Corneal Endothelium Viability Assay Using Trypan Blue Dye After Preloaded Descemet Membrane Endothelial Keratoplasty Graft Preparation.

PURPOSE: The purpose of this study was to establish a validated method, consistent with Eye Bank Association of America medical standards, for evaluating endothelial cell loss (ECL) from an entire Descemet membrane endothelial keratoplasty (DMEK) graft using trypan blue dye as an alternative to specular microscopy. METHOD: Twenty-nine corneas were prepared for preloaded DMEK by a single technician, and the endothelium was stained with trypan blue dye for 30 seconds. The technician estimated total cell loss as a percentage of the graft and captured an image. Images were evaluated by a blinded technician using ImageJ software to determine ECL and compared with endothelial cell density from specular microscopy. Tissue processing intervals were analyzed for 4 months before and after implementation of this method. RESULTS: For the 29 grafts, there was no statistically significant difference ( t test, P = 0.285) between ECL estimated by a processor (mean = 5.8%) and ECL calculated using an ImageJ software (mean = 5.1%). The processor tended to estimate greater ECL than the actual ECL determined by ImageJ (paired t test, P = 0.022). Comparatively, postprocessing endothelial cell density measured by specular microscopy were higher compared with the preprocessing endothelial cell density (mean = 4.5% P = 0.0006). After implementation of this evaluation method, DMEK graft processing time intervals were reduced by 47.9% compared with specular microscopy evaluation ( P < 0.001). CONCLUSIONS: Our results show that visual ECL estimation using trypan blue staining by a DMEK graft processor is a reliable and efficient method for endothelial assessment. Unlike specular microscopy, this method achieves comprehensive visualization of the entire endothelium, reduces total time out of cold storage, and decreases total time required to prepare and evaluate DMEK grafts.

Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings.

Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34+ human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34+ stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34+ stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r = .03; P = .5). The median CD34+ stem cell count in the post-thaw samples was 9.13 × 106/kg (range, .44 to 26.27 × 106/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34+ stem cell levels (r = .4; P = .05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.

Evaluation of the Efficacy of Sodium Chloride Nanoparticles on the Vitality of Leishmania Major (in vitro).

Leishmaniasis is one of the most important zoonotic diseases transmitted to humans by sand flies (Phlebotomus spp). Leishmania major promastigote causes Cutaneous Leishmaniasis in humans. The study aimed to investigate the effectiveness of Sodium Chloride nanoparticles (NaCl NPs) on the vitality of Leishmania major promastigote compared with the standard dose of Pentostam under laboratory conditions. Various concentrations of 2, 4, 6, and 8 µg/ml of the NaCl NPs were prepared. These concentrations were tested in vitro on L. major growth by the culture of the parasite in the cell culture microplate. After the fourth day, a different concentration of NaCl NPs was added with three replicates for each concentration. Later, the numbers of promastigotes were counted daily using a Haemocytometer stained by Trypan blue solution stain duration of the study, which continued for four days. The results showed that the Growth Index (GI) rate of L. major promastigote was decreased with increasing NaCl NPs concentration. The Growth Index rates were 1.32×106, 1.31×106, 0.95×106, and 0.78×106 for the mentioned concentrations. These values were compared with the rate of the Pentostam group and control group, which were 1.09×106 and 3.43×106, respectively. The results revealed that the highest inhibition percentage was 92% for 8 µg/ml NaCl NPs after 96 hours, Pentostam group and control group, which were %86 and %0.00 for inhibition promastigote, respectively in the same period. The statistical analysis revealed a significant difference among concentrations at P≤0.05 compared with the Pentostam and control groups. The current study concluded that the NaCl NPs have an excellent biological effect in inhibiting L. major promastigote growth in vitro. These promising results paved the way for employing NaCl NPs to treat human cutaneous leishmaniasis.

Inhibition of Vesicular Glutamate Transporters (VGLUTs) with Chicago Sky Blue 6B Before Focal Cerebral Ischemia Offers Neuroprotection.

