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 Th11–12 segment. Afterwards, the means from L and R hemicords were calculated and presented in the <b>B–G</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 (P = 0.061); 2 to 4 Western blot performed for each sample were analyzed and data were normalized to β-actin. Bars represent means±SD (<b>B–E</b>) or ± SEM (<b>G</b>) from 5 intact, 3 SP-PBS and 4 SP-BDNF rats. Mann-Whitney U test, ^#P<0.05, ^##P<0.01.</p

AAV-BDNF-induced segmental changes of GABA and GAD67 mRNA 7 weeks after spinal cord transection.

<p>(<b>A, B</b>) BDNF overexpression leads to an increase of GABA and GAD67 mRNA levels exceeding control levels in L3–6 segments. Hatched and black bars represent their segmental levels in SP-PBS and SP-BDNF rats, respectively, expressed as a percentage of the level in intact animals. GABA concentration in intact rats equals to 2.38±0.16 µmol/100mg of protein. Asterisks above the bars indicate significant differences between spinalized rats and intact controls; asterisks above the square brackets indicate significant differences between the SP-PBS and SP-BDNF groups. Data are the means ± SEM from 5 intact, 3 SP-PBS and 4 SP-BDNF rats. Two-way ANOVA with Tukey <i>post-hoc</i> tests were used, *P<0.05, ***P<0.001. (<b>C</b>) Labeling intensity of GAD67-positive boutons terminating on large neurons of the ventral horn (insets) is lower in Th10–11 than in L1–2 segments (means ± SD measured in 26 and 29 boutons, respectively). An example. (<b>D</b>) GAD67-immunolabeling of fibers and boutons (red) terminating on motoneurons (immunolabeled for VAChT, green) in the longitudinal parasagittal section of the spinal cord of the rat that received BDNF transgene with cMYC tag. Note a gradient of GAD67 immunolabeling intensity, which is lower in the thoracic region above the transection site (Th10–11, devoid of BDNF-cMYC expression - left), than in the lumbar (L1–2) region, enriched in BDNF-cMYC. Abbreviations: vh – ventral horn, vf – ventral funiculus. Bars equal to 50 µm. (<b>E</b>) A reconstruction from fused microphotographs of a thoraco-lumbar longitudinal parasagittal section of the spinal cord from the SP-BDNF rat shows widespread distribution of BDNF-cMYC immunostaining (green) caudally to the lesion. Framed areas on <b>E</b> (showed in higher magnification in <b>F–I)</b> demonstrate that BDNF transgen expression spatially correlates with GAD67 labeling (red). BDNF-cMYC is present in fibers (arrowheads), some with varicosities (<b>F, F’, H, I</b>) and in neuronal perikarya throughout grey matter (<b>G, G’, H, J</b>). Except for the scar area devoid of GAD67 immunolabeling (<b>F</b>), in other regions BDNF-cMYC signal is associated with intense GAD67 immunoreactivity detected in cell perikarya and fibers (the area above the dashed line in <b>G</b> and the dorsal horn region in <b>H</b>). No co-localization of the two markers was observed; arrowheads indicate separate cMYC and GAD67 signals in fibers (<b>F’</b>) including boutons apposing large BDNF-cMYC negative neurons (<b>I</b>). BDNF-cMYC positive neurons are GAD67 negative and receive no inputs from BDNF-cMYC expressing projections (<b>G, G’, H, J</b>). Hoechst labeling (in blue) marks cell nuclei. Bars equal to 50 µm.</p

Summary of the effect of complete spinal cord transection and BDNF overexpression in spinal rats on the levels of GABA and transcripts of GAD67 and KCC2 in the rostral and caudal segments of the lumbar spinal cord in the late post-operative period, in conjunction with the scores of their locomotor performance with no tail stimulation, on the moving treadmill.

<p>Summary of the effect of complete spinal cord transection and BDNF overexpression in spinal rats on the levels of GABA and transcripts of GAD67 and KCC2 in the rostral and caudal segments of the lumbar spinal cord in the late post-operative period, in conjunction with the scores of their locomotor performance with no tail stimulation, on the moving treadmill.</p

Locomotor capabilities of spinal rats in PBS- (SP-PBS) and BDNF-treated (SP-BDNF) groups evaluated during hindlimb walking on a moving treadmill.

<p>Differences between scores achieved with- and without tail stimulation in SP-BDNF group in early post-operative period are significant (Wilcoxon test, P = 0.005). Differences between SP-PBS and SP-BDNF groups without tail stimulation in late post-operative period are also significant (Mann-Whitney U test; P = 0.02). NA – not analyzed.</p><p>BBB scale modified by Antri and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088833#pone.0088833-Antri1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088833#pone.0088833-Antri2" target="_blank">[55]</a> was used for assessment of bipedal treadmill locomotion in adult, spinalized rats. There are four major levels and 22 scores of recovery of motor capabilities, where level 4 and score 22 correspond to locomotion of intact animal. Animals were tested twice during the experiment, in the early and late post-operative period (exact testing days are shown).</p

The effects of spinal cord transection and BDNF overexpression on segmental vesicular glutamate transporter 1 (VGluT1) and 2 (VGluT2) transcripts level.

