Zone Of Cell Death Research Articles

The role of Escherichia coli bacterioferritin in stressed induced conditions

Escherichia coli possesses at least two iron storage proteins: FtnA (ferritin) and Bfr (Bacterioferritin). FtnA is shown to be a major iron storage protein in E. coli . Although Bfr resembles the ferritin in many of its structural and functional features, the role of Bfr in E. coli is not clear. The aim of this study was to investigate the role of E. coli Bfr in stress induced conditions. In this study, E. coli bfr - (lacking the chromosomal bfr gene) and E. coli bfr + (the same as the former but containing the E. coli bfr overexpression vector) mutants were used. In order to examine the role of E. coli Bfr in stress induced conditions, E. coli cells were grown in the presence of excess iron. In the case of hydrogen peroxide toxicity, the cells were grown in an agar plate containing hydrogen peroxide discs and the results of toxicity were expressed as the size of the cell death zone. In order to find out whether Bfr supports cell growth under iron and phosphate starvation, the E. coli cells were grown in iron and phosphate depleted media. In E. coli the overproduced Bfr did not support the growth of cells under iron and phosphate deficient conditions. The results showed that overproduced Bfr in E. coli does not contribute to the survival of the cell in iron and phosphate starvation. Furthermore, it does not enhance the resistance of E. coli against iron and hydrogen peroxide toxicity.

106 Responses of pancreatic cancer cells under cryoablative conditions

Pancreatic cancer (PaCa) incidence and mortality rates continue to increase, driving the need for improved diagnostics and more effective therapies.Combinatorial approaches in PaCa have focused on the use of multiple chemotherapeutic drugs, while cryotherapeutic options have been underutilized.Given past success in prostate cancer, in this study we investigated the potential for multi-modal treatment utilizing cryoablation in conjunction with low dose chemotherapy for the treatment of PaCa.Both standard tissue culture models and the 3-D tissue engineered (PaCaTEM) approach were studied, providing a more robust assessment of the cellular responses to cryotherapy and the associated adjuncts.The human PaCa cell line (Bx-PC3) was evaluated in vitro and in a vivo-like tissue engineered model (PaCaTEM). Bx-PC3 were exposed to a range of freezing temperatures from −10 °C to −20 °C and compared to non-frozen controls with and without pretreatment with 0.025 μg/ml 5-FU. PaCaTEM models were frozen in situ following a single 5 min freeze protocol using the supercritical nitrogen (SCN) cryoablation system with a 1.5 mm × 3 cm cryoprobe.Cell survival following the treatment regimes were assessed in the in vitro studies using the metabolic activity indicator, alamarBlue and microfludic flow cytometry (MFC). Assessment of PaCaTEM samples included monitoring of iceball size and isothermal distribution during the freezing process followed by fluorescence microscopic examination of cell death at 1 and 24 h (Vybrant apoptosis assay).Freezing exposure to −10 °C did not impact overall Bx-PC3 viability, however exposure to −15 °C and −20 °C resulted in a significant loss of viability (90% and 98%, respectively), which was found to be more effective with concomitant 5-FU . At temperatures of −25 °C and colder in vitro, no metabolic activity remained throughout the observational period.Temporal analysis of the cell death response to freezing by MFC revealed that an initiation and sustained levels of both apoptosis and necrosis.Focusing on the −15 °C isotherm, apoptotic cell death was found to peak at 4 h and necrosis at 8 h post-thaw.Within the PaCaTEM samples, a 3.0 cm (±0.2 cm) diameter iceball was observed.Fluorescence imaging of the PaCaTEM revealed complete PaCa cell ablation to a diameter of 1.9 cm (±0.2 cm)’ correlating with the −25C isotherm.A transitional zone of cell death extending to the margin of the iceball was also observed.The results of this study indicate that cryoablation, alone or as a combinatorial therapy, may serve as an effective tool for the targeted ablation of pancreatic cancer.At minimum, −25 °C should be achieved as a target tissue temperature at the margin of a tumor to assure complete PaCa destruction. Source of funding: CPSI Biotech. Conflict of interest: None declared. ksnyder@cpsibiotech.com

