Journal of Forensic Toxicology & PharmacologyISSN: 2325-9841

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Research Article, J Forensic Toxicol Pharmacol Vol: 5 Issue: 1

Biochemical and Histopathological Alterations as Forensic Markers of Asphyxiated Rats and the Modifying Effects of Salbutamol and/or Digoxin Pretreatment

Badr El-Said El-Bialy1*, Nermeen Borai El-Borai1, Amira S Abd El Latif2 and Mostafa Abd El-Gaber Mohamed3
1Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, 32897, Egypt
2Department of Pharmacology, Faculty of Veterinary Medicine, Kafr El Sheikh University, Egypt
3Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, 32897, Egypt
Corresponding author : Badr El-Said El-Bialy BVSc, MS, PhD
Forensic Medicine & Toxicology department, Faculty of Veterinary Medicine, University of Sadat City, Egypt
Tel: +20-1003811701
E-mail: [email protected]
Received: February 01, 2016 Accepted: March 10, 2016 Published: March 15, 2016
Citation: El-Bialy BES, El-Borai NB, El-Latif ASA, El-Gaber Mohamed MA (2016) Biochemical and Histopathological Alterations as Forensic Markers of Asphyxiated Rats and the Modifying Effects of Salbutamol and/or Digoxin Pretreatment. J Forensic Toxicol Pharmacol 5:1. doi:10.4172/2325-9841.1000144

Abstract

Asphyxia is mainly induced by interference with respiration, or lack of oxygen in respired air as in entrapment suffocation that may occur in animals or human. In this study 24 male albino rats were assigned into four equal groups: G1 (control negative); G2 (exposed to asphyxia); G3 (asphyxiated pretreated rats with salbutamol); G4 (asphyxiated pretreated rats with digoxin). The results revealed no change in the time intervals from onset of suffocation till coma (G2, G3 and G4). Also, all external and internal classical signs of asphyxia appeared on animals of different asphyxiated groups. Moreover, Asphyxiated rats (G2) showed significant (P ≤ 0.05) increase in serum ALT, AST, urea and uric acid and insignificant increase in serum direct and total bilirubin levels but significant reductions of serum glucose, total protein and albumin levels compared to control ones. Pretreatment with salbutamol or digoxin induced insignificant reverse effects on mentioned parameters than those of asphyxiated rats except in serum ALT and AST activities and serum urea level of digoxin pretreated rats. The histopathological examination of different organs revealed no changes in histopathological appearance between asphyxiated and/or pretreated groups but various pathological alterations mainly congestion, hemorrhage and edema were found in these groups in relation to control group. In conclusion, entrapment asphyxia induced acute and progressive biochemical alterations in liver and kidney functions with presence of classical pathological changes of asphyxia in internal organs. Pretreatment with either salbutamol or digoxin could not improve the altered parameters than asphyxiated group. We recommend further study to asses the extent of reversibility of these parameters after resuscitation of asphyxiated rats.

Keywords: Suffocation; Signs of asphyxia; Salbutamol; Digoxin

Keywords

Suffocation; Signs of asphyxia; Salbutamol; Digoxin

Introduction

In the forensic context asphyxia resulting in death and defined as a forensic situation in which the body unable to receive or utilize oxygen [1].
Asphyxia can be classified from forensic point of view into four main categories: suffocation, strangulation, mechanical asphyxia, and drowning. Suffocation subdivides by its role into smothering, choking, and confined spaces/entrapment/vitiated atmosphere [1].
The outcome of asphyxia depend on the severity of hypoxemia which has adverse insult on various body organs as liver, kidney, heart, brain and other organs, respiratory failure or disturbance resulting in insufficient brain oxygen which leads to loss of consciousness or death if severe enough and prolonged [2].
The classical signs of asphyxia include cyanosis (due to stasis and congestion), increased capillary permeability (causing tissue edema), petechial hemorrhage, persistent fluidity of blood (due to fibrinolysis) and cardiac dilatation [3].
Salbutamol bronchodilator is considered as one of a short-acting β2-adrenergic receptor agonist used for the relief of bronchospasm in conditions caused by bronchial asthma as well as chronic obstructive pulmonary disease [4] It selectively binds and activates β-2 adrenergic receptors on the surface of many cells resulting in relaxation of bronchial smooth muscles and hence opening up the airways and enabling air to flow [5]. Also, it inhibits cholinergic neurotransmission with decreasing the release of acetylcholine and hence, decreased airway spasm [6].
Digoxin is a purified cardiac glycoside extracted from the foxglove plant, Digitalis lanata [7]. Digoxin is widely used in the treatment of various heart conditions, namely atrial fibrillation and atrial flutter though β blockers and/or calcium channel blockers [8].
Digoxin’s primary mechanism of action involves inhibition of the Na+/K+ ATPase, mainly in the myocardium leads to an efflux of potassium from the cell and increase sodium ion concentration (Na+ ) at the inner face of the cardiac membranes This local accumulation of sodium causes a reversal of the action of the sodium-calcium exchanger with an increase in the availability of intracellular calcium to the contractile proteins resulting in an increase in the force of myocardial contraction [9].
Digoxin also has important parasympathetic effects, particularly on the atrioventricular node [10].
This study was aimed to investigate the biochemical and histopathological alterations in asphyxiated rats by suffocation (entrapment or environmental asphyxia) as forensic markers of asphyxiated rats and the modifying effects of salbutamol and/or digoxin pretreatment.

