Gastrointestinal afferents as targets of novel drugs for the treatment of functional bowel disorders and visceral pain.

An intricate surveillance network consisting of enteroendocrine cells, immune cells and sensory nerve fibres monitors the luminal and interstitial environment in the alimentary canal. Functional bowel disorders are characterized by persistent alterations in digestive regulation and gastrointestinal discomfort and pain. Visceral hyperalgesia may arise from an exaggerated sensitivity of peripheral afferent nerve fibres and/or a distorted processing and representation of gut signals in the brain. Novel strategies to treat these sensory bowel disorders are therefore targeted at primary afferent nerve fibres. These neurons express a number of molecular traits including transmitters, receptors and ion channels that are specific to them and whose number and/or behaviour may be altered in chronic visceral pain. The targets under consideration comprise vanilloid receptor ion channels, acid-sensing ion channels, sensory neuron-specific Na(+) channels, P2X(3) purinoceptors, 5-hydroxytryptamine (5-HT), 5-HT(3) and 5-HT(4) receptors, cholecystokinin CCK(1) receptors, bradykinin and prostaglandin receptors, glutamate receptors, tachykinin and calcitonin gene-related peptide receptors as well as peripheral opioid and cannabinoid receptors. The utility of sensory neuron-targeting drugs in functional bowel disorders will critically depend on the compounds' selectivity of action for afferent versus enteric or central neurons.     

Anti-convulsants and anti-depressants

Damage to a nerve should only lead to sensory loss. While this is common, the incidence of spontaneous pain, allodynia and hyperalgesia indicate marked changes in the nervous system that are possible compensations for the loss of normal function that arises from the sensory loss. Neuropathic pain arises from changes in the damaged nerve which then alter function in the spinal cord and the brain and lead to plasticity in areas adjacent to those directly influenced by the neuropathy. The peripheral changes drive central compensations so that the mechanisms involved are multiple and located at a number of sites. Nerve damage increases the excitability of both the damaged and undamaged nerve fibres, neuromas and the cell bodies in the dorsal root ganglion. These peripheral changes are substrates for the ongoing pain and the efficacy of excitability blockers such as carbamazepine, lamotrigine and mexiletine, all anti-convulsants. A better understanding of ion channels at the sites of injury has shown important roles of particular sodium, potassium and calcium channels in the genesis of neuropathic pain. Within the spinal cord, increases in the activity of calcium channels and the receptors for glutamate, especially the N-methyl-D-aspartate (NMDA) receptor, trigger wind-up and central hyperexcitability. Increases in transmitter release, neuronal excitability and receptive field size result from the damage to the peripheral nerves. Ketamine and gabapentin/pregabalin, again with anti-convulsant activity, may interact with these mechanisms. Ketamine acts on central spinal mechanisms of excitability whereas gabapentin acts on a subunit of calcium channels that is responsible for the release of pain transmitters into the spinal cord. In addition to these spinal mechanisms of hyperexcitability, spinal cells participate in a spinal-supraspinal loop that involves parts of the brain involved in affective responses to pain but also engages descending excitatory and inhibitory systems that use the monoamines. These pathways become more active after nerve injury and are the site of action of anti-depressants. This chapter reviews the evidence and mechanisms of drugs, both anti-depressants and anti-convulsants, that are believed to be effective in pain control, with a major emphasis on the neuropathic state.     

Peripheral and central mechanisms of pain generation

Pain research has uncovered important neuronal mechanisms that underlie clinically relevant pain states such as inflammatory and neuropathic pain. Importantly, both the peripheral and the central nociceptive system contribute significantly to the generation of pain upon inflammation and nerve injury. Peripheral nociceptors are sensitized during inflammation, and peripheral nerve fibres develop ectopic discharges upon nerve injury or disease. As a consequence a complex neuronal response is evoked in the spinal cord where neurons become hyperexcitable, and a new balance is set between excitation and inhibition. The spinal processes are significantly influenced by brain stem circuits that inhibit or facilitate spinal nociceptive processing. Numerous mechanisms are involved in peripheral and central nociceptive processes including rapid functional changes of signalling and long-term regulatory changes such as up-regulation of mediator/receptor systems. Conscious pain is generated by thalamocortical networks that produce both sensory discriminative and affective components of the pain response.     

