ARISTOTLE UNIVERSITY OF THESSALONIKI
FACULTY OF HEALTH SCIENCES
SCHOOL OF MEDICINE
Olfactory and gustatory function in patients with Multiple Sclerosis
A thesis is submitted in fulfillment
of the requirements for the degree of
Master of Science in Medical Research Methodology
By
Marini Katerina
Thessaloniki, October, 2019 Olfactory and gustatory function in patients with Multiple Sclerosis
A MSc thesis submitted in fulfillment
of the requirements for the degree of
Master of Science in Medical Research Methodology
At
The Faculty of Health Sciences
School of Medicine
Aristotle University of Thessaloniki
By
Marini Katerina
Thessaloniki, October, 2019
Supervisor
Printza Athanasia
(Assistant Professor of the First ENT Clinic of Thessaloniki Aristotle University Hospital AHEPA)
Word count: ~ 9100
2 Members of advisory committee
Printza Athanasia
(Assistant Professor of the First ENT Clinic of Thessaloniki Aristotle University Hospital AHEPA)
Constantinidis Ioannis
(Professor of the First ENT Clinic of Thessaloniki Aristotle University Hospital AHEPA)
Grigoriadis Nikolaos
(Professor the Second Department of Neurology of Thessaloniki Aristotle University Hospital AHEPA-Multiple Sclerosis Center)
3 Table of contents
Absract
General part
I. Anatomy of the nose • External and internal structures • Arteries • Veins • Lymphatic drainage • Nerves II. Histology of nose III. Physiology of nose and olfaction IV. Olfactory pathway V. Multiple Sclerosis • Introduction • Etiology • Immunology • Clinical presentation • Diagnosis • Treatment VI. Olfactory and gustatory dysfunction in Multiple Sclerosis
Specific part
I. Introduction II. Aim of the study III. Research Methodology IV. Inclusion and exclusion criteria V. Olfactory assessment 1. Odor identification test 2. Odor discrimination test 3. Odor threshold test VI. Gustatory assessment
4 VII. Questionnaires VIII. Statistical analysis IX. Results X. Discussion XI. Conclusion References Appendices
5 Abbreviations
MS: Multiple Sclerosis
CNS: Central Nervous System
EDSS: Expanded Disability Status Scale
RRMS: Relapsing Remitting Multiple Sclerosis
SPMS: Secondary Progressive Multiple Sclerosis
PPMS: Primary Progressive Multiple Sclerosis
PRMS: Progressive Relapsing Multiple Sclerosis
CIS: Clinically Isolated Syndrome
MHC: Major Histocompatibility Complex
HLA: Human Leukocyte Antigen
IL2RA: Interleukin-2 receptor alpha
IL7RA: Interleukin-7 receptor alpha
EBV: Epstein-Barr virus
IgG: Immunoglobulin G
NK: Natural Killer
Th1: T helper 1
CD4: cluster of differentiation 4
VLA-4: Very Late Antigen-4
VCAM-1: Vascular Cell Adhesion Molecule 1
APC: Antigen-presenting Cell
CD8: cluster of differentiation 8
Th17: T helper 17
6 Treg: Regulatory T Cell
MRI: Magnetic Resonance Imaging
CSF: cerebrospinal fluid
DIS: dissemination in space
DIT: dissemination in time
ACTH: Adrenocorticotropic Hormone
BBB: Blood Brain Barrier
GA: Glatiramer Acetate
DMF: DimethylFumarate
CD20: cluster of differentiation 20
ENT: Ear Nose Throat
VAS: Visual Analogue Scale
SNOT-22: Sino-nasal Outcome Test
NOSE: Nasal Obstruction Symptom Evaluation
EOG: electro-olfactogram
OERPs: Odor Event-related Potentials
UPSIT: University of Pennsylvania Smell Identification test
CCCRC: Connecticut Chemosensory Clinical Research Center
BUT/PEA: N-butanol or phenylethylalcohol
7 Abstract
Background: Multiple Sclerosis (MS) is a chronic demyelinating neurodegenerative disease that affects central nervous system (CNS). Olfactory dysfunction as a clinical manifestation of MS was until recently underdiagnosed. It is now believed that olfaction is impaired particularly at early stages of MS. The aim of the study is to evaluate the extent of smelling detriment in patients with MS, while possible gustatory impairment was investigated.
Methods: We assessed fifty-nine patients from sixteen to sixty-seven years old from the Multiple Sclerosis Center of the Second Department of Neurology of the Aristotle University Hospital AHEPA and all examined by the First ENT Clinic of the Thessaloniki Aristotle University Hospital AHEPA. The Sniffin’Sticks procedure was applied, while NOSE, SNOT-22 questionnaires, as well as Visual Analogue Scales for olfactory, gustatory disabilities and nose congestion problems were completed.
Results: Without age discrimination, patients presented a mean TDI score of 33.92, meaning that they had normal olfaction (hyposmia < 30.75). Moreover, 27.11% (sixteen out of fifty-nine) of patients had TDI score equal or below 30.75. It considered more accurate to divide patients into six subgroups according to their age for further evaluation. Interestingly, by applying different cut-offs for hyposmia at each subgroup, subgroups 21-30, 31-40, 41-50 and 51-60 years old recorded hyposmia percentages fluctuating from 11% to 33%. Concerning the gustatory dysfunction, 31.25% of hyposmic patients had affected taste according to the VAS results. Finally, comparisons between normosmic and hyposmic patients and their results of NOSE and SNOT-22 questionnaires were performed and SNOT-22 registered statistically significant difference (p value=0.001).
Conclusion: This study supports the presence of olfaction impairment in patients with MS, while gustatory dysfunction was recorded too. Patients with hyposmia had more severe nose obstruction (according to SNOT-22). Olfactory and gustatory assessment should be performed for a more complete and effective diagnosis, treatment and follow-up.
8 General part
I. Anatomy of the nose
• External and internal structures
Nose represents the part of the body responsible for the perception of sense of smell. It contains the external part (external nose) and the internal part (nasal cavity). The former is a face protrusion, which is formed to the upper one third from bony pyramid and the lower two thirds are the cartilaginous part. Pyramid contains the rhinal process of frontal bone at the top, the right and left nasal bone at the middle and laterally the frontal process of maxilla. The nasofrontal angle is developed by the frontal bone and the nasal bones and varies among individuals [1]. Concerning the cartilaginous part, at the middle third of nose are located the upper lateral cartilages, which come in contact with the nasal bones externally and the septal cartilage internally providing support to the nose. The lowest one third is formed by the major alar cartilages (each one is consisted of the lateral, middle and medial crus), shaping the tip of nose, which is the most protruded part. Columella is located from the tip of nose to the upper lip of mouth and is formed by the medial crus of the lateral cartilages. Columella separates the right from left nostril and through them airflow enters the nasal cavity. Finally, minor alar and sesamoid cartilages are also included in the cartilaginous part [2].
Internally, nose is divided by the nasal septum into the right and left nasal cavity, which extends from nostrils to the pharynx. Septum is composed of three parts, the ethmoid bone at the top, the vomer below, while the septal cartilage is located at the front. Each nasal cavity is consisted of the vestibule and the main part. Nasal vestibule is covered by skin containing hair follicle, as well as sweat and sebaceous glands. Subsequently, the main part is expanded from the vestibule to the nasal choana, which is the connection to nasopharynx.
9 It is worth mentioning that nasal cavity is consisted of the inner, upper, lower and lateral wall.
The inner wall is set by the nasal septum with its bony and cartilaginous part.
The upper wall contains the nasal, frontal, ethmoid (cribriform plate) and sphenoid bones.
The palatine process of maxilla delimits the lower wall at the anterior three quarters and at the posterior quarter the horizontal plate of palatine bone.
To the formation of the lateral wall participate the maxilla, ethmoid, palatine and sphenoid bone, as well as inferior concha and lacrimal bone. That part of nasal cavity includes the three conchae or turbinate bones-the superior, middle and inferior. About 50% of people have the supreme turbinate too, which lies above the superior. Their role is increasing, warming and humidifying the inhaled air by increasing the surface of the nasal cavity and decelerating the airflow. The inferior concha is a separate bone, while the superior and middle are extensions of the ethmoid bone.
Underneath each turbinate the corresponding meatus is formed and create four ways allowing air to flow:
The inferior meatus is located above the floor of the nasal cavity and under the inferior concha and its main function is emptying the nasolacrimal duct to the nasal cavity. It is a part of the formation of the nasal valve, which will be described below.
The middle meatus is between the inferior and middle conchae at the lateral wall of the nasal cavity, while it offers the destination where maxillary, frontal and anterior ethmoid sinuses are emptying their content. The largest of the anterior ethmoid sinuses is the so-called ethmoidal bulla. At the external wall of the middle meatus a thin bonny structure is formed-the uncinate process.
The smallest and shallowest meatus is the superior one where the posterior ethmoid sinuses emit. Between the posterior edge of the superior concha and the sphenoid bone, a space that remains is the sphenoethmoidal recess [2].
10 Moreover, a structure of paramount importance is the nasal valve area. It represents the area with the highest nasal flow resistance and even small changes of the structure modify the physiology of respiration. It is divided into external and internal nasal valve. The former is delimited by the nostrils caudally, the internal nasal valve prosteriorly, anteriolaterally there is fatty- connective tissue and the alar cartilage, while the medial boundary are the columella and the septum. The latter (internal nasal valve) consists the upper part of the nasal valve area and forms the opening between the caudal end of lateral cartilage and the medial nasal septum [3].
• Arteries (both external and internal carotid artery)
It is well known that nose has a rich blood supply. More specifically:
Externally, the nose is supplied by:
1. the angular artery, which is branch of the facial artery that arises from the external carotid artery. The angular artery is distributed at the skin of the ala, as well as at the lower septum.
2. the dorsal nasal artery, branch of the ophthalmic artery from the internal carotid artery, supplying the superior part of nose.
3. branches of the suborbital artery, distributing laterally and at the septum.
Internally, nose receives blood from the arteries below:
1. external carotid artery: sphenopalatine artery, greater palatine artery, suborbital and inferior alveolar artery, which are branches of internal maxillary artery, as well as the septal branch from superior labial artery-resulting from facial artery.
2. internal carotid artery: anterior and posterior ethmoidal arteries, both branches of ophthalmic artery.
11 At the frontal part of the septum a dense vessel network is detected- Kisselbach’s plexus (or Little’s area)-with blood supply from both internal and external carotid artery.
• Veins
The most significant nasal veins are the angular, which drains to cavernous sinus through ophthalmic vein and the facial that drains to internal jugular vein. It is of paramount importance to note that anterior and posterior ethmoidal veins drain to cavernous sinus too, constituting potential way of infections transmission.
• Lymphatic drainage
Lymph drains to submandibular and deep cervical nodes. Moreover, lymphatic vessels form a network, which through olfactory fibers connect with subdural and subarachnoid space.
• Nerves The innervation of the external nose is accomplished by the infratrochlear, supratrochlear and the anterior ethmoid nerve (skin of nasal dorsal), which are included at the first division of the trigeminal nerve (the fifth cranial nerve- V1). Furthermore, the second division of trigeminal nerve (V2) provides neurosis at the inferior and lateral nose.
