Rom J Leg Med [18] 247 – 252 [2010]
DOI: 10.4323/rjlm.2010.247
© 2010 Romanian Society of Legal Medicine
Autolytic ultrastructural changes in rat and human hepatocytes
Radovan Karadžić*1, Goran Ilić2, Aleksandra Antović3, Lidija Kostić Banović4
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Abstract: The estimation of the time since death is one of the most difficult problems in forensic pathology.
Changes in the tissue structure provide another possible means by which the time of death can be estimated. The results of
all the previous researches of post-mortem autolytic processes have not provided satisfactory results in establishing the
criteria for determining the time of death based on autolytic ultrastructural changes. In the current paper, autolytic
ultrastructural changes have been observed on experimental materials of Wistar rat liver tissue in different time intervals
after death (2, 4, 6, 12 and 24 h) and in different temperature conditions (8°C, 18°C and 28°C), as well as on human
cadaver livers (6, 12 and 24h at the temperature of 18°C). This research represents the attempt to determine the dynamics
and degree of ultrastructural changes in hepatocytes in the process of post-mortem autolysis with the aim to use the
obtained results in everyday forensic practice for more precise determination of the time of death. By comparative analysis
of the established changes between human and experimental material, it has been proven that the kind, degree and
dynamics of morphological changes are directly dependant on not only the length of post-mortem time, but also, to a
bigger extent, on the temperature of environment where autolysis is happening.
Key words: ultrastructural changes, autolysis, post mortem interval, hepatocytes
A
utolysis represents the rotting of tissue without any vital reactions (without any signs of
soreness). This phenomenon explained by Salkowski (1890) as an intracellular enzyme
activity and named “auto-digestion.” Jacobi (1900) introduced the name “autolysis.” It is a
nonbacterial post-mortem self-destruction (decomposition) of tissue by its own enzymes [1-3].
The estimation of the time since death is one of the most difficult problems in forensic
pathology [4]. The estimation of accurate post-mortem interval (PMI) has been an unfinished quest of
forensic pathologist world for over millennia [5]. The results of all the previous researches of postmortem autolytic processes indicate the polymorph intra- and extra-cellular changes, not only on the
level of one organ, but in relation to different organs as well.
Consequently, the regularities of post-mortem autolytic changes, which would serve, as criteria
for safe and precise determination of the time of death, have not been defined yet [6, 7]. The
comprehensiveness of professional literature in the area of thanatology connected to the determination
of the time of death is in reverse correlation with the importance of this issue in everyday practical
work [8]. Encouraged by the lack of similar researches in forensic medicine, we have undertaken this
preliminary examination in order to determine the correlation between the time of death and autolytic
ultrastructural changes in hepatocytes by using a morphological method.
________________________
*1) Corresponding author: Radovan Karadžić, Professor, MD, PhD, Institute of Forensic Medicine,
Medical faculty Niš; Address: Bulevar Dr Zorana Djindjića 81, 18000 Niš, Serbia;
E-mail: radovanrvs@gmail.com; Fax: +38118-4233-776, Tell: +38165-832-40-41
2. Professor, MD, PhD, Institute of Forensic Medicine, Medical faculty Niš - Serbia
3. Associate Professor, MD, PhD, Institute of Forensic Medicine, Medical faculty Niš - Serbia
4. Professor, MD, PhD, Institute of Forensic Medicine, Medical faculty Niš - Serbia
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Autolytic ultrastructural changes in rat and human hepatocytes
Material and methods
Autolytic changes have been observed on an experimental tissue material of a rat and human liver
by electron microscopy (EM) method. A hundred eight Wistar rats, 4 months old, weighting 250 g, were
sacrificed by cervical dislocation. The analysis of the human material was performed on the human liver
tissue of a 20 male and female cadavers, aged 30 to 35 years, of good health with the cause of death not
connected to liver pathology.
With the aim to observe the changes of external temperature influence in relation to post-mortem
time passage on the occurrence of autolytic changes, the analysis of ultrastructural features of rat
hepatocytes was done in three experimental groups (3 male and female animals per group): I group - 8°C,
II group - 18°C, III group - 28°C, in 6 time intervals between sacrifice and section (0 - controls, 2, 4, 6, 12
and 24 h after death) in the conditions of constant air humidity of 70%. The analysis of the human material
was obtained during the routine autopsies, at room temperature (18°C), in three time groups (6, 12, and 24
h after death). Due to legal prohibition in our country to perform autopsy on bodies before 6 hours have
elapsed since the time of death, changes in the human liver in time sub-groups 0, 2 and 4 were not
analysed.
At each post-mortem time in all groups, liver tissue were excised from the frontal part of the right
liver lobe, and fixed in cacodylate buffer with glutaraldehyde and post-fixed with 1% osmium
tetrachloride. Each sample dehydrated through alcohol and acetone and embedded in Epon 812. Ultrhrathin sections were cut from the epoxy block using a diamond knife, followed by trimming and contrasting
for EM research [9, 10]. The sections were examined and photographed under a transmission electron
microscope of “JEM-100CX” (Jeol, Tokyo, Japan).
