Axoplasmic flow during chronic experimental glaucoma. 1. Light and electron microscopic studies of the monkey optic nervehead during development of glaucomatous cupping.

The anterior optic nerve and the macular region of the retina of glaucomatous eyes of five rhesus monkeys (Macaca mulatta) have been examined by light and electron microscopy. The experimental glaucoma had been induced by argon laser treatment of the anterior chamber angle. The eyes were examined 3 to 11 weeks after the onset of sustained elevation of intraocular pressure above 20 mm Hg. Severe degenerative changes were seen in eyes with higher intraocular pressure and longer duration of glaucoma. Eyes with a lesser elevation of intraocular pressure and shorter duration of glaucoma showed changes sharply localized to the axon bundles in the scleral lamina cribrosa. Accumulation of mitochondria and dense bodies occurred anterior and posterior to collagenous septae. The location of these changes is in agreement with the localization of block of axoplasmic transport identified by autoradiographic studies. It is speculated that these cytologic changes reflect blockage of axoplasmic flow in the optic nerve of eyes with glaucoma.

Aldose reductase in retinal mural cells.

Cultured mural cells (intramural pericytes) from adult rhesus retinal capillaries were examined for the presence of the sorbitol pathway. A radioimmunoassay for human aldose reductase, cross-reactive with rhesus lens aldose reductase, showed the presence of this enzyme in our cultured cells. Mural cells grown in culture media containing normal (10 mM) and high (40 mM) levels of glucose were examined for polyol accumulation by gas-liquid chromatography. Cells incubated in high glucose medium showed a threefold increase in sorbitol concentration over cells grown at low glucose levels. After 30 days in high glucose medium, mural cells formed dense multilayered areas with extensive cellular debris. These findings suggest the presence of the sorbitol pathway in cultured retinal mural cells and cellular degeneration in high glucose medium; this may have possible implications in the pathogenesis of diabetic retinopathy.

Possible regulatory mechanisms of the cornea. I. Epithelial-stromal interaction in vitro.

Electron microscopic studies of pure epithelial and stromal cultures and of combined cultures demonstrated that keratocytes activity was inhibited by the presence of epithelial cells in culture. The degradation of collagen and the formation of clear zones around the keratocytes within the explants of pure stroma is interpreted as an indication for the production of a collagenolytic substance by the active keratocytes. The collagenolytic activity of the keratocytes was not observed within the stroma of combined cultures and was probably inhibited by the activity of epithelial cells in these cultures. Furthermore, using a microculture method for the assessment of the metabolic activity of corneal cells in vitro, supernatants of epithelial cell cultures were shown to have a marked inhibitory effect on the capacity of keratocytes to synthesize DNA. No effect of stromal cell supernatant on epithelial cell activity could be detected by the same methods. It is suggested that this "one way" influence as observed in vitro might, in certain conditions, play a role as a regulatory mechanism in vivo.

Ocular cryptococcosis.

We studied ocular cryptococcosis in a 59-year-old man who had received extensive chemotherapy for abdominal malignant lymphoma. Mucinous lesions containing abundant cryptococci, but no inflammatory cells, formed in the posterior choroid of the eye. Electron microscopy revealed that mucin, a filamentous substance, coated the encapsulated organisms. Except for a few degenerating ones in histiocytes, most cryptococci appeared in the extracellular space.

Factors in the natural history of chronic subdural hematomas that influence their postoperative recurrence.

OBJECT: Factors affecting the postoperative recurrence of chronic subdural hematomas (CSDHs) have not been sufficiently investigated. The authors have attempted to determine features of CSDHs that are associated with a high or low recurrence rate on the basis of the natural history of these lesions and their intracranial extension. METHODS: One hundred six patients (82 men and 24 women) harboring 126 CSDHs who were treated at Tokyo Kosei Nenkin Hospital between January 1989 and April 1998 were studied. Types of CSDHs were classified according to hematoma density and internal architecture, and the intracranial extension of the hematomas were investigated. The postoperative recurrence rate was calculated for each factor. Based on the internal architecture and density of each hematoma, the CSDHs were classified into four types, including homogeneous, laminar, separated, and trabecular types. The recurrence rate associated with the separated type was high, whereas that associated with the trabecular type was low. Chronic subdural hematomas are believed to develop initially as the homogeneous type, after which they sometimes progress to the laminar type. A mature CSDH is represented by the separated stage and the hematoma eventually passes through the trabecular stage during absorption. Based on the intracranial extension of each hematoma, CSDHs were classified into three types, including convexity, cranial base, and interhemispheric types. The recurrence rate of cranial base CSDHs was high and that of convexity CSDHs was low. CONCLUSIONS: Classification of CSDHs according to the internal architecture and intracranial extension may be useful for predicting the risk of postoperative recurrence.

