Detection and characterization of tree shrew retinal venous pulsations: An animal model to study human retinal venous pulsations

Experimental Eye Research 185 (2019) 107689

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Detection and characterization of tree shrew retinal venous pulsations: An animal model to study human retinal venous pulsations

T

Michael Dattiloa,b, A. Thomas Readb, Brian C. Samuelsc, C. Ross Ethiera,b,∗ a

Department of Ophthalmology, Emory University School of Medicine, 1365-B Clifton Road, Atlanta, 30322, GA, USA Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive NW, Atlanta, 30332, GA, USA c Department of Ophthalmology, University of Alabama at Birmingham School of Medicine, 1670 University Boulevard, Birmingham, 35294, AL, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Spontaneous retinal venous pulsations Intracranial pressure Intraocular pressure Translaminar pressure gradient Tree shrews

Spontaneous retinal venous pulsations (SRVPs), pulsations of branches of the central retinal vein, are affected by intraocular pressure (IOP) and intracranial pressure (ICP) and thus convey potentially-useful information about ICP. However, the exact relationship between SRVPs, IOP, and ICP is unknown. It is not easily feasible to study this relationship in humans, necessitating the use of an animal model. We here propose tree shrews as a suitable animal model to study the complex relationship between SRVPs, IOP, and ICP. Tree shrew SRVP incidence was determined in a population of animals. Following validation of a modified IOP control system to accurately and quickly control IOP, IOP and/or ICP were manipulated in two tree shrews with SRVPs and the effects on SRVP properties were quantified. SRVPs were present in 75% of tree shrews at physiologic IOP and ICP. Altering IOP or ICP produced changes in tree shrew SRVP properties; specifically, increasing IOP caused SRVP amplitude to increase, while increasing ICP caused SRVP amplitude to decrease. In addition, a higher IOP was necessary to generate SRVPs at a higher ICP than at a lower ICP. SRVPs occur with a similar incidence in tree shrews as in humans, and tree shrew SRVPs are affected by changes in IOP and ICP in a manner qualitatively similar to that reported in humans. In view of anatomic similarities, tree shrews are a promising animal model system to further study the complex relationship between SRVPs, IOP, and ICP.

1. Introduction Alterations in intracranial pressure (ICP) are implicated in the pathogenesis of a number of life- and vision-threatening conditions, such as idiopathic intracranial hypertension (Burkett and Ailani, 2018; Thurtell et al., 2010; Wall, 2000), certain forms of glaucoma (Berdahl et al., 2008a, 2008b; Ren et al., 2010), traumatic brain injury (Dawes et al., 2015; Godoy et al., 2018; Miller et al., 1977), and intracranial hemorrhages (Hasan et al., 1991; Zoerle et al., 2015). Therefore, measurement of ICP is necessary and common for diagnosis and management in these situations (Vickers et al., 2017). However, all current methods used clinically to measure ICP are invasive and require direct cerebrospinal fluid access (Zhang et al., 2017). Although lumbar puncture is the least invasive of these procedures, it carries the risk of significant side effects (Adler et al., 2001; Alstadhaug et al., 2012; Del-

Rio-Vellosillo et al., 2017; Egede et al., 1999; Oliver et al., 2003; Verslegers et al., 2017) and is contraindicated in certain situations (Engelborghs et al., 2017; Holdgate and Cuthbert, 2001). Therefore, a clear need exists for a non-invasive method to accurately measure and monitor ICP. Accordingly, a number of modalities have been proposed to noninvasively measure ICP (Zhang et al., 2017; Bruce, 2014; Kristiansson et al., 2013; Raboel et al., 2012), such as transcranial Doppler ultrasonography (Schmidt et al., 2003), measurement of otoacoustic emission (Bershad et al., 2014), and imaging of changes in optic nerve sheath diameter (Strumwasser et al., 2011). However, none of these methods are in routine clinical use, largely due to concerns about their accuracy (Zhang et al., 2017; Kristiansson et al., 2013; Raboel et al., 2012), which are a consequence of assumptions necessarily made about the mechanical properties of tissues. Such properties can vary widely

Abbreviations: AC, Anterior chamber; CRV, Central retinal vein; ICP, Intracranial pressure; IOP, Intraocular pressure; IPCON, Intraocular pressure control system; mIPCON, modified intraocular pressure control system; OCT, optical coherence tomography; ONH, optic nerve head; PE, polyethylene; RNFL, Retinal nerve fiber layer; SD-OCT, Spectral domain optical coherence tomography; SRVP, spontaneous retinal venous pulsation; TLPD, Translaminar pressure difference ∗ Corresponding author. Georgia Institute of Technology, USA. E-mail addresses: [email protected] (M. Dattilo), [email protected] (A.T. Read), [email protected] (B.C. Samuels), [email protected] (C.R. Ethier). https://doi.org/10.1016/j.exer.2019.06.003 Received 24 March 2019; Received in revised form 15 May 2019; Accepted 4 June 2019 Available online 06 June 2019 0014-4835/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Schematic depicting forces exerted on the central retinal vein (CRV) at the lamina cribrosa. At the lamina cribrosa (LC), the CRV is exposed to both intracranial pressure (ICP) and intraocular pressure (IOP). The difference between IOP and ICP, i.e. the translaminar pressure difference, is believed to be responsible for spontaneous retinal venous pulsation (SRVP) generation. Adapted from Hayreh SS, Eye 2004; 18:1188 1206.

