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D Hicks

Publications and source records attributed to D Hicks.

At least 37 records · Page 2Linked to original sources
PubMed2000

Selective transplantation of rods delays cone loss in a retinitis pigmentosa model.

BACKGROUND: Rod-cone retinal degenerations (retinitis pigmentosa) are typified by initial rod loss followed by secondary cone death. Rod death, predominantly caused by gene mutations expressed specifically in these cells, induces scotopic vision loss. Cone death, the overriding cause of blindness, has no current explanation. Disease progression and preliminary data suggest that cone survival depends on rods. OBJECTIVE: To establish whether rod transplantation into mutant rodless retinas could halt cone loss. METHODS: We transplanted pure sheets of rods isolated from normal-sighted mice into the subretinal space of recipient retinal degeneration mice lacking rods but possessing approximately 30% residual cones. Control animals were unoperated on or grafted with inner retinal cells from young normal donors, entire retinas from aged retinal degeneration mice, or gelatin. Two weeks after surgery, we quantified by an unbiased method the numbers of host retinal cones after immunolabeling with specific markers. RESULTS: Only mice receiving rod-rich transplants demonstrated statistically significant greater cone numbers, with rescue of 40% of host cones normally destined to die during this period. CONCLUSION: Cone survival depends specifically on rods. CLINICAL RELEVANCE: Such findings indicate that transplantation of rods could limit loss of cones, thus preserving useful vision in human retinitis pigmentosa. Arch Ophthalmol. 2000;118:807-811

Animals↗
PubMed2000

Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium.

The discovery of disc protein renewal in rod outer segments, in 1960s, was followed by the observation that old discs were ingested by the retinal pigment epithelium. This process occurs in both rods and cones and is crucial for their survival. Photoreceptors completely degenerate in the Royal College of Surgeons mutant rat, whose pigment epithelium cannot ingest old discs. The complete renewal process includes the following sequential steps involving both photoreceptor and pigment epithelium activity: new disc assembly and old disc shedding by photoreceptor cells; recognition and binding to pigment epithelium membranes; then ingestion, digestion, and segregation of residual bodies in pigment epithelium cytoplasm. Regulating factors are involved at each step. While disc assembly is mostly genetically controlled, disc shedding and the subsequent pigment epithelium phagocytosis appear regulated by environmental factors (light and temperature). Disc shedding is rhythmically controlled by an eye intrinsic circadian oscillator using endogenous dopamine and melatonin as light and dark signal, respectively. Of special interest is the regulation of phagocytosis by multiple receptors, including specific phagocytosis receptors and receptors for neuroactive substances released from the neuroretina. The candidates for phagocytosis receptors are presented, but it is acknowledged that they are not completely known. The main neuromodulators are adenosine, dopamine, glutamate, serotonin, and melatonin. Although the transduction mechanisms are not fully understood, attention was brought to cyclic AMP, phosphoinositides, and calcium. The chapter points to the multiplicity of regulating factors and the complexity of their intermingling modes of action. Promising areas for future research still exist in this field.

Animals↗
PubMed2000

Therapeutic monitoring of sirolimus in human whole-blood samples by high-performance liquid chromatography.

BACKGROUND: Sirolimus is a macrolide antibiotic isolated from Streptomyces hygroscopicus that has demonstrated immunosuppressive activity. Human and animal studies have shown a good correlation of trough sirolimus concentrations with immunosuppressive efficacy. OBJECTIVE: This report describes a reverse-phase high-performance liquid chromatography (HPLC) method used for therapeutic drug monitoring of sirolimus. METHODS: A reverse-phase C18 column method was developed using an automated HPLC system and ultraviolet (UV) detection. Whole-blood samples collected in ethylenediamine-tetraacetic acid (EDTA) are first hemolyzed, and an internal standard (desmethoxysirolimus) is added to 1.0 mL of sample, which is then extracted with 1-chlorobutane and, after the organic layer is removed, evaporated to dryness. The residue is reconstituted in a 70% methanol/water mixture. Reconstituted extracts are analyzed by HPLC at a column temperature of 60 degrees C and a flow rate of 1.0 mL/min. Typically, chromatography requires 35 minutes between each sample injection. The UV detector is set at 278 nm with a response sensitivity of 0.010 AUFS (absorbance units full scale). Standards and controls prepared in hemolyzed EDTA-anticoagulated whole blood are extracted and run in parallel. Identification of peaks of interest is by retention time; quantification of sirolimus in controls and clinical samples uses a peak-height ratio (sirolimus/internal standard). RESULTS: The assay's precision (coefficients of variation, 5.7%-14.4%) and sensitivity (2.5 ng/mL) were found to be appropriate for therapeutic monitoring purposes. Analytical recovery of 88.0% to 106.3% was observed throughout the assay's linear range (2.5-150.0 ng/mL). Stability studies at 20 degrees C to 25 degrees C and 2 degrees C to 8 degrees C showed an estimated recovery of sirolimus ranging from 85% to 110% of target concentrations (10-90 ng/mL). In a study comparing the results of 194 samples from kidney transplant recipients assayed by the HPLC-UV assay and by a microparticle enzyme immunoassay, the HPLC-UV method provided approximately 10% lower values. CONCLUSION: The HPLC-UV assay is analytically capable of providing useful data for the clinical assessment of patients receiving sirolimus.

