Protective role of hematopoietic prostaglandin D synthase in transient focal cerebral ischemia in mice

Cerebral ischemia/reperfusion injury is characterized by the development of inflammatory response, in which vascular macrophages and endogenous microglia are involved. Recent studies showed marked induction of hematopoietic prostaglandin D synthase (HPGDS) after ischemic/reperfusion injury and its localization in microglia, but the molecular mechanism(s) of HPGDS actions in cerebral ischemia is not clear. To clarify the role of HPGDS in cerebral ischemia, C57BL/6 mice and bone marrow chimera mice with cerebral ischemia/reperfusion injury were treated with (4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79), a specific inhibitor of HPGDS. The bone marrow chimera mice exhibit expression of enhanced green fluorescent protein (EGFP) in bone marrow/blood-derived monocytes/macrophages. Mice were subjected to ischemia/reperfusion and either treated with HQL-79 (n=44) or vehicle (n=44). Brain sections prepared at 72 h and 7 days after reperfusion were analyzed for neuronal nuclei (NeuN), HPGDS, ionized calcium-binding adapter molecule 1 (Iba1), inducible NO synthase (iNOS), nitrotyrosine, nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2). The mortality rate (80%) and infarct size were larger in HQL-79- than vehicle-treated mice (58.7±8.5 versus 45.2±4.9 mm³; mean±SEM, P<0.0001) at 7 days after reperfusion. HQL-79 reduced NeuN expression in the transition area and Iba1 expression (P<0.0001) in the ischemic peri- and penumbra area, but increased COX-2 (P<0.05) and NF-kB expression (P<0.05) in ischemic penumbra and increased formation of nitrotyrosine (P<0.0001) and iNOS (P<0.0001) in the ischemic core area at 72 h and 7 days after reperfusion. In EGFP chimera mice, HQL-79 increased the migration of Iba1/EGFP-positive bone marrow-derived monocytes/macrophages, and simultaneously upregulated iNOS expression in the ischemic core area (P<0.0001), but increased intrinsic microglia/macrophages in ischemic peri-area and penumbra (P<0.0001) at 72 h and 7 days after reperfusion, suggesting involvement of monocytes/macrophages in HQL-79-induced expansion of ischemic injury. Our results demonstrated that the neuroprotective effects of HPGDS in our model are mediated by suppression of activation and infiltration of inflammatory cells.     

Dynamics and fate of USPIO in the central nervous system in experimental autoimmune encephalomyelitis

Signal loss observed in the brain by MRI following the administration of ultrasmall superparamagnetic particles of iron oxide (USPIO) has been correlated with immune cell activity in inflammatory areas during multiple sclerosis. Uptake of USPIO by circulating monocytes and their migration towards inflammatory areas have been considered as the most important mechanism for USPIO uptake by the brain parenchyma. However, the involvement of a damaged blood–brain barrier is also debated as a possible mechanism for cerebral USPIO uptake. Compared with these uptake‐associated issues, little is known about the clearance of USPIO from the brain. The acute uptake and chronic clearance of USPIO in the brain were therefore studied with MRI in an animal model of multiple sclerosis. Lewis Hannover rats with acute experimental autoimmune encephalomyelitis received a single intravenous injection of USPIO (300 µmol Fe/kg), and repetitive MRI of the brain and cervical lymph nodes, a possible drainage pathway, was performed. USPIO were detected in the brain within 1 h after injection independent of the severity of experimental autoimmune encephalomyelitis, and histological analysis revealed extracellular iron clusters colocalising with a leaky blood–brain barrier. Loss of signal was not present 72 h after USPIO injection, irrespective of the disease state. MR images of cervical lymph nodes showed USPIO accumulation at 24 h after administration, which stabilised at 72 h. Histological analyses revealed that USPIO accumulated in infiltrated macrophages in the medulla and subcapsular sinus. The current study demonstrates that USPIO enter the central nervous system directly after administration, pointing to the involvement of a damaged blood–brain barrier in the appearance of USPIO‐associated MR abnormalities. Furthermore, a possible role of the cervical lymph nodes as a drainage pathway of USPIO is suggested. These data shed new light on the use of USPIO in neuroinflammatory diseases, identifying USPIO as a marker for both cellular infiltration and blood–brain barrier damage. Copyright © 2010 John Wiley & Sons, Ltd.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

