Assignment: Evidence-Based Project, Part 1: Identifying Research Methodologies

REGULAR ARTICLE

Effects of electroconvulsive therapy in the systemic inflammatory balance of patients with severe mental disorder

Miquel Bioque, MD, PhD,1,2,3,4† Karina S. Mac-Dowell, MSc, PhD,3,5† Ana Meseguer, RN,1,3 Elisabet Macau, RN,6

Ricard Valero, MD, PhD,7,8 Eduard Vieta, MD, PhD,2,3,4,9 Juan C. Leza, MD, PhD3,5* and Miquel Bernardo, MD, PhD 1,2,3,4*

Aim: There is a great interest in the role of the immune sys- tem and the inflammatory balance as key mechanisms involved in the pathophysiology of severe mental disorders. Previous studies have indicated that electroconvulsive ther- apy (ECT) produces changes in certain inflammatory media- tors or in the immune system response. This study aimed to explore the effects of ECT on the nuclear transcription factor κB (NFκB) pathway, a main regulatory pathway of the inflammatory/immune response.

Methods: Thirty subjects with a severe mental disorder receiving treatment with ECT in our center were included. Thirteen systemic biomarkers related to the NFκB pathway were analyzed right before and 2 h after a single ECT session.

Results: An ECT session significantly decreased the expression of NFκB (P = 0.035) and of the inducible nitric oxide synthase (P = 0.012), and the plasma levels of nitrites (P = 0.027), prostaglandin E2 (P = 0.049), and 15-deoxy- PGJ2 (P < 0.001). Decrease in plasmatic levels of nitrites was

greater in females than in males (P = 0.021). A positive corre- lation between the ECT stimulus load and changes in the expression of NFkB was found (P = 0.036). Thiobarbituric acid reactive substance levels were decreased in treatment responders and increased in non-responders (P = 0.047).

Conclusion: Our study shows the effects that a single ses- sion of ECT produces on a canonical regulatory pathway of the inflammatory/innate immune system and the inflamma- tory balance. These biomarkers could be useful as treatment response targets and could help to clarify the biological basis of ECT action. These findings warrant greater attention in future investigations and in the translational significance of these data.

Keywords: biomarkers, ECT, electroconvulsive therapy, immune

system, inflammation.

http://onlinelibrary.wiley.com/doi/10.1111/pcn.12906/full

Electroconvulsive therapy (ECT) is considered an effective and safe treatment for patients with a severe mental disorder, being used mainly in certain cases of major depression, bipolar disorder (BD), and schizophrenia-related disorders resistant to pharmacological treatments.1–5 Despite the extensive clinical experience accumulated with this technique and all the scientific knowledge acquired about its mechanisms of action at the molecular level, this field of knowledge requires more research on its biological basis to better understand its mechanistic basis and in order to help clinicians in decision-making.

Moreover, in recent years we have witnessed a renewed interest in the role of certain changes in the innate immune system and inflammatory balance as key mechanisms involved in the pathophysi- ology of these mental disorders.6,7 Thus, several meta-analyses have described inflammatory mediator alterations – such as certain cyto- kines – in patients with depression,8 BD,9 and schizophrenia.10

One of the main actors regulating the immune system response is the activation of the nuclear transcription factor κB (NFκB) path- way.6 This prototypic inflammatory pathway is mainly activated in the unspecific innate immune response through toll-like receptor 4 (TLR4) in certain scenarios of biological or psychosocial stress, cytokine release, damage-associated patterns, or as a response to lipo- polysaccharide from gram-negative bacteria.11 Through recruiting adapter proteins, such as the myeloid differentiation factor 88 (MyD88) and the TIR-domain-containing adapter-inducing interferon-β (TRIF), NFκB is activated.12 NFκB acts on the gene pro- moters of inducible forms of the enzymes nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), increasing their expression. The overactivation of these enzymes can produce an accumulation of oxidative and nitrosative mediators (i.e., nitric oxide, peroxynitrite anion, and prostaglandins, such as prostaglandin E2 [PGE2]), which

1 Barcelona Clínic Schizophrenia Unit, Neuroscience Institute, Hospital Clínic de Barcelona, Barcelona, Spain 2 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 3 Centro de Investigación Biomédica en Red en Salud Mental (CIBERSAM), Madrid, Spain 4 Department of Medicine, University of Barcelona, Barcelona, Spain 5 Department of Pharmacology & Toxicology, Faculty of Medicine, Universidad Complutense de Madrid, Instituto de Investigación I+12 y IUIN, Madrid, Spain 6 Psychiatry Department, Neuroscience Institute, Hospital Clínic de Barcelona, Barcelona, Spain 7 Anesthesia Department, Hospital Clínic de Barcelona, Barcelona, Spain 8 University of Barcelona, Barcelona, Spain 9 Barcelona Bipolar Disorder Program, Psychiatry Department, Neuroscience Institute, Hospital Clínic de Barcelona, Barcelona, Spain * Correspondence: Email: bernardo@clinic.cat; jcleza@med.ucm.es † The first and second authors contributed equally to this work.

© 2019 The Authors Psychiatry and Clinical Neurosciences © 2019 Japanese Society of Psychiatry and Neurology

Psychiatry and Clinical Neurosciences 73: 628–635, 2019

628

PCNPsychiatry andClinical Neurosciences

can cause the depletion of endogenous antioxidant defenses and attack membrane phospholipids causing cell damage in a process known as lipid peroxidation, a byproduct of which can be detected by thiobarbituric acid reactive substances (TBARS). On the other hand, some endogenous counterbalancing mechanisms, activated in response to an inflammatory/immune stimulus, have also been described, including the activation of the gamma isoform of the per- oxisome proliferator activated receptor (PPARγ). Besides, several COX-2 derived products, such as the prostaglandin 15-deoxy-PGJ2 (15d-PGJ2), act as endogenous anti-inflammatory agents by targeting PPARγ, and being involved in the endogenous compensatory mecha- nism regulating the inflammatory process.13 See a detailed scheme of the whole pathway in Figure 1.

