General Study Design
The procedure of this study consisted of an isometric handgrip exercise trial. One hundred and ninety two prehypertensives (n=192, males =105 and females =87, age, 39.04±6.4 years; body mass index, 25.45±2.72 kg/m2), were recruited for the study. All the subject were diagnosed and referred by the physician with a blood pressure level classified as prehypertension based on the classification of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. This represents a systolic blood pressure range of 120-139mmHg and diastolic blood pressure range of 80-89mmHg. The subjects had an age ranging from 30 to 50years. This is because inflammatory diseases and prehypertension have been found to have an increased risk in individuals 40years and above.
A screening session was conducted to assess the baseline parameters and blood pressure of the subjects and blood samples were collected. All blood sample collection and blood pressure measurements were done according to international guidelines. The sample population was randomly selected into any of the three groups. The subjects were asked to pick from a ballot box, concealed papers marked G1, G2 or G3 to determine which group the subjects should belong. A detailed procedure of the exercise was then given to the subjects before commencement of the exercise training.
Inclusion into the study was subject to a normal medical examination, determined by a consultant Physician. Only subjects, with no clinical evidence of infectious, inflammatory and/or chronic diseases were recruited. More so, subjects were not on medication and all the participants recruited were sedentary which was defined by a score of three or less using the Rapid Assessment of Physical Activity survey. The subjects were properly briefed and written informed consent was obtained. This study conformed to the Declaration of Helsinki and jointly received institutional ethical approval of Federal Medical Centre Asaba, Delta State (FMC/ASB/A81.VOLXII/101) and Faculty of Basic Medical Sciences, Delta State University, Abraka, Delta State (REC/FBMS/DELSU/18/16/103).
Subjects were excluded from the study if their age is below and or above the age range of 30-50years or have a queried health status with clinical evidence of chronic diseases, a blood pressure above or below the prehypertension level, on medications and/or subjects who declined to participate in the study. Other exclusion criteria for Isometric Hand Grip include individuals suffering from debilitating arthritis, carpel tunnel, peripheral neuropathy, an aneurysm, or mitral valve complications.
On arrival at the clinic on the first day, subjects were made to observe a 15 minutes seated rest after which their blood samples were collected for baseline levels of inflammatory cytokine (TNF-α, IL6 and IL10). A detailed explanation and a demonstration of the exercise protocol were given to the subjects and they were asked to report at the Physiotherapy clinic at 4.00pm, for the exercise daily. The training session for each day took place between the hours of 4.00pm and 8.00pm daily.
The subjects on arrival at the clinic were made to observe a 15 minutes seated rest after which they were asked to squeeze the dynamometer with their dominant hand twice, for a maximum of 2 seconds with a five minutes rest in between; so as to determine their respective maximum voluntary contraction (M.V.C) for each session. The mean of the two readings was taken as the MVC for each subject for that session. Subjects were thereafter instructed to squeeze and sustain the dynamometer for 2minutes at 30% M.V.C. The dynamometer pointer which read the scale gave a visual feedback to the subjects for the maintenance of the 30% M.V.C.
This procedure was repeated twice for each training session with a 5 minutes rest in between. The position adopted by the subjects throughout the exercise training was sitting with upper limbs supported on a table. The exercise protocol was done for 24 consecutive days. The group one (G1) discontinued with the exercise protocol after 24 days while the group two (G2) continued for another 24 consecutive days at 30% MVC. On the other hand, the group three (G3) continued with the exercise protocol for another 24 consecutive days but at 50%MVC. At the end of the 48 days, blood samples were collected again on the 49th day for assessment of the resting data of the inflammatory cytokines (TNFα, IL6 and IL10) parameters.
Blood Sample Collection and Analysis of Cytokines
Five milliliters of overnight fasting (10–12hr) blood samples were intravenously collected pre- and post-exercise. The pre-exercise blood sample collection was done on the on the first day of the screening prior to the commencement of the exercise between the hours of 7-9am and the post exercise blood sample was collected on the 49th day to the commencement of the exercise at the same time. After 10 min of resting in a chair, venous blood from the ante cubital vein was collected into a serum tube. Immediately following collection, blood samples were allowed to settle at room temperature for 20 minutes to 1 hour in the vacutainer tubes using standard aseptic techniques to be clotted, and then was centrifuged (1,000 g) at 4°C for 20 min to separate serum from plasma.
Serum samples were then allocated into 1.5 mL tubes and immediately frozen for further analyses. Commercially available ELISA kits were then used to measure interleukin 6 (IL-6) interleukin 10 (IL-10) and Tumor necrotic factor (TNF-α) concentrations. Samples were analyzed in duplicate and all techniques and materials were used according to the manufacturer’s instructions by qualified and licensed medical laboratory scientists. All samples for any one participant were analyzed using the same assay to eliminate inter-assay variance.
Data Collection and Analysis
Three major inflammatory cytokines were selected based on their peculiar role/characteristics and ease of laboratory assay with regard to available resources and facilities. These are interleukin 6 (IL-6) interleukin 10 (IL-10) and Tumor necrotic factor (TNF-α).which is a major pro-inflammatory cytokine and has been shown to modulate multiple signaling pathways with wide-ranging down streams effect [18,19]. TNF-α plays a vital role in the typical immune response, through the modulation of pathways that involve an immediate inflammatory response with subsequent proliferation and programmed cell death. It has been fingered in a number of inflammatory diseases, particularly in rheumatoid arthritis, ankylosing spondylitis, and chrohn’s disease  while IL-6 exert pro-inflammatory effect when the signaling receptor protein which activates the membrane-bound signaling receptor protein, thus, higher levels of cardiopulmonary fitness have been associated with lower circulating concentrations of both IL-6 and CRP at rest [1,2,9].
The collected data were statistically analyzed using the descriptive and inferential statistics. The descriptive statistics employed in this study were the mean and standard deviation. The inferential statistics used in the analysis of the data included a one tailed student’s t-Test to determine the intra-groups differences in the initial and final values of the parameters, of the three groups. One way analysis of variance was thereafter used to compare the means of the three groups to determine their level of relationship. Furthermore, a two tailed student’s t-Test was used to determine the intra-groups differences in the initial and final resting values of the parameters of group one and group two; and group two and group three to determine the effect of continuation (Duration) and increase in dosage (Intensity) of the isometric effort respectively.