- Email: [email protected]
Blood barriers in the nervous system studied with horseradish peroxidase
TINS - A u g u s t 1980
187
Blood barriers in the nervous system studied with horseradish peroxidase Jean M.Jacobs The introduction o f horseradish peroxidase (HRP) as a tracer which can be used in both light and electron micrograph studies o f vascular permeability has been a major advance over previous tracer methods using mainly dyes and fluorescent substances. While confirming the existence o f the blood-brain barrier (BB B) the high resolution o f H R P has led to the identification o f the sites o f this barrier at specialized junctions between endothelial cells o f brain capillaries. It is also clear from H R P tracer studies that many parts o f the nervous system lie 'outside' the B B B and as a consequence may escape its protective function. The earliest experimental studies on vascular permeability were made with synthetic dyes, and it has been known since the end of the last century that intravenously injected acidic dyes such as trypan blue stain all the tissues of the body except the brain, spinal cord and peripheral nerves 3. This procedure caused no ill effects in experimental animals, but when the dye was introduced into the cerebrospinal fluid-filled space which surrounds the brain, the cerebral tissue soon became diffusely stained and the animals developed severe neurological symptoms*. This experiment confirmed that the absence of staining was not due to a lack of affinity of the dye for brain tissue. Clearly there was some impedence to the passage of a circulating tracer in the brain, and Lewandowsky in 1900 was the first to suggest that the capillaries of the brain were the site of this barrier. Its precise localization was not possible, however, until a tracer became available which would be used in conjunction with electron microscopy; that tracer was horseradish peroxidase (HRP). H R P as a tracer
H R P is a plant hemoprotein with a molecular weight of 40,000 and a molecular radius of about 3 nm. It was first introduced as a systemic tracer in light microscope studies of the kidney TM when it was visualized by making use of its catalytic action in the oxidative polymerization of benzidine by hydrogen peroxide (H202), giving a blue reaction product. Graham and Karnovsky ~ later extended the use of H R P to electron microscopy by substitut-
Molecules much smaller than H R P have now been developed as tracers, for example microperoxidase (tool. wt <2000), but the use of these has not substantially changed our ideas about the sites of the BBB (or their absence), and H R P remains the most widely used of this type of tracer, The b l o o d - b r a i n barrier
With the exception of a few specialized areas of the brain which will be discussed later, cerebral capillaries of mammalian species are impermeable to circulating HRP. Tracer enters from the capillary lumen into the narrow space between endothelial cells, but specialized areas of close contact, called tight junctions, prevent any further passage of H R P between the cells (Fig. 1) 11. At these tight junctions the plasma membranes of adjoining cells, each measuring 7.5 nm, unite to form a junctional membrane 14 nm thickx.
ing 3,3'-diaminobenzidine (DAB) as the electron donor in the peroxidase reaction. zone of tight junctions The resulting polymer is osmiophilic and insoluble in organic solvents, characteristics which make it ideal for use in electron fused inner l a m i n a e ~ o f plasma membrane microscopy. A particular advantage of tight junction H R P is the 'amplifying' effect produced by plasma the enzymatic method of visualization so membrane that the final reaction product is considerably larger than the original molecule. H R P can be detected by light microscopy after treatment with D A B (and before secondary fixation in osmium tetroxide for Fig. 1. Diagram o f a brain capillary showing endoelectron microscopy) as a brown reaction thelial cells (e) joined by tight junctions, which are product. formed by fusion of the inner laminae of the plasma Most studies on the blood-brain barrier membrane. The tightjunctions are continuous and act (BBB) have used H R P as a vascular tracer, as a seal betweenadjacentcells. but in some cases H R P has been introduced into the ventricles of the brain where When H R P is injected into the cerebral it mixes with cerebrospinal fluid (CSF) and ventricles it passes into the extracellular diffuses into the extracellular spaces of the spaces of the brain where it enters the cleft brain. between adjacent capillary endothelial Since the H R P technique is used primar- cells from the 'brain' side, but is again preily for ultrastructural studies, optimum