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Elderly patients who have an acute illness or who undergo surgery often experience cognitive decline. The pathophysiologic mechanisms that cause neurodegeneration resulting in cognitive decline, including protein deposition and neuroinflammation, also play a role in animal models of surgery-induced cognitive decline. With the aging of the population, surgical candidates of advanced age with underlying neurodegeneration are encountered more often, raising concerns that, in patients with this combination, cognitive function will precipitously decline postoperatively. This special article is based on a symposium that the University of California, San Francisco, convened to explore the contributions of surgery and anesthesia to the development of cognitive decline in the aged patient. A road map to further elucidate the mechanisms, diagnosis, risk factors, mitigation, and treatment of postoperative cognitive decline in the elderly is provided.
Aβ = β-amyloid; AD = Alzheimer disease; apoE = apolipoprotein E; APP = amyloid precursor protein; DAMPs = damage-associated molecular patterns; HMGB-1 = high mobility group box chromosomal protein 1; IL = interleukin; POCD = postoperative cognitive dysfunction; PRR = pattern recognition receptor; TLR = toll-like receptor
Cognitive decline, including memory dysfunction, is a leading cause of functional impairment worldwide. The risk of cognitive decline increases with age and is further enhanced after hospitalization for critical illness and surgery, resulting in significant long-term morbidity and an overall reduced quality of life.1 The maintenance and/or restoration of functional independence, including cognition, in the elderly hospitalized patient constitutes a major challenge for the health care system. It is projected that patients older than 65 years will become the largest segment of the surgical population by 2020.2 Recent advances in technology and anesthetic care have enabled increasingly older and sicker patients to be viable candidates for elective surgery; of particular concern are surgical patients with preexisting neurodegenerative conditions in whom a precipitous decline in cognitive function may occur postoperatively. The most prevalent neurodegenerative condition is Alzheimer disease (AD); it is estimated to affect 35 million people worldwide, of which 5.5 million reside in the United States.3 Because of the steady growth of the proportion of elderly individuals in developed countries, a 4-fold increase in AD is projected to occur by 2050.4,5
Persistent memory and learning disabilities may occur postoperatively and may be related to neurodegenerative processes. The observation that after an operation some patients are more likely to experience cognitive deterioration and personality changes has long been described. In 1887, George Savage6 described how surgery and anesthesia may possibly contribute to the development of “mental insanity.” Despite these early observations of perioperative cognitive decline as a hazard of surgery, no rigorous investigations were performed until the past decade, in which reports have focused on the role of anesthesia and surgery as possible causes of cognitive dysfunction.7-9
Postoperative cognitive decline (POCD) broadly follows 2 different patterns: acute cognitive dysfunction, also known as early postoperative delirium, and a later onset and more persistent POCD.10 Delirium is often seen in older patients after hospitalization and surgery11; the classic features are an acute change in mental status, inattention, disorganized thinking, and altered consciousness. Delirious patients exhibit a spectrum of behaviors that range from hyperactive to hypoactive, with the latter being more common in postoperative patients. Although often of short duration, delirium is associated with increased mortality,12 greater care dependency, costs,13 and prolonged hospitalization.14 Persistent cognitive decline and functional impairments are also associated with hospitalization in survivors of acute illness and sepsis.15 Because of the dynamic and complex processes that underlie this condition, many strategies are being contemplated for the prevention and treatment of delirium, especially in older patients who are at higher risk16 (Table 1).
