Vascularización cerebral (parte I)

Vascularización cerebral

martes 15 de noviembre de 2011

Enrike G. Argandoña




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Sistema arterial aferente

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Sistema arterial aferente

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1% volumen cerebral

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1% volumen cerebral

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25% consumo energía

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Control de la circulación sistémica

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Autorregulación vascularización

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Autorregulación vascularización

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Distribución del flujo

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Vascularización intracerebral


Arteriolas (50-100 µm)

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Arteriolas (50-100 µm) Arteriolas terminales (10-100µm)

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Arteriolas (50-100 µm) Arteriolas terminales (10-100µm) Vénulas (± 30 µm)

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Arteriolas (50-100 µm) Arteriolas terminales (10-100µm) Vénulas (± 30 µm) Capilares (<30 µm)

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Vascularización cortical

Red capilar640 km (reducida en Alzheimer)

Un capilar por neurona

Barrera hematoencefálicaMecanismos estructurales

Mecanismos metabólicos


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Paracellular aqueouspathway

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Transport proteins Receptor-mediatedtranscytosis

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Water-solubleagents

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Glucose,amino acids,nucleosides

Vinca alkaloids,Cyclosporin A,AZT

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Adherens junctionA cell–cell junction also known as zonula adherens, which is characterized by the intracellular insertion of microfilaments. If intermediate filaments are inserted in lieu of microfilaments, the resulting junction is referred to as a desmosome.

Perivascular endfeetThe specialized foot-processes of perivascular astrocytes that are closely apposed to the outer surface of brain microvessels, and have specialized functions in inducing and regulating the BBB.

PericyteA cell of mesodermal origin, and contractile-phagocytic phenotype, associated with the outer surface of capillaries.

The brain endothelial transporters that supply the brain with nutrients include the GLUT1 glucose carrier, several amino acid carriers (including LAT1, L-system for large neutral amino acids), and transporters for nucleosides, nucleobases and many other substances10. Several organic anion and cation transporters identified in other tissues and the choroid plexus are also prov-ing to be expressed on the brain endothelium. Where compounds need to be moved against a concentration gradient, the energy may come from ATP (as in the ABC family of transporters, including P-glycoprotein (Pgp) and multidrug resistance-related proteins, MRPs), or the Na+ gradient created by operation of the abluminal Na+,K+-ATPase. Some transporters (for example, GLUT1 and LAT1) are bidirectional, moving substrates down the concentration gradient, and can be present on both luminal and abluminal membranes, or predominantly on one. Quantification of GLUT1 expression on luminal and abluminal endothelial membranes is complicated by the fact that some antibodies do not recognize the trans-porter when the C-terminal is masked, as it may be in the luminal membrane23. Among the efflux transporters, Pgp is concentrated on the luminal membrane24, whereas the Na+-dependent transporters are generally abluminal,

specialized for moving solutes out of the brain25,26. They include several Na+-dependent glutamate transporters (excitatory amino acid transporters 1–3; EAAT1–3)27, which move glutamate out of the brain against the large opposing concentration gradient (<1 µM in ISF compared with ~100 µM in plasma) (FIG. 2). The clear apical–basal polarity of brain endothelial cells noted above is hence reflected in their polarized transport function20,28.

Induction of BBB propertiesWhat causes the endothelium of blood vessels growing into the brain during development to become so specialized? It has been clear from the earliest histological studies that brain capillaries are surrounded by or closely associated with several cell types, including the perivascular endfeet of astrocytic glia, pericytes, microglia and neuronal proc-esses (FIG. 2). In the larger vessels (arterioles, arteries and veins), smooth muscle forms a continuous layer, replacing pericytes1. Neuronal cell bodies are typically no more than ~10 µm from the nearest capillary6. These close cell–cell associations, particularly of astrocytes and brain capil-laries, led to the suggestion that they could mediate the induction of the specific features of the barrier phenotype in the capillary endothelium of the brain29.

Figure 3 | Pathways across the blood–brain barrier. A schematic diagram of the endothelial cells that form the blood–brain barrier (BBB) and their associations with the perivascular endfeet of astrocytes. The main routes for molecular traffic across the BBB are shown. a | Normally, the tight junctions severely restrict penetration of water-soluble compounds, including polar drugs. b | However, the large surface area of the lipid membranes of the endothelium offers an effective diffusive route for lipid-soluble agents. c | The endothelium contains transport proteins (carriers) for glucose, amino acids, purine bases, nucleosides, choline and other substances. Some transporters are energy-dependent (for example, P-glycoprotein) and act as efflux transporters. AZT, azidothymidine. d | Certain proteins, such as insulin and transferrin, are taken up by specific receptor-mediated endocytosis and transcytosis. e | Native plasma proteins such as albumin are poorly transported, but cationization can increase their uptake by adsorptive-mediated endocytosis and transcytosis. Drug delivery across the brain endothelium depends on making use of pathways b–e; most CNS drugs enter via route b. Modified, with permission, from REF. 8 ! (1996) Elsevier Science.

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Modulation of BBB functionThe term barrier suggests a relatively fixed structure, but it is now known that many (and possibly most) fea-tures of the BBB phenotype can be subject to change (modulation)16. Some of the first examples of modula-tion were found in extreme or pathological conditions. For example, opening of the BBB’s tight junctions can occur in inflammation, contributing to brain oedema52, and upregulation of GLUT1 transporter expression at the BBB is observed in starvation and hypoxia53,54.

The protein leptin can enhance the transcytosis of the peptide urocortin across the BBB, with implications for the regulation of feeding55. Some of the inflammatory mediators that increase capillary permeability in the periphery (for example, histamine and bradykinin) also act on the brain endothelium, although in general higher concentrations are required and the effects are more localized and short-lasting in the brain56. There is some evidence that post-capillary venules are particularly vulnerable to opening by inflammatory mediators57,58, which is relevant in pathologies such as multiple sclero-sis (see below). BBB Pgp function is altered in several different conditions59. For example, upregulation of Pgp over hours to days can occur in oxidative stress60 and on treatment with glutamate61. Pgp upregulation by steroids appears to involve transcriptional regulation via the nuclear pregnane X receptor (PXR)62. More rapid modulation (on a timescale of seconds) can be achieved by specific Pgp inhibitors and competitors59, whereas endothelin 1 can cause functional inhibition63.

These observations prompted characterization of the receptors present on the brain endothelium that are capable of mediating BBB modulation. Cultured brain endothelial cells and astrocytes express functional receptors for a high proportion of the agents that act as neurotransmitters and modulators in the brain56,64 (BOX 1). As many of these are also released by astrocytes and endothelium, there is potential for complex signal-ling between cells in the neurovascular unit, including microglia and oligodendrocytes65–67 (FIG. 5). Such rapid signalling (occurring over seconds to minutes), often mediated by agents with a short half-life, is distinct from the longer-term induction processes that are outlined above (hours to days), which generally involve the regulation of gene transcription and require protein synthesis.

The fact that agents released during normal neural activity can potentially influence both astrocytes and endothelium also raised the interesting possibility that signalling involving brain endothelium and glia could occur physiologically. There could be a physio-logical advantage in transiently ‘opening’ the BBB (tight junction modulation) — for example, triggered by histamine released from nerve terminals to allow the passage of growth factors and antibodies into the brain from plasma, or to ‘sample’ plasma composition9. Conversely, mechanisms for tightening the barrier could be important in conditions of stress or hypoxia: it is known that conditions in which intracellular cyclic AMP (cAMP) concentrations are increased can lead to increased TEER and upregulation of Pgp activity68. The transcription factor NF-"B can alter tight junction pro-tein expression and hence regulate BBB permeability69. Several regulatory mechanisms influence the transport of glucose and amino acids by the brain endothelium70. In cultured brain endothelial cells, glucose transport can be increased by histamine and ATP, which could be part of a mechanism by which astrocytes sense neuro-nal firing and signal to the capillaries to supply more glucose, a form of neurobarrier coupling71–73. Indeed, brain endothelial glucose uptake has been shown to be

Box 1 | Agents modifying brain endothelial function and BBB tightness

A number of chemical agents circulating in the plasma or secreted from cells associated with the blood–brain barrier (BBB) are capable of increasing brain endothelial permeability and impairing its transport and metabolic functions56,68. Other agents have the opposite effect, improving tightness and BBB function.

Agents that impair BBB function:• Bradykinin, histamine, serotonin, glutamate.

• Purine nucleotides: ATP, ADP, AMP.

• Adenosine, platelet-activating factor.

