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Simulated Forest Immersion Therapy: Methods Development

International Journal of

Environmental Research

and Public Health

Study Protocol

Simulated Forest Immersion Therapy: Methods Development

Amy Miner Ross * and Reo Jane Francesca Jones

School of Nursing, Oregon Health and Science University (OHSU), 3455 S. W. US Veterans Hospital Road,

Portland, OR 97239, USA; jonesre@ohsu.edu

* Correspondence: rossam@ohsu.edu; Tel.: +1-503-494-2123

Citation: Ross, A.M.; Jones, R.J.F.

Simulated Forest Immersion Therapy:

Methods Development. Int. J.

Environ. Res. Public Health 2022, 19,

5373. https://doi.org/10.3390/

ijerph19095373

Abstract: Shinrin-yoku, forest bathing, may provide relief from chronic and breakthrough pain in

patients with axial spondyloarthritis and improve immune function through increasing NK cell

numbers and activity and their downstream effectors, perforin and granulysin, after chemo- or

radiation therapy in breast and prostate cancer patients. The aim of this paper is to describe the study

protocol for a simulated forest immersion therapy using virtual reality and atomized phytoncides,

volatile organic compounds found in forested areas designed to effect positive change for these two

patient populations. The setting, including the room set up and samples with inclusion/exclusion

speciﬁc to this type of intervention, is outlined. Measures and calibration procedures pertinent to

determining the feasibility of simulated forest immersion therapy are presented and include: ambient

and surface room temperatures and relative humidity in real time, ambient ultraﬁne particulate

matter, ambient droplet measurement that coincides with volatile organic compounds, speciﬁc

phytoncides, and virtual reality and atomization of phytoncide set up. Particular lessons learned

while training and setting up the equipment are presented. Simulated forest immersion therapy is

possible with attention to detail during this early phase when development of methods, equipment

testing, and feasibility in deploying the intervention become operational. The expected outcome

of the development of the methods for this study is the creation of a standardized approach to

simulating forest therapy in a controlled laboratory space.

Keywords: shinrin-yoku; virtual reality; phytoncides; exposure science methods; NK cells; Visual

Analog Scale; DASS; BASDAI

Academic Editors: Amber L.

Vermeesch, Andrew Lafrenz and

Chloé Littzen

Received: 28 February 2022

Accepted: 27 April 2022

Published: 28 April 2022

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4.0/).

1. Introduction

Shinrin-yoku, roughly translated as forest bathing, is the traditional Japanese practice

of immersing oneself in nature by mindfully using our senses, such as sight and smell [1,2].

Shinrin-yoku practices range from sitting quietly and enjoying the forest to walking or hik-

ing through forested areas [3]. The key to shinrin-yoku is connecting with the atmosphere

of the forest, taking in forest aerosols, volatile organic compounds known phytoncides [4].

Phytoncides are inhaled airborne particles that trees naturally emit during different stages

of development [5–9].

In 1982, the Japanese Ministry of Agriculture, Forestry and Fisheries began researching

the health beneﬁts of shinrin-yoku [2]. There are several therapeutic effects of shinrin-

yoku in the context of depression, including improving immune system function, such as

decreased pro-inﬂammatory cytokine activity and increased in anti-inﬂammatory cells,

decreased depressive symptoms, stress, and anxiety, improved mental relaxation and at-

tentional focus, and increased feelings of awe, gratitude, and selﬂessness [4,9–16]. The

beneﬁt of forested greenspaces for human psychology and physiology is a reduction in

stress [14,17], which in turn positively impacts mood [18] and further reduces inﬂam-

mation [19]. In the context of normal male subjects, after both shinrin-yoku forest im-

mersion and hotel/sleep-based phytoncide humidiﬁcation with α-pinene, β-pinene, and

d-limonene, the following immune system improvements were found: natural killer T-cell

numbers and activity increased, as did perforin and granulysin [6,20,21].

Int. J. Environ. Res. Public Health 2022, 19, 5373. https://doi.org/10.3390/ijerph19095373

https://www.mdpi.com/journal/ijerph

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In 2017, a research agenda for nature contact and health was published with several

research domains, including the mechanistic biomedical studies domain (1.0) for future

research development [17]. We developed a simulated forest immersion therapy (SFIT)

that includes both forest aerosols and virtual reality based on this research agenda. We are

interested in psychological and physiological variables that suit our differing populations

of interest (i.e., axial spondyloarthritis patients with chronic or breakthrough pain or

breast and prostate cancer patients who have undergone chemo- or radiation therapy and

have NK cell depletion), and these align with the mechanistic biomedical domain [17].

Speciﬁcally, our research objectives are to elucidate 1.1: to what extent stress reduction

mediates observed health beneﬁts of nature contact; 1.1b: which natural elements are most

associated with stress reduction; 1.2: to what extent improved immune function mediates

observed health beneﬁts of nature contact; 1.2b which natural elements are most associated

with improved immune function; and 1.2c which markers of immune function are the most

useful for studying this effect [17]. If there are health effects of forest immersion, then could

the same health beneﬁts be achieved with simulated forest immersion as a way of providing

therapy to patients not able to exercise or move in outdoor forests or greenspaces due to

debility or frailty from chronic illness (i.e., chronic and breakthrough pain) or acute illness

(i.e., recovery of immune system function after chemo- or radiation therapy in cancer)?

