Sauna, sweat and science II - do we sweat what we drink?

Inspired by a previous 'Sauna, sweat and science' study [Zech et al. Isot Environ Health Stud. 2015;51(3):439-447] and out of curiosity and enthusiasm for stable isotope and sauna research we aimed at answering the question 'do we sweat (isotopically) what we drink'? We, therefore, pulse-labelled five test persons in a sauna experiment with beverages that were 2H-enriched at about +25,600 ‰. Sweat samples were collected during six sauna rounds and the hydrogen isotope composition δ2Hsweat was determined using an isotope ratio mass spectrometer. Before pulse labelling, δ2Hsweat - reflecting by approximation body water - ranged from -32 to -22 ‰. This is ∼35 ‰ enriched compared to usual mid-European drinking water and can be explained with hydrogen-bearing food as well as with the respiratory loss of 2H-depleted vapour. The absence of a clearly detectable 2H pulse in sweat after pulse labelling and δ2Hsweat results of ≤+250 ‰ due to a fast 2H equilibration with body water are moreover a clearly negative answer to our research question also in a short-term consideration. Given that the recovery of the tracer based on an isotope mass balance calculation is clearly below 100 %, we finally answer the question 'where did the rest of the tracer go?'

[Human Body Water Composition Measurement:Methods and Clinical Application].

Being the largest single component of the human body,water is essential for life. Disease can lead to salt and water imbalance, and it is particularly important to measure the content and distribution of water in body. The current body water measurement methods are still not mature,and it's even hard to measure extracellular and intracellular water. Isotope dilution method(ID),bioelectrical impedance analysis(BIA),skinfold thickness measurement,and resonant cavity perturbation(RCP)are the commonly used methods for measuring human body water composition. This paper analyzes the advantages and disadvantages of these methods and concludes that all these four methods can be used to measure total body water;more specifically,ID and BIA can measure extracellular water and intracellular water,whereas BIA is more suitable for clinical applications such as monitoring of fluid balance,guiding of fluid management,assessment of lymphedema and nutritional risk,and management of obesity. Body water measurement will play more important roles in diagnosis,prevention,treatment,and prognosis of diseases.

Effect of hyperbaric oxygen conditions on the ordering of interfacial water.

Hyperbaric oxygen (HBO2) conditions are applied clinically to treat diverse conditions. There is a lack of a unifying consensus as to how HBO2 acts effectively against a broad range of medical conditions, and numerous differing biological explanations have been offered. The possibility of a mechanism dependent on the extensive ordering of interfacial water has not yet been investigated. We examined the hypothesis that zones of ordered water, dubbed "exclusion zones" or "EZ," are expanded under hyperbaric oxygen conditions. Specifically, we tested whether there are significant quantitative differences in EZ size at steady state under high-pressure and/or high-oxygen conditions, compared to normal atmospheric conditions. Oxygen concentration and mechanical pressure were examined separately and in combination. Statistically significant increases in EZ size were seen at elevated air pressures and at high oxygen concentrations. These experimental results suggest the possibility of an ordered water-mediated mechanism of action for hyperbaric oxygen therapy.

MRS water resonance frequency in childhood brain tumours: a novel potential biomarker of temperature and tumour environment.