The capability of detecting small vessels beyond the conventional MRI sensitivity using iron‐based contrast agent enhanced susceptibility weighted imaging

Imaging brain microvasculature is important in cerebrovascular diseases. However, there is still a lack of non‐invasive, non‐radiation, and whole‐body imaging techniques to investigate them. The aim of this study is to develop an ultra‐small superparamagnetic iron oxide (USPIO) enhanced susceptibility weighted imaging (SWI) method for imaging micro‐vasculature in both animal (~10 μm in rat) and human brain. We hypothesized that the USPIO‐SWI technique could improve the detection sensitivity of the diameter of small subpixel vessels 10‐fold compared with conventional MRI methods. Computer simulations were first performed with a double‐cylinder digital model to investigate the theoretical basis for this hypothesis. The theoretical results were verified using in vitro phantom studies and in vivo rat MRI studies (n = 6) with corresponding ex vivo histological examinations. Additionally, in vivo human studies (n = 3) were carried out to demonstrate the translational power of the USPIO‐SWI method. By directly comparing the small vessel diameters of an in vivo rat using USPIO‐SWI with the small vessel diameters of the corresponding histological slide using laser scanning confocal microscopy, 13.3‐fold and 19.9‐fold increases in SWI apparent diameter were obtained with 5.6 mg Fe/kg and 16.8 mg Fe/kg ferumoxytol, respectively. The USPIO‐SWI method exhibited its excellent ability to detect small vessels down to about 10 μm diameter in rat brain. The in vivo human study unveiled hidden arterioles and venules and demonstrated its potential in clinical practice. Theoretical modeling simulations and in vitro phantom studies also confirmed a more than 10‐fold increase in the USPIO‐SWI apparent diameter compared with the actual small vessel diameter size. It is feasible to use SWI blooming effects induced by USPIO to detect small vessels (down to 10 μm in diameter for rat brain), well beyond the spatial resolution limit of conventional MRI methods. The USPIO‐SWI method demonstrates higher potential in cerebrovascular disease investigations.     

Central nervous system inflammatory response after cerebral infarction as detected by magnetic resonance imaging

Brain inflammation contributes to the tissue injury caused by ischemic stroke. Macrophages as the most abundant inflammatory cell population in stroke lesions can be visualized using ultrasmall superparamagnetic iron oxide (USPIO) as a cell-specific contrast agent for magnetic resonance imaging (MRI). The aim of our present study was to delineate the inflammatory response during experimental cerebral infarction by means of USPIO-enhanced MRI and to correlate the spatial distribution of USPIO-induced MR signal alterations with cellular infiltration and iron deposition. To this end USPIOs were administered to Wistar rats 5 days after photothrombotic cerebral infarction. MR imaging at 7 T performed 24 h later displayed a rim-like signal loss around the infarction in the USPIO treated animals. On histological brain sections obtained from the same animals after MRI the distribution of iron and ED1+ phagocytes was in full spatial agreement with the signal loss seen on T2*-weighted images. Our study validates USPIO-enhanced MRI as an important tool for the noninvasive visualization of brain inflammation in stroke and other CNS pathologies.     

In vivo carotid plaque MRI using quantitative T2* measurements with ultrasmall superparamagnetic iron oxide particles: a dose-response study to statin therapy

