CSF Aquaporin-4 in Idiopathic Normal Pressure Hydrocephalus: Potential as a Biomarker for Shunt Response and Glymphatic Function

CSF Aquaporin-4 in Idiopathic Normal Pressure Hydrocephalus: Potential as a Biomarker for Shunt Response and Glymphatic Function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CSF Aquaporin-4 in Idiopathic Normal Pressure Hydrocephalus: Potential as a Biomarker for Shunt Response and Glymphatic Function Johan Eriksson De Ryst, Sofia Bergström, Sara Mravinacova, David Bäckström, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7785379/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2026 Read the published version in Fluids and Barriers of the CNS → Version 1 posted 10 You are reading this latest preprint version Abstract Background Aquaporin-4 (AQP4) is crucial for brain fluid regulation and glymphatic system function. Idiopathic normal pressure hydrocephalus (INPH), characterized by impaired CSF flow, is often treated with shunt surgery. This study investigated AQP4 levels in INPH patients to explore its role in pathophysiology and potential as a biomarker for shunt response. Methods CSF samples from 233 INPH patients and 29 controls were analysed. AQP4 levels were compared between preoperative patients and controls, before and after shunt surgery (110 patients), and between shunt responders and non-responders (204 patients). A bead-based assay was used to measure AQP4, and outcomes were assessed by postoperative changes in maximum gait velocity. Results Preoperative AQP4 levels were lower in INPH patients (1177 ± 259 AU) than in controls (1351 ± 279 AU, p = 0.003), but the difference did not persist after adjustment for confounders (p = 0.87). Postoperatively, AQP4 levels increased (1714 ± 473 vs 1186 ± 269 AU, p < 0.001), with a modest correlation between AQP4 increase and gait improvement (rₛ = 0.22, p = 0.022). Compared with non-responders, shunt responders (130 patients) had lower preoperative AQP4 levels (1144 ± 250 vs. 1238 ± 273 AU, p = 0.016) Conclusions INPH patients presented AQP4 levels comparable to those of controls. The postoperative increase in AQP4 may result from altered CSF dynamics induced by the shunt; however, the underlying mechanism remains unclear, underscoring the need for further studies to clarify this relationship. Notably, lower preoperative AQP4 levels in shunt responders suggest a potential predictive role. Aquaporin 4 Cerebrospinal fluid Normal pressure hydrocephalus idiopathic Glymphatic system Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aquaporins (AQPs) are membrane proteins that form channels enabling the free passage of water along osmotic or hydrostatic gradients(1). They play a crucial role in maintaining fluid homeostasis in the brain(1), with AQP4 being particularly implicated in facilitating CSF flow through the glymphatic system(2, 3, 4). According to this hypothesis, water from the CSF in the subarachnoid space (SAS) enters the arterial perivascular spaces and subsequently flows into the brain's interstitial fluid (ISF) through the glia limitans, a barrier formed by astrocytic endfeet. This specific transport of water is facilitated by AQP4 channels on astrocytic endfeet. After water has traversed the parenchyma, AQP4 facilitates its efflux from the interstitial fluid (ISF) into CSF within the venous perivascular space, thereby creating a bulk flow that enables the removal of soluble waste products from the interstitial space, completing the glymphatic flow pathway INPH is a neurodegenerative disorder with a reported prevalence of up to 3.7% in individuals over the age of 65(5). It is characterized by ventricular enlargement and distinctive symmetrical gait disturbance(6, 7). A key feature of INPH is impaired CSF flow(8), and the condition is treatable by CSF shunting(9). Reduced glymphatic flow has recently been proposed as a contributing factor in the pathophysiology of INPH(10). This hypothesis is supported by studies in which intrathecal administration of gadolinium-based contrast agent (GBCA), followed by serial magnetic resonance imaging (MRI), demonstrated delayed uptake in the brain parenchyma and impaired clearance from the CSF in INPH patients(11, 12). There are indications that the loss of AQP4 localization in the perivascular wall in both Alzheimer’s disease (AD)(13) and INPH(14, 15, 16, 17) contributes to disrupted glymphatic flow. Several studies analysing cortical biopsies from INPH patients have shown a reduction in the normally polarized perivascular expression of AQP4 on astrocytic endfeet(14, 15, 16, 17). Consequently, AQP4 has been hypothesized to be a potential target for future medical treatment of INPH(18). As AQP4 is in direct contact with CSF, disturbances in its expression should be reflected in its CSF levels. Therefore, CSF AQP4 could serve as a biomarker, for example, for distinguishing INPH from its mimics and predicting the effectiveness of shunt surgery(14, 17, 18, 19, 20, 21). Two studies have investigated AQP4 levels in CSF in the context of INPH. The first included 10 subjects with INPH and found no difference compared with 9 controls(21). The second study, involving 81 subjects with INPH, failed to detect any AQP4(22). To date, no studies have compared AQP4 levels before and after surgery. In this study, we measured CSF AQP4 levels in 233 patients with INPH and compared these levels to those of age-matched controls (n = 29). Additionally, we evaluated how shunt treatment influences CSF AQP4 levels. Methods In summary, this observational study included CSF samples from 233 shunted INPH patients and 29 control subjects. Preoperative CSF was collected from all INPH patients, and 110 of these also underwent a follow-up lumbar puncture after shunt surgery. All samples were promptly stored in a biobank and analysed simultaneously. Preoperative AQP4 levels were compared with those of age-matched controls. In patients with longitudinal samples, preoperative levels were also compared with postoperative levels. Furthermore, we compared AQP4 levels between shunt responders and non-responders and assessed correlations between changes in AQP4 levels and changes in maximal gait velocity (V max ) after surgery. Study population and controls The University Hospital of Umeå has a tertiary hydrocephalus unit responsible for investigation, selection for surgery, and CSF shunt surgery. The referral area is northern Sweden, with approximately one million inhabitants. In the present study we included patients with a confirmed diagnosis of INPH whose clinical investigation started in 2007--2019 and whose CSF sample was stored in our biobank. Patient flow is shown in Figure 1. During the study period, patients referred for ventriculomegaly and associated symptoms underwent a standardized diagnostic work-up, including brain MRI, large-volume lumbar puncture (the “tap test”), CSF infusion testing, blood and CSF screening, and physiotherapist-led assessments of gait and balance. In diagnostically inconclusive cases, a 3-day external lumbar drainage was performed. Eligibility for shunt surgery required fulfilment of the international criteria for INPH(7) and either clinical improvement following CSF drainage and/or pathological CSF dynamic profiles. The final study cohort comprised 233 patients with INPH (mean age 74 ± 6 years; 37% women), all of whom underwent preoperative evaluation and were deemed suitable for shunt surgery. All patients received a ventriculoperitoneal shunt with an adjustable valve, using a standardized opening pressure setting in all but four cases. Postoperative CSF samples were obtained by lumbar puncture in 110 patients, at a mean of 5.5 ± 2.8 months following surgery. Handling of the samples Directly after lumbar puncture, 10 ml of CSF was collected in polypropylene tubes. The samples collected before 2012 were stored at -80 °C immediately after collection. The samples collected thereafter were first centrifuged at 400 × g for 10 minutes before being stored at -80 °C. For controls, 10 ml of CSF was immediately stored at -80 °C after sampling. All CSF samples were stored in the same biobank. CSF total tau (t-tau), phosphorylated tau (p-tau), amyloid-β42 (Aβ42) and glial fibrillary acidic protein (GFAP) was analyzed at Sahlgrenska University Hospital, Clinical Neurochemistry, Mölndal during the preoperative clinical work up. AQP4 level determination AQP4 CSF measurements were performed at SciLifeLab in Stockholm via an antibody- and bead-based technique(23) with a polyclonal rabbit IgG antibody (HPA014784) produced within the Human Protein Atlas project (HPA, www.proteinatlas.org). In brief, the antibody was immobilized onto the surface of magnetic carboxylated beads. Fifteen microliters of each sample were diluted 1/2 into 96-well plates before labelling with a tenfold molar excess of biotin (21329, Thermo Scientific) as described previously(23). Detection of captured AQP4 was enabled by the addition of a streptavidin-coupled fluorophore (SA1004-4; Invitrogen). Read-out was performed in a FlexMap3D instrument (Luminex Corporation) and the results reported as median fluorescence intensities per bead identity and sample, calculated from at least 30 measured beads. To evaluate the technical variability of the assay, coefficients of variation were calculated across all replicate samples and determined to be 4%. More detailed explanations of the experimental procedures can be found elsewhere(23). Antibody specificity was evaluated in previous studies(24, 25). Clinical improvement after shunting The postoperative outcome measure was