Cell Tiss. Res. 157, 535--540 (1975) �9 by Springer-Verlag 1975

Diffusion of Horseradish Peroxidase Perfused through the Lateral Ventricle of the Chick Telencephalon

Pierre Delorme, Jacques Gayet and Georges Grignon*

Laboratoires de Physiologie G6n6rale, Facult6 des Sciences et de Microscopie Electronique, Facult6 de M6decine, Universit6 de Nancy I, Nancy, France

Received October 14, 1974 / in final form January 15, 1975

Summary Horseradish peroxidase, perfused into the lateral ventricle of chick brain, freely and slowly diffuses through the cerebral extracellular spaces. The layer of astrocytic end-feet surrounding blood capillaries does not constitute a barrier to the tracer which permeates the basal lamina, diffuses between the pericytic cells and finally accumulates in the intercellular space beneath the tight junctions between contiguous endothelial cells. No evidence was found for transport by micropinocytotic vesicles from the cerebral parenchyma to the capillary lumen.

Key words: Cerebral perivascular spaces - - Peroxidase - - Electron microscopy.

Injection of a relatively high concentration of horseradish peroxidase (mol. wt.--~ 40000, estimated d iameter~ 5 nm) into the blood stream and application of the method of Graham and Karnovsky (1966), has demonstrated that the tracer protein remains confined to the capillary lumen and does not enter the cerebral parenchyma in adult mice (Reese and Karnovsky, 1967; Brightman and Reese, 1969), in Neeturus and Am@stoma (Bodenheimer and Brightman, 1968) or in the chick during embryogenesis and post-hatching growth (Delorme et al., 1970; Delorme, 1971). The barrier to the penetratfon of the protein molecule into the cerebral parenchyma was essentially located at the level of the tight junc- tions (zonulae occludentes), between the plasma membranes of adjacent endothelial cells surrounding the lumen of the cerebral capillaries. Moreover, the effectiveness of this blood-brain barrier for molecules of the size of horseradish peroxidase (or larger), appeared to be reinforced by the paucity of micropinocytotie vesicles present in the cytoplasm of endothelial cells, particularly in the area facing the nervous parenehyma. In order to circumvent this morphological barrier, injection of horseradish peroxidase into the lateral ventricles has been performed in the adult mouse (Brightman, 1967; Brightman and Reese, 1969; Brightman, etal., 1970) and in Necturus and Ambystoma (Bodcnheimer and Brightman 1968), or

Send offprint requests to: Doctor Pierre Delorme, Laboratoire de Physiologie G6n6rale, Uni- versit6 de Nancy 1, Case Officielle 140, 54037-Nancy, France.

* Thanks are due to Mrs. Th6rbse Jaequot and Christiane Math and Miss Marie-Christine Thuillier for technical assistance. - - This research was supported by grants from the Centre National de la Recherche Scientifique (E.R.A. n ~ 331) and from the Fondation pour la l~echerche M6dicale Frangaise.

35 Cell Ties. Res. 157

536 P. Delorme et al.

Frontal

5quom o~

§

Bregrna

Lateral ~ Ventricle

z.lo A B

Fig. ] A and B. Drawings of the skull of a 30-day old Chick in dorsal (A) and in lateral (B) view. The slope of the longitudinal axis of the skull in the lateral view (B) was adjusted, in the stereotaxic instrument, so that the bregma point (corresponding to the intersection of the frontal and parietal bones) was located on the vertical line from the zero point. The coordinates used in our experiments, corresponding to a well-defined site in the lumen of a cerebral lateral ventricle, were: anteriority (~-1.5 mm), laterality (:~ 3.5 to 4.5 ram) and

depth (~ 10.5~ 0.5 mm)

implants of pellets of the tracer were placed in adul t ra t (Becker, et al., 1968) and h u m a n brain tissues (Foncin, 1973). W h e n the blood-endothelial barrier was thus circumvented, peroxidase react ion product was found in the basal l amina in all cases with the exception of h u m a n brain tissue. The discrepancies between these data prompted us to investigate, in the chick, whether the basal lamina surrounding the telencephalic capillaries would be penet ra ted by horseradish peroxidase when the tracer, perfused into a lateral ventricle, could c i rcumvent the blood-brain barrier.

