Evidence of aquaporin involvement in human central pontine myelinolysis

Background Central pontine myelinolysis (CPM) is a demyelinating disorder of the central basis pontis that is often associated with osmotic stress. The aquaporin water channels (AQPs) have been pathogenically implicated because serum osmolarity changes redistribute water and osmolytes among various central nervous system compartments. Results We characterized the immunoreactivity of aquaporin-1 and aquaporin-4 (AQP1 and AQP4) and associated neuropathology in microscopic transverse sections from archival autopsied pontine tissue from 6 patients with pathologically confirmed CPM. Loss of both AQP1 and AQP4 was evident within demyelinating lesions in four of the six cases, despite the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes. Lesional astrocytes were small, and exhibited fewer and shorter processes than perilesional astrocytes. In two of the six cases, astrocytes within demyelinating lesions exhibited increased AQP1 and AQP4 immunoreactivities, and gemistocytes and mitotic astrocytes were numerous. Blinded review of medical records revealed that all four cases lacking lesional AQP1 and AQP4 immunoreactivities were male, whereas the two cases with enhanced lesional AQP1 and AQP4 immunoreactivities were female. Conclusions This report is the first to establish astrocytic AQP loss in a subset of human CPM cases and suggests AQP1 and AQP4 may be involved in the pathogenesis of CPM. Further studies are required to determine whether the loss of AQP1 and AQP4 is restricted to male CPM patients, or rather may be a feature associated with specific underlying precipitants of CPM that may be more common among men. Non-rodent experimental models are needed to better clarify the complex and dynamic mechanisms involved in the regulation of AQPs in CPM, in order to determine whether it occurs secondary to the destructive disease process, or represents a compensatory mechanism protecting the astrocyte against apoptosis.

Rapid correction of chronic hyponatremia is a known cause of CPM, but the molecular pathogenesis remains elusive [3,4,6,7,12]. CPM occurs in the context of severe illness, and is commonly a complication of conditions with altered serum Na + levels: alcoholism, liver transplantation, malnutrition, hepatic cirrhosis, burns or the syndrome of inappropriate antidiuresis (SIAD) [11,[13][14][15][16]. Recognition that CPM also occurs when other serum osmolytes are altered has prompted the use of "osmotic demyelination syndrome" as alternative terminology [17].
Changes in serum osmolarity are recognized to cause cerebral edema and redistribution of water and inorganic and organic osmolytes among various central nervous system (CNS) compartments [18][19][20][21]. Astrocytes, which are five times more abundant than neurons in the CNS, enwrap synapses and blood vessels and participate in blood-brain barrier maintenance. They are therefore in a unique and critical position for controlling brain volume changes [22,23]. Aquaporins (AQPs) are key regulators of brain volume homeostasis. While aquaporin-4 (AQP4) has been considered the major CNS water channel and is confined to astrocytes and ependyma [24,25], recent studies have shown that astrocytes also express aquaporin-1 (AQP1) in human and non-human primate brains [26][27][28]. However the complementary and/or redundant roles of astrocytic AQP1 and AQP4 in regulation of water homeostasis in the human CNS have yet to be addressed. This study is the first to systematically characterize astrocyte pathology and expression of AQP1 and AQP4 proteins in human CPM lesions.

General neuropathological characteristics of CPM lesions
All lesions identified in CNS tissues examined from the 6 CPM patients exhibited active demyelination (Figure 1a

Expression of AQP1 and AQP4 in the normal pons
Astrocytes in the normal pons expressed both AQP1 and AQP4 ( Figure 5). No differences were observed between the female and male controls.
Astrocytes in the pontine white matter tracts expressed preferentially, but not exclusively, AQP1 (Figure 5a-c). AQP1 in the white matter was highly concentrated on the membrane of the astrocytic cell bodies and in all astrocytic foot processes, including those abutting blood vessels ( Figure 5d). AQP4 immunoreactivity in the white matter was mainly observed in astrocytic foot processes abutting blood vessels (Figure 5f).
Astrocytes in the pontine grey matter nuclei preferentially, but not exclusively, expressed AQP4 (Figure 5a-c). AQP4 positive astrocytic processes enveloped the blood vessels as well as the neuronal cell bodies (Figure 5g). AQP1 immunoreactivity in the pontine nuclei was preferentially observed around blood vessels and around some, but not all neurons (Figure 5e).

