- Email: [email protected]
Postmortem interval estimation using the animal model of postmortem gas volume changes
Accepted Manuscript Postmortem interval estimation using the animal model of postmortem gas volume changes Chika Iwamoto, Kenoki Ohuchida, Miki Okumura, Yosuke Usumoto, Junji Kishimoto, Masaharu Murata, Noriaki Ikeda, Makoto Hashizume PII: DOI: Reference:
S1344-6223(17)30234-1 https://doi.org/10.1016/j.legalmed.2017.12.010 LEGMED 1473
To appear in:
Legal Medicine
Received Date: Revised Date: Accepted Date:
26 June 2017 22 November 2017 11 December 2017
Please cite this article as: Iwamoto, C., Ohuchida, K., Okumura, M., Usumoto, Y., Kishimoto, J., Murata, M., Ikeda, N., Hashizume, M., Postmortem interval estimation using the animal model of postmortem gas volume changes, Legal Medicine (2017), doi: https://doi.org/10.1016/j.legalmed.2017.12.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Postmortem interval estimation using the animal model of postmortem gas volume changes
Chika Iwamotoa, Kenoki Ohuchidab, Miki Okumurac, Yosuke Usumotoc, Junji Kishimotod, Masaharu Muratae, Noriaki Ikedac, Makoto Hashizumea,*
aDepartment
of Advanced Medical Initiatives, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan bDepartment
of Surgery and Oncology, Graduate School of Medical Sciences,
Kyushu University, Fukuoka, Japan cDepartment
of Forensic Pathology and Sciences, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan dDepartment
of Research and Development of Next Generation Medicine,
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan eCenter
for Advanced Medical Innovation, Kyushu University, Fukuoka,
Japan
Keywords: Postmortem interval, Portal venous gas, Intestinal gas, Autopsy imaging, micro-CT, segmentation
* Corresponding author at: Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
E-mail address: [email protected] (M. Hashizume).
1
Abstract (243 words)
It is important to estimate the postmortem interval in forensic autopsy. Many methods to estimate the postmortem interval have been reported, and are typically associated with internal examination. However, there are issues such as rejection of autopsy by the family and a lack of forensic doctor in internal examination. Therefore, it is necessary to develop new methods, such as autopsy imaging, that can substitute for internal examination. Here, we first evaluated whether gas volume in the body increased with postmortem interval. Time-dependent X-ray CT imaging of euthanized Crl:CD (SD) rats (n = 3) was performed immediately after euthanasia and at seven subsequent time points up to 168 hours (7 days) at 24-hour intervals. The data revealed that gas volume in the body increased in a time-dependent manner. Next, we reconstructed 3D images of isolated gas and calculated the gas volume using Amira software. In all cases, the volume of both portal venous gas and intestinal gas increased in a time-dependent manner. The volume of portal venous gas increased exponentially, while the volume of intestinal gas increased in a linearly with time. These data might be suggested that the postmortem gas volume change is one of indicators for estimating the postmortem interval. In addition, it would be possible to estimate more accurate postmortem interval by combining not only gas volume changes at the above two sites but also gas volume changes of the other sites such as heart cavities, kidney parenchyma, or abdominal aorta.
2
1. Introduction
Estimation of the postmortem interval is one of the most important evaluation items in forensic autopsy and postmortem examination. However, it remains difficult to estimate the postmortem interval in forensic pathology. Many methods to estimate the postmortem interval have been investigated and reported. One such method is evaluation of the decrease in rectal temperature. In this method, the postmortem interval is calculated using a downward curve of rectal temperature [1]. Other methods, such as biochemical examination, bacteriological examination, degree of digestion, and rigor mortis, were also reported [2]. Saukko et al. [3] and Usumoto et al. [4] showed that the postmortem interval can be estimated by accurately measuring the color of postmortem lividity using spectrophotometry. In addition, ultrastructural changes, such as autolysis, based on histological examination are useful phenomena to estimate the postmortem interval and cause of death [5, 6]. A previous study revealed that rapid depletion of glycogen and chromatin clumping occurred with time during autolytic changes to the rat myocardium [7]. In analyses on postmortem autolysis in the human liver, the dynamics and degree of ultrastructural changes were found to be directly dependent on not only the length of postmortem time, but also the temperature of the environment during autolysis [8]. In an examination of postmortem autolysis in various organs derived from rats, nuclear changes occurred with postmortem time [9]. Although several morphological studies on the postmortem interval
3
have been reported [10-12], it is a major issue that these postmortem changes are markedly influenced by various factors such as temperature, air humidity, and type of environment [13]. These findings suggest that the usefulness of forensic autopsy and postmortem examination to estimate the postmortem interval remains limited. Furthermore, forensic autopsy and/or postmortem examination are extremely time-consuming and require much manpower from experts. Therefore, novel methods to estimate the postmortem interval are required as a substitute for forensic autopsy and postmortem examination. In recent years, autopsy imaging by X-ray CT scans has been rapidly expanding around the world and this type of examination has widely accepted name as post-mortem computed tomography (PMCT). Autopsy imaging is a system for determining the cause of death through performance of X-radiate, X-ray CT or MRI scans on someone’s body. In addition, autopsy imaging is useful for abuse prevention, missed crime prevention, identification of victims, and analysis of postmortem changes [14]. However, micro-CT scan is quite rarely used for autopsy imaging. These observations led us to investigate ways to estimate the postmortem interval by PMCT, especially micro CT scan. In the present study, we used rat models and performed autopsy imaging to investigate whether the volume of postmortem gas, such as portal venous gas or intestinal gas, was directly related to the postmortem interval. We found that the volume of postmortem intestinal gas is useful for estimating the postmortem interval. The combination of analyses of portal
4
venous gas and intestinal gas volume changes may be useful to calculate a more detailed postmortem interval.
