Fig 1.
Panel A: Serial H&E coronal whole brain sections from anterior (top) to posterior (bottom) of 3 randomly selected animals from each group Sham and HI group, with special emphasis on lateral and 3rd ventricle size. Scale bar 2 mm. N = 12 animals/group. Panel B: Representative H&E coronal brain sections of lateral ventricle, ependymal lining, cortex and hippocampus of the studied groups (Sham & HI group). Scale bar 10 μm. N = 12 animals/group.
Fig 2.
MRI volumetric analysis in both studied groups (Sham and HI group), using the BioSpec 94/30 imaging system is a 9.4T horizontal bore magnet operates at 400 MHz and runs ParaVision™ 4.0 software.
Panel A: Coronal (top) and axial (bottom) brain slices. Panel B: lateral ventricle volume bilaterally measured by paravision software in both studied groups. Panel C: Brain volume includes brain volume + lateral ventricle volume measured by paravision software in the studied groups. Panel D: The ratio of bilateral lateral ventricle volume to total brain volume measured by paravision software in the studied groups. Since there was no significant difference in brain volume, increased lateral ventricle volume indicates white matter loss. N = 10 animals/group. Bars represent the Mean + SE. * indicates P< 0.05.
Fig 3.
Astrogliosis and white matter volume loss in PVL model.
Immunohistochemistry of periventricular brain area at P15 (or 10 days after HI) in both studied groups (Sham vs HI group). Panel A: Astrocytes (GFAP) in green, Panel B: Oligodendroglia (CNPase) in red. Panel C: Olig 2 (oligodendroglia) in red. Scale bar = 100 μm. Panel D: Quantification of GFAP intensity as fold change where sham = 1. Panel E: Quantification of CNPase intensity as fold change where sham = 1. Panel F: Quantification of Olig2 positive cells per section. There was obvious significant reduction of oligodendroglial cells (indicating white matter loss), accompanied with significant increase of astrocytes in HI group. N = 5 animals/group. 4 sections /animal. Bars represent the Mean + SE. ** * indicates P< 0.001.
Fig 4.
Neuronal apoptosis in PVL model.
Panel A: Immunohistochemistry of periventricular brain area at P15 (or 10 days after HI) of the 2 studied groups (Sham and HI group). Panel A: Apoptosis (Caspase 3) shown in red (top), Neurons (NeuN) shown in green (middle). Co-localization is shown in orange indicates neuronal apoptosis (bottom). Scale bar = 50 μm. Panel B: Activated Caspase 3/ NeuN quantification. N = 5 animals/group & 4 sections/animal. Bars represent the Mean + SE. ** * *indicates P< 0.0001. Panel C: Western blot of periventricular area of brain protein lysate at P10 (or 10 days after HI) in both studied groups. The western blot was probed for Cleaved caspase 3 (top) and reprobed for β Actin (bottom) as loading controls. Fold change of the western blot determined using Image J to measure band intensities of cleaved caspase 3 normalized to β-Actin. Caspase 3 is upregulated by 5.4 fold in HI group as compared to sham. N = 6 animals/group. Bars represent the Mean + SE. ** * *indicates P< 0.0001.
Fig 5.
Studies of microglial M1 and M2 phenotypes at P15 (10 days after HI) in HI group versus sham.
Panel A: Immunostaining for all Microglia (Iba1) in green (top), CD 68 (marker of activated microglia only) in red (middle). Co-localization in yellow indicating amount of activated microglia. Scale Bar = 100 μm. Panel B: Immunostaining for Arginase 1 (M2 microglial marker) in green (top); for Microglia (Iba1) in blue (middle); and Co-localization (bottom) indicating the percentage of M2 microglia. Co-localization showed scare M2 microglial phenotype cells in HI group. Scale Bar = 100 μm. Panel C: Quantification of CD68/Iba1 per section Panel D: Arginase 1/Iba1 per section. There was significant increase of M1 microglia and a significant reduction of M2 microlgia in HI group. N = 5 animals/group & 4 sections/animal. Bars represent the Mean + SE. **** indicates P< 0.0001.
Fig 6.
Nitric oxide synthetase isoforms and NF-kb activation in PVL model.
Panel A: nNOS western blot (MW 160 kDa) of periventricular area of brain protein lysate at P10 (or 5 days after HI) in both studied groups and represented as a ratio for β-Actin protein. No difference. Panel B: iNOS western blot (MW 130 kDa) of periventricular area of brain protein lysate at P10 (or 5 days after HI) in both studied groups and represented as a ratio for β –Actin protein. Panel C: eNOS western blot (MW 140 kDa) of periventricular area of brain protein lysate at P10 (or 5 days after HI) in both studied groups and represented as a ratio for B-Actin protein. Panel D: Phosphorylated P65 western blot (MW 65 kDa) of periventricular area of brain protein lysate at P10 (or 5 days after HI) in both studied groups and represented as a ratio to p65 protein (MW 65 kDa). For Panels A, B, C, D: N = 6 animals/group. Bars represent the Mean + SE. * indicates P< 0.05. ** indicates P< 0.01. *** indicates P< 0.001.
Fig 7.
Nitrative stress and pro-inflammatory cytokines.
Panel A: Immunohistochemistry of coronal sections of the periventricular brain area of postnatal day 15 pups (or 10 days after HI) of the two studied groups (sham and HI group). Top panel shows Nitrotyrosine (marker of nitrative stress) in red. Middle panel shows Iba1 (microglial marker) in green. Lower panel shows Co-localization in yellow nitrotyrosine secreted by microglia. There was a significant increase of nitrative stress in HI group compared to sham. Scale bar = 200 μm. Panel B: Quantification of Nitrotyrosine/Iba1 per section. N = 5 animals/group & 4 sections/animal. Bars represent the Mean + SE. **** indicates P< 0.0001. Panel C: ELISA assay of cytokines known to peak in both human and animal PVL model. Assay from brain homogenate at P6 or 24 hrs post HI, namely IL-1β, IL 6, MIP2, of the studied groups (Sham and HI group). There was a significant increase of both IL1β & IL6 in HI group compared to sham. N = 8 animals/group. Bars represent the Mean + SE. * indicates P< 0.05.
Fig 8.
Long term outcome assessed by neurobehavioral testing performed at postnatal day 60.
Panel A: There was a reduction of total body mass at P10 (P<0.0003), but the difference was not significant at P60. Panel B: Testing for grip, showed no difference in front grip between the 2 groups, while rear grip strength was significantly lower in HI group compared to sham (P<0.05). Panel C: A significant reduction of latency to fall indicating worse co-ordination was observed in HI group compared to sham (P<0.05). Panel D: Number of beam breaks and number of rears in the open field test were significantly reduced in HI group as compared to sham (P<0.05). N = 25 animals/group. Bars represent the Mean + SE. ** indicates P< 0.01.