This double-blind placebo-controlled between-subjects study (n=23) tested the antidepressant efficacy of inhaled nitrous oxide (50% N2O|50% O2 versus 100% O2) in patients diagnosed with major depression (MDD). Across multiple treatment sessions administered across a period of 4 weeks, there were significant reductions in depressive symptoms in the acute response to treatment and accumulatively across sessions.
Papers cited by this study that are also in Blossom
Berman, R. M., Cappiello, A., Anand, A. et al. · Biological Psychiatry (2000)
Objective
Major depressive disorder (MDD) is related to glutamatergic dysfunction. Antagonists of glutamatergic N-methyl-D-aspartate receptor (NMDAR), such as ketamine, have antidepressant properties. Nitrous oxide (N2O) is also a NMDAR antagonist. Thus, this study aimed to evaluate the effects of augmenting antidepressant treatment with N2O.
Methods
This double blind, placebo-controlled randomized parallel pilot trial was conducted from June 2016 to June 2018 at the Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. Twenty-three subjects with MDD (aged 18 to 65, on antidepressants, with a score > 17 on the 17-item-Hamilton Depression Rating Scale [HAM-D17]) received 50% N2O (n=12; 37.17±13.59 years) or placebo (100% oxygen) (n=11; 37.18±12.77 years) for 60 minutes twice a week for 4 weeks. The primary outcome was changes in HAM-D17 from baseline to week 4.
Results
Depressive symptoms improved significantly in the N2O group (N2O: from 22.58±3.83 to 5.92±4.08; placebo: from 22.44±3.54 to 12.89±5.39, p < 0.005). A total of 91.7% and 75% of the N2O group subjects achieved response (≥ 50% reduction in HAM-D17 score) and remission (HAM-D17 < 7), respectively. The predominant adverse effects of N2O treatment were nausea, vomiting, and headache.
Conclusion
N2O treatment led to a statistically significant reduction in HAM-D17 scores compared to placebo.
Guimara and colleagues frame their study within limitations of monoaminergic antidepressants and growing evidence implicating glutamatergic dysfunction in major depressive disorder (MDD). Previous preclinical and clinical work has shown that antagonism of the N-methyl-D-aspartate receptor (NMDAR), notably by ketamine, can produce rapid antidepressant effects. Nitrous oxide (N2O) is another NMDAR antagonist with additional actions on opioid receptors, GABAergic sites, and serotonergic systems; a 2015 proof-of-concept crossover trial reported short-term antidepressant benefit after a single 50% N2O inhalation in treatment-resistant depression. Safety concerns with N2O include effects on vitamin B12 and homocysteine metabolism, but at therapeutic concentrations it is commonly considered safe for outpatient use. This pilot trial was designed to test whether repeated adjunctive N2O inhalation would reduce depressive symptoms in patients with MDD who remained symptomatic despite at least 4 weeks of antidepressant treatment. The investigators hypothesised that 50% N2O administered twice weekly for 4 weeks, added to ongoing antidepressant therapy, would produce greater reductions in clinician-rated depressive symptoms than placebo (100% oxygen). The study therefore examined both acute (within-session) and cumulative antidepressant effects over a 1-month course of semiweekly N2O sessions.