9. Multi-dimensional assessment of pancreatic cancer cell responses to cryoablation in vitro

As the incidence of pancreatic cancer (PaCa) continues to increase worldwide with little improvement in the overall prognosis, there is a drive to identify alternative strategies for the treatment of PaCa to prolong patient survival. To this end, several reports have described the potential benefits of the use of cryoablation for the treatment of PaCa. While promising, few studies have been reported examining the physical and molecular response of PaCa to cryoablation. An understanding of these responses is critical to serve as supportive and guidance information if the use of cryo for the treatment of PaCa is to continue to grow. In an effort to detail the effects of freezing in pancreatic cancer (PaCa), a human PaCa cell line (Bx-PC3) was evaluated in vitro and using an in vivo -like tissue engineered model ( PaCaTEM ). Bx-PC3 were exposed to a range of freezing temperatures from −10 °C to −20 °C and compared to non-frozen controls. PaCaTEM models were frozen in situ following a single 5 min freeze protocol using the supercritical nitrogen (SCN) cryoablation system with a 1.5 mm × 3 cm cryoprobe. Following freezing, cell survival was assessed in the in vitro studies using the metabolic activity indicator, alamarBlue and microfludic flow cytometry (MFC). Assessment of PaCaTEM samples included monitoring of iceball size and isothermal distribution during the freezing process as well as assessment of cell ablation at 1 and 24 h post-freeze via fluorescence microscopy (Vybrant apoptosis assay). Results from in vitro experiments showed that freezing to −10 °C did not affect Bx-PC3 viability, while −15 °C and −20 °C resulted in a significant loss of viability (90% and 98%, respectively). A complete loss of cell viability was evident at temperatures of −25 °C and colder in vitro . Temporal analysis of the cell response to freezing via MFC revealed that following exposure, there was an initiation of both apoptotic and necrotic responses. Specifically, following freezing to −15 °C, apoptotic cell death was found to peak at 4hrs and necrosis at 8hrs post-thaw. Analysis of the PaCaTEM samples revealed the creation of a 3.0 cm (±0.2 cm) diameter iceball with extension of the −25 °C isotherm to a diameter of 2.0 cm (±0.1 cm). Fluorescence imaging of the PaCaTEM revealed complete PaCa cell ablation to a diameter of 1.9 cm (±0.2 cm) with a transitional zone of cell death extending to the margin of the iceball. This zone of ablation correlated to complete cell destruction below 25 °C in in vivo -like tissue. The results of this study show that cryoablation is an effective tool for the targeted ablation of pancreatic cancer. Further, the data suggest that a temperature of −25 °C may serve as a target temperature to be reached at the margin of a tumor to assure complete PaCa destruction.

Chondrocyte death after drilling and articular screw insertion in a bovine model

Intra-articular screws are used for internal fixation of osteochondral fragments after fracture or osteochondritis dissecans. This causes cartilage injury potentially leading to chondrocyte death. We have visualised/quantified the hole and zone of cell death (ZCD) in cartilage after drilling/insertion of various articular screws. Using an exvivo bovine model with transmitted light and confocal laser scanning microscopy (CLSM), the holes and ZCD following drilling/insertion of articular screws (cortical screw, headless variable pitch metallic screw, headless variable pitch bioabsorbable screw) were evaluated. In situ chondrocyte death was determined by live/dead cell viability assay. An imaging/quantification protocol was developed to compare hole diameter and ZCD from drilling/insertion of screws into cartilage. The effect of saline irrigation during drilling on the ZCD was also quantified. Screw insertion created holes in cartilage that were significantly (P≤0.001) less than the diameters of the equipment used. With a 1.5mm drill, a ZCD of 580.2±124μm was produced which increased to 637.0±44μm following insertion of a 2mm cortical screw although this was not significant (P>0.05). The ZCD from insertion of the variable pitch headless screws (diam. 3.5mm) was lower for the metallic compared to the bioabsorbable design (800.9±159 vs 1,236.4±212μm, respectively; P<0.01). The ZCD from drilling was reduced ∼50% (P<0.001) by saline irrigation. Cartilage injury during intra-articular screw fixation caused a ZCD around the hole irrespective of screw design. Saline irrigation significantly reduced the ZCD from drilling into cartilage.

