Research on pain is focused on neurobiology studies concerning neuronal plasticity development, nociceptors molecular identity, signaling mechanisms, ionic channels involved in the generation, modulation and propagation of action potentials in all type of excitable cells. All the findings open the possibility for developing new therapeutic treatment.
The interest of researchers for receptors, neurotransmitters, second messengers, transcription factors involved in neuronal processing, in spinal cord and in cortical areas, increased dramatically. There are evidences clarifying the origin of chronic pain.
Now it is well known the existence of two different kind of persistent chronic pain: nociceptive/inflammatory pain and neuropathic pain. The first, inflammation associated, is caused by tissue damage (Fig. 1A,B). The first lesion and the inflammatory process cause Aδ and C fibers alteration. These fibers are responsible of sensitization, recruitment of nociceptors normally silent and ionic channels and membrane receptors activation. Neurogenic pain syndromes arise as consequence of central and peripheral nerve damage.
During inflammation and neuropathies there are phenotypic changes of the dorsal root ganglions (DRGs) with an increase in excitability, immune system signal alteration in the CNS, endocrine modifications. It has been demonstrated that after damage nociceptors become hyperexcitable.
IMMUNE CELL INVOLVMENT IN NEUROPATHIC PAIN
The activation of the immune system has a main role in both peripheral and central abnormal sensory processing. Zuo et al. (2003) indicated that mast cells were activated in a model of partial sciatic nerve injury.
Perkins and Tracey (2000) studied and showed an invasion of endoneural neutrophils into the damaged nerve—a process that peaked 24 h after injury. Neuropathic pain symptoms did not develop after depleting circulating neutrophilis at the time of nerve injury but established symptoms did not reverse (Perkins and Tracey, 2000). Therefore, neutrophils could have an important role in the early stage if neuropathic could have an important role in the early stage of neuropathic pain development.
Several lines of evidence indicate that macrophages and the development of allodynia or hyperalgesia (Heumann, 1987; Myers et al., 1996; Sommer and Schafers, 1998; Cui et al., 2000; Liu et al., 2000). A temporal correlation between the invasion of blood-born, macrophages and the development of allodynia or hyperalgesia was shown. Furthermore, a lack of thermal hyperalgesia in a neuropathic model in the WDl mouse, which shows delayed recruitment for non-resident macrophages, has been reported. Rutkowski et al. (2002) failed to relieve mechanical allodynia after clodronate administration.
Along with or after macrophage recruitment, T cells are infiltrated into damage nerves, but their involvement in neuropathic pain has been poorly studied.
Few studies have focused on the infiltration of immune cells into the spinal cord after peripheral nerve injury, in particular hematogenous leukocytes and resident microglia (Sweitzer et al., 2002).
Fluorocitrate treatment, which blocks astrocyte and microglia metabolism, inhibits neuropathic pain, whereas minocycline, specific microglial inhibitor, blocks the development of neuropathic pain states but does not reduce pain that is already established (Raghavendra et al., 2003).
The spinal implantation of microglia, activated in vitro, simulated signs of neuropathic pain (mechanical allodynia). Microglial activation, as new studies reveal (Zhuang et al., 2005), seems to be the first step in the activation of immune responses in CNS. In fact, microglia might be responsible for the initiation of neuropathic pain states, and astrocytes may be involved in their maintenance (Tanga et al., 2005; Zhuang et al., 2005).
ROLE OF CITOKINES AND GROWTH FACTOR
Not only immune cells but also citochines play a role in the perception and transmission of pain, for example, TNFα IL-1, and IL-6 (De Jongh, 2003). The importance of growth factors and of cytokines in the development and maintenance of pain, as a result of different kind of tissue damage, is still on debating. Mendell L. was the first to identify the relation between NGF and pain. In his laboratory he studied the mechanism of hyperalgesia after NGF stimulus, probably dependent by mastocyte degranulation and by the increasing discharge in capsacin-inducted isolated sensory neurons (Fig. 1C). The high-affinity NGF receptors (tyrosine kinase receptor A—TrkA) are expressed by about 50% of nociceptors and their activation leads to phosphorylation and sensitization of TRPV1 receptors, which might account for NGF-induced heat hyperalgesia. The intracellular signaling pathways remain controversial (Bonnington and McNaughton, 2003) NGF modulates nociceptor gene expression (such as TRPV1, BDNF, substance P) after retrograde transport of NGF-TrkA to nucleus, which might underline increases in long-term nociceptor sensitivity.