Materials and Methods The study was performed in seventeen 30-day old chicks weighing about 600 g. A 0.05 per

cent solution of horseradish peroxidase (type VII-L RZ approximately 3.0 electrophoretically purified, from Sigma Chemical Co., Saint Louis, Mo., U.S.A.), in Ringer-Locke saline medium was perfused (at a rate of 2.5 ml per hour) into a lateral ventricle (after puncture of the cisterna magna) for 50 to 90 minutes. A Perfusor type I apparatus (Braun, Melsungen, West Germany) fitted with an appropriate glass needle was used. Before the perfusion was initiated, the animals were anaesthetized with Nembutal (30 mg/kg body weight, injected intra- peritoneally) and were placed in a Horsley-Clarke stereotaxic instrument (Precision Cin6mato- graphique Fran~aise, Asni~res, France) in order to introduce the glass needle into the lateral ventricle according to the stereotaxic coordinates illustrated in Fig. 1. Immediately after the perfusion was stopped, samples were removed from the neostriatum and were immersed in 2.5 per cent glutaraldehyde in 0.1 M SSrensen phosphate buffer at pH 7.25. The tissue samples subsequently processed according to the procedure previously described (Delorme et al., 1970) for the precise localization of peroxidase at the electron microscope level (Graham and Karnovsky, 1966). The observations were performed in a Siemens Elmiskop 1A and 101 electron microscope.

Diffusion of Peroxidase through the Cerebral Intercellular and Perivascular Spaces 537

Results

Following perfusion into the lateral ventricle, horseradish peroxidase pene- trated into the nervous parenchyma of the neostriatum, through the gap junctions located between the apical portions of the ependymal cells (Brightman, 1967; Brightman and Reese, 1969), and diffused through the extracellular spaces. The reaction product, accumulated in greater amounts in some synaptic junctions, and in the extracellular spaces it was preferentially adsorbed on the coating material covering the plasma membranes (Figs. 4-6).

In the vicinity of the blood capillaries, the reaction product accumulated in the intercellular space between the perivascular astrocytic end-feet (Figs. 2, 3, 8) and within the space occupied by the basal lamina which was strongly and uniformly stained (Figs. 2, 3, 7-12). The permeation of the basal lamina by the peroxidase enabled us to distinguish the perithelium from the endothelium around the capil- laries. From the basal lamina, the molecules of the tracer reached the inter- cellular spaces between the adjacent endothelial cells, and it accumulated beneath the tight junctional complexes between them (Figs. 7, 8, 10).

Some micropinocytotic vesicles were occasionally present in the perithelial and endothelial cells (Figs. 9, 11, 12). In arterioles or venules, the micropinocytotic vesicles were relatively numerous, especially in the smooth muscle cells (Fig. 12). Those vesicles opening onto the external side of the cells were full of the reaction product. However, virtually no reaction product was seen in pinocytotic vesicles at the luminal surface of the endothelial cells. No reaction product was detectable within the lumina of the capillaries. The positive reaction of the erythrocytes is due to the peroxidase activity of its haemoglobin (Figs. 3, 7).

Discussion

Our results are in accordance with those previously obtained in some lower Vertebrates and Rodents (Brightman, 1967; Brightman and Reese, 1969; Brightman et al., 1970; Bodenheimer and Brightman, 1968; Becker et al., 1968). Horseradish peroxidase, when perfused into the lateral ventricle, diffused freely through the extracellular spaces of the cerebral parenchyma. However, as previously reported by Brightman et al. (1970), the rate of diffusion was very slow, since in our experiments it was estimated to be approximately 1 to 1.5 mm for 50 to 90 minutes of intraventricular perfusion. This slow rate of diffusion of the tracer through the extracellular space was also previously noted after intra- venous injection of peroxidase in the chick embryo at the developmental stages corresponding to the strong vascularisation of the telencephalon (from the 5th to the 10th day of incubation) (Delorme, 1971).

The apparent affinity of peroxidase for some synaptic clefts has been discussed by Becker et al. (1968), who attributed it as possibly due to the binding of the basic protein molecule with acidic sites characteristic of inhibitory synapses.

The molecules of the tracer diffused freely from the intercellular spaces between the perivascular astrocytic processes to the space occupied by the basal lamina surrounding the blood vessels. Therefore, the presence of gap junctions (Reese and Karnovsky, 1967; Brightman and Reese, 1969), or of focal tight junctions (maculae occludentes) (Kuffler and Nicholls, 1966; Stensaas and Stensaas,

35*

538 P. Delorme et al.

Figs. 2--6. Blood capillaries, neuropil and synapses in the neostriatum of 30-day old chick telencephalon, after the pcrfusion of horseradish peroxidase into a lateral ventricle for 50 minutes. Enzyme reaction product fills the extracellular spaces (thick arrow), some synaptic clefts (S), the intercellular spaces between astrocytic processes (As and single small arrow), and the basal lamina (apposed arrows) of perithelial (P) and endothelial (En) cells. Tracer also accumulates beneath the tight junctions (J) between adjacent endothelial cells. Micropino- cytotic vesicles (V), opening onto the luminal side of the capillary wall, do not contain peroxidase. The lumen of the capillary (L) contains no reaction product. Erythrocytes (CyEr) are stained because of the peroxidatic activity of haemoglobin. Figs. 2: x 11000; 3: x 9000;

