J Korean Med Sci.  2006 Apr;21(2):337-341. 10.3346/jkms.2006.21.2.337.

Neuroprotective Effect of Cycloheximide on Hypoxic-Ischemic Brain Injury in Neonatal Rats

• Park WS
• Sung DK
• Kang S
• Koo SH
• Kim YJ
• Lee JH
• Chang YS
• Lee M

• KMID: 1781846
• DOI: http://doi.org/10.3346/jkms.2006.21.2.337

Abstract

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.

Keyword

Cell Separation; Hypoxia-Ischemia, Brain; Animals, Newborn; Apoptosis; Cycloheximide

MeSH Terms

Time Factors
Rats, Sprague-Dawley
Rats
Propidium
Neuroprotective Agents/*pharmacology
In Situ Nick-End Labeling
Hypoxia-Ischemia, Brain/*drug therapy/metabolism/pathology
Cycloheximide/*pharmacology
Brain Infarction/pathology/prevention & control
Apoptosis/drug effects
Annexin A5/metabolism
Animals, Newborn
Animals

Figure

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  Fig. 1 Representative photomicrographs of Eosin staining (A) and TUNEL staining (B) at the anterior commisure from the hypoxia-ischemia control group (HI) and hypoxia-ischemia with cycloheximide treatment group (HI-CHX) 1 and 4 week after the injury. Note progression of infarction in HI, and significantly reduced infarct area and less TUNEL-positive staining cells bordering the infarct of HI-CHX, compared to HI.

• 

  Fig. 2 Percentage of normal brain area at the anterior commissure from the hypoxia-ischemia control group (HI) and hypoxia-ischemia with cycloheximide treatment group (HI-CHX) 1 and 4 week after the injury. Significantly increased infarct area at 4 week compared to 1 week after the insult in HI, and significantly reduced infarct area of HI-CHX, compared to HI, was observed. *, p<0.05 compared to HI. †, p<0.05 compared to 1 week. All values are mean±standard deviation.

• 

  Fig. 3 Representative photomicrographs of fluorescent staining for TUNEL (green) and DAPI (blue) in the ipsilateral cortex (A) and the number of TUNEL positive cells (B) from the hypoxia-ischemia control group (HI)and hypoxia-ischemia with cycloheximide treatment group (HI-CHX) 48 hr after the injury. Significantly less TUNEL-positive staining cells of HI-CHX, compared to HI, are observed. All values are mean±standard deviation. *, p<0.05 compared to HI. Original magnification×400.

• 

  Fig. 4 Representative flow cytogram of an annexin V binding (abscissa, FL1) versus propidium iodide uptake (ordinate, FL2) in the ipsilateral hemisphere of the newborn rat brain cells at 48 hr after hypoxia-ischemia. The numbers in the left upper quadrant, right upper quadrant, left lower quadrant and right lower quadrant represent the percentage of damaged (annexin V-/PI+), necrotic (annexin V+/PI+), live (annexin V-/PI-), and apoptotic cells, respectively. HI, hypoxia-ischemia control group; HI-CHX, hypoxia-ischemia with cycloheximide treatment group.

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