Revival of light signalling in the postmortem mouse and human retina.

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

Postmortem metabolomics: influence of time since death on the level of endogenous compounds in human femoral blood. Necessary to be considered in metabolome study planning?

INTRODUCTION: The (un)targeted analysis of endogenous compounds has gained interest in the field of forensic postmortem investigations. The blood metabolome is influenced by many factors, and postmortem specimens are considered particularly challenging due to unpredictable decomposition processes. OBJECTIVES: This study aimed to systematically investigate the influence of the time since death on endogenous compounds and its relevance in designing postmortem metabolome studies. METHODS: Femoral blood samples of 427 authentic postmortem cases, were collected at two time points after death (854 samples in total; t1: admission to the institute, 1.3-290 h; t2: autopsy, 11-478 h; median ∆t = 71 h). All samples were analyzed using an untargeted metabolome approach, and peak areas were determined for 38 compounds (acylcarnitines, amino acids, phospholipids, and others). Differences between t2 and t1 were assessed by Wilcoxon signed-ranked test (p < 0.05). Moreover, all samples (n = 854) were binned into time groups (6 h, 12 h, or 24 h intervals) and compared by Kruskal-Wallis/Dunn's multiple comparison tests (p < 0.05 each) to investigate the effect of the estimated time since death. RESULTS: Except for serine, threonine, and PC 34:1, all tested analytes revealed statistically significant changes between t1 and t2 (highest median increase 166%). Unpaired analysis of all 854 blood samples in-between groups indicated similar results. Significant differences were typically observed between blood samples collected within the first and later than 48 h after death, respectively. CONCLUSIONS: To improve the consistency of comprehensive data evaluation in postmortem metabolome studies, it seems advisable to only include specimens collected within the first 2 days after death.

Bridging the gap between decomposition theory and forensic research on postmortem interval.

Knowledge of the decomposition of vertebrate animals has advanced considerably in recent years and revealed complex interactions among biological and environmental factors that affect rates of decay. Yet this complexity remains to be fully incorporated into research or models of the postmortem interval (PMI). We suggest there is both opportunity and a need to use recent advances in decomposition theory to guide forensic research and its applications to understanding the PMI. Here we synthesise knowledge of the biological and environmental factors driving variation in decomposition and the acknowledged limitations among current models of the PMI. To guide improvement in this area, we introduce a conceptual framework that highlights the multiple interdependencies affecting decay rates throughout the decomposition process. Our framework reinforces the need for a multidisciplinary approach to PMI research, and calls for an adaptive research cycle that aims to reduce uncertainty in PMI estimates via experimentation, modelling, and validation.

First record of Diamesus osculans (Vigors, 1825) (Coleoptera: Silphidae) colonization on a human corpse.

A partially skeletonized human corpse was found in bushes in Selangor, Malaysia in June 2020. Entomological evidence was collected during the autopsy and sent to the Department of Medical Microbiology and Parasitology, Faculty of Medicine, Universiti Teknologi MARA (UiTM) for minimum postmortem interval (PMImin) analysis. Standard protocols were applied when processing preserved and live insect specimens of both larval and pupal stages. Entomological evidence revealed that the corpse was colonized by Chrysomya nigripes Aubertin, 1932 (Diptera: Calliphoridae) and Diamesus osculans (Vigors, 1825) (Coleoptera: Silphidae). Chrysomya nigripes was chosen as the PMImin indicator as this fly species is an earlier colonizer compared to D. osculans beetle larvae which their presence is the indicative of late stage of decomposition. For the present case, the pupae of C. nigripes were the oldest insect evidence collected and based on the available developmental data, the estimated minimum PMI was established between 9 and 12 days. It is noteworthy to highlight that this is the first record of D. osculans colonization on a human corpse.

A computational approach to estimate postmortem interval using postmortem computed tomography of multiple tissues based on animal experiments.

