Cerebrospinal fluid osmolality cannot predict development or surgical outcome of idiopathic normal pressure hydrocephalus.

BACKGROUND: The etiology of idiopathic normal pressure hydrocephalus (iNPH) is currently unknown. With no visible obstructions, altered cerebrospinal fluid (CSF) dynamics may explain the accumulation of ventricular fluid. We hypothesized that elevated osmolality in the CSF of iNPH patients could potentiate formation of ventricular fluid and thereby cause the disease progression and/or predict the surgical outcome. To address this hypothesis, we determined the lumbar and ventricular CSF osmolality of iNPH patients at different disease stages and compared with lumbar CSF samples obtained from control subjects. METHODS: The osmolality of CSF was determined on a total of 35 iNPH patients at diagnosis and at the subsequent treatment with shunt surgery (n = 20) and compared with the CSF osmolality from 20 control subjects. Simultaneously collected lumbar and ventricular CSF samples from experimental pigs were used to evaluate the compatibility between CSF from different compartments. RESULTS: We found no evidence of increased osmolality in the CSF of iNPH patients upon diagnosis or at the time of shunt treatment months after the diagnosis, compared with control individuals. CSF tapped from the lumbar space could be used as a read-out for ventricular CSF osmolality, as these were similar in both the patient group and in experimental pigs. We further observed no correlation between the CSF osmolality in iNPH patients and their responsiveness to shunt surgeries. CONCLUSIONS: The osmolality of lumbar CSF is a reliable reflection of the ventricular CSF osmolality, and is not elevated in iNPH patients. iNPH therefore does not appear to arise as a function of osmotic imbalances in the CSF system and CSF osmolality cannot serve as a biomarker for iNPH or as a predictive tool for shunt responsiveness.

Inflammatory Markers in Cerebrospinal Fluid from Patients with Hydrocephalus: A Systematic Literature Review.

OBJECTIVE: The aim of this systematic review was to evaluate existing literature on inflammatory markers in CSF from patients with hydrocephalus and identify potential markers capable of promoting hydrocephalus development and progression. METHODS: Relevant studies published before December 3rd 2020 were identified from PubMed, Embase, and reference lists. Studies were screened for eligibility using the predefined inclusion and exclusion criteria. Data from eligible studies were extracted, and sources of bias were evaluated. We included articles written in English investigating inflammatory markers in CSF from patients with hydrocephalus and control subjects. The review was conducted according to the PRISMA guidelines by three independent reviewers. RESULTS: Twenty-two studies analyzed CSF from 311 patients with idiopathic normal pressure hydrocephalus (iNPH), 178 with posthemorrhagic hydrocephalus (PHH), 151 with other hydrocephalus diagnoses, and 394 control subjects. Fifty-eight inflammatory markers were investigated. The CSF of iNPH patients had increased CSF levels of IL-6, IL-1ß, and LRG compared with control subjects, whereas the CSF of PHH patients had increased levels of IL-6, IL-18, and VEGF. CSF from patients with "other hydrocephalus diagnoses" had elevated IFN-γ compared to control subjects, and VEGF was increased in congenital hydrocephalus, spina bifida, and hydrocephalus associated with tuberculous meningitis compared with controls. CONCLUSION: IL-6, IL-1ß, LRG, IL-18, VEGF, and IFN-γ are elevated in CSF from patients with hydrocephalus and may be involved in promotion of hydrocephalus development and progression. They may serve as novel disease biomarkers, and their signaling pathways may represent targets for pharmacological management of hydrocephalus.

Cerebral influx of Na+ and Cl- as the osmotherapy-mediated rebound response in rats.