Materials and Methods

Animals
A total of 24 male albino rats (120-150 g.) purchased from ALZyade Experimental Animals Production Center, Giza, Egypt, were used. Animals were housed under standard conditions of temperature (25 ± 2°C), 12 hours light/dark cycle and fed with standard chow diet and water ad libitum. Animal rearing and handling guidelines were approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Egypt. The rats were maintained for one week prior to the start of experiments for acclimatization.
Chemicals
Digoxin (Lanoxin® tablet 0.250 mg or 250 μg) and Salbutamol (Ventolin® tablet 2 mg), GlaxoSmithKline Company, were purchased from local pharmacy, El-Sadat City, Egypt. All other chemicals and reagents were of analytical grade and commercially available
Diagnostic kits for assaying serum biochemical parameters were purchased from the Biodiagnostic Company, Dokki, Giza, Egypt.
Experimental design and animal grouping
Rats were weighted and assigned into 4 experimental groups of the same weight ranges (each of 6 rats).
Group 1: Served as a control negative group.
Group 2: Exposed to entrapment asphyxia in a closed narrow plastic jar of 500 cc volume.
Group 3: Pretreated orally with salbutamol (ventolin) 2 mg/rat dissolved in distilled water (dose of child from 6-12 years), 30 mins (onset of oral effect is 15- 30 mins) before exposure to anoxic asphyxia
Group 4: Pretreated orally with digoxin (lanoxin) 6.25 μg/kg b.wt. dissolved in distilled water (maintenance dose of children up to 10 years equal to 25% of 24-hour loading dose 25 μg/kg), 60 mins (onset of oral effect is 0.5-2 hours) before exposure to anoxic asphyxia.
The time intervals from onset of suffocation till coma were recorded for each rat. Also, all external signs appeared on animals of different groups were noticed.
Collection and preparation of samples
Just before death and when the animals enter the stage of coma, blood samples were collected by heart puncture for biochemical analysis. The animals were euthanized, examined carefully at autopsy. Tissue samples were collected for histopathological examination.
Biochemical analysis
Blood sample of each rat was collected without anticoagulant, centrifuged and stored at -20°C for serum biochemical analysis; ALT, AST, glucose, total protein, albumin, total and direct bilirubin, urea and uric acid levels according to calorimetric methods of kits.
Histopathological examination
Specimens of brain, heart, lungs, liver and kidneys of each rat were collected and fixed in 10% neutral buffered formalin solution, processed and stained by hematoxylin and eosin (H&E) stain for light microscopical examination according to Bancroft et al. [11].
Statistical analysis
Values were presented as mean ± standard error (SEM). Statistical differences were analyzed by one-way ANOVA with Duncan’s post hoc test to determine the significant differences among different groups. Using SPSS (Statistical package for Social Sciences), Version 16 released on 2007. Statistical significance was considered at P ≤ 0.05.

