Unpreserved carboxymethylcellulose artificial tears evaluated in patients with keratoconjunctivitis sicca.

In order to evaluate the therapeutic value of an unpreserved carboxymethylcellulose-based artificial tear in treatment of keratoconjunctivitis sicca (KCS), 56 patients with severe keratoconjunctivitis sicca were enrolled, at a single study center, in a randomized, double-masked, 8-week comparison with a preserved hydroxypropylmethylcellulose (HMC)-based artificial tear. Patients treated with the carboxymethylcellulose (CMC)-based tear showed significant improvement in fluorescein staining, symptoms, and impression cytology grades. Patients treated with HMC-based tears showed minimal improvement in a few variables. Impression cytology specimens were analyzed by a modified technique that maps the distribution of the various grades present on the specimen. With this technique, improvement in the cytology grades was noted in the group of patients using CMC-based tears. The improvement correlated with observed decreases in symptoms of discomfort and with scores for superficial punctate staining. This study supports the observed therapeutic value of unpreserved CMC-based artificial tears and suggests the possible reversal of squamous metaplasia in patients with KCS. Further studies are required to separate the benefit of the CMC formulation from the benefits of preservative elimination.


Deep corneal scarring in pellucid marginal corneal degeneration.

Crescent-shaped, deep corneal scars were observed in seven (39%) of 18 patients with pellucid marginal corneal degeneration. The scars most likely represent a degenerative change related to the precipitous alteration in curvature of the cornea in that area.


Epidermal growth factor and corneal wound healing. A multicenter study.

A randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted in five centers to assess safety, ocular tolerance, and efficacy of an ophthalmic solution of epidermal growth factor (EGF) for the treatment of traumatic corneal epithelial defects. One hundred four patients completed the study: 47 received EGF and 57 placebo (the drug vehicle). Mean epithelial healing time was significantly enhanced in the EGF-treated group (44.17 h) compared with the placebo-treated treated group (61.05 h) (p < 0.01). The number of epithelial defects completely healed at 24, 48, and 72 h after the onset of treatment was significantly greater in the EGF-treated group. Local tolerance was adequate in both groups. These results indicated that topical EGF is well tolerated and may be a significant addition to the ophthalmologist's armamentarium for treating corneal epithelial defects.


Two-dimensional gel electrophoretic comparison of endothelial cell-Descemet's membrane proteins in Fuchs' dystrophy and normal corneas.

Two-dimensional polyacrylamide gel electrophoresis was used to compare the proteins isolated from the combined corneal endothelial cell-Descemet's membrane complex of three pairs of corneas with Fuchs' dystrophy with three pairs of normal corneas. Normal or Fuchs' dystrophy endothelium and Descemet's membrane was documented by pathologic analysis of each cornea. Proteins were separated by isoelectric point in the first dimension and molecular weight in the second dimension. Over 300 proteins were resolved from each sample, and similar patterns were noted in both groups. No consistent differences were detected between the corneas with Fuchs' dystrophy and the normal corneas. Allelic variations of some proteins were detected in both groups.


Reversible endothelial changes in the monkey cornea.

As part of a study examining the effects of contact lens extended wear in monkeys (Macaca fascicularis), reversible endothelial changes were observed in the cornea with slit lamp biomicroscopy and contact specular microscopy. These changes were associated with anterior eye inflammation in four monkeys (as occurs in humans). However, in two monkeys no obvious cause for these changes other than the presence of a contact lens on the cornea was apparent. In these two monkeys the endothelium displayed features similar to the transient endothelial bleb response, which has been observed only in humans. The monkey (M fascicularis) may thus be an appropriate model for further investigation of the endothelial response during anterior eye inflammation and the transient endothelial bleb response.


Current practices in the prevention and treatment of corneal graft rejection.

Three hundred fourteen members of the Castroviejo Society were surveyed to determine current practices in the prevention and treatment of corneal graft rejection. One hundred thirty-seven responses were received and tabulated for routine postoperative prophylactic immunosuppression as well as treatment of epithelial rejection, endothelial rejection (three patterns), subepithelial infiltrates, acutely and progressively opacifying grafts without inflammation, and photophobia. All respondents applied topical steroids in the postoperative period, while about half also administered steroids subconjunctivally. For the treatment of obvious endothelial rejection, 100% of respondents used topical steroids, 57% used subconjunctival steroids, and 37% used systemic steroids. Prednisolone acetate, 1%, was the preferred topical preparation for all conditions (55-68% of respondents). Individual responses to the questionnaire showed a surprisingly wide variation in treatment preferences.


Intraocular penetration of ketoconazole in rabbits.

We studied penetration of the antifungal agent ketoconazole into the cornea, aqueous humor, and vitreous of rabbits after topical, subconjunctival, and oral administration. The effect of debridement of corneal epithelium on penetration was also investigated. Ketoconazole levels in the cornea and aqueous humor were high after topical or subconjunctival administration, and increased markedly (especially in the cornea) if the corneal epithelium had been debrided before administration of the drug. For example, concentration of ketoconazole in the cornea 1 h after topical drug administration with or without complete corneal epithelial debridement was 44.0 +/- 10.1 and 1,391.5 +/- 130.0 micrograms/g, respectively. Drug levels in the vitreous were not detectable after topical or subconjunctival drug administration, but were improved slightly by prior epithelial debridement (8.3 and 0.12 micrograms/mL after 1 h, respectively). Orally administered ketoconazole resulted in high corneal concentrations (45.0 +/- 7.6 micrograms/g after 1 h) that were still substantial 24 h later (55.0 +/- 7.0 micrograms/g); levels in the aqueous were low.


Age-related morphological changes in lid margin and meibomian gland anatomy.

Meibomian gland dysfunction (MGD) is responsible for recurrent irritative symptoms. Attempts to characterize MGD have largely concentrated on microbial and lipid abnormalities in meibomian gland secretions. Few reports describe histological abnormalities in this disease, and fewer still morphological changes. This article follows a previous study that established a classification for MGD. This was based on morphological changes in the meibomian gland and lid margin. Using this classification, we studied the age-related changes in 80 subjects, between 5 and 87 years of age without ocular disease. The lid margin became thicker after childhood. Lid margin vascularity and cutaneous hyperkeratinization increased with age in both lids, whereas, telangiectasia increased with age in the lower lid and squamous blepharitis and posterior lid margin rounding were more common after 50 years of age in the upper lid. Multiple rows of meibomian gland orifices occurred more frequently in the upper than lower lid, and orifice narrowing and pouting increased with age. No age-related changes in the shape or form of the mucocutaneous function, gland ducts, or acini were found. Meibomian gland secretions were less easily expressed in the elderly. We have attempted to define a normal range of lid morphology in healthy children and adults that we believe to be important for the subsequent definition of lid disease, and in particular, posterior blepharitis.


Keratoconus and bilateral lattice-granular corneal dystrophies.

The occurrence of lattice and granular corneal dystrophies in the same eye has been reported in individuals who trace their families to the province of Avellino in Italy. We present the first histopathologic report of keratoconus and bilateral lattice-granular corneal dystrophies occurring in the same patient. This individual's family also originated in Avellino, Italy.


Corneal topography of posterior keratoconus.

Posterior keratoconus is an unusual abnormality of the cornea generally classified as one of the anterior chamber cleavage anomalies. It is characterized clinically by the presence of a circumscribed or generalized corneal thinning with posterior depression of the cornea and is considered distinct from keratoconus. Although patients with posterior keratoconus may have visual complaints clearly related to their abnormal corneas, the surface topography of these corneas has not been studied in detail. Keratometry and photokeratoscopy provide an incomplete picture of the surface geometry of posterior keratoconus. We utilized computer assisted topographic analysis to study the cornea of a patient with posterior keratoconus. The Topographic Modeling System demonstrated that the patient's cornea showed a central steepened "cone" coincident with the area of circumscribed posterior keratoconus as well as paracentral flattening. This report documents the topographic abnormality in this rare disorder.


Bacterial keratitis associated with vernal keratoconjunctivitis.

We cared for two patients with longstanding vernal keratoconjunctivitis who had bacterial corneal ulcers in each eye. Both patients were young, black, and had histories of atopy. The patients came for treatment with acute symptoms of pain, redness, and reduced vision in the affected eye. On examination in each case we found an epithelial defect associated with dense stromal infiltration, a calcific plaque in the bed of the ulcer, and a severe anterior chamber reaction, including a hypopyon in two cases. Cultures of corneal scrapings from all four eyes were positive for Staphylococcus aureus, and three of the four infections were polymicrobial. All four eyes responded rapidly to intensive topical antibiotic therapy, debridement of the calcific plaque, and subsequent treatment with topical corticosteroids and/or cromolyn sodium. Bacterial keratitis can occur in patients with vernal keratoconjunctivitis, especially those with vernal corneal ulcers. The abnormalities of ocular immune mechanisms found in patients with vernal keratoconjunctivitis may predispose them to bacterial keratitis.


Persistent superficial punctate keratitis after resolution of chlamydial follicular conjunctivitis.

Two cases of follicular conjunctivitis due to Chlamydia trachomatis followed by punctate epithelial keratitis are described. Both cases were initially treated with either oral tetracycline or doxycycline with resolution of the follicles. These two patients subsequently had recurrent, bilateral grayish lesions at various levels in the corneal epithelium that stained in a punctate fashion with fluorescein. There was anterior stromal edema associated with some of these lesions in one case. The lesions were confined mostly to the central cornea. These recurrent lesions were unassociated with a conjunctival reaction, were unresponsive to oral tetracycline, but were exquisitely responsive to low doses of topical steroids. Chlamydial conjunctivitis and the associated keratitis typically shows no response or actual exacerbation of symptoms with topical steroids, and the keratitis shows a predilection for the upper half of the cornea. These patients demonstrate that chlamydial keratoconjunctivitis might result in a clinical appearance consistent with Thygeson's superficial punctate keratitis.


Corneal storage medium preservation with defensins.

