Assessments of Reactive Astrogliosis Following CNS Injuries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 54

(Bio-Rad, Hercules, CA) to determine the domain organization of
astrocytes are discussed in the Materials, Methods, and Notes
sections below.

1.3. New Functional
or Genomic Techniques
to Evaluate Reactive
Astrogliosis

Subsequent sections in this chapter provide protocols and tips on
conducting histological evaluation of GFAP expression and astro-
cyte organization as measures of reactive astrogliosis. Although
these techniques are among the most prominently used and newly
developed techniques to measure reactive astrogliosis, they are
certainly not the only methods available. Thus, a brief review of
additional selected techniques to evaluate gliosis is provided below.

1. Bioluminescence imaging in brain of Gli-luc transgenic reporter
mouse.
The glioma-associated oncogene homologue Gli was originally
characterized as a gene up-regulated in gliomas and was later
discovered to be an element of the sonic-hedgehog (SHH)
signaling pathway. Holland and colleagues genetically engineered
a mouse model to evaluate the role of SHH signaling in glioma-
induced and non-neoplastic brain injury. Details on the creation
and characterization of this mouse model are found in their
recently published work ( 12, 13 ). Briefl y, this mouse model is
based on pGL3B/8GliBS-luc plasmid that was upstream of the
fi refl y luciferase gene, and chimeric founder mice were gener-
ated by pronuclear microinjection of the linearized Gli-luc
construct into fertilized oocytes. The Gli-luc transgenic reporter
mouse has been used to evaluate reactive astrogliosis after
freeze lesion brain injury ( 13 ). In this study, transgenic mice
were subjected to brain injury, and euthanized at the appropri-
ate post-injury time point. Immediately prior to euthanasia, mice
were given a retro-orbital injection of luciferin. Luciferin-
luciferase luminescence was detected in whole mouse brain
using an in vivo bioluminescence/fl uorescence detection system
(Xenogen IVIS-200 Optical Imaging System, Xenogen Corp.
Alameda, CA). This system is based on the emission spectrum
of luciferase (at 37°C) being mainly above 600 nm and the
resultant high effi ciency of tissue penetration. Additionally,
since no excitation light is required for luciferase biolumines-
cence, the signal-to-noise is relatively high and not obfuscated
by autofl uorescence ( 14 ). In the use of this mouse model to
evaluate reactive gliosis after CNS injury, Holland's research
team reported a bioluminescence ratio of bioluminescence
values from the injured to the uninjured cortex. They showed
that the SHH pathway is expressed by reactive astrocytes and is
associated with proliferation of Olig2 + cells ( 13 ). Although this
technique requires access to specialized equipment and a trans-
genic reporter mouse line, it can be used to qualitatively and
quantitatively assess reactive astrogliosis in whole brain.

1.3.1. Novel Functional
Techniques