Animals and hypoxic treatment
Colonies of CNP-EGFP (generated by Dr. V. Gallo, Children’s National Medical Center, Washington, DC), Cdk2 −/−
null (generated by Dr. P. Kaldis, Institute of Molecular and Cell Biology, Singapore), 129-Cdkn1b tm1Mlf
later named p27 −/−
, and C57BL/6 (catalog #003122, #003548; The Jackson Laboratory) and wild-type CD1 [Crl: CD1(ICR); Charles River] mice were maintained at Children’s National Medical Center animal facility following guidelines of the Institutional Animal Care and Use Committee (Children’s National Medical Center) and the National Institutes of Health. For breeding, heterozygote CNP-EGFP +
males were backcrossed to C57BL/6 females for more than eight generations. In CNP-EGFP mice, various stages of the oligodendrocyte lineage have been visualized based on EGFP expression driven by myelin-specific 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) gene promoter (Belachew et al., 2003
). p27 −/−
transgenic mice were previously described (Fero et al., 1996
). Mice deficient in p27 Kip1
are viable, larger than normal littermates, with better-developed organs (particularly thymus and spleen), suggesting that p27 Kip1
is required in many cell lineages for normal exit from the cell cycle (Fero et al., 1996
; Belachew et al., 2003
). Cdk2 −/−
transgenic mice were previously described (Berthet et al., 2003
). Although larger than their litter-mates, the only morphological or structural differences in their organs compared with wild-type mice are sterile gonads.
CNP-EGFP, p27 −/−, and Cdk2 −/− pups [3 d of age; postnatal day 3 (P3)] were exposed to 9.5–10.5% oxygen concentration in a hypoxic chamber. Oxygen concentration was maintained and monitored continuously with the sensor inside the chamber. To maintain hypoxic conditions, nitrogen was added to displace oxygen. To optimize nutrition during hypoxia, transgenic pups were housed in the chamber with two CD1 foster mothers and their pups. At P11, mice were removed from the chamber and transferred to a room with normoxic air conditions; however, they remained with CD1 foster mothers to minimize stress. Because of the low survival rate of p27 −/− mice, we modified hypoxic parameters specifically for this mouse strain to maintain oxygen concentration at 12.5%. For all three strains, exposure to hypoxia lasted 8 consecutive days (P3–P11). After 11-d-old pups were removed from the chamber, the mice proceeded to the various experimental conditions at the specified time points. Time course analysis for immunohistochemistry was performed at P11 (immediately after mice were removed from the hypoxic chamber), at P18 (at 1 week recovery in normoxic conditions), and at P45 (34 d after hypoxia).
Genotyping Cdk2−/− and p27−/− mice
To breed Cdk2 −/−
and p27 −/−
mice, we crossed two heterozygotes. Cdk2 −/−
newborn pups were genotyped following the procedure established by Berthet et al. (2003)
. Primers used in PCR were as follows: forward, 5′-CCC GTG ATA TTG CTG AAG AGC TTG GCG-3′; reverse, 5′-GGT TTT GCT GCA TGT GGG CAT GG-3′; neo, 5′-GTG ACC CTG TGG TAC CGA GCA CCT G-3′. DNA products were loaded onto 2% agarose gel to resolve the specific bands: 150 bp for wild-type mice, 500 bp for Cdk2 −/−
, and both bands for heterozygotes.
p27 −/− mice were genotyped following the protocol of The Jackson Laboratory. Primers used in PCR were as follows: oIMR0947, 5′-CTC CTG CCA TTC GTA TCT GC-3′; oIMR0948, 5′-CTC CTG CCA TTC GTA TCT GC-3′; oIMR6916, 5′-CTT GGG TGG AGA GGC TAT TC-3′; oIMR6917, 5′-AGG TGA GAT GAC AGG AGA TC-3′. DNA products were loaded onto 2% agarose gel to resolve the specific bands: 190 bp for wild-type mice, 280 bp for mutant, and both bands for heterozygotes. For both strains, only mutant and wild-type mice were analyzed.
