Intranasal delivery of transforming growth factor-beta1 in mice after stroke reduces infarct volume and increases neurogenesis in the subventricular zone

Abstract

Background: The effect of neurotrophic factors in enhancing stroke-induced neurogenesis in the adult subventricular zone (SVZ) is limited by their poor blood-brain barrier (BBB) permeability.Intranasal administration is a noninvasive and valid method for delivery of neuropeptides into the brain, to bypass the BBB. We investigated the effect of treatment with intranasal transforming growth factor-beta1 (TGF-beta1) on neurogenesis in the adult mouse SVZ following focal ischemia. The modified Neurological Severity Scores (NSS) test was used to evaluate neurological function, and infarct volumes were determined from hematoxylin-stained sections. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling was performed at 7 days after middle cerebral artery occlusion (MCAO). Immunohistochemistry was used to detect bromodeoxyuridine (BrdU) and neuron- or glia-specific markers for identifying neurogenesis in the SVZ at 7, 14, 21, 28 days after MCAO.

Results: Intranasal treatment of TGF-beta1 shows significant improvement in neurological function and reduction of infarct volume compared with control animals. TGF-beta1 treated mice had significantly less TUNEL-positive cells in the ipsilateral striatum than that in control groups. The number of BrdU-incorporated cells in the SVZ and striatum was significantly increased in the TGF-beta1 treated group compared with control animals at each time point. In addition, numbers of BrdU- labeled cells coexpressed with the migrating neuroblast marker doublecortin (DCX) and the mature neuronal marker neuronal nuclei (NeuN) were significantly increased after intranasal delivery of TGF-beta1, while only a few BrdU labeled cells co-stained with glial fibrillary acidic protein (GFAP).

Conclusion: Intranasal administration of TGF-beta1 reduces infarct volume, improves functional recovery and enhances neurogenesis in mice after stroke. Intranasal TGF-beta1 may have therapeutic potential for cerebrovascular disorders.

Figure 1

Modified Neurological Severity Scores (NSS). Improved NSS were shown in mice treated with TGF-β1 (n = 4) than in control groups (n = 6) after middle cerebral artery occlusion (MCAO). Data are presented as mean ± SEM. *P < 0.05 vs. control.

Figure 2

Effects of TGF-β1 on infarct volume at 7, 14, 21 and 28 days after ischemia. Intranasal TGF-β1 treatment group (n = 4) has reduced infarct volume (B) compared to the control group (A) (n = 6) at 7 days after MCAO. Measurement of infarct size revealed that intranasal administration of TGF-β1 significantly reduced infarct volume at days 7, 14, 21 and 28 after MCAO compared to the saline-treated groups (C). Data are presented as mean ± SEM. *P < 0.05 vs. control.

Figure 3

TUNEL labeling in ipsilateral striatum at 7 days after MCAO. TGF-β1 treated mice (n = 4) display less TUNEL-positive cells (B) than control groups (n = 6) following brain ischemia. Quantitative analysis showed reduction of the number of TUNEL-positive cells after intranasal administration of TGF-β1 (C). Scale bars = 20 μm. Data are presented as mean ± SEM. *P < 0.05 vs. control.

Figure 4

Enhancement of BrdU incorporation by intranasal administration of TGF-β1 in adult mouse brain after MCAO. Some BrdU-immunopositive cells were detected in the SVZ (A) and the striatum (D) of the control groups at 7 days after focal cerebral ischemia. BrdU-labeled cells were increased in the SVZ (B) and the striatum (E) by TGF-β1 intranasal treatment (n = 4) at 7 days after focal cerebral ischemia. Quantitative analysis showed that intranasal administration of TGF-β1 significantly increased the number of BrdU immunopositive cells in the SVZ (C) and the striatum (F) at 7, 14, 21 and 28 days after MCAO compared with that in the control groups (n = 6). Scale bars = 15 μm. Data are mean ± SEM. * P < 0.05 vs. control.

Figure 5

BrdU and DCX expression in adult mouse brain. Double immunofluorescence staining show most of BrdU-immunoreactive cells in the ipsilateral SVZ (A-C) and striatum (D-F) co-labeled with DCX after intranasal administration of TGF-β1 (n = 4) at 14 days following stroke. Quantitative data of BrdU and DCX-immunoreactive cells in the ipsilateral SVZ and striatum of TGF-β1 treated groups at 14 days after MCAO are shown in (G) and (H), respectively. Scale bars = 25 μm in A-C; 20 μm in D-F. Data are mean ± SEM. * P < 0.05 vs. control.

Figure 6

Double immunofluorescence staining for BrdU, NeuN and GFAP. The majority of BrdU-incorporated cells expressed NeuN in the ipsilateral SVZ (A-C) and striatum (E-G) at 28 days after stroke while only a few BrdU-positive cells colabeled with GFAP (H-J). Intranasal administration of TGF-β1 increased the number of BrdU and NeuN-immunoreactive cells in the ipsilateral SVZ and striatum (D) at 28 days after stroke. Scale bars = 15 μm in A-C; 10 μm in E-J. Data are mean ± SEM. * P < 0.05 vs. control.