This was a double-blind, placebo-controlled, randomized parallel pilot trial (1:1) conducted at a university hospital between 2016 and 2018. Eligibility included adults aged 18–65 with DSM-5 major depressive disorder who had been on stable antidepressant treatment for at least 4 weeks and had a baseline 17-item Hamilton Depression Rating Scale (HAM-D17) score of ≥17. The protocol was adjusted from the initial plan to permit any class of antidepressant (mono- or combination therapy) and to shorten the requirement for no antidepressant dose adjustment from 6 to 4 weeks. Exclusion criteria included bipolar disorder, psychotic or substance use disorders (except caffeine and nicotine), prior antidepressant-induced mania/hypomania, significant suicidal ideation, relevant medical comorbidities (including chronic lung disease), pregnancy or breastfeeding, contraindications to N2O (e.g. chronic B12 deficiency, pneumothorax, elevated intracranial pressure, intestinal obstruction), and inability to breathe through the nose. Participants were recruited from outpatient services, referrals and public advertisement. Subjects were randomised to inhalation of 50% N2O (diluted in 50% oxygen) or placebo consisting of 100% oxygen, each delivered by a nasal mask for 60 minutes, twice weekly for 4 weeks (eight sessions total). The N2O concentration (50%) was chosen for outpatient safety and because it had been used in prior work. Continuous monitoring of heart rate, peripheral oxygen saturation and blood pressure was performed during sessions; women of reproductive age had a urinary pregnancy test before starting. Blinding measures included having two unblinded collaborators set the device outside the assessor's presence, covering the device display, and using flavoured nasal masks to mask N2O's slight sweet odour. The primary outcome was change in HAM-D17 score from baseline to the end of week 4. Secondary outcomes included changes in Beck Depression Inventory-II (BDI-II), rates of therapeutic response (≥50% reduction from baseline) and remission (HAM-D17 ≤7; BDI-II ≤13), suicidal ideation measured by the Columbia-Suicide Severity Rating Scale (C-SSRS), emergence of manic symptoms assessed with the Young Mania Rating Scale (YMRS), and adverse events. Assessments were performed by a psychiatrist before and after each inhalation session (16 assessments total). Randomisation used permuted blocks generated online. Statistical analysis employed repeated-measures analysis of variance (ANOVA) for HAM-D17 (factors: time, drug, time×drug), with independent t-tests when interactions were significant; similar approaches were used for BDI-II and YMRS. Non-parametric tests addressed C-SSRS, and Fisher's exact test compared response/remission rates. Effect sizes were reported using partial eta-squared (Z2p) with predefined thresholds.
Major depressive disorder (MDD) is the fifth leading cause of disability worldwide, accounting for 4.2% of disabilityadjusted life years lost. Taking into account only noncommunicable diseases, depressive disorders are the second leading cause of disability worldwide.The average 12-month prevalence of MDD is 4.7%, ranging from 1.1% in Nigeria to 10.1% in Brazil, whereas the mean lifetime prevalence is 11.2%.More than half of patients have a recurrent or chronic course. They also have reduced quality of life, increased mortality and morbidity associated with other chronic medical conditions, as well as increased suicide rates.In addition, depressive disorders cause a high economic burden.The goals in MDD treatment include complete remission of symptoms and a return to premorbid functioning and quality of life.Since the discovery of the antidepressant agents in the 1950s, pharmacological treatment has been the main approach to MDD.The monoaminergic hypothesis was based on the mechanism of action of these first generation antidepressants, postulating that depressive symptoms were a consequence of dysfunctions in monoaminergic neurotransmitter systems. Although roughly 80% of patients taking antidepressants achieve a response after four treatments,only 25-40% reach complete remission.Moreover, there is a delay of days to weeks for the therapeutic response to modern antidepressants, which prolongs the patient's suffering and burden.These limitations have led researchers to question the central role attributed to monoamines in depression.Glutamate is the main excitatory neurotransmitter in the central nervous system, and the glutamatergic system is essential for neuronal plasticity, synaptogenesis, and excitotoxicity.Trullas & Skolnickdemonstrated that antagonists of N-methyl-D-aspartate subtype receptors (NMDAR) of glutamate reverse the behavioral inhibition induced by animal models of depression. The authors then hypothesized that pathways regulated by NMDAR would be involved in the pathophysiology of depression. Later, based on pre-clinical studies that followed the pioneering study by Collingridge et al.,Berman et al.demonstrated that an intravenous infusion of 0.5 mg/kg ketamine (an NMDAR antagonist anesthetic) had rapidonset antidepressant effects. Preclinical studies have focused on other possible mechanisms underlying the antidepressant effects of ketamine, beyond its action on NMDAR. Some of the mechanisms suggested to be involved in its effects include activation of the mammalian target of the rapamycin pathway, activation of alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptors,inhibition of glycogen synthase kinase 3, and inhibition of eukaryotic elongation