the change in V max . Gait velocity was assessed as the mean speed of up to six 10-meter walks observed by a physiotherapist. Patients were classified as shunt responders if V max improved by ≥0.16 m/s. This cut-off has been shown to increase the probability of at least one step improvement in the modified Rankin scale(26), i.e., a meaningful improvement for the patient. Statistical analysis Statistical analyses were conducted using IBM SPSS version 28.01.1 (SPSS, IBM Software Group, Chicago, IL, USA). Continuous variables are presented as mean ± SD or median (interquartile range (IQR)), as appropriate. Group comparisons were performed using parametric or non-parametric tests depending on data distribution. Categorical variables were analysed using the chi-square test or Fisher’s exact test when expected cell counts were low. Group differences between INPH patients and controls were examined via a generalized linear model, adjusting for age, sex, sample centrifugation prior to storage, and storage duration. Correlations were assessed with Pearson’s (r) or Spearman’s (r s ) coefficients, depending on variable type and distribution. A p-value < 0.05 was considered statistically significant. Results Sample and patient characteristics Table 1 summarizes the sample characteristics of patients with INPH and controls. The groups were matched for age, although a male predominance was observed in the INPH group. Notable differences were identified in terms of sample processing and storage conditions. Specifically, 75% of the INPH samples underwent centrifugation prior to storage, whereas none of the control samples were centrifuged. Furthermore, there was a discrepancy in the storage duration, with INPH samples collected between 2007 and 2022, compared with the control samples, which were collected between 2003 and 2004. Table 1 Characteristics of samples from INPH patients and controls INPH Control P value Participants (n) 233 29 N/A Age (years) median (IQR) 74 (70–78) 72 (69–78) 0.36 † Female (%) 37 62 0.0078 ‡ Centrifuged prior to storage (%) 75 0 N/A Storage time (days) median (IQR) 2689 (1667–4016) 6702 (6647–6828) < 0.001 † † = Independent samples Mann-Whitney U test. ‡ = Pearson chi-square (two-sided). Abbreviations: Idiopathic normal pressure hydrocephalus (INPH), interquartile range (IQR). CSF AQP4 levels in INPH patients versus controls Without adjustment for sex or pre-analytical sample handling, preoperative INPH patients showed lower AQP4 levels (median 1142 AU, IQR 1000–1321) compared with controls (median 1366 AU, IQR 1160–1516; p < 0.001) (Fig. 2 A). However, 75% of the preoperative samples had both a markedly shorter freezer storage time and had been centrifuged prior to storage, whereas none of the control samples were centrifuged. To account for this, we repeated the analysis including only the non-centrifuged preoperative samples with storage times more comparable to controls (n = 59). In this restricted comparison, no significant difference was observed between INPH patients (mean 1261 ± 303 AU) and controls (mean 1351 ± 279 AU; p = 0.17) (Fig. 2 B). To further examine group differences while accounting for covariates, we conducted a generalized linear model with AQP4 levels as the dependent variable and diagnostic group (INPH vs. control) as the predictor. The model adjusted for sex (p = 0.13), centrifugation prior to storage (p = 0.87), storage duration (p = 0.004), and age (p < 0.001). After adjustment, no difference in AQP4 levels was found between INPH patients and controls (p = 0.87). Instead, significant positive correlations were observed between AQP4 levels and storage duration (r = 0.30, p < 0.001) (Fig. 2 C), as well as between AQP4 levels and age across INPH patients and controls (r = 0.28, p < 0.001) (Fig. 2 D). Postoperative CSF AQP4 levels In the longitudinal follow-up of INPH patients (n = 110), we observed an increase in AQP4 levels six months after surgery (median 1646 AU IQR 1347–1976) compared with the preoperative levels (median 1166 AU IQR: 976–1345; p < 0.001) (Fig. 3 ). Only nine out of the 110 patients presented reduced AQP4 levels at follow-up. CSF AQP4 levels in shunt responders Among the 233 INPH patients with preoperative samples, postoperative V max data were available in 204. Of these, 130 patients (64%) were classified as shunt responders. Preoperative AQP4 levels were lower in responders (median 1089 AU, IQR 971–1277) than in non-responders (median 1213 AU, IQR 1074–1361; p = 0.008) (Fig. 4 A). Responders and non-responders did not differ in age, sex, sample centrifugation prior to storage, storage duration, Mini-Mental State Examination score, implanted shunt type, or shunt opening pressure. In the subgroup with available CSF biomarkers (t-tau, p-tau. Aβ42 and GFAP), non-responders had significantly higher t-tau and p-tau concentrations, although the proportion of patients above the predefined clinical cut-offs did not differ between groups (Table 2 ). In the 107 patients with both pre- and postoperative samples and postoperative V max data, the change in AQP4 levels from pre- to postoperative measurements was weakly positively correlated with the change in postoperative V max (r s = 0.22, p = 0.022) (Fig. 4 B). Table 2 Patient and sample characteristics of shunt responders and non-responders Shunt responder P-value Yes No Number † 130 74 N/A Age (years) median (IQR) 74 (70–78) 76 (70–78) 0.63 ‡ Female (%) 37 32 0.55 § Centrifuged before storage (%) 72 76 0.62 § Storage duration (days) median (IQR) 2539 (1477–4115) 2741 (2072–3919) 0.38 ‡ MMSE score median (IQR) (n = 197) 26 ( 21 – 28 ) 27 ( 24 – 28 ) 0.15 ‡ Shunt type (%) : Medtronic Strata™ 91.5 94.6 0,86 ¶ Medtronic Strata MR™ 7.7 5.4 Codman Certas® Plus 0.8 0.0 Strata shunt opening pressure (%) ¤ : 1.5 98.4 98.6 1.0 ¶ > 1.5 1.6 1.4 CSF concentration median (ng/L) (IQR) : t-tau (n = 170) 200 (159–266) 228 (192–310) 0.023 ‡ p-tau (n = 163) 30 ( 24 – 38 ) 36 (29–48) 0.006 ‡ Aβ42 (n = 171) 419 (331–523) 460 (337–588) 0.31 ‡ GFAP (n = 157) 490 (350–700) 550 (390–970) 0.15 ‡ t-tau > cut-off (409 ng/L) (%) 6.5 6.5 N/A p-tau > cut-off (50 ng/L) (%) 9.6 16.9 0.17 ‡ Shunt responder = ≥ 0,16 m/s improvement in maximal gait velocity after shunt surgery. † = Data for improvement in maximal gait velocity was missing from 29 out of 233 patients with preoperative samples. ‡ = Independent-Samples Mann-Whitney U test. § = Pearson chi-square test, (two-sided). ¶ = Fisher's exact test, (two-sided). ¤ = Data for shunt opening pressure was missing from one out of 130 shunt responders. Abbreviations: Interquartile range (IQR), Mini-Mental State Examination (MMSE), total-tau (t-tau), phosphorylated tau (p-tau), amyloid-β42 (Aβ42), glial fibrillary acidic protein (GFAP). Discussion Our findings demonstrate that CSF AQP4 levels can be reliably measured via a suspension bead array technique in a substantial cohort of INPH patients. After adjusting for known group differences, such as sex and preanalytical sample handling, the AQP4 levels in INPH patients did not differ from those in age-matched, neurologically healthy controls. However, preoperative AQP4 levels were lower in shunt responders than in non-responders, suggesting a potential role for AQP4 in identifying candidates for shunt surgery. Additionally, AQP4 levels increased postoperatively, with a weak positive correlation with clinical improvement. AQP4 – from the plasma membrane to CSF The cellular mechanisms regulating AQP4 expression and its turnover into CSF remain poorly understood( 1 ). One recent study revealed that membrane-bound AQP4 is continuously recycled or internalized through vesicular trafficking( 27 ). Membrane-bound proteins are typically internalized and degraded via the ubiquitin‒lysosome pathway( 28 ), suggesting that this mechanism likely governs AQP4 degradation in astrocytes. Consequently, AQP4 is not expected to leave astrocytes under normal circumstances. However, the detection of AQP4 in CSF raises the possibility that its turnover between astrocytes and CSF is mediated by additional processes, potentially involving the release of microvesicles or exosomes( 29 ). An alternative explanation is that AQP4 enters the ISF or CSF because of astrocytic cell injury. However, histopathological studies in INPH have not demonstrated overt astrocytic injury or necrosis. Instead, reactive astrogliosis has consistently been reported as a prominent finding( 14 , 15 , 16 ), suggesting that the presence of AQP4 in CSF is less likely to result from passive leakage due to cellular damage and may instead reflect regulated or secondary processes. In summary, the exact form in which AQP4 is measured in CSF remains unclear and warrants further investigation. Previous studies of CSF AQP4 levels in INPH, AD and FTD As previously mentioned, two studies have investigated AQP4 in CSF from INPH patients. The first found no difference in AQP4 levels between INPH and controls; however, this study included only 10 subjects with INPH and 9 controls( 21 ). This sample size provides sufficient power only to detect very large group differences. A second, larger study failed to detect any AQP4 in CSF using a commercial ELISA kit for detection, raising concerns about potential methodological issues ( 22 ). Taken together, the available evidence does not provide conclusive results regarding AQP4 concentration in the CSF of INPH patients. AQP4 has however been more extensively investigated in AD, which has been proposed to share several features with INPH in terms of glymphatic dysfunction( 10 , 16 ). Arighi et al. initially reported lower AQP4 levels in the CSF in a small study including eleven subjects with AD and nine controls( 21 ); however, in a subsequent larger study, they reported contradictory results, with increased levels( 30 ). Bergström et al.