As far as the nasal cavity is concerned, anterior and posterior ethmoid nerves- branches of the V1- supply the lateral wall, parts of the middle and inferior nasal conchae and the corresponding part of septum, while the sphenopalatine ganglion-V2-supplies the posterior part. The anterior and posterior ethmoid nerves, as well as sphenopalatine ganglion provide the sensory nerve supply of nasal septum.
12 The superior cervical ganglion represents the nasal sympathetic nervous system, while the sphenopalatine ganglion (the biggest parasympathetic ganglion) represents the parasympathetic one [2].
II. Histology of nose
Nasal cavity is covered with stratified squamous and transitional epithelium mainly at the anterior one third of nasal cavity-containing the head of inferior and middle conchae-, while pseudostratified columnar epithelium (respiratory type) covers the remaining two thirds [4].
At the mucous of the inferior and middle conchae there are venous-like spaces (venous erectile tissue) whose role is to congest and decongest conchae and as a consequence the capacity and resistance of nasal cavity in response to a variety of stimuli such as inflammatory, chemical, mechanical, psychological etc. These reactions are regulated by Vidi’s nerve [5].
III. Physiology of nose and olfaction
The main functions of the nose are respiration, contribution to the immunological defense, olfaction, development of various reflexes and-in combination with parasinuses-the configuration of voice quality. It is worth mentioning that about 80% of adults are nose breathers.
The inhaled air is warmed from the capillaries of mucus membrane, venous erectile tissue of conchae and the already warmed exhaled air, humidified, cleaned firstly by the hair follicle of vestibule and subsequently by the ciliated epithelium and transferred from nostrils to choanae impacting the nasal valve, where maximum velocity is occurred. After air whirl and deceleration of the airflow, air finally reaches the rhinopharyngeal wall, changes direction and continues its route with final destination the lungs. Concerning the exhaled air, it follows a slightly inferior route inside the nasal cavity [6].
13 Moreover, rhinal mucosa contributes to the immunological defense containing lymphocytes, eosinophils, mast cells, macrophages, lysozymes, cytokines such as interferon and immunoglobulins IgA, IgM and IgG. The formation of conchae is responsible for the modification of the width of the nasal cavities under the effect of inflammatory, thermic, chemical or even psychological stimuli. There is also the physiologic phenomenon called nasal cycle, which is characterized by congestion and decongestion of nasal cavities-without changes of the total airflow- with mean duration of 1.5-4 hours [7,8].
Olfaction is considered to be an important and vital sense for mammals, although humans have that sense less evolved, something that is proved by the quite smaller area of the cortex that olfaction possesses. That fact does not underestimate its contribution to humans’ quality of life. It has been investigated that different neurons and synapses recognize specific odorants- a wide range of odorants each one of them.
When a chemical stimulus-among the thousands of odorants that humans can perceive-reach a normal nasal cavity and epithelium, it travels through the olfactory bulb to specific brain areas after connecting with specific receptors via odorant-binding proteins [9]. Humans have the ability to discriminate approximately one trillion olfactory stimuli-although that number is still under debate and investigation [10]. It is worth mentioning that chemosensation involves trigeminal nerve too- (the fifth cranial nerve) and it is responsible for detecting and avoiding harmful stimuli, such as ammonia, ethanol, acetic acid, menthol, capsaicin and carbon dioxide. There is also the retronasal olfaction. Nasopharynx releases a stimulus and then it moves upward through the choanae to result to the olfactory epithelium [11].
Another interesting and important function is the formation of reflexes under the effect of sympathetic and parasympathetic innervation of nasal mucous. Psychological, hormonal, inflammatory, pharmaceutical, chemical stimuli or even exercise are responsible for congestion and decongestion of conchae or the enhanced secretory activity. More specifically trigeminal sensory neurons could cause reflexes that affect the heart and blood flow, as well as lacrimal and sneeze reflexes. Also, through autonomic innervation, olfaction has the
14 ability to provoke positive or negative reactions and sentiments such as increased appetite or modifications in sexual desire, while the quick adjustment of the sensory epithelium to unpleasant smells is another indubitably significant characteristic [12,13].
Finally, nose and sinuses have the ability to amplify sounds and form the quality of speech. That function could become comprehensible by the remarkable changes in voice quality when there is obstruction of nasal cavities (closed rhinolalia) or velopharyngeal insufficiency that results in excessive emission of air through the nose (open rhinolalia) [14,15].
IV. Olfactory pathway
The olfactory part of nasal mucosa is extended from the inner surface of superior concha to the upper half of middle concha laterally and the facing space of nasal septum (upper one third). Various airborne chemical stimuli- the so-called odorants-come in contact with the olfactory epithelium, which is pseudostratified columnar epithelium (figure 11). There the olfactory receptor cells are located (the first neurons of the olfactory tract), which are differentiated from basal cells they have the unique property to regenerate throughout life (figure 12). Other types of cells of the olfactory epithelium except from receptor cells are the stem (basal) cells and among them the supporting cells with mainly secreting properties contributing to transport of odorants as well as olfactory glands (Bowman’s glands). The receptor cells are bipolar neurons-approximately six million-and at their peripheral edge they extend knob-like dendrites to the epithelial surface and form sensory cilia. Subsequently, cilia come in contact with the odors from nasal cavity through receptors. As bipolar cells, they have a central projection too, which is an unmyelinated axon. Odorants are transported to the olfactory receptors by the odorant-binding proteins. Bowman’s glands humidify the olfactory mucous, while their secretion dissolves the odorants in order to stimulate the sensory cilia. Nerves responsible for the transmission of the olfaction pass through the small foramina of the cribriform plate-part of the ethmoid bone-in order to
15 form, reach and be coded in the olfactory bulb and thus the cranial cavity with a G protein-mediated path. The olfactory nerve’s branches or fibers are consisted of the combination of different receptor cell axons and thus olfactory nerve (the first cranial nerve) is arisen. Olfactory bulb is located at the inferior part of the frontal lobe and contains the layers below: granule cell, internal plexiform, mitral cell (mitral cell neurons are the second order neurons), external plexiform and the glomerular cell externally. As the olfactory pathway proceeds, a very significant synapsis is formed at the glomerular layer between the mitral cell neurons and the branches of the olfactory nerve (about 1000 branches with each mitral cell). The lateral fibers of mitral cells come in contact directly with the third order neurons at the piriform, periamygdaloid and entorhinal areas of the primary olfactory cortex (conscious smell perception) and projections to the medial dorsal nucleus of thalamus, basal forebrain and limbic system are sent (limbic system contains structures as hippocampus, hypothalamus, amygdala, entorhinal area etc and one of its functions is olfaction). It is worth mentioning that the olfactory bulb is connected with the hemispheres via the olfactory tract [16,17,18].
Concerning the olfactory cortex, its location is the base of the frontal lobe along with the temporal lobe (middle part). It is composed of distinct areas such as olfactory tubercle, anterior cortical amygdaloid nucleus, entorhinal cortex, periamygdaloid cortex, piriform cortex, mediodorsal nucleus of thalamus, orbitofrontal areas. At the piriform cortex representations referring to the memory, quality and identity of odors are encoded, while it makes the connection between the olfactory and limbic system. It processes visual information before the perception of an odor, leading to the detection of a pleasant or unpleasant stimulus [19,20].
16 V. Multiple sclerosis
Introduction
Multiple sclerosis (MS) is a chronic demyelinating and neurodegenerative disease of the central nervous system (CNS) with a variety of clinical manifestations. Its etiology remains contentious; the activation of the autoimmune system due to genetic, environmental and other factors is the most widespread theory for the cause of demyelination and axonal loss [28]. It was first described in 1421 and -since the 20th century when MS became one of the most common causes of visiting a neurologist- many neurologists, pathologists, anatomists, as well as illustrators and artists contributed to the approach of the disease’s progress of comprehension [29]. The involvement of the autoimmunity with genetic and environmental trigger effects proposed during the 20th century because of concerted studies, experiments in mammals and the use of techniques such as electrophoresis [29]. One of the most important researchers in MS, Jean-Martin Charcot (1825-1893) proposed the name “disseminated (cerebrospinal) sclerosis” (“la sclérose en plaques disseminées” in French) and recognized it as a distinct disease [30,31].
As for its epidemiology, MS has an interesting non-homogeneous world distribution. It is estimated that approximately 2.3 million people suffer from MS and in Europe around 700.000, with the highest prevalence detected in north Europe (Denmark and Sweden) while in Greece it is 70 per 100.000 [32,33]. China, South Africa and South America are the countries with the lowest prevalence (about 0-20/100.000 patients), while most patients worldwide are found in central and northern Europe, North America and Australia (about 60 to >100/100.000) [33]. The mean age of disease onset is 30 years (20-50 years) with a female predominance (females:males=3:1) [32], albeit MS onset in childhood is often observed [34].
MS presents with a variety of clinical manifestations. Four types have been described according to the progression of the disease (figure 1): the most common clinical presentation is the relapsing remitting (RRMS), which affects
17 around 85% of the patients [35,36]. Relapse in MS is the development of new neurological symptoms lasting more than 24 hours in the absence of fever. It represents a biphasic disease development characterized by intermittent episodes of neurological disability followed by improvement of symptoms or complete recovery over days or weeks and the deficit is often reversible because of brain’s ability to compensate [35,36].
When brain cannot respond to the axonal loss, progressive neurological decline is detected (secondary progressive MS-SPMS) and it is developed within 20-25 years at 60-70% of patients with RRMS [36,37].
The third type refers to patients with steadily progressive disease from the onset (about 10% of diagnoses), the primary progressive MS (PPMS) and about 40% of them experience the progressive relapsing MS (PRMS) type with incomplete recovery between relapses (it is the fourth type and affects 5% of all patients) [36,37,38].
Figure 1. Subtypes of Multiple Sclerosis
Newer classification (Lublin et al 2014) stratifies relapsing and progressive disease into active, non-active, and progressive and non- progressive while the fourth type (PRMS) is not used any more [39].
Clinically isolated syndrome (CIS) is a condition worth mentioning, referring to an isolated CNS demyelinating episode with more than 24 hours duration and the possibility of converting to MS is defined by radiological, clinical, laboratory and epidemiologic factors [40].
18 Etiology
It is undisputable that MS is characterized by immune dysregulation and involves both inflammation and neurodegeneration. To connect this information with the abovementioned clinical manifestations we could underline that inflammation and demyelination has to do with disease relapses and early stages. Nevertheless, progression and disability are affected mainly by neurodegeneration. Various factors have been incriminated as trigger effects, such as environmental and genetic [41]. More specifically, as for the genetic factors, it has been proved that among monozygotic twins the risk of MS development to both of them is 25-30%, dizygotic twins 2-5%, while a first-degree relative of a patient has a higher risk of about 10-50 times than a non-relative [42].
Moreover, much research has been devoted to reveal the potential role of genes, with Major Histocompatibility Complex (MHC-in humans it is called Human Leucocyte Antigen (HLA)) alleles, that represent a part of the adaptive response and surface cell proteins capable of recognizing antigens and present them to T-cells as the strongest evidence. Chromosome 6 includes the gene that is responsible for the expression of HLA. Each geographic population is not associated with the same HLA complex. Europeans and North Americans for example have a strong correlation to the HLA- DRB1*15.01 allele [43,44]. Many studies try to discover other genes and much effort needs to be made in order to clarify the exact role of each one of them, while interleukin-2 receptor alpha gene (IL2RA) and interleukin-7 receptor alpha gene (IL7RA) alleles are two examples [45].