1.1. Signiﬁcance

An estimated 1,806,590 new cases of cancer will be diagnosed in the United States

each year, of which 281,550 will be breast cancer and an estimated 248,530 will be prostate

cancer [22]. Additionally, up to 1% of the population of the United States, an estimated

2.7 million people, may have axial spondyloarthritis [23], and 50% of them suffer from

chronic widespread pain [24,25] and breakthrough pain after standard treatment in approx-

imately 60% of people [26]. Using complementary interventions to improve outcomes in

patients who are seriously ill is paramount to extending healthcare to vulnerable popu-

lations. We hope to accelerate the translation of ﬁndings for healthy individuals for the

implementation of a novel minimally invasive immune therapy for cancer patients with

solid tumors where NK cells are depleted, both in number and activity [27,28], and to deter-

mine clinically meaningful protocols for the management of pain, and comorbid symptoms

of pain, in patients with axial spondyloarthritis with chronic and breakthrough pain.

Forest bathing, virtual reality (VR), the use of phytoncides, and their separate or

combined effects constitute a new research avenue. It is intriguing to explore the effects

of nature-based interventions on chronic and breakthrough pain in patients with axial

spondyloarthritis, as well as the effects on the immune system, and how we might harness

them to beneﬁt acutely ill patients who are immunocompromised.

1.2. Background

1.2.1. Psychological Pathways of Interest

Contemporary theories, such as Kaplan’s Attention Restoration Theory [29,30], Ul-

rich’s Stress Reduction Theory [31–33], and Kellert and Wilson’s Biophilia Hypothe-

sis [34,35] provide a conceptual framework for the practice of shinrin-yoku and engaging

with nature in various forms of nature therapy. Shinrin-yoku researchers Song, Ikei, and

Miyazaki (2016) developed a conceptual framework based on an extensive review that de-

scribes how the restorative effects of nature increase physiologic immune system recovery

from stress as well as physiologic relaxation [36].

Kaplan and Kaplan hypothesized that exposure to natural settings through the ﬁve

senses has a direct effect on parasympathetic nervous system activation, thus leading to

states of greater awareness achieved through relaxation [29]. Ulrich’s Stress Reduction

Theory [31] was developed from observational studies wherein patients in hospitals with

patient room windows facing nature-laden scenery (e.g., trees, green foliage) experienced

marked improvement in health and recovery with shortened hospital stays compared to

patients in rooms with an urban view [31,32]. Wilson’s Biophilia Hypothesis [35], suggests

Int. J. Environ. Res. Public Health 2022, 19, 5373

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that humans have a developmental afﬁnity for natural surroundings, and being immersed

in nature is therefore innately appealing. This research suggests that a disconnect from

nature has adverse health impacts [34], and therefore ﬁnding effective means for individuals

to access nature is crucial [37,38].

1.2.2. Pain

Pain reduction is an emerging ﬁeld of study in greenspace interventions [39], particu-

larly the relationships between pain, stress, and the burden of chronic illness [40–44]. To

date, several studies describe the pain-reducing effects of viewing nature (e.g., simulated

greenspaces) in clinical settings for acute and chronic pain populations [37,41,45–49].

Simulated nature and greenspace exposure has been applied in clinical settings for

the treatment of acute [41,46,47] and chronic pain [37,44,50]. Virtual reality (VR)-based

therapies for pain reduction are not new, and several theories for how and why VR-based

therapies improve pain outcomes center on the element of “distraction”, such that the

virtual viewing experiencing distracts an individual from feeling their pain [51–54]. This

is largely based on the Gate Control Theory proposed by Melzack and Wall [55], which

suggests that the attention paid to the pain experience, as well as the emotion tied to

the experience of pain, which includes past emotional memories, play a role in pain

interpretation; therefore, directing attention away from the experience of pain may reduce

the sensation of pain [47,53,56–59].

Research on VR-based greenspaces or nature exposure for pain reduction describes

VR as a tool for delivering nature, and that nature is the crucial element within the inter-

ventional design [47]. In a repeated-measures design, 50 patients attending chemotherapy

sessions were evaluated for pain and stress during intravenous port access. While ﬁndings

were insigniﬁcant after one nature-based VR session, participants reported feeling relaxed,

peaceful, and distracted by positive thoughts [47]. Potential beneﬁts of virtual nature di-

rectly link to the theories describing the health effects of shinrin-yoku, including improved

relaxation, restoration, and alertness, improved functioning of the immune system, and

reduced exposure to air pollution and urbanicity [37]. Exposure to greenspaces can induce

relaxation via psychoendocrine pathways, including the function of the hypothalamic–

pituitary–adrenal (HPA) axis and resulting cortisol secretion [60,61]. Further, exposure to

greenspaces, which include greenery in the form of foliage, trees, and vistas, such as with

shinrin-yoku, improves health outcomes whether the exposure involves “live” nature or

virtual nature [62,63].

