(2024) 21:194
Pearson et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03173-w

Journal of Neuroinflammation

Open Access

RESEARCH

PPARγ activation ameliorates cognitive
impairment and chronic microglial activationin the aftermath of r‑mTBI
Andrew Pearson1,2*, Milica Koprivica1, Max Eisenbaum1,2, Camila Ortiz1,2, Mackenzie Browning1,
Tessa Vincennie1, Cooper Tinsley1, Michael Mullan1, Fiona Crawford1,2,3 and Joseph Ojo1,2,3

Abstract
Chronic neuroinflammation and microglial activation are key mediators of the secondary injury cascades and cognitive impairment that follow exposure to repetitive mild traumatic brain injury (r-mTBI). Peroxisome proliferatoractivated receptor-γ (PPARγ) is expressed on microglia and brain resident myeloid cell types and their signaling plays
a major anti-inflammatory role in modulating microglial responses. At chronic timepoints following injury, constitutive
PPARγ signaling is thought to be dysregulated, thus releasing the inhibitory brakes on chronically activated microglia.
Increasing evidence suggests that thiazolidinediones (TZDs), a class of compounds approved from the treatment
of diabetes mellitus, effectively reduce neuroinflammation and chronic microglial activation by activating the peroxisome proliferator-activated receptor-γ (PPARγ). The present study used a closed-head r-mTBI model to investigate
the influence of the TZD Pioglitazone on cognitive function and neuroinflammation in the aftermath of r-mTBI
exposure. We revealed that Pioglitazone treatment attenuated spatial learning and memory impairments at 6 months
post-injury and reduced the expression of reactive microglia and astrocyte markers in the cortex, hippocampus,
and corpus callosum. We then examined whether Pioglitazone treatment altered inflammatory signaling mechanisms
in isolated microglia and confirmed downregulation of proinflammatory transcription factors and cytokine levels.
To further investigate microglial-specific mechanisms underlying PPARγ-mediated neuroprotection, we generated a novel tamoxifen-inducible microglial-specific PPARγ overexpression mouse line and examined its influenceon microglial phenotype following injury. Using RNA sequencing, we revealed that PPARγ overexpression ameliorates
microglial activation, promotes the activation of pathways associated with wound healing and tissue repair (such as:
IL10, IL4 and NGF pathways), and inhibits the adoption of a disease-associated microglia-like (DAM-like) phenotype.
This study provides insight into the role of PPARγ as a critical regulator of the neuroinflammatory cascade that follows
r-mTBI in mice and demonstrates that the use of PPARγ agonists such as Pioglitazone and newer generation TZDs
hold strong therapeutic potential to prevent the chronic neurodegenerative sequelae of r-mTBI.

*Correspondence:
Andrew Pearson
apearson@roskampinstitute.org
1
The Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, USA
2
The Open University, Walton Hall, Kents Hill, Milton Keynes MK7 6AA, UK
3James A. Haley Veterans’ Hospital, 13000 Bruce B Downs Blvd, Tampa, FL
33612, USA

Introduction
Traumatic brain injury (TBI) is a leading cause of mortality, morbidity, and disability in both developed anddeveloping nations [54, 143]. TBI affects approximately
2.8 million people in the USA every year [124]. Even in
injuries diagnosed as mild TBI (mTBI), it is estimatedthat between 10 and 40% of mTBI patients live withcognitive and functional impairments that may persist© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by-​nc-​nd/4.​0/.

Pearson et al. Journal of Neuroinflammation(2024) 21:194

for months to years after injury [21, 78, 105]. Exposure
to repeated mTBIs (r-mTBIs) sustained within a shorttime window can exacerbate the pathological and functional consequences of injury [6, 19, 23, 38]. Exposure
to r-mTBI, often observed amongst military populations and athletes participating in high-contact sports isstrongly associated with the development of later-life
neurodegenerative pathologies seen histologically by thepresence of neurofibrillary tangles of hyperphosphorylated tau and amyloid beta (Aβ) plaques [8, 70, 71, 73, 103,104]. Despite this, there are no therapeutics available toclinicians to reduce the chronic sequelae of TBI and current treatment strategies are limited to symptom alleviation and rehabilitative care [120].
TBI is a dynamic disease process that is broadly separated into two distinct phases referred to as primary andsecondary injury [72]. Primary injury is a direct consequence of the biomechanical forces imparted on thehead that cause direct physical damage to tissues [51,103, 104]. The damage of tissues such as vessels and axon
tracts induced by the primary injury initiate a cascadeof secondary injurious effects [116]. Secondary injury
is more prolonged and widespread, involving pathophysiological mechanisms such as neuroinflammation,
metabolic dysfunction, oxidative stress, and changes insynaptic plasticity and integrity [103, 104, 110]. Chronic
microglial activation, a key component of neuroinflammation is one of the cardinal features observed following
exposure to repetitive head trauma [31, 32, 81, 83, 84, 86,
96, 97, 100]. The chronic nature of the secondary injury
cascade make it an opportunistic target for the successful treatment of post-TBI symptoms. However, the progression of these secondary injurious processes over time
are complex and incompletely understood. Moreover, itremains unknown whether these biochemical cascades of
events are reversible.
Peroxisome
proliferator-activatedreceptor-γ(PPARγ), an isoform of the PPAR family, is a ligandactivated transcription factor that once activated, forms
a heterodimer with retinoid x (RXR) and subsequently
binds to DNA to regulate the expression of genes which
are essential for various metabolic processes and celldifferentiation [35, 129]. PPARγ is well expressed by
microglia and myeloid cells in the CNS and PPARγ activation by agonists have also been shown to exert antiinflammatory and anti-oxidant properties after brain
injury or in models of neurodegenerative disorders suchas Alzheimer’s and Parkinson’s diseases [9, 91, 93, 102,123]. Our previous analyses of the chronic pathobiology of r-mTBI have identified the chronic dysregulationof PPARγ signaling as a potential therapeutic target[95]. In this study, we examine the effects of a delayed
Pioglitazone treatment on cognitive and pathologicalPage 2 of 24

outcomes such as microglial activation at the chronic
stages of r-mTBI pathophysiology. To further examine
how PPARγ activation affects microglial activation at
the chronic stages of injury, we developed a microglialspecific model of PPARγ overexpression and examinedthe effects on the microglial transcriptomic phenotype
following exposure to our r-mTBI paradigm.

Methods and materials
Culture and in vitro treatment of IMG cells

Adult mouse-derived IMG microglial cells (EF4001;
Kerafast, Boston, MA, USA) were maintained in
DMEM/F12 (Sigma-Aldrich) culture medium supplemented with 10% heat-inactivated fetal bovine serum(FBS001-HI Neuromics), 2 mM L-glutamine and 1%
anti-biotic & anti-mycotics. Cells were maintained at
37 °C and 5% ­CO2 in a humidified culture incubator.
The immortalized microglial (IMG) cell line was firstcharacterized by McCarthy et al. [68]. These cells havebeen shown to more accurately recapitulate the in vivo
microglial phenotype compared to other microglial celllines such as BV2 cells [37, 58, 68, 69]. IMG cells wereseeded into 24 well culture plates 24 h before exposure
to Lipopolysaccharide (LPS) or Pioglitazone. Cells wereincubated with control media, media containing only
LPS (10 ng/ml), or media containing LPS (10 ng/ml)
and Pioglitazone (10 µM) for 24 h.
Animals

Male C57BL6J mice at 12 weeks of age were obtained
from Jackson laboratories (Bar Harbor, ME).
To generate the microglial-specific conditional overexpression of PPARγ mice, we crossed homozygous FloxPPARγ-VP16(+/+) mice previously described by Kleinhenz
and colleagues [59], with homozygous CX3CR1 CreERT2EYFP reporter mice (Jackson Laboratories, #021160) togenerate hemizygous C­ X3CR1+/CreERT2- PPARγ-VP16(+/fl)
OE(mcg-PPARγ ) mice. Hemizygous mcg-PPARγOE andhomozygous ­CX3CR1CreERt2+/+ (Cre genotype control)
mice were used for experimental studies. Both male andfemale mice were used in each group in an approximate
50:50 ratio. The PPARγ-VP16 mice were a generous giftfrom Dr. Michael Hart.
Animals were housed in groups of three to four per
cage under standard laboratory conditions (23 ◦C ± 1 °C,
50 ± 5% humidity, 14-hour light/10-h dark cycle) withad libitum access to food and water throughout the study.
All procedures involving mice were carried out underInstitutional Animal Care and Use Committee approvaland in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals.

