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Loss of Endothelial Barrier Antigen Immunoreactivity as a Marker of Clostridium perfringens Type D Epsilon Toxin-induced Microvascular Damage in Rat Brain
J. Comp. Path. 2014, Vol. 151, 153e156
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE
Loss of Endothelial Barrier Antigen Immunoreactivity as a Marker of Clostridium perfringens Type D Epsilon Toxin-induced Microvascular Damage in Rat Brain J. W. Finnie*,†, J. Manavis* and G. Chidlow‡,x * SA Pathology, Hanson Institute Centre for Neurological Diseases, Frome Road, † School of Veterinary Science, University of Adelaide, ‡ Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Frome Road and x Department of Ophthalmology and Visual Sciences, University of Adelaide, Frome Road, Adelaide, South Australia, Australia
Summary The epsilon toxin elaborated by Clostridium perfringens type D in the intestine of domestic livestock is principally responsible for the neurological disease produced after its absorption in excessive quantities into the systemic circulation. The fundamental basis of the cerebral damage induced by epsilon toxin appears to be microvascular injury with ensuing severe, diffuse vasogenic oedema. Endothelial barrier antigen (EBA), which is normally expressed by virtually all capillaries and venules in the rat brain, was used in this study as a marker of bloodebrain barrier (BBB) integrity. After exposure to high levels of circulating epsilon toxin, there was substantial loss of EBA in many brain microvessels, attended by widespread plasma albumin extravasation. These results support microvascular injury and subsequent BBB breakdown as a key factor in the pathogenesis of epsilon toxin-induced neurological disease. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: Clostridium perfringens type D epsilon toxin; endothelial barrier antigen; microvascular injury; rat brain
Clostridium perfringens type D enterotoxaemia is an important, worldwide neurological disorder of lambs and goats and, less commonly, calves caused by epsilon toxin. The disease in sheep spans a continuum from peracute intoxication with rapid death to a subacute form characterized by a more protracted clinical course. When the brain is exposed to large doses of circulating epsilon toxin absorbed from the intestine, the clinical course is generally brief and believed to be associated with microvascular endothelial damage and loss of bloodebrain barrier (BBB) integrity, leading to severe, generalized vasogenic oedema, a marked increase in intracranial pressure, convulsions, coma and often death (Finnie, 2003, 2004; Uzal et al., 2004).
*Correspondence to: J. W. Finnie (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.010
The permeability properties of the BBB are those of capillary endothelial cells, which differ in important structural detail from endothelia elsewhere, and the effectiveness of the BBB depends largely on interendothelial tight junctions, abetted by the inductive influence of astrocytic end feet, which cover most of the capillary surface and modulate the composition of the perineuronal fluid in a highly localized manner (Vinters and Kleinschmidt-Demasters, 2008). In rats, endothelial barrier antigen (EBA) is a membrane protein expressed by virtually all the cerebral microvasculature, which includes capillaries and venules (Sternberger and Sternberger, 1987), and its expression is considered to be a good indicator of a competent BBB (Ghabriel et al., 2000, 2002). EBA is specific to the rat BBB (Sternberger and Sternberger, 1987). Although the precise function of EBA is largely unknown, its role as a specific marker Ó 2014 Elsevier Ltd. All rights reserved.
