CGRP and Oxidative Stress in Migraine Pathophysiology: Mechanisms, Clinical Evidence, and Therapeutic Implications

Headaches are a common ailment, leading to considerable global disability, surpassing any other neurological condition. They refer to discomfort in the face, head, or neck, and can present in multiple forms. The International Headache Society categorizes headaches into more than 150 types, organized into primary and secondary classifications. Primary headaches encompass tension-type, the most common type often associated with stress; migraines, which involve intense, pulsating pain commonly linked with nausea and sensitivity to light; idiopathic stabbing headaches, characterized by sharp, brief pain; exertional headaches that occur with physical activity; and cluster headaches, which result in severe, one-sided pain typically around one eye. Secondary headaches arise from other underlying conditions, such as systemic infections, head trauma, vascular issues, subarachnoid hemorrhage, or brain tumors. Accurate identification and categorization are essential for establishing the correct treatment and effectively managing the condition. [1] It is widely acknowledged that the onset of migraine headaches are believed to be linked to the stimulation of sensory afferent fibers in the ophthalmic division of trigeminal nerve. (TGV). Although the precise mechanisms that trigger this activation are not fully understood, Cortical spreading depression (CSD) is thought to be an important factor, especially in migraines accompanied by aura and possibly in those that occur without aura. CSD refers to a wave of depolarization that spreads across the cortex, which may initiate the activation of the TGV system. This activation results in the release of neuropeptides from the trigeminal ganglionic neurons, leading to neurogenic inflammation in the dura mater, contributing to the headache's pain and its related symptoms.[2] CGRP is one of the neuropeptides released by trigeminal ganglion neurons. Studies have shown a dense concentration of CGRP-containing fibers around the cerebral vessels, originating from the trigeminal ganglion. CGRP’s role in migraine was solidified when the infusion of CGRP triggered migraine-like attacks in patients suffering from migraine without aura (MO). [3] Metabolic activities in mammalian cells, including respiration and digestion, generate millions of free radicals (FRs) daily. Our body possesses natural antioxidant mechanisms, plus consuming dietary antioxidants boosts their effectiveness, in addition to the natural antioxidant characteristics found in food. Oxidative stress (OS) is associated with the onset of human diseases like cerebrovascular accidents and conditions affecting neuron degeneration. Reactive species (RS) encompass reactive oxygen, nitrogen, and halogen species. A disruption between these processes and the antioxidant defense results in strain on cellular proteins, lipids, and DNA. Oxidative stress is a significant contributor to the genesis of migraines.[4]. By reviewing the various research articles, this manuscript contributes to a deeper understanding about the role of CGRP and oxidative stress in migraine.

5. Calcitonin gene related peptide and migraine

Calcitonin gene-related peptide (CGRP) is a neuropeptide composed of 37 amino acids that is a significant regulatory molecule and a strong vasodilator for microvessels, first identified in 1982. It is part of the calcitonin family, which includes calcitonin, adrenomedullin, adrenomedullin 2 (intermedin), and amylin. In humans, there are two variants, α-CGRP and β-CGRP, which exhibit structural similarities, boasting 94% homology along with the same affinity in binding and strength.

α-CGRP:  It is found in both the central and peripheral nervous systems, primarily synthesized and kept in in sensory neurons of Aδ and C fibers located within trigeminal (TG) and dorsal root ganglia (DRG). With the help of Calcitonin I gene this production originates. Initially, a pre-mRNA transcript is generated, after which exon 4 is removed. The final transcript is translated into a pro-hormone consisting of 121 amino acids. This pro-hormone is then processed to create a mature peptide of 37 amino acids (α-CGRP), which is kept in dense-core vesicles for delivery to the axon endings.