5
2. Materials and methods
2.1. Rats Male Crl:CD (SD) rats at 6 weeks of age were purchased from Charles River Laboratories Japan Inc. (Kanagawa, Japan). All experiments were conducted according to the guidelines of the Institutional Animal Committee of Kyushu University.
2.2. XX-ray CT scanning X-ray CT imaging of whole rats including the intestine and the liver was performed using a micro X-ray CT scanner (Rigaku Corporation, Tokyo, Japan) immediately after euthanasia. After 24 hours in an 18°C chamber, X-ray CT imaging was similarly performed. Time-dependent X-ray CT images were obtained at 24- hour intervals until 168 hours (7 days) after euthanasia. We used the following parameters of micro-CT examination, FOV: 73, tube voltage: 90V, radiation dose: 200μA, slice thickness: 295.2μm, total scan time: 34sec. For all rats used in this experiment (n = 3), X-ray CT imaging was performed under the same conditions as presented above.
2.3. 3D reconstruction and determination of postmortem portal portal venous gas The part where the liver appeared on the X-ray CT images of the whole rats was extracted. The region of gas was selected automatically by Amira 3D analysis ver. 6.1.1 software (Maxnet Co., Ltd., Tokyo, Japan), and any gas other than portal venous gas was removed manually. The extracted regions
6
of the portal venous gas were reconstructed in 3D and the volume of portal venous gas was measured by the Amira software. We calculated the volume of gas at each measurement point in a similar manner.
2.4. Quantification of intestinal gas volume using 3D reconstruction from CT images The part where the bowels appeared on the X-ray CT images of the whole rats was extracted. The region of gas was selected automatically by the Amira software, and any gas except for intestinal gas was removed manually. 3D reconstruction and quantification of intestinal gas volume were performed as described above.
2.5. Statistical analysis To examine the association between intestinal gas volume and postmortem interval, the regression expressions in each case were estimated by simple linear regression analysis. The R2 values and p-values for the correlation were obtained using a bivariate analysis in JMP version 11 software (SAS Institute Japan Inc., Tokyo, Japan) with the cooperation of statistical expert. To investigate the association between portal venous gas volume and time after death, the approximate curves and R2 values in each case were obtained by logistic regression analysis in the JMP software.
7
3. Results
3.1. CT images show a timetime -dependent increase in gas volume in the body after death First, we evaluated whether gas volume in the body increased with postmortem interval. Time-dependent X-ray CT images of euthanized rats (n = 3) were performed immediately after euthanasia and at seven subsequent time points until 168 hours (7 days) at 24-hour intervals. As shown in Fig. 1A, postmortem portal venous gas increased with time after death. The star indicates portal venous gas. Postmortem intestinal gas also increased with time after death (Fig. 1B). 3D-CT images were reconstructed using the Amira software, focusing on portal venous gas or intestinal gas. Fig. 2A and 2B show representative 3D-CT images of postmortem portal venous gas (red dots), while Fig. 2C and 2D show representative 3D-CT images of postmortem intestinal gas (blue dots). The data revealed that both gas volumes in the body increased in a time-dependent manner.
3.2. Volume changes in postmortem gas in the body are significantly correlated with postmortem interval We measured portal venous gas or intestinal gas on CT images at each time point. Using the CT data, we first manually removed any unrelated gas, such as intragastric gas, to isolate the portal venous gas and intestinal gas. We then reconstructed each isolated gas in 3D-CT images and calculated the gas volume using the Amira software (Tables 1 and 2). In all cases, we found that
8
the volume of both portal venous gas and intestinal gas increased over time. As shown in Table 1, the increase rate of portal venous gas showed similar behavior in Case 1 and Case 2, but was significantly higher in Case 3. Interestingly, the increase rate of intestinal gas exhibited a similar tendency to the increase rate of portal venous gas (Table 2). The volume of intestinal gas increased by about 2–3 times compared with the initial value.