The experiment was carried out following the Regulation book on working with experimental
animals of the Institute for Biomedical Research at the Medical Faculty University of Nis (Serbia), based
on European Convention for the Protection of Vertebrate Animals used for Experimental and Other
Scientific Purposes [11]. Research on the human cadavers was approved by the Internal Ethic Committee,
and conducted at the Institute of Forensic Medicine of this Faculty.
Results
1. In relation to time passage, the following ultrastructural changes in rat hepatocytes were
determined: Group I at 8°C temperature: mitochondrial swelling and slight granularity of chromatin (4h);
initial homogenisation of mitochondrial matrix (6h); detaching of the inner and outer nuclear membrane,
initial vacuolisation of sarcoplasm (12h); peripheral chromatin migration, significant mitochondrial cristae
fragmentation, moderate vacuolisation of sarcoplasm, initial reduction of the number of glycogen granules
(24h). Group II at 18°C temperature: mitochondrial swelling, lysis and fragmentation of mitochondrial
cristae (2h); slight granularity and clumping of chromatin, crenation of karyolemma, reduced transparency
of mitochondrial matrix (4h); peripheral migration of chromatin, partial lysis of the outer nuclear
membrane, vacuolisation of sarcoplasm (6h); initial lysis of the inner nuclear membrane with occasional
leakage of chromatin in sarcoplasm, amorphous deposits in mitochondrial matrix, prominent sarcoplasm
vacuolarity, swelling and turbidness of Golgi apparatus, initial lysis of endoplasmic reticulum (ER) (12h);
diffuse leakage of chromatin in sarcoplasm due to lysis of the inner nuclear membrane in the majority of
rat hepatocytes, shape deformation with dense amorphous deposits in mitochondrial matrix, decrease in
number of glycogen granules, diffuse moderate lysis of Golgi apparatus and ER membranes in the
majority of hepatocytes (24h).
Group III at 28°C temperature: noticeable crenation and vacuolar detaching of nuclear membranes,
granulation and clumping of chromatin, fragmentation of mitochondrial cristae, amorphous deposits in
mitochondrial matrix, marked swelling of ER and Golgi apparatus membrane (2h); lysis of the outer
nuclear membrane, dislocation of nucleolus, initial vacuolisation of sarcoplasm (4h), lysis of the inner
nuclear membrane with consequent leakage of chromatin in sarcoplasm, significant loss of mitochondrial
cristae, moderate vacuolisation of sarcoplasm, decrease in number of glycogen granules (6h); dense
amorphous deposits in mitochondrial matrix, significant vacuolisation and turbidness of sarcoplasm,
complete lysis of ER and Golgi apparatus membrane in the majority of hepatocytes (12h); lysis of the
mitochondrial outer membrane in the majority of mitochondria, marked reduction of the number of
glycogen granules (24).
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Figure 1. Transmission electron micrograph of rat hepatocyte 6 h after death at 8°C temperature. Nucleus (Nu) with granularity
of chromatin (Chr); moderate swollen mitochondria with initial homogenisation of mitochondrial matrix (Mt); regular shaped
karyolemma (Klm) and endoplasmatic reticulum (Er); diffuse granular material representing glycogen (Gly). 8300 X.
Figure 2. Transmission electron micrograph of rat hepatocyte 12 h after death at 18°C temperature. Nucleus (Nu) with
peripheral migration and occasional leakage of chromatin in sarcoplasm (arrowhead); arrows indicate lysis of the inner
nuclear membrane; irregular shaped evidently swollen mitochondria with amorphous deposits in mitochondrial matrix
(Mt); initial lysis of endoplasmatic reticulum (Er). 10000 X.
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Karadžić R et al
Autolytic ultrastructural changes in rat and human hepatocytes
Figure 3. Transmission electron micrograph of rat hepatocyte 6 h after death at 28°C temperature. Nucleus (Nu) with lysis
of the inner nuclear membrane and leakage of chromatin in sarcoplasm (arrowheads); dislocation of nucleolus (Nc),
swelling of mitochondria with loss of christae (Mt). 10000 X.
Figure 4. Transmission electron micrograph of human hepatocyte 12 h after death at 18°C temperature. Nucleus (Nu) with
crenation and vacuolar detaching of nuclear membranes; peripheral migration of chromatin (arrows); swelling of
mitochondria (Mt); slightly swollen but regular shaped endoplasmatic reticulum (Er). 13000 X.
