The specific architecture of the anterior stroma accounts for maintenance of corneal curvature.

AIM: To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability. METHODS: Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer. RESULTS: After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 microm) was much thicker than that of PBS corneas (650 microm) and PM corneas (450 microm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100-120 microm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states. CONCLUSIONS: The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.

The effects of organ-culture on the density of keratocytes and collagen fibers in human corneas.

PURPOSE: Keratocytes are important in regaining corneal transparency during wound healing after surgery or trauma. Hitherto, there are still controversies concerning the effects of organ culture on the density and integrity of keratocytes and collagen fibers. The current study aimed at a systematic analysis of the effects of organ-culture on the morphology and density of keratocytes and collagen fibers. METHODS: Human corneas were organ-cultured in MEM for 7 (n = 17, 3 pairs), 14 (n = 18, 9 pairs) and 21 days (n = 18, 9 pairs). Of the pairs one cornea was processed in swollen condition and the fellow cornea after reversal of swelling in MEM plus Dextran. Eleven post-mortem corneas (PM) and 11 fresh corneas obtained from melanoma patients were used as controls. Stromal thickness, number of keratocyte profiles (corrected for swelling), number and diameter of collagen fibers were measured in light microscopical sections and electron micrographs. RESULTS: Stromal swelling due to organ-culture resulted in large keratocyte profiles with many vacuoles and large distances between collagen fibers in the posterior stroma. In contrast both keratocytes and distances between collagen fibers were not affected in the anterior stroma. After reversed-swelling the posterior corneal stroma was similar to that in fresh controls, indicating that the swelling process is largely reversible. The initial decrease in keratocyte density (18%) in the early post-mortem period did not progress during 21 days of organ culture. CONCLUSION: With respect to the morphology and density of keratocytes and collagen fibers it can be concluded that donor corneas remain suitable for transplantation up to at least 21 days after organ-culture.

Corneal morphology and sensitivity in lattice dystrophy type II (familial amyloidosis, Finnish type).

PURPOSE: To describe the corneal abnormalities and to measure different modalities of corneal sensitivity in corneal lattice dystrophy type II (familial amyloidosis, Finnish type, also known as gelsolin-related amyloidosis and originally as Meretoja syndrome). METHODS: Twenty eyes of 20 patients were examined by in vivo confocal microscopy and noncontact gas esthesiometry. RESULTS: Pleomorphism of, and dense deposits between or posterior to, the basal epithelial cells were frequently observed, as well as a reduction of long nerve fiber bundles in the subbasal nerve plexus. The anterior stroma was altered in most cases, with fibrosis and abnormal extracellular matrix. In 15 corneas, thick anterior and midstromal filaments, corresponding to lattice lines, and in 11 corneas, thin undulated structures were observed. The average mechanical sensitivity threshold of 12 subjects was increased, and in the remaining 8 subjects there was no response, even to the highest intensity of stimuli used. Three patients did not respond to CO(2), 11 to heat, and 2 to cold, but those patients who responded had normal thresholds. Patients with more long nerve fiber bundles per confocal microscopic image had better mechanical and cold sensitivity than patients with fewer nerve fiber bundles. CONCLUSIONS: Lattice lines seem to be related to amyloid material and not to corneal nerves. However, the subbasal nerve density appears reduced, which results mainly in a decrease in mechanical and, to a lesser extent, thermal sensitivity. The location of stromal filaments and undulated structures changes with increasing age.

Architecture of human corneal nerves.

PURPOSE: The corneal innervation, mainly analyzed in light microscopical studies, has been described as radially oriented stromal nerve bundles that ramify as leashes in the subbasal plexus. The current study aims to determine the orientation, the size, and the postmortem changes of the nerve fibers in the subbasal plexus of the human cornea. METHODS: Before processing for light and electron microscopy, the position of the corneas within the enucleated eyes of persons with melanoma and pairs of postmortem eyes was marked. The orientation and postmortem changes of the fibers were studied in serial "en face" semithin sections, and the size was determined in random, ultrathin cross-sections. RESULTS: Thirteen and a half hours after death, the majority of the nerve fibers were degenerated or gone. Nerve fiber bundles in the subbasal plexus run first in the 9-3 hours direction, then after bifurcation in the 12-3 hours direction and after a second bifurcation again in the 9-3 hours direction. From the main straight bundles, single-beaded fibers branch and run obliquely. Quantification of the nerve fibers shows an equally dense innervated central and central-peripheral cornea (mean fiber diameter, 0.4 micron) and a five to six times lower innervated peripheral cornea (mean fiber diameter, 0.67 micron). CONCLUSIONS: The nerve bundles in the subbasal plexus of the human cornea form a regular dense meshwork with equal density over a large central and central-peripheral area. Because of their size, the majority of the fibers can be classified as C-fibers.

