calcitonin gene-related peptide). The diminished or reversed hyperalgesia in chronic inflammatory neurokinins have a unique function in mitigating the process conditions in which the two established pain treatments, of inflammation by contributing to neurogenic inflammation NSAIDs and opiates, either have limited efficacy or adverse and hyperalgesia at the periphery, while their release from side effects” (Adapted, Scott T. Brady et al., In: Basic central terminals in the spinal cord is involved in the Neurochemistry: Principlesof Molecular, Cellular, and transmission of pain signals and integration of changes in Medical Neurobiology, 8th edition, 2012, Academic Press central excitability [1] (Fig. 5). Publishers). The mechanistic pathways depicting the intricate affiliation of inflammatory pain and hyperalgesia

pathways are schematically shown in Fig. (6).

Fig. (6). Major pathways for pain sensation. (A) The spinothalamic system pathways. (B) The trigeminal pain and temperature thermosystem (thermal hyperalgesia) pathways, which carry information about those sensations from nociceptors. (Adapted, courtesy of Purves et al., Neuroscience, 5th Edition, 2012, Sinauer Publishers). 284 Current Immunology Reviews, 2012, Vol. 8, No. 4 John J. Haddad

5. CONCLUDING REMARKS AND A PERSPECTIVE modality. Because of the importance of warning an animal about dangerous circumstances, the mechanisms and Inflammatory-induced hyperalgesia is one of the most pathways that subserve nociception are widespread and common aspects of pain-relatedinflammation and therapeutic redundant. The major nociceptive pathway, like other approach to this pain should aim at interfering with various somatic sensory modalities, comprises a three-neuron relay mediators of the inflammatory reactions including, from periphery to cortex. This arrangement differs from the neuropeptides, eicosanoids and cytokines [21-29, 40-43]. mechanosensory pathway primarily in that the central axons Nociception and peripheral sensitization after inflammation of dorsal root ganglion cells synapse on second-order pathways are shown in Fig. (7). neurons in the spinal cord, which then cross the midline and In retrospect, “whether from a structural or functional project to brainstem and thalamic nuclei in the contralateral perspective, pain is an extraordinarily complex sensory spinal cord. The thalamic neurons, in turn, project to the

Fig. (7). Nociception and peripheral sensitization after inflammation. Distinct ionotropic receptor molecules at the peripheral terminals of a nociceptor transduce noxious stimuli into depolarizing membrane currents. Upon depolarization, voltage-gated sodium channels (Nav) generate action potentials that propagate from the periphery to the cell body of the nociceptor in the dorsal root ganglion and its central terminal in the spinal cord. Inflammation leads to an enhancement of stimulus transduction and signal propagation (peripheral sensitization). Sensitization is mediated by protein kinases (PKA, PKC) that are activated in the presence of prostaglandins (PGs), bradykinin and adenosine triphosphate (ATP), which bind to G protein–coupled receptors, and interleukins and nerve growth factor (NGF), which activate tyrosine kinase (TRK) receptors. ASIC, acid sensing ion channel; B2, bradykinin receptor; EP, prostaglandin E receptor; KCNK, potassium channel, subfamily K; P2X and P2Y, purinergic receptors; TRP, transient receptor potential channel. (Adapted, courtesy of Scott T. Brady et al., Basic Neurochemistry: Principlesof Molecular, Cellular, and Medical Neurobiology, 8th edition, 2012, Academic Press Publishers).

Table 2. Chemical Mediators of Nociception and Inflammation After Damage to Tissues*

