Study On Effects And Mechanisms Of Ischemia Reperfusion Injury On Nerve Regeneration In Organotypic Brain Slices

Cerebrovascular disease is one of the most common diseases which greatly threaten human health, especially in our country. A multicenter study containing more than 15 million person-years （aged 35 to 60） with over 20 centers in 12 countries, named MONICA, showed the mortality of stroke was highest in former Soviet Union, while the male mortality of our country ranked fifth, and the female mortality of Beijing ranked second. The disease monitoring materials of tens of million people from Statistical center of Ministry of health showed that the cerebrovascular disease mortality from 1988 to 2001 in our country ranged from 105 to 135 of 100 thousand persons. Cerebrovascular disease is well known as a disease with high incidence, high prevalence, high mortality and high disability rate, so that it attracts more attention of prevention and treatment work, and it has been listed as key research projects from Six Five-Year Plan to Ninth Five-Year Plan. In the study, we paid more attention to exploring the effects and mechanisms of ischemia reperfusion injury on nerve regeneration of rat brains with methods of animal experiment, cell culture and organotypic brain slice culture in order to find a way for treatment of ischemia reperfusion injury with stem cells.Ischemia reperfusion brain injury is caused by brain ischemia and subsequent reperfusion, and reperfusion can aggravate the injury caused by ischemia. The mechanisms of neuron death due to cerebral ischemia or ischemia reperfusion brain injury contain excitatory amino acid toxicity, mitochondrial damage, apoptosis, inflammation and intracellular calcium overload. Most patients with cerebrovascular disease have definite etiology, which orders specific treatment due to different causes, except that life-saving therapy and ischemic-penumbra-saving therapy are performed in the early phase. More and more kinds of treatments and drugs have been involved in curing cerebral ischemia. The main therapies include: anticoagulant therapy, thrombolytic therapy, expansion treatment, vasodilator therapy, calcium antagonist therapy, antiplatelet therapy and Traditional Chinese Medicine （TCM） therapy. In addition, preventing brain edema, neurotrophic therapy, primary diseases’ treatment, surgery, and rehabilitation therapy also plays roles in curing cerebral ischemia. Nowadays, a new treatment, neural stem cell transplantation, has showed a promising future for curing cerebral ischemia due to its characteristics of self-renew, multipotency, low immunogenicity and ability to construct normal connections with host cells around easily. How to realize neural stem cell transplantation to cure nervous system diseases, and how temporal treatments influence neural stem cells （NSCs） come to be research hotspots of neuroscience.Most of NSCs can be found in embryonic nervous system, although in adult nervous system, there are two major neurogenesis zones, known as subventricular zone （SVZ） and hippocampal dentate gyrus （HDG）. Although NSCs can theoretically proliferate and differentiate into all kinds of neural cells in nervous system unlimitedly, how to utilize NSCs to restore injured neural function can not be performed clinically. Therefore, a lot of researches are made on this aspect. These researches can be divided into two directions, exogenous NSCs therapy and endogenous NSCs therapy. The former can be divided into 3 classes. Firstly, transplantation first then differentiation, which means transplanting proper density and quantity NSCs to target location, then promoting proliferation and differentiation of NSCs transplanted through focal intrinsic signals in order to supplement lost neural cells and restore injured neural function. Secondly, differentiation first then transplantation, which means promoting proliferation and differentiation of NSCs cultured in vitro through exogenous signal system, then transplanting those NSCs to target location in order to restore injured neural function due to neural trauma and neural degenerative disease. Finally, as gene vectors, which means using NSCs as gene vectors to carry therapeutic genes, such as neurotrophic factors, in order to reform focal microenvironment for cell survival, proliferation and differentiation. Endogenous NSCs therapy means activating endogenous NSCs in GO phase to enter cell recycle, then proliferate, differentiate and generate all kinds of neural cells to replace lost cells, reconstruct neural network and restore neural function.As known, the effector cells in NSCs transplantation therapy are not NSCs themselves, but those functional neural cells differentiated from NSCs. Therefore, how to control NSCs proliferation and differentiation is a research hotspot of neuroscience currently. More and more researches showed the complexity of NSCs modulation. The modulating process is mainly affected by exogenous factors, known as cytokines, and endogenous factors, known as genes. The exogenous factors composed of cytokines controlling NSCs proliferation and differentiation include: first, epidermal growth factor （EGF）, which is