Intranasal administration of mesenchymal stem cells ameliorates the abnormal dopamine transmission system and inflammatory reaction in the R6/2 mouse model of Huntington disease

Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy

Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Cell age- and concentration-dependent effects of EPO on glutamine synthetase activity.

<p>(<b>A</b>) DIV7 APC under normoxia treated with (+1mM Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>B</b>) DIV7 APC under 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>C</b>) DIV14 APC under normoxia treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO; (<b>D</b>) DIV14 APC upon 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>E</b>) DIV21 APC under normoxia treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>F</b>) DIV21 APC under 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively). At all three time points in culture, Glu increased the activity of GS when compared to respective controls culture without Glu (-Glu). Treatment with EPO increased the glutamate-induced activation of GS in concentration-dependent manner when compared to control culture (cf. +Glu vs +Glu+1U E and +Glu+5U E). *, p<0.05, **, p<0.01, ***, p<0.001.</p

Expression of EPOR/GFAP in young and culture aged APC under normoxia and hypoxia.

<p>(<b>A</b>) Combined picture of double immunostaining of EPOR (green) and GFAP (red) in 7-day old APC under normoxia; (<b>B</b>) Corresponding picture of EPOR in green; (<b>C</b>) Double immunostaining of EPOR (green) and GFAP (red) of APC DIV 7 upon hypoxia; (<b>D</b>) Corresponding picture to C of EPOR (green) only; (<b>E</b>) EPOR (green) and GFAP (red) expression in APC on DIV21 under normoxia; (<b>F</b>) EPOR (green) picture corresponding to E; (<b>G</b>) EPOR(green) and GFAP(red) expression in APC at DIV21 upon hypoxia; (<b>H</b>) corresponding to G picture of EPOR (green) only in DIV21 APC under hypoxia. A-H Nuclear staining with DAPI shown in blue, scale bar 200µm.</p

The significance of EPOR for astroglial cell survival and utilization of glutamate by astroglial cells.

<p>(<b>A</b>) Immunostaining for EPOR (green) and β-tubulin III (red) in DIV14 APC; (<b>B</b>) Immunostaining of EPOR (green) and GFAP (red) of DIV14 APC; (<b>C</b>) Immunostaining for EPOR (green) and /ß-tubulin III (red) in DIV14 APC transfected with EPOR siRNA; (<b>D</b>) Immunostaining of and EPOR (green) and /GFAP (red) of 14-day-old APC transfected with EPOR siRNA. Cell nuclei are stained with DAPI shown in blue. Scale bar 200µm. (<b>E</b>) Caspase 3/7 activity in untreated 14-day-old APC (white bars) and those transfected with siRNA for EPOR upon normoxia (patterned white bars) (<b>F</b>) Caspase 3/7 activity in hypoxic untreated 14-day-old APC (grey bars) and those transfected with siRNA for EPOR (patterned grey bars) Treatment of original APC (EPOR+) with transfection reagent (TR) did not influence significantly the survival of APC under both normoxic (white bars) and hypoxic (grey bars) conditions. (<b>G</b>) Glu uptake in untreated (EPOR+) APC at DIV14 and those transfected with EPOR siRNA (EPOR-) under normoxia (white bars) and hypoxia (grey bars) (<b>H</b>) GS-activity in untreated (EPOR+) APC at DIV14 and those transfected with EPOR siRNA (EPOR-) under normoxia (white bars) and hypoxia (grey bars). Treatment of original APC (EPOR+) with transfection reagent (contr TR) did not influence significantly the GS-activity of APC. * p<0.05, ** p<0.01, *** p<0.001.</p

Cell age-dependent Glu-uptake and the effects of EPO on the expression of GLAST by astroglial primary cultures (APC) under normoxic and hypoxic culture conditions.

<p>(<b>A</b>) Quantification of GLAST-positive astrocytes (GLAST+/GFAP+cells) under normoxia shows a significant (**p<0.01; ***p<0.001) increase in GLAST+/GFAP+cells upon EPO-treatment in both young and aged APC (D7 and D21); (<b>B</b>) hypoxia enhanced the effect of EPO on GLAST/GFAP+ cells. In both young and aged cells the number of GLAST/GFAP cells was increased (**p<0.01 at d7 and ***p<0.001 at d21). (<b>C</b>) The uptake of 1 mM glutamate by APC (shown in absolute values) was increased on day 14 in comparison with day 7 under normoxia (white bars, **p<0.01) and hypoxia (grey bars, ***p<0.001) and strongly decreased in 21-day old cultures (d21) exposed to hypoxia (grey bars) when compared to those from day 7 in hypoxia (grey bars d21vs. d7, p***<0.001).</p

Age- and region-dependent distribution of glutamine synthetase in human and rat skin sections.

<p>The cells were labelled with polyclonal antibodies against GS. The intensity of staining was estimated visually as absent (−), weak (+), moderate (++), strong (+++) or very strong (++++).</p

Quantification of GFAP/PCNA-positive cells in APC in young and culture aged APC upon hypoxia/Glu/EPO-exposure.

<p>(<b>A</b>) GFAP/PCNA+ cell counts in APC at DIV7 exposed to Glu under normoxia (N, white bars) and hypoxia (H, grey bars) showed an increase of proliferating astrocytes upon exposure to 5U/ml EPO (5U E) only under hypoxic conditions. Hypoxia decreased the proliferation of APC (cf- N+Glu vs. H+Gu); (<b>B</b>) Quantification of GFAP/PCNA+ cells on DIV21 shows no changes in PCNA+ astrocytes exposed to EPO under normoxia (white bars), while a significant increase (**p<0.01) is detected in EPO-treated APC (H+Glu+5U E) under hypoxia (grey bars), as compared with the hypoxic control (H+Glu). The proliferation of APC in normoxic (N+Glu) and hypoxic control conditions (H+Glu) remained unchanged.</p

Age-dependent distribution of GS and GFAP in rat scalp.

<p>(A–C) Double labelling of newborn (2–3-day-old) rat scalp. (A) staining for GS (green, pAb), (B) staining for GFAP (red, mAb), (C) merge. (D–F) Double staining of adult rat scalp sections. (D) staining for GS, (E) staining for GFAP, (F) merge. The insert in (C) shows single keratinocytes strongly stained for GS. White arrowheads in the insert point to GS-positive cells migrating towards the outer layer of the skin. The insert in (F) shows the prominent differences in the ratio of GS and GFAP in neighboring regions of the epidermis of adult rat skin evidenced by green, yellow or orange staining. Red arrows in (A–F) point to stratum basale, white arrows to stratum spinosum, magenta arrows to stratum granulosum, yellow arrows to stratum corneum. (G–L) Co-expression of GS and GFAP in adult rat leg skin. (G–I) Double labelling of skin section from the lateral surface of adult rat leg skin. (G) staining for GS (green, pAb), (H) staining for GFAP (red, mAb), (I) merge. (J–L) Double staining of skin section from the medial surface of adult rat leg skin. (J) staining for GS, (K) staining for GFAP, (L) merge. White arrows in (G–L) point to the portion of the skin in which GS can be barely detected, whereas red arrows point to the portion of the skin in which GS is strongly expressed. Binding of specific antibodies was visualized with FITC-conjugated goat anti-rabbit IgG (A, D, G, J) and Cy3-conjugated goat anti-mouse IgG (B, E, H, K). Scale bar: (A–C) 10 μm; (D–F) 20 μm.</p