As yet, no therapeutic interventions are available that prevent the onset of postoperative delirium. Marcantonio et al32 and others33 have developed and validated a scoring system to predict the possible onset of delirium before surgery. They report a 9% incidence of delirium in elective noncardiac, nonorthopedic patients within the first 5 postoperative days; after orthopedic procedures, the incidence increases to 41%. Emergency surgery in the elderly is also associated with a higher incidence of acute cognitive decline, ranging from 42.3% after surgery for lower limb ischemia to 73% after lung transplant.34,35
Postoperative cognitive dysfunction is a more subtle and prolonged change in cognition, with clinical manifestations similar to those seen in neurodegenerative disorders, and is diagnosed by neuropsychological testing. Although POCD is not listed in the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition), it has been defined as a “more than expected” postoperative deterioration in cognitive domains, including short- and long-term memory (ie, reduced ability to learn or recall information), mood, consciousness, and circadian rhythm.36 The precise threshold of deterioration and the number of domains that need to be affected are matters of debate.37 This neurocognitive impairment has been correlated with a longer hospitalization time, greater comorbidities, and higher mortality risk in patients who develop this complication compared with those who do not.38 Neurologic and cognitive complications were first described after cardiac surgery with cardiopulmonary bypass; however, POCD also affects patients after noncardiac procedures.39 An international multicenter study on POCD (ISPOCD) reported memory impairments in 25.8% of patients 1 week after noncardiac surgery and in 9.9% after 3 months in patients older than 60 years.8 Johnson et al40 reported a similar incidence, with almost 20% of middle-aged surgical patients experiencing cognitive decline 1 week after surgery compared with 4.0% in the nonsurgical control group. More recently, similar distribution patterns in type and severity of cognitive deficits were reported in a study group of 308 patients undergoing major surgery.41 At 3 months after surgery, 231 patients (75%) had normal levels of cognitive functions, 42 (13.6%) experienced memory decline but 26 (8.4%) showed only executive function impairment, and 9 (2.9%) experienced decline in both executive and memory domains. Some reports have described cognitive decline persisting up to 1 year after surgery; this may indicate a possible progression to dementia (Table 2).43 Of note, there is no certainty that this rate of cognitive dysfunction does not occur in surgical patients randomized not to have surgery.
Studies have sought to identify factors that may contribute to POCD, including exposure to general anesthesia,50 hyperventilation, hypotension, hypoxia, psychoactive drugs, and patient-related factors, including aging, genetic polymorphisms (APOE4),46,48 and comorbidities such as cancer and underlying neurodegenerative and neurovascular diseases.37 In a prospective study, patients who had previously experienced a stroke were more at risk for POCD even though they had no neurologic sequelae from the remote stroke event.42 Although age and surgery are consistently reported as important risk factors in the development of cognitive dysfunction, the etiology of perioperative cognitive decline remains unclear, and even its very existence is being debated.51 Studies exploring contributory factors have had methodological problems, including being underpowered and lacking in appropriate controls; furthermore, because of the difference in neurocognitive testing and thresholds for diagnosing cognitive dysfunction, comparison of studies and hence prioritization of risk factors have been difficult.37
Although perioperative cognitive decline and AD may share certain neuropathologic and biochemical mechanisms, there is no direct evidence linking the involvement of AD-type pathogenic mechanisms and POCD in humans and only weak epidemiological evidence associating surgery with onset of AD.52 It remains controversial whether perioperative cognitive decline increases the risk of further cognitive decline resulting in dementia, although neuropsychologists in clinical practice continually hear remarks that “the family member/friend has never been the same since the operation.” Epidemiological studies have suggested that neurodegenerative disorders, including AD, may be accelerated by surgery53 and that delirium exacerbates dementia in AD patients.54 However, Avidan et al,55 in a retrospective cohort study, reported no evidence of long-term cognitive decline after a surgical event and were unable to associate surgery or anesthesia with further dementia and AD. There are no data from studies to explore the possibility that surgery aggravates existing AD.
To examine the possible contribution of surgery and anesthesia to the development of cognitive changes and possible long-term consequence, the University of California, San Francisco, convened a state-of-the-science symposium on October 13, 2010, to discuss the putative relationship between neurodegeneration and perioperative cognitive changes in the aging population and to address the pathophysiology of these 2 conditions. Speakers in the symposium reviewed data from preclinical models and clinical studies and opined on translational approaches to understand the factors affecting postoperative cognitive decline. Additionally, to supplement this review with further up-to-date information, the authors undertook a literature search using PubMed with the following key words: Alzheimer's disease, anesthesia, delirium, dementia, postoperative cognitive dysfunction, and surgery.
