The Role of miRNAs as New Molecular Biomarkers for Dating the Age of Wound Production: A Systematic Review

Background:The timing of wounds production is a significant issue in forensic pathology. Although various methods have been evaluated, obtaining an accurate dating of lesions is still a challenge. The pathologist uses many parameters to value wound age, such as histological and immunohistochemical. In recent years, there have been many studies regarding the use of miRNAs in wound-age estimation; indeed, miRNAs have multiple potential uses in forensic pathology.Scope:This review aims to verify the efficacy and feasibility of miRNAs as a tool for determining the timing of lesions.Materials and Methods:The authors conducted the systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. PubMed was used as a search engine to find articles published between January, 1st 2016 and October, 1st 2021, to evaluate the current state of the art regarding wound-age estimation.Results:A total of 256 articles were collected; after screening according to PRISMA guidelines, the systematic review included 8 articles. The studies included in this review were all Original articles evaluating the use of biomarkers for wound-age determination.Discussion and Conclusion:The literature review showed that analysis of miRNA is an innovative field of study with significant potentiality in forensic pathology. There are few studies, and almost all of them are at an early stage. The challenge is to understand how to standardize the samples' selection to obtain reliable experimental data. This observation represents a necessary prerequisite to planning further clinical trials.

Wound Vitality in Decomposed Bodies: New Frontiers Through Immunohistochemistry

Background: The question about wound vitality and the estimation of wound age of production are two of the classic investigation fields of forensic sciences. To answer this, the techniques most frequently used in research studies are immunohistochemistry (IHC), molecular biology, and biochemistry. Despite the great data on the literature about the usefulness of IHC in forensic pathology, there is always a request for further studies, especially on tissues altered by putrefactive phenomena. In fact, the degradation of the tissues is intended as the main limiting factor to the use of this technique.Scope: The aim of this pilot study was to evaluate the immunohistochemical behavior of samples collected from decomposed bodies (in different putrefaction phases) and to relate these findings to wound vitality and postmortem interval.Materials and Methods: Samples of skin and soft tissues were collected during autopsies, which were executed on decomposed bodies, whose cause of death was concluded to be traumatic. An immunohistochemical study was performed using antibodies against CD15, CD45, IL-15, tryptase, and glycophorin-A MMPs (endopeptidases involved in degrading extracellular matrix proteins: MMP-9 and MMP-2). An immunohistochemistry (IHC) reaction was evaluated according to a qualitative method as the following legend: (0): not expressed, (+): isolated and disseminated expression, (++): expression in groups or widespread foci, and (+++): widespread expression.Results: Most of the tested markers (tryptase, glycophorin, IL15, CD 15, CD 45, and MMP9) showed to be highly expressed in the tissue of putrefied skin for 15 days.Discussion and Conclusion: Although certainly inconclusive, this experimental application demonstrated that a nonexclusive but combined use of multiple antibodies is appropriate to verify wound vitality in decomposed bodies. Among them, GPA exhibited major reliability.

Identifying biomarkers for evaluating wound extent and age in the contused muscle of rats using microarray analysis: a pilot study

Wound age estimation is still one of the most important and significant challenges in forensic practice. The extent of wound damage greatly affects the accuracy and reliability of wound age estimation, so it is important to find effective biomarkers to help diagnose wound degree and wound age. In the present study, the gene expression profiles of both mild and severe injuries in 33 rats were assayed at 0, 1, 3, 24, 48, and 168 hours using the Affymetrix microarray system to provide biomarkers for the evaluation of wound age and the extent of the wound. After obtaining thousands of differentially expressed genes, a principal component analysis, the least absolute shrinkage and selection operator, and a time-series analysis were used to select the most predictive prognostic genes. Finally, 15 genes were screened for evaluating the extent of wound damage, and the top 60 genes were also screened for wound age estimation in mild and severe injury. Selected indicators showed good diagnostic performance for identifying the extent of the wound and wound age in a Fisher discriminant analysis. A function analysis showed that the candidate genes were mainly related to cell proliferation and the inflammatory response, primarily IL-17 and the Hematopoietic cell lineage signalling pathway. The results revealed that these genes play an essential role in wound-healing and yield helpful and valuable potential biomarkers for further targeted studies.