Previous works have identified differences in the expression of certain components of this pathway comparing subjects affected by a serious mental disorder with matched healthy controls. These studies included nearly 400 subjects with schizophrenia-related disorders, 674 with BD, 148 with major depressive disorder (MDD), and 643 healthy subjects. In particular, patients in an early phase of a psy- chotic disorder showed increased levels of pro-inflammatory NFκB, iNOS, and COX-2, while the anti-inflammatory inhibitory subunit of NFκB, 15d-PGJ2, and PPARγ expression and transcriptional activity were lower.13,14 In patients with established schizophrenia, NFκB, iNOS, COX-1, COX-2, TLR4, and PGE2 expression were found to be elevated,15–18 while the anti-inflammatory prostaglandin 15d-PGJ2 and its nuclear receptor PPARγ were decreased.19 A recent study found higher levels of COX-1 in BD patients compared with healthy controls,16 while TLR4 genetic variations have been associated with this disorder.20 Finally, patients with MDD showed higher expression of TLR421,22 and elevated TRIF and MyD88.23 A study with post- mortem brain samples also showed an altered TLR4 immune response in the brains of subjects with MDD.24

Following this hypothesis, a successful treatment would help to normalize the inflammatory imbalance that some of these individuals present.14,25

Previous studies have indicated that an ECT treatment associates certain changes in inflammatory mediators or in the immune system response in patients with various mental disorders, principally major depression and schizophrenia. Most of these studies are heterogeneous

in the number of ECT sessions received (one vs 9–12), the biomarkers measured, and the pathways implicated.26,27

In patients with MDD (see review by Guloksuz et al.26), increased activity of the NK cells28–30 and interleukins (IL)-6 and IL- 1β have been described after an ECT session.31–33 Lehtimaki et al. reported that the elevation of both IL normalized at 24 h.31 Fluitman and colleagues also showed that an ECT session produced a decrease in interferon-γ production and an acute increase in the total number of leukocytes, monocytes, granulocytes, and NK cells,32 returning to baseline at 30 min. They did not find an additive effect of these changes with successive ECT sessions. Finally, Arts and colleagues described an increase in serum levels of the S100B protein, a marker of glial injury, 1 h and 3 h after an ECT session,33 but another study did not find any changes.34

In addition to these acute effects to a single ECT session, long- term effects of a treatment with repeated sessions of ECT have been described in patients with MDD (see review by Guloksuz et al.26). In a recent study, Järventausta and colleagues showed how high levels of IL-6 normalized only in patients who remitted clinically after several sessions of ECT.35 Rush et al. did not find this normalization in IL-6 or transforming growth factor (TGF)-β levels after obtaining a clinical response to several sessions of ECT.36 Albrecht et al. reported a reduction in phytohemagglutinin and concanavalin A in T lymphocyte cultures.28 Another study showed the activation of the immune system mediated by an increase in the number of lymphocytes expressing activation antigens (CD25 and CD38).29 The increase in NK cell activity in the acute phase was not sustained after completing the treatment with several sessions of ECT.29,30,32 Hestad et al. reported that high levels of tumor necrosis factor (TNF)-α normalized after a period of treatment with ECT, but not if the treatment was exclusively with drugs.37 After completing 12 sessions of ECT, they found a decrease in the signaling of proinflammatory cytokines IL-5 and eotaxin-3, together with an increase in TNF-β.38 Finally, several stud- ies have shown that S100B concentrations in serum and cerebrospinal fluid are not altered after several sessions of ECT.33,34,39

There is a single study in patients with resistant schizophrenia,27

in which after nine sessions of ECT the authors found that an increase in the anti-inflammatory response was demonstrated by increases in IL-4 and TGF-β, without altering the activation of myeloperoxidase

BIOLOGICAL/PSYCHOSOCIAL

STRESS

PBMC (cellular

machinery)

PLASMA (soluble mediators)

NUCLEUS

CYTOKINE

RELEASE

TLR4

MyD88 TRIF

NFκB

iNOS

NITRITES

CELL DAMAGE

OXIDATIVE/NITROSATIVE STRESS

LIPID PEROXIDATION PGE 2

COX-2

15dPGJ 2

PPARγ

LIPOPOLYSACCHARIDE

Fig.1 Detailed scheme of the nuclear tran- scription factor κB (NFκB) pathway. Proinflammatory components are highlighted in blue and solid lines; anti- inflammatory pathways in magenta and dotted lines. COX-2, cyclooxygenase-2; 15d-PGJ2, 15-deoxy-prostaglandin J2; iNOS, inducible nitric oxide synthase; MyD88, myeloid differentiation factor 88; PBMC, peripheral blood mononuclear cells; PGE2, prostaglandin E2; PPARγ, gamma isoform of the peroxisome proliferator activated receptor; TLR4, toll- like receptor 4; TRIF, TIR-domain- containing adapter-inducing interferon-β.

Psychiatry and Clinical Neurosciences 73: 628–635, 2019 629

PCNPsychiatry andClinical Neurosciences Effects of ECT in inflammatory balance

and NFkB. The same group had previously shown that treatment with ECT did not affect parameters of oxidative stress in patients with schizophrenia, neither after one nor after nine treatment sessions.40

As far as we know, there are no studies that have analyzed the effect of ECT related to inflammatory changes in BD.

Taking into account this heterogeneous background (in the bio- markers studied, clinical setting, and timing of the disease course), this study aimed to explore the effects of a single ECT session on the inflammatory balance in a selected group of patients with a severe mental disorder by studying the main intercellular (citoplasmatic and nuclear) and intercellular components of the canonical innate immune response and the inflammatory/oxidative consequences of their over- activation. It also aimed to explore whether there are differences in these effects depending on clinical variables of the patients and parameters related to ECT administration.

