Fig 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Longitudinal sections of C57BL/6 mouse (A,B), and Aquaporin 4 null (AQP4 null) mouse (C). Cryopreserved mouse optic nerve head tissues were labeled with anti-AQP4 (green) and DAPI (blue), then divided into 4 regions (A): pre-lamina (PL, from vitreoretinal surface to a line joining the two endpoints of BMO), unmyelinated optic nerve (UON, from BMO to 200 μm posteriorly), myelin transition zone (MTZ, from 200 μm to 350 μm posteriorly), and the myelinated optic nerve (MON, from 350 μm to the end of the section). Minimal label for AQP4 is visible in PL and the anterior portion of the UON in B6 mouse (between white arrows). AQP4 label is prominent in the myelinated portion of optic nerve in each species, as well as in retina. No AQP4 seen in the AQP4 null tissue (C). Scale Bar: 100 μm (A), 50 μm (B,C,E,F), 200 μm (E). https://doi.org/10.1371/journal.pone.0244123.g001
Source publication
Purpose
To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null).
Methods
C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporin...
Contexts in source publication
Context 1
... control and IOP elevation, longitudinal ON sections were obtained. Ki67 positive nuclei within each of the 4 regions denoted above ( Fig 1A) were counted manually to calculate density (positive cells/mm 2 ). Double labeling was performed on some sections to determine whether the Ki67 positive nuclei associated with astrocytes (expression of glial fibrillary acidic protein, GFAP, ten B6 control samples and and ten B6 3D-IOP samples) or with microglia (expression of ionized calcium binding adaptor molecule 1, Iba1, nine AQP4 null control samples and nine AQP4 null 3D-IOP samples). ...
Context 2
... regions were within the unmyelinated portion of the nerve, prior to the MTZ. For each region, measurements were made in 3 zones: 1) a zone of peripheral ON (outer area, S1A Fig); 2) a zone more centrally (inner area); and 3) the total area (S1B Fig). We calculated the mean and median PIV in the overall optic nerve and in the inner and outer zones, as well as in the choroid, the PL, the MTZ and the MON. ...
Context 3
... images of control B6 (Fig 1B) mice demonstrated abundant AQP4 expression in retinal Müller cells, retinal nerve fiber layer astrocytes, and MON astrocytes. Minimal AQP4 labeling was found in the PL and the anterior UON. ...
Context 4
... AQP4 labeling was minimally present in the posterior UON region, and substantially greater in the MTZ and MON. There was no AQP4 labeling in any region of the AQP4 null eyes (Fig 1C). control B6 and AQP4 nulls to quantify the presence of AQP4 throughout the retina and ON (Fig 3). ...
Context 5
... antibody labeling was used to assess axonal transport obstruction in B6 and AQP4 null 3D-IOP eyes, compared to contralateral control eyes, using mean fluorescent intensity, mean intensity of brightest pixels, and fraction of brightest pixels in 4 regions: retina, PL, UON and MON ( Fig 1A). In both B6 and AQP4 null 3D-IOP eyes, there were significant increases in the parameters indicating localized transport obstruction (brightest pixel mean intensity and fraction of brightest pixels), but the increases were not significantly different between the two mouse types (S2 Fig). ...
Citations
... Interestingly, AQP4 is not expressed in the rodent glial lamina or the lamina cribrosa in humans [49,52,53]. We speculate that the lack of AQP4 expression [52,53] aids to maintain the translaminar pressure gradient and restricts the fluid exchange between intraocular and intracranial fluid, which are vital aspects for maintaining fluid homeostasis by keeping IOP relatively isolated from physiological fluctuations in ICP. ...
... Mathieu et al. documenting that intracisternally administered tracers were excluded from entering the eye under physiological conditions, as was more recently replicated independently [51]. Interestingly, AQP4 is not expressed in the rodent glial lamina or the lamina cribrosa in humans [49,52,53]. We speculate that the lack of AQP4 expression [52,53] aids to maintain the translaminar pressure gradient and restricts the fluid exchange between intraocular and intracranial fluid, which are vital aspects for maintaining fluid homeostasis by keeping IOP relatively isolated from physiological fluctuations in ICP. ...
... Interestingly, AQP4 is not expressed in the rodent glial lamina or the lamina cribrosa in humans [49,52,53]. We speculate that the lack of AQP4 expression [52,53] aids to maintain the translaminar pressure gradient and restricts the fluid exchange between intraocular and intracranial fluid, which are vital aspects for maintaining fluid homeostasis by keeping IOP relatively isolated from physiological fluctuations in ICP. We conclude that the AQP4 dependence of the ocular glymphatic flow must be a consequence of the dense perivascular AQP4 expression in the optic nerve. ...