obtained ICP, making the use of SRVPs to non-invasively determine ICP of little clinical utility. The poor correlation between invasively measured ICP and the ICP predicted by observation of SRVPs is likely due, in part, to incomplete understanding of the relationship between SRVPs, IOP, and ICP. Unfortunately, there are unique challenges in studying SRVPs in humans; specifically, it is not feasible to easily and accurately manipulate and record IOP and ICP in humans (Golzan et al., 2011; Feldman et al., 1992; Schwarz et al., 2002). However, it is possible to easily and accurately control and record both IOP and ICP in animal model systems. Therefore, in order to further understand the relationship between SRVPs, IOP, and ICP, it is necessary to use an animal model. The ideal model should have orbital and ocular anatomies similar to humans and should have spontaneously occurring SRVPs with properties similar to those of human SRVPs. Tree shrews (Tupaia belangeri) are small para-primates, closely related to humans phylogenetically (Miller et al., 2007; Murphy et al., 2007). In addition, tree shrews share similar orbital and ocular anatomies with humans (Zhan et al., 2015), which is in contrast to most lower order species, such as mice, rats, and rabbits. However, SRVPs have not been previously reported or studied in tree shrews. Here we show that tree shrews exhibit SRVPs at physiologic IOP and ICP. In addition, we describe the effects of altering IOP and ICP on tree shrew SRVP properties and compare them to the reported effects of IOP and ICP on human SRVPs.

between individuals, leading to appreciable inter-subject variability. One proposed approach to non-invasively measure ICP leverages a unique property of the central retinal vein (CRV), namely spontaneous retinal venous pulsations (SRVPs), which occur on the CRV at or near the optic nerve head (ONH). SRVPs are believed to be caused by an interaction between venous blood pressure, intraocular pressure (IOP), and ICP at the lamina cribrosa (Jacks and Miller, 2003; Morgan et al., 2016; Levine and Bebie, 2016), and thus depend on the translaminar pressure difference (TLPD; TLPD = IOP – ICP; Fig. 1). This mechanism is supported by a variety of observations. For example, alterations in IOP (Jacks and Miller, 2003; Levin, 1978; Walsh et al., 1969) and ICP (Legler and Jonas, 2009; Seo et al., 2012; Abegao Pinto et al., 2013; Lee et al., 2017; Morgan et al., 2004) are well-known to affect SRVP characteristics, such as SRVP incidence (Jacks and Miller, 2003; Levin, 1978; Walsh et al., 1969; Legler and Jonas, 2009; Seo et al., 2012; Abegao Pinto et al., 2013; Morgan et al., 2004) and amplitude (Golzan et al., 2011, 2015; Donnelly and Subramanian, 2009). Similarly, alterations in ICP have been reported to affect the venous pulse pressure (Morgan et al., 2008; Motschmann et al., 2001; Querfurth et al., 2010), the IOP necessary to generate SRVPs at a given ICP. Despite this information, the exact relationship between SRVPs, IOP, and ICP remains uncertain. Although the quantitative relationship between SRVPs, IOP, and ICP is not known, SRVPs have been used clinically for > 40 years to qualitatively assess ICP status, with the presence of SRVPs suggesting normal ICP (Jacks and Miller, 2003; Levin, 1978; Walsh et al., 1969). In addition, a number of researchers have attempted to use SRVPs to noninvasively measure ICP (Motschmann et al., 2001; Querfurth et al., 2010; Firsching et al., 2000; Jonas et al., 2008). However, these studies have shown varying correlations between predicted ICP and invasively

2. Methods Prior to performing in vivo experiments in tree shrews, we validated a modified version of the IOP Control (IPCON) system previously

Fig. 2. Schematic of the modified intraocular pressure control system (mIPCON). A computer-controlled syringe pump controlled intraocular pressure (IOP) by injecting or withdrawing fluid from the anterior chamber of an eye via a 30-gauge needle inserted near the corneal limbus. 2

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described by Stockslager et al. (2016) The modified IPCON (mIPCON) system (Fig. 2) was first validated in a model eye and then in an ex vivo experiment using an enucleated rat eye. A rat eye was chosen for the ex vivo validation experiments because enucleated tree shrew eyes were not readily available or easily obtainable for ex vivo experimentation, while the size and reported ocular biomechanical properties of the rat eye (Ficarrotta et al., 2018) were closer than mouse or pig eyes to the size and reported ocular biomechanical properties of tree shrew eyes (Stockslager et al., 2016). 2.1. IOP control The mIPCON system (Fig. 2) was simplified from a 2-needle (Stockslager et al., 2016) to a 1-needle system to facilitate cannulation of the tree shrew anterior chamber (AC) and to allow for easier visualization of the tree shrew retinal vasculature. In the modified system, a 1000 μL glass syringe (Hamilton Company, Reno, NV) was actuated by a Harvard Apparatus PHD Ultra syringe pump (Harvard Apparatus, Holliston, MA) to control IOP. The syringe was connected in parallel to a calibrated Honeywell 142PC01G pressure transducer (Newark element, Chicago, IL) to record IOP in real time. The syringe pump was controlled using a custom written LabVIEW program (National Instruments, Austin, TX) on a Samsung tablet (4 GB RAM, 64-bit, 1.6 GHz IntelCore i5). Syringe pump commands were sent at 10 Hz via USB. Pressure transducer data was acquired at 10 Hz using a 12-bit USB-6008 DAQ (National Instruments, Austin, TX). Pressure transducer data was transferred to a Hewlett-Packard Laptop (8 GB RAM, 64-bit, 2.4 GHz IntelCore i3) running MATLAB 2017b (MathWorks, Natick, MA) and Microsoft Excel 365 ProPlus (Microsoft, Redmond, WA) for data analysis. Fluid connections were made using semi-rigid polyethylene tubing (PE-100) with an inner diameter of 0.86 mm (Fischer Scientific, Pittsburgh, PA) and three-way Luer connectors (Fischer Scientific, Pittsburgh, PA). The syringe pump and pressure transducer were connected to a single piece of PE-100 tubing, which was either directly connected to a “model eye” or to a 30-gauge beveled needle for AC cannulation. Two adjustable-height fluid reservoirs (25 mL each; Fischer Scientific, Pittsburgh, PA) were used to calibrate the pressure transducer or refill the glass syringe. For all experiments, the fluid reservoirs and fluid connections were filled with Dulbecco's phosphate buffered saline (Fischer Scientific, Pittsburgh, PA) with added 5.5 mM glucose.

