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Section 1Chapter

1Etiology, pathophysiology and imaging

Neuropathologyand pathophysiology of strokeKonstantin A. Hossmann and Wolf-Dieter Heiss

The vascular origin ofcerebrovascular diseaseAll cerebrovascular diseases (CVD) have their originin the vessels supplying or draining the brain.Therefore, knowledge of pathological changes occur-ring in the vessels and in the blood is essential forunderstanding the pathophysiology of the varioustypes of CVD and for the planning of efficient thera-peutic strategies. Changes in the vessel wall lead toobstruction of blood flow, by interacting with bloodconstituents they may cause thrombosis and blockadeof blood flow in this vessel. In addition to vascularstenosis or occlusion at the site of vascular changes,disruption of blood supply and consecutive infarctscan also be produced by emboli arising from vascularlesions situated proximally to otherwise healthybranches located more distal in the arterial tree orfrom a source located in the heart. At the site ofocclusion, the opportunity exists for thrombus todevelop in anterograde fashion throughout the lengthof the vessel, but this event seems to occur only rarely.

Changes in large arteries supplying the brain,including the aorta, are mainly caused by athero-sclerosis. Middle-sized and intracerebral arteries canalso be affected by acute or chronic vascular diseasesof inflammatory origin due to subacute to chronicinfections, e.g. tuberculosis and lues, or due to colla-gen disorders, e.g. giant cell arteriitis, granulomatousangiitis of the CNS, panarteritis nodosa, and evenmore rarely systemic lupus erythematosus, Takayasusarteriitis, Wegener granulomatosis, rheumatoid arterii-tis, Sjgrens syndrome, or Sneddon and Behcetsdisease. In some diseases affecting the vessels of thebrain the etiology and pathogenesis are still unclear,e.g. moyamoya disease and fibromuscular dysplasia,but these disorders are characterized by typicallocations of the vascular changes. Some arteriopathiesare hereditary, such as CADASIL (cerebral autosomal

dominant arteriopathy with subcortical infarcts andleukoencephalopathy), and in some such as cerebralamyloid angiopathy a degenerative cause has beensuggested. All these vascular disorders can causeobstruction, and lead to thrombosis and emboliza-tions. Small vessels of the brain are affected byhyalinosis and fibrosis; this small-vessel diseasecan cause lacunes and, if widespread, is the substratefor vascular cognitive impairment and vasculardementia.

Atherosclerosis is the most widespread disorderleading to death and serious morbidity includingstroke. The basic pathological lesion is the atheroma-tous plaque, and the most commonly affected sites arethe aorta, the coronary arteries, the carotid artery atits bifurcation, and the basilar artery. Arteriosclerosis,a more generic term describing hardening andthickening of the arteries, includes as an additionaltype Mnkebergs sclerosis and is characterized bycalcification in the tunica media and arteriolosclerosiswith proliferative and hyaline changes affecting thearterioles. Atherosclerosis starts at a young age, andlesions accumulate and grow throughout life andbecome symptomatic and clinically evident whenend organs are affected [1].

Atherosclerosis: atheromatous plaques, most com-monly in the aorta, the coronary arteries, the bifur-cation of the carotid artery and the basilar artery.

The initial lesion of atherosclerosis has beenattributed to the fatty streaks and the intimal cellmass. Those changes occur in childhood and adoles-cence and do not necessarily correspond to the futuresites of atherosclerotic plaques. Fatty streaks are focalareas of intracellular lipid collection in both macro-phages and smooth muscle cells. Various conceptshave been proposed to explain the progression of suchprecursor lesions to definite atherosclerosis [1, 2], themost remarkable of which is the response-to-injury 1

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hypothesis postulating a cellular and molecularresponse to various atherogenic stimuli in the formof an inflammatory repair process [3]. This inflam-mation develops concurrently with the accumulationof minimally oxidized low-density lipoproteins [4, 5],and stimulates vascular smooth muscle cells(VSMCs), endothelial cells and macrophages, and asa result foam cells aggregate with an accumulation ofoxidized LDL. In the further stages of atheroscleroticplaque development VSMCs migrate, proliferate, andsynthesize extracellular matrix components on theluminal side of the vessel wall, forming the fibrouscap of the atherosclerotic lesion [6]. In this complexprocess of growth, progression and finally rupture ofan atherosclerotic plaque a large number of matrixmodulators, inflammatory mediators, growth factorsand vasoactive substances are involved. The complexinteractions of these many factors are discussed in thespecialist literature [46]. This fibrous cap covers thedeep lipid core with a massive accumulation of extra-cellular lipids (atheromatous plaque) or fibroblasts andextracellular calcifications may contribute to a fibrocal-cific lesion. Mediators from inflammatory cells at thethinnest portion of the cap surface of a vulnerableplaque which is characterized by a larger lipid coreand a thin fibrous cap can lead to plaque disruptionwith formation of a thrombus or hematoma or even tototal occlusion of the vessel. During the development ofatherosclerosis the entire vessel can enlarge or constrictin size [7]. However, once the plaque enlarges to>40%

