ILAR J
Volume 48, Number 4, 2007
Animal Models for the Study of Movement Disorders

Kaas.  Introduction: The Use of Animal Research in Developing Treatments for Human Motor Disorders: Brain-Computer Interfaces and the Regeneration of Damaged Brain Circuits, pp. 313-316.

SUMMARY: This introductory article discusses some current and potential animal models for human motor disorders.  Animals make good models for movement disorders because they have large brains in order to guide and control motor systems.  Currently, most research is being done in rats, but emerging research utilizes NHPs as models because they more closely mimic the large human brain and have fine motor control.  Research studies include those that provide a better understanding of how sensorimotor systems work, promote brain systems to reorganize with training, study self-repairing of brain circuits, promote therapy through electrode placement in the brain to stimulate motor systems, and provide understanding of the genetic, molecular, and cellular components of motor disorders.  One particular model explores the use of a brain computer interface in order to translate arrays of neurons to prompt communication and to manipulate robotic arms.  Further research is being done to decode neuron signals and to determine where to place electrodes in the brain.  Electrodes are currently being placed in Parkinson's disease patients to stimulate neurons.  Another model of focus utilizes animals to study spinal cord injury and regeneration of damaged neural pathways.  Often, spinal cord injuries do not involve complete transections, and the remaining neurons can be studied to determine how partial recovery can occur and how to enhance this recovery.  Current studies involve the application of anti-Nogo A and digesting the perineuronal network that might otherwise inhibit the sprouting of neurites.  Scientists are exploring the use of induced injury vs. spontaneous (accidental) injury in animal models and must take into account the resultant quality of life and to minimize the adverse effects in lab animals.

QUESTIONS:
1.	Utilizing computers to read the neuron arrays and translate them to mechanical function is called:
a) 	Brain-computer programming
b) 	Brain-computer interface
c) 	Brain-computer translation
d) 	Brain-computer alignment 
2.	NHPs are increasingly used as animal models in motor disorders for all of the following reasons, except:
a) 	Their brain structure mimics that of humans
b) 	They have finer motor control than rats
c) 	They are more prone to spontaneous injury
d) 	They are cheaper and easier to obtain
e) 	c & d
3.	All of the following may promote the sprouting of new nerve cell components, except:
a) 	Anti-Nogo-A
b) 	Transplantation of cells to the lesion site
c) 	Digestion of the perineuronal network
d) 	Stretching of the lesion site

ANSWERS:
1. 	B
2.	E
3.	D

Wallace and Sikoski.  Animal Care and Use Issue in Movement Disorder Research, pp. 317-322.

Species: All
RDD: Task 2,3,5,8 and 9.

SUMMARY: Animal models of movement disorders can be challenges for laboratory animal veterinarians and the Institutional Animal Care and Use Committee. These animal models may require additional care to maintain them as a healthy, humane model.

Animal models for stroke, brain and spinal injury, dystonia, Parkinson's disease (PD) and Huntington's disease are examples of animal models of movement disorders.

For research involving animal models of movement disorders an active, engaged and proactive approach by the investigators, veterinarians and IACUC is necessary.

There is a text, Animal Models of Movement Disorders (LeDoux 2005) which should be consulted if there is a question as to which animal model to use. The ILAR journal

48(4) Animal Models Used in the Study of Movement Disorders, 2007 has many excellent review articles on animal models.

Protocol Review: Appropriate justification of number of animals is required.

If the researcher is creating a genetically engineered mouse model they also need to calculate then minimum number of animals needed to generate the experimental population. IACUC's must also ensure the animals are placed and reported in the appropriate pain/distress category. Very important in protocol review is criteria for removal or euthanasia of animals. The challenge is to balance the humane treatment of animals with the scientific goals of the study. Researchers', IACUC's and veterinarians need to reach a consensus on how to treat health issues before the study starts. Animal models of movement disorders frequently require special husbandry and veterinary care procedures. These requirements need to be clearly described in the protocol. IACUC's also need to give special attention to the training of all personnel involved in the study.

Everyone should understand how to differentiate from "normal", "expected presentation" and '"unexpected presentation". If compounds such as MPTP will be used all personnel need to be trained on human health concerns and safety associated with the use of the compound.

Post- Approval Monitoring: This is always important, but especially in animal models of movement disorders.

Reporting Requirements: When the species is a USDA covered species, the IACUC must track and report appropriate pain/distress category. If there are differences in opinions it is important to document and report minority views. Any animals listed in Category E need a detailed statement explaining the procedure and the rationale for withholding pain-relieving medications

The annual report to the USDA must also include exceptions to the USDA regulations.

Animal Care Considerations

Environment:  Usually in lab animal medicine it is relatively easy to ensure that the animal's environment, correct temperature, humidity, size of cage, bedding is appropriate. In the case of specific models, such as the mouse model of Parkinson's disease, MPTP injection in mice and cause a drop in temperature that will lead to acute death unless the environmental temperature is elevated.

Caging: This is usually also very straightforward, dictated by regulations or guidelines.

Animals which have been surgically manipulated to produce spinal cord injuries may need a different size cage. Monkeys that have had MPTP to create a model of Parkinson's disease will do better in a cage rather than a large pen where they can move around a lot.

Usually a perch is considered enrichment for a monkey but may be contraindicated in monkey models of Parkinson's disease, as they may fall of the perch and get hurt. There may be need to justify withholding enrichment if the enrichment is known to attenuate the effects of MPTP on mice. Additional precautions may be needed in animal cages such as padding the edges of PD monkeys so they do not injure themselves on edges that would not be a problem for a "normal" monkey. Cleaning and sanitization schedules may need to be modified for these animal models.

Access to Food and Water: All animals should have daily access to water and food that is palatable, free of contaminants and nutritionally adequate. Animals that may have difficulty accessing food and water due to the experimental procedures performed on it may need help. Monkeys receiving MPTP require supplemental feeding and watering for a certain time after the drug is administered. Rodents with spinal cord lesions may not be able to access food and water at the top of the cage and need to have it provided at the bottom. If animals are being used in behavioral conditioning experiments which include regulating food or water intake, body weight and hydration status need to be monitored regularly. In some cases restricting water access during the evening may be needed to assure humane care. Monkeys with a spinal cord injury model may be unable to micturate during the early stages of recovery.  During the day they may have free access to water and their bladder is expressed manually as often as needed, at night their access to water is limited so that the bladder will not distend overnight.