fix- vented from passing between the cells (and ation of tissues is important and this is best into the lumen of the capillaries) by tight achieved by perfusion fixation of the whole junctions. body through the aorta. Unfortunately this Vascular permeability studies have procedure also washes out any circulating shown that in other tissues such as skeletal H R P in the vascular system. Graham and muscle, H R P passes readily between the Karnovsky's original method for H R P capillary endothelial cells. These cells are employed a mixture of glutaraldehyde and also joined by specialized regions of close paraformaldehyde as a fixative, but since apposition but they are restricted to localparaformaldehyde has been shown to ized areas, unlike the tight junctions of reduce H R P activity, glutaraldehyde alone cerebral capillaries which form a continuis preferred as a fixative. Penetration of ous seal around the capillary. Muscle D A B and H202 into tissues is extremely endothelial cells contain large numbers of limited and it is essential to use very thin pinocytotic vesicles which are now recogpieces of tissue, preferably frozen sections nized as a means of intracellular transport of 50-80/zm. D A B is carcinogenic and across cells. By contrast, in brain capillaries must be used with care. the number of vesicles is very small and
~ •
=
"
©Elsevier/North-Holland BiomedicaPress l 1980
TINS -August 1980
188
tight ~ junctions
/
/
~_
"~'~ ~
tight
junctions
ermeurium
/ "%open junctions open junctions
~.~ineurium ~
/ /
Fig. 2. Diagram o f a peripheral nerve showing nerve fibres (dark rings) and an endoneurial capillary (endo cap) with tight junctions, lying in the endoneurial space. The inner sheath (perineurium) and outer sheath (epineurium) both have capillaries (peri cap and endo cap) with open or "leaky' junctions; tight junctions between the perineurial sheath cells prevent leakage into the endoneurium.
tracer studies show no evidence of a transcellular pathway. T h e morphological basis of the B B B is therefore associated with two features of brain capillaries, namely, the presence o f tight junctions between cells, and the relative absence of pinocytotic vesicles within these cells. It is interesting to note that before the characteristic features of cerebral capillaries were discovered, the site of the B B B was commonly held to be associated with the processes of astroglial cells which closely invest the entire brain capillary surface. Brightman, Reese and Feder 1 have shown that in the shark it is indeed at the astrocyte processes that systemically injected H R P is prevented from entering into brain tissue. H R P readily moves between the endothelial cells of the shark capillaries; moreover, these cells are full of vesicles apparently forming a transcellular pathway. Presumably, tight junctions between shark astrocyte processes perform a similar function to those in m a m m a l i a n brain endothelial cells.
Theblood-nervebarrier The structure of a peripheral nerve is shown diagrammatically in Fig. 2. Nerve fibres lie within an endoneurial space and are enclosed by two sheaths, an inner perineurium and an outer epiheurium. Endoneurial capillaries are impermeable
to HRP, but vessels in both peri- and epineurium are 'leaky'. Cells forming the perineiarium are joined by tight junctions, so that this sheath acts as a barrier to m o v e m e n t into the endoneurial space, of extravasated tracer from the peri- and epi-neurial sheaths9. The morphology of endoneurial capillaries has been less well studied than that of brain capillaries but it is most likely that they share c o m m o n characteristics.
Regionsof thenervoussystemwithouta bloodbarrier Certain well-defined areas of the brain contain cells that produce hormones or act as hormonal or chemoreceptors. These are functions which require a close association with the vascular system. Since these regions are grouped around the third and fourth ventricle, they are often called the circumventricular organs; they include the pineal, the posterior pituitary, and the area postrema. Morphological studies have shown that in these regions the capillaries have fenestrations, or pores covered by a thin diaphragm, which allow a rapid exchange of materials to and from the circulation. Tracer studies with H R P have confirmed the permeability of these vessels. Permeable blood vessels are also associated with choroid plexuses. These are highly folded sheets of epithelial cells
