17 research outputs found
Distribution of NT-3 mRNA and BDNF mRNA expression in the tissues involved in circuitry of the H-reflex and their changes caused by continuous bursts of low-threshold, unilateral stimulation of the tibial nerve.
<p><b>A.</b> Relative expression of transcripts in the control group. In the tibial nerve (shaded grey bars), mRNA expression was evaluated by means of high-fidelity reverse transcriptase and random hexamers as primers. The tibial nerve segment enwrapped in the cuff-electrode was analyzed. <b>B.</b> The changes of NT-3 and BDNF level in the spinal cord segments, soleus muscle and tibial nerve after low-threshold stimulation of the tibial nerve. NT-3 mRNA expression tended to increase in the caudal lumbar segments of the spinal cord and the effect was bilateral, similarly as that of BDNF mRNA. In the soleus muscle NT-3 mRNA and BDNF mRNA level decreased comparing to control level. NT-3 mRNA expression in the tibial nerve increased after stimulation but that of BDNF mRNA tended to decrease. Asterisks indicate statistically significant effects (***P<0.001; **P<0.01; *P<0.05, Mann-Whitney U and Wilcoxon tests).</p
Time course of amplitude changes of the M- response and H- reflex evoked by electrical stimulation of the tibial nerve in the rat.
<p>Two stimulation patterns were used: continuous bursts of stimuli (solid lines: black â H reflex, grey â M response) or single stimuli (dashed lines: black - H reflex and grey â M response). The averaged responses to the single stimuli consisted of 30 reflexes collected at the beginning of the first and at the end of the last stimulation sessions daily in one animal. The averaged responses to burst of stimuli were collected during 3 min periods at the beginning and the end of every daily session. The strength of stimulation was established near the threshold of activation of the motor fibers, which is higher than that of Ia afferents, therefore stimulation elicited a moderate H-reflex as the majority of Ia fibers are already excited when the direct motor response (M) is at its threshold.</p
Enhancing Proprioceptive Input to Motoneurons Differentially Affects Expression of Neurotrophin 3 and Brain-Derived Neurotrophic Factor in Rat Hoffmann-Reflex Circuitry
<div><p>The importance of neurotrophin 3 (NT-3) for motor control prompted us to ask the question whether direct electrical stimulation of low-threshold muscle afferents, strengthening the proprioceptive signaling, could effectively increase the endogenous pool of this neurotrophin and its receptor TrkC in the Hoffmann-reflex (H-reflex) circuitry. The effects were compared with those of brain-derived neurotrophic factor (BDNF) and its TrkB receptor. Continuous bursts of stimuli were delivered unilaterally for seven days, 80 min daily, by means of a cuff-electrode implanted over the tibial nerve in awake rats. The H-reflex was recorded in the soleus muscle to control the strength of stimulation. Stimulation aimed at activation of Ia fibers produced a strong increase of NT-3 protein, measured with ELISA, in the lumbar L3-6 segments of the spinal cord and in the soleus muscle. This stimulation exerted much weaker effect on BDNF protein level which slightly increased only in L3-6 segments of the spinal cord. Increased protein level of NT-3 and BDNF corresponded to the changes of NT-3 mRNA and BDNF mRNA expression in L3-6 segments but not in the soleus muscle. We disclosed tissue-specificity of TrkC mRNA and TrkB mRNA responses. In the spinal cord TrkC and TrkB transcripts tended to decrease, whereas in the soleus muscle TrkB mRNA decreased and TrkC mRNA expression strongly increased, suggesting that stimulation of Ia fibers leads to sensitization of the soleus muscle to NT-3 signaling. The possibility of increasing NT-3/TrkC signaling in the neuromuscular system, with minor effects on BDNF/TrkB signaling, by means of low-threshold electrical stimulation of peripheral nerves, which in humans might be applied in non-invasive way, offers an attractive therapeutic tool.</p></div
Distribution of TrkC and TrkB mRNA expression in the regions involved in H-reflex circuitry and their changes caused by continuous bursts of low-threshold, unilateral stimulation of the tibial nerve.
