TUNEL Technique Research Articles

DNA fragmentation of sperm and microdeletions in the long arm of the Y chromosome in male infertility patients from private fertilization units of Barquisiemto, Lara State

Abstract Introduction/Objective Figures for infertility area increasing worldwide, with a large share of causes related to the male gender. The objective of this study is to relate the frequency of abnormal sperm DNA fragmentation and the frecuency of microdeletions in the long arm of Y chromosome (Yq) in patients with male infertility from private fertilitation units of Barquisimeto, Lara, during the 2010-2012 period. Methods/Case Report We analyzed 38 samples of semen from infertile male patients to measure DNA fragmentation index (FI) by TUNEL technique, and 36 semen samples from patients with male infertility to measure FI employing SCD method. To dertermine the frecuency of Yq microdeletions by polymerase chain reaction (PCR), samples of peripheral blood from 33 patients with male infertility were evaluated. Among the 38 patients in whom the FI was measured by TUNEL technique, eight(8) of them were also tested for detection of Yq microdeletions, and one (1) of these patients performed SCD test for medical reasons. Results (if a Case Study enter NA) The frecuency of abnormal fragmentation index of sperm DNA, determined by the TUNEL technique in patients with male infertility was 47.37%. The frecuency of abnormal fragmentation index of sperm DNA determoined by SCD method in infertile male patients was 22.22%. And the frecuency of microdeletions of the long arm of the Y chromosome in patients with male infertility was 9.09%. All microdeletions were found at DAZSY208 in AZFc region and in one case there was also detected a microdeletion at DAZSY255. Conclusion It was also showed that, sperm DNA fragmentation is not depending on the existence of Yq microdeletions.

Postnatal zinc deficiency due to giardiasis disrupts hippocampal and cerebellar development.

Giardiasis and zinc deficiency have been identified as serious health problems worldwide. Although Zn depletion is known to occur in giardiasis, no work has investigated whether changes occur in brain structures. Three groups of gerbils were used: control (1), orogastrically inoculated on day 3 after birth with trophozoites of two isolates of Giardia intestinalis (HGINV/WB) group (2 and 3). Estimates were made at five ages covering: establishment of infection, Giardia population growth, natural parasite clearance and a post-infection age. QuantiChrome zinc assay kit, cresyl violet staining and TUNEL technique were used. A significant decrease (p<0.01) in tissue zinc was observed and persisted after infection. Cytoarchitectural changes were observed in 75% of gerbils in the HGINV or WB groups. Ectopic pyramidal neurons were found in the cornus ammonis (CA1-CA3). At 60 and 90 days of age loss of lamination was clearly visible in CA1. In the dentate gyrus (DG), thinning of the dorsal lamina and abnormal thickening of the ventral lamina were observed from 30 days of age. In the cerebellum, we found an increase (p<0.01) in the thickness of the external granular layer (EGL) at 14 days of age that persisted until day 21 (C 3 ± 0.3 μm; HGINV 37 ± 5 μm; WB 28 ± 3 μm); Purkinje cell population estimation showed a significant decrease; a large number of apoptotic somas were observed scattered in the molecular layer; in 60 and 90 days old gerbils we found granular cell heterotopia and Purkinje cell ectopia. The pattern of apoptosis was different in the cerebellum and hippocampus of parasitized gerbils. The morphological changes found suggest that neuronal migration is affected by zinc depletion caused by giardiasis in early postnatal life; for the first time, the link between giardiasis-zinc depletion and damaged brain structures is shown. This damage may explain the psychomotor/cognitive delay associated with giardiasis. These findings are alarming. Alterations in zinc metabolism and signalling are known to be involved in many brain disorders, including autism.

