δ-Opioid Receptor Activation Rescues the Functional TrkB Receptor and Protects the Brain from Ischemia-Reperfusion Injury in the Rat
Xuesong Tian 1,2, Jingchun Guo 1,3*, Min Zhu 1, Minwei Li 1, Gencheng Wu 1, Ying Xia 2*
1 State Key Laboratory of Medical Neurobiology, Department of Integrative Medicine and Neurobiology, Shanghai Medical College, Fudan University, Shanghai,China,
2 Department of Neurosurgery, University of Texas Medical School at Houston, Houston, Texas, United States of America,
3 Laboratory of Molecular Neurology, Shanghai Research Center for Acupuncture and Meridians, Shanghai, China
Introduction
Cerebral ischemia/hypoxia causes neuronal injury and leads to severe neurological disorders with few effective therapies available. Both clinicians and scientists have
set forth enormous efforts towards exploring new clues for neuroprotection against ischemic/hypoxic injury [1,2,3,4,5].Recent studies have demonstrated that the activation of the δ-opioid receptor (DOR) elicits a neuroprotective effect against such injuries. DOR is a type of G protein-coupled receptor and is widely distributed in the mammalian central nervous system,especially in the cortex and striatum [6,7]. Our initial work found that activation of DOR is protective against hypoxic/excitotoxic injury in the cortical neurons [8,9,10,11]. For example, DOR agonist [D-Ala2, D-Leu5]-enkephalin (DADLE) reduced glutamate-induced injury in neocortical neurons and this protection is selectively blocked by δ-, but not by μ- or κ-opioid receptor antagonists [9]. DOR activation with DADLE also increases the tolerance of cultured cortical neurons against hypoxia [10]. Furthermore, we showed that DOR provides
neuroprotection against hypoxic/ischemic insults in various models including neurons under hypoxia, brain slices in hypoxia or oxygen-glucose deprivation and in vivo brain exposed to cerebral ischemia [12,13,14,15,16,17,18,19,20,21,22,23]. Intracerebroventricular treatment with the DOR agonist TAN-67 (60 nmol) significantly reduced the infarct volume and attenuated neurological deficits, while Naltrindole, a DOR antagonist, aggravated ischemic damage after forebrain ischemia in rats [12]. Similar data generated from different independent laboratories further demonstrates that DOR is indeed neuroprotective against ischemic stress in the in vivo models of the brain [24,25,26,27,28]. Systemic administration of DOR agonist DADLE or Deltorphin-D (variant) reduces infarct volume after transient middle cerebral artery occlusion (MCAO) [24,25]. However, the mechanisms underlying DOR neuroprotection against ischemic insults are still poorly understood.
Previous studies showed that a DOR agonist, (+) BW373U86, increased mRNA expression of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family [29,30], in the frontal cortex, and this effect was specifically blocked by Naltrindole, but not by μ- or k-opioid receptor antagonists [30]. Recent evidence also shows that BDNF plays a significant role in neuroprotection against ischemic injury [31,32]. The BDNF-mediated effect is very likely mediated through activation of TrkB, a high-affinity tyrosine kinase receptor [33,34,35]. TrkB has two major types of isoforms, i.e., a full-length TrkB protein that possesses a tyrosine kinase domain, and a truncated isoform that lacks this domain [36]. Upon activation by BDNF, full-length TrkB undergoes autophosphorylation to regulate Erk/MAPK signaling, which may increase cAMP and activate cAMP-response-element-binding protein (CREB)-regulated gene transcription, which further promotes transcription of BDNF. This is a potential positive feedback mechanism that could produce a BDNF-induced synthesis of BDNF itself [37]. On the other hand, there is also evidence demonstrating that the DOR agonist [D-Pen2,5] enkephalin (DPDPE) produced a dose-dependent increase in the phosphorylation of cAMP-response-element-binding protein (CREB), and this effect was reversed by DOR antagonist Naltrindole [38]. All of these results prompt us to hypothesize that the mechanism of DOR neuroprotection against ischemic injury involves a BDNF-TrkB-pCREB pathway in the ischemic brain. However, there is currently no published data in this aspect. We therefore performed this work in order to investigate such a possibility.
Experimental Procedures
Animals and reagents
Adult male Sprague-Dawley (SD) rats (230±5g; Shanghai Experimental Animal center and Charles River Laboratories) were used in these studies. The rats were kept on a 12 h light–dark cycle and under controlled temperature. The animal procedures were approved by The Medical Experimental Animal Administrative Committee of Fudan University and the University of Texas medical school at Houston Animal Care and Use Committee (Animal Welfare Assurance Number: HSC-AWC-11-066) and conformed to the National Institutes of
Health Guide for the Care and Use of Animals in Research. Cresyl violet, Hoechst 33258, TAN-67 and Naltrindole were purchased from Sigma (Cat: C5402, 861405, T5824 and N115-10MG). Rabbit polyclonal anti-DOR, rabbit polyclonal anti-phospho-CREB (Ser133), mouse monoclonal anti-phospho-CREB (Ser133), sheep polyclonal anti-BDNF, and donkey anti-sheep IgG-FITC were obtained from Millpore (Cat: AB1560, 06-519, 05-807, AB1513 and AP-184F). Rabbit monoclonal anti-CREB and TrkB was obtained from Cell Signaling Technology (Cat: 9197S, 4603S). Rabbit polyclonal anti-BDNF was purchased from Alomone Labs Ltd (Cat: ANT-010). Rabbit polyclonal anti-CD11b was obtained from Abcam Inc (Cat: ab75476). Anti-mouse IgG-FITC and anti-rabbit IgG-Rhodamine were purchased from Jackson Biotechnology (Cat: 115-097-003, 111-025-003). Horseradish peroxidase conjugated goat anti-rabbit/mouse IgG (H+L) were purchased from Invitrogen (Cat: G-21234, G-21040). Laemmli sample buffer, Precision Plus Protein? Dual Color Standards and mini-protean precast gels were obtained from Bio-rad (Cat: 161-0737, 161-0374, 456-1083).
Experimental groups
The animals were randomly allocated to 14 groups. The doses of drugs to be administered via intracerebroventricular injection were determined based on the protocol followed in previous studies [39]. The control group, comprising of rats that did not undergo MCAO, was given 10 μl of artificial cerebrospinal fluid (aCSF) containing 60 nmol TAN-67 or 100 nmol naltrindole to evaluate the effects of these drugs on cerebral blood flow and neurons (n=4 per group). In ischemic groups, 10 μl of aCSF containing 60 nmol TAN-67 or 100 nmol naltrindole was injected 30-min prior to inducing MCAO (n = 16 per group). Sham operation was performed following the same procedures as in the above groups (n = 4 per group). All drugs were injected using a microinfusion pump and infusions were delivered in ~5 min.
Intracerebroventricular administration of drugs
The following drugs were used in the experiments: aCSF (pH 7.4), TAN-67 (60 nmol/10μl), and Naltrindole (100 nmol/10μl). The drugs were delivered stereotaxically into the ipsilateral lateral ventricle. In brief, guide cannulas were implanted into the right lateral ventricle (stereo coordinates: A -0.8 mm for the bregma level of anterior-posterior, L 1.4 mm for lateral, H 4.0 mm for dorsoventral on the basis of the rat brain atlas [40] and secured to the skull with dental cement. Correct placement of guide cannulas was confirmed at the time of sectioning. The lyophilized agonist and antagonist were first reconstituted (60/100 nmol/10μl) in distilled water and then in aCSF containing (in mM) 119 NaCl, 3.1 KCl, 1.2 CaCl
2
, 1 MgSO
4
, 0.5
KH
2
PO
4
, 25 NaHCO
3
, 5 D-glucose, and 2.2 urea and then filtered (0.22μm). Freshly prepared drugs were used for intracerebroventricular administration by dissolving agonists or antagonists in 10 μl aCSF and injecting into the right lateral ventricle 30 min before MCAO. The animals were sacrificed after 24 hrs of reperfusion after ischemia. The brain sections were used for immunohistochemical and western blot analysis as indicated below.
Monitoring regional cerebral blood flow
A laser Doppler flowmeter (Periflux System 5000, PERIMED, Sweden) was used to monitor the regional cerebral blood flow. Using a stereotaxic device (SR-6N, Narishige Scientific Instrument, Tokyo, Japan) and a low speed dental drill, a burr hole of 1 mm in diameter was made over the skull at a point 1 mm posterior and 5 mm lateral to the bregma on the right side [41]. A needle shaped laser probe was placed on the dura away from visible cerebral vessels. The regional blood flow was continuously recorded beginning 10 mins before inducing ischemia until 30-min after starting reperfusion without repositioning the laser Doppler probes or the animals. Transient Focal Cerebral Ischemia
Rats were anesthetized with 10% chloral hydrate (360 mg/kg i.p.), and arterial blood samples obtained via femoral catheter
were collected to measure pO
2, pCO
2
and pH with an AVL 990
Blood Gas Analyzer (AVL Co, Graz, Austria). The rectal temperature was maintained at 37 ± 0.5°C during the surgery and MCAO via a temperature-regulated heating lamp [42]. Rats with physiological variables within normal ranges were subjected to transient focal cerebral ischemia induced by right MCAO as previously described [40,42,43]. A 4-0 nylon monofilament suture with a rounded tip (diameter 0.22 mm) was introduced into the internal carotid artery through the stump of the external carotid artery and gently advanced for a distance of 22 mm from the common carotid artery bifurcation in order to block the middle cerebral artery at its origin, for 60 mins. Withdrawal of this suture restored MCA blood flow during reperfusion.
