Loss of NB-3 Aggravates Cerebral Ischemia by Impairing Neuron Survival and Neurite Growth
Background and Purpose—NB-3 is a member of the F3/contactin family of neural recognition molecules, which are crucial for cell morphogenesis and motility. NB-3 is expressed in neurons and plays an important role in axonal extension and neuronal survival. However, the role of NB-3 in cerebral ischemic injury remains unknown.
Methods—Adult male wild-type and NB-3 knockout mice were subjected to ischemic injury by unilateral middle cerebral carotid artery occlusion for 3 hours, 6 hours, and 12 hours. Ischemic infarction volumes were then determined by 2, 3, 5-triphenyltetrazolium chloride staining. Neurological dysfunction analysis was also performed. Primary culture of neuronal cells from wild-type and knockout animals was also used for analysis of neuronal survival and neurite outgrowth.
Results—NB-3 expression in the ischemic hemisphere was decreased after transient middle cerebral artery occlusion (MCAO). NB-3-knockout mice developed a 2.6-fold larger infarct volume and exhibited increased neurological deficit scores after transient middle cerebral artery occlusion compared with control mice. Substrate with NB-3 promoted neuronal survival and neurite outgrowth in vitro, whereas neurite outgrowth and neuronal survival were significantly reduced in NB-3-deficient neurons. In addition, NB-3 deficiency renders neurons more susceptible to oxygen–glucose deprivation-induced damage and NB-3 as substrate could partially through homophilic mechanisms.
Conclusions—These data demonstrate that NB-3 deficiency may aggravate brain damage after middle cerebral artery occlusion by impairing neuronal survival and neurite growth.
Neural recognition molecules at the cell surface play important roles in cell communication not only during ontogenetic development, but also in the adult during modulation of synaptic efficacy and regeneration after central and peripheral nervous system injury.1,2 Neural recognition molecules of the immunoglobulin superfamily are classified into several subgroups, including the NCAM, L1, and contactin/F3 subgroups. NB-3, also termed contactin-6, is a member of the contactin/F3 subgroup in the immunoglobulin superfamily.3 The extracellular part of NB-3 consists of 6 immunoglobulin-like domains and 4 fibronectin type III repeats and provides the structural basis for the homophilic nature of NB-3 as well as its numerous heterophilic interactions.4 A greater understanding of the functional roles of NB-3 is important to improve the targeting of recognition molecules that exert adverse effects on neuroregeneration after an acute lesion.
NB-3 is expressed exclusively in the nervous system and is predominantly upregulated at the early postnatal stage during mouse brain development.4 High expression of NB-3 mRNA was also detected in the cerebrum, cerebellum, and spinal cord in the adult brain. NB-3 knockout (KO) mice exhibit impaired cerebellum-related motor coordination.5 In addition, NB-3 as a glycosyl phosphatidylinositol-anchored neural recognition molecule, can trigger nuclear translocation of the Notch intracellular domain and promoted oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells through Deltex1.6 Recent data suggest that NB-3 directly bound to CHL1 can regulate apical dendrite projection in the developing caudal cortex through PTPα signaling.7 It is well established that neural recognition molecules such as CHL1, NCAM, and F3/contactin can promote neuronal survival in neurodegenerative diseases and nerve injury8; however, the role of NB-3 in regeneration after brain injury remains unknown.
Thus, in the present study, we were interested in examining the role of NB-3 in mediating the brain's susceptibility to ischemic injury. We detected the expression of NB-3 in the cortex after ischemic injury in wild-type mice and then confirmed that NB-3 deficiency aggravated brain damage after ischemia by impairing neurite growth and neuronal survival. These data demonstrate that NB-3 plays an essential protective role, suggesting a therapeutic potential against cerebral ischemic injury.
Materials and Methods
NB-3-deficient mice were obtained from Dr. Kazutada Watanabe in Japan. All experiments were performed in 2-month-old male mice (20 to 25 g body weight). Generation of NB-3-deficient mice was reported previously.5 Heterozygous mice were mated to generate wild-type and KO pairs. Mice were maintained at the Beijing Basic Medical Institute Animal Facility in accordance with institutional guidelines.