Immunocytochemical analysis was performed at three time points: P11, P18, and P45. Time course analysis allows identification of specific stages of oligodendrocyte development vulnerable to hypoxic damage. In each experiment, normoxic mice served as controls. Hypoxic and normoxic mice were anesthetized with isoflurane and transcardially perfused with 0.1 M PBS, pH 7.4, followed by 4% paraformaldehyde. Brains were postfixed overnight in 4% paraformaldehyde. Serial coronal and sagittal sections (50 μm) were cut using a cryostat microtome, collected in PBS, pH 7.4, and stored at 4°C until use.
Immunocytochemistry was performed on floating sections using antibodies against the following antigens: NG2, Doublecortin, GFAP, Olig2 (Millipore Bioscience Research Reagents), Ki67 (Novocastra), c-Caspase3 (Cell Signaling), CC1 (Calbiochem), S100β
, BrdU (Sigma-Aldrich), myelin basic protein (MBP) (Covance), Mash1, PDGFR (both from BD Biosciences), and Iba1 (Wako). All antibody dilutions were as previously described (Aguirre and Gallo, 2004
). Sections were incubated overnight at 4°C in primary antibodies diluted in 0.1 M
PBS, pH 7.4, containing 0.1% Triton and 5% normal goat serum. Appropriate secondary antibodies were used as follows: TRITC-conjugated AffiniPure goat anti-mouse IgG (H+L), FITC-conjugated AffinitiPure goat anti-rabbit IgG, and TRITC-conjugated AffiniPure goat anti-mouse IgM (Jackson ImmunoResearch). Sections were incubated with secondary antibodies for 1 h at room temperature and mounted.
Quantitative cellular and biochemical analysis
We used a confocal LSM (Zeiss 510) optical (magnification, 40×; step size, 1 μm) of 20- to 30-μm-thick immunostained tissue sections with a volume of 225 × 225 × 10 μm (x, y, z). The stacks were then z-axis collapsed, allowing us to analyze all elements of the cellular morphology and localize fluorescent labels to specific compartments. Four different lasers were used to image localization of FITC (488 nm excitation), CY3 (580 nm excitation), CY5 (647 nm excitation), and DAPI (400 nm excitation).
Analysis of the white matter was performed in three different areas: corpus callosum, external capsule, and cingulum. The analysis was limited within the boundaries of the white matter, as detected by DAPI distribution, to account for changes in white matter volume after hypoxia. Consistent imaging of all tissue sections and high number of sections (15–25 per group per antigen) used for quantification contributed to minimize bias. Total and relative numbers of cells expressing different antigens were estimated by scoring the number of cells double-labeled with the markers. Data were usually obtained from at least five to eight tissue sections from three to four mice per group. For CNP-EGFP mice, results are presented as mean ± SEM, and t tests were performed to establish statistical significance. In Cdk2 −/−and p27 −/− mice, statistical analysis used two-way ANOVA to determine significant differences in oligodendrocyte proliferation and differentiation.
Western blot analysis and immunoprecipitation
White matter and SVZ areas were precisely dissected from 300-μm-thick, coronal sections from hypoxic and normoxic CNP-EGFP and p27 −/− mice at P11, P18, and P45. Tissues were homogenized in RIPA lysis buffer with proteinase inhibitors (Santa Cruz Biotechnology). Protein extracts were boiled for 5 min before loading onto 4–20% gradient gels (GeneMate; 20 μg of protein per lane). Gels were electrotransferred to a 0.2 μm nitrocellulose membrane (Millipore). Blots were blocked in 5% milk in TBST for 1 h, then incubated at 4°C overnight with one of the following antibodies: anti-Cdk2, -Cdk4, -cyclin E, -p27 KIP1, -MBP, -MAG (Santa Cruz Biotechnology), -Rb, -pRb(Ser780), -pRb(Ser795), -pRb(Ser807/811), -E2F1, -p107, -E2F4, -FoxO1, -FoxO3a, -FoxO4, -Skp2 (Cell Signaling), -myelin oligodendrocyte glycoprotein (MOG), -proteolipid protein (PLP) (Abcam), -Neurofilament 200 (NF200) (Sigma-Aldrich), Neurofilament H Nonphosphorylated (SMI32; Covance), and -actin (Millipore Bioscience Research Reagents; MAB). Bands were detected with appropriate horseradish peroxide-conjugated secondary antibodies, reacted with chemiluminescent ECL substrate (GE Healthcare), and visualized by x-ray exposure. Band intensity was measured using the ImageJ program (NIH). Western blots were obtained from white matter and SVZ from three to four animals in each group and age. Data were averaged and represented as means ± SEM.
