Endothelial F-actin changes in the alkali burned rabbit cornea

The healing mechanism of corneal endothelium after alkali burn was not completely understood. Rabbit cornea was burned with 1N sodium hydxoside for 1 minute. Endothelial F-actin was stained with NBD-phallacidin in regular sequence to find out the details of endothelial healing after alkali burn. F-actin was completely destroyed leaving a sharp margin against the unaffected area 1 hour after the burn. In the 3, 5 and 7 day specimens, highly active F-actin reactions were noted at the wound margin. New multiple F-actin layers, arising from the intact endothelium near the wound margin, were noted in the 9 day specimen. In the 8 1/2 month specimen, the endothelial defected area was covered by large primitive cells, each of which showed F-actin fiber bundles in the cytoplasm with a large nuclear shadow. Nearly all of the large primitive cells showed F-actin fibers arranged in shapes of cell junctions. Twelve months after the burn, endothelial defects were not found. Nearly all of the endothelial cells were normal in size and shape except for some mushroom-like projections toward the anterior chamber in some areas. Nineteen months after the burn, the endothelial cells were normal. Endothelial wound healing process can be continued even 1 year after the alkali burn in rabbit cornea.     

Immunolocalisation of thrombospondin 1 in human, bovine and rabbit cornea

Light- and electron-microscopic immunohistochemical techniques were used to investigate the distribution of the matricellular protein thrombospondin 1 in normal human, bovine and rabbit cornea. Light-microscopic immunoreactivity for thrombospondin 1 was observed in the epithelial basement membrane, posterior Descemet's membrane and endothelium of human and bovine cornea. The bulk of the stroma, the stromal cells (keratocytes) and the anterior part of Descemet's membrane in human and bovine cornea were devoid of detectable thrombospondin 1 and the protein could not be demonstrated in any of the layers of the rabbit cornea. Electron-microscopic immunogold studies of human and bovine cornea revealed that thrombospondin 1 labelling of corneal endothelial (and basal epithelial) cells included focal deposits at cell membranes. It is postulated that thrombospondin 1 regulates interactions between cells and their basement membrane, and perhaps cell-to-cell interactions, in the normal human and bovine corneal endothelium and basal epithelium.     

Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea

In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34+/-11 % dead cells after 2 weeks, and 15+/-4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others. including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead cells observed in the first instance indicate a normal injury pattern. However, the presence of a small but significant proportion of invading inflammatory cells 12 weeks after implantation confirms a characteristic pattern of wound healing within the sponges.     

Preliminary evaluation of a hydrogel core-and-skirt keratoprosthesis in the rabbit cornea

BACKGROUND: We developed a core-and-skirt keratoprosthesis, with both components made from poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels. The identical chemical nature of both spongy skirt and transparent core assures a permanent union between them. We have previously shown that PHEMA sponges, within a certain range of pore size, can support cellular invasion and neovascularization when implanted into the rabbit cornea. The present study is the first to evaluate the behavior of the whole prosthesis after implantation into the rabbit cornea. METHODS: Hydrogel keratoprostheses were inserted intrastromally into the corneas of seven rabbits and histologically examined by light microscopy in five eyes enucleated at 8, 12, and 14 weeks. RESULTS: None of the implants extruded over this period. Both clinical and histopathologic examination showed that the keratoprostheses were well tolerated by the host tissue. The porous skirt was fully integrated into the stroma by fibrovascular invasion, and no capsule formed around the implants. Stromal melting anterior to the implant occurred in two cases, but this did not affect the fixation of the keratoprostheses. CONCLUSIONS: This study indicates that our keratoprosthesis can prevent extrusion in the short term when inserted into an intrastromal pocket of the rabbit eye.     

The effects of isoxsuprine on non-myelinated nerve fibres

A study has been made of the effect of isoxsuprine on non-myelinated nerve fibres of the desheathed rabbit vagus. Isoxsuprine did not affect the membrane potential, the membrane resistance, or the sodium pump activity. Isoxsuprine diminished the amplitude and rise time of the compound action potential. This indicates that isoxsuprine decreased the peak sodium current during the action potential. This local anesthetic action of isoxsupring is more pronounced than that of lidocaine.     