Relationship between drainage catheter location and postoperative recurrence of chronic subdural hematoma after burr-hole irrigation and closed-system drainage.

OBJECT: This study was conducted to determine the best position for the subdural drainage catheter to achieve a low recurrence rate after burr-hole irrigation and closed-system drainage of chronic subdural hematoma (CSDH). METHODS: The authors studied 63 patients with CSDH in whom the drainage catheter tip was randomly placed and precisely determined on postoperative computerized tomography (CT) scans and 104 patients with CSDH in whom CT scans were obtained 7 days postsurgery. The location of the subdural drainage catheter, the maximum postoperative width of the subdural space, and the percentage of the ipsilateral subdural space occupied by air postoperatively were determined and compared with the postoperative recurrence and reoperation rates. Patients with parietal or occipital drainage had a higher rate of CSDH recurrence and much more subdural air than those with frontal drainage. In addition, patients with residual subdural air demonstrated on CT scans obtained 7 days postsurgery also had a higher recurrence rate than those without subdural air collections. Furthermore, patients with a subdural space wider than 10 mm on CT scans obtained 7 days postsurgery had a higher recurrence rate than those with a space measuring 10 mm or less. CONCLUSIONS: The incidence of postoperative fluid reaccumulation seems to be reduced by placing the tip of the drainage catheter in the frontal convexity and by removing subdural air during or after surgery.

Vasospasm assessed by angiography and computerized tomography.

In 44 consecutive cases of ruptured cerebral aneurysm, vasospasm was demonstrated pre- or postoperatively. These cases were examined by bilateral carotid angiography and computerized tomography (CT), and the relationship between the angiographically visualized distribution of vasospasm, the neurological symptoms, and infarction seen on CT was evaluated. Vasospasm occurred in only some intracranial portions of the cerbral arteries that were immersed in blood-stained cerebrospinal fluid. Angiographically, diffuse vasospasm extensively involving bilateral carotid systems indicated the gravest prognosis for patients. Vasospasm affecting one carotid system and the anterior cerebral arteries on the opposite side often produced permanent neurological deficits. On the contrary, when vasospasm was restricted to one carotid system or to bilateral anterior cerebral arteries, it was usually associated with temporary neurological symptoms; however, it always produced residual neurological symptoms if it extended to the ascending branches (M3) of the middle cerebral arteries. Computerized tomography definitely demonstrated a low-density area or infarction in the territory of the spastic arteries in 25 (71%) of 35 cases with vasospasm. A low-density area was always detected when vasospasm occurred in M3 segments.

Lipid peroxidation as a cause of cerebral vasospasm.

In the present study, the vasocontractile activities of purified oxyhemoglobin, methemoglobin, peroxides of linoleic and arachidonic acid, and hydrogen peroxide were examined in vitro, using the canine basilar artery. It was shown that all of them possess a vasocontractile capacity in a dose-dependent fashion. Fresh canine arterial blood was incubated at 37 degree C for 2 weeks, and the daily change of its vasocontractile capacity and the amount of TBA-reactive substance was studied. This study revealed a gradual and parallel increase in both of them. In a clinical study with 32 SAH patients, the amount of TBA-reactive substance in the CSF was more elevated when angiographically confirmed vasospasm was present. The angiographical and histological responses of the basilar artery to the cisternal injection of 15-HPAA were studied in dogs. The cisternal injection of 15-HPAA (0.2 and 2.0 mg dissolved in bovine serum) caused a mild initial contraction of the basilar artery that lasted about 7 hours. On the third day, a stronger contraction occurred, persisting thereafter until sacrifice. Electronmicroscopy of those arterial samples revealed the disappearance of myofibrils, pyknotic changes of nuclei, and the appearance of vacuoles as well as electron-dense granules in the tunica media. The prolonged arterial contraction was always associated with remarkable destruction of the endothelial cells. These changes were essentially the same as those in experimental and clinical vasospasm. These data strongly indicate that free radical reactions initiated by clot lysis, represented by lipid peroxidation, plays an important role in the genesis of chronic vasospasm in subarachnoid hemorrhage.

Development and prevention of frozen shoulder after acute aneurysm surgery.