( 24 ) conducted a multicentre study comparing AQP4 levels in AD patients and controls and reported elevated AQP4 levels in CSF of AD patients across all four studied cohorts. These findings were recently corroborated by another study in which CSF AQP4 levels were quantified via ELISA( 25 ). Notably, increased AQP4 levels in CSF have also been reported in frontotemporal dementia (FTD) patients( 31 ). If AQP4 levels in the CSF are indeed elevated in AD and FTD patients but remain within the normal range, as our findings suggest, or are low in INPH patients, this could indicate distinct mechanisms of fluid flow dysfunction in INPH patients. This finding suggests that AQP4 is a promising biomarker with the potential to identify AQP4 dysregulation in AD and FTD, which is not present in INPH. AQP4 expression in astrocytes in AD and INPH Zeppenfeld et al.( 13 ) investigated the relationship between perivascular AQP4 expression and disease stage in AD and cognitively intact controls of different ages using postmortem frontal cortex tissue. They found that loss of AQP4 polarization to the perivascular space was associated with greater amyloid-β burden and more advanced Braak stage. In addition, global AQP4 expression in astrocytes increased with age in cognitively intact elderly individuals and with advancing Braak stage in AD. Taken together with previous reports of elevated CSF AQP4 in AD, these results suggest that altered perivascular polarization, combined with enhanced global astrocytic AQP4 expression, may be reflected in increased CSF AQP4 levels in AD patients. In line with these findings, our study also demonstrated an age-related increase in CSF AQP4 levels. A correlation between reduced perivascular AQP4 expression and disease stage, or an increase in global AQP4 expression, has not yet been demonstrated in INPH. However, several studies have analysed cortical biopsies from INPH patients( 14 , 15 , 16 , 17 ). These studies reported reduced expression of AQP4 in astrocytic endfeet facing the perivascular space, accompanied by astrogliosis and blood‒brain barrier dysfunction. Notably, owing to the invasive nature of brain biopsy sampling, the reference patients in these studies were not age-matched and had other neurological conditions, such as epilepsy, tumours, and aneurysms. Previous studies have shown altered AQP4 expression in astrocytes in relation to tumours( 32 ), hippocampal sclerosis in medial temporal lobe epilepsy( 33 , 34 ) and aging( 13 , 35 ). CSF AQP4 levels in INPH and glymphatic function We did not observe a significant difference in CSF AQP4 levels between INPH patients and controls. This finding suggests that CSF AQP4 levels may remain within the normal range in INPH. On the basis of the findings discussed above, CSF AQP4 levels may reflect astrocytic expression. If this is the case, our findings suggest that global AQP4 expression remains normal in INPH. However, even if overall AQP4 expression remains intact, it is still possible that the polarization of AQP4 to the perivascular space is disrupted. Nonetheless, AQP4 levels within the normal range in CSF do not support a reduced capacity for AQP4-mediated glymphatic flow. This stands in partial contrast to the conclusions of previous MRI studies using intrathecal GBCA, which revealed a delayed GBCA peak in the brain tissue of INPH patients, interpreted as reduced glymphatic clearance( 11 , 12 ). However, this delay might also be attributed to a primary delay in GBCA distribution within the SAS, reflecting altered CSF circulation patterns. Such changes could be a natural consequence of known CSF dynamics disturbances in INPH, including elevated outflow resistance( 8 ) and disproportionate enlargement of the ventricles and SAS( 36 ). CSF AQP4 levels and shunt surgery We observed a general increase in CSF AQP4 levels following shunt surgery, with responders displaying lower preoperative levels than non-responders. Moreover, postoperative increases in AQP4 levels correlated weakly with clinical improvement, as measured by changes in V max . One possible explanation for the postoperative rise in CSF AQP4 is a concentration effect due to reduced CSF volume. However, earlier studies of other potential CSF biomarkers in INPH, including amyloid-β42, t-tau, and p-tau( 37 ) and ten different proteins( 38 ) did not identify significant concentration effects from reduced CSF ventricular volume after shunt surgery. Two Japanese groups have investigated changes in intracranial brain and CSF volumes after shunt surgery, observing primarily a redistribution of CSF between different compartments of the SAS, with only minor reductions in total intracranial CSF volume at 1–12 months postoperatively (11% and 5%, respectively)( 39 , 40 ). These modest changes are unlikely to explain the increase in AQP4 levels observed here. Another potential mechanism is a sedimentation effect along the cranial–lumbar axis due to altered CSF circulation caused by shunting from the ventricles near the choroid plexus. Yet shunt surgery has been shown to produce variable effects across CSF proteins, with both increases and decreases in lumbar CSF concentrations( 38 ) making predictions for AQP4 behaviour uncertain, particularly in the absence of ventricular CSF samples. A further hypothesis is that elevated postoperative AQP4 levels reflect altered glymphatic flow due to decreased intracranial pressure and increased compliance after shunt surgery. Such changes might stimulate AQP4 expression in astrocytes and/or enhance recirculation of ISF, promoting transport of proteins, including non–membrane-bound AQP4, into CSF. The finding that responders had lower preoperative CSF AQP4 levels than non-responders, together with the weak correlation between improvement in V max and postoperative AQP4 changes, is intriguing. The present study found that non-responders had higher tau and phosphorylated tau concentrations. This may indicate a more severe comorbidity of neurodegeneration in this subgroup, consistent with previous reports of higher levels of CSF AQP4 in AD and FTD( 24 , 25 , 30 , 31 ). INPH is a heterogeneous condition, often complicated by comorbid neurodegenerative disorders such as subcortical vascular dementia, parkinsonism, and AD. Thus, the responders in our study may represent a more “pure INPH” phenotype with less neurodegenerative burden, with normal or slightly lower preoperative AQP4 levels. In these patients, the disturbed CSF dynamics typical of INPH were improved by shunting, leading to the clinical improvement. However, as a sign of functioning shunt, this change in CSF circulation also resulted in a postoperative rise in AQP4, likely due to mechanisms previously discussed. In summary, this suggests that INPH entails a CSF disturbance that may not be a AQP4-related glymphatic dysfunction. At the same time, it supports the notion that elevated AQP4 levels may indicate comorbid neurodegenerative disease, which is known to be associated with higher levels of AQP4 in CSF( 24 , 25 , 30 , 31 ) and reduced clinical effect from shunting( 41 ). Strengths and weaknesses This study offers valuable insights into the relationship between AQP4 levels in CSF and INPH. Notably, its strengths include the large sample size of INPH patients, thorough postoperative follow-up, and the significant number of patients who underwent postoperative lumbar punctures. However, several limitations must be acknowledged. Preanalytical handling of samples varied, as the control group samples were collected several years earlier than those from INPH patients were, and some samples underwent centrifugation before storage. Additionally, the control group was relatively small and had a greater proportion of females, whereas the INPH group included more males, potentially influencing the results. Conclusion This study analysed CSF AQP4 levels in a large cohort of INPH patients via a bead-based assay. After adjusting for confounders, no difference was found between INPH patients and controls. Postoperative increases in AQP4 may suggest enhanced fluid recirculation from the ISF to the CSF due to altered CSF dynamics after shunt placement. Lower preoperative AQP4 levels were linked to better shunt outcomes, suggesting its potential as a biomarker for predicting shunt response. This motivates further studies to clarify the relationship, such as examining correlations between AQP4 levels and CSF outflow resistance, comparing lumbar and ventricular CSF AQP4 levels, and exploring associated MRI characteristics. Abbreviations AQP4 – Aquaporin-4 INPH – Idiopathic normal pressure hydrocephalus SAS – Subarachnoid space ISF – Interstitial fluid GBCA – Gadolinium-based contrast agent MRI – Magnetic resonance imaging AD – Alzheimer’s disease V max – Maximal gait velocity t-tau – Total tau p-tau – Phosphorylated tau Aβ42 – Amyloid-β42 GFAP – Glial fibrillary acidic protein IQR – Interquartile range HPA – Human Protein Atlas AU – Arbitrary Units MMSE – Mini-Mental State Examination FTD – Frontotemporal dementia Declarations Ethics approval and consent to participate All patients provided written consent to save CSF for future research purposes at the time of lumbar puncture. Participants in this study were informed about the study via a letter, which included an option to opt out if they did not wish to participate. Individuals deceased at the time inclusion began (2020) were automatically incorporated in the cohort. The study was approved by the Swedish ethical review board and the ethical review board at Umeå University (reference number 2020-04469). This study was performed in accordance with the guidelines of the Declaration of Helsinki Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no competing interests to declare Funding This work was supported by the Swedish Foundation for Strategic Research (grant numberRMX18-0152) and a regional agreement between Umeå University and Västerbotten County Council (ALF). Author’s contributions J.E.D.R., S.Q., A.E., and J.M. conceived the study.J.E.D.R., S.B., S.M., D.B., A.E., and J.M. designed the work.Data were acquired by J.E.D.R., S.B., S.M., S.Q., A.E., and J.M.Data analysis and interpretation were performed by J.E.D.R., S.B., S.M., A.E., and J.M.J.E.D.R., A.E., and J.M. drafted the manuscript.All authors critically revised the manuscript and approved the final version . Acknowledgents Not applicable References MacAulay N. Molecular mechanisms of brain water transport. Nat Rev Neurosci. 