Fig 1.
Effect of DM, and/or M. charantia on the (I) number of corpuses lutea; (II) number of neonates, (III) weight of neonates; (IV) length of neonates levels in the control and different maternal groups.
Values are expressed as means ± SEM; (n = 6); (Significant, * P < 0.05), (High significant, **P < 0.01).
Fig 2.
Effect of DM, and/or M. charantia on the (I) glucose; (II) HOMA IR, (III) QUICKI levels; (IV) GLUT4; (V) AMPK; (VI) insulin; (VII) leptin levels in the control and different maternal group in the different groups.
Values are expressed as means ± SEM; (n = 6); (a: Significant as compared with the control, P < 0.05), (ab: Significant as compared with DM group, P < 0.05).
Fig 3.
Effect of DM, and/or M. charantia on the neurotransmitters levels, acetylcholine (I) and dopamine (II).
Oxidative stress markers, Malondialdehyde (III), glutathione (IV), superoxide dismutase (V) and catalase (VI). Apoptotic markers, B-cell lymphoma 2 (VII) and B-cell lymphoma-2(VIII) associated activities in cerebellum tissue homogenate in the control and different treated maternal groups. Values are expressed as means ± SEM; (n = 6); (a: Significant as compared with the control, P < 0.05), (ab: Significant as compared with DM group, P < 0.05).
Fig 4.
Photomicrographs of H&E-stained cerebellum sections from control and different pregnant mothers: control (A & B) and BM (C & D) groups, showing normal histological structure of the cerebellar cortex three layers; the molecular layer (M) was pale eosinophilic with few cells, the Purkinje cell layer (P) revealed one row of large pear-shaped cells with vesicular nuclei and prominent nucleoli and the granular cell layer (G) exhibited densely packed rounded cells and darkly stained nuclei, and normal white matter (WM).
(E & F) Cerebellar sections of diabetic mothers showing Vaculation of the neuropil (yellow arrow), a significant loss of Purkinje cells leaving clear remnant spaces (green arrow), a marked pyknosis (red arrow) thin granular layer (black arrow) with marked loss of granule cells. M: molecular layer, P: Purkinje cell layer and G: granular layer. (G & H) Cerebellar sections of BM+DM treated group showing overall improvement in the different cerebellar layers The molecular layer (M), the Purkinje cell layer (P), the granular cell layer (G) and the white matter (WM). However, degeneration and pyknosis (red arrow) of a few Purkinje cells were noticed. (I): represents approximate Purkinje cells number in different experimental groups. * p < 0.05. Right panel 100X, left panel 400X.
Fig 5.
Photomicrographs of H&E-stained cerebellum sections from control and different offspring’s’ groups.
Control (A&B) and BM (C&D) groups, showing the normal four-layered structure of the cerebellar cortex; the external granular layer (EG), the molecular layer (M), the Purkinje cell layer (P) and the internal granular layer (IG). (E&F) Cerebellar cortex sections from diabetic mothers showing Focal loss of Purkinje cells leaving empty baskets (green arrow), marked necrosis with hypereosinophilic, shrunken, angular neuroplasm and nuclear pyknosis (red arrow). EG: external granular layer, M: molecular layer, P: Purkinje cell layer and IG: internal granular layer. (G & H) Cerebellar cortex sections from DM+BM group showing quite normal external and internal granular cell layer (EG&IG), molecular layer (M), while the Purkinje cell layer (P) displayed a few numbers of degenerated cells with pyknotic nuclei (red arrow). (I): represents approximate Purkinje cells number in different experimental groups. Right panel 100X, left panel 400X.
Fig 6.
Effect of DM, and/or M. charantia on the immuno-expressional level of GFAP protein in maternal cerebellar tissue of different groups.
(A & B): The control group revealed tiny astrocytes with short processes and a mild brown stain in the granular and molecular layers, as a typical positive GFAP cells. (C & D): BM group displaying normal positive GFAP cells and small astrocytes with short processes and weak to moderate staining of astroglial cells. (E & F): diabetic group demonstrates a higher prevalence and increased expression of GFAP positive cells and astrocytes. These astrocytes exhibit larger size and greater quantity, with longer processes primarily in the granular layer. Additionally, there are brownish astroglial cells scattered across several levels of the cortex that have a positive reactivity for GFAP. (G & H): BM+DM group revealed a moderate level of GFAP expression, accompanied by a reduction in the quantity and duration of astrocyte processes, in comparison to the diabetic untreated mothers. (I): GFAP labelling index using image j analysis program. left panel 100X, Right panel 400X. The values are expressed as the means±SEM of 5 microscopic fields/tissue samples of GFAP immune-expression, **P < 0.01.
Fig 7.
