Real-time dynamics of neutrophil clustering in response to phototoxicity-induced cell death and tissue damage in mouse ear dermis

ABSTRACT

Neutrophils are highly motile innate immune cells; they actively migrate in response to inflammatory signals. Using two-photon intravital microscopy, we discovered that neutrophils form stable clusters upon phototoxicity at a certain threshold. Without significant damage to the collagen structure of mouse dermis, neutrophils aggregated together with nearby neutrophils. Surprisingly, this in situ neutrophil clustering resulted in rigorous changes of migratory direction. The density of residing neutrophils was also a critical factor affecting clustering. Additionally, we found that the triggering point of neutrophil aggregation was correlated with the structure of the extracellular matrix in the ear dermis, where autofluorescence was strongly observed. This swarming behavior of neutrophils may reflect an unknown communication mechanism of neutrophils during migration under sterile injury.     

光片荧光显微镜特点及其应用

传统荧光显微镜由于对某些荧光分子存在光毒性、光损伤等方面的缺陷,无法满足对部分活体样本进行长时间观测的需求。光片荧光显微镜（light sheet fluorescence microscope,LSFM）是一种新型荧光显微镜,有别于激光共聚焦显微镜,其特殊的正交光路设计和高效的信号采集装置,使其具备低光毒性、低光漂白、低光损伤和高时空分辨率等优良特性,从而能对细胞及大尺度生物组织样本进行时空连续性较好的记录,尤其适宜于活体生物样品。基于此,概述了光片荧光显微镜的成像原理、成像优势、成像效果的改进与优化历程及其在生命科学领域应用所取得的研究成果,重点对近三年相关应用进行了汇总,并简要介绍了其在神经生物学、发育生物学、动物细胞生物学和植物科学领域中一部分代表性研究内容,最后,总结了光片荧光显微镜的优点与发展至今仍存在的不足,并对其在光遗传学和多组学研究中的潜在应用进行了展望,以期为研究人员提供较为系统的光片荧光显微镜相关基础知识、最新的研究应用进展以及未来的潜在应用方向,为研究人员提供参考。     

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Effects of BAPTA-AM, Forskolin, DSF and Z.VAD.fmk on PDT-induced apoptosis and m-THPC phototoxicity on B16 cells

As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.     

Vorkommen und biologische Bedeutung von Benzopyranen (Chromenen) und Benzofuranen in denAsteraceae

Benzopyrans (chromenes) and benzofurans are characteristic natural products of theAsteraceae and are found together with their biogenetic precursors primarily in the tribesAnthemideae, Astereae, Eupatorieae, Heliantheae, Inuleae, andSenecioneae. The accumulation of benzopyrans and benzofurans supports an arrangement of the tribes of theAsteraceae into two groups as suggested recently based on morphological characters. The methods for analysing this class of natural products are demonstrated with the American genusEncelia. Experiments on the localization of benzopyrans and benzofurans inEncelia species indicate that these compounds are stored in resin ducts. Several benzopyrans and benzofurans are biologically active and are repellent as well as toxic towards herbivorous insects. Experiments on structure — activity relationships indicate the biological activities as being influenced by the heterocycle (pyranvs. furan) as well as by the substituents of the aromatic ring. In addition, several tested compounds showed phototoxic activities.

Vorgetragen auf der Tagung der Deutschen Botanischen Gesellschaft, Wien, September 1984.     

Phototoxicity of plant secondary metabolites: Insect and mammalian perspectives

Phototoxic chemicals produced by plants have been studied in a number of contexts, most notably as protective agents against mammalian and insect herbivores. Although there are commonalities in the responses of these two groups of herbivores to plant phototoxins, there are differences as well. Whereas a greater range of chemical classes has been demonstrated to display phototoxicity against insects, considerably more information is available on symptomology of phototoxicity and mechanisms of action in mammals. The commonalities include alterations in behavior following ingestion, notably photophobia, disruption of integumentary integrity following contact or ingestion, and metabolic detoxification following ingestion, in the case of furanocoumarins involving cytochrome P450 monooxygenases. Not yet known to exist in insects are phototoxin-mediated effects on sensory (particularly visual) systems and phototoxicity resulting from abnormal chlorophyll metabolism. In order to gain greater understanding of the ecological significance of phototoxin-mediated plant defense against both insects and mammals, there is a need for more studies centered on natural associations. © 1995 Wiley-Liss, Inc.     

