Integration of Transplanted Neural Precursors with the Injured Cervical Spinal Cord

Abstract

Cervical spinal cord injuries (SCI) result in devastating functional consequences, including respiratory dysfunction. This is largely attributed to the disruption of phrenic pathways, which control the diaphragm. Recent work has identified spinal interneurons as possible contributors to respiratory neuroplasticity. The present work investigated whether transplantation of developing spinal cord tissue, inherently rich in interneuronal progenitors, could provide a population of new neurons and growth-permissive substrate to facilitate plasticity and formation of novel relay circuits to restore input to the partially denervated phrenic motor circuit. One week after a lateralized, C3/4 contusion injury, adult Sprague-Dawley rats received allografts of dissociated, developing spinal cord tissue (from rats at gestational days 13-14). Neuroanatomical tracing and terminal electrophysiology was performed on the graft recipients 1 month later. Experiments using pseudorabies virus (a retrograde, transynaptic tracer) revealed connections from donor neurons onto host phrenic circuitry and from host, cervical interneurons onto donor neurons. Anatomical characterization of donor neurons revealed phenotypic heterogeneity, though donor-host connectivity appeared selective. Despite the consistent presence of cholinergic interneurons within donor tissue, transneuronal tracing revealed minimal connectivity with host phrenic circuitry. Phrenic nerve recordings revealed changes in burst amplitude after application of a glutamatergic, but not serotonergic antagonist to the transplant, suggesting a degree of functional connectivity between donor neurons and host phrenic circuitry that is regulated by glutamatergic input. Importantly, however, anatomical and functional results were variable across animals, and future studies will explore ways to refine donor cell populations and entrain consistent connectivity.

Keywords: interneurons; plasticity; respiration; spinal cord injury; transplantation.

FIG. 1.

Schematic of the phrenic circuit and C3/4 lateralized contusion. Respiratory drive is initiated in supraspinal neurons that project to the phrenic motor pool either directly or indirectly by pre-phrenic interneurons. Post-injury, there is compromise of descending white matter projections in addition to phrenic associated interneurons and motoneurons. At 1 week post-injury, FSC suspension is injected into the cavity at the site of injury. By 3–4 weeks after transplantation, donor neurons have survived, proliferated, and filled the cavity. A cross-section through the transplant epicenter reveals the presence of mature neurons (magenta) and glia (white) within the transplant (cyan) after immunohistochemical labeling of NeuN, GFAP, and GFP respectively (C; scale = 500 μm). At this point, a variety of tracing methods are used to determine host-to-transplant connectivity (BDA injection into C1/2 host gray matter or PRV injection into the transplant) and transplant-to-host connectivity (PRV delivered to the diaphragm). Terminal phrenic neurograms are conducted to determine the contribution of donor neurons to respiratory function. BDA, biotin dextran amine; FSC, fetal spinal cord; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclear protein; PRV, pseudorabies virus. Color image is available online at www.liebertpub.com/neu

FIG. 2.

Cross-section through the epicenter of a GFP-FSC transplant, 4 weeks after grafting. Donor tissue has filled the cavity, become confluent with host gray matter and contains mature astrocytes (white) and neurons (red), seen at high power in (C) and (D), respectively. In a different donor recipient, Ki-67 immunohistochemistry reveals a large number of cells still actively dividing at 1 month post-transplant (F–I). Whereas Ki-67–positive cells likely include both neurons and glia, one dividing, PRV-positive neuron (retrogradely labeled from the host phrenic motor pool) is visible (red arrow, I). (J–M) Transplanted tissue is well vascularized, and blood vessels appear to be continuous with those in the host, as seen by RECA immunolabeling (L, white). Scale is 500 um (A), 50 um (B–E), 50 um (F–I), and 500 um (J–M). 5HT, serotonin; FSC, fetal spinal cord; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; NeuN, neuronal nuclear protein; PRV, pseudorabies virus; RECA, rat endothelial cell antigen. Color image is available online at www.liebertpub.com/neu

FIG. 3.

Host to donor connectivity. (A) After injection of 1 μL, 10,000 molecular-weight BDA into the ipsilateral host gray matter at C1/2, a few BDA-positive fibers (red) can be seen near the center of the transplant (green). High magnification of a BDA fiber is seen in (B). (C) Cross-section through the epicenter of the transplant that has been immunohistochemically stained for the presence of serotonin and counterstained with cresyl violet. (D) A high-magnification image shows host serotonergic axons innervating the transplant near the graft-host border. Scale is 500 um for low power images (A and C) and 20 um for high-power images (B and D). Dashed lines outline the cross-section of the spinal cord (A) and delineate the graft-host border (C). 5HT, serotonin; BDA, biotin dextran amine; GFP, green fluorescent protein. Color image is available online at www.liebertpub.com/neu

FIG. 4.

Longitudinal section through the injury epicenter after injection of PRV into the transplant (A). The discrete injection site is denoted with a yellow arrow. A high degree of PRV-positive cells (indicated with red arrows) can be seen within the transplant (C), demonstrating the high degree of interconnectivity between grafted neurons. PRV labeling of host neurons is seen throughout the cervical spinal cord, from C1 (B) through C7 (D). In addition, labeling is seen within various nuclei in the brainstem (E), including the reticular nucleus (F). Orientation is rostrocaudal (left to right, A–D), dorsal ventral (top to bottom, E and F), and scales are as indicated. PRV, pseudorabies virus. Color image is available online at www.liebertpub.com/neu

FIG. 5.