Pearson et al. Journal of Neuroinflammation(2024) 21:194

Genotyping

Genotyping for Cre recombinase, CX3CR1, andPPARγ-VP16 was performed by Transnetyx, using tailsnips as previously described by Jackson Labs [126].
Injury protocol

The r-mTBI paradigm was administered as previously
described [100]. Briefly, all mice were anesthetized with
1.5 L/min of oxygen and 3% isoflurane prior to r-mTBI
or sham injury. Once anesthetized, mice had their heads
shaved, but no incisions were made. Mice were subsequently placed in a stereotaxic frame (Just for Mice™
Stereotaxic, Stoelting, Wood Dale, IL) mounted with anelectromagnetic controlled impact device (Impact One™
Stereotaxic Impactor, Richmond, IL). The stereotaxic
frame was fitted with a heating pad to maintain bodytemperature at 37 °C, and non-invasive rubber pads were
fixed in place on either side of the head to prevent lateral
movement during impact. A 5 mm blunt metal impactor
tip was positioned above the sagittal suture before each
impact. Injury was delivered to the closed skull with an
impact velocity of 5 m/s, strike depth of 1.0 mm, anddwell time of 200 ms; the force applied to the mousehead at the time of impact is approximately 72 N underthese conditions. All mice experienced short-term apnea(< 20 s) and showed no presence of skull fractures. All
mice were then allowed to recover on a heating pad
until the mouse became ambulatory and subsequently
returned to their cages with access to soft food and water.
Mice assigned to the r-mTBI cohort underwent thisinjury protocol daily with a 24-h inter-injury interval (5hits/week, Monday to Friday) for a total of 20 injuries.
To control for the effects of repeated anesthesia, miceassigned to the sham group underwent the same anesthesia protocol but did not receive an injury. Throughout the injury protocol and following injury, mice weremonitored daily for any health concerns or behavioral
abnormalities.
Pioglitazone treatment

Pioglitazone hydrochloride (#P1901) was purchasedfrom TCI America and processed into standard soy protein free rodent chow (#TD190035) (Teklad, Envigo) at
12.5 ppm and 50 ppm. Pioglitazone doses were selectedbased on allometric scaling of commonly prescribedhuman doses [88]. Following treatment, all animals wereeuthanized 24 h after the final administration.
Tamoxifen treatment

Mcg-PPARγOE and ­CX3CR1CreERt2+/+ mice were treatedwith 50 mg/kg (I.P) tamoxifen treatment for 5 consecutive days starting at 8 weeks of age. Following tamoxifen

Page 3 of 24

treatment, a four week “wash-out” phase was performed
to allow for the turnover of peripheral monocytes before
exposure to the r-mTBI paradigm.
Behavioral analysis

Barnes maze (BM) was performed to assess spatial learning and memory. Beginning 7 days before the start of the
BM trials, mice were handled daily to reduce the potential effect of novel stressors on mouse behavior. BM wasinitiated 8 days before euthanasia and lasted for 7 consecutive days. During each day of Barnes maze testing
all mice were carefully transferred from the vivarium tothe behavioral analysis suite, at which point the cages
were left undisturbed for 30 min to allow the animals
time to recover from stress. The walls of the BM room
were equipped with visual spatial cues which were kept
constant for all 7 days, room lighting, and motivation
to escape the maze was provided by high intensity LED
floodlights and again were kept constant across all 7 daysof testing. For the first 6 days (Acquisition phase), animals
were trained to locate a target hole with a black escapebox securely fixed underneath. The BM arena was a white
polymer table 1.2 m in diameter with 18 equally spaced
holes around its perimeter. Before beginning behavioral analysis, a still image of the BM table was taken andstored in the Noldus Ethovision XT visual tracking software. A mask was generated defining the target hole andall incorrect holes. This mask was overlayed onto a live
camera image to enable the quantification of number of
errors. Mice performed 4 trials per day during the acquisition phase, each lasting 90 s. The starting position ofeach trial was rotated, beginning at one of four cardinal
positions (North, East, South, West) of the maze rotating clockwise. If an animal was unsuccessful in locating
the target box or escaping the maze, animals were gently
guided to the target hole by hand. Regardless of success,all mice spent 30 s in the escape box before returning totheir cage. On day 7 (Probe phase), the escape box was
removed, mice were placed in the middle of the maze andhad 60 s to locate the correct hole.
Euthanasia

Mice were anesthetized with 1.5 L/min of oxygen and
3% isoflurane and were then perfused transcardially withphosphate-buffered saline (PBS), pH 7.4. Following perfusion, brains were either placed in a sterile petri dish for
glial isolation, post-fixed in a solution of 4% paraformaldehyde (PFA) at 4 °C for 48 h and paraffin-embedded for
immunohistochemistry, or flash frozen in liquid nitrogenand then stored at − 80 °C for subsequent biochemical
analysis.

Pearson et al. Journal of Neuroinflammation(2024) 21:194

Tissue processing and immunohistochemistry

Brain samples fixed in PFA were paraffinized using the
Tissue-Tek VIP (Sakura, USA). Sagittal sections were cut
at 6 μm using a Leica RM2235 microtome and mounted
on positively charged glass slides. Prior to staining, sections were deparaffinized in xylene and rehydrated insequential ethanol solutions of decreasing concentration.
Immunohistochemical staining for Iba1, CD68, and GFAP

Reactive microglia and astrocytes were stained using
anti-Iba1 (Wako) or anti-CD68 (Cell Signaling #97778s)
and anti-GFAP (Dako) antibodies, respectively. Following
tissue rehydration, slides were sub-merged in hydrogen
peroxide for 15 min to remove endogenous peroxidases,
followed by antigen retrieval in citric acid buffer (pH 6.0).
Slides were then blocked with normal goat serum raised
at room temperature for 1 h and subsequently incubatedwith primary antibody (Iba1-1:1000, GFAP-1:5000) at
4 ◦C overnight. The next day, slides were washed threetimes in PBS and incubated with their respective secondary antibody (VectaSTAIN ABC Kit) and developed
using 3,3′-Diaminobenzidine (DAB).
Imaging

Imaging of Iba1, CD68, and GFAP was performed on an
Olympus DP72 microscope at 20 × magnification for thecortex and hippocampus, and 40 × magnification for the
corpus callosum. Three non-overlapping regions of interest (ROI) per section were selected for the hippocampus,
the body of the corpus callosum, and the cortex (under
the injury site). ROI were selected in identical brain
regions regardless of the presence or absence of any pathological signs. Images were subsequently analyzed using
the optical segmentation tool in ImageJ. Images wereseparated into individual color channels (hematoxylin
counterstain and DAB chromogen) using a color deconvolution algorithm. Reactive coverage area was analyzed
in ­mm2 and were subsequently calculated as a percentage (%) of total area for each image. The mean values foreach region and mouse were used for statistical analysis.
Imaging and image analysis were performed by individuals who were blinded to the study groups.
Microglial isolation

Following transcardial perfusion of mice during euthanasia, mice were decapitated. The brain was removed,
rinsed in PBS, and placed into a Petri dish on ice. Enzymatic tissue digestion was performed using the Adult
Brain Dissociation Kit (130-107-677, Miltenyi Biotec)
as previously described [29, 100]. Briefly, after removing
the meninges, the brain was cut into small pieces using
a sterile scalpel blade, and samples were transferred to a
15 mL tube where 1950 μL of enzyme mix 1 (enzyme P

Page 4 of 24

and buffer Z) and then 30 μL of enzyme mix 2 (enzyme
A and buffer Y) were added, and brains were incubatedin the enzyme mix for 30 min. Brains were then further
digested using repeated trituration. Samples were briefly
centrifuged and filtered through a 70 μm cell strainer toachieve a single-cell suspension. Single cells were resuspended in 900 μL of Debris removal solution and mixed
with 3.1 mL of PBS containing 0.5% fetal bovine serum(FBS) (PB buffer), samples were then transferred to a
fresh 15 mL falcon tube, and 4 mL of PB buffer was carefully overlayed. Samples were then centrifuged at 3000 ×g
for 10 min. Cells were rinsed with PB buffer to remove
any remaining debris removal solution and centrifuged
for 10 min. The supernatant was aspirated, and cells werelabeled with anti-CD11b magnetic particles for 10 min at
4 °C; the sample was then loaded onto a pre-conditioned
LS separation column (130–042-401, Miltenyi Biotec)
and rinsed three times to remove unlabeled cells. To elute­CD11b+ve cells, the LS column was removed from themagnet, and PB buffer was used to elute the sample.
Nuclear protein isolation

