الفهرس 
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Abstract
The present study was conducted to investigate the effect of the most commonly used soft tissue filler Botulinum toxin (Botox) used separately or coupled with epidermal growth factor on the submandibular salivary glands of adult female Albino rats. Structural, ultrastructural and immunohistochemical investigations were performed on the examined groups.
Sixty healthy adult female Albino rats, three months old and 200-220 gm body weight were used in this study and were divided into three groups:
group 1.1: consisted of 20 rats and served as controls.
group 2.1:consisted of 20 rats that were subjected to subcutaneous injection of 2.5 unit botulinum toxin type A in the region of the submandibular salivary gland.
group 3.1:consisted of 20 rats injected subcutaneously with 2.5 unit botulinum toxin in 0.1 ml saline as in group 2.1 and on the next day they were subjected to daily intra peritoneal injection of epidermal growth factor (EGF)in a dose of 10 µg/Kg body weight [2] for sixty days.
At the end of the experiment, the animals of the whole groups were sacrificed by cervical dislocation and their submandibular salivary glands of the right and left sides were dissected out.
Those of the right side were fixed in 10% neutral buffered formalin for 24 hours, washed, processed, embedded in paraffin, then sectioned.
Six microns thick sections were stained with:
3- Hematoxylin and eosin stain for structural examination to find out any possible microscopic changes.
4- Immunohistochemical localization of cytokeratin, myosin and E Cadherin in the glandular tissue.
The specimens of the left sides were used for Ultrastructural examination withtransmission electron microscope.
The results of this study revealed the following:
Histological results:
Histological sections of submandibular salivary glands of rats injected with BTX lacked the normal architecture. The acini appeared to be shrunken with apparent decrease in size and number and the cells were filled with many cytoplasmic vacuoles. Ducts lost their normal histological features specially the striated duct which lost its basal striations and contained many cytoplasmic vacuolations. The granular convulated tubules showed marked decrease in their granularity.
As compared to submandibular glands of group II, those of group III showed that the glands almost regained their normal appearance. However, minimum degree of atrophic and degenerative changes among the acini and the ducts were encountered in some regions.
Ultrastructural results:
BTX group ultrastructural results revealed that the gland suffered from degeneration in its acinar and ductal cells. Nuclei were pyknotic, darkly stained with loss of continuity of the nuclear membrane which was often irregular. The cytoplasm of the serous acinar cells revealed numerous large vaculations. RER were dilated and mitochondria showed signs of degeneration manifested as loss of their cristae and even total rupture. Intracytoplasmic vacuolizations were also detected. Decrease in desmosmal junctions between cells was encountered in many areas.
Ultrastructure examination of submandibular glands of rats treated with both botox and EGF revealed that the glands almost regained its normal architecture. The acinar cells appeared pyramidal in shape with rounded basally situated normal nuclei. Nearly normal RER and mitochondria were observed. Very few, if any, cytoplasmic vaculations were found. Normal desmosomal junctions between cells were present.
Immunohistochemical findings:
E cadherin, cytokeratin and myosin expression in the experimental groups were detected using immunohistochemical methods. Analysis of the serous acini and ducts of the submandibular gland of both BTX and EGF groups revealed that the expression of all three proteins was weak in the BTX group while the EGF showed a strong reactivity.
The difference in the reactivity was statistically confirmed where a significant difference was found when comparing expression of E cadherin, myosin and cytokeratin in groups 2.1 and 3.1.