The Effect of the MNRI Method on Neurotransmitter Biomarkers of Individuals with Neurodevelopmental Disorders

Introduction: The MNRI (Masgutova Neurosensorimotor Reflex Integration) method was developed in 1989 in Russia and has spread world-wide to treat individuals with certain types reflex development deficits, behavior disorders, disorders of speech or language development, and learning disabili-ties. MNRI is based on techniques called “repatterning” or remodulation, meaning re-education, recoding the reflex nerve pathways specific for dynamic and postural reflex schemes. Objectives: Repatterning activates the extra pyramidal nervous system responsible for automatic mechanisms and processes, the extension of links between neurons, the growth of neural nets, myelination, and the creation of new nerve routing. This potential result was tested utilizing urinary measurements of the following neurotransmitters: epinephrine, norepinephrine, dopamine, DOPAC, serotonin, 5-HIAA, glycine, taurine, GABA, glutamate, PEA, and histamine. Methods: Neurological impact of the Masgutova Neurosensorimotor Reflex Method on the magni-tude of changes in neurotransmitters was assessed by an external controlled and double-blind method using patients from one of the four diagnosis groups: 1) global developmental disorders; 2) cerebral palsy, Traumatic Brain Injury (TBI), Acute Brain Injury (ABI), and seizures; 3) ADD/ADHD; and 4) anxiety disorders. Results: The post-MNRI results in participants show a tendency for regulation of the above neurotransmitters resulting in their calming down, decrease of hypervigilance, stress resilience increase, improvements in behavioral and emotional regulation, positive emotions, and cognitive pro-How cesses control. Conclusion: The application of the Masgutova Neurosensorimotor Reflex Method as a therapy modality offers a novelty paradigm for the treatment using neuro- and immune-modulation technologies presenting a non-pharmaceutical approach, based on use of neurosensorimotor reflex circuit concept. This paper presents a study of the effectiveness of MNRI therapy using neurotransmitter analysis. The aim was to investigate whether the neurotransmitters markers improve as a result of MNRI Reflex Integration treatment modality for individuals with global developmental disorders, cerebral palsy, traumatic brain injury (TBI), acute brain injury (ABI), seizures, ADD/ADHD, and anxiety disorders.