McMahon focused his work on neurotopic factors which during inflammation could play a role of neurotransmitters/neuromodulators in the dorsal horn neurons of the spinal cord, where they are released by small caliber afferent roots, increasing the excitability of dorsal horn neurons. The whole sensory neurons, coming along with damaged fibers, in neuropathic models show different gene expression. These axons, close to progressive degenerated nerves, in some amyelinic afferent fibers, recruit an elevated number of macrophages, which are cause of an increase of neuroactive molecules such as growth factors and chemokines. In this way intact nerves along with degenerated afferent fibers, could receive abnormal molecular signals from target and nerves. After nerve damage some afferent neurons fire spontaneously.
Na+ CHANNEL AND GENE EXPRESSION AFTER PERIPHERAL NERVE INJURY
Until few years ago electrophysiological data showed that peripheral damage was the origin of abnormal activity (Fig. 1D). Only recently it has been established that only some fibers become origin of abnormal activity. Primary sensory neurons could be divided in two functionally different groups: nociceptors with fibers C, amyelinic with low conduction, and mechanoreceptors with large myelinic fibers and rapid conduction Aδ. We can easily imagine how an abnormal or ectopic activity derives from nociceptors, which could determinate the onset of pain in many neuropathies. We have to consider the Aδ activity, which can be the cause of pain in presence of central sensitization. Fibers C activity starts central sensitization and following this event the Aδ activity plays an important role inducing allodynia in human neuropathy. Usually after lesion of spinal nerve L5 spontaneous activity arises exclusively from myelinic fibers, in particular during the first and the second week after damage, when neuropathic pain stabilizes (Boucher et al., 2000). Many data show that intact nerves as well as L4 nerve, after L5 lesion, have many plastic changes including the presence of spontaneous activity.
Therefore, myelinic fibers show changes similar to the damaged afferent nerves. Many Aδ afferents begin to produce high frequency impulses or trains of potentials action against the spinal cord (Michaelis and Liu, 2000). Besides electrophysiological changes already described, models of experimental neuropathy show changes of primary sensory neuron gene expression. Peripheral axons of damaged sensory fibers reveal a progressive degeneration.
Consequently, damaged neuron cells lose their binding with target and show a different gene expression. Some damaged fibers A with a phenotypic change express molecules normally associated to nociceptors (substance P, BDNF).
Altered gene expression of afferent intact nerves could be explained by an increasing of NGF available (Fokuoka et al., 2000). Moreover, substance P and VR1, abundant in intact fibers C, are regulated by NGF (Fig. 2A).
A different expression of ionic channels could contribute to explain the mechanism of spontaneous neuronal firing. Waxman et al. (1999) demonstrated that different type of damage causes alteration in sodium channel expression: axonal lesion is associated with a downregulation of sodium channel TTX-resistant expression, whereas inflammatory damage is associated with upregulation. Porreca et al. (1999) indicated that sodium channels TTX-resistant, called SNS/PN3, trigger and maintain hyperalgesia and allodynia nerve damage induced. Gold et al. demonstrated the importance of sodium channels TTX-resistant during inflammation: the discharge is modulated by mediators as prostaglandin E2, 5-HT, and adenosin, responsible of peripheral sensitization (Fig. 2B).
Several neuron specific sodium channels have been cloned and sequenced using molecular techniques. The latest revealed new nociceptive mechanisms, involving molecules receptors and a neuronal network in the spinal cord and in the brain, developed after peripheral nerve injury or nerve damage.
Almost 10 years ago, studying memory in Aplysia, Kandel (1986) found that oncogenes, first identified in the virus, were present also inside stimulated neurons. One of the genes, identical to the oncogene v-fos, is involved in neuron final changes after temporary stimuli. This gene is constitutive and is called c-fos (Fig. 2C). Hunt et al. (1987) observed that nociceptive stimuli induce the expression of c-fos in the DRG and that c-fos and other early genes, as c-jun, bind to regulator factors in the nucleus of many activated cells. Curran and Franza (1988) described the binding of fos and jun proteins to a regulatory region of AP-1, as “third messenger” in many cells already activated by second messenger as c-AMP or ions. Ji and Rupp (1997) emphasized that c-AMP responsive binding element (CREB) was involved in the transcription of many genes, in spinal neuron “long-term potentiation” and in c-fos induction after formalin test. Draisci and Iadarola (1989) demonstrated that c-fos mRNA is present in rat dorsal horn neurons few minutes after the beginning of a peripheral inflammation. During peripheral chronic inflammation, the continuous activation of fibers C result in gene transcription alteration in the DRG and in the posterior horn neurons. Following peripheral lesion, changes in the neuron excitability and in mRNA levels in sensory neurons are substrate for chronic pain.