Biotransformation↗
PubMed2000

An immunoassay for the measurement of sirolimus.

OBJECTIVE: This study assessed the performance characteristics of a new microparticle enzyme immunoassay (MEIA) for the determination of sirolimus in whole blood. BACKGROUND: In clinical investigatory studies, dose adjustments of the immunosuppressive drug sirolimus have been carried out using either high-performance liquid chromatography (HPLC) or, more recently, this investigational immunoassay kit based on the MEIA technique. METHODS: Calibration was made over the linear range 0 to 30 ng/mL. Inaccuracy and imprecision were assessed by means of 3 control samples supplied with the kit (5, 11, and 22 ng/mL) and dilution of an above-quantitation-limit sample (154 ng/mL). Specificity was determined by the addition of 2 sirolimus metabolites to sirolimus-free human whole blood or to I of the control samples supplied with the kit. In addition, whole-blood samples from patients receiving either cyclosporine or tacrolimus (N = 24) were analyzed for sirolimus. A comparison of the MEIA and a validated HPLC/MS/MS assay analyzed both pooled samples from patients receiving sirolimus and spiked samples (sirolimus 2-60 ng/mL). In a more extensive comparison of patient samples measured by the MEIA assay, a validated HPLC assay with UV detection (HPLC-UV) was used (HPLC-UV sirolimus 7-64 ng/mL). RESULTS: Inaccuracy (between-run) was < or =16.2% at all 4 concentrations (N = 5). Within-assay imprecision (repeatability) was <6% (N = 5), and between-assay imprecision (reproducibility) for the same samples was < 11% (N = 5). Recovery, assessed by means of 3 in-house control samples prepared in both fresh and previously frozen sirolimus-free human whole blood, ranged from 93.9% to 109.5%. The limit of detection, determined by dilution of the lowest nonzero calibrator (3 ng/mL), was set at 1 ng/mL, at which repeatability was 20.5% (N = 5). Five ng/mL of hydroxysirolimus cross-reacted with the assay by a mean of between 44% and 50% (N = 4); 5 ng/mL of 41-O-demethylsirolimus cross-reacted with the assay by a mean of between 86% and 127% (N = 4). Assay specificity was further challenged by ethylenediamine-tetraacetic acid (EDTA)-whole-blood samples from transplant patients not receiving sirolimus. These samples had tacrolimus and cyclosporine concentrations of 7.8 to 15.9 ng/mL and 38 to 485 microg/L, respectively. The median result was 0 ng/mL (third quartile, 0.7 ng/mL; maximum, 1.4 ng/mL); no value was above the lowest nonzero calibrator. The results of the comparison between the MEIA and the HPLC/MS/MS assay showed mean positive biases of 21% and 8% for the MEIA in measuring sirolimus in pooled patient samples and spiked samples, respectively. The results of the comparison of the MEIA and HPLC-UV median sirolimus concentrations were 18.2 and 20.1. Whole-blood samples anticoagulated with EDTA and containing sirolimus were stable for analysis by MEIA for 3 freeze-thaw cycles when stored at -20 degrees C and for 10 days when stored at 4 degrees C or at ambient temperature. A decline in sirolimus concentration occurred when samples were stored at 37 degrees C. CONCLUSION: The MEIA showed suitable precision across a clinically relevant concentration range. In terms of patient management, the practical significance of cross-reactivity with sirolimus metabolites remains to be assessed.