Introduction
Many interventions addressing developmental disorders in the medical sciences, psychology, and pediatric developmental programs use an interdisciplinary approach. The intervention used in this study with the four specific groups of participants differs from the traditional forms of medical support (medications, surgical procedures, orthotics, etc.), PT (physiotherapy), OT (occupational therapy), SP (speech therapy), cognitive-behavioral therapy, neurofeedback [1] massage therapy, hydrotherapy, dietary advice, alternative treatments such as Bobath Neuro-Developmental Techniques [2] Doman-Delacato [3] Vojta [4], Respiratory Therapy, as well as many others.
Masgutova Neurosensorimotor Reflex Method (MNRI) addresses the neurosensorimotor aspect of early sensory-motor patterns and reflexes to support sensory-motor integration and neurodevelopment of children and adults with neurodeficits and learning challenges: CP, TBI [5] [6] [7] [8], ASD [9] [10] Down syndrome [11] [12] [13] [14] [15] and other [16] [17]. MNRI therapy meets the ever-increasing demands for neurorehabilitation of individuals with impaired sensorimotor functions due to damage or dysfunctions in central nervous systems. Previous studies of the MNRI Method and its different sub-programs demonstrated a positive effect on immune markers [13] [18] [19], neurophysiological functions [7] [8] and different developmental aspects including the regulation of behavior and emotions, language, and communication [9] [17] [20] [21]. This paper presents a study of the effectiveness of MNRI therapy using neurotransmitter analysis. The aim was to investigate whether the neurotransmitters markers improve as a result of MNRI Reflex Integration treatment modality for individuals with global developmental disorders, cerebral palsy, traumatic brain injury (TBI), acute brain injury (ABI), seizures, ADD/ADHD, and anxiety disorders.
The MNRI method was developed in 1989 in Russia and further elaborated in Eastern Europe to treat individuals with certain types of sensorimotor or reflex  [13]. The MNRI therapy program is based on the theory that impaired reflex circuits can be reconstructed and re-integrated, which involves awakening the genetic sensorimotor memory in individuals even with severe diagnosis, for example, with CP and brain damage [5] [6]. MNRI is an evidence and research-based system of a therapeutic program oriented to analyze the effect of the MNRI reflex integration techniques for neurosensorimotor-cognitive development of children and adults with neurodeficits and learning problems [22]. The studies were conducted by Dr. S. Masgutova and her team and scientific colleagues from different countries between 1989 and 2016 [22].
MNRI is based on exercises and techniques called "repatterning", meaning re-education, recoding, rerouting of and paving the reflex nerve pathways specific for dynamic and postural reflex schemes (e.g., Babinski, Automatic Gait, Bauer Crawling, Hands Grasp, and others) (Reflexes: Portal to Neurodevelopment and Learning 2015). The stimulation of reflex pathways is aimed at strengthening and stabilizing the traces of genetic sensory-motor memory and activation of innate defensive mechanisms of the body's brain "alarm" system (HPA-stress axis or "hypothalamus-> pituitary gland-> adrenals" cycle activation) in times of stress or danger [23]. MNRI exercises stimulate innate neuro-regulation mechanisms and resilience in the stress and immune systems [8] [9] [24]. Repatterning activates the extra pyramidal nerve system (peripheral nerves, spinal cord, brain stem, and diencephalon) responsible for automatic mechanisms and processes, the extension of links between neurons, the growth of neural nets, myelination, and the creation of new nerve routing, as described by Sechenov [25] [26], Pavlov [27], Anokhin [28], Haines [29], Virella [30].
There are various ways to assess neurotransmitters in the body. The three most common ways to achieve this include: 1) The commonly accepted method of testing the neurotransmitters is cere- 3) Given the invasiveness and instability issues of methods (1) and (2), urine testing provides the most optimal alternative. For nearly 60 years, studies have utilized urinary measurements of neurotransmitters and neurotransmitter metabolites (Marc, et al. 2010). Since the 1950s, increased urinary excretion of dopamine (DA), norepinephrine (NE), and epinephrine (E), the three naturally occurring catecholamines, has been used to diagnose pheochromocytoma. Early days concerns about the limitations of urinalysis for neurotransmitters, were alleviated by development of fluorometric methods. High-Performance Liquid Chromatography (HPLC) methodology has expanded parameters of even greater specificity and sensitivity, allowing an even wider span of clinical application [31] [32] [33]. Lynn-Bullock examined the effects of oral ingestion of the serotonin substrate (5-hydroxytryptophan (5-HTP)) on specific brain regions in rats [34]. Serotonin is a monoamine neurotransmitter and is implicated in many physiological and behavioral functions including: affect, aggression, appetite, cognition, emesis, endocrine function, gastrointestinal function, motor function, neurotropism, perception, sensory function, sex, sleep, and vascular function [34] [35]. Serotonin levels were measured using brain tissue immunoreactivity and urinalysis and observed maximum serotonin immunoreactivity in the serotonergic dorsal raphe nucleus within 2 h of 5-HTP administration. Urinary analysis of serotonin, 5-HTP, and 5-hydroxyindolacetic acid (5-HIAA), a serotonin metabolite, had parallel changes in immunoreactivity to the dorsal raphe nucleus, demonstrating a positive correlation between CNS serotonergic activity and urinary serotonin levels [35] [36].
