Int J Legal Med DOI 10.1007/s00414-013-0876-x

ORIGINAL ARTICLE

Markers of mechanical asphyxia: immunohistochemical study on autoptic lung tissues R. Cecchi & C. Sestili & G. Prosperini & G. Cecchetto & E. Vicini & G. Viel & B. Muciaccia

Received: 8 March 2013 / Accepted: 15 May 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Forensic pathologists are often asked to provide evidence of asphyxia death in the trial and a histological marker of asphyxiation would be of great help. Data from the literature indicate that the reaction of lung tissue cells to asphyxia may be of more interest for forensic purposes than migrating cells. The lungs of 62 medico-legal autopsy cases, 34 acute mechanical asphyxia (AMA), and 28 control cases (CC), were immunostained with anti-P-selectin, anti-E-selectin, anti-SP-A, and anti-HIF1-α antibodies, in order to verify if some of them may be used as markers of asphyxia death. Results show that P- and E-selectins expression in lung vessels, being activated by several types of trigger stimuli not specific to hypoxia, cannot be used as indicator of asphyxia. Intra-alveolar granular deposits of SP-A seem to be related to an intense hypoxic stimulus, and when massively present, they can suggest, together with other elements, a severe hypoxia as the mechanism of death. HIF1-α was expressed in small-, medium-, and largecaliber lung vessels of the vast majority of mechanical asphyxia deaths and CO intoxications, with the number and intensity of positive-stained vessels increasing with the duration of the hypoxia. Although further confirmation studies are required, these preliminary data indicate an interesting potential utility of HIF1-α as a screening test for asphyxia deaths. R. Cecchi (*) : C. Sestili : E. Vicini : B. Muciaccia Department of Anatomical, Histological, Legal Medical and Orthopaedic Sciences, Faculty of Medicine and Pharmacology, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy e-mail: [email protected] G. Prosperini CASPUR, Consortium for Supercomputing Applications, Via dei Tizii 6/B, 00185 Rome, Italy G. Cecchetto : G. Viel Institute of Legal Medicine, Department of Molecular Medicine, University of Padova, Via Falloppio 50, 35121 Padova, Italy

Keywords Forensic histopathology . Mechanical asphyxia . Hypoxia . Selectins . Surfactant protein-A . Hypoxiainduced factor 1-α . Evidence

Introduction Asphyxiation can present challenging issues in terms of expert opinions and may generate debate regarding evidence in court when the death is homicidal. As is well-known from the forensic literature [1], homicidal asphyxiation with soft objects is currently a subject of discussion, and histological examinations are required to exclude any relevant acute disease because of the uncharacteristic morphological findings. Toxicological effects have to be excluded, and differentiation of asphyxia-related or hypoxic tissue and organ damage from autolytic changes is not always easy. Histomorphological changes in pulmonary tissue of individuals who die from asphyxia are considered of great importance [2, 3] and constitute one of the central fields of interest for forensic histopathology. Nevertheless, their correlation to asphyxia has to consider ventilation and perfusion alterations due to age, underlying diseases, and external factors, such as smog and smoke. The hemorrhagic edema, which occurs in cases of drug addiction, may also cause problems in morphological differential diagnosis with asphyxia. For this reason, classical histological alterations of lung tissue do not always solve problems of differential diagnosis, especially when intensive care measures or slow death has occurred, and have to be excluded. Therefore, over the decades, thanks to newly available methods, such as immunohistochemistry, attention has gradually shifted to focus on the possibility of dating the time of asphyxia or to characterizing differential diagnosis among various types of asphyxia death [4–8]. Forensic pathologists are often asked to provide evidence of asphyxia death in trial and a good marker of asphyxiation