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2. In relation to time passage, the following ultrastructural changes in human hepatocytes were
determined at 18°C temperature: slight mitochondrial swelling (6h); crenation and vacuolar detaching
of nuclear membranes, dislocation of nucleolus, clumping and peripheral migration of chromatin,
prominent swelling of mitochondria, fragmentation of mitochondrial cristae in the greater number of
hepatocytes (12); partial lysis of the outer and intactness of the inner nuclear membrane, peripheral
localisation of chromatin in the shape of an irregular homogenous ring of a snowflake form, significant
lysis of mitochondrial cristae, clearly visible diffuse presence of dense glycogen granules, moderate
vacuolisation of sarcoplasm, initial lysis of Golgi apparatus membrane and ER membranes (24h).
Discussions
Numerous methods have been proposed in the last 60 years for the determination of the time since
death. However, none of the suggested methods has provided satisfactory results, which would be widely
used in everyday forensic practice [7, 8]. The main reason lies in the fact that there are an extreme number
of factors, which influence the post-mortal degradation of tissue in each concrete case [12].
It is known that after death or tissue/body removal, anoxic post-mortem effects (ischemia,
glycolysis, and proteolysis) make changes in enzyme activity and ultrastructural cells [1, 2]. Autolytic
process including changes in shape, size, electron density, and localisation of cell structures and
generally causes gradual loss of highly arranged structural organisation of cells [13]. However, not
only different types of tissues, but also different types of cells in a tissue or organ, as well as different
organelles within one cell, can express different degree of sensitivity to autolytic process [14].
Scarpelli et al. established that the occurrence of autolysis is quick in tissues, which have a high
concentration of autolytic enzymes, such as pancreas and gastric mucosis; it is moderate in the heart,
liver, and kidney tissue, whereas it is slow in fibroblasts, which are poor in lysosomes and hydrolytic
enzymes [3]. Similar results were obtained by other authors who have studied the autolytic tissue
changes [13, 15-18].
In our research, simultaneously with observing changes in hapatocytes cell structure of
experimental and human material in different temperature conditions in relation to the duration of
PMI, a comparative analysis of the set changes between human and experimental material was
conducted. The comparison of our research results with the results of other authors’ researches is
objectively hindered by the fact that numerous experiments whose goal is to observe post-mortal
ultrastructural changes are conducted under different conditions (a type of experimental animal, a type
of examined tissue, applied methodology, conditions for conducting the study – temperature, postmortal time interval and the like) [12, 18-20]. Nevertheless, it is doubtless that, subsequent to death, all
tissues succumb to autolysis and observing those changes can be useful in determining the time of
death, especially in combination with other methods [4, 12].
According to results in the present study, it has been established that ultrastructural changes of
hepatocytes are conditioned by the duration of post-mortal time and temperature conditions of outer
environment. The degree of ultrastructural changes of rat hepatocytes indicates the existence of
significant differences between the group at temperature of 8°C and the one at 28°C, while
ultrastructural changes in the group at 18°C represent the clearest mean value of these changes.
In the present study, significant nuclear morphological degeneration in the hepatocyte of the
animal and human tissue was generally observed later than in the mitochondria, but earlier than in the
organelles of sarcoplasm. The earliest and most prominent nuclear changes are clumping and
condensation of chromatin along the nuclear membrane of hepatocyte. Karyolemma showed relatively
delay in post-mortem change, so integrity of nucleus remains preserved in all examined groups. Initial
post-mortem changes in mitochondrial structures of liver tissue are seen in mitochondria swelling
because of increased membrane permeability as well as progressive fragmentation of cristae.
However, it is the fact that mitochondria of different cell types show differences in autolytic
changes during the same time after death [21, 22]. Shape deformation and progressive reduction of the
number of mitochondria as well as vacuolisation of sarcoplasm progressively grew with the increase of
temperature and with the passage of PMI, which is in accordance with the results of other authors [19,
21]. Diffusely arranged glycogen granules in sarcoplasm became rougher, denser, and more
transparent, with the vivid reduction of their number as the time elapsed. Other authors who studied
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autolysis on the rat and the human liver tissue have published similar ultrastructural changes in nuclei,
mitochondria, and sarcoplasm [18-20, 22].
Contrary to the results obtained from the animal experimental material, EM analysis of human
hepatocytes did not show obvious autolytic changes in the first 6 hours of PMI at external temperature
of 18°C. Having compared the results from the analysed animal and human material at temperature of
18°C it has been established that ultrastructural autolytic changes in the human material develop more
slowly than the changes in the rat liver, by approximately 6 to 12 hours.
Conclusions
By EM analysis of the liver tissue sample on the human and experimental material, established
ultrastructural changes indicate regularity dependant on post-mortal time and temperature. At the same
temperature conditions (18°C), ultrastructural post-mortal autolytic changes in the rat liver manifest
themselves faster in relation to the same ones in the human liver.
These regularities in ultrastructural changes in hepatocytes are determined within other
parameters as possible indicators of more precise time of death in forensic practice. In addition,
morphological changes stated in this paper and the dynamics of their occurrence could facilitate the
evaluation of liver quality for the needs of transplantations as well as optimal time for liver
preservation for the same needs.
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