Infarction Measurement and Fluorescence Immunolabeling
After 24 hours of reperfusion, rats were sacrificed with an overdose of 10% chloral hydrate and transcardially perfused with 0.9% saline solution followed by 4% ice-cold phosphate-buffered paraformaldehyde (PFA). The brains were then removed and post-fixed in 4% PFA for 12 h and then immersed sequentially in 20% and 30% sucrose solutions in 0.1 M phosphate buffer (pH 7.4) until they sank. Coronal sections were cut on a freezing microtome (Jung Histocut, Model 820-II, Leica, Germany) at a thickness of 30 μm at 1.60 to 0?4.80 mm from bregma and stored at ?20°C in cryoprotectant solution. Sections at 1.60 to ?4.80 mm from bregma were used for cresyl violet staining, and sections at 1.0 to 0.48 mm from bregma were used for immunohistochemical staining. Cresyl violet staining was performed on slices at 360-μm intervals to identify viable cells. Fluorescence immunolabeling was used to delineate the cellular localization of DOR, CREB and BDNF. Free-floating sections from each rat brain were fixed in 4% paraformaldehyde for 15 min, followed by three washes in 0.01 M phosphate-buffered saline (PBS). Sections were incubated
with 0.3% H
2O
2
for 30 min and then placed in blocking buffer
containing 10% normal goat serum and 0.3% Triton X-100 in 0.01 M phosphate-buffered saline (PBS, pH 7.2) for 30 min at 37°C and incubated with antibodies against rabbit polyclonal anti-DOR (1:200), p-CREB (1:100), CREB(1:100), and sheep polyclonal anti-BDNF(1:200), overnight at 4°C, respectively, or with antibody combinations (anti-DOR/anti-BDNF; anti-BDNF/ anti-CREB; anti-CREB/anti-p-CREB). After washing with PBS, the sections were incubated with corresponding secondary antibodies (1:100) for 1 h at 37°C. Negative control sections received an identical process without the primary or secondary antibodies, and showed no specific staining. After washing with PBS, sections were then incubated in Hoechst 33258 (1μg/ml; Sigma) for 10 min in dark. Finally, these sections were mounted on glass slides and coverslipped using fluorescence mounting media. The fluorescent signals were detected by confocal laser scanning microscope (TCS SP2, Leica, Germany) at excitation 535 nm and 565 nm (Rhodamine), 490 nm and 525 nm (FITC), 352 nm and 461nm (Hoechst). Western Blot Analysis
A separate cohort of rats was sacrificed at 24 hrs of reperfusion after MCAO+aCSF, MCAO+TAN-67(60 nmol/10μl), naltrindole (100 nmol/10μl), and sham operation (aCSF only) (n=4 per group). The tissues of the ipsilateral striatum and cortex were quickly collected on ice, frozen immediately in dry ice, and kept at -70°C until use. Afterwards, brain tissues were homogenized in 2% CHAPS buffer because urea-saturated buffer and RIPA buffer showed similar expression of proteins whereas a higher relative expression of DOR proteins was observed after preparation using CHAPS buffer [44], [2% CHAPS (Sigma, St. Louis, MO, USA), 10 mM sodium phosphate, pH 7.2, 1% sodium deoxycholate, 0.15 M sodium chloride and protease inhibitor cocktail], and centrifuged at 12,000g for 10 min at 4°C. Protein concentration was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). Tissue homogenates (50μg protein equivalent each) from the entire ipsilateral cortex, striatum and hippocampus of each rat were boiled at 100°C in sodium dodecyl sulfate (SDS) sample buffer for 5 mins, electrophoresed on 10% SDS-polyacrylamide gel, and transferred to the polyvinyldifluoridine membrane (Bio-Rad). Membranes were blocked with 5% nonfat dry milk in 0.1% Tween 20 (TBS-T; 2 mmol/L Tris-HCl, 50 mmol/L NaCl, pH 7.5) for 2 hours at room temperature and subsequently incubated overnight at 4°C in the blocked buffer with the 1:2000 antibody for p-CREB, CREB, and the 1:500 antibody for BDNF. After that, membranes were washed with 0.1% Tween 20, and then treated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (1:5,000) for 1 hr at 37°C. Peroxidase activity was visualized with an enhanced chemiluminescence substrate system (ECL, Santa Cruz Biotechnology). Stripping filters and reprobing for β-actin was carried out for normalization. Controls for nonspecific binding were determined by omission of the primary antibody. Films were scanned with a film scanner (Image Master VDS; Amersham Biosciences Inc., Piscataway, NJ) and subsequently analyzed by measuring optical densities of immunostained bands on the film using an image-processing and analysis system (Q570IW; Leica). For each brain area, the ratios of the values obtained from ischemic and sham-operated animals were averaged from 4 different animals sacrificed at that point of time.
Statistical analysis
Two independent and blinded investigators examined the end point assessments. Whole of the data was expressed as
mean ± SE. ANOVA was utilized to analyze the difference between various groups, and p values < 0.05 were considered statistically significant.
Results
Effects of DOR activation and inhibition on cerebral blood flow and ischemic infarction
By intracerebroventricular administration, we separately applied TAN-67 and Naltrindole as well as aCSF and tested their effect on cerebral blood flow in the non-ischemic brain.There were no significant changes in the blood flow in response to DOR activation or inhibition (Figure 1). Also, these treatments had no appreciable effect on the blood flow before,during and after induction of ischemia (MCAO) (data not shown). However, DOR activation largely reduced ischemic infarction induced by MCAO, while DOR inhibition further promoted such infarction. As shown in Figure 2, all ischemic groups showed infarction typically in the striatum and https://www.doczj.com/doc/1116136673.html,pared with MCAO only group (25.0% of the whole brain,P <0.05 vs. the sham control level), MCAO+TAN-67 group showed a smaller area of infarction (16.8% of the sham control level, P <0.05 vs. MCAO only), whereas the total infarction volume increased in MCAO+naltrindole group (35.8% of the sham control level, P <0.05 vs. MCAO only) (n=10).
Effects of DOR activation and inhibition on cortical and striatal BDNF protein expression
Firstly, we performed fluorescence immunolabeling to determine the localizing distribution of DOR and BDNF in the cortex and striatum. The BDNF-labeled cells, exhibiting neuronal-like morphology, were found in the cortex and striatum with an abundant distribution in the frontoparietal cortex and lateral caudate putamen in sham-operated group (Figures 3A and 4A). Triple-labeled confocal images
also
Figure 1. Effect of DOR activation and inhibition on cerebral blood flow. Administration of aCSF, TAN-67 or Naltrindole had no significant effect on cerebral blood flow in normal rats (P >0.05).
doi: 10.1371/journal.pone.0069252.g001
showed that BDNF and DOR/MAP-2 protein were co-localized in the cytosol of neuronal-like cells in the frontoparietal cortex (Figure 3B and C).
After 1-hr ischemia and 24-hrs of reperfusion, BDNF expression remained at a similar level as that of the prior ischemia in the ipsilateral cortex. DOR activation or inhibition did not change the level of BDNF (101.9% in MCAO+aCSF,95.0% in MCAO+TAN-67, 97.7% in TAN-67 alone, 94.3% in MCAO+Naltrindole, 102.9% in Naltrindole, P>0.05 among groups) (Figure 3 D, E).
Also in the striatum, we did not detect any significant change in the expression of BDNF after MCAO and after either DOR activation or inhibition in the condition of ischemia/reperfusion (104.6% in MCAO+aCSF, 108.3% in TAN-67, 105.7% in MCAO+Naltrindole, 108.3% in MCAO+TAN-67 and 97.0% in Naltrindole, P >0.05) (Figure 4 B, C).
Effects of DOR activation and inhibition on cortical and striatal TrkB expression
We then examined the expression of both full-length and truncated TrkB receptors, depicted in the Figure 3. The expression of full-length (140KDa) TrkB significantly decreased after 1-hr ischemia and 24-hrs of reperfusion (56.5% of the sham level, P <0.05) (Figure 3 D, G). However, DOR activation greatly attenuated such a loss. As shown in Figure 3 D, G, its expression did not show any significant decrease in the MCAO +TAN-67 group after 1-hr ischemia and 24 hrs of reperfusion (90.4% of the sham level, P >0.05). In contrast, Naltrindole significantly worsened the MCAO-induced reduction of 140KDa TrkB protein expression (decreased to 34.8% of the sham level, P <0.05 vs. the sham control and MCAO+TAN-67) (Figure 3 D, G). In non-ischemic conditions, TAN-67 or Naltrindole alone did not change the expression of full-length TrkB in the sham group (102.2% in TAN-67 and 107.8% in Naltrindole,P >0.05) (Figure 3 D, G).