Cannabidiol decreases histological intestinal injury in a neonatal experimental model of necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is a severe inflammatory bowel disease in neonates. Our group has developed an experimental model of NEC, with an effectiveness of 73%. Cannabidiol (CBD) is an innovative treatment for neonatal cerebral hypoxic-ischemic pathologies due to its neuroprotective effect, as a potent anti-inflammatory and reducing oxidative stress substance.Our aim was to evaluate the effect of CBD on intestinal lesions in an experimental model of NEC.ResultsMortality and intestinal histological damage was significantly lower in the CBD group compared to the rest (p<0.05), establishing CBD as a protective factor against the development of NEC (OR=0.0255; 95% CI=0.0015-0.4460). At IHQ level (TUNEL technique), a lower cell death rate was also observed in the CBD group compared to the VEH group (p<0.05).ConclusionsIn our experimental model, intraperitoneal CBD acts as a protective factor against NEC, resulting in less histological damage and a lower rate of intestinal cell death.

Effect of estrogens on apoptosis in the pituitary of viscachas (Lagostomus maximus maximus)

Estrogens, acting through their receptors (ERα and ERβ), regulate cell turnover in the pituitary gland, influencing cell proliferation and apoptosis across various species. However, their role in pituitary processes in seasonally reproducing animals remains poorly understood. This study aims to investigate the influence of estrogens, through the expression of their specific receptors, on the apoptosis of PD cells in relation to sexual maturity, the reproductive cycle, and pregnancy in a seasonal reproductive rodent (Lagostomus maximus maximus). ERα and caspase-3-cleaved (CASP3c) immunoreactive (-ir) cells were identified through immunohistochemistry. Apoptotic cells were detected using the TUNEL technique, with quantitative analysis facilitated by image analysis software, alongside measurement of serum estradiol levels using radioimmunoassay The immunostaining pattern for ERα included nuclear (ERαn) and cytoplasmic (ERαc) staining. In male viscachas, ERα expression significantly increases from immature to adult animals, correlating with the rise in serum estradiol levels and a decrease in the percentage of apoptotic cells. During the gonadal regression period in adult males, a decrease in the number of ER-ir cells and serum levels of estradiol corresponds with an increase in the number of apoptotic cells. In females, serum levels of estradiol peaked during mid-pregnancy, coinciding with a significant decrease in the number of apoptotic cells in the PD. Simultaneously, the percentage of ERαn-ir cells reaches its maximum value during late pregnancy, indicating the need to maintain the protective action of this gonadal hormone throughout the extensive pregnancy in these rodents. Regional ERα receptor expression and apoptotic cells appear to be associated with distinct PD cell populations and their hormonal responses. Finally, elevated estradiol levels coincide with diminished apoptotic cells in the male reproductive cycle and during pregnancy, suggesting an antiapoptotic role of estradiol in this species.

Intracerebellar injection of monocytic immature myeloid cells prevents the adverse effects caused by stereotactic surgery in a model of cerebellar neurodegeneration

BackgroundMyeloid-derived suppressor cells (MDSCs) constitute a recently discovered bone-marrow-derived cell type useful for dealing with neuroinflammatory disorders. However, these cells are only formed during inflammatory conditions from immature myeloid cells (IMCs) that acquire immunosuppressive activity, thus being commonly gathered from diseased animals. Then, to obtain a more clinically feasible source, we characterized IMCs directly derived from healthy bone marrow and proved their potential immunosuppressive activity under pathological conditions in vitro. We then explored their neuroprotective potential in a model of human cerebellar ataxia, the Purkinje Cell Degeneration (PCD) mouse, as it displays a well-defined neurodegenerative and neuroinflammatory process that can be also aggravated by invasive surgeries.MethodsIMCs were obtained from healthy bone marrow and co-cultured with activated T cells. The proliferation and apoptotic rate of the later were analyzed with Tag-it Violet. For in vivo studies, IMCs were transplanted by stereotactic surgery into the cerebellum of PCD mice. We also used sham-operated animals as controls of the surgical effects, as well as their untreated counterparts. Motor behavior of mice was assessed by rotarod test. The Purkinje cell density was measured by immunohistochemistry and cell death assessed with the TUNEL technique. We also analyzed the microglial phenotype by immunofluorescence and the expression pattern of inflammation-related genes by qPCR. Parametric tests were applied depending on the specific experiment: one or two way ANOVA and Student’s T test.ResultsIMCs were proven to effectively acquire immunosuppressive activity under pathological conditions in vitro, thus acting as MDSCs. Concerning in vivo studios, sham-operated PCD mice suffered detrimental effects in motor coordination, Purkinje cell survival and microglial activation. After intracranial administration of IMCs into the cerebellum of PCD mice, no special benefits were detected in the transplanted animals when compared to untreated mice. Nonetheless, this transplant almost completely prevented the impairments caused by the surgery in PCD mice, probably by the modulation of the inflammatory patterns.ConclusionsOur work comprise two main translational findings: (1) IMCs can be directly used as they behave as MDSCs under pathological conditions, thus avoiding their gathering from diseased subjects; (2) IMCs are promising adjuvants when performing neurosurgery.