<p>(<b>A</b>) Spinal cord transection leads to a significant decrease in VGluT1 mRNA level in L1–2 segments and to less pronounced decrease in L3–6 segments (hatched bars). In SP-BDNF rats VGluT1 mRNA goes through similar to SP-PBS rats reductions, both in L1–2 and in L3–6 segments (black bars). (<b>B</b>) Spinal cord transection causes a significant decrease in VGluT2 mRNA levels in rostral and a tendency to decrease in caudal spinal cord segments (hatched bars). In SP-BDNF rats VGluT2 transcript level is significantly higher than in SP-PBS rats both in L1–2 and L3–6 segments, where it tends to be higher than in control rats (black bars). Data are the means ± SD from 5 intact, 3 SP-PBS and 4 SP-BDNF rats. Mann-Whitney U test was used to compare VGluT1 mRNA values: # P<0.05, ## P<0.02; Two-way ANOVA with Tukey post-hoc tests were used to compare VGluT2 mRNA values: *P<0.05, ***P<0.001. Asterisks above the bars indicate significant differences between spinalized rats and intact controls; asterisks put above the square brackets indicate significant differences between the SP-PBS and SP-BDNF groups.</p

Effect of spinal cord transection and BDNF overexpression on segmental potassium-chloride co-transporter 2 (KCC2) transcript and protein level.

<p>(<b>A</b>) Spinal cord transection leads to a significant decrease in KCC2 mRNA level in L1–2 segments (hatched bars). In SP-BDNF rats KCC2 mRNA is equally reduced in L1–2 segments and tends to be lower in L3–6 segments than in SP-PBS rats (black bars). (<b>B</b>) Similar tendencies were observed at the protein level. Data are the means ± SD (qPCR) or means ± SEM (WB) from 5 intact, 3 SP-PBS and 4 SP-BDNF rats. Two-way ANOVA with Tukey <i>post-hoc</i> tests were used; **P<0.01, ***P<0.001. (<b>C</b>) The representative confocal microscopy images (upper panel) and the same images tresholded (lower panel) show the pattern and intensity of KCC2 immunostaining of large diameter neurons and surrounding neuropil in the ventral horn of the spinal cord of the rats from intact, SP-PBS and SP-BDNF groups. Note a remarkable reduction of KCC2 labeling in both spinalized groups, with a loss of continuity of the cell membrane signal and torn up appearance of the processes.</p

Summary of the status of animals and tissue analysis.

<p>*Two animals from this group had died before the end of the experiment and thus were tested only behaviorally in the early post-operative period (see text for details).</p><p>Numbers in brackets mark those animals which were used for both behavioral and biochemical analyses.</p

BDNF overexpression leads to an early recovery in locomotor function. Comparison of the treadmill locomotion of the intact, SP-PBS and SP-BDNF rats.

<p>Upper panel: kinematic analysis of the gait of an intact rat during locomotion on the moving treadmill (<b>A</b>) Stick figure showing the angular excursions of the knee, ankle and toe joints during one step cycle during slow (0.05 m/s) treadmill locomotion in an intact rat. To measure the angular excursion of the hindlimb joints, the black markers were glued to the shaved skin overlying the femur and tibia heads, tibiofibular articulation, distal metatarsus and distal phalanx of the third toe (bottom, <b>A</b>). A digital Panasonic camera (NV-GS400 3CCD) was used to capture video images of the hindlimbs during treadmill locomotion. Software based on Image–Pro Plus was used to create the stick figures of the hindlimb movements with a resolution twice as fast as that of the camera (i.e., 50 images/s). Every stick image was artificially separated from the next by the same coefficient to avoid superimposing neighboring stick images. (<b>B</b>) The footprints of both hindlimbs of an intact rat corresponding to the beginning of the foot contact with the treadmill during locomotion as taken from the video. Black dots - left leg; squares - right leg. (<b>C</b>) Angular excursions in the knee, ankle and toe joints during 10 s of treadmill locomotion. Downward deflection of the angular traces indicates flexion movement. (<b>D</b>) A framed plot from <b>C</b> of the angular excursions during one step was enlarged to indicate the phases of locomotion (F-E1 correspond to the swing and E2–E3 to the stance phase). Middle panel<i>:</i> examples of treadmill locomotion during the early post-operative period (the second week after surgery) of the spinal rats injected with PBS or with AAV-BDNF. None of the SP-PBS rats were able to perform locomotor movements when their hindlimbs were placed on the moving treadmill (exemplified in the left column; rat 5.2). Addition of tactile stimulation of the tail produced some agitation in both hindlimbs but did not evoke locomotor movements in the PBS-treated rats (central). Contrary to SP-PBS treated rats, no tail stimulation had to be used to trigger locomotion with body weight support in SP-BDNF rats. Periods of alternating, treadmill walking with body weight support and plantar foot placement but reduced rhythmicity were observed in eight out of the eleven rats overexpressing BDNF (exemplified in the right column, rat 4.10). Bottom panel: examples of treadmill locomotion of the spinal rats in the late (about 40 days) postoperative period. None of the SP-PBS rats could support their body weight or perform plantar foot placement (left); tail stimulation triggered locomotor movement in the PBS-treated rats (central). The locomotor capabilities of the hindlimbs in the SP-BDNF group improved profoundly in rats that were previously classified at the lowest level of the mBBB scale, whereas worsened in rats that walked well in the early post-surgery period (exemplified in the right column, rat 4.10). In that group, stimulation of the tail attenuated the quality of locomotion in rats that walked well without tail stimulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088833#pone-0088833-t002" target="_blank"><b>Table 2</b></a>, rats with scores 16–19). Weight support is defined as an elevation of the hindquarter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088833#pone.0088833-Basso1" target="_blank">[56]</a>.</p

The changes of segmental concentration of γ-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 (Th9–10) 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 ×10 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 – ventral horn, vf – ventral funiculus.</p