Methylene blue - Educational materials

Methylene blue (MB) is a chemical compound (3,7-bis(dimethylamino)-phenothiazine-5-ium chloride) with a wide range of medical applications [1]. It was first produced by Heinrich Caro as a textile dye, but scientists quickly discovered that it could be useful in medicine.

Early studies showed that MB could be used as a medical dye to illuminate cells under a microscope, and later scientists such as Ehrlich and Guttman discovered that it was effective in treating malaria. This discovery made MB an important drug in many military campaigns, even though it had the strange side effect of tinting urine blue. Although this side effect was not popular among soldiers, it had a surprising use in psychiatry. Doctors added MB to medications to check whether patients were taking their prescriptions, since the blue color of the urine confirmed compliance [2, 3].

Eventually, scientists discovered that MB itself had a sedative effect, which led to its use in psychiatric treatment and contributed to the development of early antipsychotic drugs.

MB is currently approved by the Food and Drug Administration for the treatment of methemoglobinemia, a blood disorder in which oxygen delivery is impaired, and is also used to treat ifosfamide-induced encephalopathy, a side effect of some cancer therapies.

Other uses of MB include the treatment of urinary tract infections in elderly patients, malaria in children, and cases of vasoplegic shock where epinephrine-based treatment has failed. In addition to therapeutic uses, MB is commonly used as a tracer dye in surgery to help visualize tissues [1-3].

In recent years, methylene blue has been extensively studied for its potential in neurological treatment, showing benefits in treating psychosis and improving memory and cognitive function in conditions such as Alzheimer's disease.

Methylene blue for brain health (human and animal studies)

Recent studies have shown that methylene blue (MB) can help with brain-related conditions by protecting neurons, increasing antioxidant activity and improving mitochondrial function. Originally used in other medical therapies, MB improves memory, protects brain cells and reduces inflammation in diseases such as Alzheimer's, brain injuries and strokes. It supports brain energy and combats oxidative stress, making it a useful option for brain health and protection.

MB effectively reaches the brain, especially after intravenous (IV) administration, which provides higher concentrations than oral doses. MB accumulates in various tissues, including the brain, where its levels can be as much as ten times higher than in the blood within just an hour of injection. In the body, it quickly spreads to the lungs, liver, kidneys and heart. Researchers have also developed a modified form of MB that penetrates the brain even better and is currently being tested in clinical trials.

Both human and animal studies have shown that methylene blue supports brain health in a variety of ways. These include increasing mitochondrial function, improving oxygen metabolism and protecting against age-related cognitive decline. In a clinical trial, Rodriguez et al (2016) conducted a randomized, double-blind clinical trial to evaluate the effects of MB on attention and memory in healthy individuals. After administering low doses of MB, functional MRI revealed increased activity in brain areas associated with attention and memory processing, such as the insular cortex and prefrontal cortex. Interestingly, participants also showed 7% improvements in memory retrieval accuracy [4]. These results confirm the potential of MB to improve brain function and memory in healthy populations.

In addition, Rodriguez et al (2017) in another study found that MB reduced blood flow in certain task-related brain areas. More importantly, MB strengthened connections in regions related to perception and memory during rest [5]. This suggests that MB may modulate brain networks, potentially improving cognitive function. In addition, Telch et al (2014) conducted a a human clinical trial to study the effects of MB on fear extinction and memory. Adults with claustrophobia were randomly assigned to receive 260 mg of MB or placebo immediately after exposure therapy sessions [6]. One month later, participants who initially had low levels of fear showed significantly less fear if they received MB compared to placebo. MB also enhanced incidental contextual memory, suggesting better memory retention. However, those with higher levels of fear after training experienced less benefit or even deterioration, indicating that MB may be most effective when administered after successful exposure therapy.

Moreover, Alda et al (2017) conducted a six-month double-blind crossover study to investigate methylene blue (MB) as an additional treatment for residual symptoms in bipolar disorder [7]. Thirty-seven participants treated with lamotrigine received either a low dose (15 mg) or an active dose (195 mg) of MB. The study found that the active dose (195 mg) of MB significantly reduced depressive symptoms on both the Montgomery-Åsberg and Hamilton scales (P = 0.02 and P = 0.05). Anxiety symptoms also improved significantly (P = 0.02), while mania symptoms remained stable throughout. Although MB had no significant effect on cognitive symptoms, it was well tolerated with mild side effects. These findings suggest the potential of MB in alleviating depression and anxiety in bipolar disorder when used along with standard treatment

Domínguez-Rojas et al (2022) reported the use of MB as a life-saving therapy in a pediatric patient with refractory septic shock due to Listeria meningitis [8]. MB rapidly improved hemodynamics, enabling effective vasopressor withdrawal and normalization of lactate levels. Although the patient had neurological sequelae associated with meningitis, no adverse effects of MB were reported. This case underscores the potential of MB to treat severe vasoplegia when other therapies fail, although further research is needed.

