Systemic Diclofenac Sodium Reduces Postoperative rhBMP-2 Induced Neuroinflammation: A Rodent Model Study.

STUDY DESIGN: This is a basic science, animal research study. OBJECTIVE: This study aims to explore, in rodent models, the effectiveness of systemic nonsteroidal anti-inflammatory drugs in reducing recombinant human bone morphogenetic protein-2 (rhBMP-2) induced neuroinflammation. SUMMARY OF BACKGROUND DATA: rhBMP-2 is increasingly used to augment fusion in lumbar interbody fusion surgeries, although it can cause complications including postoperative radiculitis. MATERIALS AND METHODS: Eighteen 8-week-old Sprague-Dawley rats underwent Hargreaves testing to measure the baseline thermal withdrawal threshold before undergoing surgical intervention. The L5 nerve root was exposed and wrapped with an Absorbable Collagen Sponge containing rhBMP-2. Rats were randomized into 3 groups: (1) Low dose (LD), (2) high dose (HD) diclofenac sodium, and (3) saline, receiving daily injection treatment. Hargreaves testing was performed postoperatively on days 5 and 7. Seroma volumes were measured by aspiration and the nerve root was then harvested for hematoxylin and eosin, immunohistochemistry, Luxol Fast Blue staining, and real-time quantitative polymerase chain reaction. The Student t test was used to evaluate the statistical significance among groups. RESULTS: The intervention groups showed reduced seroma volume, and a general reduction of inflammatory markers (MMP12, MAPK6, GFAP, CD68, and IL18) compared with controls, with the reduction in MMP12 being statistically significant ( P = 0.02). Hematoxylin and eosin and immunohistochemistry of the nerve roots showed the highest macrophage density in the saline controls and the lowest in the HD group. Luxol Fast Blue staining showed the greatest extent of demyelination in the LD and saline groups. Lastly, Hargreaves testing, a functional measure of neuroinflammation, of the HD group demonstrated a minimal change in thermal withdrawal latency. In contrast, the thermal withdrawal latency of the LD and saline groups showed a statistically significant decrease of 35.2% and 28.0%, respectively ( P < 0.05). CONCLUSION: This is the first proof-of-concept study indicating that diclofenac sodium is effective in alleviating rhBMP-2-induced neuroinflammation. This can potentially impact the clinical management of rhBMP-2-induced radiculitis. It also presents a viable rodent model for evaluating the effectiveness of analgesics in reducing rhBMP-2-induced inflammation.

Combination therapy with budesonide and N-acetylcysteine ameliorates LPS-induced ALI by attenuating neutrophil recruitment through the miR-196b-5p/Socs3 molecular axis.

BACKGROUND: Neutrophil infiltration accelerates the inflammatory response and is highly correlated to the development of acute lung injury (ALI). Budesonide (BUD) and N-acetylcysteine (NAC) both inhibit the inflammatory response to alleviate ALI, so we further investigated whether their combination is better for ALI. METHODS: In this study, we investigated the effect and mechanism of Combined BUD and NAC therapy on LPS-induced ALI. Rat ALI model and neutrophil abnormal activation model were established by lipopolysaccharide (LPS). BUD and NAC were treated alone or in combination, or cells were transfected with miR-196b-5p mimic or si-Socs3 to evaluate the efficacy and mechanism of BUD and NAC alone or in combination. Histopathological observation of lungs was performed by Hematoxylin Eosin (HE) staining. The quantity of neutrophils and inflammatory factors level in bronchoalveolar lavage fluid (BALF) were determined by Richter-Gimza complex stain and Enzyme-Linked Immunosorbnent Assay (ELISA), respectively. ReverseTranscription-PolymeraseChainReaction (RT-qPCR) was utilized to assess miR-196b-5p and inflammatory factor mRNA levels. The expression level of Socs3 was detected by immunohistochemistry or Western Blot. RESULTS: BUD and NAC combined treatment had a better effect on neutrophil recruitment and inflammatory response in LPS-induced ALI than did BUD and NAC alone. Transfection of the miR-196b-5p mimic reversed the effect of combined BUD and NAC. In conclusion, the combination of BUD and NAC is a better treatment for ALI. CONCLUSIONS: Combination therapy with BUD and NAC ameliorates LPS-induced ALI by attenuating neutrophil recruitment through the miR-196b-5p/Socs3 molecular axis.

