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M R Hernandez

Publications and source records attributed to M R Hernandez.

At least 37 records · Page 2Linked to original sources
PubMed1999

Differential expression of neural cell adhesion molecule isoforms in normal and glaucomatous human optic nerve heads.

Type 1B astrocytes of the human optic nerve head (ONH) constitutively express neural cell adhesion molecule (NCAM) in vivo and in vitro. Increased synthesis of NCAM has been detected in reactive astrocytes in the glaucomatous ONH of human donor eyes. Several NCAM isoforms are generated through alternate RNA splicing in tissue- and disease-specific patterns. In this study, we analyzed expression of NCAM isoforms in ONH of normal donors at different ages and in glaucoma. Total RNA was extracted from ONH of fetal, normal adult and glaucomatous eyes, and cultured human ONH astrocytes, fetal brain astrocytes and an astrocytoma cell line, for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. To distinguish between NCAM 180 and 140 isoforms, exon-specific primer sets covering exons 13-19 were used. Isoform-specific riboprobes were used for in situ hybridization (ISH) in glaucomatous and in age-matched ONH. By RT-PCR, NCAM 140 was the predominant isoform in adult ONH as well as in all cultured cells. NCAM 180 mRNA was strongly expressed in glaucoma, whereas in normal adult tissues it was not detectable. ISH confirmed expression of NCAM in normal adult ONH and localized NCAM 140 mRNA to astrocytes. ISH demonstrated expression of NCAM 180 mRNA in reactive astrocytes in glaucomatous ONH. Our results demonstrate that the NCAM 180 isoform is induced in glaucoma. NCAM 180 may play a role in astrocyte interaction with extracellular matrix (ECM), vessels, axons and other astrocytes and, through its expanded cytoplasmic domain, serve as a signaling molecule for reactive astrocytes during remodeling of the ONH in glaucoma.

Adolescent↗
PubMed1999

Assessment of potential thrombogenicity of coagulation factor IX concentrates in an in vitro model of human thrombogenesis.

We have investigated the potential use of perfusion techniques in the evaluation of the thrombogenic profile of factor IX concentrates. Blood from healthy donors was anticoagulated with low molecular weight heparin and incubated with one of the following: (a) diluent (DIL); (b) a prothrombin complex concentrate (PCC); (c) an intermediate-purity concentrate (FIX/X); or (d) a high-purity concentrate (HPFIX). The thrombogenic potential was assessed as: (1) fibrin formation on subendothelium (Baumgartner's perfusion) and (2) prothrombin activation fragment 1+2 (F1+2, nM) determination. The percentage of fibrin deposition on the subendothelium was only significantly increased after incubation with PCC (62.0+/-3.6% vs. DIL 35.0+/-6.1%;p<0.05). None of the FIX concentrates modified platelet interaction versus control blood (DIL: 26.7+/-2.1%). F1+2 baseline values in anticoagulated blood were 0.6+/-0.1 nM. Preperfusion levels of F1+2 reached values of 4.4+/-0.1 nM for PCC and 5.4+/-0.1 nM for FIX/X. After perfusion, F1+2 values were 2.7+/-0.2 nM for DIL, 5.6+/-0.1 nM for PCC and FIX/X, and 3.3+/-0.2 nM for HPFIX. While measurement of F1+2 was influenced by residual contaminants present in the concentrates, the morphometric evaluation of fibrin deposition on perfused vascular surfaces could be more closely related to the net thrombogenic profile of each FIX preparation.

Animals↗
PubMed1999

Enhanced tenascin expression associated with reactive astrocytes in human optic nerve heads with primary open angle glaucoma.