Use of Tumor Necrosis Factor–alpha-coated Gold Nanoparticles to Enhance Radiofrequency Ablation in a Translational Model of Renal Tumors

Radiofrequency ablation (RFA) has been most effective when the tumors are small, exophytic, and away from vital structures. We enlarged the size of the ablation kill zone by infusing a 30-nm tumor necrosis factor-alpha and polyethylene glycol-coated gold nanoparticle (CYT-6091, CytImmune Sciences, Inc.) before ablation in a rabbit kidney tumor model. A total of 37 New Zealand White rabbits had VX-2 tumors implanted into their bilateral kidneys; they were then split into 3 treatment groups of 10 rabbits each and a sham group of 7 rabbits as follows: (1) CYT-6091 only, (2) RFA only, (3) CYT-6091 followed 4 hours later by RFA. Gross and microscopic measurements of the ablation size as well as histologic analysis using hematoxylin and eosin staining were performed to determine the effect of CYT-6091 on the ablation. The RFA + CYT-6091 group had a larger zone of complete cell death than the RFA-only group when measured on microscopic examination (0.30 +/- 0.07 vs 0.23 +/- 0.03 mL, P = .03). The zone of partially ablated tissue was smaller in the RFA + CYT-6091 group than in the RFA-only group (0.08 +/- 0.02 vs 0.13 +/- 0.05 mL, P = .01). We have demonstrated the efficacy of CYT-6091 in enhancing RFA in a translational kidney tumor model. The potential usage of CYT-6091 to improve RFA of renal cell carcinoma merits further study.

Immunohistochemical evaluation of the heat shock response to nonablative fractional resurfacing

Despite the emergence of nonablative fractional resurfacing (NFR) as a new therapeutic modality for skin photoaging, little is known about the molecular events that underlie the heat shock response to different treatment parameters. Human subjects are treated with a scanned 1550-nm fractional laser at pulse energies spanning 6 to 40 mJ and a 140-μm spot size. The heat shock response is assessed immunohistochemically immediately through 7 days posttreatment. At the immediately posttreatment time point, we observe subepidermal clefting in most sections. The basal epidermis and dermal zones of sparing are both found to express HSP47, but not HSP72. By day 1, expression of HSP72 is detected throughout the epidermis, while that of HSP47 remains restricted to the basal layer. Both proteins are detected surrounding the dermal portion of the microscopic treatment zone (MTZ). This pattern of expression persists through day 7 post-NFR, although neither protein is found within the MTZ. Immediately posttreatment, the mean collagen denaturation zone width is 50 μm at 6 mJ, increasing to 202 μm at 40 mJ. The zone of cell death exceeds the denaturation zone by 19 to 55% over this pulse energy range. The two zones converge by day 7 posttreatment.

Molecular Enhancement of Thermal Ablation Therapies Using TNF-α-Coated Gold Nanoparticles in a Translational Model of Renal Tumors

