Brain and Pharyngeal Responses Associated with Pharmacological Treatments for Oropharyngeal Dysphagia in Older Patients

Impaired pharyngo-laryngeal sensory function is a critical mechanism for oropharyngeal dysphagia (OD). Discovery of the TRP family in sensory nerves opens a window for new active treatments for OD. To summarize our experience of the action mechanism and therapeutic effects of pharyngeal sensory stimulation by TRPV1, TRPA1 and TRPM8 agonists in older patients with OD. Summary of our studies on location and expression of TRP in the human oropharynx and larynx, and clinical trials with acute and after 2 weeks of treatment with TRP agonists in older patients with OD. (1) TRP receptors are widely expressed in the human oropharynx and larynx: TRPV1 was localized in epithelial cells and TRPV1, TRPA1 and TRPM8 in sensory fibers mainly below the basal lamina. (2) Older people present a decline in pharyngeal sensory function, more severe in patients with OD associated with delayed swallow response, impaired airway protection and reduced spontaneous swallowing frequency. (3) Acute stimulation with TRP agonists improved the biomechanics and neurophysiology of swallowing in older patients with OD TRPV1 = TRPA1 > TRPM8. (4) After 2 weeks of treatment, TRPV1 agonists induced cortical changes that correlated with improvements in swallowing biomechanics. TRP agonists are well tolerated and do not induce any major adverse events. TRP receptors are widely expressed in the human oropharynx and larynx with specific patterns. Acute oropharyngeal sensory stimulation with TRP agonists improved neurophysiology, biomechanics of swallow response, and safety of swallowing. Subacute stimulation promotes brain plasticity further improving swallow function in older people with OD.


Introduction
The World Health Organization defined oropharyngeal dysphagia (OD) as the difficulty or inability to move a bolus safely and effectively from the oral cavity to the esophagus, and can include aspirations, choking, and residue.Its prevalence is very high in the older population, affecting between 27 and 91% of people of 70 years or older depending on the phenotype and the health condition.Although the diagnosis of OD can be easly performed by well-established clinical methods and instrumental evaluations, in the healthcare facilities it is rarely systematically screened and treated, and awareness among healthcare professionals is still insufficient [1].The etiology of OD on older patients is associated with several concomitant risk factors with neurogenic and neurodegenerative processes, and sarcopenia.Older people have many other factors associated with OD such as decreased cortical plasticity, olfaction and taste dysfunction, poor dental status, impaired muscular function, decreased saliva secretion and tissue elasticity, and skeletal changes predisposing to swallowing dysfunction [2].
OD is related with the development of several health complications and worsens quality of life.Impaired efficacy of swallow (oral and pharyngeal residue), related to weak tongue thrust and delayed time to upper esophageal sphincter opening (UESO), is associated with the development of malnutrition and dehydration as patients are not able to ingest the total amount of nutritional and hydration requirements to maintain a healthy status.On the other hand, 1 3 impaired safety of swallow (penetrations and aspirations to the airway), mainly related to a delay in the time to laryngeal vestibule closure (LVC), is associated with the development of respiratory infections, aspiration pneumonia and an increase in mortality [3].Despite the high prevalence of OD and its negative consequences, there is still no specific pharmacologic active treatment for the clinical practice.
OD was recognized as a geriatric syndrome by the Dysphagia Working Group, a committee of members from the European Society for Swallowing Disorders and the European Union Geriatric Medicine Society, due to its high prevalence, its relationship with many comorbidities and their poor outcomes, including malnutrition, respiratory infections and aspiration pneumonia, functional disability and frailty, institutionalization, increased hospital readmissions, and mortality [4].
Our studies on the pathophysiology of OD started with the century.In 2004 we developed and patented (US Patent US8251927B2) a computer-implemented system for the analysis of the swallowing process in humans during VFS that allows us to obtain robust metrics of the normal vs impaired swallow response [5].Our metrics were developed from those previously described [6].With this methodology, we first explored the pathophysiology of OD in frail older people and found a high prevalence of aspirations and residue that were associated with weak tongue bolus propulsion forces and delayed LV closure, respectively [7].These results were later confirmed in several phenotypes of older patients [8,9].Accordingly, we thought that treatment of OD in these patients should be targeted to improve these critical biomechanical events.The next step was to explore the impaired neurophysiology of the swallow response and we found aging caused a decline in pharyngeal sensitivity to electrical stimulation, a reduction in the salivary concentration of SP and CGRP, and increased latency and reduced amplitude of pharyngeal sensory evoked potentials (pSEP) peaks, and -for the first time-we proved all of these impairments were more severe in older patients with OD [10,11].We found the impairment of the oropharyngeal sensory function is closely related to the alteration of the OSR seen in older people and patients with neurologic or neurodegenerative disease and OD.There is a correlation between oropharyngeal sensory threshold and age [12], that could be explained by the reduction of the number of small diameter, myelinated fibers in the internal superior laryngeal nerve observed in older people [13], although more research is still needed in this area.Oropharyngeal sensory inputs travel through the afferent pathway of the neurophysiological response of swallowing, which transmits the stimulus from the oropharynx to the sensory cortex and the central pattern generator (CPG), located in the upper medulla, and triggers the OSR through the efferent pathway [14] (Fig. 1).According to this paradigm, we hypothesize that if the afferent pathway is impaired, as has been described in several phenotypes of patients with swallowing disorders, the efferent output may also be affected, resulting in impaired OSR [15], suggesting that the stimulation of the afferent pathway could be a good therapeutic target to improve the OSR in older patients with OD.
Another important direction was to discover a peripheral biomarker in saliva that facilitated both the diagnosis and the degree of treatment response.Similar to our results, studies described a lower basal concentration of SP in the saliva of patients with OD, as a consequence of aging or neurodegenerative disease, compared to those who do not have OD [16].In addition, a higher concentration of these neuropeptides (SP and CGRP) in saliva has been demonstrated to be a good marker for a positive response to neurostimulation treatments such as capsaicin [17,18].
Main effective and evidence-based treatment for OD in the older population is compensatory, and aims to compensate biomechanical swallow impairments through the use of thickeners for fluids (viscosity adaptation) and texture-modified diets for solids (textural adaptation) in order avoid aspiration and choking, and improve nutritional status.In addition, oral health and hygiene also plays a key role to avoid respiratory infections.This therapeutic strategy has been defined as the minimal effective treatment to be provided to this population and has already shown positive results [1,19].Finally, systematic reviews provided evidence that swallow function exercises can improve deglutition function, especially Shaker exercise, chin tuck against resistance exercise, and expiratory muscle strength training [20].New treatments aiming at recovering the swallowing function are under research showing promising results, and the near future will provide us with methods to stimulate the swallow response with pharmacological or physical stimuli [21].
The Nobel Prize in Physiology or Medicine 2021 was awarded jointly to David Julius and Ardem Patapoutian "for their discoveries of receptors for temperature and touch" [22].David Julius utilized capsaicin, a pungent compound from chili peppers that induces a burning sensation, to identify a sensor in the nerve endings of the skin that responds to heat that was named TRPV1.TRPV1 was the first but not the only receptor discovered at Julius' lab: the TRPA1 receptor was identified with another natural compound, wasabi [23] and, independently of one another, David Julius and Ardem Patapoutian used the chemical substance menthol to identify TRPM8, a receptor that is activated by cold [24,25].These natural compounds provide a window into molecular mechanisms of somatosensation and pain, and open up a potential field for agonists of these TRP receptors to be used in clinical practice such as the treatment of swallowing disorders in aging.
The aim of this review is to summarize the experience of our research group on the localization and expression of TRP receptors in the human oropharynx and larynx, and the therapeutic effects of pharyngeal sensory stimulation by pharmacological stimuli (TRPV1, TRPA1, TRPM8 agonists) and to discuss their pharmacodynamics, rationality, action mechanism and research perspectives in patients with neurogenic oropharyngeal dysphagia and older people.