Brain ischemia is one of the leading causes of death and long-term disability in the world. Interruption of the blood supply to the brain is a direct stimulus for many pathological events. The massive vesicular release of glutamate (Glu) after ischemia onset induces excitotoxicity, which is a potent stress on neurons. Loading of presynaptic vesicles with Glu is the first step of glutamatergic neurotransmission. Vesicular glutamate transporters 1, 2, and 3 (VGLUT1, 2, and 3) are the main players involved in filling presynaptic vesicles with Glu. VGLUT1 and VGLUT2 are expressed mainly in glutamatergic neurons. Therefore, the possibility of pharmacological modulation to prevent ischemia-related brain damage is attractive. In this study, we aimed to determine the effect of focal cerebral ischemia on the spatiotemporal expression of VGLUT1 and VGLUT2 in rats. Next, we investigated the influence of VGLUT inhibition with Chicago Sky Blue 6B (CSB6B) on Glu release and stroke outcome. The effect of CSB6B pretreatment on infarct volume and neurological deficit was compared with a reference model of ischemic preconditioning. The results of this study indicate that ischemia upregulated the expression of VGLUT1 in the cerebral cortex and in the dorsal striatum 3 days after ischemia onset. The expression of VGLUT2 was elevated in the dorsal striatum and in the cerebral cortex 24 h and 3 days after ischemia, respectively. Microdialysis revealed that pretreatment with CSB6B significantly reduced the extracellular Glu concentration. Altogether, this study shows that inhibition of VGLUTs might be a promising therapeutic strategy for the future.

The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues.

PURPOSE: The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium-Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK). METHODS: Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-ß1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB. RESULTS: Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-ß1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts. CONCLUSION: Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting.

Evaluation of mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity, and acute toxicity of ethanolic extract of Cissus quadrangularis.

Recent studies have focused on exploring the efficacy of Cissus quadrangularis extract (EECQ) against various metabolic disorders involving the liver as the prime target organ, suggesting a considerable threat of hepatotoxicity in the person encountering it. Consequently, the current study was aimed to unravel the mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity in HepG2 cells, and acute toxicity of EECQ. MTT, SRB, trypan blue dye exclusion, and lactate dehydrogenase (LDH) assay were performed in HepG2 cell lines to determine the cytotoxicity of the extract. The mutagenic potential was determined by the Ames test using various strains of Salmonella typhimurium. Acute toxicity was done at a dose of 2000 mg/kg in Sprague Dawley rats. MTT and SRB cytotoxicity assays demonstrated dose-dependent cytotoxicity of extract. The three highest noncytotoxic doses from the above assay, investigated by trypan blue dye exclusion and LDH assay, did not reveal cytotoxicity. Besides, mitochondrial dysfunction was determined by measuring cellular and mitochondrial ROS, ATP, NAD, mitochondrial membrane potential, Bax/Bcl2 ratio, mitochondrial and cytoplasmic cytochrome c, and apoptosis-inducing factor, were found to be equivalent in both extract exposed and unexposed cells. Moreover, the apoptotic cell morphology and the expression of pro-apoptotic mRNAs and proteins were equivalent in both the group. In acute toxicity, EECQ in rats did not cause any significant change in body weight, liver index, and liver function test. All-encompassing, the present study unraveled that EECQ is not mutagenic, cytotoxic, nor apoptotic in human hepatic cells, as well as neither acute toxicity.

Anticancer Effect of Sargassum oligocystom Hydroalcoholic Extract Against SW742, HT-29, WiDr, and CT-26 Colorectal Cancer Cell Lines and Expression of P53 and APC Genes.

