Brain Research, 100 (1975) 645--649
645
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
Short Communications
Fenestrated CNS blood vessels in chronic experimental allergic encephalomyelitis
DAVID H. SNYDER, ASAOHIRANOANDCEDRIC S. RAINE Department of Pathology ( Neuropathology), the Saul R. Korey Department of Neurology, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N. Y. 10461 and ( A.H.) Department o f Pathology ( Neuropathoiogy) , Montefiore Hospital and Medical Center and the Albert Einstein College o f Medicine, Bronx, N.Y. 10467 (U.S.A.)
(Accepted September 10th, 1975)
During the past two decades, the anatomical substrate of the mammalian bloodbrain barrier has been studied extensively by a variety of techniques4. In combination with electron-dense tracers, ultrastructural investigations have shown that in most mammals the paucity of pinocytotic vesicles within endothelial cells and the presence of tight junctions between adjacent endothelial cells constitute the major structural barrier to the movement of material across cerebral blood vessels5,7. A further contributory factor to the blood-brain barrier is the absence of fenestrations within the endothelial lining, a common feature of the vasculature of several other organ systems. Although changes in vascular permeability have been documented in a number of neuropathologic situations by the demonstration of leakage of tracer molecules into the extravascular space, the morphologic correlates of such disruptions have been difficult to document. The present communication reports a previously unobserved change in central nervous system (CNS) parenchymal vessels occurring in animals with chronic experimental allergic encephalomyelitis (EAE), an alteration of relevance to studies on vascular permeability in the CNS. The present observations form part of an ongoing study on chronic and recurrent EAE in juvenile strain 13 guinea pigse, s. The animals were inoculated in the nuchal area with 0.5 ml of an emulsion containing isologous spinal cord tissue in complete Freund's adjuvant. After about 8-12 weeks, clinical signs of encephalomyelitis (e.g., hind limb paresis) were apparent, and the animals were maintained up to 12 months after the onset of signs. Animals were anesthetized and sacrificed at various stages post-inoculation, by perfusion through the heart with phosphate-buffered 5 % glutaraldehyde for 15 min. One millimeter slices of spinal cord tissue were post-fixed for 1 h in Dalton's buffered 2 ~o osmium tetroxide, dehydrated, and embedded in Epon. One micrometer Epon sections were taken and stained with toluidine blue and examined
646
O
Fig. 1. A low power electron micrograph showing part ofa parenchymal blood vessel from the spinal cord of a Strain 13 guinea pig with chronic EAE. The endothelium is attenuated and displays fenestrated pores (arrows). Note the multi-layered basal lamina. The Virchow-Robin space (VR) contains extensive collagen deposition and mononuctear cells. L, lumen. ": 10,000.
by light microscopy. Thin sections of selected areas were stained with uranyl acetate and lead citrate and examined by electron microscopy. Light microscopy of lumbo-sacral spinal cord in these inbred guinea pigs with chronic EAE showed that a common feature of demyelinated lesions was the presence of fibrotic changes around parenchymal blood vessels. In addition to collagen deposition and mononuclear cells in the Virchow-Robin space, electron microscopy of some of these vessels revealed zones of extensive attenuation of the endothelial celt lining, thus creating fenestrated pores (Fig. 1). The fenestrae varied from 55 to 70 nm in diameter and were irregularly distributed around the endothelial wall. Often, several pores would be grouped together. Each fenestra consisted of a leaflet approximately 5 nm thick (Fig. 2). The attenuated endothelial cells were surrounded by several basal laminae (Fig. 1). The vascular lumen of affected vessels varied between 12 and 20/~m and the perivascular space was greatly increased. An additional finding in these endothelial cells was the presence of supernumerary pinocytotic vesicles particularly on the basilar aspect (Fig. 3). The observation of fenestrated endothelial cells in the parenchymal vessels of spinal cord tissue represents a finding not documented within the normal m a m m a l i a n CNS 4. However, in certain specialized areas of the CNS, a blood-brain barrier is
647
Fig. 2. High power field from a similar blood vessel. Note the fenestrated pores in the endothelium (arrows). L, lumen, x 70,000.