Effect of DM, and/or M. charantia on the immuno-expressional level of NGF protein in maternal cerebellar tissue of different groups.
Figs (A & B): the control group exhibiting typical expression of NGF protein in the molecular layer, Purkinje cells, and molecular layer. Figs (C & D): The BM-treated group had normal levels of NGF protein throughout all three layers. Figs (E & F): Diabetic mothers have a significant decrease in the expression of NGF protein across all three layers of the cerebellar cortex. Figs (G & H): The BM+DM combination therapy group demonstrated protection against synaptic damage induced by diabetes, with NGF protein levels approaching normalcy. (I): NGF labelling index using image j analysis program. left panel 100X, Right panel 400X. The values are expressed as the means±SEM of 5 microscopic fields/tissue samples of NGF immune-expression, **P < 0.01.
Fig 8.
Electron micrographs of cerebellar cortex sections from control mothers (A-D).
(A): Displaying typical Purkinje cells characterized by a viable nucleolus (nu) and eukaryotic nucleus (N), the cytoplasm of these cells is replete with rough endoplasmic reticulum (rer), mitochondria (m), and nerve fibers (nf). (B): granular layer shows granulocytes (Gc) with cytoplasm rich in rough endoplasmic reticulum (rer) and mitochondria (m). (C & D): showing intact neuropil with granulocytes (Gc), microglial cells (Mg), myelinated neural axons (*) forming synapsis, blood capillary (orange arrow) with endothelial cell (E) and red blood corpuscle (RBC) (green arrow) represents neuropil multivesicular body. (E–H) Cerebellar cortex sections from BM mothers showing, (E): exhibit normal Purkinje cells with normal eukaryotic nucleus (N) and noticeable nucleolus (nu), cytoplasm contains rough endoplasmic reticulum (rer) and mitochondria (m) and nerve fibers (nf) around it. (F): The granular layer exhibits granulocytes (Gc) characterized by cytoplasm with abundant mitochondria (m), neural synapsis and neuropil multivesicular body (colored arrow). (G & H): The image displays an undamaged neuropil with granulocytes (Gc), microglial cells (Mg), mitochondria (m), myelinated neural axons establishing synapses, a blood capillary (shown by an orange arrow) with an endothelial cell (E), and a red blood corpuscle (RBC).
Fig 9.
Electron micrographs of cerebellar cortex sections from diabetic mothers (I–L).
Cerebellar cortex sections from diabetic mothers showing, (I): reveals apoptotic Purkinje cell with indefinite nucleus and dilation in (rer) and dark lysosomal patches (L), swallowed mitochondria (m) could be noticed. (G–L): showing granular layer suffering from neuropil degenerative changes (blue arrow), abnormal granulocytes (Gc) and microglial cells (Mg), apoptotic bodied with pyknotic nuclei (red arrows) and dilated neural axons (*). Astrocyte of fibre-type (Ac) could be distinguished. (M–P) Cerebellar cortex sections from DM+BM group showing, (M): Combined treatment shows quite normal Purkinje cell with a typical eukaryotic nucleus (N) and a discernible nucleolus (nu), its cytoplasm is surrounded by nerve fibers (nf), mitochondria (m), and rough endoplasmic reticulum (rer). (N–P): showing the granular layer with a complete neuropil comprising granulocytes (Gc), microglial cells (Mg), myelinated neural axons (*) that have formed synapses, (green arrow) Denotes the multivesicular body of the neuropil.
Fig 10.
Electron micrographs of cerebellar cortex sections from different offspring’s groups.
The cerebellar cortex of both control (A–C) and BM-extract treated offspring (D–F) revealed intact neurons (blue arrow) with a distinct eukaryotic nucleus and prominent electron dense nucleoli, surrounded by numerous mitochondria (m). Clearly, there were neural axons (*) and active Ependymal cells (Ep) with distinct nucleoli and microvilli (green arrow) scattered throughout the neuropil. (G–I) Cerebellar fetal cortex from diabetic mothers depicts affected neurons with atypical nuclei (yellow arrow) or apoptotic with pyknotic nuclei (red arrow), neuropil showed vacuolated (v) with a fewer number of mitochondria (m). Neural axons showed not affected (*), while Ependymal cells (Ep) showed irregular with fewer microvilli (green arrow) in-between neurons (blue arrow). (J–L) Cerebellar fetal cortex from diabetic mothers treated with BM-extract revealed remarkable protection of the fetal cerebellar cortex characterized by an abundance of normal neurons displaying active nuclei and well-defined nucleoli (shown by the blue arrow), which were surrounded by a multitude of mitochondria (m). Evidently, neuropil included neuronal axons (*) and active Ependymal cells (Ep) exhibiting discernible nucleoli and microvilli (shown by the green arrow).