Endothelial Nitric Oxide Synthase Regulates White Matter Changes via the BDNF/TrkB Pathway after Stroke in Mice

Stroke induced white matter (WM) damage is associated with neurological functional deficits, but the underlying mechanisms are not well understood. In this study, we investigate whether endothelial nitric oxide synthase (eNOS) affects WM-damage post-stroke. Adult male wild-type (WT) and eNOS knockout (eNOS-/-) mice were subjected to middle cerebral artery occlusion. Functional evaluation, infarct volume measurement, immunostaining and primary cortical cell culture were performed. To obtain insight into the mechanisms underlying the effects of eNOS-/- on WM-damage, measurement of eNOS, brain-derived neurotrophic factor (BDNF) and its receptor TrkB in vivo and in vitro were also performed. No significant differences were detected in the infarction volume, myelin density in the ipsilateral striatal WM-bundles and myelin-based protein expression in the cerebral ischemic border between WT and eNOS-/- mice. However, eNOS-/- mice showed significantly: 1) decreased functional outcome, concurrent with decreases of total axon density and phosphorylated high-molecular weight neurofilament density in the ipsilateral striatal WM-bundles. Correlation analysis showed that axon density is significantly positive correlated with neurological functional outcome; 2) decreased numbers of oligodendrocytes / oligodendrocyte progenitor cells in the ipsilateral striatum; 3) decreased synaptophysin, BDNF and TrkB expression in the ischemic border compared with WT mice after stroke (n = 12/group, p<0.05). Primary cortical cell culture confirmed that the decrease of neuronal neurite outgrowth in the neurons derived from eNOS-/- mice is mediated by the reduction of BDNF/TrkB (n = 6/group, p<0.05). Our data show that eNOS plays a critical role in WM-damage after stroke, and eNOS-/--induced decreases in the BDNF/TrkB pathway may contribute to increased WM-damage, and thereby decrease functional outcome.


Introduction
White matter (WM) is composed of bundles of myelinated axons that connect various grey matter areas of the brain to each other [1]. Cerebral WM damage is frequently observed in human ischemic cerebrovascular disease, and is increasingly recognized as contributing to cognitive impairment and long-term disability [2,3]. Endothelial nitric oxide synthase (eNOS) is a key target in molecular stroke research [4]. Previous studies have shown that eNOS reduces acute ischemic injury and promotes recovery following cerebral ischemia by regulation of cerebral blood flow, maintaining cerebral homeostasis, exerting anti-inflammatory effects, and by increasing angiogenesis as well as neurogenesis [5][6][7][8]. However, to our knowledge, there are no reports whether eNOS regulates WM changes post-stroke. eNOS knockout (eNOS -/-) mice showed a reduced expression of neurotrophin brain-derived neurotrophic factor (BDNF) [5], suggesting that eNOS may impact WM by regulating BDNF. BDNF and its receptor tropomyosin-related kinase B (TrkB) have been implicated in regulating central nervous system (CNS) axon growth [9][10][11] and supporting a promyelinating role in vivo [12]. In vitro studies have shown that BDNF exerts direct effects upon oligodendroglia, variously promoting oligodendrocyte progenitor cell (OPC) proliferation and differentiation, as well as myelination via activation of endogenous TrkB receptors on oligodendroglia [13]. Thus, in this study using a model of stroke in mice, we investigate whether eNOS impacts WM-damage after stroke and the possible role of BDNF in this process.

Materials and Methods
All experimental procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.

Middle Cerebral Artery Occlusion (MCAo) model
Adult male C57BL/6 wild-type (WT) and eNOS -/mice (2 months old, weighting 25-30g, Jackson Laboratory) were employed in this study. All aminals were subjected to permanent right MCAo by a filament method [5]. Briefly, mice were initially anesthetized with 3.5% isoflurane and maintained with 1.0% to 2.0% isoflurane in 70% N 2 O and 30% O 2 using a facemask. The rectal temperature was controlled at 37uC with a feedbackregulated water heating system. The right common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A length of 6-0 monofilament nylon suture (8.0-9.0 mm), determined by the animal weight, with its tip rounded by heating near a flame, was advanced from the ECA into the lumen of the ICA until it blocked the origin of the MCA.