We used a synthetic defensin (rabbit neutrophil peptide-1; NP-1) as a microbicide in a corneal storage medium, Optisol without antimicrobial compounds. We established growth curves in Optisol for Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa. Each organism was evaluated separately at 4 degrees C, 23 degrees C, and 37 degrees C in Optisol with NP-1 at each of four different concentrations including 1, 10, 100, and 200 micrograms/ml. When the three organisms were evaluated in Optisol containing NP-1, we found that a concentration of 200 micrograms/ml of NP-1 successfully killed 99.9% (the limits of our assay) of all three organisms at all temperatures tested. We conclude that NP-1 exhibits promise as a nonantibiotic preservative agent in corneal storage media, since it was effective in killing organisms at all temperatures, including 4 degrees C.


Corneal toxicity study of two perfluorocarbon liquids in rabbit eyes.

Perfluorocarbon liquids are fully fluorinated, synthetic, transparent compounds with a high specific gravity. These compounds are being increasingly used as an intraoperative tool for repair of complicated retinal detachments. A potential complication of their use, however, is liquid entering the anterior chamber in aphakic patients. In the study described herein we evaluated the effects of two of these liquids when placed in the anterior chamber of the rabbit eye. Sixteen rabbit eyes underwent anterior chamber injection of 0.05 ml of either perfluoroctane, perfluoropolyether, or balanced salt solution (control eyes). Animals were monitored clinically by biomicroscopy and external photography for up to 14 days, after which they were sacrificed and the corneas processed for light and for scanning electron microscopy. The animals injected with perfluoropolyether showed more intense stromal inflammation and corneal vascularization (p less than 0.02) than did those that received perfluoroctane. However, the perfluoroctane group showed more of the "fish-egging" phenomenon (p less than 0.03). Loss of endothelial cells was similar in the two groups, as determined by light and scanning electron microscopy. These results suggest that the corneal toxicity of these two perfluorocarbon liquids is such that their use as vitreous substitutes should be limited to short-term replacement.


Effect of Harderian gland-derived growth factor on the growth of cornea stromal cells.

To investigate the physiological role of Harderian gland-derived growth factor (HGDGF), the effects of HGDGF and various other growth factors on the growth of cultured guinea pig cornea stromal cells were examined. HGDGF increased the incorporation of [3H]thymidine to 150% of the control (5% fetal calf serum). The combination of HGDGF with fibroblast growth factors (FGFs) or platelet-derived growth factor enhanced the cell growth over that of either growth factor alone, increasing the incorporation of [3H]thymidine to 180 and 190% of the control, respectively. The combination of HGDGF with transforming growth factor (TGF-beta) decreased the growth to 60% of the control, and epidermal growth factor had no effect on the activity of HGDGF. The growth-stimulating activity of HGDGF was inhibited by suramin in a different manner from that of FGFs. These findings suggest that HGDGF binds a specific cell-surface receptor and plays a role in the repair of injured parts of the cornea and in the maintenance of the cornea stromal cells.


Isoelectric focusing pattern of human corneal aldehyde dehydrogenase.

The major soluble protein in the bovine cornea (BCP 54) has recently been identified as a class 3 aldehyde dehydrogenase. An enzymatic and even a possible structural role of this protein in the mammalian cornea has been proposed. Earlier we showed that the human cornea contains the same enzyme but with a different substrate specificity, compared to the bovine. Moreover, the enzymatic activity was harbored in the dimeric 88-kD species. Genetic variants have been found for several mammalian ocular aldehyde dehydrogenases. In this study we investigated whether such variants were also present in the human cornea by using a zymography technique for aldehyde dehydrogenase activity and immunoblotting with rabbit anti-BCP 54 and lectin staining after isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We investigated 50 individual human corneal epithelial extracts and found one common IEF variant (n = 47) and two "rare" IEF variants in three individuals. Analysis of these patterns indicates that the observed IEF profiles may be caused by posttranslational events.


Fibrin-enmeshed tobramycin liposomes: single application topical therapy of Pseudomonas keratitis.

Treatment of bacterial keratitis requires frequent application of topical antibiotics. We studied the efficacy of a single topical administration of tobramycin incorporated in large multivesicular liposomes and enmeshed in a fibrin sealant on rabbit corneas infected with Pseudomonas aeruginosa. One cornea each of 25 New Zealand albino rabbits was infected with P. aeruginosa. Twenty-four hours later, the animals were randomly divided into five groups of five. Group A received single hourly drops (50 microliters) of fortified tobramycin (14.5 mg/ml, total of 17.4 mg). Group B received a single topical application of 3.5 mg tobramycin, in 0.1 ml multivesicular liposomes, enmeshed in a fibrin sealant with an overlaying bandage contact lens. Group C was treated in the same manner as group B without the addition of fibrin sealant. Groups D and E served as nondrug-treated controls, with group D receiving topical fibrin-enmeshed liposomes devoid of tobramycin and group E receiving hourly topical balanced salt solution (BSS) drops. All animals were killed 24 h after initiation of therapy. Significantly fewer colonies of Pseudomonas were present in corneas of all three treated groups, as compared with the two nondrug-treated control groups (p less than 0.02). There were significantly fewer colonies of Pseudomonas in groups A and B as compared with group C (p less than 0.02). No significant difference was noted between a single administration of topical fibrinen-meshed tobramycin-encapsulated liposomes (group B) and 24 doses of hourly fortified topical tobramycin (group A, p greater than 0.05). Tobramycin-encapsulated megaliposomes may serve as a useful adjunct in treatment of Pseudomonas keratitis.


Anti-inflammatory therapy and outcome in a guinea pig model of Pseudomonas keratitis.

Corneal scarring as a consequence of bacterial keratitis is an important cause of visual loss and a major indication for penetrating keratoplasty. Anti-inflammatory agents might be useful in this condition for limiting corneal damage, but benefit from adjunctive anti-inflammatory therapy has never been demonstrated. In this limited pilot study, we compared the effect on clinical outcome of treating Pseudomonas keratitis in guinea pigs with prednisolone (a corticosteroid), flurbiprofen (a cyclo-oxygenase inhibitor), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and a leukotriene antagonist, SKF104353 [R-(R*, S*)]-beta-[(2-carboxyethyl) thio-alpha-hydroxy-2-(8-phenyloctyl) benzenepropanoic acid, zinc salt]. None of the anti-inflammatory agents prevented sterilization of ulcers with antibiotic (ofloxacin) therapy. Therapy with the leukotriene antagonist appeared to reduce infiltrate size more quickly and produce a more rapid reduction in lesion size, but the differences were not statistically significant. Sample size calculations suggest that very large numbers of animals would be required to prove efficacy. The role of anti-inflammatory agents in reducing the stromal destruction caused by bacterial keratitis remains controversial.


Recurrence of microbial keratitis concomitant with antiinflammatory treatment in an animal model.

Recurrence of Pseudomonas keratitis during treatment with corticosteroids has been reported previously in humans. Rabbits with keratitis due to Pseudomonas aeruginosa or Streptococcus pneumoniae were treated with antibiotics and either vehicle, methylprednisolone acetate, flurbiprofen, or nordihydroguaiaretic acid (NDGA). Cultures performed after 7 days were negative, and antibiotics were discontinued. Two weeks later, Pseudomonas keratitis recurred in 6 of 7 (85.7%) steroid-treated rabbits, 1 of 8 (12.5%) flurbiprofen-treated rabbits, 1 of 8 (12.5%) NDGA-treated rabbits, and none of 8 vehicle-treated rabbits. None of the 31 rabbits infected with Streptococcus pneumoniae experienced recurrence. These data confirm the clinical observation that Pseudomonas keratitis may recur if antibiotic therapy is discontinued and corticosteroids are administered; the risk of recurrence appears to be much less with nonsteroidal antiinflammatory agents.


Subconjunctival injection of purified blood mononuclear cells into alkali-injured rabbit eyes.

Monocytes transform into tissue macrophages that scavenge necrotic debris from wounds but also release substances stimulatory to fibroblasts. In an effort to encourage the healing process, monocytes, purified from rabbit blood, were injected subconjunctivally 1 or 2 days and 6 or 7 days after alkali injury to the rabbit eye. The injected monocytes failed to alter the course of the alkali-injured eyes. Specifically, the incidence of corneal ulceration in the experimental group was not statistically different from that of the control group. A byproduct of this study was a presentation of the technique to purify platelet-free mononuclear leukocytes from rabbit peripheral blood.


Inhibition of human corneal epithelium with immunotoxin 454A12-rRA.

We examined the effects of the immunotoxin 454A12-rRa on proliferating and confluent human corneal epithelium (HCE) in vitro. Proliferating HCE was sensitive to 454A12-rRA in a dose-dependent fashion. At immunotoxin concentrations of 1,000 ng/ml for 7 days we observed an 86% reduction in cell counts. Confluent HCE was not sensitive to 454A12-rRA at equivalent concentrations of immunotoxin. These data confirm previous observations regarding selective sensitivity of proliferating ocular tissue to immunotoxin, but suggest that HCE is less sensitive to 454A12-rRA than other ocular cell types.


Cytologic evaluation of conjunctival epithelium after cataract surgery.

Although numerous studies have examined corneal epithelial wound healing, few reports describe conjunctival wound healing. Thus, we evaluated the conjunctival epithelium by brush cytology, in which a miniature brush was used to collect many cells relatively noninvasively. The temporal and superior conjunctivae were examined before and after cataract extraction on the first, third, seventh, and fourteenth days and then the first, second, and third months postoperatively, in a group of 20 patients (9 men and 11 women with a mean age of 69 years 3 months +/- 10 years). The cells collected were stained by the Papanicolaou method. Each postoperative specimen contained keratinized, polygonal, round, columnar, elongated, and inflammatory cells, as well as cells with nuclear alterations. No keratinized, elongated, or inflammatory cells were observed preoperatively. In contrast, columnar cells, which were present preoperatively, decreased during the wound healing process. Such nuclear changes as chromatin formation or the appearance of snake-like figures were observed in several patients postoperatively. These cellular changes may represent altered differentiation during the healing process. However, the conjunctival epithelium returned to normal within 2 months postoperatively in all patients.


