YEBEH-03695; No. of pages: 5; 4C: 2, 3, 4 Epilepsy & Behavior xxx (2014) xxx–xxx
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Review
Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy Lidia Alonso-Nanclares ⁎, Javier DeFelipe Instituto Cajal (CSIC), Avda. Doctor Arce, 37, Madrid 28002, Spain Laboratorio Cajal de Circuitos Corticales (Centro de Tecnología Biomédica), Universidad Politécnica de Madrid, Campus Montegancedo s/n, Pozuelo de Alarcón, 28223 Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
a r t i c l e
i n f o
Article history: Accepted 9 December 2013 Available online xxxx Keywords: Angiogenesis Blood–brain barrier Blood vessels Electron microscopy Vascular pathology
a b s t r a c t Hippocampal sclerosis is the most frequent pathology encountered in resected tissue obtained from patients with temporal lobe epilepsy. The main hallmarks of hippocampal sclerosis are neuronal loss and gliosis. Several authors have proposed that an increase in blood vessel density is a further indicator, based on interpretations from staining of markers related to both blood–brain barrier disruption and the formation of new blood vessels. However, previous studies performed in our laboratory using correlative light and electron microscopy revealed that many of these “blood vessels” are in fact atrophic vascular structures with a reduced or virtually absent lumen and are often filled with processes of reactive astrocytes. Thus, “normal” vasculature within the sclerotic CA1 field is drastically reduced. Since this decrease is consistently observed in the human sclerotic CA1, this feature can be considered another key pathological indicator of hippocampal sclerosis associated with temporal lobe epilepsy. This article is part of a Special Issue entitled “NEWroscience 2013”. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Temporal lobe epilepsy (TLE) is a common form of intractable epilepsy, and hippocampal sclerosis is the most frequent pathology encountered in resected mesial temporal tissue obtained from patients with TLE [1]. In general, hippocampal sclerosis is characterized by gliosis and neuronal loss, most prominently in the CA1 field, followed by the CA3 and CA2 fields, and the dentate gyrus [1,2]. These alterations are commonly accompanied by a dispersion of the dentate granular cell layer, with ectopic neurons appearing in the molecular layer [3]. There are also changes in the expression of a variety of molecules in the surviving cells, as well as axonal reorganization involving both excitatory and inhibitory circuits [e.g., 4–12]. Early studies on hippocampal sclerosis performed at the end of the XIXth-century, such as those conducted by Sommer [13] and Bratz [14], also described vascular alterations in the sclerotic regions, and it was suggested that these alterations contribute to epileptogenicity.
Abbreviations: BBB, blood–brain barrier; CA, cornu ammonis; Collagen-IV, collagen type IV; Ir, immunoreactive; TLE, temporal lobe epilepsy; Vv, volume fraction. ⁎ Corresponding author at: Instituto Cajal (CSIC), Avda. Doctor Arce 37, 28002 Madrid, Spain and Laboratorio Cajal de Circuitos Corticales (Centro de Tecnología Biomédica), Universidad Politécnica de Madrid, Campus Montegancedo s/n, Pozuelo de Alarcón, 28223 Madrid, Spain. E-mail address: [email protected] (L. Alonso-Nanclares).