In the cortex, the expression of the truncated isoform of TrkB (90 KDa) receptors did not show any significant change (P >0.05) after 1-hr ischemia and 24-hr reperfusion in the ipsilateral cortex (102.4% in MCAO+aCSF, 99.8% in MCAO +TAN-67, 92.9% in MCAO+naltrindole, 97.7% in TAN-67 and 103.0% in Naltrindole) (Figure 3 D, F).
Similarly as in the cortex, the expression of 140KDa TrkB largely decreased after 1-hr ischemia and 24-hr reperfusion (39.7% of the sham level, P <0.05), while DOR activation with TAN-67 greatly reversed such a decrease (80.1% of the sham level, P <0.05 vs. MCAO alone) and DOR inhibition with naltrindole tended to worsen the MCAO-induced decrease (25.9% of the sham level). TAN-67 and naltrindole did not induce any significant change in the expression of the full-length TrkB though naltrindole tended to decrease it (94.6% for TAN-67 and 85.8% for naltrindole, P >0.05 vs. the sham control) (Figure 4 B, E).
The expression of truncated TrkB (90 KDa) did not show any significant difference in the ipsilateral striatum after MCAO or exposed to DOR activation or inactivation (105.0% in MCAO +aCSF, 103.0% in MCAO+TAN-67, 100.7% in MCAO +Naltrindole, 104.6% in TAN-67 and 94.6% in Naltrindole as compared to the level of the sham control, P>0.05) (Figure 4 B,D).
Effect of DOR activation and inhibition on hippocampal BDNF and TrkB expression
There was no statistically significant difference (P >0.05) in BDNF after 1-hr ischemia and 24-hr reperfusion (94.2% in MCAO+aCSF, 95.6% in MCAO+TAN-67, 90.7% in MCAO +Naltrindole, 97.7% in TAN-67 and 101.8% in Naltrindole)(Figure 5 A, B).
In sharp contrast to the cortex and striatum, there was no statistical significance in the differences of the full-length and truncated isoforms of expression (P >0.05) among all groups studied (full-length TrkB: 94.6% in MCAO+aCSF, 100.9% in MCAO+TAN-67, 91.9% in MCAO+Naltrindole, 101.9% in TAN-67, and 102.6% in Naltrindole; truncated TrkB: 98.0% in MCAO+aCSF, 100.7% in MCAO+TAN-67, 97.2% in MCAO +Naltrindole 97.2% in MCAO+Naltrindole, 101.4% in TAN-67and 104.5% in Naltrindole) (Figure 5 A, D and C).
Effects of DOR activation and inhibition on cortical CREB/p-CREB and pATF-1
In order to explore the role of CREB/p-CREB in the DOR-mediated regulation of BDNF-TrkB signaling, we further determined their expression in the same brain regions. We observed that CREB and p-CREB were widely found in the cortex in separate staining. For example, CREB and p-CREB positive cells were abundant in the frontoparietal cortex in both sham-operated and MCAO group (Figure 6A). However, the cells co-labeling CREB and p-CREB were in a relatively small number in the cortex. Indeed, they were rarely found in the ischemic cortex in the group of MCAO alone and could be seen in the ischemic penumbra only after DOR activation . Figure 6B shows the co-localization of CREB and p-CREB with nuclear staining in the ischemic penumbra of the cortex treated with DOR activation with TAN-67. Triple-labeled confocal images show that CREB is co-localized with BDNF in penumbra of ischemic cortex (Figure 6C).
In western blots, we did not detect any significant change in total/phosphorylated CREB in response to ischemia with
or
Figure 2. Effects of TAN-67 and Naltrindole on cerebral ischemic infarction. The area of pallor delineates the ischemic core (lateral caudate putamen), and the penumbra (frontoparietal cortex). S, Sham control. M, MCAO. Note that TAN-67 treatment decreased the ischemic size, while Naltrindole increased the infarction.
doi: 10.1371/journal.pone.0069252.g002
without DOR activation or inhibition (p-CREB/CREB: 101.4%/103.1% in MCAO+aCSF, 98.9/101.3% in MCAO+TAN-67,100.5%/98.0% in MCAO+Naltrindole, 100.6/102.2% in TAN-67and 99.9/104.0% in Naltrindole, P >0.05) (Figure 6D, F and G).As shown in Figure 6E, the pATF-1 protein level did not change in any significant way after MCAO (92.1% of the sham control level, P>0.05 vs. the sham control). DOR activation with TAN-67 did not induce any appreciable change in the he pATF-1 protein (96.5% of the sham control level, P >0.05 vs.the sham control). However, DOR inhibition with Naltrindole significantly decreased the pATF-1 expression in the ischemic cortex (53.3% of the sham control level, P <0.05 vs. that of MCAO+aCSF).
Effects of DOR activation and inhibition on total CREB and p-CREB in the striatum
As shown in Figure 7A, lateral caudate putamen were abundant in CREB and p-CREB positive cells in the sham group, but were relatively lesser populated in MCAO group.Western blotting analysis showed that p-CREB/CREB protein level decreased in the ischemic striatum of the MCAO group (64.7% of the sham control level, P <0.05 vs. the sham control;Figure 7). TAN-67 tended to up-regulate total CREB protein expression in ipsilateral striatum of the MCAO group though not statistically different in our sample size (83.4% of the sham control level). In contrast, Naltrindole promoted a further decrease in the expression of total CREB protein (57.1% of the sham control, P <0.05 vs. MCAO alone) and p-CREB (73.3% of the sham control, P <0.05) of the MCAO group. In contrast
to
Figure 3. Effects of TAN-67 and Naltrindole on cortical expression of BDNF and TrkB at 24 hrs after MCAO. A,Representative fluorescent micrographs of cortical BDNF positive cells in sham group. Bar = 30 μm. B, Representative fluorescent micrographs of cortical BDNF/MAP-2 double-labeled positive cells in sham group. Bar = 15 μm. C, Representative fluorescent micrographs of cortical BDNF/DOR double-labeled positive cells in sham group. Bar = 15 μm. D, Representative Western blot images of BDNF and TrkB expression in different groups. E, Quantitative analysis of BDNF. F, Quantitative analysis of 90 KDa TrkB. G, Quantitative analysis of 140 KDa TrkB. n=4. *P<0.05 vs. the sham. #P <0.05 vs. M+TAN67. S, Sham control. M, MCAO.Note that MCAO significantly reduced the expression of 140 KDa TrkB but not of BDNF and 90 KDa TrkB, while DOR activation with TAN67 largely reversed the ischemic reduction of 140 KDa TrkB expression.
doi: 10.1371/journal.pone.0069252.g003
the changes in the ischemic conditions, Naltrindole or TAN-67had no appreciable effect on CREB protein level in the brain without MCAO.
We did not detect sufficient expression of pATF-1 protein in the tissue of striatum, unlike in the cortex.
Effects of DOR activation and inhibition on CREB/p-CREB and pATF-1 expression in the hippocampus
Using triple-labeled confocal images showing CREB and MAP 2, a neuron marker labeling mature cells exhibiting neuronal-like morphology, in the sham-operated group, we observed abundant co-localized staining in the hippocampus such as hippocampal CA1 region as shown in Figure 8A,
suggesting the existence of CREB in hippocampal neurons. In western blotting studies, we found that neither total/phosphorylated CREB nor pATF-1 changed significantly in response to ischemia (Figure 8), unlike in the cortex and striatum (pCREB/CREB: 98.9/97.1% in MCAO+aCSF,95.9/97.1% in MCAO+TAN-67, 98.3/98.0% in TAN-67,99.9/98.3% in MCAO+Naltrindole and 100.2/96.4% in Naltrindole, P >0.05; pATF-1: 104.0% in MCAO+aCSF, 98.6%in MCAO+TAN-67, 106.9% in TAN-67,108.3% in MCAO +Naltrindole, and 106.7 in Naltrindole, P
>0.05)
Figure 4. Effects of TAN-67 and Naltrindole on striatal expression of BDNF and TrkB at 24 hrs after MCAO. A,Representative fluorescent micrographs of striatal BDNF positive cells in sham group. Bar = 30 μm. B, Representative Western blot images of BDNF and TrkB expression in different groups. C, Quantitative analysis of BDNF. D, Quantitative analysis of 90 KDa TrkB. E, Quantitative analysis of 140 KDa TrkB. n=4. *P <0.05 vs. the sham. #P <0.05 vs. M+TAN67. S, Sham control. M, MCAO.Note that MCAO significantly reduced the expression of 140 KDa TrkB but not of BDNF and 90 KDa TrkB, while DOR activation with TAN67 largely reversed the ischemic reduction of 140 KDa TrkB expression.
doi: 10.1371/journal.pone.0069252.g004
Effects of DOR activation and inhibition on CD11b protein expression in the brain
We further examined whether ischemia affected CD11b expression in the cortex, striatum and hippocampus. The expression of CD11b largely increased in the MCAO group (174.6%, 171.0% and 135.2% of the sham level in cortex,striatum and hippocampus, respectively, P <0.05 vs. the sham control) (Figure 9). This increase was seemly enhanced by DOR inhibition with naltrindole in the cortex (192.3% of the sham level, P <0.05 vs. the sham control), but not in the striatum and hippocampus (180.7% and 137.7% of the sham level, respectively, P <0.05 vs. the sham control (Figure 9). In contrast, DOR activation with TAN-67 induced a significant decrease in the ischemia-induced expression of CD11b in the cortex (124.9% of the saline control level, P <0.05 vs. MCAO alone) (Figure 9), but not in the striatum and hippocampus (178.1% and 127.2% of the saline control level, respectively),suggesting that DOR activation specifically attenuated the ischemia-induced increase in CD11b expression in the cortex.
