Functional and anatomical characterization of corticotropin-releasing factor receptor subtypes of the rat spinal cord involved in somatic pain relief

Abstract Corticotropin-releasing factor (CRF) orchestrates our body’s response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. This study examined CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund’s complete adjuvant-induced hindpaw inflammation using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, paw pressure algesiometry examined antinociceptive effects of intrathecal (i.t.) CRF and their possible antagonism through CRF-R1 and/or CRF-R2 selective antagonists as well as the opioid receptor antagonist naloxone. Our results demonstrated predominantly CRF-R2 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonist elicited potent, dose-dependent anti-nociceptive effects which were antagonized selectively by i.t. CRF-R2 (K41498) antagonist but not by CRF-R1 (NBI35965) antagonist. Moreover, the opioid antagonist naloxone dose-dependently reversed the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Supporting these findings, double immunofluorescence confocal microscopy showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonist inhibit inflammatory somatic pain, occurring predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons, which results in endogenous opioid-mediated pain inhibition.

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Functional and anatomical characterization of corticotropin-releasing factor receptor subtypes of the rat spinal cord involved in somatic pain relief

10.21203/rs.3.rs-290650/v1 ◽

2021 ◽

Author(s):

Shaaban Mousa ◽

Mohammed Shaqura ◽

Baled Khalefa ◽

Li Li ◽

Mohammed Al-madol ◽

...

Keyword(s):

Spinal Cord ◽

Western Blot ◽

Opioid Receptor ◽

Radioligand Binding ◽

Corticotropin Releasing Factor ◽

Receptor Subtypes ◽

Inhibitory Interneurons ◽

Rat Spinal Cord ◽

Somatic Pain ◽

Antinociceptive Effects

Abstract Corticotropin-releasing factor (CRF) orchestrates our body’s response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptors of subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. This study aimed to examine CRF-R1 and CRF-R2 expression within spinal cord of rats with Freund’s complete adjuvant-induced hindpaw inflammation using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis, Western blot, immunohistochemistry, and radioligand binding. Moreover, paw pressure algesiometry examined antinociceptive effects of intrathecal (i.t.) CRF and their possible antagonism through CRF-R1 and/or CRF-R2 selective antagonists as well as opioid receptor antagonist naloxone. Our results demonstrated mainly CRF-R2 mRNA, protein, binding sites and immunoreactivity in dorsal horn of rat spinal cord. In parallel, i.t. CRF as well as CRF-R2 agonists elicited potent antinociceptive effects which are dose-dependent and antagonized exclusively by i.t. CRF-R2 (K41498), but not CRF-R1 (NBI35965) antagonist. Moreover, i.t. CRF elicited inhibition of somatic pain that was dose-dependently reversed by the opioid antagonist naloxone. Consistently, double immunofluorescence confocal microscopy showed CRF-R2 on enkephalin (ENK) containing inhibitory interneurons in close opposition of incoming, mu-opioid receptor-immunoreactive nociceptive neurons but not on pre- nor on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonist inhibits inflammatory somatic pain which occurs most predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons resulting in endogenous opioid-mediated pain inhibition.

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Functional and anatomical characterization of corticotropin-releasing factor receptor subtypes of the rat spinal cord involved in somatic pain relief

10.21203/rs.3.rs-268281/v1 ◽

2021 ◽

Author(s):

Shaaban A. Mousa ◽

Mohammed Shaqura ◽

Baled Khalefa ◽

Li Li ◽

Mohammed Al-madol ◽

...

Keyword(s):

Spinal Cord ◽

Western Blot ◽

Opioid Receptor ◽

Radioligand Binding ◽

Corticotropin Releasing Factor ◽

Receptor Subtypes ◽

Inhibitory Interneurons ◽

Rat Spinal Cord ◽

Somatic Pain ◽

Antinociceptive Effects

Abstract Background: Corticotropin-releasing factor (CRF) orchestrates our body’s response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptors of subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial.Methods: This study aimed to examine CRF-R1 and CRF-R2 expression within spinal cord of rats with Freund’s complete adjuvant-induced hindpaw inflammation using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis, Western blot, immunohistochemistry, and radioligand binding. Moreover, paw pressure algesiometry examined antinociceptive effects of intrathecal (i.t.) CRF and their possible antagonism through CRF-R1 and/or CRF-R2 selective antagonists as well as opioid receptor antagonist naloxone.Results: Our results demonstrated mainly CRF-R2 mRNA, protein, binding sites and immunoreactivity in dorsal horn of rat spinal cord. In parallel, i.t. low, systemically inactive doses of CRF as well as CRF-R2 agonists elicited potent antinociceptive effects which are dose-dependent and antagonized exclusively by i.t. CRF-R2 (K41498), but not CRF-R1 (NBI35965) antagonist. Moreover, i.t. CRF elicited inhibition of somatic pain that was dose-dependently reversed by the opioid antagonist naloxone. Consistently, double immunofluorescence confocal microscopy showed CRF-R2 on enkephalin (ENK) containing inhibitory interneurons in close opposition of incoming, mu-opioid receptor-immunoreactive nociceptive neurons but not on pre- nor on postsynaptic sensory neurons of the spinal cord.Conclusion: Taken together, these findings suggest that i.t. CRF or CRF-R2 agonist inhibits inflammatory somatic pain which occurs most predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons resulting in endogenous opioid-mediated pain inhibition.

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Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief

Molecular Neurobiology ◽

10.1007/s12035-021-02481-z ◽

2021 ◽

Author(s):

Shaaban A. Mousa ◽

Mohammed Shaqura ◽

Baled I. Khalefa ◽

Li Li ◽

Mohammed Al-Madol ◽

...

Keyword(s):

Spinal Cord ◽

Dorsal Horn ◽

Corticotropin Releasing Factor ◽

Receptor Subtypes ◽

Inhibitory Interneurons ◽

Rat Spinal Cord ◽

Nociceptive Neurons ◽

Somatic Pain ◽

Selective Antagonist ◽

Antinociceptive Effects

AbstractCorticotropin-releasing factor (CRF) orchestrates our body’s response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund’s complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

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