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.

Figure 1. Short-term analysis of PBC reconstitution by donor cells using doses of 60, 80 or 100 mg/kg BU and GFP+ accumulation in control spinal cord.

(A) Left: FACS plot of negative control (blue peak) and GFP+ control (red peak) blood. Centre: An example of a FACS plot of blood collected at 3 weeks post-BM transplant following treatment with 100 mg/kg BU. Roughly 48% of PBCs were GFP+. Right: Immunolabeling of blood for myeloid markers (CD11b-APC, Gr1-APC) indicate high level of donor chimerism in this blood cell population by 3 weeks post-transplant. (B) Levels of PBC and lymphoid chimerism increased over the 4-week observation period. Levels of chimerism in circulating myeloid cells increased at a rate higher than that of lymphoid cells. (C) By 4 weeks post-transplant, GFP+ cells were observed in the lumbar spinal cords of BM chimeric mice. Significantly greater numbers of GFP+ cells were observed in the spinal cords of mice treated with 80 or 100 mg/kg BU compared to mice treated with 60 mg/kg. Scale bar = 50 µm. (D) The majority of GFP+ cells in lumbar spinal cord exhibited a rod-shaped morphology and were associated with blood vessels immunolabelled with antibody to CD31. Scale bar = 50 µm.

Figure 2. PBC reconstitution by donor cells and distribution of GFP+ cells in lumbar spinal cord.

(A) Donor PBC reconstitution in mice treated with 60 mg/kg BU and 80 mg/kg BU was assessed weekly for 11 weeks post-transplant using flow cytometry. Levels of PBC chimerism achieved were similar between 60 mg/kg BU and 80 mg/kg BU treatment groups. Reconstitution of myelomonocytic cells was rapid in comparison to lymphoid populations, owing to the shorter half-life of circulating myelomonocytic cells and the minimal immunosuppressive effects of BU. Error bars represent standard deviation. (B) Distribution of GFP+ cells in control and mSOD lumbar spinal cord sections 11–14 weeks after transplantation following BU treatment or myeloablative irradiation. GFP+ cells accumulated in the grey and white matter of lumbar spinal cord sections, especially in the surrounding leptomeninges 11–14 weeks after transplantation following BU treatment or irradiation. Significantly greater numbers of BMDCs accumulated in mSOD lumbar spinal cords compared to age-matched controls using BU (60 or 80 mg/kg) or irradiative myelosuppression (p<0.05). Scale bar = 500 µm.

Figure 3. BMDCs in the spinal cord acquire the anatomical location and immunophenotype of various CNS-associated macrophages.

(A) Upper Panel: GFP+ cells with an elongated morphology were located near blood vessels within mSOD and control lumbar spinal cord sections. Lower Panel: 3D-rendered confocal image rotated backwards at 70 degrees indicating the elongated GFP+ cell is located on the abluminal side of the blood vessel. Scale bar = 50 µm. (B) Control and mSOD lumbar spinal cord sections immunolabelled with antibody to Iba1. Increased numbers of microglia were observed in the mSOD spinal cord and exhibited an active morphology characterized by hypertrophied cell bodies and retracted, thickened cellular processes. Scale bar = 10 µm (C) GFP+ cell possessing stellate morphology immunolabelled with antibody to Iba1 identifying these cells as BM-derived parenchymal macrophages. Scale bar = 10 µm.