Inhibition and recovery of retrograde axoplasmic transport in rat optic nerve during and after elevated IOP in vivo

Horseradish peroxidase (HRP) injected into one lateral geniculate nucleus of male inbred PVG/Mol hooded rats is taken up by terminals of the optic nerve and transported retrogradely towards the opposite retina. One hr after injection, the eyes were cannulated and set at an intraocular pressure (IOP) of either 35 mmHg or 15 mmHg. The IOP were set for 4 hr at which time the trial was terminated and retinal HRP content measured. It was found that in eyes set at 35 mmHg (18 eyes) the axoplasmic transport was partially blocked compared with that in eyes set at 15 mmHg (10 eyes), absorbances were 0.034 +/- 0.003 (S.E.) and 0.044 +/- 0.003 (S.E.), respectively, P less than 0.05. In a third group of eyes (nine eyes) set at 50 mmHg for 2 hr (beginning 1 hr after the intrageniculate injection), succeeded by another 2 hr of 15 mmHg IOP, there was no statistically significant difference in retinal HRP content compared to that in eyes set at 15 mmHg throughout, absorbances were 0.040 +/- 0.006 and 0.044 +/- 0.003, respectively. Two hr of 50 mmHg IOP blocks the axonal transport in the rat optic nerve (Johansson, 1986a). The result shows that also moderately increased IOP blocks axonal transport in the rat optic nerve. It also shows the presence of a rapid recovery when the pressure is normalized. A direct mechanical factor underlying axonal transport blockage is proposed.     

Blockage at two points of axonal transport in glaucomatous eyes

Blockage of axonal protein transport in intraocular hypertensive primates (Macaca irus) was studied autoradiographically and quantitatively and the findings compared with our previous work on rabbits. Fast axonal transport was blocked at two points, at the lamina scleralis and at the edge of posterior scleral foramen, and reduced by 25% when intraocular pressure of 50 mmHg continued for 6 h. The importance of the blockage at the lamina scleralis and at the edge of scleral foramen for the explanation of paracentral scotomas and the peripheral nasal step (Rønne) is discussed.     

Retrograde axoplasmic transport in rat optic nerve in vivo. What causes blockage at increased intraocular pressure?

The effect of intraocular pressure (IOP) on retrograde axonal transport of horseradish peroxidase (HRP), from the geniculate body to the retina, was studied in the rat in vivo. In 2-hr experiments (17 eyes at 50 mmHg; 13 eyes at 15 mmHg), the pressures were set just prior to the expected HRP arrival into the eyes. In 4.5-hr experiments (24 eyes at 50 mmHg; 21 eyes at 15 mmHg), the pressures were set 2.5 hr before expected HRP arrival. HRP was measured in the retinas 5 hr after the intrageniculate injection. Transport blockage occurred at an IOP of 50 mmHg in both series of experiments. In an earlier study of axonal transport in vitro, an IOP of 50 mmHg also blocked retrograde HRP transport. In a third series of experiments, 15 eyes were set at an IOP of 180 mmHg for 10 min, 10-20 min before expected arrival of HRP into the eye, while 17 control eyes were set at 15 mmHg. After the 10 min, both groups of eyes were set at 15 mmHg for another 2 hr and the HRP content in the retinas measured, 5 hr after HRP injection. There was no significant difference between these two groups of eyes, suggesting either no rapidly occurring block or a rapid recovery of transport after the high-pressure period. It is proposed that optic nerve fibers are stretched and narrowed near or at their exit, by the high IOP, but recover their shape soon after IOP is normalized.(ABSTRACT TRUNCATED AT 250 WORDS)     

