The application of a new laminitis scoring method to model the rate and pattern of improvement from equine endocrinopathic laminitis in a clinical setting.

BACKGROUND: Endocrinopathic, or hyperinsulinaemia-associated laminitis (HAL) is a common and debilitating equine foot disease, and although no pharmacological treatments are registered, several are under development. To evaluate the effect of such treatments, an accurate and consistent method is needed to track the clinical signs of laminitis over time, and the natural history of the disease, in terms of a 'normal' pattern of improvement, needs to be understood. This study examined the improvement pattern in clinical cases of naturally-occurring HAL subjected to a range of best-practice interventions, using two different scoring methods. Eighty horses and ponies with suspected HAL were enrolled in a study conducted at 16 veterinary practices across Germany. The severity of laminitis was assessed by independent veterinarians using both the traditional Obel method and a modified Obel method developed by Meier and colleagues. Assessments were made on the day of diagnosis (d 0), then on days 4, 9, 14, 25 and 42 during the intervention period. Pain medications were withheld for 24 h prior to clinical examination in all cases. RESULTS: Time to marked improvement from laminitis varied between individuals, but was difficult to monitor accurately using the Obel method, with the median grade being 2/4 on days 0 and 4, then 0/4 from d 9 onwards. More subtle changes could be identified using the Meier method, however, and the median scores were seen to follow the form of an exponential decay model in most horses, improving from 8/12 on d 0, to 0/12 on d 25. Within this composite scoring method, considerable variation was observed in the rate of improvement of individual clinical signs, with the average time taken for each sign to reach a median score of 0 ranging from 4 days (foot lift and weight shifting) to 25 days (gait when turned in a circle) across all 80 horses. CONCLUSIONS: The Meier method provides a reliable and consistent method for monitoring the clinical status of horses with HAL, and despite the variability, the pattern of improvement described here should provide a useful benchmark against which individual cases and new treatments can be assessed.

Contemporary anthropogenic silver cycle: a multilevel analysis.

Anthropogenic cycling of silver in 1997 is presented using three discrete governmental units: 64 countries encompassing what we believe to be over 90% of global silver flows, 9 world regions, and the entire planet. Using material flow analysis (MFA) techniques, the country level cycles are aggregated to produce the regional cycles, which are used to form a "best estimate" global cycle. Interesting findings include the following: (1) several silver-mining countries export ore and concentrate but also import silver-containing semiproducts and products; (2) the level of development for a country, as indicated by the gross domestic product, is a fair indicator of silver use, but several significant outliers exist; (3) the countries with the greatest mine production include Mexico, the United States, Peru, and China, whereas the United States, Japan, India, Germany, and Italy lead in the fabrication and manufacture of products; (4) North America and Europe's use of silver products exceed that of other regions on a per capita basis; (5) global silver discards, including tailings and separation waste, totaled approximately 57% of the silver mined; (6) approximately 57% of the silver entering waste management globally is recycled; and (7) the amount of silver entering landfills globally is comparable to the amount found in tailings. The results of this MFA lay the basis for further analysis, which in turn can offer insight into natural resource policy, the characterization of environmental impact, and better resource management.

Multilevel cycle of anthropogenic copper.

A comprehensive contemporary cycle for stocks and flows of copper is characterized and presented, incorporating information on extraction, processing, fabrication and manufacturing, use, discard, recycling, final disposal, and dissipation. The analysis is performed on an annual basis, ca. 1994, at three discrete governmental unit levels--56 countries or country groups that together comprise essentially all global anthropogenic copper stocks and flows, nine world regions, and the planet as a whole. Cycles for all of these are presented and discussed, and a "best estimate" global copper cycle is constructed to resolve aggregation discrepancies. Among the most interesting results are (1) transformation rates and recycling rates in apparently similar national economies differ by factors of two or more (country level); (2) the discard flows that have the greatest potential for copper recycling are those with low magnitude flows but high copper concentrations--electronics, electrical equipment, and vehicles (regional level); (3) worldwide, about 53% of the copper that was discarded in various forms was recovered and reused or recycled (global level); (4) the highest rate of transfer of discarded copper to repositories is into landfills, but the annual amount of copper deposited in mine tailings is nearly as high (global level); and (5) nearly 30% of copper mining occurred merely to replace copper that was discarded. The results provide a framework for similar studies of other anthropogenic resource cycles as well as a basis for supplementary studies in resource stocks, industrial resource utilization, waste management, industrial economics, and environmental impacts.

