Spontaneous Elevations Research Articles

Thrombin Differentially Modulates the Acute Inflammatory Response to Escherichia coli and Staphylococcus aureus in Human Whole Blood

Thrombin plays a central role in thromboinflammatory responses, but its activity is blocked in the common ex vivo human whole blood models, making an ex vivo study of thrombin effects on thromboinflammatory responses unfeasible. In this study, we exploited the anticoagulant peptide Gly-Pro-Arg-Pro (GPRP) that blocks fibrin polymerization to study the effects of thrombin on acute inflammation in response to Escherichia coli and Staphylococcus aureus Human blood was anticoagulated with either GPRP or the thrombin inhibitor lepirudin and incubated with either E. coli or S. aureus for up to 4 h at 37°C. In GPRP-anticoagulated blood, there were spontaneous elevations in thrombin levels and platelet activation, which further increased in the presence of bacteria. Complement activation and the expression of activation markers on monocytes and granulocytes increased to the same extent in both blood models in response to bacteria. Most cytokines were not elevated in response to thrombin alone, but thrombin presence substantially and heterogeneously modulated several cytokines that increased in response to bacterial incubations. Bacterial-induced releases of IL-8, MIP-1α, and MIP-1β were potentiated in the thrombin-active GPRP model, whereas the levels of IP-10, TNF, IL-6, and IL-1β were elevated in the thrombin-inactive lepirudin model. Complement C5-blockade, combined with CD14 inhibition, reduced the overall cytokine release significantly, both in thrombin-active and thrombin-inactive models. Our data support that thrombin itself marginally induces leukocyte-dependent cytokine release in this isolated human whole blood but is a significant modulator of bacteria-induced inflammation by a differential effect on cytokine patterns.

Hedgehog signaling plays a crucial role in hyperalgesia associated with neuropathic pain in mice.

Neuropathic pain is a debilitating chronic syndrome of the nervous system caused by nerve injury. In Drosophila, the Hedgehog (Hh) signaling pathway is related to increased pain sensitivity (hyperalgesia) but does not affect the baseline nociceptive threshold. In general, the contribution of the Hh signaling pathway to neuropathic pain in vertebrates is a highly debated issue. Alternatively, we investigated the potential role of Hh signaling in mechanical allodynia using a mouse model of neuropathic pain. Seven days after spinal nerve-transection (SNT) surgery, microglial activation increased in the ipsilateral spinal dorsal horn compared with that in the sham group; however, 21 days after surgery, microglial activation decreased. Contrastingly, astrocyte activation in the spinal cord did not differ between the groups. On day 21 of postsurgery, the SNT group showed marked upregulation of sonic hedgehog expression in peripheral glial cells but not in dorsal root ganglion (DRG) neurons. Intrathecal administration of the Hh signaling inhibitor vismodegib attenuated the mechanical allodynia observed on day 21 postsurgery. Conversely, intrathecal treatment with the Hh signaling activator smoothened agonist in naive mice induced mechanical allodynia, which was abolished by the ATP transporter inhibitor clodronate. Moreover, inhibition of Hh signaling by pretreatment with vismodegib significantly reduced ATP secretion and the frequency/number of spontaneous elevations of intracellular calcium ion levels in cultured DRG cells. Thus, the Hh signaling pathway appears to modulate the neural activity of DRG neurons via ATP release, and it plays an important role in sustaining mechanical allodynia and hypersensitivity in a mouse model of neuropathic pain.

Spontaneous antibody production caused by regulatory T cell deficiency occurs through a germinal center-independent pathway

Foxp3+ regulatory T cells (Tregs) are essential for the prevention of autoantibody and allergen-specific IgE production. Treg deficiency causes an elevation of the serum levels of these pathogenic antibodies, accompanied by spontaneous germinal center (GC) formation. However, it remains to be determined whether excessive and pathogenic antibody production induced by Treg deficiency requires a GC response. Here, we demonstrate that spontaneous antibody production observed in Foxp3 conditional-knockout mice did not need GC formation. Foxp3 and Bcl6 conditional-double knockout mice exhibited spontaneous elevations of IgG1, IgG2c, and IgE levels even though they showed impaired production of IgG1 and IgE specific for the immunized antigen. Furthermore, the IgG1 and IgE antibodies specific for auto- and food-antigens were produced independently of GCs. These data suggested that a GC response was unnecessary for pathogenic antibody production caused by Treg deficiency.

