The neuroprotective effect of topiramate on the ultrastructure of pyramidal neurons of the hippocampal CA1 and CA3 sectors in an experimental model of febrile seizures in rats

The objective of the current ultrastructural study was to explore the potentiality of the neuroprotective effect of TPM against damage of pyramidal neurons in the hippocampal CA1 and CA3 sectors in an experimental model of febrile seizures (FS) in rats. The FS group exhibited variously pronounced submicroscopic lesions of the neuronal perikarya, including total cell disintegration. Advanced changes induced by hyperthermic stress were manifested by marked degenerative abnormalities, such as substantial swelling of the mitochondria, dilation, degranulation and disintegration of the granular endoplasmic reticulum, and vacuolar changes in the Golgi complex. The most substantially damaged pyramidal neurons showed features of aponecrosis (so-called "dark neurons"), resulting in a marked neuronal loss in the explored areas of the hippocampal cortex. The neurodegenerative changes were accompanied by distinct damage to the blood-brain barrier components. The administration of topiramate at a dose of 80/kg b.m. prior to the induction of hyperthermic stress (as prevention against febrile seizures) caused a substantial neuroprotective action - the drug efficiently lightened the neuronal damage, basically reduced cell aponecrosis and enhanced cell viability. However, TPM applied directly after FS induction did not exert any distinct neuroprotective effect on the perikarya of pyramidal neurons in the hippocampal cortex.     

Determinants of drug brain uptake in a rat model of seizure-associated malformations of cortical development

We examined the blood-brain barrier (BBB) function in methylazoxymethanol acetate (MAM)-treated rats, a model of human developmental brain malformations. We found aberrant vessels morphology and serum albumin leakage in the heterotopic (malformed) hippocampus; these changes were associated with a significant increase in endothelial P-glycoprotein (P-gp) expression. Seizures exacerbated BBB leakage and greatly augmented P-gp expression in vessels and additionally in perivascular/parenchymal astrocytes. The effects of seizures were observed to a much larger extent in malformed than in normal brain tissue. The intrinsic changes in BBB function in MAM-exposed rats were associated with increased blood-to-brain penetration of ondansetron, a P-gp substrate. However, a marked reduction in drug brain levels was provoked by seizures, and this effect was reversed by selective blockade of P-gp activity with tariquidar. Changes in BBB function may critically contribute to determine the brain uptake and distribution of P-gp substrates in epileptic tissue associated with developmental malformations.     

Spinal Cord Compression Injury in Guinea Pigs: Structural Changes of Endothelium and Its Perivascular Cell Associations after Blood–Brain Barrier Breakdown and Repair

This study examines morphological changes of the blood–brain barrier (BBB) after spinal cord compression. The lowest thoracic segment (T13) of female guinea pigs was injured and the BBB was tested from 7 days to 5.5 months postinjury using intravenously injected horseradish peroxidase (HRP) as a tracer. Tracer leakage in the injured segment was verified with the light microscope and the fine structure of capillaries was examined. Diffuse tissue staining was observed at T13 up to 2 weeks following injury. A leaky BBB correlated with expected changes in the fine structure of endothelial cell junctions. These were predominantly nonoverlapping cell junctions which, in many instances, were separated by clefts between adjacent cells. At early survival times, numerous capillary profiles with juxtaposed astrocyte foot processes were noted in addition to altered cell associations. Complete sealing of the BBB against interstitial HRP leakage was not observed until 17 days postinjury. After the first week, some of the endothelial cells were contacted by macrophages, processes of perivascular microglia, and processes of swollen and degenerating astrocytes. Perivascular spaces varied in extent and contained amorphous deposits of extracellular materials in addition to supernumerary layers of basal lamina. The early changes were followed by profound tissue restructuring due to loss of both neurons and glia. At longer survival times the BBB to HRP repaired. Endothelial cells formed complex overlapping junctions with zonulae occludentes. Most of the capillaries in the injured segment were no longer in direct contact with astrocyte foot processes, although reactive astrocytes constituted the predominant cell type in the remaining gray matter. Substantial expansion of perivascular spaces was evident. The cytoplasm of endothelial cells had numerous pinocytotic vesicles. Perivascular spaces contained layers of assembled collagen arranged perpendicularly to each other in addition to amorphous matrix materials. The findings suggest that decoupling of astrocyte foot processes from endothelial cell surfaces does not prevent reformation of tight junctions. It remains to be examined what effects the larger perivascular spaces, extracellular matrix deposits, and changes of cell associations may have on transport systems and ionic buffering. The data are relevant for estimating an opportune time for application of barrier-impermeable drugs to the lesion area.     