The cerebrovascular response to elevated potassium--role of nitric oxide in the in vitro model of isolated rat middle cerebral arteries.

We investigated the role of nitric oxide (NO) in the vascular response to high extraluminal K(+)-concentrations in the in vitro model of isolated rat middle cerebral arteries (MCA). Under control conditions, rat MCA dilated at 20, 30, 40 and 60 mM K(+). At 80 mM K(+), a slight vasoconstriction occurred. The unspecific NO synthase (NOS)-inhibitor L(omega)-nitro-L-arginine (L-NNA) increased the resting tone at 3 mM K(+) by 31+/-5% (P<0.01). While the vasodilatative effect of 20 mM K(+) was unaffected by L-NNA, NOS-inhibition resulted in vasoconstriction at > or = 40 mM K(+) (P<0.01). In presence of L-NNA, the basal vessel diameter was restored by either the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) or the cell-permeable guanosine-3',5'-cyclic monophosphate (cGMP) analogue 8-Br-cGMP. Co-application of L-NNA with either SNAP or 8-Br-cGMP resulted in partial restitution of the vasodilatative effect of 40 mM K(+), respectively. In presence of the soluble guanylyl cyclase inhibitor 1 H-[l,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the vascular response to 40 mM K(+) was abolished. Our findings together with findings from the literature indicate a modulator role of NO at K(+) > or = 40 mM K(+), involving a cGMP-dependent mechanism.

Products of hemolysis in the subarachnoid space inducing spreading ischemia in the cortex and focal necrosis in rats: a model for delayed ischemic neurological deficits after subarachnoid hemorrhage?

OBJECT: The pathogenesis of delayed ischemic neurological deficits after subarachnoid hemorrhage has been related to products of hemolysis. Topical brain superfusion of artificial cerebrospinal fluid (ACSF) containing the hemolysis products K+ and hemoglobin (Hb) was previously shown to induce ischemia in rats. Superimposed on a slow vasospastic reaction, the ischemic events represent spreading depolarizations of the neuronal-glial network that trigger acute vasoconstriction. The purpose of the present study was to investigate whether such spreading ischemias in the cortex lead to brain damage. METHODS: A cranial window was implanted in 31 rats. Cerebral blood flow (CBF) was measured using laser Doppler flowmetry, and direct current (DC) potentials were recorded. The ACSF was superfused topically over the brain. Rats were assigned to five groups representing different ACSF compositions. Analyses included classic histochemical and immunohistochemical studies (glial fibrillary acidic protein and ionized calcium binding adaptor molecule) as well as a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Superfusion of ACSF containing Hb combined with either a high concentration of K+ (35 mmol/L, 16 animals) or a low concentration of glucose (0.8 mmol/L, four animals) reduced CBF gradually. Spreading ischemia in the cortex appeared when CBF reached 40 to 70% compared with baseline (which was deemed 100%). This spreading ischemia was characterized by a sharp negative shift in DC, which preceded a steep CBF decrease that was followed by a slow recovery (average duration 60 minutes). In 12 of the surviving 14 animals widespread cortical infarction was observed at the site of the cranial window and neighboring areas in contrast to findings in the three control groups (11 animals). CONCLUSIONS: The authors conclude that subarachnoid Hb combined with either a high K+ or a low glucose concentration leads to widespread necrosis of the cortex.

Voltage-gated proton currents in microglia of distinct morphology and functional state.