of the vessel area, the artery no longer enlarges, and thelumen narrows as the plaque grows. In vulnerableplaques thrombosis forming on the disrupted lesionfurther narrows the vessel lumen and can lead to occlu-sion or be the origin of emboli. Less commonly, plaqueshave reduced collagen and elastin with a thin andweakened arterial wall, resulting in aneurysm forma-tion which when ruptured may be the source of intra-cerebral hemorrhage (Figure 1.1).

Injury hypothesis of progression to atherosclerosis:fatty streaks (focal areas of intra-cellular lipid collec-tion)! inflammatory repair processwith stimulationof vascular smooth muscle cells ! atheromatousplaque.

ThromboembolismImmediately after plaque rupture or erosion, sub-endothelial collagen, the lipid core and procoagulantssuch as tissue factor and von Willebrand factor areexposed to circulating blood. Platelets rapidly adhereto the vessel wall through the platelet glycoproteins(GP) Ia/IIa and GP Ib/IX [8] with subsequent aggre-gation to this initial monolayer through linkage withfibrinogen and the exposed GP IIb/IIIa on activatedplatelets. As platelets are a source of nitrous oxide(NO), the resulting deficiency of bioactive NO, whichis an effective vasodilator, contributes to the progres-sion of thrombosis by augmenting platelet activation,

Figure 1.1. The stages of development of an atherosclerotic plaque. (1) LDL moves into the subendothelium and (2) is oxidized bymacrophages and smooth muscle cells (SMC). (3) Release of growth factors and cytokines (4) attracts additional monocytes. (5) Macrophagesand (6) foam cell accumulation and additional (7) SMC proliferation result in (8) growth of the plaque. (9) Fibrous cap degradation and plaquerupture (collagenases, elastases). (10) Thrombus formation. (Modified from Faxon et al. [5].)

Section 1: Etiology, pathophysiology and imaging

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enhancing VSMC proliferation and migration, andparticipating in neovascularization [9]. The activatedplatelets also release adenosine diphosphate (ADP)and thromboxane A2 with subsequent activation ofthe clotting cascade. The growing thrombus obstructsor even blocks the blood flow in the vessel. Athero-sclerotic thrombi are also the source of embolisms,which are the primary pathophysiological mechan-isms of ischemic strokes, especially from carotidartery disease or of cardiac origin.

Rupture or erosion of atheromatous plaques !adhesion of platelets ! thrombus ! obstructionof blood flow and source of emboli.

Small-vessel disease usually affects the arteriolesand is associated with hypertension. It is caused bysubendothelial accumulation of a pathological pro-tein, the hyaline, formed from mucopolysaccharidesand matrix proteins. It leads to narrowing of thelumen or even occlusion of these small vessels. Oftenit is associated with fibrosis, which affects not onlyarterioles, but also other small vessels and capillariesand venules. Lipohyalinosis also weakens the vesselwall, predisposing it to the formation of miliaryaneurysms. Small-vessel disease results in two patho-logical conditions: status lacunaris (lacunar state) andstatus cribrosus (state cribl). Status lacunaris is char-acterized by small irregularly shaped infarcts due toocclusion of small vessels; it is the pathological sub-strate of lacunar strokes and vascular cognitiveimpairment and dementia. In status cribrosus smallround cavities develop around affected arteries due todisturbed supply of oxygen and metabolic substrate.These criblures together with miliary aneurysms arethe sites of vessel rupture causing typical hypertonicintracerebral hemorrhages [1013]. The etiologyand pathophysiology of the various specific vasculardisorders are discussed in specialist articles andhandbooks [14].

Small-vessel disease: subendothelial accumulationof hyaline in arterioles.

