Table_1_Advancing ecological validity and clinical utility in virtual reality-based continuous performance test: exploring the effects of task difficulty and environmental distractors.DOCX

IntroductionResearchers have highlighted concerns regarding the limited diagnostic utility and ecological validity of the Continuous Performance Test (CPT). Recent advancements in VR-based CPTs have attempted to address these concerns by simulating real-life scenarios and enhancing attention deficit hyperactivity disorder (ADHD) diagnosis; however, certain areas require improvement for obtaining reliable data from both healthy individuals and those with ADHD. To tackle these issues, we developed an enhanced VR-based CPT program featuring four distinct difficulty levels, advancing toward home-based assessment.MethodOur feasibility study involved subjects without ADHD to establish a normative profile for VR-based CPT before extending it to ADHD assessment. Our sample included 20 Korean adults. They received a VR device with the VR-based CPT program installed and were asked to perform 1-2 blocks per day at home. Participants were instructed to complete 12 blocks over the subsequent 2 weeks. Psychological assessments and electroencephalograms (EEGs) were administered before and after the program. Post-study usability measures were also collected.ResultHigher commission errors were notably evident in the “very high” difficulty level which featured complex stimuli and increased distraction. A notable correlation emerged between the overall distraction level and CPT accuracy, along with a significant link between intensity scores and commission errors. No significant differences were found in psychological assessment and there were no significant changes in the Theta-Beta Ratio (TBR) index before and after the program. The usability of our program was fair.DiscussionThe study reveals that the newly designed VR-CPT program, simulating diverse real-life environments and offering varying task difficulty levels, proved acceptable and feasible. The key point of our study was that the adjustment and segmentation of difficulty levels in the VR-based CPT were achieved, and that this effort was validated by examining the impact of different levels of difficulty on CPT measures. Implementing this experimental setup in a home-based environment increased ecological validity, as well as clinical utility. Limitations and suggested directions for further investigation are described in detail.</p

Effects of CD4⁺CD25⁺ Treg cells deficiency on the durations of immobility in the tail suspension test (n = 14–16).

<p>After 21 days of stress, mice were isolated in each chamber and suspended upside down by taping their tails to a flat metal bar so that they could not reach the top or the sides of the chamber. The mice were left in this position for 6 min and the total duration of immobility was measured. Data were analyzed using separate one-way ANOVA followed by a <i>post hoc</i> Newman-Keuls test. Vertical bars indicate the S.E.</p

Effects of CD4⁺CD25⁺ Treg cells deficiency on levels of serotonin (5-HT) (A) and its metabolite, 5-HIAA, (B) within the hippocampus (n = 5–8).

<p>After the behavioral tests, the prefrontal cortex and hippocampus were dissected from the mice and homogenized. Each supernatant sample was injected into a reversed phase HPLC column. The amounts of monoamines and metabolites were then determined using an electrochemical detector. Data were analyzed using separate one-way ANOVA followed by a <i>post hoc</i> Newman-Keuls test. * <i>p</i><0.05 <i>vs.</i> the non-stressed control group. Vertical bars indicate the S.E.</p

Effects of CD4⁺CD25⁺ Treg cells deficiency on serum concentrations of the pro-inflammatory cytokines IL-6 (A), and TNF-á (B) (n = 10–12).

<p>After the behavior tests, blood samples were collected from the mice by retro-orbital puncture. The serum prepared from each blood sample was then subjected to duplicate measurements for cytokine analysis using the mouse cytometric bead array (CBA). Data were analyzed using separate one-way ANOVA followed by a <i>post hoc</i> Newman-Keuls test. ** <i>p</i><0.01, * <i>p</i><0.05 <i>vs</i>. the stressed anti-CD25 Ab-treated group. Vertical bars indicate the S.E.</p

Correlation between the durations of immobility in the forced swim test and levels of cytokines and monoamines (B, C).