a common mitogen inducing proliferation of NSCs. EGF can activate stem cells in GO phase and make them symmetrically divide, so that increase the number of stem cells. EGF often takes effects in late development, and promotes proliferation and differentiation of NSCs or neural progenitor cells, which need interaction of insulin-like growth factor-1. Second, basic fibroblast growth factor （bFGF）, it can promote proliferation of NSCs strongly, and it can also activate the regeneration ability of neural progenitor cells in many regions of central nervous system. The effect of bFGF is concentration dependent. In a low concentration （0.1μg/L）, bFGF promotes division and proliferation of NSCs in order to form clones of neurons, while in high concentration （1-10μg/L）, bFGF promotes NSCs forming clones of glial cells. Third, erythropoietin （EPO）, it has the ability to promote nerve development, neurotrophy and neural protection. Fourth, vascular endothelial growth factor （VEGF）, it can promote both nerve regeneration and angiogenesis. In the early phase of ischemia reperfusion brain injury, VEGF promotes nerve regeneration rather than angiogenesis. Fifth, insulin-like growth factor-1 （IGF-1）, it has complex effects on nervous system, which includes cell division, development, and maturation. Sixth, neural cell adhesion molecule （NCAM）, it participates in cell adhesion, formation of nerve fibers, promoting synaptic plasticity, and neural budding growth. NCAM is indispensable molecule in nerve development, and the very important modulating factor for NSCs plasticity. The endogenous factors are the genes control proliferation and differentiation of NSCs, which include: first, bHLH transcriptional regulating factor family; second, BMP family; third, Notch signaling system; fourth, Wnt family and Pax family; fifth, PTEN gene; sixth, REST/NRSF gene; seventh, Insc gene.More researches have shown that TCM can promote proliferation and differentiation of NSCs, which may be the mechanism that TCM can make neural function restored and nerve regenerated. Tongxinluo is a new drug generated with "collateral disease" theory. In the past few years, Tongxinluo had been used to treat ischemia reperfusion injury, and the effects were approving. Here, we used Tongxinluo medicated serum as a positive control. Organotypic brain slice culture is a kind of nerve tissue culture method developed in the past 20 years. Firstly, place brain slices in particular thickness on porous membrane of Millicell-CM inserts, and then place the inserts into six-well culture plates containing 1ml brain slice culture medium in each well in order to let brain slices be in the gas-liquid interface, so that brain slices can get oxygen and nourishment together. The technique of organotypic brain slice culture has the predominance of simulating in vivo state better than cell culture, and controlling experimental conditions and realizing in situ observation better than animal experiments.The main objectives of our study focused on exploring the effects and mechanisms of ischemia reperfusion injury on nerve regeneration. We planned to conduct experiments at 3 layers. We would discuss the effects and mechanisms of ischemia reperfusion injury on nerve regeneration, NSCs, and NSCs differentiation regulation with animal experiment, NSCs culture, and organotypic brain slice culture.The objectives our research will achieve include:First, investigate visualized location of nerve regeneration zone in embryonic rat brain, and isolate and culture embryonic NSCs orientedly based on visualized location;Second, improve the rat model of ischemia reperfusion injury with suture embolic method in order to provide reliable animal model for studies on ischemia reperfusion injury;Third, observe and discuss the effects and mechanisms of ischemia reperfusion injury on nerve regeneration and change of related cytokines in rat model of ischemia reperfusion injury;Fourth, establish methods of organotypic brain slice culture and making organotypic brain slice model of ischemia reperfusion injury; Fifth, investigate the effects of ischemia reperfusion injury on nerve regeneration and related cytokines in organotypic brain slices;Sixth, explore the effects of ischemia reperfusion injury on nerve regeneration in organotypic brain slices, and difference the effects in brain slices with in embryonic nerve regeneration zone in rat brains in order to be sure of the processes of activation, proliferation, migration, and differentiation of NSCs after ischemia reperfusion injury.Brains were taken from embryonic SD rats aged 17 days, and then they were sliced into many slices sagittally. Slices were taken to undergo anti-Nestin immunohistochemical staining every 30 microns. Photos were taken through 40×microscope. All photos were transmitted to computer to reconstruct new planar images. Based on these images, three-dimensional images were reconstructed in order to achieve space location of anti-Nestin （+） cells. Brain tissues were taken from embryonic SD rats aged 14 to 17 days based on space location of anti-Nestin （+） cells, and then they were cut to trivial pieces, beaten upon into single cell suspension