NNS 2015 Program & Abstract Book

phenotypes.

In vitro

, cultured macrophages exposed to specific cy-

tokine cocktails express a relatively predictable gene expression

profile that has led to a classification system analogous to T-cell

polarization states. Specifically,

in vitro

stimulated macrophages can

be classified within activation states termed M1 and M2 (M2a, M2b,

M2c) depending upon exposure to a particular cytokine(s). Pro-

blematically, this linear classification system has been consistently

used to define microglia/macrophage response following brain

trauma, such that these responses are viewed as ‘either or’ or ‘on/

off’. We have recently shown that TBI induces a milieu of gene

expression responses associated with these defined polarization

states, which vary across time and importantly overlap such that both

M1 and M2 are expressed simultaneously. In the current study we

examined TBI-induced expression of M1 and M2 markers using

immunofluorescent labeling acutely after injury. Specifically, we

show that in the ipsilateral tissue there is simultaneous expression of

antigenic markers associated with both M1 and M2 profiles on the

same cell. Phenotypic antigen markers of M1 and M2 were CD45,

CD68, MARCO, YM1, CD206, and CD36. Co-staining using Iba1

and F4/80 defined these cells as microglia/macrophages. In the

context of trauma, the concept of microglia/macrophage polarization

is semantically and biologically incorrect due to the Janus-faced

nature of these cells.

Keywords: M1, M2, microglia, macrophage, polarization

C7-22

NOX2 REGULATION OF MICROGLIAL ACTIVATION IN

THE TBI BRAIN: A NOVEL MECHANISM FOR NEURO-

PROTECTION AND POST-TRAUMATIC REPAIR

David Loane

, James Barrett, Mariely Alvarez, Bogdan Stoica,

Flaubert Tchantchou, Alan Faden, Alok Kumar

University of Maryland, School of Medicine, Anesthesiology, Balti-

more, USA

Microglia can be polarized towards either an M1-like/classical or

M2-like/alternative activation status in response to injury. These

phenotypes can mediate chronic neuroinflammation or promote tissue

repair, respectively. Activation of NADPH oxidase (NOX2/gp91

phox

)

is an important mechanism involved in pro-inflammatory signaling in

microglia. We reported that NOX2 is chronically expressed in M1-

like microglia in the peri-lesional area 1 year following controlled

cortical impact (CCI) in mice. Here we compared wild-type (WT;

gp91

phox

) and NOX2-deficient (NOX2-KO; gp91

phox-/-

) mice to

investigate the role of NOX2 in posttraumatic microglial polarization.

Three-month old WT or NOX2-KO male mice were subjected to

CCI (6 m/sec, 2 mm depth), and cohorts were followed for 1–28 d

post-injury. M1-/M2-like polarization was analyzed by qPCR, flow

cytometry, Western blot, and immunohistochemistry. Neurogenesis

was assessed using doublecortin (DCX) immunohistochemistry; mo-

tor recovery and histology were assessed using a beam walk test and

stereological methods.

In WT TBI mice, NOX2 was expressed in reactive microglia

(CD68

/Clic1

) in peri-lesional cortex through 28d; NOX2-KO

significantly reduced CD68/Clic1 expression at 3 and 7d post-injury.

Flow cytometry analysis of isolated microglia/macrophages revealed

that IL-4R

and its downstream signaling pathway (STAT6/JAK3)

were significantly increased in CD45

high

microglia/macrophages of

NOX2-KO TBI mice compared to WT controls. M2-like polarization

(Arg1, Ym1, TGF

) was increased at 3d post-injury in NOX2-KO,

with effects sustained through 21d. There was significant reduction

M1-like polarized microglia/macrophages (IL-1

, TNF

, IL-12,

iNOS, CD16/32) in NOX2-KO TBI mice. M1/M2-like changes were

associated with increased numbers of DCX-positive cells in the sub-

ventricular zone, striatum and peri-lesional cortex, indicating in-

creased neurogenesis. NOX2-KO TBI mice also showed significantly

improved motor function and reduced cortical neurodegeneration at

21d.

Thus, after TBI NOX2-KO mice exhibit enhanced IL-4Ra-mediated

signaling, greater M2-like repolarization, and reduced neurodegen-

eration. Moreover, altering the M1-/M2-like balance appears to sup-

port increased neurogenesis. These data indicate that NOX2 drives the

M1-like polarization of microglia/macrophages after TBI, and that

inhibiting this pathway limits tissue damage and may promote tissue

repair.

Keywords: Microglial phenotypes, NOX2, neurodegeneration,

Tissue repair

C7-23

PHOSPHODIESTERASE 4B INHIBITION AS AN ANTI-

INFLAMMATORY TREATMENT FOR TRAUMATIC BRAIN

INJURY

Nicole Wilson

, Concepcion Furones

, Mark Gurney

, Dalton

Dietrich

, Coleen Atkins

University of Miami Miller School of Medicine, Neurosurgery, Mi-

ami, USA

Tetra Discovery Partners, Grand Rapids, USA

The anti-inflammatory effects of phosphodiesterase 4 (PDE4) inhibi-

tors are well established in CNS injury models. Knock out studies

have indicated that PDE4B is predominantly responsible for the anti-

inflammatory effects of pan-PDE4 inhibition. In the absence of

PDE4B, neutrophil infiltration and tumor necrosis factor (TNF) are

reduced in models of systemic inflammation. Furthermore, PDE4B2 is

upregulated in the injured cortex after traumatic brain injury (TBI). In

other models, PDE4B2 is expressed in microglia, macrophages and

neutrophils. However, whether PDE4B2 is localized in inflammatory

cells after TBI, and whether PDE4B inhibition reduces inflammation

and improves histopathological outcome after TBI is unknown. To

address this, adult male Sprague Dawley rats received moderate

parasagittal fluid-percussion brain injury (2

–

0.2 atm) or sham sur-

gery. Flow cytometry was used to analyze microglia and infiltrating

myeloid-lineage cells isolated from the injured cortex for PDE4B2

expression at 24 hrs after TBI. PDE4B2 was elevated in microglia and

infiltrating myeloid-lineage cells at 24 hrs TBI. To determine whether

PDE4B inhibition decreases TNF and neutrophil infiltration in the

injured cortex, animals received vehicle (5% DMSO in saline) or a

PDE4B inhibitor, A33 (2-(4-{[2-(5-chlorothiophen-2-yl)-5-ethyl-6-

methylpyrimidin-4-yl]amino}phenyl)acetic acid) at 0.3 mg/kg (i.p.) at

30 min and 5 hrs post-surgery. TNF was measured at 6 hrs post-

surgery using an ELISA. Neutrophil infiltration was assessed at 24 hrs

post-surgery using flow cytometry. PDE4B inhibition significantly

decreased TNF levels and neutrophil infiltration after TBI. To deter-

mine whether PDE4B inhibition reduces pathology after TBI, animals

received vehicle (5% DMSO in saline) or A33 (0.3 mg/kg, i.p.) at

30 min post-surgery and once daily for 3 days. Cortical contusion was

evaluated in serial brain sections stained with hematoxylin and eosin.

PDE4B inhibition significantly reduced cortical contusion volume at 3

days post-injury. These results suggest that inhibiting PDE4B after

TBI may be a viable treatment to reduce inflammation and pathology.

Supported by The Miami Project to Cure Paralysis and NIH/NINDS

NS056072 and NS089351.

Keywords: Phosphodiesterase 4B, PDE4B

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