Histological analysis for neurons and myelin.

<p>Luxol Fast Blue-Cresyl Violet staining was performed in serial brain sections from control and MCAO rats for examination of myelin and neuron condition. (<b>A</b>) In controls, neurons were well established with distinct cell bodies and nuclei in examined cerebral structures from both hemispheres. Myelin also appeared normal. (<b>B</b>) In the brains of rats 7 days after MCAO, neuronal pyknosis and cellular debris were observed in the border zone of peri-infarct areas of striatum and somatosensory cortex in ipsilateral hemisphere. Fewer pyknotic neurons were seen in ipsilateral motor cortex, however, cell density was decreased compared to controls. Myelin thickness was also reduced. The lateral ventricle was expanded in the ipsilateral hemisphere. In the hemisphere contralateral to MCAO insult, fewer myelin sheaths were determined in somatosensory and motor cortices and striatum. Contralateral striatosomes also showed size decreases similar to striatosomes within infarct area. Neuronal density in motor and somatosensory cortices was diminished compared to controls. Some neurons appeared pyknotic. Scale bar in full brain images is 200 µm, in striatum is 100 µm, in motor and somatosensory cortices is 50 µm. (<b>C</b>) Semi-quantitative analysis of Cresyl Violet stained brain samples demonstrated reduction of neuronal densities in ipsilateral striatum, motor and somatosensory cortices. Neuronal densities were also diminished in analyzed cerebral cortices. Of note, neuronal cell densities in ipsilateral striatum and somatosensory cortex were analyzed outside of perilesional areas. (<b>D</b>) Semi-quantitative analysis of Luxol Fast Blue stained brain samples showed decrease of myelin intensity, mainly, in ipsilateral striatum and somatosensory cortex vs. control (baseline). Reduced myelin staining was also determined in contralateral striatum.</p

Electron microscope examination of microvasculature in the rat striatum 7 days after MCAO.

<p>Representative area of control rat striatum (<b>A, B</b>)was characterized by the normal ultrastructural appearance of neurons, capillaries, neuropil, myelinated axons, and surrounding astrocytes. A single layer of endothelial cells (ECs) is surrounded by a single layer of basement membrane (BM), forming an intact BBB. (<b>C</b>) In the hemisphere ipsilateral to MCAO insult, ultrastructural abnormalities were observed in striatum capillary endothelia. ECs showed endoplasmic reticulum swelling and formation of numerous large vacuoles in their cytoplasm. Mitochondria in the cytoplasm of most cells showed disruption of cristae. In the lumen of the capillary, fragments of microvilli and mitochondria that have been shed from EC were observed. Near the capillaries were spaces created by degenerating astrocyte cell processes and protein-filled areas. (<b>D</b>) A capillary with condensed EC cytoplasm was also observed in ipsilateral striatum. An autophagosome and dilated endoplasmic reticulum and lysosomal membranes were detected in EC. The second EC appeared to be separated from the BM. (<b>E</b>) In contralateral striatum, capillaries contain a necrotic EC and a swollen EC. A degenerated pericyte is also apparent. Some areas of edema surround the capillary. A capillary (<b>F</b>) with a swollen EC layer containing enlarged mitochondria was determined. Profiles of dilated endoplasmic reticulum and autophagocytic vacuole were observed in EC cytoplasm. Another capillary contained ECs with condensed cytoplasm and vacuoles (<b>G</b>). Surrounding the capillary were astrocytes showing severe edema. <b>En</b> - endothelial cell, <b>BM</b> - basement membrane, <b>Ast</b> – astrocyte, <b>E</b> – erythrocyte, <b>m</b> – mitochondrion, <b>A</b> – axon, <b>V</b> – vacuole, <b>P</b> – pericyte, <b>Nu</b> – nucleus, <b>Aph</b> – autophagosome, <b>Cr</b> – disrupted cristae in the mitochondrion,<b>+</b>- swollen endoplasmic reticulum, <b>arrow</b> in <b>C</b> - broken microvilli, arrowheads in <b>D</b> indicate separation of EC from BM. Asterisks in (<b>C</b>), (<b>E</b>), (<b>F</b>), (<b>G</b>) indicate extracellular edema. Magnification in (<b>A</b>) is 8,900; in (<b>B</b>), (<b>C</b>), (<b>D</b>), (<b>E</b>) is 11,000; in (<b>F</b>) is 14,000, in (<b>G</b>) is 5,600.</p

Immunofluorescence and quantitative analysis of Evans Blue extravasation into the rat brain parenchyma 7 days after MCAO.

