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Breaking the riddle of amylin’s role in nociception: a comprehensive study on the action of amylin in multiple pain models

Final Report Summary - AMYLIN_ROLE_PAIN (Breaking the riddle of amylin’s role in nociception: a comprehensive study on the action of amylin in multiple pain models)

„It is easier to find men, who will volunteer to die, than to find those who are willing to endure pain with patience“ Julius Caesar. Pain is more than just a symptom; it can develop into a disease. Chronic pain is a frequent and disabling condition with high personal and social impact. Available drugs are usually ineffective in about half of chronic pain patients; therefore there is an increasing demand for efficacious, safe, non-addictive pain killers.
Amylin is a peptide from the calcitonin gene related peptides family, mainly secreted with insulin by the pancreatic beta-cells. The best studied actions of amylin are on inhibition of food intake; however, several studies indicate that amylin might have some effects in nociceptive (pain) processing mechanisms, especially as amylin was found to be expressed by neurons that mediate noxious stimuli. In order to consider amylin as a therapeutic candidate in pain management, further comprehensive studies on the exact effect of amylin in nociception, and possible side effects from its use, need to be carried out in different pain paradigms in animals.
In this project we evaluated amylin’s effects on nociception by employing a wide and well established range of pain models and nociception behavioural tests. As chronic pain involves pathophysiological mechanisms diverse from the physiological ones occurring in acute pain, we evaluated if amylin plays a different role in either condition. Also, mechanisms underlying pain of inflammatory origin or derived from lesions in the nervous system (neuropathic pain) also seem to be quite distinct.
Therefore, in this project we determined anti- or pro-nociceptive effects of amylin in multiple pain models in rats (acute, sustained, chronic inflammatory and neuropathic pain). Amylin did not significantly alter the response of normal animals to the noxious stimuli in the acute pain tests, suggesting that amylin is not involved in the mediation of acute noxious stimuli. The effect of an acute subcutaneous (SC) amylin administration was also evaluated on the formalin test, a model of acute and tonic/sustained pain. The rat formalin test is a model of pain induced by acute tissue injury with formalin injection, which evokes a biphasic pattern with an acute and a tonic/sustained pain phase, the latter being dependent on local inflammation. Between these two phases, there is an interphase period, in which the animals display hardly any pain-related behaviour. Amylin’s effect to modulate spinal cord neurons activity was also analysed. Amylin SC administration affected mainly the interphase and the beginning of the tonic pain phase of the formalin test, suggesting a role for amylin in the modulation of pain with an inflammatory component and in the autoanalgesic/inhibitory mechanisms occurring in the interphase. Amylin also reduced the number of activated spinal cord neurons and the effects were dependent on the dose administered and on the time of amylin injection. Some brain areas known to be involved in descending modulation, in affective and defensive responses to pain were activated by formalin, and amylin administration caused a further increase in the number of activated neurons, suggesting these areas could be responsible for at least part of the observed amylin effects. Hence, data suggested that amylin may act at different parts of the pain matrix, namely, spinal cord and brain, which could explain the different effects observed according to injection timing.
A study in mice suggested that amylin attenuates visceral pain by the interaction of amylin with its specific receptor located in spinal cord neurons. We therefore investigated if some of the effects observed were mediated via spinal cord. Blockade of spinal amylin-receptors abolished the effects observed upon SC amylin administration, proving that, at least part of amylin’s effects, were mediated by spinal amylin-receptors. Also, blockade of endogenously produced amylin actions attenuated tonic pain.
Overall, data suggested that amylin modulates pain and might have affected the nociceptive system at different levels of the pain matrix at distinct time points to induce the observed effects in the formalin test.
Next we tested amylin’s effect on the monoarthritis (MA) model of chronic inflammatory pain. Our hypothesis was that chronic systemic amylin administration may influence inflammation progression and chronic pain associated with the development of the MA condition. We evaluated the nociceptive responses associated with MA using several behavioural tests and monitored the evolution of the inflammation. Data indicated that amylin reduced the nociceptive scores and increased the paw pressure thresholds, suggesting reduced allodynia (pain in response to an innocuous stimulus) and hyperalgesia (increased pain response to a noxious stimulus).
Next, we examined amylin’s role in an animal model of neuropathic pain and pain development was assessed by acute pain behavioural tests. The effect of acute and chronic subcutaneous amylin administration was evaluated to detect possible changes in painful-like behaviours in an established chronic painful neuropathy. To evaluate signs of changes in noxious input/transmission, we evaluated the expression levels of two different markers of neuronal activity. Chronic amylin administration significantly and progressively aggravated cold allodynia, suggesting that amylin may have a pro-nociceptive role in neuropathic pain. Amylin modulated the activity of the spinal neuronal circuitry differently in animals with and without neuropathy, and increased the expression of one of the markers of neuronal activity. There were also signs of degeneration of sciatic nerve fibres mediating pain to the spinal cord in SNI rats.
Next, we investigated whether some amylin effects in neuropathic rats could be mediated at the spinal cord. Spinal injection of an amylin-receptor blocker tended to ameliorate mechanical allodynia but chronic spinal amylin administration had no effect, suggesting that the pro-nociceptive effects of chronic subcutaneous amylin were probably due to an action outside the spinal cord, probably at supraspinal brain levels. Overall, data suggested that amylin may have a pro-nociceptive role in neuropathic pain; however, acute amylin-receptor blockade tended to ameliorate mechanical allodynia, suggesting that spinal amylin-receptors are sensitive to pharmacological manipulation in this pain condition.
We also further characterized the amylinergic nervous system and found that some amylin-expressing neurons are of the small C-non-peptidergic nociceptor type. Expression of calcitonin-receptor, to detect amylin-receptors, was found in the lateral spinal nucleus (Lsp). The Lsp receives noxious input from nociceptive fibres, projects to spinal cord layers that have projection neurons sending the noxious information to supraspinal areas, and receives descending modulation. Therefore, amylin-receptors in this spinal area may represent a target for endogenous spinal amylin expression to modulate pain. Furthermore, the Lsp is known to be activated after formalin injection, which supports the notion that this nucleus may represent a target for some of the amylin-effects observed upon spinal administration.
Concluding, in the present project we found evidence that amylin modulates pain and might affect the system at different levels of the pain matrix at distinct time points. A role in pain of inflammatory origin was found and amylin’s effect seems to depend upon the concomitant existence of a strong continuous noxious stimulus that may unblock the amylinergic system. Amylin’s pro- or anti-nociceptive effect were dependent on the nature of the noxious insult (inflammatory vs. neuropathic). Data points to the existence of important spinal cord mediated mechanisms. This is in agreement with studies on visceral pain suggesting a spinal site of amylin action. Still, supraspinal amylin binding-sites were proposed to be responsible for some of systemic amylin effects.
Amylin is a physiological hormone and its commercial analogue (pramlintide) has proven to be safe in diabetic patients, implying that it may be suitable for treating patients with pain in case further promising results are found. The present results helped to better understand the pain system and may support the future development of improved therapies for pain treatment.