Intranasal Administration of Human MSC for Ischemic Brain Injury in the Mouse: <i>In Vitro</i> and <i>In Vivo</i> Neuroregenerative Functions

<div><p>Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs <i>in vitro</i> and <i>in vivo</i> by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10⁶ or 2×10⁶ hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The <i>in vitro</i> capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10⁶ hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs <i>in vivo</i>, as hMSCs induce mNSCs to differentiate into neurons <i>in vitro</i>. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.</p></div

hMSCs reduce the activation of glial cells at 28 days after HI.

<p>Mice were treated with either 1×10⁶ or 2×10⁶ hMSCs or vehicle intranasally at 10 days following HI. Mice were sacrificed 28 days after HI. (A) Schematic overview of fields quantified. (B) Quantification of Iba-1+ signal/mm² or (C) GFAP+ signal/mm². (D–G) Representative sections of Iba-1 (red) and GFAP (green) expression after sham-operation (D), vehicle (E), 1×10⁶ hMSCs (F) or 2×10⁶ hMSCs (G). Sections are counterstained with DAPI (blue). Scale bar  = 100 µm. Data represent mean ± SEM. * p<0.05; **p<0.01; ***p<0.001 by ANOVA and Bonferroni post-hoc test (Sham and Vehicle n = 4; 1×10⁶ and 2×10⁶ MSC n = 3).</p

<i>In vitro</i> proliferation of hMSCs.

<p>Proliferating capacity of hMSCs <i>in vitro</i>. 1000 hMSCs were plated (T0) and proliferation was assessed at 4(T0), 24(T24), 48(T48) and 96(T96) hours after plating the MSCs by adding ³H-thymidine to the culture and measuring ³H-thymidine uptake 16 hours later. Data represent mean ± SEM. ** p<0.01; *** p<0.001 by ANOVA and Bonferroni post-hoc test. (n = 10 wells for each condition).</p

Dose effect of hMSC on motor performance and lesion volume.

<p>Mice were treated intranasally with either 1×10⁶ or 2×10⁶ hMSCs or vehicle at 10 days after HI. (A) Preference to use the unimpaired forepaw in the cylinder rearing test (CRT) was assessed at 28 days after HI. Sham-operated littermates (Sham) were used as controls. (B–C) Quantification of ipsilateral MAP2 (B) and MBP (C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area) at 28 days after HI. Representative sections of MAP2 (D) and MBP (E) staining. Data represent mean ± SEM. **p<0.01; ***p<0.001 by ANOVA and Bonferroni post-hoc test. Sham n = 13; Vehicle n = 21; 1×10⁶ hMSC n = 11; 2×10⁶ hMSC n = 12. Data presented in this figure are results from pups pooled out of 11 different litters. Treatment groups were randomly distributed between litters.</p

hMSCs induce differentiation of mouse NSCs <i>in vitro</i>.

<p><i>In vitro</i> mNSC transwell differentiation assay in co-culture with hMSCs. mNSCs were fixed at 4(T0) and 96(T96) hours after co-culture with hMSCs and stained for (A) nestin (green), (B) Olig2 (red), (C) GFAP (green) and (D) βIII-Tubulin (red). Data represent mean ± SEM. *** p<0.001 by Unpaired two-tailed T-test. Scale bar  = 100 µm. (n = 4 wells per condition).</p

SAH-induced cortical cytokine/chemokine mRNA expression.

<p><b>A</b>–<b>D</b>: mRNA expression of TNFα (<b>A</b>), IL-1β (<b>B</b>), IL-10 (<b>C</b>), MCP-1 (<b>D</b>), MIP2 (<b>E</b>) and CINC-1 (<b>F</b>) in the cortex at 48 h post-SAH. *p<0.05, **p<0.01, *** p<0.001 vs sham. Data are presented compared to mRNA levels in sham-operated animals which were put at 100%. Sham n = 14; mild-SAH n = 8, severe-SAH n = 6. Data are presented as boxplots with median and minimal/maximal whiskers.</p

Presence of MSCs in the brain.