Types of acute cerebrovasculardiseasesNumbers relating to the frequency of the differenttypes of acute CVD are highly variable dependingon the source of data. The most reliable numberscome from the in-hospital assessment of stroke in

the Framingham study determining the frequencyof complete stroke: 60% were caused by athero-thrombotic brain infarction, 25.1% by cerebralemboli, 5.4% by subarachnoid hemorrhage, 8.3%by intracerebral hemorrhage and 1.2% by undefineddiseases. In addition, isolated transient ischemicattacks (TIAs) accounted for 14.8% of the total cere-brovascular events [15].

Ischemic strokes are caused by a critical reductionof regional cerebral blood flow and, if the criticalblood flow reduction lasts beyond a critical duration,they are caused by atherothrombotic changes of thearteries supplying the brain or by emboli fromsources in the heart, the aorta or the large arteries.The pathological substrate of ischemic stroke is ische-mic infarction of brain tissue; the location, extensionand shape of these infarcts depend on the size of theoccluded vessel, the mechanism of arterial obstruc-tion and the compensatory capacity of the vascularbed. Occlusion of arteries supplying defined brainterritories by atherothrombosis or embolizationsleads to territorial infarcts of variable size: they mightbe large e.g. the whole territory supplied by themiddle cerebral artery (MCA) or small, if branchesof large arteries are occluded or if compensatorycollateral perfusion e.g. via the circle of Willis orleptomeningeal anastomoses is efficient in reducingthe area of critically reduced flow [12, 13] (Figure 1.2).In a smaller number of cases infarcts can also developat the borderzones between vascular territories, whenseveral large arteries are stenotic and the perfusion inthese last meadows cannot be maintained abovethe critical threshold during special exertion [16].Borderzone infarctions are a subtype of the low-flowor hemodynamically induced infarctions which arethe result of critically reduced cerebral perfusionpressure in far-downstream brain arteries that leadsto a reduced cerebral blood flow and oxygen supply incertain vulnerable brain areas. Borderzone infarcts arelocated in cortical areas between the territories ofmajor cerebral arteries; the more common low-flowinfarctions affect subcortical structures within a vas-cular bed but with marginal irrigation [17]. Lacunarinfarcts reflect disease of the vessels penetrating thebrain to supply the capsule, the basal ganglia, thal-amus and paramedian regions of the brain stem [18].Most often they are caused by lipohyalinosis of deeparteries (small-vessel disease); less frequent causes arestenosis of the MCA stem and microembolization topenetrant arterial territories. Pathologically these

Chapter 1: Neuropathology and pathophysiology of stroke

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lacunes are defined as small cystic trabeculated scarsabout 5mm in diameter, but they are more oftenobserved on magnetic resonance images, where theyare accepted as lacunes up to 1.5 cm diameter. Theclassic lacunar syndromes include pure motor, puresensory, and sensorimotor syndromes, sometimesataxic hemiparesis, clumsy hand, dysarthria andhemichorea/hemiballism, but higher cerebral func-tions are not involved.

Territorial infarcts are caused by an occlusion ofarteries supplying defined brain territories by athero-thrombosis or embolizations.

Borderzone infarcts develop at the borderzonebetween vascular territories and are the result of acritically reduced cerebral perfusion pressure (lowflow infarctions).

Lacunar infarcts are mainly caused by small-vessel disease.

Hemorrhagic infarctions, i.e. red infarcts in con-trast to the usual pale infarcts, are defined as ische-mic infarcts in which varying amounts of blood cellsare found within the necrotic tissue. The amount canrange from a few petechial bleeds in the gray matterof cortex and basal ganglia to large hemorrhagesinvolving the cortical and deep hemispheric regions.Hemorrhagic transformation frequently appears

during the second and third phase of infarct evolution,when macrophages appear and new blood vessels areformed in tissue consisting of neuronal ghosts andproliferating astrocytes. However, the only significantdifference between pale and red infarcts is theintensity and extension of the hemorrhagic compo-nent, since in at least two-thirds of all infarcts petechialhemorrhages are microscopically detectable. Macro-scopically red infarcts contain multifocal bleedingswhich are more or less confluent and predominate incerebral cortex and basal ganglia which are richerin capillaries than the white matter [19]. If thehemorrhages become confluent intrainfarct hematomasmight develop, and extensive edema may contributeto mass effects and lead to malignant infarction. Thefrequency of hemorrhagic infarctions (HIs) in anatomicstudies ranged from 18 to 42% [20], with a high inci-dence (up to 85% of HIs) in cardioembolic stroke [21].

