Anatomy and Neuroscience - Research Publications
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ItemNo Preview AvailableReversibility of retinal ganglion cell dysfunction due to chronic IOP elevation.(Association for Research in Vision and Ophthalmology, 2018-07-01)Purpose : To determine the duration of chronic IOP elevation beyond which ganglion cell function can no longer recover using the mouse circumlimbal suture model. Methods : IOP elevation was induced in anaesthetized (isoflurane) adult male C57BL6/J mice by attaching a circumlimbal suture (nylon, 10/0) around the equator of one eye, with the contralateral eye serving as a control. The suture was left in place for 8, 12 and 16 weeks (n=27, 23 and 27), respectively, and animals underwent electroretinography and optical coherence tomography at these time points. In two other groups, the suture was removed after 8 and 12 weeks (n=26 and 28), and the capacity for recovery assessed 4 weeks later. IOP was measured weekly (Tonolab). Retinal ganglion cell (RGC) function (or integrity) was assessed with the positive scotopic threshold response (pSTR) and retinal nerve fibre layer (RNFL) thickness. Data (mean Ā± SEM) were compared using t-test (control vs. treatment) and one-way ANOVA (within groups). Results : IOP in sutured eyes was higher than control eyes (8wk: 17.1 Ā± 0.3 vs. 26.8 Ā± 0.6 mmHg, 12wk: 13.8 Ā± 0.3 vs. 19.5 Ā± 0.5 mmHg, 16wk: 17.1 Ā± 0.2 vs. 27.4 Ā± 0.6 mmHg; all P<0.001). After suture removal, IOP returned to levels comparable to control eyes (8+4wk: 16.9 Ā± 0.3 vs. 16.1 Ā± 0.3 mmHg; P=0.08, 12+4wk: 17.3 Ā± 0.2 vs. 17.1 Ā± 0.3 mmHg; P=0.5). With IOP elevation, RGC function declined to 75% Ā± 8% (8wk), 78% Ā± 7% (12wk) and 59% Ā± 4% (16wk, all P<0.001) of control eyes. RNFL thinning was also evident (8wk: 84% Ā± 4%, 12wk: 83% Ā± 5%; 16wk: 83% Ā± 3%; P<0.001) but no change in total retinal thickness was noted (P=0.33). Suture removal at week 8 facilitated full recovery of RGC function (97% Ā± 7%, P=0.9 vs. baseline) 4 weeks later. However, there was no recovery in RNFL thickness (87% Ā± 3%, P<0.001 vs. baseline). When the suture was removed at week 12, neither function (79% Ā± 9%, P<0.05) nor RNFL thickness recovered (89% Ā± 3%, P<0.01) 4 weeks later. Conclusions : RGC dysfunction can be recovered 4 weeks after an 8-week period of mild IOP elevation, but not after a 12-week period. Beyond 12 weeks, IOP reversal only served to prevent further functional decline. This identifies a critical chronic IOP duration that results in irreversible ganglion cell dysfunction. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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ItemNo Preview AvailableSelective retinal ganglion cell vulnerability in older mice exposed to acute intraocular pressure elevation and the potential involvement of the P2X7-receptor(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : P2X7-receptors may contribute to retinal ganglion cell (RGC) death in glaucoma. We examined RGC function following acute intraocular pressure (IOP) elevation in older C57BL/6 (WT) mice and P2X7-receptor knockout (P2X7-KO) mice using a multielectrode array (MEA). Methods : In 13-month-old WT (n=15) and P2X7-KO mice (n=9), the anterior chamber of one eye was cannulated (50Ī¼m glass micropipette connected to a height-adjustable Hanks balanced salt solution reservoir) to increase IOP to 50 mmHg for 30 minutes. The contralateral eye was cannulated without increasing IOP (sham). Three days following injury, mice were dark-adapted over-night and retinae were mounted onto an MEA to record RGC spontaneous activityand light-evoked responses. Full field stimuli were 1 second flashes modulated between 0 and 1066 photoisomerisations/rod/sec. To test frequency responsiveness, full field light ON and OFF components were modulated from 1 to 30 Hz. Receptive fields were mapped by calculating the spike triggered average in response to a 32x32 checkerboard stimulus (70Āµm squares) presented at 12 Hz, with mean luminance of 517 photoisomerations/rod/sec. Cells were analyzed and sorted using Spike2 and classified into ON, OFF, ON-OFF and non-responsive types based on peak firing during light on and off full-field stimuli. Results : In WT mice there was a significant reduction in spontaneous activity (p<0.05) and full-field-evoked spike rates (p<0.05) for OFF RGCs after IOP stress compared to OFF cells of sham eyes. These changes appear to be subtype-specific as ON and ON-OFF cells showed no change in response. There were no further effects of IOP at higher temporal frequencies of full field stimulus, nor were there changes in receptive field size. In P2X7-KO mice, OFF RGCs in IOP stressed eyes showed significantly reduced spontaneous rate (p<0.05) compared to OFF RGCs in WT sham eyes, much like the effect of IOP stress on WT OFF cells. Additionally, ON RGCs from P2X7-KO eyes subjected to IOP stress showed a significant decrease in peak spike rate compared to P2X7-KO sham eyes (p<0.05). Conclusions : These results suggest that even a short period of mild IOP stress can have long lasting effect on RGC function, particularly that of OFF-RGCs. In contrast to previous studies, P2X7-KO did not prevent RGC functional deficits associated with acute mild IOP elevation.
