The Journal of Neuroscience, November 2, 2005, 25(44):10321-10335

Oxidative stress leading to ischemic cell death involves the formation of reactive oxygen species/reactive nitrogen species (ROS/RNS) through multiple injury mechanisms, such as mitochondrial inhibition, Ca2+ overload, reperfusion injury, and inflammation (Coyle and Puttfarcken, 1993; Lipton, 1999; Love, 1999; Lewen et al., 2000). Although many treatment strategies have implemented antioxidants to promote neuroprotection during ischemia, their clinical efficacy has proven disappointing (De Keyser et al., 1999; Lo et al., 2003). The Cap `n' Collar (CNC) transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an expansive set of antioxidant/detoxification genes that act in synergy to remove ROS/RNS through sequential enzymatic reactions (Li et al., 2002; Thimmulappa et al., 2002; Shih et al., 2003).

Circulation. 2008;118:157-165

Statistical AnalysisAll data are presented as mean±SEM. The results were analyzed statistically with the use of the software package Statview 5.0 (Abacus Concepts Inc, Berkeley, Calif). A paired t test was performed to compare the bromodeoxyuridine (BrdU) incorporation rate of EPCs before and after hindlimb ischemia. Scheffé’s test was performed for multiple comparisons after ANOVA between each group. A P value <0.05 was considered to denote statistical significance. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Circulation. 2008;118:157-165

Evaluation of EPC Kinetics in the Hindlimb Ischemia ModelA hindlimb ischemia model was generated to evaluate in vivo EPC functions, such as capacity for blood vessel regeneration, mobilization from BM, incorporation into sites of neovascularization, and survival of endogenous cells. A more detailed and expanded description of the materials and methods used is provided in the online-only Data Supplement.

J. Clin. Invest. 118(1): 133-148 (2007).

18FDG-PET examination. To assess the metabolic activity and synaptic density of brain tissue, experimental rats were examined using microPET scanning of 18FDG to measure relative metabolic activity as previously described (57). In brief, 18F was produced by the 18O(p, n)18F nuclear reaction in a cyclotron at Tzu-Chi General Hospital and Tzu-Chi University, and 18FDG was synthesized as previously described (58) with an automated 18FDG synthesis system (Nihonkokan). Data were collected with high-resolution small-animal PET (microPET Rodent R4; Concorde Microsystems Inc.). The system parameters were described by Visnyei et al. (57). After 1 week of each treatment, animals anesthetized with chloral hydrate (0.4 g/kg i.p.) were fixed in a customized stereotactic head holder and positioned in the microPET scanner. The animals were then given an i.v. bolus injection of 18FDG (200–250 μCi/rat) dissolved in 0.5 ml saline. Data acquisition began simultaneously with injections and continued for 60 minutes in 1 bed position using a 3D acquisition protocol. The image data acquired from microPET were displayed and analyzed by IDL version 5.5 (Research Systems) and ASIPro version 3.2 (Concorde Microsystems) software. Coronal sections for striatal and cortical measurements represented brain areas between 0 and +1 mm from the bregma, while those for thalamic measurements represented areas between –2 and –3 mm from the bregma, as estimated by visual inspection of the unlesioned side. The relative metabolic activity in regions of interest of the striatum and cortex was expressed as percentage deficit, as previously described with modification (57).

J. Clin. Invest. 118(1): 133-148 (2007).

Measurement of the infarct size using MRI. MRI was performed on rats under anesthesia in a General Electric imaging system (R4; GE) at 3.0 T. Brains were scanned in 6–8 coronal image slices, each 2 mm thick without any gaps. T2-weighted imaging pulse sequences were obtained with the use of a spin-echo technique (repetition time, 4,000 ms; echo time, 105 ms) and were captured sequentially for each animal at 1, 7, and 28 days after cerebral ischemia. To measure the infarction area in the right cortex, we subtracted the noninfarcted area in the right cortex from the total cortical area of the left hemisphere. The area of infarct was drawn manually from slice to slice, and the volume was then calculated by internal volume analysis software (Voxtool; GE).

