A role of NLRP3 and MMP9 in migraine progression: a systematic review of translational study

Background Migraines affect one billion individuals globally, with a higher occurrence among young adults and women. A significant survey in the United States indicated that 17.1% of women and 5.6% of men suffer from migraines. This study seeks to investigate the potential connection between NLRP3 and MMP9 in migraine pathology. Methods The research involved searching databases such as PubMed, Scopus, Science Direct, Google Scholar, and Proquest, with the search concluding on March 31, 2024. Following PRISMA guidelines, PICO data were collected, focusing exclusively on animal models induced by Nitroglycerine (10 mg/kg), while excluding clinical studies. Results The study, originally registered in Prospero Reg. No. CRD42022355893, conducted bias analysis using SYRCLE’s RoB tool and evaluated author consensus using GraphPad v9.5.1. Out of 7,359 search results, 22 papers met the inclusion criteria. Inter-rater reliability among reviewers was assessed using Cohen’s kappa statistics. Conclusion This review summarizes 22 preclinical studies on Nitroglycerin (NTG), NLRP3, MMP9, and related biomarkers in migraine. They reveal that NTG, especially at 10 mg/kg, consistently induces migraine-like symptoms in rodents by activating NLRP3 inflammasome and stimulating proinflammatory molecule production. Systematic Review Registration https://www.crd.york.ac.uk/prospero/, CRD42022355893.


Introduction
One of the most common causes for outpatient visits to general neurologists is migraine, a prevalent and incapacitating neurological condition.Lifestyle changes are a cornerstone of treatment in addition to pharmaceutical therapy (1).Scientists have yet to find a medication that treats migraine for ten decades.They started targeting different biomarkers such as NFkB (2)(3)(4), interleukins (5-7), serotonin (8)(9)(10)(11), etc. which was released immensely in the migraineurs but unable to find a way to cure.Then the treatment for migraine has changed dramatically as a result of medications that target the CGRP pathway (12)(13)(14).Numerous articles have provided evidence for the efficacy of monoclonal antibodies targeting CGRP or its receptor in preventing difficult-to-treat migraines (15)(16)(17)(18)(19).A trial of intravenous eptinezumab in individuals with 2-4 prior failed efforts at preventive therapy was conducted in 2022 to supplement this work (20)(21)(22).During the 12-week double-blind observation period, a single infusion of 300 mg eptinezumab significantly reduced the number of monthly migraine attack days compared to the placebo group (23)(24)(25).Nowadays, researchers are especially targeting NLRP3 inflammasomes (26)(27)(28)(29)(30) which has a robust connection to migraine and predicting MMP9 additionally playing a major role in the migraine attack (31)(32)(33).
Neurological diseases, whether fatal or non-fatal, constitute a significant portion of the non-communicable disease burden in India (34).According to the 2019 Global Burden of Disease study (GBD2019), migraine was reported as the second leading cause of disability, especially among women under the age of 50.Out of 357 publications primarily from high-income countries, the estimated global prevalence of active headache disorders was 52.0% (with a 95% confidence interval of 48.9-55.4).Specifically, the prevalence of migraine was estimated at 14.0% (with a confidence interval of 12.9-15.2),tension-type headache (TTH) at 26.0% (with a confidence interval of 22.7-29.5),and other severe headache disorders (H15+) at 4.6% (with a confidence interval of 3.9-5.5)(35).These estimates aligned with the figures for migraine and tension-type headache (TTH) reported in GBD2019, the most recent available data, but they indicated a higher prevalence of headaches in the general population.Specifically, on a daily basis, 15.8% of the global population experiences headaches (35,36).A statistical report from 2019 found that headache disorders, including conditions like migraine and tension-type headaches, were the most common neurological conditions, affecting approximately 488 million individuals in India (with a 95% uncertainty interval ranging from 449 to 527 million) (37).Regardless of regional differences, the issue of headaches is a global concern, affecting people of various ages, ethnicities, income levels, and geographic locations.Between half to three-quarters of individuals aged 18 to 65 worldwide experienced a headache in the past year, with 30% or more reporting migraine of the two main categories of headache disorders, migraine has the most significant impact on overall neurological health, contributing the highest share of Disability-adjusted life years (DALYs) (38).The DALYs associated with migraine significantly surpass those linked to tension-type headaches.Between 1990 and 2019, India saw an increase in the raw prevalence and DALY rate of headache disorders, while the age-standardized prevalence and DALY rate remained relatively stable.In 2019, it was observed that females between the ages of 35 and 59 had a higher prevalence of migraine compared to males in the same age group (39).
The prevalence of this condition increases with age, peaking between 40 and 44 years, and then gradually decreases in both men and women.The proportion of non-communicable neurological disorders was 4.0% (with a 95% uncertainty interval of 3.2-5.0) in 1990, doubling to 8.2% (with an interval of 6.6-10.2) by 2019.Projections indicate that this percentage is expected to climb to 16.5% by 2040.Migraine, a widespread neurological disorder, stands as the second most significant contributor to years lived with disability globally and ranks among the top twenty most disabling conditions worldwide (36,(40)(41)(42).Migraine is a severe neurological condition diagnosed through clinical criteria (43).Many individuals are unable to work due to migraines, which are complex neurological events lasting for several hours or even extending over multiple days.The most common type of migraines is those without an aura (44).As a result, it is estimated to affect hundreds of millions annually (45).The underlying processes in migraine pathophysiology have a significant genetic component and involve the activation of pain pathways associated with the trigeminovascular system (46)(47)(48)(49)(50)(51).This systematic study is motivated by the exploration of the roles of NLRP3 and MMP9 in the onset of migraine attacks.After identifying, evaluating, and consolidating research results, a thorough examination of relevant preclinical studies is essential to establish the reliability and potential applicability of the data in future research.This hypothesis sets the stage for establishing a robust connection between NLRP3 and MMP9 in the context of migraines.

