Intramuscular Neurotrophin-3 normalizes low threshold spinal reflexes, reduces spasms and improves mobility after bilateral corticospinal tract injury in rats
- King's College London, University of London, United Kingdom
- Imperial College London, United Kingdom
Figures
Figure 1 with 4 supplements
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 …
Figure 1—figure supplement 1
Lesion cross-sectional areas were similar on the left and right of the medulla.
Eriochrome cyanine staining of transverse sections through the medulla taken 10 weeks after bilateral pyramidotomy showed lesions in bPYX GFP rats (green) relative to naïve rats (RM two-way ANOVA, …
Figure 1—figure supplement 2
Scoring sheet for spasticity and disordered sensori-motor control of the forelimb.
Abnormal forelimb movements were scored by a blinded observer after rats were videotaped in a Perspex cylinder for 3 min each fortnight. Each sign of spasticity or abnormal forelimb movement can be …
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.
(A) Schematic showing the H-reflex paradigm. The ulnar nerve was stimulated distally and EMGs were recorded from a homonymous hand muscle (abductor digiti quinti). (B–C) A single stimulus evokes an …
Figure 1—figure supplement 4
Polysynaptic reflex responses recorded from the radial nerve after ulnar nerve stimulation were not changed after injury.
(A) Stimulation of afferents in the ulnar nerve evoked few or no responses in the (antagonistic, extensor) radial nerve. (B–C) Representative traces showing recordings from (B) uninjured naïve and (C…
Figure 2 with 2 supplements
Increased levels of neurotrophin-3 in muscle and blood post-injection of AAV1 neurotrophin-3 into forelimb muscles.
(A) Schematic showing experimental set-up. Rats received bilateral pyramidotomies and were injected either AAV1-NT3 or AAV1-GFP into forelimb muscles. The monosynaptic proprioceptive reflexes are …
Figure 2—figure supplement 1
Lesion cross-sectional areas were similar on the left and right of the medulla in both bPYX groups.
(A–B) Eriochrome cyanine staining of transverse sections through the medulla taken 10 weeks after bilateral pyramidotomy. (A) Uninjured naïve rats showed intact pyramids on the left and right …
Figure 2—figure supplement 2
No increased levels of neurotrophin-3 in homogenates of triceps brachii, spinal cord or liver after injections of an AAV expressing neurotrophin-3 into the biceps brachii and other forelimb flexors.
(A) ELISAs showed that there was no significant difference between groups in the level of Neurotrophin-3 protein in the triceps brachii extensor muscles on the ipsilateral side or contralateral side …
Figure 3 with 1 supplement
Intramuscular Neurotrophin-3 treatment improved functional recovery and reduced spasms after bilateral pyramidotomy.
Please note that for clarity in describing our model of spasticity, Figure 1 contained information from Figure 3 relating to the uninjured naïve and bPYX GFP groups. (A) Neurotrophin-3 treatment …
Figure 3—figure supplement 1
Changes in polysynaptic reflexes after bilateral pyramidotomy and with Neurotrophin-3.
(A) The polysynaptic reflex responses were recorded from the ulnar while stimulating the median nerve. The polysynaptic compound action potentials were quantified by measuring the absolute integral …
Figure 4 with 1 supplement
Neurotrophin-3 treatment restored balance between excitatory and inhibitory causes of spasticity.
(A–D) Ia boutons were identified by vGluT1 immunolabelling (green) and motor neurons were traced retrogradely with Fast Blue or Cholera Toxin beta (blue) in (B) uninjured naïve rats, (C) bPYX GFP …
Figure 4—figure supplement 1
The density of inhibitory boutons directly onto motor neurons did not change with injury or neurotrophin-3 treatment.
(A) Motor neurons were retrogradely traced with Fast blue or CTb (blue) on the treated side. Transverse spinal sections of C7/8 were immunolabeled with antibodies against vGluT1 (green) and vGAT …
Figure 5 with 1 supplement
Neurotrophin-3 treatment normalised serotonergic innervation of the C7/8 spinal cord and the ion transporter KCC2 in motor neuron membranes to normal.
(A) Representative images of C7/8 spinal cords on the treated side immunolabelled for serotonin in uninjured naïve, bPYX GFP and bPYX NT3 rats. Scale bar: 1 mm. (B) Serotonergic pixel intensity in …
Figure 5—figure supplement 1
Analysis of KCC2 in the membrane of motor neurons.
(A) Images of retrogradely traced motor neurons stained for KCC2. Scale bars 20 µm. Uninjured naïve and bPYX NT3 rats had a high immunoreactivity in the membrane. This was not observed in bPYX GFP …
Figure 6
Spinal hyper-excitability causing hyperreflexia, spasms and disordered sensorimotor control is normalized by intramuscular Neurotrophin-3 treatment.
(A) In uninjured healthy conditions, there is a balance between excitatory (afferent and descending) networks, pre-synaptic inhibition and motor neuron excitability. (B) After loss of corticospinal …
Videos
Video 1
Open-field movements shown by an uninjured naïve rat (Supplementary to Figure 1 and Experimental Procedures).
Rats are placed in a 50 cm diameter Plexiglas cylinder and videotaped for 3 min every fortnight. See Supplementary Figure 1—figure supplement 2 for scoring system. During swing, forepaw digits are …
Video 2
Open-field scoring of spasticity and disordered sensorimotor forelimb movements of rats with bilateral pyramidotomies (Supplementary to Figure 1 and Experimental Procedures).
