Bilirubin induces microglial NLRP3 inflammasome activation in vitro and in vivo.

Despite current advancements in neonatal care, hyperbilirubinemia resulting in bilirubin-induced neurological dysfunction (BIND) continues to be one of the major reasons of mortality or lifelong disability. Although the exact mechanisms underlying brain injury upon bilirubin exposure remains unelucidated, inflammation is considered to be one of the major contributors to BIND. This study investigates the role of the NLRP3 inflammasome in bilirubin-induced injury using in vitro and in vivo models. We successfully demonstrated that the upregulation of NLRP3 expression is significantly associated with the release of active caspase-1 and IL-1ß in N9 microglial cells exposed to bilirubin. Functional in vitro experiments with NLRP3 siRNA confirms that bilirubin-induced inflammasome activation and cell death are mediated by the NLRP3 inflammasome. Following injection of bilirubin into the cisterna magna of a neonatal mouse, activation of the NLRP3 inflammasome and microglia were determined by double staining with Iba1-NLRP3 and Iba1-Caspase-1. Upon injection of bilirubin into the cisterna magna, neuronal loss was significantly higher in the wild-type mouse compared to Nlrp3-/- and Caspase-1-/- strains. Collectively, these data indicate that NLRP3 inflammasome has a crucial role in microglial activation and bilirubin-induced neuronal damage.

Neuroprotective Effects of Lacosamide and Memantine on Hyperoxic Brain Injury in Rats.

In neonates supraphysiological oxygen therapy has been demonstrated to cause neuronal death in hippocampus, prefrontal cortex, parietal cortex, and retrosplenial cortex. There is a need for the detection of novel neuroprotective drugs. Neuroprotective effects of lacosamide or memantine have been demonstrated in adult patients with ischemia, trauma and status epilepticus. The effects in immature brains may be different. This study aimed to evaluate neuroprotective effects of lacosamide and memantine treatment in a hyperoxia-induced brain injury model in immature rats. This study was performed in the Animal Experiments Laboratory of Dokuz Eylul University Faculty of Medicine. Neonatal Wistar strain rat pups were exposed to hyperoxia (80% oxygen + 20% nitrogen) for five days postnatally. They were divided into five groups; hyperoxia + lacosamide, hyperoxia + memantine, hyperoxia + lacosamide and memantine, hyperoxia + saline, control groups. After termination of the experiment, brain tissues were examined. Neuron counting in examined regions were found to be higher in hyperoxia + memantine and hyperoxia + lacosamide and memantine groups than hyperoxia + saline group. The presence of apoptotic cells evaluated with TUNEL and active Caspase-3 in hyperoxia + memantine and hyperoxia + lacosamide and memantine groups were found to be lower compared to hyperoxia + saline group. This study demonstrates that neuron death and apoptosis in newborn rat brains after hyperoxia is reduced upon memantine treatment. This is the first study to show the effects of memantine and lacosamide on hyperoxia-induced damage in neonatal rat brains.

Ocular toxicity of intravitreal golimumab in a rabbit model

Background/aim: To investigate the effect of intravitreal golimumab on rabbit retina histopathology. Materials and methods: Sixteen albino New Zealand rabbits were divided into three groups. The right eye of each rabbit in groups I, II, and III received a single intravitreal injection of 5 mg/0.05 mL (6 eyes), 10 mg/0.1 mL (6 eyes), or 20 mg/0.2 mL (4 eyes) golimumab, while left eyes served as controls with the same volume of a balanced salt solution injection. All animals were examined using slit-lamp biomicroscopy and indirect ophthalmoscopy before and after intravitreal injection and at days 1 and 7. Animals were euthanized on day 7 and the eyes were enucleated for immunohistochemistry evaluation and electron microscopic examination of the retinas. Results: For groups I, II, and III, the number of cells in the outer nuclear layer and the inner nuclear layer was decreased compared to those in the control groups. In group I, the percentage of caspase-3 staining of the outer nuclear layer was significantly higher than that in the control. For groups II and III, TUNEL and caspase-3 staining percentages in the outer and inner nuclear layers were found to be significantly higher than those for the control groups. In the ganglion cell layer, for groups I, II, and III, neither TUNEL nor caspase-3 staining percentages showed any significant difference between two groups. No significant dose-dependent relationship was found for increasing doses of golimumab in all layers. Myelin figures and karyorrhexis in the photoreceptor cells were prominent in electron microscopy of the golimumab-injected eyes. Conclusion: Golimumab caused apoptosis in both photoreceptors and bipolar cells of the rabbit retina. Potential retinal toxicity of intravitreal golimumab should be considered if an intravitreal administration is planned.

