Anat Cell Biol.  2020 Sep;53(3):330-341. 10.5115/acb.20.136.

Effect of caffeinated energy drinks on the structure of hippocampal cornu ammonis 1 and dentate gyrus of adult male albino rats

• Kamar SA
• Abdel Malak HW
• Adel Saad S

• KMID: 2507646
• DOI: http://doi.org/10.5115/acb.20.136

Abstract

Energy drinks are available worldwide and frequently consumed to increase energy level and compensate lack of sleep. Energy drinks consumers aim to improve their cognitive functions. Red Bull is the most popular energy drink consumed in Egypt. However, the link between the impact of energy drinks on the structure of hippocampal cornu ammonis 1 (CA1) and dentate gyrus (DG), a highly vulnerable brain regions to various insults, has not yet documented. To study the effect of energy drinks on structure of hippocampal CA1 and DG of adult male albino rats. Twenty one adult male albino rats were divided into three groups; group I control group, groups II and III received Red Bull, with a dose of 3.75 ml/kg/day orally using gastric tube for four and eight consecutive weeks respectively. At the end of the experiment, brains were dissected and hippocampal specimens were processed for histopathological and immunohistochemical studies. Histopathological examination of hippocampal sections in group II revealed vacuoles, decrease thickness of pyramidal cell layer with irregular dark or ghost nuclei. However, changes were more severe in group III with cracks in pyramidal cell layer, massive vacuolation and signet ring cells. Moreover, star shaped astrocytes and glial fibrillary acidic protein immuno-reactivity were more abundant in group III than in group II. Caffeinated energy drinks produced neurodegenerative changes and reactive astrocytosis in hippocampal CA1 and DG of adult male albino rats. These changes were duration-dependent being more severe in longer duration of intake.

Keyword

Energy drinks; Hippocampus; Cornu ammonis; Dentate gyrus; Energy drinks

Figure

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  Fig. 1 A photomicrograph of parasagittal section of hippocampus of control rats showing its 3 regions; CA1, CA2, and CA3 (H&E, ×100). The D appeared V-shaped, with its C. C, crest; CA1, cornu ammonis 1; CA2, cornu ammonis 2; CA3, cornu ammonis 3; D, dentate gyrus.

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  Fig. 2 A photomicrograph of a parasagittal section of control hippocampus showing CA1, with three layers of cells, P, R, and M (H&E, ×200). P layer is seen as a clear narrow zone with widely spaced small deeply stained oligodendroglia cells (arrowhead). R cell layer contains closely packed cells (arrow). The M layer contains widely spaced cells. CA1, cornu ammonis 1; M, molecular; P, polymorphic; R, pyramidal.

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  Fig. 3 A photomicrograph of a parasagittal section of control hippocampus showing R cells in CA1, R cells appear large closely packed triangular neurons (arrow) (H&E, ×400). Some with N and others with n. CA1, cornu ammonis 1; n, dense elongated nuclei; N, vesicular nuclei; R, pyramidal.

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  Fig. 4 Semithin section of control hippocampal CA1 showing dark closely packed large, triangular R cells with vesicular N and prominent n (Toluidine blue, ×1,000). CA1, cornu ammonis 1; n, nucleoli; N, nuclei; R, pyramidal.

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  Fig. 5 A photomicrograph of a parasagittal section of control hippocampus showing DG with packed granule layer (H&E, ×100). The G appear rounded to oval with large vesicular nuclei. S are present. DG, dentate gyrus; G, granule dark cells; S, deeply stained stem cells .

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  Fig. 6 A photomicrograph of a GFAP immunostaining of hippocampus of adult male control rat showing the normal distribution of apparently few brownish star shaped astrocytes in CA1 and D (GFAP, ×200). CA1, cornu ammonis 1; DG, dentate gyrus; GFAP, glial fibrillary acidic protein.

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  Fig. 7 Higher magnification of a GFAP immunostaining of hippocampus of adult male control rat showing the normal distribution of brown star shaped astrocytes (arrow) within the junctional zone between molecular layers of CA1 and DG (GFAP, ×400). CA1, cornu ammonis 1; DG, dentate gyrus; GFAP, glial fibrillary acidic protein.

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  Fig. 8 A photomicrograph of a parasagittal section of hippocampus from group II showing apparent preservation of general architecture with diffuse v in all its regions (H&E, ×100). v, vacuolation.

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  Fig. 9 Higher magnification of a parasagittal section of hippocampus from group II showing an apparent decreased thickness of principal neuronal cell layer of CA1 (R) (H&E, ×200). Note some neurons with dark nuclei and wide pericellular space (arrow). CA1, cornu ammonis 1; R, pyramidal cell layer.

