Jantzie, L. | bioRxiv Science

bioRxiv2026

Therapeutic Potential of Melatonin, Erythropoietin, and TRPV4 Antagonism in a Preclinical Model of Genetic Hydrocephalus

Symptomatic hydrocephalus is a neuropathological disorder of cerebrospinal fluid (CSF) homeostasis characterized by abnormal accumulation of CSF and progressive ventricular enlargement. Current treatment relies primarily on surgical CSF diversion, which is invasive and associated with substantial rates of treatment failure and surgical revisions. Congenital hydrocephalus can be caused by genetic pathologies; for example, a point mutation in the Meckel-Gruber type 3, Tmem67, gene produces a congenital hydrocephalus phenotype in rats, characterized by progressive ventriculomegaly. Here, we evaluated a combinatorial cocktail therapeutic strategy consisting of erythropoietin (EPO), melatonin (MLT), and the transient receptor potential vanilloid 4 (TRPV4) antagonist, RN1734 (RN), in the Tmem67-- rat model of congenital hydrocephalus. Magnetic Resonance Imaging (MRI) demonstrated marked progression of ventriculomegaly in untreated Tmem67-- rats between postnatal (P) 7 and P15. RN1734 and EPO+MLT+RN1734 (triple therapy) from P7-P14 prevented further ventricular expansion. Hydrocephalus is associated with structural disruption of the periventricular white matter (WM) and ependymal lining (EL), accompanied by regionally increased TLR4 and NLRP3-associated inflammatory signaling. EPO+MLT, RN1734, or triple therapy differentially attenuated these inflammatory responses. The triple combination reduced both TLR4 and NLRP3 protein abundance within the affected periventricular region and ameliorated the structural alterations. These findings support a combined therapeutic approach that addresses ventricular pathology, inflammation and periventricular structural consequences of congenital hydrocephalus.

neuroscience↗

bioRxiv2026

Proton FLASH preserves neurocognition across delivery techniques: implications for clinical translation in pediatric brain tumors

BackgroundRadiation therapy is integral to the curative treatment of childhood brain tumors but contributes to late neurocognitive impairment in survivors. FLASH (ultra-high dose rate, >40Gy/s) reduces normal-tissue toxicity in preclinical models, and proton-FLASH is currently the only modality capable of delivering ultra-high dose rates to the deep targets, such as pediatric brain tumors. However, two questions remain unresolved before clinical translation: (1) whether the FLASH effect can be achieved on synchrotron-based proton systems, which deliver protons in discrete spills that may be insufficient to cover a clinical target within a single delivery, and (2) which dose-rate metric, among the multiple definitions currently used in the field, best predicts the biological FLASH effect. MethodsC57BL/6 mice (7-8 weeks) received 10 Gy whole-brain RT via a clinical Hitachi ProBEAT synchrotron with CBCT-guided delivery, using three transmission-beam techniques: single-spill pencil beam scanning (SS PBS), multi-spill PBS with [~]2-second inter-spot delay (MS PBS), and passive scatter (PS), compared to conventional (CONV) delivery and unirradiated controls (n=24-28/group, equal sex distribution). Dose rate was quantified using three frameworks: field dose rate (FDR), PBS dose rate (PBSDR), and dose-averaged dose rate (DADR). Recognition memory was assessed by novel object recognition (NOR) at 6 weeks post-RT, and cognitive flexibility was assessed via touchscreen visual discrimination and reversal learning at 14 weeks. Hippocampal neuroinflammation was evaluated by immunofluorescence and immunohistochemistry for Iba1, NeuN, and GFAP. ResultsFLASH conditions were met by SS PBS and PS under all three dose-rate definitions, but MS PBS qualified as FLASH only by DADR. Despite this, neuroprotection was preserved across all three FLASH techniques: discrimination index was significantly higher for SS PBS (P=0.021), MS PBS (P=0.008), and PS (P<0.001) versus CONV, with no significant difference between FLASH techniques. On touchscreen testing, FLASH-treated females demonstrated preserved cognitive flexibility (P=0.047 vs. CONV on reversal learning correct trials). Iba1+ microglia were reduced in FLASH compared to CONV mice, with morphology suggestive of preserved homeostatic state. ConclusionsSynchrotron-based proton FLASH preserves neurocognitive function across all delivery techniques, including under multi-spill delivery essential for treating clinical-scale pediatric brain tumors. Critically, this neuroprotection was observed even for deliveries that qualified as FLASH only by DADR, identifying DADR as the dose-rate metric most relevant to the biological FLASH effect with direct implications for clinical trial design and dose-rate reporting standards.

cancer biology↗