Region specific oligodendrocyte transcription factor expression in a model of neonatal hypoxic injury

Abstract

White matter injury (WMI) of prematurity is associated with a spectrum of neurological disorders ranging from mild cognitive and behavioral deficits to cerebral palsy. Translational studies have implicated impaired oligodendrocyte development after hypoxia as the primary cause of WMI, but the underlying mechanisms remain poorly understood. The goal of this study was to identify alterations in the expression of oligodendrocyte precursor cell transcription factors in a mouse model of transient mild global hypoxia. Postnatal day (P) 7 mouse pups were exposed to hypoxia (7.5% O) for 60minutes. We compared oligodendrocyte differentiation and subsequent myelin formation between hypoxia and sham animals at P9, P14 and P28 by examining the expression of key transcription factor regulators of oligodendrocyte differentiation (Ascl1, Olig1, Olig2, and Nkx2.2), as well as APC, a mature oligodendrocyte marker, in the major white matter regions including the corpus callosum, external capsule and anterior commissure. We also examined the effect on myelin formation by examining two myelin specific protein constituents, myelin associated glycoprotein (MAG) and myelin basic protein (MBP), in white matter tracts and whole brain lysate respectively. We found that transient hypoxia at P7 altered the expression of Ascl1, Olig1 and Nkx2.2, resulting in delayed myelination in the external capsule. In addition, our study showed that oligodendrocyte progenitor cells specified several days prior to a hypoxic event are more susceptible to maturation arrest than those specified shortly prior to hypoxia. Our results suggest that alterations of Ascl1, Olig1 and Nkx2.2 underlie impaired oligodendrocyte differentiation and deficient myelination in WMI. These transcription factors are potential therapeutic targets for the treatment of WMI in preterm infants.

Repeated Pediatric Concussions Evoke Long-Term Oligodendrocyte and White Matter Microstructural Dysregulation Distant from the Injury

“…Ascl1-CreER: ccGFP transgenic mice (backcrossed to a CD-1 background; generously provided by Dr. Jane Johnson, University of Texas Southwestern, TX, USA) were obtained and bred in-house [27]. The animals were housed on a 12-h light/dark cycle in a temperature-controlled animal facility with their dam until weaned at P21.…”

“…Water and food were provided ad libitum. The rationale for the use of Ascl1-CreER: ccGFP animals was to establish the baseline for future efforts further examining OL development in pediatric mTBI pathology, utilizing Ascl1 as a key regulatory transcription factor involved in WM development and myelination [27]. …”

“…The following day slides were washed with the washing buffer, blocked for 60 min, and incubated with the species-appropriate secondary antibody (Alexa Fluor 594 or 488 secondary antibodies; 1: 400, Thermo Fisher Scientific, Carlsbad, CA, USA) for 60 min at room temperature. Slides were rinsed and then counterstained with DAPI for nuclear staining and mounted with Prolong Gold mounting medium (Cat#P36930 Thermo Fisher Scientific) as described previously [27]. …”

“…The AC area was measured using MAG-stained images and ImageJ software [58]. Cell counts for OLIG2 transcription factor, the mature OL marker APC, and the cell proliferation marker Ki67 were semiautomatically counted using ImagePro Premier software (64-bit, v9.1, Media Cybernetics, Rockville, MD, USA), and all cell counts were normalized to the total number of DAPI-positive cells as was done previously [27]. For confirmatory evidence, a further analysis (cells/mm 2 ) was conducted which controlled for the area of the ROIs instead of the cellular density within the ROIs.…”

“…One of the most notable time periods for WM development includes postnatal days 7–10 (P7–P10) in rodents (equivalent to 36–40 weeks’ human gestation) when immature OLs become the predominant cell type [24-26]. We have previously demonstrated the susceptibility of OLs to mild hypoxia during this critical period [27]. By P20/21 in rodents (2/3-year-old humans) the rate of myelination peaks [26, 28, 29].…”