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Brain injury due to ischemia and reperfusion in carotid endarterectomy
(Portuguese PDF version)

Daniela Mazza Sundefeld Tardini¹, Winston Bonetti Yoshida²

1. Master. Resident Physician in Angiology and Vascular Surgery, Instituto de Moléstias Cardiovasculares - São José do Rio Preto - São Paulo.
2. Associate Professor, Department of Surgery and Orthopedics, School of Medicine of Botucatu, Universidade Estadual Paulista.

Correspondence:
Daniela Mazza Sundefeld Tardini
Rua Venezuela, 575/121
CEP 09030-310 - Santo André - SP
E-mail: carlos_tardini@uol.com.br

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ABSTRACT

Some authors have suggested that carotid endarterectomy could cause brain injury related to ischemia and reperfusion episodes and to postoperative hyperperfusion syndrome. Several mechanisms, such as free fatty acid and purine metabolites, nitric oxide formation, and leukocyte action, would be involved in the production of these alterations. Brain tissue injury due to ischemia and reperfusion has been demonstrated in clinical and experimental studies. Experimental studies have also studied the pathophysiology of the brain injuries and sought adequate treatment. Therefore, these studies are useful and important, but the great variety of experimental models used and the diversity of results achieved reflect the need for a simple, reproducible and consistent model for brain ischemia and reperfusion in order to test treatments that can minimize the damage caused by this kind of surgical intervention.

Key words: carotid endarterectomy, brain ischemia, reperfusion
Palavras-chave: endarterectomia das carótidas, isquemia cerebral, reperfusão.

J Vasc Br 2003;2(2):119-28

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Diseases affecting the carotid and vertebral arteries frequently cause neurological disorders. In 90% of cases, these vascular injuries are of atherosclerotic origin. Stenosis of the arterial lumen most commonly affects the region of the carotid bifurcation (33 - 34%), followed by the origin of the vertebral arteries (18 - 22.3%). Occlusions are most common in the proximal internal carotid artery.¹ It should be noted that certain complications of the atherosclerotic plaque, such as the presence of ulcers and/or intraplaque hemorrhage, may result in plaque or plaque particle embolization when it suffers rupture, and are significant in the clinical progress of the disease.^1-3

Less frequently, other diseases may affect these arteries, including fibromuscular dysplasia, extrinsic compression, intimal dissection, kinking and inflammatory angiopathies.² Disorders of the intracranial vessels are most commonly caused by amyloidosis, fibrinoid necrosis and giant cell arteritis.¹

In the carotid region, the symptoms vary according to the degree of injury, being typically transitory ischemic attack (TIA), amaurosis fugax, headache and retinal ischemia. Damage to the vertebral area leads to dizziness, vertigo and loss of balance or to abnormalities of the cranial nerves.^1,2,4

After a TIA episode, the patient will have around 10% risk of ischemic stroke in the first year, increasing by 6% per year to the third year of post TIA follow-up. It is also important to note that asymptomatic patients with carotid stenosis of 75% or more have 3 to 5% risk of ischemic stroke per year of follow-up.^1,2 Ischemic stroke is the most feared clinical outcome and may lead to irreversible consequences including death.

Carotid atherosclerosis is thus a serious disease and should be treated accordingly. One of the alternatives for relief of symptoms and especially for ischemic stroke prevention is surgical treatment through carotid endarterectomy. Surgery should be accompanied by the use of platelet antiaggregant drugs.

INDICATIONS FOR CAROTID ENDARTERECTONY

The indications for surgical treatment have received greater consensus as a result of prospective and multicenter studies. The NASCET⁵ study was carried out in medical centers in the USA and Canada in 1991, relating atherosclerotic carotid stenosis in symptomatic patients to the risk of new symptoms. Among patients with stenoses between 70 and 99% as diagnosed by arteriography, cumulative rate of ipsilateral ischemic stroke was 26% for those undergoing only clinical treatment, compared to 9% among those who underwent surgery. On the basis of these data, the authors concluded that carotid endarterectomy was beneficial for symptomatic patients with stenoses over 70%.

In the same year, the European Carotid Trial⁶ also evaluated symptomatic patients and showed that, in three years of follow-up, the patients with stenoses between 70 and 99% who underwent surgery presented rates of ipsilateral ischemic stroke of 2.8%, compared to 16.8% for those receiving only clinical treatment.

