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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.


Introduction
Cell therapy remains a controversial approach for treating human disorders [1][2][3]. Laboratory and clinical data are divided on transdifferentiation of cells from non-neuronal origin, with studies reporting failure of hematopoietic bone marrow cells to differenti-ate into brain cells [4,5], whereas equally compelling evidence demonstrates that peripherally transplanted adult human bone marrow cells in patients with hematologic malignancies entered the brain and generated neurons [6,7]. Clinical trials of cell therapy for neurological disorders have been performed in Parkinson's disease, Huntington's disease and stroke [8][9][10]. Two major lines of mechanistic investigations on cell therapy have focussed on the concepts that either transplanted cells will replace the damaged host cells or the grafts will serve as neurotrophic factor-secreting vehicles, both pathways implicated to afford neuroprotection and/or neurorestoration. Because the absence of surviving grafts correlates with continued display of neurological deficits in transplant recipients, in both animal models of CNS disorders and patients [11,12], a scientific bias exists for dismissing transplantation results as 'placebo effects' when graft survival could not be unequivocally demonstrated in the event of functional recovery. To this end, we previously showed that transplant-mediated functional recovery could occur in stroke without CNS entry of grafted cells, provided that neuroprotective molecules secreted by these cells crossed the blood-brain barrier (BBB) and reached the injured brain site [13]. The aim of the present study was drawn from our previous report [13]

Animals
We examined the behavioural effects and histological status of early intravenous delivery of HUCB cells into neonatal rats (Table 1). NIH and IACUC guidelines for use of animals in research were followed. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury [14].

Enzyme-linked immunosorbent assay (ELISA)
Parallel groups of animals (an additional n ϭ 5 per treatment group) from the four treatment conditions were euthanized at day 3 post-transplantation for ELISA of known CNS growth factors, including GDNF, NGF and BDNF, which have been previously shown to exert neurotrophic effects in ischaemic injury. We followed the ELISA method described elsewhere [16]

Statistical analysis
Behavioural scores and trophic factor expression levels were initially analyzed using ANOVA, followed by post hoc Bonferroni t-tests for pair-wise comparisons between treatment groups. The level of significance was set at Ͻ0.05.

Mannitol enhances behavioural recovery of HI-injured animals transplanted with HUCB
Behavioural tests were conducted at post-transplantation days 7 and 14 (Fig. 1

Few HUCB cells survive in the brains of HI-injured animals
Histological assays of graft survival at day 14 post-transplantation revealed only sporadic surviving HUCB cells detected in the HI-injured hippocampus (i.e. dentate gyrus) (Fig. 2)

Mannitol up-regulates CNS growth factors in HI-injured animals transplanted with HUCB
Parallel groups of animals from the three treatment conditions (n ϭ 5 per treatment group) were euthanized at day 3 posttransplantation for ELISA (Fig. 4)

Fig. 2 Sporadic HUCB grafts survive in HI brains. Representative images of microscopically detected HUCB cells alone (A-D) and HUCB cells ϩ 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 ϭ 60 m. Individual and mean cell counts of HuNu positive cells are shown in panels L and M, respectively.

