Journal: The Journal of Neuroscience

Article Title: Tumor Necrosis Factor α But Not Interleukin 1β Mediates Neuroprotection in Response to Acute Nitric Oxide Excitotoxicity

doi: 10.1523/JNEUROSCI.4032-05.2006

Figure Lengend Snippet: Three-dimensional representation of the volume of neuronal cell death, as measured by FJB-positive staining in wild-type (WT), IL-1β, TNF-α, and IL-1β/TNF-α double (Dbl) KO mice killed 4 d after an intraparenchymal injection of the excitotoxin SNP. A, These 3D reconstructions of FJB-positive regions were performed using a C-80 Nikon microscope and super-high-pressure mercury lamp (Nikon) fitted with a Retiga EXi Fast digital camera (QImaging) feeding to a Precision 660 workstation (Dell Computer). The FJB-positive regions were traced on a Wacom pen tablet using the Neurolucida stereological software package (version 6.02.1; MicroBrightField), and 3D reconstruction and volume determination were compiled using Neuroexplorer software (version 4.01.1; MicroBrightField). Please note the larger volumes of FJB in the TNF and Dbl KO animals compared with that of wild-type and IL-1 KO mice. B, Quantitative analysis of the FJB volume in the brains of wild-type, IL-1, TNF, and Dbl KO mice over time. Please note the rapid appearance of neuronal cell death 6 h after SNP injection and the marked spread of FJB staining (**p < 0.01 compared with wild type and IL-1 KO; data presented as mean ± SEM) in the CNS of TNF and Dbl KO 4 d after the single SNP infusion. For photomicrographs of the FJB staining, refer to Figures ​Figures11 and ​and88.

Article Snippet: The FJB-positive regions were traced on a Wacom (Vancouver, WA) pen tablet using the Neurolucida stereological software package (version 6.02.1; MicroBrightField, Williston, VT), and three-dimensional (3D) reconstruction and volume determination were compiled using Neuroexplorer software (version 4.01.1; MicroBrightField).

Techniques: Staining, Injection, Microscopy, Software

Journal: Nature Communications

Article Title: An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling

doi: 10.1038/s41467-017-00583-8

Figure Lengend Snippet: MMP-9 mediates fibrotic ECM remodeling. a Gelatinase activity of MMP-9 and MMP-2 in the PBS ( N = 5) and the I-5 group ( N = 4) examined by zymography. b , c Graphs showing quantification of MMP-2 ( b ) and MMP-9 ( c ) activity. ** indicates p < 0.01 by two-tailed Student’s t -test. Error bars represent the SEM. d , e Representative images of transverse spinal cord sections from animals injected with either PBS ( d ) or I-5 ( e ). Asterisks indicate cystic cavities. A yellow dotted line indicates the boundary of the FN-rich matrix. The boxed regions in the middle panels were magnified in dʹ, eʹ. Scale bars represent 100 µm. f MMP-9 immunoreactive granules were bounded by CD11b positive membrane-like circular structures. Scale bars represent 50 µm. g , j Representative images of transverse spinal cord sections stained with eriochrome cyanine and eosin from animals with I-5 mixed with non-targeting control siRNA (NC) ( g ) or MMP-9 siRNA ( j ). Adjacent sections were stained with antibodies against FN ( h , k ). Scale bars represent 200 μm. i , l 3D reconstruction of the spinal cord tissue using the Neurolucida software. Scale bars represent 1 mm. Asterisks indicate cystic cavities. m Quantification graph of cavity volume. ** indicates p < 0.01 by two-tailed Student’s t -test. N = 5 for each group. Error bars represent the SEM

Article Snippet: Three-dimensional reconstruction of the lesion cavity was done using the Neurolucida tracing software equipped with the 3D Slide Scanning Module (MBF bioscience).