The staining procedure using X-gal solution was performed exactly as described previously.5 Sections were counterstained with a neuronal marker (Chemicon) or oligodentricytes marker (CNPase; Sigma) immunoreaction.
Middle Cerebral Artery Occlusion Model of Ischemia in Mice
Focal ischemia was induced by left middle cerebral artery occlusion (MCAO), as described previously.9 Briefly, adult male wild-type mice (n=10) and NB-3-KO mice (n=10) were anesthetized with 5% chloral hydrate (350 mg/kg intraperitoneally). Focal cerebral ischemia was induced using 8-0 nylon monofilament coated with silicone hardener mixture through the internal carotid artery. The coated filament was introduced into the left internal carotid artery through the common carotid artery and then advanced up to the origin of anterior cerebral artery through the internal carotid artery so as to occlude the middle cerebral artery and posterior communicating artery.
Measurement of Physiological Parameter
Cerebral blood flow was measured by laser Doppler flowmetry with a Biopac LDF100A laser (Biopac Systems). Adult wild-type mice (n=8) and NB-3-KO mice (n=8) were subjected to MCAO and measured at 12 hours after MCAO. The mean values of cerebral blood flow before MCAO were taken as control (100%), and the data thereafter were expressed as percentages of this value. Arterial blood gas (pH, pco2, and po2) was measured before and at 12 hours after MCAO with a Nova Statlabs Profile 5 blood gas analyzer.
Measurement of Infarction Volume
The infarction volume was measured as described previously.10 Mice were deeply anesthetized by intraperitoneal injection of 0.6 g/kg chloral hydrate, and the brains were removed and sliced into 2-mm coronal sections using a plastic mold. The sections were stained with 1% 2,3,5-triphenyltetrazolium chloride for 30 minutes at 37°C and fixed in 4% paraformaldehyde. The area (unstained) of the infarction in the left cerebral hemisphere was then traced and measured using image analysis software (Image Pro-Plus 6.0), and the infarction volume per brain (mm3) was calculated from the measured infarction area.11
Neurological Dysfunction Analysis
The behavioral tests were conducted by one blinded to the experimental groups. A score from a series of behavioral tests was used to measure the neurological functional deficits, as previous reported.9
Primary Culture of Cells From the Cortex
Cerebral cortices were removed from embryonic Day 18 mice and dissociated by trypsinization. Cells were suspended in Dulbecco modified Eagle medium/F12 medium containing 2% B27 supplement (Invitrogen), 10% fetal bovine serum, and 10% horse serum (Gibco). Cells were planted at a density of 2×104 cells/well, incubated for 24 hours, and then processed for Tuj1 immunostaining.12
Measurement of the Neurite Outgrowth
The cells were planted onto 24-well plate coated with NB-3 (2.5, 5, or 10 μg/mL) and cultured for 24 hours. As negative controls, bovine serum albumin (BSA) at the same concentrations was used. Neurite length was evaluated by measuring the length of the Tuj1+ cells as described. The length of neurite outgrowth for approximately 1000 to 2000 neurons was measured from 3 wells for each condition.
Oxygen–Glucose Deprivation Model
Cultures of cortical neurons were prepared. The original media were removed, the cell were washed with a glucose-free Earle balanced salt solution at pH 7.4, and placed in fresh glucose-free Earle balanced salt solution. Cultures were then introduced into an incubator containing a mixture of 5% CO2 and 95% N2 at 37°C for 1 hour. The cells were returned to their original culture condition and maintained for 24 hours after oxygen–glucose deprivation (OGD) treatment.
Lactate Dehydrogenase Assays
The release of lactate dehydrogenase (LDH) into the culture medium was measured using LDH assay reagent (Promega) following the instructions of the kit menu.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature. Cells were incubated with primary antibodies overnight at 4°C. The polyclonal NB-3 antibody (1:200; R&D Systems) and mouse MAP-2 antibody (1:1000; Chemicon) were used. The immunoreactivity was visualized with Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes).
