Chronic stress causes several pain conditions including fibromyalgia. Its pathophysiological mechanisms are unknown, and the therapy is unresolved. Since the involvement of interleukin-1 (IL-1) has been described in stress and inflammatory pain but no data are available regarding stress-induced pain, we studied its role in a chronic restraint stress (CRS) mouse model. Female and male C57Bl/6J wild-type (WT) and IL-1αβ-deficient (knock-out: IL-1 KO) mice were exposed to 6 h of immobilization/day for 4 weeks. Mechanonociception, cold tolerance, behavioral alterations, relative thymus/adrenal gland weights, microglia ionized calcium-binding adaptor molecule 1 (IBA1) and astrocyte glial fibrillary acidic protein (GFAP) integrated density, number and morphological transformation in pain-related brain regions were determined. CRS induced 15–20% mechanical hyperalgesia after 2 weeks in WT mice in both sexes, which was significantly reduced in female but not in male IL-1 KOs. Increased IBA1+ integrated density in the central nucleus of amygdala, primary somatosensory cortex hind limb representation part, hippocampus cornu ammonis area 3 (CA3) and periaqueductal gray matter (PAG) was present, accompanied by a cell number increase in IBA1+ microglia in stressed female WTs but not in IL-1 KOs. CRS induced morphological changes of GFAP+ astrocytes in WT but not in KO mice. Stress evoked cold hypersensitivity in the stressed animals. Anxiety and depression-like behaviors, thymus and adrenal gland weight changes were detectable in all groups after 2 but not 4 weeks of CRS due to adaptation. Thus, IL-1 mediates chronic stress-induced hyperalgesia in female mice, without other major behavioral alterations, suggesting the analgesic potentials of IL-1 in blocking drugs in stress-related pain syndromes.

Numerous in vitro and in vivo models of Parkinson’s disease (PD) demonstrate that pituitary adenylate cyclase-activating polypeptide (PACAP) conveys its strong neuroprotective actions mainly via its specific PAC1 receptor (PAC1R) in models of PD. We recently described the decrease in PAC1R protein content in the basal ganglia of macaques in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD that was partially reversed by levodopa therapy. In this work, we tested whether these observations occur also in the rotenone model of PD in the rat. The rotarod test revealed motor skill deterioration upon rotenone administration, which was reversed by benserazide/levodopa (B/L) treatment. The sucrose preference test suggested increased depression level while the open field test showed increased anxiety in rats rendered parkinsonian, regardless of the received B/L therapy. Reduced dopaminergic cell count in the substantia nigra pars compacta (SNpc) diminished the dopaminergic fiber density in the caudate-putamen (CPu) and decreased the peptidergic cell count in the centrally projecting Edinger–Westphal nucleus (EWcp), supporting the efficacy of rotenone treatment. RNAscope in situ hybridization revealed decreased PACAP mRNA (Adcyap1) and PAC1R mRNA (Adcyap1r1) expression in the CPu, globus pallidus, dopaminergic SNpc and peptidergic EWcp of rotenone-treated rats, but no remarkable downregulation occurred in the insular cortex. In the entopeduncular nucleus, only the Adcyap1r1 mRNA was downregulated in parkinsonian animals. B/L therapy attenuated the downregulation of Adcyap1 in the CPu only. Our current results further support the evolutionarily conserved role of the PACAP/PAC1R system in neuroprotection and its recruitment in the development/progression of neurodegenerative states such as PD.

The tachykinin hemokinin-1 (HK-1) is involved in immune cell development and inflammation, but little is known about its function in pain. It acts through the NK1 tachykinin receptor, but several effects are mediated by a yet unidentified target. Therefore, we investigated the role and mechanism of action of HK-1 in arthritis models of distinct mechanisms with special emphasis on pain. Arthritis was induced by i.p. K/BxN serum (passive transfer of inflammatory cytokines, autoantibodies), intra-articular mast cell tryptase or Complete Freund’s Adjuvant (CFA, active immunization) in wild type, HK-1- and NK1-deficient mice. Mechanical- and heat hyperalgesia determined by dynamic plantar esthesiometry and increasing temperature hot plate, respectively, swelling measured by plethysmometry or micrometry were significantly reduced in HK-1-deleted, but not NK1-deficient mice in all models. K/BxN serum-induced histopathological changes (day 14) were also decreased, but early myeloperoxidase activity detected by luminescent in vivo imaging increased in HK-1-deleted mice similarly to the CFA model. However, vasodilation and plasma protein extravasation determined by laser Speckle and fluorescent imaging, respectively, were not altered by HK-1 deficiency in any models. HK-1 induced Ca2+-influx in primary sensory neurons, which was also seen in NK1-deficient cells and after pertussis toxin-pretreatment, but not in extracellular Ca2+-free medium. These are the first results showing that HK-1 mediates arthritic pain and cellular, but not vascular inflammatory mechanisms, independently of NK1 activation. HK-1 activates primary sensory neurons presumably via Ca2+ channel-linked receptor. Identifying its target opens new directions to understand joint pain leading to novel therapeutic opportunities.