<p>(<b>A</b>) In the brains of control rats, EB (red) was clearly detected within capillary lumen of motor cortex (<b>a, a’</b>), striatum (<b>b, b’</b>), and somatosensory cortex (<b>c, c’</b>) using immunofluorescence technique. Collagen IV immunostaining (arrowheads, green) was strongly apparent in basement membrane of vessels. At 7 days after MCAO, significant EB extravasation (asterisks, red) was identified in hemisphere ipsilateral to initial ischemic insult in striatum (<b>h, h’</b>), somatosensory (<b>i, i’</b>) and motor (<b>g, g’</b>) cortices. Extensive EB leakage was also seen in same cerebral structures of contralateral hemisphere (<b>d–f’</b>). Diminished or weak immunoexpression for collagen IV (arrowheads, green) was observed in numerous capillaries of both ipsi- and contralateral hemispheres. In full brain images and a–i’, blue color indicates DAPI staining of cell nuclei. Some images contain a purple background due to high density of cell nuclei (blue) and extravasated EB (red). Scale bar in full brain images is 200 µm; in a, d, g is 50 µm (representative for each panel a’–i’). (<b>B</b>) Quantitative measurement of tissue EB content showed significantly (p<0.0001) higher extravasated EB levels in ipsi- and contralateral hemispheres vs. control. Significantly (p = 0.0006) elevated EB level was determined in ipsilateral hemisphere compared to contralateral.</p

Immunohistochemical analysis of activated microglia.

<p>A few OX-6 positive cells (arrowhead) were identified in motor (<b>a</b>) and somatosensory (<b>b</b>) cortices and striatum (<b>c</b>) in control rats. A large number of activated microglial cells were determined in ipsilateral MCAO hemisphere, in brain structures with EB extravasation (<b>d–f</b>). Morphologically, these cells were characterized by large cell bodies and short processes. In the contralateral hemisphere, activated microglia were observed, primarily in the striatum (<b>i</b>) and somatosensory cortex (<b>h</b>). Some OX-6 positive cells were also seen in the contralateral motor cortex (<b>g</b>). Arrowheads indicate microglial cells. Asterisks indicate EB leakage (red). Images of OX-6 expression were converted to grayscale to better display microglia processes in white on black background. Scale bar in a-i is 25 µm. (<b>B</b>) Semi-quantitative analysis showed highest degree of OX-6 immunoexpression in ipsilateral striatum vs. control (baseline).</p

Immunohistochemical analysis of astrocytes.

<p>(<b>A</b>) Immunohistochemical analysis of astrocytes in the brains of control rats showed normal appearance of cells in the motor cortex (<b>a, a’</b>), somatosensory cortex (<b>b, b’</b>), and striatum (<b>c, c’</b>) parenchyma. GFAP positive cells (green) were distinguished surrounding capillaries (arrowheads) in control brains. In the brains of rats 7 days post-MCAO, astrogliosis was noted in striatum, motor and somatosensory cortices in ipsilateral (<b>d–f</b>’) and contralateral (<b>g–i</b>′) hemispheres. Alterations of astrocytic end-feet were marked in some capillaries. Asterisks indicate EB leakage (red). Scale bar in a–i is 50 µm, in a’–i’ (higher magnification images of <b>a–i</b>) is 25 µm. (<b>B</b>) Immunoexpression for GFAP was analyzed semi-quantitatively and higher scores were determined in areas of ipsilateral brain structures vs. control (baseline). Importantly, the same high degree of GFAP expression was noted for ipsi- and contralateral striatum and motor cortex.</p