Effects of aqueous extract of kolanut (Cola nitida) on Sprague Dawley dams and exposure on the hippocampus of the progeny

Abstract

Background: Kolanut, a tropical nut eaten by people across sub-Sahara Africa, contains caffeine, theobromine, catechins, and tannins. Pregnant women often eat it to suppress morning sickness. This study investigated the effects of kolanut on the structure and functions of the developing hippocampus. Methods: Kolanut extract at 400 mg/kg of body weight infused in gel cubes was fed to 6 female and gel cubes without kolanut to control 6 female Sprague Dawley rats from the first day of mating till parturition. Several behavioral tests were administered on the pups namely surface righting (SR), cliff avoidance (CA) across different age group, post-natal day (PND) 4, 5, 6 & 7, head rising, head pivoting, locomotion, open field, novel object recognition (NOR) and location (NOL), and radial-arm maze (RAM) PND 21 and 56. Their hippocampi were subjected to histology [Nissl, Golgi-cox], immunohistochemistry (Ki67 and DCX), biochemistry [malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPX), transcription factors [brain-derived neurotrophic factor (BDNF), and acetylcholine (ACh)]. Expression of immediate (c-fos & c-jun) and memory genes (dlg3, dlg4, creb1 & creb2) were also determined. Results: Difficulty to get pregnant was observed in 33.3% of the dams; 16.7% had a still birth while 4.0% of the pups from dams fed kolanut-treated diet had paralysis of the limb. Kolanut significantly reduced the body weight (p < 0.001 – p < 0.0001) and increased brain weight, especially at PNDs 56 & 70 (p < 0.001 – p < 0.0001) of the dams and pups compared to the control. Food consumption was significantly (p < 0.05) lower for dams on kolanut-treated diet, but their water intake was significantly higher (p < 0.001). Kolanut significantly affected the behavioral indices of the animals; it significantly increased the latency of CA and SR tests across age groups (p <0.0001 and p < 0.0001 respectively). The frequency of head rising, frequency of pivoting and its latency, and locomotion were significantly (p < 0.01 – p < 0.0001) lower in animals fed kolanut-treated diet. Animals fed kolanut-treated diet showed anxiety during the open field tests on PNDs 21 and 56, and exhibited increased line crossing, corner time, distance covered and velocity (p < 0.05 – p < 0.0001). Frequencies of freezing episodes, grooming, rearing, fecal bolus and urination were also significantly (p < 0.01 – p < 0.0001) higher in animals that received kolanut-treated diet. The discrimination ratio of NOR and NOL were significantly 5 lower in animals fed kolanut-treated diet; they took longer duration to complete the tasks in RAM. Results from Golgi staining showed that kolanut caused pyknosis (p < 0.0001), inflamed soma, reduced aborization and synapses (p < 0.0001), reduction in spine quantity (p < 0.0001), change in morphology, fragmentation and constriction of the dendrites of the hippocampal neurons. When stained with DCX, the hippocampi of animals fed kolanut showed decrease in mean density of neuronal cells (p < 0.0001), increase number of pyknosis (p < 0.0001) and chromatolytic cells (p < 0.0001), neuronal atrophy, clumping, multi-layering and gliosis of the DG cells. In addition, the Ki67 showed a significant loss of proliferating cells (p < 0.0001) in the sub-granular zone of the DG. Kolanut significantly (p= 0.0067) increased the MDA and glutathione peroxidase levels. BDNF and ACh were significantly (p = 0.0001) increased by kolanut. A significant and positive correlation was found between SRT and MDA in PND 7 (r = 0.99, p = 0.0059), grooming and MDA in PND 56 (r = 0.77, p = 0.0252), grooming and BDNF in PND 21 (r = 0.76, p = 0.0297). A significant negative correlation was obtained between rearing and ACh in PND 56 (r = -0.83, p = 0.0115), Ki67 and MDA (p = 0.0213), and DCX cells and BDNF (p = 0.017). Prenatal kolanut consumption caused downregulations of cfos mRNA and cjun mRNA and creb1levels, an increase in creb2 level and a significant reduction in the levels of dlg3 mRNA and dlg4 mRNA, thus affecting the dendrites and spine morphology. Conclusion: Prenatal kolanut consumption adversely affected food intake, behavior, neuronal morphology, decreased neurogenesis and neuroplasticity and resulted in downregulation of genes important for normal development of the neurons and synapses. It exerted anti-neuroprotective effects by inducing oxidative stress, altering cholinergic system activity, stimulating over- expression of BDNF protein and concomitantly causing changes in morphology of the hippocampal neurons. Pregnant women and those of reproductive status need to be made aware of the adverse effects of kolanut consumption during pregnancy.