The estimation of postmortem interval (PMI) is a complex and challenging problem in forensic medicine. In recent years, many studies have begun to use machine learning methods to estimate PMI. However, research combining postmortem computed tomography (PMCT) with machine learning models for PMI estimation is still in early stages. This study aims to establish a multi-tissue machine learning model for PMI estimation using PMCT data from various tissues. We collected PMCT data of seven tissues, including brain, eyeballs, myocardium, liver, kidneys, erector spinae, and quadriceps femoris from 10 rabbits after death. CT images were taken every 12 h until 192 h after death, and HU values were extracted from the CT images of each tissue as a dataset. Support vector machine, random forest, and K-nearest neighbors were performed to establish PMI estimation models, and after adjusting the parameters of each model, they were used as first-level classification to build a stacking model to further improve the PMI estimation accuracy. The accuracy and generalized area under the receiver operating characteristic curve of the multi-tissue stacking model were able to reach 93% and 0.96, respectively. Results indicated that PMCT detection could be used to obtain postmortem change of different tissue densities, and the stacking model demonstrated strong predictive and generalization abilities. This approach provides new research methods and ideas for the study of PMI estimation.

Unveiling decomposition dynamics: leveraging 3D models for advanced forensic analysis.

Forensic taphonomy, the study of post-mortem processes, is pivotal in modern forensic science. This short communication illuminates limitations in traditional 2D imaging, specifically digital photographs, within forensic taphonomy, and highlights the vast potential of 3D modeling techniques. Drawing from a recent study in Hawaii's tropical savanna, we unveil disparities between real-time observations and 2D photographs when assessing decomposition, emphasizing the importance of scoring method selection and the need to scrutinize 2D imaging's accuracy in forensic taphonomy. Conversely, 3D modeling techniques, an emerging powerhouse in forensic science, offer multidimensional data, including volume, surface area, and spatial relationships, allowing for comprehensive and precise representation of decomposition dynamics. Despite concerns about texture quality, 3D models yield objective data amenable to analysis by multiple experts, thus minimizing subjectivity and augmenting the reliability of forensic assessments. The potential for 3D modeling to bridge the gap between 2D imaging and real-time decomposition requires tailored methodologies. Future research should focus on standardizing protocols and fostering collaboration among forensic experts, technologists, and researchers to unleash 3D technology's full potential in advancing forensic taphonomy.

Luminol and the postmortem interval estimation - influence of taphonomic factors.

The estimation of the postmortem interval (PMI) is one of the key challenges for forensic anthropologists. Although there are several methods referenced for this purpose, none is sufficiently effective. One of the main reasons justifying the complexity of this task is the influence of several taphonomic factors.The study of the Luminol technique has stood out as a promising method for estimating PMI, complementing the existing methods, since it is an economic, easy and reproducible method that operates as a presumptive test. However, it is not known which taphonomic factors can influence the results obtained by this technique.The aim of this study is to test the influence of taphonomic factors, such as temperature, humidity, soil type and pH, on the estimation of the PMI by the Luminol technique.In order to test the influence of the referred factors, a sample consisting of 30 clavicles, with known and similar PMI, collected from autopsies, was distributed as evenly as possible by six vases and buried with different decomposition conditions for a period of 12 months. After the exhumation and sample preparation, the Luminol technique was applied.It was possible to clearly observe differences in the results. Thus, according to our research, it is possible to conclude that the results obtained by the application of Luminol are influenced by taphonomic factors. Therefore, the context in which a body is found should always be considered for applying this technique.

Hypothermic sepsis in time since death estimation - a case report.

Both hyper- and hypothermia are problematic in temperature based forensic time since death estimation. Hyperthermia may occur in infection, traumatic brain injury, and intoxication. Hypothermia is encountered predominantly in exposure. Sepsis may present itself clinically as hypothermic. Sepsis is not uncommon in the forensic setting and mostly occurs in the context of malpractice accusations. There is usually little overlap between sepsis and typical forensic time since death estimation scenarios of violent or otherwise suspicious deaths. In the presented case, hypothermia and time since death estimations did collide. An inmate was found dead in his jail cell. Wardens claimed they had visually approached him alive relatively shortly prior. Rectal temperature measurements, using two separate crime scene thermometers as well as temperature loggers, revealed low rectal temperature at relatively high ambient temperature. These findings suggested a much longer postmortem interval and consequently raised doubts about the stated timeline. The wardens' claims were however confirmed by camera recordings, which also allowed a reasonable estimate of the true time of death. The cause of death was confirmed as septic organ failure at autopsy, which explained low rectal temperature. The presence of WISCHNEWSKI-spots was noted. When the PRISM-method was applied to the temperature recordings, low rectal temperature at the time of death was detected successfully. However, adaptation of the underlying equation for lower "starting temperature" did not produce satisfactory results. It is concluded that even though hypothermia at the time of death may possibly be detected from temperature data, attempts at time since death estimation for cases of hypothermia by adaptation of the equation should be avoided.