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  Fig. 10 Higher magnification of a parasagittal section of hippocampus from group II showing many g within CA1 (H&E, ×400). Many congested BV are also observed. BV, blood vessels; CA1, cornu ammonis 1; g, ghost neurons.

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  Fig. 11 A photomicrograph of a parasagittal section of hippocampus from group II showing L and some G within the DG (H&E, ×400). An increased number of S were observed in the C of DG. C, crest; DG, dentate gyrus; G, darkly stained cells; L, lightly stained ghost cells; S, stem cells.

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  Fig. 12 Semithin sections of hippocampus from group II showing diffuse v within the DG. L with N and other G with n are seen. Some shrunken g are also observed (Toluidine blue, ×1,000). DG, dentate gyrus; g, ghost cells; G, dark cells; L, lightly stained cells; n, dark irregular nuclei; N, vesicular nuclei; v, vacuolations.

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  Fig. 13 A photomicrograph of a GFAP immunostaining of hippocampus from group II showing an apparent increase in the brownish positive immuno-reaction in all layers (GFAP, ×200). GFAP, glial fibrillary acidic protein.

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  Fig. 14 Higher magnification of a GFAP immunostaining of hippocampus from group II showing increased distribution of brownish star shaped astrocytes (arrow) in the junctional zone between molecular layers of CA1 and DG (GFAP, ×400). CA1, cornu ammonis 1; DG, dentate gyrus; GFAP, glial fibrillary acidic protein.

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  Fig. 15 A photomicrograph of a parasagittal section of hippocampus from group III showing preservation of the general architecture of the hippocampus. However, an apparent marked decrease in thickness of N was observed in all regions (H&E, ×100). N, principal neuronal cell layers.

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  Fig. 16 Higher magnification of a parasagittal section of hippocampus from group III showing that most of CA1 neurons have N (H&E, ×400). F are observed with n. Diffuse V within the R cell layer is also seen. CA1, cornu ammonis 1; F, signet ring cells; n, pyknotic peripheral nuclei; N, dark irregular nuclei; R, pyramidal, V, vacuolation.

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  Fig. 17 A photomicrograph of a parasagittal section of hippocampus from group III showing an apparent thinning of C of DG with widespread cracks (arrow) within the layer (H&E, ×200). C, granular cell layer ; DG, dentate gyrus.

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  Fig. 18 A photomicrograph of a parasagittal section of hippocampus from group III showing granular neurons of the DG with n, and signet ring neurons (arrow) (H&E, ×400). Intense V and C in between the granular cells are observed. Marked increase in the number of S cells in the crest of DG are also seen. C, cracks; DG, dentate gyrus; n, dark pyknotic nuclei; S, stem; V, vacuolation.

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  Fig. 19 A photomicrograph of a parasagittal section of hippocampus from group III showing loss of compact organization of the granular neurons of the DG with n, and ghost cells (arrow) (H&E, ×400). DG, dentate gyrus; n, dark pyknotic nuclei.

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  Fig. 20 Semithin sections of hippocampus from group III showing L with N & other neurons with n are observed. Multiple diffuse small V are observed (Toluidine blue, ×1,000). L, lightly stained neurons ; n, dark irregular nuclei; N, pale nuclei ; V, vacuoles.

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  Fig. 21 A photomicrograph of a GFAP immunostaining of hippocampus from group III showing an apparent intense increase in the brownish positive immuno-reaction in all regions (GFAP, ×200). GFAP, glial fibrillary acidic protein.

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  Fig. 22 Higher magnification of a GFAP immunostaining of hippocampus from group III showing wide distribution of large dark brown star shaped astrocytes with multiple thick processes (arrow) in the junctional zone between molecular layers of CA1 and DG (GFAP, ×400). CA1, cornu ammonis 1; DG, dentate gyrus; GFAP, glial fibrillary acidic protein.

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  Fig. 23 A bar chart showing pyramidal cell thickness (in µm) among the three groups. Pyramidal cells’ thickness is highly significantly reduced in one-month group and two-months group respectively compared to the control group (**P<0.01, ***P<0.001), and significant reduction was observed in two-months group compared to one-month group (#P<0.05).

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  Fig. 24 A bar chart showing pyramidal cell number among the three groups. Pyramidal cells’ number are highly significantly reduced in one-month group and two-months group respectively compared to the control group (**P<0.01, ***P<0.001), with more significant decreased number in the two-months group than one-month group (#P<0.05).

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  Fig. 25 A bar chart showing the percentage area of GFAP immuno-reactivity in the three groups. The area is highly significantly increased in one-month group and two-months group respectively compared to the control group (***P<0.001), with more significant increase in the two-months group than in one-month (###P<0.001). GFAP, glial fibrillary acidic protein.

Reference

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