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of BBB-competent brain vessels is suggested by its down-regulation in pathological states in which the BBB is disrupted and its absence in regions of the brain devoid of a BBB (Saubamea et al., 2012). Laboratory rodents are useful experimental models of the ovine disease produced by epsilon toxin, as they develop cerebral lesions of a similar nature and anatomical distribution (Finnie, 2003, 2004). Although EBA down-regulation has been used in a number of pathological states in which BBB integrity or function is impaired (Saubamea et al., 2012), this BBB marker has not previously been examined in rats given a high dose of fully activated C. perfringens type D epsilon toxin to simulate the acute ovine disease. In the only other study of vascular EBA loss in rats given this clostridial toxin (Zhu et al., 2001), the inactive epsilon prototoxin was used and it was hypothesized that a small amount was activated by endogenous proteases after intraperitoneal injection to produce a mild, subacute intoxication resulting in only minimal albumin extravasation as a marker of BBB disruption. Six, 6-week-old SpragueeDawley rats were given 1 ml of a 1 in 10 dilution of trypsin-activated prototoxin prepared from filtrates of broth cultures of C. perfringens type D (Commonwealth Serum Laboratories, Melbourne, Australia) by intraperitoneal injection. Four control rats were given a similar volume of a physiological saline solution. At 2 h post injection, the brains were fixed by transcardiac perfusion with 4% paraformaldehyde containing 0.02% heparin. After remaining in situ for 2 h, brains were removed and immersion-fixed in 10% neutral buffered formalin for 4 days, then routinely processed and embedded in paraffin wax. Sections (6 mm) were stained with haematoxylin and eosin (HE). Duplicate sections were cut for immunohistochemistry (IHC). Coronal sections were taken from three levels of brain, the ventral surface landmarks being those routinely used by the National Toxicology Programme (USA): just anterior to the optic chiasm (level 1); at the caudal borders of the mammillary bodies (level 2); and at the widest portion of the cerebellum just posterior to the transverse fibres of the pons (level 3) (Radovsky and Mahler, 1999). In IHC, endogenous albumin was used as a surrogate marker for visualizing the extravasation and spread of vasogenic oedema. A goat anti-rat albumin (catalogue number 0113-0341; Cappel Laboratories, West Chester, Pennsylvania, USA) was used at a dilution of 1 in 20,000. No antigen retrieval was required for the albumin antibody. This was followed by a biotinylated anti-goat immunoglobulin secondary reagent (catalogue number BA-9500; Vector Laboratories, Burlingame, California, USA) at a 1
in 250 dilution for 30 min, then washing in phosphate buffered saline (PBS). To detect EBA, a mouse monoclonal anti-EBA antibody (catalogue number SMI71R; Covance, Princeton, New Jersey, USA) was used. Tissue sections underwent antigen retrieval using citrate buffer and were then incubated overnight with this primary reagent at a dilution of 1 in 2,000. The following day, sections were incubated with a biotinylated anti-mouse immunoglobulin secondary reagent (catalogue number BA-2000; Vector Laboratories) at a dilution of 1 in 250 for 30 min, then washed in PBS. Both the albumin- and EBA-labelled sections were then incubated with a streptavidin-conjugated peroxidase tertiary reagent (catalogue number 21127; Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 1 h at a dilution of 1 in 1,000 and washed in PBS. Labelling was ‘visualized’ with 3, 30 -diaminobenzidine tetrahydrochloride. Sections were counterstained with haematoxylin, dehydrated, cleared and mounted. A negative control omitting primary reagent, as well as a control showing the normal pattern of expression of the antigen in question, were run with each batch of slides. In control brains, albumin extravasation was confined to non-BBB regions such as the circumventricular organs and leptomeninges, while in toxintreated brains albumin leakage was widely distributed in grey and white matter of the cerebrum, cerebellum and brainstem. In the latter, perivascular albumin labelling was stronger where the BBB was disrupted and diminished with increasing distance from the affected vessel (Fig. 1). There was uniform EBA immunolabelling of the microvascular bed in control brains, with capillaries and venules, but not arterioles, being well-delineated by endothelial immunoreactivity (Fig. 2). By contrast, in all epsilon toxin-exposed brains, there was frequently partial or
Fig. 1. Epsilon toxin-treated brain. Marked perivascular immunopositivity of extravasated plasma albumin with diffuse, but less intense, immunolabelling of surrounding parenchyma. IHC. Bar, 240 mm.
Clostridial Toxin Microvascular Damage in Rat Brain
155
Fig. 2. Control brain. Uniform EBA immunoreactivity of venular endothelium. IHC. Bar, 80 mm.
even complete loss of EBA immunoreactivity in many microvessels (Figs. 3 and 4). In some microvessels showing EBA loss, there was minimal widening of the perivascular space. Vessels showing EBA depletion were randomly distributed in both grey and white matter throughout the brain, without any discernible regional predilection, and microvessels with EBA loss were often interspersed with those showing a pattern of EBA immunopositivity that was indistinguishable from that seen in controls (Figs. 3 and 4). The mean percentage of microvessels showing total EBA loss in the three levels of brain analysed was 46% for level 1 (range 42e50%), 52% for level 2 (range 48e56%) and 47% for level 3 (range 44e50%). There was no EBA immunopositivity in neurons, glia or neuropil in toxin-treated or control brains. The loss of EBA immunolabelling in many cerebral microvessels exposed to an acute dose of C. perfringens type D epsilon toxin confirmed that one of the prin-
Fig. 3. Epsilon toxin-treated brain. Many microvessels (arrows) show almost complete loss of EBA immunoexpression, while some retain uniform EBA immunopositivity resembling that seen in control brains. IHC. Bars, 160 mm.