急性高眼压模型兔眼不同眼压状态下视神经轴浆运输的改变

目的 观察急性高眼压模型兔眼不同眼压状态下视神经轴浆运输的改变。方法 成年新西兰大白兔24只,分为眼压20、30、40 mm Hg(1 mm Hg=0.133 kPa)组和眼压为10～15 mm Hg的对照组,每组均为6只兔。采用前房穿刺灌注法联合压力持续监测法建立急性高眼压模型。实验开始时,兔眼玻璃体腔中注射罗丹明异硫氰酸(RITC)标记轴浆运输。持续3h高眼压后,过量麻醉处死兔后取下视神经。荧光显微镜下观察视神经轴浆运输情况的改变。采用德国Leica公司Q500IW图像分析软件对RITC进行灰度定量分析,并对各眼压组平均灰度值和筛板前、筛板区、筛板后350 μm区域灰度值进行统计学分析处理。结果 RITC在视神经中心呈顺行标记染色。不同眼压组轴浆运输情况不同,随着眼压升高,轴浆运输能力减弱,差异有统计学意义(F=159.3,P＜0.05)。筛板前区,各眼压组间灰度值比较,差异无统计学意义(F=0.2545,P＞0.05)。40 mm Hg组灰度值与对照组灰度值比较,在筛板区（t=5.684）和筛板后350 μm区域（t=5.124）差异均有统计学意义(P＜0.05);20、30 mm Hg组灰度值与对照组灰度值比较,差异无统计学意义(t=1.747,P＞0.05)。结论 眼压40 mm Hg持续3h将导致视神经轴浆运输改变,轴浆运输障碍部位以筛板区及以后的区域为主。     

Axonal transport blockage by acute intraocular pressure elevation in monkeys]

The distribution of orthograde rapid axonal transport blockage in the optic nerve head by acute intraocular pressure (IOP) elevation in monkeys was studied by autoradiography. Tritiated leucine was injected intravenously two hours before IOP elevation, and the IOP was elevated for five hours, maintaining a perfusion pressure of 30mmHg. Serial step cross sections from the optic nerve head at the level of the lamina cribrosa were prepared for light microscopic autoradiography, and the accumulation and distribution of grains were quantitatively analyzed using computerized image analysis. The area of focal grain accumulation was expressed as a percent of the defined optic nerve area and each eight sectors, divided by axially intersecting vertical, horizontal, and diagonal lines, respectively. In eyes with IOP elevation, the mean area of focal accumulation of grains in the temporal half of the optic nerve was significantly larger, compared with the nasal half. Within axonal bundles, the focal high accumulation of grains was frequently identified to be in association with trabecular beams of connective tissue in the peripheral portion of the axonal bundle. These results suggest temporal dominant blockage of axonal transport in the optic nerve head, which may be induced by mechanical compression of the axonal bundles during IOP elevation.     

Influence of low IOP and low calcium on retrograde axoplasmic transport in rat optic nerve in vitro

Horseradish peroxidase (HRP) injected into one lateral geniculate nucleus of male inbred PVG/Mol hooded rats is taken up by terminals of the optic nerve and transported retrogradely towards the opposite retina. Four hours after injection when a small portion of HRP had reached the retina, the eye and optic nerve were excised and incubated in vitro at 38 degrees C for another 3.5 hr during which the intraocular pressure (IOP) was set at 30 or 0 mmHg. During the in vitro period additional HRP entered the retina by axonal transport if the incubation medium contained enough Ca2+. Transport occurred at 0.45-1.1 mM Ca2+, but not at 0.30mM Ca2+. When transport occurred, no significant difference in degree of transport was found between the two pressures. The amount of HRP transported at 30 and 0 mmHg was very similar to that at 20 mmHg but significantly higher than that at 50 mmHg, (values at 20 and 50 mmHg from an earlier study). Thus, fast retrograde HRP transport was equally efficient at or near a physiological IOP as at zero pressure. Also, the degree of transport inhibition was not proportional to the height of the IOP, but started to increase above 30 mmHg. This is probably due to the presence of supporting tissue in the optic nerve head and inherent strength of the nerve fibers themselves. The lamina cribrosa in the rat eye is poorly developed and a shearing force on the nerve fibers due to laminar hole misalignment can largely be excluded. Effects on blood circulation are also excluded by the in vitro situation.     

The effects of intraocular pressure elevation on optic nerve axonal transport in the monkey.

Blockage of axonal transport by intraocular pressure (IOP) elevation was studied quantitatively in monkey eyes, using liquid scintillation counting. After 5 h of IOP elevation (perfusion pressure of 30 mmHg), axonally transported protein was measured in the distal third of each optic nerve, which was divided into superotemporal, inferotemporal, superonasal, and inferonasal portions. The ratio of the amount of radioactive protein in each portion of the optic nerve to that in the whole optic nerve was calculated. In eyes with IOP elevation, the mean ratio for the temporal optic nerve was significantly lower than that for the nasal optic nerve. It appeared that axonal transport was not affected homogenously throughout the optic nerve but was more impaired by the temporal half of the optic nerve following IOP elevation.     

