Systemic multipotent adult progenitor cells protect the cerebellum after asphyxia in fetal sheep

Abstract

Involvement of the cerebellum in the pathophysiology of hypoxic-ischemic encephalopathy (HIE) in preterm infants is increasingly recognized. We aimed to assess the neuroprotective potential of intravenously administered multipotent adult progenitor cells (MAPCs) in the preterm cerebellum. Instrumented preterm ovine fetuses were subjected to transient global hypoxia-ischemia (HI) by 25 minutes of umbilical cord occlusion at 0.7 of gestation. After reperfusion, two doses of MAPCs were administered intravenously. MAPCs are a plastic adherent bone-marrow-derived population of adult progenitor cells with neuroprotective potency in experimental and clinical studies. Global HI caused marked cortical injury in the cerebellum, histologically indicated by disruption of cortical strata, impeded Purkinje cell development, and decreased dendritic arborization. Furthermore, global HI induced histopathological microgliosis, hypomyelination, and disruption of white matter organization. MAPC treatment significantly prevented cortical injury and region-specifically attenuated white matter injury in the cerebellum following global HI. Diffusion tensor imaging (DTI) detected HI-induced injury and MAPC neuroprotection in the preterm cerebellum. This study has demonstrated in a preclinical large animal model that early systemic MAPC therapy improved structural injury of the preterm cerebellum following global HI. Microstructural improvement was detectable with DTI. These findings support the potential of MAPC therapy for the treatment of HIE and the added clinical value of DTI for the detection of cerebellar injury and the evaluation of cell-based therapy.

Keywords: MAPC; asphyxia; cerebellum; hypoxic-ischemic encephalopathy; stem cells.

FIGURE 1

Overview of quantitative analysis. A,B, Immunohistochemical Nissl staining depicting regions of interest in the cerebellum. A, Thickness of the external granular layer and molecular layer was measured in the crest (*) and inserts (**) of the anterior lobe and the posterior lobe of the cerebellum. B, Immunohistochemical stainings were analyzed in lobe III and in lobe IX and in the cerebellar peduncles. The black squares indicate the regions of interest in cerebellar white matter, cerebellar cortex, and the peduncles. * = crest of the lobule; ** = inset of lobule. Scale bar = 5 mm. AL, anterior lobe; PL, posterior lobe

FIGURE 2

MAPC treatment protected HI‐induced cerebellar cortical alterations. Graphical presentation of (A) EGL/ML‐ratio, (E) calbindin⁺ cells, and (I) GFAP linear density in the cerebellar cortex. Means ± SEM and levels of significance are depicted. Immunohistochemical (B‐D), Nissl (F‐H) calbindin, and (J‐L) GFAP staining in the cerebellar cortex at ×400 magnification. Global HI increased the EGL/ML‐ratio and decreased the number of calbindin⁺ cells in the preterm cerebellum. Furthermore, global HI increased the number of pyknotic cells (arrow in insert of panel C), caused more “gaps” within the PC layer (arrowhead in panel C), and disturbed dendritic arborization of PCs (panel G). MAPC therapy significantly prevented the increase of EGL/ML‐ratio and loss of calbindin⁺ cells after global HI. HI and MAPC treatment did not induced changes in GFAP linear density. MAPC treatment significantly increased GFAP IR after global HI. Scale bar = 50 μm. EGL, external granular layer; GFAP, glial fibrillary acidic protein; HI, hypoxia‐ischemia; IR, immunoreactivity; MAPC, multipotent adult progenitor cell; ML, molecular layer; PC, Purkinje cell; SAL, saline

FIGURE 3

The anterior cerebellum displayed a morphological phenotype distinctly different from the posterior cerebellum following HI. A‐D, Graphical presentation of EGL/ML‐ratio and calbindin⁺ cells in the (A, B) anterior lobe and (C, D) posterior lobe. Means ± SEM and levels of significance are depicted. E‐H, Immunohistochemical (E, G) Nissl and (F, H) calbindin staining in the cerebellar cortex at ×100 magnification. Global HI induced more pronounced disruption of cortical layers and loss of calbindin⁺ cells in the (E, F) anterior lobe compared to the (G, H) posterior lobe of the cerebellum. More “gaps” (arrows in panel E) within the PC layer of the anterior lobe were seen as compared to the posterior lobe. MAPC therapy prevented HI‐induced calbindin⁺ cell loss in both lobes and prevented disruption of cortical strata in the posterior lobe. Scale bar = 200 μm. EGL, external granular layer; HI, hypoxia‐ischemia; MAPC, multipotent adult progenitor cell; ML, molecular layer; PC, Purkinje cell; SAL, saline

FIGURE 4

MAPC treatment region‐specifically prevented disruption of white matter and hypomyelination without modulating microgliosis in the cerebellar white matter. Graphical presentation of (A, E) MBP IR area fractions of the (A) total cerebellum and (E) posterior lobes and (I) IBA‐1 IR area fractions in the total cerebellar white matter. Means ± SEM and levels of significance are depicted. Immunohistochemical (B‐D) MBP, (F‐H) Luxol fast blue, and (J‐L) IBA‐1 staining in the cerebellar white matter. Global HI caused marked hypomyelination with disorganization of myelin sheets and microglial proliferation in cerebellar white matter. Activated microglia were colocalized with PCs or “gaps” in the PC layer (arrows in insert of panel K). MAPC therapy prevented white matter injury in the preterm cerebellum after global HI. MAPC therapy did not alter microglial proliferation in cerebellar white matter. Images are taken at ×400 magnification. Scale bar = 50 μm. HI, hypoxia‐ischemia; IR, immunoreactivity; MAPC, multipotent adult progenitor cell; MBP, myelin basic protein; PCs, Purkinje cells; SAL, saline

FIGURE 5

Microstructural changes detected by DTI in the preterm cerebellum following HI and MAPC treatment. A, Representative FA map depicting the delineation of the cerebellar region of interest. B‐D, Graphical presentation of FA values in the (B) total cerebellum, (C) cerebellar white matter, and (D) cerebellar cortex. Means ± 95% CI and levels of significance are depicted. Global HI significantly decreased FA values in the cerebellum, indicating disruption of cerebellar microstructure. MAPC therapy significantly protected the cerebellum against microstructural alterations, particularly in the cerebellar cortex. Scale bar = 5 mm. DTI, diffusion tensor imaging; FA, fractional anisotropy; HI, hypoxia‐ischemia; MAPC, multipotent adult progenitor cell; SAL, saline