[Show abstract][Hide abstract] ABSTRACT: Imaging techniques for visualizing cerebral vasculature and distinguishing functional areas are essential and critical to the study of various brain diseases. In this paper, with the X-ray phase-contrast imaging technique, we proposed an experiment scheme for the
mouse brain study, achieving both high spatial resolution and improved soft-tissue contrast. This scheme includes two steps: sample preparation and volume reconstruction. In the first step, we use heparinized saline to displace the blood inside cerebral vessels and then replace it with air making air-filled mouse brain. After sample preparation, X-ray phase-contrast tomography is performed to collect the data for volume reconstruction. Here, we adopt a phase-retrieval combined filtered backprojection method to reconstruct its three-dimensional structure and redesigned the reconstruction kernel. To evaluate its performance, we carried out experiments at Shanghai Synchrotron Radiation Facility. The results show that the air-tissue structured cerebral vasculatures are highly visible with propagation-based phase-contrast imaging and can be clearly resolved in reconstructed cross-images. Besides, functional areas, such as the corpus callosum, corpus striatum, and nuclei, are also clearly resolved. The proposed method is comparable with hematoxylin and eosin staining method but represents the studied mouse brain in three dimensions, offering a potential powerful tool for the research of brain disorders.
Full-text · Article · Nov 2015 · Computational and Mathematical Methods in Medicine
[Show abstract][Hide abstract] ABSTRACT: Bilayer poly(l-lactic acid) fibrous scaffolds consisting of a thin aligned-fiber layer (AFL) and a relatively thick random-fiber layer (RFL) were fabricated by an electrospinning technique, which uses two slowly rotating parallel disks as the collector. The morphology and structure of the bilayer scaffolds were examined by high-magnification scanning electron microscopy and confocal microscopy. The bilayer scaffolds demonstrated gradual variation in through-thickness porosity and fiber alignment and an average porosity much higher than that of conventionally electrospun scaffolds (controls) with randomly distributed fibers. The biocompatibility and biological performance of the bilayer fibrous scaffolds were evaluated by in vivo experiments involving subcutaneous scaffold implantation in Sprague–Dawley rats, followed by histology and immunohistochemistry studies. The results illustrate the potential of the bilayer scaffolds to overcome major limitations of conventionally electrospun scaffolds associated with intrinsically small pores, low porosity and, consequently, poor cell infiltration. The significantly higher porosity and larger pore size of RFL enhances cell motility through the scaffold thickness, whereas the relatively dense structure of AFL provides the scaffold with the necessary mechanical strength. The bilayer scaffolds show more than two times higher cell infiltration than controls during implantation in vivo. The unique structure of the bilayer scaffolds promotes collagen fiber deposition, cell proliferation and ingrowth of smooth muscle cells and endothelial cells in vivo. The results of this study illustrate the high prospect of the fabricated bilayer fibrous scaffolds in tissue engineering and regeneration.
No preview · Article · Nov 2014 · Acta Biomaterialia
[Show abstract][Hide abstract] ABSTRACT: The rat suture middle cerebral artery occlusion (MCAO) is a frequently used animal model for investigating the mechanisms of ischemic brain injury. During suture MCAO, transection of the external carotid artery (ECA) potentially restrains blood flow and impairs masticatory muscle and other ECA-supported territories, consequently influencing post-operation animal survival. This study was aimed at investigating the effect of ECA transection on the hemodynamic alterations using a novel synchrotron radiation (SR) angiography technique and magnetic resonance imaging in live animals. Fifteen male adult Sprague-Dawley rats were used in this study. Animals underwent MCAO, in which the ECA was transected. SR angiography was performed before and after MCAO. Rats then underwent magnetic resonance imaging (MRI) to detect the tissue lesion both intra- and extra-cranially. Animals with SR angiography without other manipulations were used as control. High-resolution cerebrovascular morphology was analyzed using a novel technique of SR angiography. The masticatory muscle lesion was further examined by hematoxylin and eosin staining. MRI and histological results showed that there was no masticatory muscle lesion at 1, 7 and 28 days following MCAO with ECA transection. In normal condition, the ECA and its branch external maxillary artery were clearly detected. Following ECA transection, the external maxillary artery was still observed and the blood supply appeared from the anastomotic branch from the pterygopalatine artery. SR angiography further revealed the inter-relationship of hemisphere extra- and intra-cranial vasculature in the rat following MCAO. Transection of the ECA did not impair masticatory muscles in rat suture MCAO. Interrupted blood flow could be compensated by the collateral circulation from the pterygopalatine artery.
