UNIVERSIDADE FEDERAL DO PAMPA

CAMPUS ITAQUI

Dear Editor,

Ms. Ref. No.:  NTRE-D-16-00075

We are returning the revised manuscript (NTRE-D-16-00075) entitled

“Intracerebroventricular administration of streptozotocin as an experimental

approach to depression: evidence for the involvement of proinflammatory

cytokines and indoleamine-2,3-dioxygenase" that was revised to incorporate the

points raised by the referees of “Neurotoxicity Research".

The manuscript was carefully revised and all comments for the attention of the

authors proposed by referees were considered. In fact, we realized that all the changes

proposed by reviewers contributed to improve considerably the quality of our

manuscript. As required, we significantly reduced the length of the discussion. In

addition, we revised the whole manuscript carefully to avoid language errors and badly

constructed sentences as well as grammatical and spelling errors.

We would like to thank to the referees for their careful reading of the manuscript

and for all their pertinent suggestions. We hope that now the paper is acceptable for

publication in “Neurotoxicity Research".

Sincerely,

Cristiano Ricardo Jesse

REVIEWER: 1

Ms. Ref. No.:  NTRE-D-16-00075

We would like to thank to the reviewer for the suggestions and corrections made

in the manuscript. We realize that your comments contribute to improve considerably

the quality of the manuscript. The responses to the questions raised are given in

sequence. The manuscript was carefully revised and all comments for the attention of

the authors proposed by referees were considered. In fact, we realized that all the

changes proposed by reviewers contributed to improve considerably the quality of our

manuscript. As required, we significantly reduced the length of the discussion. In

addition, we revised the whole manuscript carefully to avoid language errors and badly

constructed sentences as well as grammatical and spelling errors.

INTRODUCTION

Point 1) It would be interesting if the authors make clear that the hypothesis of

inflammation is much older and that the work of Dantzer et al, Leonard et al and Miller

et al are more recent in relation the the hypothesis.

Response: We agree with reviewer’s comments. We described the most recent

references in this topic. In this way, we agree that the hypothesis of inflammation is

older and more references are added. For example, Smith (1991) described the

involvement of inflammation in the depression. In addition, the pathophysiology of

depression has not been fully elucidated and numerous studies propose that

neuroinflammation may play a role in the development of this disorder (Dunn and

Welch, 1991; Smith, 1991; Dantzer et al., 2008; Anisman, 2011 and Xie et al., 2014)

(Introduction section, page 3). If other references should be included in the article, we

can add in the next review of the manuscript.

Point 2) Please correct "Thus, the available information implicated IDO as a critical

molecular mediator of inflammation-induced depressive-like behaviour." by "Thus, the

increased activity of IDO has been implicated as a critical molecular mediator of

inflammation-induced depressive-like behaviour."

Response: The correct sentence was added in the revised manuscript. Thus, the correct

sentence is on the Introduction section (page 4, lines 10-12). We also added a new

study that demonstrated that IDO and the treatment with minocycline reduced the

depression in a model of diabetes (Da Silva Dias et al., 2016).

Point 3) In the Introduction, the authors make clear that ICV-STZ could be an

important mouse model for depression studies. Given that ICV-STZ is an AD model, I

would like the authors discourse about the possibility of this depression is related to

AD, rather than a new model of depression (Tokuchi R et al., 2016, J Neurol Sci. 2016

Jun 15;365:3-8; Harrington KD et al., 2016, Int J Geriatr Psychiatry)

Response: The ICV-STZ is a mouse model produces cerebral impairments and

pathological changes in the brain that include cholinergic deficits, oxidative stress,

neurotransmitters imbalance and neuroinflammation (Souza et al., 2013; Javed et al.,

2015; Jayant et al., 2016; Liu et al., 2016). In addition, this model causes behavioral

modifications in rodents, such as memory deficit and depression (Souza et al., 2013;

Gupta et al., 2014; Majkutewicz et al., 2016).