Risk factors for the development of postkeratoplasty glaucoma.

From a database of 1,122 penetrating keratoplasties performed under the care of the surgeons of the Corneal Clinic, Moorfields Eye Hospital (London, U.K.), 153 (14%) were identified as being complicated by postkeratoplasty glaucoma. The relative risk for its development varied with the indication for keratoplasty. Keratoconus had the lowest incidence along with some dystrophies, such as macular or granular dystrophy, and these were taken as the baseline for comparison. Anterior chamber dysgenesis syndromes had the highest risk among the indications for keratoplasty. Combined cataract or lens implant surgery was also found to be a risk factor, with anterior vitrectomy, anterior segment revision, and anterior chamber lens implant removal representing a greater risk than extracapsular extraction and posterior chamber lens implantation. Postkeratoplasty glaucoma was also strongly associated with peripheral anterior synechiae formation seen after keratoplasty.


Ultrastructural effect of L-ascorbic acid 2-phosphate on cultured keratocytes.

I studied the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting derivative of L-ascorbic acid, on the fine structures of cultured rabbit keratocytes. The results showed that cells cultured with 0.1 mM P-Asc for 30 days were more markedly multilayered than those grown without P-Asc. Dilation of the cisternae of the endoplasmic reticulum in the control cells indicated an accumulation of protein (probably procollagen). In addition, many lysosome-like structures, which may degrade underhydroxylated procollagen, were observed in the cytoplasm of the control cells. A slight increment of free ribosomes was also found within the control cells. P-Asc enhanced the multilayerization of cultured keratocytes. We conclude that the changes seen in the cytoplasm are due to the effect of P-Asc acting as vitamin C.


Improved detection of disease progression in ocular cicatricial pemphigoid.

Cicatricial pemphigoid is an autoimmune systemic disease characterized by chronic conjunctival cicatrization. The natural history is one of variable degrees of progressive scarring of the ocular surface and visual loss. Previous reports have shown systemic chemotherapy to be efficacious in halting cicatrization in progressive cases. This observation makes it of critical importance to be able to detect progression of the disease as early as possible so as to allow for earlier institution of treatment. Our experience in previous studies has led us to believe that disease staging systems currently in use are relatively insensitive in detecting disease progression. We present a revised staging system that combines the strengths of the staging systems in use today and extends their sensitivity.


A fish-eye disease-like familial condition with massive corneal clouding and dyslipoproteinemia. Report of clinical, histologic, electron microscopic, and biochemical features.

Fish-eye disease (FED) is a rare familial condition characterized by progressive bilateral corneal clouding and dyslipoproteinemia previously described in one family and an unrelated woman of Swedish descent. Biochemical studies have clearly demonstrated the existence of this entity as a unique dyslipoproteinemia. We present a non-Swedish family of Mediterranean ancestry afflicted with bilateral corneal clouding and lipoprotein analysis consistent with FED-like state. This family's biochemical profile, corneal button histology, and electron microscopy of one member are reviewed. Other dyslipoproteinemias causing corneal changes are considered. Corneal tissue and familial biochemical analyses differed significantly from previous descriptions. On the basis of these findings, explanation of pathologic deposition and disease mechanism is proposed.


Corneal topographic changes over time after radial keratotomy.

Radial keratotomy flattens the cornea, but in a nonuniform fashion. We used computer-assisted topographic analysis to examine curvature changes in different regions of the cornea. Within 45 days after surgery in human eyes, the central and midperipheral cornea flattens and the periphery steepens. Shortly after surgery, the central cornea (within 1-1.5 mm of the corneal light reflex) is steeper than the adjacent midperipheral cornea (1.5-3 mm peripheral to the light reflex). With time, however, the central cornea flattens more than the midperiphery, such that it is no longer relatively steeper. This regional variability in corneal curvature after radial keratotomy helps explain phenomena such as multifocal lens effect after radial keratotomy, and the changes with time account for the conflicting results observed clinically in human corneas and experimentally in human eye bank eyes.


Endothelial decompensation in a schizophrenic patient receiving long-term treatment with tranquilizers.

Bilateral corneal edema occurred in a schizophrenic patient receiving tranquilizers. This particular corneal edema was rapidly resolved after tranquilizer discontinuation. Specular microscopic examination disclosed marked enlargement of the corneal endothelium. This endothelial impairment may have been due to long-term administration of tranquilizers.


Infectious crystalline keratopathy with ring opacity.

A 41-year-old physician was treated for 3 months with antiviral medications, antibiotics, and steroids for presumed herpetic keratitis. When seen by us, an annular infiltrate was observed, along with crystalline-like opacities in the superficial one third of the stroma. Cultures of scrapings and of subsequent biopsies were positive for Streptococcus mitis of the viridans group; histopathology demonstrated large aggregates of cocci between the stroma lamellae. Tapering of topical corticosteroids and treatment with topical penicillin resulted in resolution of the infiltrates. The clinical appearance and findings in this patient suggest that infectious crystalline keratitis can produce an annular infiltrate. Injection of the organism into rabbit corneas produced a crystalline infiltrate, but no annular opacity was observed. Corticosteroids altered the clinical and histopathologic appearance of the lesions in rabbits.


Grand challenges in biomedical computing.

Advances in computing technology (both algorithms and hardware) over the next several years promise to make increasingly sophisticated computer modeling of biomedical phenomena a routine part of biomedical research. Improvements in both the absolute speed of processors and in their programming and graphics interfaces will allow nonexpert users to bring computing power equivalent to the supercomputers of a few years ago to bear on routine research problems and to display complex data in understandable ways (visualization). Although biomedical applications have traditionally not driven the leading edge of computing and supercomputing, such applications are increasingly being ported to advanced parallel and vector processors. This paper summarizes the current state of biomedical computing, citing examples of the best practice in research today. A number of projects enabled by advanced computing from various subdisciplines are described. Trends in technology for both inexpensive (workstation) and high-end computing (vector supercomputers and parallel processors) are cited; the implications of these for biomedical computing are discussed. "Grand challenges" in biomedical computing, i.e., computational problems of major scientific importance that are beyond our current capabilities but that might be achieved in a 5-year time frame, are outlined.


Skeletal muscle grids for assessing current distributions from defibrillation shocks.

This paper utilizes a structured and an unstructured grid representation of a torso with an anisotropic skeletal muscle to assess current distributions from defibrillation shocks. The results show that a finite-element solution on an unstructured grid of 400,000 elements (60,000 nodes) achieves comparable current distributions with a finite-difference solution on a structured grid that uses approximately the same number of nodes. Moreover, a finite-element solution on a 65,000-element (10,500 nodes) unstructured grid yielded fractional percent current results within 5% of the finer grids. The structured and unstructured grid models are used to investigate recent interpretations of experimental data that concluded that more than 80% of the total defibrillation current is shunted by the anisotropic skeletal muscle thoracic cage. It is concluded that these interpretations, which were based on a one-dimensional resistive network representation of the three-dimensional defibrillation situation, overestimate by 25% the current shunted by the anisotropic thoracic cage.


The electrocardiographic inverse problem.

Using the boundary element method in conjunction with Tikhonov zero-order regularization, we have computed epicardial potentials from body surface potential data in a realistic geometry heart-torso system. The inverse-reconstructed epicardial potentials were compared to the actual measured potentials throughout a normal cardiac cycle. Potential features (maxima, minima) were recovered with an accuracy better than 1 cm in their location. In this chapter, we use these data to illustrate and discuss computational issues related to the inverse-reconstruction procedure. These include the boundary element method, the choice of a regularization scheme to stabilize the inversion, and the effects of incorporating a priori information on the accuracy of the solution. In particular, emphasis is on the use of temporal information in the regularization procedure. The sensitivity of the solution to geometrical errors and to the spatial and temporal resolution of the data is discussed.


Generalized approach to inverse problems in tomography: image reconstruction for spatially variant systems using natural pixels.

A major limitation in tomographic inverse problems is inadequate computation speed, which frequently impedes the application of engineering ideas and principles in medical science more than in the physical and engineering sciences. Medical problems are computationally taxing because a minimum description of the system often involves 5 dimensions (3 space, 1 energy, 1 time), with the range of each space coordinate requiring up to 512 samples. The computational tasks for this problem can be simply expressed by posing the problem as one in which the tomograph system response function is spatially invariant, and the noise is additive and Gaussian. Under these assumptions, a number of reconstruction methods have been implemented with generally satisfactory results for general medical imaging purposes. However, if the system response function of the tomograph is assumed more realistically to be spatially variant and the noise to be Poisson, the computational problem becomes much more difficult. Some of the algorithms being studied to compensate for position-dependent resolution and statistical fluctuations in the data acquisition process, when expressed in canonical form, are not practical for clinical applications because the number of computations necessary exceeds the capabilities of high-performance computer systems currently available. Reconstruction methods based on natural pixels, specifically orthonormal natural pixels, preserve symmetries in the data acquisition process. Fast implementations of orthonormal natural pixel algorithms can achieve orders of magnitude speedup relative to general implementations. Thus, specialized thought in algorithm development can lead to more significant increases in performance than can be achieved through hardware improvements alone.


Structured and unstructured grid generation.

Current techniques in composite-block-structured grid generation and unstructured grid generation for general 3D geometries are summarized, including both algebraic and elliptic generation procedures for the former and Delaunay tessellations for the latter. Citations of relevant theory are given. Examples of applications for several geometries are included.


Increased gut permeability early after burns correlates with the extent of burn injury.