Thermal ablation therapies are currently used for the treatment of select renal masses. Such treatments are limited to tumors that are small (&lt;3 cm diameter), exophytic, and away from vital structures such as ureter or intestine. Novel treatment approaches are geared towards increasing the size of the thermal lesion created, limiting damage to collateral normal tissues, reducing local recurrence and distant metastases as well as improving the imaging potential of the therapy. Previous studies have demonstrated the enhancement of thermal therapies in pre-clinical murine models of solid tumors by intravenously infusing 33 nm TNF-α and PEG coated gold nanoparticles (CYT-6091, Cytimmune Sciences Inc.) prior to ablation. This study investigates the enhancement of thermal ablation therapy by CYT-6091 in a translational animal model of renal tumors. New Zealand White rabbits (37 for radiofrequency ablation (RFA), 20 for cryoablation) had VX-2 tumors implanted into their bilateral kidneys. The tumors were allowed to grow for 14 days to a size of ∼1 cm. For RFA, the rabbits were split into 3 treatment groups of 10 rabbits each and a sham group of 7 rabbits. The groups were treated with CYT-6091 (200 μg/kg) only, RFA only, or CYT-6091 (200 μg/kg) followed 4 hours later by RFA. For cryoablation, 2 treatment groups of 10 rabbits each were used. The groups were treated with cryoablation only or CYT-6091 (200 μg/kg) followed 4 hours later by cryoablation. The kidneys were harvested 3 days later for RFA and 7 days later for cryoablation. Gross and microscopic measurements of the ablation size as well as histological analysis using H&amp;E staining were performed. The RFA plus CYT-6091 group had a larger zone of complete cell death than the RFA only group when measured both on gross sectioning (0.32±0.03 vs. 0.22±0.07cm3, p=0.015) and on microscopic examination (0.30±0.07 vs. 0.23±0.03cm3, p=0.03). Overall this was a 23% increase in ablation volume. This difference in ablation size was due to a replacement of partially ablated tissue at the periphery in the RFA only group by completely ablated tissue in the RFA plus CYT-6091 group. Thus this zone of partially ablated tissue was smaller in the RFA plus CYT-6091 group than the RFA only group (0.08±0.02cm3 vs. 0.13±0.05cm3, p=0.01). Excessive tumor growth into the ablation lesion at day 7 following cryoablation prevented accurate measurements in these groups; however, a significant decrease in the rate of peritoneal carcinomatosis (metastases) was obtained in the cryo plus CYT-6091 group compared to the cryoablation alone group (1/10 vs. 8/10, p=0.04). We have shown that use of CYT-6091 prior to thermal ablation therapy in a rabbit kidney tumor model can minimize the zone of partial treatment at the periphery of the thermal lesion and thus maximize the complete kill zone in RFA while significantly decreasing the rate of metastases in cryoablation. These data provide preliminary evidence for the efficacy of adjuvant use of CYT-6091 for thermal ablation therapies in a large animal translational tumor model.

Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast

Elucidating the mechanisms by which eukaryotic cells coordinate environmental signals with intracellular ;fate' decisions, such as apoptosis, remains one of the important challenges facing cell biologists. It has recently emerged that the dynamic nature of the actin cytoskeleton is an important factor in the linkage of sensation of extracellular stimuli to signalling mechanisms that regulate programmed cell death. In yeast, actin has been shown to play a role in the regulation of apoptosis as cells prepare themselves for quiescence in the face of nutritional exhaustion, by facilitating the shutdown of Ras-cAMP-PKA pathway activity. Here, we demonstrate that the loss of Whi2p function, a protein known to influence cell cycle exit under conditions of nutritional stress, leads to cell death in yeast that displays the hallmarks of actin-mediated apoptosis. We show that actin-mediated apoptosis occurs as a result of inappropriate Ras-cAMP-PKA activity in Deltawhi2 cells. Cells lacking Whi2p function exhibit an aberrant accumulation of activated Ras2 at the mitochondria in response to nutritional depletion. This study provides evidence that the shutdown of cAMP-PKA signalling activity in wild-type cells involves Whi2p-dependent targeting of Ras2p to the vacuole for proteolysis. We also demonstrate for the first time that Whi2p-dependent regulation of cAMP-PKA signalling plays a physiological role in the differentiation of yeast colonies by facilitating elaboration of distinct zones of cell death.