1.2.3. Biological Pathways of Interest

Immune suppression is a major issue for adults with a cancer diagnosis receiving

chemo- and/or radiation therapy. In particular, NK cell suppression in this population is

problematic as NK cells are the major immune cell type surveilling foreign or infectious

antigens and eliminating them [64]. Thus, implementation of a novel minimally invasive

immune therapy in cancer patients with solid tumors where NK cells are depleted, both

in number and activity, is crucial [27,28]. Patients with solid tumors that have activated

NK cells within the tumor have longer overall survival [65,66]. Blood levels of NK cells are

essential to the movement of NK cells into tumor tissue.

Research shows positive effects of forest bathing on NK cell numbers and activ-

ity [67] (NK CD3−/CD56+/ and NK CD3−/CD56+/CD69+, respectively) and on expressed

proteins, such as perforin and granulysin [6,20,68,69]. NK cells use pattern recognition

molecules (epitope) on the surface of transformed or stressed cells to accelerate detection

and elimination of problematic cells. Perforin and granulysin are key to enabling the

natural killing mechanism of the NK cell [70]. Perforin is a downstream effector related to

the number and activity of the NK cells [64]. Perforin creates a pore in the target cell once

the target cell’s epitope is recognized [64]. The pore allows granulysin to enter the cell and

effect apoptosis of the intracellular structures; the cell lyses and dies [5,64]. Perforin and

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granulysin are needed to maintain normal immune surveillance and reduction in infection,

speciﬁcally in immunocompromised cancer patients [27,28].

Two proof-of-principle studies were conducted in middle-to-older-aged healthy men.

These two studies, a 3-day forest experience (immersion experience) and a 3-night hotel

experience, measured or used humidiﬁed-forest-derived volatile organic compounds,

known as phytoncides, respectively. Of the phytoncides tested, humidiﬁed α- and β-pinene

and limonene in combination produced an increased number of NK cells and elevated

activity [6,20].

1.3. Purpose

If forest immersion can provide immune system beneﬁts in healthy men (i.e., improved

NK cell numbers and activity, increased perforin and granulysin), can dispersal of three

phytoncides (α- and β-pinene and limonene in combination) paired with a greenspace

virtual reality provide the same positive effects on NK cells in patients with solid tumor

cancer who have completed cancer therapy? Additionally, can humidiﬁed limonene

paired with virtual reality reduce pain and psychological stress in patients with axial

spondyloarthritis? Our purpose is to deploy a standardized study protocol for simulated

forest immersion intervention in cancer patients with NK cell depletion and patients with

axial spondyloarthritis with chronic and breakthrough pain to test its feasibility and rigor.

The simulated forest immersion intervention will provide greenspace/forest experience

through three of the ﬁve senses. Virtual reality will provide visual and auditory stimuli.

Humidiﬁed aromatic forest oils will provide olfactory stimuli. Virtual reality and atomized

forest oils may be used in combination or alone [71]. Two distinct studies will use this

standardized protocol for (1) cancer patients and (2) patients with axial spondyloarthritis.

The purpose of this paper is to outline the development of the study protocol for the

intervention and the control conditions of the clinical lab setting.

2. Methods/Approach

2.1. Research Design

We will use a two-arm study design with concurrent controls selected from the breast

and prostate cancer clinics and from the arthritis clinic with two measurement time periods

to test the proposed simulated forest exposure intervention. In the SFIT study with patients

with axial spondyloarthritis who have chronic or breakthrough pain, the two-time points

were before and immediately after the intervention. In the SFIT study with patients with

either breast or prostate cancer, the two time periods were before and on Day 3 after the

intervention.

2.2. Study Sample

The study sample for study #1 will be recruited from cancer patients with solid tumors

(HR + HER2- breast, or prostate cancer) who have completed cancer therapy (hormone

therapy excepted) as this population may beneﬁt the most from increases in NK cell number

and activity, perforin, and granulysin to prevent infection as patients become relatively

immunocompromised after chemo- or radiation therapy. For study #2, the study sample

will be recruited from axial spondyloarthritis patients who have chronic or breakthrough

pain, as virtual reality has been shown to reduce pain, and d-limonene administration

in animals has shown pain reduction. Since the two studies are set in the future and are

pilot studies, we expect for study #1 to recruit and enroll 25 participants and for study #2

to recruit and enroll 25 participants. For study #1, the participant number is limited by

budget and the cost of pre-clinical and clinical laboratory tests. For study #2, the participant

number is limited by budget and the cost of paper-based tools. Concurrent controls for both

studies will be identiﬁed by the clinicians in either the cancer clinics or the arthritis clinic.

Concurrent controls will meet the same study inclusion and exclusion criteria as those

who are enrolled in the study interventions. We expect to have 4 of each of the assigned

genders in the control groups. The control groups will be randomized to receive neither

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SFIT intervention, VR, or atomized phytoncides. They will be exposed to atomized water

dispersal for 1 h, the same length of time as the study participants who will receive the SFIT

interventions. All the same data will be collected for both study #1 and study #2 on these

control participants. At the end of the studies, should an effect of the SFIT intervention be

noted, the control participants will have the opportunity to complete the same intervention.

Clinicians will lead the recruitment of these patients, followed by a phone screening for

inclusion and exclusion conducted by the principal investigator and research associate.

2.2.1. Inclusion and Exclusion for SFIT—General Considerations

Since we will be using atomized phytoncides as well as virtual reality, either in

combination or separately, several exclusion criteria apply, as seen in Table 1.