Inflammation and activation of the immune system are associated with cognitive decline.56,57 Surgical patients exhibit elevations of proinflammatory cytokines in both the central nervous system and the systemic circulation, the extent of which may relate to the degree of cognitive decline.58,59 Major surgical procedures, such as cardiac surgery and orthopedic procedures in particular, expose the patient to extensive trauma, blood loss, and extensive tissue injury; products of these are capable of engaging with the innate immune system to induce an inflammatory response.60
Animal models have been useful in identifying mechanisms and putative targets for intervention and treatment of surgery-induced cognitive decline. Data from preclinical studies support the concept that inflammation is a possible pathogenic mechanism for postoperative cognitive decline and define a causative role for interleukin 1β (IL-1β) after surgery.57 Increased expression of IL-1β in the hippocampus of old mice undergoing minor surgery was associated with cognitive decline, corroborating the view that surgery-induced neuroinflammation can result in cognitive impairment.61 Sickness behavior, a related syndrome that occurs after acute illness, is characterized by a decline in cognitive function, fever, decreased food intake, somnolence, hyperalgesia, and general fatigue and is associated with a systemic cytokine release and neuroinflammation in the hippocampus, amygdala, and limbic system.62,63 On sensing changes in proinflammatory molecules and abnormal proteins, microglia (the resident immunocompetent cells) phenotypically shift from a resting state to one that is reactive.64 During activation, microglia undergo stereotypical conformational changes in both morphology and function.65 Although activated microglia may be protective through the release of neurotrophic and anti-inflammatory molecules, these cells also secrete proinflammatory cytokines (including IL-1 and tumor necrosis factor α),66 reactive oxygen species, excitotoxins (such as glutamate and adenosine triphosphate),67 chemokines, and neurotoxins, such as amyloid precursor protein (APP) and other pathologic proteins.68 Activated microglia can also inhibit neurogenesis in the hippocampus, thereby impairing synaptic plasticity and repair of neuronal dysfunction. Conditions that can convert the usually self-limiting postsurgical neuroinflammatory response into one that is persistent may include advanced age and underlying systemic disease, both of which may be associated with enhanced or prolonged inflammation after surgery.69 Furthermore, a normal neuroinflammatory response to surgery may have long-lasting detrimental effects in settings of underlying neuropathology, whether clinically evident or not.
A so-called danger signal is required to provoke the immune system into a response to the original threat70,71; surgery is a likely trigger of the danger signal (Figure). The initiating mechanism whereby surgical trauma stimulates innate immunity is likely to involve damage-associated molecular patterns (DAMPs), which are released from cells that are dead or dying due to non-apoptotic (ie, necrotic) processes.72 Among the DAMPs that can be released during surgical trauma is the high mobility group box chromosomal protein 1 (HMGB-1), which binds and signals through a family of evolutionarily conserved pattern recognition receptors (PRRs).73 Release of HMGB1 is both active and passive in many inflammatory conditions, ranging from sepsis to arthritis, acute lung injury, and stroke.74 Early activation of the innate immunity via DAMPs such as HMGB-1 and cytokines, including tumor necrosis factor α, appears to mediate the initial response to surgical trauma that leads to neuroinflammation and cognitive decline.56 Toll-like receptors (TLRs), part of the superfamily of PRRs, recognize a diverse range of ligands including pathogen-associated molecular patterns (PAMPs) and DAMPs.75,76 Each stimulated TLR activates distinct signaling pathways that increase synthesis and release of proinflammatory mediators. Toll-like receptor 4 is perhaps the most active interface that bridges microbial infection with inflammation.77,78 Its role after lipopolysaccharide endotoxemia has been extensively studied79; however, the pathways that regulate infection-induced neuroinflammation and cognitive decline appear to be different from those seen after aseptic surgical trauma.56 Endogenous inducers of the innate immune response engage not only TLRs but also other PRRs to perpetuate and amplify the inflammatory response.80 Advanced glycation end products, HMGB-1, and some S100β proteins activate the receptor for advanced glycation end products that may contribute to the signaling mechanism underlying cognitive decline, especially in the setting of comorbidities such as obesity and diabetes, which can enhance the risk of neurodegeneration including AD.81,82
Like other inflammatory conditions, the neuroinflammation that occurs after surgery probably has 2 phases: proinflammatory and anti-inflammatory. The “switch” from the one to the other is likely to be under control of both neural and humoral factors83,84; abnormalities of the switching mechanism may cause a nonresolving chronic inflammatory state that could create the circumstances for persistent cognitive decline.
