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The possibilities of using the effects of ozone therapy in neurology

Authors:

Abstract

Objectives: The beneficial effects of ozone therapy consist mainly of the promotion of blood circulation: peripheral and central ischemia, immunomodulatory effect, energy boost, regenerative and reparative properties, and correction of chronic oxidative stress. Ozone therapy increases interest in new neuroprotective strategies that may represent therapeutic targets for minimizing the effects of oxidative stress. Methods: The overview examines the latest literature in neurological pathologies treated with ozone therapy as well as our own experience with ozone therapy. The effectiveness of treatments is connected to the ability of ozone therapy to reactivate the antioxidant system to address oxidative stress for chronic neurodegenerative diseases, strokes, and other pathologies. Application options include large and small autohemotherapy, intramuscular application, intra-articular, intradiscal, paravertebral and epidural, non-invasive rectal, transdermal, mucosal, or ozonated oils and ointments. The combination of different types of ozone therapy stimulates the benefits of the effects of ozone. Results: Clinical studies on O2-O3 therapy have been shown to be efficient in the treatment of neurological degenerative disorders, multiple sclerosis, cardiovascular, peripheral vascular, orthopedic, gastrointestinal and genitourinary pathologies, fibromyalgia, skin diseases/wound healing, diabetes/ulcers, infectious diseases, and lung diseases, including the pandemic disease caused by the COVID-19 coronavirus. Conclusion: Ozone therapy is a relatively fast administration of ozone gas. When the correct dose is administered, no side effects occur. Further clinical and experimental studies will be needed to determine the optimal administration schedule and to evaluate the combination of ozone therapy with other therapies to increase the effectiveness of treatment.
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REVIEW ARTICLE
THE POSSIBILITIES OF USING THE EFFECTS OF OZONE THERAPY IN
NEUROLOGY
Ján MASAN1,2,3, Miron SRAMKA2 , Daria RABAROVA3
1 University of St. Cyril and Methodius in Trnava, Slovak Republic.
2 St. Elizabeth University of Health Care and Social Work in Bratislava, Slovak Republic.
3 Trnava University in Trnava, University Hospital Trnava, Slovak Republic.
Correspondence to: h.Doc. MUDr. Ján Mašán, PhD.
St. Elizabeth University of Health Care and Social Work in Bratislava, Slovak Republic
E-MAIL : masanjan@gmail.com
Keywords: Ozone therapy. Major ozone therapy. Neurodegenerative disorders. Antioxidant
system. Oxidative stress biomarkers. Multiple sclerosis. Stroke. Neuropathy. Phantom limb
pain. Polyneuropathy.
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Abstract
OBJECTIVES: The beneficial effects of ozone therapy consist mainly of the promotion of
blood circulation: peripheral and central ischemia, immunomodulatory effect, energy boost,
regenerative and reparative properties, and correction of chronic oxidative stress. Ozone
therapy increases interest in new neuroprotective strategies that may represent therapeutic
targets for minimizing the effects of oxidative stress.
METHODS: The overview examines the latest literature in neurological pathologies treated
with ozone therapy as well as our own experience with ozone therapy. The effectiveness of
treatments is connected to the ability of ozone therapy to reactivate the antioxidant system to
address oxidative stress for chronic neurodegenerative diseases, strokes, and other
pathologies. Application options include large and small autohemotherapy, intramuscular
application, intra-articular, intradiscal, paravertebral and epidural, non-invasive rectal,
transdermal, mucosal, or ozonated oils and ointments. The combination of different types of
ozone therapy stimulates the benefits of the effects of ozone.
2
RESULTS: Clinical studies on O2-O3 therapy have been shown to be efficient in the
treatment of neurological degenerative disorders, multiple sclerosis, cardiovascular, peripheral
vascular, orthopedic, gastrointestinal and genitourinary pathologies, fibromyalgia, skin
diseases/wound healing, diabetes/ulcers, infectious diseases, and lung diseases, including the
pandemic disease caused by the COVID-19 coronavirus.
CONCLUSION: Ozone therapy is a relatively fast administration of ozone gas. When the
correct dose is administered, no side effects occur. Further clinical and experimental studies
will be needed to determine the optimal administration schedule and to evaluate the
combination of ozone therapy with other therapies to increase the effectiveness of treatment.
Abbreviations: O3 ozone, GSH glutathione, NADPH nicotinamide adenine
dinucleotide, (SOD) superoxide dismutase, PUFA unsaturated fatty acids, LOP lipid
ozonation products, ARE antioxidant response elements, ATP adenosine triphosphate,
ROS reactive oxygen species, DNA deoxyribonucleic acid, NADH nicotinamide
adenine dinucleotide
INTRODUCTION
Van Mauren was first to discover the distinctive odor of O3 in 1785 (Altman 2007).
However, its first identification as a distinct chemical compound was made by Schönbein in
Basel in 1840. In 1896, Nikola Tesla patented a generator for producing ozone. The use of
ozone became normal practice after the studies of Dr. H.H. Wolff. In 1915, during World War
I, ozone was used to treat war wounds. Following Bocci’s studies, ozone therapy has been
incorporated into the treatment of chronic inflammatory diseases in the orthopedic field.
When used in appropriate doses, it has been shown to be effective in inducing well-tolerated
oxidative stress. Ozone is a metastable substance and must be generated on-site.
Contraindication of O3 therapy is lung inhalation, activating factors triggering an
inflammatory response (Bocci 2006; Pryor et al. 2019). Jacobs (1982) carefully examined all
the possible negative effects of ozone therapy. Despite the famous “toxicity” of ozone, it
appears that the incidence is only 0.0007%, one of the lowest in medicine. Four deaths due to
direct IV injection of the gas were included in his data but, since 1982, other deaths due to
malpractice have occurred, of which at least three were in Italy (Bocci 2010).
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It is currently applied in a wide range of medical fields: surgery, orthopedics,
neurology, oncology, dermatology, cardiology, psychiatry, radiology, rheumatology,
gynecology, gastroenterology, angiology, dentistry, etc.
The effects of medical ozone as a substance can be attributed to those defined by
hormesis (from the Greek word hormáein = to set in motion), i.e. referring to the hypothesis
regarding the beneficial effects of low doses. At the same time, it triggers a response with
high doses, thus increasing the body’s resistance.
Induction of the response to stress by short low doses usually protects the body for a
longer period of time and against other possible types of doses (Rattan et al. 2009). The
administration of O3 therapy varies based on treatment goals and treatment focus. Ozone
therapy combines a mixture of oxygen (O2)-O3 with a diverse therapeutic range (1080
μg/ml of gas per ml of blood) (Bocci 2006). Human blood contains a large number of
antioxidants such as uric acid, ascorbic acid, cysteine, glutathione, albumin, certain chelating
proteins such as albumin, and enzymes such as catalase, the redox system of GSH, NADPH
(nicotinamide adenine dinucleotide), and superoxide dismutase (SOD) (Bocci et al., 2005).
Like any other gas, ozone dissolves upon contact with body fluids according to
Henry’s Law in relation to temperature, pressure, and ozone concentration. It reacts
immediately as soon as it is dissolved: O3 + biomolecules O2 + O2 + energy. Atomic
oxygen behaves as a reactive atom. The fundamental ROS (reactive oxygen species) molecule
is hydrogen peroxide H2O2, which is a non-radical oxidant able to act as an ozone Messenger
responsible for eliciting several biological and therapeutic effects. In physiological amounts,
they act as regulators of signal transduction and represent important mediators of host defense
and immune responses (Dattilo et al. 2015).
