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Biomedical subjects

P M Munro

Publications and source records attributed to P M Munro.

At least 19 recordsLinked to original sources
PubMed2005

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy.

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Animals↗
PubMed2000

Structure of abnormal molecular assemblies (collagen VI) associated with human full thickness macular holes.

Transversely banded deposits with an approximately 100-nm periodicity have been seen in association with a number of eye pathologies (e.g., age-related macular degeneration). Recently such aggregates have also been discovered in the cortical vitreous of a patient suffering from full thickness macular holes. The aggregates in the vitreous were of sufficient size and regularity for us to attempt 3D ultrastructural studies in the electron microscope. The molecules forming this aggregate pack in a centered tetragonal unit cell of dimensions approximately 26 x 26 x 180 nm. A real-space (r-weighted back projection) 3D reconstruction was computed. The aggregate is discussed in terms of its possible protein constituents. Collagen VI has been singled out as the most likely protein to form the aggregate. Two alternative models for the molecular packing are proposed, comprising aggregates of molecular tetramers or octamers. Understanding the structure of these abnormal banded deposits in the eye should help to throw light on the pathophysiological mechanisms of the diseases, including age-related macular degeneration, in which they occur.

Bruch Membrane↗
PubMed2000

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Animals↗
PubMed1998

Absence of p53 delays apoptotic photoreceptor cell death in the rds mouse.

PURPOSE: This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. METHODS: A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. RESULTS: The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. CONCLUSIONS: We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.

Animals↗
PubMed1997

Macular hole opercula. Ultrastructural features and clinicopathological correlation.

OBJECTIVE: To investigate the ultrastructural features of idiopathic full-thickness macular hole (FTMH) opercula excised during vitrectomy and to correlate them with the outcome of surgery. METHODS: Opercula were collected from eyes undergoing vitrectomy for stage 3 FTMH using noncrushing, cupped foreign body forceps. Following immediate fixation, specimens were processed for transmission electron microscopy. The ultrastructural features were correlated with the clinical data recorded for each patient before and after surgery. RESULTS: Eighteen specimens were studied. Native vitreous collagen was identified on the surface of all 18, while fragments of internal limiting membrane were present in 11 (61%). Eleven (61%) were found to contain only glia, comprising fibrous astrocytes and Müller cells in variable proportions. The remaining 7 (39%) were found to contain, in addition to glia, neurites and synaptic complexes, of which some were typical of cone photoreceptors. The initial surgical closure rate was significantly better in eyes in which only glia were present (9/11 [82%]), compared with those with neurites (1/7 [14%]) (P = .01). Once closure had been achieved with reoperation, the median final visual acuity was 20/60 in both groups (P = .26), although the likelihood of achieving an acuity of 20/40 or better was greater in the former (50%) than the latter group (17%). CONCLUSIONS: Two distinct types of opercula occur in association with stage 3 FTMH--those containing only glia (pseudo-opercula), which are probably associated with a foveal dehiscence and little or no loss of foveal tissue, and those containing both glia and a significant number of avulsed foveal cones (true opercula), which arise from a full-thickness foveal tear. Although the loss of foveal tissue in true opercula would seem to explain the worse initial anatomical and more modest visual results in some eyes, significant visual improvement may still be achieved after successful closure. The presence of neurites in true opercula suggests that, in at least some cases, direct traction on the foveal retina leads to macular hole formation.

Aged↗
PubMed1996

Inhibition of N-acetylaspartate production: implications for 1H MRS studies in vivo.

The effect of specific irreversible inhibitors of complexes I, III, IV and V of the mitochondrial respiratory chain, (rotenone, myxothiazol, cyanide and oligomycin, respectively) on mitochondrial N-acetylaspartate production, and its relationship to oxidative phosphorylation (ATP production and oxygen consumption) were investigated in isolated rat brain mitochondria. Mitochondrial N-acetylaspartate production, ATP production and oxygen consumption were all significantly decreased in the presence of each of the inhibitors used compared with control incubations, and correlated positively with each other. It is postulated that decreased N-acetylaspartate levels seen in disease states by 1H NMR spectroscopy in vivo may reflect primarily an impaired mitochondrial energy production rather than neuronal cell loss.