Pathobiology of neuropathic pain.

This review deals with physiological and biological mechanisms of neuropathic pain, that is, pain induced by injury or disease of the nervous system. Animal models of neuropathic pain mostly use injury to a peripheral nerve, therefore, our focus is on results from nerve injury models. To make sure that the nerve injury models are related to pain, the behavior was assessed of animals following nerve injury, i.e. partial/total nerve transection/ligation or chronic nerve constriction. The following behaviors observed in such animals are considered to indicate pain: (a) autotomy, i.e. self-attack, assessed by counting the number of wounds implied, (b) hyperalgesia, i.e. strong withdrawal responses to a moderate heat stimulus, (c) allodynia, i.e. withdrawal in response to non-noxious tactile or cold stimuli. These behavioral parameters have been exploited to study the pharmacology and modulation of neuropathic pain. Nerve fibers develop abnormal ectopic excitability at or near the site of nerve injury. The mechanisms include unusual distributions of Na(+) channels, as well as abnormal responses to endogenous pain producing substances and cytokines such as tumor necrosis factor alpha (TNF-alpha). Persistent abnormal excitability of sensory nerve endings in a neuroma is considered a mechanism of stump pain after amputation. Any local nerve injury tends to spread to distant parts of the peripheral and central nervous system. This includes erratic mechano-sensitivity along the injured nerve including the cell bodies in the dorsal root ganglion (DRG) as well as ongoing activity in the dorsal horn. The spread of pathophysiology includes upregulation of nitric oxide synthase (NOS) in axotomized neurons, deafferentation hypersensitivity of spinal neurons following afferent cell death, long-term potentiation (LTP) of spinal synaptic transmission and attenuation of central pain inhibitory mechanisms. In particular, the efficacy of opioids at the spinal level is much decreased following nerve injury. Repeated or prolonged noxious stimulation and the persistent abnormal input following nerve injury activate a number of intracellular second messenger systems, implying phosphorylation by protein kinases, particularly protein kinase C (PKC). Intracellular signal cascades result in immediate early gene (IEG) induction which is considered as the overture of a widespread change in protein synthesis, a general basis for nervous system plasticity. Although these processes of increasing nervous system excitability may be considered as a strategy to compensate functional deficits following nerve injury, its by-product is widespread nervous system sensitization resulting in pain and hyperalgesia. An important sequela of nerve injury and other nervous system diseases such as virus attack is apoptosis of neurons in the peripheral and central nervous system. Apoptosis seems to induce neuronal sensitization and loss of inhibitory systems, and these irreversible processes might be in common to nervous system damage by brain trauma or ischemia as well as neuropathic pain. The cellular pathobiology including apoptosis suggests future strategies against neuropathic pain that emphasize preventive aspects.     

Central nervous system control of the airways: pharmacological implications

Autonomic innervation of the airways is derived primarily from the parasympathetic nervous system. Preganglionic fibers originating in the brainstem project to parasympathetic ganglion neurons, which regulate airway smooth-muscle tone, glandular secretion and blood-vessel diameter. Airway preganglionic nerve activity is regulated by subsets of pulmonary and extrapulmonary afferent nerve fibers, which continuously provide polysynaptic input to brainstem preganglionic nuclei. Each of these synapses in the central nervous system is a potential site for therapeutic intervention. Potential targets include increasing opioid, GABAergic and serotonergic controls on central neurons, and blockade of tachykinin and glutamate receptors. Unfortunately, much is still unknown about the control of airway nerves at the level of the central nervous system. Recently, however, interaction between vagal afferent nerve subtypes regulating airway function has been described. This interaction, made possible by their convergence at key sites of integration in the brainstem, may lead to central sensitization analogous to that described in somatic pathways regulating pain sensation. Improved understanding of the central pharmacology of airway reflexes may provide novel therapeutics for treating symptoms associated with respiratory disorders such as chronic obstructive pulmonary disease, asthma and sleep-disordered breathing.     