factor 2.All of these actions converge to synaptic density protein synthesis, which is necessary for dendritic and somatic plasticity.Like ketamine, nitrous oxide (N 2 O) is an anesthetic that acts as an NMDAR antagonist, which could indicate a possible antidepressant effect. The main mechanism proposed for the anesthetic action of N 2 O is the inhibition of glutamatergic excitatory neurotransmission through noncompetitive NMDAR antagonism.The analgesic action of N 2 O occurs in part by stimulating the release of endogenous opioid peptides in the midbrain.In addition, N 2 O plays a direct and partial agonist effect on mu, kappa, and delta opioid receptors. Regarding its anxiolytic effects, one putative mechanism is the activation of gamma-aminobutyric acid type A receptors at the benzodiazepine binding site. Another mechanism proposed for the anxiolytic effect of N 2 O is its action on serotonin neurotransmitters. A study in rats showed increased serotonergic activity in the hypothalamus and decreased activity in the cerebral cortex with the administration of N 2 O, the latter possibly as a result of inhibition of pre-synaptic 5-HT 1A serotonin autoreceptors.N 2 O is safe and free of serious adverse reactions when used in the recommended therapeutic concentrations.The main caveat concerns the interference in homocysteine metabolism through inactivation of cobalamin (vitamin B12). Vitamin B12 deficiency may result in megaloblastic anemia, leukopenia, myelopathy, memory impairment, and behavioral changes.In 2015, Nagele et al.published a proof-of-concept trial evaluating the effect of N 2 O inhalation in a placebocontrolled, double-blind, crossover trial. Twenty treatmentresistant depressive patients received a single administration of 50% N 2 O for 60 minutes. N 2 O was superior to placebo in improving depressive symptoms (a reduction of 4.8 points in 2 hours and 5.5 points in 24 hours on the 21item-Hamilton Depression Rating Scale). N 2 O was associated with a response rate of 20%, compared to 5% with placebo. No serious adverse events were reported. In their trial, only treatment-resistant patients were enrolled, and a single N 2 O treatment was administered. Assessments were made 2 and 24 hours after N 2 O inhalation. The authors indicated the need for further trials to determine optimal dosing strategies and to evaluate a more diverse population of patients. Taken together, the available data suggest that the glutamatergic system is involved in the neurobiology of depression by modulating NMDAR and other targets in this system. Therefore, glutamatergic modulators appear to have antidepressant potential. Thus, we hypothesized that N 2 O could augment the effects of antidepressant treatment and conducted a 1-month randomized controlled pilot trial to investigate the efficacy of N 2 O in reducing depressive symptoms among MDD patients currently undergoing treatment who remained symptomatic.
Create a free account to open full-text PDFs.
Papers in Blossom that reference this study
Quach, D. F., de Leon, V. C., Conway, C. R. · Journal of the Neurological Sciences (2022)
From 201 assessed for eligibility, 23 participants were randomised (12 to N2O, 11 to placebo). Two participants in the placebo group discontinued (one after session 1 for symptom worsening, another after session 5 for adherence difficulties), leaving 21 completers for end-of-trial outcome analyses. Baseline demographic and clinical characteristics, including HAM-D17 and BDI-II severity, were similar between groups. Primary outcome (HAM-D17): Both groups showed significant reductions in HAM-D17 scores over time, but the N2O group improved more. Groupwise mean HAM-D17 changed from 22.58±3.83 to 5.92±4.08 in the N2O group and from 22.44±3.54 to 12.89±5.39 in the placebo group; a significant time×drug interaction was observed (F15,285 = 2.26; p = 0.005; Z2p = 0.11). Within-session (pre- vs post-inhalation) reductions were significant in nearly all sessions in both groups, but only the N2O group reached mean values in the remission range at post-inhalation assessments in sessions 7 and 8. At the end of session 8, HAM-D17 remission occurred in 75% (9/12) of the N2O group versus 11.1% (1/9) of the placebo group (difference 0.64; p = 0.008, Fisher's exact test). Response rates were 91.7% (11/12) for N2O versus 44.4% (4/9) for placebo (difference 0.47; p = 0.046). Secondary outcomes: BDI-II scores fell significantly over time in both groups with large effect sizes, but there was no significant time×drug interaction (F15,285 = 1.12; p = 0.34; Z2p = 0.06). End-of-trial BDI-II remission rates were 66.7% (8/12) in the N2O group and 33.3% (3/9) in placebo (p = 0.20); response rates were 66.7% vs 55.6% (p = 0.67). Suicidal ideation (C-SSRS) distributions did not differ between groups at any assessment and no suicidal behaviours occurred. YMRS scores did not indicate manic or hypomanic episodes in either group; no participant exceeded a YMRS total score of 12. Adverse effects were more frequently reported with N2O. Common events included somnolence, paresthesia, nausea, vomiting and headache. In the N2O group (96 sessions total), four of 12 participants required temporary inhalation interruptions across 12 sessions; five sessions were discontinued before completion, three of them for a single participant. One participant in the N2O arm had increased blood pressure during one session. No serious adverse events were reported. In the placebo group (78 sessions), adverse effects were similar but fewer interruptions occurred.