Parasitic and predatory behavior of Alysia manducator (Hymenoptera: Braconidae) on blow fly larvae feeding on an adult pig carcass in the Western Cape Province of South Africa: preliminary observations and forensic implications.

Wasps are part of the entomofauna associated with vertebrate carrion. They are known to parasitize and prey on specific life stages of insect hosts such as eggs, larvae, pupae, and/or adults associated with vertebrate carrion. However, reports of parasitic behavior of wasps on carrion-associated insect life stages and their possible forensic implications are non-existent in the Western Cape Province of South Africa. This study is part of ongoing research investigating the entomofauna and their pattern of succession on an adult pig carcass in Cape Town, South Africa. During this study, the parasitic wasp Alysia manducator was noted parasitizing and preying on blow fly larvae associated with the decomposing carcass. The arrival of A. manducator coincides with the occurrence of blow fly eggs and/or larvae on the carcass. These wasps were seen in close association with the eggs and larvae of blow flies on various parts of the carcass and some wasps were seen dragging fly larvae attached to their ovipositors away from one part of the carcass to another. Some A. manducator were also observed walking over several larvae on the carcass while exhibiting a stabbing behavior presumably in search of a host for oviposition. We suggest that the observations recorded in this study are of considerable forensic importance as the dragging effect and predatory and stabbing behavior exhibited by A. manducator could potentially disrupt the feeding and development of the fly larvae on the carcass. This could subsequently alter the process of carcass decomposition and/or affect minimum post-mortem interval estimations.

A comparison of maceration methods for the preparation of infant skeletal remains for forensic anthropological analysis.

Very little literature currently exists prescribing which maceration method to use when preparing infant human remains, resulting in bone quality that is suitable for forensic anthropological analysis. The aim of the study was to test five maceration methods to determine which is most suitable for infant remains for forensic anthropological analysis. The sample included five neonate pig carcasses (Sus scrofa domesticus), ranging between one to three days old. Five maceration methods were tested on the pig carcasses (one pig per maceration method) to determine their effectiveness. The methods included invertebrate maceration by meal worms, chemical maceration by bleach, chemical maceration by borax solution, enzymatic maceration by laundry detergent and sodium carbonate solution, and chemical maceration by sodium hypochlorite. A scoring method was created to assess the effectiveness of each maceration method. Invertebrate maceration and chemical maceration using bleach were the least successful methods of maceration (total maceration score = 8 respectively). Chemical maceration using borax and chemical maceration using sodium hypochlorite achieved complete maceration of the skeletal remains; however, they both resulted in artifacts that are unsuitable for forensic analysis (total maceration score = 14 respectively). Enzymatic maceration using laundry detergent and sodium carbonate was the most successful method (total maceration score = 17). The detergent technique subsequently successfully macerated all five sets of infant human remains. This study has validated that the enzymatic maceration technique using laundry detergent and sodium carbonate can be used to effectively macerate the remains of infant skeletal remains for forensic anthropological analysis.

Methods for the optimal preservation of blow fly intra-puparial forms for morphological analysis in forensic casework.

Accurate minimum post-mortem interval (minPMI) estimations often rely on a precise age determination of insect developmental stages, which is significantly influenced by environmental temperature. An optimal preservation of the entomological samples collected at crime scenes is pivotal for a reliable aging of immature insect samples. For blow flies (Diptera: Calliphoridae), the most widely used insect indicators in forensic investigations, an appropriate preservation of tissues is particularly important in the case of puparial samples because aging methods for intra-puparial forms usually depend on morphological analyses; however, although informative soft tissues and structures could be discoloured and/or distorted if they are not properly fixed, there is a lack of studies to assess different methods for the optimal preservation of intra-puparial forms collected in forensic investigations. The present study compares three preservation methods for intra-puparial forms of the blow fly Calliphora vicina Robineau-Desvoidy, 1830: (i) direct immersion into 80% ethanol, (ii) puncturing of the puparium and hot water killing (HWK) prior to preservation in 80% ethanol, and (iii) HWK without puncturing before preservation in 80% ethanol. External and internal morphological analyses of intra-puparial forms of different ages were conducted to assess the quality of preservation. The results indicate that direct immersion in ethanol led to poor preservation, affecting both external and internal tissues. Both methods with HWK resulted in a better preservation, but puncturing resulted, in some cases, in physical damage of the specimens. HWK without puncturing emerged as the optimal preservation method, consistently yielding high preservation scores for both external and internal morphological analyses. These findings have practical implications for forensic practitioners and emphasise the need for updating some published guidelines and protocols in forensic entomology.