The reaction of O3 with water causes the formation of one mole of hydrogen peroxide
(H2O2) and two moles of lipid oxidation products with polyunsaturated fatty acids (PUFA),
forming a mixture of lipid ozonation products (LOP) (Barone et al. 2015) including
lipoperoxyl radicals (Inal et al. 2011). Moderate oxidative stress caused by O3 increases the
activation of the transcription factor of the pathway-mediated nuclear factor-related erythroid
factor 2 (Nrf2) (Bocci et al. 2015, Re et al. 2014), which is responsible for activating the
transcription of antioxidant response elements (ARE). After their induction, the concentration
of antioxidant enzymes increases in response to the transient oxidative stress O3 (Bocci et al.
2015).
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POTENTIAL APPLICATION OF OZONE THERAPY
Vascular and Haematological Modulation The production of antioxidant enzymes
affects the whole body (Gonzalez et al. 2004; Valacchi 2000). O3 is a stimulator of O2
transmembrane flow. Increased intracellular O2 levels secondarily form the mitochondrial
respiratory chain (Madej et al. 2007). In red blood cells, O3 increases phosphofructokinase
activity, thereby increasing the rate of glycolysis. Increased glycolytic rate increases ATP and
2,3-diphosphoglycerate (2,3-DPG) in the cell. Following a long treatment cycle of the elderly,
Bocci et al. 2011 noted a significant increase in the 2,3-diphosphoglycerate level in
oxyhaemoglobin. As a result of the Bohr effect, its dissociation curve shifts to the right,
making it easier to transfer oxygen. Under physiological conditions, the endothelium regulates
vascular tone (Molinari et al. 2017). Ozonised blood increases the release of prostacyclin
(PGI2) and angiopoietins, both important factors in improving ischemic vasculopathy
(Fernández et al. 2008; Elvis et al. 2011).
The correction of chronic oxidative stress via the increase of antioxidant enzymes can
increase erythroblast differentiation. This leads to a progressive increase in erythrocytes and
preconditions them to having resilience towards oxidative stress, to an increase of
erythrocytes with improved metabolic properties, as well as ensuring that young erythrocytes
contain more G6PDH than older cells generated prior to the treatment, which is a type of
“super-gifted erythrocytes” capable of correcting hypoxia in vascular diseases (Chang et al.
2005). An improvement in blood circulation and oxygen supply to ischemic tissues has been
recorded, leading to an increased supply of O2 to hypoxic tissues (Brigelius-Flohé et al. 2011).
Activation of the Immune System. O3 has been shown to react with antioxidants and
alter peroxidation compounds. H2O2 has been shown to act as a regulatory step in signal
transduction by diffusing into immune cells and by facilitating a myriad of immune responses
(Gulmen et al. 2013; Caliskan et al. 2011). An increase in interferon, tumor necrosis factor,
and interleukin (IL)-2 was observed. IL-2 increases were initiated by immune response
mechanisms (Elvis 2011). In addition, H2O2 activates nuclear factor-kappa B (NF-κB) and
transforms growth factor-beta (TGF-β), thus increasing the immunoactive release of cytokines
and tissue refurbishment.
After each restarted therapy, a small percentage of immune cells are activated and
these cells release cytokines into the micro-environment, activating neighboring cells and, as
a result, slowly enhancing immune responses. Only submicromolar concentrations can reach
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all organs, particularly bone marrow, liver, central nervous system, endocrine glands, etc.,
where they act as signaling molecules of an ongoing acute oxidative stress (Mancuso et al.
1997). These molecules can elicit the upregulation of antioxidant enzymes. The induction of
HO-1 has been described as one of the most important antioxidant defense and protection
enzymes. Throughout the treatments, LOP acts as an acute oxidative stressor in the bone
marrow micro-environments and thus activates the release of metalloproteinases, of which
particularly MP-9 may favor the detachment of staminal cells. These cells, once in the blood
circulation, may be attracted to sites where a previous injury has taken place (Mancuso et al.
2008).
Lahodny (2021) collected ten blood samples with 70 μg/ml of ozone and reinfusion from a
patient as part of a session to implement the therapeutic concept. This therapy generated a
significant activation of stem cells, and subsequent rapid healing of wounds and inflammation
were documented in patients (Rowen 2018). Thanks to this therapy, a several-year defect was
cured in a mere 14 days (Mašán 2018).
Ozone therapy has a neuroimmunomodulatory effect, it activates the psychosomatic
system, thus allowing the release of the growth hormone ACTH-cortisol, neurotonic
hormones, and neurotransmitters. We clarified why patients report a feeling of euphoria and
wellness during therapy: the disappearance of asthenia and depression, a reduction of
pessimism syndrome, associated with a lack of side effects, represent positive results
(Fernández et al. 2008; Mancuso 2017).
CLINICAL APPLICATION OF OZONE THERAPY IN NEUROLOGY
The first beneficial effects of ozone in the treatment of neurological disorders refer to the
treatment of headaches and facial pain associated with pathological changes in the optic
thalamus. Ozone is used to treat allodynia, neuropathic pain, and hyperalgesia (Kal et al.
2017; Hu et al. 2018).
Neurodegenerative diseases Parkinson’s disease, Alzheimer’s and Wilson’s disease, senile
and vascular dementia, amyotrophic lateral sclerosis, optic nerve dysfunction, bilateral
sensorineural hearing loss, and maculopathy, Huntington’s disease, cognitive and movement
disorders of the elderly who experience common effects of oxidative stress (Aso et al. 2012).
The process of aging is characterized by the loss of homeostasis, leading to pathologic
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formation of reactive oxygen species (ROS), mitochondrial dysfunction, and metabolic
unbalance (Dugger et al. 2017). These pathophenotypes determine abnormal aggregation of
specific proteins (Yanar et al. 2020), given the connection between excessive ROS
accumulation and impairment in the proteostasis network. A natural bioactive molecule with
an antioxidant property such as ozone (O3) can be indicated as a potential new strategy to
delay neurodegeneration. This hypothesis is based on the evidence regarding the interaction
between O3 and Nrf2 (Galie et al. 2018; Siniscalco et al. 2018; Re et al. 2014; Vaillant et al.
2013). Molecular mechanisms related to antioxidant/anti-apoptotic/pro-autophagy processes
targeted by O3 administration via an Nrf2 biological pathway.
It is best to implement ozone therapy (O2-O3) in an early phase before the potential
development of a neurodegenerative pathology (Scassellati et al. 2020). The O2 availability
affects the expression of different hypoxia-inducible factors (HIFs) and plays the role of a
cellular adapter to hypoxia (Curro et al. 2018; Zhang et al. 2014; Re et al. 2014), leading to
the activation of trophic proteins and, consequently, to specific biological processes, including
erythropoiesis and angiogenesis (Zhou et al. 2019).
Antioxidant Property of Ozone (O3): Oxidative stress is a condition where ROS production
exceeds the cellular antioxidant defense system, leading to an imbalance between the two
systems, and this may contribute to neuronal damage. It has implications on the pathogenesis
and progression of neurodegenerative diseases (Singh et al. 2019).
Oxidative damage may impair the cells in their structure and function as a result of the
reduced activity of mitochondria. The damage is not confined to the brain but is also evident
in peripheral cells and tissues, which require a high-energy source such as the heart, muscles,
brain, or liver. To function properly, neurons rely on the mitochondria, which produce the
energy required for most of the cellular processes, such as neurotransmitter synthesis,
including synaptic plasticity.
Mitochondrial dysfunctions cause an increase in ROS for lowered oxidative capacity and
antioxidant defense, resulting in increased oxidative damage to protein and lipids, decreased
ATP production, and accumulation of DNA damage. Moreover, mitochondrial bioenergetic
dysfunction and the release of pro-apoptotic mitochondrial proteins into the cytoplasm initiate
a variety of cell death pathways (Garcia-Escudero et al. 2013; Reutzel et al. 2020).