3.3. Volume of postmortem portal venous gas is useful to estimate postmortem interval at the early stage of postmortem changes We calculated the volume changes in portal venous gas and intestinal gas using 3D-CT data in line charts (Figs. 3 and 4). The volume of portal venous gas rose exponentially, while the volume of intestinal gas increased linearly with time. In the analyses of volume changes in portal venous gas, we created approximate curves for each case by logistic regression analysis in the JMP software. The logistic regression model for Case 1 was y = 104.14 / [1 + Exp[−0.04[x–117.47]]] (R2=0.998) (Fig. 3A), that for Case 2 was y = 63.29 / [1 + Exp[−0.03[x–106.23]]] (R2=0.982) (Fig. 3B), and that for Case 3 was y = 305.37 / [1 + Exp[−0.07[x–101.87]]] (R2=0.998) (Fig. 3C). These data indicated that the models were appropriate for estimating the postmortem interval. In the analyses of changes in intestinal gas volume, we determined each regression expression for intestinal gas by simple linear regression analysis in the JMP software. The regression expression for Case 1 was y = 11.62x + 3112.04 (R2=0.803, p<0.01) (Fig. 4A), that for Case 2 was y = 22.05x + 2854.46 (R2=0.941, p<0.01) (Fig. 4B), and that for Case 3 was y = 59.40x +
9
4599.43 (R2=0.993, p<0.01) (Fig. 4C). These regression models revealed that the volume change in intestinal gas had a strong association with the postmortem interval. These data suggest that the volume of postmortem intestinal gas is useful for estimating the postmortem interval. Interestingly, for the volume of postmortem portal venous gas, it was shown that the increase rate rose steeply within 72 hours (3 days) after death. In particular, the volume change in postmortem portal venous gas was found to be effective for estimating the elapsed time from 72 hours (3 days) to 120 hours (5 days) after death.
10
4. Discussion
In the present study, we found that gas volume in the body increased in a time-dependent manner. The volume of portal venous gas rose exponentially, while the volume of intestinal gas increased in linearly with time as expected. The data revealed that the volume change in intestinal gas could be obtained by a regression line expression. Thus, if we perform CT scans and measure the abdominal gas volumes at three continuous time points, we can derive a regression formula and estimate the postmortem interval using the volume changes in intestinal gas. In the present study, the gas volume in Case 3 was significantly increased compared with that in Case 1 and Case 2. We conducted the present experiments on rats of the same age and sex, and it is highly unlikely that the differences in the increase in gas volume resulted from physical build because we used rats of roughly the same weight. However, this observation remains a matter for speculation, because the accurate weight of each rat was not measured before euthanasia. As the same tendency for the abdominal gas volume to increase over time was observed among the three cases, it seems unlikely that weight was a major factor. Previous studies have shown that Staphylococcus and Neisseria were predominant in the early postmortem interval and that Lactobacillus casei was predominant in the late postmortem interval [15]. These data suggest that existing species of bacteria change with time. Such differences in the amount and species of bacteria in the rat body may contribute to the
11
different increase rates for abdominal gas. Also, in an examination of gas compositional analysis in the body, it was shown that hydrogen and carbon dioxide were consumed by methanogenic bacteria and methane was generated [16]. Previous studies have indicated that compositional analysis of intra-cadaveric gases and the carbon dioxide concentration of the cardiac gas may become useful tools for diagnosing scuba diving fatalities [17]. Therefore, measurement and identification of bacteria and compositional analysis of postmortem gases are our possible subjects for future investigations. In addition, in an investigation of air/gas distribution and content in body cavities and viscera using PMCT, total lung gas content and aeration ratio were revealed to be different by cause of death. Thus, cause of death should be considered to estimate the postmortem interval using gas volume changes in the body [18]. In conclusion, we found that the postmortem interval can be calculated from the changes of intestinal gas and portal venous gas volumes. Interestingly, in the analyses of changes to the volume of postmortem portal venous gas, the data showed that the increase rate rose steeply from 72 hours (3 days) after death. The combination of analyses of portal venous gas and intestinal gas volume changes may be useful to calculate a more detailed postmortem interval, although further examinations regarding the effects of environmental factors will be needed.
12
Acknowledgments
We thank all of our laboratory members for valuable discussions. This work was supported in part by JSPS Grants-in-Aid for Scientific Research on Innovative Areas (Grant Numbers: 26108001, 26108010). We appreciate the technical support from Itsuro Kamimura at Maxnet Co., Ltd.
13
References
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[2].
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[3].
Saukko P, Knight B. The pathophysiology of death. In: Saukko P, Knight B, editors. Knight's forensic pathology. 3rd ed. London: Arnold; 2004, p. 52-97.
[4].
Usumoto Y, Hikiji W, Sameshima N, Kudo K, Tsuji A, Ikeda N. Estimation od postmortem interval based on the spectrophotometric analysis of postmortem lividity. Leg Med (Tokyo) 2010;12:19-22.
[5].
Madea B. Is there recent progress in the estimation of the postmortem interval by means of thanatochemistry? Forensic Sci Int 2005;151:139-49.
[6].
Badonic T, Frumkina LJ, Jakovleva NI, Hornakova A. Ultrastructural changes of neurons in dependence on the death cause in human brain. Funct Dev Morphol 1992;2:231-4.
[7].
Hibbs RG, Black WC. Electron microscopy of Post-mortem changes in the rat myocardium. Anat Rec 1963;147:261-72.
[8].