Veterinary Medical Care: Just like the previous categories veterinary medical care is required for all animal research. Due to the invasive nature of the models, animals in this category most often require more intensive veterinary care. Spinal cord injury models may require bladder expression multiple times per day. Peripheral nerve regeneration studies in rats and rabbits may result in self-trauma, decubitus ulcer formation and lack of grooming. Veterinary care could include padded bandages, E-collars as well as general supportive care.

Summary: Research using animal models of movement disorder requires that IACUC's, researchers and veterinarians work well together, communicate and train, and commit to following established guidelines.

QUESTIONS:
1. 	If a compound such as MPTP is used in an animal model, what other concerns should the IACUC address?
a.  	Have the MSDS sheet on hand
b. 	Address safety issues regarding radioactivity
c. 	Include training on human health concerns and safety for all personnel
d. 	Make sure everyone understands the mechanism of action of compound
2. 	True/False  In some cases withholding water from an animal overnight could be considered part of humane care?
3. 	For all covered species sported under Category E, what is the IACUCs responsibility in annual reports?
a. 	Same as for all other Categories
b. 	Rationale for why two major operative procedures were performed needs to be outlined
c. 	Detailed explanation of procedure and reason for withholding pain medications
d. 	Minority opinions do not need to be reported

ANSWERS:
1. 	C
2. 	True  
3. 	C

Steindler.  Stem Cell, Regenerative Medicine, and Animal Models of Disease, pp. 323-338.

No summary was provided.

QUESTIONS:
1.	Stem cells may:
a.	Treat and cure most of human injures and diseases
b.	Cause tumors by themselves
c.	Both are true
d.	None are true
2.	T/F A stem cell works to build normal or abnormal tissue indistinctly
3.	The regenerative medicine should continue efforts to:
1	
2	
2	
4.	The objective of a stem/progenitor neural cell in vitro protocol is:
a.	Isolating, expanding and priming stem/precursor/neural cells 
b.	Encourage reactive cell genesis and appropriate functional integration in diseased or injured tissues 
c.	Facilitate the propagation into the right type of neural precursor cell need in the protection or replacement protocol 
d.	a and c are true
5.	A biogenic factor is: 
a.	A compound secreted by stem/progenitor cells 
b.	A compound that affect stem/progenitor cells 
c.	A compound that allows identification of stem/progenitor cells 
d.	b and c are true
6.	T/F To test stem cell-related therapies, only animal models will be useful
7.	The protocol to replace neurons that generate movement-related disorders might include:
-Exposure....
-Replace
-Promote
8.	The cells used in cell transplant approaches may come from:
a.	Hematopoietic stem/progenitor cell 
b.	Neuropoietic stem/progenitor cell 
c.	Differentiated somatic cells dedifferentiated with growth factors 
d.	All are true
9.	The way to induce cancer in mice models are:
-
-
-
	and these models are essential to testing _________ ___________ and __________ ___________
10.	T/F Regenerative medicine and stem cell biology only depend of animal donors for generating cellular reagents
11.	The transplantation models are needed to:
a.	Test the ability of the stem cells to protect at-risk cells
b.	Test the ability of the stem cells to replace cell lost
c.	Test the ability of the stem cells to overgrowth
d.	a and b are true
12.	T/F Cell culture have revealed a remarkable plasticity of stem cells from embryonic and adult tissues
13.	The use of both in vivo and in vitro regenerative models offers hope for:
a.	Stem cell phenotyping and use of stem cells as therapeutic agents 
b.	Stem cell genetic research 
c.	Gene therapy research 
d.	The no risk of any stem cell therapy
14.	The dual nature of stem/progenitor cells is revealed by:
-
-
15.	The regenerative medicine cell therapies ___________, ____________ and __________ at risk cells and tissues
16.	Research in regenerative medicine requires animals:
a.	As source of immature cells 
b.	As recipient for cells and engineered tissue grafts 
c.	a and b are true 
d.	None is true
17.	T/F once researchers understand the nature of different stem/progenitor cell population, the experimentation with mammalian models will be reduced
18.	In regenerative medicine, before going to human clinical trials, it is worthwhile to further develop
a.	Cell culture assays 
b.	Explore virtual gene and protein screens 
c.	Standardized and efficient animal models 
d.	All are true
19.	T/F New therapeutics for movement disorders and other neurogenic disorders will come from regenerative medicine
20.	Mutant and transgenic animal have increased the scientist ability to characterize _________, __________ and ___________ development
21.	T/F Research of bone marrow hematopoiesis in the 1950s gives the notion of a population of primitive cells that gives rise to other cells and tissues
22.	Neuropoiesis means
a.	Generation of neuronal cells to form the CNS 
b.	Generation of CNS in embryos and fetuses 
c.	Lifelong neurogenic potential of cells to form the CNS 
d.	Potential to adult neuronal cells to replace lost or damaged cells
23.	Four characteristics of stem cells are?
24.	T/F A precursor is a cell that is in transition between being proliferative and seeking fate and commitment
25.	T/F In vitro studies of stem/ progenitor cells have allowed knowing what occur with these cells in vivo
26.	The choice to divide or differentiate along a particular line from stem cell is influenced by:
a.	Growth factors, cytokines, morphogens 
b.	Cytokines, enzymes, hormones 
c.	Growth factors, hormones 
d.	Morphogens, cytokines
27.	SVZ means:
a.	Subventricular zone 
b.	Supravestigial zone 
c.	Subvestibular zone 
d.	Supravertex zone
28.	Adult SVZ
a.	Represents the vestigial embryonic germinal zone 
b.	Displays a high level of constitutive proliferation 
c.	Contains the greatest density of putative neural stem cells 
d.	All are true
29.	A MASC (multipotent astrocytic stem cell) can rise to neurospheres containing glia and neurons
30.	T/F Adult neural stem cells offer a potential source of cells for drug-screening bioassays as well as for transplantation in models of neurological disease
31.	Adult mouse and human neural stem /progenitor cells can survive in tissue with rather protracted postmortem intervals
32.	In vitro high-throughput screening (HTS) bioassays
a.	Might best utilize the potency of embryonic, fetal and adult stem cells 
b.	Are relevant to detect embryonic, fetal and adult stem cells 
c.	Both are rue 
d.	None are true
33.	HTS can begin following establishment of the best in vitro culture system with the most appropriate stem/progenitor or precursor cell of interest
34.	HTS and other screens, as robotic approaches, can give information on factors that affect _________, _________ ________ __________ and ___________ along with _____________ __________ approaches can enhance the generation of neural and other precursor cell.
35.	One goal of regenerative medicine is to apply to precursor cells ___________, ___________, ____________, _____________to understand how they _________, __________, ____________ and ___________
36.	Biogenic factors are only involved in cellular processes
37.	T/F The main goal of regenerative medicine is rebuild human tissues and organs that have damaged by injuries or diseases
38.	Neuropoiesis research try to establish a dependable procedure for the discovery of genes expressed during neurogenesis
39.	A neurosphere is
a.	A clone of cells derived from a single stem or progenitor cell grown under in vitro conditions that favor their survival 
b.	A vesicle originated from stem or progenitor cells containing factors that influenced the growth of a particular neuronal cell 
c.	A group of stem or progenitor cell located in a determined area of the brain 
d.	A clone of stem cells located in the fore brain that can secret neuronal growth factors
40.	Neurospheres
a.	Contain transcripts of all genes involved in stem or progenitor cell division, expansion, growth, differentiation and survival or death 
b.	Represent units that arise from stem or precursor cells in particular stages of maturation 
c.	Represent the clonal expansion of cells that originated during distinct ontological stages of neural development 
d.	All are true
41.	T/F The ability to generate neurospheres from autopsy specimens with extended postmortem intervals provides the opportunity to create panels of cDNA libraries from neurologically abnormal stem cells
42.	cDNA libraries from individual stem or progenitor cells, represents an alternative to the use of animal or human tissues for molecular fingerprinting studies
43.	the choice of which cell is best for the protection or replacement protocol is crucial, for example for Parkinson disease the best approach would be to rescue and protect at risk dopamine neurons
44.	Major advantages of stem cells as donor source include
-
-
-
-
45.	T/F The next goal of restorative neurology is to prepare he reactive brain manipulating the molecular environment to support reactive synaptogenesis and reestablishment of appropriate synaptic connections
46.	T/F the use of the SCID mouse is important for studying the human immune system in ways not possible with human subjects
47.	T/F chimeras are created by the insertion of non human cell into a human embryo or an embryo with cells from more than one human embryo
48.	A goal of chimeric studies seems to be the generation of animal species that can provide animal models for assessing donor human cell engraftment and donor cell host interactions
49.	Transgenic or knock out postnatal or adult mice provide invaluable insights into the efficacy of new cell and drug therapies
50.	The mice most suitable to understand how new stem cell transplant paradigms can lead to neural circuit protection and repair are:
a.	C57BL/6 
b.	NOD-SCID 
c.	B6D2F1 
d.	129Sv

