Apoptotic Tissues Research Articles

A molybdenum-based nanoplatform with multienzyme mimicking capacities for oxidative stress-induced acute liver injury treatment

PEGylated MoS2 nanosheets were synthesized and modified with bovine serum albumin. These nanosheets exhibited a hepatoprotective effect, which reduces the number of apoptotic and necrotic tissues and inhibits the production of pro-inflammatory factors in disease models.

A caspase-3-activatable bimodal probe for photoacoustic and magnetic resonance imaging of tumor apoptosis in vivo

Multimodal imaging probes have shown promise in both biomedical research and clinical diagnosis. However, the development of molecular probes capable of being switched on multimodality imaging signals in response to a biomarker of interest remains challenging. In this paper, we report a caspase-3-activatable small-molecule bimodal probe (Gd-IR780) for photoacoustic imaging (PAI) and magnetic resonance imaging (MRI) of tumor apoptosis via caspase-3-triggered intramolecular macrocyclization and in situ self-assembly process. Upon interaction with caspase-3, Gd-IR780 can be efficiently converted into a macrocyclic product, Gd-IR780-MC, which subsequently self-assembles into near-infrared absorptive and paramagnetic nanoparticles (Gd-IR780-NPs), allowing to concurrently switch on PAI (∼4.3-fold at 855 nm) and MRI (r1 relaxivity increases from 7.98 ± 0.03 mM−1 s−1 to 19.66 ± 0.7 mM−1 s−1 at 0.5 T) bimodal signals in caspase buffer. Noninvasive in vivo imaging results show that Gd-IR780 can enter apoptotic U87MG tumor tissues after systemic administration, and produce markedly enhanced PAI and MRI signals for high sensitivity and spatial-resolution visualization of caspase-3 activity in the doxorubicin-treated apoptotic U87MG tumor tissues. Gd-IR780 holds a good potential to report tumor apoptosis via combined PAI and MRI, which is beneficial for the early evaluation of antitumor efficacy in vivo.

Lesions in the Hippocampus and Substantia Nigra of Wistar Rats’ Brains Induced by Organophosphate Insecticide

As little is known about the neurotoxicity of the histological structure of the brain, this study focuses on the histological side of four- to six-month-old adult Wistar rat brains, which were examined after 0.1 mg\g organophosphate had been administered orally. In this study, the lesions were mainly localized at the hippocampus and substantia nigra (compacta pars) region. Distinct areas of necrotic and apoptotic tissues were detected in the CA1, CA2, and dentate gyrus of the hippocampus and compacta pars of the substantia nigra. Programmed cell death in the dentate gyrus was observed as early as 72 hours after treatment and necrosis of some brain regions. Moreover, Lewy bodies were noticed in the compacta pars of the substantia nigra. The most important symptoms of parkinsonism were observed in the substantia nigra (compacta pars). These were decreased neurons, increased neuronal melanin in the neurons, and increased glial cells. The degeneration of some neurons was reported in the polymorphic and pyramidal layers. The data showed an increase in the density of the axon membrane and several changes to the axis structure, such as the disappearance of the myelin sheath in some areas along the axis.

Mesenchymal epithelial transition factor regulates tumor necrosis factor-related apoptotic induction ligand resistance in hepatocellular carcinoma cells through down-regulation of cyclin B1.

Tumor necrosis factor-related apoptotic induction ligand can induce cell apoptosis in various tumor cells. However, many cancer cells are resistant to tumor necrosis factor-related apoptotic induction ligand. Therefore, overcoming the tumor necrosis factor-related apoptotic induction ligand resistance makes it possible for tumor necrosis factor-related apoptotic induction ligand-based anti-cancer therapies. In this study, we took mesenchymal epithelial transition factor as the research target to study its role in tumor necrosis factor-related apoptotic induction ligand-resistant hepatocellular carcinoma. Mesenchymal epithelial transition factor gene has been proved to be an effective predictor of recurrence after hepatocellular carcinoma resection. The expression of mesenchymal epithelial transition factor and cyclin B1 were measured in tumor necrosis factor-related apoptotic induction ligand-resistant and non-resistant hepatocellular carcinoma tissues. Cyclin B1-knockdown and cyclin B1-overexpression hepatocellular carcinoma cells were treated with tumor necrosis factor-related apoptotic induction ligand; mesenchymal epithelial transition factor knockout, mesenchymal epithelial transition factor re-introduction and cyclin B1 restored in hepatocellular carcinoma cells treated with tumor necrosis factor-related apoptotic induction ligand were established. And MTT, bromodeoxyuridine, flow cytometry and western blotting were performed to evaluate the effect of mesenchymal epithelial transition factor and cyclin B1 on hepatocellular carcinoma cells treated with tumor necrosis factor-related apoptotic induction ligand. In addition, subcutaneous tumor transplantation in nude mice was conducted to access the effect of mesenchymal epithelial transition factor and cyclin B1 on tumor formation in vivo. In conclusion, cyclin B1 enhanced the cell growth and inhibited apoptosis in tumor necrosis factor-related apoptotic induction ligand-resistant hepatocellular carcinoma cells. And mesenchymal epithelial transition factor promoted the cell growth and apoptosis in tumor necrosis factor-related apoptotic induction ligand-resistant hepatocellular carcinoma cells by regulating cyclin B1. Therefore, mesenchymal epithelial transition factor regulates the cyclin B1 to regulate tumor necrosis factor-related apoptotic induction ligand resistance in hepatocellular carcinoma cells. Our results suggest a novel molecular mechanism for regulating tumor necrosis factor-related apoptotic induction ligand resistance, which might be helpful to select drug targets in the treatment of liver cancer.