• Phospholipase A2, arachidonic acid, prostaglandins, leukotrienes.

• Interleukins: IL-1#, IL-1$, IL-6.

• Tumour necrosis factor-# (TNF#), macrophage-inhibitory proteins MIP1 and MIP2.

• Complement-derived polypeptide C3a-desArg.

• Free radicals, nitric oxide.

Agents that cause barrier tightening and improved function:• Steroids, elevated intracellular cyclic AMP, adrenomedullin and

noradrenergic agents.

Figure 5 | Complex cell–cell signalling at the blood–brain barrier. A portion of a brain capillary wall, showing the main cell types present with the potential to signal to each other. Pericytes are enclosed within the endothelial basal lamina and form the closest associations with endothelium. The endfeet of astrocytic glial cells are apposed to the outer surface of the basal lamina. In the perivascular space are found microglia, the synaptic terminals and boutons of nerve fibres, and (in arterioles) smooth muscle cells. In the larger vessels, cells of the meninges form a perivascular cuff or sheath that projects down from the brain surface and demarcates the Virchow–Robin space (not shown). Agents such as ATP and histamine can influence endothelial function by ligand–receptor interaction, from the blood or the brain side. Some receptors are coupled to increases in the concentration of intracellular Ca2+. The arrows indicate the ability of the endothelium to release substances to the blood or brain side after receptor activation, as part of their ‘effector’ function. Modified, with permission, from REF. 16 % (2005) Springer.

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expression and localization of other junctional proteins such asclaudin-3, zonula occludens-1 (ZO-1), ZO-2, vascular endothe-lial cadherin (VE-cadherin), and a-catenin may compensatewell for occludin loss (Saitou et al., 2000). However, hyperplasiaof the gastric epithelium, calcifications in the brain, and testicularatrophy found in occluding-deficient mice (Saitou et al., 2000)raise a possibility that occludin has some other important phys-iological roles beyond its function as a TJ protein. Indeed, recentstudies have demonstrated that occludin regulates epithelial celldifferentiation (Schulzke et al., 2005). In addition, it controls clau-din-2-dependent TJ function, as well as cell apoptosis throughinhibition of mitogen-activated protein kinases (MAPK) and Aktsignaling pathways (Murata et al., 2005). Whether occludin has

a role in the BBB differentiation during normal development,brain vascular repair, or both remains to be explored.Recent studies of mice with experimental autoimmune en-

cephalomyelitis (EAE), a model of multiple sclerosis (MS), haveshown that occludin dephosphorylation precedes visible signsof disease and happens just prior to apparent changes in theBBB permeability (Morgan et al., 2007). These findings suggestthat occludin could be a target for signaling processes in EAEand that it may regulate the response of the BBB to the inflam-matory environment, as seen in MS.Treatment ofmicewith Tat protein results in decreased expres-

sionofoccludinandZO-1 (Puet al., 2007). Tat is normally releasedfrom human immunodeficiency virus type 1 (HIV-1)-infected

Figure 3. A Simplified Molecular Atlas of the BBB(A) Tight junctions. Claudins (claudin-3, -5, and -12) and occludin have four transmembrane domains with two extracellular loops. The junctional adhesion mol-ecule A (JAM-A) and the endothelial cell-selective adhesionmolecule (ESMA) aremembers of the Ig superfamily. Zonula occludens proteins (ZO-1, ZO-2, andZO-3)and the calcium-dependent serine protein kinase (CASK) are first-order cytoplasmic adaptor proteins that contain PDZ binding domains for the C terminus of theintramembrane proteins. Cingulin, multi-PDZ protein 1 (MUPP1), and the membrane-associated guanylate kinase with an inverted orientation of protein-proteininteraction domain (MAGI) are examples of second-order adaptor molecules. The first- and second-order adaptor molecules together with signaling moleculescontrol the interaction between the intramembrane proteins and actin/vinculin-based cytoskeleton. Adherens junctions. The vascular endothelial cadherin(VE-cadherin) is the key molecule. Platelet endothelial cell adhesion molecule 1 (PECAM-1) mediates homophilic adhesion. Catenins (a, b, c) link adhesion junc-tions to actin/vinculin-based cytoskeleton.(B) Carrier-mediated transporters.GLUT1, glucose transporter, and monocarboxylate transporter 1 (MCT1) for lactate exist at both the luminal and the abluminalmembranes. All essential amino acids (AA) are transported by the L1 and y+ systems on each membrane. Five Na+-dependent transport systems mediate elim-ination of nonessential AA (ASC, A), essential AA (LNAA), the excitatory acidic AA (EAAT) (e.g., glutamate and aspartate), and nitrogen-rich AA (N) (e.g., glutamine)from the brain. Facilitative transporters xG

! and n on the luminal membrane mediate glutamate, aspartate, and glutamine efflux to blood. Ion transporters. Thesodium pump (Na+, K+-ATPase) on the abluminal membrane controls Na+ influx and K+ efflux. Sodium-hydrogen exchanger on the luminal membrane is a keyregulator of intracellular pH. Na+-K+-2Cl– cotransporter is on the luminal membrane. The chloride-bicarbonate exchanger exists on each membrane.(C) Active efflux transporters. Multidrug efflux transporters at the luminal membrane limit drug uptake into the brain. Transporters at the abluminal membranecould act in concert with luminal transporters to eliminate drugs from brain ISF. P-gp is expressed on each membrane. Breast cancer resistance protein(BCRP) is on the luminal membrane. Multidrug resistance-associated proteins (MRPs) are expressed mainly on the luminal membrane. Organic anion transport-ing polypeptide (OATP) 2 and 3 exist on the luminal and abluminal membranes, respectively. Organic anion transporter 3 (OAT3) is on the abluminal membrane.Peptide transporters and caveolae. Peptide transport system 1 (PTS-1) on the abluminal membrane mediates efflux of opioid peptides (e.g., enkephalins) frombrain. PTS-2 mediates efflux of arginine-vasopressin (AVP). PTS-4 on the luminal membrane requires the vasopressinergic receptor 1 (V1) to transport AVP intothe brain. Receptors for insulin (IR) and transferrin (TFR) are found in the caveolar membranes. Caveolin-1 (Cav-1) could be associated with receptors (e.g., TFR),tight junctions (TJ), or growth factor receptors, such as vascular endothelial growth factor receptor (Flk-1).

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buffer) when neural activity ceases. Astrocytes can also take up K+ through transporters, particularly the Na+,K+-ATPase and NKCC1 Na+,K+2Cl– co-transporters. For both channel- and transporter-mediated K+ uptake, the net ion gain results in osmotic water uptake and slight cell swelling; the high density of AQP4 water channels in perivascular astrocytic endfeet facilitates redistribution of this water. As the brain endothelium has low water

permeability (little or no aquaporin)88–90, it is likely that the excess metabolic water joins the ISF being secreted into the pericapillary space by the endothelium5. ISF out-flow involves perivascular spaces around large vessels, and clearance routes either through the CSF or following alternative pathways to neck lymphatics.

Neurotransmitter recycling can also lead to local changes in ions and water. Glutamate is the major excitatory transmitter of the brain, and astrocyte proc-esses surrounding synapses can take up glutamate through transport proteins (particularly EAAT1 and 2); the transport is Na+-dependent and accompanied by net uptake of ions and water, again contributing to water clearance at the BBB85. Glutamate is converted to glutamine within the astrocyte and recycled to the neurons. The slight astrocytic cell swelling that accom-panies neuronal activity, resulting from activation by glutamate or ion uptake, leads to several cellular mech-anisms that contribute to the recovery of ionic balance and cell volume, some of which involve elevated intra-cellular Ca2+ concentration66,91,92. Hence, there are many links between the signalling and regulatory processes that occur in the neurovascular unit.

BBB changes in pathologyIn a number of pathologies, the function of the BBB is altered (BOX 3), and several disorders appear to involve disturbances of endothelial–glial interaction. Thus, the capillaries of many glial tumours are more leaky than those of normal brain tissue, either as a result of a lack of inductive factors, or owing to the release of perme ability factors such as vascular endothelial growth factor (VEGF). Moreover, the tight junction protein claudin 1/3 is downregulated in some brain tumours93,94.

