Disease Models For Comparative Anatomy

Question 1

What is angiogenesis?

Answer 1

Formation of blood vessels during embryogenesis.

Question 2

Duchenne Muscular Dystrophy mode of inheritance

Answer 2

x-linked recessive

Question 3

Protein lost in Duchenne Muscular Dystrophy

Answer 3

dystrophin

Question 4

Duchenne Muscular Dystrophy early phase clinical presentation

Answer 4

-Slow motor development, failure to meet milestones such as running
-Develops difficulty standing from seated position, resulting in “Gower manouvre”, at 3-4yo

Question 5

Duchenne Muscular Dystrophy intermediate phase clinical presentation

Answer 5

-Difficulty standing
-Progressive loss of ambulation

Question 6

Duchenne Muscular Dystrophy late stage clinical presentation

Answer 6

-Complete loss of ambulation at around 12yo
-Scoliosis
-Inability to eat
-Inability to breathe due to loss of diaphragm muscle

Question 7

Dystrophin gene key characteristics

Answer 7

-Very large (2.3-2.5megabases)
-Large introns
-On the X chromosome
-External (brain, muscle and purkinje) promotors to produce full length protein
-Shorter protein made using internal promotors

Question 8

Where is dystrophin deposited?

Answer 8

Just below sarcolemma (muscle membrane)

Question 9

How do most mutations causing Duchenne Muscular Dystrophy result in dystrophin loss?

Answer 9

Disrupt the open reading frame, causing failure to fully translate the mRNA and produce a functioning protein

Question 10

Therapeutic targets for Duchenne Muscular Dystrophy treatment

Answer 10

-Restore dystrophin (main target)
-Disrupt downstream effects of dystrophin loss on activity induced muscle damage and cell signalling

Question 11

Pathogenesis of Duchenne Muscular Dystrophy

Answer 11

Dystrophin loss -> poor anchoring of muscle cells to sarcolemma -> activity induced damage -> calcium influx -> hypercontraction, overloading of mitochondria, hyperactivation of proteases ->oxidative stress, nitrosylative stress, necrosis, fibrosis, inflammation

Question 12

Histopathological findings in early-intermediate DMD skeletal muscle

Answer 12

-Fibres rounded instead of hexagonal
-Fibres surrounded by fibrosis instead of being densely packed
-Infiltrate of macrophages, neutrophils, fibroblasts and T cells

Question 13

Treatments to improve quality and length of life in DMD patients

Answer 13

-Corticosteroids to slow progression
-Drug treatment of developing cardiomyopathy
-Respiratory assistance by positive pressure ventilation

Question 14

Animal model definition

Answer 14

Living non-human animal used in investigation of pathophysiology and development of treatments for a human disease

Question 15

Phenotypic animal model

Answer 15

Mirrors human pathology and clinical signs

Question 16

Genotypic animal model

Answer 16

Genetically similar to human patient

Question 17

One use for computer (in silico) modelling in early drug development

Answer 17

Determining structure-activity relationships

Question 18

One use for cell culture models in early drug development

Answer 18

Establishing dose-response curves to guide in vivo studies

Question 19

Uses for wildtype animals

Answer 19

Toxicology and safety pharmacology

Question 20

Invertebrate species used to model Duchenne Muscular Dystrophy

Answer 20

-Caenorhabditis elegans (nematode)
-Drosophila

Question 21

Vertebrate species used to model Duchenne Muscular Dystrophy

Answer 21

-Zebrafish
-Mammals including dogs, pigs, rabbits, cats, rats

Question 22

Problem with cat model of Duchenne Muscular Dystrophy

Answer 22

These cats develop increased tongue and diaphragm size, making it hard to keep them alive for study duration

Question 23

Problem with pig model of Duchenne Muscular Dystrophy

Answer 23

Lots of mortality soon after birth which is not seen in humans, perhaps making this model too phenotypically dissimilar to human patients

Question 24

Three most common animal models of Duchenne Muscular Dystrophy

Answer 24

-Mdx mouse
-GRMD (golden retriever with muscular dystrophy) dog
-DeltaE50-MD dog (MD beagles)

Question 25

Does mouse model genetic background affect phenotype?

Answer 25

Yes

Question 26

Knockout crosses with Mdx mice

Answer 26

Exacerbate pathology but do not genetically resemble DMD patients (except for Cmah-/- as humans don’t have Cmah)

Question 27

Mice clinical signs and pathology severity

Answer 27

Much milder than human and dog

Question 28

Mouse model histopathology

Answer 28

-Relatively mild
-Worse in diaphragm than skeletal muscles
-Diaphragm shows fibrosis, mononuclear cell infiltrate but skeletal muscle doesn’t

Question 29

Dog model histopathology

Answer 29

-More human-like than mouse model
-Already lots of fibrosis and cell infiltrate at 3 months

Question 30

Eccentric contraction

Answer 30

Muscle experiences lengthening forces while contracting

Question 31

Pathology in DeltaE50 dog

Answer 31

-Progressive accumulation of muscle fibrosis
-Pronounced, persistent inflammatory response
-Peak inflammation/degradation at 6-9mo
-Characteristic changes in blood biomarkers
-Marked muscle atrophy
-Very sensitive to eccentric contraction induced damage

Question 32

Statistical importance of DeltaE50 dog model

Answer 32

-Therapeutic responses >25% can be detected with n=6
-Most statistically valuable data at 3-12mo
-Unnecessary to keep dog alive after 12mo
-Decrease in biopsies needed

Question 33

Common use of dog and mice models

Answer 33

-Mice used to generate statistically robust experiments
-Dogs used to confirm the results in mice, demonstrating reduction in clinical signs

Question 34

Biomechanical considerations for DMD models

Answer 34

-Humans are bipedal whereas animal models are quadrupedal
-Severity of pathology is proportional to body size

Question 35

Proof of concept studies

Answer 35

Dosed for maximal effect, often via routes that don’t translate to humans (eg peritoneal) to prove the potential of a novel treatment

Question 36

Translational studies

Answer 36

Dose and route of administration likely to translate to humans. Must have excellent experimental design to ensure this.

Question 37

A well-designed in vivo experiment must have:

Answer 37

-Positive and negative control groups
-Animals randomly assigned to experimental groups
-Researchers are blinded
-Excellent records

Question 38

What determines if a treatment is useful?

Answer 38

Statistical significance AND larger effect size

Question 39

Reasons for poor translation from experimental to clinical application

Answer 39

-Inadequate randomisation and blinding
-Failure to have independent labs confirm results
-Lack of translational testing
-Low effect size
-Poor statistical significance