Techniques for the Study of Apoptosis in Bone.

Osteocyte apoptosis has been associated with a number of clinical conditions in bone and with the targeted turnover of specific skeletal areas. There has been great interest in the identification of the mechanisms by which apoptosis is regulated in bone and in the biological role that this process plays in bone metabolism and associated bone disease or loss of structural integrity. Here we describe several methods for the detection of apoptosis in bone sections and in bone cell cultures.

Methods for Assessing Apoptosis and Anoikis in Normal Intestine/Colon and Colorectal Cancer.

Caspase-dependent apoptosis, including its distinct cell death subroutine known as anoikis, perform essential roles during organogenesis, as well as in the maintenance and repair of tissues. To this effect, the continuous renewal of the human intestinal/colon epithelium is characterized by the exfoliation by anoikis of differentiated cells, whereas immature/undifferentiated cells may occasionally undergo apoptosis in order to evacuate daughter cells that are damaged or defective. Dysregulated epithelial apoptosis is a significant component of inflammatory bowel diseases. Conversely, the acquisition of a resistance to apoptosis represents one of the hallmarks of cancer initiation and progression, including for colorectal cancer (CRC). Furthermore, the emergence of anoikis resistance constitutes a critical step in cancer progression (including CRC), as well as a limiting one that enables invasion and metastasis.Considering the implications of apoptosis/anoikis dysregulation in gut physiopathology, it therefore becomes incumbent to understand the functional determinants that underlie such dysregulation-all the while having to monitor, assess, or evidence apoptosis and/or anoikis. In this chapter, methodologies that are typically used to assess caspase-dependent apoptosis and anoikis in intestinal/colonic normal and CRC cells, whether in vivo, ex vivo, or in cellulo, are provided.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Detection of end-stage apoptosis by ApopTag® TUNEL technique.

DNA fragmentation, the end stage of apoptosis, is the measure of ultimate demise of the cell. A convenient method for examining apoptosis via DNA fragmentation is by the Terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay where the DNA strand breaks are detected by enzymatically labeling the free 3'-OH termini with modified nucleotides. ApopTag(®) kits detect single-stranded and double-stranded breaks associated with apoptosis. This technique is also helpful to distinguish between apoptotic and necrotic cell death where the latter is associated with random DNA fragment lengths producing a DNA smear. Apoptotic cells stained positive with ApopTag(®) kits are easier to detect and their identification is more certain, as compared to the examination of simply histochemically stained tissues. In addition, quantitative results can be obtained using flow cytometry and apoptotic cells can be differentiated from necrotic cells with greater than tenfold sensitivity.

Albumin ameliorates tissue plasminogen activator-mediated blood-brain barrier permeability and ischemic brain injury in rats.

OBJECTIVE: Tissue plasminogen activator (tPA) may aggravate ischemic neuronal damage after focal cerebral ischemia and increase blood-brain barrier permeability. Human serum albumin has neuroprotective effects on ischemic stroke. However, whether albumin can attenuate the deleterious effects of tPA is yet unknown. METHODS: In the present study, we attempted to determine the effects of albumin on cerebral injury and blood-brain barrier disruption induced by middle cerebral artery occlusion for 2 hours followed by 24 hours of reperfusion and tPA. RESULTS: We found that infarct volume in rats which received saline and tPA was 35.6 +/- 3.8% (mean +/- SEM) and 50.9 +/- 4.5%, respectively. There was significant difference between the two groups (p<0.01). The infarct volume in rats that received tPA with albumin was significantly decreased to 29.2 +/- 3.3% (p<0.05), compared with tPA-only-treated group. Combination therapy using tPA with albumin also improved neurological deficits and reduced brain edema significantly (p<0.05). Relative to tPA-treated group, rats that received combination therapy using tPA with albumin had significantly reduced blood-brain barrier permeability, evaluated by quantitation of Evans blue leakage (p<0.05). CONCLUSION: In acute ischemic stroke, combination therapy using tPA with albumin can attenuate the deleterious effects of tPA.

Histological hair removal study by ruby or alexandrite laser with comparative study on the effects of wavelength and fluence.

BACKGROUND: Several different laser systems are currently used to remove unwanted hairs. In this study, we studied follicular changes following hair removal with ruby or alexandrite lasers at different fluences. METHODS: Unwanted hairs were treated with a ruby laser (Chromos 694, ICN PhotonIcs, UK) at 10, 14 or 18 J/cm2 or with an alexandrite laser (LPIR, Cynosure, USA) at 11, 14 or 17 J/cm2. A 3 mm skin punch biopsy was taken immediately after each laser exposure and also 1 month later. Specimens were stained for histological observation. They were observed using immunohistochemistry with antibodies recognizing factor VIII related antigen or PCNA, and also by the TUNEL method. Similarly, electron microscopic observation was examined. RESULTS: Immediately after the laser exposure, moderate follicular damage was observed following treatment with either type of laser. One month later, cystic formation of hair follicles and foreign body giant cells were observed in skin treated with either type of laser. A similar fluence with either laser treatment resulted in similar histological changes. CONCLUSION: In this study, the histological changes following treatment with a ruby or an alexandrite laser at the same fluence are similar.

[Protective effects of growth hormone en cell cultures of the central nervous system]. / Efecto protector de la hormona de crecimiento en cultivos de células del sistema nervioso central.

OBJECTIVE: We examine a possible protective effect from growth hormone (GH) against radiation in cell cultures of rat embryon brain cortex. MATERIALS AND METHODS: Brain cortexes were removed from rat embryos of 17 days development and prepared for cell culture. The culture medium of half the plates was enriched with 500 ng/ml GH. All plates received a radiation dose of 3 Gy per plate and were again incubated for 24 hours. The TUNEL technique was employed to verify cell apoptosis in the irradiated plates and compare its level in plates with and without GH. CONCLUSIONS: We observed that irradiated cultures with GH had significantly less cell apoptosis than those without GH and concluded that this hormone excercised a protective effect on the cell cultures. The present study demonstrated the protective effect from GH in rat embryonary cells and suggests the need for future in vitro and in vivo studies using central nervous system cells.

Automated analysis of global ischemia-induced CA1 neuronal death using terminal UTP nick end labeling (TUNEL).

In the brain, DNA fragmentation is associated with apoptotic cell death following ischemic/excitotoxic damage. Fragmented DNA can be detected in situ by labeling the 3'OH termini of the internucleosomal generated fragments with deoxynucleotides, through a process known as terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling, or TUNEL. TUNEL is frequently being used to assess neuronal death following cerebral ischemia in a number of animal models. However, conventional techniques for TUNEL can be time consuming, and are often subjective and thus can lead to inconsistencies among investigators. Moreover, the lack of tools for its quantification and standardization limits the use of this technique in assessing the magnitude of cell death. In the present report, we describe an improved higher throughput technique for TUNEL staining at room temperature on a BioGenex automated stainer, and its subsequent quantitative analysis using NORTHERN ECLIPSE, an imaging analysis program. Its implementation allows us to effectively quantify TUNEL positive cells in the CA1 region of the hippocampus following global forebrain ischemia in rats. We conclude that this general histological technique can be applied to the study of cell death in numerous other experimental models.