- Email: [email protected]
Loss of Endothelial Barrier Antigen Immunoreactivity in Rat Retinal Microvessels is Correlated with Clostridium perfringens Type D Epsilon Toxin-induced Damage to the Blood–Retinal Barrier
J. Comp. Path. 2018, Vol. 158, 51e55
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Loss of Endothelial Barrier Antigen Immunoreactivity in Rat Retinal Microvessels is Correlated with Clostridium perfringens Type D Epsilon Toxin-induced Damage to the BloodeRetinal Barrier K. A. Mander* and J. W. Finnie*,† * Discipline of Anatomy and Pathology, Adelaide Medical School, University of Adelaide and † SA Pathology, Hanson Institute Centre for Neurological Diseases, Frome Road, Adelaide, SA, Australia
Summary Clostridium perfringens type D epsilon toxin (ETX) is a potent neurotoxin producing a severe, and often fatal, neurological disorder in ruminant livestock. Microvascular damage appears to be the fundamental action of ETX in the brain and, recently, similar vascular injury, with subsequent severe vasogenic oedema, has been reported in the retina of rats given ETX. Endothelial barrier antigen (EBA) is a useful marker of an intact bloodebrain barrier in rats and it has been shown that loss of EBA immunoreactivity is correlated with ETX-induced cerebral microvascular damage in this species. This paper reports, for the first time, that loss of EBA immunoexpression also occurs in rat retinal microvessels exposed to ETX, the marked reduction in EBA immunopositivity acting as a useful marker for blooderetinal barrier breakdown produced by this neurotoxin. Ó 2017 Elsevier Ltd. All rights reserved. Keywords: Clostridium perfringens type D epsilon toxin; endothelial barrier antigen; microvascular injury; rat retina
The principal target of Clostridium perfringens type D epsilon toxin (ETX) in ruminant livestock and laboratory rodent models appears to be the cerebral microvasculature, with bloodebrain barrier (BBB) disruption leading to severe, diffuse vasogenic oedema, increased intracranial pressure, convulsions, coma and, often, death (Finnie, 2004; Uzal et al., 2016). It has also recently been shown that ETX produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro (Mander et al., 2016). However, ETX also accumulates in the eye (Tamai et al., 2003) and the bloode retinal barrier (BRB) resembles the BBB in many
Correspondence to: K. Mander (e-mail: Kimberley.mander@adelaide. edu.au). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2017.11.003
important respects (Kaur et al., 2008). The BRB is comprised of inner and outer components, the inner being formed by tight junctions (zonula occludens) between adjacent microvascular endothelial cells, supported by ensheathing pericytes and foot processes of astrocytes and Muller cells. The outer BRB is formed by tight junctions between retinal pigment epithelial cells. It has been suggested (Lawrenson et al., 1995) that the BRB may be more permeable than the BBB, due to an increased number of micropinocytotic vesicles and shorter interendothelial junctions in the former. The retina has a dual circulation: the blood supply from the nerve fibre layer to the external aspect of the inner nuclear layer is supplied by retinal vessels, while the remainder of the retina is dependent on the choroidal circulation (Kaur Ó 2017 Elsevier Ltd. All rights reserved.
