Patients with MCI, dementia, including AD, VaD and combined AD and no cognitive impairment (NCI) were recruited from the Geriatric and Memory clinic in Rumailah Hospital, Doha, Qatar between 18/09/16 and 31/07/19. Patients with severe anxiety, severe depression, Parkinson’s disease, frontotemporal dementia and Lewy body dementia, hypomania, and severe dementia who were unable to cooperate were excluded. Additionally, patients with other potential causes of peripheral neuropathy including vitamin B12 deficiency, hypothyroidism, HIV infection and hepatitis C were excluded. Diabetes was not excluded because there is high prevalence of diabetes in patients aged ≥50 years in Qatar.(30) Patients with dry eyes, corneal dystrophies, ocular trauma or surgery in the preceding 6 months were excluded. This study was approved by the Institutional Review Board of Weill Cornell Medicine in Qatar and Hamad Medical Corporation and all participants gave informed consent to take part in the study. The research adhered to the tenets of the declaration of Helsinki.
Demographic and metabolic measures
Age, gender, ethnicity, blood pressure, weight, body mass index (BMI), HbA1c, cholesterol, triglycerides, thyroid stimulating hormone (TSH), free thyroxine (FT4) and vitamin B12 were recorded.
Cognitive screening
Cognitive screening was performed using the Montreal Cognitive Assessment (MoCA) test. The MoCA assesses seven cognitive domains including visuospatial/executive, naming, memory, attention, language, abstraction and delayed recall giving a total score of 30. A score of £ 26 indicates cognitive impairment. A point was added for individuals who had formal education ≤6th grade. Cognitive symptom duration was estimated from the clinical history obtained from relatives and participants.
Diagnosis
The diagnosis of MCI and dementia were based on the ICD-10 criteria.(31) The diagnosis was made according to consensus decision by geriatricians, geriatric psychiatrists and neurologists to exclude reversible, complex and young-onset dementia. The diagnosis of MCI and dementia were based on a patient history and examination, which include (1) presenting complaint and history of illness; (2) comprehensive history of each of the cognitive domains using MoCA; (3) psychiatric history for ruling out depression, mood disorders, and psychosis; (4) medical history including episodes of delirium and other medical comorbidities; (5) medication history; (6) functional history of basic daily living activities. A comprehensive organic work-up including blood tests and brain imaging was undertaken to exclude other potentially reversible causes of cognitive decline such as tumors, subdural hematoma or normal pressure hydrocephalus. Radiological evidence for AD, included volume loss of hippocampi, entorhinal cortex, and amygdala on MRI, based on the criteria described by Dubois et al.(32) The diagnosis of probable or possible VaD was based on the NINDS-AIREN criteria(33), which include multiple large vessel infarcts or a single strategically placed infarct including angular gyrus, thalamus, basal forebrain, or posterior (PCA) or anterior cerebral artery (ACA) territories, and multiple basal ganglia and white matter lacunes, extensive periventricular white matter lesions, or combinations thereof.
MRI brain procedures
MRI was performed on a superconductive magnet operated at 3T (Skyra, Siemens). A T1-weighted 3D magnetisation prepared rapid acquisition gradient echo sequence (MPRAGE) was obtained in the sagittal plane with a 1 mm slice thickness, repetition time of 1900 ms, echo time of 2.67 ms and 2.46 ms, inversion time of 1100 ms and 900 ms, flip angle of 9 degree and 15 degree, and FOV= 240 x 100. Coronal and axial reformatted MPRAGE images were made from the sagittal 3D sequence.
Medial temporal lobe atrophy visual rating
Two board certified neuroradiologists blinded to diagnosis and clinical data assessed MRI images. T1-coronal images at the level of the midbrain were used to score for right and left medial temporal lobe atrophy (MTA). The right and left hippocampi, entorhinal cortices, perirhinal cortices were separately rated according to the five-point scale developed and validated by Duara et al, and a combined visual MTA score for each hemisphere was calculated averaging the three measurements.(11) The coronal reformatted MRI slice at the level of the mammillary bodies seen in the sagittal plane was used to define the outline of the medial temporal lobe. The outline of the entorhinal cortex in this slice was defined by the anterior parahippocampal gyrus and adjacent white matter (seen medial to the collateral sulcus and inferior to the hippocampus). The outline of the perirhinal cortex was defined by the fusiform gyrus and adjacent white matter (seen lateral to the collateral sulcus and medial to the occipitotemporal sulcus).
Corneal confocal microscopy
CCM analysis was performed with the Heidelberg Retinal Tomograph III Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany). The cornea was locally anesthetized by instilling 1 drop of 0.4% benoxinate hydrochloride (Chauvin Pharmaceuticals, Chefaro, UK) and Viscotears (Carbomer 980, 0.2%, Novartis, UK) was used as the coupling agent between the cornea and the TomoCap as well as between the TomoCap and the objective lens. Subjects were instructed to fixate on a target with the eye not being examined. Several scans of the sub-basal nerve plexus in the central cornea were captured per eye for ~2 min. The field of view of each image is 400X400 µm. At a separate time, three high clarity images per eye were selected by one researcher blind to the patient diagnosis using established criteria based on depth, focus position and contrast.(29) Corneal nerve fiber density (CNFD) (fibers/mm2), branch density (CNBD) (branches/mm2) and fiber length (CNFL) (total fiber length mm/mm2) were quantified using CCMetrics, a validated image analysis software.(27)
Power analysis
Based on data from a previous study showing a reported difference in population means of CNFD 10 fibers/mm2, CNBD 4.96 branches/mm2 or CNFL 9.3 mm/mm2,(21) a sample size of 6, 22 and 7 subjects, respectively was calculated to have 80% power to detect a significant difference between the NCI, MCI and dementia group.
Statistical analysis
Patients’ demographics and clinical characteristics were summarized using means and standard deviations for numeric variables and frequency distribution for categorical variables. Variables were compared between the NCI, MCI and dementia group using one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test for pairwise comparisons and Chi-square test, respectively.
To assess inter-rater reliability, the neuroradiologists independently scored for MTA in 30 subjects with NCI (n=10), MCI (n=10), and dementia (n=10), blind to the identity and diagnosis of the subjects. To assess intra-rater reliability, one of the neuroradiologists repeated ratings in all 30 subjects after an interval of approximately four weeks. Inter-rater and intra-rater reliability was assessed using kappa statistics.
Receiver operating characteristic (ROC) curve analysis was used to determine the ability of CNFD, CNBD, CNFL, MTA-R, and MTA-L to distinguish between patients with MCI and dementia from NCI. The area under the ROC curve (AUC) and a cut-off point with the maximal sensitivity and specificity were calculated.
All analyses were performed using IBM-SPSS (version 23; SPSS Inc, Armonk NY). Dot plots were generated using GraphPad Prism, version 6.05. A two-tailed P value of ≤0.05 was considered significant.