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Biomedical subjects

R W Beuerman

Publications and source records attributed to R W Beuerman.

At least 19 recordsLinked to original sources
PubMed2005

The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells.

AIM: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE). METHODS: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically. RESULTS: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation. CONCLUSIONS: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.

Animals↗
PubMed2003

Confocal microscopy of Aspergillus fumigatus keratitis.

AIM: To use a confocal microscope to characterise the treated and untreated courses of fungal keratitis. METHODS: In the first experiment, Aspergillus fumigatus stromal keratitis was produced in both eyes of seven New Zealand white rabbits. In the second experiment, keratitis was induced in right eyes of 20 rabbits. Group 1 rabbits were treated with topical fluconazole, group 2 rabbits received oral fluconazole, and group 3 rabbits were used as controls. The rabbits were examined with a slit lamp and confocal microscope 2, 6, 10, 14, and 20 days after inoculation. The corneal cultures were taken on days 2, 14, and 20 and biopsies were taken on days 2 and 22. RESULTS: On days 14 and 22 confocal microscopy was more sensitive than culture technique in both treated and untreated animals, since not all cases of fungal keratitis can be cultured. CONCLUSION: This study indicates that confocal microscopy is a rapid and sensitive diagnostic tool for both the early diagnosis and non-invasive follow up of fungal keratitis

Administration, Oral↗
PubMed2002

Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry.

BACKGROUND: The axolemmal distribution and density of voltage-gated sodium channels largely determines the electrical excitability of sprouting neurites. Recent evidence suggests that accumulation of sodium channels at injured axonal tips may be responsible for ectopic axonal hyperexcitability and the resulting abnormal sensory phenomena of pain and paresthesias. For future improvement in pain management it is necessary to identify structurally significant generators of autorhythmicity. A first step in this regard will be to determine the predominant types of sodium channels in injured axons. The opportunity to test human specimens from painful and non-painful neuroma is of great value. METHODS: We employed immunocytochemical methods to investigate if two types of highly specific voltage-gated sodium channel subtypes could be detected in sections of human neuroma. FINDINGS: Both subtypes of sodium channels PN1 and PN3 accumulated abnormally in human neuromas. The immunoreactive pattern was more pronounced in painful neuromas. This is in contrast to previous reports that focused either on PN1 or PN3 as main generators of hyperexcitability induced pain. INTERPRETATION: Both, PN1 and PN3 seem to be involved in hyperexcitability induced pain. It can be expected that a variety of other highly specific voltage gated sodium channel subtypes will be detected in regenerating peripheral nerve in the near future, which contribute to the development of neuropathic pain states. Thus, in order to therapeutically control hyperexcitability induced neuropathic pain, it might be worthwhile to develop pharmaceuticals that can selectively block different sodium channel subtypes and subunits.A review of the role of sodium channels in neuropathic pain is implemented in the discussion.

Humans↗
PubMed2002

Dimensional growth of the rabbit eye.

The rabbit eye has been used experimentally in research in vision and ophthalmology. However, there is little information about postnatal growth. The purpose of this study was to determine growth patterns of the rabbit globe from birth to young adult status focusing in particular on the fibrous outer tunic of the globe, the sclera. Eyes of both sexes of New Zealand White rabbits were examined on postnatal days 1, 7, 14, 28, 42 and 56. Axial length, equatorial diameter and corneal diameter were measured using a digital caliper. Plastic-embedded tissue was used to quantify scleral thickness and cellular density at anterior, equatorial and posterior sites. Axial length increased 230% between postnatal days 1 and 56. The rate of growth was not linear and revealed two peaks. The first peak occurred between postnatal days 1-7, and the second between days 14-28. Eye opening occurred on days 9-11. Sex differences were seen, with the male eye being larger (p = 0.00001). Surface area measurements of sclera and cornea revealed that the sclera occupies approximately 5/6 of the outer surface of the globe and the cornea 1/6. Scleral thickness increased from day 1 to day 42 (70-155 microm at the equator) while fibroblast density was found to decrease (0.32 to 0.05 cells/microm(2)). The results suggested that eye opening precedes a unique increase in the rate of eye growth and visual processes may be playing a role in modulating the growth pattern between days 14 and 28.