Radiopharmacological evaluation of a caspase-3 responsive probe with optimized pharmacokinetics for PET imaging of tumor apoptosis.

Early evaluation of the therapy efficiency can promote the development of anti-tumor drugs and optimization of the treatment method. Caspase-3 is a key biomarker for early apoptosis. Detection of caspase-3 activity is essential for quick assessment of the curative effect. We have reported a PET probe that could image drug-induced tumor apoptosis in vivo. However, high liver uptake limits its application. In order to optimize the pharmacokinetics of the previous probe, we introduced a hydrophilic peptide sequence to minimize liver uptake. The structure of the new probe was confirmed by mass spectrometry and nuclear magnetic resonance. This probe was able to cross the cell membrane freely and could be converted into a dimer through the condensation reaction of 2-cyano-6-aminobenzothiazole (CBT) and cysteine in response to intracellular activated caspase-3 and glutathione (GSH). The hydrophobic dimers further self-assembled into nanoparticles, which could enhance the probe aggregation in apoptotic tumor tissues. In vivo experiments showed that the tumor uptake of the new probe was higher than that of the previous probe, while the liver uptake of the new probe was significantly reduced. The new probe might be promising in imaging apoptotic tumors with suitable pharmacokinetics.

Serrate/Notch Signaling Regulates the Size of the Progenitor Cell Pool in Drosophila Imaginal Rings.

Drosophila imaginal rings are larval tissues composed of progenitor cells that are essential for the formation of adult foreguts, hindguts, and salivary glands. Specified from subsets of ectoderm in the embryo, imaginal ring cells are kept quiescent until midsecond larval instar, and undergo rapid proliferation during the third instar to attain adequate numbers of cells that will replace apoptotic larval tissues for adult organ formation. Here, we show that Notch signaling is activated in all three imaginal rings from middle embryonic stage to early pupal stage, and that Notch signaling positively controls cell proliferation in all three imaginal rings during the third larval instar. Our mutant clonal analysis, knockdown, and gain-of-function studies indicate that canonical Notch pathway components are involved in regulating the proliferation of these progenitor cells. Both trans-activation and cis-inhibition between the ligand and receptor control Notch activation in the imaginal ring. Serrate (Ser) is the ligand provided from neighboring imaginal ring cells that trans-activates Notch signaling, whereas both Ser and Delta (Dl) could cis-inhibit Notch activity when the ligand and the receptor are in the same cell. In addition, we show that Notch signaling expressed in middle embryonic and first larval stages is required for the initial size of imaginal rings. Taken together, these findings indicate that imaginal rings are excellent in vivo models to decipher how progenitor cell number and proliferation are developmentally regulated, and that Notch signaling in these imaginal tissues is the primary growth-promoting signal that controls the size of the progenitor cell pool.

Target challenging-cancer drug delivery to gastric cancer tissues with a fucose graft epigallocatechin-3-gallate-gold particles nanocomposite approach

Inhibiting component of therapy with (−)-epigallocatechin-3-gallate (EGCG) is low bioavailability of fresh tea polyphenols that outcome from insecurity under stomach related circumstances, insufficient transcellular transport. As needs are, fucose- carboxymethyl chitosan (FU-CMC) graft EGCG with gold nanoparticles (GNPs) (FU-CMC-EGCG-GNPs) nanocomposites were prepared and managed peritumorally to assess their anticancer action. The physicochemical properties of as-prepared nanocomposite were evaluated by FTIR spectroscopy, UV–visible absorption spectra, XRD, FESEM-EDX and HRTEM-SAD. Additionally, the viability and cell uptake assays revealed that the as-prepared nanocomposite successfully repressed the propagation of gastric tumor cells. In vivo anticancer treatment of FU-CMC-EGCG-GNPs nanocomposites showed more anticancer action contrasted with pure EGCG. Immuno-histological investigations established a superior numeral of apoptotic tissues in the as-prepared FU-CMC-EGCG-GNPs nanocomposites contrasted with pure EGCG. Overall, the as-prepared FU-CMC-EGCG-GNPs nanocomposite affords a proficient medicine delivery stage for chemotherapy.

Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo.

Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and it has been widely used to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employed an optimized first-order bioorthogonal cyclization reaction to control self-assembly of a fluorescent small molecule, and demonstrated its in vivo applicability by imaging of casapae-3/7 activity in human tumor xenograft mouse models of chemotherapy. The in situ assembled fluorescent nanoparticles have been successfully imaged in both apoptotic cells and tumor tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo.

Decreased Levels Of Procoagulant Phospholipids In Bleeding Patients With Overcoagulation By Vitamin K Antagonists

Background The major adverse effect of vitamin K antagonists (VKA) is the increased risk for bleeding complications. In addition to well-identified risk factors such as age, prior gastrointestinal tract bleeding, or hypertension, the intensity of anticoagulation evaluated by the International Normalized Ratio (INR) measurement is a major determinant of VKA-induced bleeding. However, for the same degree of VKA overcoagulation and comorbidities, some patient will bleed, whereas others remain asymptomatic. Therefore, identifying specific biological markers that identify patients at high risk of bleeding would have great clinical impact. Microparticles, derived from different cellular origins (endothelium, red blood cells, leukocytes, platelets or apoptotic tissues), can be detected in plasma and express procoagulant phospholipids (PPL). The presence of PPL has been associated with various diseases complicated by an hypercoagulable state. Therefore, we hypothesize that the procoagulant activity of PPL could protect against haemorrhage in patients with VKA overcoagulation. Patients and methods 53 consecutive patients who were referred to the emergency department of our institution and with an INR > 5 were enrolled in the study: 22 (10 females; 12 males, median age 82 years) were symptomatic (20 cases of minor bleeding, 2 cases of non-fatal major bleeding), whereas 31 (18 females, 13 males, median age 78 years) were asymptomatic. Median INR was 7.36 (range: 5 – 22.6) and 6.3 (range: 5 – 10.7) in symptomatic and asymptomatic patients, respectively (p = 0.17, not significant). PPL were evaluated using a factor Xa-based coagulation assay (STA-Procoag-PPL, Diagnostica Stago) in which shortened clotting times are associated with increased levels of PPL. We also quantified thrombomodulin (TM) by an ELISA assay (Asserachrom Thrombomodulin, Diagnostica Stago) and by a functional assay based on the ability of TM to activate Protein C in the presence of thrombin, since high plasma levels of TM were previously identified as a predictor of bleeding complications. Results Clotting times were significantly lower in asymptomatic patients than in bleeding patients [respective median values 36.5 seconds (range: 27.1 – 72.2) and 47.2 seconds (range : 30.5 – 72.8); p = 0.03. In contrast, there were no significant differences for TM levels, whatever the assay used (functional or immunological). Conclusion Increased PPL could contribute to decrease the haemorrhagic risk of patients treated by VKA. It is not clear if the decrease of PPL is directly responsible of the hemorrhagic syndrom, or if PPL are decreased because of a consumption during the hemorrhagic episode. In order to answer this question, it could be of interest to analyse if a prospective follow-up of this parameter could help to identify patients with an increased hemorrhagic risk when treated by VKA. Disclosures: No relevant conflicts of interest to declare.

Activation of Drosophila hemocyte motility by the ecdysone hormone

SummaryDrosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton.

IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.

In vivo fluorescence imaging of apoptosis during foreign body response

Quantification of apoptotic tissues during inflammatory processes induced by biomaterials is challenging in vivo. Here we present a non-invasive method using a fluorescence imaging system which facilitates intermittent snap shots of the current state of local apoptotic tissue. For this purpose, apoptotic cells around two different subcutaneously implanted materials (titanium discs and copper-coated titanium discs) in hairless but immunocompetent mice were quantified after 4, 8 and 23 days of implantation. For validation, the results of fluorescence signals were compared to the histology of the inflammatory tissue using apoptotic-specific TUNEL-, macrophage-specific F4/80-, neutrophile-specific NIMP-R14- and chloroacetate esterase-staining. We could demonstrate that the fluorescence signals were well suited to quantify the extent of apoptosis in vivo and this is a good indication for the biocompatibility of biomaterials. This study shows that non-invasive monitoring of tissue processes following the implantation of biomaterials is possible in vivo and may help to reduce the number of animals in studies addressing biocompatibility.