Optic nerve head axonal transport in rabbits with hereditary glaucoma

Rabbits with hereditary glaucoma develop ocular changes that resemble human congenital glaucoma and buphthalmia. The inheritance is autosomal recessive (bu). Previous research was performed primarily on albino bu/bu rabbits that were unhealthy and bred poorly. We have bred pigmented bu/bu rabbits to determine if this would improve hardiness and provide a better model for the disease in humans. First-generation offspring from matings of bu/bu albino with bu/bu pigmented rabbits were all affected, indicating that the bu gene is found at the same locus in both strains. The pigmented bu/bu offspring had a high degree of mortality, as reported previously for albino bu/bu rabbits. Newborn bu/bu rabbits initially had normal intraocular pressure (IOP; 15-23 mmHg); after 1- to 3 months, the IOP increased to 26-48 mmHg. The eyes became buphthalmic and the IOP returned to normal or sub-normal levels after 6-10 months. Since the lamina cribrosa is absent or poorly formed in the rabbit optic nerve head (ONH), this model was used to test the role of mechanical factors in the etiology of ONH pathology caused by increased IOP. Orthograde axonal transport was evaluated in both eyes from eight normal and 24 bu/bu rabbits of different ages, using intravitreal injections of [3H]leucine to mark orthograde axonal transport, followed by light- and electron-microscopic radioautography of the ONHs and superior colliculi. Normal rabbits of all ages showed no blockage of axonal transport in the ONH. All optic axons from young bu/bu rabbits with normal IOP and most axons from older buphthalmic rabbits that previously had elevated IOP were normal morphologically. Small zones of transport blockage occurred in bu/bu eyes while IOP was elevated; most affected axons lay immediately adjacent to ONH connective tissue beams that radiate outward from the central retinal vessels to the optic-nerve sheath. Thus, the rabbit, which lacks a true lamina cribrosa, does not show marked blockage of axonal transport as occurs in the LS of the monkey and cat ONH when IOP is elevated acutely. This anatomic difference appears to be protective against axonal damage, since bu/bu rabbits with chronic IOP elevation did not show significant loss of optic axons. These results are consistent with the proposed 'mechanical' theory of ONH damage resulting from increased IOP. Electron-microscopic radioautography revealed that chronically elevated IOP in bu/bu rabbits, which caused small foci of blocked ONH axonal transport against ONH beams, also caused degeneration of a few optic nerve terminals in the superior colliculi as the disease progressed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of chronic demyelination on axonal transport in experimental allergic optic neuritis

Axonal transport studies were undertaken to determine the effect of chronic demyelination on axonal function in experimental allergic optic neuritis in the guinea pig, an animal model for multiple sclerosis. Fast and slow components of axonal transport over the prelaminar, laminar, and retrolaminar portions of the optic nerve head and at the foci of demyelination in the retrobulbar optic nerve were evaluated by the autoradiographic grain-counting technique. At 6 hr there was a significant increase in grain counts over the demyelinated foci and in the regions proximal to the demyelination, including the swollen disc. At day 1 there was no significant difference in the grain counts at the site of demyelination when compared to the myelinated portion of the nerve. However, at days 3 and 7 there was a decrease in the number of grains over the demyelinated areas. These results indicate impairment of axonal function in chronic demyelination. Moreover, in this pathologic process, most of the synthesized materials appear to move in the fast transport phase, unlike in the normal optic nerve where the bulk of materials move by slow transport.     

Distribution of axonal transport blockade by acute intraocular pressure elevation in the primate optic nerve head.

We studied the degree of axonal transport blockade in various areas of the optic nerve head with acute intraocular pressure (IOP) elevation in 19 squirrel monkey eyes. When IOP was raised to 20 to 50 mm. Hg for 7 hr., mild axonal transport blockade occurred in each area of the disk, most prominently in nerve fiber bundles of the superior pole. With 7 hr. IOP elevations between 50 and 90 mm. Hg, a somewhat greater degree of transport blockade occurred throughout the nerve head, although again the superior and inferior poles were somewhat more affected. The distribution of short-term transport blockade over the entire nerve head corresponds to the diffuse damage of acute glaucoma, but the pattern hints at the preference for damage near the poles of the disk seen in chronic glaucoma. However, before these results can be fully evaluated, further information is needed on axonal pathways through the optic nerve head and on the relationship between transport obstruction and ganglion cell death.     