Antibody Specificity↗
PubMed2000

Human platelet suspension stimulates porcine retinal glial proliferation and migration in vitro.

PURPOSE: To characterize the cellular and molecular mechanisms underlying the efficacy of autologous platelet suspension adjuvant therapy in the treatment of macular hole. METHODS: Platelet suspensions were: paid from whole blood samples obtained from informed volunteers. For proliferation assays, platelet suspensions or purified growth factors were added to semi-confluent cultures of porcine real glial cells for 24 hours, followed by [3H]thymidine for 15 hours, after which time cells were washed, solubilized, and counted for uptake of radioactive tracer. For cell migration assays, confluent glial cultures were scrape wounded and maintained in the presence or absence of platelet suspension or identified platelet constituents. Cell migration into the denuded area was scored as a function of time. In certain cases, specific pharmacologic inhibitors of growth factor action were added at the same time as platelet adjuvant or growth factors. RESULTS: Platelet suspension adjuvant induced strong mitogenic and chemotactic responses in cultured glia, in a dose-dependent manner. Maximal incorporation of thymidine was two- to threefold that of control levels, with an ED50 approximately 5 x 10(6) platelets/ml, and migration was enhanced up to 80-fold after 48 hours. Platelet suspension-induced proliferation was completely blocked by addition of 25 microM genistein, a tyrosine kinase receptor inhibitor. However, the same concentration only partially blocked the cell migration response. Addition of any single growth factor or protein identified from ELISA analysis, or a combination of all factors, did not significantly stimulate proliferation or cell migration. CONCLUSIONS: Human platelet suspensions exert both proliferative and chemotactic influences on retinal glial cells in vitro, suggesting that the same responses may occur in platelet-induced macular hole repair in humans. Growth factors or proteins that have been identified within the suspensions do not mimic these responses in vitro, implying that additional currently unidentified trophic activities are also present.

Animals↗
PubMed1999

Cell death in age-related macular degeneration.

The cellular and molecular mechanisms underlying the death of photoreceptors and other retinal cells in age-related macular degeneration (AMD) remain poorly understood. Some of the questions for which answers need to be sought, and which are explicitly or implicitly addressed in this article include: (1) how do patterns of cell death in AMD compare, qualitatively and quantitatively, with "normal" cell death in aging retinas, and with cell death in retinitis pigmentosa (RP) and its animal models; (2) is apoptosis involved in AMD; (3) is there any evidence that rods are necessary for cone survival; (4) if the answer is yes, is there evidence that rods produce one or more survival-promoting factor(s) that act directly on cones; (5) are the effects of rods upon cones exclusively mediated by diffusible factors, or do they also involve contact-mediated interactions; (6) is there any evidence that photoreceptors regulate the survival and/or function of RPE and Müller cells, as well as the interactions between these cells and cones; (7) are trophic factors and their receptors in the macula different from those in other parts of the retina; and (8) are toxic mechanisms involved in the onset and progression of cell death in AMD? Clear cut answers to most of these (and related) questions about cell death in AMD are not yet available. The goal of this article is to summarize discussion that should help in the formulation of suitable hypotheses, amenable to experimental analysis. To provide a platform for such discussion, we present an overview of progress made in recent years in the analysis of other retinal degenerations and of neuronal degenerations in other regions of the CNS. We conclude with an overview of concepts and speculation derived from our current research.

Aging↗
PubMed1999

Ability of retinal Müller glial cells to protect neurons against excitotoxicity in vitro depends upon maturation and neuron-glial interactions.