Tenascin is a large extracellular matrix glycoprotein expressed in neural and non-neural tissues. In the central nervous system, tenascin is synthesized by astrocytes during development and wound healing, forming barriers and affecting neurite outgrowth. In this study we examined tenascin expression in optic nerve heads of normal and glaucomatous eyes and found that there is upregulation of tenascin mRNA and protein in reactive astrocytes from human glaucomatous optic nerve heads compared to normal age-matched controls. In the prelaminar region there was a band of tenascin immunoreactivity around the blood vessels of glaucomatous, but not in normal eyes. However, tenascin mRNA was only localized to astrocytes, suggesting that astrocytes are the cellular source of tenascin. In the lamina cribrosa, tenascin immunoreactivity and gene expression were localized to astrocytes in the cribriform plates and inside the nerve bundles. In the post-lamina region, tenascin immunoreactivity and gene expression were localized to astrocytes lining the pial septum immediately adjacent to the lamina cribrosa. In normal optic nerve heads, tenascin expression at the mRNA and protein levels was confined to clusters of astrocytes at the level of Bruch's membrane in the prelaminar optic nerve head. In glaucoma, enhanced expression of tenascin may be protective to the axons of the retinal ganglion cells by providing a barrier for humoral and/or blood-borne factors that may cause further neural damage. However, the precise role of tenascin in glaucomatous optic neuropathy is not yet elucidated.

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PubMed1999

Transforming growth factor beta isoforms in human optic nerve heads.

AIM: To determine if the isoforms of transforming growth factor beta (TGF-beta) are present in fetal, normal adult, and glaucomatous optic nerve heads. METHODS: To localise cells synthesising TGF-beta, optic nerve heads were stained using antibodies to TGF-beta 1, TGF-beta 2, and TGF-beta 3. To demonstrate synthesis, human optic nerve heads from fetal, glaucomatous, and normal age matched subjects were explanted, cultured overnight, and the culture supernatant was assayed for the presence of TGF-beta 1 and TGF-beta 2 by bioassay. In addition, semiquantitative RT-PCR was performed to determine the gene expression pattern of TGF-beta 2. RESULTS: Immunohistochemistry of glaucomatous samples revealed the presence of intense staining for TGF-beta 2 primarily in astrocytes, whereas TGF-beta 1 was localised to blood vessels. No TGF-beta 3 immunoreactivity was observed. There was little or no expression of TGF-beta in normal optic nerve heads. Optic nerve heads from glaucomatous eyes released 70-100-fold more TGF-beta 2 than normal age matched optic nerve heads. Fetal optic nerve heads released 90-100-fold more TGF-beta 2 than normal adult optic nerve heads. TGF-beta 1 was undetectable by bioassay in all samples tested. There was no apparent increase in TGF-beta 2 gene expression in glaucomatous and fetal eyes, suggesting post-transcriptional regulatory mechanisms. CONCLUSIONS: These results demonstrate that TGF-beta 2 is produced in high levels in the fetal and glaucomatous optic nerve heads, perhaps by a mechanism of post-transcriptional regulation. TGF-beta may be important during development of the optic nerve head and, in glaucoma, TGF-beta 2 may be a mediator of astrocyte reactivation and extracellular matrix remodelling in the lamina cribrosa.

Aged↗
PubMed1998

Elastosis of the lamina cribrosa in glaucomatous optic neuropathy.

The purpose of this study was to determine whether elastotic degeneration of the elastin component of the lamina cribrosa occurs in optic neuropathy associated with different types of glaucoma. Human optic nerve heads with primary open-angle, neovascular, chronic angle closure and pseudoexfoliation glaucoma, and with varying duration of disease were compared with age-matched normal eyes, using electron microscopy and immunogold labeling of elastin. The percent area occupied by immunogold-labeled elastin material was determined using a digital image analysis system. In all eyes with a history of glaucoma, elastosis was found in the lamina cribrosa and there was a significantly greater percentage of area occupied by elastin compared with age-matched control eyes (P<0.0001). Among the glaucomatous eyes, pseudoexfoliation glaucoma had the largest area of elastosis, followed by primary open-angle and secondary glaucoma (neovascular and chronic angle closure). In all glaucoma samples, large, confluent elastin aggregates of irregular and varied shapes (elastosis) were observed in the lamina cribrosa and insertion region. These results demonstrate that glaucomatous optic neuropathy is associated with elastosis of the lamina cribrosa, which may contribute to the changes in compliance of the optic nerve heads of glaucomatous eyes.

Aged↗
PubMed1997

Nitric oxide synthase in the human glaucomatous optic nerve head.

