Figure 1. Bilateral transection of the corticospinal tracts in the pyramids resulted in forelimb spasms, impaired walking and caused hyperreflexia.
(A) Schematic of experimental set-up. Rats received a bilateral pyramidotomy (bPYX) and were treated 24 hr post-injury with AAV1-NT3 or AAV1-GFP injections into biceps brachii and distal forelimb flexors and hand muscles. For clarity in describing the consequences of bPYX, Figure 1 shows only data from uninjured naïve rats and from bPYX rats treated with AAV1-GFP (bPYX GFP). (B) Transverse sections of the medullary brainstem were stained with eriochrome cyanine to quantify the cross-sectional area of the pyramids in (upper panel) uninjured naïve rats and (lower panel) bPYX GFP rats. Pyramids are absent in the lower panel. Scale bar: 200 μm. (C) The left and right pyramids were almost completely absent in bPYX GFP rats versus uninjured naïve rats (RM two-way ANOVA, group F = 107.4, p<0.0001; bPYX GFP versus naïve p<0.001). (D) A greater percentage of bPYX GFP rats exhibited flexor spasms than the percentage that exhibited extensor spasms two weeks post-injury. (E) Rats showed abnormal forelimb movements and signs of spasticity in the open field after bPYX (RM two-way ANOVA, group F = 19.8, p<0.001; bPYX GFP baseline versus all post-injury Weeks, paired t-test p-values<0.05). (F) Bilateral pyramidotomy caused rats to make many errors on the horizontal ladder with their treated forelimb as a percentage of the total steps taken (RM two-way ANOVA, group F = 123.4, p<0.001; bPYX GFP vs naïve p<0.0001; bPYX GFP vs naïve at Weeks 2, 4, 6, 8 and 10, p-values<0.05). (G) Unilateral Grip Strength Test. bPYX rats had reduced grip strength with their treated forepaw at Weeks 2, 4, 6, 8 and 10 versus uninjured naïves (RM two-way ANOVA, group F = 145.0, p<0.001; bPYX GFP vs naïve p<0.0001; bPYX GFP vs naïve at Weeks 2, 4, 6, 8 and 10, p-values<0.05). (H) Responses to mechanical stimulation of the treated forepaw were assessed using the automated von Frey test. Bilateral pyramidotomy caused slight mechanical hypersensitivity (RM two-way ANOVA, group F = 5.2, p=0.019; bPYX GFP vs naïve p = 0.003; bPYX GFP versus naïve at Week 4 and 8, p-values<0.05) (I) Montoya Staircase Pellet Reaching Test. This test assesses fine motor function of the distal forelimb. The sucrose pellets that were eaten on the treated side were counted. Bilateral pyramidotomy led to a persistent deficit in dexterity (RM two-way ANOVA, group F = 100.3, p<0.0001; bPYX GFP vs naïve p<0.0001; bPYX GFP versus naïve at weeks 1, 2, 4, 6, 8 and 10 post-injury p-values<0.0001). (J) Schematic showing the H-reflex paradigm. The ulnar nerve was stimulated distally and EMGs were recorded from a homonymous hand muscle (abductor digiti quinti). (K) Frequency-dependent depression: The H-wave was depressed at short inter-stimulus intervals in uninjured naïve rats. Less H-wave depression was observed in bPYX GFP rats two weeks post injury (RM two-way ANOVA, group F = 9.8, p<0.0001; uninjured versus bPYX GFP at 2 s, 1 s, 0.5 s, 0.2 s and 0.1 s inter-stimulus interval p-values<0.05). (L) Data for each rat at each week was analysed by measuring the area under each curve which gives an electrophysiological correlate of hyperreflexia. Bilateral pyramidotomy caused an increase in hyperreflexia relative to uninjured naïve rats at week 2, 4, 6, 8 and 10 (RM two-way ANOVA, group F = 5.9, p<0.001; bPYX GFP versus naïve p = 0.003; bPYX GFP versus naïve at weeks 2, 6 and 10 post-injury, p-values<0.05). (M) At week 10, the radial, median and ulnar nerves were exposed for stimulation and recording. The radial nerve (blue) innervates extensor muscles (blue) whereas the median and ulnar nerves (pink and magenta) innervate synergist flexor muscles (magenta). (N) Stimulation of afferents in the median nerve evoked responses in the (synergist, flexor) ulnar nerve. (O–P) Example traces show recordings from (O) uninjured naïve and (P) bPYX GFP rats. The boxed area highlights polysynaptic compound action potentials which were analysed for Figure 1Q. (Q) The polysynaptic compound action potentials were quantified by measuring the absolute integral (area under the rectified curve) from 3.5 ms to 12 ms. bPYX GFP rats had an increased polysynaptic reflex response (one-way ANOVA F-value = 4.8, p=0.02; bPYX GFP versus naïve, p-value = 0.02). (R) Stimulation of afferents in the radial nerve evoked polysynaptic responses in the (antagonistic) ulnar nerve. (S–T) Representative traces showing recordings from (S) uninjured naïve and (T) bPYX GFP rats. The boxed area highlights polysynaptic compound action potentials which were analysed for Figure 1U. (U) The polysynaptic compound action potentials were quantified by measuring the absolute integral (area under the rectified curve) from 3.5 ms to 12 ms. bPYX GFP rats had increased polysynaptic responses versus uninjured naïve rats (one-way ANOVA F-value = 4.2, p=0.03; bPYX GFP versus naïve, p-value = 0.01). (A–U) n = 10 or 11 per group. Data are represented as mean ± SEM.
Figure 1—figure supplement 1. Lesion cross-sectional areas were similar on the left and right of the medulla.
Figure 1—figure supplement 2. Scoring sheet for spasticity and disordered sensori-motor control of the forelimb.
Figure 1—figure supplement 3. The H reflex undergoes frequency-dependent depression in uninjured naïve rats whereas this is attenuated in rats with bilateral pyramidotomy.
Figure 1—figure supplement 4. Polysynaptic reflex responses recorded from the radial nerve after ulnar nerve stimulation were not changed after injury.