Nuclear protein isolation was performed using the NEPER nuclear and cytoplasmic extraction reagent kit(Thermo Scientific) according to manufacturer instructions. Isolated ­CD11b+ve cells were centrifuged for 5 min
at 500 ×g to pellet cells. 100 µL of ice-cold cytoplasmic
extraction reagent 1 (CER1) supplemented with proteinase and phosphatase inhibitors (Thermo Scientific) was
added to all samples. Samples were then mixed vigorously and incubated on ice for 10 min. 5.5 µL cytoplasmic
extraction reagent 2 (CER2) was added to all samples,
which were subsequently vortexed and centrifuged at
16,000 ×g for 5 min at 4 °C to separate nuclear and cytoplasmic proteins. The supernatant (cytoplasmic fraction)
was transferred to a fresh tube and stored at − 80 °C. The
pellet (nuclear fraction) was re-suspended in 50 µL ofchilled nuclear extraction reagent (NER) and incubatedon ice for 40 min with intermittent mixing (15 s every
10 min). The nuclear fraction was then centrifuged at
16,000 ×g for 10 min at 4 °C to remove insoluble material
and the supernatant was transferred to a fresh tube andstored at − 80 °C.
Transcriptional activity assay

Analysis of PPARγ transcriptional activity was assessedusing PPAR gamma Transcription Factor Assay Kit(ab133101, Abcam) and nuclear protein extracts from
­CD11b+ve cells. Assay was performed according to manufacturer instructions. 90 µL of complete transcription
factor binding assay buffer (CTFB) and 10 µL of sample
nuclear extracts was added to each well (90 µL of CTFB
and 10 µL of manufacturer supplied positive control

Pearson et al. Journal of Neuroinflammation(2024) 21:194

sample was added to control wells). Samples were incubated overnight at 4 °C, the following morning wells
were washed five times with 200 µL of 1X wash bufferand 100 µL of PPARγ primary antibody was added toappropriate wells. Samples were incubated with primary antibody for one hour at room temperature without agitation, then washed five times with 200 µL of
1X wash buffer. 100 µL of secondary was then added toappropriate wells, followed by a one-hour incubation atroom temperature. Samples were again washed five times
with 200 µL of 1X wash buffer and 100 µL of developing
solution was added to all wells. Samples were incubatedin the dark for 45 min at room temperature and 100 µLof stop solution was added to all wells and were immediately read at 450 nm using a Cytation 3 (Biotek, Winooski, VT) plate reader. Following quantification, all data
were normalized to protein content of sample lysates,
determined by Bicinchoninic acid (BCA) protein assay(ThermoFisher, Waltham, MA).
Cytokine quantification

Cytokine levels from isolated C­ D11b+ve cells (n = 4/group) were analyzed using the V-PLEX proinflammatory Panel 1 mouse cytokine kit (MSD, Rockville, MD)
according to the manufacturer’s instructions. Briefly,
samples were diluted 1:2 in Diluent 41 and incubated atroom temperature for 2 h. Wells were washed to remove
unbound analyte; secondary antibody was added, and thesamples were left to incubate at room temperature for 1 h
and quantified using the MESO QuickPlex SQ120 plate
reader. Following quantification, all data were normalized to the protein content of sample lysates, determined
by Bicinchoninic acid protein assay (ThermoFisher,
Waltham, MA).
Immunoblotting

For immunoblotting analysis, isolated ­CD11b+ve cells
were homogenized in MPER supplemented with protease and phosphatase inhibitors (ThermoFisher) using aprobe sonicator. Homogenized samples were centrifuged
at 21,000 ×g for 20 min at 4 °C, and the supernatant wastransferred to a fresh Eppendorf tube. Supernatant fractions were denatured by boiling at 95 °C for 10 min in
Laemmli buffer (Bio-Rad) containing β-mercaptoethanol.
Samples were then resolved on a 4–15% gradient polyacrylamide criterion gel (Bio-Rad). Proteins were thentransferred to a Polyvinylidene Fluoride (PVDF) membrane (BioRad) overnight at 90 mAh. Transferred membranes were blocked in a 5% non-fat milk buffer in
Tris-buffered saline (TBS) containing 0.05% sodium
azide (NaN3) for 1 h at room temperature and then incubated with different target-specific primary antibodies
overnight. The following primary antibodies were used:

Page 5 of 24

anti-NLRP3 (1:1000, Adipogen, #AG-20B-0014-C100),
anti-Phospho STAT3(Tyr705) (1:1000, Cell signaling,#9145), anti-STAT3 (1:1000, Cell signaling, #4904), antiPhospho NFκB(Ser536) (1:1000, Cell signaling, #3033 s),anti- NFκB (1:1000, Cell signaling, #8242 s), anti-PPARγ(1:500, ThermoFisher, #PA3-821A), anti-PGC1α (1:1000,
Novus Bio, NBP1-0676SS), anti-Tubulin (1:1000, Cellsignaling, #2144), anti-β actin (1:1000, Cell signaling,#4967).
RNA isolation

RNA isolation from isolated cells was performed as previously described [29]. Briefly, 500μL of TRIzol (Fisher
Scientific, Waltham, MA) was added under RNase freeconditions to isolated CD11b + ve cells (N = 4–5/group),and cells were lyzed through trituration. Following
homogenization, samples were incubated at room temperature for 5 min to allow for dissociation of nucleoprotein complexes and 100 μL of 1-bromo-3-chloropropane
reagent (Sigma, St. Louis, MO) was added to each sample. Samples were briefly vortexed and centrifuged at
12,000×g for 15 min. The aqueous phase was carefully
removed, and the organic phase was transferred to a
fresh eppendorf tube and stored at − 80 °C for subsequent protein precipitation. 20 ng of ultrapure glycogen(Fisher Scientific, Waltham, MA # AM9510) was added,
samples were again briefly mixed, and RNA was subsequently precipitated using 500 μL of ice-cold 100% Isopropanol. The RNA pellet was washed twice with 75%
ethanol in Diethyl pyrocarbonate (DEPC) treated water(Fisher Scientific, Waltham, MA, R0601). Pellets werebriefly air-dried and then resuspended in DEPC water,
RNA concentrations (ng/μL) and RNA purity (260:280)
were calculated using a Cytation 3 (Biotek, Winooski,
VT). At least 500 ng/sample of RNA were sent to Azenta
LLC (South Plainfield, NJ, USA) for subsequent RNA
library preparation and total RNA sequencing (20–30
million reads). RNA samples received by Azenta LLC
were quantified using Qubit 2.0 Fluorometer (LifeTechnologies, Carlsbad, CA, USA) and RNA integrity waschecked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).
RNA sequencing

Full details of RNA library preparation can be obtained
from Azenta LLC (South Plainfield, NJ, USA). Briefly,
RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina using
manufacturer’s instructions (NEB, Ipswich, MA, USA).
Briefly, mRNAs were initially enriched with Oligo(dT)beads. Enriched mRNAs were fragmented for 15 min
at 94 °C. First and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired

Pearson et al. Journal of Neuroinflammation(2024) 21:194

and adenylated at 3’ends, and universal adapters wereligated to cDNA fragments, followed by index additionand library enrichment by PCR with limited cycles. The
sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), andquantified by using Qubit 2.0 Fluorometer (Invitrogen,
Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on a single lane of a flow cell. After
clustering, the flowcell was loaded on the Illumina HiSeq
4000 according to manufacturer’s instructions. The samples were sequenced using a 2 × 150 bp Paired End (PE)configuration. Image analysis and base calling wereconducted by the HiSeq Control Software (HCS). Raw
sequence data (.bcl files) generated from Illumina HiSeq
were converted into fastq files and de-multiplexed using
Illumina’s bcl2fastq 2.17 software. One mismatch wasallowed for index sequence identification.
Bioinformatics

Bioinformatic processing was performed as previously
described [29]. Breifly, sequencing data were uploaded
to the galaxy web platform, and the public server usegalaxy.org was used to analyze the data [1]. Raw data in
the fastq format were first pre-processed using the sample quality control (QC) program FastQC [3], QC data
were then aggregated using MultiQC [30]. All data werethen processed through Trimmomatic [11], to remove
adapter sequences, poly-N-containing reads, and lowquality reads (Phred score < 25). Paired end reads werethen aligned to the mouse genome using HiSAT2 and the
GRCm38 (mm10) mouse reference genome [57]. Duplicate reads were removed, and gene expression in FPKM
(Fragments Per Kilobase Million) was measured using
FeatureCounts [63]. Sample data were aggregated into asingle data frame using column join, and gene IDs wereannotated using the AnnotateMyIDs program [27]. Batch
correction was performed using RUVSeq [107], and differential expression analysis was performed using the
DESeq2 program [65]. Resulting p values were adjusted
using the Benjamini and Hochberg approach to control
for false discovery rate. Genes identified using DESeq2
that reported a false discovery rate corrected p valueof < 0.05 were regarded as differentially expressed genes(DEGs).
Gene enrichment analysis

Data from all experiments were subsequently uploaded
to Ingenuity pathway analysis (IPA, QIAGEN). Datasets were filtered for differentially expressed genes(FDR < 0.05), and both significantly up and downregulated genes were analysed together to identify enrichedcanonical pathways. The ingenuity knowledge base wasPage 6 of 24

used as a reference set to identify significantly enrichedpathways following analysis by Fisher’s exact test and theactivation or inhibition of enriched pathways was determined with the use of a Z-score.
Statistical analysis

GraphPad PRISM software (GraphPad Software Inc.) wasused to generate the graphs and perform statistical analysis. The results are expressed as mean ± SEM. Statisticaltests performed are stated in the corresponding figurelegend.

