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Background: Ophthalmodynamometric studies can provide useful clinical information regarding glaucoma, cerebrospinal fluid pressure, and vascular disease but are affected by variable reproducibility and unknown calibration of force in terms of intraocular pressure (IOP). The aim of this study was to calculate calibration factors and identify key design principles using three different types of ophthalmodynamometers., Methods: We constructed a modified ophthalmodynamometer named OcuDyn using a large contact lens inside a ring force transducer with continuous signal acquisition and averaging. OcuDyn and Sisler ophthalmodynamometers were applied to isolated pig eyes which were cannulated and connected to a fluid reservoir and a pressure transducer to measure induced IOP at increasing force application. Using these two devices and a Meditron ophthalmodynamometer, we measured the minimum ophthalmodynamometric force (ODF) required to induce pulsation in the hemi-veins and diastolic retinal artery of each eye in glaucoma patient volunteers and family members., Results: Blood pressure was measured with the sphygmomanometer cuff held at eye level. In pigs, the relationship between induced IOP and ODF was strong (minimum r > 0.98, p < 0.001). In humans, the pressure increment (blood pressure - baseline IOP) correlated highly with human arterial ODF (r = 0.83, n = 75, p < 0.001, mean slope 0.32). Mean coefficients of variation were 12.4% (n = 66) for veins and 5.6% (n = 39) for arteries in diastole; these results did not differ significantly with those from the Meditron but were significantly lower than results with the Sisler (20%, p = 0.0014)., Conclusions: Linearity between induced IOP and ODF is strong, suggesting that calibration of 0.32 mmHg/g for OcuDyn or using two point blood pressure measures allows for valid interpolation. The better optics of the Meditron and OcuDyn result in more repeatable end-point determination and outweigh benefits conferred by signal averaging or slit lamp mounting. We calculate calibration factors of 0.89 mmHg/arbitrary unit for Meditron and 0.82 mmHg/g for Sisler. These factors may be useful in the estimation of ocular vascular, orbital, and cerebrospinal fluid pressures.

A disrupted oxygen environment in the retina of severely premature neonates is thought to be a key factor in the development of retinopathy of prematurity (ROP). This review describes our understanding of intraretinal oxygen distribution and consumption in a range of animal models, including species with naturally avascular retinas and models of induced occlusion of the retinal vasculature. The influence of graded systemic hyperoxia on retinal oxygenation is also discussed along with modulation of retinal oxygen metabolism. The differences in retinal oxygenation between developing and mature retinas are also described. Comparisons are made with studies in the monkey retina in order to assess possible similarities in behaviour between rat and human retinas. Pathogenesis mechanism and possible intervention strategies during the diseased processes in ROP are proposed based on our current knowledge.

Purpose: To develop a new technique for detailed study of the spatial distribution of retinal and choroidal microvasculature and their relationship to neurons and glial cells at the cellular level in human cadaveric eyes., Methods: Twenty-six human donor eyes were used. Wherever possible, the central retinal artery and a branch of the posterior ciliary artery were individually cannulated and perfused with oxygenated Ringer's solution with 0.5% bovine serum albumin. The perfusion pressure was continuously monitored. Once residual blood was washed out, the perfusate solutions were switched to fixative, membrane-permeabilizing solution and selected labeling solutions. The eyes were then immersion fixed and the retina and choroid flat-mounted for immunolabeling and confocal imaging before cryosectioning. The microstructures of vascular, glial, and neuronal cells in the retina and the stroma in the choroid were studied., Results: The retinal microvasculature was fully perfused and stained by cannulation of the central retinal artery. Regional distribution of choroidal vasculature perfusion was dependent on the specific feeder artery cannulated. The detailed spatial relationship between endothelial cells, glial cells, and neurons at the cellular and subcellular levels was identified with confocal microscopy and immunohistochemical labeling of retinal sections. In the choroid, endothelial cells were clearly identifiable down to the level of the intracellular cytoarchitecture of the choriocapillaris, along with their relationship to Bruch's membrane and the feeding and drainage vessels., Conclusions: A microperfusion fixation and staining technique has been developed that allows studies of the structural relationships of vascular, glial, and neuronal elements at the cellular level in human donor eyes.

This study was designed to determine if low power laser therapy can achieve amelioration of vasoproliferation yet preserve useful vision in the treated area in a transgenic mouse model of retinal neovascularisation. The mice were anaesthetised and the pupils dilated for ERG and fundus fluorescein angiography on postnatal day 32. The left eyes were treated with approximately 85 laser spots (532 nm, 50 ms, 300 microm diameter) at a power level of 20 mW at the cornea. The eyes were examined using ERG and fluorescein angiography, one, four and six weeks later. Flat mounts of FITC-dextran infused retinas, retinal histology and PEDF immunohistochemistry was studied one or six weeks after laser treatment. In untreated eyes the expected course of retinal neovascularisation in this model was observed. However, retinal neovascularisation in the laser treated eye was significantly reduced. The laser parameters chosen produced only mild lesions which took 10-20 s to become visible. ERG responses were comparable between the treated and untreated eyes, and histology showed only partial loss of photoreceptors in the treated eyes. PEDF intensity corresponded inversely with the extent of neovascularisation. Low power panretinal photocoagulation can inhibit retinal neovascularisation and yet preserve partial visual function in this transgenic mouse model of retinal neovascularisation.

Intraocular pressure and cerebrospinal fluid (CSF) pressure are important determinants of the trans-laminar pressure gradient which is believed to be important in the pathogenesis of glaucomatous optic nerve degeneration. Computational models and finite element calculations of optic nerve head biomechanics have been previously used to predict pressures and stresses in the human optic nerve. The purpose of this report is to morphometrically compare the optic nerve laminar and pia mater structure between humans and dogs, and to use previously reported tissue pressure measurements in the dog optic nerve to estimate individual-specific human optic nerve pressures and pressure gradients. High resolution light microscopy was used to acquire quantitative histological measurements from sagittal sections taken from the middle of the optic nerve in 34 human cadaveric eyes and 10 dog eyes. Parameters measured included the pre-laminar and lamina cribrosa thickness, distance from posterior boundary of lamina cribrosa to inner limiting membrane (ILM), shortest distance between anterior lamina cribrosa surface and subarachnoid space, shortest distance between ILM and inner surface of pia mater in contact with the subarachnoid space and optic nerve diameter. Pia mater thickness in the proximal 4 mm of post-laminar nerve was also determined. There was no significant difference in lamina cribrosa thickness between dog and human eyes (P = 0.356). The distance between the intraocular and subarachnoid space was greater in dogs (P < 0.001). Pia mater thickness was greatest at the termination of subarachnoid space in both species. In humans, pia mater thickness decreased over the proximal 500 mum to reach a constant value of approximately 60 mum. In dogs this decrease occurred over 1000 mum to reach a constant diameter of approximately 30 mum. Using previous measurements of optic nerve pressures and pressure gradients in dogs we estimate that at an IOP of 15 mmHg and a CSF pressure of 0 mmHg the mean pressure difference across the human pia mater will be 4.8 +/- 2.2 mmHg. If we assume that the pressure difference between the intraocular space and post-laminar tissue falls across the entire thickness of the human lamina cribrosa then an estimate of the trans-laminar pressure gradient is 2.0 +/- 0.8 mmHg/100 mum. If we assume that this pressure difference only occurs across the dense collagenous plates of the posterior lamina cribrosa then a trans-laminar pressure gradient high estimate of 3.3 +/- 1.4 mmHg/100 mum is calculated. Changes in tissue pressure gradients in the optic nerve may be an important factor in the pathogenesis of glaucomatous optic neuropathy.

The paucity of suitable experimental models has made it difficult to isolate the pathogenic role of mitochondria in central nervous system diseases associated with absolute pressure elevation and increased pressure gradients. Experimental models of traumatic brain injury (TBI) and hydrocephalus have been useful for examining the mitochondrial response following pressure increase in the central nervous system; however, the presence of multiple pathogenic factors acting on the brain in these previous studies has made it difficult to determine whether the induced changes were a result of mechanical damage, intracranial pressure elevation, or other pathogenic factors. By direct monitoring and control of pressures in the intraocular, intracranial, and vascular compartments, we use the pig optic nerve, a typical central white matter tract, to compare the temporal sequence of cytochrome c oxidase (CcO) levels between regions of absolute pressure elevation and pressure gradient increase. We demonstrate that a rise in pressure gradient without traumatic injury up-regulates CcO levels across the site of the gradient, in a manner similar to what has been previously reported for hydrocephalus. We also demonstrate that CcO changes do not occur following an absolute pressure rise. These findings taken together with our recent reports suggest that mitochondria initiate an early compensatory response to axonal damage following pressure gradient increase. Extrapolation of our results also suggests that decreased CcO levels in TBI may be secondary to mechanical damage. This study emphasises the importance of pressure gradients in regulating mitochondrial function in the central nervous system., (2009 Wiley-Liss, Inc.)

