Legal Medicine 11 (2009) S244–S247

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Legal Medicine

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Scanning electron microscopic observation of erythrocytes and endothelial cellsof electrified death rabbits

Ye Wang a,b, Lan Yang a,c, Weibo Cheng b, Min Liu a, Xiaogang Chen a, Kui Zhang a, Hong-min Chen a,Zhigang Liao a,*

a West China Preclinical and Forensic Medicine School of Sichuan University, Chengdu 610041, Chinab West China School of Public Health, Sichuan University, Chengdu 610041, Chinac Chengdu University of Traditional Chinese Medicine, 610075 China

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 December 2008Accepted 14 January 2009Available online 3 March 2009

Keywords:Electrified deathErythrocyteEndothelial cellPerforationScanning electron microscope

1344-6223/$ - see front matter � 2009 Elsevier Irelandoi:10.1016/j.legalmed.2009.01.097

* Corresponding author. Tel.: +86 28 85501551.E-mail address: liaozhigang@scu.edu.cn (Z. Liao).

To investigate the evidence of electrified death without electric mark, four animal models were devel-oped, which were electrified to death without electric mark left, contrasted with four non-electrifiedto death models. Erythrocyte and endothelium of aorta and pulmonary artery were observed by scanningelectron microscope. It was suggested that some pores can only be seen on the surface of erythrocytesand endothelial cells of aorta and pulmonary artery of those death models group two electrified. How-ever, there were no pores were found on the erythrocytes and endothelial cells of aorta and pulmonaryartery of the non-electrified group and normal control group animals. This study indicates that the per-foration phenomena might be supposed to diagnose the electrified death cases that no electric mark isleft.

� 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

In the forensic practice, there are many cases that the victimdied of electrocution but no electric mark can be detected [1], itis very difficult to determine whether they died of electrocution.In order to find an effective way for diagnosing these cases, foren-sic pathologists has been making a lot of efforts to attempt resolv-ing this problem. Pierucci and Danesino applied chemical reagentto detect the metal ion to find the latent current mark [2] and Jaku-beniene and his co-worker thought that atomic absorption spec-troscopy can be applied for detection of multimetal traces inlow-voltage electrical marks [3] and Kinoshita and his co-workerapplied a variable-pressure scanning electron microscope with en-ergy dispersive X-ray microanalyser to the diagnosis of electrocu-tion [4]. They thought if metal ions were detected in the skin ofvictim, it would support that the victim died of electrocution.Unfortunately, there are many cases died of electrocution withoutelectric mark and metal ions left. Therefore, we have to study fur-ther to find the morphological evidence that can confirm electricshock. Previous research showed that cell membrane can be perfo-rated induced by pulsed electric field [5,6] called electroporation.Since transient electric field can induce electroporation, somescholar thought that perforation should exist in cells of the bodyinjuried by electrification [7,8] but they did not confirm their idea.

d Ltd. All rights reserved.

Our previous research showed that 220 alternating current cancause red blood cells perforating in vitro. We presume that the per-forating is specificity by electric field. To confirm the presumption,we set up four animal models that were electrified to death with-out electric mark left, and four animal models that some othercauses of death as the control. The erythrocytes and endothelialcells were observed by scanning electron microscopy to find theevidence to diagnose electrocution.

2. Material and methods

2.1. Material

2.1.1. InstrumentScanning electron microscopy (Amary, model-1000, Academia

Sinica instrument factory); Voltage regulator (Chengdu instrumentfactory, China); critical point dryer (BALZERs CPD030, Liechten-stein); metal ions spattering instrument (KYKY-12, Academia Sini-ca instrument factory); Amperemeter and Voltmeter (Chengduinstrument factory, China).

2.1.2. Reagentsodium dihydrogen phosphate; dibasic sodium phosphate; so-

dium chloride; ethanol; glutaraldehyde; isoamyl acetate; liquidcarbon dioxide; liquid nitrogen; sodium nitrite. All of these re-agents were produced in China.

Fig. 1. Erythrocytes of normal control group. No perforated cell could be seen.

Y. Wang et al. / Legal Medicine 11 (2009) S244–S247 S245

2.1.3. ExperimentalAnimal New Zealand white rabbits were 5–6 months old

weighted from 2 to 2.5 kg. All animal work was conducted underthe institutional guidelines by West China experimental animalcenter of Sichuan university.

2.2. Methods

2.2.1. Animal group and modelForty-five healthy male New Zealand white rabbits were ran-

domly divided into nine groups. One group as normal controlwas put to death by dislocating the cervical vertebra directly andundergone autopsy and sampled as experimental groups. Theexperimental groups were dealt with as follows:

Model 1 and 2: Five animals of group one treated like this: Atfirst, right anterior limb and left hind limb of rabbits weresoaked with 0.1 M PBS. Then, two pieces of wet gauze soakedin the phosphate buffer saline were winded on the right anteriorlimb and left hind limb, then winded with wires and connectedto the alternating current of 220 V and electrified to death. An-other five animals were treated as described as above exceptvoltage of 110.

