Phosphate-buffered Solution Control Research Articles

Repurposing of cholesterol-lowering agents in status epilepticus: A neuroprotective effect of simvastatin

The increase of cholesterol synthesis after a status epilepticus may lead to excitotoxic processes, neuronal loss and favor the appearance of spontaneous epileptic seizures. Lowering cholesterol content could be a neuroprotective strategy.Here, we evaluated the protective effect of simvastatin administrated daily for 14 days, after the induction of a status epilepticus by intrahippocampal injection of kainic acid in mice. The results were compared to those obtained from mice showing a kainic acid-induced status epilepticus, treated daily with a saline solution, and from mice injected with a control phosphate-buffered solution without any status epilepticus. We first assessed the antiseizure effects of simvastatin by performing video-electroencephalographic recordings during the first three hours after kainic acid injection and continuously between the fifteenth and the thirty-first days. Mice treated with simvastatin had significantly fewer generalized seizures during the first three hours without a significant effect on generalized seizures after two weeks. There was a trend for fewer hippocampal electrographic seizures after two weeks. Secondly, we evaluated the neuroprotective and anti-inflammatory effects of simvastatin by measuring the fluorescence of neuronal and astrocyte markers on the thirtieth day after status onset. We found that simvastatin reduced CA1 reactive astrocytosis, demonstrated by a significant 37% decrease in GFAP-positive cells, and that simvastatin prevented the neuronal loss in CA1, demonstrated by a significant 42% increase in the NeuN-positive cells, as compared to the findings in mice with kainic acid-induced status epilepticus treated by a saline solution.Our study confirms the interest of cholesterol-lowering agents, and in particular simvastatin, in status epilepticus and paves the way for a clinical pilot study to prevent neurological sequelae after status epilepticus. This paper was presented at the 8th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures held in September 2022.

Transcriptome Analysis of Immune-Related Gene in Hybrid Yellow Catfish in Reaction to Peptidoglycan Challenge

Hybrid yellow catfish (<i>Tachysurus vachelli x Tachysurus fulvidraco</i>) is one of the most important freshwater fish in China, intensive culturing has caused increased susceptibility to bacteria, viruses, and parasites. However, little is known about its adaptation mechanisms to pathogen infection. To better understand its immune system and immune related gene under pathogen infection, the transcriptome was analyzed by comparing the data of hybrid yellow catfish spleen stimulated by PGN and PBS. Fish in experimental group were injected with 100 μl baccilus subminis PGN (1mg/ml, 69,554, Sigma) intraperitoneally, and the fish in control group injected with 100 ul phosphate buffered solution (PBS). After 24 h injection, spleens were collected and immediately frozen in liquid nitrogen, then stored in -80°C refrigerator. Total RNA from spleens of hybrid yellow catfish were extracted using Trizol reagent (Sangon, China) according to the manufacturer’s instructions. The libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, USA) and sequenced on an Illumina HiSeq X Ten platform. There were 285 differentially expressed genes (DEGs) between PGN treated experimental groups and PBS control groups at 24 h, including 213 up regulated and 72 down regulated DEGs. The expression of qRT-PCR from five up regulated DEGs and five down regulated DEGs were consistent with RNA-Seq. In the analysis of GO enrichment, DEGs in the spleens of PGN treated hybrid yellow catfish were significantly enriched in GO items, including Biological Process, Cellular Component, and Molecular Function items. The identified DEGs and enriched GO terms and KEGG pathways were useful for understanding the physical and biochemical performances, and the transcriptome data greatly enriched the genetic information of hybrid yellow catfish.

