Animal models of spinal cord injury

1. Dr Lawrence Moon lawrence.moon@kcl.ac.uk www.lawrencemoon.co.uk Animal models of spinal cord injury Please take two minutes to fill in the quick questionnaire During the lecture, do interrupt with questions if you have any

2. 1. Define an animal model and discuss why and how they are used. 2. Describe the neuropathology of spinal cord injury (SCI) in humans. 3. Give examples of different animal models of SCI. 4. Critically evaluate different animal models of SCI (pros, cons). After this lecture and appropriate studying* you should be able to * • Write lecture notes! Read them soon, to refresh your memory. • I will cover the key issues but you need to read the recommended reviews and papers. Write notes. Test a classmate on their knowledge and understanding. • Be critical. Question what you are told! • Before any exams, find and read additional up-to-date papers (e.g. by authors on the reading list) • Think about how animal models are sufficient and where they fail. • Cite authors (e.g. Smith et al,. 2007) to substantiate written claims.

3. Whereas good looking humans can be supermodels, an “Animal Model” is NOT a beautiful, photogenic pet. Thus, the following are NOT examples of good animal models What is an animal model?

4. Dogs as bad supermodels

5. An “animal model” refers to the use of a non-human animal to simulate a human disease or injury. They are used where it is practically or ethically difficult to use humans. What is an animal model? • They can be • Naturally occurring • In a normal animal, e.g. after road traffic accident • In an abnormal (spontaneous) mutant • Induced experimentally • Surgical • Genetically engineered

6. We want to discover safe and effective therapies for various diseases and injuries. Why should you learn about animal models? Many potential therapies require testing for safety and efficacy in animals before it is possible to move to a clinical trial. If you understand the pros and cons of each model, you can better evaluate the research (e.g. criticise the papers) Ethical implications.

7. Approval of Personal and Project Licence from Home Office (UK) Training & supervision What do models of SCI typically involve? Development of animal model if necessary Genetic engineering / breeding of mutant, etc. Pre-training / habituation Surgery under anaesthetic Spinal cord injury Delivery of a therapy Postoperative care (analgesia, antibiotics, etc.) Post-injury behavioural testing Electrophysiology / imaging Terminal anaesthesia, removal of tissues (e.g. fixation) Cutting of tissues Staining of tissues to reveal injury site / regenerating axons, etc.

8. Overview Introduction to spinal cord injury Incidence, prevalence Pathology Types of animal model Surgical / naturally occurring / genetic engineering Species of animals used Rats / mouse / cats / non-human primates / dogs Outcome measures Behavioural tests Histology

9. Prevalence in USA 250,000 • Incidence in USA 11,000 Spinal cord injury CAUSES: • Motor dysfunction below the injury site • Loss of sensation below the injury site • Pain • Bladder, bowel, sexual dysfunction SEQUELAE: I’ll limit discussion to animal models of locomotor dysfunction after SCI.

10. Some (v limited) spontaneous recovery / compensation • Few acute therapies • steroids (SCI) – contraversial • Few chronic therapies • rehabilitation (locomotor) • adaptation (sexual, bladder, bowel) • None fully restorative • So we need to develop safe and effective therapies Spinal cord injury

11. SFN video

12. Anatomy of human spinal cord emphasise CST and sensory axons quad v para

13. VARIANTS: • Contusion • Compression / Maceration • Laceration (cut) • Solid core injuries Pathology

14. Very few new neurons are born (neurogenesis) • Spontaneous failure of CNS axon regeneration • Limited endogenous repair (adult vs neonate) • Insufficient compensatory plasticity • Poor intrinsic axon growth • Pro-growth molecules down-regulated • Anti-growth pathways switched on • Inhospitable extrinsic environment • Cysts, cavities • Fibrotic scar • Growth-inhibitory molecules (intact & injured) • Lack of growth factors, permissive substrates Why only some spontaneous recovery?

15. Reduce cell death (neuroprotection) Promote regrowth of injured axons (regeneration) Promote compensatory regrowth by uninjured axons (collateral sprouting) Demyelination (?) Rehabilitation Goals for spinal cord repair

16. Model cell death (neuroprotection) • Induce characteristic pattern of cell death • Sparing • Allow measurement of any neuroprotection Goals for animal models of SCI • Model axon injury • Full transection of some axons • Sparing of other axons • Allow measurement of any regeneration • Allow measurement of any collateral sprouting Model demyelination (if any) • Model locomotor (or other) deficit • Allow measurement of any recovery

17. Different surgical methods • Species (rat, human, cat, dog, non-human primate) • Outcome measures • Other variables • Time until therapy is delivered • Level of spinal cord Overview of animal models

18. Various impactors: NYU OSU IH Hill et al., 2001 Exp Neurol 171:153-169. Usually at midline, mid thoracic (T9) in rats or mice Basso et al., 1996 Exp Neurol 139:244-256 Also unilateral in cervical spinal cord (C5) Contusion (weight drop)

19. Usually midthoracic Surgical knife cut Advantages True regeneration Disadvantages Very harsh Scar Postoperative care Complete transection de Winter et al 2002

20. Cut or crush with forceps Often done in rats / mice There may be no ventral CST in mice (Steward et al., 2007) Partial section: dorsal hemisection

21. Advantages Deficits are mild – loss of fine but not gross motor control Easy to trace Cuts ascending sensory fibers completely Partial section: dorsal hemisection Disadvantages Does not cut corticospinal tract (CST) completely Thus cannot be used rigorously to assess true axon regeneration Steward et al., 2003