52
K.A. Mander and J.W. Finnie
et al., 2008; Runkle and Antonetti, 2011; Campbell and Humphries, 2012). It was recently demonstrated (Finnie et al., 2014a) that ETX damages retinal microvessels in the rat in a similar manner to that found in ETX-injured brain vasculature, leading to extravasation of endogenous albumin as a surrogate immunohistochemical marker of increased vascular permeability and severe retinal oedema. In the rat, endothelial barrier antigen (EBA) is a membrane protein triplet of 23.5, 25 and 30 kDa, respectively (Pelz et al., 2013), which is uniformly expressed on the luminal aspect of brain microvessels (capillaries and venules) (Sternberger and Sternberger, 1987). EBA is considered to be a sensitive and specific marker of an intact and functionally competent BBB in this species (Ghabriel et al., 2002; Pelz et al., 2013). The permeability properties of the BBB are those of the capillary endothelium, particularly interendothelial tight junctions, but also a paucity of micropinocytotic vesicles and the inductive influence of astrocytic end feet that cover most of the capillary surface. Some brain areas, such as the circumventricular organs, are devoid of a BBB, and capillaries here are more permeable and contain fenestrations and discontinuous tight junctions (Ballabh et al., 2004). In rats given ETX (Finnie et al., 2014b), there was loss of EBA immunoreactivity in rat brain microvessels, which was attended by increased vascular permeability and widespread albumin extravasation. Since retinal microvessels constituting the BRB strongly and uniformly express EBA immunopositivity in the rat (Lawrenson et al., 1995; Dartt et al., 2011), we wished to determine, for the first time, whether ETX-induced microvascular injury in these vessels was correlated with loss of EBA immunoreactivity. A group of six, 6-week-old SpragueeDawley rats was given an intraperitoneal injection of 1 ml of a 1 in 10 dilution of trypsin-activated prototoxin prepared from filtrates of broth cultures of C. perfringens type D (Commonweath Serum Laboratories, Melbourne, Australia) (Finnie et al., 2014b). Four control rats were given a similar volume of physiological saline. The rat eyes examined in the present study were from the same animals that had previously been used to describe retinal microvascular damage (Finnie et al., 2014a) produced by ETX and EBA immunohistochemistry (IHC) in toxin-treated brains (Finnie et al., 2014b). At 1 h post-injection, rats were depressed, huddled and unresponsive to external stimuli and, at this time, were anaesthetised with isoflurane and killed by perfusion fixation of the brain (and eyes) with 4% paraformaldehyde. The eyes were then immersion fixed in Davidson’s fixative,
embedded in paraffin wax, and coronal sections (6 mm) were subjected to IHC. In order to detect EBA, a mouse monoclonal antiEBA antibody (catalogue number SMI-71R, Covance, Princeton, New Jersey, USA) was used. This was followed by a biotinylated goat anti-rabbit immunoglobulin secondary reagent (Vector Laboratories, Burlingame, California, USA) at a 1 in 250 dilution for 30 min, after which sections were washed in phosphate buffered saline (PBS). For both antibodies, slides 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 then washed in PBS. Labelling was ‘visualized’ with 3,30 -diaminobenzidine tetrahydrochloride as chromogen. Slides were then counterstained with haematoxylin, dehydrated, cleared and mounted. Positive and negative controls were included in this protocol. It was difficult to visualize extended lengths of a sufficient number of these blood vessels in a single plane at low power as mammalian retinal vascularization is sparse, and sometimes largely (rabbits) or completely (guinea pigs) absent, in order to facilitate a relatively unobstructed light path to the photoreceptors (Yu and Cringle, 2001). It was found that oil immersion (100) enabled the most convincing demonstration of EBA immunoreactivity, and its loss after exposure to ETX, in a given microvessel and, accordingly, representative images of both control and ETX-treated retinas at this magnification are shown. In control rat retinas, microvessel endothelium was uniformly immunolabelled with EBA (Fig. 1). By contrast, in ETX-exposed eyes, as in similarly exposed brains (Fig. 2), there was loss, albeit heterogeneous, of EBA immunoreactivity. Some vessels
Fig. 1. Control retina. Microvessel shows strong endothelial EBA immunopositivity. IHC. Bar, 10 mm.
Clostridium perfringens Toxin Damage to BloodeRetinal Barrier
Fig. 2. ETX-treated brain. A few microvessels are EBA immunopositive, but many (arrows) show complete loss of EBA immunoreactivity. A higher power of an immunopositive control capillary is shown as an inset. IHC. Bar, 160 mm.
