NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury

Abstract

Spinal cord injury (SCI) often leads to permanent loss of motor, sensory, and autonomic functions. We have previously shown that neurotrophin3 (NT3)-loaded chitosan biodegradable material allowed for prolonged slow release of NT3 for 14 weeks under physiological conditions. Here we report that NT3-loaded chitosan, when inserted into a 1-cm gap of hemisectioned and excised adult rhesus monkey thoracic spinal cord, elicited robust axonal regeneration. Labeling of cortical motor neurons indicated motor axons in the corticospinal tract not only entered the injury site within the biomaterial but also grew across the 1-cm-long lesion area and into the distal spinal cord. Through a combination of magnetic resonance diffusion tensor imaging, functional MRI, electrophysiology, and kinematics-based quantitative walking behavioral analyses, we demonstrated that NT3-chitosan enabled robust neural regeneration accompanied by motor and sensory functional recovery. Given that monkeys and humans share similar genetics and physiology, our method is likely translatable to human SCI repair.

Keywords: CST regeneration; NT3; chitosan; nonhuman primate; spinal cord injury repair.

Fig. 1.

Histological analyses demonstrating NT3-chitosan elicited de novo neural tissue reconstruction after a severe case of SCI. (A) A schema illustrating spinal cord hemisection and extraction leaving a 1-cm gap in monkey right spinal cord at thoracic level T8 (eighth vertebra), which was either left alone (lesion control) or inserted with NT3-chitosan matrix-containing tubular biomaterial (NT3-chitosan). (B) Gross anatomical analysis demonstrated regeneration of neural cable-like tissues in the NT3 group at the lesion/regeneration site, while only scar tissues filled the injury site in the LC group. (B, Right) NT3-chitosan–induced regeneration of neural cable-like structure in rat 9 mo after complete transection and extraction of rat thoracic spinal cord is shown. Results demonstrate the similarity between monkey and rat de novo regenerated neural tissues. (C) Immunofluorescent neurofilament staining (green) detected in regenerated neural tissues with NT3-chitosan treatment, while the LC group showed a lack of NF immunoreactivity in a major part of the lesion area. Quantitative measures of the bridge tissue diameter and the NF staining fiber density for the NT3 group are shown in SI Appendix, Table S4. (D) Over 24 mo after the operation, 1-mm-long spinal cord tissue was selected around the middle point of the regenerated tissue in the tube, and then semithin slices were made by staining with toluidine blue. The light micrographs show a large amount of regenerated blood vessels and myelinated and unmyelinated axons surrounded by epineurium- or perineurium-like structures in the regenerated cable tissue.

Fig. 2.

CST tracking with unilateral BDA injections. (A) A diagram of BDA injections in uninjured and NT3-chitosan monkeys. (B–D) Longitudinal sections of monkey spinal cord 11 wk after BDA injections in normal (uninjured, animal 30) (B), lesion control (animal 5) (C), and NT3-chitosan (animal 18) (D) monkeys more than a year after the initial operation. DAPI (blue), BDA (red), and GFAP (green) fluorescent images are shown. ROI, region of interest; small white arrows marked regenerated BDA-positive fibers.

Fig. 3.

SEPs demonstrating partial restorations with NT3-chitosan treatment. SEPs detected in left S1 (red) when right anterior tibial muscle was stimulated, and in right somatosensory cortex (green) when left hind limb muscle was stimulated, over 12 mo after the SCI operation. Lesion to the right spinal cord at T8 completely abolished SEP in left S1 cortex in the LC group, while it attenuated SEP signals in right S1, probably due to a spread of inflammation or secondary lesion. SEP reappeared in left S1 in the NT3 group; signals in the right S1 were also enhanced, likely due to the antiinflammatory effect of NT3-chitosan. In NT3 group animals, further lesion of the contralateral half (left) of the intact spinal cord completely abolished SEP signals in the right S1, while signals in the left S1 persisted. Resection of the regenerated neural cable, on the other hand, abolished SEP in left S1, and attenuated SEP signals in the right S1. Quantification and statistical analyses of amplitude and latency period for the aforementioned experiments are shown. Resection results of each animal are also displayed. Technical measurements were repeated twice. Shown are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA or two-tailed independent sample t test. ND, not determined; n.s., not significant. See SI Appendix, Tables S10 and S11 for exact n and P values.

Fig. 4.

MEPs demonstrating partial restorations with NT3-chitosan treatment. Stimulation of the right motor cortex evoked MEPs in left anterior tibial muscles (blue), and stimulation of the left motor cortex evoked MEPs in the right hind limb muscles (orange). The relationship between the LC and NT3 groups on MEPs is similar to that on SEPs shown in Fig. 3. Both contralateral (major; thick waveform lines) and ipsilateral (minor; thin waveform lines) responses were analyzed. Quantification and statistical analyses of amplitude and latency period for the aforementioned experiments are shown. Resection results of each animal are also displayed. Technical measurements were repeated twice. Shown are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA or two-tailed independent sample t test. See SI Appendix, Tables S10 and S11 for exact n and P values.