Fig. 4. Epsilon toxin-treated brain. Many microvessels (arrows) show almost complete loss of EBA immunoexpression, while some retain uniform EBA immunopositivity resembling that seen in control brains. Swelling of astrocytic end feet around an EBA-depleted vessel is shown (*). IHC. Bars, 160 mm.
cipal actions of this neurotoxin is a direct and damaging effect on the cerebral microvasculature, probably after binding to specific endothelial receptor sites (Finnie, 2003, 2004; Uzal et al., 2004). Extravasation of plasma albumin in epsilon toxintreated rats was widespread throughout the brain and did not favour any particular topographical site. However, the intensity of perivascular albumin immunopositivity was greater in some blood vessels where BBB breakdown was more severe. Similarly, loss of microvascular EBA immunopositivity, either partial or total, did not appear to selectively affect any neuroanatomical region, unlike the focal damage sustained by certain vulnerable brain areas when epsilon intoxication follows a more protracted clinical course (Finnie, 2003, 2004; Uzal et al., 2004). It is evident from the results of the present study that, when the brain of rats is exposed to high circulating levels of epsilon toxin, BBB breakdown with EBA depletion and a rapid extravasation of fluid and plasma proteins (the latter indicated by widespread albumin extravasation) produces severe, generalized cerebral oedema. Moreover, the depletion of EBA appears to cause, rather than be the consequence of, BBB opening (Ghabriel et al., 2000), possibly explaining why some microvessels showing EBA loss had not yet developed evidence of perivascular oedema in the form of astrocytic end feet swelling. Loss of microvascular EBA immunopositivity has proven to be a useful marker of BBB damage in rats given epsilon toxin and could be a valuable aid in future studies of the pathogenesis of this vascularbased, clostridial neurotoxicity.
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Acknowledgments We thank M. Daymon, SA Pathology for technical assistance and Dr M. N. Ghabriel, School of Medical Sciences, University of Adelaide, for the anti-EBA antibody used in this study.
References Finnie JW (2003) Pathogenesis of brain damage produced in sheep by Clostridium perfringens type D epsilon toxin: a review. Australian Veterinary Journal, 81, 219e221. Finnie JW (2004) Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe, 10, 145e150. Ghabriel MN, Zhu C, Hermanis G, Allt G (2000) Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the bloodebrain barrier. Brain Research, 878, 127e135. Ghabriel MN, Zhu C, Leigh C (2002) Electron microscope study of bloodebrain barrier opening induced by immunological targeting of the endothelial barrier antigen. Brain Research, 934, 140e151. Radovsky A, Mahler JF (1999) Nervous system. In: Pathology of the Mouse, RR Maronpot, Ed., Cache River Press, Vienna, Illinois, pp. 445e447. Saubamea B, Cochois-Guegan V, Cisternino S, Scherrmann J-M (2012) Heterogeneity in the rat brain
vasculature revealed by quantitative confocal analysis of endothelial barrier antigen and P-glycoprotein expression. Journal of Cerebral Blood Flow and Metabolism, 32, 81e92. Sternberger NH, Sternberger LA (1987) Bloodebrain barrier protein recognised by monoclonal antibody. Proceedings of the National Academy of Sciences USA, 84, 8169e8173. Uzal FA, Kelly WR, Morris WE (2004) The pathogenesis of peracute experimental Clostridium perfringens type D enterotoxaemia in sheep. Journal of Veterinary Diagnostic Investigation, 16, 403e411. Vinters HV, Kleinschmidt-Demasters BK (2008) General pathology of the central nervous system. In: Greenfield’s Neuropathology, 8th Edit., DN Louis, S Love, DW Ellison, Eds., Edward Arnold, London, pp. 45e47. Zhu C, Ghabriel MN, Blumbergs PC, Reilly PL, Manavis J et al. (2001) Clostridium perfringens prototoxin-induced alteration of endothelial barrier antigen (EBA) immunoreactivity at the bloodebrain barrier (BBB). Experimental Neurology, 169, 72e82.