BACKGROUND: Cerebral edema can cause life-threatening increase in intracranial pressure. Besides surgical craniectomy performed in severe cases, osmotherapy may be employed to lower the intracranial pressure by osmotic extraction of cerebral fluid upon intravenous infusion of mannitol or NaCl. A so-called rebound effect can, however, hinder continuous reduction in cerebral fluid by yet unresolved mechanisms. METHODS: We determined the brain water and electrolyte content in healthy rats treated with osmotherapy. Osmotherapy (elevated plasma osmolarity) was mediated by intraperitoneal injection of NaCl or mannitol with inclusion of pharmacological inhibitors of selected ion-transporters present at the capillary lumen or choroidal membranes. Brain barrier integrity was determined by fluorescence detection following intravenous delivery of Na+-fluorescein. RESULTS: NaCl was slightly more efficient than mannitol as an osmotic agent. The brain water loss was only ~ 60% of that predicted from ideal osmotic behavior, which could be accounted for by cerebral Na+ and Cl- accumulation. This electrolyte accumulation represented the majority of the rebound response, which was unaffected by the employed pharmacological agents. The brain barriers remained intact during the elevated plasma osmolarity. CONCLUSIONS: A brain volume regulatory response occurs during osmotherapy, leading to the rebound response. This response involves brain accumulation of Na+ and Cl- and takes place by unresolved molecular mechanisms that do not include the common ion-transporting mechanisms located in the capillary endothelium at the blood-brain barrier and in the choroid plexus epithelium at the blood-CSF barrier. Future identification of these ion-transporting routes could provide a pharmacological target to prevent the rebound effect associated with the widely used osmotherapy.

The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms.

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2 , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+ /H+ exchanger NHE1, the Na+ - HCO3- cotransporter NBCn1, and the lactate-H+ cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA-mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line significantly reduced steady-state intracellular pH (pHi ) and capacity for pHi recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA-MB-231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor-free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.

Roles of acid-extruding ion transporters in regulation of breast cancer cell growth in a 3-dimensional microenvironment.

BACKGROUND: The 3-dimensional (3D) microenvironment of breast carcinomas is characterized by profoundly altered pH homeostasis, reflecting increased metabolic acid production and a confined extracellular space characterized by poor diffusion, yet the relative contributions of specific pH-regulatory transporters to 3D growth are poorly understood. The aim of this work was to determine how 3D spheroid growth of breast cancer cells impacts the expression and spatial organization of major acid extruding proteins, and how these proteins in turn are required for spheroid growth. METHODS: MCF-7 (Luminal-A) and MDA-MB-231 (Triple-negative) human breast cancer cells were grown as ~700-950 µm diameter spheroids, which were subjected to Western blotting for relevant transporters (2- and 3D growth), quantitative immunohistochemical analysis, and spheroid growth assays. Individual transporter contributions were assessed (i) pharmacologically, (ii) by stable shRNA- and transient siRNA-mediated knockdown, and (iii) by CRISPR/Cas9 knockout. RESULTS: In MCF-7 spheroids, expression of the lactate-H(+) cotransporter MCT1 (SLC16A1) increased from the spheroid periphery to its core, the Na(+),HCO3 (-) cotransporter NBCn1 (SLC4A7) was most highly expressed at the periphery, and the Na(+)/H(+) exchanger NHE1 (SLC9A1) and MCT4 (SLC16A3) were evenly distributed. A similar pattern was seen in MDA-MB-231 spheroids, except that these cells do not express MCT1. The relative total expression of NBCn1 and NHE1 was decreased in 3D compared to 2D, while that of MCT1 and MCT4 was unaltered. Inhibition of MCT1 (AR-C155858) attenuated MCF-7 spheroid growth and this was exacerbated by addition of S0859, an inhibitor of Na(+),HCO3 (-) cotransporters and MCTs. The pharmacological data was recapitulated by stable knockdown of MCT1 or NBCn1, whereas knockdown of MCT4 had no effect. CRISPR/Cas9 knockout of NHE1, but neither partial NHE1 knockdown nor the NHE1 inhibitor cariporide, inhibited MCF-7 spheroid growth. In contrast, growth of MDA-MB-231 spheroids was inhibited by stable or transient NHE1 knockdown and by NHE1 knockout, but not by knockdown of NBCn1 or MCT4. CONCLUSIONS: This work demonstrates the distinct expression and localization patterns of four major acid-extruding transporters in 3D spheroids of human breast cancer cells and reveals that 3D growth is dependent on these transporters in a cell type-dependent manner, with potentially important implications for breast cancer therapy.