Methods Subjects Thirty subjects were recruited according to the following criteria: (i) patients who attended an ECT session in the Hospital Clínic de Barcelona following the usual practice in this center; (ii) patients who met the DSM-5 criteria for a severe mental disorder (MDD, BD, schizophrenia, or schizoaffective disorder); (iii) patients aged 18 to 70 years; and (iv) patients able to sign the informed consent.

The exclusion criteria were: (i) being pregnant; (ii) being in treat- ment with anti-inflammatory drugs (including steroids), antioxidants, antibiotics, or immunological therapies; (iii) presenting fever (>38�) or leukocytosis (>10 000 leukocytes/mm3) in the previous 24 h; (iv) having received a vaccine in the previous 4 weeks; and (v) presenting a history of neurological diseases, a history of trau- matic brain injury with loss of consciousness, mental retardation, or generalized developmental disorders.

Members of the research team informed all the potential candi- dates who came to receive an ECT session during the period of recruitment about the study and obtained the informed consent from all the participants. In an interview prior to the ECT session, and together with the medical records, the sociodemographic and clinical data of the patients were collected. The study had been approved by the Ethics Committee of the Hospital Clínic de Barcelona (record HCB/2017/0369).

ECT treatment All treatment stimuli were administered using a MECTA SPEC- TRUM 5000Q device (MECTA, Lake Oswego, OR, USA). Electro- encephalographic (EEG) and motor seizure manifestations were monitored to ensure that an adequate ictal response occurred (>20 s) or to detect prolonged seizure activity. Patients were continuously monitored (noninvasive arterial blood pressure, electrocardiogram, and heart rate and pulse oximetry) using a PHILIPS MP-20 Anesthe- sia (PHILIPS, Boeblingen, Germany). Succinylcholine (30–120 mg), atropine (0–1 mg), and sodium thiopental (75–450 mg) were used for general anesthesia. Ventilation was supported with a facemask at high oxygen concentration before and after the stimulus. The characteris- tics of the electrical stimulus were automatically recorded by the MECTA EMR software connected to the stimulator. Patients could be in any ECT regimen (acute: first 6–12 ECT sessions; continuation: first 6 months of treatment after acute regimen; maintenance: after first 6 months).

Demographic characteristics of the patients (age, sex), diagnosis, ECT regimen (acute, continuation, or maintenance), ECT electrode placement, EEG time of convulsion, and total number of lifetime ECT sessions were recorded.

All medical records were reviewed to determine patients’ responses to ECT treatment. Adapting to our sample the methodology used in previous studies,41 we scored the Clinical Global Impressions (CGI)-Improvement scale of all participants in the studied episode.42

CGI-I scores of 1 and 2 (very much improved and much improved)

were considered ‘responders,’ while scores of 3 or more (from mini- mally improved to much worse) were labeled as ‘non-responders.’

Sample collection Venous blood samples (10 mL) were collected between 08:30 hours and 09:30 hours after overnight fasting and 2 h after the stimulation. Samples were maintained at 4�C until preparation after approximately 1 h. Blood tubes were centrifuged (641 g × 10 min, 4�C). The resul- tant plasma samples were collected and stored at −80�C. The rest of the sample was 1:2 diluted in culture medium (RPMI 1640, LifeTech, Merck KGaA, Darmstadt, Germany) and a gradient with Ficoll-Paque (GE Healthcare, Madrid, Spain) was used to isolate mononuclear cells by centrifugation (800 g × 40 min, room temperature [RT]). A peripheral blood mononuclear cells (PBMC) layer was aspired and resuspended in RPMI and centrifuged (1116 g × 10 min, RT). The supernatant was removed and the mononuclear cell-enriched pellet was stored at −80�C. Once the whole sample recruitment was fin- ished in the Hospital Clínic de Barcelona, all samples were sent to Universidad Complutense de Madrid for subsequent determinations.

Biochemical measurements in plasma Prostaglandin levels

Plasma levels of COX by-products PGE2 and 15d-PGJ2 were mea- sured by a commercially available enzyme immunoassay following the manufacturer’s instructions (PGE2 ELISA Kit and 15-deoxy- Δ12,14-PGJ2 ELISA Kit, Enzo, Lausen, Switzerland). The sensitivity of the assay for PGE2 was 13.4 pg/mL; intra- and interassay coeffi- cients of variation (CV) were 5.8% and 5.1%, respectively, and for 15d-PGJ2 the sensitivity was 36.8 pg./mL; intra- and interassay CV were 5.7% and 13%, respectively.

Lipid peroxidation

Lipid peroxidation was determined by TBARS assay (Cayman Europe, Tallinn, Estonia), based on the reaction of malondialdehyde and thiobarbituric acid under high temperature (95�C) and acidic con- ditions. Intra- and interassay CV were 5.5% and 5.9%, respectively.

Nitrites

NO−2, the final and stable product of nitric oxide, was measured using the Griess method. Briefly, in an acidic solution with 1% sulfa- nilamide and 0.1% N-(1-Naphthyl)ethylenediamine, nitrites convert into a pink compound that is photometrically calculated at 540 nm in a microplate reader (Synergy 2; BioTek, Bad Friedrichshall, Germany).

Measurements in PBMC To carry out all biochemical determinations on PBMC, samples were first fractionated in cytosolic and nuclear extracts, using a modified procedure previously described.13 Protein levels were measured using the Bradford method based on the principle of protein–dye binding.

Determination of proinflammatory p65 NFκB subunit and anti- inflammatory PPARγ activity were carried out in nuclear extracts from PMBC using commercially available enzyme immunoassays NFκB (p65) and PPARγ transcription factor assay (Cayman Europe, Tallinn, Estonia) following the manufacturer’s instructions.

On the other hand, inflammatory enzymes and elements of TLR4 pathways were measured in cytosolic extracts by the western blot method.