Ozone therapy stimulates the Krebs cycle by enhancing the oxidative carboxylation of
pyruvate and stimulating the production of adenosine triphosphate (ATP) (Guven et al. 2008).
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It causes a significant reduction of nicotinamide adenine dinucleotide (NADH), an increase of
the coenzyme A levels to fuel the Krebs cycle, and oxidizes cytochrome C (Brigelius-Flohe et
al. 2011; Elvis et al. 2011).
Ozone(O3)-influenced pro-oxidative and antioxidant defense biomarkers and their role in
aging processes and neurodegenerative disorders (ND). Twenty-nine biomarkers implicated in
oxidative stress, in endogenous antioxidant, and in vitagene systems have been identified.
These biomarkers have been studied and found modulated after O3 therapy performed in in
vivo (human and animal models) samples. Neurodegenerative disorders are characterized by
progressive loss of cognitive and behavioral deterioration (Scassellati et al. 2020).
Molecular Mechanisms Involving Ozone Therapy and Their Biological Relevance in
Neuroprotection of Nrf2, and the Vitagene Network. Oxidative stress is a condition where
ROS and nitrogen (RNS) production exceeds the cellular antioxidant defense system, leading
to an imbalance between the two systems and this may contribute to neuronal damage and
abnormal neurotransmission. ROS and RNS are also major factors in cellular senescence that
leads to an increase in the number of senescent cells in tissues on a large scale (Liguori et al.
2018). Cellular senescence is a physiological mechanism that stops cellular proliferation in
response to damage that occurs during replication. Senescent cells acquire an irreversible
senescence-associated secretory phenotype (SASP), involving secretion of soluble factors
(interleukins, chemokines, and growth factors), degradative enzymes such as matrix
metalloproteases (MMPs), and insoluble proteins/extracellular matrix (ECM) components.
When O3 is administrated, it dissolves immediately in the plasma/serum and it reacts with
PUFA (polyunsaturated fatty acids), leading to the formation of the two fundamental
messengers: hydrogen peroxide (H2O2) as a ROS and 4-hydroxynonenal (4HNE) as a lipid
oxidation product (LOP) (Bocci et al. 1998).
LOPs diffuse into all cells and inform them of minimal oxidative stress. After the
oxidative/electrophilic stress challenge (Ishii et al. 2004), other aldehydes (Levonen et al.
2004) induced by O3 (Galie et al. 2018, Siniscalco et al. 2018, Re et al. 2014, Vaillant et al.
2013) inhibit the Nrf2 conjugation, provoking the nuclear accumulation of Nrf2. This leads to
the decreased expression of pro-inflammatory cytokines.
Another mechanism involves casein kinase 2 (CK2), another regulator of Nrf2 activity
through its phosphorylation. It has been demonstrated that O3 influences CK2 levels together
with Nrf2 phosphorylation, reducing oxidative stress and pro-inflammatory cytokines in
multiple sclerosis patients (Delgado-Roche et al. 2017). Similarly, O3 inhibits oxidative stress
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through inhibition of the mitogen-activated protein kinase phosphatase (MAPK) 1 signaling
pathway (Wang et al. 2018a).
Oxidative stress is one of the major drivers of protein misfolding that, accumulating and
aggregating as insoluble inclusions, can determine neurodegeneration (Hohn et al.
2020; Knowles et al. 2014). It is known that Nfr2 promotes the clearance of oxidized or
otherwise damaged proteins through the autophagy mechanism (Tang et al. 2019),
Interestingly, O3 can also modulate the degradation protein systems, not only via the Nrf2
pathway but also via activation of the AMP-activated protein kinase (AMPK)/mammalian
target of rapamycin (mTOR) signaling pathway, as demonstrated (Zhao et al. 2018).
O3 can protect against the overproduction of nitric oxide (NO) when NO is a toxic oxidant.
NO can rapidly react with other free radicals such as O2 − the highly reactive oxidant
peroxynitrite (ONOO) - and other RNS, which in turn damage the biomolecules (e.g. lipids,
protein, DNA/RNA), playing a key role in chronic inflammation and neurodegeneration
(Massaad, 2011; Toda et al. 2009), It has been demonstrated that O3 downregulates inducible
nitric oxide synthase (iNOS), which generates NO (Manoto et al. 2018; Smith et al.
2017) via NF-κB signaling.
CO acts as an inhibitor of another important pathway, NF-κB (nuclear factor kappa B subunit
1) signaling, which leads to the decreased expression of pro-inflammatory cytokines, while
bilirubin also acts as an important lipophilic antioxidant. Furthermore, HO-1 directly inhibits
pro-inflammatory cytokines and activates anti-inflammatory cytokines, leading to a balancing
of the inflammatory process (Ahmed et al. 2017). Our research group confirmed that mild
ozonization, tested on in vitro systems, induced modulation of genes including HO-1
(Scassellati et al. 2017),
In addition, Nrf2 also regulates the constitutive and inducible expression of antioxidants
including, but not limited to, Superoxide Dismutases (SOD), Glutathione Peroxidase (GSH-
Px), Glutathione-S-Transferase (GST), Catalase (CAT), and NADPH quinone oxidoreductase
1 (NQO1), phase II enzymes of drug metabolism and HSP (Galie et al. 2018, Bocci et al.
2015, Pedruzzi et al. 2012).
In this context, Nrf2 is considered a hormetic-like pathway (Calabrese et al. 2010). It
has widely been reported that the activation of Nrf2 by several different mechanisms (calorie
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restriction, physical exercise, polyphenols) can be a way to improve health due to its
transcriptional modulation on the vitagene network. Nfr2 is strongly implicated in aging
processes (Zhang et al. 2015; Schmidlin et al. 2019; Silva-Palacios et al. 2018). These
conditions share common mechanisms, and the results represent a first attempt to structure
Nrf2 as a common therapeutic medicine approach. Research on the antioxidant activities of
O3 correlated with the interaction with Nrf2 (Galie et al. 2018; Siniscalco et al. 2018; Re et al.
2014; Vaillant et al. 2013). Different antioxidants can combat many associated pathologies,
including neurodegenerative disorders (Leri et al. 2020; Calabrese 2020). The mechanisms of
the positive effects of O3 are attributed not only to up-regulation of cellular antioxidant
enzyme activity, but also to the activation of the immune and anti-inflammatory systems,
modulation of NPRL3 inflammasome, action on the proteasome, enhancement in the release
of growth factors from platelets, improvement in blood circulation and O2 delivery to
damaged tissues, and enhancement of general metabolism (Scassellati et al. 2020).
The gradual escalation of the ozone dose enhances cerebral blood flow, improves metabolism,
and corrects chronic oxidative stress. Neuronal cells may reactivate the synthesis of
antioxidant enzymes, which is crucial to normalize the redox state and avoid cell death. The
local induction of haeme oxygenase-1 played a critical role in reducing oxidative damage.
The enzyme caused the local release of CO and bilirubin that acts as a potent antioxidant of
peroxynitrite (Clavo et al. 2004). The trace amounts can pass through the blood-brain barrier
to reach the sites of neurodegeneration and upregulate the cellular synthesis of antioxidant
enzymes, which is a crucial step towards readjusting the impaired cell redox system.