TRPA1: A Transducer and Amplifier of Pain and Inflammation

The transient receptor potential ankyrin 1 (TRPA1) ion channel on peripheral terminals of nociceptive primary afferent nerve fibres contributes to the transduction of noxious stimuli to electrical signals, while on central endings in the spinal dorsal horn, it amplifies transmission to spinal interneurons and projection neurons. The centrally propagating nociceptive signal that is induced and amplified by TRPA1 not only elicits pain sensation but also contributes to peripheral neurogenic inflammation through a peripheral axon reflex or a centrally mediated back propagating dorsal root reflex that releases vasoactive agents from sensory neurons in the periphery. Endogenous TRPA1 agonists that are generated under various pathophysiological conditions both in the periphery and in the spinal cord have TRPA1‐mediated pro‐nociceptive and pro‐inflammatory effects. Among endogenous TRPA1 agonists that have been shown to play a role in the pathogenesis of pain and inflammatory conditions are, for example, methylglyoxal, 4‐hydroxynonenal, 12‐lipoxygenase‐derived hepoxilin A3, 5,6‐epoxyeicosatrienoic acid and reactive oxygen species, while mustard oil and cinnamaldehyde are most commonly used exogenous TRPA1 agonists in experimental studies. Among selective TRPA1 antagonists are HC‐030031, A‐967079, AP‐14 and Chembridge‐5861528. Recent evidence indicates that TRPA1 plays a role also in transition of acute to chronic pain. Due to its location on a subpopulation of pain‐mediating primary afferent nerve fibres, blocking the TRPA1 channel is expected to have antinociceptive, antiallodynic and anti‐inflammatory effects.     

Tissue Injury and Related Mediators of Pain Exacerbation

Tissue injury and inflammation result in release of various mediators that promote ongoing pain or pain hypersensitivity against mechanical, thermal and chemical stimuli. Pro-nociceptive mediators activate primary afferent neurons directly or indirectly to enhance nociceptive signal transmission to the central nervous system. Excitation of primary afferents by peripherally originating mediators, so-called “peripheral sensitization”, is a hallmark of tissue injury-related pain. Many kinds of pro-nociceptive mediators, including ATP, glutamate, kinins, cytokines and tropic factors, synthesized at the damaged tissue, contribute to the development of peripheral sensitization. In the present review we will discuss the molecular mechanisms of peripheral sensitization following tissue injury.     

Homomeric and heteromeric P2X3 receptors in peripheral sensory neurons

ATP contributes to nociceptive sensory processing by activating a family of ligand-gated ion channels, the P2X receptors. One of these, the P2X3 receptor, is highly localized on primary afferent neurons. In sensory neurons, P2X3 receptors function as homomeric (P2X3) and heteromeric (P2X2/3) channels. Exogenous application of ATP and related agonists excites peripheral and central nerves, and increases sensitivity to noxious stimuli. Specific targeting of P2X3 receptors by gene deletion and knockdown results in a hypoalgesic phenotype. In animal models of pain, pharmacological blockade of P2X3 receptors fully blocked specific types of chronic inflammatory and neuropathic pain. Peripheral nerve injury differentially alters functional expression of P2X3 receptors on small and large diameter primary afferent neurons. These data have delineated discrete roles for homomeric P2X3 and heteromeric P2X2/3 receptor activation in acute and chronic pain. Similar findings have also been generated from extensive research of the bladder urothelial-sensory neuron system. The urinary bladder is richly innervated by P2X3 receptor-containing neurons. Heteromeric P2X2/3 channels in the bladder contribute to both mechanosensitivity and nociceptive responses. Thus, both genetic and pharmacological approaches have provided converging evidence that activation of P2X3-containing channels is an important mediator of acute and persistent nociceptive signaling in the peripheral nervous system.     

Neuropeptides and gastric mucosal homeostasis

The role of central nervous system (CNS) in regulation of gastric function has long been known. The dorsal vagal complex (DVC) has an important role in regulation of gastric mucosal integrity; it is involved both in mucosal protection and in ulcer formation. Neuropeptides have been identified in DVC, the origin of these peptides are both intrinsic and extrinsic. Neuropeptides are localized also in the periphery, in afferent neurons. The afferent neurons also have efferent-like function in the gastroinetestinal tract, and neuropeptides released from the peripheral nerve endings of primary afferent neurons can induce gastric mucosal protection. Centrally and /or peripherally injected neuropeptides, such as amylin, adrenomedullin, bombesin, cholecystokinin, neurotensin, opioid peptides, thyreotropin releasing hormone and vasoactive intestinal peptide, influence both the acid secretion and the gastric mucosal lesions induced by different ulcerogens. The centrally induced gastroprotective effect of neuropeptides may be partly due to a vagal dependent increase of gastric mucosal resistance to injury; activation of vagal cholinergic pathway is resulted in stimulation of the release of mucosal prostaglandin and nitric oxide. Furthermore, release of sensory neuropeptides (calcitonin gene-related peptide, tachykinins) from capsaicin sensitive afferent fibers are also involved in the centrally induced gastroprotective effect of neuropeptides.     