This study investigates T₂* quantification in carotid plaques before and after the administration of ultrasmall superparamagnetic iron oxide particles (USPIOs) in a cohort of patients receiving statin therapy. Phantom studies were performed using gels with varying concentrations of USPIOs. In the phantom study, 12 gels were prepared with a range of freely distributed concentrations of USPIO nanoparticles (0–0.05 mg/mL). Relative signal intensity measurements were obtained from a T₂*‐weighted sequence as well as quantitative T₂* (qT₂*) measurements. In the patient study, 40 patients with >40% carotid stenosis were randomised to low‐ and high‐dose statin therapy (10 and 80 mg of atorvastatin). Pre‐ and post‐ (36 h) USPIO‐enhanced MRI were performed at baseline, and at 6 and 12 weeks. A linear mixed‐effects model was applied to account for the inherent correlation of multiple‐plaque measurements from the same patient and to assess dose–response differences to statin therapy. In the phantom study, the T₂*‐weighted sequence demonstrated an initial increase (T₁ effect), followed by a decrease (T₂* effect), in relative signal intensity with increasing concentrations of USPIO. The qT₂* values decreased exponentially with increasing concentrations of USPIO. In the patient study, there was a highly significant difference in post‐USPIO T₂* measurements in plaques between the low‐ and high‐dose statin groups. This was observed for both the difference in qT₂* measurements (post‐USPIO minus pre‐USPIO) (p < 0.001) and for qT₂* post‐USPIO only (p < 0.001). The post‐USPIO qT₂* values were as follows: baseline: low dose, 13.6 ± 5.5 ms; high dose, 12.9 ± 6.2 ms; 6 weeks: low dose, 13.3 ± 6.7 ms; high dose, 14.3 ± 7.7 ms; 12 weeks: low dose, 14.0 ± 7.6 ms; high dose, 18.3 ± 11.2 ms. It can be concluded that qT₂* measurements provide an alternative method of quantifying USPIO uptake. These results also demonstrate that changes in USPIO uptake can be measured using post‐USPIO imaging only. Copyright © 2010 John Wiley & Sons, Ltd.     

The effect of bone marrow‐ and adipose tissue‐derived mesenchymal stem cell transplantation on myocardial remodelling in the rat model of ischaemic heart failure

This study aimed to investigate the effect of bone marrow‐ and adipose tissue‐derived mesenchymal stem cell (BM‐MSC and AD‐MSC respectively) transplantation on left ventricular function and infarct area (IA) in the rat model of ischaemic heart failure. In anaesthetized Wistar rats, the left coronary artery (LCA) was occluded for 40 min with subsequent reperfusion for 7 days. Seven days following surgery, the animals with LCA occlusion/reperfusion were randomized into three groups: (i) Controls received intramyocardial injection of vehicle at three different locations within the peri‐infarct zone, (ii) BM‐MSC: cells were injected in the same way as in previous group (10⁶), (iii) AD‐MSC: using the same protocol as used in the BM‐MSC group. In addition there was also a sham‐treated group that had no injection. Two weeks following MSC transplantation, the hearts were isolated and perfused according to the Langendorff method followed by 30‐min global ischaemia and 90‐min reperfusion. After this IA was determined histologically. During Langendorff perfusion initial and postischaemic LV functions were the same in all groups although LV pressure at the 10th minute of reperfusion was higher in the AD‐MSC group compared to controls. However, LV pressure during 30‐min global ischaemia was significantly higher in BM‐MSC as compared to controls and AD‐MSC. The sham treated animals showed the same results as those seen with BM‐MSC. Thus, BM‐MSC transplantation, in contrast to transplantation of AD‐MSC, resulted in better preservation of the LV ability to contract during ischaemia. Furthermore, IA was significantly smaller in BM‐MSC group as compared to the controls and the AD‐MSC groups. Thus this study has demonstrated that treatment with BM‐MSC both ameliorates LV function and reduces histological scar size.     

In-vivo visualization of phagocytotic cells in rat brains after transient ischemia by USPIO

Cerebral ischemia provokes tissue damage by two major patho-physiological mechanisms. Direct cell necrosis is induced by diminished access of neurons and glia to essential nutrients such as glucose and oxygen leading to energy failure. A second factor of cellular loss is related to the activation of immune-competent cells within and around the primary infarct. While granulocytes and presumably monocytes are linked to the no-reflow phenomenon, activated microglia cells and monocytes can release cytotoxic substrates, which cause delayed cell death. As a consequence the infarct volume will increase, despite restoration of cerebral perfusion. In the past, visualization of immune competent cells was only possible by histological analysis of post-mortem tissue. However, contrast agents based on small particles of iron oxide are known to accumulate in organs rich in cells with phagocytotic function. These particles can be tracked in vivo by MRI methods based on their relaxation properties. In the present study, the spatio-temporal distribution of USPIO particles was monitored in a rat model of transient cerebral infarction using T1- and T2-weighted MRI sequences. USPIO were detected in vessels at 24 h after administration. At later time points specific accumulation of USPIO was observed within the infarcted hemisphere, with maximal signal enhancement on day 2. Their detectability based on T1-contrast disappeared between day 4 and day 7. Immuno-histochemically (IHC) stains confirmed the presence of macrophages, presumably blood-derived monocytes within areas of T1 signal enhancement. Direct visualization of iron-burdened macrophages by IHC was only possible later than day 3 after occlusion.     