Epileptogenesis in the Developing Brain: What Can We Learn from Animal Models?

Summary:  Knowledge of the processes by which epilepsy is generated (epileptogenesis) is incomplete and has been a topic of major research efforts. Animal models can inform us about these processes. We focus on the distinguishing features of epileptogenesis in the developing brain and model prolonged febrile seizures (FS) that are associated with human temporal lobe epilepsy. In the animal model of FS, epileptogenesis occurs in ∼35% of rats. Unlike the majority of acquired epileptogeneses in adults, this process early in life (in the febrile seizures model as well as in several others) does not require "damage" (cell death). Rather, epileptogenesis early in life involves molecular mechanisms including seizure-evoked, long-lasting alterations of the expression of receptors and ion channels. Whereas transient changes in gene expression programs are common after early-life seizures, enduring effects, such as found after experimental FS, are associated with epileptogenesis. The ability of FS to generate long-lasting molecular changes and epilepsy suggests that mechanisms, including cytokine activation that are intrinsic to FS generation, may play a role also in the epileptogenic consequences of these seizures.     

Astrocytic β1-integrin affects cellular composition of murine blood brain barrier in the cerebral cortex

The blood brain barrier (BBB) is composed of endothelial cells, astrocytes, and pericytes and maintains functional homeostasis by regulating transport of ions, fluid and cells between blood and neural tissue. The cellular and molecular pathways that contribute to the formation of the BBB in the developing brain have not been fully deciphered. β1-integrin (β1-itg) within endothelial cells is known to play a critical role in vasculogenesis. However, the role of astrocytic β1-itg in BBB development is not known. Our study used a mouse glial fibrillary acidic protein (GFAP)-cre transgenic line to selectively ablate β1-itg within astrocytes. We found that deletion of astrocytic β1-itg had a striking effect on the different cell types that form the BBB. Mutant mice had a decreased density of aquaporin-4 immunoreactivity within the perivascular astrocytic end-feet. We also found decreases in immunoreactivity for vimentin and CD-31 within endothelial cells. These changes were not accompanied by functional changes in BBB under physiological conditions as assessed by extravasation of large and small molecular weight molecules. However, mutant mice had an increased incidence of severe cystic injury in response to neonatal hypoxia. Our findings show that astrocytic β1-itg has an important role in defining cellular properties of the blood brain barrier in the cerebral cortex.     

Blood-brain barrier is impaired in the hippocampus of young adult spontaneously hypertensive rats

A causative role of blood-brain barrier (BBB) impairment is suggested in the pathogenesis of vascular dementia with leakage of serum components from small vessels leading to neuronal and glial damage. We examined the BBB function of young adult spontaneously hypertensive rats (SHR) in order to determine earlier changes in the BBB in chronic hypertension. SHR and stroke-prone SHR (SHRSP) were injected with horseradish peroxidase (HRP) as an indicator of BBB function and compared with Wistar Kyoto rats (WKY). The brain tissues were further examined with cationized ferritin, a marker for evaluating glycocalyx. The staining for HRP was distributed around the vessels in the hippocampal fissure of SHR and SHRSP, but not in WKY. With electron microscopy, the extravasated reaction product of HRP appeared in abluminal pits of the endothelial cells of arterioles and within the basal lamina in the hippocampus, but not the cerebral cortex, of SHR and SHRSP. On the contrary, the reaction product of HRP was never seen in the abluminal pits of the endothelial cells or the basal lamina of vessels in WKY. The number of cationized ferritin particles binding to the endothelial cells of capillaries was decreased in the hippocampus of SHR and SHRSP, while the number decreased in the cerebral cortex of SHRSP compared with those in WKY. However, the cationized ferritin binding was preserved in the endothelial cells of the arterioles with an increased vascular permeability. These findings suggest that the chronic hypertensive state induces BBB dysfunction in the hippocampus at an early stage.     