J. Clin. Invest. 118(1): 133-148 (2007).

In vivo brain ischemia/reperfusion. Adult male Sprague-Dawley rats (250–300 g) were used for this study. The rats were anesthetized with chloral hydrate (0.4 g/kg i.p.) and subjected to right MCA ligation and bilateral common carotid artery (CCA) clamping as previously described (50). Briefly, the bilateral CCAs were clamped with nontraumatic arterial clips. Using a surgical microscope, a 2-mm × 2-mm craniotomy was drilled at the point where the zygoma fuses to the squamosal bone, the right MCA was then ligated with a 10-0 nylon suture. Cortical blood flow was measured continuously with a laser Doppler flowmeter (PF-5010, Periflux system; Perimed AB) in anesthetized animals. A photodetector probe (0.45 mm in diameter) was stereotaxically placed through a skull burr hole (1 mm in diameter) in the frontoparietal cortex (l.3 mm posterior, 2.8 mm lateral to the bregma, and l.0 mm below the dura). Experimental rats were then injected i.v. with recombinant human SN (50 μg in 500 μl saline; ProSpec-Tany TechnoGene) or vehicle (500 μl saline) 30 minutes after MCA ligation through a 26-gauge syringe into the right femoral vein. After 90 minutes of ischemia, the 10-0 suture on the MCA and arterial clips on CCAs were removed to allow for reperfusion. During recovery from anesthesia, body temperature was maintained at 37°C with a heat lamp.

J. Clin. Invest. 118(1): 133-148 (2007).

SN enhances stem cell mobilization and homing to brain. BrdU labeling and analysis by transgenic GFP-chimeric mice were used to demonstrate the homing and engraftment of intrinsic neural progenitor cells (INPCs) and BMSCs to the brain. Seven days after cerebral ischemia, in SN-treated rats (n = 6), cumulative BrdU labeling revealed a few BrdU-immunoreactive cells in the ipsilateral cortex near the infarct boundary (Figure 6, A–D) and subventricular region of ischemic hemisphere (Figure 6, E–H). BrdU-immunoreactive cells were also found around the lumen of varying calibers of blood vessels in the perivascular portion of the ischemic hemisphere (Figure 6, I–L). BrdU pulse labeling showed significantly more BrdU-immunoreactive cells in the penumbral region in SN-treated rats than saline control rats (n = 8 per group; Figure 6M). Moreover, in transgenic GFP-chimeric mice, a significant increase of GFP+ cells (showing green fluorescence) was observed in the penumbral region of SN-treated mice compared with controls (Figure 6N)

J. Clin. Invest. 118(1): 133-148 (2007).

SN protects neural tissues from apoptosis. Cellular apoptosis in ischemic rat brain was studied in SN-treated and control rats (n = 6 per group) using TUNEL analysis. The control animals, which did not receive MCA ligation, showed almost no TUNEL staining in their brain sections. The penumbral region surrounding the ischemic cores of SN-treated rats contained fewer TUNEL-positive cells than did that of vehicle controls (Figure 5C). Semiquantitatively, animals treated with vehicle had significantly more TUNEL-positive cells than those treated with SN (Figure 5C). TUNEL-positive cells were distributed mainly in the ischemic core of the cerebral cortex, with the labeling essentially found in the nucleus of neuronal cells.

J. Clin. Invest. 118(1): 133-148 (2007).

Immunoreactivity of SN colocalizes to neurons and endothelial cells after cerebral ischemia. In order to identify which cerebral cells expressed SN after cerebral ischemia, double immunofluorescence was performed on brain specimens of ischemic rats with laser-scanning confocal microscopy. Ischemic cortical areas of the rats revealed many SN-IR cells coexpressing a neuronal nucleus–positive (Neu-N+) neuronal phenotype (163 ± 26 cells/mm2; Figure 1G). Some SN-IR cells showing SMA+ vascular phenotypes were also found around the perivascular and endothelial regions (Figure 1G) of the ischemic hemispheres.