Focal cerebral ischemia/reperfusion injury in mice induces hematopoietic prostaglandin D synthase in microglia and macrophages

Hematopoietic prostaglandin D synthase is a key enzyme in synthesis of prostaglandin D. Hematopoietic prostaglandin D synthase is expressed in microglia of the developing mouse brain. This study determined the serial changes and cellular localization of hematopoietic prostaglandin D synthase, and its role in cerebral ischemia/reperfusion injury using C57BL/6 mice (n=84) and bone marrow chimera mice (n=16). The latter mice were selected based on their expression of enhanced green fluorescent protein in bone marrow/blood-derived monocytes/macrophages. The middle cerebral artery was occluded for 60 min, followed by reperfusion. Hematopoietic prostaglandin D synthase expression was examined by immunohistochemistry and Western blotting. Hematopoietic prostaglandin D synthase-positive cells were mainly expressed in the peri-ischemic area at 12 h (P<0.05) and 24 h (P<0.001) after reperfusion, while they were mostly found in the transition area at 48-72 h postreperfusion (P<0.001). There was a significant increase in staining intensity as well as number of hematopoietic prostaglandin D synthase-positive cells in the ischemic core at 5-7 (P<0.001) days postreperfusion. Hematopoietic prostaglandin D synthase-positive cells also co-expressed ionized calcium-binding adapter molecule 1, a marker of microglia/macrophages, and cyclooxygenase-2, but not markers of neurons, oligodendrocytes and astrocytes. Until 72 h postreperfusion, many enhanced green fluorescent protein-positive cells were negative for hematopoietic prostaglandin D synthase, but the number of hematopoietic prostaglandin D synthase-enhanced green fluorescent protein coexpressing cells increased significantly at 5-7 days after reperfusion. Our results indicate that hematopoietic prostaglandin D synthase is mainly produced by endogenous microglia until 72 h after reperfusion, but at 7 days after reperfusion, it is also produced by migrating bone marrow/blood-derived macrophages in the ischemic brain tissue. We speculate that hematopoietic prostaglandin D synthase in the brain has different functions during early and late phases of ischemia.     

Stereological assessment of the blood‐air barrier and the surfactant system after mesenchymal stem cell pretreatment in a porcine non‐heart‐beating donor model for lung transplantation

