Change in the number of mitochondria after I-R.

<p>(A) Transmission electron microscopy images of a section from the cortex showing the nucleus (Nu) surrounded by relatively uniform and compact mitochondria (arrowheads). Scale bars: 2 μm. (B) A histogram quantifying the number of mitochondria in the cortical cells. Magnification of the brain sections, 8200x. *P<0.05 versus the sham group.</p

Representative histological characteristics of the cortical brain sections assessed using HE staining.

<p>(A and B) The cortical sections from the sham group revealed normal cortical tissue. (C and D) The cortical sections from the I-R 0 h group displayed cortical tissue injury. (E and F) The cortical sections from the I-R 24 h group demonstrated aggravated cortical tissue injury. (G and H) The cortical sections from the I-R 72 h group showed improved residual neurons in the damaged cortex. (I and J) The cortical sections from the I-R 7 d group showed an increased number of cells similar to glial cells in that they appeared round or elongated (arrowheads).</p

mitochondrial DNA Sequences.

<p>mitochondrial DNA Sequences.</p

Reperfusion Promotes Mitochondrial Biogenesis following Focal Cerebral Ischemia in Rats

<div><p>Background and Purpose</p><p>Reperfusion after transient cerebral ischemia causes severe damage to mitochondria; however, little is known regarding the continuous change in mitochondrial biogenesis during reperfusion. Mitochondrial biogenesis causes an increase in the individual mitochondrial mass of neurons and maintains their aerobic set-point in the face of declining function. The aim of this study was to examine mitochondrial biogenesis in the cortex during reperfusion following focal cerebral ischemia.</p><p>Methods</p><p>Male Wistar rats were subjected to transient focal cerebral ischemia. The relative amount of cortical mitochondrial DNA was analyzed using quantitative real-time PCR at 0 h, 24 h, 72 h, and 7 d after reperfusion. Three critical transcriptional regulators of mitochondrial biogenesis were measured by semi-quantitative reverse-transcription PCR. The protein expression of cytochrome C oxidase subunits I and IV was detected by Western blotting.</p><p>Results</p><p>Evidence of increased mitochondrial biogenesis was observed after reperfusion. The cortical mitochondrial DNA content increased after 24 h, peaked after 72 h, and maintained a high level for 7 d. The cortical expression of three critical genes for the transcriptional regulation of mitochondrial biogenesis, namely, peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcription factor A, also increased at 24 h and 72 h. The expression of peroxisome proliferator-activated receptor coactivator-1α returned to the baseline level at 7 d, but two other factors maintained higher levels compared with the controls. Moreover, the expression of cytochrome C oxidase subunits I and IV was increased in the cortex.</p><p>Conclusions</p><p>These results indicate that reperfusion increased mitochondrial biogenesis following focal cerebral ischemia, and this tendency was exacerbated as the reperfusion time was extended. Reperfusion-induced mitochondrial biogenesis was mediated through up-regulation of critical transcriptional regulators of mitochondrial biogenesis.</p></div

RT-PCR primer sequences.

<p>RT-PCR primer sequences.</p

mRNA expression of the mitochondrial biogenesis factors in the cortex.

<p>(A) Representative agarose gel of PGC-1α, NRF-1, and TFAM RT-PCR products prepared from the mRNA of the sham group or the I-R 0 h, I-R 24 h, I-R 72 h, and I-R 7 d groups. (B) The histogram shows the semi-quantitative measurement of the PCR products in (A), which were obtained using densitometric analysis. The data are expressed as the means±SD. *P<0.05 versus the I-R 0 h and sham groups; #P<0.05 versus the I-R 24 h and I-R 7 d groups.</p

Changes in the citrate synthase activity.

<p>The citrate synthase activity was expressed as (μmols of substrate transformed)×min⁻¹×(mg protein)⁻¹ of citrate synthase. The data are expressed as the means±SD. *P<0.05 versus the I-R 0 h and sham groups; #P<0.05 versus the I-R 24 h and I-R 7 d groups.</p

Change in the relative amounts of mtDNA after I-R.

<p>(A) Cortical tissue samples indicated by dotted line were used for PCR, Western Blot examination, citrate synthase activity assay, and morphology observation. (B) The relative amounts of cortical mtDNA were measured using quantitative real-time PCR in the sham group and the I-R 0 h, I-R 24 h, I-R 72 h, and I-R 7 d groups. The data are expressed as the means±SD. *P<0.05 versus the I-R 0 h and sham groups; #P<0.05 versus the I-R 24 h and I-R 7 d groups.</p

Physiological parameters.

<p>All values are presented as the mean±SD. Arterial blood gas tensions include PaO₂, PaCO₂, and pH.</p><p>MAP, mean arterial pressure; PaO₂, arterial oxygen pressure; PaCO₂, arterial carbon dioxide pressure.</p

Expression of COX-I and COX-IV in the cortex.

<p>(A) Western blots showing representative COX-I and COX-IV protein expression in the whole-cell protein. β-actin was used as a control. (B) Relative analysis of the COX-I and COX-IV protein levels in the sham group and the I-R 0 h, I-R 24 h, I-R 72 h, and I-R 7 d groups. (C) Western blots showing the representative COX-I and COX-IV protein expression in the non-synaptic mitochondrial fraction. (D) Relative analysis of the COX-I and COX-IV protein levels of mitochondrial fraction in the sham group and in the I-R 0 h, I-R 24 h, I-R 72 h, and I-R 7 d groups. The data are expressed as the means±SD. *P<0.05 versus the I-R 0 h and sham groups; #P<0.05 versus the I-R 24 h and I-R 7 d groups.</p