Background
Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological condition first described by Hakim and Adams in 1965 [
1]. INPH primarily affects adults above 65 years of age [
2,
3] and the condition is characterized by a clinical triad of symptoms; gait disturbances, urinary incontinence, and cognitive deterioration [
1,
4]. Patients with iNPH accumulate excessive amounts of cerebrospinal fluid (CSF) in their brain ventricles causing ventricular enlargement [
4] and drainage by shunt insertion often improves the patient’s clinical status [
5‐
11]. Such direct reversibility of the clinical symptoms points to excessive CSF accumulation as part of the etiology, rather than permanent brain damage. The intracranial pressure (ICP) in iNPH patients is generally below 15 mmHg [
9,
10,
12,
13] which is believed to resemble the ICP in healthy individuals [
14,
15], hence the term
normal pressure hydrocephalus. However, since reference ICP values in healthy individuals remain sparse and pseudo-healthy patients are often used instead [
16] it is still debated whether the ICP in this patient group is truly resembling the ICP of healthy individuals. The etiology of iNPH remains unresolved but impaired CSF drainage or CSF hypersecretion may underlie the excessive CSF accumulation and possible ICP elevation. Although no macroscopic obstruction is discernible on diagnostic imaging [
4,
17] some iNPH patients display elevated CSF outflow resistance [
12,
18,
19] suggesting impaired CSF clearance and absorption. On the other hand, the CSF flow in iNPH patients appears hyperdynamic as demonstrated by an increased aqueductal CSF flow [
20‐
24]. This hyperdynamic CSF flow may originate from a decreased intracranial compliance [
13,
25‐
29] but could alternatively be explained by CSF hypersecretion, a potential mechanism generally neglected in the pathogenesis of hydrocephalus. CSF hypersecretion is sufficient to promote hydrocephalus development in humans [
30‐
32] and animals [
33] and has been demonstrated to occur in rats upon activation of an inflammatory pathway involving toll-like receptor 4 and NFκβ in the CSF-producing tissue, the choroid plexus [
33]. Altered abundance of select inflammatory markers has been detected in CSF from various hydrocephalic patients [
34]. Elevated levels of inflammatory markers may potentially correlate with several factors affecting the prognosis; e.g. the risk of development of some forms of hydrocephalus, the severity of the condition, and the need for surgical intervention [
35‐
38], while treatment with anti-inflammatory drugs reduces the incidence of hydrocephalus and improves clinical outcome [
39,
40]. We hypothesize that inflammation-induced CSF hypersecretion may underlie the CSF accumulation seen in iNPH patients, in whom there is no visible CSF drainage obstruction and in whom the underlying pathological mechanism therefore remains unresolved. In the present study, we sought to elucidate the molecular coupling between inflammatory markers and development of iNPH by revealing the inflammatory marker profile in CSF from iNPH patients alongside the delineation of the abundance of the corresponding receptors in the choroid plexus. The connection between the inflammatory marker profile in this patient group and hyperactivity of the choroidal Na
+/K
+/2Cl
− (NKCC1) transport mechanism, illustrated to sustain approximately 50% of the CSF secretion by the choroid plexus [
33,
41,
42], was determined in an ex vivo rodent animal model. If inflammatory markers are involved in the pathogenesis of iNPH, pharmacological targeting of their receptors or downstream signaling pathways could prove a novel treatment strategy.
Discussion
This study, based on the novel PEA technique, uncovers the inflammatory profile of the CSF from iNPH patients investigating the largest number of inflammatory markers so far and providing novel insight into the inflammatory marker profile of CSF from iNPH patients. We here demonstrate that the CSF from iNPH patients contains elevated levels of a subset of inflammatory markers. The corresponding inflammatory receptors are, in general, expressed in the choroid plexus of rats and humans, but their activation did not stimulate NKCC1 that is highly expressed in the luminal membrane of choroid plexus in mice, rats, and humans [
41,
68,
69] and demonstrated to support approximately half of the CSF secreted in the tested species (mice, rats, dogs; [
33,
41,
42]). Note that the NKCC1 inhibitor bumetanide only exerts its inhibitory action on NKCC1-mediated CSF secretion when delivered directly into the cerebral ventricles of the experimental animal (to target NKCC1 at the luminal membrane of choroid plexus [
33,
41,
42]) and not upon administration of bumetanide i.v. or i.p., in which cases, the inhibitor fails to reach its choroidal target [
70,
71]. Inflammatory markers therefore appear
not to promote iNPH via induction of choroidal CSF hypersecretion by promotion of NKCC1 activation, at least not in the rat ex vivo model here employed. Based on the present data set, however, we cannot rule out that other transport mechanisms involved in CSF secretion (see [
71] for review) could be modulated by these inflammatory agents.
