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. 2020 Jul:329:113275.
doi: 10.1016/j.expneurol.2020.113275. Epub 2020 Mar 5.

Human neural stem cells improve early stage stroke outcome in delayed tissue plasminogen activator-treated aged stroke brains

Austin C Boese  1 , Auston Eckert  1 , Milton H Hamblin  2 , Jean-Pyo Lee  3
Affiliations

Human neural stem cells improve early stage stroke outcome in delayed tissue plasminogen activator-treated aged stroke brains

Austin C Boese et al. Exp Neurol. 2020 Jul.

Abstract

Introduction: Clinically, significant stroke injury results from ischemia-reperfusion (IR), which induces a deleterious biphasic opening of the blood-brain barrier (BBB). Tissue plasminogen activator (tPA) remains the sole pharmacological agent to treat ischemic stroke. However, major limitations of tPA treatment include a narrow effective therapeutic window of 4.5 h in most patients after initial stroke onset and off-target non-thrombolytic effects (e.g., the risk of increased IR injury). We hypothesized that ameliorating BBB damage with exogenous human neural stem cells (hNSCs) would improve stroke outcome to a greater extent than treatment with delayed tPA alone in aged stroke mice.

Methods: We employed middle cerebral artery occlusion to produce focal ischemia with subsequent reperfusion (MCAO/R) in aged mice and administered tPA at a delayed time point (6 h post-stroke) via tail vein. We transplanted hNSCs intracranially in the subacute phase of stroke (24 h post-stroke). We assessed the outcomes of hNSC transplantation on pathophysiological markers of stroke 48 h post-stroke (24 h post-transplant).

Results: Delayed tPA treatment resulted in more extensive BBB damage and inflammation relative to MCAO controls. Notably, transplantation of hNSCs ameliorated delayed tPA-induced escalated stroke damage; decreased expression of proinflammatory factors (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), decreased the level of matrix metalloprotease-9 (MMP-9), increased the level of brain-derived neurotrophic factor (BDNF), and reduced BBB damage.

Conclusions: Aged stroke mice that received delayed tPA treatment in combination with hNSC transplantation exhibited reduced stroke pathophysiology in comparison to non-transplanted stroke mice with delayed tPA. This suggests that hNSC transplantation may synergize with already existing stroke therapies to benefit a larger stroke patient population.

Keywords: Blood-brain barrier; Inflammation; Neural stem cells; Stem cell transplantation; Stroke; Tissue plasminogen activator.

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Conflict of interest statement

Declaration of Competing Interest The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Human NSCs reduce infarct volume of delayed tPA-treated stroke mouse brains.
(A) Infarct volume was significantly reduced in hNSC-engrafted brains that received delayed tPA (MCAO/R+tPA+Tx). ***P < .001 vs. sham group; ##P < .01 vs. MCAO/R group; §§P < .01 vs. MCAO/R + tPA group. (B) TTC staining (white, infarct). Shown are two representative samples of different mouse brains. (n = 8, Sham; n = 6, MCAO/R; n = 7, MCAO/R+tPA; n = 12, MCAO/R+tPA+Tx). Scale bar = 2 mm. Data are presented as mean ± SEM.
Figure 2.
Figure 2.. Human NSCs reduce expression of MMP-9 and proinflammatory genes.
(A) Diminished expression of inflammatory markers within the ipsilesional hemisphere of transplanted MCAO/R+tPA+Tx brains. RT-PCR was utilized to evaluate inflammatory gene expression, then normalized to that of GAPDH. Transcript levels of proinflammatory cytokines (TNF-α and IL-6) are upregulated in MCAO/R and MCAO/R+tPA brains; in transplanted MCAO/R+tPA+Tx brains, however, these levels are downregulated in comparison to the MCAO/R+tPA group. ***P < .001 vs. sham; #P < .05, ##P < .01 vs. MCAO/R; §P < .05, §§P < .01 vs. MCAO/R+tPA group (n = 5). (B) In MCAO/R and non-transplanted MCAO/R+tPA brains, MMP-9 is significantly upregulated. When hNSCs are transplanted (MCAO/R+tPA+Tx), however, MMP-9 is significantly reduced. ***P < .001 vs. sham; #P < .05 vs. MCAO/R; §P < .05 vs. MCAO/R+tPA group (n = 5). Data are presented as mean ± SEM.
Figure 3.
Figure 3.. hNSC transplantation increases brain-derived neurotrophic factor level.
(A) BDNF levels are shown via western blotting in the ipsilesional hemisphere of sham, MCAO/R, MCAO/R+tPA, and MCAO/R+tPA+Tx mice. (B) Quantification of A (n = 4, ****P < .0001, **P < .01 vs. sham group; §§P < .01 vs. MCAO/R+tPA group). Data are expressed as mean ± SEM.
Figure 4.
Figure 4.. BBB leakage is reduced by hNSC transplantation.
(A) IgG levels are displayed via western blotting in the ipsilesional hemispheres of sham, MCAO/R, MCAO/R+tPA, and MCAO/R+tPA+Tx brains. (B) Quantification of A (n = 4, **P < .01, ****P < .0001 vs. sham group; ##P < .01 vs. MCAO/R group; §§§§P < .0001 vs. MCAO/R+tPA group). (C) Western blot evaluation of the MMP-9 protein level in the ipsilesional hemisphere. (D) Quantification of C (n = 4, *P < .05, ****P < .0001 vs. sham group; #P < .05 vs. MCAO/R group; §§§§P < .0001 vs. MCAO/R+tPA group). (E) Zymography assay displays MMP-9 activity in the ipsilesional hemisphere. (F) Quantification of E (n = 4, ##P < .01 vs. MCAO/R group; §§§§P < .0001 vs. MCAO/R+tPA group). (G) Western blot evaluation of ZO-1. (H) Quantification of G (n = 4, *P < .05, ****P < .0001 vs. sham group; #P < .05 vs. MCAO/R group; §P < .05 vs. MCAO/R+tPA group). Data are expressed as mean ± SEM.
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