Western blot analysis

After determining and adjusting protein levels, cytosolic and nuclear extracts were mixed with loading sample buffer and 15 μg was loaded into an electrophoresis gel. Once separated on the basis of molecular weight, proteins from the gels were blotted onto a nitrocellulose mem- brane with a semi-dry transfer system (Bio-Rad, Madrid, Spain) and were incubated with specific antibodies: (i) iNOS, rabbit polyclonal

Psychiatry and Clinical Neurosciences 73: 628–635, 2019630

Effects of ECT in inflammatory balance PCNPsychiatry andClinical Neurosciences

antibody dilution of 1:750 in 1% BSA (sc651; Santa Cruz Biotech- nology, Dallas, TX, USA); (ii) COX-2, goat polyclonal antibody dilu- tion of 1:750 in 2.5% BSA (sc-1747; Santa Cruz Biotechnology); (iii) TLR4, rabbit polyclonal antibody dilution of 1:750 in BSA 1% (sc10741; Santa Cruz Biotechnology); (iv) TRIF, rabbit polyclonal antibody dilution of 1:1000 in TBStween (ab13810, abcam); (v) MyD88, rabbit monoclonal antibody dilution of 1:1000 in TBStween (ab133739, abcam); (vi) β-actin mouse monoclonal in a dilution 1:10000 (A5441, Sigma, Madrid, Spain). Proteins were rec- ognized by the respective horseradish peroxidase-linked secondary antibodies and visualized using an Odyssey Fc System (Li-COR Bio- sciences, Lincoln, NE, USA) and quantified by densitometry (NIH ImageJ software; <https://imagej.nih.gov/ij/>). Values were normal- ized to the loading control (β-actin, the blots are shown in Fig. 2). All western blots were performed at least three times in separate assays.

Statistical analysis Differences in biomarker expression before and after the ECT session were assessed using a paired t-test on those variables where distribu- tion met the assumption of normality in the Kolmogorov–Smirnov (with Lillierfors correction) test. For the rest of the biomarkers, a non- parametric Wilcoxon signed rank test was used. A value of P < 0.05 was taken to be statistically significant in all analyses.

We carried out a series of sub-analyses with the aim of exploring whether the changes in the expression of inflammatory biomarkers were associated with some of the sociodemographic, clinical, or treat- ment characteristics with ECT. To limit the number of post-hoc ana- lyses, we only explored the changes in those biomarkers with significant changes in the activity. Thus, a mixed between/within- subjects analysis of variance was conducted to assess the impact of categorical variables (sex, diagnosis, ECT regimen, electrodes place- ment, response/non-response) on the expression of these biomarkers. The relations between the biomarker changes and continuous vari- ables were investigated using the Pearson correlation coefficient. Being an exploratory analysis, corrections were not made for multiple comparisons.43

Data were managed and analyzed with the IBM SPSS Statistics v.23 (SPSS, Chicago, IL, USA).

Results Demographic/clinical characteristics and ECT parameters from the 30 participants in this study are presented in Table 1. Eighty percent of the subjects were in the continuation or maintenance ECT program, having received a mean of around 60 ECT sessions. No incidences were recorded during the ECT procedures in these patients.

Table 2 summarizes the differences in the biomarker expression between baseline and 2 h after the ECT session. ECT decreased the expression of NFĸB, iNOS, nitrites, PGE2, and 15d-PGJ2 activity in a statistically significant level (P-value < 0.05). A graphic representa- tion of the main results is summarized in Figure 2.

We carried out a series of exploratory sub-analyses to detect whether the significant changes in NFKB, iNOS, nitrites, PGE2, and 15d-PGJ2 expression were associated with certain sociodemographic, clinical, or ECT treatment characteristics. We found that the decrease in nitrites plasmatic levels was greater in females compared to males (1.41 vs 0.25 μM; F = 5.977, P = 0.021). We also found a positive correlation between the ECT stimulus load and changes in the expres- sion of NFĸB (r = 0.385, P = 0.036). Finally, we found differences in the changes of PGE2 and 15d-PGJ2 levels regarding the ECT regimen (acute, continuation, or maintenance). In the case of PGE2, patients under the continuation regimen showed a mean increase of 747.5 pg./ mL for this biomarker, while the acute and maintenance ECT regimens were associated with a decrease (−1556.62 and −765.12 pg./mL, respectively; F = 0.359, P = 0.041). Regarding 15d-PGJ2 levels, all three regimens were associated with a decrease of this anti- inflammatory marker, but it was more accentuated in the acute regimen

than in continuation or maintenance (−9.01, −6.72, and − 9.26 pg./ mL, respectively; F = 4.879, P = 0.016; F = 4.879, P = 0.016).

We searched for significant differences between responders and non-responders on the analyzed biomarkers expression. We found that TBARS levels were decreased after the ECT session in patients who were considered responders, while this lipid peroxidation determina- tion was increased in non-responders (F = 4.3, P = 0.047; Fig. 3).

We did not find significant differences in the expression of any of these biomarkers associated with diagnosis, ECT electrode placement, EEG time of convulsion, or total number of lifetime ECT sessions.

Discussion Our findings highlight the effects that a single ECT session produces on the inflammatory balance, concretely on the NFĸB pathway, a canonical regulator of the inflammatory/immune system response. Two hours after an ECT session, we found a statistically significant decrease in the levels of various proinflammatory components of this pathway: NFĸB, iNOS, nitrites, and PGE2. Overall, these findings suggest that ECT produces a rapid systemic anti-inflammatory effect on patients with severe mental disorders. ECT treatment was also associated with a significant decrease of the expression of the 15d- PGJ2, an endogenous anti-inflammatory regulator. Furthermore, patients considered as responders to ECT treatment showed decreased TBARS levels, while these levels increased in non-responders. To our knowledge, this is the first time that such effects on the inflammatory balance have been reported in a sample of patients with a severe men- tal disorder after receiving an ECT session, when such a number of intra- and intercellular components of this innate-immune and inflam- matory pathway are measured.