Eleven inflammatory markers were elevated in CSF obtained from iNPH patients compared to elderly control subjects. Three inflammatory markers (OPG, 4E-BP1, and Flt3L) were not investigated experimentally, as they were unlikely to directly influence choroidal CSF secretion: OPG acts as a decoy receptor [
72], 4E-BP1 represses translation and plays a predominant role in cell proliferation [
73], while Flt3L, a hematopoietic growth factor, promotes differentiation of hematopoietic stem cells, particularly dendritic cells [
74]. The remaining eight inflammatory markers were investigated for their ability to alter choroidal CSF secretion: CCL28, CCL23, CCL3, CXCL1, IL-18, IL-8, OSM and CXCL6.
CCL28 is a chemokine constitutively expressed by epithelial cells at mucosal sites [
75], which promotes chemotaxis of immune cells through the receptors CCR3 and CCR10 [
75,
76] and is inducible upon presence of inflammatory or infectious stimuli [
77]. CCL28 is elevated in CSF from patients with Parkinson’s disease (PD) [
78], who have normal CSF production [
79], and its presence in iNPH patients may instead indicate similar pathological features of neurodegeneration and/or inflammation [
80]. The chemokine CCL23 is expressed mainly by macrophages and exerts its chemotactic effects on immune cells through CCR1 [
81,
82]. Elevated CCL23 levels are reported in various inflammatory diseases [
83,
84] and is, in addition, associated with progression from mild cognitive impairment to Alzheimer’s disease (AD) [
85]. Elevated CCL23 in both AD and iNPH aligns well with the overlapping clinical characteristics of iNPH and AD [
86] and the concomitant AD pathology found in iNPH [
87]. CCL3 is a chemokine that exerts its proinflammatory actions through the receptors CCR1 and CCR5, and with less affinity through CCR4 [
88]. CCL3 is secreted by various cells such as monocytes, macrophages, and epithelial cells [
89] and is inducible by proinflammatory agents such as lipopolysaccharides, tumor necrosis factor α, and IL-1β [
90,
91]. CCL3 is elevated in CSF from patients with posthemorrhagic hydrocephalus [
92] and hydrocephalus associated with tuberculous meningitis [
93], which both associate with inflammation [
94]. Elevated CCL3 in iNPH patients suggests similar inflammatory conditions in this patient group.
The chemokines CXCL1, CXCL6, and IL-8 exert their actions through the receptors CXCR1 and CXCR2 [
67]. CXCL1 is elevated in brain tissue from hydrocephalic mice with genetically induced ciliary dysfunction, in which hydrocephalus develops concurrent with neuroinflammation and tissue injury [
95]. As no antecedent proinflammatory insult occurs in these mice, the authors conclude that neuroinflammation arises as a consequence of the intracranial changes associated with hydrocephalus development [
95]. Elevated CXCL1 in iNPH patients may therefore reflect presence of neuroinflammation and it may arise concomitantly with development of hydrocephalus. Like CXCL1, CXCL6 serves as a neutrophil chemoattractant [
96] and is elevated in bacterial and viral meningitis [
97].
Several studies have investigated the proinflammatory chemokine IL-8 in relation to hydrocephalus. While some studies report elevated IL-8 in CSF from patients with different hydrocephalic conditions [
93,
98,
99], others report unaltered levels [
92,
98‐
101]. Our findings of elevated IL-8 in CSF from iNPH patients are in agreement with the findings by Czubowicz [
98] but contradict others [
99‐
101]. Although the contradicting findings may result from methodological differences, additional studies are required to determine whether elevated CSF levels of IL-8 are characteristic of iNPH. IL-18 is a proinflammatory cytokine released in response to inflammatory and infectious stimuli which signals through the receptors IL-18R1 and IL-18RAP and is involved in various neurological conditions [
102]. IL-18 is elevated in CSF from preterm infants with posthemorrhagic hydrocephalus and hydrocephalus associated with spina bifida and aqueductal stenosis [
103,
104]. As these conditions are associated with inflammatory changes including astrogliosis, microgliosis, and white matter damage [
94,
103‐
105], it can be speculated whether similar changes occur in iNPH patients.
OSM is a pleiotropic interleukin-6-type cytokine that utilizes two receptor complexes, IL-6ST/LIFR and IL-6ST/OSMR, to mediate a broad range of homeostatic activities, some of which include hematopoiesis, liver regeneration, and homeostasis of neural precursor cells [
106,
107]. Overproduction of OSM is associated with skin and airway inflammation, inflammatory bowel disease, and other pathological conditions [
106,
108,
109]. OSM was recently identified as a potential CSF marker of general CNS inflammation, although interestingly, the study included iNPH patients and patients with idiopathic intracranial hypertension as the non-inflamed controls [
110]. The elevated OSM levels in CSF from iNPH patients in the present study are indicative of inflammation, but the inflammatory condition may be less pronounced in comparison to other patient groups with prominent signs of inflammation such as those suffering from meningitis or subarachnoid hemorrhage [
110].
