The detection of perturbations in the brain is often employed as a key tool in safety assessments. The presence of a perturbation signal is an undesirable finding in safety testing and represents a degree of harm has been caused to the brain. In some approaches, perturbations are used as an indicator to search for signs of permanent damage, serving as a “canary in a coal mine.” In the context of some studies, the detection of a perturbation in itself is enough to warrant a conclusive safety concern. A perturbation represents a stress to the health of the brain, but a perturbation signal alone does not indicate the infliction of permanent damage to the brain. Other endpoints discussed in the next section are able to differentiate an injury/perturbation that resolves versus an injury that leads to permanent damage.
GFAP IHC reveals all astrocytes “resting” or reactive and Nestin IHC reveals only reactive astrocytes.
Iba1 IHC and NSA’s internally developed Reactive Microglia stain are capable of revealing activated microglia. Reactivity in Iba1 stained sections is determined by hypertrophy of the cells. The Reactive Microglia stain shows cells that are in a reactive state due to an acute perturbation. Microglia in a reactive state due to a chronic perturbation are not visible with this method.
Iba1 hypertrophied microglia
Iba1 normal microglia
NSA’s Reactive Microglia Stain
Resident microglia of the brain become reactive following various forms of insult, including chemical trauma, viral infection and physical trauma. The NSA protocol stains reactive microglia but not resting microglia. After staining sections from animals that had a broad range of insults, it was realized that this ‘reactive microglia’ protocol favors the staining of microglia where the injury was acutely induced. Microglia in animals with a chronic state of perturbation, such as in EAE (experimental allergic encephalitis), failed to stain—later, using the Iba1 antibody, staining revealed abundant microglia in a state of hypertrophy. Consequently, NSA’s reactive microglia staining protocol should only be used in animals that have suffered acute injury. In acutely injured brains, NSA’s reactive microglia stain reveals the same reactive features found by using the mouse and rat specific CD68 antibodies (FA11 and ED1, respectively) and the isoB4 lectin staining.
NSA’s reactive microglia stain
Iba1 vs CD68
Not all hypertyrophied microglia stain positive with CD68
Iba1-hypertrophied microglia in rat cortex
CD68 clone ED-1 (rat)
Iba1-hypertrophied microglia in mouse cortex
CD68 clone FA-11 (mouse)
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