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should be of great help. In this case, an ideal parameter should be one which is absent in physiological conditions and appears regularly after asphyxia, growing with time before death [9, 10]. This goal is far from being realized, but in specific cases the immunohistochemical findings can be of indicative significance, together with all other available information [1]. Chronic alveolar hypoxia induces an inflammatory reaction with macrophage recruitment, an increase in albumin leakage, and enhanced expression of inflammatory mediators, which seems to be mainly macrophage dependent [11]. Macrophage recruitment was investigated as a marker of prolonged asphyxia, and authors supported the proliferation and mobilization of interstitial and alveolar macrophages and the increased number of giant cells. This argument is still being debated, however, since it can also be found in other pathological conditions (inhalation of dust, cigarette smoke, etc.) [4–8]. At the present time, it is well-known that the lung exhibits several adaptation mechanisms to conditions of low O2 tension. Acute exposure to hypoxia results in vasoconstriction of resistance-sized pulmonary arteries, increased pulmonary arterial pressure, and a redistribution of the blood flow from the basal to the apical portion of the lung [12, 13]. It is therefore expected that the acute inflammatory response to asphyxia of the lung involves mediators of the vessel response to inflammatory insult, and that some of them may represent a good marker for forensic purposes. Ortmann and Brinkmann [14] compared the expression of P-selectin in inflammatory and non-inflammatory lung tissue. P-selectin is a glycoprotein stored for rapid release in Weibel–Palade bodies of the endothelium and in the alpha granules of platelets [15] and represents an adhesion molecule involved in the process of leukocyte rolling on the endothelium. The authors found that in acute deaths (hanging, carbon monoxide and cyanide intoxication) lung vessels showed an overall occurrence of P-selectin with an intense homogeneous staining pattern. The cases of drowning showed a slightly weaker intensity, whereas protracted deaths, due to pneumonia and septic shock, showed an irregular distribution and weak intensity of the marker. Zhu et al. [16–18] and Maeda et al. [19] focused on alveolar surface integrity in asphyxia cases and found that granular staining of surfactant protein-A (SP-A) increases in intra-alveolar spaces in cases of hyaline membrane syndrome, drowning, aspiration of amniotic fluid, and mechanical asphyxia. SP-A is the major surfactant protein and its deficiency causes respiratory distress syndrome [20, 21]. It is produced by type II alveolar cells and normally covers the alveolar surface. A hypoxic condition increases the secretion of SP-A, probably because of the strong forced breathing, and then SP-A precipitates in the alveolar space in aggregates. Zhu et al. [16] showed no significant differences

between mechanical asphyxia and control groups in the intensity of staining of the alveolar surface and in the number of the type II pneumocytes, but many prominent massive intra-alveolar aggregates stained positive with SP-A were found prevalently in the mechanical asphyxia group (approximately 60 % of all cases in this group). It was concluded that this may be a result of enhanced secretion of the pulmonary surfactant. The above-mentioned articles indicate that the reaction of lung tissue cells to asphyxia may be of more interest for forensic purposes rather than migrating cells. In this context, interesting information about time of asphyxia could be given by investigating the rule of E-selectin and hypoxiainduced factor 1-α (HIF1-α) in lung tissue cells. E-selectin is an adhesion molecule that recognizes and binds to sialylated carbohydrates present on the surface proteins of leukocytes and mediates their adhesion to the endothelium. It is produced by the endothelium only after cytokine activation and needs a protein synthesis [22]. It is the first molecule activated by TNF-α. In the skin, its expression is detected after 1–2 h from insult up until 48 h. It is not constitutively expressed in tissues and is activated later with respect to P-selectin. For these reasons, its expression in lung tissue is expected to give information on more prolonged asphyxia. To our knowledge, no study with E-selectin was performed in asphyxia deaths. HIF-1a is a transcription factor expressed in response to hypoxia that activates expression of the genes involved in erythropoiesis, angiogenesis, glycolysis, and modulation of vascular tone. It is a potential mediator of pulmonary responses to hypoxia. HIF-1 is a heterodimer consisting of HIF1-α and HIF1-β subunits. HIF-1α accumulates in the nuclei of mammalian cells exposed to reduced O2 tension in a time and O2 concentration-dependent manner [23]. The HIF1-β subunit is constitutively expressed, whereas the expression and activity of the HIF-1α subunit is precisely regulated by the cellular O2. The HIF1-α subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tension. Few experimental studies suggest that levels of HIF-1 mRNA increase in rats subjected to hypoxia and that hypoxia induces HIF1-α but not HIF1-β mRNA expression in pulmonary artery endothelial cells of rats exposed to chronic hypoxia [24–26]. A study by Yu et al. [12] shows that HIF-1α protein expression is induced maximally when the lungs are ventilated with 0 or 1 % O2 for 4 h, and the highest expression occurs in the bronchial epithelium, bronchial smooth muscle, alveolar epithelium, and vascular endothelium. On reoxygenation, HIF1-α is rapidly degraded. These findings show that HIF1-α expression is tightly coupled to O2 concentration in vivo and are consistent with the involvement of HIF1-α in the physiological and pathophysiological responses