OBJECTIVES: To investigate the hypothesis that nitric oxide contributes to neurotoxicity in the optic nerve heads of patients with primary open-angle glaucoma, we have determined the presence of the 3 isoforms of nitric oxide synthase (NOS) in the tissue. METHODS: Histological sections of optic nerve heads from normal subjects and patients with glaucoma who have moderate to advanced nerve damage were studied by immunohistochemistry. Polyclonal antibodies to NOS-1, NOS-2, and NOS-3 were localized with immunoperoxidase staining. RESULTS: In normal eyes, NOS-1 is sparsely present in astrocytes throughout the optic nerve head. In glaucomatous optic nerve heads, almost every astrocyte is positive for NOS-1. Nitric oxide synthase-1 immunoreactivity is abundantly present throughout the prelaminar region and the lamina cribrosa and is localized inside the diminished nerve fiber bundles. Nitric oxide synthase-2 is present in a few cells in the disorganized lamina cribrosa of the glaucomatous eye and is not present at all in normal tissue. Nitric oxide synthase-3 is present in normal eyes in the vascular endothelia of small blood vessels of the prelaminar region. In glaucomatous tissue, NOS-3 is present in astrocytes and in the vascular endothelia of large and small vessels. CONCLUSIONS: The 3 isoforms of NOS are present in apparently increased amounts in the optic nerve head of patients with primary open-angle glaucoma. The increased presence of NOS-1 and the induction of NOS-2 in astrocytes of the lamina cribrosa suggest that the glaucomatous optic nerve head is exposed to excessive levels of nitric oxide, which may be neurodestructive, locally, to the axons of the retinal ganglion cells. Conversely, the increased presence of NOS-3 in vascular endothelia may be neuroprotective by causing vasodilation and increased blood flow in the tissue.

Aged↗
PubMed1997

Expression of neural cell adhesion molecule (NCAM) characterizes a subpopulation of type 1 astrocytes in human optic nerve head.

The human optic nerve contains a heterogeneous population of astrocytes. In situ, a specialized subpopulation of astrocytes was distinguished in the adult optic nerve head by expression of neural cell adhesion molecule (NCAM). To further study the biology of astrocytes, we have developed and characterized cells grown from explanted optic nerve heads and myelinated optic nerves as in vitro model systems. Second or third passage cells were processed for immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP) and cell surface epitopes: CD56/NCAM, HNK-1/NCAM, A2B5, and O4. Synthesis and gene expression of NCAM were characterized by Western blot analysis and RNase protection assay. Cells grown from myelinated optic nerves expressing GFAP, but not NCAM or A2B5, were identified as type 1A astrocytes, and cells expressing GFAP and A2B5, but not NCAM, were identified as type 2 astrocytes. Cells grown from explanted optic nerve head expressing GFAP, NCAM, and O4 were identified as type 1B astrocytes. Expression of NCAM by type 1B astrocytes may provide these cells with adhesion properties that allow them specialized responses in their microenvironment. Astrocytes from the lamina cribrosa may form a functional barrier to prevent myelination of the retina. In glaucoma, these astrocytes may be exposed to stresses due to fluctuation in intraocular pressure and therefore participate in the optic nerve changes associated with glaucomatous optic neuropathy.

Adult↗
PubMed1997

Hyaluronic acid in the normal and glaucomatous optic nerve.

