Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Influence of the Lower Jaw Position on the Running Pattern

Introduction

The effects of manipulated dental occlusion on body posture has been investigated quite often and discussed controversially in the literature. Far less attention has been paid to the influence of dental occlusion position on human movement. If human movement was analysed, it was mostly while walking and not while running. This study was therefore designed to identify the effect of lower jaw positions on running behaviour according to different dental occlusion positions.

Methods

Twenty healthy young recreational runners (mean age = 33.9±5.8 years) participated in this study. Kinematic data were collected using an eight-camera Vicon motion capture system (VICON Motion Systems, Oxford, UK). Subjects were consecutively prepared with four different dental occlusion conditions in random order and performed five running trials per test condition on a level walkway with their preferred running shoes. Vector based pattern recognition methods, in particular cluster analysis and support vector machines (SVM) were used for movement pattern identification.

Results

Subjects exhibited unique movement patterns leading to 18 clusters for the 20 subjects. No overall classification of the splint condition could be observed. Within individual subjects different running patterns could be identified for the four splint conditions. The splint conditions lead to a more symmetrical running pattern than the control condition.

Discussion

The influence of an occlusal splint on running pattern can be confirmed in this study. Wearing a splint increases the symmetry of the running pattern. A more symmetrical running pattern might help to reduce the risk of injuries or help in performance. The change of the movement pattern between the neutral condition and any of the three splint conditions was significant within subjects but not across subjects. Therefore the dental splint has a measureable influence on the running pattern of subjects, however subjects individuality has to be considered when choosing the optimal splint condition for a specific subject.     

Looking for Progenitors: A Molecular Approach to Finding the Origins of An Invasive Weed

One of the major problems in determining the origin of invasive species is that often their arrival is unnoticed. Only when population levels increase is their presence noticed but by this time determining the point of arrival is confounded by the extensive spread of the species. Here we use molecular markers (ISSRs) to determine the origin of an invasive weed in the Kalahari region, a species for which several origins could be possible. We show that molecular markers can be useful tools in determining the origin of invasive species     

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure¹. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury¹. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized^1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules^1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types³. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely^1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals^4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish⁶. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish^7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.     

Proliferative Regeneration of Zebrafish Lateral Line Hair Cells after Different Ototoxic Insults

Sensory hair cells in the zebrafish lateral line regenerate rapidly and completely after damage. Previous studies have used a variety of ototoxins to kill lateral line hair cells to study different phenomena including mechanisms of hair cell death and regeneration. We sought to directly compare these ototoxins to determine if they differentially affected the rate and amount of hair cell replacement. In addition, previous studies have found evidence of proliferative hair cell regeneration in zebrafish, but both proliferation and non-mitotic direct transdifferentiation have been observed during hair cell regeneration in the sensory epithelia of birds and amphibians. We sought to test whether a similar combination of regenerative mechanisms exist in the fish. We analyzed the time course of regeneration after treatment with different ototoxic compounds and also labeled dividing hair cell progenitors. Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days. However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla. The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases. Inhibiting proliferation with flubendazole blocked the production of new hair cells and prevented the accumulation of additional precursors, indicating that proliferation has a dominant role during regeneration of lateral line hair cells.     

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.     

Sequential and Opposing Activities of Wnt and BMP Coordinate Zebrafish Bone Regeneration

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Morphological and Functional Divergence of the Lower Jaw Between Native and Invasive Red Foxes

Journal of Mammalian Evolution - The introduction of European red foxes in Australia in the late mid-nineteenth century has resulted in the spread of this invasive species across the continent. The...     

Neurocognitive Function in HIV-Infected Patients: Comparison of Two Methods to Define Impairment

Objective

To compare two definitions of neurocognitive impairment (NCI) in a large clinical trial of effectively-treated HIV-infected adults at baseline.

Methods

Hopkins Verbal Learning test-Revised (HVLT-R), Colour Trail (CTT) and Grooved Pegboard (GPT) tests were applied exploring five cognitive domains. Raw scores were transformed into Z-scores and NCI defined as summary NPZ-5 score one standard deviation below the mean of the normative dataset (i.e. <−1SD) or Z-scores <−1SD in at least two individual domains (categorical scale). Principal component analysis (PCA) was performed to explore the contribution of individual tests to the total variance.

Results

Mean NPZ-5 score was −0.72 (SD 0.98) and 178/548 (32%) participants had NPZ-5 scores <−1SD. When impairment was defined as <−1SD in at least two individual tests, 283 (52%) patients were impaired. Strong correlations between the two components of the HVLT-R test (learning/recall) (r = 0.73), and the CTT and (attention/executive functioning) (r = 0.66) were observed. PCA showed a clustering with three components accounting for 88% of the total variance. When patients who scored <−1SD only in two correlated tests were considered as not impaired, prevalence of NCI was 43%. When correlated test scores were averaged, 36% of participants had NPZ-3 scores <−1SD and 32% underperformed in at least two individual tests.

Conclusion

Controlling for differential contribution of individual test-scores on the overall performance and the level of correlation between components of the test battery used appear to be important when testing cognitive function. These two factors are likely to affect both summary scores and categorical scales in defining cognitive impairment.

Trial registration

EUDRACT: 2007-006448-23 and ISRCTN04857074.     

两种分离斑马鱼卵母细胞方法的比较

对培养斑马鱼卵母细胞体外成熟中的两种分离方法进行了比较。结果表明,在卵母细胞的分离过程中,机械分离法获得的完整卵细胞的比例(87.0%)显著多于EDTA-胰酶消化法(33.2%)(P<0.01)。但机械分离法较费时,其平均分离每个卵细胞所用的时间(0.69min)显著多于EDTA-胰酶消化法(0.17min)(P<0.01)。此外,经过18h 27℃培养,机械分离法获得的卵细胞的存活率(88.2%)显著高于EDTA-胰酶消化法所获卵细胞的存活率(60.1%),而采用机械分离法和消化分离法对所获卵细胞的成熟度-GVBD%分别为83.9%、77.5%,无显著差异(P>0.05)。基于数据所示,机械分离法可以获得存活率高、完整性好的卵细胞,但所需操作时间较长;EDTA-胰酶消化法操作便捷,但其分离出的卵细胞的活力较差;两种方法对卵细胞体外培养成熟率的影响无明显差异。     

Characteristics of Rod Regeneration in a Novel Zebrafish Retinal Degeneration Model Using N-Methyl-N-Nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.     

Rabies Cases in the West of China Have Two Distinct Origins

In China, rabies remains an ongoing threat to public health. Although control efforts have been effective in reducing the number of annual cases, the virus continues to spread into new areas. Tibet, Qinghai, Gansu and Ningxia in western China have, until recently, reported only a handful of events. However, since 2011, there have been increasing numbers of cases recorded in these areas. In this study, we report the collection and analysis of samples collected from these regions. We find that cases originate from two different sources. Strains collected from Gansu and Ningxia are closely related to the primary lineage associated with the current epizootic, whereas those from Tibet and Qinghai are related to the Arctic-like-2 lineage that is most commonly associated with wildlife cases in China. Thus, it appears that while the epizootic is beginning to encroach into Gansu and Ningxia, Tibet and Qinghai a significant number of rabies cases originate from wildlife.