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to hypoxia in the lung. In contrast, HIF1-β levels do not change with an increase in time of hypoxia or a decrease in O2 concentration. Not only hypoxia, but also oxidative stress might play an important role in HIF1 activity [27, 28]. HIF-1α also has an important role in inflammatory response. To our knowledge, HIF1-α was studied in human lung tissue only for tumor or inflammation [29–32] and never in asphyxia death. The present work focuses on the reaction of lung tissue components to asphyxia, particularly the vessels and alveoli, through the study of P- and E-selectins, SP-A, and HIF1-α, in order to verify if some of them may be useful in the diagnosis of asphyxia death.

Materials and method Cases Sixty-two (62) medico-legal autopsy cases performed at the Department of Anatomical, Histological, Legal Medical and Orthopaedic Sciences of the Sapienza University of Rome, Italy, and at the Institute of Legal Medicine of the University of Padua (Italy) between 24 and 72 h post-mortem or, in cases of drowning, between 24 and 72 h after the discovery of the corpse were examined. Primary pulmonary diseases were excluded by histological investigations. Causes of death were based on routine macromorphological, micropathological, histological, and toxicological findings, and two groups were identified as: acute mechanical asphyxia (AMA) [total n=34: drowning (n=15), hanging (n=7), strangulation (n=3), smothering (n=3), and aspiration (n=6)]; control cases (CC) total n=28: carbon monoxide (CO) intoxication (n=6), heroin-related deaths (n=9), pneumonia (n=2), head trauma (n=5), natural death (n=6). Histology and immunohistochemical analysis Lung samples from each case were fixed in 4 % PBS– formaldehyde solution, dehydrated through alcohol and paraffin-embedded; 5-μm-thick paraffin-embedded sections were cut from each lung specimen and processed for morphological evaluation using hematoxylin–eosin (HE) staining and for immunohistochemistry (IHC). IHC analysis was performed using the following specific antibodies: – – –

Human anti-CD62P antigen (P-selectin) mouse monoclonal antibody (1:50 clone C34 by Novocastra); Human anti-CD62E antigen (E-selectin) mouse monoclonal antibody (1:100 clone 16G4 by Novocastra); Human anti-pulmonary surfactant protein A (SP-A) mouse monoclonal antibody (1:100 clone 32E12 by Novocastra);

–

Human anti-hypoxia-induced factor 1-α (HIF-α) mouse monoclonal antibody (1:1000 clone ESEE122 by Abnova).

For the IHC, samples were de-waxed, hydrated, and subjected to heat-induced epitope retrieval (HIER) using Target Retrieval Solution (Dako) (20′) in a thermostatic bath at 98 °C. The staining procedure was performed using a Dako Autostainer Instrument, as follows [10, 33]: incubation in peroxide solution (10′); incubation at room temperature (RT) with primary antibody, for P-selectin and Eselectin (30′) and for SP-A (60′), in Dako REAL™ EnVision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse; incubation with Dako DAB-AWAY detection system (5′); washed in distilled water; and nuclear staining with hemalaum. The primary antibody was replaced with Dako wash buffer 10× in the negative control test, while tonsil samples were used for the positive control experiment for selectins and SP-A-positive staining of intra-alveolar surface was used as internal control for SP-A. A manual immunohistochemical technique was used, as previously described [34], only for immunohistochemical detection of HIF1-α antibody. Briefly, paraffin sections were dewaxed, re-hydrated, subjected to the antigen retrieval procedure [citrate buffer pH 6.0 (15′) in microwave] and, after quenching endogenous peroxidase and blocking nonspecific binding sites, incubation was performed (60′) with the primary antibody at RT; (15′) with a biotinylated secondary antibody; (15′) with avidin-biotin-peroxidase-complex (ABC of UltreTek HRP Anti-Polyvalent kit, ScyTek Laboratories, USA); (1′) with DAB substrate (Roche, Italy); then slides were washed and nuclear counterstaining (30″) with hematoxylin was performed. Negative controls were carried out using affinity-purified mouse-IgG or by omitting the primary antibody. Optimal dilution of anti-HIF1-α antibody was set up and established using serial sections from human placenta, as positive control tissue. Since the state of morphological preservation of different samples varied according to the type of asphyxia death, our experimental protocol for HIF1-α immunodetection was designed to avoid antibody overnight incubations and the color reaction, with DAB substrate, was developed very quickly (max 1– 2 min). These precautions were able to greatly reduce background interference from all the lung samples. Analysis of immunostaining Five random microscopic fields of 1.76 mm2 for each immunostained tissue section were assessed by two independent observers, using ×10 and ×40 original magnification. The first observer using ×10 magnification randomly chose the fields and marked them with a felt-tip pen, while the second observer re-examined the marked areas in blind.

