Real-time MRI for precise and predictable intra-arterial stem cell delivery to the central nervous system

Abstract

Stem cell therapy for neurological disorders reached a pivotal point when the efficacy of several cell types was demonstrated in small animal models. Translation of stem cell therapy is contingent upon overcoming the challenge of effective cell delivery to the human brain, which has a volume ∼1000 times larger than that of the mouse. Intra-arterial injection can achieve a broad, global, but also on-demand spatially targeted biodistribution; however, its utility has been limited by unpredictable cell destination and homing as dictated by the vascular territory, as well as by safety concerns. We show here that high-speed MRI can be used to visualize the intravascular distribution of a superparamagnetic iron oxide contrast agent and can thus be used to accurately predict the distribution of intra-arterial administered stem cells. Moreover, high-speed MRI enables the real-time visualization of cell homing, providing the opportunity for immediate intervention in the case of undesired biodistribution.

Introduction

The interest in stem cell-based regenerative medicine has steadily increased, driven by the rising needs of aging societies and further spurred by numerous successful applications in small animal models. Neurological disorders are at the forefront of this interest because of the severe and long-term disability that occurs with cell loss and because of the paucity of therapeutic options. Therefore, neurological disorders are prime candidates for the application of regenerative medicine. The progress in stem cell biology is remarkable, as evidenced by several Nobel prizes over the last decade.¹ In contrast, the overall progress in developing accurate cell delivery techniques, especially for the brain, has been very slow. While the intravenous route of cell delivery is used most frequently in neurological disorders, there is general agreement that the engraftment in the brain is probably much smaller and the therapeutic effect is mediated indirectly from the peripheral location of injected cells.² Direct intra-parenchymal injection is also used frequently, but it has several drawbacks. The injected cell mass increases local pressure, which stresses both the transplanted cells, as well as the surrounding host tissue. It also results in local tissue damage, including frequent microbleeding, which, in turn, facilitates the activation of the immune system, resulting in aseptic brain inflammation.³ Thus, there is a search for more accurate and efficient ways of cell delivery to the CNS. The use of body fluids as gateways for cell delivery and transient cell reservoirs is an attractive option, but needs further technological development to make the procedure of cell transplantation highly precise.

Catheter-based intra-arterial (IA) infusion is a promising method for cell delivery to the desired regions of the CNS. The interest in that route is rapidly growing as an excellent safety and efficacy profile has been recently proven for IA re-perfusion therapy in patients with stroke.⁴ Thus, IA stem cell infusion could easily be added to endovascular intervention in patients with stroke, what makes the IA route of cell therapy even more attractive. The obstacles to appropriate cell homing^5,6 and safety issues^7,8 have been largely addressed. While the IA method is potentially powerful and clinically applicable, it requires real-time image-guidance to precisely direct the cell flow to areas of interest; otherwise, cells can reach undesired destinations. While MR tracking of superparamagnetic iron oxide (SPIO)-labeled cells has been widely pursued,^9–12 including clinical studies,¹³ to date, all MRI stem cell-tracking studies have detected cells only after the transplantation was completed, which is too late.^5,14,15

We have recently shown that MRI guidance is excellent for improving precision of IA infusion-based opening of the blood–brain barrier and chemotherapeutic drug delivery, while X-ray fluoroscopy lacks sufficient sensitivity.^16,17 Here, we show that this method is pivotal for imaging of stem cell infusion in real-time, which, in turn, enables outstanding precision of IA cell transplantation. In this study, we used therapeutic stem cells of different sizes—small glial restricted precursors (GRPs), and large mesenchymal stem cells (MSCs)—to emphasize the value and benefits of real-time image-guidance. First, we tested the predictive value of image-guidance for cell destination using selective catheterization in large animal models, and then, we investigated how the pathology changes the biodistribution of intra-arterially injected cells in small animal models. The implementation of this image-guidance into cell therapy protocols may improve the safety and efficiency of IA injection, making this the method of choice for cell delivery to the brain.

Methods

Outline of overall experimental design

Experiments were performed in accordance with guidelines for the care and use of laboratory animals approved by the Institutional Animal Care and Use Committee of Johns Hopkins University, Baltimore, MD, Mossakowski Medical Research Centre, Warsaw, Poland and University of Warmia and Mazury in Olsztyn, Poland and reported according to the ARRIVE (Animal Research: Reporting In Vivo Experiments). All experimental procedures were approved by our Institutional Animal Care and Use Committees. Initial experiments were performed to demonstrate that real-time imaging of cell transplantation is feasible, and that cell destination can be precisely predicted using a highly translational large animal model and a clinical MR scanner. Then, we showed that near-single-cell flow can be captured in real-time. Subsequently, we showed that real-time imaging is also critical for proper cell infusion in small animal models of brain diseases, and the velocity of cell inflow can be measured and potentially used to manipulate cell destination (Figure 1).

