Application Of Inflammatory Soup Research Articles

P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine

Migraine is the second most common form of headache disorder and the second leading cause of disability worldwide. Cognitive symptoms ranked second resulting in migraine-related disability, after pain. P2X7 receptor (P2X7R) was recently shown to be involved in hyperalgesia in migraine. However, the role of P2X7R in migraine-related cognitive impairment is still ill-defined. The aim of this study was to explore the molecular mechanisms underlying migraine-related cognitive impairment and the role of P2X7R in it. Here we used a well-established mouse model of migraine that triggered migraine attacks by application of inflammatory soup (IS) to the dura. Our results showed that repeated dural IS stimulation triggered upregulation of P2X7R, activation of NLRP3 inflammasome, release of proinflammatory cytokines (IL-1β and IL-18) and activation of pyroptotic cell death pathway. Gliosis (microgliosis and astrogliosis), neuronal loss and cognitive impairment also occurred in the IS-induced migraine model. No significant apoptosis or whiter matter damage was observed following IS-induced migraine attacks. These pathological changes occurred mainly in the cerebral cortex and to a less extent in the hippocampus, all of which can be prevented by pretreatment with a specific P2X7R antagonist Brilliant Blue G (BBG). Moreover, BBG can alleviate cognitive impairment following dural IS stimulation. These results identified P2X7R as a key contributor to migraine-related cognitive impairment and may represent a potential therapeutic target for mitigating cognitive impairment in migraine.

Trigeminal activation patterns evoked by chemical stimulation of the dura mater in rats

BackgroundAlthough migraine is one of the most common primary headaches, its therapy is still limited in many cases. The use of animal models is crucial in the development of novel therapeutic strategies, but unfortunately, none of them show all aspects of the disease, therefore, there is a constant need for further improvement in this field. The application of inflammatory agents on the dura mater is a widely accepted method to mimic neurogenic inflammation in rodents, which plays a key role in the pathomechanism of migraine. Complete Freund’s Adjuvant (CFA), and a mixture of inflammatory mediators, called inflammatory soup (IS) are often used for this purpose.MethodsTo examine the activation pattern that is caused by chemical stimulation of dura mater, we applied CFA or IS over the right parietal lobe. After 2 h and 4 h (CFA groups), or 2.5 h and 4 h (IS groups), animals were perfused, and c-Fos immunoreactive cells were counted in the caudal trigeminal nucleus. To explore every pitfall, we examined whether our surgical procedure (anesthetic drug, stereotaxic apparatus, local lidocaine) can alter the results under the same experimental settings. c-Fos labeled cells were counted in the second-order neuron area based on the somatotopic organization of the trigeminal nerve branches.ResultsWe could not find any difference between the CFA and physiological saline group neither 2 h, nor 4 h after dural stimulation. IS caused significant difference after both time points between IS treated and control group, and between treated (right) and control (left) side. Stereotaxic frame usage had a substantial effect on the obtained results.ConclusionsCounting c-Fos immunoreactive cells based on somatotopic organization of the trigeminal nerve helped to examine the effect of chemical stimulation of dura in a more specific way. As a result, the use of IS over the parietal lobe caused activation in the area of the ophthalmic nerve. To see this effect, the use of lidocaine anesthesia is indispensable.In conclusion, application of IS on the dura mater induces short-term, more robust c-Fos activation than CFA, therefore it might offer a better approach to model acute migraine headache in rodents.

Wider range of allodynia in a rat model of repeated dural nociception compared with infraorbital nerve chronic constriction injury

BackgroundTo identify differences in allodynia and grooming behaviours between rat models of either repeated dural nociception with inflammatory soup (IS) or infraorbital nerve chronic constriction injury (IoN-CCI). MethodsRepeated dural nociception was induced via the application of IS to the dural meninges and IoN-CCI was applied to model neuropathic pain. All surgeries were performed on the right side and a sham operation was performed on the control group. Mechanical and thermal withdrawal thresholds were tested on different facial areas and hindpaw during the interictal period and grooming behaviours were recorded. ResultsA significant decreases was found in the mechanical withdrawal thresholds of the bilateral vibrissa pad and right periorbital area in both the IS and the IoN-CCI groups, but only in the left periorbital area of the IS group. Hindpaw thermal allodynia was evident only in the IS group. Ipsilateral hindpaw grooming behaviour increased in the IS group and facial grooming behaviour increased in the IoN-CCI group. ConclusionsRepeated dural nociception induced by IS and IoN-CCI in rats effectively simulated chronic migraine (CM) and trigeminal neuralgia (TN), respectively. The IS group exhibited a wider range of allodynia than the IoN-CCI group, but further studies are necessary to determine underlying mechanisms.