Studies have further determined that those struggling with depression, ADD, and ADHD have a direct correlation to neurotransmitters in the CNS and the urine. There are several reasons preventing the use of urinary neurotransmitter levels as a sole diagnostic tool. However, the utility of urinary neurotransmitter levels as indicators and biomarkers to gauge the realm of impact and treatment is unquestionable [37]. Clinicians are already utilizing urinary histamine markers as an accurate assessment tool and treatment directive. Studies have also demonstrated a direct correlation between neurotransmitter levels and acute PTSD in children [38].
Some of the studied neurotransmitters are related biochemically: 5HIAA is a metabolite of serotonin, and DOPAC is a metabolite of dopamine. The neurotransmitters can be divided into two categories by: inhibitory vs. excitatory. This classification is complicated, since transmission of neurotransmitters is a complex system and any neurotransmitter can have a secondary function. For in- Immune and central nervous systems are integrative and interdependently maintaining. The central nervous system modulates the immune system by hardwiring sympathetic and parasympathetic nerves (autonomic nervous system) to lymphoid organs. Neuroendocrine hormones (such as corticotrophin-releasing hormone or substance P) regulate the cytokine balance. The immune system, in turn, affects brain activity including inhibition-excitation processes, as well as sleep, and body temperature. Working in this highly reciprocal manner the immune-nervous system creates a unique functional and anatomical link for the organism to act at multiple levels [29] [39]. Stress can disrupt interaction between the nervous and immune system [39].
There are three major forms of neurotransmitters functioning in a human organism, all of which communicate via signals targeting specific cells. These are: acetylcholine neurotransmitter, biogenic amines, and amino acids. Below we present a general overview of the analyzed transmitters.
Gamma Amino Butyric Acid (GABA) is the major inhibiting or calming transmitter in a human organism. The role of GABA is to inhibit or reduce the activity of the neurons or nerve cells, and the calming effect is achieved by restricting neuroelectric activity [40]. The body relies on GABA to reduce anxiety. The activity of GABA is increased during sleep, making GABA responsible for setting the circadian rhythms in humans. Other functions are impacted by GABA, for example, female hormonal cycles are affected by circadian rhythm. GABA is prominent in the hippocampus, this part of the brain is responsible for memory formation, by allowing the brain to reconfigure neural connections [40] [41]. Excess of GABA may lead to depression or apathy; low levels of GABA are known to affect sleep. There is also a potential influence of GABA on immunity and physiology wellness [42].
Glutamate is an excitatory neurotransmitter that enhances signal transduction; it is tied to learning and memory; it is also one of the most common neurotransmitters: there is more glutamate in the brain than any other amino acid. Tissue extracted from the brain contains 5 -15 mmol glutamate per kg [43].
Glutamate is not only involved in cognitive functions, but also learning function.
Being an amino acid, it has a role in metabolic function and ammonia detoxification. Traumatic brain injury or stroke may create physical damage which in turn causes glutamate to bring hyper excitation and thereby killing neurons, thus resulting in brain damage [44].
Norepinephrine assists the body to go into "high alert" and is responsible for pre but post synaptic adrenergic receptors. Norepinephrine is released in the brain as the locus coeruleus is stimulated and action potential occurs. It is also important for the long-term memory by influencing surges in specific neural circuits. When norepinephrine binds to a receptor and stimulates a nerve to respond, it works as a neurotransmitter. When it is originated in the adrenal medulla as a product of dopamine, it works as a hormone (Epinephrine and Norepinephrine, https://www.boundless.com/blog/biology/).