Fig. 3. Comparison of IOP and flow rate measurements in a model eye that replicates relevant properties of a tree shrew eye. The plot shows IOP versus flow rate data obtained in the model eye using the mIPCON system (black dots) and the iPerfusion system (red dots). The slope of the line represents the outflow facility. The outflow facility was 78.1 nL/min/mmHg for the mIPCON system and was 79.4 nL/min/mmHg for the iPerfusion system. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.2.2. Ex vivo IOP control Prior to performing in vivo tree shrew studies, we used an enucleated rat eye to determine the speed and accuracy of IOP control with the mIPCON system. Similar to the study by Stockslager et al. (2016) where the IPCON system was first validated, the enucleated eye was immersed up to the limbus in phosphate buffered saline plus 5 mM glucose and the AC was cannulated at the limbus with a 30-gauge needle attached to the mIPCON system. The cornea was covered by a piece of phosphate buffered saline-soaked gauze to prevent corneal dehydration. IOP was set at 10 mmHg and increased in increments of 5 mmHg to a maximum of 30 mmHg. The syringe pump flow rate was controlled via proportional feedback control. The IOP measured by the pressure transducer was compared to target IOP. The mIPCON system offered very accurate and precise IOP control with an IOP settling time of 1.80 ± 0.38 s and average IOP steady-state errors of < 0.1 mmHg (Fig. 4), similar to the reported IOP settling time (1.23 ± 0.35 s) and IOP steady-state errors (< 0.1 mmHg) reported by Stockslager et al. for the dual needle system (Stockslager et al., 2016).

2.2. Validation of the mIPCON system 2.2.1. Model eye A “model eye” mimicking key ocular biomechanical properties of a true eye was constructed from a piece of compliant Tygon tubing (length 430 mm, inner diameter 3.15 mm; Fischer Scientific, Pittsburgh, PA) and a glass capillary tube (length 70 mm, inner diameter 0.4 mm; Vitrocom, Mountain Lakes, NJ) connected in parallel with a three-way Luer connector. IOP versus flow rate data was obtained from the model eye with the mIPCON system and then with the iPerfusion system, a well-established, high-accuracy standard system for intraocular perfusion and ocular biomechanical measurements (Madekurozwa et al., 2017; Sherwood et al., 2016). For the mIPCON measurements, flow rate was initially set to 500 nL/min and increased in 500 nL/min increments to 2500 nL/min. The steady-state IOP was recorded for each flow rate. For iPerfusion measurements, the IOP was initially set to 5 mmHg and increased in 5 mmHg increments to 25 mmHg. The flow rate at each IOP was recorded. The measured outflow facility (the slope of the flow rate versus IOP plot) was consistent between the two systems (mIPCON: 78.1 nL/min/mmHg, 95% CI [77.4, 78.4]; iPerfusion: 79.4 nL/min/ mmHg, 95% CI [78.0, 81.2]; p = 0.81), confirming the accuracy of the mIPCON system to measure and study ocular biomechanics under steady conditions (Fig. 3).

Fig. 4. IOP control using the mIPCON system. Prior to performing in vivo experiments on tree shrews, the IOP control of the mIPCON system was tested on enucleated rat eyes. A representative IOP recording from a rat eye is shown above. The anterior chamber of a rat eye was cannulated and the IOP was increased in 5 mmHg steps from 10 mmHg to 30 mmHg. The target (desired) IOP is shown in and the true (recorded) IOP is shown in . There was little delay in reaching the target IOP with the mIPCON system (IOP settling time of 1.80 ± 0.38 s), demonstrating the speed and accuracy of the mIPCON system to control IOP. 3

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animal holder.

2.3. In vivo tree shrew experiments This study was carried out in strict accordance with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee and were performed under anesthesia in a dedicated animal surgical and imaging suite at the University of Alabama at Birmingham.

2.3.4. AC cannulation (IOP control) After the tree shrew was placed in the holder, the tree shrew AC was cannulated with a 30-gauge beveled needle near the lateral corneal limbus using a needle holder attached to a three-axis micromanipulator arm, to an insertion depth of approximately 1 mm. Cannula placement was confirmed by direct visualization of the needle in the AC and patency of the needle was confirmed by observing physiologic IOP oscillations (the ocular pulse). The 30-gauge needle was attached to the mIPCON system to control and monitor IOP. As in the in vitro tests, syringe pump flow rate was controlled via proportional feedback control. During experimental manipulation of tree shrew IOP, it was presumed that there was a water-tight seal around the 30-gauge needle placed in the anterior chamber. Although physiologic IOP pulsations (the ocular pulse) were noted throughout the tree shrew experiments, suggesting a sealed system, it is possible that a very small undetected leak could have been present at the needle insertion site. If this were the case, then the IOP measurements obtained in our experiments may differ somewhat from the actual intraocular pressure. However, since the potential corneal leak would have likely been very small, then the difference between actual and measured IOP would also be small.