In another case study, Gharaibeh et al (2019) examined a regimen to prevent ifosfamide-induced encephalopathy (IIE) in a cancer patient [9]. The regimen combined methylene blue (50 mg every 6 hours), thiamine and hydration before chemotherapy. MB successfully reduced neurological complications, allowing the patient to complete chemotherapy without significant encephalopathy. This case demonstrates the potential role of MB in preventing IIE and facilitating ongoing cancer treatment.

In a 2016 study by Gureev et al. researchers found that treating mice with methylene blue for 60 days reduced age-related decline in physical activity, exploration and anxiety behaviors [10]. The treatment also increased reactive oxygen species (ROS) in brain mitochondria, which activated the Nrf2/ARE signaling pathway. This activation improved mitochondrial biogenesis and function, and restored important mitochondrial genes such as NRF1, MTCOX1, TFAM and SOD2, increasing overall mitochondrial immunity. These results underscore the potential of methylene blue as a protective agent against age-related brain decline. In another animal study Riha et al (2005) evaluated the effects of different doses of MB on memory and brain oxygen consumption in rats [11]. A dose of 4 mg/kg was optimal, improving object recognition and habituation without behavioral side effects, while higher doses produced nonspecific effects. MB also increased brain oxygen consumption in a dose-dependent manner, correlating with increased memory retention. The results confirm that MB improves memory by affecting oxygen metabolism in the brain.

Moreover, Callaway et al (2004) studied the effects of methylene blue on mitochondrial activity and memory in rats [12]. A low dose of 1 mg/kg significantly increased cytochrome c oxidase activity 24 hours after injection and improved spatial memory retention. MB-treated rats showed 66% correct maze responses compared to 31% in the control group. These findings indicate MB's ability to improve cognitive function by increasing mitochondrial efficiency.

Moreover, Lin et al (2012) examined the effects of MB on mitochondrial function and brain metabolism in vitro and in animal models [13]. The results showed that MB increases mitochondrial oxygen consumption, glucose uptake and cerebral blood flow (CBF), especially in the hippocampus and motor cortex. Under low-oxygen conditions, MB was found to increase oxygen extraction (OEF) by 49% and reduce oxidative damage associated with ischemic stroke. These findings support MB as a brain metabolic enhancer with potential applications in neurodegenerative diseases and post-stroke recovery. In another study, Tucker et al (2018) reviewed the role of methylene blue in supporting mitochondrial function and neuroprotection. MB acts as a "redox cycler" in mitochondria, helping cells produce energy more efficiently, even when some mitochondrial pathways are impaired. It reduces oxidative stress and enhances antioxidant defenses [14]. Clinically, MB has been used to treat methemoglobinemia by restoring normal hemoglobin function, as seen in cases such as the "Blue Fugates" family.

In another study, Wrubel et al (2007) demonstrated the potential of MB to improve learning and memory through its metabolic benefits [15]. At a dose of 1 mg/kg, rats treated with MB learned to distinguish between baited and unbaited holes within three days, in contrast to saline-treated controls. The study also linked MB's cognitive effects to increased activity of cytochrome c oxidase, a key mitochondrial enzyme, which was 70% higher in MB-treated rats. These results suggest that MB promotes memory retention by increasing brain energy metabolism, making it a promising intervention for learning challenges

In addition, Haouzi et al (2020) evaluated MB as a treatment for hydrogen sulfide (H2S) poisoning, which causes severe brain and heart damage [16]. MB's redox properties help restore mitochondrial energy production, counteracting the effects of H2S, which blocks normal cellular processes. In animal studies, MB reduced neurological damage, improved motor skills and reduced mortality. MB's ability to restore oxygen utilization and reduce reactive oxygen species (ROS) positions it as a potential universal antidote to mitochondrial toxins such as H2S and cyanide. In addition, Zhang et al (2006) examined the neuroprotective effects of MB in a rotenone-induced optic nerve neuropathy model, simulating the mitochondrial dysfunction observed in diseases such as Leber's optic nerve neuropathy [17]. Rotenone caused significant retinal cell loss, but simultaneous MB treatment at different doses prevented this degeneration in a dose-dependent manner. MB was found to reverse oxidative stress and restore oxygen consumption disrupted by rotenone. These results suggest the potential of MB as a therapeutic agent in optic nerve neuropathy and other neurodegenerative conditions associated with mitochondrial dysfunction.

