Molecular, cellular and functional events in axonal sprouting after stroke

Abstract

Stroke is the leading cause of adult disability. Yet there is a limited degree of recovery in this disease. One of the mechanisms of recovery is the formation of new connections in the brain and spinal cord after stroke: post-stroke axonal sprouting. Studies indicate that post-stroke axonal sprouting occurs in mice, rats, primates and humans. Inducing post-stroke axonal sprouting in specific connections enhances recovery; blocking axonal sprouting impairs recovery. Behavioral activity patterns after stroke modify the axonal sprouting response. A unique regenerative molecular program mediates this aspect of tissue repair in the CNS. The types of connections that are formed after stroke indicate three patterns of axonal sprouting after stroke: reactive, reparative and unbounded axonal sprouting. These differ in mechanism, location, relationship to behavioral recovery and, importantly, in their prospect for therapeutic manipulation to enhance tissue repair.

Keywords: Astrocyte; Behavior; Cortex; GDF10; Recovery; Regeneration; Rehabilitation; Spinal cord; TGFβ.

Figure 1. Patterns of Axonal Sprouting or Sensorimotor Map Plasticity in Peri-Infarct Cortex

(A) Axonal sprouting after stroke in the monkey occurs between premotor and somatosensory areas, establishing novel long-distance connections after stroke. This is a long distance axonal sprouting process, spanning a centimeter of tissue and occurring between frontal and parietal lobes (Dancause et al., 2005). (B) In rat and mouse models of stroke, axonal sprouting occurs in motor, premotor, somatosensory and posterior parietal areas after stroke (Brown et al., 2009; Li et al., 2010; Overman et al., 2012: Li, Nie et al., 2015). (C) Human motor and sensory maps reorganize after stroke into new representations in peri-infarct and connected cortical areas, in a process correlated with recovery (Buma et al., 2010; Kantak et al., 2012; Grefkes and Ward, 2014)

Figure 2. Reactive Axonal Sprouting after Stroke

(A–E). The images depict maps of the location of axons labeled from a BDA neuroanatomical tracer injection into the forelimb motor cortex of the mouse. Each map is the plot of 5–6 mice (separate brain maps). Thus each map shows the aggregate motor system connections in the cortical hemisphere. The inset between (A) and (B) shows a schematic of the location of the neuroanatomical tracer injection in the mouse brain. (A) is a map of the forelimb motor system connection after stroke. The stroke is schematically shown as the grey ellipse. This is one type of stroke model, as distal middle cerebral artery stroke. (B) shows the location of motor system connections in the control (non-stroke) mouse. (C) shows the overlap of the two motor system networks in control (magenta) and stroke (blue). Note the loss of projections near the stroke (arrowhead) and the increase in projections near the stroke site and immediately outside of the control motor system connectional network (arrows). (D) shows a similar map from a different stroke model and different set of studies. This is a photothrombotic stroke in motor cortex (grey ellipse). The tracer injection (clear circle) is placed in surviving motor cortex anterior to the stroke. In light blue the projections of motor cortex in the control (non-stroke) brain are shown. In red, the projections of the motor cortex region after stroke are shown. The dark blue shows the region of dense overlap of the stroke and non-stroke projection. The arrows show the increase in motor system projections around the stroke site. (E) shows another set of studies with a tracer injection into forelimb motor cortex in control (blue) and stroke (red) in a distal middle cerebral artery stroke model (grey ellipse). Axons that project from forelimb motor cortex after stroke (red) can be seen near the stroke site at a site that has only sparse projection in the control brain (blue). (F) shows maps of cell bodies in control (magenta) and stroke cases (blue). The previous figures mapped the axonal projections. In these two maps of control (sham+vehicle) and stroke (stroke+vehicle) the motor system projections after stroke come from a population of neurons near the stroke site that are not present in the control motor system network (arrows). The stroke is schematically depicted in the ellipse. In this map the quantitative plots of cortical projections have been warped back onto a tissue section of the brain, to localize the projections within the sensorimotor cortex (see Li et al., 2010; Overman et al., 2012). (G) shows maps of the location of neurons labeled from tracer injections into the somatosensory cortex, in the representation of the facial vibrissae (or the “barrel field”). In this model of stroke in the rat, small strokes are placed in the barrel cortex. In control (non-stroke) most of the cells project in a posterior-medial direction, establishing a direction vector for the population of projections After stroke, a new population of neurons projects within the somatosensory cortex (red arrow). These are located near the stroke. This new population establishes a different vector for the connections within the somatosensory cortex. The summary of this process of post-stroke axonal sprouting in somatosensory cortex of the rat is that new projections are formed adjacent to the stroke site. (H) Summary of the process of reactive axonal sprouting after stroke. The control state is shown at the top. At the bottom, stroke induces axonal sprouting and new projections (red) toward the stroke site. (A–C) are modified from Overman et al., 12). (D) is from Omura et al., 2015. (E) is from Li et al., 2010. (F) is unpublished data from Andrew Clarkson and S. Thomas Carmichael. (G) is from Carmichael et al., 2001.

