Glial Fibrillary Acidic Protein

This study was carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the National Institutes of
Health. All procedures used were approved by the University of Texas
Southwestern Medical Center Institutional Animal Care and Use Committee APN
2007-0065. All efforts were made to minimize suffering.
Ascl1^CreERT2 knock-in mice were generated by
replacing the Ascl1 coding region with
CreER^T2[8] (link) and
Frt-Neo-Frt cassettes. The targeting strategy was the same used to generate
Ascl1^GFP knock-in mice [9] (link). The endogenous ATG was
replaced by a short sequence containing a PacI site and a consensus Kozak site.
The correct targeting event was identified by Southern analysis of EcoRI
digested DNA using 5′ and 3′ probes. After obtaining germ line
transmission in the Ascl1^{CreERT2-Frt-Neo-Frt} mice,
they were crossed with FLPe mice [10] (link) to remove the neomycin
cassette resulting in Ascl1^CreERT2 mice.
For PCR genotyping, the following primers were used: 5′-AAC TTT CCT CCG GGG CTC GTT
TC-3′ (Sense Ascl1 5′UTR) and 5′-CGC CTG GCG ATC CCT GAA CAT
G-3′ (Anti sense Cre) giving a PCR product of 247 bp.
Tamoxifen (TAM) induction of Cre recombinase was accomplished by intraperitoneal
injection of
Ascl1^CreERT2/+;R26R^YFP/YFPpostnatal day 50 (P50) mice with 180 mg/kg/day TAM (Sigma, T55648) in sunflower
oil on five consecutive days. Brains were harvested at the times specified after
TAM and processed as described [7] (link), [11] (link). R26R^YFP and
Nestin::GFP mice have been previously described [12] (link), [13] (link).
For immunofluorescence staining, free floating sections or sections mounted on
slides were incubated in the appropriate dilution of primary antibody in
PBS/3% donkey (or goat) serum/0.2% NP-40 (or 0.2% Triton
X-100), followed by appropriate secondary antibody conjugated with AlexaFluor
488, 568, or 594 (Molecular Probes). Mouse monoclonal antibodies used were:
Ascl1 (1∶750, RDI Fitzgerald, 10R-M106B), NeuN (1∶1000, Chemicon,
MAB377), GFAP (1∶400, Sigma, G3893). Rabbit polyclonal antibodies used
were: GFP (1∶500, Molecular Probes, A6455), GFAP (1∶500, DAKO,
Z0334), Ki67 (1∶500, Neomarker), Sox2 (1∶2000, Millipore). Goat
polyclonal antibodies used were: DCX (1∶200, Santa Cruz) and NeuroD1
(1∶200, Santa Cruz). Chick GFP (1∶500, Aves Lab) was also used.
Confocal imaging was carried out on a Zeiss LSM510 confocal microscope.
Ascl1⁺ fluorescence intensity levels were classified as high
or low using ImageJ and setting a threshold of pixel intensity for
Ascl1^Low (314–599 units) and Ascl1^High (>600
units). For cell number counts, three Nestin::GFP mice were
analyzed to place Ascl1⁺ progenitors in the adult neural stem
cell lineage. For in vivo genetic tracing experiments using the
Ascl1^CreERT2 knock-in line, at least two
Ascl1^CreERT2/+;R26R^YFP/YFPmice per each harvest time point (7, 30, or 180 days post-TAM) were used. For
co-localization data with each stage-specific marker, 150–500
YFP⁺ cells per animal were counted.

