Comparative Study of Two-Dimensional (2D) vs. Three-Dimensional (3D) Organotypic Kertatinocyte-Fibroblast Skin Models for Staphylococcus aureus (MRSA) Infection

The invasion of skin tissue by Staphylococcus aureus is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant Staphylococcus aureus (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains.

Anti-Inflammatory, Antioxidant, and Antifibrotic Effects of Gingival-Derived MSCs on Bleomycin-Induced Pulmonary Fibrosis in Mice

Background: Mesenchymal stem cell (MSC) intervention has been associated with lung protection. We attempted to determine whether mouse gingival-derived mesenchymal stem cells (GMSCs) could protect against bleomycin-induced pulmonary fibrosis. Methods: Mice were divided into three groups: control (Con), bleomycin (Bl), and bleomycin + MSCs (Bl + MSCs). Mice were treated with 5 mg/kg bleomycin via transtracheal instillation to induce pulmonary fibrosis. We assessed the following parameters: histopathological severity of injury in the lung, liver, kidney, and aortic tissues; the degree of pulmonary fibrosis; pulmonary inflammation; pulmonary oedema; profibrotic factor levels in bronchoalveolar lavage fluid (BALF) and lung tissue; oxidative stress-related indicators and apoptotic index in lung tissue; and gene expression levels of IL-1β, IL-8, TNF-α, lysophosphatidic acid (LPA), lysophosphatidic acid receptor 1 (LPA1), TGF-β, matrix metalloproteinase 9 (MMP-9), neutrophil elastase (NE), MPO, and IL-10 in lung tissue. Results: GMSC intervention attenuated bleomycin-induced pulmonary fibrosis, pulmonary inflammation, pulmonary oedema, and apoptosis. Bleomycin instillation notably increased expression levels of the IL-1β, IL-8, TNF-α, LPA, LPA1, TGF-β, MMP-9, NE, and MPO genes and attenuated expression levels of the IL-10 gene in lung tissue, and these effects were reversed by GMSC intervention. Bleomycin instillation notably upregulated MDA and MPO levels and downregulated GSH and SOD levels in lung tissue, and these effects were reversed by GMSC intervention. GMSC intervention prevented upregulation of neutrophil content in the lung, liver, and kidney tissues and the apoptotic index in lung tissue. Conclusions: GMSC intervention exhibits anti-inflammatory and antioxidant capacities. Deleterious accumulation of neutrophils, which is reduced by GMSC intervention, is a key component of bleomycin-induced pulmonary fibrosis. GMSC intervention impairs bleomycin-induced NE, MMP-9, LPA, APL1, and TGF-β release.

Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 μM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 μM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 μM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

PENGARUH FLAVONOID EKSTRAK MAHKOTA DEWA (PHALERIA MACROCARPA) TERHADAP PENINGKATAN INDEKS APOPTOSIS PADA PERITONEAL MENCIT MODEL ENDOMETRIOSIS