Results

The recorded symptoms
The rats of different asphyxiated groups passed through various stages prior to entering in coma at different time intervals. The most prominent symptoms of asphyxiated rats in different groups were recorded as air gasping and hyperpnoea with marked abdominal movements. After that, the rats were struggled and became cyanosed with congestion of conjunctiva and dilatation of eye pupils. The rats entered in depressant state then finally complete loss of consciousness was occurred.
Effect of asphyxia and/or salbutamol or digoxin pretreatment on time intervals till entering in coma:
Time intervals from onset of asphyxia till coma were recorded for asphyxiated and pretreated asphyxiated rats (Table 1). There were no significant changes between different groups. However, the shortest time interval was recorded in asphyxiated rats pretreated with digoxin.
Table 1: Mean values ± SEM of time intervals from asphyxia induction (minutes) till coma of the different asphyxiated rat groups.
Post mortem findings
The noticed internal post mortem findings of different asphyxiated rat groups included congested and voluminous lungs; dilated heart; congested brain and other visceral organs. However, rats of the control group showed no gross postmortem changes in their visceral organs.
Effect of asphyxia and/or salbutamol or digoxin pretreatment on serum biochemical analysis
The effects of asphyxia induced by entrapment in close place on some serum biochemical parameters are recorded in Table 2. Asphyxiated rats (G2) showed significant (P ≤ 0.05) increase in serum ALT and AST activities and insignificant increase in serum direct and total bilirubin levels but significant reductions of serum glucose, total protein and albumin levels were recorded in asphyxiated rats compared to control ones. From renal functional standpoints, serum urea and uric acid levels significantly increased in asphyxiated group compared to those of control.
Table 2: Mean values ± SEM of serum biochemical parameters in control, asphyxiated and asphyxiated pretreated with either salbutamol or digoxin rat groups.
Pretreatment with either salbutamol (G3) or digoxin (G4) induced insignificant reverse effects on mentioned parameters compared with asphyxiated rats except serum ALT and AST activities and serum urea level which were significantly reduced in digoxin pretreated group (G4) compared to asphyxiated rats (G2).
Histopathological changes
The histopathological pictures of internal organs (brain, heart, lungs, liver and kidneys) of asphyxiated rats of groups (1, 2 and 3) illustrate mainly presence of congestion, hemorrhage and edema as classical signs of asphyxia (Figure 1-5).
Figure 1: a. Brain of control group showed normal tissue; b. Brain of group 2 showed congestion (arrow) and hemorrhage with perineuronal edema; c. Brain of group 3 showed sever hemorrhage (arrow); d. Brain of group 3 showed congestion (arrow) with perivascular and perineuronal edema; e. Brain of group 4 showed congestion (arrow) with perivascular and perineuronal edema, (H&E X200).
Figure 2: a. heart of control group showed normal tissue; b. heart of group 2 showed sever congestion and hemorrhage (star) with myocardial edema (arrow); c. heart of group 3 showed hemorrhage (arrow); d. heart of group 4 showed sever hemorrhage (arrow), (H&E X200).
Figure 3: a. lung of control group showed normal tissue; b. lung of group 2 showed hemorrhage (arrow) and emphysema; c. lung of group 3 showed sever hemorrhage (arrow) and emphysema; d. lung of group 4 showed hemorrhage, hemolysis (arrow) and emphysema (star); e. lung of group 4 showed sever congestion of blood vessels (arrow) and emphysema; (H&E X100).
Figure 4: a. liver of control group showed normal hepatic parenchyma (H&E X100); b. liver of group 2 showed sever congested blood vessels (arrow) (H&E X100); c. liver of group 3 showed sever congested blood vessels (arrow) (H&E X100); d. liver of group 3 showed intravascular hemolysis (star) with vacuolar degeneration of hepatocytes (arrow) (H&E X200); e. liver of group 4 showed sever congested blood vessels (arrow) (H&E X200); f. liver of group 3 showed intravascular hemolysis (arrow) (H&E X200).
Figure 5: a. kidney of control group showed normal renal tissue; b. kidney of group 2 showed sever congestion (arrow) and hemorrhage (star); c. kidney of group 3 showed sever congested blood vessels (arrow); d. kidney of group 4 showed sever hemorrhage (arrow), (H&E X200).