Discussion
We have made a series of interesting observations in this work, i.e., (1) DOR activation with Tan-67 or inhibition with Naltrindole attenuated or increased ischemic infarction without any change in cortical blood flow in the ischemic hemisphere;(2) the level of BDNF remained unchanged in the cortex,
striatum and hippocampus at 24 hours after MCAO, which could not be changed by DOR activation or inhibition; (3)though no significant change in truncated TrkB receptor expression, the level of full-length TrkB was greatly decreased in the cortex and striatum but not in the hippocampus by MCAO, which could be largely reversed by DOR activation,while DOR inhibition further worsened the ischemic reduction;(4) MCAO decreased total CREB expression in the striatum but not in the cortex, while DOR inhibition decreased pATF-1expression in the cortex and reduced both total and phosphorylated CREB in the striatum; and (5) though MCAO increased C11b expression in all of the cortex, striatum and hippocampus, DOR activation specifically attenuated the ischemic increase in the cortex but not in the striatum and hippocampus. These results suggest a DOR-mediated regulation of TrkB pathway as a protective mechanism in the ischemic brain.
Although the primary cause of ischemic infarction is insufficient blood supply to the brain region involved and an increase in the blood flow can greatly relieve the ischemic infarction [45,46,47], this work demonstrated that DOR protection against brain ischemia does not rely on the regulation of cerebral blood flow because either DOR activation or inhibition had no appreciable effect on cerebral blood flow in non-ischemic and ischemic conditions. It is very likely that DOR exerts its protection through other mechanisms, especially the molecules for membrane and intercellular signaling.
Although
Figure 5. Effects of TAN-67 and Naltrindole on hippocampal expression of BDNF and TrkB at 24 hrs after MCAO. A,Representative Western blot images of BDNF and TrkB expression in different groups. B, Quantitative analysis of BDNF. C,Quantitative analysis of 90 KDa TrkB. D, Quantitative analysis of 140 KDa TrkB. S, Sham control. M, MCAO. Note that MCAO did not induce any appreciable change in BDNF and TrkB expression in the hipcampus. DOR activation or inhibition also had no significant effect on the expression of these proteins in this region exposed to MCAO.
doi: 10.1371/journal.pone.0069252.g005
reduced or increased brain infarction may directly or indirectly affect the molecule expression levels among the groups, we found that the signaling proteins we studied changed differentially in the same group of brain tissues, e.g., no change in BDNF vs. major change in TrkB in the same MCAO group,as discussed below.
BDNF is an important neuroprotective factor that has been proven to increase the tolerance of neurons against the ischemia in both in vitro and in vivo studies. Evidence from recent studies links DOR to BDNF gene transcription and shows that the expression of BDNF is probably regulated by DOR. For instance, intracerebroventricular administration of non-peptidic DOR agonist (+) BW373U86 increased BDNF mRNA expression in the frontal cortex through DOR-mediated mechanism because the effect was blocked by Naltrindole, but not by μ- or k-opioid receptor antagonists [29,30]. Indeed, we demonstrated in this work that BDNF and DOR protein are co-localized in the cortical cytosol of neuronal cells. However, to our surprise we did not find any significant change in BDNF expression after MCAO, suggesting that BDNF is not very sensitive to ischemic stress, at least in the first 24 hours after ischemia/reperfusion. In fact, this is beneficial to the ischemic brain since BDNF is potentially neuroprotective against ischemic injury.
BDNF has been recognized to bind to TrkB receptors and thus display its signaling regulation [33,34,35]. In our measurements, there clearly existed two types of TrkB signal bands, i.e., 140 KDa and 90 KDa bands. They respectively refer to full-length and truncated TrkB receptors. BDNF activates intracellular signaling cascades through
full-length
Figure 6. Effects of TAN-67 and Naltrindole on cortical expression of pATF-1 and p-CREB/CREB at 24 hrs after MCAO. A,Representative fluorescent micrographs of cortical p-CREB (green) and CREB (red) positive cells in sham and ischemic penumbra of the MCAO groups. Bar = 20 μm. B, Representative fluorescent micrographs of cortical p-CREB/CREB double-labeled positive cells in ischemic penumbra of the cortex after DOR activation with TAN67. C, Representative fluorescent micrographs of cortical BDNF/CREB double-labeled positive cells in penumbra. Bar = 15 μm. D, Representative Western blot images of pATF-1 and p-CREB/CREB expression levels in different groups. E, Quantitative analysis of pATF-1. F, Quantitative analysis of p-CREB. G,Quantitative analysis of total CREB. n=4. *P <0.05 vs. the sham. #P <0.05 vs. M+TAN67. &P <0.05 vs. the MCAO. S, Sham control. M,MCAO. Note that Naltrindole significantly reduced the expression of pATF-1 with MCAO but not of p-CREB and CREB in the cortex.
doi: 10.1371/journal.pone.0069252.g006
trkB to induce differentiation, proliferation and survival.Activation of the full-length trkB receptors is followed by receptor dimerization and transphosphorylation on tyrosine residues [35,36,37]. The truncated TrkB receptor, though expressed abundantly in the brain, lacks the catalytic tyrosine kinase domain. It is actually a dominant-negative receptor that inhibits full-length TrkB signaling [48]. Increased truncated TrkB receptors may contribute to the reduced BDNF-TrkB signaling and may lead to neuronal injury [49]. In fact, only the full-length TrkB receptor is a functional unit and can be phosphorylated by BDNF and signal the down-stream pathways [34,36]. One of the major findings of this work is that such functional TrkB is very sensitive to ischemic stress and could decrease by 50-60% at 24 hours after ischemia/reperfusion. Therefore, it could be a crucial factor limiting the ability of the brain to overcome the ischemic stress although the level of BDNF remains at the normal level as mentioned above. Fortunately,as we demonstrated in this work, DOR activation could largely prevent such an ischemia-induced decrease, which may be an important mechanism behind the DOR-induced brain against cerebral ischemia. The role of DOR in the up-regulation of the functional TrkB receptor was further supported by the following facts: (1) DOR inhibition worsens the ischemia-induced reduction of the functional TrkB receptor and (2) DOR up-regulation of the functional TrkB receptor was evident in the DOR-rich regions of the cortex and striatum, but not in the hippocampus that has less density of DOR [7]. All of the evidence built up the ground for us to hypothesize that DOR activation protects the full-length TrkB from loss under ischmic condition. Therefore, the BDNF-TrkB pathway is likely a novel signaling mechanism for the DOR-mediated neuroprotection against ischemia stress. However, this needs to be further varified because we cannot role out the possibility that the rescured full-length TrkB receptor resulted from better neuronal survival under DOR protection.
A growing body of evidence supports an important role of the transcriptional factor CRE
B in mediating opioid-induced signaling [38], while phosphorylated CREB is a constitutive transcription factor and possibly mediates neuroprotection [50].The administration of opioid drugs may regulate CREB and the subsequent signal changes in gene expression. For example,DOR agonist [D-Pen 2,5] enkephalin (DPDPE) produced a dose-dependent increase in CREB phosphorylation and this effect could be reversed by naltrindole [38]. Tanaka et al [51,52]showed that CREB phosphorylation at Ser133 of p-CREB protein undergoes a very rapid and transient increase (3.5 hr)within the ischemic core territory following transient focal ischemia and a marked decrease in p-CREB positive nuclei at 12 and 24 hr reperfusion. In the present study, we thoroughly examined DOR’s effect on total and phosphorylated CREB proteins in all three major regions, i.e., the cortex, striatum and hippocampus, with and without ischemic condition. We used specific and well-documented CREB antibodies (Millpore,06-519) [53,54]) that recognize both p43 phosphorylated CREB and phosphorylated ATF-1, another CRE-binding protein that has a similar sequence structure as CREB, especially in
the
Figure 7. Effects of TAN-67 and Naltrindole on striatal expression of p-CREB/CREB at 24 hrs after MCAO. A,Representative fluorescent micrographs of striatal p-CREB (green) and CREB (red) positive cells in sham and the core of MCAO groups. Bar = 20 μm. B, Representative Western blot images of p-CREB/CREB expression in different groups. C, Quantitative analysis of p-CREB. D, Quantitative analysis of total CREB. N=4. *P <0.05 vs. the sham. #P <0.05 vs. M+TAN67. S, Sham control. M,MCAO. Note that MCAO significantly reduced the expression of CREB and p-CREB. Although DOR inhibition could not further decrease their levels, DOR activation with TAN67 tended to reverse the ischemic reduction.
doi: 10.1371/journal.pone.0069252.g007
phosphorylation domain. The total CREB was dramatically reduced in the ischemic striatum, while DOR inhibition with Naltrindole significantly reduced the expression of total CREB and phosphorylated CREB protein, implying that DOR may play a role in CREB signaling in this particular region. H owever,we did not find any relationship between DOR’s activity and total/phosphorylated CREB in the cortex and hippocampus except for the notion that DOR inhibition decreased the level of cortical pATF-1, another CRE-binding protein that has a similar sequence structure as CREB, especially in the phosphorylation domain. It seems that CREB may differentially and complexly involve in DOR signaling in different brain regions, which needs further investigation to clarify the role of CREB signaling in the DOR protection against brain ischemia. On the other hand, our data could not provide any clue as to whether CREB is
upstream or downstream of BDNF, which remains unclear in the literature.