Molecular cloning of AKT1 and AKT2 and their divergent responses to insulin and glucose at transcriptional level in the liver of Japanese flounder Paralichthys olivaceus

The serine-threonine kinase AKT (protein kinase B) plays a vital role in insulin-mediated signaling transduction in mammals, but its role in carnivorous fish is still unclear. In the present study, AKT1 and AKT2 were cloned from Japanese flounder (Paralichthys olivaceus), and the full-length sequences of cDNA of AKT1 and AKT2 were 2335 bp and 3340 bp, encoding 473 and 482 amino acids, respectively. Tissue distribution results showed that AKT1 was most abundantly expressed in gill, intermediate in liver, spleen and brain. AKT2 was most highly expressed in liver, followed by gill, brain and eye. Compared with those intraperitoneally injected with phosphate buffer solution (PBS), after injected with 4 IU/kg of bovine insulin or 0.6 g/kg of glucose, the insulin-mediated insulin receptor (IR) / insulin receptor substrate (IRS) / phosphoinositide 3-kinase (PI3K) /AKT pathway and its downstream glucose metabolism pathways in the liver of Japanese flounder were significantly activated. AKT2 expression was significantly increased after insulin or glucose injection, but AKT1 not. Similar results were also found in the primary hepatocytes of Japanese flounder after 24-hour incubation with different doses of bovine insulin (50 nM, 100 nM and 200 nM) or PBS (Control). Knockdown of AKT2 in the hepatocytes of Japanese flounder inhibited glycolysis, glucose transport and glycogen synthesis. Meanwhile, knockdown of AKT2 promoted the phosphoenolpyruvate carboxykinase (PEPCK) expression and inhibited the glucose-6-phosphatase (G6P) expression in the gluconeogenesis pathway. It was confirmed in the present study that AKT2 was more sensitive to the insulin-mediated pathway than AKT1 in the liver of Japanese flounder after insulin or glucose administration. In addition, the glucose metabolism pathways could be mediated by AKT2 in the liver of Japanese flounder.

Ultrafine particles altered gut microbial population and metabolic profiles in a sex-specific manner in an obese mouse model

Emerging evidence has highlighted the connection between exposure to air pollution and the increased risk of obesity, metabolic syndrome, and comorbidities. Given the recent interest in studying the effects of ultrafine particle (UFP) on the health of obese individuals, this study examined the effects of gastrointestinal UFP exposure on gut microbial composition and metabolic function using an in vivo murine model of obesity in both sexes. UFPs generated from light-duty diesel engine combustion of petrodiesel (B0) and a petrodiesel/biodiesel fuel blend (80:20 v/v, B20) were administered orally. Multi-omics approaches, including liquid chromatography–mass spectrometry (LC–MS) based targeted metabolomics and 16S rRNA gene sequence analysis, semi-quantitatively compared the effects of 10-day UFP exposures on obese C57B6 mouse gut microbial population, changes in diversity and community function compared to a phosphate buffer solution (PBS) control group. Our results show that sex-specific differences in the gut microbial population in response to UFP exposure can be observed, as UFPs appear to have a differential impact on several bacterial families in males and females. Meanwhile, the alteration of seventy-five metabolites from the gut microbial metabolome varied significantly (ANOVA p < 0.05) across the PBS control, B0, and B20 groups. Multivariate analyses revealed that the fuel-type specific disruption to the microbial metabolome was observed in both sexes, with stronger disruptive effects found in females in comparison to male obese mice. Metabolic signatures of bacterial cellular oxidative stress, such as the decreased concentration of nucleotides and lipids and increased concentrations of carbohydrate, energy, and vitamin metabolites were detected. Furthermore, blood metabolites from the obese mice were differentially affected by the fuel types used to generate the UFPs (B0 vs. B20).

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Study on mechanisms of interleukin-17A regulating the expressions of interleukin-1β and interleukin-23 in mouse keratinocytes

