Soman-induced seizures: limbic activity, oxidative stress and neuroprotective proteins.

Soman, a potent acetylcholinesterase inhibitor, induces status epilepticus in rats followed by conspicuous neuropathology, most prominent in piriform cortex and the CA3 region of the hippocampus. Cholinergic seizures originate in striatal-nigral pathways and with fast-acting agents (soman) rapidly spread to limbic related areas and finally culminate in a full-blown status epilepticus. This leads to neurochemical changes, some of which may be neuroprotective whereas others may cause brain damage. Pretreatment with lithium sensitizes the brain to cholinergic seizures. Likewise, other agents that increase limbic hyperactivity may sensitize the brain to cholinergic agents. The hyperactivity associated with the seizure state leads to an increase in intracellular calcium, cellular edema and metal delocalization producing an oxidative stress. These changes induce the synthesis of stress-related proteins such as heat shock proteins, metallothioneins and heme oxygenases. We show that soman-induced seizures cause a depletion in tissue glutathione and an increase in tissue 'catalytic' iron, metallothioneins and heme oxygenase-1. The oxidative stress induces the synthesis of stress-related proteins, which are indicators of 'stress' and possibly provide neuroprotection. These findings suggest that delocalization of iron may catalyze Fenton-like reactions, causing progressive cellular damage via free radical products.

Endovascular stents and stent-grafts: is heparin coating desirable?

Heparins are glycosaminoglycans that, in addition to their anticoagulant activity, have interactions with growth factors and other glycoproteins. These interactions may stimulate neointimal hyperplasia when heparin is delivered locally on stents and stent-grafts. Modifying the structure of heparin to retain anticoagulant activity while minimizing these stimulatory effects on the vascular endothelium is desirable and may be achieved by understanding the relationships between the structure and function of the various parts of the heparin molecule.

The aging brain, metals and oxygen free radicals.

In this overview we bring together certa in facts and concepts that support the theory that the aging of "disease-free" brain is a consequence of the accumulated cellular-molecular modifications caused by oxygen free radicals. The relevance of transition metals, especially iron ions, in the production of oxygen free radicals, initiation of oxidative chain-reactions and in site-specific molecular modifications is documented. Mitochondria are identified as the major source of oxygen free radicals, and mitochondrial DNA is a likely target. Special attention is given to iron-sulfur clusters as sources of reactive iron and sites of modifications. Potential mechanisms by which oxygen free radicals can alter membrane receptors and intracellular signaling are cited. Although the evidence is still correlative, the oxygen free radical theory has strong experimental support and has promise for facilitating a better understanding of the "disease-free", aging brain.

Measurement of loosely-bound iron in brain regions using redox cycling and salicylate.

A sensitive iron assay was developed for measuring non-heme and loosely bound iron in regions of rat brain. The method is based on the salicylate trapping of hydroxyl radicals generated from ascorbate-driven redox cycling of Fe3+-EDTA. This assay has high sensitivity (about 20 nM) because of amplification obtained with redox-cycling and fluorescent detection of the salicylate hydroxylation product, 2,5-dihydroxybenzoate. The assay detects iron as Fe2+ and Fe3+ combined. Values of non-heme and loosely bound iron are given for three areas of cortex, caudate, hippocampus, thalamus and brainstem of the rat brain.

Iron is a regulatory component of human IL-1beta production. Support for regional variability in the lung.

The human lung accumulates iron with senescence. Smoking escalates the accumulation of iron, and we have demonstrated regional variability in the accumulation of iron in smokers' lungs. Iron has been reported to influence the production of a number of proinflammatory mediators, including human interleukin (IL)-1beta. We postulated that we could (1) demonstrate regional differences in the release of IL-1beta from human alveolar macrophages and (2) influence the production of IL-1beta in human macrophages by altering intracellular iron concentrations. To test these hypotheses, alveolar macrophages were obtained by independent lavage of the upper and lower lobes of healthy volunteers (both smokers and nonsmokers), after which the ability of each population to secrete IL-1beta was quantified, together with their ability to produce tumor necrosis factor-alpha, IL-6, and IL-8. Additionally, we established an in vitro model of "iron-loaded" cells of the human myelomonocytic cell line THP-1 in order to examine more directly the effect of iron and its chelation on the secretion of IL-1beta. We report here that an intracellular, chelatable pool of iron expands with exogenous iron-loading as well as with lipopolysaccharide (LPS) stimulation and appears to suppress transcription of IL-1beta, whereas shrinkage of this pool by early chelation augments transcription of IL-1beta beyond that induced by LPS alone. And finally, we demonstrate a regional relationship in the lung between excess alveolar iron and the production of human alveolar macrophage-derived IL-1beta, suggesting a partnership between iron and inflammation that may have clinical significance, especially in relation to lung diseases with a regional predominance.

