Neuronal and Astrocytic Structural Changes in the Prefrontal Cortex of the Male Wistar Rat Following Chronic Khat Use

Abstract

Background Chronic and Long-term use of khat may cause neuro-cognitive changes, which have been elucidated in behavioural studies. These may correlate with structural changes in the cytoarchitecture and histomorphometry of neuronal cells. With current research showing the centrality of astrocytes and other glial cells in neuronal signalling, there is possibility that these cells are also affected by chronic khat use. There’s little literature on the structural changes in the prefrontal cortex neuronal and astrocytic cytoarchitecture and morphometry in chronic khat users. Objective To describe the changes in neuronal architecture and density, as well as astrocyte morphology in rats after long-term use of khat (miraa). Study design Randomized experimental study design. Materials and Methods Young adult male Wistar rats, aged 2-3 months, weighing 200-300 grams were used in this study. They were randomized into four groups of 11 each (control, K500, K1000 and K2000) to

correspond with those used as controls, those that received 500mg/kg, 1000mg/kg and 2000mg/kg body weight khat extracts respectively. We purchased fresh khat leaves from Mikinduri market in Meru and prepare crude extract using a validated method. The control rats were fed on normal diet, while experimental groups were fed on normal diet and khat extracts using oral gavage for 6 weeks. The animals were sacrificed and their brains removed. They were processed with Haematoxylin and Eosin and Toluidine blue for general histology. We performed immunohistochemical visualization of individual neurocellular populations across the four animal groups as follows: Glial Acidic Fibrillary Protein for astrocytes, 2’, 3’ cyclic nucleotide phosphodiesterase for oligodendrocytes and doublecortin for immature neurons. Data Analysis Photomicrographs of the stained sections were transferred to Image J-Fiji software to study normal and apoptotic pyramidal neuronal cell density, astrocyte density, oligodendrocyte density and the density of immature neurons. Data was entered into SPPS, IBM version 28.0 for analysis. We used Kruskal-Wallis H test to correlate the four animal groups in terms of neuronal densities and astrocyte densities. Results The mean body weight, average brain weight and maximum cortical length of the rat brains demonstrated a significant decrease with increasing khat doses compared to controls. We observed a significant increase in the density of apoptotic pyramidal neurons in experimental

groups compared to controls (p=0.027), while a decrease in normal pyramidal neurons was noted with increasing doses of khat. Further, we observed a non-significant increase in immature doublecortin staining neurons in khat-fed rats compared to controls. There was observed a significant increase in immunoreactive astrocytes with high khat doses (2000mg/kg), coupled with increase complexity of astrocytic processes and gliosis. No group differences were noted in immunoreactive oligodendrocyte density with khat use although there was discernible reduction in myelination with increasing doses of khat. Conclusions This study has demonstrated a reduction in gross cortical indices, an increase in pyramidal neuronal apoptosis, a reduction in pyramidal neuronal density with increasing khat doses. This underscores the potential neurotoxic effects of high khat doses. The increase in astrocyte complexity, reduction in myelination and increase in immature neuroblasts in high khat doses compared to controls adds insights into the potential mechanisms involved in khat-induced brain changes, as well as the role of adult neurogenesis in substance use. Recommendations We recommend that a longitudinal study should be designed to identify the histological and immunohistochemical changes in the prefrontal cortex along the continuum of khat use so as to identify the time when changes start occurring. To eludicate the process of neuronal loss, ki-67 and caspase staining should be performed.