Formyl Peptide Receptor‐1 Activation is Crucial for Spontaneous and Salt‐Induced Hypertension in Dahl Salt Sensitive Rats: Mitochondria vs. Microbiota

AimsMitochondria evolved from bacteria and carry the same hallmarks that are an important source of inflammatogenic damage‐associated molecular patterns. One hallmark is the use of N‐formylated peptides (NFPs) as an initiator of protein synthesis. NFPs activate formyl peptide receptor (FPR‐1) and lead to vascular injury. When we first worked with Dahl Salt Sensitive (S) rats model, we observed that Dahl S fed a low‐salt (LS) diet presented signs of vascular dysfunction and spontaneous elevation of BP compared to Dahl Salt Resistant (R) rats. High‐salt (HS) diet worsened the hypertensive phenotype. Intriguingly, we observed that FPR‐1 is upregulated in arteries, and mitochondrial NFPs are elevated in the circulation of S rats (LS). We hypothesized that cell death‐derived mitochondrial NFPs and FPR‐1 activation leads to vascular dysfunction and the genesis of spontaneous elevation of BP in S rats. Because of the dual source of NFPs (from microbiota and host mitochondria), we also hypothesized that HS diet leads to gut barrier disruption, and a synergistic action of mitochondria and bacterial‐derived leaky gut NFPs are the major driving force in the maintenance of hypertension.MethodsMale S and R rats (6‐week old) were given a LS (0.3% NaCl) or HS diet (2% NaCl). Blood pressure (BP) was measured by telemetry. After 3 weeks on LS or HS diets, rats were treated (osmotic mini‐pump) with FPR‐1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, 14 days]. In another group, rats received water with amoxicillin (AMO, 5 mg/kg/day) for 3 weeks to deplete bacteria. Mesenteric resistance arteries (MRA) were collected to assess vascular function via wire myograph. Blood samples were collected to measure zonulin, a leaky gut biomarker via Elisa. t‐test p<0.05 *vs. R; #vs. absence of treatment; n=8–11 for R and S; n=6 for R and S+AMO; n=5–6 for R and S+CsH.Results and ConclusionS rats (LS) have a spontaneous elevation in BP. Treatment with FPR‐1 antagonist (CsH) decreased this response [BP (mmHg): Diastolic: R: 77±2.7 vs. R+CsH: 81±1.2 vs. S: 126±3.0* vs. S+CsH:115±2.7#]. Also, CsH decreased hypercontractility observed in arteries from S LS when compared to R rats [KCl 120mM (mN): R: 9.4±1.0 vs. R+CsH: 10.2±0.4 vs. S: 15.5±0.9* vs. S+CsH:11.7±0.8#). This suggests that FPR‐1 serves as a causative agent to the spontaneous elevation of BP. AMO did not decrease BP in S‐LS diet [BP (mmHg): Diastolic: R: 77±2.7 vs. R+AMO: 82±2.7 vs. S: 126±3.0* vs. S+CsH: 124±1.3]. In addition, zonulin is not increased in plasma in S‐LS, suggesting that leaky gut is not associated with spontaneous elevation of BP in S‐LS. However, zonulin is increased in plasma during HS diet (severe hypertension) [Zonulin (ng/mL): R‐LS: 4.09±0.1 vs. S‐LS: 4.01±0.3 vs. R‐HS: 4.31±0.1 vs. S‐HS: 6.3±0.2#]. Overall, FPR‐1 activation due to increased levels of mitochondrial NFPs is associated with the genesis of spontaneous elevations of BP in Dahl S rats. High‐salt diet leads to gut barrier disruption and, subsequently, a synergistic action of mitochondria and bacterial‐derived leaky gut NFPs are the major driving force in the maintenance of hypertension.Support or Funding InformationNSF AGEP:1432878, NIH:R00GM118885, AHA:18POST34060003

Blood pressure changes after aneurysmal subarachnoid hemorrhage and their relationship to cerebral vasospasm and clinical outcome

