[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cell (MSC) transplantation has been shown to be beneficial intreating cerebral ischemia. However, such benefit is limited by the low survival oftransplanted MSCs in an ischemic microenvironment. Previous studies showed thatmelatonin pretreatment can increase MSC survival in the ischemic kidney. However, whetherit will improve MSC survival in cerebral ischemia is unknown. Our study examined the effectof melatonin pretreatment on MSCs under ischemia-related conditions in vitro and aftertransplantation into ischemic rat brain. Results showed that melatonin pretreatment greatlyincreased MSC survival in vitro and reduced their apoptosis after transplantation intoischemic brain. Melatonin-treated MSCs (MT-MSCs) further reduced brain infarction andimproved neurobehavioral outcomes. Angiogenesis, neurogenesis and the expression ofvascular endothelial growth factor (VEGF) were greatly increased in the MT-MSC-treatedrats. Melatonin treatment increased the level of p-Erk1/2 in MSCs, which can be blocked bythe melatonin receptor antagonist luzindole. Erk phosphorylation inhibitor U0126 completelyreversed the protective effects of melatonin, suggesting that melatonin improves MSCsurvival and function through activating the Erk1/2 signaling pathway. Thus, stem cellspretreated by melatonin may represent a feasible approach for improving the beneficialeffects of stem cell therapy for cerebral ischemia.
[Show abstract][Hide abstract] ABSTRACT: Detection of microvascular changes in experimental stroke models is limited by current technologies. Using state-of-the-art synchrotron radiation (SR), we explored the feasibility of detecting the normal morphological variations of lenticulostriate arteries (LSAs) and the changes to LSAs following middle cerebral artery occlusion (MCAO). Cerebral microvessels of ICR mice were imaged with synchrotron radiation microangiography using nonionic iodine and barium sulfate as contrast agents. Using SR we reproducibly observed the detailed cerebral microvasculature of LSAs arising from the origin of middle cerebral artery (MCA) with a resolution of approximately 5 micrometers, at least a 20-fold greater resolution compared to CT or MRI imaging. Notably, SR microangiography was able to reveal ischemia/reperfusion induced leakage in the lenticulostriate artery territory. To our knowledge this is the first time that the three-dimensional morphology of LSAs and real time visualization of LSA hemorrhage have been characterized in live mice. This work demonstrates that SR microangiography can provide a unique tool for furthering experimental stroke research to examine the efficacy of neuroprotective therapies on parameters such as angiogenesis and vascular integrity.
Frontiers in bioscience (Elite edition) 01/2013; E5:517-524.
[Show abstract][Hide abstract] ABSTRACT: Transient middle cerebral artery occlusion (tMCAO) model is widely used to mimic human focal ischemic stroke in order to study ischemia/reperfusion brain injury in rodents. In tMCAO model, intraluminal suture technique is widely used to achieve ischemia and reperfusion. However, variation of infarct volume in this model often requires large sample size, which hinders the progress of preclinical research. Our previous study demonstrated that infarct volume was related to the success of reperfusion although the reason remained unclear. The aim of present study is to explore the relationship between focal thrombus formation and model reproducibility with respect to infarct volume. We hypothesize that suture-induced thrombosis causes infarct volume variability due to insufficient reperfusion after suture withdrawal. Seventy-two adult male CD-1 mice underwent 90 minutes of tMCAO with or without intraperitoneal administration of heparin. Dynamic synchrotron radiation microangiography (SRA) and laser speckle contrast imaging (LSCI) were performed before and after tMCAO to observe the cerebral vascular morphology and to measure the cerebral blood flow in vivo. Infarct volume and neurological score were examined to evaluate severity of ischemic brain injury. We found that the rate of successful reperfusion was much higher in heparin-treated mice compared to that in heparin-free mice according to the result of SRA and LSCI at 1 and 3 hours after suture withdrawal (p<0.05). Pathological features and SRA revealed that thrombus formed in the internal carotid artery, middle cerebral artery or anterior cerebral artery, which blocked reperfusion following tMCAO. LSCI showed that cortical collateral circulation could be disturbed by thrombi. Our results demonstrated that suture-induced thrombosis was a critical element, which affects the success of reperfusion. Appropriate heparin management provides a useful approach for improving reproducibility of reperfusion model in mice.
PLoS ONE 01/2013; 8(9):e75561. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Netrin-1 (NT-1) stimulates endothelial cell proliferation and migration in vitro and promotes focal neovascularization in the adult brain in vivo. This in vivo study in mice investigated the effect of NT-1 hyperexpression on focal angiogenesis and long-term functional outcome after transient middle cerebral artery occlusion (tMCAO).
Adeno-associated viral vectors carrying either the NT-1 gene (AAV-NT-1) or GFP (AAV-GFP) were generated and injected into the brains of separate groups of 93 mice. Seven days later, tMCAO followed by 7-28 days of reperfusion were carried out. Histological outcomes and behavioral deficits were quantified 7-28 days after tMCAO. Small cerebral vessel network and angiogenesis were assessed 28 days after tMCAO, using synchrotron radiation microangiography and immunohistochemistry.