Epinephrine is another flight/fight hormone released when the body is under extreme stress. It is closely related to norepinephrine since it is synthesized from norepinephrine in the adrenal medulla. Norepinephrine and epinephrine act on beta and alpha receptors, the difference is that norepinephrine is primarily focused on alpha receptors, whereas epinephrine stimulates all three beta receptors as well as alpha receptor. Dopamine is the precursor molecule to both norepinephrine and epinephrine in the catecholamine synthesis pathway. This important neurotransmitter is produced several areas of the brain. The midbrain is host to the substantia nigra, which is a part of the basal ganglia and plays an important role in the reward system, voluntary motor movements, cognition and emotions. High levels of neuromelanin in dopaminergic neurons make substantia nigra appear darker than surrounding areas [45]. One function of dopamine is movement; the caudate and putamen create the striatum, which is a bundle of dopaminergic neurons that form the nigrostriatal pathway considered a facilitator of movement. The second function is that of the reward system, which originates in the ventral tegmental area of the brain. Wolfram Schultz's 1990 experiment demonstrated that dopamine release was associated more with unpredictability rather than with mere experience or a reward system. There are two main metabolites of dopamine: Norepinephrine and DOPAC, which is correlated with oxidative stress. Serotonin (produced in the enteric nervous system of the gastrointestinal tract and in the central nervous system) has many important roles in overall health and wellbeing. It is a chemical product of nerve cells and serves in the modulation of glutamate excitation through voltage potential regulation. Serotonin is a metabolite of 5-HTP; up to 90% of serotonin is produced in the gastrointestinal tract, although it is also found in platelets and throughout the central nervous system. Serotonin is involved with smooth muscle contraction, regulating cyclic body processes and nerval impulse transmission. Most agree serotonin is the primary neurotransmitter for mood regulation [46]. Serotonin must originate in the brain if it is to be utilized by the brain. It is widespread in its roles and influences mood, digestion, ambition, social behavior, memory and sexual function. Serotonergic hypo function has been demonstrated to be a neurochemical trait associated with or precursor to impulsivity and aggression [46]. Serotonin also acts as a precursor to melatonin, which plays a major role in the body's circadian rhythm and sleep cycles. Both dopamine and serotonin have a close relationship  [48]. The main metabolite of serotonin is 5-HIAA. In our study, we analyzed 5-HIAA to obtain insight into the production of serotonin and the potential absorption concerns due to the tryptophan metabolites.
Taurine is a trophic factor and assists in the survival of synaptic neurons. Taurine is a neuromodulator and is protective against glutamate-induced excitotoxicity through limiting increase of intracellular calcium levels. This is achieved by shifting the ratio of BCL-2 and BAD ratio in favor of cell survival and reducing endoplasmic reticulum stress. Taurine plays an integral part in regulation of the amount of sensory information received by the cortex. Taurine is partly responsible for helping the brain to turn off at night by activating GABA, it is stopping the thalamus (processing input from the senses) from waking up a sleeping person [49] [50]. Taurine is also involved in maintaining metabolic homeostasis in the body by reducing blood glucose levels and helps restore insulin sensitivity. Studies have demonstrated that taurine can reverse tinnitus, since many issues with hearing are a result of nerve cell communication with the brain as opposed to mechanical issues. Taurine assists in calcium restoration and control of auditory cells. Taurine is also involved in cortisol elimination, therefore mitigating stress responses within the body [49] [51] [52].
Histamine has many physiological roles within the body, one of which is the ability to increase excitability of the central nervous system. Studies have demonstrated that histamine can be a regulator of "whole brain" activity. Studies have also confirmed the potential of histamine to assist in mediation of alertness and electrographic arousal [53] [54] [55]. The hypothalamus's ventral preoptic center assists in turning off histaminergic tuberomammillary cells which connects to the sleep/wake cycle resulting in diurnal changes in brain function.
Many functions of the hypothalamus are regulated by histamine. Vasopressin is physiologically regulated by histaminergic neurons [54] [55]. Additional studies demonstrate the potential of histamine for regulating levels of ACTH, prolactin, and oxytocin [55].
Glycine is another inflammation regulator; it plays an important role in overall health and wellness of the body. Glycine can be produced by the liver in limited amounts and is used for detoxification. Glycine helps with synthesis of bile acid, amino acids, and assists in the building of DNA, RNA and protein. By being involved in the manufacturing of immune-related hormones, it also plays a role in immunological function. Glycine is found in the spinal cord, brain stem, retina, and throughout the central nervous system. It acts on inflammatory cells such as macrophages to suppress activation of transcription factors and the formation of free radicals and inflammatory cytokines. Glycine is not just an anti-inflammatory agent, but also an immunomodulator and cytoprotective agent [56]. Excessive amounts of glycine can lead to decreased energy, anxiousness, sleep difficulties, as well as immune dysregulation and digestive stress.  [57]. PEA has a tremendous impact on overall body alertness and physiological performance.