The large, multicenter ACAS^7-9 study was carried out in the USA and Canada between 1987 and 1993, involving patients with asymptomatic carotid injuries. This study selected only patients with stenoses greater than 60% in at least one carotid artery in the region of the carotid bifurcation, and was restricted to qualified medical centers with less than 3% complication rates for surgery and diagnostic arteriography.⁸ Incidence of ipsilateral ischemic stroke was 5.1% in operated patients and 11% in patients receiving only clinical treatment with platelet antiaggregant drugs.⁹ These results were not reproduced in female patients, because the ACAS study found a complication rate of 3.6% in women, compared to 1.7% in men. In a review study, Goldstein et al.¹⁰ found even higher complication rates in female patients. The review by Rockman et al.,¹¹ however, identified no difference between the sexes under the same conditions.

With the aim of finding better surgical indications for asymptomatic patients with carotid injuries, Hobson et al.¹² published the results of the multicenter study of the Veterans Affairs Cooperative Study Group, which evaluated asymptomatic patients with atherosclerotic carotid injuries causing stenosis of greater than 50%. This study found a reduction in the incidence of neurological events in patients who underwent endarterectomy of the compromised carotid, with rates of TIA and ipsilateral ischemic stroke of 2.8% and 4.7%, respectively, compared to 6.4% and 9.4% for patients who were not operated.

In general, these studies show the advantage of carotid endarterectomy over clinical treatment in symptomatic and asymptomatic patients with severe stenosis of the carotid bifurcation.

SURGICAL TECHNIQUE

Differences of opinion exist regarding the use of local or general anesthetic, the use of a shunt and the use of patching during arteriography after resection of the plaque.^1,13 Local anesthetic has the advantage of allowing verbal contact with the patient, with the disadvantage of patient anxiety and its effect on the surgical team.¹³ Patching is recommended in cases of reduced arterial diameter, especially in women, but some authors do not make routine use of it.¹³

The use of a shunt has the aim of maintaining blood for the brain hemisphere corresponding to the carotid under operation during the resection of the atheromatous plaque. A number of surgeons prefer routine use of a shunt, others use it selectively. For the latter, indications for use are: intraoperative electroencephalographic abnormalities, observation and evaluation of clinical behavior of patients under local anesthetic and retrograde pressure in the internal carotid.¹³ In the latter case, shunt use is indicated in cases of pressures lower than 25 mmHg, cited by Moore¹³ as the lowest value acceptable for the safety of the patient. Other authors¹⁴ have shown that non-use of shunt results in increased risk of neurological complications in patients with internal carotid retrograde pressure lower than 50 mmHg and with contralateral carotid occlusion.

ISCHEMIA AND REPERFUSION IN CAROTID ENDARTERECTOMY

With or without a shunt, carotid endarterectomy always involves a variable period of brain ischemia followed by reperfusion. Studies of pre-, intra- and postoperative brain blood flow in carotid endarterectomy have drawn attention to the importance of postoperative hyperperfusion syndrome. This is characterized by the presence of a state of chronic cerebral vasodilatation associated with the loss of cerebrovascular autoregulation after the removal of severe carotid stenosis, leading to the postoperative appearance of brain edema. Clinically, this manifests as serious headache, eye and facial pain and convulsions, and may result in intracranial hemorrhage. Associated risk factors for this syndrome are: long-term systemic arterial hypertension, carotid stenosis greater than 90%, poor collateral circulation and presence of contralateral carotid occlusion.^15-17

Using transcranial Doppler ultrasound (TCD), Naylor et al.¹⁵ found that patients with the lowest pre-clamping retrograde pressure presented the highest blood flow velocities in the middle cerebral artery after the release of the carotid artery clamp, this being characteristic of hyperperfusion syndrome. These patients had poor collateral circulation and thus required the use of a shunt to prevent ischemic complications to the brain.

Sbarigia et al.¹⁶ and Jorgensen et al.,¹⁷ using TCD together with acetazolamide and CO₂ reactivity tests (both with brain vasodilatory action), also found abnormalities in the cerebrovascular autoregulation of patients with stenotic injury to the carotid arteries, especially those with stenosis greater than 90%. The CO₂ reactivity test and acetazolamide test were used to evaluate cerebrovascular autoregulation capacity, that is, to determine whether there was additional vasodilatation or whether the cerebral circulation was in a state of chronic maximum dilatation. Sbarigia et al.¹⁶ concluded that those patients with reduced cerebral reserve (low cerebrovascular autoregulation) probably had blood supply to the affected brain hemisphere only from poor collateral circulation. In the study of Jorgensen et al.,¹⁷ the patients who developed postoperative hyperperfusion syndrome presented lower retrograde pressures and CO₂ reactivity than patients without postoperative complications.