Objective: To investigate the mechanisms of interleukin-17A (IL-17A) regulating the expressions of IL-1β and IL-23 in mouse keratinocytes (KCs). Methods: Primary KCs were isolated from the skin of 400 newborn male and female wild type C57BL/6 mice and cultured in 24-well plates with Roswell Park Memorial Institute 1640 medium containing fetal bovine serum in the volume fraction of 10% for the following experiments. (1) The cells were divided into phosphate buffer solution (PBS) control group and IL-17A stimulation group according to the random number table (the same grouping method below), which were cultured with 10 μL PBS or 10 μL IL-17A in the mass concentration of 100 ng/mL for 6 hours, respectively. The expression levels of IL-1β and IL-23 mRNA in cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with 3 samples in each group. (2) The cells were divided into dimethyl sulfoxide (DMSO) control group, IL-17A+ DMSO group, IL-17A+ nuclear factor κB (NF-κB) inhibitor group, IL-17A+ signal transduction and activator of transcription 3 (STAT3) inhibitor group, IL-17A+ extracellular signal-regulated kinase 1 (ERK1) inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ c-Jun N-terminal kinase (JNK) inhibitor group. The reagents were added to cells in corresponding groups respectively and cultured for 6 hours. The volume of each reagent was 10 μL, the mass concentration of IL-17A was 100 ng/mL, and the molarity concentrations of NF-κB, STAT3, ERK1, ERK2, JNK signal pathway inhibitors PDTC, S3I-201, SCH772984, SCH772984, SP600125 were 5 μmol/L, 100 μmol/L, 4 nmol/L, 1 nmol/L, and 10 μmol/L, respectively. The expression levels of IL-1β mRNA and IL-23 mRNA in cells were detected by real-time fluorescence quantitative RT-PCR, with 3 samples in each group. (3) The cells were grouped and treated the same as those in experiment (1). The levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation were detected by Western blotting, with 3 samples in each group. Data were statistically analyzed with two-tailed Student t test, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) After culture of 6 hours, compared with those in PBS control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A stimulation group were significantly increased (t=13.46, 6.72, P<0.01). (2) After culture of 6 hours, the expression levels of IL-1β and IL-23 mRNA in cells in DMSO control group, IL-17A+ DMSO group, IL-17A+ NF-κB inhibitor group, IL-17A+ STAT3 inhibitor group, IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group were 1.00±0.11, 4.01±0.32, 0.32±0.06, 1.76±0.43, 3.62±0.24, 3.80±0.43, 4.26±0.74 and 1.03±0.29, 4.08±0.34, 4.76±0.38, 4.70±0.21, 1.06±0.42, 0.92±0.21, 0.39±0.05, respectively. Compared with those in DMSO control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A+ DMSO group were significantly increased (t=9.24, 12.60, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-1β mRNA was significantly decreased in cells in IL-17A+ NF-κB inhibitor group and IL-17A+ STAT3 inhibitor group (t=11.34, 6.91, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-23 mRNA was significantly decreased in cells in IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group (t=12.44, 13.03, 15.21, P<0.01). (3) After culture of 6 hours, compared with those in PBS control group, the levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation in cells in IL-17A stimulation group were significantly increased. Conclusions: IL-17A promotes the transcription of IL-1β in mouse KCs through the phosphorylation of NF-κB and STAT3 pathways and IL-23 through the phosphorylation of ERK and JNK pathways.

Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice

Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice

Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice

Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice

Atmospheric dust as a possible survival factor for bioaerosols

In this study, we present some of the laboratory measurements of the viability of bioaerosols together with different environmental dust to understand the interaction among those components. Model airborne bacteria, DH5α Escherichia coli, was used to assess the dust affecting a viability reduction rate of the DH5α bacteria in a Teflon reaction chamber. The viability reduction rate of the DH5α model airborne bacteria was measured with a culture method. The DH5 α bacteria were nebulized into the chamber and airborne dust materials 1) Phosphate Buffer Solution (PBS) as a control, 2) desert sand from Mongolia and 3) sludge dust from the coastal area of Japan. The result indicated that the co-existence of DH5α with desert dust from Mongolia significantly decreased the viability and with the sludge dust from Japan significantly increased the viability of the airborne DH5α compare to the control PBS dust (p&lt;0.05). Furthermore, soot as a model air pollutant was generated by a candle and mixed with a model airborne bacteria Mycobacterium smegmatis. The results indicated that the different types of airborne environmental dust influenced the viability of airborne bacteria with the DH5α experiments. Furthermore, the presence of soot indicated a possible protective effect of increasing the survival rate of Mycobacterium smegmatis.

MicroRNA-155 reduces inflammatory response induced by lipopolysaccharide in alveolar macrophages

To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. (1) The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. (2) Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. (1) After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. (2) Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.