Fig. 3. Detail from an affected vessel shows increased pinocytosis (arrows) in the endothelium. L, lumen, x 105,000.
648 known not to exist (e.g., choroid plexus, area postrema, pineal and pituitary gland) and fenestrated endothelial cells are common features here 4. As previously noted by Hirano 1, the appearance of fenestrations has not been reported in most cases of experimentally induced alterations of the blood-brain barrier, the exception to this being their presence in experimental CNS neoplasms. It is well known that during acute EAE there is in affected areas increased vascular permeability visualized morphologically by leakage of plasma proteins and mononuclear cells into the parenchyma. Using electron-dense tracers, it has been suggested that such leakage occurs via altered endothelial tight junctions 2,3. In addition, the possibility of transendothelial pinocytotic transport of plasma constituents has been raisedL This latter phenomenon is consistent with the present observation of increased pinocytotic vesicles. It should be emphasized that none of the many ultrastructural studies on acute EAE and other experimental vascular disorders has been associated with fenestrated pores. The significance of endothelial fenestrations in the CNS of the present animals with chronic EAE is obscure but might be related to a mechanical breakdown in the blood-brain barrier effected by repeated passage of cellular and humoral factors of hematogenous origin. The presence of these changes only around vessels with chronic fibrosis (a sequela of prior inflammation) and perivascular demyelination further supports the concept of a specific alteration related to this disease. Previous morphologic studies on CNS tissue during the acute phase of EAE have unequivocally demonstrated passage of hematogenous cells and plasma proteins across the endothelial lining2, 3. Our observation of fenestrated CNS endothelial cells, not previously reported, may be related to the uniquely chronic, recurrent disease pattern of the present model 2,6,8. Their absence in the numerous reports on acute EAE might be related to the shortterm nature of the disease. Further investigation is currently under way to determine the permeability properties of these vessels in chronic EAE. The authors thank Dr. R. D. Terry for constructive criticism and Dr. Saaford H. Stone for helpful assistance during this study. The excellent technical assistance of Everett Swanson, Howard Finch and Miriam Pakingan is acknowledged. Supported in part by USPHS Grants NS 08952 and NS 03356; and by Grant R G 1001-A-1 from the National Multiple Sclerosis Society (NMSS). Dr. Snyder was a postdoctoral fellow of the NMSS during the course of the work. Dr Raine is the recipient of a Research Career Development Award from the USPHS, Grant NS 70265.
1 HIRANO,A., Fine structural alterations of small vessels in the nervous system. In J. CERVOSNAVA~RO(Ed.), Pathology of Cerebral Microcirculation, de Gruyter, Berlin, 1974, pp. 203-217. 2 HmANO,A., DEMSITZEg,H. M., BECKER,N. H., LEVlNE,S., ANDZIMMERMAN,H. M., Fine structural alterations of the blood-brain barrier in experimental allergic encephalomyelitis J. Neuropath. exp. Neurol., 29 (1970) 432-440. 3 LAMPERT,P., ANDCARPENTER,S., Electron microscope studies on the vascular permeability and the mechanism of demyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. NeuroL, 24 (1965) 11-24. 4 LEE,J. C., Evolution in the concept of the blood-brain barrier phenomenon. In H. M. ZIMt,U~R~N
649 (Ed.), Progress in Neuropathology, Vol. I, Grune and Stratton, New York, 1971, pp. 84--145. 5 MAJNO, G., Ultrastructure of the vascular membrane. In W. F. HAMILTON(Ed.), Handbook of Physiology, Section 2, Circulation, VoL HL American Physiological Society, Washington, D.C., 1965, pp. 2293-2375. 6 RAINE, C. S., SNYDER, D. H., VALSAMIS,M. P., AND STONE,S. H., Chronic experimental allergic encephalomyelitis in inbred guinea pigs - - an ultrastructural study, Lab. Invest., 31 (1974) 369-380. 7 REESE, T. S., AND KARNOVSKY,i . J., The structural localization of a brain barrier to exogenous peroxidase, J. Cell BioL, 34 (1967) 207-217. 8 SNYDER,D. H., VALSAMIS,M. P., STONE,S. H., AND RAINE, C. S., Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis, J. Neuropath. exp. NeuroL, 35 (1975) 209-221.