Pathophysiology of Oropharyngeal Dysphagia in Older People
In recent years, we have demonstrated that the pathophysiology of OD in older people involves not only biomechanical impairments but also neurophysiological ones.

The Impaired Biomechanics of Swallowing in Older People
In terms of biomechanics, the total duration of the oropharyngeal swallow response (OSR) during voluntary swallow in young healthy subjects is < 740 ms with fast time to LVC (< 160 ms, measured from the opening of the glossopalatal junction (GPJ) as time 0 to the closure of the LV) and to UESO (< 220 ms, measured from the opening of the GPJ to the opening of the UES), and strong lingual propulsion force (> 0.33 mJ) leading to high bolus velocity in the hypopharynx (> 35 cm/s) [5].
The impairment in biomechanics of swallowing in patients with OD is characterized by a delay in the initial deglutition phase (reconfiguration from the respiratory to the digestive pathway) which increases the overall duration of the OSR [5,26,27].The most significant changes are the delay in the time to LVC, the main biomechanical event that protects the respiratory tract during swallowing, and also a delay in time to UESO, both contributing to aspirations and penetrations of the bolus into the respiratory tract during the pharyngeal phase [5,26].In addition, other common alterations observed in older patients with OD is decreased lingual propulsion force, and reduced bolus velocity and kinetic energy as a consequence of sarcopenia of the tongue and pharyngeal muscles, further increasing prevalence of oropharyngeal residue [5,8,28,29].We found impairment of the biomechanics of swallow response in older patients with OD was mainly characterized by prolonged total duration of swallowing (1013 ± 53 ms) and severe delay in time to LVC (476 ± 48 ms) and to UESO (403 ± 45 ms) (Fig. 2a) [5,8,30].Similarly, patients with post-stroke, dementia and Parkinson's disease and OD also showed a similarly delayed time to LVC (post-stroke patients: 416 ± 129 ms; patients with dementia: 398 ± 117 ms; patients with Parkinson's disease: 293 ± 90 ms) [28,31,32].We found time to LVC was the critical biomechanical event of the swallow response that protects the airway during deglutition, and a LVC cutoff time > 340 ms in older and post-stroke patients (diagnòstic accuracy AUC = 0.78; sensitivity = 0.96; specificity = 0.52) and those with dementia (AUC = 0.71; sensitivity = 0.64; specificity = 0.33) [28,31,33] and > 260 Parkinson disease patients (AUC = 0.80; sensitivity = 0.93; specificity = 0.57) [32] predicts unsafe swallows in these phenotypes of patients.
Finally, we also described that the spontaneous swallowing frequency (SSF) is significantly reduced by up to 50% in independently living older people from the community and with a significant negative correlation between SSF and age [34].In addition, patients with post-stroke OD of several typologies of stroke showed a significant reduction in SSF compared with post-stroke patients without OD [35].Spontaneous swallowing physiologically contributes to the protection of the respiratory tract by clearing saliva.Its reduction could lead to an increase in pharyngeal secretions and the risk of oropharyngeal aspiration.In addition, we hypothesize that this could be an indicator of sensory dysfunction as we have previously found that stimulation of the sensory pathway through TRPV agonists (Capsaicin 10 µM) improves SSF in post-stroke patients with OD (pre-capsaicin: 0.41 ± 0.32 swallows/min vs. post-capsaicin: 0.81 ± 0.51 swallow/min; p = 0.0003) [34].
In summary, the two main biomechanical alterations in swallowing function associated with OD in older persons are prolonged swallow response with delayed time to LVC, and reduced SSF, relevant factors increasing the risk of aspirations and unsafe swallow in older patients.