2.3.1. Detection of SRVPs Sixteen adult tree shrews (9 males, 7 females; age 11–18 months) were tested to determine whether tree shrews have SRVPs at physiologic IOP and ICP, and to determine the incidence of tree shrew SRVPs. Anesthesia was induced and maintained with continuous inhalation of 1% isoflurane. Tree shrew irises were dilated by topical application of 1% tropicamide and 2.5% phenylephrine onto the cornea. Tree shrews were placed in a custom-built small animal holder to facilitate ocular imaging. A hard contact lens was placed over the cornea of the experimental eye to prevent corneal dehydration. Video OCT images were obtained of the tree shrew optic nerve head and retinal vasculature. Video OCT images were analyzed offline to determine the presence and incidence of tree shrew SRVPs (see below, Video Recording and Analysis). 2.3.2. Manipulation of IOP and ICP in tree shrews with SRVPs Since we found that tree shrews have SRVPS, we went on to observe the effects of altering IOP and ICP on tree shrew SRVP properties in a subset of animals with SRVPs. Anesthesia was induced in 2 tree shrews (1 male, 1 female; age 10–14 months) with SRVPs by a combination of inhaled isoflurane (1%) and intramuscular xylazine (7.5 mg/kg), and maintained with continuous inhalation of 1% isoflurane. The cisterna magna, a cerebrospinal fluid-containing space at the base of the cerebellum, was cannulated in 2 tree shrews to alter and monitor ICP (described below, Cisterna Magna cannulation (ICP control)). Following cannulation of the cisterna magna, the tree shrews were placed in a custom-built small animal holder specifically designed to facilitate cannulation of the tree shrew anterior chamber and ocular imaging. The animal holder was equipped with a Harvard apparatus homeothermic blanket, rectal core body temperature feedback, and heart rate monitor (Holliston, MA). Tree shrew irises were dilated by topical application of 1% tropicamide and 2.5% phenylephrine onto the cornea. Next, the AC of the 2 tree shrews was cannulated at the limbus with the mIPCON system, to alter and monitor IOP (see below, AC cannulation (IOP control)). A hard contact lens was placed over the cornea of the experimental eye to prevent corneal dehydration. Realtime en face video OCT images of the tree shrew optic nerve head and retinal vasculature were obtained prior to and during alteration of IOP and/or ICP (described below). Video OCT images were analyzed offline to determine the effect of altering IOP and ICP on SRVP properties (see below, OCT Recording and Analysis).

2.4. Video recording and analysis In both the studies to detect tree shrew SRVPS (16 tree shrews) and the studies to determine the effects of altering IOP and ICP on tree shrew SRVP properties (2 tree shrews), the tree shrew optic nerve head and retinal vasculature were imaged using a Spectralis SD-OCT (Heidelberg Engineering, Franklin, MA) connected to a desktop computer. Real-time en face video output of the tree shrew optic nerve head and retinal vasculature was recorded at 30 Hz using Movavi video suite 18 (Movavi Software, Inc, St. Louis, MO). OCT videos were converted to AVI format, imported into FIJI (version 1.52e), and stabilized using the FIJI template matching plug-in. Stabilized images were saved as a stack of TIF files which were analyzed in a custom MATLAB program to detect changes in tree shrew retinal vein diameter and tree shrew SRVPs. Tree shrew retinal veins and arteries were differentiated based upon their ophthalmoscopic appearance; similar to humans, tree shrew veins are slightly larger than arteries, with the average vein diameter being 30.9 ± 3.0 pixels (n = 8 veins, average ± standard deviation) and the average artery diameter being 20.3 ± 3.2 pixels (n = 8 arteries, average ± standard deviation). Changes in retinal vein diameter were determined by manually selecting a region of interest surrounding a vein segment of interest. The region of interest was composed of 20 lines, with each line oriented orthogonal to the vein axis and separated from adjacent lines by 2 pixels (Fig. 5, upper panel). The pixel intensity across each of the 20 lines was determined and an average pixel intensity for the lines within the region of interest was extracted as a function of position (measured in pixels) for each video frame (Fig. 5, second panel). The half maximal pixel intensity within each frame was taken as the vein diameter, which was plotted as a function of frame number (Fig. 5, third panel). Frame number was directly proportional to time. To determine whether changes in the vein diameter represented SRVPs, a fast Fourier transform was performed on the vein diameter data (Fig. 5, bottom panel). SRVPs were considered to be present if a discrete peak was seen at ∼2 Hz on the fast Fourier transform, which correlated with the anesthetized tree shrews’ heart rates (∼120 beats per minute). SRVP amplitude (in pixels) was determined by averaging the difference between the minimum and maximum vessel diameter for each 1 s interval (i.e. 2 SRVP cycles) for the duration of the IOP step.