Int J Legal Med Fig. 1 Presence of P-Selectin (a) and E-selectin (b) immunostaining in pulmonary vessels (arrows) in a case of hanging. The intensity of the staining is scored +++. Nuclei were counterstained using hematoxylin solution (×200; bar=20 μm)

A

Grading of intensity and vessel counting was performed at high-power view (×40 original magnification). No interobserver variability was evidenced for grading of intensity, whereas only little inter-observer variability emerged in vessel counting. In these cases the medium value (between the first and the second observer) was reported and utilized for statistical analyses. Atelectatic areas were not considered in order to avoid interference with the results. Areas presenting an emphysema aquosum were considered not to interfere with the total amount of vessels/HPF and were therefore evaluated. Since Pselectin, E-selectin, and HIF1-α are absent in the capillaries, this vascular region was not considered in the assessment. Expression of the three markers was scored by a semi-quantitative method, evaluating the intensity and number of positively stained vessels [4]. The intensity of the marker expression was graded as absent (0), mild (+), moderate (++), and intense (+++). The number of reactive vessels was graded as absent (0), less than 10 (1), 10– 50 % (2), and more than 50 % (3). Because of possible false positives on the margins of lesions due to post-mortal artifacts, these were excluded from count. SP-A was scored according to Zhu et al. [16] evaluating density and distribution of SP-A in intra-alveolar spaces as follows: negative (−); grade I (−/+), weakly positive; grade II (+), positive with a few massive aggregates of stained granules; grade III (++), intensely and diffusely positive with many massive aggregates of stained granules. Statistical analysis A student t test with Welch correction was applied to evaluate the differences in grading and number of vessels stained with HIF1-α between CC versus AMA and CO intoxication versus AMA. Statistical significance was fixed at p<0.05.

Results Hematoxilin–eosin Local atelectasis and/or local emphysema and alveolar capillary congestion were found in the lung samples from controls and asphyxia deaths. In the asphyxia group, CO intoxication, heroin intoxication, and pneumonia cases

B

*

*

*

*

different degrees of intra-alveolar hemorrhages with intraalveolar edema intra- and interstitial alveolar edema were present (data not shown). In 13 cases (six drowning, one strangulation, one smothering, two aspirations, one CO intoxication, and two heroin intoxication cases), signs of putrefaction were visible. These cases were excluded from HIF1-α analysis. Immunohistochemistry P-selectin and E-selectin Intense overlapping and diffuse expression of P-selectin and E-selectin were found in all types of vessels, predominantly Table 1 Grading of P- and E-selectins activation in AMA and CC group. For the grading score see text. The number of vessels expressing P- and E-selectin and the intensity of the expression showed a similar trend and there was an overlap of results between grading score and cause of death Cause of death

Number of cases=62

Grading of intensity

Grading of the number of activated vessels

Hanging (AMA)

7

III

Strangulation (AMA) Smothering (AMA) Aspiration (AMA) Drowning (AMA)

3

III

3 (5 cases) 2 (2 cases) 3

3

III

3

6

III

3

15

3 (10 cases) 2 (5 cases)

