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Biomedical subjects

H M Shapiro

Publications and source records attributed to H M Shapiro.

At least 109 records · Page 6Linked to original sources
PubMed1981

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y.

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.

Acridine Orange↗
PubMed1981

Intracranial hypertension and brain oedema in albino rabbits. Part 2: Effects of acute therapy with diuretics.

Increased intracranial pressure due to brain oedema was produced in albino rabbits by combining a cryogenic lesion in the left hemisphere with the intraperitoneal administration of 6-aminonicotinamide (cytotoxic agent). The most effective reduction in ICP (74%) was achieved when furosemide and mannitol were used in combination. When either mannitol or furosemide was employed alone, the average ICP reduction was approximately 53%. Peak ICP reduction occurred at 45 minutes with furosemide, 30 minutes with mannitol and furosemide combined, and at 60 minutes with a combination of mannitol and acetazolamide. Also studied simultaneously in these animals were investigated elastance (Em), brain water content, hemispheric water volume content, electrolytes, EEG, and gross pathology. Following therapy there was a statistically significance reduction of water content in the left hemisphere (cryogenic lesion) by all therapeutic modalities except with furosemide alone. In the right hemisphere the water content was reduced by furosemide and the furosemide-mannitol combination but not by the association of mannitol with acetazolamide. A significant decrease of brain sodium was noted only for the combination of mannitol and furosemide. This study indicates that effective reduction of cytotoxic-cryogenic brain oedema and intracranial hypertension can be obtained with a variety of diuretic agents. From the standpoint of tissue dehydration, restoration of tissue electrolyte balance, and rate of ICP reduction, the combination of furosemide-mannitol appears to offer advantages over furosemide alone, or acetazolamide-mannitol.

Acetazolamide↗
PubMed1981

Selective metabolic activation of the hippocampus during lidocaine-induced pre-seizure activity.

Neurophysiologic studies indicate that local anesthetic-induced seizures are generated in subcortical brain structures. The authors utilized a quantitative autoradiographic technique to measure cerebral metabolism during lidocaine-induced seizure activity in rats anesthetized with nitrous oxide. Local cerebral metabolic rate for glucose (l-CMRg) was determined when lidocaine infusion resulted in sustained electroencephalographic patterns consisting of approximately 100--125-mu volt discharges with a frequency of about 9 Hz, lasting 1-2 sec, and superimposed upon almost isoelectric periods lasting 1-3 sec. Significant reductions in 1-CMRg (30-70 per cent decreases) occurred in 19 of 26 regions surveyed. All areas of cerebral cortex had decreased glucose uptake following lidocaine administration. The hippocampus developed a striking increase in 1-CMRg of 237 per cent, while the amygdala and other related nuclei sustained metabolic rates similar to those present before lidocaine was given. This study demonstrates a coupling of metabolic activity with functional activity in subcortical structures recognized to be involved in the generation of local anesthetic seizure activity. Additionally, it reveals a heterogeneous response of cerebral metabolism to lidocaine infusion in the presence of subcortically localized seizures.

Animals↗
PubMed1981

The effects of high-frequency positive-pressure ventilation on intracranial pressure and brain surface movement in cats.

Compared to traditional, low-frequency ventilation (LFV), mechanical ventilation at high frequencies (60-200 breaths/min) and low tidal volumes (HFV) is known to: 1) eliminate respiratory-synchronous variations in blood pressure; 2) minimize ventilatory effects on the cardiovascular system; 3) reduce peak airway pressures; and 4) suppress spontaneous respiratory efforts. Since these and other properties may make HFV useful in patients with acute intracranial pathology, we studied the effects of HFV on intracranial pressure (ICP) in cats. Compared with LFV (rate 11/min, tidal volume equals 15 ml/kg), HFV (rate 100/min, VT equals 3.3 ml/kg) had little effect on mean arterial pressure, heart rate, right atrial pressure, mean ICP or mean cerebral perfusion pressures, even if baseline ICP was raised using an epidural balloon. However, HFV effectively eliminated ventilator-linked fluctuations in both blood pressure and ICP, and at all levels of mean ICP studied (4.8, 15, and 30 torr), significantly reduced the peak ICP seen during a single respiratory cycle. The reduction in ICP fluctuation and peak pressure was more pronounced as intracranial complicance fell. However, the physiologic significance of such a change in the ICP pressure waveform is unknown. Because of the observed influence of HFV on ICP fluctuations, we also examined its effects on the physical movement of the exposed brain, using a non-contact, inductive displacement measuring device. During LFV, the cortical surface moved "in and out" by 0.36 plus or minus 0.1 (plus or minus SD) mm, a distance sufficient to make microscopic focusing difficult. Changing to HFV reduced surface movement to 0.05 plus or minus 0.01 mm, producing a very stable surface. These results suggested that HFV may play a very important role in the intraoperative management oif patients undergoing certain neurosurgical procedures, particularly those requiring microsurgical techniques where reduced brain movement may facilitate surgery.