Accumulation of abnormal proteins in the nervous system appears to be a pivotal mechanism for causing cognitive decline and memory disruption. Aggregation of naturally occurring peptides in the brain remains a hallmark for neurodegeneration, especially because these factors strongly correlate with both neurotoxicity and cell death.85 A relationship between anesthesia, β-amyloid (Aβ), and tau protein phosphorylation in particular has been suggested (and reviews have been published86-89). Specifically, the commonly used inhalation anesthetic isoflurane has been shown to enhance Aβ oligomerization in cultured cells.87 Isoflurane88,90 and another commonly used inhalation anesthetic, sevoflurane,91 but not desflurane,92 have been shown to enhance Aβ accumulation in vivo and in vitro. Interestingly, low concentration of isoflurane alone did not increase Aβ accumulation, but the combination of this low concentration of isoflurane with nitrous oxide did increase Aβ accumulation.93 Anesthetic-induced Aβ accumulation also is associated with caspase activation and apoptosis,94 and anesthesia-induced hypothermia has been shown to induce tau protein phosphorylation.89,95 However, the anesthetic propofol can also directly increase tau protein phosphorylation.96 Alternative findings that suggest that anesthesia is not associated with neurodegenerative diseases and/or that anesthetics do not promote neurodegeneration are summarized in 2 recent reviews.97,98
In addition to anesthesia-induced neurotoxicity in the newborn,99 elderly patients may be at greater risks from anesthetic exposure after either general or regional anesthesia.9 Evidence from animal models and cell culture studies suggests that exposure to anesthetics induces pathologic changes normally associated with AD, including increase in Aβ peptide and β-actin cleavage enzyme88; anesthesia-induced hypothermia also increased tau hyperphosphorylation by decreasing phosphatase 2A activity.95 In a transgenic mouse model of AD, exposure to halothane increased Aβ plaque deposition but was not associated with further deterioration of cognitive function.100 Anesthesia also appeared to have no effect on APP transcription and expression.101 Surgery under general anesthesia was associated with increased APP transcription and expression, Aβ expression, γ-secretase activity, and the hyperphosphorylated form of tau protein in aged mice.102
Animal studies suggest that anesthetics may be responsible for modifications in the brain that outlast their tenure within the body.103 Anesthetics directly affect memory processes and behavior.104,105 From preclinical studies, it is apparent that cognitive dysfunction can persist for several weeks after exposure to a commonly used anesthetic regimen106,107 and is associated with changes in gene expression in brain areas such as the hippocampus.108 Yet, some anesthetics do not produce any postexposure disturbance in cognition even in aged rats.109 Furthermore, randomized clinical trials in older patients undergoing major noncardiac surgery have reported similar incidence in POCD after regional or general anesthesia, thereby challenging the concept of anesthesia as a causative factor.7,9 However, anesthesia and surgery may potentiate each other's neurotoxic effects, leading to an increase in the severity of POCD.
Recent data suggest that tau levels can directly affect axonal transports, thus requiring Aβ as a copathogen to mediate neurotoxicity and cognitive impairment.110 Resulting neurotoxicity from the uncontrolled aggregation of these peptides ultimately disrupts brain homeostasis by limiting availability of nutrients and trophic factors to the neuronal population. Neuroinflammation, including activation of microglia cells, and release of proinflammatory mediators and serum proteins contribute to disease development in AD.111-113 Both environmental and genetic risk factors remain strong determinants for AD development.114 In particular, mutations in APP, APOE4, and presenilin 1 and 2 are predisposing genetic factors for abnormal Aβ production or deposition. Presence of the ApoE4 allele increases the abnormal accumulation of Aβ and tau and affects a number of pathways that affect neuronal activity and cell survival.115 Recently, compelling evidence from genome-wide association studies identified novel key genetic variants of molecular species involved in inflammatory signaling pathways that were associated with the development of AD and increased susceptibility for the late-onset variety.116,117
Preclinical studies are now uncovering novel mechanisms for surgical trauma, anesthesia, and neurodegeneration in triggering cognitive decline. Translational studies are required to confirm these findings in humans and test these new hypotheses.
Transformational research addressing postoperative cognitive decline will benefit from coordinated investigations using preclinical, clinical, and epidemiological experimental paradigms. The following sections describe the types of questions that can be addressed and the settings in which these can best be addressed; to comprehensively understand the causes and management of this important and ever-increasing clinical problem, bidirectional translation of studies at both preclinical and clinical levels will be required. As a result of this symposium we have been able to (1) advance our understanding of the pathogenesis of perioperative cognitive decline; (2) identify the risk factors associated with cognitive decline, including the contributions of neurodegeneration and of different disease states, including those with “hyperinflammatory diatheses” from either a heightened proinflammatory or an underperforming anti-inflammatory resolving arm; (3) identify biomarkers and standardize neuropsychological tests and imaging techniques that can predict vulnerable surgical patients; and (4) apply preemptive therapeutic strategies to thwart the devastation wrought by postoperative cognitive decline (Table 3).