Interestingly, it has been reported that migration at an early age from areas with high risk for MS to areas with low risk reduced the possibility to develop MS [46]. Among the environmental factors, cigarette smoking (no other forms of tobacco), levels of vitamin D and infection with Epstein-Barr virus (EBV) are the most adequately studied. Cigarette smoking affects and increases interestingly not only the susceptibility to MS, but also the progress of the disease (higher risk for faster swing from RRMS to SPMS or PPMS onset
19 instead of RRMS) and it is dose-related, while we should not forget the multifactorial nature of MS and possible association between smoking and genetic or other environmental factors. The role of vitamin D in preventing and/or modifying the progression of MS is under investigation, while its contribution to calcium levels, brain function and control of immune responses are well established. The two sources for vitamin D intake are the sun exposure and diet, so latitude, type of food and vitamin supplements have potential responsibility for susceptibility to MS, with higher risk in people with deficiency. It is interesting that no difference in MS risk detected among the different skin types and variations among genes that influence the expression of vitamin D can affect the disease’s risk. As far as EBV infection is concerned, although a biomarker for EBV (anti-EBNA IgG seropositivity) has proved a strong association between infection and MS susceptibility, there are ambiguities that cannot classify EBV infection as an independent risk factor for MS [46].
Immunology
In MS, innate immune response (defense mechanisms that are not antigen- specific) with neutrophils, macrophages, natural killer (NK) cells, compounds of the complement system and other factors as long as adaptive response (antigen specific defense mechanisms) with the T-cells and B-cells (differentiation into plasma cells and immunoglobulins) are of paramount importance in formatting the immunological profile of the different types of the disease [47]. MS is associated with HLA-DRB1*15.01 allele as mentioned above.
The most important factors in the immunology of MS are the T-helper 1 (Th1) cells and more specifically those that express the CD4 protein on their surface (CD4+ T-cells) from the adaptive immunity. Their role is mainly in the periphery and at the early phases of the disease. They become activated when they recognize peripherally antigens from CNS and subsequently secrete pro-inflammatory cytokines that contribute and lead to the migration of
20 the molecules across the blood brain barrier as a result of endothelial changes-interaction between very late antigen-4 (VLA-4) on T lymphocytes and vascular cell adhesion molecule -1 (VCAM-1) on endothelial cells.
After entering the CNS, elements such as antigen presenting cells (APC) and macrophages play their role in further inflammation process by expressing MHC class II molecules. They recognize and present local antigens followed by reactivation of T-cells, secretion of various cytokines and activation of microglial cells (macrophage cells located in brain and spinal cord) and astrocytes. It is worth mentioning that cytotoxic T-cells (they express CD8 protein on their surface-CD8+ T-cells), as well as B-cells play an important role in the inflammatory cascade and the myelin and axons injury [47]. Other factors playing vital role in the maintenance or obstruction of injury have to be established, with T helper 17 (Th-17-secrete interleukin 17-part of the adaptive immunity) cells as a resent example. They lead to production and recruitment of neutrophils and contribute to autoimmunity [48]. As far as the impaired defense against inflammation is concerned, there are the regulatory T-cells (T-reg cells) that in normal immune systems suppress attacks to antigens (auto- and foreign), so when they are down regulated there is susceptibility to autoimmunity. In MS T-reg cells have inadequate regulation [49].
There have been described two models that try to explain the pathophysiology of MS: the outside-in model and the inside-out. The first one supports the theory that myelin is affected before axons, while the opposite happens to the second model. Both of them end up with axonal and myelin damage [50].
All the aforementioned processes result in demyelination and secondarily loss of oligodendrocytes within the plaque (lesions that are formed in MS). Oligodendrocytes are responsible for the insulation and protection of the axons in the CNS by producing myelin. Schwann cells account for the equivalent action in the peripheral nervous system. The remaining ones try to remyelinate the axons to some extend and at early phases of the disease it makes an impact, but later it becomes ineffective and followed be atrophy.
21 CNS contains the white and the gray matter. The former contains the axons, while the latter is formed by cell bodies and nuclei. MS primarily affects the white matter, without omitting the cortex too (cortex includes grey matter).
Clinical presentation
Multiple Sclerosis has various clinical manifestations and patients develop different symptoms depending on the location of lesions in CNS and the type of MS at the onset of the disease.
Some of the most frequent symptoms that awake and motivate patients to visit a doctor are unilateral optic neuritis, opthalmoplegia, cerebellar ataxia, Lhermitte’s sign (it is described from patients as an electric shock when they have their neck flexed and can affect both trunk and limbs), limb weakness, heat intolerance, sensory loss or partial myelopathy. Less typical symptoms have been described too, such as fatigue, headache, bilateral optic neuritis, hearing loss and mental impairment. As mentioned above, most patients experience relapsing remitting episodes of such symptoms with partial to complete remission, until they convert to SPMS or they deal with progressive disability (PPMS). However, relapses can occur even in SPMS and PPMS but the main driver of disability is progression due to neurodegeneration [51].
Diagnosis
So far, there is not a single and pathognomonic biomarker or test that could establish diagnosis in MS, although several of them have been proposed with controversial results among studies. Diagnostic, predictive, clinically useful and disease activity biomarkers have been investigated. Clinical presentation and subjective symptoms are combined with imaging methods (Magnetic Resonance Imaging-MRI) and cerebrospinal fluid (CSF) analysis in order to reach diagnosis as early and effectively as possible. To contribute to that
22 direction, in 2001 diagnostic criteria were established by McDonald and revised in 2017 with emphasis given to dissemination of lesions in space and time (DIS and DIN) and to the early diagnosis of PPMS. Their revised edition is relevant to pediatric populations as well as Latin Americans and Asians [52,53].
The most important first approach of diagnosis is a complete medical history of the patients-not only the neurological symptoms that forced them visit a doctor, but also previous symptoms that could have been mistaken as less important.
MRI has a fundamental role in contributing to diagnosis, not only by detecting characteristic abnormalities, but also by excluding other conditions. Findings that exist to the majority of patients with MS are: T2-hyperintense lesions in the white matter of CNS, while T1-weighted imaging is presented hypoinstense, the so-called ‘black holes’. On the contrary, despite its great contribution the previous years, CSF analysis is not always required to establish diagnosis.
Concerning the McDonald’s diagnostic criteria, they established in order to approach an early diagnosis by excluding alternative diseases and characterized by quite high sensitivity and specificity leading to more effective treatment and quality of life for patients. Below there are some useful definitions followed by an aggregate table with some additional revisions applied by researchers in 2017.
McDonald (2017 revised) MRI criteria for DIS in MS (table 1):
• Demonstration of diagnosis requires at least one T2-hyperintense lesion in at least two locations characteristic for MS. Specific areas are the cortical, juxtacortical, periventricular, infratentorial and spinal cord. • Presence of an additional attack involving a different CNS location.
McDonald (2017 revised) MRI criteria for DIT in MS:
• Development of a new T2-hyperintense lesion and/or gadolinium- enhancing lesion(s) when compared to a baseline scan, without
23 restriction of the timing of the baseline scan. Moreover, detection of gadolinium enhancing and nonenhancing MRI lesions simultaneously is an alternative to demonstrate DIT. • Positive oligoclonal bands in CSF can substitute dissemination in time (DIT). • Presence of a new clinical attack. It is worth mentioning that both asymptomatic and symptomatic lesions in MRI are used in order to ascertain dissemination in space or time.
McDonald (2017 revised) criteria for diagnosis of PPMS: necessarily one year of disease progression and two of the following three: 1. DIS in brain with at least one T2-hyperintense lesion in at least one location specific for MS, 2. DIS in spinal cord with at least two T2 lesions and 3. Positive CSF analysis (CSF-specific oligoclonal bands).
An “attack” is defined as an event with duration 24 hours or more that is a characteristic inflammatory attack of the CNS, either objective after physical examination or described by patients, without fever or infection. The event could be current or historical and patient description should contain convincing information about a demyelinating CNS episode [53].
24 Table 1. McDonald’s diagnostic criteria (2017 revised)
Clinical presentation Additional data required for diagnosis of MS At least 2 attacks, at least 2 T2- none hyperintense lesions or 1 lesion with strong evidence for a previous demyelinating episode
At least 2 attacks or an DIS as described above (2017 objectively-observed lesion revision: cortical lesions can be used too).
An additional episode with a different CNS site is required.
One attack, at least 2 DIT as defined above. objectively-observed lesions An additional clinical attack is required.
Clinically isolated syndrome: 1 DIS with a future attack and attack, evidence for 1 lesion different CNS site and DIT with a second episode.
2017 revision: symptomatic and asymptomatic lesions can be used in DIT and DIS.
Primary progressive MS Criteria as described above, with the aforementioned 2017 revisions.
25 Treatment
The medication that is used and has induced a better quality of life by slowing the long-term progression of the disease is therapy with immunomodulatory agents, while methylprednisolone and adrenocorticotrope hormone (ACTH) are preferred in disease (acute) relapses. Except from their anti-inflammatory characteristics, they are quite effective in diminishing edema [54]. Moreover, it is of great importance to relief each patient’s symptoms, such as pain and weakness, spasticity, fatigue, so appropriate medication is used in each case. Physicians’ aim is to reduce relapsing episodes, in addition to decrease the possibility for new MRI T2 lesions. There have been developed injectable (subcutaneous, intramuscular, intravenous) and oral MS treatments.
The first disease-modifying drug developed about 25 years ago, was interferon beta 1-alpha and it constituted the most effective treatment for MS. Interferons are cytokines-proteins that are produced in response to viruses, bacteria or even tumor cells. Their contribution to MS is their ability to stabilize the blood brain barrier (BBB) and prevent inflammatory cells from invading into CNS leading to reduction of neuroinflammation, along with the increase of neurons’ endurance. Moreover, the role of Th17 cells in autoimmunity investigated quite recently and it is worth mentioning that interferon beta decreases their production [54].
Another effective immunomodulatory agent is glatiramer acetate (GA) with a different way of action. It simulates one of the main targets of T-cells in MS- myelin basic protein, so patients’ immune system reacts against GA. As a result, inflammation and myelin damage are limited, relapses are reduced, as well as MRI lesions [55].
Interferons’ route of administration is subcutaneous and intramuscular, while GA’s is subcutaneous. Interferons have positive results to SPMS and contribute to the decrease of disability progression, an effect that GA does not provide but both of them are appropriate for RRMS [56].
26 The so-called second era of MS treatment contains mainly two agents- natalizumab and fingolimod. The former is a monoclonal antibody that inhibits migration of inflammatory cells through BBB by binding lymphocytes’ α4β1- integrin followed by prevention of interaction with endothelial cells. Despite its remarkable benefits in relapses and disease progression, there is quite high risk for multifocal leukoencephalopathy, a potentially fatal condition, so its use is limited. Concerning the latter drug, fingolimod consisted the first oral drug that released and prevents migration of T-cells to CNS by binding to a receptor of them (sphingsosine-1-phosphate receptor). Its use is limited too, due to severe side effects that have recorded [57].
From 2009, newer drugs were developed and approved such as teriflunomide and dimethylfumarate (DMF)-both of them orally administered. Teriflunomide inhibits the dihydro-orotate dehydrogenase enzyme and its mechanism of action depends on its ability to decrease the activity and proliferation of T and B lymphocytes [58]. DMF activates nuclear factor erythroid 2-related factor 2 resulting in prevention of inflammatory processes [59].
