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NEUROPHARMACOLOGY
Department of Pathology and Laboratory Medicine (C.L.K., S.P.Y., L.W.) and Department of Pharmaceutical and Biological Sciences (S.P.Y.), Medical University of South Carolina, Charleston, South Carolina
Received February 20, 2007; accepted May 8, 2007.
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Abstract |
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; Ozduman et al., 2004). The developing brain differs from the adult brain both structurally and functionally (Johnston et al., 2001; Wei et al., 2006) and in its response to hypoxia and ischemia (Derugin et al., 2000). Newborn cells in parts of the brain may differ morphologically and functionally from cells produced in the adult brain (Lemasson et al., 2005). Compared with the adult brain, the developing brain exhibits higher plasticity and is relatively resistant to excitotoxicity (Johnston et al., 2001; Blomgren et al., 2003).
The type of cell death that occurs after a hypoxic and/or ischemic event varies substantially between the adult and the neonatal brain. Comparisons of adult and immature stroke models suggest that apoptosis and an apoptosis/necrosis continuum may be more prevalent in the neonatal brain and that apoptotic cell death is most probably responsible for delayed cell death (Johnston et al., 2001; Northington, 2006; Wei et al., 2006). The developing brain and immature cells are more susceptible to apoptotic death, which has been defined as an important pathological process in a variety of neonatal models, including hypoxia-ischemia (Hill et al., 1995; Nakajima et al., 2000) and transient or permanent focal cerebral ischemia (Wen et al., 2004; Wei et al., 2006). The functional and developmental differences described between the neonatal and adult brain indicate that extrapolating adult data to the neonate is not recommended.
Angiogenesis, the growth of new blood vessels from an existing vasculature, may contribute to neuroprotection and functional recovery after stroke. Recent studies suggest that vascular endothelial cells (ECs), neurons, and astrocytes that are in physical proximity to the endothelium form a functional unit that serves to maintain cerebral homeostasis. Physiological interactions among all these components of the neurovascular unit control cerebral microcirculation; the structural and functional integrity of the unit is critical for normal brain function, as well as tissue repair after brain damage (Blomgren et al., 2003; Curin et al., 2006). Therefore, in addition to neural cells, protection and regeneration of ECs and ultimately the neurovascular unit have emerged as a potential approach in the treatment of ischemic stroke. The idea of the neurovascular unit has been further supported by studies showing that proteins originally thought to be only involved in angiogenesis, such as the Tie/Ang system, may also be involved in modulating neurogenesis (Ohab et al., 2006; Zhang et al., 2007).
Erythropoietin (EPO) is a hematopoietic glycoprotein that induces homodimerization of two molecules of the EPO receptor (EPOR) on the cell surface (Farrell and Lee, 2004), initiating the Janus kinase/signal transducers and activators of transcription signal transduction cascade (Wei et al., 2006; Zhang et al., 2007). Despite the differences between the adult and the developing brain, studies in both adult and neonatal humans and rodent stroke models have shown the safety and efficacy of recombinant human erythropoietin (rhEPO) treatment (Ehrenreich et al., 2002; Gumy-Pause et al., 2005; McPherson et al., 2007). In our previous investigations, we showed that rhEPO was protective against ischemia-induced neuronal cell death in neonatal rats (Wei et al., 2006) and stimulated angiogenesis, which led to restoration of local blood flow after ischemia in adult rodents (Li et al., 2007). rhEPO has also been implicated in vascular protection (Chong et al., 2002; Li et al., 2007). In addition, EPO administration significantly increased neurogenesis in the dentate gyrus after brain injury (Grote et al., 2005). The up-regulation of EPO and EPOR in blood vessels, neurons, and astrocytes of adult human brains infers their role in endogenous protective mechanisms (Sola et al., 2005).
Ischemia increases expression of hypoxia-inducible factor-1
and results in the transcriptional activation of VEGF (Shweiki et al., 1992) and basic fibroblast growth factor (bFGF) (Folkman and Klagsbrun, 1987). Angiopoietins (Ang-1, Ang-2) and their receptors (Tie-1, Tie-2) are angiogenic factors involved in later stages of angiogenesis (Wei et al., 2005). By recognizing the importance of the integrity of the neurovascular unit and the multiple protective effects of EPO in the ischemic brain, the present investigation examined the hypothesis in neonates that rhEPO postischemia treatment not only protected neuronal cells as shown previously (Wei et al., 2006) but also protected vascular ECs as a mechanism of repairing the neurovascular unit after ischemic stroke. The effects of rhEPO was tested in an ischemiaonly neonatal model of whisker-barrel cortex stroke that mimics common clinical cases of small ischemic strokes (Wei et al., 2006).