ObjectiveCerebral vasospasm (VS) and resulting delayed ischemic brain injury constitute the most severe secondary complication after subarachnoid hemorrhage (SAH). Identification of early clinical predictors of developing vasospasm and poor outcome has remained a major challenge in neurointensive care medicine. Aim of the present study was analyze the relevance of spontaneous changes in blood pressures and their predictive value for predicting vasospasm as well as adverse clinical outcome. Methods98 aneurysmal SAH patients were analyzed retrospectively. Patients were divided into two study groups: (1) VS+ (developing VS) and (2) VS− (not developing VS). Repeat-angiography was routinely performed on day 8 after SAH or earlier if clinical signs were suggestive for overt vasospasm. Systolic, diastolic and mean blood pressures were averaged hourly and plotted over time. Secondly, blood pressure (BP)-progression was analyzed with respect to clinical outcomes as assessed by the Glasgow outcome scale. ResultsMean, systolic, and diastolic blood pressure values progressed in both VS− and VS+ cohorts over time. However, as early as 4 days after SAH a significant dissociation of RR curves was observed between the groups with patients in the VS+ group displaying a significantly higher slope coefficient of blood pressure elevation. An increase of mean arterial pressure >20% within the first 4 days was predictive of developing vasospasm. Elevation of mean arterial blood pressure in the VS+ group was mainly attributable to changes in diastolic pressure. Elevation of mean arterial blood pressure >25% within the first week after SAH was associated with unfavorable outcome. ConclusionsSAH leads to spontaneous and progressive elevations in mean arterial blood pressure. Vasospasm might be anticipated by identifying early elevations of mean arterial blood pressure. Finally, spontaneous elevations of mean arterial blood pressure correlate with poorer outcomes.

Increased STAT5 signaling in the ring dove brain in response to prolactin administration and spontaneous elevations in prolactin during the breeding cycle

Prolactin acts on target cells in the central nervous system (CNS) to stimulate behavioral changes associated with parental care in birds, but the signaling mechanisms that mediate these actions have not been characterized. In mammals, the Janus Kinase 2–Signal Transducer and Activator of Transcription 5 (JAK2–STAT5) signaling pathway mediates many of the actions of prolactin. To assess the importance of this pathway in prolactin-sensitive target cells in the avian brain, we measured changes in activated (phosphorylated) STAT5 (pSTAT5) in the forebrain of female ring doves sampled as plasma prolactin levels change during the breeding cycle and in prolactin-treated, non-breeding females. The anatomical distribution of cells exhibiting pSTAT5 immunoreactivity in dove brain closely paralleled the distribution of prolactin receptors in this species. The density of pSTAT5 immunoreactive (pSTAT5-ir) cells was highest in the preoptic area, the suprachiasmatic, paraventricular, and ventromedial hypothalamic nuclei, the lateral and tuberal hypothalamic regions, the lateral bed nucleus of the stria terminalis, and the lateral septum. Mean pSTAT5-ir cell densities in these eight brain areas were several fold higher in breeding females during late incubation/early post-hatching when plasma prolactin levels have been observed to peak than in non-breeding females or breeding females sampled at earlier stages when prolactin titers have been reported to be lower. Similar differences were observed between prolactin-treated and vehicle-treated females in all three of the forebrain regions that were compared. We conclude that JAK2–STAT5 signaling is strongly activated in response to prolactin stimulation in the ring dove brain and could potentially mediate some of the centrally-mediated behavioral effects of this hormone.

Imaging and Manipulating Calcium Transients in Developing Xenopus Spinal Neurons

Many forms of electrical excitability expressed in the embryonic nervous system depend on Ca(2+) influx. This discovery has stimulated investigation of the functions of spontaneous elevations of intracellular Ca(2+) and their roles in neuronal development. We present a protocol for imaging different classes of intracellular Ca(2+) transients in embryonic Xenopus (amphibian) spinal neurons grown in dissociated cell culture and in the intact neural tube (the developing spinal cord), focusing on early stages of neuronal differentiation around the time of neural tube closure. The protocol describes methods for gain-of-function and loss-of-function experiments to reveal the functions of these Ca(2+) transients. The methods can also be applied to explant and organotypic cultures. The procedures are sufficiently simple that they can be further adapted for dissociated neuronal cell cultures from other developing embryos, embryonic spinal cords of vertebrates such as zebrafish, and ganglia in the developing nervous systems of invertebrates.

