Shock-induced bubble jetting into a viscous fluid with application to tissue injury in shock-wave lithotripsy.

Shock waves in liquids are known to cause spherical gas bubbles to rapidly collapse and form strong re-entrant jets in the direction of the propagating shock. The interaction of these jets with an adjacent viscous liquid is investigated using finite-volume simulation methods. This configuration serves as a model for tissue injury during shock-wave lithotripsy, a medical procedure to remove kidney stones. In this case, the viscous fluid provides a crude model for the tissue. It is found that for viscosities comparable to what might be expected in tissue, the jet that forms upon collapse of a small bubble fails to penetrate deeply into the viscous fluid "tissue." A simple model reproduces the penetration distance versus viscosity observed in the simulations and leads to a phenomenological model for the spreading of injury with multiple shocks. For a reasonable selection of a single efficiency parameter, this model is able to reproduce in vivo observations of an apparent 1000-shock threshold before wide-spread tissue injury occurs in targeted kidneys and the approximate extent of this injury after a typical clinical dose of 2000 shock waves.

[Calcium oxalate stone formation. New pathogenetic aspects of an old disease]. / Kalziumoxalatsteinbildung. Neue pathogenetische Aspekte einer alten Erkrankung.

Calcium oxalate (CaOx) urolithiasis is a very common disorder. Surprisingly, the pathogenetic mechanisms leading to CaOx stone formation have been largely unknown so far. The long-accepted simple explanation by an exceeding of the solubility product of lithogenic substances in the urine cannot sufficiently describe the complex processes. Deviating from the hypothesis that proclaims that the initial crystal deposition takes place in the lumens of renal tubules, new insights suggest a primary plaque formation in the interstitial space of the renal papilla. Initially, calcium phosphate (CaPh) crystals and organic matrix are deposited along the basement membranes of the thin loops of Henle and extend further in the interstitial space to the urothelium, constituting the so-called Randall's plaques that can be regularly found during endoscopy of CaOx-stone-forming patients. These CaPh crystals seem to be the origin for the development of future CaOx stones, which form by the attachment of further matrix molecules and CaOx from the urine to the plaque. The driving forces, the exact pathogenetic mechanisms, and the involved matrix molecules remain largely unknown. Possibly, completely different pathomechanisms lead to the common clinical diagnosis of"CaOx stone former."

Glomerular endothelial cells in uranyl nitrate-induced acute renal failure in rats.

In uranyl nitrate (UN)-induced acute renal failure (ARF) glomerular ultrafiltration coefficient (K(f)) decreases because of unknown reasons. Since transport of water across the glomerular capillary wall occurs predominantly extracellularly through the endothelial fenestrae (EF), a reduction in the diameter and/or the density of EF can reduce the extracellular filtration area and the glomerular K(f). To examine this possibility, ARF was induced in rats by intravenous administration of UN in low (15 mg/kg) and high doses (25 mg/kg). Fenestral density ( x+/-SEM) per 5 cm(2) from the scanning electron micrographs (x30,000) was 107+/-10, 103+/-9, and 101+/-11 at 2, 7, and 17 h after the intravenous administration of bicarbonate saline to the control rats. In the low-dose UN group the EF density was 91+/-2, 52+/-8, and 45+/-11 at 2, 7, and 17 h after the injection, whereas for the high-dose group at corresponding time intervals the EF density was 95+/-3, 54+/-9, and 44+/-10. Fenestral diameters, in Angstrom units ( x+/-SEM), were 751+/-53, 765+/-43, and 764+/-37 at 2, 7, and 17 h after the injection of bicarbonate saline to control rats. At corresponding intervals after the administration of UN, the fenestral diameters were 501+/-61, 472+/-28, and 438+/-98 for the low-dose group and 525+/-43, 470+/-39, and 440+/-56 for the high-dose group. 2, 7, and 17 h after the injection of UN, fenestral area of the low-dose group decreased to 52.1, 30.1, and 24.6% of the controls, whereas in the high-dose group, the fenestral area declined to 54.3, 30.2, and 23.6% of the controls. Administration of UN (15 mg/kg) to sodium-loaded rats did not alter renal function or endothelial cell morphology. It is suggested that in UN-induced ARF the morphological alterations in endothelial cells reduce the K(f) of glomerular capillaries by reducing the filtration area.

Function and structure in the diphenylamine-exposed kidney.