Guimara and colleagues interpret their findings as indicating that adjunctive 50% N2O inhalation twice weekly for 4 weeks produced greater clinician-rated antidepressant benefit than placebo in symptomatic patients on stable antidepressant therapy. The HAM-D17 showed both acute within-session reductions and a cumulative effect across sessions in the N2O group, whereas the placebo group showed more limited cumulative change. Reported response and remission rates on the HAM-D17 were substantially higher in the N2O arm (response 91.7%, remission 75%) and reached statistical significance versus placebo. The authors note an inconsistency between clinician-rated (HAM-D17) and self-rated (BDI-II) measures: both instruments improved, but the superiority of N2O over placebo was evident only on the HAM-D17. They attribute this divergence in part to differences between clinician and self-rated scales and to known only-moderate correlations between them. Possible mechanisms for N2O's antidepressant effect are discussed, including NMDAR antagonism, interactions with the opioid system (as suggested by ketamine–naltrexone data), and effects on nitric oxide signalling. Acknowledged limitations include the small sample size, short 4-week duration, lack of post-treatment follow-up and absence of an intention-to-treat analysis. The investigators also recognise potential unblinding due to N2O-associated adverse effects and the use of 100% oxygen as placebo rather than an active comparator. Practical constraints led to sessions being scheduled at varying times; treatment-resistance status was not systematically recorded, though the high response rates suggest few participants were treatment-resistant. Measurement issues are highlighted: HAM-D and BDI-II include items (sleep, appetite, weight) that do not change rapidly, which complicates within-session comparisons. The chosen 50% N2O concentration prioritised outpatient safety, but optimal dosing and frequency remain unknown; the trial also lacked capnography or gas analysis to measure expired N2O or CO2. Finally, the authors call for larger, longer, and methodologically rigorous trials—preferably with active controls, intention-to-treat analyses, clearer distinction of treatment-resistant samples, and exploration of dose and administration schedules—to determine whether N2O is an effective antidepressant alone or as adjunctive therapy.
This double-blind placebo-controlled randomized parallelgroup (1:1) pilot trial was conducted at the Laborato ´rio de Psicofarmacologia, Hospital das Clı ´nicas, Faculdade de Medicina de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo (HCFMRP-USP). The eligibility criteria from the initial trial protocol required two adjustments to increase the number of eligible subjects. Instead of including only subjects on selective serotonin reuptake inhibitors, we decided to include subjects using any class of antidepressant, alone or in combination, in addition to reducing the period without antidepressant dose adjustment from 6 to 4 weeks. The remaining criteria were maintained. Eligible participants were men or women aged between 18 and 65 years who had been diagnosed with MDD according to DSM-5 criteria, were on antidepressant treatment without dose adjustments for at least 4 weeks, and who scored 4 17 on the 17-item-Hamilton Depression Rating Scale (HAM-D 17 ). Exclusion criteria consisted of 1) diagnosed bipolar, psychotic, or substance use disorder (except caffeine and nicotine), a clinical history of antidepressant-induced hypomania/mania episodes; 2) psychotic symptoms; 3) significant suicidal ideation; 4) chronic lung diseases or other relevant clinical conditions; 5) pregnancy or breastfeeding; 6) contraindications to the use of N 2 O (chronic vitamin B12 deficiency, pneumothorax, elevated intracranial pressure, intestinal obstruction); and 7) difficulty breathing through the nose. Participants were recruited from the outpatient services of the university hospital, referred by private clinics, or spontaneously sought out the trial through a public advertisement campaign.
Subjects were randomly allocated to receive either N 2 O or placebo. N 2 O was administered at a concentration of 50% diluted in 50% oxygen for 60 minutes in eight sessions (twice a week over 4 weeks). Intervals between trial sessions were 3 or 4 days, alternately. We chose the 50% concentration due to its safety for outpatient administration, since this concentration produces minimal sedation and has no impact on cardiorespiratory function, according to American Anesthesiology Association guidelines.Furthermore, Nagele et al. used this concentration in their pilot trialN 2 O was inhaled through a disposable nasal mask coupled to a portable device for nitrous oxide/oxygen sedation and analgesia (Mandala-Matrix MDM, Porter Instruments, Hatfield, PA, USA). The air volume was between 5 and 7 L/min, which is the respiration volume for a healthy adult.The placebo condition consisted of 100% oxygen inhaled for the same period through a nasal mask coupled to the same device. This was chosen based on the limitations of our equipment and for blinding purposes. Evidence suggests that 1 hour of normobaric 100% O 2 is safe, and central nervous system toxicity does not occur during normobaric exposures.Physiological parameters (heart rate, oxygen peripheric saturation, and blood pressure) were continuously monitored during the session as a safety measure. The sessions lasted approximately 3 hours, including the inhalation period (N 2 O or oxygen) and the clinical assessment. The sessions were performed individually under the supervision of the staff anesthesiologist. For safety issues, the subjects were instructed not to eat food or drink liquids (except water) for at least 2 hours before each session. As an additional safety measure, women of reproductive age took a urinary pregnancy test during the first session before beginning the study procedures.
