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Biomedical subjects

K R Wagner

Publications and source records attributed to K R Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1994

Efficacious experimental stroke treatment with high-dose methylprednisolone.

BACKGROUND AND PURPOSE: Recent studies reveal success in treating spinal cord trauma with early, high-dose methylprednisolone. As in spinal cord research, failure to find therapeutic effects with steroids in studies of acute stroke treatment may reflect institution of treatment too late and at too low dosage. We presently test the efficacy of stroke treatment with methylprednisolone administered early and at high doses using a cat temporary middle cerebral artery occlusion model. METHODS: We occluded the middle cerebral artery for 4 hours in 24 pentobarbital-anesthetized cats. To enhance the probability of brain injury, we maintained the cats' serum glucose concentrations at high levels both during occlusion and for 6 hours afterward. Using a blinded, randomized study design, we treated 12 cats with methylprednisolone (30 mg/kg IV infused over 15 minutes starting 30 minutes after occlusion followed by 5.4 mg.kg-1.h-1 IV for the next 23 hours) and 12 control cats with vehicle. During and for 8 hours after occlusion, we monitored cerebral blood flow, brain and rectal temperatures, and multiple cardiovascular and blood compositional parameters. We assessed brain pathological outcome after animal survival for 4 days or after acute death from hemispheric edema. RESULTS: Experimental and control animals showed similar early mortality rates (treated, 3/12; controls, 4/12). However, surviving methylprednisolone-treated cats (n = 9) showed a mean infarct size more than six times smaller than in the control animals (n = 8) (mean +/- SEM, 2.4 +/- 0.7% versus 15.6 +/- 6.2% of the ischemic territory, respectively; P < .05). The methylprednisolone-treated animals also showed less marked reduction in cerebral blood flow during ischemia than did the controls (mean +/- SEM, 58 +/- 5% versus 74 +/- 4%; P < .005). CONCLUSIONS: Administering methylprednisolone at high doses early after onset of ischemia significantly reduces tissue injury in cats that survive 4 days of temporary middle cerebral artery occlusion. This improvement in outcome occurs in the setting of significant increases in ischemic cerebral blood flow. However, methylprednisolone treatment did not reduce hemispheric edema in animals that died early after temporary middle cerebral artery occlusion.

Animals↗
PubMed1993

The 87K postsynaptic membrane protein from Torpedo is a protein-tyrosine kinase substrate homologous to dystrophin.

Postsynaptic peripheral membrane proteins at the neuromuscular junction have been proposed to participate in the immobilization of the nicotinic acetylcholine receptor at the synapse. An 87 kd cytoplasmic peripheral membrane protein has been demonstrated to colocalize with the nicotinic acetylcholine receptor in the Torpedo electric organ and at the mammalian neuromuscular junction. We have cloned the cDNA encoding the 87K protein from Torpedo electric organ, and the predicted protein sequence is homologous to the C-terminal domains of dystrophin, the protein product of the Duchenne muscular dystrophy gene. The 87K protein displays a restricted pattern of expression detected only in electric organ, brain, and skeletal muscle. Analysis of the in vitro and in vivo phosphorylation of the 87K protein indicates that it is multiply phosphorylated on serine, threonine, and tyrosine residues. The 87K protein is in a complex with other proteins associated with the postsynaptic membrane, including dystrophin and a 58 kd protein. These results suggest that the 87K protein is involved in the formation and stability of synapses and is regulated by protein phosphorylation.

Amino Acid Sequence↗
PubMed1993

Bumetanide decreases canine cerebrospinal fluid production. In vivo evidence for NaCl cotransport in the central nervous system.

