الفهرس 
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Abstract
Cisplatin as a chemotherapeutic agent has been indicated for the treatment of various tumors or prior to radiotherapy and surgery. However, chemotherapeutic agents can lead to morphologic damage in salivary gland tissue (Mittal et al., 2015).
There are many clinical studies and researches which have looked into salivary gland damage-regeneration through ADSCs application, by means of systemic and local use. Both animal and human experiments have verified that ADSCs could represent a safe treatment strategy for salivary gland damage (Lombaert et al., 2017).
Platelet rich plasma has been the subject in different studies in medicine, but there was lack in studies handling the effect of PRP injection on SMG after chemotherapy.
- AIM OF THE WORK:
In the present study we aimed to test the hypothesis that PRP could play a role in the improvement of the structural changes that could occur in the cisplatin treated SMGs. In addition, we aimed to compare the possible treatment effect of both ADSCs and PRP and find out which is more effective and profound.
- METHODS:
- Experimental design:
The animals were housed in wire mesh cages, 5 rats each, in the Medical Research Centre, Faculty of Medicine, Ain Shams University, under controlled temperature and dark-light cycle. They were fed standardized diet and tap water was available ad libtium, as reviewed and approved by institution guide lines of Ain Shams University Ethical Committee with authorization number (688).
Sixty two adult male albino rats, weighing between (250-300 grams) were used in the present study. Ten rats were used as a source of PRP and the rest were included in the experimental design as the 4 main groups; each group was then subdivided into two subgroups; subgroup A (was sacrificed at day 10) and subgroup B (was sacrificed at day 18).
group I: (control subgroups): This group was consisted of 10 rats and was considered as a negative control which received intraperitoneal injection of saline (5mg/kg) on day 1 and 8 corresponding to the cisplatin buffer. Then rats were divided into 2 subgroups (IA, IB).
group II: (Cisplatin.gp): This group consisted of 14 rats which received intraperitoneal injection of cisplatin at a dose of (5mg/kg) on day 1 and 8 and was considered as positive control group, according to (Lin et al., 2018). Then rats were divided into 2 subgroups (IIA, IIB).
group III: (Cisplatin + ADSCs): This group consisted of 14 cisplatin treated rats at the same dose of (group II), ADSCs (2 × 106 cells/rat) were injected once intravenously via tail vein into the cisplatin treated rats after 72h from cisplatin injection, according to (Hany et al., 2017). Then rats were divided into 2 subgroups (IIIA, IIIB).
group IV: (Cisplatin + PRP): This group consisted of 14 cisplatin treated rats at the same dose as (group II), then rats received PRP in a dose of (0.5 mL/kg) by intraperitoneal injection (3 times/week), starting from 72 h after cisplatin injection. Rats of this group were divided into 2 subgroups: (IVA, IVB).
-Blood samples for osmotic fragility test: Blood samples were taken from each group at day 10 and 18 respectively, to monitor the systemic effect of cisplatin, ADSCs and PRP to support and confirm our histological results. We chose the median osmotic fragility as a systemic parameter. Two ml of blood from each rat in all subgroups were taken from the retro-orbital plexus. Hematocrit and erythrocytes mean osmotic fragility were determined on heparinized blood using Sysmex hemocytometer according to (Karale et al., 2017).
-Preparation of the specimens: At the end of the experimental period of each subgroup (10 or 18 days), the rats were anesthetized and killed by over dose of anesthesia. The submandibular glands were excised freely. Half the number of the specimens were prepared for examination by H&E, Caspase-3 and fixed in 10% buffered formaldehyde for 48 hours [The formaldehyde is buffered in PH 7.2 PBS]. The other half was prepared for transmission electron microscope and was fixed for 1 hour in buffered glutaraldehyde (2.5%) at 4°C temperature followed by 2 hours in osmium tetroxide (1%).
