J Korean Med Sci.  2006 Jun;21(3):490-494. 10.3346/jkms.2006.21.3.490.

Therapeutic Window for Cycloheximide Treatment after Hypoxic-Ischemic Brain Injury in Neonatal Rats

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

• KMID: 1778434
• DOI: http://doi.org/10.3346/jkms.2006.21.3.490

Abstract

We have previously shown that cycloheximide significantly inhibited apoptosis, and reduced ensuing cerebral infarction in a newborn rat model of cerebral hypoxiaischemia. This study was performed to determine the therapeutic window for cycloheximide therapy. Seven day-old newborn rat pups were subjected to 100 min of 8% oxygen following a unilateral carotid artery ligation, and cycloheximide was given at 0, 6, 12 and 24 hr after hypoxia-ischemia (HI). Apoptosis or necrosis was identified by performing flow cytometry with a combination of fluorescinated annexin V and propidium iodide, and the extent of cerebral infarction was evaluated with triphenyl tetrazolium chloride (TTC) at 48 hr and 72 hr after HI, respectively. With cycloheximide treatment at 0 hr after HI, both apoptotic and necrotic cells by flow cytometry were significantly reduced, only necrotic cells were significantly reduced at 6 and 12 hr, and no protective effect was seen if administration was delayed until 24 hr after HI compared to the HI control group. Infarct volume, measured by TTC, was significantly reduced by 92% and 61% when cycloheximide was given at 0 or 6 hr after HI respectively; however, there was an insignificant trend in infarct reduction if cycloheximide was administered 12 hr after HI, and no protective effect was observed when administration was delayed until 24 hr after HI. In summary, cycloheximide was neuroprotective when given within 6 hr after HI in the developing newborn rat brain.

Keyword

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

MeSH Terms

Rats, Sprague-Dawley
Rats
Protein Synthesis Inhibitors/therapeutic use
Oxygen/metabolism
Neuroprotective Agents/*therapeutic use
Necrosis
Hypoxia-Ischemia, Brain/*drug therapy
Hypoxia, Brain
Humans
Flow Cytometry
Cycloheximide/*therapeutic use
Brain Ischemia
Apoptosis
Animals, Newborn
Animals

Figure

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  Fig. 1 Representative flow cytogram of an annexin V binding (abscissa) versus propidium iodide (PI) uptake (ordinate) (A) and regional cell percentage (B) in the ipsilateral hemisphere of the newborn rat brain cells at 48 hr after hypoxia-ischemia. The numbers in the left upper quadrant (Q1), right upper quadrant (Q2), left lower quadrant (Q3) and right lower quadrant (Q4) represent the percentage of damaged (annexin V-/PI+), necrotic (annexin V-/PI+), live (annexin V-/PI-), and apoptotic (annexin V+/PI-) cells, respectively. HI represents hypoxia-ischemia control group; HI_0, 6, 12, 24 represent hypoxia-ischemia with cycloheximide treatment group at 0, 6, 12 and 24 hr post HI, respectively. All values are mean±standard deviation.*, p<0.05 compared to HI;†, p<0.05 compared to HI_0;‡, p<0.05 compared to HI_6.

• 

  Fig. 2 Representative photomicrographs of triphenyl tetrazolium chloride staining (A) and percentage of infarct volume (B) from the hypoxia-ischemia control group (HI) and hypoxia-ischemia with cycloheximide treatment group at 0, 6, 12, 24 and 24 hr after HI (HI_0, 6, 12, 24), respectively. Note significantly reduced infarct volume both in HI_0 and 6 compare HI. All values are mean±standard deviation.*, p<0.05 compared to HI;†, p<0.05 compared to HI_0;‡, p<0.05 compared to HI_6.

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