The injection of STZ is more used to induce alterations in brain neurochemistry,

which resemble to those found in the brain of sporadic Alzheimer’s Disease (AD). The

main differences observed in the ICV-STZ, when compared the model of AD and

depression, are the dose, the site, the time of injections and the days of evaluation after

the injection.

Our study and

Souza et al., 2013

Liu et al., 2016 Javed et al.,

2015

Dose 0.1 mg/site; 4 µl 3 mg/kg 2.57 mg/kg, b.

wt. in

saline,

Site The bregma

coordinates used for

injection were −1.0

mm lateral, −0.3 mm

posterior, and −2.5

mm below

0.8mm posterior to

bregma, 1.0mm

lateral to sagittal

suture, and 3.0mm

beneath the surface

of the brain

anterioposterior

−0.8 mm, lateral

±1.0 mm and

dorso-ventral

−3.0 mm

relative to bregma

and ventral from

dura with the

tooth bar set at

0 mm.

Injections 1 time day 1 and 3 (2

times)

2 times (two

ventricules)

Behavioral

observations

1 hour – 1 week

(depression)

1 month

(memory)

15- 21 days

(memory)

Moreover, we agree with the possibility that the mechanism of toxicity in both

models is very similar, with involvement of oxidative stress, neurotransmitters

imbalance, neuroinflammation and consequently, behavioral changes. In our study, we

emphasize the depression and the neurochemistry alterations. In conclusion, we believe

that depression is an early sign of the AD and the cognitive alterations is a late

symptom. Thus, the ICV-STZ is a good model to study the mechanisms and strategy in

brain-related diseases (memory deficit, depression and AD).

MATERIAL AND METHODS

Point 1: Justify the dose used in the present study. 

Point 2: Justify also if the surgery procedure would influence or not the behavioral

responses measured 6 hours after the icv injection of STZ. 

Responses: In the first study in our laboratory (Souza et al., 2013), the main line of

study was the protocol to investigate the depression and the neuroinflammation. Thus,

in this first study, we performed a dose-curve response at all points (1h, 6h, 24h and 1

week). In this way, the proposed model in this study was developed using different

doses. The dose used was based in the study published by Souza et al., 2013 that

demonstrated the depression like activity and produced inflammation in hippocampus of

mice.

Our study and Souza

et al., 2013

Liu et al., 2016 Javed et al., 2015

Dose 0.1 mg/site; 4 µl 3 mg/kg 2.57 mg/kg, b.

wt. in

saline,

Injections 1 time day 1 and 3 (2

times)

2 times (two

ventricules)

Behavioral 1 hour – 1 week 1 month 15- 21 days

observations

(depression)

(memory)

(memory)

One of the main care in our study was the behavioral responses measured 1 and

6 hours after the ICV injection of STZ. Thus, we utilized a low dosage of anesthetic and

observed that locomotor, ambulatory and grooming activities (to discard false positive

in the depressive-like activity) was unaltered. Therefore, we considered that performed

analyses (behavioral and neurochemistry) in this study at 6 hours did not influence the

final results. In conclusion, the surgery procedure did not influence the behavioral

response measured 6 h after the ICV-STZ.

RESULTS

Point 1) As the ICV-STZ is considered a model of AD, it would be interesting if the

authors had undergone the animals to some behavioral test related to memory to

correlate or not with the AD. What do the authors think about that?

Response: The main objective of our study was to explore the mechanism involved in

the beginning of the process after the ICV-STZ. Thus, we investigated the depressive

behavior induced by ICV-STZ and related to inflammation, serotonin levels and

kynurenine pathway. We considered that AD has similar symptoms, such as memory,

depression and injury. In this way, we emphasize the depression in this model of brain

injury with the elucidation of the mechanisms involved.

In future studies, the analyses included the memory and the correlations with

AD, depression and inflammation. Conversely, in this study, the focus is the depression

and the mechanisms involved.

Point 2) In many studies, using the same model, the various deleterious effects

(oxidative stress, neuroinflammation, etc.) persist longer than 7 days (1 week). As

explain this reduction in the levels of pro-inflammatory cytokines observed in the

present study after 7 days?