BACKGROUND: We conducted a study on periarthritis humeroscapularis or "frozen shoulder," a postoperative complication of aneurysm surgery. The purpose of this study was to seek the cause of this complication and the methods of preventing it in patients who undergo aneurysm surgery. METHODS: The diagnosis of frozen shoulder was based on the clinical presence of shoulder pain and difficulty in raising arms that developed within 3 months of surgery. Sixty-four patients who underwent aneurysm surgery with no motor deficit were examined and classified into three groups: (1) early surgery (29 patients in the acute stage after subarachnoid hemorrhage); (2) delayed surgery (19 patients in the chronic stage); and (3) elective surgery (16 patients with unruptured aneurysms). RESULTS: The incidence of frozen shoulder was 41% in the early surgery group, 16% in the delayed surgery group, and 13% in the elective surgery group. The highest incidence of frozen shoulder was found to occur in the early surgery group and was attributed to the immobility of their upper extremities during postoperative treatment. Since patients who undergo surgery in the acute stage are often delirious and confused for several days after surgery, their arms are tied down by their sides in order to prevent them from inadvertently removing catheters such as the one for ventricular drainage. It seems that this manner of immobilizing the patient's arms is the cause of the development of frozen shoulder: Our study showed that if each arm was passively raised by turns above the patient's shoulder, the patient was able to maintain the range of motion of the upper arms and was less likely to develop frozen shoulder. CONCLUSION: Inactivity of the shoulder joints due to immobilizing the upper extremities of patients after acute aneurysm surgery seemed to cause the development of frozen shoulder. The incidence of this complication was greatly reduced by keeping the patient's upper arms raised alternately to maintain their range of motion after acute aneurysm surgery.

A technique for prevention of donor site pain associated with harvesting iliac bone grafts.

Patients who have undergone anterior cervical spine fusion with an iliac bone graft frequently experience donor site pain after surgery. We found that an effective technique to avoid donor site pain is to round off the corners of the iliac crest with an air drill after harvesting the graft.

Intramastoid packing for CSF rhinorrhea following acoustic neurinoma removal.

A case is presented of cerebrospinal fluid rhinorrhea following operation on an acoustic neurinoma by suboccipital transmeatal approach. The rhinorrhea was cured by sealing the mastoid air cells with muscle pieces. Possible route of CSF rhinorrhea and significance of its treatment are discussed.

Actin in wound-healing of rabbit corneal endothelium. II. Study by nitrobenzoxadiazole-phallacidin method.

The distribution of F-actin in the rabbit corneal endothelial cells was studied in vivo and in culture using nitrobenzoxadiazole-conjugated phallacidin. In the normal cornea, the fluorescence showing the presence of F-actin was observed along the membrane of the endothelial cells, but little fluorescence was seen in the cytoplasm. During wound-healing processes after transcorneal freezing, the endothelial cells migrating to the wound area showed abundant fiber-like fluorescence in the cytoplasm. In about 28 days after the injury, the endothelial cells recovered normal shape and the pattern of actin localization became normal, ie, fiber-like fluorescence localization along the cell membrane. The endothelial cells were cultured for about one week and the explants were removed. After further culture for about two weeks the cultured cells became confluent forming a monolayer. At the center of this monolayer, a small wound was made, and changes in the cell shape and actin distribution were studied. The actin distribution in the undisturbed monolayer cells was similar to that seen in vivo, ie, fiber-like fluorescence along the cell membrane. After the wound production, many cells were seen to migrate toward the wound center, and abundant fluorescent fiber-like structures were observed throughout the cytoplasm. Addition of cytochalasin B to the culture medium suppressed cell migration in a dose-dependent manner. At a high cytochalasin B concentration the fiber-like fluorescence was not formed and scattered fluorescent speckles were observed. Further culture in cytochalasin B-free medium after exposure to this agent permitted a recovery of cell migration and formation of the fiber-like actin fluorescence. It was suggested that polymerization of actin filaments is activated in the migrating cells during wound-healing, and that cytochalasin B reversibly blocks this polymerization, thereby suppressing cell migration. Actin filament polymerization would constitute a significant part of the mechanism underlying cell migration and wound-healing.

Actin in wound-healing of rabbit corneal endothelium. I. Study by immunoperoxidase method.