<p>PKH-26 labeled 1.0×10⁶ MSCs were administered intranasally at 3, 10 and 17 days post-HI. Because no significant difference was found between Veh groups treated at different time points we pooled all animals into one group. (A, B, C) Notice the severe HI-induced damage, as the layer structure of the ipsilateral cortex and hippocampus are lost. (A) MSCs (red) in the ipsilateral hippocampus (see arrow heads) 24 h after administration at 3 days post-HI. (B) MSCs (see arrow heads) in the ipsilateral damaged cortex 24 h after administration at 10 days post-HI. (C) Lack of MSCs (see arrow heads) in the ipsilateral cortical areas surrounding the lesion site when MSCs are given at 17 days post-HI. Contralateral pictures depict hippocampal area (in A) and cortical area (in B and C). (D) Control groups showing lack of MSCs in the hippocampal area and cortical area at 3 and 10 days, respectively, after MSC administration in sham-operated animals and HI-Vehicle treated brain without MSC treatment. Asterisk = lesion site. Blue = Dapi staining. Scale bar 50 µm. Data presented in this figure are results from pooled experiments out of 10 different litters. Treatment groups were randomly distributed between litters.</p

Long-term macrophage/microglia activation after SAH.

<p><b>A</b>: Schematic coronal brain view showing where the photographs were taken in the cortex. Photographs are taken at 21 days post-SAH. <b>B</b>–<b>G</b>: Representative photographs of macrophage/microglia activation by Iba-1 staining in the ipsilateral hemisphere of sham-operated (C), mildly affected SAH (D), severely affected SAH (F) animals and the contralateral hemisphere of a mildly affected SAH animal (E) and severely affected SAH animal (G). B shows a negative control (NC) of the severely affected SAH animal used in F is which the primary antibody was omitted. Insets show a higher magnification. <b>H</b>: Quantification of the number of Iba-1 positive pixels in the contralateral and ipsilateral cortex of sham-operated (black), mildly affected SAH (pink) and severely affected SAH (red) animals. Data are presented as a boxplot with median and minimal/maximal whiskers *p<0.05. Scale bar represents 300 µm in the low magnification photograph and 30 µm in the inset.</p

Dose effect of MSCs on motor performance and lesion volume at 35 days post-HI.

<p>Mice received 0.25×10⁶, 0.5×10⁶, 1×10⁶ MSCs or Vehicle (Veh) treatment at 10 days after induction of HI. (A) Paw preference to use the unimpaired forepaw in the cylinder rearing test (CRT) was assessed at 5 weeks post-HI. Sham-operated littermates (Sham) were used as controls. Quantification of ipsilateral MAP2 (B) and MBP(C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area) at 5 weeks post-HI. (D) Representative sections of MAP2 loss. Insets show higher magnification of corresponding MAP2 sections. Scale bar = 100 µm. (E) Representative sections of MBP area loss. Data represent mean ± SEM. Sham n = 8; Veh n = 10; 0.25×10⁶ MSC n = 11; 0.5×10⁶ MSC n = 10; 1×10⁶ MSC n = 13. *p<0.05; **p<0.01 vs Veh. Data presented in this figure are results from pooled experiments out of 8 different litters. Treatment groups were randomly distributed between litters.</p

Long-term astrocyte activation after SAH.

<p><b>A</b>: Schematic coronal brain view showing where the photographs were taken in the cortex. Photographs are taken at 21 days post-SAH. <b>B</b>–<b>G</b>: Representative photographs of astrocyte activation by GFAP staining in the ipsilateral hemisphere of sham-operated (C), mildly affected SAH (D) and severely affected SAH (F) animals and the contralateral hemisphere of a mildly affected SAH (E) and severely affected SAH animal(G). B shows a negative control (NC) of the severely affected SAH animal used in F is which the primary antibody was omitted. Insets show a higher magnification. <b>H</b>: Quantification of the number of GFAP positive pixels in the contralateral and ipsilateral cortex of sham-operated (black), mildly affected SAH (pink) and severely affected SAH (red) animals. Data are presented as a boxplot with median and minimal/maximal whiskers. Scale bar represents 300 µm in the low magnification photograph and 30 µm in the inset.</p