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Biomedical subjects

D J Gould

Publications and source records attributed to D J Gould.

At least 19 recordsLinked to original sources
PubMed1999

The effects of mitotic inhibition on the spinal cord response to the superimposed injuries of spinal cord hemisection and peripheral axotomy.

The present study was carried out to test the hypothesis that dividing microglia are responsible for the depression of crossed phrenic nerve activity documented at 2 weeks postphrenicotomy in an injury model which superimposes the effects of spinal cord injury on peripheral axotomy. Crossed phenic nerve activity is defined as the respiratory activity recorded from the phrenic nerve during the crossed phrenic phenomenon (CPP) which is a respiratory reflex induced by respiratory stress following an ispsilateral spinal cord hemisection. Young adult female Sprague-Dawley rats were subjected to left intrathoracic phrenicotomies. Cytarabine (Cyt-A, a powerful antimitotic drug) or saline-filled miniosmotic pumps were then implanted into the cisterna magna and 2 weeks were allowed to pass at which time the CPP was induced by a left C2 spinal cord hemisection and transection of the contralateral phrenic nerve. Control studies including bromodeoxyuridine labeling of mitotic cells and a triple immunofluorescent protocol were carried out to verify that microglial cells were the primary cell type undergoing mitosis in the current injury model and that Cyt-A completely inhibited cellular proliferation. Quantitative electrophysiological analysis of crossed phrenic nerve activity showed that there is a statistically significant depression of activity at 2 weeks postphrenicotomy when animals were infused with saline compared to controls. Crossed phrenic nerve activity levels were not significantly different, however, from control levels when 2-week postphrenicotomized rats were infused with Cyt-A. Immunofluorescent studies showed that the majority of cells dividing in response to phrenicotomy were microglia. Furthermore, there were no astrocytes seen dividing at any time. From the results, we conclude that activated microglial cells may be responsible for the depression in crossed phrenic activity normally seen 2 weeks postphrenicotomy. Further, the activation of microglia may be related to the astrocytic response to injury. The activated microglial cell may be acting as a coordinator of various aspects of the injury response. Alternatively, the activation of microglia may be a necessary step in the cascade of multiple events that take place in the spinal cord after injury.

Animals↗
PubMed1998

Potentiation of TNF-alpha toxicity by conjugation with ricin A-chain.

Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor alpha (TNF-alpha) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-alpha can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-alpha cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-alpha and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC50=4 pM) not sensitive to native TNF-alpha, and sensitive WEHI cells with substantially increased lethality (LD50=0.01 fM). This increased TNF-alpha cytotoxic activity suggests that hybrid toxins containing TNF-alpha may have therapeutic applications in the treatment of cancer.

Cell Line↗
PubMed1998

Petrified ears.

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Ear Diseases↗
PubMed1997

Pathway-specific effects of calcitonin gene-related peptide on irideal arterioles of the rat.

1. Arteriolar diameter and membrane voltage have been measured to investigate the actions of calcitonin gene-related peptide (CGRP) in rat irideal arterioles. 2. Activation of sensory nerves inhibited sympathetic vasoconstriction, reduced the accompanying 40-50 mV depolarization by 90% and caused a 4 mV hyperpolarization. 3. The inhibition of vasoconstriction was prevented by either preincubation in L-NAME (10 microM), to inhibit nitric oxide production, by preincubation in the cell-permeant adenylate cyclase inhibitor dideoxyadenosine (1 mM) or by preincubation in the ATP-sensitive potassium channel blocker glibenclamide (10 microM). The subsequent addition of a nitric oxide donor to the glibenclamide solution inhibited nerve-mediated vasoconstriction, suggesting that the potassium channel involvement preceded the production of nitric oxide. The small hyperpolarization was not affected by L-NAME. 4. Nerve-mediated vasodilatation persisted in the presence of L-NAME (10 microM) but was abolished with the CGRP1 receptor antagonist CGRPS-37. 5. In arterioles preconstricted with the alpha 2-adrenoceptor agonist UK-14304 (100 nM), exogenous CGRP caused a hyperpolarization and a dose-dependent vasodilatation, neither of which was affected by L-NAME (10 microM). 6. In arterioles preconstricted with 30 mM KCl, CGRP (10 nM) caused vasodilatation but not hyperpolarization, suggesting that the hyperpolarization was not causal to the vasodilatation. 7. Forskolin (30 nM), in the presence of L-NAME to present effects due to nitric oxide, caused vasodilatation. 8. These results suggest that CGRP inhibits sympathetic nerve-mediated vasoconstriction through sequential increases in cyclic AMP and nitric oxide, while vasodilatation results from increases in cyclic AMP alone. The production of nitric oxide, but not its mechanism of action, appears to be dependent on the activation of ATP-sensitive potassium channels. The possible sites of action of these two pathways are discussed.

