Proteomics of the aqueous humor in healthy New Zealand rabbits

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.     

THE MECHANISM OF AQUEOUS HUMOR FORMATION INFERRED FROM CHEMICAL STUDIES ON BLOOD-AQUEOUS HUMOR DYNAMICS

The importance of considering the effect of a possible flow out of the anterior chamber before inferring any mechanism of aqueous humor formation from the relative concentration of a substance in the aqueous humor and plasma under equilibrium conditions has been stressed. Several processes to account for the chemical equilibria between aqueous humor and blood based on the ultrafiltration and secretion hypotheses with a possible simultaneous loss of aqueous humor by flow have been outlined. On the basis of these processes, equations were formulated which would relate the rates of transfer into and out of the anterior chamber to the ratio of concentration of a substance in the aqueous to that in the blood at various intervals after its introduction into the blood. The explanation of equilibrium ratios above and below one for aqueous constituents is made apparent from the mathematical formulations. For each substance tested a determination was made of the best fit when the concentration in the aqueous humor is plotted against time. This fit was obtained by plotting the rate of transfer in against the rate of transfer out of the anterior chamber for all of the experimentally found concentration ratios on the basis of both the ultrafiltration and secretory hypotheses. Two sets of values were obtained from these calculations, one set for each hypothesis. The substantial agreement of all the experimental data with an assumed rate of leakage out of the anterior chamber of approximately 4 c. mm. per minute was shown to be compatible only with the idea that all the monovalent electrolytes tested entered the anterior chamber as a result of secretory process. It could not be decided from these chemical studies whether the non-electrolytes and the one multivalent electrolyte tested enter the anterior chamber by ultrafiltration or secretion. Experimental findings from other sources were cited which would suggest that non-electrolytes enter the anterior chamber as a result of ultrafiltration. The implications of the mechanism outlined in the paper with respect to intraocular pressure have been discussed. Supplementary evidence from the literature has been given in support of the conclusions presented here.     

兔先天性青光眼的临床研究

目的 总结兔先天性青光眼的临床特点。方法 对先天性青光眼兔和正常兔进行临床观察,研究其在眼压、眼球结构、视功能方面的变化。结果 青光眼兔的眼压明显升高,角膜直径变大,前房变深,眼轴变长,房角变宽,眼底视乳头损害明显,视觉诱发电位明显异常。结论 兔先天性青光眼的房水排泄障碍部位可能在小梁,兔眼球壁对高眼压的耐受力弱,在高眼压下容易出现眼球扩张,视功能损害。     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

慢性高眼压下兔视网膜组织蛋白质组变化的初步研究

应用蛋白质组学技术对兔青光眼慢性高眼压视网膜组织的蛋白进行初步分析。左眼前房注入0.2mL复方卡波姆溶液制作成慢性高眼压模型,右眼为对照眼。28d后分离各组视网膜组织,用双向电泳分离试验组和对照组的蛋白,然后分析电泳图谱,对比、分析其表达蛋白质点的差异,寻找兔视网膜中与慢性高眼压相关的蛋白质。结果表明,慢性高眼压诱导视网膜组织3种蛋白质出现明显差异表达。质谱鉴定出3个蛋白质,分别为热休克蛋白70(heat shock 70 kD protein,HSP70),丙酮酸激酶(Pyruvate kinase)和烯醇酶(enolase)。通过双向电泳,发现兔视网膜蛋白质表达与对照眼相比有质和量的变化,这些变化涉及与神经节细胞(retinal ganglion cells,RGCs)糖酵解及应激反应有关的几组蛋白质,提示上述蛋白质组改变可能参与了慢性青光眼神经节细胞凋亡的过程。     

Aqueous humor dynamics in the enucleated deer eye

Facility of outflow of aqueous humor was determined in ten deer (Odocoileus virginianus) eyes enucleated within 1 hr post-mortem. Although both outflow facility and anterior chamber volume were much greater than values reported in the literature for a variety of other species, calculated values for aqueous humor turnover were within the reported range for other animals as well as for man. These results strengthen the hypothesis that the rate of turnover of aqueous humor is quite similar in eyes of vastly differing dimensions.     

Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye

 This report describes the distribution and localization of thrombomodulin (TM) in the rat eye by light and electron microscopic immunocytochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures lining the cavities of the posterior and anterior chambers and the limbus. TM was localized on the basal, lateral, and apical plasma membranes of the inner and outer ciliary epithelium, and the posterior iris epithelium in which there was no polarized expression of TM. In the anterior chamber, TM was localized on the luminal surface of the corneal endothelium, but was negative on the anterior border layer of the iris, which is composed of a discontinuous layer of fibroblasts and collagen fibers. Thus, TM was present at sites of cell-to-cell contact. TM was also present on the endothelia of the trabecular meshwork and the Schlemm’s canal in the limbus. TM was localized not only on the luminal plasma membrane, but also on the cytoplasmic giant vacuoles in the endothelial cells of the Schlemm’s canal. These findings extend the importance of anticoagulant mechanisms to the systems of secretion, circulation, and drainage of the aqueous humor. Accepted: 18 March 1997     

Antioxidant and anticataractogenic effects of topical captopril in diquat-induced cataract in rabbits.

1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive and free radical scavenger, protected the rabbit lens from peroxidative and oxidative damage induced by 1 mM diquat in vitro. To evaluate the anticataract efficacy of captopril, an experimental group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 microliters was instilled onto both eyes four times a day for a total of 8 weeks. Following the same procedure, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30 microliters of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits were injected with the diluent, 30 microliters per eye. The intravitreal diquat or its diluent injection was only for one time. From slit-lamp biomicroscopic observation of the diquat-injected right eyes, the anticataract effect of captopril in the treatment group was indicated by the finding that in four of five rabbits the cataract did not advance; whereas in four of five rabbits treated with the diluent the cataract progressed to grade 3. The lenses in the diluent-injected control left eyes of the rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O2- production was about 50% less (p less than .001) in the aqueous humor, vitreous humor, and lens, compared with O2-, 33.49 +/- 2.26 microM (mean +/- SEM) in the aqueous humor, 17.12 +/- 0.75 microM in the vitreous humor, and 31.44 +/- 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p less than .01) differences in the production of .OH and H2O2 in eye tissues were also observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization and ontogeny of aquaporin-1 and -4 expression in iris and ciliary epithelial cells in rats

The precise localization of aquaporin (AQP)1 and AQP4 was studied in iris and ciliary epithelial cells, in both mature and developing rats, to elucidate the molecular mechanisms underlying aqueous humor balance. Anterior segments of eyes dissected from embryonic day (E)13, E15, E18, and E20, postnatal day (P)0, P7, and P14, and postnatal week 8 rats were subjected to immunofluorescence analysis with AQP isoform-specific antibodies. In adult rat eye, AQP1 was localized to the apical and basolateral plasma membranes of iris epithelial cell layers and of anterior ciliary non-pigmented epithelial (NPE) cells. Conversely, AQP4 was localized to the basolateral plasma membrane of NPE cells in ciliary epithelium and the posterior iris. Developmentally, AQP1 was detected as early as E15 in immature iris and ciliary epithelial cells, and expression persisted throughout development up to adulthood. In contrast, AQP4 was first observed at P7 in the developing pars plicata, and the AQP4-positive area gradually spread to cover the entire pars plicata as development proceeded. These findings indicate that both AQP1 and AQP4 contribute to aqueous humor secretion in the rat eye, thereby maintaining proper intraocular pressure. Moreover, AQP appears to play a major role in aqueous humor secretion in early eye development. This study thus provides a basis for understanding the molecular mechanisms of aqueous humor secretion in pathological and physiological conditions.     

Role of interferon-like viral inhibitor in endotoxin-induced corneal resistance to Newcastle disease virus

Oh, J. O. (University of British Columbia, Vancouver, B.C., Canada), and E. J. Gill. Role of interferon-like viral inhibitor in endotoxin-induced corneal resistance to Newcastle disease virus. J. Bacteriol. 91:251-256. 1966.-A state of marked resistance to the toxic corneal effects of Newcastle disease virus (NDV) was observed in rabbit eyes after intravenous injection of 10 or 100 mug of typhoid endotoxin. By use of tissue cultures of rabbit corneal endothelial cells for assay, high titers of an interferon-like viral inhibitor were detected in serum and in ocular aqueous humor of these animals. The pretreatment of eyes with aqueous humor or serum containing the inhibitor markedly suppressed the production of corneal toxicity by NDV. The intravenous injection of 1 mug of the endotoxin had a negligible effect on the corneal reaction, and little or no inhibitor was found in either serum or aqueous humor. Normal aqueous humor or serum contained no inhibitor and had no suppressive effect on the NDV-induced reactions. The results indicated that the inhibitor played an important role in the induction of corneal resistance to NDV in vivo. The inhibitor in aqueous humor of endotoxin-injected rabbits was found to be derived from blood after an increase in the permeability of "blood-aqueous barrier" of iris due to the endotoxin. Therefore, intravenously administered typhoid endotoxin induced corneal resistance to NDV in rabbits through its dual action on host: (i) release of an interferon-like viral inhibitor into the blood stream and (ii) disruption of the blood-aqueous barrier of the iris, thus allowing the passage of the viral inhibitor from blood into the anterior chamber, where it modified the corneal endothelial cells to render them resistant to NDV.     