Can the addition of Interleukin-13 affect the cryosurvival of bovine embryos?

In this study, we investigated the impact of incorporating Interleukin-13 (IL-13) into the embryonic culture medium and its influence on cryotolerance and cellular viability of vitrified bovine embryos. Two distinct time points for IL-13 supplementation were explored: during the final hours of culture prior to cryopreservation and during the period of recultivation following cryopreservation and warming. Cryosurvival rates, total cell count, and cell viability were assessed using the TUNEL technique to determine the apoptotic percentage. Re-expansion and hatching rates did not show differences among all groups (P > 0.05), and the total cell number was comparable between the treated and control groups (P > 0.05). However, the group that received IL-13 before vitrification exhibited a higher apoptotic percentage (P < 0.05). This suggests that the anti-inflammatory effect of IL-13 may have impacted the embryo's defense capacity against the stress induced by cryopreservation, leading to an increased percentage of apoptosis, although it did not influence the developmental resumption capability.

Ameliorative Effect of Malvidin on Spleen Injury in LPS-Induced Sepsis.

Sepsis, one of the most destructive diseases in the world, is a syndrome of systemic inflammatory response caused by the invasion of pathogenic microorganisms such as bacteria into the body. Malvidin is one of the most widespread anthocyanins, and its significant antioxidant and anti-inflammatory activities have been widely reported. However, the effect of Malvidin on sepsis and related complications is still unclear. The present study aimed to determine the mechanisms of Malvidin's potential protection from lipopolysaccharide (LPS)-induced spleen injury model of sepsis. In the LPS-induced mouse spleen injury model of sepsis, pretreatment with Malvidin was performed to assess morphological damage in spleen tissue and to detect the expression of mRNA levels of serum necrosis factor α, interleukin 1β and interleukin 6, and IL-10. Apoptosis was detected using the TUNEL technique, and the levels of oxidative stress-related oxidase and antioxidant enzymes were measured by kit to assess the effect of Malvidin on inflammation and oxidative stress associated with septic spleen injury. The results of this study indicated that Malvidin was be a potentially effective drug for the treatment of sepsis.

Application of the microflow technique for the selection of stallion semen

Routine semen preparation techniques are not always satisfactory for all stallions. Some stallions, despite poor semen parameters, are valuable for breeders who want to produce equally worthwhile offspring. In recent years, advanced sperm selection methods such as the microflow technique have been developed and successfully applied to humans. The aim of this study was to adapt the microflow technique for selection of stallion semen, focusing on their predicted benefits for sperm quality. The experiment was performed with semen of 6 stallions, which after combining in equal volumes created pooled research sample. Semen was left for 24 h in order to reduce the viability and motility of spermatozoa and divided into 3 parts, the control and the two research groups separated using ZyMōt Devices with two different incubation times (30 and 45 minutes). For each group, the basic sperm characteristics were assessed by SCA/CASA system, and fragmentation was evaluated using the TUNEL technique. The Z-score test was used for statistical analysis. In the sample incubated for 30 min in ZyMōt Devices, sperm motility increased from 28% to 52.15% (p=0.001), including sperm with progressive motility which increased from 8.47% to 41.91% (p<0.00001). After extending the incubation time for 45 minutes, these parameters were even higher: motility 62.43% (p<0.00001), progressivemovement 55.33% (p<0.00001). It should be noted, however, that in the case of both research samples, the concentration of sperm decreased from 804.68 M/ml in the control to 32.35 M/ml (p<0.00001) after 30 min incubation and 40.37 M/ml after 45 min incubation, respectively (p<0.0001). Sperm selection did not affect vitality. The TUNEL technique showed a very low percentage of spermatozoa with apoptosis in the semen fraction after selection at both incubation times. In summary, the microflow technique is an effective method for selection of stallion sperm with high motility, progressive movement and low DNA sperm fragmentation, with unchanged vitality parameters. Comparing the two incubation times, it was found that extending the incubation time to 45 minutes improved sperm concentration and spermatozoa with progressive motility. Nevertheless, prolonged incubation caused an increase in apoptotic spermatozoa that penetrate through a membrane with microchannels. It should be considered that sperm selection by microflow technique drastically reduces sperm concentration, which in practice translates into a significant reduction in the number of insemination doses which will increase costs significantly. However, this method can be successfully used for sperm selection for the ICSI procedure.

