1
2	Case Study 29 Rt h m had diving accident at age 20. C4 quadriplegic with some sensory sparing and some motor function at C4-5. MRI and XR spine: fx C5 lamina and C6 body w.o. cord compression. R_X high dose methyl-prednisolone and halo neck brace. Trf after 10 days to Rehab, still quadriplegic, move R toe. After 1 month of inpatient rehab, he was walking, wearing halo, and had recovered substantial strength in hands R > L. Seen in Neurology OPD at age 22, c/o mild L hand wkness and stiffness, poor stamina, spasms, throbbing and dysesthesias in legs, neck pain, partial erectile and ejaculatory dysfunction. Neuro Exam: nl CN, almost nl motor excpt 4.5/5 L finger abd, sl RAM L fingers, sl vibr toes bilat, sl pin and temp R T2-T8, MRs 2-3 excpt 4 at L ankle, bilat Hoffmann’s and L Babinski. Gait and station nl. Urologic w/u and EMG showed only penile tumescence in snap test. R_X nortriptyline 75 mg/d improved pain and stiffness but worsened sexual symptoms. Sildenafil improved sexual function. Went to medical school and now functioning well socially and professionally.
3	Epidemiology of Spinal Cord Injury More than 10,000 new cases of traumatic SCI in the US each year More than 230,000 persons currently living with traumatic SCI in the US Many more cases of SCI due to diseases Tumors Infections Neurodegenerative Demyelinating
4	ASIA Adaptation of Frankel Classification
5	Prognosis of Spinal Cord Injury The probability that a person will regain walking with or without assistive device depends on the initial ASIA scale score: ASIA A (complete loss of motor and sensory function below the level of injury) - 3% {futility in clinical trials?} ASIA B (Incomplete, preserved sensation only) with intact peri-anal pin sensation - 50% ASIA C (Incomplete, preserved nonfunctional motor) - 75% ASIA D (Incomplete, preserved functional motor) - 95% {ceiling effect in clinical trials} (Consortium for Spinal Cord Medicine. Outcomes following traumatic spinal cord injury: Clinical Practices Guidelines for Health-care Professionals. Washington, DC: Paralyzed Veterans of America,1999)
6	Requirements for Functional Recovery Minimize Neuronal and Axonal Loss Replace Lost Neurons Remyelinate Demyelinated and Regenerated Axons Promote Regeneration of Interrupted Axons Translate Restored/Preserved Connections into Function Pharmacological interventions Physical therapeutic interventions
7	Response to Axotomy in CNS
8	Peripheral Nerve Environment Supports Axon Regeneration
9	Factors Limiting Regeneration in CNS Environmental Growth cone collapsing factors myelin associated (Nogo, MAG, oligodendrocyte-myelin glycoprotein, tenascin-R, ephrinB3) neuron associated (semaphorins, netrins, ephrins) Astrocyte-derived (chondroitin-6-S proteoglycans) Fibroblast-derived (semaphorin 3A) Mechanical properties of the scar? Intraneuronal (GAP-43, L1, NF, tubulins, c-jun, ATF-3, Rho GTPases, cAMP, netrin receptors)
10	In Response to Injury, Axons in Tissue Culture Make a Growth Cone
11	Myelin-Associated Growth Inhibitors Neurons (embryonic) cultured on bed of Schwann cells or extracts of PNS myelin grow axons well. Neurons cultured on oligodendrocytes or extracts of CNS myelin fail to grow axons well. Several molecular constituents of myelin block axon growth in tissue culture. The three most interesting are: Nogo Myelin-Associated Glycoprotein (MAG) Oligodendrocyte Myelin Glycoprotein (OMGP, MOG) The Nogo receptor mediates the effects of all three of these proteins
12	Anti-NOGO Promotes Regeneration and/or Sprouting in CNS Transplant IN-1-secreting hybridoma subventricularly. Perform dorsal over-hemisection to cut the corticospinal tract. After 30-60 days, inject anterograde label in motor cortex. Detect axon terminals up to 11 mm caudal to the hemisection in IN-1 treated rats, but only 1 mm in control hybridoma recipients. Subsequent studies suggested that many of the nerve terminals had not regenerated but had sprouted across the midline from unlesioned ipsilateral corticospinal pathways.
