Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

This study follows the policies and guidelines of Human Research Protection Program of Indiana University Institutional Review Board.

1. Location selection

1. Select the number of sites (n) based on different levels of nature.
   NOTE: We chose three sites for our work. Using a continuum based on levels of "naturalness", Site A was considered the most natural and is comprised of approximately 1,200 acres of wooded ridges bordering a lake and set within a deciduous forest. The most common activities include walking and wildlife watching. Site B was a 33-acre municipal park featuring walking paths, places for gatherings, playgrounds, and open field space for causal recreational activities. Site C was an urban, indoor exercise facility with the lowest level of naturalness. All three sites are in relatively close proximity to a medium-size city (estimated population of 46,000 people) in the midwestern United States.

2. Participant screening and preparation

1. Complete participant recruitment before subjects engage in recreational experiences.
   NOTE: For Sites A and B, subjects were approached by the researcher in the parking lot of the park entrance. For Site C, subjects were approached at the front desk of the indoor exercise facility. To control for differences in activity type, subjects recruited for both Sites A and B were predominately hiking, while for Site C the primary activity was running or walking on the indoor track.
2. During recruitment, remind subjects to comply with the guidelines related to collecting salivary cortisol samples. They must not eat or drink 10 min before providing saliva samples.
   NOTE: It should be noted that in this case, the saliva samples were not normalized for volume or dilution (concentration) - i.e. whether the participants were adequately hydrated and/or did hydration change between the sample collections.
3. Give red armbands (or equivalently noticeable clothing) to each participant to allow for easy identification when emerging at the end of their engagements.
4. Assign a 30-40 min time allotment so that all subjects across the three sites spend a similar amount of time in each site. Exclude subjects who spend substantially more or less time than the rest of the sample.
5. Keep the type of activity engaged consistent.
   NOTE: In this case, the "medium" activity was walking or hiking. Subjects who engaged in activities substantially different from walking or hiking were excluded from the sample. For example, subjects who were fishing, picnicking, or weight lifting were not included in the respective samples.

3. Conditions and experimental design

1. Use a quasi-experimental pre-test/post-test design.
2. After identifying subjects and upon their agreeing to participate, ask each subject to read and sign an IRB form explaining the voluntary nature, purpose, and procedures of the study.
3. Following this process, give subjects a piece of clothing (e.g., red armband) for future identification and obtain physiological and psychological measures of stress levels (i.e., PSQ, 1-2 mL of saliva that is spit or drooled into a test tube). Collect samples from the late afternoon to early evening.
   NOTE: These data were collected by researchers both 1) just before subjects entered the site and 2) immediately upon ending visitation to the site.

4. Saliva samples

1. To avoid sample dilution, ask subjects not to eat, drink, or rinse mouths 10 min before providing saliva samples.
2. Ask subjects to provide 1-2 mL saliva samples (excluding foam) just prior to the recreational experience and immediately following conclusion of the experience.
3. Collect saliva samples using a passive drool method:
   1. Provide subjects with a 2 in plastic drinking straw to drool into a 2 mL cryovial (see Table of Materials).
   2. Instruct subjects to allow saliva to pool in their mouths, then drool down the straw and into the cryovial. According to the Saliva Collection and Handling Advice (2011), 1 mL (excluding foam) is adequate for most tests.
   3. Label samples with an assigned 3-digit ID number (i.e., 001) and letter to indicate the timing of data collection (i.e., A represents pre-test, B represents post-test; where 001A represents the saliva sample provided by participant 001 before conducting the recreational experience).
   4. After the sample is collected and labeled, it should then be stored frozen temporarily in a steroid foam box full of dry ice for no more than 2 h.
   5. Transport the marked samples to a laboratory and store at -80 °C until analyzed.

5. Quantification of α-amylase

NOTE: In this assay, α-amylase hydrolyzes 2-chloro-p-nitrophenyl-α-D-maltotrioside to 2-chloro-nitrophenol and forms glucose, 2-chloro-p-nitrophenyl-α-D-maltoside, maltotriose, and glucose. The reaction is monitored at an absorbance of 405 nm, which corresponds to α-amylase activity in the sample. This assay demonstrates linearity between 0 and 2000 U/L.

1. Materials
   1. Used a liquid amylase reagent set (see Table of Materials) to quantify α-amylase in saliva samples. All reagents are provided as ready-to-use liquids and stored at 0-4 °C.
   2. Use a multi-mode reader (see Table of Materials) capable of reading an optical density at 405 nm with a temperature controlled to 37 °C during the assay.
2. Analysis
   1. Thaw the samples on ice prior to analysis.
   2. Dilute samples 1:10 with 1x PBS (10 µL of saliva + 90 µL of PBS).
   3. Analyze each sample in duplicate.
   4. Equilibrate the amylase reagent to 20-25 °C for at least 30 min.
   5. Add 0.1 mL of amylase reagent to a 96 well microplate for each sample.
   6. Pre-incubate the microplate at 37 °C for a minimum of 5 min.
   7. Add 2.5 µL of the sample to the amylase reagent.
   8. Take an initial reading after 60 s.
   9. Continue readings every 60 s for an additional 2 min.
   10. Calculate the mean absorbance difference per minute (ΔAbs/min).
3. Calculation
   1. To calculate amylase activity, use the following formula:
      