Clostridium perfringens prototoxin-induced alteration of endothelial barrier antigen (EBA) immunoreactivity at the blood-brain barrier (BBB)

It has been reported that the severe cerebral edema produced in experimental animals by Clostridium perfringens (Cl p) type D epsilon toxin can be prevented by prior treatment with its precursor prototoxin due to competitive binding to endothelial cells (ECs) at the blood-brain barrier (BBB). In this study we investigate the effects of the prototoxin on the BBB, without added toxin. The integrity of the BBB was assessed by its ability to prevent leakage of endogenous albumin. ECs at the BBB were studied by immunocytochemistry for any alteration in the endothelial barrier antigen (EBA), a molecular marker for the intact BBB. Immunocytochemistry showed rapid but mild opening of the BBB to endogenous albumin. Light and electron immunocytochemistry showed qualitative and quantitative reduction in EBA immunoreactivity, with a spectrum of changes at time intervals from 1 h to 14 days post-prototoxin injection. Some vessels with ultrastructural changes and widening of the perivascular space retained EBA immunoreactivity. Many vessels showed partial or complete loss of EBA staining with minimal widening of the perivascular space and edema. Recovery of EBA expression was still incomplete at 14 days postinjection. This is the first report to show endothelial cell damage, mild reversible cerebral edema, and alteration in BBB markers following administration of Cl p prototoxin. This model of mild brain edema may be useful for BBB studies.     

Heterogeneity in the rat brain vasculature revealed by quantitative confocal analysis of endothelial barrier antigen and P-glycoprotein expression

While phenotypic endothelial heterogeneity is well documented in peripheral organs, it is only now being explored in the brain. We used confocal imaging of thick sections of rat brain to qualitatively and quantitatively examine the expression of two key markers of the blood–brain barrier (BBB) in the rat, P-glycoprotein (P-gp), and endothelial barrier antigen (EBA). We found that these markers were not uniformly distributed throughout the whole vasculature of the cortex and hippocampus. P-glycoprotein displayed a gradient of expression from an almost undetectable level in large penetrating arterioles to a high and uniform level in capillaries and venules. While EBA was lacking in all cerebral arterioles, regardless of their size, its expression varied greatly among endothelial cells in capillaries and venules, yielding a striking mosaic pattern. A detailed quantitative analysis of the distribution of these markers at the single cell level in capillaries is provided. These results challenge the view of a uniform BBB and suggest that regulatory mechanisms might differentially modulate BBB features not only among arterioles/capillaries/venules but also at the single cell level within the capillaries. Hypotheses are made regarding the underlying mechanisms and physiopathological consequences of this heterogeneity.     

The effect of bile salts on the permeability and ultrastructure of the perfused, energy-depleted, rat blood-brain barrier

The action of bile salts upon the rat blood-brain barrier (BBB) was assessed in the absence of energy-yielding metabolism. Brains were perfused in situ with a Ringer solution for 5 min followed by a 1 min perfusion containing either sodium deoxycholate (DOC), taurochenodeoxycholate (TCDC), or Ringer/DNP. The integrity of the BBB was then determined by perfusing with the radiotracer [14C]mannitol for 2.5 min. Alternatively, the brains were perfusion fixed for ultrastructural assessment. At 0.2 mM DOC, the BBB remained intact and the cerebral ultrastructure was similar to the controls. At 1 mM and above, disruption of the BBB became evident. At 2 mM, the cerebral cortex became severely vacuolated, with damaged endothelium and collapsed capillaries. With TCDC, BBB disruption occurred at 0.2 mM without any apparent ultrastructural damage to the microvasculature. Following 2 mM TCDC, similar, but less widespread, structural changes to the 2 mM DOC-perfused animals was apparent. Opening of the BBB occurred at a concentration lower than that required to cause lysis of either red blood cells or cultured cerebral endothelial cells. It is proposed that the effect of bile salts at concentrations of 1.5 mM and above is largely due to their lytic action as strong detergents on endothelial cell membranes, but that at lower concentrations a more subtle modification of the BBB occurs.     