(1)H MRS thermometry has been investigated for brain trauma and hypothermia monitoring applications but has not been explored in brain tumours. The proton resonance frequency (PRF) of water is dependent on temperature but is also influenced by microenvironment factors, such as fast proton exchange with macromolecules, ionic concentration and magnetic susceptibility. (1)H MRS has been utilized for brain tumour diagnostic and prognostic purposes in children; however, the water PRF measure may provide complementary information to further improve characterization. Water PRF values were investigated from a repository of MRS data acquired from childhood brain tumours and children with apparently normal brains. The cohort consisted of histologically proven glioma (22), medulloblastoma (19) and control groups (28, MRS in both the basal ganglia and parietal white matter regions). All data were acquired at 1.5 T using a short TE (30 ms) single voxel spectroscopy (PRESS) protocol. Water PRF values were calculated using methyl creatine and total choline. Spectral peak amplitude weighted averaging was used to improve the accuracy of the measurements. Mean PRF values were significantly larger for medulloblastoma compared with glioma, with a difference in the means of 0.0147 ppm (p < 0.05), while the mean PRF for glioma was significantly lower than for the healthy cohort, with a difference in the means of 0.0061 ppm (p < 0.05). This would suggest the apparent temperature of the glioma group was ~1.5 °C higher than the medulloblastomas and ~0.7 °C higher than a healthy brain. However, the PRF shift may not reflect a change in temperature, given that alterations in protein content, microstructure and ionic concentration contribute to PRF shifts. Measurement of these effects could also be used as a supplementary biomarker, and further investigation is required. This study has shown that the water PRF value has the potential to be used for characterizing childhood brain tumours, which has not been reported previously.

Efeito do extrato aquoso de cabelo de milho (Zea mays L. ) sobre a excreção renal de água e eletrólitos e pressão arterial em ratos Wistar anestesiados / Effect of aqueous extract of corn silks (Zea mays L. ) on the renal excretion of water and electrolytes and arterial pressure in anesthetized Wistar rats

O milho (Zea mays L.) possui nos estigmas (cabelo de milho) substâncias que o tornam diurético, podendo ser importante no controle da hipertensão. No presente estudo, foi investigado o efeito do extrato aquoso (EA) do cabelo de milho sobre o fluxo renal de água (V) e eletrólitos e a pressão arterial (PA) em ratos Wistar anestesiados. Foram realizados 3 grupos: I) Controle - administração intragástrica (AI) de 1mL de água destilada; II) AI de 1 mL de EA de cabelo de milho a 20 por cento e III) AI de 1 mL de solução contendo furosemida. Canulou-se a artéria carótida esquerda para mensuração da PA, de 10 em 10 minutos, e a bexiga urinária, para mensuração de V de 30 em 30 minutos e da carga excretada dos íons sódio (Qe (Na+)) e potássio (Qe (K+)). Protocolo experimental: quatro períodos de 30 minutos cada: basal (avaliação dos parâmetros basais) e experimentais (Ex) 1, 2 e 3 (30, 60 e 90 minutos após a AI, respectivamente). O Grupo I não apresentou alterações significativas entre os períodos nos parâmetros analisados (p>0,05). O Grupo II apresentou aumento significativo (p<0,05) em V, em Qe (Na+) e em Qe (K+) nos períodos Ex2 e Ex3, com redução significativa na PA (p<0,05) em Ex2 e Ex3. Conforme esperado, o Grupo III apresentou aumento significativo em V nos períodos Ex2 (p<0,05) e Ex3 (p<0,001), aumento em Qe (Na+) em Ex1 (p<0,05), Ex2 (p<0,001) e Ex3 (p<0,001) e aumento em Qe (K+) em Ex2 (p<0,05) e Ex3 (p<0,001), com redução significativa na PA (p<0,05) em Ex2 e Ex3. Os dados mostram que o EA do cabelo de milho possui efeito diurético, porém não age como um diurético "de alça", uma vez que não levou à expoliação de potássio e nem a uma excreção tão acentuada de sódio quanto à furosemida.