Experiment Groups
Neurological functional outcome was measured in all of the survival animals. Animals were sacrificed under deep ketamine/ xylazine anesthesia at 7 days after MCAo, among which, 8 mice (n = 4 for WT-MCAo and eNOS -/--MCAo, respectively) were employed for tissue protein and RNA extraction, which were used for Western blot and real-time PCR (RT-PCR) assays. The remaining 24 mice (n = 12 for WT-MCAo and n = 12 eNOS -/--MCAo, respectively) were fixed by transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde. The brains were then coronally sectioned, paraffin-embedded for infarct volume measurement, histochemistry and immunohistochemistry staining.

Functional Tests
The single pellet reaching test was performed before MCAo and at 7 days after MCAo [14]. Briefly, all animals were trained 30 min daily for 5 days before MCAo, and subjected to a restricted diet overnight prior the training and experimental testing. Animals were trained to use their left forepaw to extend through the slot from Plexiglas reaching box and reach the food pellets (Bioserve Inc.). The reaching was scored as a success when the animal reached and obtained a food pellet. Otherwise, the reach was scored a miss when the animal knocked the food away or dropped the food after grasping. Each animal was provided with 20 pellets each day during the testing period. The number of the left forepaw attempts and the number of successes were counted for each animal during a 10 min testing period. Performance was defined by the success rate = (number of success/number of left forepaw attempts)*100. Functional evaluation was measured by an investigator who was blinded to the experimental groups.

Immunostaining Quantification
For quantitative measurement of Bielschowsky silver, pNFH, LFB, CNPase, PDGFRa, synaptophysin, BDNF and TrkB, 5 slides from each brain with 4 fields of view on each slide from the striatum of the ischemic boundary zone (IBZ) were digitized under a 406 objective (Olympus BX40; Olympus) using a 3-CCD color video camera (Sony DXC-970MD; Sony) interfaced with an micro computer imaging device (MCID) analysis system (Imaging Research). Quantification methods included: 1) Synaptic protein, BDNF and TrkB expression -the percentage of positive area of synaptophysin, BDNF and TrkB to the total selected scan area in the IBZ; 2) Axon or myelin damage -the percentage of Bielschowsky silver-, pNFH-and LFB-positive areas to the total selected scan area in the ipsilateral striatal WM bundles in the IBZ; 3) the number of Oligodendrocytes and OPCs -the total numbers of CNPase-or PDGFRa-immunoreactive cells with the selected scan area in the 406 magnified field in the ipsilateral striatum in the IBZ were counted, and the average number of Oligodendrocytes and OPCs from 5 slides each brain with 4 fields of view on each slide were obtained.

Primary Cortical Cell Culture
In addition to the endothelium of cerebral blood vessels, eNOS is expressed in astrocytes in the CNS [16,17]. To investigate whether eNOS deletion decreases BDNF and TrkB expression in cultured cortical cells, we employed primary mixed cortical cell cultures containing neurons and glial cells.
Cortical cells were prepared from embryonic day 15 pregnant WT or eNOS -/mice. Briefly, embryos were removed, and the cerebral cortex dissected, stripped of meninges, and dissociated by a combination of Ca 2 /Mg 2 -free HBSS containing 0.125% trypsin digestion for 15 min. The triturated cells were passed through a 40 mm cell strainer and counted. The cells were plated in poly-Dlysine-coated (Sigma-Aldrich) dishes (35 mm, Corning) at a density of 2610 6 cells/ml in DMEM with 5% FBS and incubated for an initial 24 h. After 24 h, the culture medium was changed to neurobasal growth medium (Invitrogen) containing 2% B-27 (Invitrogen), 2 mM GlutaMax, and 1% antibiotic-antimycotic. Mitotic inhibitors were not added, as glial cell growth was arrested by confluence, and the paucity of growth factors in the medium used for supporting neuron survival. On day in vitro (DIV) 3, the culture medium was replaced with HBSS and the neurons were subjected to oxygen-glucose deprivation (OGD) for 2 h in the anaerobic chamber, and then returned to normal culture conditions. The cultures were harvested after 24 h for Western blot and RT-PCR assay.