ANSWERS:
1.	a
2.	F
3.	Discover the best stem/progenitor cell in vitro protocol to facilitate the massive propagation in a type of neural precursor cell

Discover biogenic factors that encourage reactive cell genesis and appropriate functional integration in diseased or injured tissues

Establish the best animal models for the testing of new stem cell related therapies
4.	d
5.	b
6.	F
7.	Exposure to combination of drugs that encourage neuroprotection and selecting cell genesis to thwart further degenerative process

Replace cells already lost

Promote reconnection between existing and newly generated cells
8.	d
9.	Genetic manipulation
Chemical carcinogenesis
Transplantation of cancer cells
Cell therapies, chemotherapy interventions
10.	F
11.	c
12.	T
13.	a
14.	Generation (poiesis)
	Overgeneration of cells and tissues
15.	Protect, repair, replace
16.	c
17.	T
18.	d
19.	T
20.	Normal, abnormal, restorative
21.	T
22.	c
23.	The ability to self renew
	Give rise to more committed progeny (progenitors)
	Respond to tissue injury or disease with reparative attempts
	Maintain all of these characteristics after serial transplantation
24.	T
25.	F
26.	a
27.	a
28.	d
29.	T
30.	T
31.	T
32.	a
33.	T
34.	Stemness, stem cell proliferation, neurogenesis, gene discovery
35.	Genomic, proteomic, transcriptomics, metabolomics
	Multiply, growth, differentiate, survive
36.	F
37.	T
38.	T
39.	a
40.	d
41.	T
42.	F
43.	T
44.	Pluripotency
	Potential for virtually unlimited proliferation
	Amenability to genetic modification
	Possibility to differentiate them into purified neural cell population
45.	T
46.	T
47.	T
48.	T
49.	T
50.	b

Emborg.  Nonhuman Primate Models of Parkinson’s Disease, pp. 339-355.

Primary species: Macaque spp.
Secondary species: squirrel monkey, baboon
Tertiary species: African greens, common marmoset, capuchin monkey.
Tasks: 1, 2, 9, 10

SUMMARY: This article reviews the non-human primate models of Parkinson’s disease (PD).  PD is the second most common progressive neurodegenerative disease (first being Alzheimer’s disease) affecting 1 million people in the United States by conservative measures.  The cause remains unclear and is likely multi-etiologic including genetic and environmental factors.   Risk factors include pesticide exposure, head trauma and aging.  Primary symptoms include resting tremor, rigidity, bradykinesia and postural instability.  Non-motor symptoms are also common including emotional changes.  Variability in disease progression and intensity is hallmark.  Pathologic findings include loss of dopaminergic neurons in the substantia nigra pars compacta and intracytoplasmic inclusion bodies (Lewy bodies).  Treatment remains symptomatic with dopamine replacement and surgical interventions (i.e. Targeted ablations, deep brain stimulation).

An animal model that encompasses all aspects of PD including its progressive nature, motor and non-motor symptoms and pathological findings has not been identified.  Researchers rely on models that represent one stage of the disease.  The core PD models result in the motor deficit “paralysis agitans” and associated nigrostriatal dopaminergic loss.   Models of PD are induced by three different mechanisms: aging, targeted gene transfection and neurotoxins.  

