Aquaporin-4 inhibition mediates piroxicam-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rodents
Authors: Dileep R Yavagal
Anand Kumar Pandey
Pallab Bhattacharya
Sudip Paul
Ranjana Patnaik
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Aquaporin-4; AQP4; cerebral edema; Piroxicam; neuroprotection
Affiliations: School of Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, (U.P.), India ; Department of Neurology, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida, United States of America.

Abstract:  Background and purpose: Aquaporin-4(AQP4) is an abundant water channel protein in brain that regulates water transport to maintain homeostasis. Cerebral edema resulting from AQP4 over expression is considered to be one of the major determinants for progressive neuronal insult during cerebral ischemia. Although, both upregulation and downregulation of AQP4 expression is associated with brain pathology, over expression of AQP4 is one of the chief contributors of water imbalance in brain during ischemic pathology. We have found that Piroxicam binds to AQP4 with optimal binding energy value. Thus, we hypothesized that Piroxicam is neuroprotective in the rodent cerebral ischemic model by mitigating cerebral edema via AQP4 regulation. Methods: Rats were treated with Piroxicam OR placebo at 30 min prior, 2 h post and 4 h post 60 minutes of MCAO followed by 24 hour reperfusion. Rats were evaluated for neurological deficits and motor function just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, biochemical analysis, RT-PCR and western blot experiments. Results: Piroxicam pretreatment thirty minutes prior to ischemia and four hour post reperfusion afforded neuroprotection as evident through significant reduction in cerebral infarct volume, improvement in motor behavior, neurological deficit and reduction in brain edema. Furthermore, ischemia induced surge in levels of nitrite and malondialdehyde were also found to be significantly reduced in ischemic brain regions in treated animals. This neuroprotection was found to be associated with inhibition of acid mediated rise in intracellular calcium levels and also downregulated AQP4 expression. Conclusions: Findings of the present study provide significant evidence that Piroxicam acts as a potent AQP4 regulator and renders neuroprotection in focal cerebral ischemia. Piroxicam could be clinically exploited for the treatment of brain stroke along with other anti-stroke therapeutics in future. 

