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Biomedical subjects

B D Watson

Publications and source records attributed to B D Watson.

At least 73 records · Page 4Linked to original sources
PubMed1986

Photochemically induced spinal cord injury in the rat.

We have developed in the rat a minimally invasive model of reproducible spinal cord injury initiated photochemically. With the exposed spinal column intact, 560 nm irradiation of the translucent dorsal surface induces excitation of the systemically injected dye, rose Bengal, in the spinal cord microvasculature. The resultant photochemical reaction leads to vascular stasis. Histopathological changes at 7 days include hemorrhagic necrosis of the central gray matter, edematous pale-staining white matter tracts and vascular congestion. At the level of cord irradiation (T8) the entire cord thickness is necrosed except for the periphery of the anterior funiculus. Voluntary motor function is consistently lost in the subacute phase of injury.

Animals↗
PubMed1986

Photochemically induced cortical infarction in the rat. 1. Time course of hemodynamic consequences.

Alterations in local CBF (LCBF) were assessed autoradiographically in the rat at several time points following photochemically induced cortical infarction. Cortical infarction of consistent size and location was produced by irradiating the brain with green light through the intact skull for 20 min following the systemic injection of rose bengal. A consistent pattern of altered LCBF was recorded in both ipsilateral and contralateral brain regions over the course of the study. At 30 min, a severely ischemic zone surrounded by regions of cortical hyperemia was apparent. LCBF was also depressed relative to control values in ipsilateral cortical regions remote from the irradiated area, while contralateral cortical structures were mildly hyperemic. By 4 h, the zone of severe ischemia had enlarged and its margins were no longer hyperemic. Ipsilateral cortical and some subcortical structures demonstrated significantly depressed levels of LCBF. At 5 days, LCBF throughout both ipsilateral and contralateral cortices was depressed compared with control values. By 15 days, LCBF had returned to control levels in most brain structures shown histopathologically not to be irreversibly damaged. The temporal sequence and magnitude of these hemodynamic alterations are consistent with findings in clinical studies in which repeated measurements of CBF have been carried out in patients with acute stroke. The ability to produce a cortical infarct that results in a consistent pattern of altered CBF should facilitate the investigation of stroke mechanisms responsible for these hemodynamic abnormalities.

Animals↗
PubMed1986

Photochemically induced cortical infarction in the rat. 2. Acute and subacute alterations in local glucose utilization.

Local CMRglu (LCMRglu) values were measured by [14C]2-deoxyglucose autoradiography in the rat at 4 h and 5 days following photochemically induced cortical infarction, and these data were compared with neuropathological findings in adjacent serial sections. At both time periods, LCMRglu was markedly reduced within the lesion center, and irregular regions of moderate-to-marked glucose hypermetabolism were noted within the marginal zone of the developing infarct. At 4 h, the hypermetabolic zones were shown by pathological examination to be characterized by normal-sized, moderately hyperchromatic neurons scattered among occasional dark, shrunken neurons within preserved neuropil. In contrast, the hypermetabolic zones at 5 days coincided with foci of intense macrophage infiltration, with dissolution of the neuropil. Significant decreases in glucose utilization were also demonstrated at 4 h within brain structures remote from the site of focal injury. These structures included the lateral and auditory cortices ipsilaterally, the striatum and thalamus ipsilaterally, and the hippocampus bilaterally. In addition to these remote metabolic effects, depressed metabolism occurred within the homologous cortical region contralateral to the site of infarction. By 5 days, glucose utilization was severely depressed in all ipsilateral cortical regions but not within any contralateral cortical region. Analysis of these data suggests that more than one mechanism is responsible for the metabolic alterations occurring within brain regions remote from the site of irreversible damage. Results are discussed in light of the hemodynamic alterations occurring in this stroke model, which are presented in the accompanying report.

Animals↗
PubMed1986

Vascular aspects and hemodynamic consequences of central nervous system injury.

