Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus,  Actinomycetales order includes bacteria that are markedly divergent in their infectious potential. They range from deadly pathogens, like Mycobacterium tuberculosis, to facultative pathogens and those living solely in the environment The Corynebacterineae contain mycolic acids, which form into an outer membrane in the cell envelope and confer characteristic properties of bacteria in this suborder. Mycolic acids are α-alkyl-β-hydroxy fatty acids, which are divided into subgroups based on functional groups in the meromycolate chain, including oxygen modifications, unsaturations and cyclopropyl groups. The variability in carbon chain length, as well as the number and nature of meromycolate substitutions, are the result of specific fatty acyl synthases, cyclopropanation enzymes, and other multi-enzyme complexes Genera in the suborder Mycobacterium have the longest reported mycolic acid chains and so can be distinguished from other actinobacteria based on this property. Mycobacterial mycolates are typically C60–88 and display the greatest chemical functionality within the meromycolate chains. The other mycolata demonstrate a varied degree of unsaturation and a diverse carbon chain length, including Corynebacterium (C22–36), Rhodococcus (C34–54), Nocardia (C50–62), Gordonia (C54–66) and Tsukamurella (C64–74) The different chemical functional groups and carbon chain lengths of the mycolate structure are valuable as chemotaxonomic markers. For example, species in the genus Rhodococcus species. However, reversed phase HPLC of mycolates showed late elution, suggesting that these untypable species produce mycolic acids that were unusually hydrophobic and likely of longer chain length than those normally found in Rhodococcus species. Whereas most rapidly growing mycolata stain weakly acid fast, the high intensity of the acid-fast stain was surprising, a second finding that suggested possibly unique mycolate structures in these unusual bacteria Segniliparus rotundus and Segniliparus rugosus. These findings from human isolates, along with isolation of a strain from a seal, suggest that Segniliparus rugosus may represent an emerging infectious disease associated with environmental water sources or cystic fibrosis Recently, rapidly growing, acid-fast staining bacteria generated scientific interest because they were isolated from sputa or bronchial washings of human patients, suggesting natural colonization or respiratory infection, yet they were untypable by standard criteria Segniliparus mycolates. The overall length of mycolic acids, which reaches C100, is distinctly atypical of rapid growing mycolata. To our knowledge, these lipids represent the longest mycolic acids known and are among the longest lipids known in cell biology. Discovery of new extremes in mycolate chemical diversity support new models for the spatial distribution and folding of mycolic acids in the Segniliparus cell envelope.Here we describe high accuracy mass spectrometry and nuclear magnetic resonance to establish the chemical composition and structure of 65 molecular species belonging to three classes of Segniliparus rotundus strain CDC 1076T ( = ATCC BAA-972T = CIP 108378T), Segniliparus rugosus sp. nov. type strain CDC 945T ( = ATCC BAA-974T = CIP 108380T) Mycobacterium tuberculosis strain H37Rv ( = ATCC 27294T).Strains used in this study include Bacteria were grown in sterile filtered (0.2 µm) Middlebrook 7H9 medium containing 0.2% glycerol (w/v) and 10% (v/v) Middlebrook ADC enrichment. Cultures were incubated at 37°C for 4 days after which cells were collected by centrifugation at 3,500 rpm, 20°C for 15 min. Cells were washed three times, resuspended in 10 ml fresh growth medium, and cellular debris was allowed to settle. Five ml of suspended fine growth culture was transferred to 45 ml medium and incubated for 4 days, after which time cells were collected by centrifugation and sterilized with gamma irradiation at 2.0 megarad.2 Electron Microscope For scanning electron microscopy, cells were fixed with 5% glutaraldehyde in cacodylate buffer  for 4 hours at room temperature. Suspensions were filtered using a 0.1 um polycarbonate filter . Filters were dehydrated in a graded series of ethanol washes and immersed in Hexamethyldisilazane  overnight at room temperature. Finished specimens were mounted on aluminum stubs with silver paint, coated with 30 nM of gold and observed using a Philips XL Environmental Scanning Electron Microscope . For transmission electron microscopy, cells were placed into formalin for 1 hr, washed 3 times for 2 min each in 0.1 M phosphate buffer and transferred to a 1% solution of OsO4 for 1 hour. Cells were washed with water and dehydrated in a graded series of ethanol washes, collected and placed into embedding molds with 100% resin solution. The resin was polymerized at 50°C for 8 hours, cooled and cut into sections on a copper grid. Uranyl acetate and lead citrate were added, dried and the specimens were observed with a Phillips Tecnai™ GTotal mycolates were isolated by saponification as previously described, using new, clean glassware and HPLC grade solvents a.m.u. Collision induced dissociation (CID)-MS experiments were performed on a LXQ 2 dimensional ion-trap mass spectrometer (Thermo Scientific) equipped with an electrospray ionization (ESI) source. For the positive-ion mode analysis, 1-D TLC purified samples were dissolved in chloroform/methanol  at ∼10 µg/ml. Five µl of sample were mixed with a solution of 1 mM sodium acetate in methanol and loaded into a nanospray tip and analyzed by electrospray ionization-MS. Bacterial pellets of S. rugosus and S. rotundus were extracted with chloroform, which were subsequently diluted with methanol to a final concentration of ∼10 µg/ml, loaded into a nanospray tip (5 µl), and analyzed with a spray voltage and capillary temperature were set to 0.8 kV and 200°C. The collision energy was 28∼35% of maximum and the trapping of product ions were carried out with a q value of 0.2 or 0.25.Total and preparative MAMEs were analyzed by LC-ESI0 MS using an Agilent Technologies 6520 Accurate-Mass Q-Tof  connected to an Agilent 1200 series HPLC system using a Varian Monochrom 3 µm×150 mm×2 mm diol column. The samples were suspended at 1 mg/ml in solvent A  and filtered. Ten µg of lipid was injected, and the column was eluted at 0.15 mL/min with a binary gradient from 0% to 100% solvent B : 0–10 min, 0% B; 17–22 min, 50% B; 30–35 min, 100% B; 40–44 min, 0% B, followed by additional 6 min 0% B post-run as described in detail cis unsaturations, trans unsaturations and α-branched, β-hydroxyl systems were based on comparison of the spectra obtained with prior published spectra 1H-NMR spectra were recorded on a Barnett Instrument 500 MHz at room temperature in 100 µg/ml deuterated chloroform in 200×5 mm 535-pp Wilmad NMR tubes with tetramethyl silane as a proton reference. Chemical shifts are expressed in ppm downfield from the signal of the TMS (0 ppm). The peak assignment for protons in cyclopropyl, S. rotundus and S. rugosus show differing smooth and rough colony forms, and display somewhat distinct appearance by electron microscopy  or methoxy (m/z 1268.314) functional groups, as well as ammoniated α-mycolates (m/z 1169.219)  classes using normal phase, one-dimensional thin layer chromatography (1D TLC). As expected, e chains , left. M169.219) , inset. Segniliparus rotundus, we detected three spots, but these did not precisely co-migrate with mycobacterial mycolates. For S. rugosus, we consistently detected two spots. To determine the chemical basis for TLC separation of Segniliparus MAMEs into two or three subclasses, we carried out positive-ion mode LC-ESI-MS with high accuracy TOF detection , C(X+1), H[2(X+1-Y)+1], where X is the number of carbons and Y is the number of unsaturations and cyclopropyl groups. Ammoniated adducts correspond to O(3), N(1), C(X+1), H[2(X+1-Y)+4]. For example, S. rotundus ions detected at m/z 1374.427 and 1391.454 were deduced as C95H185O3 and C95H188NO3, respectively , α-mycolates  and short α’-mycolates . The average length of Segniliparus α-mycolates (C94) greatly exceeded those lengths of mycolates in M. tuberculosis (C79), which are normally known as long chain mycolates , 1330 (C92 MA), and 1136 (C78 MA), which were deduced as [M-H]− of C96 MA, C92 MA and C78 MA. Additionally, for S. rotundus, we detected m/z 969, which was deduced as C66 MA. Each of these ions was subjected to CID-MS analysis. S. rugosus mycolates all generated single product ion 339 and no detectable chain length series  analysis . In the h series . In controtundus . We inteS. rotundus doublet, there is overlap in chain length of the α+ and α mycolates. Therefore, these two samples are considered enriched for homogenous chain length, but are not pure class-specific mixtures  protons in the mycolate chains were seen at 1.2–1.5 ppm, and the terminal methyl (CH3) protons appeared at 0.88 ppm. Characteristic protons of the Cα branched , the hydroxylated Cβ , and the terminal methyl group in ester linkage to the mycolate  were identified in all five lipid samples, confirming the presence of the α-branch, β-hydroxyl and carboxylate, which define these lipids as mycolic acids.These initial conclusions, based on mass measurements, were further tested by 1H-NMR analysis of each class. For the high migrating mixtures . Therefoycolates . The pro1H-NMR chemical shifts and the integration of proton signals. As predicted by mass spectrometry, signals for methoxy-mycolate protons  and for keto-mycolate, protons of CH2  and CH , were absent from all 5 preparations of Segniliparus MAMEs, confirming the lack of oxygenated structures in the meromycolate chain.The presence, number and stereochemistry of oxygen-containing R groups, unsaturations or cyclopropyl groups in the meromycolate chain are potentially important because these deviations from otherwise straight alkane structure influence the packing and thereby affect the fluidity of the mycolate membrane cis-double bond protons  were at 5.35 ppm and trans-double bond protons  were at 5.31 and 5.22 ppm. Methylene protons adjacent to the double bonds were located at 2.0 ppm and 1.98 ppm for cis and trans, respectively  and methyl group (3.71 ppm), provide the basis for integration to establish the number and configuration of double bonds in each class . Based oarations  and S5.2H4 in length. Trans- but not cis-double bonds introduce an extra methyl branch, which contributes to C3H6. Therefore, mixed MAMEs comprised of molecules possessing and lacking trans unsaturations or cis-cyclopropanations typically appear as two overlapping alkane series in which each molecule differs by CH2. For the molecules only containing double bonds, these rules predict that odd chain mycolates are seen only in trans unsaturated molecules. Cross comparison of mass spectra  and Tsukamurella (C64–78). The observed variance is also distinctly atypical for any bacterium in the suborder Corynebacterineae. More typically, mycolyl lipids vary by 8 to 20 methylene groups, rather than 42 as seen here. Thus, intraorganismal variation in chain length raises new questions about the native conformations of mycolic acids.The term segnilomycolates is suggested for this class of molecules, whose distinguishing features include lack of oxygen-containing substitutions on the meromycolate chains, high degree of Segniliparus α-mycolic acids is unknown, but likely influences mycolate membrane fluidity and permeability. Biochemical and structural data show that the unusual mycolates observed here form a mycolate membrane that is analogous to those found among other actinobacteria. For example, previous studies of S. rugosus and S. rotundus revealed brightly staining acid fast cells The precise role for such a structural diversity of cis unsaturation, which bend meromycolate chains and have the largest effects to raise transition temperatures, which might represent a natural modification to mitigate overly tight packing + mycolates are associated with one, two or three unsaturations or cyclopropyl groups, respectively Click here for additional data file.Figure S2S. rotundus and S. rugosus mycolates derived from protonated adducts. MAMEs derived from S. rotundus and S. rugosus were analyzed by positive-ion mode Q-Tof mass spectrometry. The empiric formulas were deduced from the accurate mass of MAME proton adducts. The difference between the detected and the calculated m/z is reported as the error.Elemental composition of (TIF)Click here for additional data file.Figure S3Segniliparus rotundus that were separated and recovered from 1D TLC as high, middle or low bands, as illustrated in Positive-ion mode of Q-Tof mass spectrometry of MAMEs derived from (TIF)Click here for additional data file.Figure S4Segniliparus rugosus were separated and recovered from 1D TLC as high or middle bands as illustrated in Positive-ion mode Q-Tof mass spectrometry of MAMEs derived from (TIF)Click here for additional data file.Figure S5S. rotundus MAMEs isolated from the 1D TLC high migrating band and middle migrating band were analyzed by 1H NMR spectroscopy. As indicated in (TIF)Click here for additional data file.


Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory. Drug discovery is a constant need for biomedical research and clinical progress. Particularly mega-biodiversity areas such as the tropical rainforests of Central America are very promising for the discovery of new pharmaceutical lead compounds. Therefore, we chose an ethnomedical approach to drug discovery and focussed on traditional remedies of the Maya to preselect plants with proven health effects.Pluchea odorata which are relevant for anticancer treatment. P. odorata grows in the USA, Mexico, Belize, Guatemala, Panama, Cayman Islands, Guadeloupe, Jamaica, Puerto Rico, St. Lucia, Venezuela, and Ecuador  and is still used by the Maya to treat common cold, fever, flu, head colds, headache, hypertension, neuralgia, ophthalmia, palsy, pneumonia, snake bite, swellings, inflammation, and bruises of the skin [Traditional medicine plants are often used for hundreds of years , which ithe skin . The medthe skin , as wellthe skin . Gridlinthe skin  and Bauea priori successful therapies of all kinds of cancer and other diseases is the acquisition of drug resistance. However, it has not been reported so far that complex plant mixtures induce treatment resistances in patients, most likely because several distinct active principles target various intra- and intercellular signalling pathways simultaneously thereby preventing that the organism develops an “escape mechanism.” Thus, the development of complex extracts should be considered as a strategy to treat cancer. Standardisation procedures for such mixtures must be individually developed to provide therapeutics which are effective and safe and do not exhibit batch to batch variations. The fractionation and accompanying testing in bioassays and by appropriate analyses are a feasible concept for standardisation and remedies emerging along such procedures can be approved by national agencies : “Use of a fermented wheat germ extract as anti-inflammatory agent” by Hidvegi and Resetar).Pharmaceutical drugs are prepared under standardised conditions and usually contain just one active principle. In contrast, ethnopharmacologic remedies are mixtures of a vast number of compounds, which are in many cases unknown, and vary in their composition and activity depending on the growth area and the time of collection. This makes them difficult for application and trading. Moreover, plant extracts can also contain compounds that are systemically toxic or counteract the desired effect of the active principle(s). On the other hand, some of the different compounds in a plant extract can synergise. The present work will give examples of this phenomenon. A common reason to terminate P. odorata, which was constantly controlled regarding its activity by bio-assays and analyses that can be standardised. This resulted in fractions causing distinct cellular responses indicative for wound healing or anti-neoplastic properties.Here we describe a fractionation approach of the dichloromethane extract of Pluchea odorata (L.) Cass. were collected in Guatemala, Departamento Petén, near the north-western shore of Lago Petén Itzá, San José, within an area of four-year-old secondary vegetation ~1 km north of the road from San José to La Nueva San José . Voucher specimens  were archived at the Museum of Natural History, Vienna, Austria. 6 kg of air-dried plant material has been extracted with dichloromethane by Björn Feistel , and the extract stored in an exsiccator in the dark at 4°C until use.The aerial parts  of Plant extraction with dichloromethane was essentially as described earlier , 7. 6000 Vacuum Liquid Chromatography (VLC) was used for the separation of large amounts of extract. 36 g dichloromethane extract was redissolved in dichloromethane, mixed with 70 g silica gel and evaporated to dryness, and ground in a mortar to obtain a homogenous powder. A 12 × 40 cm column was packed with 900 g silica gel, on top the silica gel-containing extract, and covered with sea sand to ballast the sample. The mobile phase was passed through by application of reduced pressure. After checking the collected fractions by TLC, those with similar bands were recombined, and this resulted in three combined fractions  Fractionation of F2.6 (Less Than 2 g of Residue was fractionated by CC). 1.6 g F2.6, which was gained by fractionation of F1.1 was dissolved in dichloromethane, mixed with 3 g silica gel, evaporated to dryness, placed on top of a 5 × 50 cm silicia gel column and eluted with mobile phases shown in  CC Fractionation of F3.3. 1.08 g of F3.3, which was derived from F2.6, was dissolved in dichloromethane and placed on top of a 2 × 30 cm silica gel column. Mobile phases were used as illustrated in  CC Fractionation of F4.3.7. 146 mg F4.3.7 was dissolved dichloromethane and applied on a 80 × 1.5 cm silica gel column and fractionated with one litre solvent . Fractions were collected in tubes, ten drops per minute, and every 30 minutes the tubes were changed. Afterwards the column was washed with 300 mL methanol. Fractions were checked by TLC, and those with similar band patterns were recombined resulting in twelve fractions , F4.6.4 (55 mg). CC Fractionation of F3.6. 0.32 g F3.6, which was derived from F2.6, was dissolved in methanol and applied on top of a 3.5 × 40 cm Sephadex column (2.5 × 40 cm for 3.6). Methanol was used as mobile phase, and fractions were collected and checked by TLC, and those with similar band patterns were recombined to yield four combined fractions: F4.6.1 (9 mg), F4.6.2 (5 mg),  CC Fractionation of F4.6.3. 20 mg F4.6.3 (very oily), which was derived from F3.6, was dissolved in dichloromethane, mixed with silica gel, and applied on top a 2.5 × 15 cm dichloromethane conditioned silica gel column. Successful elution was achieved with dichloromethane : ethylacetate (50 : 50). Fractions were checked by TLC and those with similar band patterns were recombined yielding six combined fractions . ASR consisted of 0.5 mL anisaldehyde, 10 mL glacial acetic acid, 85 mL methanol and 5 mL H2SO4 (sulfuric acid). The sprayed plate was heated at 100°C for five minutes, and then compounds were detected under UV254,365 and visible light. Unless otherwise stated 8 μL extract or fraction solution was applied to the plate.TLC was used for control of fractionation. Stationary phase and mobile phases are described in 2.HL-60 (human promyelocytic leukaemia cell) cells were purchased from American Type Culture Collection (ATCC). The cells were grown in RPMI 1640 medium which was supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% Glutamax, and 1% Penicillin-Streptomycin. Both medium and supplements were obtained from Life Technologies . The cells were kept in humidified atmosphere at 37°C containing 5% COP. odorata. Extract and fractions were dissolved in ethanol . HL-60 cells were seeded in 24-well plates at a concentration of 1 × 105 cells per mL RPMI medium allowing logarithmic growth within the time of treatment with plant extract or fractions. The control was treated with solvent. After 24 and 48 hours, the number of cells was determined using the Sysmex Cell Counter . Experiments were done in triplicate. Percentage of cell division progression compared to the untreated control was calculated by applying the following formula:Proliferation assays , 9 were 5 cells per mL RPMI medium. Cells were treated with fractions or extract of P. odorata. The cells were incubated for 8, 24, 48, and/or 72 hours, depending on the experiment. At each time point, 100 μL cell suspension of each well was transferred into separate wells of a 96-well plate, and Hoechst 33285 and propidium iodide were added at final concentrations of 5 μg/mL and 2 μg/mL, respectively. After one hour of incubation at 37°C, stained cells were examined and photographed with an Axiovert fluorescence microscope  equipped with a DAPI filter. Type and number of cell deaths were evaluated by visual examination of the photographs according to the morphological characteristics revealed by HOPI staining. Experiments were done in triplicate.Determination of cell death by Hoechst 33258 (HO) and propidium iodide (PI) double staining  allows identifying the amount and the type of cell death (early or late apoptosis or necrosis) –12. Ther6 cells per mL RPMI medium. P. odorata fractions F1, F4.6.3, and F4.3.7 were analysed by western blots. HL60 cells were either incubated with 40 μg/mL F1 or with 10 μg/mL of one of the other two P. odorata fractions for the indicated times. At each time point, 4 × 106 cells were harvested, placed on ice, and centrifuged . Then, the supernatant (medium) was discarded, and the pellet was washed twice with cold phosphate buffered saline  and centrifuged . The cell pellet was lysed in a buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 1% Triton-X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM protease inhibitor cocktail (PIC) . Afterwards the lysate was centrifuged at 12000 rpm for 20 min at 4°C. Supernatant was transferred into a 1.5 mL tube and stored at −20°C for further analyses.Preparation of lysates. HL60 cells were seeded in a tissue culture flask at a concentration of 1 × 10Gel electrophoresis (SDS-PAGE) and blotting. Equal amounts of protein samples (lysate) were mixed with sodium dodecyl sulfate (SDS) sample buffer (1 : 1) and loaded onto a 10% polyacrylamide gel. Proteins were separated by polyacrylamide gel electrophoresis (PAGE) at 120 Volt for approximately one hour. To make proteins accessible to antibody detection, they were electrotransferred from the gel onto a polyvinylidene difluoride (PVDF) Hybond membrane  at 95 Volt for 80 minutes. Membranes were allowed to dry for 30 minutes to provide fixing of the proteins on the membrane. Methanol was used to moist the membranes. Equal sample loading was checked by staining the membrane with Ponceau S .Protein detection. After washing with PBS or TBS , membranes were blocked in PBS- or TBS-milk (5% nonfat dry milk in PBS containing 0.5% Tween 20 or TBS containing 0.1% Tween 20) for one hour at room temperature. Then membranes were washed with PBS/T (PBS containing 0.5% Tween 20) or TBS/T (TBS containing 0.1% Tween 20), changing the washing solution four to five times every five minutes. Then every membrane was incubated with a primary antibody (1 : 500) in blocking solution , at 4°C over night gently shaking. Subsequently the membrane was again washed with PBS/T or TBS/T and incubated with the second antibody (peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG) diluted 1 : 2000 for one hour at room temperature. After washing the membranes, chemiluminescence was developed with enhanced chemiluminescence (ECL) plus detection kit  (two seconds to ten minutes), and membranes were exposed to the Lumi-Imager F1 (Roche) for increasing times.α-tubulin (6-11B-1) and β actin (AC-15) antibodies were from Sigma . Monoclonal mouse α-tubulin (DM1A), β-tubulin (H-235), Cdc25A (F-6), and polyclonal rabbit paxillin (H-114), ROCK-1 (C8F7), c-Jun (H-79), Jun b (210) were from Santa Cruz Biotechnology, Inc. . Monoclonal rabbit cleaved caspase 8 (Asp391) (18C8) and phospho-Stat3 (Tyr705)(D3A7) antibodies and polyclonal rabbit cleaved caspase 3 (Asp175), human-specific cleaved caspase 9 (Asp330), phospho-Chk2 (Thr68), Chk2, phospho-myosin light chain 2 (MLC2-Ser19), myosin light chain 2, Stat3, and MYPT1 were from Cell Signaling . Polyclonal rabbit phospho-Cdc25A (Ser177) antibody was from Abgent , polyclonal rabbit phospho-MYPT1 (Thr696) from Upstate , mouse monoclonal γH2AX (pSer139) (DR 1017) from Calbiochem , and mouse monoclonal c-Myc Ab-2 (9E10.3) from Thermo Fisher Scientific, Inc. . The secondary antibodies peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Dako .Monoclonal mouse ascites fluid anti-acetylated t-test was applied to compare differences between control samples and treatment groups. Statistical significance level was set to P < 0.05.For statistical analyses, Excel 2003 software and Prism 5 software package  were used. The values were expressed as mean ± standard deviation, and Student's P. odorata exhibits strong anti-neoplastic activity . 