A global hypoxia preconditioning model: neuroprotection against seizure-induced specific gravity changes (edema) and brain damage in rats.

Hypoxia preconditioning states that a sublethal hypoxia episode will afford neuroprotection against a second challenge in the near future. We describe and discuss a procedure for the development of global hypoxia preconditioning in adult male Wistar rats, using a mildly hypoxic (9% O(2), 91% N(2)) atmospheric exposure of 8 h. The persistence of neuroprotection was analyzed using a kainic acid (KA) model of brain injury. Rats were challenged with KA (14 mg/kg, i.p.) on 1-14 days post-hypoxia. The effects of hypoxia preconditioning on seizure score, weight loss, brain edema and histopathology were assessed. Brain edema, predominantly of vasogenic origin, was measured 24 h after KA administration using a reproducible and quantitative method based on the specific gravities of tissue samples. A density gradient column (1.0250-1.0650 g/cm(3)) comprised of kerosene and bromobenzene was used to assess the presence of edema in regions involved in seizure initiation and propagation that are normally extensively damaged (i.e., piriform cortex and hippocampus). Specific gravities of tissues were calculated through extrapolation with known NaCl standards. We found that hypoxia preconditioning prevented the formation of edema in these brain regions when KA challenge was given 1, 3, and 7, but not 14 days post-hypoxia exposure. Furthermore, neuroprotection was observed in animals that had robust seizures. The described procedure may be used to examine the neuroprotective mechanisms induced by global hypoxia preconditioning against many subsequent challenges reflecting a variety of experimental models of brain injury, and will provide a better understanding of the brain response to hypoxia and stress.

Hypoxia preconditioning attenuates brain edema associated with kainic acid-induced status epilepticus in rats.

Kainic acid (KA)-induced seizures elicit edema associated with necrosis in susceptible brain regions (e.g., piriform cortex and hippocampal CA1 and CA3 regions). To test the hypothesis that hypoxia preconditioning protects against KA-induced edema formation, adult male rats were exposed to a 9% O2, 91% N2 atmosphere for 8 h. KA (14 mg/kg, i.p.) was administered 1, 3, 7, or 14 days later. Regional analysis of edema indicated that hypoxia exposure attenuated edema formation in piriform and frontal cortices and hippocampus when KA was given 1, 3, or 7 days later but not 14 days after hypoxia. Cycloheximide (2 mg/kg s.c.) given 1 h prior to hypoxia prevented the protective effect of hypoxia on KA-induced edema attenuation in the piriform cortex and hippocampus. Thus, hypoxic challenge induces a general adaptive response that protects against the seizure-associated pathophysiology, with no direct relationship to seizure intensity. This response may involve stress-related transcription factors and effector proteins.

Genistein sensitivity of calcium transport pathways in serotonin-activated vascular smooth muscle cells.

Recent studies showed that serotonin-activated increases in intracellular Ca2+ in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhibition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+ and tyrosine phosphorylation are functionally coupled. Therefore, we sought to characterize genistein-sensitive Ca2+ transport pathways in rat aortic A10 cells loaded with fura-2. In the presence of extracellular Ca2+, serotonin evoked a transient increase in [Ca2+]i that was followed by a smaller sustained increase. The transient was inhibited 25-40% by L-type Ca2+ channel antagonists and inhibited 90-95% by genistein. The sustained response was unaffected by L-channel antagonists and only slightly inhibited by genistein. In the absence of extracellular Ca2+, the transient was reduced by 50%, while the sustained component was virtually abolished. These results suggest that influx and release pathways are major contributors to the transient component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably involves an L-type Ca2+ channel that is regulated by tyrosine kinase activity. The pathways that participate in the sustained response are different because they are insensitive to l-channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free media was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+ was unaffected by genistein, reduced by caffeine, and essentially eliminated by thapsigargin. Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+ from the sarcoplasmic reticulum, whereas genistein-mediated suppression probably reflects inhibition of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+ and interference with release. Each A10 cell expressed at least two different ryanodine receptors and two different receptors for inositol 1,4,5-trisphosphate.

Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium.