Zona Pellucida Ablation for Day 3 Embryo Hatching in Poor Prognosis Cases Prior to Acellular Fragment Removal: Acidified Tyrodes Vs. Non-Contact 500μS Laser Pulsation

Assisted hatching (AH) of human embryos has commonly been performed using the acidified media technique (AAH), but the use of infrared laser ablation (LAH) has become a popular technique due to its' efficiency (time), elimination of acidic media and precise control of zona pellucida opening (size). Fragment removal (FR) of cytoplasmic extrusions can be performed to increase cell-cell contact by eliminating apoptotic tissues within the embryo. Our study aimed to compare the clinical pregnancy outcomes (gestational sac) in patients undergoing FR during the laboratory's transition from using standard AAH to the LAH technique.

Tomato polyphenol oxidase B is spatially and temporally regulated during development and in response to ethylene.

Plant polyphenol oxidases (PPOs) are ubiquitous plastid-localized enzymes. A precise analysis of PPO function in plants has been complicated by the presence of several family members with immunological cross reactivity. Previously we reported the isolation of genomic clones coding for the seven members of the tomato (Solanum lycopersicum) PPO family (A, A’, B, C, D, E, and F). Here we report the complex spatial and temporal expression of one of the members, PPO B. The PPO B promoter was sequenced and subjected to homology analysis. Sequence similarities were found to nucleotide sequences of genes encoding enzymes/proteins active in the following systems: phenylpropanoid biosynthesis, signal transduction and responsiveness to hormones and stresses, fruit and seed proteins/enzymes, and photosynthesis. Chimeric gene fusions were constructed linking PPO B 5′ flanking regions to the reporter gene, β-glucuronidase (GUS). The resultant transgenic plants were histochemically analyzed for GUS activity in various vegetative and reproductive tissues, and evaluated for PPO B responsiveness to ethylene induction. It was shown that PPO B expression was tissue specific, developmentally regulated, ethylene induced, and localized predominantly to mitotic or apoptotic tissues.

Biological studies in animal models using [99mTc](CO)3 recombinant annexin V as diagnostic agent of apoptotic processes

There are many diseases associated with variations in the expression of apoptosis such as organ rejection after transplantation, myocardial ischemia or infarct and neurodegenerative diseases. For this reason, the early visualization of this process is relevant to set fast and effective therapeutic strategies. The precursor was prepared according to the procedure reported by R. Alberto, R. Schibli, P. Schubiger, U. Abram, and T. Kaden [Reactions with the technetium and rhenium carbonyl complexes (NEt(4))[MX(3)(CO)(3)]. Synthesis and structure of Tc(CN-But)(3)(CO)(3)](NO(3)) and (Net(4))[Tc(2)(μ-SCH(2)CH(2)OH)(3)(CO)(3)], Polyhedron 1996;15: 1079-89]. Recombinant annexin V was incubated with [(99m)Tc](H(2)O)3(CO)(3)(+) solution, previously neutralized with buffer. Biodistribution studies were performed in 8-week-old female Wistar rats. Animals were housed and treated in compliance with institutional guidelines related to animal experimentation. Work protocol was previously approved by the Animal Ethics Committee of the university. Two groups of rats were defined. One was used as control and the other group was previously injected with 150 mg/kg ip of cyclophosphamide to induce apoptosis. The synthesis of carbonyl precursor achieved yields higher than 90%, and the radiolabeled protein was obtained with 92% of radiochemical purity and high stability in vitro. An important uptake in apoptotic tissues was confirmed by biodistributions, scintigraphic images and histological studies. Biodistribution studies revealed hepatobiliary elimination, high stability in vivo and important uptake in the reticuloendothelial system. In the pathologic model, higher uptake values correspond to the liver, spleen, lungs and femur. Histological studies confirmed the development of apoptosis at 8 and 24 h postinduction in the spleen and lymphocyte bulks in the peribronchial area. Scintigraphic images confirmed high uptake both the spleen and the lungs.

Homing of annexin-labeled stem cells to apoptotic cells

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues.

