PubMed Health⌕ Search

Biomedical subjects

Robert M Zucker

Publications and source records attributed to Robert M Zucker.

16 recordsLinked to original sources
PubMed2006

Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis.

BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. METHODS: The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. RESULTS: Tissues as thick as 500 microm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). CONCLUSIONS: The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-microm thick.

Amines↗
PubMed2006

Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy.

BACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy. METHODS: Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths. RESULTS: Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image. CONCLUSIONS: Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.

Algorithms↗
PubMed2006

Quality assessment of confocal microscopy slide-based systems: instability.

BACKGROUND: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. METHODS: A number of tests have been devised to evaluate equipment performance and instability. The following four instability tests are described: galvanometer scanning, stage drift, correct wavelength spectral detection, and long-term laser power. RESULTS: Quality assurance tests revealed that a confocal microscope can become unstable in the following parameters, yielding inaccurate data: laser power, PMTs functionality, spectrophotometer accuracy, galvanometer scanning and laser stability, and stage drift. Long-term laser power stability has been observed to vary greatly. CONCLUSIONS: Confocal systems can become unstable in the following parameters: long-term laser power, galvanometer scanning, spectrophotometer accuracy, and stage stability. Instability in any of these parameters will affect image quality. Laser power fluctuations result from either a defective Acousto-optic tunable filter or improper heat dissipation. Spectrophotometer instability will generate unreliable spectra data, extra light reflections, and poor image quality. Galvanometer scanning instability yields poor image quality while microscope stage drift results in a sample going out of the plane of focus. With minor modifications, these tests may be applicable to other slide-based systems.

Animals↗
PubMed2006

Quality assessment of confocal microscopy slide based systems: performance.

BACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection device consisting of a CCD camera or a PMT. The optical principles employed is slide-based systems are similar to those of confocal microscopes (CLSM). METHODS: The following tests evaluated confocal equipment performance: dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, PMT reliability, and system noise. RESULTS: Quality assurance tests provide a basis to determine if the equipment is operating correctly. Laser power, PMTs function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance and laser stability were tested colocalization of UV and visible peaks of a bead should be less than 210 nm. Interference contrast optics decrease fluorescence resolution. CONCLUSIONS: QA tests that assess CLSM system performance are also applicable to other slide-based systems. By utilization this type of testing approach, the subjective nature of assessing the CLSM may be eliminated. These tests serve as guidelines for other investigators to ensure that their machines are providing data that is accurate with the necessary resolution, sensitivity and precision.

Animals↗
PubMed2006

Evaluation of confocal microscopy system performance.

The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a "pretty picture." Without the use of functional tests, many of the machines could be working at suboptimal performance levels, delivering suboptimum performance and possibly misleading data. To replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, photomultiplier tube quality, overall machine stability, and system noise. These tests will help serve as a guide for other investigators to ensure that their machines are working correctly to provide data that are accurate with the necessary resolution, sensitivity, and precision. Utilization of this proposed testing approach will help eliminate the subjective nature of assessing the CLSM and allow different machines to be compared. These tests are essential if one is to make intensity measurements.

Animals↗
PubMed2005

Wavelength and alignment tests for confocal spectral imaging systems.

Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference spectra within a particular system as well as between related and unrelated instruments. We also find evidence of instability that, if not diagnosed, could lead to inconsistent data. This variability confirms the need for diagnostic tools to provide a standardized, objective means of characterizing instability, evidence of misalignment, as well as performing calibration and validation functions. Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emit distinct, stable spectral features, in place of a sample. An MIDL characterization verifies the accuracy and consistency of a CSI system and validates acquisitions of biological samples. We examined a total of 10 CSI systems, all of which displayed spectral inconsistencies, enabling us to identify malfunctioning subsystems. Only one of the 10 instruments met its optimal performance expectations. We have found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrument errors and measure accuracy and reproducibility under normalized conditions. Using this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. It is evident that if CSI systems of a similar make and model were standardized to reveal the same spectral profile from a standard light source, then researchers could be confident that real-life experimental findings would be repeatable on any similar system.