Whole-cell patch-clamp measurements were performed to investigate voltage-gated proton currents (I(PR)) in cultured murine microglia of distinct morphology and functional state. We studied I(PR) in ameboid microglia of untreated cultures, in ameboid microglia which had been activated by lipopolysaccharide, and in ramified microglia which had been exposed to astrocyte-conditioned medium. Proton currents of these three microglia populations did not differ regarding their activation threshold or the voltage dependence of steady-state activation. Moreover, pharmacological properties of I(PR) were similar: proton currents were sensitive to extracellularly applied Zn2+ or La3+, and could be abolished by each of those at a concentration of 100 microM. In the presence of extracellular Na+, I(PR) was decreased to a similar small extent due to activity of the Na+/H+ exchanger in all microglial populations. In contrast, proton currents of microglia differed between the three cell populations with respect to their current density and their time-course of activation: in comparison with untreated microglia, the current density of I(PR) was reduced by about 50% in microglia after their treatment with either lipopolysaccharide or astrocyte-conditioned medium. Moreover, I(PR) activated significantly more slowly in cells exposed to lipopolysaccharide or astrocyte-conditioned medium than in untreated cells. It can be concluded that the distinct H+ current characteristics of the three microglial populations do not correlate with the functional state of the cells.

Involvement of stretch-activated Cl- channels in ramification of murine microglia.

A stretch-activated Cl- current (ICl) was investigated in cultured murine microglia using the whole-cell configuration of the patch-clamp technique. After application of membrane stretch, a Cl- current appeared within seconds, and its amplitude increased further within 3-8 min. ICl underwent rundown, which was prevented by addition of 4 mM ATP to the intracellular perfusing solution. The stretch-activated Cl- current exhibited outward rectification and did not show any voltage-dependent gating. Lowering the concentration of extracellular Cl- from 142 to 12 mM by equimolar substitution of Cl- with gluconate shifted the reversal potential of ICl by 41.6 +/- 1.8 mV in the depolarizing direction. 4, 4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) blocked ICl in a voltage- and time-dependent manner. At a test potential of +40 mV, a half-maximal blockade at 16.1 microM DIDS and at 71.0 microM SITS was determined for ICl. At a concentration of 200 microM, 5-nitro-2-(3-phenylpropylamino)benzoic acid or flufenamic acid blocked ICl by 88% and 75%, respectively. Each of these four Cl- channel blockers reversibly inhibited the ramification process of microglia, whereas blockers of voltage-gated Na+ and K+ channels did not affect the transformation of microglia from their ameboid into the ramified phenotype. It is suggested that in microglia functional stretch-activated Cl- channels are required for the induction of ramification but not for maintaining the ramified shape.

Changes in proton currents in murine microglia induced by cytoskeletal disruptive agents.

Voltage-gated proton currents (IPR) were investigated in cultured murine microglia using the whole-cell configuration of the patch clamp technique. At a gradient of 1.5 between intracellular (pHi = 6.0) and extracellular pH (pHo = 7.5) values, outward IPR were detected at depolarizing potentials, while the activation threshold of IPR was -40 mV. Time-dependent activation of IPR was fitted by a single exponential with a time constant of 661 ms at +40 mV. An increase in the activation time constant of IPR was seen after exposure of microglia to the cytoskeletal disruptive agents cytochalasin D or colchicine. Moreover, the current density of IPR was significantly reduced by 49% in cells treated with cytochalasin D and by 27% in cells treated with colchicine for 24 h. In contrast, voltage-dependence of steady-state activation of IPR was unchanged after disruption of the cytoskeleton. Exposure of microglia to the cytoskeletal stabilizers phalloidin and taxol did not affect IPR of microglia.

Age-dependent variations in potassium sensitivity of A-currents in rat hippocampal neurons.

Hippocampal pyramidal neurons were either cultured from prenatal rats or acutely isolated from the brain of newborn and juvenile rats. The influence of lowering the concentration of the extracellular potassium concentration ([K+]o) on isolated fast transient outward K+ currents (I(A)) was studied in these neurons using the patch clamp technique in the whole cell configuration. With respect to the response of I(A) to lowering [K+]o, three types of cells were observed. The first subpopulation of neurons was characterized by a complete suppression of I(A) over the whole voltage range under potassium-free solutions (type A neurons). A second proportion of cells showed an increase of I(A) at test pulses below -0 mV and a decrease of I(A) at voltages above -0 mV (type B neurons). In a third group of neurons, amplitudes of I(A) increased at all potentials tested during omission of potassium ions from the extracellular superfusate (type C neurons). Whereas type A and type B neurons were preferentially found in freshly plated cultures and newborn rats, the majority of type C cells was detected in long-term cultures and in animals of older ages. Thus, hippocampal A-currents lose their sensitivity to extracellular potassium ions during early ontogenesis.