The ITGB2 subunit A, commonly referred to as CD11b or OX42, and the antibodies against this subunit are widely used as microglial markers. It recognizes both the resting and the activated microglia [55]. Microglial activation labeled by CD11b/OX42 was highly expressed in white matter regions such as the corpus callosum, external capsule, and internal capsule,rather than in the gray matter of rat brains [56]. It’s increase is an index of microglial activation [57].
Ischemia may activate microglia in the brain, which exerts both beneficial and deleterious effects on neurons. For example, activated microglia can express a variety of proinflammatory cytokines including interleukin-1β (IL-1β),interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which induce neuroinflammation and neurotoxicity [58,59,60].
For
Figure 8. Effects of TAN-67 and Naltrindole on hippocampal expression of pATF-1 and p-CREB/CREB at 24 hrs after MCAO. A, Representative fluorescent micrographs of hippocampal MAP 2 (green) and CREB (red) positive cells in CA1 in sham group. Bar = 20 μm. B, Representative Western blot images of pATF-1 and p-CREB/CREB expression in different groups. C,Quantitative analysis of pATF-1. D, Quantitative analysis of p-CREB. E, Quantitative analysis of total CREB. N=4. Note that neither MCAO nor DOR activation had any significant effect on the expression of CREB, pCREB and pATF-1 in the hippocampus.
doi: 10.1371/journal.pone.0069252.g008
example, post-ischemic inflammation is characterized by a rapid activation of resident microglial cells and by infiltration of macrophages in the injured parenchyma where they release neurotoxic substances, including pro-inflammatory cytokines,chemokines, and oxygen/nitrogen free radicals to exacerbate the injury [61]. On the other side, microglia activation may result in an increase in neurotrophins such as BDNF [58],which is beneficial to the brain. Therefore, microglial activation may partially contribute to the maintenance of BDNF at a normal level in ischemic condition. This may not be the case,however, in our model because neither DOR activation nor inhibition did not change the level of BDNF despite significantly altering the level of CD11b in the cortex as well as other brain regions. We therefore rather believe that DOR activation may reduce microglial activation and decrease proinflammatory cytokines, thus attenuating ischemic injury in the cortex since DOR activation specifically attenuated the increase in CD11b in the cortex.
In summary, our data shows the importance of DOR in neuroprotection against ischemic insult. DOR activation rescues TrkB signaling by reversing ischemia-reperfusion induced reduction of the full-length TrkB receptor and down-regulates microglial activation, while having nothing to do with the regulation of cerebral blood flow. It is likely that DOR activation rescues/upregulates BDNF-TrkB-CREB signaling and suppresses microglia-released proinflammatory cytokines,thus protecting the brain against ischemic injury. Our study provides a translational clue for a potential application in clinical settings. However, the present results are limited to the pretreatment of the DOR agonist via ICV injection for transient focal cerebral ischemia. It is needed to further elucidate the effects of DOR activation in other conditions (e.g., during
or
Figure 9. Effects of TAN-67 and Naltrindole on expression of CD11b at 24 hrs after MCAO. A, C and E, Representative Western blot images of cortical, striatal and hippocampal CD11b expression in different groups. B, Quantitative analysis of CD11b in the cortex. D, Quantitative analysis of CD11b in the striatum. F, Quantitative analysis of CD11b in the hippocampus. N=4. *P <0.05vs. the sham. #P <0.05 vs. M+TAN67. S, Sham control. M, MCAO. Note that MCAO significantly increased the expression of CD11b in the cortex, striatum and hippocampus. However, DOR activation specifically attenuated such ischemic increase in the cortex, but not in the striatum and hippocampus.
doi: 10.1371/journal.pone.0069252.g009
