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Biomedical subjects

D Franklin

Publications and source records attributed to D Franklin.

At least 19 recordsLinked to original sources
PubMed1992

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.

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PubMed1992

Generation of digoxigenin-labelled double-stranded and single-stranded probes using the polymerase chain reaction.

As the polymerase chain reaction (PCR) can be used for the generation of vector-free probes, the optimum conditions for incorporation of digoxigenin-11-dUTP into hepatitis B virus (HBV) probes have been investigated. High yields of double-stranded or single-stranded probes can be obtained by utilizing a pair of primers or one primer alone. The probes were tested by dot-blot hybridization on HBV plasmid DNA, slot-blot hybridization on total cellular RNA of Alexander cells and Southern blot hybridization on cellular DNA of Alexander cells and HBV plasmid DNA. They were also tested by in situ hybridization (ISH) on HBV-positive biopsy liver tissue. A ratio of dig-dUTP:dTTP of 1:3 gave highest sensitivity in DNA hybridization. No loss of amplification efficiency and sensitivity was observed when the final concentration of dig-11-dUTP and dTTP was reduced to 20 microM and 60 microM respectively, compared to 200 microM each of dATP, dCTP, dGTP. Several different sizes of double-strand probes were compared by dot-blot hybridization. Longer probes were more sensitive. Strong signal could also be obtained by combination of two or three small probes, which have overlapping sequences. Single-stranded DNA probes had advantages of simplicity of use, high sensitivity and strand specificity.

Base Sequence↗
PubMed1991

Bidirectional function of coronary collateral channels in conscious dogs.

STUDY OBJECTIVE: The aim was to investigate the bidirectional functional adequacy of collateral perfusion in conscious dogs. DESIGN: Left circumflex coronary artery (LCCA) occlusions of 1 or 2 min duration were repeated to stimulate the development of collateral perfusion to the LCCA area, and the left anterior descending coronary artery (LAD) was occluded once daily to evaluate the development of retrograde LCCA-LAD flow. SUBJECTS: 7 male mongrel dogs were used, weight 25-28 kg. MEASUREMENTS AND MAIN RESULTS: Coronary collateral flow from the LCCA to the LAD perfusion area was measured as the abrupt decrease in the LCCA flow (implanted Doppler transducer) upon release of a brief LAD occlusion. Measurements were repeated daily during the development of collaterals induced by repeated, brief occlusions of the LCCA. After 35(SD17) days of such occlusions; there was no sustained reduction in LCCA regional myocardial function during an LCCA occlusion, and reactive hyperaemic repayment following the occlusion was negligible. Before and after collateral development, the LCCA to LAD collateral flow increased from 1.1(0.2) to 8.6(5.1) cm.s-1. LAD systolic segment shortening during the LAD occlusion increased from 2.1(2.0)% (first occlusion) to 19.3(8.6)% (last occlusion). CONCLUSIONS: LAD to LCCA collaterals serve as functionally significant bidirectional perfusion conduits, and monitoring of collateral perfusion development is practical by measuring the step reduction in LCCA flow upon abrupt release of an LAD occlusion.

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PubMed1991

Effects of coronary occlusion duration on reactive hyperemia in conscious dogs and ponies.

Coronary reactive hyperemia duration (RHD) and coronary blood flow debt repayment (BFDR) were compared in conscious dogs and ponies instrumented with coronary artery Doppler flow probes and pneumatic occluders. Additional ponies were instrumented with pacing electrodes. With the use of a Latin square design, eight animals of each species were subjected to a randomized series of nine coronary occlusions ranging from 5 s to 2 min in duration. In both species, postocclusion blood flow velocity rose rapidly and plateaued at similar peak levels relative to control, but in ponies this plateau lasted significantly longer. The interspecies difference in plateau duration increased as a function of coronary occlusion duration (COD). RHD ranged from 19.5 +/- 5.9 to 139.7 +/- 5.9 s in dogs and from 26.6 +/- 9.0 to 395.0 +/- 9.0 s in ponies. The slope of the RHD vs. COD curve was steeper in ponies. BFDR was similar in dogs and ponies at the shortest COD (418.1 +/- 26 vs. 451.4 +/- 58%) but declined in dogs as a function of COD to 232.3 +/- 26%. In ponies, BFDR increased as a function of COD to a maximum of 945.4 +/- 58% with a 60-s occlusion and then declined to 614.3 +/- 58%. RHD was not significantly altered in ponies when heart rate was changed to match that in dogs. Although the underlying basis for these interspecies differences in RHD and BFDR was not determined, the differences were considered to be too large to be explained by animal model differences in coronary conductance, collateral blood flow, or myocardial oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

