Neuropathological and Motor Impairments after Incomplete Cervical Spinal Cord Injury in Pigs

Abstract

Humans, primates, and rodents with cervical spinal cord injury (SCI) show permanent sensorimotor dysfunction of the upper/fore limb as consequence of axonal damage and local neuronal death. This work aimed at characterizing a model of cervical SCI in domestic pigs in which hemisection with excision of one centimeter of spinal cord was performed to reproduce the loss of neural tissue observed in human neuropathology. Posture and motor control were assessed over 3 months by scales and kinematics of treadmill locomotion.Histological measurements included lesion length, atrophy of the adjacent spinal cord segments, and neuronal death. In some animals, the retrograde neural tracer aminostilbamidine was injected in segments caudal to the lesion to visualize propriospinal projection neurons. Neuronal loss extended for 4-6 mm from the lesion borders and was more severe in the ipsilateral, caudal spinal cord stump. Axonal Wallerian degeneration was observed caudally and rostrally, associated to marked atrophy of the white matter in the spinal cord segments adjacent to the lesion. The pigs showed chronic monoplegia or severe monoparesis of the foreleg ipsilateral to the lesion, whereas the trunk and the other legs had postural and motor impairments that substantially improved during the first month postlesion. Adaptations of the walking cycle like those reported for rats and humans ameliorated the negative impact of focal neurological deficits on locomotor performance. These results provide a baseline of behavior and histology in a porcine model of cervical spinal cord hemisection that can be used for translational research in SCI therapeutics.

The Cortical Motor System in the Domestic Pig: Origin and Termination of the Corticospinal Tract and Cortico-Brainstem Projections

“…Domestic piglets have the adult brain neuronal numbers and sulcal pattern established at birth (Jelsing et al, 2006;Ernst et al, 2018), and show a perinatal brain growth spurt like that of humans (Dickerson and Dobbing, 1967). These facts, together with evidence that the human CST arrives to the lower cervical segments before birth (Eyre et al, 2000), and our observation of axonal Wallerian degeneration in the dorsolateral and ventromedial spinal fascicles, where the porcine CST is located, caudal to a cervical C6 spinal cord hemisection in 2-month-old piglets (Cerro et al, 2021), support the notion that all porcine brain-spinal cord axons have reached their target regions in the perinatal period. On the other hand, M1 thickness increases by 60% during the early postnatal life, but it reaches 95% of its adult value by 5-6 months of age (Desantis et al, 2021).…”

“…A profuse innervation was consistently detected in the IB nucleus, which is located in the medial region of lamina IV from C1 to C6 and contains sensory relay neurons projecting to the thalamus ( Granum, 1986 ; Kemplay and Webster, 1986 ). Some axonal terminations were also present in lamina VIII, frequently arising from decussating commissural fibers ( Figure 4B ), and in the ventromedial region surrounding putative motoneurons for axial muscles ( Cerro et al, 2021 ). The finding of corticospinal axons arriving to lateral laminae IX was rare ( Figure 4D ).…”

“…Maximal retrograde labeling of corticospinal neurons in primates and rodents was obtained by applying neural tracers to the cut axonal fibers after a high cervical spinal cord hemisection ( Nudo and Masterton, 1990 ). However, we have shown that pigs with cervical C5-C6 spinal cord hemisection develop severe postural and motor deficits and become demanding for health care and basic behaviors ( Cerro et al, 2021 ). Because those impairments are expected to be even greater after C1 hemisection, in the present work we attempted a less traumatic while still efficient method for retrograde neuronal tracing, namely injecting a relatively large total volume (48 μl) of the fluorescent tracer aminostilbamidine into the lateral funiculus of the right, caudal part of C1, at the approximate location of the dorsolateral component of the CST.…”

“…Aminostilbamidine differs from hydroxystilbamidine (Fluoro-Gold) only in an amino group substituting for a hydroxyl group, but otherwise these molecules are identical. The use of aminostilbamidine in the present work was based on our previous studies with this neural tracer in rodents ( Collazos-Castro et al, 2005 ; Lucas-Osma and Collazos-Castro, 2009 ; López-Dolado et al, 2013 ) and pigs ( Cerro et al, 2021 ) showing that: (1) it produces an intense, durable, and bleach-resistant fluorescent neuronal labeling in paraformaldehyde-fixed tissue; (2) it is suitable for tracing neurons with long axons and can be easily combined with Alexa-conjugated dextran and other tracers for multiple cell labeling; (3) neuronal uptake of aminostilbamidine is very efficient and occurs by multiple ways including healthy axonal terminals and apparently fibers of passage, as well as by axons damaged by the needle or tissue necrosis at the injection site, similarly to Fluoro-Gold ( Schmued and Fallon, 1986 ; Dado et al, 1990 ).…”

“…Aminostilbamidine differs from hydroxystilbamidine (Fluoro-Gold) only in an amino group substituting for a hydroxyl group, but otherwise these molecules are identical. The use of aminostilbamidine in the present work was based on our previous studies with this neural tracer in rodents (Collazos-Castro et al, 2005;Lucas-Osma and Collazos-Castro, 2009;López-Dolado et al, 2013) and pigs (Cerro et al, 2021) showing that: (1) it produces an intense, durable, and bleach-resistant fluorescent neuronal labeling in paraformaldehyde-fixed tissue;…”