Enzyme-synthetic approach to demonstration of phosphorylase activity in the living rabbit cornea

An enzyme-synthetic method of demonstrating phosphorylase was applied to the living rabbit cornea, and polyglucose particles synthesized from glucose-1-phosphate in vivo were studied electron microscopically. In the corneas in which the medium for phosphorylase was applied from the anterior chamber or the bulbar subconjunctiva, synthesized polyglucose particles were found in the cytoplasmic matrices of the epithelium. When the medium was deposited in the conjunctival sac, a few synthesized polyglucose particles were found in the cytoplasmic matrices of only the superficial layer of the corneal epithelium. These findings suggest that metabolites for glycogen metabolism come mainly from the aqueous humor in the anterior chamber. The polyglucose particles synthesized by the enzyme-synthetic method in vivo resemble native glycogen particles. In addition, these particles were not overproduced because the synthesis of polyglucose is probably regulated in vivo.     

Effect of aflatoxin B and rubratoxin B on bacteriophage and rabbit cornea cells

Rabbit cornea cells exhibited a sensitivity to 1 mug aflatoxin B1 and 5 mug rubratoxin B per ml of growth medium. No changes were observed in the bacteriophages tested in the presence of 25 mug aflatoxin B1 or 100 mug rubratoxin B per ml of medium by the plaque-forming unit method or single-step growth curves.     

Ocular penetration and bioconversion of prostaglandin F2alpha prodrugs in rabbit cornea and conjunctiva

The objective of this study was to identify prostaglandin F2alpha (PGF2alpha) prodrugs that have an optimal ocular absorption profile and therefore could be potentially useful for the treatment of glaucoma. Rabbit cornea, conjunctiva, and iris/ciliary body were mounted in a flow-through chamber to evaluate the permeability and bioconversion of PGF2alpha and its prodrugs. The prodrugs tested were PGF2alpha 1-isopropyl, 1,11-lactone, 15-acetyl, 15-pivaloyl, 15-valeryl, and 11,15-dipivaloyl esters. After 4 h in the donor or acceptor compartments, the products and formation of PGF2alpha were analyzed by HPLC. Effects on intraocular pressure and ocular surface hyperemia were also determined. All prodrugs penetrated the rabbit cornea faster than PGF2alpha by 4- to 83-fold. All prodrugs penetrated conjunctiva faster than PGF2alpha, except the 15-acetyl ester prodrug, which was equally permeable. No direct correlation between drug lipophilicity and permeability across the cornea or conjunctiva was apparent. The most metabolically stable prodrug was the 1,11-lactone, followed by the 11,15-dipivaloyl, 15-pivaloyl, 15-acetyl, 1-isopropyl, and the 15-valeryl esters, the latter of which was extensively converted to PGF2alpha. A separation index for various prodrugs was calculated from the ratio of the bioavailable PGF2alpha for ocular hypotension to the bioavailable PGF2alpha for hyperemia. The highest separation index was observed for the 1,11-lactone prodrug (2.33), followed by the 11,15-dipivaloyl ester prodrug (1.80). Thus the 1,11-lactone and 11,15-dipivaloyl ester prodrugs appeared to be superior to the others in providing bioavailable PGF2alpha for ocular hypotension, while minimizing hyperemia. The favorable separation index for these compounds appeared to be due to their metabolic stability at the corneal surface and conjunctiva combined with sufficient bioavailability for ocular hypotension.     

Growth-associated protein GAP-43 and nerve cell adhesion molecule in sensory nerves of cornea

We used dual-wavelength fluorescence microscopy and monoclonal antibodies to growth-associated protein (GAP-43) and nerve cell adhesion molecule (N-CAM) to identify these proteins in nerve fibers of normal rat and rabbit corneas. Overlapping immunoreactivity of GAP-43 and N-CAM was evident along nerve fibers of rabbit corneal sections, suggesting that GAP-43 is constitutively expressed in these sensory nerves. The immune reaction of monoclonal antibody to GAP-43 and [125I]protein A was used to quantitate relative amounts of GAP-43 in the normal cornea and in a cornea subjected to a de-epithelializing wound. Collectively these findings imply that GAP-43 is axoplasmically transported from cells in the trigeminal (or superior cervical) ganglion to the cornea. Moreover, these data indicate that GAP-43 appears to be involved in the remodeling of corneal nerves that is necessary for normal innervation.     

