Lab Anim Res.  2016 Dec;32(4):194-199. 10.5625/lar.2016.32.4.194.

Ischemic brain injury decreases dynamin-like protein 1 expression in a middle cerebral artery occlusion animal model and glutamate-exposed HT22 cells

• Jang AR
• Koh PO

• KMID: 2362910
• DOI: http://doi.org/10.5625/lar.2016.32.4.194

Abstract

Dynamin-like protein I (DLP-1) is an important mitochondrial fission and fusion protein that is associated with apoptotic cell death in neurodegenerative diseases. In this study, we investigated DLP-1 expression in a focal cerebral ischemia animal model and glutamate-exposed hippocampal-derived cell line. Middle cerebral artery occlusion (MCAO) was surgically induced in adult male rats to induce focal cerebral ischemic injury. Brain tissues were collected 24 hours after the onset of MCAO. MCAO induces an increase in infarct volume and histopathological changes in the cerebral cortex. We identified a decrease in DLP-1 in the cerebral cortices of MCAO-injured animals using a proteomic approach and Western blot analysis. Moreover, glutamate treatment significantly decreased DLP-1 expression in a hippocampal-derived cell line. The decrease in DLP-1 indicates mitochondrial dysfunction. Thus, these results suggest that neuronal cell injury induces a decrease in DLP-1 levels and consequently leads to neuronal cell death.

Keyword

Brain ischemia; MCAO; dynamin-like protein 1; hippocampal-derived cell line

MeSH Terms

Adult
Animals
Blotting, Western
Brain Injuries*
Brain Ischemia
Brain*
Cell Death
Cell Line
Cerebral Cortex
Glutamic Acid
Humans
Infarction, Middle Cerebral Artery*
Male
Middle Cerebral Artery*
Mitochondrial Dynamics
Models, Animal
Neurodegenerative Diseases
Neurons
Rats
Glutamic Acid

Figure

• 

  Figure 1 Representative photos of TTC stain (A and B) and hematoxylin-eosin stain (C) in the sham-operated and middle cerebral artery-occluded (MCAO) animals. The ischemic area remained white, while the intact area was stained red (A). The percentage of ischemic lesion area was calculated by the ratio of the infarction area to the whole slice area (B). Data (n=5) are represented as mean±S.E.M. *P<0.01. Arrows indicate pathological changes with scalloped shrunken form and intensive dark masses (C). Scale bar=100 µm.

• 

  Figure 2 Dynamin-like protein 1 (DLP-1) protein spots identified by MALDI-TOF in the sham-operated and middle cerebral artery-occluded (MCAO) animals. Circles indicate DLP-1 protein spots. The intensity of spots was measured using PDQuest software. The ratio of intensity is described as spots intensity of MCAO animal to spots intensity of sham-operated animal. Data are shown as mean±S.E.M. *P<0.05. (vs. Sham) Mw and pI indicate molecular weight and isoelectrical point, respectively.

• 

  Figure 3 Western blot analysis of dynamin-like protein 1 (DLP-1) in the cerebral cortices from sham-operated and middle cerebral artery-occluded (MCAO) animals. Densitometric analysis is represented as intensity of these proteins to intensity of actin. Molecular weight markers (kDa) are depicted at left. Data (n=6) are represented as mean±S.E.M. *P<0.05.

• 

  Figure 4 Cell viability (A) and Western blot analysis of dynamin-like protein 1 (DLP-1) in HT22 cells. Glutamate (5 mM) or vehicle was exposed to HT22 cells for 24 hours. Cellular viability was assessed using the MTT assay. Cell survival was expressed as percentage of neuroprotection vs. vehicle set at 100% (A). Densitometric analysis is represented as intensity of DLP-1 to intensity of actin (C). Data (n=5) are represented as mean±S.E.M. *P<0.05 (vs. Vehicle)

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