Pathogenesis of block of rapid orthograde axonal transport by elevated intraocular pressure.

The subject was investigated in 40 rhesus monkeys. The perfusion pressure (PP) in the eye was altered by changing the blood pressure (BP) and/or intraocular pressure (IOP). Rapid orthograde axoplasmic transport was studied by intravitreal injection of 100 μCi of [³H]leucine and light microscopic autoradiography. The study was planned to determine whether the presence and degree of rapid orthograde axoplasmic flow blockage relate more closely to the level of IOP (suggesting a mechanical basis) or of PP (suggesting an ischemic mechanism). It proved technically impossible to maintain IOP and PP at desired levels for the necessary period ($\text{6–7}\text{1}\text{2}\text{hr}$)—this difficulty has never been admitted in the literature. Several biological and technical variables also confounded the interpretation of the data, e.g. non-availability of a reliable technique to ascertain the exact PP in the vessels of the optic nerve head, lack of information on the blood flow and the nutritive capability of a given PP, possible artefacts by the perfusion pump used to alter BP, and marked limitations of routine microscopic autoradiography in quantifying exactly the axoplasmic flow blockage. As a result an intensive and sustained study offers only inconclusive results, and cannot resolve the controversy as to whether the axoplasmic flow blockage is due to ischemia or mechanical compression of the nerve fibers in the optic nerve head. Although no single observation is absolutely decisive one way or the other, the sum total of the various evidence put together is somewhat suggestive that the axonal blockage is probably mediated by ischemic mechanism.     

Failure of unilateral carotid artery ligation to affect pressure-induced interruption of rapid axonal transport in primate optic nerves

Previous experiments showed that optic nerve axonal transport can be blocked at the level of the lamina cribrosa by elevated intraocular pressure. In an effort to discover if this blockage might be secondary to pressure-induced ischemia, we studied the effect of unilateral common carotid artery ligation upont the pressure-induced interruption of axonal transport. In 13 owl monkeys (Aotus trivirgatus), the right common carotid artery was ligated within the anterior cervical triangle. Three days later, ophtalmodynomometry was performed on all experimental eyes. In nine of the 13 animals, this estimate of ophthalmic artery pressure was 10 to 20 mm Hg less in the right compared to the left eye. Optic nerve axonal transport was studied in right and left eyes during 5 hours of increased intraocular pressure (ocular pressure 35 mm Hg less than mean femoral artery blood pressure). No significant difference in the extent to which the transport mechanisms were interrupted could be demonstrated when comparing right and left eyes of the experimental animals. These observations fail to support a vascular mechanism for this pressure-induced interruption of axonal transport.     

Axonal transport and cytoskeletal changes in the laminar regions after elevated intraocular pressure

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.     

Time-dependent effects of elevated intraocular pressure on optic nerve head axonal transport and cytoskeleton proteins

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.     

A pilot study of in vivo venous pressures in the pig retinal circulation

Retinal venous pressure was examined in six pigs using a balanced servo‐nulling micropuncture technique. The mean transmural venous pressure was 0.95 mmHg (SD 1.50 mmHg). The transmural venous pressure was lower at the optic disc than away from the disc (0.32 ± 1.46 mmHg vs 1.69 ± 1.19 mmHg, P < 0.01). At the disc a transmural pressure of zero or less (0 to –0.5 mmHg) was demonstrated in 10/21 (48%) of the disc readings. Over an intraocular pressure range of 15–26 mmHg there was a strong correlation between intraocular pressure and retinal venous pressure (Pearson coefficient r = 0.92). The results are compatible with the Starling resistor theory of venous outflow from the eye.     

XXXIV Edward Jackson Memorial Lecture: drusen of the optic disc and aberrant axoplasmic transport.

