Mexico, prevalence reported for major depressive disorder (MDD) is of 7.2%. It is currently in the top 5 causes of disability worldwide. One third of patients will not achieve remission after two treatments, being classified as treatment-resistant. In a neurochemical level, evidence shows dysregulation of the excitatory neurotransmitter Glutamate in patients with MDD. Chronic stress has been related to this dysregulation. Ketamine, has shown to regulate glutamatergic neurotransmission, and specially promote the release and production of neurotrophic factors key in the causes of MDD inhibited by glutamate dysregulation), and allow restoration of areas affected.

Clinical studies of ketamine in MDD have shown robust, durable , and rapid effects (during the first 4-24 hours), allowing a great opportunity for patients who do not achieve benefits from antidepressants or patients with suicidal ideation . These results have been reported in metaanalysis.

To our knowledge, there are no studies using Magnetic Resonance Spectroscopy, in areas related to MDD, after a series of ketamine administrations, which we think may show changes after this chronic administration and explain its antidepressant properties.

Goals: Provide clinical evidence of responseas well as a neurological basis or biomarker of response to a series of ketamine infusions.

1. Background 1.1 Major depressive disorder (MDD) MDD is a clinical syndrome characterized by the presence of low modo, anhedonia, appetite and weight changes, sleep disturbances, psychomotor alterations, fatigue, guilt and low self-esteem, ideas related to death or suicide, and concentration difficulties.

MDD represents one of the first causes of disability worldwide. In Mexico, prevalence is estimated in 7.2% of the population. In accordance to the largest clinical trial of MDD, the STAR*D (Sequenced Treatment Alternatives to Relieve Depression), up to one third of the patients will not achieve remission after 4 treatment strategies. These obligates research of new treatments for MDD and treatment-resistant depression TRD.

1.2 Treatment-resistant Depression (TRD) There is a lack of consensus to define TRD, with multiple criteria used by distinct authors. However, the most used definition is the failure to achieve response or remission after two consecutive treatments at an adequate dose and duration, considering the last episode.

1.3 Physiopathology of MDD. Historically, serotonin and noradrenaline disturbances in production, metabolism and reuptake have been implicated in MDD, as well as dopamine. However, this hypothesis seems insufficient in explaining the lack of immediate response, and the lack of response of up to one third of patients.

It is necessary to understand MDD as a multifactorial disorder (biological, psychological, and environmental agents). At a genetic level, some polymorphisms have been related to the appearance of MDD, such as the gen associated with the glucocorticoid receptor NR3C1, the one related to the monoaminooxidase-A, and the one related to the glucogen kinase-synthase 3. Heredability for MDD has been calculated around 37%.

In a molecular basis, there are three principal factors implicated in the genesis of MDD: neurotrophic factors such as brain-derived neurotrophic factor (BDNF), proinflammatory cytokines (interleukin-1 beta, 6, tumoral necrosis factor alpha), and a dysregulation in the hypothalamus-hypophysis-adrenal axis.

Anatomically speaking, the majority of neuroimaging studies converge in the existence of a hyperactive amygdala, ventral striatum and medial prefrontal cortex to negative stimuli. Among regions of major interest are the amygdala, prefrontal cortex, the cingulate gyrus in its subgenual area and anterior or pregenual area (pgACC), ventral striatum, medial thalamus, posterior cingulate gyrus and anterior insulae.

1.5 Glutamate and GABA in MDD y GABA Glutamatergic and GABAergic dysfunction in affective disorders has increased interest in the last years, evidenced by clinical neuroimaging studies that demonstrate disturbances in such systems in patients with MDD, animal models of stress, and the role of glucocorticoids in the glutamatergic regulation secondary to chronic stress, as well as studies about the action of antidepressants in these systems.

1.6 Magnetic Resonance Spectroscopy H1-MRS in MDD H1-MRS studies coincide with the diminution of glutamate, glutamine and Glx (a composite measure of the previous two) in patients with MDD compared to controls in the pgCCA. Such finding has been correlated with the severity of MDD and anomalous connectivity with the anterior insulae.