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Materials and Methods |
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). In brief, postnatal day 7 (P7) pups were anesthetized by hypothermia. Hypothermia anesthesia was chosen because many of the drugs used to anesthetize adult animals provided inadequate anesthesia for neonates or were associated with problems such as excessively high mortality (Danneman and Mandrell, 1997). In this regard, hypothermia (immersion in ice) has been judged as a humane, safe, and effective anesthesia method for survival surgeries of neonatal rats (Danneman and Mandrell, 1997). The hypothermia procedure was kept the same for all pups in different experimental groups. Pups were placed in a noninvasive head-holder to allow for a 2.5- to 3.0-mm-diameter craniectomy through the right parietal skull. The transparent dura was left intact over the whisker-barrel cortex area. Multiple branches of the middle cerebral artery were permanently ligated with sterile 11 silk sutures that were passed through the dura under a dissecting microscope. This was combined with permanent cauterization of the common carotid artery. Animal care and handling followed Institute of Laboratory Animal Resources (1996). rhEPO and BrdU Administration. rhEPO (5000 U/kg, volume 0.10 ml i.p.; Amgen Inc., Thousand Oaks, CA) or saline (volume 0.10 ml i.p.) was administered 60 min after ischemia and once daily after surgery for 3 days. Animals were given i.p. injections of 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg; Sigma-Aldrich, St. Louis, MO) daily beginning on P7 until the day of sacrifice. Animals were anesthetized by an inhaled overdose of isoflurane and sacrificed at various time points after surgery.
TUNEL Staining. The DeadEnd Fluorometric TUNEL System kit (Promega, Madison, WI) was used as described previously (Whitaker et al., 2007). In brief, fresh frozen brains were mounted and sectioned in a cryostat Vibratome tissue sectioning system (14 µm, Ultrapro 5000; Warner Instruments, Hamden, CT). Sections were fixed in 10% buffered formalin (Fisher Scientific, Pittsburgh, PA), washed in phosphate-buffered saline, and processed as recommended by the company's protocol.
Immunohistochemistry. Frozen brain sections were fixed in 10% buffered formalin (10 min; Fischer Scientific). Immunohistochemistry proceeded as described previously (Whitaker et al., 2007). After blocking in 1.0% fish gelatin (Sigma-Aldrich), the slides were incubated overnight at 4°C with the primary antibody rabbit anti-GLUT-1, mouse anti-NeuN (1:400–1:1000, AB1340, MAB377; Chemicon, Temecula, CA), or rat anti-BrdU (1:800; Abcam, Cambridge, UK). Slides were washed and incubated with secondary antibody Alexa Fluor 488 anti-rabbit, anti-mouse IgG, Cy5-conjugated anti-mouse IgG, or Cy3-conjugated goat anti-rat IgG (1:200–1:1000; Invitrogen, Carlsbad, CA), washed in phosphate-buffered saline, and mounted with ProLong AntiFade (Invitrogen). Slides were visualized by fluorescent and confocal microscopy (BX61; Olympus, Tokyo, Japan).
Western Blot. The penumbra region of the brain was removed and frozen. Lysis buffer with protease inhibitor was added, and samples were prepared with a Dounce homogenizer and centrifuged at 14,000 rpm at 4°C for 25 min, and supernatant was collected. Protein concentration was determined by the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane in a Hoefer Mini-Gel system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Membranes were blocked in 7% nonfat-evaporated milk (Carnation; Nestle USA, Solon, OH) diluted in Tris-buffered saline, 0.1% Tween 20 (TBST) at pH 7.4 and incubated overnight at 4°C with a primary antibody against Tie-1, Ang-2, VEGF, or bFGF (1:500–1:4000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted in 1% bovine serum albumin. Mouse 
-actin (1:4000; Sigma-Aldrich) was used for loading controls. Blots were then washed in TBST and incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG (1:5000; Promega, Madison, WI) diluted in 3% milk TBST and washed, and the signal was detected using alkaline phosphatase substrate 5-bromo,4-chloro,3-indolylphosphate nitroblue tetrazolium (Sigma-Aldrich). Western blotting was performed at least three times to confirm data reproducibility and for quantification.