10 The subfractionation of F3.3 on a silica gel column yielded the potent antiproliferative and proapoptotic fraction F4.3.7 , which caused also the degradation of α-tubulin and β-actin as consequence of swift onset of cell death.In search of molecular causes for reduced proliferation, we found that the fractionation steps enriched a spindle toxin or an indirect microfilament-targeting activity. This was reflected by induced ilaments , 17, witilaments ), becausilaments . EvidentThe low or absent activation of Chk2 (resp.) suggested that a genotoxic activity, which was formerly present in the crude extract  and in oP. odorata is used also as a wound healing remedy, this property also involves tissue regeneration and the regulation of a process called “epithelial to mesenchymal transition” (EMT) [P. odorata is successfully used for tissue recovery and healing of skin bruises, the increased mobility of cells  is required to close the tissue disruption. This very property, however, is detrimental throughout cancer treatment, and elimination of this activity might be beneficial for this purpose. Therefore, future research has to address the question whether F4.3.7 is advantageous for cancer cell treatment, whereas F4.6.3 exhibits advanced wound healing properties. Since n” (EMT) , 24. Then” (EMT) , 26. If n” (EMT) –29. In cn” (EMT) . The mobn” (EMT) , 31, andn” (EMT) , 33, ROCn” (EMT) , MLC2 fon” (EMT)  and MYPTn” (EMT) , are indn” (EMT) –39, and α-tubulin acetylation followed by the activation of caspases 8 and 3 and the typical nuclear morphology of apoptosis. The impact on apoptosis and the orchestration of apoptosis regulator activation were similar for both fractions. In comparison to earlier work, genotoxic components activating Chk2 were eliminated.A spindle-damaging activity became enriched by fractionations, which was reflected by increased Importantly, F4.6.3 induced mobility markers, whereas F4.3.7 inhibited the expression of mobility markers or did not interfere with their constitutive expression. This property implicates an inhibitory effect of F4.3.7 on tumour progression which was also reflected by the downregulation of the Jun family of oncogenes and the suppression of Stat3 activity.These findings can provide a basis for the development of remedies (1) supporting wound healing in case of F4.6.3 or (2) interfering with tumour progression in case of F4.3.7.


Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bβ3 (GP IIb/IIIa), α5β1, αvβ3, αvβ1, and αvβ5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice.Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake  Intravenous (IV) administration of recombinant tissue plasminogen activator (rt-PA) is an effective treatment for ischemic strokes if administered within 4.5 hours of symptom onset ,2. Howev2bβ3 (glycoprotein [GP] IIb/IIIa), α2β1, α5β1, and αvβ3. Among these integrins, the GP IIb/IIIa receptor, which mediates the final common pathway of platelet aggregation by binding specifically to fibrinogen [Adhesion and aggregation of platelets are mediated by interactions of ligands with multiple integrins, including integrins αbrinogen , is the brinogen -16. Thesbrinogen ,15,17-19Gloydius saxatilis, has the tripeptide sequence RGD [3)-induced arterial thrombosis model in mice and platelet aggregometry [3-induced arterial thrombosis model and platelet aggregometry. Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake ence RGD ,21. Saxaence RGD  and plategometry ,24. We aAll animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University College of Medicine  and were performed in strict accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).2O and 30% O2. Anesthesia was maintained with 2% isoflurane. During operative procedures, body temperature was monitored continuously with a rectal probe and maintained at 37.0 ± 0.2°C using a homeothermic blanket control unit and a heating pad . A FeCl3-induced carotid thrombosis model was used to test the thrombolytic activity of saxatilin in vivo. A midline cervical incision was made, and the left common carotid artery was carefully dissected under a surgical microscope. An ultrasonic Doppler ﬂow probe  was placed around midportion of the common carotid artery (CCA). Carotid blood flow was obtained with a Transonic TS420 Blood Flow Meter  and an iWorx IX-304T data acquisition system . Computer-based analysis by iWorx Labscribe 2 software (version 2.045000) was performed to minimize any bias when assessing results. CCA baseline flow was measured for 5 minutes. Oxidative vascular injury with chemical stress was induced by placing a filter paper (700 μm × 500 μm) saturated with 50% FeCl3  on the adventitial surface of the midpoint of the exposed CCA for 5 minutes. After removing the filter paper, the CCA was washed with normal saline and blood flow was recorded. Thrombus formation and arterial occlusion were determined by decreased blood flow, and complete occlusion was deﬁned as absence of blood flow for 10 minutes.Eight-week-old male Institute of Cancer Research (ICR) mice weighing 32-34 g were used. Animals were housed in a temperature-controlled animal facility under a 12/12 hours reversed light and dark cycle, in a plastic cage with soft bedding, and given with a free access to food and water. For operative procedures, animals were anesthetized with 5% isoflurane in a mixture of 70% NConsistency of the mouse model for thrombus formation and size was assessed. Ten minutes after complete occlusion, injured CCA segments were excised, immediately immersed in 4% paraformaldehyde for fixation, and embedded in paraffin for histological analysis. Paraffin blocks were consecutively sectioned longitudinally into 3 μm slices. Sectioned slices were mounted on glass slides and stained with hematoxylin and eosin. Thrombus size  for each animal was determined using a light microscope  and Zeiss AxioVision software  for a slice representing the largest part of the thrombus.2) overnight at 4°C, washed in 0.1 M phosphate buffer (pH 7.4) and post-fixed in 1% osmium tetroxide in the same buffer for 2 hours. Specimens were then dehydrated through a series of ascending ethanol concentrations, exchanged through propylene oxide, and incubated with a 1:1 mixture of EPON  and propylene oxide for 18 hours, embedded in an EM oven, and trimmed. Subsequently, 0.25 μm semi-thin sections were stained with toluidine blue and observed under a light microscope to determine the FeCl3-damaged region. After retrimming, ultrathin sections (80 nm) were obtained by ultramicrotome  with a diamond knife, double stained with uranyl acetate and lead citrate, and examined by TEM  at 80 kV [For transmission electron microscopy (TEM), arteries were immediately fixed with Karnovsky solution , specimens were fixed and dehydrated as described above, replaced with isoamyl acetate, dried in a critical point dryer , coated with a thin layer of gold (100 nm) in an ion coater , and examined with by field-emission SEM  at 20 kV. 3-induced arterial thrombosis model for evaluation of thrombolytic effects, 10%, 20%, 30%, 40%, and 50% (w/v) concentrations of the FeCl3 were tested using five mice for each test concentration. Carotid blood flow was measured continuously for 150 minutes from complete occlusion.To determine the optimal condition of the FeClExpression and mass production of saxatilin have been described ,26. Ten To evaluate the dose response to saxatilin, animals were randomly divided into seven groups, with five mice in each group: normal saline (control group), 1, 1.75, 2.5, 3.75, 5.0, or 10.0 mg/kg saxatilin. Ten percent of the dose was administered by IV bolus, and the rest was infused continuously for 60 minutes.The thrombolytic effects of saxatilin were assessed after different administration methods. A total dose of 5 mg/kg of saxatilin was used in each animal, and animals were divided into the following four groups with five mice in each group: 1) bolus injection of total dose (5 mg/kg) at 10 minutes after occlusion; 2) double bolus injection of saxatilin with a half dose (2.5 mg/kg) of saxatilin at 10 minutes after occlusion and 60 minutes after the first bolus injection; 3) half-dose bolus injection (2.5 mg/kg) at 10 minutes after occlusion, then continuous infusion for 60 minutes of the remaining dose; or 4) bolus injection of 10% of the total dose (0.5 mg/kg) at 10 minutes after occlusion, and then continuous infusion of the remaining dose (4.5 mg/kg) for 60 minutes. The presence and degree of recanalization were assessed by measuring blood flow. Immediately after 2 hours of blood-flow monitoring, the CCA was removed from all mice, fixed with 4% paraformaldehyde solution, and embedded in paraffin for histological examination. Paraffin blocks were consecutively sectioned in a transverse direction into 3 μm slices, mounted on glass slides, and stained with hematoxylin and eosin.Carotid blood flow was determined by calculating the area under time-flow curves. All measured values were standardized by the minimum blood flow of each animal to avoid differences from variations in physiological condition between animals. Thrombolytic effects were calculated as described below and expressed as percent of mean control baseline blood flow: (mean blood flow during 2 hours of monitoring/mean baseline blood flow) × 100. Mean values of each group in the dose-response study were calculated and graphed on a standard thrombolytic activity curve (mean ± SD).Average blood flow was calculated for each animal each minute to achieve a representative time-dependent pattern during monitoring. Values for mean and standard deviation of all animals in each group were calculated, and temporal changes are shown as continuous bar graphs.Time from administration of saxatilin to effective recanalization was assessed. Effective recanalization was defined as restoration of blood flow to at least 50% of the baseline level, maintained for longer than 30 minutes.Blood (900 μl) was drawn by cardiac puncture from five mice anesthetized with isoflurane into a syringe containing 100 μl of 150 USP sodium heparin solution, resulting in a final heparin concentration of 15 USP/ml. A total of 500 μl of heparinized whole blood was mixed with the same volume of normal saline. Saxatilin concentrations were adjusted by dilution with normal saline, and 0.1, 1, or 2 μg of saxatilin against adenosine diphosphate (ADP) and 5, 50, or 100 μg of saxatilin against collagen were added into each test cuvette and preincubated with magnetic stirring at 37°C for 5 minutes. The same volume of normal saline was added as a control. Agonists for platelet aggregometry were 20 µM ADP or 5 μg/ml of collagen . Platelet aggregation activity was measured with an impedance method in a platelet aggregometer . To test saxatilin effects on preformed platelet aggregates, 10, 50, and 250 μg of saxatilin or the same volume of normal saline as a control were added to cuvettes after maximal aggregation in response to 20 μM ADP or 5 μg/ml of collagen. Platelet disaggregation effects were calculated as percentage of restoration to baseline levels. 