الفهرس 
INTRODUCTION AND AIM OF THE WOEKOnly 14 pages are availabe for public view
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Abstract
The present study had been performed to investigate the adverse effects of tramadol treatment on the chick embryos. To attain this goal, 240 Freshly fertile chicken eggs (Gallus gallus domesticus) weighing (50 ± 5 g.) were obtained from the Faculty of Agriculture Ain shams University breeder farm. Before incubation, eggs were cleaned with distilled water and 70% ethanol. After that, they were randomly positioned within a hatching incubator chamber that was set to a temperature of 38 ᵒC while horizontal. Using a drip pan filled with sterile water, a constant humidity in the range of 60% to 70% was kept. Every four hours, the incubator’s automated turner rotated the eggs to maintain equal environmental conditions and simulate the hens’ normal nesting behavior.
Drug administration and doses selection:
The acute toxicity of tramadol was determined by calculation of LD50 that is found to be 195 mg/kg egg-wt. Then, different doses of tramadol were selected ranging from the maximum tolerated dose (MTD) to the lowest dose with no observed adverse effect levels (NOAEL). Therefore, doses 12.5, 25, 50, and 100 mg/kg egg wt of tramadol seemed to be the best doses for the induction of congenital malformation in the tested chick embryos. As well, dose (50 mg /kg egg wt) of tramadol is equivalent to the human-recommended daily dose.
After 24 hours of incubation, chick embryos received a single dose of tramadol (12.5, 25, 50 or 100 mg/kg egg wt) dissolved in 0.5 ml of distilled water /egg by direct injection into the air chamber. Embryos were extracted on day 3, 6 ,12 and 18 of incubation for examination.
The results can be summarized as follow:
I-Morphological investigations:
The results revealed that, tramadol treatment caused increase mortality rate, heartbeats, reduction of crown-rump length. Besides remarkable external anomalies as exencephaly, acephaly, enlargement in the head region, hematoma, ectopiacordis, undeveloped beak, reduced external auditory aperture, ectopia visceral, anophthalmia, scanty feathers and limb deformities in embryos extracted on days 3, 6 ,12 and 18 of incubation as compared to untreated control groups in a dose dependent manner.
II-Angiogenesis evaluation:
On day 3 of incubation. A window of about 25 mm by 25 mm was made in the eggshell to allow microscopic imaging in the yolk sac membrane, (YSM). High-resolution images were captured using LCD digital microscope. Images were acquired from the yolk sac membrane of the embryos and their area vasculosa. The result showed that, tramadol exposure results in a significant (p< 0.05, p<0.0001) reduction in the fractal dimension (Df) value of the vessel plexus, total vessel length, and vessel percentage area, which could lead to a disruption and reduction in vascular branch patterning at all selected doses when compared with the controls.
III-Pax1 gene expression:
Pax1 gene was detected using the quantitative real time methylation specific polymerase chain reaction (QRT-PCR) assay in tissue homogenates of chorioallantoic membrane (CAM) of chick embryo at day 3 of incubation. The results recorded significant reduction (p<0.05, p<0.0001) in the mean values after drug administration at all doses as compared to control groups.
IV-Biochemical investigations:
On day 12 of incubation, a significant (p<0.05, p<0.0001) declination was documented in the volume of amniotic fluid exposed to all doses of the drug (12.5, 25, 50 and 100 mg /kg egg wt) and the percentage of change recorded -9.7%, -16%, -25% and -40% respectively when compared with the controls.
Liver enzymes in amniotic fluid of chick embryo on day 12 of incubation revealed a highly significant increase (P <0.0001) in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) enzyme levels with the administration of tramadol at all doses compared to control groups. While, total protein concentrations revealed a significant (p<0.01, p<0.0001) reduction at all doses of the tramadol treatment compared to controls.
Data of urea and creatinine concentrations showed a significant
(p< 0.01) increase in tramadol treated groups than control groups in a dose dependent manner, whereas amylase and glucose levels at all doses were significantly (p< 0.01, p<0.001) higher than controls.
Sodium concentration levels in the amniotic fluid of 12 days- old chick embryo significantly (p<0.001) increased after single injection with tramadol at all doses compared to controls. On contrast, potassium, calcium and phosphorus concentrations has been shown a significant (p<0.05 to p<0.0001) decrease at all doses of the drug administration related to control groups.