which project into the ventricles and secrete CSF. A n underlying network of permeable blood vessels provides a plasma filtrate from which the CSF is produced. Circulating H R P passes through choroidal vessels and into the surrounding stroma (Fig. 3), but tight junctions between choroidal epithelial cells prevent it from passing into the ventricles. Sensory ganglia are collections of nerve cells lying outside the CNS but connected to it by nerve fibres which form the sensory roots. Blood vessels in the ganglia are permeable to circulating H R P which can penetrate into the extracellular spaces surrounding the ganglion cells6. Autonomic ganglia also have permeable blood vessels, and in both types of ganglia leakage probably takes place mainly through fenestrated endothelial cells. Ganglia and interconnecting nerves forming part of the autonomic nervous system lie between muscle layers along the entire length of the alimentary tract. Tracer studies with H R P show that there is no blood barrier throughout this 'enteric nervous system 'v which represents a considerable volume of nervous tissue (Fig. 4). H R P can be taken up at the exposed ends of nerve fibres and transported in a retrograde direction back to the cell body. Olsson and Malmgren have already discussed this useful technique for tracing neuroanatomical pathways 1°. H R P can also enter axons at nerve terminals: the tracer that leaks from skeletal muscle capillaries after large intravenous injections of H R P is taken up into axons at the neuromuscular junctions and is found in the cells of origin in the brain or spinal cord some 12-24 h later 2. Similarly in areas of vascular permeability in the brain, extravasated tracer enters axons that terminate there, and can later be localized in their cell bodies. The function of the BBB is to maintain a special internal environment within the brain and also to protect nervous tissue from potentially damaging substances whilst at the same time hindering the escape of essential substances such as neurotransmitters. It is perhaps surprising, then, that so m u c h nervous tissue lies 'outside' the BBB. We have seen that in some special brain regions, endocrine flmctions dictate the necessity of vascular permeability, but in other parts, for example the dorsal root ganglia, the functional significance of blood vessel permeability remains unknown. It is important to be aware, therefore, of the potential vulnerability of some parts of the nervous system to damaging substancess.
TINS -August
1980
189
ventricle
Fig. 3. Electron micrograph o f choroid plexus from a rat given intravenous HRP 5 rain before killing. In this fortuitous section the blood vessel (b.v.) is filled with electron dense HRP (it is usually washed out during perfusion fixation). H R P has passed across fenestrated endothelial cells (arrows) and fills the extracellular spaces: it has also entered the gap between adjacent choroidal epithelial cells (ch.ep.) (arrow heads), outlining the complex folding at the base o f these cells. A tight junction at the apex o f the cell prevents HRP from passing into the ventricle (my = microvilli o f choroidal epithelial cell) inset below. Fenestrations in a choroidal blood vessel seen at a higher magnification.
Reading list 1 Brightman, M. W., Reese, T. S. and Feder, N. (1979) in Capillary Permeability. Alfred Benzon Symposium H, pp. 468-476, Munksgaard, Copenhagen 2 Broadwell, R. D. and Brightman, M. W. (1976) J. Comp. Neurol. 166, 257-283 3 Ehrfich, P.(1885)inDasSauerstoff-Bedi~rfnisdes organisms, Hirschwald, Berlin 4 Goldmann, E. E. (1913)Abh. PreussAkad. Wiss. Phys-Math Kl, 1-60 5 Graham, R. C. and Karnovsky, M. J. (1966)
Fig. 4. Electron micrograph from the small intestine of a rat killed 5 rain after intravenous injection of HRP. Part o f the myenteric plexus is seen lying between longitudinal and circular muscles. H R P has passed out of blood vessels in the muscle layers into the extracellular spaces. It has also spread into the plexus, filling the gaps around a nerve cell (arrow heads) and outlining nerve and glial processes, and synaptic endings.
J. Histoehem. Cytochem. 14,291-302 6 Jacobs, J. M., MacFarlane, R. M. and Cavanagh, J. B. (1976) J. Neurol. Sci. 29, 95-107 7 Jacobs, J. M. (1977)J. Neurocytol. 6,607-618 8 Jacobs, J. M. (1980) in Experimental and Clinical Neurotoxicology (Spencer, P. S. and Schaumburg, H. H. eds), Williams and Wilkins, Baltimore (in preparation) 90lsson, Y. (1975) in Peripheral Neuropathy (Dyck, P. J., Thomas, P. K. and Lambert, E. H. eds), pp. 190-200, W.B. Saunders Company, Philadelphia, London and Toronto
10 Olsson, Y. and Malmgren, L. T. (1978) Trends NeuroSci. 1, i05-107 11 Reese, T. S. and Karnovsky, M. J. (1967) J. Cell Biol. 34, 207-217 12 Straus, W. (1957)J. Biophys. Biochem. Cytol. 3, 1037-1040
Jean M. Jacobs is a post-doctoral Brain Research Trust Fellow in the Department of Neuropathology, Institute of Neurology, Queen Square, London W C I N 3BG, U.K.