Efficacy of Laser-Activated Gold Nanoshells in Ablating Prostate Cancer Cells in Vitro

Nanoshells (NS) are nanoparticles consisting of a dielectric silica core covered by a thin gold shell. Nanoshells can be designed to absorb near-infrared (NIR) light strongly to generate heat and provide optically guided hyperthermic ablation. Laser-activated gold nanoshells (LAGN) may offer a minimally invasive targeted ablative treatment for prostate cancer. We studied the in-vitro effectiveness of LAGN ablation on human prostate cancer cells. Two human prostate cancer (PCa) cell lines, PC-3 and C4-2, were grown to 80% confluency in T medium with 5% fetal bovine serum. In order to determine a threshold concentration of gold nanoshells (GNS) needed to achieve full cellular ablation, dose titration was performed. In subsequent experiments, GNS were added to PCa cells in phosphate-buffered saline at concentrations above the predetermined threshold. The cells were then exposed to NIR light (810 nm, 88 W/cm2) for 5 minutes and stained immediately for viability using the Calcein AM assay. For determining long-term cell survival, the crystal violet assay was employed. The GNS could be evenly distributed across the culture plates. A ratio of 5000 GNS per PCa cell was critical for achieving cell kill. Cells treated with GNS + NIR demonstrated a laser-specific zone of cell death. The crystal violet viability assay confirmed consistent cell death rather than induction of cell dormancy. Cells treated with GNS alone or with NIR light alone demonstrated no toxicity. Laser-activated gold nanoshells can ablate human PCa cells in vitro. This nanoparticle technology is an attractive therapeutic agent for selective tumor ablation.

Distribution of macrophages, osteoclasts and the B-lymphocyte lineage in osteolytic metastasis of mouse mammary carcinoma

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.

In situ analysis of Raidd-beta-galactosidase fusion gene expression in transgenic mouse midgestation embryos

Apoptosis and differentiation are tightly intertwined processes occurring at organ formation and remodelling during embryonic development. RAIDD (receptor-interacting protein [RIP]-associated ICH-1/CED-3-homologous protein with a death domain), a dual-domain adaptor protein has been shown to mediate the recruitment of CASPASE-2 to tumour necrosis factor receptor-1 (TNF-R1) signalling complex through RIP kinase. However, Raidd overexpression studies suggest that apart from the established role in apoptosis, Raidd may have an additional function in cell differentiation. In this study, we could not generate Raidd null adult mice suggesting that lack of function of Raidd might be embryonic lethal. Thus, to elucidate the role of Raidd during mouse embryogenesis when the processes of organogenesis are most dynamic, we studied the Raidd expression pattern in midgestation mouse embryos. We generated Raidd+/- transgenic mice with a reporter transgene encoding the bacterial Beta-galactosidase (beta-gal) under the control of Raidd promoter. During the midgestation period (E8.5-E12.5), Raidd is expressed in developing organs derived from the ectoderm such as lens, structures of the inner ear and the fourth brain ventricle in regions where differentiation takes place implicating Raidd role in this process. In addition, Raidd expression was found in developing mesenchyme organs like heart and kidney and in the endothelial lining of the midgut at the time when profound morphological changes take place in these organs. In developing heart and kidney Raidd expression patterns overlapped with known zones of cell death suggesting Raidd may be involved in apoptosis-mediated remodelling. The observed lethality of mice targeted at both Raidd alleles and Raidd expression patterns during midgestation period strongly suggest that Raidd plays an important role in mammalian development.

Cryosurgical changes in the porcine kidney: histologic analysis with thermal history correlation

Advances in minimally invasive renal cryosurgery have renewed interest in the relative contributions of direct cryothermic and secondary vascular injury-associated ischemic cell injury. Prior studies have evaluated renal cryolesions seven or more days post-ablation and postulated that vascular injury is the primary cell injury mechanism; however, the contributions of direct versus secondary cell injury are not morphologically distinguishable during the healing/repair stage of a cryolesion. While more optimal to evaluate this issue, minimal acute (⩽3 days) post-ablation histologic data with thermal history correlation exists. This study evaluates three groups of porcine renal cryolesions: Group (1) in vitro non-perfused ( n=5); Group (2) in vivo 2-h post-ablation perfused ( n=5); and Group (3) in vivo 3-day post-ablation perfused ( n=6). The 3.4 mm argon-cooled cryoprobe’s thermal history included a 75 °C/min cooling rate, −130 °C end temperature, 60 °C/min thawing rate, and 15-min freeze time. An enthalpy-based mathematical model with a 2-D transient axisymmetric numerical solution with blood flow consideration was used to determine the thermal history within the ice ball. All three groups of cryolesions showed histologically similar central regions of complete cell death (CD) and transition zones of incomplete cell death (TZ). The CD had radii of 1.4, 1.1, and 1.0 cm in the non-perfused, 2-h and 3-day lesions, respectively. Capillary thrombosis was present in the 2-h perfused cryolesions with the addition of TZ arteriolar/venous thrombosis in the 3-day perfused lesions. Thermal modeling revealed the outer CD boundary in all three groups experienced similar thermal histories with an approximately −20 °C end temperature and 2 °C/min cooling and thawing rates. The presence of similar CD histology and in vitro/in vivo thermal histories in each group suggests that direct cryothermic cell injury, prior to or synchronous with vascular thrombosis, is a primary mediator of cell death in renal cryolesions.