Lead-induced abnormalities in blood-brain barrier permeability in experimental chronic toxicity

The aim of this paper was to determine whether prolonged drinking of lead acetate-containing water by adult rats, which imitates environmental exposure to lead (Pb), affects some morphological and biochemical properties of rat brain microvessels. We noted a significant increase of lead level in capillaries and synaptosomes obtained from brains of rats under chronic toxicity conditions. Intravenously injected horseradish peroxidase (HRP) was used to evaluate the functional state of the blood-brain barrier (BBB). The results indicate that, systematically administered at low doses, lead induces BBB dysfunction. The changes, revealed in light microscopy and confirmed by electron microscopic studies, are typical for “leaky” microvessels, reported for variety of neuropathological conditions associated with BBB damage. Enhanced pinocytotic activity of the endothelial cells and the opening of interendothelial tight junctions, together with enormous phagocytizing action of the pericytes, are the most characteristic ultrastructural features noted. The presence of specific type of perivascular cells containing droplets of lipids in the cytoplasm, together with changes in phospholipid profile in brain capillaries, suggest that altered lipid composition of membranes may, at least in part, be responsible for changes in observed membrane permeability.     

The blood-brain barrier to horseradish peroxidase at the onset of bicuculline-induced seizures in hypothalamus,pallidum, hippocampus,and other selected regions of the rabbit

Summary Rabbits were subjected to bicuculline-induced generalized seizures of 15-min duration to elucidate the mechanism by which the macromolecule horseradish peroxidase (HRP) traverses the blood-brain barrier (BBB) in specific brain areas. Transendothelial pinocytosis at the level of arterioles was the main route of passage. In addition, in thalamus and hippocampus pinocytotic vesicles were observed in capillaries. In thalamus, hypothalamus and septum vesicles in the endothelium of venules were also present. Repeatedly, pinocytotic vesicles were ejecting their content into the interendothelial clefts, so that the presence of HRP reaction product between adjacent tight junctions cannot be considered a conclusive evidence for their opening.The HRP, which had reached the neuropil due to the seizure-evoked BBB opening, accumulated in the interstitial spaces and penetrated the synaptic cleft. Uptake of the tracer in vesicular form into presynaptic boutons, presumably excitatory ones as diagnosed by their ultrastructural features, was observed in all brain regions. The uptake was rare in septum, periaqueductal gray, hypothalamus, and cerebellar cortex; frequent in pallidum, hippocampus, and medulla oblongata; and very intense in thalamus. Uptake in postsynaptic dendrites was present mostly in the vicinity of boutons. Incorporation into glial processes was rare and confined to perivascular astrocytes.It is suggested, that HRP traverses the BBB by regionally selective, transmitter-controlled pinocytotic transport and that the neuronal uptake of the foreign protein is at least partially dependent on the involvement of synapses of particular brain regions in the paroxysmal activity during the generalized seizures.     

Compromised blood–brain barrier competence in remote brain areas in ischemic stroke rats at the chronic stage

Stroke is a life‐threatening disease leading to long‐term disability in stroke survivors. Cerebral functional insufficiency in chronic stroke might be due to pathological changes in brain areas remote from the initial ischemic lesion, i.e., diaschisis. Previously, we showed that the damaged blood–brain barrier (BBB) was involved in subacute diaschisis. The present study investigated BBB competence in chronic diaschisis by using a transient middle cerebral artery occlusion (tMCAO) rat model. Our results demonstrated significant BBB damage mostly in the ipsilateral striatum and motor cortex in rats at 30 days after tMCAO. The BBB alterations were also determined in the contralateral hemisphere via ultrastructural and immunohistochemical analyses. Major BBB pathological changes in contralateral remote striatum and motor cortex areas included 1) vacuolated endothelial cells containing large autophagosomes, 2) degenerated pericytes displaying mitochondria with cristae disruption, 3) degenerated astrocytes and perivascular edema, 4) Evans blue extravasation, and 5) appearance of parenchymal astrogliosis. Discrete analyses of striatal and motor cortex areas revealed significantly higher autophagosome accumulation in capillaries of ventral striatum and astrogliosis in dorsal striatum in both cerebral hemispheres. These widespread microvascular alterations in ipsilateral and contralateral brain hemispheres suggest persistent and/or continued BBB damage in chronic ischemia. The pathological changes in remote brain areas likely indicate chronic ischemic diaschisis, which should be considered in the development of treatment strategies for stroke. J. Comp. Neurol. 522:3120–3137, 2014. © 2014 Wiley Periodicals, Inc.     