We document here microvascular alterations occurring in models of mild, moderate, and severe cerebral ischemic injury. The relationship of the vascular abnormalities to the generation of hemodynamic alterations was also evaluated. Following periods of severe incomplete ischemia, scanning electron microscopic analysis of cerebral microvessels revealed the widespread production of cerebral endothelial microvilli. These microvilli increased in frequency as the ischemic insult was prolonged and remained prominent during periods of recirculation. Although these luminal projections would not be expected to inhibit reperfusion completely, they might increase microvascular resistance, leading to moderate hemodynamic impediments extending into the post-ischemic period. Similar periods of complete ischemia resulted in more severe microvascular alterations. Light and electron microscopic studies revealed a high frequency of compressed capillary lumina with vascular stasis. These compressed vessels were consistently surrounded by swollen astrocytic foot processes. When recirculation was instituted for 1 hr following 1 hr of complete ischemia, regions of non-perfusion were detected autoradiographically within brain regions destined to undergo ischemic infarction. Finally, in an attempt to determine the consequences of a primary microvascular insult on brain structure and function, the endothelial layer of microvessels in the cerebral cortex was injured using a noninvasive photochemical method. Endothelial damage led to platelet aggregation in both pial and intraparenchymal vessels. Occlusive thrombi were frequently detected with perivascular edema associated with vascular compression and severe focal ischemia. Ultrastructural blood-brain barrier studies using the horseradish peroxidase tracer demonstrated that protein leakage at the site of primary vascular injury resulted in tracer material in brain regions remote from the pathological lesion. The widespread leakage of protein tracer was associated with decreased blood flow in remote brain regions at several postirradiation periods. These data emphasize the importance of injury-induced microvascular dysfunction in the generation of brain lesions and hemodynamic abnormalities.

Animals↗
PubMed1986

Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System.

Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were transformed with a kanamycin resistance marker (neomycin phosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant.

Journal Article↗
PubMed1985

Induction of reproducible brain infarction by photochemically initiated thrombosis.

We have used a photochemical reaction in vivo to induce reproducible thrombosis leading to cerebral infarction in rats. After the intravenous injection of rose bengal, a potent photosensitizing dye, an ischemic lesion was formed by irradiating the left parietal convexity of the exposed skull for 20 minutes with green light (560 nm) from a filtered xenon arc lamp. Animals were allowed to survive from 30 minutes to 15 days after irradiation. Early microscopic alterations within the irradiated zone included the formation of thrombotic plugs and adjacent red blood cell stasis within pial and parenchymal vessels. Scanning electron microscopy revealed frequent platelet aggregates adhering to the vascular endothelium, often resulting in vascular occlusion. Carbon-black brain perfusion demonstrated that occlusion of vascular channels progressed after irradiation and was complete within 4 hours. Histopathological examination at 1, 5, and 15 days revealed that the associated infarct evolved reproducibly through several characteristic stages, including a phase of massive macrophage infiltration. Although cerebral infarction in this model is initiated by thrombosis of small blood vessels, the fact that the main pathological features of stroke are consistently reproduced should permit its use in assessing treatment regimens. Further, the capability of producing infarction in preselected cortical regions may facilitate the study of behavioral, functional, and structural consequences of acute and chronic stroke.

Animals↗
PubMed1985

New cloning vehicles for transformation of higher plants.

We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage lambda; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.

Journal Article↗
PubMed1985

Postischemic cerebral lipid peroxidation in vitro: modification by dietary vitamin E.

Using an in vitro system, we studied the effect of postischemic reoxygenation on cerebral lipid peroxidation in relation to the dietary intake of vitamin E (VE) in rats. Homogenates prepared from VE-deficient, -normal, and -supplemented brains, which were previously rendered ischemic for 30 min by decapitation, were incubated under air or nitrogen gas for 60 min. The extent of peroxidation in brain tissue was estimated by a thiobarbituric acid (TBA) test and by diene conjugation in total lipid extracts. The brain levels of alpha-tocopherol and of total and free fatty acids (FAs) were also determined. Aerobic incubation increased TBA reactants in all dietary groups; the effect was largest in the VE-deficient group, intermediate in the VE-normal group, and smallest in the VE-supplemented group. In contrast, nitrogen incubation did not alter the basal levels of TBA reactants except for a small rise associated with VE deficiency. Conjugated dienes changed in parallel with TBA reactants. alpha-Tocopherol decreased after aerobic incubation and also, to a lesser degree, after nitrogen incubation in each dietary group. Only in the reoxygenated samples of the VE-deficient group was there a significant fall in total polyunsaturated FAs. The levels of free FAs continuously increased throughout ischemia and subsequent incubation. However, the level of free polyunsaturated FAs was similar after aerobic and nitrogen incubation in each dietary group, and was not affected by VE. Thus, cerebral reoxygenation after ischemia propagates peroxidative reactions within esterified polyunsaturated FAs. The modification by VE of reoxygenation-induced lipid peroxidation suggests free radical mediation.

