Fig 1.
Mean body weights of the control group and the kola nut-treated groups at PND 1, 7, 14, 21 and 28 for n = 5 pups.
*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 for the indicated comparison.
Fig 2.
Mean brain weights of the control group pups and the kola nut-treated group pups at PND 1, 7, 14, 21 and 28 for n = 5 pups.
*P<0.05, **P<0.01 and ****P<0.0001 for the indicated comparison.
Fig 3.
Demarcations of layers of the cerebellum of control and kola nut extract-treated pups.
A microphotograph of the mid-sagittal section of a hematoxylin and eosin-stained folium surface of the cerebellar cortex from a control (PND14) rat pup. The red line shows the cortical thickness of the folium surface comprising the molecular (M), purkinje (P) and granular (G) layers, while the blue double arrow shows the molecular layer (M) thickness. Scale bar = 40 μm.
Fig 4.
Mid-sagittal sections of formalin-fixed, paraffin-embedded, H&E stained cerebellum of control (CON) and kolanut extract-treated (TRT) PND 21 pups.
The three layers of the cerebellar cortex are shown (ML is Molecular Layer; P is Purkinje; GL is Granular Layer). CON shows a normal cytoarchitecture of the cerebellum with viable Purkinje cells (black arrow). TRT1 and TRT3 show a Purkinje cell layer that is comprised of pyknotic and degenerated Purkinje cells (black arrow) and TRT2 shows a sparsely populated Purkinje cell layer (black arrow). Scale bar = 40 μm.
Fig 5.
Mid-sagittal sections of formalin-fixed, paraffin-embedded, H&E stained cerebellum of PND 14 pups.
CON shows a normal molecular layer (ML) with few cells scattered in the parenchyma. TRT1 shows thinner ML with a mild separation between the ML and granular layer (black arrow) with pyknotic and degenerated purkinje cells (black arrow heads). TRT2 shows thinner ML with sparse Purkinje cells (black arrow). TRT3 shows relatively normal ML with various morphologies (red thin arrow) in the parenchyma, slight separation of the ML and granular layer (black arrow), and pyknotic purkinje cells with central chromatolysis (black arrow head) Scale bar = 40 μm.
Fig 6.
Midsagittal sections of formalin-fixed, paraffin-embedded, H&E stained cerebellum of PND 21 pups.
Slide CON shows the absence of external granular layer (EGL) (arrow). Slides TRT1, TRT2 and TRT3 show retention of external granular layer (EGL) (arrow) following the ingestion of kola nut extract. The insets show the lower magnification of the photomicrographs. Scale bar = 40 μm for main image and 60 μm for inset.
Fig 7.
Mid-sagittal sections of formalin-fixed, paraffin-embedded, Nissl-stained cerebellum of PND 28 pups.
CON shows a normal cytoarchitecture of the cerebellum with viable Purkinje cells and normal flask-shaped morphology (black arrow). TRT1 shows loss of Purkinje cells and pyknotic cells (red arrow). TRT2 and TRT3 show Purkinje cell layer with pyknotic and degenerated cells and loss of normal morphology (red arrow). Scale bar = 40 μm.
Fig 8.
Midsagittal sections of the cerebellar cortex of PND 21 pups immunostained with anti-GFAP antibody.
CON shows astrocytes, most of which are not expressing detectable levels of GFAP (red arrowhead) within the granular layer. TRT1 shows upregulation of GFAP with astrocyte hypertrophy (red arrow head) and mild overlap of domain, while TRT2 and TRT3 show severe diffuse reactive astrogliosis with pronounced upregulation of GFAP, disruption of individual astrocyte domain, astrocytic hypertrophy and proliferation (red arrow) as well as pronounced overlap of astrocyte processes (black arrow). Scale bar = 40 μm.
Fig 9.
Midsagittal sections of the cerebellum of PND 21 pups stained with anti-Glial Fibrillary Acidic Protein (GFAP) antibody.
Slide CON shows absence of spongiosis in the white matter of the cerebellar folium of control pups. Slides TRT1, TRT2 and TRT3 show the presence of mild to severe spongiosis caused by degeneration of white matter following the ingestion of kola nut extract (red arrow). Scale bar = 40 μm.
Fig 10.
Photomicrographs of the cerebellar cortex of PND 28 pups immunostained with anti-GFAP antibody.
CON shows proper layering of the Bergmann glia (arrow) in the molecular layer (ML) of the control pups. TRT1 shows a loss of the Bergmann glia depicted by upregulation of GFAP reactivity (black arrow). TRT2 shows an upregulation of GFAP reactivity as well as a severe disruption in the layering of the Bergmann (black arrow). TRT3 shows a mild disruption in the normal layering of the Bergmann glia (black arrow). Note the hypertrophic astrocyte with overlapping processes (black arrow head). Scale bar = 40 μm.
Fig 11.
Formalin-fixed, paraffin-embedded cerebellum stained with anti-Bcl-2 monoclonal antibody.
CON shows mild expression of Bcl-2 and normal architecture of Purkinje cells. TRT1 shows layering of the Purkinje cells (rectangular box) with an overexpression of Bcl-2 protein in neurones. TRT2 shows necrotizing and necrotized Purkinje cells (red arrow head). TRT3 shows necrotized Purkinje cells with high expression of Bcl-2 protein. Scale bar = 40 μm.
Fig 12.
Purkinje cell densities of the cerebella of the control group (CON) and the kola nut-treated groups (400, 600, 800 mg/kg) at PND 14, 21 and 28 (n = 5 pups).
*P<0.05, **P<0.01 and ****P<0.0001 for the indicated comparison.
Fig 13.
The thickness of the molecular layer of the cerebellum in the control (CON) and kola nut-treated (400, 600, 800 mg/kg) groups at PND 7, 14, 21 and 28 (n = 5 pups).
*P<0.05 and **P<0.01 for the indicated comparison.
Fig 14.
Changes in GFAP expression of both the control (CON) and kola nut-treated (400, 600, 800 mg/kg) groups at PND 1, 14 and 28 (n = 5 pups).
*P<0.05, **P<0.01 and ***P<0.001 for the indicated comparison.
Fig 15.
Dose-dependent Bcl-2 protein expression in both the control (CON) and kola nut-treated (400, 600, 800 mg/kg) groups (n = 5 pups).
*P<0.05, **P<0.01 and ***P<0.001 for the indicated comparison.