In BBB disruption, agrin is lost from the abluminal surface of the brain endothelial cells adjacent to astro-cytic endfeet11; this may contribute to BBB damage in Alzheimer’s disease95, and to the redistribution of astro-cytic AQP4 in glioblastomas96. Astrocytic AQP4 expres-sion is upregulated in brain oedema triggered by BBB breakdown. Such upregulation could be adaptive in helping to clear the accumulating fluid, but the associ-ated cell swelling would tend to exacerbate the problem under extreme conditions. Indeed, AQP4–/– mice show protection against ischaemic brain oedema48. Some chronic neuropathologies such as multiple sclerosis may involve an early phase of BBB disturbance (involving the downregulation of claudin 1/3 (REF. 11)) that precedes neuronal damage, which suggests that vascular damage can lead to secondary neuronal disorder97.

In epilepsy, the normal pattern of brain ABC trans-porter expression may change, with upregulation of Pgp on astrocytes and brain endothelium98,99; this may be an adaptive response to barrier opening (and hence a less efficient BBB), which is often seen during seizure activity.

In animal models of Alzheimer’s disease, amyloid-! (A!) accumulation is often first seen in the neighbour-hood of blood vessels, with toxicity on endothelium and astrocytes observed before significant neuronal loss1;

Box 3 | Pathological states involving BBB breakdown or disorder

Several pathologies of the CNS involve disturbance of blood–brain barrier (BBB) function, and, in many of these, astrocyte–endothelial cooperation is also abnormal.

Stroke • Astrocytes secrete transforming growth factor-! (TGF!), which downregulates brain

capillary endothelial expression of fibrinolytic enzyme tissue plasminogen activator (tPA) and anticoagulant thrombomodulin (TM)150.

• Proteolysis of the vascular basement membrane/matrix151.

• Induction of aquaporin 4 (AQP4) mRNA and protein at BBB disruption152.

• Decrease in BBB permeability after treatment with arginine vasopressin V1 receptor antagonist in a stroke model153.

Trauma• Bradykinin, a mediator of inflammation, is produced and stimulates production and

release of interleukin-6 (IL-6) from astrocytes, which leads to opening of the BBB102.

Infectious or inflammatory processesExamples include bacterial infections, meningitis, encephalitis and sepsis.

• The bacterial protein lipopolysaccharide affects the permeability of BBB tight junctions. This is mediated by the production of free radicals, IL-6 and IL-1!154.

• Interferon-! prevents BBB disruption155.

Multiple sclerosis • Breakdown of the BBB97.

• Downregulation of laminin in the basement membrane156.

• Selective loss of claudin 1/3 in experimental autoimmune encephalomyelitis94.

HIV• BBB tight junction disruption157,158.

Alzheimer’s disease• Increased glucose transport, upregulation of glucose transporter GLUT1, altered

agrin levels, upregulation of AQP4 expression95,159.

• Accumulation of amyloid-!, a key neuropathological feature of Alzheimer’s disease, by decreased levels of P-glycoprotein transporter expression160.

• Altered cellular relations at the BBB, and changes in the basal lamina and amyloid-! clearance100.

Parkinson’s disease• Dysfunction of the BBB by reduced efficacy of P-glycoprotein101.

Epilepsy• Transient BBB opening in epileptogenic foci, and upregulated expression of

P-glycoprotein and other drug efflux transporters in astrocytes and endothelium98,99.

Brain tumours• Breakdown of the BBB161,162.

• Downregulation of tight junction protein claudin 1/3; redistribution of astrocyte AQP4 and Kir4.1 (inwardly rectifying K+ channel)20,93,96.

Pain• Inflammatory pain alters BBB tight junction protein expression and BBB

permeability108.

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on the endothelial surface, particularly loss of theterminal sialic group on the luminal side of the endo-thelial plasma membrane.

4.2. Capillaries at the CircumventricularOrgans Are Permeable to Circulating

MacromoleculesThe circumventricular organs are seven small, well-

circumscribed areas located at the ependymal borderof the third and fourth ventricles (Fig. 14), where capi-llaries are permeable to hydrophilic solutes. These sitesare the pineal body, median eminence, neurohypophy-seal-hypothalamic axis, subcommissural organ, areapostrema, subfornical organ, and organum vasculo-sum of laminae terminalis.

The circumventricular organs are endowed withpermeable capillaries that have fenestrated endo-thelium, with the exception of the subcommissuralorgan. The functions of the circumventricular organsare uncertain, although some investigators suggestthat macromolecular permeability at these sites maybe related to the involvement of the respective neu-ronal groups in the regulation of neuroendocrinefunctions.

4.3. Immune and Inflammatory Mediators Playan Important Role in a Variety of

Pathophysiologic Pathways Such as in CerebralIschemia

After cerebral ischemia, leukocytes—through theirinitial interactions with the microvascular endothe-lium—play an important role in the development ofthe infarct. Within hours of the onset of ischemia, poly-morphonuclear (PMN) leukocytes accumulate andobstruct the microvasculature, then enter the parench-yma, followed by cells of the monocyte/macrophagelineage. Adhesion to the endothelium and transmigra-tion through the vessel wall are influenced by inflamma-tory mediators, including cytokines. Upon activation,under conditions of ischemia/reperfusion, PMN leuko-cytes and the endothelium generate free radicals andproteases that contribute to microvascular and tissueinjury.

4.3.1. ADHESION RECEPTORS

Adhesion receptors that mediate cell-cell and cell-matrix interactions in the cerebrovasculature belong tothree families: selectins, integrins, and immunoglobulin-related receptors. Their inhibition may modulate cellu-lar inflammation and reduce infarction. The access ofPMN and other leukocytes to perivascular cells in thedeveloping infarction requires their direct contact withthe microvascular endothelium. After middle cerebralartery occlusion (and reperfusion), PMN leukocytescontribute tomicrovascular obstruction and edema for-mation during their adherence to the endothelium.Adherence and transmigration of PMN leukocytesthrough the postcapillary endothelium involve thesequential interaction of P-selectin, intercellular adhe-sion molecule (ICAM-1), and E-selectin. The selectinfamily consists of P-selectin found on platelets andendothelial cells, E-selectin (endothelial cells), andL-selectin (leukocytes). P-selectin on endothelial cellsand platelets mediates their interaction with granulo-cytes and monocytes. Integrins are heterodimeric adhe-sion molecules with a ubiquitous distribution. Theadhesion properties of certain integrins are central toleukocyte transmigration.Firmadhesion ismediated bythe interaction of granulocyte b2-integrins withendothelial cell ICAM-1 (integrin aM b2 MAC-1), orendothelial cell ICAM-1 and ICAM-2 (integrin aL b2,LFA-1).

4.3.2. CYTOKINES

Ischemic cerebral tissues generate cytokines, super-oxide free radicals, biogenic amines, and thrombinThese are stimulators for endothelial cells, granulocyte,

Fig. 14.Midline sagittal schematic drawing of the brain show-ing circumventricular organs (dark shaded structures): NH,neurohypophysis; ME, median eminence; OVLT, organumvasculosum of lamina terminales; SFO, subfornicial organ;PI, pineal gland or body; SCO, subcommissural organ; AP,area postrema; CP, choroid plexus; OC, optic chiasm; AC,anterior commissure; CC, corpus callosum (lightly shadedareas).

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disturbances of CNS homeostasis as a result of barrier deficiencies could contribute to and exacerbate the later neuropathology100. Recently, Kortekaas et al.101 showed an elevated uptake of the Pgp substrate [11C]verapamil using positron emission tomography (PET) in the midbrain of patients with Parkinson’s disease, which is consistent with disturbed Pgp function in the BBB.

The ability of agents released during inflammation to increase the permeability of the brain endothelium may depend on associated cell types (FIG. 6). Thus, as well as acting on endothelial bradykinin B2 receptors to raise intracellular Ca2+ concentrations and open tight

junctions, bradykinin can activate NF-$B in astrocytes, leading to the release of interleukin-6 (IL-6), which can amplify the effect by acting back on the endothelium102. Tumour necrosis factor-# (TNF#) can increase BBB permeability by direct actions on the endothelium103 and indirect effects involving endothelial endothelin 1 production and IL-1! release from astrocytes, in a complex immunoregulatory loop104. Systemic infection can exacerbate CNS inflammatory pathologies such as multiple sclerosis by several mechanisms, including activation of already primed central macrophages, with some mechanisms effective even with an intact BBB105. Indeed, the ability of the BBB to transport cytokines may contribute to the link between central and peripheral disease106.