Table 1. General exclusion criteria related to intervention only.

Exclusion Criterion

History of asthma [72]

Inability to detect common odors from

commercial fragrances [73]

History of smoking within 15 min before the

start of SFIT [74]

Allergy to pine or citrus aroma [74,75]

History of intractable seasickness [76]

History of seizures [77]

Limitations of vision and hearing not corrected

by eye lenses or hearing aids

Inability to complete study requisites

Rationale

Inhaled phytoncides may produce airway

irritation, asthma exacerbation, or

bronchoconstriction

Inhaled phytoncides provide half of the

intervention and smell of the forest

Smoking within 15 min before therapy will

alter the ability of the participant to detect

commercial fragrances or the aroma of the

phytoncides

Inhaled phytoncide aromas are pine and citrus

and may cause dermatitis

VR may cause nausea/vomiting without relief

after 5–10 min

VR may heighten susceptibility to

photosensitive seizure due to changing light in

the forest video

VR requires good vision and hearing correction

with eye lenses or hearing aids

Intervention directions must be followed;

speciﬁc to follow up measurements

Inclusion and Exclusion for SFIT for Breast and Prostate Patients

Participants will be included if they are willing and able to provide informed consent,

are of either biological sex, older than 18 years of age, and have completed cancer therapy

for HR + HER2 breast cancer or prostate cancer, Stage I–III, with no evidence of metastasis.

Participants will be excluded if they have a history of autoimmune disease, are on immune

modulating therapies (endocrine therapy allowed), have had surgery or an invasive proce-

dure in the past two months, and recent infection in the past two weeks (these are known

confounding variables in immune system measures of interest).

Inclusion and Exclusion for SFIT for Axial Spondyloarthritis Patients

Participants diagnosed with axial spondyloarthritis (axSpA) will be included if they

are willing and able to provide informed consent and are at least 18 years of age or older,

and they may be any sex or gender. Additional inclusion criteria are: a score of 4 or higher

on the 10-point Bath Ankylosing Spondylosis Disease Activity Index (BASDAI) (a standard

criterion for suboptimal control of symptoms and disease [78] with a correlation between

patient-reported BASDAI scores and measurable disease) [79], and a rheumatologist over-

seeing their care. Participants will be excluded if they are in an active phase of treatment

with biologic cytokine inhibitors (which may confound the effects of the intervention on

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outcomes measures) [80]. Use of commonly prescribed painkillers is acceptable, and we

will control for their use in the analysis.

2.3. Setting

The SFIT Lab is located in our Integrated Bio-Behavioral Lab space within our school.

The lab room, where the instrumentation for the SFIT is set up, is 20 feet × 15 feet with a

12-foot ceiling. The room has temperature control, so a consistent temperature between

deployments of SFIT with participants can be maintained, as well as lighting control, so the

lights can be dimmed when patients are using VR.

2.4. Intervention—Procedure

Our two studies are novel as no one to date has used simulated forest immersion

in patients with acute or chronic morbid conditions. The principal investigator (PI) and

research associate (RA) will implement SFIT in a separate room from the room used to

cross-check inclusion and exclusion, obtain informed consent, collect baseline measures,

and allow the participant to rest. Prior to the arrival of the participant, the SFIT intervention

space will be prepared. Preparation of the intervention space includes calibration of the

instruments that will measure volatile organic compound particles (phytoncides are volatile

organic compounds) and droplets, room temperature and humidity, and room surface

temperatures, followed by measurement of the ambient particles and droplets and room

temperature and humidity prior to the implementation of the atomized phytoncides [71,81].

Phytoncides α-pinene, β-pinene, and d-limonene (Floraplex Terpenes, Ypsilanti, MI, USA)

will be prepared for atomization with a commercially available atomizer (Asakuki 500 mL

Premium Atomizer, Tronhon Co., Ltd., Chongqing, China) that can emit phytoncides for

up to 3 h. Once the dose expected (0.80 ppm) reaches the dose published by Li [6], the

participant will be brought into the intervention room. To date, the Li study, which was

conducted with healthy men, has been the only study to record phytoncide dose in a

controlled setting. We will use this concentration as the target dose for our humidiﬁed

phytoncide set up. Both at the beginning and at the end of 1 h of exposure to the simulated

forest immersion therapy intervention, ambient phytoncide in the contained space will

be measured indirectly by measuring both the increase in ambient air particle mass and

number, as well as by measuring the change in total volatile organic compounds (VOCs).

Total particle numbers will be measured with a continuous ultraﬁne particle counter (P-Trak

8525, TSI, Shoreview, MN, USA), and the total VOCs will be measured with the portable

handheld monitor (Mini ppbRAE 3000, Honeywell International INC.; San Jose, CA, USA).

The P-Trak and Mini ppbRAE 3000 will measure the increase in particle numbers and

water droplets (aerosol), respectively, which will serve as a surrogate of relative exposure

to phytoncide. Continuous monitoring of room temperature and relative humidity will

be measured by the HOBO MX2301 Temperature/RH Data Logger (ONSET, Bourne, MA,

USA). Room surface temperature will be measured by ADC Adtemp Mini 432 Non-Contact

Infrared Thermometer (American Diagnostic Corporation, Hauppauge, NY, USA). VR

will be provided by VIVE Pro Eye, HTC (high-tech computer) Corporation, (New Taipei

City, Taiwan) with digital rendering of forested greenspace. Once 1 h of SFIT concludes,

the participant will be removed to the preparation area and given instructions related to

reporting unanticipated problems, adverse events, and serious adverse events, and an

appointment to return for follow up on Day 3. See Figure 1, which illustrates the SFIT

process/procedure.