Discussion

In asphyxia, organs and tissues are deprived of oxygen together with failure to eliminate CO2 causing loss of consciousness and then death [1].
Although, brain is the first affected organ by the deficiency of oxygen and its functions are disturbed even by mild oxygen lack [12], asphyxia also was recorded to induce damage to almost every tissue and organ of the body including kidneys [13], liver [14] and heart [15].
Hypoxic hepatic injury was previously recorded [16] and could be reflected by elevation of liver enzymes and bilirubin in association with hypoproteinemia.
Concerning the effect of asphyxia on liver, our results revealed a significant increase in serum ALT and AST levels of asphyxiated rats. Chhavi et al. [17] and Choudhary et al. [18] were also recorded elevation in liver enzymes in birth asphyxia. ALT is the most specific serum marker of liver injury as it only present in liver, but AST is widely present in the liver, kidney, lungs and heart so elevation in serum ALT and AST activities gives indication about the damaging effects of asphyxia on the liver and other different body organs.
The ALT and AST enzyme activities increase as a result of the damaging effect of hypoxia on the liver parenchyma, which needs oxygen, glucose, and nutrients for its use. Also, ALT and AST are sensitive markers of impaired liver membrane with decreased intake of energetic substrates into the cell [17,19]. Moreover, hepatic dysfunction in asphyxia is caused by redistributing cardiac output away from non vital viscera to the heart, brain and adrenal glands [20].
In our study, asphyxiated rats showed a significant reduction in serum glucose level as previously recorded by Jayaprakash and Murali [21], that could be attributed to the increase in glucose metabolism [22] or to the temporary hyperinsulinism [23] induced by asphyxia.
Estimation of serum total protein and albumin levels can indicate hepatic insult. In this study, the significant reduction in serum total protein and albumin levels of asphyxiated rats was also previously recorded by Islam et al. [19] who found a significant decrease in their levels in asphyxiated neonates. On the other hand, Tarcan et al. [24] showed that hypoproteinemia is an imprecise index of the severity of liver damage in birth asphyxia because of the long life of serum proteins, and referring hypoalbuminemia to capillary leakage rather than liver damage because its levels do not change rapidly with hepatic involvement.
Bilirubin is a breakdown product of hemoglobin. However liver is responsible for clearing of bilirubin, serum bilirubin levels may not always rise following hepatic injury due to relative non-specificity of serum bilirubin as a marker for liver injury and relatively normal or minor bilirubin elevation in ischemic hepatitis [25].
Our results revealed insignificant increase in serum total and direct bilirubin levels of asphyxiated rats. Similarly, total serum bilirubin levels were elevated in asphyxiated babies [26]. In addition, Mukesh et al. [27] recorded a significant rise of serum total bilirubin and insignificant rise of serum direct bilirubin in asphyxiated newborns.
As kidneys are very sensitive to oxygen deprivation, renal insufficiency may occur as a result of hypoxic ischemia, which if prolonged, may even lead to irreversible cortical necrosis [13].
Our results revealed hypoxic renal damage that reflected by the significant increase in serum urea and uric acid levels of asphyxiated rats. Gupta et al. [13] found that serum creatinine values were higher in asphyxiated infants compared to non-asphyxiated infants. In addition, our results are supported by Naithani and Simalti [28] who concluded that serum uric acid levels appear promising in identifying patients with a risk of developing hypoxic-ischemic encephalopathy.
Blood urea and serum creatinine were significantly higher in asphyxiated babies that could be attributed to obstruction of renal tubular lumen and back leak mechanism [13,28]. Moreover, Tissue hypoxia is a recognized cause of adenine nucleotide catabolism with appearance of oxypurines (hypoxanthine and xanthine) intermediate catabolites. These intermediates used as metabolic markers of ATP degradation in tissues followed by uric acid formation [29].
Although salbutamol possesses a rapid relieving effect of acute dyspnea [30] and digoxin has a stimulating effect on heart muscles by increasing their force of contraction without increasing their oxygen demand [31], their use as protective drugs before induction of asphyxia in rats induced insignificant changes in time intervals till occurrence of coma. Also, the tested drugs induced insignificant reductions in almost of elevated biochemical parameters of asphyxiated rats except ALT, AST and urea levels that were significantly reduced in digoxin pretreated group. Various studies proved that cardioprotectivede plants (including cardiac glycosides derived from digitalis plant) significantly reversed the altered biochemical variation such as marker enzymes serum ALT and AST [32,33]. Also the short time interval till rats entering in coma of digoxin pretreatment induced less deleterious changes.
Although cardiac glycosides still enjoy significant role in treatment of heart failure, they have a drawback of narrow therapeutic index and the most severe adverse effects on heart are heart block, arrhythmia, tachycardia and serious heart consequences so, it needs constant therapeutic drug monitoring [34]. We think that in hypoxic condition, the cardiac glycosides dose should be monitored before their use.
It was proved that administration of salbutamol during hypoxia causes significant cardiovascular effects due to hypokalemia and systemic vascular side effects and so asthmatic patients in respiratory distress should be given Beta-2 adrenoceptor agonists in concomitant with oxygen [35,36]. Also, salbutamol lung concentration decreased in chronic hypoxia and so reduced salbutamol effect on pulmonary resistance [37].
The histopathological examination of different organs revealed no changes in histopathological appearance between asphyxiated and/or pretreated groups but various pathological alterations were found in these groups in relation to control group.
In conclusion entrapment asphyxia induced acute and progressive biochemical alterations in liver and kidney functions that go along with the histopathological changes of these organs of rats beside classical pathological changes of asphyxia in brain, lungs and heart. These alterations could be used as medico legal or forensic markers of asphyxiated rats. The pretreatment with either salbutamol or digoxin could not significantly improve such altered parameters compared to the asphyxiated group.
We recommend further study to asses the extent of reversibility of these parameters after resuscitation of asphyxiated rats.

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