PURPOSE: Colorectal cancer (CRC) is the third most common cancer in the world, with enhancing morbidity and mortality each year. Due to the drug resistance against CRC, the use of novel compounds besides chemotherapy is required. Natural seafood contains large amounts of biologically active substances with new chemical structures and new medicinal activities. The aim of this study was to evaluate the effects of hydroalcoholic extract of Sargassum oligocystom algae on SW742, HT-29, WiDr, and CT-26 CRC cell lines, and to evaluate the expression of P53 and APC genes using quantitative real-time PCR (RT-qPCR). METHODS: The cytotoxicity of S. oligocystom hydroalcoholic extract was determined by MTT and trypan blue methods in six different concentrations including 0.1, 0.2, 0.5, 1, 2, and 4 mg/mL on various CRC cell lines and a control group. The expression of P53 and APC genes in exposure to 2 mg/mL of the extract was also evaluated using RT-qPCR. RESULTS: The LD50 and LD90 of S. oligocystom included 0.5-1 and > 2 mg/mL, respectively mostly affecting SW742 and CT-26 cells. In the trypan blue test, 90% viability and death of cells were observed at 0.1 and 4 mg/mL of extract, respectively. The 2 mg/mL was a safe cytotoxic concentration. A significant viability decrease was observed at concentrations ≥ 1 mg/mL (p < 0.001). Sargassum oligocystom extract at 2 mg/mL significantly increased the expression of APC ranging 1.98-2.2-fold (p < 0.001) but not P53 gene which ranged 0.5-0.68-fold (p = 0.323) after 24 h. CONCLUSION: These results indicated that the brown algae S. oligocystom extract had significant antitumor effects against the SW742, HT-29, WiDr, and CT-26 CRC cell lines and especially CT-26, suggesting that it may be a potential candidate for further studies and therefore designing drugs of natural anticancer origin. The S. oligocystom had an anticancer effect via an increase in the APC gene expression.

Effects of 2-methoxyestradiol on hydrogen peroxide induced neuronal cell death and tau hyperphosphorylation.

AIMS: Alzheimer's Disease (AD) is characterized by progressive cognitive impairment, and memory loss. It has been shown that depletion of estrogens renders women vulnerable to AD with menopause women presenting higher risk for AD development than men. However, women under hormone replacement therapy (HRT) with 17ß-estradiol (E2) show lower risk for AD, implying that E2 may be protective. It has been shown that E2 exerts its effects through the estrogen receptor (ER) but also via its biologically active metabolites, 2-hydroxyestradiol (2OH), and 2-methoxyestradiol (2ME). We hypothesized that the neuroprotective effects of E2 are partly attributed to its metabolites. MATERIALS AND METHODS: SH-SY5Y neuronal cells were subjected oxidative stress (OS) cell death by hydrogen peroxide (H2O2), in the presence or absence of E2, 2ME and 2OH. Viability was assessed by trypan blue and thiazolyl blue tetrazolium bromide assays, intracellular OS with the Dichlorodihydrofluorescein Diacetate (DCFDA) assay, and Bax, p53 and PUMA quantified by RT-PCR. Tau hyperphosphorylation was studied by western blot. KEY FINDINGS: E2 and its metabolites 2OH and 2ME protect from cell death as assessed by the viability assays. Their effect was partly attributed to their antioxidant properties evidenced by the reduction of intracellular OS. Treatment with 2ME resulted in a reduction of Bax, but not p53 or PUMA in cells challenged with OS. Finally, 2ME was able to inhibit tau hyperphosphorylation as well. SIGNIFICANCE: E2 protects neuron cells partly through its metabolites. Further studies are needed to fully delineate the mechanism for this protection.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

BACKGROUND: Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells. AIM: To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models. METHODS: For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)_nu mice received daily intratumoral injections of PTHrP (40 µg/kg) in 100 µL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05. RESULTS: By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (P < 0.01) and at 20 h (P < 0.01) whereas the levels diminished at 3 min (P < 0.05), 10 min (P < 0.01), and 1 h to 5 h (P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased (P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis (P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation (P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation (P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation (P < 0.05), migration (P < 0.05), and the EMT program (P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 (P < 0.001), OXA (P < 0.01), and DOXO (P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively (P < 0.05). CONCLUSION: PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

MSC-Derived exosomes suppress colorectal cancer cell proliferation and metastasis via miR-100/mTOR/miR-143 pathway.

