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Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.
Our experimental plan is schematically diagrammed in Figure 1.
Figure 1. Schematic representation of our experimental plan.
Figure 2. Fusion of CT (bony anatomy) and MRI (soft-tissue) images within the ECLIPSE software. Matching of axial, coronal, and sagittal sections allow for the co-registry of the critical anatomical features of the rat brain derived from each imaging modality.
Figure 3. Contoured hippocampal regions of the brain. Subsequent to image fusion, hippocampi and brain excluding hippocampi are identified and contoured axially defining specific volumetric regions for these organs. These regions provide anatomical and volume information necessary for adjusting the dose distribution to the desired target(s).
Figure 4. High precision irradiation options using intensity-modulated radiation therapy (IMRT) or volumetrically-modulated arc therapy (VMAT) in the form of RapidArc. These techniques deliver 6 MV photon beams either as multiple static trajectories that can converge on very small target volumes (IMRT), or dynamically as one or more arcs focused on the target region (RapidArc).
Figure 5. ECLIPSE calculated doses superimposed to the axial images. Doses shown are for single or dual hippocampi irradiation treatment plans.
Figure 6. ECLIPSE calculated dose-volume histogram. Data contrasts the percentage of the irradiated versus the non-irradiated hippocampal volume under the single hippocampus treatment plan shown in Fig. 5.
Figure 7. Image guided rat positioning for radiotherapy. Orthogonal digitally-reconstructed radiograph (DRR) images generated from the CT treatment planning data in ECLIPSE are fused to orthogonal x-ray images of the rat on the treatment table taken with the Trilogy's on-board imaging (OBI) system. The DRRs are weighted on bone density that highlights the skull and other bony landmarks which facilitate co-registration with the OBI images. Matching of these sets provide treatment table position shifts that must be made to achieve the co-registration of the CT and x-ray images.
Figure 8. Location of transplanted NSCs following stereotaxic surgery. At 1-month post-transplantation, animals were perfused, brains were sectioned and stained with BrdU (to detect transplanted NSCs) and counter stained with hematoxylin. The needle track (Nt, red line), indicates the injection trajectory that deposited NSCs at the transplant-release site (Tr), just below the corpus callosum (CC) and above the CA1. Transplanted NSCs showed extensive migration from Tr throughout the host hippocampus (dentate gyrus, DG; dentate hilus, DH; CA1 and CA3 subfields; magnification x4). Scale bar, 200 μm.
Considerable research is underway exploring the myriad of ways that stem cells can be used clinically to restore normal functions to damaged, aged and diseased tissues8. The eventual realization of these efforts will require a detailed understanding of the behavior of engrafted cells within unique microenvironments that are distinct from undamaged normal tissue. Our work has demonstrated that within the irradiated tissue bed, cranially engrafted NSCs can functionally restore cognition, where they survive, migrate and differentiate along neural and glial lineages2. Precisely how these cells mediate recovery of cognition is uncertain at present, but does depend on conducting a series of carefully controlled experimental procedures in a reproducible manner. We have detailed these critical procedures here in efforts to expedite the translational potential of stem cell therapies for ameliorating adverse cognitive effects associated with the clinical management of brain and other forms of cancer. Additional considerations that are likely to have a significant impact of the quality of data are highlighted below.
Transplantation based therapies depend on stem cells as the critical reagent, and accordingly, care must be exercised to properly characterize cultures, maintain sterility and use matched passage numbers for reliability of results. Transplanted human stem cells were labeled with BrdU prior to surgery, to provide one means for tracking them in vivo. Under our experimental conditions, transplanted NSCs did not undergo extensive proliferation, so that dilution of the BrdU label was not problematic. Alternatively, transplanted human stem cells can be distinguished from host cells by immunostaining for human specific markers such as nuclear matrix protein (h-NUC or hNUMA)9 or human-specific nuclear antigen (HuNu)2. Human stem cells could also be labeled with a variety of fluorescent markers to facilitate their identification within the host brain.
Attention to irradiation parameters define precise dose delivery techniques, and those described here model current clinical practices in radiation oncology. Reproducible transplantation of stem cells in the brain is also critical, and is accomplished using a digital stereotaxic instrument. The capability to precisely micromanipulate a micro-syringe for stem cell implantation in small brain structures like the hippocampus greatly reduces human error. With this apparatus, NSCs were transplanted in four distinct sites spanning the anterior to posterior regions of the rat hippocampus. Dorso-ventral (DV) coordinates were determined based on experience with athymic nude (ATN) rats such that surgical procedures would not cause damage to the hippocampal formation2. A coronal section through the rat brain reveals key structures of the hippocampal formation including the dentate gyrus (DG), the dentate hilus (DH) and CA1 and CA3 subfields (Fig. 8). Transplanted NSCs, introduced dorsally from the visible needle tract (Nt, redline), are visualized as darkly staining (brown) cells deposited at the transplant-release site (Tr) just below the corpus callosum (CC), that subsequently migrate throughout the septo-temporal axis of the hippocampus.
No conflicts of interest declared.
This work was supported by NIH NINDS grant R01 NS074388 581 (C.L. Limoli), California Institute for Regenerative Medicine (CIRM) Grant RS1-00413 (C.L.L.), CIRM Training Grant TG2- 0115 (M.M.A.), and a CIRM Grant to JOVE in support of the video documentation.