2021;22(6):326–44. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med. 2012;4(147):147ra11. Mestre H, Hablitz LM, Xavier AL, Feng W, Zou W, Pu T et al. Aquaporin-4-dependent glymphatic solute transport in the rodent brain. Elife. 2018;7. 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Disproportionately Enlarged Subarachnoid Space Hydrocephalus in Idiopathic Normal-Pressure Hydrocephalus and Its Implication in Pathogenesis. Acta Neurochir Suppl. 2016;122:287–90. Liden S, Farahmand D, Laurell K. Ventricular volume in relation to lumbar CSF levels of amyloid-beta 1–42, tau and phosphorylated tau in iNPH, is there a dilution effect? Fluids Barriers CNS. 2022;19(1):59. Tullberg M, Blennow K, Mansson JE, Fredman P, Tisell M, Wikkelso C. Ventricular cerebrospinal fluid neurofilament protein levels decrease in parallel with white matter pathology after shunt surgery in normal pressure hydrocephalus. Eur J Neurol. 2007;14(3):248–54. Hiraoka K, Yamasaki H, Takagi M, Saito M, Nishio Y, Iizuka O, et al. Changes in the volumes of the brain and cerebrospinal fluid spaces after shunt surgery in idiopathic normal-pressure hydrocephalus. J Neurol Sci. 2010;296(1–2):7–12. Yamada S, Ishikawa M, Yamaguchi M, Yamamoto K. Longitudinal morphological changes during recovery from brain deformation due to idiopathic normal pressure hydrocephalus after ventriculoperitoneal shunt surgery. Sci Rep. 2019;9(1):17318. Thavarajasingam SG, El-Khatib M, Vemulapalli KV, Iradukunda HAS, Laleye J, Russo S, et al. Cerebrospinal fluid and venous biomarkers of shunt-responsive idiopathic normal pressure hydrocephalus: a systematic review and meta-analysis. Acta Neurochir (Wien). 2022;164(7):1719–46. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2026 Read the published version in Fluids and Barriers of the CNS → Version 1 posted Editorial decision: Revision requested 10 Nov, 2025 Reviews received at journal 10 Nov, 2025 Reviews received at journal 26 Oct, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviewers invited by journal 21 Oct, 2025 Editor assigned by journal 21 Oct, 2025 Submission checks completed at journal 21 Oct, 2025 First submitted to journal 05 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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01:17:58","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118751,"visible":true,"origin":"","legend":"","description":"","filename":"82e317f0a752440e9f27b97ec6b2ef451structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/ad051aae1a21f0faaf85894b.xml"},{"id":95222932,"identity":"03f8d704-a3ca-4767-a094-7255f4790be8","added_by":"auto","created_at":"2025-11-05 16:21:23","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128465,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/1dd071219f0a2c5171d385e0.html"},{"id":95063512,"identity":"67e64e37-ba72-4779-931e-968145f8e9f2","added_by":"auto","created_at":"2025-11-04 01:17:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94531,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of patient inclusion: Patients evaluated for suspected hydrocephalus at our center between 2007 and 2019 were selected for analysis. Abbreviations: Idiopathic normal pressure hydrocephalus (INPH), cerebrospinal fluid (CSF).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/526976edde7e8d4d45720bdf.jpg"},{"id":95063514,"identity":"c41baa80-c465-4cd7-a84a-baccf9a0305e","added_by":"auto","created_at":"2025-11-04 01:17:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81998,"visible":true,"origin":"","legend":"\u003cp\u003eA. CSF AQP4 levels among all preoperative INPH samples (n = 233) compared with non-centrifuged controls (n = 29). B. CSF AQP4 levels among non-centrifuged preoperative INPH samples (n = 59) compared with non-centrifuged controls (n = 29). C. Correlation between CSF AQP4 levels and freezer storage duration across all preoperative INPH samples and controls (n = 262). A positive correlation was observed (r = 0.30, p \u0026lt; 0.001). D. Correlation between CSF AQP4 levels and age across all preoperative INPH samples and controls (n = 262). A positive correlation was observed (r = 0.28, p \u0026lt; 0.001). The values are in arbitrary units (AUs). Abbreviations: Aquaporin-4 (AQP4); idiopathic normal pressure hydrocephalus (INPH); not significant (NS).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/65455a26a7884e4ae8fcd445.jpg"},{"id":95223312,"identity":"008953a8-492e-4191-81bf-5b4ee778bc14","added_by":"auto","created_at":"2025-11-05 16:22:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17666,"visible":true,"origin":"","legend":"\u003cp\u003ePreoperative and postoperative CSF AQP4 levels in INPH patients (n = 110). The values are presented in arbitrary units (AUs). Abbreviations: Aquaporin-4 (AQP4); idiopathic normal pressure hydrocephalus (INPH).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/2165966c6ff4ab27a8f636ce.jpg"},{"id":95063515,"identity":"74eb03f3-6adb-4ae7-b333-3338844bd7f5","added_by":"auto","created_at":"2025-11-04 01:17:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38135,"visible":true,"origin":"","legend":"\u003cp\u003eA. Preoperative CSF AQP4 levels in shunt responders (n = 130) compared with non-responders (n = 74).\u003c/p\u003e\n\u003cp\u003eB. Correlation between the preoperative-to-postoperative change in CSF AQP4 levels and the preoperative-to-postoperative change in Vmax in INPH patients (n = 107). A weak positive correlation was observed (rₛ = 0.22, p = 0.022). The values are presented in arbitrary units (AUs). Abbreviations: Idiopathic normal pressure hydrocephalus (INPH); maximal gait velocity (Vmax).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/31c3b382d66cf2b367ca73cb.jpg"},{"id":107928034,"identity":"4621f2d3-0aa7-4086-85bb-51def5b88abb","added_by":"auto","created_at":"2026-04-27 16:06:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":540857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7785379/v1/948485d4-ae0c-4d7e-bd7b-faa7d7f2d593.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CSF Aquaporin-4 in Idiopathic Normal Pressure Hydrocephalus: Potential as a Biomarker for Shunt Response and Glymphatic Function","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAquaporins (AQPs) are membrane proteins that form channels enabling the free passage of water along osmotic or hydrostatic gradients(1). They play a crucial role in maintaining fluid homeostasis in the brain(1), with AQP4 being particularly implicated in facilitating CSF flow through the glymphatic system(2, 3, 4). According to this hypothesis, water from the CSF in the subarachnoid space (SAS) enters the arterial perivascular spaces and subsequently flows into the brain's interstitial fluid (ISF) through the glia limitans, a barrier formed by astrocytic endfeet. This specific transport of water is facilitated by AQP4 channels on astrocytic endfeet. After water has traversed the parenchyma, AQP4 facilitates its efflux from the interstitial fluid (ISF) into CSF within the venous perivascular space, thereby creating a bulk flow that enables the removal of soluble waste products from the interstitial space, completing the glymphatic flow pathway\u003c/p\u003e\n\u003cp\u003eINPH is a neurodegenerative disorder with a reported prevalence of up to 3.7% in individuals over the age of 65(5). It is characterized by ventricular enlargement and distinctive symmetrical gait disturbance(6, 7). A key feature of INPH is impaired CSF flow(8), and the condition is treatable by CSF shunting(9). Reduced glymphatic flow has recently been proposed as a contributing factor in the pathophysiology of INPH(10). This hypothesis is supported by studies in which intrathecal administration of gadolinium-based contrast agent (GBCA), followed by serial magnetic resonance imaging (MRI), demonstrated delayed uptake in the brain parenchyma and impaired clearance from the CSF in INPH patients(11, 12).\u003c/p\u003e\n\u003cp\u003eThere are indications that the loss of AQP4 localization in the perivascular wall in both Alzheimer’s disease (AD)(13) and INPH(14, 15, 16, 17) contributes to disrupted glymphatic flow. Several studies analysing cortical biopsies from INPH patients have shown a reduction in the normally polarized perivascular expression of AQP4 on astrocytic endfeet(14, 15, 16, 17). Consequently, AQP4 has been hypothesized to be a potential target for future medical treatment of INPH(18). As AQP4 is in direct contact with CSF, disturbances in its expression should be reflected in its CSF levels. Therefore, CSF AQP4 could serve as a biomarker, for example, for distinguishing INPH from its mimics and predicting the effectiveness of shunt surgery(14, 17, 18, 19, 20, 21). Two studies have investigated AQP4 levels in CSF in the context of INPH. The first included 10 subjects with INPH and found no difference compared with 9 controls(21). The second study, involving 81 subjects with INPH, failed to detect any AQP4(22). To date, no studies have compared AQP4 levels before and after surgery.