In summary, we believe that axoplasmic transport alteration is the anatomic substrate for formation of drusen of the optic disc. In familial cases the cause of axoplasmic transport alteration may be related to the presence of a genetically determined, small, crowded optic nerve head. We believe that these congenitally elevated nerve heads evolve over a period of many years through stages of atrophy and drusen formation. It appears that vascular malformations in the familial cases are primarily developmental; however, secondary vascular alterations may occur as the drusen enlarge. In retinitis pigmentosa, the drusen may be cuased by diminished production of axoplasmic material by the ganglion cell. As a general principle, chronic alterations in axonal transport from any cause seem to produce aggregates of swollen nerve fibers which impart a yellow-white appearance to the disc tissue, and account for the yellow filled-in appearance of the disc in patients with drusen, chronic atrophic papilledema and melanocytomas and, in part, for the "waxy yellow" appearance of the disc in retinitis pigmentosa.     

Studies of hemorrhage on the optic disc.

The general medical and laboratory findings in 60 patients having a sector hemorrhage on the optic disc were compared with the findings in 102 patients with intraocular pressure elevation but without a disc hemorrhage. Of 22 parameters studied, systemic hypertension was the only abnormality which occurred significantly more frequently in the hemorrhage group. A study of the intraocular pressures suggests that hemorrhage may not be related to the level of intraocular pressure. Examinations of 50 patients with systemic hypertension and of 50 patients with cardiac abnormalities revealed no hemorrhage on the optic disc. These factors by themselves may not be responsible for the occurrence of hemorrhages.     

视神经挫伤轴浆运输和超微结构变化的实验研究

目的 研究实验性视神经挫伤轴浆运输与超微结构的变化。方法采用自行设计的弹簧冲击器对家兔视神经进行定量损伤，术后1、3、7、14d应用辣根过氧化物酶(HRP)顺行标记和透射电镜观察视神经轴浆运输以及超微结构的变化。结果 不同时间实验组HRP反应产物较正常对照组明显降低，差异有显著性，HRP反应产物随观察时间延长逐渐增加，但至术后14d仍明显低于正常。电镜观察见损伤后1d大部分轴突颗粒状变性，线粒体肿胀，髓鞘松解，3d时损伤最重，14d部分轴突恢复正常。结论 视神经挫伤后局部轴浆运输发生障碍，同时轴突变性，14d部分轴突功能恢复。     

外伤性睫状体脱离手术治疗

目的探讨外伤性睫状体脱离行睫状体缝合复位术的效果。方法外伤性睫状体脱离范围超过60°、经散瞳及皮质类固醇等保守治疗无效、低眼压持续时间超过15d者31例（31眼）,进行直接睫状体缝合复位术,分析临床资料并评价其手术效果。结果术后观察3～6个月。术后第1天,14眼眼压〉21mmHg;眼压在10～21mmHg者15眼;〈10mmHg者2眼。对眼压〉21mmHg者给予局部降眼压药1～2种,眼压均控制在24mmHg以下,所有病例在2月内眼压均控制良好并停药。视力均有提高,其中〈0．1者2眼,0．1～0．25有6眼,0．3～0．5有9眼,0．6～0．9有12眼．≥1．0有2眼。视盘及视网膜水肿减轻或恢复正常。其中2眼因低眼压持续时间〉2个月。视盘色淡,视力分别为0．06和数指／50cm。结论睫状体脱离缝合复位术是治疗眼挫伤后睫状体脱离的有效方法,对脱离范同大、保守治疗无效者,应及早手术,有利于恢复眼压,保护视功能。     

Intraocular pressure-dependent dynamic changes of optic disc cupping in adult glaucoma patients.

The authors performed a study of intraocular pressure-dependent changes in optic disc cupping in 17 adults with chronic open-angle glaucoma. Analyses with the Rodenstock Optic Nerve Head Analyzer were performed at baseline low intraocular pressure during therapy, after elevation of intraocular pressure (from therapeutic failure or noncompliance), and after reduction of intraocular pressure with successful therapy. Optic disc cupping increased significantly upon short-term increase of intraocular pressure from baseline of 20.4 +/- 2.5 mmHg to 31.1 +/- 5.9 mmHg. Optic disc cupping reverted to baseline after persistent intraocular pressure reduction to 19.3 +/- 4.8 mmHg. These data demonstrate intraocular pressure-dependent dynamic changes of optic disc cupping in patients with demonstrable glaucomatous optic nerve damage. They underscore the detrimental effect of elevated intraocular pressure and the beneficial effect of intraocular pressure reduction on optic disc cup changes.