Disturbed GABA neurotransmission has been also found in the occipital cortex in patients with MDD and TRD, suggesting a possible biomarker for differential diagnosis. Specifically in TRD patients, Glx in pgACC have been found to be altered, however more studies are needed.

1.7 pgACC in MDD pgACC in MDD refers to the rostral portion of the ACC that englobes the anterior portion of the corpus callosum, nominated sometimes as the medial prefrontal cortex. It comprises Brodmann's areas 24, 25, 32 and 33. The pgACC corresponds to the area 33. Through models of meta-analytic connectivity, its role in the production of interception and subjective feelings, coordinating responses appropriate to internal and external events along with the insulae, and its involvement in the representation of interoceptive information have been confirmed. It seems also to represent the area in with the distinction of cognition and affect takes place39. Such function is supported by evidence of structures connected to the pgACC (lateral and ventromedial prefrontal cortices, and limbic regions).

pgACC activity has been shown to be a predictor of response of some depression treatments such as pharmacological treatments and transcranial magnetic stimulation.

Functional MRI (fMRI), has demonstrated an increased connectivty between pgACC and dorsolateral prefrontal cortex, as well as a diminution in the connectivity of the pgACC and caudate. Such findings may be explained by an intense cognitive control over emotional regulation in MDD patients.

There is evidence of abnormal glutamatergic abnormalities in cerebral activity in resting state in MDD patients, finding a correlation between lower glutamate levels in the pgACC and a diminished connectivity of the same area with the anterior insulae compared to controls through H1-MRS and BOLD techniques.

Because of these findings, it is of great interest for the investigators to study this region in relation to ketamine interventions as an antidepressant therapy.

1.9 Ketamine as an antidepressant Ever since the first clinical study reporting the use of ketamine as an antidepressant for TRD patients, showing a rapid (in hours) and robust (in days) response after a unique administration, the literature has grown exponentially. However, mechanisms of action remain inconclusive.

Its antidepressant properties have been vinculated by ketamine's capacity to stimulate the release and expression of BDNF. Contrary to the result of chronic stress, inhibiting these results, ketamine seems to stimulate it. This molecule is involved in the modulation of neuroplasticity, specifically in the prefrontal cortices. Another of the explanations in a molecular level has been its regulation in the glycogen kinase-synthase 3 (GSK3), required for the pruning and synaptic reconsolidation50. Finally, ketamine has been shown to regulate the lateral habenula (implicated in MDD), probably also explaining its role in the down-regulation of monoamines.

Its clinical effects make ketamine a candidate to solve problems related to MDD in public health, confirmed by various systematic reviews and meta-analysis.

There are clinical trials reporting the efficacy of repeated administrations of ketamine (from 7 days up to 83 days after the last administration) with IV ketamine and intranasal esketamine. However, studies vary in the number of interventions, intervals, conditions to continue treatment, times of measurements, follow-up, making it impossible to obtain standardized results. Also, repeated doses have not been reported with H1-MRS technique to explore if there are durable changes after chronic administration of subanesthetic doses of ketamine in MDD.

1.10 Glutamatergic and GABAergic chances in pgACC before ketamine administration H1-MRS data concerning glutamate levels in healthy subjects show that after ketamine administration, there is a significative increase in glutamate in the pgACC, correlating with psychopathology scales such as the PANSS, supporting the idea that ketamine exerts an important effect in the neurotransmission related to its mechanism of action in this region of interest. Such findings propose the difference in this region of interest pre and postinfusion of ketamine and represent an important antecedent for the current proposal. In a similar way, in healthy subjects, hippocampal augments of Glx and a decreased of the fronto-temporal connectivity and temporo-parietal after the administration measured at 10 minutes after ketamine administration has been shown.

When measuring glutamate, Glx and GABA in the pgACC after ketamine in MDD patients, there have been mixed results. Some authors have found a significative increase of the same, correlating with clinical response when measuring during the infusion53. However, using major tesla MRI and measuring the effect of ketamine in glutamate at 24 hours after administration show no differences against placebo. Both findings are discussed concerning the time of measure, concluding that there is a rapid and robust increase, but a transitory one. However these changes do not persist.