Results
Pioglitazone treatment inhibits LPS‑induced microglial
NF‑κB activation in vitro

PPARγ activation has been suggested to exert antiinflammatory effects on microglia and macrophages. As
such, we hypothesized that prolonged PPARγ activation
would ameliorate microglial activation and inflammatory signaling following r-mTBI. But first, we assessed
the anti-inflammatory potential of PPARγ agonism in
microglia, by exploring the effects of the well-known
PPARγ agonist Pioglitazone on LPS-exposed immortalised microglia (IMG) in vitro. Following LPS exposurefor 24 h, PPARγ transcriptional activity was significantly
reduced compared to controls. However, co-administration of LPS and Pioglitazone to IMG cells prevented the
LPS-induced decrease in PPARγ transcriptional activity(Fig. 1A). One such mechanism through which PPARγ isthought to reduce inflammation is via the inhibition of
NFκB signaling [17, 24]. As such we examined the lysatesof IMG cells exposed to either LPS (10 ng/mL), Pioglitazone (10 µM), or both for 24 h and assessed the effects
on NFkB activation. As expected, LPS exposure alone
significantly increased NFkB phosphorylation compared
to control samples. In line with the Pioglitazone-induced
increase in PPARγ transcriptional activity we observedthat Pioglitazone treatment was able to significantly
reduce the NFkB phosphorylation relative to cells treatedwith LPS alone (Fig. 1B).
Pioglitazone treatment ameliorates chronic deficits
in spatial learning and memory

After observing prominent anti-inflammatory effects of
Pioglitazone treatment in vitro, we next sought to assess
how PPARγ agonism may influence the chronic outcomes of r-mTBI. To this end, mice were exposed to our
20-hit r-mTBI paradigm and were treated with either low(12.5 ppm) or high (50 ppm) Pioglitazone for 3 months
starting at 3 months post injury. Seven days before theend of treatment mice underwent Barnes maze testing toassess spatial and learning memory (Fig. 2).

Pearson et al. Journal of Neuroinflammation(2024) 21:194

Page 7 of 24

Fig. 1 PPARγ activation reduces the LPS-induced inflammatory response in microglia. Immortalised microglia (IMG) were exposed to LPS(10 ng/ml) or LPS (10 ng/ml) and Pioglitazone (10 µM) for 24 h before harvesting. A Assessment of PPARγ transcriptional activity in IMG cells
following control, LPS, or LPS + Pioglitazone treatment. B Quantification of the Phosphorylated NFκB (Ser536) to total NFκB ratio in microglial celllysates following exposure to control, LPS, or LPS + Pioglitazone treatment for 24 h. C Representative immunoblot image showing the protein
expression of Phospho and total NFκB in microglia. α-Tubulin served as a loading control. Data were analyzed by a one-way ANOVA followed
by Tukey’s correction for multiple comparisons (n = 3/group). Asterisks denote statistical significance as follows: *p < 0.05, ***p < 0.001

Fig. 2 Schematic representation of the experimental timeline. At 12 weeks of age, male C57BL6/J mice were exposed to our 20-hit r-mTBI
paradigm consisting of five injuries per week for four weeks. Injuries were separated by 24 h. At three months post-last injury, sham and injured
mice were randomly selected for low or high dose Pioglitazone treatment for three months. At one week prior to the end of treatment, spatial
memory and learning function was assessed using the Barnes maze

Spatial learning was tested by assessing cumulative
distance to the target hole (CD) over the 6-day acquisition phase (Fig. 3A). A three-way mixed ANOVA wasrun to investigate the effects of injury, treatment, andtime on cumulative distance. At six months post-injury
there was a statistically significant three-way interaction between treatment, r-mTBI, and time (F(7.694,
42) = 3.285, p = 0.002). Further analysis using a two-way
ANOVA found a significant interaction of injury andtreatment on spatial learning on days 3 (F(2,62) = 4.099,
P = 0.021), 5( F(2,62) = 4.229, P = 0.019), and 6(F(2,62) = 10.399, P ≤ 0.001) of the acquisition phase.
Post-hoc analysis revealed a significant main effect of

injury on day 3, and significant effects of injury andtreatment days 5 and 6. We observed a greater recorded
CD in r-mTBI animals who received either vehicle(p = 0.011) or low dose Pioglitazone (p = 0.013) treatment relative to their sham counterparts respectively.
Pairwise comparison analysis revealed that injured animals treated with high dose Pioglitazone demonstrated
a significantly reduced CD relative to injured animals
who received vehicle treatment on both days 5 and 6.
Interestingly, while Pioglitazone treatment had no significant effect on sham animals regardless of dose, we
observed an indication of a dose–effect in r-mTBI animals, with those treated with high dose Pioglitazone

Pearson et al. Journal of Neuroinflammation(2024) 21:194

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Fig. 3 PPARγ activation ameliorates r-mTBI associated memory and learning deficits. Acquisition data shows learning across the six acquistion
phase by the mean cumulative distance to the target hole in cm (A), and the mean latency to find the target hole (B). Probe data shows the meanlatency to find the target hole (C), number of errors (D), average velocity (E), and total distance traveled (F). Acquisition trials (A, B) were analyzed
using a repeated measures three-way ANOVA followed by Tukey’s correction for multiple comparisons (n = 10–12/group). Probe data (C-F) wereanalyzed using a two-way ANOVA followed by by Tukey’s correction for multiple comparisons. Asterisks denote statistical significance between TBI
vehicle and sham vehicle treated mice as follows: *p < 0.05, **p < 0.01, ***p < 0.001. $ denote statistical significance between TBI High dose and TBI
vehicle treated mice as follows: $p < 0.05, $$p < 0.01, $$$p < 0.001. Values are expressed as mean ± SEM

performing significantly better than low dose treated
r-mTBI animals (p = 0.025).
Spatial learning and cognitive function were further
assessed by recording the latency to first acquire the target hole (Fig. 3B). Mice exposed to our r-mTBI paradigm
took significantly longer to locate the target hole compared to sham animals, but no significant effect of treatment was observed over the 6 day.
After 6 days of training, the hidden target box was
removed, and spatial memory was assessed by their ability to locate the target hole using the visual clues in theirenvironment. Consistent with our data from the acquisition phase, injured mice took significantly longer to findthe target hole than their sham counterparts (Fig. 3C).
Two-way ANOVA analysis found no significant main
effect of treatment. However, a significant interaction
between r-mTBI and treatment was recorded. Post-hoc

analysis revealed that injured mice who received vehicle
treatment took significantly longer to find the target holethan vehicle-treated sham animals (p ≤ 0.001). While ourdata indicate that low dose Pioglitazone had no effecton latency to find the target hole, injured mice treatedwith high-dose Pioglitazone demonstrated a substantial
reduction in target hole latency compared to vehicletreated r-mTBI mice (P = 0.039). No significant difference
was noted between injured mice treated with high-dose
Pioglitazone and sham animals who received high-dose
Pioglitazone (P = 0.977) or vehicle treatment (P = 0.619).
Notably, while some mice would acquire the target
hole as expected, analysis of the footage indicated somemice would circle the maze moving from hole to hole in
an attempt to locate the target box. Therefore, in addition to latency, we also analyzed number of entry errors(Fig. 3D). We observed that vehicle-treated injured micePearson et al. Journal of Neuroinflammation(2024) 21:194

made significantly more errors during the probe trial
than their vehicle-treated sham counterparts (p ≤ 0.001).
However, injured mice who received high dose Pioglitazone demonstrated a significant decrease in the number of errors relative to vehicle-treated r-mTBI mice(p = 0.002), indicating improved retention of spatial
memory.
Throughout behavioral analysis, we recorded the average velocity and distance travelled to assess motor function (Fig. 3E, F). Statistical analysis found no significant
effects of injury or treatment on average velocity or distance travelled suggesting exposure to r-mTBI does not
induce motor deficits.
Pioglitazone treatment rescues chronic reactive gliosis