Ultrastructural organization of human corneal nerves.

PURPOSE: Although the human cornea is densely innervated, observations of the nerve fiber distribution and ultrastructure are scarce. This study aimed to provide a detailed electron microscopic analysis of nerve fibers in the central and peripheral human cornea. METHODS: Samples from seven fresh corneas, obtained from the eyes of persons with melanoma, were processed for light and electron microscopic examinations. Both frontal and cross-sections were studied. Furthermore, serial ultrathin sections from the mid-epithelium to the anterior stroma were used. RESULTS: Unmyelinated nerve fiber bundles (as many as 30 nerve fibers and cross-section as large as 20 micrometers) run parallel to the stromal collagen fibers. Nerve fibers contain clear, dense cored and dense vesicles and are ensheathed by thin rims of Schwann cell protrusions and amorphic matrix. Some nerve fibers invaginate the cytoplasm of keratocytes. After passing through Bowman's membrane, bundles of straight fibers (cross-section 0.1 to 0.5 micrometers) and single-beaded nerve fibers, which both lack Schwann cell ensheathment, run parallel in an alternating manner. Beaded nerve fibers, containing many mitochondria and glycogen (cross-section as large as 2 micrometers), turn upward and invaginate both basal and wing cells. Except for the presence of myelinated nerve fibers in the peripheral stroma, no differences in the central cornea were observed. CONCLUSIONS: Nerve fibers invaginating epithelial cells and keratocytes suggest that both cell types are directly innervated. The presence of vesicles, mitochondria, and glycogen in stromal and epithelial nerve fibers suggest that classical and peptidergic transmitters, probably of sensory origin, innervate the human cornea. Peptidergic transmitters in nerve fibers may be involved in neuroimmunomodulation of the cornea.

Ultrastructural neuropathologic effects of Taxol on neurons of the freshwater snail Lymnaea stagnalis.

Cerebral ganglia of the freshwater snail Lymnaea stagnalis were incubated in vitro in 10 microM Taxol for 8 and 24 h. Cremophor EL (0.1%) was used as a diluant. The tissue was processed for electron microscopy. Various ultrastructural parameters were assessed quantitatively. Cremophor EL appeared to seriously affect the cell somata of the multipeptidergic caudodorsal cells. In the Cremophor-controls the mean area of Golgi zones, the percentage dense material (neuropeptides) in these zones, the number of large electron dense granules (these are involved in neuropeptide processing) and the mean nuclear heterochromatin clump size, were significantly smaller than in the Ringer-controls, whereas the number of lipid droplets was higher. All these parameters, except for the lipid droplets, were not different in the Cremophor-controls and the Taxol-treated specimens. After 24 h treatment, but not after 8 h, Cremophor EL furthermore induced an increase in the number of axonal microtubules. It is argued that the results might signify activation of the neurons by Cremophor EL. Taxol induced a significant increase in the number of microtubules in axons and cell somata. Furthermore an increase in the number of Golgi zones was observed, suggesting activated neuropeptide synthesis. In all groups immunostaining with antibodies to neuropeptides produced by the caudodorsal cells was normal. Release of neuropeptide (exocytosis) from axon endings was elevated after Taxol treatment, and exceptionally high in specimens cotreated with Taxol and Org 2766 (incubation time 22 h). The effect of Org 2766 and Taxol on the number of microtubules was cumulative.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the ACTH(4-9) analogue, ORG 2766, on vincristine cytotoxicity in two human lymphoma cell lines, U937 and U715.

The use of cytotoxic drug vincristine (VCR) is limited by the occurrence of peripheral neuropathy. A neurotrophic ACTH(4-9) analogue, ORG 2766, is being studied for its protective effect. Possible modulatory effects of ORG 2766 on tumour cell growth and interference with the cytotoxic efficacy of VCR were studied in two human lymphoma cell lines, U937 and U715. The effects of ORG 2766 on cell growth and survival and on VCR-mediated cytotoxicity were investigated using two MTT-based assays to study direct cytotoxic effects and to assess residual growth after pretreatment. Treatment with ORG 2766 alone had no effect on cell growth and survival. Neither did this drug affect VCR cytotoxicity. However, after 96 h pretreatment with ORG 2766 and a culture period of 7 days, a reduction in residual growth and a potentiation of VCR-induced inhibition of growth capacity was observed in U715 cells, and to some extent also in U937 cells. It is concluded that ORG 2766 has no stimulatory effects on tumour growth and does not negatively interfere with VCR-mediated cytotoxicity. Rather it enhances the cytostatic effect of VCR. It is suggested that ORG 2766 can safely be used in clinical trials investigating the ability of ORG 2766 to counteract VCR-induced neurotoxicity.