To our knowledge no H1-MRS studies after a series of ketamine infusions to know if there are durable changes in glutamate of GABA levels that may explain the durable antidepressant effects of ketamine.

1.11 Experience in the National Institute of Neurology and Neurosurgery (NINN), Mexico Clinical experience with ketamine as an antidepressant and augmentation agent to conventional antidepressants has shown similar clinical results as reported previously, and even have shown longer times until relapse. There is extensive experience in the utilization of H1-MRS in other disorders. The investigators consider that this institution is ideal for the development of the current proposal.

2.1 Key questions

1. Will patients with TRD have a clinical response (50%) after the infusion of ketamine as measured by the Hamilton Depression Rating Scale (HDRS) and the Montgomery-Asberg Depression Rating Scale (MADRS) during the first 24 hours, and different to patients receiving placebo?
2. Will TRD patients presenting response, present a significant increase in Glutamate and GABA in the pgACC measured by H1-MRS 24 hours after the last intervention, compared to the basal measure?
3. Will this response, if achieved, be different than changes in Glutamate and GABA in the pgACC among patients receiving placebo?

3. METHODOLOGY 3.1 Design A double-blind randomized clinical trial will be performed 3.2 Sample During the sample selection, inclusion and exclusion criteria defined afterwards will be used. Sampling will be non-probabilistic in a consecutive case manner. Patients will participate voluntarily after informed consent is achieved.

Subjects treated in the NINN will be divided in two groups:

1. Subjects with TRD, receiving ketamine infusions from start.
2. Subjects with TRD, receiving placebo (saline solution 0.9%), and afterwards

3.3 Procedure

1. Sampling.

2. Previous evaluation:

   1. Psychiatric evaluation (HDRS-17, MADRS).
   2. Medical evaluation

3. Randomization

4. Basal 1H-MRS for GABA:

5. Basal 1H-MRS for Glutamate:

   Similar to the GABA acquisition.

6. Interventions

   1. All interventions will be done as an out-patient basis by a psychiatrist.
   2. A 0.5 mg/kg infusion of ketamine or placebo IV along 40 minutes will be performed. Vigilance will be strict (vital signs, adverse effects, subjective experience, clinimetry).
   3. After every intervention, the patient will be observed for 1 hour or more if considered necessary by the clinician, returning to their normal routine afterward.
   4. Such intervention will be done twice weekly in a prior of 4 weeks (day 1, 4, 8, 11, 15, 19, 23, and 27) for a total of 8 infusions in patients receiving ketamine.

   i. In case of having received placebo, patients will then receive 8 infusions of ketamine.

   ii. In case of having received ketamine, they will continue until completing 8 infusions, and then receive 4 infusions of placebo.

7. Posterior evaluation

   1. Psychiatric evaluation as explained earlier at 4, 24, 72 hours and weekly up to 12 weeks after the last infusion of relapse.
   2. Glutamate and GABA in pgACC measures with the parameters after 10 minutes of starting the infusion, 24 hours after, and 1 week after the last infusion of ketamine or placebo.

8. Follow-up a. 12-week follow-up after last intervention or relapse, after which patients will end participation and their care will continue as usual.

Inclusion Criteria:

1. Age: 18-65 years
2. MDD diagnosis as provided by DSM-5 criteria
3. TRD as defined by failure to achieve response to two consecutive antidepressant therapies at an adequate dose and duration
4. Patients approving inclusion by signing the informed consent

Exclusion Criteria:

1. Comorbidity with other mental and neurological disorders (except generalized anxiety disorder)
2. Substance use disorders at least 3 months prior to enrollment
3. Evidence of structural abnormalities in basal MRI
4. Pregnancy or lactation
5. Hypersensitivity to ketamine
6. Cardiac failure
7. Personal history of psychosis
8. First-degree relatives with history of psychosis
9. Uncontrolled close-angle glaucoma
10. Neurological disease (present)
11. Uncontrolled Hypertension
12. Contraindications for the realization of H1-MRS.

Undecided.

Double-blind