Authorizations for reporting these three cases were granted by the Eastern Ontario Regional Forensic Unit and the Laboratoire de Sciences Judiciaires et de Médecine Légale du Québec.
The sampling followed a relatively standardized protocol for all TBI cases: samples were collected from the cortex and underlying white matter of the pre-frontal gyrus, superior and middle frontal gyri, temporal pole, parietal and occipital lobes, deep frontal white matter, hippocampus, anterior and posterior corpus callosum with the cingula, lenticular nucleus, thalamus with the posterior limb of the internal capsule, midbrain, pons, medulla, cerebellar cortex and dentate nucleus. In some cases, gross pathology (e.g. contusions) mandated further sampling along with the dura and spinal cord if available. The number of available sections for these three cases was 26 for case1, and 24 for cases 2 and 3.
For the detection of ballooned neurons, all HE or HPS sections, including contusions, were screened at 200×.
Representative sections were stained with either hematoxylin–eosin (HE) or hematoxylin-phloxin-saffron (HPS). The following histochemical stains were used: iron, Luxol-periodic acid Schiff (Luxol-PAS) and Bielschowsky. The following antibodies were used for immunohistochemistry: glial fibrillary acidic protein (GFAP) (Leica, PA0026,ready to use), CD-68 (Leica, PA0073, ready to use), neurofilament 200 (NF200) (Leica, PA371, ready to use), beta-amyloid precursor-protein (β-APP) (Chemicon/Millipore, MAB348, 1/5000), αB-crystallin (EMD Millipore, MABN2552 1/1000), ubiquitin (Vector, 1/400), β-amyloid (Dako/Agilent, 1/100), tau protein (Thermo/Fisher, MN1020 1/2500), synaptophysin (Dako/Agilent, ready to use), TAR DNA binding protein 43 (TDP-43) ((Protein Tech, 10,782-2AP, 1/50), fused in sarcoma binding protein (FUS) (Protein tech, 60,160–1-1 g, 1/100), and p62 (BD Transduc, 1/25). In our index cases, the following were used for the evaluation of TAI: β-APP, GFAP, CD68 and NF200; for the neurodegenerative changes: αB-crystallin, NF200, ubiquitin, tau protein, synaptophysin, TDP-43, FUS were used.
For the characterization of the ballooned neurons only, two cases of fronto-temporal lobar degeneration, FTLD-Tau, were used as controls. One was a female aged 72 who presented with speech difficulties followed by neurocognitive decline and eye movement abnormalities raising the possibility of Richardson’s disorder. The other was a male aged 67 who presented with a primary non-fluent aphasia progressing to fronto-temporal demαentia. In both cases, the morphological findings were characteristic of a corticobasal degeneration.

Immunofluorescence staining was performed as previously described with modifications [45 (link), 46 (link)]. Mice were euthanized by isoflurane overdose at each time point. The brains were sectioned at 20 µM of thickness, fixed with 4% paraformaldehyde (Thermo Fisher) for 15 min, then permeabilized with 0.1% Triton X-100 for 10 min. After washing with the phosphate-buffered saline (PBS) for 15 min, the sections were blocked for 1 h and incubated overnight with primary antibodies for ZO-1 (1:100, Thermo Fisher) claudin-5 (1: 100, Thermo Fisher) and GFAP (1:100, Cell Signaling), respectively. Alexa fluorescent secondary antibodies (Thermo Fisher) were used at 1:400 dilutions for 1 h. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) for nucleus and washing with PBS, the sections were mounted with Permount (Thermo Fisher). The whole sections were scanned with a Leica Stellaris SP8 Falcon microscope (Leica Microsystem) and the images (20X magnitude) were captured with the same microscope. Mean total fluorescence intensity was calculated for each color channel and intensity of green color (ZO-1/GFAP) and red color (claudin-5) was expressed relative to blue color (DAPI). Cortex and hippocampus of both hemispheres of each brain section were used to evaluate the expression levels of ZO-1, claudin-5 and GFAP. To minimize the subjective bias, all images for ZO-1, claudin-5 and GFAP expression analysis were captured under the same microscopic parameter (laser power, pinhole size, exposure time) setting.

Paraffin-embedded tissue was sectioned with a thickness of 6 μm. One slide per animal was used for staining, each containing 5 equally spaced sections. Cresyl violet staining was performed to visualize neurons^{22 (link)}. Antibody staining was performed as previously described^{114 (link)}, except that all primary antibodies were incubated overnight at RT, followed by another overnight at 4 °C. For Aβ staining, the Mouse On Mouse detection kit (Vector labs) was used according to manufacturer’s instructions. The following primary antibodies were used: mouse anti-human Aβ (1:150; Covance); chicken anti-GFAP (1:150; Abcam). Cy2/Cy3-conjugated anti-mouse/chicken secondary antibodies (1:150; Jackson Immunoresearch) were used. For counterstaining, 4’,6-diamidino-2-phenylindole (1:5000; Biolegend) was used.