Quantitative ultrastructural effects of cisplatin (Platinol), carboplatin (JM8), and iproplatin (JM9) on neurons of freshwater snail Lymnaea stagnalis.

Qualitative and quantitative ultrastructural effects of the platinum compounds cisplatin (Platinol), carboplatin (JM8), and iproplatin (JM9) were studied on two types of identified peptidergic neuron (caudodorsal cells, light green cells) in the pond snail Lymnaea stagnalis. Depending on the parameter under investigation, either one or both cell types were studied. Central nervous systems of the snail were incubated for 5 and 20 h in various identical and equitoxic drug concentrations. Cisplatin had the most severe effects. Platinol, i.e., cisplatin dissolved in NaCl solution with the addition of HCl (pH 2.0-3.0), as well as cisplatin dissolved in snail Ringer's solution (pH 7.8), caused swelling of axons and distensions of the intercellular spaces. This drug induced an increase in chromatin clump size in the caudodorsal cells (20-h incubation), while carboplatin and iproplatin induced the formation of many small chromatin clumps. Incubation in snail Ringer's solution (controls) and cisplatin affect the morphology of the nucleoli. At high dosages of cisplatin, the nucleoli of light green cells were transformed into homogeneous dense structures. The data indicate that platinum compounds react with nuclear and nucleolar DNA. All three drugs affected the activity and organization of the rough endoplasmic reticulum and the Golgi apparatus of the peptidergic neurons studied (qualitative observations). These effects, which point to a reduced neuropeptide synthesis, may be secondary, i.e., exerted via inhibition of RNA synthesis and ribosome formation (nucleoli). The fact that the number of neuropeptide granules in the cytoplasm of the cells remained constant (both cell types) may indicate that granule transport was also inhibited. Cisplatin and iproplatin induced an increase in the number of lysosomes in the light green cells. The number of lipid droplets in these cells was not affected by drug treatment. The results corroborate clinical data indicating that cisplatin is highly neurotoxic. Despite conflicting clinical data, observations on the snail neurons suggest that iproplatin is also neurotoxic, although less than cisplatin. Carboplatin is minimally neurotoxic, which is in accordance with clinical data. The central nervous system of Lymnaea is a suitable model for studying possible neurotoxic effects of platinum compounds.

Morphological and electrophysiological study of the effects of cisplatin and ORG.2766 on rat spinal ganglion neurons.

Eleven- to 12-wk-old rats were treated twice a week with cisplatin/saline or with cisplatin plus ORG.2766 during 12.5 wk. Cisplatin and ORG.2766 were administered at a final concentration of 0.04 mg/ml (i.p.) and 10 micrograms/ml (s.c.), respectively. Control animals were treated with saline. In this period the cisplatin-treated animals developed a peripheral neuropathy resulting in impairment of sensory functions. Estimates of the motor (MNCV) and sensory (SNCV) nerve conduction velocity were made after 0, 7.5, 10, and 12.5 wk. It appeared that the MNCV of the control, cisplatin-, and cisplatin plus ORG.2766-treated rats increased from 50 to 59 m/s. In contrast, the SNCV of the cisplatin-treated rats decreased significantly (P less than 0.001) from 63 to 56 m/s, whereas that of the control animals increased from 62 to 84 m/s. Rats which received cisplatin plus ORG.2766 showed an increase in SNCV up to control levels. After 12.5 wk the animals were perfused with a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer. At the level of L5 and L6, 5 mm of spinal cord tissue and three dorsal root ganglia were removed and processed for electron microscopy. With the point-counting method the volume fraction (v/v) of somata and myelin in spinal ganglia was estimated. No significant change in the volume fraction of somata of the control (0.42), cisplatin (0.33)-, and cisplatin plus ORG.2766 (0.39)-treated rats was found. The same held true for the volume fraction of myelin of the control (0.53), cisplatin (0.59)-, and cisplatin plus ORG.2766 (0.58)-treated rats. In addition, the number of lysosomes per 100 microns 2 was estimated in spinal ganglion neurons and in spinal cord motor neurons of a total of 120 randomly chosen neurons. It was found that the number of lysosomes in the spinal ganglion neurons of the control animals was lower (10 per 100 microns 2) than in cisplatin-treated (30 per 100 microns 2) and in cisplatin plus ORG.2766-treated rats (28 per 100 microns 2) (P less than 0.05). No difference was observed in the number of lysosomes between cisplatin- and cisplatin plus ORG.2766-treated rats. The number of lysosomes in spinal cord tissue of cisplatin-treated rats (2.4 per 100 microns 2) did not differ from controls (0.1 per 100 microns 2) and from cisplatin plus ORG.2766-treated rats (0.8 per 100 microns 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Use of snail neurons in developing quantitative ultrastructural parameters for neurotoxic side effects of Vinca antitumor agents.