The effect of atorvastatin on lung histopathology in a murine model of chronic asthma.

INTRODUCTION: Atorvastatin is a statin group medicine that reduces the level of serum cholesterol; thus it is used to treat hypercholesterolaemia. Independent of the cholesterol-lowering property of statins they also have anti-inflammatory and immunomodulating effects. This study aimed to investigate the effect of atorvastatin on histological changes in the lungs in a murine model of chronic asthma. MATERIALS AND METHODS: Twenty-eight BALB/c mice in Group I, II, III and IV were divided into four groups. All the mice except the control group (Group I) were sensitised with ovalbumin. Intraperitoneal injection with saline, atorvastatin (10mg/kg), dexametazon (1mg/kg) was administered to Group II, Group III, and Group IV respectively for five consecutive days. Mice were sacrificed 24h after the last drug administration. All the histological properties of lung tissue samples from all groups were evaluated with light and electron microscopy. In addition, IL-4 and IL-5 levels of the lung tissue were measured. RESULTS: When Group II and Group III (atorvastatin) were compared, thicknesses of basement membrane and subepithelial smooth muscle layer, height of epithelium, number of mast and goblet cells were significantly lower in Group III. In comparing Group III (atorvastatin) and Group IV (dexamethasone), all the improvements in histological parameters were similar. In addition, the IL-4 and IL-5 levels of the lung tissue were significantly lower in atorvastatin group (Group III) compared to placebo-treated group. CONCLUSION: Atorvastatin had a beneficial effect on histological changes in a chronic murine model of asthma.

The effects of dexmedetomidine on secondary acute lung and kidney injuries in the rat model of intra-abdominal sepsis.

In the present study, the effects of dexmedetomidine on secondary lung and kidney injuries were studied in the rat model of intra-abdominal sepsis by immunohistological and biochemical examinations. We measured serum creatinine, kidney tissue malondialdehide and plasma neutrophil gelatinase-associated lipocalin levels. In order to evaluate tissue injury we determined kidney tissue mononuclear cell infiltration score, alveolar macrophage count, histological kidney and lung injury scores and kidney and lung tissue immunoreactivity scores. We demonstrated that dexmedetomidine attenuates sepsis-induced lung and kidney injuries and apoptosis in the rat model of sepsis. There is still need for comparative studies in order to determine the effects of dexmedetomidine on organ functions in early human sepsis.

Effect of the selective mitochondrial KATP channel opener nicorandil on the QT prolongation and myocardial damage induced by amitriptyline in rats.

OBJECTIVES: The aim of this study is to evaluate the protective effect of nicorandil, a selective mitochondrial KATP channel opener, on QT prolongation and myocardial damage induced by amitriptyline. METHODS: The dose of amitriptyline (intraperitoneal, i.p.) that prolong the QT interval was found 75 mg/kg. Rats were randomized into five groups the control group, amitriptyline group, nicorandil (selective mitochondrial KATP channel opener, 3 mg/kg i.p.) + amitriptyline group, 5-hdyroxydecanoate (5-HD, selective mitochondrial KATP channel blocker, 10 mg/kg i.p.) + amitriptyline group and 5-HD + nicorandil + amitriptyline group. Cardiac parameters, biochemical and histomorphological/immunohistochemical examinations were evaluated. p < 0.05 was accepted as statistically significant. KEY FINDINGS: Amitriptyline caused statistically significant prolongation of QRS duration, QT interval and QTc interval (p < 0.05). It also caused changes in tissue oxidant (increase in malondialdehyde)/anti-oxidant (decrease in glutathione peroxidase) parameters (p < 0.05), myocardial damage and apoptosis (p < 0.01 and p < 0.001). While nicorandil administration prevented amitriptyline-induced QRS, QT, QTc prolongation (p < 0.05), myocardial damage and apoptosis (p < 0.05), it did not affect the changes in oxidative parameters (p > 0.05). CONCLUSIONS: Our results suggest that nicorandil, a selective mitochondrial KATP channel opener, plays a protective role in amitriptyline-induced QT prolongation and myocardial damage. Mitochondrial KATP channel opening and anti-apoptotic effects may play a role in the cardioprotective effect of nicorandil.

