Changes in Glial Fibrillary Acidic Protein-Immunoreactive Astrocytes in the Prefrontal Cortex of the Male Rat following Chronic Khat Use

Abstract

Background: Long-term khat consumption is associated with significant neurocognitive changes, which have been elucidated in behavioral studies. With current research showing the centrality of astrocytes and other glial cells in neuronal signaling, there is possibility that these cells are also affected by chronic khat use. There is little literature on the structural changes in the prefrontal cortex neuronal and astrocytic cytoarchitecture and morphometry in chronic khat users.

Objective: The objective of this study was to describe the changes in astrocyte morphometry and structure in rats after long-term use of khat (miraa).

Materials and methods: Adult male Wistar rats, aged 2-3 months, weighing 200-300 g were randomized into four groups of 10 each (control, Group 1, Group 2, and Group 3) to correspond with those used as controls and those that received 500 mg/kg, 1000 mg/kg, and 2000 mg/kg body weight khat extracts, respectively. Fresh khat leaves were purchased from Maua market in Meru, and crude extract was prepared using lyophilization. The control rats were fed on normal diet, while the experimental groups were fed on normal diet and khat extracts using oral gavage for 6 weeks. The animals were sacrificed and their brains were removed. We performed immunohistochemical visualization of astrocytes using glial fibrillary acidic protein. Photomicrographs of the stained sections were transferred to ImageJ Fiji software to study the astrocyte density and astrocytic processes. We used Kruskal-Wallis test to correlate the four animal groups in terms of astrocyte densities.

Results: We observed an increase in the average number of astrocytes with increasing doses of khat compared to controls, with those in Group 3 (2000 mg/kg) having an exuberant reactive astrocytosis. Further, escalating khat doses resulted in increased glial fibrillary acidic protein immunoreactivity in the nuclei and astrocytic processes, gliotic changes, and increased complexity of astrocytic processes.

Conclusion: Chronic khat use, especially at high doses, results in reactive astrocytosis and astrogliosis, which may be part of the mechanisms involved in the cognitive changes associated with its use.

Keywords: Chronic consumption; glial fibrillary acidic protein; khat; reactive astrocytes.

Figure 1

Photomicrograph showing the square grids used in counting of astrocytes using ImageJ Fiji software (GFAP stain, ×400). GFAP: Glial fibrillary acidic protein

Figure 2

Photomicrograph of a coronal section through the frontal lobe of the right cerebral hemisphere. The PFC and its associated WM are located adjacent to the OC (toluidine blue staining, ×40). PFC: Prefrontal cortex, WM: White matter, OC: Olfactory cortex

Figure 3

Photomicrograph showing arrangements of astrocytes in the layers of the prefrontal cortex, with predominance of astrocytes in layers II and III (magnification ×100, GFAP Stain). GFAP: Glial fibrillary acidic protein

Figure 4

Graph depicting the general trend of astrocyte densities in each study group

Figure 5

Box plots of the astrocyte densities in each study group

Figure 6

Representative photomicrograph of the prefrontal cortex showing an increase in density GFAP-immunoreactive astrocytes with increasing doses of khat with relatively more cells and more exuberant branching in Group 2 (1000 mg/kg) and Group 3 (2000 mg/kg) (GFAP stain, ×400). GFAP: Glial fibrillary acidic protein

Figure 7

Photo micrograph of the prefrontal cortex of the control group (a). Note the appearance of a normal astrocyte having a relatively smaller nucleus (N) with indistinct nucleolus and fewer and shorter processes (P). In group 1 (b) (500mg/kg), the numerous astrocytes with stout ramifying processes (P). Nucleolus (N) become noticeable and evidence of mild astrogliosis is seen in the area delimited by a rectangle. While in group 2 (c) (1000mg/kg) an area of moderate gliosis is delimited by the rectangle. Note the densely staining cytoplasm of the astrocytes in the rectangle. In the experimental group 3 (d) (2000 mg/kg), there is a greatly increased number of astrocytes with severe astrogliosis in the area delimited by the white rectangle. Magnification = x 400 (GFAP)

Figure 8

Photomicrographs comparing the astrocytic processes in control group and group 3 (2000 mg/kg). Note the increased complexity and thickness of astrocytic processes in the experimental group. The black arrows show astrocytic processes in the control group while the red arrows demonstrate increased complexity and thickness of astrocytic processes in group 3 rats (Magnification ×400, GFAP)