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connection on the regulator, meaning that the threading has to be compatible between the

cylinder and the regulator. If 500 psi is exceeded during calibration or survey mode, the

diaphragm within the RAE 3000 may be damaged, leading to inaccurate data collection. In

survey mode, data will be updated every 60 s and a data log will be created. The data log

will be downloaded using software speciﬁc to the ppbRAE 3000 and found online [82].

HOBO MX2301 Temperature/RH Data Logger, ONSET, Bourne, MA, USA

The HOBO temperature and relative humidity (HOBO T/RH) data logger, which

is suitable for both indoor and outdoor application, is a small portable unit that uses an

application (HOBOConnect) loaded onto a mobile device. The app will use a Bluetooth

connection to the HOBO T/RH that is easily conﬁgurable and logs temperature and relative

humidity in real time that you will view on our mobile device, in this case an iPhone. The

HOBO T/RH will be placed within a 30 m line of sight towards the participant. Due to

the size of the room within which we will set up the intervention, we will use one HOBO

T/RH. Data download will be accomplished when the iPhone (mobile device) is within

100 m of the HOBO T/RH unit. Data updates every 2 min with an accuracy of ±0.2 ◦C and

±3.5% RH [86]. Data software will be downloaded online [84].

Measurement of Ambient Room Conditions

Room temperature and humidity may alter the overall measurement of particle num-

ber and droplets. Measuring all four of these ambient conditions will allow for consistency

in the experimental condition between participants. The room has a set temperature of

70 ◦F, and since the room is located against a foundation wall, humidity may vary; there-

fore, it is important to monitor both of these ambient conditions and use cutoff criteria

based on average temperature and humidity of the controlled lab setting. We will also use

a non-contact infrared surface thermometer to measure radiant heat of room surfaces that

may add to the perceived comfort of the SFIT intervention room [81]. The ADC Adtemp

Mini 432 Non-Contact Infrared Thermometer (American Diagnostic Corporation, Haup-

pauge, NY, USA) with a range of 59–77 ◦F will be used for this purpose. The ambient room

air temperature, humidity, and surface temperature date will be collected as a mean prior

to, during, and at the end of the intervention.

Asakuki 500 mL Premium Atomizer, Tronhon Co., Ltd., Chongqing, China

The atomizer holds 500 mL of liquid that can be atomized over 3 h. Of that 500 mL, a

portion will be reduced that coincides with the amount of phytoncide that will be added.

We expect that we will add 30 mL per phytoncide to the atomizer to achieve the detectable

published amount [6,21]. Mist will be created by an ultrasonic plate within the atomizer,

and the mist will be adjusted for a weak mist or a strong mist. Choice of weak or strong

mist will be adjusted to ﬁt the published detectable amount of phytoncide. Mist time can

be regulated to maintain 60, 120, and 180 min of operation. We will use a 60 min mist time

with the participants of study #1 and study #2. A fan within the atomizer will disperse the

mist into the room. Room temperature and humidity will be monitored continuously as

low temperature and high humidity may condense the mist into water droplets [87], which

is to be avoided to allow for accurate dose calculations.

Phytoncides, Floraplex Terpenes, Ypsilanti, MI, USA

α-pinene, β-pinene, and d-limonene are the phytoncides (forest oils) of interest for

SFIT. Interestingly, all three in combination have been tested in normal males in both

forest immersion and hotel/sleep contexts and have shown effective elevations in NK cell

numbers and activity as well as increased expression of perforin and granulysin [5,6,20,21].

However, these three phytoncides have not been tested in the SFIT context with breast

and prostate cancer patients. D-limonene alone has been tested in the context of pain

and shown to be effective in an animal model when not paired with VR [88]. All three

phytoncides are available as puriﬁed isolates in containers of 4, 8, or 32 ounces. The

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phytoncides will be added to the Asakuki atomizer with an easy calculation of ounces to

ml, and that amount will be subtracted from the 500 mL total container in the atomizer so

as to maintain a standardized addition of phytoncides:water ratio. Measurement of this

mixed mist by ultraﬁne particulate and VOC survey will be the method of determining the

dose of the phytoncide.

VIVE Pro Eye, HTC (High-Tech Computer) Corporation, New Taipei City, Taiwan

The VIVE Pro Eye is capable of delivering digitally rendered greenspace visual record-

ings from forested or park-like greenspaces. The headset has sensors that coordinate the

virtual greenspace with the participants’ visual gaze (native eye tracking) to move the

surroundings of the digital greenspace through interaction with the base stations using

motion sensors mounted on tripods in front of the participant [89,90]. Set up will include

downloading the VIVE and SteamVR software onto a computer speciﬁcally dedicated for

the VIVE system (e.g., using Windows 10 operating system). Tracking will be performed

on the computer software and saved for later review during data entry [91]. The computer

will be located behind the chair in which the participant will be sitting. Sounds will be

adjusted for those that are a part of the virtual greenspace; ambient sounds in the room

will be muted. Although software comes with the purchase of the VIVE system, training

videos can be found online and will be completed before use [87–89].

