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Biomedical subjects

R F Keep

Publications and source records attributed to R F Keep.

At least 19 recordsLinked to original sources
PubMed1996

Choroid plexus taurine transport.

The putative osmoregulatory agent, taurine, is lost from the brain during hypo-osmotic stress or ischemia, but the regulatory mechanisms involved in this loss have not been fully elucidated. In this study, we have examined taurine transport by the isolated rat choroid plexus, one element of the brain-blood interface, and examined how it may be regulated as part of brain volume regulation. Choroid plexus taurine uptake was Na- and Cl-dependent with a Vmax and Km of 6.5 +/- 0.3 pmol/mg/min and 232 +/- 33 microM. The latter is substantially greater than the normal CSF taurine concentration and this may be important in removing taurine released into the CSF during parenchymal cell swelling. Taurine uptake also appears calmodulin dependent as it was reduced by 84 and 91% in the presence of 25 microM trifluoperazine and 100 microM W-7, two calmodulin inhibitors. Taurine efflux from choroid plexus was stimulated by trifluoperazine, taurine, and hypo-osmotic stress. The latter two effects were reduced by niflumic acid, suggesting that taurine and hypo-osmotic stress act on the same pathway. The stimulation of efflux by hypo-osmotic stress decreased with time, whereas the effect of external taurine was sustained. If this efflux pathway is involved in the movement of taurine from choroid plexus to blood, these results suggest that changes in extracellular taurine may be more important than the direct effect of hypo-osmolality in the long-term loss of taurine from the brain.

Animals↗
PubMed1996

The role of the coagulation cascade in brain edema formation after intracerebral hemorrhage.

The coagulation cascade has a potential role in brain edema formation due to intracerebral hemorrhage. In this study blood and other solutions were injected stereotactically into the right basal ganglia in rats. Twenty-four hours following injection, brain water and ion contents were measured to determine the amount of brain edema. Intracerebral blood resulted in an increase in brain water content. The amount of brain edema surrounding the intracerebral hematoma was reduced by a thrombin inhibitor N alpha-(2-Naphthalenesulfonylglycyl)-4-amidino-DL-phenylalaninep iperidide, (alpha-NAPAP) infused into the hematoma after the clot had been allowed to solidify. The inhibitor did not alter the actual size of the clot mass. An artificial clot composed of fibrinogen, thrombin, and styrene microspheres also produced brain edema. A fibrin clot led to edema formation even in the absence of mass effect provided by the microspheres. The single component responsible for production of brain edema in all these models was thrombin. The edema was formed in response to a fibrinogen-independent pathway. These results indicate that the coagulation cascade is involved in brain edema that develops adjacent to an intracerebral hematoma.

Animals↗
PubMed1996

Edema from intracerebral hemorrhage: the role of thrombin.

The mechanism by which intracerebral hemorrhage leads to the formation of brain edema is unknown. This study assesses the components of blood to determine if any are toxic to surrounding brain. Various solutions were infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later; brain edema and ion contents were measured. Whole blood caused an increase in brain water content and ion changes consistent with brain edema. Concentrated blood cells, serum from clotted blood, and plasma from unclotted blood all failed to provoke edema formation when infused directly into the brain. On the other hand, activation of the coagulation cascade by adding prothrombinase to plasma did produce brain edema. The edema response to whole blood could be prevented by adding a specific thrombin inhibitor, hirudin, to the injected blood. This study indicates that thrombin plays an important role in edema formation from an intracerebral blood clot.

Animals↗
PubMed1996

Thrombin-soaked gelatin sponge and brain edema in rats.

Previous work from this laboratory has shown that injection of thrombin into rat basal ganglia causes brain edema. This study investigates the effect on rat brain of thrombin-soaked gelatin sponge (used for intraoperative hemostasis in clinical situations) at a concentration similar to that used in humans. Three models were developed to evaluate this effect. In the first model, a gelatin sponge soaked with vehicle or thrombin (100 U/cm3) was placed on the intact pia of the right frontal lobe in rats without cortical lesions. In the second model, frontal cortex was excised (3 mm3) and the exposed brain was cauterized with electrocoagulation. Gelatin sponge was soaked with vehicle or thrombin (1000, 100, 10, or 1 U/cm3) and placed in the lesion site. In the third model, hirudin, a specific thrombin antagonist, was added to the thrombin-soaked gelatin sponge and placed in a similar cortical lesion to determine if the observed effects were specific to thrombin. The dose-response range for thrombin was determined qualitatively by magnetic resonance (MR) imaging and quantitatively by brain edema formation 24 hours after exposure. We found no edema in the cortically intact rats. The rats given cortical lesions developed significant edema when subjected to 1000, 100, and 10 U/cm3 thrombin as seen on MR imaging and at 100 and 10 U/cm3 thrombin as revealed by wet/dry weight and ion studies of brain tissue. Topical hirudin prevented thrombin-induced edema. It is concluded that thrombin-soaked gelatin sponges cause or enhance significant brain edema in rats at concentrations typically used for human neurosurgery.