Animals↗
PubMed1980

Cardiopulmonary resuscitation in the nonhuman primate.

Seventeen adult male Cynamolgus monkeys were subjected to potassium chloride-induced cardiopulmonary arrests of 10 to 12 minutes. Pupils became dilated and fixed at a mean interval of 30 +/- 11 seconds, and the electroencephalogram became isoelectric at a mean interval of 34 +/- 13 seconds. The monkeys were resuscitated by means of oxygen, external cardiac message, epinephrine, calcium, and bicarbonate. The mean time required to resuscitate the monkeys was 140 +/- 70 seconds. After resuscitation, the monkeys were kept in an intensive care unit. Pupillary responsiveness and electroencephalographic activity were slow to return, with mean intervals of 90 +/- 85 and 108 +/- 36 minutes, respectively. Although all 17 monkeys were resuscitated, only 9 survived 96 hours or more. Of the 9 survivors, 2 appeared neurologically normal, 3 were moderately impaired, and 4 were severely impaired.

Animals↗
PubMed1980

Changes in endoneurial fluid pressure, permeability, and peripheral nerve ultrastructure in experimental lead neuropathy.

The dynamics of endoneurial edema were studied by quantifying endoneurial fluid pressure (EFP) during the development of lead neuropathy and correlating these data with changes in blood-nerve barrier permeability and with morphological alterations in nerves, capillaries, and Schwann cells. EFP measured from the sciatic nerve in control Long-Evans rats was 2.1 +/- 1.0 cm H2O. EFP was significantly elevated 7 weeks after animals were started on a diet containing 6% lead carbonate, and it increased progressively until a plateau in pressure was reached between weeks 9 and 11. Thereafter, EFP gradually returned to normal values. The progressive increase in EFP was highly correlated with the extravasation of osmotically active macromolecules, traced by fluorescein isothiocyanate-dextran compounds of graded molecular weight and by horseradish peroxidase (HRP). Electron microscopy revealed extravasation of HRP between endothelial cells, intranuclear inclusions characteristic of lead poisoning in Schwann cell nuclei, demyelination, and remyelination. The observation of intranuclear inclusions consistent with lead deposition in Schwann cells strengthens the hypothesis that extravasated lead in the interstitial fluid causes direct injury to Schwann cells, giving rise to demyelination. Nerve compliance was determined.

Animals↗
PubMed1980

Electrophysiology of T lymphocyte cholinergic receptors.

The presence of functional muscarinic-cholinergic receptors on at least some T lymphocytes is suggested by the existence of saturable, high-affinity, specific muscarinic binding in T cell-enriched, but not in B cell-enriched, cell suspensions and by observed cholinergic effects on lymphocytes, (e.g., increased lytic capacity of cytotoxic lymphocytes preincubated with muscarinic agents). In this study, we used flow cytometry and a fluorescent probe of membrane potential, the cyanine dye 3,3'-dihexyloxacarbocyanine iodide, to examine the effects of cholinergic agonists and antagonists on the membrane potentials of lymphocytes in T cell-enriched and B cell-enriched suspensions. Acetylcholine (AcCho) and carbamoylcholine (CbmCho) depolarized the membranes of T cells, but not of B cells; the maximal depolarization was produced by 10 nM AcCho or by 1 nM CbmCho. Depolarization following exposure to these concentrations of agonists was maximal by 5-8 min; T cell membrane potentials returned to control values by 13-15 min. Less marked depolarization was produced by 100 nM AcCho and 10 nM CbmCho; 100 pM CbmCho was only slightly less effective than 1 nM CbmCho, and the depolarization persisted 12 min after exposure. Depolarization induced by 1 nM AcCho was abolished when AcCho was combined with 10 nM atropine but not when AcCho was combined with 1 nM atropine or 200 nM d-tubocurarine. The time course of the membrane potential response and its dependence on the relative concentrations of AcCho and specific cholinergic blocking agents correlate well with both binding studies and biological effects. Our results provide evidence that T lymphocytes have functional muscarinic receptors; the flow cytometric method should be generally applicable to studies of the electrophysiology and pharmacology of receptor-ligand interactions.