Data retrieval
In this process, one reviewer (RR) collected data from each study, which encompassed information such as the animal strain, weight, route of administration (ROA), dose, and the specific biomarkers addressed in the study.This data was then documented in an Excel Sheet for the purpose of data management.Subsequently, three reviewers (ASS, AS and RR) cross-validated the gathered information to ensure its coherence and relevance.

Risk of bias
Using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias (SYRCLE's RoB) method (53-55), five reviewers (IK, CV, RR, ASS, and AS) conducted individual assessments of bias risk for all 22 studies.This assessment questionnaire comprises ten categories associated with six types of bias, evaluating the methodological quality of preclinical studies.These categories encompass elements like sequence generation, baseline characteristics, allocation concealment, random housing, random outcome assessment, incomplete outcome data, selective outcome reporting, and other factors, such as drug pooling, funder influence, designspecific bias risk, unit of analysis errors, and replacement of dropouts from the original population.Each study's risk of bias was evaluated by all reviewers using the designations 'yes' , 'no, ' and 'unclear' to denote high risk, low risk, and insufficient information to determine the risk of bias, respectively.Any disparities in these assessments were resolved through discussion among the reviewers or by reaching a consensus.The author agreement is assessed based on kappa values with reference values of less than zero No agreement; 0.00-0.20 Slight agreement; 0.21-0.40Fair agreement; 0.41-0.60Moderate agreement; 0.61-0.80Substantial agreement; 0.81-1.000Almost perfect agreement.The level of agreement was quantified using kappa, employing the GraphPad by Dotmatics tool. 2

Data analysis
This review was officially registered with PROSPERO under the reference CRD42022355893.We conducted a thorough examination of the literature to ascertain the extent and prevalence of the utilization of particular concepts, objectives, and correlated outcome measures in preclinical trials focused on migraine treatments.The methodology protocol adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (56), which offer consensus-based standards for developing and carrying out highquality systematic literature reviews.The PRISMA checklist provides guidelines for various stages of conducting a literature search and review.This includes specifying eligibility criteria for published papers, determining the dataset for the search, formulating search terms, establishing a standardized review process for identified publications, implementing record tracking and data management systems, outlining the data to be extracted from each publication meeting inclusion criteria, and devising a strategy for data extraction from qualifying publications.

Study selection
The database search yielded 7,359 results, with 47 records from PubMed, 323 from Science Direct, 803 from Scopus, 6,180 from Google Scholar, and 6 from Proquest.Initially, 737 titles were 2 https://www.graphpad.com/quickcalcs/kappa1/?K=3 screened, and after eliminating duplicates, book chapters, other than NTG induction model, clinical studies, and conducting abstract screening, at last 22 remained.All of the studies that were included adhered to ethical guidelines and received approval from the relevant institutional bodies.The characteristics and outcomes of these selected studies are listed in Table 2. Various animal species were represented among the rodents in the study, with the Nitroglycerin (10 mg/kg, b.wt) model being the most frequently employed for inducing migraine-related conditions, and it was the primary focus.The timeline of the systematic review is presented in Figure 1.

Population
Albino Sprague Dawley rats and mice, CD1 mice, Wistar rats, Knockout mutant mice, Neonatal Mice, and C57BL6/J mice were included, and other than these were excluded from this study.Of these 22 studies, 9 studies used C57BL6/J mice; 6-Sprague Dawley rats studies; 3-CD1 mice studies; Wistar rats-2; Knockout mutant mice-1; Neonatal mouse-1 study for pharmacological evaluation.They used both genders for the study, and mostly Nitroglycerin (10 mg/kg, b.wt, i.p) chemical induction model was selected and utilized to create migraine-like conditions.

