Brain Water Channels May Finally Help Forensics Tell Freshwater From Saltwater Drowning

Drowning remains one of the most elusive diagnoses in forensic pathology, and a new study published in the International Journal of Legal Medicine suggests that the answer may lie in a family of microscopic water channels buried deep within the cerebral cortex. Researchers from the University of Genova in Italy and the University Center of Legal Medicine Lausanne-Geneva in Switzerland examined the immunohistochemical expression of five proteins—aquaporin 4, aquaporin 5, aquaporin 9, vasopressin, and glial fibrillary acidic protein—in brain tissue from people who died by drowning in fresh water and in salt water. Their findings point to a distinctive molecular fingerprint of freshwater drowning that could one day help pathologists distinguish between the two forms of death by submersion, a distinction that has practical consequences in real casework.

The difficulty of diagnosing drowning is well known in the forensic community. Unlike many causes of death, drowning leaves behind no pathognomonic autopsy feature, no single sign that definitively proves a person died from liquid entering the airways. Instead, forensic pathologists arrive at the diagnosis by exclusion, combining circumstantial evidence such as witnessed submersions or suicide notes with macroscopic findings like external foam, pleural effusion, acute pulmonary emphysema, and Paltauf’s spots, alongside histological and toxicological investigations. The World Health Organization estimates that approximately 372,000 drowning deaths occur worldwide each year, making it the third-leading cause of accidental injury death, so the stakes for improving diagnostic accuracy are considerable.

Pathophysiologically, drowning is divided into two principal forms depending on the medium. When a person drowns in fresh water, the hypotonic liquid passes rapidly into the pulmonary circulation, producing haemodilution, hyperkalaemia, and hypervolaemia, and death generally ensues faster than in salt water. In saltwater drowning, by contrast, the hypertonic sea water draws fluid from the bloodstream into the lungs, causing hypokalaemia, hypovolaemia, and haemoconcentration. These opposite haemodynamic trajectories were the central hypothesis of the new study: if freshwater and saltwater drowning drive the body’s water balance in opposite directions, the proteins that regulate water movement in brain cells might respond in measurably different ways.

The research team, led by Rosario Barranco and Francesco Ventura of the University of Genova together with Tony Fracasso of the Lausanne-Geneva center, conducted a retrospective analysis of ten freshwater drowning cases drawn from Lake Geneva and saltwater drowning cases from the Mediterranean Sea, using ten deaths from acute external bleeding as a control group. The exclusion criteria were deliberately strict: cases involving people over 65 years of age, neurological or psychiatric disease, cardiopulmonary resuscitation, putrefaction, a postmortem interval exceeding 72 hours, or ethanol and drug intoxication were all removed from consideration. Tissue samples were taken from the frontal cerebral cortex, fixed in formaldehyde, embedded in paraffin, and cut into five-micrometer sections for staining with antibodies against each of the five target proteins.

Slides were evaluated under blinded conditions, with seven randomly selected microscopic fields per slide scored on a semi-quantitative scale from absent to intense. Inter-observer agreement was complete for 85 percent of the slides and intra-observer agreement reached 95 percent, giving the investigators confidence in the reproducibility of their scoring. Statistical comparisons were performed with the Kruskal-Wallis and Mann-Whitney U tests for global comparisons between the three groups, supplemented by Student’s t-test, with significance set at p less than 0.05.

The headline result concerns aquaporin 4, the most abundant water channel protein in brain parenchyma, which is localized primarily in astrocytes and the basolateral membrane of the ependyma. Freshwater drowning cases showed a particularly intense AQP4 expression, with seven of the ten cases reaching the maximum grade of 3, and the difference between freshwater drowning and both the saltwater and control groups was statistically significant. By contrast, no significant difference emerged between saltwater drowning cases and controls. The authors interpret this as activation of AQP4 in response to the osmotic stimulus and haemodilution that occur when hypotonic fresh water floods the pulmonary circulation, a terminal vital reaction that helps the brain eliminate excess water through the subarachnoid spaces in cooperation with proteins such as connexin-43 and the potassium channel Kir4.1.