Acquisition and the immunogenicity of the outer membrane FomA protein of Fusobacterium nucleatum

Objective: To express and purify outer membrane protein FomA of Fusobacterium nucleatum (Fn) through gene recombination technique with Escherichia coli (Ec) expression system, and to detect the immunogenicity and the immune effects of the recombinant protein on gingival tissues. Methods: The gene recombination technique and Ec expression system were used to express and purified the FomA protein. Totally 20 C57 mice were immuned with the protein or the phosphate buffer solution (PBS) buffer by subcutaneous injection (each 10 mice), and the specific FomA antibody was detection in mice serum. The immunogenicity of FomA protein was assessed by comparing the differences between groups. Furthermore, the model of mice gum abscess was constructed with Fn or Fn and Porphyromonas gingivalis (Pg) mixed suspension used the above mice. The score of the gingival abscess was recorded and the interleukin (IL)-1β in gum tissue and mice serum was determined by enzyme-linked immunosorbent assay method, and the differences of the indexes between groups were compared to evaluate the effect of the FomA protein immunization. Results: Totally 1.0-1.5 g FomA protein were successfully obtained and the protein purity was over 90%. The FomA specific antibody was detected in the serum of mice by subcutaneous injection of the protein, and the antibody titer reached the highest level in 2 weeks after secondary immunization. The model of submaxillary gingival abscess was successfully constructed. In the Fn model, the score of the FomA protein immune group was (1.82±0.35), and the PBS control group was (2.62±0.71), with statistically significant difference between the two groups (P=0.049). In the Fn+Pg mixture model, the score of gingival abscess in the FomA immune group (2.31±0.55) was lower than that in PBS group (3.63±0.45), and the difference was statistically significant (P=0.003). Both in Fn and Fn+Pg injection group, the concentration of IL-1β in the serum of FomA immune mice and gingival tissues was lower than that of PBS control mice (P<0.001). Conclusions: The recombinant FomA protein can be acquired by Ec expression system, and it can produce a certain level antibodies in the mice serum. The way of mice subcutaneously injected with the recombinant FomA protein can reduce the severity of periodontal infections caused by Fn and Pg.

Effect of Biomineralization Ability on Push-out Strength of Proroot Mineral Trioxide Aggregate, Mineral Trioxide Aggregate Branco, and Calcium Phosphate Cement on Dentin: An In vitro Evaluation.

Context:Biomineralization is a process which leads to the formation of an interfacial layer with tag-like structures at the cement-dentin interface. It is due to interaction of mineral trioxide aggregate (MTA) and Portland cement with dentin in phosphate-buffered solution (PBS). This study is aimed to evaluate the effect of influence of biomineralization process on push-out bond strength of ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK, USA), MTA Branco (Angelus Soluc¸o˜es Odontolo´gicas, Londrina, PR, Brazil) and calcium phosphate cement (BioGraft CPC).Aim:The aim of this study was to evaluate the effect of biomineralization process on the push-out strength of ProRoot MTA, MTA Branco, and CPC after mixing with 0.2% chlorhexidine gluconate solution (0.2% CHX) and 2% lidocaine solution (2% LA) on the bond strength of MTA-dentin.Materials and Methods:Dentin discs with uniform cavities were restored with ProRoot MTA, MTA Branco, and calcium phosphate cement after mixing with 0.2% CHX solution and 2% lidocaine solution. The samples were uniformly distributed into two groups. Experimental group being immersed in PBS solution and control group being immersed in saline for 2 months. Instron testing machine (Model 4444; Instron Corp., Canton, MA, USA) was used to determine the bond strength.Statistical Analysis Used:A two-way analysis of variance and post hoc analysis by Bonferroni test.Results:All samples immersed in experimental group displayed a significantly greater resistance to displacement than that observed for the samples in control group (P < 0.05). MTAs displayed a significantly greater resistance to displacement than calcium phosphate cements.Conclusion:The main conclusion of this study was that the push-out bond strength of the cements, mainly the MTA groups, was positively influenced by the biomineralization process.

