Korean J Physiol Pharmacol.  2018 Mar;22(2):145-153. 10.4196/kjpp.2018.22.2.145.

Toll-like receptor 2 promotes neurogenesis from the dentate gyrus after photothrombotic cerebral ischemia in mice

• Seong KJ
• Kim HJ
• Cai B
• Kook MS
• Jung JY
• Kim WJ

• KMID: 2410104
• DOI: http://doi.org/10.4196/kjpp.2018.22.2.145

Abstract

The subgranular zone (SGZ) of hippocampal dentate gyrus (HDG) is a primary site of adult neurogenesis. Toll-like receptors (TLRs), are involved in neural system development of Drosophila and innate immune response of mammals. TLR2 is expressed abundantly in neurogenic niches such as adult mammalian hippocampus. It regulates adult hippocampal neurogenesis. However, the role of TLR2 in adult neurogenesis is not well studied in global or focal cerebral ischemia. Therefore, this study aimed to investigate the role of TLR2 in adult neurogenesis after photochemically induced cerebral ischemia. At 7 days after photothrombotic ischemic injury, the number of bromodeoxyuridine (BrdU)-positive cells was increased in both TLR2 knock-out (KO) mice and wild-type (WT) mice. However, the increment rate of BrdU-positive cells was lower in TLR2 KO mice compared to that in WT mice. The number of doublecortin (DCX) and neuronal nuclei (NeuN)-positive cells in HDG was decreased after photothrombotic ischemia in TLR2 KO mice compared to that in WT mice. The survival rate of cells in HDG was decreased in TLR2 KO mice compared to that in WT mice. In contrast, the number of cleaved-caspase 3 (apoptotic marker) and the number of GFAP (glia marker)/BrdU double-positive cells in TLR2 KO mice were higher than that in WT mice. These results suggest that TLR2 can promote adult neurogenesis from neural stem cell of hippocampal dentate gyrus through increasing proliferation, differentiation, and survival from neural stem cells after ischemic injury of the brain.

Keyword

Hippocampus; Ischemia; Neural stem cell; Neurogenesis; Toll-like receptor 2

MeSH Terms

Adult
Animals
Brain
Brain Ischemia*
Bromodeoxyuridine
Dentate Gyrus*
Drosophila
Hippocampus
Humans
Immunity, Innate
Ischemia
Mammals
Mice*
Neural Stem Cells
Neurogenesis*
Neurons
Survival Rate
Toll-Like Receptor 2*
Toll-Like Receptors*
Bromodeoxyuridine
Toll-Like Receptor 2
Toll-Like Receptors

Figure

• 

  Fig. 1 Role of TLR2 in differentiation of neural stem cells in hippocampal dentate gyrus (HDG) after photothrombotic ischemia.(A) Representative confocal image of Doublecortin (DCX) positive cells in hippocampal dentate gyrus (DG) of wild type and TLR2 KO mice at 0 and 10 days after ischemic injury. DCX (Green) stained cells show newly generated immature neurons. (B) The number of DCX positive cell in hippocampal DG was lower in TLR2 KO mice than that in WT mice after photothrombosis induced cerebral ischemia. Quantitative analysis of the number DCX-positive cells (n=5 each). Data are expressed as mean±SEM. *p<0.05; ***p<0.001. Scale bar, 100 µm.

• 

  Fig. 2 Role of TLR2 in proliferation of neural stem cells in HDG after photothrombotic ischemia.WT and TLR2 KO mice received a single dose of BrdU (50 mg/kg) at three hours before sacrifice. The number of BrdU-positive cells (green) in the SGZ of WT mice and TLR2 KO mice from 1 day to 10 days after ischemia was determined. (A) Newly proliferated cells in the subgranular zone (SGZ) of the DG were immunostained with anti-BrdU. (B) Quantitative analysis of the number of BrdU-positive cells in the DG of the hippocampus. The number of BrdU-positive cells was gradually increased in TLR2 KO mice and WT mice after photothrombotic ischemia. However, the increasing rate of BrdU-positive cells was lower in TLR2 KO mice than that in WT mice. Data are expressed as mean±SEM. **p<0.01; ***p<0.001. Scale bar, 100 µm.

• 

  Fig. 3 Role of TLR2 in the survival of new generated cells in HDG after photothrombotic ischemia.(A) BrdU-positive cells (green) in the hippocampal dentate gyrus (DG) were determined at three hours and 28 days after BrdU injection for five consecutive days after ischemia. (B) Survival rate of BrdU-positive cells was lower in TLR2 KO mice than that in WT mice. (C) Representative confocal image of activated caspase-3 positive cells (red) in the subgranular zone (SGZ) from six sections after ischemia. (D) The number of caspase-3 positive cells was increased in TLR2 KO mice than that in WT mice after photothrombosis induced cerebral ischemia. Data are expressed as mean±SEM. *p<0.05. Scale bar, 200 µm.

• 

  Fig. 4 Role of TLR2 in mature neuronal differentiation of newly generated cells in the HDG after photothrombotic ischemia.(A) Representative microphotograph of BrdU/NeuN double positive cells in the hippocampal dentate gyrus at 28 days after BrdU injection for five consecutive days. BrdU (green) and NeuN (red) double stained cells (yellow, arrow) represent newly generated mature neurons. (B) Quantitative analysis of the number of BrdU- and/or NeuN-positive cells. (C) The percentage of NeuN/BrdU double positive cells was lower in TLR2 KO mice than that in WT mice. Data are presented as mean±SEM. *p<0.05; **p<0.01. Scale bar, 100 µm.

• 

  Fig. 5 Role of TLR2 in glia differentiation from newly generated cells in HDG after photothrombotic ischemia.(A) Representative confocal image of BrdU/GFAP double-positive cells in subgranular zone (SGZ) of 28 days after BrdU injection. BrdU (green) and GFAP (red) double stained cells (yellow) represent newly generated astrocytes. (B) The percentage of BrdU/GFAP positive cells ratio was higher in TLR2 KO mice than that in WT mice after photothrombosis-induced cerebral ischemia. Data are presented as mean±SEM. *p<0.05. Scale bar, 100 µm.

• 

  Fig. 6 Role of TLR2 in microglia activation in the hippocampus after photothrombotic ischemia.(A) Representative confocal image of Iba-1 (red) positive cells in the DG at 7 days after photothrombotic ischemia. (B) Quantification of the number of Iba-1-positive cells. It was significantly increased in TLR2 KO mice compared to that in WT mice. Data are presented as mean±SEM. **p<0.01. Scale bar, 100 µm.

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