Blood Brain Barrier Compromise Detection
- Anti-IgG Stain reveals the locations of blood brain barrier compromise in the brain.
- Allows for visualization of BBB compromise that is not accompanied by a loss in cells.
- Does not require the injection of a foreign protein marker (i.e., horseradish peroxidase (HRP)) into the vasculature.
- Perfusion fixation is preferred with overnight in perfusion fix then transfer to PBS. CAUTION: sacrifice with CO2 suffocation and not immediate brain removal and placement in fix can allow serum proteins to extravasate into the brain parenchyma and yield a false positive of BBB compromise (see image bottom right on p 71).
The locations of BBB compromise can be revealed with immunohistochemistry by using an antibody against the animal’s own IgG. Areas with “leaky” BBB allow serum proteins to pass into the brain parenchyma, e.g., IgG’s and albumin.
BBB Compromise by Physical Trauma
In the MultiBrain® section below (derived from freeze-sectioning a MultiBrain® Block of 16 rat brains), forebrain sections are shown from rat brains that had been injected with a test article. Different amounts were injected, then the rats were sacrificed at different times following the injection. The result was different degrees of leaky BBB as indicated by the brown coloration (the blue is a Nissl counterstain). Note the halo of BBB immunostaining extends beyond the margin of the frank lesion.
The images in each panel below show sections from the brains of two different rabbits that were subjected to fine air emboli to create multiple infarctions. In the top panel, the locations of BBB compromise are seen as deep amber-colored areas. In the bottom panel, the lesion sites are seen by an absence of staining of cell bodies. The arrows illustrate some of the infarction sites in each of the differently stained, near adjacent sections. In the section on the left, the rather large halo of IgG staining goes beyond the lesion site near the third ventricle, as revealed with the Nissl stain below it. On the other hand, in the section on the right, the angled arrows show two distinct sites of IgG staining that correspond in size to the unstained patches in the Nissl section.
Other zones of IgG staining do not correspond to a loss of Nissl staining, perhaps illustrating BBB compromise which is unaccompanied by the loss of cells. For example, note the large area in the right section that encompasses the left external capsule, as well as the area adjacent to the left optic nerve (vertical arrows).
NSA staff hypothesizes that the BBB may not merely be open or shut but may be opened to varying degrees such that molecules of different sizes would accordingly penetrate different degrees. For a given time after BBB compromise, small serum proteins may be found at a greater radius from a given site than larger proteins, i.e., IgGs.
BBB Compromise due to CO2 Sacrifice
There is evidence that sacrifice by CO2 causes the BBB to open during the sacrifice process. When conducting a BBB compromise study, the impact of this event could serve to give a false indication of brain penetration for a particular compound, as the compound may not have crossed the BBB under normal circumstances. We surmise that the extreme stress, induction of acidosis and lack of oxygen during the several minutes before death result in a breakdown of the BBB. If the brain is not removed immediately or soon after cessation of breathing, IgG serum proteins may enter the brain parenchyma. The images below depict the difference between a normal IgG antibody staining pattern of an anesthetized, perfused mouse versus abnormal IgG antibody staining seen from a mouse sacrificed by CO2 suffocation. The brain was not removed immediately after death.
The appearance of IgG staining in normal (mouse) brain shows only some tincture in periventricular structures and other areas known to have leaky BBB such as the median eminence and area postrema.
Brain from a mouse sacrificed by CO2 suffocation and brain was not removed and placed in fix soon after death. Widespread IgG staining results due to extravasation of serum proteins including IgGs.
NSA RECOMMENDATION: If the intactness of the BBB is to be studied, the animals should be anesthetized with a barbiturate such as Nembutal, then perfused transcardially with a suitable aldehyde fix. Note that use of gaseous anesthetics such as Fluothane tends to prevent flushing of red blood cells from the brain vasculature.
References of potential interest:
Use of anti-IgG to detect BBB breakdown:
Richmond, J.D., et al. Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier. Br Research 780: 108–118, 1998
References of interest within this paper:
37) Rapoport, S.I., et al. Quantitative aspects of reversible osmotic opening of the blood-brain barrier. Am J Physiol 238: R421–R431, 1980
45) Tanno, H., et. al. Breakdown of the blood-brain barrier after fluid percussive head injury in the rat. Part I. Distribution and time course of protein extravasation. J Neurotrauma 9: 21–32, 1992
Other serum proteins (albumin, fibrinogen, fibronectin):
Yu, W.R., et al. Systemic hypothermia following compression injury of rat spinal cord: reduction of plasma protein extravasation demonstrated by immunohistochemistry. Acta Neuropath (Berl) 98: 15–21, 1999
Entry of other agents:
Muldoon, L.L., et al. Comparison of intracerebral inoculation and osmotic blood-brain barrier disruption for delivery of adenovirus, herpes virus, and iron oxide particles to normal rat brain. Am J Path 147: 1840–1851, 1995