2.3.3. Cisterna magna cannulation (ICP control) After successful induction of anesthesia in the 2 tree shrews, each animal was moved to the surgical area. The hair overlying the skull, neck, and down to the shoulder blades was removed. The skin and muscles at the skull base were incised and reflected to expose the atlanto-occipital membrane, which was incised with a 25-gauge needle to expose the cisterna magna. A cannula connected to an adjustableheight fluid reservoir and Honeywell pressure transducer was carefully inserted into the cisterna magna; ICP was controlled by adjusting the height of the fluid reservoir. Proper placement of the cannula was ensured by observing normal physiologic cerebrospinal fluid pressure oscillations associated with the cardiac and respiratory cycles. Once proper placement of the cannula was confirmed, it was secured in place with superglue (cyanoacrylate). After allowing time for the cyanoacrylate to cure, the tree shrew was moved to the custom-built small 4

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Fig. 5. Schematic diagram of objective and automatic analysis of video frames to determine the presence and amplitude of SRVPs. Video OCT was acquired at 30 Hz and each frame of the video was saved as an individual TIF file. The TIF files were imported into Matlab (top panel) and an area of the vessel of interest was selected (blue solid box). The average pixel intensity across the area of interest for each frame was plotted (second panel). The halfway point between the maximum and minimum pixel intensity plot for each frame was determined and taken as the vessel width (in pixels). The change in the vessel width is plotted as a function of frame number (third panel). A fast Fourier transform was performed on the vein diameter data. SRVPs were considered present if a peak was seen at ∼2 Hz, which corresponded to the average heart rate of anesthetized tree shrews. A representative fast Fourier transform is shown (bottom panel).

2.5. Tree shrew optic nerve histology Enucleated tree shrew eyes were immersion fixed in 4% electron microscopy grade paraformaldehyde (EMS, Hatfield, PA), cryoprotected in a 1:1 mixture of 30% sucrose in PBS: optimal cutting temperature compound (EMS, Hatfield, PA), embedded in optimal cutting temperature compound, and then flash frozen in 2-methylbutane cooled with liquid nitrogen. Tissue was cut away (and sections collected) on a cryostat until the midpoint of the optic nerve was reached. Tissue blocks were removed and allowed to thaw while simultaneously being fixed for several hours in a refrigerated glutaraldehyde solution. Samples were next washed, dehydrated in an ethanol series, infiltrated and embedded in Histocryl resin (EMS, Hatfield, PA) according to a slightly modified protocol. Specifically, the lens was drilled out and the resulting cavity filled with resin to overcome poor infiltration of the intact lens, which caused the sections to collapse. After embedding, 3 μm thick sagittal sections were cut on a Leica UC7 ultramicrotome (Leica USA), stained with toluidine blue and imaged on a Leica DM6 microscope (Leica USA). 3. Results 3.1. SRVPs in tree shrews Since SRVPs have not been previously reported in tree shrews, we screened both eyes of 16 tree shrews for the presence of SRVPs under normal physiologic conditions, i.e. without manipulation of IOP and ICP. We found that twelve of 16 (75%) tree shrews exhibited SRVPs, similar to the reported incidence of SRVPs in humans (Levin, 1978; Harder and Jonas, 2007; Legler and Jonas, 2007; Lorentzen, 1970). When SRVPs were present in a tree shrew, they were present in both eyes. However, the SRVP amplitude typically differed between eyes of the same tree shrew (data not shown). Since we have shown that tree shrews have SRVPs and that tree shrew SRVPs occur with an incidence similar to the reported incidence in humans, we wanted to determine whether tree shrew SRVPs were affected by changes in IOP and ICP in a manner similar to that reported in humans. 3.2. Effect of IOP on tree shrew SRVP amplitude To determine the effects of changes in IOP on tree shrew SRVPs, the cisterna magna and AC were cannulated in a tree shrew with SRVPs. ICP was measured to be 10 mmHg and was subsequently pressure clamped at 10 mmHg. IOP was initially pressure clamped at 5 mmHg with the mIPCON system and then increased in 3 mmHg steps up to 20 mmHg. After reaching an IOP of 20 mmHg, IOP was further increased to supraphysiologic levels (25 mmHg and 35 mmHg). At each IOP step, the IOP was held constant for 1 min to allow IOP stabilization, optimization of the video OCT images and to allow enough data to be obtained for analysis. SRVPs were not detectable until IOP was increased to 17 mmHg in 5

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Fig. 6. Effect of IOP on SRVPs at a constant ICP (10 mmHg). The plotted quantity is change in vessel diameter (in pixels) minus the mean vessel diameter for each IOP step, i.e. the deviations of vessel diameter from the mean, versus time. At an ICP of 10 mmHg, IOP was initially pressure clamped at 5 mmHg and IOP was increased in 3 mmHg steps to 20 mmHg. IOP was subsequently increased to 25 mmHg and 35 mmHg. No SRVPs were present at or at lower IOPs (A). SRVPs were an IOP of first detected at an IOP of 17 mmHg (B). As IOP was increased, SRVP amplitude increased. The average SRVP amplitude was 1.54 pixels at an IOP of at an IOP of (C), 17 mmHg (B), at an IOP of (D), and at an IOP of (E).

