The abundance and localization of claudin-1 and -5 in the adult tomcats (Felis catus) testis, tubules rectus, rete testis, efferent ductules, and epididymis.

Tight junctions (TJ) are the anatomical component of blood-testis (BTB) and blood-epididymis (BEB) barriers and contain many proteins, including claudins. The presence of claudins in domestic cat testis and epididymis has not been previously described. This study aimed to determine whether claudin-1 and claudin-5 participate in the structure of BTB and BEB and whether their amounts differ between the testis and epididymal segments of adult cats, using Western blotting (WB) and immunohistochemistry. WB results demonstrated that claudin-1 was significantly lower in the testis than in all epididymal segments and higher in the corpus epididymis than in the cauda, while claudin-5 in the testis was significantly lower than in the caput and corpus. Claudin-1 was absent at the Sertoli-Sertoli junctions, while claudin-5 was detected at the level of the BTB during stages I and VIII. Both claudins were observed in the pachytene spermatocytes and the developing acrosome of the round and elongating spermatids. Claudin-5 was also detected in the cytoplasm of some spermatogonia, Sertoli cells, and late spermatid acrosome. In the epididymal segments, both claudins were localized to the area of the tight junctions and along the entire length of the lateral plasma membranes of adjacent principal cells and between principal and basal cells. These results may indicate that in the domestic cat, claudin-1 and -5 participate as both tight junction proteins and adhesion molecules in the BEB's structure, claudin 5 is a component of the BTB, and both proteins may be involved in postmeiotic germ cell development, especially acrosome development.

Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain Barrier Tight Junctions.

The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between the adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for TJ formation and assemble into multiprotein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB.

Molecular Insights into the Thrombotic and Microvascular Injury in Placental Endothelium of Women with Mild or Severe COVID-19.

Clinical manifestations of coronavirus disease 2019 (COVID-19) in pregnant women are diverse, and little is known of the impact of the disease on placental physiology. Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has been detected in the human placenta, and its binding receptor ACE2 is present in a variety of placental cells, including endothelium. Here, we analyze the impact of COVID-19 in placental endothelium, studying by immunofluorescence the expression of von Willebrand factor (vWf), claudin-5, and vascular endothelial (VE) cadherin in the decidua and chorionic villi of placentas from women with mild and severe COVID-19 in comparison to healthy controls. Our results indicate that: (1) vWf expression increases in the endothelium of decidua and chorionic villi of placentas derived from women with COVID-19, being higher in severe cases; (2) Claudin-5 and VE-cadherin expression decrease in the decidua and chorionic villus of placentas from women with severe COVID-19 but not in those with mild disease. Placental histological analysis reveals thrombosis, infarcts, and vascular wall remodeling, confirming the deleterious effect of COVID-19 on placental vessels. Together, these results suggest that placentas from women with COVID-19 have a condition of leaky endothelium and thrombosis, which is sensitive to disease severity.

Brain endothelial cell junctions after cerebral hemorrhage: Changes, mechanisms and therapeutic targets.

Vascular disruption is the underlying cause of cerebral hemorrhage, including intracerebral, subarachnoid and intraventricular hemorrhage. The disease etiology also involves cerebral hemorrhage-induced blood-brain barrier (BBB) disruption, which contributes an important component to brain injury after the initial cerebral hemorrhage. BBB loss drives vasogenic edema, allows leukocyte extravasation and may lead to the entry of potentially neurotoxic and vasoactive compounds into brain. This review summarizes current information on changes in brain endothelial junction proteins in response to cerebral hemorrhage (and clot-related factors), the mechanisms underlying junction modification and potential therapeutic targets to limit BBB disruption and, potentially, hemorrhage occurrence. It also addresses advances in the tools that are now available for assessing changes in junctions after cerebral hemorrhage and the potential importance of such junction changes. Recent studies suggest post-translational modification, conformational change and intracellular trafficking of junctional proteins may alter barrier properties. Understanding how cerebral hemorrhage alters BBB properties beyond changes in tight junction protein loss may provide important therapeutic insights to prevent BBB dysfunction and restore normal function.

Chronic Exposure to the Fusarium Mycotoxin Deoxynivalenol: Impact on Performance, Immune Organ, and Intestinal Integrity of Slow-Growing Chickens.