Forensic taphonomic experimental design matters: a study assessing clothing and carrion biomass load on scavenging in Cape Town, South Africa.

The identification of unknown human remains is a significant and ongoing challenge in South Africa, worsened by the country's high murder rate. The rate of decomposition in South Africa is significantly influenced by vertebrate scavenging, which, if not considered, can impede the accurate estimation of the post-mortem interval. Scavenging patterns vary greatly depending on the environment and ecological region, and there is limited data for the Western Cape province. To address this gap, two clothed and uncaged pig carcasses weighing 60 kg each were placed in the field in July 2021 and January 2022, respectively. Motion-activated infrared-capable trail cameras were used to observe decomposition, scavenger species, and their activities. Additionally, a comparative sample of 16 unclothed carcasses deployed between 2014 and 2016 in the same habitat were analyzed to assess the impact of clothing and biomass load. The study found three main results: (1) Regardless of habitat or biomass load, it took significantly less time to reach decomposition milestones (25%, 50%, and 75%) during the summer season; (2) the presence of mongoose scavengers had a greater impact on the time required to reach milestones during winter compared to summer; and (3) single carcass deployments reached the milestones faster than multi-carcass deployments in both seasons. This research highlights the potential inaccuracy of current methods for estimating the post-mortem interval when scavenging activity is not considered or documented in the underlying experimental data, particularly for environments or ecological biomes where scavengers actively impact decomposition rates.

Catch me if you can-emission patterns of human bodies in relation to postmortem changes.

The present study examines for the first time the emission patterns and olfactory signatures of 9 complete human corpses of different stages of decomposition. Air sampling was performed inside the body bags with solid sorbents and analysed by coupled gas chromatography-mass spectrometry after thermal desorption (TD-GC-MS). Furthermore, odour-related substances were detected by gas chromatography-olfactometry (GC-O). Sulfurous compounds (mainly dimethyl di- and trisulfide) were identified as most important to the odour perception. Around 350 individual organic substances were detected by TD-GC-MS, notably sulfurous and nitrogenous substances as well as branched alkanes, aldehydes, ketones, alcohols, carboxylic acids, carboxylic acid esters and ethers. A range of terpenes was detected for the first time in a characteristic emission pattern over all decomposition stages. Concentrations of the substances varied greatly, and no correlation between the emission patterns, the stage of decomposition and the cause of death could be found. While previous studies often analysed pig cadavers or only parts of human tissue, the present study shows the importance of analysing complete human corpses over a range of decomposition stages. Moreover, it is shown that using body bags as a kind of "emission test chamber" is a very promising approach, also because it is a realistic application considering the usual transport and store of a body before autopsy.

Postmortem CT analysis of paranasal sinuses using an experimental model of drowning.

Fluid-filled paranasal sinuses are suggested to be a valuable tool to distinguish between drowning and non-drowning postmortem, yet the mechanisms governing fluid entry remains unknown. We investigate if fluid-filled paranasal sinuses are caused by a passive influx from submersion or an active aspiration mechanism during drowning. The ovine nasal cavity and maxillary sinuses are remarkably similar anatomically to humans, and have been used for endoscopic surgical training in recent decades. We submerged 15 decapitated ovine heads from agricultural waste at a depth of 2 m in flowing water for 1, 8, and 24 h and 7 days. Paranasal sinuses were CT imaged and compared pre- and post-submersion to non-submerged controls. Furthermore, we examined the paranasal sinuses of a single homicide case of a non-drowned submerged subject. Results demonstrate that fluid passively enters the maxillary sinus postmortem in the non-drowned ovine heads following 1 h of submersion. Fluid volume was independent of submersion time and influenced by time out of water as well as handling, since volume was reduced between consecutive CT scans. In contrast to our hypothesis, the filling of the paranasal sinuses is due to passive influx of fluid from submersion rather than an active aspiration during drowning. The observation that paranasal sinuses were fluid-filled in a single medico-legal case of postmortem submersion supports the finding of passive influx. Consequently, careful interpretation of fluid-filled paranasal sinuses is required when bodies are found in water, as the finding cannot distinguish between postmortem submersion and drowning.