Retracted: Yes

PubPeer comments:
 Figure 3. Effect of Piroxicam (10 mg kg) pre and post treatment on (a) TTC stained coronal brain sections of sham, vehicle and Piroxicam treated rats Unexpected similarities between images of different treatment![file] , 
 Thank you for identifying.We will check this with the then research students coauthors engaged in performing experiments and data collection if any image was mis-uploaded inadvertently during the assembly of final images among numerous images obtained from multiple experiments. We emphasize that mere stained image mis-upload(if at all)u00a0does not affect the validity of other data,interpretation, conclusions or any other aspect of the robust study in the published paper. Nevertheless,if at all any mis-upload is found by counter checking with the raw data files, as a part of our scientific responsibility and commitmentu00a0we will surely contact the journal Editorial Office to put a correction. u00a0u00a0 , 
 #2 Another similar image in the article but different treatment![file] , 
 Similar images of TTC staining from one of the authors previous research article in Brain Research. Images dimensions and shade is altered. ![file] , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0234828 Following the publication of this article [1], concerns were raised regarding similarities in text and figures in this article and previously published articles in Brain Research [2, 3], Neuropharmacology [4], Cellular and Molecular Neurobiology [5], Medical Hypotheses [6] and Anesthesia Analgesia [7]. Furthermore, the editors noted similarities between figures in this article and figures later published in The Journal of Physiology and Biochemistry [8] and Life Sciences [9]. Specifically: Within Fig 3A: u25cb. The first brain section in the Vehicle panel looks similar to the second brain section in the Vehicle + Drug (2 hr post) panel. u25cb. The second brain section in the Vehicle panel looks similar to the fourth brain section in the Vehicle panel. u25cb. The third brain section in the Vehicle panel looks similar to the fourth brain section in the Vehicle + Drug (4 hr post) panel. u25cb. The second brain section in the Vehicle + Drug (30 min pre) panel looks similar to the third brain section in the Vehicle + Drug (2 hr post) panel. The second brain section in the Vehicle + Drug 5mg panel of Fig 2A looks similar to the first brain section in the Vehicle + Drug (30 min pre) panel of Fig 3A. Similarities were also noted between images reported in this article and in other published articles [2u20134, 8, 9]. In total, 19 out of 24 panels in Fig 2A and 11 out of 20 panels in Fig 3A appear similar to other images within this article and or to images published in [2u20134, 8, 9] that represent the results of different experiments. The following text excerpts overlap with text published in previous articles: u25cb. Materials and Methods section, subsections similar to [2] which was not cited as an original source for the procedures: u201cEvaluation of motor coordination and neurological scoringu201d (paragraph 2), u201cEstimation of cerebral infract volume and brain swellingu201d, u201cBiochemical Analysisu201d, u201cMeasurement of Nitriteu201d, and u201cEstimation of Malondialdehyde (MDA)u201d. u25cb. Materials and Methods section, subsections similar to [5] which was not cited as an original source for the procedures: u201cIsolation of Total RNAu201d, u201cReverse Transcriptionu201d, u201cPolymerase Chain Reactionu201d, u201cTissue Lysate Preparationu201d, u201cSDS-Polyacrylamide Gel Electrophoresisu201d, and u201cImmunoblottingu201d. u25cb. Results section, subsections similar to [2]: u201cDose optimization of Piroxicamu201d, u201cEffect of Piroxicam on pre and post treatment on cerebral infract volume and neurological deficitu201d (paragraph 1), u201cEffect of Piroxicam on brain nitrite levelsu201d, and u201cEffect of Piroxicam on brain MDA levelsu201d. u25cb. Discussion sections similar to [6]: Paragraphs 1, 2, 5, 6. u25cb. Discussion sections similar to [2]: Paragraphs 5, 6. u25cb. Discussion sections similar to [10]: Paragraphs 9, 10. u25cb. Discussion sections similar to [7]: Paragraphs 12, 13. The authors stated that the similarities between panels within [1] and between panels in [1] and their own articles resulted from inadvertent mis-uploading of panels during figure preparation. They have offered replacement panels to address some of the issues outlined above. The authors also stated that they cannot comment on images published in different papers by different authors. The authors provided underlying data to support results in Figs 2A and 3A. However, the underlying data provided were not sufficient to resolve the concerns noted above, including the similarities between panels depicting different experimental conditions. Regarding text overlap, the authors stated that the similarity in methodology results from both labs using the same methods and that the overlap in the Results and Discussion sections was due to the authorsu2019 unawareness of guidelines. In light of the text overlap and the image concerns that call into question the reliability of the reported results and conclusions, the PLOS ONE Editors retract this article. The panels of Figs 2A and 3A that are listed below appear visually similar to material published in 2010u20132011 which are not offered under a CC-BY license. The indicated panels are therefore excluded from the PLOS ONE article s [1] license. At the time of retraction, the article [1] was republished to note this exclusion in the Figs 2 and 3 legends and the articleu2019s copyright statement. Visually similar to images in Figures 1C and 2C of [4], published in 2010 by Elsevier: The first and fourth brain sections in the Sham panel of Fig 2A The first and fourth brain sections in the Vehicle panel of Fig 2A The first, third, and fourth brain sections in the Vehicle + Drug 5mg panel of Fig 2A The first, third, and fourth brain sections in the Vehicle + Drug 10mg panel of Fig 2A The third and fourth brain sections in the Vehicle + Drug 20mg panel of Fig 2A Visually similar to images in Figure 1 of [2], published in 2011 by Elsevier: The second, third, and fourth brain sections in the Vehicle panel of Fig 3A The fourth brain section in the Vehicle + Drug (2 hr post) panel of Fig 3A The third and fourth brain sections in the Vehicle + Drug (4 hr post) panel of Fig 3A Visually similar to images in Figures 2C, 3C, and 4C of [3] published in 2011 by Elsevier: The second brain section in the Sham panel of Fig 3A The third brain section in the Vehicle + Drug (30 min pre) panel of Fig 3A The authors did not agree with retraction. References 1.Bhattacharya P, Pandey AK, Paul S, Patnaik R, Yavagal DR (2013) Aquaporin-4 Inhibition Mediates Piroxicam-Induced Neuroprotection against Focal Cerebral Ischemia Reperfusion Injury in Rodents. PLoS ONE 8(9): e73481. pmid:24023878 2.Pandey AK, Hazari PP, Patnaik R, Mishra AK. The role of ASIC1a in neuroprotection elicited by quercetin in focal cerebral ischemia. Brain Res. 2011;1383:289u2010299. pmid:21281608 3.Mishra V, Verma R, Singh N, Raghubir R. The neuroprotective effects of NMDAR antagonist, ifenprodil and ASIC1a inhibitor, flurbiprofen on post-ischemic cerebral injury. Brain Research. 2011 May;1389:152u2013160. pmid:21396924 4.Mishra V, Verma R, Raghubir R. Neuroprotective effect of flurbiprofen in focal cerebral ischemia: the possible role of ASIC1a. Neuropharmacology. 2010;59(7u20138):582u2010588. pmid:20813117 5.Modi PK, Kanungo MS. Age-dependent expression of S100beta in the brain of mice. Cell Mol Neurobiol. 2010;30(5):709u2010716. pmid:20099023 6.Bhattacharya Pallab Pandey Anand Paul Sudip. (2011). Minocycline and magnesium in combination may be a good therapeutic intervention for cerebral ischemia. Medical hypotheses. 77. 1129u201331. pmid:21985758 7.Zheng YY, Lan YP, Tang HF, Zhu SM. Propofol pretreatment attenuates aquaporin-4 over-expression and alleviates cerebral edema after transient focal brain ischemia reperfusion in rats. Anesthesia and Analgesia. 2008 Dec;107(6):2009u20132016. pmid:19020152 8.Bhattacharya P., Pandey A.K., Paul S. et al. Alleviation of glutamate mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism. J Physiol Biochem 70, 901u2013913 (2014). pmid:25292423 9.Bhattacharya P, Pandey AK, Paul S, Patnaik R. Melatonin renders neuroprotection by protein kinase C mediated aquaporin-4 inhibition in animal model of focal cerebral ischemia. Life Sciences. 2014 Apr;100(2):97u2013109. pmid:24530291 10.Migliati E. Role of Aquaporin-4 Water Channels in Cerebral Edema After Ischemic Stroke [dissertation]. University of Arizona; 2006. https: repository.arizona.edu handle 10150 194063 , 
 See also https: retractionwatch.com 2022 05 24 the-internet-may-be-a-challenging-venue-biomedical-engineering-group-up-to-four-retractions ,