Gap-Junction Suppression of Electrical Activity in Normal and Diabetic Pancreatic Islets

The pancreatic islets of Langerhans are multicellular microorgans which play a central role in maintaining blood glucose homeostasis, through secretion of hormone insulin. Interactions between cells within the islet are critical to enhance and coordinate the β-cell insulin secretion response to glucose. Gap junction channels coordinate membrane potential between β-cells in the islet. This serves to coordinate oscillations in free-calcium activity ([Ca2+]i) and insulin secretion, but also to suppress spontaneous [Ca2+]i elevations under basal glucose. Here we quantify the gap junction suppression of β-cell excitability and describe this with a percolation model of cell-cell coupling. We utilize islets from wild-type mice and 2 transgenic mouse models in which mutations to the KATP channel either increase or decrease excitability in a population of β-cells in the islet. In each set of islets, we quantify the variation of [Ca2+]i and insulin secretion with gap junction coupling, at low glucose and high glucose. In all cases reduced gap junction coupling resulted in elevated [Ca2+]i; with the variation of [Ca2+]i with gap junction conductance showing a similar non-linear relationship which can be described using the percolation model. This variation could also describes insulin release and glucose homeostasis: large elevations in [Ca2+]i predict significant reductions in blood glucose levels. Importantly, in mice expressing KATP channel mutations that reduce insulin secretion and cause severe hyperglycemia and diabetes, a reduction in gap junction coupling elevates insulin release and prevents diabetes. Thus the relationship governing gap junction coupling and the suppression of spontaneous elevations in [Ca2+]i is dependent on the distribution of excitability across β-cells of the islet and the level of coupling between β-cells, and this governs the overall islet response. Utilizing this may be a promising therapeutic treatment for specific monogenic forms of diabetes.

Androgens regulate adenylate cyclase activity and intracellular calcium in stromal cells derived from human prostate

Increased smooth muscle tone is a significant component of benign prostatic hyperplasia, the onset of which correlates with age and declining serum testosterone levels. This study investigates the effects of androgens on key regulators of smooth muscle tone: intracellular calcium ([Ca(2+)](i)) and cyclic adenosine monophosphate (cAMP) in human cultured prostatic stromal cells (HCPSC). HCPSC were cultured in the absence or presence of dihydrotestosterone (DHT; 3, 30, and 300 nM) or testosterone (0.3-300 nM) alone or in the presence of flutamide (10 microM). Changes in [Ca(2+)](i) were determined in FURA-2AM (10 microM) loaded cells. Changes in cAMP were determined by Alpha Screen(R) assay. Up to 32% of cultured cells exhibited spontaneous elevations of [Ca(2+)](i). The frequency of these elevations was reduced by nifedipine (10 microM), ryanodine (1 microM), and the adenylate cyclase inhibitor MDL 12,330A (20 microM). Compared to steroid-free cells, a 3-day incubation of cells with testosterone (only 3 nM) elevated basal, but not peak [Ca(2+)](i). In the presence of flutamide, all concentrations of testosterone tested elevated basal, but not peak [Ca(2+)](i). DHT (30 and 300, but not 3 nM) lowered peak and basal [Ca(2+)](i). Increased testosterone concentration dependently decreased resting cell cAMP (pIC(50): 7.64 +/- 0.29 nM). These findings demonstrate that some HCPSC have the ability to spontaneously and transiently elevate [Ca(2+)](i). The magnitude of these [Ca(2+)](i) peaks, along with resting levels of calcium and cAMP, appear to be regulated by androgens.

Spontaneous oscillations in intracellular Ca2+ concentration via purinergic receptors elicit transient cell swelling in rat parotid ducts

Using multiphoton microscopy, we established that rat parotid ductal cells exhibit spontaneous oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). These oscillatory Ca(2+) responses were observed during continuous perfusion with a physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. The timing and patterns of these spontaneous Ca(2+) oscillations varied among individual ductal cells, and the average number of Ca(2+) responses in a single responding ductal cell was 2.1 in a 10-min recording period. High-speed scanning (0.6 s/image) revealed that most spontaneous elevations in [Ca(2+)](i) were initiated at the luminal side of ductal cells and spread toward the basal side within 2 s. Electron microscopic analysis after Ca(2+) imaging indicated that spontaneously oscillating ducts contained numerous granules at the luminal side, which is characteristic of granular ducts. These Ca(2+) oscillations were completely blocked by the purinergic receptor inhibitors 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) and suramin but were not blocked by the muscarinic antagonist atropine or the alpha-adrenergic antagonist phentolamine. Simultaneous observation of fura-2 fluorescence and differential interference contrast (DIC) images showed that spontaneous elevations of [Ca(2+)](i) were well correlated with changes in shape of ductal cells. Using a plasma membrane fluorescence probe, SynaptoGreen C4, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Our findings present the possibility that purinergic receptors mediate spontaneous Ca(2+) oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva in the resting state.