Protective Effect of Combined Moxibustion and Decoction Therapy on Bleomycin-Induced Pulmonary Fibrosis in Rats under Nuclear Factor-κB/Transforming Growth Factor-ß1/Smads Signaling Pathway.

it was aimed to discuss the effect of moxibustion (Mox) combined with Bu Fei Qu Yu (BFQY) decoction under the nuclear factor-κB (NF-κB)/transforming growth factor-ß1 (TGF-ß1)/Smads signaling pathway in the treatment of pulmonary fibrosis (PF). The PF rat models were prepared with bleomycin (BLM). They were divided into the normal (Nor) group, the PF model group (BLM puncture perfusion), the Mox group (grain-sized Mox at the back-shu points and Xuxiao points), the BFQY group (intragastrical BFQY decoction), and the Mox combined with BFQY decoction (Mox+BFQY) group. Lung tissue sections were prepared, and the hematoxylin-eosin (HE) staining and Masson staining were performed to observe the inflammatory response and the degree of PF. The contents of hydroxyproline (HYP), glutathione (GSH), and malondialdehyde (MDA), and the expressions of NF-κB p65, TGF-ß1, Smad2, and Smad7 in lung tissues were detected. Compared with those in the Nor group, the inflammatory response score, PF degree score, HYP, GSH, and MDA contents, NF-κB p65, TGF-ß1, and Smad2 expressions were significantly increased in the PF group, but Smad7 expression decreased (P<0.05). The above symptoms were significantly improved in the Mox, BFQY, and Mox+ BFQY groups (P<0.05). The effect was more remarkable in the Mox+BFQY group, and there was no significant difference in each index compared with those in the Nor group (P>0.05). Thus, the combined therapy of Mox and decoction had an effect on PF through the NF-κB/TGF-ß1/Smads pathway.

Inhibition of inflammasome activation via sphingolipid pathway in acute lung injury by Huanglian Jiedu decoction: An integrative pharmacology approach.

BACKGROUND: Acute lung injury (ALI) is a serious health issue which causes significant morbidity and mortality. Inflammation is an important factor in the pathogenesis of ALI. Even though ALI has been successfully managed using a traditiomal Chinese medicine (TCM), Huanglian Jiedu Decoction (HLD), its mechanism of action remains unknown. PURPOSE: This study explored the therapeutic potential of HLD in lipopolysaccharide (LPS)-induced ALI rats by utilizing integrative pharmacology. METHODS: Here, the therapeutic efficacy of HLD was evaluated using lung wet/dry weight ratio (W/D), myeloperoxide (MPO) activity, and levels of tumor necrosis factor (TNF-α), interleukin (IL)-1ß and IL-6. Network pharmacology predictd the active components of HLD in ALI. Lung tissues were subjected to perform Hematoxylin-eosin (H&E) staining, metabolomics, and transcriptomics. The acid ceramidase (ASAH1) inhibitor, carmofur, was employedto suppress the sphingolipid signaling pathway. RESULTS: HLD reduced pulmonary edema and vascular permeability, and suppressed the levels of TNF-α, IL-6, and IL-1ß in lung tissue, Bronchoalveolar lavage fluid (BALF), and serum. Network pharmacology combined with transcriptomics and metabolomics showed that sphingolipid signaling was the main regulatory pathway for HLD to ameliorate ALI, as confirmed by immunohistochemical analysis. Then, we reverse verified that the sphingolipid signaling pathway was the main pathway involed in ALI. Finally, berberine, baicalein, obacunone, and geniposide were docked with acid ceramidase to further explore the mechanisms of interaction between the compound and protein. CONCLUSION: HLD does have a better therapeutic effect on ALI, and its molecular mechanism is better elucidated from the whole, which is to balance lipid metabolism, energy metabolism and amino acid metabolism, and inhibit NLRP3 inflammasome activation by regulating the sphingolipid pathway. Therefore, HLD and its active components can be used to develop new therapies for ALI and provide a new model for exploring complex TCM systems for treating ALI.

Abnormal hyperplasia of chondrocytes in a rat model of glucocorticoid-induced osteonecrosis of the femoral head.