The corn (Z. mays) has in its stigmas (corn silks) substances that make it diuretic, which may be important in hypertension control. In this study, the effect of aqueous extract (AE) of corn silks on the renal flow of water (V) and electrolytes and arterial pressure (AP) was investigated in anesthetized Wistar rats. Three groups were tested: I) Control - intragastric administration (IA) of 1mL of distilled water, II) IA of 1 mL of AE of corn silks at 20 percent and III) IA of 1 mL of a solution containing furosemide. Cannulation was performed in the left carotid artery to measure AP, at every 10 minutes, and in the urinary bladder to measure V, at every 30 minutes, and the excreted load of ions sodium (Qe (Na+)) and potassium (Qe (K+)). Experimental protocol: four periods of 30 minutes each: basal (evaluation of basal parameters) and experimental (Ex) 1, 2 and 3 (30, 60 and 90 minutes after IA, respectively). Group I had no significant differences between periods for the analyzed parameters (p>0.05). Group II presented a significant increase (p<0.05) in V, Qe (Na+) and Qe (K+) in periods Ex2 and Ex3, with significant reduction in AP (p<0.05) in Ex2 and Ex3. As expected, Group III had a significant increase in V in periods Ex2 (p<0.05) and Ex3 (p<0.001), an increase in Qe (Na+) in Ex1 (p<0.05), Ex2 (p<0.001) and Ex3 (p<0.001) and an increase in Qe (K+) in Ex2 (p<0.05) and Ex3 (p<0.001), with an important reduction in AP (p<0.05) in Ex2 and Ex3. These data show that AE of corn silks has a diuretic effect but does not act as a loop diuretic since it did not lead to potassium loss or marked sodium loss, compared to furosemide.

Fat and water 1H MRI to investigate effects of leptin in obese mice.

Leptin is known to be associated with regulation of body weight and fat content. The effects of exogenous leptin on abdominal visceral (VS) and subcutaneous (SC) fat volume and hepatic fat-to-water ratio in leptin-deficient obese mice were investigated by (1)H magnetic resonance imaging (MRI). Chemical shift-selected fat and water (1)H MRI of control and leptin-treated mice were obtained 1 day before treatment and after 7 days of treatment (0.3 mg/kg/day). Hepatic fat-to-water ratio and VS fat volume decreased significantly with treatment, whereas SC fat volume did not change. Noninvasive measurement of fat and water content in different body regions using MRI should prove useful for evaluating new drugs for the treatment of obesity and other metabolic disorders.

Proton MR spectroscopy in neonates with perinatal cerebral hypoxic-ischemic injury: metabolite peak-area ratios, relaxation times, and absolute concentrations.

BACKGROUND: Results from cerebral proton (1)H-MR spectroscopy studies of neonates with perinatal hypoxic-ischemic injury have generally been presented as metabolite peak-area ratios, which are T1- and T2-weighted, rather than absolute metabolite concentrations. We hypothesized that compared with (1)H-MR spectroscopy peak-area ratios, calculation of absolute metabolite concentrations and relaxation times measured within the first 4 days after birth (1) would improve prognostic accuracy and (2) enhance the understanding of underlying neurochemical changes in neonates with neonatal encephalopathy. METHODS: Seventeen term infants with neonatal encephalopathy and 10 healthy controls were studied at 2.4T at 1 (1-3) and 2 (2-4) (median [interquartile range]) days after birth, respectively. Infants with neonatal encephalopathy were classified into 2 outcome groups (normal/mild and severe/fatal), according to neurodevelopmental assessments at 1 year. The MR spectroscopy peak-area ratios, relaxation times, absolute concentrations, and concentration ratios of lactate (Lac), creatine plus phosphocreatine (Cr), N-acetylaspartate (NAA), and choline-containing compounds (Cho) from a voxel centered on the thalami were analyzed according to outcome group. RESULTS: Comparing the severe/fatal group with the controls (significance assumed with P < 0.05), we found that Lac/NAA, Lac/Cho, and Lac/Cr peak-area ratios increased and NAA/Cr and NAA/Cho decreased; Lac, NAA, and Cr T2s were increased; [Lac] was increased and [Cho], [Cr], and [NAA] decreased; and among the concentration ratios, only [Lac]/[NAA] was increased. Comparison of the normal/mild group with controls revealed no differences in peak-area ratios, relaxation times, or concentration ratios but decreased [NAA], [Cho], and [Cr] were observed in the infants with normal/mild outcome. Comparison of the normal/mild and severe/fatal groups showed increased Lac/NAA and Lac/Cho and decreased NAA/Cr and NAA/Cho peak-area ratios, reduced [NAA], and increased Lac T2 in the infants with the worse outcome. CONCLUSIONS: Metabolite concentrations, in particular [NAA], enhance the prognostic accuracy of cerebral (1)H-MR spectroscopy-[NAA] was the only measurable to discriminate among all (control, normal/mild, and severe/fatal outcome) groups. However, peak-area ratios are more useful prognostic indicators than concentration ratios because they depend on metabolite concentrations and T2s, both of which are pathologically modulated. Concentration ratios depend only on the concentrations of the constituent metabolites. Increased Cr T2 may provide an indirect marker of impaired cellular energetics, and similarly, NAA T2 may constitute an index of exclusively neuronal energy status. Our recommendation is to collect data that enable calculation of brain metabolite concentrations. However, if time constraints make this impossible, metabolite peak-area ratios provide the next best method of assigning early prognosis in neonatal encephalopathy.