Brain network alterations in the inflammatory soup animal model of migraine

Advances in our understanding of the human pain experience have shifted much of the focus of pain research from the periphery to the brain. Current hypotheses suggest that the progression of migraine depends on abnormal functioning of neurons in multiple brain regions. Accordingly, we sought to capture functional brain changes induced by the application of an inflammatory cocktail known as inflammatory soup (IS), to the dura mater across multiple brain networks. Specifically, we aimed to determine whether IS alters additional neural networks indirectly related to the primary nociceptive pathways via the spinal cord to the thalamus and cortex. IS comprises an acidic combination of bradykinin, serotonin, histamine and prostaglandin PGE2 and was introduced to basic pain research as a tool to activate and sensitize peripheral nociceptors when studying pathological pain conditions associated with allodynia and hyperalgesia. Using this model of intracranial pain, we found that dural application of IS in awake, fully conscious, rats enhanced thalamic, hypothalamic, hippocampal and somatosensory cortex responses to mechanical stimulation of the face (compared to sham synthetic interstitial fluid administration). Furthermore, resting state MRI data revealed altered functional connectivity in a number of networks previously identified in clinical chronic pain populations. These included the default mode, sensorimotor, interoceptive (Salience) and autonomic networks. The findings suggest that activation and sensitization of meningeal nociceptors by IS can enhance the extent to which the brain processes nociceptive signaling, define new level of modulation of affective and cognitive responses to pain; set new tone for hypothalamic regulation of autonomic outflow to the cranium; and change cerebellar functions.

Dural administration of inflammatory soup or Complete Freund's Adjuvant induces activation and inflammatory response in the rat trigeminal ganglion.

BackgroundMigraine is a painful disorder with a huge impact on individual and public health. We hypothesize that migraine pain originates from a central mechanism that results secondarily in hypersensitivity in peripheral afferents associated with the cerebral and cranial blood vessels. It has previously been shown that application of inflammatory or algesic substances onto the dura mater or chemical stimulation of the dural receptive fields causes hypersensitivity to mechanical and thermal stimulation together with direct activation of the TG. We asked whether local inflammation of dura mater induces inflammatory activation in the trigeminal ganglion.MethodsWe performed topical administration of inflammatory soup (IS) or Complete Freund’s Adjuvant (CFA) onto an exposed area of the rat dura mater in vivo for 20 min. The window was closed and the rats were sacrificed after 4 h and up to 7 days. Myography was performed on middle meningeal arteries. The trigeminal ganglia were removed and processed for immunohistochemistry or Western blot.ResultsBoth CFA and IS induced enhanced expression of pERK1/2, IL-1β and CGRP in the trigeminal ganglia. The pERK1/2 immunoreactivity was mainly seen in the satellite glial cells, while IL-1β reactivity was observed in the neuronal cytoplasm, close to the cell membrane, seemingly as sign of neuro-glial interaction. The CGRP expression in the neurons and nerve fibres was enhanced after the application of either inflammatory agent. Myography resulted in a strong vasoconstrictor response to IS, but not to CFA.ConclusionsThese results suggest that the application of IS or CFA onto the dura mater causes long-term activation of the TG and demonstrate the importance of the neuro-glial interaction in the activation of the trigeminovascular system.Electronic supplementary materialThe online version of this article (doi:10.1186/s10194-015-0564-y) contains supplementary material, which is available to authorized users.

Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease–derived stimuli

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9−/− mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus–response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9−/− afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9−/− mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics.