β-CGRP: This variant is mainly present in the enteric nervous system and the pituitary gland. With the help of calcitonin II gene it is produced, which is found on chromosome 11.  CGRP can be categorized into four parts: N-terminus (Amino acids 1–7), α-helix (amino acids 8–18), β- or γ-twist (Amino acids 19–27), and C-terminus (Amino acids 28–37).   The CGRP receptor is a membrane-associated heterodimer consisting of the receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). The CLR belongs to the class B "secretin-like" group of G protein-coupled receptors (GPCR). RAMP1 is a part of the RAMP family, which includes RAMP1, RAMP2, and RAMP3. The amylase 1 (AMY1) receptor is formed by the interaction of the calcitonin receptor (CTR) with RAMP1, resulting in another CGRP receptor. [5] The calcitonin gene-related peptide (CGRP) pathway has been identified as a crucial factor in the genesis of migraines. Groundbreaking studies have shown that during an intense migraine episode, CGRP is released in large quantities into the extra cerebral circulation. [6] Calcitonin Gene-Related Peptide (CGRP) is a neuropeptide that plays a important role in various pathways within the central nervous system (CNS), especially in the development of migraines. CGRP and its receptors play a role in how pain is perceived, sensory processing, and the regulation of neurovascular functions. The trigeminal ganglion neurons are crucial in the pathophysiology of migraines, linking the peripheral nervous system to the central nervous system. CGRP, which is abundant in these neurons, is emitted from both peripheral and central nerve endings, as well as from within the trigeminal ganglion itself. When peripheral nerve endings releases CGRP, it initiates a series of events, such as enhancing the production of nitric oxide and increasing the sensitivity of trigeminal nerves. Furthermore, CGRP released within the trigeminal ganglion interacts with nearby neurons and satellite glial cells, leading to peripheral sensitization. This mechanism can also induce central sensitization in second-order neurons located within the caudalis region of the trigeminal nucleus. Over time, a transition of central sensitization from activity-dependent to activity-independent may take place, which can lead to the progression from episodic to chronic migraine.[7] The Posterior Thalamic Area (PTA) also receives retinal ganglion cell’s signals, which are crucial for vision. This integration of signals from both the trigeminal and visual pathways in the PTA helps clarify the increased sensitivity to brightness (light) observed during migraines. Research conducted on rodents indicates that the PTA is integral to the genesis of photophobia. When the PTA is activated, it escalates light sensitivity, mirroring the sensory hypersensitivity experienced in a migraine episode. CGRP appears to play a significant role in these mechanisms as it is present in specific nuclei within the PTA. Studies indicate that injecting CGRP within the PTA results in increased neuronal firing, reinforcing its involvement in modulating thalamic activity. Furthermore, nociceptive and somatosensory stimuli the gathering of neurons within the central nervous system that produce CGRP in the thalamus, thereby enhancing sensory signals. This implies that CGRP is a contributing factor to the increased sensory sensitivity associated with migraines.[8]. Early research indicated that stimulating the dura in humans resulted in localized headaches, linking the trigeminal nerve's innervation to migraine pain. Studies in animals verified that trigeminal fibers do innervate the dura, and stimulation activates nociceptive neurons within the trigeminocervical complex. Inflammation amplifies the responses, indicating peripheral sensitization, although neurogenic inflammation has not been documented in humans. While CGRP-mediated degranulation of mast cell has been seen in animal studies, human mast cells lack CGRP receptors, which limits CGRP’s involvement in human migraine pathology. Nonetheless, in vitro experiments utilizing human dural tissue indicates the substance P, which is co-expressed with CGRP in certain afferent fibers, can trigger mast cell degranulation. Although this points to a possible involvement of substance P in neuroinflammation, it is doubtful that it alone can generate enough neurogenic inflammation to have a substantial impact on the beginning of migraines. Thus, while substance P might contribute, it is not regarded as a primary factor in the genesis of migraines when not co-expressed with CGRP.[7]  Additionally, Parts of the CGRP receptor have been identified in vascular smooth muscle cells., highlighting CGRP's significance in vasodilation, which is pertinent to migraines. The The vessels that carry blood in dura are supplied with CGRP-positive nerve fibers.