This review addresses a rapidly growing body of evidence suggesting that enhanced protein tyrosine phosphorylation may be a previously unrecognized mechanism for coupling receptor activation of vascular smooth muscle cells to increases In the intracellular concentration of Ca2+ and contraction. The hypothesis proposes that activation of diverse types of receptors that are not tyrosine kinase promotes stimulation of a cytosolic tyrosine kinase. In turn, the activated kinase induces tyrosine phosphorylation of substrates that are linked to regulatory mechanisms for release of intracellular Ca2+ stored in the sarcoplasmic reticulum and to regulatory mechanisms for influx of extracellular Ca2+. Within this framework, we examine some relevant functional aspects of receptor and nonreceptor tyrosine kinases in different types of cells, the emerging relationships between tyrosine kinase activity and regulation of intracellular Ca2+. We review studies of nonreceptor tyrosine kinase activity in vascular smooth muscle cells suggesting that a physiologically relevant kinase may be the enzyme called pp60. Data that appear to link tyrosine phosphorylation to contraction of smooth muscle are examined, particularly with respect to results obtained with tyrosine kinase inhibitors and measures of changes in tyrosine phosphorylation. Next, we review studies with cultured vascular smooth muscle cells that point to potential coupling between receptor activation, enhanced tyrosine phosphorylation of substrates such as the GTPase activating protein for ras, and the gamma-1 isoform of phospholipase C, and mechanisms controlling Ca2+ influx and release. Emphasis is placed on examining the strengths and weaknesses of different experimental approaches. Lastly, a summary of the data is provided which calls attention to some major issues requiring resolution to permit acceptance or rejection of the underlying hypothesis, and we briefly address some of its possible pathophysiological implications.

Nitric oxide detection with intracerebral microdialysis: important considerations in the application of the hemoglobin-trapping technique.

Nitric oxide (NO.) is involved in processes such as neurotransmission, memory, brain injury, vessel relaxation, etc. To study the functional and pathological roles of NO. in the brain, a reliable method to monitor NO. directly is needed. Since oxyhemoglobin (Hb) has a high affinity for NO. and upon binding is converted quantitatively to methemoglobin (MetHb), spectrophotometry of Hb conversion to MetHb can give a credible measurement of NO. concentration. Although this method is especially promising for in vivo microdialysis, factors can influence the reproducibility and stability, making it difficult to obtain reliable results at low NO. levels. Evaluation of the diffusion rates of NO. and sodium nitroprusside across the microdialysis membrane indicates that NO. readily diffuses through the membrane. By taking into account protein degradation and Hb autoxidation as well as integrating the difference spectra, this assay has a practical differential detection limit of about 7 nM (0.4 pmol) in vivo. We evaluated this method in anesthetized and awake rats by measuring the release of NO. induced by the excitotoxin kainic acid (13 mg/kg, i.p.). A protocol with detailed analytical parameters for NO. monitoring in neurobiological research is given.

Vestibular and sensory interaction deficits assessed by dynamic platform posturography in patients with multiple sclerosis.

Vestibular impairments have not been routinely identified in patients with multiple sclerosis (MS), because of the confounding effects of deficits in other neural systems. In this study, 35 patients with MS were evaluated by means of a systematic alteration of the sensory environment (dynamic posturography) in order to identify those patients who became unstable when vestibular inputs were needed to maintain stance. Subjects were assigned to either a high-function (HF) or a low-function (LF) group on the basis of a functional status assessment score obtained prior to the posturography test. For the HF group, 30% (7/23) had abnormal posturography scores. Of those subjects, 3 had a vestibular dysfunction pattern or a somatosensory-vestibular impairment. In contrast, 58% of the LF group (7/12) had abnormal posturography scores. Nearly all of these LF patients (6/7) had a vestibular dysfunction pattern or a combined visual-vestibular or somatosensory-vestibular impairment. Posturography might serve as one method to evaluate the functional consequences of a vestibular deficit in patients with MS.

Human serum amyloid P component is an invariant constituent of amyloid deposits and has a uniquely homogeneous glycostructure.

Human serum amyloid P component (SAP) is a normal plasma protein and the precursor of amyloid P component (AP), a universal constituent of the abnormal tissue deposits in amyloidosis, including Alzheimer disease. We show here that its single N-linked biantennary oligosaccharide does not display the microheterogeneity usually characteristic of glycoproteins. The protein and the glycan structures of AP were also invariant, their resistance to degradation suggesting a role in persistence of amyloid deposits. Asialo-SAP was rapidly cleared from the circulation in mice by a mechanism dependent on terminal galactose residues and was catabolized in hepatocytes. However blockade of this pathway did not affect the clearance of native SAP. Rapid hepatic uptake and catabolism of human asialo-SAP in man were also directly demonstrated. The protein and glycan homogeneity of SAP and the integrity of AP suggest that the complete glycoprotein structure is important for the normal and the pathophysiological functions of this molecule.