187
Blood barriers in the nervous system studied with horseradish peroxidase Jean M.Jacobs The introduction o f horseradish peroxidase (HRP) as a tracer which can be used in both light and electron micrograph studies o f vascular permeability has been a major advance over previous tracer methods using mainly dyes and fluorescent substances. While confirming the existence o f the blood-brain barrier (BB B) the high resolution o f H R P has led to the identification o f the sites o f this barrier at specialized junctions between endothelial cells o f brain capillaries. It is also clear from H R P tracer studies that many parts o f the nervous system lie 'outside' the B B B and as a consequence may escape its protective function. The earliest experimental studies on vascular permeability were made with synthetic dyes, and it has been known since the end of the last century that intravenously injected acidic dyes such as trypan blue stain all the tissues of the body except the brain, spinal cord and peripheral nerves 3. This procedure caused no ill effects in experimental animals, but when the dye was introduced into the cerebrospinal fluid-filled space which surrounds the brain, the cerebral tissue soon became diffusely stained and the animals developed severe neurological symptoms*. This experiment confirmed that the absence of staining was not due to a lack of affinity of the dye for brain tissue. Clearly there was some impedence to the passage of a circulating tracer in the brain, and Lewandowsky in 1900 was the first to suggest that the capillaries of the brain were the site of this barrier. Its precise localization was not possible, however, until a tracer became available which would be used in conjunction with electron microscopy; that tracer was horseradish peroxidase (HRP). H R P as a tracer
H R P is a plant hemoprotein with a molecular weight of 40,000 and a molecular radius of about 3 nm. It was first introduced as a systemic tracer in light microscope studies of the kidney TM when it was visualized by making use of its catalytic action in the oxidative polymerization of benzidine by hydrogen peroxide (H202), giving a blue reaction product. Graham and Karnovsky ~ later extended the use of H R P to electron microscopy by substitut-
Molecules much smaller than H R P have now been developed as tracers, for example microperoxidase (tool. wt <2000), but the use of these has not substantially changed our ideas about the sites of the BBB (or their absence), and H R P remains the most widely used of this type of tracer, The b l o o d - b r a i n barrier
With the exception of a few specialized areas of the brain which will be discussed later, cerebral capillaries of mammalian species are impermeable to circulating HRP. Tracer enters from the capillary lumen into the narrow space between endothelial cells, but specialized areas of close contact, called tight junctions, prevent any further passage of H R P between the cells (Fig. 1) 11. At these tight junctions the plasma membranes of adjoining cells, each measuring 7.5 nm, unite to form a junctional membrane 14 nm thickx.
ing 3,3'-diaminobenzidine (DAB) as the electron donor in the peroxidase reaction. zone of tight junctions The resulting polymer is osmiophilic and insoluble in organic solvents, characteristics which make it ideal for use in electron fused inner l a m i n a e ~ o f plasma membrane microscopy. A particular advantage of tight junction H R P is the 'amplifying' effect produced by plasma the enzymatic method of visualization so membrane that the final reaction product is considerably larger than the original molecule. H R P can be detected by light microscopy after treatment with D A B (and before secondary fixation in osmium tetroxide for Fig. 1. Diagram o f a brain capillary showing endoelectron microscopy) as a brown reaction thelial cells (e) joined by tight junctions, which are product. formed by fusion of the inner laminae of the plasma Most studies on the blood-brain barrier membrane. The tightjunctions are continuous and act (BBB) have used H R P as a vascular tracer, as a seal betweenadjacentcells. but in some cases H R P has been introduced into the ventricles of the brain where When H R P is injected into the cerebral it mixes with cerebrospinal fluid (CSF) and ventricles it passes into the extracellular diffuses into the extracellular spaces of the spaces of the brain where it enters the cleft brain. between adjacent capillary endothelial Since the H R P technique is used primar- cells from the 'brain' side, but is again preily for ultrastructural studies, optimum fix- vented from passing between the cells (and ation of tissues is important