OBJECTIVE: The aim of our study was to determine whether abnormal hyperplasia of chondrocytes occurs in glucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) using a well-established rat model. MATERIALS AND METHODS: Rats were injected with lipopolysaccharide and methylprednisolone to induce GC-ONFH, while control animals were injected with saline (12 animals per group). Establishment of the disease model was confirmed using micro-computed tomography and hematoxylin-eosin (HE) staining of femoral head tissue sections. Chondrocyte hyperplasia was detected using HE staining and semi-quantitated using toluidine blue and saffron O staining. Expression of the autophagy marker LC3B was assessed in cartilage tissues of femoral head using immunohistochemistry. RESULTS: GC-ONFH animals showed significantly greater area of abnormal chondrocyte hyperplasia in femoral head tissue sections than control animals. They also showed significantly higher expression of LC3B in articular cartilage of the femoral head. CONCLUSIONS: GC-ONFH may be associated with abnormal chondrocyte hyperplasia in articular surface cartilage, which may be related to glucocorticoid-induced overactivation of autophagy.

[Effect of Jinzhen Oral Liquid on cough after lipopolysaccharide-induced infection in rats and mechanism].

This study aims to explore the effect of Jinzhen Oral Liquid(JOL) on cough after infection in rats and the mechanism. To be specific, a total of 60 male SD rats were classified into 6 groups: normal group(equivalent volume of distilled water, ig), model group(equivalent volume of distilled water, ig), Dextromethorphan Hydrobromide Oral Solution group(3.67 mL·kg~(-1), ig), high-, medium-, and low-dose JOL groups(11.34, 5.67, and 2.84 mL·kg~(-1), respectively, ig). Lipopolysaccharide(LPS, nasal drip), smoking, and capsaicin(nebulization) were employed to induce cough after infection in rats except the normal group. Administration began on the 19 th day and lasted 7 days. Capsaicin(nebulization) was used to stimulate cough 1 h after the last administration and the cough frequency and cough incubation period in rats were recorded. The pathological morphology of lung tissue was observed based on hematoxylin-eosin(HE) staining. Immunohistochemistry(IHC) was used to detect the specific expression of transient receptor potential vanilloid 1(Trpv1), nerve growth factor(NGF), tropomyosin receptor kinase A(TrkA), and phosphorylated-p38 mitogen-activated protein kinase(p-p38 MAPK) in lung tissue, Western blot the protein expression of Trpv1, NGF, TrkA, and p-p38 MAPK in lung tissue, and real-time fluorescent quantitative polymerase chain reaction(real-time PCR) the mRNA expression of Trpv1, NGF, and TrkA. The results showed that model group demonstrated significantly high cough frequency, obvious proliferation and inflammatory cell infiltration in lung tissue, significantly enhanced positive protein expression of Trpv1, NGF, TrkA, and p-p38 MAPK in lung tissue and significant increase in the mRNA expression of Trpv1, NGF, and TrkA compared with the normal group. Compared with the model group, JOL can significantly reduce the cough frequency, alleviate the pathological changes of lung tissue, and decrease the protein expression of Trpv1, NGF, TrkA, and p-p38 MAPK in lung tissue, and high-dose and medium-dose JOL can significantly lower the mRNA expression of Trpv1, NGF, and TrkA. This study revealed that JOL can effectively inhibit Trpv1 pathway-related proteins and improve cough after infection. The mechanism is that it reduces the expression of NGF, TrkA, and p-p38 MAPK in lung tissue, thereby decreasing the expression of Trpv1 and cough sensitivity.

[Mechanism of "Ephedrae Herba-Descurainiae Semen Lepidii Semen" combination in treatment of bronchial asthma based on network pharmacology and experimental verification].

