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Mohamed Ahmed

on 30 October 2016

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Pentoxifylline Prevents Cognitive and Molecular Impairment in a Rat Model of Posttraumatic Stress Disorder
Mohammed Ahmed, B.Pharm, M.Sc. in Clinical Pharmacy
American Fellowship in Research Bioethics

Significance of our study
Posttraumatic Stress Disorder (PTSD):
Animals Housing and Grouping:
Eighty Wistar rats were obtained from animal house at JUST, randomly grouped (12/group) and housed (4/cage) under animal care standards of Animal Care and Use Committee at JUST.
Group 1
: post traumatic stress disorder (PTSD).
Group 2
: control (CON).
Group 3
: Pentoxifylline (100mg/kg) Group (PTX).
Group 4
: vitamin E (100mg/kg)
Group 5
: PTX with PTSD.
Group 6
: VitE with PTSD.
Experiment Timetable:
A. Behavioral Tests:
D. Brain Deived Neurotrophic Factor:
Behavioral Test:
Many protocols have been established to test different forms of learning and memory.
Radial Arm water maze (RAWM) was used to evaluate cognitive function -learning and memory in our rat model.
RAWM was used due to its availability in our laboratory, in addition to advantages over other types of animal mazes.
RAWM consist of circular tank divided
into six arms radiating from the center
filled with worm water.

A hidden platform is located at the end
of one arm (the goal arm).

Experiments were done in dimly light room with two different pictures on the walls which serve as visual cues for the rats.

RAWM Protocol:
An acquisition phase six learning trials followed by 5 min rest then the other six trials.
Thirty minutes after the acquisition phase rats were testes for short-term memory test.
long-term memories were evaluated after 5hrs and 24 hrs.
Animal Brain's Dissection and Molecular Analsysis:
At the end of our experiment, rats were sacrificed by decapitation followed by brain extraction and hippocampus isolation.
Tissues were stored at -40 until processing.
Enzyme-Linked Immunosorbent Assay (ELISA), was used to determine oxidative stress biomarkers and Brain Derived Neurotrphic Factor (BDNF) in the hippocampus.
1-Catalase (CAT).
2-Glutathione peroxidase (GPx).
3-Reduced glutathione (GSH).
4-Oxidized glutathione (GSSG).
5-Thiobarbituric acid reactive substances (TBARS).
Levels of Histones Acetylations:
Histone proteins were extracted from the hippocampus using available commercial Kit.
After histone extraction, the levels of global histone 3 and Histone 4K8 acetylations were detected using ELISA.
PTSD and Oxidative Stress:
Learning and Memory:
Histone acetylation :
PTSD is a syndrome which can be developed after exposure to severe traumatic event as those occurring during wars or natural disasters.
Despite the variety of pharmacological and psychological therapies, up to 30% of patients remain symptomatic 10 years after initial diagnosis.
PTSD is characterized mainly by persistent experience of trauma, avoidance, hyperarousal and impaired cognitive functions.
It is believed that these major disabilities are related to oxidative stress which is generated through the psychological or physical traumatic events.
can be defined as a process of acquiring information and knowledge, while
is the process by which information is encoded, stored and retrieved.
Memories can be divided according to duration of their storage into:
1- Short-term memory (STM): which last up to two hours.
2- Long-term memory (LTM): which last for several hours, days or even throughout life.
Aims of our Study:
Pentoxifylline (PTX)
To show that PTSD impairs both short-term and long-term memories, and reduce antioxidant mechanisms in the hippocampus.
To explore whether PTX improves PTSD-induced cognitive impairments.
Spatial memory
is responsible for recording information about the environment and its spatial orientation, which is highly dependent on hippocampus.
Try to understand the real mechanisms by which PTX improves PTSD-induced cognitive impairment.
Significance of Work:
Results of this study may:
2. Improve our understanding of the possible therapeutic effect of PTX and the mechanism(s) by which PTX improves cognitive impairments induced by PTSD.
3. Improve our understanding of enviromental-epigenetic interaction.
Single Prolonged Stress (SPS) as PTSD Animal Model:
1. Immobilization:
SPS is composed of one day prolonged stress which applied as follow:
Validity of SPS as PTSD Animal Model:
Validity of an animal model is usually tested using three criteria:
1-Face validity:
2-Construct validity:
3- Predictive validity:
Several recent studies showed that SPS carry the three types of validity especially predictive validity.
Immobilization for two hours in a transparent plastic bag, this act as psychological stress.
2. Forced swimming
Rats were forced to swim for 20 min, this act as a physical stress.
3. Ether anesthesia
Rats were placed in a covered glass container with saturated ether until loss of consciousness 1.5-2 minutes.
PTX; a methylxanthine derivative and a phosphodiesterase inhibitor, is a potent antioxidant that can readily pass BBB.
PTX was shown to reduce cognitive impairments associated with brain ischemia, status epilepticus, sleep deprivation and aging process.
Several studies had shown that the variety of pharmacological benefits are related to antioxidant and anti-inflammatory effect of PTX
We hypothesize that chronic administration of PTX can prevent cognitive impairments induced by PTSD.
About 5-8 % of men and 10-14% of women may develop PTSD at some point of their life.
1. Provide a new neuroprotective effect of PTX in PTSD-induced cognitive impairments.
Several studies concluded that oxidative stress in the brain, adrenal glands, and systemic circulation may play a critical role in the development and further exacerbation of PTSD.
Recent studies showed that PTSD may facilitate neurodegenration probably by oxidaitve stress.
Is the study of heritable changes in gene expression caused by mechanisms other than alterations in the underlying DNA sequence.

Since epigenetic changes do not involve DNA sequence alterations, they are reversible, unlike mutations of DNA
Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation as well as neural cell aging.
Histone proteins are subject to various post-translational modifications that affect gene regulation, including acetylating , methylation, and phosphorylation on many different amino acid residue.
Histone acetylation plays an important role in the activation of gene transcription.
Each point is the mean ± SEM of 12-14 rats. * indicates significant difference from control, (P < 0.05).
Each point is the mean ± SEM of 12-14 rats. * indicates significant difference from control, (P < 0.05).
Each point is the mean ± SEM of 12-14 rats. * indicates significant difference from control, (P < 0.05).
Each point is the mean ± SEM of 12-14 rats. * indicates significant difference from control, (P < 0.05).
Each point is the mean ± SEM of 12-14 rats. * indicates significant difference from control, (P < 0.05).

A bilateral limbic structure plays an important role in memory consolidation and spatial memory.
Human and animal studies showed that extensive hippocampal damage may lead to
-the inability to form or retain new memories.
Hippocampal function may be reduced by several disease processes, one of them is posttraumatic stress disorder in which symptoms severity of the disease is directly related to hippocampal hypofunction.
The Man with No Memory:
"Henry Molaison (HM) taught us about memory by losing his". Jenni Ogden PhD.

Two-way ANOVA; followed by Bonferroni posttest; was used to compare the number of errors during RAWM between groups.

One-way ANOVA; followed by Bonferroni posttest; was used to compare the results of immunoassays.

All statistical tests were carried out using GraphPad Prism software.

Significance levels were considered at P < 0.05.

Statistical Analysis:
Each point is the mean ± SEM of 12-14 rats. * indicates significant difference from control, (P < 0.05).
B. Oxidative Stress Biomarkers
C. Epigenetic Modifications
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