Changes in HAM-D 17 scores from baseline to the end of week 4 were considered the primary outcome. Secondary outcomes included changes in Beck Depression Inventory-II (BDI-II) scores from baseline to the end of week 4, therapeutic response and remission at the end of the trial according to HAM-D 17 and BDI-II scores, the effects on suicidal ideation, which were assessed with the Columbia-Suicide Severity Rating Scale (C-SSRS), the occurrence of manic symptoms, which was assessed with the Young Mania Rating Scale (YMRS), and any adverse effects reported by the subjects or observed by the clinicians. All measurements were taken by a psychiatrist at the beginning (pre-inhalation) and end (post-inhalation) of each session, totaling 16 evaluations throughout the trial.
For subject allocation, a randomization plan was generated at Randomization.com, with randomly permuted blocks in a 1:1 ratio. The subjects and the psychiatrist in charge of the assessments were kept blind to group allocation. Two collaborators not blinded to group allocation were responsible for setting the equipment parameters to provide 50% N 2 O or 100% oxygen according to the randomization plan while the psychiatrist in charge of the assessment was absent from the room. This procedure was done at the beginning and end of the 60minute inhalation period. Other measures to strengthen blinding included covering the device's digital display with a dark cloth and masking the slightly sweet odor of N 2 O by using flavored nasal masks.
Statistical analysis was performed in SPSS version 23.0. Clinical and demographic characteristics were compared through non-parametric analysis for categorical data (w 2 test), and a parametric test was used for normallydistributed nominal data (t-test for independent samples). To assess the effects of adjuvant N 2 O or placebo to antidepressant treatment, we performed repeated measures analysis of variance for HAM-D 17 scores considering the factors time, drug (N 2 O vs. placebo), and timedrug interaction. When significant time-drug interactions were detected, independent sample t-tests were applied for each evaluation point. HAM-D 17 scores were also analyzed for each group separately using repeatedmeasures analysis of variance and paired t-tests with pre-inhalation and post-inhalation data from each session. To assess the effect size of the differences found, we used Z partial square (Z 2 p ) tests and classified values according to Maroco's method.In this classification table, Z 2 p values 4 0.5 correspond to very high effect size, 0.25-0.5 to high effect size, 0.05-0.25 to medium effect size, and p 0.05 to small effect size. The same analyses were performed for BDI-II and YMRS data. The severity of suicidal ideation was assessed using the ordinal subscale of the C-SSRS and non-parametric Mann-Whitney U tests to detect differences between the two groups at each assessment point. As an additional analysis, we calculated the number of subjects in each group who fulfilled criteria for remission and therapeutic response at the end of the trial. We compared the rates of both groups using Fisher's exact test. For the HAM-D 17 , remission was defined as scores o 7 and therapeutic response as reductions X 50% against baseline scores. For the BDI-II, remission was defined as scores p 13 and therapeutic response as reductions X 50% against baseline scores.
The subjects were given a complete description of the trial and written informed consent was obtained from those who agreed to participate. The study was conducted in accordance with the terms of the declaration of Helsinki, approved by the HCFMRP-USP board of ethics (CAAE: 46352015.5.0000.5440), and registered in the Brazilian Register of Clinical Trials (; number RBR-5rz5ch).
Subjects were recruited between June 2016 and June 2018, and the trial sessions were held between August 2016 and June 2018. A total of 201 subjects were assessed for eligibility, of whom 23 were included in the final sample. Eleven subjects were randomly allocated to the placebo group and 12 were allocated to the N 2 O group. Two subjects (both female) in the placebo group did not complete the eight sessions of the trial. One subject left the trial after the first session, reporting worsening of symptoms. The other subject was excluded after the fifth session due to poor adherence to the treatment protocol because of difficulties leaving home (Figure). The 23 subjects who began the trial were included in the analysis of demographic and baseline clinical characteristics. For the analysis of outcome measures at the end of the trial, only the 21 subjects who completed the eight sessions were considered. The exclusion of these subjects did not affect group matching.