Standard micropuncture and microdissection techniques were used to examine the function and structure of nephrons in rats whose kidneys were made cystic by dietary exposure to diphenylamine. Heterogeneity characterized the lesion, with dilation and frank cyst formation occurring in 5-30% of nephrons. Elevated intraluminal hydrostatic pressures, occurring in the absence of increased glomerular filtration or decreased net water reabsorption, were recorded in dilated, but not in nondilated nephrons. Structural studies demonstrated communication of dilated nephrons with cysts, concretions of debris within tubular lumens, evidence of extrinsic pressure by cysts on adjacent tubules, and apparent luminal narrowing of some proximal tubules. These observations were used to explain prolonged loop of Henle transit times and occasional failure to detect [3H]inulin excretion after microperfusion into dilated tubules. It was concluded that the elevated hydrostatic pressures in the dilated nephrons of diphenylamine-exposed kidneys were the consequence of variably severe and frequently incomplete tubular occlusion. These findings support the hypothesis that cyst formation is a consequence of partial obstruction and elevated intratubular pressure in this model and perhaps in other susceptible mammalian kidneys.

Structural changes of isolated hepatocytes during treatment with digitonin.

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.

Strategies for improved shock wave lithotripsy.

Research in lithotripsy that started with the effort to characterize acute shock wave damage to the kidney has led to advances on several fronts, including discovery of strategies that have improved clinical treatment. It is appreciated now that shock wave trauma is primarily a vascular lesion, that injury is dose dependent, and that hemorrhage can be severe and can lead to a permanent loss of functional renal mass. Studies of the renal functional response to lithotripsy have shown that shock wave treatment triggers vasoconstriction in the kidney. This finding has been turned to advantage, and it is now known that when treatment is begun using low amplitude pulses, subsequent high amplitude shock waves are far less damaging. Thus, when shock waves are delivered judiciously, treatment can have a protective effect. The finding that cavitation is a key mechanism in vessel rupture has led to the development of novel experimental methods of shock wave delivery that can suppress bubble expansion and minimize tissue damage. Progress has also been made in understanding the physical mechanisms involved in stone comminution, and it is seen that the forces generated by cavitation, shear stress and circumferential squeezing act synergistically to fragment stones. Recent work suggests that a broad focal zone may be an advantage, allowing stones to be broken with lower amplitude pulses. Cavitation has been shown to play a critical role in reducing stone fragments to a size that can be voided. Cavitation is also the factor that limits the rate at which treatment can be performed, as stones break significantly better at slow rate than at fast ratean observation from basic research that is now appreciated in clinical practice. The current environment in lithotripsy research is encouraging. There is great interest in developing new technology, and in finding ways to improve how lithotripsy is performed.

Cell and wall morphology of intestinal arterioles from 4- to 6- and 17- to 19-week-old Wistar-Kyoto and spontaneously hypertensive rats.

To determine whether vascular smooth muscle cells around intestinal arterioles of various sizes undergo comparable changes in spontaneously hypertensive rats, 4- to 6-week-old (n = 10) and 17- to 19-week-old (n = 10) rats from the Wistar-Kyoto and the spontaneously hypertensive strains were used to study the external morphology of vascular smooth muscle cells by scanning electron microscopy and the vessel wall cross-sectional characteristics by light microscopy. At the time of fixation all vascular tone had been abolished. Scanning electron microscopy analysis revealed that all Wistar-Kyoto and spontaneously hypertensive rats at a given age have spindle-shaped vascular smooth muscle cells of comparable length and longitudinal width for a given branching order of arterioles. However, normal maturation is associated with elongation and widening of the vascular smooth muscle cells. Light and scanning electron microscopy indicated that a monolayer of vascular smooth muscle cells, wrapped at almost 0 degree to the vessel's radial axis, is maintained in adult spontaneously hypertensive rats. The radial thickness of this vascular smooth muscle cell monolayer was significantly (p less than 0.025) increased for only the largest arterioles of young and adult spontaneously hypertensive rats. This radial thickening of individual vascular smooth muscle cells increased the muscular component of the wall area for the largest arterioles by about 50% in adult spontaneously hypertensive rats. Other smaller submucosal arterioles of young and adult spontaneously hypertensive rats had normal vessel wall and vascular smooth muscle cell characteristics. These data indicate that hypertrophy in the smooth muscle cell's radial dimension is the primary morphological change in intestinal arterioles of spontaneously hypertensive rats. However, the vascular smooth muscle cell hypertrophy is confined to the largest arterioles such that the remaining smaller arteriolar vessels in the spontaneously hypertensive rat retain a normal smooth muscle cell and overall wall morphology.

Renal afferent arteriole in the spontaneously hypertensive rat.