mechanically. All cell suspension was filtrated, followed by centrifugation and resuspension. The cell density of cell resuspension was adjusted to 3×10⁵/ml by NSCs culture medium, and then inoculated into culture flasks, which were cultured in CO₂ incubator. The cultured cells were identified with Nestin and passaged.Ischemia reperfusion injury rat models were made by modified suture embolic method, and all rats were graded at 2 hours after surgery. The rat models, graded greater than 2, underwent subsequent experiments. Rats’ brains of each group were prepared for slicing at ultra-early stage （2h）, acute stage （3d）, and chronic stage （7d）. Each slice underwent anti-Nestin immunohistochemical staining in order to observe the changes of NSCs at different time. Prepare ischemia reperfusion injury animal models, and divide them to three groups of 3 day group, 5 day group, and 7 day group. At corresponding time points, brains were taken from those successful rat models. The brain tissue was used to examine mRNA quantities of four kinds of cytokines of EPO, VEGF, EGF, and IGF-1 by RT-PCR. So we can observe the effects of ischemia reperfusion injury on proliferation, differentiation of NSCs and related cytokines.Brains of postnatal 3 days SD rats were sliced into brain slices, containing hippocampus, characterized by the thickness of 300 microns, and then brain slices were placed on porous membrane of Millicell-CM inserts. Place the inserts into six-well culture plates containing 1ml brain slice culture medium in each well in order to let brain slices be in the gas-liquid interface. All the six-well culture plates were cultured in CO₂ incubator. Well-status brain slices were selected for preparing ischemia reperfusion brain slices. Substitute normal saline for brain slice culture medium. All the brain slices were incubated in 37℃and full of N₂ for 1 hour in order to mimic ischemia. Substitute brain slice culture medium for normal saline, and the ischemic brain slices were incubated in normal conditions in order to mimic reperfusion. The normal brain slices and ischemia reperfusion bran slices were divided into four groups of ischemia reperfusion group （IRG）, ischemia reperfusion therapy group （ITG）, positive control group （PCG）, and negative control group （NCG）. All brain slices were incubated in CO₂ incubator. Observe the changes of organotypic brain slices. Five visual fields were randomly selected, and the number of new born cells was counted. At 1 day, 3 days, and 7 days, the corresponding brain slices underwent triple immunofluorescent staining. The brain slices would be incubated with BrdU for 24 hours. The number of new born cells was conducted statistical analysis with SPSS 10.0. Prepare organotypic brain slices as mentioned above. The brain slices were incubated in CO₂ incubator. Observe the brain slices everyday. At 1 day, 3 days, and 7 days, the corresponding brain slices were gotten out, and the changes of EPO and VEGF would be examined by ELISA, while the changes of EGF, bFGF, IGF-1, and NCAM would be examined by qPCR. The outcome was conducted statistical analysis with SPSS 10.0.The results showed: Nestin（+） cells were distributed abroad in the embryonic nervous system, especially in olfactory bulb, rostral migration stream, subventricular zone, hippocampus, pons, cerebella, spinal cord, part of nervous nuclei. Embryonic NSCs could be isolated and cultured. The primary NSCs formed neurospheres by means of division proliferation and polymerization, and polymerization was the main manner of NSCs cultured forming neurospheres.Ischemia reperfusion injury animal models could be successfully made with suture embolic method. At ultra-early stage, vascular erythrocytes’ stasis could be only seen. At acute stage, encephalomalacia zone formed. At chronic stage, the cells in encephalomalacia zone dissolved and occurred ballooning degeneration. At ultra-early stage, there were no Nestin（+） cells. At acute stage, Nestin（+） emerged and distributed around encephalomalacia zone. At chronic stage, Nestin（+） cells disappeared. The organization structure was complete in control group, and there were no Nestin（+） cells. The expressions of EPO, VEGF, EGF, and IGF-1 increased at 3 days after ischemia reperfusion injury. The expressions of EPO, VEGF, and EGF were highest at 5 days, and decreased after that, while the expression of IGF-1 increased as time went on.General observation of organotypic brain slices, the interface of air was humid and glossy. The peripheral brain slices adhered to the porous membrane. All brain slices had homogeneous color, and gradually turned thinner till a particular thickness about 150nm. Observed by microscope, brain slices had uniform light transmission, and could be distinguished the structures of hippocampus, dentate gyrus, and ventricles. At 3 days, there were few new born cells appeared in HDG and SVZ. Most of the new born cells were relatively uniform round and ellipse. After undergoing ischemia reperfusion injury, the number of new born cells was significantly increased. The new born cells gathered mainly in HDG and SVZ. Compared with the new born cells of PCG, the new born cells were characterized by