Response: The reduction in the levels of pro-inflammatory cytokines is due to the dose

of STZ (0.1 mg/site) and the number of administration (1 injection) (based on the study

of Souza et al., 2013). In this way, the inflammatory response was acute with a peak at 6

hours. After this point, the inflammatory response (demonstrated in reduction of levels

of cytokines) decreased in the other points (24 h and 1 week) with correlations with the

behavioral analyses. Moreover, the maximum effect observed in the cytokines is also

observed in the IDO activity/kynurenine levels. Thus, there are relations between the

reduction of cytokines, the normalization of the kynurenine pathway (IDO activity and

kynurenine levels) and the behavioral test on the time-course.

DISCUSSION

Point 1) "The depressive like-behaviour induced by STZ had already been reported by

other laboratories (Hirano et al., 2007; Ho et al., 2012)." In both studies the authors used

STZ as a Type 1 diabetes model (a single injection ip). Are the effects of STZ injected

ip and icv equal? Please, make an explanation about the differences between the ip or

icv injections.

Response: The ip injection of STZ induced a diabetes in animals (Gupta et al., 2014). It

inhibits insulin secretion by specific necrosis of pancreatic beta cells, resulting insultin

depedent diabetes (Lenzen, 2008). In this way, there are preclinical reports indicating

that diabetes results in reduced metabolism, such as monoaminergic activity in the

brain, which can mediate behavioral abnormalities that resemble depressive and anxiety

symptoms in humans (Gupta et al., 2014; Yamoto et al., 2004). In addition, animal

studies revealed that diabetes (being a metabolic stressor) impedes ensuing impairment

of hippocampal neurogenesis that may be correlated with depression alterations (Zhang

et al., 2008).

The icv-STZ injection increased the brain nitroso-oxidative stress,

acetylcholinerase activity and production of cytokines (Paidi et al., 2015; Hassanzadeh

et al., 2015). In this model, the damage was caused by the direct action of STZ on the

brain and it did not involve the alteration of glucose and metabolic consequence of

diabetes. Thus, in the icv-STZ injection, the inflammation and oxidative stress was

caused by STZ that was verified in our study, such as involvement of cytokines and

kynurenine pathway. In conclusion, the effects of STZ injected by ip and icv routes are

different and there are several studies evidencing these models.

Point 2) It should be interesting to cite the manuscript from da Silva Dias et al (Mol

Neurobiol. 2015 Dec 15. [Epub ahead of print]) that showed by ip injection of STZ a

depressive like-behavior and changes in inflammatory cytokines in the hippocampus,

IDO expression and also changes in neurotransmitter levels. The treatment with

minocycline was also performed in these animals. 

Response: We agree with reviewer’s comments. This study was added in the

manuscript (Introduction section, page 4), thus it contributed to the hypothesis that

minocycline, inflammation and kynurenine pathway is involved in the depression. In the

study published by Da Silva Dias et al (2016), the model utilized was the injection by

the ip route of STZ (diabetes) and the treatment with minocycline and IDO direct

inhibitor 1-methyl-tryptophan (1-MT) reduced the neuroinflammation in the HIP

followed by IDO activation with a consequent decrease in the 5-HT levels can be a

possible pathophysiological mechanism that links depression to diabetes.

Our study also utilized the treatment with minocycline with similar results when

compared to the Da Silva Dias: reduction of neuroinflammation and kynurenine

pathway and protection against behavioral alterations. We emphasized that models are

different: the injection of STZ was made by ICV or IP (diabetes) routes.

Point 3) It seems that depression as a single pathology and as comorbid with type-1

diabetes or AD have common pathophysiological mechanisms. Authors should get

deeper this discussion. Moreover, depression could then lead to diabetes and/or AD?

Response: The present study and the literature demonstrated the depression as single

pathology with pathophysiological mechanisms with type-1 diabetes and AD

(Alzheimer’s disease).

In this study and in the literature are not conclusive to that depression lead to

diabetes and/or AD. There are demonstrated in animal studies that diabetes modify the

metabolism and it induced depression and AD.