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ItemNo Preview AvailableFractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy( 2020)Local blood flow control within the CNS is critical to proper function and is dependent on coordination between neurons, glia and blood vessels. Macroglia such as astrocytes and MĆ¼ller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial involvement, since mice lacking Cx3cr1, exhibited no fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan, abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 weeks due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.
Significance Statement
This work identifies a novel role for microglia, the innate immune cells of the CNS, in the local control of the retinal vasculature and identifies deficits early in diabetes. Microglia contact neurons and vasculature and express several vasoactive agents. Activation of microglial fractalkine-Cx3cr1 signalling leads to capillary constriction and blocking the renin-angiotensin system (RAS) with candesartan abolishes microglial-mediated vasoconstriction in the retina. In early diabetes, reduced retinal blood flow is coincident with capillary constriction, increased microglial-vessel association, loss of microglial-capillary regulation and altered microglial expression of the RAS pathway. While candesartan restores retinal capillary diameter early in diabetes, targeting of microglial-vascular regulation is required to prevent coincident dilation of large retinal vessels and reduced retinal blood flow. -
ItemFractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy(NATL ACAD SCIENCES, 2021-12-21)Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and MĆ¼ller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.
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ItemReversibility of Retinal Ganglion Cell Dysfunction From Chronic IOP Elevation(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2019-09)PURPOSE: To test the hypothesis that the capacity for retinal ganglion cells to functionally recover from chronic IOP elevation is dependent on the duration of IOP elevation. METHODS: IOP elevation was induced in one eye in anesthetized (isoflurane) adult C57BL6/J mice using a circumlimbal suture. Sutures were left in place for 8 and 16 weeks (n = 30 and 28). In two other groups the suture was cut after 8 and 12 weeks (n = 30 and 28), and ganglion cell function (electroretinography) and retinal structure (optical coherence tomography) were assessed 4 weeks later. Ganglion cell density was quantified by counting RBPMS (RNA-binding protein with multiple splicing)-stained cells. RESULTS: With IOP elevation (ā¼10 mm Hg above baseline), ganglion cell function declined to 75% Ā± 8% at 8 weeks and 59% Ā± 4% at 16 weeks relative to contralateral control eyes. The retinal nerve fiber layer was thinner at 8 (84% Ā± 4%) and 16 weeks (83% Ā± 3%), without a significant difference in total retinal thickness. Ganglion cell function recovered with IOP normalization (suture removal) at week 8 (97% Ā± 7%), but not at week 12 (73% Ā± 6%). Ganglion cell loss was found in all groups (-8% to -13%). CONCLUSIONS: In the mouse circumlimbal suture model, 12 weeks of IOP elevation resulted in irreversible ganglion cell dysfunction, whereas retinal dysfunction was fully reversible after 8 weeks of IOP elevation.
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ItemRetinal ganglion cell dysfunction in mice following acute intraocular pressure is exacerbated by P2X7 receptor knockout(NATURE PORTFOLIO, 2021-02-18)There is increasing evidence for the vulnerability of specific retinal ganglion cell (RGC) types in those with glaucoma and in animal models. In addition, the P2X7-receptor (P2X7-R) has been suggested to contribute to RGC death following stimulation and elevated IOP, though its role in RGC dysfunction prior to death has not been examined. Therefore, we examined the effect of an acute, non-ischemic intraocular pressure (IOP) insult (50 mmHg for 30 min) on RGC function in wildtype mice and P2X7-R knockout (P2X7-KO) mice. We examined retinal function using electroretinogram recordings and individual RGC responses using multielectrode arrays, 3 days following acute IOP elevation. Immunohistochemistry was used to examine RGC cell death and P2X7-R expression in several RGC types. Acute intraocular pressure elevation produced pronounced dysfunction in RGCs; whilst other retinal neuronal responses showed lesser changes. Dysfunction at 3 days post-injury was not associated with RGC loss or changes in receptive field size. However, in wildtype animals, OFF-RGCs showed reduced spontaneous and light-elicited activity. In the P2X7-KO, both ON- and OFF-RGC light-elicited responses were reduced. Expression of P2X7-R in wildtype ON-RGC dendrites was higher than in other RGC types. In conclusion, OFF-RGCs were vulnerable to acute IOP elevation and their dysfunction was not rescued by genetic ablation of P2X7-R. Indeed, knockout of P2X7-R also caused ON-RGC dysfunction. These findings aid our understanding of how pressure affects RGC function and suggest treatments targeting the P2X7-R need to be carefully considered.
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ItemCharacterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.(Frontiers Media SA, 2017)To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 Ā± 3 mmHg. After 1 day, IOP had recovered to 27 Ā± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 Ā± 1 mmHg, ocular hypertensive 19 Ā± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (-15 Ā± 4%), bipolar cell (-15 Ā± 4%) and ganglion cell responses (-19 Ā± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior -12 Ā± 5%; temporal, -7% Ā± 2%; inferior -9 Ā± 4%; nasal -8 Ā± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 Ā± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.
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ItemRelationship between the Magnitude of Intraocular Pressure during an Episode of Acute Elevation and Retinal Damage Four Weeks later in Rats(PUBLIC LIBRARY SCIENCE, 2013-07-29)PURPOSE: To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats. METHODS: Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; nā=ā4 to 8 each, 10-100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (-6.04 to 2.72 log cd.s.m(-2)). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m(-2)) after 15 min of light adaptation (150 cd/m(2)). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology. RESULTS: All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density. CONCLUSIONS: Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a 'threshold' for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more.