The central nervous system of the snail Lymnaea stagnalis was studied in order to develop a test system to predict the neurotoxic side effects of the three cytostatic Vinca alkaloids, vincristine (VCR), vindesine (VDS), and vinblastine (VLB). Vinca alkaloids appear to interfere with microtubule formation by the induction of paracrystalline inclusions. After in vitro incubation the numbers of these inclusions were counted in cross-sections of the cerebral commissure using electron microscopy. For each compound the number of paracrystalline profiles increases with increasing concentrations and incubation times. At equimolar concentrations (0.15 mM), VCR induces more paracrystals than VDS, and VDS induces more than VLB. These effects are clear after short periods of incubation (e.g., after 2 h, VCR:VDS:VLB = 5:2:1). Equitoxic concentrations of VCR, VDS, and VLB induce similar numbers of paracrystals. Furthermore, morphological changes in the cell bodies of identified neurons (light green cells) in the cerebral ganglia were observed. Quantitative analysis shows that at equimolar concentrations the surface area of nuclear chromatin of all Vinca alkaloid-treated cells is approximately 30% lower than that of the controls. The lamellae of the rough endoplasmic reticulum are swollen and have lost their regular arrangement. For VDS and VLB this swelling is accompanied by a strong increase (about 3-fold) in the total surface area of the rough endoplasmic reticulum. No increase was observed for VCR. The compounds do not affect the number of secretory granules. In contrast to the controls, all Vinca-treated cells show lipid droplets. After VCR treatment they are about 5-fold as numerous as after treatment with VDS or VLB. The total surface area of lysosomes increases about 1.3-fold by VDS and VLB treatment and about 3-fold by VCR treatment. From these quantitative data it is concluded that VCR is more neurotoxic than VDS and VLB. VDS appears to be more neurotoxic than VLB as judged from the data on paracrystal induction. On the basis of a comparison of these data with clinical data on Vinca-induced neurotoxicities, it is proposed that neurons of the snail L. stagnalis may be suitable for the development of a test system to predict the degree of clinical neurotoxicity induced by Vinca antitumor drugs.

Snail neurons as a possible model for testing neurotoxic side effects of antitumor agents: paracrystal formation by Vinca alkaloids.

The suitability of neurons of the freshwater snail Lymnaea stagnalis as a test system for the neurotoxic side effects of antitumour Vinca alkaloids has been investigated, by studying the process of paracrystal induction by Vinca antitumour agents. Three Vinca alkaloids have been compared: the natural vinblastine and vincristine and the semisynthetic vindesine. They appear to induce two types of inclusion. The first type is paracrystalline and has a rod-like shape with a width of 0.3-2.5 micron and a length of 1-10 micron. It consists of hexagonally arranged tubules with a lattice constant of approximately 28 nm. The second type appears as ladder-like profiles with a periodicity of approximately 30 nm. It is proposed that the ladder-like profiles are in fact helical structures and are precursors of the paracrystals. Both types of inclusion may fill up large parts of the axons; they are rare in axon terminals and almost absent from the neuronal somata. It has been concluded that the process of paracrystal induction by Vinca alkaloids in Lymnaea neurons is very much the same as in mammalian neurons and may be largely responsible for the neurotoxic effects of the Vinca drugs, because it impairs axonal transport of neuronal secretory granules. Apparently, in this respect vindesine behaves in a similar way as the conventional vincristine and vinblastine drugs. Vincristine induces clearly more paracrystals than vindesine, whereas the least paracrystals occur in vinblastine-treated material. These differences correlate with the different clinical neurotoxicities of these drugs. Therefore, because Lymnaea neurons can be considered as excellent model systems for studies of the functioning of neurons in general, it is expected that counting the number of paracrystals in Lymnaea nervous tissue will prove to be a good method to predict the degree of clinical neurotoxicity of newly developed antitumor Vinca alkaloids.