Animals↗
PubMed2004

Developmental toxicity of methanol: Pathogenesis in CD-1 and C57BL/6J mice exposed in whole embryo culture.

BACKGROUND: Methanol causes axial skeleton and craniofacial defects in both CD-1 and C57BL/6J mice during gastrulation, but C57BL/6J embryos are more severely affected. We evaluated methanol-induced pathogenesis in CD-1 and C57BL/6J embryos exposed during gastrulation in whole embryo culture. METHODS: Conceptuses with five to seven somites were exposed to 0, 1, 2, 3, 4, or 6 mg methanol/ml culture medium for 24 hr and embryonic morphology was assessed. Cell death was evaluated by histology and LysoTracker red staining, and cell-cycle distribution was evaluated by flow cytometry. RESULTS: In C57BL/6J embryos, craniofacial defects were observed at 3 mg methanol/ml and greater. The response for CD-1 embryos was different, with increased dysmorphology only at 6 mg/ml. However, protein content in CD-1 embryos was reduced at 3 mg methanol/ml and above, indicating growth retardation. Yolk sac toxicity occurred only at 6 mg methanol/ml in both strains. Methanol caused only small changes in cell-cycle distribution, while cell death was induced at 4 and 6 mg methanol/ml in both strains after 8 hr. The extent of cell death after 8 hr was greater in C57BL/6J embryos, and increased over time through 18 hr; in contrast, CD-1 embryos showed less cell death at 18 than at 8 hr, suggesting recovery. CONCLUSIONS: Cell death plays a prominent role in methanol-induced dysmorphogenesis, while cell-cycle perturbation may not. Differences in the extent of cell death between CD-1 and C57BL/6J embryos correlated with differences in the severity of dysmorphogenesis.

Abnormalities, Drug-Induced↗
PubMed2004

Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice.

BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.

Animals↗
PubMed2004

Methanol exposure during gastrulation causes holoprosencephaly, facial dysgenesis, and cervical vertebral malformations in C57BL/6J mice.

BACKGROUND: Exposure of pregnant outbred CD-1 mice to methanol during the period of gastrulation results in exencephaly, cleft palate, and cervical vertebra malformations [Rogers and Mole, Teratology 55: 364, 1997], while inbred C57BL/6J mice are sensitive to the teratogenicity of ethanol. C57BL/6J fetuses exhibit the holoprosencephaly spectrum of malformations after maternal exposure to ethanol during gastrulation, but the sensitivity of C57BL/6J mice to methanol-induced teratogenesis has not been previously described. METHODS: Pregnant C57BL/6J mice were administered two i.p. injections totaling 3.4 or 4.9 g/kg methanol or distilled water four hrs apart on gestation day 'GD' 7. On GD 17, litters were examined for numbers of live, dead and resorbed conceptuses, fetuses were weighed as a litter and examined externally, and all fetuses were double stained for skeletal analysis. RESULTS: No maternal intoxication was apparent, but the high dosage level caused a transient deficit in maternal weight gain. The number of live fetuses per litter was reduced at both dosages of methanol, and fetal weight was lower in the high dosage group. Craniofacial defects were observed in 55.8% of fetuses in the low dosage group and 91.0% of fetuses in the high dosage group, including micro/anophthalmia, holoprosencephaly, facial clefts and gross facial angenesis. Skeletal malformations, particularly of the cervical vertebrae, were observed at both dosages of methanol, and were similar to those previously reported in the CD-1 mouse following methanol exposure. CONCLUSIONS: The types of craniofacial malformations induced in the C57BL/6J mouse by methanol indicate that methanol and ethanol have common targets and may have common modes of action.

Abnormalities, Drug-Induced↗
PubMed2004

Calibration and validation of confocal spectral imaging systems.