Finally, in the second line treatment along with natalizumab, fingolimod, belong mitoxantrone, alemtuzumab, anti -CD 20 monoclonal antibodies such as ocrelizumab and rituximab, as well as a T cell apoptotic agent, cladribine. Mitoxantrone is an antineoplastic drug causing suppression of macrophages and lymphocytes, so the risk of leukemia and cardiotoxicity is apparent. The latter agent is a monoclonal antibody, which suppresses immune cells, with high risk for thrombocytopenia and malignancies [60].
VI. Olfactory and gustatory dysfunction in Multiple Sclerosis
The prevalence of olfactory dysfunction among patients with Multiple Sclerosis (MS) is about 40%, even though literature lacks large sample sizes [21]. It is of paramount importance the fact that there is an increasing interest about the role of olfaction in patients with MS. Olfaction is harmed early in many neurodegenerative diseases such as MS, Alzheimer’s, Parkinson’s and
27 Huntington’s disease and that observation could become a parameter of early manifestation of these conditions. The need for prognostic markers is highlighted by the crucial role of the right and punctual treatment plan [22].
It is well known that MS is an inflammatory and demyelinating disease of the central nervous system (CNS) and more and more studies come to the conclusion that both electrophysical (for example olfactory event-related potentials) and psychophysical (for example Sniffin’Sticks test) methods show correlation between olfaction impairment and higher scores of Expanded Disability Status Scale (EDSS), disease duration or stage [23].
There are also studies that correlate olfaction with cognitive impairment, showing that many regions responsible for cognition are affected. Memory, attention and information processing speed are some of the functions that related with olfactory impairment [25,26].
Olfaction is inextricably linked to taste and therefore the latter is affected in patients with MS (the four classic taste qualities) [27].
28 Specific part
I. Introduction
Multiple Sclerosis is a well-described neurodegenerative disease affecting over two million people on a worldwide scale [61]. As an autoimmune disorder, it combines genetic susceptibility, environmental, psychological and a variety of other factors in order to appear and cause axonal loss and demyelination. Maybe the most characteristic compounds of MS are the MS lesions, which represent areas of demyelination of the white matter of the central nervous system (CNS), with quite resent evidence involving the cerebral cortex too.
Concerning the subtypes of the disease, about eight out of ten patients confront the relapsing-remitting one (RRMS), with new or recurrent symptoms which resolve after a varied period of time.
The impairment of olfaction in patients suffering from Multiple Sclerosis (MS) became quite recently accepted from the scientific community. It has been proved that at earlier stages of MS threshold is harmed and reflects the disease activity and reversible inflammatory events for about a year before the examination, while both discrimination and identification are related to more progressive stages and neurodegeneration when permanent damages have occurred. The latter are linked with cognitive dysfunction and affected Expanded Disability Status Scale (EDSS) [62]. Moreover, the irreversible impairment of discrimination and identification are linked with a more general physical disability of the patients, while threshold seems to return to normal values when relapses are dissolved [63,64]. It is worth mentioning that due to the recent investigation and correlation between MS and olfaction disorders, differences between studies have been detected, with olfaction’s impairment prevalence fluctuating between 30 to 70 per cent [65,66].
As smell and taste are closely related to each other, there are studies that prove the gustatory impairment too [67].
29 II. Aim of the study
Study’s main purpose is to investigate both olfactory and gustatory function of patients with Multiple Sclerosis.
III. Research Methodology
This study constitutes a cross-sectional one, in which participated 59 patients diagnosed with Multiple Sclerosis (carried out from December of 2018 to July of 2019). All participants were examined-checked by the First ENT Clinic of the Thessaloniki Aristotle University Hospital AHEPA. The aim was to record patients’ olfactory abilities and detect possible dysfunction. Also, by using a Visual Analogue Scale (VAS), we tried to detect possible gustatory impairment. It is worth mentioning that all patients came from the Multiple Sclerosis Center of the Second Department of Neurology of the Aristotle University Hospital AHEPA. They were either hospitalized at the Neurology Department or they visited the outpatient clinic of the Multiple Sclerosis Center. The scientific council of the Aristotle University of Thessaloniki, as well as the bioethics committee approved the study. All participants were evaluated as appropriate for entering the study and written informed consent was obtained after extensive information about the procedure.
IV. Inclusion and exclusion criteria
According to the inclusion criteria, the study included patients up to 16 years old with certain diagnosis of MS according to the McDonald Criteria (2017 revised) that were either hospitalized or they visited the MS center for their scheduled control or therapy. The Expanded Disability Status Scale (EDSS) score ranged from 1 to 8. Subtypes of MS were classified as RRMS, PPMS, SPMS and clinical isolated syndrome (CIS) and participants could belong to any stage and subtype of the disease. Moreover, demographic data for all participants were recorded.
30 Concerning the exclusion criteria, they included: active or recent upper airway infections, allergic rhinitis, acute or chronic rhinosinusitis, nasal polyposis, previous nasal surgery, head trauma that affected the olfactory pathway, as well as systemic diseases that could affect olfaction.
For the evaluation of the symptoms stemming from nose each participant completed a visual analogue scale (VAS) with values from zero (no symptom) to ten (very severe symptom), as well as the SNOT-22 (SinoNasal Outcome) and NOSE (Nasal Obstruction Symptom Evaluation) questionnaires. They are published and valid questionnaires, while the validation for Greek patients was used.
V. Olfactory assessment
At the present study, the Sniffin’Sticks test ® procedure will be used [68, 69]. Olfactory dysfunction is a quite common problem and it is considered to be an underdiagnosed one, while many people and professionals depend their work, career or even well-being on their ability and sensitivity of smelling [70]. It has been investigated and established that different odorants share similar neurological pathways, namely receptor cells. By virtue of that commonality it is unneeded and unattainable to examine the great range of odorants in each patient with olfactory problems. There is requirement for quantitative ways and clinical tests to assess and measure the smelling abilities of a person with olfactory difficulties that visits an otolaryngologist.
The two major categories of olfactory tests are the psychophysical and electrophysiological, while there are imaging procedures that contribute to more complete and accurate observation and diagnosis [69]. We are able to examine the sense of smell and possible impairments from low-cost and easy methods to more expensive and precise, which reflect and depict the olfactory epithelium, bulbs etc. Examples of electrophysiological tests are the electro- olfactogram (EOG) and the odor event-related potentials (OERPs) [71]. Most clinicians prefer and use the psychophysical measurements, due to their
31 practicality and after the notice and evidence that although “objective”, electrophysiological tests are not infallible and it is time and money- consuming to submit a person to a test with doubtful results [71]. Concerning the most used olfactory tests, they are the [69]:
1. odor identification test: either the University of Pennsylvania Smell Identification test (UPSIT) or the Identification part of Sniffin’Sticks procedure. Both of them rely their function on the use of specific odorants at a suprathreshold level that examinees are asked to identify. They are widely used due to their practicality and convenience in administering and validating. It only takes a few minutes to complete and their high sensitivity makes them superior to other similar procedures. Answers are forced, meaning that patients have to give a response for each odor, even if they are not sure. That makes them more reliable and is an indirect way to detect the malingerers, if responses are unexpected and dubious. Sniffin’Sticks test was first developed in 1997 by Hummel and is the procedure that will be used in our survey [68]. It belongs to the psychophysical category that mentioned above and is a semi-objective tool. It contains three subtests; the identification, discrimination and threshold test, with specific score for each one (I for identification, D for discrimination and T for threshold-TDI score in total). All subtests contain pen-like items with diameter of 1.3cm and length of about 15cm. They are impregnated with 4mL of an odorant fluid or substance (dissolved in propylene glycol), as well as an antibacterial factor. There is also the “Identification” part of the Connecticut Chemosensory Clinical Research Center (CCCRC), which uses 10 odorants that the examinee has to recognize in jars, among a list of 20 choices.
32
2. odor discrimination test: the second subtest of the Sniffin’Sticks procedure that will be used contains 16 triplets of pens, thus 48 pens. Each triplet includes a red, a blue and a green pen. It is more time- consuming than the identification test and the examinees cover their eyes in order to extract more safe results. They are asked to detect which of the three pens of each triplet smells different from the other two (red and blue pens are impregnated with the same odor). The green pen is the one that diverges and examiners present the green pen in different order for every triplet to prevent falsely correct responses. They give a forced-choice answer too, even if they are not sure. About 3 seconds are inserted between different pens of the same triplet, while between consecutive triplets about 30 seconds are needed in order to perform the test as aptly as possible. “D score” represents the number of correct responses among the sixteen triplets.
3. odor threshold test: many physicians identify that test as being similar to the more widespread pure-tone auditory threshold test, in the sense that both are aiming to detect the lowest amount of tone or odorant respectively that a person has the ability to discriminate. Referring to the Sniffin’Sticks procedure-again- it comprises of 48 pens divided in triplets of a green, a blue and a red one. At that subtest, the correct response is the red specimen, as it is impregnated with N-butanol or phenylethylalcohol (BUT/PEA). The green and blue are containing a solvent and have no smell. It is possibly the most time-consuming and complex part of the olfaction ability measurements for both the examinees to undertake and the examiners to evaluate and grade it. The first red pen contains the highest concentration of BUT/PEA and it is presented only once to the individual (who has blindfolded eyes). In
33 order to comprehend the difference, one of the odorless pens is presented about 30 seconds later. After 30 seconds, number sixteen triplet is performed with the less intensely odor. As a whole, triplet number one has the red pen with the highest concentration of BUT/PEA and as moving forward to number sixteen, concentration is progressively decreasing. For example, after having explained the procedure and given the triplet number one as reference triplet, examiner performs the lowest concentration, namely pen sixteen (interval between pens of the same triplet about 3 seconds and among different triplets 30 seconds). If wrong response is given, triplet 14 (and not 15) follows, then 12, 10 etc in order to save time until the first right identification. An alternative row could be triplets 15,13,11 and so forth. Examiner should pay attention to the order that reveals each pen of the triplets, so that patients do not memorize a specific order and lead to corrupted results. As for the scoring, for the so-called “T score” a grid is used. When the patient offer the first correct answer, the same triplet but in different order is presented and if the response is right again, then two “+” are written down next to the corresponding triplet number, beginning from the left of the grid. After two consecutive proper replies, the examiner continues with the immediately higher number of triplet of pens that contain BUT/PEA at lower concentration. The second column is completed when the first wrong response is noted (“-“). Then an immediately higher concentration is performed (lower number) and the third turning point as the process proceeds from left to the right is when two consecutive correct answers are recorded (“++”). Then a lower concentration is given until the first incorrect identification and so on and so forth. When seven turning points are completed, the last four are added and then divided by four (mean value).
34 At the present study, we will use the Sniffin’Sticks procedure as mentioned above, so the score will be formed from the sum of the individual I, D and T scores (TDI score). In order to define normosmia, hyposmia and anosmia we use TDI score >30.75, from 16.5 to 30.75 and <16.5 respectively, according to the latest normative data [72]. It is worth mentioning that test’s sensitivity and specificity values are quite high, approximately 85%. Most physicians avoid performing all three subtests in clinical practice in order to save time and by excluding the discrimination test, they achieve similar levels of sensitivity and specificity [69].
The examination room should be well ventilated without intense smells. Participants should not have consumed any food or drink except from water, while smoking is not allowed 15 minutes before examination. Also, the examiner is recommended to wear odorless cotton gloves in order not to confuse examinees with additional smells.