In a study, Singh et al (2023) examined the effects of methylene blue (MB) on brain metabolism in humans and rats, using imaging to measure blood flow and metabolic changes [18]. MB was administered intravenously at doses of 0.5 and 1 mg/kg in humans and 2 and 4 mg/kg in rats. Surprisingly, MB reduced global cerebral blood flow and oxygen metabolism in humans, as well as glucose metabolism in rats, with dose-dependent effects. These findings underscore a potential hormetic effect in which MB, at higher doses, may inhibit rather than stimulate metabolism. The study suggests that MB's metabolic effects may be more pronounced in conditions of impaired brain metabolism than in healthy subjects.

In addition, Rojas et al (2009) examined the effects of MB on neurotoxin-induced damage in rats [19]. When co-administered with rotenone (Rot), a neurotoxin that causes "metabolic strokes" in the striatum, MB significantly reduced lesion size and oxidative stress. MB also preserved cytochrome oxidase activity in motor-related brain areas and maintained connectivity in basal ganglia-thalamocortical circuits. Behaviorally, MB improved the motor asymmetry caused by Rot. These results confirm the neuroprotective role of MB by reducing oxidative stress, preserving energy metabolism and protecting neural networks.

In addition, Gonzalez-Lima and Bruchey (2004) found a significant role for MB in improving fear extinction memory in rats [20]. MB (4 mg/kg, intraperitoneally) was administered daily for five days after extinction training, resulting in significantly lower freezing responses to conditioned sounds compared to the control group. MB also increased brain metabolic activity in key prefrontal areas, such as the infralimbic cortex, correlating with better memory retention. This suggests that MB improves extinction memory by increasing brain energy metabolism and cytochrome oxidase activity.

Moreover, Bhurtel et al (2018) examined the effects of MB in Parkinson's disease (PD) models using MPTP and MPP+ neurotoxins [21]. Pretreatment with MB significantly reduced dopaminergic neuron loss, glial activation and dopamine deficiency. It also increased brain-derived neurotrophic factor (BDNF) levels and activated the Erk signaling pathway, both of which are important for neuronal survival and dopamine production. Blocking these pathways reversed the protective effects of MB, underscoring their importance in the neuroprotection mediated by MB.

In addition, Abdel-Salam et al (2014) evaluated the neuroprotective effects of methylene blue (MB) against rotenone-induced damage in rats [22], a model of Parkinson's disease. Rotenone (1.5 mg/kg, three times a week) caused significant oxidative stress, inflammation, apoptosis and loss of dopaminergic neurons. Concomitant administration of MB (5, 10 or 20 mg/kg daily) reduced oxidative stress markers such as malondialdehyde (MDA) and nitric oxide (NO), restored antioxidant levels such as glutathione, and increased protective enzymes (AChE and PON1). MB also reduced markers of inflammation (TNF-α) and apoptosis (caspase-3), while preserving dopaminergic neurons. These findings suggest that MB protects against oxidative damage, inflammation and neuronal loss in models of Parkinson's disease.

In another study by Abdel-Salam et al. (2016), rats exposed to malathion, a pesticide that causes significant oxidative stress and brain damage, were treated with MB (5 or 10 mg/kg) [23]. Malathion increased lipid peroxidation (MDA by 32.8%), nitric oxide levels (by 51.4%) and neuronal degeneration. It was found that simultaneous administration of MB significantly reduced oxidative stress, restored antioxidant levels (GSH increased by up to 67.7%) and improved enzyme activity (PON1 by 30.9%). Histopathology showed that MB minimized neuronal damage and glial cell activation. These results indicate the potential of MB in counteracting neurotoxicity caused by pesticide exposure

In 2016. Abdel-Salam et al. also examined the effects of MB on oxidative stress and brain damage caused by toluene, a neurotoxic solvent [24]. Exposure to toluene increased markers of oxidative damage, decreased glutathione (GSH) levels, and induced inflammation (elevated NF-κB). MB treatment decreased markers of oxidative stress (MDA, nitrites), reduced inflammation and restored levels of neurotrophic factor (BDNF). It also inhibited apoptotic pathways by decreasing caspase-3 activity and improved glial cell function (normalized GFAP levels). These results indicate that MB protects against chemical-induced neurotoxicity by reducing oxidative stress, inflammation and cell death