PEA (phenylethylamine) is an excitatory neurotransmitter that assists in
Emotions are regulated in limbic region and having a very high concentration of PEA makes an impact on motivation, emotions and socialization. Because it is an endogenous stimulant, PEA enhances dopamine, norepinephrine and serotonin. PEA causes fast impulse-mediated release of catecholamines (dopamine and epinephrine) and serotonin in the brain, resulting in rapid improvement of cognitive performance, attention, awareness, pleasure, libido, and overall sense of wellbeing [58]. Furthermore, increase in PEA improves alertness and higher cognitive functioning [48]. PEA can increase catecholamine levels and block re-uptake of dopamine, norepinephrine, and serotonin. This can bring restoration of energy as well as mood elevation. However, too much PEA can lead to anxiety. Studies demonstrate that those with ADHD will show either low or high levels of PEA. High levels can also be associated with anxiety, insomnia and mind racing [57] [59].

Demographic Data
Two basic cohorts of individuals were involved in present research-the Study

Analysis of Neurotransmitters
The urine test panel for study included 12 neurotransmitters applied to Study (n = 80) and Control (n = 117) groups. The urine collection was performed on the first day of the conference and again at the last day of the conference, urine col- and abstain from tobacco, avoid strenuous workouts, and not take supplements or medications (unless instructed otherwise by a physician). The urine collected was the second urine of the day collected 2 hours following wake up while still fasting. Two 5 ml tubes were collected to accommodate the volume needs. One vial contained a stabilizer as specified to maintain the integrity of the sample. The samples were kept refrigerated until processed in the reference lab. Urinary samples were processed blindly by a CLIA accredited reference laboratory, Pharmasan Labs (Osceola, WI) to quantitatively measure twelve urinary neurotransmitters: epinephrine (1), norepinephrine (2), dopamine (3), DOPAC (4), serotonin (5), 5-HIAA (6), glycine (7), taurine (8), GABA (9), glutamate (10), PEA (11), and histamine (12). The reference laboratory-generated participant reports and provided external control data (n = 117) with a comparative statistical analysis between control and study participants. In short, descriptive statistics of mean, standard deviation (SD), and Cohen's d (ɗ) effect size were calculated in Excel (Office 2013 v15, Microsoft, Redmond, WA). The effect size for each parameter between groups was assessed by calculating Cohen's ɗ. A ɗ value between ± [0.2 -0.5] is a small effect, ± [0.5 -0.8] a medium effect, and a value greater than +0.8 or less than −0.8 is a large effect. An ANOVA was performed using GraphPad Prism (v7, GraphPad Software, San Diego, CA) employing the Holm-Sidak's multiple comparison model to calculate the statistical significance (p value, α = 0.05) between control and sample for each group.

The MNRI Reflex Integration Therapy Modality
Each session was focused on a process of neurodevelopment called reflex integration session or module. The MNRI process included the following MNRI modules: 1) Reflex Repatterning-focuses on paving and improving the connectivity between the sensory and motor neurons in a reflex circuit [6] [7] that influence the sensory-motor milestones, motor programming, planning and control, and also cognitive skills [9] [20] [21].
2) Neuro-Structural Reflex and Immune System Integration-focuses on improving the functions of reflexes responsible for postural control, spine flexibility, abdomen, and neck and limbs musculature tone regulation, release of core tendon guard creating positive protection and the feeling of being secure, and immune system, creating the immunomodulatory effect aimed at improvement of functions of the T-1 immunity, cytokinesis, CD-4, CD-8 and other immune cells functions, anti-inflammatory effect, regulation of immunoglobulins (IgE, IgG and other) [13] [14] [15].
3) Neuro-Tactile Integration-focuses on the regulation and normalization of tactile sensitivity (hyper-or hypo-), coordination and integration of receptors, skin dermatomes, and overall peripheral and central nervous system for support of reflex repatterning and integration [21].

Results
The baseline data on analysis of neurotransmitters markers for individuals with global developmental disorders (Group 1), cerebral palsy, TBI, and seizures (Group 2), ADD and ADHD (Group 3), and anxiety disorders (Group 4) are presented in Table 2. The effect size was assessed using Cohen's ɗ (see Methods) to determine the change in neurotransmitter levels between pre-and posttreatment intervention.

Study Group 3
Study range from small to large effects (see Table 2, Table 4 and Figure 3).
Histamine (−0.6 medium effect) acts as an excitatory neurotransmitter and also serves an important role in the body's immune response. Elevated levels are commonly associated with fatigue, sensitivities, and immune stress. Histamine had a pre-mean score of 57 with standard deviation of 41. The post mean score was 38 with standard deviation of 17. Histamine received a p value of 0.0365, meaning that there is less than 4% probability that the difference in outcomes would be observed if the intervention had no benefit (see Table 2, Table 4, and Figure 3).
Taurine (−0.9 large effect). Taurine is a calming amino acid and can act as an agonist for both glycine and GABA. Elevated levels can be associated with symptoms of excessive energy, anxiousness, sleep difficulties, and oxidative stress [51] [62]. Taurine had a pre-mean score of 648 with standard deviation of 440. The post-mean score was 357 with standard deviation 170. The p value was statistically significant at 0.0407.
Taurine had a −0.9 change which would indicate a large Cohen's effect [51] [52] [63]. Taurine pre-mean score was 604 with standard deviation of 535. The post treatment mean was 230 with standard deviation of 190. The p value was 0.0153 which would translate into a 2% probability that our findings were due to chance (see Table 2, Table 5, and Figure 4).