Administration, Topical↗
PubMed1995

Developmental changes in blood-brain barrier potassium permeability in the rat: relation to brain growth.

1. The potassium permeability of the blood-brain barrier (BBB) was determined in anaesthetized rats aged between 21 days gestation and adult using 86Rb+ as a marker for potassium. 2. The brain influx rate constant for 86Rb+ was high in fetal cortex at 21 days gestation (42.5 +/- 4.3 microliters g-1 min-1) but had decreased markedly by just after birth (12.2 +/- 0.6 microliters g-1 min-1). There was a further, gradual, postnatal decline to 7.0 +/- 0.3 microliters g-1 min-1 by 50 days after birth. 3. Developmental changes in passive BBB permeability were examined over the same age range using 14[C]urea. These studies showed similar developmental changes in influx rate to those found for 86Rb+. Specifically, a marked perinatal decline followed by a more gradual postnatal fall. Thus, the changes in potassium permeability probably reflect a decrease in the BBB paracellular leak during development. 4. The changes in BBB permeability coincide with changes in the rate of brain growth and the associated rate of brain potassium accumulation. As the potassium permeability properties of the adult BBB would provide insufficient potassium influx to meet the requirement associated with fetal brain growth, it is suggested that need for potassium may be the reason for the greater BBB permeability early in development.

Animals↗
PubMed1995

Blood-brain barrier taurine transport during osmotic stress and in focal cerebral ischemia.

Little is known about blood to brain taurine transport despite substantial evidence suggesting a role of taurine in brain volume regulation during osmotic stress or conditions inducing cell swelling, such as ischemia. We have made measurements of the taurine influx rate constant (K1) with [3H]taurine in three conditions: raised plasma taurine concentrations induced by infusion with 50 mM taurine (10 microliters/100 g/min); osmotic stress induced by i.p. injections of 1.5 M NaCl (2 ml/100 g) or distilled water (10 ml/100 g); and 4 h of middle cerebral artery occlusion (MCAo). In rats with MCAo, additional determinations were made of tissue water and taurine contents, and blood-brain barrier passive permeability with [3H]alpha-aminoisobutyric acid. Taurine infusion increased plasma taurine from 110 +/- 63 microM (SD) to 407 +/- 63 (p < 0.001) and decreased taurine K1 at the blood-brain barrier by 70% (p < 0.001), signifying saturable uptake that maintained unidirectional influx constant. Similarly, although hypo- and hyperosmolality increased and decreased plasma taurine concentration, respectively, a reciprocal relationship between K1 and plasma taurine in these experiments ensured that unidirectional fluxes of taurine into brain were unchanged by osmotic stress. During MCAo, the taurine K1 was reduced 80% in the ipsilateral ischemic tissue compared with the contralateral nonischemic tissue (p < 0.001). This decline may be due to a release of taurine into the brain circulation, because there was a concomitant loss of tissue taurine of 7.4 +/- 2.4 mmol/g dry weight (p < 0.05). Alternately, if taurine uptake is sodium dependent, the decline might reflect a disruption of the endothelial sodium gradient.

Aminoisobutyric Acids↗
PubMed1995

Mechanisms of brain ion homeostasis during acute and chronic variations of plasma potassium.