Figure 1.

Schematic representation of overall experimental design. The numbers indicate the sequence of experiments. © JHU I-Hsun Wu 2016.

>For the large animal studies, we used the minimal number of animals that would enable proper demonstration of the feasibility of our methods. The sample size for the procedures in rodents was set to reach statistical significance for the acquired data. We performed initial transplantation studies for two animals in each group, providing means and SD values, which were then used to perform a power analysis and assign groups accordingly. We hypothesized that there would be differences in the speed of inflow, depending on the delay between stroke induction and cell infusion, and that these differences can be detected by real-time MRI. As our studies did not include a therapeutic arm or different treatments between groups, no randomization or blinded group allocation was performed.

Cell preparation and labeling

Several cell types were used for this study, including commercially available human mesenchymal stem cells (hMSCs, PT-2501, Lonza), human glial-restricted precursors (hGRPs; Q cells®, Q Therapeutics), and porcine (p)MSCs, and mouse (m)GRPs derived from transgenic luciferase-expressing mice (Luc⁺mGRP; Taconic). pMSCs were isolated from iliac crest bone marrow following Ficoll density gradient centrifugation. Nucleated cells were collected and cultured in MSC medium (Lonza) until a monolayer reached 70% confluence. mGRPs were isolated according to a previously established protocol.¹⁸ Cells from passage two to five were used in all experiments.

Mostly, hMSCs were used in experiments, but for porcine transplantations, we used easily obtainable pMSCs to lower the cost of experiments. GRPs were used to show the difference in cell flow through cerebral vessels, and hGRPs were used to visualize them on a single cell level in MRI, while mGRPs were used when bioluminescent cells were desired.

Labeling of cells with SPIO nanoparticles (Molday ION-Rhodamine B, BioPAL, Inc.) was performed by overnight incubation with 20 µg Fe/ml of the contrast agent formulation. We have previously reported that this protocol is neutral for MSC characteristic (including viability).¹⁹ We have also successfully used this protocol in our previous studies with neural stem cells²⁰ or GRPs.³ Prior to injection, cells were trypsinized, centrifuged at 1000 r/min for 4 min, resuspended in 10 mM PBS, pH = 7.4, filtered through a 70 µm nylon cell strainer (BD Falcon™), counted, checked for viability (viability at least 90% qualified cells for transplantation) and adjusted to 0.2–1 × 10⁶ cells/ml.

Cannulation of arteries under fluoroscopic guidance in large animal models

Swine (male, weight = 30 kg, n = 6) were anesthetized with propofol (3 mg/kg/h) and a 5-French femoral arterial sheath was surgically introduced. A 5-French angled glide catheter was advanced over a 0.035-inch glide wire and the right CCA was selectively catheterized using real-time fluoroscopy. Since pigs do not have an extracranial portion of the internal carotid artery (ICA), the catheter was advanced distally into the ascending pharyngeal artery proximal to the rete mirabile (route of major blood supply to the brain) using roadmap guidance.

For cerebral IA injection in dogs (male greyhound dogs, weight = 20–25 kg, n = 2), a 5-French femoral arterial sheath was surgically introduced, and a 5-French angled glide catheter was advanced over a 0.035-inch glide wire into the ICA under fluoroscopic guidance. A 1.7-French microcatheter was then advanced over a 0.014-inch microwire, and the origin of the MCA was selectively catheterized under roadmap guidance.

For spinal IA injection in dogs (male greyhound dogs, weight = 20–25 kg, n = 2), a 5-French femoral arterial sheath was surgically introduced. A 5-French reverse curve catheter was advanced over a 0.035-inch guide wire and a limited spinal digital subtraction angiogram was performed on a single plane angiography unit (Artis, Siemens). Intersegmental arteries were selectively catheterized and injected with 2 ml of iodinated contrast (Omnipaque 300, GE Healthcare) to identify the origin of the Adamkiewicz artery (great radicular artery). Using the 5-French catheter as a guide catheter, a 1.7-French microcatheter was co-axially advanced over a 0.014-inch microwire to selectively catheterize the Adamkiewicz artery.