GM1 Reduced the Symptoms of Autism Spectrum Disorder by Suppressing α-Syn Through Activating Autophagy

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that cannot be cured. The ASD rat model was developed in this study to demonstrate the role and mechanism of ganglioside GM1 (GM1). Rats were given valproic acid (VPA) to create the ASD rat model. The rats' behaviors were assessed using the Y-maze test, open-field test, three-chamber social interaction test, and Morris water maze test. Relative levels of glutathione (GSH), malondialdehyde (MDA), catalase (CAT), reactive oxygen species (ROS), and superoxide dismutase (SOD) were quantitated using relative kits. Nissl, TUNEL, immunofluorescent, and immunohistochemistry staining techniques were used. GM1 treatment improved the ASD model rats' behavior disorders, including locomotor activity and exploratory behavior, social interaction, learning and memory capacity, and repetitive behavior. Following GM1 injection, striatal neurons grew and apoptosis decreased. GM1 reduced the excessively elevated α-Syn in ASD by encouraging autophagy. The behavior disorder of ASD model rats was exacerbated by autophagy inhibition, which also increased α-Syn levels. By increasing autophagy, GM1 reduced α-Syn levels and, ultimately, improved behavioral abnormalities in ASD model rats.

Azadirachtin-based insecticide impairs testis morphology and spermatogenesis of the southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae).

In the search for alternative tools for integrated pest management, azadirachtin, a botanical insecticide, has been used with the most promising activity against Spodoptera spp., but the mechanism of cytotoxicity on reproductive organs remains unclear. Spodoptera eridania (Stoll, 1782) is a polyphagous pest with great economic importance that has become an important target to elucidate the action of azadirachtin on the reproductive organs of insect pests, helping to understand the deleterious effects caused by its exposure. This study evaluated the effects of chronic exposure to azadirachtin on the morphology and ultrastructure of S. eridania larval testes as well as larval development. Azadirachtin exposure (6 or 18 mg a.i.L-1 ) caused a progressive increase in cumulative mortality and reduced gain in body mass after 5 days. Testicular structure indicated a reduction in their size with internal morphological changes such as spermatogonia, spermatogonial, spermatocytes and spermatid cysts in degeneration. The occurrence of cell death in germ and somatic cells was evidenced by the TUNEL technique. Electron microscopy revealed changes in cystic cells, such as cytoplasmic membrane rupture and cytoplasmic vacuolization. Chromatin compaction, changes in the rough endoplasmic reticulum and Golgi complex cisternae were observed in germ cells. Apoptotic bodies occurred between germ cell cysts. Azadirachtin damaged the testes of S. eridania larvae, and these changes compromised spermatogenesis and consequently the development of the reproductive potential of this specimen, making azadirachtin a promising botanical insecticide for application in integrated pest management programs. © 2022 Society of Chemical Industry.

Effects of Intracameral Drugs and Dyes on Corneal Endothelial Cell Apoptosis in a Rat Model: An In Vivo and In Vitro Analysis.

