A study of alfa feto protein parameter in serum and histological effects of pups liver by SEM in pregnant rats exposed to overdose of aminoglycoside antibiotics

Imprint

Ashraf raoof mohammed ali, Noor I.Abdul-Zahra, Doaa Amer Kadhim. A study of Alfa Feto Protein parameter in Serum and Histological Effects of Pups Liver by SEM in Pregnant Rats Exposed to Overdose of Aminoglycoside antibiotics. Cardiometry; Issue No. 28; August 2023; p. 48-52; DOI: 10.18137/cardiometry.2023.28.4852; Available from:

• I.    INTRODUCTION

  During pregnancy many pregnants are on medications for acute infections related with pregnancy (e.g. heart burn and morning sickness), and also utilized for chronic illness management, predating or developing during pregnancy (e.g. epilepsy, asthma, depression, psychosis, chronic hypertension and genitourinary bacterial infections), sometime a treatment is endorsed to the mother to treat fetal diseases (e.g. fetal dysrhythmia) (Yates and Thomas, 2016).Little is thought about the impacts of human fetal exposure when another medication is approved unless it is specifically prescribed for use in pregnancy, Since numerous factors may add to adverse fetal impacts, having comprehensive information about in utero exposures will enhance our capacity to make correct determinations about causality (Clemow et al., 2015).The pregnancy duration represent critical periods in which the pregnant mother is exposed to external impacts and the most likely effects are the period called the embryonic period, which extends in the human fertilized stage until the end of the eighth week of pregnancy, during this period the three germinal layers are formed which included ectoderm, mesoderm and endoderm and also neural crest cells, it is known that different body organs originate from these germinal layers (Deye et al., 2016).It has been confirmed that antibiotics can damage developing fetal organs during pregnancy (Romero et al., 2007; Sachdeva et al., 2009). Placenta is permeable to the most antibiotics, for example gentamicin and cephalotin preserve in amniotic fluid and embryonic tissues in high concentration, it is worthy to note that permeability of placenta to various antibiotics varies during pregnancy, as it is reduced to Gentamicin in the last days of the pregnancy but increase under Cephalotin effect (Schuenemann, et al., 2007).Gen-tamicin is antibiotic an aminoglycoside derived from Micomono- sporapurpurea, it has a bactericidal effect

  on gram-negative microorganisms by preventing the production of protein in the bacterial cell therefore it is used in the treatment of a number of infections caused by a number of bacterial species such as respiratory infection, skin, gallbladder, bile ducts, urinary tracts, meningitis, septicemia diseases (Narayana, 2008).Gentamicin is quickly absorbed after intra muscular injection and mainly diffused in extracellular fluid (peak serum levels usually reached within 30 to 90 minutes) and it accumulates in the renal proximal convoluted tubules (50 to 100 times more than that in serum). Although gentamicin passes through the placenta and is excreted in breast milk, it is not absorbed from the gastrointestinal tract. Gentamicin concentration in umbilical cord blood and the placenta reaches 50% and 8% of mother’s blood serum respectively, after 1-2 hours of intra muscular injection (Pacifici and Marchini, 2017).In mammals, the peri-implantation is considered as a developmental period, implantation and signaling for pregnancy maintenance, which are a periods for floating time after zona pellucida hatch, but the cells growth and differentiation during embryonic development (Elmetwally et al.,2019).Implantation is defined as the process by which the embryo attaches to the endometrial surface of the uterus and invades the epithelium and then the maternal circulation to form the placenta (Mesiano, 2019).

• II.    MATERIAL AND METHODS

  • 2.1    Animal Model:

• 2.2    Drug used

• 2.3    Experimental croups:

This study achieved on pregnant white rat Rattus norvegicus females (15) and males(5) for mating. All rat weights ranging from 200-250 g. They should be in good health. The rats are placed in plastic cages with metal covers, 48 cm wide, 15 cm wide and 7 cm wide. The sawdust, which should be replaced three times a week, is considered in its care to clean the hatching of the special diet and plastic bottles can be used to make a watering tough with a cork equipped with metal pipes. The animals are placed under suitable laboratory conditions in terms of temperature 18-26 Cº and light/dark cycle 10/14 and ventilation rate time/hour 10-15 and also the relative humidity 30-70 (Tan and Tan, 2017).

From the ampoule of gentamicin, required dosages can be administered to animals of varying body weights as illustrated in table below:

Standard dose	Stock solution	Animals body weight (g)	Calculated dose in mg	Equivalent dose in ml
Gentamicin, 80 mg/kg for Rats	80 mg/2ml =40 mg/ml	100 g	8 mg	0.20 ml
		150 g	12 mg	0.30 ml
		250 g	20 mg	0.5 ml

Single dose= 20mg /kg/day (equivalent 0.5 ml of gentamicin).

Double dose= 40 mg /kg/day (equivalent 1 ml of gentamicin).

The average weight of animals = 250 g equivalent 0.25 kg (Erhirhie et al., 2004 ).