this tree shrew. At an IOP of 17 mmHg, small amplitude SRVPs (1.54 pixels; 4.98% of average vein diameter) were detected (Fig. 6A). As IOP was incrementally increased to a maximum of 35 mmHg, SRVP amplitude increased at each IOP step; average SRVP amplitude was 1.77 pixels (5.73% of average vein diameter) at an IOP of 20 mmHg (Figs. 6B), 5.87 pixels (19.00% of average vein diameter) at an IOP of 25 mmHg (Fig. 6C), and 9.15 pixels (29.61% of average vein diameter) at an IOP of 35 mmHg (Fig. 6D). Thus, changes in IOP affect tree shrew SRVP amplitude, with acute increases in IOP causing increased SRVP amplitudes. 3.3. Effect of ICP on SRVPs To further study the effects of changes in IOP and ICP on tree shrew SRVPs, the AC and cisterna magna were cannulated in another tree shrew with SRVPs. ICP was measured to be 7 mmHg and was initially pressure clamped at 7 mmHg. IOP was initially pressure clamped at 4 mmHg and was subsequently increased in 3 mmHg steps. At an ICP of 7 mmHg, no SRVPs were detected at IOPs of 4 mmHg (data not shown) or 7 mmHg (Fig. 7A, black trace, Video 1A). However, when IOP was further increased to 10 mmHg, SRVPs were detected in a single vein (Fig. 7A and C, green trace, Fig. 8A & Video 1B). Further increases in IOP caused multiple veins to exhibit SRVPs (Fig. 8B & Video 1C). In the same tree shrew, IOP was lowered to 6 mmHg and ICP was increased to 30 mmHg to simulate elevated ICP, after which IOP was again increased in 3 mmHg steps. No SRVPs were detected at IOPs of 15 mmHg or lower (Fig. 7B, blue trace, Video 2A). However, SRVPs were present in one vein at an IOP of 18 mmHg (Fig. 7B and C, red trace, Fig. 8C & Video 2B), the same vein that first developed SRVPs at an ICP of 7 mmHg (Fig. 8A & Video 1B). Similar to the effect of increasing IOP at an ICP of 7 mmHg, further increasing the IOP at an ICP of 30 mmHg caused multiple veins to exhibit SRVPs (Fig. 8D & Video 2C). In addition to the effect of ICP on tree shrew venous pulse pressure, ICP also affected tree shrew SRVP amplitude. As ICP was increased from 7 mmHg to 30 mmHg, the SRVP amplitude at the venous pulse pressure decreased. At an ICP of 7 mmHg and an IOP of 10 mmHg, the SRVP amplitude was 10.5 pixels (33.98% of average vein diameter); however at an ICP of 30 mmHg and an IOP of 18 mmHg, the SRVP amplitude was 3.9 pixels (12.62% of average vein diameter) (Fig. 7C). Therefore, similar to IOP, changes in ICP also affect tree shrew SRVP properties. In this tree shrew, an acute elevation of ICP caused an

Fig. 7. Effects of ICP and IOP on tree shrew SRVPs. The plotted quantity is change in vessel diameter (in pixels) minus the mean vessel diameter for each IOP step, i.e. the deviations of vessel diameter from the mean, versus time. A. At ( ), an ICP of 7 mmHg, SRVPs were present at an IOP of but not at an IOP of 7 mmHg (black tracing). B. At an ICP of 30 mmHg, SRVPs ( ), but not an IOP of were present at an IOP of ( ). C. Comparison of the SRVPs at an ICP of ( , same as in A) and ( , same as in B). The SRVP amplitude at an ICP of 30 mmHg (3.9 pixels) was smaller than the SRVP amplitude at an ICP of 7 mmHg (10.5 pixels).

increased venous pulse pressure and a decreased SRVP amplitude, similar to the reported effect of increased ICP on human SRVPs (Motschmann et al., 2001; Querfurth et al., 2010; Jonas et al., 2008; Firsching et al., 2011).

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elevating IOP with manual pressure on the globe caused an increase in SRVP amplitude. In addition to the effect of IOP on SRVPs, we also showed that changes in ICP affected the venous pulse pressure and the tree shrew SRVP amplitude. The relationship between venous pulse pressure and SRVPs that we found in tree shrews is qualitatively identical to that described by Morgan et al. (2008) in dogs and by multiple authors in humans during ophthalmodynamometry (Motschmann et al., 2001; Querfurth et al., 2010; Jonas et al., 2008; Firsching et al., 2011), namely that a higher IOP is necessary to produce SRVPs when ICP is elevated. However, in this tree shrew, the venous pulse pressure was lower than the ICP, which is in contrast to what has previously been reported in monkeys (Rios-Montenegro et al., 1973), dogs (Gibbs, 1936), and humans (Querfurth et al., 2010), where the venous pulse pressure was 8–10 mmHg higher than the ICP. One possible explanation for the difference between our data and the previously published data is that the above-mentioned studies used direct observation (ophthalmoscopy) to detect SRVPs. In contrast, we used objective detection software to detect SRVPs, which has been shown to be markedly more sensitive than direct observation for detecting SRVPs (Jenkins et al., 2017). Therefore, it is possible that the actual venous pulsation pressure in prior studies was markedly lower than what was reported. However, since our data was obtained from one tree shrew, experimental data from multiple tree shrews are necessary to better understand the relationship between tree shrew venous pulse pressure and ICP. Similar to the effects of altering IOP on tree shrew SRVP amplitude, altering ICP also affected tree shrew SRVP amplitude; specifically, acutely increasing ICP caused tree shrew SRVP amplitude to decrease. Although the effect of increasing ICP on human SRVP amplitude has not been described, we speculate that human SRVP amplitude will respond to acute elevations of ICP similar to tree shrew SRVPs, based on the assumption that the TLPD is responsible for the generation of SRVPs. Since the TLPD equals IOP minus ICP (TLPD = IOP – ICP), as ICP increases, the TLPD decreases. It is possible that smaller TLPDs may produce “weaker” (lower amplitude) SRVPs compared to SRVPs generated at higher TLPDs. A likely explanation for the similarity between tree shrew SRVPs and human SRVPs is that tree shrews and humans share similar orbital and ocular anatomies. Tree shrews have a collagenous, load-bearing lamina cribrosa similar to the human lamina cribrosa (Zhan et al., 2015). This is in contrast to lower order species, such as rats and mice, which lack a well-developed, load-bearing lamina cribrosa (May and Lutjen-Drecoll, 2002; Morrison et al., 1995). In addition, like the human CRV, the tree shrew CRV passes through the central region of the lamina cribrosa (Albon et al., 2007) and travels within the substance of the retro-laminar optic nerve (Fig. 9) for a short distance after exiting the eye, where it is exposed to ICP. In contrast, the CRV of lower order species does not travel within the optic nerve after exiting the eye and is therefore not exposed to ICP in the same way (May and LutjenDrecoll, 2002). Although tree shrews and humans share similar orbital and ocular anatomies, there are a number of anatomical differences between tree shrew and human ocular anatomy which may differentially affect how tree shrew SRVPs and human SRVPs respond to alterations in IOP and ICP. Although both tree shrews and humans have a collagenous, loadbearing lamina cribrosa, the collagen beams in the human lamina cribrosa are arranged in a meshwork pattern (Albon et al., 2007; Dandona et al., 1990; Elkington et al., 1990; Jonas et al., 1991; Samuels et al., 2018), whereas the collagen beams of the tree shrew lamina cribrosa are radially oriented (Albon et al., 2007; Samuels et al., 2018). If the structure of the lamina cribrosa has an impact on SRVP amplitude and generation, as suggested by the work of Golzan et al. (2015), then it is possible that the different lamina cribrosa organization may affect the response of SRVPs to changes in IOP and ICP. In addition, the orientation of the retinal vasculature is different between tree shrews and humans. In humans, there are curved vascular arcades with 4 main