Karadzic R, Ilic G, Antovic A, Banovic LK. Autolytic ultrastructural changes in rat and human hepatocytes. Rom J Leg Med 2010;18:247-52.
[9].
Tomita Y, Nihira M, Ohno Y, Sato S. Ultrastructural changes during in situ early postmortem autolysis in kidney, pancreas, liver, heart and skeletal muscle of rats. Leg Med (Tokyo) 2004;6:25-31.
[10].
Collan Y, Salmenpera M. Electron microscopy of postmortem autolysis of rat muscle tissue. Acta Neuropath (Berlin) 1976;35:219-33.
[11].
Armiger LC, Seelye RN, Phil D, Carnell VM, Smith CU, Gavin JB, Herdzson PB. Morphologic and biochemical changes in autolysing dog heart muscle. Lab Invest 1976;34:357-62.
[12].
Nevalainen TJ, Anttinen J. Ultrastructural and functional changes in pancreatic acinar cells during autoltsis. Virchows Arch [Cell Pathol] 1977;24:197-207.
[13].
Janssen W. Forensic histopathology. Berlin: Springer; 1984. p. 15-47.
[14].
Okumura M, Usumoto Y, Tsuji A, Kudo K, Ikeda N. Analysis of postmortem changes in internal organs and gases using computed tomography data. Leg Med (Tokyo) 2017;25:11-15.
14
[15].
Zhang L, Guo JJ, Telet-Siyit, Peng YL, Xie D, Guo YD, Yan J, Zha L, Cai JF. Bacterial succession on rat carcasses and applications for PMI estimation. Fa Yi Xue Za Zhi 2016;32:1-6.
[16].
Varlet V, Bruguier C, Grabherr S, Augsburger M, Mangin P, Uldin T. Gas analysis of exhumed cadavers buried for 30 years: a case report about long time alteration. Int J Legal Med 2014;128:719-24.
[17].
Varlet V, Dominguez A, Augsburger M, Lossois M, Egger C, Palmiere C, Vilarino R, Grabherr S. Understanding scuba diving fatalities: carbon dioxide concentrations in intracardiac gas. Diving Hyperb Med 2017;47:75-81.
[18].
Sogawa N, Michiue T, Ishikawa T, Kawamoto O, Oritani S, Maeda H. Postmortem volumetric CT data analysis of pulmonary air/gas content with regard to the cause of death for investigating terminal respiratory function in forensic autopsy. Forensic Sci Int 2014;241:112-7.
15
Figure legends
Fig. 1. Postmortem changes to the rat liver and bowels (A, B) CT images of rats (n = 3) were taken at 24-hour intervals until 168 hours (7 days) after euthanasia. Representative CT images of postmortem portal venous gas (A) and intestinal gas (B) are shown. The star indicates portal venous gas.
Fig. 2. 3D reconstruction of postmortem portal venous or intestinal gas volume (A, B) 3D-CT reconstructed images of postmortem portal gas at 72 hours (3 days) and 168 hours (7 days) after euthanasia. (C, D) 3D-CT reconstructed images of postmortem intestinal gas at 72 hours and 168 hours after euthanasia. Portal venous gas is shown in red and intestinal gas is shown in blue. These representative results were reconstructed by Amira 3D analysis software.
Fig. 3. Volume changes in postmortem portal venous venous gas The volume of portal venous gas was measured on 3D-CT reconstructed images. The volume showed a sharp increase from 72 hours (3 days) after death. Representative line plots of three independent experiments are shown.
Fig. 4. Volume changes in postmortem postmortem intestinal gas
16
Time-dependent straight lines for the postmortem interval and intestinal gas volume interaction determined on 3D-CT reconstructed images were calculated. Data shown are representative of three independent experiments.
Table 1 Volume of portal venous gas is associated with postmortem interval The volume of portal venous gas in rats was determined on 3D-CT reconstructed images taken at 24-hour intervals from 0 to 168 hours (7 days) after euthanasia.
Table 2 Volume of intestinal gas is is associated with postmortem interval The volume of intestinal gas in rats was determined on 3D-CT reconstructed images taken at 24-hour intervals from 0 to 168 hours (7 days) after euthanasia.
17
18
Table 1 Volume of portal venous gas is associated with postmortem interval
Portal venous gas (mm3)
Postmortem interval (h)
0
24
48
72
96
120
144
168 92.531
Increase rate (-fold at 168h vs at 0h)
Case 1
1.749
5.274
6.534
11.808
29.197 54.433
80.415
Case 2
0.309
0.103
10.701
18.547
25.031 40.362
44.838 57.314
185.5
Case 3
0.978
8.721
10.393
36.014 116.404 245.799 282.353 306.509
313.4
19
52.9
Table 2 Volume of intestinal gas is associated with postmortem interval
Intestinal gas (mm3)
Postmortem interval (h)
0
24
48
72
96
120
144
168
Increase rate (-fold at 168h vs at 0h)
Case 1
2656.5 3643.7
4106.1 4118.0 4084.8
4139.9 4763.5 5194.1
1.96
Case 2
3386.4 3228.0
3725.8 4071.5 4796.6
5426.6 6328.0 6689.8
1.98
Case 3
5126.0 5556.4
7268.5 8813.0 10394.3 11730.9 13163.1 14662.6
2.86
20
Highlights
・The postmortem gas volume were calculated using time-dependent CT images. ・The volume of postmortem portal venous gas rose exponentially with time after death. ・The volume of postmortem intestinal gas increased linearly with time after death. ・Estimation of the postmortem interval by postmortem gas volume changes in the body.