However, neuropathological analysis has mainly focused on the loss of neurons and gliosis. In recent years, there has been renewed interest in the study of vasculature alterations. For example, studies on sclerotic hippocampal tissue have reported changes in the permeability of the blood–brain barrier (BBB), with this disruption being proposed as a trigger for epileptiform activity [15–21, for a review, see 22]. It has been proposed that inflammatory processes are activated if normal BBB permeability is disrupted [23], thereby damaging the adjacent brain tissue, producing ion imbalances, and inducing cell death [24]. In addition, an increase in the density of blood vessels in the sclerotic hippocampus of patients with TLE has been reported [25–27; reviewed in 28], which is in line with the activation of expression of angiogenic factors observed in animal models after electroconvulsive seizures [29]. However, other studies on the microvasculature of sclerotic hippocampal tissue obtained from patients with epilepsy by surgical resection have found an impressive reduction in the blood vessels and a variety of alterations in the microanatomy of the remaining blood vessels [30]. In this review, we will discuss the controversy surrounding angiogenesis in the human sclerotic hippocampus and its relationship to epileptogenicity. 2. Study of the microvasculature in hippocampal sclerosis Brain microvasculature can be histologically analyzed by numerous approaches, including histochemical and immunocytochemical methods,
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Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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which provide indirect labeling of the blood vessels by specific staining of some of their components. Histochemical methods have been applied, such as the detection of NADPH–diaphorase activity, which is an enzyme located in the endothelium whose staining is reduced in epilepsy animal models [31]. Staining of the endogenous enzyme alkaline phosphatase (AP) has been widely used (reviewed in [32]). Alkaline phosphatase is an enzyme present in the endothelial cells of the BBB, and it has been used to visualize the capillary network of the cerebral cortex and subcortical structures [33–36]. In particular, AP staining labeled numerous blood vessels in all fields of the normal hippocampal formation, and a significant decrease in the density of AP-positive blood vessels has been described in the CA1 of the sclerotic hippocampus [30]. It has also been shown that AP activity varies in the rat brain when seizures are induced [37]. Furthermore, uniform AP staining was observed in all hippocampal fields of patients with nonsclerotic epilepsy, suggesting that the changes in AP activity are specifically related to hippocampal sclerosis and not to epileptic activity per se [30]. Moreover, genetic defects in AP lead to epileptic seizures due to alterations in the normal AP activity [38–42], which has been hypothesized to be due to the inability of AP to regulate GABA synthesis and neurotransmission in the cerebral cortex [43,44]. Thus, the reduction of AP staining would suggest a modification in its activity, and this might in turn imply alterations to the GABAergic system. Such alterations have been proposed as being responsible for the development of TLE [reviewed in 45–47]. The detection of plasma components outside the blood vessels has been used as an indicator of BBB disruption related to epileptogenicity [20,25,48]. For example, it has been described that vascular endothelial growth factor (VEGF) is upregulated in pathological conditions of the brain tissue, including seizures, and that this growth factor promotes BBB disruption and formation of new microvessels [25, reviewed in 27,49].
3. Angiogenesis or vascular reduction? In recent years, a number of laboratories have described an increase in the number of blood vessels in both patients with epilepsy and animal models, using markers related to angiogenesis [reviewed in 27,49]. Studies performed using collagen type IV (collagen-IV) immunoreactivity (-ir) in the sclerotic hippocampus of patients with epilepsy showed a significantly higher density (increase of 160%) in the CA1 field when compared with the nonsclerotic hippocampus [30]. Collagen-IV is an essential component of the basal lamina in terms of maintaining the endothelial permeability barrier, and it is present in most blood vessel types [50]. Collagen-IV-ir has been largely used to study the microvascular network in several brain disorders [51–54]. It seems clear that the higher density of positive collagen-IV-ir elements in the sclerotic hippocampus suggests that angiogenesis has occurred. This would be in keeping with studies using immunostaining for VEGF which have reported an increase in blood vessel density in patients with TLE [25,27]. However, we observed that closer examination of the collagen-IV-ir elements revealed that in the stratum pyramidale of the CA1 field from the sclerotic hippocampi, many of the collagen-IV-ir blood vessels were notably altered (Figs. 1–3). These blood vessels had a rough surface with numerous small, spine-like protrusions compared with the typically smooth surface found in the nonsclerotic CA1. These protrusions gave a “spiny” appearance to the surface of the blood vessels. Furthermore, the most notable alteration was that instead of the homogeneous staining of the blood vessel surface, numerous abnormal tubular or “vascular-like” structures appeared with a vacuolar or reticulated appearance [30].
Fig. 1. Low-power photomicrographs of collagen-IV immunostained hippocampal sections from control (A) and sclerotic hippocampus (B) to illustrate the distribution of collagen-IV-ir blood vessels. Arrows indicate the CA1 areas also shown at a higher magnification in Figs. 2A and B. C: Photomicrograph of Nissl-stained section showing the hippocampal formation from a patient with sclerotic epilepsy. Note that the sclerotic hippocampal formation is smaller than the control hippocampus. Sub: subiculum. Scale bar (in C): 1000 μm.
