Biomedical subjects

R W Beuerman

Publications and source records attributed to R W Beuerman.

At least 19 recordsLinked to original sources

PubMed1992

Vancomycin-enriched corneal storage medium.

Antibiotics in a corneal preservation solution probably have little effect during storage at 4 C, but are effective as the tissue is warmed. The tissue acts as a sponge, soaking up the antibiotic from the solution and releasing it into the eye, where the bactericidal effect is achieved. Currently, high concentrations of gentamicin (relative to the minimal inhibitory concentration) are used in the preserving solution for this purpose. Presumably, proportionately high concentrations of any proposed new antibiotic added to supplement the bactericidal effect of gentamicin, such as the vancomycin used in this study, would be required. However, neither the ability of donor tissue to tolerate high concentrations of vancomycin nor the stability of vancomycin at neutral pH in appropriate storage media has been documented. We evaluated the addition of vancomycin (100 micrograms/ml) to two corneal storage media that contained gentamicin in terms of stability of the antibiotic in solution and the effect on the endothelial cells of donor tissue stored for two weeks at 4 C. Vancomycin was stable in solution at neutral pH (7.2) during the five-month period of the study; the concentration exceeded 90 micrograms/ml for the first five weeks. The endothelial cells from donor tissue stored in the vancomycin-enriched media showed no notable differences from those stored in the same media without vancomycin in terms of cell shape, cell borders, cell swelling, and apical holes. The stability of vancomycin in storage and the absence of endothelial toxicity in vitro support the potential use of this antibiotic as a supplement to gentamicin for the prevention of endophthalmitis in patients receiving corneal transplants.

Aged↗

PubMed1992

Molecular and cellular responses of the corneal epithelium to wound healing.

The corneal epithelium responds rapidly to injury, repairing defects with a layer of cells that covers the denuded corneal surface and prevents infection and loss of vision. After a wound, reorganization of the remaining epithelial cells occurs over several hours, resulting in the formation of a migratory leading edge. However, expression of genes such as c-fos occurs within minutes of wounding. This early expression may be important for directing epithelial reorganization and the later mitotic burst. Our results show that receptors for epidermal growth factor are upregulated in the migratory cell population. Proliferation through a mitotic burst was observed in cells surrounding the original wound margin after 36 hours. The interaction between gene expression and cell surface receptors for growth factors and cell proliferation suggests that wound healing occurs in a complex, but tightly controlled process in the corneal epithelium.

Animals↗

PubMed1992

Action potential response of the corneal nerves to irritants.

The cornea, in addition to its refractive function for the eye, and by way of its very dense sensory innervation, serves a very important protective function for the visual organ. The cornea receives mainly sensory innervation from the first division of the trigeminal ganglion and a sparse amount of sympathetic fibers. The sensory nerves carry out their protective function by responding to various types of stimuli in a way so that they are all perceived psychologically as painful. Neurophysiological data indicates that, despite the morphological similarity of free-nerve endings in the cornea, they are differentiated functionally. A concentration series, (0.005 to 10% solution in saline), of various potential irritants (phosphate detergent, baby shampoo, liquid chlorine bleach, herbal shampoo, onion juice, SDS, and sodium chloride) was applied directly to the cornea of the anesthetized rabbit. Neural activity was assessed from extra-cellular records of long ciliary nerve over a ten second application period, and for ten seconds following stimulus removal. Baby shampoo was non-stimulatory over the applied concentration range. Sodium chloride, on the other hand, exhibited linear response dynamics over the range of 0.01 to 5% (p < 0.001). SDS was highly stimulatory, but showed no predictable concentration or response relationship. All of the other irritants tested responded in a logarithmic fashion. This suggests that the application of neurophysiological techniques to assess the pain and potential inflammatory aspects of a substance for human use can be monitored in this fashion. Moreover, response profiles for various classes of compounds and homologous series, as well as pH and osmolality, can be established.

Action Potentials↗

PubMed1992

In vivo assessment of corneal stromal toxicity by tandem scanning confocal microscopy.