Age Factors↗
PubMed2001

Differential expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin and hypoxanthine phosphoribosyltransferase (HPRT) in postnatal rabbit sclera.

PURPOSE: GAPDH, beta-actin, HPRT and 18S rRNA are constitutively expressed in all mammalian cells. In accordance with the nature of invariant control, these genes have been used to standardize genes of interest in expression studies. Recent studies have suggested that GAPDH, beta-actin and HPRT in special situations may come under temporary regulatory control, but that 18S rRNA may be more likely to remain constitutive. However, little is known about the quantitative expression of these genes in fibroblasts and in particular during early postnatal development, a time of rapid changes in cell metabolism. In this study we have examined the differential expression of these genes in association with scleral development from an early postnatal age up to young adult status. METHODS: GAPDH, beta-actin, HPRT, and 18S rRNA gene expression were analyzed in the rabbit sclera from 1 day to 8 weeks postnatally by real-time, comparative PCR. RESULTS: Real-time PCR analysis showed that the expression levels of GAPDH, beta-actin, and HPRT were higher in the first postnatal week and then declined. However, from 2 to 8 weeks, the mRNA levels of these three genes underwent significant variations (P < 0.01) in their levels of expression. In contrast, the expression level of 18S rRNA showed no significant variation (P >or= 0.5) over this time period. Conclusions. The present study shows that GAPDH, beta actin and HPRT gene were differentially expressed in early postnatal scleral development. It also suggests that these gene products could be implicated in the developmental process and have a crucial role in the early postnatal period. This study demonstrates that 18S rRNA may be preferable to normalize genes of interest in studies of early development.

Actins↗
PubMed2000

Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys.

OBJECTIVE: To study the effects of lacrimal gland removal on basal and reflex tear production and on the ocular surface in the squirrel monkey. METHODS: Unilateral main lacrimal gland removal in 6 squirrel monkeys was followed by Schirmer testing, slit-lamp examination with fluorescein, and collection of basal and reflex (stimulated) tears for analysis of tear protein spectra between 0 and 20 kd, as well as histological evaluation. RESULTS: Schirmer test results showed an 80% decrease in basal tears and a 90% decrease in reflex tears during week 1, and a 32.2% and 33.3% decrease, respectively, at week 20 after surgery, compared with the contralateral control side. However, no gross abnormalities or fluorescein staining were seen in 5 of the 6 monkeys, and the conjunctival surfaces remained normal. The main and accessory lacrimal glands appeared to secrete similar types of proteins. No histological changes were seen in corneal, conjunctival, or eyelid tissues 20 weeks after surgery. CONCLUSIONS: Tears from accessory lacrimal glands were sufficient to maintain a stable tear layer on the cornea, suggesting that so-called basal tear flow is made up of fluid from both main and accessory lacrimal glands and that decreased tear production by the main lacrimal gland is not a causative factor in keratoconjunctivitis sicca. CLINICAL RELEVANCE: This study shows that total removal of the main lacrimal gland does not in itself lead to keratoconjunctivitis sicca. However, the nature of neural control of the accessory glands is not yet clear.

Animals↗
PubMed2000

Model for avulsion injury in the rat brachial plexus using passive acceleration.

We have developed an experimental model for brachial plexus injuries in the rat that closely simulates the characteristics of human injury. This model produces avulsion injuries in a noninvasive manner. A prototype apparatus was designed that allowed a force to be transmitted to a restrained limb by passive acceleration. Reproducible results were obtained in 32 rats. A significant correlation was found between the test weight and the number of roots avulsed (r = 0.92; P < 0.05). The amount of force also correlated to the pattern of avulsion injury: a 230-g weight produced either C6 (54%), C7 (15%), or C6 and C7 (31%) avulsions; a 330-g weight produced C6 (18%), C7 (9%), or C6 and C7 (73%) avulsions; a 530-g weight produced C5 through C8 (75%) or C6 through T1 (25%) avulsions. This model of brachial plexus injury may be useful to further our understanding of the cellular response to this incapacitating injury and to develop therapeutic strategies with behavioral correlates.

Acceleration↗
PubMed1999

Confocal microscopy in lattice corneal dystrophy.