6

III (4 cases) II (9 cases) I (2 cases) III

9

II

2

2 5

II II (4 cases) I (1 case) II (4 cases) I (2 cases)

2 3 (4 2 (1 3 (4 2 (2

CO intoxication (CC) Heroin intoxication (CC) Pneumonia (CC) Head trauma (CC) Natural death (CC)

6

3

cases) case) cases) cases)

Int J Legal Med Table 2 Immunohistochemical distribution of intra-alveolar SP-A. For grading score, see text

SP-A

Cause of death

Total

Grading

Hanging (AMA)

7

Strangulation (AMA)

3

2 3 2 3

(5 (2 (2 (1

cases) cases) cases) case)

Smothering (AMA)

3

Aspiration (AMA)

6

Drowning (AMA)

15

CO intoxication (CC)

6

Heroin intoxication (CC)

9

Pneumonia (CC)

2

(2 (1 (4 (2 (9 (6 (5 (1 (6 (3 (1 (1

cases) case) cases) cases) cases) cases) cases) case) cases) cases) case) case)

Head trauma (CC) Natural death (CC)

5 6

1 2 2 3 2 3 1 2 1 2 1 2 1 1

In all cases, linear staining spreading on the intra-alveolar inner surface and staining of the type II alveolar cells were found. Significant differences in relation to the cause of death were found in the distribution of SP-A-positive granular precipitates in the alveolar spaces, which were classified in three grades as previously explained (Table 2). In the AMA group, grade III (intense and diffuse positive staining with many massive aggregates of granular stains) was found in 11/34 cases (32.4 %), whereas grade II (few massive aggregates of stained granules) was found in 21/34 cases (61.8 %). In the CC group, grade II was found in 5/27 cases (18.5 %), while the weakly positive grade I in 23/27 (cases 85.2 %; Fig. 2a–b).

in veins, apart from the capillaries, which were always negative. Where present, platelets and megakaryocytes stained positive with P-selectin and showed no difference in staining intensity, acting as a further internal control. Endothelial cells of vessels in AMA group showed a highly diffuse (grade 3) in 23/34 (67.6 %), intense (+++) and homogeneous activation with P- and E-selectins (Fig. 1a–b). Only in drowning was the intensity of staining moderate (++; Table 1). With regard to the CC group, in heroin intoxication and in pneumonia the expression of P- and E-selectins was irregularly distributed in 100 % of cases, less diffused (10–50 %, grade 2) and mild/moderate (+/++), while CO intoxication showed a 100 % of diffuse (grade 3) and intense (III) reaction, and head trauma and natural death a grade 2/3 diffusion and a mild/moderate (I/II) intensity (Table 1). Fig. 2 A representative pattern of SP-A immunostaining distribution in lung tissue of a control case (a) with a grading score of I/II (scattered), and of a drowning case (b) with a grading score of II/III (dense but localized). Nuclei were counterstained using hematoxylin solution (×100; bar=50 μm)

A

HIF 1-α HIF1-α proved to be very sensitive to putrefaction, thus the 13 cases (six drowning, one strangulation, one smothering, two aspirations, one CO intoxication, and two heroin intoxications) with histological signs of putrefaction were not evaluated for HIF1-α. The analysis of HIF1-α refers, therefore, to a total of 49 cases in spite of 62: AMA group n=24 [drowning (n=9), hanging (n=7), strangulation (n=2), smothering (n=2), and aspiration (n=4)] and CC group n= 25 [CO intoxication (n=5), heroin intoxication (n=7), pneumonia (n=2), head trauma (n=5), and natural death (n=6)]. HIF1-α was found to be expressed on endothelial cells prevalently in activated arteries and in few activated veins, never in capillaries (Fig. 3). In some cases, arteries expressed HIF1-α also in smooth muscle cells (Fig. 3b). Pneumocytes and leukocytes proved to be able to express HIF1-α (data not shown). The number of vessels activated with HIF1-α never exceeded 30 % of all vessels and was strongly correlated with the cause of death. In CC group, 16/25 cases (64 %, corresponding to all natural deaths, head trauma, pneumonia, and 4/7 heroin-related deaths) were scored as grade 0 (absent), while the remaining 3/7 heroin-related deaths and all CO intoxication deaths were scored as grade 1 (<10 %). AMA group was scored mainly as grade 1 and 2 (<10–50 %; Fig. 4).

