|Home | About | Journals | Submit | Contact Us | Français|
Neuronal replacement has recently gained attention as a potential therapeutic target under ischemic conditions. However, the oligodendrogenic infrastructure is equally critical for restoration of brain function, and is also sensitive to ischemic injury. Erythropoietin (EPO) is a neuroprotective molecule that stimulates neuronal replacement following neonatal hypoxia/ischemia (H/I) when delivered shortly after the onset of reperfusion. Since EPO can improve recovery of neurological function in the absence of tissue protection we hypothesize that EPO may improve neurological function via enhancement of white matter recovery following H/I. Thus, we sought to determine the effects of delayed administration of EPO on white matter injury as well as recovery of neurological function following neonatal H/I.
EPO (1000 U/kg) was injected intraperitoneally at multiple timepoints beginning 48 hours after H/I in postnatal day 7 rats. The effects of EPO on oligodendrogenesis, white matter injury and neurogenesis were evaluated using bromodeoxyuridine incorporation and cell-specific immunohistochemistry. Neurological function was assessed by sensorimotor behavioral tests.
Delayed administration of EPO was incapable of reducing brain volume loss, but significantly increased oligodendrogenesis and maturation of oligodendrocytes, and attenuated white matter injury following H/I. These effects occurred concurrently with enhanced neurogenesis. Delayed EPO treatment improved behavioral neurological outcomes 14 days after H/I injury.
Our study demonstrates that delayed administration of EPO promotes oligodendrogenesis and attenuates white matter injury concurrently with increased neurogenesis. These effects likely contribute to the observed improvement in neurological functional outcomes.
Cerebral white matter is highly vulnerable to ischemic injury in both adults1 and neonates,2 with the latter in particular leading to permanent impairment of the brain. During development, oligodendrocyte progenitor cells (OPCs) undergo rapid differentiation into mature oligodendrocytes, which are more susceptible to ischemic injury.2 Following brain ischemia in adults there is a delayed increase in the number of mature oligodendrocytes in peri-infarct areas, while immature oligodendrocytes proliferate in the regions surrounding the lateral ventricles,3 indicating that ischemic damage may be compensated for, at least in part, by increasing replacement of oligodendrocytes. Thus, it is plausible to propose that post-ischemic interventions geared toward improving OPC survival and differentiation may greatly improve outcomes in the neonatal ischemic brain.
Erythropoietin (EPO) has emerged as a promising candidate for neuroprotection in both animal models of ischemia4, 5 and stroke patients.6 When administered acutely following neonatal ischemia, EPO is neuroprotective and also stimulates angiogenesis and neurogenesis.4 The more clinically relevant delayed (>24 hours post-ischemia) administration of EPO does not decrease infarct, but has recently been reported to result in a reorganization of white matter in adult rats as detected by MRI.7 The effects of delayed EPO administration on oligodendrogenic replacement and functional recovery in the ischemic neonate are currently unknown. The objectives of this study were to investigate whether delayed administration of EPO stimulates oligodendrogenesis and attenuates white matter injury following neonatal H/I, and whether these effects result in improved neurological outcomes.
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. The neonatal H/I model was performed in postnatal day 7 Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA) as described.4 In brief, the left common carotid artery was ligated under anesthesia with 3% isoflurane. After a 1.5-h recovery period, the pups were placed in glass chambers containing a humidified atmosphere of 8% O2/92% N2 and submerged in a 37°C water bath to maintain normothermia. After 2.5 h of hypoxia, the pups were returned to their dam for the indicated time. Naïve (nonischemic) animals served as controls. Recombinant human EPO was produced4 and injected in a phosphate-buffered saline (PBS) intraperitoneally at 1000 U/kg body weight on days2, 4, 6, 9 and 13 after H/I. Injection of PBS served as the vehicle control. Bromodeoxyuridine (BrdU, 50 mg/kg; Sigma) was injected intraperitoneally according to the indicated regimen. All animals were transcardially perfused with 4% paraformaldehyde in PBS and their brains removed. After postfixation and cryoprotection, coronal sections were cut using a microtome. Brain volume loss was determined by calculating the amount of surviving tissue using cresyl violet staining as previously described.8
Newborn cells were labeled by injection of BrdU prior to sacrifice according to the illustrated regimens. For visualization of newborn cells, sections were pretreated with 1N HCl followed by 0.1mol/L boric acid (pH 8.5), blocked and then incubated with anti-BrdU (1:2000, BD Biosciences). Oligodendrogenesis and neurogenesis were analyzed 5 and 14 d after H/I. Oligodendrocytes were visualized with anti-NG2 (1:200; Cell Signaling) and anti-adenomatous polyposis coli (APC; 1:200; Santa Cruz Biotechnology) immunostaining for OPCs and mature oligodendrocytes, respectively. Migrating immature neural progenitors were visualized using anti-doublecortin (DCX; 1:1000; Santa Cruz Biotechnology) immunostaining. Secondary IgG antibodies included AlexaFluor 488 (1:500; Molecular Probes), and Cy3 (1:2000; Jackson Immunoresearch). Immunopositive cell densities were calculated as the number of cells in the designated area divided by the area measured by the MCID image analysis system. To evaluate white matter injury, coronal sections were stained with myelin basic protein (MBP; 1:400; Santa Cruz Biotechnology) and neurofilament 200 (NF-200; 1:200; Sigma) antibodies and images were digitized using confocal microscope (Olympus Fluoview FV1000). The mean intensity value of MBP and NF-200 staining was calculated in the corpus callosum (CC), cortex (CTX) and striatum (ST) as previously reported 9 and expressed as the relative ratio of MBP to NF-200 staining.