Techniques: Activity Assay, Zymography, Two Tailed Test, Injection, Membrane, Staining, Control, Software

Journal: Nature Communications

Article Title: An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling

doi: 10.1038/s41467-017-00583-8

Figure Lengend Snippet: Interaction between macrophages and I-5 hydrogel. a Representative images of Nile Red fluorescence in a macrophage cell line. Cultured macrophages were treated with Nile Red (NR) alone or nanoparticles consisting of NR and either CP-2 or I-5. In addition, JNJ7777120, a histamine receptor 4 inhibitor, or mepyramine maleate, a histamine receptor 1 inhibitor, was added to the culture medium 30 min before treatment with I-5 polymer nanoparticles. Scale bar represents 50 µm. b Graph showing quantification of NR fluorescence intensity. *** indicates p < 0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. N = 4 replicate experiments per group. Error bars represent the SEM. c , d Representative images of transverse spinal cord sections from animals injected with CP-2 hydrogel lacking the imidazole group. c Eriochrome cyanine and eosin staining revealed prominent cystic cavities (*) at the center of the lesion ( c ). d FN staining showed a smaller area of FN-rich matrix. Scale bars represent 200 μm. e 3D reconstruction of the spinal cord tissue from an animal injected with CP-2 hydrogel using the Neurolucida software. Scale bar represents 1 mm. f Graph showing the quantification of the cavity volumes. The data set for the I-5 group was the same as the one used in Fig. . ** indicates p < 0.01 by two-tailed Student’s t -test. N = 8 for the I-5 group and N = 5 for the CP-2 group. Error bars represent the SEM

Article Snippet: Three-dimensional reconstruction of the lesion cavity was done using the Neurolucida tracing software equipped with the 3D Slide Scanning Module (MBF bioscience).

Techniques: Fluorescence, Cell Culture, Polymer, Injection, Staining, Software, Two Tailed Test

Journal: Nature

Article Title: Required growth facilitators propel axon regeneration across complete spinal cord injury

doi: 10.1038/s41586-018-0467-6

Figure Lengend Snippet: a, Schematics show demarcation of SCI lesion center (Cn) and evenly spaced lines beyond the Cn placed by image analysis software (Neurolucida®, Microbrightfield) for quantification of axon intercepts in horizontal tissue sections of mice with SCI and one (D 1 ) or two (D 1 +D 2 ) hydrogel depots. b, Multi-fluorescent, survey images show BDA-labeled axons and GFAP-labelled astrocytes that demarcate astrocyte scar proximal borders (PB) and distal borders (DB) around the non-neural lesion core (LC) after SCI. The hydrogel of the empty depot (left) was tagged with a blue fluorescent label for visualization. Note the essential absence of axons passing the astrocyte scar (AS) proximal border (PB) to reach the lesion center (Cn) or beyond in the mouse with SCI plus empty depot (left), in striking contrast with the large number of axons that regrew through the lesion core (LC) and passed beyond the distal astrocyte scar border (DB) into spared grey matter (gm) in the mouse with full treatment of stimulatory AAV plus growth factors (right). GFAP staining shows that the SCI lesions are anatomically complete across the entire width of the spinal cord in both cases. Note that the second depot was placed at 9 days after SCI, by which time the distal astrocyte scar border was essentially formed . Note also that astrocytes do not migrate into the depots, potentially giving the mistaken impression of cavity formation when looking only at the GFAP channel alone. Nevertheless, examination of other fluorescence channels shows that depot sites clearly contain DAPI-stained stromal cells and BDA-positive axons. c, Large area survey images of BDA-labeled axons in composite mosaic scans of horizontal sections. In a control mouse (top) that received SCI plus empty depot, few axons reach the lesion center, almost none pass beyond, and no axons are present at 3mm. In the a treated mouse (middle) that received stimulatory AAV plus growth factors, many axons regrow through the lesion core and reach or pass 1.5mm beyond the lesion center, which is the equivalent length of a full thoracic spinal segment in mice . Note also that there are no axons present at 3mm, demonstrating that the SCI lesion was complete and that axons that are found past the lesion center represent axon regrowth after SCI in response to the experimental manipulations. In an uninjured mice (bottom), there are many labeled axons at the distance equivalent to 3mm beyond the location of SCI in injured mice. d, Graph shows mean±SEM numbers of axon intercepts at lesion centers for all experimental groups (dots in graphs show numbers and distribution of individual mice per group). (ns not significant versus SCI-only, # P <0.01 versus SCI-only and ns versus each other, ** P <0.01, *** P <0.001 versus all other groups, one-way ANOVA/Bonferroni, F (12, 57) = 22.3).

Article Snippet: Cell counts were performed using stereological image analysis software (StereoInvestigator®, 9.14.5 32 Bit, and NeuroLucida®, 9.14.5 32 Bit, MicroBrightField, Williston, VT) operating a computer-driven microscope regulated in the x, y and z axes (Zeiss).

Techniques: Software, Labeling, Staining, Fluorescence, Control