after ischemia) on different models of ischemia (e.g., global ischemia). Also, it is equally important to evaluate a long-term outcome of DOR activation in ischemia/reperfusion. All these issues should be carefully addressed in future investigations.Author Contributions
Conceived and designed the experiments: X-ST J-CG G-CW YX. Performed the experiments: X-ST. Analyzed the data: X-ST MZ M-WL. Contributed reagents/materials/analysis tools: G-CW YX. Wrote the manuscript: YX X-ST J-CG.
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10.1038/nrn2038. PubMed: 17180163.
()五固溶体的结构 固溶体的结构发生了变化： 虽然固溶体仍保持着溶剂的晶格类型，但若与纯组元相比，结构还是发生了变化，有的变化还相当大，主要表现在以下凡个方面：晶格畸变；偏聚与有序；有序固溶体 ()A晶格畸变 造成晶格畸变形成弹性应力场 由于溶质与溶剂的原子大小不同，因而在形成固溶体时，必然在溶质原子附近的局部范围内造成晶格畸变，并因此而形成一个弹性应力场。 晶格畸变的大小可由晶格常数的变化所反映 对置换固溶体来说，当溶质原子较溶剂原子大时，晶格常数增加；反之，当溶质原子较溶剂原子小时,则晶格常数减小。形成间隙固溶体时，晶格常数总是随着溶质原子的溶入而增大。 ()B偏聚与有序 1.长期以来，人们认为溶质原于在固溶体中的分布是统计的、均匀的和无序的， 如图3-8a所示。 2.但经X射线精细研究表明，溶质原子在固溶体中的分布，总是在一定程度上 偏离完全无序状态，存在着分布的不均匀性 3.当同种原子间的结合力大于异种原子间的结合力时，溶质原子倾向于成群地 聚集在一起，形成许多偏聚区图3.8
4. 反之，当异种原子（即溶质原子和溶剂原子）间的结合力较大时，则溶质原 子的近邻皆为溶剂原子，溶质原子倾向于按一定的规则呈有序分布，这种有序分布通常只在短距离小范围内存在，称之为短程有序(图3-8c) ()C 有序固溶体 有序固溶体的概念 ● 具有短程有序的固溶体，当低于某一温度时，可能使溶质和溶剂原子在整个 晶体中都按―定的顺序排列起束，即由短程有序转变为长程有序，这样的固溶体称为有序固溶体。 ● 当溶质原子按适当比例并按一定顺序和一定方向，围绕着溶剂原子分布时， 这种固溶体就叫有序固溶体 有序固溶体有确定的化学成分可以用化学式来表示 ● 例如在Au Cu -合金中，当两组元的原子数之比()Au Cu :即等于1:1()CuAu 和 3:1()Au Cu 3时，在缓慢冷却条件下，两种元素的原子在固溶体中将由无序排列转变为有序排列，铜、金原子在晶格中均占有确定的位置，如图3.9所示 ● 对CuAu 来说，铜原子和金原于按层排列于()001晶面上，一层晶面上全部是 铜原子，相邻的一层全部是金原子 ● 由于铜原子较小，故使原来的面心立方晶格略变形为93.0=a c 的四方晶格 ● 对Au Cu 3来说，金原子位于晶胞的顶角上，铜原子则占据面心位置 固溶体的有序化温度： 当有序固溶体加热至某一临界温度时，将转变为无序固溶体,而在缓慢冷却至这一温度时，又可转变为有序固溶体。这一转变过程称为有序化.发生有序化的临界温度称为固溶体的有序化温度。
1.置换固溶体中，被置换的溶剂原子哪里去了 答：溶质把溶剂原子置换后，溶剂原子重新加入晶体排列中，处于晶格的格点位置。 2.间隙固溶体和间隙化合物在晶体结构与性能上的区别何在举例说明之。 答：间隙固溶体是溶质原子进入溶剂晶格的间隙中而形成的固溶体，间隙固溶体的晶体结构与溶剂组元的结构相同，形成间隙固溶体可以提高金属的强度和硬度，起到固溶强化的作用。如：铁素体F是碳在α-Fe中的间隙固溶体，晶体结构与α-Fe相同，为体心立方，碳的溶入使铁素体F强度高于纯铁。 间隙化合物的晶体结构与组元的结构不同，间隙化合物是由H、B、C、N等原子半径较小的非金属元素（以X表示）与过渡族金属元素（以M表示）结合，且半径比r X/r M＞时形成的晶体结构很复杂的化合物，如Fe3C间隙化合物硬而脆，塑性差。 3.现有A、B两元素组成如图所示的二元匀晶相图，试分析以下几种说法是否正确为什么 （1）形成二元匀晶相图的A与B两个相元的晶格类型可以不同，但是原子大小一定相等。 （2）K合金结晶过程中，由于固相成分随固相线变化，故已结晶出来的固溶体中含B 量总是高于原液相中含B量. （3）固溶体合金按匀晶相图进行结晶时，由于不同温度下结晶出来的固溶体成分和剩余液相成分不相同，故在平衡态下固溶体的成分是不均匀的。 答：（1）错：Cu-Ni合金形成匀晶相图，但两者的原子大小相差不大。 （2）对：在同一温度下做温度线，分别与固相和液相线相交，过交点，做垂直线与成分线AB相交，可以看出与固相线交点处B含量高于另一点。 (3)错：虽然结晶出来成分不同，由于原子的扩散，平衡状态下固溶体的成分是均匀的。 4.共析部分的Mg-Cu相图如图所示：
《材料结构与性能》试题2011级硕士研究生适用 一、名词解释（20分） 原子半径，电负性，相变增韧、Suzuki气团 原子半径：按照量子力学的观点，电子在核外运动没有固定的轨道，只是概率分布不同，因此对原子来说不存在固定的半径。根据原子间作用力的不同，原子半径一般可分为三种：共价半径、金属半径和范德瓦尔斯半径。通常把统和双原子分子中相邻两原子的核间距的一半，即共价键键长的一半，称作该原子的共价半径（r c）；金属单质晶体中相邻原子核间距的一半称为金属半径（r M）；范德瓦尔斯半径（r V）是晶体中靠范德瓦尔斯力吸引的两相邻原子核间距的一半，如稀有气体。 电负性：Parr等人精确理论定义电负性为化学势的负值，是体系外势场不变的条件下电子的总能量对总电子数的变化率。 相变增韧：相变增韧是由含ZrO2的陶瓷通过应力诱发四方相（t相）向单斜相（m相）转变而引起的韧性增加。当裂纹受到外力作用而扩展时，裂纹尖端形成的较大应力场将会诱发其周围亚稳t-ZrO2向稳定m-ZrO2转变，这种转变为马氏体转变，将产生近4%的体积膨胀和1%-7%的剪切应变，对裂纹周围的基体产生压应力，阻碍裂纹扩展。而且相变过程中也消耗能量，抑制裂纹扩展，提高材料断裂韧性。 Suzuki气团：晶体中的扩展位错为保持热平衡，其层错区与溶质原子间将产生相互作用，该作用被成为化学交互作用，作用的结果使溶质原子富集于层错区内，造成层错区内的溶质原子浓度与在基体中的浓度存在差别。这种不均匀分布的溶质原子具有阻碍位错运动的作用，也成为Suzuki气团。 二、简述位错与溶质原子间有哪些交互作用。（15分） 答：从交互做作用的性质来说，可分为弹性交互作用、静电交互作用和化学交互作用三类。 弹性交互作用：位错与溶质原子的交互作用主要来源于溶质原子与基体原子间由于体积不同引起的弹性畸变与位错间的弹性交互作用。形成Cottrell气团，甚至Snoek气团对晶体起到强化作用。弹性交互作用的另一种情况是溶质原子核基体的弹性模量不同而产生的交互作用。 化学交互作用：基体晶体中的扩展位错为保持热平衡，其层错区与溶质原子间将产生相互作用，该作用被成为化学交互作用，作用的结果使溶质原子富集于层错区内，造成层错区内的溶质原子浓度与在基体中的浓度存在差别，具有阻碍位错运动的作用。 静电交互作用：晶体中的位错使其周围原子偏离平衡位置，晶格体积发生弹性畸变，晶格畸变将导致自由电子的费米能改变，对于刃型位错来讲，滑移面上下部分晶格畸变量相反，导致滑移面两侧部分的费米能不相等，导致位错周围电子需重新分布，以抵消这种不平衡，从而形成电偶极，位错线如同一条电偶极线，在它周围存在附加电场，可与溶质原子发生静电交互作用。 三、简述点缺陷的特点和种类，与合金的性能有什么关系（15分） 答：点缺陷对晶体结构的干扰作用仅波及几个原子间距范围的缺陷。它的尺寸在所有方向上均很小。其中最基本的点缺陷是点阵空位和间隙原子。此外，还有杂质原子、离子晶体中的非化学计量缺陷和半导体材料中的电子缺陷等。 在较低温度下，点缺陷密度越大，对合金电阻率影响越大。另外，点缺陷与合金力学性能之间的关系主要表现为间隙原子的固溶强化作用。
《材料结构与性能》习题 第一章 1、一25cm长的圆杆，直径2.5mm，承受的轴向拉力4500N。如直径拉细成 2.4mm，问： 1)设拉伸变形后，圆杆的体积维持不变，求拉伸后的长度； 2)在此拉力下的真应力和真应变； 3)在此拉力下的名义应力和名义应变。 比较以上计算结果并讨论之。 2、举一晶系，存在S14。 3、求图1.27所示一均一材料试样上的A点处的应力场和应变场。 4、一陶瓷含体积百分比为95%的Al2O3（E=380GPa）和5%的玻璃相（E=84GPa），计算上限及下限弹性模量。如该陶瓷含有5%的气孔，估算其上限及下限弹性模量。 5、画两个曲线图，分别表示出应力弛豫与时间的关系和应变弛豫和时间的关系。并注出：t=0,t=∞以及t=τε（或τσ）时的纵坐标。
6、一Al2O3晶体圆柱（图1.28），直径3mm，受轴向拉力F ，如临界抗剪强度τc=130MPa，求沿图中所示之一固定滑移系统时，所需之必要的拉力值。同时计算在滑移面上的法向应力。 第二章 1、求融熔石英的结合强度，设估计的表面能为1.75J/m2；Si-O的平衡原子
间距为
