Quick links to information on this page:

• Applications
• Advantages
• Specifications
• BBB Compromise by Physical Trauma
• BBB Compromise by CO2 Sacrifice
• NSA Recommendations
• References of interest

Applications:

• Stain reveals the locations of blood brain barrier compromise in the brain.

Advantages:

• 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. HRP) into the vasculature.

Specifications:

• Use either perfusion solution 2 or 3, found in NSA's perfusion protocols for optimized staining

• Sacrifice by CO2 suffocation will totally obscure areas of compromise.

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 albumen.

 

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 and 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 stainless patches in the Nissl section.

Other zones of IgG staining do not correspond to a loss of Nissl staining, perhaps illustrating BBB compromise that 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 hypothesize that the BBB may not merely be open or shut but can 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

Although not fully researched at the time of this printing, there seems to be 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 allowing the IgG serum proteins to enter the brain parenchyma. The images below depict the difference between a "normal" IgG antibody staining pattern of an anesthetized, perfused rat, vs. "abnormal" IgG antibody staining seen from a rat sacrificed by CO2 suffocation.

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.	In this brain from a mouse sacrificed by CO2 suffocation, there is widespread IgG staining.

NSA Recommendations:

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
  Richmon,JD et.al. 1998. Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier.
  Br. Research 780: 108-118.
  • References of interest within this paper:
    37) Rapoport, SI et.al. 1980 Quantitative aspects of reversible osmotic opening of the blood-brain barrier. Am J Physiol 238: R421-R431.
    45) Tanno, H. et.al. 1992. 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.
• Other serum proteins (albumin, fibrinogen, fibronectin)
  Yu,WR et.al. 1999. Systemic hypothermia following compression injury of rat spinal cord: reduction of plasma protein extravasation demonstrated by immunohistochemistry. Acta Neuropath (Berl) 98: 15-21.
• Entry of other agents
  Muldoon,LL et.al. 1995. Comparison of intracerebral inoculation and osmotic blood-brain barrier disruption for delivery of adenovirus, herpes virus, and iron oxice particle to normal rat brain. Am J Path. 147: 1840-1851.

For futher reference, please see: Stains

return to top