Fig. 5.

fMRI analyses indicating recovery of sensory function with NT3-chitosan treatment. Diagram illustrating fMRI experimental design with heat/temperature stimuli applied to the left hind limb. The lateral spinothalamic tract is transmitting the corresponding sensory signal to the somatosensory cortex as illustrated. Averaged fMRI signals within each group were superimposed onto a 3D monkey brain and coronal structural images, respectively (P < 0.05, GRF-corrected). Uninjured and NT3 groups displayed significant activation in the S1 area representing the left hind limb receptor field upon thermal stimulation, but LC animals showed no signal. Quantitative BOLD signal changes of the three groups are shown in a bar graph (n = 3 to 5 animals). Intergroup comparisons demonstrate obvious differential activation between the uninjured and LC groups and between the NT3 and LC groups, with no difference between uninjured and NT3 groups (quantitative data are also demonstrated in a bar graph). The schema of brain structures is superimposed onto coronal images. Color scales indicate t values. MNI coordinates are shown. Technical measurements were repeated twice. Error bars represent the mean ± SEM. **P < 0.01 by ANOVA. See SI Appendix, Tables S10 and S11 for exact n and P values. cs, central sulcus; FFG, fusiform gyrus; ITG, inferior temporal gyrus; ls, lateral sulcus; MNI, Montreal Neurological Coordinates; MTG, middle temporal gyrus; PCG, posterior cingulate cortex; PE, sensory association cortex; R, right; S1, primary somatosensory cortex; SMG, supramarginal gyrus; STG, superior temporal gyrus; T, thoracic vertebra; TS, temperature stimulation; NaN, not a number.

Fig. 6.

Improvement of animal walking behavior after NT3-chitosan treatment. (A) Lesion areas were similar in size and comparable between the LC and NT3 groups. (B–D) Bilateral hind limb kinematics, joint trajectory, and end point trajectory during bipedal locomotion in uninjured (B), LC (C), and NT3 (D) monkeys are displayed. The arrow indicates the direction of stepping (swing phase). Stick figures represent the hind limb (left, LH; right, RH) and precisely describe the animal’s stepping movements in the swing. The joint trajectory showed continuous movement of representative joints during stepping. Successive trajectories (gait cycles, 5) of the hind limb end point during stance (gray), swing (blue), and dragging (red) are shown for each condition together. The repeated trajectories were superimposed to show the gait consistency. (E) Clustering matrix of 12 clinically relevant gait parameters for three groups of animals is shown. These parameters were chosen to evaluate the overall gait performance. The color scale indicates relative values of each parameter. In the matrix, relative values in the NT3 group are much closer to that of the uninjured group. Raw data are also shown in bar graphs (Left) (n = 3 to 5 animals). Technical measurements were repeated at least three times. Shown are mean ± SEM. **P < 0.01, ***P < 0.001 by ANOVA or Kruskal–Wallis test or two-tailed Mann–Whitney U test. See SI Appendix, Tables S10 and S11 for exact n and P values. (F) Hind limb models displayed a representative gait cycle in uninjured and NT3 monkeys. Step phases of the right hind limb: ① the end of the stance phase; ② mid swing near the vertical line of the body; ③ the end of the swing phase; ④ mid stance with contralateral hind limb (LH) near the vertical line of the body; and ⑤ the end of the stance phase. An LC animal (Middle) showed a dragging right hind limb in the whole gait cycle.

Fig. 7.

MRI results indicating NT3-chitosan enabled neural tissue regeneration after SCI. (A) Diagram illustrating the spinal cord hemisection and extraction model. (B–D) Typical fiber tract reconstruction for the uninjured (B), LC (C), and NT3 (D) groups is displayed. In the LC group, fiber tracts were interrupted, deformed, and distorted at the border rostral and caudal to the lesion, with no fiber tracts present within the lesion (C). In contrast, in the NT3 group, numerous regenerated fiber tracts extend across the surgical site, reconnecting the rostral and caudal ends of the injured cord (D). Arrowheads indicate the surgical sites. Red dashed lines indicate the damaged zone, and tubular structures in the NT3 group represent positions of chitosan tubes. (E) Graph of averaged FA values and percentages of rostral–caudal voxel numbers of the three groups in three locations (I to III). Results of two levels in the middle site were gathered as location II. The NT3 group showed significantly higher FA values and percentages of voxels compared with the LC group (n = 3 to 5 animals). (F) Diffusion tensor fiber tracking tractography showed longitudinal changes of lesion (Top) and treated (Bottom) spinal cord with time after operation. LC animals showed a lack of neural fibers in the damaged zone, while NT3 animals displayed regenerated fiber bundle growth progressively with time and eventually connecting the two ends of the severed cord at 6 mo. I, inferior; L, left; R, right; S, superior. (G) Time courses of changes in FA values and percentages of rostral–caudal voxels at the center of the surgical site (location II) from LC and NT3 groups up to 6 mo post operation (n = 3 or 4 animals). Technical measurements were repeated twice. Shown are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA or two-tailed independent sample t test or two-tailed Mann–Whitney U test. See SI Appendix, Tables S10 and S11 for exact n and P values.