A checklist is associated with increased quality of reporting preclinical biomedical research: A systematic review

<div><p>Irreproducibility of preclinical biomedical research has gained recent attention. It is suggested that requiring authors to complete a checklist at the time of manuscript submission would improve the quality and transparency of scientific reporting, and ultimately enhance reproducibility. Whether a checklist enhances quality and transparency in reporting preclinical animal studies, however, has not been empirically studied. Here we searched two highly cited life science journals, one that requires a checklist at submission (<i>Nature</i>) and one that does not (<i>Cell</i>), to identify <i>in vivo</i> animal studies. After screening 943 articles, a total of 80 articles were identified in 2013 (pre-checklist) and 2015 (post-checklist), and included for the detailed evaluation of reporting methodological and analytical information. We compared the quality of reporting preclinical animal studies between the two journals, accounting for differences between journals and changes over time in reporting. We find that reporting of randomization, blinding, and sample-size estimation significantly improved when comparing <i>Nature</i> to <i>Cell</i> from 2013 to 2015, likely due to implementation of a checklist. Specifically, improvement in reporting of the three methodological information was at least three times greater when a mandatory checklist was implemented than when it was not. Reporting the sex of animals and the number of independent experiments performed also improved from 2013 to 2015, likely from factors not related to a checklist. Our study demonstrates that completing a checklist at manuscript submission is associated with improved reporting of key methodological information in preclinical animal studies.</p></div

Prednisolone and salbutamol synergistically suppress HMGB1-induced TNFα secretion.

<p>RAW 264.7 cells were pretreated with prednisolone and salbutamol at the indicated concentrations and exposed to HMGB1 (5 µg/ml) for 18 hours. TNFα secretion (<b>A, B</b>) and LDH release (<b>E, F</b>) were measured in the supernatant. Cell viability (<b>C, D</b>) was measured by the MTT assay. (^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group, ^#p<0.05 compared to the respective first compound treatment).</p

List of hit compounds identified in the primary screen.

<p>Non-toxic compounds that reduced the HMGB1-induced TNFα production by 2 standard deviation values are listed in order of potency, according to their inhibitory potency for TNFα secretion. The source library of the compounds, their known biological activity and the respective viability values are shown. Viability was measured by the MTT assay. (Abbreviations: MAP kinase: Mitogen-activated protein kinase, U0126∶1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene, MEK: mitogen-activated protein kinase kinase, STAT3: Signal transducer and activator of transcription 3, ST057244∶1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]piperidin-2-one, Bay 11-7085: (2<i>E</i>)-3-[[4-(1,1-dimethylethyl)pheny?l]sulfonyl]-2-propenenitrile, Bay 11-7082∶3- [(4- methylphenyl)sulfonyl]- (2E)- propenenitrile, ST009819: (2R,3R,13R,14R)-3-(phenylcarbonyl)-17,19-dioxa-4-azapentacyclo[14.2.1.0<2,14>. 0<4,13>.0<7,12>]nonadeca-5,7(12),8,10-tetraen-15-one, MNS: 3,4-methylenedioxy-β-nitrostyrene, IkB: inhibitor of nuclear factor κB kinase, NF-κB: nuclear factor κB, HIV: human immunodeficiency virus, Src: sarcoma tyrosine kinase, Syk: Spleen tyrosine kinase).</p

Concentration- and time-dependence of the HMGB1-induced inflammatory response and reduction in cell viability in RAW 264.7 macrophages.

<p>RAW 264.7 cells were treated with the indicated amount of HMGB1 for 24, 48 or 72 hours. <b>A:</b> Cell viability was measured with the MTT assay and <b>B:</b> TNFα secretion was measured in the supernatant.</p

Prednisolone and salbutamol inhibit the HMGB-induced TNFα production.

