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Updated: Jun 21, 2025
Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes
Published on: December 11, 2015
Changes in Glial Fibrillary Acidic Protein-Immunoreactive Astrocytes in the Prefrontal Cortex of the Male Rat
Paul Bundi Karau1,2, Paul Odula2, Moses Obimbo2
1Department of Human Anatomy, School of Medicine and Health Sciences, Kenya Methodist University, Meru, Nairobi, Kenya.
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.
