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microglia in an area of interest, and presenting Immunohistochemistry
activation as the percentage of microglia that score Transcardial perfusion sacrificed animals with saline
above a certain level. This technique provides a containing 0.1% ethylene diamine tetraacetic acid, under
more specific and sensitive measurement for pentobarbital anesthesia (6%, 2 mL/kg). Half of each
microglia activation than densitometry, but is very brain was immersion fixed in 4% paraformaldehyde for
labor intensive and rather depends on a subjective 4 days followed by cryoprotection with 30% sucrose in
interpretation of the activation stage. Moreover, this 0.01 mol/L phosphate-buffered saline (PBS). Free floating
analysis method is solely based on the classical view sections of 30 µm containing the prefrontal cortex (PFC)
on microglial activation and as such does not take and the dorsal hippocampus were pretreated with 0.3%
alternatively morphological changes, such as hyper- H O for 20 min. Sections were incubated for 3 days
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ramification into account. [12,13] with 1:2500 rabbit-anti IBA-1 (Wako, Neuss, Germany)
in 2% bovine serum albumin, 0.1% triton X-100 at 4°C,
In the past years several other methods have been followed by a 1 h incubation with 1:500 goat-anti rabbit
developed that involve morphological characteristics, secondary antibody (Jackson, Wet Grove, USA) at room
such as morphological complexity, [14] the extent of temperature. The sections were then incubated for 2 h
ramification [13,15,16] or the length of the dendritic with avidin-biotin peroxidase complex (Vectastain ABC
processes. [13,15,16] Although these methods allow a kit, Vector, Burlingame, USA) at room temperature.
more detailed and accurate analysis of microglial Labeling was visualized by using a 0.075 mg/mL
morphology, they still tend to be time consuming and diaminobenzidine (DAB) solution activated with 0.1%
depend on specialized equipment (e.g. a confocal H O . All dilutions were made in 0.01 mol/L PBS. All
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microscope) and software. sections were thoroughly rinsed 4 times with 0.01
mol/L PBS between staining steps. Sections were
Considering the disadvantages of the aforementioned mounted onto glass slides in a 1% gelatin solution
methods, we were in search of a method to determine and dehydrated through gradients of ethanol and xylol
changes in microglial morphology that is quick and easy solutions.
to perform, reproducible, and reliable as a microglial
activation marker. In this paper, we describe a newly Analysis of microglial activation
developed semi-automatic image analysis method for Three immunohistochemical labeled sections per area
IBA-1 stained brain sections of rat. We compare this for each rat were analyzed by an operator blinded
novel method to the most commonly used methods to the treatment. Average microglial activation was
today, namely densitometry and visual characterization determined in the apical dendritic field (stratum
of the activation stage, using IBA-stained sections of radiatum) of the dentate gyrus inner blade (DGib) and
rat brains from a previously published experiment. [17] the cornu ammonis 1 (CA1) hippocampal region and
in layer III of the PFC (Zilles’s Cg1).
METHODS
Densitometry
Animals and interventions The OD of the IBA-1 stained sections was measured
To investigate whether our method could be applied to using quantitative imaging software (Leica QWin, Leica
rat brain sections with a range of activation stadia we re- Microsystems, Rijswijk, The Netherlands), at ×100
analyzed IBA-1 stained brain sections from a previously magnification. The OD was corrected according to the
performed experiment in young and aged rats. [17] Male background staining measured in the corpus callosum
Wistar rats (HsdCpb: WU) of 3 months (young, n = 10) of each individual section. The resulting average OD in
and 18-20 months (old, n = 10) were obtained from each brain area was taken as a measure for microglial
a colony of the Semmelweis University (Budapest, activation.
Hungary). In each age group, 5 rats were subjected to
abdominal surgery under anesthesia (3% sevoflurane Visual characterization
in O at 0.7 L/min) and buprenorphine analgesia Images were taken of the DGib, CA1 and PFC of
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(0.003 mg/kg s.c.) and the other 5 remained naïve as the IBA-1 stained sections at ×400 magnification
control, yielding 4 experimental groups (young control, using quantitative imaging software (Leica QWin,
young surgery, old control and old surgery). Between Leica Microsystems). In each image, a circle was
week 1 and 6 following surgery, the rats underwent drawn which covered 0.06 mm of the original
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several behavioral tests. [17] Animals were sacrificed section. The microglia in this area were counted and
6 weeks after the surgical intervention. All experiments morphologically characterized based on the activation
[4]
were approved by the local animal experiment and stages 1-2 or 3-5 as described by Kreutzberg.
welfare committee (Dier Experimenten Commissie, Microglia characterized as being in stages 3-5 were
Groningen, The Netherlands). considered to be activated. The number of activated

Neuroimmunol Neuroinflammation | Volume 1 | Issue 2 | September 2014 83