Animals↗
PubMed1997

Glial changes in the phrenic nucleus following superimposed cervical spinal cord hemisection and peripheral chronic phrenicotomy injuries in adult rats.

The objective of the present study was to characterize the microglial and astroglial reaction in the phrenic nucleus following either an ipsilateral C2 spinal cord hemisection, a peripheral phrenicotomy, or a combination of the two injuries in the same adult rat. The present study used three different fluorescent markers and a confocal laser image analysis system to study glial cells and phrenic motoneurons at the light microscopic level. Young adult female rats were divided into one combined injury group (left phrenicotomy and left C2 spinal hemisection with periods of 1 to 4 weeks between injuries, N = 12) and three other groups consisting of noninjured animals (N = 3), animals that received C2 hemisection only (N = 3), and animals with phrenicotomy only (survival periods of 2 (N = 3) and 4 (N = 3) weeks after phrenicotomy). Fluorogold was injected into the diaphragm to label phrenic motoneurons in all animals. Microglia and astrocytes were labeled with Texas red and fluorescein, respectively, and were visualized simultaneously along with phrenic motoneurons. The results suggest that the microglial and astrocytic response in the superimposed injury model are similar to the glial reactions characteristically seen in a peripheral axotomy alone model. These reactions include proliferation and migration of microglial cells along the perineuronal surface (peaking at 2 weeks) and the hypertrophy of astrocytes (peaking at 4 weeks). In addition, the increase in astrocytic tissue, which is characteristically seen in response to axotomy alone, is significantly enhanced in the superimposed injury model. Also, there is a large and rapid increase in GFAP-positive astrocytes within 24 hours after hemisection alone. The information gained from the present study will aid in determining, predicting, and eventually manipulating central nervous system responses to multiple injuries with the objective of reestablishing function in the damaged CNS.

Animals↗
PubMed1997

Distribution of bacteria on hands and the effectiveness of brief and thorough decontamination procedures using non-medicated soap.

Our perception of the role of hand washing in the clinical situation is based on experimental studies in which test-bacteria are usually inoculated onto the skin surface and removed using hand washing preparations containing antiseptics. In this study, we have investigated the distribution of bacteria on the hands of volunteers and the effectiveness of long (3 minute) and brief (10 second) washes in removing both naturally-occurring and artificially-inoculated bacteria (Micrococcus sp.), using only soap and water. There was a tenfold reduction in median counts of artificially inoculated bacteria following both long and brief washes. However, less than 50% of naturally-occurring bacteria were removed and, for hands previously disinfected by immersion in 70% ethanol, the washing procedure increased bacterial counts. In both unwashed hands, and hands washed following a strict protocol, the mean variation in counts of naturally-occurring bacteria at different sites (wrists, dorsal surface, palmar surface, fingertips and interdigital spaces) was only two-fold. The efficiency of recovery of naturally-occurring organisms was estimated by repeated swabbing, to be more than 60%. The data question the value of typical hand wash procedures recommended by many authorities for use in clinical situations and of the perfunctory hand washes frequently adopted by nursing staff in busy wards. Experimental evidence is required to justify procedures and to identify the precise circumstances in which they are of value.

Disinfectants↗
PubMed1997

Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs.

Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1, a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.

Animals↗
PubMed1997

Electrical properties of iridial arterioles of the rat.

When intracellular recordings were made from iridial arterioles, the cells had membrane potentials of about -65 mV and perivascular nerve stimulation evoked a membrane depolarization. When these cells were labeled with lucifer yellow, all cells that responded to perivascular nerve stimulation had the morphological characteristics of smooth muscle cells. Cells with the morphological characteristics of endothelial cells were never stained. When impaled with two separate recording electrodes, the smooth muscle layer was shown to form an electrical syncytium with a membrane time constant of approximately 80 ms and an electrical length constant of approximately 900 microns. At the ultrastructural level, areas of close apposition were frequently observed between adjacent smooth muscle cells and between adjacent endothelial cells. On the other hand, at contacts between smooth muscle and endothelial cells, the membranes characteristically had much larger separations. The observations show that individual smooth muscle cells are electrically coupled to their neighbors, but the morphological studies raise the possibility that in these arterioles the endothelial and muscle layers are electrically separate.

