282 - Astrocyte neuropathology following hypoxic ischemic injury in the developing brain

Publication Number: 4278.282

Anna C. Bitners, Johns Hopkins Children's Center, Baltimore, MD, United States; Andrew Cavanagh, Johns Hopkins University School of Medicine, Baltimore, MD, United States; Benjamin Sollinger, Johns Hopkins University School of Medicine, Baltimore, MD, United States; Christian Clarke, Johns Hopkins University School of Medicine, Baltimore, MD, United States; Debra L.. Flock, Johns Hopkins University School of Medicine, Fallston, MD, United States; Frances Northington, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Background: Astrocytes are multifunctional glial cells that play a critical role in the response to brain injury. Following hypoxic ischemia (HI), perturbations in water flux precipitate the development of cerebral edema via increased abundance of the water channel protein Aquaporin-4 (AQP4) at the astrocytic cell membrane. Magnetic resonance imaging signatures of microstructural fluid flux in post-HI brains over time correlate with regional tissue viability, suggesting that the astrocytic responses to injury that mediates edema formation may participate in the varying regional responses to neonatal HI.

Objective: To assess temporal and regional variations in astrocyte neuropathology and AQP4 abundance following HI in regions of the brain shown to be relatively sensitive (somatosensory cortex [SSCx]) or resistant (cingulum) to HI utilizing a murine model of neonatal hypoxic ischemic encephalopathy.

Design/Methods: HI was induced in P10 mouse pups utilizing the Rice-Vannucci model with a sham group for comparison (n=5-7/group). Pups were sacrificed 3 or 48 hours after HI. Immunohistochemistry for GFAP and immunofluorescence (IF) for GFAP/AQP4 were utilized to identify reactive astrocytes and AQP4 abundance. GFAP+ astrocytes were manually identified, counted and compared between HI vs shams in the cingulum and SSCx ipsilateral to the injury. Western blots (WB) were performed with a homogenized hemi cortex of fresh frozen tissue and AQP4 antibody (GTX133151) to detect AQP-4 monomers (~35 kDa) and dimers (~70 kDa). Mann Whitney or paired t-tests were used to assess differences between groups as appropriate and p < 0.05 was considered significant.

Results: Figure 1. In the ipsilateral HI cingulum, the median astrocyte count at 48h was significantly increased at 117 (IQR 76.5) vs 77.0 (IQR 46.8) in shams (p = 0.0455). Cell counts did not differ significantly at 3h or at either timepoint in the SSCx. Figure 2. AQP4 expression intensity at 48h increased following HI in both regions. Figure 3. At 48, in a n=3 subset of tissues, a difference in abundance of neither the monomer nor dimer forms of AQP4 reached significance (p=0.10) between HI-ipsi and sham.

Conclusion(s): In these experiments thus far, a combination of upregulated numbers of GFAP+ astrocytes and increased AQP-4 expression at 48h after injury marked the more resistant ipsilateral cingulum, suggesting that multifactorial astrocyte responses may participate in regional vulnerability to injury. Ability to handle rapid changes in cerebral water and ion fluxes likely resides upstream in determining brain tissue survival vs death after neonatal HI.

Figure 1. Cell counts of GFAP+ astrocytes
Graphs showing number of GFAP+ astrocytes stratified by region (1A,B cingulum or 1C,D somatosensory cortex [SSCx}) and time point (3 hours or 48 hours) in sham vs hypoxic ischemia (HI) groups. Data presented as median with error bars indicating interquartile range.

Figure 2. Aquaporin-4 abundance determined by immunofluorescence
Slope chart showing mean gray value of aquaporin-4 immunofluorescence of paired cortical regions (2A,B cingulum, 2C,D somatosensory cortex [SSCx]) contralateral (contra) and ipsilateral (ipsi) to the hypoxic ischemia (HI) insult at 48 hours post-HI.

Figure 3. Aquaporin-4 abundance determined by Western blot
3A Western blot for AQP4 in sham, HI Contra, and HI Ipsi cortical homogenates. 3B,C Scatter plot displaying relative adjusted intensity values comparing shams to hypoxic ischemia (HI) brains ipsilateral (ipsi) to the injury. Relative adjusted intensity values of the bands of interest (3B 35 kDa, 3C 70 kDa) were calculated as a percent of the average mean grey value in sham tissue. Data are presented as median with error bars indicating interquartile range.