Aerobiosis↗
PubMed1984

Concurrent measurement of (Na+, K+)-ATPase activity and lipid peroxides in rat brain following reversible global ischemia.

Lipid peroxides, quantitated as lipid conjugated dienes, and (Na+, K+)-ATPase activity were assayed concurrently in brains of control rats and in three groups subjected to 30 min of reversible forebrain ischemia followed by 0, 1, and 4 hr of recirculation. Multiple small samples were taken from lateral, dorsolateral and medial cortex, hippocampus, thalamus and striatum following in situ freezing. (Na+, K+)-ATPase activity was elevated in hippocampus, dorsolateral and lateral cortex (P less than 0.10) and in thalamus (P less than 0.05) following 30 min ischemia. ATPase activity in medial cortex continued to increase during the first 1 hr of recirculation (P less than 0.10). Following 4 hr of recirculation, decreased enzyme activities were observed in all of these regions (lateral cortex and hippocampus, P less than 0.10). No changes in ATPase activity were observed in samples from striatum. Of the regional samples assayed for lipid peroxide content, the incidence of conjugated dienes as a function of recirculation time was 6% (0 hr), 23% (1 hr), and 17% (4 hr). For these samples, plots of normalized ATPase activity vs. tissue conjugated diene concentration revealed that normalized ATPase activity varied with recirculation time, but was independent of the magnitude of the lipid peroxidative process (expressed in terms of tissue conjugated diene concentration). These results suggest that disturbances in membrane structure and function presumed to arise from lipid peroxidation are not responsible for the behavior of the ATPase under the current in vivo conditions.

Animals↗
PubMed1984

Lipid peroxidation in vivo induced by reversible global ischemia in rat brain.

It has been hypothesized that ischemia, followed by reperfusion, facilitates peroxidative free-radical chain processes in brain. To resolve this question, rats were subjected to reversible global ischemia. From coronal sections of brains frozen in situ, small (ca. 2 mg) amounts of tissue were sampled from neocortex, hippocampus, and thalamus of both cerebral hemispheres of four groups of rats exposed to 30 min cerebral ischemia followed by 0, 30, 60, and 240 min of reperfusion, and from a control group subjected to the same operative procedures, except for the induction of ischemia. Heptane-solubilized total lipid extracts from these samples were analyzed spectroscopically in the 190-330 nm range for content of isolated (nonconjugated) double bonds and of conjugated diene structures; the latter are formed from isolated double bonds during peroxidation of unsaturated fatty acids. Spectra derived from tissue regions of rats subjected to ischemia, or ischemia followed by reperfusion, were compared to averaged, region-specific control spectra and were normalized to the original content of isolated double bonds in the peroxidized samples. The resultant difference spectra were analyzed in terms of ratios of conjugated diene concentration to the concentration of isolated double bonds originally at risk in the specific tissue zones considered. The peak representing conjugated diene formation was centered at 238 +/- 1 nm and was usually well resolved when the molar ratio [conjugated diene]/[isolated double bonds], expressed as a percentage [( CD]/[IDB]), was greater than 0.25%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

alpha-Tocopherol and ubiquinones in rat brain subjected to decapitation ischemia.

Levels of alpha-tocopherol (alpha-Toc), reduced ubiquinones (QH2) and oxidized ubiquinones (Q) were assayed in rat forebrain subjected to decapitation ischemia. Post-decapitation levels of alpha-Toc decreased by 16% at 3 min and 20% at 15 min. Increases in Q9H2 (83%) and in Q10H2 (107%) were observed immediately following decapitation; thereafter their levels began to decrease and approached to the pre-ischemic values at 15 min. In contrast, Q9 and Q10 tended to increase continuously during ischemia. The data indicate that complete ischemia results in distinct changes in the cerebral content of lipid-soluble antioxidants. The decrease of alpha-Toc may make the brain prone to peroxidative attack when cerebral tissue is subsequently reoxygenated.

