Keratocyte density and size in conscious humans by digital image analysis of confocal images.

PURPOSE: Confocal microscopy can give images of high magnification and resolution in undisturbed living tissue. It provides new information about the cellular structure of the cornea. Our aim was to measure the density, size and distribution of keratocytes. METHODS: Healthy cornea in four subjects was examined using tandem scanning confocal microscopy. Methods for digital analysis of images were developed. RESULTS: Keratocyte density in confocal cross-sections was greatest immediately under Bowman's membrane (maximum 800 cells/mm2) and decreased sharply towards posterior cornea (minimum 65 cells/mm2). Cross-sectional cell size ranged from 78 to 211 microns2, but did not correlate with depth in the tissue. CONCLUSIONS: Results are consistent with those of earlier work using histological and biochemical techniques in isolated tissue. The methods we have developed enable studies of ongoing processes in conscious humans and can be used to examine diseased tissue as well as the response to injury.

Confocal microscopy using oblique sections for measurement of corneal epithelial thickness in conscious humans.

PURPOSE: Thickness measurements by confocal microscopy in conscious human subjects may be liable to error as a result of instability of the eye or instrument. Our aim was to evaluate a technique which was expected to be less sensitive to such problems. METHOD: Thickness of corneal epithelium was determined from oblique confocal sections through cornea. A contact lens of known thickness worn by subjects was used to calibrate images. RESULTS: There were two layers in images which could have corresponded to the stromal/epithelial interface. The mean result in each subject ranged from 38 to 53 microm using the more superficial layer and 46 to 60 microm using the deeper one. The smaller values gave the distance between the epithelial surface and the sub-epithelial nerve plexus and thus seemed to correspond to epithelial thickness. CONCLUSIONS: Measurements of epithelial thickness by our new method are comparable with results of earlier studies.

Neurosecretory endings and target cells in the synaptic cleft of the human vocal cord: an electron microscopic study.

The action of neurotransmitters is of interest in the normal function of the vocal cord since they regulate mucous secretion and vessel caliber. The intersynaptic space between glandular and myoepithelial cells and the corresponding neuroendocrine endings is the area in which these substances are delivered. This area has not been previously studied in the human vocal cord. Specimens from vocal cords have been studied by electron microscopy. In our study, it seems to be clear that there exists a very close contact between myoepithelial cells and secretory nerve endings. This close relationship is likewise observed between these cells and glandular cells.

Effects of air injection into the corneal stroma of man: an in vitro study.

BACKGROUND: Corneal stromal air injection has been described for assisting the deep dissection of the recipient corneal bed in lamellar grafting. The consistent finding of air entering the anterior chamber is considered an undesirable complication of the procedure. The route of air entry into the anterior chamber has not been investigated previously. METHODS: The route of air passage through the cornea was studied in 10 human cadaver eyes by direct viewing of the inner corneal surface with an operating microscope and by electron microscopy. RESULTS: Air entered the anterior chamber peripheral to Descemet's membrane in every case in the absence of pre-existing corneal pathology. The air expansion of the cornea occurred only along planes occupied by keratocytes and their cell processes and this expansion caused disruption of the keratocytes. CONCLUSIONS: Air injection into the cornea caused bubbles to enter the anterior chamber peripheral to Schwalbe's line, through the trabecular meshwork. The air produced cleavage of the collagen lamellae along those planes occupied by keratocytes and their cell processes since this provides a path of least resistance. Disruption of the keratocytes is a direct mechanical effect. Descemet's membrane and Bowman's layer act as effective barriers to air passage in the normal cornea.

Comparison of tear film thickness in three species determined by the glass fibre method and confocal microscopy.

Using optical methods to measure tear film thickness we previously found that values were substantially larger than earlier estimates by mechanical and chemical techniques. The predominant component was found to be mucus, not aqueous fluid. This article describes the results of using confocal microscopy to re-examine an earlier method of measuring thickness, the glass fibre technique. Confocal microscopy enabled visual identification of tear and epithelial surfaces and accurate measurement of the separation between them, giving the full film thickness. Consecutive measurements were made by each method in eyes of 22 animals of three species. Measurements using fibres in rabbits gave results of 7 microns, replicating earlier findings. However, the fibre technique gave the same result in all species. Values were not correlated with measurements by microscopy, which ranged from 10 to 14 microns. We propose that fibres were too flexible to be pressed through all mucus layers and contact epithelium, and as a result this method underestimated film thickness.

Pathological changes in levator palpebrae superioris muscle treated with botulinum toxin in a case of carotico-cavernous fistula.

We describe the case of a patient with carotico-cavernous fistula who had botulinum toxin A to induce a protective ptosis 4.5 days before death. The levator palpebrae superioris muscle from both sides and the superior rectus muscle from the injected side were obtained for examination. The preserved samples were stained with haematoxylin and eosin, Martius scarlet blue, Glees, S100, dehydrogenase, ATPase, and toluidine blue as well as being examined by electron microscopy. Inflammation and oedema were found that were probably due to the carotico-cavernous fistula. Axonal and some myelin sheath damage were also seen.

Bacterial adhesion to intraocular lenses.

