An Ultrastructural Study on Hyperosmotic Opening of Tight Junction by Lanthanum Nitrate Tracing

Wang Zhenyu，Wang Yi ，Yi Shengyu

Department of Neurosurgery, Peking University Hospital,

Wzy502@mailchina.com

Abstract: A model of hyperosmotic opening of the blood-brain barrier(BBB)was successfully made by infusion of 1.4M mannitol into the internal carotid artery of rabbit. We selected lanthanum nitrate as a tracer. Ultrastructural changes of hyperosmotic opening of the BBB were observed through injection of lanthanum nitrate into blood vessels or through impregnation of small blocks of brain tissue in lanthanum solution. Electron microscopy showed that lanthanum nitrate passed through the opening of endothelial tight junction from the blood into the subendothelial layers and the extracellular spaces of the brain, or from the outside into the inside of blood vessels. The data suggest that the morphological basis of osmotic opening of BBB is the reversible widening of the tight junctions between endothelial cells, and that lanthanum nitrate

infusion has an advantage over block impregnation.

Key words: blood-brain barrier: lanthanum nitrate; mannitol; ultrastructure; animal model

The blood-brain barrier (BBB)blocks water-soluble drugs passing from blood into the brain[1],Recently, method of osmotic BBB opening has been used with initial success to increase the delivery into the brain of antibiotic and anticancer agents that are normally excluded from the brain[2,3]. However, the structural basis of the barrier alteration at the level of cerebral

endothelium has not been fully elucidated. In the present study, the ultrastructural changes of osmotic BBB opening was observed by injection of lanthanum nitrate into blood vessels of impregnation of small blocks of brain tissue in lanthanum solution.

MATERIALS AND METHODS

Model of osmotic opening of the BBB. Twelve white rabbits weighing 2.5 to 3 kg were used in this study. The model of osmotic BBB opening was made with the modified technique of Rapoport, et al[1]. Each animal received sodium pentobarbital (30mg/Kg) intravenously and atropine (0.1mg/Kg) intramuscularly preoperatively. The right common carotid artery was isolated and the external carotid was ligated at its origin. An 4 1/2 needle was used to puncture the right common carotid and 1.4M mannitol (8-10ml, 37℃) was injected into it within 40 sec. In the control group, normal saline was infused instead of mannitol at the same rate and volume. Two percent Evans blue(EB,2ml/kg) was administered via a marginal vein of ear at 5 min prior to carotid infusion. EB was used as a marker dye because it is known to bind tightly to plasma albumin and therefore does not normally penetrate the tight junction between cerebral endothelial cells[4].

Preparation of fixative solutions.

1. Low concentration fixative solution contained 1% freshly prepared para-formaldehyde and 1.25% purified gluta-raldehyde in 0.1N sodium cacodylated buffer(pH7.4) with 5% sucrose.

2. High concentration fixative solution contained 2% paraformaldehyde, 2.5%glutaraldehyde and 4% lanthanum nitrate in the same buffer as described above. Lanthanum nitrate was prepared by dissolving in water and slowly adding 0.01N NaOH to pH7.7

3. Block impregnation fixative solution contained 2% lanthanum nitrate and 2.5% glutaraldehyde in 0.1N sodium cacodynate buffer.

Preparation of specmen for electron microscopy. Ten minutes after mannitol or normal saline was infused into the carotid, regional fixation of the brain was initiated by perfusing first with low and then with high concentration fixative solutions for 15 min each, through bilateral carotids at the pressure of 16.0 kPa (120 mmHg), while the fixative solution was allowed to escape from the cervical veins. The brain was removed and the localization and the density of EB stain was evaluated. Five biopsies for electron microscopic examinations were taken from representativeareas of each brain based on the degree of EB staining. The specimens were diced into 1 mm3 blocks and foxed in the high concentration solution for 2 h. They were washed three times in sodium cacodylate buffer containing 2% lanthanum nitrate, and postfixed with OsO4 in sodium cacodylate buffer containing 2%lanthanum nitrate for 2 h. The tissue was embedded in epon. Thin sections were cut with a LKB Ⅲ microtome and observed under a China-made DXA-4-10microscope, either unstained or following lead citrate staining.Some animals did not undergo perfusion. The brain tissues for electron microscopy were impregnated in fixative solution containing lanthanum nitrate for 3-4 h. The remaining procedures were the same as indicated above.

RESULTS

Infusion of 1.4 mannitol into the internal carotid artery via the right common carotid produced EB staining of ipsilateral brain regions. Stainging was absent in the brain of the animal perfused with isotonic saline (Fig. 1,Fig.1-6 are placed in interposed pages).

In the mannitol group, ultrastructur-al observation identified that lanthanum of high density entered the subendothelial space through widened tight junctions and formed black encirclement after its perfusion into the blood vessels. Part of the tracer passed through the basement membrane to the adjacent neuropil(Fig 2, Fig.3). In the control animals that received lanthanum nitrate via vascular infusion, the tracer did not enter the interendothelial junctions or subendothelial layers.

Lanthanum was still in the lumen of blood vessels (Fig. 4) In lanthanum permeated tissue blocks derived from animals infused with mannitol, colloidal lanthanum was found in arteries and capillaries, But the traversing of tracer through consecutive

membrane appositions of the tight junctions could not be observed clearly. In animals infused with normal saline, lanthanum was only visualized extravascularly (Fig.5,Fig.6).

DISCUSSION

The unilateral reversible osmotic opening of the BBB may be induced by perfusion of hypertonic solutions into the internal carotid artery. This BBB opening is not associated with any brain damage[1].In our experiment, a model of heyperosmotic opening of the BBB was Successfully made by infusion of 1.4M mannitol into the carotid artery of rabbits. Osmotic BBBopening technique has been generally acknowledged[5]. It may be of significance in neurophysiology and neuropharmacology. Rapoport, et al[6] reported that hypertonic infusion increased cerebrovascular permeability by shriking the vascular endothelial cells, thereby widening the interendothelial tight junctions. However, this postulate lacks morphological evidence. Brightman, et al[7] studied the ultrastructure of hyperosmotic opening of the BBB using infusion of horseradish peroxidase(HRP) into vessels, and confirmed the above hypothesis.

However, HRP method is complex and expensive, and it is limited in use. Lanthanum nitrate is a heavy metal of high density (MW433dalton) and its particle is about 40 in size. Lanthanum can be used as a tracer for observing ultrastructural changes of extracellular spaces because it can not pass through the interendothelial tigh