Mechanisms for hemorrhagic transformation aremanifold and vary with regard to the intensity ofbleeding. Petechial bleeding results from diapedesisrather than vascular rupture. In severe ischemic tissuevascular permeability is increased and endothelialtight junctions are ruptured. When blood circulationis spontaneously or therapeutically restored, bloodcan leak out of these damaged vessels. This can alsohappen with fragmentation and distal migration of an

a b c d eFigure 1.2. Various types and sizes of infarcts due to different hemodynamic patterns. (a) Total territorial infarct due to defective collateralsupply. (b) Core infarct, meningeal anastomosis supply peripheral zones. (c) Territorial infarct in center of supply area, due to branch occlusion.(d) Borderzone infarction in watershed areas due to stenotic lesions in arteries supplying neighboring areas. (e) Lacunar infarctions due tosmall-vessel disease. (Modified from Zlch [13].)


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embolus (usually of cardiac origin) in the damagedvascular bed, explaining delayed clinical worsening insome cases. For the hemorrhagic transformation thecollateral circulation might also have an impact: insome instances reperfusion via pial networks maydevelop with the diminution of peri-ischemic edemaat borderzones of cortical infarcts. Risk of hemor-rhage is significantly increased in large infarcts, withmass effect supporting the importance of edemafor tissue damage and the deleterious effect of latereperfusion when edema resolves. In some instancesalso the rupture of the vascular wall secondary toischemia-induced endothelial necrosis might causean intrainfarct hematoma. Vascular rupture canexplain very early hemorrhagic infarcts and earlyintrainfarct hematoma (between 6 and 18 hours afterstroke), whereas hemorrhagic transformation usuallydevelops within 48 hours to 2 weeks.

Hemorrhagic infarctions (HI) are defined as ische-mic infarcts in which varying amounts of bloodcells are found within the necrotic tissue. They arecaused by leakage from damaged vessels, due toincreased vascular permeability in ischemic tissueor vascular rupture secondary to ischemia.

Intracerebral hemorrhage (ICH) occurs as a resultof bleeding from an arterial source directly into thebrain parenchyma and accounts for 515% of allstrokes [22, 23]. Hypertension is the leading riskfactor, but in addition old age and race, and alsocigarette smoking, alcohol consumption and highserum cholesterol levels, have been identified. In anumber of instances ICH occurs in the absence ofhypertension, usually in atypical locations. The causesinclude small vascular malformations, vasculitis,brain tumors and sympathomimetic drugs (e.g.cocaine). ICH may also be caused by cerebral amyloidangiopathy and rarely damage is elicited by acutechanges in blood pressure, e.g. due to exposure tocold. The occurrence of ICH is also influenced bythe increasing use of antithrombotic and throm-bolytic treatment of ischemic diseases of the brain,heart and other organs [24, 25].

Spontaneous ICH occurs predominantly in thedeep portions of the cerebral hemispheres (typicalICH). Its most common location is the putamen(3550% of cases). The subcortical white matter isthe second most frequent location (approx. 30%).Hemorrhages in the thalamus are found in 1015%,in the pons in 512% and in the cerebellum in 7%

[26]. Most ICHs originate from the rupture of small,deep arteries with diameters of 50 to 200 mm,which are affected by lipohyalinosis due to chronichypertension. These small-vessel changes lead toweakening of the vessel wall and miliary micro-aneurysm and consecutive small local bleedings,which might be followed by secondary ruptures ofthe enlarging hematoma in a cascade or avalanchefashion [27]. After active bleeding starts it can con-tinue for a number of hours with enlargement ofhematoma, which is frequently associated with clinicaldeterioration [28].

Putaminal hemorrhages originate from a lateralbranch of the striate arteries at the posterior angle,resulting in an ovoid mass pushing the insular cortexlaterally and displacing or involving the internal cap-sule. From this initial putaminal-claustral location alarge hematoma may extend to the internal capsuleand lateral ventricle, into the corona radiata and intothe temporal white matter. Putaminal ICHs wereconsidered the typical hypertensive hemorrhages.

Caudate hemorrhage, a less common form ofbleeding from distal branches of lateral striate arter-ies, occurs in the head of the caudate nucleus. Thisbleeding soon connects to the ventricle and usuallyinvolves the anterior limb of the internal capsule.