It has recently been proposed that activated astro-cytes and microglial cells could maintain neuropathic pain107. As astrocytes have extensive gap junctional connectivity and form glial networks, it has been sug-gested that glia may be involved in the spreading of pain sensation. In injury, several substances are released from central and peripheral neurons, connective tis-sue cells and blood cells. Many of these substances, such as substance P, calcitonin gene-related peptide (CGRP), serotonin, histamine and ATP, can affect the BBB from both the blood and the nervous tissue sides. For example, the release of IL-1! leads to a decreased concentration or altered localization of the tight junc-tion protein occludin, and increased BBB permeability. TNF#, histamine and interferon-& released in inflam-matory pain can also cause changes in brain endothelial permeability108.

The involvement of microglia in signalling within the pathological neurovascular unit has been men-tioned above66,67. It is possible that damage to the endothelium and basal lamina allows expression of endothelial receptors that are normally downregu-lated (for example, receptors for nucleotides such as ATP), opening new communication loops between endothelium, pericytes, astrocytes and microglia that are important in barrier repair.

Targeting the BBB to fight diseaseThe BBB as a therapeutic target. We have seen how information on the routes across the BBB (FIG. 3) needs to be taken into account in developing drug delivery strate-gies to target sites in the CNS in treating neural disorders.Given the evidence for involvement of BBB damage as an early event in many neurological conditions, it is not surprising that there is growing interest in the BBB as a therapeutic target in its own right109–112. The underlying logic is that if BBB dysfunction can be reduced, halted or reversed, this could be valuable therapy in conditions in which neuronal damage is secondary to, or exacer-bated by, BBB damage. Steroids such as dexamethasone are widely used to reduce inflammation, and are an accepted treatment for brain oedema113. It is now known that dexamethasone can improve barrier function not only by increasing the tightness of the brain endothelial tight junctions114, but also by upregulating BBB Pgp59. Ca2+ channel blockers hold promise for reducing brain

Figure 6 | Astroglial–endothelial signalling under pathological conditions. Examples of astroglial–endothelial signalling in infection or inflammation, stroke or trauma, leading to opening of the blood–brain barrier (BBB) and disturbance of brain function. bradykinin, produced during inflammation in stroke or brain trauma, acts on endothelial and astroglial bradykinin B2 receptors, leading to an increase in the concentration of intracellular Ca2+. In astrocytes, this can trigger the production of interleukin-6 (IL-6) through activation of nuclear factor-$B (NF-$B) (1). Bradykinin, substance P, 5-hydroxytryptamine (5-HT, serotonin) and histamine acting on astrocytes can lead to the formation of ATP and prostaglandins (PGs), with effects on vascular tone and endothelial permeability (2) by mechanisms that are known to involve endothelium. Lipopolysaccharide (LPS), formed in infections, leads to the release from microglia of tumour necrosis factor-# (TNF#), IL-1! and reactive oxygen species (including O2

•–), all of which have the ability to open the BBB (3). Astrocytes downregulate tissue plasminogen activator (tPA) production via transforming growth factor-! (TGF!), but there is still sufficient tPA to open the BBB, leading to an influx of tPA from the blood (4). Following disruption of the BBB involving a decrease in agrin expression, K+ and glutamate (Glu) from the blood can reach the brain extracellular space. Aquaporin 4 (AQP4) is upregulated on the astroglial endfeet, leading to astroglial swelling (5). ET1, endothelin 1.

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artery originating from the internal carotid arteryinstead of the basilar artery.

Anastomoses among branches of the internal andexternal carotid arteries exist through the ophthalmicartery (e.g., the large branch of the internal carotidartery) and its end-to-end connections with facialbranches of the ipsilateral external carotid artery.There are extensive collateral anastomoses betweenpial meningeal branches of the internal carotid andmeningeal branches of the external carotid artery.

4. CAPILLARIES

The perivascular sheath of CSF surrounding pene-trating arteries and arterioles (perivascular space orVirchow-Robin space) disappears where the glia lim-itans merges with the basal lamina of the brain capil-laries. These vessels, which in humans are 4 to 7 mm indiameter, are composed of one endothelial-cell layer,resting on a basal lamina that completely encircles apericyte. Pericytes do not form a continuous layeraround the endothelial-cell layer; individual pericytesare found at infrequent intervals on the luminal sideof the capillary wall. Encircling the basal lamina ofthe endothelial cell or the pericyte are numerous pro-cesses of astrocytes joined to one another by gapjunctions.

4.1. The Blood-Brain Barrier Refers to aComplex Array of Physical, Metabolic, and

Transport Properties of the CapillaryEndothelium

The blood-brain barrier is a complex anatomic ormechanical, physiologic and osmotic barrier protect-ing the brain. Circulating macromolecules, such asglobulins and albumin, do not cross the endotheliallining of brain capillaries. This contrasts with theready escape of circulating macromolecules that nor-mally occurs inmost extracranial tissues. The originaldescription of the blood-brain barrier is attributedto Ehrlich who, in 1885, observed that intravenousinjections of Evans blue, a dye that circulates boundto albumin, result in the diffuse distribution of the dyeto almost every organ and tissue except the brain andspinal cord.

The concept of a blood-brain barrier describes theinability of circulating macromolecules to enter theextracellular space or interstitial fluid of the brainand spinal cord. The mechanical component of thebarrier has been traced primarily to structural charac-teristics of the endothelial capillary lining of the brainand spinal cord that are lacking in the endothelial

lining of capillaries in other organs. A first importantfeature is that endothelial cells lining capillaries andvenules in the CNS are joined at the luminal portion byzonulae occludentes or pentalaminar structures thatrepresent the fusion of the outermost layers of twoapposing endothelial-cell membranes (Fig. 13). Thesecond factor preventing the escape of circulatingmacromolecules in the brain is the paucity of endocy-totic pits in the endothelium of most vessels in theCNS. In contrast, the endothelial lining of capillariesand venules in extraneural tissues has abundant endo-cytotic pits and sizable gaps or fenestrae throughwhich circulating particles such as 40-kDa horseradishperoxidase or 445-kDa apoferritin readily escape intothe surrounding interstitial fluids.

Cerebral endothelium may become abnormallypermeable to circulating macromolecules by severalmechanisms: enhanced transcytosis or transport ofmolecules across the endothelial cytoplasm by meansof endothelial vesicles; separation of the endothelialjunctions; formation of tubular channels by fusion ofendothelial vesicles; and loss of the negative charge

Fig. 13. Normal rat brain capillary (original magnification!7000). The inset shows a close-up view of the capillary wallto demonstrate a tight junction (arrows) (original magnifica-tion !32,200).

Chapter 7 / Vasculature of the Human Brain 159


Astroglia

44


Astroglia

Inducción de la BHE

44


Astroglia


Mantenimiento de la BHE

44


Astroglia



Control del tono vascular

44


Astroglia



Control del tono vascular

Estructura de la BHE?

44


Pericitos

45


Pericitos

Identidad oscura

Células pluripotenciales

Participación en inducción y maduración de la BHE

45


46LETTERdoi:10.1038/nature09522

Pericytes regulate the blood–brain barrierAnnika Armulik1, Guillem Genove1, Maarja Mae1, Maya H. Nisancioglu1, Elisabet Wallgard1{, Colin Niaudet1, Liqun He1{,Jenny Norlin1, Per Lindblom2, Karin Strittmatter1{, Bengt R. Johansson3 & Christer Betsholtz1

Theblood–brainbarrier (BBB) consists of specific physical barriers,enzymes and transporters, which together maintain the necessaryextracellular environment of the central nervous system (CNS)1.The main physical barrier is found in the CNS endothelial cell,and depends on continuous complexes of tight junctions combinedwith reduced vesicular transport2.Otherpossible constituents of theBBB include extracellular matrix, astrocytes and pericytes3, but therelative contribution of these different components to the BBBremains largely unknown1,3. Here we demonstrate a direct role ofpericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increasesthe permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeabilityoccurs by endothelial transcytosis, a process that is rapidly arrestedby the drug imatinib. Furthermore, we show that pericytes functionat the BBB in at least two ways: by regulating BBB-specific geneexpression patterns in endothelial cells, and by inducing polariza-tion of astrocyte end-feet surrounding CNS blood vessels. Ourresults indicate a novel and critical role for pericytes in the integ-ration of endothelial and astrocyte functions at the neurovascularunit, and in the regulation of the BBB.Platelet-derived growth factor (PDGF)-B/PDGF receptor-b (PDGFR-

b) signalling isnecessary for pericyte recruitmentduringangiogenesis4,5.Perinatal lethality precludes analysis of postnatal processes in Pdgfb orPdgfrb null mice6,7, but several othermousemutants of this pathway areviable postnatally. Two suchmutantswereusedhere: PDGF-B retentionmotif knockouts (Pdgfbret/ret) where PDGF-B binding to heparan sul-phate proteoglycans was disrupted8; and mutants in which Pdgfb nullalleles were complemented by one or two copies of a conditionally silenthumanPDGF-B transgene targeted to theRosa26 locus andactivated byendothelial Cre recombinase (hemizygous R26P1/0 or homozygousR26P1/1 mice; Supplementary Fig. 2a–d).We quantified pericyte coverage in different regions of the CNS by

