The results of this study demonstrate that, while a single dose of exogenous EPO immediately following transient MCAO did not preserve tissue volume in the long term, 3 doses of EPO did improve histology, with increased regional and total hemispheric brain volumes. In addition, these histological findings correlated with behavioral performance, with multiple-dose-treated rats not differing from shams and performing better in most components of spatial learning and memory performance.
EPO has previously been shown to preserve brain volume following neonatal hypoxia-ischemia [13
], and to decrease infarct volume following transient MCAO in P7 rats [23
]. We have previously demonstrated that P10 rats treated with a single dose of EPO following MCAO had an increase in hemispheric brain volume and improved sensorimotor function at 2 weeks after injury [24
]. We initially chose a single dose of EPO immediately following injury, as opposed to during or prior to injury, to more closely approximate a clinical scenario where EPO may be used for treatment. Similarly, this treatment regimen resulted in a marked increase in hemispheric brain volume and increased neurogenesis in the striatum of injured rats at 6 weeks after injury, possibly also demonstrating a proliferative and reparative effect [30
While these results for single-dose EPO suggest a role for EPO in cell survival and repair, this may not be sufficient for long-term improvement. At later time points after injury, this single-dose EPO treatment was not as effective. At 3 months after neonatal stroke, there was no longer a difference in hemispheric volume between rats treated with a single dose of EPO and vehicle-treated rats. There was also no cognitive improvement, with no differences in any components of the Morris water maze between rats treated with a single dose and vehicle-treated rats. The underlying mechanisms responsible for neuroprotection at earlier time points may not persist this far out after a single dose of EPO. Endogenous EPO is upregulated after injury [10
], and this single dose of EPO immediately after injury may not be sufficient to overcome any endogenous mechanisms at this late time point. In addition, newly generated cells may not maintain the intracellular signaling or perfusion to survive.
EPO appears to function via multiple mechanisms, and while single-dose treatment may decrease apoptosis and increase cell survival initially, it may have little to no effect on long-term cell repair and incorporation into neural networks, or on later neurogenesis and cell proliferation. Following hypoxia and stroke, neuronal transcription factor HIF-1α expression is stabilized and increases expression of downstream targets that include EPO and VEGF [10
], initiating pathways for neuroprotection, angiogenesis, and repair. EPO leads to phosphorylation of Janus kinase 2 (JAK2), and eventually, phosphorylation and activation of the mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), as well as the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) pathway and signal transducer and activator of transcription 5 (STAT5), which are critical in cell survival. For example, EPO increases the rate and quantity of STAT5 expression, resulting in upregulation of anti-apoptotic genes such as Bcl-xL [23
] and NF-κB [33
]. Akt also inactivates Bcl2 family member Bad and limits inflammation [35
]. In addition to anti-apoptotic effects, the ERK pathway has anti-inflammatory effects and increases neurogenesis while altering cell fate commitment [36
], implicating this pathway in repair processes. Following injury, tissue protective and reparative effects of EPO-stimulated ERK activation have been shown in adult models of brain injury [35
]. Increases in angiogenesis following injury may be necessary for long-term survival of injured or newly generated cells, which appears to be effects of both EPO and its interaction with VEGF [39
]. For example, EPO-treated rats had increased VEGF and brain-derived neurotrophic factor levels after stroke, as well as angiogenesis and neurogenesis, with induced neuroblast migration to these regions [7
]. PI3K also mediates increased angiogenesis in the ischemic boundary following ischemic stroke [40
]. However, the effect and time course of phosphorylation and activation of these kinases in the immature brain and their effect on cytokine production following stroke are not clear.
EPO does not appear to protect against early injury in the first 6 h [18
], rather at later time points, suggesting a delay is required for the responsible mechanisms. This may be related to upregulation of EPO-R, synthesis of protein, or activation of a cascade that results in increases in cell number, function, and perfusion. For example, EPO has also been found to inhibit neointimal hyperplasia after arterial injury by acting on injured vessels and mobilizing endothelial progenitor cells to the neo-endothelium [41
], promoting angiogenesis and repair of blood vessels. In addition to decreased apoptosis, increased neurogenesis and angiogenesis, cell repair may be an important component of these histological changes and long-term cognitive improvements. EPO may work by mechanisms similar to those seen with environmental enrichment, which is known to have effects on neuronal plasticity and cognitive function. Enrichment includes exposing these animals to tunnels, platforms, toys, and running wheels that potentiate social interaction and increase stimulation [42
]. These factors result in structural changes in neural cells, and possibly volume of brain structures, with increased neurogenesis, synaptogenesis, dendritic branching, and increased neurotrophic factors in the hippocampus, striatum, and cortex [43
]. Enrichment increases brain-derived neurotrophic factor following brain injury [46
], although some studies suggest females may benefit from this more than males [48
]. Daily enrichment also prevents spatial memory deficits after adult hypoxia-ischemia [49
] and stroke [47
], and early housing in an enriched environment prevented some cognitive deficits after neonatal hypoxia-ischemia [51
]. Young adult mice develop more differentiating neuroblasts, with increased neuronal survival in the dentate gyrus [52
], and physical activity and enriched environments also increased proliferation of precursor cells in the adult hippocampus [53
The Morris water maze is a validated test for spatial learning and memory [54
]. Performance in the water maze often correlates with hippocampal function [55
], but also requires intact vision and motor ability for completion of tasks. While there was no difference in cognitive function in rats treated with a single dose, EO-3 rats did show a trend toward escaping to the hidden platform in the place condition, suggesting improved learning over the 6 days of testing relative to other injured groups, and these rats also had significant improvement in memory, remembering the previous location of the platform in the probe trials. EO-3 rats also demonstrated improved vision and motor ability in the cue condition.
Performance in these cognitive tests correlated with total hippocampal volume, as it appears that this hippocampal injury resulted in spatial memory deficits that persisted into adulthood. In addition, behavioral findings on most components of the water maze correlated with striatal, neocortical, and primary visual cortical volume. The striatum is also involved with acquisition in complex maze tasks [56
], while the neocortex was used as a marker of the sensorimotor cortex. It is difficult to determine how injuries to particular brain regions relate to spatial learning or memory deficits because of the strong intercorrelations among regional areas.
Given the evolution of injury over time, and the improvement seen with additional and later doses of EPO, it remains to be seen what therapeutic regimen results in the best long-term outcomes. Recent findings suggest that up to 3 doses of 5 U/g show short-term improvement in apoptosis and gliosis after rat neonatal hypoxia-ischemia, with dose-dependent protection in a U-shaped manner [57
]. Taken together with previous studies, our observations suggest that EPO may be useful both early, for its direct neuroprotective effect by preventing cell death, and late, influencing cell repair, progenitor cell fate, neurogenesis and migration. Perhaps by clarifying the mechanisms and timing of both repair and neurogenesis, the optimal dosing regimen to maximize long-term outcomes will become clear.