Effect on healing rates of wounds treated with direct cold atmospheric plasma: a case series

Objective: The response of different critical acute and hard-to-heal wounds to an innovative wound care modality—direct application of cold atmospheric plasma (CAP)—was investigated in this clinical case series. Method: Over an observation period of two years, acute wounds with at least one risk factor for chronification, as well as hard-to-heal wounds were treated for 180 seconds three times per week with CAP. CAP treatment was additional to standard wound care. Photographs were taken for wound documentation. The wound sizes before the first CAP treatment, after four weeks, after 12 weeks and at wound closure/end of observation time were determined using image processing software, and analysed longitudinally for the development of wound size. Results: A total of 27 wounds (19 hard-to-heal and eight acute wounds) with a mean wound area of 15cm2 and a mean wound age of 49 months were treated with CAP and analysed. All (100%) of the acute wounds and 68% of the hard-to-heal wounds healed after an average treatment duration of 14.2 weeks. At the end of the observation period, 21% of hard-to-heal wounds were not yet closed but were reduced in size by >80%. In 11% of the hard-to-heal wounds (n=2) therapy failed. Conclusion: The results suggested a beneficial effect of additional CAP therapy on wound healing. Declaration of interest: This work was carried out within the research projects ‘Plasma for Life’ (funding reference no. 13FH6I04IA) with financial support from the German Federal Ministry of Education and Research (BMBF). In the past seven years AFS has provided consulting services to Evonik and has received institutional support by Heraeus, Johnson & Johnson and Evonik. There are no royalties to disclose. The Department for Trauma Surgery, Orthopaedics and Plastic Surgery received charitable donations by CINOGY GmbH. CINOGY GmbH released the di_CAP devices and electrodes for the study. WV and AH were involved in the development of the used di_CAP device (Plasmaderm, CINOGY GmbH). WV is shareholder of the outsourced start-up company CINOGY GmbH.

Complex challenges of estimating the age and vitality of muscle wounds: a study with matrix metalloproteinases and their inhibitors on animal and human tissue samples

The estimation of wound age and wound vitality is a recurring task in forensic routine work and has been subject of forensic research for a long time. By now, an unrestrictedly reliable marker or set of markers has not been found. In a study on myocardial infarctions, matrix metalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) were detected immunohistochemically in mechanically wounded myocardium (ECG electrodes, vessel ligations). Against this background, the potency of MMP-9, MMP-2, and TIMP-1 as markers for the estimation of wound age and wound vitality was tested in a broad approach with human tissue samples drawn during autopsies and with an animal model, the isolated perfused Langendorff heart. The study comprised samples of injured human skeletal muscle, injured human myocardium, rats’ hearts with vital wounds, and rats’ hearts with postmortem-inflicted wounds that were all stained immunohistochemically. The results showed great scattering, leading to the conclusion that MMP-2, MMP-9, and TIMP-1 are not suitable for wound age estimation. Merely the results for TIMP-1 suggested that this marker might be able to differentiate between vital and postmortem-inflicted wounds. With a view to the promising results of the preceding study, the results underline the necessity to test possible markers of wound age/wound vitality on a large and diverse sample set.