CD13 or PDGFR-b staining (Fig. 1a, b, e and Supplementary Fig. 3).Pdgfbret/ret, R26P1/0 and R26P1/1 mice displayed pericyte coveragecorresponding to 26%, 40% and 72%, respectively, compared to con-trols (Fig. 1a, b). Quantification of absolute numbers of mural cells(pericytes and vascular smooth muscle cells) using the transgenicreporter XLacZ49 confirmed low mural cell densities in Pdgfbret/ret

and R26P1/0 mice, and close to normal levels in R26P1/1 mice (Sup-plementary Fig. 3b–p). We extended previous observations4,5,10,11 thatreduced pericyte densities correlate with increased vessel diameterand reduced vessel density (Fig. 1c–e and Supplementary Fig. 4).Importantly, these phenotypes were almost completely normalizedin R26P1/1 mice (Fig. 1d, e and Supplementary Fig. 4a–c).Increased water content in brains of Pdgfbret/ret mice (Fig. 1f) indi-

cated impairment of the BBB. We tested the BBB integrity in the differ-ent mutants using a panel of tracers (Supplementary Table 1). The azodye Evans blue12 accumulated in mutant brain parenchyma in a time-dependent fashion (Fig. 1i) and in correlation with pericyte density: itwas largest in Pdgfbret/ret mice followed by R26P1/0 and R26P1/1

(Fig. 1g, h, j and Supplementary Fig. 5a–c). Similarly, the fluorescentdye cadaverine Alexa Fluor-555 accumulated significantly in the brainparenchyma of Pdgfbret/ret and R26P1/0 mice (Fig. 1j and Supplemen-tary Fig. 5d, h, i). Additionally, fluorescently labelled albumin, 70 kDadextran and IgGpassed theBBB inPdgfbret/ret andR26P1/0mice, butnotin controls or in R26P1/1 mice (Fig. 1j and Supplementary Fig. 5e–g).These experiments establish a close correlation between pericyte densityand permeability across the BBB for a range of tracers of differentmolecular masses (Supplementary Table 1).Permeability in CNS vessels is impeded by continuous complexes of

endothelial junctions13,14. We studied such complexes in adult pericyte-deficient mutants using markers for adherens (VE-cadherin) and tight(ZO-1 and claudin 5) junctions. Pdgfbret/ret, R26P1/0 and controlsshowed junctional marker expression at similar levels as judged byimmunostaining and western blotting (Supplementary Fig. 6a–c anddata not shown). The junctional markers were distributed in a patternconsistent with continuous junction complexes in both mutants andcontrols; however,mutants displayed focally increased junctional widthand undulation. These patterns were confirmed by transmission elec-tron microscopy, which failed to reveal any apparent abnormalities inthe ultrastructure of endothelial junctions, with the exception thatlonger and irregular stretches of endothelial overlap were commonlyfound inpericyte-deficientmutants (Fig. 2c andSupplementary Fig. 6e).Because continuity, ultrastructure andmarker expressionwere con-

sistentwith retained integrity of endothelial junctions in the absence ofpericytes, we took advantage of the fixable nature of the fluorescenttracers to explore the route of extravasation in Pdgfbret/ret and R26P1/0

mice in more detail. Cadaverine Alexa Fluor-555 accumulated inendothelial cells and in the brain parenchyma in Pdgfbret/ret andR26P1/0 mice, but not in controls or in R26P1/1mice (Fig. 2a). Extra-vasated cadaverine Alexa Fluor-555 localized mainly to neurons(Figs 2a and 3d and Supplementary Fig. 7a). Similar patterns of dis-tribution were observed for fluorescent albumin, IgG and 70 kDadextran (Fig. 2b and Supplementary Fig. 7b). We also studied thedistribution of horseradish peroxidase (HRP, 44 kDa) by transmissionelectron microscopy. We found increased uptake of HRP specificallyin macrovesicular structures in the endothelium in Pdgfbret/ret mice incomparison with controls (Fig. 2c, d and Supplementary Fig. 6d, e).Pdgfbret/ret microvessels also showed marked accumulation of HRPreactivity at the vascular basement membrane, without apparent co-localization with endothelial junctions (Fig. 2c, d and SupplementaryFig. 6d, e). Together, these observations indicate that macromolecularpermeability across the BBB in pericyte-deficient vessels occursthrough a transcytosis route. Pericyte deficiency was not associatedwith changes in the polarization or with signs of fenestration in theendothelial cells (Supplementary Figs 4d, 6d, e and 7c), features thatcharacterize the BBB defects observed as a result of impaired Wnt/b-catenin signalling15,16.The BBB breaks open in conjunction with stroke, leading to life-

threatening CNS oedema. A recent study demonstrated that the tyro-sine kinase inhibitor imatinib counteracts oedema in experimental

1Department of Medical Biochemistry andBiophysics, Division of Vascular Biology, Karolinska Institute, Scheeles vag 2, SE-171 77 Stockholm, Sweden. 2AstraZeneca AB, Clinical Development, SE-431 83Molndal, Sweden. 3The Electron Microscopy Unit, Institute for Biomedicine, The Sahlgrenska Academy, University of Gothenburg, PO Box 420, SE-405 30 Gothenburg, Sweden. {Present addresses:DepartmentofGenetics andPathology, RudbeckLaboratory, DagHammarskjolds vag20,UppsalaUniversity, SE-75185Uppsala, Sweden (E.W.); AppliedBiosystemsSweden, Lindhagensgatan76, POBox12650, SE-112 92 Stockholm, Sweden (L.H.); German Cancer Research Center DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany (K.S.).

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Su deficit incrementa permeabilidad agua y otras moléculas mediante transcitosis

Regula la expresión génica de genes endoteliales de BHE

Induce polarización de pies astrocitarios

Participación en inducción y maduración de la BHE regulando la relación astrocito-endotelio

46LETTERdoi:10.1038/nature09522

Pericytes regulate the blood–brain barrierAnnika Armulik1, Guillem Genove1, Maarja Mae1, Maya H. Nisancioglu1, Elisabet Wallgard1{, Colin Niaudet1, Liqun He1{,Jenny Norlin1, Per Lindblom2, Karin Strittmatter1{, Bengt R. Johansson3 & Christer Betsholtz1

Theblood–brainbarrier (BBB) consists of specific physical barriers,enzymes and transporters, which together maintain the necessaryextracellular environment of the central nervous system (CNS)1.The main physical barrier is found in the CNS endothelial cell,and depends on continuous complexes of tight junctions combinedwith reduced vesicular transport2.Otherpossible constituents of theBBB include extracellular matrix, astrocytes and pericytes3, but therelative contribution of these different components to the BBBremains largely unknown1,3. Here we demonstrate a direct role ofpericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increasesthe permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeabilityoccurs by endothelial transcytosis, a process that is rapidly arrestedby the drug imatinib. Furthermore, we show that pericytes functionat the BBB in at least two ways: by regulating BBB-specific geneexpression patterns in endothelial cells, and by inducing polariza-tion of astrocyte end-feet surrounding CNS blood vessels. Ourresults indicate a novel and critical role for pericytes in the integ-ration of endothelial and astrocyte functions at the neurovascularunit, and in the regulation of the BBB.Platelet-derived growth factor (PDGF)-B/PDGF receptor-b (PDGFR-

b) signalling isnecessary for pericyte recruitmentduringangiogenesis4,5.Perinatal lethality precludes analysis of postnatal processes in Pdgfb orPdgfrb null mice6,7, but several othermousemutants of this pathway areviable postnatally. Two suchmutantswereusedhere: PDGF-B retentionmotif knockouts (Pdgfbret/ret) where PDGF-B binding to heparan sul-phate proteoglycans was disrupted8; and mutants in which Pdgfb nullalleles were complemented by one or two copies of a conditionally silenthumanPDGF-B transgene targeted to theRosa26 locus andactivated byendothelial Cre recombinase (hemizygous R26P1/0 or homozygousR26P1/1 mice; Supplementary Fig. 2a–d).We quantified pericyte coverage in different regions of the CNS by

