Abstract
The authors sought to use radiolabeled annexin V, a marker of phosphatidylserine expression, to image Alzheimer dementia (AD). Four of five patients with AD had multifocal cortical annexin V uptake, whereas all seven non-AD and six control patients had normal SPECT. The mean cortex/cerebellar activity in patients with AD (1.4 ± 0.6) was higher than that of non-AD dementia patients (0.7 ± 0.2; p = 0.02). Radiolabeled annexin V may be useful for imaging AD.
Since its initial description, there have been no reports on the use of 99mTc-HYNIC-annexin V, a marker of phosphatidylserine (PS) expression, for imaging patients with Alzheimer dementia (AD).1 Annexin V is a ubiquitous 35-kDa endogenous intracellular protein that binds to the anionic membrane phospholipid, PS.2 PS is selectively and massively expressed on cells immediately after caspase-3 activation during the early phases of apoptosis. Under conditions of mild ischemia/reperfusion related stress, however, low to intermediate levels of PS have been found which can be reversed with timely reperfusion and normal levels of oxygen.3 In addition, 99mTc-HYNIC-annexin V has been found to localize to sites of ischemic injury in animal models even in regions in which the blood-brain barrier is functionally intact.4,5
In this study, we examined PS expression within the cerebrum of patients with AD as defined by 99mTc-HYNIC-annexin V SPECT. We also studied patients with non-AD dementia and nondiseased healthy age-matched controls.
Methods.
Patient population.
Twelve patients with clinical dementia were enrolled in the study (table). Five patients had AD (four men and one woman, mean age 74 years, range 59 to 85 years), and the remaining seven patients were diagnosed with non-AD dementia, including four with vascular dementia6 and three with Lewy body disease7 (4 men and 3 women, mean age 71 years, range 52 to 83 years). All AD and non-AD dementia patients diagnosed according to the National Institute of Neurological and Communicative Diseases and Stroke-Alzheimer's Disease and Related Disorders Association work group criteria8 had a severe degree of dementia as defined as a range of 10 to 18 on the Mini-Mental State Examination (MMSE). All patients had a brain MRI scan. Patients with other neurologic diseases, including stroke, surgery, or tumor, were excluded. Patients with AD were receiving treatment with acetylcholinesterase inhibitors (rivastigmine 3 to 4.5 mg BID or donepezil 10 mg QD), with two also receiving memantine (10 mg BID). The study was approved by the local ethics committee of the hospital, as well as by the Ministry of Health. All participants of the study (or authorized guardian) signed a consent form. The control group consisted of six healthy individuals (three men and three women, mean age 76) with no reports of cognitive decline and no evidence of dementia.
Table Patient characteristics
Preparation of 99mTc-HYNIC annexin V for human use.
NAS 2020 Kits were generously supplied by Theseus Imaging Corporation (Boston, MA). Radiolabeling was performed according to package insert instructions prepared by the Theseus Imaging Corporation. Radiochemical purity of the drug product was verified to be greater than 90% as determined by instant thin-layer chromatography (ACD) in each dose administered. Each 15 to 20 mCi (555 to 740 MBq) of 99mTc-HYNIC-rh-annexin V was administered through an indwelling IV line in a volume of approximately 2.0 mL.
Annexin V SPECT imaging.
SPECT images were obtained using a dual-head gamma camera (Elscint-Helix, Haifa, Israel) equipped with a pair of low-energy, high-resolution collimators. Images were acquired in a 128 × 128 matrix at 6 ° angular steps, with 40 seconds in each step. Acquired images were transferred to a dedicated Xeleris workstation (General Electric Medical Systems) for processing. Raw SPECT data were reconstructed using a commercially available Ordered Subset Expectation Maximization algorithm (OSEM; two iterations, 10 subsets) and post-filtered using a Butterworth filter (cutoff frequency 0.5, order 10.0). The final reconstructed pixel size was 3.87 by 3.87 mm.
The mean of the total cortical activity in a transaxial slice at the level of the orbitomeatal line was determined by two experienced nuclear medicine physicians for all patients and compared to the mean activity of the cerebellum (cortex/cerebellar ratio).
Statistical analysis.
Analysis of data was performed using an online Fisher exact calculator for tables up to 2 × 5 (Simple Interactive Statistical Analysis, 2000). The test was two-sided and considered significant at p < 0.05.
Results.
Two hours after injection of 99mTc-HYNIC-annexin V, SPECT imaging of healthy age-matched controls revealed the physiologic distribution of tracer activity, with virtually no observable tracer uptake in the normal brain parenchyma.
Four of the five patients with AD had abnormal foci of increased annexin uptake (figure), whereas all seven non-AD patients had an unremarkable SPECT. Annexin uptake in the patients with AD was scattered throughout the cortex but spared the region of the cerebellum. The mean cortex/cerebellar activity ratio in the patients with AD (1.4 ± 0.6) was higher than that of non-AD patients (0.7 ± 0.2; p = 0.02). The only AD patient with no annexin uptake was first diagnosed 5 years before the scan and had a MMSE score of 18.
Figure. Representative transaxial SPECT slice from a patient with Alzheimer dementia (AD) (A) shows multiple foci of annexin uptake (Mini-Mental State Examination [MMSE] score = 11) and a corresponding T2-weighted magnetic resonance (MR) image (D) showing mild atrophy. Another patient (B) with non-AD dementia (multi-infarct dementia) shows minimal background activity (MMSE score = 13), and the corresponding T2-weighted MR image (E) shows abnormal signals in the periventricular white matter consistent with diffuse ischemic changes. The SPECT image of a control subject is also presented (C).
Discussion.
Our pilot study shows the feasibility of imaging PS expression in the brain of patients with AD using 99mTc-HYNIC-annexin V SPECT. These results also suggest that annexin V may provide complementary information useful for differentiating AD from non-AD dementia.
Although increased levels of PS have been documented in animal models4,5 and humans with acute stroke,9 it is unclear what the mechanism of annexin V uptake is or its relation to apoptotic neuronal death (irreversible PS expression) within the brain. A protein specific form of endocytosis has been found to be activated within mildly stressed cardiomyocytes and neurons that can pump annexin V into the cells of the heart and brain (even in the presence of a functional blood-brain barrier). This pump is activated in the context of low to intermediate levels of PS expression that are reflective of reversible cellular stress (or territories at risk) that can be defined by annexin V imaging.
In our study, it is possible that neurons in regions of increased annexin V uptake were under stress (reversible low to intermediate levels of PS expression) or were undergoing apoptosis (high levels of irreversible PS expression) thereby blurring the distinction between salvageable and dying neurons. Although we cannot conclude that we are imaging apoptosis, it is intriguing that the one AD patient without increased annexin V uptake also had long-standing dementia in which little ongoing neuronal cell death would be expected.
The main limitations of our study are the relatively small number of patients that must be reaffirmed in a larger test group, that PS expression due to apoptosis and neuronal stress cannot be distinguished by annexin V imaging, and that PS expression will more than likely be seen in other forms of neurodegenerative disease and acute ischemia and not be specific to AD.
Acknowledgment
The authors thank Ms. Shula Calman and Ms. Vered Pilosof for technical assistance.
Footnotes
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*The first and second authors contributed equally to the manuscript.
Supported in part by Theseus Imaging Corporation, Cambridge, MA.
Disclosure: The authors report no conflicts of interest.
Received September 23, 2005. Accepted in final form January 18, 2006.
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