This study investigates the long-term effects of deoxynivalenol (DON) consumption on avian growth performance, on the proliferation, apoptosis, and DNA damage of spleen cells, and on intestinal integrity. Two hundred and eight 5-day-old black-feathered Taiwan country chickens were fed diets containing 0, 2, 5, and 10 mg/kg of DON for 16 weeks. Body weight gain of male birds in the 2 mg/kg group was significantly lower than that in the 5 mg/kg group. At the end of trial, feeding DON-contaminated diets of 5 mg/kg resulted in heavier spleens. Moreover, the increase in DON induced cellular proliferation, apoptosis, and DNA damage signals in the spleen, the exception being female birds fed 10 mg/kg of DON showing reduced proliferation. Expression of claudin-5 was increased in jejunum of female birds fed 2 and 5 mg/kg of DON, whereas decreased expression levels were found in male birds. In conclusion, our results verified that DON may cause a disturbance to the immune system and alter the intestinal barrier in Taiwan country chickens, and may also lead to discrepancies in growth performances in a dose- and sex-dependent manner.

Chronic type 2 diabetes reduces the integrity of the blood-brain barrier by reducing tight junction proteins in the hippocampus.

In the present study, we investigated the effects of type 2 diabetes-induced hyperglycemia on the integrity of the blood-brain barrier and tight junction markers in the rat hippocampus. Forty-week-old diabetic (Zucker diabetic fatty, ZDF) rats and littermate control (Zucker lean control, ZLC) rats were used in this study. We evaluated the integrity of the blood-brain barrier by measuring sodium fluorescein extravasation and blood vessel ultrastructure. In addition, tight junction markers, such as zona occludens-1, occludin and claudin-5, were quantified by western blot analysis. ZDF rats showed significantly increased sodium fluorescein leakage in the hippocampus. Tight junction markers, such as occludin and claudin-5, were significantly decreased in the hippocampi of ZDF rats compared to those of ZLC rats. In addition, ZDF rats showed ultrastructural changes with phagocytic findings in the blood vessels. These results suggest that chronic untreated diabetes impairs the permeability of the hippocampal blood-brain barrier by down-regulating occludin and claudin-5, indicating that chronic untreated diabetes may cause hippocampus-dependent dysfunction.

TGFß1 exacerbates blood-brain barrier permeability in a mouse model of hepatic encephalopathy via upregulation of MMP9 and downregulation of claudin-5.

Recent studies have found that vasogenic brain edema is present during hepatic encephalopathy following acute liver failure and is dependent on increased matrix metalloproteinase 9 (MMP9) activity and downregulation of tight junction proteins. Furthermore, circulating transforming growth factor ß1 (TGFß1) is increased following liver damage and may promote endothelial cell permeability. This study aimed to assess whether increased circulating TGFß1 drives changes in tight junction protein expression and MMP9 activity following acute liver failure. Blood-brain barrier permeability was assessed in azoxymethane (AOM)-treated mice at 6, 12, and 18 h post-injection via Evan's blue extravasation. Monolayers of immortalized mouse brain endothelial cells (bEnd.3) were treated with recombinant TGFß1 (rTGFß1) and permeability to fluorescein isothiocyanate-dextran (FITC-dextran), MMP9 and claudin-5 expression was assessed. Antagonism of TGFß1 signaling was performed in vivo to determine its role in blood-brain barrier permeability. Blood-brain barrier permeability was increased in mice at 18 h following AOM injection. Treatment of bEnd.3 cells with rTGFß1 led to a dose-dependent increase of MMP9 expression as well as a suppression of claudin-5 expression. These effects of rTGFß1 on MMP9 and claudin-5 expression could be reversed following treatment with a SMAD3 inhibitor. AOM-treated mice injected with neutralizing antibodies against TGFß demonstrated significantly reduced blood-brain barrier permeability. Blood-brain barrier permeability is induced in AOM mice via a mechanism involving the TGFß1-driven SMAD3-dependent upregulation of MMP9 expression and decrease of claudin-5 expression. Therefore, treatment modalities aimed at reducing TGFß1 levels or SMAD3 activity may be beneficial in promoting blood-brain barrier integrity following liver failure.

Expression of the ALS-causing variant hSOD1(G93A) leads to an impaired integrity and altered regulation of claudin-5 expression in an in vitro blood-spinal cord barrier model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood-spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood-spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood-spinal cord barrier endothelial cells of presymptomatic hSOD1(G93A) mice and bEnd.3 cells stably expressing hSOD1(G93A). In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the ß-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1(G93A) cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood-spinal cord barrier.

