Inosine induces axonal rewiring and improves behavioral outcome after stroke

Abstract

Cerebral infarct (stroke) often causes devastating and irreversible losses of function, in part because of the brain's limited capacity for anatomical reorganization. The purine nucleoside inosine has previously been shown to induce neurons to express a set of growth-associated proteins and to extend axons in culture and in vivo. We show here that in adult rats with unilateral cortical infarcts, inosine stimulated neurons on the undamaged side of the brain to extend new projections to denervated areas of the midbrain and spinal cord. This growth was paralleled by improved performance on several behavioral measures.

Figure 1

Schematic illustration of the stroke and major cortical efferent pathways. (a) The sensorimotor cortex and other right hemisphere structures were damaged unilaterally by occluding the right MCA; in the second set of studies, parts of the anterior cerebral artery were occluded as well. The pathways originating from the intact left hemisphere that were traced with BDA include the corticorubral (green) and corticospinal (blue) tracts. (b) Coronal section through the forebrain showing the extent of injury (black) and the cells of origin of the intact corticospinal (blue) and corticorubral (green) tracts. (c) Projections from the sensorimotor cortex to the ipsilateral red nucleus (R). Compensatory growth to the denervated (Right) red nucleus, as investigated here, is shown in red. (d) CST projections from the intact hemisphere decussate in the caudal medulla, course in the contralateral dorsal funiculus, and synapse primarily on layer 4–6 interneurons in the cervical and lumbar enlargements of the spinal cord. Inosine-induced collateral projections to the denervated side are shown in red.

Figure 2

Behavioral recovery after stroke. (a) In the first study, forelimb placing was evaluated by using a 10-point scale as described (4, 17). Animals treated with inosine performed better than saline-treated controls from day 7 onward. (b) Hindlimb placing of control and inosine-treated animals was scored on a six-point scale (4, 17). (c) Body swing, a measure of basal ganglion dysfunction, is scored by the percentage of times animals turn more than 10° to the side ipsilateral to the lesion when suspended from the tail (20). (d) Food retrieval: constrained. Score represents the percent of animals that used their impaired (left) paw when the right paw was constrained. (e) Food retrieval: free choice. Percent of animals using the impaired paw when free to use either paw. (f) Inosine-treated rat spontaneously using its impaired left paw (yellow arrow) to retrieve food pellets even when the intact right paw was unconstrained. *, P < 0 05; **, P <0.01; ***, P < 0.001.

Figure 3

Axon crossing at the level of the red nucleus. (a) Quantitation of axons crossing from the intact side of the brain to the denervated red nucleus (averaged from four sections per case). (b) Schematic coronal section through the midbrain at the level of the red nucleus. Box (red) indicates approximate area shown in c and d. Labeled axons in the vicinity of the red nucleus are shown on the normal side (Left) and on the denervated (Right) side in an animal treated with saline after stroke (c), and in an animal treated with inosine after stroke (d). *, P < 0.05; **, P < 0.01. (Bar in d = 500 μm.)

Figure 4

Axon crossing in the cervical spinal cord. (a–g) Transverse sections through the cervical enlargement showing the area of the denervated CST. The midline and medial-most fibers of the intact CST are on the far right side of each frame. (h) Low-power photo through the cervical spinal cord showing the approximate area enlarged in a–g (red frame). (a–c) Double-labeled section from a control animal treated with saline after stroke. (d–f) Similarly stained sections from an animal treated with inosine after stroke. (a, d) BDA-labeled fibers originating in the intact hemisphere, stained red here, projecting to the denervated side of the spinal cord (arrows) in saline- (a) and inosine-treated (d) animals. (b, e) Fibers with high levels of the growth-associated protein GAP-43 (green fluorescence) in the same sections shown in a and d, respectively. The intense fluorescence in the neuropil lateral to the CST (asterisks) is unaffected by treatment. (c, f) Merged images showing that crossed, BDA-labeled fibers contain high levels of GAP-43 (yellow staining: arrows). (g) Section from an inosine-treated animal stained with horseradish peroxidase-conjugated avidin–biotin complex followed by diaminobenzidine. Arrows show axons crossing the midline. (Bar a–f, 100 μm; g, 50 μm.)