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Statistics. Data were graphed using Prism, version 4 (GraphPad Software, Inc., San Diego, CA). All analyses were performed using SAS, version 9.1 (SAS Institute Inc., Cary, NC) as described previously (Whitaker et al., 2007). As a result from the hierarchical nested experimental design, the data were clustered. To properly account for this correlation, a Poisson regression using generalized estimating equations was used with the natural log of the count data for each slide, clustering on rat and slide within rat; counts within a slide were considered approximately independent of one another, whereas slides within a rat were considered to be correlated. This correlation was accommodated by the model. An alpha level of 0.05 was used for all counts. Data are presented as mean ± S.E.M. Experimental/animal numbers are reported in corresponding figure legends.
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RESULTS |
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Colocalization of TUNEL and GLUT-1, a marker for cerebral microvessel ECs, indicated injured ECs (Fig. 1D–F) (Li et al., 2007; Whitaker et al., 2007). In rhEPO-treated animals 12 to 72 h after stroke, there were significantly fewer GLUT-1/TUNEL-positive vessels compared with those in saline controls (Fig. 1H).
rhEPO Modulated Expression of Neurovascular Regulatory Proteins. To detect rhEPO-mediated potential effects on cell responses to ischemia, protein expression of several key factors in neurogenesis and angiogenesis/vascular proliferation was investigated. Western blotting revealed significantly increased expression of Tie-1, Ang-2, and bFGF in rhEPO-treated ischemic pups (Fig. 2). There was a consistent trend of VEGF increase in the rhEPO-treated group (Fig. 2), whereas no change was found in the expression of Tie-2 and nerve growth factor (data not shown).
rhEPO Enhanced Neurogenesis, Vasculogenesis, and Repair of the Neurovascular Unit. The focal ischemic insult reduced total neurons labeled by the specific marker NeuN in the penumbra region 7 to 21 days after stroke compared with no-stroke control animals (Fig. 3A). In rhEPO-treated pups, the number of NeuN-positive cells remained stable for up to 21 days after ischemia (Fig. 3A).
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To elucidate whether the increased BrdU-positive cells included naive neurons, neurogenesis was identified by the costaining of BrdU and NeuN (Fig. 3, C and D). Colabeled BrdU/NeuN cells in no-stroke pups were detected at a low level (Fig. 3E). Ischemia elevated the neurogenesis activity, but the activity subsided 21 days after ischemia (Fig. 3E). In ischemic pups that received rhEPO, there was a much greater increase in the number of BrdU/NeuN-positive cells; the elevation of neurogenesis remained at 21 days after ischemia when neurogenesis in the saline group declined back to a low level (Fig. 3E).
Among other cells, EC proliferation can be demonstrated by BrdU incorporation into endothelial cells marked by specific marker GLUT-1 (Wei et al., 2006; Whitaker et al., 2007) (Figs. 4 and 5). Immunohistochemical staining allowed for analysis and counting of proliferating microvessels (angiogenesis) (Fig. 4). Although ischemia stimulated proliferation of total cells (see Fig. 2), the ischemic insult in fact hampered proliferating activity of ECs (Figs. 4, A–C, and 5). In contrast, 7 to 21 days after ischemia, the rhEPO group showed increased vessel proliferation compared with saline controls (Fig. 5A). The largest increase was seen 14 days after ischemia. The increases in BrdU-positive vessels were consistent with a steady increase in microvessels labeled with GLUT-1 in the region 7 to 21 days after ischemia (Fig. 5B).
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In an effort to elucidate a possible effect of rhEPO on repairing the neurovascular unit, we specifically examined the relative proximity of NeuN-positive cells to proliferating vascular ECs in the postischemic penumbra region. Immunohistochemical fluorescent imaging and confocal imaging confirmed that rhEPO-treated animals had significantly more NeuN-positive cells located proximal to proliferating microvessels compared with saline controls (Fig. 5C).