Neurite Outgrowth Measurement
To test whether eNOS -/decreases dendrite outgrowth and whether the mechanisms underlying the decreased neurite outgrowth are mediated by the BDNF/TrkB pathway, primary cortical cell culture was utilized. Briefly, cortical cells were plated at a density of 3610 3 cells/chamber in 8-chamber slides and cultured with neurobasal medium containing 2% B27 and antibiotics without mitotic inhibitors. At DIV3, the cultures were subjected to 2 h of OGD, and were grouped into (6 well/group): 1) WT-OGD; 2) eNOS -/--OGD; 3) WT-OGD + K252a 200 nM (tyrosin protein kinase inhibitor, Calbiochem); 4) eNOS -/--OGD + BDNF 50 ng/ml. The cultures were then returned to normal culture conditions for an additional 24 hours. For measurement of neurite outgrowth, neuron-specific class III b-tubulin (TUJ1) immunostaining was performed to present neuronal bodies and dendrites. TUJ1-fluorescently labeled neurons were photographed at 106. Total dendrite length was measured in 20 neurons in each well using the MCID analysis system, and the total length was averaged.

RT-PCR
The total RNA was isolated with TRIzol (Invitrogen). Quantitative PCR was performed using the SYBR Green RT-PCR method on an ABI 7000 PCR instrument (Applied Biosystems). The following primers for RT-PCR were designed using Primer Express software (ABI). BDNF Fwd: TAC TTC GGT TGC ATG AAG GCG; Rev: GTC AGA CCT CTC GAA CCT GCC. TrkB Fwd: TCA TCA AGT CAG AGG TGA CAG G; Rev: ACT GGG TAC ACT CCT TCT CTC G. GAPDH: Fwd: AGA ACA TCA TCC CTG CAT CC; Rev: CAC ATT GGG GGT AGG AAC AC. Each sample was tested in triplicate, and samples were obtained from six independent experiments that were used for analysis of relative gene expression data using the 2 2DDCT method.

Statistical Analysis
Independent two-sample t-test was used to assess the neurological functional outcome, lesion volume, immunostaining, Western blot and RT-PCR measurement. Correlations between the success of single pellet reaching and the density of Bielshowsky silverstained axons were tested by Pearson's correlation coefficients. One-way ANOVA and Tukey test after Post Hoc test were performed for analyzing neurite outgrowth from cortical neurons. All data are presented as mean 6 Standard Error (SE). Statistical analysis was performed in a blinded manner.

Mortality Rate
Within 7 days after stroke, 9 mice died out of the 41 subjected to MCAo (3 in 19 WT group and 6 in 22 eNOS -/group). The mortality rate in eNOS -/mice with stroke (27.3%) was significantly higher than in WT stroke mice (15.8%).

Lesion Volume and Neurological Functional Outcome
No significant difference was found in the ischemic lesion volume between WT-MCAo and eNOS -/--MCAo groups (Fig.1A, p = 0.436, n = 12/group).
The single pellet reaching test measures the ability of skilled forepaw use [18]. There was no significant difference in the percentage of successful single pellet reaching between WT and eNOS -/mice prior to MCAo, but the degree of functional deficits in eNOS -/mice tested 7 days after MCAo was significantly worse than WT mice (Fig.1B, p,0.05, n = 12/group). Independent two-sample t-test was used for the statistical analysis of lesion volume and functional outcome.

eNOS -/-Increased WM-damage after MCAo
Compared with the WT-MCAo mice, eNOS -/--MCAo mice exhibited a significant decrease in the density of Bielschowsky sliver-stained axons (Fig.1C, p = 0.031) and the density of pNFHimmunoreactive neurofilament (Fig.1D, p,0.01) in the ipsilateral striatal bundles in the IBZ (n = 12/group). Correlation analysis showed that the success rate of single pellet reaching was significantly positive correlated with the density of Bielschowsky sliver-stained axons (Fig.1E, r = 0.75).
Independent two-sample t-test was used for the statistical analysis of WM-damage between WT-MCAo and eNOS -/--MCAo mice. Correlations between the success of single pellet reaching and the density of Bielshowsky silver-stained axons were tested by Pearson's correlation coefficients.