VI. Gustatory assessment
Gustatory function was assessed subjectively by the completion of a Visual Analogue Scale (VAS). Final score was emerged from further quantification (values ranging from zero to ten -asymptomatic to very severe gustatory disability).
VII. Questionnaires
SinoNasal Outcome Test 22 (SNOT-22) questionnaire consists of 22 questions examining except from nasal symptoms, symptoms not rising from nose dysfunction, quality of sleep, functionality and emotional status. Participants assess their symptoms with values from zero (no symptom) to five (very severe symptom) and final score is their sum. With this questionnaire we are able to evaluate the impact of nasal obstruction on participants’ quality of life.
35 Nasal Obstruction Symptom Evaluation (NOSE) questionnaire is investigating the severity of nasal obstruction and contains five questions. Participants grade them from zero (no symptoms) to four (severe symptoms) and the emerging result is multiplied with five for the final score, which could range from zero (no symptoms) to one hundred (very severe symptomatology of nasal obstruction).
VIII. Statistical analysis
Data were analyzed with IBM SPSS Statistics for Windows (IBM Corp., Armonk, NY, USA) version 25.0, while descriptive statistics were recorded. Qualitative variables are presented as frequencies and quantitative as means (with standard deviation). Kolmogorov-Smirnov test was used in order to confirm normality of data. Mann-Whitney U test was used for the not normally distributed data. Values <0.05 were considered statistically significant.
IX. Results
Fifty-nine patients [19 male (32.2%) and 40 female (67.8%); mean age: 39.5±13.24 years], fulfilled the inclusion criteria and participated at the study. Their demographic data are depicted at table 2.
36 Table 2. Characteristics of the 59 participants of the study
Characteristics [n, (%)] Patients
Gender
Male 19 (32.2%)
Female 40 (67.8%)
Age (years) 39.5±13.24 (min=16.5, max=67)
MS subtype
RRMS 43 (72.88%)
SPMS 7 (11.87%)
PPMS 9 (15.25%)
CIS 0 (0%)
Disease duration 10.13±7.9 (min=0, max=31) (years)
EDSS 4±1.97(min=1, max=8) It can be observed that our sample of patients had a female predominance which is consistent with the existing literature about the epidemiology of MS, as well as the frequency of the subtypes, with the relapsing remitting one (RRMS) representing the most common.
Concerning patients’ (without age discrimination) olfactory abilities and status of nose congestion, descriptive statistics were recorded and presented at table 3. More specifically, for threshold, discrimination, identification, total TDI score, as well as SNOT-22 and NOSE questionnaires mean, standard deviation, range and quantiles were registered.
37 Table 3. Results without age discrimination
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE SNOT-22 NOSE
Mean 7.83 12.12 13.97 33.92 21.25 11.6
Standard 2.44 2.7 1.87 5.5 16.86 16.95 deviation
Range 3-13.75 5-16 7-16 15-42.25 0-85 0-80 The most significant deduction that derives from the table above, is that patients as a whole presented a mean value of 33.92 for TDI score, meaning that they are normosmics according to the 30.75 cut-off.
Furthermore, Oleszkiewicz et al. provided normative data by examining the smelling ability of about 9000 healthy individuals using the Sniffin’Sticks procedure [72]. As it was the first study including such a big population, it became possible to make deductions and comparisons between smaller subgroups, each containing participants of the same decade of life. The first subgroup was from five to ten years old, the second from eleven to twenty, the third from twenty-one to thirty and proceeding until the last one, which included people over eighty-one years old. That study offered the possibility to place different cut-offs for hyposmia at each group of subjects, while the subsample with the best odor performance is that from twenty-one to thirty years old. That group decided to be used to establish hyposmia, with TDI score below 30.75.
Consequently, patients at the current study divided into six subgroups according to their age and at each subgroup descriptive statistics computed separately for male and female participants.
38 Results with age and gender discrimination
Table 4. Age group 1: 11-20 years [all=4, male=1, female=3]
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE
Mean 10.69 13.5 13.5 37.69
All subjects
Male 9.75 14 12 35.75
Female 11 13.3 12 38.33
Median 10.12 13.5 13.5 37.62
All subjects
Variance 4.68 0.33 3 13.02
All subjects
Standard 2.16 0.58 1.73 3.6 deviation
All subjects
Male
Female 2.54 0.57 1.73 4.12
Minimum 8.75 13 12 33.75
All subjects
Male
Female 8.75 13 12 33.75
Maximum 13.75 14 15 41.75
All subjects
Male
Female 13.75 14 15 41.75
Range 5 1 3 8
All subjects
Interquartile 3.94 1 3 6.94 range
All subjects
39 Table 5. Age group 2: 21-30 years [all=14, male=3, female=11]
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE
Mean 9.09 13.86 14 36.95
All subjects
Male 6.75 11.66 13 31.41
Female 9.73 14.45 14.27 38.45
Median 10 14.5 14 38.87
All subjects
Variance 6.83 6.13 2.92 36.83
All subjects
Standard 2.61 2.48 1.7 6.07 deviation
All subjects
Male 3.13 3.21 1.73 7.95
Female 2.2 2.02 1.68 4.86
Minimum 3.5 8 11 22.5
All subjects
Male 3.5 8 11 22.5
Female 3.75 10 11 24.75
Maximum 11.5 16 16 42.25
All subjects
Male 9.75 14 14 37.75
Female 11.5 16 16 42.25
Range 8 8 5 19.75
All subjects
Interquartile 2.75 3.25 3 5 range
All subjects
40 Table 6. Age group 3: 31-40 years [all=12, male=5, female=7]
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE
Mean 6.81 11.75 14.33 32.89
All subjects
Male 7.1 11.6 15.2 33.9
Female 6.61 11.86 13.71 32.18
Median 6.62 12 15 33.62
All subjects
Variance 2.51 6.57 2.61 18.95
All subjects
Standard 1.58 2.56 1.61 4.35 deviation
All subjects
Male 1.46 2.7 0.84 1.66
Female 1.75 2.67 1.8 5.61
Minimum 5 7 11 25.5
All subjects
Male 5.5 7 14 31.5
Female 5 8 11 25.5
Maximum 9.5 16 16 41.5
All subjects
Male 9.5 14 16 36
Female 9.5 16 16 41.5
Range 4.5 9 5 16
All subjects
Interquartile 2.81 3.5 2.5 5.44 range
All subjects
41 Table 7. Age group 4: 41-50 years [all=16, male=7, female=9]
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE
Mean 7.08 10.94 14 32.01
All subjects
Male 7.39 10.57 13.43 31.39
Female 6.83 11.22 14.44 32.05
Median 7.5 11.5 15 32.87
All subjects
Variance 5.94 8.73 6.13 36.75
All subjects
Standard 2.44 2.95 2.48 6.06 deviation
All subjects
Male 2.62 3.15 3.2 8.05
Female 2.41 2.95 1.81 4.29
Minimum 3 5 7 15
All subjects
Male 3 5 7 15
Female 3 7 10 27
Maximum 11.75 16 16 39.75
All subjects
Male 11.75 14 16 39.75
Female 9.25 16 16 38.5
Range 8.75 11 9 24.75
All subjects
Interquartile 3.5 4 2.5 7 range
All subjects
42 Table 8. Age group 5: 51-60 years [all=9, male=2, female=7]
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE
Mean 7.53 11.78 13.89 33.19
All subjects
Male 6.25 10.5 15 31.75
Female 7.9 12.14 13.57 33.6
Median 8.25 12 14 34
All subjects
Variance 4.69 5.44 2.86 16.59
All subjects
Standard 2.16 2.33 1.69 4.07 deviation
All subjects
Male 2.83 2.12 1.41 3.53
Female 2.05 2.41 1.72 4.37
Minimum 4.25 9 11 25.25
All subjects
Male 4.25 9 14 29.25
Female 4.25 9 11 25.25
Maximum 10 16 16 38.25
All subjects
Male 8.25 12 16 34.25
Female 10 16 16 38.25
Range 5.75 7 5 13
All subjects
Interquartile 4.13 4 3 6.13 range
All subjects
43 Table 9. Age group 6: 61-70 years [all=4, male=1, female=3]
THRESHOLD DISCRIMINATION IDENTIFICATION TDI SCORE
Mean 7.44 11.25 13.25 31.87
All subjects
Male 9.5 12 12 33.5
Female 6.75 11 13.6 31.3
Median 7.5 11.5 13 39.34
All subjects
Variance 3.02 6.25 2.25 22.02
All subjects
Standard 1.74 2.5 1.5 4.7 deviation
All subjects
Male
Female 1.3 3 1.53 5.6
Minimum 5.25 8 12 25.25
All subjects
Male
Female 5.25 8 12 25.25
Maximum 9.5 14 15 36.25
All subjects
Male
Female 7.5 14 15 36.25
Range 4.25 6 3 11
All subjects
Interquartile 3.19 4.75 2.75 8.5 range
All subjects
44 It is worth mentioning that hyposmia is diagnosed according to different ages and TDI score as followed: from eleven to twenty years old: 28.5, from twenty- one to thirty: 30.75, from thirty-one to forty: 30.5, from forty-one to fifty: 28.15, from fifty-one to sixty: 27.25 and from sixty-one to seventy: 24.88. The aforementioned subsamples are those with the greatest significance for our study [72]. Moreover, at the table below we present the percentages of patients from each subgroup that recorded scores below the 10th percentile according to Oleszkiewicz’s study.
Table 10. Proportions of patients with scores below the 10th percentile
Threshold Discrimination Identification TDI score
11-20 years old 0 0 0 0
21-30 years old 14.28 14.28 14.28 14.28
31-40 years old 33.33 16.66 33.33 16.66
41-50 years old 25 37.5 12.5 18.75
51-60 years old 0 33.33 11.11 11.11
61-70 years old 0 25 0 0 It seems that olfactory discrimination and identification were affected more than threshold and TDI score. Our study carried out at the MS Center and most of the examinees were outpatient, so it is reasonable that threshold would be less impaired. That is because of the fact that both identification and discrimination are irreversibly affected at disease’s progressive stages.
Taking into consideration the cut-off of 30.75, fifteen out of the fifty-nine patients performed values lower than that, while one met the 30.75. With percentiles, 25.4% of the overall sample had TDI score lower than 30.75 or 27.11% had TDI score equal with or below 30.75.
Further analysis will be carried out according to hyposmia, as differences
45 emerged by dividing the sample into the six subgroups mentioned above.
Table 11. Hyposmia according to different cut-offs of TDI score relating to age subgroups
Subgroup (years) Cut-off (TDI score) Hyposmia (%)
11-20 28.5 0
21-30 30.75 14.25
31-40 30.5 33.33
41-50 28.15 18.75
51-60 27.25 11.11
61-70 24.88 0 It is obvious that by dividing patients according to their age, significant differentiations were recorded. For example, if the more general cut-off of 30.75 was used, the percentage of 27% could not reflect the interestingly low percentages that recorded to the youngest and the oldest patients.
Besides the assessment of smelling abilities through the Sniffin’Sticks procedure, patients with MS were additionally evaluated with the use of two visual analogue scales (VAS); one pertaining to the sense of olfaction and the other referring to gustation-both of them reflect a subjective way of assessment. According to the aforementioned VAS, 33.33% of the patients that characterized hyposmic due to TDI score (less than 30.75), had already realized their problem, while 31.25% of the hyposmics detected also impaired gustatory function.