More frequent utilization of non‐heart‐beating donor (NHBD) organs for lung transplantation has the potential to relieve the shortage of donor organs. In particular with respect to uncontrolled NHBD, concerns exist regarding the risk of ischaemia/reperfusion (IR) injury‐related graft damage or dysfunction. Due to their immunomodulating and tissue‐remodelling properties, bone‐marrow‐derived mesenchymal stem cells (MSCs) have been suspected of playing a beneficial role regarding short‐ and long‐term survival and function of the allograft. Thus, MSC administration might represent a promising pretreatment strategy for NHBD organs. To study the initial effects of warm ischaemia and MSC application, a large animal lung transplantation model was generated, and the structural organ composition of the transplanted lungs was analysed stereologically with particular respect to the blood–gas barrier and the surfactant system. In this study, porcine lungs (n = 5/group) were analysed. Group 1 was the sham‐operated control group. In pigs of groups 2–4, cardiac arrest was induced, followed by a period of 3 h of ventilated ischaemia at room temperature. In groups 3 and 4, 50 × 10⁶ MSCs were administered intravascularly via the pulmonary artery and endobronchially, respectively, during the last 10 min of ischaemia. The left lungs were transplanted, followed by a reperfusion period of 4 h. Then, lungs were perfusion‐fixed and processed for light and electron microscopy. Samples were analysed stereologically for IR injury‐related structural parameters, including volume densities and absolute volumes of parenchyma components, alveolar septum components, intra‐alveolar oedema, and the intracellular and intra‐alveolar surfactant pool. Additionally, the volume‐weighted mean volume of lamellar bodies (lbs) and their profile size distribution were determined. Three hours of ventilated warm ischaemia was tolerated without eliciting histological or ultrastructural signs of IR injury, as revealed by qualitative and quantitative assessment. However, warm ischaemia influenced the surfactant system. The volume‐weighted mean volume of lbs was reduced significantly (P = 0.024) in groups subjected to ischaemia (group medians of groups 2–4: 0.180–0.373 μm³) compared with the sham control group (median 0.814 μm³). This was due to a lower number of large lb profiles (size classes 5–15). In contrast, the intra‐alveolar surfactant system was not altered significantly. No significant differences were encountered comparing ischaemia alone (group 2) or ischaemia plus application of MSCs (groups 3 and 4) in this short‐term model.     

Periadventitial delivery of anti‐EGF receptor antibody inhibits neointimal macrophage accumulation after angioplasty in a hypercholesterolaemic rabbit

Monocyte recruitment and their differentiation into macrophages are both early events in native and accelerated atherosclerosis that follows angioplasty. We have investigated the putative functional role of the epidermal growth factor receptor (EGFR) present on rabbit monocytes/macrophages. The impact of periadventitial delivery of an EGFR‐specific, blocking monoclonal antibody (ICR62, which inhibits EGF‐binding to its receptor) was investigated in a rabbit model of accelerated atherosclerosis induced by a combination of carotid injury and 4 weeks of a 2% cholesterol‐diet. Two weeks after the initiation of the diet, a balloon‐catheter angioplasty of the left common carotid artery was performed and a collar placed around the injured carotid artery immediately, for the delivery of ICR62 antibody, isotype‐matched antibody or saline control. Monocyte/macrophage accumulation, cell proliferation and neointimal thickening were determined 2 weeks after the delivery of the antibodies. The function of the EGFR on rabbit monocytes was also investigated in vitro, using chemotaxis assays. Treatment with ICR62 was associated with a significant reduction in macrophage accumulation and neointimal thickening and a 76% reduction in neointimal area of the vessel wall compared with controls. In vitro ICR62 inhibited macrophage and smooth muscle cell migration towards EGFR ligands including EGF and HB‐EGF. These findings suggest that EGFR ligation may be important in the development of early atherosclerotic lesions following balloon‐catheter angioplasty, and periadventitial delivery may provide a feasible approach for administration of the inhibitors of EGFR‐binding such as ICR62.     

Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells

We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M‐CSF or GM‐CSF. When differentiated in GM‐CSF (GM‐MØP) the resultant cells resemble GM‐CSF bone marrow‐derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM‐MØP to effectively present antigen to a T‐cell hybridoma, these cells are comparatively poor at priming the expansion of IFN‐γ responses from naïve CD4⁺ T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM‐MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM‐CSF represents a unique in vitro model of inflammatory monocyte‐like cells, with important differences from bone marrow‐derived dendritic cells, which will facilitate functional studies relating to the many ‘sub‐phenotypes’ of inflammatory monocytes.     

Collaboration of cancer‐associated fibroblasts and tumour‐associated macrophages for neuroblastoma development

Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour‐associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer‐associated fibroblast (CAF) marker, alpha smooth muscle actin (αSMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the αSMA‐staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and peripheral blood mononuclear cell (PBMC)‐derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up‐regulated in PBMC‐derived macrophages treated with NBCM. The expression of αSMA by BM‐MSCs was increased in NBCM‐treated cells. Co‐culturing with CAF‐like BM‐MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co‐cultured with TAM‐like macrophages had the opposite effect. Intriguingly, TAM‐like macrophages enhanced not only the invasive abilities of tumour cells and BM‐MSCs but also the proliferation of BM‐MSCs. CXCL2 secreted from TAM‐like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC‐derived macrophages and BM‐MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