This study aims to investigate mechanism of "Ephedrae Herba-Descurainiae Semen Lepidii Semen" combination(MT) in the treatment of bronchial asthma based on network pharmacology and in vivo experiment, which is expected to lay a theoretical basis for clinical application of the combination. First, the potential targets of MT in the treatment of bronchial asthma were predicted based on network pharmacology, and the "Chinese medicine-active component-target-pathway-disease" network was constructed, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the potential targets. Molecular docking was used to determine the binding activity of key candidate active components to hub genes. Ovalbumin(OVA, intraperitoneal injection for sensitization and nebulization for excitation) was used to induce bronchial asthma in rats. Rats were classified into control group(CON), model group(M), dexamethasone group(DEX, 0.075 mg·kg~(-1)), and MT(1∶1.5) group. Hematoxylin and eosin(HE), Masson, and periodic acid-Schiff(PAS) staining were performed to observe the effect of MT on pathological changes of lungs and trachea and goblet cell proliferation in asthma rats. The levels of transforming growth factor(TGF)-ß1, interleukin(IL)6, and IL10 in rat serum were detected by enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein levels of mitogen-activated protein kinase 8(MAPK8), cyclin D1(CCND1), IL6, epidermal growth factor receptor(EGFR), phosphatidylinositol 3-kinase(PI3 K), and protein kinase B(Akt) by qRT-PCR and Western blot. Network pharmacology predicted that MAPK8, CCND1, IL6, and EGFR were the potential targets of MT in the treatment of asthma, which may be related to PI3 K/Akt signaling pathway. Quercetin and ß-sitosterol in MT acted on a lot of targets related to asthma, and molecular docking results showed that quercetin and ß-sitosterol had strong binding activity to MAPK, PI3 K, and Akt. In vivo experiment showed that MT could effectively alleviate the symptoms of OVA-induced asthma rats, improve the pathological changes of lung tissue, reduce the production of goblet cells, inhibit the inflammatory response of asthma rats, suppress the expression of MAPK8, CCND1, IL6, and EGFR, and regulate the PI3 K/Akt signaling pathway. Therefore, MT may relieve the symptoms and inhibit inflammation of asthma rats by regulating the PI3 K/Akt signaling pathway, and quercetin and ß-sitosterol are the candidate active components.

FLUOROFENIDONE ATTENUATES PULMONARY INFLAMMATION AND FIBROSIS BY INHIBITING THE IL-11/MEK/ERK SIGNALING PATHWAY.

ABSTRACT: Idiopathic pulmonary fibrosis is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. Interleukin (IL)-11 plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. In this study, we explore whether a potential antifibrotic agent fluorofenidone (FD) exerts its anti-inflammatory and antifibrotic effects through suppressing activation of the IL-11/MEK/ERK signaling pathway in vivo and in vitro. Male C57BL/6 J mice were intratracheally injected with bleomycin or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with hemotoxylin and eosin, and Masson trichrome. Cytokines were measured using the enzyme-linked immunosorbent assay. The α-smooth muscle actin (α-SMA), fibronectin, and collagen I were measured using immunohistochemistry, and the phosphorylated extracellular signal-regulated kinase, phosphorylated mitogen-activated protein kinase, IL-11RA, and gp130 were measured using Western blot. The RAW264.7 cells and the normal human lung fibroblasts were treated with IL-11 and/or FD, IL-11RA-siRNA, or MEK inhibitor. The expressions of phosphorylated extracellular signal-regulated kinase, phosphorylated mitogen-activated protein kinase, IL-11RA, gp130, α-SMA, fibronectin, and collagen I were measured using Western blot and/or real-time polymerase chain reaction, and the cytokines were measured using enzyme-linked immunosorbent assay. Results showed that FD markedly reduced the expressions of IL-8, IL-18, IL-11, monocyte chemotactic protein-1, α-SMA, fibronectin, and collagen I in mice lung tissues. In addition, FD attenuated IL-11-induced expressions of α-SMA, fibronectin, and collagen I and inhibited IL-11RA, gp130, and phosphorylation of the ERK and MEK protein expression, as well as reduced the expressions of IL-8, IL-18, and monocyte chemotactic protein-1 in vitro. This study demonstrated that FD attenuated bleomycin-induced pulmonary inflammation and fibrosis in mice by inhibiting the IL-11/MEK/ERK signaling pathway.

LncRNA H19 alleviates sepsis-induced acute lung injury by regulating the miR-107/TGFBR3 axis.

