Introduction
Approximately 60,000 preterm infants weighing less than 1500 g are born each year in the USA. Although survival rates are improving, long-term complications in such very low birth weight (VLBW) infants are commonly observed that involve the respiratory, cardiovascular, intestinal, and central nervous systems [
1]. Cerebral palsy and other neurological sequelae in VLBW infants are associated with damage to cerebral white matter tracts [
2] and cerebellum [
3]. Cerebellar abnormalities are found in 20% of VLBW infants and are characterized as hemorrhagic or hypoplastic [
4]. Clinical features associated with cerebellar abnormalities include postnatal glucocorticoid exposure, intraventricular hemorrhage, and chronic lung disease [
5]. In particular, chronic lung disease can lead to intermittent hypoxemia and is associated with cerebellar hypoplasia in MRI studies [
6,
7].
Although the cerebellum acts principally in the regulation of neural circuits for motor control and coordination [
8], it also has roles in control of higher order cognitive functions [
9‐
11]. Regions of the brain, undergoing extensive neurogenesis, are particularly vulnerable to hypoxic and/or ischemic insults during the third trimester and early neonatal periods [
6,
7]. In humans, the cerebellum undergoes rapid growth during the third trimester through the first year of life [
3,
6], whereas in rodents this phase of development is primarily post-natal.
The major driver of cerebellar growth is proliferation of cerebellar granule neuron precursors (CGNPs) [
12], which depends on Sonic hedgehog (Shh) signaling [
12‐
18]. Shh is a secreted protein that inhibits the transmembrane repressor Patched, which in turn, de-represses activity of Smoothened (Smo), a G-protein coupled receptor. Smo activation in the cilia of CGNP upregulates target genes
Gli1 and
N-
myc that drive cell cycle progression [
15‐
17]. Thus, mutations affecting Shh production in Purkinje cells or Smo function on CGNP result in cerebellar hypoplasia [
19].
Postnatal glucocorticoids are administered to preterm infants for indications of severe chronic lung disease and hypotension [
3,
20,
21]. In the preterm lung, glucocorticoids promote production of pulmonary surfactant protein B and regulate the inflammatory response by interacting with transcription factors, such as nuclear factor kappaΒ (NF-κΒ) and activated protein 1 [
22‐
24]. Although glucocorticoids help promote lung surfactant production and lung epithelial differentiation [
22,
25], and physiological concentrations of these hormones are essential for normal brain development [
26], high level exposure to potent glucocorticoids in the postnatal period causes brain injuries, including impaired cognition, cerebral palsy, and cerebellar hypoplasia [
3,
6,
26‐
31].
11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), a NAD-dependent high affinity enzyme involved in the local metabolic inactivation of endogenous glucocorticoids into inert 11-keto derivatives, acts in opposition to 11βHSD type 1, which converts its substrate into active corticosterone. Dexamethasone and betamethasone can cross the placenta to the fetus because they have a low affinity for cortisol binding globulin and are not inactivated by 11βHSD2, which is expressed at high levels in the placenta. In contrast, corticosterone and prednisolone are susceptible to inactivation by 11βHSD2 activity. 11βHSD2 is expressed in the developing CNS, including cerebellar granule neuron precursors (CGNPs) [
32] where its function is necessary for normal cerebellar development [
33]. Indeed, Shh signaling is protective against prednisolone-induced cerebellar injury through upregulation of 11βHSD2 specifically in CGNPs.
Chronic lung disease, airway instability, and apnea of prematurity can lead to an intermittent hypoxemic environment in the brain, which has been shown to affect cortical development, oligodendrocytes [
34], and interneurons [
35‐
37]. Certain cellular responses to hypoxia are mediated by hypoxia-inducible factors (HIFs) [
38,
39], which are transcription factors with an unstable subunit (HIF1α or HIF2α) that is degraded in the presence of oxygen, and a constitutively expressed subunit (HIF1β or HIF2β) [
40,
41]. HIFs coordinate the response to low oxygen by stimulating genes involved in metabolism and angiogenesis. In normoxia, HIFα becomes modified by prolyl-hydroxylase (PHD) and is recognized by the E3 ubiquitin ligase von Hippel Lindau factor (VHL), which then targets HIFα to the proteasome for degradation [
42,
43]. Conversely in hypoxia, PHD is inactive, allowing HIFα to become stabilized, which then binds to HIFβ in the nucleus to activate target genes such as VEGF, BNIP3, glycolytic enzymes, and LEF-1/Tcf-1 [
44,
45]. Previous studies show that glucocorticoid administration in neonatal mice causes cerebellar hypoplasia by downregulating Shh signaling and CGNP proliferation [
29,
30] as well as cell death [
46]; moreover, these effects are rescued by systemic administration of a small-molecule Smoothened agonist (SAG) [
29]. The effect of hypoxia on cerebellar growth and its relationship to Shh signaling, however, is unclear. Moreover, cell type-specific roles of HIF in the developing cerebellum have so far not yet been defined.