Effects of Cross-linked Hyaluronic Acid in a Rat Model of Vestibular and Cochlear Toxicity.

Objective: To investigate the effects of cross-linked hyaluronic acid (CLHA) in an experimental model of vestibulotoxicity and cochleotoxicity. Methods: Twenty-eight female Wistar albino rats (200-250 g) were divided into four groups. Group A received 0.06 mL of 13.33 mg/mL gentamicin, Group B received 0.06 mL of CLHA, Group C received 0.03 mL of 26.66 mg/mL gentamicin and 0.03 mL CLHA, and Group D received 0.06 mL of 0.09% saline. All groups underwent auditory brainstem response testing at 4-32 kHz, signal-to-noise ratio of distortion product otoacoustic emission measurements at 1.5-8 kHz and vestibular tests on days 0,1,7,10. The rats were sacrificed, and their labyrinths were histologically assessed and scored. Results: The hearing thresholds of Groups A and C were similar and significantly higher than those of the other groups at all frequencies, beginning from day 1. The vestibular and histological scores of Groups A and C were similar and significantly higher than those of the other groups from day 1. The audiological results, vestibular scores, and histological scores of Groups B and D were similar, except for a temporary middle ear effusion and hearing threshold shift in Group B. No significant deterioration was observed in the audiological, vestibular, and histological analyses of Groups B and D. Conclusion: That both Group A and Group C similarly showed worsening audiological, vestibular, and histological tests suggests that CLHA did not alter the pharmacokinetics and histologic results of gentamicin.

The effects of perinatal steroid therapy on growth factor levels during different stages of the developing brain.

OBJECTIVE: Excess glucocorticoid (GC) exposure on the fetal brain during critical stages of development has considerable effects on the development of the central nervous system (CNS). This study thus aimed to evaluate the differential effects of GC exposure on critical growth factor levels during different stages of brain maturation. METHODS: For this purpose, forty-two rat pups were divided into six groups based on the timing of betamethasone administration. Rats in the treatment groups were exposed to intraperitoneal betamethasone injections beginning at different time points (postnatal days 1, 2, and 3). Rats in the placebo group received the same volume of 0.9% saline via the same fashion. Pups were sacrificed at 24 h following the final injection for determining the neuronal density and immunohistochemical evaluation of critical growth factors. RESULTS: In the groups treated with betamethasone on postnatal day 1 (P1) and P2, which correspond to 22-24 and 24-28 gestational weeks in humans, the neuronal count in the hippocampal regions was significantly lower than their control groups. However, if steroid therapy was administered on P3, corresponding to 28-32 weeks in humans, no difference was observed between the two groups. Growth factors were affected in different ways depending on the steroid administration time and evaluated region. CONCLUSIONS: The results suggest that the modulating effect of steroids on neuron count and growth factor response depends on the stage of brain development at the time of exposure. Therefore, this may be one of the key determinants affecting the deleterious and beneficial effects of GCs on the CNS.

Immunomodulatory activities of alpha lipoic acid with a special focus on its efficacy in preventing miscarriage.