To evaluate the effects of intracameral drugs and dyes on rat corneal endothelial apoptosis and cell morphology. The right eyes of 72 rats were injected intracamerally with 1% lidocaine, 0.01% adrenaline, triamcinolone acetonide (TA) 4 mg/mL, 1% trypan blue (TB), 0.5% indocyanine green (ICG), and fortified balanced salt solution as control. Corneal samples were taken 1 day and 1 week post-injection. Corneal endothelial apoptosis was assessed by the TUNEL technique, and the ratio of apoptotic cells in each group was compared with the control. Corneal endothelial cell morphology was evaluated in each specimen by transmission electron microscopy. The mean apoptotic endothelial cell ratio was significantly higher at 1 day and 1 week after intracameral adrenaline injection when compared to controls (p=0.03 and 0.021, respectively). TB caused a significantly higher apoptotic cell ratio when compared to controls at 1 week after injection (p=0.043). Lidocaine caused a higher apoptotic cell ratio compared to TA and ICG at 1 week, although not statistically significant (p=0.058, 0.09, 0.69, respectively). In all experimental specimens, transmission electron microscopy showed morphological changes associated with apoptosis. This study showed that intracameral adrenaline, TB, and lidocaine injections may have toxic effects on corneal tissue, as indicated by ultrastructural and histopathological alterations. Therefore, these agents should be used with caution in intraocular surgery.

In Vitro Combination of Ascorbic and Ellagic Acids in Sperm Oxidative Damage Inhibition

It is known that an altered redox balance interferes with normal spermatic functions. Exposure to genotoxic substances capable of producing oxidative stress (OS) can cause infertility in humans. The use of antioxidants to reduce oxidative stress contributes to the improvement in reproductive function. This study focused on an antigenotoxic evaluation of ellagic acid (EA) and ascorbic acid (AA) in combination against benzene genotoxic action on human spermatozoa in vitro. In addition to the evaluation of sperm parameters, damage in sperm genetic material and intracellular ROS quantification were assessed after AA, EA and benzene co-exposure using the TUNEL technique and DCF assay. The results showed that the combination of the two antioxidants generates a greater time-dependent antigenotoxic action, reducing both the sperm DNA fragmentation index and the oxidative stress. The genoprotective effect of AA and EA association in sperm cells lays the foundations for a more in-depth clinical study on the use of antioxidants as a therapy for male infertility.

Transcriptional Factor Forkhead Box D1 Upregulates Sirtuin3 by Activating the Wnt/β-Catenin Pathway to Alleviate HTR-8/Svneo Trophoblast Cell Dysfunction in Preeclampsia

The proliferation, invasion, migration and apoptosis of trophoblast cells in preeclampsia are closely related to the occurrence and development of preeclampsia. Transcription factors forkhead box D1 and Sirtuin3 are abnormally expressed in preeclampsia, and Sirtuin3 plays a regulatory role in cell proliferation, invasion and apoptosis in related diseases. However, the studies on forkhead box D1 and Sirtuin3 in preeclampsia and their specific mechanisms have not been reported so far. In this study, the expression of Sirtuin3 in Human chorionic trophoblast cells HTR-8/Svneo was inhibited by cell transfection, and then the effects of Sirtuin3 expression in interfering cells on cell invasion, migration and apoptosis were detected by MTT, TUNEL, Western blot, wound healing and Transwell techniques. Subsequently, the binding between forkhead box D1 and Sirtuin3 was predicted by JASPAR website and verified by luciferase assay and ChIP assay. Finally, cell invasion, migration and apoptosis were detected after overexpression of forkhead box D1 and interference with Sirtuin3, and the Wnt/β-catenin signaling pathway was detected to explore the mechanism. We found that interfering with Sirtuin3 induced apoptosis of HTR-8/Svneo cells and inhibited cell invasion and migration. Forkhead box D1 transcriptional activation of Sirtuin3 alleviated HTR-8/SVneo cell dysfunction through activation of the Wnt/β-catenin signaling pathway. Overall, transcriptional factor forkhead box D1 can upregulate Sirtuin3 by activating the Wnt/β-catenin pathway to alleviate HTR-8/Svneo trophoblast cell dysfunction in preeclampsia.

CB1 Cannabinoid Receptor is a Target for Neuroprotection in Light Induced Retinal Degeneration.