Neurodegenerative disorders affect approximately 50 million people globally and have a
terrific and increasingly negative social-economic impact on families and society. A better
understanding of degenerative events and the effects of ozone therapy during the early stage
of the disease may be able to slow down the demise of critical populations of neurons and
thus provide patients with a better quality of life through ozone therapy. The use of ozone in
the treatment of various diseases can have a therapeutic effect, provided that the correct dose
is administered at the right time interval and depending on the antioxidant capacity of the
tissue exposed, as well as ensuring the concentration is used within a non-toxic range. The
versatility of ozone therapy is due to the cascade of ozone-derived compounds able to act on
several targets leading to a multifactorial correction of various pathological conditions. Ozone
therapy can improve well-being and delay the negative effects of aging. The aging process is
essentially linked to the balance of oxidants and antioxidants, advanced glycation end
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products (AGE), the role of genes and the immune system, the importance of telomeres and
telomerase, hormones, nutrition, environmental factors, and certain other factors. Stem cell
therapy, virtual reality rehabilitation, electromagnetic fields, and ozone therapy are among the
therapeutic approaches in the treatment of neurodegenerative disorders (Mitrečić et al. 2020).
Ozone Autohaemotherapy Induces Cerebral Metabolic Changes in Multiple Sclerosis
Patients.
The oxygenated hemoglobin concentration is increased and the chronic oxidative stress level
typical of MS sufferers is reduced (Molinari et al. 2014). Ozone has an effect on biomarkers
of oxidative stress and inflammation, it significantly improves the activity of antioxidant
enzymes and increases the reduced cellular glutathione levels, and demonstrates antioxidant
and anti-inflammatory effects.
Ozone promotes Nrf2 phosphorylation, reducing oxidative stress and pro-inflammatory
cytokines in multiple sclerosis patients (Delgado-Rosche et al. 2017).
Mechanisms of vascular pathophysiology in multiple sclerosis patients treated with ozone
therapy demonstrate revascularisation and regeneration of the blood-brain barrier as a result
of immunoreactive glial cells coming into contact with blood vessel walls during ozone
therapy (Ameli et al. 2019; Biomed 2019). The cerebrovascular system enhanced by ozone
autohemotherapy in multiple sclerosis patients increases brain metabolism and helps them
recover from the lower activity levels that predominate in MS patients (Molinari et al. 2017).
At both primary and chronic stages of MS, antioxidant therapy is an active approach to
disease progression. O3 therapy increases total tissue-oxygen levels in patients with multiple
sclerosis. Ozone therapy is a therapeutic alternative for patients with multiple sclerosis
(Molinari et al. 2014; Simonetti et al. 2014).
Ozone therapy applied in acute ischemic stroke increases blood oxygen saturation,
improves blood circulation, activates erythrocyte metabolism, improves tissue oxygenation
and oxygen supply, restores cell function, promotes oxygen metabolism, and induces
thrombolysis by hydrogen peroxide (H2O2) formation (Qiu et al. 2021). Ozone was
administered daily via rectal inflation (at an ozone concentration of 40 mg/l and 200 ml) for
15 days. In the clinical phase, an improvement in the quality of life was recorded in 80% of
patients treated with ozone therapy Qiu et al. 2021). Ozone therapy has certain therapeutic
benefits for ischemic stroke patients. Jing Qiu, Hui-sheng Chen (2016) reported that ozonated
autohemotherapy substantially improved neurological function in stroke patients.
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Neurological Deficits Treated with Ozone Therapy. Trigeminal neuralgia, postherpetic
neuralgia, meningitis, cervical myelopathy, demyelinating of nerves. Acoustic neuroma,
muscular neuropathy, and parkinsonism have shown satisfactory improvements
(Kakkad 2018).
The treatment of trigeminal neuralgia with percutaneous ozone and injections into the
Gasserian ganglion has been shown to have positive long-term effects on pain (An et al. 2018;
Gao et al. 2020).
The treatment of phantom limb pain with ozone injection into the nerve root in the stump
was met with positive results (Li et al. 2020).
Ozone therapy in patients with pain secondary to chemotherapy-induced peripheral
neuropathy with cancer of the colon and rectum treated with oxaliplatin and rectal
insufflation sessions of O3/O2 as part of the ongoing randomized controlled trial
(O3NPIQ) (O3NPIQ) 2020 (Clavo et al. 2019). Chemotherapy-induced peripheral
neuropathy decreases the quality of life of patients and can lead to a decrease in and
interruption of chemotherapy treatment. Potential pathophysiological mechanisms involved in
chemotherapy include chronic oxidative stress and consequent increases in free radicals and
pro-inflammatory cytokines. Several antioxidant‐based therapies have been tested. On the
other hand, ozone therapy can elicit an adaptive antioxidant and anti-inflammatory response
that could prove to be useful (Clavo et al. 2021).
Ozone therapy in cerebral ischemia and hypometabolism in meningioma treated by
stereotactic radiosurgery. Following ozone autohemotransfusion treatment, there was an
improvement in brain perfusion and metabolism, as demonstrated by SPECT and PET scans
(Clavo et al. 2011). Rectal ozone therapy showed positive effects of improving
neurorehabilitation in children with infratentorial ependymoma (Mašán & Golská 2017).
Ozone therapy of resistant meningitis in infants a mixture of ozone gas with pure oxygen
was used to treat resistant meningitis in infants with hydrocephalus and the infection was
cured (Dahhan 2015).
Endolumbal ozone therapy in the treatment of patients with a complicated spinal injury
in the acute period improved neurological symptoms (Yuldashev et al. 2020).
In the treatment of lumbar discopathy, ozone therapy is used with verified clinical benefits
in the treatment of lumbar disc damage. Ozone application methods can be administered
12
paravertebrally, juxtaforaminally, intradiscally (De Oliveira Magalhaes et al. 2012; Mašán
2017; Li et al. 2020; Yuldashev et al. 2020).
CONCLUSION
Ozone therapy is a biological treatment method with a wide range of applications in
medicine. The versatility of ozone therapy is due to the cascade of ozone-derived compounds
able to act on several targets leading to a multifactorial correction of pathological conditions.
O3 therapy induces moderate oxidative stress when interacting with lipids, increases
endogenous production of antioxidants, local perfusion, and oxygen delivery, as well as
enhances immune responses. Oxidative stress occurs when there is an imbalance between free
radical formation and antioxidant defense and is associated with damage to lipids, proteins,
and nucleic acids. Ozone is being examined as a master regulator of multiple cytoprotective
responses, as a key actor across a wide range of diseases, and as a therapeutic objective for
aging and aging-associated disorders. It provides scientific evidence for the application of
oxygen-ozone (O2-O3) in the treatment of neurological diseases. Oxidative stress is currently
thought to play a significant role in the development of inflammatory diseases, ischemic
diseases, hypertension, Alzheimer’s disease, Parkinson’s disease, muscular dystrophy, and
many others.
The versatility of ozone therapy is due to the cascade of ozone-derived compounds able to act
on several targets leading to a multifactorial correction of various pathological conditions, as
well as cardiovascular, peripheral vascular, neurological, orthopedic conditions, skin diseases,
wound healing, diabetes, and lung diseases, including the pandemic disease caused by the
COVID-19 coronavirus. Ozone therapy also promotes tissue perfusion, immunomodulatory
effect, energy effect of the body, and has regenerative and reparative properties. It appears to
be a potentially effective treatment method. When the correct dose is administered, no side
effects occur. Further clinical and experimental studies will be needed to determine the
optimal administration schedule and to evaluate the potential combination of ozone therapy
with other therapies to increase the effectiveness of treatment.
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... Oxidative stress can lead to protein misfolding and insoluble inclusions, which are associated with ALS (9,10). As a potent oxidizer, ozone can transiently worsen oxidative stress (11). However, this worsening in turn can activate the nuclear factor-related erythroid factor 2 (Nrf2) pathway, ultimately leading to the transcription of antioxidant response elements (AREs, 11,12). ...
... As a potent oxidizer, ozone can transiently worsen oxidative stress (11). However, this worsening in turn can activate the nuclear factor-related erythroid factor 2 (Nrf2) pathway, ultimately leading to the transcription of antioxidant response elements (AREs, 11,12). In small study of patients with multiple sclerosis, ozone therapy (20 ug/ml delivered rectally three times per week) was associated with increased markers of antioxidant activity and decreased markers of oxidative damage to lipids and proteins (12). ...
