Korean J Physiol Pharmacol.  2019 Jul;23(4):281-289. 10.4196/kjpp.2019.23.4.281.

Increased expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 after pilocarpine-induced status epilepticus in mice

• Cho KO
• Kim JY
• Jeong KH
• Lee MY
• Kim SY

• KMID: 2450495
• DOI: http://doi.org/10.4196/kjpp.2019.23.4.281

Abstract

Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.

Keyword

Hippocampus; Mouse; Vascular endothelial growth factor-C; Vascular endothelial growth factor receptor-3; Status epilepticus

MeSH Terms

Adult
Animals
Astrocytes
Brain
Embryonic Structures
Epilepsy
Epilepsy, Temporal Lobe
Hippocampus
Humans
Immunohistochemistry
Lymphangiogenesis
Mice*
Microglia
Pyramidal Cells
Receptors, Vascular Endothelial Growth Factor
Seizures
Status Epilepticus
Vascular Endothelial Growth Factor A*
Vascular Endothelial Growth Factor C*
Vascular Endothelial Growth Factor Receptor-3*
Receptors, Vascular Endothelial Growth Factor
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor C
Vascular Endothelial Growth Factor Receptor-3

Figure

• 

  Fig. 1 Temporal profiles of neuronal death assessed by Fluoro-Jade staining in the hippocampus following pilocarpine injection. Compared to sham-manipulated hippocampi with no Fluoro-Jade positive neurons, at 1 day after pilocarpine-induced status epilepticus (SE), hilar neurons started to express Fluoro-Jade reactivity. At 4 days and 7 days after pilocarpine-induced SE, a number of Fluoro-Jade positive pyknotic cells were observed in CA1 and CA3 subfields of the hippocampus, representing degenerating neurons. Scale bar in far left column = 200 µm; same magnification was used for photomicrographs labeled as hippocampus. Scale bar in far right column = 50 µm; same magnification was used for photomicrographs labeled as CA1 and CA3. N = 6 per each time-point.

• 

  Fig. 2 Temporal profiles of reactive gliosis in hippocampus following pilocarpine injection. (A) Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed increased GFAP immunoreactivity at 1 day after status epilepticus (SE) onset and further elevation at 4 days and 7 days after pilocarpine-induced SE, whereas minimal GFAP expression was observed in sham-manipulated animals. A square in each low magnification photomicrograph was visualized in the next panel. Scale bar in bottom left column = 200 µm; same magnification was used for entire left column. Scale bar in bottom right column = 20 µm; same magnification was used for entire right column. N = 6 per each time-point. (B) Immunohistochemistry for Ox42 showed markedly increased Ox42 immunoreactivity from 1 day after SE compared to sham-manipulated animals. A square in each low magnification photomicrograph was visualized in the next panel. Scale bar in bottom left column = 200 µm; same magnification was used for entire left column. Scale bar in bottom right column = 20 µm; same magnification was used for entire right column. N = 6 per each time-point.

• 

  Fig. 3 Spatiotemporal profiles of vascular endothelial growth factor (VEGF)-C and vascular endothelial growth factor receptor (VEGFR)-3 immunoreactivity after pilocarpine-induced status epilepticus (SE). (A) Immunoreactivity to VEGF-C and VEGFR-3 was observed in pyramidal cell layer and granule cells in the hippocampus of sham-manipulated animals. However, from 1 day after SE, small cells in the statum radiatum started to express VEGF-C and VEGFR-3, which was enhanced at 4 days and 7 days after pilocarpine-induced SE. A rectangle in each low magnification photomicrograph is visualized in the inset. Scale bar in bottom left of VEGFR-3-stained image at 7 days after SE = 200 µm; same magnification was used for all lower magnification photomicrographs. Scale bar in bottom right of VEGFR-3-stained inset at 7 days after SE = 50 µm; same magnification was used for all insets. (B) Schematic indicating the subpyramidal zone where VEGF-C- and VEGFR-3-expressing cells were quantitatively analyzed. (C) Graph showing number of VEGF-C-immmnoreactive cells in subpyramidal zone. VEGF-C-positive cells were significantly increased at 4 days after SE and peaked at 7 days after SE compared to sham-controls. Values are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test was performed. N = 6 for each group, *p < 0.05 vs. sham. (D) Graph showing number of VEGFR-3 immmnoreactive cells in subpyramidal zone. VEGFR-3 positive cells were significantly increased at 4 days and 7 days after SE compared to sham-controls. Values are expressed as mean ± SEM. One-way ANOVA followed by Dunnett's post-hoc test was performed. N = 6 mice for each group, *p < 0.05 vs. sham.

• 

  Fig. 4 Phenotypic analysis of vascular endothelial growth factor (VEGF)-C immunoreactive cells in hippocampus following pilocarpine injection. In sham-manipulated animals, most VEGF-C-positive cells were pyramidal neurons and a few glial fibrillary acidic protein (GFAP)-colabeled astrocytes in the stratum radiatum, without Ox42 colabeled microglia. At 7 days after status epilepticus (SE), VEGF-C positive cells co-expressed GFAP or Ox42 immunoreactivity in the subpyramidal zone of the hippocampus. White arrows indicate double-labeled cells magnified in the insets. Among GFAP expressing cells in the subpyramidal zone, 82.9% showed VEGF-C immunoreactivity, whereas 13.6% of all Ox42 positive cells demonstrated VEGF-C expression. Scale bar = 50 µm for low magnified images. Scale bar = 10 µm for insets. N = 6 per each group.

• 

  Fig. 5 Phenotypic analysis of vascular endothelial growth factor receptor (VEGFR)-3 immunoreactive cells in hippocampus following pilocarpine injection. In sham-manipulated animals, VEGFR-3 positive cells were pyramidal neurons without glial fibrillary acidic protein (GFAP) or Ox42 expressing glial cells. At 4 days after status epilepticus (SE), VEGFR-3 positive cells co-expressed GFAP or Ox42 immunoreactivity in the subpyramidal zone of the hippocampus. White arrows indicate a representative double-labeled cell magnified in the insets. Among GFAP expressing cells in subpyramidal zone, 81.3% showed VEGFR-3 immunoreactivity, whereas 6.8% of all Ox42 positive cells demonstrated VEGFR-3 expression. Scale bar = 50 µm for low magnified images. Scale bar = 10 µm for insets. N = 6 per each group.

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