<p>Correlations in immobility are shown in the scatter-plat with IL-6 and TNF-á (<i>Closed circles = </i>IL-6: <i>closed triangles = </i>TNF-á) (A), 5-HT and 5-HIAA (<i>Closed circles = </i>5-HT : <i>closed triangles = </i>5-HIAA) (B) and DA and DOPAC (<i>Closed circles = </i>DA : <i>closed triangles = </i>DOPAC) (C) plotted separately. Data were analyzed using Pearson’s correlation coefficients (2-tailed).There were significant correlation at the level of 0.05 between the stress condition and changes in cytokines (IL-6, r = 0.48 and TNF-á, r = 0.46) and monoamines (5-HT, r = −0.46; 5-HIAA, r = −0.46; DA, r = −0.47) concentrations.</p

Schedule of experimental procedures.

<p>Mice were divided into non-stressed WT mice, stressed WT mice, non-stressed CD4⁺CD25⁺ regulatory T (Treg) cells depleted mice, stressed CD4⁺CD25⁺ Treg cells depleted mice; from day 1 to 22, the stressed WT mice and stressed CD4⁺CD25⁺ Treg cells depleted mice received daily immobilization stress for 4 h and then were moved back to the home cage (three mice to a cage). An anti-CD25 antibody (Ab) was injected intraperitoneally once 24 h before CIS and then on the 8^th day after the first CIS. On day 22, EPM, FST and TST were conducted in the afternoon (22 day) to determine depression and anxiety related behaviors after the previous day’s morning CIS.</p

Effects of CD4⁺CD25⁺ Treg cells deficiency upon chronic immobilization stress in the elevated plus maze (n = 14–16).

<p>After 21 days of stress, mice were individually placed in a central platform facing a closed arm and allowed to freely explore the maize for 5 min. Time spent in the open arms (A) and closed arms (B) of the maze and the frequency of entry into both arms (C) in the elevated plus maze. The time spent in the respective arms was recorded. Data were analyzed using separate one-way ANOVA followed by a <i>post hoc</i> Newman-Keuls test. ***<i>p</i><0.001, *<i>p</i><0.05 <i>vs.</i> the non-stressed control group. Vertical bars indicate the S.E.</p

Anti-CD25 Ab-induced depletion of CD4⁺CD25⁺ Treg cells in mice (n = 8–10).

<p>Representative dot plots are shown for CD4⁺CD25⁺FOXP3⁺ cells. The efficacy of CD4⁺CD25⁺ Treg cell depletion was confirmed by flow cytometry analysis using PE-anti-mouse CD25 and fluorescent isothiocyanate- anti-mouse CD4 (A). Summaries of the percentage of positive cells are shown in (B) for CD4⁺CD25⁺FOXP3⁺ cells and are presented as the mean ± SE. *** <i>p</i><0.001 <i>vs.</i> the wild-type mice.</p

Effects of CD4⁺CD25⁺ Treg cells deficiency on serum concentrations of the Th1 cytokines IL-2 (A) and IFN-γ (B) (n = 10–12).

<p>After the behavior tests, blood samples were collected from the mice by retro-orbital puncture. The serum prepared from each blood sample was then subjected to duplicate measurements for cytokine analysis using the mouse cytometric bead array (CBA). Data were analyzed using separate one-way ANOVA followed by a <i>post hoc</i> Newman-Keuls test. ** <i>p</i><0.01, * <i>p</i><0.05 <i>vs</i>. the stressed anti-CD25 Ab-treated group. Vertical bars indicate the S.E.</p

Effects of CD4⁺CD25⁺ Treg cells deficiency on levels of dopamine (DA) (A) and its metabolite, DOPAC (B) within the prefrontal cortex (n = 5–8).

<p>After the behavioral tests, the prefrontal cortex and hippocampus were dissected from the mice and homogenized. Each supernatant sample was then injected into a reversed phase HPLC column, after which the amounts of monoamines and metabolites were determined using an electrochemical detector. Data were analyzed by separate one-way ANOVA followed by a <i>post hoc</i> Newman-Keuls test. Vertical bars indicate S.E.</p