Therefore, we used correlative light and electron microscope (EM) techniques to analyze these collagen-IV-ir elements in the sclerotic CA1. These correlative light and EM methods allowed the unequivocal identification of blood vessels and its ultrastructure. The surface of many blood vessels displayed abnormal protrusions of the basal lamina that were responsible for the “spiny” appearance of the blood vessels in this region (Fig. 2,3). Interestingly, the vascular-like collagen-IV-ir processes with a vacuolar appearance were indeed atrophic branches of blood vessels with the peculiarity that their reduced lumen was mainly filled with the processes of reactive astrocytes. The nature of these structures suggested collapse of the blood vessels in the CA1 field of the sclerotic hippocampus [30]. Remarkably, these altered blood vessels were mainly found in those areas where neuronal loss was so severe that virtually no
Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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Fig. 2. Photomicrographs of the CA1 field from a control (A) and sclerotic (B) hippocampus illustrating the apparent increase in collagen-IV-ir blood vessels in the sclerotic tissue. C: Higher magnification to illustrate the smooth surface of the collagen-IV-ir blood vessels in the control tissue. D: Higher magnification to illustrate the presence of small spine-like protrusions on the surface of the collagen-IV-ir blood vessel (arrowheads). E: Higher magnification to show the abnormal tubular or “vascular-like” collagen-IV-ir structures with a vacuolar or reticulated appearance. Scale bar (in E): 75 μm in A, B and 10 μm in C–E.
neurons were present [55]. It is well known that neurons, glia, and blood vessels are closely coupled, both anatomically and functionally [reviewed in 56–58]. Indeed, neurons, glia, and blood vessels have been considered together as a functional entity called the neurovascular unit [24]. This is in line with the fact that perturbations in the integrity of neurovascular function are thought to initiate several cascades associated with injury, which may damage the adjacent brain tissue, producing ion imbalances and inducing cell death [24]. In order to confirm these observations, we used plastic semithin sections (2–5 μm thick) stained with toluidine blue (Fig. 4). This method allows the visualization not only of neurons and glia but also of blood vessels, regardless of their possible molecular alterations. We estimated the volume fraction (Vv) occupied by blood vessels in the sclerotic CA1 field where immunostaining for collagen-IV appeared to show an increase of blood vessels. We observed a significant decrease in the volume occupied by blood vessels in the CA1 field (approximately 60%) compared with nonsclerotic tissue [30], confirming that many of the structures that had been interpreted as blood vessels in the preparations immunostained for collagen-IV were in fact fragments resulting from degenerating of blood vessels. Finally, it should be noted that the significance of the local reduction of blood vessels is not clear. In order to define the functional significance of specific changes in the microvascular network of patients with TLE, it will be necessary to perform electrophysiological studies in conjunction with correlative microanatomical and neurochemical characterization of the epileptic tissue.
4. Conclusions Angiogenesis in the sclerotic hippocampus has been proposed previously, based on the apparent increase in blood vessel density, presumed from staining of markers related to BBB disruption and formation of new blood vessels (such as VEGF). However, correlative light and electron microscopy reveals that many of these “blood vessels” are in fact atrophic vascular structures with a reduced or virtually absent lumen and are often filled with processes of reactive astrocytes. Thus, “normal” blood vessels within the sclerotic CA1 are drastically reduced as confirmed by volume fraction analysis in semithin section (which showed a 60% reduction). It is striking that this kind of relatively simple and powerful analysis had gone unnoticed in previous histopathological assessment of the sclerotic hippocampus. Since this decrease is consistently observed in the human sclerotic CA1, this feature can be considered as another histopathological hallmark of hippocampal sclerosis.
Acknowledgments This work was supported by grants from the following entities: the Spanish Ministry of Economy and Competitiveness (BFU2012-34963 to JDF) and the Cajal Blue Brain Project, Spanish partner of the Blue Brain Project initiative from EPFL. Conflict of interest The authors declare that there are no conflicts of interest.
Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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Fig. 3. Correlative light and electron microscopy photomicrographs of a collagen-IV-ir blood vessel in the CA1 field of a sclerotic hippocampus. A: Photomicrograph of a plastic-embedded section showing collagen-IV-ir blood vessels. B: Photomicrograph of a 2-um-thick semithin plastic section obtained from the same section shown in A. V1 and V2 indicate the same blood vessels labeled in A. C: Low-power electron micrograph taken after the resection of the semithin section shown in B illustrating blood vessel V1. The arrow and arrowhead indicate small spine-like protrusions on the surface of the collagen-IV-ir blood vessel and an atrophic branch, respectively. D, E: Higher magnification of C to illustrate the morphological alterations to the blood vessel V1. Arrow in D indicates the same protrusion as in C. E is a higher magnification of the atrophic branch in the blood vessel, which appears as an abnormal protrusion of the endothelial membrane. Scale bar (in E): 150 μm in A, 75 μm in B, 10 μm in C, 2.5 μm in D, and 1 μm in E.
Fig. 4. Photomicrograph of semithin sections (2 μm) stained with 1% toluidine blue, from control (A) and sclerotic (B) CA1 field to illustrate the Cavalieri method used to estimate the Vv of blood vessels. Note the presence of abundant pyramidal cells and blood vessels in the nonsclerotic CA1 (A) when compared with the sclerotic tissue (B). A grid of small intersections overlying the tissue is displayed. Adjacent grid points are 20 μm apart, providing an area enclosed by any 4 given grid points of 400 μm2 (20 × 20). Yellow asterisks indicate the intersections in the grid that lies within blood vessels (n = 5, 5 × 400 = 2000 μm2 in nonsclerotic and n = 1, 400 μm2 in sclerotic). Note that the number of intersections (yellow asterisks) in the grid that lies within the blood vessels is higher in the control tissue than in the sclerotic tissue. Scale bar (in B): 20 μm.
Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
Contents lists available at ScienceDirect
Epilepsy & Behavior journal homepage: www.elsevier.com/locate/yebeh
Review
Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy Lidia Alonso-Nanclares ⁎, Javier DeFelipe Instituto Cajal (CSIC), Avda. Doctor Arce, 37, Madrid 28002, Spain Laboratorio Cajal de Circuitos Corticales (Centro de Tecnología Biomédica), Universidad Politécnica de Madrid, Campus Montegancedo s/n, Pozuelo de Alarcón, 28223 Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
a r t i c l e
i n f o
Article history: Accepted 9 December 2013 Available online xxxx Keywords: Angiogenesis Blood–brain barrier Blood vessels Electron microscopy Vascular pathology
a b s t r a c t Hippocampal sclerosis is the most frequent pathology encountered in resected tissue obtained from patients with temporal lobe epilepsy. The main hallmarks of hippocampal sclerosis are neuronal loss and gliosis. Several authors have proposed that an increase in blood vessel density is a further indicator, based on interpretations from staining of markers related to both blood–brain barrier disruption and the formation of new blood vessels. However, previous studies performed in our laboratory using correlative light and electron microscopy revealed that many of these “blood vessels” are in fact atrophic vascular structures with a reduced or virtually absent lumen and are often filled with processes of reactive astrocytes. Thus, “normal” vasculature within the sclerotic CA1 field is drastically reduced. Since this decrease is consistently observed in the human sclerotic CA1, this feature can be considered another key pathological indicator of hippocampal sclerosis associated with temporal lobe epilepsy. This article is part of a Special Issue entitled “NEWroscience 2013”. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Temporal lobe epilepsy (TLE) is a common form of intractable epilepsy, and hippocampal sclerosis is the most frequent pathology encountered in resected mesial temporal tissue obtained from patients with TLE [1]. In general, hippocampal sclerosis is characterized by gliosis and neuronal loss, most prominently in the CA1 field, followed by the CA3 and CA2 fields, and the dentate gyrus [1,2]. These alterations are commonly accompanied by a dispersion of the dentate granular cell layer, with ectopic neurons appearing in the molecular layer [3]. There are also changes in the expression of a variety of molecules in the surviving cells, as well as axonal reorganization involving both excitatory and inhibitory circuits [e.g., 4–12]. Early studies on hippocampal sclerosis performed at the end of the XIXth-century, such as those conducted by Sommer [13] and Bratz [14], also described vascular alterations in the sclerotic regions, and it was suggested that these alterations contribute to epileptogenicity.