Animals↗
PubMed1996

Sensory nerves play an efferent role in the function of the arterioles, but not the dilator muscle, of the rat iris.

We have studied the expression, distribution and function of receptors for the sensory neurotransmitters, substance P (SP) and calcitonin gene-related peptide (CGRP) in the dilator muscle and arterioles of the rat iris. Immunohistochemical studies showed that the sensory fibres containing these peptides are distributed throughout the connective tissue stroma of the iris and in association with the larger arterioles, but do not come into close association with the dilator muscle cells. Using reverse-transcription polymerase chain reaction, we have shown that both NK1 and NK3 receptor message is expressed by iris tissue, comprising both dilator muscle and stromal tissue. Binding sites for the NK1 agonist, [Sar9, Met(O2)11]-substance P (SarSP), and for CGRP are confined to the stromal layer and to the larger arterioles within that layer and do not appear to be associated with the dilator muscle itself. Application of either SarSP or CGRP produced both a vasodilatation and an inhibition of sympathetic nerve-induced vasoconstriction of the larger arterioles. Neither SarSP nor CGRP altered the resting tone of the dilator nor were they capable of modulating the contractions due to sympathetic nervous activity. These results suggest that the sensory fibres perform an efferent role in the larger irideal arterioles while their presence in the irideal stroma appears not to modulate the activity of the dilator muscle.

Adrenergic Fibers↗
PubMed1996

Recent advances in understanding the spectrum of canine generalised progressive retinal atrophy.

Canine generalised progressive retinal atrophy (gPRA) is a large and ever-increasing collection of naturally occurring, heterogeneous, progressive disorders. Most are inherited in an autosomal recessive manner and new, breed-specific forms continue to be described. The gPRAs cause photoreceptor cell death and subsequent retinal degeneration, culminating in blindness. In humans, similar inherited retinal dystrophies are recognised as retinitis pigmentosa and macular dystrophy. Molecular biological studies have revealed disease-causing mutations in several genes in humans and also in mice with retinal dystrophies. Recently, molecular genetic techniques have identified the cause of one form of gPRA in Irish setters while important candidate genes have been investigated in other breeds. Identification of mutations responsible for different forms of gPRA allows carrier and predegenerate animals to be detected using DNA-based tests. Such genetic tests will greatly facilitate the eradication of these diseases in different breeds.

Animals↗
PubMed1996

Alpha-adrenoceptor activation of a chloride conductance in rat iris arterioles.

Changes in membrane potential associated with alpha-adrenoceptor-mediated contraction of rat iris arterioles after nerve stimulation (10 Hz, 1 s) have been measured with conventional intracellular recording techniques. Two different types of intracellular responses were recorded. Cells that show a depolarization are proposed to represent the arteriolar smooth muscle cells because the characteristics of the depolarization are correlated with those of the contraction. Cells that show no response or a small hyperpolarization in response to nerve stimulation are proposed to represent the endothelial cells of the arteriole. Both the depolarization and the contraction were abolished by tetrodotoxin (1 microM), benextramine (10 microM), and prazosin (0.1 mM), indicating that they result from nerve-mediated activation of alpha-adrenoceptors. A small but significant part of the contraction (30%) and the depolarization (11%) was nifedipine sensitive (10 microM). Caffeine (1 mM) abolished the contraction and reduced the depolarization by one-half. Reducing the external chloride concentration also abolished the contraction and reduced the depolarization by 90%. Flufenamic acid (250 mM) abolished both the contraction and the depolarization. It is suggested that, in iris arterioles, the activation of synaptic alpha-adrenoceptors leads to the release of intracellular calcium that activates both the chloride channels in the cell membrane leading to depolarization and the intracellular contractile apparatus leading to vasoconstriction.

Animals↗
PubMed1995

Investigation of the role of opsin gene polymorphism in generalized progressive retinal atrophies in dogs.

The generalized progressive retinal atrophies (gPRAs) form a group of retinal degenerations of pedigree dogs and cats, which have a variety of genetic origins (mostly unknown). We have examined the opsin gene for polymorphisms in several breeds of pedigree dog suffering from distinct forms of gPRA, by methods including single-strand conformation polymorphism analysis, microsatellite analysis and direct sequencing. The breeds examined included the Tibetan terrier, the miniature schnauzer, the Irish setter, the miniature poodle, the Labrador retriever and the English cocker spaniel, as well as individuals from breeds in which PRA has not been described and of mixed breed. Individuals from each of the named breeds suffering from PRA were compared with clinically normal dogs. Two polymorphisms were found. One, segregating within the Tibetan terrier population, but not seen in other breeds, was a synonymous transition at nucleotide position 780 in exon 3. Inheritance of this polymorphism suggests that opsin is unlikely to contain mutations causative of gPRA in this breed. The other polymorphism occurred between all miniature schnauzers examined and dogs of other breeds. It consisted of a single base insertion in intron 2. No polymorphisms in the opsin sequence were detected in any other breed. DNA sequencing allowed rigorous exclusion of mutations in opsin as a cause of gPRA in miniature poodles, English cocker spaniels or Labrador retrievers.