In another animal study, Wu et al (2024) showed that methylene blue (MB) effectively alleviated cognitive and neuronal impairment caused by repeated neonatal exposure to isoflurane (ISO) in rats [25]. Administered at a dose of 1 mg/kg intraperitoneally three times before each exposure to ISO, MB improved learning and memory in behavioral tests such as the Barnes maze. It also reduced neuronal damage, apoptosis, mitochondrial fragmentation and neuroinflammation, while maintaining the integrity of the blood-brain barrier. These findings support MB as a promising intervention to protect developing brains from anesthesia-induced damage. In addition, Goma et al (2021) investigated the protective role of MB against copper oxide nanoparticle (CuO-NP)-induced neurotoxicity in rats [26]. MB (1 mg/kg) preserved neurobehavioral functions, reduced oxidative damage and prevented mitochondrial dysfunction and neuronal apoptosis. It significantly counteracted the toxic effects of CuO-NPs, including elevated markers of oxidative stress and brain damage. These results suggest the antioxidant and mitochondrial protective potential of MB against environmental neurotoxins.

Methylene blue for mood disorders

Research suggests that methylene blue (MB) can help treat mood disorders such as depression and anxiety. Narsapur and Naylor (1983) were among the first to study MB in patients with manic-depressive psychosis who did not respond to standard treatment [27]. They found that 14 of 22 patients improved after taking oral MB (100 mg two or three times a day), and two patients saw short-term benefits from intravenous MB. Later, Naylor et al (1986) conducted a two-year study comparing a low dose of MB (15 mg/day) with a higher dose (300 mg/day) [27]. The higher dose significantly reduced depressive symptoms, but even the low dose reduced hospital admissions, indicating a benefit even at lower doses.

Another study by Naylor et al (1987) confirmed that MB at a dose of 15 mg/day helped alleviate severe depression in 35 patients [27]. Animal studies further support the antidepressant and anti-anxiety effects of MB. Eroglu and Caglayan (1997) found that MB improved symptoms in rats at doses of 7.5-30 mg/kg, but higher doses (60 mg/kg) were less effective, showing a U-shaped response curve [27].

Similarly, Kurt et al (2004) found that MB reversed sildenafil-induced anxiety in rats. Guimarães et al (1994) and de-Oliveira and Guimarães (1999) showed that injection of MB into specific brain areas reduced anxiety in a dose-dependent manner [27]. Research on MB analogs is also promising. Harvey et al (2010) showed that methylene green, a similar compound, has antidepressant-like effects like MB in animals [27]. Delport et al (2014) found that azure B (a metabolite of MB) and ethylthionine chloride (ETC) reduced depression-like behavior in rats without significant MAO-A inhibition, suggesting fewer side effects [27]. These studies also showed that MB appears to act through multiple mechanisms, including MAO-A inhibition, mitochondrial enhancement and modulation of the NO pathway.

Methylene blue supports mitochondrial function in brain/neurological disorders

Mitochondrial dysfunction is a key factor in many brain diseases, leading to inflammation, oxidative stress and cellular energy deficiencies [28]. Methylene blue (MB), an FDA-approved drug traditionally used for conditions such as methemoglobinemia and cyanide poisoning, has recently shown potential in addressing these mitochondrial problems in neurological conditions.

MB acts as a helper for the mitochondrial energy-producing parts of cells. Its action is to transfer electrons in the mitochondrial electron transport chain, especially in the case of blockages in complex I and complex III [28]. This action helps restore normal electron flow, allowing mitochondria to produce energy more efficiently. In this way, MB reduces the production of harmful molecules called reactive oxygen species (ROS), which are often responsible for cell damage and inflammation.

In diseases such as Alzheimer's disease, Parkinson's disease, stroke and traumatic brain injury (TBI), mitochondrial dysfunction and energy deficits are common.

Here's how MB can help in such conditions:

• Alzheimer's disease (AD): MB has been shown to reduce the levels of beta-amyloid proteins, which are associated with AD [28]. This prevents interference of these proteins with mitochondrial enzymes and helps preserve mitochondrial function. MB also inhibits the clumping of tau proteins, another hallmark of AD, and has been observed to improve memory and cognitive function in both animal studies and human clinical trials.
• Traumatic Brain Injury (TBI): After TBI, MB can reduce brain swelling, protect the blood-brain barrier and reduce cell death in the brain [28]. Studies have shown that low doses of MB administered soon after injury can significantly improve neuronal survival and promote regeneration by improving mitochondrial function and energy production.
• Stroke: In models of ischemic stroke, MB improves the activity of key mitochondrial complexes, increases glucose uptake and enhances oxygen consumption [28]. These effects help restore energy balance in brain cells and reduce the area of stroke damage.
• Parkinson's disease: MB has shown protective effects on dopamine-producing neurons affected by Parkinson's disease [28]. By reducing oxidative stress and promoting mitochondrial health, MB helps preserve neuronal function in models where mitochondrial toxins are present.