Exosomes derived from mesenchymal stem cells (MSCs) are mostly responsible for the therapeutic effects of MSCs. To show the therapeutic effects of the human bone marrow MSC-derived exosomes (MSC-Exos) on colorectal cancer (CRC) and explore the molecular cross-talks between them, CRC cells were treated with the MSC-Exos. We found that MSC-Exos were enriched with miR-100 and miR-143, which effectively downregulated mTOR, Cyclin D1, K-RAS, HK2 while upregulated p-27 expression. All these effects were reversed by concurrent treatment with MSC-Exos and antagomiR-100, confirming that they were caused by exosomal transfer of miR-100 into recipient CRC cells. Moreover, exosomal miR-100 promoted endogenous miR-143 expression. The flow cytometry, MTT and trypan blue assays revealed that MSC-Exos could efficiently suppress proliferation and induce apoptosis of the CRC cells. Furthermore, wound healing, transwell migration and invasion assays confirmed their inhibitory effects on the migration and invasiveness of SW480 cells. We further confirmed these effects by analyzing the expression levels of epithelial to mesenchymal transition (EMT) factors and metastasis-related genes. Results showed that MSC-Exos significantly suppressed the expression of MMP2 and MMP9 (metastasis-related genes), SNAIL and TWIST (EMT-inducing transcription factors), Vimentin and N-cadherin (mesenchymal cell markers), whereas E-cadherin (epithelial cell marker) was remarkably up-regulated. Collectively, our data indicated that MSC-Exos could suppress proliferation, migration, invasion and metastasis while inducing the apoptosis of the CRC cells via miR-100/mTOR/miR-143 axis. Our findings highlight that MSC-Exo treatment as well as miR-100 restoration might be considered as potential therapeutic strategies for CRC.

Subconjunctival Lymphatics Respond to VEGFC and Anti-Metabolites in Rabbit and Mouse Eyes.

Purpose: To characterize and pharmacologically influence subconjunctival lymphatics in rabbit and mouse eyes. Methods: Rabbits received subconjunctival injections of trypan blue or fixable fluorescent dextrans. Bleb-related outflow pathways were quantified. Immunofluorescence for vessel-specific markers (lymphatics [podoplanin and LYVE-1] and blood vessels [CD31]) were performed in native rabbit conjunctiva and after fixable fluorescent dextran injection. Vascular endothelial cell growth factor-C (VEGFC) was injected subconjunctivally in rabbits. mRNA and protein were assessed for the above markers using RT-PCR and Western blot. Alternatively, mouse studies used Prox1-tdTomato transgenic reporter mice. Subconjunctival injection conditions included: no injection, balanced salt solution (BSS), VEGFC, 5-fluorouracil (5FU) and two concentrations of mitomycin-C (MMC). Two mouse injection protocols (short and long) with different follow-up times and number of injections were performed. Mouse eyes were enucleated, flat mounts created, and subconjunctival branching and length assessed. Results: Rabbit eyes demonstrated clear bleb-related subconjunctival outflow pathways that were distinct from blood vessels and were without nasal/temporal predilection. Immunofluorescence against vessel-specific markers showed lymphatics and blood vessels in rabbit conjunctiva, and these lymphatics overlapped with bleb-related subconjunctival outflow pathways. Subconjunctival VEGFC increased lymphatic (P = 0.004-0.04) but not blood vessel (P = 0.77-0.84) mRNA or protein in rabbits. Prox1-tdTomato transgenic reporter mice demonstrated natively fluorescent lymphatics. Subconjunctival VEGFC increased murine lymphatic branching and length (P ≤ 0.001-0.004) while antimetabolites (P ≤ 0.001-0.043) did the opposite for the long protocol. Discussion: Subconjunctival lymphatics are pharmacologically responsive to both VEGFC and antimetabolites in two animal models studied using different methodologies. These results may be important for bleb-forming glaucoma surgeries or ocular drug delivery.

Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

The Transcriptome Profile of Retinal Pigment Epithelium and Müller Cell Lines Protected by Risuteganib Against Hydrogen Peroxide Stress.

