Axon density in corpus callosum of wild-type and Hx^−/− mice.

<p>The number of myelinated (axons containing compact myelin) and unmyelinated axons were counted in a 30 µm² area from electron micrographs of corpus callosum sections. There were no changes in axon density between wild-type and Hx^−/− mice. Values are the average number of axons per field ± SD. Wild-type, n  = 3; Hx^−/−, n  = 3.</p

Alteration of myelin ultrastructure in the absence of Hx.

<p>EM analysis was performed on the corpus callosum of wild-type and Hx^−/− mice at twelve month of age. <b>A</b>) Electron micrographs show that in Hx^−/− mice the axons are hypomyelinated and the number of small myelinated axons is reduced in comparison to wild-types. Bar  = 1 µm. <b>B</b>) The distribution of myelin thickness in wild-type and Hx^−/− mice fibers demonstrated that myelin sheath was thicker in Hx^−/− fibers. P<0.0001. <b>C</b>) g-ratio scatter diagram in wild-type and Hx^−/− mice fibers. Elevated <i>g</i> ratio values were observed for all axons in Hx^−/− mice, indicating that impaired myelination affected axon of all sizes. P<0.001. <b>D</b>)The distribution of axonal size in wild-type and Hx^−/− mice fibers showed that Hx^−/− mice had bigger axons compared to controls. P<0.0001. n = 5 mice for each genotype.</p

Hx promotes OL differentiation.

<p>OPCs were grown with or without Hx and the differentiation process was analyzed. <b>A</b>) Representative images showing the different developmental stages taken into consideration: stage I, OPCs (bipolar); stage II: pre-OL (primary branched); stage III: immature OL (secondary branched); stage IV: mature OL (secondary branched cells with membranous processes). Cells at stage I and II are PDGFRα positive, CNPase negative, cells at stage III are PDGFRα negative, CNPase positive and cells at stage IV are PDGFRα negative, CNPase and MBP positive. <b>B</b>) Kinetics of OL differentiation. Cells were cultured for 48 h in the absence (NT) or presence of Hx (Hx) or heme-Hx complex (Hx-heme), and the number of cells at each differentiation stage was counted as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020173#s2" target="_blank">Materials and Methods</a>. Cells were scored by morphology and immunoreactivity to PDGFα and CNPase as shown in (A). Hx treatment accelerated the differentiation process whereas the heme-Hx complex was ineffective. *  =  P<0.05. Results shown are representative of three independent experiments.</p

Impaired OL development in Hx^−/− mice.

<p>Brain sections of wild-type and Hx^−/− mice were immunoreacted to discriminate between OPCs and mature OLs and OPCs and OLs counted as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020173#s2" target="_blank">Materials and Methods</a>. <b>A</b>) Quantification of PDGFRα-positive cells demonstrated similar numbers of OPCs in both cerebral cortex and corpus callosum in Hx^−/− and wild-type mice at P10. On the contrary, the number of CC1-positive, GFAP-negative mature OLs in Hx^−/− mice was strongly reduced compared to wild-type animals at P10 and P20. Data represent mean ± SEM, n = 3 mice for each genotype. **  = P<0.01, ***  = P<0.001. B) Maps, obtained with Neurolucida/Neuroexplorer, of brain sections of PDGFRα- (left) and CC1- (right) positive cells, respectively, in Hx^−/− and wild-type mice at P10. Red  =  OPCs, blue  =  mature OLs. Note the reduced number of mature OLs in the supragranular layer of cortex in Hx^−/− mice. C) Representative pictures of CC1/GFAP double staining for CC1 (brown) and GFAP (grey) in brain sections of a wild-type and a Hx^−/− mouse. The latter shows a strong reduction in the number of CC1 positive cells in the cortex (arrows) and in corpus callosum, CC, (arrow-heads) compared to wild-type animal. Bar  = 50 µm.</p

Reduction of MBP protein production in Hx^−/− brain.

<p><b>A</b>) Western blot analysis of MBP expression in brain extracts of wild-type and Hx^−/− mice. Cerebral cortex and basal ganglia region lysates were analyzed at two and twelve months of age. Representative experiments are shown. <b>B)</b> Band intensities were measured by densitometry and normalized to actin expression (AU: Arbitrary Unit). The overall MBP production was obtained by summing the relative intensities of the four isoforms recognized by the antibody (indicated by arrows in scanned gels). Densitometry data represent mean ± SEM; n = 3 for each genotype. *  =  P<0.05. Results shown are representative of three independent experiments.</p

The cortex of Hx^−/− mice is hypomyelinated.

<p><b>A</b>) Coronal sections of a wild-type and a Hx^−/− mouse at twelve months of age stained with Black-Gold reaction to detect myelinated fibers. Hx^−/− mouse shows reduced myelination in cerebral cortex compared to wild-type (a, b) and the hypomyelination mainly affects the supragranular layers in motor and somatosensory cortex (arrows in c, d). Higher magnification shows that in layer I of Hx^−/− mouse the staining is very weak compared to wild-type (e, f). Bar (c, d)  = 500 µm; Bar (e, f)  = 100 µm. <b>B</b>) Quantification of fiber density in motor cortical area, assessed at 2, 6 and 12 months of age, shows a severe reduction in Hx^−/− mice. Data represent mean ± SEM, n = 3 mice for each genotype. *  = P<0.05, ***  = P<0.001.</p

Flow chart of experiment 2.

<p>In experiment 2, animals were exposed to X-Ray irradiation on day 1, 7, 15 and 23. Twenty-four hours after the last irradiation, animals started the 4-week period of bortezomib chemotherapy. The Dynamic Aesthesiometer Test was performed on day 3, 12, 18, 25, 32, 46 and the cytofluorimetric analysis of PB and BM CD45 positive cells on days 26, 40 and 46. On day 46 animals underwent the neurophysiologic analysis and, once euthanized, the sample collection for the neuropathological analysis.</p

Body weight, experiment 2.

<p>Body weights were measured twice a week to monitor X-Ray irradiation and bortezomib-induced toxicity. X-Ray-treated animals showed an initial slight body weight decrease on day 4 and 8 then resumed a normal growth trend similar to naïve animals. Starting from the second bortezomib administration (day 32) animals showed a marked and statistically significant decrease of body weight that persisted till the end of the experimental period.</p

Neurophysiology, experiment 1.

<p>Nerve Conduction Velocity (NCV) and action potential amplitudes were tested with an electromyographic apparatus in the caudal and digital nerves two days after the last bortezomib administration. Bortezomib induced a significant decrease in the caudal (A) and digital (C) NCV compared to the naïve and vehicle-treated mice. Bortezomib induced a statistically significant reduction in the caudal action potential amplitude (B). There was no difference between naïve/vehicle and bortezomib-treated animals in the digital action potential amplitude (D).</p

Bone marrow and peripheral blood and bone marrow white blood cells (WBC) count and FACS analysis, experiment 2.

<p>Black arrows indicate the treatment given to mice, such as the X-ray irradiation doses (350 RAD induction and 100 RAD maintenance), and bortezomib administrations. For each group of animals, the WBC concentration within the BM (A) or peripheral blood (B) is indicated for the corresponding analysis time point. The number of hematopoietic cells in the peripheral blood expressing the pan-leukocyte antigen CD45 is also reported (C). A comparable immune-suppression was observed in both X-Ray and X-Ray-bortezomib-treated animals after the irradiation induction and maintenance doses, as shown by the decreased cellularity and leukopenia in the BM and spleen of irradiated mice, regardless of bortezomib treatment.</p