Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Abstract

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. Video LinkThe video component of this article can be found at https://www.jove.com/video/3107/ ProtocolOur experimental plan is schematically diagrammed in Figure 1. Growth and preparation of human neural stem cells (NSCs) for transplantation1. The EnStem-A cell line (EMD Millipore) was used in this study. NSCs are routinely validated by the manufacturer for high levels of expression of the multipotent markers nestin and Sox2, and low-level expression of the pluripotent marker Oct-4, along with the ability to differentiate into multiple neuronal phenotypes and to maintain a normal karyotype after multiple passages. Under our growth conditions, these NSCs displayed abundant expression of Sox2 and nestin and retained their ability to differentiate, as described previously 1 . NSCs were grown on poly-L-ornithine (20 μg/ml) and laminin (5 μg/ml) coated tissue culture treated flasks. Cells were grown ...

Pre-clinical research in small animals using radiotherapy technology – a bidirectional translational approach

“…On the other hand, free positioning of anaesthetised rats is described (see Fig. 1A-B) [16,[60][61][62][63]. Bolus material or acrylic plates may be used to assure proper dose build up [60,61] or to shift the depth dose curve of an electron beam [63].…”

“…As an example, irradiation of rat brains is used to investigate necrosis, vessel dilation and demyelination by means of histological methods [14]. Recent publications showed highly conformal irradiation techniques to specifically evaluate toxicity in subvolumes of the brain, such as in the hippocampus [15,16]. In mice and rats, radiation-induced lung toxicities were investigated as a function of survival, histopathological alterations and breathing rates [17].…”

“…More sophisticated, individualised multi-field treatments are used in only few single fraction experiments. Here, techniques known from human radiation oncology, such as whole-brain radiotherapy with fixed lateral opposing photon beams [62], intensity-modulated radiation therapy and volumetrically-modulated arc therapy [16], are translated for application in rats. Treatment planning is commonly carried out only for an exemplary irradiation setup by means of a clinical treatment planning system (TPS).…”

“…Image courtesy of Gabriel Charest; D: Tongue irradiation of a mouse using an orthovoltage X-ray unit [97]. Image courtesy of Wolfgang Dörr. accelerator, which are the multi-leaf-collimator (MLC) and electron applicators [16,[60][61][62][63]. Additionally, a customised block inserted into the accessory holder at the gantry or electron aperture made from MCP alloys (MCP Group, Tilly, Belgium) may be utilised [56][57][58].…”

“…For internal targets the animal positions may be verified on film [58] or by fluoroscopy using a radiotherapy simulator [57]. If only a single animal is examined, precise (automatic) position adjustment is feasible using the on-board imaging capabilities to acquire orthogonal radiographs or a cone beam CT (CBCT) [16,62].…”