<p>RAW 264.7 cells were pretreated with prednisolone (1 µM) and salbutamol (1 µM) and then exposed to HMGB1 (5 µg/ml) for various time up to 18 hours. <b>A</b>: TNFα secretion measured in the supernatant is plotted versus exposure length. (MEAN±SD values are shown) <b>B</b>: TNFα mRNA expression, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is shown as fold expression values of vehicle treated cells. (CTL: vehicle treated control, HMGB: cells exposed to HMGB1, Pred: cells pretreated with prednisolone and exposed to HMGB1, Salb: cells pretreated with salbutamol and exposed to HMGB1, Pred+Salb: cells pretreated with both prednisolone and salbutamol and exposed to HMGB1. ^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group, ^#p<0.05 compared to single compound treatment).</p

HMGB1 induces time-dependent caspase activation in RAW 264.7 macrophages.

<p>RAW 264.7 cells were exposed to HMGB1 (5 µg/ml) for 24, 48 or 72 hours. Activated Caspase-3 was detected in cell extracts by Western blotting. Tubulin was used for loading control. The graph shows relative Caspase-3 activation values, normalized to tubulin. (**p<0.01 shows significant caspase activation compared to vehicle-treated cells).</p

Inhibition of the HMGB-induced TNFα production by catecholamines and glucocorticoids <i>in vivo</i>.

<p>Balb/c male mice (Charles River Laboratories) were injected with 0.5 mg/kg HMGB1 in the presence of 60 min pretreatment of either vehicle, or 20 mg/kg prednisolone, 10 mg/kg salbutamol, the combination of prednisolone and salbutamol (doses as above), or the glucocorticoid receptor blocker mifepristone (30 mg/kg) or the β-receptor antagonist propranolol (10 mg/kg). At 8 hours after HMGB1 injection, animals were sacrificed and serum levels of TNFα were measured. ^#p<0.05 represents a significant increase in TNFα serum levels in response to HMGB1; *p<0.05 represents significant inhibition of HMGB1-induced TNFα production by the various pharmacological agents indicated. n = 7 animals per group.</p

Combined screening to identify pharmacological potentiators of dexamethasone-mediated inhibition of the HMGB1-induced pro-inflammatory response.

<p>RAW 264.7 cells were pre-treated with dexamethasone (3 µM) in combination with test compounds and exposed to HMGB1 for 18 hours. TNFα production was measured from the supernatant and the viability of the cells was measured by the MTT assay. <b>A:</b> TNFα responses measured in the combination screen are plotted versus the TNFα production values measured in the single compound screen. TNFα production values higher than MEAN+2SD are shown in red (“steroid inhibitors”) and values lower than MEAN+2SD in green boxes (“potentiators of steroids) for the combination screen. Red dots denote the toxic compounds, green the steroid potentiators and purple those that increase the TNFα production. Compounds that inhibited the HMGB-induced TNFα production in the single compound screen, but failed to potentiate the action of steroids are shown in yellow. <b>B:</b> TNFα responses relative to the activity of dexamethasone are plotted versus the viability values. Red and green boxes indicate the upper and lower 2 SD limits.</p

Screening for compounds that reduce the HMGB1-induced pro-inflammatory response.

<p><b>A:</b> Timeline of the cell-based screening: RAW 264.7 cells were pre-treated with test compounds and exposed to HMGB1 for 18 hours. TNFα production was measured from the supernatant and the viability of the cells was measured by the MTT assay. <b>B:</b> Dot graph showing the individual TNFα/viability results of the tested 5,646 compounds. TNFα responses are shown as % values of the HMGB1-induced TNFα production. Values lower than MEAN-2SD are shown in red (viability) and green (TNFα response) boxes to denote “toxic” and “Hit” compounds. <b>C–D:</b> Distribution of viability (C) and TNFα response (D) data with superimposed Gaussian distribution curves fitted to the data points.</p

Inhibition of the HMGB-induced inflammatory response by endogenous catecholamines and glucocorticoids at physiological concentrations.

<p>RAW 264.7 cells were pretreated with cortisol (0.7 µM), noradrenaline (0.5 ng/ml), adrenaline (0.5 ng/ml), dexamethasone (1 µM) and salbutamol (1 µM) and exposed to HMGB1 (5 µg/ml) for 18 hours. TNFα secretion was measured in the supernatant. (^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group, ^#p<0.05 cells treated with all compounds in combination versus treated with a combination of two.).</p