Pharmacological properties of Ca2+-activated K+ currents of ramified murine brain macrophages.

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I(K,Ca)) were investigated in ramified murine brain macrophages. In order to induce I(K,Ca) the intracellular concentration of nominal free Ca2+ was adjusted to 1 microM. The Ca2+-activated K+ current of brain macrophages did not show any voltage dependence at test potentials between -120 and +30 mV. A tenfold change in extracellular K+ concentration shifted the reversal potential of I(K,Ca) by 51 mV. The bee venom toxin apamin applied at concentrations of up to 1 microM did not affect I(K,Ca). Ca2+-activated K+ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3 nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I(K,Ca) at concentrations between 1 and 50 nM. Tetraethylammonium (TEA) blocked 8.0% of I(K,Ca) at a concentration of 1 mM, whereas 31.4% of current was blocked by 10 mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2 mM for their ability to block I(K,Ca). La3+ reduced I(K,Ca) by 72.8%, whereas Cd2+ decreased I(K,Ca) by 17.4%; in contrast, Ni2+ did not have any effect on I(K,Ca). Ba2+ applied at a concentration of 1 mM reduced I(K,Ca) voltage-dependently at hyperpolarizing potentials.

Developmental changes of inward rectifier currents in neurons of the rat entorhinal cortex.

A slice preparation was used to investigate inward rectifier currents (I(H)) of entorhinal cortex (EC) neurons. Using the whole-cell configuration of the patch-clamp technique, I(H) was studied in pyramidal cells from layer IV of the rat EC and in stellate cells from layer II of the EC. Inward rectifier currents were analyzed in neurons of newborn (P1-3), juvenile (P8-14) and adult (>P28) rats. Pyramidal cells of juvenile rats possessed a significantly larger current density of I(H) than pyramidal cells of newborn rats, whereas no differences in the current density of I(H) were found between pyramidal neurons of juvenile and of adult animals. In contrast, the current density of I(H) of stellate cells was significantly increased in juvenile rats compared with newborn rats as well as in adult rats compared with juvenile rats. Moreover, in adult rats the current density of I(H) was larger in stellate cells than in pyramidal cells, whereas opposite data were obtained in juvenile animals.

Distinct soluble astrocytic factors induce expression of outward K+ currents and ramification of brain macrophages.

Isolated cultured murine brain macrophages (BM) were treated with supernatants of enriched astrocytic cultures. The astrocyte-conditioned medium (ACM) induced ramification of BM. In parallel, BM expressed voltage-gated outward K+ currents (I(K)) during the first 2 days after the application of ACM. However, in ramified BM which were treated once with ACM, I(K) disappeared 5 days after that treatment. In contrast, BM expressed I(K) over a period of more than 5 days when cells were treated daily with ACM. A blockade of I(K) by charybdotoxin or by kaliotoxin did not inhibit ramification of the cells. Furthermore, after application of low-concentrated ACM BM exhibited I(K) but did not change their morphology. It is suggested that in murine BM the ramification and the expression of I(K) are induced by distinct soluble factors derived from astrocytes.

Blockade of voltage-gated outward K+ currents of ramified murine microglia by scorpion peptide toxins.

Microglial cells were cultured from murine neonatal brain. Ramification of isolated microglia could be induced by the application of astrocyte-conditioned medium (ACM). Voltage-gated outward potassium currents (IK) were measured in ramified microglial cells 12-24 h after their treatment with ACM. The effects of the specific K+ channel blockers charybdotoxin (CTX), noxiustoxin (NTX) and kaliotoxin (KTX) on IK of ramified microglia were studied. All these peptide toxins blocked IK in a concentration-dependent manner, while showing a high sensitivity for IK. A half-maximal effective concentration (IC50) of CTX was estimated to be 1.13 nM, while IC50 values of 1.24 nM and of 0.81 nM were calculated for KTX and for NTX, respectively. In contrast, dendrotoxin (DTX) did not show any effect on IK. It is suggested that ramified microglial cells express outward K+ currents exhibiting pharmacological properties similar to that of outward K+ currents in cytokine-activated ameboid microglia.