Na/K/2Cl cotransport carrier plays an important role in fluid absorption and secretion in many epithelial tissues. The role of the carrier, however, in mammalian choroidal cerebrospinal fluid (CSF) production has been controversial. We used ventriculo-cisternal perfusion (VCP) labeled with blue dextran with or without bumetanide and measured choroidal CSF production in anesthetized, and paralyzed, mechanically ventilated dogs. During 3 h of VCP, mean intracerebroventricular and arterial pressures, PaCO2, pH, [HCO3-], and serum osmolality remained normal in both groups (n = 9 in each group). Beginning 90 min after the start of VCP, choroidal CSF production was measured every 15 min. In group I (control group), values for CSF production (means +/- SD) were 49 +/- 20, 49 +/- 21, 51 +/- 21, 51 +/- 23, 48 +/- 20, 56 +/- 24, and 48 +/- 20 microliters/min, at 90, 105, 120, 135, 150, 165, and 180 min, respectively. These values did not differ significantly from each other. In group II (bumetanide group), after baseline control CSF production had been determined at 90 and 105 min, bumetanide (10(-4) mol/liter) was added to VCP. Mean values for CSF production were 54 +/- 15 and 52 +/- 17 microliters/min before, and 39 +/- 25, 34 +/- 19, 28 +/- 10, 30 +/- 17, and 30 +/- 18 microliters/min after addition of bumetanide at 90, 105, 120, 135, 150, 165, and 180 min, respectively. Comparing the two groups, baseline values for CSF production measured at 90 and 105 min did not differ significantly. After addition of bumetanide (group II), however, decrements in CSF production varied from 30 +/- 27% at 120 min to 47 +/- 14% at 150 min, which were significantly different from changes in group I. The results of this study indicate that NaCl cotransport carrier is involved in secretion of CSF in dogs, and inhibition of the transporter results in approximately 50% reduction in CSF production.

Animals↗
PubMed1992

Hemorrhagic infarct conversion in experimental stroke.

STUDY OBJECTIVE: This study investigated the relations between hemorrhagic infarction and occlusion, release, levels of glycemia, brain energy state, and lactate content after cerebrovascular occlusion. DESIGN: Prospective, controlled laboratory investigation. TYPE OF PARTICIPANTS: One hundred six pentobarbital-anesthetized cats. INTERVENTIONS: The middle cerebral artery was occluded with a Yasargil clip transorbitally either temporarily (0.5, four, and eight hours) or permanently. Normoglycemic and hyperglycemic animals were closely monitored for eight hours. Brain pathology was assessed after two weeks' survival or at the time of spontaneous animal death. Topographic brain metabolite studies were carried out after four hours of middle cerebral artery occlusion. MEASUREMENTS AND MAIN RESULTS: Morphometric quantitation of cerebral hemorrhage and infarction and fluorometric determinations of blood and brain tissue, glucose, glycogen, lactate, adenosine triphosphate, and phosphocreatine from 16 topographic brain sites were carried out. Twenty-one of 82 (25.6%) animals evaluated neuropathologically showed hemorrhagic infarcts. Occluding the artery in hyperglycemic animals caused fivefold more frequent and 25-fold more extensive hemorrhage into infarcts than in normoglycemic animals. Temporary occlusion with clip release after four hours in hyperglycemic animals caused the most extensive hemorrhage into infarcts. Most hemorrhages into infarcts (81%) took place in animals that died within a few hours after they experienced ischemia and that showed infarction and marked edema of the entire middle cerebral artery territory. Linear regression analyses demonstrated a close relation between hemorrhage into infarcts and near-total energy depletion (adenosine triphosphate, less than 0.3 microM/g; phosphocreatine, less than 0.5 microM/g) in brain sites that showed extremely high tissue lactate concentrations (more than 30 microM/g). The biochemical changes that correlated with hemorrhage into infarcts were more marked than those with infarcts without hemorrhage. CONCLUSION: Hyperglycemia and restoration of blood flow to ischemic territories were strong risk factors for hemorrhagic infarct conversion. Concomitant tissue metabolic changes suggest that marked tissue energy depletion accompanied by acidosis damages brain vessels and renders them penetrable for edema fluid and, ultimately, red blood cell extravasation.