Surgically induced brain injury in rats: the effect of erythropoietin.

Brain tissue at the edge of a surgical resection site is at risk for damage from direct trauma, retractor stretch, hemorrhage, edema, and electrocautery. In this study we used a new rodent model of surgically induced brain injury (SBI) to study this tissue at the edge of a resection site. The SBI model entails stereotaxic resection of part of the right frontal lobe. We tested pretreatment with erythropoietin, a known neuroprotectant, for protective effects in this model. Three groups of male Sprague-Dawley rats (280-330g) were used: SBI without treatment (n=63), SBI with EPO treatment (n=76), and Sham surgery (n=12). Rats were sacrificed 24h, 72h, and 7 days after SBI or Sham surgery. Postoperative assessment included mortality, histology, immunohistochemistry, Evans blue exudation, brain water content, and magnetic resonance imaging. No difference was found between untreated and EPO-treated groups in mortality, histology, TUNEL, magnetic resonance imaging, or blood-brain-barrier breakdown. The EPO-treated group had statistically more brain water content at 24h than the untreated group. Immunohistochemistry demonstrated a qualitative increase in VEGF in the EPO-treatment group. We conclude that EPO does not ameliorate damage in SBI, and may increase brain edema early after surgery.

Measurement of fat/water ratios in rat liver using 3D three-point dixon MRI.

Hepatic steatosis, or fatty liver, is commonly observed during the animal phase of drug safety studies. A noninvasive three-dimensional (3D) three-point Dixon method was used to quantitatively evaluate the fatty livers of rats induced by an experimental microsomal transfer protein (MTP) inhibitor, in an effort to develop a safety biomarker that could be translated to human studies. The method was implemented at 2.0 T for in vivo studies, and at 7.1 T for higher-resolution magnetic resonance (MR) histologic studies. In three separate protocols to study dose response and longitudinal evolution, intrahepatic fatty accumulation was detected by this method and confirmed by chemical and histologic assessments. Consistent with the pathologic changes, the fat/water ratios estimated by the MR technique increased significantly at doses of 1 mg/kg and 100 mg/kg of MTP inhibitor after 14 days of continuous administration. Among the more important findings were: 1). with the 3D three-point Dixon method, in vivo longitudinal studies of liver fat distribution can be conducted at significantly higher resolution than has previously been reported; 2). MR histology allows delineation of distribution at the microscopic scale of 0.0024 mm(3) resolution; and 3). the 3D three-point Dixon technique provides relative estimates of liver fat content and distribution at a high confidence level. This technique will be applicable in future studies in which fatty liver is a potential safety issue.

[Biophysical measurement of cutaneous hydration in vivo]. / La mesure biophysique de l'hydratation cutanée in vivo.