Thalamic hemorrhages can involve most of thisnucleus and extend into the third ventricle mediallyand the posterior limb of the internal capsule laterally.The hematoma my press on or even extend into themidbrain. Larger hematomas often reach the coronaradiata and the parietal white matter.

Lobar (white matter) hemorrhages originate atthe cortico-subcortical junction between gray andwhite matter and spread along the fiber bundlesmost commonly in the parietal and occipital lobes.The hematomas are close to the cortical surface andusually not in direct contact with deep hemispherestructures or the ventricular system. As atypicalICHs they are not necessarily correlated withhypertension.

Cerebellar hemorrhages usually originate in thearea of the dentate nucleus from rupture of distalbranches of the superior cerebellar artery and extendinto the hemispheric white matter and into the fourthventricle. The pontine tegmentum is often com-pressed. A variant, the midline hematoma, originatesfrom the cerebellar vermis, always communicateswith the fourth ventricle and frequently extends bilat-erally into the pontine tegmentum.


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Pontine hemorrhages from bleeding of small para-median basilar perforating branches cause mediallyplaced hematomas involving the basis of the pons.A unilateral variety results from rupture of distal longcircumferential branches of the basilar artery. Thesehematomas usually communicate with the fourth ven-tricle and extend laterally and ventrally into the pons.

The frequency of recurrent of ICHs in hyperten-sive patients is rather low (6%) [29]. The recurrencerate is higher with poor control of hypertension andalso in hemorrhages due to other causes. In someinstances multiple simultaneous ICHs may occur, butalso in these cases the cause is other than hypertension.

In ICHs, the local accumulation of blood destroysthe parenchyma, displaces nervous structures anddissects the tissue. At the bleeding sites fibrin globesare formed around collections of platelets. After hoursor days extracellular edema develops at the peripheryof the hematoma. After 4 to 10 days the red bloodcells begin to lyse, granulocytes and thereafter micro-glial cells arrive and foamy macrophages are formed,which ingest debris and hemosiderin. Finally, theastrocytes at the periphery of the hematoma pro-liferate and turn into gemistocytes with eosinophiliccytoplasm. When the hematoma is removed, theastrocytes are replaced by glial fibrils. After thatperiod extending to months the residue of thehematoma is a flat cavity with a reddish liningresulting from hemosiderin-laden macrophages [26].

Intracerebral hemorrhage (ICH) occurs as a result ofbleeding from an arterial source directly into thebrain parenchyma, predominantly in the deep por-tions of the cerebral hemispheres (typical ICH).Hypertension is the leading risk factor, and themost common location is the putamen.

Cerebral venous thrombosisThrombi of the cerebral veins and sinuses can developfrom many causes and because of predisposing con-ditions. Cerebral venous thrombosis (CVT) is oftenmultifactorial, when various risk factors and causescontribute to the development of this disorder [30].The incidence of septic CVT has been reduced toless than 10% of cases, but septic cavernous sinusthrombosis is still a severe, however rare, problem.Aseptic CVT occurs during puerperium and lessfrequently during pregnancy, but may also be relatedto use of oral contraceptives. Among the non-infection causes of CVT are congenital thrombophilia,

particularly prothrombin gene and factor V Leidenmutations, and prothrombin mutation, as well asantithrombin, protein C and protein S deficienciesmust be considered. Other conditions with risk forCVT are malignancies, inflammatory diseases andsystemic lupus erythematodes. However, in 2035%of CVT the etiology remains unknown.

The fresh venous thrombus is rich in red bloodcells and fibrin and poor in platelets. Later on, it isreplaced by fibrous tissue, occasionally with recanali-zation. The most common location of CVT is thesuperior sagittal sinus and the tributary veins.

Whereas some thromboses, particularly of thelateral sinus, may have no pathological consequencesfor the brain tissue, occlusion of cerebral veins usuallyleads to a venous infarct. These infarcts are located inthe cortex and adjacent white matter and often arehemorrhagic. Thrombosis of the superior sagittal sinusmight lead only to brain edema, but usually causesbilateral hemorrhagic infarcts in both hemispheres.These venous infarcts are different from arterialinfarcts: cytotoxic edema is absent or mild, vasogenicedema is prominent, and hemorrhagic transformationor bleeding is usual. Despite this hemorrhagiccomponent heparin is the treatment of choice.