In our study, we demonstrated the ICV-STZ, independent of glucose levels and

diabetes, cause inflammation and behavioral alterations (depressive-like tests).

Moreover, these alterations are similar to other studies that observed symptoms of AD

(Gupta et al., 2014; Santos et al., 2015; Zhang et al., 2015; Deshmukh et al., 2016;

Majkutewicz et al., 2016). Thus, the model utilized of ICV-STZ demonstrated similar

mechanisms involved in the disease of depression. All these points are included in the

discussion section to improve the study.

Point 4) The authors really believe ICV-STZ can be an animal model for studying

depression? It would not be a model to study depression associated with Alzheimer's

disease?

Response: The model demonstrated with several behavioral tests and analyses of

neurotransmitters that ICV-STZ is a model for studying depression in mice. The

alterations verified in the levels of serotonin, cytokines and kynurenine pathway

contributed to add the ICV-STZ such as model of depression. Another hypothesis is that

depression and the AD have similar mechanisms such as oxidative stress, inflammation

and imbalance in neurotransmission in the brain. Thus, this model can be study the

depression associated with AD.

REVIEWER: 2

Ms. Ref. No.:  NTRE-D-16-00075

We would like to thank to the reviewer for the suggestions and corrections made

in the manuscript. We realize that your comments contribute to improve considerably

the quality of the manuscript. The responses to the questions raised are given in

sequence. The manuscript was carefully revised and all comments for the attention of

the authors proposed by referees were considered. In fact, we realized that all the

changes proposed by reviewers contributed to improve considerably the quality of our

manuscript. As required, we significantly reduced the length of the discussion. In

addition, we revised the whole manuscript carefully to avoid language errors and badly

constructed sentences as well as grammatical and spelling errors.

Point 1) Please, explain why minocycline was administered before ICV-STZ and not

after?

Response: The time was administration of minocycline was chosen based in the

literature (O’connor et al., 2009). In addition, the schedule of administration of

minocycline was made to determine the role of inhibition of cytokine production and

this mechanism was demonstrated with previous ICV-STZ. In the study of our group

(Souza et al., 2013) the peak effect was 6 h and to obtain an effect in this point, the

administration of minocycline was carried out before the ICV-STZ.

In future studies, if the analyses in the brain and in the behavioral tests will be

made in other points of time, the administration of minocycline should be after the ICV-

STZ. In conclusion, in the second study of our group, the main interest is the

mechanism that ICV-STZ caused depressive-like effect. In this way, the administration

of minocycline was before the ICV-STZ to emphasize the mechanism. Thus, with this

protocol, the involvement of cytokines and kynurenine pathway was evidenced by the

administration of minocycline before the ICV-STZ.

Point 2) Methods' section: how was the hippocampal tissue processed before the

different analyses (ELISA, liquid chromatograph)? Please, explain.

Response: We agree with reviewer’s comments. The processing of tissue before the

analyses was added in the Method’s section. In this way, we included in section “Pro-

inflammatory cytokines levels: The hippocampus (S1) was diluted in a solution

containing bovine serum albumin (BSA 10 mg/ml), EGTA (2 mM), EDTA (2 mM)

and PMSF (0.2 mM) in phosphate-buffered saline (PBS, pH 7.4). Levels of tumour

necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in the hippocampus were

determined using commercially available ELISA assays, following the instructions

supplied by the manufacturer (DuoSet Kits, R&D Systems; Minneapolis). The

concentration of cytokines was normalized to the protein concentration contained in

the samples. Results are shown as pg/mg tissue” and “Tryptophan (TRP) and

kynurenine (KYN) levels and Serotonin (5-HT) and 5-Hydroxyindoleacetic acid (5-

HIAA) levels: The tissues were diluted (1:5 w/v for HPLC analysis in buffer containing

20 mM KH2PO4, 0.1 mM PMSF, 10 mM EDTA, 0.4 M NaCl, protease inhibitors and

140 mM KCl). This tissue preparation were sonicated in ultrasonic cooling bath for

15 min at 40 kHz following by centrifugation at 16,000g during 10 min at 4 °C, and

then 10 μL of supernatant was used to HPLC to carry out the determination of

neurotransmitters” (Materials and methods section, pages 8-11).