The Impaired Neurophysiology of Swallowing in Older People
Neurophysiological impairments in the swallow response in older people have been less studied, and in 2015 we started a line to study the neurophysiology of the afferent sensory pathways involved in pharyngeal sensory function and that of efferent motor pathways controlling the motor output of the swallow response.
To study the sensory pathway, we used pharyngeal sensory evoked potentials (pSEPs) to intrapharyngeal electrical stimulation [10] that are characterized by having four peaks: the early peaks (N1 and P1) represent the conduction of the sensory stimuli from the pharynx to the brain cortex, while the late peaks (N2 and P2) represent the integration of the stimuli in the brain (Fig. 2b).In healthy young people the latency from the stimulus to each peak is about 80 ms for N1, 140 ms for P1, 190 ms for N2, and 300 ms for P2.Regarding the amplitudes, N1-P1, P1-N2 and N2-P2 are about 6 μV, 3 μV and 10 μV, respectively.In addition, deglutition sensory cortex representation is symmetric and bilateral [10,36].In contrast, motor cortex representation in young people is asymmetrical and bilateral, which means that the voluntary motor control of swallowing depends on hemispheric dominance, which is independent of handedness and unique for each person, however more studies are needed in this field [37].
We found the aging process impairs pharyngeal sensory function and increases the pharyngeal sensory threshold to electrical stimuli, being significant in older patients with OD (11 mA) compared with young healthy people (6 mA).This impairment in pharyngeal sensitivity negatively correlates with the concentration in saliva of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) suggesting these peptides could be used as biomarkers of reduced pharyngeal sensory function in older patients [11].In addition, the aging process increases the latency and reduces the amplitude of the pSEPS peaks, more so in older people with OD in which the latency of N1 and N2 peaks increases to 80 ms and 250 ms respectively, and the amplitudes of P2-N2 are reduced to 4 μV [10] (Fig. 2b).For the first time, we demonstrated that older people present a significant decline in pharyngeal sensory function, more severe in older patients with OD.This sensory impairment might be a critical pathophysiological element and a potential target for treatment of swallowing dysfunction in older patients.We found similar results in older patients with chronic post-stroke OD.In this case, patients showed an increased latency and reduced amplitude of pSEPs, but also a loss in the symmetry of the sensory potentials and their cortical representation when comparing both brain hemispheres, the ipsilesional hemisphere being the one that presented the reduced values [36].In addition, the pharyngeal motor evoked potentials remained asymmetric in 26.7% of post-stroke patients with chronic OD without physiologic hemispheric dominance and with a significant reduction of cortical excitability of efferent pathways [38].Finally, our group has described, also for the first time, that these impairments in the efferent pathway and brain excitability have also been observed in older patients with OD.In summary, the main alterations in the neurophysiology of the swallow response in older patients with OD are characterized by reduced oropharyngeal sensitivity, increased conduction and cortical integration latency of sensory inputs and reduced brain activation and excitability of cortical swallow motor areas.

Oropharyngeal Sensory Innervation and Structures
The human oropharynx is covered by three types of mucosa depending on their main function: (1) masticatory mucosa, covered by a stratified parakeratinized epithelium with a very thin submucosa.It covers hard structures that support high tensile and stretching forces, such as the gums or the hard palate; (2) lining mucosa, covered by a non-keratinized stratified epithelium with a thick submucosa, sometimes divided by the lamina propria.This type of mucosa covers most of the structures of the oral cavity and oropharynx, and (3) specialized mucosa, containing filiform papillae, covered by parakeratinized stratified epithelium, and fungiform, calciform and foliate papillae, covered by nonkeratinized stratified epithelium.It covers the dorsum and sides of the tongue and the soft palate (Fig. 3) [21,39,40].
The human oropharynx and larynx are innervated by afferent fibers of the cranial nerves (CN) V, VII, X and IX: two thirds of the tongue are innervated by the lingual nerve, formed by the lingual branch of the CN V and the chorda tympani of the CN VII; the base of the tongue, the tonsils and the foliate papillae are innervated by two of the four subdivisions of the lingual branch of the CN IX, which also innervates the lingual face of the epiglottis; the lateral and posterior pharynx walls are innervated by the pharyngeal plexus (formed by the pharyngeal branch of the CNs IX and X); and finally, the hypopharynx and the larynx are innervated by the internal superior laryngeal nerve of the CN X [21,[41][42][43][44].
In addition, there are several structures and receptors located in the oropharyngeal mucosa that have an important role in the integration of different sensory stimuli (such as chemical, mechanical and thermal).Initiation and modulation of swallowing requires sensory feedback from the afferent stimuli from the alimentary bolus and its physicochemical characteristics: (a) the Meissner corpuscles perceive painless light mechanical stimuli and are disposed within the papillae in different directions [45][46][47]; (b) the Merkel cells perceive the lightest touch without pain and are found alone or in groups in the basal layer of the epithelium, always bound to the basal lamina [48][49][50][51]; (c) the Pacini corpuscles perceive deep mechanical stimuli without pain and are found in the deepest layer of the submucosa [52,53]; (d) the Krause bulbs perceive light touch and cold stimuli and are found within the papillae [54,55]; (e) the free nerve endings are located below the basal lamina and, to a lesser extent, penetrating the epithelium.They are generally Aδ and C type and have the role of perceiving painful, chemical and thermal stimuli [48,56,57]; and (f) the taste buds, which distinguish between the five basic tastes: salt, sweet, bitter, sour and umami, and are found on the fungiform papillae of the two anterior thirds of the tongue and the soft palate, in the rifts of the circumvallate papillae and on the foliate papillae of the tongue base (Fig. 4) [58][59][60].And we are Fig. 3 Representation and immunohistochemistry images of the two main types of epithelium found in the human oropharyngeal mucosa: keratinized epithelium, such as the lingual mucosa (at the top) and nonkeratinized epithelium, like the epiglottis mucosa (at the bottom) especially interested in these free sensory nerves that could potentially express TRP receptors and this was unknown in humans when we started our studies on their location and expression in the human oropharynx and larynx.