MB's potential to increase mitochondrial efficiency, reduce oxidative stress and improve cellular energy production makes it a promising option for treating various brain disorders associated with mitochondrial problems. Its ability to cross the blood-brain barrier and target neuronal mitochondria increases its therapeutic potential.

Methylene blue in Alzheimer's disease (human and animal studies)

Methylene blue actively fights tau aggregation, protects mitochondria and improves cognitive function, making it a potential candidate for treating Alzheimer's disease.

Preclinical and clinical studies demonstrate its ability to slow disease progression, especially when combined with advanced delivery methods or optimized dosing. In a study by Liu et al (2024), they developed an optimized approach using methylene blue (MB) in combination with black phosphorus (BP) to combat Alzheimer's disease (AD) [29]. MB, a tau aggregation inhibitor, was delivered intranasally using a BP-based hydrogel formulation. This method bypassed the blood-brain barrier (BBB), ensuring sustained release and direct delivery to the brain. In mouse models, this strategy inhibited tau aggregation, restored mitochondrial function, reduced nervous system inflammation and improved cognitive performance. These findings suggest the potential of MB in the fight against Alzheimer's disease, especially when combined with advanced drug delivery systems.

Moreover, Zakaria et al (2016) evaluated the ability of MB to protect mitochondria from beta-amyloid (Aβ) toxicity, a key factor in AD progression [30]. Specifically, MB reduced Aβ levels and its binding to amyloid-binding alcohol dehydrogenase (ABAD), preserving mitochondrial function. In addition, MB improved cell survival, reduced oxidative stress and restored levels of estradiol, a hormone essential for brain health. These effects underscore MB's role in protecting neurons and slowing the progression of AD.

During a clinical trial, Wilcock et al (2018) investigated leuco-methylthionine (LMTM), a form of MB, as a stand-alone therapy for mild AD in a Phase III study [31]. Patients receiving LMTM (100 mg or 4 mg twice daily) showed significant improvements in cognitive and functional outcomes, reduced brain atrophy and increased glucose uptake. Interestingly, low doses (4 mg) were as effective as higher doses, making LMTM a promising and safer therapeutic option for AD.

In addition, Wischik et al (2015) conducted a study on 321 patients with mild to moderate AD to evaluate the optimal dosage of methylthionine (MT, the active ingredient in MB) [32]. They identified an optimal daily dose of 138 mg of MB, as this dose significantly improved cognitive performance and cerebral blood flow, maintaining the benefits for 50 weeks. In contrast, higher doses (228 mg/day) were less effective due to absorption problems, underscoring the importance of dose optimization in MB-based therapies. MT has been shown to inhibit tau protein aggregation and reduce tau pathology in preclinical models. By targeting this hallmark of AD, MT not only slows cognitive decline, but also protects against neurodegeneration. Clinical studies support its role as an inhibitor of tau aggregation, highlighting its potential to modify AD progression.

Moreover, MB shifts between its reduced form, leukomethylthioneine (LMT), and its oxidized form, stabilizing as methylthioneine chloride (MTC). In clinical trials, particularly a phase 2 study, MTC proved effective at a dose of 138 mg/day. It improved cognitive function and brain imaging results in patients with mild to moderate AD. However, a higher dose of 228 mg/day did not show the same efficacy, which was attributed to problems with dissolution and absorption of the drug. To improve the drug's delivery, researchers developed a new formulation, LMTX, which provides stable delivery of LMT and has shown more consistent results in both preclinical and clinical studies. This was noted in a study by Baddeley et al. (2015), who noted the important role of timely release of MT in the stomach for its efficacy [33].

Further research has confirmed MB's potential to treat not only psychiatric conditions, but also broader neurodegenerative diseases such as Alzheimer's disease. MB can improve brain health by strengthening the blood-brain barrier, reducing inflammation and promoting mitochondrial function. Clinical trials, such as one noted by Alda (2019), have shown mixed results; however, specific doses, such as 138 mg, which proved beneficial in one study, continued to show positive effects on cognitive function up to 50 weeks later [34].

Moreover, a review by Atamna and Kumar (2010) evaluated potential mechanisms of action of MB in AD [35]. Such as its ability to improve mitochondrial health and protect against amyloid-β toxicity - central issues in AD. MB facilitates mitochondrial function and reduces oxidative stress. In addition, combining MB with osmolytes such as carnosine may provide a dual approach to combating AD by stabilizing proteins and preventing harmful amyloid-β aggregation.