Epithelial wound closure following superficial corneal abrasions precipitates a complex series of cellular changes in the stroma. Transmission electron microscopy and light microscopy have demonstrated that keratocytes in the anterior stroma show prominent cytoplasmic process and granularity within an hour after this injury. Cell degeneration follows rapidly, with almost complete depletion of these keratocytes by 6-12 hours. Regeneration then occurs by 24-48 hours. These findings were confirmed in-vivo by tandem-scanning confocal light microscopy in rabbits. The optical sectioning capability of this instrument allowed us to demonstrate the three-dimensional array of fibroblast process, their subsequent condensation, and loss by sequential examinations on the same animals. Cell shrinkage and other fixation artifacts common in corneal histological sections were avoided by this technique. Using this method, we investigated the corneal cytotoxicity of an antimitotic agent, mitomycin-C and a muscarinic antagonist, atropine sulphate. Mitomycin-C, an antimetabolite used in pterygium treatment, led to irreversible keratocyte depletion even after four days of observation. However, cellular reaction to the wound were attenuated by atropine sulphate suggesting that it may be useful as a novel inhibitor of fibroblast proliferation.

Animals↗

PubMed1992

Early diagnosis of infectious keratitis with in vivo real time confocal microscopy.

The tandem scanning confocal microscope (TSM) was adapted for in vivo examination of the cornea in rabbits with experimental bacterial and fungal keratitis. Compared to slit lamp biomicroscopy, the TSM provides superior lateral and axial resolution and serial optical sectioning capability, which may be useful for identification of corneal pathogens in the early stages of infection. We used the TSM to examine normal rabbit eyes infected with bacteria (Bacillus cereus) and a filamentous fungus (Aspergillus). We also examined a human cornea removed by penetrating keratoplasty after a clinical diagnosis of amoebic keratitis. In the early stages of bacterial infection, slit lamp examination revealed a nonspecific minimal stromal haze and limbal injection indistinguishable from sterile ulcers and epithelial defects. With the TSM, bacteria were visible as highly refractile bodies in the epithelium and superficial stroma. Branching fungal hyphae were also easily identified by the TSM, as were Acanthamoeba cysts and parasites in the subepithelial stroma. Our results indicate that this technique may provide a new modality for quickly and accurately identifying the agent of corneal infection, thereby facilitating prompt and appropriate treatment.

Acanthamoeba Keratitis↗

PubMed1992

EGF cell surface receptor quantitation on ocular cells by an immunocytochemical flow cytometry technique.

A method is presented for the rapid flow cytometric determination of epidermal growth factor (EGF) receptor densities on the surface of cultured ocular cells. The technique uses a biotinylated monoclonal antibody directed against the EGF receptor in conjunction with a streptavidin-bound fluorochrome and requires the specific fluorescence per cell to be measured as a function of ligand and receptor concentration. Because the measurement is noninvasive and restricted to cell surface-bound material, the cells can be kept in a physiologic environment, even at the moment of assay. Calculated receptor densities ranged from 5142/cell (infant human corneal endothelium) to 35,678/cell (infant human keratocytes) to greater than 5 x 10(5)/cell for an A431 control cell line. Species and donor age differences were noted, as was transient receptor downregulation after EGF administration. Flow cytometry represents a valuable time saving procedure for large scale applications while providing the same level of sensitivity as standard radioimmunoassays. This technique is applicable to quantitation of other growth factor cell surface receptors and could greatly expand the use of flow cytometry in the research laboratory.

Animals↗

PubMed1991

Optisol corneal storage medium.