经鼻腔长期输送骨髓基质细胞改善大鼠缺血再灌注性脑损伤

背景：经鼻腔黏膜给药是一种可以克服血脑屏障、减少外周不良反应、效能高的脑内给药方式。 目的：验证经鼻腔长期输送骨髓基质细胞治疗大鼠缺血再灌注性脑损伤的可行性。 方法：贴壁培养法分离大鼠骨髓基质细胞。缺血再灌注脑损伤大鼠随机分成实验组和对照组。实验组经鼻腔滴入骨髓基质细胞,对照组同法给予磷酸盐缓冲液,隔天1次,共4周。每周进行1次行为学评价,最后1次行为学评价后进行脑病理学检测。 结果与结论：与对照组比较,从第2周开始神经病学严重程度评分和Morris水迷宫实验检测结果表明实验组行为学逐步改善(P < 0.05,P < 0.01)。与行为学改善相一致,缺血再灌注导致对照组CA1区海马细胞数量减少了66%,而实验组CA1区细胞丢失明显要少,只丢失了25% (P < 0.01)。说明了经鼻腔长期输注骨髓基质细胞具有改善大鼠缺血再灌注脑损伤的作用。     

Femoral head histology subsequent to ischemia, reperfusion and steroid treatment

An episode of ischemia followed by reperfusion of the femoral head (FH) is thought to be a common pathway in the pathogenesis of femoral head necrosis (FHN). Femoral head histology was investigated after short-term high-dose steroid treatment and femoral head ischemia and reperfusion in a large animal model. Twenty-two pigs were randomized to receive methylprednisolone 20 mg/day/kg bodyweight intamuscularly for 3 days followed by methylprednisolone 10 mg/day/kg bodyweight for 11 days (n=11), whereas the control group (n=11) received no treatment. After 6 h of unilateral hip-joint pressure increase to 250 mmHg, the pressure was discontinued and reperfusion was allowed for 4 h. Undecalcified histology was performed for the femoral head subchondral region, the mid-region, and the region adjacent to the growth plate. Congestion phenomena were predominantly discerned in femoral head sections of the tamponaded hips. Histomorphometry revealed fat cell hyperthrophy and reduced hemopoetic marrow cells in the femoral heads of the steroid-treated group of animals. The number of blood vessels and bone trabeculae remained unchanged. These characteristics may correlate with early-stage femoral head necrosis.     

Multiparametric CMR imaging of infarct remodeling in a percutaneous reperfused Yucatan mini‐pig model

To further understanding of the temporal evolution and pathophysiology of adverse ventricular remodeling over the first 60 days following a myocardial infarction (MI) in both the infarcted and remote myocardium, we performed multi‐parametric cardiac magnetic resonance (CMR) imaging in a closed‐chest chronic Yucatan mini‐pig model of reperfused MI. Ten animals underwent 90 min left anterior descending artery occlusion and reperfusion. Three animals served as controls. Multiparametric CMR (1.5T) was performed at baseline, Day 2, Day 30 and in four animals on Day 60 after MI. Left ventricular (LV) volumes and infarct size were measured. T₁ and T₂ mapping sequences were performed to measure values in the infarct and remote regions. Remote region collagen fractions were compared between infarcted animals and controls. Procedure success was 80%. The model created large infarcts (28 ± 5% of LV mass on Day 2), which led to significant adverse myocardial remodeling that stabilized beyond 30 days. Native T₁ values did not reliably differentiate remote and infarct regions acutely. There was no evidence of remote fibrosis as indicated by partition coefficient and collagen fraction analyses. The infarct T₂ values remained elevated up to 60 days after MI. Multiparametric CMR in this model showed significant adverse ventricular remodeling 30 days after MI similar to that seen in humans. In addition, this study demonstrated that remote fibrosis is absent and that infarct T₂ signal remains chronically elevated in this model. These findings need to be considered when designing preclinical trials using CMR endpoints.     