B

Int J Legal Med Fig. 3 HIF1-α was immunodetected in endothelial and smooth muscle cells in pulmonary arteries of different sizes (arrows) in a case of hanging (b) and drowning (c) while no staining was found in control cases (a). Sections were hematoxylincounterstained [×200 (a) and (b); ×100 (c); bar=50 μm (a), 100 μm (b), 20 μm (c)]

B

A

C

Intensity of staining was intense (+++) in 1/7 hanging; moderate (++) in 6/7 hanging, 4/4 aspiration, 1/2 smothering, 3/5 CO intoxication, and 1/7 heroin intoxication; mild (+) in 4/9 drowning, 2/5 CO intoxication, and 2/7 heroin intoxication. The mean values of HIF1-α positively stained vessels and their differences in number and intensity were evaluated using Student’s t test with Welch correction [35] to evaluate the difference between CC versus AMA and CO intoxication versus AMA (reported in Tables 3 and 4, respectively), and p values<0.05 were considered significant. A comparison in relation to the caliber of vessels activated shows in both groups a statistically significant higher number

Fig. 4 Vessel distribution: Number of vessels stained positive to HIF1α in relation to the causes of death (n=49). 1, hanging (n=7); 2, strangulation (n=2); 3, smothering (n=2); 4, aspiration (n=4); 5, drowning (n=9); 6, CO intoxication (n=5); 7, heroin intoxication (n=7); 8, pneumonia (n=2); 9, head trauma (n=5); 10, natural death (n=6). The 13 cases in which putrefaction was present are not included in the table

of activated pre-capillary arterioles/post-capillary venules (defined small-caliber vessels), as respect to arterioles/venules (defined medium-caliber vessels) and arteries/veins (defined large-caliber vessels; Table 3). A comparison of the results concerning CO intoxication and the AMA group shows the activation of HIF1-α also in CO intoxication; no statistical significant difference was found between these two groups (Table 4).

Discussion In the present study, IHC was used in order to investigate potential markers of asphyxia in a case history containing, in addition to cases of mechanical asphyxia, also CO intoxication (in which there is a gradual depletion of oxygen in the blood) and heroin intoxication (which are mostly deaths from respiratory depression due to inhibition of the central respiratory bulbar center). Natural deaths and head trauma cases were used as a point of reference for defining the basic levels of the markers. P-selectin was used in a previous study for the differentiation between inflammatory and non-inflammatory lung diseases [14] and proved to be intensely expressed in a higher percentage of vessels in non-inflamed tissue exposed to lack of oxygen, such as cases of hanging, drowning, and CO intoxication, which was confirmed by our results. Nevertheless, the above-mentioned study did not investigate healthy lungs and therefore it does not permit the verification of whether P-selectin has a basic expression in lung

Int J Legal Med Table 3 Intensity and number of vessels stained positive to HIF1-α: comparison between AMA and CC groups. The 13 cases in which putrefaction was present are not included in the table Vessel

p value

CC Mean

AMA SD

Mean

SD

Number

Total

0.00031

3.40

6.19

15.38

13.22

Intensity

Large caliber Medium caliber Small caliber Total Large caliber Medium caliber Small caliber

0.00228 0.00203 0.00066 0.00005 0.00027 0.00029 0.00052

0.44 0.64 2.32 0.64 0.24 0.48 0.60

1.04 1.29 4.88 0.76 0.60 0.77 0.76

1.75 3.13 10.58 1.54 1.21 1.38 1.54

1.67 3.37 9.68 0.66 1.02 0.82 0.98

vessels. Our casuistry contains 11 cases in which lungs were healthy (head trauma and natural deaths) and shows in these samples the diffused P-selectin immunodetection, with a mild and moderate intensity, which corresponds to a basal and constitutive expression of the marker in the lungs. This prevents P-selectin from being used in the diagnosis of mechanical asphyxia. In our study, E-selectin was investigated for the first time in healthy human lungs and in asphyxia cases, and showed a comparable reaction as with P-selectin. Unfortunately, also E-selectin cannot provide evidence of asphyxia deaths. The constant and overlapping positivity of both markers (P- and E-selectins) in lung vessels leads us to believe that, in the lungs, internal and external factors may act as a continuous trigger for the activation of selectins in the endothelium of the lung vessels, resulting in a similar expression. Nevertheless, this would result in a continuous recruitment of leukocytes that, however, does not occur. Therefore, it can be argued that selectins may have a different role in pulmonary endothelium, or that, at the end of life, whatever the cause of death, there is an activation of the pulmonary selectin system.