Action potential fatigue in nonmyelinated nerve fibers: garfish olfactory and rabbit vagus nerve

With the new method the fecal flora of 13 clinically healthy dogs aged 3 to 42 months was analysed qualitatively and quantitatively. It was characterized by that bacteroidaceae constituted the most prodominant flora, catenabacteria, streptococci, peptostreptococci, lactobacilli and bifidobacteria were the next most numerous, enterobacteria consisted the accompanying flora. The numbers of Clostridium perfringens were remarkably fluctuated and seem to be influenced by the composition of the food ingested by the host. The total cultivable counts averaged log 10.8 +/- 0.2/g. The composition of the Lactobacillus flora and Bifidobacterium flora in the feces of 34 dogs from 3 different age groups was analysed. In the feces of all age groups L. acidophilus, L. salivarius and L. fermenti with 9 different types were found. Within the lactobacilli L. acidophilus type VI c, L. salivarius type VI a and L. fermenti type IV b were most frequently found. L. acidophilus types VI c and XI, L. salivarius types VI a and VI b could not be placed in any established types of each species, and were described as new types. Bifidobacteria were regularly found in large numbers in the feces of dogs aged 2 to 24 months. B. adolescentis and B. pseudolongum consisted the main Bifidobacterium flora.     

Distribution and origin of peptide-containing nerve fibres in the rat and human mammary gland

The structures in the mammary gland involved in milk ejection have been investigated with regard to their relation to different types of peptidergic nerve fibres and their origin. Lactating rats were studied with immunohistochemistry focusing on the nipple, the parenchyma, the mammary blood vessels and the mammary nerve. The human mammary gland was also analysed. In the mammary gland from rat and human, nerve endings in the subepidermis, around smooth muscle cells in the nipple, in the connective tissue surrounding lactiferous ducts and alveoli in the nipple and in the parenchyma of the mammary gland showed immunoreactivity for calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, peptide histidine isoleucine, neuropeptide Y, galanin and tyrosine hydroxylase, whereas dynorphin-positive nerve fibres could not be detected. The mammary nerve contained calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y and tyrosine hydroxylase immunoreactivities; the adventitia of the mammary artery contained nerve fibres immunoreactive for neuropeptide Y and tyrosine hydroxylase, while vasoactive intestinal polypeptide-, peptide histidine isoleucine-, calcitonin gene-related peptide- and substance P-positive fibres were found in the tissue surrounding the artery. The wall of the mammary vein had nerve terminals immunoreactive for neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P. With the help of retrograde tracing using wheat germ agglutinin in combination with immunohistochemistry, projections of calcitonin gene-related peptide-immunoreactive cells in the dorsal root ganglia to the nipple were established. Neurons in the sympathetic stellate ganglion containing neuropeptide Y and tyrosine hydroxylase also projected to the mammary gland. Moreover retrogradely-labelled cells were found in the nodose ganglion, and they were vasoactive intestinal polypeptide-immunoreactive. These results demonstrate a rich distribution of different types of nerve fibres in structures of the mammary gland related to milk ejection. These nerve fibres and their peptides may be involved in the local control of milk ejection.     

The effect of fibronectin on re-epithelialization of rabbit cornea after alkali burn

As in other organ systems, there are ocular disorders largely unique to HIV such as Kaposi's sarcoma and cytomegalovirus retinitis. Other syndromes, such as acute retinal necrosis, although not unique to HIV infection, are well recognised in this group of patients and are sufficiently uncommon to make one consider HIV infection. However, most ocular signs and symptoms of HIV infection are common and non-specific, and require other clinical clues to raise the suspicion of HIV infection.     

Natural complex of cytokines is a potent stimulant to posttraumatic regeneration in rabbit cornea

We elaborated an original technique based on local application of natural complex of cytokines (NCC) secreted by autologous peripheral leukocytes. In this study, we evaluated NCC influence on the healing of penetrating corneal wounds. NCC was derived from supernatants of PHA-stimulated rabbit peripheral leukocytes. Biological tests revealed the presence of IL-1, TNF, IL-6, MIF, and LIF in the complex. Chinchilla rabbits with standard penetrating corneal wounds received daily NCC instillation. The controls were instilled with cultural media 199 with antibiotics. At 24 hours, 3, 7, 14 and 30 days post-wounding, the rabbits were euthanized. We performed morphometry of corneal cross-sections stained with hematoxylin and eosin. In NCC-treated animals, we observed more vigorous migration and activation of neutrophils and macrophages followed by augmented resorption of fibrin. The later post-injury period (14-30 days) was marked with complete healing of the endothelial defect (in the controls, the mass of proliferating cells projected into the anterior eye chamber) and mature scar tissue with a higher content of fibrous component. NCC-treated eye scars were 1.6-fold thinner than the controls'. Local application of NCC promotes effective healing of posttraumatic cornea. It regulates all stages of regeneration and prevents rude scarring.     