13	Anti-Nogo Promotes Collateral Sprouting in CNS
14	Nogo Transgenic Mouse Models Give Differing Results on Regeneration Trials Of three laboratories making mice lacking Nogo and/or NgR, two have found that these mice show enhanced regeneration after spinal cord injury, and one has found no effect. The differences could be due to: Differences in the locus and extent of genetic deletions. Differences in experimental protocols. Failure to differentiate between sprouting and regeneration. At a minimum, the experiments using anti-Nogo antibodies reported more robust results and much of the rationale rests on the ability of Nogo and other NgR agonists to inhibit axon elongation in tissue culture, which is an assay of growth cone collapse in embryonic neurons.
15	Chondroitin-6-Sulfate Proteoglycans
16	Chondroitinase Enhances Regeneration
17	Chondroitinase-ABC Promotes Regeneration and Functional Recovery after Spinal Cord Injury
18	Neurotrophins Promote Regeneration in CNS
19	Influence of Age Axons grow during embryonic development. Axons can regenerate early in development but not in the adult CNS. What is the reason for this developmental loss of regenerative ability? Environment? Neuron-intrinsic?
20	Regeneration in Optic System May Depend on Neuron Age, not Environment Age
21	Cyclic-AMP and Neurotrophins Mimic Conditioning Lesion Effect in Regeneration of Central Axons of DRGs
22	Role of cAMP in Regeneration of DRG and RGC Axons Intraneuronal cAMP levels fall during early postnatal development, corresponding to a loss of ability of axons to regenerate on myelin in vitro. Central axotomy leads to increased cAMP in early postnatal DRG but decreased cAMP in older DRG neurons. Conditioning lesion of peripheral but not central axon leads to increased cAMP levels in DRG and regeneration of the central branch in the dorsal columns. Neurotrophins mimic effect of a conditioning lesion on ability of axons to grow on myelin. Injection of db-cAMP into DRG mimics the effect of peripheral conditioning lesion in enhancing regeneration of the central axon.
23	Injection of db-cAMP into DRG Increases Dorsal Column Axon Regeneration
24	Rho Mediates Effects of Growth Cone Collapsing Molecules and is Inactivated by cAMP
25	Proposed Links Between cAMP and Regeneration
26	Large Larval Sea Lamprey
27	Regenerating Axons at the Scar (Adult Lamprey)
28	Regenerating vs. Developing Axon Tips
29	Axon Regeneration in Lamprey Spinal Cord is Accelerated by cAMP without Growth Cones
30	Clinical Trials in Spinal Cord Injury - Approaches Neuroprotection Enhance Physiological Conductivity Promote Axon Remyelination Promote Axon Regeneration Replace Lost Neurons
31	Clinical Trials in Spinal Cord Injury - Neuroprotection Methylprednisolone (only approved drug) Minocycline. Phase I in Canada (mechanism not certain; may reduce axon retraction and oligodendrocyte apoptotis by inhibiting several aspects of the inflammatory response through inhibition of the p38 mitogen-activated protein kinase pathway) Gacyclidine (GK-11, Neureva, France). Phase II/III (NMDA antagonist) - negative Skin-Activated Macrophages (Proneuron Biotechnologies). Phase II (inhibit release of proinflammatory cytokines, e.g., TNF, and release IL-1β BDNF) – suspended for lack of funds. CSF Drainage (Kwon, U. British Columbia) (?reduce cytokine release by decreasing CSF pressure)
32	Clinical Trials in Spinal Cord Injury- Overcome Partial Conduction Block Spinal cord injury results in apoptosis of oligodendrocytes. Some axons that are not interrupted by the injury nevertheless show conduction block. Acutely, conduction block may be relative if only one internode is demyelinated, or the oligodendrocyte degeneration is incomplete, but total if two consecutive internodes are completely demyelinated.