This review considers the critical role of the conjunctiva in determining the success or failure of glaucoma filtration surgery. Glaucoma filtration surgery can be defined as an attempt to lower intraocular pressure (IOP) by the surgical formation of an artificial drainage pathway from the anterior chamber to the subconjunctival space. Many types of glaucoma filtration surgery have been developed since the first attempts almost 180 years ago. The wide range of new techniques and devices currently under investigation is testament to the limitations of current techniques and the need for improved therapeutic outcomes. Whilst great attention has been paid to surgical techniques and devices to create the drainage pathway, relatively little attention has been given to address the question of why drainage from such artificial pathways is often problematic. This is in contrast to normal drainage pathways which last a lifetime. Furthermore, the consequences of potential changes in aqueous humour properties induced by glaucoma filtration surgery have not been sufficiently addressed. The mechanisms by which aqueous fluid is drained from the subconjunctival space after filtration surgery have also received relatively little attention. We propose that factors such as the degree of tissue damage during surgery, the surrounding tissue reaction to any surgical implant, and the degree of disruption of normal aqueous properties, are all factors which influence the successful formation of long term drainage channels from the conjunctiva, and that these channels are the key to successful filtration surgery. In recent years it has been suggested that the rate of fluid drainage from the subconjunctival space is actually the determining factor in the resultant IOP reduction. Improved knowledge of aqueous humour induced changes in such drainage pathways has the potential to significantly improve the surgical management of glaucoma. We describe for the first time a novel type of drainage surgery which attempts to minimise surgical trauma to the overlying conjunctiva. The rationale is that a healthy conjunctiva allows drainage channels to form and less opportunity for inflammation and scar tissue formation which are a frequent cause of failure in glaucoma filtration surgery. Successful drainage over extended periods of time has been demonstrated in monkey and rabbit eyes. Long lasting drainage pathways were clearly associated with the presence of lymphatic drainage pathways. A new philosophy in glaucoma drainage surgery is proposed in which minimisation of surgical trauma to the conjunctiva and the encouragement of the development of conjunctival drainage pathways, particularly lymphatic pathways, are central pillars to a successful outcome in glaucoma filtration surgery.

Purpose: To explore the tissue ablation properties of pulsed 266-nm and 213-nm laser radiation in porcine retina and the potential for 213-nm laser radiation to cut through human trabecular meshwork., Methods: Segments of porcine retinas were used, and localized areas of tissue were exposed to 266-nm or 213-nm laser. Human trabecular meshwork from donor eyes was also ablated using pulsed 213-nm laser. Ocular tissue was bathed in fluid to mimic the intraocular environment. Single or multiple pulses at various fluence levels were delivered with a tapered fiber-optic probe. The tissue was then fixed for histologic examination. Ablation depth and extent of damage were measured and related to fluence level and number of pulses applied., Results: Ablation of the inner retina was achieved by single pulses at fluence levels of 0.6 J/cm(2) and higher with 266-nm laser radiation and 0.2 J/cm(2) and higher with 213-nm laser radiation. At the same fluence, ablation depth was greater (P < 0.001) with 213-nm than 266-nm laser, with less collateral damage. Ablation of human trabecular meshwork using 213-nm laser was highly dependent on fluence after exposure to single and multiple pulses, allowing good control of ablation depth., Conclusions: Laser radiation at 213 nm has greater ablation efficiency, less collateral damage, and a more linear dose-response than ablation at 266 nm. Precise removal of human trabecular meshwork was demonstrated using pulsed 213-nm laser radiation.

Purpose: Cytoskeleton proteins play a critical role in maintaining retinal ganglion cell structure, viability, and function. This study documents the distribution of cytoskeleton protein subunits in the various regions of the normal human optic nerve and identifies important relationships among mitochondria, myelin, and neurofilament proteins., Methods: Twenty-three optic nerves from human cadavers were used. Confocal microscopy was used to examine the distribution of neurofilament light, neurofilament medium, neurofilament heavy (phosphorylated and unphosphorylated), neurofilament heavy (phosphorylated only), actin, and microtubule associated protein (MAP)-1 along the sagittal plane of the optic nerve. Comparisons were made among superior, middle, and inferior regions and also among temporal, central, and nasal portions of the optic nerve. Colocalization of neurofilament light, mitochondrial cytochrome c oxidase (COX), and myelin was also performed., Results: There are significant differences in the pattern and distribution of neurofilament protein subunits, actin, and MAP-1 along the sagittal plane of the optic nerve. Cytoskeleton proteins and COX mitochondria are found in highest concentrations in the prelaminar and lamina cribrosa regions. COX and neurofilament light occurs predominantly in unmyelinated nerve, with a significant decrease in concentration occurring on optic nerve myelination., Conclusions: The heterogeneous distribution of cytoskeleton proteins along the sagittal plane may be an important functional adaptation that reflects the nonuniform nature of the physiological and structural environment of the optic nerve. The heterogeneous distribution of cytoskeleton proteins may also partly account for the asymmetric pattern of optic nerve damage after intraocular pressure elevation.

Aim: Retinal vein ophthalmodynamometric force (ODF) is predictive of future optic disc excavation in glaucoma, but it is not known if variation in ODF affects prognosis. We aimed to assess whether a change in ODF provides additional prognostic information., Methods: 135 eyes of 75 patients with glaucoma or being glaucoma suspects had intraocular pressure (IOP), visual fields, stereo optic disc photography and ODF measured on an initial visit and a subsequent visit at mean 82 (SD 7.3) months later. Corneal thickness and blood pressure were recorded on the latter visit. When venous pulsation was spontaneous, the ODF was recorded as 0 g. Change in ODF was calculated. Flicker stereochronoscopy was used to determine the occurrence of optic disc excavation, which was modelled against the measured variables using multiple mixed effects logistic regression., Results: Change in ODF (p = 0.046) was associated with increased excavation. Average IOP (p = 0.66) and other variables were not associated. Odds ratio for increased optic disc excavation of 1.045 per gram ODF change (95% CI 1.001 to 1.090) was calculated., Conclusion: Change in retinal vein ODF may provide additional information to assist with glaucoma prognostication and implies a significant relationship between venous change and glaucoma patho-physiology.

Although intracranial pressure (ICP) elevation can induce significant structural and functional changes within the central nervous system (CNS), almost complete neuronal recovery is possible if ICP and associated pathogenic factors are restored in the acute phase of the disease process. Nitric oxide synthase (NOS) isoforms have been implicated in the pathogenesis of many CNS diseases and may play an important role in the development of neuronal tolerance in the early stages of pressure elevation. In this paper we use the pig optic nerve, a typical central white matter tract, to study the time-dependent sequence of NOS isoform change following pressure elevation. The timing of NOS isoform change in relationship to structural and functional changes to axons and glial cells is also discussed. This study demonstrates that endothelial cell nitric oxide synthase (ecNOS), an enzyme that plays a protective role in the CNS, is up-regulated in a time-dependent manner after pressure elevation. ecNOS levels increase after axonal and astrocyte injury, suggesting that it might be a compensatory response that is initiated in an effort to preserve CNS function. Inducible NOS (iNOS) and neuronal NOS (nNOS), which are known to have a deleterious effect on the CNS, were not detected in this study. The increase in ecNOS demonstrated in this study is significantly different to the increase in iNOS and nNOS previously demonstrated following traumatic brain injury. Changes in ecNOS levels may therefore be important in the development of neuronal tolerance in the early stages of CNS diseases such as hydrocephalus.

Astrocytes maintain an intimate relationship with central nervous system (CNS) neurons and play a crucial role in regulating their biochemical environment. A rise in neural tissue pressure in the CNS is known to lead to axonal degeneration however the response of astrocytes during the early stages of neural injury has not been studied in great detail. The optic nerve is a readily accessible model in which to study CNS axonal injury. Previous work from our laboratory has shown that an acute increase in intraocular pressure (IOP) results in axonal cytoskeleton changes and axonal transport retardation within the optic nerve head. Axonal changes occurred in a time-dependent manner with the magnitude of change being proportional to the duration of the IOP rise. Using glial fibrillary acidic protein (GFAP) as a marker of astrocytes we have now studied pressure induced changes in astrocyte structure in the optic nerve head. Using confocal microscopy we found that an increase in IOP resulted in morphological changes in the astrocytes that were consistent with previous reports of swelling. In addition there was also a decrease in GFAP intensity within these astrocytes. These changes occurred in a time-dependent manner with the chronology of change coinciding with that of axonal change. There was no evidence of apoptosis in regions where astrocyte changes were found. The present results provide evidence that in the early stages of neural tissue pressure rise there are both astrocyte and axonal injury.