Sensorimotor neurological function was evaluated using foot fault tests and the negative geotaxis test as described.4 Coordination of contralateral limbs was determined by foot fault tests, which analyze the successful rate of the animal using the right foot. Overall motor behavior was evaluated using grid walking to determinetotal steps taken over 1 minute. Negative geotaxis was determined by testing the time needed for pups to successfully turn and climb up an incline board with their forelimbs.
All values are expressed as mean ± SD. Statistical comparisons among groups were determined using analysis of variance followed by post hoc analysis using Fisher’s probable least-squares difference tests.
Neurotoxic stimuli, such as H/I, and neurotherapeutics may affect the normal course of oligodendrogenesis due to injury and recovery. Following H/I in neonates, the number of immature OPC (NG2+) cells was significantly higher than in age-matched controls at 5 d in the CC and CTX in the ipsilateral hemispheres (Supplemental Fig. 1C, E). Similar to total NG2+ cell counts, the number of BrdU-labeled NG2+ cells increased significantly at 5 d in the ischemic CTX and CC compared to control animals (Supplemental Fig. 1C, F), suggesting that severe stress induces transient proliferation or repair of OPCs.
Several studies have found heterogeneity in NG2+ cell populations, including the capacity to give rise to both oligodendrocytes and astrocytes.10 In order to determine the fate of OPC maturation into oligodendrocytes, we extended the survival time to 14 d post-H/I and immunostained brain sections with the APC antibody, which labels only the cell bodies of mature oligodendrocytes, and counterstained with BrdU to identify newly generated oligodendrocytes. Interestingly, although H/I increased numbers of NG2+ cells, the number of mature APC+ oligodendrocytes – both total and newly generated – significantly decreased at 14 d following H/I in both the CC and CTX of the ipsilateral hemisphere region peripheral to the infarct zone (Fig. 1F, G, H). This decrease was not observed in the contralateral hemisphere, suggesting that the effects of H/I on mature oligodendrocytes are specific to H/I conditions. Taken together, these results indicate that early OPCs have a delayed capacity to proliferate but fail to mature or survive following H/I, and may therefore afford an extended time window for intervention within one week after H/I injury.
We have previously demonstrated that administration of EPO prior to or at the onset of reperfusion effectively enhances neuronal proliferation, migration and replacement following neonatal H/I, along with diminished brain volume loss.4 Given that increased BrdU-labeled OPCs are observed within a week following H/I (Supplemental Fig. 1F), and acute treatment with EPO promotes cellular proliferation in the neonatal brain,4 we sought to determine whether delayed EPO administration exerted effects on oligodendritic replacement following H/I. Multiple injections of EPO (1000 U/kg BW) and BrdU were administered intraperitoneally into pups as diagrammed (Fig. 1A). Consistent with other models,7 delayed administration of EPO did not reduce brain volume loss (Fig. 1B). However, delayed EPO administration significantly increased the total number of NG2+ cells in the CC, CTX and ST of both the ipsilateral and contralateral hemispheres, well above that induced by H/I alone or naïve control (Fig. 1C, D, E). The increase in NG2-reactive cells extended to newly proliferating cells, as cells immunoreactive for both NG2 and BrdU were significantly increased above control or H/I tissue. In the ipsilateral hemisphere, delayed EPO treatment following H/I restored the number of APC+ cells (both total and newly generated BrdU+) to levels similar to those in control brain (Fig. 1F, G, H). Delayed EPO treatment also significantly increased APC-immunoreactive cells in all regions of the contralateral hemisphere above control (Fig. 1H). Delayed EPO administration thus promotes generation and survival of oligodendrocytes under both lethal and sublethal stress.
