ttt 2015 academic conference presentation
TRANSCRIPT
Christine Yen [鄧思婷] Taiwan Tech Trek Academic Conference August 14, 2015
Modeling Neurodegenerative
Diseases in Mice
1
Parkinson’s Disease (PD)
2
• Characteristics
- Loss dopaminergic neurons in substantia nigra pars compacta (SNpc)
• Causes: unknown, sporadic
- Genetic, environmental factors
• Prevalence
- Primarily among elderly
- 1-3% of population over 55 yrs
http://what-when-how.com/wp-content/uploads/2012/04/tmp3675_thumb_thumb.jpg
Parkinson’s Disease
• Pathophysiology
-SNpc releases dopamine-> receptors in striatum
- Dopamine deficiency
- Striatum cannot signal
motor thalamus, basal ganglia
• Symptoms - Tremors, rigidity, bradykinesia
difficulty walking
- Dementia
http://file.scirp.org/Html/2-2620020/24f49151-d68f-4540-bd71-c1b45e35562d.jpg http://discovermagazine.com/~/media/Images/Issues/2014/March/dopamine-chart.jpg
Striatum
SNpc
3
6-OHDA Model
4
• What is 6-OHDA?
‒ Neurotoxin induces SNpc neurodegeneration
• Dopaminergic selectivity - 6-OHDA structural analog:
dopamine -Taken into SNpc via dopamine transporters (DAT)
http://openi.nlm.nih.gov/imgs/512/250/2957224/2957224_PD2010-427810.002.png
Before
After
• Mechanism of neurotoxicity
- Degradation by MAO
- Generates H2O2, free radicals
- Inhibits mitochondrial complex I
• Why model PD?
- No cure, limited treatments
- Better understand pathology
- Develop drugs: pre-clinical trials
6-OHDA Model of PD
http://www.sciencedirect.com/science/article/pii/S030100820100003
Method of implementation
• Stereotaxic surgery
– Direct injections striatum
– Unilateral vs bilateral injections
• Retrograde degeneration of SNpc
– Mimics PD: humans
• Subjects
– 9 mice injected w/ 3 mg/kg 6-OHDA
– 3 controls injected with saline
6
Motor Outputs
Left Brain Right Brain
Controls left side of body
Controls right side of body
6-OHDA lesion
Motor impairments develop
Motor function unaffected
7
Onset: PD Phenotypes
• Successful model: replicates phenotypes
- Symptoms typically emerge within weeks
• Conduct behavioral assays
- Assess degree motor deficits develop
• Establish SOP’s: behavioral tests
-Ensure protocols run smoothly
http://www.nature.com/nrn/journal/v10/n7/fig_tab/nrn2652_F1.html 8
Behavioral Assays: Overview
Rotarod Test
Adhesive Removal Test
Forelimb Rearing Test
9
Rotarod Test
• Assesses coordination, balancing abilities
• Rod acceleration: 4-40 rpm
• Walked 300s, rested: 15 min intervals
• Record time spent continuously walking
10
(3 weeks post injection)
Rotarod in Action
11
Data Collection/Results
12
13
Rotarod Performance: 6-OHDA groups vs control (training period)
180
190
200
210
220
230
240
250
260
270
280
7/29 7/30 7/31
6-OHDAgroups
Control group
Ave
rage
Tim
e w
alke
d (
s)
Dates
14
215
220
225
230
235
240
245
250
255
260
265
7/29 8/5
6-OHDA groups
Control group
Rotarod Performance: 6-OHDA groups vs control A
vera
ge T
ime
wal
ked
(s)
Dates
Adhesive Removal Test
• Measure sensorimotor deficits
• Adhesives: alternately applied
• Left, right forepaw
• Measure time: sense, remove adhesives
• Mice affected by 6-OHDA
15
Sticker
Forelimb Rearing Test
• Assess forelimb motor impairment
• Placed glass cylinder
• Note forelimb favored: when rearing
• Record frequency use:
• Left, right or both forelimbs
• Mice affected by 6-OHDA:
• Ipsilateral forelimb dependence
Forelimb Rearing Test
16
Modeling Alzheimer’s Disease (AD)
17
Morris Water Maze
• Alzheimer’s disease behavioral assay
• Tests spatial learning/memory
• Developed SOP’s
• Protocol runs smoothly
• Establish appropriate environment
18
MWM Procedure
▪ Mice swim in tub: opaque water
▪ Find submerged platform
▪ Visual cues posted: walls
▪ Released: four different directions
▪ Factors of importance
NW
W SE
SW
19
Conclusions
▪ Early stages: developing PD phenotypes
▪ SOP’s: identify, correct interfering factors
▪ Lack training
▪ Timing: trials
▪ Mouse behaviors
20
Acknowledgements
▪ My appreciation to:
Dr. Huang for her guidance
National Animal Laboratory Center, NARLabs
The mouse subjects
▪ Thank you all!
21