2.5. Baseline Fidelity Measures—Linkages to Equipment

Perception of air quality prior to introduction of phytoncides into the room’s air will

be evaluated repeatedly prior to each intervention day using two healthy individuals of

both assigned sexes each time to assess the air quality of the room at the set temperature of

70 ◦F while monitoring the relative humidity. We will use the facial exposure method as

described by Fang, Clausen, and Fanger [92]. In order to ensure ﬁdelity of the measures of

phytoncide dose prior to, during, and at the end of the intervention period, the equipment

mentioned above will be zero and span-calibrated before introduction of phytoncides into

the room’s air. Recording temperature and humidity using the HOBO T/RH before and

during the intervention will ensure that the reliability of the survey data from the P-Trak

and the ppbRAE 3000 has not been affected by changes in temperature and humidity.

Feasibility and Reliability of Intervention Stability

We expect to determine the ease of use of VR and phytoncide atomization, the drop off

of phytoncide over the intervention period, and engagement in VR leading to a standard-

ization of the procedure and protocol. Data will be collected from the participant, and the

research associate and principal investigator will use ﬁeld notes and will include challenges

and facilitators related to the delivery method of VR and phytoncides and the ability of

the participant to engage in VR for the duration of the study intervention. Atomized

phytoncides prior to and after 1 h of dose delivery will be used to measure by the P-trak

and the Mini ppbRAE3000 to determine dose drop off during the intervention delivery.

Quantitative data related to dose drop off will be analyzed by t-test with an α level of 0.05.

We will collect deviations from the standardized procedures, including the deployment

of VR and atomized phytoncides. We will monitor the timing, preservation, and delivery

of specimens to specialized labs on the academic healthcare campus in order to track the

ability to maintain the expected optimized rigor in testing immune cells.

2.6. Data Collection

Since the participants will be recruited from the breast and prostate cancer clinics

and the rheumatology clinic, the medical history that appears in the EPIC electronic

medical record will be available for review prior to enrollment per IRB approval (OHSU

IRB#00023183) and cross-checked with the participants after we have obtained informed

consent on the day of the SFIT intervention. This will serve as the start of the data collection

process for determining inclusion/exclusion of participants (see Section 2.2.1, Table 1; In-

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clusion and Exclusion for SFIT for Breast and Prostate Patients and Inclusion and Exclusion

for SFIT for Axial Spondyloarthritis Patients) and baseline data collection (see Section 2.6.1,

Section 2.7, and its subsections, and Section 2.8 and its subsections). Case report forms will

be used to capture participant baseline and data collected at all pertinent time points per

protocol and will serve as a hard-copy record of data, which we will enter into a research

electronic data capture system, as required by our university.

2.6.1. Baseline

Demographic data collection will occur prior to the start of both SFIT intervention

studies and will be speciﬁc to each population of interest [93]. The behavioral/psychological

measures, biological measures, and feasibility measures will be collected prior to placement

of the participant in the SFIT intervention room.

2.6.2. Follow Up

Follow-up data collection will be within the 3–4-day period after the SFIT intervention

and in study #1 participants with breast or prostate cancer and will include blood specimens

for CBC with differential counts of leukocytes, NK cell phenotyping and plasma for perforin

and granulysin ELISAs [6]. We will survey the participants on events that might affect

immune response. Every effort will be made to minimize the amount of blood drawn.

Data collection will occur immediately after the intervention for study #2 participants

with AxSpA and include the same measures as baseline as well as the intervention ﬁdelity

measures. Follow up will also include collection of adverse events, serious adverse events,

and unanticipated problems, per IRB protocol for intervention studies.

2.7. Measures—Behavioral/Psychological

To measure the impact of SFIT on patients with chronic or breakthrough pain due

to axial spondyloarthritis (axSpA), scales which interpret the direct effect on symptoms

of pain, psychological distress, and physical functionality speciﬁc to axSpA will be used.

These include the Visual Analog Scale for pain [94,95], the Depression, Anxiety, and Stress

Scale [96,97], and the Bath Ankylosing Spondylitis Disease Activity Scale [98].

2.7.1. Demographics

Demographic characteristics of participants will include clinically relevant ethno-

graphic details speciﬁc to assigned sex (male or female), race, and ethnicity. Diagnostic

information speciﬁc to axSpA, including date of diagnosis and onset of symptoms (date),

chronicity of symptoms (in months), and non-biologic medication management (name

of medication/last date of use), will be ascertained. Since culture, religion, and personal

belief systems inﬂuence perception of pain, depression, stress, and functionality, we will

include questions about these three pertinent individual characteristics in our baseline

demographic data collection [99].

2.7.2. Visual Analog Scale (VAS)

The VAS is a widely used self-reported tool measuring present-state perceived pain

intensity [100]. Patients will be asked to indicate their perceived pain intensity along a

10 cm horizontal line (which can be on paper or computerized), and this rating will then

be measured from the left edge up to the indicated marking to represent the level of pain

intensity. The line represents a continuum between “no pain” and “worst pain”. The VAS

is often used in clinical settings and is sensitive in determining the effect of comfort or

pharmacological interventions [94]. The VAS has performed well on psychometric tests of

validity (for example, η2 = 0.47; F = 0.44 [94,101]), and reliability (rs,VAS = 0.52–0.89 [102])

for measuring pain clinically. VAS scores will be treated as ratio data [103].