CD13 or PDGFR-b staining (Fig. 1a, b, e and Supplementary Fig. 3).Pdgfbret/ret, R26P1/0 and R26P1/1 mice displayed pericyte coveragecorresponding to 26%, 40% and 72%, respectively, compared to con-trols (Fig. 1a, b). Quantification of absolute numbers of mural cells(pericytes and vascular smooth muscle cells) using the transgenicreporter XLacZ49 confirmed low mural cell densities in Pdgfbret/ret

and R26P1/0 mice, and close to normal levels in R26P1/1 mice (Sup-plementary Fig. 3b–p). We extended previous observations4,5,10,11 thatreduced pericyte densities correlate with increased vessel diameterand reduced vessel density (Fig. 1c–e and Supplementary Fig. 4).Importantly, these phenotypes were almost completely normalizedin R26P1/1 mice (Fig. 1d, e and Supplementary Fig. 4a–c).Increased water content in brains of Pdgfbret/ret mice (Fig. 1f) indi-

cated impairment of the BBB. We tested the BBB integrity in the differ-ent mutants using a panel of tracers (Supplementary Table 1). The azodye Evans blue12 accumulated in mutant brain parenchyma in a time-dependent fashion (Fig. 1i) and in correlation with pericyte density: itwas largest in Pdgfbret/ret mice followed by R26P1/0 and R26P1/1

(Fig. 1g, h, j and Supplementary Fig. 5a–c). Similarly, the fluorescentdye cadaverine Alexa Fluor-555 accumulated significantly in the brainparenchyma of Pdgfbret/ret and R26P1/0 mice (Fig. 1j and Supplemen-tary Fig. 5d, h, i). Additionally, fluorescently labelled albumin, 70 kDadextran and IgGpassed theBBB inPdgfbret/ret andR26P1/0mice, butnotin controls or in R26P1/1 mice (Fig. 1j and Supplementary Fig. 5e–g).These experiments establish a close correlation between pericyte densityand permeability across the BBB for a range of tracers of differentmolecular masses (Supplementary Table 1).Permeability in CNS vessels is impeded by continuous complexes of

endothelial junctions13,14. We studied such complexes in adult pericyte-deficient mutants using markers for adherens (VE-cadherin) and tight(ZO-1 and claudin 5) junctions. Pdgfbret/ret, R26P1/0 and controlsshowed junctional marker expression at similar levels as judged byimmunostaining and western blotting (Supplementary Fig. 6a–c anddata not shown). The junctional markers were distributed in a patternconsistent with continuous junction complexes in both mutants andcontrols; however,mutants displayed focally increased junctional widthand undulation. These patterns were confirmed by transmission elec-tron microscopy, which failed to reveal any apparent abnormalities inthe ultrastructure of endothelial junctions, with the exception thatlonger and irregular stretches of endothelial overlap were commonlyfound inpericyte-deficientmutants (Fig. 2c andSupplementary Fig. 6e).Because continuity, ultrastructure andmarker expressionwere con-

sistentwith retained integrity of endothelial junctions in the absence ofpericytes, we took advantage of the fixable nature of the fluorescenttracers to explore the route of extravasation in Pdgfbret/ret and R26P1/0

mice in more detail. Cadaverine Alexa Fluor-555 accumulated inendothelial cells and in the brain parenchyma in Pdgfbret/ret andR26P1/0 mice, but not in controls or in R26P1/1mice (Fig. 2a). Extra-vasated cadaverine Alexa Fluor-555 localized mainly to neurons(Figs 2a and 3d and Supplementary Fig. 7a). Similar patterns of dis-tribution were observed for fluorescent albumin, IgG and 70 kDadextran (Fig. 2b and Supplementary Fig. 7b). We also studied thedistribution of horseradish peroxidase (HRP, 44 kDa) by transmissionelectron microscopy. We found increased uptake of HRP specificallyin macrovesicular structures in the endothelium in Pdgfbret/ret mice incomparison with controls (Fig. 2c, d and Supplementary Fig. 6d, e).Pdgfbret/ret microvessels also showed marked accumulation of HRPreactivity at the vascular basement membrane, without apparent co-localization with endothelial junctions (Fig. 2c, d and SupplementaryFig. 6d, e). Together, these observations indicate that macromolecularpermeability across the BBB in pericyte-deficient vessels occursthrough a transcytosis route. Pericyte deficiency was not associatedwith changes in the polarization or with signs of fenestration in theendothelial cells (Supplementary Figs 4d, 6d, e and 7c), features thatcharacterize the BBB defects observed as a result of impaired Wnt/b-catenin signalling15,16.The BBB breaks open in conjunction with stroke, leading to life-

threatening CNS oedema. A recent study demonstrated that the tyro-sine kinase inhibitor imatinib counteracts oedema in experimental

1Department of Medical Biochemistry andBiophysics, Division of Vascular Biology, Karolinska Institute, Scheeles vag 2, SE-171 77 Stockholm, Sweden. 2AstraZeneca AB, Clinical Development, SE-431 83Molndal, Sweden. 3The Electron Microscopy Unit, Institute for Biomedicine, The Sahlgrenska Academy, University of Gothenburg, PO Box 420, SE-405 30 Gothenburg, Sweden. {Present addresses:DepartmentofGenetics andPathology, RudbeckLaboratory, DagHammarskjolds vag20,UppsalaUniversity, SE-75185Uppsala, Sweden (E.W.); AppliedBiosystemsSweden, Lindhagensgatan76, POBox12650, SE-112 92 Stockholm, Sweden (L.H.); German Cancer Research Center DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany (K.S.).

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Basal lamina

Apical membrane

Cingulin, JACOP, PAR3/6,CASK, 7H6, Itch, MUPP1,MAGI-1–3, ZONABAF6, RGS5

!-, "-, #-Catenin,Desmoplakin,p120ctn, ZO-1

Actin/vinculin-basedcytoskeleton

ZO-1

ZO-2

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VE-cadherin

PECAM

JAMs,ESAM

Claudin 3, 5, 12Occludin

Basolateralmembrane

Tightjunction

Adherensjunction

Orthogonal arrays of particles(OAPs). The organized arrays (square lattice) of intramembranous particles detected by the freeze–fracture technique in certain astrocyte processes. First identified on the polarized endfeet on blood vessels and in the outer glial layer (glia limitans) below the pia, they have subsequently been shown to contain specific protein complexes held together by structural proteins.

Basal laminaThe extracellular matrix layer produced by the basal cell membrane, used as an anchoring and signalling site for cell–cell interactions.

Astrocytes show a number of different morpho-logies, depending on their location and association with other cell types. Of the ~11 distinct phenotypes that can be readily distinguished, 8 involve specific interactions with blood vessels30. There is now strong evidence, particularly from studies in cell culture, that astrocytes can upregulate many BBB features, leading to tighter tight junctions (physical barrier)31,32, the expression and polarized localization of transporters, including Pgp24 and GLUT1 (REF. 33) (transport barrier), and specialized enzyme systems (metabolic barrier)9,34–36. More recently, some of the other cell types present at the BBB, including pericytes, perivascular macrophages and neurons, have also been shown to contribute to barrier induction37–43. Given the complexity of the bar-rier properties of the BBB, and the anatomical relation-ships of the associated cells, it is not surprising to find synergistic inductive functions involving more than one cell type. For example, astrocytes are necessary for

the correct association of endothelial cells and peri-cytes in tube-like structures in vitro38, which suggests that interactions between the three cell types are also required for proper cerebral capillary differentiation in vivo.

The converse induction, in which brain endo-the lium enhances the growth and differentiation of associated astrocytes, has also been shown44,45. Indeed, upregulation of the endothelial enzyme #-glutamyl transpeptidase (#GTP) involves a two-way induction with astrocytes46, and co-culture results in the upregu-lation of antioxidant enzymes in both endothelial cells and astrocytes47.

