Effect of cisplatin and metformin on dendritic branching.

<p>Sholl-analysis of pyramidal cells in the cingulate cortex of Golgi-stained brains was performed. (A) Examples of actual and (B) reconstructed pyramidal cells. (C) Plot of intersection number vs radius. (D) Areas under the curve of plots in (C) were analyzed Two-way ANOVA (cisplatin×metformin, F(1,31) = 34.81, p<0.05) followed by LSD. Scale bar: 20μm. All data are expressed as mean±SEM. *, p<0.05. n = 8–11 per group.</p

Effect of cisplatin and metformin on dendritic spine density Brains of mice treated with cisplatin and metformin were stained using a Golgi-staining kit and sliced were analyzed for dendritic spine density on pyramidal neurons in the cingulate cortex.

<p>(A-D) representative examples of actual images, an actual dendrite and a skeletonized dendrite. (E) Quantified data were analyzed by two-way ANOVA (cisplatin×metformin, F(1,17) = 22.26, p<0.05) followed by Tukey. Scale bar: 20μm. All data are expressed as mean±SEM. *, p<0.05. n = 5–6 per group</p

Effects of cisplatin and metformin on cognitive function.

<p>Mice received three cycles (5 daily injections of 2.3 mg/kg i.p followed by 5 days without injections) of cisplatin treatment with or without metformin (100 mg/kg i.p.). (A) Set up of novel object/place recognition test. (B) Effect of cisplatin and metformin on performance in the novel object/place recognition test. Data were analyzed by two-way ANOVA (interaction effect: Cisplatin×metformin, F(1,46) = 6.465, p<0.05) followed by LSD. (C) The total interaction time with novel and familiar object did not differ between groups. Data were analyzed by two-way ANOVA. (D) Set up of the social discrimination test. (E) Effect of cisplatin and metformin on performance in the social discrimination test. Data were analyzed by two-way ANOVA (Interaction: Cisplatin×metformin, F(1,44) = 4.42, p<0.05) followed by LSD. (F) The total interaction time during the test did not differ between groups. Data were analyzed by two-way ANOVA. (G) Effect of cisplatin and metformin on body weight (Time×Cisplatin, F(3,95) = 48, p<0.05). All data are expressed as mean±SEM. *, p<0.05. n = 10–14 per group.</p

Astrocyte activation in brain of mice treated cisplatin and metformin.

<p>Expression of GFAP as a measure of astrocyte activation was analyzed in hippocampus. (A). Green: GFAP; Blue: DAPI. (B) Quantification of GFAP expression. Data were analyzed by two-way ANOVA (cisplatin×metformin, F(1,16) = 0.8265, p>0.05). Scale bar: 20um. All data are expressed as mean±SEM. *, p<0.05. n = 5–6 per group</p

Effects of cisplatin and co-administration of metformin on the organization of myelin basic protein-positive fibers in the cingulate cortex.

<p>Mice were treated with cisplatin and metformin and brains were collected after behavioral analysis. MBP staining patterns in the cingulate cortex were analyzed as a measure of integrity of myelinated fiber networks. (A) Representative examples. (B) Pictures of all sections were captured using a Leica microscope and quantified by Fuji ImageJ with a coherency plugin. Data were analyzed by two-way ANOVA (cisplatin×metformin, F(1,15) = 17.36, p<0.01) followed by Tukey post hoc testing. Scale Bar: 20μm. All data are expressed as mean±SEM. *, p<0.05; **,p<0.01. n = 4–5 per group</p

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

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

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

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 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