Diabetes Mellitus and microRNA as Risk Factors for Mild Cognitive Impairment: Impact of Life Style Modification
Condition: Diabetes Mellitus, Type 2
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 2; Cognitive Dysfunction
Study type: Interventional

Summary: 
      This is a 2-step study; 1st step was a cross sectional study carried out on 163 consecutive
      Type 2 Diabetes Mellitus (T2DM) patients employee attending outpatient clinics of the
      National Research Center (NRC), Egypt. A face-to-face interview was carried out to collect
      data about demographic data and medical history. Cognitive function assessment was carried
      out using Adenbrooke's Cognitive Examination III (ACE III) test and the Trail Making Test
      (TMT) which consists of two parts (A and B). Quality of life and depression were also
      assessed using Short Form-36 (SF-36) and Hamilton Depression Rating Scale (HRDS)
      questionnaires respectively. The second step was an interventional study for health education
      and life style modification. The main outcome measures were Fasting Blood Glucose (FBG),
      HbA1c, total blood cholesterol, HDL cholesterol, LDL cholesterol and triglycerides, oxidant
      and antioxidant (Malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase
      (GR)) which were assessed at baseline and reassessed after the intervention.
    

Detailed description: 
      The current study was carried out during the period from February 2016 to June 2019 on two
      steps.

      1. First step (baseline assessment): It was a cross sectional study carried out on 163 Type 2
      Diabetes Mellitus (T2DM) patients attending outpatient clinics at NRC. Patients were 65 males
      and 98 females; aged from 40 to 65 years. All patients were literate and were able to
      complete the tests of cognitive function. The exclusion criteria were T2DM patients with
      history of head trauma, stroke, transient ischemic attack, brain tumor, epilepsy, psychiatric
      disease, cardiac or liver failure and visual or hearing disabilities. Approval of NRC ethical
      committee was obtained with registration number 15131. Participants signed a written informed
      consent before being included in the study.

      1.1 A face to face interview was carried out with all participants to collect data about
      demographics, medical history and cognitive function. Data included age, gender, level of
      education, and tobacco smoking. Detailed medical history of diabetes was taken from patients
      including age of diagnosis of diabetes, frequency of hypoglycemic attacks, type of treatment
      (insulin or oral hypoglycemic drug), history of hypertension, dyslipidemia or other diseases
      and medications taken. Any complaint suggestive for presence of sensory peripheral neuropathy
      such as tingling, burning pain or numbness in hands and feet was recorded. All studied
      participants were subjected to thorough clinical examination, systolic and diastolic blood
      pressure and anthropometric assessment (weight and height measurements).

      1.2 Assessment of cognitive function with its domains was carried out using Adenbrooke's
      Cognitive Examination III (ACE III) test which assesses five cognitive domains: attention,
      memory, verbal fluency, language and visuospatial abilities. It is the most reliable and
      validated Arabic form cognitive scale and freely available to be applied. Objective mild
      cognitive impairment (MCI) was considered if ACE III score is less than 88. The Trail Making
      Test (TMT) was also used. TMT is one of the most widely used instruments in
      neuropsychological assessment as an indicator of speed of cognitive processing and executive
      functioning. It assesses attention, concentration, psychomotor speed, cognitive shifting and
      complex sequencing function by measuring the time required to connect a series of
      sequentially numbered and lettered circles. The test consists of two parts (A and B). The
      direct score of each part is represented by the time of completion of the tasks. Shorter time
      indicates better cognitive function.

      1.3 Hamilton Rating Scale for Depression (HRSD) was used, it is one of the most widely used
      scales that objectively rate depression severity. The scale includes 17 items, each one is
      scored on a 3 or 5 points scale, with the final score indicative of the severity of the
      patient's depression. The classification of depression level was carried out depending on the
      National Institute for Health & Clinical Excellence of the United Kingdom (UK) (0-7 = Normal,
      8-13 = Mild depression, 14-18 = Moderate depression, 19-22 = Severe depression and ≥ 23 =
      Very severe depression).