Localization and Expression of TRP in the Human Oropharynx and Larynx
The presence of molecular receptors in the sensory nerves that innervate the oropharyngeal mucosa enable the perception of thermal and chemical stimuli.The TRP are the main family of sensory receptors found in the human oropharynx.Activation of these receptors, due to their capacity to perceive a wide spectrum of sensory stimuli, initiates the transmission of the sensory input from the oropharynx to several regions of the CNS (the sensory cortex and the dorsal swallowing group, DSG, located in the CPG) through the sensory nerves V, VII, IX and X.
The first TRP receptor identified was the subfamily vaniloid member 1 (TRPV1) [61].TRPV1 is mainly expressed in the afferent Aδ and C fibers and has been found in many human nerves (such as dorsal root ganglia and trigeminal ganglia) and non-nerve (epithelial cells) tissues.In addition, co-expression with some pro-inflammatory neuropeptides like SP and CGRP has been reported [62][63][64].In the human oropharynx and larynx TRPV1 are found in the plasma membrane of the mucosa epithelial cells, in greater amounts in the superficial layers and decreasing through the intermediate layer cells to the basal lamina.In addition, they are found in the Aδ sensory fibers innervating the mucosa and in some leukocytes in the submucosa.We found TRPV1 in the lingual mucosa, specifically on the membranes of the stratum basale and stratum spinosum cells of the filiform papillae epithelium and in the more superficial cell layers of the stratum granulosum and stratum corneum with a weaker signal expression (Fig. 5).Finally, we found the expression levels of TRPV1 receptors decreased from the tongue to the epiglottis: 25.43 × 10 -5 ± 2.12 × 10 -5 cycle threshold (ct) (tongue), 20.97 × 10 -5 ± 2.06 × 10 −5 ct (pharynx) and 16.59 × 10 -5 ± 1.49 × 10 −5 ct (epiglottis) [65].
Following the description of TRPV1, the noxious cold stimuli receptor was identified: the transient receptor potential channel subfamily ankyrin member 1 (TRPA1), formerly called ANKTM1.TRPA1 co-localizes with TRPV1, SP and CGRP in a subpopulation of C-fiber sensory neurons from the nodose, trigeminal and dorsal root ganglia (DRG) [66,67].TRPA1 can also be found in fibroblasts and epithelial cells and bronchial walls [68,69].Within the human oropharynx, we found TRPA1 in the sensory nerve fibers below the basal lamina and the nerve fibers that penetrate the epithelium, the Langerhans cells of the stratum spinosum of the lingual mucosa, the lingual surface of the epiglottis mucosa and the intermediate layer of the pharyngeal mucosa.In addition, TRPA1 is located in the sensory fibers that innervate the blood vessels that irrigate the submucosa (Fig. 5).We found TRPA1 was similarly expressed in the different oropharynx regions studied: 2.74 × 10 -5 ± 7.81 × 10 −6 ct (tongue), 1.49 × 10 -5 ± 2.68 × 10 −6 ct (pharynx) and 2.43 × 10 -5 ± 7.97 × 10 −6 ct (epiglottis) [65].
Unlike TRPA1, TRPM8 is found in a subpopulation of small diameter peripheral sensory nerves without coexpression with TRPV1 or CGRP [25].Regarding the oropharynx, we found TRPM8 was mainly expressed in the nerve fibers innervating the surface mucosa of the tongue, the oropharynx and the lingual face of the epiglottis.These fibers, either in bundles or individually, are generally located below the epithelium but can occasionally cross the basal lamina and penetrate the deeper layers of the epithelium.They can also be found in the nerve fibers innervating the blood vessels that irrigate the submucosa (Fig. 5).It is important to note that we did not find mRNA expression of this receptor in any of the studied oropharynx regions as we analyzed this using mucosa samples and the soma of the sensory nerves are located in the brainstem [70].