We have previously demonstrated that while exposure to
our 20-hit r-mTBI paradigm does not induce gross neuronal loss, it does result in chronic reactive gliosis up to
3 months post-injury [100]. As inflammation is linked tobehavioural impairments [7, 46], we examined the effectof Pioglitazone treatment on chronic microglia and astrocyte reactivity following r-mTBI. As behavioural testing
found a limited effect of low dose Pioglitazone treatment,
our subsequent analyses focused on the assessment ofhigh dose Pioglitazone.
Brain tissue was collected at 6 months post-last injury,
tissue sections were subsequently labeled with anti-Iba1,

Page 9 of 24

anti-CD68, and anti-GFAP and immunoreactive populations of microglia and astrocytes were quantified in thecortex (underneath the impact site), the hippocampus,
and the corpus callosum. Building upon our prior work,
we observed that exposure to r-mTBI induced a significant increase in Iba1 immunoreactivity in all three
regions assessed (Fig. 4A–O). Treatment with Pioglitazone in injured mice resulted in a significant reduction in Iba1 in the hippocampus but showed no effect in
the Cortex or Corpus Callosum. We next assessed theexpression of the marker CD68, a lysosome-associatedmembrane protein which has been implicated in theregulation of phagocytic activity [34, 47]. We observed asignificant injury-dependent increase in CD68 immunoreactivity in the Cortex, and Corpus Callosum which wassignificantly ameliorated following Pioglitazone treatment (Fig. 5A–O).
Our findings also demonstrate an impact of Pioglitazone treatment on reactive astrocyte populations inthese brain regions. Specifically, we observed that exposure to r-mTBI induced a significant increase in GFAP
immunoreactivity in Cortex, Hippocampus, and Corpus
Callosum (Fig. 6A–O). However, r-mTBI mice treatedwith Pioglitazone demonstrated a significant decreasein GFAP immunoreactivity in all regions assessed suggesting that PPARγ agonism may be an effective target to
reduce chronic reactive glial changes following r-mTBI.

Fig. 4 Effect of PPARγ activation by Pioglitazone on microglial activation (Ionized calcium binding adaptor molecule 1 [Iba1]) at 6 months
post r-mTBI. Quantitation and representative microscope images of ­Iba1+ve immunoreactive area in the cortex (A–E), hippocampus (F–J),
and Corpus Callosum (K–O) (n = 7/8 per group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction for multiple
comparisons. All data are expressed as mean ± SEM percentage reactive area

Pearson et al. Journal of Neuroinflammation(2024) 21:194

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Fig. 5 Effect of PPARγ activation by Pioglitazone on microglial activation (Cluster of differentiation 68 [CD68]) at six months post r-mTBI.
Quantitation and representative microscope images of ­CD68+ve immunoreactive area in the cortex (A–E), hippocampus (F–J), and Corpus Callosum(K–O) (n = 7/8 per group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction for multiple comparisons. All data areexpressed as mean ± SEM percentage reactive area

Fig. 6 Effect of PPARγ activation by Pioglitazone on astrocyte activation (Glial fibrillary acid protein [GFAP]) at six months post r-mTBI. Quantitation
and representative microscope images of ­GFAP+ve immunoreactive area in the cortex (A–E), hippocampus (F–J), and Corpus Callosum (K–O)(n = 7/8 per group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction for multiple comparisons. All data are expressed
as mean ± SEM percentage reactive area

Pearson et al. Journal of Neuroinflammation(2024) 21:194

Pioglitazone treatment rescues microglial PPARγ signaling

With promising evidence of beneficial effects observed
during behavioral and neuropathological analyses, wenext sought to better understand the microglial specific neuroinflammatory mechanism that drives r-mTBI
pathophysiology and how PPARγ agonism via Pioglitazone treatment may beneficially influence these pathogenic cascades. Herein, we isolated ­CD11b+ve microglial
cells from the brains of vehicle or Pioglitazone treatedsham and injured mice at six months post-injury andassessed the activation of PPARγ signaling and inflammatory signaling pathways.
To assess how exposure to r-mTBI may affect microglial PPARγ signaling we assessed the DNA binding
activity of PPARγ via transcriptional activity assay andquantified the expression of PPARγ and its co-factor
PGC1α by immunoblotting. In line with our in vitro
findings, PPARγ transcriptional activity was inhibitedin microglia isolated from vehicle-treated r-mTBI micerelative to sham animals. However, we observed no effectof Pioglitazone treatment on brain myeloid cell specific
PPARγ transcriptional activity in either sham or injured
animals relative to their respective vehicle-treated controls (Fig. 7A).

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We next asked if the changes in transcriptional activity also reflected changes in PPARγ protein expression. Immunoblotting revealed that PPARγ expression
was reduced in microglia isolated from vehicle-treated
r-mTBI mice relative to their sham vehicle-treatedcounterparts. Post-hoc analysis revealed that Pioglitazone treatment had no significant effect on PPARγ
expression in sham animals relative to sham vehicle-treated controls. However, in injured animals,
we observed that pioglitazone treatment markedly
increased PPARγ expression relative to vehicle treatedanimals (Fig. 7B).
To further develop these findings, we assessed if thebeneficial effects of Pioglitazone treatment observed
may be mediated through changes in the protein
expression of PGC1α (Fig. 7C). PGC1α is a transcriptional coactivator of PPARγ and has been implicated in
the regulation of several genes associated with microglial polarization, neuroinflammation, and mitochondrial biogenesis. In accordance with our findings on
PPARγ expression, we observed that microglial PGC1αexpression was chronically reduced in mice exposed
to r-mTBI but was rescued following Pioglitazone
treatment.

Fig. 7 Pioglitazone treatment rescues microglial PPARγ signaling and reduces inflammatory signaling. A Quantitation of PPARγ transcriptional
activity in isolated microglia. Quantitation and representative immunoblots of PPARγ (B), PGC1α (C), Phospho NFκB (Ser536) (D), Phospho STAT3(Tyr705) (E), and NLRP3 (F) in microglia isolated at six months post last injury from sham and injured mice who received Pioglitazone or vehicle
treatment (n = 4/group). (B) (n = 3/group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction for multiple comparisons. Alldata are expressed as mean ± SEM percentage of control

Pearson et al. Journal of Neuroinflammation(2024) 21:194

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Pioglitazone treatment reduces chronic microglial
pro‑inflammatory signaling

significantly reduce STAT3 phosphorylation comparedto vehicle-treated r-mTBI mice.
In addition to upregulating the expression of proinflammatory cytokines such as IL-6, IFNγ, and TNFα,
NFκB activation has received increasing attention as apivotal step in the activation of to the nucleotide-binding domain and leucine-rich repeat (NLR) family pyrin
domain-containing protein 3 (NLRP3) inflammasome[2, 67]. This multimeric protein complex is an important
link in the production of interleukin 1β (IL-1β), a proinflammatory cytokine that has repeatedly been implicated in secondary neuronal injury and the developmentof cognitive impairment and neurological disorders [13,98, 108]. NLRP3 expression was significantly increased in
microglia isolated from mice exposed to r-mTBI relative
to sham mice. However, NLRP3 expression was markedly inhibited in injured mice who received pioglitazone
relative to injured mice who received vehicle treatment(Fig. 7F). However, we did note that NLRP3 expression
was significantly increased amongst sham animals who
received Pioglitazone relative to sham vehicle-treatedcontrols.

