Nerve growth factor (NGF), a trophic factor involved in the development, maintenance and survival of the peripheral nervous system and the cholinergic neurons of the central nervous system, is significantly reduced in Alzheimer’s Disease (AD) patients (Aloe et al., 2012). Particularly basal forebrain complex (BFC) neurons are highly affected in AD patients. NGF protects BFC neurons in experimental trauma models and in age-associated cholinergic decline. Therefore, administration of NGF is investigated as a therapeutic option for AD patients (Aloe et al., 2012). NGF therapy is limited by hyperalgesia, limited bioavailability, and use of invasive methods including intracranial injection of adeno-associated virus-based gene delivery vector, which constitutively expresses human NGF. In this context, non-pharmacological modes of treatment such as meditation and Yoga have been considered as alternative approaches to treat neurological disorders. Yogic breathing (YB, also called Pranayama) is a collection of techniques to regulate breathing voluntarily. YB induces a strong relaxation response via vagal and parasympathetic stimulation (Jerath et al., 2006). Such responses are causally linked to rapid changes in gene expression, particularly to those genes controlling stress, inflammation, and metabolism. Saliva is a known source of NGF, and incidentally, Yoga and relaxation stimulate salivation. Saliva (0.75-1.5 liters/day in humans) contains numerous biologically active molecules to be potentially useful as diagnostic markers and as therapeutic clues. Salivary secretion regulates the digestive, nervous, immune, and respiratory systems. We conducted a pilot study to investigate whether YB could potentially stimulate salivary expression of NGF in cognitively normal healthy volunteers. This could serve as a model for future studies in AD patients.
A total of 20 volunteers (male and female), age 18 and above were recruited from Charleston Metro area following written informed consent. They were randomized (1:1) into YB and Attentional Control (AC) groups. (Exclusion criteria: breathing problems (inability to breath through nostrils, chronic bronchitis, emphysema and asthma), speech problems that would prevent chanting, inability to listen and follow exercise instructions, sinus congestion, Sjogren’s syndrome, chronic dry mouth due to medication or other conditions, and use of drugs that cause dry mouth). The study protocol was approved by the Institutional Review Board of the Medical University of South Carolina, Charleston, SC. In a single, 20-minute one-on-one session with the Yoga Instructor, the AC group quietly read a scientific text of their choice for 20 minutes whereas the YB group performed 10 minutes of Om Chanting followed by 10 minutes of Thirumoolar Pranayamam (TP) (Verse 568) (Rajasekaran and Narayana, 2006; Thirumoolar, Himalayan Academy Publications, Hawaii) as explained in the Supplementary material. Whole saliva samples were collected at 0 and 20 min from all participants and were used to measure NGF in duplicate by ELISA (enzyme-lined immunosorbent assay). The average pre NGF values (0 min) were compared to the average post NGF values (20 min) between groups (AC and YB). In the AC group, the average difference between pre and post measures increased by a mean(SD) of 0.67(1.40) points. In the YB group, the average difference between pre and post measures increased by a mean (SD) of 6.34(11.02) points. A mixed model with repeated measures was used to estimate the between-group differences over time. Both duplicate post measures were used (as the repeated measures), and the model was adjusted for the baseline (average of the two duplicate pre NGF values). We also allowed for the variances within the AC and YB groups to be different. This modeling process suggested that the control group had NGF post values 7.7 points lower than the YB group (95% CI: −18.0, 2.7). A scatter-plot of NGF from these two groups is presented in Figure S1. The results were further evaluated by Western blotting which showed that 60% of the samples showed a marked increase in NGF level only in the YB group but not in the AC group (Figure S2). These results indicate that NGF could be stimulated in saliva by acute YB practice, and that further study may be warranted.
Chanting Om (Pranava Pranayamam) is an ancient cultural practice and believed by some to be associated with physical and emotional well-being (Kumar et al., 2010). In ancient Tamil texts and in present day among several populations worldwide, the symbol Om assumes a great significance. It has been suggested that chanting Om increases the cutaneous peripheral vascular resistance, auricular branch vagal nerve stimulation (VNS), and improves mid-latency auditory evoked potential (Jerath et al., 2006), which is lacking in about 40% of Alzheimer’s patients. VNS was beneficial in AD patients as it enhanced cognitive functions. Electrical pulsing for VNS showed deactivation of limbic brain regions. Chanting Om also caused a similar deactivation of the limbic brain regions, amygdala, hippocampus, parahippocampal gyrus, insula, orbitofrontal, and anterior cingulate cortices and thalamus. TP is from one of the ancient Tamil texts referring to the benefits of breathing exercise, Thirumanthiram (Rajasekaran and Narayana, 2006; Thirumoolar, Himalayan Academy Publications, Hawaii). Yogic breathing increases vagal tone, increases parasympathetic dominance, decreases sympathetic discharges, and produces theta waves in brain (Jerath et al., 2006). This is the first time the molecular expression of a neurotrophic factor NGF is reported in response to chanting Om and Thirumoolar Pranayamam. How could the salivary NGF be transported to the CNS? We hypothesize that, NGF produced by the salivary glands is taken up by 1) the innervating sensory and motor neurons of the oral organs for retrograde axonal transport to the cell body and transcytosis to reach the central neurons, and 2) the sublingual absorption route into the bloodstream to reach other distal target organs. These mechanisms are well within the theoretical framework of production and transport mechanisms established for neuromodulators. It is to be emphasized here that this pilot study is the first ever attempt to stimulate endogenous NGF expression by non-invasive methods. Our findings could lead to future studies on using salivary neuromodulators and biomarkers as outcome measures of Yoga interventions.
Supplementary Material
Figure S1. Induction of Salivary Nerve Growth Factor by Yogic Breathing. Equal volume of saliva from Attention Control (n=10) and Yogic Breathing group (n=10) were subjected to ELISA in duplicates (Abnova). The amount of NGF (pg/mL) from AC (Blue) and YB (Red) groups at 0 minutes (Circle) and 20 minutes (Triangle) are plotted.
Figure S2. Induction of Salivary Nerve Growth Factor by Yogic Breathing. Equal volume of saliva from Yogic Breathing group (n=10) (A) and Attention Control (n=10) (B) were subjected to Western Blotting analysis using anti-NGF antibody (Cell Signaling Technology). This antibody detected both the mature NGF (~13kDa) and pro-NGF (~26kDa). After Western blotting, the membranes were stained with Ponceau S to show the level of protein loading (shown at the bottom of each blot).
Footnotes
Conflict of Interest: None
References
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Supplementary Materials
Figure S1. Induction of Salivary Nerve Growth Factor by Yogic Breathing. Equal volume of saliva from Attention Control (n=10) and Yogic Breathing group (n=10) were subjected to ELISA in duplicates (Abnova). The amount of NGF (pg/mL) from AC (Blue) and YB (Red) groups at 0 minutes (Circle) and 20 minutes (Triangle) are plotted.
Figure S2. Induction of Salivary Nerve Growth Factor by Yogic Breathing. Equal volume of saliva from Yogic Breathing group (n=10) (A) and Attention Control (n=10) (B) were subjected to Western Blotting analysis using anti-NGF antibody (Cell Signaling Technology). This antibody detected both the mature NGF (~13kDa) and pro-NGF (~26kDa). After Western blotting, the membranes were stained with Ponceau S to show the level of protein loading (shown at the bottom of each blot).