Targeted Photodynamic Therapy Agent with a Built-In Apoptosis Sensor for in Vivo Near-Infrared Imaging of Tumor Apoptosis Triggered by its Photosensitization in Situ

Imaging apoptotic cells or tissues after cancer therapy in situ would be a very useful tool for assessing proper treatment conditions and therapeutic outcome. By combining therapeutic and imaging functions, we have designed a multifunctional, membrane-permeable, and cancer-specific agent that triggers and images apoptosis in targeted cells. We chose photodynamic therapy (PDT) as an appropriate cancer treatment modality and caspase 3 as an apoptosis-specific imaging target. This targeted photodynamic therapy agent with a built-in apoptosis sensor (TaBIAS) induces photodamage only to target cells and simultaneously identifies those that are apoptotic by its near-infrared fluorescence. It contains a fluorescent photosensitizer used as an anticancer drug and a cancer-associated folate receptor homing molecule connected to a caspase 3 cleavable peptide linker that has a fluorescence quencher on the opposing site. We demonstrated that PDT-triggered cleavage of the peptide linker by caspase 3, one of the key executioner caspases, results in a detectable increase in fluorescence in folate receptor-overexpressing cancer cells and tumors. The presence of apoptosis was confirmed in vitro by flow cytometry and ex vivo by Apoptag assay, supporting the ability of TaBIAS to specifically induce and image apoptosis in situ.

Apoptotic Cell Debris and Phosphatidylserine-Containing Lipid Vesicles Induce Apolipoprotein J (Clusterin) Gene Expression in Vital Fibroblasts

The molecular events in cells undergoing programmed cell death (apoptosis) are well studied; however, the response of the surviving neighbor cells to local cell death is largely uncharacterized. Apolipoprotein J (clusterin) is an 80-kDa glycoprotein that has been implied in cytoprotection of the vital cells, presumably by assisting in the clearance of apoptotic vesicles and membrane remnants. Its mRNA is specifically up-regulated in the vital cells of apoptotic tissues. The molecular mechanisms, however, leading to this response are not known. We here show that exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces apoJ mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphosphatidylcholine (PC), but not liposomes with PC alone nor with dimyristoylphosphatidylethanolamine or phosphatidic acid, did elevate apoJ mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process.

Detection of Apoptotic Cells in a Dexamethasone-Induced Thymic Apoptosis Model in Rats Using A Modified Warthin-Starry Silver Impregnation Method to Prevent Fading.

A modified Warthin-Starry (mWS) method was developed to stain apoptotic cells in tissue sections. Essentially the classical WS method is applied except for a minor change to prevent fading. In order to clarify the relationship between mWS-positive cells and apoptosis, apoptotic thymic tissues were examined on the rats treated with dexamethasone. The results clearly showed the distribution of mWS-positive cells to coincide well with that for apoptotic cells observed by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) method. An additional characteristic is that nuclei of mitotic thymocytes, with highly condensed chromatin, are always stained. These observations suggest that the mWS method might be useful and convenient to detect condensed chromatin during apoptosis.

Apoptosis in the Rat Penis After Penile Denervation

Purpose Despite the advances in nerve sparing prostatectomy for prostate cancer, some patients develop impotence or subjectively complain of a decrease in penile size. We hypothesized that these clinical observations may be explained by injury to the cavernous nerves resulting in programmed cell death (apoptosis) within the penis. We utilized a rat model of penile denervation in order to demonstrate apoptosis after denervation. Methods and Materials Fifteen male Sprague Dawley rats underwent abdominal exploration and bilateral cavernous neurotomy. Fifteen sham operations were performed as normal controls. The rats were sacrificed on postoperative day 1, 2, 3, 6, and 10 and their penises were harvested. Messenger RNA was extracted and probed on a northern blot for sulfated glycoprotein-2 (SGP-2). SGP-2 is a gene product reported to be elevated in apoptotic tissues. Separate denervated and sham rats were used for DNA extraction (sacrificed postoperative day #2) in order to demonstrate the internucleosomal DNA fragmentation (laddering) found in apoptotic tissues. In addition, in situ histology was performed with ISEL techniques (in situ end labeling) to stain for apoptotic nuclei in denervated rats. Results Northern blot analysis showed a large increase in SGP-2 mRNA expression in the denervated rats with little detected in the sham operated group. DNA extraction studies revealed the presence of internucleosomal DNA fragmentation on agarose gel (a marker for apoptosis) in the denervated group versus intact high molecular weight DNA in the sham rats. In addition, in situ staining of denervated penile erectile tissue demonstrated apoptotic nuclei in the cavernous tissue. Conclusion Apoptosis of penile erectile tissue occurs after denervation of the rat penis. This has not been previously described in the literature and may offer some explanation at the molecular level concerning the mechanism of impotence and/or decrease in penile size after radical prostatectomy.