Bromodeoxyuridine (BrdU) cell proliferation tracking in piglet forebrain SVZ.

<p>Confocal microscope images showing that subsets of BrdU⁺ cells in the forebrain SVZ can be immunophenotyped (arrows) as cells positive for the neuroblast marker doublecortin (A-C, Dcx), the astrocyte marker GFAP (D-F), the oligodendrocyte marker Olig2 (G-I), and the neuron marker NeuN (J). The lateral ventricle is identified (v). Scale bars: (in I, applies to A-I) = 12 µm; (in J) = 23 µm. <b>K</b>. Graph showing the proportions of the total BrdU⁺ cells that are either GFAP⁺ Olig2⁺, Dcx⁺, or NeuN⁺. Values are mean ± SD (n=4). Asterisk denotes Dcx significant difference (p < 0.05) compared to GFAP, Olig2, and NeuN.</p

Bromodeoxyuridine (BrdU) cell proliferation tracking in piglet OB SVZ.

<p><b>A</b> and <b>B</b>. Confocal microscope images showing that the OB contains numerous BrdU⁺ cells (A, green) and subsets of these cells are newly born neurons as shown by the colocalization (B, seen as yellow, arrows) with the neuron marker NeuN. <b>C</b>. Confocal microscope images showing the co-labeling (arrows) of the DNA synthesis marker BrdU (red) and glial fibrillary acidic protein (GFAP, green) in the OB SVZ. Some BrdU⁺ nuclei are associated with GFAP⁺ cytoplasm. Scale bars = 33 µm (A), 17 µm (B), 20 µm (C). <b>D</b>. Graph showing the proportions of the total BrdU⁺ cells that are either GFAP⁺ or NeuN⁺. Values are mean ± SD (n=4). Some BrdU⁺ cells were not positive for either marker. Asterisk denotes NeuN significant difference (p < 0.001) compared to GFAP.</p

Localization of NSC and NPC markers in piglet forebrain SVZ and white matter.

<p><b>A</b> and <b>B</b>. Immunohistochemical localization of nestin (A) and musashi (B) shows enrichment of these two NSC markers in piglet forebrain SVZ. Immunoreactivity is seen as orange-brown staining. Arrows identify the SVZ. The lateral ventricle is seen at right. The opaque layer at the interface of the SVZ and the ventricular cavity is the ciliated ependymal layer. Scale bar = 33 µm. <b>C</b> and <b>D</b>. Immunohistochemical localization of nestin (C) and Dlx2 (D), a neuroprogenitor marker, shows enrichment in individual cells (arrows) in piglet subcortical white matter. Scale bar in C (same for D-F) = 17 µm. <b>E</b> and <b>F</b>. Nestin primary antibody (E) was preadsorbed against nestin synthetic peptide, and Dlx2 primary antibody (F) was preadsorbed against recombinant Dlx2. No staining is seen in either preparation. <b>G</b>. Graph showing the number of nestin⁺ and Dlx2⁺ cell bodies in the subcortical white matter of the parasagittal gyrus in primary somatosensory cortex in piglets at postnatal day (PND) 5 and 30. Values are mean ± SD (n= 3-4 piglets/group). Asterisk denotes significant difference (p < 0.05) compared to PND5. </p

Newborn piglet OB-SVZ cells form multipotent neurospheres.

<p><b>A</b>. Floating neurosphere formed by notch-1-immunopanned NSCs isolated from the OB. Scale = 12 µm. <b>B</b>-<b>D</b>. Attached neurospheres dispersed as monolayers are multipotent by differentiating into cells that are neurons identified by MAP2, astrocytes identified by GFAP, and oligodendrocytes identified by O4. <b>E</b>. Neurosphere density increased when medium was supplemented with FGF2. Scale bars = 12.5 (B), 20 (C), 20 (D), 50 (E) µm.</p

Characterization of the lateral migratory stream (LMS) of newly born neurons and identification of immature neurons in piglet forebrain.