      Where ΔAbs/min = changes in absorbance difference per minute; TV = total assay volume (0.1025 mL); *1000 = conversion of U/mL to U/L; MMA = millimolar absorptivity of 2-chloro-p-nitrophenol = 12.9; SV = sample volume (0.0025 mL); and LP = light path (1 cm). Substituting gives:
      
      Therefore, multiply the ΔAbs/min by 3178x the dilution factor (10) to obtain amylase in U/L.
   2. For samples above 2000 U/L (assay linearity) dilute further (at least 2x using PBS) and re-assay, then multiply the α-amylase result by the additional dilution factor.

6. Quantification of cortisol

NOTE: In this assay, free cortisol is quantified in saliva using a cortisol standard curve. Standards and diluted samples are added to a microtiter plate that is pre-coated with an antibody. A cortisol-peroxidase conjugate is added to the wells, followed by addition of a monoclonal antibody to cortisol. The amount of cortisol/peroxidase conjugate binding decreases as the concentration of cortisol in the sample increases.

1. Materials
   1. Use a Cortisol Enzyme Immunoassay Kit (see Table of Materials) to quantify cortisol in saliva samples. All reagents needed to perform this assay are included in the kit. All components of the kit are stored at 0-4 °C prior to reaching the expiration date.
   2. Use a spectrophotometer able to read an optical density (OD) at 450 nm (see Table of Materials), as well as software capable of using OD recordings from the plate reader to perform four-parameter logistic curve (4 PLC) fitting.
2. Reagent preparation
   1. Allow all reagents to equilibrate to 20-25 °C for a minimum of 30 min.
   2. Dilute the cortisol assay buffer 1:5 using deionized water.
   3. Dilute the wash buffer 1:20 using deionized water.
      NOTE: Assay and wash buffers are stable for 3 months when stored at 0-4 °C.
3. Sample preparation
   1. Thaw the samples on ice prior to analysis.
   2. Dilute samples 1:10 with cortisol assay buffer (20 µL of saliva + 180 µL of buffer) and use within 2 h of preparation.
4. Preparation of standards
   1. Label glass test tubes #1-#7.
   2. Pipet 225 μL of assay buffer into tube #1 and 125 μL of buffer into tubes #2-#7.
   3. Add 25 μL of the cortisol stock solution to tube #1 and vortex.
   4. Remove 125 μL of buffer from tube #1 and add it to tube #2, then vortex.
   5. Repeat the serial dilutions for tubes #3-#7.
      NOTE: The final cortisol concentrations of each standard is shown in Table 1.
   6. Standards must be used within 2 h of preparation.

Table 1: Standard curve preparation table.

5. Analysis
   1. Use the plate layout below in Figure 1 as a guide to set up the microplate.
   2. It is recommended to use a multichannel pipette for addition of reagents.
   3. Add 50 μL of samples or standards into the appropriate number of wells in the plate. Samples and standards should be run in duplicate.
   4. Add assay buffer (75 μL) into the non-specific binding (NSB) wells.
   5. Add assay buffer (50 μL) into the maximum binding (B0) wells and zero standard (blank) wells.
   6. Add 25 μL of the cortisol conjugate to each well.
   7. Add 25 μL of the cortisol antibody to each well, except for the NSB wells.
   8. Gently tap the side of the plate to mix the reagents.
   9. Cover with the plate sealer and shake at room temperature (RT) at 20-25 °C for 1 h.
   10. Remove the well contents and rinse each well 4x with wash buffer (300 Μl). Tap the plate to dry on absorbent towels between washes.
   11. Add the TMB substrate to each well (100 μL).
   12. Incubate the plate at 20-25 °C for 30 min without shaking.
   13. Add stop solution to each well (50 μL).
   14. Read the optical density in each well of the microplate at 450 nm.
   15. Average the optical densities for each standard, then sample and subtract the mean optical density for the NSB wells.
   16. Calculate the % bound (B/B0) for all samples using maximum binding (B0) controls.
   17. Create a standard curve using software capable of four-parameter logistic regression curve fitting, calculated from the %B/B0 curve.
   18. Multiply the result by the dilution factor (10) to obtain cortisol values in pg/mL.
   19. Samples with optical densities falling above the highest standard should be further diluted with assay buffer and re-assayed, then the result should be multiplied by the additional dilution factor.

Figure 1: Example plate layout. Please click here to view a larger version of this figure.

7. Psychological measurement (Perceived Stress Questionnaire)

1. Measure psychological levels of subjects using the PSQ published by Fliege et al.¹³, which includes four factors (worry, tension, tension, joy) and utilizes 20 items.
2. Ask the subject to fill out the PSQ just prior to their recreational experience and immediately following conclusion of the experience.
3. Tag questionnaires with a 3-digit ID number identical to each subject's biophysiological level of stress.