Figure 1.
Inosine improves open-field behavior and skilled use of hindlimbs.
Behavioral studies were carried out in 2 separate cohorts of rats. General locomotor function was evaluated using the Basso-Beattie-Bresnahan (BBB) scale, whereas fine motor coordination was tested using the ladder rung-walking test, scoring the percentage of steps in which either hindlimb was placed on a rung without error. A, B. Study 1. Inosine was delivered either i.c.v. (black) or i.v. (blue); saline-treated controls are shown in red. Note the superior performance of animals treated with either i.c.v. or i.v. inosine on the BBB (A) and the ladder rung walking test (B). C, D, Study 2. Inosine and saline were delivered i.v. Inosine-treated rats again performed better than saline-treated controls on both the BBB (C) and ladder-rung walking test (D) at weeks 3 and 4. *P < 0.05, **P < 0.01, ***P < 0.001. Data in all figures represent group means ± SEM (Two-way ANOVA, Bonferroni a priori test).
Figure 2.
Interruption of the dorsal corticospinal tract (CST) after spinal cord injury (SCI).
A. Hindlimb CST axons were labeled by injecting biotinylated dextran amine (BDA) into 9 sites in the hindlimb motor area of each hemisphere 4 weeks after SCI. Rats survived 2 more weeks to allow for BDA transport. B. Identity of brain structures (from the atlas of Paxinos and Watson, 1997). C. Parasagittal section through the thoracic cord shows labeled CST axons (arrowhead) rostral to the lesion (dashed lines). Note the absence of labeled fibers distal to the injury. D, E. Transverse sections of the spinal cord at the cervical (D) and lumbar (E) enlargements. Note heavy labeling at the cervical level (D, D') but absence of labeling in the lumbar cord (E, E'). Scale bar, 500 µm.
Figure 3.
Inosine does not affect lesion size.
A. Depth of lesions for all animals in Studies 1 and 2 (bars show group means; dashed lines show exclusion limits). B. lesion depth after excluding rats in which the lesion was either ≤ 50% or > 85% the depth of the spinal cord. C. Area of lesions (percentage of tissue lost within an 8 mm segment of spinal cord centered at the lesion epicenter). Inosine and saline-treated groups did not differ from each other in lesion depth or area (One-way ANOVA, Dunnet a priori test).
Figure 4.
Behavioral outcome depends upon treatment but not depth of lesion.
Scattergrams show open field behavior (A) and ladder rung scores (B) as a function of lesion depth. Blue dots represent rats treated with either i.c.v. or i.v. inosine, and red dots are rats treated with saline; dotted lines show the upper and lower bounds of our exclusion criteria. Within these bounds, inosine-treated rats out-performed controls, but there is no significant correlation (n.s.) between performance and lesion size. Performance on both tests falls off for lesions outside the lower limit for inclusion in the study.
Figure 5.
Inosine increases CST axon branching in the cervical spinal cord.
A. Transverse section through the cervical enlargement. B. BDA-labeled CST collaterals were quantified at the border of the main (dorsomedial) CST (B), the midline (M), and at distances I and II from the midline. C - E, SCI alone increases CST sprouting in the gray matter compared to a sham-treated controls (D vs. C), and inosine enhances it further (E). F - H, Quantitation of CST sprouting into the gray matter at the indicated loci. #P < 0.05, ##P < 0.01 compared to sham-operated controls; *P < 0.05, ** P < 0.01 compared to saline-treated rats with SCI. Axon counts were normalized by the total number of axons counted in the main CST at the cervical level (Mann-Whitney test).
Figure 6.
Inosine increases CST contacts onto LPSNs.
A. Transverse section of an inosine-treated animal though the cervical enlargement shows position of LPSNs (boxes). B, C. Enlargement of areas in (A) show LPSNs (red) that project to the lumbar enlargement. B', C'. Higher magnification of cells in B and C. Arrowheads show BDA-labeled CST collaterals (green) contacting an LPSN soma (B') and proximal dendrites (C'). D, E, Quantitation of CST synapses upon LPSN somata and within 20 µm of labeled dendrites. SCI alone increases axosomatic synapses compared to sham-operated controls, and inosine increases the number of these to a greater extent (D). Inosine also increases putative axodendritic contacts compared to normal animals (E). *P < 0.05 compared to saline treatment, #P < 0.05, ##P < 0.01 compared to sham-operated controls (Mann-Whitney).
Figure 7.
Inosine increases synaptic contacts between CST collaterals and LPSNs.
A. Spinning-disc confocal image shows BDA-labeled boutons (red) in the cervical enlargement that arise from CST axon collaterals and express the presynaptic protein synaptophysin (green). B. Confocal image shows CST boutons co-localized with synaptophysin (green)-positive contacts upon a CTB-labeled LPSN (blue). Side images show co-localization in the x-z and y-z planes. C. Quantitation of synaptophysin- and BDA-positive boutons on LPSN somata. SCI alone increases synaptic contacts on LPSNs, and inosine causes a further 4-fold increase. Data are normalized by the total number of labeled axons in the main bundle of the CST. *P < 0.05 compared to saline treatment, ##P < 0.01 compared to normal control (Mann-Whitney test).
Figure 8.
Inosine increases serotonergic input to the lumbar spinal cord.
A. Schematic diagram of the lumbar enlargement. Box delineates area of 5-HT analysis in the ventral horn. B. Integrated intensity of 5-HT immunofluorescence. SCI decreases 5-HT immunofluorescence compared to normal controls. i.v. inosine restores 5-HT levels back to normal (F = 10.93; **P < 0.01 compared to saline treatment, †P < 0.05 compared to sham-operated controls: One-way ANOVA, Dunnet a priori test). C - E. Photomicrographs show serotonergic fibers in the lumbar enlargement of sham-operated rats (C) and after SCI in rats treated with i.v. saline (D) or i.v. inosine (E).
Figure 9.
Dose-response relationship for intravenous inosine.
A, B. Behavioral results 4 weeks after SCI. The effects of inosine reach a plateau at 100 mM in both the open-field (A) and ladder rung walking (B) tests. C, D. Behavioral test results over the 4 week test period. *P < 0.01; **P < 0.01; ***P < 0.01 (One-way ANOVA, Dunnet a priori test (a, b); two-way ANOVA, Bonferroni a priori test (c, d)).