Abbreviations: BBB, blood–brain barrier; CA, cornu ammonis; Collagen-IV, collagen type IV; Ir, immunoreactive; TLE, temporal lobe epilepsy; Vv, volume fraction. ⁎ Corresponding author at: Instituto Cajal (CSIC), Avda. Doctor Arce 37, 28002 Madrid, Spain and Laboratorio Cajal de Circuitos Corticales (Centro de Tecnología Biomédica), Universidad Politécnica de Madrid, Campus Montegancedo s/n, Pozuelo de Alarcón, 28223 Madrid, Spain. E-mail address: [email protected] (L. Alonso-Nanclares).
However, neuropathological analysis has mainly focused on the loss of neurons and gliosis. In recent years, there has been renewed interest in the study of vasculature alterations. For example, studies on sclerotic hippocampal tissue have reported changes in the permeability of the blood–brain barrier (BBB), with this disruption being proposed as a trigger for epileptiform activity [15–21, for a review, see 22]. It has been proposed that inflammatory processes are activated if normal BBB permeability is disrupted [23], thereby damaging the adjacent brain tissue, producing ion imbalances, and inducing cell death [24]. In addition, an increase in the density of blood vessels in the sclerotic hippocampus of patients with TLE has been reported [25–27; reviewed in 28], which is in line with the activation of expression of angiogenic factors observed in animal models after electroconvulsive seizures [29]. However, other studies on the microvasculature of sclerotic hippocampal tissue obtained from patients with epilepsy by surgical resection have found an impressive reduction in the blood vessels and a variety of alterations in the microanatomy of the remaining blood vessels [30]. In this review, we will discuss the controversy surrounding angiogenesis in the human sclerotic hippocampus and its relationship to epileptogenicity. 2. Study of the microvasculature in hippocampal sclerosis Brain microvasculature can be histologically analyzed by numerous approaches, including histochemical and immunocytochemical methods,
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Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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which provide indirect labeling of the blood vessels by specific staining of some of their components. Histochemical methods have been applied, such as the detection of NADPH–diaphorase activity, which is an enzyme located in the endothelium whose staining is reduced in epilepsy animal models [31]. Staining of the endogenous enzyme alkaline phosphatase (AP) has been widely used (reviewed in [32]). Alkaline phosphatase is an enzyme present in the endothelial cells of the BBB, and it has been used to visualize the capillary network of the cerebral cortex and subcortical structures [33–36]. In particular, AP staining labeled numerous blood vessels in all fields of the normal hippocampal formation, and a significant decrease in the density of AP-positive blood vessels has been described in the CA1 of the sclerotic hippocampus [30]. It has also been shown that AP activity varies in the rat brain when seizures are induced [37]. Furthermore, uniform AP staining was observed in all hippocampal fields of patients with nonsclerotic epilepsy, suggesting that the changes in AP activity are specifically related to hippocampal sclerosis and not to epileptic activity per se [30]. Moreover, genetic defects in AP lead to epileptic seizures due to alterations in the normal AP activity [38–42], which has been hypothesized to be due to the inability of AP to regulate GABA synthesis and neurotransmission in the cerebral cortex [43,44]. Thus, the reduction of AP staining would suggest a modification in its activity, and this might in turn imply alterations to the GABAergic system. Such alterations have been proposed as being responsible for the development of TLE [reviewed in 45–47]. The detection of plasma components outside the blood vessels has been used as an indicator of BBB disruption related to epileptogenicity [20,25,48]. For example, it has been described that vascular endothelial growth factor (VEGF) is upregulated in pathological conditions of the brain tissue, including seizures, and that this growth factor promotes BBB disruption and formation of new microvessels [25, reviewed in 27,49].
