Biochemical Markers and Pathological Features of Postmortem Time Interval Distinguishing Freshwater and Saltwater Drowning Induced Death in Albino Rats

Identification of drowning in freshwater or saltwater still controversial. The recovered bodies form water and movement of cadavers made the task of differentiation of two types of drowning so difficult...


Introduction
Drowning is the type of violent asphexia of respiratory dysfunction from submersion/immersion in liquid resulted in death [1][2][3][4]. Diagnosis of the drowning of recovered animals from fresh water is very important in forensic medicine, legal and insurance [3]. There several effects of drowning on the corpse of drowned rats. The organ weights for the lung and brain of drowned rats were increased in the experimental drowning group compared to 24-hour post-mortem immersion group. while the mean heart weight was not [5]. Differentiation between fresh water and saltwater drowning on forensic medicine still controversial [6] as the external examination and the corpse gross findings are in most of the reported cases not specific and there are no clear laboratory investigations for diagnosis of drowning especially when dead is moved away of water resources (Figure 1). The diagnosis of drowning considers one of the major issues in forensic medicine, especially when there is a delay in recovering the case [7]. The diagnosis of drowning based on pathological changes is often a difficult task, as the typical features of pulmonary edema and congestion are nonspecific [8,9]. Recently, immune-reactive proteins like IL-6, aqp5 and others were used for identification of bodies recovered from seawater [10][11][12][13][14]. The rationale of this study is exploring that biochemical and cytokines could be distinguished seawater and freshwater drowning and these changes may be differing when cause of death is changed and so biochemical marker still controversial up to now.

Materials and Methods
Animal procedures were conducted with the accordance of the animal care committee of the ethics Board of the faculty of veterinary medicine Mansoura university, Egypt. 36 male Albino rats (weight, 150-200GM was purchased from faculty of pharmacy, Mansoura University, Egypt). They were managment on a 12-h light-dark cycle with available of food and water for ad libitium ( Figure 2). Rats were exposed to drowning by keeping them in a wire box in collected freshwater (Egyptian nil river) or saltwater (mediterranean sea). Time from start of drowning till complete death was recorded. Recovered dead rats were kept in fixed supine position on research lab. Temperature (20oC) for 48hrs hours postmortem. Blood samples of each 6 rats for each time point were collected from heart and great vessels at autopsy [15] and suffer postmortem changes in 0, 24 and 48hrs hours. Samples were centrifuged immediately for 20 min at 2000 rpm. Serum samples was stored at -20 deep freezer until measurement ( Figure 3).

Histopathology Examination
The 12 male rats were anesthetized with thiopental and natural death was induced [16]. 24 recovered corpse of drowned rats were kept for 0, 24 and 48hrs and their tissues like heart, lung, and brain were separated at different time point after death and fixed at 10% neutral buffered formalin. Fixed tissue were procedures were described previously before in details for eosin and hematoxylin staining [17].

Immunohistochemically localization IL-6
The tissue of brain, heart and lung paraffin sections that were mounted on positively charged glass slides were processed for single immunohistochemistry with an antibody to IL-6 according to method described earlier [18]. The mean area percentage of collagen fiber content between the brain, heart and lung of drowned rats either in fresh water or slat water was quantified for each group. Atomic absorption spectrometry analysis of stomach content of trace mineral in fresh and saltwater drowning ( Figure   4). The collected stomach content was collected after recovered rats from water either fresh or salt one. The samples were kept in polyethylene bags till digestion step by acid. Liquid digestion: All samples were digested by acid mixture. the mixture of Nitric-Perchloric acids were used in the liquid digestion of samples (Sigma-Aldrich, Egypt). Combined acids (10 mL, 65% high purity HNO3 and 3:1 v/v) was added to the beaker containing 2g fresh meat for liquid digestion of samples ( Figure 5).

Statistical Analysis
Statistical analysis was carried out using the student's t-test and one way annova. P<0.05 was considered significant [19] (spss version13).

Pathological Features of Salt water Drowning
Immediately after recovering dead rat from saltwater, the brain is showing angular shaped neurons, and minute vacuolation in the brain parenchyma, Heart is showing normal cardiomyocytes and slightly increases in interstitial spaces and lung displays over distended alveoli with air with cellular death and desquamation of intrapulmonary bronchiole. After 24hrs, the brain is showing postmortem changes represented by death of neurons (arrow), and vacuolation in the brain parenchyma, heart is showing normal cardiomyocytes and slightly increases in interstitial spaces, lung displays over distended alveoli with air with death pneumocytes with rupture of capillaries and cellular death of interstitial cells. After 48hrs, the brain is showing postmortem changes represented by death of neurons, and severe vacuolation in the brain parenchyma, heart is showing death of cardiomyocytes and increases in interstitial spaces, lung displays pulmonary atelectasis and over distended alveoli with air with rupture of capillaries and cellular death of interstitial cells (Figure 2).

Figure 2:
Show pathological feature of salt water drowning in rats, the brain is showing changes represented by death of neurons, and marked vacuolation in the brain parenchyma, heart is showing death of cardiomyocytes and increases in interstitial spaces, lung displays pulmonary atelectasis and over distended alveoli with air with rupture of capillaries and cellular death of interstitial cells at 48 hrs than 24hrs .

Biochemical modulation in drowned rats in saltwater drowning
Notably, drowning in saltwater retard autolysis and protein       Notably, biochemical, trace mineral, heavy metal of stomach content, histopathology, collagen and IL-6 share a difference in fresh water and more prominent in salt water than those recorded in natural induced experimental rats. There is significant linear correlation between creatine kinase (CPK) and calcium with the time of blood putrefaction [21] similar to what recorded at 72 hrs postmortem. Notably, the increase in the serum calcium level was observed in freshwater drowning [22]. the Postmortem changes started earlier in the heart 3 to 4 hours compared with other organs after death [23]. While collagen deposition at onset of death is prominent factor for diagnosis of death especially in drowning cases in forensic Autopsies [24]. Up with standing with current detection of abundance of IL-6 in tissues of seawater drowned rats than freshwater as seen previously [10,13]. Finally, as biochemical markers and trace mineral different in vitreous humor in seawater and freshwater [25] also could be differ in blood.

Conclusion
Changes of biochemical of postmortem times may dependent on cause of death and notably IL-6 expression could distinguish freshwater and seawater drowning.