C-reactive Protein Levels in Plasma and Chronic Venous Ulcer Exudate of Persons Who Inject Drugs: A Pilot Study

Background. Persons who inject drugs (PWID) in the groin, legs, and/or feet are at high risk for chronic venous ulcers (CVUs). The plasma C-reactive protein (CRP) level is a marker of systemic inflammation. Objective. This pilot study examined CRP levels in plasma and CVU exudate of PWID. The aims were to (1) compare levels of CRP in plasma and exudate; (2) examine if the CRP level in exudate changed over 4 weeks with wound treatment; and (3) examine the relationship of the exudate CRP level with CVU area, CVU age, number of CVUs, and number of comorbidities. Materials and Methods. Persons who inject drugs seeking wound care were enrolled in this Institutional Review Board approved prospective, longitudinal, descriptive study. A blood sample was collected on the first visit (week 1); the plasma was then separated. Wound exudate was collected on swabs during the first visit (week 1) and 4 weeks later (week 4). All samples were stored at -80° C. Samples were eluted from swabs using mass spectrometry grade water then aliquoted for CRP analysis. Results. The participants of the study included 14 PWID (mean age, 62.14 ± 4.52 years; mean number of comorbidities, 5.71 ± 1.90; and mean number of ulcers 2.07 ± 1.07 that were present for a mean of 7.96 ± 11.91 years without healing). C-reactive protein level in plasma was a mean of 6.47 ± 8.56 mg/L, with lower levels found in wound exudate but highly correlated (rho = .925). Exudate CRP levels decreased from week 1 to week 4, and the 2 were highly correlated (rho = .895). Exudate CRP level week 1 was not significantly related to wound area, wound age, number of ulcers, or number of comorbidities. Conclusions. Plasma and exudate CRP levels were highly correlated. Exudate CRP levels decreased across time. Future large-scale wound healing studies should examine CRP levels over a longer duration and as they correlate to wound healing.

Determination of Sex in Ungulate Herbivores via near Infrared Spectroscopy of Hair: Growing Cattle as a Surrogate Model

Near infrared spectroscopy (NIRS) has been used in a variety of medical and veterinary science applications. In particular, NIRS calibrations have been developed in livestock for steroid content in cattle hair, and wound age or stage of healing in hot iron cattle brands. These NIRS applications also have potential utility in forensic science. Portable NIRS instruments facilitate measurements on live animals and or animal samples in the field. The objective of this study was to evaluate the efficacy of determining sex in growing cattle via NIRS of hair utilizing a portable spectrometer. In two consecutive years, log 1/R spectra (350–2500 nm) were collected using an ASD Field Spec fitted with a contact probe. Experimental subjects were Bos taurus cross calves (n = 12, yr 1; n = 14, yr 2) born to cows grazing central Arizona rangeland. Calf age was approximately 60, 90 and 210 d at branding, estrus synchronization, and weaning, respectively. As cattle were gathered for these routine working events, a total of 7 M and 19 F calves were scanned 3 times each over the left ribcage. A linear discriminant function was applied to spectral data in order to determine sample membership in M or F groups at each collection date. Chi-square procedures were used to determine differences (P < 0.05) in proportion of correct identifications per group and collection date. Overall, 86% of F and 72% of M were correctly (P < 0.05) identified. Corresponding values were 82% for F and 71% for M at branding, 100% for F and 89% for M at estrus synchronization, and 86% for F and 64% for M at weaning. Calf sex was successfully determined using portable NIRS in this proof of concept study. Efficacy of this method should be evaluated for different ungulate herbivores and under additional collection scenarios.

An observational study protocol to explore loneliness and systemic inflammation in an older adult population with chronic venous leg ulcers