Cell Survival Assay (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl Tetrazolium Bromide Assay)
Cells (1×105) were cultured in 96-well plates for 24 hours. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution was then added and cells were cultured at 37°C for 4 hours. The absorbance of each well at 450 nm was recorded on an enzyme-linked immunosorbent assay reader (Bio-Rad), and the percentage of surviving cells was calculated.
Cell Death Assay (Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End Labeling Staining)
Cell death was determined by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) method (In Situ Cell Death Detection Kit; Roche Biochemicals), per the manufacturer's instructions. Cells were then counterstained with 4′,6-diamidino-2-phenylindole. One thousand to 2000 neurons were counted per coverslip for each condition from 4 wells.
Data are presented as means±SD. Differences between control and experimental groups were examined by 1-way analysis of variance. P<0.05 was considered statistically significant. Three independent experiments were performed for each datum.
Expression and Distribution of NB-3 in the Adult Mouse Brain
The spatiotemporal expression of NB-3 mRNA in the developing brain was previously reported.3,5 To investigate the role of NB-3 after ischemic injury, we examined NB-3 expression in the adult mouse brain. NB-3-deficient mice were generated by substituting a part of the NB-3 gene with the galactosidase (LacZ) gene. Complete overlap of LacZ expression with the NB-3 immunostaining pattern was reported in the heterozygous mouse brain.5 We performed double staining of X-gal with a neuronal marker (neurons) and CNPase (oligodendrocytes). LacZ expression was high in the II/ III layer of cortex and in the hippocampus (Figure 1A) and was colocalized with neuronal marker-positive neurons in the cortex (Figure 1B) but not with CNPase+ cells in cortex (Figure 1C), as previously reported.5,13
NB-3 Deficiency Aggravates Brain Damage After Cerebral Ischemia
To evaluate the role of NB-3 in brain injury, we used the well-established mouse MCAO model for focal cerebral ischemia. Protein was extracted from cortical brain tissues at 3 hours, 6 hours, and 12 hours after MCAO and examined by Western blot. The expression levels of NB-3 protein in the ischemic hemisphere were markedly decreased after MCAO when compared with the intact side. There were approximately 2-fold decrease at 6 hours and a 5-fold reduction at 12 hours after MCAO (Figure 2A), whereas there was no change in β-actin expression after ischemia.
Next, we assessed the effects of NB-3 losing on the brain injury after insulting in MCAO. NB-3-KO and wild-type mice were subjected to MCAO for 12 hours. We found that the infarction area in NB-3-KO mice developed a 2.6-fold larger infarct volume (75±11 mm3) than NB-3+/+ control (29±9 mm3; Figure 2A–C). Increased brain damage in the NB-3-deficient mouse was accompanied by increased neurological deficit scores (Figure 2D). There was no significant difference in ischemic-related mortality between NB-3-KO and control mice after MCAO (control group: 15% mortality, n=12; NB-3-KO group: 12% mortality, n=15). In addition, the cerebral blood flow significantly decreased after MCAO at 12 hours; however, there were no differences between wild-type mice and NB-3 KO mice before and after MCAO. The value of body weight, blood pCO2, pO2, and pH were all in the normal range (see Supplemental Table I; http://stroke.ahajournals.org). These data suggest that NB-3 deficiency increases vulnerability to ischemic damage.
NB-3 Enhances Neuron Survival and Neurite Outgrowth
To determine whether NB-3 affects neuronal survival and neurite outgrowth, primary cultured cortical neurons from embryonic Day 18 embryos were then used. All Tuj1-positive neurons expressed NB-3 and colocalized with Tuj1 immunoreactivity (Figure 3A). Addition of NB-3 caused a dramatic dose-dependent increase in the percentage of Tuj1+ neurons with neurite length >2 soma diameters (10 μg/mL NB-3, 57.02%±1.75%) compared with BSA-treated cells (48.80%±1.72%; Figure 3B–C). The mean neurite length was 137.4±3.340 μm in NB-3-coated neurons versus 116.3±2.798 μm in BSA-coated neurons (data not shown). Furthermore, the percentage of TUNEL-positive cells in NB-3 (5 μg/mL) -coated wells was significantly decreased compared with control cells (data not shown). These results indicated that NB-3 addition increased the neurite outgrowth and decreased the cell apoptosis.
