Morphological changes in C6 glioma cells induced by shikonin.

<p>(A) Images under transmission electron microscope. A1, normal C6 glioma cells; A2, enlarged cellular image labeled with asterisk in A1; A3, C6 cells treated 3h with 6.0 μmol/L shikonin; A4, enlarged cellular image labeled with asterisk in A3. Compared with normal C6 glioma cells, morphological features of necrosis such as electron-lucent cytoplasm, swollen or disrupted organelles, intact nuclear membrane and loss of plasma membrane were found in the C6 cells treated with shikonin. (B) Nuclear images under fluorescence microscope. Blue color represented being stained with Hoechst 33342, and red color represented PI staining. The nucleus in C6 glioma cell treated with 0 µmol/L shikonin or Nec-1 alone showed round figure and homogenous blue color. In the shikonin treated groups, part of the nuclei stained with red color showed no changes in the nuclear figure or size and density of blue color, indicating they were necrotic cells (arrow). By contrast, another part of the nuclei with red staining displayed increased fluo-density in blue color and condensed nuclear size, indicating that they were apoptotic cells (arrow head). Pretreatment with Nec-1 decreased necrotic cells, but apoptotic cells still could be found. Scale bar  = 20 µm.</p

Long-term effects of shikonin on the cellular viability of C6 glioma cells.

<p>MTT assay showed that the decreased cellular viability of C6 glioma cells caused by shikonin was dependent on shikonin concentration and incubation time. *: <i>p</i><0.01 versus control group.</p

Short-term effects of shikonin on the cellular viability of C6 and U87 glioma cells.

<p>MTT assay showed that the viability of C6 (A) and U87 (B) glioma cells decreased in a dose dependent manner when incubated 3 hours with shikonin. However, the inhibitory effects of shikonin on the viability of C6 (C) and U87 (D) glioma cells was suppressed significantly by pretreatment with Nec-1, but not by z-VAD-fmk.</p

Flow cytometry analysis of glioma cell death mode caused by shikonin.

<p>(A) Representative image of flow cytometry of C6 (A) and U87 glioma cells (B). After 3 hours incubation either with lower or higher concentration of shikonin, both the percentage of necrotic cells and late apoptotic cells increased significantly. The necrotic and late apoptotic cells were significantly inhibited by pretreatment with Nec-1, but did not suppress by pre-incubation with z-VAD-fmk. These data indicated that Nec-1 blocked shikonin induced necrosis in glioma cells. Additionally, late apoptotic cells did not reduce when they were pretreated with z-VAC-fmk but attenuated by Nec-1, indicating they were necrotic cells as well. Further, the necroptotic cells were suppressed by antioxidant NAC.</p

Changes in ROS production and cellular viability in glioma cells caused by shikonin.

<p>The left panels in A and D were the images of C6 and U87 glioma cells under phase contrast microscope, and the right panel were the fluorescence microscopic images of ROS generation by shikonin using DCFH-DA staining (4X). B and E were statistical analysis of cellular viability in C6 and U87 glioma cells. C and F were statistical analysis of fluodensity. The images showed that the ROS level increased significantly in both C6 and U87 glioma cells treated with shikonin while cellular viability decreased when compared with control group. However, no significant difference in fluodensity was found either in C6 or U87 cells between lower or higher concentration of shikonin groups. By contrast, pretreatment with Nec-1 or NAC inhibited the production of ROS and rescued C6 and U87 glioma cell death caused by shikonin. Scale bar  = 40 μm. *: <i>p</i><0.01 versus control group; #: <i>p</i><0.01 versus control group.</p

CT and ESWAN images of the brain of a 50-year-old man (A, B) and a 59-year-old man (C, D).

<p>The Fisher grades of the SAH based on the CT images were grade 2 (A) and grade 4 (C), respectively. Follow-up MR (1.5T) images were obtained at 34 weeks (B) and 177 weeks (D) after the head injury. The <i>arrows</i> denote areas of SS-CNS, which is indicated by a rim of hypointensity on the surface of the brain. Note the extent of deposition in each case. The CT and ESWAN images display a degree of correlation, patients with a higher Fisher grade exhibit more regions containing SS-CNS deposits.</p

An MRI performed on a healthy subject using spin-echo T1-weighted imaging.

<p>We obtained axial slices of the normal brain. The anatomical structures of the subarachnoid space were labeled according to the following method. The subarachnoid area of each cerebral hemisphere was divided into 5 regions (frontal lobe, temporal lobe, occipital lobe, parietal lobe, and Sylvian fissure), and the subarachnoid areas of the cerebellum and the brainstem were divided into two relatively independent regions. Then, the number of regions, ranging from 0 to 12, of the subarachnoid area of the entire brain, excluding the ventricular system, was determined.</p

Univariate analyses of factors associated with extent of SS-CNS deposition based on ESWAN in 31 patients.

<p>SS, superficial siderosis; ESWAN, enhanced gradient echo T2 star-weighted angiography.</p><p>*Student’s t-test, t = −0.434, P>0.05.</p><p>**Fisher's exact test, P<0.05.</p><p>†3 (3 patients classified as grade 3), 13 (11 patients classified as grade 2 and 2 patients as grade 1).</p><p>‡ 11 (2 patients classified as grade 3and 9 patients as grade 4), 4 (4 patients classified as grade 2).</p><p>Univariate analyses of factors associated with extent of SS-CNS deposition based on ESWAN in 31 patients.</p

Chi-square contingency table analysis reveals a correlation between the number of regions exhibiting tSAH (%) based on CT images and the number of regions exhibiting SS-CNS (%) based on ESWAN images in 31 patients (<i>χ</i>^<i>2</i> = 17.73, <i>P</i><0.05).

<p>Chi-square contingency table analysis reveals a correlation between the number of regions exhibiting tSAH (%) based on CT images and the number of regions exhibiting SS-CNS (%) based on ESWAN images in 31 patients (<i>χ</i>^<i>2</i> = 17.73, <i>P</i><0.05).</p

CT and enhanced gradient echo T2 star-weighted angiography (ESWAN) images of the brain of a 54-year-old man who experienced a traumatic brain injury.

<p>An axial head CT image displays right frontotemporal SAH (Fisher grade 4) with bilateral frontal contusions and intracerebral hematoma (A). A follow-up CT image 26 weeks after the brain injury indicates that the hemorrhages were completely resolved and the lateral ventricles were mildly enlarged (B). A follow-up MRI (1.5T) image was obtained 26 weeks following the head injury (C,D). The axial ESWAN image displays a rim of hypointensity (<i>arrowheads</i>), with hemosiderin deposits forming along the cerebral convexity (C, D).</p