Water and ion regulation in cicadas in relation to xylem feeding

Cicadas feed on xylem fluid. This is hypotonic to the haemolymph and contains high concentrations of potassium, sodium, calcium, magnesium, chloride, and phosphate ions. The urine contains the same ions in the same proportions but in slightly lower concentrations. Amino acids and sucrose are present in xylem fluid and traces of amino acids are also found in urine.Water is rapidly shunted from foregut to hindgut via the filter chamber. Injection of xylem fluid into the oesophagus results in an immediate tenfold increase in flow rate in the ileum. The osmotic pressure of xylem fluid in the filter chamber rapidly rises whilst the osmotic pressure in the anterior part of the ileum rapidly falls.Absorption of nutrients and ions into the haemolymph probably occurs in the conical segment and anterior tubular midgut. Storage excretion of divalent ions occurs in the mid-midgut and ions may be transported from the haemolymph into the posterior tubular midgut.The Malpighian tubules secrete a fluid slightly hypertonic to blood containing K⁺ (42 mM/l.] and Na⁺ (14 mM/l.).The osmotic pressures within the internal Malpighian tubules and internal midgut in the filter chamber are considerably higher than the osmotic pressure of the xylem fluid when it first enters the filter chamber proper. Passive osmosis will occur and water will be shunted into the ileum.Reabsorption of K⁺ and Na⁺ occurs in the ileum.     

Increased iris-lens contact following spontaneous blinking: Mathematical modeling

The purpose of this work was to study in silico how iris root rotation due to spontaneous blinking alters the iris contour. An axisymmetric finite-element model of the anterior segment was developed that included changes in the iris contour and the aqueous humor flow. The model geometry was based on average values of ocular dimensions. Blinking was modeled by rotating the iris root posteriorly and returning it back to the anterior. Simulations with maximum rotations of 2°, 4°, 6°, and 8° were performed. The iris-lens contact distance and the pressure difference between the posterior and anterior chambers were calculated. When the peak iris root rotation was 2°, the maximum iris-lens contact increased gradually from 0.28 to 0.34mm within eight blinks. When the iris root was rotated by 6° and 8°, the pressure difference between the posterior and anterior chambers dropped from a positive value (1.23Pa) to negative values (-0.86 and -1.93Pa) indicating the presence of reverse pupillary block. Apparent iris-lens contact increased with steady blinking, and the increase became more pronounced as posterior rotation increased. We conclude that repeated iris root rotation caused by blinking could maintain the iris in a posterior position under normal circumstances, which would then lead to the clinically observed anterior drift of the iris when blinking is prevented.     

Inhibition of the ocular effects of sodium arachidonate by anti-inflammatory compounds

Topical administration of aqueous solutions of sodium arachidonate to the eyes of rabbits increases intraocular pressure, constricts the pupil and increases both the protein and prostaglandin content of aqueous humor. Arachidonic acid itself dissolved in arachis oil is less effective than sodium arachidonate, although addition of polysorbate mono-oleate greatly increases the effects produced by arachidonic acid. Pretreatment with topically applied non-steroidal anti-inflammatory agents prevents the ocular effects of sodium arachidonate, indomethacin being 2–4 times as potent as either indoxole or pirprofen. Dexamethasone was without effect in these experiments. The results suggested that non-steroidal anti-inflammatory agents deserve serious consideration for topical use in the treatment of ocular inflammation.     