BACKGROUND: The purpose of the study was to assess the appearance of lattice corneal dystrophy by means of white-light confocal microscopy. METHODS: Two consecutive patients with lattice corneal dystrophy were prospectively examined. In vivo white-light tandem-scanning confocal microscopy was performed in the right eye of the first patient. Her left eye had undergone penetrating keratoplasty 4 years earlier. Histologic findings of the corneal button were compared with confocal microscopic findings of the right eye. The other patient was monocular and confocal microscopy was performed only in the non-seeing eye. RESULTS: In both patients, linear and branching structures with changing reflectivity and poorly demarcated margins were visualized in the stroma. The linear structures measured approximately 40-80 microm in width. CONCLUSION: Lattice corneal dystrophy presents characteristic linear images on confocal microscopy and should not be misdiagnosed as fungal hyphae in cases of corneal infection.

Aged↗
PubMed1999

Characterization of immortalized rabbit lacrimal gland epithelial cells.

To establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm2 plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 microM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.

Animals↗
PubMed1999

Characterization of epithelial downgrowth by confocal microscopy.

A 43-year-old white woman with a history of multiple ocular surgeries, including 4 penetrating keratoplasties, developed a concentric retrocorneal membrane at the graft periphery in the right eye. A white-light, tandem, scanning confocal microscope using a 24x/0.60 contact objective was used to examine the right eye in vivo. At the endothelial layer, confocal microscopic images similar to corneal epithelial cells were detected at the graft periphery. Unlike normal endothelial cells, the imaged cells demonstrated easily recognizable nuclei.

Adult↗
PubMed1999

Denervation of rabbit lacrimal gland increases levels of transferrin and unidentified tear proteins of 44 and 36 kDa.

PURPOSE: The main lacrimal gland secretes proteins and fluid that make up the aqueous component of the tears. Previous reports indicate that the parasympathetic innervation of the gland influences the secretion of protein from the lacrimal gland. We investigated the effect of lacrimal nerve transection on the levels of individual proteins and overall protein concentration in the tear fluid. METHODS: The main lacrimal gland was unilaterally denervated in adult rabbits at the site of nerve entry to the gland. The contralateral gland (sham-operated) had identical surgical manipulations, excluding nerve transection. Tears were collected daily from both eyes for up to 9 days, after which lacrimal glands were collected. SDS-PAGE, densitometric and image analysis, and Western blot were performed. RESULTS: Consistently measurable tear protein bands ranged from 6 kDa to 85 kDa, using densitometric analysis. Lacrimal gland denervation produced a sustained increase in proteins of 85, 44, and 36 kDa in tears and lacrimal gland tissue from the denervated side, compared with the sham-operated side (0.025 > p > 0.001). The band at 85 kDa was identified as transferrin by Western blot. Tears from the denervated glands also showed transient decreases in low molecular weight tear proteins (18, 12/10, and 6 kDa), as well as a decrease in overall protein concentration, compared with tears from sham-operated glands and non-operated glands (p < 0.001). CONCLUSIONS: These results demonstrate that, in rabbit tears, the quantities of transferrin and two unidentified tear proteins, as well as overall protein concentration, are influenced by the sensory and/or autonomic innervation to the lacrimal gland. The decrease in overall tear protein concentration after lacrimal gland denervation may be related to a loss of nerve-regulated secretagogue-induced protein secretion.

Animals↗
PubMed1999

Confocal microscopy in cornea guttata and Fuchs' endothelial dystrophy.

AIMS: To report the appearances of cornea guttata and Fuchs' endothelial dystrophy from white light confocal microscopy. METHODS: Seven eyes of four consecutive patients with cornea guttata were prospectively examined. Of the seven eyes, three also had corneal oedema (Fuchs' dystrophy). In vivo white light tandem scanning confocal microscopy was performed in all eyes. Results were compared with non-contact specular microscopy. RESULTS: Specular microscopy was precluded by corneal oedema in one eye. In the remaining six eyes, it demonstrated typical changes including pleomorphism, polymegathism, and the presence of guttae appearing as dark bodies, some with a central bright reflex. In all seven eyes, confocal microscopy revealed the presence of round hyporeflective images with an occasional central highlight at the level of the endothelium. Changes in cell morphology and size were readily appreciated. CONCLUSION: By comparison with specular microscopy, the hyporeflective images with an occasional central highlight seen on confocal microscopy are consistent with the presence of guttae. Confocal microscopy may confirm the diagnosis of cornea guttata and Fuchs' endothelial dystrophy by demonstrating the presence of guttae. This technique is especially valuable in cases of corneal oedema, where specular microscopy may fail to visualise the endothelium. However, specular microscopy should remain the method of choice to evaluate the endothelium, principally because it is easier to use.

