Effects of Post-Session Administration of Methylene Blue on Fear Extinction and Contextual Memory in Adults With Claustrophobia

Participants (N=42) reporting marked claustrophobic fear (peak fear score >50 on a 100-point scale) while performing two consecutive behavioral approach tests and meeting DSM-IV criteria for claustrophobia (excluding the requirement of functional impairment, criterion E) were recruited from a large participant pool of undergraduate students (N=1,163) through a two-stage screening process. Detailed information on exclusion criteria is presented in the data supplement accompanying the online version of this article.

Randomly assigned participants ranged in age from 18 to 36 years (mean age=19.3 years [SD=1.46]) and were predominantly female (82%), white (76.19%), and non-Hispanic (71.43%). A detailed description of the sample characteristics is presented in Table 1 (35, 36).

Potential study participants completed a brief online assessment of claustrophobia (stage 1). Those reporting marked fear of enclosed spaces were invited to the laboratory for a face-to-face screening visit (stage 2), which also served as their formal pretreatment assessment. This consisted of 1) the Structured Clinical Interview for DSM-IV Axis I Disorders (37), 2) completion of self-report patient-rating scales, and 3) behavioral approach tests involving exposure to two distinct claustrophobia chambers. Eligible participants were stratified on gender and pretreatment claustrophobia severity and randomly assigned in double-blind fashion to one of two treatment arms: extinction training plus postsession methylene blue or extinction training plus postsession placebo. All extinction training was completed in one session and was identical in the two treatment arms. Ratings of peak fear and end fear after each of six 5-minute exposure trials were obtained. Outcome assessments identical to the pretreatment assessment were obtained immediately after extinction training and at a 1-month follow-up visit (for a graphical depiction of the experimental design and participant flow chart, see Figure S1 in the online data supplement). All study procedures were approved by the institutional review board at the University of Texas at Austin.

The extinction training paradigm we utilized has been used in several published basic studies evaluating the mechanisms of change in exposure therapy (38–42). In brief, the paradigm consists of the following elements: brief education about the nature of claustrophobia; presentation of a therapy rationale emphasizing the fear-reducing effects of direct confrontation with the feared target; six 5-minute in vivo exposure trials in which the participant enters a tightly enclosed wooden chamber and remains inside in a supine position for a duration of 5 minutes; and completion of self-report rating scales after each exposure trial.

United States Pharmacopeia-grade methylene blue powder (ScienceLab, Houston) was put into gelatin capsules, which were identical in appearance to the placebo capsules that contained food dye indigo carmine powder (FD&C blue no. 2, ScienceLab, Houston). Capsules were provided to each participant in a sealed, numbered envelope prepared by an unblinded pharmacist at the University of Texas at Austin. All other study personnel were blind to the drug condition. The 260-mg methylene blue dose corresponds to the 4-mg/kg dose shown to be effective in previously published preclinical studies of object recognition memory and fear extinction (22, 24, 29). Methylene blue and placebo were administered on three occasions, at a dose of one 86.66-mg capsule each. One capsule was administered immediately following the completion of extinction training. The participants were instructed to take the second capsule before going to bed that night (6–10 hours later) and to take the last capsule after waking up (another 6–10 hours later). This divided dosing schedule was intended to reduce possible urinary tract irritation sometimes associated with methylene blue excretion through the urine. Participants were instructed to take the capsules with a large glass of water in order to further minimize the chance of urinary tract irritation and to reduce the intensity of urine discoloration. Since the average half-life for urinary excretion of methylene blue is 6.6 hours (43), our administration procedure served to maintain methylene blue in the circulation throughout the critical memory consolidation period following extinction training.

Twenty-four hours after completing extinction training, participants were administered a telephone interview to assess medication adherence and side effects.

Consistent with recommendations outlined by Kraemer et al. (47) for testing putative moderators in clinical trials, we performed a multiple regression analysis in which peak fear at the 1-month follow-up was predicted by drug condition (methylene blue compared with placebo), end fear (fear level at the last exposure trial), and their interaction. To enhance confidence that end fear was responsible for this effect and not other third variables associated with end fear, we followed the suggestions of Steiner et al. (48) and controlled for other relevant variables that may be related to both end fear and peak fear at follow-up. These control variables were initial fear at the first exposure trial, postexposure fear in the generalization context, and the presence (at baseline) of other axis I disorders.

To test the hypothesis that methylene blue would enhance contextual memory at the 1-month follow-up, we performed two additional regression analyses in which drug condition (methylene blue compared with placebo) was the predictor of participants’ scores on our context memory index (modeled separately for posttest and follow-up). For consistency with our analyses of peak claustrophobic fear at follow-up, the presence of another axis 1 disorder was dichotomously coded and included as a covariate in the model.