Effects of nitroglycerin and diltiazem on well-developed coronary collateral circulation in conscious dogs.

The purpose of the present study was to compare the effects of nitroglycerin and diltiazem on coronary collateral circulation. Studies were conducted in 8 conscious dogs instrumented for the measurement of left circumflex coronary artery (LCCA) flow, subendocardial segment lengths in areas perfused by the LCCA, and left anterior descending coronary artery (LAD). Brief, repeated LCCA occlusions sufficiently developed collateral vessels for the resting metabolic requirement in the LCCA region. One week following the cessation of repeated LCCA occlusions, two-minute coronary occlusions with and without drug pretreatment were performed on separate days. The ischemic responses to coronary occlusions were not altered by diltiazem (50 micrograms/kg, IV), but nitroglycerin (5 micrograms/kg, IV) attenuated myocardial ischemia definitely. The authors conclude that nitroglycerin produces greater effects than diltiazem in attenuating myocardial ischemia in the collateral dependent zone when effects of each drug on systemic and coronary circulation were minimized by pretreatment with small doses.

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PubMed1978

Measurement of specific laryngeal muscle function by ultrasound.

Ultrasonic dimension gauges were implanted in the crico-arytenoideus dorsalis muscle in a mature horse. Resting and contracted muscle length and average contraction rate were measured serially in the conscious state over a period of 25 days. Results suggest that specific laryngeal muscle function in the horse may be defined with this approach. Since man and horse are both known to suffer from laryngeal hemiplegia, this experimental preparation may help provide information of benefit to both species.

Animals↗
PubMed1978

Myocardial infarction in the baboon: regional function and the collateral circulation.

Occlusion of the anterior descending coronary artery was produced in sedated baboons 7-15 days after implantation of a micromanometer and ultrasonic crystals for measurement of regional left ventricular dimensions in ischemic, marginal, and control segments. One minute after coronary occlusion (CO), ischemic segments exhibited a marked systolic bulge with wall thinning, and percent systolic shortening of marginal segments decreased. Over the ensuing weeks, there was a progressive increase of end-diastolic lengths in marginal and ischemic segments, whereas systolic shortening in these segments did not improve significantly. Control segments did not change. In control baboons, the coronary collateral index was 55 +/-25 (SE) compared to 560 +/- 74 in normal dogs. One month after CO, the collateral index was 543 +/- 144 in baboons compared to 6,685 +/- 716 in dogs, regions of normal tissue were seen in the infarct (14.2 +/- 2% of left ventricular mass). Minimal coronary collateral development in the baboon provides a likely explanation for differences from the dog in regional functional responses and in the character of the infarct.

Animals↗
PubMed1978

Measurement of multiple simultaneous small dimensions and study of arterial pressure-dimension relations in conscious animals.

This paper describes the development of several important modifications that were incorporated into the ultrasonic, transit-time dimension system in order to obtain multiple simultaneous, instantaneous, and continuous measurements of the external dimensions of the aorta and its major branches in conscious, unrestrained animals. At operation a pair of small piezoelectric crystals was sutured to arterial adventitia, and a miniature pressure gauge was implanted in the vessel at the same cross-sectional plane. After recovery from surgery, wall motion was not altered appreciably and scarring was minimal. This technique allows long-term monitoring of aortic pressure-dimension relations and is applicable for small (fetal and neonatal) as well as large (adult dogs and sheep) animals. When vessel wall thickness is measured, stress-radius analysis can be performed so as to compute vascular elastic stiffness as a function of stress. Moreover, the suitability for radiotelemetry of the pressure and dimension signals measured with this technique enables the study of these parameters in unrestrained animals, e.g., during spontaneous severe exercise.