Animals↗
PubMed1983

Compression-induced brain edema: modification by prior depletion and supplementation of vitamin E.

We studied the degree of edema resulting from focal brain compression in rats raised on vitamin E-deficient, -normal, or -supplemented diets. After release of 24 hours of epidural compression, edema developed ipsilaterally and was characterized by extravasation of serum protein, increased water and sodium content, and little change in potassium. The degree of swelling and increase of sodium in the previously compressed area were most pronounced in the vitamin E-deficient group and mildest in the vitamin E-supplemented group. Degradative processes of biomembranes seem to participate in the pathogenesis of brain edema; vitamin E may stabilize membranes by physicochemical interactions between the phytyl side chain and polyunsaturated phospholipids, or vitamin E may disrupt chains of free radical reactions.

Animals↗
PubMed1982

Potentiation of lipid peroxides by ischemia in rat brain.

Post-ischemic changes in energy metabolites and natural antioxidant compounds have been measured in rat brain in vitro concurrent with two different assays for peroxidized lipids. No exogenous free radical initiators were employed. In vitro oxygenation of minced brain preparations for periods of 10 minutes to 4 hours, following 5 minutes of preparatory ischemia, yielded increased levels of lipid conjugated dienes and TBA-reactive material, in contrast to anaerobically incubated preparations. However, either aerobic or anaerobic incubation of brain minces facilitated increased ratios of lactate:pyruvate and glutathione (oxidized):glutathione (reduced), as well as increased total ubiquinone content and loss of alpha-tocopherol. Observation of lipid radical formation in vivo was then attempted using rats given embolic stroke in one hemisphere and left in the post-ischemic condition for times up to 24 hours. Conjugated dienes were found in lipids extracted from the ipsilateral hemisphere but not from the contralateral hemisphere. These observations of conjugated dienes in vivo (formed presumably during post-ischemic reperfusion) and in vitro (facilitated by oxygenation of brain minces), indicate that lipid radical intermediates and associated chain peroxidation processes are potentiated by ischemia and occur during tissue reoxygenation.

Aerobiosis↗
PubMed1982

Structural and functional degradation of Ca2+:Mg2+-ATPase rich sarcoplasmic reticulum vesicles photosensitized by erythrosin B.

Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+:Mg2+-ATPase pump protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 +/- 0.19) X 10(-3) and (1.25 +/- 0.18) X 10(-3). Damage to vesicle function was assayed by measurements of increases in passive Ca2+ permeability. Rates of passive Ca2+ movement into the SR lumen were increased by dye photosensitization in proportion to radiation absorbed. Active Ca2+ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 microM and 1.16 microM, respectively. The photochemical lability of the Ca2+ pump protein and alterations in passive and active Ca2+ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.

Animals↗
PubMed1980

Measurement of surface potential and surface charge densities of sarcoplasmic reticulum membranes.

The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS-) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS- is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(e psi o/kT), where psi o is the membrane surface potential, e is the electronic charge, and kT has its usual meaning. The potential measured is characteristic of the ANS- bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS-, referred to as the aqueous phase bathing the surface, were in the range -10 to -15 mV. This was observed for the outside and inside surfaces of the Ca2+-ATPase-rich fraction of the SR and for both surfaces of the SR fraction rich in acidic Ca2+ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS- binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca2+ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca2+ permeability. The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types of SR. Values for the Ca2+-ATPase rich SR fraction were between 2.9 X 10(3) and 3.8 X 10(3) esu/cm2, (0.96 X 10(-6) and 1.26 X 10(-6) C/cm2) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6 X 10(3) and ca. 2.2 X 10(3) esu/cm2 (2.2 X 10(-6) and 0.73 X 10(-6) C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in the SR. The acidic Ca2+ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of A from the membrane surface. The experiments gave evidence for a Donnan effect on the K+ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in this SR fraction. The equilibrium constant for ANS- was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in the SR lumen.

Anilino Naphthalenesulfonates↗