Western blot and immunohistochemistry showed that on the day of tMCAO, NT-1 hyperexpression had been achieved in both normal and ischemic hemispheres. Immunofluorescence imaging showed that NT-1 expression was primarily in neurons and astrocytes. Ischemia-induced infarction in the NT-1 hyperexpression group was attenuated in comparison to saline or AAV-GFP-treated groups (P<0.01). Similarly, neurological deficits were greatly improved in AAV-NT-1-treated mice compared with mice in saline or AAV-GFP-treated groups (P<0.05). In addition, angiogenesis was increased in AAV-NT-1-treated mice compared with the other 2 groups (P<0.05). In vivo synchrotron radiation microangiography 28 days after tMCAO revealed more branches in AAV-NT-1-treated mice than in other groups.
AAV-NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovascularization, and improved long-term functional recovery.
[Show abstract][Hide abstract] ABSTRACT: Recent studies indicate that overexpression of adiponectin (APN) could attenuate ischemic brain injury. However, the mechanism of APN effect remains unclear. In this study, we investigated the cellular mechanisms of APN action during cerebral ischemia. Adult mice (n=120) received an intracerebral injection of adeno-associated viral vector carrying the APN gene (AAV-APN). The mice were subjected to a transient ispilateral middle cerebral artery occlusion (tMCAO) after 7-day AAV-APN gene transfer. Cortical atrophy volume, neurological function, microvessels counts, phospho-AMPK and downstream angiogenic factor vascular endothelial growth factor (VEGF) were examined. Overexpression of APN was observed in the mouse brain following AAV-APN gene transfer. Cortical atrophy volume was attenuated in the AAV-APN-transduced mice compared with the AAV-GFP and saline-treated mice (7.9±0.6%, 19.8±0.3% and 20.3±1.1%, respectively, P<0.05), with significant improvement in neurological function and an increased number of microvessels (199±5 vs 151±4 and 148±4 mm(-2), P<0.01). Furthermore, the expression of phospho-AMPK and VEGF were increased in the AAV-APN-transduced compared with the control mice (P<0.01), whereas inhibiting phospho-AMPK, reducing VEGF expression and attenuating the effect of APN on brain atrophy and angiogenesis (P<0.01). APN overexpression attenuates ischemia-induced brain atrophy and has improvement in neurological function. The consequence is related to promotion of focal angiogenesis. The AMPK signaling pathway has an important role in upregulating angiogenic factor VEGF.Gene Therapy advance online publication, 23 February 2012; doi:10.1038/gt.2012.7.
[Show abstract][Hide abstract] ABSTRACT: Precise in vivo evaluation of cerebral vasospasm caused by subarachnoid hemorrhage has remained a critical but unsolved issue in experimental small animal models. In this study, we used synchrotron radiation angiography to study the vasospasm of anterior circulation arteries in two subarachnoid hemorrhage models in rats. Synchrotron radiation angiography, laser Doppler flowmetry-cerebral blood flow measurement, [(125)I]N-isopropyl-p-iodoamphetamine cerebral blood flow measurement and terminal examinations were applied to evaluate the changes of anterior circulation arteries in two subarachnoid hemorrhage models made by blood injection into cisterna magna and prechiasmatic cistern. Using synchrotron radiation angiography technique, we detected cerebral vasospasm in subarachnoid hemorrhage rats compared to the controls (p<0.05). We also identified two interesting findings: 1) both middle cerebral artery and anterior cerebral artery shrunk the most at day 3 after subarachnoid hemorrhage; 2) the diameter of anterior cerebral artery in the prechiasmatic cistern injection group was smaller than that in the cisterna magna injection group (p<0.05), but not for middle cerebral artery. We concluded that synchrotron radiation angiography provided a novel technique, which could directly evaluate cerebral vasospasm in small animal experimental subarachnoid hemorrhage models. The courses of vasospasm in these two injection models are similar; however, the model produced by prechiasmatic cistern injection is more suitable for study of anterior circulation vasospasm.
PLoS ONE 01/2012; 7(3):e33366. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intraluminal suture technique for producing middle cerebral artery occlusion in rodents is the most commonly used method for modeling focal cerebral ischemia associated with clinical ischemic stroke. Synchrotron radiation angiography may provide a novel solution to directly monitor the success of middle cerebral artery occlusion.
Twenty adult Sprague-Dawley rats for middle cerebral artery occlusion models were prepared randomly with different suture head silicone coating. In vivo imaging was performed at beam line BL13W1, Shanghai Synchrotron Radiation Facility, Shanghai, China.
Silicone-coated suture was superior to uncoated suture for producing consistent brain infarction. Additionally, silicone coating length was an important variable controlling the extent of the ischemic lesion: infarcts affected predominantly the caudate-putamen with large variability (<2 mm), both the cortex and caudate-putamen (2-3.3 mm), and most of the hemisphere, including the hypothalamus (>3.3 mm).
Synchrotron radiation angiography provides a useful tool to observe hemodynamic changes after middle cerebral artery occlusion, and the physical properties of suture are critical to the success of the middle cerebral artery occlusion model.
