APPLICATION NUMBER: 71e8c723e61011e9813097f462703207 NAME : AKSHAYAKEERTH A AFFILIATION : SASTRA DEEMED TO BE UNIVERSITY

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ACADEMIC WRITING
APPLICATION NUMBER: 71e8c723e61011e9813097f462703207
NAME : AKSHAYAKEERTH A
AFFILIATION : SASTRA DEEMED TO BE UNIVERSITY
COURSE CODE: UGC19_GE03

2. NOX2 Mediated-Parvalbumin Interneuron Loss Might Contribute
to Anxiety-Like and Enhanced Fear Learning Behavior in a Rat
Model of Post-Traumatic Stress Disorder
Fang-fang Liu1 • Lin-dong Yang2 • Xiao-ru Sun1 • Hui Zhang1 • Wei Pan1 •
Xing-ming Wang1 • Jian-jun Yang1 • Mu-huo Ji1 • Hong-mei Yuan3
1 Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China • 2 Department of Obstetrics
and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China • 3 Department of Anesthesiology,
Nanjing Maternity and Child Health Care Hospital, Nanjing Medical University, Nanjing, China
Journal: Molecular Neurobiology • Publisher: Springer • Published Online: 09 December 2015•
Volume: 53 • Issue: 10 • Pages: 6680-6689 • DOI: 10.1007/s12035-015-9571-x

3. BACKGROUND OF THE STUDY:
AFTER
EFFECTS
• DEFINITION: Chronic psychiatric disease following severe traumatic event or physiological stress.
IMAGE SOURCE:
https://www.123rf.com/photo_117009977_stock-vector-
post-traumatic-stress-disorder-ptsd-signs-and-symptoms-
illustrations-depict-man-with-post-traumatic-.html
POST TRAUMATIC STRESS DISORDER (PTSD)
• AFTER EFFECTS:
 Inability to concentrate
 Expresses aggressive behaviour
 Has frequent flashbacks of the traumatic event.
• RESEARCH GAPS:
 Mechanisms underlying the intricate biological and psychological
symptoms remain to be determined.
 No specific pharmacological agent is currently available to treat
PTSD symptoms.
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4. RESEARCH GAP ADDRESSED:
PTSD
ROS and
Inflammation
• Cues for determining mechanism behind how ROS and inflammation causes PTSD from other studies :
 By impairing neural function and synaptic plasticity in animal model (Wilson CB, 2013).
 Upregulated lipid peroxidation levels and proinflammatory cytokines are observed in PTSD patients (Gola H,2013).
• HYPOTHESIS: NOX2 mediated oxidative stress induced PV interneuron loss causes behavioural changes in the PTSD
patients.
Mechanism
?
NOX2 - Nicotinamide adenosine dinucleotide phosphate
(NADPH) oxidase 2
PV- Parvalbumin
PV loss in
neurodegen
er-ative
diseases
Increase of
oxidative
stress
NOX2
(Schiavone S, 2009)24-10-2019 4

5. • To investigate the possible role of NOX2-mediated PV interneuron
loss in the development of behavioral alteration in a rat model of
PTSD induced by Single prolonged stress (SPS).
OBJECTIVES:
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6. Development of PTSD model by Single Prolonged Stress (SPS)
METHODOLOGY:
Image
source:https://www.criver.com/reso
urces/sprague-dawley-rats-sas-sd-us-
pricing
(250–300 g)
Image source: (Oh et al., 2018)
Restrained-2h forced to
swim - 20
min
Rest -15 min exposed to
diethyl
ether until loss
of
consciousness
Left undisturbed for 7 days
Control
Rats were left undisturbed in their home cage.
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7. EXPERIMENTAL GROUPS
Image
source:https://www.criver.com/
resources/sprague-dawley-rats-
sas-sd-us-pricing
CONTROL SPS MODEL
APOCYNIN
TREATED
(5mg/kg, sc)
APOCYNIN
UNTREATED
APOCYNIN
TREATED
(5mg/kg, sc)
APOCYNIN
UNTREATED
EARLY
TREATMENT
GROUP
DELAYED
TREATMENT
GROUP
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8. EXPERIMENTAL DESIGN
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9. BEHAVIOURAL TESTS
OPEN FIELD TEST – tests locomotor activity and anxiety
Image source:https://www.youtube.com/watch?v=gJDV2cp8w9E
More time the
animal spends in
outer edge: It is
MORE ANXIOUS
Less time it spends
in outer edge: It is
LESS ANXIOUS
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10. FEAR CONDITIONING TEST
• Rats were placed in a conditioning chamber (32
cm×28 cm×50 cm) consisted of four vertical
Plexiglas sides, with a floor consisting of horizontal
metal bars (0.5-cm diameter, spaced 1.5 cm apart)
connected to an electric shock generator.
• The rats were initially allowed to explore for 3 min.
• 30s of auditory tone followed by 2s of foot shock
was given.
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FEAR CONDITIONING TEST
CONTEXTUAL CONDITIONING TONE CONDITIONING
Freezing behaviour ( absence of movement except that involves breathing and
heart beats) was measured as magnitude of fear conditioning for 3min.
Rats were placed in the same cage
and were observed for freezing
behaviour without tone or shock
provided.
The auditory-cued fear test was performed 2 h
after the contextual fear conditioning. Rats
were placed in an altered chamber and
freezing behavior was observed.