In another important finding, Medina et al (2011) conducted a study on 3xTg-AD mice [36]. They found that MB not only reduced amyloid-β levels, but also improved memory and learning ability. This was attributed to MB's ability to stimulate proteasome activity, helping to remove harmful proteins and offering a potential therapeutic pathway for treating AD.

Also, Auchter et al (2014) evaluated the potential of MB in improving cognitive function impaired by reduced blood flow to the brain, a risk factor for AD [37]. In their study, rats subjected to carotid artery occlusion to simulate reduced cerebral blood flow were administered a low daily dose of 4 mg/kg MB. The treatment significantly improved memory and learning in these rats. These results demonstrate the potential of MB to improve brain energy utilization and support cognitive function under difficult conditions. In addition, Paban et al (2014) conducted a study on a transgenic mouse model of AD [38]. They investigated whether MB could prevent or treat cognitive impairment by affecting beta-amyloid deposition. Their results showed that MB, whether delivered in drinking water or by injection, significantly improved cognitive function and reduced amyloid deposits in the brain. These findings suggest the dual utility of MB in both the preventive and therapeutic contexts of AD.

Moreover, Stelmashook et al (2023) evaluated the effects of MB in an experimental model of sporadic AD induced by streptozotocin administration [39]. Their results showed that MB treatment alleviated memory impairment, reduced nervous system inflammation and moderated autophagy markers in rats. These results confirm the neuroprotective and anti-inflammatory properties of MB against Alzheimer's disease. In another animal study, Zhou et al (2019) examined the effects of MB on caspase-6-related cognitive decline in a mouse model of AD [40]. Their study showed that MB effectively inhibited caspase-6 activity in neurons and significantly improved memory and synaptic function. The results indicate the potential of MB to reverse AD-related cognitive deficits.

Methylene blue (MB) in the treatment of traumatic brain injury (TBI)

Methylene blue shows great potential as a neuroprotective agent in traumatic brain injury. It reduces inflammation, enhances mitochondrial function, protects the blood-brain barrier and improves regeneration. Traumatic brain injury (TBI) often disrupts limbic function, increases inflammatory markers and damages the blood-brain barrier (BBB). A study examining the effects of MB administered intravenously (1 mg/kg) 30 minutes after TBI showed that it significantly improved limbic function, reduced inflammation (as seen by lower levels of S100 protein) and restored BBB integrity [41].

In addition, laboratory experiments have confirmed MB's ability to protect neurons from inflammatory toxins such as lipopolysaccharides. These findings suggest that MB reduces inflammation and protects the BBB, making it a promising treatment for TBI. Moreover, in a mouse model, MB administered 15-30 minutes after injury reduced brain swelling and inflammatory markers, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), while increasing anti-inflammatory markers such as IL-10 [42]. Behaviorally, MB improved recovery and reduced depressive symptoms within a week of injury. Although MB did not prevent weight loss or motor function, its anti-inflammatory and mood-stabilizing effects show therapeutic potential in the treatment of TBI.

In another study using a rat model of mild TBI, rats treated with MB showed smaller lesion volumes on MRI scans compared to the control group [43]. Behavioral tests showed better recovery of motor function, with improvements in forelimb function and coordination within two weeks. In addition, histological results confirmed fewer degenerating neurons in MB-treated animals. These results underscore the effectiveness of MB in reducing brain damage and improving recovery from mild TBI. A study by Shen et al. showed that MB restores mitochondrial membrane potential, increases ATP production and reduces neuronal apoptosis [44]. MB enhanced the BBB and improved cognitive and motor recovery after TBI. These findings support MB as a potential treatment for mitochondrial dysfunction and cell death caused by brain injury.

Moreover, Zhao et al. confirmed in an animal study that MB reduces brain swelling and promotes autophagy, a process that removes damaged cells [45]. It also lowered microglia activation, which can exacerbate inflammation. Neurological deficits and lesion volume were significantly reduced in animals treated with MB in both the acute and chronic phases of injury, indicating its long-term protective effects. Moreover, TBI can lead to long-term brain damage and neurodegeneration, similar to Alzheimer's disease [46]. Common mechanisms include oxidative stress, chronic inflammation and mitochondrial dysfunction. Specifically, MB addresses these issues by reducing oxidative damage, controlling autophagy and improving mitochondrial function. Its protective effects make it a promising therapy not only for TBI, but also for other neurodegenerative diseases.

Neuropsychiatric benefits of methylene blue (MB)

Methylene blue (MB) has a long history in psychiatry, first studied in the early 20th century for mood disorders, and later reconsidered in the 1970s as an alternative to lithium in bipolar disorder. Modern studies have confirmed its antidepressant and anti-anxiety effects both in animal studies and in patients with mood disorders, particularly bipolar disorder [23].