Fig. 8. Still images from en face video OCT recordings of tree shrew SRVPs. In A & B, the ICP was pressure clamped at 7 mmHg. In C & D, the ICP was pressure clamped at 30 mmHg. A. IOP pressure clamped at 10 mmHg. The vein at 3 o'clock ( ) exhibited SRVPs. B. IOP pressure clamped at 16 mmHg in the same tree shrew as in A. SRVPs were seen in many retinal veins. Three of the . C. IOP pressure clamped pulsating retinal veins are indicated by at 18 mmHg in a tree shrew with ICP pressure clamped at 30 mmHg. The vein at ) exhibited SRVPs. D. IOP pressure clamped at 21 mmHg in 3 o'clock ( the same tree shrew as in C. SRVPs were seen in many retinal veins. Three of the . The yellow asterisk (*) in pulsating retinal veins are indicated by A & C indicates the retinal vessel that was analyzed to generate the graphs in Fig. 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion In this study we have shown that tree shrews exhibit SRVPs at physiologic IOP and ICP and that alterations in IOP and ICP affect tree shrew SRVP properties, similar to what has been reported in humans. We found that SRVPs occurred in 12/16 (75%) tree shrews at physiologic IOP and ICP. In humans, the reported incidence of SRVPs varies between 20 and 85% (Levin, 1978; Harder and Jonas, 2007; Legler and Jonas, 2007; Lorentzen, 1970). Most of these studies used ophthalmoscopy to detect RVPs, and the wide range of reported SRVPs likely is due to a combination of the subtle nature of SRVPs and inherent limitations of ophthalmoscopy, such as observer and patient motion, making it difficult to accurately identify SRVPs. Indeed, when objective detection methods are used to assess SRVPs, such as fundus videos or video OCT recordings, the reported incidence of SRVPs increases to 80–90% (Jenkins et al., 2017), very similar to the incidence of SRVPs we found in tree shrews. In addition to tree shrews having a similar SRVP incidence as reported in humans, we found that tree shrew SRVPs were affected by both IOP and ICP, also similar to the situation in humans. With ICP pressure clamped at 10 mmHg, tree shrew SRVP amplitude increased as IOP was increased, similar to the relationship between IOP and human SRVP amplitude described by Golzan et al. (2011), where pharmacologically lowering IOP acutely with apraclonidine or timolol in normal subjects caused a decrease in human SRVP amplitude and acutely 7