Brain and CSF potassium concentrations are well regulated during acute and chronic alterations of plasma potassium. In a previous study, we have shown that during chronic perturbations, regulation is achieved by appropriate adaptation of potassium influx, but that the degree of such adaptation during acute perturbations is much less. To elucidate further potential regulatory mechanisms, rats were rendered acutely or chronically hyper- or hypokalemic (range 2.7-7.6 mM). Measurements were made of brain and CSF water and ion contents to examine whether regulation occurred by modulation of K+ uptake into parenchymal cells. Furthermore, the permeability-surface area products (PSs) of 22Na+ were determined, because changes in K+ efflux fia Na+,K(+)-ATPase on the brain-facing side of the blood-brain barrier might be reflected in modified Na+ permeability. Brain and CSF K+ concentrations and Na PS were all independent of chronic changes in plasma K+ and acute hypokalemia, suggesting that neither modulation of parenchymal K+ uptake nor K+ efflux via the Na+,K(+)-ATPase is involved in extracellular K+ regulation in these conditions. In contrast, Na PSs were increased by 40% (p < 0.05) in acute hyperkalemia. This was accompanied by a slight loss of tissue K+ and water from the intracellular space. These results suggest that increased potassium influx in acute hyperkalemia is compensated by stimulation of K+ efflux via Na+,K(+)-ATPase. A slight degree of overstimulation, as indicated by a net loss of tissue K+, leads us to hypothesize that other factors, apart from the kinetic characteristics of Na+,K(+)-ATPase, may regulate this enzyme at the blood-brain barrier.

Animals↗
PubMed1995

Choroid plexus potassium cotransport: modulation by osmotic stress and external potassium.

The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide-sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 +/- 0.02, 0.44 +/- 0.04, and 0.84 +/- 0.06 min-1 in 240, 300, and 424 mOsm/kg solutions, respectively (p < 0.001). This increase in efflux with osmolality, an opposite effect to that found in many cells, was solely due to enhanced K cotransport. The increased cotransport may be involved in limiting brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide-sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 +/- 1, 19 +/- 3, 13 +/- 2, and 6 +/- 1 nmol/mg/min after 0, 10, 30, and 60-min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide-sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.

Animals↗
PubMed1995

N-system amino acid transport at the blood--CSF barrier.

Despite L-glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in L-[14C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na(+)-dependent mechanism. The Na(+)-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na(+)-dependent uptake was inhibited neither by the A-system substrate alpha-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and L-glutamate gamma-hydroxamate, three N-system substrates. Replacement of Na+ with Li+ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The Vmax and Km for glutamine transport by this system were 8.1 +/- 0.3 nmol/mg/min and 3.3 +/- 0.4 mM, respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.

Amino Acids↗
PubMed1995

Chronic central potassium infusion prevents deoxycorticosterone-salt hypertension in rats.

Although it has long been established that cerebrospinal fluid potassium concentration (CSF [K]) is very tightly regulated, it has been reported that rats made hypertensive by central infusions of aldosterone have significantly lower CSF [K] compared with normotensive controls. We investigated whether reduced CSF [K] is also present in another animal model of hypertension, the deoxycorticosterone acetate (DOCA)-salt rat, and we hypothesized that chronic intracerebroventricular (IVT) infusion of potassium with miniosmotic pumps might attenuate the rise in blood pressure observed in these rats. DOCA-salt rats without IVT infusions or with control CSF infusions (0.5 microliter/h of 2.9 mM K for 2 wk) had a significantly increased systolic blood pressure and a significantly lower CSF [K] compared with their respective sham groups. In contrast, DOCA-salt rats receiving IVT infusions with elevated [K] (10, 30, or 150 mM) had significantly lower blood pressures compared with those receiving control CSF. They also did not exhibit decreased CSF [K] compared with their respective sham groups. At 10 and 150 mM K, the blood pressure rise in DOCA-salt rats was not significantly different from shams. At 30 mM K, there was a slight, but significant, increase in blood pressure in the DOCA-salt rats compared with their shams, but this rise was still much less than in DOCA-salt rats infused with 2.9 mM K. Infusions with elevated [K] had no effect on blood pressure in the sham animals. These studies suggest that altered brain potassium homeostasis may play an important role in the development of DOCA-salt hypertension.

Animals↗
PubMed1995

Intracerebral infusion of thrombin as a cause of brain edema.