Astrocytic pathology in CPM lesions
Loss of AQP1 and AQP4 was evident within demyelinating lesions in four of the six cases (Table 1, Figure 1b Demographics and clinical characteristics of the CPM patients ( Table 1) Blinded review of medical records revealed that all 4 CPM cases with lesional loss of AQP1 and AQP 4 immunoreactivities were male, whereas the 2 cases with increased lesional AQP1 and AQP4 immunoreactivities were female. Median age at hospitalization was 49 years (range . Median disease duration (interval to death from arrival at hospital or from clinical onset of CPM) was 9 days (range 1-60).

CPM lesions are characterized by astrocytopathy
Apoptosis and loss of oligodendrocytes are traditionally considered the pathological hallmarks of CPM. However we found in a subset of CPM cases that astrocytes were variably reduced in numbers, were small with fewer and shorter processes, and AQP1 and AQP4 immunoreactivities were lost within actively demyelinating regions with relative preservation of myelin. These findings suggest that prominent degenerative changes in astrocytes may precede demyelination. Astrocyte damage has been described in a single previous report [8], however this is the first study characterizing in detail the predominant astrocytic injury in human CPM. Although autopsy studies do not permit definite determination of whether this astrocytic damage is primary or secondary, a rat model of osmotic demyelination suggests that rapid correction of hyponatremia triggers astrocytic damage in male rats, and that ensuing loss of trophic communication between astrocytes and oligodendrocytes results in inflammation, microglial activation, oligodendrocyte injury, and subsequent demyelination [35]. The constellation of neuropathological findings we observed, particularly among four of the CPM patients, implies a similar sequence of pathogenic events, with two notable differences. First, GFAP immunoreactivity is lost in rat lesional astrocytes, but is retained in lesional astrocytes of patients with CPM, despite a loss of both AQP1 and AQP4. This argues against the loss of AQPs being strictly due to a loss of astrocytes. Second, in the rat CPM model, lesional astrocytes show signs of extensive apoptosis, whereas apoptotic astrocytes were not evident in human CPM. Interspecies and interregional astrocytic heterogeneity may explain these discrepancies. Most human CPM lesions reside in the pons, while rodent CPM lesions typically reside in the external capsule, claustrum, corpus striatum, neocortex, hippocampus and anterior commissure [3,4]. Furthermore, unlike rodent astrocytes which only express AQP4, astrocytes in the human brain express both AQP1 and AQP4 [27,28]. Species and CNS differences with respect to astrocyte morphology, properties and functions therefore exist and have been described previously in both the normal and diseased brain [36][37][38].

Relevance of AQPs to CPM
A unique finding in our study is that immunoreactivity for AQP proteins is lost in some actively demyelinating lesions in human CPM. This finding was not universal. Two of the six cases had an increase of AQP1 and AQP4 immunoreactivities within actively demyelinating CPM lesions. Complex dynamic processes that maintain water homeostasis likely contribute to the discordance of these findings. As the principal water channels in human CNS astrocytes, both AQP1 and AQP4 are involved indirectly in osmolyte movement through functional and molecular interactions with numerous ion channels and osmolyte transporters [18][19][20][21].
The observed loss of AQP1 and AQP4 in four of six cases may reflect a compensatory astrocytic response to a hypotonic milieu. The astrocyte response anticipated in a hypotonic environment is swelling due to osmotically- driven water influx through AQP channels. The astrocyte's physiological regulatory volume decrease preserves cell volume homeostasis by releasing inorganic and organic osmolytes, which in turn are followed by water [19,20,39,40]. The therapeutic use of a hyperosmolar solution to rapidly correct chronic hypotonicity causes endothelial cell shrinkage and blood-brain barrier disruption, vasogenic edema and increased osmolarity of the CNS extracellular space [41,42]. Water efflux from astrocytes to compensate for intracellular and extracellular osmolarities, further accentuates the shrinkage of astrocytes. Loss of AQP4 and AQP1 could represent a protective mechanism whereby astrocytes restrict water loss and prevent the triggering of apoptosis precipitated by a loss of cell volume [40,[43][44][45][46][47][48][49]. The reduced number and small size of GFAP-positive (non-apoptotic) astrocytes that we observed in regions of AQP1 and AQP4 loss within CPM lesions are compatible with this hypothesis.
In contrast, two of the six CPM cases were characterized by astrogliosis associated with increased AQP1 and AQP4 immunoreactivities. In the setting of vasogenic edema accompanying CPM, an increase in AQP1 and AQP4 should facilitate water removal [21,50]. Administration of urea or reinduction of hyponatremia during inadvertent rapid correction of hyponatremia are both known to decrease the number and severity of CPM lesions and to increase AQP4 expression [51][52][53][54][55][56]. However, AQPs are only one of several factors that may be involved in the genesis and resolution of CPM. Although this was a small series, we did not observe differences between the severity of lesions, nor did the clinical outcomes differ in the patients regardless of observed tissue AQP status.