2, and 0.02% NaN3) for 12 hours at 37°C. Gels were stained with 0.1% amido black containing acetic acid, methanol, and distilled water (volume ratio 1:3:6) for 1 hour and destained by four washes with the same solution without amido black for 130 minutes. Gels were scanned using a flatbed scanner  [Fibrin/fibrinogen zymography was performed to determine fibrinolytic activity. Fibrinogen gels were prepared using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 1.2% fibrinogen  and 0.1 NIH unit/ml of thrombin . A total of 100 μg saxatilin or 3 ng rt-PA  with an equal volume of sample buffer  was loaded into wells. Gels were rinsed in 150 ml of 2.5% Triton X-100 (15 minutes) and incubated with 250 ml reaction buffer  .2bβ3, αvβ3, α5β1, αvβ1, αvβ5, α1β1, and α2β1  (100 ng) were immobilized in 96-well immunoplates for 16 hours at 4°C. Each well was blocked with skim milk and washed three times with 0.05% PBS-T. Saxatilin-Fc (~100 nM) was added to wells and incubated for 2 hours at room temperature. After washing three times with PBS-T, anti-HuFc antibody conjugated to HRP  (1:2000) was added to wells and incubated for 1 hour at room temperature. Nonadherent antibodies were removed by washing three times with PBS-T. Substrate was o-phenylenediamine tablets solubilized in phosphatidylcholine buffer; 100 μl was added to each well and incubated for 10 minutes. Absorbance at 490 nm was read on a spectrophotometer. We determined the Kd values from the titration curve using the simple linearization method [i gives a straight line and evaluates the apparent dissociation constant. The slope of the plot is 1/Kd.To investigate the interaction of saxatilin with integrins, Fc-tagged recombinant protein was generated because there was no available antibody against saxatilin. The vector encoded the Fc region of human IgG1 (~230 amino acids). Interaction with integrins was evaluated using enzyme-linked immunosorbent assay (ELISA). Integrins for αn method , the ploWe evaluated thrombolytic effects of other well-known plasminogen activators: rt-PA , urokinase-type PA (u-PA) , and the GP IIb/IIIa receptor antagonists abciximab  and tirofiban . Agents were administrated as 10% IV bolus injection with continuous infusion of the remaining 90% for 1 hour at 0.9, 1.8, 2.7, 4.8, 7.2, 9, or 18 mg/kg for rt-PA; 100, 500, 1000, 5000, 10,000, or 50,000 IU/kg for u-PA; 0.25, 0.5, 1, 2.5, 5, 10, 20, or 40 mg/kg for abciximab; and 0.5, 1.25, 2.5, 3.75, 5, or 10 mg/kg for tirofiban. An equal volume of normal saline was administered to control animals. in vivo, 8-week-old male ICR mice (31-36 g) were used. Saxatilin was conjugated with NHS-Rhodamine  according to manufacturer’s instruction. NHS-Rhodamine was dissolved in 10 mg/ml of dimethylsulfoxide . Saxatilin and NHS-Rhodamine were mixed and incubated at room temperature for 1 hour, and then non-reacted NHS-Rhodamine was removed with PD-10 Desalting Columns . Rhodamine-labeled saxatilin was diluted in 0.5 mg/ml and was administered intravenously at 5 mg/kg. To isolate serum, blood was collected from the jugular vein at 0, 5, 10, 20, 40, and 80 minutes after administration of Rhodamine-labeled saxatilin. The blood was incubated at room temperature for 30 minutes in dark and serum was separated by centrifugation at 10,000 rpm for 10 minutes. The serum was placed in black 96-well microplate . Fluorescence was acquired via LS 50B Fluorescence Spectrometer .To evaluate half-life of saxatilin Thrombosis was induced in the same manner as it was performed in the dose-response study. To avoid spontaneous recanalization, distal site of the CCA was ligated with Silkam 5-0 thread  immediately after achieving complete occlusion. After 1, 3, 6, 12, or 24 hours, the CCA ligation was gently removed and then 5 mg/kg saxatilin was administered intravenously (10% bolus and then continuous infusion of the remaining dose for 60 minutes). Thrombus at a time of complete occlusion was used as control fresh thrombus. Randomly selected five mice were used in each group. To compare thrombolytic efficacy between saxatilin and rt-PA on aged thrombus, 9 mg/kg rt-PA was administered in the same way at 3 or 6 hours after complete occlusion.To assess whether saxatilin administration causes thrombocytopenia or neutropenia, hematologic analyses were performed with blood collected from 5 mice after sham operation. Immediately after infusion of 5 mg/kg saxatilin or same volume of normal saline for 60 minutes, 1 ml of blood was collected in 10% EDTA via the lateral saphenous vein. Complete blood count (CBC) with differential counts was obtained using the automatic cell counter . Randomly selected four mice were used in each group. Immediately after infusion of 5 mg/kg saxatilin or same volume of normal saline for 60 minutes, bleeding time was assessed by transection of the tail at 1 cm from the tail tip using a sharp scalpel. Blood was blotted every 15 seconds and the time period from tail transection to cessation of bleeding was defined as the bleeding time. Bleeding times exceeding 600 seconds were considered as 600 seconds.Statistical analyses were performed using SPSS . Normality of distributions was verified using the Kolmogorov-Smirnov test. Differences among groups in dose response studies were compared with a one-way ANOVA test, followed by a post-hoc Tukey method. Differences between groups in hematological assessment were compared with a Mann-Whitney U test. Values were presented as a mean ± standard deviation (SD). P < 0.05 was considered significant.3. Two of the five mice by 30% and all of the five mice by 40% achieved complete occlusion, but all of them showed spontaneous recanalization during monitoring for 150 minutes except one mouse by 40%. All mice by 50% FeCl3 achieved complete occlusion, which was maintained without spontaneous recanalization during the monitoring . After 5 minutes of FeClexamined . CCA threxamined . Thrombu3 was applied showed relatively normal vascular structure, with normally shaped endothelial cells, smooth muscle cells, erythrocytes, and discoid platelets . Endothelial cells appeared empty with a loss of organelles. Some platelets were activated and aggregated and adhered to the luminal surface in mildly damaged regions . At the thrombus border, activated and aggregated platelets adhered to the damaged luminal surface, and no intact endothelial cells were observed . In severely damaged regions, platelet-rich thrombi containing erythrocytes completely occluded the vascular lumen . FeCl3 treatment caused a loss of vascular undulation with a flattened and disconnected arrangement of smooth muscle cells. The internal elastic lamina remained intact even in severely damaged regions.The regions farthest from where FeClSaxatilin treatment did not cause any notable changes in blood flow at a dose of 1 mg/kg (2.36 ± 0.78%) or 1.75 mg/kg (5.14 ± 2.92%) compared to the normal saline group (2.42 ± 1.07%) , 9A. BloHalf-life of saxatilin, which was measured using saxatilin labeled with NHS-Rhodamine, was 4.1 minutes . Saxatilin at 5 mg/kg was effective in dose-response studies, so this dosage was used to determine the optimal method of intravenous administration of saxatilin . Mean peDecreased thrombolytic effects were observed at different times according to administration method. Abrupt reocclusion was observed approximately 50 minutes after the first bolus injection in mice treated with double bolus injection, approximately 100 minutes after a total dose bolus injection, and approximately 110 minutes after a half-dose bolus injection with continuous infusion of the remaining half dose. Reocclusion was not observed with a bolus injection of 10% of the total dose and continuous infusion of the remaining dose .Effective recanalization was not observed in mice treated with normal saline, 1 mg/kg saxatilin, or 1.75 mg/kg saxatilin. Only two of five mice treated with 2.5 mg/kg of saxatilin and three of five mice treated with 3.75 mg/kg of saxatilin showed effective recanalization. Effective recanalization was observed in all mice treated with saxatilin at 5 or 10 mg/kg . The timEffective recanalization was also evaluated according to saxatilin administration method. All mice treated with 5 mg/kg saxatilin achieved effective recanalization except for two mice administered a bolus injection of the total dose. Time to effective recanalization was 2.86 ± 0.22 minutes in mice treated with a bolus injection of the total dose, 13.44 ± 26.31 minutes in mice that received a double bolus injection, 19.48 ± 25.94 minutes in mice that received a half-dose bolus injection followed by continuous infusion of the other half dose, and 13.92 ± 6.02 minutes in mice treated with a bolus injection of 10% of the total dose with continuous infusion of the remaining dose . Platelet aggregometry showed that saxatilin has a strong inhibitory effect on platelet aggregation . SaxatilExperiments using preformed thrombi showed dose-dependent disaggregating effects of saxatilin . ADP-indBoth fibrin and fibrinogen zymograms showed clear bands of rt-PA. However, saxatilin did not demonstrate a clear fibrinolysis band, which suggested that saxatilin had no fibrinolytic activity .2bβ3 with the highest affinity (Kd = 6.8 × 10-12 M). Saxatilin-Fc also showed high affinity for integrins αvβ3 (Kd = 2.0 × 10-11 M), αvβ5 (Kd = 3.5 × 10-9 M), α5β1 (Kd = 4.4 × 10-9 M), and αvβ1 (Kd = 5.9 × 10-9 M). Saxatilin-Fc did not bind to integrins α1β1 or α2β1.We investigated the binding affinity of saxatilin-Fc to various integrins . SaxatilDose-dependent effects of thrombolytic agents were determined by calculating the area under time-flow curves . After rMean percentages of blood flow compared to baseline blood flow were 94.50 ± 20.47% in the group that received 5 mg/kg saxatilin at 1 hour; 17.71 ± 27.27% at 3 hours; 4.48 ± 1.37% at 6 hours; 10.67 ± 6.06% at 12 hours; and 4.51 ± 3.04% at 24 hours . Mean peThrombolytic effects of saxatilin were evaluated using 71 animals. Two animals had bleeding at the cervical incision site, and one died of bleeding approximately 90 minutes after a bolus injection of saxatilin. The two animals with bleeding complications had received a single bolus dose of 5 mg/kg. None of the mice in the other groups showed bleeding complications. No significant differences in hematologic parameters were observed between normal saline-infusion group and 5 mg/kg saxatilin-infusion group . The taiin vivo and in vitro. The thrombolytic effects of saxatilin were dose dependent. Time to recanalization decreased with increasing saxatilin dosage. Reponses to saxatilin varied with administration method. Administration of the total dose in a bolus resulted in fast effective recanalization (approximately 3 minutes). However, reocclusion was frequently observed, and was also observed in mice treated with a bolus injection of 50% of the total dose. The regimen of a bolus injection of 10% of the total dose with continuous infusion of the remaining dose for 60 minutes resulted in a longer time to effective recanalization than treatment with a high-dose bolus injection. However, this regimen did not show reocclusion. These findings might result from the relatively short half-life of saxatilin, which was 4.1 minutes in mice.We demonstrated that saxatilin has strong thrombolytic effects 2bβ3, αvβ3, α5β1, αvβ1, and αvβ5, with the highest affinity for α2bβ3. Administration of GP IIb/IIIa antagonists not only prevents thrombus formation, but also disaggregates preformed fresh thrombi through competitive binding with the platelet GP IIb/IIIa receptor to the RGD domain of fibrin or fibrinogen [2bβ3 does not act alone in mediating platelet adhesion/aggregation, but as part of a synergistic, multiple integrin-ligand association during platelet adhesion and aggregation [2bβ3, saxatilin showed high affinity for αvβ3 and α5β1, which mediate platelet function