Background: Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and anti-inflammatory effects via its receptor subtype 4 (SST 4 ) without influencing endocrine functions. Therefore, SST 4 is considered to be a novel target for drug development in pain, especially chronic neuropathy which is a great unmet medical need. Purpose and Experimental Approach: Here, we examined the in silico binding, SST 4 -linked G protein activation and β-arrestin activation on stable SST 4 expressing cells and the effects of our novel pyrrolo-pyrimidine molecules (20, 100, 500, 1,000, 2,000 µg·kg −1 ) on partial sciatic nerve ligation-induced traumatic mononeuropathic pain model in mice. Key Results: The novel compounds bind to the high affinity binding site of SST 4 the receptor and activate the G protein. However, unlike the reference SST 4 agonists NNC 26-9100 and J-2156, they do not induce β-arrestin activation responsible for receptor desensitization and internalization upon chronic use. They exert 65–80% maximal anti-hyperalgesic effects in the neuropathy model 1 h after a single oral administration of 100–500 µg·kg −1 doses. Conclusion and Implications: The novel orally active compounds show potent and effective SST 4 receptor agonism in vitro and in vivo . All four novel ligands proved to be full agonists based on G protein activation, but failed to recruit β-arrestin. Based on their potent antinociceptive effect in the neuropathic pain model following a single oral administration, they are promising candidates for drug development.

Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and anti-inflammatory effects via the somatostatin sst4 receptor without endocrine actions. Therefore, sst4 is considered to be a novel target for drug development in pain including chronic neuropathy, which is an emerging unmet medical need. Here, we examined the in silico binding, the sst4-linked G-protein activation on stable receptor expressing cells (1 nM to 10 μM), and the effects of our novel pyrrolo-pyrimidine molecules in mouse inflammatory and neuropathic pain models. All four of the tested compounds (C1–C4) bind to the same binding site of the sst4 receptor with similar interaction energy to high-affinity reference sst4 agonists, and they all induce G-protein activation. C1 is the more efficacious (γ-GTP-binding: 218.2% ± 36.5%) and most potent (EC50: 37 nM) ligand. In vivo testing of the actions of orally administered C1 and C2 (500 µg/kg) showed that only C1 decreased the resiniferatoxin-induced acute neurogenic inflammatory thermal allodynia and mechanical hyperalgesia significantly. Meanwhile, both of them remarkably reduced partial sciatic nerve ligation-induced chronic neuropathic mechanical hyperalgesia after a single oral administration of the 500 µg/kg dose. These orally active novel sst4 agonists exert potent anti-hyperalgesic effect in a chronic neuropathy model, and therefore, they can open promising drug developmental perspectives.

The Tac4 gene-derived hemokinin-1 (HK-1) is present in pain-related regions and activates the tachykinin NK1 receptor, but with binding site and signaling pathways different from Substance P (SP). NK1 receptor is involved in nociception, but our earlier data showed that it has no role in chronic neuropathic hyperalgesia, similarly to SP. Furthermore, NK1 antagonists failed in clinical trials as analgesics due to still unknown reasons. Therefore, we investigated the role of HK-1 in pain conditions of distinct mechanisms using genetically modified mice. Chronic neuropathic mechanical and cold hyperalgesia after partial sciatic nerve ligation (PSL) were determined by dynamic plantar aesthesiometry and withdrawal latency from icy water, motor coordination on the accelerating Rotarod. Peripheral nerve growth factor (NGF) production was measured by ELISA, neuronal and glia cell activation by immunohistochemistry in pain-related regions. Acute somatic and visceral chemonocifensive behaviors were assessed after intraplantar formalin or intraperitoneal acetic-acid injection, respectively. Resiniferatoxin-induced inflammatory mechanical and thermal hyperalgesia by aesthesiometry and increasing temperature hot plate. Chronic neuropathic mechanical and cold hypersensitivity were significantly decreased in HK-1 deficient mice. NGF level in the paw homogenates of intact mice were significantly lower in case of HK-1 deletion. However, it significantly increased under neuropathic condition in contrast to wildtype mice, where the higher basal concentration did not show any changes. Microglia, but not astrocyte activation was observed 14 days after PSL in the ipsilateral spinal dorsal horn of wildtype, but not HK-1-deficient mice. However, under neuropathic conditions, the number of GFAP-positive astrocytes was significantly smaller in case of HK-1 deletion. Acute visceral, but not somatic nocifensive behavior, as well as neurogenic inflammatory mechanical and thermal hypersensitivity were significantly reduced by HK-1 deficiency similarly to NK1, but not to SP deletion. We provide evidence for pro-nociceptive role of HK-1, via NK1 receptor activation in acute inflammation models, but differently from SP-mediated actions. Identification of its targets and signaling can open new directions in pain research.