1.6×10-8cm；弹性模量值从60到75GPa。 2、融熔石英玻璃的性能参数为：E=73GPa；γ=1.56J/m2；理论强度。如材料中存在最大长度为的内裂，且此内裂垂直于作用力的方向，计算由此而导致的强度折减系数。 3、证明材料断裂韧性的单边切口、三点弯曲梁法的计算公式： 与 是一回事。 4、一陶瓷三点弯曲试件，在受拉面上于跨度中间有一竖向切口如图2.41所示。如果E=380GPa，μ=0.24，求KⅠc值，设极限载荷达50㎏。计算此材料的断裂表面能。 5、一钢板受有长向拉应力350 MPa，如在材料中有一垂直于拉应力方向的中心穿透缺陷，长8mm（=2c）。此钢材的屈服强度为1400MPa，计算塑性区尺寸r0及其与裂缝半长c的比值。讨论用此试件来求KⅠc值的可能性。
《材料结构与性能》 习题
《材料结构与性能》习题 第一章 1、一25cm长的圆杆，直径2.5mm，承受的轴向拉力4500N。如直径拉细成2.4mm，问： 1)设拉伸变形后，圆杆的体积维持不变，求拉伸后的长度； 2)在此拉力下的真应力和真应变； 3)在此拉力下的名义应力和名义应变。 比较以上计算结果并讨论之。 2、举一晶系，存在S14。 3、求图1.27所示一均一材料试样上的A点处的应力场和应变场。 4、一陶瓷含体积百分比为95%的Al2O3（E=380GPa）和5%的玻璃相（E=84GPa），计算上限及下限弹性模量。如该陶瓷含有5%的气孔，估算其上限及下限弹性模量。 5、画两个曲线图，分别表示出应力弛豫与时间的关系和应变弛豫和时间的关系。并注出：t=0,t=∞以及t=τε（或τσ）时的纵坐标。
6、一Al2O3晶体圆柱（图1.28），直径3mm，受轴向拉力F ，如临界抗剪强度τc=130MPa，求沿图中所示之一固定滑移系统时，所需之必要的拉力值。同时计算在滑移面上的法向应力。 第二章
1、求融熔石英的结合强度，设估计的表面能为1.75J/m2；Si-O的平衡原子间距为1.6×10-8cm；弹性模量值从60到75GPa。 2、融熔石英玻璃的性能参数为：E=73GPa；γ=1.56J/m2；理论强度。如材料中存在最大长度为的内裂，且此内裂垂直于作用力的方向，计算由此而导致的强度折减系数。 3、证明材料断裂韧性的单边切口、三点弯曲梁法的计算公式： 与 是一回事。 4、一陶瓷三点弯曲试件，在受拉面上于跨度中间有一竖向切口如图2.41所示。如果E=380GPa，μ=0.24，求KⅠc值，设极限载荷达50㎏。计算此材料的断裂表面能。 5、一钢板受有长向拉应力350 MPa，如在材料中有一垂直于拉应力方向的中心穿透缺陷，长8mm（=2c）。此钢材的屈服强度为1400MPa，计算塑性区尺寸r0及其与裂缝半长c的比值。讨论用此试件来求KⅠc值的可能性。
固溶体
固溶体 所谓固溶体是指溶质原子溶入溶剂晶格中而仍保持溶剂类型的合金相。 这种相称为固溶体，这种组元称为溶剂，其它的组元即为溶质。工业上所使用的金属材料，绝大部分是以固溶体为基体的，有的甚至完全由固溶体所组成。例如，广泛用的碳钢和合金钢，均以固溶体为基体相，其含量占组织中的绝大部分。因此，对固溶体的研究有很重要的实际意义。 ● 固溶体的分类 按溶质原子在晶格中的位置不同可分为置换固溶体和间隙固溶体。 1、置换固溶体溶质原子占据溶剂晶格中的结点位置而形成的固溶体称置换固溶体。当溶剂和溶质原子直径相差不大，一般在15％以内时，易于形成置换固溶体。铜镍二元合金即形成置换固溶体，镍原子可在铜晶格的任意位置替代铜原子。 2、间隙固溶体溶质原子分布于溶剂晶格间隙而形成的固溶体称间隙固溶体。间隙固溶体的溶剂是直径较大的过渡族金属，而溶质是直径很小的碳、氢等非金属元素。其形成条件是溶质原子与溶剂原子直径之比必须小于0.59。如铁碳合金中，铁和碳所形成的固溶体――铁素体和奥氏体，皆为间隙固溶体。 ●按固溶度来分类：可分为有限固溶体和无限固溶体。无限固溶体只可能是转换固溶体。 ● 按溶质原子与溶剂原子的相对分布来分；可分为无序固溶体和有序固溶体 这两点各位有时间补充说明下 ● 固溶体的性能 当溶质元素含量很少时，固溶体性能与溶剂金属性能基本相同。但随溶质元素含量的增多，会使金属的强度和硬度升高，而塑性和韧性有所下降，这种现象称为固溶强化。置换固溶体和间隙固溶体都会产生固溶强化现象。 适当控制溶质含量，可明显提高强度和硬度，同时仍能保证足够高的塑性和韧性，所以说固溶体一般具有较好的综合力学性能。因此要求有综合力学性能的结构材料，几乎都以固溶体作为基本相。这就是固溶强化成为一种重要强化方法，在工业生产中得以广泛应用的原因。 第二节金属学及热处理基本知识 一、金属晶体结构的一般知识 众所周知，世界上的物质都是由化学元素组成的，这些化学元素按性质可分成两大类： 第一大类是金属，化学元素中有83种是金属元素。固态金属具有不透明、有光泽、有延展性、有良好的导电性和导热性等特性，并且随着温度的升高，金属的导电性降低，电阻率增大，这是金属独具的一个特点。常见的金属元素有铁、铝、铜、铬、镍、钨等。 第二大类是非金属，化学元素中有22种，非金属元素不具备金属元素的特征。而且与金属相反，随着温度的升高，非金属的电阻率减小，导电性提高。常见的非金属元素有碳、氧、氢、氮、硫、磷等。 我们所焊接的材料主要是金属，尤其是钢材，钢材的性能不仅取决于钢材的化学成分，而且取决于钢材的组织，为了了解钢材的组织及对性能的影响，我们必须先从晶体结构讲起。 (一)晶体的特点
《材料结构与性能》 第一章金属及合金的晶体结构 1．重要名词晶体非晶体单晶体多晶体晶粒晶界各向异性假等向性（伪各向同性）空间点阵阵点（结点）晶胞简单晶胞（初级晶胞）布拉菲点阵晶系晶面晶面指数晶向晶向指数密勒指数晶面族晶向族晶带晶带轴面间距配位数致密度点阵常数面心立方（A1）体心立方(A2) 密排六方(A3) 同素异构现象四面体间隙八面体间隙多晶型性(同素异构转变) 原子半径合金相固溶体间隙固溶体置换固溶体有限固溶体无限固溶体电子浓度无序分布偏聚短程有序短程有序参数维伽定律中间相金属间化合物正常价化合物电子化合物(Hume-Rothery相) 间隙相间隙化合物拓扑密堆相(TCP相) PHACOMP方法超结构（有序固溶体，超点阵）长程有序度参数反相畴（有序畴） 2．试述晶体的主要特征。 2]。3．画出立方晶系中的下列晶面和晶向：(100), (111), (110), (123), (130)), (121), (225), [112], [312], [11 画出六方晶系中的下列晶面：(0001), (1120), (1011)。 4．画出立方晶系(110)面上的[111]方向，(112)上的[111]方向。在其(111)面上有几个<110>方向？ 5．计算面心立方、体心立方、密排六方点阵晶胞的晶胞内原子数、致密度。其中原子的配位数是多少？6．面心立方和密排六方点阵的原子都是最密排的，为什么它们形成了两种点阵？ 7．画图计算面心立方和体心立方点阵的四面体、八面体间隙的半径r B与原子半径r A之比。 8．铜的面心立方点阵常数为3.608?，计算其（122）晶面间距。 9．立方晶系中晶面指数和晶向指数有什么关系？ 10．写出立方晶系{112}晶面组的全部晶面和<123>晶向族的全部晶向。 11．已知点阵常数a=2 ?，b=6 ?, c=3 ?, 并已知晶面与三坐标轴的截距都是6 ?，求该晶面的指数。12．若γ-Fe晶胞中的八面体间隙都被C原子填满，试计算C原子的原子百分数和重量百分数。另外，这样的事情能否发生，为什么？ 13．试画出面心立方点阵中(001), (011) 和（111）晶面的原子排列，并标出原子间距。 14．判断下列晶向是否属于相应的晶面或平行于该晶面：[112]与(111)；[110]与(121)；[210]与(101)。15．下列晶向是否是两个晶面的交线？(1)[112]与(111)及(110)；(2)[101]与(111)及(111)；(3)[101]与(111)及(111)。 16．银属面心立方点阵，若其原子半径为1.44 ?，求其晶格常数，并根据其原子量求其密度。 17．α-Fe→γ-Fe转变发生在910℃，该温度下其点阵常数分别为2.892 ?和3.633 ?，试求转变前后的体积变化。若转变前后原子半径未变化，体积变化又有多大？ 18. Al和Ag均属面心立方点阵，已知r Ag= 1.441?, r Al=1.428?, 它们在固态下是否可能无限互溶，为什么？19．固溶体的溶解度主要取决于哪些因素？ 20．碳原子在γ-Fe晶胞中存在于什么位置？碳原子溶入后其点阵常数如何变化？为什么？碳原子溶入α-Fe 中又如何？ 21．计算含1-wt%C的γ-Fe中多少个晶胞中溶入一个碳原子？ 22．中间相一般具有什么特点？ 23．以黄铜为例说明什么是电子化合物及电子化合物的类型。 24．电子化合物为什么可以具有一定的成分范围？25．试述间隙固溶体、间隙相、间隙化合物的异同。26．试述短程有序和长程有序的关系。27．影响有序化的因素有哪些？ 28．有序化对合金的性能有何影响？