February 4th, 2014 ½ Received, Accepted, April 19th, 2014
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE
Loss of Endothelial Barrier Antigen Immunoreactivity as a Marker of Clostridium perfringens Type D Epsilon Toxin-induced Microvascular Damage in Rat Brain J. W. Finnie*,†, J. Manavis* and G. Chidlow‡,x * SA Pathology, Hanson Institute Centre for Neurological Diseases, Frome Road, † School of Veterinary Science, University of Adelaide, ‡ Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Frome Road and x Department of Ophthalmology and Visual Sciences, University of Adelaide, Frome Road, Adelaide, South Australia, Australia
Summary The epsilon toxin elaborated by Clostridium perfringens type D in the intestine of domestic livestock is principally responsible for the neurological disease produced after its absorption in excessive quantities into the systemic circulation. The fundamental basis of the cerebral damage induced by epsilon toxin appears to be microvascular injury with ensuing severe, diffuse vasogenic oedema. Endothelial barrier antigen (EBA), which is normally expressed by virtually all capillaries and venules in the rat brain, was used in this study as a marker of bloodebrain barrier (BBB) integrity. After exposure to high levels of circulating epsilon toxin, there was substantial loss of EBA in many brain microvessels, attended by widespread plasma albumin extravasation. These results support microvascular injury and subsequent BBB breakdown as a key factor in the pathogenesis of epsilon toxin-induced neurological disease. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: Clostridium perfringens type D epsilon toxin; endothelial barrier antigen; microvascular injury; rat brain
Clostridium perfringens type D enterotoxaemia is an important, worldwide neurological disorder of lambs and goats and, less commonly, calves caused by epsilon toxin. The disease in sheep spans a continuum from peracute intoxication with rapid death to a subacute form characterized by a more protracted clinical course. When the brain is exposed to large doses of circulating epsilon toxin absorbed from the intestine, the clinical course is generally brief and believed to be associated with microvascular endothelial damage and loss of bloodebrain barrier (BBB) integrity, leading to severe, generalized vasogenic oedema, a marked increase in intracranial pressure, convulsions, coma and often death (Finnie, 2003, 2004; Uzal et al., 2004).
*Correspondence to: J. W. Finnie (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.010
The permeability properties of the BBB are those of capillary endothelial cells, which differ in important structural detail from endothelia elsewhere, and the effectiveness of the BBB depends largely on interendothelial tight junctions, abetted by the inductive influence of astrocytic end feet, which cover most of the capillary surface and modulate the composition of the perineuronal fluid in a highly localized manner (Vinters and Kleinschmidt-Demasters, 2008). In rats, endothelial barrier antigen (EBA) is a membrane protein expressed by virtually all the cerebral microvasculature, which includes capillaries and venules (Sternberger and Sternberger, 1987), and its expression is considered to be a good indicator of a competent BBB (Ghabriel et al., 2000, 2002). EBA is specific to the rat BBB (Sternberger and Sternberger, 1987). Although the precise function of EBA is largely unknown, its role as a specific marker Ó 2014 Elsevier Ltd. All rights reserved.
154
J.W. Finnie et al.