Model 3 and 4: Five animals of group one treated like this: Twowires were fixed at the bilaterals of a oblong container with a smallquantities PBS about 1.5 cm deep. Then, one rabbit was put in eachtime. Finally, the two wires were connected to the alternating cur-rent of 220 V and electrified to death. Another five animals weretreated as described as above except voltage of 110.

Model 5 to 8 were the cause of death inflicted by falling fromheight, burn to death, the poisoning and asphyxia other than theelectrocution.

Model 5: The rabbits were put into a bag and fallen from 40 mhigh one by one.

Model 6: Rabbits were burned to death using ethanol.Model 7: The cause of death in this group is poisoning. Rab-

bits were intragastric administration with sodium nitrite todeath.

Model 8: The cause of death in this group is asphyxia. Rabbitswere put into a small container and carbon dioxide was filled toit till animal died.

All animals were performed autopsy. The blood and pulmonaryand ascending aorta were taken.

2.2.2. Samples treatmentThe blood was centrifuged for 3 min by 3000 rpm and re-

moved the supernatant. The process was repeated for three times.Then the erythrocytes was fixed by 3%(v/v) glutaraldehyde solu-tion for 3 h. The samples were centrifuged for 3 min by3000 rpm once again and removed the supernatant. The speci-mens were dehydrated with gradient ethanol alcohol of 40%,60%, 70%, 80%, 90%, 100% and 100%(v/v). The samples wereparked to slides and subsequently dried by carbon dioxide criticalpoint and coated with gold using a metal ions spattering instru-ment. The samples were examined by a scanning electronmicroscope.

The samples of pulmonary and ascending aorta were fixed inthe 3%(v/v) glutaraldehyde for 3 h. Then they were dehydratedwith gradient ethanol alcohol of 40%, 60%, 70%, 80%, 90%, 100%and 100%(v/v). The samples then were treated as the blood beforeobserved by scanning electron microscope.

2.2.3. Result assessmentFive hundred cells were counted randomly on the screen of the

scanning electron microscope and figured out perforated cellsnumber. Then calculated the mean numbers of perforated cellsper one hundred cells, called perforation index.

2.2.4. Statistical analysisThe measurement data were demonstrated by mean ± SD, ana-

lysed by one-way ANOVA and SNK test, a = 0.05.

3. Results

The control group and model 5–8 groups: The shape of theendothelial cells of pulmonary artery and aorta of the controlgroup were normal (Figs. 1–4). There were no pores on the cellsin all of the groups. Sometimes, deformity of erythrocytes can befound, but no perforation. Their perforated cell indexes are almostequal to zero.

Model 1 and 2 groups (110 V and 220 V): Some perforatederythrocytes could be seen. The pores are oval or round in shapes,most pores locating the center of the red blood cells (Fig. 5). Thepores diameters were ranging from 0.8 to 2.4 l. And the distribu-tion of perforated cells are nonuniformity and not consecutive.Sometimes some fragment of cells can be seen. The shape of eryth-rocytes were deformed and projected. The perforated index inerythrocytes was 30.4 ± 5.1 and 25.5 ± 3.6 respectively. Some per-forated cells can be seen in the pulmonary artery endothelial cellsand ascending aorta endothelial cells. Most pores are oval or roundin shapes locating the center of endothelial cells (Fig. 6). The perfo-rated index of pulmonary endothelial cell was 25.4 ± 4.5 and20.7 ± 3.4, respectively. The perforated index of aorta endothelialcell was 15.7 ± 4.0 and 10.8 ± 3.2, respectively. Compared with nor-mal control and model 5–8 groups, there is obvious significance indiversity (P < 0.05). However, there is no significance in diversitybetween model 1 and 2 group (P > 0.05).

Model 3 and 4 groups (110 and 220 V): The shape of pores aresimilar to model 1 and 2 groups. And the distribution of perforatedcells are nonuniformity and not consecutive either. The perforatedcell index of erythrocytes was 27.3 ± 4.2 and 20.8 ± 3.3, respec-tively. The perforated index of pulmonary endothelial cell was19.64 ± 3.1 and 17.2 ± 2.6 respectively. The perforated index of aor-ta endothelial cell was 12.2 ± 2.8 and 10.4 ± 2.1, respectively. Com-pared with normal control and model 5–8 groups, there is obvioussignificance in diversity (P < 0.05). However, there is no signifi-cance in diversity among model 3 and 4 (P > 0.05) (see Table 1).