Cerebral venous thrombosis can lead to a venousinfarct. Venous infarcts are different from arterialinfarcts: cytotoxic edema is absent or mild, vaso-genic edema is prominent, and hemorrhagic trans-formation or bleeding is usual.

Cellular pathology of ischemic strokeAcute occlusion of a major brain artery causes astereotyped sequel of morphological alterations whichevolve over a protracted period and which dependon the topography, severity and duration of ischemia[31, 32]. The most sensitive brain cells are neurons,followed in this order by oligodendrocytes, astro-cytes and vascular cells. The most vulnerable brainregions are hippocampal subfield CA1, neocorticallayers 3, 5 and 6, the outer segment of striate nucleus,and the Purkinje and basket cell layers of cerebellarcortex. If blood flow decreases below the threshold ofenergy metabolism, the primary pathology is necrosisof all cell elements, resulting in ischemic brain infarct.If ischemia is not severe enough to cause primaryenergy failure, or if it is of so short duration thatenergy metabolism recovers after reperfusion, adelayed type of cell death may evolve which exhibits


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the morphological characteristics of necrosis, apop-tosis or a combination of both. In the following,primary and delayed cell death will be describedseparately.

Cellular pathology of ischemic strokePrimary ischemic cell deathIn the core of the territory of an occluded brain arterythe earliest sign of cellular injury is neuronal swellingor shrinkage, the cytoplasm exhibiting microvacuolat-ion (MV), which ultrastructurally has been associatedwith mitochondrial swelling [33]. These changesare potentially reversible if blood flow is restoredbefore mitochondrial membranes begin to rupture.One to two hours after the onset of ischemia, neuronsundergo irreversible necrotic changes (red neuron or

ischemic cell change (ICC)), characterized by con-densed acidophilic cytoplasm, formation of triangularnuclear pyknosis and direct contact with swollenastrocytes. Electronmicroscopically mitochondriaexhibit flocculent densities which represent denatu-rated mitochondrial proteins. After 24 hours, ische-mic cell change with incrustrations appears, whichhas been associated with formaldehyde pigments de-posited after fixation in the perikaryon. Ischemic cellchange must be distinguished from artifactual darkneurons which stain with all (acid or base) dyes andare not surrounded by swollen astrocytes (Figure 1.3).

With ongoing ischemia, neurons gradually losetheir stainability with hematoxylin; they becomemildly eosinophilic and, within 4 days, transform intoghost cells with a hardly detectable pale outline. Inter-estingly, neurons with ischemic cell change are mainly

Light microscopical characteristics of rat brain infarction

ControlAcute ischemic changes

Necrotic changes

sham surgery

red neuron ghost neuron

4 hours 2 hours

Dark neuron artifact

1 day 3 days sham surgery

swelling shrinkage

Figure 1.3. Light-microscopicalevolution of neuronal changes afterexperimental middle cerebral occlusion.(Modified from Garcia et al. [94].)


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located in the periphery and ghost cells in the centerof the ischemic territory, which suggests that mani-festation of ischemic cell change requires someresidual or restored blood flow, whereas ghost cellsmay evolve in the absence of flow [32].

Primary ischemic cell death induced by focalischemia is associated with reactive and secondarychanges. The most notable alteration during the ini-tial 12 hours is perivascular and perineuronal astro-cytic swelling; after 46 hours the bloodbrain barrierbreaks down, resulting in the formation of vasogenicedema; after 12 days inflammatory cells accumulatethroughout the ischemic infarct, and within 1.5 to3 months cystic transformation of the necrotic tissueoccurs together with the development of a peri-infarctastroglial scar.

Delayed neuronal deathThe prototype of delayed cell death is the slowlyprogressing injury of pyramidal neurons in the CA1

sector of the hippocampus after a brief episode ofglobal ischemia [34]. In focal ischemia delayed neur-onal death may occur in the periphery of corticalinfarcts or in regions which have been reperfusedbefore ischemic energy failure becomes irreversible.Cell death is also observed in distant brain regions,notably in the substantia nigra and thalamus.