Purified thrombin from an exogenous source is a hemostatic agent commonly used in neurosurgical procedures. The toxicity of thrombin in the brain, however, has not been examined. This study was performed to assess the effect of thrombin on brain parenchyma, using the formation of brain edema as an indicator of injury. Ten microliters of test solution was infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later, and the extent of brain edema and ion content were measured. Concentrations of human thrombin as low as 1 U/microliter resulted in a significant increase in brain water content. Rats receiving 10 U/microliters had a mortality rate of 33% compared to no mortality in the groups receiving smaller doses. Thrombin-induced brain edema was inhibited by a specific and potent thrombin inhibitor, hirudin. A medical grade of bovine thrombin commonly used in surgery also caused brain edema when injected at a concentration of 2 U/microliters. Edema formation was prevented by another highly specific thrombin inhibitor, N alpha-(2-Naphthalenesulfonylglycyl)-4-DL-phenylalaninepiperidid e (alpha-NAPAP). Thrombin-induced brain edema was accompanied by increases in brain sodium and chloride contents and a decrease in brain potassium content. Changes in brain ions were inhibited by both hirudin and alpha-NAPAP, corresponding to the inhibition of brain water accumulation. This study shows that thrombin causes brain edema when infused into the brain at concentrations as low as 1 U/microliter, an amount within the range of concentrations used for topical hemostasis in neurosurgery.

Animals↗
PubMed1994

Developmental stages in experimental liver metastases: relation to invasiveness.

We have previously reported that an invasive morphotype can be evoked in a rat colon carcinoma by transplanting it into pre-induced subcutaneous granulation tissue. We have now studied the interaction of the same tumor with liver tissue, which is extremely poor in connective tissue in comparison with the subcutaneous site. Tumor cells were injected into the portal system and the resulting experimental liver metastases were examined by electron microscopy and immunohistochemistry. Early metastases consisted of well-differentiated acini, fully surrounded by connective tissue that was derived from the periportal stroma. In a later stage, this connective tissue was overgrown by tumor cells and, almost immediately, acinar differentiation was lost. Most metastases eventually reached the liver capsule, which reacted by forming a layer of granulation tissue. Only in this layer, we observed invasion by thin tumor cell strands, which were often intimately associated with fibroblasts or with blood capillaries. The tumor cells remained smooth and rounded during this process. After fully penetrating the granulation tissue, the tumor cell strands reached the liver surface, where they formed poorly structured papillary masses that were nearly devoid of stroma. Our observations indicate that, even in a relatively homogeneous organ like the liver, the tumor-host interaction is highly complex and dynamic. They also confirm the notion that granulation tissue stimulates tumor invasiveness. Finally, they show that tumor cells can actively invade host tissues without exhibiting a "fibroblastic" morphology.

Animals↗
PubMed1994

Blood-brain barrier permeability and brain concentration of sodium, potassium, and chloride during focal ischemia.

Brain edema formation during the early stages of focal cerebral ischemia is associated with an increase in both sodium content and blood-brain barrier (BBB) sodium transport. The goals of this study were to determine whether chloride is the principal anion that accumulates in ischemic brain, how the rate of BBB transport of chloride compares with its rate of accumulation, and whether the stimulation seen in BBB sodium transport is also seen with other cations. Focal ischemia was produced by occlusion of the middle cerebral artery (MCAO) in anesthetized rats. Over the first 6 h after MCAO, the amount of brain water in the center of the ischemic cortex increased progressively at a rate of 0.15 +/- 0.02 (SE) g/g dry wt/h. This was accompanied by a net increase in brain sodium (48 +/- 12 mumol/g dry wt/h) and a loss of potassium (34 +/- 7 mumol/g dry wt/h). The net rate of chloride accumulation (16 +/- 1 mumol/g dry wt/h) approximated the net rate of increase of cations. Three hours after MCAO, the BBB permeability to three ions (22Na, 36Cl, and 86Rb) and two passive permeability tracers ([3H]alpha-aminoisobutyric acid ([3H]AIB) and [14C]urea) was determined. Permeability to either passive tracer was not increased, indicating that the BBB was intact. The rate of 36Cl influx was 3 times greater and the rate of 22Na influx 1.8 times greater than their respective net rates of accumulation in ischemic brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Potassium cotransport at the rat choroid plexus.

The choroid plexuses are involved in cerebrospinal fluid (CSF) secretion and CSF K homeostasis. We examined K transport mechanisms present in the isolated rat choroid plexus that may be involved in these functions, predominantly using 86Rb as a marker for K. The study demonstrates that there are two primary uptake mechanisms. Ouabain-sensitive Na-K-adenosinetriphosphatase and bumetanide-sensitive cotransport, probably of the Na-K-2Cl form, account for 48 and 46% of uptake, respectively. Efflux studies demonstrate that the primary K efflux mechanism is also bumetanide-sensitive cotransport with the other major component probably being by K channels as it is inhibitable by barium or quinidine. Efflux via the cotransporter was not inhibited by R(+)-butylindazone, a KCl cotransport inhibitor, but it was enhanced in the presence of ouabain (P < 0.001) or increased extracellular Na concentration (P < 0.01). Furthermore, Na efflux was bumetanide sensitive (P < 0.05). In all, these data suggest that the efflux cotransporter is also of the Na-K-Cl form and that it is the same transporter as the influx mechanism operating in both directions. The evidence presented leads us to hypothesize that this cotransporter is on the apical membrane of the choroid plexus and that it may have a central role in CSF secretion and perhaps CSF K homeostasis.