The aim of the study was to determine whether the Heidelberg Retinal Flowmeter (HRF), a confocal scanning laser Doppler flowmeter, can measure choroidal blood flow in pig eyes.An HRF was used to obtain flow maps from in vitro pig eyes under a range of perfusion flow rates (0 - 500 microL/min) under conditions in which only the choroid was perfused. In some cases choroidal blood flow was also measured simultaneously using a conventional fiberoptic based Laser Doppler Perfusion Monitor (LDPM) which used the same laser wavelength (780 nm). The relationship between perfusion flow, HRF measured flow and LDPM measured flow was determined. HRF flow maps were also obtained in vivo as a function of focal plane setting through the retina and choroid.Across the range of perfusion flow rates through the isolated eyes there was a poor correlation with HRF measured choroidal flow and perfusion flow. In contrast, there was a strong linear relationship between perfusion flow and LDPM measured blood flow. Both in vitro and in vivo, no choroidal vessels could be visualised in the HRF flow maps, even when the focal plane was in the choroid.The HRF is unable to measure choroidal blood flow in pig eyes. This is not due to an inability of the 780 nm laser to penetrate into the choroid or due to red blood cell velocities in the choroid being higher than the measurement range of the instrument.

Purpose: To study the time-dependent effects of elevated intraocular pressure (IOP) on axonal transport and cytoskeleton proteins in the porcine optic nerve head., Methods: Fifteen pigs were used for this study. Rhodamine-beta-isothiocyanate was injected into the vitreous of each eye to study axonal transport. IOP in the left eye was elevated to 40 to 45 mm Hg, and IOP in the right eye was maintained between 10 and 15 mm Hg. Cerebrospinal fluid pressure was also continually monitored. IOP was elevated for 3 hours (n = 7) or 12 hours (n = 8) before animal euthanatization. Antibodies to phosphorylated neurofilament heavy (NFHp), phosphorylation-independent neurofilament heavy (NFH), neurofilament light, neurofilament medium (NFM), microtubule, and microtubule-associated protein (MAP) were used to study the axonal cytoskeleton. Confocal microscopy was used to compare axonal transport and cytoskeleton change between control and high IOP eyes in different laminar regions and quadrants of the optic nerve head. Results from these experiments were also compared with 6-hour elevated IOP data from an earlier study., Results: Three hours of IOP elevation caused a decrease in NFH, NFHp, and NFM within laminar regions, with no demonstrable change in axonal transport. Changes to MAP and microtubules were only seen after 12 hours of IOP elevation. Axonal transport change occurred in a time-dependent manner with peripheral nerve bundle changes occurring earlier and being greater than central nerve bundle changes., Conclusions: Time-dependent changes in axonal transport and cytoskeletal structure in the optic nerve head provide further pathogenic evidence of axonal damage caused by elevated IOP.

Purpose: To investigate the axonal cytoskeleton changes occurring in the prelaminar region, lamina cribrosa, and postlaminar region of the porcine optic nerve after an acute increase in intraocular pressure (IOP) and whether this corresponds with axonal transport abnormalities., Methods: Six white Landrace pigs were used. The left eye IOP was elevated to 40 to 45 mm Hg for 6 hours, and the right eye IOP was maintained between 10 and 15 mm Hg. Rhodamine-beta-isothiocyanate (RITC) was injected into the vitreous of each eye at the beginning of the experiment, to study axonal transport. After euthanasia, optic nerves were removed and prepared for axonal transport and cytoskeleton studies. Antibodies to phosphorylated neurofilament heavy (NFHp), phosphorylation-independent neurofilament heavy (NFH), neurofilament light (NFL), neurofilament medium (NFM), microtubule, and microtubule-associated protein (MAP) were used to study the axonal cytoskeleton. Montages of confocal microscopy images were quantitatively analyzed to investigate simultaneous changes in optic nerve axonal transport and cytoskeletal proteins in the high-IOP and control eyes., Results: Axonal transport of RITC was reduced in the prelaminar, lamina cribrosa, and proximal 400 mum of the postlaminar optic nerve regions in the high-IOP eye. NFHp, NFM, and NFH were significantly reduced in the prelaminar, lamina cribrosa, and proximal postlaminar regions in the high-IOP eye. No differences in NFL, MAP, and tubulin staining were detected., Conclusions: Elevated IOP induced both axonal transport and cytoskeleton changes in the optic nerve head. Changes to the cytoskeleton may contribute to the axonal transport abnormalities that occur in elevated IOP.

Purpose: To determine intraretinal oxygen distribution and consumption in a rat model of retinal artery occlusion during air breathing and stepwise systemic hyperoxia., Methods: Laser occlusion of the pair of retinal arteries feeding the area of retina under investigation was performed. Oxygen-sensitive microelectrodes were then used to measure oxygen tension as a function of depth through the retina. Breathing mixtures were manipulated to produce stepwise increments in systemic oxygen levels, and the measurement of intraretinal oxygen distribution was repeated. Oxygen distribution in the retina was analyzed by an established eight-layer mathematical model of retinal oxygen consumption., Results: Intraretinal oxygen distribution in the occluded area confirmed that the choroid was the only source of retinal oxygenation. Under air-breathing conditions, the oxygen supply from the choroid was sufficient to support the photoreceptor inner segments. Any remaining oxygen was consumed by the outer plexiform layer. Increases in inspired oxygen level reduced the extent of inner retinal anoxia. However, some degree of anoxia in the innermost retina was usually present., Conclusions: Occlusion of the retinal circulation renders most of the inner retina anoxic. Ventilation with 100% oxygen does not generally avoid some degree of intraretinal anoxia. With 100% oxygen ventilation, the oxygen consumption of the inner retina was more than four times that of the outer retina. A marked degree of heterogeneity in oxygen uptake of different retinal layers was evident. The dominant oxygen consumers were the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer.

Background: Retinal vein pulsation is often absent in glaucoma, but can be induced by applying a graded ophthalmodynamometric force (ODF) to the eye, which is elevated in glaucoma., Aim: To assess whether ODF has a predictive value in determining glaucoma progression., Methods: 75 patients with glaucoma and suspected glaucoma were examined prospectively in 1996, and then re-examined at a mean of 82 months later. All subjects had intraocular pressure, visual fields, stereo optic disc photography and ODF measured on their initial visit. When venous pulsation was spontaneous, the ODF was said to be 0 g. At re-examination, central corneal thickness and blood pressure were also measured. Initial and subsequent optic disc photographs were compared and graded into those that had increased excavation and those that had remained stable. The relationship between increased excavation (recorded as a binary response) and the measured variables was modelled using a multiple mixed effects logistic regression., Results: ODF at the initial visit was strongly predictive of increased excavation (p = 0.004, odds ratio 1.16/g, range 0-60 g), with greater predictive value in women than in men (p = 0.004). Visual field mean deviation was predictive of increased excavation (p = 0.044), as was optic nerve haemorrhage in association with older age (p = 0.038). Central corneal thickness was not significantly predictive of increased excavation (p = 0.074) after having adjusted for other variables., Conclusion: ODF measurement seems to be strongly predictive of the patient's risk for increased optic disc excavation. This suggests that ODF measurement may have predictive value in assessing the likelihood of glaucoma progression.

We report on a method for delivering high fluence pulsed 266 nm laser radiation to the target tissue via an optical fiber. The fourth harmonic of a Nd:YAG laser was concentrated using a hollow glass taper and launched into an optical fiber. Fluences of up to 2 J/cm(2) were routinely output at the tapered optical fiber tip. The maximum fluence generated before failure of the optical fiber was between 3.5 and 8 J/cm(2). Ablation of ocular tissue was demonstrated using fluences of 1.0 and 0.4 J/cm(2). The delivery system has the potential for use in intraocular surgical procedures.

