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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.