Background: Endometriosis is one of the main reproductive problems today because the incidence is quite high. The development and progression of endometriosis cells results from an abnormal balance between cell proliferation and apoptosis. Cell apoptosis is triggered by an imbalance between positive signals (cell growth factors) and negative signals (DNA damage) in endometriosis. The anti-cancer effect of Mahkota Dewa has been shown to suppress cancer growth and inhibit cancer cell invasion through inhibition of proliferation and anti-apoptotic activity, Purpose: This study aimed to determine the effect of giving flavonoid extract from Mahkota Dewa fruit on the apoptotic index in endometriosis model mice. Methods: This research is an experimental study conducted at the Laboratory of the Faculty of Veterinary Medicine, Airlangga University, Surabaya. The sample used mice (Mus musculus) endometriosis model. The study was divided into 6 groups: negative control, positive control and treatment with the flavonoid extract of the Mahkota Dewa fruit at a dose of 3.75 mg/day, 7.5 mg/day, 11.25 mg/day and 15 mg/day. Measurement of apoptotic index using Tunel kit immunohistochemistry. Observational data were analyzed by ANOVA dan Tukey. Results: There was significant difference in the mean apoptotic index of the five groups of observational samples, the mean value of the apoptotic index was found between the KP group (7.96 ± 1.02) and the group given flavonoid extract from the Mahkota Dewa fruit group P1 at a dose of 3.75 mg (5.36 ± 0.91), P2 dose 7.5 mg (3.4 ± 0.49), P3 dose 11.25 mg (3.88 ± 0.59), P4 dose 15 mg (3.96 ± 0.75). Conclusion: The administration of flavonoid extract of the Mahkota Dewa fruit had a significant effect on increasing cell apoptotic index in endometriosis model mice.Suggestion Further study is needed to see the effect of flavonoid extract of Mahkota Dewa on rabbit experimental animals. Keywords: endometriosis, flavonoid extract of Mahkota Dewa fruit, apoptotic index ABSTRAK Latar Belakang: Endometriosis menjadi salah satu masalah reproduksi utama saat ini karena angka kejadian cukup tinggi. Perkembangan dan progresi sel endometriosis akibat terjadinya abnormalitas keseimbangan antara proliferasi dan apoptosis sel. Apoptosis sel dipicu karena adanya ketidakseimbangan antara sinyal positif (faktor pertumbuhan sel) dan sinyal negatif (kerusakan DNA) pada kondisi endometriosis. Efek anti kanker mahkota dewa terbukti dapat menekan pertumbuhan kanker dan menghambat terjadinya invasi sel kanker melalui penghambatan aktivitas proliferasi dan anti apoptosis.Tujuan: Penelitian ini bertujuan untuk mengetahui efek pemberian flavonoid ekstrak dari buah mahkota dewa terhadap indeks apoptosis pada mencit model endometriosis.Metode: Penelitian ini merupakan penelitian eksperimental yang dilakukan di Laboratorium Fakultas Kedokteran Hewan Universitas Airlangga Surabaya. Sampel menggunakan mencit (Mus musculus) model endometriosis. Penelitian dibagi menjadi 6 kelompok, yaitu : kontrol negatif, kontrol positif dan perlakuan pemberian ekstrak flavonoid buah mahkota dewa dosis 3,75 mg/hari, 7,5 mg/hari, 11,25 mg/hari dan15 mg/hari. Pengukuran indeks apoptosis menggunakan imunohistokimia Tunel kit. Data hasil pengamatan dianalisis dengan uji ANOVA dan Tukey. Uji statistik dikatakan bermakna bila p<0,05.Proses penghitungan dilakukan dengan bantuan piranti lunak (soft-ware) SPSS for windows 19.0.Hasil: Terdapat perbedaan yang bermakna rerata indeks apoptosis kelima kelompok sampel pengamatan didapatkan nilai rerata indeks apoptosis antara kelompok K- (7.96 ± 1.02) dengan kelompok pemberian ekstrak flavonoid dari buah mahkota dewa kelompok P1 dosis 3,75 mg (5.36 ± 0.91), P2 dosis 7,5 mg (3.4 ± 0.49), P3 dosis 11,25 mg (3.88 ± 0.59), P4 dosis 15 mg (3.96 ± 0.75).Kesimpulan: Pemberian ekstrak flavonoid buah mahkota dewa memberikan pengaruh yang signifikan terhadap peningkatan indeks apoptosis sel pada mencit model endometriosis.Saran Perlu studi lanjut untuk melihat pengaruh flavonoid ekstrak mahkota dewa pada hewan coba kelinci. Kata Kunci: endometriosis, flavonoid ekstrak buah mahkota dewa, indeks apoptosis

Study on the Relationship between Cardiomyocyte Apoptosis and Left Ventricular Function in Spontaneously Hypertensive Rats

At present, hypertension is a relatively common cardiovascular disease. It not only affects the normal operation of target organs such as the heart and kidneys, but also causes cardiovascular and cerebrovascular diseases, which can lead to death. The apoptosis of cardiomyocytes is widespread in the cardiovascular system and is closely related to vascular diseases. Therefore, the purpose of this article is to explore the relationship between changes in left ventricular function, myocardial multidimensional strain and interstitial fibrosis in spontaneously hypertensive rats (SHR) during cardiomyocyte apoptosis. Whether the research is consistent in terms of order, as the age of hypertensive rats increases, whether there is a close connection between myocardial cell apoptosis and the structure and function of the left heart. The method in this article is to use the method of experimental comparison to randomly group 60 experimental mice and observe the changes of various indicators of rats of different ages, from 12 weeks to 84 weeks. The observations include left ventricular mass index (LVMI), left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (FS), LVEDP, LV+dp/dtmax, LV-dp/dtmax. At the same time, using the TUNEL labeling method, the left ventricular myocardial tissue was sliced, and the apoptosis index of subendocardial and subepicardial myocardial cells was calculated. Then 3 groups were randomly selected from the experimental group, and Western blotting was used to quantitatively detect apoptosis-related the expression of the proteins Bcl-2, Bax, and Fas were compared between the groups. After analysis and determination, it can be found that the apoptosis index of cardiomyocytes is positively correlated with LVMI, CVF, and PVCA (r is 0.83, 0.89, 0.72, respectively, p=0.00). Corresponding conclusions are drawn from the comparison of data. As hypertensive rats grow older, the apoptotic index of cardiomyocytes will continue to increase, and when the myocardial hypertrophy is severe to heart failure, the apoptotic index of cardiomyocytes will increase significantly. This shows that the increase in cardiomyocyte apoptosis is closely related to left heart remodeling, the development of myocardial fibrosis and overall cardiac dysfunction.