To determine if increased gut permeability within 48 hrs after burn injury correlates with the extent of injury, before sepsis and pulmonary disorders have complicated the clinical course. Nonrandomized, controlled study. Consecutive patients admitted with burn injuries on > 20% of body surface area. Intestinal absorption and renal excretion of polyethylene glycol 3350 was used as the macromolecule to determine gut permeability; polyethylene glycol 400 intestinal absorption was used as an internal control for abnormal motility and malabsorption. Polyethylene glycol 3350 (40 g) and polyethylene glycol 400 (5 g) were administered enterally. Gut permeability was significantly increased early after the injury. The patients excreted 0.56 +/- 0.34% (n = 11) of polyethylene glycol 3350, compared with the amount (0.12 +/- 0.04%) (p < .05) previously reported in normal volunteers. There was no significant difference in the excretion of polyethylene glycol 400 in the patients (27.0 +/- 4.6%, n = 11) vs. the normal volunteers previously reported (26.3 +/- 5.1%, n = 12), suggesting normal intestinal motility and absorption. The percentage of excretion of polyethylene glycol 3350 correlated with the percentage body surface burned; patients with smaller injuries excreted 0.32 +/- 0.17% (n = 6), which was greater than normal and less than those values from patients with larger injuries, 0.84 +/- 0.25% (n = 5) (p < .001 by Turkey test). Using our newly developed method to separate and quantify polyethylene glycols in urine, gut permeability was found to be increased early after burn injury, which confirms a previous study using lactulose as the permeability probe. Furthermore, this increased gut permeability to polyethylene glycol 3350 correlated with the extent of the burn injury.


Value of postprocedural chest radiographs in the adult intensive care unit.

To evaluate the necessity for postprocedural chest radiographs after catheterization of central veins, insertion of pulmonary artery catheters, and placement of endotracheal tubes. Prospective, controlled study. Two academic tertiary adult ICUs. Consecutive patients (n = 316) requiring central vein cannulation or endotracheal intubation in the ICUs. After each invasive procedure, the physician was instructed to complete a detailed evaluation sheet. Criteria based on the details of the procedure and immediate postprocedural clinical evaluation of the patient were used to determine the likelihood of a radiologically detectable complication. Actual radiologic findings were subsequently compared against clinical predictions. Ability of housestaff to correctly predict the absence of radiologically detectable postprocedural complications (predictive negatives). Ability to predict the absence of complications after cordis catheter insertions via the subclavian vein or internal jugular vein was very high (151/152; p < .001). Unsuspected complications were more frequent with central vein multilumen catheter insertions (3/24; p < .001). Ability to predict uncomplicated pulmonary artery catheterization was also high (110/111; p < .001). Physicians were unable to predict the majority of complications associated with endotracheal intubations (28/32; p > .50). The use of a protocol that includes an evaluation of the characteristics of the procedure and postprocedural physical examination can greatly reduce the need for routine chest radiographs after subclavian and internal jugular vein cordis catheterizations and pulmonary artery catheter placement. Chest radiographs should be performed after endotracheal intubation and multilumen catheter insertion.


Prevalence of, and risk factors for, upper gastrointestinal tract bleeding in critically ill pediatric patients.

To determine the occurrence of, and risk factors for, the development of upper gastrointestinal (GI) tract bleeding in critically ill pediatric patients. Prospective, descriptive, comparative study. ICU in a tertiary care pediatric hospital affiliated with a university. All patients < 19 yrs of age who were admitted to the ICU for a 4-month period (n = 429) were eligible for inclusion. A total of 221 patients were excluded for reasons listed below. Thus, 208 patients were studied. None. Patients were evaluated for overt upper GI bleeding as indicated by coffee ground material or bright red blood in gastric aspirates or black, tarry stools. Excluded were patients who were transferred out of the ICU within 24 hrs of admission, were receiving medications that would alter their risk for upper GI bleeding, or had a GI tract surgical procedure. Patients were categorized by diagnoses and analyzed for relative risk for upper GI bleeding. Of the 208 patients included, 25% had evidence of upper GI bleeding. There was no association between upper GI bleeding and age, weight, race, or sex. Diagnoses independently associated with an increased risk for upper GI bleeding were: circulatory shock, an operative procedure > or = 3 hrs in duration, and trauma. No clinically important sequelae were directly attributable to upper GI bleeding in this group of patients; however, intervention with antacids and histamine-2 receptor (H-2) antagonists likely decreased the progression of GI bleeding. Overt evidence of upper GI bleeding is not uncommon in critically ill pediatric patients. Certain diagnoses or risk factors may predispose these patients to develop upper GI bleeding.


Thermodilution right ventricular ejection fraction measurement reproducibility--a study in patients undergoing coronary artery bypass graft surgery.

To assess the effects of heart rate, right ventricular systolic performance (ejection fraction), chamber dimensions, and flow rate (cardiac index) on the reproducibility of algorithm-derived triplicate thermodilution right ventricular ejection fraction measurements. Prospective study; combined hemodynamic and echocardiographic clinical evaluation. Operating room in a university hospital. Twenty-one coronary artery bypass graft patients. The right atrial delivery site was positioned by analysis of transduced pressure waveform and echocardiographic imaging of tracer agitated saline cavitations. Measurement reproducibility was quantified by determining the variation (standard deviation) within 101 triplicate thermodilution measurement sets. There was no significant relationship between measurement reproducibility and estimates of right atrial area (21.6 +/- 6.9 cm2), diameter (5.1 +/- 0.8 cm) and supero-inferior length (5.1 +/- 0.9 cm) and right ventricular maximal minor axis diastolic diameter (4.21 +/- 1.05 cm). Reproducibility was also unrelated to right ventricular end-diastolic volume index (97.9 +/- 32.7 mL/m2) and cardiac index (2.9 +/- 0.9 L/min/m2). Measurement reproducibility was directly related to mean right ventricular ejection fraction (0.39 +/- 0.14) and inversely related to heart rate (80.8 +/- 18.6 beats/min) (p < .01 and < .001, respectively). Thermodilution-derived right ventricular ejection fraction measurement reproducibility was unrelated to estimates of right atrial and ventricular dimensions and cardiac index. Measurement reproducibility was a direct function of right ventricular systolic performance and an indirect function of heart rate. Measurement should be interpreted with these constraints in mind.


Right ventricular systolic time intervals determined by means of a pulmonary artery catheter.

To evaluate the right ventricular systolic time interval as an index of right ventricular function and also to ascertain whether the right ventricular ejection fraction may be determined by means of a conventional pulmonary artery catheter. Prospective study. Intensive care unit. Eight, consecutive critically ill adult patients. Simultaneous blind measurements, performed by two investigators, of the right ventricular systolic time interval and right ventricular ejection fraction, determined by means of a pulmonary artery catheter. Two studies, separated by an interval of 24 hrs, per patient. Linear regression analysis. Multiple regression test. Of the 16 studies performed, two determinations of right ventricular systolic time intervals were technically inadequate. In the remaining 14 valid studies, we found one close linear correlation between the right ventricular ejection fraction and the preejection period/ejection time quotient measured using the simultaneous display of the electrocardiogram (EKG) and pulmonary arterial pressure curve (r2 = .90, p < .001, right ventricular ejection fraction = 68.96-60.59 x [right ventricular preejection period/right ventricular ejection time]). The method proved to be simple, very accurate, with little interobserver variation (8.09 +/- 10.6% interobserver variation for right ventricular preejection period/right ventricular ejection time) and provided adequate information regarding situations in which the performance of the right ventricle is modified in a given patient. The right ventricular preejection period/right ventricular ejection time quotient was the only variable that displayed a significant relationship with the right ventricular ejection fraction in the multivariate analysis (p < .001). Right ventricular systolic time intervals, measured using the simultaneous display of the pulmonary artery catheter curve and EKG, provide adequate information regarding right ventricle performance in critically ill patients. The close linear correlation between the right ventricular preejection period/right ventricular ejection time quotient and the right ventricular ejection fraction enables the investigator to estimate, with a high degree of accuracy, the right ventricular ejection fraction and the values derived from the preload of the right ventricle, without the need for a modified pulmonary artery catheter.


Experience with hemoperfusion for organophosphate poisoning.

To evaluate the usefulness of extracorporeal clearance techniques in the treatment of organophosphate poisoning, particularly hemoperfusion. Retrospective study. An ICU of a general hospital. Ten patients with organophosphate poisoning initially received classic treatment with gastric lavage and washing of the whole skin surface, as well as the administration of cathartics, activated charcoal, atropine, and obidoxime or pralidoxime. All patients underwent one to three hemoperfusions. Plasma insecticide concentrations and cholinesterase activity were determined daily. Two to three biopsies of fat tissue were carried out at 1- to 2-wk intervals. The amount of the insecticide removed during hemoperfusions was also determined. Five patients presented with a prolonged nicotinic syndrome. Two of these patients showed sequelae of delayed neurotoxicity. Another two of the five patients died and the remaining patient recovered without sequelae. In none of the patients could > 0.1% of the total absorbed poison be removed by hemoperfusion. No changes in symptoms were observed after these procedures. Fat tissue concentrations of the insecticide were 20 to 50 times higher than the concentrations in plasma. Atropine decreased the intestinal transit time, and 10 days after poisoning, a powerful cathartic treatment indicated the persistence of gut content, which probably caused prolonged absorption of the toxin. Extracorporeal cleansing mechanisms did not remove any clinically important amount of insecticide from our patients due to the high lipid solubility of these agents and no changes in symptoms were observed after these methods. The use of atropine decreases bowel peristalsis. Early use of powerful cathartics could avoid protracted absorption of the poison stored in the gut, although the potential benefits of this therapy require confirmation by the performance of prospective, controlled investigations.


Bacterial and fungal colonization of endotracheal tubes in children: a prospective study.