Aged rhesus and squirrels monkeys have been studied for age-related changes in the nigrostriatal and dopaminergic system.  A genetic mutation in the alpha-synuclein has been identified in the rare familial form of PD (represents 5% of all cases of PD).  Direct gene transfection of mutated alpha-synuclein in the common marmoset has resulted in degeneration of dopaminergic fibers in the striatum with associated motor impairments.   Neurotoxins are the most commonly used model and include 6-OHDA, a catecholaminergic neurotoxin, and MPTP, a mitochondrial complex I inhibitor.  6-OHDA must be injected directly into the brain and involves significant technical skill in delivery to induce the desired symptoms.  MPTP can be given systemically with the time course of symptom onset dependent on dosing (intermittent vs. continuous) and technique (peripheral or central access).  

Each model described does not represent PD in its entirety.  They each target certain symptom, pathologic lesion or functional lesion.  There is no one perfect model which requires that careful thought go into choosing a model that is appropriate for the question being asked.  The future requires more development of multi-systemic, multi-symptom models.  

QUESTIONS:
1.	List the 4 hallmark symptoms of PD.
2.	What are the three main modalities for creating PD in NHP?
3.	What are the three modalities for evaluating PD in NHPs?

ANSWERS:
1.	Resting tremor, rigidity, bradykinesia, postural instability
2.	Aging, targeted gene transfection, neurotoxins
3.	Behavioral testing, in vivo imaging, post-mortem analysis

Ramaswamy et al.  Animal Models of Huntington’s Disease, pp. 356-373.

RDL Task 9: Collaborate on the Selection and Development of Animal Models
Primary Species: Mouse (Mus musculus), Rat (Rattus norvegicus), and non-human primates (species not indicated)

SUMMARY: Huntington’s Disease (HD) is a progressive neurological disorder in which a genetic mutation in the IT15 gene leads to cell death in the striatum and cerebral cortex causing declines in cognitive, motor and psychiatric function.  The genetic mutation is an expanded trinucleotide repeat (CAG) leading to the production of mutant htt protein that causes cell death by mechanisms that remain unclear.  Symptoms of this invariably fatal disease include involuntary hyperkinetic (or choreaform) movements of the arms, legs and face, as well as debilitating cognitive and personality changes.  This review article outlines the animal models available for scientific study of HD and evaluation of potential therapeutics.  In addition to HD models in invertebrates (Caenorhabditis elegans and Drosophila melanogaster), there are models of the disease in rodents (rats and mice) and nonhuman primates; these models can be categorized as non-genetic/toxin-induced or genetic models.

Toxin-induced HD models (Table 1) induce neuronal death either by excitotoxic mechanisms or by the disruption of mitochondrial machinery.  Quinolinic acid is the most commonly employed excitotoxic agent, and localized administration results in both motor and cognitive deficits in rodents and nonhuman primates.  3-Nitropropionic acid inhibits mitochondrial enzymes leading to motor and cognitive deficits when administered systemically or locally in rodents and monkeys; this agent has the advantage of causing dose-dependent hyper or hypokinetic symptoms of HD.  The predictability of toxin induced cell death has made them useful in the testing of neuroprotective therapeutics such as coenzyme Q10 and creatine.  Critics of toxin-induced HD models point to acute non-progressive lesions that are not linked to a genetic cause as not being representative of the natural history of HD, a genetic disease.

Genetic models can be further broken down into transgenic (See Table 2) and knock-in (See Table 3) models.  Transgenic HD models are produced by random insertion of a portion of the htt gene into the rodent genome and recapitulate both motor and cognitive symptoms; the R6/2 mouse is the most commonly used transgenic HD model.  One transgenic rat model is available, allowing for testing of long-term therapeutic interventions owing to rats’ longer lifespans.  The production of mouse knock-in models involves the targeted insertion of a portion of the human htt gene in the mouse gene locus on chromosome 7; the CAG150 mouse is the most promising knock-in mutant.  Concerns about the random nature of genomic insertion and exogenous promoter contributions in transgenic models incited the need for knock-in models, but the subtle phenotypes in knock-in animals make therapeutics testing difficult.  A non-transgenic genetic model of HD involving the site-specific induction of mutant htt by viral vectors has recently been developed; this approach will circumvent genetic drift in mutant lines and has great potential for use in nonhuman primates-species in which complex cognitive and motor deficits can be studied, and in which genetic engineering has proven difficult.

QUESTIONS:
1.	Which of the following has not shown promise as a therapeutic in animal models of HD?
a.	Coenzyme Q10
b.	RNA interference (RNAi)
c.	Creatine
d.	Kainic acid
e.	GDNF
2.	Which of the following animal species has not been used to model HD?
a.	Nonhuman primates
b.	Chinchillas
c.	Mice 
d.	Rats
e.	Drosophila
3.	True or False?  The most commonly used transgenic model of HD is the R6/1 mouse.
4. 	True or False?  The phenotypes of knock-in mouse models of HD are subtle.
5.	True or False?  Although HD was first described in 1872, the genetic mutation responsible for the disease was not identified until 1993.
6.	Which of the following is not a symptom of HD?
a.	Hypokinesia
b.	Hyperkinesia
c.	Visuospatial deficits
d.	Poor memory recall
e.	All of the above are symptoms of HD
7.	True or False?  Hypokinesia is effectively modeled in toxin models of HD.
8.	Clinical care and monitoring of animal models of HD may include all but which of the following?
a.	Gavage or hand feeding
b.	Weekly bodyweights
c.	Analgesics
d.	Euthanasia of moribund animals
e.	All of the above may be components of care necessary for HD animal models
9.	True or False?  Mouse lines with large polyglutamine (CAG) repeats are predisposed to CAG repeat instability, such that repeat length increases in subsequent generations leading to earlier onset of HD symptoms.
10.	True or False?  The CAG150 mouse is the most promising knock-out mutant model of HD.