The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems.

Cell death in gliomas may occur either by apoptosis, or, in the case of high grade tumours, by necrosis, but questions remain as to the pathogenesis and relationship between these processes. The development of cell death was investigated in multicellular glioma spheroid cultures. Spheroids model the development of cell death due to diffusion gradients in a three-dimensional system without confounding influences of immune response, pressure gradients, etc. Spheroid cultures were established from four malignant glioma cell lines: U87, U373, MOG-G-CCM and A172; harvested from culture at weekly intervals and stained with Haematoxylin and Eosin (H&E), TdT-mediated dUTP-X nick end labelling (TUNEL) and by immunohistochemistry for vimentin, Glial Fibrillary Acidic Protein (GFAP) and Ki67. Annexin V flow cytometry and counts of apoptotic cells on H & E stained sections were performed to assess levels of apoptosis. Modes of cell death were also characterized by electron microscopy. Spatially separate zones of proliferation, differentiation and central cell death developed with increasing spheroid diameter. Central cell death developed at a predictable radius (300-400 microm) for each cell line. Ultrastructural examination showed this to be necrotic in type. Apoptosis was most reliably assayed by morphological counts using H & E. Basal levels of apoptosis were low (< 0.5%), but increased with increasing spheroid diameter (> 2% in U87). In particular, levels of apoptosis rose following development of central necrosis and apoptoses were most abundant in the peri-necrotic zone. There were quantitative differences in the levels of apoptosis and necrosis between glioma cell lines. The predictable onset of necrosis in the spheroids will allow us to investigate the pathogenesis of necrosis and events in prenecrotic cells. There is a relationship between the development of necrosis and apoptosis in this model and these processes can be separately assayed. Further in vitro and genetic studies will enable us to study these events and interactions in greater detail than is possible using other cell culture and in vivo systems.

Differential tissue growth and patterns of cell death in mouse limb autopod morphogenesis.

Programmed cell death (PCD) is considered one of the most important cellular processes in the morphogenesis of organs and tissues during animal development. Although the embryonic limb has been established as a classic model for the study of PCD, detailed studies on this process' contribution to morphogenesis are still lacking. In the present work, using modern computer-aided techniques, we estimated the contribution of PCD to mouse limb morphogenesis. For the detection of apoptotic cell death, we stained whole embryonic limbs with acridine orange or, in some instances, used the TUNEL technique, and visualized the tissues by confocal laser scanning microscopy. We found that cell death patterns are dynamic during limb development, and occur in gradients oriented with the main limb axes, anteroposterior, dorsoventral and distoproximal. Interdigital apoptosis in the autopod was initially detected at the most distal region, and then more proximally as development proceeded. Interestingly, we found that digit separation is more pronounced on the dorsal side, contrary to what is expected from the apoptotic cell distribution, which shows more abundant cell death in the ventral region. Using 2-D and 3-D models, we found that most digit individualization occurs rather by digit growth than by interdigital cell death. Therefore, digits do not mainly individualize by degeneration of preformed interdigital tissue, but probably by a dynamic balance between proliferation and cell death, reducing interdigital growth, which results in protrusion of digits. We determined the expression pattern of fgf-8 during the period of digit individualization, as the product of this gene could participate in defining the limb growth pattern. Initially, fgf-8 expression was coincident with the apical ectodermal ridge, but when cell death was first detected in the interdigits, fgf-8 expression became restricted to the tip of the growing digits. Therefore, FGF-8 could be one of the factors responsible for differential digit-interdigit growth, and might also act as a survival factor on interdigital tissue. We also found that the expression patterns of rar-beta, bmp-2, bmp-4, bmp-7, msx-1, and msx-2 genes, proposed to be involved in the activation of interdigital cell death, did not overlap with, or were not highly expressed in the major zones of cell death in the developing limb.