While delayed EPO administration is ineffective at decreasing total infarct volume, subtle cellular effects may still occur, leading to improved outcomes. Thus, we examined the extent of white matter injury at the cellular level in affected brain areas (Fig. 2A) using MBP as a marker of myelination and NF-200 as a marker of axons. Following H/I, a significant and prolonged decrease in MBP immunoreactive density was observed in the CTX and ST (Fig. 2B, C) persisted for 14 d post-H/I, suggesting severe and long-term axonal demyelination. Reduced MBP staining in the CC seen at 5 d post-H/I was transient, recovering to control levels by 14 d post H/I (Fig. 2B, C, D). Prolonged white matter injury was found only in ischemic tissue, as the CTX and ST of the contralateral hemisphere were unaffected (Fig. 2E). These results demonstrate that H/I damages white matter in a prolonged fashion.
Delayed EPO treatment following H/I significantly increased the ratio of MBP to NF-200 staining 14 d following H/I in the CC and CTX (Fig. 2D). Since there was no effect of EPO on white matter at the earlier 5 d timepoint, this result indicates that restoration of white matter by EPO is a significantly delayed event. These data illustrate that the timecourse of white matter restoration following H/I parallels the timeframe for EPO-mediated oligodendrogenesis.
We have previously shown that EPO administered immediately after H/I injury enhances neuronal migration and replacement and also improves neurological outcomes.4 Therefore, we next sought to determine if similar effects could also be observed in the more clinically relevant delayed EPO administration and correlate to oligodendrogenesis. Consistent with our previous report, a significant increase in the number of newly generated migrating progenitor cells (BrdU+/DCX+) occurred at 10 and 14 d after H/I in the CTX (Fig. 3A, B) and ST (Fig. 3C, D). Delayed administration of EPO following H/I significantly increased DCX+ and BrdU+/DCX+ cells in ischemic CTX (Fig. 3A, B) and ST (Fig. 3C, D) compared to both controls and vehicle-treated H/I animals.
Finally, to test whether the increased oligodendrogenesis and neurogenesis could be translated into neurofunctional improvement, sensorimotor neurological function was evaluated using foot fault tests and negative geotaxis test. A decrease in the total steps taken in vehicle-and EPO-treated H/I groups compared to controls was only evident 10 d after H/I, which recovered by 14 d (Fig. 4A). Delayed EPO treatment significantly decreased the fault rate in H/I animals at the later timepoints of 10 and 14 d following H/I for both the contralateral forelimb (Fig. 4B) and hindlimb (Fig. 4C). EPO-treated H/I animals significantly improved their negative geotaxis response at later timepoints compared to vehicle-treated H/I animals (Fig. 4D). These data suggest that delayed administration of EPO following H/I does not affect short-term motor outcomes, but rather significantly improves outcomes at an extended period 14 d following injury, in a timeframe concurrent with oligodendrogenesis and neuronal replacement.
The present study investigated the state of brain oligodendrogenesis and neurological functional outcomes following delayed administration of EPO to H/I treated neonatal rats. The findings demonstrate that EPO administration delayed by 24 h was able to: 1) induce a prolonged increase in oligodendrogenesis and maturation in the ischemic hemisphere and in the corpus callosum; 2) attenuate white matter injury concomitant with migration of neuronal precursors into the ischemic region; and 3) improve neurological functional outcomes following neonatal H/I.
Administration of EPO to ameliorate damage to the ischemic brain has proven effective if acutely administered within 6 hours of ischemia. However, its potential clinical value as a post-ischemic therapeutic has remained questionable due to its inability to reduce infarct size in the adult brain.7 Interestingly, in the neonatal brain, delayed administration of EPO was also ineffective at decreasing infarct volume, but significantly improved neurological function outcomes. These findings demonstrate that gross preservation of neural tissue is not a definitive requirement for the recovery of neurological function. Rather, the restoration of neurological function may be influenced by a variety of factors, such as reorganization of surviving tissue7 and, as suggested by the current data, restoration of myelin and oligodendrocytic architecture. Our data shows a correlation between increased oligodendrogenesis, neuronal migration and white matter recovery with improved neurological function outcomes. Promotion of oligodendrogenesis may thus function in two manners: prevention of further degradation of existing neurons via remyelination and promotion of neuronal migration.
