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There is an absolute dependence of the concept of development on supply of adequately balanced nutrients especially during the perinatal age which is critical to development. Therefore, an upgraded nutrition is specially required during gestation and lactation, as this is the critical period of neurodevelopment. This study sought to investigate the effect of protein deficiency during gestation (F0) and lactation through to adolescence on neurological functions of subsequent (F1 and F2) generations, establishing the possible consequential mechanistic association. Rats in four groups were fed different rations of protein diets (PD) as formulated: 21% PD, 10% PD, 5% PD and control diet (standard rat chow, containing 16–18% protein), from adolescent through to gestation and lactation, next generations were weaned to the maternal diet group. Neurobehavioral studies (which include; Surface righting reflex, Negative geotaxis, Learning and Memory tests), brain oxidative stress and quantification of serotonin and dopamine levels in the brain were conducted. Result shows significantly altered neurobehavior, reflected in the reduction of reflex response and postural reaction score at P ≤ 0.05. There was also a transgenerational cognitive impairment of brain function in the F-generations, following perinatal protein malnutrition as shown in the Y-maze result, measuring spatial memory and Morris water maze result (cognition), providing a background for the observed sensorimotor response. The significant increase in dopamine level, decrease in the antioxidant capacity of the protein deficient brain groups are consistent with significantly altered serotonin system, critical to neurodevelopment and functional activities of learning and memory. Therefore, persistent early life protein deficiency mediates dysfunction in neurodevelopment and this involves life-long changes in key neurotransmitters and the brain redox status underlying the neurobehavioral display.
This study was carried out in accordance with the guidelines of health research Act 2004, for standard care and use of laboratory animal models. Ethical approval was given by the Health Research Ethics Committee (HREC) of College of Medicine University of Lagos (REC 11), Nigeria. Forty virgin female Sprague Dawley rats (aged 6–8 weeks) were obtained from the animal house of the College of Medicine University of Lagos, Lagos Nigeria (Figure 1). Following the ethics of the standard care of animal models in research, rats were kept in groups in clean and capacious plastic cages (seven per cage) under standard laboratory conditions including well aerated room, good lighting, with suitable temperature (30° ± 2°C) in a neat environment and at a 12-h light/dark cycle. The animals were divided into four (4) groups and acclimatized for 2 weeks, where they had access to standard rat chow and water ad libitum. Experimental Timeline of the events, from the study initiation to termination. PND, Postnatal days. The grouping was based on the diet received by the rat. Grouped rats received ad libitum formulated diets which include either of these diet formulations: 21% PD (protein diet), 10% PDD (Protein deficient diet 1), 5% PDD (Protein deficient diet 2), and CP (standard rat chow, containing 16–18% protein) throughout gestation and lactation, forming Groups 1 2, 3 and 4, respectively, and pups were weaned to maternal diet. The varying protein quality diet for rat was formulated using local materials such as: Maize, SBM, GNC, Fish meal cassava starch, oil etc., all in varying percentages toward the attainment of the desired percentage of protein in the diet. This feed was formulated by the help of some experts at federal institute of industrial research Oshodi (FIIRO). Formulated feeds were given to the rats. Vaginal cytology was evaluated 3 weeks pre-breed period, and the rats were time-mated with a certified reproductive male in each treatment group, the confirmed pregnant rats were separated to produce F1 generation, this process was repeated for F1 generation to produce F2 generation, while the different diet group feeding continues. Presence of spermatozoa in vagina smear confirms day 1 of pregnancy. Weaning was done at postnatal day 21–28. The test was evaluated on postnatal day 2 through to 6. This test was carried out to measure the motor function and coordination by placing pups in a supine position and the time taken to adopt normal position was recorded, scored within cut-off time of 0–3 s (Naik et al., 2015). The vestibular and proprioceptive functions were evaluated in rats (postnatal day 2–14, every other day) were placed head down on an inclined board (about 45°), the time it took the animal to show a postural reaction by turning upright normally was recorded and scaled to score (Naik et al., 2015). At six (6 weeks), offspring were randomly selected (n = 5) for different neurobehavioral trainings and trials. The test was conducted as a modified method described by Yarube et al. (2016), The number and the sequence of arms entered were also recorded. The parameters were activity, defined as the number of arms entered, and percent alternation, calculated as the number of alternations or triads. A wide plastic cylindrical tank of about 120 cm width, that is surrounded by a wall (45 cm) and filled with water that was made opaque to reduce light penetration (25°C) was set up for the experiment. A rescue platform, 10 cm high was hidden at the center of the tank, below water level. Rats were given three swimming sessions on each of five consecutive days. At trial, the time taken to locate the platform (escape latency) was measured (Wang et al., 2017). The brain serotonin and dopamine concentration in the rat were determined using high performance liquid chromatography (HPLC), Agilent 100 series with VWD detector degasser, Quat Pump, Col Com and a manual injector system. Striatum tissues in the brain were collected, homogenized, deproteinized by chilled acetonitrile and filtered using syringe filter in preparation for injection. Serotonin and dopamine contents were determined by comparing the peak height ratios of the standards (5-HT and dopamine) used as calibrators and the chromatogram of unknown (Ebuehi and Abey, 2016). The brain antioxidant capacity was measured using the following biomarkers. The reduced glutathione level was determined based on the yellow color formed after reacting with 5,5’dithiobis- 2-nitrobenzoic acid (DTNB), which is then read at 412 nm (Ellman, 1978). The activity of the enzyme catalase was analyzed by measuring the initial rate of hydrogen peroxide (50 mM) decomposition, where one unit is the amount of enzyme that hydrolyses 1 mol. of H2O2 per minute, following method described by Cohen et al. (1996). The super oxide dismutase (SOD) activity was determined according to the modified method of Sun and Zigman (1978), determined by the ability of SOD to inhibit the autoxidation of epinephrine, taken as the difference between superoxide anion production and decomposition, measured by the increase in absorbance at 320 nm. Malondialdehyde is red specie absorbing at 535 nm (Buege and Aust, 1978). It is formed from the breakdown of polyunsaturated fatty acids, serving as a convenient index for determining the extent of lipid peroxidation that reacts with thiobarbituric acid. Absorbance taken at 535 nm, corresponds to the concentration of MDA per mg protein. Nitric Oxide was assayed by the Griess reaction method (Bryan and Grisham, 2007). 0.2 ml of the Griess reagent mixed with 3 ml of the sample and incubated at room temperature for 10 min. Absorbance was read using the spectrophotometer at 540 nm. This procedure is not stoichiometric and thus, a standard curve of sodium nitrite was plotted in order to extrapolate the values for unknown samples. All data were analyzed with maternal and weaning diets as factors. Results are presented as the mean ± SEM. Statistical analysis was performed using GraphPad Prism 7.0 for analysis of variance (ANOVA) followed by post hoc Turkey’s test, when appropriate; P ≤ 0.05 is considered significant.
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