Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia

Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.


Synthesis of dextran coated superparamagnetic nanoparticles
D-MNPs were prepared following a previously described protocol 1

Characterization of D-MNPs
A structural analysis of the D-MNPs was performed by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Samples were measured on a TEM microscope model Philips CM-12 operating at 120 kV with a Philips powder diffractometer fitted with Philips PW1710 control unit, vertical Philips PW1820/00 goniometer, and a FR590 EnrafNonius generator. Magnetic properties were recorded on a vibrating sample magnetometer (VSM) (Quantum Design TM, CA, USA). Iron content was determined by inductive coupled plasma optical emission spectroscopy (ICP-OES) (Varian Inc., Palo Alto, CA, USA), and the mean hydrodynamic particle size was measured using a dynamic light scattering (DLS) ALV-5000F instrument (ALV-GmbH, Germany) with vertically polarized incident light (θ = 488 nm) supplied by a diode-pumped Nd:YAG solid-state laser (Coherent Inc., CA, USA). Zetapotentials were measured using a Zetasizer NanoZS instrument (Malvern, UK) equipped with a red laser (633 nm) in backscatter mode at 25 ºC. Polymer adsorption onto the cores was confirmed by Fourier-transform infrared (FTIR) spectroscopy with a FTIR spectrometer (FTIR 670, Varian Inc., Palo Alto, CA, USA) and quantified by thermal gravimetric analysis (TGA) on a Q5000IR (TA instruments, New Castle, DE, USA).

Cell labeling with D-MNPs combined with Poly-L-Lysine
MSCs were labeled following the protocol previously described. 2

Characterization of D-MNP labeling of MSCs
Total cell counts were performed using Trypan Blue stain (STEMCELL Technologies, Grenoble, France) and a Neubauer counting chamber (Blaubrand, Sigma-Aldrich, St. Louis, MO, USA). Samples were diluted 1:5 with PBS and 1:2 with Trypan Blue. Cell counts were performed using an inverted microscope (Olympus IX51, Shinjuku, Tokyo, Japan).
Cell viability assays were conducted using the lactate dehydrogenase assay (LDH) kit (Sigma-Aldrich, St Louis, USA), following the manufacturer's protocol. Lysed cell supernatants were also included (negative control). In brief, supernatants were centrifuged at 1000 rpm for 5 min and further incubated with LDH-reagents for 20 min. Then, the plate was read in a Synergy2 plate reader (Biotek Instruments, VT, USA) at 490 nm, and the viability rate (%) was calculated with respect to control and lysate values.
Iron uptake was quantified by dissolving 1×10 5 cells in 1 mL HCl 37% extra pure (Merck, Darmstadt, Germany). Distilled water was added to achieve a total volume of 5 mL.

Magnetic resonance imaging
All studies were conducted on a 9.4T horizontal bore magnet (Bruker BioSpin, Ettlingen, Germany) with 440 mT/m gradients and a combination of a linear birdcage resonator (7 cm in diameter) for signal transmission and a 2 × 2 surface coil array for signal detection. A quadrature volume coil (7 cm in diameter) was also used in phantom studies.

In vitro magnetic resonance imaging
Agar phantoms loaded with D-MNPs were made following a previously 2  Agar phantoms of labeled and non-labeled cells were made to assess the detection capabilities of D-MNPs by magnetic resonance following a previously described procedure 2 with 1X10 5 cells per condition. T 2 *-weighted images were acquired using a multi gradient echo (MGE) sequence with 4.44 ms echo time, 1.8 s repetition time, 16 echoes with 6.75 ms echo spacing,100 KHz spectral bandwidth, FA= 30º, 14 slices of 1 mm, 2 averages, 75 × 75 mm 2 FOV (with saturation bands to suppress signal outside this FOV),a matrix size of 256 × 256 (inplane resolution of 293 μm/pixel × 293 μm/pixel) and implemented with fat suppression option.

In vivo magnetic resonance imaging
Basal ischemic lesion during MCA occlusion was determined by counting pixels with apparent diffusion coefficient (ADC) values below a threshold in the ipsilateral brain hemisphere. The values of ADC in the healthy rat brain normally do not fall below 0.55 x10 -3 mm 2 /s; therefore, this threshold provides a convenient means of segmenting abnormal tissue. 4

Transmission electronic microscopy study
Animals were sacrificed under sevofluorane anesthesia (8%), and 6 samples of brain cortex and striatum were taken immediately after death. Fixation and post-fixation were performed in 2% glutaraldehyde or 1% OsO 4 in sodium cacodylate buffer. Inclusion was performed in Spurr's epoxy resin. Semithin sections (0.5 μm) were stained with Toluidine blue and examined under an optical microscope to select brain regions for further TEM analysis, and ultrathin sections (100 nm) were stained with uranyl acetate and lead citrate.

Immunohistology
Animals were anesthetized and perfused transcardially with 100 mL PBS 0.1M pH 7.4 and 150 mL 4% formaldehyde (VWR Chemicals, Leuven, Belgium). Brain, lung and heart were removed carefully from the skull and sectioned in 2 mm slices in a matrix, post fixed by immersion in 4% formaldehyde overnight, dehydrated, and embedded in 4% paraffin (VWR International a/s, Albertslund, Denmark).