Purpose: To determine whether clinically relevant doses of sodium fluorescein produce changes in vascular tone in retinal arterioles and veins and whether any such effects were light dependent., Methods: Segments of porcine retinal arterioles and veins were dissected, cannulated, and perfused and their outer diameter monitored during intraluminal application of increasing doses (10(-10) to 10 (-3) g/ml) of sodium fluorescein under either brightly lit (350 lux) or dimly lit (4 lux) conditions. The significance of any induced change in vessel diameter was assessed in relation to the initial vessel diameter., Results: At the higher light level, sodium fluorescein produced a significant dose-dependent contraction in porcine retinal arterioles and veins with a threshold of 10 (-5) g/ml and 10 (-9) g/ml sodium fluorescein, respectively. At the maximal dose tested (10(-3) g/ml), vessel diameter was reduced to approximately 85% of the initial vessel diameter in retinal arterioles and veins. Under dimly lit conditions, the vasoconstrictive effect of sodium fluorescein was still evident but the constriction was significantly smaller (p < 0.05) in both arteries and veins, reaching approximately 91% and 93%, respectively., Conclusions: Sodium fluorescein induced light-dependent vasoconstrictive effects on the retinal vasculature of the pig. Should a similar effect be present in human retinal vessels, then reduced illumination level may limit the vasoconstrictive effects of sodium fluorescein when used in routine ophthalmic examinations.

Purpose: To determine the intraretinal oxygen distribution and oxygen consumption in the rat before eye opening and maturation of the retina., Methods: Oxygen-sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized Sprague-Dawley rats at postnatal day (P)15. Measurements were made under air-breathing conditions and at increasing levels of systemic oxygenation (20%, 40%, 60%, 80%, and 100% oxygen) under light-adapted conditions. Oxygen consumption in the outer retina and in the predominantly avascular region of the inner retina was assessed by fitting the oxygen profiles to a mathematical model of oxygen consumption. The retinas were processed for histology and compared with retinas from mature animals., Results: Under normal conditions, the intraretinal oxygen distribution at P15 was significantly different in some respects from that in mature animals. Oxygen consumption analysis indicated an average outer retinal oxygen consumption rate of 103 +/- 15 nL/min per cm2 and an inner retinal oxygen consumption rate of 42.4 +/- 11.7 nL/min per cm2. Inner retinal oxygen consumption was significantly (P < 0.001) lower than that previously measured in mature rats, but outer retinal oxygen consumption was similar. Systemic hyperoxia increased the oxygen level throughout the retina, but choroidal PO2 in particular remained significantly lower than in adult rats (P < 0.001). At P15 there were marked differences in the relative thickness of some retinal layers when compared with adult rats. In particular the inner and outer nuclear layers were much thicker at P15, the outer segments of the photoreceptors and the inner and outer plexiform layers were not fully developed., Conclusions: At P15, before eye opening, the oxygen consumption of the inner retina is lower than in mature retinas, presumably reflecting the immaturity of the retina in such young animals so soon after their first exposure to light stimuli.

Purpose: To explore the ablation potential of 266 nm laser pulses, with an intact porcine retina preparation., Methods: Segments of porcine eyes were used in an in vitro preparation in which localized areas of intact retina and choroid could be exposed to 266 nm laser irradiation. The segments of ocular tissue were bathed in fluid, to mimic the intraocular environment. Contact between the probe and the retinal surface was established before the first laser pulse. Single or multiple pulses (5-7 ns duration) at fluence levels of 0.4 to 1.2 J/cm2 were delivered via a tapered fiber optic probe with a tip size of approximately 110 microm. The retinal tissue was then fixed and sectioned for histologic examination. The ablation depth and extent of damage were measured and related to fluence level and the number of pulses applied., Results: Ablation of the inner retina was achieved by single pulses at fluence levels of 0.6 J/cm2 and higher. The depth of retinal ablation was highly dependent on fluence for lesions generated with a single pulse but less so for multiple pulses (3-10), particularly at lower fluence levels. Higher numbers of pulses (50-100) did not increase ablation depth in a predictable manner., Conclusions: Pulsed laser (266 nm) irradiation at low pulse counts and high fluence levels is a possible alternative for localized retinal ablation with minimal collateral damage in a fluid environment.

Purpose: To make the first measurements of intraretinal oxygen distribution in the mouse, an animal model of increasing importance in ophthalmic research., Methods: Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of depth through the retina and choroid in anesthetized mice (n = 8). All measurements were performed under light-adapted conditions, with the animals spontaneously inspiring room air. The oxygen distribution in the avascular portion of the outer retina was analyzed by an established three-layer mathematical model that determines outer retinal oxygen consumption., Results: The intraretinal oxygen distribution in the inner retina in individual profiles was often characterized by sharp peaks associated with elements of the retinal microvasculature, but, in the outer retina, the oxygen distribution was much more predictable and reflected the high oxygen uptake of the photoreceptors. Average choroidal oxygen tension was 42.0 +/- 1.2 mm Hg (mean +/- SE), and P(O2) at the surface of the retina was 21.7 +/- 0.8 mm Hg. The average minimum oxygen tension in the outer retina was 4.2 +/- 0.5 mm Hg. Average outer retinal oxygen consumption in the mouse was 193.3 +/- 10.6 nL O2/min per square centimeter, which is similar to that previously reported in the outer retina in vascularized areas of retina in the rat and monkey using similar techniques., Conclusions: The intraretinal oxygen distribution in the mouse is qualitatively and quantitatively similar to that in other species with vascularized retinas. The rate of oxygen consumption in the outer retina is also similar. These baseline data can now be used in studies employing mouse models of retinal disease.

Objective: To investigate the origin of signals from scanning laser Doppler flowmetry (SLDF) and the influence of axial scan depth on the measurement of blood flow in the rat retina., Methods: We performed SLDF in 5 adult Sprague-Dawley rats using a specially modified Heidelberg retina flowmeter. Axial scans were obtained from +2 diopters (D) to -3 D (in steps of 0.25 D) or from +1 D to -2 D (in steps of 0.125 D) relative to the retinal surface. Fluorescein isothiocyanate-dextran angiograms were obtained in whole-mounted retinas to visualize the angioarchitecture and identify measurement locations in the SLDF flow maps. Axial SLDF flow profiles were obtained in an artery, vein, arteriole, venule, and capillary bed using the mean blood flow values in 2 x 2-, 4 x 4-, and 10 x 10-pixel measurement windows., Results: The SLDF images showed good correspondence with the angiograms and resolution to third-order arterioles and venules; however, neither the superficial nor deep capillary circulations were visualized. Flow was imaged from large choroidal vessels. Measured flow from capillaries was independent of depth and indistinguishable from background levels., Conclusion: The technique of SLDF images blood flow in larger retinal vessels but not in capillaries. Clinical Relevance Scanning laser Doppler flowmetry may not reliably measure capillary blood flow.

The rabbit eye presents a valuable model to study the effects of vascular occlusion on the function and structure of myelinated nerve fibers. The rabbit eye has a band of myelinated nerve fibers within the intraocular compartment that are supplied by a narrow band of retinal vasculature. These vessels were transiently occluded ( approximately 8 hours) using laser photocoagulation and the transmission of electrical signals along the nerve fibers was assessed by recording the visual evoked response (VER). Morphological damage was assessed by histological techniques. The ischemic insult produced no permanent change in retinal function as assessed by electroretinography, but the VER was suppressed, indicating failure of nerve fiber transmission. Histologically, the visible damage to the region supported by the retinal vasculature worsened following reperfusion, showing evidence of demyelination and necrosis followed by macrophage responses and gliosis. This rabbit model of ischemia/reperfusion of the retinal vasculature offers a rare opportunity to reliably study the response of myelinated nerve fibers to ischemia/reperfusion insults and has demonstrated the susceptibility of myelinated nerve fibers to such insults.

The aim of this study was to improve our ability to interpret and validate Heidelberg Retina Flowmeter (HRF) flow images by recording flow measurements from specific regions of the retinal vasculature by taking advantage of the ability to precisely regulate perfusion flow in an isolated eye preparation. The retinal vasculature in 16 isolated perfused pig eyes was perfused with a 50%/50% Krebs/RBC solution at known flow rates ranging from 0 to 300 microl min(-1). At each flow rate, HRF images were obtained at a location approximately two disc diameters from the disc. After HRF image acquisition, the retinal vasculature was perfused with fluorescein isothiocyanate for fluorescence microscopy. Using the standard HRF software and a 10 x 10pixel measurement window, flow rates were measured from a retinal artery, vein, arteriole, venule, and the retinal capillary bed and a capillary-free-zone. The relationship between HRF measured flow and perfusion flow in the different measurement locations was determined. At zero perfusion flow the measured HRF flow was consistently greater than zero ( approximately 170 arbitrary units (AU)), and not significantly different at each measurement location except for the retinal vein, which had a significantly higher HRF flow value ( approximately 230AU). At higher perfusion flow rates the flow signal from the larger vascular elements (arteries and veins) increased rapidly thereafter to reach several thousand AU at a total perfusate flow of 50 microlmin(-1) and increased less rapidly at higher flow rates. In arterioles, the HRF flow was more linear over a broader range of perfusate flow rates but the peak flow signal was an order of magnitude smaller than that from the retinal artery. Both the linearity and magnitude of the flow signal in venules was less than that in arterioles. In capillary areas and in the capillary free zone, the HRF flow showed only a very weak relationship to perfusion flow when compared to the background noise. The choice of location for HRF flow analysis greatly influences the ability of the technique to measure changes in retinal blood flow. The major arteries and veins provide the strongest signal and greatest signal to noise ratio. However, the retinal arterioles produce an HRF signal that is more linear over a wider range of perfusate flow rates.

