| BACKGROUND:Intracerebral hemorrhage (ICH) is one of the most common cerebrovascular diseases with relatively high rates of incidence, morbidity and mortality, demonstrating its harmfulness to human health. The incidence of ICH is caused by vascular rupture in long-term hypertension eventually leading to brain hemorrhage. ICH always has the following features: compression of adjacent brain tissue due to hematoma, reduction of cerebral blood flow, disruption of blood-brain barrier (BBB) function, and increased brain edema, which all contribute to neurological deterioration. Secondary injury, such as brain edema, is one of the main factors determining disease progression in patients with ICH as the clinical prognosis of ICH patients is closely related to the extent of brain edema. Vasogenic brain edema and cytotoxic brain edema are two major types of brain edema after ICH. The disruption of the BBB can evoke a vasogenic brain edema which is the leading factor in the poor prognosis.The BBB is a structure composed of tight junctions between capillary and endothelial cells, subendothelial basement membrane, pericytes, astrocytes and extracellular matrix. Since the BBB can prevent some of the substances in the blood from entering the brain, it is commonly regarded to have a protective function between blood and brain tissue. The BBB maintains the neural microenvironment by regulating the passage of molecules into and out of the brain and protects the brain against microorganisms and toxins in the blood. The tight junctions of the BBB consist of cytoplasmic adhesion proteins and transmembrane proteins, with cytosolic adhesion proteins consisting of ZO-1, ZO-2 and ZO-3 subtypes, and transmembrane protein mainly constituting claudins, occludins and junction associated molecules (JAM). In the case of pathological conditions such as ICH, the destruction of these structures can cause an increase of BBB permeability. Disruption of the BBB is an important pathophysiological change after ICH and contributes to formation of vasogenic brain edema, which plays an important role in secondary neuronal death and neurological dysfunction.Some studies indicate that nitric oxide (NO) is closely related to BBB damage and brain edema formation. NO is an endothelium-derived factor that is, among others, released upon pressure, shear and platelet aggregation. Under some circumstances, microglia and astrocyte in the central nervous system can generate NO radicals from inducible NOS (iNOS) activation. In some pathological conditions of the nervous system, such as cerebral ischemia, ICH and subarachnoid hemorrhage, the formation of NO is closely linked to BBB permeability. Especially when produced in large amounts, NO can combine with the superoxide anion (02-) and form a metabolite called peroxynitrite (0N00-), which is a strong biological oxidant and a destructive product. In the presence of ONOO-, radical ions can mediate various forms of tissue damage by way of disrupting the tyrosine residues in proteins. Studies also found that the extent of ONOO- damage in ICH is closely related to BBB disruption, ONOO- can disrupt BBB integrity by several mechanisms such as impairing cellular energy metabolism, inhibiting Na+/K+-ATPase activity, which lead to cytotoxic brain edema, and activating the matrix metalloproteinases (MMPs), which can compromise BBB integrity. Under the intervention of the related drug, the inhibition of ONOO- generation can effectively reduce the extent of BBB disruption.Bone marrow mesenchymal stem cells (BMSCs) are adult stem cells mainly derived from bone marrow tissue which have the capability of self-replication and differentiation into bone cells, cardiac cells, liver cells and glial cells. These cells possess the capacity of relatively easy to isolate, expand rapidly in culture. Due to their properties, MSCs appear to be an ideal cellular source for the repair of disesases including ICH. Previous studies have shown that MSCs can migrate to the site of injury and achieve a therapeutic effect in ICH by increased endogenous cell proliferation and promotion of the extent of angiogenesis. However, recent studies indicated that the primary mechanism for the therapeutic effect of MSCs may involve the secretion trophic factors, growth factors and a variety of adhesion molecules, commonly referred to as bystander mechanisms.Along with increasing numbers of studies, paracrine effects of MSCs are attracting increasing attention and were found to exert a therapeutic effect in various diseases, such as myocardial infarction, acute tubular injury, corneal injury, liver injury, traumatic brain injury and other experimental models. Therapeutic effects were mostly related to the suppression of apoptosis, promotion of wound tissue cell differentiation, induction of angiogenesis, restoration of tissue function and reduction of inflammation, along with a formation of microvessels in myocardial infarctions. In addition, some studies indicated that MSCs can relieve the cerebral infarct penumbra by providing nutritional support, inhibiting apoptosis and promoting angiogenesis via paracrine mechanisms. Moreover, MSCs were reported to exert anti-inflammatory and immunomodulatory mechanisms in brain injury treatment.Recent studies indicate that the capacity of MSCs is related to some soluble factors such as interleukin (IL)-10, indoleamine 2,3-dioxygenase, prostaglandin E2, which is a so-called bystander mechanism of MSCs and more recently, TSG-6, one of the anti-inflammatory factors, has attracted increased attention. Although numerous studies have shown that MSC transplantation can promote nerve cell proliferation and differentiation and is able to reduce inflammatory reaction in ICH treatment, the fact that it can also reduce BBB damage and brain edema after ICH, and ultimately achieve the therapeutic effect in ICH via paracrine mechanism, has not been reported so far. Therefore the present experiment aim to study the impact of MSCs on the BBB and brain edema after ICH and to explore the possible mechanisms of MSCs using SD rats in an ICH model. This study provides deeper knowledge to achieve a more complete understanding of MSC transplantation in the treatment of ICH.Chapter Ⅰ:Collection, culture and characterization of rats’ BMSCsObjective:BMSCs were harvested from femur and tibia of SD rats and seeded as qualified seed cells.Methods:Femur and tibia from 5-week-old SD rats were collected and MSCs were seeded at a density of 1×106 cells/25cm2. At 90% confluence, adherent cells were digested and expanded. Cells of passage three were used in this experiment. Flow cytometry was used for identification of cell phenotype in CD29, CD34, CD44, CD45 and CD90.Results:Flow cytometry of third passage MSCs showed the following results: CD29:99.52%, CD44:94.63%, CD90:99.65%; CD34:1.61%, CD45:0.95% suggesting that the phenotypes of CD29, CD44 and CD90 were highly expressed, while CD34 and CD45 were lowly expressed.Conclusion:In this study, bone marrow was separated and cultured as seed in order to obtain high purity and detailed biological characteristics of stable MSCs. Related phenotypes are in line with international standards on MSCs.The obtained MSCs can serve as a suitable research seed for stem cell transplantation in this experiment.Chapter Ⅱ:The protective effect of MSCs on the BBB after intracerebral hemorrhageObjective:Using a model of ICH, we detected substances which are closely related to the destruction of BBB at different time points, including the expression of iNOS, ONOO-, MMP-9, tight junction protein of claudin-5 and ZO-1. We then studied the change in substance formation and nerve function after administration of MSCs to provide a new reference for MSC treatment in clinical ICH patients.Methods:Type IV collagenase was used in the rat ICH model and SD rats were randomly divided into sham group, ICH model (ICH+PBS) group and MSC transplantation (MSCs+ICH) group. Procedures for both ICH+PBS and sham groups were similar:The ICH+PBS group received 2ul of phosphate buffer solution (PBS) containing 0.5IU type IV collagenase that was injected into the right striatum using 10-uL syringe, the sham group was injected with 2ul of PBS without type IV collagenase. Two hours following ICH,200ul of 5×106/ul MSCs were intravenously transplanted into rats of MSCs+ICH group, an equal volume of PBS was injected into rats of the ICH+PBS group. Density of Iba-1+ microglia/macrophages and neutrophil MPO+ cells was measured with immunohistochemistry, while the detection of apoptosis was measured with TUNEL. Cytokines including IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and transforming