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2.7.3. Depression, Anxiety, and Stress Scale (DASS)

The DASS comprises a set of three self-report scales, which are intended to measure

clinically signiﬁcant symptoms of emotional states of depression, anxiety, and stress [96,104].

Each of the three DASS scales (depression, anxiety, and stress) contains 14 items, divided

into subscales of 2–5 items measuring the same construct, for a total of 42 items. The

participants will be asked to complete the DASS prior to and immediately after the SFIT

intervention. The DASS, which is intended to measure symptom severity of self-reported

negative emotional states, including depression, anxiety, and stress, shows good psychome-

tric validity and reliability (Cronbach’s α = 0.89; test–retest and split-half reliability scores

are rDASS = 0.99 and 0.96, respectively [96]) as a dimensional measurement of psychological

distress associated with chronic conditions [104].

2.7.4. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)

The BASDAI is commonly used to measure clinical symptoms of AS and axSpA,

including fatigue, spinal pain, joint pain related to swelling, and enthesitis, or inﬂammation

of the tendons and ligaments, as well as morning stiffness duration and severity [105]. It

consists of 6 self-report questions, with each question scored from 1, representing “none” or

no symptoms, to 10, representing “the worst”, with the score from the questions pertaining

to morning stiffness and duration averaged such that 5 questions in total are scored. The

participants will be asked to complete the BASDAI prior to and immediately after the SFIT

intervention. The resulting score (from 0 to 50) is divided by 5 to give a ﬁnal BASDAI score

of 0–10, with scores of 4 or greater indicating signiﬁcant disease [106]. The BASDAI has

demonstrated extraordinary reliability at p < 0.001 [105]. In a test of validity of the BASDAI

for AS patients, Cronbach’s α = 0.786 [98].

2.8. Measures—Biological/Immune System

We will use established pre-clinical and clinical measures to characterize the immune

responses before and after the simulated forest exposure intervention.

2.8.1. Demographics

Demographic characteristics of participants will include clinically relevant ethno-

graphic details speciﬁc to age, assigned sex (male or female), race, and ethnicity, and history

of smoking.

2.8.2. CBC and Differential Cell Count

Complete blood count (CBC) and differential cell counts will be measured at baseline

(prior to implementation of the simulated forest exposure intervention) and at Day 3.

Correlation between this clinical measure and the data from ﬂow cytometry and ELISA

(outlined below) will be conducted to translate the pre-clinical ﬁndings into clinical use. A

whole blood sample for a CBC with a differential cell count will be collected two times using

a 4 mL EDTA tube, prior to the SFIT intervention and on Day 3 after the SFIT intervention.

This measure will include WBC count and percentages of 100 cell counts and absolute

counts for neutrophils, lymphocytes, and monocytes. The clinical core laboratory at OHSU

complies with established inter- and intra-assay parameters as it is accredited by Clinical

Laboratory Improvement Amendments.

2.8.3. Flow Cytometry for NK Cell Number and Activity

Flow cytometry is used to monitor immune system changes tied to speciﬁc dis-

ease states, which makes it ideal for deﬁning cellular responses of interest [107]. NK

CD3−/CD56+ and NK CD3−/CD56+/CD69+, (i.e., NK number and activity, respectively)

will be measured by ﬂow cytometry immunophenotyping using freshly collected periph-

eral whole blood (approximately 4 mL). Cells will be prepared for ﬂow cytometry using

the standard ﬂuorescence-activated cell sorting method. Data analysis will be performed

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by gating on live cells based on forward versus side scatter proﬁles, then on singlets using

forward scatter area versus height, followed by cell-subset-speciﬁc gating [107].

2.8.4. Perforin Expression

Perforin expression will be measured by an enzyme-linked immunosorbent assay

(ELISA) and will be used to monitor downstream perforin secretion due to NK cell activ-

ity [27]. Perforin will be measured using plasma extracted from whole blood, which will

be frozen at −80 ◦C and stored until needed for the assay. Optimized ELISA kits from

ThermoFisher Scientiﬁc, Waltham, MA, USA, will be used per manufacturer instructions to

detect perforin levels. The enzyme-dependent color change will be read out on a Multi-

Mode Mircroplate Reader. Perforin concentration will be extrapolated from the standard

curve [108].

2.8.5. Granulysin Expression

Granulysin expression will be measured by an enzyme-linked immunosorbent assay

(ELISA), which will be used to monitor downstream granulysin secretion due to NK cell

activity [27]. Granulysin will be measured using plasma extracted from whole blood, which

will be frozen at −80 ◦C and stored until needed for the assay. Optimized ELISA kits

from AbCam, Cambridge, MA, USA, will be used per manufacturer instructions to detect

granulysin levels. The enzyme-dependent color change will be read out on a Multi-Mode

Mircroplate Reader. Granulysin concentration will be extrapolated from the standard

curve. For both perforin and granulysin expression, we will need 4 mL of freshly collected

peripheral whole blood [108].