ANSWERS:
1.	d. Kainic acid
2.	b. chinchillas
3.	False; R6/2 mice are most commonly used
4.	True
5.	True
6.	e. all of the above
7.	False; hypokinesia, while a feature of late stage HD, is generally not seen in toxin models of HD
8.	e. all of the above
9.	True
10.	False; the CAG150 mouse is a knock-in model

Kleim et al.  Rat Models of Upper Extremity Impairment in Stroke, pp. 374-384.

Task 9: Collaborate on the selection and development of animal models

This article reviews commons methods of producing stroke in rats including devascularization, photothrombosis, chemical vasoconstriction and hemorrhage and describes testing to assess forelimb function after stroke.  Analysis of limb movement in the rat reveals similar motor components during reaching behavior which has led to sensorimotor tests that measure aspects of both motor impairment and recovery.

Methods of inducing stroke in a rat
1	Middle cerebral artery occlusion: temporary or permanent restriction of blood flow to the cortex and striatum.  Most widely used method involves insertion of a suture into the common carotid and advancing the suture into the internal carotid until it lodges in the junction of the anterior and middle cerebral arteries.  Alternatively microspheres injected into the carotid may be used in place of the suture.
2	Endothelin-1: Injected or topically applied endothelin-1 on brain tissue inducing vasoconstriction.
3	Photothrombosis: injection of photosensitive dyes into the bloodstream followed by induction of photo-oxidation by irradiating specific areas of tissue.
4	Devascularization: Using electrocautery or surface vessels or pial stripping inducing cortical infarctions.
5	Focal hemorrhagic damage: infusion of bacterial collagenase or whole blood into cortical and/or subcortical areas disrupting vessels integrity.

Methods of measuring forelimb impairment
1	Cylinder task: examines forelimb use for postural support by encouraging rodent to support self in a specially designed cylinder.
2	Forelimb placing: holding the animal by the torso with the forelimbs hanging freely, the rat is moved laterally toward the edge of a table until the vibrissae make contact with the edge in an effort to stimulate a placing response which is recorded.
·	Swim task: Pre-lesion and post-lesion forelimb use during swimming is recorded and compared.
·	Adhesive dot removal: Adhesive labels are placed on the forelimb and removal of labels is recorded.
·	Montoya staircase test: Rats are required to reach from a central platform with their forelimbs to retrieve food pellets at variable distances on a descending “staircase”.
·	Foot fault: Rats placed on an elevated grid or ladder and placement on limbs is recorded.
·	Sunflower seed opening: Rats are given a set number of seeds and the time to open and consume the seeds is recorded.
·	Pasta matrix reaching task: Rats reach through a high vertical slot for pasta pieces on a shelf with performance determined by the number of pieces retrieved.
·	Tray reaching: Rats placed in training boxes reach through metal bars on the front of the box to grasp food on a tray in front of the box. Successful reaches are recorded.
·	Single pellet reaching: Similar to the tray reaching task except only a single pellet is placed in front of the training box instead of a tray filled with food.
·	Skilled reaching movement analysis: This video analysis is used to identify enduring impairments or compensatory strategies used for reaching and grasping.

Considerations for study design.
·	Stroke model: No general agreement on best model although middle cerebral artery occlusion may be most clinically relevant. 
·	Using a battery of sensorimotor tests: Use of tasks that measure pre-existing and acquired motor abilities.  Time to administer tests and sensitivity of the testing are important considerations.
·	Establishing stable prestroke measures of motor performance.
·	Motor testing is a form of rehabilitation: If testing rehabilitation strategies be aware testing procedures can act as rehabilitative procedures especially affecting control groups.
·	Verifying lesion 

QUESTIONS:
1. 	Which of the following is not a method of producing stroke in the rat?
a.  	Photothrombosis
b.  	Middle cerebral artery occlusion
c.  	Intracerebral injections of myelin basic protein
d.  	Devascularization
e.  	Chemical vasoconstriction
2.  Which of the following is not a task used to assess forelimb function in rat stroke models?
a.  	Swim task
b.  	Acquisition of a maze
c.  	Sunflower seed opening
d.  	Foot fault
e.  	Single pellet reaching 
3.  T/F  Post-stroke testing can serve as a form of rehabilitation and can confound results especially in situations where animals are over tested.

ANSWERS
1. 	c
2. 	b
3. 	T

Onifer et al.  Rat Models of Traumatic Spinal Cord Injury to Assess Motor Recovery, pp. 385-395.

Following primary mechanical spinal cord injury (SCI), there is a complex cascade of events which lead to progressive death of viable axons and cells which impairs endogenous recovery processes.

Studies on curative measures are focused on intervention of the secondary injury cascade, enhancement of endogenous repair mechanisms, axon regeneration, cell replacement, and rehabilitation.  These studies have led to the development of several animal models of traumatic SCI in man. This article describes the adult rat model of SCI and the deficits that each model reproduces.

Rats have been chosen to study traumatic SCI because they are readily available and the morphological, biochemical, and functional changes that occur after SCI are similar to those seen in man.

SCIs have historically been classified into three categories:
1	A contusion evolving to cavity formation
2	Massive compression
3	A laceration.  

The most frequent contusion injuries are focal spinal cord compression.  Laceration injuries are focal and over time lead to a connective tissue mass in the spinal cord.

Assessments of Motor Deficits and Recovery in Adult Rats with SCI
There are several modalities for assessing behavioral and electrophysiological function of adult rats with traumatic SCI.  These assessments evaluate sensorimotor function rather than sensory or motor functions individually.  Pre-SCI assessment helps establish baseline values.

Behavior is assessed via:
1	Staircase Test platform and chamber which enables cervical spinal cord-injured rats to avoid having to support themselves on three dysfunctional limbs while reaching for , grasping, and retrieving pellets.
2	Forelimb grip strength- The cervical SCI rat is pulled away while grasping a bar.
3	Evaluating forelimb and hind limb placing and footfalls- assessed while rat walks or locomotes on a horizontal or inclined grid, horizontal ladder, beam, or rope.
4	Open-field locomotor rating scale to assess functional outcome using paw placement, joint movement, and truncal stability as important factors in determining the level of functional recovery. Scores range from zero (no hind limb movements) 
5	 to 21 (normal coordinate gait).
6	Others: footprint analysis, CatWalk-assisted gait analysis, and kinematics, and inclined plane to evaluate functional outcome in rats after SCI.  Several studies correlate these data to electromyographic recordings made at the same time from electrodes implanted in limb muscles.
 