Intervention
By decreasing microglial and NLRP3 inflammasome activity, TREM1 regulation demonstrates potential as a therapy approach for chronic migraine.Through stimulating the Nrf2 pathway, which in turn decreases oxidative stress and neuroinflammation, NBP lessens the severity of migraines.One possible mechanism by which Wuzhuyu Decoction reduces inflammation and oxidative stress in migraine sufferers is via activating the NRF2 pathway.A possible treatment target in chronic migraine could be AMPK activation, which lowers neuroinflammation.UA has the ability to reduce migraine symptoms by reducing mitochondrial dysfunction in different parts of the brain.Vestibular dysfunction and increased fibrinogen levels are the results of NTG-induced migraines in rats.Possible migraine treatment targets include the trigeminal nucleus caudalis (TNC), which shows increased expression of PACAP and PAC1R.One novel approach to studying migraine in rats is the use of postauricular injection.In mice that have peripheral hypersensitivity caused by NTG, acupuncture at the GV16 acupoint alleviates mechanical and cold allodynia.RUT alleviates migraine symptoms by triggering the antioxidant mechanism of Nrf2.Possible relief from migraine symptoms may be achieved through the inhibition of the NGF/TRPV1/COX-2 pathway by Shaoyao Gancao Decoction.A possible therapeutic target could be the suppression of PACAP/ PAC1R, since repeated intranasal PACAP treatments in CM rats cause hyperalgesia.The fact that perampanel blocks PACAP expression suggests it may be useful in treating migraines.It has been suggested that PAR2 could be a therapeutic target for migraines, as its activation and IL-6 signaling could prime NO donors.Metabolite and target influences on MXFD's efficacy in migraine treatment provide light on areas for future investigation.Novel migraine therapy techniques may be available via BAY-117082's modulation of the Erk/CREB/Akt pathways.One mechanism by which W146 reduces chronic  (Continued) Based on these results, RUT, a natural medicine, may be a good choice for migraine treatment.(Continued)

Comparison
The comparison of findings across the 22 studies underscores the complexity of migraine pathophysiology and highlights the diverse approaches to intervention and treatment.Many studies focus on specific molecular pathways implicated in migraine pathophysiology, such as neuroinflammation (57, 60, 65), oxidative stress (58,59,61), and mitochondrial dysfunction (61,65).These studies suggest that targeting these pathways could offer therapeutic benefits for migraine patients.Various pharmacological agents are explored across the studies, including natural compounds (58,65), traditional Chinese medicines (59,66), and novel drugs (62,67).These interventions aim to modulate molecular targets associated with migraine pathogenesis, highlighting the potential of diverse treatment modalities.Studies utilize different animal models of migraine induction, including NTG-induced models (57, 62, 68, 71), GTN-induced models (31,73), and others.Variations in animal species, strains, and induction methods contribute to the diversity of findings and insights into migraine mechanisms.Behavioral tests are commonly employed to evaluate migraine symptoms and treatment outcomes across studies, including assessments of mechanical hypersensitivity (59,61), light aversion (61), and vestibular function (62).These assessments provide valuable insights into the efficacy of interventions in alleviating migraine-associated symptoms.Studies investigate a range of molecular targets implicated in migraine pathogenesis, such as CGRP (58,64), NLRP3 inflammasome (57, 71), and PACAP/ PAC1R signaling (62).Targeting these specific molecules offers potential avenues for developing novel migraine therapies.While the specific mechanisms vary, many interventions exert their therapeutic effects by modulating key signaling pathways associated with migraine, including the NF-κB pathway (57), Nrf2 antioxidant pathway (58,65), and ERK/CREB/BDNF pathway (62).Understanding these mechanisms provides insights into the underlying biology of migraine and informs the development of targeted therapies.Migraine is one of several neurological illnesses studied using C57BL/6 mice in preclinical settings.Researchers frequently opt to use pre-existing models because they allow them to make use of prior knowledge and procedures, which in turn makes it easier to analyze data and compare it to other studies.Although there is not a foolproof mouse model of migraine because the condition is complex, some mouse strains, such C57BL/6, can experience migraine-like symptoms in response to certain environmental factors.Behavioral abnormalities, nociceptive reactions, and neurochemical changes similar to migraine patients may be among these symptoms.There is a gender difference in migraine prevalence, with women experiencing more attacks than men.To get a full view of the pathogenesis and treatment responses to migraines, it is essential to research both sexes.Researchers may be able to simplify trial design by minimizing any confounding effects due to hormonal fluctuations in females or study sex-specific differences in antimigraine activity by focusing on male mice in this circumstance.Migraine sensitivity and intensity are both affected by hormonal changes, especially changes in estrogen levels.Because male mice do not experience these hormonal changes, researchers can use them to their advantage when designing experiments to determine the effectiveness of antimigraine drugs.

Outcome
Across the research included in this review, a variety of outcome measures were observed, such as behavioral tests (n = 9; 40.09%) (   The systematic review reveals a significant association between NLRP3 and MMP9 expression levels and migraine progression.Elevated levels of both NLRP3 and MMP9 are consistently observed in migraine patients compared to controls, suggesting their potential involvement in the pathophysiology of the condition.Furthermore, the review highlights the inflammatory cascade initiated by NLRP3 activation and subsequent MMP9 release as a potential mechanism underlying migraine pathogenesis.These findings underscore the importance of targeting NLRP3 and MMP9 pathways for the development of novel therapeutic strategies aimed at mitigating migraine progression and improving patient outcomes.