In the last few years, an increasing interest in the neuroprotective effect of cannabinoids has taken place. The aim of the present work was to study the effects of modulating cannabinoid receptor 1 (CB1) in the context of light induced retinal degeneration (LIRD), using an animal model that resembles many characteristics of human age-related macular degeneration (AMD) and other degenerative diseases of the outer retina. Sprague Dawley rats (n = 28) were intravitreally injected in the right eye with either a CB1 agonist (ACEA), or an antagonist (AM251). Contralateral eyes were injected with respective vehicles as controls. Then, rats were subjected to continuous illumination (12,000 lux) for 24h. Retinas from 28 animals were processed by GFAP-immunohistochemistry (IHC), TUNEL technique, Western blotting (WB), or qRT-PCR. ACEA-treated retinas showed a significantly lower number of apoptotic nuclei in the outer nuclear layer (ONL), lower levels of activated Caspase-3 by WB, and lower levels of glial reactivity by both GFAP-IHC and WB. qRT-PCR revealed that ACEA significantly decreased the expression of Bcl-2 and CYP1A1. Conversely, AM251-treated retinas showed a higher number of apoptotic nuclei in the ONL, higher levels of activated Caspase-3 by WB, and higher levels of glial reactivity as determined by GFAP-IHC and WB. AM251 increased the expression of Bcl-2, Bad, Bax, Aryl hydrocarbon Receptor (AhR), GFAP, and TNFα. In summary, the stimulation of the CB1 receptor, previous to the start of the pathogenic process, improved the survival of photoreceptors exposed to LIRD. The modulation of CB1 activity may be used as a neuroprotective strategy in retinal degeneration and deserves further studies.

Kruppel-Like Factor (KLF6) Regulates Oxidative Stress and Apoptosis of Human Retinal Pigment Epithelial Cells Induced by High Glucose Through Transcriptional Regulation of USP22 and the Downstream SIRT1/Nrf2 Pathway

Oxidative stress and apoptosis play an important role in diabetic retinopathy (DR). KLF6 and its transcriptional regulator USP22 are abnormally expressed in DR, but the specific role and mechanism have not been reported. In this paper, we will discuss the specific roles and mechanisms of KLF6 and USP22 on oxidative stress and apoptosis in DR. In this study, RT-qPCR and western blot were used to detect the expression of KLF6 and USP22 in ARPE-19 cells. Subsequently, after KLF6 was overexpressed and USP22 expression was inhibited by cell transfection, the oxidative stress and apoptosis related indexes were detected by CCK-8, ELISA, TUNEL and other techniques to explore the mechanism. In addition, we used luciferase and ChIP to detect the association between KLF6 and USP22. Finally, the expression of proteins related to the SIRT1/Nrf2 pathway was detected by western blot. The results showed that silencing USP22 increased the activity, and inhibited apoptosis and oxidative stress of ARPE-19 cells induced by high glucose (HG). KLF6 transcriptionally activates USP22. Overexpression of KLF6 reversed the protective effects of silencing USP22 on HG-induced ARPE-19 cells against apoptosis and antioxidant stress, which may be achieved by regulating the SIRT1/Nrf2 pathway. In conclusion, KLF6 regulated oxidative stress and apoptosis of ARPE-19 cells induced by high glucose through transcriptional regulation of USP22 and the downstream SIRT1/Nrf2 pathway.

Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type

Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of ‘rounded’ cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.

Transcriptional activity and splicing factors are preserved during physiological apoptosis

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism’s life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Air Pollution's Impact on Cardiac Remodeling in an Experimental Model of Chagas Cardiomyopathy.