Expression of the transient receptor potential ankyrin 1 (TRPA1) receptor has been demonstrated not only in the dorsal root and trigeminal ganglia but also in different brain regions (e.g., hippocampus, hypothalamus, and cortex). However, data concerning their role in neurodegenerative and age-related diseases of the CNS is still indistinct. The aim of our study was to investigate the potential role of TRPA1 in a mouse model of senile dementia. For the investigation of changes during aging, we used male young (3-4-month-old) and old (18-month-old) wild-type (TRPA1(+/+);WT) and TRPA1 receptor gene-deleted (TRPA1(-/-)) mice. Novel object recognition (NOR) test as well as Y maze (YM), radial arm maze (RAM), and Morris water maze (MWM) tests were used to assess the decline of memory and learning skills. In the behavioral studies, significant memory loss was detected in aged TRPA1(+/+) mice with the NOR and RAM, but there was no difference measured by YM and MWM tests regarding the age and gene. TRPA1(-/-) showed significantly reduced memory loss, which could be seen as higher discrimination index in the NOR and less exploration time in the RAM. Furthermore, young TRPA1(-/-) animals showed significantly less reference memory error in the RAM and notably higher mobility in NOR, RAM, and YM compared with the age-matched WTs. Our present work has provided the first evidence that TRPA1 receptors mediate deteriorating effects in the old age memory decline. Understanding the underlying mechanisms could open new perspectives in the pharmacotherapy of dementia.

We have provided evidence that the inhibitory neuropeptide somatostatin is released from the activated capsaicinsensitive peptidergic sensory nerves, and mediate analgesic and anti-inflammatory actions via its subtype 4 (sst4) Giprotein-coupled receptor.Although the native peptide cannot be therapeutically used due to its short half-life and broad range of endocrine actions via its sst2, sst3, sst5 receptors, small molecule sst4 agonists might be promising candidates, as novel analgesic and anti-inflammatory drugs.We investigated the expression pattern of sst4 in the brain using LacZ immunohistochemistry, as well as inflammatory and pain processes mediated by different mechanisms of neurogenic and neuropathic origins using sst4 gene-deleted mice and synthetic agonists.Special emphasis was put on chronic disease models mimicking unmet medical needs.Sst4 is present in several brain areas involved in pain and mood regulation, such as neocortical areas, hippocampus, amygdala and the dorsal spinal root.Sst4-deficient mice showed increased inflammation in arthritis and airway inflammation, enhanced nocifensive behaviors, inflammatory and neuropathic hyperalgesia, anxiety and depression-like behaviors, as well as had greater susceptibility to chronic variable stress.Neuronal activation patterns were different in stress-and pain-related brain regions of wildtype and sst4-deficient mice both under acute and chronic stress/pain conditions suggesting that the central actions are mediated by sst4-dependent modulation of neuronal plasticity and sensitization.The synthetic heptapeptide sst4/sst1 agonist TT-232 and the selective non-peptide sst4 superagonist J-2156, but not the sst2,3,5 agonist octreotide, inhibited non-neurogenic and neurogenic inflammation and pain behaviors both in acute models and chronic diabetic neuropathy, traumatic nerve injury and arthritis.Our 66 patented, orally active small molecule sst4 agonists were screened by in silico receptor binding models, competitive binding and G-protein activation assays in primary sensory neurons and sst4-expressing cell lines.In vivo testing of the 3 most promising candidates revealed that they all inhibited neurogenic inflammation, neuropathic hyperalgesia, anxiety and depression-like behaviors in 50-500 migrog/kg oral dose range, and one had sedative effect after the highest dose.Our results provide evidence that sst4 is a good target for treating neurogenic inflammation, neuropathic pain, anxiety and depression.Our small molecule agonists are promising drug candidates for drug development.