Bone marrow cell transplantation restores olfaction in the degenerated olfactory bulb

Abstract

Bone marrow contains heterogeneous cell types including end-lineage cells, committed tissue progenitors, and multipotent stem/progenitor cells. The immense plasticity of bone marrow cells allows them to populate diverse tissues such as the encephalon, and give rise to a variety of cell types. This unique plasticity makes bone marrow-derived cells good candidates for cell therapy aiming at restoring impaired brain circuits. In the present study, bone marrow cells were transplanted into P20 mice that exhibit selective olfactory degeneration in adulthood between P60 and P150. These animals, the so-called Purkinje Cell Degeneration (PCD) mutant mice, suffer from a progressive and specific loss of a subpopulation of principal neurons of the olfactory bulb, the mitral cells (MCs), sparing the other principal neurons, the tufted cells. As such, PCD mice constitute an interesting model to evaluate the specific role of MCs in olfaction and to test the restorative function of transplanted bone marrow-derived cells. Using precision olfactometry, we revealed that mutant mice lacking MCs exhibited a deficit in odorant detection and discrimination. Remarkably, the transplantation of wild-type bone marrow-derived cells into irradiated PCD mutant mice generated a large population of microglial cells in the olfactory bulb and reduced the degenerative process. The alleviation of MC loss in transplanted mice was accompanied by functional recovery witnessed by significantly improved olfactory detection and enhanced odor discrimination. Together, these data suggest that: (1) bone marrow-derived cells represent an effective neuroprotective tool to restore degenerative brain circuits, and (2) MCs are necessary to encode odor concentration and odor identity in the mouse olfactory bulb.

Figure 1.

Cellular effects of bone marrow cell transplantation in the olfactory bulb of PCD mice. A–C, GFP+ BMDCs (green) expressed microglial marker (Iba1 in red) in the olfactory bulb (counterstaining with DAPI, blue) and display a highly ramified morphology typical of microglia. Scale bar, 200 μm. D, E, MC somata (arrows) in PCD (D) and grafted PCD mice (E) labeled with antibodies against reelin (red). GL, Glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer. Scale bars, 100 μm. F, Evolution of MC density in PCD and grafted PCD animals. Student's t test, **p < 0.01. G–I, Coronal view of a transplanted mouse OB. GFP+ BMDCs (green; G, I) were spread throughout the entire olfactory bulb, displayed microglial morphology (G, I, inset), but never colocalized with reelin (red; H, I). Scale bar, 250 μm; inset, 100 μm. J, K, Proliferative Ki67-positive cells in the subventricular zone in PCD (J) and grafted PCD [K, counterstaining with propidium iodide (PI)]. LV, Lateral ventricle. Scale bars, 75 μm. L, Quantification of the proliferation rate (percentage of Ki67+ cells) in the SVZ and RMS in WT (white), grafted WT (green), PCD (hatched white), and grafted PCD (hatched green). Student's t test, **p < 0.01.

Figure 2.

Olfactory performance improvement in PCD mice after BMDC transplantation. A, Schematic drawing of the go/no-go procedure. A trial is initiated when the animal breaks the infrared (IR) beam by entering his snout into the sampling port (1). After 1 s, the odor stimulus is presented to the odor sampling port (2). For S+ odor, animals get a water reward if they lick the water tube during the 2 s of odor presentation (3). For S− odor, the animal learns to retract his head from the sampling port (3′). B, C, Mean percentage of correct responses for decreasing odor concentrations of (+)-carvone (B) and increasing binary mixtures of (+)-carvone and (−)-carvone (C). Wild-type (white squares, n = 8), PCD mutant (hatched white circles, n = 7), grafted PCD mutant (hatched green circles, n = 6), and grafted wild-type (green squares, n = 12). Student's t test, **p < 0.01; ^#p < 0.05.