Voltage-operated calcium channels: characteristics and their role in the mechanism of action of psychotropic drugs.

Ion channels can be divided in two main groups, receptor-operated channels (ROC) and voltage-operated channels (VOC). The function of ROC depends on the action of agonists and antagonists, the function of VOC is closely connected with the activity of enzymes and the processes of phosphorylation of membrane proteins. Electrophysiological studies indicate the existence of three types of VOC (K+, Na+, Ca2+ channels). In number of neurons various subtypes of Ca2+ channels (P, T, N and L-type) occur together. Among them, the L-type Ca2+ channel has been first described and most studied. The L-type calcium channel is localized on nerve terminals in the pre- and postsynaptic parts, as well as on cell bodies and may be involved in the mechanism of action of psychotropic drugs. Our own experiments have shown that chronic treatment with various psychotropic drugs changes the density of L-type Ca2+ channels in the central nervous system. We have found the involvement of L-type VOC in responsiveness to pain, morphine tolerance and dependence, and adaptation changes induced by several chronic administration of psychotropic drugs. Thus, according to pharmacological and also clinical data, L-type Ca2+ channels may be involved in etiology of variety of psychiatric disorders.     

The dynamic TRPA1 channel: a suitable pharmacological pain target?

Acute pain detection is vital to navigate and survive in one's environment. Protection and preservation occur because primary afferent nociceptors transduce adverse environmental stimuli into electrical impulses that are transmitted to and interpreted within high levels of the central nervous system. Therefore, it is critical that the molecular mechanisms that convert noxious information into neural signals be identified, and their specific functional roles delineated in both acute and chronic pain settings. The Transient Receptor Potential (TRP) channel family member TRP ankyrin 1 (TRPA1) is an excellent candidate molecule to explore and intricately understand how single channel properties can tailor behavioral nociceptive responses. TRPA1 appears to dynamically respond to an amazingly wide range of diverse stimuli that include apparently unrelated modalities such as mechanical, chemical and thermal stimuli that activate somatosensory neurons. How such dissimilar stimuli activate TRPA1, yet result in modality-specific signals to the CNS is unclear. Furthermore, TRPA1 is also involved in persistent to chronic painful states such as inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis and emphysema. Yet how TRPA1's role changes from an acute sensor of physical stimuli to its contribution to these diseases that are concomitant with implacable, chronic pain is unknown. TRPA1's involvement in the nociceptive machinery that relays the adverse stimuli during painful disease states is of considerable interest for drug delivery and design by many pharmaceutical entities. In this review, we will assess the current knowledge base of TRPA1 in acute nociception and persistent inflammatory pain states, and explore its potential as a therapeutic pharmacological target in chronic pervasive conditions such neuropathic pain, persistent inflammation and diabetes.     

Spinal astrocytes as therapeutic targets for pathological pain

Development of next-generation analgesics requires a better understanding of the molecular and cellular mechanisms underlying pathological pain. Accumulating evidence suggests that the activation of glia contributes to the central sensitization of pain signaling in the spinal cord. The role of microglia in pathological pain has been well documented, while that of astrocytes still remains unclear. After peripheral nerve inflammation or injury, spinal microglia are initially activated and subsequently sustained activation of astrocytes is precipitated, which are implicated in the induction and maintenance of pathological pain. Astrocytic activation is caused by the production of diffusible factors from primary afferent neurons (neuron-to-astrocyte signals) and activated microglia (microglia-to-astrocyte signals). Although astrocyte-to-neuron signals implicated in pathological pain is poorly understood, activated astrocytes, as well as microglia, produce proinflammatory cytokines and chemokines, which lead to adaptation of the dorsal horn neurons. Furthermore, it has been suggested that glial glutamate transporters in the spinal astrocytes are down-regulated in pathological pain and that up-regulation or functional enhancement of these transporters prevents pathological pain. This review will briefly discuss novel findings on the role of spinal astrocytes in pathological pain and their potential as a therapeutic target for novel analgesics.     