Analysis of Variance↗
PubMed1995

Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.

Adaptation, Physiological↗
PubMed1994

Seawater effects on various Vibrio species.

This study compared the effects of sea water on Vibrio cholerae and six other Vibrio spp. Survival in seawater microcosms as well as uptake of a carbonated substrate in marine or non-marine conditions were investigated. Except for V. vulnificus becoming non-culturable, all the other selected species survived in sea water for at least 15 days at 20 degrees C. Depending on the species tested, the substrate was better transported in a high salt medium (V. cholerae, V. fluvialis and V. metschnikovii), than in a low salt medium (V. fluvialis, V. furnissii, V. parahaemolyticus and V. vulnificus). In terms of the response of the species to marine conditions, no correlation was found between survival in sea water and substrate uptake.

Culture Media↗
PubMed1993

Functional properties of retinal Müller cells following transplantation to the anterior eye chamber.

Two types of glial cells occur in the retina, Müller cells and astrocytes. These cells share several structural features such as extending endfeet onto blood vessels of the retina. Retinal vessels express a tight blood-retinal barrier which is comparable to the blood-brain barrier (BBB) of the CNS. While astrocytes have been implicated in the induction of the BBB, the role of Müller cells in the blood-retinal barrier is unknown. To determine if Müller cells are capable of influencing vascular permeability, we have prepared Müller cells that are free of astrocytes and transplanted them to a peripheral target, the anterior eye chamber. Müller cells were identified 2 weeks to 3 months after injection and were predominantly localized within the connective tissue of the ciliary body. The Müller cells occurred as dense clusters of cells closely associated with ciliary blood vessels. The ciliary vessels adjacent to Müller cells were freely permeable to circulating horseradish peroxidase (HRP), suggesting that Müller cells did not induce tight barrier properties from these leaky peripheral vessels. In contrast, cortical astrocytes injected into the anterior eye chamber preferentially formed a monolayer on the anterior surface of the iris, a region known to contain blood vessels that are impermeable to circulating tracers (e.g., Raviola, Exp Eye Res [Suppl] 25:27, 1977). Müller cells were rarely associated with the iris and the few cells that were present were located deep within the iris stroma rather than on the surface. The behaviour of guinea pig Müller cells transplanted to the anterior eye chamber contrasts sharply with that of cortical astrocytes in terms of: 1) the ocular compartment to which Müller cells migrate; 2) the tissue invasiveness of the cells; and 3) the degree of permeability of blood vessels adjacent to transplanted cells. The results of this study emphasize the functional distinctness of the two types of retinal glia and suggest that Müller cells from guinea pig retina may not be active in modifying the permeability properties of peripheral blood vessels, a function that has been suggested for astrocytes.

Animals↗
PubMed1993

The proton NMR spectrum in acute EAE: the significance of the change in the Cho:Cr ratio.

We have studied by nuclear magnetic resonance spectroscopy the evolution of the proton spectral changes in acute experimental allergic encephalomyelitis. We found an in vivo elevation in the ratio between the peaks assigned to "choline containing compounds" (Cho) and creatine plus phosphocreatine (Cr). This was associated with an increase in choline, betaine, and phosphorylcholine (PC) as well as a reduction in N-acetylaspartate (NAA), aspartate, N-acetylaspartatylglutamate and inositol in vitro. Histological examination revealed inflammation with no evidence of demyelination or neuronal loss. We conclude that the increase in the ratio of Cho:Cr was due to an increase in the concentrations of PC, betaine, and choline in association with inflammation, and not as others have suggested, with demyelination. The reported reduction in NAA may be due to dysfunction of neurones rather than their loss.