along with α2bβ3 [5β1 and α2bβ3 [5β1 and α2bβ3 share a fibrinogen-binding site [5β1 is involved in contraction of fibrin clots as well as platelet adhesion [vβ3 is involved in thrombus formation by adhering not only to ligands for α2bβ3 , but also to collagen [The findings of this study indicated that saxatilin had thrombolytic effects on platelets but not fibrin/fibrinogen. Saxatilin is an RGD-containing protein. RGD-containing proteins from snake venom are characterized by strong conservation of the tripeptide RGD and by disulfide bond arrangements. These structural features might be critical for potential inhibition of platelet adhesion/aggregation and antagonism of integrin-mediated adhesion . In an ibrinogen . Saxatilregation . In addith α2bβ3 . Strong nd α2bβ3 . Integriing site . Integriadhesion . Integricollagen ,36. Thus50 of saxatilin (400 mg/kg) in ICR mice [vβ5 [vβ5 in our study; thus, saxatilin might be beneficial as an αvβ5 antagonist for preventing bleeding complications induced by vascular leakage. Additionally, αvβ5 inhibition following administration of saxatilin might be advantageous for stroke outcomes. In β5-deficient mice, infarction volume and blood-brain barrier breakdown is dramatically reduced after permanent middle cerebral artery occlusion [Saxatilin appears to be safe because its effective dose was low and it rarely caused bleeding complications. In addition, saxatilin did not cause any notable changes in hematological parameters including platelet and neutrophil counts compared with control group. The most efficient dose in our study (5 mg/kg) was much lower than the known LDICR mice . In tailice [vβ5 . Saxatilcclusion . We also determined the effect of saxatilin on aged thrombus. Thrombolytic effects of saxatilin decreased as thrombus age increased. Decreased thrombolytic effects in aged thrombi were also seen in rt-PA. While effects of thrombolytic drugs on aged thrombi have not well known, our findings suggest that effects of drugs acting on platelets as well as fibrinogen may be decreased as thrombus age increases and that reducing time from onset to treatment is important to enhance effects of the thrombolytic drugs.3 than previous studies (2.5–65%) [3 such as 10-40%, blood flow restored spontaneously during 150 minutes of monitoring period after initially complete occlusion. Low concentrations of FeCl3 are usually used to evaluate thrombosis mechanisms or the effects of antithrombotic drugs, which do not require the long-term monitoring needed to evaluate thrombolytic effects. Scanning and transmission electron microscopy showed that 50% FeCl3 causes a loss of undulation and severe damage to the endothelium and smooth muscle but did not damage internal elastic lamina. These findings were consistent with previous observations using a lower dose of FeCl3 (7.5-20%) [Our methods had distinct features compared to other experimental models evaluating thrombolytic effects. First, to produce arterial thrombosis, we used a smaller filter paper saturated with a higher concentration (50%) of FeCl2.5–65%) ,24,38,39in vivo efficacy of new thrombolytic drugs. Second, the assessment methods we used were different from previous studies in which recanalization is typically assessed to determine thrombolytic effects. Our study determined and calculated the degree of flow restoration on a minute-by-minute basis using ultrasound blood flow measurements. Histological examination showed that blood flow measurements were a good representation of the degree of recanalization and thrombus resolution. The assessment method we used provided quantitative data on degree of recanalization, time to effective recanalization, and real-time occurrence of reocclusion. Our method also enabled group comparisons as well as quantitative data on each animal. We found a dose-dependent thrombolytic effect for rt-PA. The effective dose of rt-PA in rodents is about 10-fold higher than for humans because of different PA systems -42. Therin vitro thrombolytic effects. Third, thrombolytic effects were determined using platelet aggregometry. Platelet aggregometry has been used to test inhibition of thrombus formation and is typically used to assess platelet anti-aggregating drugs. However, we used platelet aggregometry to assess platelet disaggregation after administering a thrombolytic drug to preformed thrombi. We found that platelet aggregometry could also be used to assess In conclusion, disaggregation of platelets from fibrin is a potential approach to dissolving thrombi ,12. Seve


Sleepiness and cognitive dysfunction are recognized as prominent consequences of sleep deprivation. Experimentally induced short-term sleep fragmentation, even in the absence of any reductions in total sleep duration, will lead to the emergence of excessive daytime sleepiness and cognitive impairments in humans. Tumor necrosis factor (TNF)-α has important regulatory effects on sleep, and seems to play a role in the occurrence of excessive daytime sleepiness in children who have disrupted sleep as a result of obstructive sleep apnea, a condition associated with prominent sleep fragmentation. The aim of this study was to examine role of the TNF-α pathway after long-term sleep fragmentation in mice.The effect of chronic sleep fragmentation during the sleep-predominant period on sleep architecture, sleep latency, cognitive function, behavior, and inflammatory markers was assessed in C57BL/6 J and in mice lacking the TNF-α receptor (double knockout mice). In addition, we also assessed the above parameters in C57BL/6 J mice after injection of a TNF-α neutralizing antibody.Mice subjected to chronic sleep fragmentation had preserved sleep duration, sleep state distribution, and cumulative delta frequency power, but also exhibited excessive sleepiness, altered cognitive abilities and mood correlates, reduced cyclic AMP response element-binding protein phosphorylation and transcriptional activity, and increased phosphodiesterase-4 expression, in the absence of AMP kinase-α phosphorylation and ATP changes. Selective increases in cortical expression of TNF-α primarily circumscribed to neurons emerged. Consequently, sleepiness and cognitive dysfunction were absent in TNF-α double receptor knockout mice subjected to sleep fragmentation, and similarly, treatment with a TNF-α neutralizing antibody abrogated sleep fragmentation-induced learning deficits and increases in sleep propensity.Taken together, our findings show that recurrent arousals during sleep, as happens during sleep apnea, induce excessive sleepiness via activation of inflammatory mechanisms, and more specifically TNF-α-dependent pathways, despite preserved sleep duration. Sleep fragmentation (SF), unlike prolonged sleep deprivation, is a common consequence of many sleep disorders in humans ,2, of naIn the brain, expression of Homer1a is increased after sleep loss, suggesting a role for sleep in the regulation of intracellular calcium homeostasis, particularly in protection and recovery from the calcium-pool changes induced by the prolonged neuronal activation imposed by extended wakefulness . AccordiSF is one of the hallmark characteristics of sleep apnea. Experimentally induced short-term SF, even in the absence of any reductions in total sleep duration, will lead to the emergence of excessive daytime sleepiness and cognitive impairments in humans ,6. HowevThe experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the National Institutes of Health Guide in the Care and Use of Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.ad libitum.Male C57BL/6 J mice TNF-α receptor double knockout mice , matched for age and weight (20–25 g), were housed with a 12 hour light/dark cycle (07.00 to 19.00 hours) at a constant temperature (26 ±1°C), and allowed access to food and water The SF device used to induce SF in rodents has been previously described  , which allows simultaneous monitoring of two biopotential channels (temperature and locomotor activity) were chronically implanted to record an electrencephalogram (EEG) from the frontal area and an electromyogram (EMG) from the superior nuchal muscle  .Sleep recordings, SF, and sleep scoring were performed as previously described ,44. BehaFor the neutralizing antibody studies (TNF-α antibody (Ab) + SF), we gave the C57BL/6 J mice an intraperitoneal injection of either vehicle  or TNF-neutralizing antibody  10 minutes before starting the SF procedures) .The Morris water maze was used to assess spatial reference learning and memory, and working memory. The maze protocol is similar to that described by Morris , with mo2 within 20 seconds and were pulverized on LN2 for nuclear acid extraction using ice-cold 5% trichloroacetic acid (TCA). The mixture was sonicated with a probe sonicator for 10 pulses and separated by centrifugation at 10,000 g and 4°C for 15 minutes. The supernatant was collected and neutralized with Tris buffer . ATP levels were then measured using a commercial kit )ATP Bioluminescence Assay Kit CLS II; Roche Applied Science, Indianapolis, IN, USA). Protein concentrations in the supernatant were determined using a protein assay kit  to normalize ATP levels.Cortical tissues were snap-frozen in liquid N2 and immediately homogenized in 1% SDS preheated to 92°C. The homogenate was separated by centrifugation at 14,000 g for 15 minutes, and the supernatant containing total cellular proteins was collected. This method has been shown previously to effectively inhibit activation of phosphorylation and dephosphorylation processes associated with many routine protein preparation protocols [Snap-frozen cortical tissues were pulverized on liquid Nrotocols ,53. The Nuclear extracts from hippocampal tissues harvested from mice exposed to SF or control sleep conditions were prepared , in accordance with the manufacturer’s instructions, using a dounce homogenizer. For the DNA binding assays, six animals per condition were studied. CREB nuclear binding was assessed using . This enzyme-linked immunosorbent assay employs an antibody that selectively recognizes the phosphorylated epitope of CREB when the latter is specifically bound to a specific oligonucleotide coated onto a 96-well plate. Specificity of the binding was further monitored by competition with free wild-type and mutated oligonucleotides. Samples were assessed by a spectrophotometer at 450 nm.The mRNA expression  was determined by quantitative RT-PCR using commercially available specific primers.TNF-α brain cortical levels were measured in triplicate using a commercially available ELISA assay in accordance with the manufacturer’s instructions . This method has a minimum detection level of 0.25 pg/ml with intra-assay and inter-assay coefficients of variability of 7.4% and 7.8% respectively, and a dynamic linear range of 2.2 to 2,500 pg/ml. Assays were deemed acceptable if triplicate values were within 10% of each other.2O2 for 30 minutes, washed in PBS, and blocked with a buffer of PBS containing 0.4% Triton X-100, 0.5% tyramide signal amplification (TSA) blocking reagent  containing 10% normal goat serum  for 1 hour. Sections were then serially incubated with either TNF-α  or pCREB antibody  at 40 C for 48 hours with a biotinylated anti-rabbit antibody (1:600 in the PBS/0.5% TSA/10% NGS blocking reagent described above) , and then with streptavidin-horseradish peroxidase (diluted 1:100 in the blocking reagent as before) followed by tetramethyl rhodamine tyramide diluted 1:50 for 2 minutes (Perkin Elmer Life Sciences). Sections were subsequently incubated with serum raised against the neuronal marker Neu-N , followed by a biotinylated anti-rat antibody (1:200 l Vectastain Elite ABC Kit), and by fluorescein