4: x 15000; 5--6: x 30000

Figs. 7--12. Walls of neostriatal blood capillaries (Figs. 7--10) and of arterioles or venules (Figs. 11 and 12) of 30-day old Chick telencephalon, after the perfusion of horseradish peroxidase into a lateral ventricle for 50 minutes. The basal lamina is completely stained and the reaction product fills the intercellular spaces between the endothelial cells up to the t ight junctions (J and thick arrow). In Figs 7 and 12, lysosomal bodies (Ly) strongly stained, are present in the cytoplasm of an astrocytic end-foot or of a muscle cell. In endothelial cells (Figs. 9, 11--12, En), in pericytes (Fig. 11, P) and in smooth muscle cells (Fig. 12, SMC) the micropinocytotic vesicles (V) which are full of the reaction product, are only located a t the abluminal surface of the cells. The micropinocytotic vesicles present in the cytoplasm of endothelial cells (Figs. 9, 12) or which open into the lumen of the blood vessels (Fig. 9) do not

contain peroxidase. Figs. 7 - -9 : • 11: • 24500; 10: • 67500; 12: • 23000

540 P. Delorme et al.

1968), be tween the p lasma membranes of some as t rocyt ic processes, does not create an imped imen t to the pene t ra t ion of molecules of this size.

The basal l amina surrounding the blood vessels offered no bar r ie r to the diffusion of peroxidase , since i t was uni formly s ta ined b y the reac t ion produc t . This observa t ion agrees wi th previous s tudies (Brightman, 1967; B r igh tman and Reese, 1969; Br igh tman et al., 1970; Bodenhe imer and Br igh tman, 1968; Becker et al., 1968). Ar res t of the enzyme molecule benea th the endothel ia l t igh t junct ions confirms, for the chick brain, the results previous ly ob ta ined b y the above- ment ioned authors and our own earl ier conclusions (Delorme et al., 1970; Delorme, 1971) concerning the role of a b ra in -bar r ie r s t ruc ture p layed b y these t igh t junc- tions.

The presence of microp inocyto t ic vesicles, on only the ex te rna l side of smooth muscle cells, and of per i the l ia l and endothel ia l cells sur rounding the te lencephal ic b lood vessels, does not suggest t h a t peroxidase is t r anspo r t ed in vesicles from the ex te rna l side of the wall of the blood vessels to the i r lamina .

References Becker, N. H., Hirano, A., Zimmerman, H. M. : Observations of the distribution of exogenous

peroxidase in the rat cerebrum. J. Neuropath. exp. Neurol. 27, 439-452 (1968) Bodenheimer, T. S., Brightman, M. W.: A blood-brain barrier to peroxidase in capillaries

surrounded by perivascular spaces. Amer. J. Anat. 122, 249-267 (1968) Brightman, M. W. : The intracerebral movement of proteins injected into blood and cerebro-

spinal fluid of mice. Progr. Brain Res. 29, 19-37 (1967) Brightman, M. W., Klatzo, I., Olsson, Y., Reese, T. S.: The blood-brain barrier to proteins

under normal and pathological conditions. J. neurol. Sci. 10, 215-239 (1970) Brightman, M. W., Reese, T. S. : Junctions between intimately apposed cell membranes in the

vertebrate brain. J. Cell Biol. 40, 648-677 (1969) Delorme, P. : Etude ultrastructurale du d~veloppement des capillaires, de leur environnement

et des modifications de leur perm~abilit6 s la peroxydase du Raifort dans le t~lenc~phale du Poulet au cours de l'embryog~n~se et de la croissance postnatale. Th~se de Doctorat ~s-Sciences Naturelles, Novembre 1971, Nancy, France

Delorme, P., Gayet, J., Grignon, G. : Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken. Brain Res. 22, 269-283 (1970)

Foncin, J.-F.: Comportement des macromol@ules de peroxydase au voisinage des capillaires c~r6braux humains. Acta neurol. 18, 242-245 (1973)

Graham, R. C., Karnovsky, M. J. : The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-301 (1966)

Kuffler, S.W., Nicholls, J. G.: The physiology of neuroglial cells. Ergebn. Physiol. 57, 1-90 (1966)

Reese, T. S., Karnovsky, M.J.: Fine structural localization of a blood-brain barrier to exo- genous peroxidase. J. Cell Biol. 34, 207-217 (1967)

Stensaas, L.J. , Stensaas, S. S. : Astrocytic neuroglial cells, oligodendrocytes and microgliocytes in the spinal cord of the toad. II. Electron microscopy. Z. Zellforsch. 86, 184 213 (1968)