Glutamate is the most abundant excitatory amino acid in the central nervous system. It has also been described as a potent toxin when present in high concentrations because excessive stimulation of its receptors leads to neuronal death. Glial influence on neuronal survival has already been shown in the central nervous system, but the mechanisms underlying glial neuroprotection are only partly known. When cells isolated from newborn rat retina were maintained in culture as enriched neuronal populations, 80% of the cells were destroyed by application of excitotoxic concentrations of glutamate. Massive neuronal death was also observed in newborn retinal cultures containing large numbers of glia, or when neurons were seeded onto feeder layers of purified cells prepared from immature (postnatal 8 day) rat retina. When newborn retinal neurons were seeded onto feeder layers of purified glial cells prepared from adult retinas, application of excitotoxic amino acids no longer led to neuronal death. Furthermore, neuronal death was not observed in mixed neuron/glial cultures prepared from adult retina. However, in all cases (newborn and adult) application of kainate led to amacrine cell-specific death. Activity of glutamine synthetase, a key glial enzyme involved in glutamate detoxification, was assayed in these cultures in the presence or absence of exogenous glutamate. Whereas pure glial cultures alone (from young or adult retina) showed low activity that was not stimulated by glutamate addition, mixed or co-cultured neurons and adult glia exhibited up to threefold higher levels of activity following glutamate treatment. These data indicate that two conditions must be satisfied to observe glial neuroprotection: maturation of glutamine synthetase expression, and neuron-glial signalling through glutamate-elicited responses.

Animals↗
PubMed1999

Glial cell line-derived neurotrophic factor induces histologic and functional protection of rod photoreceptors in the rd/rd mouse.

PURPOSE: To evaluate the neuroprotective potential of glial cell line-derived neurotrophic factor (GDNF) in the retinal degeneration (rd/rd) mouse model of human retinitis pigmentosa. METHODS: Subretinal injections of GDNF were made into rd/rd mice at 13 and 17 days of age and electroretinograms (ERGs) recorded at 22 days. Control mice received saline vehicle injections or underwent no procedure. At 23 days of age, retinas from treated and control mice were fixed and processed for wholemount immunohistochemistry using an anti-rod opsin antibody, and rod numbers were estimated using an unbiased stereological systematic random approach. Subsequent to counting, immunolabeled retinas were re-embedded and sectioned in a transverse plane and the numbers of rods recalculated. RESULTS: Although ERGs could not be recorded from sham-operation or nonsurgical rd/rd mice at 22 days of age, detectable responses (both a- and b-waves) were observed in 4 of 10 GDNF-treated mice. Stereological assessment of immunolabeled rods at 23 days showed that control rd/rd retinas contained 41,880+/-3,890 (mean +/- SEM; n = 6), phosphate-buffered saline (PBS)-injected retinas contained 61,165+/-4,932 (n = 10; P < 0.001 versus control retinas) and GDNF-injected retinas contained 89,232+/-8,033 (n = 10; P < 0.001 versus control retinas, P < 0.002 versus PBS). This increase in rod numbers after GDNF treatment was confirmed by cell counts obtained from frozen sections. CONCLUSIONS: GDNF exerts both histologic and functional neuroprotective effects on rod photoreceptors in the rd/rd mouse. Thus rescue was demonstrated in an animal model of inherited retinal degeneration in which the gene defect was located within the rods themselves, similar to most forms of human retinitis pigmentosa. GDNF represents a candidate neurotrophic factor for palliating some forms of hereditary human blindness.

Animals↗
PubMed1998

Survival of purified rat photoreceptors in vitro is stimulated directly by fibroblast growth factor-2.

Basic fibroblast growth factor (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal growth factor (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

Animals↗
PubMed1998

GABAA and GABAC receptors in adult porcine cones: evidence from a photoreceptor-glia co-culture model.

1. Edge contrast enhancement is an integrated visual function based on the complex centre-surround organization of the cone photoreceptor light response. While centre responses result from direct light activation, surround responses are thought to result from lateral inhibition mediated by horizontal cells. This feedback signal has been attributed to GABAA receptors which have been found in lower vertebrate cones. 2. In order to study the GABA response of adult mammalian photoreceptors, we designed a culture system consisting of isolated photoreceptors seeded on a layer of retinal glial cells. Mature rods and cones required the presence of Muller glial cells to survive and develop neurites; they degenerated in the absence of glial cells. 3. Cone photoreceptors generated large GABA responses whereas rod photoreceptors did not respond to GABA applications. 4. Cone GABA responses consisted of two distinct components, one suppressed by the GABAA receptor blockers bicuculline and SR95531, and the second by the GABAC receptor antagonists TPMPA and imidazole-4-acetic acid (I4AA). Pentobarbital greatly increased the GABAA receptor component whereas it did not affect, or even reduced, the GABAC receptor component. During long GABA applications, GABAA receptor currents desensitized by 78%, contrasting with the sustained GABAC response. 5. Expression of GABAC receptors in cone photoreceptors was confirmed by anti-rho-subunit immunolabelling of porcine retinal sections. 6. These results indicate that both GABAA and GABAC receptors may participate in the feedback synapse from horizontal cells to cone photoreceptors in the mammalian retina.