2.9. Follow-Up Measures Day 3

In addition to collecting whole-blood specimens for CBC with differential, NK cell

number and activity, perforin, and granulysin, we will note any unanticipated problems,

adverse events and serious adverse events affecting the participants per IRB protocol for

intervention studies. Unanticipated problems will be determined with the assistance of

the clinicians and the study team as these are determined through a ranking procedure

speciﬁed by our university’s Ofﬁce of Human Research Protections. Adverse events will

include subjective or objective symptoms occurring spontaneously, signiﬁcant clinical lab

abnormalities, a worsening of the participants condition from baseline, are recurrence or

increase in signs and symptoms of original disease that occur after the SFIT intervention

and are worsened or changed in quality. Serious adverse events will include death, life-

threatening adverse event, new hospitalization or prolongation of current hospitalization, or

a new signiﬁcant incapacity or new substantial inability to complete activities of daily living.

3. Discussion

We have presented our lab set up for the SFIT intervention. The SFIT intervention as

described may be used in a multi-arm design with a control group, using VR only, phyton-

cide atomization only, or both in combination. We expect to use whatever combination is

proven to be most effective as a minimally invasive intervention for all of the following

designs in a stepwise progression: longer intervention duration to optimize dose effect;

intermittent, but repeated intervention to optimize dose effect drop off; and home use

application to move the optimized intervention into practical use.

An intervention using VR and humidiﬁed phytoncides, α- and β-pinene, and limonene,

in a simulated forest exposure intervention as a substitute for forest bathing in AXSpA

patients with chronic or breakthrough pain and cancer patients with early-stage solid

tumors (HR + HER2- breast cancer and prostate cancer) who have completed surgery or

chemo- and/or radiation therapy (exclusive of hormone therapy) is possible in a lab setting.

Moving SFIT to a home setting may be challenging, but not insurmountable.

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3.1. Expected Outcomes

The expected outcome for the future two studies is the creation of a standardized

protocol for deploying SFIT. This will include calibration and measurement set points,

cutoff criteria and describing how to maintain a consistent dose of phytoncide and ease the

use of VR for participants. We expect to uncover pertinent adverse events, severe adverse

events, and unanticipated problems. For study #1, we expect that the combined use of our

three phytoncides of interest and VR will improve NK cell numbers and activity and blood

levels of perforin and granulysin in patients with breast or prostate cancer. For study #2,

we expect that the use of d-limonene and VR will reduce pain, stress, and depression in

patients with axial spondyloarthritis.

3.2. Lessons Learned

We have had challenges with procurement of supplies during the current development

and delivery backlog produced by delays in shipping due to COVID-19 conditions in our

current world state. Some of these were delivered relatively quickly, within one week;

however, we procured span gas of the wrong concentration, and the next week all span

gas was out of stock nationwide with a delay in the expected delivery of 1–2 months.

Phlebotomy supplies have been challenging to obtain through our medical supply process

within the university due to the increased usage of the supplies to care for COVID-19

patients. At times, our academic medical center announced requests for a reduction in the

usage of various supplies for laboratory work. The lesson learned here is to start early

after IRB approval and be aware of potential delays that might encumber grant funding

previously awarded.

The cylinder containing the span gas connection and the ﬁtting to the regulator must

match. If the cylinder has a male threading and the regulator has female threading, these

may match, but care needs to be taken in looking at the speciﬁcation of the cylinder

connection with respect to the regulator connection. In our case, we needed to have male

threading on the replacement span gas with a connection that was a CGA 600 speciﬁcation

in order to match our existing regulator. The replacement price differential is 12:1 with the

regulator being much more expensive than the span gas cylinder.

4. Conclusions

We have presented the theoretical conceptions and established the foundation for the

move to the pragmatic operations of the SFIT intervention. SFIT is in its early stages as a

potential therapy in the two populations of interest to us presented here. We expect further

development in building this novel lab set up in the immediate foreseeable future as we

work to move this therapy into the home setting under the control of patients needing

this minimally invasive therapy. This is relevant to healthcare science because healthcare

providers are responsible for optimizing patient healing and recovery, while reducing the

harmful effects of therapies that deleteriously affect the patient’s ability to thrive with their

chronic or temporarily morbid conditions.

Author Contributions: Conceptualization, A.M.R. and R.J.F.J.; methodology, A.M.R.; writing—

original draft preparation, A.M.R. and R.J.F.J.; writing—review and editing, A.M.R.; funding acquisi-

tion, A.M.R. All authors have read and agreed to the published version of the manuscript.

Funding: This research is funded by Sigma Theta Tau International, Beta Psi Chapter, under grant

number GSONO0529A; the OHSU School of Nursing Small Grants Program—Innovation Grants,

under grant number GSONO0530A; and Hartford Center of Nursing Excellence—Hartford Award

for Research and Practice, under grant number GSONO0531A.

Institutional Review Board Statement: The OHSU Institutional Review Board approved this study,

10.4.21, IRB#00023183.

Informed Consent Statement: Informed consent will be obtained from all subjects involved in the

study, once we are enrolling participants.

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Data Availability Statement: Not applicable.

Acknowledgments: Equipment was donated in kind by Hector A. Olvera-Alvarez from the OHSU

School of Nursing Integrated Bio-behavioral Laboratory.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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