Risk of bias
The results of the risk of bias evaluation, conducted using SYRCLE's RoB tool, are summarized in Figure 2. Notably, there was a high risk of selection-bias, performance-bias, and detection-bias identified in the assessed studies.While all studies addressed sequence generation and allocation concealment, clarity regarding random grouping was absent in nine of them.Most studies adequately presented baseline characteristics, although three studies lacked clarity in this regard.All included studies explicitly reported random housing of animals.For the assessment of random outcome, 12 studies exhibited some degree of clarity, while it remained unclear in two studies.In most instances, there were efforts to blind investigators during behavioral outcome assessment, and histological examination was consistently blinded across all 22 studies.An overall unclear risk of bias was found in the category of attrition bias.While all studies reported complete outcome data, they did not offer clear explanations for the reasons behind or methods for handling missing data.To assess the interrater reliability among the reviewers, Cohen's kappa statistics were employed.To ensure impartiality in the study, Mrs. Pavithra, who was blinded to it, was randomly assigned the selection of a subset of studies for evaluation.The kappa test was utilized to gauge the degree of agreement and potential discrepancies among the reviewers.

Quality assessment
We meticulously reviewed and assessed all reports using the standard checklist provided by SYRCLE's-RoB tool.Cohen's-kappa statistic was employed to ascertain the agreement between two/more raters.Among the 22 studies, it was noted that 8 of them incorporated a random component in the sequence-generation processes, whereas only 9 studies are employed C57/BL6 mice weighing between 20 and 25 g; the remaining studies used a variety of weight ranges for evaluation.The issue of investigator blinding was insufficiently addressed in all the studies, and allocation concealment was not blinded in 2 of them.Nearly all studies mentioned the random housing of animals, and 1 study provided clear documentation of blinding the outcome assessors.All the studies were ensured the random assignment of animal outcome evaluation.However, two studies inadequately addressed the handling of incomplete outcome data.There was a general lack of documentation regarding other potential causes of bias including medication dropouts, pooling, unit of analysis errors, design specific bias, and conflicts of interest.

Discussion
All 22 preclinical investigations suggested that based on the different behavioral tests, biochemical parameters, histological and immunohistochemistry reports revealed that Nitroglycerine has the capacity to damage the neuronal cells, over expression of NLRP3, Brain derived neurotrophic factor (BDNF), Nerve growth factor (NGF), RIPK1 activation, neuroinflammation, cognitive impairment, NFkB, TXNIP, ADAR3, p-ERK, c-Fos, HMGB1 and Neurotrophin-3 (NT3) may be responsible for the migraine attack.After extensive literature reviews and discussions, we concluded that the NTG-inducing model (10 mg/kg) is the most suitable for inducing the production of NLRP3, MMP9, and other proinflammatory molecules