INTRODUCTION: Alpha lipoic acid (ALA) is an essential mitochondrial co-factor and, as a free molecule, it can exert multi-level immunomodulatory functions. Both ALA and its reduced form, dihydrolipoic acid (DHLA), are believed to be able to chelate heavy metals, to regenerate essential antioxidants and to repair important molecules damaged by oxidation. The largest part of the effects of ALA/DHLA couple can be explained by a specific stimulatory activity on Nrf2-dependent gene transcription and by the inhibition of NF-kB activity. These features have prompted its use as a drug for several diseases. Areas covered: This article surveys the main features of ALA/DHLA and its therapeutic effects. Its complex and differentiated function cannot simply be reduced to anti-inflammatory, antioxidant and detoxifying action. We highlight its capability to finely modulate several physiological pathways when unbalanced. In particular, we focus our attention on pregnancy, in relation to ALA administration by oral route and by a new formulation for vaginal delivery, in patients with threatened miscarriage. Expert opinion: Future efforts should be devoted to explaining carefully ALA/DHLA mechanism of action to reactivate the physiological balance when modified during pregnancy. On the other hand, ALA safety in pregnant women and its pharmacokinetics by vaginal route, have to be studied in depth. Moreover, ALA efficacy has to be confirmed in a much larger sample of patients.

Doxycycline down-regulates matrix metalloproteinase expression and inhibits NF-κB signaling in LPS-induced PC3 cells.

INTRODUCTION: Matrix metalloproteinase enzymes (MMPs) play important role in inflammation, malignant cell proliferation, invasion and angiogenesis by mediating extracellular matrix degradation. Doxycycline, a synthetic tetracycline, behaves as a MMP inhibitor at a subantimicrobial dose and inhibits tumor cell proliferation, invasion and angiogenesis. The aberrant activity of nuclear factor kappa B (NF-κB) causes activation of MMPs and thereby proliferation and invasion of cancer cells. The aim of this study was to investigate the effects of doxycycline on the expression of MMPs in lipopolysaccharide (LPS)-induced PC3 human prostate cancer cells and the possible role of NF-κB signaling. MATERIAL AND METHODS: PC3 cells were incubated with LPS (0.5 µg/mL) for 24 h in the presence or absence of doxycycline (5 µg/mL). The effects of LPS and doxycycline on the expressions of MMP-2, MMP-8, MMP-9, MMP-10, NF-κB/p65, IκB-α, p-IκB-α, IKK-ß were examined by Western blotting and immunohistochemistry in PC3 cells. Furthermore, relative proteinase activities of MMP-2 and MMP-9 were determined by gelatin zymography. RESULTS: LPS increased expression and activity of MMP-9 and expression of MMP-8, MMP-10, NF-κB /p65, p-IκB-α, IKK-ß and doxycycline down-regulated its effects with the exception of MMP-10 expression. The expression of MMP-2 and IκB-α was affected by neither LPS nor doxycycline. CONCLUSIONS: Our findings indicate that doxycycline inhibits the expression of various MMPs and NF-κB signaling may play a role in the regulation of MMPs expression in LPS-induced PC3 human prostate cancer cells.

Resveratrol ameliorates 2,4-dinitrofluorobenzene-induced atopic dermatitis-like lesions through effects on the epithelium.

Background. Resveratrol is a natural polyphenol that exhibits anti-inflammatory effects. The aim of this study was to investigate the effects of resveratrol treatment on epithelium-derived cytokines and epithelial apoptosis in a murine model of atopic dermatitis-like lesions. Material and Methods. Atopic dermatitis-like lesions were induced in BALB/c mice by repeated application of 2,4-dinitrofluorobenzene to shaved dorsal skin. Twenty-one BALB/c mice were divided into three groups: group I (control), group II (vehicle control), and group III (resveratrol). Systemic resveratrol (30 mg/kg/day) was administered repeatedly during the 6th week of the experiment. After the mice had been sacrificed, skin tissues were examined histologically for epithelial thickness. Epithelial apoptosis (caspase-3) and epithelium-derived cytokines [interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP)] were evaluated immunohistochemically. Results. Epithelial thickness and the numbers of IL-25, IL-33, TSLP and caspase-3-positive cells were significantly higher in group II compared to group I mice. There was significant improvement in epithelial thickness in group III compared with group II mice (p < 0.05). The numbers of IL-25, IL-33, and TSLP-positive cells in the epithelium were lower in group III than in group II mice (p < 0.05). The number of caspase-3-positive cells, as an indicator of apoptosis, in the epithelium was significantly lower in group III than in group II mice (p < 0.05). Conclusion. Treatment with resveratrol was effective at ameliorating histological changes and inflammation by acting on epithelium-derived cytokines and epithelial apoptosis.