Stem cell transplantation in a large animal model

After fixation of catheter in desired position, the pigs were transferred from X-ray table to a clinical 3T MRI (TRIO, Siemens) scanner and pre-transplantation T2 and SWI scans were acquired. Next, MSCs were injected IA at 5 ml/min while acquiring dynamic GE-EPI scans for real-time monitoring of cell inflow into the brain. The unique vascular anatomy of swine, with the network of arterioles on the skull base (rete mirabile), prohibits advancing an intravascular catheter into selective brain arteries. The non-selective catheterization results in transcatheter cell distribution to the entire hemisphere, what allows for determination of feasibility of real-time cell visualization of cell flow, but is insufficient to assess the predictability of cell distribution.

Thus, selective catheterization experiments were performed in dogs, which are characterized by an intracranial circulation that is more similar to that of humans. With the microcatheter at the origin of the MCA, we initially injected three separate short boluses of iron oxide-based MRI contrast agent: ferumoxytol (0.03 mgFe/ml),¹⁶ while acquiring dynamic GE-EPI (parameters are in a section below) to predict the area of cell distribution. Then, the MSCs were intra-arterially infused at the same speed. To confirm that the cerebral blood flow (CBF) was not altered by transplanted cells, three boluses of ferumoxytol followed cell delivery.

Middle cerebral artery occlusion stroke model

Sprague-Dawley rats (male, 250 g, Harlan, n = 4) were anesthetized with 2% isoflurane and an optic fiber was attached to the temporal bone after surgical cut-down to measure blood flow with laser Doppler (moorVMS-LDF, Moore Instruments). ICA was exposed with ligation of the extracranial branches.⁵After temporary closure of the ICA and the CCA by surgical clips, the arterial wall of the ECA was microdissected and an Ethilon 4.0 suture with a silicon tip was manually advanced into the ICA. After removal of the clip, the suture was further advanced into the cerebral arteries until a decrease in CBF of at least 70% (as measured by LDF) was reached. The suture was held in place for 45 min and then removed. The stump of the ECA was ligated and the clip was removed from the CCA, restoring blood flow in the CCA and the ICA wounds, which were sutured and closed.

Lacunar stroke model

Wistar rats (male, 250 g, n = 16) were anesthetized with ketamine i.p. (90 mg/kg) and xylazine (10 mg/kg), immobilized in a stereotactic apparatus (Stoelting Inc.). A small burr hole was drilled in the skull over the right hemisphere. The needle, connected to a 10 -μl syringe (Hamilton), was inserted into the right striatum (A = 0.0, L = 3.0, D = 3.5 mm). An injection of 3 μl of 5 mM ouabain (Sigma) in saline was given at a rate of 1 μl/min via a microinfusion pump (Stoelting Inc.) mounted on the stereotactic apparatus. The needle was then withdrawn and the skin was closed with a nylon suture.

Carotid artery cannulation in rats

For the lacunar stroke model, under general isoflurane anesthesia, the animals were positioned supine and the CCA and ICA were dissected with the extracranial branches permanently ligated. For the middle cerebral artery occlusion (MCAO) stroke model, the surgical wound was re-opened and the carotid arteries were dissected within the post-operative scar, without approaching the PA or the stump of the ECA. The CCA was permanently ligated at the proximal site. For both stroke models, a catheter (VAH-PU-C20, Instech Solomon Inc.) connected to #30 PTFE tubing was introduced into the ICA and secured in place to the ICA and the CCA. Temporary sutures were applied to the surgical wound and the animal was then transferred to the MRI scanner. After transplantation and imaging, the catheter was removed and the surgical wound was permanently closed with sutures.

IA stem cell delivery in the MCAO and lacunar stroke model

SPIO-labeled hMSCs were injected IA in Sprague-Dawley rats at speeds 0.2 and 0.4 ml/min a day after induction of MCAO stroke. The MRI and bioluminescent imaging was performed during and after transplantation (n = 2).

SPIO-labeled MSCs were injected IA in Wistar rats (n = 16) at a speed 0.2 ml/min at various time points (1, 2, 3, and 7 days) after the induction of lacunar stroke.²¹ Following the acquisition of baseline T2 and T2* sequences, SPIO-labeled hMSCs were loaded into the syringe attached to the catheter and infused. Dynamic acquisition of the MR images using GE-EPI was started 10 s before initiating the infusion of SPIO-labeled cells. We then evaluated IA delivery of SPIO-labeled small-size hGRPs (diameter ca. 13 µm) compared to that of larger-sized MSCs (diameter ca. 25 µm).