The ocular glymphatic system subserves the bidirectional polarized fluid transport in the optic nerve, whereby cerebrospinal fluid from the brain is directed along periarterial spaces towards the eye, and fluid from the retina is directed along perivenous spaces following upon its axonal transport across the glial lamina. Fluid homeostasis and waste removal are vital for retinal function, making the ocular glymphatic fluid pathway a potential route for targeted manipulation to combat blinding ocular diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Several lines of work investigating the bidirectional ocular glymphatic transport with varying methodologies have developed diverging mechanistic models, which has created some confusion about how ocular glymphatic transport should be defined. In this review, we provide a comprehensive summary of the current understanding of the ocular glymphatic system, aiming to address misconceptions and foster a cohesive understanding of the topic.
... The existence of a glymphatic system is controversial [13,35] and it is not known how AQP4, a channel that transports water in response to osmotic gradients, might regulate transport of large molecules such as dextrans and antibodies. It has been proposed that AQP4 regulates transport from the eye to the optic nerve via perivascular spaces as part of the ocular glymphatic system [8], however the absence of AQP4 expression in astrocytes of the optic nerve head [14,36] demonstrates that AQP4 is not necessary for fluid movement through this area. We did not find any evidence that AQP4 regulates transport of dextrans from the CSF into the optic nerve, however we have not directly tested the possibility that AQP4 regulates fluid transport along the optic nerve, between the optic nerve fibers [8]. ...
It has been proposed that cerebrospinal fluid (CSF) can enter and leave the retina and optic nerve along perivascular spaces surrounding the central retinal vessels as part of an aquaporin-4 (AQP4) dependent ocular ‘glymphatic’ system. Here, we injected fluorescent dextrans and antibodies into the CSF of mice at the cisterna magna and measured their distribution in the optic nerve and retina. We found that uptake of dextrans in the perivascular spaces and parenchyma of the optic nerve is highly sensitive to the cisternal injection rate, where high injection rates, in which dextran disperses fully in the sub-arachnoid space, led to uptake along the full length of the optic nerve. Accumulation of dextrans in the optic nerve did not differ significantly in wild-type and AQP4 knockout mice. Dextrans did not enter the retina, even when intracranial pressure was greatly increased over intraocular pressure. However, elevation of intraocular pressure reduced accumulation of fluorescent dextrans in the optic nerve head, and intravitreally injected dextrans left the retina via perivascular spaces surrounding the central retinal vessels. Human IgG distributed throughout the perivascular and parenchymal areas of the optic nerve to a similar extent as dextran following cisternal injection. However, uptake of a cisternally injected AQP4-IgG antibody, derived from a seropositive neuromyelitis optica spectrum disorder subject, was limited by AQP4 binding. We conclude that large molecules injected in the CSF can accumulate along the length of the optic nerve if they are fully dispersed in the optic nerve sub-arachnoid space but that they do not enter the retina.
... The injection of microbeads into the anterior chamber produces IOP elevation known to cause RGC death that is maximal by six weeks [23] (Figures 4A and S4, Table S1). Mice exposed to elevated IOP followed prior experience with bead injection [23,24], having significant mean IOP elevation at three days, decreasing at two weeks, and with minimal difference from baseline at six weeks ( Figure S4, Table S1). Micro-dissected UON, MON, and retinal tissues were collected at three days (early, 3 D), two weeks (middle, 2 W), and six weeks (late, 6 W) post-injection to characterize gene expression changes spanning the time course of this model ( Figure 4A). ...
... An important pathway common between UON and MON in our analysis was axon guidance. In the mouse UON, the vast majority of cells locally generating RNA are astrocytes [24]. The major cellular content difference between UON and MON is the presence of oligodendrocytes in the latter region. ...
... The MON had more increases in cell cycle genes. Both astrocytes and microglia proliferate in the mouse glaucoma model [24]. In both mouse and rat ocular hypertension models, there was a loss of oligodendrocytes in the MON, and oligodendrocyte precursor cells proliferated, while activation of microglia was detected only in advanced damaged nerves [36]. ...
A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor β pathways, as well as extracellular matrix–receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.
... C1 and C12 were both most abundant in the optic nerve and expressed AQP4 at high levels ( Fig. 3C and D). They were present at far higher levels in the myelinated parts of the ON than ONH (Fig. S2B), consistent with a previous report on AQP4 expression (37). Despite similarities in their transcriptomic profile and tissue distribution, they were molecularly distinct; C1 selectively expressed SLC14A1 and SOX9, whereas C12 expressed higher levels of GAP43 and CHI3L1 (Fig. 3C). ...
