Fengfeng Tian

Tumorigenic development of induced pluripotent stem cells in ischemic mouse brain.

Induced pluripotent stem (iPS) cells may provide cures for various neurological diseases. However, undifferentiated iPS cells have high tumorigenicity, and evaluation of the cells fates, especially in pathologic condition model, is needed. In this study, we demonstrated the effect of ischemic condition to undifferentiated iPS cells fates in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells were characterized with immunofluorescent staining. The iPS cells (5 × 105) were injected into ipsilateral striatum and cortex after 24 h of MCAO. Histological analysis was performed from 3 to 28 days after cell transplantation. iPS cells in ischemic brain formed teratoma with higher probability (p < 0.05) and larger volume (p < 0.01) compared with those in intact brain. Among the four transcriptional factors to produce iPS cells, c-Myc, Oct3/4, and Sox2 strongly expressed in iPS-derived tumors in ischemic brain (p < 0.01). Additionally, expression of matrix metalloproteinase-9 (MMP-9) and phosphorylated vascular endothelial growth factor receptor2 (phospho-VEGFR2) were significantly increased in iPS-derived tumors in the ischemic brain (p < 0.05). These results suggest that the transcriptional factors might increase expression of MMP-9 and activate VEGFR2, promoting teratoma formation in the ischemic brain. We strongly propose that the safety of iPS cells should be evaluated not only in normal condition, but also in a pathologic, disease model.

In vivo imaging of autophagy in a mouse stroke model

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.

Expression of Keap1-Nrf2 system and antioxidative proteins in mouse brain after transient middle cerebral artery occlusion

Reactive oxygen species and their detrimental effects on the brain after transient ischemia have been implicated in the pathogenesis of the ischemic injury. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system is currently recognized as the major cellular defense mechanism under oxidative stress, but the involvement of the Keap1-Nrf2 system in the ischemic brain injuries has not been fully investigated to date. In the present study, we investigated temporal changes of Keap1, Nrf2, and their downstream antioxidative proteins in post-ischemic mice brains with respect to spacial differences between the peri-infarct regions and the regions destined to infarct. In the peri-infarct regions, a steady level of Keap1 showed a decremental expression started at 2h of reperfusion after 60 min of transient middle cerebral artery occlusion (tMCAO). In contrast, Nrf2 began to show a significant increase at 2h with a peak at 8h of reperfusion after tMCAO. Both Keap1 and Nrf2 are mainly expressed in neuronal cells but not in glial cells. In the same peri-infarct region, downstream antioxidative proteins such as thioredoxin, glutathione, and heme oxygenase-1 showed significant increases at later time-points of 24-72 h of reperfusion after tMCAO. In the regions destined to infarct, a similar trend of expression changes to those in the peri-infarct regions was observed in Keap1, Nrf2, and 3 downstream antioxidative proteins with much less reactions. The changes found in this study suggest that the induced antioxidative stress proteins after cerebral ischemia may play an important endogenous neuroprotective response under oxidative stress after ischemic stroke.

Strong neurogenesis, angiogenesis, synaptogenesis, and antifibrosis of hepatocyte growth factor in rats brain after transient middle cerebral artery occlusion.

Hepatocyte growth factor (HGF) and glial cell line‐derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL‐positive cells, but only HGF significantly increased the number of BrdU‐positive cells in the subventricular zone, and 5′‐bromo‐2′‐deoxyuridine ‐positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF‐treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO.

Antiapoptotic and antiautophagic effects of glial cell line‐derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in rats

Glial cell line‐derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule‐associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser2448 (p‐mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3‐I plus LC3‐II (relative to β‐tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p‐mTOR‐positive cells. GDNF and HGF significantly decreased the numbers of TUNEL‐, LC3‐, and LC3/TUNEL double‐positive cells. LC3/TUNEL double‐positive cells accounted for about 34.3% of LC3 plus TUNEL‐positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways.