The morphological appearance of neurons duringthe interval between ischemia and cell death exhibits acontinuum that ranges from necrosis to apoptosiswith all possible combinations of cytoplasmic andnuclear morphology that are characteristic of thetwo types of cell death [35]. In its pure form, necrosiscombines karyorrhexis with massive swelling of endo-plasmic reticulum and mitochondria, whereas inapoptosis mitochondria remain intact and nuclearfragmentation with condensation of nuclear chroma-tin gives way to the development of apoptotic bodies(Figure 1.4). A frequently used histochemicalmethod for the visualization of apoptosis is terminaldeoxyribonucleotidyl transferase (TdT)-mediated

Inflammation and cavitation of ischemic infarctionNecrotic neurons and PMN leukocytesNecrotic neurons, ghosts and PMN leukocytes

Cavitation with sparing of outer cortical layerLipid-laden macrophages and necrosis1.5 days 1.5 days

subacute infarct cystic infarct

Figure 1.4. Transformation of acute ischemic alterations into cystic infarct. Note pronounced inflammatory reaction prior to tissue cavitation.(Modified from Petito [32].)


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biotin-16-dUTP nick-end labeling (TUNEL assay),which detects DNA strand breaks. However, as thismethod may also stain necrotic neurons, a clear dif-ferentiation is not possible [36].

A consistent ultrastructural finding in neuronsundergoing delayed cell death is disaggregation ofribosomes, which reflects the inhibition of proteinsynthesis at the initiation step of translation [37].Light-microscopically, this change is equivalent totigrolysis, visible in Nissl-stained material. Disturb-ances of protein synthesis and the associated endo-plasmic reticulum stress are also responsiblefor cytosolic protein aggregation and the formationof stress granules [38]. In the hippocampus, stacksof accumulated endoplasmic reticulum may becomevisible but in other areas this is not a prominentfinding.

Severe ischemia induces primary cell death due tonecrosis of all cell elements.

Not so severe or short-term ischemia inducesdelayed cell death with necrosis, apoptosis or acombination of both.

Pathophysiology of strokeAnimal models of focal ischemiaAccording to the Framingham study, 65% of strokesthat result from vascular occlusion present lesions inthe territory of the middle cerebral artery, 2% in theanterior and 9% in the posterior cerebral artery terri-tories; the rest are located in brainstem or cerebellum,or in watershed or multiple regions. In experimentalstroke research, this situation is reflected by thepreferential use of middle cerebral artery occlusionmodels.

Transorbital middle cerebral artery occlusion:this model was introduced in the seventies forthe production of stroke in monkeys [39], andlater modified for use in cats, dogs, rabbits and evenrats. The procedure is technically demanding andrequires microsurgical skills. The advantage of thisapproach is the possibility of exposing the middlecerebral artery at its origin from the internal carotidartery without retracting parts of the brain. Vascularocclusion can thus be performed without the riskof brain trauma. On the other hand, removal of theeyeball is invasive and may evoke functional disturb-ances which should not be ignored. Surgery may also

cause generalized vasospasm which may interferewith the collateral circulation and, hence, inducevariations in infarct size. The procedure thereforerequires extensive training before reproducible resultscan be expected.

The occlusion of the middle cerebral artery at itsorigin interrupts blood flow to the total vascularterritory, including the basal ganglia which are sup-plied by the lenticulo-striate arteries. These MCAbranches are end-arteries which, in contrast to thecortical branches, do not form collaterals with theadjacent vascular territories. As a consequence, thebasal ganglia are consistently part of the infarct corewhereas the cerebral cortex exhibits a gradient ofblood flow which decreases from the peripheraltowards the central parts of the vascular territory.Depending on the steepness of this gradient, a cor-tical core region with the lowest flow values in thelower temporal cortex is surrounded by a variablysized penumbra which may extend up to the para-sagittal cortex.

Transcranial occlusion of the middle cerebralartery: post- or retro-orbital transcranial approachesfor middle cerebral artery occlusion are mainly usedin rats and mice because in these species the main stemof the artery appears on the cortical surface rather closeto its origin from the internal carotid artery [40]. Incontrast to transorbital middle cerebral artery occlu-sion, transcranial models do not produce ischemicinjury in the basal ganglia because the lenticulo-striatebranches originate proximal to the occlusion site.Infarcts, therefore, are mainly located in the tem-poro-parietal cortex with a gradient of decliningflow values from the peripheral to the central partsof the vascular territory.