showed complete EBA loss (Fig. 3), others partial depletion and, a much smaller number, showed no apparent reduction in EBA immunopositivity. In contrast to retinas in non-treated, control rats, in which microvessels were strongly immunolabelled with EBA, most of those in ETX-treated animals showed a marked reduction in EBA immunoreactivity, which was sometimes complete. This diminution of EBA immunopositivity correlated with severe toxin-induced microvascular injury demonstrated at the ultrastructural level, marked extravasation of plasma albumin as a vascular tracer and marker of increased vascular permeability, and vasogenic retinal oedema. However, as in ETX-exposed rat brains (Finnie et al., 2014b), endothelial EBA depletion was not uniform and ranged from complete loss to, uncommonly, no apparent decrease in immunopositivity. This heterogeneity in the microvascular response to ETX suggested that retinal blood vessels were not equally susceptible to ETX-induced endotheliotoxicity, a pattern resembling that found in ETX-exposed brains (Finnie et al., 2014b). The cerebral endothelium has often been regarded as a homogeneous cell population, but it is now recognized that the BBB is comprised of a more heterogeneous population of endothelial cells, although our understanding of the basis of this diversity is still in its infancy. This heterogeneity in blood vessels comprising the BBB is evident, not only between different neuroanatomical regions as a reflection of their functional diversity, but also amongst capillaries and venules, and even at the single cell level within individual microvessels. Most BBB properties are expressed in capillaries and venules, but capillaries are preferentially involved in structural and transport
53
functions, while venules have a greater role in inflammatory-related tasks and enzymatic and metabolic functions of the BBB, the looser interendothelial venular tight junctions, for example, more readily permitting leucocyte extravasation (Macdonald et al., 2010; Wilhelm et al., 2016). It is likely that there is a similar heterogeneity of endothelial cells comprising the BRB, but this has not been studied to date. In sheep exposed to ETX, pooling of an extravasated, protein-rich fluid around blood vessels is an important diagnostic neuropathological feature of the acute disease (Uzal et al., 2016), but not all vessels appear to be damaged as there is considerable variability in the neuroanatomical distribution of leaked plasma protein (Garcia et al., 2015). This finding suggests that, as in retinas exposed to ETX in the present study, not all microvessels are equally susceptible to ETX injury. It is accepted that different neuroanatomical regions may be uniquely sensitive to a given xenobiotic, reflecting variations in the local circulation, neuron and neurotransmitter distribution, presence of absence of specific receptors and compartmentalization of certain biochemical or metabolic functions (Bolon et al., 2008). EBA in rats is uniformly expressed on the luminal face of the entire cerebral capillary bed (Sternberger and Sternberger, 1987) and is also believed to be an important component of tight junctions in these vessels (Jin et al., 2014). Its expression is correlated with intact cerebral microvessels and reduced EBA expression is characteristic of diseases in which BBB integrity or function is compromised, such as stroke, traumatic brain injury, exposure to neurotoxic agents and experimental allergic encephalopathy (Saubamea et al., 2012; Pelz et al., 2013). It is unclear whether downregulation of EBA has a
Fig. 3. ETX-treated retina. Microvessel shows marked loss of EBA immunopositivity. IHC. Bar, 10 mm.
54
K.A. Mander and J.W. Finnie
causative role in opening the BBB or is merely a consequence of its altered integrity. However, administration of an anti-EBA antibody resulted in opening of the BBB and extravasation of a vascular tracer, suggesting the former is likely to be operative (Ghabriel et al., 2002). EBA is found in an endothelial cytoplasmic pool, whose function may be to replenish luminal membrane EBA. ETX diminishes endothelial cytoplasmic immunolabelling of EBA, which precedes the reduction in luminal labelling, implying that this toxin might impair cytoplasmic synthesis of EBA, leading to impaired EBA production and its progressive depletion at the endothelial cell luminal surface (Zhu et al., 2001). It has been observed (Saubamea et al., 2012) that individual capillaries are sometimes composed of a mix of EBA-positive and EBA-negative endothelial cells, suggesting that its expression may alter over time in a given endothelial cell. EBA expression appears to be highly regulated, being temporally downregulated in response to a changed microenvironment, then gradually returning to its pre-existing level (Saubamea et al., 2012). In conclusion, loss of retinal microvascular EBA immunoreactivity appears to act as a reliable indicator of BRB damage produced by ETX in the rat.
Acknowledgment This research did not receive any specific grant from funding agencies in the public, commercial or notfor-profit sectors.