645
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
Short Communications
Fenestrated CNS blood vessels in chronic experimental allergic encephalomyelitis
DAVID H. SNYDER, ASAOHIRANOANDCEDRIC S. RAINE Department of Pathology ( Neuropathology), the Saul R. Korey Department of Neurology, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N. Y. 10461 and ( A.H.) Department o f Pathology ( Neuropathoiogy) , Montefiore Hospital and Medical Center and the Albert Einstein College o f Medicine, Bronx, N.Y. 10467 (U.S.A.)
(Accepted September 10th, 1975)
During the past two decades, the anatomical substrate of the mammalian bloodbrain barrier has been studied extensively by a variety of techniques4. In combination with electron-dense tracers, ultrastructural investigations have shown that in most mammals the paucity of pinocytotic vesicles within endothelial cells and the presence of tight junctions between adjacent endothelial cells constitute the major structural barrier to the movement of material across cerebral blood vessels5,7. A further contributory factor to the blood-brain barrier is the absence of fenestrations within the endothelial lining, a common feature of the vasculature of several other organ systems. Although changes in vascular permeability have been documented in a number of neuropathologic situations by the demonstration of leakage of tracer molecules into the extravascular space, the morphologic correlates of such disruptions have been difficult to document. The present communication reports a previously unobserved change in central nervous system (CNS) parenchymal vessels occurring in animals with chronic experimental allergic encephalomyelitis (EAE), an alteration of relevance to studies on vascular permeability in the CNS. The present observations form part of an ongoing study on chronic and recurrent EAE in juvenile strain 13 guinea pigse, s. The animals were inoculated in the nuchal area with 0.5 ml of an emulsion containing isologous spinal cord tissue in complete Freund's adjuvant. After about 8-12 weeks, clinical signs of encephalomyelitis (e.g., hind limb paresis) were apparent, and the animals were maintained up to 12 months after the onset of signs. Animals were anesthetized and sacrificed at various stages post-inoculation, by perfusion through the heart with phosphate-buffered 5 % glutaraldehyde for 15 min. One millimeter slices of spinal cord tissue were post-fixed for 1 h in Dalton's buffered 2 ~o osmium tetroxide, dehydrated, and embedded in Epon. One micrometer Epon sections were taken and stained with toluidine blue and examined
646
O
Fig. 1. A low power electron micrograph showing part ofa parenchymal blood vessel from the spinal cord of a Strain 13 guinea pig with chronic EAE. The endothelium is attenuated and displays fenestrated pores (arrows). Note the multi-layered basal lamina. The Virchow-Robin space (VR) contains extensive collagen deposition and mononuctear cells. L, lumen. ": 10,000.
by light microscopy. Thin sections of selected areas were stained with uranyl acetate and lead citrate and examined by electron microscopy. Light microscopy of lumbo-sacral spinal cord in these inbred guinea pigs with chronic EAE showed that a common feature of demyelinated lesions was the presence of fibrotic changes around parenchymal blood vessels. In addition to collagen deposition and mononuclear cells in the Virchow-Robin space, electron microscopy of some of these vessels revealed zones of extensive attenuation of the endothelial celt lining, thus creating fenestrated pores (Fig. 1). The fenestrae varied from 55 to 70 nm in diameter and were irregularly distributed around the endothelial wall. Often, several pores would be grouped together. Each fenestra consisted of a leaflet approximately 5 nm thick (Fig. 2). The attenuated endothelial cells were surrounded by several basal laminae (Fig. 1). The vascular lumen of affected vessels varied between 12 and 20/~m and the perivascular space was greatly increased. An additional finding in these endothelial cells was the presence of supernumerary pinocytotic vesicles particularly on the basilar aspect (Fig. 3). The observation of fenestrated endothelial cells in the parenchymal vessels of spinal cord tissue represents a finding not documented within the normal m a m m a l i a n CNS 4. However, in certain specialized areas of the CNS, a blood-brain barrier is
647
Fig. 2. High power field from a similar blood vessel. Note the fenestrated pores in the endothelium (arrows). L, lumen, x 70,000.
Fig. 3. Detail from an affected vessel shows increased pinocytosis (arrows) in the endothelium. L, lumen, x 105,000.
