I will now begin to talk about molecular pathways of persistent pain conditions. And I'd like to talk about really, two fundamental processes that, that are neurologically based. And that contribute towards the sensitization of especially C fibers, and these pain transmitting neurons that I spoke about earlier. So these are very important mechanisms that contribute to pathological pain. Pain that we experience in inflammatory conditions. Pain that we experience under conditions of nerve injury. And under metabolic disorders such as diabetes. So they're two phenomenons that we'll spend a little time with. Phenomenon known as peripheral sensitization and it's second is central sensitization. Now if we talk about peripheral sensitization you may recall from one of the previous slides that I presented that there are cells of the immune system that become resident to an area where there's injury. And when there, where there is chronic inflammation or even acute inflammation, those cells of the, immune system invade an area of injury. There are a number of, chemotactic and chemokines that are, that are attractants to, of these cells. There are vascular changes that permit these cells to migrate to, the site of injury. And these cells when they arrive, are generally involved in, in tissue healing but in the process they release a whole series of substances which sensitize, C fibers. And this, as I mentioned earlier, may be adaptive in the sense that it post injury, it is promoting an immobilization, a pain signal that, that causes the individual to immobilize the injury, to go into a quiescent stage, and to become in a, more of, of a recovery stage then an active stage promoting healing. But these substances released by mast cells and macrophages and, and from surrounding tissues including the the dying back of schwan cells or even schwan cells that become active in the inflammatory soup. They're a whole series of substances such as nerve growth factor and ethilium, histamine, bradychinin just a host of agents different purines like ATP, adenosine which make their way to these proteins I spoke about earlier. Surface receptors on these C fiber terminals. And when these receptors are stimulated this produces the transduction of signals into the intracellular space, where a variety of second messenger cascades are activated. Many of these are, are kinases that can Influence the excitability of ion channels, and actually in some of these receptors, many of which are called g protein-coupled receptors, that once this cascade begins to develop, that the phosphorylation of many of these proteins changes their biological characteristics so in many cases they can be inhibited or activated depending upon the nature of the false relation effect on the protein. But the point is, is that many of these chemicals released by these endogenous cells of the immune system are able to activate these C fibers. The second messenger pathways will feed back to alter the transduction properties of these [UNKNOWN] so they become more excitable. So that when there's a given stimulus, they actually show greater activity greater discharge sending more signals to the brain. In fact they can lower their threshold for activation to the point where they become spontaneously active, so that in the presence of inflammatory soup. In an inflammatory milieu you'll see that the terminals of the C fibers can take on spontaneous generator activities, sending signals in a spontaneous way to the central nervous system, very much like uh,cardiac pacemakers. So that all of a sudden we have a peripheral generator, of C5 input making there way to the it's way to the central nervous system. A second consequence of, of the activation of this second messenger cascade is that it causes gianormic induction or changes in the. In these terminals such that certain types of transcriptional factors are activated leading to changes in gene expression and protein expression, which feedback, these protein feedback to these terminals. And, either become surface proteins. Or can influence again the biological transduction properties of these proteins. So there are some longer-term events that actually change the characteristics of these cells so that they take on a different phenotype or a different trait, so that they become now very active for long periods of time. In a temporally dependent manner. So acutely we see that the second [UNKNOWN] cascade can produce almost immediate effects on some of these proteins. And then with gene induction, we see that genetic production, genomic production of proteins can influence the actual phenotypic characteristics of the cell. Now I mentioned on this previous slide, that when this cell depolarizes it sends signals to the brain. But you can see here that when it depolarizes, it also depolarizes this nerve ending. And when that nerve ending is depolarized, a number of very interesting events occur which is shown on this slide. It produces a phenomenon known as neurogenic inflammation. And so here, again is a C fiber. A noxious nociceptive stimulus is coming in. The second messengers are being activated and signals are being sent to the dorsal root ganglion where, where, genes are being up-regulated or down-regulated. But concomitantly, at the same time, the terminal arbors are being depolarized. So not only is this afferent sending signals to the brain, there is an anti-dromic activation of