[9]. One of the findings indicates that CGRP receptors, which may be vital in migraine and other headache occurrences, are located in the meninges, trigeminal ganglion, and spinal trigeminal nucleus. The trigeminal ganglion is particularly noteworthy, as our comprehension of CGRP signaling and neuronal interactions in this region is limited. The presence of CLR and RAMP1 components of the CGRP receptor supports the idea that CGRP is involved in regulating blood flow, modulating mast cell secretion, and affecting neuronal and glial functions.[10]. The animal portion of the study demonstrates that trigeminal ganglion stimulation triggers CGRP release in cats, akin to what is observed in humans. This aligns with human research and underscores the importance of animal models. In particular, superior sagittal sinus stimulation in animals results in enhanced cerebral blood flow and CGRP release, without substance P (SP), replicating the CGRP-specific release noted in migraines.[11] Human research indicates that the impact of CGRP are more intricate. The interaction of CGRP with receptors within the trigeminal ganglion and its function in controlling blood circulation, mast cell secretion, and neuronal functions highlight its importance in the initiation and progression of migraines. Migraine occurs more frequently in women, with hormonal changes significantly contributing to its onset and intensity. The research involved 124 women diagnosed with episodic migraine and endometriosis to examine CGRP levels throughout the menstrual cycle. Blood samples were collected during menstruation (on day 2) and during the preovulatory phase (on day 15). The main outcome measured was the variation in CGRP levels between these two phases. The results indicated that women with both migraine and endometriosis experienced an elevation in levels of CGRP during menstruation (+6.32) compared to the periovulatory phase. Conversely, healthy controls exhibited a lower levels of CGRP (-10.14). These results imply that CGRP levels undergo significant fluctuations during the menstrual cycle.[12]. Estrogens and progesterone can elevate CGRP production and affect its role in transmitting pain. Research has demonstrated that estrogen therapy can enhance CGRP release at sensory nerve endings lead to the expansion of dural arteries in rats. Investigations involving ovariectomized rats indicate that treatments with estrogen and progesterone lead to elevated CGRP levels in the dorsal root ganglion. Blood levels of CGRP are lower in postmenopausal women, higher in those who are pregnant, and elevated in individuals using contraceptives or hormone replacement therapy. Generally, women have higher CGRP levels compared to men. Progesterone has an antagonistic effect on estrogen’s influence on CGRP along with its receptor expression. Increased CGRP levels are associated with migraines but may also indicate vascular changes, particularly during pregnancy. [13]. In a recent investigation involving human vascular tissue samples, notable differences were found in how CGRP-induced relaxation responses vary between human middle meningeal arteries and coronary arteries. These findings indicates that the differences may result from varying receptor expression across different vascular areas, along with the effects of hormonal fluctuations and age on CGRP’s influence. Importantly, heightened CGRP levels might lead to receptor desensitization, which could clarify the diminished CGRP response observed in young females compared to a more pronounced response in young males. These findings highlight the necessity of taking into account sex-specific factors and receptor behavior when analyzing CGRP-related vascular responses, especially regarding hormonal effects and aging.[14]. The research carried out by Bianca Raffaelli and colleagues examines how sex hormones affect the release of calcitonin gene-related peptide (CGRP) in female individuals experiencing episodic migraine (EM) at various hormonal stages. Levels of CGRP in plasma and tear fluid were assessed in participants with a typical menstrual cycle, those on combined oral contraception (COC), and postmenopausal women, and compared to age-matched controls without EM. The findings revealed that women with EM who had a regular menstrual cycle showed significantly elevated CGRP levels during their menstruation in comparison with control group, in both plasma and tear fluid. However, no variation in CGRP levels were noted between migraine participants and the control group among those using COC or postmenopausal women. Additionally, CGRP concentrations in tear fluid were notably higher in menstruating migraine participants than in those on COC. These results underscore the potential influence of hormonal changes on CGRP levels and their potential involvement in migraine pathophysiology during varying hormonal phases.