Korean J Perinatol.  2016 Mar;27(1):15-27. 10.14734/kjp.2016.27.1.15.

Brain-Derived Neurotrophic Factor (BDNF) Exerts a Protective Effect via an Anti-Apoptotic Mechanism on Hypoxic-Ischemic Injury in the Rat Brain

• Kim BJ
• Lee HS
• Han YH
• Jeong JE
• Lee EJ
• Choi EJ
• Kim WT

• KMID: 2164265
• DOI: http://doi.org/10.14734/kjp.2016.27.1.15

Abstract

PURPOSE
Perinatal hypoxic-ischemic (HI) brain injury remains a common cause of chronic handicapping conditions of cerebral palsy, mental retardation, learning disability, and epilepsy. HI brain injury induces cell death via either necrosis or apoptosis. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. It plays key roles in survival, differentiation, and maintenance of neurons. This study was to investigate the neuroprotective effects of BDNF via the mechanisms of anti-apoptosis in HI brain injury by using cortical astrocyte and neuronal cell culture.
METHODS
Cortical astrocytes culture of 1-day-old Sprague-Dawley (SD) rat pups and embryonic cortical neuronal cell culture of SD rats at 14-day gestation were done. The Normoxia group was prepared in 5% CO2 incubators and the Hypoxia group and Hypoxia+BDNF group (after treatment with BDNF for 24 hours) were placed in 1% O2 incubators (94% N2, 5% CO2) for 6 or 18 hours. The expression of Bcl-2 and Bax were assessed by real-time PCR and western blot. The caspase-3 activation was evaluated by caspase activity assay kit.
RESULTS
In astrocyte and neuronal cell, the expressions of Bcl-2 in the hypoxia groups were reduced compared to the normoxia groups, whereas, those in the Hypoxia+BDNF groups were increased compared to the hypoxia groups. However, the expressions of Bax and caspase-3 and the ratio of Bax/Bcl-2 were revealed reversely. In astrocyte, Hypoxia group for 6 hours was not significantly altered in Bcl-2, Bax expressions.
CONCLUSION
BDNF neuroprotective effects on HI brain injury in neonatal rats may occur via anti-apoptotic mechanism.

Keyword

Anti-apoptosis; Brain-derived neurotrophic factor; Hypoxia-ischemia; Neuroprotection

MeSH Terms

Animals
Anoxia
Apoptosis
Astrocytes
Blotting, Western
Brain Injuries
Brain*
Brain-Derived Neurotrophic Factor*
Caspase 3
Cell Culture Techniques
Cell Death
Cerebral Palsy
Epilepsy
Humans
Incubators
Intellectual Disability
Learning Disorders
Necrosis
Neurons
Neuroprotective Agents
Pregnancy
Rats*
Rats, Sprague-Dawley
Real-Time Polymerase Chain Reaction
Brain-Derived Neurotrophic Factor
Caspase 3
Neuroprotective Agents

Figure

• 

  Fig. 1 Fluorescence images (x 200) of rat cortical astrocyte and neuronal cell cultured for 10 days and stained for the appropriate phenotypic markers. Nuclei were stained with DAPI (blue). Approximately 95% of the cells stain positive for astrocyte marker GFAP (green) (A), more than 95% of the cells are positive for the neuronal cell marker MAP2 (green) (B).

• 

  Fig. 2 Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay. Cultured dispersed astrocytes or neuronal cells were prepared with different concentrations of brain-derived neurotrophic factor (BDNF) for 24 hours before a hypoxic insult for 6 or 18 hours. The concentration of drug was 5, 100, and 200 ng/mL. The damaged cells were restored following administration of BDNF. The effective doses were 100 ng/mL in the HB groups both astrocyte and neuronal cell. N, normoxia; 6H, hypoxia for 6 hours; 6HB; hypoxia for 6 hours after treatment with BDNF; 18H, hypoxia for 18 hours; 18HB; hypoxia for 18 hours after treatment with BDNF. ∗P<0.05, statistically significant vs. N.

• 

  Fig. 3 Real-time PCRs of Bcl-2 (A; N, 100±5.0; 6H, 107.2±5.3; 6HB, 112.9±5.6; 18H, 52.3±2.6; 18HB, 73.2±3.7) and Bax (B; N, 100±2.0; 6H, 102.1±2.9; 6HB, 103.8±2.1; 18H, 115.6±1.7; 18HB, 113.7±3.0) mRNAs and the ratio of Bax/ Bcl-2 were revealed in the cortical astrocyte culture. BDNF was administered at 100 ng/mL. N, normoxia; 6H, hypoxia for 6 hours; 6HB; hypoxia for 6 hours after treatment with BDNF; 18H, hypoxia for 18 hours; 18HB; hypoxia for 18 hours after treatment with BDNF. ∗P<0.05, statistically significant vs. H.

• 

  Fig. 4 Real-time PCRs of Bcl-2 (A; N, 100±4.5; 6H, 42.3±6.7; 6HB, 76.3±3.9; 18H, 30.8±4.0; 18HB, 62.9±5.5), Bax (B; N, 100±3.7; 6H, 121.0±5.2; 6HB, 95.9±7.1; 18H, 173.5±2.9; 18HB, 126.1±6.4) mRNAs and the ratio of Bax/Bcl-2 were revealed in the embryonic cortical neuronal cell culture. BDNF was administered at 100 ng/mL. N, normoxia H, hypoxia for 6 hours; 6HB; hypoxia for 6 hours after treatment with BDNF; 18H, hypoxia for 18 hours; 18HB; hypoxia for 18 hours after treatment with BDNF. ∗P<0.05, statistically significant vs. H.

• 

  Fig. 5 Western blots (A) of Bcl-2 (B; N, 100±2.0; 6H, 94.9±1.8; 6HB, 98.0±0.6; 18H, 75.4±0.73; 18HB, 89.8±2.1) and Bax (C; N, 100±5.5; 6H, 105.8±4.8; 6HB, 102.5±4.2; 18H, 151.5±6.1; 18HB, 111.9±5.6) and the ratio of Bax/Bcl-2 were revealed in the cortical astrocyte culture (n=4). BDNF was administered at 100 ng/mL. N, normoxia; 6H, hypoxia for 6 hours; 6HB; hypoxia for 6 hours after treatment with BDNF; 18H, hypoxia for 18 hours; 18HB; hypoxia for 18 hours after treatment with BDNF. ∗P<0.05, statistically significant vs. H.

• 

  Fig. 6 Western blots (A) of Bcl-2 (B; N, 100±2.0; 6H, 42.1±3.2; 6HB, 82.3±1.9; 18H, 30.9±4.9; 18HB, 70.4±5.2), Bax (C; N, 100±5.7; 6H, 145.6±6.9; 6HB, 105.5±7.2; 18H, 164.3±8.1; 18HB, 121.9±3.7) and the ratio of Bax/Bcl-2 were revealed in the embryonic cortical neuronal cell culture (n=4). BDNF was administered at 100 ng/mL. N, normoxia; 6H, hypoxia for 6 hours; 6HB; hypoxia for 6 hours after treatment with BDNF; 18H, hypoxia for 18 hours; 18HB; hypoxia for 18 hours after treatment with BDNF. ∗P<0.05, statistically significant vs. H.

• 

  Fig. 7 Astrocyte (A) and neuronal cell (B) were plated dish 24 hours before the induction of apoptosis. After treatment with 100 ng/mL BDNF for 24 hours before a hypoxic insult, the activity of caspase-3 was assayed using a caspase-3/CPP32 colorimetric assay kit. ∗P<0.05, statistically significant vs. N.

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