Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells

Abstract

Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9-T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism.

Fig. 1

Photograph through surgical microscope of scaffold implanted into T9–T10 hemisection lesion. Arrows indicates scaffold position. Scale bar = 10mm.

Fig. 2

Scanning electron microscope image of scaffold architecture.

Fig. 3

Composite scaffold design.

Fig. 4

Seeding of hNSC on PLGA scaffolds. (A): Microscope image of toluidine blue staining of a cross-section of a PLGA scaffold seeded with hNSCs (purple dots). (B): Montage of multiple microscope images of the different areas of the same scaffold cross-section; inverted greyscale image of DAPI fluorescence (black dots are hNSC nuclei stained for DAPI). Scale bar = 500 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

Temporal profile of functional recovery post-injury. (A): Video neuromotor scores in ipsilateral (left) hindlimb. (B): Video neuromotor scores in contralateral (right) hindlimb. Designations are as follows: Scaffold + hNSC: mean and standard deviation for Y430 and X992; n = 2 except for day 1 and day 44 where n = 1 due to sacrifice of X992 at 40 days post-injury. Scaffold alone: Y464. Control (no treatment): Y156.

Fig. 6

Hematoxylin and eosin staining of lesion margins. (A and B): Rostral (A) and caudal (B) lesion margins of scaffold + hNSC treated animal 40 days post-injury (X992). Arrows indicate preserved polymer matrix. (C and D): Rostral (C) and caudal (D) lesion margins of scaffold + hNSC treated animal 82 days post-injury (Y430). There was no appreciated polymer matrix, indicating substantial degradation and clearance of the scaffold within 82 days in vivo. Glial cell proliferation and mononuclear cell infiltration adjacent to the lesion margin is visible in both subjects (A and D). Magnification = 40×.

Fig. 7

Silver staining for axonal degeneration of lateral corticospinal tracts (CST) in thoracic cross-sections caudal to the injury site. (A and B): Ipsilateral (A) and contralateral (B) images of control subject 111 days post-injury. C and D: ipsilateral (C) and contralateral (D) images of scaffold treated subject 83 days post-injury. E and H: Ipsilateral (E and G) and contralateral (F and H) images of scaffold + hNSC treated subjects 40 (E and F) and 82 (G and H) days post-injury. The lesion is unilateral with respect to degeneration of axons in the CST. Dors = dorsal. Vent = ventral. Magnification = 10×.

Fig. 8

Silver staining and Iba1 staining of the dorsal funiculus in thoracic cross-sections rostral to the injury site. (A and B): Silver (A) and Iba1 (B) staining of control subject 111 days post-injury. The lesion crosses the midline slightly in the control subject in the dorsal funiculus. (C and D): silver (C) and Iba1 (D) staining of scaffold treated subject 83 days post-injury. The images show total bilateral damage to the dorsal funiculus in the scaffold treated subject. E and H: silver (E and G) and Iba1 (F and H) staining of scaffold + hNSC treated subjects 40 (E and F) and 82 (G and H) days post-injury. The lesion is predominately unilateral, with some axonal degeneration of contralateral dorsal funiculus afferents with spatially correlating microglial and macrophage activation. Dors = dorsal. Vent = ventral. Magnification = 10×.

Fig. 9

Silver staining for axonal degeneration in thoracic sagittal sections through the hemisection lesions. Sections shown are closest to the middle of the cord in the rostral–caudal direction. (A): Control subject 111 days post-injury. B: scaffold treated subject 83 days post-injury. (C and D): Scaffold + hNSC treated subjects 40 (C) and 82 (D) days post-injury. The lesion crosses the midline, in reference to the central canal where present, into the contralateral gray matter. In these sections the contralateral lateral funiculi appear to be preserved from the surgical lesion in all subjects. However, some degenerative staining is visible in contralateral efferents. Ipsi = ipsilateral lesioned side. Contra = contralateral unlesioned side. The cords are oriented rostral to caudal from left to right. Scale bar = 1mm.

Fig. 10

Silver staining for axonal degeneration of ventral funiculus tracts in thoracic cross-sections caudal to the injury site. (A): Control subject 111 days post-injury. B: scaffold treated subject 83 days post-injury. C and D: scaffold + hNSC treated subjects 40 (C) and 82 (D) days post-injury. The lesion is bilateral with respect to degeneration of axons in the ventral–medial tracts. Dors = dorsal. Vent = ventral. Magnification = 10×.

Fig. 11

Iba1 staining for microglial and macrophage activation around lateral corticospinal tracts (CST) in thoracic cross-sections caudal to the injury site. (A and B): Ipsilateral (A) and contralateral (B) images of control subject 111 days post-injury. (C and D): Ipsilateral (C) and contralateral (D) images of scaffold treated subject 83 days post-injury. E and H: ipsilateral (E and G) and contralateral (F and H) images of scaffold + hNSC treated subjects 40 (E and F) and 82 (G and H) days post-injury. The lesion is unilateral with respect to microglial and macrophage activation in the cord around the CST. Dors = dorsal. Vent = ventral. Magnification = 10×.