We conducted morphometric studies on the afferent arteriole of spontaneous hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats to gain a better understanding of its changes with the development of hypertension. Differences may be related to the SHRs' increased renal vascular resistance. Methacrylate vascular casts were made of the renal vasculature after perfusion fixation with glutaraldehyde. These vascular casts were then examined and measurements made with the scanning electron microscope. Results from this examination of the scanning electron microscope demonstrated a smaller afferent arteriolar diameter in the SHR, compared to the WKY, for both the inner and outer cortical glomeruli. This difference was seen in the 6-week-old SHR, prior to a statistically different blood pressure from the WKY controls, as well as in the 12-week-old hypertensive SHR. However, this afferent diameter difference between rat strains was more pronounced in rats at 12 weeks of age. The tapering of the afferent arteriole (difference between proximal and distal afferent diameters) was greater in the 12-week-old SHR than in the age-matched WKY or 6-week-old SHR. We conclude that the smaller caliber afferent arterioles of the SHR may predispose and play a role in the pathogenesis of the subsequent hypertension. The increased afferent arteriolar tapering seen in the hypertensive SHR relates to the already present increased blood pressure. Wall thickness/radius ratios are not different between rat strains (SHR and WKY) at either 6 or 12 weeks of age. These results suggest increased vascular constriction or hypoplastic vessels as the cause of the smaller caliber vessels in the SHR rather than increased wall thickness.

The glomerular filtration barrier in the spontaneously hypertensive rat.

To determine whether the differences in the physiological characteristics of SHR and WKY kidneys might be related to differences in renal structure, we studied the kidneys of SHR and WKY rats utilizing scanning and transmission electron microscopy and latex perfusion of the glomerular vasculature. Scanning and transmission electron microscopy revealed a smaller diameter in the glomerular endothelial fenestrae of SHR compared to that in WKY rats even prior to the development of differences in blood pressure. With age, the density and diameter of SHR endothelial fenestrae progressively decreased, which was not true in WKY rats. Latex casts of glomerular vasculature showed the frequent presence of afferent arteriolar constriction in 12-week-old SHR. Thus, the filtration barrier of the SHR is abnormal. THe pathogenesis of hypertension in the SHR is complex and probably multifactorial; however, renal structural changes may contribute to the development and maintenance of hypertension. The afferent arteriolar constrictions may be the structural basis for the increased vascular resistance described in the SHR.

The structure of the inferior lobe of the teleost hypothalamus.

Electron microscopic and Golgi studies on the inferior lobes of sunfish and goldfish are described. The inferior lobe consists primarily of a nucleus ventricularis of densely packed cells surrounding the lateral recess of the third ventricle, and a peripherally situated nucleus diffusus consisting mostly of scattered neurons. A cell-sparse zone of dense neuropil is located between the two cellular areas. Neurons of both nuclei have spiny dendrites and axons which originate from basal dendrites. In some cases axons are found to send a collateral into the cell-sparse zone. Neurons of the nucleus diffusus possess collaterals that extend a considerable distance within the nucleus itself. The ultrastructure of cells of both nuclei reveals cytoplasmic organelles typical of most neurons. Synapses containing dense-cored and clear vesicles are present on the spines and shafts of the dendrites of both neuronal types. In only rare cases synapses were observed on the soma of neurons of the nucleus ventricularis. Possible anatomical substrates involved in the control of feeding and aggression in teleosts are considered in light of the present findings. Morphological similarities of the inferior lobes and related areas in various fishes and amphibians are discussed and their possible significance for the understanding of the evolution of hypothalamic mechanisms is considered.

Developing renal innervation in the spontaneously hypertensive rat: evidence for a role of the sympathetic nervous system in renal damage.

The spontaneously hypertensive rat (SHR) exhibits increased renal sympathetic nerve activity and neurotransmitter levels compared with the control Wistar-Kyoto rat (WKY). These renal nerve abnormalities have been implicated as the cause of hypertension in the SHR. The aims of the present study were to characterize the ontogeny of renal sympathetic innervation in SHR in order to determine any functional implications. Glyoxylic acid histofluorescent and radio-enzymatic norepinephrine assays demonstrated an accelerated development of renal innervation in newborn and 1-, 2-, 3- and 6-week-old SHR compared with WKY. Sympathetic nervous system function was blocked in developing male SHR by treating pups from days 0 to 14 with: (1) guanethidine, (2) combined alpha- and beta-receptor antagonists (prazosin and timolol), or (3) vehicle (5% sucrose). Blood pressure (mean), renal function (plasma creatinine) and histologic renal damage were assessed at 42 weeks of age. Although the blood pressure of the drug-treated rats remained elevated, renal damage was reduced and renal function was improved compared with control (sucrose-treated) SHR. The data demonstrate that the SHR kidney develops a precocious sympathetic innervation and that inhibition of the development of sympathetic function ameliorates renal damage independently of systemic hypertension.