      1.4 Assessment of health related quality of life (HRQOL) was carried out using Short Form-36
      (SF-36) questionnaire. The questionnaire contains 36 items comprising eight domains of health
      status which are physical functioning (PF), role physical (RP), bodily pain (BP), general
      health perception GH), vitality (VT), social functioning (SF), role emotional (RE) and mental
      health (MH). Scoring of each domain is from 0 to 100, with a score of '100' as the best
      rating of health. The Arabic version of the questionnaire was used.

      1.5 Assessment of T2DM patients' life style. Patients were given a designed questionnaire to
      assess their life style regarding nutritional habits, physical, social and mental activities
      and the opinion about the best way to motivate them for changing their lifestyle.

      1.6 Baseline Laboratory analysis. Five ml blood sample was withdrawn under complete aseptic
      technique from each participant, after fasting for at least 10 hours at the beginning of the
      study. It was divided into dry plain and lavender ethylene diamine tetra acetic acid (EDTA)
      vacutainer tubes. Level of glycated hemoglobin (HbA1c) and total blood cholesterol were
      assessed using Olympus-au-400 chemistry auto-analyzer. Malondialdehyde (MDA) concentrations
      in serum was determined using ELISA kit based on the competitive binding enzyme immunoassay
      technique. Erythrocytes were examined for the enzymatic antioxidant activity of catalase,
      glutathione peroxidase (GPx) and glutathione reductase (GR).

      1.7 For miRNAs expression analysis: Blood was drawn into special RNA preservation tubes;
      inverted 8-10 times, and then stored frozen at -80°C for further RNA extraction. RNA
      isolation and estimation of RNA quality was employed prior to initiating the characterization
      of miRNA expression. Only 1-10 nano grams of purified total RNA or equivalent was enough to
      reliably quantify the miRNAs of interest. The concentrations of miRNAs selected in this study
      was determined by the single target TaqMan miRNA quantitative Real Time (qRT) Polymerase
      Chain Reaction (PCR) assay (Applied Biosystems) in the plasma samples according to the
      manufacturer's instructions. A two-step protocol were applied which requires only reverse
      transcription with a miRNA-specific primer, followed by real-time PCR with TaqMan probes. The
      TaqMan MicroRNA Assays is a target-specific stem-loop structure reverse-transcription primer.
      The stem-loop accomplishes two goals: 1) specificity for only the mature miRNA target, and 2)
      formation of a Reverse Transcriptase (RT) primer/mature miRNA-chimera, extending the 5' end
      of the miRNA. RT reactions containing RNA samples, looped- primers, 1 X buffer, reverse
      transcriptase, and RNase inhibitor will be incubated for 30 min each, at 16°C and at 42°C.
      The resulting longer RT amplicon presents a template amenable to standard real-time PCR,
      using TaqMan assays. Then Real-time PCR was performed on Quant Studio 12 K Flex Real-Time PCR
      Instrument.

      Second step (interventional trial): This step was an interventional study for health
      education and life style modification. It was carried out to Implementing the life style
      modification program on T2DM patients.

      2.1 Designing two health educational booklets After identifying the T2DM participants' needs,
      the research team designed two health education booklets. The first booklet titled "Diabetes
      mellitus and how to control its complications". The booklet includes information about T2DM
      disease and, how it affects different body systems leading to Diabetes Mellitus (DM)
      complications. It includes also information on normal and abnormal level of fasting blood
      glucose and glycated hemoglobin (HbA1c). It demonstrates how to control and prevent DM
      complications, stressing on healthy nutrition and carbohydrate counting, physical activities,
      strict adherence to medical regimen and avoiding stress. The second booklet titled "Mild
      cognitive impairment and how to prevent" it includes information about definition of
      cognitive function, MCI and Alzheimer and its relation with T2DM and risk factors for
      cognitive impairment and how to control and improve cognitive function.

      2.2 Patient education program implementation: Only 144 participants accepted the invitation
      to attend the health education (HE) sessions. The actual number who attended were only 121.
      The reasons for non-participation include: lack of interest and they refused commitment to
      the life style program.

