Thoracic 9 Spinal Transection-Induced Model of Muscle Spasticity in the Rat: A Systematic Electrophysiological and Histopathological Characterization

Abstract

The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABAB receptor agonist), tizanidine (α2-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity.

Fig 1. 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.

(A, 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. (C, D)- 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).

Fig 2. Potentiation of spinal hyper-reflexia by increased angle and velocity of ankle rotation.

(A, B, C, D)—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.

Fig 3. Post-spinal transection time-dependent increase in spinal hyper-reflexia.

(A, B, C, D)—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. (E, F)—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).

Fig 4. Increase in Hoffmann reflex and loss of rate-dependent depression (RDD) in spinally transected rats at 3 months after transection.

(A, 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). (C)—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).

Fig 5. Development of tactile hypersensitivity in rats at chronic stages after spinal transection.

(A)—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, C)- 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). (D, E)- 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).

Fig 6. Effective suppression of ankle dorsiflexion-evoked EMG response and muscle resistance after systemic treatment with baclofen, tizanidine and NGX424.

(A)- Representative recording of EMG response (EMG) and corresponding ankle resistance (PMR) in an animal injected systemically with saline, Tizanidine (1mg/kg), baclofen (10mg/kg) or NGX 424 (1mg/kg). (B, C)- Statistical analysis showed significant suppression of EMG response (B) and PMR (C) after treatment will all three drugs if compared to saline treated animals (repeated measures two-way ANOVA, Bonferroni post hoc; *-p< 0.05; **-p< 0.01; ***-p< 0.001).

Fig 7. 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.

(A, 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). (C, D)—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).

Fig 8. Quantitative and qualitative analysis of VGluT1 and GlyT2 expression in lumbar spinal cord (L2-L6) at 3 months post-spinal transection.

(A-H)- 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. (I-J)- 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).

Fig 9. Immunofluorescence analysis of lumbar spinal cord sections at 3 months after spinal cord transection.

(A, B)- Qualitative analysis of GAD65 and synaptophysin immuoreactivity showed normally appearing double-labeled synaptophysin/GAD65+ puncta (white arrows; confocal microscopy). (C, D, E, F)- 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. (G, H, I)- 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).