[15]. CGRP is crucial in the genesis of migraines, with its levels being affected by hormonal changes, particularly in women. Research indicates that during menstruation, CGRP levels rise in women experiencing episodic migraines, highlighting a strong relationship between hormonal shifts and CGRP release. Estrogen and progesterone have an impact on both CGRP production and receptor expression, which contributes to the occurrence and intensity of migraines. Increased levels of CGRP are linked to vascular alterations, especially in females, and may indicate changes in receptor responsiveness or desensitization. These insights emphasize the need to consider hormonal variations when examining CGRP's involvement in migraine pathophysiology and imply that treatments targeting CGRP should take into account sex-specific variables and hormonal conditions to enhance therapeutic effectiveness. In another investigation, it was observed that CGRP infusion triggered headaches in people who suffer from migraines, exhibiting a distinct temporal pattern reminiscent of glyceryl trinitrate (GTN) and histamine. The initial headache was mild, but a more intense headache began five hours later, resembling a typical migraine without aura. CGRP led to the dilation of the middle cerebral artery, although there were no significant modifications in cerebral blood circulation. It is proposed that CGRP generates migraine-like headaches via endothelial receptors and nitric oxide (NO) pathways.16]  An increase in CGRP release during headache-free intervals may be attributable to a generalized dysfunction in the release of CGRP from sensory nerve endings, potentially linked to abnormal vascular regulation. This reinforces the notion that CGRP plays a role in migraine pathophysiology even outside of acute attacks.[17]. A pivotal study conducted by Olesen and his team provided compelling evidence connecting CGRP to migraines. They discovered that intravenous administration of CGRP provoked moderate to severe migraines in individuals with headaches, often resembling typical migraine episodes. Approximately 65% of the participants experienced a migraine-like episode, and 84% developed a delayed headache alongside other migraine symptoms. Conversely, control individuals only reported mild headaches. These findings indicate that those prone to migraines exhibit heightened sensitivity to CGRP. Subsequent investigations, including studies involving CGRP-sensitive mice, further validated this association.[18]. Research data strongly indicates the crucial function of CGRP in the genesis of migraines. Evidence demonstrates that the administration of CGRP stimulates headache episodes resembling migraines, underscoring its role during both active migraine attacks and during headache-free intervals. Elevated levels of CGRP in individuals with migraines imply a continuous dysfunction in CGRP release, which could be linked to vascular regulation. This solidifies CGRP as an important target for migraine therapies and further validates its role in the beginning of the condition. Migraine therapies can be categorized into two primary groups: non-specific treatments and specific treatments. Non-specific options encompass analgesics and NSAIDs, including acetaminophen, aspirin, ibuprofen, and naproxen sodium, which aid in symptom relief. For more intense episodes, combination medications that contain aspirin, paracetamol, and caffeine are also beneficial. Specific treatments consist of ergot derivatives such as ergotamine and dihydroergotamine, which interact with serotonin receptors to alleviate acute migraines. Ergotamine is cost-effective and has a long-lasting effect; however, it is contraindicated for individuals with hypertension, coronary disease, and during pregnancy, and may lead to nausea and cramps. Dihydroergotamine (DHE) is generally better tolerated, though it is less effective in oral form and works better when given intranasally or via injections. Triptans, including sumatriptan, zolmitriptan, and rizatriptan, have mostly supplanted ergot derivatives because of their swift pain relief and reduced side effects.[19]. Nevertheless, these medications were not specifically formulated for treating migraines and often come with side effects or fail to provide sufficient relief. Focusing on CGRP has arisen as a novel treatment approach to offer pain relief and assist migraine sufferers in resuming their daily lives. Considering the peripheral aspect is crucial to comprehend that an anti-migraine medication may not necessarily have to traverse the blood-brain barrier (BBB) or access the central nervous system (CNS) to be effective. Increased levels of CGRP, unlike other neuropeptides, were detected in the external jugular vein during the phase of the headache of a migraine and returned to normal as the headache improved. Additionally, administering human CGRP has been demonstrated to induce a migraine episode in sensitive individuals, while triptan treatment led to the adjustment of CGRP levels to a standard range.