These results are in line with most previous studies that have examined the inflammatory effects of acute treatment with ECT, find- ing that this treatment has a relatively fast impact on the systemic inflammatory and immune response, taking into account that there are

Table 1. Demographic, clinical characteristics, and electroconvulsive therapy parameters

n = 30

Age 52.97 (16.81) Sex

Female 19 (57.7%) Male 11 (33.3%)

Diagnosis Schizophrenia 6 (18.2%) Schizoaffective disorder 7 (21.2%) Bipolar disorder 13 (39.4%) Major depressive disorder 4 (12.1%)

ECT regimen Acute 6 (20.0%) Continuation 7 (23.3%) Maintenance 17 (56.7%)

ECT application Bitemporal 27 (90%) Right unilateral 3 (10%)

Lifetime ECT number of sessions 60.77 (66.27) Duration (s) of EEG convulsion 40.27 (12.49)

Data expressed as number of patients (percentage) or mean (SD). In ECT regimen: ‘Acute’ refers to the first 6–12 ECT sessions, ‘Continuation’ for the following sessions during the first 6 months, and ‘Maintenance’ for the sessions received after this first 6-month period. ECT, electroconvulsive therapy; EEG, electroencephalographic.

Psychiatry and Clinical Neurosciences 73: 628–635, 2019 631

PCNPsychiatry andClinical Neurosciences Effects of ECT in inflammatory balance

important methodological differences among the relatively few avail- able studies. This set of studies suggests that certain inflammatory parameters could be used as biomarkers of response to ECT treatment and that certain subpopulations of patients might benefit most from

this treatment. Seeing the effects of this technique on the immune sys- tem, these results also increase the interest in knowing ECT’s effects on certain psychiatric disorders mediated by immunity, such as auto- immune encephalitis.44,45

PRE

T L

R 4

/β -a

c ti n

( %

O D

)

β actin

0

50

100

150

POST

PRE

TLR4 89 kDa

42 kDa

POST

PRE

T R

IF /β

-a c ti n

( %

O D

)

β actin

0

50

100

150

POST

PRE

TRIF 66 kDa

42 kDa

POST

PRE

M y D

8 8

/β -a

c ti n

( %

O D

)

β actin

0

50

100

150

POST

PRE

MyD88 35 kDa

42 kDa

POST

PRE

A U

0

0.2

0.4

0.8

0.6

POST

NFκB (p65) Activity

*

PRE

iN O

S /β

-a c ti n

( %

O D

)

β actin

0

50

100

150

POST

PRE

iNOS 130 kDa

42 kDa

POST

PRE

C O

X -2

/β -a

c ti n

( %

O D

)

β actin

0

50

100

150

POST

PRE

COX-2 72 kDa

42 kDa

POST

PRE

P la

s m

a (

p g

/m L

)

0

1000

2000

4000

3000

POST

PGE 2

TBARS Nitrites

*

*

PRE

M a

lo n

d ia

ld e

h y d

e p

la s m

a

(μ M

)

0

5

10

15

POST PRE

P la

s m

a N

O – 2 (

μ M

)

0

4

2

6

8

POST

15d-PGJ 2

***

PRE

P la

s m

a (

p g

/m L

)

0

20

10

30

40

POST

PPARγ Activity

*

PRE

A U

0

0.2

0.4

0.6

POST

(a) (b) (c)

(d) (e) (f)

(g) (h)

(j) (k)

(i)

Fig.2 Mean differences (SD) on biomarkers at baseline and 2 h after an electroconvulsive therapy session (univariate analysis). (a) Western blot analysis of the toll-like receptor 4 (TLR4) activity in peripheral blood mononuclear cells (PBMC); (b) western blot analysis of the TIR-domain-containing adapter-inducing interferon-β (TRIF) activity in PBMC; (c) western blot analysis of the myeloid differentiation factor 88 (MyD88) activity in PBMC; (d) p65 nuclear transcription factor κB (NFκB) subunit activ- ity assay in nuclear extracts from PMBC; (e) western blot analysis of the inducible nitric oxide synthase (iNOS) activity in PBMC; (f) western blot analysis of the cyclooxygenase-2 (COX-2) activity in PBMC; (g) plasma levels of prostaglandin E2 (PGE2); (h) lipid peroxidation determined by thiobarbituric acid reactive substances (TBARS); (i) plasma levels of nitrites; (j) plasma levels of 15-deoxy-prostaglandin J2 (15d-PGJ2); (k) gamma isoform of the peroxisome proliferator activated receptor (PPARγ) activity assay in nuclear extracts from PMBC. AU, arbitrary units; OD, optical density.

Psychiatry and Clinical Neurosciences 73: 628–635, 2019632

Effects of ECT in inflammatory balance PCNPsychiatry andClinical Neurosciences

Apart from decreasing some proinflammatory parameters, an interesting result observed was the decrease in the expression of 15d- PGJ2. Though the general approach of our study was exploratory, this was the most robust finding from a statistical point of view, being sig- nificant even after applying multiple testing corrections. The decrease of such anti-inflammatory mediators could be related to the appear- ance of some of the most common side-effects of ECT, such as cog- nitive effects. Our group has recently reported an association between the expression of this biomarker and the presence of certain cognitive deficits in patients with a first psychotic episode.46 These results sug- gest that future research will identify the role of inflammatory bio- markers in relation to the cognitive deficits that some people under treatment with ECT present.

Interesting differences in lipid peroxidation results were found; this was one of the most relevant parameters of the pathway studied

and was determined by the TBARS assay. Specifically, those patients who were considered responders showed decreased TBARS levels after the ECT session, while non-responders showed increases of this lipid peroxidation determination. These results indicate that in the subpopulation of responders, ECT could mitigate cell damage pro- cesses mediated by lipid peroxidation, while in non-responders these processes could get worse.