Purpose: To measure the intraretinal oxygen distribution and consumption in the fovea, the parafovea, and the inferior retina in the monkey eye and determine the influence of graded systemic hyperoxia., Methods: Oxygen sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized monkeys (n = 8) under normoxic and hyperoxic conditions. Oxygen consumption rates in the avascular regions of retina were determined by fitting the oxygen profiles to established oxygen consumption models., Results: Under normoxic conditions, in the foveal area, the intraretinal oxygen distribution reflected the absence of retinal capillaries and the predominantly choroidal origin of retinal oxygenation. A similar shape of oxygen distribution was seen in the parafoveal retina with the addition of local perturbations in the inner retina attributed to the presence of retinal capillaries. In the inferior retina the same general shape was found. Oxygen consumption in the outer retina was higher in the parafoveal region, and the minimum oxygen tension was lower. During hyperoxia, choroidal oxygen levels in all areas increased dramatically, but the increase in oxygen tension in the inner retina was much less. The avascular nature of the foveal area allowed oxygen consumption analysis of both the inner and outer retina and showed that inner retinal oxygen consumption increased significantly during hyperoxic ventilation to a level equivalent to that of the outer retina., Conclusions: In the outer retina of the monkey the P(O2) minimum is lower, and the oxygen consumption rate is higher in the parafoveal region. During systemic hyperoxia, outer retinal oxygen consumption is unaffected, but in the foveal area, total oxygen consumption increases. This regulation of oxygen consumption in the monkey retina is comparable to that reported in lower mammals and may represent an important mechanism in retinal homeostasis.

Purpose: To determine whether a sphincter-like control point is present in 90 degrees branches of the retinal vasculature. Such a control point could selectively control retinal blood flow to the deeper capillary layers., Methods: A microperfusion system was used in which porcine retinal arteries with intact branches were cannulated and perfused intraluminally at physiological flow rates. The vasoactive response of the proximal and distal regions of either 90 degrees -or Y-type branches to intraluminal potassium (124 mM) were monitored simultaneously in real-time., Results: The proximal region of the 90 degrees branches demonstrated a localized vasoconstriction when compared to the more distal region (p < 0.0001). In contrast, the Y branches contracted evenly along their length., Conclusions: The localized vasoactivity near the branch point of the 90 degrees -type branches may explain previous observations of so-called sphincter-like activity in these vessels. These sphincters may selectively regulate blood flow to the deeper retinal capillary layer.

Vision loss due to various forms of outer retinal degeneration remains a major problem in clinical ophthalmology. Most retinal degenerations are precipitated by genetic mutations affecting the retinal pigment epithelium and sensory retina, but it is becoming increasingly evident that resultant metabolic changes within the retina may also contribute to the further progression of photoreceptor cell loss. In particular, a role for the local oxygen environment within the retina has been proposed. The correct balance between retinal oxygen supply and oxygen consumption in the retina is essential for retinal homeostasis, and disruption of this balance is a factor in many retinal diseases. In animal models of photoreceptor degeneration, manipulation of environmental oxygen levels has been reported to be able to modulate the rate of photoreceptor degeneration. Clinically, hyperbaric oxygen therapy has already been used in retinitis pigmentosa patients and other types of oxygen therapy have been proposed. It therefore seems appropriate to review our current understanding of the oxygen environment in the normal and degenerating retina, and to build a clearer picture of how the retinal oxygen environment can be modulated. We focus on techniques that have been, or may be, applied clinically, such as modulation of systemic oxygen levels and modulation of retinal oxygen metabolism by light deprivation. Data from direct measurements of intraretinal oxygen distribution in rat models at different stages of photoreceptor degeneration will be reviewed. These models include the Royal College of Surgeons (RCS) rat, and the P23H rat model of outer retinal degeneration. Microelectrode based techniques have allowed the intraretinal oxygen distribution to be measured as a function of retinal depth under well-controlled systemic conditions at different stages of the degeneration process. Both models showed changes in the intraretinal oxygen distribution during the degenerative period, with the changes reflecting the gradual loss of oxygen metabolism of the degenerating photoreceptors. This results in higher than normal oxygen levels in the remaining outer retina and a significant alteration in the oxygen flux from the choroid to the inner retina. The maintenance of normal oxygen levels in the inner retina implies that inner retinal oxygen uptake is well preserved, and that there is also reduced oxygen input from the deeper capillary layer of the retinal circulation. Choroidal oxygen tension and the oxygen tension in the pre-retinal vitreous were unaffected at any of the time periods studied prior to, and during, the degeneration process. It is well known that both hypoxia and hyperoxia can cause neural cell stress and damage. Logically, any therapeutic intervention based on oxygen therapy should attempt to restore the oxygen environment of the remaining retinal cells to within the physiological range. Before any oxygen based therapies for the treatment of retinal degeneration should be seriously considered, the oxygen environment in the degenerating retina should be determined, along with clinically usable methods to restore the oxygen environment to the critical cell layers.

Purpose: To determine the factors associated with central retinal vein pulsation changes in glaucoma and identify any hemiretinal vein pulsation changes and their association with sectoral visual field loss., Methods: One hundred twenty-six patients with glaucoma and 40 normal subjects had automated perimetry, blood pressure, and intraocular pressure measured. A hemifield sensitivity loss was calculated from the upper and lower halves of each field. Those without spontaneous venous pulsation on the optic disc had an ophthalmodynamometer applied, to measure the minimum ophthalmodynamometric force (ODF) necessary to induce venous pulsation. When ODF was restricted to the hemiveins, the force needed to induce pulsation in each hemivein was measured., Results: Eighty-three patients with glaucoma had no spontaneous venous pulsation. The minimum ODF was strongly correlated with mean deviation (Spearman rank r = -0.475, P < 0.0001). Mixed linear regression analysis showed that mean deviation (P < 0.0001) and pulse blood pressure (P < 0.0001) were significantly associated with minimum ODF. There was a strong association between differences in hemifield sensitivity loss and in hemivein ODF (rank r = 0.369, P < 0.0001, n = 80). Multiple linear regression modeling demonstrated that lower hemivein ODF was independently associated with upper field loss (P = 0.003) and upper hemivein ODF with lower field loss (P < 0.0001)., Conclusions: These venous pulsation findings in glaucoma are independent of blood pressure. The hemifield and hemivein association suggests that the major hemivein change is adjacent to the site of major disc damage.

Purpose: The purpose of this study was to characterize the endothelial (EC) F-actin cytoskeleton at different orders of the retinal microvasculature in the normal and diabetic rat and determine if changes in F-actin are associated with different stages of diabetes., Methods: The EC F-actin cytoskeleton distribution, nuclei shape, and capillary diameter in the retinal vasculature of rats after 5 and 28 weeks of streptozotocin (STZ)-induced diabetes were compared to those in age-matched controls. The eyes were enucleated, arterially perfused, and labeled for F-actin cytoskeleton and nuclei (YO-PRO-1) or microvascular leakage (FITC-dextran). Retinal whole mounts were then examined by confocal microscopy., Results: The EC F-actin distribution and nuclear size and shape were highly dependent on the location down the vascular tree. The retinal arterial system in the rat shows a high level of F-actin stress fibre (SF) staining. Peripheral border (PB) staining was present in the ECs of all vessels. Diffuse F-actin staining was observed in endothelial cytoplasm in capillaries, venules, and veins. EC nuclei became distinctly less elongated down the vascular tree. In diabetic rats at 5 weeks, at the capillary level the F-actin staining was more diffuse, and areas of F-actin loss were evident. Both dot-like and diffuse leakage was detected in retinal capillaries, and these leakage types were closely associated with the degree of F-actin changes. In diabetic rats at 28 weeks, there was an increased level of SF staining in the arterial system in addition to capillary F-actin changes., Conclusions: The EC F-actin cytoskeleton and nuclei shape retinal microvasculature of the normal rat change with location along the vascular tree. In the early stages of diabetes, there are changes to the F-actin cytoskeleton that are clearly associated with microvascular leakage. F-actin distribution could indicate important structural changes in the pathogenesis of diabetic retinopathy.