<p><b>A</b>. Confocal image showing the colocalization of BrdU (red) and NeuN (green) in subsets of neurons in the piriform cortex. Single-labeled neurons are green, single-labeled BrdU⁺ nuclei are red, and double-labeled neurons are yellow. <b>B</b>. Immunofluorescence showing the colocalization of BrdU (red) and doublecortin (Dcx, green) in the LMS. One Brdu⁺ cell (top cell) does not express Dcx, but another Brdu⁺ cell (bottom cell) is Dcx⁺ as seen by the yellow around the nucleus and the trailing Dcx-labeled process (arrow). <b>C</b>. Immunofluorescence showing the colocalization of BrdU (red) and β-tubulin III (βTubIII, green) in the LMS. <b>D</b>. Immmunoperoxidase staining for β-tubulin III (βTubIII) revealed a column of βTubIII⁺ immature neurons (brown cells) below the anterior striatum within the LMS. Inset shows a βTubIII⁺ LMS neuron with a fusiform morphology and leading and trailing processes. <b>E</b>. In piriform cortex, βTubIII⁺ immature neurons were found in cellular islands in layer II (arrow). Boxed area is shown at right at higher magnification and rotated 90°. <b>F</b>. In OB, βTubIII⁺ immature neurons (arrows) were found in the granule cell layer (GCL). These cells possessed extensive local vertical-oriented arborizations (inset) consistent with an interneuron morphology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081105#B46" target="_blank">46</a>]. EPL, external plexiform layer; MCL, mitral cell layer. Scale bars = 25 µm (A), 5 µm (B), 4 µm (C), 25 µm (D), 8 µm (D inset), 32 µm (E), 12.5 µm (E right), 100 µm (F), 25 µm (F inset).</p

In vivo BrdU labeling in piglet forebrain reveals a new corridor of newly born cells named the lateral migratory stream.

<p><b>A</b>-<b>H</b>. Images of a piglet forebrain section are shown with BrdU incorporation detected by the immunoperoxidase method (brown labeling) and counterstaining with cresyl violet. A low magnification image of piglet ventral forebrain is shown for perspective (A). Regions delineated by black rectangles in A are shown as higher magnification montage images in B-F that illustrate the distributions of BrdU labeled cells (hatched arrows, brown dots). Rostral migratory stream (RMS), and lateral ventricle (lv).<b>G and H</b>. Higher magnification images showing BrdU⁺ cell labeling (hatched arrows, brown nuclei) in the lateral migratory stream (G) beneath the striatum and in the piriform cortex (H). Unlabeled nuclei are pale violet. Scale bars: A = 300 µm, B (same for C-F) = 100 µm, G (same for H) = 25 µm.</p

Iron chelation decreases BDNF expression in human neuroblastoma cells.

<p><b>A.</b> SH-SY5Y cells were treated with either vehicle or 100 µM deferoxamine (DFO) for 24 hours. RT-PCR measures of BDNF transcripts show a decrease in total BDNF transcripts (tBDNF, variants 1–14, 16–18 (*p<0.01, n = 4–5). <b>B.</b> Iron chelation did not cause changes in cell survival as assessed by trypan blue exclusion 24 hours post-treatment (n = 5). <b>C.</b> SH-SY5Y cells were transfected with luciferase reporters for the promoter regions of BDNF I–IV and were then treated with 100 µM deferoxamine for 24 hours. No significant differences were observed between control and treated cells (n = 3–4).</p

The expression of BDNF is reduced in the cerebral cortex and striatum of ceruloplasmin-deficient mice.

<p><b>A.</b> RT-PCR measures of the 4 BDNF transcripts show a decrease in BDNF transcripts 1, 2 and 4 in the cortex of the CpKO mouse (*p<0.04, n = 5). <b>B.</b> RT-PCR measures of the 4 BDNF transcripts show a decrease in BDNF transcripts 1, 2, 3 and 4 in the striatum of the CpKO mouse (*p<0.05, n = 5). <b>C.</b> Examples of immunoblots of BDNF protein levels in the cortex and striatum (top panel) and densitometry results showing significant decreases BDNF protein levels (normalized to actin level) using Image J software. (*p<0.03; n = 4 WT and 3 CpKO mice).</p

CpKO mice have increased cerebral infarct volume after focal ischemic stroke.

<p><b>A.</b> Representative images of TTC-stained brain sections in mice subjected to 45 min MCAO with a 72 hours post-stroke survival. <b>B.</b> Calculation of percent infarct volume show a significant increase in the cortex and hemisphere of the CpKO mice compared to WT mice. (*p<0.002; n = 8 WT and 6 KO mice).</p

CpKO mice have decreased concentrations of iron in the cerebral cortex and striatum.

<p>Atomic absorption measures of cortical and striatal biopsy punches show significant decreases in iron concentrations in CpKO compared to wild type mice in both the cerebral cortex ( n = 18; *p<0.02) and the striatum (n = 7; *p<0.03).</p