Novel Approach for Evaluation of Bacteroides fragilis Protective Role against Bartonella henselae Liver Damage in Immunocompromised Murine Model

Bartonella henselae is a gram-negative facultative intracellular bacterium and is the causative agent of cat-scratch disease. Our previous data have established that Bacteroides fragilis colonization is able to prevent B. henselae damages through the polysaccharide A (PSA) in an experimental murine model. In order to determine whether the PSA is essential for the protection against pathogenic effects of B. henselae in immunocompromised hosts, SCID mice were co-infected with B. fragilis wild type or its mutant B. fragilis ΔPSA and the effects of infection on murine tissues have been observed by High-Frequency Ultrasound (HFUS), histopathological examination, and Transmission Electron Microscopy (TEM). For the first time, echostructure, hepatic lobes length, vascular alterations, and indirect signs of hepatic dysfunctions, routinely used as signs of disease in humans, have been analyzed in an immunocompromised murine model. Our findings showed echostructural alterations in all infected mice compared with the Phosphate Buffer Solution (PBS) control group; further, those infected with B. henselae and co-infected with B. henselae/B. fragilis ΔPSA presented the major echostructural alterations. Half of the mice infected with B. henselae and all those co-infected with B. henselae/B. fragilis ΔPSA have showed an altered hepatic echogenicity compared with the renal cortex. The echogenicity score of co-infected mice with B. henselae/B. fragilis ΔPSA differed significantly compared with the PBS control group (p < 0.05). Moreover the inflammation score of the histopathological evaluation was fairly concordant with ultrasound findings. Ultrastructural analysis performed by TEM revealed no significant alterations in liver samples of SCID mice infected with B. fragilis wild type while those infected with B. fragilis ΔPSA showed the presence of collagen around the main vessels compared with the PBS control group. The liver samples of mice infected with B. henselae showed macro-areas rich in collagen, stellate cells, and histiocytic cells. Interestingly, our data demonstrated that immunocompromised SCID mice infected with B. henselae and co-infected with B. henselae/B. fragilis ΔPSA showed the most severe morpho-structural liver damage. In addition, these results suggests that the HFUS together with histopathological evaluation could be considered good imaging approach to evaluate hepatic alterations.

Antioxidant and renoprotective effects of sphingosylphosphorylcholine on contrast-induced nephropathy in rats

Contrast induced nephropathy (CIN) is a major cause of morbidity, and increased costs as well as an increased risk of death. This study was evaluated effects of exogenous sphingosylphosphorylcholine (SPC) administration on CIN in rats. Eight animals were included in each of the following eight groups: control, control phosphate-buffered solution (PBS), control SPC 2, control SPC 10, CIN, CIN PBS, CIN SPC 2 and CIN SPC 10. The induced nephropathy was created by injected with 4 g iodine/kg body weight. SPC was administered 3 d at a daily two different doses of 2 μm/mL and 10 μm/mL intraperitoneally. The severity of renal injury score was determined by the histological and immunohistochemical changes in the kidney. Malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) were determined to evaluate the oxidative status in the renal tissue. Treatment with 2 and 10 μM SPC inhibited the increase in renal MDA, NO levels significantly and also attenuated the depletion of SOD in the renal injuryCIN. These data were supported by histopathological findings. The inducible nitric oxide synthase positive cells and apoptotic cells in the renal tissue were observed to be reduced with the 2 and 10 μM SPC treatment. These findings suggested that 2 and 10 μM doses can attenuate renal damage in contrast nephropathy by prevention of oxidative stress and apoptosis. The low and high dose SPC may be a promising new therapeutic agent for CIN.

