Mannitol facilitates neurotrophic factor up-regulation and behavioural recovery in neonatal hypoxic-ischaemic rats with human umbilical cord blood grafts

Abstract

We recently demonstrated that blood-brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood-brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic-ischaemic (HI) injury model. Seven-day-old Sprague-Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post-transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme-linked immunosorbent assay at day 3 post-transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up-regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor-mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood-brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.

Fig 1

HUCB grafts ameliorate HI-induced behavioural deficits. EBST (A), Rotarod (B). Significant behavioural recovery of locomotor tasks in transplanted HI-injured animals (P < 0.05 versus vehicle or mannitol alone) was detected at both post-transplant testing days. Both groups of HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in EBST and Rotarod test compared with those that received vehicle alone or mannitol alone at both post-transplant testing days 7 and 14, but the HI animals that received combined HUCB cells and mannitol displayed significantly better improvement than those that received HUCB cells alone (Ps < 0.01 versus*vehicle/mannitol or **HUCB alone). Data are shown as mean values + S.E.

Fig 2

Sporadic HUCB grafts survive in HI brains. Representative images of microscopically detected HUCB cells alone (A–D) and HUCB cells + mannitol (E–H; adjacent section with high magnification images shown in I–K). Very few HuNu-positive HUCB cells (green) were detected in the HI-injured hippocampal dentate gyrus, which co-labelled with the nuclei marker Hoechst (blue). These data indicate that only a handful of intravenously transplanted HUCB cells reached the ischaemic hippocampus. Bar = 60 μm. Individual and mean cell counts of HuNu positive cells are shown in panels L and M, respectively.

Fig 3

HUCB grafts partially normalize hippocampal dendritic density. Transplantation of HUCB alone or in combination with mannitol led to partial normalization of hippocampal dendritic density in HI-injured animals. Data are presented as percentage + S.E. of control (A). (B) Vehicle, (C) Mannitol, (D) HUCB alone and (E) HUCB + mannitol. Quantitative analyses are shown in (F). *Ps < 0.01 versus vehicle or mannitol. Bar = 60 μm.

Fig 4

HUCB grafts promote neurotrophic factor up-regulation in HI brains. ELISA revealed that both groups of HI animals that received HUCB cells alone or when combined with mannitol significantly increased GDNF, NGF and BDNF brain levels at day 3 after HI compared with animals treated with vehicle alone or mannitol alone, but the HI animals that received combined HUCB cells and mannitol displayed significantly better improvement than those that received HUCB cells alone (Ps < 0.0001 versus*vehicle/mannitol or **HUCB alone). Data are shown as mean values + S.E.