A Preclinical Murine Model for the Early Detection of Radiation-Induced Brain Injury Using Magnetic Resonance Imaging and Behavioral Tests for Learning and Memory—with Applicationsforthe Evaluation of Possible Stem Cell Imaging Agents and Therapies.
EthelJ. Ngen, Lee Wang, Nishant Gandhi, Yoshinori Kato,Michael Armour,Wenlian Zhu, John Wong, Kathleen L. Gabrielson, Dmitri Artemov.
EthelJ. Ngen, Lee Wang,Yoshinori Kato,Wenlian Zhu, Dmitri Artemov
Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Traylor Building 217, Baltimore, MD 21205, USA. Email:; Telephone: 410-614-2703; Fax: 410-614-1948.
Nishant Gandhi,Michael Armour, John Wong
Department of Radiation Oncology and MolecularRadiation Sciences, TheJohns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Kathleen L. Gabrielson
Department ofMolecular and Comparative Pathobiology, TheJohns Hopkins University School of Medicine, Baltimore, Maryland, USA.
EthelJ. Ngen and Lee Wang contributed equally to this study.
Supplementary Methods
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Supplementary Figures
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Supplementary Methods
S1. Image-guided irradiation: reasons for the choice of the hippocampus as the irradiation target
The hippocampus was chosen as the irradiation target because of its involvement in memory and learning [1]. Recently, it was demonstrated that irradiation of the hippocampus with a single beam, delivered perpendicularly (90°) to the hippocampus, inhibited neurogenesis in the dentate gyrus [2]. Thus, we decided to investigate whether the inhibition of neurogenesis in the dentate gyrus would translate into deficits in memory and learning, and whetherthese deficits could be detected noninvasively and early-on, with behavioral tests, as reported for other hippocampal pathologies [3].
Given the ionizing nature of the high-energy X-ray beams used in radiation therapy, it is difficult to prevent the irradiation of structures along the beam’s path. Although several targeted radiation delivery techniques have been developed to minimize exposure to non-targeted structures, tissues along the beam path still receive some irradiation [4, 5]. In order to minimize the irradiated brain volume and also to minimize the potential variability of the irradiated volume between the mice, a single-beam treatment plan was chosen over a multiple-beam treatment plan. However, as determined by the treatment plan calculations, structures along the beam’s path all received some radiation. This was consistent for all the mice.
S2. Behavioral studies: reasons for the experimental design
Although it has been shown that increased training increases conditioned learning in mice [6], this has been demonstrated to occur through the hippocampal/amygdala-independent plasticity of the thalamus–cortical areas [3]. Furthermore, since all behavioral experiments were conducted at least three weeks apart, throughout the study, it was expected that the mice had sufficient time to recover before the next set of experiments. This was especially true since delayed fear-conditioned tests (with one training session) were performed, as opposed to trace fear-conditioned tests (with multiple training sessions) [1].
S3.Human mesenchymal stem cell culture and SPIO labeling
Cells were cultured in mesenchymal stem cell growth medium (MSCGM), at 37 °C and in 5% CO2. MSCGM was prepared by mixing mesenchymal stem cell basal medium supplemented with 10% mesenchymal stem cell growth supplements, including 2% L-glutamine; and 0.1% gentamicin and amphotericin, all obtained from LonzaPoietics. The cells were cultured in plastic-bottomed flasks at a density of 5000 cells/cm2. Prior to being used,mycoplasma assays were performedon the cells at passage 3, by the Johns Hopkins University Genomic Core facility. All cells used tested negative for mycoplasma. All experiments were carried out with stem cells before passage 5, in accordance with the supplier’s recommendations.
The cells were then labeled with superparamagnetic particles (BionizedNanoferrite [BNF] particles), obtained from MicromodPartikeltechnologie GmbH, using a standard stem cell labeling protocol [7]. Briefly, 9 µL of BNF particles (27.4 mg/mL) and 2.5 µL of poly-L-lysine (PLL, 1.5 mg/mL) were gently stirred in 10 mL of cell culture media, at room temperature for one hour. The cells were next incubated with the mixture for 24 h at 37°C, thenrinsed thoroughly with PBS, and fresh media was placed in the dishes. Under this well-established and standardized labeling protocol, the stemness of the labeled stem cells is maintained, as described in various reports [8-12].
S4.Quantification of stem cell migration
The migration of the transplanted stem cells, from the implantation site to the radiation-induced brain injury site, was next quantified by comparing theratio of low intensity (black) pixels in the irradiated brain hemisphere to that in the non-irradiated hemisphere. Regions of interests (ROIs) were drawn over both brain hemispheres. A threshold pixel value from the non-irradiated hemisphere before stem cell implantation was then established, and pixels below this threshold were classified as black pixels. The number of black pixels indicative of SPIO-labeled cells in both ROIs was then calculated.This was done using the histogram function in the NIH ImageJ software. This method used to quantify migration does not take into account exact distances, but rather a fixed threshold distance from the implantation site to the radiation-induced injury site. Since the cells were implanted 1 mm from the sagittal suture, in the hemisphere contralateral to the radiation-induced injury site, for the transplanted stem cells to be classified as havingmigrated to the radiation-induced brain injury hemisphere, they needed to have travelled at least the 1mm threshold distance from the implantation site to the irradiated hemisphere.