these terminals. They're depolarizing. And as they depolarize they release. A variety of neuropeptides from these terminals. Substance P, calcetone and gene related peptide, neurochinan a and glutamate are some of the substances that are antidromically released with the activation of these C-fibers. These substances produce profound inflammatory effects. They're able to serve as chemokines to bring cells into the environment the immune cells shown here, mast cells and, and other immune cells possess receptors for these neuropeptides. And when these receptors are activated these cells degranulate, releasing histamine, serotonin, prostaglandins, cyt a variety of cytokines, which produce this excitatory effect the spontaneous generating effect of these C fibers, but they also produce proofound vasoldilation. So CGRP is one of the most potent vasodilators that we know. And this peptide produces changes in capular, cap, capillary permeability, changes in blood flow, and vascular compliance so that we get edema, redness and swelling in the area. As well as in the change in temperature that occurs in the inflamed site. So these C fibers not only convey information to the brain, but they're profoundly, effective in, in, in propagating inflammatory response at the site of tissue injury. And, these may be very important mechanisms that contribute to both acute and persistent pain conditions. Finally, I'd like to, to speak briefly about some of the events that occur peripherally again, that are conveying this peripheral sensitization. So, as I've already mentioned, under normal physiological conditions when there is a painful stimulus some of these ion channels and G protein couple receptors are activated leading to sodium influx into the, into the a neuron, and then there's a discharge, electrical discharge of the axon making its way to, to the brain. When there is peripheral sensitization due to inflammatory substances again,. The pH is lowered in the tissue. ATP is is released, and many of the proteins and chemical substances I mentioned earlier released that are causing activation of the second messenger system. Again, the phosphorylation and then the, the effects on gene expression leading to phenotypic changes. And in combinate with that, a much lower threshold for activation of these cells. And when they're activated, they are very active and again they can take on spontaneous nerve activity. So this is a case again for inflammatory pain. In the case of nerve injury where there's been a physical injury to the nerve. In the periphery what we find is that again there is excitation of the terminals, and there is at the dorsal root ganglion again change in gene expression, where certain proteins again are transported to the area of injury. And, they are oftentimes expressed in the neuromacite, an area that forms a neuroma. And these, neuromas, take on, pacemaker activity, in part due to, changes in potassium channels and so the neuroma can take on pacemaker activity driving pain signals to the brain. So nerve injury results in ex, excitatory effects, in part through what's called ectopic activity, where the ectopic activity is mediated by new proteins that are being synthesized and integrated into the membrane. And that these new proteins have the capabilities of producing excitatory effects that are now being transmitted to the nervous system. Now much of what I just talked about, these new proteins and the expression of these new proteins result from the activation again of these channels, a variety of channels which cause the second messenger, certain kinases to be activated. Leading to the activation of transcriptional factors. Leading to the ultimate expression of new proteins that make their way into the membrane and change the physical characteristics. In phenotypic patterns of these primary afferents. So these are the, the primary basic mechanisms, molecular mechanisms that we're aware of, that contribute to peripheral sensitization. The, the last aspect that is relatively new, leading to peripheral sensitization. Is a role for cytokines. These are cell, these are substances which are released by the immune system. And can greatly impact the biology and the activity of a delt. I'm sorry, of C fibers in particular. I speak mostly of C fibers in this presentation because we have a, still a relatively poor understanding of the molecular mechanisms that I think contribute to A delta mediated pain. I think most of what we understand relatively well relates to C fiber mediated pain. And in this specific case, in response to tissue injury resident cells and immune cells release bradykinin, which leads to sort of a master regulator. A cytokine TNF alpha, which, is a almost the master regulator of other cytokines. Activating at least two pathways that can impact pain. One pathway signals on the IL-1 Beta, IL-6 Pathway, which leads to the up-regulation of the enzyme that makes prostaglandins, Cox-2. And so, the eicosanoids, which make prostaglandins. Can sensitize these nociceptors by specific surface receptors, as I've shown previously in the other slides. Another pathway which is becoming I think, of great interest, especially where there's widespread pain in the body is a pathway that activates IL-8, interleukin 8. Interleukin A is in circulation or in organ tissues can stimulate the terminals of the sympathetic nervous system to cause a release of catecholamines like