The skin, because of its involvement in health, comfort and aesthetics, is a particular "organ". Cutaneous hydration is essential for the dermatologist and the cosmetician. For the dermatologist, "dry skin" is a clinical sign of an often unclear pathogenetic disorder and for the cosmetician it is a discomfort related to aging, climatic effects or sun exposure and should be compensated or prevented. The essential measurement and objective quantification of cutaneous hydration has led to the development of various techniques, based on differing biophysical principles and studies aimed at providing better understanding of the mechanisms involved. Faced with an abundance of information and means, the aim of this review is to briefly present the various techniques and instrumental measurements of hydration in vivo, their biophysical principles and their pertinence for the clinician.

Use of magnetic resonance imaging for in vivo measurements of water content in human brain: method and normal values.

OBJECT: The authors present a quantitative in vivo magnetic resonance (MR) imaging method and propose its use for the accurate assessment of brain water in humans. METHODS: With this technique, a pure T1-weighted image of a selected brain slice in a patient is generated, and the image is subsequently converted to a pure water image by means of an equation derived from a tissue relaxation model. The image intensity in the resulting water map directly yields absolute measures of water expressed in grams of water per gram of tissue at a given anatomical location. The method has been validated previously in a series of phantom experiments and in an infusion model of brain edema in cats. In this report, the authors evaluate the method by using samples of tissue harvested from patients who underwent surgery for brain tumor removal and apply the technique to a series of normal volunteers, providing average regional brain water content (f(w)) values for a range of tissues. Application of the method in pathological conditions such as head trauma, tumor, and hydrocephalus allows quantification of regional or global increases in f(w) that result from edema. CONCLUSIONS: It is now possible to obtain accurate brain water measurements with the anatomical resolution of MR imaging. This permits monitoring of the development and resolution of edema in a variety of clinical circumstances, thus enhancing understanding of the underlying pathophysiological processes.

Magnetic resonance imaging-based compartmentation and its application to measuring metabolite concentrations in the frontal lobe.

Partial volume mixing of water compartments within a spectroscopy voxel (e.g. cerebrospinal fluid within a "brain" voxel) may, if not corrected for, lead to underestimation of brain metabolite concentrations. To correct for this source of bias, a new imaging-based method of compartmentation analysis is presented. Brain water, cerebrospinal fluid and solid matter content were obtained from proton density- and T2-weighted images of the brain and an external standard in 10 healthy young males (21 to 30 years), and results compared with a previously-described technique based on spectroscopy. Mean (SD) fractional water content (betaMR) of the 2 x 2 x 2 cm3 voxel in the frontal lobes was 0.79 (0.03) by imaging, slightly but significantly (p = 0.03) smaller than the value of 0.83 (0.03) obtained by spectroscopy. From water-suppressed spectra recorded at five echo times, using betaMR determined by imaging, the T2-corrected concentrations of compounds containing N-acetylaspartate, creatine, choline and myo-inositol were 10.6 (1.0), 8.0 (0.9), 1.6 (0.3) and 3.7 (0.7) mmol.l(-1) of brain, respectively. Imaging-based compartmentation is a rapid and straightforward technique, and can be performed on standard MR systems.

Does growth hormone augment brain edema caused by brain injury? A study with a freeze brain injury model in the rat.

OBJECTIVE: Because of the known sodium and water retention associated with growth hormone (GH) therapy, it is crucial to evaluate the safety of GH after brain injury. To clarify this issue, we investigated whether GH affects brain edema in a rat brain freeze-injury model. METHODS: Male Wistar rats (n = 29) were divided into four groups according to the substance injected (GH vs. normal saline) and whether the brain was injured or not. The subcutaneous injections of GH (0.8 IU/kg) or saline were given 24 hours apart. In the injury groups, after the second injection, an aluminum rod (4-mm diameter) cooled to -50 degrees C was placed on the exposed dura mater in the right parietal region for 4 minutes, under anesthesia. At 4 hours after the insult, brain and skeletal muscle were excised and their water content was measured by drying. RESULTS: Freeze injury of the brain caused an increase in water and sodium content in skeletal muscle. GH injection augmented this edema in skeletal muscle. Freeze injury of the brain also caused an increase in water and sodium content in the injured hemisphere of the brain. GH injection did not exacerbate this edema in injured brain tissue. Neither freeze injury nor GH injection caused brain edema in the noninjured hemisphere or in the cerebellum. CONCLUSION: GH administration did not augment brain edema caused by brain injury in our model.