There were no significant differences between the two groups regarding age, sex, and severity of symptoms according to the HAM-D 17 (with scores corresponding to a moderate episode in both groups) or the BDI-II (with scores corresponding to a severe episode in both groups). The demographic and clinical characteristics of the subjects are presented in Table.
Significant reductions in HAM-D 17 scores occurred over time in both groups, with very high and high effect sizes in the N 2 O and placebo groups, respectively (N 2 O: F respectively). Group comparisons showed a significant time-drug interaction effect with a high effect size (F 15, 285 = 2.26; p = 0.005; Z 2 p = 0.11). Independent sample t-tests for each of the 16 assessments throughout the trial showed significant differences with the post-inhalation assessment at the fourth session, although this effect disappeared in sessions 5 and 6 until reappearing in the final two sessions, as shown in Figure. Within each session, paired t-tests showed significant reductions in post-vs. pre-inhalation scores in all sessions in both groups, except for the sixth session of the placebo group. However, considering the group means, only the N 2 O group reached the remission range, which occurred in the post-inhalation assessment of the seventh and eighth sessions. Likewise, only the N 2 O group reached means within the therapeutic response range compared to baseline scores, which occurred in the post-inhalation assessment of the third and fourth sessions, and again in the sixth, seventh, and eighth sessions. At the end of the eighth session, 75% (9 of 12) of subjects in the N 2 O group achieved remission vs. 11.1% (1 of 9) in the placebo group, a difference of 0.64 (p = 0.008; Fisher's exact test). The response rates were 91.7% (11 of 12) in the N 2 O group vs. 44.4% (4 of 9) in the placebo group, a difference of 0.47 (p = 0.046; Fisher's exact test).
Significant reductions in BDI-II scores with large effect sizes occurred in both groups (N 2 O: F 16.3369.22); however, there were no timedrug interaction effects (F 15, 285 = 1.12, p = 0.34, Z 2 p = 0.06). At the end of the trial, 66.7% of subjects in the N 2 O group (8 of 12) reached remission criteria vs. 33.3% (3 of 9) in the placebo group, with a non-significant difference of 0.33 (p = 0.2; Fisher's exact test). The response rates were 66.7% (8 of 12 subjects) in the N 2 O group vs. 55.6% (5 of 9 subjects) in the placebo group, with a non-significant difference between proportions of 0.11 (p = 0.67; Fisher's exact test).
The Mann-Whitney U test showed that the score distributions were similar for both groups in all assessments. We found no significant differences in the median suicide ideation scores between groups. None of the subjects presented suicidal behavior throughout the trial (Table).
There were also no significant differences in YMRS variation over time in either group (N 2 O: 1.4261.24 for 0.7561.29; placebo: 2.4463.00 for 1.0062.65, F 15, 286 = 1.036, p = 0.42). The temporal distribution of mean YMRS scores is shown in Figure. No subject had a total YMRS score 4 12 during the trial, which indicates the presence of manic episodes.
The mean inhalation time per session in the N 2 O group was 59.164.93 minutes in a total of 96 sessions. Somnolence, paresthesia, nausea, and headache were the most frequent adverse effects. In four of the 12 subjects in this group, temporary inhalation interruptions were required in 12 different sessions for the following reasons: nausea, vomiting, emotional discomfort, and regurgitation. Five sessions were discontinued before the end, three for the same subject (at 56, 47, and 18 minutes due to nausea and regurgitation at 52 minutes, increased blood pressure at 40 minutes, and severe headache and distress soon after beginning the last inhalation session). Other adverse effects included confusion, psychomotor retardation, emotional discomfort, hearing hypersensibility, restlessness, laughter, and difficulty keeping the nasal mask on. In the placebo group, a total of 78 sessions were performed, with a mean inhalation time per session of 59.9660.34 minutes. Adverse effects included headache, somnolence, nausea, and difficulty keeping the nasal mask on. One subject required a temporary interruption due to nausea and severe headache, and this session was ended at 57 minutes. Regarding the monitored physiological parameters, one patient in the N 2 O group had increased blood pressure in one session.