The distribution of actin in the rabbit cornea was examined by the indirect immunoperoxidase method using mouse monoclonal anti-actin IgM and goat anti-mouse IgM serum conjugated with peroxidase, which was stained with 4-chloronaphthol. Flat preparation of the cornea was made and the actin distribution was examined in the normal endothelium and also during wound-healing of the endothelium that took place after transcorneal freezing. In the normal cornea, the epithelial, stromal and endothelial cells contained actin in the cytoplasm. Diffuse actin staining in the endothelial cytoplasm was observed in the flat preparation of the normal endothelium. After endothelial damage, the cells adjacent to the damaged area showed a fiber-like staining of actin around the nucleus; this occurred 12 to 24 hours after the freezing. Punctate staining was seen on the 2nd day, and the intensity of the staining was maximum on the 3rd day when the wound was completely covered. On the 3rd day, fibroblast-like cells were encountered and a long fiber-like staining of actin was seen in the cytoplasm, but the staining disappeared on the 4th day. The long fiber-like and punctate staining reappeared on the 14th day. Normal staining pattern was recovered on the 28th day. These findings suggest that actin production was accelerated in the endothelial cells during the wound-healing, and that this productive activity returned to normal levels as healing was completed.

Wound-healing of the corneal endothelium in the monkey: a morphometric study.

Topographical aspects and the time course of the healing processes in the corneal endothelium were studied in 31 cynomolgus monkey eyes. The corneal endothelium was damaged at the center by transcorneal freezing, using a probe with a tip diameter of 2.5 mm. Flat preparations of the endothelium stained with silver nitrate were made at various time intervals after freezing. Panoramic photographs of the corneal endothelium along the entire length of one meridian were made, and the sizes of individual cells were calculated by a computerized image analyzer. The size and shape of the endothelial cells showed marked changes around the wound edge, and the cells migrated to the duodenal area, which was covered by the endothelial cells in 3 days. Subsequently, the cell size distribution recovered toward the normal pattern, but multinucleated giant cells persisted in the center of the wound after one year. A slow enlargement of the cells occurred in the periphery, where final average cell size was about 25% larger than the original level. It was thought that the cells of a very wide area were mobilized in the wound-healing processes and that cell proliferation probably played a minor role.

Wound-healing of corneal endothelium in monkey: an autoradiographic study.

The mitotic activity of the corneal endothelium during wound-healing was studied in 8 eyes of 5 cynomolgus monkeys. By transcorneal freezing, the endothelium was damaged at the corneal center over an area with a diameter of 2.5 mm. At various intervals, 3H-thymidine was injected into the anterior chamber and its incorporation into the endothelial cells was studied by autoradiography. On flat preparations of the endothelium, the total number of cells labeled with 3H-thymidine was counted on all specimens. The labeling of the cells occurred exclusively within the zone of about 2 mm from the center of the wound. The maximum labeling occurred 2 days after freezing and it decreased abruptly after 4 days, when endothelial covering of the central denuded area had been completed. No labeled cells were encountered after 30 days. The percentage of labeled cells to all cells present in a given area was computed and its regional distribution studied. On the second day after freezing, the maximum percentage of 12-19% was found in the center of the wound in one cornea and about 2 mm from the center in another cornea. On the third day, the maximum percentage of only 3-7% was found in the center and it decreased toward the periphery. It was thought that the number of labeled cells was too small to cover the damaged area by cell multiplication. It is suggested that the endothelial defect in the monkey cornea is covered mainly by migration of the adjacent cells.

Transmission and scanning electron microscopic studies on endothelial cells in macular corneal dystrophy.

Macular corneal dystrophy of both eyes in a 50-year-old man was studied by light microscopy and transmission and scanning electron microscopies. The endothelial cells contained empty vacuoles and numerous granules stained with alcian blue and periodic acid fuchsin. The endothelial cells were thin, especially at the guttata excrescences of the Descemet's membrane. Transmission electron microscopy revealed two types of vacuoles in the endothelium. The first type, which has been reported before, contained fine fibrillar material. The second type, described for the first time in this paper, was larger in size than the first type, and contained mostly amorphous material which appeared at the same electron density as the empty space of the anterior chamber, while a small amount of fibro-membranous materials were occasionally seen in the vacuoles. The second type of vacuoles sometimes appeared to be flattened. Cytoplasmic organelles of the endothelial cells and endothelial junctions were normal. Endothelial cells at the guttata excrescences were thin and contained only fine filaments. Slight depressions were observed on the endothelial surface facing the anterior chamber. Scanning electron microscopy of the endothelium showed an irregular surface which was composed of craters, nodules and pits. The cytoplasmic membrane of the endothelial cells at the top of the craters were continuous with that at the bottom of the craters. Marginal folds of the endothelial cells were obscure. Vacuoles of the first type were identical to those seen in the keratocytes of this disease. The second type of vacuoles and the guttata excrescences of the Descemet's membrane were thought to represent the craters and nodules on the endothelial surface.

Electron microscopic observations of Descemet's membrane of peripheral cornea.