After demonstrating in vivo target engagement, wesought to assess how PPARγ agonism influences the
microglial inflammatory signaling cascades in the aftermath of r-mTBI. As such we assessed the expression
and activation of the pro-inflammatory transcription
factors NFκB and STAT3. Both NFκB and STAT3 are
well-acknowledged pro-inflammatory mediators andhave been heavily implicated in the pathogenesis ofneurodegenerative disorders [16, 55, 74, 119].
NFκB is a central component of the inflammatorycascade, as an early response transcription factor theactivation of NFkB is regulated by phosphorylation anddoes not require the synthesis of new proteins enablingits immediate response to injury. As such, we assessed
NFκB phosphorylation (Serine 536) to determine theeffect of Pioglitazone treatment on the activation of
NFκB signaling in isolated microglia (Fig. 7D). In linewith our in vitro evidence, NFκB phosphorylation
was significantly increased in microglia isolated fromvehicle-treated r-mTBI mice compared to sham vehicle mice. Notably, the injury-induced increase in NFκB
phosphorylation was reduced in mice treated with
pioglitazone compared to vehicle treated mice.
Several studies have previously implicated microglial STAT3 pathway activation with the initiation ofseveral pro-inflammatory processes in the aftermathof experimental brain trauma. As such, we assessed
the effect of PPARγ agonism on STAT3 pathway activation (Fig. 7E). We observed that exposure to r-mTBI
induced a chronic increase in microglial STAT3 phosphorylation compared to sham mice. However, our data
demonstrate that pioglitazone treatment was able toPioglitazone treatment reduces microglial
pro‑inflammatory cytokine expression

To further examine the effects of r-mTBI and pioglitazone treatment on the inflammatory response to
r-mTBI, we measured the intracellular concentrationsof the pro-inflammatory cytokines IL-6, and TNFα in
microglia isolated from sham and injured mice using a
multiplex cytokine array. In line with our previous findings, microglia isolated from the brains of injured vehicle-treated mice expressed significantly higher levels of
IL-6 (p = 0.004) (Fig. 8A), and TNFα (p ≤ 0.001) (Fig. 8B)
relative to sham vehicle-treated animals. Furthermore,

Fig. 8 PPARγ activation reduces chronic microglial pro-inflammatory cytokine production following exposure to r-mTBI. Microglia were isolated
from sham and injured mice who received Pioglitazone (50ppm chow) or vehicle treatment and the concentrations of IL-6 (A) and TNFα (B) in celllysates were assessed using the MSD multiplex cytokine array (n = 4/group). Data were analyzed using a two-way ANOVA followed by Tukey’s
correction for multiple comparisons. All data are expressed as mean ± SEM percentage of control

Pearson et al. Journal of Neuroinflammation(2024) 21:194

post-hoc analysis indicated that the injury-induced
increase in TNFα was significantly blunted in injured
mice who received Pioglitazone treatment relative to
vehicle treated animals. However, Pioglitazone treatment in injured mice had no effect on IL-6 expression
relative to injured mice who received vehicle treatment(p = 0.158).
Generation of a mouse model of microglial specific PPARγ
overexpression

Our results demonstrate that delayed Pioglitazone treatment is effective in ameliorating the chronic microglial
activation associated with exposure to r-mTBI. However, the broad expression of PPARγ by multiple CNS
cell types [130], and the off-target effects of pharmacological PPARγ agonists make it difficult to disentangle themicroglial specific effects of PPARγ activation to r-mTBI
outcomes [136]. As such, we generated a microglial-specific tamoxifen-inducible mouse model of PPARγ activation by crossing the VP16-PPARγ LoxP mouse line with
the CX3CR1-Cre recombinase mouse line (Fig. 9A, B).
In this model, the constitutive overexpression and activation of PPARγ in CX3CR1-expressing cells is driven

Page 13 of 24

by the CAG-VP16 cytomegalovirus (CMV) immediate early enhancer/ chicken β-actin (CAG) promoter.
Tamoxifen administration enables the translocation of
CreER recombinase to the nucleus where it excises thefloxed stop sequence. With the stop cassette removed,
the PPARγ gene is now under the control of the CAG
promoter. The CAG promoter is a strong, constitutive
promoter composed of the CMV enhancer element, thechicken β-actin promoter, and the rabbit β-globin splice
acceptor site, which induces high levels of gene expression. Furthermore, the VP16 sequence fused to PPARγ isa transcriptional activation domain derived from the herpes simplex virus that enhances the transcriptional activity of PPARγ. This activation is ligand-independent andresults in a more potent and constant activation, bypassing the regulatory control of PPARγ activation exerted byligand binding and co-factor interactions.
To validate the model, 8-week-old mcgPPARγOE mice
were treated with tamoxifen or vehicle for 5 days. At
28 days post-last treatment, mice were euthanized and­CD11b+ve microglia were isolated and PPARγ expression
and transcriptional activity was assessed (Fig. 9C, D). We
observed a significant increase in the protein expression

Fig. 9 Generation of a tamoxifen inducible microglial-specific PPARγ overexpression mouse line. A, B Graphic showing the generationof conditional PPARγ overexpressing mice and how tamoxifen treatment induces cre recombination in mcgPPARγOE mice. C To validatethat tamoxifen treatment was able to induce cre-recombination, non-injured mcgPPARγOE mice received Tamoxifen (50mg/kg) or vehicle treatment
for 5 days and the transcriptional activity of PPARγ in isolated microglia was assessed. D Quantitation and representative immunoblot of PPARγ
expression in microglia isolated from vehicle and Tamoxifen treated mice (n = 5–11/group) E Graphic showing the experimental schedule. After
validating that our treatment paradigm was sufficient to induce microglial PPARγ overexpression, 8 week old mcgPPARγOE and ­CX3CR1CreERt2+/+(Cre) mice received Tamoxifen for 5 days. At 12 weeks old, mice were exposed to our 20-hit r-mTBI paradigm or sham procedure. At 30 days post last
injury all mice were euthanised and the brain tissue was processed for histopathology or microglia were isolated from the brain tissue for RNA
sequencing

Pearson et al. Journal of Neuroinflammation(2024) 21:194

and transcriptional activity of PPARγ in microglia isolated from mice treated with tamoxifen compared tocontrols. Following validation of the mouse model, wesought to investigate how microglial-specific PPARγ
overexpression influenced the microglial response to
r-mTBI. PPARγOE and Cre-littermate mice were treatedwith tamoxifen at 8 weeks old for one week, then allowed
3 weeks for peripheral monocyte turnover. Mice were
exposed to our 20-hit paradigm at 12 weeks old andthen we assessed the microglial phenotype at 1 month
post last injury by path and microglial RNA sequencing(Fig. 9E).
Microglial‑specific PPARγ overexpression rescues chronic
reactive gliosis

To assess the effect of microglial-specific PPARγ overexpression on the response to r-mTBI we assessed Iba1
and CD68 immunoreactivity. In accordance with ourfindings from wild type mice, we observed that exposure
to r-mTBI increased Iba1, CD68, and GFAP immunoreactivity in the cortex, hippocampus and corpus callosum
regardless of genotype. However, injured mcgPPARγOE
mice exhibited significant reductions in Iba1, CD68,
and GFAP immunoreactivity compared to injured­CX3CR1CreERt2+/+ controls in all areas assessed (Figs. 10,
11, 12A–O).

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Microglial‑specific PPARγ overexpression ameliorates
the pro‑inflammatory phenotype in response to r‑mTBI

To further investigate the effect of mcgPPARγOE on
the microglial phenotype following r-mTBI we performed unbiased RNA-sequencing on microglia isolated from both Injured and sham mcgPPARγOE miceand ­CX3CR1CreERt2+/+ littermates at one month post
last-injury.
We firstly analysed the effect of r-mTBI on the­CX3CR1CreERt2+/+ mice to establish a baseline effect of
injury on the microglial phenotype (Fig. 13A, B). Differential expression analysis identified 1078 (708 Up, 370Dn) differentially expressed genes (DEGs) in microglia
isolated from Cre TBI and Cre sham mice. Exposure to
r-mTBI induced the upregulation of several genes commonly observed in disease associated microglia (DAM)(Itgax, Cst7, Lpl, Clec7a, Hif1a, ApoE, Spp1) [14, 22, 62].
To better understand the altered signaling pathways andbiological functions of microglia isolated from injured
mice we performed gene enrichment and pathway analysis using IPA. Enrichment analysis revealed the significant upregulation of genes associated with Cytokine
signaling, phagosome formation, and inflammasome
pathway activation. Furthermore, we observed the downregulation of biological pathways associated with LXR/
RXR, TGF-b, and AMPK signaling.