OBJECTIVE: Acute lung injury (ALI) increases sepsis morbidity and mortality. LncRNA H19 plays a critical role in sepsis. miR-107 is highly-expressed and TGFß type III receptor (TGFBR3) is poorly-expressed in sepsis, yet their roles in sepsis development require further investigation. This study aimed to investigate the mechanism of H19 in alleviating sepsis-induced ALI through the miR-107/TGFBR3 axis. METHODS: Mice were intravenously injected with Ad-H19 adenovirus vector or control vector one week before establishing the mouse model of cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with oe-H19 or oe-NC plasmids and then stimulated by lipopolysaccharide (LPS). Lung injury was assessed via hematoxylin-eosin staining, measurement of wet-to-dry (W/D) ratio, and TUNEL staining. Levels of H19, miR-107, and TGFBR3 were determined by RT-qPCR. Apoptosis of PMVECs was evaluated by flow cytometry. Levels of Bax and Bcl-2 in lung tissues and PMVECs were measured using Western blot. Total protein concentration and the number of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) were quantified. Levels of TNF-α, IL-1ß, IL-6, and IL-10 in BALF, lung tissues, and PMVECs were measured by ELISA. Cross-linking relationships among H19, miR-107 and TGFBR3 were verified by dual-luciferase and RIP assays. RESULTS: H19 was poorly-expressed in CLP-operated mice. H19 overexpression attenuated sepsis-induced ALI, which was manifested with complete alveolar structure, decreased lung injury score and lung W/D ratio, and inhibited apoptosis in CLP-operated mice, which was manifested with decreased number of TUNEL-positive cells and Bax level and increased Bcl-2 level. CLP-operated mice had increased concentration of total protein and number of total cells, neutrophils, and macrophages in BALF, which was nullified by H19 overexpression. H19 overexpression declined levels of TNF-α, IL-1ß, and IL-6 and elevated IL-10 levels. H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production. H19 targeted TGFBR3 as the ceRNA of miR-107. miR-107 overexpression or silencing TGFBR3 partially averted the inhibition of H19 overexpression on LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production. CONCLUSION: LncRNA H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production and attenuated sepsis-induced ALI by targeting TGFBR3 as the ceRNA of miR-107.

Moxifloxacin induces aortic aneurysm and dissection by increasing osteopontin in mice.

Fluoroquinolones are one of the most frequently prescribed antibiotics. However, their use increases the risk of Aortic aneurysm and dissection (AAD). The mechanism underlying this effect remains unclear. AAD are caused by weakening of the aortic wall and loss of vascular smooth muscle cells. Osteopontin is involved in the occurrence and development of AAD. The aim of the present study was to examine the role of moxifloxacin, a fluoroquinolone, in the occurrence of AAD using a moderate-severity AAD mouse model. Four-week-old male C57BL/6J mice were fed a high-fat diet. At 8 weeks of age, the mice were infused with saline or angiotensin II (1000 ng kg-1 min-1) via osmotic minipumps for 4 weeks, and then orally administered water (vehicle) or moxifloxacin (30 and 100 mg kg-1 day-1) for another 3 weeks. Moxifloxacin (30 and 100 mg kg-1 day-1) induced AAD and elastin degradation in aortic tissues, as revealed by hematoxylin and eosin staining and elastica-van Gieson staining. Additionally, immunohistochemical staining and Western blot analyses showed that moxifloxacin 100 mg kg-1 day-1 decreased the protein expression of smooth muscle protein 22α, one of the markers of the contractile phenotype of vascular smooth muscle cells, in aortic tissues compared to vehicle and moxifloxacin 30 mg kg-1 day-1. Furthermore, moxifloxacin (100 mg kg-1 day-1) increased the protein expression of osteopontin and matrix metalloproteinases-2 in the aortic tissues when compared to control. Moxifloxacin may induce the onset of AAD and weakening of the aortic media by increasing the expression of osteopontin and matrix metalloproteinase-2 and decreasing that of smooth muscle protein 22α in aortic tissue.

Perillyl alcohol attenuates chronic restraint stress aggravated dextran sulfate sodium-induced ulcerative colitis by modulating TLR4/NF-κB and JAK2/STAT3 signaling pathways.