Discussion
Our study demonstrated the impact on the neurological system from the MNRI treatment. The tested neurotransmitters are chemical messengers in our brain and body, delivering signals from one synapse to another. To ensure proper transmission from the presynaptic cell to the receptors of the postsynaptic cell the neurotransmitters must have appropriate genetic and chemical structure [29] [64]. Neurotransmitters participating in inhibitory or excitatory synapses determine functional properties of the underlying circuit. Depending on the specific pair of neurotransmitters and corresponding receptors, qualitative and quantitative features of the synapse vary due to the distinct kinetics and signaling cascades imprinted in the identity of the synaptic partners [64] [65]. This mechanism is determined by function of sensory neurons and, on other side, defines Neuroscience & Medicine   Taurine   Pre  Tx   N  10  10  10  10  10  10  10  10  3  10  10  3  10  3  3  10   Mean  7015 1003  263  3  8  37  1326  42  22  49  102  53  214  15  45  604   SD  8399 1240  113  1  3  24  638  18  10  31  102  17  151  11  31  535 Post Tx We have tested the following neurotransmitters: epinephrine (1), norepinephrine (2), dopamine (3), DOPAC (4), serotonin (5), 5-HIAA (6), glycine (7), taurine (8), GABA (9), glutamate (10), PEA (11), and histamine (12). Below the interpretation of neurotransmitter levels in pre-and post-intervention MRNI therapy for all study group (n = 80) individually compared to the Healthy Control group (n = 117) is presented.

Study Group 1
Study Group 1 consisted of participants with developmental disorders (n = 45).
The results of the neurotransmitters analysis show improvement in levels of the DOPAC, dopamine, norepinephrine, PEA, serotonin, and taurine, all having a 0.2 -0.3 impact, which would categorize as a small effect according to Cohen's method (see Table 1, Table 2 and Figure 1). This effect of reduction in excitatory neurotransmitter levels results in a decrease of inflammation in the neurological system, as well the reduction of neurodegenerative tendencies and positive tendency of restoration of nerve cell, which is similar to the findings of other authors [68] [69]. For instance, if serotonin as one of the modulators for glutamate causes significantly high level of glutamate, this could result in nerve dam-  [71]. There is also a known widespread negative effect when it concerns serotonin level increase influencing mood, digestion, ambition, social behavior, and memory [70] [72]. In the limbic region high concentrations and levels of PEA can lead to anxiety, and lower levels may lead to depression, focus issues, and lack of ability to think clearly. This imbalance may have a profound impact on also social interactions [73].
We found a small effect size increase in Epinephrine (see Table 1, Table 2 and  Table 1, Table 2 and Figure 1).
The impact of the MNRI intervention in this group was seen, particularly, in glycine and norepinephrine, at a 0.2 -0.3 effect, which would categorize as a small effect according to Cohen's method (see Table 1, Table 6 and Figure 2).
Glycine plays a role with the inflammatory response and immune response of the organism [74]. This outcome can be estimated as significant as the impact of glycine due to its function to act as an immunomodulator and being cytoprotective [74]. The effect of the MNRI therapy is found in the improvement of the cytokinesis processes corresponding to immune studies done in MNRI in 2008-2016 [13] [19] [75]. Norepinephrine is a part of the sympathetic response, so a reduction as our study demonstrates can be considered as an impacting change in the body's physical response to stress, which is coherent to other researches in the area of this neurotransmitter [76]. Subsequently, changes in histamine considered to be a "powerful" regulator of "whole brain" activity and many hypothalamic functions [54] [77] [78] also can be interpreted as a factor for higher integrative brain functions, improvement in brain connectivity and neuroplasticity, novelty-induced attention and arousal.
Similar data also was found in some studies of this neurotransmitter as decisive for brain development and basis for major adaptation to changing environments by comparing news with the remembrance of things past, also for physiology of danger recognition, and survival [78].  The data on regulating effect of the norepinephrine after the MNRI treatment was noted in reduction of negative chronic stress and clinically seen improvements in muscle tone regulation and in decrease of motor rigidity and pathology in reflex patterns (see Table 1, Table 6 and Figure 2). Clinical observation has also demonstrated regulatory effect of norepinephrine on mood, motivation for rehabilitation and more mobility and flexibility, better posture control and motor coordination. A significant reduction of seizure episodes during and after the MNRI intervention also took place, which corresponds to studies of the effects of this neurotransmitter on improvements on this health condition by other authors [79].