P2X receptors and nociception.

The potential importance for nociception of P2X receptors, the ionotropic receptors activated by ATP, is underscored by the variety of pain states in which this endogenous ligand can be released. Several important findings have been made recently indicating that P2X receptors can be involved in pain mechanisms both centrally and in the periphery. The roles of ATP at these two sites and the P2X receptor subtypes involved appear to be different. In the periphery, ATP can be released as a result of tissue injury, visceral distension, or sympathetic activation and can excite nociceptive primary afferents by acting at homomeric P2X(3) or heteromeric P2X(2/3) receptors. Centrally, ATP released from central afferent terminals or second order neurons can modulate neurotransmitter release or postsynaptically activate neurons involved in central nociceptive transmission, with P2X(2), P2X(4), P2X(6), and some other receptors being potentially involved. Evidence from in vivo studies suggests that peripheral ATPergic mechanisms are most important under conditions of acute tissue injury and inflammation whereas the relevance of central mechanisms appears to be more limited. Furthermore, the release of ATP and P2X receptor-mediated afferent activation appear to have been implicated in visceral and neuropathic pain; the importance of the ATPergic component in these states needs to be investigated further. Thus, peripheral P2X receptors, and homomeric P2X(3) and/or heteromeric P2X(2/3) receptors in particular, constitute attractive targets for analgesic drugs. The development of selective antagonists of these receptors, suitable for a systemic in vivo use although apparently difficult, may prove a useful strategy to generate analgesics with a novel mechanism of action.     

Decreased expression of glial cell line-derived neurotrophic factor signaling in rat models of neuropathic pain

1. In an attempt to clarify whether glial cell line-derived neurotrophic factor (GDNF), a survival factor for subpopulations of primary afferent neurons, is involved in the states of neuropathic pain, we observed changes in the expressions of GDNF and its signal-transducing receptor Ret after nerve injury in two rat models of neuropathic pain. 2. In the rats treated with sciatic nerve ligation (chronic constrictive injury (CCI) model) or spinal nerve ligation at L5 (SNL model), the thresholds of paw withdrawal in response to mechanical or heat stimuli began to decrease on the injured side within the first week after the operation and the decreases in the thresholds persisted for more than 2 weeks. 3. In CCI-treated rats, the GDNF contents in L4 and L5 dorsal root ganglia (DRGs) on the injured side were markedly decreased at day 7 after the operation and stayed at low levels at day 14. In SNL-treated rats, comparable reductions of GDNF levels in L4 and L5 DRGs on the injured side were observed at 14 postoperative days. 4. Significant decreases of the percentages of DRG neurons expressing Ret were also observed at L4 DRGs in CCI-treated rats at 7 and 14 postoperative days and in SNL-treated rats at 14 days. 5. In CCI- or SNL-treated rats, continuous intrathecal administration of GDNF (12 microg day-1) using an osmotic pump suppressed the increased sensitivities to nociceptive stimuli to control levels. 6. The present results suggested that the dysfunction of GDNF signaling in the nociceptive afferent system may contribute to the development and/or maintenance of neuropathic pain states.     

The Role of Cytokines in the Initiation and Maintenance of Chronic Pain

Recent advancements in the pain field have identified a central nervous system (CNS) neuroimmune response that may act as the driving force for neuronal hypersensitivity, the pathological correlate to chronic pain following peripheral nerve injury. Neuroimmune activation involves the activation of nonneuronal cells such as endothelial and glial cells, which when stimulated leads to enhanced production of a host of inflammatory mediators, such as cytokines. The central production of proinflammatory cytokines, such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor have been found to play a key role in the propagation of persistent pain states. In addition, chemotactic cytokines, chemokines, have also been recently identified in the CNS neuroimmune cascade that ensues after injury to a peripheral nerve. The extravasation of leukocytes from the blood to the site of perceived injury is defined as the neuroinflammatory aspect of this cascade. Chemokines directly control this leukocyte transmigration process. They are synthesized at the site of injury and establish a concentration gradient through which immune cells migrate. Recent studies have demonstrated leukocyte trafficking into the CNS following peripheral nerve or lumbar nerve root injury. With the use of selective cytokine inhibitors and neutralizing antibodies, tactile and thermal hypersensitivity is attenuated in animal models of neuropathy. A further understanding of the role of nonneuronal cells, the physiological mechanisms of CNS cytokines and chemokines, and their relevance to neuro- immune activation and neuroinflammatory processes may lead to the development of novel pharmacological agents for the treatment and prevention of chronic pain. (c) 2002 Prous Science. All rights reserved.     