Leak from damaged cells or stimulated/induced release: Ach (Acetylcholine): Released from damaged cells, causes pain. Protons: In-migrating leukocytes secrete lactic acid; key aspect of inflammatory ‘‘soup’’ is low pH. 5-HT: Released from platelets, intradermal application produces pain, activates nociceptors. ATP: Application produces pain, activates nociceptors. Histamine: Released by mast cells, produces itch not pain, but along with 5-HT, BK (Bradykinin), stimulates synthesis ofprostaglandins. Synthesized locally by enzymes from substrates released by damage/insult or that enter area as part of inflammatory process (e.g., leukocyte migration): Bradykinin: Synthesized from protein precursor in plasma (kininogen), activates nociceptors to produce pain, sensitizesnociceptors to other inputs (including heat), triggers other elements of inflammatory process (including synthesis ofprostaglandins and release of cytokines). Prostaglandins: Synthesized from arachidonic acid after injury. Contributes to inflammation by inducing vasodilationand plasma extravasation, attracts immune cells. Sensitizes nociceptors. Released by activated nociceptors themselves: Substance P: Acts on other afferent fiber terminals to activate and/or sensitize, contributes to inflammation by causingvasodilation, plasma extravasation. Stimulates mast cells to release histamine. CGRP (Calcitonin gene-related peptide): Contributes to inflammation, dilates arterioles, synergizes with substance P inproducing plasma extravasation. *Adapted, P. Michael Conn, In: Neuroscience in Medicine, 3rd edition, 2008, Humana Press Publishers. Hyperalgesia and Inflammatory Responses Current Immunology Reviews, 2012, Vol. 8, No. 4 285

Primary Hyperalgesia/Nociception

Secondary Hyperalgesia/Nociception Fig. (8). Mechanisms of primary and secondary hyperalgesia. (A) Hyperalgesia within an area of injured is referred to as primary hyperalgesia, and that in surrounding tissue is called secondary hyperalgesia. (B) Pain rating and activity in primary afferents before and after injury to the tissue. Sensitized primary afferents explain increased pain in the injured region. (C) Pain rating and activity in primary afferents and dorsal horn neurons with receptive fields in the area of secondary hyperalgesia. Primary afferents innervating the area of secondary hyperalgesia are not sensitized, whereas dorsal horn neurons responding to stimulation of that area show increased responsiveness (central sensitization). (Adapted, courtesy of P. Michael Conn, Neuroscience in Medicine, 3rdedition, 2008, Humana Press Publishers). same cortical areas as other somatic sensory modalities. The Summary of inflammatory mediators involved with nocice- molecular basis of pain modulation is particularly intricate ption and hyperalgesia is given in Table 2. Mechanisms of and is only beginning to be deciphered. The major features primary and secondary hyperalgesia are shown in Fig. (8). are the modulation of pain peripherally by the release of a variety of agents at the injury site, and the central CONFLICT OF INTEREST modulation of afferent pain pathways by endogenous opioids that act at the level of both the spinal cord and the The author confirms that this article content has no brainstem. Tremendous progress in understanding pain has conflict of interest. been made in the last 25 years, and much more seems likely, given the importance of the problem. No patients are more ACKNOWLEDGEMENTS distressed—or more difficult to treat—than those with chronic pain. Indeed, some aspects of pain seem much more The author’s work therein cited was, in part, supported by destructive to the sufferer than required by any physiological the Anonymous Trust (Scotland), the National Institute for purposes; consider, for example, the pain of a chronic illness Biological Standards and Control (England), the Tenovus Trust such as invasive cancer. Perhaps such seemingly excessive (Scotland), the UK Medical Research Council (MRC, London), effects are a necessary but unfortunate by product of the the National Institutes of Health (NIH), and the Wellcome Trust protective benefits of this vital sensory modality”. (Adapted, (London). Dr. John J. Haddad held the distinguished Georges Scott T. Brady et al., In: Basic Neurochemistry: Principlesof John Livanos fellowship (London, UK), and the National Molecular, Cellular, and Medical Neurobiology, 8th edition, Institutes of Health postdoctoral fellowship (NIH; UCSF). This 2012, Academic Press Publishers). review was written by the author who is officially affiliated with the aforementioned institution and formally declare that there Current studies are tailoring their design into unraveling isn’t any financial interest or conflict in relation with writing up the intricacy of pain-inflammatory responses connection for this manuscript. their clinical and physiological relevance [1-3, 43-45]. 286 Current Immunology Reviews, 2012, Vol. 8, No. 4 John J. Haddad

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Received: October 7, 2012 Revised: November 2, 2012 Accepted: November 5, 2012