Aqueous-humor/plasma-chloride ratios in rabbits,dogs, and human beings

The ratio of concentration of chloride in the aqueous humor compared with that in the plasma of rabbits, dogs, and human beings, was determined by the Schales and Sendroy methods. Consistent results were obtained in all the experiments by the Schales method with or without protein, and by the Sendroy method without protein. In the presence of protein, however, lower chloride concentrations were found in the plasma of dogs and human beings by the Sendroy method. The ratios in this instance were higher than values predicted on the basis of Gibbs-Donnan equilibrium and similar to those obtained by several previous investigators using the same method. With rabbits both methods gave essentially the same results under all conditions. The ratios (Schales and Sendroy) for the rabbits based on arterial plasma averaged 0.98, on venous plasma 1.00; the ratio (Schales) for the dog for venous plasma was 1.00; the ratio (Schales, protein present or removed; Sendroy, protein removed) for human beings, based on venous plasma, was approximately 1.03. Ratios of 1.08 for the dog and 1.07 for human beings were obtained by the Sendroy method in the presence of protein. Possible explanations for these apparent discrepancies are discussed. In dogs and in human beings, the major evidence supports the contention that the aqueous humor/plasma chloride ratio is not in excess of that predicted by the Gibbs-Donnan equilibrium and therefore requires no special explanation. In the rabbit all the evidence indicates that the chloride ratios are actually less than the value predicted by the Gibbs-Donnan equilibrium.     

Changes in leucine uptake in the retina of the hamster after traumatic detachment

Protein metabolism was investigated in detached hamster retinas. By sucking off 0.2 ml of aqueous humor from the anterior chamber through limbic insertion of a 27-gauge needle, a tractional force pulled off the neural retina from the retinal pigment epithelium and created a simple detachment without retinal breaks in the right eyes of the hamsters. The left eyes were left untouched as normal controls and sham controls were induced by simple limbic insertion without suction. The animals were sacrificed at selected intervals of 1, 3, 6, 9, 16, 24, 32 days after the operation. Subsequently, scintillation counting and autoradiography were employed to study retinal protein metabolism using leucine uptake as an index. After tritiated leucine uptake, scintillation counting of radioactive substance indicated that detached retinas had taken in less tritiated leucine than normal controls from day 1 to 6 after the operation, but this change had normalized by day 9. For autoradiography, the change in leucine uptake rate was shown to be different in different layers. All the retinal cells seemed to show a decreased leucine uptake with the exception of the outer nuclear layer, in which leucine appeared to be significantly upregulated. This paper illustrates the patterns of protein metabolism and their change after traumatic detachment as well as their possible recovery.     

Parasite-specific antibody profile in the aqueous humor of rabbits with ocular toxocariasis

Human ocular toxocariasis is diagnosed using ophthalmologic and immunologic examinations. Many researchers have suggested that intraocular parasite-specific antibody levels are indicative of ocular toxocariasis, but little is known about the time course of the changes in these levels. We therefore investigated the anti-Toxocara canis antibody profile in the aqueous humor in an animal model of ocular toxocariasis. We intravitreally injected T. canis larvae into the right eye of 4 rabbits; 2 rabbits were orally administered T. canis eggs. We collected serum, aqueous humor, and tear samples weekly and determined the serum and aqueous humor levels of anti-T. canis immunoglobulin (Ig)G, IgA, IgM, and IgE antibodies and the tear IgG antibody level by enzyme-linked immunosorbent assay (ELISA). The severity of vitreous opacity and the aqueous humor IgG levels (measured using optical density [OD]) changed concordantly in the larvae-injected eyes; the OD exceeded 0.1 from 2–4 weeks after infection and remained elevated during active intraocular inflammation. However, the aqueous humor IgG levels were also elevated in 6 out of 8 eyes without intraocular larvae in both groups, and were low in 1 eye with live intravitreal larvae. In contrast, the serum IgG and IgM levels and the tear IgG levels increased in all rabbits, regardless of the presence of intraocular inflammation. Vitreous opacity occurred in all intravitreally infected eyes, but significant histopathological evidence of retinal damage was not detected. Thus, besides the presence of intraocular larvae, some other factors in the host may be required for the development of retinal lesions.     