In this preliminary study, our findings show that based on HAM-D 17 scores, the antidepressant effect of N 2 O was superior to placebo. Both groups had lower HAM-D 17 scores at the end of the trial, although the reduction was greater in the N 2 O group (mean reduction of 16.66 points from baseline vs. 9.55 points in the placebo group). In addition, only the N 2 O group reached a mean score within the remission range (5.92 vs. 12.89 in the placebo group). As for the placebo group, reductions in HAM-D 17 scores vs. baseline did not fulfill the criteria for a therapeutic response. In addition to changes in mean group scores, a therapeutic response occurred in 91.7% of the N 2 O group (vs. 44.4% of the placebo group), and the remission criterion was fulfilled by 75% of the N 2 O group (vs. 11.1% in the placebo group). Both of these rates were significantly higher than those in the placebo group. These findings agree with the only published study on the antidepressant effects of N 2 O, in which 25% of the patients in the N 2 O group achieved a response (vs. 5% in the placebo group), and 15% of the patients in the N 2 O group achieved remission (vs. 0% in the placebo group).That study included only patients with treatment-resistant depressive disorder, which could explain the lower remission and response rates in both the active drug and placebo groups. It is interesting to note that the antidepressant effect was both acute and cumulative. The amplitude of in-session score reduction in the N 2 O group was greater than that of the placebo group in all sessions, demonstrating the acute effect of the drug, although differences in mean postinhalation scores between the two groups were statistically significant only in the fourth, seventh, and eighth sessions (p = 0.027, p = 0.008, and p = 0.003, respectively). On the other hand, considering pre-inhalation assessments from the second to the eighth session as a follow-up of the effects in previous sessions, we found progressive antidepressant effects over time in the N 2 O group, which demonstrates a cumulative effect, while in the placebo group this occurred only until the third session. Unlike N 2 O and ketamine, other glutamatergic modulators, such as memantine, have shown no antidepressant effects.Determining which actions differentiate ketamine and N 2 O from other glutamatergic modulators is important both for understanding the neurobiology of depressive disorders and for the development of accurate targets for new treatment agents. One study demonstrated that the antidepressant effect of ketamine is reversed by naltrexone, an antagonist of the opioid system.Furthermore, Zarate & Machado-Vieiraproposed that the antidepressant effect of N 2 O may result from its ability to increase concentrations of nitric oxide, a messenger that has been linked to antidepressant, anxiolytic, and analgesic effects. Mean BDI-II scores also decreased in the N 2 O and placebo groups, which indicates improvement in depressive symptoms (mean score reduction of 23.5 from baseline in the N 2 O group vs. 19.97 in the placebo group). Both groups achieved mean scores within the therapeutic response range. However, changes in BDI-II scores did not reflect the same intensity of symptom improvement or the superiority of N 2 O over placebo as HAM-D 17 scores. Here, a fundamental difference between the HAM-D and BDI-II should be highlighted: the first is a clinician-rated instrument, whereas the latter is a self-rating inventory, which might result in different perceptions regarding symptom severity. Discrepancies between the perceived severity reported in these two types of instruments are frequent, and several possible explanations have been discussed in the literature. Carter et al.found only a moderate correlation between symptom severity measured with clinician-rated scales vs. self-rating scales. Nevertheless, clinician-rated scales remain the standard for studies assessing antidepressant treatments. Evidence in the literature points out a tendency for patients to perceive their symptoms as more severe than clinicians, which is in accordance with our findings.As for the improvement in depressive symptoms found in the placebo group (both through HAM-D 17 and BDI-II scores), high response rates to placebo have been described in clinical trials of antidepressant treatments. Our placebo group showed results on par with previous findings, including BDI-II scores that did not differ significantly between groups.This could lead to bias, which could result in masking or underestimation of the effects of active drugs. Among the possible explanations for the placebo effect seen in our trial, we highlight the possibility that it may derive from the care and attention received by the participants or from the self-limited nature of depressive episodes. More importantly, we cannot rule out a delayed antidepressant effect in the placebo group, since only 4 weeks without dose adjustment were an inclusion requirement. Suicidal ideation improved in both groups by the end of the study according to the C-SSRS data, although with no significant difference between groups, unlike the improvement reported in a trial with ketamine.It should be noted, however, that none of the subjects presented suicidal behavior or increased suicidal ideation during the trial. According to the YMRS results, no manifestations of mania or hypomania were found following N 2 O treatment. It is important to emphasize that a diagnosis of bipolar disorder or previous history of antidepressant-induced mania or hypomania were used as exclusion criteria in this trial. Although we clinically evaluated the presence of dissociative and psychotomimetic symptoms in the sample, no standardized tests were used for this purpose. These effects could have been assessed