J. Clin. Invest. 118(1): 133-148 (2007).

Secretoneurin promotes neuroprotection and neuronal plasticity via the Jak2/Stat3 pathway in murine models of strokeJ. Clin. Invest. Woei-Cherng Shyu, et al. 118:133 doi:10.1172/JCI32723 [Go to this article.] Figure 1Cerebral ischemia increases expression of SN in human and rat brain. (A) Representative brain of a stroke patient; boxed region shows infarct area. IHC studies of the penumbral area showed markedly increased SN-IR. (B) SN-IR in the stroke patients’ brains significantly increased at 1 and 3 days after cerebral infarction compared with that of controls. (C and D) IHC and quantitative analysis of SN-IR of animal cerebral ischemia (boxed region) showed greater numbers of SN-IR cells compared with controls. (E and F) Measurement of SN level using ELISA at the indicated time points showed a significant increase of SN expression in both ischemic rats’ brains and stroke patients’ sera compared with controls. (G) Double immunofluorescence with laser-scanning confocal microscopy of ischemic rat brain specimens. The ischemic cortical areas of the rats revealed SN-IR cells coexpressing Neu-N+. Some SN-IR cells showing vascular phenotypes (SMA+ cells) were also found around the perivascular and endothelial regions of the ischemic hemispheres. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. control. Scale bars: 50 μm

The Journal of Neuroscience, March 17, 2004, 24(11):2750-2759

Ischemic preconditioning and global ischemia. Age-matched male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were maintained in a temperature- and light-controlled environment with a 14/10 hr light/dark cycle and were treated in accordance with the principles and procedures of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. Animals were subjected to global ischemia by four-vessel occlusion (4 min for preconditioning; 10 min for global ischemia), followed by reperfusion as described (Kinouchi et al., 1993; Calderone et al., 2003). For sham operation, animals were subjected to the same anesthesia and surgical exposure procedures, except that the carotid arteries were not occluded. Body temperature was monitored and maintained close to 37.5 ± 0.5°C with a rectal thermistor and heat lamp until the animal had fully recovered from anesthesia. The rare animals that exhibited obvious behavioral manifestations (abnormal vocalization when handled, generalized convulsions, loss of >20% body weight by 3–7dor hypoactivity) and animals that failed to show complete loss of the righting reflex from 2 min after occlusion was initiated and anesthesia was discontinued until the end of occlusion were excluded. Histology and neuronal counts from six to eight rats per group (four sections per animal) were as described (Calderone et al., 2003). Statistical analysis was assessed by ANOVA, followed by Scheffe's post hoc tests. Ischemic preconditioning and global ischemia in gerbils were as described (Tanaka et al., 2002).

The Journal of Neuroscience, January 1, 1998, 18(1):205-213

Figure 5. Histological analysis after 24hr of MCA occlusion. A, Photomicrograph showing the histological changes after 24hr of MCA occlusion in wild-type (Wt) and knock-out mutant mice (Sod2 /+). The infarct area was localized in the caudoputamen and MCA territory cortex in both mice groups. However, cortical infarction extended to the boundary zone of the anterior cerebral artery territory, and brain swelling was extremely severe in the knock-out mutant mice. Also shown are infarct volume (B) and hemisphere enlargement (C) in wild-type and knock-out mutant mice at 24hr ischemia. Values are mean±SE; **p<0.01and ***p<0.001,Student's t test. Cerebral infarction and hemisphere enlargement were significantly more severe in knock-out than in wild-type mice. Scale bar, 1 mm.