[20] . In line with the widespread presence of 5-HT1 receptors, triptans can prevent the vasodilation of intracranial vessels and inhibit the release of CGRP to obstruct neurogenic inflammation and the central transmission of pain signals from trigeminal nerves.[21]  Rizatriptan, an anti-migraine medication belonging to the triptan class, is understood to function as abortive treatment works by inhibiting the peripheral release of neuropeptides in the meninges and the central release of glutamate and CGRP in the spinal trigeminal nucleus. [22]. Given the role of CGRP in migraine pain signaling, small molecule CGRP receptor antagonists, known as gepants, were created for migraine treatment. The initial gepants did not succeed in reaching the market due to cases of hepatotoxicity and issues with pharmacokinetics. While new gepants are in development with no toxicity reported so far, the latest method for CGRP blockade involves antibodies against CGRP (eptinezumab, fremanezumab, galcanezumab) or its receptor (erenumab), every one of which possess demonstrated efficacy in the preventative care for migraines.[23]. At present, two gepants (rimegepant and ubrogepant) have been accepted for treating acute migraines, and two others (atogepant and rimegepant) are approved for preventive care. These gepants are administered orally, with zavegepant (BHV-3500), a nasal spray formulation, undergoing clinical trials. It is crucial to recognize that gepants and antibodies likely diminish rather than completely eliminate CGRP actions. This limitation stems from the bolus release of peptides, and if these treatments do not entirely block both CGRP receptors, their efficacy could be affected.[18] Certain gepants, like telcagepant, showed comparable or even superior effectiveness to triptans such as rizatriptan. Although telcagepant exhibited mixed results as a preventive option, it successfully reduced migraine days in certain post hoc analyses. In summary, CGRP receptor blockade, utilizing both gepants and monoclonal antibodies, provides substantial evidence of efficacy in managing migraines, both acutely and preventively. CGRP blockers exhibit effectiveness that is equal to or slightly better than topiramate for migraine prevention, lowering the number of monthly migraine days by 2.9 to 4.3 days, with responder rates of 50% or more ranging from 56.3% to 62.3%. They also generally experience less severe and less frequent side effects, making them a favorable choice for preventing migraines. Clinical study evidence indicates that CGRP blockade generally leads to mild to moderate side effects, such as nausea, headache, and fatigue. Although serious side effects like liver toxicity were observed with some medications (e.g., telcagepant), others have demonstrated no such issues. Monoclonal antibodies directed at CGRP have presented minimal adverse effects, with no reports of liver or cardiovascular complications. While early research indicates that inhibiting CGRP could affect cardiovascular health, wound healing, gastrointestinal function, and pituitary hormone regulation, no significant negative effects have been noted; however, long-term risks necessitate additional investigation.[24]. Inhibiting CGRP significantly reduces the occurrence of migraines and has a lower incidence of side effects compared to conventional therapies. Nevertheless, there are ongoing worries regarding the long-term effects on cardiovascular health, the possibility of developing resistance to the medication, and the elevated costs associated with it, which require additional research.

5. Oxidative stress in migraines

1. Evidence for oxidative stress in migraines

Migraine is a common neurological condition that leads to considerable disability and associated societal costs. Various factors that elevate oxidative stress within the brain trigger migraine attacks, resulting in neuronal alterations and pain. Research indicates that the brain of migraine sufferers experiences an energy deficit, which may amplify oxidative stress and reduce the threshold for attack initiation. Studies using magnetic resonance spectroscopy (MRS) reveal an energy shortage in the migraine-affected brain, marked by diminished phosphocreatine-to-creatine ratios and heightened ADP levels, which correlate with the the intensity and occurrence of attacks. Reduced ATP levels, especially in the occipital lobe, point to compromised energy production, further supported by studies on sleep deprivation that connect energy deficits to the onset and occurrence of migraines.