eNOS -/-Decreases Synaptic Protein Expression after MCAo
To further elucidate whether eNOS -/decreases synaptic protein after stroke, synaptophysin protein expression was measured by immunostaining and Western blot analysis. eNOS -/--MCAo mice exhibit significantly decreased synaptophysin expression in the ipsilateral ischemic border compared with WT-MCAo mice ( Fig.3A-C, p,0.001, n = 12/group). Western blot assay also showed eNOS -/significantly decreased synaptophysin protein levels in the IBZ (Fig.3D, p,0.05, n = 4/group). These data suggest that eNOS -/mice have decreased synaptic protein expression in the ischemic brain after stroke compared with WT mice. Independent two-sample t-test was used for the statistical analysis of synaptophysin between WT-MCAo and eNOS -/--MCAo mice.

eNOS -/-Mice Exhibit Decreased BDNF/TrkB Expression in the Ischemic Brain
To investigate the mechanism of eNOS -/increased WM damage, BDNF/TrkB and eNOS expression in the brain were measured. Decreased eNOS-expression in both ischemic ipsilateral and contralateral brain of eNOS -/mice was confirmed by Western blot assay (Fig.4, p,0.05, n = 4/group). eNOS -/--MCAo mice exhibit significantly decreased BDNF protein expression in the ipsilateral IBZ, measured by immunostaining and Western blot analysis (Fig.5A-C and H, p,0.05, n = 12/group for immunostaining; n = 4/group for Western blot), but BDNF mRNA level measured by RT-PCR was negative (Fig.5G, n = 4/group), compared with WT-MCAo mice.
TrkB protein expression measured by immunostaining and TrkB mRNA expression in the IBZ were also significantly decreased in eNOS -/--MCAo mice compared with WT-MCAo mice (Fig.5D-G, p,0.05, n = 12/group for immunostaining; n = 4/group for Western blot). There are two major isoforms of TrkB, full-length functional (140 KDa) and truncated non-functional (90 KDa) isoforms, and they exhibit different characteristics. The production of truncated isoform of TrkB receptors is induced in response to an injury [19]. Therefore, increased truncated TrkB receptors also contribute to BDNF-TrkB signaling in neuronal injury. We examined the expression of both full-length and truncated TrkB by Western blot. As shown in Fig.5H, the expression of 140 kDa TrkB in the IBZ in eNOS -/--MCAo mice was significantly decreased; however, the relative levels of 90 kDa TrkB compared to 140 kDa TrkB (Ratio of 90/140 kDa TrkB) was significantly increased compared with WT-MCAo mice (p,0.05, n = 4/group). These data indicate that eNOS -/--MCAo mice exhibit decreased full-length BDNF, but increased truncated BDNF compared with WT mice 7 days after stroke.
Independent two-sample t-test was used for the statistical analysis of BDNF and TrkB expression between WT-MCAo and eNOS -/--MCAo mice.

eNOS -/-Decreased BDNF/TrkB Expression and Neurite Outgrowth in Cortical Cell Cultures Derived from eNOS -/-Mice after OGD
To complement the in vivo data that eNOS -/mice exhibit decreased BDNF/TrkB expression and increased WM-damage, the BDNF and TrkB protein and mRNA expression in the cortical cell cultures (containing neurons, asctrocytes and oligodendrocytes) were measured by Western blot and RT-PCR assays. Fig.6A shows that eNOS, BDNF and TrkB protein expression were significantly decreased in the eNOS -/--cortical cell cultures compared with in the WT-cortical cell cultures (p,0.05, n = 6/ group). Fig.6B shows BNDF mRNA level was significantly decreased (p,0.05, n = 6/group) and TrkB mRNA level was marginally (p = 0.068, n = 6/group) decreased in the eNOS -/-cortical cell cultures compared with WT-cortical cell cultures. Independent two-sample t-test was used for the statistical analysis of BDNF and TrkB expression between the cortical cell cultures derived from WT and eNOS -/mice, respectively.
To further test whether BDNF/TrkB mediates the eNOS -/induced reduction of neurite outgrowth, neurite outgrowth was measured in cortical neurons. Fig.6C shows the neurite outgrowth significantly decreased in eNOS -/--neurons compared to WTneurons, and inhibition of TrkB in WT-neurons significantly decreased neurite outgrowth compared with the non-treatment WT-neurons (p,0.05, n = 6/group). In addition, BDNF treatment in eNOS -/--neurons significantly attenuated the eNOS -/induced decreased neurite outgrowth compared with the eNOS -/--neurons. eNOS is not only expressed in cerebral vascular endothelial cells, but also in astrocytes [16]. One-way ANOVA and Tukey test after Post Hoc test were performed for analyzing neurite outgrowth from cortical neurons.
Taken together, these data indicate that eNOS -/significantly decreased BDNF expression in astrocytes, and the low BNDF secretion from astrocytes may further decrease TrkB level in neurons and thereby decrease neurite outgrowth. Therefore, eNOS -/--induced reduction of neurite outgrowth is, at least partially, mediated by BDNF/TrkB pathway.

