Study subjects
Subjects were prospectively recruited from Weihai Municipal Hospital between February 2019 and February 2020. The Mini-Mental Status Examination(MMSE) was used as a general cognitive screening with a cut off of 27 for controls and patients with diabetes mellitus. There were 274 subjects without dementia included in the study: 81 control participants (controls), 101 normotensive patients with DM without OH, and 92 patients with DM and neurogenic OH (DMOH). All groups were matched for age, male: female ratio, and education. Controls were not excluded for taking antihypertensive medications as long as they were normotensive at the time of testing and had no evidence of OH. OH was defined as an orthostatic drop in the systolic blood pressure (BP) of at least 20 mmHg and/or in the diastolic BP of at least 10 mmHg during the first 3 minutes of standing or being positioned with a head-up tilt on a 60-degree tilt table [12]. Personal backgrounds, any medications, and current and past medical histories were recorded for all subjects. Participants also received the Hamilton Anxiety Rating Scale (Ham-A) and the17-item version of the Hamilton Rating Scale (Ham-D17). The Toronto Clinical Neuropathy Score (TCNS) was used to evaluate neuropathy. The exclusion criteria were as follows: (1) a concomitant neurological disorder that could potentially affect cognitive function or a family history of dementia; (2) a history of cardiovascular problems and stroke or other factors that may influence cerebral blood flow; (3) an abnormal finding on routine transcranial Doppler (TCD), such as middle cerebral artery (MCA) stenosis or vasospasm; (4) a poor temporal window on conventional TCD; (5) patients who were unable to continue TCD monitoring with head-up tilting(HUT) due to severe symptoms associated with orthostasis, such as syncope/presyncope, headache, faintness, dizziness, or significant tachycardia (> 150 beats per minute); and (6) other serious heart, lung, liver, kidney, or brain diseases that affect quality of life.
This study was approved by the Institutional Review Board of Weihai Municipal Hospital. In addition, written informed consent was obtained from every participant.
Assessment of orthostatic hypotension
Participants were instructed to remain on all medications as prescribed and to eat a light breakfast the morning of testing. Testing was scheduled for an 11 A.M. start time to minimize diurnal effects on hemodynamics. Before testing, all participants were allowed a 20-minute period of rest in the supine position to establish physiological and psychological equilibration. The assessments were performed in a quiet room by the same examiner. Blood pressure and heart rate were measured in the supine position after lying down for at least 5 minutes, and measurements were repeated at 1 minute and 3 minutes after standing using a fully automatic electronic sphygmomanometer (Omron HBP-1300; Omron Healthcare, Inc, Dalian, China). OH can be categorized into early OH occurring only at 1 minute after standing and delayedand/or prolonged OH occurring at 1 and 3 minutes after standing [13].
Head-up tilt test with cerebral blood flow measurements
All Doppler measurements were continuously monitored by a Digi - Lite TCD (RIMED, Israel). The 2 - MHz probes were fixed bilaterally over the temporal bone windows using a stable headset (RIMED PW SN12 - 2516). Cerebral blood flow velocity (CBFV) was taken from the mean values of the envelope curves registered simultaneously in the M1 segment of the left MCA at a depth of 50 - 60 mm as well as in the P2 segment of the right posterior cerebral artery (PCA) at a depth of 60 - 65 mm. After a resting period of 15 minutes in the supine position, the recording of CBFV was performed. Then, the subjects were tilted to an 80°head-up position with the use of a tilt table. Measurements of CBFV were repeated 3 minutes after the head-up positioning. Testing was performed at 10 A.M. and 12 P.M. in a quiet air - conditioned room at 23°C standard temperature.
Measurement of serum concentrations
Blood samples were obtained from patients between 6 and 7A.M. after overnight fasting. Samples were centrifuged (402 g, 10 min) to segregate serum and then stored at -70°C until assayed. The serum hypoxia-inducible factor-1𝛼 (HIF-1𝛼) levels were measured according to a standard enzyme-linkedimmunosorbent assay (ELISA) kit (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer’s instructions. The interassay and intraassay precisions were < 10%.