of BBB-competent brain vessels is suggested by its down-regulation in pathological states in which the BBB is disrupted and its absence in regions of the brain devoid of a BBB (Saubamea et al., 2012). Laboratory rodents are useful experimental models of the ovine disease produced by epsilon toxin, as they develop cerebral lesions of a similar nature and anatomical distribution (Finnie, 2003, 2004). Although EBA down-regulation has been used in a number of pathological states in which BBB integrity or function is impaired (Saubamea et al., 2012), this BBB marker has not previously been examined in rats given a high dose of fully activated C. perfringens type D epsilon toxin to simulate the acute ovine disease. In the only other study of vascular EBA loss in rats given this clostridial toxin (Zhu et al., 2001), the inactive epsilon prototoxin was used and it was hypothesized that a small amount was activated by endogenous proteases after intraperitoneal injection to produce a mild, subacute intoxication resulting in only minimal albumin extravasation as a marker of BBB disruption. Six, 6-week-old SpragueeDawley rats were given 1 ml of a 1 in 10 dilution of trypsin-activated prototoxin prepared from filtrates of broth cultures of C. perfringens type D (Commonwealth Serum Laboratories, Melbourne, Australia) by intraperitoneal injection. Four control rats were given a similar volume of a physiological saline solution. At 2 h post injection, the brains were fixed by transcardiac perfusion with 4% paraformaldehyde containing 0.02% heparin. After remaining in situ for 2 h, brains were removed and immersion-fixed in 10% neutral buffered formalin for 4 days, then routinely processed and embedded in paraffin wax. Sections (6 mm) were stained with haematoxylin and eosin (HE). Duplicate sections were cut for immunohistochemistry (IHC). Coronal sections were taken from three levels of brain, the ventral surface landmarks being those routinely used by the National Toxicology Programme (USA): just anterior to the optic chiasm (level 1); at the caudal borders of the mammillary bodies (level 2); and at the widest portion of the cerebellum just posterior to the transverse fibres of the pons (level 3) (Radovsky and Mahler, 1999). In IHC, endogenous albumin was used as a surrogate marker for visualizing the extravasation and spread of vasogenic oedema. A goat anti-rat albumin (catalogue number 0113-0341; Cappel Laboratories, West Chester, Pennsylvania, USA) was used at a dilution of 1 in 20,000. No antigen retrieval was required for the albumin antibody. This was followed by a biotinylated anti-goat immunoglobulin secondary reagent (catalogue number BA-9500; Vector Laboratories, Burlingame, California, USA) at a 1
in 250 dilution for 30 min, then washing in phosphate buffered saline (PBS). To detect EBA, a mouse monoclonal anti-EBA antibody (catalogue number SMI71R; Covance, Princeton, New Jersey, USA) was used. Tissue sections underwent antigen retrieval using citrate buffer and were then incubated overnight with this primary reagent at a dilution of 1 in 2,000. The following day, sections were incubated with a biotinylated anti-mouse immunoglobulin secondary reagent (catalogue number BA-2000; Vector Laboratories) at a dilution of 1 in 250 for 30 min, then washed in PBS. Both the albumin- and EBA-labelled sections were then incubated with a streptavidin-conjugated peroxidase tertiary reagent (catalogue number 21127; Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 1 h at a dilution of 1 in 1,000 and washed in PBS. Labelling was ‘visualized’ with 3, 30 -diaminobenzidine tetrahydrochloride. Sections were counterstained with haematoxylin, dehydrated, cleared and mounted. A negative control omitting primary reagent, as well as a control showing the normal pattern of expression of the antigen in question, were run with each batch of slides. In control brains, albumin extravasation was confined to non-BBB regions such as the circumventricular organs and leptomeninges, while in toxintreated brains albumin leakage was widely distributed in grey and white matter of the cerebrum, cerebellum and brainstem. In the latter, perivascular albumin labelling was stronger where the BBB was disrupted and diminished with increasing distance from the affected vessel (Fig. 1). There was uniform EBA immunolabelling of the microvascular bed in control brains, with capillaries and venules, but not arterioles, being well-delineated by endothelial immunoreactivity (Fig. 2). By contrast, in all epsilon toxin-exposed brains, there was frequently partial or
Fig. 1. Epsilon toxin-treated brain. Marked perivascular immunopositivity of extravasated plasma albumin with diffuse, but less intense, immunolabelling of surrounding parenchyma. IHC. Bar, 240 mm.
Clostridial Toxin Microvascular Damage in Rat Brain
155
Fig. 2. Control brain. Uniform EBA immunoreactivity of venular endothelium. IHC. Bar, 80 mm.
even complete loss of EBA immunoreactivity in many microvessels (Figs. 3 and 4). In some microvessels showing EBA loss, there was minimal widening of the perivascular space. Vessels showing EBA depletion were randomly distributed in both grey and white matter throughout the brain, without any discernible regional predilection, and microvessels with EBA loss were often interspersed with those showing a pattern of EBA immunopositivity that was indistinguishable from that seen in controls (Figs. 3 and 4). The mean percentage of microvessels showing total EBA loss in the three levels of brain analysed was 46% for level 1 (range 42e50%), 52% for level 2 (range 48e56%) and 47% for level 3 (range 44e50%). There was no EBA immunopositivity in neurons, glia or neuropil in toxin-treated or control brains. The loss of EBA immunolabelling in many cerebral microvessels exposed to an acute dose of C. perfringens type D epsilon toxin confirmed that one of the prin-
Fig. 3. Epsilon toxin-treated brain. Many microvessels (arrows) show almost complete loss of EBA immunoexpression, while some retain uniform EBA immunopositivity resembling that seen in control brains. IHC. Bars, 160 mm.