Fig. 10 Microglial-specific PPARγ overexpression reduces microglial Iba1 immunoreactivity at one month post r-mTBI. Quantitation
and representative microscope images of ­Iba1+ve immunoreactive area in the cortex (A–E), hippocampus (F–J), and Corpus Callosum (K–O)
of mcg-PPARγOE and ­CX3CR1CreERt2+/+ (Cre) (n = 6 per group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction
for multiple comparisons. All data are expressed as mean ± SEM percentage reactive area

Pearson et al. Journal of Neuroinflammation(2024) 21:194

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Fig. 11 Microglial-specific PPARγ overexpression reduces microglial CD68 immunoreactivity at one month post r-mTBI. Quantitation
and representative microscope images of ­CD68+ve immunoreactive area in the cortex (A–E), hippocampus (F–J), and Corpus Callosum (K–O)
of mcg-PPARγOE and ­CX3CR1CreERt2+/+ (Cre) (n = 6 per group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction
for multiple comparisons. All data are expressed as mean ± SEM percentage reactive area

Fig. 12 Microglial-specific PPARγ overexpression reduces astrocyte GFAP immunoreactivity at one month post r-mTBI. Quantitation
and representative microscope images of ­GFAP+ve immunoreactive area in the cortex (A–E), hippocampus (F–J), and Corpus Callosum (K–O)
of mcg-PPARγOE and ­CX3CR1CreERt2+/+ (Cre) (n = 6 per group). Data were analyzed using a two-way ANOVA followed by Tukey’s correction
for multiple comparisons. All data are expressed as mean ± SEM percentage reactive area

Pearson et al. Journal of Neuroinflammation(2024) 21:194

Page 16 of 24

Fig. 13 PPARγ overexpression modifies the microglial response to r-mTBI. Volcano plots of injury-induced differential gene expression
and histograms showing IPA gene enrichment of altered biological pathways in isolated microglia between injured and sham­CX3CR1CreERt2+/+- VP16-PPARγWT/WT (Cre) mice (A, B), injured and sham mcgPPARγOE mice (C, D), and injured mcgPPARγOE vs injured C­ X3CR1CreERt2+/+
mice (E, F), respectively (n = 4–5/group). Histograms show activation and inhibition of biological pathways by Z-score, corresponding heatmaps
show the significance of pathway enrichement as − ­Log10 pValue

We next analysed the effect of r-mTBI exposure on
microglia transcriptomic phenotype in mcgPPARγOE
mice (Fig. 13C, D). Differential expression analysis identified only 118 (112Up, 16Dn) differentially expressed
genes between injured and sham mice (approximately
tenfold less DEG’s than TBI vs sham ­CX3CR1CreERt2+/+mice). However, analysis of these differentially expressed
genes revealed that injured mcgPPARγOE mice still
exhibited a marked increase in some inflammatory andDAM genes (Lpl, Itgax, Spp1, Cst7, ApoE, Clec7a). This
inflammatory response was also identified following
gene enrichment analysis with cytokine signaling and
IL-12 signaling pathways being significantly upregulated.
Downregulated biological pathways between microglia isolated from injured and sham mcgPPARγOE mice
included NAD signaling, FAK signaling, and Anandamide signaling.
We then compared the transcriptome of microglia isolated from injured mcgPPARγOE and­CX3CR1CreERt2+/+ mice to examine how PPARγOE influences the inflammatory response in the chronic aftermath of r-mTBI (Fig. 13E, F). Differential expression
analysis identified 1971 DEGs (603Up, 1368Dn). Top
upregulated genes in injured PPARγOE mice included
Gprasp2, Plcg1, Ide, Cfap141, and Ccr5. Downregulated

genes included Cstc, Siglecf, Tlr2, Cstl, and Rpl19. Geneenrichment analysis revealed that injured PPARγOE
mice exhibited an upregulation of genes associated with
reparative pathways, such as IL-10 signaling, PPARγ
signaling, and myelination signaling pathways and adownregulation of genes associated with Cytokine signaling, Neuroinflammation, and EIF2 signaling.
Microglial‑specific PPARγ overexpression reduces
pro‑inflammatory, and Disease associated microglia Gene
expression

To assess how the microglial responses to r-mTBI and
PPARγ overexpression relate to other neurological diseases, we used a hypothesis driven approach to compare
the microglial transcriptomes from sham and injured
mcgPPARγOE and ­CX3CR1CreERt2+/+ mice against thegene signature of DAM microglia seen in certain neurodegenerative disorders (Fig. 14). We observed that
exposure to r-mTBI results in the increased expression of several DAM genes and the downregulation of
homeostatic genes. Notably, mcgPPARγOE was able toprevent this transition towards a “DAM-like” state suggesting a neuroprotective phenotype.

Pearson et al. Journal of Neuroinflammation(2024) 21:194

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Fig. 14 Microglial PPARγ overexpression prevents the adoption of a disease associated microglia-like (DAM-like) phenotype at 30 days post r-mTBI.
Heatmap depicting the expression of DAM genes (rows) across ­CX3CR1CreERt2+/+ (Cre) and mcgPPARγOE mice (n = 4–5/group) who received r-mTBI
or sham procedures (columns). Data are expressed as row z-score (red corresponds to gene upregulation and blue to downregulation)

Discussion
Chronic neuroinflammation and microglial activation
are hallmark features of traumatic brain injury [44, 64,
117, 134]. However, the underlying mechanisms driving microglial pathobiology in the chronic aftermath of
r-mTBI remain unclear [77]. PPARγ is a nuclear transcription factor that is well expressed in immune cells
such as microglia [9, 10, 121]. As a nuclear transcription factor, PPARγ signaling has been implicated inseveral cellular functions, including lipid and glucose
metabolism, cell proliferation, and inflammation [12,35, 80, 112, 115, 129]. While several studies have investigated the potential of PPARγ agonism to beneficiallyinfluence the pathogenic cascades that mediate secondary tissue damage following brain trauma [24, 49, 53,
137, 139, 142], dissecting the microglial specific effectsof PPARγ agonism has thus far been difficult to achievein vivo. Therefore, in the present study, we utilized
both pharmacological studies and a conditional transgenic mouse overexpressing PPARγ in microglia toinvestigate the beneficial effects of PPARγ agonism on
microglial pathobiology in the aftermath of r-mTBI. We
demonstrated that activation of PPARγ reduces microglial inflammatory pathway activation and inhibits proinflammatory cytokine production. Furthermore, we
also present evidence to demonstrate the delayed Pioglitazone treatment can ameliorate r-mTBI neuropathology and cognitive impairment.
In the present study we found that exposure to r-mTBI
or the pro-inflammatory stimuli LPS inhibited theexpression and activity of PPARγ in microglia. While asimilar decrease in PPARγ expression and activity has
been observed in several studies of CNS and peripheral

Pearson et al. Journal of Neuroinflammation(2024) 21:194

inflammation [15, 43, 45, 79, 95, 125, 132], the exact
mechanism driving the downregulation of PPARγ expression and activity in response to inflammatory stimuli is
still unclear. Furthermore, while the true cause of thisdownregulation is likely disease specific, Shibuya and colleagues proposed that the increased production of superoxide and Nitric oxide (NO) radicals in response to LPS,
TNFα, or peroxynitrite exposure results in the nitrationof PPARγ tyrosine residues, preventing ligand interaction
and subsequent nuclear translocation. Furthermore, theauthors noted that due to the central roles of PPARγ in
inflammation and immune responses, the suppression of
PPARγ activity by ROS might promote chronic or recurrent inflammation [114]. Together these data support the
hypothesis that impaired PPARγ signaling in microglia isa critical driver of chronic dysregulated microglial activation and therefore represents a promising therapeutic target to ameliorate CNS inflammation and improve
r-mTBI outcomes.
Cognitive and functional impairments are amongst
the most commonly reported clinical symptoms of TBI[26, 113]. The remediation of these learning and memory
deficits remains a major aspiration of both clinical andpreclinical TBI researchers. However, no therapeuticinterventions have thus far demonstrated substantial efficacy [36, 66, 120]. Our laboratory has previously developed several preclinical models of repetitive brain trauma
using closed head mid-line impact model of r-mTBI
using the same impact parameters but varying the quantity and chronicity of impacts [28, 29, 81, 83–86, 94, 96,100]. Consistent with our previous work, here we demonstrated that exposure to our 20-hit r-mTBI paradigm
induces chronic spatial learning and memory deficits. We
observed that Pioglitazone treatment markedly improved
performance in both the acquisition and probe phases of
the Barnes maze. These results are in line with the workof several others who have reported improved cognitive
performance in preclinical models of CNS trauma, neurodegeneration, and aging in response to PPARγ agonist treatment [20, 89, 101, 128]. The close link between
diabetes and the development of cognitive decline has
led many researchers to investigate how Pioglitazone
may prevent cognitive dysfunction. While the complete
mechanisms underlying the neuroprotective activity of
Pioglitazone have not yet been fully elucidated, multiple
studies have shown that pioglitazone treatment increased
short- and long-term memory through the preservation of synaptic plasticity and integrity [18, 111]. Somestudies have suggested that the neuroprotective effect ofPioglitazone is mediated through its ability to preventthe mis-sorting and hyperphosphorylation of tau via theinhibition of CDK5 [79]. While tau pathology is highly
relevant to TBI and is considered a pathognomic feature