Proteome alterations in human autopsy tissues in relation to time after death.

Protein expression is a primary area of interest for routine histological diagnostics and tissue-based research projects, but the limitations of its post-mortem applicability remain largely unclear. On the other hand, tissue specimens obtained during autopsies can provide unique insight into advanced disease states, especially in cancer research. Therefore, we aimed to identify the maximum post-mortem interval (PMI) which is still suitable for characterizing protein expression patterns, to explore organ-specific differences in protein degradation, and to investigate whether certain proteins follow specific degradation kinetics. Therefore, the proteome of human tissue samples obtained during routine autopsies of deceased patients with accurate PMI (6, 12, 18, 24, 48, 72, 96 h) and without specific diseases that significantly affect tissue preservation, from lungs, kidneys and livers, was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the kidney and liver, significant protein degradation became apparent at 48 h. For the lung, the proteome composition was rather static for up to 48 h and substantial protein degradation was detected only at 72 h suggesting that degradation kinetics appear to be organ specific. More detailed analyses suggested that proteins with similar post-mortem kinetics are not primarily shared in their biological functions. The overrepresentation of protein families with analogous structural motifs in the kidney indicates that structural features may be a common factor in determining similar postmortem stability. Our study demonstrates that a longer post-mortem period may have a significant impact on proteome composition, but sampling within 24 h may be appropriate, as degradation is within acceptable limits even in organs with faster autolysis.

Autopsy-Based Comparative Study of Gross and Histopathological Findings at Bone Fracture Surfaces Before and After Death.

ABSTRACT: Fractures of bones are commonly encountered in traumatic injuries, and distinguishing between antemortem (AM) and postmortem (PM) bone fractures is crucial for estimating the time since injury and has important medicolegal implications. Correct differentiation enables understanding the dynamics of the injury and, in some cases, the cause of death. The present study aims to evaluate the gross morphological and histopathological characteristics of bony fracture surfaces to determine whether they occurred before or after an individual's death.Fifty-seven sets of bone samples, including both antemortem and artificially created postmortem fractures, were collected from cadavers during medicolegal autopsies, meeting the inclusion criteria. Gross morphological and histopathological features were examined after staining the bony fracture edges with hematoxylin and eosin stain. The study revealed distinct morphological characteristics at fracture surfaces for both antemortem and artificially created postmortem fractures. In addition, signs of vital reaction were observed exclusively in antemortem fracture cases.In conclusion, the findings emphasize the importance of meticulous morphological and histopathological examination of bony fracture surfaces to differentiate between antemortem fractures and artificially created postmortem artifacts. This differentiation holds significant value in forensic investigations and medicolegal cases.

The Role of Protein Degradation in Estimation Postmortem Interval and Confirmation of Cause of Death in Forensic Pathology: A Literature Review.

It is well known that proteins are important bio-macromolecules in human organisms, and numerous proteins are widely used in the clinical practice, whereas their application in forensic science is currently limited. This limitation is mainly attributed to the postmortem degradation of targeted proteins, which can significantly impact final conclusions. In the last decade, numerous methods have been established to detect the protein from a forensic perspective, and some of the postmortem proteins have been applied in forensic practice. To better understand the emerging issues and challenges in postmortem proteins, we have reviewed the current application of protein technologies at postmortem in forensic practice. Meanwhile, we discuss the application of proteins in identifying the cause of death, and postmortem interval (PMI). Finally, we highlight the interpretability and limitations of postmortem protein challenges. We believe that utilizing the multi-omics method can enhance the comprehensiveness of applying proteins in forensic practice.

CT analysis of skull contents in naturally mummified human corpses, a multicentric study.