Investigation of the sensitivities of two different serotypes of Streptococcus pneumoniae in C57BL/6 mice with acute otitis media

To compare the sensitivities of two different serotypes of Streptococcus pneumoniae in acute otitis media of C57BL/6 mice. The middle ear cavity of C57BL/6 mice were inoculated via intratympanic injection of Streptococcus pneumoniae TIGR4 or 6B 1×10(8) CFU, and the control group were inoculated equivalent phosphate buffered solution (PBS). The incidence of mice, development of otitis media and the middle ear lavage fluid pathological changes by HE staining, as well as cell counts and cytokine levels were investigated. After inoculated with Streptococcus pneumoniae TIGR4, 6B and PBS, the survival rate of 6B group was significantly less than TIGR4 group and PBS control group (P < 0.01). Inflammatory cells in the middle ear cavity were mainly neutrophils, and the inflammatory cells recruitment in TIGR4 group were more than 6B group; The levels of IL-6 and TNF-α in the middle ear lavage fluid in TIGR4 group and 6B group were significantly increased compared with PBS control group, while the TIGR4 group were significantly increased compared with 6B group;6B group had delayed bacterial clearance in the middle ear. The study implied that Streptococcus pneumoniae 6B had higher pathogenicity for acute otitis media in C57BL/6 mice than TIGR4.

The effects of celecoxib-poly lactide-co-glycolide microparticles on rat retina after intravitreai injection

Objective To investigate the effects of （CEL-PLGA MS） on rat retina after intravitreal injection. celecoxib-poly lactide-co-glycolide microparticles Methods A total of 32 male Brown Norway rats were randomly divided into CEL-PLGA-MS group and celecoxib group, 16 rats in each group. The rats in CEL-PLGA-MS group were divided into four dosage group, four rats in each group, which received intravitreal injection of PLGA with celecoxib at the concentration of 40, 80, 160, 320μmol/L, respectively. The rats in celecoxib group were divided into four dosage group, four rats in each group, which receivedintravitreal injection of celecoxib at the concentration of 40, 80, 160, 320 μmol/L, respectively. Phosphate buffer solution （PBS） was injected in two rats as PBS control group. Two rats as normal control group received no treatment. The difference of retinal thickness among groups was measured by optical coherence tomography （OCT）. The morphological and histological change of retina was evaluated under light microscope and transmission electron microscope. Results There was no difference of retinal thickness between normal control group and PBS control group （F = 0.12, P~〉 0.05）. At the first week after injection, the retinal thickness of CEL-PLGA-MS group and celecoxib group were thicker than that in normal control group and PBS control group （F = 9.62, 46.13; P〈 0.01）. The retinal thickness of celecoxib group was thicker than that in CEL-PLGA-MS group （F = 165.15, P〈 0.01）. The retinal thickness was estimated equal among 40, 80, 320 μmol/L dosage groups in CEL-PLGA MS group （F= 4.79, P~0.01）. The retinal thickness of 160, 320 μmol/L dosage group were thicker than that in 40, 80 μmol/L dosage group in celecoxib group （F=28.10, P〉0.01）. At the second week after injection, there was no difference of retinal thickness between CEL-PLGA-MS and celecoxib group （F= 3.79, P〈0. 05） ; the retinal thickness of CEL-PLGA-MS and celecoxib group became thinner gradually compare to the first week after injection （F=7.28, 103.99; P〈0.01）. At the fourth week after injection, the retinal thickness of celecoxib group was thicker than that in CEL-PLGA-MS group （F= 19. 11, P〈0. 01）. The retinal thickness of CEL-PLGA-MS group was approximately the same to normal control group and PBS control group （F=2.02, P〉0.05）. The retinal thickness of celecoxib group was thicker than that in normal control group and PBS control group. No considerable abnormality of the retina was seen by light microscope and the retinal thickness corresponded with the values measured by OCT at the first week after injection. The abnormal structures of the retina were seen in 160, 320 μmol/L dosage group of celecoxib group and inner changed evidently by the transmission electron microscope. Disordered arrangement of microfilaments, dilated microtubule and some mitoehondria vacuolation were observed in 320 μmol/L dosage group of celecoxib group. Others changed slightly. Conclusions CEL-PLGA-MS has less toxicity on the retina than free-celecoxib after intravitreal injection. The safety of intravitreal injection with CEL-PLGA-MS is better than celecoxib. Key words: Cyclooxygenase 2 inhibitors/toxicity; Cyclooxygenase 2 inhibitors/diagnostic use; Delayed-action preparations/toxicity; Choroidal neovascularization/drug therapy

