Modulation of Cerebrospinal Fluid Dysregulation via a SPAK and OSR1 Targeted Framework Nucleic Acid in Hydrocephalus

Abstract Hydrocephalus is one of the most common brain disorders and a life‐long incurable condition. An empirical “one‐size‐fits‐all” approach of cerebrospinal fluid (CSF) shunting remains the mainstay of hydrocephalus treatment and effective pharmacotherapy options are currently lacking. Macrophage‐mediated ChP inflammation and CSF hypersecretion have recently been identified as a significant discovery in the pathogenesis of hydrocephalus. In this study, a pioneering DNA nano‐drug (TSOs) is developed by modifying S2 ssDNA and S4 ssDNA with SPAK ASO and OSR1 ASO in tetrahedral framework nucleic acids (tFNAs) and synthesis via a one‐pot annealing procedure. This construct can significantly knockdown the expression of SPAK and OSR1, along with their downstream ion channel proteins in ChP epithelial cells, thereby leading to a decrease in CSF secretion. Moreover, these findings indicate that TSOs effectively inhibit the M0 to M1 phenotypic switch of ChP macrophages via the MAPK pathways, thus mitigating the cytokine storm. In in vivo post‐hemorrhagic hydrocephalus (PHH) models, TSOs significantly reduce CSF secretion rates, alleviate ChP inflammation, and prevent the onset of hydrocephalus. These compelling results highlight the potential of TSOs as a promising therapeutic option for managing hydrocephalus, with significant applications in the future.

The samples were kept within a refrigerator at a temperature of 4°C, and on a daily basis over the course of a week, they were retrieved to undergo PAGE gel electrophoresis testing.

pH and salt solutions stability
To analyze the pH stability, TSOs were incubated in TM buffer with different pH values at 37 ℃ for 2h, and analyzed by PAGE.In order to analyze the salt solution stability, TSOs was incubated in 10% NaCl and MgCl2 solution at 37℃ for 2h, and then analyzed by PAGE.

Transwell assay:
The cell density was adjusted to an optimal level using a serum-free high-sugar DMEM medium, and subsequent inoculation took place within a Transwell chamber.Following cell adhesion to the chamber walls, distinct compounds were introduced based on the aforementioned groupings, and a 24-hour cultivation ensued.After sample collection, they were fixed with 4% paraformaldehyde fixative solution for 15 min.After PBS cleaning for twice, the transwell chambers were stained with crystal violet for at least Subsequently, they were immediately placed under a positive fluorescence microscope for observation and images acquisition.

Hematoxylin-Eosin staining:
The paraffin-embedded samples were prepared into 3 μm thick paraffin sections.Then the tissues are first deparaffinized and rehydrated, followed by immersion in hematoxylin dye for 10 min.Subsequently rinsed with water, and then introduced into a saturated lithium carbonate solution for 30 seconds.Then the slices were washed again and subjected to eosin staining for a duration of 2 minutes.A gradient alcohol immersion facilitated dehydration and transparency after cleansed with water.Finally, Permount TM Mounting Medium was added for storage.

Measurement of CSF production rates:
The method for measuring the rate of CSF secretion was described by Karimy et al 9 .
Briefly, the rats were anesthetized and positioned on a stereotactic apparatus, then a cranial burr hole(1mm) was drilled over the left ventricle (coordinates:0.6mm posterior and 1.6mm lateral to bregma).A ventricular infusion tube, as previously mentioned, was then inserted and fixed on the burr hole, with a depth of 4.5 mm.Next, the rat's head was gently rotated by 90° using the ear bars, positioning it with the nose pointing downward.Suboccipital muscles were dissected to expose the atlantooccipital ligament.
A 29-gauge needle was punctured through the ligament and advanced 5mm through the foramen of Magendie to the fourth ventricle.To create a closed CSF circulation in the lateral ventricles, we then used sterile molecular-grade mineral oil (100 μl; Sigma-Aldrich) to occlude the aqueduct Sylvius, thereby preventing CSF flow from the third ventricle to the fourth ventricles.Once the rat remained in the same position, a straight PE tube (OD:0.5mm,ID:1mm, length:20 cm) was connected to the fix tube.The PE tube should be flush with the fixed tube and should be perpendicular to the rat's head.
The volume of CSF collected over a given time (40 min) was calculated using the formula V (mm3) = π•r 2 •d, where "r" represents the radius of the PE tube and "d" indicates the distance that CSF traveled within the tube.Consequently, the rate of CSF formation(ul/min) was the calculated.

Transcriptome sequencing analysis
The NR8383 cells were cultured in 12-well plate and divided into two groups.The first group received stimulation with LPS(1ug/ml) (n=3), while the second group was exposed to both LPS (1 μg/ml) and TSOs (250 nM) (n=3).After 24 hours, the cells were harvested, and total RNA was isolated using the TRIzol reagent (Invitrogen, USA).
Subsequently, quantification and purity assessment of the RNA were performed using a NanoDrop ND-1000 instrument (NanoDrop, USA).After detecting the RNA integrity, the RNA samples underwent processing at Novogene Bioinformatics Technology Co., Ltd (Tianjin, China).RNA-Seq libraries were generated utilizing the Illumina standard mRNA-seq library preparation kit and sequenced by the Illumina NovaSeq 6000.The thresholds of significantly different expression were p < 0.05 and | log2foldchange | ＞0.6 .The GO and KEGG databases were used to explore the functions and biological pathways in which the differentially expressed genes were involved.

Table S2 .
Primer Sequence of Gene designed for Q-PCR.