Although the visual system extends through the brain, most vision loss originates from defects in the eye. Its central element is the neural retina, which senses light, processes visual signals, and transmits them to the rest of the brain through the optic nerve (ON). Surrounding the retina are numerous other structures, conventionally divided into anterior and posterior segments. Here we used high-throughput single nucleus RNA sequencing (snRNA-seq) to classify and characterize cells in the extraretinal components of the posterior segment: ON, optic nerve head (ONH), peripheral sclera, peripapillary sclera (PPS), choroid, and retinal pigment epithelium (RPE). Defects in each of these tissues are associated with blinding diseases, for example, glaucoma (ONH and PPS), optic neuritis (ON), retinitis pigmentosa (RPE), and age-related macular degeneration (RPE and choroid). From ~151,000 single nuclei, we identified 37 transcriptomically distinct cell types, including multiple types of astrocytes, oligodendrocytes, fibroblasts, and vascular endothelial cells. Our analyses revealed a differential distribution of many cell types among distinct structures. Together with our previous analyses of the anterior segment and retina, the new data complete a Version 1 cell atlas of the human eye. We used this atlas to map the expression of >180 genes associated with the risk of developing glaucoma, which is known to involve ocular tissues in both anterior and posterior segments as well as neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable.
... AQP4 expression can also be found in the rodent optic nerves (Mathieu et al., 2017(Mathieu et al., , 2018Kimball et al., 2021), while chronic intraocular pressure elevation may not only accompany with altered AQP4 expression (Dibas et al., 2008), but also reduced cerebrospinal fluid entry into the optic nerve (Mathieu et al., 2018;Faiq et al., 2021b). In addition, knocking out AQP4 may reduce amyloid clearance from the optic nerve (Wang et al., 2020). ...
... It is worth noting that difference between sexes exists in Alzheimer's disease (Mielke, 2018) while there is a reported spike in the incidence of glaucoma in postmenopausal women (Dewundara et al., 2016). On the other hand, rodents do not have lamina cribrosa and their AQP4 expression in the optic nerve is different from that of humans (Kimball et al., 2021). Thus, cautions are warranted in the context of direct clinical translations of the current results. ...
Abstract
Central insulin resistance, the diminished cellular sensitivity to insulin in the brain, has been implicated in diabetes mellitus, Alzheimer's disease and other neurolagical disorders. However, whether and how central insulin resistance plays a role in the eye remains unclear. Here, we performed intracerebroventricular injection of S961, a potent and specific blocker of insulin receptor in adult Wistar rats to test if central insulin resistance leads to pathological changes in ocular structures. 80 mg of S961 was stereotaxically injected into the lateral ventricle of the experimental group twice at 7 days apart, whereas buffer solution was injected to the sham control group. Blood samples, intraocular pressure, trabecular meshwork morphology, ciliary body markers, retinal and optic nerve integrity, and whole genome expression patterns were then evaluated. While neither blood glucose nor serum insulin level was significantly altered in the experimental or control group, we found that injection of S961 but not buffer solution significantly increased intraocular pressure at 14 and 24 days after first injection, along with reduced porosity and aquaporin 4 expression in the trabecular meshwork, and increased tumor necrosis factor a and aquaporin 4 expression in the ciliary body. In the retina, cell density and insulin receptor expression decreased in the retinal ganglion cell layer upon S961 injection. Fundus photography revealed peripapillary atrophy with vascular dysregulation in the experimental group. These retinal changes were accompanied by upregulation of pro-inflammatory and pro-apoptotic genes, downregulation of anti-inflammatory, antiapoptotic, and neurotrophic genes, as well as dysregulation of genes involved in insulin signaling. Optic nerve histology indicated microglial activation and changes in the expression of glial fibrillary acidic protein, tumor necrosis factor a, and aquaporin 4. Molecular pathway architecture of the retina revealed the three most significant pathways involved being inflammation/ cell stress, insulin signaling, and extracellular matrix regulation relevant to neurodegeneration. There was also a multimodal crosstalk between insulin signaling derangement and inflammation-related genes. Taken together, our results indicate that blocking insulin receptor signaling in the central nervous system can lead to trabecular meshwork and ciliary body dysfunction, intraocular pressure elevation, as well as inflammation, glial activation, and apoptosis in the retina and optic nerve. Given that central insulin resistance my lead to neurodegenerative phenotype in the visual system, targeting insulin signaling may hold promise for vision disorders involving the retina and optic nerve.