Discussion
eNOS plays a crucial role in vascular function and neuroprotection from ischemic stroke [20,21]. eNOS is predominantly expressed by vessels endothelial cells [22][23][24] and are also located in Purkinje cell bodies in the cerebellar cortex, olfactory bulb, dentate nucleus in granular layer and hippocampal pyramidal cells, and astrocytes surrounding the cerebral blood vessels [16,17,[25][26][27]. Astrocytes detect neuronal activity and can release neurotransmitters, which in turn control synaptic activity [28][29][30][31]. eNOS-positive astrocytic perisynaptic sheaths on neuronal somas in the cortex may influence neuronal transmission directly at axonsomatic synapses in the cortex [16]. In addition, the presence of eNOS in astrocytes and in their processes that contact blood vessels suggests that the link between local cortical activity and changes in cerebral blood flow could be mediated by astrocytic release of nitric oxide [16].
There is substantial evidence that many of the therapeutic approaches to the treatment of stroke, such as statins, rosiglitazone and physical activity, exert their effects by increasing eNOS [4,21,32,33]. Simvastatin treatment of stroke protects against cerebral injury by upregulating eNOS, increasing functional protein expression, and augmentation of cerebral blood flow. However, the effects of simvastatin are completely absent in eNOS -/mice [21,34]. Mevastatin, also increased levels of eNOS mRNA and protein, reduced infarct size, and improved neurological deficits in a dose-and time-dependent manner in mice [32]. eNOS also mediates T090317, a liver X receptor agonist, treatment-induced angiogenesis and improved functional outcome after stroke in mice [6]. Voluntary physical activity improves longterm stroke outcome by eNOS-dependent mechanisms related to improved angiogenesis and cerebral blood flow [33]. These findings support the importance of targeting eNOS as a means to induce stroke protection and enhance postischemic neuroregeneration [17]. In this study, we extend the role of eNOS in mediating neuroprotection. We demonstrate that eNOS -/mice exhibit no significant difference in lesion volume 7 days after MCAo but increased mortality compared with WT mice, these data are consistent with our previously reported studies [5,35]. However, eNOS -/mice exhibit more severe WM alterations, including decreases in phosphorylated-neurofilament and axon density, and neurological functional outcome after focal brain ischemia compared to WT mice. Moreover, axonal damage is positively significantly correlated with the functional outcome. Synaptophysin is an indicator of presynaptic plasticity and synaptogenesis [36]. Dysregulation of synaptogenesis also plays an important role in the development of pathologies associated with stroke [3,37]. We also observed that synaptophysin, a synaptic protein, is significantly decreased in eNOS -/mice.
Oligodendrocytes are responsible for the formation of myelin sheaths surrounding axons, and myelin acts as an insulator, increasing the speed of transmission of all nerve signals [38]. Following cerebral ischemia, WM damage identified by a decrease in synapses, axonal density and myelination, is coincident with decreases of neural progenitor cells and OPCs [37,[39][40][41]. In the present study, there was no significant difference in demyelination presented by LFB-stained myelin density and MBP expression between WT-MCAo and eNOS -/--MCAo mice; however, eNOS -/mice exhibited reduced numbers of oligodendrocytes/ OPCs post stroke. This might result because oligodendrocytes and OPCs are particularly sensitive to ischemic insult [42]. Stroke induces significantly oligodendrocyte and OPC death 7 days after stroke [43]. However, demyelination usually does not change much in the early stage after ischemia, but is significantly decreased 10 days to 56 days after stroke [44]. Our data indicate, for the first time, eNOS not only regulates vascular changes and neurogenesis, but eNOS plays a vital role in mediating WM integrity and function alterations after stroke.