Bioluminescence imaging

Whole-body bioluminescence imaging (BLI) was performed in rats to determine the biodistribution of transplanted Luc⁺mGRPs. Anesthesia was induced with 5% isoflurane and maintained with 2% isoflurane/98% oxygen. Luciferin was administered i.p. at 150 mg/kg. Male rats (weighing ca. 250 g, n = 2) were placed inside a Spectrum/CT optical imager (PerkinElmer) and imaged every 5 min for a period of 30 min, with the acquisition time set to 1 min. The acquired images were processed using LivingImage® software (PerkinElmer).

Histological analysis

Animals were transcardially perfused with 10 mM phosphate-buffered saline (PBS) and then with PBS-buffered 4% paraformaldehyde. Brain tissue was cryopreserved in 30% sucrose and cryosectioned at 30 µm. Fluorescent images of SPIO rhodamine-tagged cells were acquired using an inverted microscope (Zeiss, Axio Observer Z1). Fluorescent images were analyzed with NIH Image J software. For quantitative assessment of cell targeting, images of 2.8 × 2.8 mm (4.19 × 10⁶ pixels in each dimension) were converted to 8-bit datasets and a minimum threshold of 30 (dog tissue samples) or 15 (rat tissue samples) was applied. Higher thresholds for dog tissue were related to a higher endogenous background, which is often observed for the brain in older subjects. The “analyze particles function” was used to select fluorescent objects sized between 40 and 450 pixels in each dimension to enable cell counting. The number of cells for each field of view was counted using stereological methods. The number of cells present in areas corresponding to the areas of cell homing on the MR images was compared to areas in which no cell inflow was visible on the MR images.

Magnetic resonance imaging

Large animals

For dogs and swine, a clinical 3T Siemens Trio was used to acquire pre-transplantation baseline T2 and T2* scans and dynamic GE-EPI (TE = 36 ms, TR = 3000 ms, FOV = 1080, matrix = 128, and acquisition time = 3 s and 50–100 repetitions). For brain imaging, a quadrature head coil was employed, and, for spinal imaging, a coil built within the scanner bed was used. These fast sequences offer MR images in real-time, providing immediate feedback, which enables continuous monitoring of cell delivery. Standard T2-w and gradient-echo images were also acquired after cell injection.

Small animals

After cannulation, imaging was performed using a horizontal bore Bruker 7T or 11.7T scanner with a 15 mm planar surface coil. Following acquisition of pre-transplantation baseline T2 and T2* images, real-time dual MRI of ferumoxytol perfusion and SPIO-labeled cell injections was performed using a standard echo planar imaging (EPI) sequence with TE = 17 ms, TR = 2000 ms, FOV = 26 × 26 mm, matrix = 96 × 96, and acquisition time = 2 s with 200 repetitions. Standard T2-w and gradient-echo images were also acquired after labeled cell injection.

Image processing

OsiriX and Amira software were used for image visualization. Quantitative image analysis was achieved using MATLAB. A square-type waveform was assumed, representing the time course of injection. Reference waveforms were correlated with the pixel time course to form an array of p values. P maps were calculated for each continuous dynamic acquisition. The p values were normalized to the contralateral hemisphere (which did not receive injection) to mitigate the internal noise of each dataset.

Statistical analysis

Regression analysis is reported as Type III tests. The least square mean (LSM) values were used to detect differences from baseline and for comparison between means (PROC MIXED, SAS 9.4).

Results

Visualization of IA cell infusion procedure to the brain using real-time MRI in a porcine model

Successful cannulation of the ascending pharyngeal artery with visualization of the rete mirabile was confirmed by X-ray fluoroscopy (Figure 2(a)) with reference to the anatomy of cerebral vessels (Figure 2(b)). Serially acquired images of the inflow of SPIO-labeled cells into the brain exhibited a gradual, focal reduction of pixel intensities (PI) on consecutive GE-EPI images over the period of cell injection. This corresponded to the process of MSC accumulation within the porcine brain (Figure 2(c) to (h)), with the cell location subsequently confirmed by high resolution ex vivo MRI (Figure 2(i)). Notably, follow-up MR imaging performed the next day (n = 3) and after one week (n = 1), using T2-w sequences, did not detect ischemia as a possible result of cell-induced microemboli (Figure 2(j)).

