title: A 2 × 2 factorial design for the combination therapy of minocycline and remote ischemic perconditioning: efficacy in a preclinical trial in murine thromboembolic stroke model.
abstract: BACKGROUND
  After the failure of so many drugs and therapies for acute ischemic stroke, innovative approaches are needed to develop new treatments. One promising strategy is to test combinations of agents in the pre-hospital setting prior to the administration of intravenous tissue plasminogen activator (IV-tPA) and/ or the use of mechanical reperfusion devices in the hospital.
  
  
  METHODS
  We performed a 2 × 2 factorial design preclinical trial where we tested minocycline (MINO), remote ischemic perconditioning (RIPerC) and their combination treatment in a thromboembolic clot model of stroke in mice, without IV-tPA or later treated with IV-tPA at 4 hours post-stroke. Cerebral blood flow (CBF) was measured by laser speckle contrast imaging (LSCI), behavioral outcomes as neurological deficit score (NDS) and adhesive tape removal test, and infarct size measurement were performed at 48 hours post-stroke. Mice within the experimental sets were randomized for the different treatments, and all outcome measures were blinded.
  
  
  RESULTS
  RIPerC significantly improved CBF as measured by LSCI in both with and without tPA treated mice (P < 0.001). MINO and RIPerC treatment were effective alone at reducing infarct size (p < 0.0001) and improving short-term functional outcomes (p < 0.001) in the tPA and non-tPA treated animals. The combination treatment of MINO and RIPerC significantly reduced the infarct size greater than either intervention alone (p < 0.05). There were trends in favor of improving functional outcomes after combination treatment of MINO and RIPerC; however combination treatment group was not significantly different than the individual treatments of MINO and RIPerC. There was no "statistical" interaction between minocycline and RIPerC treatments indicating that the effects of RIPerC and MINO were additive and not synergistic on the outcome measures.
  
  
  CONCLUSIONS
  In the future, combining these two safe and low cost interventions in the ambulance has the potential to "freeze" the penumbra and improve outcomes in stroke patients. This pre-clinical 2 × 2 design can be easily translated into a pre-hospital clinical trial.
authors.affiliation: Department of Neurology, Georgia Regents University, Augusta, GA 30912, USA ; Department of Medical Laboratory, Imaging and Radiologic Sciences, Georgia Regents University, Augusta, GA 30912, USA ; Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, USA ; Charlie Norwood VA Medical Centre, Augusta, USA.
authors.forename: Md Nasrul
authors.initials: MN
authors.lastname: Hoda
authors.affiliation: Department of Neurology, Georgia Regents University, Augusta, GA 30912, USA ; Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, USA ; Charlie Norwood VA Medical Centre, Augusta, USA.
authors.forename: Susan C
authors.initials: SC
authors.lastname: Fagan
authors.affiliation: Department of Neurology, Georgia Regents University, Augusta, GA 30912, USA ; Charlie Norwood VA Medical Centre, Augusta, USA.
authors.forename: Mohammad B
authors.initials: MB
authors.lastname: Khan
authors.affiliation: Department of Medical Laboratory, Imaging and Radiologic Sciences, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Kumar
authors.initials: K
authors.lastname: Vaibhav
authors.affiliation: Department of Neurology, Georgia Regents University, Augusta, GA 30912, USA ; Department of Medical Laboratory, Imaging and Radiologic Sciences, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Aizaz
authors.initials: A
authors.lastname: Chaudhary
authors.affiliation: Department of Psychiatry, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Phillip
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Krishnan M
authors.initials: KM
authors.lastname: Dhandapani
authors.affiliation: Department of Biostatistics and Epidemiology, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Waller
authors.affiliation: Department of Neurology, Georgia Regents University, Augusta, GA 30912, USA ; Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Hess
keywords: Intravenous tPA; Minocycline; Neurovascular protection; Remote ischemic perconditioning; Thromboembolic stroke