The total antioxidant capacity (TAC) recorded a highly significant (p<0.0001) decrease at all doses of tramadol administration when compared with the control groups in the amniotic fluid of 12 days- old chick embryo. This process leads to free radical formation or reactive oxygen species (ROS) which causes damage to the cell membrane and DNA fragmentation, resulting in cell death.
V-P53 and caspase 3 activites:
The levels of p53 and caspase 3 were determined by using a solid phase enzyme linked immunosorbent assay (ELISA) in the whole tissue embryos on day12 of incubation. All treated groups showed a highly significant increase (p<0.0001) in activities of both p53 and caspase 3 when compared to the control groups
VI-Histological investigations:
a. Neural tube defects (NTDs):
Examination of transverse sections through cranial regions of chick embryos at 3 days of incubation stained with Hematoxylin-Eosin showed that, Embryos treated with tramadol at doses of 25, 50, and 100 mg/kg egg wt had an open neural tube. Increasing drug dose was linked with increasing numbers of embryos with cranial neural tube defects. The changes were the highest in the group of embryos treated with a dose of 100 mg/kg egg wt as compared to the other groups. The percentages of neural tube defects were recorded at 20% (2/10), 50% (5/ 10), and 60% (6/10) in groups exposed to the drug at doses of 25, 50, and 100 mg/kg egg wt, respectively.
b. Eye:
Transverse sections in the head region of 6 days old chick embryos experimental groups showed more or less abnormalities in eye development. Groups treated with doses of (25 and 50 mg/kg egg wt) of tramadol showed unilateral microphthalmia indicated by optic cups growth retardation and underdeveloped lenses.
The histological structure of the eyes of chick embryos treated with high doses (50 and 100 mg/kg egg wt) of tramadol showed eye dysplasia represented by abnormal growth and differentiation of the embryonic retina, wide separation of the intraretinal space, anophthalmia, absence of lens and thickness of the inner nervous layer.
c. Liver:
The histological examination of the hepatic tissues of chick embryos at 18 days incubation period treated with 12.5 &25 mg/kg egg wt of tramadol revealed congestion and dilation in central veins, mild sinusoidal inflammatory cellular exudate, and the hepatic cells possessed a vacuolated cytoplasm.
Besides, in the experimental groups, 50 and 100 mg/kg egg wt tramadol-treated chick embryos showed prominent destruction of the normal pattern of the hepatic strands and appeared as coagulative masses of degenerative necrosis. The nuclei of these injured hepatocytes have also displayed marked symptoms of deterioration, some of them showed clear features of pyknosis, karyorrhexis, and karyolysis.
d. Kidney:
The histological examination of the kidney tissues of chick embryos at 18 days of incubation treated with 12.5&25 mg/kg egg wt of tramadol revealed shrinkage and contracted glomeruli and destruction of the parietal layer of Bowman’s capsule. Also, interstitial hemorrhage and the epithelial cells lining of the convoluted tubules exhibit cloudy swelling and hydropic degeneration.
Additionally, in the experimental groups, 50 and 100 mg/kg egg wt tramadol treated chick embryo showed hypertrophied, dilation and congestion of the glomerulus and Bowman’s capsule. Some of the convoluted tubules fused into coagulative mass (necrosis) with many pyknotic nuclei and lysis of renal tubules.
e. Heart:
The cardiac tissues of the chick embryo at 18 days of incubation treated with tramadol at doses of 12.5&25 mg/kg egg wt revealed unremarkable muscle fibers degeneration, Drug at dose 50 mg/kg egg wt exhibited vacuolar muscular degeneration, focal inflammatory cell infiltration, and necrosis of some cardiomyocytes. Heart tissues treated with 100 mg/kg egg wt of tramadol showed loss of the normal cardiac tissue configuration, distorted cardiac muscle fibers, and necrotic myocarditis.
VII-Skeletal abnormalities:
The endo-skeletal system of 18-day- old chick embryo of the control groups showed numerous bony elements stained with alizarin red and cartilages stained with alcian blue. The treatment with tramadol caused increase skeletal malformations, including cervical scoliosis, kinked tail and pygostyle, curved scapula, and retardation in the degree of ossification of some long bones in a dose dependent manner.