Authors :
Presenting Author: Juhee Oh, MD – Catholic University of Korea, St. Vincent's Hospital
young-Min Shon, MD – Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea; Sungchul Lim, MD – Catholic University of Korea, St. Vincent's Hospital
Rationale:
Calbindin is a 28kDa calcium-binding protein expressed in restricted neuronal populations in the mammalian brain where it may play a role in protecting neurons against excitotoxic insults. Recent findings indicate that vitamin D can induce the expression of calbindin D28k in kidney, but chronic treatment results in a clinically mild hypervitaminosis can not affect the content of calbindin-D28k in the cerebral cortex and hippocampus. Calbindin D28k immunoreactivity decreased in the CA1/CA2 fields one and three days after kainic acid-induced seizure, and was lost extensively in the pyramidal layer ten days after seizure, but the exact role of calbindin D28k in the hippocampus has been unknown.
Methods:
Vitamin D or vehicle (ethanol) was administered every 24h for seven days. Lithium chloride followed 24h later by pilocarpine was administered intraperitoneally 7days after vitamin D treatment. The expression of calbindin D28k was assessed by immunohistochemistry (N=3) and Western blot (N=3) in the hippocampus isolated at various times (0, 4, 8, 24 hours) after LPSE. Neuronal injuries were assessed by cresyl violet stain (N= 6).
Free-floating tissue sections was used for immunohistochemistry, using Dako EnVison TM system. Sections were incubated in blocking solution (normal goat serum) for one hour. Incubation in primary antibody (anti-calbindin D28k antibody) and in PBS-TX was done overnight at 4°C. The following day, calbindin D28k-immunolabeling of sections was done using DAKO EnVison kit.
Proteins were separated on 10% denaturing gels, transferred onto nitrocellulose membranes, and blocked for one hour with 1% non-fat dry milk in TBS/0.1% Tween 20. Incubation with the antiserum was continued in the same buffer for one hour. Blots were washed with TBS/Tween (3X) and then incubated with secondary anti-rabbit antibody conjugated to horse-radish peroxidase for one hour. Following 3X washes in TBS/Tween, blotted proteins were detected by chemiluminescence.
Cresyl violet stains were analyzed to assess neuronal damage by consensus among three blinded observers. Hippocampus was divided into three sectors for damage analysis.
Results:
Calbindin D28k immunoreactivity was increased in dentate granule cells, the pyramidal cells of the CA1/CA2 area of the hippocampus in the vitamin D group than that of the control group. The control group showed decreased calbindin D28k immunoreactivity in the CA1/CA2 areas four and eight hours after LPSE, and extensively in the pyramidal layer 24 hours after seizure, whereas the vitamin D group revealed that Calbindin D28k immunoreactivity maintained in CA1/CA2 areas until 24 hours after seizure. The neuronal injury by cresyl violet stain at 72 hours after the LPSE was more severe in CA1 area of the control group than that of the vitamin D group.
Conclusions:
Vitamin D induced calbindin D28k in the pyramidal cells of CA1/CA2 areas of the hippocampus and delayed the seizure-induced loss of calbindin D 28k. Also vitamin D had neuroprotective effect in the pyramidal cells of CA1/CA2 areas of the hippocampus. These findings suggest that neuroprotective effect of vitamin D may be mediated partially by induction of calbindin D28k.
Funding: None