Boldine increases functional recovery and response to physical rehabilitation in a mouse model of contusion spinal cord injury

Membrane channels such as connexins (Cx), pannexins and P2X7 receptors are permeable to calcium, ATP and other small molecules. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels and pannexins. To test if boldine improved function after SCI, boldine or vehicle was administered to mice with a moderate severity contusion SCI. Boldine increased spared white matter and locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Increases in locomotor function observed after treadmill walking at a slow speed were further augmented by boldine. Boldine reduced immunostaining for activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked hemichannels, specifically Cx26 and Cx30, on cultured astrocytes and blocked calcium entry through activated P2X7R. Studies by RT-qPCR showed that boldine reduced expression of chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes SNAP25 and Grin2b, as well as GAP-43. Bulk RNA sequencing revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI and that numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine spares tissue and increases locomotor function through mechanisms that may include reduced expression of pro-inflammatory molecules resulting in greater remodeling of surviving circuits Overall design: At 4 months of age, male and female animals of similar weight were randomly assigned to SCI or a sham-SCI. Each group was then randomly assigned to boldine or vehicle treated groups. For treadmill training studies, SCI animals were randomly assigned to vehicle with no treadmill, vehicle with treadmill or boldine with treadmill. The experimental design and timeline for the study procedures is shown in Figure 1 of the article