To evaluate both the frequency and route of endotracheal colonization of intubated children by pathogens and to assess the usefulness of Pediatric Risk of Mortality scoring and measurement of gastric pH in predicting this colonization. Prospective data collection. A multidisciplinary pediatric ICU. Nineteen children with medical/surgical problems who were intubated for at least 4 days. Buccal mucosa, and endotracheal and gastric aspirates were cultured for pathogens immediately after endotracheal intubation and daily for 4 to 5 days. Pediatric Risk of Mortality scores were calculated at the time of endotracheal intubation; gastric pH was measured each time that the cultures were obtained. The buccal mucosae of ten (53%) of 19 children were colonized with Candida species at the time of endotracheal intubation. This colonization correlated with the degree of physiologic instability as measured by Pediatric Risk of Mortality scores (12.9 +/- 2.8 SEM for those patients colonized vs. 4.9 +/- 1.0 for those patients not colonized; p = .01), but not with preintubation antibiotic therapy. Eleven (58%) children acquired 27 endotracheal pathogens after the day of intubation. The buccal mucosa was the initial site of colonization of 19 (70%) of 27 of the acquired pathogens. A gastric pH of > 3.0 at the time of intubation correctly predicted colonization in nine of 11 children (p < .025, Fisher's exact test); a Pediatric Risk of Mortality score of > 8 at the time of endotracheal intubation correctly predicted colonization in eight of 11 patients (p < .05, Fisher's exact test). Colonization of the buccal mucosa appears to be the crucial antecedent to endotracheal colonization in children. In children, gastric pH and Pediatric Risk of Mortality scores at the time of endotracheal intubation can predict pathogenic endotracheal colonization within 4 days of intubation. Selective decontamination regimes may be appropriate for these patients, especially those regimes that are directed to the buccal mucosa.


Oxygen consumption, energy expenditure, and substrate utilization after cardiac surgery in children.

To determine the oxygen consumption (VO2), resting energy expenditure, and substrate utilization after cardiac surgery in children. Prospective, observational, cross-sectional study with factorial design. Pediatric ICU at a university hospital. Twenty-six consecutive children during the first 3 days after open-heart surgery. Patients were mechanically ventilated and received routine therapeutic interventions. VO2, resting energy expenditure, and substrate utilization were determined by indirect calorimetry. Cardiac index was calculated using the Fick equation from the measured VO2 and the arterial-mixed venous oxygen content difference, and this cardiac index value was compared with a simultaneous cardiac index value that was measured by thermodilution whenever possible. There were excellent correlation and agreement between cardiac index measurements by Fick equation and thermodilution, indicating accurate VO2 measurements. VO2 was consistent with predicted values in healthy resting children. Resting energy expenditure was consistent with the predicted basal metabolic rate. The mean caloric intake was 19% of the mean energy expenditure. The respiratory quotient was 0.74 +/- 0.05. The substrate utilization showed a shift toward fat oxidation and either gluconeogenesis or impaired carbohydrate utilization. Cardiovascular surgery in children does not significantly alter resting energy expenditure but influences the substrate utilization. Perioperative hormonal stress responses and therapeutically administered catecholamines may explain the shift toward fat oxidation.


Intensive care treatment of patients with bleeding esophageal varices: results, predictors of mortality, and predictors of the adult respiratory distress syndrome.

To determine the factors predicting mortality from bleeding esophageal varices and to examine the possibility of an association between the development of adult respiratory distress syndrome (ARDS) and the use of ethanolamine oleate as an esophageal variceal sclerosant. Retrospective review. ICU in a teaching hospital. A total of 101 patients with endoscopically confirmed bleeding esophageal varices were admitted on 124 occasions from 1985 to 1990. Mean age was 50 +/- 13.5 (SD) yrs. There were 62 males and 39 females. Using the Child-Pugh classification, 21.8% patients were class A, 38.6% class B, and 39.6% class C. Mean ICU and hospital lengths of stay were 5.4 +/- 5.1 and 19.6 +/- 16.1 days, respectively. Mean Acute Physiology and Chronic Health Evaluation (APACHE II) score on admission was 16.5 +/- 7.6. Endoscopic variceal sclerotherapy was performed in 99 (79.8%) of 124 ICU admissions in the 101 patients. Esophageal balloon tamponade was performed in 64 (51.6%) and a vasopressin infusion was administered in 47 (37.9%) of the 124 ICU admissions. A variety of factors was studied to find predictors of mortality and the development of ARDS. Forty-eight (48.5%) of the 101 patients died during the hospital stay. Independent predictors of mortality (by stepdown logistic regression) were total volume of ethanolamine oleate injected during sclerotherapy, multiple blood transfusions, Glasgow Coma Scale score, International normalized ratio for prothrombin test, and the presence of circulatory shock on ICU admission. Age, sex, Child-Pugh score, APACHE II score, serum bilirubin, albumin, and creatinine concentrations, use of esophageal balloon tamponade or vasopressin infusion, sepsis, pneumonia, congestive cardiac failure, aspiration, and ARDS were not statistically independent predictors of outcome. There was no difference in the mortality rates for the various causes of liver disease. Pulmonary complications occurred in 44 (43.6%) patients; sepsis occurred in 31 (25%) patients. ARDS developed in 14 patients (11.3% admissions, 13.9% patients). Statistically independent predictors of ARDS were sepsis, low plasma albumin concentration, use of esophageal balloon tamponade, and more than one sclerotherapy session. The volume and type of sclerosant used were not statistically independent predictors. Outcome is poor for patients with bleeding esophageal varices requiring ICU admission and is related to the severity of liver failure, the degree of blood loss, and failure of therapy to stop the bleeding. The findings do not support an association between the use of the sclerosant ethanolamine and the development of ARDS.


Bulk diffusion apnea test in the diagnosis of brain death.

To assess the efficacy of bulk diffusion in maintaining oxygenation during apnea testing for brain death. Case series. ICU in a primary care hospital. Twenty-four consecutive patients with suspected brain death. Most patients suffered cerebral trauma from vehicular accidents. Patients were preoxygenated with an FIO2 of 1.0 and were maintained during apnea testing with bulk flow of an FIO2 of 1.0 at 40 to 60 L/min in adults and 15 L/min in children. The pre-apnea PaCO2 was between 35 to 45 torr (4.7 to 6.0 kPa) in all patients. Twenty-three patients completed the test. Five patients had a PaO2 < 100 torr (< 13 kPa) during the 10-min ventilator withdrawal time period. No patient breathed spontaneously during the apnea test. Twenty-two patients achieved a PaCO2 > 60 torr (> 8 kPa). One patient had a postapnea PaCO2 of 59 torr (7.8 kPa). The test was stopped in one patient who became hypotensive. The bulk diffusion technique has several advantages, including ease of performance over other methods of supplying oxygen during apnea testing, but this method does not prevent hypoxemia in patients with lung disease.


Nitric oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia.

Nitric oxide synthesis occurs both in vitro and in vivo in response to inflammatory stimuli and can have profound effects on the local cellular environment. Hepatocytes, Kupffer cells, and endothelial cells produce nitric oxide in vitro, but the in vivo role of this reactive mediator in the liver is unknown. We assessed the role of nitric oxide synthesis during endotoxemia in mice by inhibiting its synthesis with NG-monomethyl-L-arginine after lipopolysaccharide injection and by determining the effects of this inhibition on hepatic damage. Injection of lipopolysaccharide in mice increased plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, and caused mild hepatic damage as measured by increased circulating hepatocellular enzyme levels. NG-monomethyl-L-arginine decreased plasma nitrite and nitrate values, but increased the lipopolysaccharide-induced hepatic injury. NG-monomethyl-L-arginine caused no hepatic damage when given without lipopolysaccharide. The extent of hepatic damage with NG-monomethyl-L-arginine was proportional to the dose of lipopolysaccharide used and could be reduced with concurrent administration of L-arginine but not D-arginine. Nitric oxide synthesis provides a protective function against lipopolysaccharide-induced liver injury that increases in importance as the degree of endotoxemia increases. The production of nitric oxide is, therefore, an important part of the liver's response to a systemic inflammatory stimulus.


Effect of extracorporeal membrane oxygenation on cerebral hemodynamics in newborn lambs.

Neurologic injury caused by changes in cerebral blood flow is a concern in infants treated with extracorporeal membrane oxygenation (ECMO). To investigate the hypothesis that cerebral hemodynamics would be influenced by bypass flow rates, eight ketamine anesthetized lambs (age range 1 to 8 days) had measurements of cerebral hemodynamics before and after institution of venoarterial bypass. Cerebral blood flow (whole brain and regional) were measured using the radiolabeled microsphere technique. A left carotid artery catheter (placed without sacrificing the vessel) reference sample was used for calculation of brain blood flows. Arterial and venous (sagittal sinus) oxygen contents and blood gases were measured across the cerebral circulation. Cerebral oxygen consumption, oxygen delivery, and fractional oxygen extraction were calculated. Measurements were made pre-ECMO and after placement on venoarterial ECMO at flow rates of 50 and 100 mL/kg/min. In addition, four other lambs were studied at a flow rate of 150 mL/kg/min, and seven additional animals were used to address methodologic issues with the microsphere technique in this model. Cerebral blood flow and oxygen delivery significantly decreased, compared with control at both flow rates of 100 and 50 mL/kg/min (87.8 +/- 5.2 vs. 36.4 +/- 6.2; and 35.1 +/- 5.8 mL/100 g/min; 5.82 +/- 0.37 vs. 2.14 +/- 0.35; and 2.19 +/- 0.39 microM/100 g/min, respectively). Cerebral fractional oxygen extraction increased significantly at a flow of 50 mL/kg, and cerebral oxygen consumption decreased significantly at a flow of 100 mL/kg. At a flow rate of 150 mL/kg/min, cerebral hemodynamic variables did not differ from control measurements. These findings demonstrate that changes in cerebral blood flow and cerebral oxygen metabolism can occur in the newborn lamb with the initiation of ECMO, and that these changes are dependent on bypass flow rate.


Epinephrine blood concentrations after peripheral bronchial versus endotracheal administration of epinephrine in dogs.