In-Vitro Bioassay of the Pharmacology of TRPV1 Receptors. In Vitro Desensitization of TRP Receptors
Once we had identified these receptors as potential pharmacological targets, the aim of our next study was to explore the pharmacodynamics and the potential mechanisms of desensitization of agonists for TRPV1 receptors, in a bioassay using human PC-3 cells prior to performing a clinical study.In this bioassay, we compared the pharmacodynamics of capsaicin (Tocris, Bristol, UK), natural capsaicinoids (McIlhenny Co, Avery Island, LA, USA), and piperine (Sigma Aldrich, St Louis, MO, USA), studied the effect of desensitization after repeated exposure and described the effect of a specific TRPV1 antagonist, SB366791 (Tocris, Bristol, UK).
We found TRPV1 receptors are successfully stimulated by capsaicin, piperine, and natural capsaicinoids in a concentration-dependent manner and determined that the half maximal effective concentration (EC 50 ) needed to stimulate the TRPV1 receptor and was 4.14 × 10 −4 M for piperine, 1.90 × 10 −6 M for capsaicin and 4.36 × 10 −6 M for natural capsaicinoids.With these results we inferred that the in vitro therapeutic potency of these three agonists would be capsaicin and natural capsaicinoids were at a similar level and both more than piperine (capsaicin≈natural capsaicinoids > piperine) (Fig. 6).In addition, we demonstrated that the activation induced by the agonists was caused by specific stimulation of the TRPV1 receptors as no response was observed after 5-min pre-incubation with the specific TRPV1 receptor antagonist SB366791, Activation induced by capsaicin was reduced to 74.7 ± 2.9% and by piperine to 100.0 ± 0.005%.Finally, we also observed a significant reduction in the specific activation, suggesting in vitro desensitization effects in isolated human PC-3 cells, after repetitive stimulation with capsaicin (38.3 ± 4.1%) and piperine (67.6 ± 5.3%) [71].

The Discovery of TRP and the 2021 Nobel Prize in Physiology or Medicine
The 2021 Nobel Prize in Physiology or Medicine was jointly awarded to Drs David Julius and Ardem Patapoutian "for their discoveries of receptors for temperature and touch".[22].Prior to their discoveries, our understanding of how the nervous system perceived our environment still contained a fundamental unsolved seminal question: How are temperature and touch sensed and converted into electrical signals in the nervous system?.And this is the research question that the two Nobel Laureates in Medicine or Physiology 2021 have answered during their scientific careers.
In the latter part of the 1990s, Dr. Julius began to use capsaicin, a pungent compound found naturally in chillies which induces a burning sensation, to identify a receptor in the nerve endings of the skin that responds to heat.Dr. Julius and his co-workers created a library of millions of DNA fragments corresponding to genes that are expressed in sensory neurons that can react to pain and heat and they hypothesized that this library would include the DNA fragment that would encode the protein capable of reacting to capsaicin.They introduced individual genes from this collection into cultured cells that normally do not react to capsaicin and, after laborious research, they identified a single gene that was able to make cells sensitive to capsaicin [61].This and other experiments revealed that the identified gene encoded a new protein, and that this new receptor, called TRPV1, detected heat and was activated at temperatures perceived as painful.In addition, they saw that TRPV1 channels were receptors that could be activated simultaneously by a wide range of harmful ligands such as temperature, acid and capsaicin, and this aspect of polymodal sensor made this receptor extremely important for its role in the detection of somatosensation and pain.The TRPV1 was the first but not the only receptor discovered in the Julius Laboratory.Independently of each other, David Julius and Ardem Patapoutian used another natural compound, menthol, to identify TRPM8, a receptor that was shown to be activated by cold [24,25].They also discovered TRPA1, the wasabi receptor [23], a fascinating receptor known as a sensor for pain and itching in humans, as well as a sensor for environmental irritants and involved in various protective responses such as tears, respiratory tract resistance and cough.A common feature of all these TRP receptors is that they have very powerful natural agonists such as capsaicin for TRPV1, piperine or cinamaldehyde for TRPA1, and menthol for TRPM8.Last June 2 and 3, 2022, we had the honor to receive the visit of Dr. David Julius in Barcelona, Catalonia, Spain (Fig. 7).On June 2, Dr Julius gave a webinar offered by CIBER, the Spanish research consortium, to all its members, and moderated by the scientific director of the CIBERehd [72], Dr. Rafael Bañares.To the 300 attendees, Dr. Julius explained his discovery, focusing on the use of natural TRP receptor agonists for the study of the molecular mechanisms of pain, and on his advances in the study of the TRPA1 receptor, which plays an important role in the perception of a wide variety of irritant substances and in the transmission of sensory impulses related to neurogenic inflammation.In addition, Dr. Pere Clavé explained his experience in the use of the TRPV1, TRPA1 and TRPM8 receptors as therapeutic targets for the active treatment of pharyngolaryngeal sensitivity alterations in older and neurological patients with OD.In the afternoon, at an academic event, part of the celebrations of the 150th anniversary of the Acadèmia de Ciències Mèdiques de Catalunya i Balears (ACMCB), Dr. Julius gave a lecture on translating the role of TRP receptors into clinical practice, especially in the field of pain.He discussed the possibility of developing new strategies to treat chronic pain more accurately and effectively and described biomarkers that allow us to objectively classify pain and measure the analgesic effect of TRP-based compounds [73].We collected the following main messages from his visit: (a) the presence of TRP receptors in the sensory nerves that innervate the oropharyngeal mucosa allow the perception of thermal and chemical stimuli that are involved in protective reflexes, for instance a bolus that is too hot or too cold; (b) TRP channels are very sophisticated biophysical signaling structures that can integrate information that is really important for the sensitization of the afferent nerve fibers; (c) a common feature of these TRP receptors is that they have powerful natural agonists; (d) natural products such as capsaicin, wasabi and menthol provide a window into molecular mechanisms of somatosensation and pain, (e) TRPV1 is a polymodal receptors activated by several factors such as temperature > 43 °C, protons and pH < 5.5 and capsaicin, (f) molecules like capsaicin (chili pepper), menthol (mint), isothiocyanates (cruciferous vegetables) or thiosulfinates (garlic) found in natural products are actually perceived as pain and not as taste following a gustatory response, as an exemple, when you eat chili pepper what you experience is pain, (g) the neuropeptides, such as substance P and CGRP, secreted peripherally as a consequence of the activation of C-fibers expressing TRPA1 and other nociceptors, decrease the activation threshold of TRP channels and also enhance the sensitivity of neurofibers.
Finally, on June 3rd 2022, our research group organized a scientific meeting at the Hospital de Mataró, where the group was able to discuss its line of research on the potential of TRP receptors in the human oropharynx with Dr. Julius.