[Show abstract][Hide abstract] ABSTRACT: Cerebral aneurysms of traditional animal models are usually too small to be imaged in vivo. A novel rat model induced by ligation of the unilateral common carotid artery and contralateral pterygopalatine and external carotid arteries was investigated. To evaluate the morphological changes of the cerebral arteries, synchrotron radiation angiography was utilised at 1 month, 2 months and 3 months after induction. Cerebral aneurysms and expansion of anterior cerebral arteries in these rats were identified when the animals were alive. This novel model is useful for cerebral aneurysm research.
Journal of Clinical Neuroscience 12/2011; 19(1):135-8. · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The suture middle cerebral artery occlusion (MCAO) model is used worldwide in both academia and industry. However, the variable occurrence of dysplasia in posterior communicating arteries (PcomAs) induces high mortality and instability in permanent MCAO models, limiting the model's application to transient focal ischemia. In particular, high mortality in intraluminal suture MCAO models is associated with the dysplasia of PcomAs in C57BL/6 mice. Optimization of silicone coating length is critical for reducing mortality and generating stable infarct in this model. The aim of our study is to reduce mortality and improve the reproducibility of the intraluminal suture MCAO model in C57BL/6 mice, which have high variation in PcomA dysplasia. Adult male C57BL/6 mice (n=38) underwent MCAO using sutures with various diameters and silicone coating lengths. The occlusion of cerebral vessels was examined by synchrotron radiation live angiography. The morphology of PcomAs was examined under a microscope after MICROFIL(®) infusion. Neurological outcome, infarct volume, and mortality were examined within 28 days. Optimizing the silicone coating on an 8-0 suture tip, we were able to reduce the model mortality to zero after permanent occlusion in C57BL/6 and produce stable brain infarct volume independent of the patency of PcomAs.
Journal of neurotrauma 11/2011; 29(7):1499-505. · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Netrin-1 (NT-1) is one of the axon-guiding molecules that are critical for neuronal development. Because of its structural homology to the endothelial mitogens, NT-1 may have similar effects on vascular network formation. NT-1 was shown to be able to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and also promote focal neovascularization in adult brain in vivo. In the present study, we reported the delivery of NT-1 using an adeno-associated virus (AAV) vector (AAV-NT-1) into mouse brain followed by transient middle cerebral artery occlusion (tMCAO). We found that AAV vectors did not elicit a detectable inflammatory response, cell loss or neuronal damage after brain transduction. The level of NT-1 was increased in the AAV-NT-1-transduced tMCAO mice compared with the control mice. Furthermore, the neurobehavioral outcomes were significantly improved in AAV-NT-1-transduced mice compared with the control animals (P<0.05) 7 days after tMCAO. Our data suggests that NT-1 plays a neuronal function recovery role in ischemic brain and that NT-1 gene transfer might present a valuable approach to treat brain ischemic disorders.
[Show abstract][Hide abstract] ABSTRACT: Traditionally, there are no methods available to detect the fine morphologic changes of cerebrovasculature in small living animals such as rats and mice. Newly developed synchrotron radiation microangiography can achieve a fine resolution of several micrometers and had provided us with a powerful tool to study the cerebral vasculature in small animals. The purpose of this study is to identify the morphology of cerebrovasculature especially the structure of Lenticulostriate arteries (LSAs) in living mice using the synchrotron radiation source at Shanghai Synchrotron Radiation Facility (SSRF) in Shanghai, China. Adult CD‐1 mice weighing 35–40 grams were anesthetized. Nonionic iodine (Omnipaque, 350 mg I ∕mL) was used as a contrast agent. The study was performed at the BL13W1 beam line at SSRF. The beam line was derived from a storage ring of electrons with an accelerated energy of 3.5 GeV and an average beam current of 200 mA. X‐ray energy of 33.3 keV was used to produce the highest contrast image. Images were acquired every 172 ms by a x‐ray camera (Photonic‐Science VHR 1.38) with a resolution of 13 μm∕pixel. The optimal dose of contrast agent is 100 μl per injection and the injecting rate is 33 μl∕sec. The best position for imaging is to have the mouse lay on its right or left side, with ventral side facing the X‐ray source. We observed the lenticulostriate artery for the first time in living mice. Our result show that there are 4 to 5 lenticulostriate branches originating from the root of middle cerebral artery in each hemisphere. LSAs have an average diameter of 43±6.8 μm. There were no differences between LSAs from the left and right hemisphere (p<0.05). These results suggest that synchrotron radiation may provide a unique tool for experimental stroke research.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy offers a novel approach for the treatment of experimental stroke. The adeno-associated virus (AAV) mediated vascular
endothelial growth factor (VEGF) gene transfer into the ischemic brain is described in detail in this chapter. Other methods
are also illustrated here, including the generation of mouse middle cerebral artery occlusion (MCAO) model, injection of viral
vector into mouse brain, and standard assays for determining the successes of brain ischemia and gene transfer.
Key wordsGene therapy-ischemia-adeno-associated virus-VEGF-middle cerebral artery occlusion-neurons