Animals↗
PubMed1998

Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse.

The role of cellular interactions in the mechanism of secondary cone photoreceptor degeneration in inherited retinal degenerations in which the mutation specifically affects rod photoreceptors was studied. We developed an organ culture model of whole retinas from 5-week-old mice carrying the retinal degeneration mutation, which at this age contain few remaining rods and numerous surviving cones cocultured with primary cultures of mixed cells from postnatal day 8 normal-sighted mice (C57BL/6) retinas or retinal explants from normal (C57BL/6) or dystrophic (C3H/He) 5-week-old mice. After 7 days, the numbers of residual cone photoreceptors were quantified after specific peanut lectin or anti-arrestin antibody labeling by using an unbiased stereological approach. Examination of organ cultured retinas revealed significantly greater numbers of surviving cones (15-20%) if cultured in the presence of retinas containing normal rods as compared with controls or cocultures with rod-deprived retinas. These data indicate the existence of a diffusible trophic factor released from retinas containing rod cells and acting on retinas in which only cones are present. Because cones are responsible for high acuity and color vision, such data could have important implications not only for eventual therapeutic approaches to human retinal degenerations but also to define interactions between retinal photoreceptor types.

Animals↗
PubMed1998

Gangliosides and neurotrophic growth factors in the retina. Molecular interactions and applications as neuroprotective agents.

Polypeptide growth factors and gangliosides can both be considered as trophic agents involved in almost all stages of neural cell development, differentiation, survival, and pathology. In most cases their physiological roles are still not clear due to the considerable complexity in their regulation. Several growth factors [e.g., basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)] and one species of ganglioside (GM1) have been shown to exert interactions with each other and also to exhibit neuroprotective effects against retinal ischemia in vivo and cerebral excitotoxicity in vitro. Different experimental models are used to investigate their relevance to ischemic and excitotoxic conditions in the retina, and it is shown that (1) both bFGF and EGF show very effective neuroprotection for rat retinal neurones exposed to toxic levels of glutamate or its nonphysiological agonist kainate in vitro; (2) GM1 (10(-5M) used under the same conditions does not afford protection; (3) retinal glial cells also suffer morphological perturbations following glutamate or kainate treatment, but this effect is dependent on neuron-glial interactions, indicating the existence of intermediate neuron-derived messenger molecules; (4) these glial changes can be corrected by posttreatment with either bFGF or EGF in vitro; (5) using an in vivo animal model involving anterior chamber pressure-induced ischemia in adult rats, it is shown that either pretreatment by intraperitoneal injection of GM1, or posttreatment by intraocular injection of the same ganglioside, reduces significantly histological damage to inner nuclear regions; and (6) in cultured retinal Müller glial cells the existence of molecular and metabolic interactions between both types of trophic factors is demonstrated. Hence both these groups of trophic molecules show interesting features for retinal ischemic treatment.

Animals↗
PubMed1998

Excitotoxic damage of retinal glial cells depends upon normal neuron-glial interactions.

Glutamate, the principal retinal neurotransmitter, can also act as a toxin when present in excessive concentrations as may occur in pathologies such as retinal ischemia or more generally in cerebral neuronal degenerative disease. As glial cells play pivotal roles in transfer of blood-borne molecules and in glutamate clearance, we investigated the effects of the excitatory amino acids glutamic and kainic acid on different in vitro preparations of retinal Müller glial cells. Glial viability or morphology were not influenced by excitatory amino acid exposure in either pure glial cultures or in monolayer cultures of mixed neonatal neurons and glia, whereas kainic acid specifically lysed amacrine cells in mixed or pure neuronal cultures. When retinal fragments were pre-incubated in excitatory amino acids prior to dissociation and seeding into culture, under these conditions Müller glial cells exhibited a dramatic loss of their normal epithelioid form to a retracted morphology. However, glial cell viability was not compromised, and rapid restoration of epithelioid in vitro glial morphology could be achieved by addition of exogenous epidermal and basic fibroblast growth factor to the culture medium. This study demonstrates that glial cells are structurally perturbed by excitotoxic conditions and that such effects are dependent on normal glial-neuronal interactions.

Animals↗