growth factor (TGF)-β1 were analyzed with ELISA. Using immunofluorescence, we detected the expression of ZO-1, iNOS and 3-NT, while protein expression in ZO-1, Claudin-5, iNOS,3-NT and MMP-9 was assessed by Western Blot.Results:Immunohistochemistry and TUNEL results indicate that the density of Iba-1+ microglia/macrophages and MPO+ neutrophils was significantly increased three days after ICH in the PBS+ICH group when compared with the sham group, which could be reduced by transplantation of MSCs. ELISA analysis showed that cytokines associated with inflammation were also significantly reduced in the ICH+MSC group at days 1,3 and 7 following ICH. The results of immunofluorescence indicated that MSC treatments effectively elevated ZO-1 levels, obviously reduced in PBS+ICH group at 3 days after ICH, and reduced the levels of iNOS and 3-NT, which were increased in the PBS+ICH group. Similarly, the results of Western Blot analysis suggested that MSC transplantation effectively reduced the levels of iNOS,3-NT and MMP-9 which increased in the PBS+ICH group and increased the levels of ZO-1 and Claudin-5 which decreased in the PBS+ICH group.Conclusion:The disease progress of ICH triggered accompanying cellular changes, such as iNOS,3-NT, MMP-9 and BBB damage, which is associated with the extent of brain edema. These products can disrupt endothelial cells’ tight junctions, such as claudin-5 and ZO-1. The transplantation of MSCs can effectively reduce the damaging effects described above as well as the extent of BBB disruption.Chapter Ⅲ:Mechanisms of MSC transplantation with regard to protective effects of the BBB in ICH ratsObjective:Although some studies have found that MSC transplantations enjoy a therapeutic effect in ICH, the main mechanism is associated with the promotion of nerve cell differentiation and the induction of angiogenesis. There is little attention focus on the effect of MSCs on BBB permeability and brain edema after ICH, where the brain edema is closely related to BBB permeability in ICH. Therefore we established an ICH model and intravenously transplanted MSCs to study their effect on BBB permeability after ICH.Methods:ICH model establishment and group setting procedures are similar to chapter Ⅱ. Type Ⅳ collagenase was used in the rat ICH model, and SD rats were randomly divided into sham group, ICH model (PBS+ICH) group and MSC transplantation (MSCs+ICH) group. The motor function of rats was assessed with the mNSS scoring system at days 1 and 3 after ICH. Brain edema and Evan’s blue were assessed to detect BBB damage at the same time points. RT-PCR and Western Blot were used to assess the expression of anti-inflammatory factor TSG-6. In addition to TSG-6, Western Blot was also used for NF-κB signaling pathway detection.Results:Using the mNSS scoring system to assess the extent of brain edema and Evan’s blue, we found that the permeability of the BBB was significantly increased in the PBS+ICH group, whereas the treatment with MSCs improved mNSS score, BBB permeability and brain edema degree. The results of the Western Blot analysis showed that MSC treatment in ICH has upregulated the expression of the inhibitory factor TSG-6 at 24 and 72 h after ICH, similar results were obtained on mRNA levels. Compared with a basal level of IκB-α in the sham-operated group, the levels of IκB-α in the PBS-treated group were reduced. On the contrary, treatment with MSCs inhibited the degradation of IκB-α in the MSCs+ICH group. Unlike the levels of IκB-α, phosphorylation of Ser536 on p65 was increased in the PBS+ICH group when compared with the sham-operated group, whereas MSCs treatment inhibited this increase. Similar to the phosphorylation of Ser536 on p65, MSC treatment reduced NF-κB p65 levels in the nuclear fractions of the ICH tissue when compared with the PBS-treated group.Conclusion:Our results demonstrated that intravenous transplantation of MSCs decreased the levels of ONOO- and the degree of BBB leakage and improved neurological recovery in a rat ICH model. The results indicated that the mechanism of MSCs in ICH rats was related to TSG-6, which improved BBB disruption by inhibiting the NF-κB signaling pathway. The levels of iNOS and ONOO- decreased after the inhibitory effect of TSG-6 in the NF-κB signaling pathway. |
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