To confirm these results, the effects of NB-3 deficiency on the neuronal survival and neurite outgrowth were investigated by using cultured neurons derived from NB-3-KO and wild-type mice. We found that the percentage of neurons with neurites in NB-3-deficient cells (42.87%±1.3%) showed a significant reduction compared with NB-3+/+ cells (54.12%±1.1%; Figure 4A–B), and the neurite length was shorter in NB-3−/− cells (70.51±1.8 μm) than NB-3+/+ cells (94.25±2.2 μm; Figure 4C). Neuronal survival was also reduced in NB-3−/− cells compared to NB-3+/+ cells (Figure 3D), whereas the percentage of TUNEL+ cells was increased in NB-3−/− cells (8.81%±1.1%) compared with control (4.28%±0.4%; Figure 4E). These data clearly demonstrated NB-3-deficient neurons exhibit relatively fewer neurite outgrowth and more cell death than control cells, suggesting NB-3 increased neurite outgrowth and neuronal survival.
NB-3-Deficient Neurons Render Increased Susceptibility to OGD-Induced Damage
To further confirm the relationship between NB-3 and neuronal susceptibility to ischemic injury, cortical neurons derived from NB-3-KO and wild-type mice were exposed to OGD for 1 hour after 7 day culture in vitro. The gross morphological features of MAP-2 immunohistochemistry were observed. OGD-treated neurons from NB-3-deficient mice displayed more cell body shrinkage, neurite retraction, and neural process fragmentation compared with those from NB-3+/+ control (Figure 5A). Then, OGD-induced cell death was quantified by measuring LDH release in the culture medium. LDH release in the medium from NB-3−/− neurons significantly increased, and there was an approximately 1.4-fold increase in cell death after OGD treatment compared with the control (Figure 5B). In addition, the percentage of TUNEL+ cells in NB-3−/− neuron was significantly increased after exposure to OGD compared with control (Figure 5C–D). Consistent with the data in in vivo experiments, NB-3−/− neurons in response to OGD showed more severe damage than the control neurons, which suggested NB-3 had a neuroprotective role in ischemic injury.
The Homophilic Interaction of NB-3 Is Essential for Neuronal Survival
It has been reported that homophilic or heterophilic interaction of recognition molecule plays a crucial role in the regulation of neuronal survival and growth.1 To investigate whether NB-3 interacts by homophilic or heterophilic binding mechanisms, NB-3 as substrates was coated on the coverslips. Cortical neurons from NB-3+/+ and NB-3-KO mice were planted separately onto coverslips coated with NB-3 (5 μg/mL) or BSA (5 μg/mL) as controls. The data showed that percentage of neurons with neurite outgrowth NB-3 as substrate (52.04%±2.0%) increased markedly compared with those on BSA (42.49%±2.0%). There was no obvious change in NB-3−/− neurons with neurite outgrowth treated with NB-3 substrate (45.83%±1.7%) compared with those on BSA (43.95%±2.1%; Figure 6A). Furthermore, there was an increase in mean neurite length in NB-3+/+ neurons treated with NB-3 substrate (182.37±9.1 μm) compared with those on BSA (150.78±8.8 μm), whereas a significant difference in the mean neurite length of NB-3 deficient neurons with NB-3 substrate was not observed compared with those treated with BSA (Figure 6B). These observations indicate that NB-3 enhances neurite outgrowth, most likely through homophilic mechanisms.