648 known not to exist (e.g., choroid plexus, area postrema, pineal and pituitary gland) and fenestrated endothelial cells are common features here 4. As previously noted by Hirano 1, the appearance of fenestrations has not been reported in most cases of experimentally induced alterations of the blood-brain barrier, the exception to this being their presence in experimental CNS neoplasms. It is well known that during acute EAE there is in affected areas increased vascular permeability visualized morphologically by leakage of plasma proteins and mononuclear cells into the parenchyma. Using electron-dense tracers, it has been suggested that such leakage occurs via altered endothelial tight junctions 2,3. In addition, the possibility of transendothelial pinocytotic transport of plasma constituents has been raisedL This latter phenomenon is consistent with the present observation of increased pinocytotic vesicles. It should be emphasized that none of the many ultrastructural studies on acute EAE and other experimental vascular disorders has been associated with fenestrated pores. The significance of endothelial fenestrations in the CNS of the present animals with chronic EAE is obscure but might be related to a mechanical breakdown in the blood-brain barrier effected by repeated passage of cellular and humoral factors of hematogenous origin. The presence of these changes only around vessels with chronic fibrosis (a sequela of prior inflammation) and perivascular demyelination further supports the concept of a specific alteration related to this disease. Previous morphologic studies on CNS tissue during the acute phase of EAE have unequivocally demonstrated passage of hematogenous cells and plasma proteins across the endothelial lining2, 3. Our observation of fenestrated CNS endothelial cells, not previously reported, may be related to the uniquely chronic, recurrent disease pattern of the present model 2,6,8. Their absence in the numerous reports on acute EAE might be related to the shortterm nature of the disease. Further investigation is currently under way to determine the permeability properties of these vessels in chronic EAE. The authors thank Dr. R. D. Terry for constructive criticism and Dr. Saaford H. Stone for helpful assistance during this study. The excellent technical assistance of Everett Swanson, Howard Finch and Miriam Pakingan is acknowledged. Supported in part by USPHS Grants NS 08952 and NS 03356; and by Grant R G 1001-A-1 from the National Multiple Sclerosis Society (NMSS). Dr. Snyder was a postdoctoral fellow of the NMSS during the course of the work. Dr Raine is the recipient of a Research Career Development Award from the USPHS, Grant NS 70265.
1 HIRANO,A., Fine structural alterations of small vessels in the nervous system. In J. CERVOSNAVA~RO(Ed.), Pathology of Cerebral Microcirculation, de Gruyter, Berlin, 1974, pp. 203-217. 2 HmANO,A., DEMSITZEg,H. M., BECKER,N. H., LEVlNE,S., ANDZIMMERMAN,H. M., Fine structural alterations of the blood-brain barrier in experimental allergic encephalomyelitis J. Neuropath. exp. Neurol., 29 (1970) 432-440. 3 LAMPERT,P., ANDCARPENTER,S., Electron microscope studies on the vascular permeability and the mechanism of demyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. NeuroL, 24 (1965) 11-24. 4 LEE,J. C., Evolution in the concept of the blood-brain barrier phenomenon. In H. M. ZIMt,U~R~N
649 (Ed.), Progress in Neuropathology, Vol. I, Grune and Stratton, New York, 1971, pp. 84--145. 5 MAJNO, G., Ultrastructure of the vascular membrane. In W. F. HAMILTON(Ed.), Handbook of Physiology, Section 2, Circulation, VoL HL American Physiological Society, Washington, D.C., 1965, pp. 2293-2375. 6 RAINE, C. S., SNYDER, D. H., VALSAMIS,M. P., AND STONE,S. H., Chronic experimental allergic encephalomyelitis in inbred guinea pigs - - an ultrastructural study, Lab. Invest., 31 (1974) 369-380. 7 REESE, T. S., AND KARNOVSKY,i . J., The structural localization of a brain barrier to exogenous peroxidase, J. Cell BioL, 34 (1967) 207-217. 8 SNYDER,D. H., VALSAMIS,M. P., STONE,S. H., AND RAINE, C. S., Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis, J. Neuropath. exp. NeuroL, 35 (1975) 209-221.