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Discussion |
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; Northington, 2006). Studies have shown that particular treatments exert different results when comparing neonatal and adult models. Comparing a neonatal hypoxia-ischemia and an adult middle cerebral artery occlusion model, Cimino et al. (2005) found that treatment with simvastatin to prevent ischemic damage only protected neonatal ischemic damage when given as a pretreatment, whereas the statin was protective in the adult model at all of the tested application times. Moreover, statin-dependent activation of endothelial nitric-oxide synthase was found only in the adult middle cerebral artery occlusion model (Cimino et al., 2005). Comprehensive reviews investigating the differences between the adult and neonatal brain and stroke should be referenced for further clarification (Johnston et al., 2001; Northington, 2006).
Experiments with rhEPO treatment in stroke models have shown a multifaceted role of rhEPO on neuroprotection, neurogenesis, angiogenesis, and anti-inflammatory responses. The neuroprotective effect of EPO has been well documented in previous investigations (Demers et al., 2005). Although many pathways have been implicated in rhEPO-afforded neuroprotection, fewer studies have defined a role for rhEPO in vascular protection in vivo. The present investigation demonstrates in neonatal ischemic stroke that, at a clinically relevant dosage administered 60 min after ischemia, rhEPO is not only protective against neuronal cell death but also shows significant protection of vascular ECs. As a result, rhEPO increases the number of neurons and microvessels from days to several weeks after stroke. In addition, rhEPO stimulates neurogenesis and angiogenesis/vasculogenesis, probably mediated by increased expression of several key molecules in these processes. The increased distribution of newly formed neurons close to ECs suggests an enhanced repair process of the neurovasculature or the neurovascular unit. This orchestrated regeneration of vascular ECs and neuronal cells may serve as an important mechanism of rhEPO-mediated therapeutic effects. In the present investigation, rhEPO was administrated for 3 to 4 days, which is unlikely to trigger a hematopoietic effect; in humans, for example, stimulation of hematopoietic response is usually achieved after several weeks of rhEPO administration (Dührsen and Hossfeld, 1994).
We have previously demonstrated that rhEPO significantly attenuated cell death, specifically neuronal cell death, in the same model of neonatal whisker-barrel cortex stroke (Wei et al., 2006). This neonatal mini-stroke model presents with an initial necrotic component of cell death followed with primary features of apoptosis as neuronal injury induced by focal ischemia (Wei et al., 2006). With the advent of the phenomenon of the neurovascular unit, the inextricable relationship between neurons and the vasculature is becoming apparent. Hypoxic conditions stimulate expansion of the microvascular system in the brain (Ogunshola et al., 2000). The EPO-mediated protective effect on ECs was shown in vitro where increased numbers of ECs were attributed to an increase in proliferation and reduced apoptosis (Muller-Ehmsen et al., 2006). We hypothesized that rhEPO treatment would have a similar effect on vascular proliferation in vivo. Increased GLUT-1-positive microvessels were confirmed after rhEPO treatment, probably resulting from rhEPO-mediated protective and proliferative effects. We observed an overall increase in cell proliferation after rhEPO administration. Seven and 14 days after ischemia, there was significantly more cell proliferation in the rhEPO group compared with controls, suggesting a period of active regeneration in the ischemic and surrounding area.
An interesting observation is that, although rhEPO enhanced neurogenesis (BrdU/NeuN-positive cells) and angiogenesis (BrdU/GLUT-1-positive cells), the total BrdU-positive cells increased at 14 days but not 21 days after stroke in the rhEPO-treated group (Fig. 3B). Two possibilities are most likely to explain the observation. 1) EPO neuroprotection in the neonate has been shown to involve the blocking of a secondary delayed rise in interleukin- and other inflammatory mediators (Sun et al., 2005). Proliferation of nonneuronal cells, such as those in inflammatory reactions, may be prohibited by rhEPO at this time. 2) Subsided effect of rhEPO may be due to the short administration time (4 days).
Normal neonatal angiogenesis peaks between P13 and P24, stabilizes between P24 and P33, and decreases after stability (Ogunshola et al., 2000). The neonatal controls in our study followed a similar trend, as did the rhEPO-treated group. We observed an increase in microvessel proliferation 7 and 14 days after stroke (P14 and P21) following rhEPO treatment and a similar decrease and stabilization of angiogenesis by 21 days after ischemia (P28) (Fig. 4 and 5). Although BrdU proliferation in the saline control group was observed, this did not translate into increased microvessels. The postischemia brain may have been too harsh an environment for successful vascular regeneration, which suggests the importance of a protective as well as regenerative therapy after stroke.