Figure 2.

Use of real-time MRI to predict IA transcatheter perfusion territory in the swine brain. (a) Placement of catheter in the ascending pharyngeal artery feeding the carotid rete of many intertwining arteries that supply cerebral blood flow. (b) Schematic of the cerebral vasculature in swine. T2-weighted MRI (c,f), real-time GE-EPI (d,g), and SWI MRI (e,h), pre- (c–e) and post (f–h) injection of 5 × 10⁶ MSCs at 1 ml/min. Cell engraftment occurred in nearly the entire hemisphere, with SWI and GE-EPI sequences being the most sensitive. (i) Ex vivo high-resolution MRI showing a punctate pattern of cell distribution within the brain. (j) T2-weighted image obtained after one week post cell injection reveals normal brain anatomy with no signs of infarct/ischemia. Figure 2(b) © 2013 Lydia Gregg.

Prediction of cell destination using real-time MRI in a canine model depends on catheter tip location

While pig experiments were instrumental in showing that real-time imaging of IA stem cell infusion is feasible, the inability to selectively cannulate the cerebral arteries due to the presence of the rete mirabile did not allow for the investigation of spatial prediction of cell destination. Thus, for this purpose, we used a canine model, as the dog carotid system is similar to that of the human.

A 0.3 mg/ml concentration of ferumoxytol was sufficient to detect a change in signal intensity on MRI. The observed perfusion area in the dog brain was found to correspond to the MCA territory (Figure 3(a) and (b)). We then injected MSCs at the same speed and compared their spatial distribution to that of the ferumoxytol bolus and found an exact overlap (Supplementary Video 1). There was a moderate-to-strong correlation between the two p-maps derived from the ferumoxytol perfusion territory and cell engraftment territory (r = 0.58, p < 0.05). Since MSCs lodged in the brain capillaries, we confirmed that homing of cells to the brain does not compromise blood perfusion. Microscopic analysis of post-mortem brain tissue revealed SPIO-rhodamine-labeled red fluorescent cells in the targeted vascular territorial area, but not in the non-targeted areas (Figure 3(c)). Quantitative analysis of the number of localized fluorescent cells revealed that the difference was statistically significant (p < 0.05).

Figure 3.

Use of real-time MRI to predict IA transcatheter perfusion territory in dog brain. Three bolus injections of 300 μl Ferumoxytol® (0.3 mg Fe) were given 15 s apart at an injection speed of 1 ml/min within a 0–170 s interval, with subsequent clearance. Subsequent IA injection of SPIO-labeled cells infused at the interval between 170 and 400 s resulted in a gradual reduction of PI in the same region previously highlighted by ferumoxytol infusion. To confirm that the CBF was not compromised by the engrafted cells, ferumoxytol was injected a second time as three boluses after cell delivery, between the interval of 400–600 s, which resulted in clearance of ferumoxytol but not of transplanted cells (a). Red ROI represents the brain region of transplanted cell engraftment as predicted by ferumoxytol infusion. Blue ROI represents the region outside the targeted cell engraftment area, also as predicted by ferumoxytol infusion. Green ROI represents the contralateral hemisphere used as a control to validate the temporal stability of the image. Graph lines and ROIs are shown in corresponding colors (b). The darkening of MR image due to SPIO is illustrated at the graph as the drop of signal. The difference between the ipsilateral targeted hemisphere (C), and the contralateral non-targeted hemisphere (D) was also statistically significant on post-mortem evaluation (e, <0.0001). Localized cells are visible as red (rhodamine⁺).

Visualization of IA delivery procedure of MSCs to the spinal cord using real-time MRI in a canine model

While we have shown that real-time MRI of cell infusion to the brain is feasible, that organ is relatively homogeneous magnetically, and thus, easy to image. Therefore, we tested whether real-time MRI of cell infusion is also possible in the spinal cord, as the spine is difficult to image due to magnetic field inhomogeneity.

We demonstrated a broad distribution of infused cells within the lumbar and thoracic spinal cord. These images showed more cells engrafting to the regions of the proximal artery of Adamkiewicz (Figure 4(a) and (b); ROI 1 and 2) compared to the distal part (Figure 4(a) and (b); ROI 3 and 4). The dynamics of cell inflow are visualized in Figure 4(c) and Supplementary Video 2. A rapid and extensive cell inflow to the segments proximal to the artery of Adamkiewicz was observed, with a slower and reduced cell inflow in distant regions of the spinal cord.