Page 18 of 24

of neurodegenerative disorders such as chronic traumatic
encephalopathy (CTE), previous studies of r-mTBI fromour laboratory have shown little evidence of tau pathology out to one-year post-injury in wild-type mice suggesting that the development of toxic tau species is likely
not a driving factor of the cognitive impairment seen inthis study [83].
In both our protein and transcriptome analysis of
isolated microglia we observed that microglial PPARγ
agonism reduces chronic pro-inflammatory activation
following r-mTBI via the suppression of NFκB signaling.
In the CNS, NFkB signaling has been implicated in regulating several physiological and pathological processes.
Under baseline conditions, NFkB resides in the cytoplasm in a complex with inhibitory IkB proteins [41, 42].
However, following stimulation by DAMPs, cytokines, oroxidative stress NFkB rapidly becomes activated and istranslocated to the nucleus, where it initiates transcriptional programs which mediate the expression of several
inflammatory genes [61]. NFkB is thought to be criticalin the propagation of inflammatory responses, as manyof the NFkB-induced gene expression products act on
receptors that, in turn, also activate NFkB. This feed forward mechanism of NFkB is thought to be a major mediator of the chronic inflammation that follows TBI [92].
PPARγ agonism has been shown to inhibit NFkB throughseveral potential mechanisms. In addition to the inhibition of NFkB via direct binding, PPARγ agonism has beenshown to suppress NFkB pathway signaling through IL-4
and IL-10 signaling [56, 106, 127]. This finding agrees
with our transcriptomic analysis of mcgPPARγOE mice
which found that PPAR activation increased IL-10 signaling suggesting a potential neuroprotective mechanism
for the observed improvements in cognitive and neuropathological outcomes.
While NFkB has long been acknowledged as a central
molecule in inflammation following TBI [92], the contributions of the NLRP3 inflammasome, or the adoption of
a DAM-like phenotype to TBI pathobiology, and importantly, their regulation by PPARγ have only recently
become acknowledged and are still under investigation[50, 117, 138]. NLRs are cytosolic pattern recognition
receptors that, when activated, form the inflammasome
complex, and mediate the production of IL-1β and IL-18
through caspase-1 processing [52, 122]. While theseinterleukins do play critical roles in normal wound healing, they have been shown to promote the accumulation of reactive oxygen and nitrogen species [5, 75, 118].
This feed-forward mechanism of oxidative damage andinflammasome activation has been suggested to promoteinflammatory neuronal cell death, known as pyroptosis,
and is commonly seen in neurodegenerative disorders[39, 40, 48]. Studies of both TBI and spinal cord injury

Pearson et al. Journal of Neuroinflammation(2024) 21:194

have shown that PPARγ agonism was able to inhibit the
NLRP3 mediated production of IL-1β through the inhibition of caspase-1, with these studies noting that reduced
NLRP3 activity was strongly correlated with improved
functional and pathological outcomes [76, 138]. Together,
the intricate interplay between PPARγ, NFκB, and inflammasome signaling underscores the complexity of neuroinflammatory mechanisms and the potential of PPARγ
modulation as a multifaceted therapeutic strategy.
In the present study, Tamoxifen treatment was administered before animals were exposed to our 20-hit r-mTBI
paradigm. While this prophylactic treatment paradigm
demonstrated effective target engagement and has beenused in several other studies to investigate the mechanisms of experimental brain injury [33, 135, 141], it ispossible that the effects we observed in this study may
have been even more pronounced if Tamoxifen treatment
was administered after exposure to experimental brain
injury. In addition to the increased clinical relevance ofa post-injury treatment paradigm, following exposure to
r-mTBI microglia rapidly proliferate and migrate towards
the injury site [25, 77, 133]. These “new-born” microglia which have not been exposed to Tamoxifen will nothave undergone Cre recombination and therefore will
not express constitutively active PPARγ, potentially limiting our analysis of the therapeutic efficacy of microglial
PPARγ overexpression. Therefore, future studies shouldcarefully consider the timing of gene recombination
when designing such studies.
It has to be acknowledged that in the present study
we did observe some negative side effects in shamanimals treated with Pioglitazone. Specifically, weobserved that some sham animals treated with Pioglitazone exhibited an increase in the microglial expressionof NLRP3. While the mechanism driving this phenomenon requires further investigation, the clinical use of
Pioglitazone is associated with multiple complications
including weight gain, fluid retention, congestive heart
failure, and accelerated bone loss [90]. The associatedside effects and poor pharmacokinetic profile of earlier generation thiazolidinediones such as Pioglitazone
and Rosiglitazone limit their potential clinical utility.
However, later generation PPARγ agonists such as therecently developed Leriglitazone may have the potential
to reduce the side effects associated with Pioglitazone.
Leriglitazone is a novel active metabolite of Pioglitazone which has been shown to promote neuroprotection in both neurons and astrocytes, improves axon
myelination, and preserves BBB integrity in models of
adrenoleukodystrophy [87]. Furthermore, pharmacokinetic and pharmacodynamic studies have demonstrated
that Leriglitazone has a greater ability to cross the BBB

Page 19 of 24[109], and better transcriptional efficacy than Pioglitazone [82]. Together, these factors mean that lower
doses of Leriglitazone could be able to ameliorate thechronic deleterious sequelae of r-mTBI while minimizing any potential negative side effects. Leriglitazone iscurrently being evaluated for the treatment of cerebral
adrenoleukodystrophy [109], adrenomyeloneuropathy[60], and Friedreich’s ataxia [99]. These conditions all
share a neuroinflammatory aspect which shows overlap with TBI [4, 131, 140]. As such, the investigation of
Leriglitazone in r-mTBI may hold strong clinical utility
for the treatment of traumatic brain injury.
In conclusion, our results highlight the potential of
Pioglitazone and microglial-specific PPARγ overexpression as promising strategies to modulate neuroinflammation and improve cognitive outcomes following
r-mTBI. It is particularly noteworthy that Pioglitazone’s
effects were not limited to the acute phase but extended
to chronic phases of r-mTBI, suggesting that there is a
prolonged timeframe in which treatment can elicit therapeutic benefits. The microglial-specific PPARγ overexpression model reinforced these findings, emphasizing
the importance of targeting microglial-specific pathways for long-term neuroprotection. While this study
provides promising insights, further investigations are
warranted to elucidate the precise molecular mechanisms underlying PPARγ-mediated neuroprotection.
As such, the development of mcgPPARγOE mice in thisstudy provides a valuable platform to interrogate themicroglial-specific therapeutic mechanisms of not only
PPARγ activation but also the activation of its downstream cellular mediators. Additionally, clinical translation of these findings may hold significant implications
for developing novel therapeutic interventions for individuals with a history of repeated brain injuries.
Acknowledgements
Dr. Crawford is a Research Career Scientist at the James A. Haley Veterans
Hospital, Tampa, FL. We would like to thank the Roskamp Institute for theirgenerosity in helping to make this work possible.
Author contributions
Conceptualization: AP, MM, FC, and JO. Methodology: AP, FC, and JO.
Investigation, AP, MK, ME, CO, MB, TV, and JO. Data analysis: AP, CT, ME, and
JO. Resources: MM, FC, and JO. Writing—original draft, AP and JO. Writing—review and editing: AP, ME, FC, and JO. Supervision: MM, FC, and JO. Funding
acquisition: FC and JO.
Funding
This work was supported by the National Institute of Health (NIH) under award
number 5R03AG075365-02 and by the U.S. Department of Defense underaward number W81XWH-17-1-0638 to FC and JO. Opinions, interpretations,conclusions, and recommendations are those of the author and are not necessarily endorsed by the National Institute of Health.
Availability of data and materials
The raw data that support the findings of this study are available upon reasonable request to the corresponding author.

Pearson et al. Journal of Neuroinflammation(2024) 21:194

Declarations
Ethics approval and consent to participate
The current study does not involve human participants, human tissue, ordata. All animal experiments were reviewed and approved by the Institutional
Animal Care and Use Committee (IACUC) at the Roskamp Institute (Protocol#R092). All procedures were performed in accordance with IACUC and the NIH
Guidelines for the Care and Use of Laboratory Animals.
Competing interests
The authors declare they have no competing interests.
Received: 15 February 2024 Accepted: 12 July 2024

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