Optisol is an investigational, intermediate-term corneal storage medium containing chondroitin sulfate and dextran to enhance corneal dehydration during storage. We used scanning electron microscopy to grade endothelial cell morphologic characteristics in terms of cell shape, cell borders, cell swelling, and apical holes in pairs of corneas stored in Optisol and Dexsol. Optisolstored corneas showed significantly fewer morphologic changes after 14 days at 4 degrees C than did Dexsol-stored corneas. No significant differences were seen after 1 to 4 days at 26 degrees C. Temperature-reversal analysis showed no significant change in corneal thickness with warming after 2-week storage at 4 degrees C in either medium, although Optisol-stored corneas were significantly thinner than those stored in Dexsol at all times. The results of scanning electron microscopy suggest that preservation at refrigerator temperature for 2 weeks in Optisol is superior to preservation in Dexsol. Both media may be useful in preserving endothelial structure for limited periods at room temperature, which could provide a measure of safety in shipping or storage where refrigeration is unreliable.

Adult↗

PubMed1991

Expression of tenascin and cellular fibronectin in the rabbit cornea after anterior keratectomy. Immunohistochemical study of wound healing dynamics.

Anterior keratectomy (AKE) was done on rabbits, and the appearance of immunohistochemically demonstrable tenascin (TN) or cellular fibronectin (cFN) was studied at different times (5 min to 14 months) after the operation. The substance TN was first observed 12 hr after wounding in the posterior stroma; cFN appeared with the same localization 12 hr later. During postoperative week 1, both TN and cFN immunoreactions shifted to more anterior parts of the cornea, and 9 days after wounding, they were localized in the most anterior part of the stroma only. Thereafter the reactions gradually decreased in intensity but still were visible 3 months after AKE. No reaction for TN or cFN was present 14 months postoperatively.

Animals↗

PubMed1991

Human recombinant epidermal growth factor in experimental corneal wound healing.

Human recombinant epidermal growth factor (hEGF) was evaluated in various corneal wound healing models in the rabbit. Human EGF accelerated epithelial wound healing in corneal reepithelialization, anterior-keratectomy, and alkali-burn models at concentrations of 10-500 micrograms/ml given four times daily (qid). In the corneal reepithelialization model, 100 micrograms/ml of hEGF qid produced a 45% increase in the wound-healing rate compared with control (0.13 versus 0.09 mm/hr) with a similar response at 500 micrograms/ml qid. In the anterior-keratectomy model, 500 micrograms/ml of hEGF qid accelerated healing by 40% (0.07 versus 0.05 mm/hr), although the 100 micrograms/ml dose was not active in this model, and 1 microgram/ml of hEGF actually slowed the healing rate. In the alkali-burn model, 10 and 100 micrograms/ml of hEGF qid for 32 days appeared to produce faster initial healing of the wound compared with control, although the wound recurred in both hEGF and control groups. These results suggest that hEGF may be helpful in some epithelial disorders in humans, although considerations of dose response and optimal dosing regimens must be addressed.

Animals↗

PubMed1991

Flow cytometry measurements of the DNA content of corneal epithelial cells during wound healing.

This study presents the first application of flow cytometry (FCM) techniques to the assessment of cell cycle dynamics in the corneal epithelium after experimental wounding. Anterior keratectomies 6 mm in diameter were created in the central corneas of albino rabbits. The authors sampled the epithelial tissue obtained outside the wound at 12-hr intervals until wound closure at 72 hr. Regenerated epithelium from the surface of the wounded area was collected at 78 hr. The percentages of nuclei in the G0/G1 (growth), S (DNA synthesis), and G2/M (tetraploid/mitosis) phases were determined by FCM. An increase in the percentage of nuclei in the G2/M phase at 36 hr was seen, compared with cell populations in samples from unwounded control corneas. The authors found an increase in mitotic activity in the corneal epithelium during the period of cell migration before wound closure.

Animals↗

PubMed1990

One-year refractive results of central photorefractive keratectomy for myopia in the nonhuman primate cornea.