Figure 4.

Use of real-time MRI to predict IA transcatheter perfusion territory in the dog spinal cord. Digital subtraction angiogram of selective catheterization of the artery of Adamkiewicz. 1=5-French reverse curve guide catheter located in the inter-segmental artery branching off the aorta, 2=1.7-French microcatheter located in the Adamkiewicz artery (great radicular artery), 3=Adamkiewicz artery on the angiogram, and 4=anterior spinal artery directly supplying blood to the spinal cord (a). Time course of cell injection visualized by GE-EPI MRI (b). Several ROIs were drawn (green and red=proximal to the Adamkiewicz artery, blue and orange=distal to the artery, white=control below the artery), and their pixel intensity quantified as shown in C. Control ROI (white) validates the stability of the GE-EPI MR image. Graph lines and ROIs are shown in corresponding colors.

Near-single-cell real-time visualization of stem cells in a small animal model of stroke under high-field MRI

While the infusion of large MSCs results in spontaneous accumulation of MSCs in cerebral vessels, the small GRPs flow through the cerebral vasculature, being stopped only after appropriate cell engineering.⁵ Thus, we were curious whether these distinct cell behaviors could be captured in real-time by MRI. As expected, large-size MSCs gradually accumulated in the cerebral vasculature (Supplementary Video 3). Real-time MRI of cell infusion clearly demonstrated that small-size hGRPs were able to flow through the brain capillary bed, with only a few cell aggregates lodging within the vasculature (Supplementary Video 4, white arrow). To avoid cell aggregation, the cells were infused immediately after preparation and the real-time MRI was the only proof that cells flowed through the cerebral vessels (Supplementary Figure 1).

Application of real-time MRI to adjust the speed of IA cell injection in small animals

Although we have previously shown that the speed of 0.2 ml/min is effective in IA delivery of stem cells to the intact rat brain,²² real-time MRI failed to show any inflow of cells to the brain subjected to MCAO when infused at the same speed (Figure 5). Due to the small size of the surface coil, imaging of extracerebral tissues was unfeasible. Since the transplanted cells were luciferase (+),^3,23 we used whole-body BLI to identify homing areas presumably outside the brain. BLI showed that cells homed to the ocular region, indicating that the injected cells were exclusively routed to the ophthalmic artery, an intracranial branch of the ICA, and failed to reach the cerebral arteries (Figure 5(f)). We hypothesized that this might be due to a blood pressure imbalance as the catheter fills the entire lumen of the artery. With no blood flow in the cannulated artery, a slow transcatheter flow can be directed to the ophthalmic artery as a result of the blood pressure transferred through the circle of Willis. Our results indicated that the blood pressure in the cerebral arteries might be higher in a stroke model, preventing the cells from being infused into the brain at low infusion speeds (0.2 ml/min). Thus, we doubled the injection speed to 0.4 ml/min and this yielded efficient targeting of the cells to the brain, as confirmed by MRI (Figure 5). P-value maps revealed that only 40 pixels were changed in the slow infusion mode, which was not significantly different from the baseline (p = 0.35). Fast cell infusion resulted in a change of 715 pixels, which was statistically significant compared to baseline (p < 0.05). There was also a difference between slow cell infusion and fast cell infusion images (p < 0.05). Consistently, the BLI signal that represented engrafted cells was detected from the brain region (Figure 5(k)).

Figure 5.

Use of real-time MRI to adjust the cell injection speed during IA injection to ensure efficient stem cell delivery to the brain in rats. (a) Schematic of the cerebral circulation in the rat. (b–e) Injecting cells at 0.2 ml/min does not result in cerebral engraftment, as no difference in pixel intensity (red circle = ROI, quantified in d) between the pre- (0 s) and post-injection (400 s) images was observed. (f) BLI of the same animal shows signal only above the ocular area. (g–j) In contrast, injecting cells at 0.4 ml/min results in effective cerebral engraftment, with a marked decrease in pixel intensity (i). (k) BLI confirms signal in the cerebral area without ocular localization. Color-coded subtraction images (e,j) highlight the difference in cell engraftment. Figure 5(a) © 2013 Lydia Gregg.