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ALSUntangled reviews alternative and off-label treatments for people living with amyotrophic lateral sclerosis (PALS). Here we review ozone therapy. Ozone therapy has possible mechanisms for slowing ALS progression based on its antioxidant, anti-inflammatory, and mitochondrial effects. A non-peer-reviewed report suggests that ozone treatment may slow progression in a mTDP-43 mouse model of ALS. One verified “ALS reversal” occurred on a cocktail of alternative treatments including ozone. There are no ALS trials using ozone to treat PALS. There can be potentially serious side effects associated with ozone therapy, depending on the dose. Based on the above information, we support an investigation of ozone therapy in ALS cell or animal models but cannot yet recommend it as a treatment in PALS.
... After five months, neurological disturbances appeared along with major depression and seizures (Nuzzo et al. 2021). The emergence of individual disorders and musculoskeletal syndromes from the use of computers and smartphones during COVID, as well as inappropriate working conditions in the home environment, highlighted the consequences of post-COVID syndrome (Masan et al. 2021). Special attention should be paid to inflammatory markers in the peripheral blood, especially neutrophil to lymphocyte ratio, C-reactive protein, D-dimers, serum ferritin (Wijeratne et al. 2020). ...
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Objective: The aim of this research was to investigate the prevention, diagnosis, and treatment of patients after COVID-19 with the possibility of using artificial intelligence and virtual reality in combination with traditional approaches to patient rehabilitation. Materials and methods: Statistical methods were used to evaluate the situation of COVID-19 worldwide and in Slovakia until March 2022. We investigated the rehabilitation options of breathing exercises, upper and lower limb rehabilitation, and cognitive tasks in patients with post-COVID syndrome who met the criteria for a combined rehabilitation program using virtual reality. Using artificial intelligence, we can predict in advance the evolution of the pandemic according to the records of infected patients and the evolution of the pandemic in the world, taking into account nearby territories. In the treatment of post-COVID syndrome, parameters have been identified that can be measured to objectively assess the improvement of the patient's condition and to continue personalizing individual rehabilitation scenarios. Results: In the patients who underwent the combined rehabilitation method, we observed progress in their ability to improve breathing, limb motor skills and also cognitive function of the patients. We identified different categories of parameters that can be evaluated by artificial intelligence methods, and we evaluated different scenarios using the exterior of nature and the interior of the room of the rehabilitation method of virtual reality, as well as the key elements of the "WOW" effect creating emotional changes in the patient for their motivation. Conclusion: We showed that artificial intelligence and virtual reality methods have the potential to accelerate rehabilitation and increase motivation in patients with post-COVID syndrome.
... Not until a couple of years ago that both clinical and epidemiological studies provided for the rst time evidence that ozone (O 3 ), a highly oxidative gas, may be a potential novel therapeutic strategy in the treatment of degenerative disorders [2][3][4] . In this context, clinical trials evidenced the effectiveness of O 3 therapy in the treatment of multiple sclerosis 2,3,5 , due to its potential to induce controlled oxidative stress 6 , through mechanism of action involving its interaction with the nuclear factor erythroid-derived 2-like 2 (Nrf2) 3 , a key regulator of inducible antioxidant responses 7 . ...
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Amyotrophic lateral sclerosis (ALS), a devastating progressive neurodegenerative disease, has no effective treatment. Recent evidence supports a strong metabolic component in ALS pathogenesis, raising additional therapeutic targets against ALS. At this respect, improvements in motor de cits and disease-associated weight loss after repeated exposures to ozone (O 3) in the mouse model of ALS based on TDP-43 proteinopathy (TDP-43 A315T mice) have been reported. Here, the underlying molecular mechanisms to determine whether O 3 exposure induces metabolic changes in ALS have been investigated. Molecular biology analysis demonstrated that O 3 signi cantly modi ed the expression pro le of hypothalamic neuropeptides, altering phosphorylation levels of the signal transducer and activator transcription 3 (STAT3) and protein kinase B (Akt), concomitantly to increase the expression of genes involved in metabolism and thermogenesis in the brown adipose tissue (BAT) of TDP-43 A315T mice. Composition of fecal gut microbiome of exposed TDP-43 A315T mice varied signi cantly compared to wild type (WT) controls. Densitometric analysis of neuromuscular junction, indicated that O 3 does not impaired the progression of disease in the skeletal muscle. Our ndings suggest the effectiveness of O 3 exposure to induce metabolic effects in the hypothalamus and BAT of this ALS mouse model and may be a new complementary non-pharmacological approach for ALS therapy.
... Ozone gas (O 3 ) was discovered in 1840, and its expansion into the medical field has given rise to compelling research in the recent decades to validate its clinical value [1]. Despite some controversies, several papers [2][3][4][5][6][7][8][9][10][11] have proposed relevant medical features, including bactericidal and virucidal effects, inflammatory modulation and circulatory stimulation, with considerable applications in several medical fields such as wound healing, documented that knee pain could be decreased after O 2 O 3 intra-articular management in patients affected by knee osteoarthritis (KOA). Likewise, tendon disorders are another conceivable focus for O 2 O 3 therapy, and a recent randomized controlled trial (RCT) evaluated the usefulness of O 2 O 3 therapy in patients with shoulder impingement, indicating that it might be assumed an intriguing alternative intervention in case of contraindication to corticosteroids [49]. ...
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To date, the application of oxygen-ozone (O2O3) therapy has significantly increased in the common clinical practice in several pathological conditions. However, beyond the favorable clinical effects, the biochemical effects of O2O3 are still far from being understood. This comprehensive review aimed at investigating the state of the art about the effects of O2O3 therapy on pro-inflammatory cytokines serum levels as a modulator of oxidative stress in patients with musculoskeletal and temporomandibular disorders (TMD). The efficacy of O2O3 therapy could be related to the moderate oxidative stress modulation produced by the interaction of ozone with biological components. More in detail, O2O3 therapy is widely used as an adjuvant therapeutic option in several pathological conditions characterized by chronic inflammatory processes and immune overactivation. In this context, most musculoskeletal and temporomandibular disorders (TMD) share these two pathophysiological processes. Despite the paucity of in vivo studies, this comprehensive review suggests that O2O3 therapy might reduce serum levels of interleukin 6 in patients with TMD, low back pain, knee osteoarthritis and rheumatic diseases with a concrete and measurable interaction with the inflammatory pathway. However, to date, further studies are needed to clarify the effects of this promising therapy on inflammatory mediators and their clinical implications.
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Oxygen‐ozone (O2‐O3) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2‐related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative‐stress‐related neurodegenerative diseases, act through the Nrf2‐pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2‐O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright‐field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)‐activated microglial cells human microglial clone 3 (HMC3) and in DMF‐treated LPS‐activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS‐activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro‐inflammatory cytokines, Interleukin‐6, and tumor necrosis factor‐α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS‐activated and LPS + DMF cells, whereas the cell motility and the secretion of pro‐inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2‐O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. Low‐dose ozone (O3) does not damage activated microglial cells in vitro Low‐dose O3 decreases cell motility and pro‐inflammatory cytokine secretion in activated microglial cells in vitro Low‐dose O3 potentiates the effect of an anti‐inflammatory drug on activated microglial cells Low‐dose ozone (O3) does not damage activated microglial cells in vitro, but is able to decrease cell motility and pro‐inflammatory cytokine secretion, and potentiate the effect of anti‐inflammatory drugs. Modulation of microglia activity may contribute to the beneficial effects of O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation.