Adenosine Triphosphate↗
PubMed1992

Hyperglycemic versus normoglycemic stroke: topography of brain metabolites, intracellular pH, and infarct size.

Hyperglycemia aggravates brain pathologic outcome following middle cerebral artery (MCA) occlusion in cats. We presently determined if hyperglycemia during occlusion leads to high lactic acid accumulations in the ischemic MCA territory. We measured brain metabolite concentrations in 14 MCA territory sites at 0.5 and 4 h following occlusion in hyper- (20 mM) and normoglycemic (5 mM) cats and correlated these results with previous brain pathologic findings. Hyper- versus normoglycemia during MCA occlusion resulted in significantly higher lactate concentrations in the ischemic territory and more numerous loci with lactates greater than 17 mumol/g. At 0.5 h of occlusion, ATP levels were lower in normoglycemic cats, while at 4 h, ATP was similarly reduced (40%) in both glycemia groups. At 4 h, PCr was more reduced in hyperglycemics secondary to a greater brain tissue acidosis. Carbohydrate substrates at 0.5 h were more markedly depleted in normoglycemics, likely limiting lactate accumulation (34.3% versus only 5.0% of sites in hyperglycemics with glucose less than 0.5 mumol/g). Although lactate was markedly elevated at both 0.5 and 4 h in hyperglycemic ischemic territories, clip release at 4 versus 0.5 h yields a significantly poorer brain pathologic outcome. Correspondingly, intracellular pH, calculated from the creatine kinase equilibrium, was more markedly depressed at 4 than at 0.5 h of occlusion, demonstrating a time-dependent dissociation between tissue lactate and hydrogen ion accumulations. The present findings show that following MCA occlusion (a) hyperglycemia increases the magnitude and topographic extent of marked tissue lactic acidosis, (b) infarct size following 0.5 h of clip release correlates more closely with tissue acidosis than with lactate concentrations, (c) ischemic tissue ATP concentrations correlate poorly with infarct size, (d) normoglycemia limits lactate accumulation during focal ischemia because tissue glucose is depleted, and (e) early during ischemia, tissue buffering or antiport mechanisms may prevent marked increases in intracellular hydrogen ion activity.

Adenosine Diphosphate↗
PubMed1991

Tissue metabolite levels in different types of skeletal muscle during sepsis.

The effect of sepsis on energy and metabolite levels in the white, fast-twitch extensor digitorum longus (EDL) and the red, slow-twitch soleus (SOL) muscles was studied in rats. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham-operated. Sixteen hours later, metabolite levels in muscle tissue were determined. Adenosine triphosphate (ATP) levels and energy charge were reduced during sepsis in SOL, but were unchanged in EDL muscles. In contrast, phosphocreatine (PCr) concentration was reduced during sepsis in EDL, but not in SOL. Tissue glycogen levels were reduced and lactate concentrations were increased in both muscles during sepsis. Results suggest that sepsis affects energy metabolism differently in different types of skeletal muscle. Tissue lactate accumulation may be consistent with muscle hypoperfusion following CLP, although other mechanisms may also be involved.

Adenosine Triphosphate↗
PubMed1991

Protein tyrosine kinases and phosphatases in the nervous system.

Evidence in the past year has provided support for a prominent role of tyrosine phosphorylation in the regulation of neuronal function. The discovery that many novel forms of protein tyrosine kinases and phosphatases are expressed in the brain has revealed that the regulation of tyrosine phosphorylation is highly complex. The recent identification of substrate proteins in the brain for the protein tyrosine kinases and phosphatases has begun to clarify the functional role of tyrosine phosphorylation in the development and modulation of the nervous system.

Animals↗
PubMed1991

Folding of influenza hemagglutinin in the endoplasmic reticulum.

