Stanford University
Department of Comparative Medicine
Developing Best Care Practices and
Endpoint Guidelines for
Rodent Models of Neurologic Disease
Lisa Heath, DVM, DACLAM
Rodent Models
• Experimental Allergic Encephalomyelitis
(EAE)
• Amyotrophic Lateral Sclerosis (ALS)
• Huntington’s Disease
Guidelines and Endpoints
Developing Guidelines
• Resources
• Working with Researchers
Multiple Sclerosis in People

Fatigue

Numbness

Gait, balance,
coordination
problems
• Bowel/bladder
dysfunction
• Cognitive
dysfunction
• Pain
Multiple Sclerosis
Health.howstuffworks.com
Multiple Sclerosis
•http://www.elements4health.com/images/stories/conditions/myelin-sheath.jpg
EAE Models

Active EAE:
immunized with myelin antigen or peptide
emulsified in CFA

Monophasic or relapsing-remitting disease

Disease process commences 10-15 days postimmunization

75-80% incidence of disease
EAE Models

Passive or adoptively transferred EAE:
Recipient mice injected with activated,
myelin-specific T cells (harvested from
immunized donor mice)

Relapsing-remitting disease
EAE Guidelines Benchmarking

We benchmarked with other institutions to
see if their IACUC had specific guidelines to
cover EAE rodent models

64 institutions were surveyed regarding
whether or not they had rodent EAE models
•

19 institutions responded
Survey Results
Data and graphs courtesy of Sonja Wallace
EAE Benchmarking Survey
Breakdown of Respondents
5%
5%
53%
University
Biotech/Pharma
Private Institution
11%
Government
26%
CRO
EAE Benchmarking Survey
IACUC Guidelines
Specific EAE
Guideline
6%
68%
26%
Humane Endpoint
Guideline
No Guideline
Specified
EAE Benchmarking Survey
% Weight Loss Endpoint
5%
10% Loss
5%
15% Loss
11%
20% Loss
69%
5%
5%
25% Loss
30% Loss
Not Specified
EAE Benchmarking Survey
Separate Debilitated Animals
16%
63%
Separate
Don't Separate
Not Specified
21%
Summary

Majority of respondents had an IACUC guideline
specifically for EAE models

Some respondents used their humane endpoint
guideline for EAE models

Majority of respondents used 20% percent weight loss
as an endpoint

Majority of respondents separated
symptomatic from asymptomatic animals
Refining Guidelines
• Intervention/Monitoring:
When and how often
• Nutrition and Hydration:
What, when and how often
• Include the Researchers
EAE Scoring
Score 0 = No overt signs of disease
Requirements:
 Label Special Request Card with
“EAE Model” on Experimental Day 1

Baseline Body Weight recorded on
Day 1

Assess and record scoring
observations at least 3 times per
week.
EAE Scoring
Score 1 = Limp tail, flaccidity of tail
Requirements (same as for Score 0)
EAE Scoring
Score 2 = Paraparesis; limp tail and hindlimb
weakness, waddling gait.

Provide gel pack and/or moistened food on
cage floor, or other appropriate supplements.

Weigh and/or assess body condition score at
least 3 times per week.
Tail Suspension Test
A.
B.
Normal Plantar Reflex
Hindlimb Clasping
Animal Welfare Information Center Bulletin, Winter 1999/2000,
Vol. 10 No. 3-4
EAE Scoring
Score 3 = Hindlimb paralysis. Total loss of moment in hindlimbs.
Mouse moves itself with forelimbs.

Daily Scoring and monitoring.

Weigh and/or assess BCS at least once daily.

Assess hydration status (body weight and skin turgor).

Assess bladder for evidence of urinary retention. Express
bladder as needed.

Provide fluid supplementation, SC or oral gavage.
EAE Scoring
Score 4: Subdivided into 4A and 4B
(for guidelines only*)

4a = Hindlimb paralysis and mild forelimb
weakness

4b. Hindlimb paralysis and moderate to
severe forelimb paralysis; lateral
recumbency.
EAE Scoring
Score 4b. Hindlimb paralysis and moderate to
severe forelimb paresis; lateral recumbency.

Mice must be monitored at least twice daily for
no more than 24 hours.

Any animal that does not remit to score 3
within the time frame must be euthanized.
EAE Scoring
Score 5 : Moribund, or complete hindlimb
paralysis with moderate to severe forelimb
paresis with no remittance after 24 hrs and
intensive supportive care.

Euthanize immediately.
EAE Scoring

Any animal which loses >20% body weight
or has a body condition score <2 must also
be euthanized, regardless of EAE score.
A Sidenote on Body Weight Recording

Each mouse’s body weight data is unique

No averages!

