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. 2012 Oct 5;7(28):2221-6.
doi: 10.3969/j.issn.1673-5374.2012.028.008.

Expression of Clock genes in the pineal glands of newborn rats with hypoxic-ischemic encephalopathy

Bin Sun  1 , Xing Feng  1 , Xin Ding  1 , Li Bao  2 , Yongfu Li  3 , Jun He  4 , Meifang Jin  1
Affiliations

Expression of Clock genes in the pineal glands of newborn rats with hypoxic-ischemic encephalopathy

Bin Sun et al. Neural Regen Res. .

Abstract

Clock genes are involved in circadian rhythm regulation, and surviving newborns with hypoxic-ischemic encephalopathy may present with sleep-wake cycle reversal. This study aimed to determine the expression of the clock genes Clock and Bmal1, in the pineal gland of rats with hypoxic-ischemic brain damage. Results showed that levels of Clock mRNA were not significantly changed within 48 hours after cerebral hypoxia and ischemia. Expression levels of CLOCK and BMAL1 protein were significantly higher after 48 hours. The levels of Bmal1 mRNA reached a peak at 36 hours, but were significantly reduced at 48 hours. Experimental findings indicate that Clock and Bmal1 genes were indeed expressed in the pineal glands of neonatal rats. At the initial stage (within 36 hours) of hypoxic-ischemic brain damage, only slight changes in the expression levels of these two genes were detected, followed by significant changes at 36-48 hours. These changes may be associated with circadian rhythm disorder induced by hypoxic-ischemic brain damage.

Keywords: Bmal1; Clock; brain; brain hypoxia; cerebral ischemia; mRNA; neonatal rats; neural regeneration; pineal gland; protein.

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Conflict of interest statement

Conflicts of interest: None declared.

Figures

Figure 1
Figure 1
mRNA expression of the Clock gene in the rat pineal gland after hypoxic-ischemic brain damage (HIBD). (A) Representative reverse transcription-PCR image. (B) Quantification of Clolck mRNA expression. Data were expressed as the gray scale ratio of target gene to β-actin gene (mean ± SD from six rats per group). M: Marker; 0 h, 2 h, 12 h, 24 h, 36 h, 48 h: Clock mRNA levels at 0, 2, 12, 24, 36, 48 hours after HIBD or sham operation. The expression of Clock mRNA in the pineal gland does not show rhythmic variation in both groups (P > 0.05, t-tests)
Figure 2
Figure 2
Changes in the expression levels of CLOCK protein in the pineal gland after hypoxic-ischemic brain damage (HIBD). (A) Representative western blot image. GAPDH was used as an internal control. (B) Quantification of CLOCK protien expression. The relative expression levels of CLOCK protein are represented as the fold change compared with the control group. The expression levels of CLOCK protein were significantly higher than that of the control group at 48 hours after HIBD. Data are expressed as mean ± SD of six independent experiments and were analyzed by t-tests. aP < 0.05, vs. control group. C0: normal group. A0, A2, A12, A24, A36, A48: CLOCK protein levels at 0, 2, 12, 24, 36, 48 hours after HIBD. B0, B2, B12, B24, B36, B48: CLOCK protein levels at 0, 2, 12, 24, 36, 48 hours in the control group.
Figure 3
Figure 3
mRNA expression of the Bmal1 gene in the rat pineal gland after hypoxic-ischemic brain damage (HIBD). (A) Representative reverse transcription-PCR image. (B) Quantification of Bmal1 mRNA expression. Bmal1 mRNA expression increased at 36 hours and decreased at 48 hours after HIBD compared with that of the control group. Data are expressed as the gray scale ratio of target gene to β-actin gene (mean ± SD from six rats per group). aP < 0.05, vs. control group (t-tests). M: Marker. 0 h, 2 h, 12 h, 24 h, 36 h, 48 h: Bmal1 mRNA levels at 0, 2, 12, 24, 36, 48 hours after HIBD or sham operation.
Figure 4
Figure 4
Changes in the expression levels of BMAL1 protein in the pineal gland after hypoxic-ischemic brain damage (HIBD). (A) Representative image of a western blot analysis. GAPDH was used as the internal control. (B) Quantification of BMAL1 protien expression. The relative expression level of BMAL1 protein is represented as the fold change compared with the control group. The expression levels of BMAL1 protein were significantly higher than that of the control group at 48 hours after HIBD. Data were expressed as mean ± SD of six independent experiments and analyzed by t-tests. aP < 0.05, vs. control group. C0: normal group. A0, A2, A12, A24, A36, A48: BMAL1 protein levels at 0, 2, 12, 24, 36, 48 hours after HIBD. B0, B2, B12, B24, B36, B48: BMAL1 protein levels at 0, 2, 12, 24, 36, 48 hours in the control group.
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