Aquaporin 9 delivered a second encouraging result. This channel, expressed in astrocytes, glial cells of the pineal gland, subpial vessels, and neurons, plays a role in regulating water movement, cerebrospinal fluid production, astrocyte migration, and neuronal energy balance. Intense AQP9 expression was found in four of the freshwater cases, and the difference between freshwater drowning and the other two groups was statistically significant with the t-test, while the global tests showed a trend in the same direction. The researchers propose that the hypotonic stimulus of freshwater immersion activates both AQP4 and AQP9 to counteract haemodilution, facilitating the transport of water and small solutes and supporting neuronal metabolic adaptations during the final minutes of life.

The remaining markers proved harder to interpret. Aquaporin 5, found mainly in neurons and astrocytes of the grey matter, and vasopressin, the hormone that stimulates aquaporin activity largely through the V1a receptor, both showed significantly higher expression in freshwater drowning cases than in controls, but no significant differences separated freshwater from saltwater drowning or saltwater from controls. The authors suggest that saltwater drowning may represent an intermediate situation between freshwater drowning and the control group, with osmotic alterations activating these proteins to a lesser extent, and they caution that vasopressin is subject to endocrine and temporal fluctuations that are not consistently reflected in postmortem immunohistochemical signals. Glial fibrillary acidic protein, a marker of glial reaction associated with traumatic or chronic lesions, showed no significant differences between groups, a result the team regarded as expected given that GFAP is not specific to acute osmotic change.

The study only partially confirms the influential earlier work of An and colleagues, who in 2011 demonstrated increased intracerebral aquaporin 4 expression in freshwater drowning but also reported reduced expression in saltwater drowning. The Genoa and Geneva team did not reproduce that decrease, finding no statistically significant difference between their saltwater group and controls, and they hypothesize that the discrepancy may relate to the different chemical and salinity characteristics of the Mediterranean Sea compared with the ocean. To their knowledge, the new study is the first to analyze the immunohistochemical expression of the major brain aquaporins alongside vasopressin in drowning cases, extending a line of research that the same group has previously pursued in lung and kidney tissue with markers including aquaporin 2, aquaporin 5, vasopressin receptor 2, and renin.

The practical implications reach beyond the laboratory. In maritime cities crossed by rivers, a drowning may occur in fresh water before currents drag the body out to sea, and establishing where the fatal immersion actually took place can matter for reconstructing the circumstances of death. The authors are careful to frame AQP4 and AQP9 as promising complementary markers for the differentiation of freshwater and saltwater drowning rather than as independent diagnostic markers, and they acknowledge that their strict exclusion criteria, while minimizing confounding variables, restricted the number of cases in each group. Validation on larger samples, they conclude, will be essential before these molecular signatures can enter routine forensic practice, but the study marks a significant step toward an objective, protein-level diagnosis of one of forensic medicine’s most stubborn diagnostic challenges.

Subject of Research: Immunohistochemical cerebral cortex expression of aquaporins, vasopressin, and GFAP in freshwater and saltwater drowning for forensic diagnosis.

Article Title: Immunohistochemical cerebral cortex expression of aquaporin 4, aquaporin 5, aquaporin 9 Vasopressin and GFAP in saltwater drowning and freshwater drowning

Article References: Barranco, R., Ventura, F., & Fracasso, T. (2026). Immunohistochemical cerebral cortex expression of aquaporin 4, aquaporin 5, aquaporin 9 Vasopressin and GFAP in saltwater drowning and freshwater drowning. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04021-7

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04021-7

Keywords: drowning, freshwater drowning, saltwater drowning, aquaporin 4, aquaporin 9, vasopressin, GFAP, forensic pathology, immunohistochemistry, cerebral cortex, osmotic stimulus, forensic histopathology