Hosoda et al.¹⁸ conducted a study using single photon emission computed tomography (SPECT) together with acetazolamide before and after carotid endarterectomy. As in the studies cited above, the authors found reduced cerebrovascular reactivity in patients who developed postoperative hyperperfusion syndrome.

The results of these studies show that the vascular restoration of carotid stenoses greater than 90% in patients with limited cerebral blood flow reserves can lead to brain edema and the risk of cerebral hemorrhage in the postoperative period.

Carotid endarterectomy can thus cause brain tissue injuries that may be related to intraoperative ischemia-reperfusion episodes. On the basis of these studies, it can be presumed that the use of a shunt during carotid clamping would be associated with shorter, lower intensity brain ischemia, but not with protection against injuries related to postoperative hyperperfusion syndrome.

BRAIN ISCHEMIA AND REPERFUSION

As is the case in a range of other organs, a number of mechanisms are involved in producing brain tissue injury under ischemia and reperfusion.^19,20 These mechanisms include free fatty acid metabolites, purine metabolites, nitric oxide formation and leukocyte action. The study of antioxidant drugs and free radical blockers will aid the understanding of the range of chemical reactions and the possible paths followed by these metabolites in causing lesions.

During the ischemic phase, oxygen supply to the affected tissue is reduced, leading to inhibition of mitochondrial oxidative phosphorylation and reduced production of adenosine triphosphate (ATP). Stored ATP is, however, still consumed and degraded to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and, at a later stage, to adenosine, inosine and hypoxanthine.²¹ Lack of cellular energy causes a failure of the sodium-potassium pump (Na⁺/K⁺), which leads to greater accumulation of intracellular Na⁺ and loss of intracellular K⁺, with consequent edema of the cell and its organelles. There is a simultaneous flow of Ca⁺⁺ and chloride into the cell.^22-24

This accumulation of Ca⁺⁺ in the cytosol causes the activation of the protease calpain, which breaks one peptide bond of the enzyme xanthine dehydrogenase (XD), forming xanthine oxidase (XO).^23-27 Unlike XD, XO requires oxygen to convert hypoxanthine into xanthine. In the ischemic phase, there will therefore be an accumulation of these two substances. With reperfusion, hypoxanthine will be oxidized to xanthine and thus to uric acid, with the formation of the anion superoxide²⁸ as a by-product (Figure 1).

Figure 1 - Sequence of events related to ischemia and reperfusion.²¹

According to Kontos,²⁸ production of the free radical superoxide may have a number of origins: the cyclooxygenase route, the oxidation of small molecules of hemoglobin and myoglobin, of mitochondrial components and of unsaturated fatty acids and the action of enzymes such as xanthine oxidase. Superoxide is a weak oxidizing agent^29,30 and its toxic action occurs more through the function of its reduction products, such as hydrogen peroxide (H²O²), the peroxyl radical (HO²º) and the hydroxyl radical (OHº), the latter being a strong oxidizing agent. These free radicals are the reactive oxygen species (ROS).

Increased intracellular calcium and increased extracellular potassium also lead to the activation of the C and A phospholipases, encouraging the breakdown of phospholipids in the cell membrane and liberating large quantities of free fatty acids (FFA), especially arachidonic acid (AA),^22-24,31 the liberation of which is directly related to the duration of ischemia and to the brain area affected.^22,23 In reperfusion, the AA accumulated during ischemia is metabolized via the lipoxygenase and cyclooxygenase routes, forming thromboxanes (vasoconstrictors), prostaglandins (vasodilators) and superoxide.

Lipid peroxidation is also an important consequence of free radical action. It is a chain of reactions that begins with the removal of a hydrogen atom from a carbon-methylene group on the lateral chain of a free fatty acid molecule, transforming it into a free radical (Lº).^26,29,31 To stabilize its configuration during reperfusion, this Lº reacts with O², forming a peroxyl radical, which is acted upon by a range of agents, including the ferrous ion (Fe II), forming the ferric ion (Fe III) and Lº (Fenton's reaction). Through this chain of reactions, the free fatty acids are transformed into lipid hydroperoxides (Figure 2).

Figure 2 - Lipid peroxidation reactions.

Another consequence of increased intracellular calcium in ischemia is the formation of nitric oxide (NO) from L-arginine through the action of the Ca⁺⁺ dependent constitutive nitric oxide synthase (cNOS). It is known that only cNOS is found in neurons and in the cerebrovascular endothelium, being known as nNOS in neurons and ecNOS in the endothelium³⁵.