Application of real-time dual MRI to predict cell biodistribution in small animals depends on the infusion speed

While we have above shown, in a large animal model, that the dependence of cell distribution territory on the catheter tip position can be predicted, here, we investigated, in a small animal model, whether the dependence of cell distribution on the infusion speed could also be predicted. Using transcatheter ferumoxytol injection, we observed a strong association between infusion speed and perfusion territory. In a single experiment, an infusion speed of 0.2 ml/min in the MCAO stroke model failed to route blood flow to the brain, with signal detectable only at the skull base, which can be assigned to the OA contribution area. Ineffective transcatheter perfusion into the brain was evidenced by a low number of pixels with reduced signal intensity compared to baseline (283 pixels, p = 0.4) (Figure 6). Similar results were found for the IA cell injections at this rate. Increasing the injection speed to 0.4 ml/min was sufficient to obtain successful brain perfusion, as evidenced by a significant number of pixels with reduced intensity compared to baseline (1720 pixels, p < 0.05) (Figure 6). Again, the pixel mapping of perfusion results was consistent with the cell injection experiment described above, demonstrating the predictive value of this method. The difference between the two rates of infusion was statistically significant (p < 0.05). Importantly, IA-infused ferumoxytol completely clears from the brain vessels within seconds, enabling repetition of the procedure during the same imaging session until desired brain coverage is achieved.

Figure 6.

Use of real-time MRI to predict IA transcatheter perfusion territory in the rat brain. Two bolus injections of 0.03 mg Fe were given 10 s apart at an injection speed of 0.2 ml/min (a,b) or 0.4 ml/min (c,d). Color-coded scale shows changes in signal intensity resulting from the transient ferumoxytol inflow. An inefficient cerebral perfusion is achieved at 0.2 ml/min speed (black circle = ROI, quantified in b), with a flow of contrast agent visible solely at the skull base near the ICA prior to the circle of Willis (green circle = ROI, quantified in b). In contrast, injecting ferumoxytol at 0.4 ml/min resulted in effective cerebral perfusion, with a 75% decrease in pixel intensity (d). Note that the ferumoxytol perfusion is transient and completely clears at 20 s after each injection.

The measurement of cell inflow velocity after real-time MRI of IA cell delivery in a lacunar model of stroke

The real-time MRI was capable of revealing the time course of cell distribution within the stroke lesion (Supplementary Figure 2). PI graphs were generated to depict the time course for selected ROIs (Supplementary Figure 2(b)). No change of signal was observed in the contralateral hemisphere, demonstrating that the cells did not re-enter the circulation with secondary lodging in the contralateral brain. Real-time MRI demonstrated that cells initially are located within the stroke periphery, with a delayed inflow into the core of the infarct. However, at the end of the infusion, the cell distribution within the overall infarcted area was quite homogeneous.

By comparing different time intervals between stroke induction and cell injection, it became apparent that the temporal–spatial pattern of engraftment is interval-dependent, with the slowest and lowest amount of cell homing at day 1 post injection compared to later time points (Figure 7). Histological analysis revealed that rhodamine-labeled cells indeed localized specifically in the regions previously shown as hypointense on MRI. Cell localization in regions that previously exhibited no MRI hypointensity was negligible (Figure 7) (p < 0.001). Interestingly, real-time monitoring of cell infusion also indicated the existence of an extensive extracranial-intracranial anastomosis system in some animals, resulting in cells being carried from the brain to the muscles of the head (Supplementary Video 5).

Figure 7.

Dependence of velocity and number of infused cells upon the interval between stroke induction and cell transplantation. Visualization of the velocity of cerebral cell inflow for different intervals between stroke induction and cell injection (red=high velocity, blue=low velocity) (a). Graph representing the dependence of cell inflow velocity on the time delay after stroke induction (b). Graph representing the percentage of entire brain-cell engraftment as a function of the time delay after stroke induction (c). Note that the maximum brain engraftment that can be achieved was 50% (dashed line), as no contralateral flow was observed. Fluorescent images of tissue slices were used as a reference standard to validate targeted delivery. The difference between the ipsilateral targeted hemisphere (d), and the contralateral non-targeted hemisphere (e), was statistically significant on post-mortem images (f, p < 0.001). Asterisks represent statistical significance (p < 0.05).