Filament occlusion of the middle cerebral artery:the currently most widely used procedure formiddle cerebral artery occlusion in rats and miceis the intraluminal filament occlusion technique,first described by Koizumi et al. [41]. A nylonsuture with an acryl-thickened tip is inserted intothe common carotid artery and orthogradelyadvanced, until the tip is located at the origin ofthe middle cerebral artery. Modifications of theoriginal technique include different thread typesfor isolated or combined vascular occlusion, adjust-ments of the tip size to the weight of the animal,poly-L-lysine coating of the tip to prevent incom-plete middle cerebral artery occlusion, or the use ofguide-sheaths to allow remote manipulation of the


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thread for occlusion during polygraphic or mag-netic resonance recordings.

The placement of the suture at the origin ofthe middle cerebral artery obstructs blood supplyto the whole MCA-supplied territory, includingthe basal ganglia. It may also reduce blood flow inthe anterior and posterior cerebral arteries, particu-larly when the common carotid artery is ligated tofacilitate the insertion of the thread. As this minim-izes collateral blood supply from these territories,infarcts are very large and produce massive ischemicbrain edema with a high mortality when experimentslast for more than a few hours. For this reason,threads are frequently withdrawn 12 hours fol-lowing insertion. The resulting reperfusion salvagesthe peripheral parts of the MCA territory, andinfarcts become smaller [42]. However, the patho-physiology of transient MCA occlusion differs basic-ally from that of the clinically more relevantpermanent occlusion models, and neither themechanisms of infarct evolution nor the pharmaco-logical responsiveness of the resulting lesions arecomparable.

Transient filament occlusion is also an inappro-priate model for the investigation of spontaneous orthrombolysis-induced reperfusion. Withdrawal of theintraluminal thread induces instantaneous reperfu-sion whereas spontaneous or thrombolysis-inducedrecanalization results in slowly progressing recircula-tion. As post-ischemic recovery is greatly influencedby the dynamics of reperfusion, outcome andpharmacological responsiveness of transient filamentocclusion is distinct from most clinical situations ofreversible ischemia where the onset of ischemia ismuch less abrupt.

Clot embolism of middle cerebral artery:middle cerebral artery embolism with autologousblood clots is a clinically highly relevant but alsoinherently variable stroke model which requirescareful preparation and placement of standardizedclots to induce reproducible brain infarcts [43].The most reliable procedure for clot preparationis thrombin-induced clotting, which results in cylin-drical clots that can be dissected into segments ofequal length. Selection of either fibrin-rich (white)or fibrin-poor (red) segments influences the speedof spontaneous reperfusion and results in differentoutcomes. Clots can also be produced in situ bymicroinjection of thrombin [44] or photochemically

by UV illumination of the middle cerebral arteryfollowing injection of rose Bengal [45].

The main application of clot embolism is for theinvestigation of experimental thrombolysis. Thedrug most widely used is human recombinant tissueplasminogen activator (rt-PA) but the dose requiredin animals is much higher than in humans, whichmust be remembered when possible side effectssuch as t-PA toxicity are investigated. The hemody-namic effect, in contrast, is similar despite thehigher dose and adequately reproduces the slowlyprogressing recanalization observed under clinicalconditions.

Various procedures for artery occlusion models,mostly middle cerebral artery occlusion models,were developed to study focal ischemia in animals.

Regulation of blood flowIn the intact brain, cerebral blood flow is tightlycoupled to the metabolic requirements of tissue(metabolic regulation) but the flow rate remainsessentially constant despite alterations in bloodpressure (autoregulation). An important require-ment for metabolic regulation is the CO2 reactivityof cerebral vessels, which can be tested by theapplication of carbonic anhydrase inhibitors orCO2 ventilation. Under physiological conditions,blood flow doubles when CO2 rises by about30mmHg and is reduced by approximately 35%when CO2 falls to 25mmHg. The vascular responseto CO2 depends mainly on changes in extracellularpH, but it is also modulated by other factors such asprostanoids, nitric oxide and neurogenic influences.

Autoregulation of cerebral blood flow is theremarkable capacity of the vascular system to adjustits resistance in such a way that blood flow is keptconstant over a wide range of cerebral perfusionpressures (80150mmHg). The range of autoregula-tion is shifted to the right, i.e. to higher values, inpatients with hypertension and to the left duringhypercarbia. The myogenic theory of autoregulationsuggests that changes in vessel diameter are caused bythe direct effect of blood pressure variations on themyogenic tone of vessel walls. Other influences aremediated by metabolic and neurogenic factors butthese may be secondary and are not of greatsignificance.


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acv physiopathology cambridge.pdf