In the initial ischemic periods, Ca⁺⁺ is important in the increased liberation of glutamate, an excitatory neurotransmitter present in increasing quantities in the extracellular environment. Increased extracellular glutamate in ischemia is a source of free radicals and thus a precursor of abnormalities that occur in reperfusion.^24,35,36 The activation of glutamate N-methyl-D-aspartate (NMDA) receptors leads to the activation of nNOS, and thus to generation of NO and subsequent vasodilation.²⁸ On the other hand, the stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors results in the generation of superoxide, which may lead to the interaction of NO with superoxide radicals,³⁷ resulting in the formation of peroxynitrite, which breaks down into the hydroxyl radical.

There is evidence that leukocytes also participate in brain injury under ischemia and reperfusion. During ischemia, it is supposed that leukocytes trapped in the cerebral vasculature are activated, liberating chemotactic factors for leukotrienes in reperfusion. In addition to interacting with platelets, leukocytes may also metabolize arachidonic acid in reperfusion, leading to the formation of lipid peroxides. Mechanical obstruction by leukocytes does not, however, seem to have the same importance in the cerebral capillaries as it does in injuries caused by ischemia-reperfusion in other tissues. Studies on the treatment of brain ischemia-reperfusion with vasodilator drugs and with heparin show that the accumulation of leukocytes is not responsible for hyperperfusion.³⁸

In addition to leukocytes, the glia cells are also important in brain injury from ischemia-reperfusion. It is supposed that these cells, like macrophages, possess inducible nitric oxide synthase (iNOS), and are thus NO producers through two enzymatic routes: one non-Ca++ dependent (iNOS) and the other Ca⁺⁺ dependent (cNOS). These two classes of phagocytes - macrophages and glia cells - are also responsible for cytotoxic activity in the brain.

In summary, increased intracellular Ca⁺⁺, caused by failure of the Na⁺/K⁺ pump in ischemia due to lack of O² and ATP, may cause:

• stimulation of the protein kinase C, leading to the liberation of superoxide by the endothelium and neutrophils;
• stimulation of phospholipase C, leading to breakdown of phospholipids in the membrane and increased quantities of free fatty acids, which enter the metabolism of lipoxygenase and cyclooxygenase;
• action in smooth muscle cells, leading to vasoconstriction in the reperfusion phase. Activation of cNOS, transforming L-arginine into NO;
• breaking the xanthine dehydrogenase peptide bond, forming xanthine oxidase and the by-product superoxide.
  


As in a number of other tissues, the brain also has an antioxidant status, that is to say, a balance between the formation of oxidation agents, cited above, and the action of antioxidant substances that are responsible for combating the ROS formed in brain ischemia-reperfusion. Many antioxidants are known to exist, including the superoxide dismutase enzymes, responsible for the conversion of the superoxide anion into H²O², and catalase, which transforms H²O² into O² and H²O. Both act as "preventive" antioxidants, preventing the formation of OHº. Like catalase, hemoglobin and myoglobin act to convert H²O² into O² and H²O.³⁹ Another antioxidant group is the glutathione system, which can act preventively or as a chain breaking antioxidant, that is, it reduces groups oxidized by ROS and forms oxidized by other antioxidants.³⁹

In view of its specific characteristics, brain tissue is susceptible to injury by oxidizing agents because it contains large iron reserves and high polyunsaturated lipid levels and because of its poor antioxidant defense.⁴⁰ Some authors draw attention to the fact that abnormalities in the functioning of the glutathione system may be related to a greater susceptibility of the brain tissue to ischemia-reperfusion injuries. Almeida et al.⁴¹ noted glutathione depletion in the context of exposure to glutamate neurotoxicity through the NO formation mechanism. Anderson and Sims⁴² and Lievre et al.⁴³ also found reduced glutathione activity related to ischemia-reperfusion.

ROS AND CAROTID ENDARTERECTOMY

In a clinical study with the aim of studying possible brain injuries during carotid endarterectomy and related to the presence of oxidative stress, Rabl et al.⁴⁴ found that preoperative venous administration of a vitamin complex in patients undergoing carotid endarterectomy led to reduced postoperative presence of plasma lipid peroxides, when compared to patients receiving placebo.

Bacon et al.⁴⁵ found reduction of plasma antioxidants in operated patients, both during clamping of the carotid artery (ischemic phase) and in the reperfusion phase, demonstrating consumption of antioxidants and potential production of free radicals in these situations.

The study of Weigand et al.⁴⁰ found that the plasma antioxidant status showed significant decline during carotid artery clamping in endarterectomy. The authors also found increased arterial and venous dosage of malondialdehyde (MDA), a product of the breakdown of lipid peroxides, both during ischemia and after 15 minutes of reperfusion. They conclude that the appearance of the products of lipid peroxidation (MDA) and the depletion of plasma antioxidants indicate the presence of free radical production during carotid endarterectomy.