21
Fig. 1. A 0h
24h
48h
72h
*
96h
120h
144h
168h
Fig. 1. B 0h
24h
48h
72h
96h
120h
144h
168h
Fig. 2. A
B
C
D
Fig. 3.
80
Case 1 104.14
y=
50
1+Exp[-0.04[x-117.47]]
R2=0.998
40
Case 2 63.29 1+Exp[-0.03[x-106.23]]
R2=0.982
40
60
C
門脈ガス量(mm3) aaaaaaaaaa
y=
B
門脈ガス量(mm3) aaaaaaaaaa
aaaaaaaaaa
Gas volume (mm3) 門脈ガス量(mm3)
A
30
20
Case 3 305.37
300
y=
250
R2=0.998
1+Exp[-0.07[x-101.87]]
200 150 100
10
20
50 0
0
0 -10 0
50
100 死後経過時間(h)
aaaaaaaaaa
150
-50 0
50
100 死後経過時間(h)
150
aaaaaaaaaa
Postmortem interval (h)
0
50
100 死後経過時間(h)
aaaaaaaaaa
150
Fig. 4. A
B Case 2
Case 3
y= 11.62x + 3112.04
y= 22.05x + 2854.46
y= 59.40x + 4599.43
R2=0.803
R2=0.941
R2=0.993
aaaaaaaaaa
Gas volume (mm3)
Case 1
C
aaaaaaaaaa
aaaaaaaaaa
Postmortem interval (h)
aaaaaaaaaa
S1344-6223(17)30234-1 https://doi.org/10.1016/j.legalmed.2017.12.010 LEGMED 1473
To appear in:
Legal Medicine
Received Date: Revised Date: Accepted Date:
26 June 2017 22 November 2017 11 December 2017
Please cite this article as: Iwamoto, C., Ohuchida, K., Okumura, M., Usumoto, Y., Kishimoto, J., Murata, M., Ikeda, N., Hashizume, M., Postmortem interval estimation using the animal model of postmortem gas volume changes, Legal Medicine (2017), doi: https://doi.org/10.1016/j.legalmed.2017.12.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Postmortem interval estimation using the animal model of postmortem gas volume changes
Chika Iwamotoa, Kenoki Ohuchidab, Miki Okumurac, Yosuke Usumotoc, Junji Kishimotod, Masaharu Muratae, Noriaki Ikedac, Makoto Hashizumea,*
aDepartment
of Advanced Medical Initiatives, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan bDepartment
of Surgery and Oncology, Graduate School of Medical Sciences,
Kyushu University, Fukuoka, Japan cDepartment
of Forensic Pathology and Sciences, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan dDepartment
of Research and Development of Next Generation Medicine,
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan eCenter
for Advanced Medical Innovation, Kyushu University, Fukuoka,
Japan
Keywords: Postmortem interval, Portal venous gas, Intestinal gas, Autopsy imaging, micro-CT, segmentation
* Corresponding author at: Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
E-mail address: [email protected] (M. Hashizume).
1
Abstract (243 words)
It is important to estimate the postmortem interval in forensic autopsy. Many methods to estimate the postmortem interval have been reported, and are typically associated with internal examination. However, there are issues such as rejection of autopsy by the family and a lack of forensic doctor in internal examination. Therefore, it is necessary to develop new methods, such as autopsy imaging, that can substitute for internal examination. Here, we first evaluated whether gas volume in the body increased with postmortem interval. Time-dependent X-ray CT imaging of euthanized Crl:CD (SD) rats (n = 3) was performed immediately after euthanasia and at seven subsequent time points up to 168 hours (7 days) at 24-hour intervals. The data revealed that gas volume in the body increased in a time-dependent manner. Next, we reconstructed 3D images of isolated gas and calculated the gas volume using Amira software. In all cases, the volume of both portal venous gas and intestinal gas increased in a time-dependent manner. The volume of portal venous gas increased exponentially, while the volume of intestinal gas increased in a linearly with time. These data might be suggested that the postmortem gas volume change is one of indicators for estimating the postmortem interval. In addition, it would be possible to estimate more accurate postmortem interval by combining not only gas volume changes at the above two sites but also gas volume changes of the other sites such as heart cavities, kidney parenchyma, or abdominal aorta.