For immunoprecipitation, white matter and SVZ tissue extracts from hypoxic and normoxic CNP-EGFP mice were prepared in RIPA buffer containing 2% Triton X-100 and 0.2% SDS. Aliquots (270 μg of tissue) were incubated overnight with antibodies against E2F1 (Cell Signaling) for white matter and E2F4 (Santa Cruz Biotechnology) for SVZ, together with 15 μl of agarose A (Santa Cruz Biotechnology). Immunocomplexes bound to agarose A were collected by centrifugation and washed twice in 500 μl of RIPA buffer containing inhibitors. Precipitated proteins were analyzed by immunoblotting with an anti-Rb Abs (Cell Signaling) for white matter and anti-p107 antibody (Sigma-Aldrich) for SVZ. Bands were detected using HRP-labeled polyclonal anti-mouse Ig (BD Biosciences) and developed with a chemiluminescent substrate (ECL; GE Healthcare).
Cell culture preparation and analysis
White matter areas were dissected from 300-μm-thick coronal sections prepared from hypoxic and normoxic mice at P18 and digested for 30 min at 37°C in HBSS (Invitrogen) containing papain (13 U/ml; Sigma-Aldrich), DNase (5 U/ml; Sigma-Aldrich), and trypsin (Sigma-Aldrich). White matter cells were dissociated by trituration and resuspended in Hanks buffer containing 1 M HEPES (BioSource), 15% sucrose, and penicillin/streptavidin. For differentiation, cells were plated onto laminin-coated dishes (Invitrogen) with a density of 650 cells/μl. Equal numbers of cells from hypoxic and normoxic CNP-EGFP mice were used in all experiments. For cell differentiation assays, cells from hypoxic and normoxic white matter were cultured for 5 d and incubated with growth factors [10 μg/ml PDGF, 10 μg/ml T3 (Millipore)]. To establish the cellular composition of the cultures, we cultured white matter cells from normoxic and hypoxic CNP-EGFP mice for 24 h, and labeled them with various cell-specific markers, including MAP2 for neurons, NG2 for oligodendrocyte progenitors, GalC for mature oligodendrocytes, GFAP for astrocytes, and Iba1 for microglia. We found that neurons, astrocytes, and microglia were not affected by hypoxia and together represented a constant fraction of the total cultured cells (22.7% for normoxia and 20.9% for hypoxia). However, the percentage of mature oligodendrocytes substantially decreased after hypoxia (from 36.6 to 14.4%), while the percentage of NG2 progenitor cells increased from 40.7 to 64.7%.
To assess the proliferative potential of cells, BrdU was added to the culture medium at 10 μ
g/ml, followed by 60 min incubation at 37°C. Cells were then fixed in 4% paraformaldehyde and kept in PBS until use. BrdU incorporation was visualized by immunofluorescence using anti-mouse BrdU antibody and TRITC-conjugated AffiniPure goat anti-mouse IgG (H+L). To study whether proliferating cells belonged to the oligodendrocytic lineage, we costained NG2 +
, Olig2 +
, O4 +
, GalC +
, and GFAP +
cells with anti-BrdU or anti-Ki67 antibodies. Standard protocols were used to immunolabel differentiated cells (Aguirre and Gallo, 2004
) with primary antibodies against O1 (Millipore) and O4 (R&D). For quantification, the percentages of positive cells were counted in random fields captured at 10× magnification (>250 cells per condition and per stain) from at least three different samples and subjected to statistical analysis. We found that, in normoxia, mainly NG2 +
(69.0%) and Olig2 +
(74%) cells were proliferating in the cultures. We also found only a small fraction of GalC +
(3%), O4 +
(8%), GFAP +
(9%), and Iba1 +
(8%) cells. In hypoxia, still mainly NG2 +
(85%) and Olig2 +
(54%) cells were proliferating, whereas only a small fraction of GalC +
(2%), O4 +
(3%), GFAP +
(8%), and Iba1 +
(8%) cells were detected.
