Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP(-/-)Vim(-/-)) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP(-/-)Vim(-/-) mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.We subjected GFAP(-/-)Vim(-/-) and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP(-/-)Vim(-/-) and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN(+) neurons in the ischemic and contralateral hemisphere was comparable between GFAP(-/-)Vim(-/-) and wild-type mice. In wild-type mice, the number of S100(+) astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0+/-50.1 vs. 85.6+/-34.0, p<0.05). In the GFAP(-/-)Vim(-/-) mice, the number of S100(+) astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP(-/-)Vim(-/-) mice showed an increase in NeuN(+)BrdU(+) (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7+/-7.7; n = 29 versus 2.9+/-3.6; n = 28, respectively, p<0.05), but a comparable number of S100(+)BrdU(+) (surviving newly born) astrocytes.Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons

Experimental Studies on the Influence of Plasma Treatment of Polyethylene in Carbon Fiber Composites: Mechanical and Morphological Studies

This research focused on enhancement of mechanical properties in carbon fiber (CF)-filler-reinforced linear low-density polyethylene (PE) matrix composites. A hand layup method using an oven was used as the fabrication method. Improvement in adhesion was achieved by oxygen plasma treatment to the PE matrix. CF and PE were initially mixed by normal stirring, ultrasonication and mechanical stirring before being filtered and dried for fabrication. Better tensile results were observed with a plasma-treated polyethylene (PEP)/10 wt.% CF combination, with a maximum tensile strength of 21.5 MPa and improvement in the properties of up to 12.57% compared to non-plasma PE with the same CF addition. The addition of carbon fibers at 13 and 15 wt.% resulted in a reduction in the tensile strength properties to 18.2 MPa and 17.7 MPa, respectively. This reduction in tensile strength was due to agglomeration of CF with plasma- and non-plasma-treated PE. The fabrication condition of 180 &deg;C temperature for 20 min showed better tensile properties than other conditions. The SEM results following tensile testing revealed enhanced CF filler adherence with plasma PE results, as well as fewer surface deformations. A higher flexural strength of 25.87 MPa was observed for the plasma treated PE/7 wt.% CF combination

Hemisphere and infarct volume in <i>GFAP^−/−Vim^−/−</i> and wild-type mice after hypoxia-ischemia.

<p>Volume measurements in MAP-2 stained sections from <i>GFAP^−/−Vim^−/−</i> (GV) (n = 23) and wild-type (WT) (n = 19) mice at P12 and P31, i.e. 3 and 22 days after hypoxia-ischemia. (A) Ipsi- and contralateral hemisphere volume in <i>GFAP^−/−Vim^−/−</i> and wild-type mice. (B) Infarction volume in <i>GFAP^−/−Vim^−/−</i> and wild-type mice. Data are mean ± SD.</p

GFAP mRNA expression in mice after hypoxia-ischemia, assessed by quantitative rt-PCR.

<p>Cortex from wild-type mice exposed to hypoxia-ischemia on P9, examined by quantitative rt-PCR immediately after (n = 6), 6 h (n = 4), 24 h (n = 3), 3d (n = 4), 7d (n = 4), and 21d (n = 4) after hypoxia-ischemia. The values represent a fold increase compared to 0 hours after HI. Data are mean ± SD (** p<0.01).</p

Quantification of S100⁺ and S100⁺BrdU⁺ cells at 22 days after hypoxia-ischemia.

<p>(A) Number of S100⁺ cells per optical field in ipsi- and contralateral subventricular zone, striatum and cortex, in <i>GFAP^−/−Vim^−/−</i> (GV) (n = 29) and wild-type (WT) (n = 28) mice at P31, i.e. 22 days after hypoxia-ischemia. (B) Number of S100⁺BrdU⁺ cells per optical field in ipsi and contralateral subventricular zone, striatum and cortex, in <i>GFAP^−/−Vim^−/−</i> (n = 29) and wild-type (n = 28) mice at P31, i.e. 22 days after hypoxia-ischemia. The numbers represent cells counted on 8 µm thick stacks of confocal images within the 450 µm×300 µm optical fields in the respective brain regions. (C) Orthogonal sections showing S100⁺BrdU⁺ cells. Data are mean ± SD. (* p<0.05, ** p<0.01). Scale bar  = 15 µm.</p

Quantification of NeuN⁺ and NeuN⁺BrdU⁺ cells at 22 days after hypoxia-ischemia.

<p>(A) Number of NeuN⁺ cells per optical field in the ipsi- and contralateral subventricular zone, striatum and cortex, in <i>GFAP^−/−Vim^−/−</i> (GV) (n = 29) and wild-type (WT) (n = 28) mice at P31, i.e. 22 days after hypoxia-ischemia. (B) Number of NeuN⁺BrdU⁺ cells per optical field in ipsi- and contralateral subventricular zone, striatum and cortex, in <i>GFAP^−/−Vim^−/−</i> (n = 29) and wild-type (n = 28) mice at P31, i.e. 22 days after hypoxia-ischemia. The numbers represent cells counted on 8 µm thick stacks of confocal images within the 450 µm×300 µm optical fields in the respective brain regions. (C) Orthogonal sections showing NeuN⁺BrdU⁺ cells. Data are mean ± SD. (* p<0.05, ** p<0.01, *** p<0.001). Scale bar = 15 µm.</p

Visualization of neuronal axons in the infact area and in the contralateral hemisphere.

<p>Neuronal axons were visualized with antibodies against neurofilament proteins on sections from wild-type (A-D) and <i>GFAP^−/−Vim^−/−</i> (E-H) mice at P12, i.e. 3 days after hypoxia-ischemia. The frames indicate respective fields for higher magnification. Scale bar for A, C, E, G = 500 µm; for B, D, F, H = 50 µm.</p