Photorefractive keratectomy for the correction of myopia was performed in 32 eyes of 16 green monkeys. The corneas healed satisfactorily, with normal formation of basal lamina and hemidesmosomal attachments visible in 14-week histologic specimens. No recurrent erosions were observed clinically. After a transient period of faint haze, all corneas were clear at 17 weeks and remained clear through the 1-year follow-up. In terms of accuracy, all corneas demonstrated a significant flattening compared with preoperative values, but no significant difference was seen between the groups with different intended corrections (1.5 and 3 diopters). The changes in corneal shape stabilized by 17 weeks, as measured by keratometry. The clinical results suggest that mechanical removal of the epithelium is preferable to laser ablation of the epithelium. Overall, the results demonstrate that excimer laser ablation of the corneal stroma can produce a stable diptric change in the primate cornea with good healing and long-term corneal clarity.

Animals↗

PubMed1990

Central photorefractive keratectomy for myopia. The blind eye study.

Prior to undertaking a study in sighted human eyes, we performed photorefractive keratectomy with the 193-nm excimer laser for the correction of myopia in nine legally blind eyes to evaluate safety, efficacy, predictability, and stability. In most cases, reepithelialization was complete by 5 days after surgery; no recurrent erosions were seen. By the end of the 6-month study, all of the corneas had a 0 or 1+ clarity score, on a scale of 0 (clear) to 5+ (opaque). Keratometry and pachometry demonstrated stable flattening of the corneas. One month after surgery, changes in refraction evaluated by retinoscopy showed fair predictability, with no significant increase in refractive or keratometric astigmatism, followed by some regression of effect by the end of the study, possibly caused by anatomical remodeling. The amount of regression appeared to be directly related to the amount of correction intended, suggesting that this effect would not be clinically important in the treatment of mild to moderate myopia.

Adult↗

PubMed1990

Initial evaluation of variable graft lengths and lesion lengths in the repair of nerve gaps.

This experiment evaluated the electrical and histologic differences between two groups of rats, one of which underwent same-length bilateral resection of posterior tibial nerves prior to being repaired with grafts of different lengths, while the other group underwent different-length resections with same-length graft repair. In this rat model, 18 animals were used and divided into two groups. The first group of animals underwent bilateral resection of 8-mm segments of posterior tibial nerve. To repair these nerves, one leg received two 8-mm sural nerve grafts (Group A), while the other leg received two 16-mm sural nerve grafts (Group B). The second group of rats underwent posterior tibial nerve resections of 8 mm and 16 mm, respectively. The leg with the 8 mm of posterior tibial nerve resected, received two side-by-side 16-mm sural nerve grafts (Group C); the other leg with 16 mm resected, received two 16-mm sural nerve grafts (Group D). Electrophysiologic comparison of nerve conduction velocity for Groups A and B showed a significant difference (p less than 0.05), as did the same comparison for Groups C and D (p less than 0.05). Histologic studies showed that Groups A and D had marked extrafascicular escape of the regenerating nerve axons, disorganizational growth of minifascicles, and loss of integrity of the donor fascicles, while Groups B and C had very minimal extrafascicular escape of regenerating axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1990

Corneal nerve disruption reactivates virus in rabbits latently infected with HSV-1.

Trauma, inflammation, and neuronal stimulation or damage can reactive latent herpes simplex virus type 1 (HSV-1). The innervation density of the corneal epithelium is 300-600 times that of skin and, therefore, corneal nerve disruption could provide a strong stimulus for HSV-1 reactivation. This study has documented HSV-1 ocular reactivation following three methods of corneal nerve disruption in rabbits. Twenty HSV-1 latently infected rabbits (26 eyes) were divided into three groups: 7 rabbits received uniocular cryogenic injury, 7 rabbits underwent uniocular anterior superficial keratectomy, and 6 rabbits had binocular transection of the corneal nerves at the corneoscleral limbus which, in contrast to the other treatments, produced minimal epithelial change. Opposite eyes in the first two groups of rabbits were left undisturbed to serve as HSV-1 infected controls. Three additional rabbits, not infected with HSV-1, underwent gold chloride impregnation of the corneal nerves for light microscopic documentation of corneal nerve damage induced by each procedure. On all HSV-1 infected eyes, daily HSV-1 ocular cultures were obtained for 7 consecutive days. All three procedures resulted in marked corneal nerve destruction and degeneration. HSV-1 shedding occurred in 5/7 (71%) of the eyes that underwent cryogenic lesioning; in 5/7 (71%) of the eyes that underwent anterior keratectomy; and in 8/12 (67%) of the eyes that had the corneal nerves transected at the corneoscleral limbus. Only 4 (29%) of the 14 control eyes had positive HSV-1 ocular cultures. This investigation provides strong evidence that corneal nerve disruption is correlated with ocular HSV-1 reactivation.