SP-A was confirmed to be a potential indicator of AMA because massive intra-alveolar aggregates of granular stains were found exclusively in the AMA group, while in the CC group few precipitates were found in one case of pneumonia, one of CO intoxication, and two cases of heroin intoxication. The absence of huge intra-alveolar aggregates in healthy lungs (head trauma and natural deaths), makes SPA a promising marker of hypoxic conditions. However, the most interesting results were provided by the presence of HIF1-α in lung vessels. It is interesting that all CC lung tissues (except CO intoxication cases and 3/7 heroin intoxications) did not show HIF1-α immunostaining in the endothelium of arterioles, arteries, and veins, but only in pre-capillary arterioles. On the contrary, different grades of HIF1-α expression were found in AMA cases in small-, medium-, and large-caliber vessels. This indicates that in normal conditions, lung vessels, constitutively, do not express a detectable level of HIF1-α antigen, while after acute lack of O2 they are activated and express, especially in endothelial cells, a detectable HIF1-α level very quickly. Activated vessels within AMA group cases expressed HIF1-α in all vessel types. Pre-capillary arterioles/post-capillary

Table 4 Intensity and number of vessels stained positive to HIF1-α: comparison between AMA and CO intoxication cases. The 13 cases in which putrefaction was present are not included in the table Vessel

p value

CO intoxication Mean

Number

Intensity

Total Large caliber Medium caliber Small caliber Total Large caliber Medium caliber Small caliber

0.31 0.52 0.11 0.46 0.84 0.22 0.47 0.45

10.2 1.2 1.6 7.4 1.6 0.6 1.6 1.2

AMA SD

Mean

SD

8.76 1.64 1.34 7.9 0.55 0.89 0.55 0.84

15.38 1.75 3.13 10.58 1.54 1.21 1.38 1.54

13.22 1.67 3.37 9.68 0.66 1.02 0.82 0.98

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venules showed the highest number of vessels expressing HIF1-α, followed by arterioles/venules and, finally, arteries/veins. This particular and specific distribution pattern of immunostaining could indicate a role of HIF1-α in the vasoconstriction of resistance-sized pulmonary arteries induced by hypoxia in the lungs [12, 13]. Arteries were activated particularly in hanging deaths, in which the number of vessels and the intensity of the staining was also higher in comparison with the other AMA deaths. A reason for this may be that in hanging hypoxia is often associated with hypoxemia, which emphasizes the reaction of the endothelial cells. Apart from hanging, medium- and small-sized vessels were activated particularly in CO intoxication, which may be dependent on the progressive hypoxemia determined by the increased accumulation of carboxyhemoglobin.

Conclusion To evaluate different types of morphological changes in combination is particularly useful when determining the mechanism of death in suspected asphyxiations, especially when external signs undergo atypical changes [36]. Our results indicate that P- and E-selectin expression in lung vessels, being activated by several types of trigger stimuli, cannot be used as indicator of asphyxia. Intra-alveolar granular deposits of SP-A seem to be related to an intense hypoxic stimulus, and when massively present, they can suggest, together with other elements, a severe hypoxia as the mechanism of death. The most important results of our study concern, however, the presence, localization, and distribution pattern of HIF1-α in lung vessels after hypoxic stimuli. HIF1-α was expressed in small-, medium-, and large-caliber lung vessels of the vast majority of mechanical asphyxia deaths and CO intoxications, with the number and intensity of positivestained vessels increasing with the duration of the hypoxia. Although further confirmation studies are required, these preliminary data indicate an interesting potential utility of HIF1-α as a screening test for asphyxia deaths.

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