High bias Low bias U nclear
The risk of bias was analysed for all the authors.The proportion of studies with a high risk of bias categorised as 'Yes' is indicated in orange.The blue colour represents the proportion of studies with a low risk of bias labelled as 'No,' while the purple colour represents the proportion of study with an unclear response to the stated issue.The stimulation of NLRP3 inflammasomes was linked with elevated levels of IL1β and IL18 in the brain, potentially contributing to neuroinflammation (82).An intriguing discovery is that the modulation of NLRP3-IL1β signaling plays an important role in the transition from acute to chronic neuroinflammation is represented in Figure 4, particularly driven by brain microglia.Upon activation of the Trigeminal nerve system pathway, microglia release an array of chemokines, cytokines, as well as free radicals including nitric oxide and reactive oxygen species.Nevertheless, further investigation into brain microglia is necessary to advance therapy (76).Excessively active microglia can potentially lead to neuroinflammation and tissue damage.Interestingly, both PAMP s and DAMP s are implicated in the process of neuroinflammation (93-95).PAMP s initiate the priming signaling molecule for the NLRP3inflammasome, while DAMP s serve as the second signaling molecule, fostering neuro inflammation and enabling the transmission of pain signal from primary to higher centers (79,96).TXNIP NLRP3 plays a critical role in microglia mediated neuroinflammation.The buildup of NLRP3 leads to increased caspase1 activation and maturation (97-99).Consequently, caspase1 has the capacity to cleave pro-IL1β and pro-IL18, amplifying the proinflammatory response (78,(100)(101)(102).The Li et al. explored the presence of ADARs genes in various human tissues, which exhibit a specific down-regulation of the NLRP3 inflammasome.This discovery could potentially be identified as a novel target for migraine treatment (83,103).MMP9 is typically regarded as an inflammatory mediator (104).Elevated MMP9 levels intensify the interconnected inflammatory processes involving CCL2 and CXCL1 (105).MMP9 is among the biomarkers that might serve as an alternative therapeutic target to disrupt BBB integrity in astrocytes, and its role in migraine pathogenesis is of significant importance to explore (31,(106)(107)(108)(109).
Several key discoveries from this systematic review were emphasized, notably the association between PX7R, RIPK, P2X4 receptor activation, NLRP3, and MMP9 in the genesis of migraines.The interconnected relationship between NLRP3 and MMP9 was depicted in Figure 5. Concentrating on these markers could offer potential solutions to alleviate the ongoing challenges faced by individuals dealing with migraines.Activation of NLRP3 and upregulation of MMP9 contribute to neuroinflammation, which is increasingly recognized as a key factor in migraine pathogenesis.Neuroinflammation can sensitize trigeminal nociceptive pathways, leading to the generation and propagation of migraine pain.MMP9-mediated disruption of the blood-brain barrier can facilitate the entry of inflammatory cells and molecules into the brain parenchyma, exacerbating neuroinflammation and promoting migraine attacks.Both NLRP3 activation and MMP9 upregulation have been implicated in the sensitization of pain pathways, enhancing the perception of pain associated with migraine attacks and potentially contributing to the development of chronic migraine.Cortical spreading depression (CSD), a wave of neuronal depolarization followed by suppression of neuronal activity, is believed to underlie the aura phase of migraine (110).NLRP3 inflammasome activation and MMP9-mediated neuroinflammation may modulate the susceptibility to CSD, influencing the frequency and severity of migraine attacks.The roles of NLRP3 and MMP9 in migraine pathophysiology not only sheds light on the underlying mechanisms of the disease but also identifies potential therapeutic targets.Targeting these molecules or their downstream pathways could offer new strategies for the treatment and management of migraine, particularly in patients who do not respond to currently available therapies.Treatment with TREM1 modulation in C57BL/6 J male mice and BV2 cells revealed potential therapeutic insights for chronic migraine, while interventions including Dl-3-n-butylphthalide (NBP), Wuzhuyu Decoction, AMPK activator, UA, and RUT demonstrated varied mechanisms in reducing migraine severity and neuroinflammation across different mouse models and cell lines.SYRCLE's RoB tool was used by reviewers IK, RR, ASS, CV, and AS to thoroughly assess all the studies included in the analysis.The majority of these studies effectively reported critical aspects such as sequence generation, allocation concealment, baseline characteristics, blinding, animal housing, randomization of outcome assessment, and comprehensive outcome data, which enhanced the reliability of their findings.However, it's worth noting that a few studies failed to provide adequate details regarding blinding and the proper execution of outcome assessment randomization (n = 14).Several factors led us to conclude that nitroglycerin models alone would be sufficient for our purposes.To begin, nitroglycerininduced migraine models have shown to be a reliable way to replicate migraine symptoms in the lab, making them a popular choice for migraine studies.An effective tool for studying the biology of migraines and assessing possible treatments, this has undergone thorough characterization and validation in clinical and preclinical investigations.The second benefit of nitroglycerin-induced migraine models is that they can reproduce both the acute and chronic phases of migraine, including the onset of headaches, pain sensitization, and the emergence of migrainerelated symptoms like photophobia and nausea.Since nitroglycerin models capture all the hallmarks of migraine pathophysiology, they are ideal for investigating different facets of this condition and evaluating potential new treatments.We are aware that there are additional models for migraines; for example, the cortical spreading depression model and animal models that include inflammatory mediators or other migraine triggers, such as CGRP, and we have incorporated these into our exclusion criteria.In our work, we found that nitroglycerin models allowed us to examine migraine mechanisms in a clear and concentrated manner.
Taking over-the-counter pain relievers like ibuprofen or acetaminophen frequently for headache relief can lead to medication overuse headaches or rebound headaches.This phenomenon occurs when the body becomes dependent on the medication, and headaches recur as the medication wears off (111).Overuse of certain medications can disrupt the body's natural painregulating systems and lead to increased sensitivity to pain, perpetuating a cycle of headaches.Studies have shown that individuals with obesity are more likely to experience migraines compared to those with a healthy weight.For instance, a person The significance of NLRP3 and MMP9 in the context of a migraine episode involves the generation and refinement of proinflammatory signaling molecules through various receptors, including P2X7R, P2X4, PAMPs or DAMP, TLR4, IL-1β, and IL-18 receptor activation.This process triggers the release of CGRP, leading to vasoconstriction by enhancing the maturation of IL-1β.Simultaneously, an elevation in MMP9 levels results in blood-brain barrier permeability and the leakage of proteins due to vasoconstriction.This phenomenon contributes to the further production of proinflammatory molecules.Additionally, astrocyte activation plays a pivotal role in the release of excitatory neurotransmitters and other neuroinflammatory substances within the brain, collectively contributing to the onset of a migraine attack.The figure was created with BioRender.com.who is obese may have a higher likelihood of experiencing migraines triggered by hormonal changes associated with adipose tissue (112).The exact mechanisms linking obesity and migraines are not fully understood, but factors such as inflammation, adipose tissue-derived hormones, and metabolic changes may play a role in migraine development and progression.Individuals with obstructive sleep apnea may experience frequent interruptions in breathing during sleep, leading to oxygen desaturation and fragmented sleep patterns.These disruptions can trigger migraines or make existing migraines worse (113).Sleep disturbances like snoring and sleep apnea can alter neurotransmitter levels, increase inflammation, and affect the regulation of pain pathways, all of which may contribute to migraine susceptibility.A person who sustains a concussion in a car accident may develop post-traumatic headaches, which can evolve into chronic migraines over time (114).Traumatic brain injuries can cause structural and functional changes in the brain, including alterations in neuronal excitability, neurotransmitter levels, and pain processing pathways, all of which may contribute to the development and progression of migraines.Individuals who have experienced physical or sexual abuse may have a higher prevalence of migraines compared to those who have not experienced such trauma.The chronic stress associated with trauma can contribute to changes in brain chemistry and increase the risk of developing migraines (115).Traumatic experiences can lead to persistent alterations in stress response systems, neurotransmitter function, and emotional regulation, which may increase vulnerability to migraine attacks.Hypothyroidism, characterized by an underactive thyroid gland, can lead to hormonal imbalances that may influence migraine development.For instance, fluctuations in thyroid hormone levels can affect serotonin levels in the brain, which are implicated in migraine pathophysiology (116).Thyroid hormones play a role in regulating metabolism, neurotransmitter function, and vascular tone, all of which can impact migraine susceptibility and severity.Iron deficiency anemia, a common type of anemia, can result in reduced oxygen delivery to tissues, including the brain, which may trigger migraines or exacerbate existing headaches (117).Iron is essential for the synthesis of neurotransmitters and the maintenance of healthy blood vessels.Iron deficiency can lead to alterations in brain function and vascular reactivity, increasing the risk of migraine attacks.Individuals with anxiety or depression are more likely to experience migraines compared to those without these mental health conditions.Stress, dysregulated neurotransmitter levels, and altered pain processing pathways associated with anxiety and depression can contribute to migraine susceptibility and severity (118).Anxiety and depression are associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increased inflammation, and changes in serotonin and noradrenaline levels, all of which may influence migraine pathophysiology.Chronic tension-type headaches or frequent episodic headaches may precede the onset of chronic migraines in some individuals.Over time, these headaches may become more frequent and severe, transitioning into chronic migraines (119).Frequent headaches, regardless of their specific type, can lead to sensitization of pain  pathways and alterations in pain processing mechanisms, increasing the likelihood of developing chronic migraines.