Adult↗
PubMed1999

Confocal microscopy in the iridocorneal endothelial syndrome.

AIMS: To report the appearances of iridocorneal endothelial (ICE) syndrome from real time, white light confocal microscopy. METHODS: Three consecutive patients, each with ICE syndrome, were examined prospectively. Corneal specular and confocal microscopic examinations were performed in all three patients. In the first patient, a penetrating keratoplasty was performed and the cornea was examined by light and scanning electron microscopy. No surgery was performed in the remaining two patients. RESULTS: In the first patient corneal oedema prevented endothelial specular microscopy. Confocal microscopy performed before penetrating keratoplasty successfully revealed abnormal epithelial-like endothelial cells. Histological examinations of the cornea following penetrating keratoplasty revealed the presence of multilayered endothelial cells with epithelial features (microvilli). In the remaining two patients, specular microscopy showed the presence of ICE cells with typical dark/light reversal. Confocal microscopy demonstrated groups of endothelial cells with epitheloid appearances. In all three patients, the contralateral endothelial appearance was normal by specular and confocal microscopy, except for moderate endothelial polymegathism in one patient. Epithelial-like endothelial cells were characterised by prominent nuclei on confocal microscopy. CONCLUSIONS: The application of confocal microscopy indicates that the ICE syndrome is characterised by epitheloid changes in the endothelium. Confocal microscopy may be used to diagnose the ICE syndrome by demonstrating epithelial-like endothelial cells with hyperreflective nuclei. This technique is especially of value in cases of corneal oedema, since specular microscopy may fail to image the endothelium in such cases.

Aged↗
PubMed1999

Confocal microscopy in posterior polymorphous corneal dystrophy.

PURPOSE: To report the distinguishing characteristics of posterior polymorphous corneal dystrophy (PPMD) using confocal microscopy. MATERIAL AND METHODS: Two consecutive patients with PPMD were prospectively examined using a white-light tandem scanning confocal microscope with a 24x/0.60 contact objective. RESULTS: At the level of Descement's membrane, roundish hyporeflective images were found in 1 patient. In the other patient, hyporeflective bands were detected. In both patients, patchy hyperreflective areas were identified. CONCLUSION: Confocal microscopy may allow diagnosis of PPMD by demonstrating the alterations in Descement's membrane. This technique is especially valuable in cases of endothelial decompensation, where slit-lamp and specular microscopy may fail to demonstrate changes in Descement's membrane.

Aged↗
PubMed1999

Differential diagnosis of linear corneal images on confocal microscopy.

PURPOSE: This study aimed to detect corneal conditions presenting with linear images on white light confocal microscopy and to analyze their distinguishing characteristics. METHODS: In 1996 and 1997, 153 eyes of 110 patients with various corneal conditions were examined. In vivo examination of the cornea was performed by using a white-light tandem scanning confocal microscope. Images were captured by using a video camera and stored on S-VHS video tapes. In this retrospective study, patient charts and confocal microscopic video records were reviewed. Conditions with linear images were looked for, and the images were analyzed and compared. RESULTS: The only structures presenting as linear images on confocal microscopy in normal subjects consisted of corneal nerves. The following pathologic conditions also had linear images on confocal microscopy: corneal vascularization, mycotic keratitis, lattice corneal dystrophy, and posterior polymorphous dystrophy. Each condition could be identified based on its reflectivity, delineation, size, branching pattern, and location in the cornea. CONCLUSION: Different corneal conditions present with linear images on confocal microscopy. Correct identification is critical to avoid misdiagnosis.

Cornea↗