Because exposure to glucocorticoid and hypoxia are clinically associated with cerebellar abnormalities [
29,
30], we modeled such compound injury in neonatal mice using a chronic hypoxia model (10% FiO
2) combined with administration of the synthetic glucocorticoid prednisolone. Although chronic hypoxia would model a relatively extreme form of clinical injury, it represents a robust and reproducible injury model that has been previously used and described extensively in the literature [
34,
47,
48]. Intermittent hypoxia, while clinically more relevant, may confound results as it can precondition animals to neuroprotection from further hypoxic damage. We chose to use prednisolone rather than dexamethasone, which is more commonly given to infants with chronic lung disease, as dexamethasone is not metabolized by 11βHSD2 and therefore not affected by SAG [
30]. As both prednisolone and hydrocortisone as sensitive to 11βHSD2, SAG therapy is therefore effective against these compounds.
Here we address the novel aspect of cross-talk between hypoxia/HIF signaling and glucocorticoid pathways in the developing cerebellum. We observed that chronic hypoxia resulted in cerebellar hypoplasia but that Purkinje cell populations were well preserved. When combined, however, hypoxia and glucocorticoids caused Purkinje cell death and enhanced cerebellar volume deficits. Both hypoplasia and Purkinje cell death were rescued in part by administration of SAG, even when given days after administration of the dual insults. To determine cell type-specific roles of the HIF pathway, we performed conditional knockout of VHL to induce and hyperactivate HIF1α in CGNPs or Purkinje cells. In the presence of active HIF in CGNP, prednisolone administration resulted in cerebellar hypoplasia. In contrast, prednisolone with active HIF in Purkinje cells resulted in cell death. Together, these findings indicate that hypoxia/HIF with postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. In particular, these results demonstrate for the first time the ill effects of HIF and glucocorticoid signaling in Purkinje cells. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.
Materials and Methods
Animals
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee and Laboratory Animal Resource Center at University of California, San Francisco (UCSF). Mouse colonies were maintained in accordance with NIH and UCSF guidelines.
C57Bl6/J mouse lines were obtained from the Jackson Laboratories.
Gli-
luciferase [
49],
VHL floxed [
50],
Atoh1-
cre (
Math1-
cre) [
51], and
L7-
cre [
19] mice have been previously described.
Preparation of SAG
Synthesis of SAG has been previously described [
52]. SAG was dissolved in dimethyl sulfoxide (DMSO) to 5 mM and further diluted with normal saline or culture medium. These experiments used SAG as a free-base form. Vehicle controls comprised saline containing an equivalent concentration of SAG.
Chronic Hypoxic Rearing and Systemic Administration of Prednisolone and SAG
Chronic hypoxic rearing was performed as previously described [
35,
48,
53,
54]. Briefly, litters of the
C57Bl6/
J strain were culled to a size of, at most, ten pups and co-fostered with CD1 or Swiss Webster strain dams then reared at 10% O
2 in a hypoxic chamber (Biospherix, Inc., Laconia, NY) from postnatal day 3 (P3) to P11. Tissue from P4, P7, P11, P22, or P40 was then harvested acutely for analysis. On P3, pups received daily intraperitoneal injections of prednisolone (0.67 g/kg body weight, Sigma-Aldrich), SAG (20 g/kg body weight), prednisolone in combination with SAG, or vehicle (DMSO), for 8 days, or the duration of the hypoxic experiment. For acute treatment, a one-time dose of SAG was given at P11.
Tissue Processing and Immunohistochemistry
Following intracardial perfusion of 4% paraformaldehyde, tissue was post-fixed for 1–2 h at 4 °C, cryoprotected in 30% sucrose, and embedded in OCT. Frozen sections were cut on a cryostat (20 μm) and stored at −80 °C. For staining, sections were thawed and then rehydrated in PBS. If necessary, tissues underwent antigen retrieval with citrate buffer (pH 6.0) at 95 °C for 10 min. Sections were blocked in 10% goat or donkey serum in 0.1% TritonX-100-containing PBS (blocking solution). Primary antibodies were diluted in blocking solution, and tissues incubated at 4 °C overnight or 2-h room temperature. For primary antibodies, we used PH3 (mouse monoclonal, Cell Signaling), Calbindin (mouse monoclonal or rabbit polyclonal, Swant), Cleaved Caspase 3 (rabbit polyclonal, Cell Signaling), NeuN (mouse monoclonal, Millipore), Iba1 (rabbit polyclonal, Wako), HIF1a (rabbit polyclonal, Cayman Chemicals), BNIP3 (rabbit polyclonal, Cell Signaling), and Cre (rabbit polyclonal, Millipore). Following primary incubation, tissues were washed with 0.1% Tween20-containing PBS, then incubated with proper Alexa Fluor secondary antibodies (Invitrogen) in blocking solution for 1 h at room temperature. Sections were mounted with fluoromount containing DAPI (Southern Biotech).