[25]. Findings from case-control research suggest a significant function of oxidative stress in migraines. In a study involving 46 patients with migraines and 45 healthy controls, those with migraines displayed notably higher serum levels of malondialdehyde (MDA), total oxidant status (TOS), and oxidative stress index (OSI) in comparison to the controls. Moreover, migraine patients had lower concentration of total thiols (T–SH), glutathione (GSH), and total antioxidant capacity (TAC).[26] Additionally, Mansoureh Togha's research revealed that chronic migraine (CM) sufferers had decreased antioxidant levels (CAT, SOD, TEAC) and elevated oxidative stress markers (NO, MDA) when compared to episodic migraine (EM) patients and control subjects. The correlations indicated that an increase in headache days was associated with heightened oxidative stress and a disruption in antioxidant enzyme balance.[27]. Lana Othman Mahmood's research, which included 60 patients with migraines and 30 controls, indicated that individuals suffering from migraines exhibited significantly elevated serum levels of markers for oxidative stress, such as malondialdehyde (MDA), nitric oxide (NO), and asymmetric dimethyl arginine (ADMA), in comparison to the control group. Additionally, a positive association was identified between the number of headache days and the levels of these markers, implying that heightened oxidative and nitrosative stress, along with endothelial dysfunction, plays a role in the pathophysiology of migraines.[28] Gyanesh M. Tripathi's investigation found that individuals suffering from migraines experienced notably lower levels of Glutathione (GSH), Glutathione-S-Transferase (GST), and Total Antioxidant Capacity (TAC) when in comparison to the control group. Following treatment with Repetitive Transcranial Magnetic Stimulation (rTMS) or Amitriptyline (AMT), levels of GSH and TAC showed an increase. The rTMS treatment led to improvements in headache frequency, Visual Analog Scale (VAS) scores, and Migraine Index (MI) in 50.8%, 58.3%, and 74.2% of patients, respectively.[29]. The analysis uncovered significantly higher levels of 4-hydroxy-2-nonenal (HNE), a marker for lipid peroxidation, in people experiencing migraines relative to healthy controls (P < 0.05). Moreover, HNE levels were strongly correlated with insulin, BMI, and various lipid markers, including oxidized LDL. The logistic regression analysis further emphasized that higher HNE levels were linked to an increased likelihood of migraines, especially among women in the top quartile of HNE (OR = 4.55, 95% CI: 1.33–15.56, P < 0.05). ).[30]  The findings indicate that oxidative stress markers such as HNE play a role in the onset of migraines. R. ALP and S. SELEK examined the imbalance of oxidative stress in individuals suffering from migraine without aura (MWoA). Their research showed that the Total Antioxidant Status (TAS) in MWoA patients was significantly lower when compared to healthy individuals. Conversely, levels of Total Oxidant Status (TOS) were significantly elevated in those with MWoA. Additionally, the Oxidative Stress Index (OSI), was found to be higher in MWoA patients. The study also identified a negative correlation between headache duration and Total thiol (SH) levels, while a positive relationship was identified between OSI and headache frequency.[31] These results suggest an increased level of oxidative stress in patients with MWoA, reinforcing the idea that oxidative imbalance contributes to the advancement of migraines.  Oxidative stress can result in the genesis of reactive oxygen species (ROS), which can harm cellular components, such as DNA. This DNA damage is often indicated by rising levels of biomarkers like 8-hydroxy-2′-deoxyguanosine (8-OHdG). In a study, plasma samples from 50 migraine patients (11 with aura, 39 without aura) and 30 healthy individuals were analyzed for 8-OHdG levels. The findings showed that plasma levels of 8-OHdG were notably higher in individuals suffering from migraines in comparison with the controls. The increase in 8-OHdG was more significant in individuals experiencing migraine without aura, indicating DNA damage linked to oxidative stress in migraines cases. [32]. Evidence consistently indicates that oxidative stress contributes to the process of genesis of migraines. Research has found increased degrees of oxidative stress markers, including MDA, NO, and HNE, along with decreased antioxidant levels (such as GSH and TAC) in individuals suffering from migraines in comparison with healthy controls. The observed correlations between markers of oxidative stress and the frequency or intensity of headaches further imply that an oxidative imbalance may contribute to both the onset and progression of migraines. Treatments like rTMS and AMT, which influence oxidative stress levels, have demonstrated a reduction in migraine symptoms, highlighting the significance the role of oxidative stress in the mechanisms of migraine development and suggesting possible therapeutic approaches focused on restoring oxidative balance.