Study Group 3
Study Group 3 consisted of participants diagnosed with ADD/ADHD (n = 11).
The neurotransmitters post-tests after the MNRI intervention found large effect of decrease in taurine, medium effect decreases were found in epinephrine, glutamate, glycine, histamine and PEA. Small effect decreases were found in 5-HIAA, norepinephrine, and GABA. A medium effect increase was found in DOPAC levels (see Table 1, Table 3 and Figure 3). Interesting that our findings in this study group are more closely aligned to the overall down regulatory neu- Cohen's effect sizes were much larger in Study Group 3 (see Table 1, Table 3 and Figure 3). As before mentioned, these neurotransmitters can have an impact of increasing immune response and inflammation in the body resulting in a reduction of improper excitability in the physiological system. This result is similar to those that are described in studies carried by other authors [72] [73] [74].
As before mentioned in Study Group 1, theoretically regulation for PEA could reduce symptoms of anxiety [73]. We can conclude that the MNRI treatment intervention had more profound neurophysiolocial changes in this subpopulation of patients. Clinical observations show that the participants of this Study Group 3 demonstrated remarkably improved ability to focus, enhanced memory, improved visual-auditory processing, and more readily self-regulate reactivity and emotional responses. Administration of standardized behavioral and symptom assessment tools at school and home would be useful to further detect the potential correlation between the treatment intervention, neurotransmitter level changes, and behavioral and ADD/ADHD symptom changes. MNRI therapy may be considered as an evident informed manual therapy intervention for those suffering from ADD/ADHD.

Study Group 4
Study Group 4 consisted of participants diagnosed with anxiety disorders (n = 10). The post-test of neurotransmitters after the MNRI intervention found large effect decreases in taurine and medium effect decreases were found in histamine levels. Small effect decreases were found in DOPAC, GABA, and PEA (see Table   1, Table 4 and Figure 4). This study group had several pre-treatment neurotransmitter levels which were closer to those levels of the Healthy Control Group, thus, we would not expect to detect as many significant effect changes in these neurotransmitters in participants of this study group. The decrease in taurine and histamine as it is known, is especially beneficial to those patients suffering from anxiety disorders [80]. Histamine is believed to be a major neuroexcitation regulator and a significant decrease in levels of it would theoretically reduce excitability of the central nervous system [71] [79]. The impact of taurine in the cortex is significant as it plays a role in how much sensory information the brain receives [80] [81]. Thus, decreases in these particular neurotransmitters will give neurological homeostasis and thereby reduction in anxiety and improved quality of life in individuals with anxiety who undergo the MNRI treatment. The long-term work with post-trauma individuals shows highly significant results [16] [24] [81].
Small effect decreases were found in PEA, DOPAC and GABA.
High levels of PEA, as known, are associated with anxiety, insomnia and mind racing [57] [59]. Thus, small effect decreases in PEA after the MNRI therapies can explain the observed easier regulation of emotions and improvement in behavioral control, also increased the ability to calm down and stress resiliency. GABAergic and dopaminergic pathways are balancing excitatory-inhibitory process and neural network reconfigurations [40] [41] [82]. This explains the "calming down effect" and reduction of anxiety in participants of this Study Group. The effect of the DOPAC as a metabolite of dopamine, was in balance with the PEA metabolites [57] [83] was evidently allowing for rapid improvement of motivation, cognitive performance, ability to enjoy and play, and overall sense of wellbeing that we were observing in these participants. The normalization effect of these neurotransmitter levels lead to the restoration of energy, and mood elevation also is similarly described by other authors [84].
Overall, the post-MNRI results in participants in this group show tendency for regulation of the above neurotransmitters (see Tables 1-4, Table 6 and Figures   1-4) resulting in their calming down, decrease of hypervigilance, stress resilience increase, improvements in behavioral and emotional regulation, positive emotions, and cognitive processes control-attention, thinking, creativity, goal setting and easier decision making.

Conclusion
The present findings suggest that the MNRI treatment positively impacts central nervous, immune, and endocrine systems by regulation of the stress hormones and neurotransmitters regulation. The results indicate that through treatment using reflex patterns there is a reduction in the excitability within the nervous system, and reduction in the inflammatory response. All four study groups demonstrated positive changes that were generally small scale. The anxiety disorders group showed medium effect in the histamine response, and a large effect on the neurotransmitter taurine. The ADD/ADHD group had remarkable me-

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper. The scientists performing the qualitative and quantitate analysis of these research results were not privy to details regarding the treatment or suspected outcomes. They also had no vested interest in MNRI.