Effects of Quercetin Treatment on Epithelium-derived Cytokines and Epithelial Cell Apoptosis in Allergic Airway Inflammation Mice Model.

Quercetin is a dietary flavonoid which has anti-inflammatory effects. This study aimed to evaluate the influence of quercetin on histopathological aspects and airway epithelium in  allergic airway  inflammation mice model. Twenty-eight BALB/c mice were randomly divided into four groups: Group I (control), Group II (untreated mice with allergic airway inflammation), Group III (allergic airway inflammation quercetin-treated [16mg/kg/day]), Group IV (allergic airway inflammation dexamethasone-treated [1mg/kg/day]). Ovalbumin was administered intraperitoneally and via inhalation to achieve allergic airway inflammation mice model and treatments were also given intraperitoneally. Epithelium thickness, subepithelial smooth muscle thickness, number of mast and goblet cells, and basement membrane thickness were examined on samples isolated from lung. Immunohistochemical evaluationof lung tissues was performed using  IL-25, IL-33, thymic stromal lymphopoietin (TSLP), terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) and cysteine-dependent aspartate-specific proteases(caspase)-3 antibodies. IL-4, IL-25, IL-33, TSLP were quantified in bronchoalveolar lavage (BAL) and OVAspecific IgE levels was measured in serum by standard ELISA protocols. IL-25, IL-33, thymic stromal lymphopoietin (TSLP) and cysteine-dependent aspartate-specific proteases (caspase)-3. Quercetin treatment led to lower epithelial thickness, subepithelial smooth muscle thickness, goblet and mast cell numbers compared to untreated  mice with allergic airway inflammation (p<0.05). However, quercetin treatment was not effective on improving basal membane thickness. Immunohistochemical scores of IL-25, IL-33, TSLP, caspase-3 and TUNEL were lower in quercetin-treated mice  t compared to untreated mice with allergic airway inflammation (p<0.05). IL-4, IL-25, IL-33, TSLP levels in BAL and OVA-specific IgE in serum were lower in quercetin treated mice compared to untreated mice (p<0.05). These findings suggest that quercetin improves chronic histopathological changes except basal membrane thickness in lung tissue and its beneficial effects on inflammation might be related to modulating epithelium derived cytokines and epithelial apoptosis.

The effects of α-lipoic acid on aortic injury and hypertension in the rat remnant kidney (5/6 nephrectomy) model.

OBJECTIVE: The present study was designed to investigate the effects of α-lipoic acid on the abdominal aorta and hypertension in a remnant kidney model histomorphometrically, immunohistochemically, and ultrastructurally. METHODS: We surgically reduced the renal tissue mass to 5/6 by applying a remnant kidney model. The rats were divided into 4 groups: Group 1- control group, Group 2- lipoic acid group, Group 3- 5/6 nephrectomy group, and Group IV: 5/6 nephrectomy+lipoic acid-treated group. Lipoic acid solution 100 mg/kg was administered by oral gavage for 8 weeks to Groups II and IV. At the end of the experiment, systemic mean blood pressure was monitored. Then, aortic tissues were removed and fixed. After routine histological procedures, tissue sections were examined histochemically, immunohistochemically (type I angiotensin receptor, vascular endothelial growth factor, alpha-smooth muscle actin), and ultrastructurally. RESULTS: The blood pressure measurements in 5/6 nephrectomy group were significantly higher compared to other groups. In the 5/6 nephrectomy+lipoic acid group, measured blood pressure values and tunica media thickness were significantly lower than in the 5/6 nephrectomy group. In the 5/6 nephrectomy+lipoic acid group, decreased aortic wall thickness, regularity in the structure of elastic fibrils, and more organized elastic lamellae were seen. The expression of type I angiotensin receptor, vascular endothelial growth factor, alpha-smooth muscle actin in the 5/6 nephrectomy+lipoic acid group was decreased compared to the 5/6 nephrectomy group. CONCLUSION: In the present study, we found that α-lipoic acid could be a favorable agent for the target organ effects of secondary hypertension.