References Ballabh P, Braun A, Nedergaard M (2004) The bloode brain barrier: structure, regulation, and clinical implications. Neurobiology of Disease, 16, 1e13. Bolon B, Anthony DC, Butt M, Dorman D, Green MV et al. (2008) ‘Current pathology techniques’ symposium review: advances and issues in neuropathology. Toxicologic Pathology, 36, 871e889. Campbell M, Humphries P (2012) The blooderetina barrier: tight junctions and barrier modulation. Advances in Experimental Medicine and Biology, 763, 70e84. Dartt DA, Dana R, D’Amore P, Niederkorn JY (2011) Immunology, Inflammation and Diseases of the Eye. Elsevier, San Diego, p. 229. Finnie JW (2004) Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe, 10, 145e150. Finnie JW, Manavis J, Casson RJ, Chidlow G (2014a) Retinal microvascular damage and vasogenic oedema produced by Clostridium perfringens type D epsilon toxin in rats. Journal of Veterinary Diagnostic Investigation, 26, 470e472.
Finnie JW, Manavis J, Chidlow G (2014b) Loss of endothelial barrier antigen (EBA) immunoreactivity as a marker of Clostridium perfringens type D epsilon toxininduced microvascular damage in rat brain. Journal of Comparative Pathology, 151, 153e156. Garcia JP, Giannitti F, Finnie JW, Manavis J, Beingesser J et al. (2015) Comparative neuropathology of ovine enterotoxaemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives. Veterinary Pathology, 52, 465e475. 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. Jin X, Chen Z, Liu X, Liang B, Zhang H et al. (2014) The expression of endothelial barrier antigen (EBA) and S100B in the rat parietal cortex following brain irradiation. Brain Research, 1558, 84e89. Kaur C, Foulds WS, Ling EA (2008) Blooderetinal barrier in hypoxic ischaemic conditions: basic concepts, clinical features and management. Progress in Retinal Eye Research, 27, 622e647. Lawrenson JG, Ghabriel MN, Reid AR, Gajree TN, Allt G (1995) Distribution of a putative endothelial barrier antigen in the ocular and orbital tissues of the rat. British Journal of Ophthalmology, 79, 462e466. Macdonald JA, Murugesan N, Pachter JS (2010) Endothelial cell heterogeneity of bloodebrain barrier gene expression along the cerebral microvasculature. Journal of Neuroscience Research, 88, 1457e1474. Mander KA, Williams R, Finnie JW (2016) Clostridium perfringens type D epsilon toxin produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro. In: 5th Cold Spring Harbor Blood Brain Barrier Conference, Cold Spring Harbor, New York, USA. Pelz J, Hartig W, Weise C, Hobohm C, Schneider D et al. (2013) Endothelial barrier antigen-immunoreactivity is conversely associated with bloodebrain barrier dysfunction after embolic stroke in rats. European Journal of Histochemistry, 57, e38. Runkle EA, Antonetti DA (2011) The blooderetinal barrier: structure and functional significance. In: The BloodeBrain and Other Neural Barriers, S Nag, Ed., Humana Press, New York, pp. 133e148. 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 is recognised by monoclonal antibody. Proceedings of the National Academy of Science of the United States of America, 84, 8169e8173. Tamai E, Ishida T, Miyata S (2003) Accumulation of Clostridium perfringens epsilon toxin in the mouse kidney and its possible biological significance. Infection and Immunity, 71, 5371e5375.
Clostridium perfringens Toxin Damage to BloodeRetinal Barrier
Uzal FA, Giannitti F, Finnie JW, Garcia JP (2016) Diseases produced by Clostridium perfringens type D. In: Clostridial Diseases of Animals, FA Uzal, JG Songer, JF Prescott, MR Popoff, Eds., Wiley-Blackwell, Ames, pp. 157e172. Wilhelm I, Nyul-Toth A, Suciu M, Hermenean A, Krizbai JA (2016) Heterogeneity of the bloodebrain barrier. Tissue Barriers, 4, e1143544. Yu DY, Cringle SJ (2001) Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease. Progress in Retinal and Eye Research, 20, 175e208.
