J Korean Neurosurg Soc.  2014 Jun;55(6):307-312. 10.3340/jkns.2014.55.6.307.

Semi-Quantitative Analyses of Hippocampal Heat Shock Protein-70 Expression Based on the Duration of Ischemia and the Volume of Cerebral Infarction in Mice

• Choi JI
• Kim SD
• Kim SH
• Lim DJ
• Ha SK

• KMID: 2191109
• DOI: http://doi.org/10.3340/jkns.2014.55.6.307

Abstract

OBJECTIVE
We investigated the expression of hippocampal heat shock protein 70 (HSP-70) infarction volume after different durations of experimental ischemic stroke in mice.
METHODS
Focal cerebral ischemia was induced in mice by occluding the middle cerebral artery with the modified intraluminal filament technique. Twenty-four hours after ischemia induction, both hippocampi were extracted for HSP-70 protein analyses. Slices from each hemisphere were stained with 2,3,5-triphenyltetrazolium chloride (2%), and infarction volumes were calculated. HSP-70 levels were evaluated using western blot and enzyme-linked immunosorbent assay (ELISA). HSP-70 subtype (hsp70.1, hspa1a, hspa1b) mRNA levels in the hippocampus were measured using reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS
Cerebral infarctions were found ipsilateral to the occlusion in 10 mice exposed to transient ischemia (5 each in the 30-min and 60-min occlusion groups), whereas no focal infarctions were noted in any of the sham mice. The average infarct volumes of the 2 ischemic groups were 22.28+/-7.31 mm3 [30-min group+/-standard deviation (SD)] and 38.06+/-9.53 mm3 (60-min group+/-SD). Western blot analyses and ELISA showed that HSP-70 in hippocampal tissues increased in the infarction groups than in the sham group. However, differences in HSP-70 levels between the 2 infarction groups were statistically insignificant. Moreover, RT-PCR results demonstrated no relationship between the mRNA expression of HSP-70 subtypes and occlusion time or infarction volume.
CONCLUSION
Our results indicated no significant difference in HSP-70 expression between the 30- and 60-min occlusion groups despite the statistical difference in infarction volumes. Furthermore, HSP-70 subtype mRNA expression was independent of both occlusion duration and cerebral infarction volume.

Keyword

Cerebral infarction volume; Heat shock protein; Occlusion time

MeSH Terms

Animals
Blotting, Western
Brain Ischemia
Cerebral Infarction*
Enzyme-Linked Immunosorbent Assay
Heat-Shock Proteins
Hippocampus
Hot Temperature*
HSP70 Heat-Shock Proteins
Infarction
Ischemia*
Mice*
Middle Cerebral Artery
RNA, Messenger
Shock*
Stroke
HSP70 Heat-Shock Proteins
Heat-Shock Proteins
RNA, Messenger

Figure

• 

  Fig. 1 A : Infarct areas are shown as pale (unstained) regions after 2,3,5-triphenyltetrazolium chloride staining. B : Quantitative analyses of infarct volumes at 30-min or 60-min after middle cerebral artery occlusion. The infarct volumes at 60-min are significantly larger than the volume at 30 min (Mann-Whitney U-test, p=0.032). Lt : left.

• 

  Fig. 2 Western blot (A) and quantitative analysis (B) show statistical differences in HSP-70 expression between the ischemic groups (30- and 60-min) and the sham group in the ipsilateral (left, Lt) hippocampi of the mice (Kruskal-Wallis tests, p=0.034), whereas that between the 30- and 60-min groups does not (p=0.730). No statistical differences are noted in the contralateral (right, Rt) side (Kruskal-Wallis tests, p=0.792). Data are expressed as the mean±SD. *Significance at p≤0.05. HSP-70 : heat shock protein 70, SD : standard deviation.

• 

  Fig. 3 ELISA quantitative analyses. Statistical differences in HSP-70 expression between the ischemic groups (30- and 60-min) and the sham group in the ipsilateral (left, Lt) hippocampi of mice are evident (Kruskal-Wallis tests, p=0.011), whereas that between the 30- and 60-min groups are not (p=0.971). No statistical differences are noted in the contralateral (right, Rt) side (Kruskal-Wallis tests, p=0.058). Data are expressed as the mean±SD. ELISA : enzyme-linked immunosorbent assay, HSP-70 : heat shock protein 70, SD : standard deviation.

• 

  Fig. 4 HSP mRNA expression (hsp70.1, hspa1a, and hspa1b) is not dependent on MCA occlusion time (Kruskal-Wallis tests, p>0.05). Data are expressed as the mean±SD. No significant differences in HSP mRNA expression (hsp70.1, hspa1a, hspa1b) in the bilateral (Rt : contralateral, Lt : ipsilateral) hippocampi of mice are evident (Kruskal-Wallis tests, p>0.05). Data are expressed as the mean±SD. HSP : heat shock protein, mRNA : messenger RNA, MCA : middle cerebral artery, SD : standard devicetion.

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