Free Radical Scavenger Edaravone Administration Protects against Tissue Plasminogen Activator Induced Oxidative Stress and Blood Brain Barrier Damage

One of the therapeutics for acute cerebral ischemia is tissue plasminogen activator (t-PA). Using t-PA after 3 hour time window increases the chances of hemorrhage, involving multiple mechanisms. In order to show possible mechanisms of t-PA toxicity and the effect of the free radical scavenger edaravone, we administered vehicle, plasmin, and t-PA into intact rat cortex, and edaravone intravenously. Plasmin and t-PA damaged rat brain with the most prominent injury in t-PA group on 4-HNE, HEL, and 8-OHdG immunostainings. Such brain damage was strongly decreased in t-PA plus edaravone group. For the neurovascular unit immunostainings, occludin and collagen IV expression was decreased in single plasmin or t-PA group, which was recovered in t-PA plus edaravone group. In contrast, matrix metalloproteinase-9 intensity was the strongest in t-PA group, less in plasmin, and was the least prominent in t-PA plus edaravone group. In vitro data showed a strong damage to tight junctions for occludin and claudin 5 in both administration groups, while there were no changes for endothelial (NAGO) and perivascular (GFAP) stainings. Such damage to tight junctions was recovered in t-PA plus edaravone group with similar recovery in Sodium-Fluorescein permeability assay. Administration of t-PA caused oxidative stress damage to lipids, proteins and DNA, and led to disruption of outer parts of neurovascular unit, greater than the effect in plasmin administration. Additive edaravone ameliorated such an oxidative damage by t-PA with protecting outer layers of blood-brain barrier (in vivo) and tight junctions (in vitro).

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T3-S1 level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.

Dynamic changes of mitochondrial fusion and fission proteins after transient cerebral ischemia in mice

With fusion or fission, mitochondria alter their morphology in response to various physiological and pathological stimuli, resulting in elongated, tubular, interconnected, or fragmented forms. Immunohistochemistry and Western blot analysis were performed at 2 days, 7 days, 14 days, and 28 days after 90 min of transient middle cerebral artery occlusion (tMCAO) in mice. This study showed that mitochondrial fission protein dynamin‐related protein 1 (Drp1) and fusion protein optic atrophy 1 (Opa1) were both upregulated in the ischemic penumbra, with the peak at 2 days after tMCAO, whereas phosphorylated‐Drp1 (P‐Drp1) progressively increased with a peak at 14 days after tMCAO. Double‐immunofluorescence analysis showed many Drp1/cytochrome c oxidase subunit l (COX1) double‐positive cells and Opa1/COX1 double‐positive cells in the ischemic penumbra and also showed some double‐positive cells with Drp1/terminal deoxynucleotidyl transferase‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) and Opa1/TUNEL in the ischemic penumbra. In contrast, both Drp1 and Opa1 showed progressive decreases until 2 days after tMCAO in the ischemic core because of necrotic brain damage. The present study suggests that there was a continuous mitochondrial fission and fusion during these periods in the ischemic penumbra after tMCAO, probably in an effort toward mitophagy and cellular survival.

In vivo optical imaging for evaluating the efficacy of edaravone after transient cerebral ischemia in mice

Detection and protection of apoptosis, autophagy and neurovascular unit (NVU) are essentially important in understanding and treatment for ischemic stroke patients. In this study, we have conducted an in vivo optical imaging for detecting apoptosis and activation of matrix metalloproteinases (MMPs), then evaluated the protective effect of 2 package types of free radical scavenger edaravone (A and B) on apoptosis, autophagy and NVU in mice after transient middle cerebral artery occlusion (tMCAO). As compared to vehicle treatment, edaravones A and B showed a significant improvement of clinical scores and infarct size at 48 h after 90 min of tMCAO with great reductions of in vivo fluorescent signal for MMPs and early apoptotic annexin V activations. Ex vivo imaging of MMPSense 680 or annexin V-Cy5.5 showed a fluorescent signal, while which was remarkably different between vehicle and edaravone groups, and colocalized with antibody for MMP-9 or annexin V. Edaravone A and B ameliorated the apoptotic neuronal cell death in immunohistochemistry, and activations of MMP-9 and aquaporin 4 with reducing autophagic activations of microtubule-associated protein 1 light chain 3 (LC3) in Western blot. In this study, edaravone in both packages showed a similar strong neuroprotection after cerebral ischemia, which was confirmed with in vivo and ex vivo optical imagings for MMPs and annexin V as well as reducing cerebral infarct, inhibiting apoptotic/autophagic mechanisms, and protecting a part of neurovascular unit.

Temporal and spatial differences of multiple protein expression in the ischemic penumbra after transient MCAO in rats

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.