Some limitations should be taken into account when analyzing these results. First, the relatively small sample size could be limiting the statistical power. Second, the sample was heterogeneous regarding certain clinical variables (i.e., diagnosis, psychopharmacological treat- ment, or ECT regimen). Third, we studied the effects of an ECT ses- sion, while it would be also very informative to have retested the patients 24 h after the ECT session and at the end of the whole acute treatment (generally 9–12 sessions). Fourth, as the main aim of our study was to explore the effects of a single session of ECT on the inflammatory balance in a selected group of patients with a severe mental disorder, we did not have a group of healthy controls paired with the study sample with which to compare the expression of bio- markers at baseline. Fifth, we do not know the effects of anesthesia, the apnea time, or the tonic–clonic seizures on the biomarkers studied.

Despite these limitations, we believe that our study has identified significant effects of ECT treatment over PBMC pro-/anti- inflammatory pathways in a group of patients with severe mental disorders; and these findings warrant greater attention in future inves- tigations. Furthermore, key strengths of the study deserve mention: (i) we included a wide spectrum of biochemical inflammatory markers both in plasma samples and peripheral blood mononuclear cells, all- owing in-depth insights and relations between multiple components of the pro- and anti-inflammatory signaling pathways; and (ii) the diagnostic evaluation was performed with a very comprehensive pro- tocol and inclusion–exclusion criteria were applied in a strict manner.

In conclusion, and regarding the implications for clinical prac- tice, in this study we have identified the effects that an ECT session produces on the inflammatory balance of patients with a severe men- tal disorder. Although more scientific evidence is needed to expand this preliminary data, the determination of multiple components of pro- and anti-inflammatory cellular pathways, including the activity of nuclear receptors, have interesting potential as biological markers and potential targets. These results can also be helpful to clarify the

Table 2. Mean differences (�SD) in the biomarker levels between pre-ECT and post-ECT session (2 h after stimulus) in 30 patients with severe mental disorders

Marker Pre-ECT Post-ECT Statistics P-value

TLR4 WB, % OD 100.13 � 17.15 93.24 � 21.22 t = 1.763 0.089 TRIF WB, % OD 100.22 � 18.26 96.42 � 15.92 t = 1.175 0.250 MyD88 WB, % OD 105.32 � 41.29 102.81 � 46.84 z = −0.205 0.837 NFKB AAnucl, AU 0.58 � 0.26 0.45 � 0.17 z = −2.108 0.035 iNOS WB, % OD 99.77 � 10.44 93.37 � 14.64 t = 2.698 0.012 COX-2 WB, % OD 100.44 � 17.31 104.56 � 29.81 t = −0.845 0.405 PGE2 plasma, pg/ml 2976.21 � 1891.15 2405.73 � 2475.15 z = −1.964 0.049 15d-PGJ2 plasma, pg/ml 35.78 � 7.49 28.30 � 7.22 t = 4.633 <0.001 Nitrites plasma, μM 7.09 � 2.60 6.11 � 3.27 z = −2.211 0.027 TBARS plasma, μM MDA 11.44 � 4.98 11.04 � 5.24 z = −0.936 0.349 PPARγ AAnucl, AU 0.477 � 021 0.37 � 0.14 z = −1.861 0.063 Data expressed as mean � SD. Immune/inflammatory/oxidative parameters shown in white cells; anti-inflammatory/antioxidative parameters in gray cells. See Methods section for detail. AAnucl, activity assay in nuclear extracts; AU, arbitrary units; COX-2, cyclooxygenase-2; 15d-PGJ2, 15-deoxy-prostaglandin J2; iNOS, inducible nitric oxide synthase; MyD88, myeloid differentiation factor 88; NFκB, nuclear transcription factor κB; OD, optical density; PGE2, prostaglandin E2; Plasma, plasma levels; PPARγ, gamma isoform of the peroxisome proliferator activated receptor; TBARS, thiobarbituric acid reactive substances; TLR4, toll-like receptor 4; TRIF, TIR-domain-containing adapter-inducing interferon-β; WB, western blot.

PRE-ECT

8

9

10

11

12

13

POST-ECT

TBARS

Fig.3 Differences between treatment ( ) responders and ( ) non- responders in thiobarbituric acid reactive substance (TBARS) levels at baseline and 2 h after an electroconvulsive therapy (ECT) session.

Psychiatry and Clinical Neurosciences 73: 628–635, 2019 633

PCNPsychiatry andClinical Neurosciences Effects of ECT in inflammatory balance

mechanisms of actions of ECT. Such findings warrant greater atten- tion in future investigations and in the translational significance of these data.

Acknowledgments The authors wish to acknowledge Sonia Hernández, Marc Valentí, and all members of the Neuroanesthesia Section at the Hospital Clínic de Barcelona. This study was supported by Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional, Unión Europea, ‘Una manera de hacer Europa’, MINECO SAF2016/75500-R, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), and by Generalitat de Catalunya and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement (2014SGR441 and 2014SGR398).

Disclosure statement Dr Bernardo has been a consultant for, has received grant/research support and honoraria from, and has been on the speakers/advisory board of ABBiotics, Adamed, Angelini, Casen Recordati, Eli Lilly, Janssen-Cilag, Lundbeck, Otsuka, Takeda, and Somatics; and has obtained research funding from the Ministry of Education, Culture and Sport, the Spanish Ministry of Economy, Industry and Competi- tiveness (CIBERSAM), by the Government of Catalonia, Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement (2017SGR1355), Foundation European Group for Research In Schizophrenia (EGRIS), and the 7th Framework Program of the European Union. Dr Bioque has received honoraria from talks and consultancy from Adamed, has received honoraria from consultancy for Ferrer, has received research support and honoraria from talks and consultancy from Janssen-Cilag, has received honoraria from talks and consultancy from Lundbeck, has received honoraria from talks and consultancy from Otsuka, and has received a research prize from Pfizer. Dr Vieta has received grants and served as consultant, advisor or CME speaker for the following entities: AB-Biotics, Allergan, Angelini, AstraZeneca, Bristol-Myers Squibb, Dainippon Sumitomo Pharma, Farmindustria, Ferrer, Forest Research Institute, Gedeon Richter, Glaxo-Smith-Kline, Janssen, Lundbeck, Otsuka, Pfizer, Roche, Sanofi-Aventis, Servier, Shire, Sunovion, Takeda, the Brain and Behaviour Foundation, the Spanish Ministry of Science and Inno- vation (CIBERSAM), the Seventh European Framework Programme (ENBREC), and the Stanley Medical Research Institute. The rest of authors report no competing interests for this study.