33	Clinical Trials in Spinal Cord Injury- Overcome Partial Conduction Block Loss of myelin results in conduction block because the inward current carried by Na⁺ leaks out of the axon through the capacitance of the bared axolemma exposed K⁺ channels that occupy the juxtanodal membrane
34	Clinical Trials in Spinal Cord Injury-Overcome Partial Conduction Block Chronically, axon conduction may be partly restored through remyelination by surviving oligodendrocytes increased density of Na⁺ channels in the demyelinated segments
35	Clinical Trials in Spinal Cord Injury-Overcome Partial Conduction Block K⁺ channel blockers 4-Aminopyridine (4-AP; Fampridine-SR, Acorda) HP-184 (Sanofi-Aventis) Both Phase III
36	Clinical Trials in Spinal Cord Injury- Enhance Regeneration Intrathecal C3 transferase (BA-210, BioAxone Therapeutic) Phase I/II (inactivates Rho by ADP ribosylation; also neuroprotective) Oligodendrocyte progenitor transplants. Phase I planned (Hans Keirstead with Geron) Olfactory ensheathing cell transplants (contorversial; small Phase I in Australia) Anti-Nogo66 antibodies (Novartis; Phase I in Europe - not toxic; Phase II to begin in US) (promotes collateral sprouting and/or regeneration)
37	Clinical Trials in Spinal Cord Injury-Enhance Regeneration C3 transferase (Rho inhibitor) Dorsal over-hemisection to cut CST in mice C3 in fibrin applied to lesion Anterograde label with WGA-HRP from motor cortex Count nerve terminals caudal to lesion C3-treated animals showed longer distance regeneration and enhanced locomotor recovery. Phase I – not toxic.
38	Clinical Trials in Spinal Cord Injury-Enhance Remyelination Oligodendrocyte Precursor Cells Derive OPCs from human embryonic stem cells (medium containing EGF, bFGF, retinoic acid) Transplant into rat spinal cord contusion at 7d or 10 mo post-lesion Count axons in area of lesion remyelinated by OPCs or by Schwann Cells Measure behavioral recovery (BBB; kinematic analysis) OPCs survive and differentiate into oligodendrocytes (GalC, RIP and O4 immunohistochemistry) at both 7 d and 10 mo p-injury However, OPC transplantation results in axon remyelination by oligodendrocytes and functional recovery only at 7 d post-injury Thus there is a time window for OPC effectiveness
39	Clinical Trials in Spinal Cord Injury-Enhance Regeneration Autologous skin-activated macrophages Isolate rat macrophages by FACS for ED1 and incubate them with rat dermis. Measure trophic factors and cytokines in supernatants. Perform T9 spinal cord contusion. At 4-9 d post-injury, inject macrophages into caudal border of lesion. Assess locomotion (BBB score, 1-21) weekly. Count score 6 as recovered. Sacrifice for morphology at 5-6 mo. Phase II clinical trial suspended for lack of funds.
40	Clinical Trials in Spinal Cord Injury-Enhance Regeneration Olfactory ensheathing cells The olfactory nerve turns over throughout life, so axons are constantly regenerating. The ensheathing glia of the ON can be obtained by nasal biopsy and may provide a supportive environment for regeneration. Animal studies have differed as to whether transplants of OECs meylinate axons (olfactory axons are unmyelinated) and whether they promote axon regeneration in the injured spinal cord. The differences may relate to heterogeneity in OEC biology and whether they were obtained from the lamina propria (of the nasal mucosa) or the olfactory bulb. Hundreds of SCI patients have been transplanted by Dr. Hongyun Huang, a neurosurgeon in Beijing, but the benefits are debated and no controls have been performed. A single blind, controlled Phase I trial of 3 transplanted patients and 3 controls is under way in Brisbane, Australia.
41	Automatic Treadmill Stepping
42	Partial Body Weight-Supported Treadmill Training (Courtesy B. Dobkin, MD, UCLA)
43	Conclusions Most preclinical data providing the rationales for clinical trials in SCI are based on: Effects on embryonic neurons and axon growth cones in cell culture, which may not be a relevant model for regeneration of mature CNS axons Effects on hemisection or contusion models of partial spinal cord injury, in which assessment of regeneration is often ambiguous Experiments in rodents, which may not provide the scale of required regeneration distances to assess efficacy in humans Nevertheless, after many years of basic research, clinical trials for spinal cord repair are now under way. Most trials are for neuroprotection. Even when there is a regeneration rationale, as in activated macrophages and Rho inhibitors, the strongest evidence is still for neuroprotection. Bioethics of highly invasive procedures are being addressed vigorously in the scientific community.