2
1. Introduction
Estimation of the postmortem interval is one of the most important evaluation items in forensic autopsy and postmortem examination. However, it remains difficult to estimate the postmortem interval in forensic pathology. Many methods to estimate the postmortem interval have been investigated and reported. One such method is evaluation of the decrease in rectal temperature. In this method, the postmortem interval is calculated using a downward curve of rectal temperature [1]. Other methods, such as biochemical examination, bacteriological examination, degree of digestion, and rigor mortis, were also reported [2]. Saukko et al. [3] and Usumoto et al. [4] showed that the postmortem interval can be estimated by accurately measuring the color of postmortem lividity using spectrophotometry. In addition, ultrastructural changes, such as autolysis, based on histological examination are useful phenomena to estimate the postmortem interval and cause of death [5, 6]. A previous study revealed that rapid depletion of glycogen and chromatin clumping occurred with time during autolytic changes to the rat myocardium [7]. In analyses on postmortem autolysis in the human liver, the dynamics and degree of ultrastructural changes were found to be directly dependent on not only the length of postmortem time, but also the temperature of the environment during autolysis [8]. In an examination of postmortem autolysis in various organs derived from rats, nuclear changes occurred with postmortem time [9]. Although several morphological studies on the postmortem interval
3
have been reported [10-12], it is a major issue that these postmortem changes are markedly influenced by various factors such as temperature, air humidity, and type of environment [13]. These findings suggest that the usefulness of forensic autopsy and postmortem examination to estimate the postmortem interval remains limited. Furthermore, forensic autopsy and/or postmortem examination are extremely time-consuming and require much manpower from experts. Therefore, novel methods to estimate the postmortem interval are required as a substitute for forensic autopsy and postmortem examination. In recent years, autopsy imaging by X-ray CT scans has been rapidly expanding around the world and this type of examination has widely accepted name as post-mortem computed tomography (PMCT). Autopsy imaging is a system for determining the cause of death through performance of X-radiate, X-ray CT or MRI scans on someone’s body. In addition, autopsy imaging is useful for abuse prevention, missed crime prevention, identification of victims, and analysis of postmortem changes [14]. However, micro-CT scan is quite rarely used for autopsy imaging. These observations led us to investigate ways to estimate the postmortem interval by PMCT, especially micro CT scan. In the present study, we used rat models and performed autopsy imaging to investigate whether the volume of postmortem gas, such as portal venous gas or intestinal gas, was directly related to the postmortem interval. We found that the volume of postmortem intestinal gas is useful for estimating the postmortem interval. The combination of analyses of portal
4
venous gas and intestinal gas volume changes may be useful to calculate a more detailed postmortem interval.
5
2. Materials and methods
2.1. Rats Male Crl:CD (SD) rats at 6 weeks of age were purchased from Charles River Laboratories Japan Inc. (Kanagawa, Japan). All experiments were conducted according to the guidelines of the Institutional Animal Committee of Kyushu University.
2.2. XX-ray CT scanning X-ray CT imaging of whole rats including the intestine and the liver was performed using a micro X-ray CT scanner (Rigaku Corporation, Tokyo, Japan) immediately after euthanasia. After 24 hours in an 18°C chamber, X-ray CT imaging was similarly performed. Time-dependent X-ray CT images were obtained at 24- hour intervals until 168 hours (7 days) after euthanasia. We used the following parameters of micro-CT examination, FOV: 73, tube voltage: 90V, radiation dose: 200μA, slice thickness: 295.2μm, total scan time: 34sec. For all rats used in this experiment (n = 3), X-ray CT imaging was performed under the same conditions as presented above.
2.3. 3D reconstruction and determination of postmortem portal portal venous gas The part where the liver appeared on the X-ray CT images of the whole rats was extracted. The region of gas was selected automatically by Amira 3D analysis ver. 6.1.1 software (Maxnet Co., Ltd., Tokyo, Japan), and any gas other than portal venous gas was removed manually. The extracted regions
6
of the portal venous gas were reconstructed in 3D and the volume of portal venous gas was measured by the Amira software. We calculated the volume of gas at each measurement point in a similar manner.
2.4. Quantification of intestinal gas volume using 3D reconstruction from CT images The part where the bowels appeared on the X-ray CT images of the whole rats was extracted. The region of gas was selected automatically by the Amira software, and any gas except for intestinal gas was removed manually. 3D reconstruction and quantification of intestinal gas volume were performed as described above.
2.5. Statistical analysis To examine the association between intestinal gas volume and postmortem interval, the regression expressions in each case were estimated by simple linear regression analysis. The R2 values and p-values for the correlation were obtained using a bivariate analysis in JMP version 11 software (SAS Institute Japan Inc., Tokyo, Japan) with the cooperation of statistical expert. To investigate the association between portal venous gas volume and time after death, the approximate curves and R2 values in each case were obtained by logistic regression analysis in the JMP software.
7
3. Results
3.1. CT images show a timetime -dependent increase in gas volume in the body after death First, we evaluated whether gas volume in the body increased with postmortem interval. Time-dependent X-ray CT images of euthanized rats (n = 3) were performed immediately after euthanasia and at seven subsequent time points until 168 hours (7 days) at 24-hour intervals. As shown in Fig. 1A, postmortem portal venous gas increased with time after death. The star indicates portal venous gas. Postmortem intestinal gas also increased with time after death (Fig. 1B). 3D-CT images were reconstructed using the Amira software, focusing on portal venous gas or intestinal gas. Fig. 2A and 2B show representative 3D-CT images of postmortem portal venous gas (red dots), while Fig. 2C and 2D show representative 3D-CT images of postmortem intestinal gas (blue dots). The data revealed that both gas volumes in the body increased in a time-dependent manner.