Modulation of A-currents by [K+]o in acutely isolated pyramidal neurones of juvenile rat entorhinal cortex and hippocampus.

Pyramidal neurones of the entorhinal cortex and of hippocampal areas CA1 and CA3 were acutely isolated from juvenile rats. The effect of lowering the concentration of extracellular potassium ions ([K+]o) on fast transient A-currents (IA) was studied using the whole-cell configuration of the patch-clamp technique. Upon lowering [K+]o from 5.4 to 0 mM amplitudes of IA in all pyramidal neurones were reduced only at test potentials positive to -20 mV, but were increased at potentials below -20 mV. Omission of magnesium ions from the intracellular perfusate resulted in an increase of IA in external K+-free solution at any potential tested. Moreover, when [K+]o was reduced steady-state activation and inactivation curves of IA were shifted in the hyperpolarizing direction.

Comparison of voltage-dependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3.

1. The properties of voltage-gated potassium currents were studied in acutely isolated rat hippocampal pyramidal cells from area CA1 and CA3 at postnatal ages of day 6-8, 9-14, and 26-29 (P6-8, P9-14, and P26-29) with the use of the whole cell version of the patch-clamp technique. 2. The outward current pattern of all cells under investigation could be separated in a fast transient A current (IA) and a delayed rectifier-like current (IK). 3. In both preparations, IA activated and inactivated rapidly. Vh describing steady-state inactivation was -84.5 mV in CA3 cells and -85.5 mV in CA1 cells. The activation behavior was characterized by Vh = -23.8 mV in CA3 cells and -27.2 mV in CA1 cells. The removal of inactivation was monoexponential both in CA1 and CA3 neurons with time constants of 32.1 and 28.5 ms, respectively. IA was insensitive to tetraethylammonium (TEA), dendrotoxin (300 nM), and mast cell degranulating peptide (200 nM), but could be blocked with 5 mM 4-aminopyridine (4-AP) by approximately 80%. In both preparations, A currents did not depend on Ca2+ influx. 4. Delayed rectifier currents (IK) in CA1 and CA3 pyramidal neurons decayed along a double exponential time course. Steady-state inactivation was described by Vh = -79.5 mV in CA3 cells and -76.0 mV in CA1 cells. The activation curves were characterized by midpoints of -3.8 mV in CA3 cells and of -1.4 mV in CA1 cells. The removal of inactivation was monoexponential in CA1 and CA3 neurons with time constants of 210.3 and 202.4 ms, respectively. All kinetic properties were identical for the differentially decaying components of IK. In CA1 cells IK was blocked by TEA at +30 mV with an IC50 of 0.98 mM. In CA3 cells the corresponding IC50 value was 1.05 mM. About 20% of IK were insensitive to TEA. IK was partially blocked by approximately 30% with 100 microM 4-AP. Mast cell degranulating peptide (100-200 nM) and dendrotoxin (50-300 nM) had no effect on IK. 6. Perfusion of charybdotoxin (30 nM), Cd2+ (300 microM), La3+ (10 microM), or Ca(2+)-free solutions resulted in the isolation of a small noninactivating outward current component. Around 10% of IK appeared to be Ca2+ dependent in CA1 neurons. In CA3 pyramidal cells Ca(2+)-dependent outward currents seemed to be somewhat larger with approximately 20%. 7. In CA1 as well as in CA3 cells, the kinetic and pharmacological properties of IA and IK remained stable during postnatal development. However, the contribution of IA and IK to the whole cell current varied with age. IA was more prominent in CA1 cells of age group P6-8 than in age-matched CA3 cells. CA3 cells had smaller A currents and larger delayed rectifier currents than CA1 pyramidal cells. Current densities of IA and IK were analyzed during development to assess changes in the expression of these currents. With increasing postnatal age, the expression of IA was downregulated in both preparations. This effect was more pronounced in CA3 than in CA1 cells. In contrast, IK was upregulated during the same developmental period. This increase in the expression of IK was with approximately 300% much larger in CA1 cells than in CA3 cells with only approximately 50%.