Figure 3. Reparative Axonal Sprouting after Stroke

(A–D). The cortical mapping conventions are the same in this figure as in the previous figure. Each map shows the quantitative plot of axonal projections from forelimb motor cortex in 5–6 mice per condition. In this map, all conditions are stroke (light blue) or stroke+growth promoting agent (red). Dark blue is an area of dense overlap of the two projections. (A) Anti-Lingo-1 antibody treatment in stroke (red) induces a significant increase in motor system connections to prefrontal cortex and to the second somatosensory cortex (SII) (arrows) compared to the motor system connections of stroke-alone (blue). (B) Blockade of EphrinA signaling (EphA5-Fc delivery) causes shifts of motor system axonal projection to premotor cortex and an increase in motor projections to SII. (C) Blockade of EphA4 signaling (EphA4-Fc) (red) produces a substantial axonal sprouting from motor cortex to premotor cortex, SII and SI (arrows). As noted in the text this substantial axonal sprouting response is likely because blocking EphA4 blocks astrocyte EphrinA signaling and also myelin inhibition through Ephrin B3. (D) Delivery of the stroke-induced brain growth factor GDF10 induces motor system axonal sprouting into premotor cortex and SII (arrows) compared to the stroke-alone condition (blue). In all of these maps, axonal sprouting is stimulated by blockade of an axonal growth inhibitor or stimulation of an axonal growth program after stroke but remains confine to the sensorimotor system. (E) Schematic of reparative axonal sprouting after stroke. In reparative axonal sprouting stimulation of the post-stroke brain produces new patterns of connections within the sensorimotor system (green) that are associated with recovery, on top of the normal post-stroke reactive axonal sprouting (red axons). (F) Schematic view of the mouse brain with the normal motor system projection (blue) and the reparative post-stroke axonal projection (red). The location of two stroke sites in the models used in these studies is shown in the grey ellipses. (G–I) Axonal sprouting in the cervical spinal cord in normal (untreated or unstimulated) stroke and after stimulation of axonal sprouting. (G) Drawings of one half of the cervical spinal cord grey matter showing the half of the spinal cord that has lost its projection from a stroke in the sensorimotor cortex. The axons projecting from the hemisphere contralateral to the stroke are traced in black. In stroke-only, these axons are sparse and located near the midline. After stroke plus treatment with the axonal growth stimulating molecule inosine, there is an increase in axonal projections that extend further into the spinal cord and toward the ventral horn (motor spinal cord). (H) Maps of the same region of cervical spinal cord in a different set of studies in a large stroke has been placed in the sensorimotor cortex and the projection of the contralateral corticospinal neurons is mapped. The color coding represents density of axonal projections (red = high, blue = low). In stroke alone (left image) there is a sparse projection into contralateral spinal cord that is close to the midline. In stroke plus treatment with a Nogo antagonist, there is an increase in contralateral axonal projections, seen as a greater density and an extension in all directions into the ventral, lateral and dorsal laminae of the spinal cord. In the case of both inosine and anti-Nogo treatments, functional recovery is enhanced in association with these patterns of cervical spinal cord axonal sprouting. (I) Schematic view of the cervical spinal cord with representative zones of axonal projections in the region of the cord that is contralateral to the normal corticospinal projection. In control (blue) there are few axonal projections to the contralateral spinal cord. After stroke (dark orange) there is a small increase in axonal projections into the contralateral spinal cord near the central canal and sparsely into the middle layers of the spinal cord (reactive axonal sprouting). In stroke plus treatment, in this case inosine or with Nogo blockade, there is a further increase in axonal sprouting into the ventral horn and partially into the dorsal horn (reparative axonal sprouting). (A) is from Li et al., 2010. (B, C) are from Overman et al., 2012. (D) is from Li, Nie et al., 2015. (G) is from Zai et al., 2009. (H) is from Lindau et al., 2014.

Figure 4. Unbounded Axonal Sprouting after Stroke

When axonal growth is stimulated by blockade of a glial growth inhibitor at the same time that behavioral activity of the motor system is manipulated by neurorehabilitative activities, axonal sprouting can extend beyond the sensorimotor areas to widespread brain systems. (A) connectional map of motor system axonal projections in stroke with forced overuse of the affected forelimb. Compared to normal post-stroke axonal sprouting (Figure 3A) there is an increase in projections within the motor cortex itself (blue). With blockade of the astrocyte growth inhibitor EphrinA5, there is a massive increase in axonal projections to orbital prefrontal cortex, lateral prefrontal cortex and temporal and parietal areas (red). (B) schematic rendering of post-stroke axonal sprouting with behavioral overuse of the affected limb (blue) and with behavioral overuse of the affected limb and blockage of a glial growth inhibitor (red). (C, D) Studies from Dr. Martin Schwab of axonal sprouting the spinal cord from neurons in the contralateral hemisphere from the stroke. The left panel in (C) shows axons that project into the ipsilateral spinal cord when the glial growth inhibitor Nogo is blocked and simultaneously the animal uses its affected forelimb in a daily repetitive reach task. The right panel in (C) shows the axons in this same projection when Nogo is blocked first, and then skilled reach training is used. There is a substantial increase in axonal projections when behavioral activity is manipulated at the same time as axonal growth is molecularly stimulated. (D) Schematic of the spinal cord grey matter, with the laminae. Red columns indicate simultaneous Nogo blockade and behavioral activity; blue columns indicate first Nogo blockade then behavioral activity. When Nogo is blocked and skilled reach training implemented simultaneously, there is axonal sprouting throughout the spinal cord grey matter, including to non-motor areas (such as the dorsal spinal cord, section “A”). The region of the photomicrographs in (C) is highlighted with a red dotted ellipse in (D). (E) Schematic of unbounded axonal sprouting after stroke (blue axons). In conditions of manipulation of behavioral activity, such as with repetitive skilled reach or forced overuse of the affected limb, plus blockade of a glial growth inhibitor, axonal sprouting occurs over a very widespread brain or spinal regions. This occurs on top of the reactive (red) and reparative (blue) axonal sprouting after stroke.