and this is best into the lumen of the capillaries) by tight achieved by perfusion fixation of the whole junctions. body through the aorta. Unfortunately this Vascular permeability studies have procedure also washes out any circulating shown that in other tissues such as skeletal H R P in the vascular system. Graham and muscle, H R P passes readily between the Karnovsky's original method for H R P capillary endothelial cells. These cells are employed a mixture of glutaraldehyde and also joined by specialized regions of close paraformaldehyde as a fixative, but since apposition but they are restricted to localparaformaldehyde has been shown to ized areas, unlike the tight junctions of reduce H R P activity, glutaraldehyde alone cerebral capillaries which form a continuis preferred as a fixative. Penetration of ous seal around the capillary. Muscle D A B and H202 into tissues is extremely endothelial cells contain large numbers of limited and it is essential to use very thin pinocytotic vesicles which are now recogpieces of tissue, preferably frozen sections nized as a means of intracellular transport of 50-80/zm. D A B is carcinogenic and across cells. By contrast, in brain capillaries must be used with care. the number of vesicles is very small and
~ •
=
"
©Elsevier/North-Holland BiomedicaPress l 1980
TINS -August 1980
188
tight ~ junctions
/
/
~_
"~'~ ~
tight
junctions
ermeurium
/ "%open junctions open junctions
~.~ineurium ~
/ /
Fig. 2. Diagram o f a peripheral nerve showing nerve fibres (dark rings) and an endoneurial capillary (endo cap) with tight junctions, lying in the endoneurial space. The inner sheath (perineurium) and outer sheath (epineurium) both have capillaries (peri cap and endo cap) with open or "leaky' junctions; tight junctions between the perineurial sheath cells prevent leakage into the endoneurium.
tracer studies show no evidence of a transcellular pathway. T h e morphological basis of the B B B is therefore associated with two features of brain capillaries, namely, the presence o f tight junctions between cells, and the relative absence of pinocytotic vesicles within these cells. It is interesting to note that before the characteristic features of cerebral capillaries were discovered, the site of the B B B was commonly held to be associated with the processes of astroglial cells which closely invest the entire brain capillary surface. Brightman, Reese and Feder 1 have shown that in the shark it is indeed at the astrocyte processes that systemically injected H R P is prevented from entering into brain tissue. H R P readily moves between the endothelial cells of the shark capillaries; moreover, these cells are full of vesicles apparently forming a transcellular pathway. Presumably, tight junctions between shark astrocyte processes perform a similar function to those in m a m m a l i a n brain endothelial cells.
Theblood-nervebarrier The structure of a peripheral nerve is shown diagrammatically in Fig. 2. Nerve fibres lie within an endoneurial space and are enclosed by two sheaths, an inner perineurium and an outer epiheurium. Endoneurial capillaries are impermeable
to HRP, but vessels in both peri- and epineurium are 'leaky'. Cells forming the perineiarium are joined by tight junctions, so that this sheath acts as a barrier to m o v e m e n t into the endoneurial space, of extravasated tracer from the peri- and epi-neurial sheaths9. The morphology of endoneurial capillaries has been less well studied than that of brain capillaries but it is most likely that they share c o m m o n characteristics.
Regionsof thenervoussystemwithouta bloodbarrier Certain well-defined areas of the brain contain cells that produce hormones or act as hormonal or chemoreceptors. These are functions which require a close association with the vascular system. Since these regions are grouped around the third and fourth ventricle, they are often called the circumventricular organs; they include the pineal, the posterior pituitary, and the area postrema. Morphological studies have shown that in these regions the capillaries have fenestrations, or pores covered by a thin diaphragm, which allow a rapid exchange of materials to and from the circulation. Tracer studies with H R P have confirmed the permeability of these vessels. Permeable blood vessels are also associated with choroid plexuses. These are highly folded sheets of epithelial cells