3.2. Volume changes in postmortem gas in the body are significantly correlated with postmortem interval We measured portal venous gas or intestinal gas on CT images at each time point. Using the CT data, we first manually removed any unrelated gas, such as intragastric gas, to isolate the portal venous gas and intestinal gas. We then reconstructed each isolated gas in 3D-CT images and calculated the gas volume using the Amira software (Tables 1 and 2). In all cases, we found that
8
the volume of both portal venous gas and intestinal gas increased over time. As shown in Table 1, the increase rate of portal venous gas showed similar behavior in Case 1 and Case 2, but was significantly higher in Case 3. Interestingly, the increase rate of intestinal gas exhibited a similar tendency to the increase rate of portal venous gas (Table 2). The volume of intestinal gas increased by about 2–3 times compared with the initial value.
3.3. Volume of postmortem portal venous gas is useful to estimate postmortem interval at the early stage of postmortem changes We calculated the volume changes in portal venous gas and intestinal gas using 3D-CT data in line charts (Figs. 3 and 4). The volume of portal venous gas rose exponentially, while the volume of intestinal gas increased linearly with time. In the analyses of volume changes in portal venous gas, we created approximate curves for each case by logistic regression analysis in the JMP software. The logistic regression model for Case 1 was y = 104.14 / [1 + Exp[−0.04[x–117.47]]] (R2=0.998) (Fig. 3A), that for Case 2 was y = 63.29 / [1 + Exp[−0.03[x–106.23]]] (R2=0.982) (Fig. 3B), and that for Case 3 was y = 305.37 / [1 + Exp[−0.07[x–101.87]]] (R2=0.998) (Fig. 3C). These data indicated that the models were appropriate for estimating the postmortem interval. In the analyses of changes in intestinal gas volume, we determined each regression expression for intestinal gas by simple linear regression analysis in the JMP software. The regression expression for Case 1 was y = 11.62x + 3112.04 (R2=0.803, p<0.01) (Fig. 4A), that for Case 2 was y = 22.05x + 2854.46 (R2=0.941, p<0.01) (Fig. 4B), and that for Case 3 was y = 59.40x +
9
4599.43 (R2=0.993, p<0.01) (Fig. 4C). These regression models revealed that the volume change in intestinal gas had a strong association with the postmortem interval. These data suggest that the volume of postmortem intestinal gas is useful for estimating the postmortem interval. Interestingly, for the volume of postmortem portal venous gas, it was shown that the increase rate rose steeply within 72 hours (3 days) after death. In particular, the volume change in postmortem portal venous gas was found to be effective for estimating the elapsed time from 72 hours (3 days) to 120 hours (5 days) after death.
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4. Discussion
In the present study, we found that gas volume in the body increased in a time-dependent manner. The volume of portal venous gas rose exponentially, while the volume of intestinal gas increased in linearly with time as expected. The data revealed that the volume change in intestinal gas could be obtained by a regression line expression. Thus, if we perform CT scans and measure the abdominal gas volumes at three continuous time points, we can derive a regression formula and estimate the postmortem interval using the volume changes in intestinal gas. In the present study, the gas volume in Case 3 was significantly increased compared with that in Case 1 and Case 2. We conducted the present experiments on rats of the same age and sex, and it is highly unlikely that the differences in the increase in gas volume resulted from physical build because we used rats of roughly the same weight. However, this observation remains a matter for speculation, because the accurate weight of each rat was not measured before euthanasia. As the same tendency for the abdominal gas volume to increase over time was observed among the three cases, it seems unlikely that weight was a major factor. Previous studies have shown that Staphylococcus and Neisseria were predominant in the early postmortem interval and that Lactobacillus casei was predominant in the late postmortem interval [15]. These data suggest that existing species of bacteria change with time. Such differences in the amount and species of bacteria in the rat body may contribute to the
11
different increase rates for abdominal gas. Also, in an examination of gas compositional analysis in the body, it was shown that hydrogen and carbon dioxide were consumed by methanogenic bacteria and methane was generated [16]. Previous studies have indicated that compositional analysis of intra-cadaveric gases and the carbon dioxide concentration of the cardiac gas may become useful tools for diagnosing scuba diving fatalities [17]. Therefore, measurement and identification of bacteria and compositional analysis of postmortem gases are our possible subjects for future investigations. In addition, in an investigation of air/gas distribution and content in body cavities and viscera using PMCT, total lung gas content and aeration ratio were revealed to be different by cause of death. Thus, cause of death should be considered to estimate the postmortem interval using gas volume changes in the body [18]. In conclusion, we found that the postmortem interval can be calculated from the changes of intestinal gas and portal venous gas volumes. Interestingly, in the analyses of changes to the volume of postmortem portal venous gas, the data showed that the increase rate rose steeply from 72 hours (3 days) after death. The combination of analyses of portal venous gas and intestinal gas volume changes may be useful to calculate a more detailed postmortem interval, although further examinations regarding the effects of environmental factors will be needed.