Early metabolic alterations in edematous perihematomal brain regions following experimental intracerebral hemorrhage.

OBJECT: The authors previously demonstrated, in a large-animal intracerebral hemorrhage (ICH) model, that markedly edematous ("translucent") white matter regions (> 10% increases in water contents) containing high levels of clot-derived plasma proteins rapidly develop adjacent to hematomas. The goal of the present study was to determine the concentrations of high-energy phosphate, carbohydrate substrate, and lactate in these and other perihematomal white and gray matter regions during the early hours following experimental ICH. METHODS: The authors infused autologous blood (1.7 ml) into frontal lobe white matter in a physiologically controlled model in pigs (weighing approximately 7 kg each) and froze their brains in situ at 1, 3, 5, or 8 hours postinfusion. Adenosine triphosphate (ATP), phosphocreatine (PCr), glycogen, glucose, lactate, and water contents were then measured in white and gray matter located ipsi- and contralateral to the hematomas, and metabolite concentrations in edematous brain regions were corrected for dilution. In markedly edematous white matter, glycogen and glucose concentrations increased two- to fivefold compared with control during 8 hours postinfusion. Similarly, PCr levels increased several-fold by 5 hours, whereas, except for a moderate decrease at 1 hour, ATP remained unchanged. Lactate was markedly increased (approximately 20 micromol/g) at all times. In gyral gray matter overlying the hematoma, water contents and glycogen levels were significantly increased at 5 and 8 hours, whereas lactate levels were increased two- to fourfold at all times. CONCLUSIONS: These results, which demonstrate normal to increased high-energy phosphate and carbohydrate substrate concentrations in edematous perihematomal regions during the early hours following ICH, are qualitatively similar to findings in other brain injury models in which a reduction in metabolic rate develops. Because an energy deficit is not present, lactate accumulation in edematous white matter is not caused by stimulated anaerobic glycolysis. Instead, because glutamate concentrations in the blood entering the brain's extracellular space during ICH are several-fold higher than normal levels, the authors speculate, on the basis of work reported by Pellerin and Magistretti, that glutamate uptake by astrocytes leads to enhanced aerobic glycolysis and lactate is generated at a rate that exceeds utilization.

Serial experimental and clinical studies on the pathogenesis of multiple organ dysfunction syndrome (MODS) in severe burns.

These serial clinical and experimental studies were designed to clarify the pathogenesis of postburn MODS. Both animal and clinical studies were performed. In animal experiments, 46 male cross-bred dogs were cannulated with Swan-Ganz catheters and 39 of them were inflicted with 50% TBSA third degree burns (7 were used as controls). The burned dogs were randomly divided into 4 groups: immediate infusion, delayed infusion, delayed fast infusion and delayed fast infusion combined with ginsenosides. All dogs were kept under constant barbiturate sedation during the whole study period. Hemodynamics, visceral MDA, mitochondrial respiratory control rate (RCR) and ADP/O ratio, ATP, succinic dehydrogenase (SDH), organ water content as well as light and electron microscopy of visceral tissues were determined. In the clinical study, 61 patients with extensive deep burns were chosen, of which 16 sustained MODS. Plasma TXB2/6-keto-PGF1alpha ratio, TNF, SOD, MDA, circulatory platelet aggregate ratio (CPAR), PGE2, interleukin-1, total organ water content and pathological observations of visceral tissues from patients who died of MODS were carried out. Results demonstrated that ischemic-reperfusion damage due to severe shock, sepsis and inhalation injury are three main causes of postburn death. All inflammatory mediators increased markedly in both animals and patients who sustained organ damage or MODS. SDH, RCR, ADP/O and ATP decreased significantly. These findings suggested that ischemic damage and systemic inflammatory response syndrome (SIRS) initiated by mediators or cytokines might be important in the pathogenesis of postburn MODS.