Collection of neuronal-derived exosomes from blood, the detection of exosomes, and the quantification of exosomal proteins
Fasting blood was sampled between 6 and 7A.M. and stored in a polypropylene tube containing EDTA. After drawing, the blood samples were centrifuged at 4000 g for 10 min to obtain the plasma. Specific neuronal-derived exosomes were immediately separated for consistency according to a published protocol [14]. Then, 0.5 ml of plasma was incubated with 0.15 ml of thromboplastin-D (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 60 minutes, and 0.35 ml of calcium- and magnesium-free Dulbecco’s phosphate-buffered saline (DPBS)(Thermo Fisher Scientific, Waltham, MA, USA) with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) was added. After centrifugation at 3000 g for 20 minutes at 4°C, supernatants were incubated with ExoQuick exosome precipitation solution (SEXOQ; System Biosciences, CA) and incubated at 4°C for 1 hour. After centrifugation at 1500 g for 30 minutes at 4°C, each pellet was resuspended in 250 µl of DPBS. Each exosome suspension received 100 μl of 3% bovine serum albumin (BSA) (Thermo Fisher Scientific, Waltham, MA, USA) and was incubated for 2hours at 4°C each with3 μlof rabbit anti-L1 cell adhesion molecule (L1CAM) antibody (clone 5G3; eBiosciences, San Diego, USA). Then, 25 µl of streptavidin - agarose resin (Thermo Fisher Scientific, Waltham, MA, USA) containing 50 μl of 3% BSA was added. After centrifugation at 400g for 10 minutes at 4°C and removal of the supernatant, each pellet was suspended in 50 μl of 0.05 M glycine - HCl (pH 3.0) by vortexing for 10 minutes. Each suspension then received 0.4 ml of M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) that had been adjusted to pH 8.0 with 1 M Tris - HCl (pH 8.6).These suspensions were incubated at 37°C for 10 minutes and vortexed for 15 seconds before storage at -80°C until use in enzyme-linked immunosorbent assays (ELISAs).
Western blotting was used to detect the protein marker of exosomes, namely, TSG101, using a monoclonal rabbit anti - human TSG101 antibody according to the manufacturer’s instructions (1:500, Abcam, Cambridge - UK). Centrifuged samples and immunoprecipitated samples were used to identify plasma neuronal - derived exosomes, and supernatants were used as negative controls.
Transmission electron microscopy (TEM) was used to identify the exosomes according to a published protocol with minor modifications [15]. After immunoprecipitation, the isolated neuronal-derived exosomes were stored in 1% paraformaldehyde, dehydrated through a graded series of ethanol and embedded in Epon. Ultrathin sections (65 nm) were stained with uranyl acetate and Reynold’s lead citrate. Finally, the samples were analyzed by a JEM - 1400 plus transmission electron microscope.
Neuronal-derived exosomal proteins were measured by ELISA kits for human Aβ42 (Thermo Fisher Scientific kit), total tau (Abcam kit), and tau phosphorylated at threonine 181 (Abcam kit). The amount of CD81 protein was measured to normalize the exosomal content. The mean value for all determinations of CD81 in each assay group was set at 1.00, and the relative values for each sample were used to normalize their recovery [11]. Exosomal protein assays were performed by investigators blinded to clinical and OH data.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics 22.0 (IBM, Armonk, NY). Categorical variables were analyzed using the chi-squared test. Tests on the homogeneity of variances were performed. Numerical data, such as the concentrations of amyloid-β and tau proteins in exosomes and group differences, were analyzed by using analyses of variance with Tukey post hoc analysis. Correlative analysis was performed using a linear regression model. All tests were two-tailed, and the threshold for statistical significance was P < 0.05.