1、离子键及其形成的离子晶体陶瓷材料的特征。 答：当一个原子放出最外层的一个或几个电子成为正离子，而另一个原子接受这些电子而成为负离子，结果正负离子由于库仑力的作用而相互靠近。靠近到一定程度时两闭合壳层的电子云因发生重叠而产生斥力。这种斥力与吸引力达到平衡的时候就形成了离子键。此时原子的电中性得到维持，每一个原子都达到稳定的满壳层的电子结构，其总能量达到最低，系统处于最稳定状态。因此，离子键是由正负离子间的库仑引力构成。由离子键构成的晶体称为离子晶体。离子晶体一般由电离能较小的金属原子和电子亲和力较大的非金属原子构成。离子晶体的结构与特性由离子尺寸、离子间堆积方式、配位数及离子的极化等因素有关。 离子键、离子晶体及由具有离子键结构的陶瓷的特性有： A、离子晶体具有较高的配位数，在离子尺寸因素合适的条件下可形成最密排的结构； B、离子键没有方向性 C、离子键结合强度随电荷的增加而增大，且熔点升高，离子键型陶瓷高强度、高硬度、高熔点； D、离子晶体中很难产生自由运动的电子，低温下的电导率低，绝缘性能优良； E、在熔融状态或液态，阳离子、阴离子在电场的作用下可以运动，故高温下具有良好的离子导电性。 F、吸收红外波、透过可见波长的光，即可制得透明陶瓷。 2、共价键及其形成的陶瓷材料具有的特征。 答：当两个或多个原子共享其公有电子，各自达到稳定的、满壳层的状态时就形成共价键。由于共价电子的共享，原子形成共价键的数目就受到了电子结构的限制，因此共价键具有饱和性。由于共价键的方向性，使共价晶体不密堆排列。这对陶瓷的性能有很大影响，特别是密度和热膨胀性，典型的共价键陶瓷的热膨胀系数相当低，由于个别原子的热膨胀量被结构中的自由空间消化掉了。 共价键及共价晶体具有以下特点： A、共价键具有高的方向性和饱和性； B、共价键为非密排结构； C、典型的共价键晶体具有高强度、高硬度、高熔点的特性。 D、具有较低的热膨胀系数； E、共价键由具有相似电负性的原子所形成。 3、层状结构材料的各向异性。 答：层状结构中范德华力起着重要的作用，陶瓷的层状结构间有较强的若键存在使得层与层之间连接在一起。蒙脱石和石墨的结构层内键合类型不同于层间键合类型，因此材料显示出较高的各向异性。所有的这些层状结构的层与层之间很容易滑移，粘土矿物中的这种层状结构使它在有水的情况下容易发生塑性变形。 4、影响陶瓷材料密度的因素。 答：密度是指单位体积的质量，陶瓷材料的密度有四种表示方式，分别是：结晶学密度、理论密度、体积密度、相对密度。前三种在制作过程中没有形成气孔，在结构内的原子间只有间隙。陶瓷材料的密度主要取决于元素的尺寸，元素的质量和结构堆积的紧密程度。相对原子质量大的元素构成的陶瓷材料显示出较高的密度，如碳化钨、氧化铪等。金属键合和离子键合陶瓷中的原子形成紧密堆积，会使其密度比共价键键合陶瓷（较开放的结构）的密度更
《材料结构与性能》习题 第一章 1、一25cm 长的圆杆，直径2.5mm ，承受的轴向拉力4500N 。如直径拉细成2.4mm ，问： 1) 设拉伸变形后，圆杆的体积维持不变，求拉伸后的长度； 2) 在此拉力下的真应力和真应变； 3) 在此拉力下的名义应力和名义应变。 比较以上计算结果并讨论之。 2、举一晶系，存在S 14。 3、求图1.27所示一均一材料试样上的A 点处的应力场和应变场。 4、一陶瓷含体积百分比为95%的Al 2O 3（E=380GPa ）和5%的玻璃相（E=84GPa ），计算上限及下限弹性模量。如该陶瓷含有5%的气孔，估算其上限及下限弹性模量。 5、画两个曲线图，分别表示出应力弛豫与时间的关系和应变弛豫和时间的关系。并注出：t=0,t=∞以及t=τε（或τσ）时的纵坐标。 6、一Al 2O 3晶体圆柱（图1.28），直径3mm ，受轴向拉力F ，如临界抗剪强度τc =130MPa ，求沿图中所示之一固定滑移系统时，所需之必要的拉力值。同时计算在滑移面上的法向应力。
第二章 1、求融熔石英的结合强度，设估计的表面能为1.75J/m2；Si-O的平衡原子间距为1.6×10-8cm；弹性模量值从60到75GPa。 2、融熔石英玻璃的性能参数为：E=73GPa；γ=1.56J/m2；理论强度。如材料中存在最大长度为的内裂，且此内裂垂直于作用力的方向，计算由此而导致的强度折减系数。 3、证明材料断裂韧性的单边切口、三点弯曲梁法的计算公式： 与 是一回事。
4、一陶瓷三点弯曲试件，在受拉面上于跨度中间有一竖向切口如图 2.41所示。如果E=380GPa，μ=0.24，求K Ⅰc 值，设极限载荷达50㎏。计算此材料的断裂表面能。 5、一钢板受有长向拉应力350 MPa，如在材料中有一垂直于拉应力方向的中心穿透缺陷，长8mm（=2c）。此钢材的屈服强度为1400MPa，计算塑性区尺寸 r 0及其与裂缝半长c的比值。讨论用此试件来求K Ⅰc 值的可能性。 6、一陶瓷零件上有以垂直于拉应力的边裂，如边裂长度为：①2mm；② 0.049mm；③2μm，分别求上述三种情况下的临界应力。设此材料的断裂韧性为 1.62 MPa〃m2。讨论诸结果。 7、画出作用力与预期寿命之间的关系曲线。材料系ZTA陶瓷零件，温度在 900℃，K Ⅰc 为10MPa〃m2，慢裂纹扩展指数N=40，常数A=10-40，Y取π。设保证实验应力取作用力的两倍。 8、按照本章图2.28所示透明氧化铝陶瓷的强度与气孔率的关系图，求出经验公式。 9、弯曲强度数据为：782，784，866，884，884，890，915，922，922，927，942，944，1012以及1023MPa。求两参数韦伯模量数和求三参数韦伯模量数。 第三章 1、计算室温（298K）及高温（1273K）时莫来石瓷的摩尔热容值，并请和安杜龙—伯蒂规律计算的结果比较。 2、请证明固体材料的热膨胀系数不因内含均匀分散的气孔而改变。
固溶体 所谓固溶体是指溶质原子溶入溶剂晶格中而仍保持溶剂类型的合金相。 这种相称为固溶体，这种组元称为溶剂，其它的组元即为溶质。工业上所使用的金属材料，绝大部分是以固溶体为基体的，有的甚至完全由固溶体所组成。例如，广泛用的碳钢和合金钢，均以固溶体为基体相，其含量占组织中的绝大部分。因此，对固溶体的研究有很重要的实际意义。 ● 固溶体的分类 按溶质原子在晶格中的位置不同可分为置换固溶体和间隙固溶体。 1、置换固溶体溶质原子占据溶剂晶格中的结点位置而形成的固溶体称置换固溶体。当溶剂和溶质原子直径相差不大，一般在15％以内时，易 于形成置换固溶体。铜镍二元合金即形成置换固溶体，镍原子可在铜晶格的任意位置替代铜原子。 2、间隙固溶体溶质原子分布于溶剂晶格间隙而形成的固溶体称间隙固溶体。间隙固溶体的溶剂是直径较大的过渡族金属，而溶质是直径很小的碳、氢等非金属元素。其形成条件是溶质原子与溶剂原子直径之比必须小于0.59。如铁碳合金中，铁和碳所形成的固溶体――铁素体和奥氏体，皆为间隙固溶体。 ●按固溶度来分类：可分为有限固溶体和无限固溶体。无限固溶体只可能是转换固溶体。 ● 按溶质原子与溶剂原子的相对分布来分；可分为无序固溶体和有序固溶体 这两点各位有时间补充说明下 ● 固溶体的性能 当溶质元素含量很少时，固溶体性能与溶剂金属性能基本相同。但随溶质元素含量的增多，会使金属的强度和硬度升高，而塑性和韧性有所下降，这种现象称为固溶强化。置换固溶体和间隙固溶体都会产生固溶强化现象。 适当控制溶质含量，可明显提高强度和硬度，同时仍能保证足够高的塑性和韧性，所以说固溶体一般具有较好的综合力学性能。因此要求有综合力学性能的结构材料，几乎都以固溶体作为基本相。这就是固溶强化成为一种重要强化方法，在工业生产中得以广泛应用的原因。 第二节金属学及热处理基本知识 一、金属晶体结构的一般知识 众所周知，世界上的物质都是由化学元素组成的，这些化学元素按性质可分成两大类：第一大类是金属，化学元素中有83种是金属元素。固态金属具有不透明、有光泽、有延展性、有良好的导电性和导热性等特性，并且随着温度的升高，金属的导电性降低，电阻率增大，这是金属独具的一个特点。常见的金属元素有铁、铝、铜、铬、镍、钨等。 第二大类是非金属，化学元素中有22种，非金属元素不具备金属元素的特征。而且与金属相反，随着温度的升高，非金属的电阻率减小，导电性提高。常见的非金属元素有碳、氧、氢、氮、硫、磷等。 我们所焊接的材料主要是金属，尤其是钢材，钢材的性能不仅取决于钢材的化学成分，而且取决于钢材的组织，为了了解钢材的组织及对性能的影响，我们必须先从晶体结构讲起。 (一)晶体的特点 对于晶体，大家并不生疏。食盐、水结成的冰，都是晶体。一般的固态金属及合金也都是晶体。并非所有固态物质都是晶体。如玻璃、松香之类就不是晶体，而属于非晶体。 晶体与非晶体的区别不在外形，而在内部的原子排列。在晶体中，原子按一定规律排列得很整齐。而在非晶体中，原子则是散乱分布着，至多有些局部的短程规则排列。 由于晶体与非晶体中原子排列不同，因此性能也不相同。 (二)典型的金属晶体结构 金属的原子按一定方式有规则地排列成一定空间几何形状的结晶格子，称为晶格。金属
《材料结构与性能》思考题 第一章金属及合金的晶体结构 1．重要名词晶体非晶体单晶体多晶体晶粒晶界各向异性假等向性（伪各向同性）空间点阵阵点（结点）晶胞简单晶胞（初级晶胞）布拉菲点阵晶系晶面晶面指数晶向晶向指数密勒指数晶面族晶向族晶带晶带轴面间距配位数致密度点阵常数面心立方（A1）体心立方(A2) 密排六方(A3) 同素异构现象四面体间隙八面体间隙多晶型性(同素异构转变) 原子半径合金相固溶体间隙固溶体置换固溶体有限固溶体无限固溶体电子浓度无序分布偏聚短程有序短程有序参数维伽定律中间相金属间化合物正常价化合物电子化合物(Hume-Rothery相) 间隙相间隙化合物拓扑密堆相(TCP相) PHACOMP方法超结构（有序固溶体，超点阵）长程有序度参数反相畴（有序畴） 2．试述晶体的主要特征。 3．画出立方晶系中的下列晶面和晶向：(100), (111), (110), (123), (130)), (121), (225), [112], [312], [11 2]。画出六方晶系中的下列晶面：(0001), (1120), (1011)。 4．画出立方晶系(110)面上的[111]方向，(112)上的[111]方向。在其(111)面上有几个<110>方向 5．计算面心立方、体心立方、密排六方点阵晶胞的晶胞内原子数、致密度。其中原子的配位数是多少 6．面心立方和密排六方点阵的原子都是最密排的，为什么它们形成了两种点阵 7．画图计算面心立方和体心立方点阵的四面体、八面体间隙的半径r B与原子半径r A之比。 8．铜的面心立方点阵常数为?，计算其（122）晶面间距。 9．立方晶系中晶面指数和晶向指数有什么关系