Differences in the expression of AQPs in CPM lesions
An unexpected finding in our study was that all CPM cases exhibiting lesional loss of AQP1 and AQP4 (d) AQP1 in the white matter is highly concentrated on the membrane of the astrocytic cell bodies (arrow heads) and in all astrocytic foot processes, including those abutting blood vessels (arrows) (AQP1, scale bar = 100 μm); (e) AQP1 immunoreactivity in the pontine nuclei is preferentially observed around blood vessels (arrows) and around some, but not all neurons (AQP1, scale bar = 50 μm); (f) AQP4 immunoreactivity in the white matter is mainly observed around blood vessels (arrows) (AQP4, scale bar = 100 μm); (g) AQP4 positive astrocytic processes in the pontine nuclei envelop the blood vessels (arrow) as well as all neuronal cell bodies (AQP4, scale bar = 50 μm).
immunoreactivity were male patients and that female cases exhibited a lesional increase in astrocytic AQP immunoreactivity. The small number of patients precludes definitive conclusions concerning sexual dimorphism in astrocytic AQP regulation. However, female and male sex hormones have been reported to have opposite effects on ion transporters, and therefore could exert similar effects on the expression of water channels. For instance, it is known that estrogens impair brain adaptation to hyponatremia whereas androgens enhance it through respective inhibition and stimulation of Na + -K + -ATPase [77][78][79][80][81][82][83][84]. Furthermore, it is known that estrogen induces AQP1 expression by activating the estrogen-response element in the promoter of the Aqp1 gene during angiogenesis in human breast and endometrial carcinomas [85]. Thus, it is conceivable that in the setting of osmotic stress, estrogens and androgens could have opposite effects on AQP1 and AQP4 expression in female and male patients. If downregulation of AQP1 and AQP4, and/or failure of their upregulation were an androgen-dependent mechanism, this would, on the one hand protect the astrocyte from apoptosis, but also be expected to worsen the osmotic disturbance. This outcome could plausibly explain the higher incidence of CPM observed in the male population [5,16].CPM is also known to occur in association with a multitude of several underlying conditions, with alcoholism, liver transplantation, malnutrition, hepatic cirrhosis, burns and SIAD being among the most common. Although most of these conditions cause changes in serum Na + levels, changes in other serum osmolytes have also been associated with CPM including hypokalemia, hyperglycemia, hypophosphatemia, and correction of hyperammonemia [11,[13][14][15][16]. Furthermore, hypoxia and arginine vasopressin, in addition to estrogen, affects the brain's ability to adapt to cellular edema [39]. Therefore the differences observed in the expression of AQP1 and AQP4 among the human CPM lesions may alternatively be explained by different underlying disease processes that contributed to the development of CPM.