      2.2.1 Face to face and group discussion HE sessions to encourage life style modification: All
      the studied patients were invited to attend the HE sessions. Seven sessions were held at NRC
      over a period of nearly 2 months explaining the two designed booklets. HE lectures were
      presented by the public health team and a diabetic expert who shared in the group discussion
      to explain and answer the questions related to management of T2DM. HE program was provided
      for controlling DM, information about foods important to brain health and different
      intellectual games to improve their cognition.

      2.2.2 Dissemination of Educational Materials The prepared booklets were distributed to all
      attendants. Participants were informed about the importance of these two booklets in
      improving their scientific information on T2DM and MCI.

      2.2.3 Reassessment of laboratory analysis. Three ml of blood sample was withdrawn under
      complete aseptic technique from each participant, after fasting for at least 10 hours at the
      time of health education. It aimed to assess FBG, HbA1c and total blood cholesterol just
      before the intervention and were reassessed three months during the intervention to be an
      incentive for the patients as a monitoring the progress they attained due to life style
      modification. Each participant was asked to receive his laboratory results personally from
      the research team. This allowed to discuss the interpretation of his results and thus
      re-encourage or give him any needed advice to maintain his adherence to the lifestyle
      modification program.

      2.2.4 Clinical follow-up and management were carried out by specialist in endocrinology and
      neurology depending on laboratory results and patient compliance.

      2.2.5 Individualized Medical Nutrition program: Each participant was asked to complete a
      3-day food diary. These were discussed with each participant individually through a
      face-to-face interview in order to advise and convince them to change their dietary habits .
      Every patient was taught how he can regulate his diet depending on the result of laboratory
      analysis and carbohydrate count.

      2.2.6 Individualized biweekly follow-up scheme was also distributed as a guide for the
      targeted healthy behaviors needed to be changed which allowed self-monitoring of their
      achieved progress. Over a period of 6 months, the participants were followed up through
      mobile phone, using designed sheet, to assess patients' compliance and adherence to the life
      style modification program.

      2.2.7 WhatsApp phone application group: The participants were welcomed to contact the
      research team whenever they had any inquiry concerning life style or treatment modifications.
      Through this group, 12 audio messages were recorded and distributed. These messages
      summarized the content of the booklet, to be an easily accessible way for the patients to
      help them follow the health education. Tips for healthy habits as well as exercises for
      mental activities were also uploaded to this group.

      2.3.8 Final reassessment of clinical status and laboratory profile. Six months after
      recruitment, a face-to-face interview was carried out with the 98 participants who continued
      till the end of the study. They fulfilled a final sheet to assess their compliance to the
      program. ACE III test and TMT with its two parts A and B tests were reassessed with clinical,
      weight and blood pressure reassessment to evaluate the impact of the lifestyle modification
      program on cognitive function. Quality of life and depression were reassessed using SF-36 and
      HRDS questionnaires respectively. Laboratory analysis for the level of HbA1c, lipid profile,
      oxidant and antioxidant were reassessed after the intervention.
    

Intervention name(s): Health education program
Intervention type(s): Behavioral
Intervention description(s): Seven face to face sessions and group discussion health education sessions. Two prepared booklets were distributed to all attendants titled "Diabetes mellitus and how to control its complications" and "Mild cognitive impairment and how to prevent it".
Lab results were discussed individually with each participant to discuss his adherence to the lifestyle modification program.
Clinical follow-up and management with endocrinology and neurology specialists. Individualized Medical Nutrition. Participants were then counselled on healthy eating habits.
Individualized biweekly follow-up scheme through mobile phone. A WhatsApp group was created for all participants to contact the research team whenever they have any inquiry concerning life style modifications. Through this group, 12 HE audio electronic messages were shared with participants.
Final reassessment of clinical status and laboratory profile was carried out with participants who continued till the end of the study.
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Supportive Care
Keywords: Diabetes mellitus; Health education; Depression; Quality of life; Healthy life style; Cognitive function