Presentation on Traumatic brain injury

1. Study On The Neuroprotective Potential of Vitamin B1
(Thiamine) In Traumatic Brain Injury (TBI) In Adult Mice
Department of Chemistry
Presented by: Hafiza Mubaraka Rahat (SU-17-01-114-014)
Mansoor Husn (SU-17-01-114-007)
Palwasha (SU-17-01-114-003)
Sarhad University of Science and Information Technology,
Peshawar, Pakistan
Supervised by: Dr. Shahid Ali Shah
(Assistant Professor)

2.  Literature Review
 Aim & Objectives
 Observations and Data
 Expected Outcome
 Methodology

5.  To investigate the Vitamin B1 neuroprotective ability
against TBI in mice.
 To analyze the antineuroinflammatory effect of Vitamin B1
against TBI.
 To ensure the neuroprotective mechanism of Vitamin B1
against TBI.

6. OBSERVATIONS AND DATA
Y-Maze Test
GROUP: TOXIC (TBI)
Mouse I Mouse II
Trail I (%) 50 57
Trail II (%) 52.6 72.2
Trail III (%) 64 66
Average (%) 55.53 65.07
GROUP: TOXIC+DRUG (TBI+B1)
Mouse I Mouse II
Trail I (%) 30.7 33.6
Trail II (%) 85.7 50
Trail III (%) 66 65
Average (%) 60.80 49.43
Percentage of spontaneous alteration =
𝑵𝒐 𝒐𝒇 𝑻𝒓𝒊𝒑𝒍𝒊𝒄𝒂𝒕𝒆𝒔
𝑻𝒐𝒕𝒂𝒍 𝑵𝒐 𝒐𝒇 𝒂𝒓𝒎 𝒆𝒏𝒕𝒓𝒊𝒆𝒔 −𝟐
× 100

7. Observations and Data
GROUP: TOXIC (TBI)
Mouse I Mouse II
Trail I (Sec) 41 9
Trail II (Sec) 11 5
Trail III (Sec) 11 21
Average (Sec) 21 11.7
GROUP: TOXIC+DRUG (TBI+B1)
Mouse I Mouse II
Trail I (Sec) 6 15
Trail II (Sec) 9 8
Trail III (Sec) 7 6
Average (Sec) 7.3 9.7
Morris Water Test

9. Methodology

10. 8 Albanian white male mice were selected and divided into 4 categories.
MICE SELECTION
CONTROL
• Healthy Mice
with no injuries
TOXIC+
DRUG
• Induced TBI and
injected with
Thiamine
TOXIC
• Induced TBI with
no drugs
injected
DRUG
• Injected
Thiamine to
healthy mice
with no injuries

11.  2 out of 4 groups that were to be traumatized
were separated.
 To induce the injury, The mice were
anesthetized with chloroform.
 The brain of the anesthetized mice were
exposed by opening the skull with surgical blade.
 The exposed brain was then hit with a weighted
rod to induce traumatic effects.
INDUCING TBI

12. INJECTIONS OF THIAMINE
 Toxic and Toxic + Drug categories were injected
with thiamine solution.
 Injection of thiamine was prepared with following
composition;
 Thiamine: μg.
 Saline Sol: μL.
 Total 7 injections were injected per mouse every
3rd day over the period of 3 weeks.
 Intra peritoneal pathway was used and 300 μL of
thiamine sol was injected to each mouse.

13. BEHAVIORAL TESTS
 Y-Maze Test.
 Morris Water Test.
Two different behavioral tests are performed to study the
neurotoxic effects of TBI. And neuroprotective effects of
thiamine (vitamin B1). These tests are;
These tests are performed to study the memory and brain
functions of the mice.

14. Setup consists of a Y-shaped maze which have
three identical arms at a 120° angle from each
other.
The mouse is set free in the center of the maze
to explore tall three arms freely for 8 minutes
and arm entries are recorded
At the end, the percentage of alternation is
calculated using recorded arm entries and
number of triplicates made.
 Y Maze Spontaneous Alternation is a behavioral test for studying spatial learning and
memory of the mice.
 A healthy mouse usually tend to inspect a new arm of the maze rather than returning to one
that was previously visited.
Y-MAZE TEST

15. MORRIS WATER TEST
 This test is performed to assess the effects of
test compounds on both learning and memory
of the mice. By recording the time to find the
hidden platform form the starting point. We can
evaluate their ability to learn and remember the
location of the platform.
 Morris water test is used to evaluate and
compare learning and memory in mice.

16. BRAIN EXTRACTION
The mouse is first
dipped into a beaker
having chloroform to
anesthetize it.
Then the anesthetize
mouse is placed in
dissecting tray and the
head of the mouse is cut
off with dissecting
scissors.
The skull of the mice is
removed to expose the
brain of the mouse.
The exposed brain is
left off with tweezers
and placed in an
epindorph tube.

17. HOMOGENIZATION
 Homogenization is defined as process of reducing a substance to
extremely small particles and distributing it uniformly throughout a fluid.
Step 1:
Half of the mouse brain is taken into a test tube
and 1mL TPER is added into it.
Step 2:
Tissue homogenizer is set up and cleaned with 1x
TBST and distilled water respectively.
Step 3:
Test tube having brain and TPER is placed under the
tissue homogenizer and ran until the brain is
completely homogenized.

18. CENTRIFUGATION
 Centrifugation is the process that uses centrifugal force for the
separation of two liquids in a mixture. In this process, the denser
component of the mixture sediment and the lighter component
will be supernatant.
 The brain is transferred into Epindorph tube and placed into the
rotor of the centrifuge.
 The led of the centrifuge is closed and the parameters are set to
3 0C at 14000 RPM and ran for 25 minutes.
 At the completion of the process, The E. Tubes are taken out and
two distinct layers can be noticed.
 The upper liquid layer that is protein, is transferred into another
E. Tube with micropipette and refrigerated for future use in gel
electrophoreses.

19. GEL ELECTROPHORESIS
Gel electrophoresis is a process used to separate mixtures of proteins according to
molecular size. In gel electrophoresis, the molecules to be separated are pushed by an
electrical field through a gel that contains small pores. The molecules travel through
the pores in the gel at a speed based on their size. This means that a small protein
molecule will travel a greater distance through the gel than the larger protein molecule.

20.  Gel electrophoresis involves an electrical field that separates the molecules
with electric charge. Proteins, however, are not negatively charged; thus, when
we want to separate proteins using gel electrophoresis, we must first mix the
proteins with a detergent called sodium dodecyl sulfate.
 This treatment makes the proteins unfold into a linear shape and coats them
with a negative charge, which allows them to migrate toward the positive end
of the gel and be separated.
 Finally, after the protein molecules have been separated using gel
electrophoresis, bands representing molecules of different sizes can be
detected.
PROCESS OF
GEL ELECTROPHORESIS