3. Angiogenesis or vascular reduction? In recent years, a number of laboratories have described an increase in the number of blood vessels in both patients with epilepsy and animal models, using markers related to angiogenesis [reviewed in 27,49]. Studies performed using collagen type IV (collagen-IV) immunoreactivity (-ir) in the sclerotic hippocampus of patients with epilepsy showed a significantly higher density (increase of 160%) in the CA1 field when compared with the nonsclerotic hippocampus [30]. Collagen-IV is an essential component of the basal lamina in terms of maintaining the endothelial permeability barrier, and it is present in most blood vessel types [50]. Collagen-IV-ir has been largely used to study the microvascular network in several brain disorders [51–54]. It seems clear that the higher density of positive collagen-IV-ir elements in the sclerotic hippocampus suggests that angiogenesis has occurred. This would be in keeping with studies using immunostaining for VEGF which have reported an increase in blood vessel density in patients with TLE [25,27]. However, we observed that closer examination of the collagen-IV-ir elements revealed that in the stratum pyramidale of the CA1 field from the sclerotic hippocampi, many of the collagen-IV-ir blood vessels were notably altered (Figs. 1–3). These blood vessels had a rough surface with numerous small, spine-like protrusions compared with the typically smooth surface found in the nonsclerotic CA1. These protrusions gave a “spiny” appearance to the surface of the blood vessels. Furthermore, the most notable alteration was that instead of the homogeneous staining of the blood vessel surface, numerous abnormal tubular or “vascular-like” structures appeared with a vacuolar or reticulated appearance [30].
Fig. 1. Low-power photomicrographs of collagen-IV immunostained hippocampal sections from control (A) and sclerotic hippocampus (B) to illustrate the distribution of collagen-IV-ir blood vessels. Arrows indicate the CA1 areas also shown at a higher magnification in Figs. 2A and B. C: Photomicrograph of Nissl-stained section showing the hippocampal formation from a patient with sclerotic epilepsy. Note that the sclerotic hippocampal formation is smaller than the control hippocampus. Sub: subiculum. Scale bar (in C): 1000 μm.
Therefore, we used correlative light and electron microscope (EM) techniques to analyze these collagen-IV-ir elements in the sclerotic CA1. These correlative light and EM methods allowed the unequivocal identification of blood vessels and its ultrastructure. The surface of many blood vessels displayed abnormal protrusions of the basal lamina that were responsible for the “spiny” appearance of the blood vessels in this region (Fig. 2,3). Interestingly, the vascular-like collagen-IV-ir processes with a vacuolar appearance were indeed atrophic branches of blood vessels with the peculiarity that their reduced lumen was mainly filled with the processes of reactive astrocytes. The nature of these structures suggested collapse of the blood vessels in the CA1 field of the sclerotic hippocampus [30]. Remarkably, these altered blood vessels were mainly found in those areas where neuronal loss was so severe that virtually no
Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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Fig. 2. Photomicrographs of the CA1 field from a control (A) and sclerotic (B) hippocampus illustrating the apparent increase in collagen-IV-ir blood vessels in the sclerotic tissue. C: Higher magnification to illustrate the smooth surface of the collagen-IV-ir blood vessels in the control tissue. D: Higher magnification to illustrate the presence of small spine-like protrusions on the surface of the collagen-IV-ir blood vessel (arrowheads). E: Higher magnification to show the abnormal tubular or “vascular-like” collagen-IV-ir structures with a vacuolar or reticulated appearance. Scale bar (in E): 75 μm in A, B and 10 μm in C–E.
