Intravenous grafts recapitulate the neurorestoration afforded by intracerebrally delivered multipotent adult progenitor cells in neonatal hypoxic-ischemic rats

Abstract

Once hypoxic-ischemic (HI) injury ensues in the human neonate at birth, the resulting brain damage lasts throughout the individual's lifetime, as no ameliorative treatments are currently available. We have recently shown that intracerebral transplantation of multipotent adult progenitor cells (MAPCs) results in behavioral improvement and reduction in ischemic cell loss in neonatal rat HI-injury model. In an attempt to advance this cellular therapy to the clinic, we explored the more practical and less invasive intravenous administration of MAPCs. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, then 7 days later received intracerebral or intravenous injections of allogeneic rat MAPCs. On post-transplantation days 7 and 14, the animals that received MAPCs via the intracerebral or intravenous route exhibited improved motor and neurologic scores compared with those that received vehicle infusion alone. Immunohistochemical evaluations at day 14 after transplantation revealed that both intracerebrally and intravenously transplanted MAPCs were detected in the ischemic hippocampal area. The degree of hippocampal cell preservation was almost the same in the two treatment groups and greater than that in the vehicle group. These results show that intravenous delivery of MAPCs is a feasible and efficacious cell therapy with potential for clinical use.

Figure 1

The EBST and Rotarod test at 1 and 2 weeks after intracerebral or intravenous injection of MAPC. (A) EBST, (B) Rotarod. Significant behavioral recovery of locomotor tasks in transplanted HI-injured animals (P<0.05 versus control) appears by week 2. Intracerebrally (IC) and intravenous (IV) transplanted HI-injured animals did not differ significantly in their behavioral recovery. Data are shown as mean values+s.e. *P<0.05 versus vehicle-treated rats.

Figure 2

Multipotent adult progenitor cell (MAPC) graft survival in HI brains of behaviorally recovered animals. Representative images of βgal and MAP2 staining of MAPC after intracerebral and intravenous transplantation are shown (A–C: intracerebral graft; D–L: intravenous graft). βgal-Positive allogeneic grafts were detected in the hippocampal CA3 region and the adjacent CA2 area, which colabeled with the nuclei marker Hoechst in rats receiving both intracerebral (A–C: A, βgal; B, MAP2; C, merged with Hoechst) and intravenous (D–F: D, βgal; E, MAP2; F, merged with Hoechst) grafts. A higher magnification, using confocal microscopy, further reveals the colocalization of intravenously delivered βgal-labeled MAPC with the neuronal marker MAP2 (G–I). Some MAPCs (J) colocalized with MAP2 (K) seemed to reside inside vessels in the hippocampus, which was confirmed by confocal z-stacked imaging (L). These data indicate that transplanted allogeneic MAPCs survived in the ischemic hippocampus (in the case of intravenous route, migrated into the ischemic hippocampus), and expressed a neuronal phenotypic marker, which could have promoted recovery from motor deficits associated with HI injury. Bars: A–F, 30μm; G–I, 10μm; J–L, 2.5 μm.

Figure 3

Intracerebral (IC) and intravenous (IV) MAPC grafts reduce CA3 cell loss. Representative images of low (A1–D1) and high-magnification (A2–D2) Nissl staining of CA3 are shown (A: intact; B: vehicle; C: intravenous; D: intracerebral; A1–D1: bar=50 μm; A2–D2: bar=15 μm). Cell counts of Nissl-stained cells along the ischemic hippocampal CA3 region revealed that intracerebral and intravenous grafts significantly rescued the damaged neuronal cells compared with vehicle-infused HI-injured animals (E). Data are shown as mean values+s.e. expressed as percentages relative to the intact side. *P<0.05 versus vehicle-treated rats.