<p><b>A.</b> Relative expression of transcripts in the control group. In the tibial nerve (shaded grey bars), the mRNA expression was evaluated by means of high-fidelity reverse transcriptase and random hexamers as primers. <b>B.</b> The changes of TrkC and TrkB level in the spinal cord segments, soleus muscle and tibial nerve after low-threshold stimulation. TrkC mRNA expression was high in the spinal cord of the control group and negligible in the soleus muscle. It tended to decrease after stimulation in the spinal cord and increased in the soleus muscle. Stimulation caused clear decrease of NT-3 mRNA in the tibial nerve. TrkB mRNA expression decreased both in the spinal cord and soleus muscle after stimulation but in the tibial nerve it increased on the sham-side and tended to decrease on the stimulated side. Asterisks indicate statistically significant effects (***P<0.001; **P<0.01; *P<0.05, Mann-Whitney U and Wilcoxon tests).</p
Schema showing the circuitry of the monosynaptic Hoffmann (H) reflex (an analog of the muscle stretch reflex) showing the location of the stimulating and recording electrodes.
<p>Low-threshold continuous burst stimulation of the tibial nerve, addressed to group Ia fibers, was applied to enhance the proprioceptive input to the extensor motoneurons innervating the ankle muscles. The amplitudes of H-reflex and direct M-response were used to control the strength of stimulation.</p
NT-3 and BDNF protein level in the tissues involved in the H-reflex circuitry.
<p><b>A.</b> The protein level of NT-3 and BDNF in the intact animals (control group), measured in the lumbar segments (L1-2; L3-6) and the soleus muscle (sol), by means of ELISA. NT-3 level was the highest in the soleus muscle whereas that of BDNF predominated in the spinal cord. <b>B.</b> The changes of NT-3 and BDNF level in the spinal cord segments and soleus muscle after low-threshold, unilateral stimulation of the tibial nerve. Stimulation caused a clear increase of NT-3 protein level in the L3-6 segments of the spinal cord, where the motoneurons innervating the soleus muscles and its synergists acting at the ankle joints are located, and in the soleus muscle. The effect of stimulation on the protein level of BDNF was weaker and detected only in the caudal lumbar segments of the spinal cord. Asterisks indicate statistically significant effects (***P<0.001; **P<0.01; *P<0.05, Mann-Whitney U and Wilcoxon tests).</p
An example of averaged H- and M- waves in the soleus muscle elicited by continuous bursts of pulses applied to the tibial nerve every 25 ms.
<p>Each burst was composed of three pulses (pulse widthâ=â200 ”s) with 4 ms inter-pulse interval. The mean latencies (± SD) of the H-and M-responses recorded in the soleus muscle were 5.9±0.6 ms and 1.9±0.2 ms, respectively. During the burst stimulation only the first M<sub>1</sub> and the last H<sub>3</sub> response (framed) were analyzed as they were not contaminated by the accompanying responses.</p
AAV-BDNF counteracts thoraco-lumbar BDNF deficits and causes BDNF overproduction in spinal segments 7 weeks after spinal cord transection.