Retroviral injection of p27Kip1 into white matter
Dividing cells in white matter were directly labeled with CMV-GFP retrovirus in hypoxic and normoxic brains at P18. The virus plasmid pNIT contains a cDNA fragment of EGFP downstream of the tetracycline operon enhancer-promoter. For in vivo experiments, wild-type mice (P18) were stereotaxically injected in the white matter with p27 Kip1 EGFP retrovirus stock (2 μl; titer, 1–2 × 10 6 cfu ml −1). For a control, mock virus was injected into white matter. The following coordinates were used: 1 mm anteroposterior, 1 mm mediolateral, and 1.5 mm dorsoventral. Brains were processed for immunohistochemical staining using anti-CC1 (Calbiochem) and S100β antibodies (Sigma-Aldrich).
For ultrastructural investigation of myelinated axons in hypoxic and normoxic white matter, brains from P18 and P45 mice were perfused with 4% paraformaldehyde containing 15% picric acid and 0.2% glutaraldehyde. After washing in 0.1 M PBS, brains were sectioned at 200 μm on the vibratome. Sections were osmicated 60 min in 1% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M phosphate buffer, washed several times in distilled water, contrast enhanced for 30 min with 1% uranyl acetate (Electron Microscopy Sciences), and dehydrated in ascending concentrations of acetone. Finally, sections were soaked 60 min in a mixture of Araldite 502 (5.4 g), DDSA (4.6 g), DMP-30 (0.2 g) (all from Electron Microscopy Sciences), and flat-embedded between glass slides and coverslips at 65°C for 48 h. Prepared sections were cut into semithick sections (90 μm), stained with toluidine blue for anatomical identification of white matter, and resectioned into 70 nm ultrathin sections. These sections were examined with a JEOL transmission electron microscope (JEM-1400), and pictures were taken with a Gatan SC1000 ORIUS CCD camera. Electron-microscopic images were prepared using Adobe Photoshop CS2.
siRNA-induced FoxO1 and Skp2 knockdown in white matter cells
Cell transfection was performed using the NeuroPORTER Transfection Reagent (Genlantis, T400750) according to the manufacturer’s instructions. After white matter dissection, cells were plated in 12-well cell culture dishes at a density of 50 cells/μl for 24 h. At the time of transfection, cell cultures were ~60% confluent. Commercially available siRNAs directed toward FoxO1 and Skp2 were purchased from Dharmacon. A mixture of siRNAs (20 pM each) produced specific knockdown of FoxO1 and Skp2 at 7 h after transfection. Briefly, 2 μl of 20 pM of each FoxO1 or Skp2 siRNA solution and 12 μl of the transfection reagent were incubated in 100 μl of OptiMEM medium (Invitrogen) for 20 min, to facilitate complex formation. The siRNA transfection mix was added to the cells cultured in 10% FBS. Control consisted of nonspecific siRNA (Silencer negative). Cells were transfected for 7 h at 37°C, washed with Hanks buffer, and cultured in MEM with 10% FBS for an additional 24 h. The medium was then changed to stem cell medium (SCM) (20 ng/ml EGF and 10 ng/ml FGF), and cells were cultured for 4 d. To assess the proliferative potential of cells, BrdU was added to the culture medium at a concentration of 10 μg/ml, followed by 60 min incubation at 37°C. Cells were then fixed in 4% paraformaldehyde and kept in PBS until use. BrdU incorporation was visualized by immunofluorescence using anti-mouse BrdU antibody. Percentages of BrdU + cells were quantified from at least three different experiments and subjected to statistical analysis. To demonstrated FoxO1 and Skp2 knockdown, Western blots analysis was performed on transfected CNP-EGFP cells from normoxic and hypoxic mice. After transfection, cells were lysed in 50 μl of ice-cold RIPA buffer. Protein samples were prepared and processed as described in Western blots analysis and immunoprecipitation. Membranes were incubated with anti-FoxO1, anti-Skp2, and anti-p27 Kip1 antibodies, and results were normalized relatively to actin.