Author contributions M. Bioque conducted the literature review, recruited the participants, applied the ECT session, collected data, conducted the main statistical analysis, wrote the first draft of the manuscript, and handled subse- quent drafts after receiving coauthors’ feedback. K.S.M.-D., per- formed all biochemical determinations in plasma and in cells and prepared subcellular samples, assisted with the analysis, and wrote the first draft of the manuscript. A.M. collected the biological samples and separated plasma and PBMC. E.M. recruited the participants, col- lected the blood samples, and applied the ECT session. The rest of the coauthors participated in the ECT procedures, collected data, and commented on drafts. All of the authors contributed to the final ver- sion of the paper.

References 1. UK ECT Review Group. Efficacy and safety of electroconvulsive therapy

in depressive disorders: A systematic review and meta-analysis. Lancet 2003; 361: 799–808.

2. Dierckx B, Heijnen WT, van den Broek WW, Birkenhäger TK. Efficacy of electroconvulsive therapy in bipolar versus unipolar major depression: A meta-analysis. Bipolar Disord. 2012; 14: 146–150.

3. Tharyan P, Adams CE. Electroconvulsive therapy for schizophrenia. Cochrane Database Syst. Rev. 2005. https://doi.org/10.1002/14651858. CD000076.pub2

4. Bernardo M, Urretavizcaya M. Dignifying electroconvulsive therapy based on evidence. Rev. Psiquiatr. Salud Ment. 2015; 8: 51–54.

5. Vieta E, Berk M, Schulze TG et al. Bipolar disorders. Nat. Rev. Dis. Primers. 2018; 4: 18008.

6. Leza JC, Garcia-Bueno B, Bioque M et al. Inflammation in schizophre- nia: A question of balance. Neurosci. Biobehav. Rev. 2015; 55: 612–626.

7. Garcia Bueno B, Caso JR, Madrigal JL, Leza JC. Innate immune recep- tor toll-like receptor 4 signalling in neuropsychiatric diseases. Neurosci. Biobehav. Rev. 2016; 64: 134–147.

8. Dowlati Y, Herrmann N, Swardfager W et al. A meta-analysis of cyto- kines in major depression. Biol. Psychiatry 2009; 67: 446–457.

9. Modabbernia A, Taslimi S, Brietzke E, Ashrafi M. Cytokine alterations in bipolar disorder: A meta-analysis of 30 studies. Biol. Psychiatry 2013; 74: 15–25.

10. Miller BJ, Buckley P, Seabolt W, Mellor A, Kirkpatrick B. Meta- analysis of cytokine alterations in schizophrenia: Clinical status and anti- psychotic effects. Biol. Psychiatry 2011; 70: 663–671.

11. MacDowell KS, Pinacho R, Leza JC, Costa J, Ramos B, Garcia- Bueno B. Differential regulation of the TLR4 signalling pathway in post-mortem prefrontal cortex and cerebellum in chronic schizophrenia: Relationship with SP transcription factors. Prog. Neuropsychopharmacol. Biol. Psychiatry 2017; 79: 481–492.

12. Takeuchi O, Akira S. Toll-like receptors: Their physiological role and signal transduction system. Int. Immunopharmacol. 2001; 1: 625–635.

13. Garcia-Bueno B, Bioque M, Mac-Dowell KS et al. Pro−/anti- inflammatory dysregulation in patients with first episode of psychosis: Toward an integrative inflammatory hypothesis of schizophrenia. Schizophr. Bull. 2013; 40: 376–387.

14. Garcia-Bueno B, Bioque M, MacDowell KS et al. Pro-/antiinflammatory dysregulation in early psychosis: Results from a 1-year follow-up study. Int. J. Neuropsychopharmacol. 2014; 18: pyu037.

15. Kaiya H, Uematsu M, Ofuji M et al. Elevated plasma prostaglandin E2 levels in schizophrenia. J. Neural Transm. 1989; 77: 39–46.

16. García-�Alvarez L, Caso JR, Garcíaa-Portilla MP et al. Regulation of inflammatory pathways in schizophrenia: A comparative study with bipo- lar disorder and healthy controls. Eur. Psychiatry 2018; 47: 50–59.

17. Müller N, Wagner JK, Krause D et al. Impaired monocyte activation in schizophrenia. Psychiatry Res. 2012; 198: 341–346.

18. McKernan DP, Dennison U, Gaszner G, Cryan JF, Dinan TG. Enhanced peripheral toll-like receptor responses in psychosis: Further evidence of a pro-inflammatory phenotype. Transl. Psychiatry 2011; 1: e36.

19. Martínez-Gras I, Pérez-Nievas BG, García-Bueno B et al. The anti- inflammatory prostaglandin 15d-PGJ2 and its nuclear receptor PPARgamma are decreased in schizophrenia. Schizophr. Res. 2011; 128: 15–22.

20. Oliveira J, Busson M, Etain B et al. Polymorphism of toll-like receptor 4 gene in bipolar disorder. J. Affect. Disord. 2014; 152-154: 395–402.

21. Hung Y-Y, Kang H-Y, Huang K-W, Huang T-L. Association between toll-like receptors expression and major depressive disorder. Psychiatry Res. 2014; 220: 283–286.