Once animal models are better developed to address the multifactoriality of cognitive decline, they can be used to explore molecular mechanisms that can yield putative therapeutic targets that are amenable to manipulation and testing for efficacy (and safety) in patients. It will be necessary to confirm that factors that increase the likelihood of postoperative cognitive dysfunction in humans (including advanced age and postoperative pulmonary complications, especially of an infectious nature) are similar in animals. The exacerbating effect of the type of surgery (eg, orthopedic vs abdominal procedures) needs to be confirmed. Further development of animal models is needed to faithfully recapitulate the clinical setting by introducing the realism of the postoperative state, including sleep deprivation and the addition of sedatives and analgesics that have their own cognition-altering effects. Key changes noted in the animal models need to be sought in surgical patients to establish that the pathophysiologic processes are similar. Animal models can be used to address whether underlying neurodegenerative and vascular causes of dementia increase the risk of postoperative cognitive decline. Animal studies are needed to test the influence of diabetes, obesity, and underlying acute neurologic disease, such as stroke, on the development of postoperative cognitive decline. In the same vein, answering the question of the potential influence of a surgical procedure on disease progression in animal models of neurodegenerative disease becomes tractable. Perhaps the biggest controversy tying animal models of postoperative cognitive decline to the human illness is to address why animals recover from postoperative cognitive decline, whereas more than 10% of elderly patients appear to experience a progressive deterioration of cognitive function. At best, the existing animal models portray the acute delirium end of the postoperative cognitive decline spectrum, leaving the question of the existence of long-term cognitive decline after surgery for further model development.55 Even if one narrowly confines the applicability of the existing animal model, one still needs to understand why delirium does not occur after each surgical procedure in humans as it does in the animal models. In this respect the influence of factors that enhance the protective effect of “cognitive reserve” or exacerbate the pathophysiologic mechanisms can be directly explored. For example, the pathophysiology of “double hits” in the causation of postoperative delirium (produced by simulating 2 bouts of trauma as occur in hip fracture patients undergoing surgical stabilization) can be examined with greater temporal resolution to inform appropriate timing of the fracture stabilization procedure. The animal models can be used to determine the influence of different anesthetics (including regional vs general as well as different general anesthetic agents) in light of the differential neuroimmodulatory effects of anesthetic agents. Because we anticipate that preemptive neuromodulation is a possible therapeutic strategy, further understanding will be required to distinguish between inflammatory processes that facilitate healing and those that mediate cognitive dysfunction. In all likelihood, this discrimination will require very precise temporal resolution of the inflammatory processes initiated after surgical trauma and a careful elucidation of the proinflammatory vs the anti-inflammatory phase of the immune response to aseptic trauma.
As mentioned in the preceding section, it will be necessary to confirm that the changes that occur in animal models also occur in surgical patients. To progress in the clinical realm, both standardized testing and a definition of what constitutes an abnormality will be needed.37 It will be necessary to identify patients particularly at risk to develop an “enriched” patient population that will “power” therapeutic trials designed to test the safety and efficacy of an intervention. This will require identification of a characteristic phenotype either by sifting through routinely collected clinical information or by defining a biomarker associated with a higher risk. Although the former can be amenable to retrospective data mining of large clinical and administrative databases, the latter will require prospective studies. In this regard, 2 domains, genetic and neuroimaging, hold the greatest promise. The potential of genome-wide association studies is now being realized in the field of neuropsychiatric disorders with reduction in the costs of genetic testing.97,98 Neuroimaging can now be used to establish the preclinical and prodromal AD pathology for the development of neurodegenerative conditions, and its role in postoperative cognitive decline requires further investigation.118,119 Apart from the use of neuroimaging biomarkers (including amyloid load, as well as alterations in white matter and connectivity, and inflammatory markers), this powerful investigative modality may assist in the diagnosis (with different parameters including T1, T2, diffusion tensor imaging, arterial spin labeling perfusion magnetic resonance imaging, resting-state functional magnetic resonance imaging, contrast-enhancing studies, and positron emission tomography) of a condition whose existence is yet to be acknowledged by the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition).