Emergency endotracheal drug administration has become an acceptable route for drug delivery during cardiopulmonary resuscitation. The purpose of the present study was to determine whether the site of endotracheal epinephrine injection is an important factor in its absorption. Epinephrine (1:1000), in a dose of 0.02 mg/kg diluted in 2 mL of saline, was given to ten anesthetized mongrel dogs. Each dog was studied twice: once when the epinephrine was injected into the endotracheal tube, and on another day, through the endotracheal tube via a flexible catheter wedged into a peripheral bronchus. Arterial blood samples for plasma epinephrine concentration determinations were collected, before and at 1, 2, 5, 10, 15, and 30 mins after each intratracheal drug administration. Both routes of epinephrine administration significantly increased plasma concentrations within 1 min of injection. Higher plasma epinephrine concentrations were achieved after peripheral bronchial epinephrine administration (maximal concentration 8.9 +/- 3.2 vs. 2.0 +/- 0.4 ng/mL), and the total dose absorbed was significantly (76.5 +/- 13.5 vs. 36.7 +/- 6.5 ng/min/mL, p < .05) higher. The time interval to reach maximal concentration was significantly shorter with the peripheral bronchial dosing than with the endotracheal route (1.3 +/- 0.2 vs. 2.7 +/- 0.5 min, p < .05). Neither group demonstrated a significant change in heart rate, and both had similar, minor decreases in BP for > 2 to 5 mins. There were no significant differences between the arterial blood gases of the two groups at various stages of the experiment. In dogs, epinephrine administered via the peripheral bronchial route has a clear pharmacologic advantage over the endotracheal route. This advantage may be more important during cardiopulmonary resuscitation conditions and other low flow states, and may account for the failure observed with the endotracheal route in recently published clinical reports.


Hyperbaric oxygen therapy accelerates neurologic recovery after 15-minute complete global cerebral ischemia in dogs.

Although hyperbaric oxygen therapy is clinically used for the treatment of several types of ischemic brain injury, few basic animal studies are available that provide a rationale for this therapy for complete global brain ischemia. Therefore, we investigated the effect of hyperbaric oxygen therapy on neurologic recovery after 15-min complete global cerebral ischemia in a canine model. Complete global ischemia was induced in 19 dogs by occlusion of the ascending aorta and the caval veins. Nine dogs were randomized to treatment with hyperbaric oxygenation (3 atmospheres absolute, 100% oxygen for 1 hr) at 3, 24, and 29 hrs after ischemia under spontaneous respiration, while the other ten dogs served as the control group without hyperbaric oxygen therapy (group C). Neurologic recovery was evaluated based on the electroencephalogram (EEG) activity score (1 = normal; 5 = isoelectric) and the neurologic recovery score (100 = normal; 0 = brain death) over a 14-day postischemic period. The survival rates were 3/10 (30%) in the control group vs. 7/9 (78%) in the group treated with hyperbaric oxygen (p < .05). Over the 14-day postischemic period, the best (lowest) EEG scores of each dog were significantly (1.7 +/- 0.2 vs. 2.9 +/- 0.3; mean +/- SE, p < .01) lower in the hyperbaric oxygen-treated group. The best neurologic recovery scores of each dog were significantly (69 +/- 6 vs. 48 +/- 5; mean +/- SE, p < .05) higher in the treated animals. The number of dogs that recovered to a neurologic recovery score of > 65 (assessed as a slight disability) were 1/10 in the control group and 6/9 in the group treated with hyperbaric oxygen (p < .02). Hyperbaric oxygen therapy performed in the early postischemic period accelerated neurologic recovery and improved the survival rate in dogs after 15-mins of complete global cerebral ischemia.


Undergraduate education in critical care medicine.

To review the current status and future needs of medical students with respect to education in critical care medicine. Literature review and questionnaire administered at the 1990 Educational and Scientific Symposium of the Society of Critical Care Medicine. Medical students are presently not required to master even the basic aspects of critical care medicine before licensing. The results of the questionnaire demonstrated that critical care medicine professionals believe a critical care core curriculum should be established and its completion should be made a requirement for graduation from medical school. A reference set of critical care medicine learning objectives and a strategy for their implementation are presented. There is a gap between what student physicians are required to learn and what they need to know to effectively initiate critical care interventions and to appropriately refer patients to ICUs. This gap can and should be remedied with wide implementation of a critical care core curriculum.


Mean airway pressure: physiologic determinants and clinical importance--Part 2: Clinical implications.

To discuss the theoretical relationship of mean alveolar pressure to its most easily measured analog, mean airway pressure, and to describe the key determinants, measurement considerations, and clinical implications of this index. Relevant articles from the medical and physiological literature, as well as mathematical arguments developed in this article from first principles. Theoretical, experimental, and clinical information that elucidates the physiologic importance, measurement, or adverse consequences of mean airway pressure. Mathematical models were used in conjunction with data from the published literature to develop a unified description of the physiological and clinical relevance of mean airway pressure. Geometrical and mathematical analyses demonstrate that shared elements comprise mean airway pressure and mean alveolar pressure, two variables that are related by the formula: mean alveolar pressure = mean airway pressure + (VE/60) x (RE - RI), where VE, RE, and RI are minute ventilation and expiratory and inspiratory resistances, respectively. Clear guidelines can be developed for selecting the site of mean airway pressure determination, for specifying technical requirements for mean airway pressure measurement, and for delineating clinical options to adjust the level of mean airway pressure. Problems in viewing mean airway pressure as a reflection of mean alveolar pressure can be interpreted against the theoretical basis of their interrelationship. In certain settings, mean airway pressure closely relates to levels of ventilation, arterial oxygenation, cardiovascular function, and barotrauma. Because mean airway pressure is associated with both beneficial and adverse actions, a thorough understanding of its theoretical and practical basis is integral to formulating an effective pressure-targeted strategy of ventilatory support. Mean airway pressure closely reflects mean alveolar pressure, except when flow-resistive pressure losses differ greatly for the inspiratory and expiratory phases of the ventilatory cycle. Under conditions of passive inflation, mean airway pressure correlates with alveolar ventilation, arterial oxygenation, hemodynamic performance, and barotrauma. We encourage wider use of this index, appropriately measured and interpreted, as well as its incorporation into rational strategies for the ventilatory management of critical illness.


Effects of storage temperature on viable bioprosthetic heart valves.

Long-term in vivo success of bioprosthetic allografts is dependent upon retention of cellular functions, such as protein synthesis. The purpose of the experiments presented in this report was to determine the storage conditions necessary for retention of protein synthetic functions in human allograft heart valve leaflets. Tissue viability was assessed by measurement of tritiated-glycine incorporation into proteins. Comparison of short-term (less than 3 month)- and long-term (1 and 2 years)-cryopreserved heart valve leaflet storage in a liquid nitrogen freezer below -135 degrees C demonstrated preservation of fibroblast protein synthesis. In contrast, storage in a mechanical freezer at -80 degrees C resulted in a time-dependent loss of fibroblast protein synthesis. There was no statistically significant effect on protein synthesis in leaflets stored for 1 week at 4 degrees C compared to control cryopreserved liquid nitrogen-stored leaflets. After 2 weeks of 4 degrees C storage leaflet protein synthesis declined significantly to 15% that of cryopreserved controls. These results demonstrate that liquid nitrogen storage of valve bioprostheses is required for long-term preservation of cellular functions.


Parthenogenetic activation pattern and microtubular organization of the mouse oocyte after exposure to 1,2-propanediol.

We have studied the effect of 1,2-propanediol (PROH) on cumulus-oocyte complexes from the mouse. We determined the morphological survival rate, the pattern of parthenogenetic activation, and the microtubular and chromosomal organization. Cumulus-oocyte complexes were collected at 16 h post hCG from superovulated female hybrid mice. These cumulus-intact oocytes were exposed to 1.5 or 3 M PROH for 6, 12, or 18 min at 0, 22, or 37 degrees C. The cryoprotectant was diluted out in a 1 M sucrose solution at 22 degrees C. After 5-6 h at 37 degrees C, oocytes were denuded and examined under Nomarski optics. The results show that PROH can induce degeneration and parthenogenetic activation in the mouse oocyte in a concentration, temperature, and time-dependent way. As the activation stimulus was strengthened, an increasing proportion of oocytes shifted from parthenogenetic activation with polar body extrusion to parthenogenetic activation with polar body retention and even to immediate cleavage. Nontoxic and nonactivating conditions involved mainly exposure to 1.5 M PROH at 0 degrees C. Spindle integrity and chromosomal organization were analyzed for exposure to 1.5 and 3 M PROH for 12 min at 0 degrees C. The separate effect of cooling and exposure to 1 M sucrose were also evaluated. Microtubules were visualized by monoclonal anti-alpha-tubulin labeling followed by immunogold-silver staining. Cooling and exposure to 1 M sucrose or to 1.5 M PROH did not induce major abnormalities in the microtubular or chromosomal organization. On the other hand, a significant percentage of deformities such as spindle size reduction and loss of bipolarity were observed after exposure to 3 M PROH. The results of the present study demonstrate that the use of PROH as a single cryoprotectant for the freezing of mature unfertilized oocytes cannot be recommended in procedures involving ambient temperature or concentrations exceeding 1.5 M PROH. On the other hand, the potential beneficial effect of low temperatures may outweigh the effect of concentration at subzero temperatures and could be explored further in the tailoring of conditions for slow controlled freezing.


Rheological measurements of the influence of 1,2-propanediol on actin/alpha-actinin gel structure: the effects of temperature and protein concentrations.

In previous studies, we demonstrated that 1,2-propanediol induces shortening and bundling of actin filaments, both in vitro and in vivo, and that it enhances actin/alpha-actinin interaction, especially at low temperature. 1,2-Propanediol also promotes homogeneous microporous networks which can be vitrified by rapid cooling. In the present study, dynamical rheological measurements were performed under various sets of experimental conditions including temperature (4 or 20 degrees C), protein concentrations (actin and alpha-actinin), and 1,2-propanediol presence or absence. Gelation kinetics were monitored, and the resulting actin mechanical properties investigated, in order to untangle the respective effects of the experimental parameters. Whether in the presence or absence of solvent, low temperature brings about a rigidification of the sample, as does high protein concentration, as expected. However, 1,2-propanediol itself involves either softening of the sample (at high temperature and low protein concentration or at low temperature and high protein concentration) or rigidification in the case of low temperature and low protein concentration. These effects result from the competition between actin/alpha-actinin affinity (enhanced by both low temperature and 1,2-propanediol), bundling of filaments (fostered by alpha-actinin for alpha-actinin/actin ratios used), rate of actin polymerization (higher at high temperature), shortening effect of 1,2-propanediol on actin filaments, and chain mobility (lower at high protein concentration). As discussed, only the combination of low temperature and low protein concentration induces full crosslinking of the system into a viscoelastic solid under the influence of 1,2-propanediol.