The concept of the neurovascular unit led us to investigate angiogenesis/vascularization and neurogenesis. rhEPO treatment resulted in an increase in neurogenesis 7 to 21 days after stroke in the rhEPO group. ECs secrete soluble factors that stimulate the self-renewal of neural stem cells in the vascular niche (Shen et al., 2004). Administration of human cord blood-derived CD34+ cells after stroke induced angiogenesis, which led to secondary neurogenesis (Taguchi et al., 2004). Our study may support the idea that an angiogenic response results in the establishment of a favorable environment for the regeneration of neurons (Taguchi et al., 2004). We observed an increase in the number of neurons in close proximity to angiogenic microvessels in the rhEPO-treated animals (Figs. 4G and 5C). EC and neuronal proliferation and viability intimately affect each other. Angiogenic and neurogenic factors are important in forming and maintaining a functional neurovascular system. In the present investigation, both processes increased between 7 and 21 days after ischemia, further implying that this is the period of high proliferative activities and a potential target for therapeutic treatments. Recently, Ang-1 and Tie-2 have been implicated to mediate neuroblast migration to the penumbra region following adult mouse stroke (Ohab et al., 2006). The intimate and complicated relationship between the angiopoietins and the corresponding receptor tyrosine kinases Tie-1 and Tie-2 allows for the possibility that this system may be having an effect in progenitor proliferation and differentiation in our model of rhEPO-mediated neurovascular remodeling following neonatal focal ischemia. Furthermore, as previously stated, bFGF and various fibroblast growth factor receptors have also been implicated in neural progenitor modulation (Mudò et al., 2007; Xiao et al., 2007). Although we have not determined precisely where the neural and vascular progenitor cells are derived from in our model, we presume that a majority of the cells come from the subventricular zone. The simultaneous effect of rhEPO on protection and subsequent proliferation may, in turn, allow for a neurogenic and angiogenic effect that is modulated by the expression of the Tie/Ang system and/or bFGF. The nurtured brain microenvironment that results from rhEPO administration may further promote repair of the neurovascular unit in the penumbra region, thus eventually translating to potential functional recovery.
EPO and EPOR are also expressed in glial cells, and their expression increases following cerebral ischemia (Bernaudin et al., 1999). rhEPO markedly reduces astrocyte activation, the recruitment of leukocytes and microglia into ischemic area, and the production of the proinflammatory cytokines in the ischemic rat brain (Villa et al., 2003). These events should benefit the protection and repair of the neurovascular unit, although one study reported that EPO (30 pM) in cortical cultures did not show protective effects against hypoxia- or -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced glial cell death (Sinor and Greenberg, 2000).
Our study seems to be the first to investigate the effect of rhEPO on vascular protection and neurovasculature repair in an in vivo neonatal stroke model. rhEPO has recently emerged as a hopeful neuroprotective drug. We now present results that, in addition to neuroprotection, rhEPO is also protective against ischemia-induced EC death in the neonatal brain. The multifaceted effects of rhEPO, including cell protection, neurogenesis, and angiogenesis, are essential for preservation of the neurovascular unit that is the fundamental element of the brain circulation and neuronal activities. The possibility that EPO may show similar comprehensive effects in adult animals remains to be tested.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EC, endothelial cell; EPO, erythropoietin; rhEPO, recombinant human erythropoietin; BrdU, 5-bromo-2'-deoxyuridine; GLUT-1, glucose transporter-1; NeuN, neuronal nuclear protein; EPOR, erythropoietin receptor; bFGF, basic fibroblast growth factor; Ang, angiopoietin; VEGF, vascular endothelial growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; P, postnatal; TBST, Tris-buffered saline, 0.1% Tween 20.
Address correspondence to: Dr. Ling Wei, Department of Pathology and Laboratory Medicine, 165 Ashley Ave. Medical University of South Carolina, Charleston, SC 29425. E-mail: weil{at}musc.edu
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6 animals per group; *, P <0.05 compared to saline control.
, P < 0.05 compared to no-stroke controls.