Moreover, 2.32% of the patients with normosmia completed the VAS as if they had olfactory disability, but none of them mentioned gustatory problem. Of the overall sample, 11.86% of the participants detected reduction of their smelling ability and 8.47% mentioned diminished gustation.
46 Hyposmia established at TDI score lower than 30.75 Taking that distinction into consideration, further analysis could be presented. Sixteen patients suffering from MS were characterized as hyposmics according to the Sniffin’Sticks procedure.
Table 12. Descriptive statistics concerning patients with hyposmia
Threshold Discrimination Identification TDI Score SNOT-22 NOSE
Mean 5.12 9 12.62 26.73 31.44 17.81
Median 4.87 9 12 27 25 15
Variance 3.17 2.66 6.52 15.87 398.13 443.23
Standard 1.78 1.63 2.55 3.98 19.95 21.05 deviation
Minimum 3 5 7 15 10 0
Maximum 8.75 11 16 30.75 85 80
Range 5.75 6 9 15.75 75 80
Interquartile 2.87 2 4 4.47 16.5 28.75 range
First quartile 3.56 8 11 25.21 19.25 0
Third quartile 6.44 10 15 29.69 35.75 28.75
The same process was followed for the forty-three remaining normosmic patients with the results presented below:
47 Table 13. Descriptive statistics concerning patients with normosmia
Threshold Discrimination Identification TDI Score SNOT-22 NOSE
Mean 8.85 13.28 14.46 36.59 17.32 9.3
Median 8.75 13 15 36.25 15 0
Variance 3.21 4.06 1.54 9.23 194.89 218.55
Standard 1.79 2.01 1.24 3.04 13.96 14.78 deviation
Minimum 5.25 7 12 31.5 0 0
Maximum 13.75 16 16 42.25 58 55
Range 8.5 9 4 10.75 58 55
Interquartile 2.5 2 1 5.25 23 20 range
First quartile 7.5 12 14 34 5 0
Third quartile 10 14 15 39.25 28 20
It considered interesting and significant to proceed with comparisons between the hyposmic and normosmic patients according to NOSE and SNOT-22 scores, in order to investigate if hyposmics had nasal congestion too.
Table 14. Comparison between NOSE and hyposmic - normosmic patients
Number of patients NOSE Score p value
Hyposmics 16 17.81 (±21.05) 0.06
Normosmics 43 9.3 (±14.78)
48 Table 15. Comparison between SNOT-22 and hyposmic - normosmic patients
Number of patients SNOT-22 Score p value
Hyposmics 16 31.44 (±19.95) 0.01
Normosmics 43 17.32 (±13.96)
From the two tables above, it could be deducted that there is statistically significant difference between the hyposmic and normosmic patients according to their scores at the SNOT-22 test. In other words, hyposmic patients suffered from nasal congestion too, compared to normosmic ones.
Concerning the NOSE test comparison, p value (0.06-close to 0.05) pointed out that although there is not statistically significant difference, hyposmics performed quite worse scores, so it is possible that many of them suffered from congestion too.
49 X. Discussion
It has been noted several times that MS is a disease that attacks the CNS, so the extent of the affected structures reflects the disability status of patients. Inflammation is responsible for the relapses of MS, while more progressive stages are characterized of neurodegeneration. Moreover, is has been proved that olfactory threshold records lower scores at earlier stages of the disease, while discrimination and identification are impaired at progressive stages. Having the above into consideration, discrimination and identification involve more complex connections of the olfactory pathway or even more central structures, so more extended damage is needed in order for them to be decreased. It has been proved that olfactory pathway involves many areas among the olfactory epithelium and the frontal and temporal lobes. Consequently, the more widespread the damage is (axonal loss, demyelination), the more severe the smell perception becomes. By carrying out the Sniffin’Sticks procedure, the frequency of patients with hyposmia (TDI score equal or below 30.75) was 27.11% and results conform with other studies [65,73]. Gustatory function was impaired in 31.25% of patients with hyposmia and 8.47% of the overall sample. Olfaction is connected with gustation as they share similar structures such amygdala and orbitofrontal cortex. That could explain the coexistence of olfactory and gustatory disability, but our proportion (31.25%) is quite lower than other studies, maybe because our method was subjective [74].
With further analysis and division to subgroups per decade according to TDI score cut-offs, slight to significant differences were detected. The proportions were considerably lower at the extremity subgroups, from 0 to about 15%, while for ages 30 to 50, proportions of hyposmia maintained at about 20-30%. That division was first applied by Oleszkiewicz et al. When comparisons between nose problems and patients with hyposmia and normosmia carried out, we noticed that patients with hyposmia had more severe nose congestion too (p<0.05 for SNOT-22 questionnaire). One explanation for that could be the fact that SNOT-22 questionnaire contains questions that reflect patients’ functionality and quality of life except from nasal symptoms. As a result,
50 higher scores could emerge from answers having to do with sleep disorders, morning fatigue, difficulties in concentrating etc.
Researchers dealt with the correlation between regions that affected olfaction and MS. For many years the prevailing opinion was that olfactory bulb and tract had no evidence of MS plaques, the olfactory nerve could not be affected due to its unmyelination and thus olfaction was considered to be at the same levels with the normal population [75]. But since then many researches have been carried out and proved that regions responsible for the sense of smell were found demyelinated and harmed too. Smelling disability does not depend on a single mechanism and to that opinion is demonstrated by the fact that not only olfactory but also trigeminal nerve is affected and the evidence that co-morbidities of the disease reduce patients’ olfaction. For example, higher EDDS scores or psychological parameters (many patients suffer from depression) that stem from MS are responsible for lower identification and discrimination results. Another interesting observation is that patients with MS suffer more often from chronic sinusitis, so the impairment of the more peripheral olfactory epithelium could be responsible for some cases of low TDI scores. That could be an explanation to our observation about the hyposmic patients’ nose congestion problems. Further investigation should be done in order to correlate MS with nasal obstruction. It is important that olfactory bulb and tract demyelination is also met at other similar diseases such as Alzheimer’s and Parkinson’s disease and reduction of smelling ability could be used as a biomarker for them [76]. Olfaction depends not only on olfactory bulb and tract but also on cerebellum, orbitofrontal and visual cortex, insula and other specific areas of the CNS, whose inflammation and demyelination are the reasons for MS’s symptoms. From the above it could be deducted that olfactory dysfunction is reasonable and reflects the extent of the CNS damage [76]. It should not be omitted that findings from fMRI such as the reduction to olfactory bulb’s volume and the characteristic plaques at the olfactory cortex, have contributed to more completed comprehension of disease’s pathophysiology [65,77].
51 Sniffin’Sticks test represents a semi-objective procedure, so a variety of factors could influence the results and examiners should take each one of them into consideration in order to decide about patients’ level of olfactory dysfunction.
XI. Conclusion
• Multiple sclerosis represents a CNS disease with a variety of clinical manifestations.
• Olfaction is affected from the early stages of Multiple sclerosis, so its dysfunction could be used as a biomarker.
• To the perception of smell contribute central areas and structures (olfactory cortex), as well as more peripheral (olfactory epithelium).
• Olfactory identification and discrimination reduction reflect a more widespread and progressive damage, while threshold is affected at earlier stages and when more peripheral areas are under inflammation.
• Olfactory and gustatory perception share some networks, so it is reasonable that they interact.
• Multiple sclerosis is a complex disease with many co-morbidities, so Sniffin’Sticks test’s results should be interpreted carefully and according to each patient’s disability status and general condition.
52 References
1. Patel, R. G. Nasal Anatomy and Function. Facial Plast. Surg. 33, 3–8 (2017). 2. Jones N. The nose and paranasal sinuses physiology and anatomy. Adv Drug Deliv Rev 2001; 51(1-3):5-19. 3. Wexler DB, Davidson TM. The nasal valve: a review of the anatomy, imaging, and physiology. Am J Rhinology. 2004;18:143–150.
4. Park KS, Wells JM, Zorn AM, Wert SE, Whitsett JA. Sox17influences the differentiation of respiratory epithelial cells. DevBiol. 2006; 294: 192-202
5. Van Cauwenberge P, Sys L, De Belder T, Watelet JB. Anatomy and physiology of the nose and the paranasal sinuses. Immunol Allergy Clin North Am 2004; 4(1):1-17. 6. Doorly DJ, Taylor DJ, Schroter RC. Mechanics of airflow in the human nasal airways. Respir Physiol Neurobiol 2008; 163: 100-110. 7. Beule AG. Physiology and pathophysiology of respiratory mucosa of the nose and the paranasal sinuses. GMS Curr Top Otorhinolaryngol Head Neck Surg. 2010;9:Doc07. doi:10.3205/cto000071 8. Kahana-Zweig R, Geva-Sagiv M, Weissbrod A, Secundo L, Soroker N, Sobel N. Measuring and Characterizing the Human Nasal Cycle. PLoS One. 2016;11(10):e0162918. Published 2016 Oct 6. doi:10.1371/journal.pone.0162918 9. Zhao K, Scherer PW, Hajiloo SA, Dalton P. Effect of anatomy on human nasal air flow and odorant transport patterns: Implications for olfaction. Chem Senses 2004; 29(5):365-379. 10. Bushdid C, Magnasco MO, Vosshall LB, Keller A. Humans can discriminate more than 1 trillion olfactory stimuli. Science. 2014;343:1370–2. 11. Özay H, Çakır A, Ecevit MC. Retronasal Olfaction Test Methods: A Systematic Review. Balkan Med J. 2019;36(1):49–59. doi:10.4274/balkanmedj.2018.0052
53 12. Baraniuk JN, Merck SJ. Nasal reflexes: implications for exercise, breathing, and sex. Curr Allergy Asthma Rep. 2008;8(2):147–153. doi:10.1007/s11882-008-0025-7 13. Baraniuk, J. N., & Kim, D. Nasonasal Re fl exes , the Nasal Cycle , and Sneeze. Curr Allergy Asthma Rep (2007) 7: 105. https://doi.org/10.1007/s11882-007-0007-1 14. Behrman A, Shikowitz MJ, Dailey S. The effect of upper airway surgery on voice. Otolaryngol Head Neck Surg. 2002;127(1): 36-42. 15. Rosanowski F, Eysholdt U. Phoniatric aspects in cleft lip patients. Facial Plast Surg. 2002;18(3):197-203. 16. de Castro F. Wiring Olfaction: The Cellular and Molecular Mechanisms that Guide the Development of Synaptic Connections from the Nose to the Cortex. Front Neurosci. 2009;3:52. Published 2009 Dec 4. doi:10.3389/neuro.22.004.2009 17. Lancet, D. (1988) Molecular components of olfactory reception and transduction. In Margolis, F.L. and Getchell, T.V. (eds), Molecular Neurobiology of the Olfactory System Plenum Press, New York, pp. 25-50. 18. Scott, J.W. (1986) The olfactory bulb and central pathways. Experientia , 42, 223-232 19. Zatorre, R. J., Jones-Gotman, M., Evans, A. C., & Meyer, E. (1992). Functional localization and lateralization of human olfactory cortex. Nature, 360(6402), 339–340. https://doi.org/10.1038/360339a0 20. A. K. Afifi, R. A. Bergman: Functional Neuroanatomy, Text and Atlas, 2nd edition, McGraw-Hill (2005), p. 139-143. 21. Lucassen, E. B., Turel, A., Knehans, A., Huang, X., & Eslinger, P. (2016). Olfactory dysfunction in Multiple Sclerosis : A scoping review of the literature. Multiple Sclerosis and Related Disorders, 6, 1–9. https://doi.org/10.1016/j.msard.2015.12.002 22. Jordy, S. S., Junior, A. S., André, F., Macedo, B., Manica, G. R., Tilbery, C. P., & Carabetta, E. G. (2016). Olfactory alterations in patients with multiple sclerosis, (January), 697–700. https://doi.org/10.1590/0004-282X20160128