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Canonical transforming growth factor-beta (TGF-β) signaling exerts neuroprotection and influences memory formation and synaptic plasticity. It has been considered as a new target for the prevention and treatment of depression. This study aimed to examine its modulatory role in linking prenatal maternal depressive symptoms and the amygdala volumes from birth to 6 years of age. We included mother–child dyads (birth: n = 161; 4.5 years: n = 131; 6 years: n = 162) and acquired structural brain images of children at these three time points. Perinatal maternal depressive symptoms were assessed using the Edinburgh Postnatal Depression Scale (EPDS) questionnaire to mothers at 26 weeks of pregnancy and 3 months postpartum. Our findings showed that the genetic variants of TGF-β type I transmembrane receptor (TGF-βRI) modulated the association between prenatal maternal depressive symptoms and the amygdala volume consistently from birth to 6 years of age despite a trend of significance at 4.5 years of age. Children with a lower gene expression score (GES) of TGF-βRI exhibited larger amygdala volumes in relation to greater prenatal maternal depressive symptoms. Moreover, children with a lower GES of the TGF-β type II transmembrane receptor (TGF-βRII), Smad4, and Smad7 showed larger amygdala volumes at 6 years of age in relation to greater prenatal maternal depressive symptoms. These findings support the involvement of the canonical TGF-β signaling pathway in the brain development of children in the context of in utero maternal environment. Such involvement is age-dependent.
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(1) Background: Chemotherapy-induced peripheral neuropathy (CIPN) decreases the quality of life of patients and can lead to a dose reduction and/or the interruption of chemotherapy treatment, limiting its effectiveness. Potential pathophysiological mechanisms involved in the pathogenesis of CIPN include chronic oxidative stress and subsequent increase in free radicals and proinflammatory cytokines. Approaches for the treatment of CIPN are highly limited in their number and efficacy, although several antioxidant-based therapies have been tried. On the other hand, ozone therapy can induce an adaptive antioxidant and anti-inflammatory response, which could be potentially useful in the management of CIPN. (2) Methods: The aims of this works are: (a) to summarize the potential mechanisms that could induce CIPN by the most relevant drugs (platinum, taxanes, vinca alkaloids, and bortezomib), with particular focus on the role of oxidative stress; (b) to summarize the current situation of prophylactic and treatment approaches; (c) to describe the action mechanisms of ozone therapy to modify oxidative stress and inflammation with its potential repercussions for CIPN; (d) to describe related experimental and clinical reports with ozone therapy in chemo-induced neurologic symptoms and CIPN; and (e) to show the main details about an ongoing focused clinical trial. (3) Results: A wide background relating to the mechanisms of action and a small number of experimental and clinical reports suggest that ozone therapy could be useful to prevent or improve CIPN. (4) Conclusions: Currently, there are no clinically relevant approaches for the prevention and treatment of stablished CIPN. The potential role of ozone therapy in this syndrome merits further research. Randomized controlled trials are ongoing.
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The questions of the one-value solvability of an inverse boundary value problem for a mixed type integro-differential equation with Caputo operators of different fractional orders and spectral parameters are considered. The mixed type integro-differential equation with respect to the main unknown function is an inhomogeneous partial integro-differential equation of fractional order in both positive and negative parts of the multidimensional rectangular domain under consideration. This mixed type of equation, with respect to redefinition functions, is a nonlinear Fredholm type integral equation. The fractional Caputo operators' orders are smaller in the positive part of the domain than the orders of Caputo operators in the negative part of the domain under consideration. Using the method of Fourier series, two systems of countable systems of ordinary fractional integro-differential equations with degenerate kernels and different orders of integro-differentation are obtained. Furthermore, a method of degenerate kernels is used. In order to determine arbitrary integration constants, a linear system of functional algebraic equations is obtained. From the solvability condition of this system are calculated the regular and irregular values of the spectral parameters. The solution of the inverse problem under consideration is obtained in the form of Fourier series. The unique solvability of the problem for regular values of spectral parameters is proved. During the proof of the convergence of the Fourier series, certain properties of the Mittag-Leffler function of two variables, the Cauchy-Schwarz inequality and Bessel inequality, are used. We also studied the continuous dependence of the solution of the problem on small parameters for regular values of spectral parameters. The existence and uniqueness of redefined functions have been justified by solving the systems of two countable systems of nonlinear integral equations. The results are formulated as a theorem.
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Systems medicine is founded on a mechanism-based approach and identifies in this way specific therapeutic targets. This approach has been applied for the transcription factor nuclear factor (erythroid-derived 2)–like 2 (Nrf2). Nrf2 plays a central role in different pathologies including neurodegenerative disorders (NDs), which are characterized by common pathogenetic features. We here present wide scientific background indicating how a natural bioactive molecule with antioxidant/anti-apoptotic and pro-autophagy properties such as the ozone (O3) can represent a potential new strategy to delay neurodegeneration. Our hypothesis is based on different evidence demonstrating the interaction between O3 and Nrf2 system. Through a meta-analytic approach, we found a significant modulation of O3 on endogenous antioxidant-Nrf2 (p < 0.00001, Odd Ratio (OR) = 1.71 95%CI:1.17-2.25) and vitagene-Nrf2 systems (p < 0.00001, OR = 1.80 95%CI:1.05-2.55). O3 activates also immune, anti-inflammatory signalling, proteasome, releases growth factors, improves blood circulation, and has antimicrobial activity, with potential effects on gut microbiota. Thus, we provides a consistent rationale to implement future clinical studies to apply the oxygen-ozone (O2-O3) therapy in an early phase of aging decline, when it is still possible to intervene before to potentially develop a more severe neurodegenerative pathology. We suggest that O3 along with other antioxidants (polyphenols, mushrooms) implicated in the same Nrf2-mechanisms, can showed neurogenic potential, providing evidence as new preventive strategies in aging and in NDs.
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This paper demonstrates that ginseng mixtures and individual ginseng chemical constituents commonly induce hormetic dose responses in numerous biological models for endpoints of biomedical and clinical relevance, typically providing a mechanistic framework. The principal focus of ginseng hormesis-related research has been directed toward enhancing neuroprotection against conditions such as Alzheimer’s and Parkinson’s Diseases, stroke damage, as well as enhancing spinal cord and peripheral neuronal damage repair and reducing pain. Ginseng was also shown to reduce symptoms of diabetes, prevent cardiovascular system damage, protect the kidney from toxicities due to immune suppressant drugs, and prevent corneal damage, amongst other examples. These findings complement similar hormetic-based chemoprotective reports for other widely used dietary-type supplements such as curcumin, ginkgo biloba, and green tea. These findings, which provide further support for the generality of the hormetic dose response in the biomedical literature, have potentially important public health and clinical implications.
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A continuous rise in life expectancy has led to an increase in the number of senior citizens, now amounting to a fifth of the global population, and to a dramatic increase in the prevalence of diseases of the elderly. This review discusses the threat of dementia, a disease that imposes enormous financial burden on health systems and warrants efficient therapeutic solutions. What we learned from numerous failed clinical trials is that we have to immediately take into account two major elements: early detection of dementia, much before the onset of symptoms, and personalized (precision) medicine treatment approach. We also discuss some of the most promising therapeutic directions, including stem cells, exosomes, electromagnetic fields, and ozone.