Electronic health records, as well as the longitudinal databases that have been established by consortia such as the Alzheimer Disease Research Centers, will prove to be a valuable source of information on which hypotheses can be formulated for subsequent testing both in the preclinical and prospectively in the clinical settings. In particular, the contribution of ethnic, lifestyle, and socioeconomic factors and comorbidities to the development of postoperative cognitive decline can be interrogated. With the establishment of the American Society of Anesthesiologists–supported Anesthesia Quality Improvement database, there is a strong likelihood that associations will provide the grist for future hypotheses-testing studies.
We are likely to be confronted by an epidemic of postoperative cognitive decline that we are ill-equipped to address. We are only now beginning to scratch the surface of its proclivity, diagnosis, and causation and to contemplate possible strategies to prevent and treat this postoperative complication. It is axiomatic that for postoperative cognitive decline to be successfully counteracted, coordinated efforts will be required from experts in aging, neurodegeneration, vasculopathy, inflammation, neuroimaging, and the human genome working in conjunction with surgeons, anesthesiologists, and primary care physicians. Therefore, it is pivotal to establish multidisciplinary collaborations to confront a clinical problem with enormous patient suffering and societal consequences.
Dr Weiner reports the following financial disclosures: served on the following scientific boards in 2009, Elan/Wyeth Alzheimer's Immunotherapy Program North American Advisory Board, Novartis Misfolded Protein Scientific Advisory Board Meeting, Banner Alzheimer's Institute Alzheimer's Prevention Initiative Advisory Board Meeting, and Research Advisory Committee on Gulf War Veterans' Illnesses; in 2010, Lilly, Araclon and Institut Catala de Neurociencies Aplicades, Gulf War Veterans Illnesses Advisory Committee, VACO, and Biogen Idec; and in 2011, Pfizer. He did consulting for the following in 2009: Elan/Wyeth, Novartis, Forest, Ipsen, and Daiichi Sankyo, Inc; in 2010, Astra Zeneca, Araclon, Medivation/Pfizer, Ipsen, TauRx Therapeutics Ltd, Bayer Healthcare, Biogen Idec, Exonhit Therapeutics, SA, Servier, and Synarc; in 2011, Pfizer. He received funding for travel in 2009 from Elan/Wyeth Alzheimer's Immunotherapy Program North American Advisory Board, Alzheimer's Association, Forest, University of California, Davis, Tel-Aviv University Medical School, Colloquium Paris, Ipsen, Wenner-Gren Foundations, Social Security Administration, Korean Neurological Association, National Institutes of Health, Washington University at St. Louis, Banner Alzheimer's Institute, Clinical Trials on Alzheimer's Disease, Veterans Affairs Central Office, Beijing Institute of Geriatrics, Innogenetics, and New York University; in 2010, NeuroVigil, Inc, CHRU-Hopital Roger Salengro, Siemens, AstraZeneca, Geneva University Hospitals, Lilly, University of California, San Diego-ADNI, Paris University, Institut Catala de Neurosciencies Aplicades, University of New Mexico School of Medicine, Ipsen, and CTAD (Clinical Trials on Alzheimer's Disease); in 2011, Pfizer, AD PD meeting, Paul Sabatier University, and Novartis. Dr Weiner serves on the Editorial Advisory Board for Alzheimer's & Dementia, and MRI; received honoraria in 2009 from American Academy of Neurology and Ipsen; in 2010, from NeuroVigil, Inc, and Insitut Catala de Neurociencies Aplicades. He receives Commercial Entities Research Support from Merck and Avid; Government Entities Research Support from DOD and VA; and has stock options in Synarc and Elan. The following organizations contribute to the Foundation for N1H and thus to the NIA—funded Alzheimer's Disease Neuroimaging Initiative: Abbott, Alzheimer's Association, Alzheimer's Drug Discovery Foundation, Anonymous Foundation, AstraZeneca, Bayer Healthcare, BioClinica Inc (ADNI 2), Bristol-Myers Squibb, Cure Alzheimer's Fund, Eisai, Elan, Gene Network Sciences, Genentech, GE Healthcare, GlaxoSmithKline, Innogenetics, Johnson & Johnson, Eli Lilly & Company, Medpace, Merck, Novartis, Pfizer Inc, Roche, Schering Plough, Synarc, and Wyeth.