12. RESULTS:
BEHAVIOUR
TESTING
EXPERIMENTAL
GROUPS
ELISA for:
• (TNF-α)
• Interleukin (IL)-
1β, IL-6, and IL-
10
Malondialdehyde
and Superoxide
Dismutase Assay
Western Blot of PV,
NOX2, 4-hydroxynonenal
(4-HNE), and β-actin
Immunofluoresence
for PV
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13. RESULTS:
BEHAVIOURAL TESTS
Data are presented as mean±SEM (n=12),*P<0.05
For the total distance traveled, there
was no significant difference among
the groups.
SPS exposure did
not affect gross motoric
behavior
Early treatment but not delayed treatment with apocynin increased the time in the center after
SPS.
Increase in anxiety
Less time spent in
center after SPS
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14. Decreased the freezing time to
context and tone after SPS
Early treatment but not
delayed treatment with APO
Decrease in fear learning
after SPS
FEAR CONDITIONING
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15. Data are presented as mean±SEM (n=8), *P<0.05
• Early treatment with APO attenuated hippocampal IL-6 levels induced by SPS.
• No difference in TNF-α, IL-1β, and IL-10 expressions at day 7 after SPS.
EVALUATION OF NEUROINFLAMMATION
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16. EVALUATION OF OXIDATIVE STRESS:
MALONDIALDEHYDE AND SUPEROXIDE DISMUTASE LEVELS
• Increased MDA levels at day 1 and day 3 after SPS when compared with the
control groups
• Early treatment with apocynin significantly decreased MDA levels at day 3 after
SPS.
no difference in SOD activity over time after SPS
Data are presented as mean ± SEM (n=8), *P<0.05
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17. • SPS progressively decreased
PV
expression, which was
accompanied by a significant
increase in NOX2 and 4-HNE
Expressions.
• Early treatment of APO blocked
oxidative stress and reversed PV
expressions to normal levels.
Data are presented as mean±SEM (n=4), # P<0.05 versus the control+ vehicle group;*P<0.05 versus the SPS+vehicle grou
EVALUATION OF OXIDATIVE STRESS: WESTERN BLOT
4- HNE : 4-Hydroxynonenal
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18. Positive correlation Negative correlation
CORRELATIONS DRAWN:
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19. • SPS reduced the PV levels in the CA1 and
CA3 regions of the hippocampus at 14 days.
• Early treatment with apocynin attenuated
the decreased PV expression induced by SPS.
Data are shown as mean±SEM (n=6),scale bar=100 μm. *P<0.05.
IMMUNOFLUORESCENCE:
TO DETECT PV LEVELS
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DISCUSSION
1
• SPS enhanced anxiety and fear learning behavior.
• SPS increased expressions of NOX2, oxidative
stress, neuroinflammation and decreased PV
expression.
2
• Early treatment with apocynin reversed all the
abnormalities.

21. 1
• NOX2 activation plays a key role in oxidative stress and
inflammation.
• This caused PV interneuron loss and consequent PTSD symptoms.
2
• Early pharmacological inhibition of NOX2 represents a potential
novel treatment strategy to reduce the harmful effects of traumatic
stress.
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CONCLUSION

22. • Wilson CB, McLaughlin LD, Nair A, Ebenezer PJ, Dange R,Francis J (2013) Inflammation and
oxidative stress are elevated in the brain, blood, and adrenal glands during the progression of
posttraumatic stress disorder in a predator exposure animal model.PLoS One 8:e76146.
doi:10.1371/journal.pone.0076146.
• Gola H, Engler H, Sommershof A, Adenauer H, Kolassa S,Schedlowski M, Groettrup M, Elbert T
et al (2013) Posttraumatic stress disorder is associated with an enhanced spontaneous production of
pro-inflammatory cytokines by peripheral blood mononuclear cells. BMC Psychiatry 13:40.
doi:10.1186/1471-244X-13-40.
• Schiavone S, Sorce S, Dubois-Dauphin M, Jaquet V, Colaianna M, Zotti M, Cuomo V, Trabace L et
al (2009) Involvement of NOX2 in the development of behavioral and pathologic alterations in
isolated rats. Biol Psychiatry 66(4):384–392. doi:10.1016/j.biopsych.2009.04.033
• Oh, J., Kim, Y., Kim, S., Lee, B., Jang, J., Kwon, S. and Park, H. (2018). Acupuncture modulates
stress response by the mTOR signaling pathway in a rat post-traumatic stress disorder
model. Scientific Reports, 8(1).
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ADDITIONAL REFERENCES:

23. FEEDBACK OF THE COURSE:
• The course Academic writing helped me a lot to know more about
research writing, which will be undeniably helpful for me, when I will
be publishing my original research articles or review articles.
• The course also aided me in the process of literature survey and also in
finding the best suitable journal for publication of our research work in
a more suitable journal.
• The course will definitely help me in becoming a better researcher and
in exhibiting the results obtained by me all over the globe.
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THANK YOU