Background Chronic venous leg ulcers (CVLUs) are the most common type of lower extremity wound. Even when treated with evidenced-based care, 30–50% of CVLUs fail to heal. A specific gap exists about the association between psychosocial stressors, particularly loneliness, and biomarkers of inflammation and immunity. Loneliness is highly prevalent in persons with CVLUs, has damaging effects on health, and contributes to the development of multiple chronic conditions, promotes aberrant inflammation, and diminishes healing. However, the confluence of loneliness, inflammation and the wound healing trajectory has not been elucidated; specifically whether loneliness substantially mediates systemic inflammation and alters healing over time. This study seeks to address whether there is a specific biomarker profile associated with loneliness, CVLUs, and wound healing that is different from non-lonely persons with CVLUs. Methods An observational prospective study will identify, characterize and explore associations among psychosocial stressors, symptoms and biomarkers between 2 CVLU groups, with loneliness+ (n = 28) and without loneliness- (n = 28) during 4 weeks of wound treatment, measured at 3 time points. We will examine psychosocial stressors and symptoms using psychometrically-sound measures include PROMIS® and other questionnaires for loneliness, social isolation, depression, anxiety, stigma, sleep, fatigue, pain, quality of life, cognition, and function. Demographics data including health history, sex, age, wound type and size, wound age, and treatment will be recorded from the electronic health record. We will characterize a biomarker panel of inflammatory genes including chemotaxic and growth factors, vascular damage, and immune regulators that express in response to loneliness to loneliness and CVLUs using well-established RNA sequence and PCR methods for whole blood samples. In an exploratory aim we will explore whether age and sex/psychological stressors and symptoms indicate potential moderation/mediation of the effect of loneliness on the biomarker profile over the study period. Discussion This study will provide insight into the influence of psychosocial stressors, symptoms, and biological mechanisms on wound healing, towards advancing a future healing prediction model and interventions to address these stressors and symptoms experienced by persons with CVLUs.

Insight into Molecular Profile Changes after Skeletal Muscle Contusion using Microarray and Bioinformatics Analyses

Muscle trauma frequently occurs in daily life. However, the molecular mechanisms of muscle healing, which partly depend on the extent of the damage, are not well understood. This study aimed to investigate gene expression profiles following mild and severe muscle contusion, and to provide more information about the molecular mechanisms underlying the repair process. A total of 33 rats were divided randomly into control (n = 3), mild contusion (n = 15), and severe contusion (n = 15) groups; the contusion groups were further divided into five subgroups (1, 3, 24, 48, and 168 h post-injury; n = 3 per subgroup). A total of 2,844 and 2,298 differentially expressed genes were identified using microarray analyses in the mild and severe contusions, respectively. From the analysis of the 1,620 coexpressed genes in mildly and severely contused muscle, we discovered that the gene profiles in functional modules and temporal clusters were similar between the mild and severe contusion groups; moreover, the genes showed time-dependent patterns of expression, which allowed us to identify useful markers of wound age. The function analyzes of genes in the functional modules and temporal clusters were performed, and the hub genes in each module–cluster pair were identified. Interestingly, we found that genes downregulated at 24−48 h were largely associated with metabolic processes, especially of the oxidative phosphorylation, which has been rarely reported. These results improve our understanding of the molecular mechanisms underlying muscle repair, and provide a basis for further studies of wound age estimation.

MicroRNAs as Useful Tools to Estimate Time Since Death. A Systematic Review of Current Literature

Estimating the time of death remains the most challenging question in forensic medicine, because post-mortem interval (PMI) estimation can be a remarkably difficult goal to achieve. The aim of this review is to analyze the potential of microRNAs (miRNAs) to evaluate PMI. MiRNAs have been studied as hallmarks and biomarkers in several pathologies and have also showed interesting applications in forensic science, such as high sensible biomarkers in body fluid and tissue, for wound age determination and PMI evaluation due to their low molecular weight and tissue-specific expression. The present systematic review was carried out according to the Preferred Reporting Items for Systematic Review (PRISMA) standards. We performed an electronic search of PubMed, Science Direct Scopus, and Excerpta Medica Database (EMBASE) from the inception of these databases to 12 August 2020. The search terms were (“PMI miRNA” or “PMI micro RNA”) and (“miRNA” and “time of death”) in the title, abstract and keywords. Through analysis of scientific literature regarding forensic uses of miRNAs, has emerged that the intrinsic characteristics of such molecules, and their subsequent resistance to degradation, make them suitable as endogenous markers in order to determine PMI. However, further and larger studies with human samples and standardized protocols are still needed.