Animals↗
PubMed1994

Rubidium entry into brain and cerebrospinal fluid during acute and chronic alterations in plasma potassium.

To elucidate whether K+ uptake across the blood-brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier is subject to acute or chronic regulation, rats were rendered acutely or chronically hyper- or hypokalemic (range: 2.8-7.2 mM). Measurements were made of the permeability-surface (PS) products of 86Rb+, a marker for K+, and alpha-[3H]aminoisobutyric acid (AIB), a passive permeability marker, and of CSF K+ concentration ([K+]CSF). [K+]CSF decreased by 8% in chronic hypokalemia P < 0.01), but otherwise remained unchanged. The AIB PS products were unaltered in any group, excluding a change in passive permeability. The Rb PS product, however, increased by 31% for brain tissue (P < 0.01) and by 46% for CSF (P < 0.05) during acute hypokalemia, but was unchanged during acute hyperkalemia. During chronic hypokalemia the Rb PS product increased by 40% for brain (P < 0.01) and 55% for CSF (P < 0.01) and decreased during chronic hyperkalemia by 37% for brain (P < 0.01) and 49% (P < 0.01) for CSF. Unidirectional K+ fluxes were calculated, revealing greater regulation of K+ influx into both brain tissue and CSF during chronic compared with acute changes of plasma K+ concentration ([K+]pl). Our results suggest that K+ transport is saturable at both the BBB and the blood-CSF barriers under normal conditions and that both barriers adapt to chronic changes in [K+]pl by modifying specific, transcellular routes of K+ entry.

Aminoisobutyric Acids↗
PubMed1993

Transplantation of colon carcinoma into granulation tissue induces an invasive morphotype.

The stroma surrounding many malignant tumors resembles granulation tissue. To test the hypothesis that such stroma stimulates tumor invasiveness, we compared, by electron microscopy and immunohistochemistry, the growth patterns of CC531 rat colon adenocarcinoma in 2 experimental situations: (i) after transplantation into the undisturbed subcutaneous connective tissue of rats, and (ii) after transplantation into experimentally induced subcutaneous granulation tissue in rats. For the latter experimental situation, a subcutaneous "tissue chamber" was designed allowing fragments of tumor tissue to be transplanted into the very center of developing granulation tissue. In the undisturbed subcutaneous tissue, the whole tumor was generally encapsulated, and the tumor cells were arranged in compact groups with a strong tendency to form acini. In the pre-formed granulation tissue, on the other hand, the tumor tissue closely matched descriptions of invasive colon carcinomas in the literature and met the criteria for the "invasive morphotype". In this situation, the tumor consisted of thin, unorganized, widely dispersed strands of irregular tumor cells with numerous protrusions that deeply penetrated the surrounding matrix. Our results show that an invasive morphotype can be evoked by pre-inducing granulation tissue at the transplantation site.

Adenocarcinoma↗
PubMed1993

Potassium transport at the blood-brain and blood-CSF barriers.

Figure 5 gives a summary of K transporters at the BBB based on the available evidence. It appears that the cerebral endothelial cells have an array of potassium channels, although the degree to which each is open under physiological conditions is uncertain. Different channels are present on the luminal and abluminal membranes, and the opening and closing of these channels may allow modulation of the brain K influx and efflux rates and play a role in brain K homeostasis. These channels may also play a role in hyperosmotic brain volume regulation by increasing the entry rate of potassium into brain and may be involved in volume regulation of the endothelial cell itself. The nature of fluid transport at the BBB remains to be fully elucidated, with the presence of a Na/K/2Cl co-transporter being uncertain. The abluminal inwardly-rectifying channel may act as a leak pathway to allow modulation of fluid secretion by the Na/K ATPase without altering the K concentration of that fluid. Finally, there is some evidence that K transport at the BBB is under hormonal and neuronal control. The cerebral capillaries possess receptors for many of the hormones present in blood and brain.

Animals↗