54 23. Ciurleo, R., Bonanno, L., Salvo, S. De, Romeo, L., Rifici, C., Sessa, E., et al. (2018). Olfactory dysfunction as a prognostic marker for disability progression in Multiple Sclerosis : An olfactory event related potential study, 1–9. https://doi.org/10.1371/journal.pone.0196006 24. Bsteh, G., Berek, K., Hegen, H., Teuchner, B., Auer, M., Wurth, S., et al. (2018). Smelling multiple sclerosis : Different qualities of olfactory function reflect either inflammatory activity or neurodegeneration. https://doi.org/10.1177/1352458518814113 25. Atalar, A. Ç., Erdal, Y., Tekin, B., Yıldız, M., Akdoğan, Ö., & Emre, U. (2018). Author ’ s Accepted Manuscript. Multiple Sclerosis and Related Disorders. https://doi.org/10.1016/j.msard.2018.02.032 26. Carotenuto, A., Costabile, T., Moccia, M., Falco, F., Scala, M. R., Russo, C. V, et al. (2018). PT US CR. https://doi.org/10.1016/j.msard.2018.09.024 27. Doty, R. L., Tourbier, I. A., Pham, D. L., Cuzzocreo, J. L., Udupa, J. K., Karacali, B., et al. (2016). Taste dysfunction in multiple sclerosis. Journal of Neurology. https://doi.org/10.1007/s00415-016-8030-6 28. Garg, N., & Smith, T. W. (2015). An update on immunopathogenesis, diagnosis, and treatment of multiple sclerosis.Brain and Behavior, 5(9).https://doi.org/10.1002/brb3.362 29. Milo, R., & Miller, A. (2014). Revised diagnostic criteria of multiple sclerosis. Autoimmunity Reviews, 13(4–5), 518–524. https://doi.org/10.1016/j.autrev.2014.01.012 30. Compston, A. (2005). The story of multiple sclerosis.McAlpine’s Multiple Sclerosis: Fourth Edition, 3–68. https://doi.org/10.1016/B978- 0-443-07271-0.50003-3 31. Charcot M. Histologie de la sclerose en plaques.Gaz Hosp. 1868; 141:554–5. 557–8. 32. Ludwin, S. K., Antel, J., & Arnold, D. L. (2016). Multiple sclerosis. Neuroscience in the 21st Century: From Basic to Clinical, Second Edition. https://doi.org/10.1007/978-1-4939-3474-4_103 33. Browne, P., Chandraratna, D., Angood, C., Tremlett, H., Baker, C., Taylor, B. V., & Thompson, A. J. (2014). Atlas of multiple sclerosis
55 2013: A growing global problem with widespread inequity. Neurology, 83(11), 1022–1024. https://doi.org/10.1212/WNL.0000000000000768
34. Otallah, S., &Banwell, B. (2018).Pediatric Multiple Sclerosis: an Update. Current Neurology and Neuroscience Reports, 18(11), 4–11. https://doi.org/10.1007/s11910-018-0886-7
35. Corral-Verdugo, V., Frías-Armenta, M., &García-Cadena, C. H. (2013).Introduction to the psychological dimensions of sustainability.Psychological Approaches to Sustainability: Current Trends in Theory, Research and Applications, 11(3), 3–18. https://doi.org/10.1038/NRNEUROL.2015.14
36. Dutta, R., & Trapp, B. D. (2015). Relapsing and progressive forms of multiple sclerosis – insights from pathology, 27(3), 271– 278.https://doi.org/10.1097/WCO.0000000000000094
37. Kamm, C. P., Uitdehaag, B. M., &Polman, C. H. (2014). Multiple sclerosis: Current knowledge and future outlook. European Neurology, 72(3–4), 132–141. https://doi.org/10.1159/000360528
38. Tullman MJ, Oshinsky RJ, Lublin FD, Cutter GR. Clinical char- acteristics of progressive relapsing multiple sclerosis. MultScler. 2004;10(4):451-454. 39. Lublin, F. D. (2014). New Multiple Sclerosis Phenotypic Classification. European Neurology, 72(s1), 1–5.doi:10.1159/000367614
40. Marcus, J. F., &Waubant, E. L. (2013). Updates on Clinically Isolated Syndrome and Diagnostic Criteria for Multiple Sclerosis.TheNeurohospitalist, 3(2), 65–80. https://doi.org/10.1177/1941874412457183 41. Huang WJ, Chen WW, Zhang X. Multiple sclerosis: Pathology, diagnosis and treatments. ExpTher Med. 2017;13(6):3163–3166. doi:10.3892/etm.2017.4410 42. Kenealy, S. J., Pericak-Vance, M. A., & Haines, J. L. (2003). The genetic epidemiology of multiple sclerosis. Journal of Neuroimmunology, 143(1-2), 7–12.doi:10.1016/j.jneuroim.2003.08.005
56 43. Ramagopalan SV, Maugeri NJ, Handunnetthi L, Lincoln MR, Orton SM, et al. (2009) Expression of the Multiple Sclerosis-Associated MHC Class II Allele HLA-DRB1*1501Is Regulated by Vitamin D. PLOS Genetics 5(2): e1000369. https://doi.org/10.1371/journal.pgen.1000369 44. International Multiple Sclerosis Genetics Consortium; Wellcome Trust Case Control Consortium 2, Sawcer S, et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature. 2011;476(7359):214–219. Published 2011 Aug 10. doi:10.1038/nature10251 45. Liu, H., Huang, J., Dou, M., Liu, Y., Xiao, B., Liu, X., & Huang, Z. (2017). Variants in the IL7RA gene confer susceptibility to multiple sclerosis in Caucasians: evidence based on 9734 cases and 10436 controls. Scientific Reports, 7(1), 1207. https://doi.org/10.1038/s41598- 017-01345-8 46. Milo, R., &Kahana, E. (2010). Autoimmunity Reviews Multiple sclerosis : Geoepidemiology , genetics and the environment. Autoimmunity Reviews, 9(5), A387–A394. https://doi.org/10.1016/j.autrev.2009.11.010 47. Sospedra, M., & Martin, R. (2005). I MMUNOLOGY OF M ULTIPLE S CLEROSIS ∗. https://doi.org/10.1146/annurev.immunol.23.021704.115707 48. Jadidi-Niaragh, F., &Mirshafiey, A. (2011). Th17 Cell, the New Player of Neuroinflammatory Process in Multiple Sclerosis. Scandinavian Journal of Immunology, 74(1), 1–13.doi:10.1111/j.1365- 3083.2011.02536.x 49. Zozulya, A. L., &Wiendl, H. (2008). The role of regulatory T cells in multiple sclerosis. Nature Clinical Practice Neurology, 4(7), 384– 398.doi:10.1038/ncpneuro0832 50. Tsunoda, I., &Fujinami, R. S. (2002). Inside-Out versus Outside-In models for virus induced demyelination: axonal damage triggering demyelination. Springer Seminars in Immunopathology, 24(2), 105– 125.doi:10.1007/s00281-002-0105-z 51. Gelfand, J. M. (2014). Multiple sclerosis. Multiple Sclerosis and Related Disorders, 269–290.doi:10.1016/b978-0-444-52001-2.00011-x
57 52. McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121–127. 53. Thompson, A. J. et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol. 17, 162–173 (2017). The most recent MS criteria in which the role of MRI in documenting dissemination in time and space are discussed. 54. Current Disease-Modifying Therapies in Multiple Sclerosis. (2003). Seminars in Neurology, 23(2), 133–146. doi:10.1055/s-2003-41138 55. Doshi, A., & Chataway, J. (2017). Clinical Medicine, 17(6), 530–536. Multiple sclerosis, a treatable disease doi:10.7861/clinmedicine.17-6- 530 56. La Mantia L, Di Pietrantonj C, Rovaris M, Rigon G, Frau S, Berardo F, et al., Interferons‐beta versus glatiramer acetate for relapsing‐remitting multiple sclerosis. Cochrane Database of Systematic Reviews 2016, Issue 11. Art. No.: CD009333. DOI: 10.1002/14651858.CD009333.pub3. 57. La Mantia, L., Tramacere, I., Firwana, B., Pacchetti, I., Palumbo, R., & Filippini, G. (2016). Fingolimod for relapsing-remitting multiple sclerosis. Cochrane Database of Systematic Reviews. doi:10.1002/14651858.cd009371.pub2 58. Miller, A. E. (2017). Oral teriflunomide in the treatment of relapsing forms of multiple sclerosis: clinical evidence and long-term experience. Therapeutic Advances in Neurological Disorders, 10(12), 381–396. doi:10.1177/1756285617722500 59. Wingerchuk, D. M., & Carter, J. L. (2014). Multiple Sclerosis: Current and Emerging Disease-Modifying Therapies and Treatment Strategies. Mayo Clinic Proceedings, 89(2), 225–240. doi:10.1016/j.mayocp.2013.11.002 60. Wingerchuk, D. M., & Weinshenker, B. G. (2016). Disease modifying therapies for relapsing multiple sclerosis. BMJ, i3518. doi:10.1136/bmj.i3518
58 61. Weinshenker BG. Epidemiology of multiple sclerosis. Neurol. Clin. 1996;14:291–308. 62. Bsteh, G., Berek, K., Hegen, H., Teuchner, B., Auer, M., Wurth, S., et al. (2018). Smelling multiple sclerosis : Different qualities of olfactory function reflect either inflammatory activity or neurodegeneration.https://doi.org/10.1177/1352458518814113) 63. Lutterotti A, Vedovello M, Reindl M, et al. Olfactory threshold is impaired in early, active multiple sclerosis. MultScler2011; 17(8): 964– 969. 64. Bsteh G, Hegen H, Ladstatter F, et al. Transient impairment of olfactory threshold in acute multiple sclerosis relapse. MultSclerRelatDisord2018; 23: 74–77. 65. Doty RL, Li C, Mannon LJ, et al. Olfactory dysfunction in multiple sclerosis. Relation to plaque load in inferior frontal and temporal lobes.Ann N Y AcadSci1998; 855: 781–786. 66. Bsteh G, Hegen H, Ladstätter F, et al. Change of olfactory function as a marker of inflammatory activity and disability progression in MS. MultScler. Epub ahead of print 1 November 2017. DOI: 10.1177/1352458517745724. 67. Fleiner, F., Dahlslett, S. B., Schmidt, F., Harms, L., & Goektas, O. (2010). Olfactory and Gustatory Function in Patients with Multiple Sclerosis. American Journal of Rhinology & Allergy, 24(5), e93– e97.doi:10.2500/ajra.2010.24.3506 68. Hummel T, Sekinger B, Wolf SR et al (1997) “Sniffin” sticks’: olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold. Chem Senses 22:39–52 69. Rumeau C, Nguyen DT, Jankowski R (2015) How to assess olfactory performance with the Sniffin’ Sticks test(®). Eur Ann Otorhinolaryngol Head Neck Dis. [pii]: S1879-7296(15)00103-9 70. A. Rolet, E. Magnin, J.L. Millot, E. Berger, C. Vidal, G. Sileman, L. Rum bachOlfactory dysfunction in multiple sclerosis: evidence of a decrease in different aspects of olfactory function Eur. Neurol., 69 (2013), pp. 166-170
59 71. Doty RL. Olfactory dysfunction and its measurement in the clinic. World J Otorhinolaryngol Head Neck Surg. 2015;1(1):28–33. Published 2015 Oct 26. doi:10.1016/j.wjorl.2015.09.007 72. Oleszkiewicz, A.; Schriever, V.A.; Croy, I.; Hähner, A.; Hummel, T. Updated Sniffin’ Sticks normative data based on an extended sample of 9139 subjects. European Archives of Oto-Rhino- Laryngology2019,276, 719–728.doi:10.1007/s00405-018-5248-1. 73. Zivadinov R, Zorzon M, MontiBragadin L, Pagliaro G, Cazzato G. Olfactory loss in multiple sclerosis. J NeurolSci, 1999;168(2):127-30. doi:10.1016/S0022-510X(99)00189-6 74. Uecker FC, Olze H, Kunte H, Gerz C, Göktas Ö, et al. (2017) Longitudinal Testing of Olfactory and Gustatory Function in Patients with Multiple Sclerosis. PLOS ONE 12(1): e0170492. https://doi.org/10.1371/journal.pone.0170492 75. Ansari KA. 1976. Olfaction in multiple sclerosis. With a note on the discrepancy between optic and olfactory involvement. Eur Neurol 14:138–145. 76. Lucassen, E. B., Turel, A., Knehans, A., Huang, X., & Eslinger, P. (2016). Olfactory dysfunction in Multiple Sclerosis: A scoping review of the literature. Multiple Sclerosis and Related Disorders, 6, 1– 9. doi:10.1016/j.msard.2015.12.002 77. Doty, R. L., Li, C., Mannon, L. J., & Yousem, D. M. (1997). Olfactory Dysfunction in Multiple Sclerosis. New England Journal of Medicine, 336(26), 1918–1919. doi:10.1056/nejm199706263362617
Appendices
60 • Appendix 1
Written informed consent
ΕΝΤΥΠΟ ΣΥΓΚΑΤΑΘΕΣΗΣ ΑΣΘΕΝΟΥΣ
Βεβαιώνω ενυπόγραφα ότι μετά από αναλυτική ενημέρωση για τους σκοπούς της μελέτης:
Μελέτη της όσφρησης, της γεύσης και της σχετιζόμενης με αυτές ποιότητας ζωής σε ασθενείς με χρόνια ρινοκολπίτιδα, πολλαπλή σκλήρυνση και γνωστικές διαταραχές με σταθμισμένα ερωτηματολόγια, κλινική αξιολόγηση και σταθμισμένες δοκιμασίες όσφρησης και γεύσης.