Electron microscope study of blood-brain barrier opening induced by immunological targeting of the endothelial barrier antigen

Barrier vessels in the central nervous system are lined with endothelial cells which constitute the blood-brain barrier (BBB) and show selective expression of certain biochemical markers. One of these, the endothelial barrier antigen (EBA), is specific to the rat. The exact role of EBA in the BBB is not known, although several studies have shown a correlation between the reduction in EBA expression in endothelial cells and the opening of the BBB. However, in these studies it was not possible to determine if EBA reduction was a primary event or was secondary to opening of the BBB. A recent light microscope study demonstrated that immunological targeting of EBA in vivo, by intravenous injection of a monoclonal antibody (anti-EBA), leads to acute and widespread opening of the BBB. In the current study we have employed this model together with tracer application and immunoperoxidase electron microscopy to determine the site of binding of the injected antibody and the route of opening of the BBB. The results showed that (a) the anti-EBA injected in vivo became bound to brain endothelial cells, principally to luminal membranes. (b) Endothelial cells showed widened intercellular junctions and increased cytoplasmic vesicles and vacuoles. (c) Many perivascular astrocytic processes were swollen. (d) The macromolecular tracer HRP was present in vesicles, vacuoles, widened paracellular clefts, the perivascular space and brain parenchyma. In conclusion, the in vivo targeting of EBA leads to opening of the BBB apparently via paracellular and transcellular routes. This model is useful for the study of vascular permeability in the CNS and experimental manipulation of the BBB. It may have a potential application in experimental studies on drug delivery throughout the CNS.     

血脑屏障内皮细胞粘附分子表达与癫痫发病关系

目的研究血脑屏障内皮细胞上细胞间粘附分子-1(ICAM-1)表达与癫癎发病的关系.方法用免疫细胞化学法,观察Wistar大鼠皮质和海马区ICAM-1免疫反应阳性表达.结果对照组大鼠皮质、海马区血脑屏障内皮细胞及神经细胞上均有少量ICAM-l免疫反应阳性表达;美解眠致癎组大鼠,上述脑区ICAM-1免疫反应阳性表达显著增强(P＜0.01);致癎前给予地塞米松组大鼠,相应脑区ICAM-I免疫反应阳性表达与对照组比较无显著差异(P＞0.05).结论表达于血脑屏障内皮细胞及神经细胞上的ICAM-1通过影响血脑屏障通透性及调节脑内免疫反应参与癫癎发病.糖皮质激素治疗癫癎机制之一与其抗粘附效应有关.     

米诺环素早期干预脑出血：对大鼠脑内血管内皮生长因子、神经生长因子、热休克蛋白70表达及血脑屏障通透性的影响

Abstract Inflammatory factor aggregation and blood-brain barrier (BBB) damage occur around hematoma foci following intracerebral hemorrhage. Minocycline is lipophilic, can pass through the BBB, and shows anti-inflammatory effects in models of central nervous system disease. We found that minocycline application at 6 hours after intracerebral hemorrhage reduced BBB permeability, decreased vascular endothelial growth factor expression, and increased nerve growth factor and heat shock protein 70 expression, primarily in neurons and microglia. Early intraperitoneal injection of minocycline attenuated BBB damage possibly by reducing vascular endothelial growth factor expression and enhancing nerve growth factor and heat shock protein 70 expression. Key Words: blood-brain barrier; intracerebral hemorrhage; vascular endothelial growth factor; nerve growth factor; heat shock protein 70; therapy     

The large apparent work capability of the blood-brain barrier: a study of the mitochondrial content of capillary endothelial cells in brain and other tissues of the rat