The mean fear level was 73.0 (SD=20.0; range: 20–100) at the first exposure and 23.5 (SD=23.3; range: 0–90) at the last exposure (end fear). As hypothesized, there was a significant drug condition-by-end fear interaction (b=−0.74; t=2.71, df=34, p=0.01; Cohen's d=0.93). We probed this interaction using procedures recommended by Aiken and West (49). This entailed two follow-up analyses, with end fear centered alternatively at low (end fear=0; 1 standard deviation below the mean) and high (end fear=47; 1 standard deviation above the mean) levels of fear. These analyses showed that for participants with low end fear (end fear=0), those given methylene blue had significantly lower levels of fear at the 1-month follow-up (peak fear=10.7) compared with those given placebo (peak fear=29.8) (b=19.2; t= 2.21, df=34, p=0.04; Cohen's d=0.76) (Figure 1). For those with higher levels of end fear, the opposite finding was observed; those given methylene blue had marginally higher levels of fear at the 1-month follow-up (peak fear=33.0) compared with those given placebo (peak fear=17.4) (b=−15.6; t=1.78, df=32, p=0.08; Cohen's d=0.63).

We used this same procedure to examine the model-based relation between end fear at the last exposure trial and peak fear at the 1-month follow-up for those given methylene blue compared with those given placebo. For those who received methylene blue, end fear at the last exposure trial significantly predicted peak fear at the 1-month follow-up (b=0.48; t=2.43, df=32, p=0.02; Cohen's d=0.86), with higher end fear associated with higher fear 1 month later. However, for those who received placebo, there was no significant relationship between end fear and peak fear at the 1-month follow-up (b=−0.27) (t=1.10, df=32, p=0.28; Cohen's d=0.39).

We also hypothesized that scores on our incidental contextual memory test given at the 1-day and 1-month follow-up assessments would reveal enhanced performance for those who received methylene blue compared with placebo. Although those in the methylene blue group did not have greater recall 1 day after completing extinction training (p=0.40), they did demonstrate better free recall performance at the 1-month follow-up (recall score=1.4) compared with those in the placebo group (recall score=0.7) (b=0.69; t=2.06, df=37, p<0.05; Cohen's d=0.68) (Figure 2).

We next attempted to verify that recall assessed by our incidental context memory recall index was not merely a result of lower fear among participants in the methylene blue group. We found that peak fear at posttreatment was unrelated to incidental recall at posttreatment and at follow-up, across drug conditions (all p values >0.26) and within drug conditions (all p values >0.12). Similarly, peak fear at follow-up was unrelated to free recall at follow-up, both across drug conditions (all p values >0.57) and within each drug condition (all p values >0.23). The reverse possibility also received no support, since incidental recall at posttreatment was unrelated to peak fear at follow-up, both across (p=0.88) and within (all p values >0.33) drug conditions.

Furthermore, because incidental recall could be associated with overall memory functioning and hence could be related to emotional memory of end fear at the last exposure trial, we investigated whether memory recall moderated the effect of end fear on peak fear at follow-up (i.e., end fear could be highly related to peak fear at follow-up for those with greater recall but less related to peak fear for those with poor recall). No evidence was found for this possibility (all p values >0.24). Thus, it appears that the memory facilitation effects of methylene blue at follow-up were independent of any differential changes in fear between the methylene blue and placebo groups.

The frequency and mean severity of side effects reported during the study are summarized in Table 2. Minor side effects were reported in each drug condition. The three most commonly reported side effects in the methylene blue group were urine discoloration, increased frequency of urination, and dizziness, whereas the three most common side effects in the placebo group were urine discoloration, headache, and fecal discoloration. These side effects were generally mild, and no serious adverse reactions were observed. No problems with medication adherence were reported.

Our study includes some limitations. First, our sample was relatively small and largely comprised of young, female university students. Thus, replication with a larger, more diverse sample is warranted. Second, conclusions as to whether methylene blue enhances exposure therapy in other anxiety disorders await further investigation. Third, single-item fear ratings (i.e., self-reported subjective units of distress) are a ubiquitous measure of fear responding in the phobia treatment literature. However, additional indices of fear, including physiological measurements, would have strengthened conclusions drawn from the present findings. Fourth, extinction training was delivered in a single session. Although single-session extinction-based treatments have been shown to be efficacious (42, 56), it remains unclear as to whether methylene blue enhances exposure therapy when delivered in a multisession treatment format. Fifth, a 4-mg/kg posttraining dosing strategy was selected because it has been shown to be the most reliable in rats for enhancing posttraining fear extinction memory retention (22, 29), as well as long-term behavioral habituation and object memory recognition (24). Future studies are needed to determine the optimal methylene blue dosing strategies for enhancing exposure therapy. Finally, given the small sample size, it is unclear how effectively the control variables corrected the potential differences between groups and between participants with varying levels of end fear.

Our findings have several implications for clinical practice. First, because methylene blue appears to strengthen the memory for fear extinction learning (or lack thereof) that occurs during exposure therapy, administration of methylene blue at the beginning of a therapy session should be avoided, given the inability to predict whether a session will be successful. Second, our findings suggest that postsession methylene blue administration should be done judiciously, after careful consideration of the patient’s level of fear attenuation achieved insession. Based on our findings, patients who continue to show moderate to high levels of fear at the conclusion of an exposure therapy session may have their fear inadvertently strengthened by methylene blue administration, thus leading to a less favorable therapeutic outcome. Finally, these findings highlight the need for research on the development of empirically based decision rules for administering methylene blue and other cognitive enhancers in the context of exposure therapy.