Restoring Function After Severe Spinal Cord Injury Through BioLuminescent-OptoGenetics

Abstract

The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through the application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuron-specific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either neuron population below the level of injury significantly improved locomotor recovery lasting beyond the treatment window. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI.

Summary: Bioluminescent optogenetic activation of spinal neurons results in accelerated and enhanced locomotor recovery after spinal cord injury in rats.

Keywords: bioluminescence; chemogenetic; optogenetic; spinal cord injured (SCI); stimulation.

Figure 1

Spinal cord injury model. (A) Schematic of the experimental model with viral injection for BL-OG stimulation in the lumbar enlargement and contusion injury in the thoracic region. (B) Timeline of experimental procedures with the first surgery for lateral ventricle cannula placement and virus injection 3 weeks prior to injury. (C) Expression of AAV 2/9 hSyn-LMO3 in the lumbar spinal cord (arrow pointing to expressing interneurons). The highest levels of expression are restricted to interneuron populations in lamina IV-VIII and X with some expression more dorsal and in lamina IX. (D) Expression of AAV 2/9 Hb9-LMO3 in the lumbar spinal cord (arrow pointing to expressing motor neurons). The Hb9 promoter restricts expression to motor neurons in lamina IX. Some low level of expression does occur throughout other laminae of the cord.

Figure 2

Bioluminescent optogenetic stimulation of spinal cord neurons. (A) Example of in vivo bioluminescent imaging of a rat expressing LMO3 in the lumbar spinal cord following CTZ infusion through the lateral ventricle. Luminescence (pseudocolored) is localized over the lumbar region of the cord. (B) A representative trace of luminescence over time following CTZ infusion in the lateral ventricle. (C) Single unit electrophysiological response in the lumbar spinal cord of rats expressing AAV 2/9 hSyn-LMO3 compared to non-expressing animals when CTZ is infused through the lateral ventricle. Similar to luminescence over time, activity increases and peaks between 10 and 30 min following CTZ infusion. n = 7 for LMO3 expressing and n = 4 for non-expressing animals that received CTZ. Shading = SEM. (D) Raster plot of the response to CTZ in an LMO3 expressing rat.

Figure 3

Accelerated and enhanced locomotor recovery with BL-OG. (A) BBB locomotor scores following injury and treatment for animals expressing LMO3 in neurons (hSyn) or specifically in motor neurons (Hb9) or expressing just the luciferase in neurons (sbGLuc-B7). Animals received either CTZ or vehicle following injury. Those which received neural stimulation regardless of neuronal subpopulation (hSyn-LMO3 + CTZ, Hb9-LMO3 + CTZ) recovered at a faster rate, to a greater extent, and maintained their status following the treatment period compared to the non-stimulated vehicle treated group (LMO3+veh) as well as the groups expressing only the luciferase. For Bonferroni post-hoc: hSyn-LMO3+CTZ vs vehicle *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Hb9-LMO3 + CTZ vs. vehicle ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; hSyn-LMO3 + CTZ vs. Hb9-LMO3 + CTZ; %p < 0.05. n = 6 for hSyn-LMO3 + CTZ, n = 6 for Hb9-LMO3 + CTZ, n = 11 for vehicle treated animals. Animals expressing the luciferase only were not significantly different from the vehicle treated controls. (B) Comparison of the percentage of weight supporting animals at the endpoint (28 days) for those that received BL-OG stimulation (hSyn-LMO3 + CTZ, Hb9-LMO3 + CTZ) compared to all three control groups (LMO3 + veh, sbGluc-B7 + CTZ, sbGluc-B7 + veh).

Figure 4

No sparing of white matter at the injury site. (A–D) Cross sections of spinal cords stained with eriochrome cyanin which stains white matter blue. (A) hSyn-LMO3 + CTZ; (B) Hb9-LMO3 + CTZ; (C) Vehicle treated; (D) Example of the white matter present in the same region of the spinal cord in a non-injured rat. (E) Comparison of the cross sectional area of spared white matter following injury and treatment. There were no differences in the amount of degeneration that occurred as a result of the contusion injury with or without neural stimulation.