Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.     

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.     

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using ''gold standard'' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

Plasticity of haemoglobin concentration and thermoregulation in a mountain lizard

The plastic capability of species to cope with the new conditions created by climate change is poorly understood. This is particularly relevant for organisms restricted to high elevations because they are adapted to cold temperatures and low oxygen availability. Therefore, evaluating trait plasticity of mountain specialists is fundamental to understand their vulnerability to environmental change. We transplanted mountain lizards, Iberolacerta cyreni, 800 m downhill to evaluate the plastic response in body condition, thermoregulation traits, haemoglobin level, and haemoparasite load. Initial measurements of body mass, total haemoglobin concentration ([Hb]), hematic parasite intensities, dorsal luminance, and thermoregulatory behaviour were resampled after two and four weeks of acclimation. We also tested whether an anti-parasitic drug reduced haemoparasite intensity. After only two weeks of acclimation to a lower elevation, lizards decreased 42% in [Hb], had 17% less parasite intensities, increased body condition by 25%, and raised by ~3% their mean preferred temperatures and their voluntary thermal maximum. The anti-parasitic treatment had no significant effect on the intensity of hematic parasites, but our results suggest that negative effects of haemoparasites on [Hb] are relaxed at lower elevation. The rapid plastic changes observed in thermal preferences, body condition, [Hb], and parasite intensity of I. cyreni demonstrate a potential plastic response of a mountain specialist. This may be adaptive under the climatic extremes typical of mountain habitats. However, there is uncertainty in whether the observed plasticity can also help overcome long term environmental changes.     

Teaching ethnic and racial relations: some comments on Michael Banton

Banton's critique of race and ethnic relations highlights differences between the U.K. and the U.S.A. The interplay between ethnic politics and the teaching of the subject in the universities reflects differing reactions to the demise of Marxism, the impact of globalization and the growth of transnationalism. In America, anthropology, economics, cultural studies and conflict resolution have had a mixed impact on the field, but an examination of textbooks over the past thirty years - as well as Banton's prolific scholarship - suggests that most of the core problems remain the same.     

暗腹雪鸡与海兰褐鸡血液生理生化指标的比较

为了解暗腹雪鸡（Tetraogallus himalayensis）正常血液生理生化特性,本文对暗腹雪鸡21项血液生理生化指标进行了测定,并与海兰褐鸡（Gallus gallus domesticus var. Hyline）进行了比较。测定结果,暗腹雪鸡的红细胞纵径及横纵径乘积、平均红细胞容积及平均红细胞血红蛋白含量极显著大于海兰褐鸡（P<0.01）;暗腹雪鸡天冬氨酸氨基转移酶和胆固醇含量均极显著高于海兰褐鸡（P<0.01）。其他各项血液生理生化指标两禽种间之间差异不显著。实验结果表明,暗腹雪鸡血液生理生化指标与家养蛋鸡之间有一定差异,暗腹雪鸡对高原低氧环境有较强的适应性可能与其红细胞体积较大、平均红细胞容积及平均红细胞血红蛋白含量较高有一定关系。     

PBL联合CBL教学方法在胃肠外科临床见习教学中的应用

目的:探讨PBL联合CBL教学模式在医学生胃肠外科见习教学中的应用效果.方法:选取50名2008级胃肠外科见习医学生作为研究对象,将其随机分成两组:实验组采用PBL联合CBL教学模式,对照组则仅采用传统LBL教学模式.通过出科考核及问卷调查两种方式评价胃肠外科见习教学效果.结果:显示PBL+ CBL组的考试成绩要明显优于LBL组,差异有统计学意义(P＜ 0.05),并且见习后的问卷调查结果提示学生对PBL+ CBL的教学模式满意度较高.结论:我们的研究结果表明PBL+ CBL双轨教学模式受到学生欢迎,在提高学生理论知识水平方面比LBL模式更为有效.     

青年教师对医学临床课程教学能力现状调查及应对策略研究

如何提高我国临床课程青年教师团队的素质与教学能力,是近年来伴随国内医学教育发展扩大而日益突出的问题。作为当前医学临床课程的教学主力,青年教师的教学能力会对医学临床课程教学效果产生重大影响。但绝大部分临床课程青年教师都毕业于医学院校,未曾接受专业的师范类培训,导致尽管自身临床知识丰富,但教学水平明显受限的情况发生。本研究采取对我院各科室青年临床教师进行问卷调查的方法,深入分析青年教师的临床教学能力现状与问题,并提出改进方案,以使他们能够更快完成青年临床医师与青年临床教师之间的角色变换,提升自身教学能力,进而提高我院临床课程的教学质量。