BACKGROUND: Ulcerative colitis (UC) is the most prevalent chronic inflammatory immune bowel disease. The modernization of lifestyle accompanied by the stress to cope with the competition has resulted in a new range of complications where stress became a critical contributing factor for many diseases, including UC. Hence there is an urgent need to develop a dual role in curtailing both systemic and neuroinflammation. Perillyl alcohol (POH) is a natural essential oil found in lavender, peppermint, cherries etc and has been widely studied for its strong anti-inflammatory, antioxidant and anti-stress properties. HYPOTHESIS/PURPOSE: POH regulates the various inflammatory signaling cascades involved in chronic inflammation by inhibiting farnesyltransferase  enzyme. Several studies reported that POH could inhibit the phosphorylation of  NF-κB, STAT3 and promote the endogenous antioxidant enzymes like Nrf2 via farnesyltransferase enzyme inhibition.  Also, the effects of POH against UC is not known yet. Thus, this study aims to explore the anti-ulcerative properties of POH on stress aggravated ulcerative colitis in C57BL/6 mice. METHODS: Ulcerative colitis was induced by duel exposure of chronic restraint stress (day 1 to day 28) and 2.5% dextran sulphate sodium (day8 to day14) in mice. POH treatment 100 and 200 mg/kg was administred from day14 ti day28 following oral route of administration. Disease activity index, colonoscopy, western blot analysis and histological analysis, neurotransmitter analysis and Gene expression studies were perofomerd to asses the anti-colitis effects of POH. RESULTS: The treatment reversed the oxidative stress and inflammatory response by inhibiting TLR4/NF-kB pathway, and IL-6/JAK2/STAT3 pathway in both isolated mice colons and brains. The inhibition of these pathways resulted in a decrease in pro-inflammatory cytokines like IL-6, IL-1ß and TNF-α. The treatment improved the physiological and histological changes with decreased ulcerations as examined by colonic endoscopy and Haematoxylin and Eosin staining. The treatment also improved the behavior response as it increased mobility time which was reduced by chronic restrained stress. This was due to increased satiety neurotransmitters like dopamine and serotonin and decreased cortisol in mice brains. CONCLUSION: These results infer that POH has significant anti-colitis activity on chronic restraint stress aggravated DSS-induced UC in mice.

Evaluation of clinical and histological effects of KGF-2 and NGF on corneal wound healing in an experimental alkali burn rabbit model.

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-ß (TGF-ß) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-ß was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

Limbal stem cell deficiency (LSCD) in rats and mice following whole body exposure to sulfur mustard (SM) vapor.

Ocular injuries following sulfur mustard (SM) exposure are characterized by an acute phase expressed by corneal erosions and inflammation of the anterior segment that after a clinically silent period may be followed by irreversible corneal injuries. The latter includes epithelial defects, chronic inflammation and neovascularization (NV), and were defined in rabbits and in humans as Limbal Stem Cell Deficiency (LSCD), that derived from a delayed loss of corneal epithelial stem cells (ESC), due to secondary processes most likely in the epithelial stem cell (SC) niche. The present study expands our research on SM-induced ocular injury to rodents (rats and mice) following whole body vapor exposure, aiming to define whether the delayed development of LSCD is a general characteristic of SM ocular toxicity. Freely moving rats and mice were exposed to SM vapor (155 µg/l, 10 min). Clinical examination was carried out in rats and included a slit-lamp bio-microscopy, up to 6 months. Eyes were taken for histology at different time points following exposure and evaluation included hematoxylin and eosin (H&E) staining for general morphology, PAS for identification of goblet cells and p63 immunohistochemistry for progenitor epithelial cells. Whole body exposure to SM vapor in rats and mice resulted in acute ocular injury characterized by corneal erosions and ocular inflammation. Following a brief recovery period, 80-90% of the exposed eyes developed corneal NV associated with abnormal corneal epithelium, stromal inflammation and endothelial damage. The late injury was accompanied by migration of conjunctival goblet cells to the cornea and a loss of limbal epithelial progenitor cells, indicating LSCD. The long-term ocular injury shown hereby in rats and mice was consistent with the lesions described in rabbits and in human casualties and demonstrated the general phenomenon of limbal epithelial stem cells deficiency in SM ocular toxicity. The delayed manifestation of this pathology points towards a therapeutic window for the development of medical countermeasures in small animals following exposure in a real life scenario.

Prospective Single-Center Observational Study of the Allergenic Potential of Mercromina Film and Other Common Antiseptics in Patients With Contact Dermatitis. / Estudio observacional de seguridad, prospectivo y unicéntrico para determinar la capacidad alergogénica de Mercromina Film® y otros antisépticos de uso común en pacientes con dermatitis de contacto.