norepinephrine. And also It's believed that it may actually activate the release of epinephrine, from adrenal tissues. Which, together, these catecholamines activate a receptor subtype called a beta two adrenergic receptor. And the beta two adrenergic receptor, when activated, sensitizes nose [INAUDIBLE] as well. So ILA is prominently present in patients with fibromyalgia, in patients with widespread pain, and it maybe that ILA is activating this pathway that ultimately activates the beta-adrenergic system. Leading to C fiber sensitization, and some of the widespread muscle pain that we see in, in these patients. So this is a, a relatively new pathway and there will be substantial new work that will be conducted on understanding. How cytokines and chemokines influence of pain processing through peripheral sensitization. The next section of this vignette relates to central sensitization. And it can be seen from a mechanistic perspective in this portrait. So again, the nociceptor, a delta C fiber, but we'll focus again primarily on C fiber and the A beta fiber which is sending out a branch again to the pain transmitting neuron. And conveys a sense of touch. And so, with central sensitization there is an injury to the site, it could be skin or viscera, it could be you know, an included coronary artery. It could be a variety of events that lead to the activation of these C fibers, which are now depolarizing, sending signals to the, pain neuron, and we now know that like these peripheral sites, these neurons also begin to change their excitatory capabilities. They can take on pacemaker activity, and they can change their phenotype through genetic mechanisms. And [COUGH] we know that the primary initiating stimulus for this is a peripheral input which leads to excitation of these C fibers. Leading to these changes in the pain transmitting neuron, conveying excitatory events of, of the neuraxis into the brain. So when we have injury, we have hyperalgesia, enhanced responses to uh,innocuous stimulus. Concomitantly when we have an injury to the skin or the viscera, whether it be inflammatory in nature or neuropathic in nature, we find that once this central neuron becomes sensitized,. Now the touch system, which was under normal physiological conditions conveying touch, now there is a disinhibition, there's actually a facilitation in this pathway, in part because of a removal of an inhibitory drive But, also, because this cell now becomes extremely sensitive to the neurotransmitters being released by A-beta fibers. So now, this cell, is not only responding to noxious stimuli, capable of activating the sensitized nociceptors. but now it's responding to touch because of the unmasking of this synaptic pathway as a result of sensitization of this pain neuron. So now we have what's called alodenia where a traditionally non-noxious touch stimulus. Is able to evoke pain. And I think you know to put this into I think a common experience is sunburn. When you have a sunburn, when you touch the area of a sunburn you evoke pain. And it's thought that a part a good portion of that touch evoke pain is being mediated by these a beta fibers. That are now conveying and activating these pain transmitting neurons which are sending signals to the higher regions of the brain. And also ,these cells the c fibers have lowered their threshold to such an extent. The touch is also very likely to activate these fibers as well. So, in the contexts of a sunburn or in an area that's been injured, when we touch that area, the beta-fibers are activating these jazzed up sensitized pain neurons. And these jazzed up C-fibers are also activating these neurons. So you see that there's a very dynamic and a very complex intrinsic mechanism that we have that signals tissue injury and danger. And finally, in talking about central sensitization,. I would just like to highlight that some of the same neural mechanisms, the same biochemical mechanisms that I talked about earlier about, neurotransmitters being released and second messengers being activated, producing changes in excitation and in phenotypic, also exist on these pain-transmitting neurons. so here we have the C-fiber coming in. It releases certain transmitters, like BDNF, substance P, glutamate and these neurotransmitters are released, they make their way across the synaptic cleft, they interact with specific receptors on the pain transmitting neuron. Activating these secondary cascades, second messenger cascades. Activating different types of kinases, which in turn, influence the biological activity of these channels, making them more active. At the same time, this second messenger cascade is inducing transcriptional factors in the nucleus that then lead to phenotypic changes in expression of novel mechanisms that are leading to more persistent pain states. So this is reminiscent of what happens to the C fiber, but now this is occurring at the level of the spinal cord with these pain transmitting neurons. And there is more and more evidence that this type of redundancy exists all the way up to cortical pathways so that we see sensitization not only in the spinal cord, but there's evidence of sensitization in many of the ascending pathways. That I talked about being mediated by similar types of biochemical events. So these are some of the biochemical events between C fibers and pain transmitting neurons which lead to sensitization. There are two others that