One inflammatory marker, CDCP1, was significantly decreased in the CSF obtained from iNPH patients compared to elderly control subjects. CDCP1 is a transmembrane glycoprotein chemotactic for T-cells [
65] that has been associated with cell adhesion and cancer development [
111]. Although its physiological role in iNPH patients remains unclear, it can be speculated whether CDCP1 may serve as a diagnostic marker, potentially distinguishing iNPH from other types of hydrocephalus.
Inflammatory markers such as cytokines, interleukins, and chemokines signal through various intracellular pathways and are capable of acting synergistically or antagonistically [
112]. In the present study, the elevated inflammatory markers were combined into cocktails and the ability of the inflammatory markers to modulate choroidal CSF secretion was thus not assessed individually. The combination of the inflammatory markers into cocktails may have compromised the effect of each inflammatory marker. However, as the inflammatory markers were elevated collectively in the CSF from iNPH patients, we sought to mimic the nature of iNPH by combining the inflammatory markers. In the present study, we did not obtain evidence that inflammatory markers can act directly on the choroid plexus to promote such hyperactivation of NKCC1, as was observed in an in vivo rodent model of intraventricular hemorrhage [
33]. The excised choroid plexuses were kept in tissue culture conditions for 16 h to allow the inflammatory agents to serve their purpose via modulation of transcriptional and translational processes, including post-translational modifications such as phosphorylation of proteins, which has been described earlier for NKCC1-mediated CSF hypersecretion [
33]. However, it is possible that all elevated inflammatory markers have to be included in the same experiment, that additional time is required for the inflammatory markers to have an effect on the choroidal transport protein NKCC1 (and/or target other choroidal transporters), or that lesser time is required for optimal initiation of the inflammatory machinery: Other studies have shown that upon an inflammatory stimulus, the choroid plexus transcriptome responds rapidly within 3–6 h [
113,
114] followed by gradual return to baseline within 72 h [
113]. However, whereas these studies only applied an inflammatory stimulus once, we exposed the choroid plexus to the inflammatory markers continuously for 16 h and would therefore not expect the effects of the inflammatory markers on the choroidal CSF secretion to subside. Although we document choroid plexus epithelial cell viability and bumetanide-sensitive
86Rb
+ efflux following the 16 h incubation time, prolonged presence of the inflammatory markers may have initiated oxidative stress, apoptosis, or other cellular mechanisms that could have compromised potential effects of the inflammatory markers on choroidal NKCC1-mediated ion transport. However, other research groups have reported successful exposure of choroid plexus epithelial cell lines to inflammatory stimuli for longer time periods [
115].
The inability of the inflammatory markers to modulate choroidal NKCC1 transport activity ex vivo and thus potentially contribute to development of iNPH leaves the molecular coupling between inflammatory markers and iNPH unresolved. Elevated levels of inflammatory markers in CSF from iNPH patients indicate presence of an ongoing inflammatory condition. However, whether this inflammatory condition promotes iNPH development or arises secondarily to the pathological changes observed in the iNPH brain remains uncertain. Some studies demonstrate that CSF levels of inflammatory markers decrease following CSF shunting in parallel with clinical improvement [
116,
117] suggesting that the inflammatory condition is intrinsically related to disturbed CSF dynamics. Interestingly, we did not find any significant differences in the CSF distribution of inflammatory markers when comparing iNPH shunt responders and iNPH shunt non-responders. Our findings therefore illustrate that the inflammatory profile of iNPH patient CSF obtained during the diagnostic workup cannot be used to predict the outcome of VP shunting in this patient group. One must, however, bear in mind that shunt response may be affected by other factors not accounted for in the present study, including clinical complications and comorbidities. In the future, studies should aim to resolve the role of inflammation in the pathogenesis of iNPH and thereby elucidate whether alleviation of the inflammation condition may prove beneficial in treatment of iNPH.
The present study is associated with certain limitations. Although all included iNPH patients were in moderate to severe stage of disease (see “
Methods”), the exact time between onset of symptoms and time of CSF sampling is unknown and could affect the inflammatory profile of the patients. The elderly control group is of an average age of 10 years below that of the iNPH patients. Since inflammation tends to increase with age, such age difference could affect the obtained results. We quantified the inflammatory marker content in CSF from human iNPH patients sampled at one time point only. Since an animal model of iNPH does not currently exist, the ability of (the rat version of) these elevated inflammatory markers to modulate choroidal NKCC1 activity was investigated in rat choroid plexus, as these experiments would not be possible with human tissue. Although our RNA-seq analysis revealed that the majority of the corresponding inflammatory receptors were expressed in the choroid plexus of rats and humans, we cannot exclude that differences between species (humans and rats) may have compromised the findings of our study. Direct modulation of choroidal CSF secretion requires that the receptors for the elevated inflammatory markers are present on the CSF-facing membrane of the choroid plexus. As our RNA-seq analysis only allowed for determination of choroidal receptor expression, not membrane localization, we cannot exclude that some of the inflammatory receptors were absent from the CSF-facing membrane and thus not activated by inclusion of the respective inflammatory marker.
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