Effects of changing culture medium on preimplantation embryo development in rabbit

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

Image-Guided Peri-Tumoral Radiofrequency Hyperthermia-Enhanced Direct Chemo-Destruction of Hepatic Tumor Margins

PurposeTo validate the feasibility of using peri-tumoral radiofrequency hyperthermia (RFH)-enhanced chemotherapy to obliterate hepatic tumor margins.Method and MaterialsThis study included in vitro experiments with VX2 tumor cells and in vivo validation experiments using rabbit models of liver VX2 tumors. Both in vitro and in vivo experiments received different treatments in four groups (n=6/group): (i) RFH-enhanced chemotherapy consisting of peri-tumoral injection of doxorubicin plus RFH at 42°C; (ii) RFH alone; (iii) doxorubicin alone; and (iv) saline. Therapeutic effect on cells was evaluated using different laboratory examinations. For in vivo experiments, orthotopic hepatic VX2 tumors in 24 rabbits were treated by using a multipolar radiofrequency ablation electrode, enabling simultaneous delivery of both doxorubicin and RFH within the tumor margins. Ultrasound imaging was used to follow tumor growth overtime, correlated with subsequent histopathological analysis.ResultsIn in vitro experiments, MTS assay demonstrated the lowest cell proliferation, and apoptosis analysis showed the highest apoptotic index with RFH-enhanced chemotherapy, compared with the other three groups (p&lt;0.01). In in vivo experiments, ultrasound imaging detected the smallest relative tumor volume with RFH-enhanced chemotherapy (p&lt;0.01). The TUNEL assay further confirmed the significantly increased apoptotic index and decreased cell proliferation in the RFH-enhanced therapy group (p&lt;0.01).ConclusionThis study demonstrates that peri-tumoral RFH can specifically enhance the destruction of tumor margins in combination with peri-tumoral injection of a chemotherapeutic agent. This new interventional oncology technique may address the critical clinical problem of frequent marginal tumor recurrence/persistence following thermal ablation of large (&gt;3 cm) hepatic cancers.

Tetrahydropalmatine reduces cell death and improves functional recovery after traumatic spinal cord injury in ratsTetrahydropalmatine reduces cell death and improves functional recovery after traumatic spinal cord injury in rats

Purpose: To investigate the effectiveness of tetrahydropalmatine (THP) in the treatment of spinal cord injury (SCI) in rats. Methods: Adult Sprague Dawley rats were divided into 3 groups: normal control group, SCI group, and SCI group treated with THP (100 mg kg-1). The effect of THP on spinal cord water content, levels of inflammatory mediators, oxidative stress and apoptosis were determined. Locomotor activity in rats was measured using Basso, Beattie and Bresnahan (BBB) scores. Various oxidative stress markers as well as cytokine levels (NF-ĸB, IL-6, IL-1β and TNF-α were determined. Apoptotic index was measured using TUNEL assay. Results: After 72 h of treatment with THP, BBB scores in SCI group of rats significantly increased from 4.19 ± 0.41 to 8.89 ± 0.47 (p < 0.05). Tunel assay revealed a higher apoptotic index (42.50 ± 3.19) in the tissues of SCI rats than in SCI rats treated with THP (31.48 ± 1.19, p < 0.01). Expressions of inflammatory cytokines were significantly upregulated in SCI rats. However, THP administration resulted in significant downregulation of the expressions (p < 0.01). Conclusion: These results indicate that THP attenuates traumatic SCI in rats via modulation of the levels of anti-inflammatory mediators. Thus, THP has a promising potential for the management of SCI.