The posterior portion of the Descemet's membrane was studied by scanning and transmission electron microscopy; the materials comprised 87 human peripheral corneas with ages from 2 to 98 years, 5 monkey corneas and 4 rabbit corneas. In some specimens, the endothelium was removed by ultrasonication. After removal of the endothelium, "curly structures" were recognized on the surface of the Descemet's membrane, where the membrane showed a gradual thinning. These structures appeared along the whole circumference of the cornea with variable width in the human specimens, but in monkey and rabbit corneas, the extent of these structures was less than in the human cornea. The "curly structures" were not encountered in young subjects, and they increased with age. There was a positive correlation (r = 0.60, P less than 0.001) between the age and the extent of these structures. Other aging products of the Descemet's membrane, ie, Hassall-Henle bodies, were partly surrounded by the "curly structures". The human "curly structures" consisted of collagen fibrils, halo structures in the collagen bundles, wide-spacing fibers, microfibrils, ground substances containing minute filaments and a structure resembling the Descemet's membrane. Components of "curly structures" of the monkey and rabbit were almost the same as those of the human except for the Descemet's membrane-like structure.

Influence of argon laser treatment of glaucoma on corneal endothelium.

The morphometric study of the corneal endothelium was carried out by means of specular microscopy prior to and for one year following laser trabeculoplasty (LTP) or laser iridotomy. A significant increase in the endothelial cell size was demonstrated in 6 months after trabeculoplasty and in 3 months following iridotomy. The magnitude of increase in cell size following laser iridotomy was significantly correlated with the amount of laser energy delivered to the eye. However, such correlation was not demonstrated following LTP where the applied energy was kept fairly constant among the present treated cases. The increase in cell size failed to correlate with clinical factors including IOP before and after laser therapy, age and the presence or absence of endothelial burn. The treatment protocols using reduced energy are indicated to minimize endothelial damage induced by LTP or laser iridotomy.

A fluorophotometric study of the barrier functions in the anterior segment of the eye after intracapsular cataract extraction.

The transfer coefficient of fluorescein across the corneal endothelium, kc.ac, was determined by a method of oral dye administration in 54 normal eyes. The kc.ac averaged 0.46 +/- 0.13 (SD) hour-1, and the fluorescein distribution ratio between the anterior chamber and the cornea, rac, was 0.57 +/- 0.20. There were no significant differences in these values between young subjects and those older than 50 years. A total of 56 eyes which underwent intracapsular cataract extraction were divided into three groups, 1) those operated on with routine medications, 2) with topical indomethacin and 3) with alpha-chymotrypsin of 7,500 times dilution. The value of rac was fixed at 0.57 and kc.ac was calculated taking the fraction, X, of unbound fluorescein in the aqueous into account. In addition, the apparent transfer coefficients of the dye between the blood and anterior chamber, k'in and k'out, were also calculated from the apparent fluorescein concentrations in the whole plasma and aqueous. The kc.ac was significantly greater in patients than in normal eyes, showing damage of the corneal endothelium by the surgery. The value of X was less than unity, indicating an increase in aqueous protein in the patients, but it was closer to unity in the indomethacin group than in other groups. The value of k'in was significantly less in the indomethacin group than in other groups. Between the groups with routine medication and with chymotrypsin, no significant difference was found in X or in k'in. It was concluded that topical indomethacin suppressed disruption of the blood-aqueous barrier and also that chymotrypsin at the present dilution did not cause serious disruption of the barrier.

Permeability of the corneal endothelium to fluorescein--a follow-up of keratoplasty cases.

The permeability of the corneal graft endothelium to fluorescein was determined, by the oral administration method, in 47 eyes with penetrating keratoplasty. The endothelial permeability was expressed as the transfer coefficient of the dye between the anterior chamber and the cornea. In the early postoperative period, an increase in the aqueous protein was noted and a correction was made for fluorescein binding to aqueous protein. The measurements of the graft thickness and morphometry of the endothelial cells by specular microscopy were also carried out concurrently. The transfer coefficient was high in the early postoperative period, and was reduced by time. Similarly the graft was thick in the early postoperative period but it thinned to a normal level over a period of several months. A significant correlation was found between the transfer coefficient and the corneal thickness. The mean cell size of the graft endothelium was significantly greater than that of the normal cornea. The mean cell size and the postoperative period were correlated using the present data and those reported by Sato previously. A significant positive correlation was found, indicating progressive enlargement of the graft endothelial cells over a long period of time. The mean cell size was not significantly correlated with the transfer coefficient nor with the graft thickness.