Purpose: To make the first measurements of intraretinal oxygen distribution and consumption after laser photocoagulation of the retina and to compare the efficiency of micropulsed (MP) and continuous wave (CW) laser delivery in achieving an oxygen benefit in the treated area., Methods: Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of retinal depth before and after laser treatment in anesthetized, mechanically ventilated, Dutch Belted rabbits (n = 11). Laser lesions were created by using a range of power levels from an 810-nm diode laser coupled with an operating microscope delivery system. MP duty cycles of 5%, 10%, and 15% were compared with CW delivery in each eye., Results: Sufficient power levels of both the CW and MP laser reduced outer retinal oxygen consumption and increased oxygen level within the retina. At these power levels, which correlated with funduscopically visible lesions, there was histologically visible damage to the RPE and photoreceptors. Retinal damage was energy dependent but short-duty-cycle MP delivery was more selective in terms of retinal cell damage, with a wider safety range in comparison with CW delivery., Conclusions: The relationship between laser power level and mode of delivery and the resultant changes in oxygen metabolism and oxygen level in the retina was determined. Only partial destruction of RPE and photoreceptors is necessary, to produce a measurable oxygen benefit in the treated area of retina.

Purpose: To improve the interpretation of Heidelberg Retina Flowmeter (HRF; Heidelberg Engineering GmBH, Dosselheim, Germany) flow maps by examining the influence of specific vascular structures and focus depth in the presence and absence of retinal blood flow., Methods: HRF flow maps were recorded from the inferior retina of anesthetized Brown Norway rats over a wide range of focus levels, before and after laser occlusion of the retinal circulation. Analysis of the resultant flow maps showed that the sample window was positioned on a retinal artery, arteriole, or vein, or in a retinal capillary area, with or without a visible underlying choroidal vessel. The relationship between HRF-measured flow (arbitrary units) and focus depth was determined for each location. At the conclusion of each experiment, the effect of reduction of systemic blood pressure on the choroidal circulation and the level of background signal in the HRF flow map with no ocular blood flow were assessed., Results: The strongest flow signals came from the retinal arteries, veins, and arterioles and were reduced to choroidal background level after occlusion of the central retinal artery. Larger choroidal vessels also contributed strong flow signals. In contrast, the flow signal from the retinal capillary area was weak and unaffected by retinal artery occlusion. Changing the depth of focus significantly altered the contribution from the major retinal arteries, arterioles, and veins, but no significant depth effect was seen for retinal capillaries or choroidal vessels. The HRF flow signal remaining when systemic blood pressure was reduced to zero was not significantly different from the capillary sampling location when the eye was normally perfused., Conclusions: In the pigmented rat eye, the HRF signal from retinal capillaries is not significantly different from the background noise unrelated to blood flow. Strong flow signals can be obtained from the retinal arteries, retinal arterioles, retinal veins, and choroidal vessels. Current HRF flow maps in the rat therefore reflect blood flow in the larger elements of the microvasculature rather than the capillary network.

Purpose: To determine the ability of the retina and choroid in the avascular region of the rabbit retina to regulate intraretinal oxygen levels during periods of acute systemic hyperoxia., Methods: Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of depth through the retina and choroid in anesthetized rabbits before and after stepwise incremental increases in inspired oxygen level. The extent of any oxygen increases throughout the retina and choroid was determined, and retinal oxygen consumption was determined by analyzing intraretinal oxygen distribution., Results: Increases in systemic arterial oxygen in the rabbit resulted in significant increases in oxygen tension in the choroid and in all retinal layers. There was no apparent regulation of choroidal oxygen tension, as seen in the avascular retina of the guinea pig. Neither was there any increase in oxygen consumption within the retina, which has been shown to dampen the extent of raised oxygen levels in the inner retina in other mammals. Consequently, all retinal layers in the avascular area of the rabbit retina were exposed to high oxygen levels during systemic hyperoxia. The magnitude of the oxygen changes in all retinal layers was consistent with that in the preretinal vitreous., Conclusions: Unlike other mammals studied to date, the rabbit does not possess any regulatory mechanisms for controlling the intraretinal oxygen environment during acute increases in systemic arterial oxygen levels. This may well account for the reported vulnerability of the rabbit retina to toxic damage during extended periods of systemic hyperoxia., (Copyright Association for Research in Vision and Ophthalmology)

Purpose: To determine whether changes in central retinal vein pulsation characteristics occur in glaucoma, and how these are related to indices of glaucoma severity., Design: A large, consecutive, prospective, case-controlled study., Participants: Ninety-four consecutive glaucoma patients and 105 glaucoma suspects seen in a tertiary referral clinic were examined. Forty-one age-matched normal subjects also were examined., Methods: The presence or absence of spontaneous venous pulsation was observed in these 3 groups. The ophthalmodynamometric force (ODF) required to induce venous pulsation at the optic disc was measured in those without spontaneous pulsation. Optic disc photographs were obtained and visual field testing was performed for all subjects., Main Outcome Measures: The prevalence of spontaneous venous pulsation between these 3 groups was compared. The relationship between ODF and visual field mean deviation, neuroretinal rim area, age, intraocular pressure (IOP), gender, and diagnosis of glaucoma was investigated using linear mixed models fitted by Gibb's sampling., Results: Significantly fewer (chi-square, 27.7; P<0.001) glaucoma patients (54%) were observed to have spontaneous venous pulsation than suspects (75%) or normals (98%). A worse visual field mean deviation was shown to be the most significant predictor of a higher ODF (P<0.000), with younger age (P<0.000) also predictive of a higher ODF. A strong relationship between ODF and mean deviation was found in the glaucoma patients (r = 0.59; n = 52; P<0.001)., Conclusions: Spontaneous venous pulsation is less common in glaucoma. The ODF required to induce venous pulsation is increased in glaucoma, and this ODF is greater in those with more severe field loss.

The vasoactive effect of endothelin-1 applied intraluminally or extraluminally was studied in vitro in isolated perfused porcine pulpal arterioles using a microperfusion system. Pulpal arterioles (outer diameter, 94.2 +/- 2.8 microm, n = 12) were cannulated and perfused at a constant flow rate in an environment-controlled bath on the stage of an inverted microscope. The vessel diameters were measured online. Both intraluminal and extraluminal application of endothelin-1 (10(-16) M to 10(-8) M) induced dose-dependent constrictions, reaching 82.3 +/- 1.7% (n = 12) and 70.5 +/- 1.3% (n = 12) at 10(-8) M, respectively. Nifedipine reversed endothelin-1-induced constriction dose-dependently at 10(-7) M and above. These data demonstrate that endothelin-1 induces calcium-dependent vasoconstriction in porcine pulpal arterioles, with extraluminal application more potent, which seems to reflect the possible modulation of vascular endothelium in the control of vascular tone.

The oxygen requirements of different retinal layers are of interest in understanding the vulnerability of the retina to hypoxic damage in retinal diseases with an ischemic component. Here, we report the first measurements of retinal oxygen consumption in the visual streak of the rabbit retina, the region with the highest density of retinal neurons, and compare it with that in the less-specialized region of the retina underlying the vascularized portion of the rabbit retina. Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of retinal depth in anesthetized animals. Measurements were performed in the region of the retina containing overlying retinal vessels and in the center of the visual streak. Established mathematical analyses of the intraretinal oxygen distribution were used to quantify the rate of oxygen consumption in the inner and outer retina and the relative oxygen contributions from the choroidal and vitreal sides. Outer retinal oxygen consumption was higher in the visual streak than in the vascularized area (means +/- SE, 284 +/- 20 vs. 210 +/- 23 nl O2.min(-1) x cm(-2), P = 0.026, n = 10). However, inner retinal oxygen consumption in the visual streak was significantly lower than in the vascular area (57 +/- 4.3 vs. 146 +/- 12 nl O2 x min(-1) x cm(-2), P < 0.001). We conclude that despite the higher processing requirements of the inner retina in the visual streak, it has a significantly lower oxygen consumption rate than the inner retina underlying the retinal vasculature. This suggests that the oxygen uptake of the inner retina is regulated to a large degree by the available oxygen supply rather than the processing requirements of the inner retina alone.