The Journal of Neuroscience, March 15, 1999, 19(6):1988-1997

Ischemia model. All experimental procedures were approved by the Subcommittee on Animal Studies of the Veterans Affairs Medical Center (San Diego, CA). Male Wistar rats (250-300 gm) were fasted overnight. Anesthesia was induced with 3% halothane followed by maintenance with 1-2% halothane in an oxygen/nitrous oxide (30/70%) gas mixture. Catheters were inserted into the external jugular vein, tail artery, and tail vein to allow blood sampling, arterial blood pressure recording, and drug infusion. Both common carotid arteries were exposed and encircled by loose ligatures. Fifteen minutes before ischemia induction and 15min after ischemia, blood gases were measured and adjusted to PaO2 >90 mmHg and PaCO2 35-45 mmHg, pH 7.35-7.45, by adjusting tidal volume of the respirator. Bipolar EEG was recorded every 5-10 min before ischemia, continuously during the ischemic insult, and every 5min after ischemia until the rat recovered from the anesthesia. At the beginning of a 30min steady-state period before induction of ischemia, the inspired halothane concentration was decreased to 0.5%, and 150IU/kg heparin was administered intravenously. Blood was withdrawn via the jugular catheter to produce a mean arterial blood pressure of 50mmHg, and ischemia was induced by clamping both carotid arteries. Blood pressure was maintained at 50mmHg during the ischemic period by withdrawing or infusing blood through the jugular catheter. At the end of the ischemic period, the clamps were removed, and the blood was reinfused through the jugular catheter, followed by 0.5ml of 0.6M sodium bicarbonate. In all experiments, temperature was maintained at 37°C before, during, and after ischemia (15min of reperfusion). Halothane was discontinued at the end of ischemia, and all wounds were sutured. At 4or 24hr, 3d, or 1week after the ischemic episode, the animals were reanesthetized, tracheotomized, and artificially ventilated. For electron microscopic studies, the brains were perfused with ice-cold 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer. Sham-operated control rats were subjected to the same surgical procedures but without induction of ischemia.

Am J Pathol. 2008 July; 173(1): 57–67.

Murine Ischemia/Reperfusion ProtocolsAll animal studies were approved by the animal protocol review committee at Baylor College of Medicine. IL-1RI−/− mice15 and WT C57/BL/6 controls (purchased from The Jackson Laboratories, Bar Harbor, ME) were used for myocardial infarction experiments. Female mice, 8 to 12 weeks of age (18.0 to 22.0 g body weight) were anesthetized by an intraperitoneal injection of sodium pentobarbital (60 μg/g). A closed-chest mouse model of reperfused myocardial infarction was used to avoid the confounding effects of surgical trauma and inflammation, which may influence the baseline levels of chemokines and cytokines.16 The left anterior descending coronary artery was occluded for 1 hour then reperfused for 6 hours to 7 days. At the end of the experiment, the chest was opened and the heart was immediately excised, fixed in zinc-formalin, and embedded in paraffin for histological studies, or snap-frozen and stored at −80°C for RNA isolation. Sham animals were prepared identically without undergoing coronary occlusion/reperfusion. Animals used for histology underwent 24-hour, 72-hour, and 7-day reperfusion protocols (eight animals per group). Mice used for RNA extraction underwent 6 hours, 24 hours, and 72 hours of reperfusion (eight animals per group). To examine matrix metalloproteinase (MMP) and TIMP mRNA expression in the infarcted and remodeling myocardium hearts from animals undergoing 1 hour of ischemia and 7 days of reperfusion were used (eight mice per group). mRNA was extracted separately from the infarct and the remote noninfarcted myocardium. Additional animals [knockout (KO), n = 10; wild type (WT), n = 8] were used for perfusion-fixation after 7 days of reperfusion to assess remodeling-associated parameters.

The Journal of Neuroscience, November 24, 2004, 24(47):10763-10772

Cerebral hypoxia and ischemia model. Adult C57BL/6 mice (25-30 gm body weight) were anesthetized with isoflurane and subjected to permanent occlusion of the unilateral common carotid artery. After recovering from the anesthesia, mice were exposed to hypoxia inside a controlled atmosphere chamber (model 855-AC; PlasLabs) that was infused with 7.5% oxygen. An oxygen analyzer (model 600; ESD) was used to monitor the oxygen concentration inside the chamber. To induce cerebral hypoxia-ischemia in rats, both common carotid arteries were reversibly occluded in 1-month-old rats followed by exposure to 7.5% oxygen inside a computer-controlled hypoxic chamber (In Vivo 400 workstation and Ruskinn gas mixer). The animal procedures were approved by the Institutional Animal Care and Use Committee and conform to the National Institutes of Health Guide for Care and Use of Laboratory Animals.