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Background: Ozone injection around Gasserian ganglion (OIAGG) has been reported to be an effective treatment for trigeminal neuralgia (TN); however, there remain areas for improvement. To overcome one of these limitations, a multicenter examination of application would be extremely helpful. Objective: The goal of this report was to assess the efficacy of OIAGG for refractory TN across multiple centers and to explore factors predictive of successful treatment. Design: A multicenter, retrospective study. Setting: The study was conducted across 3 pain centers across China. Patients and methods: A total of 103 subjects from 3 pain centers were enrolled in the study. An ozone-oxygen mixture gas at a concentration of 30 µg/mL was injected into the area around the Gasserian ganglion performed under C-arm X-ray guidance. Primary outcome measures included a pain assessment using a visual analog scale (VAS) and the Barrow Neurological Institute (BNI) pain intensity scale. Clinical assessment of patients for these outcome measures was performed at pretreatment, post-treatment, 6 months, 1 year and 2 years after the OIAGG. Results: Successful pain relief was defined as a score within BNI grades I-IIIa. The pain relief rates at post-treatment, 6 months, 1 year and 2 years after the procedure were 88.35%, 86.87%, 84.46% and 83.30%, respectively. The VAS at each observation time point was significantly different from the preoperative levels (P<0.05). Logistic regression analysis showed that previous nerve damage had a significant effect on the treatment results. No significant complications or side effects were found during or after treatment. Conclusion: This multicenter research confirms our previous single center results that OIAGG is both effective and safe for patients with TN.
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Brain aging is one of the major risk factors for the development of several neurodegenerative diseases. Therefore, mitochondrial dysfunction plays an important role in processes of both, brain aging and neurodegeneration. Aged mice including NMRI mice are established model organisms to study physiological and molecular mechanisms of brain aging. However, longitudinal data evaluated in one cohort are rare but are important to understand the aging process of the brain throughout life, especially since pathological changes early in life might pave the way to neurodegeneration in advanced age. To assess the longitudinal course of brain aging, we used a cohort of female NMRI mice and measured brain mitochondrial function, cognitive performance, and molecular markers every 6 months until mice reached the age of 24 months. Furthermore, we measured citrate synthase activity and respiration of isolated brain mitochondria. Mice at the age of three months served as young controls. At six months of age, mitochondria-related genes (complex IV, creb-1, β-AMPK, and Tfam) were significantly elevated. Brain ATP levels were significantly reduced at an age of 18 months while mitochondria respiration was already reduced in middle-aged mice which is in accordance with the monitored impairments in cognitive tests. mRNA expression of genes involved in mitochondrial biogenesis (cAMP response element-binding protein 1 (creb-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), nuclear respiratory factor-1 (Nrf-1), mitochondrial transcription factor A (Tfam), growth-associated protein 43 (GAP43), and synaptophysin 1 (SYP1)) and the antioxidative defense system (catalase (Cat) and superoxide dismutase 2 (SOD2)) was measured and showed significantly decreased expression patterns in the brain starting at an age of 18 months. BDNF expression reached, a maximum after 6 months. On the basis of longitudinal data, our results demonstrate a close connection between the age-related decline of cognitive performance, energy metabolism, and mitochondrial biogenesis during the physiological brain aging process. 1. Introduction The average life expectancy has increased considerably to over 80 years in developed countries [1], and the multifactorial aging process is characterized by several changes on the cellular level [2, 3]. Mitochondria are cell organelles with central functions such as energy metabolism, including ATP production and generation of reactive oxygen species (ROS); however, mitochondrial dysfunction has been identified as an important hallmark of aging [4–9]. In addition, there are many studies that describe the close relationship between various age-related diseases and impaired mitochondrial function, which makes mitochondria interesting as a potential target for the treatment and prevention of neurodegenerative diseases [10, 11]. Mitochondrial dysfunction is characterized by a reduced efficiency of the respiratory chain system diminishing the synthesis of high-energy molecules such as ATP and the expression of genes involved in mitochondrial biogenesis, cellular longevity, and the antioxidant defense systems [12]. Evidences point out that the activity of complex I and complex IV of the respiratory chain system is impaired in aged brains which leads to a reduced capability to produce ATP [13, 14]. In 1956, the “free radical, theory of aging” was postulated by Harman which states that cellular aging is a direct consequence of free radicals, especially the superoxide anion radical (O2⋅−), attacking cells and tissue [3, 15–17]. This framework has been refined over the last years. In 1979, mitochondria were identified as the key producer of ROS that significantly contribute to aging processes [18–21]. However, low “physiological” ROS levels are known to have important functions for signaling mechanisms in the cell [22]. Under physiological conditions, the antioxidative defense system, including superoxide dismutase (SOD) and the glutathione (GSH) system, is able to eliminate highly reactive molecules [23]. However, if there is an imbalance between the generation of ROS and the cellular defense system, oxidative damage occurs which can initiate apoptosis and trigger neurodegenerative diseases. Furthermore, aging is characterized by changes in mitochondrial dynamics [24]: these organelles are able to fuse and to divide. The later process, the so-called fission, is part of the cellular quality control and results in fragments of different sizes that are cleared by mitophagy [25, 26]. The fission processes also help to regulate the cellular ATP levels. Fusion leads to enrichment of mtDNA and finally reduces mutations, [24]. Most of the aging studies in rodents conducted so far compared aged animals to young ones but did not collect longitudinal data over the entire lifetime. Thus, studies on cognitive performance and bioenergetic parameters in the brain covering the lifespan are rare. Therefore, we measured the development of the energy metabolism and mRNA expression of genes involved in mitochondrial biogenesis, antioxidant capacity, and synaptic plasticity in the brain as well as the cognitive performance every six months in the same cohort of female NMRI mice. Female NMRI mice are a well-described outbred mouse model for the physiological, “normal” aging process which reflects a high variability of the genome [27–29]. This model has been described as most suitable for studies on physiological aging compared to inbred or genetically modified mouse models with accelerated aging or reduced lifespans [30, 31]. 2. Material and Methods 2.1. Animals and Treatment Female NMRI mice (Navar Medical Research Institute) were purchased at the age of 3 weeks from Charles River (Sulzbach, Germany) and kept in the animal station until they reached the ages of 3, 6, 12, 18, and 24 months. All mice had ad libitum access to a standard pelleted diet (cat. no.1324; Altromin, Lage, Germany) and drinking water. Behavioral testing was performed before all time points. Mice were sacrificed by decapitation. The brain was quickly dissected on ice after the removal of the cerebellum, the brain stem, and the olfactory bulb. All experiments were carried out by individuals with appropriate training and experience according to the requirements of the Federation of European Laboratory Animal Science Associations and the European Communities Council Directive (Directive 2010/63/EU). Experiments were approved by the regional authority (Regierungspraesidium Darmstadt; #V54–19 c 20/15–FU/1062). 