CGNP Primary Cell Culture, Transfection, and Luciferase Assay
Primary cultures were performed as previously described [
30]. Briefly, either
C57Bl6/
J or
Gli-
luciferase pups were euthanized at P4 or P5, and their cerebella were dissected and dissociated. Cultures were maintained in serum-free medium containing only vehicle or ShhN for 24 h prior to treatments. Cultures were then transfected with a HIF1α overexpressing plasmid or an empty vector for 12 h using the Piggyback transposon system to allow high efficiency expression as previously described [
55]. Cells were collected 12 h afterward for protein extract or luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). For analysis of Shh targets under hypoxic conditions, cultures were incubated in a 1% O
2 incubator for 24 h, or treated with dimethyloxalylglycine (DMOG) (Sigma), a PHD4 inhibitor that upregulates HIF.
Western Blot
Preparation of protein extracts, immunoblots, and fluorescent detection was done as previously described using the Li-Cor Odyssey system (Li-Cor, Lincoln, NE) [
34]. Antibodies used include HIF1α (rabbit polyclonal, Cayman Chemicals), Cyclin D1 (rabbit polyclonal, ThermoScientific), Patched 1 (goat polyclonal, Santa Cruz), Gli1 (rabbit polyclonal, Santa Cruz), Gli3 (goat polyclonal, R and D Systems), N Myc (mouse monoclonal, Millipore), and β-Actin (mouse ascites, Sigma).
Single-Molecule Fluorescent In Situ Hybridization Against Gli1 and Atoh1
To assess Gli1 mRNA expression in the cerebellum, we used the RNAscope LS Multiplex Assay (Advanced Cell Diagnostics). P11 mouse brain sections were baked at 65 °C for 45 min, post-fixed in 4% paraformaldehyde for 15 min, and dehydrated prior to the assay. Multiplexed single-molecule fluorescent in situ hybridization (smFISH) against Gli1 and Atoh1 mRNA was performed on the BOND RX automated stainer (Leica) using the RNAscope reagents. The Gli1 probe consists of 20 double-Z probes targeting nucleotides 25–1025 on the mouse transcript NM_010296.2 and was developed with the fluorophore Opal 520 (Perkin Elmer). The Atoh1 probe consists of 20 double-Z probes targeting nucleotides 847–2088 on the mouse transcript NM_007500.4 and was developed in Opal 570.
Microscopy and Quantification
Surface area of the entire cerebellum was calculated as previously described [
30]. Briefly, the cerebellum from immunostained sections with DAPI was outlined, and surface area was measured in Adobe Photoshop CS6 using the Record Measurements function, with measurement scale set to match the objective lens from the microscope (e.g., for the Zeiss × 10 objective, 1 pixel = 0.65 μm). For area measurement and cell counting of immunohistochemistry (IHC) from sagittal brain sections of P4, P7, P11, and P22 wild-type and transgenic animals, tiled images comprising the entire cerebellar vermis were taken with a Zeiss AxioImager.Z2 microscope equipped with a motorized stage using either the × 10 or × 20 objective. The mean area per section for each animal was determined in mm
2 from measurements of five parasagittal sections from the midline region of the cerebellum, and is used as a proxy for volume. Quantification of cerebellar cross-sectional area resulted from an average of
n = 3 animals in normoxic and hypoxic conditions for P4 and P22;
n = 3 in normoxic, and
n = 4 in hypoxic conditions at P40. For the hypoxia + prednisolone experiments, quantification of the cerebellar vermis is resulted from
n = 3 animals for the Nx and Hx conditions, and
n = 5 animals for the Hx + prednisolone and Hx + prednisolone + SAG conditions. For experiments using transgenic animals, quantification of the cerebellar IGL resulted from
n = 3 animals for the
Math1Cre;
Vhl(
fl/+) and
Math1Cre;
Vhl(
fl/
fl) conditions,
n = 7 for
Math1Cre;
Vhl(
fl/+) + prednisolone condition, and
n = 6 for the
Math1Cre;
Vhl(
fl/
fl) + prednisolone condition;
n = 3 for
L7Cre;
Vhl(
fl/+),
L7Cre;
Vhl(
fl/
fl), and
L7Cre;
Vhl(
fl/+) + prednisolone conditions, and
n = 4 for
L7Cre;
Vhl(
fl/
fl) + prednisolone condition. The area of the cerebellum was measured using either Adobe Photoshop CS6 or Stereo Investigator (MBF Biosciences).
The numbers of immunopositive or double-immunopositive cells were quantified by a blinded investigator using the Count function on Adobe Photoshop CS6, or the Spot Detection function on Imaris (Bitplane). The average cell count for each animal was determined from measurements of five to seven whole parasagittal sections from the midline region of the cerebellum. Quantification of Calbindin-positive cells resulted from n = 3 animals in normoxic and hypoxic conditions, n = 6 in the hypoxia + prednisolone condition, and n = 5 in the hypoxia + prednisolone + SAG condition.
Statistical Analyses
For all quantified data, mean + SEM values are presented. Statistical analysis was performed using unpaired, two-tailed Student’s t tests, and with an ANOVA (single factor). For a significant difference (p < 0.05), a Tukey’s post-hoc test was performed (GraphPad Prism).