22. Partial section: lateral hemisection Freund et al., 2006 - primate • Interrupts tracts unilaterally (e.g. CST in primate; RST in rodents) • Primate CST is lateral • Rat CST is mostly dorsal but also lateral and ventral • Advantage • Deficits are unilateral • Contralateral tract / limb serves as within-animal control -> power

23. Rat • Most common; cheap, friendly • Anatomy well understood • Disadvantage: CST lesions aren’t very disabling • Quadruped; does this model us as bipeds? Species

24. Mice • Smaller – behaviour can be tougher to measure • Transgenics / knockouts exist • Zheng et al,. 2006 • Anomaly - very little cyst formation • Disadvantage: CST lesions aren’t very disabling Species

25. Non-human primate • Similar anatomy to human • Similar pathology • Disadvantages: Expensive; ethically challenging Species • Cats • Used less often nowadays • Earliest trials based on cat studies

26. Naturally occurring injuries • Road traffic accidents • Spinal disc herniation (hernia / prolapse / “slipped”) • Chondrodystrophic dogs • Bassets, dachshunds, bulldogs Dogs • Commonly cervical or thoracolumbar

27. Compression / contusion • Use autopsy material to understand SCI Dogs • Test out new therapies in dogs? • Ethical opportunity • Jeffery et al,. 2006a,b • Disadvantages • Sporadic • Not controlled – variable (but this models human)

28. Most species are not bipedal (but consider birds). • Species can have different musculoskeletal arrangements • e.g. rat has fused radius and ulnar bones • Species can have different neuroanatomical arrangements • e.g. amount of direct cortico-motorneuronal synapses Phylogenetic differences Lemon & Griffiths (2005)

29. Behavioural testing • Locomotion (forelimb, hindlimb) • Pain • Bladder function Overview of outcome measures • Electrophysiology / imaging • Histology (Tissue processing) • Size of injury • Axon growth • Myelination (traditional stains) • Transplant characteristics

30. Grid walk / beam – show Schallert video Behavioural testing • Rats / mice • Easy • Sensitive to deficits (e.g. after CST injury) • Quantitative (count faults) • Forelimb and / or hindlimb

31. Forelimb reaching • Non-human primates • Rats Behavioural testing Show rat reaching video from Tim Schallert

32. Open field locomotion • BBB test • Basso, Beattie, Bresnahan, 1995 A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 12:1-21. • Contusion / weight drop • Transection • Forelimb and hindlimb • Rats (BMS for mice) Behavioural testing

33. Connectivity / electrical properties of axons • MEP – motor evoked potential • SSEP – somatosensory evoked potential • TCMS – transcranial magnetic stimulation • EMG - electromyography Electrophysiology / imaging • CT – computed tomography • MRI – magnetic resonance imaging • fMRI – functional MRI • PET – positron emission tomography • Allows repeated measurements (longitudinal)

34. To study tissue / cells / molecules • Traditional histology • H & E • Nuclei - cresyl violet • Solochrome cyanine – Myelin • Modern histology • Tract tracing – anterograde / retrograde / transsynaptic • Protein expression • Immunolabelling • Western blotting / proteomics • Gene expression • In situ hybridisation • Northern blotting / microarray / real time PCR Histology (study of tissue)

35. What do I want to model or measure? • Cell loss / Neuroprotection? • Contusion / compression best? • True axon regeneration? • Complete section of tract required • Collateral sprouting? • Partial sparing of tract required How to select a model.... • What animal should I use? • What axon tracts do I want to cut? • Primate CST is in different location to rodent CST

36. Animal models allow controlled simulation of a human SCI and testing of therapies • Different types exist to model different aspects of SCI Conclusions • Majority use surgical, a few use naturally occurring • Pros and cons to each model. • Any questions?

37. Anatomy of human spinal cord: Kandel, Schwartz & Jessell, Principles of Neural Science Spinal cord injury statistics: http://www.spinalcord.uab.edu/show.asp?durki=21446 Reviews on animal models for spinal cord injury Courtine et al., 2007 Can experiments in nonhuman primates expedite the translation of treatments for spinal cord injury in humans? Nat. Med. 13(5):561-6 Moon & Bunge, 2005. From animal models to humans. Journal of Neurological Physical Therapy 29:55-70. Brosamle & Huber, 2006 Cracking the black box. Drug Discovery Today: Disease Models. Jeffery et al., 2006. Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy. Spinal Cord. 44:584-593. Zheng et al., 2006. Genetic models for studying inhibitors of spinal axon regeneration. TINS 29:640-6. Steward et al., 2003. False resurrections. J Comparative Neurol. 459:1-8. Lemon & Griffiths, 2005. Comparing the function of the corticospinal system in different species: organizational differences for motor specialization? Muscle and Nerve. 32:261-79 Key papers to read critically Jeffery et al., 2006. Autologous Olfactory Glial Cell Transplantation Is Reliable and Safe in Naturally Occurring Canine Spinal Cord Injury. J Neurotrauma 22:1282-1293 Freund et al., 2006. Nogo-A-specific antibody treatment enhances sprouting and functional recovery after cervical lesion in adult primates. Nature Medicine. 12:790-2 and pages 1220, 1231-1233. Include Supplementary Materials. Reading list

38. Articles on ethics of using animals in research (optional) The animal ethics reader (eds. Susan Armstrong and Richard Botzler) Articles on ethics of using non-human primates in research (very optional) The Great Ape Project (eds. Paola Cavalieri and Peter Singer) Optional reading list