Acetylcholine↗
PubMed1980

Cerebral circulatory and metabolic responses to intravenously administered lorazepam.

Cerebral vascular and metabolic effects of lorazepam were evaluated in ten awake monkeys by use of a modification of the Kety-Schmidt technique. Five received ketamine, 10 mg/kg, im, five to eight hours prior to the study, but all animals were otherwise treated identically. Monkeys receiving ketamine had significantly greater (P < 0.05) cerebral blood flow (CBF) values before lorazepam was given (46 +/- 1 ml/100 g/min) than did monkeys not receiving ketamine (41 +/- 1 ml/100 g/min), but in all other respects, premedicated and unpremedicated animals did not differ. Lorazepam administration did not significantly alter systemic arterial blood pressure or blood-gas values. However, it did decrease CBF by 26 per cent and increase cerebral vascular resistance (CVR) by approximately 25 per cent (P < 0.01). The cerebral metabolic rate for glucose (CMRg) decreased 42 per cent (P < 0.05). Following lorazepam administration, the cerebral metabolic rate for oxygen (CMRO2) decreased by 21-30 per cent. When combined CMRO2 data for the two anesthetic groups are pooled, this decrease is significant (P < 0.05). This study indicates that sedative doses of lorazepam decrease cerebral blood flow and metabolism with minimal effects on blood pressure and blood-gas values. Lorazepam administration did not produce any change in cerebral metabolism indicative of brain hypoxia or ischemia.

Anesthesia, Intravenous↗
PubMed1980

Heterogeneity of local cerebral blood flow-PaCO2 sensitivity in neonatal dogs.

Local cerebral blood flow (1-CBF) sensitivity to changes in arterial carbon dioxide tension (PaCO2) was measured in nitrous oxide-anesthetized newborn puppies using a quantitative autoradiographic technique and [14C]antipyrine as a tracer. 1-CBF was determined in four experimental groups at PaCO2 levels of 22, 34, 48, and 65 Torr, respectively. All seven brain areas studied demonstrated 1-CBF sensitivity to altered PaCO2. There were no significant differences (P greater than 0.05) in three subcortical white matter regions (frontal, parietal, occipital) in either 1-CBF or 1-CBF-CO2 sensitivity. With the exception of the thalamus, a reciprocal relationship existed between changes in local cerebral vascular resistance and blood flow. In the thalamus, 1-CBF increased at a greater rate than the 1-CVR reduction. The results show that CBF is heterogeneous in different brain areas of the neonatal dog at normocapnia and that differences in 1-CBF-CO2 sensitivity exist.

Animals↗
PubMed1980

A technique for the flow cytometric analysis of lymphocytes bearing histamine receptors.

Histamine receptors have been demonstrated on lymphocyte membranes by a variety of techniques. We now report a method that allows for the flow cytometric analysis of histamine receptors on human peripheral T cells. Histamine is conjugated to fluoresceinated human albumin by the coupling agent ECDI. This conjugated histamine compound (FHA-his) binds to approximately 45% of T cells. Fluoresceinated human albumin alone (FHA), not conjugated to histamine, does not bind to T cells. In addition, unconjugated histamine can inhibit completely the binding seen with FHA-his. We conclude that this technique demonstrates specific FHA-his binding to histamine receptors on T cells and can be used to determine the number of cells bearing such receptors. In addition, the reagent could be used with a cell sorter to isolate distinct histamine-receptor-bearing (HR+) cells for further immunologic study.

Blood Cell Count↗