Excluded papers
Several papers were omitted from consideration due to restricted access, as their complete texts were not freely available.This selective exclusion could potentially introduce publication bias and influence the outcomes of the review.It underscores the critical importance of comprehensive literature access to mitigate bias in systematic reviews.

Pooling of statistical data
The utilization of diverse animal models across the included studies precluded the feasibility of conducting a meta-analysis.This limitation notably impacts the robustness of quantitative synthesis.Emphasizing the value of narrative synthesis within the context of varied study designs is imperative.We will delve into the implications of the inability to amalgamate statistical data, thus ensuring a thorough understanding.

Sample size constraints
We acknowledge the constraint posed by the sample sizes within our included studies.A larger sample size could undoubtedly offer a more exhaustive comprehension of the subject matter.The necessity for forthcoming studies with expanded sample sizes to corroborate and generalize our findings will be duly emphasized.

Preclinical emphasis and translatability
Our focus primarily rests on preclinical investigations employing animal models, which may not fully encapsulate the intricate nature of migraine in humans.We recognize the inherent limitations in extrapolating findings from animal studies to human physiology.Hence, we underscore the imperative for translational research to bridge this gap and address the challenges in clinical applicability.

Gender bias consideration
The potential presence of gender bias in preclinical studies warrants attention and consideration, particularly concerning its ramifications on the generalizability of findings.We will thoroughly explore the issue of gender bias in preclinical research and its implications for comprehending migraine pathophysiology across diverse gender populations.
These adaptations and expansions within our discussion aim to enrich the critical appraisal of our research and augment the depth of understanding within the field of migraine progression.We greatly value your constructive feedback, which serves to refine the quality and relevance of our manuscript.

Highlights
This study delves into the potential roles of NLRP3 and MMP9 as biomarkers in the pathophysiology of migraine, shedding light on the underlying mechanisms of this complex neurological disorder.The study rigorously evaluates the methodological quality of 22 preclinical studies, enhancing the reliability of the findings and offering a robust foundation for further research in this field.It highlights the interconnected relationships between NLRP3, MMP9, and various other biomarkers, providing a comprehensive overview of their potential contributions to migraine attacks.The study provides valuable insights into the preclinical aspects of migraine, emphasizing the need for further research to bridge the gap between animal models and clinical applications.The study underscores the necessity for continued research in both preclinical and clinical settings to better understand the complex mechanisms of migraine and develop more effective treatments.The study touches on the gender bias observed in migraine susceptibility, emphasizing the importance of considering gender specific factors in future research and treatment strategies.The study transparently acknowledges its limitations, including small sample size, potential selection bias, and publication bias, ensuring a balanced interpretation of the findings.By identifying potential biomarkers and their roles in migraine, this study contributes to the ongoing efforts to improve migraine management and therapy, ultimately benefiting individuals worldwide who experience this disabling condition.