Specializations of astrocytic perivascular endfeet. Astrocytes are derived from ependymoglia of the developing neural tube, and retain some features of their original apical–basal polarity, together with more specific polarization of function in relation to particular cell–cell associations of the adult28,30. The perivascular endfeet of astrocytes, which are closely applied to the microvessel wall, show several special-ized features characteristic of this location, including a high density of orthogonal arrays of particles (OAPs) containing the water channel aquaporin 4 (AQP4) and the Kir4.1 K+ channel, which are involved in ion and volume regulation (see below). The OAPs/AQP4 polarity of astrocytes correlates with the expression of agrin, a heparin sulphate proteoglycan, on the basal lamina11,48. Agrin accumulates in brain microvessels at the time of BBB tightening, and is important for the integrity of the BBB20. The agrin splice variant Y0Z0 is a specific component of the endothelial basal lamina of CNS capillaries. Agrin is required for the segregation of AQP4 to the perivascular astrocytic endfeet, mediated by agrin binding to !-dystroglycan (a member of the dystrophin–dystroglycan complex, DDC), which couples to AQP through !1-syntrophin, another member of the DDC. !-Syntrophin also binds to Kir4.1, which explains the co-localization of Kir4.1 and AQP4. The precise localization of this complex array of membrane proteins in the astrocytic endfeet, anchored by agrin in the basal lamina, provides part of the evidence that this extracellular matrix makes an important contribution to the inductive influences between the endothelium and astrocytes.

Inducing factors. Astrocytes are able to secrete a range of chemical agents9,28,36,49. Several of these glia-derived factors, including transforming growth factor-" (TGF"), glial-derived neurotrophic factor (GDNF)50, basic fibroblast growth factor (bFGF) and angiopoetin 1 (ANG1, acting on the TIE2 endothelium-specific recep-tor tyrosine kinase 2), can induce aspects of the BBB phenotype in endothelial cells in vitro51. Conversely, endothelium-derived leukaemia inhibitory factor (LIF) has been shown to induce astrocytic differentiation45. The defects in BBB function in some neuropathologies, especially those that involve glia (see below), suggest that continuing induction during adult life is necessary for normal function.

Figure 4 | Molecular composition of endothelial tight junctions. Simplified and incomplete scheme showing the molecular composition of endothelial tight junctions. Occludin and the claudins — proteins with four transmembrane domains and two extracellular loops — are the most important membranous components. The junctional adhesion molecules (JAMs) and the endothelial selective adhesion molecule (ESAM) are members of the immunoglobulin superfamily. Within the cytoplasm are many first-order adaptor proteins, including zonula occludens 1, 2 and 3 (ZO-1–3) and Ca2+-dependent serine protein kinase (CASK), that bind to the intramembrane proteins. Among the second-order adaptor molecules, cingulin is important, and junction-associated coiled-coil protein (JACOP) may also be present. Signalling and regulatory proteins include multi-PDZ-protein 1 (MUPP1), the partitioning defective proteins 3 and 6 (PAR3/6), MAGI-1–3 (membrane-associated guanylate kinase with inverted orientation of protein–protein interaction domains), ZO-1-associated nucleic acid-binding protein (ZONAB), afadin (AF6), and regulator of G-protein signalling 5 (RGS5). All of these adaptor and regulatory/signalling proteins control the interaction of the membranous components with the actin/vinculin-based cytoskeleton. In epithelial cells, tight and adherens junctions are strictly separated from each other, but in endothelial cells these junctions are intermingled. The most important molecule of endothelial adherens junctions is vascular endothelial cadherin (VE-cadherin). In addition, the platelet–endothelial cell adhesion molecule (PECAM) mediates homophilic adhesion. The chief linker molecules between adherens junctions and the cytoskeleton are the catenins, with desmoplakin and p120 catenin (p120ctn) also involved. Itch, E3 ubiquitin protein ligase. Modified, with permission, from REF. 20 $ (2005) Wiley-VCH.

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Tight junctionA belt-like region of adhesion between adjacent cells. Tight junctions regulate paracellular flux, and contribute to the maintenance of cell polarity by stopping molecules from diffusing within the plane of the membrane.

Abluminal membraneThe endothelial cell membrane that faces away from the vessel lumen, towards the brain.

MeningesThe complex arrangement of three protective membranes surrounding the brain, with a thick outer connective tissue layer (dura) overlying the barrier layer (arachnoid), and finally the thin layer covering the glia limitans (pia). The sub-arachnoid layer has a sponge-like structure filled with CSF.

Circumventricular organs(CVOs). Brain regions that have a rich vascular plexus with a specialized arrangement of blood vessels. The junctions between the capillary endothelial cells are not tight in the blood vessels of these regions, which allows the diffusion of large molecules. These organs include the organum vasculosum of the lamina terminalis, the subfornical organ, the median eminence and the area postrema.

Receptor-mediated transcytosisThe mechanism for vesicle-mediated transfer of substances across the cell, the first step of which requires specific binding of the ligand to a membrane receptor, followed by internalization (endocytosis).

Adsorptive-mediated transcytosisThe mechanism for vesicle-mediated transfer of substances across the cell, the first step of which involves nonspecific binding of the ligand to membrane surface charges, followed by internalization (endocytosis).

Our understanding of the molecular structure of tight junctions derives from studies of both epithelia and endothelia (FIG. 4). Among the molecules identified as making important contributions to tight junction structure are the transmembrane proteins occludin and the claudins. Occludin is a 60–65 kDa protein with a car-boxy (C)-terminal domain that is capable of linking with zonula occludens protein 1 (ZO-1; see below). The main function of occludin appears to be in tight junction regu-lation12,22. In the BBB, expression of the proteins claudin 3 (originally misidentified as claudin 1, now also referred to as 1/3), claudin 5 and possibly claudin 12 appears to contribute to the high TEER11,20. Junctional adhesion mole cules JAM-A, JAM-B and JAM-C are present in brain endothelial cells, and are involved in the formation and maintenance of the tight junctions. The transmem-brane proteins are connected on the cytoplasmic side to a complex array of peripheral membrane proteins that form large protein complexes, the cytoplasmic plaques. Within the plaques are adaptor proteins with many protein–protein interaction domains, including ZO-1, ZO-2 and ZO-3; the Ca2+-dependent serine protein kinase (CASK); MAGI-1, MAGI-2 and MAGI-3 (membrane-associated

guanylate kinase with inverted orientation of protein–protein interaction domains); the partitioning defective proteins PAR3 and PAR6; and MUPP1 (multi-PDZ-protein 1). These help to organize the second class of plaque proteins, the regulatory and signalling molecules (including the small GTPases) and their regulators, such as the regulator of G-protein signalling 5 (RGS5), and the transcription regulator the ZO-1-associated nucleic acid-binding protein (ZONAB). A newly identified protein, junction-associated coiled-coil protein (JACOP), may anchor the junctional complex to the actin cytoskeleton. Cell–cell interaction in the junctional zone is stabilized by adherens junctions.

The tight junction has a valuable function not only in restricting paracellular permeability (gate function), but also in segregating the apical and basal domains of the cell membrane (fence function) so that the endothelium can take on the polarized (apical–basal) properties that are more commonly found in epithelia, such as those of the gastrointestinal tract and kidney20. The PAR3–atypical protein kinase C (aPKC)–PAR6 complex appears to be involved in regulating tight junction formation and in establishing cell polarity.

Figure 2 | Cellular constituents of the blood–brain barrier. The barrier is formed by capillary endothelial cells, surrounded by basal lamina and astrocytic perivascular endfeet. Astrocytes provide the cellular link to the neurons. The figure also shows pericytes and microglial cells. a | Brain endothelial cell features observed in cell culture. The cells express a number of transporters and receptors, some of which are shown. EAAT1–3, excitatory amino acid transporters 1–3; GLUT1, glucose transporter 1; LAT1, L-system for large neutral amino acids; Pgp, P-glycoprotein. b | Examples of bidirectional astroglial–endothelial induction necessary to establish and maintain the BBB. Some endothelial cell characteristics (receptors and transporters) are shown. 5-HT, 5-hydroxytryptamine (serotonin); ANG1, angiopoetin 1; bFGF, basic fibroblast growth factor; ET1, endothelin 1; GDNF, glial cell line-derived neurotrophic factor; LIF, leukaemia inhibitory factor; P2Y2, purinergic receptor; TGF!, transforming growth factor-!; TIE2, endothelium-specific receptor tyrosine kinase 2. Data obtained from astroglial–endothelial co-cultures and the use of conditioned medium8,10,24–27,33,45,50,51.