Point 3) Methods' section: "Protein determination": Please, explain better this paragraph

(which proteins were analyzed?, ....)

Response: We agree with reviewer’s comments. We modified the section “Protein

determination”. Thus, we explain better this paragraph: “Protein concentration was

measured by the method of Bradford (1976), using bovine serum albumin (1 mg/ml) as

the standard (Sigma). The Bradford assay is a protein determination method that

involves the binding of Coomassie Brilliant Blue G-250 to proteins (Bradford 1976). It

is this blue protein form that is detected at 595 nm in the assay using a microplate

reader.” (Page 11).

Point 4) Introduction/discussion: Please, introduce/discuss why did you focused your

studies in the hippocampus and not in other areas more relate to depression such us the

amygdala or the prefontal cortex?

Response: The focus on studies using the ICV-STZ model is the hippocampus. The

main modifications observed in the article published by Souza et al., (2013) and in this

study are in the hippocampus and not in amygdala or prefrontal cortex. Thus, the model

of depression induced by ICV – STZ demonstrated the alterations in cytokines and

kynurenine pathway in the main area involved (hippocampus). In addition, other studies

using STZ in animals models also utilized the hippocampus to demonstrate the

mechanisms (Gupta et al., 2014; Santos et al., 2015; Zhang et al., 2015; Deshmukh et

al., 2016; Majkutewicz et al., 2016; Da Silva Dias et al., 2016). Moreover, in the

hippocampus from patients suffering uni- and bipolar depression a modification in

kynurenine-immunoreactive microglia was observed (Busse et al., 2015; Réus et al.,

2015).

To demonstrate that hippocampus is the main area utilized in depression, we

added other studies. Thus, intracerebroventricular administration of LPS in C57BL/6J

mice induced depressive-like behavior and increased IDO expression, synthesis and

secretion of TNF-a and IL-6, and activation of inducible isoform of nitric oxide

synthase (iNOS) in the hippocampus (Fu et al., 2010). LPS injection in metabolic

syndrome (MetS) mice was associated with a decrease in hippocampal brain-derived

neurotrophic factor (BDNF) (Dinel et al., 2014), an important protein involved in

neuroplasticity and the pathophysiology of depression (Ign_acio et al., 2014; Reus et al.,

2013). Mice subjected to UCMS demonstrated a higher peripheral KYN pathway

activity in hippocampus (Laugeray et al., 2010). Viral mimetic Poly I:C induced

symptoms of depression and anxiety in rats accompanied by increased expression of IL-

1b, IL-6, TNF-a and CD11b and by decreased expression of BDNF and it's receptor

TrkB in the hippocampus (Gibney et al., 2013). SpragueeDawley rats subjected to

unpredictable chronic mild stress (UCMS), an animal model of depression, and

quinolinic acid- microinjection into the hippocampus exhibited depressive-like

behavior, increased levels of glutamate, and altered levels of the glutamate receptors

subunit (Chen et al., 2013). Danzhi Xiaoyao San (DXS), which is used in the prevention

and treatment of affective disorders in China, was administrated to rats subjected to

animal models of unpredictable chronic mild stress (UCMS). Treatment was effective in

reversing anhedonic behavior (Zhu et al., 2015). In the same study, DXS decreased IL-6

and TNF-α in the serum, and downregulated IDO activity and KYN production,

and upregulated TRP in the hippocampus (Zhu et al., 2015).

Finnaly, to confirm the interrelationship between neuroinflammation and STZ-

induced depression states, we sought to assay the levels of proinflammatory cytokines

TNF-α and IFN-γ in the hippocampus of mice, a brain region implicated with

depression (Piser, 2010).

Point 5) Discussion: is too long. Please, be more concise and avoid repetition as much

as possible.

Response: We agree with reviewer’s comments. In this way, the manuscript was

revised and more concise to improve the study.