Data recording: pre-disease weight and
pre-calculated 20% loss cut-off
EAE Scoring: Mouse Posture
0 : Tail has tone.
1: Tail no longer stands up, loss of tail
tone. Walks normally.
2: Limp tail, hindlimb dysfunction.
3: No longer holds onto underside of
cagelid with hindlimbs. Moves
hindlimbs.
Current Protocols in Neuroscience
4: Hindlimbs drag behind, not used for
movement.
EAE Scoring
Models of Multiple Sclerosis. ACNR. Vol 6. No 6. 2007
Amyotrophic Lateral Sclerosis

Fatal neurodegenerative disease

Familial and Sporadic
Lou Gehrig’s Disease

Loss of upper and lower motor neurons

Paralysis of muscles
www.alsa.org
ALS Models
SOD1 mice
ALS Mouse Models





Muscle atrophy
Weight loss
Limb weakness and paralysis
Oral and pharyngeal dysphagia
Respiratory weakness and paralysis
ALS Scoring System
Score Full extension of hind legs away from lateral
of 0
midline when mouse is suspended by its tail,
and mouse can hold this for two seconds,
suspended two or three times.
Score Collapse or partial collapse of leg extension
of 1
towards lateral midline (weakness) or
trembling of hind legs during tail suspension.
Score Toes curl under at least twice during walking of
of 2
12 inches, or any part of foot is dragging
along cage bottom/table.*
Score Rigid paralysis or minimal joint movement, foot
of 3
not being used for generating forward motion.
Score Mouse cannot right itself within 30 seconds after
of 4
being placed on either side.
Working with ALS Mice: Guidelines for preclinical testing & colony management. The Jackson Laboratory
ALS Scoring System

*If one hind leg is scored as 2, food pellets
are left on bedding.

If both hind legs are scored as 2, Nutra-Gel
is provided as food in addition to food
pellets on bedding and a long sipper tube is
placed on the water bottle.
Working with ALS Mice: Guidelines for preclinical testing & colony management. The Jackson Laboratory
ALS Scoring System:: 8 Point Scale
CS 0
CS 1
CS1.5
CS2*
No evidence of disease
Shaking or splaying of hind legs when
suspended by tail
Weakness in one hind leg
Change in gait (used as disease onset when
attained on two consecutive days
*disease onset
CS 2.5 Extreme weakness in one hind leg (inability to
dorsiflex)
CS 3
Extreme weakness in both hind leg
CS 3.5 Functional paralysis in one hind leg
Solomon, et.al. One Universal Common Endpoint in Mouse Models of Amyotrophic Lateral Sclerosis. June 2011, vol. 6,
issue 6.
Endpoint Scoring for ALS: 8 Point Scale
CS4
Both hind legs functionally paralyzed
CS4+
CS4 plus the earliest time mice attained one of the
following:
a) weight loss > 20% vs. body weight immediately
prior to a clinical score of 2*
b) weight loss > 20% vs. peak body weight
c) body condition score <2
d) righting reflex > 20s (clinical score of 5)
CS5
CS 4 plus a righting reflex >20s
Huntington’s Disease
Huntington’s Disease Scoring:
Part 1
Score of 0 Normal
Score of 1 Clasps hind limbs within 30s of being
suspended
Score of 2 Clasps hind limbs within 5s but
recovers quickly after 30s
Score of 3 Clasps hind limbs within 5s, difficulty
recovering when released after 30s
Auerbach, et.al. Human Molecular Genetics, 2001, Vol. 10, No. 22.
Huntington’s Disease Scoring: Movement
Abnormalities






Tail stiffness or paralysis
Hind-limb stiffness or paralysis
Reduced left-right alteration of
hindlimbs (“hopping”)
Resting tremors
Difficulties walking after handling
Seizure-like episodes
Huntington’s Disease Scoring:
Part 2
Score of 0
Normal
Score of 1
One movement abnormality
Score of 2
Two movement abnormalities
Score of 3
Three movement abnormalities
Huntington’s Disease Scoring: Combining
Part 1 and 2

Scores range from 0:0 (normal, no disease)
to 3:3 (greatest expression of clinical signs)

Choose score/score combinations for adding
care :
•
•
food and water accessibility, supplements
heat source, nesting materials
Huntington’s Disease
Reference:
Guyenet, et.al. (2010).
A Simple Composite Phenotype Scoring System for
Evaluating Mouse Models of Cerebellar Ataxia.
JoVE. 39. ID 1787.
Tests: ledge test, hindlimb clasping, gait, kyphosis
Huntington’s Disease

Noise sensitivity: construction, cage changes,
hoods and ventilation

Seizure-like activity and deaths related to noise
and other disturbances

Hypothermia in late stage disease:
external heat source;
nestlets vs. paper nesting material
Enviro-dri® (Shepherd Specialty Papers) www.ssponline.com
Summary

Meet with investigators: look at their scoring
and weight data

Provide specific solutions and options

Include investigators on guidelines
development