Epidural Stimulation Combined with Triple Gene Therapy for Spinal Cord Injury Treatment

Abstract

The translation of new therapies for spinal cord injury to clinical trials can be facilitated with large animal models close in morpho-physiological scale to humans. Here, we report functional restoration and morphological reorganization after spinal contusion in pigs, following a combined treatment of locomotor training facilitated with epidural electrical stimulation (EES) and cell-mediated triple gene therapy with umbilical cord blood mononuclear cells overexpressing recombinant vascular endothelial growth factor, glial-derived neurotrophic factor, and neural cell adhesion molecule. Preliminary results obtained on a small sample of pigs 2 months after spinal contusion revealed the difference in post-traumatic spinal cord outcomes in control and treated animals. In treated pigs, motor performance was enabled by EES and the corresponding morpho-functional changes in hind limb skeletal muscles were accompanied by the reorganization of the glial cell, the reaction of stress cell, and synaptic proteins. Our data demonstrate effects of combined EES-facilitated motor training and cell-mediated triple gene therapy after spinal contusion in large animals, informing a background for further animal studies and clinical translation.

Keywords: adenoviral vector; cell-mediated gene therapy; epidural electrical stimulation; glial cell-derived neurotrophic factor; human umbilical cord blood mononuclear cell; neural cell adhesion molecule; pigs; spinal cord injury; vascular endothelial growth factor.

Figure 1

Study design. Upper panel: (A) Implantation of stimulating electrodes followed by recovery and training of animals to walk on a treadmill. (B) Spinal contusion followed by intrathecal administration of saline (0.9% NaCl) 4 h after injury. (C) Training on a treadmill every 2nd day started 2 weeks after injury. (D) Termination of the experiment at 60 days after injury. The caudal part of the spinal cord relative to the epicenter of injury (red cross) was divided into three segments (CS1, CS2, and CS3) for histological and molecular studies. Low panel: (A’) Implantation of stimulating electrodes followed by recovery and training of animals to walk on a treadmill. (B’) Spinal contusion followed by intrathecal administration of gene modified umbilical cord blood mononuclear cells (UCBC) 4 h after injury. (C’) Epidural electrical stimulation (EES) every 2nd day combined with training on a treadmill started 2 weeks after injury. (D’) Termination of the experiment at 60 days after injury and harvesting of the spinal cords for investigation. Intact healthy animals (not shown) were used to collect basic behavioral, electrophysiological, and histological data for comparative analysis.

Figure 2

Evaluation of the motor recovery. (A) Porcine thoracic injury behavioral scale (PTIBS) scores in control (green) and treated (pink) groups. (B) Video recording of the hip (A), knee (B), and ankle (C) joint kinematics during stepping on a treadmill was performed in intact healthy pigs 1 week before surgery and at different time points after spinal cord contusion injury. (C) The angle (degree) of movements measured in the hip, knee, and ankle joints in experimental pigs before surgery (I, yellow bar) and week 5, 6, 7, and 8 after spinal cord injury (SCI) in control (C, green bar) and treated (GT-ES, pink bar) groups during training on the treadmill. The blue bar (GT-ES when stimulation) represents data of animals from the GT-ES group during training on the treadmill enabled with epidural stimulation at the L2 segment. Average angles of five sequential step cycles for each animal are presented as color points; bars correspond to group-wise average values. The colors of points in I (yellow bars) correspond to the colors of C and GT-ES bars.

Figure 3

Assessment of hind limb skeletal muscles and an electrophysiological evaluation of M- and H-reflex. (A) Tibialis anterior muscles weighing analysis. (B) Evaluation of muscle fibers area in the tibialis anterior muscle. (C,C’,C’’) Cross sections of the tibialis anterior muscle stained with hematoxylin and eosin from I, C, and GT-ES groups, correspondingly. (D) Electrophysiological study of M- and H-reflex in the soleus muscle. (E) Parameters of the electromyography (EMG) signal. Data obtained for each animal are presented as points; bars represent group-wise average values.

Figure 4

The area of the preserved gray matter in spinal cords at 60 days after SCI in CS1 and CS2 segments. (A,A’) Cross sections of spinal cords stained with hematoxylin and eosin in control (C) and treated (GT-ES) animals from CS1. (B,B’) Spinal cords of control and treated pigs from CS2. (C) An intact spinal cord. (D) The relative area of preserved gray matter in control (C) and treated (GT-ES) pigs. Values averaged within animals are presented as points; bars represent group-wise average values.

Figure 5

Immunofluorescent analysis of cell stress molecules in spinal cord ventral horns at 60 days after SCI in the CS2 segment. (A) Immunoexpression of a heat shock protein of 27 kDa (Hsp27). (B) Immunoexpression of a neuron specific K⁺-Cl⁻ co-transporter (KCC2). (C) Immunoexpression of a pro-apoptotic protein Caspase3. Nuclei are counterstained with DAPI (blue). (D) KCC2 mean intensity and the number of Hsp27 and cCaspase3-positive cells in intact (I), control (C), and treated (GT-ES) pigs. Caspase3-positive cells in the intact (I) group were not observed. Values averaged within animals are presented as points; bars represent group-wise average values.

Figure 6

Immunoexpression of synaptic proteins in spinal cord ventral horns at 60 days after SCI. (A) Immunofluorescent staining with antibodies to Synaptophysin (synaptic vesicles protein). (B) Immunofluorescent staining with antibodies to postsynaptic density protein 95 kDa (PSD95). (C) Merged images representing Synaptophysin and PSD95. Nuclei are counterstained with DAPI (blue). Values averaged within animals are presented as points; bars represent group-wise average values.

Figure 7

Immunofluorescent analysis of glial cells reorganization in spinal cord ventral horns at 60 days after SCI. (A) Astrocytes were visualized with antibodies against glial fibrilar acidic proteins (GFAP). (B) Microglial cells against ionized calcium binding adaptor molecule 1 (Iba1). (C) Oligodendroglial cell against transcription factor Olig2. Nuclei were counterstained with DAPI (blue). (D) Mean number of cells in intact (I), control (C), and treated (GT-ES) pigs. Values averaged within animals are presented as points; bars represent group-wise average values.

Figure 8

Cytokines profiling and synaptic gene expression in the spinal cord at 60 days after SCI. (A) Comparison of target genes expression. The results are represented as an average log2(fold change) in control (C, green points) and treated (GT-ES, pink points) pigs relative to the expression level in the intact group. Threshold cycles (Ct) were obtained for two animals (two repeats per animal) in each group and normalized by corresponding Gapdh values. (B) Heatmap representing a log-transformed cytokines absolute concentration in spinal cord samples, where each column represents tissue samples (two samples per animal). Columns are bunched by experimental groups (I, C, and GT-ES). Cytokines are ordered by the results of hierarchical agglomerative clustering.