Discussion
The present study extends the utility of mannitol in potentiating the therapeutic efficacy of cell therapy in adult stroke to neonatal HI injury animal model. The permeation of the BBB via mannitol enhanced the behavioural recovery achieved by intravenously transplanted HUCB cells in HI-injured animals. In addition, such mannitol treatment when combined with HUCB transplantation further increased the CNS levels of at least three neurotrophic factors, namely, GDNF, NGF and BDNF. Although mannitol treatment did not increase HUCB graft survival, the combined BBB permeation and HUCB transplantation significantly elevated trophic factor levels in the HI brain, which could have mediated the robust functional improvement. These results support our concept that the therapeutic outcome produced by cell therapy can be recognized not solely based on graft survival in the injured brain but also through improved entry of neuroprotective and/or neurorestorative molecules from the periphery into the CNS. We previously reported that ELISA revealed partially increased (15% above control non-stroke animals) levels of trophic factors from circulating blood in stroke animals that received intravenous HUCB grafts plus mannitol [13]. In contrast, no detectable elevations in trophic factors in circulating blood were obtained from stroke animals injected either with intravenous HUCB grafts alone, mannitol alone or vehicle alone compared with controls. These observations further strengthen the notion that significant elevation of trophic factors in the brain, instead of the peripheral blood circulation, likely mediated the functional recovery of HI-injured animals that received the combination treatment of HUCB and mannitol. Cerebral palsy accounts for a number of neurological disorders that occur in infancy or early childhood, with majority due to genetic syndromes [17,18] and others caused by brain infections [19], such as bacterial meningitis or viral encephalitis, or head injury from perinatal brain damage [20], vehicular accident [21] or child abuse and neglect [22]. There is no cure for cerebral palsy, but physical therapy [23] along with other rehabilitation treatments (e.g. occupational therapy, speech therapy), as well as drugs for seizure and muscle spasm regulation, and pain medication may improve the child's daily activities [24][25][26]. The brain damage produced by the rat neonatal HI model recapitulates some of the CNS malformations seen in cerebral palsy infants. We previously reported consistent hippocampal cell loss following HI insult in the neonatal rat [27], thereby allowing investigations into the potential of cell therapy for cerebral palsy.
Intravenous delivery of HUCB alone promoted behavioural recovery in HI-injured animals, but the functional improvement was more pronounced when HUCB transplantation was combined with mannitol, which is used clinically for hyperosmolar therapy. The amelioration of behavioural abnormalities represents improvement in both motor asymmetry and motor coordination as revealed by EBST and Rotarod test, respectively, which persisted in the two test periods of days 7 and 14 post-transplantation. The combination of HUCB transplantation and mannitol treatment enhanced the behavioural recovery by about 40% in EBST and 50% in Rotarod compared with HUCB transplantation alone. Long-term monitoring of these motor tasks to reveal stable functional outcome and to determine the need for booster HUCB transplantation and mannitol treatment should further provide guidance on the optimal therapeutic regimen for HI. In addition, in view of the established involvement of the hippocampus in learning and memory, the assessment of cognitive performance in HI-injured animals undergoing this treatment should be pursued in subsequent studies.
The present HUCB cell dose of 15k is dramatically low compared with previously reported therapeutically active doses for HUCB. In our previous study in adult stroke [13], we found that 200,000 HUCB cells (already considered a low dose) when combined with mannitol mimics those seen in other reports using much higher doses (Ͼ500,000 cells without mannitol) of IV HUCB in stroke [28,29]. A plausible explanation for the ability of a much lower cell dose of HUCB to exert therapeutic benefits in HI is likely due to the neonatal brain being more plastic than the adult brain, thereby allowing the young host microenvironment to respond more appropriately to therapeutic intervention. Although our initial concept for BBB permeation was to allow for the donor cells to be injected acutely after brain injury, we recognized that the diagnosis of cerebral palsy normally takes place after days or a few weeks after birth. Accordingly, we adjusted the timing of cell transplantation and mannitol, to augment the clinical application of this treatment for cerebral palsy. Here, we show that even with a 1-week delay post-injury, mannitol was able to potentiate the therapeutic effects of HUCB, suggesting that BBB permeabilization in neonates is critical for optimizing the functional outcome of cell therapy when the chosen route of cell administration is from the periphery. With the mannitol enhancement of HUCB-induced behavioural recovery, such minimally invasive intravenous cell delivery offers a more practical and a less traumatic approach than directly injecting cells into the brain, especially when the transplant recipient is an infant.