It's worth noting that early clinical studies have shown that even low doses of MB can stabilize mood without inducing mania, a common side effect of traditional antidepressants. For example, a two-year study using a 15 mg daily dose significantly reduced depressive symptoms and hospitalizations for bipolar disorder [23].

In addition to mood stabilization, MB has potential benefits in other psychiatric conditions. In schizophrenia, MB may act by reducing nitric oxide (NO), which has been linked to psychotic symptoms [23]. Although human studies are limited, animal studies have shown that MB can counteract the effects of drugs that cause psychosis-like symptoms. MB has also been tested as a cognitive enhancer in treatments for fear-based disorders such as claustrophobia and post-traumatic stress disorder (PTSD), showing sustained reductions in fear [23].

The neuroprotective role of MB extends beyond psychiatry. A study in rats exposed to malathion, a pesticide that causes oxidative stress and brain damage, found that MB significantly reduced oxidative damage and brain inflammation [23]. Rats treated with MB experienced lower levels of lipid peroxidation and nitric oxide and had better activity of protective enzymes such as PON1 and AChE. Higher doses of MB further minimized neuronal damage in memory-related brain areas such as the cerebral cortex and hippocampus [23]. These findings suggest that MB is a neuroprotective and therapeutic agent in many psychiatric and neurological conditions. By reducing oxidative stress, inflammation and symptoms associated with psychosis, MB offers benefits for mental health and cognitive function.

How does methylene blue (MB) support brain health?

Methylene blue (MB) plays many roles in supporting brain health. It acts on various pathways that help treat brain and mood disorders [47-49]. These include;

• A shot of energy for brain cells: MB acts as a redox agent, switching between oxidized and reduced forms to bypass blockages in the mitochondrial electron transport chain, especially in complex I and complex III. By restoring electron flow, MB increases the production of ATP, the main source of energy for brain cells. This is particularly beneficial in conditions of low oxygen levels (hypoxia), such as stroke or neurodegenerative diseases, in which brain cells have difficulty producing enough energy.
• Focusing on brain cells: MB has the unique ability to cross the blood-brain barrier and accumulate in brain tissues. This selective targeting ensures that its action is concentrated in the nervous system. This property makes MB effective in treating conditions specifically associated with brain cell dysfunction, such as Alzheimer's disease and brain injury.
• Improves mood: MB inhibits monoamine oxidase (MAO), an enzyme that breaks down neurotransmitters such as serotonin, norepinephrine and dopamine. By preventing the breakdown of these mood-regulating chemicals, MB increases their levels, helping to stabilize mood and reduce symptoms of depression and anxiety.
• Protects against oxidative stress: MB reduces the production of reactive oxygen species (ROS) by acting as a mitochondrial electron carrier. ROS are harmful molecules that cause oxidative damage to cells. MB also reduces levels of nitric oxide (NO), which contributes to oxidative stress and inflammation in large amounts. By regulating NO levels, MB protects neurons from damage and maintains healthy brain function.
• Regulates brain cell signals: MB inhibits guanylyl cyclase, an enzyme involved in the generation of cyclic GMP (cGMP), a signaling molecule in brain cells. Overactive cGMP signaling can lead to harmful neuronal overactivity. MB helps modulate this activity, preventing damage and promoting normal communication in the brain.
• Prevents the formation of Tau protein clumps: In Alzheimer's disease, tau proteins fold and aggregate, disrupting cellular function. MB directly inhibits tau aggregation, slowing the progression of neurodegeneration. This mechanism helps protect brain cells from structural and functional damage associated with Alzheimer's disease.
• Supports neurotransmitters: MB increases the release of neurotransmitters such as serotonin, norepinephrine and dopamine, which are essential for mood regulation, focus and overall cognitive function. By maintaining these chemicals, MB promotes emotional well-being and mental clarity.
• Lowers amyloid beta levels: Amyloid-beta is a toxic protein that accumulates in Alzheimer's disease, leading to neuronal damage and memory loss. MB reduces the production of amyloid-beta and prevents its interaction with mitochondrial enzymes such as amyloid-binding alcohol dehydrogenase (ABAD). This preserves mitochondrial function and prevents cell death.
• Improves memory and learning: MB increases the activity of acetylcholine, a neurotransmitter essential for learning and memory. This enhancement supports cognitive processes and can help alleviate memory deficits in conditions such as Alzheimer's disease and traumatic brain injury.