Leakage of a trapped fluorescent marker from liposomes: effects of eutectic crystallization of NaCl and internal freezing.

Leakage of trapped carboxyfluorescein from DL-alpha-dipalmitoylphosphatidylcholine multilamellar liposomes (diameter 1-2 microns) in NaCl solutions was measured after rapid freezing to temperatures between -15 and -55 degrees C. Leakage was low after freezing between -15 and -35 degrees C, but increased steeply between -35 and -45 degrees C. From DSC measurements it was found that the increase in leakage was associated with two crystallization processes: Eutectic crystallization of NaCl and freezing of undercooled solvent trapped in the interior of the liposomes ("internal freezing"). Damage caused by the former process could effectively be prevented by small amounts of trehalose (1% less than or equal to w less than or equal to 1.5%). Trehalose in these concentration also decreased damage due to internal freezing, but to a minor degree. In addition to these damaging transitions, a time-dependent process was found to cause leakage from the liposomes at -25 degrees C. The association between leakage and thermal activity suggests that DSC supplements cryomicroscopy and leakage measurements in the characterization of cryostability of liposomes.


Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides.

The technique of isothermal emulsion differential scanning calorimetry was used to determine time-temperature-transformation (TTT) curves for aqueous glycerol and butane-2,3-diol in the presence of various polysaccharides and sugars. The critical cooling rate required to avoid the crystallization of ice in these solutions was then calculated from the experimental TTT curves. The polysaccharides used in this study included starch hydrolysis products and dextrans of various molecular weights. The sugars used here were sucrose, glucose, trehalose, and raffinose. The results show that the critical cooling rates of butane-2,3-diol and glycerol are reduced by varying amounts by the addition of such materials but that the reduction is not as great as is achieved by the addition of polyethylene glycol with a molecular weight of 400.


Hydration of oligosaccharides: anomalous hydration ability of trehalose.

The disaccharide trehalose extensively exists in anhydrobiotic organism and is considered to play an important role in preserving the integrity of biomembrane. However, the preserving mechanism remains unclear. In this report, we examine the hydration abilities of trehalose and several oligosaccharides composed of alpha-D-glucopyranosyl residues. The unfrozen water fraction per molecule was determined from differential scanning calorimetry measurements of their aqueous solutions. Further, the NMR relaxation time of the natural abundance 17O of water is measured for several saccharide solutions. These results indicate that trehalose has the highest hydration ability among the saccharides studied. In other words, trehalose can effectively lower the mobility of water molecules hydrogen-bonded with saccharides. It is thus reasonable that, among the disaccharides studied, trehalose exhibits the maximum stabilizing effect on the bilayer structure of lipid whose acyl chains are bonded with each other by the apolar interaction, because the apolar interaction is strengthened with the stabilization of the surrounding water structure.


Hibernation alters the frog's immune system.

The lymphomyeloid organs and blood leukocyte populations of the leopard frog, Rana pipiens, undergo conspicuous changes during hibernation at 4 degrees C. Within the blood, spleen, thymus, jugular bodies, and bone marrow there was a progressive loss of hemopoietic populations resulting in a marked lymphocyte depletion. Termination of the 135-day hibernation period resulted in the restoration of all hemopoietic elements in the blood and lymphomyeloid organs within 30 days. Frogs subjected to experimental hibernation and immunized showed weakened immune responses when brought from the hibernaculum. Plaque-forming cells (PFC) were lower in spleen, jugular bodies, and bone marrow, and serum antibody titers were also lower. Although the kinetics of the primary responses were essentially the same, the secondary responses differed suggesting major rearrangements with respect to the numbers of cells and their function in secreting antibody. The apparent lymphocyte aplasia may contribute to the absence of immunological responsiveness during periods of hibernation.


The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo.

Using standardized freeze wounds in cat corneas, we tested the efficacy of basic Fibroblast Growth Factor (bFGF) solubilized in phosphate buffered saline (PBS) to promote endothelial healing when injected intraocularly at doses ranging from 0.01 microgram to 10 micrograms. After 6 days, animals were humanely sacrificed and corneal tissues were fixed and stained for light microscopy and computation of remaining wound areas. A significant dose-response relationship was found between the dosages of 0.01, 0.1, and 1.0 microgram of bFGF/eye and the stimulation of more completely healed endothelium six days after transcorneal freeze wounding. Significantly larger endothelial wounds were present six days after wounding when the eyes were treated with 10 micrograms of bFGF/eye compared with controls treated with PBS only.


Systemic and topical protection studies using Pseudomonas aeruginosa flagella in an ocular model of infection.

Purified flagella from P. aeruginosa ATCC 19660 were used for active, passive, or topical immunization prior to corneal challenge with strain 19660. At 30 days post-infection, a significant number of mice actively or passively immunized with flagella and infected with the homologous bacterial strain were protected from ocular disease when compared to control animals. In topical immunization studies, premixing of 19660 flagella with the bacterial inoculum prior to ocular challenge with strain 19660, provided results similar to those of the active or passive immunization studies. A reduced lipopolysaccharide (LPS:1 E.U./mg) flagella preparation was also produced and used similarly. Again, significant protection was achieved in mice immunized by flagella regardless of the immunization route. An in vitro adherence assay also was performed to examine quantitatively the effect of exogenously applied flagella, or an antiflagella monoclonal antibody (MAb) on bacterial adhesion. Premixing of the bacterial inoculum with flagella or the MAb prior to applying it topically to corneas in organ culture all significantly inhibited bacterial binding. These results strongly suggest that significant ocular protection is achieved with either active or passive immunization, or premixing of the bacterial inoculum with flagella from strain 19660 prior to ocular challenge with the homologous bacterial strain. They also indicate that topical application of flagella or antiflagella MAb provide protection against ocular disease by decreasing bacterial adhesion to cornea.


Examination of a lens 'native' plasma membrane fraction and its associated crystallins.

A bovine lens "native" plasma membrane fraction containing its full compliment of extrinsic proteins was prepared by sucrose density centrifugation of the water insoluble fraction. The major membrane fraction was found at the 25/45% sucrose interface. This fraction contained 73% of the total water insoluble phospholipid, 74% of the total water insoluble cholesterol and 58% of the total urea-insoluble protein. Only 9% of the total urea-soluble protein was membrane associated (extrinsic protein), most (75%) was recovered from the pellet. The major intrinsic protein (8 M-urea-insoluble) of the membrane fraction was MIP28, with lesser amounts of MP17. Extrinsic proteins (8 M-urea-soluble) were examined by SDS-PAGE, isoelectric focusing, immunoblotting and amino acid composition analysis. Approximately 70% of the total extrinsic protein appeared to be alpha A-crystallins and modified alpha A-crystallins. About 20% of the extrinsic protein was apparently beta- and gamma-crystallins. The remainder contained presumed cytoskeletal proteins and perhaps other unidentified polypeptides. The native plasma membrane was found distributed throughout the lens with only minor differences in the quantitative composition of the membrane fraction. We have concluded that the native membrane fraction represents the lens plasma membrane with its extrinsic proteins which exist in vivo. These extrinsic proteins appeared to be primarily acidic alpha-crystallin polypeptides with minor amounts of beta- and gamma-crystallins, and presumed cytoskeletal elements. We speculate that these extrinsic proteins may serve as a nucleation site for the association of other water insoluble protein through protein-protein interactions such as those found in the non-membrane associated urea-soluble protein. Together, these interactions may form a structured cytoplasmic matrix important for the maintenance of lens transparency.


Proteoglycan synthesis by scleral chondrocytes is modulated by a vision dependent mechanism.

Proteoglycan synthesis was measured in chick sclera at the onset of form-deprivation myopia, as well as in the period immediately following removal of the occluder. Two day-old chicks were monocularly form vision deprived for periods from one to ten days and proteoglycan synthesis was determined after placing posterior scleral buttons in organ culture and measuring 35SO4 incorporation into glycosaminoglycans. Following 24 hrs of form-deprivation, proteoglycan synthesis was 33% higher in myopic eyes as compared with paired control eyes. The rate of proteoglycan synthesis further increased to levels 83% higher than controls after four days of form-deprivation and remained elevated throughout the ten day period of deprivation. Removal of the occluder after 10 days of form-deprivation resulted in a rapid drop in the rate of proteoglycan synthesis to control levels within 24 hrs. Proteoglycan synthesis was also measured in scleral chondrocytes isolated from control and myopic eyes after 10 days of form-deprivation. Proteoglycan synthesis by chondrocytes from myopic eyes did not return to control levels until 48 hrs after plating. Since the rate of proteoglycan synthesis returns to control levels more quickly during the recovery period ex vivo than when scleral chondrocytes from myopic eyes are placed in cell culture, we suggest that a mechanism is present within the eye which rapidly lowers the rate of proteoglycan synthesis in response to form vision.


Enrichment of polyunsaturated fatty acids from rat retinal pigment epithelium to rod outer segments.