Angiotensins↗
PubMed1978

Alteration of the left ventricular diastolic pressure-segment length relation produced by the pericardium. Effects of cardiac distension and afterload reduction in conscious dogs.

Left ventricular pressure and segment length were measured in seven conscious chronically instrumented dogs with the pericardium intact and 3-9 days after pericardiectomy. Diastolic pressure-length plots were obtained under control conditions and after acute volume loading followed by sodium nitroprusside infusion. In all dogs with intact pericardium, volume loading displaced the entire diastolic pressure-length curve upwards and sodium nitroprusside shifted it toward control. After pericardiectomy the pressure-segment length data during control, volume loading and sodium nitroprusside fell on a single curve (intercepts and slopes not statistically different); After dextran infusion, intrapericardial pressure rose from control 1.5 +/- 0.7 mm Hg to 8.2 +/- 0.5 mm Hg, and it fell to 4.8 +/- 0.1 mm Hg after nitroprusside. Therefore, in acute cardiac dilatation the pericardium contributed significantly to the increased left ventricular diastolic pressure and to the fall during sodium nitroprusside infusion and appeared responsible for shifts in the diastolic pressure-segment length relation.

Animals↗
PubMed1978

[Nitroglycerin in experimental myocardial infarction in the baboon. The effect of reducing the pre-charge and post-charge on regional myocardial function].

A left anterior descending coronary artery occlusion was produced in the sedated state in baboons previously instrumented with a left ventricular micromanometer and ultrasonic crystals measuring segmental myocardial lengths of control, marginal and ischemic regions. 3-8 days after the onset of the ischemia, nitroglycerin (0.4 mg IV) was injected. Following the injection (NG), heart rate increased significantly compared to the control ischemic state (CIS); end diastolic pressure and systolic pressure decreased significantly. In control segments, VCF increased from 0.99 +/- 0.11 circ/s to 1.26 +- 0.11 circ/s (p less than 0.01); in marginal segments VCF increased (CIS: 0.32 +/- 0.07 circ/s; NG: 0.42 +/- 0.09/s; p less than 0.01); in ischemic segments, the bulge observed before the injection persisted after nitroglycerin. Increased performances of control and marginal regions were attributed mainly to afterload reduction although preload reduction tended to decrease this response. The contribution of improved marginal function was small indicating that improvement of active hemodynamic function was largely due to changes in control segments function.

Animals↗
PubMed1977

Hyperfunction with normal inotropic state of the hypertrophied left ventricle.

Conscious dogs were instrumented with an inflatable cuff around the ascending aorta, a high-fidelity micromanometer in the left ventricle (LV), and pairs of ultrasonic crystals for measurements of LV wall thickness and internal LV diameter. Wall stress (WSt) and mean velocity of wall shortening (VCF) were calculated. Mean force-velocity relations and WSt-diameter loops in single contractions were then analyzed over a range of matched systolic pressures during acute aortic constrictions both before and after induction of chronic hypertrophy by sustained aortic constriction. At normal LV systolic pressures and at each matched level of systolic LV pressure, wall shortening velocity was increased in the hypertrophied ventricle. However, force-velocity relations obtained by relating mean VCF to mean WSt at various stress levels fell on the same relation as during control. The linear relation between LV diameter and pressure at the end of ventricular ejection was shifted to the left in the hypertrophied ventricle, indicating enhanced shortening. However, linear WSt-diameter relations at end-ejection were not different in control and hypertrophied hearts. These findings indicate that the ventricle hypertrophied by pressure overload exhibited hyperfunction as a pump but that its myocardium had a normal level of inotropic state.

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