Platonin preserves blood-brain barrier integrity in septic rats.

OBJECTIVES: Platonin possesses potent anti-inflammatory and antioxidative capacities. Because systemic inflammation and oxidative stress are crucial in mediating sepsis-induced blood-brain barrier (BBB) integrity loss, this study elucidated the effects of platonin on preserving BBB integrity in septic rats. METHODS: A total of 72 adult male rats (200-250 g) were randomized to receive cecal ligation and puncture (CLP), CLP plus platonin, sham operation, or sham operation plus platonin (n = 18 in each group). Systemic inflammation and oxidation levels and BBB integrity in the surviving rats were determined after 24-hour monitoring. RESULTS: Plasma levels of interleukin-6 (IL-6) and malondialdehyde (MDA)-markers of systemic inflammation and oxidation-and the grading of Evans blue staining of the brains, BBB permeability to Evans blue dye, and brain edema levels-markers of BBB integrity-in rats that received CLP were significantly higher than rats that received sham operation (all p < 0.001). By contrast, the plasma levels of IL-6 (p < 0.001) and MDA (p < 0.001), and the grading of Evans blue staining (p = 0.015), BBB permeability to Evans blue dye (p = 0.043), and brain edema levels (p = 0.034) in rats that received CLP plus platonin were significantly lower than rats that received CLP. Experimental data further revealed that the concentration of tight junction protein claudin-5, a major structural component of BBB, in rats that received CLP was significantly lower than rats that received CLP plus platonin (p = 0.023). CONCLUSION: Platonin could attenuate sepsis-induced BBB integrity loss in rats.

Dabigatran abrogates brain endothelial cell permeability in response to thrombin.

Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell ß1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.

Modulation of VEGF-induced retinal vascular permeability by peroxisome proliferator-activated receptor-ß/δ.

PURPOSE: Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARß/δ in VEGF-induced retinal hyperpermeability. METHODS: Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation. RESULTS: Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 µM) and PPARß/δ-directed siRNA (20 µM). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor ß/δ siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice. CONCLUSIONS: These data suggest a protective effect for PPARß/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARß/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation.

Strong claudin 5 expression is a poor prognostic sign in pancreatic adenocarcinoma.

We investigated the expression of claudin 5 in 88 ductal adenocarcinomas of the pancreas. The results were correlated with patient prognosis, with claudin 5 expression in blood vessels, with the expression level of bcl2 and bax and with apoptosis. Claudin 5 expression was detected in 24 (38%) cases. It was not associated with tumour size or spread, but strong claudin 5 expression correlated with a worse survival (p = 0.005). Claudin 5 also associated with a higher extent of apoptosis and greater expression of bax protein. In the tumour vasculature, some vessels displayed a loss of claudin 5 expression. The presence of this loss was associated with tumour grade and the presence of nodal metastases (p = 0.02, p = 0.022, respectively). These results indicate that claudin 5 is upregulated in a proportion of pancreatic ductal adenocarcinomas. The association of strong claudin 5 expression with a worse survival is in line with some earlier reports indicating that this protein is involved with increased locomotion and more aggressive spread of carcinomas. The association of claudin 5 with apoptosis and bax might be due to stronger cellular kinetics found in such tumours. The loss of claudin 5 expression in the tumour vasculature points to a leaky vessel type; this might also ease the access of tumours to vessels and be reflected in its association with the presence of nodal metastases.

Phospholipid transfer protein (PLTP) deficiency impaired blood-brain barrier integrity by increasing cerebrovascular oxidative stress.

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood-brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.

Claudins 1, 2, 3, 4, 5 and 7 in solar keratosis and squamocellular carcinoma of the skin.