The observed changes in the contralateral hemisphere demonstrate that EPO may also potentially aide brain tissue that is only indirectly stressed by H/I. Increased oligodendrogenesis was observed in the contralateral hemisphere, while EPO treatment further enhanced oligodendrogenesis in all regions of the contralateral hemisphere above control. This result is intriguing, as very little damage is observed within the contralateral hemisphere. One possible explanation is that severe stress (i.e., ipsilateral hemisphere) damages oligodendrocytes, while mild injury (i.e., contralateral) acts as a preconditioning signal to induce the production of protective factors, such as endogenous EPO, which in turn stimulates neurogenesis as well as oligodendrogenesis. In line with this, upregulation of EPO and EPOR was observed after hypoxia in the CNS11, 12 and EPO induces neurogenesis in response to hypoxia-only insult and in the normal brain. 13 The contralateral hemisphere undergoes a sublethal hypoxic challenge, and, due to transhemispheric diaschisis, may have alterations in electrical activity, cerebral blood flow and metabolites.14 Further studies examining the effects of EPO in sublethal stress conditions will be interesting, as in addition to provide protection to severe injury, sublethal injury may also be a potential target of EPO.
The current study supports other works demonstrating that neonatal H/I has differential effects on the various stages of maturing oligodendrocytes.2 In particular, immature OPCs are more resistant to neonatal H/I as compared to mature oligodendrocytic populations, and the number of late OPCs is increased by EPO treatment. EPO has been demonstrated to possess oligodendro-protective capacity;15 however, we also demonstrate that delayed EPO treatment induces OPC proliferation (BrdU+) as opposed to simple cellular protection. This is consistent with the observed effects of EPO on neuronal populations, where EPO is capable of exerting both neuroprotection and stimulation of neurogenesis. While the mechanism for EPO-stimulated cell replacement is currently unknown, both neurons and OPCs express EPO receptors.16 The signaling mechanisms underlying cell replacement in the brain may further identify potential therapeutic targets and points for intervention.5
Interestingly, the effects of delayed EPO administration were not detectable until a later (14 d) timepoint. The observation that EPO exerts effects on white matter injury is novel, as until now no effects of delayed EPO administration had been observed at earlier timepoints in neonatal H/I. The longer 14 d timeframe extends a recent report that described EPO as ineffective at attenuating white matter injury following neonatal H/I with an endpoint of 72 hours.17 Consistently, we found that shorter survival timepoints (5 d) exhibited no significant differences between H/I and EPO-H/I groups in terms of cell replacement, whereas longer survival (14 d) resulted in significant differences in white matter cell counts and sensorimotor behavior. These data argue that EPO facilitates post-ischemic white matter restoration rather than a direct protective effect on pre-existing oligodendrocytes. Current and previous studies4, 18 suggest that oligodendrogenesis and neurogenesis are closely related to neurological outcome improvement after ischemia. Conceptually, oligodendrogenesis and neurogenesis may have a delayed rather than immediate effect on neurological outcomes, based on its long temporal and complex spatial pathophysiological processes. This was supported by our study showing that longer survival (14 d) rather than shorter survival timepoints (5 d) resulted in improved sensorimotor behavior. It is tempting to speculate that delayed EPO administration also has effects on oligodendrogenesis/neurogenesis and functional profiles at even longer periods, and this warrants further study.
The present study indicates that therapeutics such as EPO, with positive effects on oligodendrocytic replacement, also yield improved functional outcomes in the absence of gross tissue protection. The interplay between white matter restoration, neurogenesis and neurological function outcomes following H/I in the neonate may effectively improve post-ischemic therapies.
A, BrdU labeling diagram. B, Indication of selected fields. C, Immunofluorescent staining of NG2 (red) and BrdU (green) in the CC and CTX. Scale bar, 100 μm. D, Confocal (left) and 3D (right) image of NG2 and BrdU staining 3d post-H/I. Quantification of NG2+ (E) and BrdU+/NG2+ (F) cells in the CC (left) and CTX (right). *p<0.05, **p<0.01
We thank Carol Culver and Armando P. Signore for editorial assistance and Pat Strickler for secretarial support.
This project was supported by National Institutes of Health/NINDS grants NS43802, NS45048, NS36736, NS56118 (to J.C.), and NS053473 (to G.C.), VA Merit Review grants (to J.C. and G.C.), and AHA Scientist Development Grant 06300064N (to G.C.).