Intermittent perfusion protects the brain during deep hypothermic circulatory arrest.

BACKGROUND: Deep hypothermic circulatory arrest (DHCA) has been shown to cause impairment in recovery of cerebral blood flow (CBF) and cerebral metabolism (CMRO2) proportional to the duration of the DHCA period. This effect on CMRO2 may be a marker for brain injury, because CMRO2 recovers normally after cardiopulmonary bypass (CPB) when DHCA is not used. The aim of this study was to investigate the effects of intermittent perfusion during DHCA on the recovery of CMRO2 after CPB and to correlate these findings with electron microscopy (EM) of the cerebral microcirculatory bed. METHODS: Fifteen neonatal piglets were placed on CPB and cooled to 18 degrees C. Each animal then underwent either: (1) 60 minute continuous CPB (control), (2) 60 minute uninterrupted DHCA (UI-DHCA), or (3) 60 minute DHCA with intermittent perfusion (1 minute every 15 minutes) (I-DHCA). All animals were then rewarmed and weaned from CPB. Measurements of CBF and CMRO2 were taken before and after CPB. A further 9 animals underwent CPB without DHCA (2 animals) or with DHCA (7 animals), under various conditions of arterial blood gas management, intermittent perfusion, and reperfusion time. RESULTS: UI-DHCA resulted in significant impairment to recovery of CMRO2 after CPB (p < 0.05). Regardless of the blood gas strategy used, the EM after UI-DHCA revealed extensive damage characterized by perivascular intracellular and organelle edema, and vascular collapse. I-DHCA, on the other hand, produced a pattern of normal CMRO2 recovery identical to controls, and the EM was normal for both these groups. CONCLUSIONS: Intermittent perfusion during DHCA is clinically practical and results in normal cerebral metabolic and ultrastructural recovery. Furthermore, the correlation between brain structure and CMRO2 suggests that monitoring CMRO2 during the operation may be an outstanding way to investigate new strategies for neuroprotection designed to reduce cerebral damage in children undergoing correction of congenital cardiac defects.

In vivo accumulation of iron on crocidolite is associated with decrements in oxidant generation by the fiber.

In vivo exposures to fibrous silicates are characterized by the formation of asbestos bodies. These structures consist of the original fiber with a coating of inexact composition, but it will include iron and protein. We tested the hypothesis that this iron, accumulated on asbestos bodies, participates in electron transport and oxidant generation. Thirty-day-old, male guinea pigs were intratracheally instilled with 1.0 mg crocidolite. Six months later, the animals were anesthetized, euthanized, and the fibers were isolated from the lungs. Energy-dispersive x-ray analysis and x-ray photoelectron spectroscopy confirmed an accumulation of metal onto the fiber after in vivo exposure. Stains for iron demonstrated a heterogeneous distribution of the metal on the silicate, while the uptake of a commercially available polyclonal antibody to ferritin localized to beaded enlargements along the coated fibers. Chelatable [Fe3+] associated with the fiber increased after in vivo exposure. However, oxidant generation by asbestos bodies was decreased relative to uncoated fibers despite the elevation in the concentration of metal associated with the crocidolite. We conclude that iron is accumulated onto fibers in the lungs of guinea pigs. Some portion of this accumulation of iron is in the form of ferritin, and this metal is not chemically reactive in oxidant production. Asbestos bodies may represent a successful attempt by the host to sequester the metal adsorbed to the surface of a fiber and diminish the oxidative challenge introduced by a fibrous silicate. Subsequently, the generation of free radicals by the fibrous silicate is diminished.

Elemental composition of Na pump inhibited rabbit aorta VSM cells by electron probe X-ray microanalysis.

The Na pump in vascular smooth muscle (VSM) is likely to influence not only intracellular Na content but also the content and distribution of other cations and anions measured by electron probe X-ray microanalysis (EPXMA). The hypothesis we tested was that EPXMA of pump-inhibited VSM would yield a characteristic cellular elemental profile, providing insight into the contribution of the Na pump to the intracellular milieu and an approach to identifying when VSM operates under the constraints of pump inhibition. We assessed the contractile state and elemental EPXMA profile of rabbit aorta that was either quiescent or contracted by serotonin (10(-6) M) or ouabain (10(-6) M). VSM cytoplasm showed the anticipated low Na (28 +/- 2 mM) and high K (182 +/- 5 mM) content. With ouabain, Na rose and K fell to reverse the Na-to-K ratio (0.15 +/- 0.01 vs. 6.6 +/- 0.3; P < 0.01). With serotonin, the ratio rose slightly (0.28 +/- 0.2; P < 0.05). Nuclei and mitochondria showed a similar pattern. CI showed a small increase (56 +/- 2 to 102 +/- 4 mM) with ouabain, a shift that could not be accounted for on the basis of charge redistribution to maintain neutrality because the change in Na and K were essentially offsetting. EPXMA measures total and not ionized Ca. If changes in cytoplasmic Ca occurred, they were too small to be measured by the imaging methods employed. The sustained, myogenic contractile response of VSM to Na pump inhibition shows a characteristic elemental profile that could prove useful in its identification. Direct measurement of membrane potential during the myogenic response to Na pump inhibition should have a high priority.