Animals↗

PubMed1990

Ocular leprosy in nine-banded armadillos following intrastromal inoculation.

Leprosy shows a higher percentage of ocular involvement than any other systemic infection. In humans, the cornea is the first ocular tissue affected. Our previous studies in armadillos with naturally acquired and experimental disseminated leprosy showed that 44% had corneal infection. Mycobacterium leprae is found in armadillo burrows in Louisiana, U.S.A., and ocular abrasions may be the portal of entry for these organisms in wild armadillos. To test the cornea as a route of infection, we injected eight armadillos intrastromally with 2 x 10(6) M. leprae in 1 microliters. Two and 4 months later, the armadillos were sacrificed and their eyes processed for light- and electron-microscopy. After 2 months, M. leprae were found in histiocytes mainly in the corneal limbus, sclera and bulbar conjunctiva. At 4 months, however, there was a visible corneal leproma in one animal. Microscopically, it was found to be a histiocytic granuloma with heavy M. leprae invasion. In addition, cells were seen in the anterior chamber. Leprosy is endemic in regions where other corneal infections which compromise the epithelial barrier property are prevalent and where leprosy bacilli are found in the environment. The entry of leprosy bacilli into the cornea may produce lesions which spread posteriorly in the eye.

Animals↗

PubMed1989

The effect of collagen shields on epithelial wound healing in rabbits.

We evaluated the effect of a collagen shield on epithelial healing in keratectomy wounds in rabbit eyes. Superficial keratectomies 6 mm in diameter were created in 12 eyes of six rabbits. Six eyes received collagen shields every eight hours; six eyes received no treatment (control group). Epithelial healing was significantly faster (P less than .01) in corneas treated with collagen shields (47.8 +/- 1.1 microns/hr) compared to untreated control corneas (40.8 +/- 1.6 microns/hr). Regression analysis gave a projected time for closure of treated corneas of 73.96 hours, compared to 83.41 hours for untreated corneas. Scanning electron microscopy of collagen shields after eight hours of wear showed a large number of polymorphonuclear leukocytes entrapped in the collagen matrix. Light microscopy of healed corneas showed that the appearance of the regenerated epithelium in treated and untreated corneas was similar. These results demonstrate that collagen shields speed reepithelialization of keratectomy wounds in the rabbit cornea.

Animals↗

PubMed1989

Endothelial cell damage in human and rabbit corneas stored in K-Sol without antioxidants.

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology.

Animals↗

PubMed1989

Cytomegalovirus replication in cultured human retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) was isolated from the globe of a donor positive for human immunodeficiency virus (HIV) who had cytomegalovirus (CMV) retinitis secondary to acquired immunodeficiency syndrome (AIDS). In culture, the cells exhibited normal epithelioid morphology by phase contrast microscopy. After two weeks the cells developed cytomegaly and dense intranuclear and cytoplasmic inclusions and, eventually, died. Transmission electron microscopy (EM) demonstrated intranuclear nonenveloped virus particles 80-120 nm in diameter consistent with a herpes type infection. Immunofluorescence staining demonstrated the presence of CMV antigens. Conditioned medium from the infected cells caused infection in RPE cells isolated from normal donors. Hybridization assay demonstrated the presence of CMV DNA and indicated that the time course of the infection was similar, but not identical to infection in MRC-5 and HEL cells. We conclude that cultured human RPE is a permissive host for CMV.

Acquired Immunodeficiency Syndrome↗