neurons were present [55]. It is well known that neurons, glia, and blood vessels are closely coupled, both anatomically and functionally [reviewed in 56–58]. Indeed, neurons, glia, and blood vessels have been considered together as a functional entity called the neurovascular unit [24]. This is in line with the fact that perturbations in the integrity of neurovascular function are thought to initiate several cascades associated with injury, which may damage the adjacent brain tissue, producing ion imbalances and inducing cell death [24]. In order to confirm these observations, we used plastic semithin sections (2–5 μm thick) stained with toluidine blue (Fig. 4). This method allows the visualization not only of neurons and glia but also of blood vessels, regardless of their possible molecular alterations. We estimated the volume fraction (Vv) occupied by blood vessels in the sclerotic CA1 field where immunostaining for collagen-IV appeared to show an increase of blood vessels. We observed a significant decrease in the volume occupied by blood vessels in the CA1 field (approximately 60%) compared with nonsclerotic tissue [30], confirming that many of the structures that had been interpreted as blood vessels in the preparations immunostained for collagen-IV were in fact fragments resulting from degenerating of blood vessels. Finally, it should be noted that the significance of the local reduction of blood vessels is not clear. In order to define the functional significance of specific changes in the microvascular network of patients with TLE, it will be necessary to perform electrophysiological studies in conjunction with correlative microanatomical and neurochemical characterization of the epileptic tissue.
4. Conclusions Angiogenesis in the sclerotic hippocampus has been proposed previously, based on the apparent increase in blood vessel density, presumed from staining of markers related to BBB disruption and formation of new blood vessels (such as VEGF). However, correlative light and electron microscopy reveals that many of these “blood vessels” are in fact atrophic vascular structures with a reduced or virtually absent lumen and are often filled with processes of reactive astrocytes. Thus, “normal” blood vessels within the sclerotic CA1 are drastically reduced as confirmed by volume fraction analysis in semithin section (which showed a 60% reduction). It is striking that this kind of relatively simple and powerful analysis had gone unnoticed in previous histopathological assessment of the sclerotic hippocampus. Since this decrease is consistently observed in the human sclerotic CA1, this feature can be considered as another histopathological hallmark of hippocampal sclerosis.
Acknowledgments This work was supported by grants from the following entities: the Spanish Ministry of Economy and Competitiveness (BFU2012-34963 to JDF) and the Cajal Blue Brain Project, Spanish partner of the Blue Brain Project initiative from EPFL. Conflict of interest The authors declare that there are no conflicts of interest.
Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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Fig. 3. Correlative light and electron microscopy photomicrographs of a collagen-IV-ir blood vessel in the CA1 field of a sclerotic hippocampus. A: Photomicrograph of a plastic-embedded section showing collagen-IV-ir blood vessels. B: Photomicrograph of a 2-um-thick semithin plastic section obtained from the same section shown in A. V1 and V2 indicate the same blood vessels labeled in A. C: Low-power electron micrograph taken after the resection of the semithin section shown in B illustrating blood vessel V1. The arrow and arrowhead indicate small spine-like protrusions on the surface of the collagen-IV-ir blood vessel and an atrophic branch, respectively. D, E: Higher magnification of C to illustrate the morphological alterations to the blood vessel V1. Arrow in D indicates the same protrusion as in C. E is a higher magnification of the atrophic branch in the blood vessel, which appears as an abnormal protrusion of the endothelial membrane. Scale bar (in E): 150 μm in A, 75 μm in B, 10 μm in C, 2.5 μm in D, and 1 μm in E.
Fig. 4. Photomicrograph of semithin sections (2 μm) stained with 1% toluidine blue, from control (A) and sclerotic (B) CA1 field to illustrate the Cavalieri method used to estimate the Vv of blood vessels. Note the presence of abundant pyramidal cells and blood vessels in the nonsclerotic CA1 (A) when compared with the sclerotic tissue (B). A grid of small intersections overlying the tissue is displayed. Adjacent grid points are 20 μm apart, providing an area enclosed by any 4 given grid points of 400 μm2 (20 × 20). Yellow asterisks indicate the intersections in the grid that lies within blood vessels (n = 5, 5 × 400 = 2000 μm2 in nonsclerotic and n = 1, 400 μm2 in sclerotic). Note that the number of intersections (yellow asterisks) in the grid that lies within the blood vessels is higher in the control tissue than in the sclerotic tissue. Scale bar (in B): 20 μm.
Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
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Please cite this article as: Alonso-Nanclares L, DeFelipe J, Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy, Epilepsy Behav (2014), http://dx.doi.org/10.1016/j.yebeh.2013.12.009
