The Effect of Intravenous Erythropoietin Treatment on Cognition During Hypoglycemia in Patients With Type 1 Diabetes.

Experimental protocol 6 days before hypoglycemic clamp subjects attended the clinic, and information about demography, lifestyle and clinical characteristics was collected. A physical examination with focus on possible thromboembolic disease was performed. Blood pressure was measured twice after 10 min rest in a sitting position. Measures of peripheral and autonomic neuropathy were assessed and a 12-lead electrocardiogram (ECG) was visually assessed. State of hypoglycemia awareness was classified according to Pedersen-Bjergaard et al [22]. The frequencies of episodes of mild and severe hypoglycemia in the preceding week were recorded in a questionnaire. A sensor for continuous glucose monitoring (CGM) was mounted (Guardian® REAL-time, Medtronic Minimed, Northridge, USA). Hereafter, the subject was re-introduced to the experimental protocol and cognitive tests were trained twice to avoid a learning effect during the experiments. Finally, study medication was injected intravenously over four minutes. Subjects were instructed to live and eat as they used to do, to avoid any rigorous exercise, use of alcohol and psychoactive drugs, for the next week. Calibration of the CGM was done by the patient with the blood glucose meter CONTOUR® LINK (Bayer Health Care, Leverkusen, Germany).

3 days before experiment A new CGM sensor was inserted and a hypoglycemia alarm was activated. Because the CGM glucose value is delayed approximately 10 min compared to plasma glucose the alarm-point was set to 4.5 mmol/l. CGM glucose profiles for the last three days were downloaded to check if any insulin dosage changes should be done to decrease the risk of hypoglycemia until the experiment 3 days later. Finally, the subjects trained the cognitive function tests again.

Day of experiment Subjects arrived at 8.00 a.m. in the laboratory after an overnight fast. Data from the CGM were downloaded. If hypoglycemia, defined by at least two consecutive CGM values below 3.5 mmol/l or one self-monitored blood glucose value below 3.5 mmol/l, had occurred during the preceding night, the experiment was postponed for at least 14 days. This was the case at one occasion. CGM warned the participants about decreasing blood glucose during the night before the experiment at three occasions before it actually occurred. In these cases the participants were instructed to eat a standardized meal of 10 grams of glucose and 35 g of dark bread. No participants had a CGM glucose > 20 mmol/l during the night before the experiments.

The subject was introduced to the experimental setting and was equipped as follows: 1) One antecubital intravenous line for infusion of insulin and glucose and one line as distally as possible for blood sampling. Thereafter, the hand and arm were placed on and under heated pads to arterialize venous blood; 2) EEG (electroencephalography) cap, two ear lobe references and two precordial ECG leads connected to a digital EEG recorder (Cadwell, Kennewick, Washington, USA); 3) an automatic blood pressure unit (UA787, A&D Medical, Abingdon, UK). Subsequently, subjects trained the cognitive tests.

The experiment was carried out in a cyclic manner with a total of three identical cycles: A baseline cycle (euglycemia), a hypoglycemic cycle and a recovery cycle (euglycemia). In the start of each cycle arterialized venous blood was drawn. Subsequently, hypoglycemic symptoms were assessed followed by measurement of blood pressure and pulse rate. Hereafter, evaluation of cognitive function was done with the California Cognitive Assessment Package (CALCAP), a trail making test and the Stroop test. All cognitive testing was conducted in the same sequence by the same person (PLK). At the end of each cycle, 5 min of EEG recording was done with eyes closed. Finally, arterialized venous blood was drawn again.