tyramide reagent (1:50) for 3 minutes (Perkin Elmer Life Sciences). Sections were then washed in PBS and mounted onto glass slides. In general, 20 sections/animal were visualized using both fluorescent and confocal microscopes.Anesthetized mice were perfused transcardially with 0.9% NaCl followed by 4% formalin, then the brains were removed, fixed in 4% formalin, and cryoprotected with 30% sucrose. Coronal sections (40 um) were incubated in 0.3% Hpost hoc Tukey tests. The same statistical approaches were used to compare probe trial, reference memory, EPM, and FST. For all comparisons, P < 0.05 was considered significant. For behavioral test assessments, all the experimental conditions, the data were divided into six blocks . We used a multivariate MANOVA model  that included latency, path length, and swim speed, and two between-group factors: 1) Groups (four levels): Sleep control (SC)-C57BL6/J, SC-TNF-KO and TNF-KO mice exposed to 15 days SF (SF-TNF-KO) ; and 2) condition (two levels): SC or SF. All F statistics are reported using Pillai’s Trace. The interaction of three different factors  were determined using this mixed-model repeated-measures MANOVA. The biochemical assays were analyzed using one-way ANOVA.To elucidate the nature of identified interactions between control baseline sleep characteristics and those emerging after 15 days of SF in C57BL/6 J mice and TNF-α receptor knockout mice, the data were analyzed by one-way repeated-measures ANOVA. First, overall statistical significance between the treatment groups (baseline and SF) was determined for the 24-hour period, followed by post-hoc Tukey tests, as needed. Delta power during SWS, wake episodes, and the latency of SWS after each episode of wake were also treated with similar statistical approaches. Similar statistical approaches were used to compare the entire training period between the treatment groups. In addition, two-way repeated measures ANOVA were used to analyze each trial blocks, followed by P < 0.001), indicating that SF had influenced state . EEG monitoring during the 12-hour LP (07.00 to 19.00 hours) showed that the C57BL6/J mice were awake for 38.52 ± 4.11% of the time; after 15 days of SF, they spent 42.66 ± 0.85% of the time awake. Analysis of SWS and REM sleep during LP showed that the animals spent 52.81 ±9.90% and 8.68 ±1.73% of the time in SWS and REM sleep respectively, during baseline, which decreased to 50.39 ±0.86% and 6.9 ± 0.33%, respectively, after 15 days of SF. During the dark period (DP), there was no significant variation in wake, SWS, or REM sleep in animals subjected to 15 days SF, who had values of 58.27 ± 0.78, 36.9 ± 0.66 and 4.83 ± 0.13%, respectively, compared with baseline values of 64.59 ± 3.66, 32.01 ± 3.51 and 3.4 ± 0.35%  showed significant differences between baseline and SF  compared with baseline (13.38 ±2.08 episodes per 2 hours) , indicating that SF had significant effects on SWS latency. The latency to SWS was significantly reduced throughout the LP during the SF procedures. On average, the control animals had a latency of 368.16 ±72.73 seconds to SWS at baseline, compared with 73.14 ±2.86 seconds on day 15 (P < 0.0001), indicating progressive increases in sleep propensity. Immediately after cessation of SF procedure, the latency to SWS during DP had a tendency to return to baseline levels. However, for a total period of 24 hours (from 0700 to 0700 hours the next day), the latency to SWS remained significantly lower after SF (230.47 ±8.88 seconds) compared with baseline  .Delta power during SWS of the EEG remained unchanged after SF  and path length, , indicating that SF adversely affected task performance. Significant differences in latencies were seen during blocks 2 , 3 , 4 , 5  and 6 . There were no significant differences in block 1. Repeated measures ANOVA showed significant differences in path lengths during blocks 2 , 3 , 4 , 5  and 6 , with no significant differences in block 1. There were no significant differences in swim speed . The magnitude of impairment was greatest in SF-C57BL6/J mice  and path length .On a standard place-discrimination task, C57BL6/J mice exposed to 15 days of SF (SF-C57BL6/J) exhibited longer latencies and path-lengths to locate the hidden platform compared with control mice (SC-C57BL6/J),  and a significantly higher percentage of time spent immobile  compared with the SC-C57BL6/J sleep controls  and in the number of entries into the closed arm    Figure B. Furthe) Figure C. Howeve) Figure A andAMP ) Figure B after S) Figure C.F = 7.639; P < 0.004)  but also in protein levels  . During DP, these percentage were 62.13 ± 0.81% and 56.56 ± 0.74%, respectively (P < 0.05). The percentage of time the TNFR-KO mice spent in SWS was 58.1 ± 1.07% before SF and 56.09 ± 1.13 after 15 days of SF (P < 0.05) for LP, and 34.64 ± 0.87% and 36.9 ± 0.66% for DP (P < 0.05). The time spent in REM was 7.68 ± 0.77% before SF and 6.6 ± 0.44% after SF during LP, and 3.47 ± 0.88% and 4.83 ± 0.13, respectively, during DP (p < 0.05). There were no changes in time spent in wake, SWS and REM sleep during the 24 hour period. After 15 days of SF, SWS latency was markedly reduced in the wild-type controls compared with the TNFR-KO mice  , the percentage change from baseline mean showed only a trend towards increase in delta.EEG monitoring of TNFR-KO mice during 12 hours of LP showed that they were awake for 34.71 ± 1.14% of the time without SF, which increased to 42.66 ± 0.85% after 15 days of SF  and path length, , indicating that SF adversely affected task performance for C57BL6/J mice only. Significant differences in latencies were seen during blocks 2 , 3 , 4 [F = 3.35; P < 0.03) and 6 . There were no significant differences in blocks 1 or 5. Repeated measures ANOVA showed significant differences in path lengths during block 6 , with no significant differences in other blocks. In the reference memory tests, SF-C57BL6/J mice exhibited significant deficits in memory retention in both latency  and path length . However, the SF-TNF-KO mice performed similarly to sleep controls  showed that SC TNF KO and SC C57BL6/J mice required significantly less time than their SF littermates to find the hidden platform in a Morris water maze  indicated that as the training progressed, the SC TNF KO and SC C57BL6/J mice reached the hidden platform and covered the shortest distance compared with their littermates exposed to SF in a Morris water maze  exhibited longer latencies and path lengths to locate the hidden platform compared with the sleep controls (SC- C57BL6/J) or the SC-TNFR-KO or SF-TNF-KO mice   compared with all other treatment groups, including SF-TNF-KO mice  . During the 12 hours of LP, untreated mice spent 57.27 ±2.7% of the time in SWS, which reduced significantly after 15 days of TNF-α Ab + SF (53.88 ±1.27%) (P < 0.02).A similar trend was also seen for REM sleep . There was no significant difference in SWS latencies between saline (−86.857 ±21.45%) and TNF-α Ab + SF (−96.53 ±18.9%) mice on day 1 during LP. On day 15 SF, the percentage difference in SWS latency was reduced in saline-treated mice (−223.84 ±35.09%) compared with mice treated with TNF-α Ab + SF  , indicating that TNF-α Ab injection prevented the SF-associated excessive sleepiness. Overall comparison of theta power during quiet wakefulness, a marker of sleepiness, showed a significant increase in C57BL/6 J after 15 days of SF  . These mice were awake for similar lengths of time at baseline (34.53 ±2.62%) and after 15 days of TNF-α Ab + SF (34.83 ±0.39%), and the difference was not significant  and path length  compared with all the other groups, indicating that TNF-α Ab injection prevented SF-associated behavioral deficits.In the reference memory tests, SF-C57BL6/J mice injected with vehicle  exhibited significant deficits in memory retention Figure E in bothet al. [In this study, we aimed to investigate the effects of SF on mice, and found that SF mimicking the recurrent arousals associated with sleep apnea induces both increased sleepiness and neurocognitive deficits. In addition, we sought to investigate the role of the TNF-α mediated pathway in SF and its relationship to measures of sleep architecture and cognitive behavior. In healthy people, serum inflammatory markers have been associated with abnormal sleep architecture . Patientet al.  suggesteDepressive and anxiety symptoms are common in patients with OSA -68, and We found that, in contrast to SD, SF does not curtail sleep duration and also does not induce reductions in ATP or intracellular energy sources, as evidenced by the absence of AMPK phosphorylation after SF. However, the SF-induced cognitive and behavioral alterations were associated with reduced CREB phosphorylation and transcriptional activity in the hippocampus, and with increased expression of PDE4. Because sleep deprivation has been associated with reduced brain ATP levels and increased phosphorylation of AMPK -76, and In the present study, we found that mice that are periodically awakened during their sleep period exhibit sustained increases in sleep propensity in the presence of globally preserved sleep patterns and duration. Indeed, sustained implementation of SF results in normalization of the total duration of sleep and waking, and in delta power. However, preserved sleep duration in the context of chronic SF is not accompanied by normal sleep latencies, thereby indicating increased sleepiness in the absence of sleep curtailment. This finding is further corroborated by the increases in theta frequency during quiet waking periods during the DP interval. In other words, normalization after long-term SF of the commonly used and widely accepted standard measures of sleep integrity did not abrogate the increases in sleepiness, suggesting that perturbations in sleep continuity play a crucial role in the maintenance of wake drive and of wake-related cognitive, mood, and behavioral tasks.Based on previous evidence suggesting that TNF-α has important regulatory effects on sleep, and that TNF-α plays a role in the occurrence of excessive daytime sleepiness in children who have disrupted sleep as a result of OSA, a condition associated with prominent SF -82, we eAlthough these findings are essentially confirmatory of our previous study , we furtA major challenge in the field of sleep research has been to determine how sleep disruptions resulting from an extensive list of perturbations included in everyday living, or those induced by illnesses such as sleep apnea, can affect cognitive function and mood. The findings presented here define a crucial role for TNF-α in this context, and further support the link between sleep and immunity , even inAMP, Adenosine monophosphate; ANOVA, Analysis of variance; ATP, Adenosine triphosphate; BL, Baseline; CREB, Cyclic AMP response element-binding protein; DP, Dark period; EEG, Electroencephalogram; EMG, Electromyogram; EPM, Elevated plus maze; FFT, Fast Fourier transform; IL-1β, Interleukin-1beta; I-L6, Interleukin-6; LP, Light period; LTP, Long-term potentiation; MANOVA, Multivariate analysis of variance; NADPH, Nicotinamide adenine dinucleotide phosphate; PDE, Phosphodiesterase; REM, Rapid eye movement; SF, Sleep fragmentation; TNFR KO, TNF receptor double knockout mice; TNF-α, Tumor necrosis factor-alpha.The authors have no competing interest to declare in relation to this manuscript.DG and YW conceived and designed the study;VR, DN, SZ, FH, NK, and DG were responsible for analysis and interpretation;, and VR, DN and DG for drafting the manuscript for important intellectual content. VR, DN, SZ, NK, FH, FK, RL, AC took part in the experimental procedures. All authors read and approved the final manuscript.