Lobar intracerebral hemorrhage model in pigs: rapid edema development in perihematomal white matter.

BACKGROUND AND PURPOSE: The mechanisms underlying brain injury from intracerebral hemorrhage (ICH) are complex and poorly understood. To comprehensively examine pathophysiological and pathochemical alterations after ICH and to examine the effects of hematoma removal on these processes, we developed a physiologically controlled, reproducible, large-animal model of ICH in pigs (weight, 6 to 8 kg). METHODS: We produced lobar hematomas by pressure- controlled infusions of 1.7 mL of autologous blood into the right frontal hemispheric white matter over 15 minutes. We froze brains in situ at 1, 3, 5, and 8 hours after hematoma induction and cut coronal sections of hematoma assessment, morphological brain examination, and immunohistochemical and water content determinations. RESULTS: At 1 hour after blood infusion, "translucent" white matter areas were present directly adjacent to the hematoma. These markedly edematous regions had a greater than 10% increase in water content (>85%) compared with the contralateral white matter (73%), and this increased water content persisted through 8 hours. In addition, these areas were strongly immunoreactive for serum proteins. Intravascular Evans blue dye failed to penetrate into the brain tissue at all time points, demonstrating that this serum protein accumulation and edema development were not due to increased blood-brain barrier permeability. CONCLUSIONS: Experimental lobar ICH in pigs models a prominent pathological feature of human ICH, ie, early perihematomal edema. Our findings suggest that serum proteins, originating from the hematoma, accumulate in adjacent white matter and result in rapid and prolonged edema after ICH. This interstitial edema likely corresponds to the low densities on CT scans and the hyperintensities on T2-weighted MR images that surround intracerebral hematomas acutely after human ICH.

Phosphoenolpyruvate/adenosine triphosphate enhances post-ischemic survival of skeletal muscle.

This study examined whether ischemia-reperfusion injury to skeletal muscle could be reduced by post-ischemic infusion of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP). The rectus femoris muscle of 54 rabbits was rendered ischemic for 3.5 hr. Eighteen rabbits received no further treatment. Thirty-six were infused intra-arterially at the end of ischemia, 18 with vehicle alone, and 18 with a mixture of PEP (80 mumol/kg) and ATP (2.6 mumol/kg). Six rabbits from each group were explored after 24 hr reperfusion and the muscles assessed for viability (by nitro blue tetrazolium), ATP (by luciferin-luciferase chemiluminescence), malonyldialdehyde (MDA) (thiobarbituric acid method), and water content. The remaining muscles in each group were examined histologically after either 1 hr or 4 days of reperfusion. At 24 hr the viability of the PEP/ATP infused muscles (78.9 +/- 15.4 percent) was significantly greater than that of untreated (41.4 +/- 27.3 percent) or vehicle-infused groups (34.0 +/- 32.7 percent). ATP stores were significantly higher and MDA (indicative of free radical activity) and water content significantly lower in the PEP/ATP treated group. At 24 hr and 4 days, muscles infused with PEP/ATP showed less necrosis and fewer infiltrating neutrophils than the untreated groups. Studies with isolated rabbit neutrophils showed that ATP alone significantly inhibited superoxide anion production by stimulated neutrophils. However, when combined with PEP at concentrations similar to those achieved in vivo, ATP did not significantly affect superoxide production. The findings indicate that post-ischemic infusion of PEP/ATP significantly reduces ischemia-reperfusion injury in rabbit skeletal muscle. The protective effect of PEP/ATP is more likely to be due to supplementation of intracellular ATP stores than to the inhibition of superoxide production by infiltrating neutrophils.