Outcome of renal transplantation in hepatitis BsAg-positive patients.

Renal transplantation of patients with previous or ongoing hepatitis B virus infection has been tempered with a concern that immunosuppression may lead to viral replication and progressive liver damage. However, renal transplantation as therapy for end-stage renal failure in these patients improves quality of life and reduces the risk of body fluid exposure to their carers. To assess the long-term outcome of renal transplantation in hepatitis-BsAg-positive patients a retrospective study was carried out on the patients transplanted in this unit since 1969. Seventy-six patients received 98 grafts up to December 1991; follow-up was available on 68. Thirty-one of the 68 patients died; the causes of death were infective 23, cardiovascular 6, liver failure 4, pancreatitis 2, aspiration 1, GI haemorrhage 1, and stopped therapy 1. Serological markers of hepatitis B virus infection did not correlate with outcome. The risk of developing liver failure after renal transplantation appears small in the hepatitis-BsAg-positive patients and no patient should be denied a renal transplant on the basis of serological tests.

Evaluation of new high-performance calcium polyphosphate bioceramics as bone graft materials.

The purpose of this study was to evaluate the ability of a recently developed porous calcium polyphosphate bioceramic (CPB) to function as a bone graft substitute. After six weeks, postsurgical extraction of the mandibular first and second molars, alveolar ostectomies were performed bilaterally in five dogs. The ridge forms were then restored using the CPB implant material on one side and the autogenous bone obtained from the contralateral ostectomy site on the other. The graft and implant sites were retrieved after 4 months and decalcified and undecalcified sections were prepared for special staining (modified Attwood) and subsequent light microscopy and histomorphometry. In addition, the undecalcified sections were prepared for histometry using backscattered electron imaging (BSEI). Histologically, the CPB implants showed extensive vascularization and cellularity within an "invading" loose connective tissue matrix. On the opposite side, the loose connective tissue of the autografts showed hypovascularity and hypocellularity. Neither the implants nor the autografts showed any histologic evidence of an inflammatory reaction. Using light microscopic histomorphometry, the implants showed a higher incidence of union than the autografts. On BSEI histometry, the CPB implants showed significantly greater new bone formation than the autografts. This study reveals that porous CPB possesses certain characteristics essential for the "ideal" implantable bone substitute necessary for the repair of craniofacial and other bony defects.

The fibrinolytic response to ancrod therapy: characterization of fibrinogen and fibrin degradation products.

Ancrod is a purified coagulant venom which renders blood incoagulable by cleaving fibrinopeptide A (FPA) from fibrinogen, but the mechanism involved in the clearance of fibrin from the circulation is unknown. To investigate the fibrinolytic response to ancrod, and to increase understanding of clearance mechanisms, six patients with peripheral vascular disease causing claudication were infused with ancrod at 2 u/kg over 6 h followed by 2 u/kg at 12 h intervals for 38 h. Venous blood samples were taken at time 0, 3, 6, 25 and 49 h for assay of fibrinogen (Fbg), fibrinopeptide A (FPA), total fibrin(ogen) degradation products (TDP), fibrin degradation products (FbDP), fibrinogen degradation products (FgDP), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (tPA), urinary type plasminogen activator (u-PA), plasminogen, alpha 2 antiplasmin (alpha 2 AP) and plasminogen activator inhibitor-1 (PAI-1). Fibrinogen (median and range) was 2.3 (1.4-3.90) g/l at time 0 and thereafter was undetectable. FPA rose from 2.5 (1.8-3.6) to 600 and 188 pmol/l at 3 h and 6 h and remained elevated. TDP, FbDP and FgDP increased greatly following ancrod while there was no evidence of XL-FDP. The surprising increase in FgDP during defibrination suggests either that fibrinogen is digested following its incorporation into circulating fibrin protofibrils or that some of the fibrin subunits in the photofibril retain one of the two fibrinopeptide A's. tPA and uPA remained unchanged. Plasminogen fell from 125 (100-155)% to 79 (40-118)% at 49 h and alpha 2 AP fell from 91 (75-107)% to 24 (10-35)% at 49 h. The level of PAI-1 was depressed during defibrination, with the exception of the 6 h data. The results demonstrate that ancrod removes FPA from fibrinogen to produce non-cross-linked (soluble) fibrin. This is cleared from the circulation without evidence of an increase in the circulating activities of the plasminogen activators, tPA or UK, but with evidence of plasminogen activation and consumption.