through scales such as the Clinician Administered Dissociative States Scale or the Brief Psychiatric Rating Scale. We considered that since the inclusion of further instruments would have had a substantial impact on the already long trial sessions and, we opted to omit them. Nevertheless, according to clinical evaluation and subjective reports, dissociative and psychotomimetic effects were neither frequent nor intense among participants. Regarding drug tolerability, reports of adverse effects were more frequent in the N 2 O group; however, the 60minute inhalation session could be completed in most cases without discomfort or only minimal discomfort for the subjects. In fact, subjects who received N 2 O reported tranquility, relaxation, and a ''sense of well-being,'' considering it a pleasant experience. Only one subject required temporary interruption or early discontinuation in most sessions (six of eight sessions) due to adverse effects (nausea and vomiting). Nevertheless, the subject did not consider this to be a severe discomfort and chose to remain in the trial. Among the others, six did not require interruption or early discontinuation, and the remaining five discontinued inhalation in less than half of the sessions. The main adverse effects were somnolence, nausea, vomiting, headache, and paresthesia. No serious adverse events occurred and the aforementioned effects improved rapidly after discontinuing inhalation, with no further intervention necessary, which suggests an acceptable risk-benefit ratio for the use of N 2 O in patients with MDD, as was also reported by Nagele et al.Our study has certain limitations that should be pointed out. Since the trial had a small sample, our results do not represent definitive measures of efficacy and should be interpreted with caution until they are replicated in trials with larger samples. Furthermore, the trial duration was short (4 weeks) and we did not include post-treatment assessments or an intention-to-treat analysis. Of note, only two subjects abandoned the trial, both in the placebo group, one of whom reported worsening symptoms and another for personal reasons. Long-term follow-up trials are needed to establish whether the potential benefits of N 2 O for depression persist over time. In addition, due to logistical issues that involved coordinating the needs of the subjects and research collaborators and the availability laboratory environment, we were unable to perform the trial sessions at the same time for all subjects. Data was not collected regarding treatment-resistant depression, but the high response rates in both groups suggest that only a few patients were treatment resistant. Finally, adverse events associated with the inhalation of N 2 O may have affected the blinding procedures and revealed the use of active drugs to the subjects and researchers. With this in mind, future investigations should consider the use of active drug controls. The most commonly used depression rating scales in clinical trials are not appropriate to detect changes in symptom severity that occur within hours or a few days. The HAM-D was developed to assess symptoms over a period of 1 week, while the BDI-II was designed to cover a period of 15 days. For example, both instruments include items related to sleep and weight that do not change rapidly. In order to circumvent this limitation, we instructed the subjects to repeat answers about sleep, appetite, and weight in scales applied within the same session, that is, before and after inhalation. A visual analogue scale or even the 6-item-Hamilton Depression Rating Scale might have been more appropriate to capture faster mood changes within the same session, which should also be kept in mind by future investigators. Finally, up to the time of writing the initial protocol of this study, we could find no data in the literature about the best concentration of N 2 O to be used in patients with depressive disorders. We chose to use a 50% concentration to avoid deep sedation and preserve participant safety in an outpatient research setting. Forthcoming studies should assess the effects of different concentrations to determine the most effective dose with the fewest adverse effects. Likewise, the frequency of N 2 O administration should also be the object of future research. It is worth mentioning that administering N 2 O/oxygen with portable devices is relatively easy and can be performed by non-anesthesiologists after proper training.Finally, we were unable to measure the expired fraction of carbon dioxide and N 2 O since the nasal masks used in the trial do not allow coupling a capnograph or gas analyzer, which could provide important evidence about the best dose to be used. In conclusion, this is the second trial of N 2 O as a potential treatment for depression and the first to apply semiweekly doses for a month in only non-treatmentresistant patients. Further studies with larger samples, a clear distinction between treatment-resistant and nontreatment-resistant patients, and an intention-to-treat analysis are needed to determine whether N 2 O is an effective antidepressant alone or as an adjuvant therapy. Our results show that 50% N 2 O administered twice a week for one month in association with antidepressant treatment in patients with persistent symptoms led to reductions in depressive symptoms as measured by the HAM-D 17 , but not the BDI-II. In addition, 91.7% of the N 2 O group achieved a therapeutic response and 75% fulfilled the criteria for remission according to the HAM-D 17 , and 66.7% achieved response or remission according to the BDI-II. These data suggest that 50% N 2 O has stronger antidepressant effects as an adjunctive treatment than antidepressants alone in patients still symptomatic after at least 4 weeks of oral pharmacotherapy.