55
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.
September 29th, 2017 ½ Received, Accepted, November 25th, 2017
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Loss of Endothelial Barrier Antigen Immunoreactivity in Rat Retinal Microvessels is Correlated with Clostridium perfringens Type D Epsilon Toxin-induced Damage to the BloodeRetinal Barrier K. A. Mander* and J. W. Finnie*,† * Discipline of Anatomy and Pathology, Adelaide Medical School, University of Adelaide and † SA Pathology, Hanson Institute Centre for Neurological Diseases, Frome Road, Adelaide, SA, Australia
Summary Clostridium perfringens type D epsilon toxin (ETX) is a potent neurotoxin producing a severe, and often fatal, neurological disorder in ruminant livestock. Microvascular damage appears to be the fundamental action of ETX in the brain and, recently, similar vascular injury, with subsequent severe vasogenic oedema, has been reported in the retina of rats given ETX. Endothelial barrier antigen (EBA) is a useful marker of an intact bloodebrain barrier in rats and it has been shown that loss of EBA immunoreactivity is correlated with ETX-induced cerebral microvascular damage in this species. This paper reports, for the first time, that loss of EBA immunoexpression also occurs in rat retinal microvessels exposed to ETX, the marked reduction in EBA immunopositivity acting as a useful marker for blooderetinal barrier breakdown produced by this neurotoxin. Ó 2017 Elsevier Ltd. All rights reserved. Keywords: Clostridium perfringens type D epsilon toxin; endothelial barrier antigen; microvascular injury; rat retina
The principal target of Clostridium perfringens type D epsilon toxin (ETX) in ruminant livestock and laboratory rodent models appears to be the cerebral microvasculature, with bloodebrain barrier (BBB) disruption leading to severe, diffuse vasogenic oedema, increased intracranial pressure, convulsions, coma and, often, death (Finnie, 2004; Uzal et al., 2016). It has also recently been shown that ETX produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro (Mander et al., 2016). However, ETX also accumulates in the eye (Tamai et al., 2003) and the bloode retinal barrier (BRB) resembles the BBB in many
Correspondence to: K. Mander (e-mail: Kimberley.mander@adelaide. edu.au). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2017.11.003
important respects (Kaur et al., 2008). The BRB is comprised of inner and outer components, the inner being formed by tight junctions (zonula occludens) between adjacent microvascular endothelial cells, supported by ensheathing pericytes and foot processes of astrocytes and Muller cells. The outer BRB is formed by tight junctions between retinal pigment epithelial cells. It has been suggested (Lawrenson et al., 1995) that the BRB may be more permeable than the BBB, due to an increased number of micropinocytotic vesicles and shorter interendothelial junctions in the former. The retina has a dual circulation: the blood supply from the nerve fibre layer to the external aspect of the inner nuclear layer is supplied by retinal vessels, while the remainder of the retina is dependent on the choroidal circulation (Kaur Ó 2017 Elsevier Ltd. All rights reserved.
