Translation of the rat thoracic contusion model; part 2 - forward versus backward locomotion testing.

STUDY DESIGN: Experimental animal study. OBJECTIVES: Locomotion analyses in rat spinal cord contusion injury (SCI) models are widely used for the evaluation of recovery of supraspinal locomotor control. However, many commonly used locomotion tests are inadequate to test for spinal cord integrity as they assess motor function that can be highly mediated through below-level propriospinal pattern-generating circuitry, independently of below-level perception. Here we report a behavioral motor test that is more sensitive for spinal cord integrity, even 6 weeks after injury: the backward locomotion rotating rod. SETTING: University of California - San Diego. METHODS: A modified rotating rod test was run in reverse. The rod diameter was increased and thin rubber lining was added. As a reference, we included commonly used motor tests: BBB score, catwalk gait analysis, motor-evoked potentials, single frame analyses, a forward rotating rod test and the 55° inclined ladder test. RESULTS: Unlike commonly used motor tests, the backward locomotion rotating rod test significantly discriminates between both sham-operated (falling latency: 20.4 s s.d.±4.5) vs mild SCI animals, and mild vs moderate SCI animals (differences between each group at acute, subacute and chronic phases: ⩾6 s, P⩽0.01). Moderate SCI animals were practically unable to make even slight backward hindpaw movements. The backward locomotion ability in the chronic phase correlates best with BBB locomotor scores from the acute phase. CONCLUSION: Our data show that backward locomotion is a highly sensitive and quick test to discriminate between sham, mild and moderate SCI, even after 6 weeks. Backward locomotion testing may improve the translational value of experimental results for the clinic.

Translation of the rat thoracic contusion model; part 1-supraspinally versus spinally mediated pain-like responses and spasticity.

STUDY DESIGN: Experimental animal study. OBJECTIVES: Stimulus-evoked below-level paw withdrawals in animal models of spinal cord injury (SCI) can be mediated solely by below-level spinal cord reflexes. Interpreting lowered thresholds for such responses as a model for chronic below-level pain after (thoracic contusion) SCI appears not appropriate, which requires reinterpretation of many prior results. However, how to reinterpret the changes in withdrawal thresholds and what can be a better alternative for pain/sensory assessments remains unclear. SETTING: University of California, San Diego. METHODS: We introduce a method using supraspinally mediated escape responses to assess pain-like sensitivity thresholds on a continuous/linear scale. To further understand the decrease in hindpaw withdrawal thresholds, we investigated whether they may be interpreted as spasticity. RESULTS: The escape response test can be used to assess SCI-induced changes in below-level sensory thresholds. These thresholds were found to increase soon after moderate or severe SCI, while, in parallel, hindpaw withdrawal thresholds decreased. However, the latter did not co-occur with spasticity, suggesting that SCI-induced increased withdrawal responses are probably best interpreted as a form of hyperreflexia with pathophysiological analogies of spasms and/or clonus, or a species-specific phenomenon. CONCLUSION: Decreased below-level withdrawal thresholds do not reflect pain-like hypersensitivity in rodent models of (thoracic contusion) SCI. A large body of previous preclinical SCI pain research needs reinterpretation. We actually found below-level thermal and mechanical hypoesthesia and we also excluded a relation between withdrawal hyperreflexia and spasticity. Withdrawal hyperreflexia might still prove useful to model spasms or clonus, which are, like hypoesthesia, also significant clinical problems after SCI.

Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.