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National Eye Institute grant numbers T32-EY007092 (MD) and R21EY026218 (BCS); and the Georgia Research Alliance (CRE). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exer.2019.06.003. References Abegao Pinto, L., Vandewalle, E., De Clerck, E., Marques-Neves, C., Stalmans, I., 2013. Lack of spontaneous venous pulsation: possible risk indicator in normal tension glaucoma? Acta Ophthalmol. 91, 514–520. Adler, M.D., Comi, A.E., Walker, A.R., 2001. Acute hemorrhagic complication of diagnostic lumbar puncture. Pediatr. Emerg. Care 17, 184–188. Albon, J., Farrant, S., Akhtar, S., et al., 2007. Connective tissue structure of the tree shrew optic nerve and associated ageing changes. Investig. Ophthalmol. Vis. Sci. 48, 2134–2144. Alstadhaug, K.B., Odeh, F., Baloch, F.K., Berg, D.H., Salvesen, R., 2012. Post-lumbar puncture headache. Tidsskr. Nor. Laegeforen 132, 818–821. Berdahl, J.P., Allingham, R.R., Johnson, D.H., 2008a. Cerebrospinal fluid pressure is decreased in primary open-angle glaucoma. Ophthalmology 115, 763–768. Berdahl, J.P., Fautsch, M.P., Stinnett, S.S., Allingham, R.R., 2008b. Intracranial pressure in primary open angle glaucoma, normal tension glaucoma, and ocular hypertension: a case-control study. Investig. Ophthalmol. Vis. Sci. 49, 5412–5418. Bershad, E.M., Urfy, M.Z., Pechacek, A., et al., 2014. Intracranial pressure modulates distortion product otoacoustic emissions: a proof-of-principle study. Neurosurgery 75, 445–454 discussion 54-5. Bruce, B.B., 2014. Noninvasive assessment of cerebrospinal fluid pressure. J. Neuro Ophthalmol. 34, 288–294. Burkett, J.G., Ailani, J., 2018. An up to date review of pseudotumor cerebri syndrome. Curr. Neurol. Neurosci. Rep. 18, 33. Dandona, L., Quigley, H.A., Brown, A.E., Enger, C., 1990. Quantitative regional structure of the normal human lamina cribrosa. A racial comparison. Arch. Ophthalmol. 108, 393–398. Dawes, A.J., Sacks, G.D., Cryer, H.G., et al., 2015. Intracranial pressure monitoring and inpatient mortality in severe traumatic brain injury: a propensity score-matched analysis. J. Trauma Acute Care Surg. 78, 492–501 discussion -2. Del-Rio-Vellosillo, M., Garcia-Medina, J.J., Pinazo-Duran, M.D., Abengochea-Cotaina, A., Barbera-Alacreu, M., 2017. Ocular motor palsy after spinal puncture. Reg. Anesth. Pain Med. 42, 1–9. Donnelly, S.J., Subramanian, P.S., 2009. Relationship of intraocular pulse pressure and spontaneous venous pulsations. Am. J. Ophthalmol. 147, 51–55 e2. Egede, L.E., Moses, H., Wang, H., 1999. Spinal subdural hematoma: a rare complication of lumbar puncture. Case report and review of the literature. Md. Med. J. 48, 15–17. Elkington, A.R., Inman, C.B., Steart, P.V., Weller, R.O., 1990. The structure of the lamina cribrosa of the human eye: an immunocytochemical and electron microscopical study. Eye (Lond) 4 (Pt 1), 42–57. 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Relationship between the pressure in the veins on the nerve head and the cerebrospinal fluid pressure. Arch. Neurol. Psychiatr. 35, 292–295. Godoy, D.A., Lubillo, S., Rabinstein, A.A., 2018. Pathophysiology and management of intracranial hypertension and tissular brain hypoxia after severe traumatic brain injury: an integrative approach. Neurosurg. Clin. 29, 195–212. Golzan, S.M., Graham, S.L., Leaney, J., Avolio, A., 2011. Dynamic association between intraocular pressure and spontaneous pulsations of retinal veins. Curr. Eye Res. 36, 53–59. Golzan, S.M., Morgan, W.H., Georgevsky, D., Graham, S.L., 2015. Correlation of retinal nerve fibre layer thickness and spontaneous retinal venous pulsations in glaucoma and normal controls. PLoS One 10, e0128433. Harder, B., Jonas, J.B., 2007. Frequency of spontaneous pulsations of the central retinal vein. Br. J. Ophthalmol. 91, 401–402. Hasan, D., Lindsay, K.W., Vermeulen, M., 1991. 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Fig. 9. Photomicrograph of the retrolaminar tree shrew optic nerve stained with toluidine blue. This image is of a longitudinal section of the nerve near the middle of the nerve. The lamina cribrosa (white arrows), the central retinal artery (red asterisk), and the central retinal vein (blue asterisk) are shown. A second, smaller vessel, adjacent to the central retinal artery and central retinal vein (unlabeled), is also present and may represent another vein or a lymphatic channel. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

vessel pairs emanating from the optic disc that sequentially branch to supply blood to the human retina. In contrast, in tree shrews there are as many as 8 pairs of vessels emanating radially from the optic disc and travelling horizontally without appreciable branching (Stockslager et al., 2016; Albon et al., 2007). Although the significance of this difference is unknown, it is possible that the different retinal vascular arrangement is related to structural and functional differences in the tree shrew and human retina, which may affect SRVP properties, just as changes in retinal nerve fiber layer (RNFL) thickness affected human SRVP amplitudes. Although our data suggests a relationship between IOP, ICP and tree shrew SRVPs, the details of this relationship are not completely understood. For example, the effect of RNFL thickness on SRVP amplitude (Golzan et al., 2015) and our data showing that multiple veins demonstrated SRVPs at IOPs above the venous pulse pressure, while only one vein had SRVPs at the venous pulse pressure (Fig. 8, Videos 1 & 2), suggest a more complex relationship between SRVPs, IOP, and ICP, which is likely affected by ocular tissue structural properties and potentially other, yet unidentified, factors, as well as IOP and ICP. However, in our current study, we were not able to further characterize this complex relationship because we were able to manipulate IOP and/or ICP in only 2 tree shrews. In addition, in the 2 tree shrews where IOP and/or ICP were manipulated, we did not measure the tree shrew peripapillary RNFL thickness and are therefore unable to comment about the effect of RNFL thickness on tree shrew SRVP properties. Therefore, further studies in tree shrews are necessary to better understand the factors governing and affecting tree shrew SRVPs. Despite the limitations of our study and the anatomic differences between tree shrew and human eyes, the similar orbital anatomy and the generally similar relevant ocular anatomy, coupled with the similar response of tree shrew SRVPs to alterations in IOP and ICP compared to humans, strongly suggest that tree shrews are a suitable animal model system to further study the complex relationship between SRVPs, IOP, and ICP and to potentially determine and characterize other factors governing SRVP generation and affecting SRVP properties.

Grant information This work was supported by the National Institutes of Health/ 8

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