In agreement with our study in adult stroke [13], the present results indicate that spontaneous BBB opening produced by HI injury was not permissive enough to allow CNS entry of endogenous or graft-derived trophic factors, suggesting the need for exogenous BBB manipulation. We concur that BBB may not be fully developed in neonatal rats, but even such immature BBB appears capable of blocking the entry of peripherally secreted growth factors as seen in our neonatal animals that received HUCB alone without mannitol treatment. In contrast, mannitol treatment in neonatal animals that received HUCB resulted in elevated CNS levels of growth factors, suggesting that even an immature BBB needs to be permeabilized in order to achieve enhanced entry of peripherally secreted growth factors by HUCB. A perturbed BBB permeability produced by mannitol facilitates mobilization of graftderived trophic factors to be present in the brain to exert therapeutic benefits. The lack of significant increments in brain trophic factor levels in HI-injured animals that received vehicle alone or mannitol alone suggests that HUCB grafts instead of the host tissues per se were likely the source of elevated CNS levels of trophic factors. In concert with preclinical evidence demonstrating the neuroprotective and neuroregenerative effects of GDNF in ischaemic injury [30,31], cell-based CNS therapies designed to deliver GDNF, in combination with a cocktail of known and yet to be determined trophic factors secreted by stem cells such as HUCB, may further enhance the therapeutic benefits produced by exogenous delivery of a single or a few specific trophic factors. Although host-derived growth factors have been shown to be up-regulated after ischaemic injury, this endogenous compensatory mechanism is not sufficient to afford neuroprotection and neuroregeneration. Accordingly, we postulate that the observed elevation of CNS growth factors following mannitol treatment is likely derived from the peripherally transplanted HUCB. Our previous study [13] showed that the use of neutralizing trophic factor antibodies by exposing HUCB cells to antibodies against GDNF, NGF and BDNF before transplantation prevented elevations in trophic factor levels, and completely blocked the behavioural and histological beneficial effects produced by combined HUCB and mannitol treatment. These observations implicate the pivotal role of mannitol permeabilization of BBB in promoting the trophic factor effects of peripherally administered HUCB grafts to be manifested in ischaemic brain injury.
The visualization of a few surviving HUCB cells in the brains of HI-injured brains is unexpected because we did not detect any grafted HUCB cells in transplanted adult stroke animals [13]. As noted earlier, it is likely the plastic neonatal brain creates a more conducive host microenvironment for the HUCB cells to survive. This favourable neonatal host brain, in tandem with mannitol treatment, might have facilitated the migration of the HUCB cells from the periphery since chemokine up-regulation, such as stromal cell-derived factor 1, accompanies neonatal HI injury [32]. We have previously shown that cultured [33,34] or grafted HUCB [35] cells express phenotypic neuronal markers, which could have contributed to the observed functional recovery. Indeed, differentiation of grafted cells into neural lineage, including neurons and astrocytes, has been implicated in reorganization of the HI-injured neonatal brain [36]. However, the very low number of HUCB cells that crossed the BBB even with mannitol treatment supports the notion that the primary mechanism underlying the functional recovery was likely mediated by neurotrophic factor secretion rather than HUCB graft survival in the brain per se. Additional immunohistochemical assays (not shown) using neuronal (MAP2), glial (GFAP) and oligodendrocytic (O4) markers reveal that none of the few cells reaching the brain expressed any of these phenotypic labelling, suggesting that HUCB cells did not differentiate into a neuronal lineage. These observations further strengthen our claim that graft survival and/or neuronal commitment only partially contributed to the therapeutic benefits, and that CNS elevation of neurotrophic factors either directly by HUCB cells or indirectly whereby HUCB cells stimulated the spared endogenous cells to up-regulate known, as well as yet to be determined, growth factors largely played the key role in affording protection from and/or repair of the HI-injured brain regions. In the end, the primary mechanistic pathway we invoke for the observed therapeutic benefits is that mannitol facilitates the up-regulation of growth factors by HUCB cells, host cells or both in the CNS where these molecules abrogated HI injury. The present cell transplant-mediated trophic factor neuroprotection and/or neuroregeneration parallels our previous reports showing that the cell dose administered, rather than the subsequent number of surviving cells, appears to correspond to the therapeutic threshold to recognize functional recovery [13,27,37,38]. This concept of 'bystander effects' (i.e. growth factor secretion) overriding the 'cell replacement' (i.e. graft survival) has recently received direct scientific support [39,40]. Of note, we observed here that HUCB grafts alone or in combination with mannitol partially normalized dendritic density in hippocampal CA1 region, suggesting neurotrophic factor effects.
These results, taken together, advance the strategy of BBB permeation via mannitol treatment as an adjunct to intravenous transplantation of HUCB for treating neonatal HI injury, as demonstrated previously in adult stroke [13]. The present therapeutic benefits of combined mannitol and HUCB were achieved without immunosuppression, which is often accompanied by deleterious side effects, thereby further paving the way for autologous, as well as allograft transplantation.