骨髓间充质干细胞移植脑梗死大鼠：神经功能恢复与突触素的表达

BACKGROUND:Synaptophysin plays an important role in the recovery of neural function after cerebral ischemia. OBJECTIVE:To investigate the effects of bone marrow mesenchymal stem cell transplantation on nervous function and expression of synaptophysin after cerebral infarction. METHODS:Totally 60 rats were equivalently randomized into four groups, including sham operation, control, model and stem cell treatment groups. Rats in the control, model and stem cell treatment groups were used for preparing cerebral infarction models, and the remaining underwent the sham operation. After 1 day of modeling, bone marrow mesenchymal stem cells were transplanted into the rat lateral ventricle in the stem cell treatment group, and rats in the control group was given the injection of the same amount of PBS. After 1, 7 and 14 days of treatment, rat’s neurological function was scored on beam-walking test, rotarod test and screen test, and expression of synaptophysin was detected by RT-PCR and immunohistochemical assay. RESULTS AND CONCLUSION:At 7 and 14 days after treatment, the beam-walking test, rotarod test and screen test scores in the stem cell treatment group were significantly lower than those in the control and model groups (P < 0.05), and the above scores showed no significant differences between the control group and model group (P > 0.05). At 1 day after treatment, the mRNA expression of synaptophysin and the number of synaptophysin-positive cells in the sham operation group were significantly higher than those in the other three groups (P < 0.05); at 7 and 14 days after treatment, the mRNA expression of synaptophysin and the number of synaptophysin-positive cells in the stem cell treatment group were significantly increased compared with the other three groups (P < 0.05), and additionally, the mRNA expression of synaptophysin and the number of synaptophysin-positive cells in the sham operation group were significantly lower than those in the model and control groups (P < 0.05). These findings suggest that bone marrow mesenchymal stem cell transplantation can effectively promote the recovery of neurological function in cerebral infarction rats, and partially promote the formation of synaptophysin.     

The tyrosine kinase Abl is a component of macrophage podosomes and is required for podosome formation and function

Myeloid leukocytes form actin‐based plasma membrane protrusions, called podosomes, that are implicated in myeloid cell recruitment into tissues and cell migration within the interstitium. In this study, we show that tyrosine kinases of the Abl family are present in podosomes formed by murine and human macrophages. Silencing of Abl expression in bone marrow‐derived macrophages and monocyte‐derived macrophages by siRNA or Abl enzymatic inhibition with imatinib resulted in the disassembly of macrophage podosomes and the reduction of their capacity to degrade an extracellular matrix and migrate through matrigel matrices and endothelial cell monolayers. Additionally, macrophages deficient in Src‐family kinases, which cross‐talk with Abl in regulating macrophage migration, also demonstrated podosome disassembly. These findings suggest that podosome disassembly induced by Abl targeting may inhibit podosome‐dependent functions such as leukocyte recruitment into inflammatory sites and osteoclast‐dependent bone resorption.     

MRI Detection of Early Bone Metastases in B16 Mouse Melanoma Models

Bone metastasis causes significant morbidity in cancer patients, including bone pain, pathologic fractures, nerve compression syndrome, and hypercalcemia. Animal models are utilized to study the pathogenesis of skeletal metastases and to evaluate potential therapeutic agents. Previously published methods for imaging bone metastasis in rodent models have focused on identifying advanced stage metastasis using simple X-rays. Here we report MRI as a method for detecting early bone metastases in mouse models in vivo. B16 mouse melanoma cells were injected into the left cardiac ventricle of C57BL/6 mice and magnetic resonance (MR) images were obtained of the left leg following the development of metastatic disease, when tumor associated bone destruction was histologically present but not visible by X-ray. T1 and T2 relaxation times of bone marrow were measured in healthy control mice and B16 melanoma tumor-bearing mice. Mean T2 values for normal marrow were 28 ms (SD 5) and for diseased bone marrow were 41 ms (SD 3). T2 relaxation time of diseased bone marrow is significantly longer than that of normal bone marrow (P < 0.0001) and can be used as a marker of early bone metastases. These studies demonstrate that MR imaging can detect bone marrow metastases in small animals prior to development of cortical bone loss identified by X-ray.