Protein movement across the blood-brain barrier in hypervolemia.

The blood-brain barrier, attributed to the presence of tight junctions linking endothelial cells of cerebral vessels 9, provides only a partial obstacle to the entry of serum proteins from blood to brain. Experiments in mice have demonstrated that intravenously injected horseradish peroxidase (HRP) moves by vesicular transport across normal cerebral arterioles 1~ and accumulates in random extracellular foci throughout the brain 1. In these studies, HRP was injected in a volume of 0.4 ml and 0.5 ml respectively; the mouse blood volume is only 1.3 ml 5. Injection of such relatively large volumes enhances HRP movement across capillary enaotheliurn in the mouse lung 1°. We decided to investigate, therefore, whether hypervolemia induced by saline or water injection significantly increases the permeability of the mouse blood-brain barrier to HRP. Experiments were conducted on 50 mice: C57BL/6J, male, 2-3 months old, weighing 24-26 g. Two mice were injected with 1.0 ml saline only as controls for endogenous peroxidase activity; the remaining 48 mice were each given 20 mg HRP. These 48 mice were divided into 12 groups of 4 mice. Nine groups were injected with HRP dissolved in varying volumes of 0.9 ~o saline: 0.1, 0.5, 1.0 ml, and killed after varying circulation times: 5, 30, 60 min. Three groups were given HRP in i .0 ml distilled water for 5, 30, and 60 min. Injections were made smoothly at about 0.2 ml/sec via the tail vein using a 30-gauge needle mounted on a 1.0 ml syringe; for the 0.1 ml injections a 250 #1 syringe was used. After anesthesia with 6 mg sodium pentobarbital i.p., the right atrium was opened and half-strength Karnovsky's fixative perfused via the left ventricle for 10 min under 50 mm Hg. The brain was stored overnight in fixative at 4 °C and serial 100 #m coronal sections (Vibratome) were collected in Tris.HCI buffer, pH 7.6 .HRP activity was visualized using diaminobenzidine according to the method of Graham and Karnovsky 4. Thirty sections from each brain, representing all levels, were selected without regard to grossly visible HRP staining and evaluated in the light microscope. Cerebral cortex and caudate-putamen from 3 mice given HRP in 1.0 ml saline and 3 mice given HRP in 1.0 ml water were examined in the electron microscope.