Electrophysiology is assessed via:
1	tcMMEP (Transcranial magnetic motor evoked potential)-involves noninvasive magnetic stimulation at the unanesthetized rat’s skull. The recording of evoked potentials with EMG electrodes temporarily inserted into hind limb muscles. Assesses supraspinal axon conduction.
2	MIER (Magnetically evoked interenlargement)- involves noninvasive magnetic stimulation at the rat’s hip or knee and the recording of evoked potentials with EMG electrodes temporarily inserted into forelimb and masseter muscles. Evaluates propriospinal axon conduction.
3	SSEP (Somatosensory evoked potential)- involves electrical stimulation of the paws with electrodes temporarily inserted into them, and the recording of evoked potentials from electrodes previously implanted in the cranium over the somatosensory cortex.
 
Rat Models of Traumatic SCI

Weight Drop
First used in dogs. A given weight is dropped from a known height on the exposed dorsal surface of the spinal cord of an anesthetized animal. In rats the spinal column is stabilized. Different degrees of injury severity and functional outcomes are necessary. Too moderate injuries can lead to spontaneous recovery, whereas, too severe of an injury may mask potentially useful therapeutic strategies.

The popular NYU/MASCIS device consists of dropping a 10g wt. from 6.25 to 50mm directly onto the exposed dorsal spinal cord dura.

Aneurysm Clip Compression
To overcome the problem some investigators find with the size of the rat for use in the weight drop method of SCI and to more closely model the anterior compression normally observed in man, modified Kerr-Lougheed aneurysm clips are used. The rat spinal cord is exposed and aneurysm clip is applied with one blade under the ventral surface of the cord and the other over the dorsal surface. The clip is rapidly released and allowed to compress the cord for a predetermined amount of time. Results indicated high correlation of all neuron outcomes to the compression force using the aforementioned behavioral assessment modalities.

Calibrated Forceps Compression
The use of forceps leads to a moderate severity injury technique in the guinea pig. This compression injury produced a considerably large volume of tissue compression and displacement versus the aneurysm clips.  A delayed functional loss in the guinea pig was not observed in the rat, indicating possible significant species differences.

Contusion
In an attempt to gain more control over the injury severity and more closely monitor biomechanical properties, several labs began experiments with controlled pneumatic compression models. New electromechanical spinal cord impactor, known as the Ohio State University device induces injury to the cord by means of a solenoid-controlled air cylinder mounted on a rigid frame with a tip that impacts the exposed dorsal spinal cord.
As with the weight drop studies, immediately following the trauma, functional deficits appeared and spontaneous recovery reached a plateau around 2-3 weeks following injury.

Laceration
The most frequently used method. Includes complete transaction, focal myelotomy, dorsal or lateral hemisection, lateral overhemisection, resection, or aspiration lesions that definitively sever spinal cord axons of passage. 
These laceration injuries are not typically seen clinically, however, they can effectively disconnect both ascending and descending axonal pathways at designated levels of SCI.

Chemical-mediated SCI
These methods induce pathology seen after traumatic spinal cord injury such as:

1	Vascular damage (hemorrhage, reduced blood flow, and ischemia)
o	IV injection of photosensitive dyes rose Bengal or erythrosib B with irradiation of exposed vertebrae
o	Vasoconstrictor endothelin
o	Spinal cord gray matter injection with nonspecific nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester=> neuronal degeneration
2	Rise in excitotoxicity(=>oligodendrocyte and neuron death)
o	Administering glutamate, glutamate and aspartate, NMDA, or kainite.
3	Free radical , peroxynitrite, and calpain-induced damage to lipids and proteins
o	Administration of paraquat, H2O2 and FeCl2, or S- or 3-morpholinosydnonimine, peroxynitrite donors (These oxidants destroy neurons, block motor axon conduction, and impair open-field locomotion, hind limb function, and inclined plane stability)
4	Oligodendrocyte degeneration and demyelination(Hallmarks of traumatic SCI)
o	Intraspinal injection of lysolecithin or L-aphalysophosphatidyl choline alone or with irradiation, ethidium bromide alone or after x-irradiation.
5	Syringomyelia (characterized by fluid-filled cavity formation, then enlargement with delayed and then progressively deteriorating function)
o	Injection of a suspension of kaolin into the rat cisterna magna to produce hydrocephalus, into the spinal cord subarachnoid space to create arachnoiditis, or into the spinal cord dorsal columns and central gray matter to occlude the central canal.
o	Microinjection of quisqualic acid in the spinal cord gray matter alone and in combo with kaolin into the spinal cord subarachnoid space to produce cavities.

No questions or answers were provided.

Darian-Smith.  Monkey Models of Recovery of Voluntary Hand Movement after Spinal Cord and Dorsal Root Injury, pp. 396-410.

ACLAM Species Designation - Primary Species – Non-human primates

SUMMARY: This is a review of different aspects involved in the recovery of voluntary hand movement after experimental spinal cord and dorsal root injury. Document gives an overview of the major pathways responsible for voluntary, manipulative behaviors on monkey, ape and human.  It also describes specific lesion models and provides an overview of the major mechanisms responsible for the behavioral recovery and reorganization after such injuries.  It can appreciated also from the document that human and macaque hand anatomy and function are very similar, here, macaque came as one of the most suitable nonhuman primate model for the study of post injury dexterity.

Two of the most remarkable characteristics of the nervous system related with the voluntary, manipulative behaviors of monkey, apes and human are:
1	Primates are the only mammals with corticospinal pathways that directly synapse with motor neurons that innervate the hand forelimb (corticomotoneuronal projections) which shape an important role in shaping hand use.
2	The rubroepinal tract is relatively insignificant in monkeys and almost nonexistent in humans.  

Some highlights of the document related with the post injury phases of regeneration are: 
1	Immediately after a nerve is cut a “silent” region is produce in the contralateral cortical hand map.
2	During the second phase (weeks a months) there is a consolidation of cortical and subcortical body maps, as spared connection have the opportunity to establish stronger new and alternate pathways.
3	The final phase involves the eventual atrophy of chronically deafferented neuronal populations and can continue for many years and even decades.