Pharmacotherapies for glaucoma

Glaucoma is a group of progressive optic neuropathies in which the axons in the optic nerve are injured, retinal ganglion cell numbers are reduced and vision is gradually and permanently lost. The only approved and effective way to treat glaucoma is to reduce the intraocular pressure (IOP). This is usually accomplished by surgical and/or pharmacological means. Drugs designed to reduce IOP target one or more of the parameters that maintain it. These parameters (collectively known as aqueous humor dynamics) are the production rate of aqueous humor, the pressure in the episcleral veins and the drainage of aqueous humor through the trabecular or uveoscleral outflow pathways. Intraocular pressure lowering drugs can be classified as inflow or outflow depending on whether they reduce aqueous humor inflow into the anterior chamber or improve aqueous humor outflow from the anterior chamber. Inflow drugs, like β adrenergic antagonists and carbonic anhydrase inhibitors, reduce the rate of aqueous humor production. Outflow drugs, like prostaglandin analogs, cholinergic agonists and sympathomimetics, increase the rate of drainage through the uveoscleral outflow pathway and/or increase the facility of outflow through the trabecular meshwork. Some drugs have mixed inflow/outflow effects. This review summarizes the pharmacological treatments for glaucoma in use today and some new drugs showing potential for use in the future.     

The eye of the magellanic penguin (Spheniscus magellanicus): structure of the anterior segment

We undertook a light and scanning electron microscopic study of the eye in the Magellanic penguin (Spheniscus magellanicus). The anatomical peculiarities of the eyeball shape in Sphenisciformes have been previously described by others; here, we show that they are accompanied by several modifications in the organization of the anterior segment of the eye. The main change was found in the portion of opaque sclera extending from the cornea to the anterior border of the scleral ossicles, which was much broader than in other avian eyes. This scleral region was made of a very dense fibrous tissue and was as difficult to cut as the ossicles. The corneo-scleral boundary was also different from that of other birds, since the aqueous humor channel and the pectinate ligament were located 1.0-1.5 mm posterior to the cornea. The osseous ring was formed by 13 bones, including three pairs of over- and underplates. There was a single ciliary muscle, with meridionally oriented striated fibers. They were inserted on a circumference along the boundary between the fibrous sclera and the ossicles, far away from the wall of the aqueous humor channel. On their posterior end, the muscle fibers formed a tendinous structure attached to the inner surface of the sclera and to the outer surface of the ciliary body. Only short zonular fibrils were observed. These anatomical features are probably relevant for the adaptation of penguin eyes to vision on land and in the aquatic environment.     

Experimental Study of Aqueous Humor Flow in a Transparent Anterior Segment Phantom by Using PIV Technique

Pupillary block is considered as an important cause of primary angle-closure glaucoma (PACG). In order to investigate the effect of pupillary block on the hydrodynamics of aqueous humor (AH) in anterior chamber (AC) and potential risks, a 3D printed eye model was developed to mimic the AH flow driven by fluid generation, the differential pressure between AC and posterior chambers (PC) and pupillary block. Particle image velocimetry technology was applied to visualize flow distribution. The results demonstrated obvious differences in AH flow with and without pupillary block. Under the normal condition (without pupillary block), the flow filed of AH was nearly symmetric in the AC. The highest flow velocity located at the central of AC when the differential pressure between AC and PC was under 5.83 Pa, while it appeared near the cornea and iris surface when the differential pressure was greater than 33.6 Pa. Once pupillary block occurred, two asymmetric vortices with different sizes were observed and the shear stress in the paracentral cornea and iris epithelium increased greatly. It can be concluded that the pupillary block and the elevated differential pressure between AC and PC could change the flow distribution and thus increase the risk of corneal endothelial cells detachment. This study could make a further understanding of the pathogenesis of PACG.     

Penetration of radioactive sodium and chloride into cerebrospinal fluid and aqueous humor

1. Experiments were performed on six dogs to determine the rate of penetration of Cl(33) and Na(24) across the blood-aqueous humor and blood-cerebrospinal fluid barriers after intravenous injection of the radioactive ions. The radioactivity measurements were made with an immersion type of Geiger-Müller counter. 2. The concentrations of the labelled ions in the anterior chamber and the cisterna magna increase slowly to approach that of plasma. The rate of penetration k is calculated from a simple exponential equation with the half-value interval t(0.5) or the time required for the labelled-ion concentration in the fluid to reach 50 per cent of that of plasma. The average t(0.5) for Cl(38) and Na(24) in aqueous humor are 34.3 +/- 9 and 27.3 +/- 9 minutes, respectively, while those for cerebrospinal fluid are 90 +/- 6 and 95 +/- 6 minutes, respectively. 3. A study of the radioactivity in plasma was made to determine the per cent remaining after a steady state was reached. By means of this determination the sodium and chloride space was calculated to be 33 +/- 5 per cent.