Our Hypothesis of Action Mechanisms in the Swallow Response in Humans and Our Methodology for Clinical Trials
With the evidence collected from all these studies, we hypothesized that oropharyngeal stimulation with Fig. 7 Photograph taken on June 2, 2022 during the visit of Dr. David Julius in commemoration of the 150 anniversary of the Academia de Ciencies Mèdiques i de la Salut de Catalunya i Balears in Barcelona, Catalonia, Spain.From left to right: Dr. Pere Clavé, Dr. David Julius and Dr. Noemí Tomsen pharmacological or natural TRP agonists, such as capsaicin (TRPV1), piperine (TRPV1/A1) or menthol (TRPM8), would stimulate the TRP channels found in the human oropharynx.This stimulation could occur in different ways depending on the type of the agonist used: (1) natural agonists for TRP receptors or chemical stimulants capable of trespassing the epithelial barrier would elicit their effect either directly on subepithelial or intraepithelial nerves, or through release of a second messenger, such as anandamide, produced by responsive epithelial cells [74]; (2) chemical stimulants unable to permeate through epithelia would elicit their effect either through second messengers [75,76] or directly on intraepithelial nerves (Fig. 8).In this way, epithelial cells could work as amplifiers of chemical stimuli.The direct contact between epithelial cells and nerves seen in studies using electron microscopy [57,77,78] could support the idea of second messengers produced by epithelial cells while it does not deny the direct activation of nerve receptors by TRP agonists or chemical substances reaching deep epithelial layers.Once excited, the sensory nerves would release pro-inflammatory factors (such as SP and CGRP) that would sensitize the area and promote swallow and cough reflex if needed [79].The evidence collected in our studies on human tissue points to a dense submucosal innervation in contrast to scarce cases of intraepithelial fibers (Fig. 8).Other authors found TRPV1 and TRPA1 receptors were co-expressed in neurons with CGRP and SP.SP is known to be a neuropeptide that enhances swallow and cough reflex and probably acts as a neurotransmitter in pharyngeal mucosa in response to local stimuli [80].However, more basic and clinical studies on the mechanism of action of TRP agonists are needed in order to confirm or refute our hypothesis and to assess their possible interactions with other body parts/systems.Following our hypothesis, first we designed a group of videofluoroscopy (VFS) studies to assess the acute effect of TRP stimulants on the prevalence of VFS signs of impaired safety and efficacy of swallow and the biomechanics of the voluntary swallow response.In short, older patients were first studied during the deglutition of a control series and two series of boluses of the same volume supplemented with the TRP pharmacologic stimulants.The first study described the therapeutic effect of natural capsaicinods on the swallow response in older patients with OD and was published in Gut in 2013, and the second one assessed the acute effect of piperine and was published in the Journal of Gastroenterology in 2014, and the last three explored the effect of TRPV1, TRPA1 and TRPM8 agonists and were published in NGM in 2017, 2019 and 2020 [81][82][83][84][85].We used the same experimental design for these five studies that included 178 patients that were treated with TRP agonists.These acute biomechanical experiments were complemented with acute studies exploring the effect of TRP agonists on SSF, and on the neurophysiology of afferent (pSEPs) and efferent (MEPs) elements of the swallow response.More recently, we developed experiments exploring the therapeutic effect of subacute stimulation (2 week) with TRP agonists in older patients with OD.The main results of all these clinical studies are:

Acute Studies
These studies consisted of acute oropharyngeal stimulation with natural capsaicinoids (150 µM) added to a medium viscosity (274.4 mPa•s) bolus.First control series (T0) of 5, 10 and 20 mL boluses without supplementation followed by two 5, 10 and 20 mL nectar series (T1 and T2) supplemented with a TRP agonist.Between T0 and T1, there was a sensitization period that consisted of two 5 mL nectar boluses containing the same agonist.This stimulation caused a significant reduction (by 50%) in the prevalence of videofluoroscopic signs of both safety and efficacy impairment.These improvements were manifested by shortening the time to laryngeal vestibule closure (LVC) by more than 100 ms and to upper esophageal sphincter opening (UESO) by up to 70 ms and increasing hyoid and laryngeal displacement in patients with OD caused by age, stroke or neurodegenerative diseases (Figs.9a, 10) [81].When the same bolus was supplemented with a lower dose of capsaicinoids (10 µM), the improvements in the biomechanics of swallowing disappeared, and there were no effects on the neurophysiology sensory pathway although we observed a small increase in cortical activation (Fig. 9c) [84].However, there was a significant increase in the SSF in post-stroke patients with OD at this lower dose, suggesting that the stimulation of TRPV1 receptor not only improves the transmission of sensory inputs to the sensory cortex during voluntary swallow but also stimulates the brainstem CPG increasing spontaneous swallow frequency (Fig. 9b) [34].

