Increase in Hoffmann reflex and loss of rate-dependent depression (RDD) in spinally transected rats at 3 months after transection.

<p><b>(A, B)</b>—Measurement of H-reflex and statistical analysis of the H/M ratio showed a significant increase in responses at 3 months after transection if compared to wild-type non-injured animals (unpaired two-tailed t-test; ***P< 0.001). <b>(C)</b>—Testing of RDD showed a significant loss of RDD in spinally-transected animals at stimulation frequencies of 1, 5 and 10 Hz (one-way ANOVA; Bonferroni post hoc; ***P< 0.001).</p

Potentiation of spinal hyper-reflexia by increased angle and velocity of ankle rotation.

<p><b>(A, B, C, D)</b>—Comparing the effect of 40° to 80°of ankle rotation if ankle is rotated at 40, 200 or 400°/sec showed the most potent EMG response and corresponding increase in peripheral muscle resistance (PMR) at 80°of ankle rotation delivered at 400°/sec.</p

Effective suppression of Hoffmann reflex and spinal transection-induced tactile hypersensitivity after systemic treatment with baclofen, NGX 424 and tizanidine in rats with chronic spinal transection.

<p><b>(A, B)</b>- Systemic treatment with baclofen (10mg/kg), NGX424 (1mg/kg) or tizanidine (1mg/kg) led to a clearly detectable suppression of H-reflex (A) and supramaximal tactile stimulus-evoked EMG response in rats at 3 months after spinal transection (B). <b>(C, D)</b>—Statistical analysis showed significant suppression of H-reflex (C) and tactile stimulus evoked EMG response if compared to saline-injected animals (one-way ANOVA; Bonferroni post hoc; *-p< 0.05; **-p< 0.01; ***-p< 0.001).</p

Development of tactile hypersensitivity in rats at chronic stages after spinal transection.

<p><b>(A)</b>—Application of tactile stimuli (von Fray filaments; 1-15g) on the plantar surface of hind paw led to a clear EMG response measured by surface EMG electrodes from gastrocnemius muscle in animals at 3 months post-spinal-transection (TSCT 1, 2, 3). No response was seen in naive non-injured animals. Application of 5 repetitive stimuli at the same pressure (15g) and delivered every 5 seconds led to a consistent responses after each stimulus (A-right panels). <b>(B, C)</b>- Statistical analysis of repetitive (5x stimuli) tactile stimulus-evoked EMG responses separated by 10–15 seconds intervals showed a significant increase in transected animals (compared to naïve controls) at paw pressures between 2–15 grams (one-way ANOVA; Bonferroni post hoc; *-p< 0.05; **-p< 0.01; ***-p< 0.001). <b>(D, E)</b>- Statistical analysis of repetitive tactile stimulus-evoked (5x stimuli) EMG responses separated by 5 seconds intervals showed a comparable significant increase in animals with transection as seen after application of stimuli separated by 10–15 sec intervals (one-way ANOVA; Bonferroni post hoc; *-p< 0.05; **-p< 0.01; ***-p< 0.001).</p

Experimental setup to permit the measurement of computer-controlled ankle dorsiflexion-evoked muscle resistance and corresponding changes in gastrocnemius muscle EMG activity in fully awake restrained animals.

<p><b>(A, B)</b>—Fully awake rats are placed into PVC tube (internal diameter: 6 cm; red arrow) and the right (or left) paw is attached to a bridging pressure transducer (blue arrow). During computer-controlled ankle rotation the changes in ankle resistance (active peripheral muscle resistance = PMR) is recorded. At the same time the EMG (active EMG response) is recorded from gastrocnemius muscle by using transcutaneously placed needle recording electrodes. <b>(C, D)</b>- An example of EMG and PMR recording before, during and after computer-controlled ankle dorsiflexion (0→80°; 400°/sec) in naïve-non-injured rat or in an animal at 3 months post-Th9 transection (D). Note a near complete EMG and PMR non-responsiveness during ankle dorsiflexion in naive-non-injured animal (C) but a clear burst like-EMG activity and corresponding increase in PMR in chronically transected animal (D).</p

Quantitative and qualitative analysis of VGluT1 and GlyT2 expression in lumbar spinal cord (L2-L6) at 3 months post-spinal transection.

<p><b>(A-H)</b>- Transverse spinal cord sections taken from L2-L6 segments in naïve and spinally transected animals (3 months post-transection) and double-stained with GlyT2/NeuN or VGluT1/NeuN antibodies. Normally appearing distribution of neurons and GlyT2/VGluT1 staining pattern can be seen in both control and spinally transected animals. <b>(I-J)</b>- Statistical analysis of GlyT2 density signal and VGluT1 puncta in the ventral horn showed a significant increase in GlyT2 expression and a significant decrease in VGluT1 puncta in chronically transected animals (t-test *-p< 0.05; **-p< 0.01; scale bar: A, B, E, F: 500 μm; C, D, G, H: 80 μm).</p

Post-spinal transection time-dependent increase in spinal hyper-reflexia.

<p><b>(A, B, C, D)</b>—Measurement of EMG activity and PMR starting at 2 weeks after spinal transection showed a progressive increase in recorded responses with the most pronounced increase seen at 12 weeks after spinal transection. <b>(E, F)</b>—Statistical analysis showed a significant increase in both EMG response and PMR if compared across all 3 time points analyzed (i.e. 1, 2 and 3 months), (one-way ANOVA; Bonferroni post hoc; *** p< 0.001, ** p< 0.01).</p

Immunofluorescence analysis of lumbar spinal cord sections at 3 months after spinal cord transection.

<p><b>(A, B)</b>- Qualitative analysis of GAD65 and synaptophysin immuoreactivity showed normally appearing double-labeled synaptophysin/GAD65+ puncta (white arrows; confocal microscopy). <b>(C, D, E, F)</b>- Immunostaining with Iba1 and GFAP antibody (C, D) and NeuN (E, F) showed normally-appearing neurons, but an increase in IB1 and GFAP immunoreactivity in activated-hypertrophic astrocytes and microglial cells in lamina VII. <b>(G, H, I)</b>- Quantitative densitometric analysis of GAD65, GFAP and IB1 immunoreactivity showed a decrease in GAD65 and increase in GFAP and IB1 staining in TSCT animals if compared to naïve controls (t-test ***-p<0.001; scale bar: A, B: 50 μm C, D, E, F: 200 μm).</p