Eighteen normal human eye-bank eyes (age: 18-81 years), five fetal eyes (16-24 weeks), 11 primary open-angle glaucoma (POAG) eyes (age: 76-89 years), and two Schnabel's cavernous optic atrophy eyes were examined using a biotinylated-hyaluronan binding protein to study the changes in the distribution of hyaluronic acid (HA) in the fetal, adult and glaucomatous optic nerve head. The vitreous body served as a positive control. Sections treated with Streptomyces hyaluronidase were used to confirm specificity. Monoclonal antibodies to myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) were used as additional controls. In fetal optic nerve, HA was localized in blood vessels, peripapillary sclera and the pial septae in the retrolaminar nerve. No staining was associated with axons. Staining for MBP was negative. In adults, HA was found surrounding the myelin sheaths in the retrolaminar nerve; staining decreased with age. In contrast, HA staining in myelinated peripheral nerves (e.g. ciliaries) remained unchanged with age. HA also was localized to the adventitia of arteries and veins throughout the posterior segment. Compared to age-matched normal eyes, HA staining was virtually absent around myelin sheaths of the retrolaminar nerve in POAG eyes. Similar changes were not found in other HA positive structures. In Schnabel's cavernous optic atrophy. HA was present in increased amount in the atrophic area, but virtually absent in the remaining retrolaminar nerve. HA staining was invariably positive in vitreous, and Streptomyces hyaluronidase treated sections were negative. In adults, staining of MBP was associated with the myelin sheath in the retrolaminar nerve. In contrast to HA, staining of MBP was unchanged with age and in POAG. In Schnabel's atrophy, MBP staining disappeared only in the atrophic area. HA in the retrolaminar optic nerve appears to be associate with the space-filling matrix between myelin sheaths. HA is not present in the axon bundles prior to myelination of the optic nerve. HA in the retrolaminar optic nerve appears to decrease with age and is further reduced in POAG; however, corresponding changes are not found in MBP or in peripheral nerves. Perhaps, decreased amounts of HA is related to a higher susceptibility to elevated intraocular pressure or to optic nerve atrophy. In Schnabel's cavernous optic atrophy, HA is present in increased amount only in the atrophic area while MBP is markedly decreased, suggesting in situ production of HA in areas of optic nerve atrophy.

Adolescent↗
PubMed1997

Cyclooxygenase-1 and cyclooxygenase-2 in the human optic nerve head.

To investigate the hypothesis that eicosanoids act as cellular mediators in the optic nerve head of normals and of patients with glaucoma, we have determined the presence of the two cyclooxygenase (COX) isoforms in human tissue. Histological sections of optic nerve heads were studied by immunohistochemistry. Age matched normal donors were compared with eyes from glaucoma patients with moderate to severe nerve damage. Polyclonal antibodies to human COX-1 and COX-2 were localized with immunoperoxidase staining. Specific antibodies for vascular endothelia and microglia were also colocalized. In normal and glaucomatous eyes. COX-1 was localized exclusively to the prelaminar and lamina cribrosa regions of the optic nerve head. No staining for COX-1 was observed in the nerve fiber layer or the myelinated optic nerve. COX-1 was associated with the astrocytes of the glial columns and the cribriform plates, but not with the endothelia lining the capillaries. In glaucoma, more astrocytes appeared to be stained with antibody to COX-1 than in normals and staining was intensely perinuclear. There was no staining for COX-2 in normal tissue. A few COX-2 positive cells were found in the prelaminar, lamina cribrosa and postlaminar regions of the glaucomatous optic nerves. Positive staining for COX-2 was not associated with microglia. COX-1 is constitutively present in astrocytes that are localized exclusively to the prelaminar and lamina cribrosa regions of the human optic nerve head. Eicosanoids, synthesized by COX-1 in this tissue, may have a homeostatic and a neuroprotective role related to the axons of the retinal ganglion cells. The sparse presence of COX-2 in glaucomatous tissue probably reflects the lack of inflammation associated with glaucomatous optic neuropathy.

Aged↗
PubMed1997

In vitro evaluation of the hemostatic effectiveness of non viable platelet preparations: studies with frozen-thawed, sonicated or lyophilized platelets.

OBJECTIVES: Because of the limited life span of platelets in regular storage systems, we were interested in investigating the effects on hemostasis of nonliving platelet derivatives. METHODS: We evaluated the effects of different platelet preparations on primary hemostasis in a well-established perfusion model. Studies were carried out with blood anticoagulated with low molecular weight heparin. Similar amounts of frozen-thawed, sonicated or lyophilized platelets were added to normal blood or to blood which had been experimentally depleted of platelets. Platelet interaction with the subendothelium and fibrin deposition were morphometrically evaluated. RESULTS: Addition of nonviable platelet preparations to thrombocytopenic blood always promoted a statistically significant increase in the deposition of fibrin on the subendothelium, but only lyophilized platelets retained some ability to interact with the subendothelium. Flow cytometry studies demonstrated the presence of GPIb, GPIIIa and P-selectin on lyophilized platelets. CONCLUSIONS: Preparations containing nonviable platelets may still retain some hemostatic properties.