Neutralization of polyethylene glycol on ocular surface toxicity induced by benzalkonium chloride-preserved latanoprost

Background Reducing ocular surface toxicity induced by topical antiglaucoma medications,and thus improving the therapeutic compliance of glaucomatous patients to treating drug become an urgent issue in the long-term antiglaucomatous therapies.Whether polyethylene glycol,a new type of artificial tear,can play a protective role from ocular surface toxicity induced by topical antiglaucoma medications remains further study.Objective The present study was to investigate the protective effect of polyethylene glycol on ocular surface toxicity induced by prolonged use of benzalkonium chloride-preserved latanoprost eye drops.Methods Thirty adult female New Zealand rabbits were randomly divided into normal control group,phosphate buffer solution (PBS) group and polyethylene glycol eye drops group.PBS with 0.005％ latanoprost was topically administered for consequent 60 days in the PBS group,and 0.005％ latanoprost with polyethylene glycol eye drops was used at the same way in the polyethylene glycol group.Schirmer Ⅰ test,fluorescein and rose bengal staining of cornea and conjunctiva,conjunctiva impression cytology specimens collecting were performed before experiment as well as 31 and 61 days after administration of drug.Histomorphological and ultrastructural changes of cornea and conjunctiva were observed under the light microscopy,scanning and transmission electron microscopy on day 61.Results The score of corneal fluorescine staining was significantly different among the control group,PBS group and polyethylene glycol group (H =25.265,P＜0.001 ;H=29.426,P＜0.001) on day 31 and day 61.Significant difference also was found in the score of rose bengal staining of cornea and conjunctiva (H=26.167,P＜0.001 ;H =29.212,P＜0.001),Schirmer Ⅰ test value (F=4.746,P＜0.05) and conjunctival goblet cell density (F=18.055,P＜0.001).Compared with the control group,corneal fluorescein scores [0(0,1.0) vs.3 (2.0,3.0)] and rose bengal scores [0(0,1) vs.2(2,3)] were significantly increased (both P＜0.001),and Schirmer Ⅰ test value scores and conjunctival goblet cell density were decreased ((5.18±0.46/)mm vs.(9.43±0.57)mm;(53.04±2.98)/HP vs.(87.73±2.34)/HP) (both P＜0.01)on day 61.Impaired microvilli and organelles of the corneal and conjunctival epithelial cells were observed.Compared with PBS-treated group,PEG-treated group showed decrease in fluorescein [3(2.0,3.0) vs.1 (0.5,1.0)] and rose bengal scores [2 (2,3) vs.1 (0,1)] (both P＜0.001),increase in Schirmer Ⅰ test value scores [(7.00±0.71)mm vs.(5.18±0.46) mm,P＜0.05],increase in goblet cell density ((63.88±3.32)/HP vs.(53.04±2.98)/HP,P＜0.05) and improvement of grading scores of conjunctival epithelium on day 61.In addition,complete mierovilli and cell organelles were found in the corneal and conjunctival epithelial cells in the polyethylene glycol group.Conclusions Topical application of polyethylene glycol can decrease the ocular surface toxicity induced by benzalkonium chloride-preserved latanoprost. Key words: Polyethylene glycol; Latanoprost; Benzalkonium chloride; Ocular surface toxicity

Recombined adenovirus of p21 inhibit the proliferation of Rhesus retinal vascular endothelial cells in vitro

Objective To observe the inhibitory effect of recombined adenovirus of p21 (rAd-p21) on the proliferation of Rhesus retinal vascular endothelial cell line (RF/6A). MethodsRF/6A cells were cultured in vitro which divided into phosphate buffered solution (PBS) group, rAd-p21 transfection group and negative control (NC) group. Plasmid vectors were transfected into RF/6A cells. The expression of p21mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively. The cell cycle distribution was analyzed by flow cytometry. Endothelial-cell tube formation assay was performed in Matrigel. Results The expression of p21 mRNA and protein in rAd-p21 transfection group were higher than that in PBS and negative control group. The cell cycle distribution showed that the proportion of G0/G1 cells in rAd-p21 transfection group[(67.45 ± 11.61) ％]was apparently higher than that in the negative control group[(41.55±8.99)％]and PBS group[(40. 76±6.66) ％](F=21.284, P =0. 000 ). The number of endothelial-cell tubes in the rAd-p21 transfection group (3.86±1.21) was apparently less than that in the negative control group (7.62±2.69) and PBS group (8.25±3.19) (F=7.138,P=0.004).Conclusions The p21 mRNA and protein can stably express in RF/6A cells after rAd-p21 transfection.RAd-p21 can inhibit the proliferation of RF/6A cells. Key words: Retinal neovascularization/ prevention & control; Gene transfer techniques; Transfection; Animal experimentation