\u003c/p\u003e\n\u003cp\u003eIn this study, we measured CSF AQP4 levels in 233 patients with INPH and compared these levels to those of age-matched controls (n = 29). Additionally, we evaluated how shunt treatment influences CSF AQP4 levels.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eIn summary, this observational study included CSF samples from 233 shunted INPH patients and 29 control subjects. Preoperative CSF was collected from all INPH patients, and 110 of these also underwent a follow-up lumbar puncture after shunt surgery. All samples were promptly stored in a biobank and analysed simultaneously. Preoperative AQP4 levels were compared with those of age-matched controls. In patients with longitudinal samples, preoperative levels were also compared with postoperative levels. Furthermore, we compared AQP4 levels between shunt responders and non-responders and assessed correlations between changes in AQP4 levels and changes in maximal gait velocity (V\u003csub\u003emax\u003c/sub\u003e) after surgery.\u003c/p\u003e\n\u003ch2\u003eStudy population and controls\u003c/h2\u003e\n\u003cp\u003eThe University Hospital of Umeå has a tertiary hydrocephalus unit responsible for investigation, selection for surgery, and CSF shunt surgery. The referral area is northern Sweden, with approximately one million inhabitants. In the present study we included patients with a confirmed diagnosis of INPH whose clinical investigation started in 2007--2019 and whose CSF sample was stored in our biobank. Patient flow is shown in Figure 1.\u003c/p\u003e\n\u003cp\u003eDuring the study period, patients referred for ventriculomegaly and associated symptoms underwent a standardized diagnostic work-up, including brain MRI, large-volume lumbar puncture (the “tap test”), CSF infusion testing, blood and CSF screening, and physiotherapist-led assessments of gait and balance. In diagnostically inconclusive cases, a 3-day external lumbar drainage was performed. Eligibility for shunt surgery required fulfilment of the international criteria for INPH(7) and either clinical improvement following CSF drainage and/or pathological CSF dynamic profiles.\u003c/p\u003e\n\u003cp\u003eThe final study cohort comprised 233 patients with INPH (mean age 74 ± 6 years; 37% women), all of whom underwent preoperative evaluation and were deemed suitable for shunt surgery. All patients received a ventriculoperitoneal shunt with an adjustable valve, using a standardized opening pressure setting in all but four cases. Postoperative CSF samples were obtained by lumbar puncture in 110 patients, at a mean of 5.5 ± 2.8 months following surgery.\u003c/p\u003e\n\u003ch2\u003eHandling of the samples\u003c/h2\u003e\n\u003cp\u003eDirectly after lumbar puncture, 10 ml of CSF was collected in polypropylene tubes. The samples collected before 2012 were stored at -80 °C immediately after collection. The samples collected thereafter were first centrifuged at 400 × g for 10 minutes before being stored at -80 °C. For controls, 10 ml of CSF was immediately stored at -80 °C after sampling. All CSF samples were stored in the same biobank. CSF total tau (t-tau), phosphorylated tau (p-tau),\u0026nbsp;amyloid-β42 (Aβ42) and glial fibrillary acidic protein (GFAP) was analyzed at Sahlgrenska University Hospital, Clinical Neurochemistry, Mölndal during the preoperative clinical work up.\u003c/p\u003e\n\u003ch2\u003eAQP4 level determination\u003c/h2\u003e\n\u003cp\u003eAQP4 CSF measurements were performed at SciLifeLab in Stockholm via an antibody- and bead-based technique(23) with a polyclonal rabbit IgG antibody (HPA014784) produced within the Human Protein Atlas project (HPA, www.proteinatlas.org). In brief, the antibody was immobilized onto the surface of magnetic carboxylated beads. Fifteen microliters of each sample were diluted 1/2 into 96-well plates before labelling with a tenfold molar excess of biotin (21329, Thermo Scientific) as described previously(23). Detection of captured AQP4 was enabled by the addition of a streptavidin-coupled fluorophore (SA1004-4; Invitrogen). Read-out was performed in a FlexMap3D instrument (Luminex Corporation) and the results reported as median fluorescence intensities per bead identity and sample, calculated from at least 30 measured beads. To evaluate the technical variability of the assay, coefficients of variation were calculated across all replicate samples and determined to be 4%. More detailed explanations of the experimental procedures can be found elsewhere(23). Antibody specificity was evaluated in previous studies(24, 25).\u003c/p\u003e\n\u003ch2\u003eClinical improvement after shunting\u003c/h2\u003e\n\u003cp\u003eThe postoperative\u0026nbsp;outcome measure was\u0026nbsp;the change in V\u003csub\u003emax\u003c/sub\u003e.\u0026nbsp;Gait velocity was assessed as the mean speed of up to six 10-meter walks observed by a physiotherapist. Patients were classified as shunt responders if V\u003csub\u003emax\u003c/sub\u003e improved by ≥0.16 m/s. This cut-off has been shown to increase the probability of at least one step improvement in the modified Rankin scale(26), i.e., a meaningful improvement for the patient.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analyses were conducted using IBM SPSS version 28.01.1 (SPSS, IBM Software Group, Chicago, IL, USA).\u0026nbsp;Continuous variables are presented as mean ± SD or median (interquartile range (IQR)), as appropriate. Group comparisons were performed using parametric or non-parametric tests depending on data distribution. Categorical variables were analysed using the chi-square test or Fisher’s exact test when expected cell counts were low. Group differences between INPH patients and controls were examined via a generalized linear model, adjusting for age, sex, sample centrifugation prior to storage, and storage duration. Correlations were assessed with Pearson’s (r) or Spearman’s (r\u003csub\u003es\u003c/sub\u003e) coefficients, depending on variable type and distribution. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSample and patient characteristics\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the sample characteristics of patients with INPH and controls. The groups were matched for age, although a male predominance was observed in the INPH group. Notable differences were identified in terms of sample processing and storage conditions. Specifically, 75% of the INPH samples underwent centrifugation prior to storage, whereas none of the control samples were centrifuged. Furthermore, there was a discrepancy in the storage duration, with INPH samples collected between 2007 and 2022, compared with the control samples, which were collected between 2003 and 2004.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of samples from INPH patients and controls\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eINPH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eParticipants (n)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (years) median (IQR)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74 (70\u0026ndash;78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e72 (69\u0026ndash;78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.36\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFemale (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0078\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCentrifuged prior to storage (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStorage time (days) median (IQR)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2689 (1667\u0026ndash;4016)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6702 (6647\u0026ndash;6828)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u0026dagger; = Independent samples Mann-Whitney U test. \u0026Dagger; = Pearson chi-square (two-sided). Abbreviations: Idiopathic normal pressure hydrocephalus (INPH), interquartile range (IQR).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCSF AQP4 levels in INPH patients versus controls\u003c/h3\u003e\n\u003cp\u003eWithout adjustment for sex or pre-analytical sample handling, preoperative INPH patients showed lower AQP4 levels (median 1142 AU, IQR 1000\u0026ndash;1321) compared with controls (median 1366 AU, IQR 1160\u0026ndash;1516; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, 75% of the preoperative samples had both a markedly shorter freezer storage time and had been centrifuged prior to storage, whereas none of the control samples were centrifuged. To account for this, we repeated the analysis including only the non-centrifuged preoperative samples with storage times more comparable to controls (n\u0026thinsp;=\u0026thinsp;59). In this restricted comparison, no significant difference was observed between INPH patients (mean 1261\u0026thinsp;\u0026plusmn;\u0026thinsp;303 AU) and controls (mean 1351\u0026thinsp;\u0026plusmn;\u0026thinsp;279 AU; p\u0026thinsp;=\u0026thinsp;0.17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further examine group differences while accounting for covariates, we conducted a generalized linear model with AQP4 levels as the dependent variable and diagnostic group (INPH vs. control) as the predictor. The model adjusted for sex (p\u0026thinsp;=\u0026thinsp;0.13), centrifugation prior to storage (p\u0026thinsp;=\u0026thinsp;0.87), storage duration (p\u0026thinsp;=\u0026thinsp;0.004), and age (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). After adjustment, no difference in AQP4 levels was found between INPH patients and controls (p\u0026thinsp;=\u0026thinsp;0.87). Instead, significant positive correlations were observed between AQP4 levels and storage duration (r\u0026thinsp;=\u0026thinsp;0.30, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), as well as between AQP4 levels and age across INPH patients and controls (r\u0026thinsp;=\u0026thinsp;0.