abnormal shape, inhomogeneity, and asymmetrical distribution. Ischemia reperfusion injury and Tongxinluo medicated sera could increase the quantity of new born cells significantly. The cells were mainly distributed in SVZ and HDZ, and labeled with DAPI/BrdU/Nestin. As culture went on, the new born cells gradually migrated to the cortex. The new born cell quantities of five visual fields were different with each other significantly. On the survival brain slices, "microflow" phenomena could be observed.The difference of EPO OD values among each group at 1 day or 3 days after ischemia reperfusion injury was significant. At 1 day, the EPO OD values of each group decreased according to the order of PCG, ITG, IRQ and NCG, while at 3 days, according to the order of NCG, PCG, IRQ and ITG At 7 days, the difference among each group was not significant. The EPO OD values decreased according to the order of 3 days. The interaction effects of each interference factors were significant. At each time point, the differences of VEGF OD values among each group were significant. The expressions of VEGF in IRG and ITG increased step by step, while the expression in NCG decreased quickly, and the expression in PCG got the highest point at 3 days. The expressions of EGF were highest at 1 day in IRQ PCQ and NCQ and then decreased gradually, while in ITQ increased gradually. At early stage, the expressions of bFGF in IRQ ITQ and PCG were higher. At 7 days, the expressions in ITG and PCG decreased. The expression of bFGF in NCG was low at early stage, but as culture went on, the expression increased gradually. Ischemia reperfusion injury inhibited the expression of IGF-1, but at 3 days, the expression of IGF-1 started increasing, while the expression of IGF-1 in ITG retained a level steadily. The expressions of NCAM in IRG and PCG all increased. At early stage, the expression of NCAM in ITG was low, but increased as culture went on.Summary of the whole article:（1） Investigate visualized location of nerve regeneration zone in embryonic rat brain. Find that the distribution of neurogenesis zone was abroad. Presume that neurogenesis zone in adult rat brain might not be restricted in HDG and SVZ. Other part of rat brain could also have potential of nerve regeneration, which would be the basis for restoring injured neural function.（2） Reveal the space-time characteristic of NSCs’ expression in embryonic rat brains: abroad distribution, sufficient expression, and being not in GO stage.（3） Find a new zone with dense NSCs in pons of embryonic rat brain.（4） Reveal the main manner of embryonic NSCs cultured forming neurospheres at early culture stage was polymerization by calculating, including polymerization between cells and cells, cells and neurospheres, and neurospheres and neurospheres.（5） Approve existence of lots of NSCs in embryonic rat brain by isolating and culturing NSCs from embryonic rat brain.（6） Reveal the facts that rat brains could endure longer ischemia, recovered earlier and better than human brains.（7） Find the time window of emerging of endogenous NSCs. Indicate that therapy with endogenous NSCs should be in the therapy time window.（8） Find the main current of endogenous NSCs activated was to form a saccate structure and to limit the injury in ischemia reperfusion injury.（9） Reveal that endogenous NSCs were activated in situ and the activated NSCs were mainly gathering around the injured part of brains.（10） Approve that ischemia reperfusion injury was the very important factor inducing emerging and proliferation of endogenous NSCs.（11） Approve that ischemia reperfusion injury promoted EPO, VEGF, EGF, and IGF-1, related to NSCs, participating restoring injured neural function and activating neurogenesis zone.（12） Reveal that ischemia reperfusion had transitory effect of inducing nerve regeneration in organotypic brain slices, but injured the potential of subsequent nerve regeneration.（13） Find the current of radial migration of neonatal cells from SVZ, HDG in organotypic brain slices.（14） Describe two-dimensional simulating graph of the current of radial migration of neonatal cells from SVZ, HDG in organotypic brain slices.（15） Find that activating and migration of NSCs could be observed directly in organotypic brain slices which would be difficult in animal experiment. Indicate that organotypic brain slices were good in vitro models for studying nerve regeneration.（16） Reveal that ischemia reperfusion injury promoted expression of EPO, but injured the expression of EPO simultaneously, the same to the effects of ischemia reperfusion injury on NSCs.（17） Reveal that ischemia reperfusion injury could delay expression of VEGF, which was related to restoring injured neural function at chronic stage.（18） Reveal that ischemia reperfusion injury promoted continuous expression of EGF with interaction of multiple factors.（19） Reveal that ischemia reperfusion injury activated nerve regeneration by promoting expression of bFGF.（20） Reveal ischemia reperfusion injury inhibits the expression of IGF-1, and the expression of IGF-1 would increase responsively and transiently at 3 days.（21） Reveal ischemia reperfusion injury promoted the expression of NCAM.