BDNF is the most abundant neurotrophin in the brain and mediates axon growth and brain plasticity [9]. BDNF knockout mice show significant synaptic fatigue, and exogenous BDNF attenuates synaptic fatigue [45,46]. Stroke induces an increase in  mature BDNF expression which coincides with the increase of synaptophysin both in ipsilateral cortex and hippocampal territories [47]. Post-stroke treatment with amphetamine facilitates behavioral recovery, which is associated with an increase in synaptogenesis and upregulation of BDNF in the lesioned cortex [48]. Voluntary exercise not only upregulates eNOS, but also leads to an endogenous upregulation of BDNF and associated proteins involved in synaptic function, and enhances functional recovery after traumatic brain injury [49]. Targeted deletion of TrkB by the nestin promoter show significant reduction in cortical myelin expression (MBP and CNPase), the number of myelinated axons, and the thickness of myelin sheath in the corpus callosum [50]. We have previously demonstrated that eNOS -/mice exhibit reduced expression of brain BDNF, eNOS by regulating BDNF expression is also a critical mediator of neurogenesis in the brain, and the reduction of BDNF in eNOS -/mice may be responsible for the deficits in functional recovery and reduced brain plasticity [5]. In the present study, we found that eNOS -/mice showed a significant decrease in BDNF, 140KDa TrkB and relative level of 90KDa/140KDa TrkB, concurrently with a decrease in axon density and synaptophysin, and functional outcome. Our new data in concert the other reports, indicate that eNOS plays an important role in regulating endogenous BDNF and TrkB, which are involved in axon growth and synaptic function, and thereby in functional recovery after stroke.
Under physiological conditions, BDNF is predominantly synthesized and secreted from pre-and postsynaptic neurons, whereas astrocytes have a role in its storage and secretion [51][52][53]. Growing evidence suggests that astrocytes synthesize BDNF after induction of a neural lesion [54] or inflammation [51]. The cerebral microvasculature has also been identified as a source of BDNF that can influence the generation and survival of neurons, and brain-derived endothelial cells under hypoxia exhibit increased secretion of BDNF [55,56]. BDNF is secreted into extracellular and then binds to its cell surface recepters TrkB or P 75 to activate its function [9,57]. In addition, BDNF exerts its effects on oligodendroglia via activation of TrkB receptors [13,58,59]). Using co-cultures of dorsal root ganglia neurons and oligodendrocyte precursor cells, phosphorylation of TrkB was highly correlated with myelination, and inhibiting TrkB signalling also inhibited the promyelinating effect of BDNF, suggesting that BDNF enhances CNS myelination via activating oligodendrocyte TrkB-full length receptors [13]. In vitro analyses of basal forebrainderived OPCs reveal that BDNF increases their proliferation as well as their differentiation into mature oligodendrocytes [58][59][60]. In the present study, BDNF/TrkB expression and neurite outgrowth reduction were also found in primary cortical cell cultures derived from eNOS -/mice. Blocking TrkB decreased neurite outgrowth in WT-cortical neurons, and exogenous administration of BDNF attenuates neurite outgrowth reduction in eNOS -/--cortical neurons. Our in vitro data also support the hypothesis that the increased WM damage post-stroke observed in eNOS -/mice is at least partially, mediated by decreasing BDNF/ TrkB signaling activity. From our data, BDNF-mRNA in the ischemic brains and TrkB-mRNA in the cortical cell cultures derived from eNOS -/--MCAo mice were not significantly decreased compared to WT-MCAo mice. RT-PCR measures transcription, and mRNA presence is not sufficient to confirm the expression of the corresponding mature/active protein since protein expression is also regulated by post-translational regulation.
In summary, our data indicate that eNOS -/mice exhibit increased neurological functional deficits after stroke. The concomitantly increased WM damage and decreased synaptic protein as well as reduction in the numbers of oligodendrocytes/ OPCs may contribute to worsening of neurological functional deficits observed in eNOS -/mice. Thus, the BDNF/TrkB pathway, at least partially, mediates eNOS -/reduced WM damage after stroke.