We further investigated the effect of NB-3 as substrate on the neurons' survival in response to OGD. OGD-induced cell death was quantified by measuring LDH release and TUNEL analysis. In NB-3+/+ neuron, there was a significant reduction in LDH release treated with NB-3 substrate after exposure to OGD compared with BSA control. There was also a little reduction in NB-3−/− neurons treated with NB-3 substrate in response to OGD compared with control but no difference between these 2 groups (Figure 6C). In addition, after exposure to OGD, the percentage of TUNEL+ cells in NB-3+/+ neurons treated with NB-3 substrate (7.36%±0.47%) was decreased compared with those on BSA-coated substrate (8.55%±0.35%; Figure 6D). There was also a reduction in the number of TUNEL+ cells in NB-3−/− neurons after treatment with NB-3 substrate (10.2%±0.7%) compared withcontrol substrate (11%±0.8%; Figure 6D). These data suggest a neuroprotective role of NB-3 after OGD injury partially through homophilic mechanisms by reducing the cell death.
In the present study, we demonstrated that NB-3 was important for neuronal survival and neurite outgrowth associated with cerebral ischemic injury. Expression of NB-3 protein in the ischemic hemisphere was decreased after MCAO at 3 hours, 6 hours, and 12 hours, and there was an increased infarct size in NB-3-KO mice after MCAO that was accompanied by increased neurological deficit scores. These data suggest that NB-3 plays an essential protective role after ischemic injury.
The NB-3 expression pattern both in neonatal and adult indicates an essential role in the formation of the complicated neuronal network.14 NB-3 has been reported wildly expressing in the brain under normal conditions; here we found that NB-3 was downregulated in the range of hours after MCAO. During this time, the infarct volume is still expanding, and the expression pattern of NB-3 is compatible with a causal role in infarct growth, suggesting that NB-3-mediated processes within neurons may provide protection from ischemic damage.
To test this hypothesis, we cultured cortical neurons from NB-3-KO mice. NB-3-KO neurons showed decreased neurite outgrowth and survival in vitro. Furthermore, in agreement with the axon-promoting activity role of NB-3, neurons substrated with NB-3 promoted neurite outgrowth and survival. These results are consistent with our in vivo study demonstrating that NB-3 is neuroprotective after brain injury.
The protective effect of neural recognition molecules in ischemic stroke have been paid extensive attention.15–17 However, the mechanism by which NB-3 protects against cell death is unknown. Neural recognition molecules engage in cell interactions with various molecules either in the extracellular matrix or on the cell surface to mediate cell contact and adhesion.2,18 NB-3 was previously reported to mediate contact and adhesion of migrating neurons and growing apical dendrites with extracellular matrix or other cells, providing a beneficial environment for movement and growth. Tenascin-C was shown to regulate neurite outgrowth in vitro through its fibronectin type III BD domains by interacting with the complementary neuronal receptor F3/contactin.19 Recently, NB-3 was found to directly associate with CHL1, a member of the L1 family of neural recognition molecules, and enhance its cell surface expression, whereas both CHL1 and NB-3 interact with protein tyrosine phosphatase a and regulate its activity.7 CHL1 expression is upregulated in both neurons and astrocytes after nervous system injury in the adult mouse. CHL1 has both conductive and inhibitory functions depending on its homophilic or heterophilic interactions.20 In our study, NB-3 expression was reduced after cerebral ischemic injury, which may play a beneficial role in acute brain injury. As such, we demonstrated that NB-3 was involved in control of neurite growth and cell survival partly by homophilic interactions. Future studies need to address NB-3 interacting with other adhesion molecules that regulate the neuronal microenvironment producing a permissive or inhibitory action on neurite outgrowth and extension process.
In conclusion, we demonstrated that NB-3 plays an essential protective role in neurons and that NB-3 deficiency may aggravate brain damage after MCAO by impairing neuronal survival and neurite growth. Thus, NB-3 may have therapeutic potential against cerebral ischemic injury.
Sources of Funding
This work was supported by grants from the Natural Sciences Foundation of China, No. 30670792 and 30870799; the Beijing Natural Science Foundation, No. 5092023; and the National Basic Research Program of China, No. 2006CB504100 and 2011CB910802.
We thank Professor Zhang Tianming for language editing.
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.110.609560/-/DC1.
- Received November 24, 2010.
- Revision received April 6, 2011.
- Accepted April 25, 2011.
- © 2011 American Heart Association, Inc.
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