These combined effects make MB a potential treatment for a range of brain conditions, including mood disorders, memory problems and even neurodegenerative diseases such as Alzheimer's. Because it was one of the first drugs used to treat the brain, MB has a long history, but new research is finding even more uses for it.

Methylene blue in ischemia reperfusion

Studies have shown that methylene blue helps and alleviates symptoms or complications associated with ischemia. In a study by Lu et al (2016), they showed that methylene blue reduces hippocampal cell death and improves memory deficits after global cerebral ischemia (GCI) in rats [50]. MB, administered at a dose of 0.5 mg/kg/day by subcutaneous minipump for seven days, significantly increased neuronal survival in the CA1 region of the hippocampus and preserved mitochondrial function, including cytochrome c oxidase activity and ATP production. Behavioral improvements in spatial learning and memory tests were also noted, indicating MB's ability to reduce cell death and promote cognitive recovery from ischemia.

In addition, Shi et al (2021) investigated how MB reduces cerebral edema caused by ischemic stroke [51]. Intravenously administered MB reduced both cytotoxic and vasogenic edema in rats, as demonstrated by MRI scans. Mechanistically, MB inhibited aquaporin 4 (AQP4) expression and reduced activation of the ERK1/2 pathway in astrocytes, which are essential for brain water balance. These findings, confirmed in cell culture models, suggest that MB reduces brain edema by modulating AQP4 and ERK1/2 and helps treat brain edema after stroke.

In another study, Huang et al (2018) evaluated the effects of chronic oral MB treatment (at a low dose) in a rat model of focal ischemia. The results showed significant behavioral and structural improvements, including reduced lesion volume and white matter damage [52].

Also, Miclescu et al (2010) studied the role of MB in protecting the blood-brain barrier (BBB) during ischemia/reperfusion induced cardiac arrest in a porcine model [53]. Infusion of MB during resuscitation reduced albumin leakage, brain water content and neuronal damage. It also reduced nitric oxide-induced damage and increased endothelial nitric oxide synthase activation. These results indicate the potential of MB to preserve BBB integrity and prevent brain damage in ischemia/reperfusion scenarios.

In addition, Zhang et al (2020) demonstrated the neuroprotective potential of MB in a neonatal rat model of hypoxic-ischemic (HI) brain injury [54]. MB preserved mitochondrial function, reduced oxidative stress and neuroinflammation, and improved blood-brain barrier integrity. In addition, behavioral tests confirmed improved motor coordination and memory in treated rats. These findings suggest that MB is a promising therapy for HI neonatal encephalopathy.

During laboratory studies, Ryou et al (2015) revealed a role for MB in increasing energy metabolism and hypoxia-inducible factor-1α (HIF-1α) activation during oxygen-glucose deprivation (OGD) and reoxygenation in neuronal cells [55]. MB improved glucose uptake, ATP production and mitochondrial enzyme activity. It also increased the nuclear translocation of hypoxia-inducible factor-1α (HIF-1α).

Dosage, pharmacokinetics and contraindications of methylene blue

Methylene blue (MB) is often taken orally in doses ranging from 15 to 300 mg per day, with peak blood concentrations usually reached 1 to 2 hours after ingestion [34]. Intravenous (IV) MB is absorbed more efficiently, making it potentially better for brain-related effects, although the best dose for psychiatric use is still uncertain. Interestingly, higher oral doses do not always lead to predictably higher blood levels.

The body removes MB mainly through the kidneys, often as leucomethylene blue, along with two related compounds, azure A and azure B. Azure B has even shown mood-enhancing effects in animal studies. The half-life of MB is about 5 to 6.5 hours [34].

The effects of MB vary depending on the dose. Low doses often improve mood and have a calming effect, while higher doses can have the opposite effect, potentially increasing oxidative stress in animal studies [34].

MB is usually well tolerated in humans, but mild side effects can occur, such as stomach discomfort, urinary problems or bluish colored urine, which some people find unpleasant [34].

There are important safety considerations regarding the use of MB. The FDA warns that combining MB, especially in intravenous form, with certain antidepressants that affect serotonin can cause serotonin syndrome, a serious reaction. However, no such cases have been reported with oral MB use [34].

In addition, people with glucose-6-phosphate dehydrogenase (G6PD) enzyme deficiency should avoid MB because it can cause hemolytic anemia, a condition in which red blood cells break down prematurely. This deficiency is more common in Mediterranean, African and Asian populations [34].

Disclaimer

This article was written for educational purposes and is intended to raise awareness of the substance being discussed. It is important to note that the substance discussed is a substance, not a specific product. The information contained in the text is based on available scientific research and is not intended to serve as medical advice or promote self-medication. The reader should consult any health and treatment decisions with a qualified health professional.

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