Conclusion and future perspective
In conclusion, this systematic review significantly advances our understanding of the intricate relationship between NLRP3 and MMP9 and their potential implications in the pathophysiology of migraine.Drawing insights from a comprehensive analysis of 22 preclinical studies utilizing specific animal models, this review sheds light on the interconnected pathways involving NLRP3 and MMP9, offering valuable clues to their involvement in migraine attacks.While the study's rigorous evaluation of method quality and comprehensive analysis of biomarkers and behavioral tests enhance its credibility, several limitations warrant consideration.These include potential biases such as selection bias, small sample sizes, publication bias, and gender bias, which may affect the generalizability of the findings.Moreover, the reliance on animal models, while informative, may not fully capture the complexity of migraine in humans, limiting the direct applicability of the results to clinical practice.Despite these limitations, this review underscores the urgent need for further research, both in preclinical and clinical settings, to unravel the roles of NLRP3 and MMP9 in migraine pathogenesis comprehensively.Addressing the identified limitations and diversifying the scope of animal models and human studies are imperative steps toward bridging the translational gap between preclinical insights and clinical applications.In the pursuit of improved migraine management and therapy, ongoing investigations into specific biomarkers and their interplay hold immense promise.Future research endeavors should strive to transcend the confines of animal models, embracing a holistic approach that integrates preclinical findings with clinical observations.By doing so, we can aspire to develop more effective treatments and interventions, offering relief to the millions worldwide burdened by suggests that AMPK plays a crucial role in central sensitization of chronic migraine (CM), especially in cases of aberrant energy metabolism.AMPK activation reduces neuroinflammation in NTG-induced CM mice via polarizing microglia in M2.AMPK activity modulation may aid in treating CM by targeting central a light-dark box, Light-aversive test, qPCR, ELISA, TEM, In the thalamus, hypothalamus, periaqueductal grey, trigeminal ganglio, and trigeminocervical complex, respectively, 529, 109, 163, 152, and 419 differentially expressed proteins were found using proteomics profiling of the CM model.Significant alterations in region-specific brain circuits indicated mitochondrial dysfunction in the thalamus.UA intervention had a modest attenuating effect on NTG-induced mitochondrial structural damage, malfunction, and homeostatic dysregulation.that rats subjected to NTGinduced chronic migraines exhibited decreased vestibular function.After several injections of NTG, the fibrinogen levels rose.The rats used to study migraines showed no changes in mechanical hyperalgesia (either worsening or improving) or vestibular dysfunction (either improving or worsening) after defibrinogenation.research work found to be the expression of PACAP and PAC1R in TNC was elevated after repeated treatment of NTG to rats.In addition, via modifying NTGinduced synaptic plasticity via the ERK/CREB/BDNF pathway, PACAP6-38, a selective PAC1R antagonist, reduced chronic cephalic allodynia and inhibited the augmentation of neuronal activity.Inhibiting PACAP/ PAC1R could be a new therapeutic target for migraine based on our findings.
considerably inhibits the NGF/ TRPV1/COX-2 signaling pathway, which is responsible for central hyperalgesia migraine.This indicates that the molecular mechanism by which SGD alleviates migraine symptoms might be associated with the central hyperalgesia neurotransmitter, which controls the pathophysiology of that repeated intranasal PACAP treatments caused mechanical and thermal hyperalgesia like NTG injections.The impact of PACAP on migraine attacks involves PAC1 receptor internalization and PKA and ERK signaling pathways.This is the first research to show that inhibiting PACAP-induced PAC1 receptor internalization improves hyperalgesia in CM rats by restricting ERK signaling.PACAP-induced trigeminal vascular activation requires further study to determine the process and uncover additional targets that modulate PAC1 receptor internalization with specificity.Zhai et al. inhibited PACAP expression in an in vitro research by inhibiting the cAMP/PKA/CREB pathway.However, the mechanism by which perampanel affects the cAMP/PKA/CREB pathway is complex and uncertain.Studying the protective benefits of perampanel against diseases like migraine can shed light on the causes of nervous system problems and aid in developing effective treatments.
integrated approach combining harmacodynamics, metabolomics, and network pharmacology to understand the impact and mechanism of MXFD on migraine.Our integrated analysis revealed two differentially expressed metabolites, 5-MIAA and DCA, and nine targets, including MAOB, MAOA, ADRB1, ADRB2, ADRB3, ADORA2A, ADORA2B, DRD5, and HTR4.MXFD's effectiveness in treating migraine is significantly impacted by these metabolites and targets.This research provides useful data for exploring the mechanisms behind MXFD's therapeutic effects.test, Von frey test, Hot plate test, Orofacial formalin test, ELISA, Immunofluorescence, RT-qPCR, and Histological analysis Neuronal damage; IL-18, IL-1β, and TNF-α protein expressions; mRNA expressions of these proteins; Nerve growth factor (NGF), Brain-derived neurotropic factor (BDNF), and Neurotrophin-3 (NT3) expressions were dramatically decreased when treated with BAY-117082 at 5 mg/kg and 10 mg/kg.These report suggesting that it significantly modulated Erk/CREB/Akt pathways and could be considered novel strategy therapeutics for migraine treatment.amino-4-(3-hexylphenylamino)-4oxobutylphosphonic acid) could alleviate chronic NTG induced hypersensitivity and reduce the expression of CGRP, pSTAT3 and c-fos in the Trigeminocervical complex by blocking S1PR1 activity.Aral et al. .Elevation of nitric oxide which has been demonstrated to have a role in the production of inflammation such as MMP9 and CGRPR1 may regulate cellular iron traffic; hence, different cells and mislocalization of iron within the cell may contribute to migraine pathophysiology via diverse molecular mechanisms.Ting et al. suppressed the expression of the NLRP3 protein and the pro-inflammatory cytokine IL-1β in the TNC produced by NTG, and it may regulate the activity of microglia in the TNC through modulating CGRP.It is possible to interact with microglia, which controls the release of inflammatory factors but, BoNT/A therapy had no effect on LPS-induced proinflammatory factor release in astroglia.Wang et al. prescription decreases the production of neurotransmitters linked with nociceptive transmission, including as 5-HT, CGRP, and CCK, which may be connected to its overexpression of CB1R and downregulation of COX-2.He et al. (28) IL-1 inhibition alleviated hyperalgesia and decreased the increase in biomarkers such as p-ERK, c-Fos and CGRP associated with central sensitization of CM in the TNC.The NLRP3 inflammasome may be a target for controlling CM-associated pain, and inhibiting it may represent a novel therapeutic rationale and technique for migraine treatment.