2.2. Passive Avoidance Test The test was carried out using a passive avoidance step-through system (cat. no. 40533/mice; Ugo Basile, Germonio, Italy) and a protocol similar to the protocol published by Shiga et al. [32]. On day one of the experiment, the mouse was put into the light chamber (light intensity 75%). The door toward the dark chamber was opened after a 30 s delay, and time was recorded until the mouse enters into the dark chamber. In the dark chamber, the mouse received an electric shock (0.5 mA, 1 s duration). If the mouse did not enter the dark chamber after 180 s, the test was stopped. The same test was repeated 24 h later. This time, the door toward the dark chamber was already opened after 5 s and time was measured until the mouse entered the dark chamber but the electric shock was turned off. The test was aborted after 300 s. 2.3. One-Trial Y-Maze Test A one-trial Y-maze test was conducted using a custom-made Y-maze (material: polyvinyl chloride; length of arms: 36 cm; height of arms: 7 cm; width of arms: 5 cm; and angle between arms: 120°). At the beginning of the test, the mouse was put into one of the three arms of the Y-maze, and the sequence of the entries was recorded for 5 min. Spontaneous alternation was determined using the formula [33]. 2.4. Preparation of Dissociated Brain Cells One hemisphere of the brain was used to prepare dissociated brain cells (DBCs) for ex vivo studies. The brain was washed once in medium 1 (138 mM NaCl, 5.4 mM KCl, 0.17 mM Na2HPO4, 0.22 mM KH2PO4, , and 58.4 mM sucrose; ). Afterwards, it was cut into small pieces in 2 ml of medium 1 using a scalpel. The chopped brain was then pressed through a 200 μm nylon mesh into a beaker containing 16 ml of medium 1 using a plastic Pasteur pipette with a wide opening. In the last step, the brain homogenate was filtered through a 102 μm nylon mesh. The resulting brain homogenate was centrifuged (2000 rpm, 5 min, and 4°C) before the pellet was redissolved in 20 ml of medium 2 (110 mM NaCl, 5.3 mM KCl, , , , 70 mM sucrose, and 20 mM HEPES). The centrifugation step was repeated twice; after the last centrifugation, the pellet was redissolved in 4.5 ml of Dulbecco’s modified without supplements. DBCs were seeded in 250 μl aliquots in 12 replicates into a 24-well plate for the measurement of the mitochondrial membrane potential. For the measurement of the ATP level, DBCs were seeded in 50 μl aliquots into a 96-well plate. Cells were incubated for 3 h in a humidified incubator (5% CO2). Respectively, 6 wells were incubated for 3 h with sodium nitroprusside (0.5 mM for ATP measurement; 2 mM for the measurement of the mitochondrial membrane potential) in DMEM. The remaining cell suspension was reserved for protein determination and stored at -80°C. 2.5. Measurement of ATP Concentrations in DBCs The ViaLight Plus bioluminescence kit (Lonza, Walkersville, USA) was used for assessing ATP concentrations in DBC. At the end of the incubation, the 96-well plate was removed from the incubator and allowed to cool to room temperature for 10 min. Afterwards, all wells were incubated with 25 μl lysis buffer in the dark for 10 min. In the next step, wells were incubated with 50 μl monitoring reagent. The emitted light (bioluminescence) was recorded using a luminometer (Victor X3 multilabel counter). The ATP concentrations in the wells were determined using a standard curve; ATP concentrations of DBC were normalized to protein content. 2.6. Measurement of Mitochondrial Membrane Potential MMP was measured in DBC using the fluorescence dye Rhodamine123 (R123). DBCs were incubated in an incubator (37°C, 5% CO2) for 15 min with 0.4 μM R123. Afterwards, the reaction was stopped by adding Hank’s Balanced Salt Solution (HBSS) into the wells. DBCs were centrifuged (914 g, 5 min, room temperature), the medium was aspirated, and DBCs were supplemented with new HBSS. DBCs were triturated to obtain a homogenous sample. Subsequently, MMP was assessed by reading the R123 fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 535 nm (Victor X3 multilabel counter). The fluorescence in each well was read in four consecutive runs. The fluorescence values were then normalized to protein content. 2.7. Isolation of Brain Mitochondria and High-Resolution Respirometry Half a brain hemisphere (the frontal part) was used to isolate brain mitochondria. The protocol is described in Hagl et al. [34]. The pellet obtained from the last centrifugation step was dissolved in 250 μl MIRO5 (0.5 mM EGTA, , 60 mM K-lactobionat, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 100 mM sucrose, 1 g/l BSA). Subsequently, 80 μl of the resulting cell suspension was injected into an Oxygraph 2k-chamber. A complex protocol was used to investigate the function of the respiratory chain complexes. The capacity of the oxidative phosphorylation (OXPHOS) was determined using complex I-related substrates pyruvate (5 mM), malate (2 mM), and ADP (2 mM) followed by the addition of succinate (10 mM). Mitochondrial integrity was measured by addition of cytochrome c (10 μM). Oligomycin (2 μg/ml) was added to determine leak respiration (leak (omy)), and afterwards, uncoupling was achieved by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, injected stepwise up to 1-1.5 μM). Complex II respiration was measured after the addition of rotenone (0.5 μM). Complex III inhibition was achieved by the addition of antimycin A (2.5 μM) and was subtracted from all respiratory parameters. COX activity was measured after ROX determination by applying 0.5 mM tetramethylphenylenediamine (TMPD) as an artificial substrate of complex IV and 2 mM ascorbate to keep TMPD in the reduced state. Autoxidation rate was determined after the addition of sodium azide (>100 mM), and COX respiration was additionally corrected for autoxidation. 2.8. Citrate Synthase Activity Citrate synthase activity was measured photometrically in isolated brain mitochondria as described in Hagl et al. [34]. 2.9. Protein Quantification Protein content was determined according to the BCA method using a Pierce™ Protein Assay Kit (Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. 2.10. Transcription Analysis by Quantitative Real-Time PCR (qRT-PCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions using ~20 mg RNAlater stabilized samples (Qiagen, Hilden, Germany). RNA was quantified measuring the absorbance at 260 and 280 nm using a NanoDrop™ 2000c spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity was assessed using the ratio of absorbance 260/280 and 260/230. To remove residual genomic DNA, samples were treated with a TURBO DNA-free™ kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA was synthesized from 250 ng total RNA using the iScript cDNA Synthesis Kit (BioRad, Munich, Germany) according to the manufacturer’s instructions and was stored at -80°C. qRT-PCR was conducted using a CfX 96 Connect™ system (BioRad, Munich Germany). Oligonucleotide primer sequences, primer concentrations, and product sizes are listed in Table 1. All primers were received from Biomol (Hamburg, Germany) or Biomers (Ulm, Germany). cDNA for qRT-PCR was diluted 1 : 5 with RNase-free water (Qiagen, Hilden, Germany), and all samples were performed in triplicates. PCR cycling conditions were an initial denaturation at 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 58°C for 45 s, and 72°C for 29 s. Gene expression was analyzed using the −(2ΔΔCq) method using BioRad CfX manager software and was normalized to the expression levels of beta 2 microglobulin (B2M) and phosphoglycerate kinase 1 (PGK1). Primer Sequence Product size (bp) Conc. (μM) AMPK (β-subunit) 5-agtatcacggtggttgctgt-3 5-caaatactgtgcctgcctct-3 190 0.1 B2M 5-ggcctgtatgctatccagaa-3 5-gaaagaccagtccttgctga-3 198 0.4 BDNF 5-gatgccagttgctttgtctt-3 5-atgtgagaagttcggctttg-3 137 0.1 CI (NADH-ubiquinone oxidoreductase 51 kDa subunit) 5-acctgtaaggaccgagaga-3 5-gcaccacaaacacatcaaaa-3 227 0.1 CIV (cytochrome c oxidase subunit 5A) 5-ctgttccattcgctgctatt-3 5-gcgaacagcactagcaaaat-3 217 0.1 Creb-1 5-tagctgtgacttggcattca-3 5-ttgttctgtttgggacctgt-3 184 0.5 CS 5-aacaagccagacattgatgc-3 5-atgaggtcctgctttgtcct-3 184 0.1 GAP43 5-agggagatggctctgctact-3 5-gaggacggggagttatcagt-3 190 0.15 Nrf-1 5-tcggagcacttactggagtc-3 5-ctagaaaacgctgccatgat-3 228 0.5 PGC1-α 5-tgtcaccaccgaaatcct-3 5-cctggggaccttgatctt-3 124 0.05 PGK1 5-gcagattgtttggaatggtc-3 5-tgctcacatggctgacttta-3 185 0.4 SOD2 5-acagcgcatactctgtgtga-3 5-gggggaacaactcaactttt-3 183 0.1 SYP1 5-tttgtggttgttgagttcct-3 5-gcatttcctccccaaagtat-3 204 0.1 Tfam 5-agccaggtccagctcactaa-3 5-aaacccaagaaagcatgtgg-3 166 0.5 bp: base pairs; conc.: concentration.
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