The purpose of this review is to outline the techniques and applications for isolated ocular vascular preparations and their significance to ophthalmic research. Various isolated ocular vascular preparations have been utilized in studies of ocular vascular biology, physiology and pharmacology, including work in both normal and diseased conditions. However, there is still significant potential for further studies to improve our understanding of the ocular circulation and its regulation. Experience has shown that there is no single preparation capable of addressing all of the questions that must be answered if a complete understanding of mechanisms of vascular regulation in the eye is to be achieved. Rather, it is necessary to select the appropriate preparation and techniques to address each individual question in the most appropriate manner. In this review, particular emphasis is placed on the applications for isolated ocular preparations and the relevance of such studies to our understanding of the pathogenesis of eye diseases involving the vasculature. Examples are given where therapeutic approaches in diabetes and glaucoma are assessed in terms of their impact on the vasoactive properties of the ocular vasculature.A significant heterogeneity is present in the different parts of the ocular vasculature, not only in the structural and functional properties of vessel itself, but also in terms of the tissue environment and innervation. A single vasoactive agent may also have different effects when applied to the inside or the outside of the same region of a vessel. The vasoactive response of the vascular system as a whole is what determines the rate of blood flow through the system, but this is regulated by a multitude of factors in different regions of the vascular network. Isolating individual components of the ocular vasculature is readily achievable for the extraocular vessels such as the ophthalmic or ophthalmocilliary arteries, which can be studied in myograph type systems measuring the mechanical vasoactive force generated by the vessel. Retinal vessels from very large animals can also be studied in this way, but the small diameter of the retinal vessels in most species requires a perfusion rather than myograph based technique. Perfusion based studies of vessel diameter in response to vasoactive stimuli can be applied to individual retinal arteries and their branches. Perfusion of more complex elements of the ocular vasculature such as isolated segments of the retina or ciliary body, or whole isolated perfused eyes may use the perfusate pressure as the determinant of vasoactive state. However, when several components of the ocular vasculature are being perfused simultaneously it may be difficult to separate out the contribution from the different vascular elements. The advantage of isolated preparations is that systemic influences can be eliminated, and vascular components can be studied that are inaccessible in vivo. The disadvantage is that no matter how well controlled the in vitro environment may be, it will always be a relatively poor mimic of the in vivo conditions. However, such in vitro work has certainly improved our understanding of the vasoactive properties of different regions of the ocular vasculature in both health and disease.

The effects of endogenous vasoactive substances were evaluated in anaesthetized rats using a laser Doppler flowmeter to monitor changes in pulpal blood flow, as well as directly in isolated pulpal arteriole preparations utilising a microperfusion and monitoring system to observe changes in vessel diameter. In anaesthetized rats, while systemic arterial blood pressure remained relatively stable, intra-arterial delivery of adrenaline (epinephrine) (A), noradrenaline (norepinephrine) (NA), phenylephrine (PHE), dopamine (DOPA), 5-hydroxytryptamine (5-HT), or endothelin-1 (ET-1) produced a dose-dependent reduction in pulpal blood flow (order of potency: ET-1>>A=NA>PHE=DOPA=5-HT); acetylcholine induced a dose-dependent increase in pulpal blood flow; histamine, isoproterenol and adenosine produced no significant changes. In isolated arteriole preparations, intraluminal delivery of A, NA, PHE, DOPA or 5-HT produced dose-dependent vasoconstriction (A=NA>PHE=DOPA=5-HT). Acetylcholine relaxed NA-precontracted vessels dose-dependently. Histamine and isoproterenol produced a small vasodilatation. Intraluminal ET-1 produced a small vasoconstriction at 10(-8)M, whereas extraluminal ET-1 produced a dose-dependent vasoconstriction from 10(-10)M and above. Intraluminal adenosine failed to dilate vessels precontracted with ET-1, whereas extraluminal adenosine caused a complete relaxation. These combined in vivo and in vitro data suggest that, in the rat incisor, the pulpal microcirculation is capable of functional regulation and that pulpal blood flow may be modulated by endothelium-related factors, metabolic (tissue-related) factors, as well as humoral (blood-borne) factors.

Purpose: To determine the effect of intraocular pressure (IOP) and cerebrospinal fluid pressure (CSFP) on optic disc movement and lamina cribrosa displacement using confocal scanning laser tomography (CSLT)., Methods: The anterior chamber and lateral ventricles were cannulated in mixed-breed dogs (n = 8) to allow modulation and control of IOP and CSFP, respectively. Optic disc topography was determined after baseline (set at IOP 15 mm Hg and CSFP of 0 mm Hg) and with each step-wise increase in IOP (steps of 3-5 mm Hg up to an average of 32 mm Hg) with CSFP fixed at 0 mm Hg. After the pressure returned to baseline, images were obtained after each step-wise increase in CSFP (steps of 2 to 4 mm Hg up to an average of 12 mm Hg) with IOP fixed at 15 mm Hg. Data were analyzed by a new probabilistic method for CSLT and global parameters generated by the instrument software. The global parameter changes from baseline were analyzed as a function of the translaminar pressure difference (IOP minus CSFP)., Results: Elevation in IOP resulted in significant posterior displacement of the disc surface, whereas elevation in CSFP resulted in significant anterior displacement. For a given degree of pressure change, an increase in CSFP resulted in larger changes than a corresponding increase in IOP. The deepest 5% of locations within the disc surface were displaced nonlinearly (with an inverse exponential function, r = 0.92) as a function of the difference in translaminar pressure. Most displacement occurred at low translaminar pressure differences, with little extra movement at differences higher than 15 mm Hg. The change in the volume subtended by the anterior lamina cribrosa showed a nonlinear relationship similar to the translaminar pressure difference (r = 0.98), with negligible volume change at high difference in pressures., Conclusions: Most optic disc movement occurs with pressure changes in the low range of translaminar pressure differences. This is consistent with the mechanical properties of collagen.

The aim was to determine the oxygen tension (P(O(2))) and rate of oxygen consumption in the pulp. Twelve rats were anaesthetised and artificially ventilated. Under an operating microscope, a recessed oxygen-sensitive microelectrode was inserted into the pulp through a small saline-covered cavity on the labial surface of the lower incisor. P(O(2)) was measured as a function of the transverse distance from the saline medium through to the middle of the pulp. Oxygen profiles were characterised by a decline of oxygen tension outside the pulp in the saline medium and a steeper gradient across the interface, before a localised oxygen consuming region corresponding to the odontoblasts. A plateau with some localised fluctuations was then followed by an increase in oxygen tension in the middle of the pulp. The average oxygen tension in the plateau region was 23.2 mmHg+/-2.1 mmHg (n=12). A mathematical model was used to extract oxygen consumption data from P(O(2)) profiles recorded from non-perfused pulp (created by reducing systemic blood pressure). The analysis revealed that there was a distinct oxygen consumption zone in the outer pulp, which anatomically corresponded to the odontoblast layer. The average oxygen consumption rate of the odontoblasts was 3.2+/-0.2 ml O(2)/min per 100g pulp tissue. The zone of high oxygen consumption was 68.7 micro m+/-6.9 micro m (n=24) thick. It is concluded that pulpal oxygen distribution is heterogeneous and that the odontoblast could be a major oxygen consumer within the rat incisor pulp.

Purpose: To make the first quantitative assessment of the rate of oxygen consumption in high oxygen-consuming layers of both the outer and inner retina of the rat in vivo., Methods: Oxygen-sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized rats. Individual PO2 profiles were fitted to a multilayer mathematical model of PO2 distribution that is able to determine the oxygen uptake in those retinal layers in which the oxygen supply is through diffusion from vascular layers of the retina. This includes the entire outer retina and the region of the inner retina containing the inner plexiform layer. Measurements were performed in the light (13 mW/cm2 at the cornea) and in the dark and the amplitude and time constant for light-induced changes in outer retinal oxygenation determined., Results: Under light-adapted conditions, the oxygen consumption of the outer retina was 148 +/- 11 nL O2/min/cm2 (n = 20) [corrected] and that of the included portion of the inner retina was 184 +/- 17 nL O2/min/cm2 [corrected]. In the dark, outer retinal oxygen consumption increased by 47.8% (P < 0.001), and the time constant for the resultant PO2 decrease in the outer retina was 14.9 +/- 1.8 seconds (n = 16). There was no significant change in inner retinal oxygen consumption between light and dark conditions (P = 0.89)., Conclusions: Under light-adapted conditions the oxygen uptake by the selected region of the inner retina (primarily the inner plexiform layer) is greater than that of the outer retina (P < 0.01). Dark adaptation rapidly and significantly increases outer retinal oxygen consumption, but the inner retina remains unaffected.