Volumes of mitochondria in capillary endothelial cells were determined stereologically from electron micrographs of rat cerebellum, cerebral cortex, spinal cord, cauda equina, choroid plexus, anterior pituitary, median eminence of the hypothalamus, renal proximal tubules, skin, cardiac and skeletal muscle, lung, and renal glomerulus. The capillaries of the first four of these tissue types exhibit blood-brain barrier (BBB) characteristics of permeability and capillary ultrastructure and were found to have mitochondrial contents amounting to 8 to 11% of the endothelial cytoplasmic volume. Tissues from non-BBB regions were determined to have mitochondrial volumes of 2 to 5% of their respective cytoplasmic volumes, with a variety of capillary ultrastructures. The apparent excess metabloic work capability of the BBB suggested by this greater number of mitochondria may be related to maintenance of ion differentials between blood plasma and brain extracellular fluid, to extrachoroidal cerebrospinal fluid formation, or to maintaining the unique structural characteristics of central nervous system capillaries.     

Neuropathological changes during generalized seizures in newborn monkeys.

The brains of four 2-week-old marmoset monkeys were perfusion-fixed immediately after bicuculline-induced seizures lasting 1.5-4.3 h and were later examined by light and electron microscopy. Mean arterial blood pressure and rectal temperature measurements during seizures did not differ significantly from baseline. Plasma glucose concentrations decreased to the 1.5 mM range at the end of seizures, and arterial pH and bicarbonate were lower than in control animals, although arterial pO2 and pCO2 were maintained. Neuropathological changes were minimal. Swollen astrocytic processes surrounded some capillaries and some neurons in cerebral cortex, hippocampus, putamen and thalamus. Almost all the neurons examined looked normal, but mitochondrial swelling was present in a few. All but the most severe mitochondrial swelling, which occurred very rarely in one of four animals, is potentially reversible. The virtual absence of neuronal necrosis in these neonatal monkeys is consistent with the resistance to seizure-induced brain damage found in immature rats, and stands in sharp contrast to the damage seen in older animals. Lack of neuronal damage, however, does not rule out potential adverse effects of prolonged seizure activity on subsequent brain growth and development.     

增龄相关性脑小血管病谱系的病理基础及其影像学特点

增龄相关性脑小血管病谱系（spectrum o f a ge-related cerebral small vessel diseases， ArCSVDS）是指随着年龄的增长，脑小动脉、微动脉、毛细血管、微静脉、小静脉出现老化的一组脑 小血管病（cerebral small vessel disease，CSVD），包括深穿支动脉病（deep perforating arteriopathy，DPA） 和β淀粉样蛋白（β-amyloid，Aβ）有关的脑淀粉样血管病（cerebral amyloid angiopathy，CAA）。以往的 研究往往将这两种疾病视为独立的实体，然而最近的研究发现，无论从年龄、高血压等相似的脑血 管病危险因素，还是从潜在的相互作用角度出发，DPA与CAA是一组ArCSVDS疾病，血脑屏障（blood brain barrier，BBB）破坏、内皮细胞损伤、血管周围Aβ是连接DPA与CAA的共同发病机制，而且有 30%～68%的患者两者可以重叠发病。     

The long-term effects of febrile seizures on the hippocampal neuronal plasticity – Clinical and experimental evidence

Febrile seizures are the most common seizure disorder in childhood, but their long-term effects on the developing brains especially neuronal injury and neurocognitive function remain unresolved. Recent epidemiological studies reassure that most febrile seizures do not adversely affect global intelligence and hippocampal function, such as memory. However, there are concerns regarding those children who experience febrile seizures during the first postnatal year, having prior developmental delay and pre- or peri-natal events. Magnetic resonance imaging (MRI) studies confirmed that prolonged and focal FS can occasionally produce acute hippocampal injury that evolves into atrophy. Animal studies have revealed that the exposure of hippocampal neurons to experimental febrile seizures early in life, particularly prolonged or frequently repetitive FS, or together with brain malformation, may lead to sustained dysfunction of these cells, in spite of the absence of neuronal damage. Genetic studies suggest that the relationship between febrile seizures and subsequent epilepsy and neurocognitive dysfunction is sometimes genetic, but there are complex interactions with genetic or environmental modifiers. Therefore, there is a small group of children in whom febrile seizures-induced hippocampal injury might occur. Identification of the target population for subsequent mesial temporal sclerosis is important for prevention and early intervention.