Effects of voltage clamping on epithelial cell composition in toad urinary bladder studied with x-ray microanalysis.

Toad urinary bladder epithelial cells were incubated in Na Ringer's with the serosal surface of the epithelium clamped at either +50 mV, 0 mV (short-circuited) or -50 mV with respect to the mucosal surface. Following incubation, portions of tissue were coated with an external albumin standard and rapidly frozen. Cryosections were freeze-dried and cell composition determined by x-ray microanalysis. Cell water and ion contents were unaffected when tissues were short-circuited rather than clamped close to their open-circuit potential difference (+50 mV). Incubation with vasopressin at +50 mV, and under short-circuit conditions, caused Na uptake without cell swelling or gain in Cl. Clamping at -50 mV resulted in uptake of water and ions, with considerable variation from cell to cell. These variations in cell composition were exacerbated by vasopressin. The greater the increase in water content, the greater the rise in cell Cl. However, there was no consistent pattern to the associated changes in cation contents. Most cells gained some Na. In some cells, this gain was accompanied by an increase in K. In others, the gain of Na was predominant and cell K content actually fell. At -50 mV with ouabain, many of the cells also gained water. As was found in our earlier study with ouabain under short circuit conditions (Bowler et al., 1991), there was considerable variation in the extent of the Na gain and K loss; some cells were largely depleted of K while in others the K content remained relatively normal. These results indicate differences between granular cells in the availabilities in the plasma membranes of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control.

Autologous lymphocyte therapy for experimental canine lymphoedema: a pilot study.

Obstructive lymphoedema, an accumulation of protein-rich fluid in interstitial spaces, was created in five dogs by a combination of the irradiation of one groin and subsequent surgical ablation of any remaining lymphatics. The lymphoedema was stable for up to 2 years. The aim was to test the efficacy of intra-arterial injection of autologous lymphocytes as a therapy for lymphoedema. The hypothesis was that cytokines produced by lymphocytes mediate proteolysis by macrophage proteinases in the lymphoedematous limb to remove the excess protein and relieve the oedema. A concentrated lymphocyte-rich preparation was isolated from blood by the Ficoll-Paque method. These preparations were injected into the femoral artery four times at approximately 4 weekly intervals. Three months after the first injection of lymphocytes, lymphoedematous limbs showed a marked 69% reduction in the mean excess circumferences compared with opposite control limbs. After treatment, skin thickness and hydroxyproline content (both measures of fibrosis) as well as water content (a measure of oedema) had reduced significantly. In specimens of interstitial fluid and in skin homogenates acidic proteinase activity increased and the protein concentration decreased significantly compared with controls. It is concluded that increased proteolysis, possibly due to activated macrophages recruited to the lymphoedematous limb, may partly explain these results.

Longitudinal (T1) and transverse (T2) relaxation times of tissue water protons in mouse developing sarcoma 180-A: effect of hyperthermia.

In vitro studies on Swiss mice, inoculated with dead or live sarcoma 180-A ascites cells, were carried out to monitor the changes, if any, in the longitudinal (T1) and transverse (T2) relaxation times of tissue water protons following hyperthermic treatment and subsequent thermotolerance. Both relaxation processes exhibited biexponential relaxation curves. With increasing size of the tumours, the longitudinal relaxation behaviour changed from bi- to mono-exponential. This was not observed for the transverse relaxation phenomenon. Inoculation with either dead or live cells caused an immediate increase in the both relaxation times. In the case of dead cell inoculation, the increase lasted for only 24 h after which the relaxation times became the same as for the uninoculated controls. The transverse relaxation time increased as a result of exposure to hyperthermia. During development of thermotolerance, both the relaxation times decreased.