Quantification of donor neurite outgrowth. A longitudinal section of the cervical spinal cord through the intermediate gray matter, immunohistochemically stained for GFP can be seen in (A). The transplant epicenter (green) has been overexposed so that GFP⁺ neurites projecting throughout the spinal cord can be visualized. High-magnification images of GFP⁺ neurites using lower, appropriate exposures were taken at 4-mm intervals along the length of the entire section, beginning at the rostral and caudal borders of the transplant and excluding GFP⁺ neurons. These images spanning the entire width of the cord were used to quantify the total area of GFP⁺ fibers (B). Outgrowth was measured in three sections per animal to include the dorsal horns (blue), intermediate gray (orange), and ventral horns (gray). Column graphs represent the average outgrowth at each distance in n = 4 animals with standard deviation. Statistical significance is marked by asterisks (p < 0.05). Scales are as indicated. GFP, green fluorescent protein. Color image is available online at www.liebertpub.com/neu

FIG. 6.

Longitudinal section through the transplant epicenter and phrenic motor pool with immunohistochemical labeling of GFP (green), PRV (red), and CHAT (white) after PRV application to the diaphragm. GFP⁺ (donor) neurites can be seen extending throughout the entire pictured cervical cord, from C4 to C6 (A). PRV labeling is seen within the host phrenic motor pool in close association with GFP axons (high magnification images seen in F–I) as well as within the transplant (B–E), indicating connectivity between donor neurons and host phrenic circuitry. Rostrocaudal orientation is top-bottom; scales are as indicated. ChAT, choline acetyltransferase; GFP, green fluorescent protein; PRV, pseudorabies virus. Color image is available online at www.liebertpub.com/neu

FIG. 7.

Immunohistochemical characterization of transplanted neurons. Low-power images of cross-sections through the GFP⁺ transplant epicenter can be seen in (A), (F), and (H), which have been immunohistochemically stained for NeuN and glutaminase (A, B, C, D, and E), TH (F and G) and GAD65/67 (H and I) in red. High-magnification insets reveal examples of glutaminase-positive, TH-positive, and GAD65/67-positive donor neurons (E, G, and I, respectively). A high degree of GAD-65/67-positive nerve terminals can be seen throughout the entire transplant, closely resembling what is seen throughout host grey matter. There are also pockets of high-density GAD-67/65 labeling in the transplant as is normally seen in the host dorsal horns, suggesting that some degree of long-term, cytoarchitecture fate is maintained. Scales are as indicated. GAD65/67, glutamic acid decarboxylase 65/67; GFP, green fluorescent protein; NeuN, neuronal nuclear protein; TH, tyrosine hydroxylase. Color image is available online at www.liebertpub.com/neu

FIG. 8.

Quantification of donor to host connectivity. (A) Longitudinal section through the transplant epicenter shows donor FSC tissue (green) that has been immunohistochemically labeled for PRV (red) and ChAT (white). A higher magnification of a co-labeled (PRV and ChAT) donor neuron can be seen in (B)–(D). Quantification of these neurons within the transplant reveals that very few ChAT⁺ donor neurons connect to host phrenic circuitry (E). ChAT-positive neurons mostly appear to be evenly dispersed throughout the transplant, and the number of donor ChAT neurons correlates with the size of the transplant (F). Conversely, donor PRV⁺ neurons often appear in clusters, and quantification appears independent of transplant size (G). Rostrocaudal orientation is top-bottom, and scale bars are as indicated. ChAT, choline acetyltransferase; GFP, green fluorescent protein; PRV, pseudorabies virus. Color image is available online at www.liebertpub.com/neu

FIG. 9.

Activity of donor neurons. (A) Cross-section through a SD-derived transplant, where dashed lines indicate the donor-host border. Immunohistochemical labeling of cfos demonstrates high degree of neuronal activity within the transplant, indicated by a large number of labeled nuclei (B). Multi-unit recordings of donor neuron activity revealed a bursting pattern (blue) correlated to phrenic motor output (green, C). Both integrated (top) and raw (bottom) activities are shown for each recording site. Scales are as indicated. Color image is available online at www.liebertpub.com/neu

FIG. 10.

Functional connectivity between donor and host phrenic circuitry. Terminal, phrenic nerve recordings were conducted to determine the extent of functional connectivity between donor and host phrenic circuitry. After collecting baseline phrenic activity, glutamatergic (NMDA and AMPA), and serotonergic (5HT-2, -5, and -7) receptor antagonists were injected directly into the transplant epicenter, separated by a washout period. The average amplitudes of left (ipsilateral to injury) integrated phrenic bursts are shown before and after application of glutamatergic (A) and serotonergic (B) antagonists to the transplant. Each point represents data from an individual animal. Changes in phrenic bursting in response to glutamatergic antagonist application was seen within 10 min in 50% of animals (blue and green, A), though minimal change was seen in response to serotonergic antagonist (B). Examples ipsilateral, integrated phrenic bursts from one animal can be seen in (C) and (D), where each trace represents a 40-sec average of phrenic activity before (red) and after (blue) antagonist application. 5HT, serotonin; 5HT Ant, 5HT antagonist; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate. Color image is available online at www.liebertpub.com/neu

FIG. 11.

Terminal, ipsilateral hemidiaphragm electromyography (EMG) activity post-injury and treatment. (A) Bar graphs represent the average burst amplitudes during baseline activity (± standard deviation) for each group: vehicle (HBSS) treated with or without immunosuppression (+CSA), Sprague-Dawley–derived fetal spinal cord tissue (SD-FSC) transplantation, and GFP-expressing Fisher–derived tissue (GFP-FSC) transplantation with immunosuppression (+CSA). Averages were calculated from 40-sec samples of integrated activity during eupneic (20% O₂) breathing. (B) The average percent changes (± standard deviation) in burst amplitude in response to a respiratory challenge (hypoxia, 10% O₂) for each group. Individual data points have been overlaid to show the extent of variability in each group. CSA, cyclosporine A; FSC, fetal spinal cord; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution. Color image is available online at www.liebertpub.com/neu