52
K.A. Mander and J.W. Finnie
et al., 2008; Runkle and Antonetti, 2011; Campbell and Humphries, 2012). It was recently demonstrated (Finnie et al., 2014a) that ETX damages retinal microvessels in the rat in a similar manner to that found in ETX-injured brain vasculature, leading to extravasation of endogenous albumin as a surrogate immunohistochemical marker of increased vascular permeability and severe retinal oedema. In the rat, endothelial barrier antigen (EBA) is a membrane protein triplet of 23.5, 25 and 30 kDa, respectively (Pelz et al., 2013), which is uniformly expressed on the luminal aspect of brain microvessels (capillaries and venules) (Sternberger and Sternberger, 1987). EBA is considered to be a sensitive and specific marker of an intact and functionally competent BBB in this species (Ghabriel et al., 2002; Pelz et al., 2013). The permeability properties of the BBB are those of the capillary endothelium, particularly interendothelial tight junctions, but also a paucity of micropinocytotic vesicles and the inductive influence of astrocytic end feet that cover most of the capillary surface. Some brain areas, such as the circumventricular organs, are devoid of a BBB, and capillaries here are more permeable and contain fenestrations and discontinuous tight junctions (Ballabh et al., 2004). In rats given ETX (Finnie et al., 2014b), there was loss of EBA immunoreactivity in rat brain microvessels, which was attended by increased vascular permeability and widespread albumin extravasation. Since retinal microvessels constituting the BRB strongly and uniformly express EBA immunopositivity in the rat (Lawrenson et al., 1995; Dartt et al., 2011), we wished to determine, for the first time, whether ETX-induced microvascular injury in these vessels was correlated with loss of EBA immunoreactivity. A group of six, 6-week-old SpragueeDawley rats was given an intraperitoneal injection of 1 ml of a 1 in 10 dilution of trypsin-activated prototoxin prepared from filtrates of broth cultures of C. perfringens type D (Commonweath Serum Laboratories, Melbourne, Australia) (Finnie et al., 2014b). Four control rats were given a similar volume of physiological saline. The rat eyes examined in the present study were from the same animals that had previously been used to describe retinal microvascular damage (Finnie et al., 2014a) produced by ETX and EBA immunohistochemistry (IHC) in toxin-treated brains (Finnie et al., 2014b). At 1 h post-injection, rats were depressed, huddled and unresponsive to external stimuli and, at this time, were anaesthetised with isoflurane and killed by perfusion fixation of the brain (and eyes) with 4% paraformaldehyde. The eyes were then immersion fixed in Davidson’s fixative,
embedded in paraffin wax, and coronal sections (6 mm) were subjected to IHC. In order to detect EBA, a mouse monoclonal antiEBA antibody (catalogue number SMI-71R, Covance, Princeton, New Jersey, USA) was used. This was followed by a biotinylated goat anti-rabbit immunoglobulin secondary reagent (Vector Laboratories, Burlingame, California, USA) at a 1 in 250 dilution for 30 min, after which sections were washed in phosphate buffered saline (PBS). For both antibodies, slides 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 then washed in PBS. Labelling was ‘visualized’ with 3,30 -diaminobenzidine tetrahydrochloride as chromogen. Slides were then counterstained with haematoxylin, dehydrated, cleared and mounted. Positive and negative controls were included in this protocol. It was difficult to visualize extended lengths of a sufficient number of these blood vessels in a single plane at low power as mammalian retinal vascularization is sparse, and sometimes largely (rabbits) or completely (guinea pigs) absent, in order to facilitate a relatively unobstructed light path to the photoreceptors (Yu and Cringle, 2001). It was found that oil immersion (100) enabled the most convincing demonstration of EBA immunoreactivity, and its loss after exposure to ETX, in a given microvessel and, accordingly, representative images of both control and ETX-treated retinas at this magnification are shown. In control rat retinas, microvessel endothelium was uniformly immunolabelled with EBA (Fig. 1). By contrast, in ETX-exposed eyes, as in similarly exposed brains (Fig. 2), there was loss, albeit heterogeneous, of EBA immunoreactivity. Some vessels
Fig. 1. Control retina. Microvessel shows strong endothelial EBA immunopositivity. IHC. Bar, 10 mm.
Clostridium perfringens Toxin Damage to BloodeRetinal Barrier
Fig. 2. ETX-treated brain. A few microvessels are EBA immunopositive, but many (arrows) show complete loss of EBA immunoreactivity. A higher power of an immunopositive control capillary is shown as an inset. IHC. Bar, 160 mm.
