Neocortical tissue recovery in severe congenital obstructive hydrocephalus after intraventricular administration of bone marrow-derived mesenchymal stem cells

Abstract

Background: In obstructive congenital hydrocephalus, cerebrospinal fluid accumulation is associated with high intracranial pressure and the presence of periventricular edema, ischemia/hypoxia, damage of the white matter, and glial reactions in the neocortex. The viability and short time effects of a therapy based on bone marrow-derived mesenchymal stem cells (BM-MSC) have been evaluated in such pathological conditions in the hyh mouse model.

Methods: BM-MSC obtained from mice expressing fluorescent mRFP1 protein were injected into the lateral ventricle of hydrocephalic hyh mice at the moment they present a very severe form of the disease. The effect of transplantation in the neocortex was compared with hydrocephalic hyh mice injected with the vehicle and non-hydrocephalic littermates. Neural cell populations and the possibility of transdifferentiation were analyzed. The possibility of a tissue recovering was investigated using ¹H High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (¹H HR-MAS NMR) spectroscopy, thus allowing the detection of metabolites/osmolytes related with hydrocephalus severity and outcome in the neocortex. An in vitro assay to simulate the periventricular astrocyte reaction conditions was performed using BM-MSC under high TNFα level condition. The secretome in the culture medium was analyzed in this assay.

Results: Four days after transplantation, BM-MSC were found undifferentiated and scattered into the astrocyte reaction present in the damaged neocortex white matter. Tissue rejection to the integrated BM-MSC was not detected 4 days after transplantation. Hyh mice transplanted with BM-MSC showed a reduction in the apoptosis in the periventricular neocortex walls, suggesting a neuroprotector effect of the BM-MSC in these conditions. A decrease in the levels of metabolites/osmolytes in the neocortex, such as taurine and neuroexcytotoxic glutamate, also indicated a tissue recovering. Under high TNFα level condition in vitro, BM-MSC showed an upregulation of cytokine and protein secretion that may explain homing, immunomodulation, and vascular permeability, and therefore the tissue recovering.

Conclusions: BM-MSC treatment in severe congenital hydrocephalus is viable and leads to the recovery of the severe neurodegenerative conditions in the neocortex. NMR spectroscopy allows to follow-up the effects of stem cell therapy in hydrocephalus.

Keywords: Bone marrow-derived mesenchymal stem cells; Hydrocephalus; Reactive astrocytes; Spectroscopy.

Fig. 1

Characterization of BM-MSC before transplantation into hydrocephalic hyh mice. a BM-MSC primary culture forming a colony unit. Phase-contrast microscopy. b BM-MSC expressing the mRFP1 (red). Merge of phase-contrast and epifluorescence microscopies. c Spindle-shaped BM-MSC under scanning electron microscopy. d Oil Red O staining of lipid droplets (arrows) in the cytoplasm of BM-MSC after adipogenic differentiation for 14 days. Merge of phase-contrast and bright-field microscopy images. e Collagen type II immunostaining of a 3D pellet culture of BM-MSC after 21 days of chondrogenic differentiation. f Osteoinducted BM-MSC stained with alkaline phosphatase (ALP) 21 days after induction. g ALP activity in control (black) and osteoinducted (gray) BM-MSC at 7, 14, and 21 days. h, i Representative immunophenotype profiles of unfixed BM-MSC for CD44 and CD34 markers by flow cytometry. j Flow cytometry analysis of DNA content (DAPI fluorescence) in fixed BM-MSC. k, l Detection of a neural cell marker (GFAP) and a neuroprotector factor (GDNF) in fixed BM-MSC by flow cytometry. m Immunofluorescence (green) in fixed BM-MSC before injection for δGFAP, nestin, NG2, αGFAP, β-III tubulin, NeuN, GDNF, NGF, BDNF, and VEGF. In the absence of labeling, the channel for red (RFP fluorescence) is also shown. Nuclear staining with DAPI (blue). Negative control represents the omission of the primary antibodies. **P < 0.02, ***P < 0.01 Wilcoxon-Mann-Whitney test