-Histo-morphometric analysis: This study was done using computerized image analyze and a digital video camera (C5060, Olympus, Japan) in order to assess:
1-Platelet concentration before and after separation: for conformation of the proper platelet rich plasma preparation.
2- Area fraction of acini (at day 10&18) under L.M, at x200 magnification.
3- Area fraction of apoptotic cells (at day 10&18) under L.M, at x200 magnification.
4- Median osmotic fragility at day (10&18): to monitor the systemic effect of cisplatin, ADSCs and PRP in order to support and confirm our histological results.
All Data was collected and tabulated.
-Results:
I- Routine examination by light microscope:
A) Cisplatin group (subgroup IIA &IIB):
Examining the H&E stained sections of this group revealed interstitial edema, shrunken serous acini with ill-define outline, atypical shape and multiple cytoplasmic vacuoles. IDs, SDs, GCTs and EDs showed signs of degeneration. Hyalinization of the fibrous connective tissue surrounding the excretory ducts as well as congested blood vessel engorged with RBCs also were detected. Subgroup IIB had the same histological appearance as subgroup IIA but was apparently worse.
B) Adipose derived stem cells group (subgroup IIIA &IIIB) & Platelet rich plasma group (subgroup IVA & IVB):
Examining the H&E stained sections of these two groups revealed apparent improvement compared to cisplatin group (subgroup IIA &IIB) in the form of less atrophied and shrunken glandular elements, less interstitial edema, serous acini with relatively regular outline and less cytoplasmic vacuoles. IDs, SDs, GCTs and EDs showed signs of regeneration. Hyalinization of the fibrous connective tissue surrounding the excretory ducts as well as congested blood vessel engorged with RBCs were also less than cisplatin group. Adipose derived stem cells subgroup IIIA had apparent better histological appearance than PRP subgroup IVA. On the other hand, PRP subgroup IVB had apparent better histological results than ADSCs subgroup IIIB.
II- Electron microscopic results:
A) Cisplatin group (subgroup IIA &IIB):
Ultra-structural examination of this group revealed interstitial edema, atrophied and shrunken acini with pyknotic nuclei, polymorphism and karyohexis. IDs, SDs, GCTs and EDs showed signs of degeneration. Dilated rER, swollen and ruptured mitochondria, multiple lysosomes, many secretory pools and abundant cytoplasmic vacuoles were detected in most glandular elements. Subgroup IIB had the same ultra-structural appearance as subgroup IIA but was apparently worse.
B) Adipose derived stem cells group (subgroup IIIA &IIIB)& PRP group (subgroup IVA & IVB):
Ultra-structural examination of these two groups revealed apparent improvement than cisplatin group (subgroup IIA &IIB) in the form of less atrophied and shrunken glandular elements, apparently less interstitial edema, serous acini with relatively regular outline, many open faced nuclei with prominent nucleoli and less cytoplasmic vacuoles. IDs, SDs, GCTs and EDs showed signs of regeneration. Well organized rER, lamellar mitochondrial cristae and regular configuration of most glandular elements were seen. ADSCs subgroup IIIA had apparent better ultrastructure appearance than PRP subgroup IVA. On the other hand, PRP subgroup IVB had apparent better ultrastructure results than ADSCs subgroup IIIB.
III) Immuno-histochemical, Hematological and Statistical analysis:
- Both ADSCs and PRP successfully enhanced the regeneration of SMG after the cisplatin induced cytotoxicity; this was represented by significant increase in area fraction of acini as well as significant decrease in area fraction of apoptotic cells in both groups compared to cisplatin group at day 10 and 18 respectively.
- Regarding comparison between ADSCs and PRP, at day 10 ADSCs showed better result represented by significant increase in area fraction of acini compared to PRP group.
- At day 18 there was a non-significant difference between ADSCs group and PRP group regarding area fraction of acini, area fraction of apoptotic cells and median osmotic fragility. These results were supported by our histological and electron microscopic findings.