Effects on the Olfactory Epithelium in the Nose and the Olfactory Nerve (Nervus Olfactorius) From Exposure to Diesel Exhaust
Phase: NA
Conditions: Alzheimer Disease (AD), Dementia Alzheimer Type, Air Pollution Exposure
Sponsor: Umeå University

 
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Study Protocol 
Title of Research Study: E5ects on the Olfactory 
Epithelium and the Olfactory Nerve from Exposure to 
Diesel Exhaust 
Lung and Allergy Department, Norrland University Hospital 
Umeå, Sweden 
Principal Investigator: Dr. Ala Muala 
Co-Investigators: Prof. Thomas Sandström, Prof. Anders Blomberg 
Date of the document: 17-06-2021 
 
 

 
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Study Protocol 
 
Effects on the Olfactory Epithelium and the Olfactory Nerve from Exposure to Diesel 
Exhaust 
 
Scientific Research Question 
The overall goal of this research project is to study and deepen our understanding of the health 
effects associated with exposure to air pollution generated by the combustion of diesel, focusing 
on the nervous system. 
Specific aim: 
• 
To study the oxidative and inflammatory effects of exposure to diesel exhaust on the 
respiratory epithelium and the olfactory nerve in healthy individuals. Additionally, the 
study aims to determine whether these exposures result in measurable effects on the 
sense of smell and cognitive ability. 
 
Background 
UNICEF recently published a report highlighting the health effects of air pollution from a global 
perspective, with a particular focus on the situation for children. It is estimated that air pollution 
causes approximately 7 million deaths each year globally, with around 600,000 of these deaths 
being children under 5 years old. Indoor and outdoor air pollution are the 3rd and 9th leading 
causes of death, respectively, contributing to about 3.5 million premature deaths in 2010. 
The Swedish Environmental Research Institute IVL has estimated the Swedish healthcare and 
societal costs due to the negative impacts of air pollution on heart attacks, strokes, asthma, 
chronic obstructive pulmonary disease (COPD), and pneumonia at 42 billion SEK annually. 
Living in areas with high levels of air pollution is a risk factor for diseases of the central nervous 
system, including Alzheimer's disease (AD) and cognitive impairment in the elderly, 
Parkinson's disease, and stroke. 
It has also been demonstrated in people living in highly polluted urban areas that fine particles 
(PM2.5) have been found in the brain, showing that iron particles (magnetite) can be 
translocated to the brain. The entry point is the olfactory nerve, where particles can enter 
directly without having to penetrate the blood-brain barrier to reach the brain. It is primarily 
ultrafine particles (with an aerodynamic diameter <100-200 nm) that can enter through the 
olfactory nerve via inhalation. Particles of this size range originate from both motor exhausts 
and biomass combustion, depending on the combustion technique. 

 
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The olfactory nerve area is thus an important region of interest for understanding how 
particulate matter (PM) damages the brain. Interestingly, olfactory dysfunction has been 
reported in elderly women exposed to high levels of PM (19). It is critical that this possible 
entry route into the central nervous system is studied in detail to better understand the health 
effects of PM exposure in humans and to identify potential new biomarkers for air pollution 
effects. 
Dementia is a major cause of morbidity and mortality worldwide. By 2050, the number of 
Alzheimer's disease (AD) patients is expected to rise to over 130 million, up from 47 million 
today. In a study from northern Sweden, researchers (Forsberg et al.) demonstrated that 
exposure to traffic-related air pollutants may be a risk factor for the development of vascular 
dementia and Alzheimer's disease. Alzheimer's disease is the most common neurodegenerative 
disease, and there is currently no cure. Current medications only alleviate symptoms. 
Neuropathological changes in AD include the deposition of protein aggregates, 
neuroinflammation, and oxidative stress. Despite the clinical evidence that olfactory 
dysfunction precedes AD-associated cognitive decline by several decades, the significance of 
air pollution effects on the olfactory area has not been studied in human trials. 
Lipophilic particles such as diesel exhaust have been shown in animal studies to penetrate the 
brain via the olfactory epithelium and the olfactory nerve, causing neurological effects. Particles 
can, depending on the hydrocarbon profile on their surface and protein interactions, be 
transported along neurons, leading to post-translational neurogenic effects. This is likely not 
unique to the central nervous system but may also occur in the airways. We have previously 
demonstrated CNS effects using quantitative EEG analysis, which is consistent with increasing 
epidemiological reports of neurogenic impacts of air pollution. 
Our colleagues in Finland have established a new physiologically relevant human model of 
olfactory epithelium from healthy non-demented controls and Alzheimer's patients that can be 
used to study the effects of air pollution. By examining the acute effects on the olfactory nerve 
following exposure to diesel exhaust, this is a crucial step in advancing knowledge about the 
health effects of these two major contributors to PM. 
Inclusion Criteria and Population 
Healthy non-smoking participants aged 20-70 years with an equal gender distribution will be 
recruited. 
The participants will be examined regarding clinical status, lung function, prick test, ECG, 
blood status, bleeding status, Na+, K+, and creatinine. 
Exclusion Criteria 
1. Allergic rhinitis 
2. Asthma 
3. Diabetes mellitus 
4. Renal or liver failure 
5. Angina or previous ventricular arrhythmia 
6. Systolic blood pressure >190 mmHg or <100 mmHg 
7. Previous blood disorders 
8. Blood donation within the past three months 