First: Control group: included five female rats injected by normal saline (Nacl 0.9%) intraperitoneally for seven days, the group sacrifice it in the end of experiment.,

Second: first treated group : included five female rats injected intraperitoneally by dose 20mg /kg/day of gentamicin for twenty days, the group sacrifice it in the end of experiment.

Third: second treated group : included five female rats injected intraperitoneally by dose 40mg /kg/day of gentamicin for twenty days, the group sacrifice it in the end of experiment.

• 2.4    Animals sacrifice and collection of pups liver

  The experimental animals of all groups were sacrifice after general anesthesia by combination of Ketamine: Xylazine (90mg/ kg: 10mg/ kg intraperitoneal), used ketamine 0.5 ml & xylazine 0.1 ml to each 250 g of body weight for anesthesia when sacrifice the female rat from the control & treated croups, after the anesthesia the females rats put in anatomical dish and made linear incision by scissors in abdominal region for extraction the liver that contains for collected, by anatomical tools. Saved in containers contains 10% formalin (AlTameemi, 2014).

• 2.5 : Scanning Electron Microscope SEM

Procedure:

• A.    Glutelaldehyde Fixation

• 1.    Prepare fixative solution and fill the scintillation vials with fixative.

• 2.    Cut specimen tissue and place in tube with glute-laldehyde solution.

• 3.    Place tube on its side to keep the air away from the tissue.

• 4.    Incubate at 4 Cº for 12-24 hours (Overnight is good).

• B.    Osmium tetroxide fixation

• 1.    The solution can be stored in the cold room or freezer for about one month. If frozen, thaw at room temperature. This solution should be straw colored. If it is purple it is no longer good.

• 2.    Dilute to a 1% solution in 25 mM phosphate buffer.

• 3.    Pour off fixative and add the 1% osmium tetroxide solution.

• 4.    Incubate in the cold room overnight to several days. The osmium turns black.

• C.    Dehydration of tissue

• 1.    Pour off osmium solution and rinse 3 times with 25mM phosphate buffer.

• 2.    Put tissue through an alcohol series. 15 to 30 minutes each step.

• 1:    Alfa Feto Protein parameter

  The physiological measurement of Alfa Feto Protein in all expimental groups show negative results due to absent of malformations in pups and this agreement with may studies.The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development (Gabant et al.,2002). The developed methods incorporating panning and multiparametric FACS to enrich for fetal hepatoblasts. The procedures yielded predominantly single cells with greater than 90% viability, and at E15,95% of the whole organ weight was recovered in the final cell preparation. The panning procedures removed 84% of the total cells, reduced the hemopoietic and endothelial cell fraction from greater than 90% to approximately 70% and enriched hepatoblasts by greater than fivefold. The increase in the parenchyma-specific gene expression of albumin and AFP was illustrated by Northern-blot analysis of the cells before and after panning. In addition, the procedure’s specificity was demonstrated by analysis of the adherent cells,which contained negligible amounts of albumin and AFP mRNA. Cell enrich-

  ment was further confirmed by in vitro studies, which revealed a dramatic increase in the number of parenchymal cell colonies compared with the original suspension. The plating efficiency after panning was significantly higher (up to 60%) compared with previous studies, in which it ranged from 6% to 10% (Sigal et al.,1994)

Table (1-1)

The results of Alfa Feto Protein in all study groups.

Study Groups	(Alfa Feto Protein)nn/ml Results positive or negative
Control group	negative	negative	negative
20mg/kg/day of gentamicin	negative	negative	negative
40mg/kg/day of gentamicin	negative	negative	negative

• 2:    Histological Study of pups liver by SEM

  Scanning electron micrographs results in pups liver in control groups showed normal external architecture structures of hepatocytes figure (1), while in treated by doses20+40mg/kg/day of gentamicin show changes in external architecture structures but in treated by dose 40mg/kg/day of gentamicin show severely effects they showed deformation of hepatocytes and high fenestrae figure (2). In scanning electron microscope, the impacts of the aminoglycoside antimicrobials are generally restricted to the apical surface of the cell, poly-l-lysine induces complete necrosis of the cell, qualitative assessment of apical surface damage allows the antibiotics to be ranked in the following order: neomycin, gentamicin, dihydrostreptomycin, neamine, amikacin, and spectinomy-cin (Hobbie et al.,2008).Liver plays an important role in the detoxification and metabolic elimination of various drugs and harmful substances, Its exposure to hazardous compounds and toxins could lead to injury and reduced function (Arjinajarn et al.,2017). The tumor necrosis factor elevated due to inflammation of liver and apoptosis were observed in gentamicin treated rats lead to deformation of hepatocytes (Arjinajarn et al.,2017).Gentamicin has actuated histological modifications of hepatocytes including hydropic degeneration of hepatocytes, fatty changes, inflammatory cell infiltration and congestion of portal vein (Galaly et al.,2014).

Figure (1): Scanning electron micrograph of pups liver control group show a normal architecture of parenchyma (hepatocytes) (    ).

Figure (2): Scanning electron micrograph of pups liver treated by dose40mgkg day of gentamicin show deformation of hepatocytes( ■>), high fenestrae( -).