The folding of influenza hemagglutinin (HA0) in the ER was analyzed in tissue culture cells by following the formation of intrachain disulfides after short (1 min) radioactive pulses. While some disulfide bonds were already formed on the nascent chains, the subunits acquired their final disulfide composition and antigenic epitopes posttranslationally. Two posttranslational folding intermediates were identified. In CHO cells constitutively expressing HA0, mature HA0 subunits were formed with a half time of 3 min and their folding reached completion at 22 min. The rate of folding was highly dependent on cell type and expression system, and thus regulated by factors other than the sequence of the protein alone. Exposure of cells to stress conditions increased the level of glucose regulated proteins, including BiP, and decreased the folding rate. The efficiency of folding and subsequent trimerization was not dependent on the rate of translation, nor on temperature between 37 and 15 degrees C; however, the rates of folding and trimerization decreased with decreasing temperature. Whereas the rate of folding was independent of expression level, trimerization was accelerated at higher levels of expression.

Animals↗
PubMed1990

Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchor-free influenza hemagglutinin.

The influenza hemagglutinin precursor (HA0) and many other glycoproteins fold and oligomerize in the endoplasmic reticulum (ER). Only correctly folded oligomers are transported to the cell surface. To analyse the rules which determine this type of ER sorting, we have extended our analysis of hemagglutinin transport to two soluble, anchor-free recombinant HA0s derived from X31/A/Aichi/68 and A/Japan/305/57 influenza A. The results showed that individual monomers rapidly acquired a folded structure similar to that of monomeric membrane-anchored HA0. They were efficiently transported and secreted, but oligomerization was not required for secretion. Trimers or higher order complexes were either not formed (X31 HA0), or appeared during passage through the late compartments of the secretory pathway, with no effect on the rate of transport (Japan HA0). However, when initial folding was disturbed by inhibition of N-linked glycosylation, anchor-free X31 HA0 was misfolded and retained in the ER as disulfide-linked complexes associated with binding protein, BiP (GRP78). The complexes were similar to those seen for the nonglycosylated membrane-bound HA0, but instead of forming immediately after synthesis they appeared with a half-time of 6 min. Taken together, the data demonstrate that the structural criteria that makes the anchor-free HA0 transport competent are less stringent than those for the membrane form; they must fold correctly but do not need to oligomerize.

Animals↗
PubMed1990

Determiners of fatal reperfusion brain oedema.

Brain oedema is an important aspect of infarction from cerebrovascular occlusion. In a cat stroke model where the middle cerebral artery (MCA) was reversibly or permanently occluded, we analyzed the incidence of fatal hemispheral oedema in 35 normo- (6 mM) and 35 hyperglycaemic (20 mM for 6 hours) animals, with (N = 45) and without (N = 25) restoration of blood flow with clip release at 4 and 8 hrs of occlusion. Fatal hemispheral oedema occurred in 23% of cats (16/70) while hyperglycaemia, for one, and restoration of blood flow, for another, each quadrupled its occurrence. Further, evidence of remote oedema in the form of posterior cingulate cortical pressure atrophy from transtentorial herniation was found in animals that were allowed to survive for 2 weeks and that exhibited infarcts that affected 12 to 95% of the MCA territory. Thus, hemispheral oedema in association with MCA occlusion developed sufficiently markedly as to cause transtentorial herniation in 47% of all cats (33/70). We carried out biochemical analyses in 14 hyper- and 10 normoglycaemic cats after 4 hrs of MCA occlusion for ATP, phosphocreatine (PCr), lactate, glucose and glycogen. The biochemical findings then were correlated with the occurrence of reperfusion oedema following clip release after 4 hrs of occlusion point-by-point in the brains. Linear regression analyses of the brain metabolic and pathologic data revealed highly significant (p less than 0.001) correlations of acute oedema with brain tissue ATP and PCr reductions less than 1.5 microM/g, with lactic acid accumulation greater than 20 microM/g and with the extents of reduction in brain tissue glucose concentrations in the ischaemic territories.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Delayed decreases in specific brain mitochondrial electron transfer complex activities and cytochrome concentrations following anoxia/ischemia.