Animals↗
PubMed1997

Astrocyte responses in human optic nerve head with primary open-angle glaucoma.

PURPOSE: To identify and characterize astrocyte responses and reactivation in human optic nerve heads from patients with primary open-angle glaucoma. METHODS: Fifteen optic nerve heads with primary open-angle glaucoma and 13 normal controls were fixed in 4% paraformaldehyde, paraffin embedded, and stained for immunofluorescence and immunoperoxidase. The antibodies used were against glial fibrillary acidic protein (GFAP) and against neural cell adhesion molecule (N-CAM). RESULTS: Two subpopulations of type 1 astrocytes exist in the normal optic nerve. Type 1A astrocytes express only glial fibrillary acidic protein and type 1B express both glial fibrillary acidic protein and neural cell adhesion molecule. These are the major cell subpopulations in the lamina cribrosa and prelaminar regions. In primary open angle glaucoma, type 1B astrocytes in the prelaminar region showed increased immunoreactivity for glial fibrillary acidic protein and neural cell adhesion molecule, and cytoplasmic enlargement with thicker and longer cytoplasmic processes. At the level of the lamina cribrosa, type 1B astrocytes appeared round and the cell bodies were no longer in the cribriform plates but located in the nerve bundles. Type 1A astrocytes were not observed in the glaucomatous optic nerve head. CONCLUSIONS: Astrocyte responses in primary open angle glaucoma may underlie cellular changes that lead to axonal damage and optic nerve head remodeling. These responses may have pathogenic significance for glaucomatous optic neuropathy.

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PubMed1996

Tropoelastin gene expression in optic nerve heads of normal and glaucomatous subjects.

Elastic fibers are a major component of the extracellular matrix in the optic nerve head (ONH) and undergo marked morphological changes during primary open angle glaucoma (POAG). Previous findings indicated that there is reactivation of tropoelastin mRNA synthesis in glaucoma. In this study, we sought to determine the alternative splicing pattern of tropoelastin in the human optic nerve head and in cultured laminar astrocytes. Furthermore, we compared the alternative splicing pattern of normal elastogenesis with that of reactivation of elastin synthesis in patients with primary open angle glaucoma. Our results demonstrate that exons 23 and 32 of tropoelastin are alternatively spliced in the normal ONH as well as in tissue from glaucomatous patients. There are no qualitative differences. We also demonstrated that astrocytes from the ONH synthesize tropoelastin in vitro. In conclusion, we have demonstrated a tropoelastin alternative splicing pattern in the human optic nerve head and laminar astrocytes. Abnormalities in elastic fibers in the ONH of patients with POAG are not due to an aberrant splice variant of tropoelastin. Astrocytes grown from ONH explants may serve as an in vitro model to study extracellular matrix changes in glaucoma.

Adult↗
PubMed1995

Heterogeneity of astrocytes in human optic nerve head.

Previous studies demonstrated regional differences in the synthesis of extracellular matrix by astrocytes during optic nerve head (ONH) maturation and in glaucomatous optic neuropathy, suggesting heterogeneity of astrocytes. To characterize different types of glial cells in human fetal and adult ONH, we used a variety of neural cell markers such as HNK-1/N-CAM, A2B5, galactocerebroside (GalC), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cryostat or paraffin sections were prepared from fetal (16-25 weeks) and mature (8 months to 75 years old) ONH and processed for standard single/double immunocytochemistry. Two subpopulations of type 7 astrocytes were present in the mature prelaminar and laminar regions. Glial cells expressing only GFAP were identified as type 1A astrocytes at the edges of the cribriform plates. Cells forming the glial columns and lining the cribriform plates expressed both GFAP and HNK-1/N-CAM and were identified as type 1B astrocytes. In the myelinated nerve, type 1A astrocytes form the glial limiting membrane. Cells labeled with GFAP and A2B5 were identified as type 2 astrocytes, and GFAP-negative cells labeled with GalC, MBP, and HNK-1/N-CAM were identified as oligodendrocytes. In fetal ONH, all glial cells expressed HNK-1/N-CAM. In older fetal ONH, some glial cells also expressed GFAP. No type 2 astrocytes or oligodendrocytes were present in the fetal ONH. In conclusion, at least two subpopulations of type 1 astrocytes exist in human ONH: Type 1A astrocytes may serve as structural support for axons; type 1B astrocytes, which retain the developmental neural marker HNK-1/N-CAM, may have a more complex function by interfacing between blood vessels and other connective tissue surfaces. These findings demonstrate the heterogeneity of astrocytes in the human ONH and suggest differential regional responses to changes in their microenvironment.