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA, 22:6n-3), are enriched in phospholipids of vertebrate rod outer segments (ROS). Retinal ROS can incorporate 22 carbon (C-22) PUFA from the plasma pool where C-20 PUFA are predominant. In this study, we analyzed the fatty acid composition of retinal pigment epithelium (RPE) and ROS from rats fed different fatty acid supplements to determine whether this enrichment is at the photoreceptor-RPE boundary or the RPE-choriocapillaris boundary. Long Evans rats were raised from birth for 13-14 weeks on a diet supplemented with 10% (wt/wt) hydrogenated coconut oil (COC; 0.2% 18:2n-6, no 18:3n-3), safflower oil (SAF; 73.8% 18:2n-6, 0.1% 18:3n-3), or linseed oil (LIN; 16.4% 18:2n-6, 52.2% 18:3n-3). These diets were chosen because they increased plasma levels of 20:3n-9, 20:4n-6, and 20:5n-3, respectively. These three fatty acids served as metabolic markers. Plasma levels of 22:6n-3 were reduced by the COC and SAF diets. The RPE incorporated 20:3n-9, 20:4n-6, and 20:5n-3 from the plasma. However, the levels of 20:3n-9 and 20:5n-3 were very low in ROS and 20:4n-6 was not significantly elevated in the ROS of the SAF diet group. The relative amount of total C-20 PUFA in phospholipids in RPE was similar to that found in plasma and was about 4-16 times (depending on different lipid classes) that in the ROS. In contrast, C-22 PUFA (22:6n-3 and 22:5n-6) showed a step-wise, average 3-5 fold increase in concentration from the plasma to the RPE to the ROS.(ABSTRACT TRUNCATED AT 250 WORDS)


Lipids of frog retinal pigment epithelium: comparison with rod outer segments, retina, plasma and red blood cells.

The glycerolipid and fatty acid compositions of frog retinal pigment epithelium (RPE) were determined and compared with rod outer segments (ROS), retina, plasma, and red blood cells (RBC). The glycerolipid class composition of RPE was similar to RBC and ROS or retina, with phosphatidylcholine and phosphatidylethanolamine being the major components. The fatty acid composition of RPE differed substantially from that of plasma or RBC; the former contained much higher levels of C-20 and C-22 polyunsaturated fatty acids (PUFAs), such as 20:4n-6 and 22:6n-3, but less C-18 mono-, dienoic, and trienoic acids. The difference between RPE and ROS or retina with respect to fatty acid profile was also dramatic; RPE had relatively less 22:6n-3, but more 20:4n-6 and 18:2n-6, than ROS or retina. These results suggest that frog RPE cells may selectively take up C-20 and C-22 PUFAs from the circulation, but preferentially deliver 22:6n-3 to the ROS and retina. Fatty acid analyses show that 20:4n-6 and 22:6n-3 were unevenly distributed among RPE glycerolipids; phosphatidic acid, diglyceride, triglyceride, and phosphatidylserine are relatively more enriched in 22:6n-3 compared with 20:4n-6. This information might imply that these two PUFAs are metabolized differently inside the frog RPE cells.


Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis.

Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.


The ability of lens alpha crystallin to protect against heat-induced aggregation is age-dependent.

Alpha crystallin was prepared from newborn and aged bovine lenses. SDS-PAGE and tryptic peptide mapping demonstrated that both preparations contained only the alpha-A and alpha-B chains, with no significant contamination of other crystallins. Compared with alpha crystallin from the aged lens, alpha crystallin from the newborn lens was much more effective in the inhibition of beta L crystallin denaturation and precipitation induced in vitro by heat. Together, these results demonstrate that during the aging process, the alpha crystallins lose their ability to protect against protein denaturation, consistent with the hypothesis that the alpha crystallins play an important role in the maintenance of protein native structure in the intact lens.


Glycation of lens membrane intrinsic proteins.

Changes occurring at the membrane are believed to be the decisive factors in the initiation of diabetic cataract. During diabetic hyperglycemia lens crystallins were shown to undergo glycation. Several studies indicated that glycation brings about protein conformational changes thus implicated in cataractogenesis. Since the membrane proteins are the first targets for glycation, in this study we measured the glycation of alkali washed urea-insoluble membrane proteins from control and diabetic rats by two different methods, phenyl-boronate affinity chromatography and [3H]NaBH4 reduction, and confirmed by amino acid analysis. There was a significant increase in the glycation of membrane proteins in diabetic cataract lenses when compared to controls. It appears that lysine is the major site of glycation. Concomitant to early glycation, there was an increase in non-tryptophan fluorescence (Ex: 350 nm/Em: 440 nm) in the diabetic lens membrane proteins suggesting the presence of advanced glycation mediated protein cross-links. In order to identify whether the major membrane intrinsic protein, MIP26, undergoes glycation, we isolated MIP26 along with its degradatory product MIP22 as one peak on molecular sieve HPLC. HPLC isolated MIP26/MIP22 was further separated on SDS-PAGE followed by slicing and counting. This analysis revealed that MIP26 and MIP22 were more or less equally glycated in controls, however, in diabetic rats glycation of MIP22 was glycated slightly higher than MIP26. Moreover, the proportion of MIP22 increased by about 2-fold in diabetic lenses compared to controls. Thus it appears that major glycation sites are still retained in MIP22 in diabetic rat lenses. In vitro glycation studies with bovine lens membranes were also done using 14C glucose, followed by SDS-PAGE and autoradiography. The major protein glycated in vitro also seems to be MIP26. Interestingly, MIP22 was less glycated than MIP26 in vitro.


The effect of chlorpromazine on endotoxin-induced uveitis in the Lewis rat.

Chlorpromazine (CPZ) has been used extensively in the treatment of psychiatric disorders, and has recently been shown to possess systemic anti-inflammatory properties as well. To investigate the potential effects of CPZ on ocular inflammation, we evaluated its action on endotoxin-induced uveitis (EIU) in Lewis rats. At three different dosage levels, CPZ produced highly significant reductions in the mean aqueous aspirate inflammatory cell counts and histological inflammatory scores as compared to controls treated with vehicle only. Analysis of aqueous fluid demonstrated a similar decrease in protein concentration and phospholipase A2 (PLA-2) activity in the treated animals. The ability of CPZ to inhibit the development of EIU may be related to its properties as a calcium channel blocker and inhibitor of the enzyme phospholipase A2.


Collagenolytic/gelatinolytic metalloproteinases in normal and keratoconus corneas.

Cells of keratoconus corneas have been reported to produce higher levels of collagenolytic/gelatinolytic enzymatic activities than do cells of normal corneas. The current study investigates the contribution of 1) specific enzyme gene products, and 2) the degree to which these proteins are present in the activated forms, to the increased enzymatic activities. We demonstrate that two neutral gelatinolytic enzymes, a 66/59 kD form and a 92 kD form, can be directly extracted from both normal and keratoconus corneas. These enzymes are identified as the pro- and activated forms of MMP-2 and as the pro-form of MMP-9, specific members of the matrix metalloproteinase family. Normal and keratoconus corneas show no significant differences in amounts or types of extractable neutral gelatinases, nor in the amounts or types that they synthesize in culture. Furthermore, in both the normal and keratoconus corneas, gelatinases are found primarily in the inactive form. These studies suggest the possible importance of changes in proteinase inhibitor levels to the characteristic biochemical features of keratoconus corneas.


The retinal pigment epithelium induces fenestration of endothelial cells in vivo.

Rodent photoreceptor dystrophies are characterized by late stage ingrowth of retinal blood vessels into the retinal pigment epithelium (RPE) where they proliferate. Some of these vessels develop the fenestrated phenotype of the choriocapillaris (CC). To determine if development of fenestrae in these endothelial cells is a function of the duration of time the endothelial cell had been encapsulated by the RPE, we did an ultrastructural morphometric study of these vessels in urethane induced photoreceptor degeneration in Long-Evans rats. Retinas of animals aged 20, 24, 40 and 56 weeks were studied. The fraction of vessel profiles within the RPE that had fenestrated endothelial cells increased from 10% to 90% between 20 to 56 weeks. The average number of fenestrae per vessel increased approximately 25 fold between 20 and 24 weeks but stabilized after that, despite a decrease in the number of vessels present at 56 weeks. A large number of degenerated retinal vessel profiles were seen in the RPE at 40 weeks. These facts support the idea that the presence of the RPE induces endothelial cell fenestrae, and also show that a complex process of remodelling including proliferation and degeneration is occurring in these vessels. Analogies between the basic cell biology of neovascularization occurring in these rodent models and that of proliferative diabetic retinopathy and age-related macular degeneration are discussed.


Retinyl ester hydrolysis in the rabbit lacrimal gland.

The lacrimal gland stores retinyl esters which are synthesized by the enzyme acyl CoA:retinyl acyl transferase. Retinol is released from retinyl ester reserves by retinyl ester hydrolase (REH). Since the lacrimal gland secretes retinol, this gland should also contain this enzyme. To identify bile salt-dependent REH activity, rabbit lacrimal glands were homogenized in 0.05 M Trismaleate buffer, and enzyme activity was determined in the tissue homogenate, in the membrane fraction and in the cytosolic fraction by measurement of production of retinol from retinyl palmitate (nmol retinol produced/mg protein/h). In the lacrimal gland, production of retinol was optimal in the presence of 200 mM CHAPS at pH 7. The REH activity in the presence of 1000 microM retinyl palmitate was 2.38 +/- 0.18 nmol/mg/h in the homogenate, 1.13 +/- 0.16/nmol/mg/h in membranes and 3.25 +/- 0.26 nmol/mg/h in cytosol. By comparison, REH activity in rabbit liver was 6.58 +/- 0.75 nmol/mg/h. The REH activity in lacrimal gland was not affected by vitamin A deficiency. These data are consistent with the presence of retinyl ester hydrolase activity in the lacrimal gland and provide further evidence that this gland is adapted for metabolism and secretion of retinol.


The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.

The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.


Cell-specific expression of LBP-32 mRNA in retina and other locations of newborn mouse eye as revealed by in situ hybridization.

LBP-32 is a cell surface and cytoplasmic protein which is thought to both mediate cell attachment to laminin and play a role in translation initiation. In the present study, antisense RNA for LBP-32 was used to document its cellular mRNA expression pattern in newborn mouse eye. In situ hybridization revealed that LBP-32 was distributed uniformly through the retina as well as over anterior oblique muscle, in corneal and lens epithelial cells and in capillary endothelial cells of the choroid. This unique cell-specific expression raises interesting questions of the role of LBP-32 in eye development.


Binding of actin to lens alpha crystallins.

Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.