Group III mGluR-selective and mGluR8-selective agonists potentiate morphine-induced inhibition of sensitized peripheral nociceptors

Opioids such as morphine are often used to treat acute and chronic pain. However, they cause significant side effects when taken in high doses or for long periods of time. Current studies aim to establish receptor groups and agonists that might potentiate analgesic efficacy of opioids, allowing treatment with lower doses in order to minimize side effects. One receptor family of interest is the metabotropic glutamate receptors (mGluRs). Current literature indicates that intrathecal and systemic applications of Group III agonists potentiate morphine analgesia. We have previously demonstrated that peripheral administration of the Group III-selective agonist L-AP-4 synergistically potentiates morphine anti-hyperalgesia. In this study, we employed the in vitro skin-nerve preparation to determine if the combination decreases sensitization at the single fiber level. Fibers were sensitized to heat via application of inflammatory soup and morphine, L-AP-4, or a Group III mGluR8-selective agonist (DCPG) was applied. Dose response curves for each drug were used to identify individual IC50s and to determine combination doses of morphine plus either L-AP-4 or DCPG. Further study will involve applying combinations and assessing responses to thermal stimuli to determine if Group III agonists potentiate morphine at the single fiber level. We also examined the anatomical basis of potentiation by double-labeling dorsal root ganglion (DRG) cells for Group III mGluR8 and the mu opioid receptor (μOR). Thus far, data indicate that the receptors are co-localized in 41% of nociceptors. This will be confirmed via triple-labeling DRG cells for mGluR8, μOR, and a nociceptor marker (TRPV1). Co-expression of mGluR8 and μOR on nociceptors suggests that potentiation occurs via an intracellular mechanism. Overall, we expect that peripheral Group III mGluR activation will potentiate morphine-induced inhibition of inflamed peripheral fibers via modulation of an intracellular pathway. Funding: NIH F30DA03229801 to ALB; NS027910/DA027460 to SMC. Opioids such as morphine are often used to treat acute and chronic pain. However, they cause significant side effects when taken in high doses or for long periods of time. Current studies aim to establish receptor groups and agonists that might potentiate analgesic efficacy of opioids, allowing treatment with lower doses in order to minimize side effects. One receptor family of interest is the metabotropic glutamate receptors (mGluRs). Current literature indicates that intrathecal and systemic applications of Group III agonists potentiate morphine analgesia. We have previously demonstrated that peripheral administration of the Group III-selective agonist L-AP-4 synergistically potentiates morphine anti-hyperalgesia. In this study, we employed the in vitro skin-nerve preparation to determine if the combination decreases sensitization at the single fiber level. Fibers were sensitized to heat via application of inflammatory soup and morphine, L-AP-4, or a Group III mGluR8-selective agonist (DCPG) was applied. Dose response curves for each drug were used to identify individual IC50s and to determine combination doses of morphine plus either L-AP-4 or DCPG. Further study will involve applying combinations and assessing responses to thermal stimuli to determine if Group III agonists potentiate morphine at the single fiber level. We also examined the anatomical basis of potentiation by double-labeling dorsal root ganglion (DRG) cells for Group III mGluR8 and the mu opioid receptor (μOR). Thus far, data indicate that the receptors are co-localized in 41% of nociceptors. This will be confirmed via triple-labeling DRG cells for mGluR8, μOR, and a nociceptor marker (TRPV1). Co-expression of mGluR8 and μOR on nociceptors suggests that potentiation occurs via an intracellular mechanism. Overall, we expect that peripheral Group III mGluR activation will potentiate morphine-induced inhibition of inflamed peripheral fibers via modulation of an intracellular pathway. Funding: NIH F30DA03229801 to ALB; NS027910/DA027460 to SMC.

Sex Differences in Behavior and Expression of CGRP‐Related Genes in a Rodent Model of Chronic Migraine

The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene-related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate-buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP. Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP-related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP-related genes were upregulated by both IS and PBS applications. This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation.

Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum

Hypersensitivity in inflammatory/irritable bowel syndrome is contributed to in part by changes in the receptive properties of colorectal afferent endings, likely including mechanically insensitive afferents (MIAs; silent afferents) that have the ability to acquire mechanosensitivity. The proportion and attributes of colorectal MIAs, however, have not previously been characterized. The distal ∼3 cm of colorectum with either pelvic (PN) or lumbar splanchnic (LSN) nerve attached was removed, opened longitudinally, pinned flat in a recording chamber, and perfused with oxygenated Krebs solution. Colorectal receptive endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. MIA endings were tested for response to and acquisition of mechanosensitivity by localized exposure to an inflammatory soup (IS). Colorectal afferents were also tested with twin-pulse and repetitive electrical stimulation paradigms. PN MIAs represented 23% of 211 afferents studied, 71% (30/42) of which acquired mechanosensitivity after application of IS to their receptive ending. LSN MIAs represented 33% of 156 afferents studied, only 23% (11/48) of which acquired mechanosensitivity after IS exposure. Mechanosensitive PN endings uniformly exhibited significant twin-pulse slowing whereas LSN endings showed no significant twin-pulse difference. PN MIAs displayed significantly greater activity-dependent slowing than LSN MIAs. In conclusion, significant proportions of MIAs are present in the colorectal innervation; significantly more in the PN than LSN acquire mechanosensitivity in an inflammatory environment. This knowledge contributes to our understanding of the possible roles of MIAs in colon-related disorders like inflammatory/irritable bowel syndrome.

Enhancement of Ectopic Discharge in Regenerating A- and C-Fibers by Inflammatory Mediators

Afferent A- and C-fibers regenerating into a nerve following peripheral nerve injury are exposed to inflammatory mediators released by Schwann cells, resident and invading macrophages, and other inflammatory cells. Here we tested the hypothesis that ongoing and evoked activity in these afferent fibers are enhanced by a mixture of inflammatory mediators [inflammatory soup (IS)] applied to the injured nerve. Using in vivo electrophysiology, regenerating afferent nerve fibers were studied 7-14 days after sural nerve crush lesion. The ectopic activity was studied before and <or=1.5 h after topical application of IS to the nerve in 73 C-fibers and 22 A-fibers that were either ectopically active before application of IS (61 C-fibers, 17 A-fibers) or recruited by IS (12 C-fibers, 5 A-fibers). More than one half of the C-fibers were activated by IS for <or=90 min after its removal. The majority of mechano- (23/38) and heat-sensitive (29/35) C-fibers as well as mechano-sensitive A-fibers (12/17) decreased their activation thresholds and/or increased the response magnitude to mechanical and/or heat stimulation of the nerve. Noxious cold sensitivity, but not nonnoxious cold sensitivity, was weakly influenced by IS. Some initially nonresponsive C- and A-fibers developed new ectopic properties, i.e., were recruited, and exhibited ongoing activity and/or could be activated by physiological stimuli after application of IS. The results suggest that inflammatory mediators may be critical to enhance ectopic excitability of regenerating afferent nerve fibers. These peripheral mechanisms may be important triggering and maintaining neuropathic pain.

Inflammatory Hyperalgesia: A Role for the C-Fiber Sensory Neuron Cell Body?

Peripheral nerve injury increases the chemosensitivity and excitability of injured afferents, resulting in ectopic activity arising from within dorsal root ganglia. Studies of dissociated sensory ganglion neurons in vitro suggest afferent somata might be sensitized by persistent inflammation. However, it is unknown whether this inflammation-induced sensitization is manifest in somata within the intact ganglia. To explore this possibility, intracellular electrophysiologic recording was used with a sciatic nerve–L4–dorsal root ganglia preparation to compare excitability and chemosensitivity of cutaneous C-fiber somata from control and inflamed rats. Cutaneous afferents were identified with the retrograde dye DiI. Excitability was assessed before and after the application of inflammatory soup (IS) containing bradykinin, serotonin, and prostaglandin E 2 all at a pH of 7.0. Persistent inflammation decreased the excitability of cutaneous afferents in intact ganglia and had no significant influence on the magnitude of IS-induced increase in excitability. Opposite to the effects observed in intact ganglia, excitability was greater in dissociated cutaneous nociceptors obtained from inflamed rats, although the magnitude of the IS-induced increase in excitability was not significantly affected by inflammation. These results suggest that the cell bodies of putative cutaneous nociceptors in the intact ganglia contribute minimally to pain and hyperalgesia associated with persistent inflammation. Perspective Results of the present study suggest that inflammation-induced changes in afferent somata are minimal. However, they also suggest that inflammatory mediator–induced increase in the excitability of sensory neuron somata might contribute to global changes in nociception observed under high systemic inflammatory mediator loads.