Page 15 - Read Online

Turner et al. LPS preconditioning neuroprotective

INTRODUCTION METHODS

Endotoxin preconditioning with lipopolysaccharide Animals
(LPS) was previously shown to be neuroprotective in All procedures involving live animals were approved
models of ischemic stroke and traumatic brain injury by the Institutional Animal Care and Use Committee
(TBI). The neuroprotective mechanisms of LPS of West Virginia University and were performed
[1]
remain unclear, particularly with regards to vascular- according to the principles of the Guide for the
mediated effects. Prior studies have studies this Care and Use of Laboratory Animals, published
using contusion-based models of neurotrauma. The by the Institute of Laboratory Resources, National
contusion model irreversibly injures a focal brain Research Council (National Institutes of Health
region, creating a gross structural void associated publication 85-23-2985). Thirty-two male Sprague
[2]
with significant vascular injury. The mechanisms Dawley rats (Hilltop) at 3-4 months of age were
of LPS preconditioning are poorly understood used in this study. Animals were given standard rat
and have not been studied in a model of diffuse chow and water ad libitum.
axonal injury. There is emerging evidence that pro-
inflammatory regulation is neuroprotective in models LPS preconditioning
of neural injury, particularly the regulation of pro- Rats were pretreated with a single intraperitoneal
[3]
inflammatory cytokines and microglial phenotype injection of either 0.2 mg/kg LPS (Sigma-Aldrich) or
changes. Microglia is pleiotropic and transition 0.9% saline (equal volume) 7 days prior to TBI.
between continuum states. These states are termed
M1 or M2 depending on the inciting events. Locomotor behavior
[4]
Microglia may have protective and destructive roles, After LPS injection, the development of sickness
[5]
depending on the transition state. The ratio of behavior was monitored using activity chambers
microglial states can predict the subacute outcome as described by Godbout et al. [10] Locomotor
following injury. M1 microglia are pro-inflammatory activity was assessed using an automated activity
and can cause long-term deficits. Although LPS monitoring system (San Diego Instruments, San
[6]
has been shown to influence astrocyte phenotypes, Diego, CA) that recorded beam breaks in the x, y,
it is not clear whether LPS preconditioning and z planes. Animals were acclimated to the room
modulates microglial phenotypes following diffuse for 1 h prior to testing. Testing chambers consisted of
axonal injury. [7] square Plexiglass housing and 16 × 16 photobeam
arrays to detect lateral movements. An 8 × 8 array,
In this study, we investigated the neuroprotective located above the 16 × 16 array, detected rearing-
effect of LPS preconditioning in a closed-head associated movements. Activity was quantified over
model of diffuse axonal injury for the first time. This 30 min and the sum of fine, ambulatory, and rearing
model of TBI has increased clinical relevance due to beam breaks was calculated to give the total number
axonal shearing seen with diffuse neurotrauma. We of beam breaks. These recordings were completed
[8]
hypothesized that LPS preconditioning would reduce at 2, 4, 24, and 48 h post-injection.
inflammation and neurodegeneration following
diffuse axonal injury. In addition, we predicted that TBI induction
LPS preconditioning would regulate the oncostatin Animals were divided into four groups: sham surgery
M receptor (OSMR) in astrocytes and activate the (n = 8), sham surgery with LPS pretreatment (n
M1 microglia phenotype after TBI. TBI promotes = 8), impact-acceleration injury following saline
pro-inflammatory cascades and increases the injection (n = 8), and impact-acceleration injury with
expression of glial fibrillary acidic protein (GFAP); [2,9] LPS pretreatment (n = 8). Anesthesia was induced
however, a link between astrocyte regulation and and maintained using isoflurane (4% induction, 2%
the microglia phenotype after TBI has not been maintenance). Body temperature was controlled
investigated. In this study, we explored the effect with a homeothermic heating blanket equipped
of LPS preconditioning on astrocytes and microglia with a rectal probe. Rats received an impact-
and addressed the relationship between cytokine acceleration injury as described previously. [11-13]
receptor expression, astrocyte reactivity, and Briefly, a 10-mm diameter and 3-mm thick stainless
microglial phenotype. Improving our understanding steel disk was affixed to the skull with cyanoacrylate
of the protective effects of LPS preconditioning may between bregma and lambda. The animal was
accelerate the identification of novel therapeutic placed in a prone position on a foam bed with a
targets that reduce damage after TBI in individuals metal disk directly beneath a 2-m tall Plexiglass tube.
at risk of concussion. A 450-g weight was dropped from the top of the
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ January 20, 2017 7