No preview · Article · Nov 2014 · Journal of Synchrotron Radiation
[Show abstract][Hide abstract] ABSTRACT: Micro-computed tomography (micro-CT) is a powerful tool for visualizing the vascular systems of tissues, organs, or entire small animals. Vascular contrast agents play a vital role in micro-CT imaging in order to obtain clear and high-quality images. In this study, a new kind of nanostructured barium phosphate was fabricated and used as a contrast agent for ex vivo micro-CT imaging of blood vessels in the mouse brain. Nanostructured barium phosphate was synthesized through a simple wet precipitation method using Ba(NO3)2, and (NH4)2HPO4 as starting materials. The physiochemical properties of barium phosphate were characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and thermal analysis. Furthermore, the impact of the produced nanostructures on cell viability was evaluated via the MTT assay, which generally showed low to moderate cytotoxicity. Finally, the animal test images demonstrated that the use of nanostructured barium phosphate as a contrast agent in Micro-CT imaging produced sharp images with excellent contrast. Both major vessels and the microvasculature were clearly observable in the imaged mouse brain. Overall, the results indicate that nanostructured barium phosphate is a potential and useful vascular contrast agent for micro-CT imaging.
No preview · Article · Feb 2014 · Journal of Nanoparticle Research
[Show abstract][Hide abstract] ABSTRACT: Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)-a subunit of H3 methyltranferase-by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.
[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.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cell (MSC) transplantation has been shown to be beneficial in treating cerebral ischemia. However, such benefit is limited by the low survival of transplanted MSCs in an ischemic microenvironment. Previous studies showed that melatonin pretreatment can increase MSC
survival in the ischemic kidney. However, whether it will improve MSC survival in cerebral ischemia is unknown. Our study examined the effect of melatonin pretreatment on MSCs under ischemia-related conditions in vitro and after transplantation into ischemic rat brain. Results showed that
melatonin pretreatment greatly increased survival of MSCs in vitro and reduced their apoptosis after transplantation into ischemic brain. Melatonin-treated MSCs (MT-MSCs) further reduced brain infarction and improved neurobehavioral outcomes. Angiogenesis, neurogenesis, and the expression
of vascular endothelial growth factor (VEGF) were greatly increased in the MT-MSC-treated rats. Melatonin treatment increased the level of p-ERK1/2 in MSCs, which can be blocked by the melatonin receptor antagonist luzindole. ERK phosphorylation inhibitor U0126 completely reversed the protective
effects of melatonin, suggesting that melatonin improves MSC survival and function through activating the ERK1/2 signaling pathway. Thus, stem cells pretreated by melatonin may represent a feasible approach for improving the beneficial effects 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.
No preview · Article · Jan 2013 · Frontiers in bioscience (Elite edition)
[Show abstract][Hide abstract] ABSTRACT: It is generally accepted that the de-differentiation of smooth muscle cells, from the contractile to the proliferative/synthetic phenotype, has an important role during vascular remodelling and diseases. Here we provide evidence that challenges this theory. We identify a new type of stem cell in the blood vessel wall, named multipotent vascular stem cells. Multipotent vascular stem cells express markers, including Sox17, Sox10 and S100β, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell-like cells that subsequently differentiate into smooth muscle cells. On the other hand, we perform lineage tracing with smooth muscle myosin heavy chain as a marker and find that multipotent vascular stem cells and proliferative or synthetic smooth muscle cells do not arise from the de-differentiation of mature smooth muscle cells. In response to vascular injuries, multipotent vascular stem cells, instead of smooth muscle cells, become proliferative, and differentiate into smooth muscle cells and chondrogenic cells, thus contributing to vascular remodelling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of multipotent vascular stem cells, rather than the de-differentiation of smooth muscle cells, contributes to vascular remodelling and diseases.
Preview · Article · Jun 2012 · Nature Communications
[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.
[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: 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.
Full-text · Article · Dec 2011 · Journal of Clinical Neuroscience
[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.
Full-text · Article · Nov 2011 · Journal of neurotrauma
[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.
No preview · Article · Mar 2011 · Frontiers of Medicine
[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
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