you should also be aware of. One relates to. Nerve sprouting, so that especially in response to nerve injury what we find is that the a beta fibers that are coming into the spinal cord actually begin to sprout to a greater extent. So more of these minor pathways become evident. And they become evident for large segments of the spinal cord reaching across several dermatomes, is the thought. And that this allows then for a touch stimulus in the one area of the body to be perceived as a rather large territory of, of pain. So these A-beta fibers sprout, and they synapse with these pain-transmitting neurons. For quite a substantial distance within the spinal cord. And then the final mechanism that can contribute to central sensitization is a loss of inhibition. So as I mentioned there are inhibitory intra neurons which are keeping the pain neuron in check in keeping it quiet. We now know when there's a profound C fiber barrage, when there's profound input to the C fiber that these inhibitory neurons go through a calcium-mediated death. They end up being destroyed. And andostin allows for a disinhibition to occur. And for activation of these pain pathways to come about. And this may then also contribute to spontaneous activity that's occurring in these pain transmission neurons. So there's several mechanisms by which, central sensitization can occur. By a chemically and anatomically associated events can contribute, to the phenomenon of central sensitization. And clinically, this is characterized by, hyperalgesia. That is enhanced responses to a painful stimulus. Allodynia that is a a painfully evoked response to a traditionally non-noxious touch stimulus. And referral pain, a pain that refers across anatomical distributions much greater that the site of injury, and mediated in part by this concept or process of,of a central sensitization of these pain transmitting neurons. Finally, with respect to central sensitization I'd like to introduce the concept of a new player. A ew nonneuronal neighbor that is contributing to the phenomenon of central sensitization. And they are glial tissues, astrocytes,and microglia, and I think many of you know that astrocytes and microglia really compose a substantial percentage of the brain and spinal cord and that these These cells have been historically viewed as supporting structures to neurons. Providing nourishment cleaning up debris and foreign objects from, from the central nervous system. Yet we now recognize that the microglia play a very important role in synaptic transmission. In part by buffering potassium levels and the excitability of neural pathways. And within the spinal chord there's been considerable work int he spinal chord that has helped identify the biological processes that these, these cells mediate. And to review and to present those to you. I'll just a back up a little bit on the mechanisms again. Again in panel a this is the traditional mechanism, C-fiber a-delta being activated and being sent, sending signals through ascending pathways. Panel b, you know, we have the C-fibers coming in, releasing neurochemicals. activating these proteins, producing the activation of the pain signal, or the pain neuron. And in panel C, we see that, again, the profound C-fiber input, we get some of these second messenger systems going off, and some novel proteins being expressed. And new chemicals being generated that sensitize this pathway. But in this situation, where there are now chemicals being released, some of these chemicals can actually influence the resident allele cells that surround, and actually form part of the synapse. With these c fibers and pain-transmitting neurons. And these chemicals actually produces changes. Changes in these boulia, so now that they become very, very active. And they begin to produce a variety of substances. So, substances such as nitric oxide. prostaglandins and fractalkine which are released from pain transmitting neurons,activate these cells so they become extremely active, and they begin to release a variety of pro pain-evoking substances. Especially cytokines and prostaglandins. And many other agents which activate these pain neurons, and cause greater sensitization between the C-fiber and these pain-transmitting neurons. And so it's now recognized that glia play an extremely important role not only as supporting structures for the nervous system,. But they're very important in mo, modulating synaptic activity. And it's also now believed that these cells, these resident cells, actually may be the process by which pain memory can express, such that after healing. These resident stale cells, these glial cells may remain fairly active for a period of time. So that sometime later, a year, two years later, a reinjury to the site leads to even greater response with respect to pain and inflammation. And it's thought that these resident cells actually are, are more primed and ready to go off to be rekindled to a greater extent to release these substances which then reactivate these pathways. So, these cells may play a very important process in pain memory. And we now believe they also play a very important role in memory at the level of the hippocampus and other pathways of the brain involving, involved in learning and memory. But the same process is involved in microglia effects on learning and memory may occur in the spinal cord. to, to produce, these sensitive events. [SOUND] [BLANK_AUDIO]
