Intraspinal transplantation and modulation of donor neuron electrophysiological activity

Abstract

Rat fetal spinal cord (FSC) tissue, naturally enriched with interneuronal progenitors, was introduced into high cervical, hemi-resection (Hx) lesions. Electrophysiological analyses were conducted to determine if such grafts exhibit physiologically-patterned neuronal activity and if stimuli which increase respiratory motor output also alter donor neuron bursting. Three months following transplantation, the bursting activity of FSC neurons and the contralateral phrenic nerve were recorded in anesthetized rats during a normoxic baseline period and brief respiratory challenges. Spontaneous neuronal activity was detected in 80% of the FSC transplants, and autocorrelation of action potential spikes revealed distinct correlogram peaks in 87% of neurons. At baseline, the average discharge frequency of graft neurons was 13.0 ± 1.7 Hz, and discharge frequency increased during a hypoxic respiratory challenge (p<0.001). Parallel studies in unanesthetized rats showed that FSC tissue recipients had larger inspiratory tidal volumes during brief hypoxic exposures (p<0.05 vs. C2Hx rats). Anatomical connectivity was explored in additional graft recipients by injecting a transsynaptic retrograde viral tracer (pseudorabies virus, PRV) directly into matured transplants. Neuronal labeling occurred throughout graft tissues and also in the host spinal cord and brainstem nuclei, including those associated with respiratory control. These results underscore the neuroplastic potential of host-graft interactions and training approaches to enhance functional integration within targeted spinal circuitry.

Keywords: Cervical spinal cord injury; Fetal spinal cord; Hypoxia; Respiratory recovery; Spinal cord repair; Transplantation.

Fig. 1. Longitudinal sections through the cervical spinal cord at the site of the the C2 hemilesion and transplant

Tissues were immunolabeled for the presence serotonin and counterstained with cresyl violet. The grafts typically filled nearly the entire C2Hx lesion cavity (A). Serotonergic immunoreactivity was robust immediately rostral to the graft (B) but staining intensity was considerably less on the contralateral side of the spinal cord (C). Scale bars indicate 1 mm (A) and 200 microns (B, C).

Fig. 2. Longitudinal sections through the cervical spinal cord immunolabeled for the presence of PRV

Sixty four hours following PRV injection into donor tissue, there was extensive PRV labeling observed throughout the transplant. Note that the donor tissue in this animal appears to be in two large pieces, separated at the center of the transplant. Labeled neurons were observed bilaterally in the cervical host spinal cord, both rostral and caudal to the transplant (A). Labeled host neurons have dendrites extending rostro-caudally and laterally (B). In some cases dendrites appear to be crossing the spinal midline. Neurons labeled within the donor tissue (C) appear highly interconnected. Smaller labeled structures may represent with transverse section through a dendritic process or labeling in small glia. Scale bars indicate 500 (A) and 200 microns (B, C).

Fig. 3. Transverse section through the caudal medulla immunolabeled using antibodies against PRV (brown), and counterstained with cresyl violet (blue)

A small incision (arrowheads) has been made on the left of the medulla to indicate the side ipsilateral to injury and transplant. Sixty four hours following labeling, there was a substantial number of PRV labeled neurons through the medulla. This representative section demonstrates labeling in the medial and lateral reticular nuclei (gigantocellular (Gi) and lateral reticular (LRt)), raphé nuclei (obscures (ROb) and pallidus (RPa)), nucleus of the solitary tract (NTS), nucleus ambiguous (Amb) and the ventral respiratory column (VRC). The pyramidal tract is indicated by ‘py’. Scale bar represents 500 (A) and 200 microns (B, C).

Fig. 4. Spontaneous firing patterns of FSC graft neurons and the associated auto-correlogram profiles

Graft neuron activity (Unit) was simultaneously recorded with the phrenic nerve originating on the uninjured side of the spinal cord. “Marker” indicates individual neural spikes sorted using Spike 2 software. The mean discharge frequency (f) of graft neurons was calculated using 200 ms bins. Auto-correlation analysis revealed three main spontaneous firing patterns of graft neurons. The neuron in panel A has a regular interspike interval and displays a periodic firing in the auto-correlogram. Panel B shows a graft neuron with an initial peak (arrow) in the auto-correlogram. Panel C depicts a graft neuron with an irregular interspike interval. The inlet in auto-correlogram represents the superimposition of individual graft neuron spikes (time scale bar: 0.2 ms). Phr and ∫Phr represents raw and integrated phrenic signals, respectively.

Fig. 5. Representative examples of graft neuron activity during baseline conditions and hypoxic challenge

Four graft neurons were simultaneously recorded by the array electrode. Note that discharge frequency increased in each neuron during hypoxia. The trace labeled as “Marker” shows individual neuronal spikes. The spikes are superimposed to the right of each trace. The superimposition waveform is identical during baseline and hypoxia suggesting spikes are from the same neuron under both conditions. The scale bar near the discharge rate (f unit) represents 0–10 Hz.

Fig. 6. Representative example of the phrenic nerve response to hypoxia along with discharge of single graft neuron

Note that hypoxia induces a progressive increase in the phrenic motor output along with an increase in the discharge frequency (Hz) of the graft neuron. The areas marked a and b in the top panel are shown at an expanded time scale trace in the lower panels. The trace labeled as “Marker” shows individual neuronal spikes. The spikes are shown superimposed to the right of each trace.

Fig. 7. The impact of hypoxia (A) and hypercapnia (B) on the average discharge rate of graft neurons

The mean (□/■) and individual (○/●) discharge frequency of graft neurons is shown during baseline as well as hypoxic (A) or hypercapnic (B) challenge. The mean discharge frequency of graft neurons significantly increased during hypoxia (p=0.001) but not hypercapnia (p=0.172).

Fig. 8. Linear regression analysis of the discharge frequency of graft neurons during baseline (abscissa) and respiratory challenge (ordinate)

Graft neurons with a lower baseline discharge frequency tended to increase bursting during hypoxia (A) but not hypercapnia (B).

Fig. 9. The ventilatory behavior of uninjured control, C2Hx and C2Hx+FSC animals at 5 and 10 weeks post-injury

Both C2Hx and C2Hx+FSC animals had similar rapid shallow breathing pattern during baseline conditions at 5 and 10 weeks post-injury. The tidal volume (left panel) of both the C2Hx and C2Hx+FSC animals was similarly blunted compared to control animals during hypoxia at 5 weeks post-injury. However, the tidal volume of FSC grafted animals was significantly larger than the C2Hx only group at 10 weeks post-injury. At the 10 week time point, the respiratory frequency (breaths/min; middle panel) of FSC grafted animals during the hypoxic challenge was lower when compared to the C2Hx only group. Minute ventilation (right panel) was similar across all groups during all conditions. **: p < 0.01 compared with control animals. ##: p < 0.01 compared with the value during the baseline.