showed complete EBA loss (Fig. 3), others partial depletion and, a much smaller number, showed no apparent reduction in EBA immunopositivity. In contrast to retinas in non-treated, control rats, in which microvessels were strongly immunolabelled with EBA, most of those in ETX-treated animals showed a marked reduction in EBA immunoreactivity, which was sometimes complete. This diminution of EBA immunopositivity correlated with severe toxin-induced microvascular injury demonstrated at the ultrastructural level, marked extravasation of plasma albumin as a vascular tracer and marker of increased vascular permeability, and vasogenic retinal oedema. However, as in ETX-exposed rat brains (Finnie et al., 2014b), endothelial EBA depletion was not uniform and ranged from complete loss to, uncommonly, no apparent decrease in immunopositivity. This heterogeneity in the microvascular response to ETX suggested that retinal blood vessels were not equally susceptible to ETX-induced endotheliotoxicity, a pattern resembling that found in ETX-exposed brains (Finnie et al., 2014b). The cerebral endothelium has often been regarded as a homogeneous cell population, but it is now recognized that the BBB is comprised of a more heterogeneous population of endothelial cells, although our understanding of the basis of this diversity is still in its infancy. This heterogeneity in blood vessels comprising the BBB is evident, not only between different neuroanatomical regions as a reflection of their functional diversity, but also amongst capillaries and venules, and even at the single cell level within individual microvessels. Most BBB properties are expressed in capillaries and venules, but capillaries are preferentially involved in structural and transport
53
functions, while venules have a greater role in inflammatory-related tasks and enzymatic and metabolic functions of the BBB, the looser interendothelial venular tight junctions, for example, more readily permitting leucocyte extravasation (Macdonald et al., 2010; Wilhelm et al., 2016). It is likely that there is a similar heterogeneity of endothelial cells comprising the BRB, but this has not been studied to date. In sheep exposed to ETX, pooling of an extravasated, protein-rich fluid around blood vessels is an important diagnostic neuropathological feature of the acute disease (Uzal et al., 2016), but not all vessels appear to be damaged as there is considerable variability in the neuroanatomical distribution of leaked plasma protein (Garcia et al., 2015). This finding suggests that, as in retinas exposed to ETX in the present study, not all microvessels are equally susceptible to ETX injury. It is accepted that different neuroanatomical regions may be uniquely sensitive to a given xenobiotic, reflecting variations in the local circulation, neuron and neurotransmitter distribution, presence of absence of specific receptors and compartmentalization of certain biochemical or metabolic functions (Bolon et al., 2008). EBA in rats is uniformly expressed on the luminal face of the entire cerebral capillary bed (Sternberger and Sternberger, 1987) and is also believed to be an important component of tight junctions in these vessels (Jin et al., 2014). Its expression is correlated with intact cerebral microvessels and reduced EBA expression is characteristic of diseases in which BBB integrity or function is compromised, such as stroke, traumatic brain injury, exposure to neurotoxic agents and experimental allergic encephalopathy (Saubamea et al., 2012; Pelz et al., 2013). It is unclear whether downregulation of EBA has a
Fig. 3. ETX-treated retina. Microvessel shows marked loss of EBA immunopositivity. IHC. Bar, 10 mm.
54
K.A. Mander and J.W. Finnie
causative role in opening the BBB or is merely a consequence of its altered integrity. However, administration of an anti-EBA antibody resulted in opening of the BBB and extravasation of a vascular tracer, suggesting the former is likely to be operative (Ghabriel et al., 2002). EBA is found in an endothelial cytoplasmic pool, whose function may be to replenish luminal membrane EBA. ETX diminishes endothelial cytoplasmic immunolabelling of EBA, which precedes the reduction in luminal labelling, implying that this toxin might impair cytoplasmic synthesis of EBA, leading to impaired EBA production and its progressive depletion at the endothelial cell luminal surface (Zhu et al., 2001). It has been observed (Saubamea et al., 2012) that individual capillaries are sometimes composed of a mix of EBA-positive and EBA-negative endothelial cells, suggesting that its expression may alter over time in a given endothelial cell. EBA expression appears to be highly regulated, being temporally downregulated in response to a changed microenvironment, then gradually returning to its pre-existing level (Saubamea et al., 2012). In conclusion, loss of retinal microvascular EBA immunoreactivity appears to act as a reliable indicator of BRB damage produced by ETX in the rat.
Acknowledgment This research did not receive any specific grant from funding agencies in the public, commercial or notfor-profit sectors.