Intraocular lenses removed because of recurrent episodes of intraocular inflammation showed bacteria adhering to the prosthesis. Infections localized to areas around intraocular lenses have been reported in the literature. Two such infections and an in vitro study of bacterial adherence to implant surfaces are reported. Adherent bacteria were found to be more numerous on the polypropylene haptic than on the polymethylmethacrylate optic in both in vitro and in vivo cases. We suggest that the intraocular lens becomes contaminated with commensal organisms of low virulence during insertion into the eye, and that these organisms are the source of the inflammatory episodes. Experiments show that lenses as supplied are sterile and that in vitro bacteria adhere preferentially to the haptic.

The autonomic innervation of the human vocal cord: neuropeptides.

Three methods have been used to study the sympathetic and parasympathetic innervation of the vocal cord: paraformaldehyde-induced fluorescence, electron microscopy, and the peroxidase-antiperoxidase technique. Each method has been successful in determining adrenergic fibers, autonomic endings, and the neuropeptides VIP and NPY, respectively. The close relationship between these neuropeptides and the autonomic endings (adrenergic and cholinergic), and the presence of the neuropeptides in the chorion of the vocal cord, around glands and vessels, gives new evidence that autonomic innervation plays a role in the functioning of this part of the larynx.

Differential staining of ocular goblet cells.

Millipore filters were used to obtain sheets of cells from the ocular surface. Using Periodic Acid Schiff-haematoxylin the intracellular neutral mucus of the goblet cells stains a brilliant, bright pink and the cell nuclei dark blue making it possible to observe the epithelial cells and the goblet cell population. In certain ocular surface diseases the size of the PAS-haematoxylin staining goblet cell population is reduced, returning towards normal as ocular health improves. These observations had previously been interpreted as showing degeneration followed by regeneration of the goblet cells. However, Alcian blue stains an acidic mucus, which does not stain with PAS-haematoxylin. Acidic mucus containing goblet cells have been revealed using Alcian blue staining when added to the PAS stained cells in conditions that have previously been shown to have reduced goblet cell population when assessed by PAS-haematoxylin staining. This suggests that the staining properties of some of the goblet cells have changed. The extra goblet cells revealed by Alcian Blue staining makes the total of goblet cells present close to normal in numbers. Improved clearing methods and cytological preservation has revealed that there is also a population of goblet cells that do not stain with either technique. These observations must cast doubts on previous claims of reduced goblet cell population associated with some extraocular diseases.

Vasoactive intestinal polypeptide and neuropeptide-Y, as possible mediators in laryngeal oedema. An immunofluorescence study.

Little attention has been paid to the autonomic innervation of the human vocal cord. Vasoactive intestinal polypeptide (VIP) and Neuropeptide-Y (NPY) are of interest because of their association with the autonomic nervous system. These neuropeptides have not been studied in the human vocal cord by immunofluorescence technique. Both neuropeptides were found in the connective tissue close to the basal layer of the mucosa, around glandular structures and arterial walls. Comments are made about the role these peptides might play in the physiopathology of vocal cord oedema.

Botulinum toxin A-induced protective ptosis in corneal disease.

Botulinum toxin A produces a temporary, flaccid ptosis when injected into the levator palpebrae superioris muscle. The resulting protective ptosis was used to aid healing in 21 cases of indolent ulceration, and, prophylactically, in 4 cases of neuroparalytic keratitis. Of the indolent ulcers, 90% healed completely. In all but one case, the cornea was covered completely by the lid and complete ptosis was produced in 75% of cases in an average of 3.6 days, lasting for 16 days on average before recovery began. Recovery of levator function was complete in 8.5 weeks on average. Superior rectus underaction was seen in 68% of cases but this recovered completely in all cases in an average of 6 weeks. Impression cytology showed a trend toward normal conjunctival morphology as healing progressed.

The superior laryngeal nerve: its projection to the dorsal motor nucleus of the vagus in the guinea pig.

The distribution of neurons in the dorsal motor nucleus of the vagus nerve (DMNV) that innervate the supraglottic and glottic areas of the larynx of the guinea pig have been studied using the horseradish peroxidase (HRP) technique. Following soaking of the superior laryngeal nerve in a solution of HRP, labeled neurons were always located ipsilaterally, at levels between the estria acustica and the caudal end of the inferior olivary nucleus. Characteristically, the neurons were small or medium in size.

Monitoring ocular disease by impression cytology.

The technique for obtaining and processing conjunctival impression cytology is described. The method of grading the stained samples is outlined. Our findings in various ocular conditions with particular emphasis on; alkali burns, uveitis, hypopyon ulcer and Botulinum toxin protective ptosis are shown. Cytology from severely damaged eyes is highly abnormal, improving as the ocular disease settles. In alkali burns a return to normal cytology does not correlate with return of corneal clarity. Loss of goblet cells is a common finding in the ocular diseases demonstrated, and appears to be a non-specific indicator of ocular problems.

The sympathetic innervation of the vocal cord. An experimental study in the guinea pig.

Formaldehyde-induced fluorescence and electron microscopy have been used to show the sympathetic innervation of the vocal cord in the guinea pig. The topographical distribution and morphology of the sympathetic fibres and endings are commented upon.