INTRODUCTION: This study aimed to assess skull contents, brain appearance, and density on postmortem computed tomography in naturally mummified corpses. MATERIAL AND METHODS: For this purpose, a retrospective multicentric study, including mummified corpses from two French centers (Brest and Nantes) and from the New Mexico Decedent Image Database (USA), was performed by analyzing postmortem computed tomography (PMCT) focused on the head and neck of partially or fully mummified corpses discovered between 2011 and 2022. The PMCT analysis provided data on the CT appearance of brains, allowing them to be classified into four different categories (desiccation, liquefaction, dura mater only (DMO), and absence), and to measure densities (HU) of the brain remains. In addition, data on postmortem intervals (PMI) from Nantes and Brest centers were collected and analyzed to test the link between brain densities and PMIs. RESULTS: 54 cases of naturally mummified corpses were included. The brains were classified as liquefied (56%), desiccated (17 %), DMO (20 %), and absent (7 %) based on their CT appearance. Dehydrated brains were significantly (p < 0.004) denser (median 102 HU, interquartile range (IQR) 41) than either liquefied brains (median 39.5 HU, IQR 9) or brains with DMO (median -25 HU, IQR 57). However, the density of brain remains was not significantly affected by where the bodies were found (p = 0,41). Analysis of PMI and brain densities was performed on 22 cases. The results showed that brain remains were significantly (p = 0.039) denser when they were found after a PMI of more than six months. CONCLUSION: Brain desiccation was the aspect with the highest densities on PMCT, and for which we were able to highlight great preservation of anatomical structures observable in living organisms.

Seasonal diversity and dynamics of entomofauna associated with the decomposition of pig (Sus scrofa domesticus L.) carcasses in a Peri-urban area of Central Burkina Faso, West Africa.

This study aimed to determine the effects of seasons on the diversity and dynamics of insects associated with pig carrion in Gampela, Plateau Central Region, Burkina Faso, West Africa. Pig carcasses (Sus scrofa domesticus L.) were exposed in an open area to monitor their decomposition process and their colonization by insects during a warm and dry period (March-April 2015), a warm and wet period (August-September 2015) and a cool and dry period (December 2015-January 2016). Monitoring and entomological data collection occurred for 60 days for each study period. The decomposition process always included five stages (fresh, bloating, putrefaction, postputrefaction, and skeletal), which varied in duration depending on the study period. In sum, 118,761 insects belonging to 4 orders, 12 families, 17 genera, and 21 species were collected during the three study periods, with approximately 61% of the individuals collected during the warm and wet period. The dynamics of insect populations associated with the pig carcasses included two peaks at each study period. Formicidae was the most abundant insect family in each study period, but Musca domestica was the most abundant species. The listed species were found to belong to four trophic groups with a predominance of predators/carnivores followed by necrophagous insects. The orders, families, and trophic groups reported in this study were significantly more abundant during the warm and wet period. The results show prospects for the use of Calliphoridae, Muscidae, and Dermestidae in criminal investigations, depending on seasonal variations in Sudano-Sahelian areas.

Immunohistochemical expression of HMGB1 and related proteins in the skin as a possible tool for determining post-mortem interval: a preclinical study.

Determining the post-mortem interval (PMI) is one of forensic pathology's primary objectives and one of its most challenging tasks. Numerous studies have demonstrated the accuracy of histomorphology and immunohistochemical investigations in determining the time of death. Nevertheless, the skin, a robust and easy-to-remove tissue, has only been partially analyzed so far. By studying 20 adult male mice, we tried to determine whether post-mortem immunohistochemical detection in the skin of HMGB1 proteins and associated components (Beclin1 and RAGE) could be used for this purpose. We discovered that nuclear HMGB1 overexpression indicates that death occurred within the previous 12 h, nuclear HMGB1 negativization with high cytoplasmic HMGB1 intensity indicates that death occurred between 12 and 36 h earlier and cytoplasmic HMGB1 negativization indicates that more than 48 h have passed since death. RAGE and Beclin1 levels in the cytoplasm also decreased with time. The latter proteins' negativization might indicate that more than 24 and 36 h, respectively, have passed from the time of death. These indicators might potentially be helpful in forensic practice for determining the PMI using immunohistochemistry.