TRP channels: potential drug target for neuropathic pain

Neuropathic pain is a debilitating disease which affects central as well as peripheral nervous system. Transient receptor potential (TRP) channels are ligand-gated ion channels that detect physical and chemical stimuli and promote painful sensations via nociceptor activation. TRP channels have physiological role in the mechanisms controlling several physiological responses like temperature and mechanical sensations, response to painful stimuli, taste, and pheromones. TRP channel family involves six different TRPs (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and TRPA1) which are expressed in pain sensing neurons and primary afferent nociceptors. They function as transducers for mechanical, chemical, and thermal stimuli into inward currents, an essential first step for provoking pain sensations. TRP ion channels activated by temperature (thermo TRPs) are important molecular players in acute, inflammatory, and chronic pain states. Different degree of heat activates four TRP channels (TRPV1–4), while cold temperature ranging from affable to painful activate two indistinctly related thermo TRP channels (TRPM8 and TRPA1). Targeting primary afferent nociceptive neurons containing TRP channels that play pivotal role in revealing physical stimuli may be an effective target for the development of successful pharmacotherapeutics for clinical pain syndromes. In this review, we highlighted the potential role of various TRP channels in different types of neuropathic pain. We also discussed the pharmacological activity of naturally and synthetically originated TRP channel modulators for pharmacotherapeutics of nociception and neuropathic pain.     

The mechanisms of immune-to-brain communication in inflammation as a drug target

There is considerable evidence that the peripheral immune system can signal the brain to elicit a sickness response during infection and inflammation. The induction of the sickness response involves the expression of proinflammatory cytokines such as interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6, both in the periphery and in the brain. The mechanisms by which peripheral cytokines can affect brain function have been the subject of much debate. The precise mechanisms by which cytokines signal the central nervous system (CNS) are unknown, but possibilities include: 1) the direct entry of cytokine into the brain across the blood-brain barrier by a saturable transport mechanism: 2) the interaction of cytokine with circumventricular organs such as the orgnum vasculosum of the lamina terminalis [OVLT] and area postrema, which lack the blood-brain barrier; and 3) activation of afferent neurons of the vagus nerve. Increasing evidence has suggested that the afferent vagus nerve is an important pathway for immune-to-brain communication. However, there are inconsistent findings for the involvement of the afferent vagus nerve in the mediation of transmitting inflammatory signals to the brain. Thus, we describe here the functional relevance of the vagal afferent nerve in mediating these effects. An understanding of the mechanisms involved in immune-to-brain communication should permit us to create new drugs as therapeutic targets to decrease sickness or promote recovery. This review focuses on recent discoveries of the multipathway mechanisms for the induction of sickness behavior mediated through neuroimmune interactions in the CNS.     

The challenge of chronic pain

Chronic pain is a complex problem with staggering negative health and economic consequences. The complexity of chronic pain is presented within Cervero and Laird's model that describes three phases of pain, including pain without tissue damage, pain with tissue damage and inflammation, and neuropathic pain. The increased afferent input in phases 2 and 3 of chronic pain produces marked changes in primary afferents, dorsal root ganglia, and spinal cord dorsal horn. These changes promote the symptoms of chronic pain, including spontaneous pain, hyperalgesia, and allodynia. Increased afferent input also evokes supraspinal input to the dorsal horn, including biphasic innervation from the ventromedial medulla and A7 catecholamine cell group, that promotes hyperalgesia and allodynia. More rostral brain structures, such as the lateral hypothalamus, amygdala, and hippocampus, may also play a role in chronic pain. Although much has been discovered about the multiple pathological mechanisms involved in chronic pain, further research is needed to fully comprehend these mechanisms.