... While Immunolabeling of human secondary glaucoma eyes found reduced AQP9 in the retina, the presence or change in AQP4 in the ONH itself was not demonstrated [38]. We exposed C57BL/6 and AQP4 knock out mice to bead-induced intraocular pressure (IOP) elevation for 3 days, 1 and 6 weeks [39]. Wild type mice had abundant AQP4 expression in Müller cells, astrocytes of the retina and myelinated optic nerve, but minimal AQP4 in prelaminar and unmyelinated optic nerve by immunolabeling and gene expression, despite the presence of the DG complex. ...
... We used our published method for semi-quantifying fluorescence [39] using the pixel intensity value (PIV), calculated using FIJI (ImageJ, Bethesda MD). Longitudinal, cryopreserved porcine sections from 4 samples immunolabeled against AQP4 were imaged using the same acquisition settings. ...
... Our results reported here showing the lack of significant expression of AQP4 in the lamina cribrosa of human and porcine eyes is consistent with our previous findings with mise and rats and suggests the more universal finding, that these channels are likely not present in the equivalent of the lamina cribrosa in any mammalian eye. The minimal presence of AQP4 at the large animal lamina and the unmyelinated nerve of rodent eyes is highly evolutionarily conserved and therefore may be either advantageous for normal ONH function, and/or a protective influence for retinal ganglion cell axons from glaucoma damage [39]. There are several possible hypotheses for the potential benefit from this regional astrocytic characteristic. ...
Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, myelin transition zone (MTZ, region just posterior to lamina) and myelinated ON (MON). Semi- quantification of AQP4 labeling & real-time quantitative PCR (qPCR) data were analyzed in retina and ON tissue. Porcine and control human eyes had abundant AQP4 in Müller cells, retinal astrocytes, and myelinated ON (MON), but minimal expression in the lamina cribrosa. AQP1 and AQP9 were present in retina, but not in the lamina. Immunolabeling of GFAP and αDG was similar in lamina, myelin transition zone (MTZ) and MON regions. Semi-quantitative AQP4 labeling was at background level in lamina, increasing in the MTZ, and highest in the MON (lamina vs MTZ, MON; p≤0.05, p≤0.01, respectively). Expression of AQP4 mRNA was minimal in lamina and substantial in MTZ and MON, while GFAP mRNA expression was uniform among the lamina, MTZ, and MON regions. Western blot assay showed AQP4 protein expression in the MON samples, but none was detected in the lamina tissue. The minimal presence of AQP4 in the lamina is a specific regional phenotype of astrocytes in the mammalian optic nerve head.
... ONH astrocytes are a unique group of astrocytes, specifically residing within the unmyelinated portion of the optic nerve (Choi et al., 2015;Kimball et al., 2021). Primary astrocytes were isolated and cultured from ONH tissue from 6 to 8 weeks old C57BL/6J mice, and cell purity was confirmed as previously described (Suppl. ...
... Cells were immunoreactive for the astrocyte marker GFAP, and negative for oligodendrocyte marker OSP and microglial/macrophage marker F4/80. ONH astrocytes, in contrast to astrocytes within the myelinated portion of the nerve, do not express aquaporin 4 (AQP4) (Kimball et al., 2021). Thus, we tested immunoreactivity for AQP4, which was negative. ...
In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF β2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF β2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF β2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF β2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury.
Although the visual system extends through the brain, most vision loss originates from defects in the eye. Its central element is the neural retina, which senses light, processes visual signals, and transmits them to the rest of the brain through the optic nerve (ON). Surrounding the retina are numerous other structures, conventionally divided into anterior and posterior segments. Here, we used high-throughput single-nucleus RNA sequencing (snRNA-seq) to classify and characterize cells in six extraretinal components of the posterior segment: ON, optic nerve head (ONH), peripheral sclera, peripapillary sclera (PPS), choroid, and retinal pigment epithelium (RPE). Defects in each of these tissues are associated with blinding diseases-for example, glaucoma (ONH and PPS), optic neuritis (ON), retinitis pigmentosa (RPE), and age-related macular degeneration (RPE and choroid). From ~151,000 single nuclei, we identified 37 transcriptomically distinct cell types, including multiple types of astrocytes, oligodendrocytes, fibroblasts, and vascular endothelial cells. Our analyses revealed a differential distribution of many cell types among distinct structures. Together with our previous analyses of the anterior segment and retina, the data presented here complete a "Version 1" cell atlas of the human eye. We used this atlas to map the expression of >180 genes associated with the risk of developing glaucoma, which is known to involve ocular tissues in both anterior and posterior segments as well as the neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable.
A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and in human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and on separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor β pathways, as well as extracellular matrix–receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.