<p>(<b>A</b>) Diagrammatic representation of spinal cord microdissection for biochemical analyses. A photograph exemplifying the lesion and injection site is shown below. AAV-BDNF was injected separately to each hemicord, and injection efficacy was analyzed for samples from right (R) and left (L) hemicords, except for the lesioned Th11â12 segment. Afterwards, the means from L and R hemicords were calculated and presented in the <b>BâG</b>. BDNF mRNA levels were evaluated with qPCR (<b>B, C</b>). BDNF concentration was measured with ELISA in the s1 fraction obtained from the homogenates of spinal Th10-L6 segments (<b>D, E</b>), and changes in BDNF mature (mBDNF) and precursor (proBDNF) forms were evaluated by Western blot analysis (<b>F, G</b>). Spinal cord transection leads to a decrease in the BDNF mRNA level (<b>B</b>; hatched bars) and protein concentration (<b>D</b>; hatched bars) in the lesion site, low thoracic and rostral lumbar segments. Black horizontal lines in <b>B</b> and <b>D</b> mark the control values for the intact animals. AAV-BDNF causes significant increase of the level of BDNF transcript (<b>C</b>; black bars) and BDNF concentration (<b>E</b>; black bars) in the transection site and in the spinal segments caudally to the transection. Bars in <b>C</b> and <b>E</b> show the ratios of the means of BDNF mRNA (<b>C</b>) and protein (<b>E</b>) concentration in spinal BDNF-treated rats (SP-BDNF) to that in the intact animals (left panels) and in SP-PBS rats (right panels). (<b>F</b>) Representative Western blots show the occurrence of mBDNF in individual intact, SP-PBS and SP-BDNF rats and indicate, that proBDNF is clearly detectable in SP-BDNF rats; in the intact and SP-PBS rats proBDNF is below the level of detection. (<b>G</b>) Relative optical density of mBDNF bands in respective groups indicate that in SP-BDNF rats mBDNF is elevated above controls in the rostral lumbar segment and tends to increase in the caudal lumbar segments (Pâ=â0.061); 2 to 4 Western blot performed for each sample were analyzed and data were normalized to ÎČ-actin. Bars represent means±SD (<b>BâE</b>) or ± SEM (<b>G</b>) from 5 intact, 3 SP-PBS and 4 SP-BDNF rats. Mann-Whitney U test, <sup>#</sup>P<0.05, <sup>##</sup>P<0.01.</p
The changes of segmental concentration of Îł-aminobutyric acid (GABA), glycine (Gly), glutamate (Glu) and aspartate (Asp) measured 5 weeks after complete spinal cord transection in the whole tissue homogenates of thoracic (Th) and lumbar (L) segments.
<p>All samples were measured simultaneously by means of HPLC and the measurements were carried out in quadruplicates. The data of four rats with complete spinal cord transection performed at thoracic segments (Th9â10) and four intact rats are presented. Data show means ± SD, Two way-ANOVA, Tukey <i>post-hoc</i> tests, *P<0.05, ***P<0.001.</p
Spinal cord transduction with an AAV1/2 vector expressing EGFP or BDNF with cMYC tag at 7 weeks after spinal cord transection.
<p>Upper panel: (A) Tiled images taken of the thoraco-lumbar segments of a spinal cord from a spinalized rat that received the EGFP transgene; micrographs were taken on a fluorescence microscope at Ă10 magnification. The dashed lines delineate the edges of the scar. (<b>B</b>) An enlarged micrograph of the framed area in (<b>A</b>) documenting the numerous EGFP-positive fibers (arrows) running along the grey matter in close proximity to the lesion site. (<b>C</b>) An enlarged micrograph of the framed area in (<b>A</b>) showing transduced cells of neuronal morphology in the ventral horn (arrows). (<b>D</b>) A merging of the EGFP expression signal (green) with vesicular acetylcholine transporter (VAChT, red) immunolabeling shows their colocalization (yellow), which confirms that the transduced cells are motoneurons (arrows). Bottom panel: The spinal cord from a rat that received the BDNF-cMYC transgene. (<b>E</b>) cMYC immunostaining detected BDNF-cMYC-positive neuronal fibers (exemplified by arrows) below the transection, in the lower thoracic segments of the spinal cord. Fibers approach and encroach on the scar from its caudal aspect. The white dashed line delineates the caudal border of the scar. A dense mesh of small caliber BDNF-cMYC-positive fibers prevails in the grey matter (<b>E</b>) whereas large caliber varicose fibers appear in the white matter (<b>F</b>). (<b>G</b>) The confocal microscope microphotograph shows two BDNF-cMYC-positive, large size neurons (arrows) of the L2 ventral horn. Hoechst nuclear labeling is shown in blue. Abbreviations: vh â ventral horn, vf â ventral funiculus.</p