Adult↗
PubMed1995

Elastosis of the lamina cribrosa in pseudoexfoliation syndrome with glaucoma.

BACKGROUND: Pseudoexfoliation syndrome is characterized by the presence of glycoprotein fibers in ocular and extraocular tissues, and often is associated with glaucoma. Pseudoexfoliation material may be associated closely with elastic microfibrillar-associated glycoprotein as well as elastin. METHODS: Four optic nerve heads of two patients with pseudoexfoliation syndrome and glaucoma were examined using electron microscopy and immunogold detection of elastin. Optic nerve heads from healthy age-matched individuals and patients with primary open-angle glaucoma were used for comparisons. RESULTS: In all eyes with pseudoexfoliation and glaucoma, there was marked and widespread elastosis in the connective tissue of the lamina cribrosa. Elastotic fibers appeared as large and irregular aggregates of electron-dense material labeled with anti-elastin antibody. Abundant microfibrils were interspersed in the elastotic aggregates, whereas no typical pseudoexfoliation fibers were observed. In contrast, there were less elastotic fibers in the lamina cribrosa from patients with primary open-angle glaucoma compared with pseudoexfoliation glaucoma. Other changes of extracellular matrix were similar to those observed in primary open-angle glaucoma: decreases in collagen fiber density, presence of basement membranes not associated with cell surfaces, and abundant bundles of microfibrils not labeled with elastin antibody. The elastic fibers appeared normal in other locations within the optic nerves of patients with pseudoexfoliation glaucoma, including in the pial septa and blood vessels of the retrolaminar myelinated optic nerve. CONCLUSION: The authors' findings demonstrate marked and site-specific elastosis in the lamina cribrosa of patients with pseudoexfoliation syndrome with glaucoma, suggesting an abnormal regulation of elastin synthesis and/or degradation in the optic nerve of patients with this disease.

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PubMed1995

ONTRAK TESTCUP: a novel, on-site, multi-analyte screen for the detection of abused drugs.

We developed a rapid, sensitive, and simple-to-use multi-analyte diagnostic device for the detection of drugs of abuse in urine: the ONTRAK TESTCUP. No sample or reagent handling is necessary with this device, and the device also serves as the sample collection cup. The TESTCUP contains immunochromatographic reagents that qualitatively and simultaneously detect the presence of benzoylecgonine, morphine, and cannabinoids (delta9-tetrahydrocannabinol [THC] in urine. It is based on the principle of competition between the drug in the sample and membrane- immobilized drug conjugate for antidrug antibodies coated on blue-dyed microparticles. Each drug assay has its own strip, which contains an antibody specific to benzoylecgonine, morphine, or THC. A sample is collected in the TESTCUP, a lid is placed on it, and a chamber at the top of the cup is filled with urine by inverting the cup for 5 s. Urine proceeds down immunochromatographic strips, and the assays are developed. In approximately 3-5 min, the Test Valid bars appear, a decal is removed from the detection window, and the results are interpreted. The appearance of a colored bar at the detection window for each drug indicates a negative result. The absence of color in any specific drug detection window indicates a positive result for that drug. If a positive result is obtained, the same device (cup) can be used for gas chromatographic-mass spectrometric (GC-MS) confirmation. When the precision of the TESTCUP was evaluated, the results obtained were as follows: for urine controls containing drug at 50% of its cutoff concentration, the results were greater than or equal to 96, 98, and 96% negative for benzoylecgonine, morphine, and THC, respectively; for urine controls containing drug at 120% of its cutoff concentration, the results were greater than or equal to 97, 100, and 98% positive for benzoylecgonine, morphine, and THC, respectively. The correlations of clinical sample results using the TESTCUP versus results by GC-MS and the ONTRAK and OnLine assays were assessed. There was 100% agreement between samples prescreened positive by GC-MS and positive by TESTCUP for all three assays. There was 100% agreement between TESTCUP and ONTRAK results and between TESTCUP and OnLine results when testing clinical samples positive and negative for cocaine (benzoylecgonine) or THC. Greater than 99% agreement was observed between TESTCUP and ONTRAK results and between TESTCUP and OnLine results when testing clinical samples positive and negative for morphine. The cross-reactivity of the TESTCUP assay to related drugs and drug metabolites was also determined, and the results were similar to those of the ONTRAK and OnLine assays.