Animals↗
PubMed1995

Treatment of basal cell carcinoma by dermatologists in the United Kingdom. British Association of Dermatologists Audit Subcommittee and the British Society for Dermatological Surgery.

Details of presentation and treatment were obtained prospectively by questionnaire for 1366 patients with basal cell carcinoma, first attending under the care of 166 consultant dermatologists in the U.K., during a 2-week period. One thousand five hundred and ninety-seven tumours were reported in these patients (median age 71 years). Most were situated on the head and neck, and their median size was 9 mm. Excision was the most common treatment used in 58% of tumours, curettage and cautery was used in 24%, cryotherapy in 8%, and radiotherapy in 8%. On average, one in four tumours were referred to other specialists for treatment (range 0-70%). Very few patients (2%) were referred to a combined dermatology/radiotherapy/plastic surgery clinic, calling into question its value and availability. There was considerable variation in practices between dermatologists, demonstrating a clear need for individual, local audit of the management of this common dermatological problem. The data collected in this study form a suitable measure with which local performance may be compared.

Adolescent↗
PubMed1995

Cross talk between receptors mediating contraction and relaxation in the arterioles but not the dilator muscle of the rat iris.

1. Sympathetic nerve stimulation causes contraction of the dilator muscle and the large arterioles of the iris via the activation of alpha 1B-adrenoceptors. We have investigated whether increases in adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and the activation of receptors in these tissues can modulate these nerve-mediated contractions. 2. Increasing intracellular cyclic AMP with dibutyryl cyclic AMP (1 mM), forskolin (50 microM) or isobutylmethylxanthine (100 microM) produced relaxation of both the dilator and the arterioles, abolished the nerve-mediated constriction of the arterioles, but potentiated the nerve-mediated contraction of the iris dilator. 3. Pretreatment of the preparations with cholera toxin, to activate Gs permanently, caused a dilatation of the arterioles and abolished the nerve-mediated constriction but had no effect on the dilator muscle. 4. The beta-adrenoceptor agonist, isoprenaline (1 microM), the adenosine-A1,-A2 agonist, N-ethylcarboxamidoadenosine NECA (100 nM), in the presence of the A1-selective antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 10 nM), and calcitonin gene-related peptide (CGRP, 10 nM) all separately caused a dilatation of the arterioles and abolished the nerve-mediated constriction, while only isoprenaline (1 microM) produced an effect on the dilator, i.e. a relaxation but a potentiation of the nerve-mediated contraction. These results suggest the presence of at least 3 types of receptor linked to Gs and an increase in cyclic AMP in the arterioles, i.e. beta-adrenoceptor, adenosine-A2 and CGRP, but only 1 Gs-linked receptor, i.e. beta-adrenoceptors, on the dilator muscle cells.2+ '

Animals↗
PubMed1995

Modulation of sympathetic vasoconstriction by sensory nerves and nitric oxide in rat irideal arterioles.

Transmural nerve stimulation of arterioles in the rat iris produces a vasoconstriction mediated via alpha-1B adrenoceptors and the mobilization of intracellular calcium. This study has investigated the role of sensory nerves in modulating this vasoconstriction using isolated preparations of iris perfused with Krebs' solution. Repeated stimulation of the transmural nerves for 1 sec (10 Hz), at intervals less than 2 min, produced a rapid and long-lasting loss of the vasoconstriction. This inhibition was not seen in arterioles taken from rats treated neonatally with capsaicin (50 mg/kg) nor after application of capsaicin (10(-5) M) to control preparations. In arterioles from control rats, both the substance P analog Sar9 and calcitonin gene-related peptide (CGRP) were effective in inhibiting nerve-mediated vasoconstriction. L-NG-nitroarginine methyl ester (10(-5) M), but not D-NG-nitroarginine methyl ester (10(-5) M), prevented the loss of vasoconstriction during repetitive stimulation, which suggested the involvement of nitric oxide. Antagonists to the NK1 and NK2 neurokinin receptor subtypes, RP 67580 and L-659877, respectively, were without effect, whereas the antagonist to the CGRP1 receptor, CGRP8-37, prevented the loss of vasoconstriction during repetitive nerve stimulation. The effects of both Sar9 and CGRP (10(-8) M) in inhibiting the nerve-mediated vasoconstriction were prevented by preincubation in L-NG-nitroarginine methyl ester (10(-5) M), which suggested that the mechanism of action of both peptides involved the release of nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Alpha 1B-receptors and intracellular calcium mediate sympathetic nerve induced constriction of rat irideal blood vessels.