Thalidomide inhibition of vascular remodeling and inflammatory reactivity in the quinolinic acid–injected rat striatum

Effects of thalidomide administration on vascular remodeling, gliosis and neuronal viability have been studied in excitotoxin-injected rat striatum. Intrastriatal injection of quinolinic acid (QUIN) caused time-dependent changes (durations of 6 h, 1 and 7 d post-injection) in vascular remodeling. QUIN excitotoxic insult was associated with increased numbers of vessels (laminin or collagen IV markers) demonstrating considerable abnormalities in morphology, including short fragments and vascular loops. Non-lesioned striatum, with injection of phosphate buffer solution (PBS) as a vehicle, showed no evidence for vascular remodeling. A maximal extent of vascular remodeling was measured at 1 d post-QUIN and was correlated with marked increases in microgliosis (ED1 marker) and astrogliosis (glial fibrillary acidic protein [GFAP] marker) relative to control PBS injection. Double staining of laminin with ED1 and GFAP demonstrated areas of close association of glial cells with blood vessels. Treatment of QUIN-injected animals with the anti-inflammatory compound, thalidomide significantly inhibited vascular remodeling (by 43%) and reduced microgliosis (by 33%) but was ineffective in modifying extents of astrogliosis. Intrastriatal QUIN injection was associated with a marked loss of striatal neurons relative to non-lesioned control with thalidomide treatment exhibiting a significant degree of neuroprotection (24% recovery) against QUIN-induced neurotoxicity. These results suggest close links between microglial-mediated inflammatory responses and vascular remodeling, with inflammatory reactivity associated with, and contributing to, neuronal damage in excitotoxically-lesioned striatum.

Effects of sphingosylphosphorylcholine against cholestatic oxidative stress and liver damage in the common bile duct ligated rats

The goal of this study was to evaluate the possible protective effects of sphingosylphosphorylcholine (SPC) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. Fifty-six animals were included in each of the following 7 groups: control, SPC control, phosphate-buffered solution control, sham operated, bile duct ligation (BDL), BDL plus phosphate-buffered solution, and BDL plus SPC. Sphingosylphosphorylcholine was administered 14 days at a daily dose of 2 μm/mL intraperitoneally. The severity of cholestasis and hepatic injury was determined by changes in the plasma enzyme activities of aspartate aminotransferase, alanine aminotransferase, gama glutamin transferase, and levels of total bilirubin and direct bilirubin. Malondialdehyde, nitric oxide, and superoxide dismutase were determined to evaluate the oxidative status in the liver tissue. Myeloperoxidase activity and levels of tissue hydroxyproline were determined to assess neutrophil activation and collagen accumulation, respectively. Treatment with SPC markedly reduced serum transaminase activities as compared to BDL rats. Sphingosylphosphorylcholine also inhibited the increase in liver malondialdehyde; nitric oxide levels significantly and also attenuated the depletion of superoxide dismutase in the liver after BDL. Similarly, the increase in tissue myeloperoxidase activity and hydroxyproline owing to BDL was also attenuated by the SPC treatment. These data were supported by histopathologic findings. The α-smooth muscle actin–positive cells in the BDL were observed to be reduced with the SPC treatment. In conclusion, these findings suggested that SPC can attenuate hepatic damage in extrahepatic cholestasis by prevention of oxidative stress, and inflammatory process. All these findings suggest that SPC may be a promising new therapeutic agent for cholestatic liver injury.