4. Discussion

The results showed that rabbits died of electrocution by model1, 2 and model 3, 4 with alternating current 110 V or 220 V withoutelectrical mark left, demonstrating that the experimental modelsare successful. The erythrocytes and the endothelial cells of pul-

Fig. 2. Pulmonary artery endothelium of control group. No perforated cell could beseen.

Fig. 3. Blood cells of model 7 rabbits. No perforated cell could be seen. Some bloodcells are deformity.

Fig. 4. Pulmonary artery endothelium in model 7 rabbits. No perforated cell couldbe found.

Fig. 5. Erythrocytes of model 1 rabbits (electrocuted with 110 V). Perforated cellcan be seen in the center of the cell. The pores are ellipse and fragments of cell couldbe seen.

Fig. 6. The pulmonary artery endothelium of model 1 rabbits. The pore could befound in the end of one endothelium cell.

S246 Y. Wang et al. / Legal Medicine 11 (2009) S244–S247

monary artery and ascending aorta of rabbits were perforated bymodel 1, 2 and model 3, 4, whereas no pores were found in the ani-mals with other causes of death and the normal control group. Inthe forensic practice, the cases that the victim died of mechanical

energy, heat energy, chemical energy, and electric energy, are fre-quent, while the cases that the victims died of light energy and nu-clear energy are seldom. Therefore, we simulated the death causedby mechanical energy, heat energy, chemical energy with model 5,6, 7 and 8 group, respectively. In the model 7, we chose the sodiumnitrite as a poison which could cause person to death. Because it isa kind of blood poisons, and can caused erythrocytes and endothe-lial cell injury directly, and be regarded as a representative of thekind of poison that can vulnerably harm to blood cells and endo-thelial cells. The results showed only the blood cells, the endothe-lial cells of pulmonary artery and ascending aorta showedperforated in the model 1, 2 and model 3, 4 group rabbits died ofelectric energy. The phenomena indicated that the perforation isspecificity to electrical shock compared with 5–8 groups.

As we know, our results were the first experimental evidencethat alternating current caused perforation of cells in vivo, and itis specificity to electric energy, relatively. Although many othercauses can perforate on the cell membrane, and some pores left,however, the pores is very small so that they could not be directlyobserved by scanning electron microscope with magnified withinten thousands multiples, they can be observed by other methods[9–11]. Therefore, in the forensic practice, when the victims weresuspected to die of electric shock, erythrocytes, endothelial cellsof pulmonary artery and ascending aorta should be detected byscanning electron microscope to find the perforation. If the perfo-

Table 1Perforated cell index of erythrocyte, pulmonary artery endothelium and aorta endothelium of rabbits when electrified.

Cells Normal control Model 5 Model 6 Model 7 Model 8 Model 3 Model 4 Model 1 Model 2

Erythrocyte 0 0 0 0 0 20.8 ± 3.3* 27.3 ± 4.2* 25.5 ± 3.6* 30.4 ± 5.1*

Pulmonary artery endothelium 0 0 0 0 0 17.2 ± 2.6* 19.6 ± 3.1* 20.7 ± 3.4* 25.2 ± 4.5*

Ascending aorta endothelium 0 0 0 0 0 10.4 ± 2.1* 12.2 ± 2.8* 10.8 ± 3.2* 15.7 ± 4.0*

* P < 0.05, vs normal control group and Model 5–8 groups.

Y. Wang et al. / Legal Medicine 11 (2009) S244–S247 S247

ration were detected, we should consider the death of cause beelectrocution. Because the pores caused by pulsed can reseal by it-self [12], and it generally is difficult to detect by scanning electronmicroscope. In other word, when the pores were detected on thecell membrane of endothelial cells and erythrocytes, electricalfatality should be diagnosed [13].

Conflict of interest statement

This paper was finished by Dr. Ye Wang, who was the doctorialpostgraduate, and Professor Zhi-Gang Liao is the tutor of Dr YeWang, therefore, first author is Ye Wang, and the correspondingauthor is Zhi-Gang Liao. Other authors also took part in the re-search. There are no conflicts of interest among the authors.

The authors sequence, from the second to eighth author, respec-tively is, Lan Yang, Weibo Cheng, Min Liu, Xiaogang Chen, KuiZhang, Hong-min Chen, and Zhi-Gang Liao, but Professor Zhi-GangLiao is corresponding author.

The purpose of this paper is to confirm the hypothesis thatalternating current can cause the cell member perforation andthe perforation is specificity. And the paper is not published atany journals.

Acknowledgement

We thank teacher Peng Xuemei for her help on the operating ofscanning electron microscope and Dr. Jianzhao Mu for perfectingthe manuscript.

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