which project into the ventricles and secrete CSF. A n underlying network of permeable blood vessels provides a plasma filtrate from which the CSF is produced. Circulating H R P passes through choroidal vessels and into the surrounding stroma (Fig. 3), but tight junctions between choroidal epithelial cells prevent it from passing into the ventricles. Sensory ganglia are collections of nerve cells lying outside the CNS but connected to it by nerve fibres which form the sensory roots. Blood vessels in the ganglia are permeable to circulating H R P which can penetrate into the extracellular spaces surrounding the ganglion cells6. Autonomic ganglia also have permeable blood vessels, and in both types of ganglia leakage probably takes place mainly through fenestrated endothelial cells. Ganglia and interconnecting nerves forming part of the autonomic nervous system lie between muscle layers along the entire length of the alimentary tract. Tracer studies with H R P show that there is no blood barrier throughout this 'enteric nervous system 'v which represents a considerable volume of nervous tissue (Fig. 4). H R P can be taken up at the exposed ends of nerve fibres and transported in a retrograde direction back to the cell body. Olsson and Malmgren have already discussed this useful technique for tracing neuroanatomical pathways 1°. H R P can also enter axons at nerve terminals: the tracer that leaks from skeletal muscle capillaries after large intravenous injections of H R P is taken up into axons at the neuromuscular junctions and is found in the cells of origin in the brain or spinal cord some 12-24 h later 2. Similarly in areas of vascular permeability in the brain, extravasated tracer enters axons that terminate there, and can later be localized in their cell bodies. The function of the BBB is to maintain a special internal environment within the brain and also to protect nervous tissue from potentially damaging substances whilst at the same time hindering the escape of essential substances such as neurotransmitters. It is perhaps surprising, then, that so m u c h nervous tissue lies 'outside' the BBB. We have seen that in some special brain regions, endocrine flmctions dictate the necessity of vascular permeability, but in other parts, for example the dorsal root ganglia, the functional significance of blood vessel permeability remains unknown. It is important to be aware, therefore, of the potential vulnerability of some parts of the nervous system to damaging substancess.
TINS -August
1980
189
ventricle
Fig. 3. Electron micrograph o f choroid plexus from a rat given intravenous HRP 5 rain before killing. In this fortuitous section the blood vessel (b.v.) is filled with electron dense HRP (it is usually washed out during perfusion fixation). H R P has passed across fenestrated endothelial cells (arrows) and fills the extracellular spaces: it has also entered the gap between adjacent choroidal epithelial cells (ch.ep.) (arrow heads), outlining the complex folding at the base o f these cells. A tight junction at the apex o f the cell prevents HRP from passing into the ventricle (my = microvilli o f choroidal epithelial cell) inset below. Fenestrations in a choroidal blood vessel seen at a higher magnification.
Reading list 1 Brightman, M. W., Reese, T. S. and Feder, N. (1979) in Capillary Permeability. Alfred Benzon Symposium H, pp. 468-476, Munksgaard, Copenhagen 2 Broadwell, R. D. and Brightman, M. W. (1976) J. Comp. Neurol. 166, 257-283 3 Ehrfich, P.(1885)inDasSauerstoff-Bedi~rfnisdes organisms, Hirschwald, Berlin 4 Goldmann, E. E. (1913)Abh. PreussAkad. Wiss. Phys-Math Kl, 1-60 5 Graham, R. C. and Karnovsky, M. J. (1966)
Fig. 4. Electron micrograph from the small intestine of a rat killed 5 rain after intravenous injection of HRP. Part o f the myenteric plexus is seen lying between longitudinal and circular muscles. H R P has passed out of blood vessels in the muscle layers into the extracellular spaces. It has also spread into the plexus, filling the gaps around a nerve cell (arrow heads) and outlining nerve and glial processes, and synaptic endings.
J. Histoehem. Cytochem. 14,291-302 6 Jacobs, J. M., MacFarlane, R. M. and Cavanagh, J. B. (1976) J. Neurol. Sci. 29, 95-107 7 Jacobs, J. M. (1977)J. Neurocytol. 6,607-618 8 Jacobs, J. M. (1980) in Experimental and Clinical Neurotoxicology (Spencer, P. S. and Schaumburg, H. H. eds), Williams and Wilkins, Baltimore (in preparation) 90lsson, Y. (1975) in Peripheral Neuropathy (Dyck, P. J., Thomas, P. K. and Lambert, E. H. eds), pp. 190-200, W.B. Saunders Company, Philadelphia, London and Toronto
10 Olsson, Y. and Malmgren, L. T. (1978) Trends NeuroSci. 1, i05-107 11 Reese, T. S. and Karnovsky, M. J. (1967) J. Cell Biol. 34, 207-217 12 Straus, W. (1957)J. Biophys. Biochem. Cytol. 3, 1037-1040
Jean M. Jacobs is a post-doctoral Brain Research Trust Fellow in the Department of Neuropathology, Institute of Neurology, Queen Square, London W C I N 3BG, U.K.