EXPERIMENTAL STUDIES

Few clinical studies have evaluated the presence of ischemia-reperfusion injuries in brain tissue, but a number of experimental studies have been developed with the aim of understanding the relevant pathophysiology and seeking appropriate treatments. These studies have been and continue to be extremely useful and important, but the wide variety of experimental models used and of results obtained suggests that none of them is ideal, resulting in difficult reproduction and preventing definitive conclusions.

The most common variations of experimental technique were related to ischemic type (transient or irreversible) and duration, reperfusion time, arteries involved, animals used and the range of associated tests with antioxidant drugs and other parameters.

Unilateral or bilateral temporary carotid occlusion is a commonly used model in studies of brain ischemia-reperfusion, with variations in ischemic and reperfusion time. Siragusa et al.⁴⁶ and Nakase et al.⁴⁷ induced transient ischemia in Wistar rats by means of the occlusion of one of the common carotid arteries. While the first used ischemic periods of five minutes and reperfusion periods of ten minutes, the second used ischemic periods of 15 minutes with four days of reperfusion. Other authors performed studies in which both carotid arteries were occluded temporarily and in isolation.^48-55

To reduce collateral circulation, other authors made use of a pressure tube around the animal's neck or induced hypotension during the ischemic period with the aim of generating a higher level of ischemia.⁵⁶ The pressure tube was also used in association with ligation of the basilar artery.⁵⁷

Seeking to intensify the brain ischemia, Pulsinelli et al.⁵⁸ describe an effective technique of cauterizing the vertebral arteries in Wistar rats that has been used by other authors, with variations of ischemic and/or reperfusion time and of the species of animal used.^56,59-62 Furlow⁶³ has developed a study of cerebral blood flow during occlusion of both carotid arteries in Sprague-Dawley rats, associated or not with cauterization of the vertebral arteries, and has found significant flow reduction in animals where the cauterization was performed.

Other authors performed studies with irreversible^38,64 or transient occlusion of the intracranial arteries, generally of the middle cerebral artery. Kochanek et al.³⁸ occluded the intracranial arteries of dogs using air embolization via the carotid artery. Bralet et al²² induced irreversible intracranial ischemia in Sprague-Dawley rats using microparticle embolization of the middle cerebral artery. Transient ischemia of the middle cerebral artery has been the object of studies by a number of other authors,^62,65-69 with variation in terms of the species and race studied. Takamatsu et al.⁶⁸ performed a study on monkeys, Anderson et al.⁶⁶ on Wistar rats Feng et al.⁶⁷ and Yuki⁶⁵ and on Sprague-Dawley rats.

Rats of other races have also been used in experimental studies of brain ischemia-reperfusion, including Lister-Hooded⁷⁰ and Swiss-Albino⁶⁴. Other species, such as guinea pigs,⁵⁴ genetically modified mice,^30,52 sheep,⁷¹ primates⁶⁸,⁷² and pigs⁵⁵ have also been used.

The methods of evaluating occurrence of injuries resulting from brain ischemia-reperfusion are also varied. The most common method has been dosage of thiobarbituric acid reactive substances (TBARS), that is, dosage of malondialdehyde (MDA), the end product of lipid peroxidation.^{46,48,49,56,64,67,70,73}

Other parameters used have been histology, enzyme dosage, dosage of direct products of oxidation or lipid peroxidation and tests with antioxidant drugs. The following methods can be cited as examples: antioxidant enzyme dosage,^48,67 nitrite and nitrate dosage,^50,74 histological study^52,53,60,66 and conjugated diene dosage^55,64,64,75 (these also being products of lipid peroxidation).

Despite the wide variety of models, animals and parameters, the experimental models have been useful in the study of the pathophysiology and treatment of brain ischemia-reperfusion syndrome. There is, however, continuing need for a simple, reproducible model. The rat model that best approximates this ideal is that of Pulsinelli,⁶⁰ although it has not always been reproducible.^56,76 Further studies are necessary to resolve this problem.

CONCLUSION

Carotid endarterectomy may lead to oxidative stress related to clamping and reperfusion, generally manifesting as brain edema, characteristic of postoperative hyperperfusion syndrome. Few clinical studies have been performed but experimental studies, although variable, generally confirm the problem.

It is thus necessary to create an experimental model for brain ischemia-reperfusion that is simple, reproducible and consistent, with the aim of testing treatments that may be able to reduce the injuries caused by this type of intervention.

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