Calcium depletion and repletion in cultured chick heart muscle cells.

Calcium-free incubation followed by exposure to calcium damages naturally occurring cardiac muscle preparations irreversibly. Whether the observed calcium overload during calcium repletion is a primary cause for, or a secondary consequence of, sarcolemmal disruption and cell injury is controversial. We used cultured embryonic chicken heart muscle cells to correlate ionic, metabolic, and ultrastructural changes during calcium depletion (0Ca, 1 mM EGTA) and repletion. After 10 min of calcium depletion, intracellular Na increased four-fold above control levels, intracellular K decreased by 26%, total cell Ca decreased by 81%, and cytosolic ionized Ca concentration decreased by 87%. Within 10 min of subsequent calcium repletion, total cell Ca transiently increased to four-fold above control, cytosolic ionized Ca concentration transiently increased to twice control, and both Na and K returned toward control levels; by 3 h of calcium repletion, physiological cation (Na, K, Ca) contents were restored and adenine nucleotide contents were normal. Long-term (i.e. 120 min) calcium depletion did not significantly reduce cell ATP levels, but increased adenine nucleotide turnover as indicated by adenosine and lactate release; after 60 min of subsequent calcium repletion, ionic and metabolic parameters were returned to control levels. During calcium depletion (both short- and long-term) and subsequent repletion, no ultrastructural changes occurred. When Mg was also removed during calcium depletion, the ionic changes during depletion and subsequent repletion were enhanced. When 10 microM CCCP was present during calcium depletion and repletion, cytosolic ionized Ca concentration increased to six-fold above control with no increase in total cell Ca content, suggesting that the increased Ca is buffered, in part, by mitochondria. These results indicate that an increase in Ca per se, occurring when high energy phosphate levels and/or cellular Ca buffering capacity are maintained, does not seem to be associated with irreversible cell injury.

Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Calcium measurements with electron probe X-ray and electron energy loss analysis.

This paper presents a broad survey of the rationale for electron probe X-ray microanalysis (EPXMA) and the various methods for obtaining qualitative and quantitative information on the distribution and amount of elements, particularly calcium, in cryopreserved cells and tissues. Essential in an introductory consideration of microanalysis in biological cryosections is the physical basis for the instrumentation, fundamentals of X-ray spectrometry, and various analytical modes such as static probing and X-ray imaging. Some common artifacts are beam damage and contamination. Inherent pitfalls of energy dispersive X-ray systems include Si escape peaks, doublets, background, and detector calibration shifts. Quantitative calcium analysis of thin cryosections is carried out in real time using a multiple least squares fitting program on filtered X-ray spectra and normalizing the calcium peak to a portion of the continuum. Recent work includes the development of an X-ray imaging system where quantitative data can be retrieved off-line. The minimum detectable concentration of calcium in biological cryosections is approximately 300 mumole kg dry weight with a spatial resolution of approximately 100 A. The application of electron energy loss (EELS) techniques to the detection of calcium offers the potential for greater sensitivity and spatial resolution in measurement and imaging. Determination of mass thickness with EELS can facilitate accurate calculation of wet weight concentrations from frozen hydrated and freeze-dried specimens. Calcium has multiple effects on cell metabolism, membrane transport and permeability and, thus, on overall cell physiology or pathophysiology. Cells can be rapidly frozen for EPXMA during basal or altered functional conditions to delineate the location and amount of calcium within cells and the changes in location and concentration of cations or anions accompanying calcium redistribution. Recent experiments in our laboratory document that EPXMA in combination with other biochemical and electrophysiological techniques can be used to study, for example, sodium and calcium compartmentation in cultured cardiac cells. Such analyses can also be used to clarify the role of calcium in anoxic renal cell injury and to evaluate proposed ionic defects in cells of individuals with cystic fibrosis.

Quantitative microchemical imaging of calcium in Na-K pump inhibited heart cells.

Quantitative electron probe X-ray imaging techniques have been utilized to determine simultaneously the element content within a single cultured embryonic chick heart cell and its intracellular compartments as well as the average elemental content of several heart cells within a population. These features of microchemical imaging have permitted establishment of data regarding: (1) the heterogeneity of calcium accumulation in mitochondrial, cytoplasmic and nuclear compartments under conditions which elevate total cell calcium without producing irreversible cell injury; and (2) the variability of calcium accumulation from cell to cell within the population sampled. The results indicate that during Na-K pump inhibition (K-free HT-BSS, 10(-4) M ouabain, 60 min) elevation of mitochondrial calcium, measured in situ by electron probe X-ray microanalysis, to levels more than 100 times greater than in the basal state, may not cause irreversible mitochondrial uncoupling and cell death.

Biochemical and structural changes in cultured heart cells induced by metabolic inhibition.

