Hydrocephalus is a brain disorder characterized by pathological accumulation of cerebrospinal fluid (CSF), leading to increased ventricular volume (VV) and potential damage to the corpus callosum (CC) and cingulate cortex (CgCt). Although surgical interventions, such as CSF shunting, are standard, their high complication rates underscore the urgent need to understand the underlying pathological process and develop pharmacological therapies. Previous studies using the Tmem67–/– genetic rat model of hydrocephalus have demonstrated pronounced VV expansion due to increased CSF production before postnatal day 10 (P10). Our laboratory previously found that treatment with the TRPV4 antagonist RN1734 ameliorates hydrocephalus in this model, which was hypothesized to work by slowing CSF production at the choroid plexus, thereby reducing VV. In this study, we investigated a novel neuroinflammatory mechanism and hypothesized that elevated VV enhances the expression of TLR4, a receptor for damage-associated molecular patterns (DAMPs) that triggers NLRP3 inflammasome activation within the CC and CgCt. We further propose that this inflammation contributes to lipid droplet (PLIN3) accumulation, 4-Hydroxynonenal (4-HNE) (oxidative stress), and an increase in mitochondrial reactive oxygen species (Mito-ROS), and that RN1734 treatment (P7-P15) would ameliorate these effects. Brain tissues from P15 Tmem67+/+ (control), Tmem67–/– (hydrocephalic), and RN1734-treated Tmem67–/– rats (n=3 litters) were analyzed by immunohistochemistry (IHC) and western blotting. Tmem67–/– rat brains showed robust IHC co-labelling of the TLR4–NLRP3 signaling pathways in CC and CgCt. Quantitative analysis confirmed a significant (p < 0.05) increase in fluorescence intensity for TLR4, NLRP3, PLIN3, 4-HNE, and Mito-He in the CgCt of Tmem67–/– rats compared to controls. Specifically, co-labelling of NLRP3 with the neuronal marker NeuN confirmed that inflammasome activation was significantly higher in CgCt neurones (p < 0.05). Alongside these findings, we observed significant neuronal swelling (via NeuN/Nissl) and cell death (via Cleaved Caspase-3) in the CgCt (p < 0.05). Interestingly, RN1734 treatment significantly ameliorated the activation of this inflammatory pathway, as well as the resultant neuronal swelling and cell death, although these levels did not return to the control baseline (Tmem67+/+). In addition, RN1734 treatment significantly reduced total NLRP3 protein levels compared to untreated Tmem67–/– rats. These findings demonstrate a critical link between innate immune activation (TLR4–NLRP3) and oxidative stress induced neuronal injury in hydrocephalus. This study reveals that while neuroinflammation may be a secondary consequence of the primary mechanical pathology (increased VV), the ability of TRPV4 antagonist to suppress this cascade in the parenchyma reveals a possibility of dual therapeutic mechanism, targeting both CSF production and cortical inflammation, and offers a powerful strategy to protect neuronal integrity. This research was conducted with the support of Congressional Directed Medical Research Program Grants #HT94252310296 and #HT94252310401 (BBY). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Audu et al. (Fri,) studied this question.