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003ePostoperative CSF AQP4 levels\u003c/h3\u003e\n\u003cp\u003eIn the longitudinal follow-up of INPH patients (n\u0026thinsp;=\u0026thinsp;110), we observed an increase in AQP4 levels six months after surgery (median 1646 AU IQR 1347\u0026ndash;1976) compared with the preoperative levels (median 1166 AU IQR: 976\u0026ndash;1345; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Only nine out of the 110 patients presented reduced AQP4 levels at follow-up.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCSF AQP4 levels in shunt responders\u003c/h2\u003e\u003cp\u003eAmong the 233 INPH patients with preoperative samples, postoperative V\u003csub\u003emax\u003c/sub\u003e data were available in 204. Of these, 130 patients (64%) were classified as shunt responders. Preoperative AQP4 levels were lower in responders (median 1089 AU, IQR 971\u0026ndash;1277) than in non-responders (median 1213 AU, IQR 1074\u0026ndash;1361; p\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Responders and non-responders did not differ in age, sex, sample centrifugation prior to storage, storage duration, Mini-Mental State Examination score, implanted shunt type, or shunt opening pressure. In the subgroup with available CSF biomarkers (t-tau, p-tau. Aβ42 and GFAP), non-responders had significantly higher t-tau and p-tau concentrations, although the proportion of patients above the predefined clinical cut-offs did not differ between groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the 107 patients with both pre- and postoperative samples and postoperative V\u003csub\u003emax\u003c/sub\u003e data, the change in AQP4 levels from pre- to postoperative measurements was weakly positively correlated with the change in postoperative V\u003csub\u003emax\u003c/sub\u003e (r\u003csub\u003es\u003c/sub\u003e = 0.22, p\u0026thinsp;=\u0026thinsp;0.022) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatient and sample characteristics of shunt responders and non-responders\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eShunt responder\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNumber\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026dagger;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (years) median (IQR)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74 (70\u0026ndash;78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76 (70\u0026ndash;78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.63\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFemale (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.55\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCentrifuged before storage (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.62\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStorage duration (days) median (IQR)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2539 (1477\u0026ndash;4115)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2741 (2072\u0026ndash;3919)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.38\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMMSE score median (IQR) (n\u0026thinsp;=\u0026thinsp;197)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26 (\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27 (\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.15\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eShunt type (%)\u003c/b\u003e:\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedtronic Strata\u0026trade;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e91.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e94.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0,86\u003csup\u003e\u0026para;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedtronic Strata MR\u0026trade;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCodman Certas\u0026reg; Plus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStrata shunt opening pressure (%)\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026curren;\u003c/b\u003e\u003c/sup\u003e:\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.0\u003csup\u003e\u0026para;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCSF concentration median (ng/L) (IQR)\u003c/b\u003e:\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003et-tau (n\u0026thinsp;=\u0026thinsp;170)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e200 (159\u0026ndash;266)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e228 (192\u0026ndash;310)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.023\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-tau (n\u0026thinsp;=\u0026thinsp;163)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30 (\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36 (29\u0026ndash;48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.006\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAβ42 (n\u0026thinsp;=\u0026thinsp;171)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e419 (331\u0026ndash;523)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e460 (337\u0026ndash;588)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGFAP (n\u0026thinsp;=\u0026thinsp;157)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e490 (350\u0026ndash;700)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e550 (390\u0026ndash;970)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.15\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003et-tau\u0026thinsp;\u0026gt;\u0026thinsp;cut-off (409 ng/L) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ep-tau\u0026thinsp;\u0026gt;\u0026thinsp;cut-off (50 ng/L) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eShunt responder\u0026thinsp;=\u0026thinsp;\u0026ge;\u0026thinsp;0,16 m/s improvement in maximal gait velocity after shunt surgery. \u0026dagger; = Data for improvement in maximal gait velocity was missing from 29 out of 233 patients with preoperative samples. \u0026Dagger; = Independent-Samples Mann-Whitney U test. \u0026sect; = Pearson chi-square test, (two-sided). \u0026para; = Fisher's exact test, (two-sided). \u0026curren; = Data for shunt opening pressure was missing from one out of 130 shunt responders. Abbreviations: Interquartile range (IQR), Mini-Mental State Examination (MMSE), total-tau (t-tau), phosphorylated tau (p-tau), amyloid-β42 (Aβ42), glial fibrillary acidic protein (GFAP).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings demonstrate that CSF AQP4 levels can be reliably measured via a suspension bead array technique in a substantial cohort of INPH patients. After adjusting for known group differences, such as sex and preanalytical sample handling, the AQP4 levels in INPH patients did not differ from those in age-matched, neurologically healthy controls. However, preoperative AQP4 levels were lower in shunt responders than in non-responders, suggesting a potential role for AQP4 in identifying candidates for shunt surgery. Additionally, AQP4 levels increased postoperatively, with a weak positive correlation with clinical improvement.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAQP4 \u0026ndash; from the plasma membrane to CSF\u003c/h2\u003e\u003cp\u003eThe cellular mechanisms regulating AQP4 expression and its turnover into CSF remain poorly understood(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). One recent study revealed that membrane-bound AQP4 is continuously recycled or internalized through vesicular trafficking(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Membrane-bound proteins are typically internalized and degraded via the ubiquitin‒lysosome pathway(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), suggesting that this mechanism likely governs AQP4 degradation in astrocytes. Consequently, AQP4 is not expected to leave astrocytes under normal circumstances. However, the detection of AQP4 in CSF raises the possibility that its turnover between astrocytes and CSF is mediated by additional processes, potentially involving the release of microvesicles or exosomes(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). An alternative explanation is that AQP4 enters the ISF or CSF because of astrocytic cell injury. However, histopathological studies in INPH have not demonstrated overt astrocytic injury or necrosis. Instead, reactive astrogliosis has consistently been reported as a prominent finding(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), suggesting that the presence of AQP4 in CSF is less likely to result from passive leakage due to cellular damage and may instead reflect regulated or secondary processes.