Traumatic brain injury (TBI) sets in motion cascades of biochemical changes that result in delayed cell death and altered neuronal architecture. Studies have demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) effectively reduces apoptosis following a number of stimuli. The Wnt family of proteins, and growth factors are two major factors that regulate GSK-3 activity. In the absence of stimuli, GSK-3 is constitutively active and is complexed with Axin, adenomatous polyposis coli (APC), and casein kinase Iα (CK1α) and phosphorylates ß-Catenin leading to its degradation. Binding of Wnt to Frizzled receptors causes the translocation of GSK-3 to the plasma membrane, where it phosphorylates and inactivates the Frizzled co-receptor lipoprotein-related protein 6 (LRP6). Furthermore, the translocation of GSK-3 reduces ß-Catenin phosphorylation and degradation, leading to ß-Catenin accumulation and gene expression. Growth factors activate Akt, which in turn inhibits GSK-3 activity by direct phosphorylation, leading to a reduction in apoptosis.Using a rodent model, we found that TBI caused a rapid, but transient, increase in LRP6 phosphorylation that is followed by a modest decrease in ß-Catenin phosphorylation. Phospho-GSK-3β immunoreactivity was found to increase three days post injury, a time point at which increased Akt activity following TBI has been observed. Lithium influences several neurochemical cascades, including inhibiting GSK-3. When the efficacy of daily lithium was assessed, reduced hippocampal neuronal cell loss and learning and memory improvements were observed. These influences were partially mimicked by administration of the GSK-3-selective inhibitor SB-216763, as this drug resulted in improved motor function, but only a modest improvement in memory retention and no overt neuroprotection.Taken together, our findings suggest that selective inhibition of GSK-3 may offer partial cognitive improvement. As a broad spectrum inhibitor of GSK-3, lithium offers neuroprotection and robust cognitive improvement, supporting its clinical testing as a treatment for TBI. Both clinical and experimental studies have shown that the pathophysiology of traumatic brain injury (TBI) is complex and involves both primary and secondary injuries. Primary pathologies develop extremely rapidly after the traumatic event, and are typically not amenable to pharmacological interventions. In addition to causing immediate physical damage to the brain, the primary injury sets in motion cascades of molecular, cellular, genomic and metabolic processes that result in secondary injury Glycogen synthase kinase3 (GSK-3) was originally identified as a regulator of glycogen metabolism. Its role has been expanded to include regulation of protein synthesis, cell proliferation, cell differentiation, microtubule dynamics, cell motility and apoptosis In the present study, we examined the consequences of TBI on GSK-3 activity and post-TBI administration of GSK-3 inhibitors on neurocognitive outcome. Treatment of injured animals with lithium, a multi-target drug that inhibits GSK-3, offered significant neuroprotection and improved learning and memory in brain injured animals. However, the GSK-3-selective small molecule inhibitor SB-216763 resulted in improved motor function, but only modest cognitive enhancement that occurred in the absence of visible neuroprotection.Guide for the Care and Use of Laboratory Animals. Protocols were designed to minimize pain and discomfort during the injury procedure and recovery period.All experimental procedures were approved by the Animal Care and Use Committee of The University of Texas Health Science Center at Houston (protocol# AWC-08-046),and were conducted in accordance with the recommendations provided in the 33/37), total ß-Catenin, phospho GSK-3β (Ser9), total GSK-3β, phospho-Erk1/2 , total Erk1/2antibodies, phospho-Akt (Ser473), total Akt, phospho-LRP6 (Ser1490), and total LRP6 were purchased from Cell Signaling Technology . Antibodies to NeuN were obtained from Millipore .Male Sprague-Dawley rats (275–300 g) were purchased from Charles River Laboratories . Lithium chloride and SB-216763 was purchased from Sigma Aldrich  and Tocris Bioscience , respectively. Antibodies to phospho ß-Catenin  device was used to cause brain injury as previously described 2/N2O mixture, mounted a stereotaxic frame, and a midline incision was made. Bilateral 6-mm craniectomies were produced midway between the bregma and lambda with the medial edges of the craniectomies 1-mm lateral to the midline. Rats received a single impact (2.7 mm deformation) on the right parietal lobe with an impact velocity of 6 meters/sec. Core body temperature was maintained at 37-38°C by use of a heating pad. The animals were given time to recuperate in a warming chamber before being returned to their home cages. Animals were weighed daily after the injury for the first 3 days, then weekly thereafter.All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the recommendations provided in the Lithium chloride was dissolved in sterile saline to a final concentration of 25 mg/ml. For testing the influence of lithium chloride on cognitive function following TBI, animals were injured then injected (s.cu.) with 0.5 ml (1 mEq/kg lithium) or an equal volume of vehicle 30 minutes post-injury, and every 24 hours thereafter for the first 5 days post-injury (n = 10/group). For biochemical analysis, naïve rats were given an s.cu. injection of lithium  or vehicle once a day for 5 continuous days (n = 3/dose). Three hrs after the last injection, rats were killed by decapitation and hippocampi removed for the preparation of protein extracts. For single dose studies, 1.0 mEq/Kg lithium was injected, and the animals killed 3 hr later. The selective GSK-3β inhibitor, SB-216763, was dissolved in DMSO to a stock concentration of 8.33 mg/mL and further diluted in 60% PEG400 and 30% distilled water for a final concentration of 0.83 mg/mL. For testing the influence of GSK-3β inhibition on cognitive function following TBI, animals were i.p. injected with 5 mg/kg SB-216763 or an equal volume of vehicle 30 min following injury and every 24 hours thereafter for the first 5 days after injury (n = 10/group). For biochemical analysis, uninjured rats were given a single i.p. injection with SB-216763 (2.5 or 5 mg/kg) and killed 1 hr later (n = 3/dose).2PO4, 3 mM KCl, 124 mM NaCl, 10 mM dextrose, 26 mM NaHCO3 and 2 mM MgCl2) containing phosphatase inhibitors . Tissues were homogenized (20 strokes) in 10 volumes of a buffer containing 10 mM Tris pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.5 mM DTT, phosphatase inhibitors (0.1 µM okadaic acid and 1 mM sodium orthovanadate) and protease inhibitors (1 mM PMSF and 10 µg/ml leupeptin) using a motorized teflon-glass homogenizer. The samples were immediately aliquoted and frozen at −80°C.Hippocampal tissues were quickly removed while submerged in ice cold artificial cerebral spinal fluid .Hippocampal tissue extracts were sonicated (5 pulses of 1 second each) using a Sonics Vibracell sonicator  and a 0.4 mm diameter probe. The amount of protein in each sample was determined by a Bradford assay using bovine serum albumin (BSA) as the standard. Samples were denatured at 95°C for three minutes in 1x NuPage SDS sample buffer . Equal amounts of protein were loaded, electrophoresed, and transferred to Immobilon-P membranes  using the NOVEX X-Cell II system  and the buffers provided by the vendor. Membranes were blocked overnight in 5%BSA in TBST, followed by a three-hour incubation in primary antibodies (0.1–0.5 µg/ml in TBST containing 2% BSA) at room temperature. Membranes were then washed in TBST and incubated at room temperature with alkaline phosphatase (AP)-conjugated secondary antibodies for one hour as recommended by the vendor . Following extensive washing, immunoreactivity was detected using a chemiluminescence system and quantified using All behavioral tests were conducted by an experimenter blind to the treatment groups. A vestibulomotor  and a motor skill task (paw placement) were used to determine animals' motor performance on days 1–4 post-injury. For beam balance, rats were pre-assessed by placing them on a narrow wooden beam (1.5 cm wide) and measuring the duration they remained on the beam for up to 60 seconds. Animals were given repeated training until capable of balancing on the beam for the entire 60 sec period for 3 consecutive trials. Following injury, animals were given three daily trials during which the length of time spent on the beam was recorded. Paw placement was evaluated by placing the animal on a wire grid (opening size of 2×2 cm) and counting the number of foot faults out of a total of 50 steps. A foot fault was defined as when a front paw misses and appears below the plane of the grid. Paw placement was repeated three times to give an average daily score.Rats were tested for their cognitive performance using the standard hidden platform version of the Morris water maze Adobe Illustrator, with the area of the resultant outlines quantified by Image J, from the National Institutes of Health. Contusion volume was calculated using the equation A1(0.5X1)+A2(0.5X1 +0.5X2)+An-1(0.5Xn-1 +0.5Xn)+An(0.5Xn) where A is the area (mm2) of the contusion for each slice, and X is the distance (mm) between two sequential slices. Once the contusion volume had been calculated for each animal, the blind code was broken and group differences assessed.Following the completion of the behavioral studies, approximately 28 days following the injury, animals (n = 10/group) were deeply anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed, post-fixed overnight in perfusant, then cryoprotected in a 30% sucrose solution. Cortical tissue loss was estimated essentially as described previously For post-TBI histological evaluation, tissue sections generated for the measurement of cortical contusion volume were used for immunohistochemistry. Free-floating slices were incubated overnight in primary antibody (0.5–1.0 µg/ml) in TBS containing 2% BSA and 2.5% normal goat serum. After extensive washing, immunoreactivity was detected using species-specific secondary antibodies coupled to Alexafluors. These fluorescently-labeled tissue