που διεξάγεται από την Α’ ΩΡΛ κλινική του Τμήματος Ιατρικής Σχολής Επιστημών Υγείας του ΑΠΘ δέχομαι εθελοντικά να συμμετάσχω στην παραπάνω μελέτη.
Ο συμμετέχων / Η συμμετέχουσα
Θεσσαλονίκη, ...../...... /......
• Appendix 2
61 Visual analogue scales
Τον τελευταίο μήνα:
1. Έχετε κάποιο πρόβλημα με την όσφρησή σας;
0 10
0 1 2 3 4 (0=κανένα πρόβλημα, 4=σοβαρό πρόβλημα)
2. Έχετε κάποιο πρόβλημα με τη γεύση σας; 0 10
0 1 2 3 4 (0=κανένα πρόβλημα, 4=σοβαρό πρόβλημα)
3. Έχετε κάποιο πρόβλημα με την αναπνοή από τη μύτη; 0 10
0 1 2 3 4 (0=κανένα πρόβλημα, 4=σοβαρό πρόβλημα)
4. Έχετε καταρροή; 0 10
0 1 2 3 4 (0=κανένα πρόβλημα, 4=σοβαρό πρόβλημα)
• Appendix 3
NOSE (Nasal Obstruction Symptom Evaluation) Questionnaire-greek validation
Κατά τον τελευταίο ΕΝΑµήνα, πόσο μεγάλο πρόβληµα αποτέλεσαν για εσάς οι παρακάτω καταστάσεις;
62 Σας παρακαλούµε βάλτε σε κύκλο τη σωστότερη δυνατή απάντηση:
NOSE (τον τελευταίο μήνα)
Κανένα Πολύ ήπιο Μέτριο Μεγάλο Σοβαρό πρόβλημα πρόβλημα πρόβλημα πρόβλημα πρόβλημα
Ρινική συμφόρηση ή 0 1 2 3 4 μπούκωμα
Ρινική απόφραξη 0 1 2 3 4
Δυσκολία στην αναπνοή 0 1 2 3 4 από τη μύτη
Δυσκολία στον ύπνο 0 1 2 3 4
Αδυναμία αναπνοής από 0 1 2 3 4 τη μύτη κατά τη σωματική δραστηριότητα
Lachanas VA, Tsiouvaka S, Tsea M, Hajiioannou JK, Skoulakis CEValidation of the nasal obstruction symptom evaluation (NOSE) scale for Greek patients.OtolaryngolHeadNeckSurg. 2014 Nov;151(5):819-23
• Appendix 4
Sino-NasalOutcomeTest-22 (SNOT-22) Questionnaire-greek validation
Παρακάτω θα διαβάσετε µια λίστα συµπτωµάτων και κοινωνικών/συναισθηµατικών επιπτώσεων του ρινικού σας προβλήµατος.Θα θέλαµε να µάθουµε περισσότερα για το πρόβληµά σας και θα το εκτιµούσαµε εάν απαντούσατε όσο το δυνατόν καλύτερα στις παρακάτω ερωτήσεις.Δεν υπάρχουν σωστές ή λάθος απαντήσεις και µόνο εσείς µπορείτε να µας δώσετε αυτές τις πληροφορίες.
SNOT-22 (τον τελευταίο μήνα)
63 Πολύ μικρό
πρόβλημα πρόβλημα πρόβλημα πρόβλημα πρόβλημα πρόβλημα
Σοβαρό Σοβαρό σοβαρό
Κανένα Κανένα
Μέτριο Μέτριο
Μικρό Μικρό
Πολύ
1. Καθαρισμός ρινός (φυσάτε τη μύτη σας) 0 1 2 3 4 5
2. Πταρμοί 0 1 2 3 4 5
3. Ρινικός κατάρρους 0 1 2 3 4 5
4. Ρινική συμφόρηση 0 1 2 3 4 5
5. Ανοσμία, αγευσία 0 1 2 3 4 5
6. Βήχας 0 1 2 3 4 5
7. Οπισθορινική βλέννα 0 1 2 3 4 5
8. Πηκτή ενδορινική βλέννα 0 1 2 3 4 5
9. Αίσθημα πλήρωσης ώτων 0 1 2 3 4 5
10. Ζάλη 0 1 2 3 4 5
11. Ωταλγία 0 1 2 3 4 5
12. Άλγος, αίσθημα πίεσης κεφαλής 0 1 2 3 4 5
13. Διαταραχές έναρξης ύπνου 0 1 2 3 4 5
14. Αφυπνίσεις κατά τον ύπνο 0 1 2 3 4 5
15. Ελλειπής ύπνος 0 1 2 3 4 5
16. Πρωινός κάματος 0 1 2 3 4 5
17. Αδυναμία 0 1 2 3 4 5
18. Μειωμένη παραγωγικότητα 0 1 2 3 4 5
19. Ελάττωση συγκέντρωσης 0 1 2 3 4 5
20. Απογοητευμένος-η/ κουρασμένος-η/ 0 1 2 3 4 5 ευερέθιστος-η 21. Αποθαρρυμένος-η 0 1 2 3 4 5
22. Κατησχυμένος-η (ντροπιασμένος-η) 0 1 2 3 4 5
Lachanas VA, Tsea M, Tsiouvaka S, Hajiioannou JK, Skoulakis CE, Bizakis JG. The sino-nasal outcome test (SNOT) - 22: validation for Greek patients. EurArchOtorhinolaryngol. 2014 Oct;271(10):2723-8.
• Appendix 5
64 Identification test (Sniffin’Sticks) – greek validation
1. ΠΟΡΤΟΚΑΛΙ ΒΑΤΟΜΟΥΡΟ ΦΡΑΟΥΛΑ ΑΝΑΝΑΣ
2. ΚΑΠΝΟΣ ΚΟΛΛΑ ΔΕΡΜΑ ΠΑΠΟΥΤΣΙΩΝ ΓΡΑΣΙΔΙ
3. ΤΥΡΙ ΒΑΝΙΛΙΑ ΣΟΚΟΛΑΤΑ ΚΑΝΕΛΑ
4. ΠΡΑΣΟ ΕΛΑΤΟ ΜΕΝΤΑ ΚΡΕΜΜΥΔΙ
5. ΚΑΡΥΔΑ ΜΠΑΝΑΝΑ ΚΑΡΥΔΙ ΚΕΡΑΣΙ
6. ΡΟΔΑΚΙΝΟ ΚΡΕΜΜΥΔΙ ΛΕΜΟΝΙ ΜΕΝΤΑ
7. ΓΡΑΠΑ ΚΕΡΑΣΙ ΔΥΟΣΜΟΣ ΜΠΙΣΚΟΤΟ
8. ΜΟΥΣΤΑΡΔΑ ΜΕΝΤΑ ΚΕΡΑΣΙ ΛΑΔΟΜΠΟΓΙΑ
9. ΚΡΕΜΜΥΔΙ ΣΚΟΡΔΟ ΞΙΝΟΛΑΧΑΝΟ ΚΑΡΟΤΟ
10. ΤΣΙΓΑΡΟ ΚΑΦΕΣ ΚΡΑΣΙ ΚΑΠΝΟΣ
11. ΠΕΠΟΝΙ ΨΑΡΙ ΤΥΡΙ ΜΗΛΟ
12. ΓΑΡΥΦΑΛΛΟ ΠΙΠΕΡΙ ΚΑΝΕΛΑ ΜΟΥΣΤΑΡΔΑ
13. ΑΧΛΑΔΙ ΔΑΜΑΣΚΗΝΟ ΡΟΔΑΚΙΝΟ ΑΝΑΝΑΣ
14. ΧΑΜΟΜΗΛΙ ΣΜΕΟΥΡΟ ΤΡΙΑΝΤΑΦΥΛΛΟ ΚΕΡΑΣΙ
15. ΟΥΖΟ ΜΕΛΙ ΡΟΥΜΙ ΕΛΑΤΟ
16. ΨΩΜΙ ΨΑΡΙ ΤΥΡΙ ΖΑΜΠΟΝ
Konstantinidis I, Printza A, Genetzaki S, Mamali K, Kekes G, Constantinidis J.Cultural adaptation of an olfactory identification test: the Greek version of Sniffin' Sticks.Rhinology. 2008 Dec;46(4):292-6
65 • Appendix 6
Discrimination test (Sniffin’Sticks)
• Appendix 7
Threshold test (Sniffin’Sticks)
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