Rats exhibiting signs of disordered sensorimotor control have their forepaw digits in a flexed position during swing phase presumably because of hypertonic flexor muscles in the forepaw. Movements …
Video 3
Additional signs and associated features of spasticity and disordered sensorimotor movements (Supplementary to Figure 1 and Experimental Procedures).
The behaviours shown in this video were not scored as part of the forelimb scale, but were frequently observed. Rats with bilateral pyramidotomy displayed prolonged forelimb muscle contractions and …
Tables
Table 1
Properties of the M-wave and H-reflex (Supplementary to Figures 1 and 3). Values (mean ± SEM) are given for motor threshold, M-wave and H-wave latencies and maximum amplitudes, maximum depression of …
| baseline | Week 2 | Week 4 | Week 6 | Week 8 | Week 10 | ||
|---|---|---|---|---|---|---|---|
| Motor threshold (mA) | naïve | 1.14 ± 0.2 | 2.25 ± 0.5 | 2.13 ± 0.5 | 2.01 ± 1.5 | 2.27 ± 0.4 | 3.45 ± 0.5 |
| bPYX GFP | 1.83 ± 0.3 | 1.26 ± 0.2 | 1.71 ± 1.4 | 2.42 ± 0.5 | 2.15 ± 0.4 | 2.05 ± 1.6 | |
| bPYX NT3 | 1.28 ± 0.2 | 1.33 ± 0.3 | 2.06 ± 0.4 | 1.62 ± 0.3 | 1.79 ± 0.5 | 2.04 ± 0.5 | |
| M-wave latency (ms) | naïve | 0.96 ± 0.0 | 0.95 ± 0.1 | 0.85 ± 0.0 | 0.88 ± 0.0 | 0.97 ± 0.1 | 1.13 ± 0.1 |
| bPYX GFP | 0.92 ± 0.1 | 0.91 ± 0.1 | 0.98 ± 0.0 | 0.99 ± 0.1 | 0.91 ± 0.0 | 1.01 ± 0.1 | |
| bPYX NT3 | 0.99 ± 0.1 | 0.89 ± 0.0 | 1.24 ± 0.3 | 0.98 ± 0.1 | 0.94 ± 0.1 | 1.12 ± 0.1 | |
| maximum M-wave (mV) | naïve | 7.23 ± 0.9 | 6.76 ± 0.5 | 5.53 ± 0.4 | 4.76 ± 0.5 | 4.83 ± 0.5 | 3.31 ± 0.4 |
| bPYX GFP | 6.34 ± 0.8 | 6.94 ± 0.9 | 8.34 ± 0.7 | 5.84 ± 0.7 | 6.92 ± 0.6 | 4.93 ± 0.7 | |
| bPYX NT3 | 7.89 ± 0.8 | 6.25 ± 0.7 | 4.82 ± 1.0 | 6.08 ± 0.7 | 3.74 ± 0.4 | 4.32 ± 0.5 | |
| H-wave latency (ms) | naïve | 5.66 ± 0.1 | 5.60 ± 0.1 | 5.39 ± 0.1 | 5.42 ± 0.1 | 5.69 ± 0.1 | 5.97 ± 0.1 |
| bPYX GFP | 5.48 ± 0.1 | 5.24 ± 0.1 | 5.67 ± 0.1 | 5.55 ± 0.4 | 5.42 ± 0.1 | 5.60 ± 0.2 | |
| bPYX NT3 | 5.22 ± 0.1 | 5.30 ± 0.2 | 5.80 ± 0.3 | 5.48 ± 0.1 | 5.36 ± 0.2 | 5.40 ± 0.1 | |
| maximum H-wave (mA) | naïve | 1.34 ± 0.2 | 1.46 ± 0.3 | 1.09 ± 0.2 | 1.22 ± 0.2 | 1.31 ± 0.2 | 0.92 ± 0.2 |
| bPYX GFP | 1.60 ± 0.3 | 3.50 ± 0.7 | 2.84 ± 0.5 | 2.48 ± 1.8 | 2.72 ± 0.6 | 1.94 ± 0.5 | |
| bPYX NT3 | 2.43 ± 0.5 | 2.15 ± 0.4 | 1.53 ± 0.3 | 2.01 ± 0.3 | 1.41 ± 0.2 | 1.63 ± 0.2 | |
| maximum H-wave (%) | naïve | 19.5 ± 8.0 | 21.7 ± 3.3 | 19.7 ± 3.5 | 29.9 ± 6.5 | 26.5 ± 3.0 | 31.2 ± 5.8 |
| bPYX GFP | 29.5 ± 6.7 | 46.2 ± 4.7 | 31.9 ± 4.8 | 41.8 ± 5.7 | 40.8 ± 6.2 | 43.8 ± 5.5 | |
| bPYX NT3 | 30.7 ± 5.7 | 38.2 ± 6.5 | 36.8 ± 4.8 | 44 ± 10.7 | 45.3 ± 9.1 | 45.0 ± 8.8 | |
| maximum depression of H-wave | naïve | 24.3 ± 4.2 | 35.9 ± 3.8 **** | 42.7 ± 6.7 | 37.9 ± 7.3 | 34.7 ± 6.6 | 41.0 ± 5.5 |
| bPYX GFP | 38.5 ± 5.9 | 56.4 ± 7.9 | 57.5 ± 8.7 | 61.0 ± 8.c2 *** | 56.3 ± 8.1 * | 62.9 ± 8.3 *** | |
| bPYX NT3 | 37.9 ± 5.9 | 58.9 ± 6.1 | 48.6 ± 8.0 | 37.6 ± 5.8 | 36.5 ± 6.3 | 29.2 ± 3.9 |
-
Table 1: Properties of the M- and H-wave.