22. Keri S, Szabo C, Kelemen O. Expression of toll-like receptors in periph- eral blood mononuclear cells and response to cognitive-behavioral ther- apy in major depressive disorder. Brain Behav. Immun. 2014; 40: 235–243.

23. Hajebrahimi B, Bagheri M, Hassanshahi G et al. The adapter proteins of TLRs, TRIF and MYD88, are upregulated in depressed individuals. Int. J. Psychiatry Clin. Pract. 2014; 18: 41–44.

24. Martín-Hernández D, Caso JR, Javier Meana J et al. Intracellular inflam- matory and antioxidant pathways in postmortem frontal cortex of subjects with major depression: Effect of antidepressants. J. Neuroinflammation 2018; 15: 251.

25. Hannestad J, DellaGioia N, Bloch M. The effect of antidepressant medi- cation treatment on serum levels of inflammatory cytokines: A meta- analysis. Neuropsychopharmacology 2011; 36: 2452–2459.

26. Guloksuz S, Rutten BP, Arts B, van Os J, Kenis G. The immune system and electroconvulsive therapy for depression. J. ECT 2014; 30: 132–137.

27. Kartalci S, Karabulut AB, Erbay LG, Acar C. Effects of electroconvul- sive therapy on some inflammatory factors in patients with treatment- resistant schizophrenia. J. ECT 2016; 32: 174–179.

28. Albrecht J, Helderman JH, Schlesser MA, Rush AJ. A controlled study of cellular immune function in affective disorders before and during somatic therapy. Psychiatry Res. 1985; 15: 185–193.

29. Fischler B, Bocken R, Schneider I, De Waele M, Thielemans K, Derde MP. Immune changes induced by electroconvulsive therapy (ECT). Ann. N. Y. Acad. Sci. 1992; 650: 326–330.

Psychiatry and Clinical Neurosciences 73: 628–635, 2019634

Effects of ECT in inflammatory balance PCNPsychiatry andClinical Neurosciences

30. Kronfol Z, Nair MP, Weinberg V, Young EA, Aziz M. Acute effects of electroconvulsive therapy on lymphocyte natural killer cell activ- ity in patients with major depression. J. Affect. Disord. 2002; 71: 211–215.

31. Lehtimaki K, Keranen T, Huuhka M et al. Increase in plasma proinflammatory cytokines after electroconvulsive therapy in patients with depressive disorder. J. ECT 2008; 24: 88–91.

32. Fluitman SB, Heijnen CJ, Denys DA, Nolen WA, Balk FJ, Westenberg HG. Electroconvulsive therapy has acute immunological and neuroendocrine effects in patients with major depressive disorder. J. Affect. Disord. 2011; 131: 388–392.

33. Arts B, Peters M, Ponds R, Honig A, Menheere P, van Os J. S100 and impact of ECT on depression and cognition. J. ECT 2006; 22: 206–212.

34. Agelink MW, Andrich J, Postert T et al. Relation between electroconvul- sive therapy, cognitive side effects, neuron specific enolase, and protein S-100. J. Neurol. Neurosurg. Psychiatry 2001; 71: 394–396.

35. Järventausta K, Sorri A, Kampman O et al. Changes in interleukin-6 levels during electroconvulsive therapy may reflect the therapeutic response in major depression. Acta Psychiatr. Scand. 2016; 135: 87–92.

36. Rush G, O’Donovan A, Nagle L et al. Alteration of immune markers in a group of melancholic depressed patients and their response to electro- convulsive therapy. J. Affect. Disord. 2016; 205: 60–68.

37. Hestad KA, Tonseth S, Stoen CD, Ueland T, Aukrust P. Raised plasma levels of tumor necrosis factor alpha in patients with depression: Nor- malization during electroconvulsive therapy. J. ECT 2003; 19: 183–188.

38. Rotter A, Biermann T, Stark C et al. Changes of cytokine profiles during electroconvulsive therapy in patients with major depression. J. ECT 2013; 29: 162–169.

39. Zachrisson OC, Balldin J, Ekman R et al. No evident neuronal damage after electroconvulsive therapy. Psychiatry Res. 2000; 96: 157–165.

40. Kartalci S, Karabulut AB, Ozcan AC, Porgali E, Unal S. Acute and chronic effects of electroconvulsive treatment on oxidative parameters in schizophrenia patients. Prog. Neuropsychopharmacol. Biol. Psychiatry 2011; 35: 1689–1694.

41. Medda P, Toni C, Mariani MG, De Simone L, Mauri M, Perugi G. Elec- troconvulsive therapy in 197 patients with a severe, drug-resistant bipolar mixed state: Treatment outcome and predictors of response. J. Clin. Psy- chiatry 2015; 76: 1168–1173.

42. Guy W. ECDEU Assessment Manual for Psychopharmacology – Revised. Department of Health, Education and Welfare, Rockville, MD, 1976.

43. Bender R, Lange S. Adjusting for multiple testing--When and how? J. Clin. Epidemiol. 2001; 54: 343–349.

44. Coffey MJ, Cooper JJ. Electroconvulsive therapy in anti-N-methyl-D- aspartate receptor encephalitis: A case report and review of the literature. J. ECT 2016; 32: 225–229.

45. León-Caballero J, Pacchiarotti I, Murru A et al. Bipolar disorder and antibodies against the N-methyl-d-aspartate receptor: A gate to the involvement of autoimmunity in the pathophysiology of bipolar illness. Neurosci. Biobehav. Rev. 2015; 55: 403–412.

46. Cabrera B, Bioque M, Penadés R et al. Cognition and psychopathology in first-episode psychosis: Are they related to inflammation? Psychol. Med. 2016; 46: 2133–2144.

Psychiatry and Clinical Neurosciences 73: 628–635, 2019 635

PCNPsychiatry andClinical Neurosciences Effects of ECT in inflammatory balance

Copyright of Psychiatry & Clinical Neurosciences is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.