Time course of glomerular endothelial injury related to pulsatile perfusion preservation.

To elucidate the time course of glomerular and arterial endothelial injury resulting from pulsatile perfusion preservation of human kidneys, we examined two kidneys, one at 16 and the other at 42 hr, for which no suitable recipient could be found. The scanning electron microscope revealed subtle changes at 16 hr in the filtration barrier. These included mild endothelial swelling with an increase in the appearance of bulbous processes, and elongated fenestrae. The visceral epithelial surface was normal as was the arterial endothelial surface. By 42 hr the glomerular endothelial surface displayed very prominent cytoplasmic ridges and clearly distorted fenestrae. The arterial endothelium exhibited a tendency to separate from the vessel wall. The proximal tubular epithelium revealed scattered loss of microvilli. These changes are similar in kind to, albeit less severe than, those described after 60 hr of perfusion. They may represent cell swelling following ischemia, or be the result of altered cell permeability engendered by low temperature. The possibility remains that such changes could be minimized by modifying the perfusate. Scanning electron microscopy provides a versatile tool in the study of vascular and other surfaces of tissues stored with perfusion preservation.

Glomerular endothelial injury related to renal perfusion. A scanning electron microscopic study.

To elucidate abnormalities in renal morphology related to perfusion preservation, we examined kidneys perfused for 60 hr with a scanning electron microscope. In addition to tubular necrosis and fused glomerular visceral epithelial foot processes, we identified changes in the glomerular endothelial and arterial endothelial surfaces. The glomerular endothelium revealed fenestrae that were smaller and more irregular than normal, as well as abnormal bulbous projections. The arterial endothelium displayed striking degenerative changes. These abnormalities may account for the altered glomerular function and intravascular coagulation that occur in some kidneys preserved by lengthy perfusion.

Calciphylaxis is associated with hyperphosphatemia and increased osteopontin expression by vascular smooth muscle cells.

Calciphylaxis or calcific uremic arteriolopathy (CUA) is a fatal disease in dialysis patients due to calcification of cutaneous blood vessels. The pathogenesis has been attributed to elevated parathyroid hormone (PTH). However, recent studies evaluating vascular calcification in nondialysis patients have found that the smooth muscle cells play an active role, including production of the bone matrix protein osteopontin. To examine the involvement of various clinical parameters and smooth muscle cells of CUA, we performed a case-control analysis comparing 10 CUA patients with our current dialysis patients. Available histologic sections were immunostained for osteopontin, markers of smooth muscle cells, endothelial cells, and macrophages. Compared with our current dialysis population, patients with CUA were more likely to be obese, white, and female (P < 0.02). Comparison of laboratory values found CUA patients with lower serum albumin, greater serum phosphorus, and greater calcium X phosphorus product (P < 0.01). In contrast, there was no difference in the concentration of PTH or calcium between the 2 groups. Immunostaining of calcified blood vessels showed that all calcified vessels stained positive for osteopontin, whereas all the noncalcifed vessels showed no osteopontin localization. Staining for smooth muscle alpha-actin decreased in the medial layer with calcification, with cells appearing to be sloughed off, leading to near occlusion of the vessel lumen. Our case-control study demonstrates that hyperphosphatemia and an elevated calcium X phosphorus product is associated with CUA. Histologic examination suggests that the calcification is associated with increased expression of osteopontin by smooth muscle cells.

Intracoronary administration of streptokinase in a canine model. Disturbance in thromboxane A2-prostacyclin balance.

The potential impact of local intracoronary infusion of streptokinase (SK) on vascular prostaglandin synthetic pathways was studied in a canine model. Control animals (n = 10) underwent left coronary artery (LCA) infusion of 50,000 units SK for 90 minutes; experimental animals (n = 10) underwent LCA infusion of normal saline. Plasma samples for radioimmunoassay (RIA) of prostacyclin (PGI-2) and thromboxane (TXA-2) were obtained from the coronary sinus (CS) as follows: one sample preinfusion, six samples during infusion, and three samples postinfusion in each animal. Comparisons between control and experimental plasma levels of PGI-2 and TXA-2 were made for each sampling time. The PGI-2 levels remained at or below the lower limits of detectability by RIA (the most sensitive assay available) in both control and experimental animals. TXA-2 levels were higher in experimental than in control animals at all sampling times, with the most significant differences occurring in samples 3 (after 30 minutes of infusion, .001 less than P less than .01), 4 (after 45 minutes of infusion, .05 less than P less than .10), and 5 (after 60 minutes of infusion, .02 less than P less than .05). We suggest (1) it is unlikely that any of the beneficial effects of coronary streptokinase infusions are PGI-2-mediated, (2) that the TXA-2 increases in our model may represent a pathophysiologic-biochemical correlate of previously identified morphologic evidence of endothelial damage in animals infused with fibrinolytic agents, and (3) that our findings may indicate that fibrinolytic infusions produce competing effects: lysis of thrombi and endothelial injury with TXA-2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioeffects of extracorporeal shock-wave lithotripsy. Strategy for research and treatment.

