Fluoxetine treatment promotes functional recovery in a rat model of cervical spinal cord injury

Manuela Scali, Tatjana Begenisic, Marco Mainardi, Marco Milanese, Tiziana Bonifacino, Giambattista Bonanno, Alessandro Sale, Lamberto Maffei, Manuela Scali, Tatjana Begenisic, Marco Mainardi, Marco Milanese, Tiziana Bonifacino, Giambattista Bonanno, Alessandro Sale, Lamberto Maffei

Abstract

Spinal cord injury (SCI) is a severe condition leading to enduring motor deficits. When lesions are incomplete, promoting spinal cord plasticity might be a useful strategy to elicit functional recovery. Here we investigated whether long-term fluoxetine administration in the drinking water, a treatment recently demonstrated to optimize brain plasticity in several pathological conditions, promotes motor recovery in rats that received a C4 dorsal funiculus crush. We show that fluoxetine administration markedly improved motor functions compared to controls in several behavioral paradigms. The improved functional effects correlated positively with significant sprouting of intact corticospinal fibers and a modulation of the excitation/inhibition balance. Our results suggest a potential application of fluoxetine treatment as a non invasive therapeutic strategy for SCI-associated neuropathologies.

Figures

Figure 1. Fluoxetine induces recovery of motor…
Figure 1. Fluoxetine induces recovery of motor functions after SCI.
(a, b) Staircase. After SCI, all rats lost grasping ability. Starting from the 2nd week, FLX rats began to retrieve significantly more pellets and showed a greater accuracy than CTR-inj. (c, d) Horizontal ladder. After SCI, the number of footslips was significantly increased in all groups. Starting from the 4th week, FLX rats showed a better performance than CTR-inj. The performance of CTR-sham rats did not change throughout the testing period. Box-whisker plot: the horizontal lines in the box denote the 25th, 50th (median), and 75th percentile values; the small square inside the box represents the mean; error bars denote the 5th and 95th percentile values. X represents max and min values. Curves: error bars represent SEM. Symbols indicate statistical difference.
Figure 2. Faster recovery of gait coordination…
Figure 2. Faster recovery of gait coordination in FLX rats.
(a) Typical footprints of animal walking 2 weeks after SCI. Gray: forepaw footprints; Black: hindpaw footprints (b) Gait analysis. After SCI, FLX rats showed a marked recovery of coordination compared to CTR-inj at the 3rd and 5th testing week. The performance of CTR-sham rats did not change throughout the testing period. Error bars represent SEM. Symbols indicate statistical difference.
Figure 3. Fluoxetine increases compensatory sprouting after…
Figure 3. Fluoxetine increases compensatory sprouting after SCI.
(a) Diagram of BDA injections in the sensorimotor cortex. The position of SCI at C4 level is also indicated. (b) Representative pictures of sagittal spinal cord sections at C4 level of CTR-inj (left) and FLX treated (right) rats. Stars indicate lesion cavity. Arrows indicate traced sprouting CST fibers. Scale bar: 50 μm. (c) Quantification of sprouting fibers in the ventral grey matter at 0.5, 1.5, 2.5, 3.5, 4.5 mm from the lesion center, normalized on total counts at medulla oblongata level (100%). FLX rats showed more sprouting fibers at 1.5 and 4.5 mm caudal to the lesion than CTR-inj. Error bars represent SEM. Asterisks indicate statistical significance.
Figure 4. Fluoxetine increases the excitation/inhibition ratio…
Figure 4. Fluoxetine increases the excitation/inhibition ratio in the motor cortex and the spinal cord.
(a) Representative Western blot from control or fluoxetine-treated rat motor cortex (left) and spinal cord (right). Please note that cropped blots are shown (For full length blots, see Supplementary Information, Fig S1–S2). (b) Schematic diagram showing the synaptosome technique. (c, d) Quantification of the expression levels of the vesicular transporter proteins for glutamate and GABA (vGluT-1 and vGAT, respectively) by Western blot analysis, and of GABA and glutamate released after a pulse of KCl (15 mM) by synaptosomes in superfusion. Both the vGluT-1/vGAT expression ratio and the Glu/GABA stimulus-evoked overflow ratio were significantly increased after three weeks of fluoxetine treatment, either in the motor cortex and in the spinal cord. Error bars indicate SEM. Asterisks indicate statistical significance.
Figure 5. Spinal cord injury.
Figure 5. Spinal cord injury.
(a) Histological sagittal sections of CTR-inj (left) and FLX (right) cervical spinal cord. The star indicates the lesion cavity. (b) Quantification of lesion size in CTR-inj and FLX rats as the percentage of max transverse width of the spinal cord. There is no statistical difference between the two groups. Box-whisker plot: the horizontal lines in the box denote the 25th, 50th (median), and 75th percentile values; the small square inside the box represents the mean; error bars denote the 5th and 95th percentile values. X represents max and min values. Scale bar: 1 mm. Abbreviations: D, dorsal; V, ventral; R, rostral; C, caudal.

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