Fig. 2

Location of BM-MSC in the hosting tissue and detection of neuroprotector factors expression. a Walls of the lateral ventricle of a hyh mouse administered at 20 days of age with BM-MSC expressing the mRFP1 (red) and labeled with a green cell tracker (white arrows), 4 days post-injection (dpi). a’ Detail of a BM-MSC (RFP fluorescence, red) colabeled with the fluorescent green cell tracker (white arrow). b, b” Colabeling of the mRFP1 (red) with an antibody against RFP (green) in the administered BM-MSC, 4 dpi, in the neocortex of a hyh mouse injected at 20 days of age. c BM-MSC (red, white arrows) entering into the brain parenchyma of a hyh mouse 20 days of age, 1 dpi, through a ventricle surface presenting a loose periventricular layer of reactive astrocytes (GFAP immunolabeling, green) in the neocortex wall. d In the neocortex walls of hyh injected at P20, BM-MSC were found in three different locations at 4 dpi: between the dense layer of reactive astrocytes covering the ventricle surface (arrowhead, GFAP immunolabeling in green), around the blood vessels (arrow), and deep into the brain parenchyma (asterisk). e–e”’ Coexpression in BM-MSC (mRFP1, red; arrows) at 4 dpi in the neocortex wall of GFAP (magenta) and nestin (green). f–f”’ Coexpression at 4 dpi in BM-MSC (red mRFP1, arrow) of GFAP (magenta) and GDNF (green). g–g”’ BM-MSC (mRFP1, red; arrow) colabeled with the green cell tracker and anti-BDNF (magenta) at 4 dpi. h–h”’ Expression in BM-MSC (red, mRFP1; arrows) of VEGF (green) at 4 dpi

Fig. 3

Levels of proinflammatory cytokines and densities of neural cells. a, b Levels of mRNA of interleukins IL-1α and IL-1β in the groups of mice: non-hydrocephalic (nh), hydrocephalic hyh non-injected (hni), hydrocephalic hyh sham-injected (sham), and hydrocephalic hyh BM-MSC-treated (BM-MSC). c–f Levels of mRNA, protein, and cell densities for CD45 and iba1 as microglia markers. g, g’ Immunofluorescence for iba1 in the neocortex of a hydrocephalic hyh mouse treated with BM-MSC and in a hydrocephalic hyh sham-injected mouse. h–j Levels of mRNA, protein, and cell densities for the GFAP astrocyte marker. k, k’ Immunofluorescence for GFAP in the neocortex of a hydrocephalic hyh mouse treated with BM-MSC and in a hydrocephalic hyh sham-injected mouse. l, m Densities of NG2+ and Olig2+ cells in tissue sections. n, n’ Immunofluorescence for NG2 (green) and Olig2 (red). o Densities of NeuN+ cells in sections of the neocortical layers 2–3 and 5. p, p’ Immunofluorescence for NeuN (green) in the neocortex. q Immunofluorescence for glutamine synthetase. The BM-MSC (mRFP1, red) present no reaction for glutamine synthetase (arrows). r Immunofluorescence for NG2. The BM-MSC present a weak immunoreaction (arrows) compared to NG2 cells (arrowheads). s Immunofluorescence for β-III tubulin (magenta) in BM-MSC (mRFP1, red) labeled with the green cell tracker in the neocortical wall. Arrowhead points to a β-III tubulin negative BM-MSC. Insets in r and s represent splitting of the channels showing immunolabeling for NG2 and cell tracker (green) and β-III tubulin (magenta) in the framed red fluorescent BM-MSC. *P < 0.05, ***P < 0.02, ***P < 0.01 Wilcoxon-Mann-Whitney test

Fig. 4

Densities of apoptotic cells in the neocortex. a TUNEL+ cells in hydrocephalic hyh mice treated with BM-MSC and hydrocephalic hyh sham-injected mice, 4 dpi. ^###P < 0.01 Student’s t test. b, c Periventricular apoptotic cells (arrows) in a hyh mouse administered with BM-MSC cells and in a hydrocephalic hyh sham-injected mouse. c’ Detail of an apoptotic cell shown in the area framed in c

Fig. 5

Levels of metabolites in the neocortex. Analysis was carried out in non-hydrocephalic mice, hydrocephalic hyh mice transplanted with BM-MSC, and hydrocephalic hyh sham-injected mice, 4 dpi. *P < 0.05, **P < 0.02, ***P < 0.01 Wilcoxon-Mann-Whitney test; ^##P < 0.02, ^###P < 0.01 Student’s t test

Fig. 6

Heat map showing the relative concentrations of cytokines secreted by BM-MSC stimulated with TNFα. C1, control without TNFα. C2, equal to C1 with the addition of 10% FBS. Cytokines were detected using an array. Colors were assigned according to the relative scale of expression. Z = 0 represents the mean value