Available information on the bioeffects of ESWL is insufficient to characterize the tissue injury induced by shock waves. The cellular mechanisms have not been elucidated, nor are there enough data to establish objective criteria for treatment.

The structure of the hypothalamic inferior lobes of the blacktip reef shark: scanning and transmission electron microscopic observations.

The inferior lobes of the shark hypothalamus were examined with light, transmission and scanning electron microscopy. The cells bordering the floor of the lateral recess appear to be typical liquor-contacting neurons. With scanning electron microscopy (SEM) the apical ends of these cells are seen to bulge into the ventricular lumen. In contrast, the roof is lined by a more typical ependymal cell characterized by numerous cilia and microvilli. In addition, SEM reveals several kinds of supraependymal cells with processes that appear to penetrate the ventricular lining. A periventricular nucleus underlies the ependymal cells. Neurons of the periventricular nucleus contain numerous lipofuchsin granules. The rest of the inferior lobe consists of many neuronal fibers. The morphology of the hypothalamic inferior lobe is discussed in relation to its possible role in feeding and aggressive behavior in both elasmobranchs and teleosts.

Cell damage by lithotripter shock waves at high pressure to preclude cavitation.

Acoustic cavitation has been implicated as a cause of cell damage by lithotripter shock waves, particularly under in vitro conditions. When red blood cells were exposed to shock waves (from an electrohydraulic lithotripter) while under high hydrostatic pressure (> 80 atm), cell lysis was dramatically reduced over that seen at atmospheric pressure, which is consistent with damage due to acoustic cavitation. However, even at > 120 atm of pressure, lysis was still 97% above that of cells not exposed to shock waves, revealing significant damage that apparently was due to mechanisms other than cavitation. Hydrostatic pressure alone did not cause cell lysis, and shock-wave-dependent damage occurred when the cells were in fluid suspension, or when they were centrifuged to the end of the vial. Shock-wave damage at high pressure increased with increasing shock-wave number, and was seen at 24 and 20 kV, but not at 16 kV. This shock-wave damage at high pressure makes up a noteworthy portion of the total cell injury seen at atmospheric pressure (about 10% at 24 kV), suggesting significant noncavitational injury to cells in vitro. Because cavitation occurs far more readily in vitro than in vivo, the noncavitational damage seen in the present study could represent a substantial portion of cell injury seen in vivo with shock-wave lithotripsy.

Quantitation of shock wave cavitation damage in vitro.

Acoustic cavitation damage was quantitated using aluminum foil targets placed within 2-mL polypropylene cryovials. The vials contained various media tested for their potential to support cavitation and were exposed to shock waves using an unmodified Dornier HM3 lithotripter. Foil damage, expressed in terms of a "damage index," was measured from digitized light microscopy images by quantitating the spread of gray-scale histograms. Target sensitivity was demonstrated by reproducible dose-response curves over the range (1-200 shock waves) commonly used for in vitro cell injury studies. Increased shock wave repetition rate reduced the damage index. Untreated foils showed a very low damage index (0.001% +/- 0.001%), while treated foils submerged in Ringer buffer yielded significant damage (2.2% +/- 0.3%, p < 0.001). Degassing the buffer reduced damage to 0.3% +/- 0.1% (p < 0.001). Foils submerged in castor oil showed virtually no damage. These results implicate acoustic cavitation in target damage. Targets immersed in biological fluids (blood and urine) had significantly less damage than in Ringer. The effect of degassing was also evaluated in a red blood cell lysis assay. Hemoglobin release in degassed preparations was significantly reduced compared to nondegassed controls (p < 0.001) and correlated with reduced foil damage index in cell-free vials. These findings characterize a sensitive method to quantitate acoustic cavitation and implicate a role for cavitation in shock wave lithotripsy-induced cell lysis.