Subacute Studies
The multiple dose strategy with the lower dose (10 μM) of capsaicinoids showed better results although it was a proof of concept study with a limited number of patients in each group.Taking 10 ml of a capsaicin solution three times a day for 10 days improved the penetration-aspiration scale (PAS) score by 1 point (p = 0.038), reducing the time to LVC by 100 ms (p = 0.042) [84].In addition, a faster (by shortening the N1 peak latency by 26 ms) and more intense (by increasing the P1-N2 and N2-P2 amplitudes by 2 µV; p = 0.038 and p = 0.05, respectively) neurophysiological response and significant changes in the activation of those brain areas that are probably related to the swallowing control (cingulate gyrus, paracentral lobule and medial frontal gyrus) were recorded.We also found a significant positive correlation between the improvement of the conductivity of the stimuli from the pharynx to the sensory cortex at the pSEP and a shortening of the time to LVC (r = 0.750, p = 0.003), suggesting that this treatment strategy could induce neuroplasticity processes that will be translated into biomechanical improvements in the swallow response.Finally, we defined the criteria (an improvement in the recommended viscosity and/or a reduction of 1 point in PAS score in the post-treatment VFS) to determine when a patient is responding to the treatment, reaching a responder rate of 70% [84].

Effect of TRPA1 Agonists Piperine, Cinnamaldehyde and Citral
We also explored the acute effect of TRPA1 and TRPV1/ TRPA1 agonists.First we explored the effect of supplementation of the alimentary bolus with a high (150 µM) or low (1 mM) dose of the TRPV1/TRPA1 agonist piperine (Sigma-Aldrich, St Louis, MO, USA)).After acute administration of the supplemented medium viscosity bolus (274.4 mPa/s), patients with OD as a consequence of aging, neurological or neurodegenerative disease showed a reduction in the prevalence of non-safe swallows by 34.5% at 150 µM and by 57.2% at 1 mM and an improvement -reduction-in the time to LVC by 90 ms at 150 µM and by 80 ms at 1 mM.In addition, those patients that received the higher dose also reduced their PAS score frome 3.3 ± 0.5 to 1.9 ± 0.3 [82].
The TRPA1 agonists studied by our group were cinnamaldehyde-zinc (756.6 µM-70 mM) and citral (1.6 mM).The acute oral stimulation with a medium viscosity bolus (182 mPa s) supplemented with one of these agonists induced a significant reduction of time to LVC by 100 ms with cinnamaldehyde-zinc and by 50 ms with citral and to UESO by 60 ms with cinnamaldehyde-zinc and 40 ms with citral, but only cinnamaldehyde-zinc also reduced the PAS score from 4.0 ± 1.5 to 3.2 ± 1.8 and the prevalence of unsafe swallows by 30%.Studies with pSEP showed a shortening of the latency of P2 peak by 40 ms and increased the activation of brain areas that are related to swallowing control (temporal and frontal gyrus), also suggesting effects on the neural response [85].

Effect of the TRPM8 Agonists
We also found that supplementation of medium viscosity bolus (274.4 mPa•s) with menthol at 10 −2 M improved the swallow response in patients that had OD due to aging, stroke or neurodegenerative diseases by reducing the time to LVC about 20%.In contrast, we did not observe any significant improvement when a lower dose of menthol (10 −3 M) was used [83].Regarding the combination of a TRPA1 with a TRPM8 agonist, citral-isopulegol (1.6-1.3 mM), we observed that it had a minor therapeutic effect as it only reduced the time to UESO by 40 ms and enlarged the latency of P2 peak of pSEPs by 5 ms [85].

Comparative Effect of Acute Stimulation with TRPV1, TRPA1 and TRPM8 Agonists
When we compared the acute effect of the different TRP agonists, we found natural capsaicinoids (150 µM) reduced the prevalence of penetrations by 50%, the time to LVC by 24.38% and pharyngeal residue by 80%, and was the most effective active treatment tested [83].Piperine (150 µM) reduced the prevalence of penetrations by 56.38% and the time to LVC by 25.55% but had no effect on pharyngeal residue.Finally, menthol only reduced the prevalence of penetrations by 37.5% at a concentration of 1 mM and time to LVC by 18.44% at 10 mM, making it the least effective of these agonists regarding their effects on VFS sign and swallow response [83].Therefore, according to our results, the therapeutic potency of TRP agonists in older patients with OD is TRPV1≈TRPA1 > TRPM8.