Pharmacokinetics of intravitreal vancomycin in normal and infected rabbit eyes

The purpose of this study was to determine the pharmacokinetics governing the distribution and elimination of intravitreally injected vancomycin in normal and infected rabbit eyes. Two groups each of 36 pigmented animals were used. Group 1 served as control. In Group 2, experimental endophthalmitis was induced in the right vitreous by inoculation with Staphylococcus aureus. Once endophthalmitis developed, a vancomycin solution was injected. Four animals from each group were killed at nine time points post-injection, the vitreous and aqueous were removed, and blood samples were taken for HPLC analysis. Data analysis was performed using the RSTRIP program. The half-lives were 69 hours in normal vitreous and 14.53 hours in infected vitreous. Therapeutic drug levels were present in the vitreous 84 hours post-injection in all eyes; they were detected from 2 to 48 hours in normal aqueous but at lower levels in the infected ones. Kv and Ca/Cv ratios suggested that the primary route of elimination was across the retina and the anterior chamber in normal eyes, and via the retina in infected eyes. Results indicate that pharmacokinetic parameters change in pathological conditions, which may help establish better treatment guidelines for endophthalmitis.     

Medullary projections of rabbit carotid sinus nerve

In New Zealand white rabbits, cholera-toxin HRP was injected into the carotid sinus nerve just proximal to the carotid sinus. After survival periods of 3-5 days the rabbits were anesthetized and the brain fixed with aldehyde solution. Transverse sections were cut on a sledge microtome and the sections reacted with the tetramethylbenzidine procedure. HRP-positive fibers entered the ipsilateral dorsolateral medulla at the level of the acoustic tubercle, joining the tractus solitarius. Positive fibres were found principally ipsilaterally in four regions of the medulla: in the caudal two thirds of the nucleus tractus solitarius, in its dorsolateral regions and, more caudally, in its commissural subdivision; in the dorsolateral aspect of the spinal nucleus of the trigeminal nerve; in the region ventral and ventrolateral to the tractus solitarius (extending beyond the nucleus tractus solitarius); and in the ventrolateral medulla oblongata.     

Chemosensitivity of nociceptive, mechanosensitive afferent nerve fibres in the guinea-pig ureter

The mechanosensitivity and chemosensitivity of afferent fibres were investigated in an in vitro preparation of the guinea-pig ureter. Electrophysiological recordings were obtained from 5 U-1 (low mechanical threshold, contraction-sensitive) and 74 U-2 units (high threshold). U-2 units had significant higher levels of spontaneous activity, lower conduction velocities, higher mechanical thresholds (U-1: 7 mmHg; U-2: 39 mmHg), less pronounced phasic responses and longer latencies in the response to distensions than the U-1 units. For chemical stimulation, guinea-pig urine (> 800 mosmol/L), bradykinin and capsaicin were applied intraluminally. The responses of U-1 units mainly corresponded to the contractions induced by the chemical stimulation. The vast majority of the U-2 units were excited by urine, bradykinin (threshold: 0.1-1 microM) and capsaicin (threshold: 0.03-0.3 microM). The responses to urine could be mimicked by high concentrations of potassium ions (> 200 mM), but not by an equiosmolar solution of NaCl, urea and mannitol. Chemical stimulation could also result in a transient sensitization of the U-2 units to mechanical stimuli. In the anaesthetized guinea-pig, pseudo-affective responses could be evoked by ureteric distension (threshold: 30-60 mmHg) and serosal application of capsaicin. Intraluminal application of urine in vivo did not evoke any reactions, suggesting that the responses of the U-2 units to urine might be due to an impaired barrier function of the urothelium in vitro. The data are in agreement with the hypothesis that U-2 units are visceral polymodal nociceptors. Since the U-1 units were also able to encode at least noxious mechanical stimuli, their involvement in visceral nociception cannot be excluded.