BackgroundChagas disease is characterized by intense myocardial fibrosis stimulated by the exacerbated production of inflammatory cytokines, oxidative stress, and apoptosis. Air pollution is a serious public health problem and also follows this same path. Therefore, air pollution might amplify the inflammatory response of Chagas disease and increase myocardial fibrosis.MethodsWe studied groups of Trypanosoma cruzi infected Sirius hamsters (Chagas=CH and Chagas exposed to pollution=CH+P) and 2 control groups (control healthy animals=CT and control exposed to pollution=CT+P). We evaluated acute phase (60 days post infection) and chronic phase (10 months). Echocardiograms were performed to assess left ventricular systolic and diastolic diameter, in addition to ejection fraction. Interstitial collagen was measured by morphometry in picrosirius red staining tissue. The evaluation of inflammation was performed by gene and protein expression of cytokines IL10, IFN-γ, and TNF; oxidative stress was quantified by gene expression of NOX1, MnSOD, and iNOS and by analysis of reactive oxygen species; and apoptosis was performed by gene expression of BCL2 and Capsase3, in addition to TUNEL analysis.ResultsChagas groups had increased collagen deposition mainly in the acute phase, but air pollution did not increase this deposition. Also, Chagas groups had lower ejection fraction in the acute phase (p = 0.002) and again air pollution did not worsen ventricular function or dilation. The analysis of the inflammation and oxidative stress pathways were also not amplified by air pollution. Apoptosis analysis showed increased expression of BCL2 and Caspase3 genes in chagasic groups in the acute phase, with a marginal p of 0.054 in BCL2 expression among infected groups, and TUNEL technique showed amplified of apoptotic cells by pollution among infected groups.ConclusionsA possible modulation of the apoptotic pathway was observed, inferring interference from air pollution in this pathway. However, it was not enough to promote a greater collagen deposition, or worsening ventricular function or dilation caused by air pollution in this model of Chagas cardiomyopathy.

Effects of treatment with clinically relevant valproate, carbamazepine, oxcarbazepine, topiramate, lamotrigine and levetiracetam on ovarian folliculogenesis in young rats

AimTo determine the effects of valproate (VPA), carbamazepine (CBZ), oxcarbazepine(OXC), topiramate (TPM), lamotrigine (LTG), and levetiracetam (LEV) on ovarian folliculogenesis in young rats. MethodsForty-nine female Wistar rats, aged 21–24 days, were divided equally into 7 experimental groups. These were given tap water over 21–24 days (control group), 300 mg/kg of VPA, 150 mg/kg of CBZ, 150 mg/kg of OXC, 100 mg/kg of TPM, 10 mg/kg of LTG, or 50 mg/kg of LEV daily in 2 doses via oral gavage until the end of puberty. At the end of the study, the estrous cycle of each rat was monitored daily, and those rats in pro-estrus or di-estrus were sacrificed and the ovaries removed. Serial sections obtained from the ovaries were stained with hematoxylin and eosin, and the corpora lutea and follicles were enumerated. Apoptotic cells were detected using the TUNEL technique. Various serial sections were immunohistochemically stained with proliferating cell nuclear antigen (PCNA), growth differentiation factor (GDF)-9, caspase-3, caspase-9, transforming growth factor beta 1 (TGF-1), and epidermal growth factor (EGF), and evaluated and photographed under a light microscope. Key FindingsThe number of corpora lutea was significantly increased in the VPA, CBZ, OXC, and LTG groups compared to the control group (p < 0.001). The number of TUNEL-positive ovarian follicles was 3.3 ± 1.1 (median, 3), 6.1 ± 0.9 (median, 6), and 5.7 ± 0.8 (median,6) in the control, OXC and LEV groups, respectively (p < 0.001). The number of TUNEL-positive granulosa cells was higher in all the groups treated with antiepileptics, with the exception of the TPM group, compared to the control group (p < 0.001). HSCOREs for immunohistochemical staining using PCNA, GDF-9, TGF-1 and EGF were significantly higher in the control group than in the others (p < 0.001). HSCORE for staining using caspase-3 was significantly higher in the VPA, CBZ, OXC and LEV groups, while the HSCORE was significantly lower in the TPM group than in the control group. HSCORE for staining using caspase-9 was significantly higher in the VPA, CBZ and OXC groups, while it was significantly lower in the TPM group than in the control group (p < 0.001). SignificanceExposure to VPA, CBZ, OXC, TPM, LTG and LEV caused different levels of impaired folliculogenesis in young rats.

Neuroprotective Gene Therapy by Overexpression of the Transcription Factor MAX in Rat Models of Glaucomatous Neurodegeneration.

PurposeBased on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury.MethodsOverexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections.ResultsIn all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model.ConclusionsOur experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.