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accumulation of neuropeptides in the brain ISF. Uptake of mostcirculating peptides by the brain can be comparedwith uptake ofdrugs such as acetaminophen, which exerts analgesic activitywith an uptake of only 0.2%/g brain, or morphine, which hasan uptake of <0.02%/g.CaveolaeRaft-dependent endocytosis is cholesterol sensitive, clathrin in-dependent internalization of ligands and receptors from theplasma membrane (Lajoie and Nabi, 2007). It encompasses en-docytosis of caveolae, smooth plasmalemmal vesicles that formsubdomains of cholesterol and sphingolipid-rich rafts that areenriched in caveolin-1. The caveolae control transcellular per-meability by regulating endocytosis, transcytosis, and signalingin lipid-based microdomains of the BBB (Parton and Richards,2003). The caveolar membranes contain receptors for transfer-rin, insulin, albumin, ceruloplasmin, RAGE, LDL, HDL, interleu-kin-1, and vesicle-associated membrane protein-2 (Wolburg,2006) (Figure 3C). Signaling complexes at caveolin-1 includeheterotrimeric G proteins, members of the MAPK pathway, srctyrosine kinase, protein kinase C, and the endothelial NO syn-thase. The involvement of caveolin-1 in NO and calcium signalinghas been demonstrated in caveolin-1-deficient mice (Drab et al.,2001). VEGFR-2 (Labrecque et al., 2003) and P-gp (Jodoin et al.,2003) are also closely associated with caveolin-1. Caveolin-1can also influence the levels of TJ proteins in BEC (Song et al.,2007a). The role of caveolae in BBB functions in health and dis-ease remains to be explored.

Enzymatic BBBEndothelial cells of the BBB provide a metabolic barrier by ex-pressing a number of enzymes that modify endogenous and ex-ogenous molecules, which otherwise could bypass the physicalbarrier and negatively affect neuronal function (Pardridge, 2005).The capillary endothelium, pericytes, and astrocytes express

a variety of ectoenzymes on the plasma membranes, includingaminopeptidases, endopeptidases, cholinesterase, and others.

Passive Transport by the Brain FluidsBrain ISF-CSF ‘‘bulk flow’’ mediates transport of molecules intothe CSF at a slow rate, irrespective of their size (Davson, 1976;Zlokovic, 2005). The CSF acts as a sink for potentially toxicmolecules and metabolic waste products. Toxic molecules andmetabolic waste products are removed from the CSF backinto the circulation by active transport or facilitated diffusionacross the choroid plexus epithelium, or by vacuolar transportacross the epithelial arachnoid granulations.

Neurovascular UnitEndothelium, the site of anatomical BBB, neurons, and non-neuronal cells (e.g., pericytes, astrocytes, andmicroglia) togetherform a functional unit, often referred to as a neurovascular unit(Figure 4A) (Lo et al., 2003; Iadecola, 2004; Hawkins and Davis,2005; Zlokovic, 2005). The close proximity of different nonneuro-nal cell typeswith eachother andwith neurons allows for effectiveparacrine regulations that are critical for normal CNS functioninganddiseaseprocesses (Boillee et al., 2006a;DeaneandZlokovic,2007; Lok et al., 2007). These include regulation of hemodynamicneurovascular coupling, microvascular permeability, matrix in-teractions, neurotransmitter inactivation, neurotrophic coupling,and angiogenic and neurogenic coupling (Figure 4B).Vascular versus Neuronal Origin of Brain DisordersBrain disorders may have a vascular origin (Figure 4A, arrow 1).Vascular cells, i.e., endothelium and pericytes, can directly affectneuronal and synaptic functions through changes in the bloodflow, the BBB permeability, nutrient supply, faulty clearance oftoxicmolecules, failure of enzymatic functions, the altered secre-tion of trophic factors and matrix molecules, abnormal expres-sion of vascular receptors, or induction of ectoenzymes.

Figure 4. Schematic of the Neurovascular Unit(A) Endothelial cells and pericytes are separated by the basement membrane. Pericyte processes sheathe most of the outer side of the basement membrane. Atpoints of contact, pericytes communicate directly with endothelial cells through the synapse-like peg-socket contacts. Astrocytic endfoot processes unsheathethe microvessel wall, which is made up of endothelial cells and pericytes. Resting microglia have a ‘‘ramified’’ shape. In cases of neuronal disorders that havea primary vascular origin, circulating neurotoxinsmay cross the BBB to reach their neuronal targets, or proinflammatory signals from the vascular cells or reducedcapillary blood flow may disrupt normal synaptic transmission and trigger neuronal injury (arrow 1). Microglia recruited from the blood or within the brain and thevessel wall can sense signals from neurons (arrow 2). Activated endothelium, microglia, and astrocytes signal back to neurons, which in most cases aggravatesthe neuronal injury (arrow 3). In the case of a primary neuronal disorder, signals from neurons are sent to the vascular cells and microglia (arrow 2), which activatethe vasculo-glial unit and contributes to the progression of the disease (arrow 3).(B) Coordinated regulation of normal neurovascular functions depends on the vascular cells (endothelium and pericytes), neurons, and astrocytes.

Neuron 57, January 24, 2008 ª2008 Elsevier Inc. 185

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Neuron. 2011. 71(3)Quaegebeur A, Lange C, Carmeliet P.


http://www.ncbi.nlm.nih.gov/pubmed/21835339#

http://www.ncbi.nlm.nih.gov/pubmed/21835339#

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Quaegebeur%20A%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Quaegebeur%20A%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lange%20C%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lange%20C%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Carmeliet%20P%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Carmeliet%20P%22%5BAuthor%5D

Sprout induction.

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“Angioneurins can be broadly defined asmolecules that affect both neural and

vascular cell functions; these effects mightinclude, but are not restricted to, the

regulation of angiogenesis, blood–brainbarrier (BBB) integrity, vascular perfusion,neuroprotection, neuroregeneration and

synaptic plasticity”

Angio(glio?)neurins

Zacchigna et al., Nature Reviews Neuroscience. 2008


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I. Inductor de:• Proliferación endotelial• Migración endotelial• Inhibición apotosis

II. Efectos neurotróficos y neuroprotectores

III. Permeabilidad vascular

VEGFVascular Endothelial Growth Factor


VEGFVascular Endothelial Growth Factor


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Mechanism of BOLD Functional MRIBrain activity

Oxygen consumption Cerebral blood flow

OxyhemoglobinDeoxyhemoglobin

Magnetic susceptibility

T2*

MRI signal intesity


Oxyhemoglobin and Deoxyhemoglobin in Veins during

Brain Activation

Oxyhemoglobin Deoxyhemoglobin

Rest Activation

Normal blood flow High blood flow


Time Series and Activation Maps

Off Off Off OffOn On On On

Scan Number

Sign

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Multi-Slice Spiral Images


Multi-Slice EPI Images


Activation Maps on Anatomical Images

MSSpiral

MSEPI

3DSpiral


Visual Activation Maps (ISI=12s)


New software that makes functional planning easier and faster

BrainLAB provides a significant leap in neurosurgical planning withthe integration of B OLD MRI (Blood Oxygen Level Dependentfunctional MRI) data directly into the OR and image-guided sur-gery. This iPlan module allows anatomical images to be enhancedwith functional maps showing perceptual, motoric and cognitiveareas, leading to more refined surgical approaches and improvedpatient treatments.

B OLD MRI imaging can be fused with other imaging modalitiesand overlaid onto other anatomical or functional images. The dis-play and selection of designated functional areas together withother objects like tumors is made easy with an intuitive user inter-face and advanced image processing.

• Automated import of DIC OM B OLD MRI data • Automatic detection of functional activations• Interactive selection and display of functional areas

and regions of interest• Flexible definition of different functional paradigms• Conversion into 3D objects for use in navigation

iPlan® BOLD MRI Mapping

All images courtesy of Johann Wolfgang Goethe University Hospital,Frankfurt, Germany

accelerated functional planning


©2006 BrainLAB AG . Printed in Germany. NS-FL-E-iPlan Bold Rev.1 0406 Q:3000 ® registered trademark of BrainLAB AG in

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BrainLAB is the only company to offer an approved combination

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MORE COMPREHENSIVE PLANNING WITH FUNCTIONAL

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Functional MRI plays an important role in surgical planning for pro-

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out the functional neuroanatomy of other brain systems devoted tomemory, attention and object recognition, and these functions will

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FMRI images, when fused with high resolution anatomical scans,show the proximity of important functioning cortical regions to

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of cortical stimulation by limiting the stimulation attempts required

to identify function. Furthermore, in those cases for which stimula-

tion mapping is not feasible, fMRI provides valuable information

regarding localization of critical functions and contributes toreduced morbidity.


Neural correlates of admiration and compassion. Immordino-Yang MH, McColl A, Damasio H, Damasio A. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8021-6.


http://www.ncbi.nlm.nih.gov/pubmed/19414310

http://www.ncbi.nlm.nih.gov/pubmed/19414310

Vascularización cerebral (parte I)

Health & Medicine

Transcript of Vascularización cerebral (parte I)