BACKGROUND: Confocal spectral imaging (CSI) microscopic systems currently on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, some CSI systems have been found to present inconsistent spectral profiles of reference spectra within a particular system and between related and unrelated instruments. This variability confirms that there is a need for a standardized, objective calibration and validation protocol. METHODS: Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg(+), Ar(+), and inorganic fluorophores that emit distinct, stable, spectral features in place of a sample. We derived reference spectra from the MIDL data to accurately predict the spectral resolution, ratio of wavelength to wavelength, contrast, and aliasing parameters of any CSI system. We were also able to predict and confirm the influence of pinhole diameter on spectral profiles. RESULTS: Using this simulation, we determined that there was good agreement between observed and theoretical expectations, thus enabling us to identify malfunctioning subsystems. We examined eight CSI systems and one nonconfocal spectral system, all of which displayed spectral inconsistencies. No instrument met its optimal performance expectations. In two systems, we established the need for factory realignment that had not been otherwise recognized. CONCLUSIONS: We found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrumental errors and measure accuracy and reproducibility under normalized conditions. With this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. We determined that, if CSI systems were standardized to produce the same spectral profile of a MIDL lamp, researchers could be confident that the same experimental findings would be obtained on any CSI system.

Animals↗
PubMed2003

Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat.

Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.

Animals↗
PubMed2003

Quantitative fluorescence of 5-FU-treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red.

BACKGROUND: LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and tissues. After cell death, a high level of lysosomal activity (acidic enzyme) is expressed in tissues resulting from phagocytosis of apoptotic bodies by neighboring cells. LT was shown previously to be an indicator of cell death in a manner similar to other standard assays (Annexin, terminal dUTP nick end labeling, Nile blue sulfate, neutral red, and acridine orange). METHODS: LT fluorescence in fetal rat hindlimbs at gestational day 14 was measured 8 h after administration of the teratogen, 5-fluorouracil (5-FU), with the use of confocal laser scanning microscopy (CLSM). Four dose levels of 5-FU (0, 20, 30, and 40 mg/kg) were studied. The preparation technique involved staining with LT, paraformaldehyde fixation, methanol dehydration, and clearance with benzyl alcohol and benzyl benzoate. After this treatment, the limb was nearly transparent and ready for CLSM analysis. RESULTS: LT staining was observed in specific regions undergoing apoptosis in normal (control) hindlimbs. After 5-FU treatment, highly fluorescent regions appeared in the progress zone (PZ) of the limb. A dose-dependent response to 5-FU treatment was observed. Compared with controls, hindlimbs treated with 20, 30, and 40 mg/kg of 5-FU exhibited more fluorescence within the highly proliferative PZ. These results showed a dose-response relation between 5-FU exposure and LT uptake. CONCLUSIONS: We found that three-dimensional volumetric regions indicating a high level of fluorescence in the embryonic limb bud can be quantified with three different computer analysis programs. The combination of a sample preparation procedure that clears tissue, a CLSM technique that addresses the equipment variables, and an application of statistical population analysis procedures enabled the visualization and quantification of fluorescence in entire fetal rat hindlimbs that were approximately 500 microm in thickness.

Animals↗
PubMed2003

Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes, zygotes, and pre-implantation stage embryos.

Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature oocytes and decline after fertilization, suggest that GSH synthesis may be associated with cell cycle events. To explore this possibility, we measured the concentrations of GSH in Golden Hamster oocytes and zygotes at specific stages of oocyte maturation and at intervals during the first complete embryonic cell cycle. Between 2 and 4 hr after the hormonal induction of oocyte maturation, GSH concentrations increased significantly (approximately doubling) in both oocytes and their associated cumulus cells. This increase was concurrent with germinal vesicle breakdown and the condensation of metaphase I chromosomes in the oocyte. GSH remained high in ovulated, metaphase II (MII) oocytes, but then declined significantly, by about 50%, shortly after fertilization, as the zygote progressed back into interphase (the pronucleus stage). GSH concentrations then plummeted by the two-cell embryo stage and remained at only 10% of those in MII oocytes throughout pre-implantation development. These results demonstrate that oocyte GSH concentrations fluctuate with the cell cycle, being highest during meiotic metaphase, the critical period for spindle growth and development and for sperm chromatin remodeling. These observations raise the possibility that GSH synthesis in maturing oocytes is regulated by gonadotropins, and suggest that GSH is more important during fertilization than during pre-implantation embryo development.