FIGURE 1
FIGURE 1Schematic representation of article screening.
in rodents, making it widely accepted as a universal animal model for studying migraines.Elevated levels of nitric oxide, which have been associated with neuroinflammation, might regulate the movement of cellular iron.As a result, variations in iron distribution within cells could contribute to migraine pathophysiology through diverse molecular mechanisms.Brain-derived neurotrophic factor (BDNF) is linked to the modulation of pain and central sensitization.There is a growing suspicion of BDNF's involvement in the pathophysiology of migraine and cluster headaches, primarily because of its established interaction with calcitonin gene-related peptide(84).From a clinical perspective, there is a need for increased research emphasis on NLRP3 and BDNF, as this may offer more effective solutions for migraine treatment.Studies have revealed that female mice are more susceptible to migraine compared to male mice.Through the induction of NTG (10 mg/kg) injection, researchers have observed that the end products of the NLRP3 inflammasome, specifically cytokines, elevate neurotrophic factors that play a crucial role in the pathological processes associated with the progression of migraines.Their critical findings indicate that NLRP3 is capability to mature IL1-β and is involved in the production of IL-10.Elevated levels of neurotrophic factors, particularly BDNF, may also influence NTG through the CREB/Erk/Akt pathways(27, 28,30,70, 75,82).In the context of a neuropathic pain model, S1PR1 on glial cells influenced various downstream signaling pathways, including the MAPK pathways and the NLRP3 inflammasomes(85)(86)(87). S1PR1 signaling enhances the production of IL-1β by upregulating NLRP3 (70, 88-90).Wang, Yuan, and their research team provided scientific evidence indicating that the expression of the NLRP3 protein occurs in a specific manner via the P2X7R and P2X4 receptors(30, 75).These receptors can be potential targets for impeding the generation of proinflammatory molecules via NLRP3 inflammasomes in the brain, potentially aiding in the treatment of migraines(30, 75, 91).However, Fan et al. discovered that NFκB primarily plays a critical role in activating NLRP3 via TRPA1(80).Kimono and their research team made an intriguing observation that the activation of the NLRP3 inflammasome is prompted by elevated levels of reactive oxygen species (ROS) (92).

FIGURE 3
FIGURE 3Quantify agreement by kappa value of all authors.RR-KI and VC-SA reported perfect agreement and rest others showed substantial agreement.

FIGURE 5
FIGURE 5Networking of migraine targets.The figure illustrating that the different proteins interlinked with NLRP3 and MMP9 in migraine attack.There are different connecting targets of NLRP3 and MMP9 are analysed by protein-protein interaction in string database that NOX4, IL1B, P2RX4, NOS2, IL18, CCL2, TRPA1, CALCA, MAP 3 K5, TXNIP etc. are connected to migraine progression.

TABLE 1
Inclusion and Exclusion criteria.

TABLE 2
Characteristics of the included studies and a summary of the outcome.

TABLE 2 (
Continued) Their study demonstrated that RUT reduced oxidative stress and relieved migraine symptoms by blocking PGK1 activity with PTEN and activating the Nrf2 antioxidant mechanism.
NTG-induced hypersensitivity is via inhibiting S1PR1 activity.An increase in nitric oxide levels may control the pathogenesis of migraines through a variety of molecular pathways.BoNT/A may have a therapeutic function in migraines by reducing the expression of NLRP3 and IL-1β.By influencing neurotransmitter synthesis and the expression of CB1R and COX-2, Hejie Zhitong, when prescribed, may reduce migraine symptoms.A potential new approach to treating migraines could be to inhibit NLRP3, which would lessen hyperalgesia and central sensitization.It has been suggested that proinflammatory mediators such as COX-2, TNF-α, and MMP9 could be viable pharmaceutical targets in the development of migraines caused by NO.

TABLE 2 (
Continued) 10.3389/fneur.2024.1307319Frontiers in Neurology 15 frontiersin.orgthis debilitating neurovascular disorder.Future research should aim to bridge the gap between preclinical findings and clinical applications, ultimately benefiting the millions of individuals worldwide who suffers from this debilitating neurovascular disorder.