Purpose: Oxidative stress contributes to the pathogenesis of vision-impairing diseases. In the retina, retinal pigment epithelium (RPE) and Müller cells support neuronal homeostasis, but also contribute to pathological development under stressed conditions. Recent studies found that the investigational drug risuteganib (RSG) has a good safety profile, provided protection in experimental models, and improved visual acuity in patients. The present in vitro study evaluated the effects of RSG in RPE and Müller cell lines stressed with the oxidant hydrogen peroxide (H2O2). Methods: Human RPE (ARPE-19) and Müller (MIO-M1) cell lines were treated with various combinations of RSG and H2O2. Trypan blue assay was used to investigate the effect of compounds on cell viability. Gene expression was measured using RNA sequencing to identify regulated genes and the biological processes and pathways involved. Results: Trypan blue assay found RSG pre-treatment significantly protected against H2O2-induced cell death in ARPE-19 and MIO-M1 cells. Transcriptome analysis found H2O2 regulated genes in several disease-relevant biological processes, including cell adhesion, migration, death, and proliferation; ECM organization; angiogenesis; metabolism; and immune system processes. RSG pre-treatment modulated these gene expression profiles in the opposite direction of H2O2. Pathway analysis found genes in integrin, AP-1, and syndecan signaling pathways were regulated. Expression of selected RSG-regulated genes was validated using qRT-PCR. Conclusions: RSG protected cultured human RPE and Müller cell lines against H2O2-induced cell death and mitigated the associated transcriptome changes in biological processes and pathways relevant to the pathogenesis of retinal diseases. These results demonstrate RSG reduced oxidative stress-induced toxicity in two retinal cell lines with potential relevance to the treatment of human diseases.

Preparation and evaluation of polycaprolactone/chitosan/Jaft biocompatible nanofibers as a burn wound dressing.

Tissue engineering is an emerging method for replacing damaged tissues. In this study, the potential application of electrospun polycaprolactone/chitosan/ the internal layer of oak fruit (Jaft) as skin scaffolds was investigated. A combination of Polycaprolactone (PCL), chitosan (CH), and the internal layer of oak fruit (Jaft) was used to incorporate mechanical properties of synthetic polymers, biological properties of natural polymers, and antibacterial activity of Jaft. Physical and morphological characteristics of prepared scaffolds were investigated using a scanning electron microscope (SEM), mechanical analysis, swelling ratio, and contact angle. Moreover, chemical and biological properties were evaluated by Fourier-transform infrared spectroscopy (FTIR), chromatography, flow cytometry, DAPI staining, MTT assay, and trypan blue exclusion assay. Obtained results demonstrated that the fabricated scaffolds have good mechanical properties. Moreover, the addition of chitosan and Jaft to the PCL scaffolds improved their water absorption capacity as well as surface hydrophilicity. MTT results showed the fabricated nanofibrous scaffolds have adequate cell viability, which is higher than the cell culture plate at each time point of culture. Furthermore, SEM images of cultured scaffolds, trypan blue exclusion assay, and DAPI staining confirmed that fibroblast cells could be well-attached and proliferate on the PCL/CH/Jaft scaffolds. Results have proven that this novel bioactive scaffold has promising mechanical properties, suitable biocompatibility in vitro, and in vivo. Consequently, it could be a promising candidate for skin tissue engineering applications.

Chicago sky blue 6B (CSB6B), an allosteric inhibitor of macrophage migration inhibitory factor (MIF), suppresses osteoclastogenesis and promotes osteogenesis through the inhibition of the NF-κB signaling pathway.

Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator involved in various pathophysiological and inflammatory states. Accumulating line of evidence suggests a role for MIF in regulating bone metabolism and therefore a prime candidate for therapeutic targeting. In this study, we showed that Chicago sky blue 6B (CSB6B) suppresses RANKL-induced osteoclast and bone resorption in vitro via the inhibition of NF-κB signaling activation and promoting proteasome-mediated degradation of MIF. Consequently, the induction of NFATc1 was impaired resulting in downregulation of NFATc1-responsive osteoclast genes. We also demonstrated that CSB6B treatment enhanced primary calvarial osteoblast differentiation and bone mineralization in vitro via the suppression of NF-κB activation and upregulation of Runx expression. Using two murine models of osteolytic bone disorders, we further showed that administration of CSB6B protected mice against pathological inflammatoryc calvarial bone destruction induced by titanium particles mice as well as estrogen-deficiency induced bone loss as a result of ovariectomy. Together, as an MIF inhibitor, CSB6B can inhibit osteoclast differentiation and bone resorption function and enhance the mineralization of osteoblasts through the inhibition of NF-κB pathway. MIF is a prime target for therapeutic targeting for the treatment of osteolytic bone disorders and the MIF inhibitor CSB6B could be potential anti-osteoporosis drug.

Application of Trypan Blue Stain in the Microbiological Diagnosis of Infectious Keratitis-A Case Series.

PURPOSE: The purpose of this study was to report 3 cases of microbial keratitis, wherein trypan blue staining was used to aid the microbiological diagnosis of fungal keratitis and Pythium keratitis in a rural setup. METHODS: Three consecutive patients who presented with a diagnosis of infectious keratitis underwent corneal scraping, and the smears were assessed using trypan blue stain and potassium hydroxide (KOH) mount. RESULTS: Of the 3 cases, the first 2 cases showed septate fungal filaments in trypan blue-stained preparation and KOH mount. Case 3 showed the presence of broad aseptate filaments with ribbon-like folds on both KOH mount and trypan blue stain, consistent with the diagnosis of Pythium keratitis. The first 2 cases improved with topical and systemic antifungals. Case 1 healed with scarring at 7 weeks with improvement in best-corrected visual acuity (BCVA) to 20/60. Case 2 healed within 2 weeks, and BCVA improved to 20/40. Case 3 received topical linezolid (0.2%), azithromycin eye ointment, and oral azithromycin. At 5 weeks the infection decreased but thinning was noted, which necessitated tissue adhesive and bandage contact lens application. Scarring was noted at 10 weeks, and BCVA improved to 20/40. CONCLUSIONS: In this case series, trypan blue staining showed promising results in the easy identification of aseptate and septate fungal elements. This is the first case series showing the utility of this stain in the management of microbial keratitis.

Trypan-Assisted Automated Endothelial Cell Loss Measurements Compared With Specular Microscopy.

PURPOSE: The aims of this study were 1) to compare area of cell loss (ACL) on trypan staining with ACL on specular imaging and 2) to evaluate the use of automated software for measuring ACL on trypan staining. METHODS: Donor corneas with transplant-grade endothelium were mechanically injured with an 18-gauge cannula and a Fogla deep anterior lamellar keratoplasty dissector tip to create an easily identifiable "bullseye" pattern of cell death. Each cornea was then stained with trypan blue 0.06% for 90 seconds and imaged at 2× magnification. ACL on staining was measured using manual (ImageJ, National Institute of Health, Bethesda, MD) versus automated software tools (custom-built Aphelion macro, ADCIS, S.A., Saint-Contest, France). The bullseye was then imaged using specular microscopy, and ACL was measured by tracing the dead cell borders. ACL was then compared between both modalities. RESULTS: Eleven donor corneas were evaluated. Both manual (0.42 mm2) and automated (0.45 mm2) measurements of ACL after trypan staining underestimated mean ACL on specular imaging (0.54 mm2) (P < 0.01). However, on regression analysis, there was a good predictive correlation between automated trypan measurements and specular imaging (R2 = 0.99, residual SE = 0.0044, P < 0.01). When ACL on specular imaging was measured by tracing cell nuclei along the margin of injury (rather than cell borders) (0.45 mm2), there was no statistically significant difference between specular and automated trypan measurements (P = 0.95). CONCLUSIONS: Trypan-assisted automated measurements of ACL correlated well with ACL on specular imaging, suggesting that automated software may be a useful tool for evaluating endothelium in donor corneas.