We examined the relationship between ionic homeostasis, ATP, and irreversible cell injury in cultured embryonic chick heart cells treated with rotenone (10(-4) M) alone or in combination with iodoacetate (IAA) (10(-3) M), in the presence of extracellular calcium (Ca0) (2.7 mM) and its nominal absence. Changes in Na, K, and total cell Ca content did not correlate with parameters indicative of irreversible injury, i.e., ultrastructural damage or lactate dehydrogenase (LDH) release. Because structural defects in the plasma membrane occurred without a significant release of LDH after exposure to rotenone plus IAA for 1 h, LDH release appears to be a relatively late event in cell injury. In addition, cells exposed to rotenone in the presence of Ca0 for 2.5 h showed a significant fall in ATP and a rise in LDH release. This response was attenuated in the nominal absence of Ca0, and the addition of rotenone caused an eightfold increase in intracellular sodium (Nai), whereas in the presence of Ca0, Nai increased only threefold in 2.5 h. Thus Ca0 appears to promote Nai-Ca0 exchange and lead to an increase in cell Ca that can then stimulate ATP breakdown by Ca-activated ATPases. Of the measured variables associated with myocardial cell injury, a decline in ATP correlates best with changes in either LDH or morphology. The apparent lack of correlation between changes in intracellular ion content, LDH release, and morphology supports the conclusion that myocardial cell injury is a multifactorial process.

Suspension of medullary thick ascending limb tubules from the rabbit kidney.

A procedure for isolating a suspension of tubules derived from the rabbit medullary thick ascending limb is described. The purity of the preparation was assessed by microscopy and enzyme assays and the viability of the preparation was assessed by measuring oxygen consumption. Microscopy revealed that the suspension contains 95% thick ascending limbs and that the isolation procedure preserves the structure of the epithelium except for the loss of the basement membrane. The preparation had a high activity of calcitonin-sensitive adenylate cyclase, a marker enzyme for the medullary thick ascending limb. Control oxygen consumption was considerably higher than that reported for proximal tubules in the literature, and nystatin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone addition produced a more than 100% increase in oxygen consumption. Furosemide inhibited the oxygen consumption by 43% and ouabain inhibited it by 42%. Furosemide inhibited sodium chloride entry without directly affecting the Na-K-ATPase or cellular metabolism. Chloride removal depressed oxygen consumption to the same extent as furosemide, but some of this action was through direct inhibition of cellular metabolism.

Effects of colchicine and cytochalasin B on vasopressin- and cyclic adenosine monophosphate-induced changes in toad urinary bladder.

Coincident with an increase in water permeability, the ridge-like surface structures of toad bladder granular cells transform to individual microvilli after stimulation with vasopressin (VP) or cyclic adenosine monophosphate (cAMP) by a mechanism that is yet to be defined. To explore the possible role of microtubules and microfilaments in this cell response, colchicine and cytochalasin B were employed to determine whether interference with the function of these components of the cytoskeletal system would prevent the VP- and cAMP-induced conversion of ridges to microvilli. Incubation of toad urinary bladders in 10(-4) M colchicine for 4 hours or 10(-5) M cytochalasin B for 90 minutes before stimulation with 20 mU. per ml. of VP markedly inhibited osmotic water flow. However, neither agent prevented the striking conversion of ridges to surface microvilli induced by VP and cAMP as seen with scanning electron microscopy. In addition, the ridges characteristic of granular cells were maintained in control bladders incubated with colchicine or cytochalasin B, but left unstimulated. Under the conditions of these experiments, these findings suggest that microtubules and microfilaments are not essential for maintenance of normal surface configuration in granular cells of toad urinary bladder, and that they are not involved in the mechanism responsible for VP- and cAMP-induced surface changes that occur in association with increased water permeability of this epithelium.

Effect of vasopressin and serosal hypertonicity on toad urinary bladder.

The mucosal surface of toad urinary bladder was examined with scanning electron microscopy following its exposure to 20 mU/ml of vasopressin (VP), 10(-4) M 8-bromo-cAMP, 1 mM acetylcholine chloride, serosal hypertonicity, or a hypotonic bathing medium. After a 30-min exposure to VP, the arborizing ridge-like surface pattern typical of granular cells was transformed into microvilli, a response that was not dependent on transepithelial osmotic water movement. An identical response occurred following a 30-min exposure of the bladder to 8-bromo-cAMP, again in the absence of an osmotic gradient. Microvillus formation was not observed when cell volume was increased by incubation of tissue in half-normal amphibian Ringer's solution for 30 min, or with exposure to acetylcholine, which caused accentuation of the convexity of the apical surface of the granular cell similar to that observed with VP-induced osmotic water flow. However, 60 min of incubation in a hypertonic serosal medium (mannitol, 240 mM) caused transformation of ridges to microvilli mimicking the picture obtained with VP. These findings establish that transepithelial osmotic water flow with cell swelling is not required for microvillus formation on the apical surface of granular cells following VP stimulation, and that the surface changes are not due to cell swelling alone or to changes in the configuration of the apical plasmalemma. The results also suggest that the response to VP is mediated via the generation of cAMP. Finally, this study demonstrates that serosal hypertonicity also causes transformation of ridges to microvilli by a mechanism that is yet to be defined.