Discussion

To the best of our knowledge, we are the first to show that high-speed MRI, based on a GE-EPI pulse sequence, enables monitoring of IA delivery of SPIO-labeled stem cells to the CNS in real-time with sufficient sensitivity and high temporal resolution. The GE-EPI is frequently used for dynamic susceptibility contrast imaging in perfusion MRI with intravenous Magnevist® administration, thus most MR scanners are equipped with this pulse sequence.²⁴ The loco-regional perfusion territory of individual arteries, although in general highly unpredictable, justifies the effort to predict cell biodistribution during catheter-based IA delivery. Real-time feedback about the temporal and spatial distribution of IA-injected ferumoxytol enables optimization of the position of the catheter and the infusion speed to ensure that cells reach the desired CNS regions with the highest possible precision. The inflow of cells and perfusion of the contrast agent into the brain depends on the local vascular resistance, with the possibility of reduced resistance in case of compromised cerebral autoregulation, as is encountered at some delay after cerebral ischemia. To overcome this resistance, it may be necessary to adjust the speed of IA cell injection according to the outcome of ferumoxytol pre-injection. The dynamic feature of GE-EPI also facilitates image processing and subtraction of background, enabling the creation of color-coded maps of cell biodistribution.

The real-time MRI was also capable of immediately detecting the potentially dangerous cell aggregates, which provides the opportunity for sudden cessation of cell infusion and the administration of a potentially de-aggregating therapy. The real-time monitoring of the procedure also answers questions about the lack of transplanted cells in the desired brain area, whether they were misrouted, or did not adhere and stopped within the cerebral vessels.

As IA therapy of brain disorders has been found effective not only in animal models,^25–27 but also in spontaneous animal disorders²⁸ as well as in a clinical trial,²⁹ it is critical to deploy this therapy in a highly controlled manner, particularly as there is some variability in results and not all authors have reported a prolonged positive effect.³⁰ It is expected that real-time monitoring of IA stem cell deliveries will further improve the therapeutic effect, as well as avoid complications in the clinical scenario. For example, it was shown that IA delivery of unmodified autologous bone marrow mononuclear cells is not superior to intravenous delivery,³¹ but, based on our results, the majority of small mononuclear cells probably went through the cerebral circulation unhindered, and more runs were necessary to accumulate in the brain parenchyma,³² which most probably was the reason for the lack of advantage of IA delivery.

Extensive evaluation of the safety of IA delivery was beyond the scope of this manuscript. However, we have previously performed detailed studies and established conditions for safe intracarotid infusion of stem cells, and we found that cell dose and the velocity of cell infusion are major determinants of safety.^7,8 We postulate that monitoring of cell transplantation procedures in real-time under MRI will redefine the method by which safety is evaluated from group-based to individual-based. This personalized approach will most likely result in much improved safety of the procedure and increase the efficacy of transplanted cells.

Currently, placement of catheters in the cerebral arteries is routinely performed under the guidance of biplane fluoroscopy. For MR-guided transcatheter cell delivery, the subject must be transferred between the angiography suite and the MRI suite. However, there are rapid advances in the development of intravascular “MR microscopy,” with MR coils built into the catheter.³³ With this approach, the entire procedure could possibly be performed in the future with real-time MRI as a “one-stop shop.”

Conclusions

High-speed MRI based on a GE-EPI pulse sequence can be used for real-time monitoring of IA delivery of SPIO-labeled stem cells to the CNS. Furthermore, a SPIO-based contrast agent is capable of predicting stem cell destination, as well as verifying vessel patency after IA cell infusion. Thus, real-time MRI allows for highly precise IA infusion of stem cells to the CNS, which may facilitate the use of this minimally invasive route to deliver cells to the vast, but defined brain regions, and is especially critical for large brains such as the human brain. Here, we have shown the feasibility of this technique in large animals using clinical MR scanners, making this approach fully clinically translatable.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by RO1 NS076573 (PW), R01 NS045062 (JWMB), MSCRFII-0193 (PW), MSCRFII-0052 (PW). NCN 2012/07/B/NZ4/01427 (JW), R21NS081544 (MJ), DOD PT120368 (PW, MJ) and the National Centre for Research and Development ERA-NET NEURON “MEMS-IRBI” project (BL, AN). MJ was supported by a Mobility Plus Fellowship from the Polish Ministry of Science and Higher Education.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

MJ – designed and performed experiments, performed data and statistical analysis and drafted the manuscript. PW – designed experiments and performed experiments and drafted the manuscript, JW, AN, AH, PH, JX, MP, PG – designed experiments, performed experiments, and edited manuscript, MC – data analysis and edited manuscript, ZA, BL, WM, JWMB – designed experiments and edited manuscript

Supplementary material

Supplementary material for this paper can be found at http://jcbfm.sagepub.com/content/by/supplemental-data