\u003c/p\u003e\u003cp\u003eIn summary, the exact form in which AQP4 is measured in CSF remains unclear and warrants further investigation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePrevious studies of CSF AQP4 levels in INPH, AD and FTD\u003c/h2\u003e\u003cp\u003eAs previously mentioned, two studies have investigated AQP4 in CSF from INPH patients. The first found no difference in AQP4 levels between INPH and controls; however, this study included only 10 subjects with INPH and 9 controls(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This sample size provides sufficient power only to detect very large group differences. A second, larger study failed to detect any AQP4 in CSF using a commercial ELISA kit for detection, raising concerns about potential methodological issues (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Taken together, the available evidence does not provide conclusive results regarding AQP4 concentration in the CSF of INPH patients.\u003c/p\u003e\u003cp\u003eAQP4 has however been more extensively investigated in AD, which has been proposed to share several features with INPH in terms of glymphatic dysfunction(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Arighi et al. initially reported lower AQP4 levels in the CSF in a small study including eleven subjects with AD and nine controls(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e); however, in a subsequent larger study, they reported contradictory results, with increased levels(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Bergstr\u0026ouml;m et al.(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) conducted a multicentre study comparing AQP4 levels in AD patients and controls and reported elevated AQP4 levels in CSF of AD patients across all four studied cohorts. These findings were recently corroborated by another study in which CSF AQP4 levels were quantified via ELISA(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Notably, increased AQP4 levels in CSF have also been reported in frontotemporal dementia (FTD) patients(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIf AQP4 levels in the CSF are indeed elevated in AD and FTD patients but remain within the normal range, as our findings suggest, or are low in INPH patients, this could indicate distinct mechanisms of fluid flow dysfunction in INPH patients. This finding suggests that AQP4 is a promising biomarker with the potential to identify AQP4 dysregulation in AD and FTD, which is not present in INPH.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAQP4 expression in astrocytes in AD and INPH\u003c/h2\u003e\u003cp\u003eZeppenfeld et al.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) investigated the relationship between perivascular AQP4 expression and disease stage in AD and cognitively intact controls of different ages using postmortem frontal cortex tissue. They found that loss of AQP4 polarization to the perivascular space was associated with greater amyloid-β burden and more advanced Braak stage. In addition, global AQP4 expression in astrocytes increased with age in cognitively intact elderly individuals and with advancing Braak stage in AD. Taken together with previous reports of elevated CSF AQP4 in AD, these results suggest that altered perivascular polarization, combined with enhanced global astrocytic AQP4 expression, may be reflected in increased CSF AQP4 levels in AD patients. In line with these findings, our study also demonstrated an age-related increase in CSF AQP4 levels.\u003c/p\u003e\u003cp\u003eA correlation between reduced perivascular AQP4 expression and disease stage, or an increase in global AQP4 expression, has not yet been demonstrated in INPH. However, several studies have analysed cortical biopsies from INPH patients(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These studies reported reduced expression of AQP4 in astrocytic endfeet facing the perivascular space, accompanied by astrogliosis and blood‒brain barrier dysfunction. Notably, owing to the invasive nature of brain biopsy sampling, the reference patients in these studies were not age-matched and had other neurological conditions, such as epilepsy, tumours, and aneurysms. Previous studies have shown altered AQP4 expression in astrocytes in relation to tumours(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), hippocampal sclerosis in medial temporal lobe epilepsy(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and aging(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCSF AQP4 levels in INPH and glymphatic function\u003c/h2\u003e\u003cp\u003eWe did not observe a significant difference in CSF AQP4 levels between INPH patients and controls. This finding suggests that CSF AQP4 levels may remain within the normal range in INPH. On the basis of the findings discussed above, CSF AQP4 levels may reflect astrocytic expression. If this is the case, our findings suggest that global AQP4 expression remains normal in INPH. However, even if overall AQP4 expression remains intact, it is still possible that the polarization of AQP4 to the perivascular space is disrupted. Nonetheless, AQP4 levels within the normal range in CSF do not support a reduced capacity for AQP4-mediated glymphatic flow.\u003c/p\u003e\u003cp\u003eThis stands in partial contrast to the conclusions of previous MRI studies using intrathecal GBCA, which revealed a delayed GBCA peak in the brain tissue of INPH patients, interpreted as reduced glymphatic clearance(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, this delay might also be attributed to a primary delay in GBCA distribution within the SAS, reflecting altered CSF circulation patterns. Such changes could be a natural consequence of known CSF dynamics disturbances in INPH, including elevated outflow resistance(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and disproportionate enlargement of the ventricles and SAS(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCSF AQP4 levels and shunt surgery\u003c/h2\u003e\u003cp\u003eWe observed a general increase in CSF AQP4 levels following shunt surgery, with responders displaying lower preoperative levels than non-responders. Moreover, postoperative increases in AQP4 levels correlated weakly with clinical improvement, as measured by changes in V\u003csub\u003emax\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eOne possible explanation for the postoperative rise in CSF AQP4 is a concentration effect due to reduced CSF volume. However, earlier studies of other potential CSF biomarkers in INPH, including amyloid-β42, t-tau, and p-tau(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and ten different proteins(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) did not identify significant concentration effects from reduced CSF ventricular volume after shunt surgery. Two Japanese groups have investigated changes in intracranial brain and CSF volumes after shunt surgery, observing primarily a redistribution of CSF between different compartments of the SAS, with only minor reductions in total intracranial CSF volume at 1\u0026ndash;12 months postoperatively (11% and 5%, respectively)(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). These modest changes are unlikely to explain the increase in AQP4 levels observed here.\u003c/p\u003e\u003cp\u003eAnother potential mechanism is a sedimentation effect along the cranial\u0026ndash;lumbar axis due to altered CSF circulation caused by shunting from the ventricles near the choroid plexus. Yet shunt surgery has been shown to produce variable effects across CSF proteins, with both increases and decreases in lumbar CSF concentrations(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) making predictions for AQP4 behaviour uncertain, particularly in the absence of ventricular CSF samples.\u003c/p\u003e\u003cp\u003eA further hypothesis is that elevated postoperative AQP4 levels reflect altered glymphatic flow due to decreased intracranial pressure and increased compliance after shunt surgery. Such changes might stimulate AQP4 expression in astrocytes and/or enhance recirculation of ISF, promoting transport of proteins, including non\u0026ndash;membrane-bound AQP4, into CSF.\u003c/p\u003e\u003cp\u003eThe finding that responders had lower preoperative CSF AQP4 levels than non-responders, together with the weak correlation between improvement in V\u003csub\u003emax\u003c/sub\u003e and postoperative AQP4 changes, is intriguing. The present study found that non-responders had higher tau and phosphorylated tau concentrations. This may indicate a more severe comorbidity of neurodegeneration in this subgroup, consistent with previous reports of higher levels of CSF AQP4 in AD and FTD(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). INPH is a heterogeneous condition, often complicated by comorbid neurodegenerative disorders such as subcortical vascular dementia, parkinsonism, and AD. Thus, the responders in our study may represent a more \u0026ldquo;pure INPH\u0026rdquo; phenotype with less neurodegenerative burden, with normal or slightly lower preoperative AQP4 levels. In these patients, the disturbed CSF dynamics typical of INPH were improved by shunting, leading to the clinical improvement. However, as a sign of functioning shunt, this change in CSF circulation also resulted in a postoperative rise in AQP4, likely due to mechanisms previously discussed. In summary, this suggests that INPH entails a CSF disturbance that may not be a AQP4-related glymphatic dysfunction. At the same time, it supports the notion that elevated AQP4 levels may indicate comorbid neurodegenerative disease, which is known to be associated with higher levels of AQP4 in CSF(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and reduced clinical effect from shunting(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStrengths and weaknesses\u003c/h2\u003e\u003cp\u003eThis study offers valuable insights into the relationship between AQP4 levels in CSF and INPH. Notably, its strengths include the large sample size of INPH patients, thorough postoperative follow-up, and the significant number of patients who underwent postoperative lumbar punctures. However, several limitations must be acknowledged. Preanalytical handling of samples varied, as the control group samples were collected several years earlier than those from INPH patients were, and some samples underwent centrifugation before storage. Additionally, the control group was relatively small and had a greater proportion of females, whereas the INPH group included more males, potentially influencing the results.