Animals↗
PubMed1993

Glutamate uptake and synthesis by Escherichia coli cells in seawater: effects on culturability loss and glycinebetaine transport.

In filtered natural seawater supplemented with potassium glutamate, the ability of Escherichia coli MC4100 cells to grow on a complex medium was enhanced as a logarithmic function of the external glutamate concentration. By comparison, a glutamate-respiring strain of E. coli exhibited a greater decline in culturability in seawater, suggesting a protective influence of the accumulated amino acid. Potassium glutamate increased the uptake of 14C-glycinebetaine by E. coli MC4100 cells in seawater and enhanced the protective effects of the betaine against culturability loss, possibly by increasing the expression of the ProU transport system. This bacterium apparently was able to synthesize glutamate because a protective effect (i.e. a lower culturability loss) was observed in seawater when supplemented with precursor compounds (2-oxoglutarate and glutamine). The combination of 2-oxoglutarate and glutamine resulted in the greatest protection of cells, possibly due to the synthesis of glutamate through glutamine 2-oxoglutarate amino transferase activity. The possible influence of glutamate and its precursors on survival of E. coli cells in the natural marine environment is considered, since glutamate, glutamine and betaines have been found in marine coastal waters and sediments.

Betaine↗
PubMed1992

Metabolically dependent blood-brain barrier breakdown in chronic relapsing experimental allergic encephalomyelitis.

We have studied chronic relapsing experimental allergic encephalomyelitis (CREAE), a model of immune-mediated demyelination, using gadolinium (Gd)-enhanced magnetic resonance imaging in vivo and the blood-brain barrier (BBB) markers, lanthanum nitrate and Gd nitrate, histologically. In regions of the spinal cord showing Gd enhancement, there was evidence for vesicular transport as a mechanism of BBB breakdown in CREAE, shown by an increased number of endothelial vesicles containing lanthanide (lanthanum or Gd, whichever had been perfused) and deposition of tracer in the perivascular space; tight interendothelial junctions remained intact. Prior perfusion with 2,4-dinitrophenol, a metabolic inhibitor, suppressed the appearance of endothelial vesicles containing lanthanide and tracer in the perivascular space. We conclude that an important contribution to BBB breakdown in CREAE is mediated by a metabolic change in the endothelial cells associated with increased vesicular transport.

2,4-Dinitrophenol↗
PubMed1992

OmpC and OmpF porins influence viability and culturability of Escherichia coli cells incubated in seawater.

The contribution of the major outer membrane porins OmpF and OmpC to the maintenance of viability and culturability of Escherichia coli cells in seawater was analyzed using isogenic mutant strains lacking one or both porins. Cells that possessed OmpF and OmpC survived better than those lacking one or both of them. However, the results differed, depending on whether the cells were adapted to high osmolarity or not before transfer to seawater. When cells were grown at low osmolarity, survival was largely influenced by porins, the OmpF+ strains surviving better than those lacking this porin. Addition of an OmpF plasmid to OmpF- OmpC- cells also improved their viability. When grown at high osmolarity, the role of porins was less critical since both the viability and culturability of the cells increased. However, cells that expressed only OmpC showed the most dramatic loss of viability. Cells lacking both OmpF and OmpC exhibited a higher loss of viability and culturability in seawater. Regarding the influence of porins on survival, these results show that the conditions that prevail during the growth of cells before their transfer to seawater are highly influential: cells that express the porin corresponding to the growth conditions they are in at the time of transfer survive better.

Colony Count, Microbial↗
PubMed1992

Sensitivity of Escherichia coli cells to seawater closely depends on their growth stage.

