Neuroprotection

All rodents were housed at 25 °C in a 12/12 h light/dark cycle with access to food and water ad libitum. The procedures used in this study were fully approved by the Institutional Animal Care and Use Committee of the Intramural Research Program, National Institute on Aging, NIH (detailed in approved animal protocols 331-TGB-2024; 488-TGB-2022). Studies were performed in accord of the ARRIVE guidelines and recommendations, and all efforts were undertaken to minimize any potential animal suffering and as well as the number of animals used. This was achieved by incorporating the outcome measures from our prior studies [44 (link)] and a power analysis [45 (link)]. In this first-in-animal evaluation of TFBP and TFNBP in a rodent model of LPS-induced systemic and neuroinflammation and of TFBP in CCI TBI, studies were conducted in male rodents alone to evaluate whether these novel agents demonstrate a signal of in vivo anti-inflammatory efficacy using the least number of animals possible. This decision was made in the knowledge that gender differences have been identified in the response of rodents to TBI, in addition to other brain insults [46 (link)–48 (link)]. In this regard, TBI incidence in young to middle-aged adults is lower in women than men, and derives primarily from different causes [46 (link)]. More confusing in human studies is the effect of gender on TBI outcome. Considerably fewer studies have focused on women challenged with TBI in relation to males, and outcome varies in relation to age, menopausal status as well as severity of TBI and chosen outcome measure. Whereas human studies often report worse outcomes in women than men, importantly animal studies largely describe the opposite [46 (link)]. In this regard, studies in ovariectomized rodents have demonstrated that estrogens provide significant neuroprotection to mitigate damage in female rodents, but not necessarily in the human species [46 (link)]. Hence, to avoid potential confounds associated with estrogen generation in young female rodents, or potentially ovariectomizing animals or aging them to a postmenopausal state, we performed our first-in-animal studies, reported herein, in young adult male rodents. Aware that there are undoubtedly gender differences in relation to the pharmacokinetics, pharmacodynamics and tolerability of TFBP and TFNBP, these could then be evaluated in future studies in the event that a promising signal of efficacy is demonstrated in the first-in-animal investigation described herein.

All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Use Committee, in compliance with the guidelines of the Association for the Assessment and Accreditation for Laboratory Animal Care, International and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.¹² Animal experiments complied with Animal Research: Reporting of In Vivo Experiments guidelines. All experiments were conducted using male C57BL/6J mice (2–3 months old; Jackson Laboratories, Bar Harbor, ME, USA).
The animals were housed five per cage, maintained in a 14 h light/10 h dark cycle, fed a balanced diet ad libitum. Animals were randomly assigned to groups, based on injury designation (sham or CCI) and treatment designation (vehicle or pioglitazone). Treatments were given in random order. All experimentation was performed blinded to treatment groups. For all outcomes, experiments were conducted with biological replicates of N = 4–8/group. Additionally, technical triplets were used in each assay.
Two separate cohorts were used for this study. The acute study was conducted to examine the effect of pioglitazone treatment after mild brain contusion on total, glia-enriched, and synaptic mitochondrial bioenergetic measures in the brain. Experimental groups for the acute study were euthanized at 48 h after injury. The subacute efficacy study was conducted to examine how modulation of acute mitochondrial bioenergetics with pioglitazone translated into cortical neuroprotection after mild brain contusion. Experimental groups for the subacute efficacy study were euthanized at 15 days after injury. Mice received a bolus (i.p.) administration of pioglitazone (100 µL volume; 1:1 DMSO and PEG400) at either 0.25, 3, 12, or 24 h after mild CCI, based on the treatment strategy outlined in Hubbard et al.^{4 (link)} The 15-day efficacy study utilized 7-day duration Alzet Mini Pump 1007D (release of 0.5 µL/h; Alzet, Cupertino, CA, USA).