S5.Quantification of histology samples
The degree of necrosis was quantified by measuring the change in cellularity of the irradiated brain hemisphere compared to the non-irradiated hemisphere in irradiated versus non-irradiated mice. Briefly, H&E images of the same regions of both brain hemispheres from both irradiated and non-irradiated mice were acquired and regions of interests (ROIs) of the same size were drawn. The number of white pixels indicative of tissue loss in both ROIs was then calculated, after establishing a threshold pixel value from the non-irradiated hemisphere. This was achieved using the histogram function in the NIH ImageJ software.
A similar approach was used to quantitatively estimate hemorrhage following irradiation. Briefly, PPB images of the same regions of both brain hemispheres from both irradiated and non-irradiated mice were acquired and regions of interests (ROIs) of the same size were drawn. The number of blue pixels indicative of hemorrhage in both ROIs was then calculated, after establishing a threshold pixel value from the non-irradiated hemisphere. This was achieved using the histogram function in the NIH ImageJ software.Samples from three mice each were used for the quantification.
Supplementary Figures
Supplementary Fig.1MRI of radiation-induced brain injury in mice, six weeks post-irradiation.T2-weighted and contrast-enhanced T1-weighted MR images of a representative irradiated mouse brain,six weeks after irradiation.
Supplementary Fig.2Correlation between the blood-brain barrier (BBB) permeability and short-term memory changes. a)Comparison ofthe area of T1contrast-enhancement in a representative irradiated mouse,at two, six, and ten weeks following irradiation. b) Quantitative comparison of contrast-enhanced pixels of a representative irradiated mouse brain, at two, six, and ten weeks following irradiation(n = 10; P < 0.05). c) Comparison of changes in short-term memory (STM), assessed during the context test sessions, before irradiation, two weeks post-irradiation, and ten weeks post-irradiation (n= 10; P < 0.05).
Supplementary Fig. 3 MRI of radiation-induced (100 Gy) brain injury.T2-weighted MR images of a representative irradiated mouse brain,two weeks after irradiation.
Supplementary Fig. 4 The weight of irradiated mice, six weeks following irradiation compared to non-irradiated mice (n= 10).
Supplementary References
1.Jarrard LE(1993) On the role of the hippocampus in learning and memory in the rat. Behav Neural Biol 60: 9-26
2.Ford EC, Achanta P, Purger D, Armour M, Reyes J, Fong J, Kleinberg L, Redmond K, Wong J, Jang MH, Jun H, Song H-J, Quinones-Hinojosa A (2011) Localized CT-guided irradiation inhibits neurogenesis in specific regions of the adult mouse brain. Radiat Res 175: 774
3.Saxe MD, Battaglia F, Wang J-W, Malleret G, David DJ, Monckton JE, Garcia ADR, Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR (2006) Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci 103: 17501-17506
4.Greene-Schloesser D, Robbins ME (2012) Radiation-induced cognitive impairment-from bench to bedside. Neuro-Oncol 14: iv37-iv44
5.Teh BS, Woo SY, Butler EB(1999) Intensity modulated radiation therapy (IMRT): A new promising technology in radiation oncology. Oncologist 4: 433-442
6.Stapley NW, Guariglia SR, Chadman KK (2013) Cued and contextual fear conditioning in BTBR mice is improved with training or atomoxetine. Neurosci Lett 549: 120-124
7.Kraitchman D, Kedziorek D, Bulte J (2009) MR imaging of transplanted stem cells in myocardial infarction. In: Shah K (ed) Molecular Imaging. Humana Press, pp 141-152
8.Balakumaran A, Pawelczyk E, Ren J, Sworder B, Chaudhry A, Sabatino M, Stroncek D, Frank JA, Robey PG(2010) Superparamagnetic iron oxide nanoparticles labeling of bone marrow stromal (mesenchymal) cells does not affect their “stemness”. PLoS ONE 5: e11462
9.Arbab AS, Yocum GT, Rad AM, Khakoo AY, Fellowes V, Read EJ, Frank JA(2005) Labeling of cells with ferumoxides–protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed 18: 553-559
10.Frank JA, Miller BR, Arbab AS, Zywicke HA, Jordan EK, Lewis BK, Bryant LH, Bulte JWM (2003) Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents. Radiology 228: 480-487
11.Gutova M, Frank JA, D'Apuzzo M, Khankaldyyan V, Gilchrist MM, Annala AJ, Metz MZ, Abramyants Y, Herrmann KA, Ghoda LY, Najbauer J, Brown CE, Blanchard MS, Lesniak MS, Kim SU, Barish ME, Aboody KS, Moats RA (2013) Magnetic resonance imaging tracking of ferumoxytol-labeled human neural stem cells: Studies leading to clinical use. Stem Cell Transl Med 2: 766-775
12.Thu MS, Bryant LH, Coppola T, Jordan EK, Budde MD, Lewis BK, Chaudhry A, Ren J, Varma NRS, Arbab AS, Frank JA (2012) Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging. Nat Med 18: 463-467
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