Sensitivity of Escherichia coli cells in seawater, considered in terms of culturability loss, was examined after different growth periods in a mineral medium supplemented with glucose (M9) at 37 degrees C under aerobic or anaerobic conditions. Their sensitivity varied considerably during the different growth phases and differed when cells were grown under aerobic or anaerobic conditions. Sensitivity of aerobic cells rapidly increased during the lag phase, then decreased during the exponential phase and became minimal during the stationary phase. Coliforms isolated from human faeces showed a similar sensitivity after incubation in wastewater at 37 degrees C for 3 h. The sensitivity phase was completely eliminated when cells were incubated with chloramphenicol. Variation of sensitivity in anaerobic cells according to their growth phase was comparable with that found for aerobic cells which had been left in seawater for a long period (6 d). However, for shorter periods in this medium (1-2 d), cells grown until the mid-exponential phase remained resistant to seawater. During the second half of the growth phase, they were as sensitive as aerobic cells at lag phase. Escherichia coli cells grown under anaerobic conditions, such as found in the intestine, progressively adapt to aerobic conditions after their transfer into aerated seawater and their sensitivity to seawater increases. On a practical level, these observations show that it is necessary to control accurately the age of cells before inoculation in seawater microcosms to conserve a comparative value in results. The importance of this factor is vital as all variations in sensitivity of cells to seawater according to their prior growth phase proved to be logarithmic functions of time.

Aerobiosis↗
PubMed1991

Autoantibodies to glycoprotein antigens mediate subacute demyelinating encephalomyelitis in the Lewis rat.

Serum autoantibodies were induced in Lewis rats by immunization with a mixture of lentil lectin-binding glycoproteins isolated from bovine brain myelin. Intraperitoneal administration of 2-10 million syngeneic myelin basic protein-activated spleen cells to these rats led within 4-5 days to paralysis which, in most cases, persisted for several weeks. The major neuropathological features of the disease were numerous macrophages in both brain and spinal cord and large areas of demyelination, generally with axon preservation, particularly adjacent to the pial surfaces of the cord. This model is easily induced and will be useful for studies of demyelination and remyelination.

Animals↗
PubMed1991

The longstanding MS lesion. A quantitative MRI and electron microscopic study.

Important questions remain unanswered about the sequence of events leading to progressive and ultimately irreversible tissue damage in MS. This study was designed to investigate the pathological characteristics of, and function of, the blood-brain barrier within longstanding MS lesions using quantitative and Gd-DTPA enhanced MRI techniques. The ultrastructural appearances of postmortem lesions from a single, separate case of MS have been correlated with the MRI findings. Both MRI and ultrastructural analysis revealed considerable heterogeneity in the chronic lesions: some are 'closed' with no detectable extracellular water, but most are 'open' and show expansion of the extracellular space to as much as 87% of tissue area. This variable expansion probably results from differing degrees of axonal loss. Evidence of blood-brain barrier damage was found in only 17% of lesions, was less severe than that seen in acute lesions, and may result from repeated previous inflammatory insults. The findings imply progressive axonal loss in lesions as they age. It is possible that this loss is related to clinical progression of the disease.

Adult↗
PubMed1990

Duration and selectivity of blood-brain barrier breakdown in chronic relapsing experimental allergic encephalomyelitis studied by gadolinium-DTPA and protein markers.

Gadolinium-DTPA (Gd-DTPA) enhancement seen with magnetic resonance imaging in chronic relapsing experimental allergic encephalomyelitis (CREAE) corresponded with sites of blood-brain barrier breakdown judged by traditional markers in areas of inflammatory demyelination. Duration of Gd-DTPA leakage for individual lesions in CREAE varied from 5 days to more than 5 wks. By contrast, in acute EAE leakage was of shorter duration (always less than 5 days). Selective enhancement was observed in CREAE lesions using Gd-protein markers. Gd-albumin enhancement was not always seen in areas of leakage of the smaller molecular weight compound Gd-DTPA. The addition of immunoglobulin to the gadolinium complex led to enhancement of lesions not seen with Gd-albumin alone. From the similarities between the histology and the patterns of Gd-enhancement in CREAE and multiple sclerosis, it is probable that Gd-enhancement reflects active inflammation (with or without demyelination) in the human disease.

Animals↗