Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Oct;14(5):688-703.
doi: 10.1007/s12975-022-01083-8. Epub 2022 Oct 1.

Dental Pulp Stem Cell-Derived Conditioned Medium Alleviates Subarachnoid Hemorrhage-Induced Microcirculation Impairment by Promoting M2 Microglia Polarization and Reducing Astrocyte Swelling

Ling-Yu Yang  1 , Yong-Ren Chen  2 , Jing-Er Lee  3 , Kuo-Wei Chen  4   5 , Hui-Tzung Luh  4   5 , Yi-Tzu Chen  1   5 , Kuo-Chuan Wang  6 , Sung-Tsang Hsieh  7   8
Affiliations

Dental Pulp Stem Cell-Derived Conditioned Medium Alleviates Subarachnoid Hemorrhage-Induced Microcirculation Impairment by Promoting M2 Microglia Polarization and Reducing Astrocyte Swelling

Ling-Yu Yang et al. Transl Stroke Res. 2023 Oct.

Abstract

Aneurysmal subarachnoid hemorrhage (SAH) can cause severe neurological deficits and high mortality. Early brain edema following SAH contributes to the initiation of microcirculation impairment and may further lead to delayed ischemic neurologic deficit (DIND). This study aimed to investigate whether dental pulp stem cell conditioned medium (DPSC-CM) ameliorates SAH-induced microcirculation impairment and the underlying mechanisms. SAH was induced via intrathecal injection of fresh autologous blood in Wistar male adult rat. DPSC-CM or DPSC-CM + insulin growth factor-1 (IGF-1) antibody was randomly administered by intrathecal route 5 min after SAH induction. To evaluate the underlying mechanisms of DPSC-CM in the treatment of SAH, primary rat astrocyte and microglia co-cultures were challenged with hemolysate or SAH-patient CSF in the presence or absence of DPSC-CM. The results showed that in vivo, DPSC-CM treatment decreased the brain water content, improved microcirculation impairment and enhanced functional recovery at 24 h post-SAH. DPSC-CM treatment also alleviated the expressions of water channel protein aquaporin-4 (AQP4) and pro-inflammatory cytokines, and enhanced the expressions of anti-inflammatory factors in the cortical region. However, all the beneficial effects of DPSC-CM were abrogated after treatment with IGF-1 neutralizing antibody. The in vitro results further showed that DPSC-CM treatment reduced hemolysate/SAH-patient CSF-induced astrocyte swelling and promoted M2 microglia polarization, partially through IGF-1/AKT signaling. The data suggested that DPSC-CM significantly reduced brain edema and rescued microcirculation impairment with concomitant anti-inflammatory benefits after SAH, and may potentially be developed into a novel therapeutic strategy for SAH.