The most dramatic behavioral changes occur during the chronic second phase, and the models reviewed in this article focus on that period.

Regarding the primates Models, authors mention some important advantages of the primate models against the rodent models such as: Although rodent models provide information on cellular, molecular and biochemical mechanism related with plastic changes, they do not provide the clinical link for the study of hand recovery after injuries, because rats do not have the sophisticated volitional sensorimotor movements of macaque and human hands. Macaque monkeys can learn to perform very sophisticated sensorimotor tasks that test tactile sensation and fine control of the digits.

The document review also the most commonly used test of post injury hand function the authors established advantages and disadvantages of Kluver board and dorsal root lesion model (DRT), and the use of precision task assessment to evaluate dexterity in monkeys with DRL. Other models described were dorsal column transection models, cervical hemisection lesion models. An important feature related with the age of the animals used in the studies is that young animals of 2.5-3.5 Kg were use, mainly because they do not yet have the back muscle bulk of older males. So recovery from the injury was quickly when the technique of rhizotomy was used. Another important feature was that different to humans is that monkeys did not exhibited signs of neuropathic pain.

Finally, authors make a description of the neuronal mechanism that mediate reorganization and recovery of hand function after injury.

QUESTIONS:
1.	Regarding innervation of hand and forelimb, a unique characteristic of primates major descending tract, the corticoespinal tract is: 
a)	Presence of corticomotoneuronal projections
b)	Well development of the rubroespinal tract
c)	Absence of direct synapsis with motor neurons
d)	Absence of corticomotorneuronal projections
2.	Why rodents can not be a good model for study hand function recovery? 
3.	Why in the dorsal root lesion model young animals are used rather than more resistant old animals? 
4.	Why during experimental dorsal root lesion (DRL) surgery, the dura madre is not sutured? 
a)	It is removed during the experimental surgery
b)	It repairs by it self
c)	Suturing produce adhesions
d)	Suturing this area is very risky
5.	Which of the next commonly used test to assess manual performance in monkeys is difficult to record and do not systematically test individual digit function? 
a)	Precision grip task assessment
b)	Kluver board
c)	Dorsal root lesion

ANSWERS:
1.	a)
2. 	Rodent do not provide the clinical link since they do not have the fractionated digit use
3. 	Young animals (2.5-3.5 Kg.) do not yet have developed the back muscle bulk. Hence surgery produces less damage and fast recovery
4. 	c)
5. 	b).

Byl.  Learning-based Animals Models: Task-specific Focal Hand Dystonia, pp. 411-431.

Primary Species- Owl monkey (Aotus nancymaae), rat

SUMMARY: This article discusses the use of the owl monkey and the Sprague-Dawley rat as two animal models of learning based mechanisms of development of focal hand dystonia (FHd) in humans.  Dystonia is characterized by disabling involuntary movement that leads to writhing, twisting end range movements and abnormal postures.  Dystonia may be generalized, focal (affecting only one part of the body) or involve only a well-learned task.  Focal dystonia is a manifestation of problems in sensorimotor integration, cortical excitability, and loss of inhibition.

There are no clinical lab tests to diagnose focal hand dystonia (FHd). Patients with focal dystonia have no structural abnormality when examined by magnetic resonance imaging.  The hand has large, orderly, somatotopic, highly differentiated representation in the thalamus, basal ganglia and cortex.  Neurons that respond to stimulation of hairy skin do not mix in functional connectivity with neurons that represent glabrous skin; neurons that represent different fingertips to not mix; and neurons that respond only to deep skin stimulation do not mix with those that respond to light cutaneous stimulation.  Neurons respond either to one modality or another, but not both.  Neurons which cluster in the dorsal columns in small modules (0.5 mm in diameter) all respond to the same stimulation modality and are similar in positional representation on the skin.  “This precise representation exists in the primary thalamic nucleus for the somatosensory inputs (the ventral posterolateral nucleus).  Modality segregation also exists in the cortex, and also in the primary motor cortex. By use of cortical microstimulation a loosely topographic map of the muscle groups was obtained in which independent muscle actions are represented in segregated modules or columns.  Microstimulation of different columns may activate different single digits or cause antagonistic muscle actions.  Independent motor actions may be represented a few hundred microns apart or a few millimeters.  The columns have output projections into the spinal motoneuronal pools, where antagonistic muscle groups have a reciprocal inhibition. The ascending sensory pathways include peripheral receptors, dorsal column nuclei, the ventral posterolateral thalamic nucleus and primary somatosensory cortex area.  A breakdown of columnar boundaries in somatosensory or motor cortices results in signal sharing in normally independent functional modules, causing loss of motor control, simulating focal dystonia. The topographic and functional neural representations in the brain may be modified by maturation and development, environmental enrichment, deprivation, drugs disease, injury, and attended practiced progressive behaviors.

The primate model involved training monkeys to perform two hand-closing tasks.  Food deprivation for 20-22 hrs before beginning each training week, with weight maintained at 80-90% of normal ) with behavioral rewards was used to train the monkeys.  By using software to control the sensorimotor apparatus, the tasks the monkeys performed for food reward were attended, rapid, rewared, stereotypical and near coincident in time and repetitive for 2 hrs/day, 5 days/week, and practiced over time for 5 wks to 12 months.  Speed of repetitions, number of repetitions, and time of training and accuracy of task performance were monitored.  After motor performance deteriorated by 50% in speed and accuracy, training continued at least another 2 wks before mapping was carried out.  Electrophysiological mapping was performed using stereotactic apparatus and craniotomy and use of microelectrodes.  Mapping continued from 15 hrs to 5 days, with 24 hr monitoring.  In the passive hand opening task after monkeys declined to poor task performance and abnormal movements, there was marked dedifferentiation, demonstrated by a breakdown in the normally separated cortical representation of the different digits. In the active hand opening task, both monkeys temporarily decreased the repetition of task performance in the third week of training.  Both monkeys resumed training after a week.  One monkey who continued to use an articulated strategy of opening and closing the hand developed degraded somatosensory organization on the trained side, with many receptive fields overlapping adjacent digits.  The other monkey resumed training using a relaxed proximal arm-trunk pulling strategy.  This monkey did not develop signs of motor dysfunction and continued to perform normally on both the target and food retrieval tasks.  This monkey only developed mild degradation of he hand representation on the trained side.

Abnormalities in receptive field size, cortical column size differentiating different digits and a mixing of normally segregated response modalities occurred in animals with movement dysfunction that simulated focal hand dystonia.; receptive fields at single sites in the primary somatosensory map increased up to 10 times normal size, with sensory physiology abnormalities largest on the digits most affected.  Also, response modalities such as single digit representation or representation of glabrous or haired skin became mixed in the hand maps of animals with the disorder.  Cortical columns leading to the adjacent digit were up to four times the normal size.  As a result of the abnormality in cortical column size, the ability of the somatosensory system to present independent inputs was reduced dramatically.  Preliminary signs of stress (e.g. a temporary reduction in task performance) may indicate that the intense repetitions of a task could be a risk factor for the development of involuntary dystonic movements. An early, temporary decline in frequency and quality of repetitive movements appeared before the observation of motor dysfunction.  In the rat model after 8 wks of high force or high rate of repetitive hand use, 100% of the rats shifted from a coordinated scooping strategy to a clumsy raking strategy to retrieve food reward.  In the primate models, after the initial slowing of practice intensity, animals either resumed or modified their strategy to retrieve food reward.  Central cortical changes were noted after the development of abnormal motor performance, and these changes were consistent with negative learning.  In both human and animal studies of FHd, there was cortical degradation on both the affected (trained side) and the unaffected side, with reduction in the area of the somatosensory hand representation, excessive large receptive fields overlapping in adjacent digits, and dorsal and glabrous surfaces with receptive fields that persist across broad columnar distances.  In the monkeys as in humans with FHd there are long thalamic trajectories on the trained side. In the rat studies, if the rate of repetition and force were low, animals did not necessarily degrade their motor performance.  In the primate studies the animals who did not develop movement problems performed the task slowly, took frequent breaks, required long training periods (45-90 minutes to perform a limited number of repetitions at the target task, performed the task several times a day for shorter training periods (30 min three times a day instead of 1.5 hr once a day) or did not use a strategy involving near simultaneous movements ( they employed a large-strategy- trunk and arm movement instead of hand movement).  Personality traits similar in primates and people who developed movement disorder (FHD) were noted.  Some primates were driven to perform tasks quickly with a lot of force whereas others performed the task slowly, took a lot of breaks or used a trunk strategy rather than a hand squeezing strategy.  Humans (musicians) with FHd were more likely to have a history of anxiety, perfectionisms, perseveration and phobias compared to musicians without hand dystonia.  In humans with FHd, people tend to have a greater restriction in finger spread, forearm rotation and shoulder rotation.  One primate that developed focal dystonia had a congenital defect of the flexor profundus and sublinmus tendon of D4 on the trained side and D3 on the untrained side.  This primate developed signs of dystonic movement of D4 after only 5 wks of training. 

One model of dystonia not detailed in this article involves genetic mutation of the DYT1 genes, associated with generalized dystonia and torsion dystonia. Dyt1 gene is an autosomal gene of low penetrance.  Individuals with a defect in this gene have increased glucose metabolism in the posterior putamen/globus pallidus, cerebellum and supplementary motor area.  These brain regions have high levels of Torsin A and Torsin B.  In animal models of spasmodic torticollic and animal models of blepharospasm (hyperactivity in frontal cortex, striatum thalamus, cerebellum); depleting dopamine increased excitability and increased dopamine decreased excitability. Additional techniques that may be helpful to image patients with FHd patients and study neurophysiology in FHd patients include non intrusive functional magnetic resonance imaging, magnetoencephalography and magnetic stimulation.

QUESTIONS:
1.	The development of task specific and other focal dystonias is considered multifactorial, with contributory factors including
a)	Genetics
b)	Anatomy
c)	Physiology: disruption of inhibition in the spinal cord, cortical dysfunction, disrupt of sensory perception, somatosensory representation and or cortical sensory activation, abnormal presynaptic desynchronization of movement or abnormal muscle spindle afferent firing, abnormal gating of somatosensory inputs
d)	Psychology :Behavioral characteristics/personality characteristics of perfection and perseverance; stressful jobs that require high levels of repetitive hand use
e)	Environment : peripheral trauma, nerve root irritation, peripheral nerve entrapment, anatomic restrictions
f)	All of the above 
2.	Progressive mental and physical practice result in 
g)	Expanded cortical representations
h)	Increased number of excitable neurons
i)	Increased myelination
j)	Strengthened synapses between coincident inputs
k)	Shortened integration time
l)	Increased complexity of dendritic branching
m)	All of the above
3.	Focal hand dystonia is  also known as
n)	Occupational hand cramps
o)	Task-specific dystonia
p)	Writer’s cramp
q)	Musician’s cramp
r)	Golfer’s yip
s)	Keyboarder’s cramp
t)	All of the above
4.	The most common type of focal dystonia is
u)	Hand dystonia
v)	Blepharospasm (involuntary spasms of bilateral eyelid closure)
w)	Spasmodic torticollis (sustained involuntary muscles contractions that rotate the head into abnormal postures)
5.	Examination of patients with focal dystonia using functional magnetic resonance imaging (fMRI) may reveal 
x)	Difference in firing patterns
y)	Difference in areas of activation
z)	Difference in topography (e.g., represtational area size, location, digit order, latency, amplitude, density of neural firing and co-contraction of agonists and antagonists)
aa)	All of the above
6.	Compared to using humans, limitations of the primate model of FHd is 
bb)	The difficulty to obtain feedback about pain, depression or anxiety
cc)	The difficulty to document nontask-related sensory ability such as specific sensory discrimination skills ( e.g. graphesthesis, kinesthesia, stereognosis) and fine motor, rapid alternating movements
dd)	Both motor and sensory electrophysiological mapping cannot be easily carried out in the same session because sensory mapping takes place under pentobarbital-induced anesthesia and motor mapping under Ketamine-induced anesthesia
ee)	All of the above

ANSWERS:
1.	f
2.	g
3.	g
4.	a
5.	d
6.	d