Animals↗
PubMed2002

Hindbrain and cranial nerve dysmorphogenesis result from acute maternal ethanol administration.

Acute exposure of mouse embryos to ethanol during stages of hindbrain segmentation results in excessive cell death in specific cell populations. This study details the ethanol-induced cell loss and defines the subsequent effects of this early insult on rhombomere and cranial nerve development. Ethanol at a teratogenic dosage (2.9 g/kg) or a comparable volume of vehicle was administered in each of two intraperitoneal injections to pregnant C57BL/6J mice on gestational day (GD) 8, 8 h, and GD 8, 12 h (defined hereafter as GD 8.5). Ethanol-exposed GD 9 embryos, visualized in three dimensions using laser scanning confocal microscopy of LysoTracker Red fluorescence or Nile blue sulphate vital staining, displayed excessive apoptosis in the rostral hindbrain, specifically within rhombomeres 1-3, as well as in cranial neural crest cells and ectodermal placodes. Comparably treated embryos examined on GD 10.5-11 illustrated a disproportionate reduction in the length of the rostral hindbrain. Examination of plastic histological sections of GD 9 embryos and via scanning electron microscopy on GD 10 revealed deficiencies in the hindbrain, with a phenotype including abnormal rhombomere segmentation and an extremely small fourth ventricular roofplate. Whole-mount antineurofilament immunohistochemistry on GD 10.5 and GD 11 illustrated a variety of cranial nerve abnormalities ranging from fused or absent ganglia to ectopic or disorganized fibers. In addition, a delay in the development of the glossopharyngeal (IX) nerve/ganglia complex was observed. These hindbrain and cranial nerve abnormalities are discussed in the context of the genesis of human alcohol-related birth defects and neurodevelopmental disorder.

Alcohol-Induced Disorders, Nervous System↗
PubMedDate not supplied

Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro.

We previously established that levels of the sperm membrane protein, SP22, are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in postmeiotic germ cells. In this study, polyclonal and monoclonal antibodies were generated to study the expression of SP22 in the testis and epididymis, and to determine whether SP22 plays a coincidental or causal role in fertility. Polyclonal antiserum was raised in sheep against full-length recombinant rat SP22 (rSP22). Hybridoma clones were generated from mice immunized with rSP22 and boosted with native SP22; positive clones were used for ascites production. Immunoblots indicated that affinity-purified anti-rSP22 immunoglobulin (Ig) and ascites Ig recognized denatured and native SP22, respectively. Linear epitope mapping of the 189-amino acid SP22 sequence revealed 3 distinct peptide sequences recognized by anti-rSP22 Ig, and 1 sequence recognized by ascites Ig. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites antibodies. Isolated rete testis sperm revealed discrete staining over the cytoplasmic droplet, whereas staining was apparent over the equatorial segment of the head by the time sperm reached the caput epididymidis. Clear cells were, interestingly, immunostained along the length of the epididymis. Ascites Ig and anti-SP22 Ig each recognized the equatorial segment of sperm heads from rat, hamster, bull, rabbit, and human. Ascites Ig and affinity-purified anti-rSP22 Ig each significantly inhibited the fertility of cauda epididymal sperm from the rat in vivo, as well as the fertilization rates of cauda epididymal sperm in vitro. Moreover, affinity-purified anti-rSP22 significantly inhibited in vitro fertilization of both zona-intact and zona-free hamster oocytes, suggesting that SP22 may play a role in both the zona penetration and membrane fusion steps of fertilization.

Amino Acid Sequence↗