Keywords: Aneurysmal subarachnoid hemorrhage; Brain edema; Conditioned medium; Dental pulp stem cells; Microcirculation impairment; Neuroinflammation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Astrocyte swelling and impaired microcirculation on the cortical surface at 24 h and 48 h after SAH induction. A After a craniotomy was performed from the sham, SAH 24 h and SAH 48 h groups, all vasculatures including the main arterioles and venules on the brain surface were clearly seen (A represents arterioles, V represents venules, bar = 50 μm). B Observation of the arteriole diameters of primary (pa), secondary (sa), and terminal (ta) arterioles using a CAM1 capillary anemometer. Quantitatively, the diameters of the sa and ta were smaller at 24 h in the SAH group than in the sham group and were increased at 48 h as compared with 24 h after SAH. C The regional cerebral blood flow and D PbtO2 were significantly lower at both 24 h and 48 h in the SAH group than in the sham group. E The arrow indicates microvessels in representative electron micrographs (bar = 2 μm). The areas marked by a square are shown in higher magnification of the lower panel (bar = 1 μm). L marks the lumen of a microvessel and asterisks mark the end-feet of astrocytes. The swollen end-feet (*) remarkably compressed the microvessels in the SAH 24 h and SAH 48 h groups. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, n = 4
Fig. 2
Fig. 2
Effects of DPSC-CM on brain edema and microcirculation at 24 h post-SAH. A DPSC-CM administration significantly reduced the brain water content in the cortex region at 24 h after SAH. B The regional cerebral blood flow and C the partial pressure of oxygen (PbtO2) at the brain surface were significantly higher in the SAH + CM rats than in the SAH + Veh rats. However, the administration of IGF-1 neutralizing antibodies moderately blunted the DPSC-CM-mediated effects on the two parameters. D DPSC-CM significantly improved the latency to fall at 7 days after SAH induction, as measured by the Rotarod test. E The arrow points to microvessels in representative electron micrographs (bar = 5 μm) from the four groups. L marks the lumen of a microvessel and asterisks mark the end-feet of astrocytes in the higher-magnification images of the lower panel (bar = 1 μm). The swollen end-feet (*) remarkably compressed the microvessels in the SAH + Veh and SAH + CMIGFab groups, whereas DPSC-CM administration attenuated astrocyte swelling. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 4–5
Fig. 3
Fig. 3
DPSC-CM administration reduced the expression of AQP4 and 4-HNE in astrocyte at 24 h post SAH. A Representative immunofluorescence images of 4-HNE (the product of lipid peroxidation; green) and GFAP (a marker for astrocyte; red) labeling in the cerebral cortex region from a SAH animal are shown. Remarkably 4-HNE accumulation was identified in astrocytes after SAH induction (bar = 50 μm). B Representative immunofluorescence images of GFAP and AQP4 labeling in the cerebral cortex region. GFAP immunoreactivity is shown in green, and AQP4 is shown in red (bar = 100 μm). C Representative HE-stained coronal sections from a sham control showing the cortex region to compare the fluorescent signals between the 4 groups of rats, as indicated by the black square boxes. Western blot analysis showed that DPSC-CM administration reduced the expressions of D 4-HNE and F AQP4 in the cortical region at 24 h after SAH. E GFAP and AQP4 expressions were quantified using the area fraction (percentage of GFAP or AQP4 immunoreactivity in the overall field). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 5
Fig. 4
Fig. 4
Effects of DPSC-CM on pro- and anti-inflammatory microglial (M1-, M2-like) states at 24 h post-SAH. A Representative immunostaining with Iba-1 in the cerebral cortex region and a morphological change from a “resting” form in the sham animal to an “activated” morphology in a SAH animal. B Real-time PCR analysis showed that DPSC-CM administration reduced the mRNA expression of M1 microglial-associated pro‐inflammatory factors, including IL-6, IL-1β, and TNF-α. C SAH induced the mRNA levels of M2 microglial-associated anti‐inflammatory factors, including TGF-β and Arg-1, which were significantly increased in the DPSC-CM group. D ELISA of the protein levels of IL-10 in the four groups showed that DPSC-CM administration significantly increased the IL-10 expression in plasma samples. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 5
Fig. 5
Fig. 5
Effects of DPSC-CM on primary astrocytes exposed to hemolysate and SAH-patient CSF for 24 h. A Immunocytochemical staining for 4-HNE (shown in brown; upper panels, bar = 100 μm; lower panels, bar = 20 μm). B There was a significant decrease in the number of 4-HNE-positive cells in the hemolysate + DPSC-CM group. C Immunofluorescence staining for GFAP (shown in red). Representative images showing the effects of exposure of astrocytes to hemolysate or SAH-patient CSF with Veh/DPSC-CM/DPSC-CMIGFab treatment for 24 h, respectively (bar = 100 μm). D Quantitative analysis of GFAP+ cells showed that the astrocyte cell perimeter significantly increased after exposure to hemolysate for 24 h. DPSC-CM treatment significantly decreased the astrocyte cell perimeter. E The level of AQP4 protein in co-cultured astrocytes and microglia was measured by western blot. Hemolysate caused an increase in AQP4 expression, which was suppressed by DPSC-CM treatment. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3
Fig. 6
Fig. 6
Effects of DPSC-CM treatment on microglial M2 polarization in astrocyte/microglia co-cultures exposed to hemolysate or SAH-patient CSF for 24 h. A Representative immunofluorescence images of Arg-1 and OX42 labeling in the astrocyte/microglia co-cultures. OX42 (a marker for activated microglia) immunoreactivity is shown in green, and Arg-1 (M2a microglia marker) is shown in red (bar = 100 μm). B Neutralization of IGF-1 moderately blunted the DPSC-CM-mediated microglia M2 polarization and decreased the number of Arg-1/OX42-positive cells after exposure to hemolysate. C Real-time PCR analysis showed that DPSC-CM markedly inhibited IL-6 and increased the IL-4 and IL-10 mRNA levels after exposure to hemolysate. Western blots and quantification showed that DPSC-CM treatment increased D pAKT (serine-473)/AKT ratio and E the expression of Arg-1 after exposure to hemolysate, which were both reversed by the neutralizing IGF-1 antibody and Ly294002 (AKT-PI3K inhibitor). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 2–3
Fig. 7
Fig. 7
DPSC-CM restored SAH-induced astrocyte swelling-mediated microcirculation impairment and promoted microglia M2 polarization via the IGF-1/AKT pathway
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Macdonald RL, Schweizer TA. Spontaneous subarachnoid haemorrhage. Lancet. 2017;389(10069):655–666. - PubMed
    1. Rosengart AJ, Schultheiss KE, Tolentino J, Macdonald RL. Prognostic factors for outcome in patients with aneurysmal subarachnoid hemorrhage. Stroke. 2007;38(8):2315–2321. - PubMed
    1. Sehba FA, Pluta RM, Zhang JH. Metamorphosis of subarachnoid hemorrhage research: from delayed vasospasm to early brain injury. Mol Neurobiol. 2011;43(1):27–40. - PMC - PubMed
    1. Zetterling M, Hallberg L, Ronne-Engstrom E. Early global brain oedema in relation to clinical admission parameters and outcome in patients with aneurysmal subarachnoid haemorrhage. Acta Neurochir (Wien) 2010;152(9):1527–1533. - PubMed
    1. Sehba FA, Friedrich V. Cerebral microvasculature is an early target of subarachnoid hemorrhage. Acta Neurochir Suppl. 2013;115:199–205. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources