The inulin space, solute concentrations, and weight changes in rat renal medullary slices incubated in iso-osmolal media, and their modification during anoxia and hypothermia.

1. The volume of distribution of [14-C]carboxyl inulin has been studied in slices of outer and inner medulla from rat kidney incubated in Krebs phosphate-bicarbonate Ringer, modified to render it iso-osmolal with the tissue fluids in these zones, under three conditions, (a) aerobically at 37 degrees C (control), (b) anoxically at 37 degrees C, and (c) aerobically at 0 degrees C. 2. Under control conditions near steady-state volumes of approximately 24 and 42 mul./100 mg wet weight slice were obtained for outer and inner medulla respectively during the period 10-30 min from the start of incubation. In the outer medulla the volumes of distribution in anoxic and hypothermic slices exceeded that in control slices during this time, but control values increased from 30 to 100 min so that after 100 min the distribution volumes were approximately 30 mul./100 mg under each set of conditions. 3. In the inner medulla control and anoxic slices had inulin distribution volumes of approximately 42 mul./100 mg during 10-30 min, rising to over 50 mul./u99 mg by 100 min. Slices incubated hypothermically reached a steady-state value of approximately 40 mul./100 mg by 30 min, which did not increase further for up to 100 min. 4. All slices lost about 10% of their initial weight during the first 3 min of incubation. Thereafter control slices maintained weight constancy for at least 30 min (outer medulla) or 100 min (inner medulla); slices incubated anoxically or hypothermically gained weight, the gains being greatest in anoxic outer and hypothermic inner medulla. 5. The K concentration within control slices (both zones), hypothermic outer and anoxic inner medulla attained equilibrium when slice [K] was approximately 8 times medium [K] (5-9 mM). In anoxic outer and hypothermic inner medullary slices [K] fell to a significantly greater extent, but interpretation of these findings in terms of slice K loss is subject to modification in respect of the increases in slice weight (water content) accompanying the [K] decreases. 6. There was a transient (1-3 min) rise in [Na] in all slices. This was followed by a [Na] decrease, which was most apparent in control slices, and finally a gradual increase towards medium [Na] (141 and 180 mM for outer and inner medulla respectively).     

Factors affecting the distribution of chloride ions in rat renal outer medulla.

1. The intracellular Cl- concentration, [Cl-]1, of rat renal outer medullary slices has been studied in iso-osmolal media containing 42, 93, 144, 189 or 225 mM chloride, [Cl-]0. Equilibrium values for [Cl-]1 were attained within 25-50 min from the start of incubation such that [Cl-]1/[Cl-]0 = 0-46. This ratio was independent of [Cl-]0 within the range studied. Intracellular Na+ was unaffected, and only minor variations of cell volume were observed (calculated from slice weight changes and [14C]carboxyl inulin spaces. 2. When [Cl-]0 = 189 mM, [Cl-]1 remained constant at 87 mM for up to 50 min, indicating that these figures may represnet the interstitial [Cl-] and mean intracellular [Cl-] respectively in outer medulla. 3. Omission of bicarbonate from medium containing 189 mM-Cl- caused an increase in [Cl-]1/[Cl-]0 to 0-58, which was not significantly affected by anoxia or by the presence of arsenite (5 X 10(-3) M) or 2,4-dinitrophenol (10(-3) M). Significant further increases were observed in the presence of iodoacetic acid (5 X 10(-3) M) (0-70), acetazolamide (10(-3) M or 5 M 10(-3) M) (0-71) and iodacetic acid plus 2,4-dinitrophenol (0-85). The addition of the diuretic agents ouabain, ethacrynic acid-cysteine and frusemide (all 10(-3) M) to 189 mM-Cl- media containing bicarbonate reduced [Cl-]1/[Cl-]0 to 0-36, 0-37 and 0-42 respectively. 4. The mean 36Cl- space of outer medulla after 50 min incubation in 189 mM Cl- medium was 49-7 +/- 2-1 micronl./100 mg wet wt. The volume of distribution was not significantly affected by ouabain, ethacrynic acid-cysteine or frusemide (10(-3) mM). 5. Net efflux of 36Cl- from slices loaded with isotope into 'cold' 189 mM Cl medium showed three components with rate constants of 69 X 10(-3), 18 X 10(-3) and 9-9 X 10(-5) sec-1 respectively. Efflux was not affected by ouabain, ethacrynic acid-cysteine or frusemide (10(-3) mM). 6. The main conclusions drawn from this study are: (i) the interstitial fluid Cl- concentration of normally hydrated rat outer medulla is approx. 189 mM; (ii) the [Cl-]1/[Cl-]0 ratio 0-46 may represent a chiefly passive distribution maintained by the opposing gradient of a second anion, probably bicarbonate; (iii) the energy required to maintain unequal distribution of Cl- in the absence of external bicarbonate is derived chiefly from glycolysis, with a small aerobic component; (iv) there may be two intracellular chloride pools whose net rates of Cl- exchange differ by a factor of approx. 180.     

Rates of oxygen consumption and of anaerobic glycolysis in renal cortex and medulla of adult and new-born rats and guinea-pigs

1. Rates of oxygen uptake and of anaerobic glycolysis were estimated in slices from the renal cortex and medulla (a) of adult rats and guinea-pigs, (b) of new-born (1-, 5- and 21-day-old) rats and of guinea-pigs of 1, 12, 21, 24 and 120 hr age.2. In the adult rat, Q(O2) values for the cortex were 12.55 +/- 0.20 (22) and for the medulla: 8.56 +/- 0.17 (22) mul./hr.mg dry weight, while in the new-born rat (24 hr old) they were 10.99 +/- 0.46 (12) and 9.33 +/- 0.18 (9) mul./hr.mg dry weight respectively.3. Values for Q(CO2) (N2) (anaerobic glycolysis) in the 14 hr old new-born rat were in the renal cortex 9.65 +/- 0.35 (5) and in the medulla 7.39 +/- 0.43 (5) mul./hr.mg dry weight; while in the adult they were 2.25 +/- 0.08 (16) and 5.76 +/- 0.14 (16) mul./hr.mg dry weight, respectively.4. In the adult guinea-pig values for Q(CO2) (N2) were of the same order as in the adult rat, though the rate of O(2) uptake was for the cortex 8.12 +/- 0.22 (12) and for the medulla 5.02 +/- 0.23 (11) mul./hr.mg dry weight.5. Though the Q(O2) values in the renal cortex and medulla were smaller in the 1 hr old new-born guinea-pig, they were already increasing in the 12 hr old neonate.6. The results are discussed in the light of enzyme changes occurring during the process of maturation of the nephron as indicated by histochemical observations.     

Extrasynaptic volume transmission and diffusion parameters of the extracellular space

Extrasynaptic communication between neurons or neurons and glia is mediated by the diffusion of neuroactive substances in the volume of the extracellular space (ECS). The size and irregular geometry of the diffusion channels in the ECS substantially differ not only around individual cells but also in different CNS regions and thus affect and direct the movement of various neuroactive substances in the ECS. Diffusion in the CNS is therefore not only inhomogeneous, but often also anisotropic. The diffusion parameters in adult mammals (including humans), ECS volume fraction alpha (alpha=ECS volume/total tissue volume) and tortuosity lambda (lambda(2)=free/apparent diffusion coefficient), are typically 0.20-0.25 and 1.5-1.6, respectively, and as such hinder the diffusion of neuroactive substances and water. These diffusion parameters modulate neuronal signaling, neuron-glia communication and extrasynaptic "volume" transmission. A significant decrease in ECS volume fraction and an increase in diffusion barriers (tortuosity) occur during neuronal activity and pathological states. The changes are often related to cell swelling, cell loss, astrogliosis, the rearrangement of neuronal and astrocytic processes and changes in the extracellular matrix. They are also altered during physiological states such as development, lactation and aging. Plastic changes in ECS volume, tortuosity and anisotropy significantly affect neuron-glia communication, the spatial relation of glial processes toward synapses, glutamate or GABA "spillover" and synaptic crosstalk. The various changes in tissue diffusivity occurring during many pathological states are important for diagnosis, drug delivery and treatment.     

3H-Inulin and electrolyte concentrations in Bowman's capsule in rat kidney

Micropuncture experiments were performed on Bowman's capsules in male and female non-diuretic Munich rats. 55 samples were collected and analysed for Na, Cl, K, Ca, P, Mg and 3H-inulin contents. Their electrolyte concentrations were compared to the corresponding concentrations obtained from plasma artificial ultrafiltrates. Compared to normal Wistar rats, our Munich rats had several special characteristics: high arterial pressure (115-155 mm Hg), high concentrating ability (phi equals 2684 mOsm/1) and high NaCl reabsorption capacity. Whole kidney GFR was low in males (0.361 ml/min/kidney/100 g B.W.). 3H-Inulin concentration was the same in plasma and glomerular ultrafiltrates (GF). The corresponding ratio (GF/P) in equals 1.03 plus or minus 0.01 (N equals 55) confirmed the lack of sieving effect for inulin. GF electrolyte concentrations were, for Na, Cl, K, Ca, P, Mg respectively 139 plus or minus 0.05, 1.46 plus or minus 0.07 and 0.52 plus or minus 0.04 ml/1 (N equals 23) in females and 141 plus or minus 2, 120 plus or minus 2, 3.84 plus or minus 0.07, 1.31 plus or minus 0.03, 1.78 plus or minus 0.05 and 0.48 plus or minus 0.01 mM/1 (N equals 32) in males. Comparison of ultrafiltration trough a cuprophan membrane and glomerular ultrafiltration led to the following conclusions: For Na, Cl and P artificial and glomerular ultrafiltration produced identical results. On the other hand, for Ca and to a lesser extent for Mg and K ions, artificial ultrafiltration did not accurately reflect the true glomerular ultrafiltrate composition. The reasons for these differences are discussed below.     

Endocrine responses to insulin hypoglycaemia in the young calf.

1. Variations in the output of glucocorticoids and catecholamines from the right adrenal gland, in response to insulin hypoglycaemia, have been investigated in calves 2-5 weeks after birth. These have been correlated with changes in the concentration of glucocorticoids and glucagon in arterial plasma. 2. Moderate hypoglycaemia for a limited period (0-1 u. insulin/kg), elicited a prompt increase in steroid output from the adrenal gland followed by a significant rise in plasma glucagon concentration. By comparison, changes in both catecholamine output and peripheral plasma glucocorticoid concentrations were found to be trivial in this group of animals. 3. Administration of a larger dose of insulin (0-5 u./kg) produced a more substantial fall in plasma glucose concentration followed by spontaneous recovery within 2-3 hr. This stimulus elicited the release of greater amounts of both cortisol and corticosterone, followed by a significant increase both in the output of adrenaline and in plasma glucagon concentration. Increase in steroid output was accompanied by an increase in adrenal blood flow and was associated with elevated concentrations of both steroids in arterial plasma. 4. The adrenal cortical response and associated changes in plasma steroid concentration were found to be transient even in response to persistent and intense hypoglycaemia (4 u. insulin/kg). The increase in plasma glucagon concentration in this group of animals was not significantly greater than that produced by smaller doses of insulin. However, substantial amounts of adrenaline (78 plus or minus 14 ng. kg-minus 1 min-minus 1; maximum; n equals 9) together with a little noradrenaline (10 plus or minus 3 ng.kg-minus 1 min-minus 1; maximum; n equals 9) were released from the right adrenal gland under these conditions. 5. Changes in adrenal blood flow could be related to adrenal glucocorticoid output in calves given 0-1 or 0-5 u. insulin/kg. In animals given the largest dose of insulin adrenal blood flow was found to increase coincidentally with rising steroid output but this hyperaemia then persisted after steroid output had subsided to values within the normal range. 6. Calves given the largest dose of insulin (4-0 u./kg) invariably collapsed and convulsed after 2-3 hr, but these symptoms could not be related to any particular endocrine response. No clinical signs of hypoglycaemia were observed in the other animals. 7. The results are discussed in relation to previous studies of adrenal function in this and other species.     

Steady-state glucose oxidation by dog kidney in vivo: relation to Na+ reabsorption.

In eight experiments at normal or slightly elevated blood glucose concentration we quantified the steady-state renal glucose oxidation rate (see article) during control, at reduced Naomega absorptive rates (raised ureteral pressure), and during respiratory alkalosis. A tracer amount of either [1-14C]glucose or or [U-14C]D(omega)-glucose was infused at a constant rate into one renal artery. (see article) was calculated from the renal 14CO2 production rate (corrected for recirculation) and the specific activity of glucose in renal arterial blood. The control (see article) (n equals 8) equals 4.40 plus or minus 0.9 mumol/100 g-min (mean plus or minus SE). When net Naomega reabsorption was decreased by 45% (n equals 6), or when the pH of extracellular fluid was raised (n equals 2), no significant effect on (see article) (9.1 plus or minus 4.2 and 3.9 plus or minus 2.3 mumol/min-100 g, respectively) occurred. The mean glucose oxidation rate for all experiments was 5.65 plus or minus 1.73 mumol g-1-min-1 and required similar to 13% of the renal O2 utilization. Glucose oxidation provides energy either for basal renal work or for some portion of renal transport work not affected by increased ureteral pressure.     

Abnormalities of volume regulation and membrane integrity in myocardial tissue slices after early ischemic injury in the dog: effects of mannitol, polyethylene glycol, and propranolol.

The authors used an in vitro myocardial tissue slice technique to quantitate the transmural distribution of alterations in cell volume regulation and membrane integrity following early ischemic injury and to evaluate directly the effects of therapeutic interventions in a system not subjects to influences of coronary blood flow. Left circumflex coronary occlusion was produced in 57 dogs for 30 or 60 minutes. After in vitro incubation in Krebs-Ringer-phosphate-succinate medium containing trace 14C-inulin, typical values (ml H2O/g dry weight) for control nonischemic myocardial slices were 3.68 +/- 0.07 (SEM) for total tissue water, 2.67 +/- 0.07 for inulin impermeable space, and 1.01 +/- 0.04 for inulin diffusible space. Ischemic myocardial slices exhibited an impaired response to cold shock (0 C for 60 minutes) and rewarming (37 C for 60 minutes). After 60 minutes coronary occlusion, respective increases in total tissue water, inulin-impermeable space and inulin-diffusible space of ischemic slices were 25.5 +/- 2.6%, 6.2 +/- 4.9% and 84.4 +/- 12.5% for papillary muscle, 22.2 +/- 2.1%, 10.4 +/- 4.2% and 52.5 %/- 10.3% for subendocardium and 9.1 +/- 1.5%, 7.2 +/- 2.3% and 15.8 +/- 5.5% for subepicardium. Significant but usually less marked alterations occurred after 30 minutes of coronary occlusion. Propranolol treatment in vivo (2 mg/kg) and/or in vitro (0.01 mg/ml medium) produced no significant changes in tissue water or inulin spaces of ischemic slices, compared with saline controls. Incubation in hyperosmolar mediums resulted in significant reductions in total tissue water and inulin-impermeable space with little change in inulin-diffusible space of both ischemic and control slices. Fifty milliosmolar polyethylene glycol (MW 6000) produced a greater reduction in tissue water and ultrastructural evidence of cell swelling than did either 40 or 100 milliosmolar mannitol (MW 182). The major effect of hyperosmolar incubation appeared to be a selective reduction in edema of cells with structurally intact membranes. Thus, in vitro studies, with myocardial tissue slices provide evidence of widespread alterations of membrane integrity after 30--60 minutes of in vivo coronary artery occlusion. In vitro abnormalities of cell volume regulation can be partially reversed by direct osmotic effects on myocardial cells.     

The effect of hypoxia, hypercapnia and hypotension upon carotid body blood flow and oxygen consumption in the cat

1. Carotid body blood flow (c.b.f.), the arterio-venous oxygen (A-V O(2)) difference and oxygen consumption were measured in forty-seven cats, anaesthetized with pentobarbitone, paralysed with gallamine and ventilated artificially. Carotid sinus and cervical sympathetic nerves were intact throughout.2. A system for perfusing the carotid body artificially with blood is described and evidence is given which shows that similar results were obtained whether the carotid body was naturally or artificially perfused.3. With arterial pressure, blood gas tensions and pH within physiological limits, c.b.f. varied between 33 and 68 mul./min, average 41.5; A-V O(2) difference between 0.21 and 0.46 ml./100 ml., average 0.34, and calculated oxygen consumption between 0.115 and 0.195 mul. O(2)/min, average 0.147.4. With constant mean arterial pressure, hypoxia (30-40 mm Hg P(a, O2)) or hypercapnia (> 50 mm Hg P(a, CO2)) resulted in a small increase of c.b.f., up to 14 mul./min above control; an average fall of A-V O(2) difference by 49% of control and an average fall of oxygen consumption by 36% of control.5. Carotid body blood flow fell linearly with mean arterial pressure over the range 100-170 mm Hg, the slope of the curve varying between 0.78 and 1.22 mul. min(-1). mm Hg(-1). M.A.P. A-V O(2) difference was unaffected so that oxygen consumption fell in proportion to c.b.f.6. It is concluded that the unique response of the carotid body to these stimuli is a fall in oxygen consumption and that this bears a closer relation to the known pattern of chemoreceptor discharge than do changes in total blood flow.     

Permeability of urinary bladder of Rana cancrivora to urea in the presence of oxytocin

1. When Rana cancrivora collected from fresh water had been exposed for 3 days to saline solutions having osmolalities from 280 to 690 m-osmole/kg, urea concentrations in plasma and urine appeared to come into equilibrium, and were from 70 to 200 m-mole/l.2. Plasma urea level of fresh water R. cancrivora (48 m-mole/l.) was doubled (82 m-mole/l.) after 8 hr of exposure to 270 m-osmolal saline. It continued the same after 24 hr of exposure.3. When isolated urinary bladders of R. cancrivora were exposed to Ringer on the serosal aspect and one-fifth Ringer on the mucosal aspect, then in response to this osmotic difference of 190 m-osmole/kg, the rate of fluid movement (mucosa to serosa), which was 10.3(+/-2) mul./cm(2).hr, was not significantly altered when up to 60% of the NaCl of the Ringer solution was substituted by urea.4. Under the same circumstances, when oxytocin (50 m-u./ml.) was present in the serosal solution, the rate of fluid movement (mucosa to serosa) was 133.2(+/-7.9) mul./cm(2).hr in the absence of urea; it was progressively decreased by the presence of urea until, when 80% of the NaCl had been substituted by urea, the rate of fluid movement was reduced to 14.5(+/-4.0) mul./cm(2).hr.5. The diminished rate of fluid movement under the above circumstances could not be correlated with serosal urea concentration, with serosal availability of Na(+), nor with Na(+) concentration difference across the bladder wall. It appeared to be directly related to the ;non-urea osmotic difference' across the bladder wall provided by solutes other than urea.6. When isolated bladders were exposed to an osmotic difference of 190 m-osmole/kg, but having 25 mM urea present in the mucosal solution, then fluid moved from mucosa to serosa at a rate of 10.4(+/-1.3) mul./cm(2).hr in the absence of oxytocin and 124(+/-9) mul./cm(2).hr when oxytocin (50 m-u./ml.) was present. In the former case no urea passed across the bladder wall, but in the latter case urea passed from mucosa to serosa at a rate of 3.16(+/-0.3) mumole/cm(2).hr. The fluid moving from mucosa to serosa thus contained urea 25.5 m-mole/l.7. Vasotocin (10(-9)M), which is equipotent with oxytocin (50 m-u./ml.) in affecting permeability of the isolated urinary bladder to water, was also equipotent in producing a reduced rate of water fluid movement in the presence of 40% urea (vasotocin, 63 mul./cm(2).hr; oxytocin, 59 mul./cm(2).hr).8. When groups of frogs were cystectomized, and other groups of frogs were sham-operated, then after 48 hr of exposure to fresh water or to 300 m-osmolal saline the sham-operated frogs had plasma urea level raised from 20 m-mole/l. (fresh water) to 42 m-mole/l. (saline), while the cystectomized frogs had 20 m-mole/l. (fresh water) and 26 m-mole/l. (saline).9. The hypothesis is presented that hormone-induced permeability of the urinary bladder to urea contributes to the immediate adjustment of plasma urea level by which R. cancrivora survives when exposed to high environmental salinity.     

Hydraulic and oncotic pressure measurements in inner medulla of mammalian kidney.

The vasa recta are thought to play an important role in the transfer of water andsolutes within the renal medulla. Hydraulic pressures were measured in vasa recta onthe surface of the exposed papilla in young Munich Wistar rats, and blood was collected from these microvessels for determination of total protein concentration and calculation of colloid oncotic pressure. In descending vasa recta at the base of the exposed papilla, mean hydraulic pressure was 9.2 plus or minus 0.4 (SE) mmHg and plasma protein concentration averaged 7.1 plus or minus 0.4 g/100 ml. Corresponding valuesin ascending vasa recta at the same level were 7.8 plus or minus 0.4 mmHg and 5.6 plusor minus 0.3 g/100 ml. respectively. The protein concentrations correspond to calculated oncotic pressures of 26 and 18 mmHg in descending and ascending vasa recta, respectively. We interpret these findings as evidence for net water uptake by the vasa recta in the renal inner medulla for which the driving forces are the transcapillary hydraulic and oncotic pressure differences.     

Adipose tissue blood flow and cellularity in the growing rabbit.

Resting blood flow was measured in isolated, innervated, epigastric fat-pads of 27 male rabbits during growth in the 1st yr of life and found to vary widely in range (7.6-28.1 ml/100 g tissue per min). Definition of adipose tissue composition and of fat-cell size and number made it possible to explain the wide range of flow and to identify two types of relationships between adipose blood flow and tissue constituents. Expressed in the usual manner (ml/100 g tissue per min), adipose blood flow declined with increasing adiposity of the fat depots, and a negative correlation was found between flow and fat-cell volume (r equals -.571, P less than .01). In contrast, when blood flow was expressed on the basis of fat-cell number (ml/108 fat cells/min), a positive and highly significant correlation was found between blood flow per fat cell and fat-cell volume (r equals .842, P less than .001). In the rabbit tissue the relationship of blood flow to fat-cell number and size was more predictable than the usual expression of flow in terms of tissue wet weight. Food deprivation for 18-24 h did not significantly alter these relationships. The results indicate that changes in adipose tissue composition and cellularity, resulting from growth and from accumulation of lipid in enlarging adipocytes, are important determinants of blood flow regulation to adipose tissue.     

Inotropic and intracellular acid-base changes during metabolic acidosis.

Experiments in isolated, Ringer-perfused isovolumic rabbit hearts showed that metabolic acidosis resulted in a decrease in peak left ventricular pressure and dP/dt. Concomitantly, the decrease in extracellular pH from 7.28 plus or minus 0.02 to 6.82 plus or minus 0.02 at constant PaCO2 was associated with a negative av HCO3- difference that lasted throughout the duration of acidosis. This negative av HCO3- difference indicated that either HCO3- moved into the intravascular space or H+ moved in the opposite direction during acidosis. During perfusion with normal pH solution av HCO3- was not significantly different from zero. Washout of the extracellular space with the acid solution can account for only 32 percent of the total amount of HCO3-recovered in the venous perfusate during the 30 min of acidosis. The remaining 68 percent (10.7 plus or minus 3.1 mmol times kg-1 of cardiac tissue) must then have originated in the cellular fluid. When intracellular fluid volume is taken into account, it can be calculated that 21.3 plus or minus 6.1 mmol of HCO3- moved into the vascular space per liter of intracellular water. The magnitude of this HCO3- flux suggests that significant myocardial cellular acid-base changes are associated with metabolic acidosis.     

Distribution of chylomicrons and albumin in dog kidney

1. Under specified experimental conditions the distribution space of labelled chylomicrons in the kidney was 13·8 ± 0·9 ml./100 g. tissue. The assumption is supported that this provides a measure for the quantity of intravascular plasma constituents.

2. Values for red blood cells and albumin distribution spaces were 5·2 ± 0·6 and 20·2 ± 1·0 ml./100 g tissue, respectively, in the whole kidney. The ratio of tissue haematocrit over simultaneous arterial haematocrit averaged 0·56. The extravascular albumin fraction amounted to about 31·0% of the total albumin in the whole kidney.

3. A statistically significant correlation was demonstrated between osmotic urine/plasma (U/P) ratios (within the approximate limits of 0·6-1·8) and quantities of extravascular albumin in the medulla.

     

Diffusion in brain extracellular space

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is 20% and the tortuosity is 1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain.     

Prolonged vasodilation following fatiguing exercise of dog skeletal muscle.

Prolonged vasodilation follows 20- and 60-min periods of stimulation (4 twitches/s) of dog skeletal muscle with blood flow held constant at 23 plus or minus 2 ml/100 g per min. During stimulation isometric tension development fell 61 plus or minus 3%, and we have operationally defined this as fatiguing exercise. During stimulation vasodilation was maximum. Following stimulation vascular resistance returned to control with average half-times of 6.9 plus or minus 0.6 and 5.8 plus or minus 0.4 min after 20 and 60 min of stimulation, respectively. This prolonged vascular recovery following fatiguing exercise is an order of magnitude slower than vascular recovery following less severe exercise. The pressor response to an intra-arterial bolus of angiotensin (0.5 mug) is not depressed during prolonged vasodilation. Prolonged vasodilation does not appear to be closely linked to changes in tissue oxygen consumption. In addition, the changes in resistance can be dissociated from changes in K+, osmolality, and lactate production following fatiguing exercise. We conclude that prolonged vasodilation following fatiguing exercise is caused by a metabolic vasodilator substance which is yet to be identified.     

Reevaluation of the needle method for measuring interstitial fluid pressure.

Inserting a needle into subcutaneous spaces should allow a subatmospheric pressure to be measured if interstitial fluid pressure is truly negative as measured by the capsule and wick techniques. Previous needle measurements of interstitial fluid pressure have produced a positive value, but in most instances fluid has been injected into the tissues prior to recording of pressure. Therefore, we measured subcutaneous needle pressure in anesthetized dogs without fluid injection into the tissues. Approximately 30 min are required for an equilibrium pressure after insertion of the needle. The mean 30-min pressure was 4.6 +/- 0.5 (SE) mmHg (n equals 41). With observable edema, interstitial fluid pressures as measured with the needle were always positive. However, the needle method for continuous recording of pressure lacks rapid sensitivity to changes in tissue fluid pressures. In order to develop a needle method that would follow changes in interstitial fluid pressure, 0.5-1 mul of saline was injected into or withdrawn from the tissue. With this method, pressure plateaued in 10-20 min. This plateau pressure increased with tissue hydration and decreased with dehydration.     

In vivo measurement of brain glucose transport and metabolism employing glucose- -11C.

The radiopharmaceutical glucose--11C was used in vivo measurement of brain-glucose transport kinetics and metabolism in the rhesus monkey. Radiotracer was injected intravenously as a bolus. Radioactivity was continuously recorded from the head and from the arterial blood via an indwelling peripheral artery catheter for a collectionperiod of 2-3 min. To correct the reading obtained from the head for radioactivitycontained in blood, a second intravenous injection of the vascular tracer -15O-labeled carboxyhemoglobin was used. The method was tested in nine phencyclidine-anesthetized monkeys in which cerebral glucose metabolism (CMRGlc) was simultaneously measured by our method and by a standard method emplying the Fick principle. A highlysignificant correlation was found between the two methods of measuring CMRGlc (r =0.929). In addition, our model predicted a ratio of forward-to-reverse glucose flux across the blood-brain barrier (BBR) (1.37 plus or minus 0.23 SD), the brain-to-bloodglucose concentration ratio across the BBB (0.633 plus or minus 0.14), the relative tissue free-glucose space (17 plus or minus 7%), the brain free-glucose concentration (13.6plus or minus 8.5 mg/100 g of tissue), and the brain free-glucose turnover time (2.96 plus or minus 1.98 min). author     

A method for the separation of lymphocytes and plasma cells from the human palatine tonsil using sedimentation in an isokinetic gradient of Ficoll in tissue culture medium.

Several methods for the dissociation of human tonsils into cell suspensions were compared. Dissociation of tonsils using 0-25 per cent trypsin gave both the largest number of total cells and the largest number of plasma cells per gram of tonsil. Lymphocytes and plasma cells were separated in a previously described isokinetic gradient of Ficoll in tissue culture medium. In the purest gradient fractions, lymphocytes were 97-2 plus or minus 1-9 per cent of nucleated cells. The purest gradient fractions contained 43-1 plus or minus 5-9 per cent plasma cells. More than 95 per cent of purified lymphocytes and plasma cells excluded Trypan Blue.     

Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3 – absorption to norepinephrine

Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55%. While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.