The present study has investigated the receptors involved in the non-cholinergic nerve mediated constriction of the larger blood vessels (30-50 microns) within the rat iris. This response was blocked by the alpha-adrenoceptor antagonist, benextramine (10(5) M). Furthermore, the response was more sensitive to blockade by the alpha 1 antagonist, prazosin (IC50 9 x 10(-10) M), than to blockade by the alpha 2 antagonist, yohimbine (IC50 2 x 10(-7) M), or the adrenergic antagonist, WB4101 (IC50 2 x 10(-8) M), and was abolished by chloroethylclonidine (10(-5) M). These results suggest the involvement of alpha 1B-adrenoceptors. The nerve mediated constriction was not blocked by the voltage-dependent calcium channel blocking drugs, nifedipine (10(-6) M), verapamil (10(-6) M) or diltiazem (10(-6) M), but was completely abolished by the intracellular calcium mobilizer, caffeine (10(-3) M), supporting the hypothesis that alpha 1B-adrenoceptors are activated following nerve stimulation. Dantrolene (10(-4) M), which interferes with calcium release from the sarcoplasmic reticulum, reduced the nerve mediated constriction by 40% as did thapsigargin (2 x 10(-6) M), which inhibits the calcium ATPase responsible for uptake of calcium into intracellular stores. When influx of calcium was blocked by verapamil (10(-6) M), thapsigargin, but not dantrolene, completely abolished the response. Noradrenaline (10(-5) M) produced a vasoconstriction in the presence or absence of external calcium although the latter response was significantly smaller than the former. Vasoconstriction produced by a submaximal concentration of noradrenaline (10(-6) M), was completely prevented by pretreatment with chloroethylclonidine. The data indicate that noradrenaline released from sympathetic nerves causes a constriction of arterioles in the iris by activating alpha 1B-adrenoceptors and releasing calcium from dantrolene sensitive and insensitive intracellular stores, followed by inflow of calcium through verapamil sensitive calcium channels. Applied noradrenaline also activates chloroethylclonidine sensitive receptors on the arteriolar surface.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1991

Inflammatory mediators and cellular infiltration of the lungs in a guinea pig model of the late asthmatic reaction.

Alterations in cell numbers, vascular permeability, and concentrations of various inflammatory mediators in the lung were measured in a guinea pig model of the late asthmatic reaction. Animals sensitized by inhalation of ovalbumin were challenged with an aerosol of ovalbumin or saline, and bronchoalveolar lavage fluid (BALF) and peripheral blood were collected after periods ranging from 5 min to 72 h. Increased vascular leakage within the lungs was indicated by elevated BALF/plasma albumin ratios at all time points, and was maximal 6 h after challenge. There were increased numbers of eosinophils in BALF by 6 h after challenge and they remained elevated at least until 72 h. A corresponding increase in the proportion of blood leukocytes represented by eosinophils was observed at 6 and 17 h, which suggests that these cells may be drawn to the lung following their release into the circulation, but by 72 h the proportion in blood had returned to normal. A transitory neutrophilia was evident in BALF and blood 6 h after allergen exposure, but there were no allergen-induced changes in BALF numbers of macrophages, lymphocytes, epithelial cells, or mast cells (as assessed by concentrations of cell-associated histamine). beta-Glucuronidase activity was significantly increased in BALF of guinea pigs at 2 h and 17 h following challenge. The degree to which eicosanoids can be recovered in BALF was investigated by instilling a range of tritiated compounds into the lungs of normal guinea pigs at the time of lavage. Ratio high-performance liquid chromatography revealed that there had been little metabolism of the eicosanoids recovered in BALF. However, there was evidence for a rapid removal of these mediators from the lung, a process which will militate against their accurate quantitation in BALF. Histamine, prostaglandin D2, and thromboxane B2 were detected in BALF but did not differ between treatment groups, and levels showed no simple relationship with the other inflammatory changes measured.

Albumins↗