CENTRAL EVENTS ASSOCIATED WITH PERIPHERAL NEUROPATHY
The prolonged activity of fibers C, even if with moderate frequency, is able to induce a synaptic conduction increase in dorsal root neurons. This central sensitization has been demonstrated in many experimental models of inflammatory pain. Few seconds of fibers C activity result in minutes of post-synaptic depolarization. The latest is caused by the activation of NMDA, glutamate, tachikinin NK-1, substance P, and neurochinin A receptors.
The importance of glutamate, substance P, neurokinin, in central sensitization has been demonstrated for their ability to prevent depolarization using antagonist of NMDA receptors in particular not competitive antagonists of NMDA receptors (Fig. 2D), which reduce nociceptive behavior formalin induced. Competitive antagonists (AP-5 for NMDA receptor) as well as not competitive (MK-801 for phencyclidin FCP) reduce the persistent activity of dorsal roots in association with peripheral formalin injection (Berrino et al., 2003).
In our study (Viggiano et al., 2004) we observed after formaldehyde injection in rat lip skin, an altered releasing of GABA in the spinal nucleus of trigeminus (Fig. 3A); moreover, NO synthesis mediates the increased releasing of amino acids in the same nucleus. Recently, it has been demonstrated that free radicals could be involved in signal transduction of pain as cellular mediators in a sub-toxicity condition (Smythies, 1999).
The strong evidence that nitric oxide is an important mediator of hyperalgesia is in the CNS, but evidence for a peripheral action is less clear. Nitric oxide is induced in tissues during inflammation, probably through both inducible and neuronal nitric oxide synthase (iNOS and nNOS). Nitric oxide donors can induce pain in humans and NOS inhibitors can reduce inflammatory hyperalgesia in PGE2 dependent manner (Omote, 2001; Thomsen and Olesen, 2001; McMahon et al., 2005).
For these reasons we decided to evaluate, in experimental model of pain, formalin injection induced, superoxide dismutase (SOD) activity in section of brain sensory nuclei using immunohystochimic technique to quantify SOD. We found a difference, statistic significant, in SOD activity between the two side of the encephalic trunk, in trigeminus sensory nuclei, confirming SOD as modulator, fully involved in pain transmission (Viggiano et al., 2005; Fig. 3B–E).
International scientific literature emphasized the prevalence of “benign” pain chronic syndromes, more intense, frequent and prolonged in women compared to men. For this reason we decided to investigate the different aspects of visceral pain, in standard condition, in rat models, male and female, with induced ureter stones. The rats showed behavior indicating visceral pain (ureteral crisis) and muscle hyperalgesia directly correlated to organ pain potential. Such a model reproduced the same condition found in clinical practice.
Moreover, the sexual dysmorphism present in painful stimuli appeared us to be an important factor in order to evaluate the role of sexual hormones in the modulation of pain.
A brief peripheral nociceptive stimulus induces c-fos expression in central nervous system within 30 min, with spikes after 1 or 2 h. Then c-fos expression disappears within 8 h. Therefore, c-fos seems to be a neuronal activation index, which could localize sensory and motoneurons involved.
We have reviewed the different mechanisms suggested for the maintenance of pain. Dubner (2004) studied descending nociceptive mechanisms and their changes after tissue damage, including suppression and facilitation of defense behavior during pain. Many researchers emphasize the role of these changes in inducing NMDA and AMPA receptors gene expression, after prolonged inflammation.
Neugebauer et al. (2004) demonstrated a relation between a persistent pain and amygdale. The amygdale is the center of negative affective status such as anxiety, depression, fear. Capsular lateral division of amygdale central nucleus has recently been defined as “nociceptive-amygdale,” since it has been demonstrated, through neuroimaging techniques, neuroplastic biochemical pharmacological, and electrophysiological changes during persistent pain.
Molecular biology is important to clarify how altered gene expression can regulate neuronal activity after inflammation or tissue damage. Since the entire genome has been studied, we will be able to find new genes involved in specific condition such as pain, hoping that in the next future it will be possible control pain through gene transfer.