MRI study of immediate cell viability in focused ultrasound lesions in the rabbit brain.

The purpose of this study was to evaluate cell viability in MR imaged focused ultrasound (FUS) lesions using cell-viability staining with triphenyl tetrazolium chloride (TTC) and both light and electron microscopy. Ten paired ultrasonic lesions were created in 5 rabbit brains in vivo with an ultrasound beam of 1.5 MHz electrical power input to the transducer of 50 W and exposure duration of 15 seconds. T2-weighted fast spin-echo (FSE) MRI was performed to detect the FUS lesions in the brain 4 hours after treatment, after which the animals were immediately euthanized. Lesion sizes were measured on TTC-stained specimens, histological sections stained with hematoxylin and eosin (H&E), and T2-weighted MR images. The differences between the lesion diameters measured with the three methods were within the range of 0.1--0.7 mm. The lesion sizes measured from MRI correlated well with those seen from H&E sections. The measurements from MRI slightly overestimated lesion sizes on TTC-stained wet tissues by approximately one MRI pixel (0.31 mm). Electron microscopy demonstrated nuclear and cytoplasmic ultrastructural damage within the grey-white, non-TTC-stained lesion zone, whereas the TTC-stained normal tissue showed preservation of neuronal ultrastructure. Therefore, MR-imaged lesions represent a cell-death zone in rabbit brain 4 hours after FUS ablation, with slight overestimation by approximately one MRI pixel. J. Magn. Reson. Imaging 2001;13:23-30.

Chemoembolization of Liver Tumor in a Rabbit Model: Assessment of Tumor Cell Death with Diffusion-Weighted MR Imaging and Histologic Analysis

To assess the efficacy of chemoembolization of liver tumors by determining the fraction of viable tumor cells remaining after treatment with use of diffusion magnetic resonance (MR) imaging and histologic analysis. VX2 tumor was grown in the livers of 12 rabbits. Animals were divided into a chemoembolization group and an untreated group. Conventional, perfusion, and diffusion MR imaging was performed on all rabbits. Histopathologic analysis of explanted livers was performed to document tumor cell death and measure Bcl-2 levels (inhibitor of apoptosis). Diffusion-weighted MR imaging delineated zones of tumor cell death as regions of lower signal intensity in both groups. Apparent diffusion coefficients were significantly greater in the area of tumor necrosis than in the area of viable tumor. Histologic analysis demonstrated a significantly lower percentage of viable cells in the treated group (<1%) than in the control group (55%). Bcl-2 expression detected within the viable areas of the tumor was greater in the treated group than in the control group. Chemoembolization causes extensive tumor cell destruction. Diffusion MR imaging can detect tumor cell death and can be used to assess the efficacy of chemoembolization. Bcl-2 was expressed in the treated group, suggesting an apoptotic pathway of cell death.

Morphological and biochemical re-evaluation of the process of cavitation in the rat knee joint: cellular and cell strata alterations in the interzone.