Hyperglycemic, but not normoglycemic cats exposed to anoxia develop neurologic signs following reoxygenation including fasciculations, focal and tonic-clonic seizures and coma after a symptom-free period. These symptomatic hyperglycemic cats may develop brain edema and will show diffuse neuronal injury or brain infarction depending on length of survival. Brain mitochondria isolated from symptomatic but not asymptomatic cats have decreased ADP- and uncoupler-stimulated oxygen consumption rates. Since impaired respiration could result from altered electron transport chain function, we measured cytochrome c, b, and aa3 concentrations and the activities of the five electron transfer complexes in isolated brain mitochondria. In symptomatic cats marked alterations were present in particular in complex IV, cytochrome oxidase, with a 57% reduction in activity and a 45% reduction in prosthetic group (cytochrome aa3) concentrations. Less marked reductions in other segments of the chain included 27% and 41% decreases, respectively, in cytochrome c concentrations and in electron transfer complex II, succinate:ubiquinone oxidoreductase activity. Cytochrome b concentrations and complex I, II and V activities were unchanged. Small but significant decreases in cytochrome aa3 concentrations (18%) and cytochrome oxidase activity (20%) were also present in mitochondria from postanoxic hyperglycemic cats prior to appearance of neurologic signs. These results indicate that delayed decreases in the activities of specific electron transfer complexes are correlated with impaired mitochondrial respiration and neurologic deterioration in postanoxic hyperglycemic cats. However, it is presently unclear if these postanoxic brain mitochondrial alterations are primary or secondary events in the development of brain injury.

Animals↗
PubMed1990

Delayed onset of neurologic deterioration following anoxia/ischemia coincides with appearance of impaired brain mitochondrial respiration and decreased cytochrome oxidase activity.

We previously demonstrated markedly inhibited brain mitochondrial respiration only in cats that (a) were hyperglycemic at anoxia and (b) had neurologic signs, i.e., fasciculations in tongue or facial muscles or focal seizures following reoxygenation. However, since the relationship between time of onset of mitochondrial dysfunction and neurologic signs was unclear, in the present study we killed postanoxic cats immediately when signs first appeared. Cerebrocortical homogenates and isolated brain mitochondria only from symptomatic cats showed markedly inhibited substrate-, ADP-, and uncoupler-stimulated respiration rates. Cytochrome oxidase activity and cytochrome aa3 concentrations were also markedly reduced in these mitochondria. Since brain mitochondrial function was impaired when neurologic signs first appeared, mitochondrial alterations are an important early organellar change correlated with development of neurologic deterioration following anoxia.

Animals↗
PubMed1989

Spinal vs. supraspinal sites of action of the alpha 2-adrenergic agonists clonidine and ST-91 on the acoustic startle reflex.

Previous work has shown that stimulation of alpha 2-adrenergic receptors depresses the startle responses in rats. The present study suggests that this depressant effect involves supraspinal rather than spinal alpha 2-adrenergic receptors because intraventricular but not intrathecal infusion of the hydrophilic alpha 2-adrenergic agonist ST-91 depressed the acoustic startle reflex. To determine the point in the acoustic startle pathway where alpha 2-adrenergic receptor activation might ultimately alter neural transmission, startle responses were elicited electrically from different points along the acoustic startle pathway after systemic administration of clonidine. Clonidine depressed acoustically-elicited startle and startle elicited by electrical stimulation of the ventral cochlear nucleus to a comparable magnitude and over a similar time course. It also partially depressed startle elicited by electrical stimulation of the nucleus reticularis pontis caudalis (RPC). Taken together, these data suggest that alpha 2-adrenergic stimulation depresses startle by acting on supraspinal receptors, but that this effect is ultimately expressed, at least in part, by actions at both spinal and brainstem levels of the acoustic startle response pathway. The results are compared to other drugs known to affect the startle reflex.