Superoxide dismutase in the anal gills of the mosquito larvae of Aedes aegypti: its inhibition by alpha-terthienyl.

The anal gills of the mosquito larvae of Aedes aegypti were shown to possess superoxide dismutase (EC 1.15.1.1) activity, which increased with the maturation of the larvae from instar 1 to instar 4. This enzyme was highly inhibited upon treatment of the larvae with alpha-terthienyl (2,2':5,2"-terthiophene) and subsequent exposure to long-wave ultraviolet light. Inhibition also occurred with treatment of the crude enzyme extract in a similar fashion. Exposure of the enzyme to the ultraviolet light alone or the treatment of the enzyme with alpha-terthienyl in darkness could not manifest this inhibition. This finding adds a new dimension to the complex mechanism(s) proposed for the photodynamic toxicity of alpha-terthienyl.     

Sediment-Associated Phototoxicity to Aquatic Organisms

Phototoxicity is a two to greater than 1000-fold increase in chemical toxicity caused by ultraviolet radiation (UV), which has been demonstrated in a broad range of marine and freshwater fish, invertebrates, and other aquatic organisms in water column exposures. Field collected sediments containing polycyclic aromatic hydrocarbons (PAHs) and other contaminants are phototoxic to sediment-dwelling organisms in laboratory tests, but in situ or field investigations of phototoxicity have not been reported. Sediment provides a pathway for the bioaccumulation of phototoxic chemicals through contact with and ingestion of bedded and suspended sediment, and maternal transfer, but risks are uncertain. Risks from sediment-associated phototoxicity will be greatest in areas of both high contaminant exposure (e.g., surficial and suspended sediments in harbors, outfall areas, and spill sites), and high UV exposure (e.g., high optical clarity or shallow depths). Organisms and life-stages most at risk will be those translucent to UV that inhabit the photic zone and near shore areas, but benthic organisms may have generally low UV exposure because of life history and morphological characteristics. Site-specific assessments are needed to characterize risks both spatially and temporally because of heterogeneous sediment contamination and large differences in species sensitivity and exposure pathways.     

Oxidative stress–mediated photoactivation of carbazole inhibits human skin cell physiology

Prolonged exposure of the earth's surface to the sun's ultraviolet radiation may result in various skin diseases and cataract. Carbazole (CBZ), as a polycyclic-aromatic hydrocarbon (PAH), is blended with a five-member nitrogen-containing ring. It is found in cigarette smoke, coal, eye kohl, tattoo ink, and wood combustion and affects various types of flora and fauna. Our findings suggest that CBZ generates reactive oxygen species (ROS) like through type-I photodynamic reaction and causes phototoxicity in the human keratinocyte cell line (HaCaT), which has been proved by mitochondrial dehydrogenase and neutral red uptake assays. CBZ induces single strand DNA damage. We have investigated the involvement of the apoptotic pattern of cell death and confirmed it by cytochrome C release from mitochondria and caspase-9 activation. Similarly, photo-micronuclei formation was associated to CBZ-induced phototoxicity. The results of this study strongly support that the upregulation of bax, cyto-C, apaf-1, casp-9 and down regulation of bcl2, keap-1, nrf-2, and hmox-1 genes cause apoptopic cell death. Downregulation of antioxidant genes showed a significant amount of ROS generation by photosensitized CBZ. Therefore, the current study will be a step forward to safeguard human beings from sunlight-induced photosensitive CBZ prolonged exposure.