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Comparison of MSC-Neurogenin1 administration modality in MCAO rat model

Abstract Intracerebral (IC) grafting of mesenchymal stem cells (MSCs) is not currently used in humans due to its potential complications. On the other hand, intra-arterial (IA) administration can be facilitated for engrafting of intensifed MSCs in the injured human brain. The study is designed to compare the two methods of MSC administration using IA and IC routes through the parameters of behavior, infarct volume, cell distribution, and MSC identification. An ischemic stroke model was generated in Sprague Dawley male rats. This experiment used MSCs/Ngn1 that express Neurogenin1 (Ngn1) to ensure grafted MSC maintenance. MSCs/Ngn1 or normal saline was administrated via the IC or IA route on day 3. All animals were randomly assigned into four groups (five rats in each group): IC-control, IA-control, IC-MSCs/Ngn1, or IA-MSCs/Ngn1. Motor behaviors, infarct volume, and distribution of superparamagnetic iron oxide (SPIO)-labeled cells on magnetic resonance imaging (MRI) were compared from each group. There were no baseline differencess in motor behaviors or infarct volume between IC-MSCs/Ngn1 and IA-MSCs/Ngn1. Hovever, the IA-MSCs/Ngn1 group showed the greatest recovery on Rotarod testing and adhesive removal tests (p = 0.003 and p = 0.009 vs. IC-MSCs/Ngn1, respectively). The IA-MSCs/Ngn1 group also had more evenly distributed SPIO-labeled cells on MRI. The results suggest that IA administration is likely to be benefcial for humans based on its ability to improve behavioral outcomes and ensure even MSC engrafting.


Introduction
Mesenchymal stem cells (MSCs) exist in adult bone marrow and occupy less than 0.01% of all nucleated bone marrow cells [1]. Even though the main MSC differentiation is of mesodermal lineage, several experimental studies have revealed that these cells can differentiate into neuronal and glial ectodermal cells [2][3][4]. Animal and human trials have suggested that MSC administration exerts beneficial effects in ischemic heart disease, stroke, and even progressive neurodegenerative diseases, regardless of their autologous or allogeneic origin [5][6][7].
Neurogenins (Ngn) are a family of basic helix-loop-helix (bHLH) transcription factors involved in neuronal or glial differentiation, and their subtypes are classified into Ngn 1, 2, or 3 [8]. Among Ngn subtypes, Ngn1 facilitates neuronal differentiation as an activator for downstream transcription factors such as NeuroD [9]. A previous study demonstrated that intracerebral (IC) administration of Ngn1expressing MSCs (MSCs/Ngn1) produced restorative augmentation of behavioral functions and induced the expression of neuron-specific proteins and voltage-gated Ca 2+ and Na + channels, suggesting survival of the grafted MSCs [10].
Animal experiments have shown that intracerebral (IC) administration is a reliable route for stem cell transplantation [11][12][13]. IC cell grafting is avoided in the clinical field due to its hazardous complications, such as seizure, chronic subdural hematoma, cortical vein occlusion, and postprocedural clinical deterioration [14][15][16]. On the other hand, with intravenous (IV) administration the majority of MSCs are entrapped within the lung and liver before they are targeted to the brain, which potentially reduces their therapeutic potential [5,17].
Theoretically, intra-arterial (IA) cell grafting is more feasible in clinical applications because of more predictable engrafting (vs. IV approach) and less invasiveness (vs. IC approach). An animal study reported that IA administration of bone marrow stromal cells can facilitate axonal sprouting and remyelination in the cortical ischemic boundary zone and improve neurological function [18]. Recent animal studies showed that IA administration is a safe and more effective method than IV administration for treating cerebral ischemia [19,20]. Abbreviations: IC = intra-cerebral, IA = intra-arterial, NS = normal saline, MSCs/Ngn1=Ngn1-expressing mesenchymal stem cell Figure 1. Routes of stem cell administration. A. Rat was anesthetized, skull exposed, and cells were intra-cerebrally transplanted into the cortex and striatum in the ipsilateral hemisphere using a stereotactic apparatus. Numbers indicate the distance (in mm) anterior to bregma and shading indicates the region of ischemic damage. B. Rat was anesthetized, common carotid artery (CCA) ligated, followed by insertion of a catheter tip to the CCA bifurcation through external carotid artery (ECA), and intra-arterial administration of cells was performed. ICA, internal carotid artery; PPA, pteryopalatine artery.

Statistical analysis
Comparisons between the two groups were achieved using Student's unpaired t-test.
Multiple comparisons for each treatment group were analyzed using one-way analysis of variance (ANOVA) with the independent variables being treatment groups and days of testing. These were followed by Tukey's post hoc tests. All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA), and the results were used for multiple comparisons.

Serial changes in infarct volume according to administration modality
The RIV of the rat brains were monitored using MRI analysis over the 28-day experimental period (Fig. 2C) and histologic assessments of ischemic rat brains were performed on day 28 (Fig. 2D). MSCs/Ngn1 stem cells via intra-cerebral or intra-arterial route were evaluated by the A. Rotarod test or B. adhesive removal test. MSC/Ngn1 stem cells showed a therapeutic effect compared with the control and the intra-arterial route had better functional recovery compared with the intra-cerebral route. C. Quantitative analysis of infarct volume was evaluated using brain magnetic resonance imaging. Statistical significant differences between the groups were determined by analysis of variance (*p < 0.05 compared with IA-control group; # p < 0.05 compared with IC-MSCs/Ngn1 group

IC-MSCs/Ngn1
Rotarod test (s)  Stem cell therapy has emerged as a promising strategy for treating neurological diseases [25,26]. MSCs are of particular clinical interest because they are easily isolated from bone marrow, adipose tissue, and umbilical cord blood and can be expanded through as many as 50 population doublings in about 10 weeks [27]. In previous studies, MSCs were shown to ameliorate neurologic dysfunction in patients with acute ischemic stroke [5,17,28]. One study reported that IC administration of Ngn1-expressing MSCs was more beneficial than parenterally delivered MSCs in an acute animal stroke model [10]. A possible   It is known that IA delivery of MSCs enables efficient cell administration to an infarcted area and it results in significant functional recovery after ischemic stroke in a rat model [29,30]. However, the major issue of IA administration is microembolization due to aggregation of the cells in the blood vessels [31]. The major determinants of the safety of IA stem cell administration are cell size, velocity of injection, and cell dose [22,32]. Janowski et al. [22] showed that administration of MSCs with an infusion velocity over 1 mL/ minute often resulted in stroke whereas a lower velocity (0.2 mL/minute) was safe. They also showed that stroke lesions occurred frequently when injecting 2 x10 6 MSCs, but not after lowering the dose to 1x 10 6 cells.
In this study, we administered a low dose of  [14][15][16]. Although IV injection is a clinically relevant and minimally invasive technique, only a small amount of injected cells reach the targeted brain area due to the first pass effect in the lungs and liver [19]. Since the IA route increases the chances of MSC migration and distribution, it facilitates efficient engrafting of stem cells to the target brain areas [33].