Overexpression of p27Kip1 and FoxO1 in white matter cells
To overexpress p27 Kip1 or FoxO1 in white matter cells from normoxic and hypoxic brains at P18, we used a p27 Kip1 retrovirus (a gift from Dr. M. Luskin, Emory University, Atlanta, GA) or mock virus and pCMV5 HA FoxO1 plasmid (Addgene), or an empty vector as a control. Plasmid constructs were introduced into cultured cells by liposomal transfection for 6 h in 12-well plates using 1.0 μg of DNA and 12 μl of NeuroPORTER Transfection Reagent in OptiMEM medium. After transfection, the cells were washed in SCM containing 20 ng/ml EGF and 10 ng/ml FGF, and plated for poly-lysine-coated dishes for culturing. After 5 d in culture, cells were labeled with anti-O4, -O1, -GalC, -Olig2, and -p27 Kip1 antibodies or collected for Western blot. Membranes were incubated with anti-FoxO1 and anti-p27 Kip1 antibodies, and results were normalized relatively to actin.
All human tissue was collected in accordance with guidelines established by the University of California San Francisco Committee on Human Research (H11170-19113-07) (). Following autopsy, all brains, except case 2, were immersed in PBS with 4% paraformaldehyde for 3 d. On day 3, the brain was cut in the coronal plane at the level of the mamillary body and immersed in fresh 4% paraformaldehyde/PBS for an additional 3 d. In case 2, the brain was immersed in 4% phosphate-buffered formalin for 1 week at room temperature. After fixation, all tissue samples were equilibrated in PBS with 30% sucrose for at least 2 d. Following sucrose equilibration, tissue was placed into molds and embedded with OCT for 30 – 60 min at room temperature or 4°C followed by freezing in dry ice-chilled ethanol or methyl butane. The diagnosis of hypoxic ischemic encephalopathy (HIE) requires clinical and pathological correlations. With respect to the pathological features, all HIE cases in this study showed consistent evidence of diffuse white matter injury, including astrogliosis and macrophage infiltration. These findings were confirmed by the increase in the number and the staining intensity of GFAP- or CD68-positive cells, respectively. In addition, the HIE case also showed evidence of neuronal injury, including the presence of ischemic neurons and variable degrees of neuronal loss, in cerebral cortex, hippocampus, and basal ganglia.
Cases 1 and 2 demonstrated clinical evidence of HIE and pathological evidence of HIE and low output state in multiple organs on postmortem examination. More specific findings in case 1 (8 weeks of age, born at full term) included hypoplastic left heart status-post procedure and diffuse white matter injury in brain with focal neuron dropout in cerebral and cerebellar cortices. Case 2 (5 months of age, born prematurely at 26 weeks gestation) with a clinical diagnosis of severe HIE who underwent therapeutic hypothermia showed diffuse white matter injury on postmortem evaluation. Case 6 (1-d-old term infant) with midgut volvulus with extensive hemorrhagic necrosis of small bowel was not found to have significant neuropathological findings of HIE.
cDNA was first synthesized from 1.5 to 2 μg of total RNA extracted from cultured cells obtained from hypoxic and normoxic white matter in a total volume of 11 μl, including 10 mM dNTP mix and 0.5 μg/μl oligo-dT (Invitrogen; 12371-019). Reaction mixtures were heated at 65°C for 5 min, and then at 42°C for 50 min. From 2 μl of cDNAs, sequences of interest were amplified in a thermocycler in a total volume of 25 μl of mixture with Taq polymerase. Primer pairs are described in the supplemental materials. PCR products were resolved by vertical electrophoresis on 2% agarose gels. Intensity of bands was measured using the Image J program (NIH). Primers were as follows: CNP, 5′-CCG GAG ACA TAG TGC CCG CA-3′; 5′-AAA GCT GGT CCA GCC CTT CC-3′; MBP, 5′-CTA CCC ACT GTC GAT GAC TTA TTG ATT AGA C-3′; 5′-CTC TAA TCA ATA AGT CAT CGA CAG TGG GTA C-3′; Olig2, 5′-GTG TCT AGT CGC CCA CGT G-3′; 5′-CGA TGT TGA GGT CGT GCA T-3′; GFAP, 5′-ACT TAA CAA ATC CCT TCC TTC ATC C-3′; 5′-CCC TCT CTC CTG TTC AGT G-3′; Actin, 5′-CGT GGG CCG CCC TAG GCA CA-3′; 5′-TTG GCC TTA GGG TTC AGG GGG-3′.