To assess the contribution of apoptosis to the mechanism of synovial joint cavitation, and to clarify morphological cellular changes during cavitation, we investigated the development of the rat knee joint by light and electron microscopy, TUNEL methods, and electrophoresis of DNA fragments. Although cavitation occurred within the interzone, which consists of 2 outer and a middle layer termed the intermediate zone, no morphological or biochemical signs of cell death, in particular apoptosis, were seen in the interzone at any embryonic stage. Microscopic and ultrastructural alterations affecting cell differentiation were clearly observed in the interzone, i.e. mesenchymal cells gradually showed elongation, cytoplasmic vacuolation and pyknosis in the intermediate zone where the elongated cells were arranged in parallel in some strata. Some of these cells were further flattened into spindle cells and the number of strata decreased to 2. The rest of the cells were incorporated secondarily into the outer layers, becoming chondroblasts. Collagen fibrils were arranged in a network structure in the outer layers, which obviously differed from the directional pattern parallel to the long axis of elongated cells in the intermediate zone. In addition, the density of collagen fibrils was higher in the outer layers than in the intermediate zone. During cavitation, the initial separation was detected between the elongated cells in the intermediate zone in paraffin sections at E16.5 and the spindle cells in epoxy sections at E18.5. The spindle cells lining the cavity, namely, the surfaces of the epiphysis and meniscus, finally became chondrocytes. The diminution of proteoglycans and collagen fibrils and the synthesis of hyaluronan in the extracellular matrix are now generally believed to be parts of the mechanism for cavitation based on the concept of 'loss of cohesion'. The microscopic and ultrastructural alterations in the interzone seemed to reflect differences in the arrangement and density of collagen fibrils and the developmental condition of the extracellular matrix between layers. Also it did not seem likely that these alterations inhibit the synthesis of hyaluronan at the presumptive joint line because this synthesis takes place at the plasma membrane. Separation between spindle cells should therefore represent the mechanism for developmentally programmed cavitation. Reorganization of the extracellular matrix is probably necessary for the cellular metamorphoses in the interzone involved in the process of cavitation.

Surgical manipulation of mammalian embryos in vitro.

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Ultrastructural and cytochemical identification of apoptotic cell death accompanying development of the fetal rat olfactory nerve layer.

It has been previously shown that the embryonic olfactory nerve contains, in addition to glial ensheathing cells, a large population of differentiated neurons that migrate from the developing olfactory epithelium, in close association with the olfactory axon fascicles. The purpose of our study was to verify the hypothesis according to which a process of physiological cell death might be involved in the progressive disappearance of these migrating neurons that has been reported during late embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in rat embryos by using light and electron microscopy, with special reference to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visualization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labeling was performed by combining the DNA-specific bisbenzimide with two neuronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of programmed cell death accompanying the morphogenesis of the olfactory nerve layer. A cell death process was observed within the olfactory nerve layer from its onset at embryonic day 13 (E13). While only few pycnotic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cells were also observed along the olfactory axon fascicles when they penetrated the olfactory nerve layer. Degenerating cells appeared strongly TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apoptotic cells were neurons. These observations indicate that apoptosis may account for the progressive decrease in the number of migrating neurons present within the embryonic olfactory nerve layer. Otherwise, a zone of massive cell death by apoptosis was observed at E14 within the nasal mesenchyme located ventrally and caudally to the olfactory nerve layer. Double-labeling experiments showed that apoptotic cells present within this zone were not neurons. Our findings strongly suggest that apoptotic cell death of migrating neurons may allow the elimination of non-functional cells whereas that of mesenchymal cells may facilitate outgrowth of the newly formed olfactory axon fascicles by pathway formation.

The effect of a light-cured periodontal dressing material on HeLa cells and fibroblasts in vitro.

The effect which a visible light-curing periodontal dressing material based upon a urethane dimethacrylate resin has upon HeLa cells and fibroblasts in its uncured and fully cured states has been studied in vitro. Fully-cured material has no effect on either cell type. Uncured material produces a surrounding zone of growth inhibition and cell death on direct contact. Inhibition is caused by the release into the medium of substances toxic to cells. However, this growth inhibition is overcome in time (5 days). It is suggested that partly-cured material containing residual free monomer in contact with a healing gingival site could impede rapid repair. Nuclear magnetic resonance microimaging has shown that in places where there is a greater depth of material (e.g., interproximal regions) this underlying material may be partly cured. Increasing the exposure to the curing light will prevent (or minimize) the presence of partly-cured material; the fully-cured material being compatible with the cells.