INTRODUCTION: Although Mercromina Film and other topical antiseptics are widely used, they are not included in the standard series recommended by the Spanish Contact Dermatitis and Skin Allergy Research Group for testing suspected allergic contact dermatitis (ACD). Furthermore, no recent studies have investigated the allergenic potential of merbromin. OBJECTIVE: To determine the allergenic potential of merbromin and compare it with that of other topical antiseptics widely used in clinical practice, including povidone-iodine, chlorhexidine, and eosin. MATERIAL AND METHODS: Prospective single-center observational safety study of 105 patients with suspected ACD seen at the dermatology department of our hospital. RESULTS: Of the 105 patients studied, 1.9% had a positive patch test to merbromin and 12.4% were sensitized to povidone-iodine. The differences in the proportion of patients with ACD to Betadine Solución Dérmica (povidone-iodine) compared with the rest of the antiseptics was statistically significant (McNemar test, P<.05). No adverse reactions were observed in any of the patients. CONCLUSIONS: Based on the patch tests conducted, Mercromina Film has very low allergenic potential. The highest allergenic potential was observed for povidone-iodine.

Survival of Pseudomonas aeruginosa in modified Romanowsky staining solutions.

BACKGROUND: Anecdotal reports suggest that rapid staining solutions can become contaminated with micro-organisms, especially Pseudomonas aeruginosa. HYPOTHESIS/OBJECTIVES: To determine whether inoculation of rapid Romanowsky-type stains with P. aeruginosa results in viable bacterial contamination, which could lead to cross-contamination of slides during cytological staining. METHODS: Pseudomonas aeruginosa was inoculated into clean and organically contaminated staining solutions (fixative, eosin and methylene blue) and positive (broth) and negative (bleach) control solutions. Subsequent viability and survival were detected by measuring colony-forming units per millilitre at various time points up to 2 weeks. Each sample was stained and microscopically examined to determine whether bacteria were visible. RESULTS: No bacteria could be cultured at any time point from the bleach or fixative solution. In clean eosin and methylene blue staining solutions, viable bacteria were recovered for up to 1 h, but by 24 h all bacteria were dead. In staining solutions contaminated with hair and dead skin cells, bacteria survived in methylene blue for up to 1 h, and viable bacteria persisted in the eosin stain for 2 weeks. In solutions containing viable organisms, the bacteria could be observed by microscopic examination; no bacteria were visible when the solutions contained no viable organisms. CONCLUSIONS AND CLINICAL IMPORTANCE: Pseudomonas aeruginosa can survive in commonly used staining solutions for variable periods of time, but is unable to proliferate. Although theoretically this might complicate cytological interpretation and subsequent diagnosis, the likelihood of this in clinical practice appears remote when the correct staining technique is used.

Allergic contact dermatitis from purified eosin.

We report on a patient, seen in 1993, who developed sensitization to eosin from topical preparations. The patient had a positive patch test reaction to pure tetrabromofluorescein but not to its impurities separated by thin-layer chromatography. Our findings suggest that the allergen was eosin itself.

Allergic contact dermatitis from eosin.

Before 1960, eosin sensitivity was not rare and lipstick cheilitis was very common. We report 4 patients seen in 1988 and 1989 who were sensitized to eosin from topical bacteriostatic preparations. All 4 patients had positive patch tests to eosin. The allergen is probably an impurity rather than eosin itself.

Mutagenicity studies on FD&C red No.3.

FD&C Red No.3 (erythrosine) has been used as a dye in foods, drugs and cosmetics since its approval by the US Department of Agriculture in 1907. In 1977 the Certified Color Manufacturers' Association (CCMA) initiated studies on FD&C Red No.3 including chronic toxicity and carcinogenicity studies in rats and mice. Data from the CCMA chronic studies revealed an increased incidence of thyroid follicular cell hyperplasia and adenomas in male rats that received 4% FD&C Red No.3 in the diet (2464 mg/kg/day) during life-time (30 months) following in utero exposure. In this report, results of published studies on the mutagenicity of FD&C Red No.3 are critically reviewed. Additional mutagenicity tests including Ames Salmonella/microsome assay, L5178Y TK+/- mouse lymphoma assay, mouse micronucleus test and mitotic recombination assay with yeast Saccharomyces cerevisiae strain D5 are described. These test results together with the literature review indicate that FD&C Red No.3 can be considered non-mutagenic across several genetic endpoints including gene mutation, chromosome aberrations, primary DNA damage and cell transformation. The results of the genotoxicity assessment generally exclude FD&C Red No.3 as a genotoxic initiator and suggest that some other mechanism is responsible for the increase in tumors.