A multi-layer mathematical model of oxygen supply and consumption in the rat retina is described. The model takes advantage of the highly layered structure of the retina and the compartmentalisation of the available oxygen sources. The retina is divided into eight layers, each with a distinct oxygen consumption or supply rate. When applied to the available data from intraretinal oxygen measurements in the rat under normal physiological conditions, a close fit between the model and the data was achieved (r(2)=0.98+0.005, n=6). The model was then used to investigate recent evidence of oxygen regulating mechanisms in the rat retina during systemic hyperoxia. Fitting our model to the experimental data (r(2)=0.988+0.004, n=25) allowed the relative oxygen delivery or consumption of the key retinal layers to be determined. Two factors combine to produce the relative stability of inner retinal oxygen levels in hyperoxia. The retinal layer containing the outer plexiform layer/deep retinal capillaries, switches from a net source to a net consumer of oxygen, and the oxygen consumption of the outer region of the inner plexiform layer increases significantly. The model provides a useful tool for examining oxygen consumption and supply in all retinal layers, including for the first time, those layers within the normally perfused inner retina.

The purpose of this study was to measure the intraretinal oxygen distribution across the retina under conditions, which maximise outer retinal oxygen consumption. In particular, we looked for evidence of increased oxygen delivery from the choroid and the deep retinal capillary layer, and whether or not this was sufficient to avoid the development of intraretinal anoxia. Under dark-adapted conditions the photoreceptors need additional energy, at least part of which is derived from increased oxidative metabolism. In earlier studies in the cat retina it was revealed that dark adaptation could render some regions of the outer retina anoxic. The present study of the in vivo oxygen distribution across the rat retina in light and dark found no evidence of outer retinal anoxia in the dark. This was despite a mean increase of 52.6+/-11.4% (n=7) in outer retinal oxygen consumption in the dark. The mean value for the minimum outer retinal PO(2) in the dark was 5.2+1.2 mmHg. Oxygen delivery from both the choroid and the deep retinal capillary layer increased in the dark (P<0.01, and P<0.001, respectively). It is argued that the ability of the deep capillary layer to compensate for changes in oxygen demand in the outer retina is an important element in the maintenance of homeostasis in the retina. This is in addition to the role of the deep capillary layer in supplying oxygen to the highly consuming plexiform layers within the inner retina. These findings in the rat retina also demonstrate that intraretinal anoxia in the dark, is not, as implied by earlier work in the cat, a general feature of mammalian retinas.

The role of oxygen in the regulation of the pulpal microcirculation is unknown. This investigation is aimed to measure tissue oxygen tension and blood-flow changes in the pulp of rat lower incisors during graded systemic hyperoxia, and to determine the response of the pulpal vasculature to various oxygen tensions. Twenty-four Sprague-Dawley rats were anaesthetized and artificially ventilated with the appropriate gas mixture. Recessed oxygen-sensitive microelectrodes were used to measure pulpal tissue oxygen tension via a small access cavity filled with saline on the labial surface of the incisor. A laser Doppler flowmeter was used to record pulpal blood-flow. Inspired oxygen was increased stepwise from 20 to 100% in 20% steps. Systemic blood-gas concentrations were measured at each step. Systemic arterial oxygen tension at 100% oxygen ventilation reached 481.2 +/- 30.7% of the baseline at 20% oxygen breathing (n=21). Pulpal tissue oxygen tension did not change significantly whereas pulpal blood-flow fell dose-dependently to 74.6 +/- 5.0% at 100% oxygen ventilation (n=21). Systemic hyperoxia, therefore, induces a significant reduction in pulpal blood-flow whereas pulpal tissue oxygen tension remains relatively stable, indicating an oxygen-dependent local regulatory mechanism.

A novel isolated perfused pulpal arteriole preparation and microperfusion system was used to evaluate the direct vasoactive responses of pulpal arterioles to selected agonists. Short lengths of porcine pulpal arterioles (101.7+/-2.2 microm o.d., n=105) were dissected out and placed in an environment-controlled bath on the stage of an inverted microscope. Both ends of the vessel were cannulated and perfused at a controlled rate through the lumen. The diameter of the vessel was measured online. Following equilibration, the vessel was challenged with various agonists: adrenaline (epinephrine), noradrenaline (norepinephrine), phenylephrine, dopamine, isoproterenol, 5-hydroxytryptamine, histamine and adenosine. The endothelium-dependent vasodilator acetylcholine was used to evaluate endothelial cell function. Adrenaline, noradrenaline, phenylephrine, 5-hydroxytryptamine and dopamine caused dose-dependent contractions (adrenaline=noradrenaline>phenylephrine>dopamine>5-hydroxytryptamine). Isoproterenol and histamine provoked a dose-dependent dilation. Adenosine produced pronounced vasodilatation in vessels precontracted with 10(-8)M endothelin-1. Functional adrenergic, histamine, 5-hydroxytryptamine and adenosine receptors are, therefore, present in porcine pulpal arterioles. The isolated perfused pulpal arteriole preparation may prove valuable in understanding local control mechanisms of pulpal microcirculation.

The presence of cholinergic mechanisms in the control of pulpal microcirculation has been a controversial issue. In this study, we aimed to determine the direct vasoactive responses of isolated pulpal arterioles to acetylcholine, and to investigate whether such responses are endothelium-dependent. Using an in vitro micro-perfusion system, we isolated pig pulpal arterioles, cannulated and perfused them intraluminally, and monitored the diameter. Following equilibration, the vessels were contracted with 10(-5) M noradrenaline, and the effect of increasing doses of acetylcholine was determined. The influence of the muscarinic antagonist, atropine, or the loss of endothelial cell function following saponin treatment was also determined. Acetylcholine induced a dose-dependent vasodilation, reaching 94.6+/-1.4% (n = 22) of the uncontracted diameter at 10(-4) M. The vascular relaxation effect of acetylcholine was abolished in the presence of atropine, and by saponin treatment. Analysis of these data suggests that, in the pig, the acetylcholine-induced vasodilation of incisor pulpal arterioles is endothelium-dependent and mediated by muscarinic receptors.

Diabetic retinopathy is the leading cause of new blindness in the working-age population. If improved treatment regimens are to be developed it is crucial that the underlying pathophysiological mechanisms responsible for diabetic retinopathy are better understood. The multifactorial nature of the many pathways implicated in diabetic retinopathy requires a very detailed approach to elucidate the key mechanisms involved and their interactions in order to develop logical strategies aimed at therapeutic intervention. Fortunately, the streptozotocin rat model of diabetes displays many of the morphological and functional changes to the retinal vasculature that are evident in human diabetic retinopathy. This study reviews some of the recent experimental work by the authors in the streptozotocin rat, compares their findings to the human pathology and outlines potential new avenues for therapeutic intervention. In particular the improved understanding of which layers of the inner retina have the most stringent metabolic demands has helped identify which retinal layers are most susceptible to metabolic or hypoxic/ischaemic insult. It is concluded that improved treatment outcomes may ensue if the therapy is targeted at the appropriate tissue at specific stages of the disease.

Purpose: To compare the vasoactive properties of the docosanoid unoprostone, its free acid, and different members of the prostanoid family on isolated perfused pig retinal arterioles to assess their potential to modulate retinal blood flow., Methods: Segments of porcine retinal arterioles were dissected, cannulated, and perfused, and their diameter monitored during either intraluminal or extraluminal application of increasing doses (10(-10)-10(-4) M) of either the docosanoid unoprostone isopropyl and its free acid or of selected prostanoids: prostaglandin (PG) F(2alpha) and thromboxane A(2) analogue (U46619). Studies were performed on arterioles in their uncontracted state, and also during precontraction with endothelin-1 (10(-9) M). The significance of any induced change in vessel diameter was assessed in relation to the initial vessel diameter or, in the case of endothelin-1 administration, to the contracted diameter with endothelin-1 alone., Results: In normal-tone arterioles without endothelin-1 contraction, PGF(2alpha) and U46619 both produced a potent dose-dependent contraction, but neither unoprostone isopropyl nor unoprostone free acid had a significant vasoactive effect. In endothelin-1-contracted arterioles, U46619 produced further contraction, PGF(2alpha) produced a slight vasodilatation, and unoprostone isopropyl and its free acid produced a pronounced dilatation., Conclusions: Of the agents tested, unoprostone isopropyl and its free acid were the most potent vasodilators of endothelin-1-contracted pig retinal arterioles. Members of the prostanoid family demonstrated a different effect on the diameter of isolated retinal arterioles compared with the docosanoids. The potential therefore exists for the docosanoid unoprostone to have a beneficial effect on retinal blood flow in addition to any reduction in intraocular pressure.