2. Mechanism and potential role of mitochondrial dysfunction

Nitric oxide (NO) is a transient molecule that induces vasodilation and is swiftly converted into nitrates and nitrites. The body continuously generates NO by oxidizing L-arginine via the enzyme NO synthase. A lack of L-arginine or the presence of inhibitors such as asymmetric dimethyl arginine (ADMA) can decrease NO production, which may result in hypertension. Research on NOx levels in migraine sufferers indicates slight increases in plasma and serum in comparison with control subjects. However, the measurements frequently overlap between migraine with aura (MA) and migraine without aura (MO) [33], implying that NO could play a part in the onset of migraines.. The nitric oxide produced contributes to the vasodilation experienced during migraines and stimulates the substance P’s release, along with protein and fluid extravasation. Additionally, in the vascular system, nitric oxide inhibits the renin-angiotensin system by reducing the production of angiotensin-converting enzyme and angiotensin-II AT1 receptors. Angiotensin elevates oxidant production by significantly activating nicotinamide adenine dinucleotide phosphate oxidase enzyme (NADPH oxidase), thus nitric oxide's down regulation has a protective antioxidant effect [34] The role of NADPH oxidase 2 (NOX2) in nitroglycerin (NTG)-induced migraines in mice highlights how oxidative stress persists in trigeminal neurons and the gastrointestinal (GI) tract during an attack, indicating that the Nrf2/NOX2 signaling pathway is crucial in migraines. The equilibrium among NOX2 and Nrf2 is essential, as the up regulation of Nrf2 can diminish NOX2 expression, thereby limiting both inflammation and oxidative stress within the brain and GI. [35]. TRPA1 is essential in the genesis of migraines, serving as a detector for reactive oxygen and nitrogen species (RONS) generated during oxidative stress. It is triggered by compounds such as acrolein found in cigarette smoke, lipid peroxides, and nitric oxide, all of which are associated with migraine triggers. Once activated, TRPA1 promotes the release of calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and the sensitization of meningeal nociceptors. This process initiates pain signaling and stimulates the trigemino-vascular system, which contributes to headaches. Consequently, TRPA1 is essential to converting oxidative stress signals into pain, thus being a key factor in migraine onset.[36]  Lipid peroxides, characterized by their long half-lives and lack of net charge, can diffuse readily to target receptors, playing an important part in oxidative stress signaling. Within the framework of migraines, these lipid peroxides activate TRPA1 ion channels located on nociceptive nerve endings within the perivascular meninges. When certain cysteine residues on TRPA1 undergo oxidation, the channel opens up, permitting the entry of calcium ions, which translates oxidative stress into a neural signal, resulting in neurogenic inflammation—a significant feature of migraines.[34]. A shortfall of brain energy also initiates the development of migraines, whether due to increased energy demands or reduced energy supply. This results in oxidative stress, as neurons depend on NADPH (produced through mitochondrial processes) for their antioxidant defenses. The generation of NADPH is contingent upon enzymes involved in the Krebs cycle and oxidative phosphorylation. During a migraine aura, cortical spreading depression (CSD) prompts a reduction in NADH levels, which disrupts antioxidant defenses through the enzyme nicotinamide nucleotide transhydrogenase (NNT). This leads to a recovery in NADH, causing an overproduction of oxidants within the electron transport chain. In migraines without aura, similar shifts in NADH are likely to produce oxidants in area of cortex distant from blood vessels, resulting in oxidative stress. These variations in energy levels activate nociceptive ion channels sensitive to oxidants, triggering a migraine attack. [25]. Michal Fila determined that epigenetic mechanisms may significantly influence the pathophysiology of migraines, particularly through mitochondrial dysfunction. Mitochondria generate reactive oxygen species (ROS) that cause to oxidative stress, which can harm cellular components and cause neuronal dysfunction, potentially triggering migraines. The abnormal expression of proteins like PGC-1α, which takes part in mitochondrial biogenesis, indicates that mitochondrial regulation may affect susceptibility to migraines. Epigenetic alterations, including mtDNA methylation, might also influence mitochondrial function and contribute to genesis of migraine. More investigation is required to investigate the epigenetic regulation of mitochondria, especially mito-miRNAs, to enhance our understanding of their involvement in migraine pathogenesis. [37]. Mitochondrial abnormalities result in calcium dysregulation, excessive production of free radicals, a diminished mitochondrial membrane potential, and an imbalance in the opening of the mitochondrial permeability transition pore (mPTP). These dysfunctions hinder oxidative phosphorylation, causing energy depletion, neuronal apoptosis, and reduced pain thresholds, which can trigger migraine attacks. Cortical spreading depression (CSD) further impairs mitochondrial function, increasing ROS accumulation and disrupting energy metabolism. Elevated levels of nitric oxide (NO) hinder mitochondrial function by inhibiting cytochrome c oxidase and NADH dehydrogenase, disrupting oxidative phosphorylation and decreasing ATP production. This energy deficit results in neuronal dysfunction, leading to the onset of migraines. NO also opens the mitochondrial permeability transition pore (mPTP), causing the release of cytochrome c (Cyt-c) and reactive oxygen species (ROS). The buildup of ROS in mitochondria exacerbates cellular damage, further impairing mitochondrial function and escalating ROS production.[38]