Acoustic Stimulation↗
PubMed1989

Delayed neurologic deterioration following anoxia: brain mitochondrial and metabolic correlates.

Hyper- but not normoglycemic cats exposed to 8 min of anoxia show neurologic signs (fasciculations, myoclonic jerks, seizures) that develop after a symptom-free period. We examined brain mitochondrial function and metabolite concentrations at 0, 1, 3, and 5 h following exposure to anoxia, to correlate biochemical findings with the presence ("symptomatic") or absence ("presymptomatic") of neurologic signs. Brain mitochondria isolated postexposure only from symptomatic cats showed markedly decreased (-50%), state 3 (ADP-stimulated), and uncoupler-stimulated respiration rates with NAD- and FAD-linked substrates. Respiratory control and ADP/oxygen (ADP/O) ratios remained unchanged, indicating, respectively, that coupling and efficiency of ATP synthesis were preserved. Thus, inhibition of electron transport chain function, not phosphorylative activity, may be rate limiting for respiration. During anoxia, hyperglycemic cats showed higher brain lactate levels (26 versus 20 mumol/g), but similar ATP and phosphocreatine concentrations, compared with normoglycemic cats. After exposure, in all animals lactate and phosphocreatine were restored to control levels, whereas ATP remained at 85%. Cats that became symptomatic demonstrated four- to sixfold increases in lactate and 50% reductions in phosphocreatine. At 3 and 5 h postexposure, symptomatic animals showed significant reductions in ATP concentrations. We conclude that although initially asymptomatic, hyperglycemic cats exposed to anoxia undergo a neurologic deterioration over several hours following reoxygenation that is correlated with inhibition of mitochondrial respiration, increases in tissue lactate, and decreases in energy state.

Adenosine Diphosphate↗
PubMed1989

Fatal strokes in hyperglycemic cats.

Hyperglycemia is associated with three- to fourfold larger infarcts than normoglycemia following permanent middle cerebral artery occlusion in cats. We investigated the effects of glycemia on brain outcome when middle cerebral artery blood flow was restored (clip release) after 4 hours of occlusion. Seven of 13 hyperglycemic (22 mM) and one of 12 normoglycemic (6 mM) anesthetized cats developed total middle cerebral artery territory infarcts and hemispheric edema and died of brainstem compression. The remaining six and 11 cats recovered fully and later showed no or only small infarcts. Compared with permanent occlusion, restoration of blood flow after 4 hours reduced infarct volume in all normoglycemic and hyperglycemic cats that survived, but caused a much higher proportion (54% vs. 17%) of hyperglycemic and, for the first time, one normoglycemic cat, to die of infarct extension, hemorrhagic infarct conversion, and total territory edema. Thus, clip release after 4 hours caused some cats to show reduced and others to show augmented tissue damage. Rendering cats hyperglycemic substantially worsened their outcome after reperfusion by increasing their death rate from total territory edema sevenfold. Our results demonstrate that risk/benefit analyses for recanalization efforts in humans should take serum glucose concentrations into account.

Animals↗
PubMed1988

Folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin.

Results from several systems indicate that correct protein folding and subunit assembly correlate with the transport of membrane and secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex. Because the site of oligomer assembly and its precise relationship to intracellular transport remain unclear, we have studied in detail the folding and trimerization of the influenza virus hemagglutinin (HA0) relative to its transport from ER to Golgi. Trimerization and transport were analyzed using several different methods, including transport inhibitors, temperature blocks, semi-intact cells, in vitro protein translocation, and immunocytochemistry. Taken together, the results clearly demonstrate that trimerization occurs at a point prior to exit from the ER. Before assembly, HA0 monomers were extensively folded and possessed intramolecular disulfide bonds, but monomers were not transported to the cis Golgi compartment. Thus, hemagglutinin progresses through at least two intermediate states before transport to the Golgi: highly folded monomers and trimers that have not yet left the ER.

Adenosine Triphosphate↗