 
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9. Previous or ongoing tobacco smoking and exposure to air pollution in the workplace 
10. Lack of informed consent 
 
Project Description 
The studies will be conducted using our well-established techniques and methods. Exposure to 
controlled levels of motor exhaust from diesel fuel has been conducted for more than two 
decades, and in recent years we have also developed a collaboration with Associate Professor 
Christoffer Boman's group at the Thermochemical Energy Conversion Laboratory (TEC-Lab) 
at Umeå University. This offers a unique opportunity to study under different combustion 
conditions. 
Hypothesis: Diesel exhaust cause cell dysfunction in the olfactory system secondary to 
mitochondrial dysfunction and oxidative stress. Additionally, we hypothesize that these 
exposures result in measurable effects on the sense of smell and cognitive ability. 
 
Project Description 
The studies will be conducted using our well-established techniques and methods. Exposure to 
controlled levels of diesel fuel exhaust has been studied for over two decades. 
Hypothesis: 
Diesel exhaust gases cause cell dysfunction in the olfactory system secondary to mitochondrial 
dysfunction and oxidative stress. The hypothesis also includes studying whether exposures lead 
to measurable effects on the sense of smell and cognitive abilities. 
 
Study Design 
Forty people will be exposed to diesel exhaust gases and filtered air. Exposures will be blinded 
and randomized. Both groups will undergo mucosal biopsies of the olfactory epithelium after 
local anesthesia following exposure. 
All samples collected during the study will be coded, and data will be processed in a blinded 
manner. 
 
ResearchParticipants: 
Forty healthy, non-smoking individuals aged 20-70 years will be included. Strict inclusion and 
exclusion criteria will be applied based on a medical assessment as outlined above. 
 
Exposure to Diesel Exhaust 

 
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Diesel exhaust will be generated using a Volvo diesel engine. More than 90% of the exhaust 
will be shunted, with the remaining portion mixed with filtered air (HEPA filter) and directed 
into the exposure chamber at a concentration of 300 µg/m³ using techniques established over 
decades. The exposure will last for 1 hour, blinded and randomized. During the exposure, 
participants will alternate between 15-minute periods of rest and exercise on an ergometer cycle 
with a load that ensures a minute ventilation of 20L/min/m² body surface area. Symptoms will 
be recorded every 30 minutes based on a modified Borg scale. 
 
Olfactory Epithelium Biopsy 
After local anesthesia with Xylocain following exposure, a small biopsy of the nasal mucosa 
will be taken by a specialist doctor. Material from the nasal biopsies will be used both for culture 
experiments and for later analyses, including PCR analysis after freezing at -80°C, and will be 
sent to our collaborators, Dr. Katja Kanninen and Dr. Pasi Jalava at the University of Eastern 
Finland. Nasal mucosa biopsies have been performed previously at the ENT department at 
Umeå University following ethical approval. Complication risks are virtually nonexistent, but 
there may be minor discomfort or slight nasal bleeding, which can be easily stopped using a 
compress applied to the biopsy site. ENT doctors are experienced in managing nasal bleeding. 
Biopsy sampling will be randomized between the left and right sides across two exposure 
sessions, with the contralateral biopsy taken after the second exposure. 
 