Cocaine↗
PubMed1995

Expression of muscarinic receptor subtype mRNA in the human ciliary muscle.

PURPOSE: To investigate the expression of the muscarinic receptor (mAChR) subtypes (m1, m2, m3, m4, and m5) in the human ciliary muscle. METHODS: cDNA probes specific for the m1 to m5 subtypes were used to characterize the expression of subtype-specific mRNAs by in situ hybridization and Northern blot analysis on a cell line derived from human ciliary muscle tissue (H7CM), sectioned tissue from four donors, and fresh human ciliary muscle tissue from three donors. RESULTS: Messenger RNA for m2, m3, and m5 was detected in situ hybridization and Northern blot analysis in the H7CM cell line and in human ciliary muscle tissue from the donors. In human ciliary muscle tissues, the expression of mRNA of the m2 and m3 subtypes was more prominent in the circular portion than in the longitudinal portion. In contrast, mRNA for the m5 subtype was greater in the longitudinal portion than in the circular portion. Additionally, Northern blot analysis showed that m1 and m4 were expressed in ciliary muscle tissue. CONCLUSION: These studies show that the human ciliary muscle definitely expresses the mRNA of subtypes m2, m3, and m5, and may also express the mRNA of m1 and m4. Although the expression of mRNA for the m2, m3, and m5 subtypes differed between the circular and longitudinal portions of the ciliary muscle, the strong expression of all three subtypes of muscarinic receptors by both portions demonstrates that the differential distribution of muscarinic subtypes probably is not responsible for the dissociation between outflow facility and accommodation that can occur under certain conditions.

Adult↗
PubMed1994

Collagen type IV gene expression in human optic nerve heads with primary open angle glaucoma.

Previous studies have demonstrated proliferation of basement membranes in the optic nerve head in primary open angle glaucoma (POAG). We used in situ hybridization (ISH) of a radiolabeled riboprobe specific for human collagen IV, a ubiquitous component of basement membranes, to identify cells actively synthesizing basement membranes in the optic nerve head in POAG. In addition, to detect and further characterize the collagen IV mRNA transcripts, we used reverse transcriptase-polymerase chain reaction (RT-PCR) in total RNA extracted from individual optic nerve heads with POAG and from age-matched normal controls. ISH results demonstrate that, in POAG, numerous astrocytes in the prelaminar region expressed collagen IV mRNA. Lamina cribrosa cells and astrocytes in the compressed lamina cribrosa hybridized the probe. Few astrocytes and lamina cribrosa cells hybridized the probe in the optic nerve head of normal age-matched controls. RT-PCR products for collagen IV and for glyceraldehyde-3-dehydrogenase (G3PDH), a reference gene, were detected by agarose electrophoresis as single bands of the expected sizes and positively identified by Southern hybridization using specific cDNA probes in normal and POAG samples. No additional products (bands) were observed in RT-PCR experiments, indicating that there was no genomic DNA contamination in the total RNA extract. The lack of additional bands suggests that, at least in the ten samples used in this study, there were no alternatively spliced RNA products in any of the amplified sequences. Semi-quantitative analyses using densitometry showed a two-fold increase in collagen type IV PCR present in POAG samples. No differences were detected in levels of G3PDH PCR products between POAG and normal samples. This investigation provides evidence of increased biosynthesis of collagen type IV at the mRNA level in optic nerve heads with POAG. Whether this phenomenon represents a response to elevated intraocular pressure or a reparative mechanism to the loss of axons remains to be determined.

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