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study analysed CSF AQP4 levels in a large cohort of INPH patients via a bead-based assay. After adjusting for confounders, no difference was found between INPH patients and controls. Postoperative increases in AQP4 may suggest enhanced fluid recirculation from the ISF to the CSF due to altered CSF dynamics after shunt placement. Lower preoperative AQP4 levels were linked to better shunt outcomes, suggesting its potential as a biomarker for predicting shunt response. This motivates further studies to clarify the relationship, such as examining correlations between AQP4 levels and CSF outflow resistance, comparing lumbar and ventricular CSF AQP4 levels, and exploring associated MRI characteristics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAQP4 – Aquaporin-4\u003c/p\u003e\n\u003cp\u003eINPH – Idiopathic normal pressure hydrocephalus\u003c/p\u003e\n\u003cp\u003eSAS – Subarachnoid space\u003c/p\u003e\n\u003cp\u003eISF – Interstitial fluid\u003c/p\u003e\n\u003cp\u003eGBCA – Gadolinium-based contrast agent\u003c/p\u003e\n\u003cp\u003eMRI – Magnetic resonance imaging\u003c/p\u003e\n\u003cp\u003eAD – Alzheimer’s disease\u003c/p\u003e\n\u003cp\u003eV\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e– Maximal gait velocity\u003c/p\u003e\n\u003cp\u003et-tau – Total tau\u003c/p\u003e\n\u003cp\u003ep-tau – Phosphorylated tau\u003c/p\u003e\n\u003cp\u003eAβ42 – Amyloid-β42\u003c/p\u003e\n\u003cp\u003eGFAP – Glial fibrillary acidic protein\u003c/p\u003e\n\u003cp\u003eIQR – Interquartile range\u003c/p\u003e\n\u003cp\u003eHPA – Human Protein Atlas\u003c/p\u003e\n\u003cp\u003eAU – Arbitrary Units\u003c/p\u003e\n\u003cp\u003eMMSE – Mini-Mental State Examination\u003c/p\u003e\n\u003cp\u003eFTD – Frontotemporal dementia\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eAll patients provided written consent to save CSF for future research purposes at the time of lumbar puncture. Participants in this study were informed about the study via a letter, which included an option to opt out if they did not wish to participate. Individuals deceased at the time inclusion began (2020) were automatically incorporated in the cohort. The\u0026nbsp;study was approved by the Swedish ethical review board and the ethical review board at Umeå University (reference number 2020-04469). This study was performed in accordance with the guidelines of the Declaration of Helsinki\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no competing interests to declare\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Swedish Foundation for Strategic Research (grant numberRMX18-0152) and a regional agreement between Umeå University and Västerbotten County Council (ALF).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor’s contributions\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eJ.E.D.R., S.Q., A.E., and J.M. conceived the study.J.E.D.R., S.B., S.M., D.B., A.E., and J.M. designed the work.Data were acquired by J.E.D.R., S.B., S.M., S.Q., A.E., and J.M.Data analysis and interpretation were performed by J.E.D.R., S.B., S.M., A.E., and J.M.J.E.D.R., A.E., and J.M. drafted the manuscript.All authors critically revised the manuscript and approved the final version\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003eAcknowledgents\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMacAulay N. 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Ventricular cerebrospinal fluid neurofilament protein levels decrease in parallel with white matter pathology after shunt surgery in normal pressure hydrocephalus. Eur J Neurol. 2007;14(3):248\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHiraoka K, Yamasaki H, Takagi M, Saito M, Nishio Y, Iizuka O, et al. Changes in the volumes of the brain and cerebrospinal fluid spaces after shunt surgery in idiopathic normal-pressure hydrocephalus. J Neurol Sci. 2010;296(1\u0026ndash;2):7\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamada S, Ishikawa M, Yamaguchi M, Yamamoto K. Longitudinal morphological changes during recovery from brain deformation due to idiopathic normal pressure hydrocephalus after ventriculoperitoneal shunt surgery. Sci Rep. 2019;9(1):17318.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThavarajasingam SG, El-Khatib M, Vemulapalli KV, Iradukunda HAS, Laleye J, Russo S, et al. Cerebrospinal fluid and venous biomarkers of shunt-responsive idiopathic normal pressure hydrocephalus: a systematic review and meta-analysis. Acta Neurochir (Wien). 2022;164(7):1719\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aquaporin 4, Cerebrospinal fluid, Normal pressure hydrocephalus, idiopathic, Glymphatic system","lastPublishedDoi":"10.21203/rs.3.rs-7785379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7785379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAquaporin-4 (AQP4) is crucial for brain fluid regulation and glymphatic system function. Idiopathic normal pressure hydrocephalus (INPH), characterized by impaired CSF flow, is often treated with shunt surgery. This study investigated AQP4 levels in INPH patients to explore its role in pathophysiology and potential as a biomarker for shunt response.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCSF samples from 233 INPH patients and 29 controls were analysed. AQP4 levels were compared between preoperative patients and controls, before and after shunt surgery (110 patients), and between shunt responders and non-responders (204 patients). A bead-based assay was used to measure AQP4, and outcomes were assessed by postoperative changes in maximum gait velocity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreoperative AQP4 levels were lower in INPH patients (1177\u0026thinsp;\u0026plusmn;\u0026thinsp;259 AU) than in controls (1351\u0026thinsp;\u0026plusmn;\u0026thinsp;279 AU, p\u0026thinsp;=\u0026thinsp;0.003), but the difference did not persist after adjustment for confounders (p\u0026thinsp;=\u0026thinsp;0.87). Postoperatively, AQP4 levels increased (1714\u0026thinsp;\u0026plusmn;\u0026thinsp;473 vs 1186\u0026thinsp;\u0026plusmn;\u0026thinsp;269 AU, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with a modest correlation between AQP4 increase and gait improvement (rₛ = 0.22, p\u0026thinsp;=\u0026thinsp;0.022). Compared with non-responders, shunt responders (130 patients) had lower preoperative AQP4 levels (1144\u0026thinsp;\u0026plusmn;\u0026thinsp;250 vs. 1238\u0026thinsp;\u0026plusmn;\u0026thinsp;273 AU, p\u0026thinsp;=\u0026thinsp;0.016)\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eINPH patients presented AQP4 levels comparable to those of controls. The postoperative increase in AQP4 may result from altered CSF dynamics induced by the shunt; however, the underlying mechanism remains unclear, underscoring the need for further studies to clarify this relationship. Notably, lower preoperative AQP4 levels in shunt responders suggest a potential predictive role.\u003c/p\u003e","manuscriptTitle":"CSF Aquaporin-4 in Idiopathic Normal Pressure Hydrocephalus: Potential as a Biomarker for Shunt Response and Glymphatic Function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 01:17:53","doi":"10.21203/rs.3.rs-7785379/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-11T04:19:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-10T11:40:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T23:48:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188857267731186435747098202628798089615","date":"2025-10-25T07:21:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132783581496980509781704150092256159004","date":"2025-10-22T16:52:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160763053920800306215823737555145284065","date":"2025-10-22T06:57:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T16:02:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-21T15:56:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-21T04:59:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fluids and Barriers of the CNS","date":"2025-10-05T14:47:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46841fdf-68e8-40fa-a370-d268c45a0f5d","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:04:32+00:00","versionOfRecord":{"articleIdentity":"rs-7785379","link":"https://doi.org/10.1186/s12987-026-00809-2","journal":{"identity":"fluids-and-barriers-of-the-cns","isVorOnly":false,"title":"Fluids and Barriers of the CNS"},"publishedOn":"2026-04-21 15:58:36","publishedOnDateReadable":"April 21st, 2026"},"versionCreatedAt":"2025-11-04 01:17:53","video":"","vorDoi":"10.1186/s12987-026-00809-2","vorDoiUrl":"https://doi.org/10.1186/s12987-026-00809-2","workflowStages":[]},"version":"v1","identity":"rs-7785379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7785379","identity":"rs-7785379","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}