Optimizing Visualization of Descemet Membrane Endothelial Keratoplasty Tissue: Assessing the Impact of Trypan Blue Exposure on Stain Duration and Corneal Endothelial Cell Function.

PURPOSE: To assess how trypan blue staining affects Descemet membrane endothelial keratoplasty (DMEK) graft visibility and corneal endothelial cell (CEC) mitochondrial respiration. METHODS: DMEK grafts (n = 20) were stained with trypan blue 0.06% for 1, 3, 5, or 10 minutes. Each graft was injected into an artificial anterior chamber. Surgery was simulated with tapping and sweeping motions on the corneal surface and injections of balanced salt solution (BSS). Graft visibility was assessed at 5, 10, 20, and 30 minutes. Effects of trypan blue on mitochondrial respiration were assessed using primary CECs cultured from donor corneas (n = 43). Treatment wells exposed to trypan blue 0.06% (1, 5, or 30 minutes) and donor-matched control wells to methylene blue 1% (1 minute) or BSS (1, 5, or 30 minutes) were assayed for key respiration parameters. RESULTS: After 5 minutes of surgical manipulation, grafts stained for 5 minutes were significantly more visible than grafts stained for 1 or 3 minutes; there was no added benefit of staining for 10 minutes. After 10 minutes of surgical manipulation, grafts stained for 3 minutes were more visible than grafts stained for 1 minute, without additional benefits of staining ≥5 minutes. No visibility differences were observed after ≥20 minutes of surgical manipulation. CEC mitochondrial respiration did not change significantly following trypan blue exposure for all intervals tested compared to BSS. CONCLUSIONS: Staining DMEK grafts with trypan blue for 3 to 5 minutes optimizes visibility during surgical manipulation without mitochondrial impairment. Corneal surgeons learning DMEK will benefit from optimizing this critical step.

Early Complications With Preloaded Descemet Membrane Endothelial Keratoplasty Are Not Dependent on Optisol-GS Washout or Trypan Blue Restaining.

PURPOSE: To describe the intraoperative and early postoperative complications using preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts with intraocular injection of the graft in Optisol-GS and omission of trypan blue restaining. METHODS: This is a retrospective case series of 132 consecutive eyes with Fuchs endothelial dystrophy or endothelial failure who underwent DMEK using preloaded donor tissue prepared as previously described. The graft was not restained with trypan blue by the surgeon, and Optisol-GS was injected with the graft into the eye instead of being rinsed from the injector. Early postoperative complications (0-8 wk) including intraoperative fibrin formation, intraocular inflammation, elevated intraocular pressure, partial graft detachment requiring rebubble, and early graft failure were recorded. RESULTS: No eyes developed intraoperative fibrin formation or postoperative inflammation (such as toxic anterior segment syndrome) or elevated intraocular pressure. For eyes with Fuchs corneal dystrophy, our rebubble rate was 21% (22/106 eyes). Early graft failure was noted in 2% (3/132 eyes), which is similar to previous reports. CONCLUSIONS: Our results suggest that injection of Optisol-GS into the anterior chamber during DMEK graft injection does not lead to increases in intraoperative or early postoperative complications. Trypan blue restaining is not necessary for intraoperative visualization. This simplification can reduce graft manipulation and save time and resources for this procedure.

Unintentional Staining of the Anterior Vitreous with Trypan Blue During Cataract Surgery.

During phacoemulsification and intraocular lens (IOL) implantation surgery, the trypan blue dye used to stain the anterior capsule passed into vitreous cavity and stained the anterior capsule and anterior vitreous in 6 patients. There was history of trauma in 2 patients, uveitis in 1 patient, mature cataract in 1 patient, and no risk factors in the other patients. IOL was implanted in-the-bag without problem in 5 patients. In the patient with iris and zonular defects due to trauma, a sutured IOL was implanted in the same session. The migration of trypan blue into the vitreous cavity through damaged or intact lens zonules is a rare but important complication that makes subsequent surgical steps substantially more difficult.