Fig. 4. Epsilon toxin-treated brain. Many microvessels (arrows) show almost complete loss of EBA immunoexpression, while some retain uniform EBA immunopositivity resembling that seen in control brains. Swelling of astrocytic end feet around an EBA-depleted vessel is shown (*). IHC. Bars, 160 mm.
cipal actions of this neurotoxin is a direct and damaging effect on the cerebral microvasculature, probably after binding to specific endothelial receptor sites (Finnie, 2003, 2004; Uzal et al., 2004). Extravasation of plasma albumin in epsilon toxintreated rats was widespread throughout the brain and did not favour any particular topographical site. However, the intensity of perivascular albumin immunopositivity was greater in some blood vessels where BBB breakdown was more severe. Similarly, loss of microvascular EBA immunopositivity, either partial or total, did not appear to selectively affect any neuroanatomical region, unlike the focal damage sustained by certain vulnerable brain areas when epsilon intoxication follows a more protracted clinical course (Finnie, 2003, 2004; Uzal et al., 2004). It is evident from the results of the present study that, when the brain of rats is exposed to high circulating levels of epsilon toxin, BBB breakdown with EBA depletion and a rapid extravasation of fluid and plasma proteins (the latter indicated by widespread albumin extravasation) produces severe, generalized cerebral oedema. Moreover, the depletion of EBA appears to cause, rather than be the consequence of, BBB opening (Ghabriel et al., 2000), possibly explaining why some microvessels showing EBA loss had not yet developed evidence of perivascular oedema in the form of astrocytic end feet swelling. Loss of microvascular EBA immunopositivity has proven to be a useful marker of BBB damage in rats given epsilon toxin and could be a valuable aid in future studies of the pathogenesis of this vascularbased, clostridial neurotoxicity.
156
J.W. Finnie et al.
Acknowledgments We thank M. Daymon, SA Pathology for technical assistance and Dr M. N. Ghabriel, School of Medical Sciences, University of Adelaide, for the anti-EBA antibody used in this study.
References Finnie JW (2003) Pathogenesis of brain damage produced in sheep by Clostridium perfringens type D epsilon toxin: a review. Australian Veterinary Journal, 81, 219e221. Finnie JW (2004) Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe, 10, 145e150. Ghabriel MN, Zhu C, Hermanis G, Allt G (2000) Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the bloodebrain barrier. Brain Research, 878, 127e135. Ghabriel MN, Zhu C, Leigh C (2002) Electron microscope study of bloodebrain barrier opening induced by immunological targeting of the endothelial barrier antigen. Brain Research, 934, 140e151. Radovsky A, Mahler JF (1999) Nervous system. In: Pathology of the Mouse, RR Maronpot, Ed., Cache River Press, Vienna, Illinois, pp. 445e447. Saubamea B, Cochois-Guegan V, Cisternino S, Scherrmann J-M (2012) Heterogeneity in the rat brain
vasculature revealed by quantitative confocal analysis of endothelial barrier antigen and P-glycoprotein expression. Journal of Cerebral Blood Flow and Metabolism, 32, 81e92. Sternberger NH, Sternberger LA (1987) Bloodebrain barrier protein recognised by monoclonal antibody. Proceedings of the National Academy of Sciences USA, 84, 8169e8173. Uzal FA, Kelly WR, Morris WE (2004) The pathogenesis of peracute experimental Clostridium perfringens type D enterotoxaemia in sheep. Journal of Veterinary Diagnostic Investigation, 16, 403e411. Vinters HV, Kleinschmidt-Demasters BK (2008) General pathology of the central nervous system. In: Greenfield’s Neuropathology, 8th Edit., DN Louis, S Love, DW Ellison, Eds., Edward Arnold, London, pp. 45e47. Zhu C, Ghabriel MN, Blumbergs PC, Reilly PL, Manavis J et al. (2001) Clostridium perfringens prototoxin-induced alteration of endothelial barrier antigen (EBA) immunoreactivity at the bloodebrain barrier (BBB). Experimental Neurology, 169, 72e82.
February 4th, 2014 ½ Received, Accepted, April 19th, 2014