Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis

Abstract

Myeloablative preconditioning using irradiation is the most commonly used technique to generate rodents having chimeric bone marrow, employed for the study of bone marrow-derived cell accumulation in the healthy and diseased central nervous system. However, irradiation has been shown to alter the blood-brain barrier, potentially creating confounding artefacts. To better study the potential of bone marrow-derived cells to function as treatment vehicles for neurodegenerative diseases alternative preconditioning regimens must be developed. We treated transgenic mice that over-express human mutant superoxide dismutase 1, a model of amyotrophic lateral sclerosis, with busulfan to determine whether this commonly used chemotherapeutic leads to stable chimerism and promotes the entry of bone marrow-derived cells into spinal cord. Intraperitoneal treatment with busulfan at 60 mg/kg or 80 mg/kg followed by intravenous injection of green fluorescent protein-expressing bone marrow resulted in sustained levels of chimerism (∼80%). Bone marrow-derived cells accumulated in the lumbar spinal cord of diseased mice at advanced stages of pathology at both doses, with limited numbers of bone marrow derived cells observed in the spinal cords of similarly treated, age-matched controls; the majority of bone marrow-derived cells in spinal cord immunolabelled for macrophage antigens. Comparatively, significantly greater numbers of bone marrow-derived cells were observed in lumbar spinal cord following irradiative myeloablation. These results demonstrate bone marrow-derived cell accumulation in diseased spinal cord is possible without irradiative preconditioning.

Submyeloablative conditioning with busulfan permits bone marrow-derived cell accumulation in a murine model of Alzheimer’s disease

“…Previously, we used a non-myeloablative BU regimen to establish sustained chimerism in both wildtype (wt) mice and an ALS model overexpressing mutant human superoxide dismutase 1 (mSOD). We observed engraftment of BMDCs in the spinal cords of both wt and mSOD mice following treatment with 60-100 mg/kg BU and transplantation of GFP + BM cells [11].…”

“…However, a limitation to this approach is the requirement for lethal total-body irradiation (TBI) to achieve adequate BM chimerism, a protocol which can affect blood-brain barrier (BBB) permeability [5], as well as the function of endogenous microglia [6,7]. Several recent studies have demonstrated the effectiveness of the milder myelosuppressive conditioning agent busulfan (BU; busulfex) for achieving BM chimerism [8][9][10][11][12], compared to BM conditioning by TBI. We evaluated BU in a murine model of AD to determine if BU was capable of producing BM chimerism, as well as BMDC accumulation in the CNS.…”

Bone marrow-derived macrophages and the CNS: An update on the use of experimental chimeric mouse models and bone marrow transplantation in neurological disorders

“…Compared with irradiated mice, engraftment of GFP + Iba1 + cells was strongly reduced in BU-treated mice both under normal conditions and after facial nerve axotomy. In another study, Lewis et al [95] reported that BU conditioning leads to a weaker infiltration of BMDC in the spinal cord of SOD1 mice in comparison with irradiation. However, the authors observed a significant recruitment of BMDC into the spinal cord of wild-type mice treated with BU under normal conditions.…”

“…Several studies in mouse came out lately comparing the effects of BU-and irradiation-based conditioning regarding microglial engraftment, BBB dysfunction and the neuroinflammatory response [94][95][96][97][98]. Our group compared a fully-myeloablative dose of 10 Gy WBI and a chemotherapeutic regimen consisting of 80 mg/kg of BU injected twice daily over four days (total 8 injections of 10 mg/kg) followed by two daily injection of 100 mg/kg of CY for a total of 200 mg/kg [94].…”

“…It also permits an important infiltration of myeloid cells under pathological conditions and is the best option to optimize the recruitment and study the therapeutic role of BMDC in CNS diseases. However, further work remains to be done to understand why BU treatment led to such discording results in term of microglial replacement/turnover [94][95][96][97][98] because treatments promoting microglia turnover are clinically promising for many rare genetic diseases [5,11,12,101,188]. Therefore, every conditioning regimen has their advantages and limitations and the best regimen should be chosen wisely depending on the purposes of the experiment and potential treatments (Fig.…”