AQP4 in CPM and NMO
Our findings, corroborated by prior studies, justify considering CPM a primary astrocytopathy with secondary demyelination [86][87][88]. Furthermore, we demonstrate for the first time that AQP1 and AQP4 contribute to human CPM pathology and pathogenesis. A primary role for AQP4 has been associated with neuromyelitis optica (NMO), an autoimmune inflammatory astrocytopathy caused by complement-activating IgG autoantibodies directed against AQP4 [88][89][90]. Early active demyelinating lesions in both NMO and a subgroup of CPM patients are characterized by a spectrum of astrocytic damage, AQP4 loss, retention of small GFAP immunoreactive astrocytes with fewer and shorter processes, intramyelinic edema and apoptosis of oligodendrocytes with secondary demyelination. Loss of AQP4 in NMO is caused in part by the internalization of AQP4 by some astrocytes following its interaction with NMO-IgG (antigenic modulation). Complement, when available, is activated by NMO-IgG remaining on internalization-resistant AQP4 [88][89][90][91] expressing astrocytes. In contrast to previously published studies [92], we did not find evidence for either complement activation or AQP4 astrocyte internalization in CPM lesions, suggesting that loss of AQP4 can occur in the absence of complement-activating antibodies. The CPM lesions characterized by AQP4 and AQP1 loss, but preserved GFAP staining of astrocytes in the absence of antibodies, complement activation or AQP4 astrocyte internalization, somewhat resemble the type 6 NMO lesions recently described which show a more pronounced loss of AQP4 and AQP1 than GFAP, and also occur in the absence of immunoglobulin deposition or complement activation [27]. We propose that loss of AQP4 and AQP1 in the setting of CPM may be due to compensatory changes in response to the local osmotic environment, resulting in astrocytic injury as a consequence of the osmotic stress. Type 6 NMO lesions may similarly mirror astrocytic injury caused by osmotic stress due to disturbances of the local osmotic environment either due to a possible direct effect of NMO-IgG on water transport [90] or triggered by astrocytes in nearby regions that are damaged via antibodyand complement-mediated mechanisms [88][89][90][91].

Conclusions
Our findings provide the first evidence that astrocytic AQP1 and AQP4 may be involved in the pathogenesis of CPM. Both are lost in a subset of CPM patients. It remains to be determined whether loss of AQPs in CPM is a protective compensatory mechanism to protect against astrocytic apoptosis. Furthermore, additional studies are needed in order to clarify whether AQP loss is a pathological characteristic of all male patients with CPM, or rather a feature associated with specific underlying causes of CPM. Since studies of autopsied human tissues offer only a single snapshot in disease evolution and since there are major differences between rodent and human astrocytes, non-rodent models are needed in order to better define the complex and dynamic relationship between regulation of AQPs and astrocyte injury in CPM.

Archival material
To characterize AQP1 and AQP4 immunoreactivity and neuropathological characteristics of CPM, we analyzed microscopic transverse sections of the pons in archival autopsied tissue from two control cases (17 year old female and 15 year old male), and six pathologically confirmed CPM cases. Clinical information was obtained from medical records. The study was approved by the Institutional Review Board of the Mayo Clinic, Rochester.

Competing interests
Dr. Popescu served as a speaker for Teva Innovation Canada, and receives research support from the Saskatchewan Health Research Foundation (principal investigator) and the Canada Research Chairs program (principal investigator). Dr. Bunyan reports no disclosures. Dr. Guo reports no disclosures. Dr. Parisi serves on scientific advisory boards for the US Government Defense Health Board and the Subcommittee for Laboratory Services and Pathology; serves as a Section Editor for Neurology; receives royalties from the publication of Principles & Practice of Neuropathology, 2nd ed. (Oxford University Press, 2003); and receives research support from the NIH (NS32352-13; co-investigator). Dr. Lennon is a named inventor on a patent relating to AQP4 as a target of pathogenic autoantibodies in NMO and related disorders and on a pending patent related to AQP4 applications to cancer; has received greater than the federal threshold for significant interest from licensing of this technology; receives no royalties from the sale of Mayo Medical Laboratories' service serological tests; however, Mayo Collaborative Services, Inc., receives revenue for conducting these tests; is named inventor on two patent applications filed by the Mayo Foundation for Medical Education and Research relating to functional assays for detecting NMO/AQP4 antibody; receives research support from the National Institutes of health (NS65829; co-investigator). Dr. Lucchinetti may accrue revenue for a patent re: Aquaporin-4 associated antibodies for diagnosis of neuromyelitis optica; receives royalties from the publication of Blue Books of Neurology: Multiple Sclerosis 3 (Saunders Elsevier, 2010); and receives