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.     

髋骨闭孔沟的临床解剖学测量

目的：对闭孔沟进行解剖学观察和测量,为相关应用提供临床解剖学依据和丰富人类学数据。方法：选取结构完整、解剖标志清晰的髋骨108例,其中男性髋骨59例（左侧36例,右侧23例）,女性髋骨49例（其中左侧20例,右侧29例）,对闭孔沟进行观察并测量其长度和闭孔沟中点宽度。结果：试验测得男性左、右侧闭孔沟长度分别为31．4±3．4mm、32．1±2．4min,女性左、右侧闭孔沟长度分别为19．4±2．2mm、20．8±2．7mm;统计学分析显示同性别左、右侧差异无统计学意义（P〉0．05）,男、女性之间差异有统计学意义（P〈0．05）。男性左、右侧闭孔沟中点宽度分别为9．7±1．6mm、10．7±1．5mm,女性左、右侧闭孔沟中点宽度分别为12．1±1．7mm、11．6±2．0mm;同性别左、右侧及男、女间差异均无统计学意义（P〉0．05）。结论：男、女性闭孔沟测量数据可为闭孔处疾病的诊断和治疗提供应用解剖学的考虑依据并丰富人类学数据。     

Anatomical aspects of angiosperm root evolution

Background and Aims

Anatomy had been one of the foundations in our understanding of plant evolutionary trends and, although recent evo-devo concepts are mostly based on molecular genetics, classical structural information remains useful as ever. Of the various plant organs, the roots have been the least studied, primarily because of the difficulty in obtaining materials, particularly from large woody species. Therefore, this review aims to provide an overview of the information that has accumulated on the anatomy of angiosperm roots and to present possible evolutionary trends between representatives of the major angiosperm clades.

Scope

This review covers an overview of the various aspects of the evolutionary origin of the root. The results and discussion focus on angiosperm root anatomy and evolution covering representatives from basal angiosperms, magnoliids, monocots and eudicots. We use information from the literature as well as new data from our own research.

Key Findings

The organization of the root apical meristem (RAM) of Nymphaeales allows for the ground meristem and protoderm to be derived from the same group of initials, similar to those of the monocots, whereas in Amborellales, magnoliids and eudicots, it is their protoderm and lateral rootcap which are derived from the same group of initials. Most members of Nymphaeales are similar to monocots in having ephemeral primary roots and so adventitious roots predominate, whereas Amborellales, Austrobaileyales, magnoliids and eudicots are generally characterized by having primary roots that give rise to a taproot system. Nymphaeales and monocots often have polyarch (heptarch or more) steles, whereas the rest of the basal angiosperms, magnoliids and eudicots usually have diarch to hexarch steles.

Conclusions

Angiosperms exhibit highly varied structural patterns in RAM organization; cortex, epidermis and rootcap origins; and stele patterns. Generally, however, Amborellales, magnoliids and, possibly, Austrobaileyales are more similar to eudicots, and the Nymphaeales are strongly structurally associated with the monocots, especially the Acorales.     

Matching roots to their environment

Background

Plants form the base of the terrestrial food chain and provide medicines, fuel, fibre and industrial materials to humans. Vascular land plants rely on their roots to acquire the water and mineral elements necessary for their survival in nature or their yield and nutritional quality in agriculture. Major biogeochemical fluxes of all elements occur through plant roots, and the roots of agricultural crops have a significant role to play in soil sustainability, carbon sequestration, reducing emissions of greenhouse gasses, and in preventing the eutrophication of water bodies associated with the application of mineral fertilizers.

Scope

This article provides the context for a Special Issue of Annals of Botany on ‘Matching Roots to Their Environment’. It first examines how land plants and their roots evolved, describes how the ecology of roots and their rhizospheres contributes to the acquisition of soil resources, and discusses the influence of plant roots on biogeochemical cycles. It then describes the role of roots in overcoming the constraints to crop production imposed by hostile or infertile soils, illustrates root phenotypes that improve the acquisition of mineral elements and water, and discusses high-throughput methods to screen for these traits in the laboratory, glasshouse and field. Finally, it considers whether knowledge of adaptations improving the acquisition of resources in natural environments can be used to develop root systems for sustainable agriculture in the future.     

Secretory structures at syconia and flowers of Ficus enormis (Moraceae): A specialization at ostiolar bracts and the first report of inflorescence colleters

The fig (Ficus L.) infructescence, called syconium, is a receptacle with an apical opening, the ostiole, closed by bracts. The ostiolar bracts produce an exudate, which is rather conspicuous in some species. It has not been histochemically analyzed yet, and the structures responsible for its production are still unknown. Some wild growing species of Ficus from Brazil produce high amounts of this ostiolar exudate. Ficus enormis (Mart. ex Miq.) Miq. grows as trees or shrubs in the Atlantic rainforest. Our goal was to identify the secretory structures present in the inflorescence and, to characterize histochemically the ostiolar tissues and exudates. Syconia samples of F. enormis were processed and stained according to the usual techniques in plant anatomy. The morphological analysis revealed different types of bracts, one type specialized in secretion, another showing transitional characteristics between secretory and non-secreting bracts, and a third one being non-secreting. They are designated as secretory ostiolar bracts, transitional bracts and wall bracts. The floral bracteoles, digital-shaped colleters present in the ostiole, at the syconium axis and at the flower receptacle, were also analyzed. All have similar structure, like finger-shaped secretory trichomes. The colleters present among ostiolar bracts may contribute to production and composition of the ostiole exudate.