In-Vitro Studies and Biomarker Analysis 
Effects at the cellular level of exposure to air pollution will be studied in olfactory epithelial 
cells 
using 
in-vitro 
tests 
at 
the 
University 
of 
Kuopio, 
Finland. 
RNA sequencing and DNA methylation will be conducted to identify biomarkers for acute 
exposure to air pollutants at the Institute of Experimental Medicine, Czech Academy of 
Science, Czech Republic. 
 
Olfactory Testing 
The Sniffin’ Sticks test (Hummel et al., 1997), a commercially available test, will be used to 
determine the participant's odor detection threshold. The odorant (n-butanol) is presented in 
marker pens filled either with n-butanol or a blank (no odor). The test uses 16 concentrations 
of n-butanol, from the weakest concentration (1.2 ppm n-butanol) to the strongest (4% n-
butanol). In a "triple-forced-choice paradigm," the detection threshold is determined using a 
staircase method (Doty, 1991). Three pens are presented in a randomized order—two with 
blank stimuli and one with the odorant at a given concentration. The participant’s task is to 
identify which pen contains the odorant. 
Participants wear eye masks to prevent visual identification of the pens. The pen is placed 
approximately 2 cm below the nostrils for about 3 seconds. The test starts at the 8th 
concentration level, and if the participant correctly identifies the odorant four times in a row, 

 
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the test continues with higher concentrations. If the participant fails to do so, the test proceeds 
to lower concentrations. 
The test has good test-retest reliability (Albrecht et al., 2008) and validity (Ribeiro et al., 2016). 
The exposure levels of n-butanol are much lower than the occupational exposure limit of 15 
ppm (45 mg/m³), so no toxic effects or discomfort risks are expected. 
Cognitive Testing 
Cognitive ability will be assessed using the Letter Digit Substitution Test, a paper-and-pencil 
test that evaluates working memory, processing speed, and attention. A key at the top of the 
page provides the correct mapping for the numbers 1 to 9, which are paired with specific letters. 
Below the key, there are 120 letters (9 different ones), each connected to a blank square. The 
participant's task is to write as many numbers as possible in the empty squares under a 60-
second time limit according to the letter-number mapping provided in the key. 
The test has good test-retest reliability (Hoex et al., 2002) and validity (Natu & Agarwal, 1995). 
There is no risk of pain or discomfort associated with the test. 
 
Blood Tests 
Blood samples will be taken before exposure and before olfactory epithelium biopsies. These 
will be analyzed for cell count, inflammation markers, and lymphocyte subtyping using flow 
cytometry. A maximum of 100 ml of blood will be drawn during each exposure. 
The effects of exposure to air pollution at the cellular level will be studied in monocytes from 
peripheral blood using in-vitro tests. Further analyses, including PCR (polymerase chain 
reaction), will be conducted for the APOE gene, a genetic factor that can influence sensitivity 
to air pollution. These analyses will be performed at Kuopio University, Finland. 
RNA sequencing and DNA methylation will also be conducted to identify biomarkers for acute 
exposure to air pollution at the Institute of Experimental Medicine, Czech Academy of Science, 
Czech Republic. 
 
COVID-19 Testing 
Participants will be tested for SARS-CoV-2 before each exposure session. 
 
 
 
Significance 

 
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Air pollution causes significant health effects on the lungs, cardiovascular system, and nervous 
system, which affects many patients and places a heavy burden on healthcare systems, with 
societal costs exceeding 40 billion SEK annually in Sweden. Our research aims to deepen the 
understanding of the multifaceted health effects of air pollution, which impacts various organ 
systems. This is especially important as we have previously shown that current medications do 
not prevent the full extent of the health effects of air pollution. Advanced research will lead to 
a better understanding and the potential development of more effective interventions, which is 
crucial for health as well as for industries and authorities. 
we are one of the few research groups with the knowledge and resources to conduct human 
exposure studies to examine this specific type of combustion and subsequently investigate its 
health effects using unique and advanced methods. Therefore, this project is highly significant 
in increasing our knowledge of how diesel exhaust negatively affects the olfactory nerve, which 
is crucial for understanding the mechanisms behind the health effects of air pollution, such as 
the development of cognitive decline and dementia. The findings may have important 
implications for public health policies and strategies to mitigate the impact of air pollution on 
cognitive health.