Summary, Conclusions and Brief Discussion on the Future of Sensory Stimulation Treatment with TRP Agonists for Older Patients With OD
Our experience on the use of TRP agonists as active treatment for oropharyngeal dysphagia could be summarized as follows: acute or subacute oropharyngeal stimulation with natural TRPV1 and TRPA1 agonists reduced the prevalence of patients with VFS signs of impaired safety and efficacy of swallow, and improved both the biomechanical and neurophysiological swallow response in older and neurogenic patients with OD.We did not observe any significant adverse event nor de-sensitization during acute and subacute clinical studies.We obtained these results after 20 years of research including 6 doctoral thesis and 15 peer-reviewed articles with basic and clinical studies that follow the scientific pathway we have included in this article.However, more studies should be performed with a higher number of patients, and more homogeneous cohorts to confirm our results.
The impairment we observed in the sensory neurophysiological response led us to hypothesize that the afferent pathway could be a good therapeutic target to improve the OSR in older patients with OD, and one of the best ways to stimulate it would be through the stimulation of TRP receptors using natural agonists.Studies by Dr. D. Julius clearly demonstrated the strong stimulation of these receptors using natural compounds [23,61,86].To test our hypothesis we launched two lines of research: basic studies on localization and expression of TRPV1, TRPA1 and TRPM8 receptors in the human oropharynx and larynx [65,70,71]; and (b) clinical trials to explore the effect of powerful natural agonists for these receptors [81][82][83][84][85]87], some of them in cooperation with the nutritional industry [85] and some of them inspired in previous studies by S. Ebihara [88,89] and using VFS or SSF to measure the effects.
What we found in the basic studies was that TRPV1, TRPA1 and TRPM8 were widely expressed in the human oropharynx and larynx with distinct patterns: TRPV1 is mainly located in the plasmatic membrane of the epithelial cells and also in the nerves ending below the basal lamina and in those penetrating the epithelium.In terms of expression, it shows a reduction in expression levels from the oral cavity to the epiglottis; TRPA1 is located in the sensory nerve endings penetrating in the epithelium and in those located below the basal lamina, its expression remains constant in all oropharyngeal structures; and TRPM8 is located in the sensory nerves below the basal lamina but also in some specialized structures such as the Kraus bulbs.We also explored the pharmacokinetics of natural agonists for TRPV1 in an in vitro bioassay with positive results, so we were able to stimulate the receptors with natural compounds (the first one, natural capsaicinoids) [71].
What we found in the clinical trials was extensively described during the presentation at the 2nd WDS and summarized in this article.Again, acute and sub-acute pharyngeal stimulation with TRP agonists improved the biomechanics and neurophysiology of swallowing in several phenotypes of older patients with OD with the following in vivo therapeutic potency: TRPV1 and TRPA1 were at a similar level and both higher than TRPM8 (TRPV1≈TRPA1 > TRPM8).Our studies with VFS during voluntary swallow suggest a stimulatory effect on cortical structures and those with SSF a stimulatory effect on the CPG in the brain stem, both critical for the safety of swallow.During our clinical studies on humans, we did not observe any significant adverse event or desensitization effect during repetitive stimulation in acute and chronic studies, and -on the contrary-our subacute studies suggest repetitive oropharyngeal stimulation with TRP agonists promote brain plasticity in the oropharyngeal sensory areas of the cortex [84].Although we have two strong groups of candidates (TRPV1 and TRPA1 receptors) and evidence of the effectiveness of TRP agonists in the short and mid-term, more studies are needed to describe the long-term effect of these treatments, including larger sample sizes, specifying characteristics of responders and non-responders, and also the physiological swallow functions that change due to treatment as well as the clinical outcomes of these change.In addition, a synergistic strategy with the combination of TRP agonists with the realization of swallowing exercises should also be explored.These studies will reveal whether long-term administration of these compounds at therapeutic concentrations leads to desensitization.Currently, behavioral interventions show positive results with larger evidence for specific exercises [20], however, the evidence of these interventions is similar to that of new treatments such as TRP stimulation [90], indicating a promising future for the translation of pharmacological treatment to clinical practice.Finally, the results explained in this summary show a hopeful future towards developing an active pharmacological treatment for patients with swallowing disorders associated with aging that will allow us to move on from compensation treatments to the improvement of the swallowing function.
Studies presented during the 2nd World Dysphagia Summit in August 2021 have been updated and complemented with what we learned from Dr. David Julius, Nobel Prize of Physiology or Medicine for the discovery of TRPV1, TRPA1 and TRPM8 during his visit to the CIBERehd-Gastrointestinal Physiology Lab at the Hospital de Mataró, Catalonia, Spain in June 2022.

Fig. 1
Fig. 1 Schematic representation of the neurophysiologic circuits of swallowing function: A afferent or sensory pathway in blue; b efferent motor pathway in red for voluntary swallows, and c efferent motor pathway for spontaneous swallows in orange.CPG central pattern generator, DSG dorsal swallow group, VSG ventral swallow

Fig. 2
Fig. 2 Biomechanical and neurophysiological swallow response in young healthy volunteers, older healthy volunteers and older patients with oropharyngeal dysphagia.A Chronograms of oropharyngeal swallow response.B Pharyngeal sensory evoked potential (top) and scalp current density maps (blue color: negative electric current; red color: positive electric current) at N1 and P2 pSEP peaks (below).HV

Fig. 4
Fig. 4 Schematic representation of the main sensory structures located in the human oropharynx: Meissner corpuscle, Merkel cell, Pacinian corpuscle, Krause bulb, taste bud and free nerve ending.Parts of this figure were drawn using pictures from Servier Medical Art.Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https:// creat iveco mmons.org/ licen ses/ by/3.0/)

Fig. 5
Fig. 5 TRPV1, TRPA1 and TRPM8 localization patterns in the human oropharynx.On the left, immunofluorescence images of the TRPV1, TRPA1 and TRPM8 (all marked in red) localization in tongue, pharynx and epiglottis.The nucleus are marked in blue and neuron-specific enolase (NSE) in green.On the right, schematic representation of the localization pattern in the human oropharynx and

Fig. 6
Fig.6 Relative activation of PC-3 cells following the activation with diferent TRPV1 agonists (capsaicin -red-, natural capsaicinoids -pinkand piperine -violet-) at increasing concentrations.At the left, frame of the in vitro activation pic

Fig. 8
Fig. 8 Hypothesis of action of chemical stimulants and natural TRP agonists on TRP receptors located in the human oropharynx and larynx

Fig. 9 Fig. 10
Fig. 9 Summary of the effect of sensory oropharyngeal stimulation with capsaicin on the oropharyngeal swallow response (A), the spontaneous swallowing frequency (B) and brain activation (C).Blue and red/yellow colors indicate increase and decrease in brain activation, respectively