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Acknowledgments
We thank all of our laboratory members for valuable discussions. This work was supported in part by JSPS Grants-in-Aid for Scientific Research on Innovative Areas (Grant Numbers: 26108001, 26108010). We appreciate the technical support from Itsuro Kamimura at Maxnet Co., Ltd.
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Figure legends
Fig. 1. Postmortem changes to the rat liver and bowels (A, B) CT images of rats (n = 3) were taken at 24-hour intervals until 168 hours (7 days) after euthanasia. Representative CT images of postmortem portal venous gas (A) and intestinal gas (B) are shown. The star indicates portal venous gas.
Fig. 2. 3D reconstruction of postmortem portal venous or intestinal gas volume (A, B) 3D-CT reconstructed images of postmortem portal gas at 72 hours (3 days) and 168 hours (7 days) after euthanasia. (C, D) 3D-CT reconstructed images of postmortem intestinal gas at 72 hours and 168 hours after euthanasia. Portal venous gas is shown in red and intestinal gas is shown in blue. These representative results were reconstructed by Amira 3D analysis software.
Fig. 3. Volume changes in postmortem portal venous venous gas The volume of portal venous gas was measured on 3D-CT reconstructed images. The volume showed a sharp increase from 72 hours (3 days) after death. Representative line plots of three independent experiments are shown.
Fig. 4. Volume changes in postmortem postmortem intestinal gas
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Time-dependent straight lines for the postmortem interval and intestinal gas volume interaction determined on 3D-CT reconstructed images were calculated. Data shown are representative of three independent experiments.
Table 1 Volume of portal venous gas is associated with postmortem interval The volume of portal venous gas in rats was determined on 3D-CT reconstructed images taken at 24-hour intervals from 0 to 168 hours (7 days) after euthanasia.
Table 2 Volume of intestinal gas is is associated with postmortem interval The volume of intestinal gas in rats was determined on 3D-CT reconstructed images taken at 24-hour intervals from 0 to 168 hours (7 days) after euthanasia.
17
18
Table 1 Volume of portal venous gas is associated with postmortem interval
Portal venous gas (mm3)
Postmortem interval (h)
0
24
48
72
96
120
144
168 92.531
Increase rate (-fold at 168h vs at 0h)
Case 1
1.749
5.274
6.534
11.808
29.197 54.433
80.415
Case 2
0.309
0.103
10.701
18.547
25.031 40.362
44.838 57.314
185.5
Case 3
0.978
8.721
10.393
36.014 116.404 245.799 282.353 306.509
313.4
19
52.9
Table 2 Volume of intestinal gas is associated with postmortem interval
Intestinal gas (mm3)
Postmortem interval (h)
0
24
48
72
96
120
144
168
Increase rate (-fold at 168h vs at 0h)
Case 1
2656.5 3643.7
4106.1 4118.0 4084.8
4139.9 4763.5 5194.1
1.96
Case 2
3386.4 3228.0
3725.8 4071.5 4796.6
5426.6 6328.0 6689.8
1.98
Case 3
5126.0 5556.4
7268.5 8813.0 10394.3 11730.9 13163.1 14662.6
2.86
20
Highlights
・The postmortem gas volume were calculated using time-dependent CT images. ・The volume of postmortem portal venous gas rose exponentially with time after death. ・The volume of postmortem intestinal gas increased linearly with time after death. ・Estimation of the postmortem interval by postmortem gas volume changes in the body.
21
Fig. 1. A 0h
24h
48h
72h
*
96h
120h
144h
168h
Fig. 1. B 0h
24h
48h
72h
96h
120h
144h
168h
Fig. 2. A
B
C
D
Fig. 3.
80
Case 1 104.14
y=
50
1+Exp[-0.04[x-117.47]]
R2=0.998
40
Case 2 63.29 1+Exp[-0.03[x-106.23]]
R2=0.982
40
60
C
門脈ガス量(mm3) aaaaaaaaaa
y=
B
門脈ガス量(mm3) aaaaaaaaaa
aaaaaaaaaa
Gas volume (mm3) 門脈ガス量(mm3)
A
30
20
Case 3 305.37
300
y=
250
R2=0.998
1+Exp[-0.07[x-101.87]]
200 150 100
10
20
50 0
0
0 -10 0
50
100 死後経過時間(h)
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150
-50 0
50
100 死後経過時間(h)
150
aaaaaaaaaa
Postmortem interval (h)
0
50
100 死後経過時間(h)
aaaaaaaaaa
150
Fig. 4. A
B Case 2
Case 3
y= 11.62x + 3112.04
y= 22.05x + 2854.46
y= 59.40x + 4599.43
R2=0.803
R2=0.941
R2=0.993
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Gas volume (mm3)
Case 1
C
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Postmortem interval (h)
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