References Ballabh P, Braun A, Nedergaard M (2004) The bloode brain barrier: structure, regulation, and clinical implications. Neurobiology of Disease, 16, 1e13. Bolon B, Anthony DC, Butt M, Dorman D, Green MV et al. (2008) ‘Current pathology techniques’ symposium review: advances and issues in neuropathology. Toxicologic Pathology, 36, 871e889. Campbell M, Humphries P (2012) The blooderetina barrier: tight junctions and barrier modulation. Advances in Experimental Medicine and Biology, 763, 70e84. Dartt DA, Dana R, D’Amore P, Niederkorn JY (2011) Immunology, Inflammation and Diseases of the Eye. Elsevier, San Diego, p. 229. Finnie JW (2004) Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe, 10, 145e150. Finnie JW, Manavis J, Casson RJ, Chidlow G (2014a) Retinal microvascular damage and vasogenic oedema produced by Clostridium perfringens type D epsilon toxin in rats. Journal of Veterinary Diagnostic Investigation, 26, 470e472.
Finnie JW, Manavis J, Chidlow G (2014b) Loss of endothelial barrier antigen (EBA) immunoreactivity as a marker of Clostridium perfringens type D epsilon toxininduced microvascular damage in rat brain. Journal of Comparative Pathology, 151, 153e156. Garcia JP, Giannitti F, Finnie JW, Manavis J, Beingesser J et al. (2015) Comparative neuropathology of ovine enterotoxaemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives. Veterinary Pathology, 52, 465e475. 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. Jin X, Chen Z, Liu X, Liang B, Zhang H et al. (2014) The expression of endothelial barrier antigen (EBA) and S100B in the rat parietal cortex following brain irradiation. Brain Research, 1558, 84e89. Kaur C, Foulds WS, Ling EA (2008) Blooderetinal barrier in hypoxic ischaemic conditions: basic concepts, clinical features and management. Progress in Retinal Eye Research, 27, 622e647. Lawrenson JG, Ghabriel MN, Reid AR, Gajree TN, Allt G (1995) Distribution of a putative endothelial barrier antigen in the ocular and orbital tissues of the rat. British Journal of Ophthalmology, 79, 462e466. Macdonald JA, Murugesan N, Pachter JS (2010) Endothelial cell heterogeneity of bloodebrain barrier gene expression along the cerebral microvasculature. Journal of Neuroscience Research, 88, 1457e1474. Mander KA, Williams R, Finnie JW (2016) Clostridium perfringens type D epsilon toxin produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro. In: 5th Cold Spring Harbor Blood Brain Barrier Conference, Cold Spring Harbor, New York, USA. Pelz J, Hartig W, Weise C, Hobohm C, Schneider D et al. (2013) Endothelial barrier antigen-immunoreactivity is conversely associated with bloodebrain barrier dysfunction after embolic stroke in rats. European Journal of Histochemistry, 57, e38. Runkle EA, Antonetti DA (2011) The blooderetinal barrier: structure and functional significance. In: The BloodeBrain and Other Neural Barriers, S Nag, Ed., Humana Press, New York, pp. 133e148. 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 is recognised by monoclonal antibody. Proceedings of the National Academy of Science of the United States of America, 84, 8169e8173. Tamai E, Ishida T, Miyata S (2003) Accumulation of Clostridium perfringens epsilon toxin in the mouse kidney and its possible biological significance. Infection and Immunity, 71, 5371e5375.
Clostridium perfringens Toxin Damage to BloodeRetinal Barrier
Uzal FA, Giannitti F, Finnie JW, Garcia JP (2016) Diseases produced by Clostridium perfringens type D. In: Clostridial Diseases of Animals, FA Uzal, JG Songer, JF Prescott, MR Popoff, Eds., Wiley-Blackwell, Ames, pp. 157e172. Wilhelm I, Nyul-Toth A, Suciu M, Hermenean A, Krizbai JA (2016) Heterogeneity of the bloodebrain barrier. Tissue Barriers, 4, e1143544. Yu DY, Cringle SJ (2001) Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease. Progress in Retinal and Eye Research, 20, 175e208.
55
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.
September 29th, 2017 ½ Received, Accepted, November 25th, 2017