sections were evaluated by a blind observer to identify any potential differences between the treatment groups. As visible neuroprotection was detected following lithium administration, stereological cell counts were carried out. For cell counts, NeuN immunostaining was performed using a colorimetric detection system consisting of a secondary antibody conjugated to horseradish-peroxidase and diaminobenzadine (DAB) as the chromagen. This was done to avoid the photobleaching often associated with high magnification imaging of fluorescent immunohistochemistry.Stereo Investigator  3 for each section was obtained from the estimated cells divided by the contour volume. The number of cells/mm3 for each animal was calculated as the average of the number of cells/mm3 from each section examined.A blind counting methodology was employed for determination of NeuN positive cells in the CA3 layer of the ipsilateral hippocampus in lithium chloride- and vehicle-treated animals (n = 5/group). Every tenth section through the ipsilateral dorsal hippocampus was processed for NeuN immunoreactivity using colorimetric detection as described above. NeuN positive cells within the CA3 subfield were counted using the optical dissector technique using post-hoc test. For data that did not pass a Shapiro-Wilk normality test, appropriate non-parametric analysis was performed. Data were considered significant at p≤0.05 and presented as Mean ± Standard Error of the Mean (S.E.M.).For evaluation of behavioral data, repeated measures analyses of variance (two-way or one-way as appropriate) and t-tests were utilized to determine statistical differences. A Holm-Sidak method for multiple comparisons post-hoc test was used to determine data points with significant differences. Dose response western data were evaluated using a one-way ANOVA followed by a Dunnet's 1490. This site has been shown to be phosphorylated in response to GSK-3 translocation to the LRP6 signalosome  = 4.297; P = 0.006). The total levels of LRP6 did not significantly change at any of the time points examined  = 0.587; P = 0.710). As described above, GSK-3 is also inhibited following phosphorylation by Akt. Previous studies have reported that cortical impact injury results in increased Akt phosphorylation (on Thr308) and activity 3-day post-injury 9 of GSK-3ß increases after injury, reaching significance by the 3-day time point  = 7.335; p<0.001). This is consistent with a previous study which showed that GSK-3 phosphorylation is increased in the hippocampus for at least 24 hr after a weight drop model of TBI  = 0.622; p = 0.684).To determine the influence of TBI on GSK-3 activity, western blots were performed to examine the phosphorylation of LRP6, GSK-3ß, and ß-Catenin in hippocampal extracts prepared at various time points following injury (n = 5/time point). 33/37 is reduced, leading to its nuclear accumulation and enhanced gene expression. To assess if the TBI-associated decreases in GSK-3 activity are associated with changes in ß-Catenin phosphorylation and levels, western blots were again carried out. 33/37 of ß-Catenin acutely following injury  = 4.948; p = 0.003). Although a significant decrease in ß-Catenin phosphorylation was observed by 3 hr post-injury, no concurrent increase in the total levels of this protein was observed  = 1.208; p = 0.336). No changes in either the phosphorylation or total levels of ß-Catenin were observed at the time point at which GSK-3ß phosphorylation was observed.Upon inactivation by either the Wnt-LRP6 or Akt cascades, GSK-3-mediated phosphorylation of ß-Catenin on Ser = 5.326, p = 0.011). No significant difference was detected in the total levels of GSK-3ß  = 1.770, p = 0.200)  = 49.283, p<0.001), with maximal accumulation observed at 1.0 mEq lithium (t-test: 0.956), total GSK-3 levels (Student's t-test: 0.130) or ß-Catenin levels (Student's t-test: 0.587). Based on these results, we tested the consequences of daily post-injury administration of 1.0 mEq lithium for the first 5 days after injury using a series of behavioral and cognitive tasks.Inhibition of GSK-3 has been shown to reduce neuronal death triggered by a number of stimuli = 0.200) . Althoug lithium . Since m = 3.267, p = 0.087). In addition, neither ipsilateral  = 2.033, p = 0.170;  = 1.589, p = 0.222;  = 14.324, p = 0.001). All animals had normal vestibulomotor and motor function by day 7 post-injury (data not shown), and had returned to their pre-injury weight.The influence of post-injury lithium injection on vestibulomotor and motor function was tested on days 1–4 post-injury using the beam balance and foot fault tasks, respectively . Rats we = 4.082, p<0.001;  = 11.50, p<0.001) while the saline-injected controls did not  = 0.62, p = 0.619)  = 6.58, p<0.001) with a significant difference in the time each group spent searching in the target quadrant (quadrant I). Short-term memory was found to be improved in animals treated with lithium, as evidenced by reduced latency to cross the original platform location (Student's t-test p = 0.034) and more number of platform crossings (Student's t-test p = 0.004) , the rats treated with lithium had learning curves that were significantly different from their vehicle-injected counterparts (significant interaction by repeated measures two-way ANOVA: F= 0.619) . Compari= 0.004)  in a proStereo Investigator. These counts revealed that animals treated with lithium had significantly more ipsilateral CA3 pyramidal neurons than those counted in vehicle-injected animals  = 8.833, p = 0.016), suggesting decreased GSK-3 activity. Associated with this was a significant decrease in the phosphorylation of LRP6 on Ser1490  = 5.891, p = 0.038; 33/37  = 4.697, p = 0.059;  = 0.381, p = 0.698;  = 0.865, p = 0.468; As our results with lithium suggested that GSK-3 inhibition may offer neuroprotection, we next tested if a GSK-3-specific inhibitor could mimic the influences of lithium. SB-216763 has been shown to be a potent inhibitor of GSK-3, while having no significant influence on the activity of other kinases  = 0.039, p = 0.846  = 4.74, p = 0.040) and contralateral  = 9.97, p = 0.004) foot faults as compared to their vehicle-injected counterparts. SB-216763 treatment did not have any significant influence on post-injury weight loss  = 0.282, p = 0.602). All animals had normal vestibulomotor and motor function by day 7 post-injury (data not shown).In order to determine the influence of SB-216763 on TBI outcome, rats were injured, then i.p. injected 30 minutes later with either 5.0 mg/kg SB-216763 or vehicle (n = 10/group). Rats continued to receive daily injections for the first 5 days after injury during which time they were tested for their vestibulomotor and motor performances . Drug ad = 0.846 , they di = 1.639, p = 0.217). Similarly, when a probe trial was given 24 hr after the completion of training, no differences were seen in the latency to cross  or the number of platform crossings  between the two groups  = 3.186, p = 0.040) and SB-216763  = 4.019, p = 0.017) treated groups displayed significant preference for the target quadrant (I) relative to the starting quadrant (III), there was no significant difference between the two groups in their quadrant preferences  = 0.971, p = 0.338). Interestingly, when a short term memory test was given at 30 minutes following training, SB-216763 injected animals crossed the platform location significantly more than the vehicle-injected controls  nor contralateral  cortical tissue loss was different between the two groups.When evaluated for their performance in the standard hidden platform version of the Morris water maze task (beginning 14 days post-injury), no difference in learning was detected between injured rats treated with 5.0 mg/kg SB-216763 and those treated with vehicle  that binds to, and regulates, ß-Catenin-mediated gene expression 9. Although the kinase responsible for this phosphorylation has not been identified, the time point at which we observed the increase (3 days post-injury), is consistent with that seen for activation of Akt. Using a cortical impact model of injury, Zhang et al., reported that TBI causes a delayed phosphorylation of Akt on Thr308 that occurs 72 hr post-injury In addition to Wnt signaling, GSK-3 has been shown to be inhibited by direct phosphorylation by a number of kinases including protein kinase A (PKA) Accumulating evidence suggests that GSK-3 may play an important role in apoptosis with its overexpression inducing, and its inhibition preventing, apoptosis Since multiple mechanisms contribute to TBI pathophysiology, it is thought that a drug specific for a single target may not give rise to meaningful improvements in neurobehavioral outcome. There is a general consensus among TBI researchers that either a combination treatment, or a drug that targets multiple TBI-associated pathological cascades, is likely to be effective as a treatment Recently, it has been shown that pretreatment of mice with lithium prior to brain injury reduces inflammation, prevents TBI-induced depression, improves spatial learning and memory, and protects against hippocampal cell loss Although oral lithium carbonate is an effective therapy of treating bipolar disorders, benefit is typically only seen following prolonged use. This delayed onset of action is in contrast to the relatively acute effects described herein. While the mechanism underlying lithium's effect following TBI is not clear, a few studies have suggested that mechanistic differences exist between acute and chronic lithium treatment paradigms. For example, chronic, but not acute, lithium treatment increases BDNF expression in the rat hippocampus and temporal cortex The therapeutic dose for lithium is in the range of 900 mg of lithium carbonate/day (24 mEq lithium). Usually, these patients receive an initial dose of 300 mg (or 8mEq) to reduce the occurrence of adverse events such as nausea and kidney injury. The dose is then gradually increased to the recommended serum therapeutic dose (0.6–1.2mEq/L). Although we did not monitor the serum level of lithium, previous studies investigating its pharmacokinetics in rats have indicated that 5 mEq/kg lithium reaches a peak serum concentration of approximately 10 mEq/L with a half-life of 7 hr