Claudins are tight junction proteins regulating the paracellular permeability of cell layers. We investigated the expression of claudins 1, 2, 3, 4, 5 and 7 in a sample set consisting of a total of 93 cases representing normal skin, actinic keratoses and squamous cell carcinomas of the skin. There were several changes found in claudin expression. Claudin 1 appeared to be progressively decreased in solar keratosis and skin squamous cell carcinomas compared to normal skin while expression of claudin 2 was increased. With claudins 3 and 5 occasional immunoreactivity was found in squamous cell carcinomas. Claudins 4 and 7 were variably expressed in skin neoplasia compared to normal skin. According to the results expression of claudins 1 and 2 change in parallel with the severity of the epidermal preneoplastic and neoplastic lesions thus probably influencing the disturbed epithelial polarity characteristic of these lesions. Claudin 1 under- and claudin 2 overexpression also lead to a leakier epithelial barrier function of the skin with a resulting damage to skin epithelial resistance. Other claudins investigated in this study did not show progressive changes even though occasional overexpression of them was found in skin squamous cell carcinoma.

Claudin expression and tight junction protein localization in the lining epithelium of the keratocystic odontogenic tumors, dentigerous cysts, and radicular cysts.

OBJECTIVE: The aim of this study was to evaluate the expression and localization of tight junction proteins (TJPs) or claudins in the keratocystic odontogenic tumor (KCOT) and to correlate with its biological behavior. STUDY DESIGN: Five claudins (-1, -3, -4, -5, and 7) were examined immunohistochemically in 25 KCOTs and compared with 10 dentigerous cysts (DCs) and 10 radicular cysts (RCs). RESULTS: Marked claudin-3 loss of expression in KCOT basal layer (n=24/25; 96%) compared with DCs (n=1/10; 10%) and RCs (n=5/10; 50%) (P<.05) suggests that claudin-3 downregulation may indicate altered or loss of basal cell polarity and impaired barrier function of KCOT lining epithelium and this might contribute indirectly to its biological behavior. In contrast, claudins-1, -4, -5, and -7 distribution patterns were less distinctive in all three entities, suggesting that these TJP molecules probably play limited roles in influencing their different growth potentials. CONCLUSION: Present findings suggest that differential claudin expressions in the lining epithelium of KCOTs, DCs, and RCs probably reflect their neoplastic or nonneoplastic nature.

Paracellular barrier and tight junction protein expression in the immortalized brain endothelial cell lines bEND.3, bEND.5 and mouse brain endothelial cell 4.

The blood-brain barrier (BBB) is formed by brain endothelial cells. Many immortalized brain endothelial cell lines have been established; these have been used as in vitro BBB models. The aim of the present study was to assess the paracellular barrier properties of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 cells, and mouse brain endothelial cell 4 (MBEC4), and those of the primary mouse brain endothelial cells pMBECs. bEND.3 cells showed low permeability to sodium fluorescein and obvious staining of tight junction proteins (claudin-5, occludin and ZO-1) similar to pMBECs; these barrier properties of MBEC4 and bEND.5 cells were low. In addition, bEND.3 cells expressed the highest level of claudin-5 among all cells. These results suggest that bEND.3 cells are a convenient and useful model for evaluating BBB function, especially the paracellular barrier.

Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels.

Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoraco-lumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.

Molecular pathology of pulmonary edema after injury in forensic autopsy cases.

The lung is vulnerable to trauma; pulmonary edema starts quickly as part of the systemic responses involved in shock. The present study investigated the molecular pathology of posttraumatic alveolar damage and responses involving pulmonary edema in forensic autopsy cases of injury (n = 66) compared with acute cardiac death cases (n = 13). Intrapulmonary mRNA and immunohistochemical expressions of matrix metalloproteinases (MMPs; MMP-2 and MMP-9), intercellular adhesion molecule-1, claudin-5, and aquaporins (AQPs, AQP-1 and AQP-5) were examined. Subacute injury deaths showed an increase in lung weight similar to that in acute cardiac death, but relative mRNA quantification using the Taqman real-time PCR assay demonstrated different findings among the causes of death; higher expressions were detected for all markers, except for AQP-5 in sharp instrument injury, for MMP-2 in blunt brain injury, and for MMP-9 in non-brain blunt injury, but these expression levels were lower in acute cardiac death. In immunostaining, only MMPs showed differences among the causes of death: MMP-2 expression was evident in most subacute deaths due to blunt brain injury and sharp instrument injury, whereas MMP-9 was intensely positive in those of non-brain blunt injury and sharp instrument injury. These findings suggest significant differences in the mechanism of pulmonary edema among fatal injuries and acute cardiac death, especially between blunt and sharp instrument injury. Systematic analysis of gene expressions using real-time PCR in combination with immunohistochemistry may be useful in evaluating pulmonary damage and responses after injury in death investigations, especially in connection with posttraumatic shock.