Spontaneous Ca2+ oscillations via purinergic receptors elicit transient cell swelling in rat parotid ducts

Rat parotid ductal cells were found to exhibit spontaneous Ca(2+) oscillations. These oscillatory Ca(2+) responses were observed during continuous perfusion with physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. These Ca(2+) oscillations were completely blocked by the purinergic receptor inhibitors, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) (PPADS) and suramin, but were not blocked by the muscarinic antagonist, atropine, nor the alpha-adrenergic antagonist, phentolamine. Simultaneous observation with fura-2 fluorescence and differential interference contrast (DIC) images showed that the spontaneous elevations of [Ca(2+)]i were well correlated with the shape changes of the ductal cells. Using a plasma membrane fluorescence probe, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Electron microscopic analysis after Ca(2+) imaging indicated that the spontaneously oscillating duct cells contained numerous granules at the luminal side, which is characteristic of the granular duct cells. These results indicate that the spontaneous [Ca(2+)]i increase occurs through purinergic receptors, and activates Ca(2+)-dependent ion transporters and/or channels. Our findings present the possibility that spontaneous Ca(2+) oscillations via purinergic receptors are involved in the regulation of the electrolyte composition of saliva in resting states.

Calcium sparks activate calcium-dependent Cl− current in rat corpus cavernosum smooth muscle cells

Spontaneous transient currents, due to activation of Ca(2+)-dependent K(+) and Cl(-) channels, occur in corpus cavernosum smooth muscle cells (CCSMC) of the penis. The Ca(2+) events responsible for triggering Ca(2+)-dependent Cl(-) channels have never been identified in vascular muscle. We used high-speed fluorescence imaging combined with patch-clamp electrophysiology to provide the first characterization of Ca(2+) events underlying these currents. Freshly isolated rat CCSMC loaded with fluo-4 exhibited localized, spontaneous elevations of intracellular Ca(2+) (Ca(2+) sparks) in 57% of cells. There was an average of 6.4 +/- 0.5 release sites/cell with a frequency of 0.9 +/- 1 Hz/cell and peak amplitude DeltaF/F(o) of 67 +/- 10%. We addressed the controversy of whether these events are mediated by ryanodine or inositol 1,4,5 trisphosphate (IP(3)) receptors. Caffeine caused either a global Ca(2+) rise at high concentrations or an increase in spark frequency at lower concentrations, whereas ryanodine dramatically reduced the amplitude and frequency of sparks. 2-Aminoethoxydiphenyl borate, an inhibitor of IP(3) receptors, had no effect on spark frequency. Combined imaging and electrophysiological recording revealed strong coupling between Ca(2+) sparks and biphasic transient currents, a relationship never before shown in vascular muscle. Moreover, spark frequency increased on depolarization, an effect abolished with the blockade of Ca(2+) channels, consistent with Ca(2+) influx regulating Ca(2+) release from stores. We establish for the first time that Ca(2+) sparks occur in CCSMC and arise from Ca(2+) release through ryanodine receptors. Moreover, the voltage dependence of spark frequency demonstrated here provides novel functional evidence for voltage-dependent Ca(2+) influx in CCSMC.

ATP Released via Gap Junction Hemichannels from the Pigment Epithelium Regulates Neural Retinal Progenitor Proliferation

The retinal pigment epithelium (RPE) plays an essential role in the normal development of the underlying neural retina, but the mechanisms by which this regulation occurs are largely unknown. Ca2+ transients, induced by the neurotransmitter ATP acting on purinergic receptors, both increase proliferation and stimulate DNA synthesis in neural retinal progenitor cells. Here, we show that the RPE regulates proliferation in the underlying neural retina by the release of a soluble factor and identify that factor as ATP. Further, we show that this ATP is released by efflux through gap junction connexin 43 hemichannels, the opening of which is evoked by spontaneous elevations of Ca2+ in trigger cells in the RPE. This release mechanism is localized within the RPE cells to the membranes facing the neural retina, a location ideally positioned to influence neural retinal development. ATP released from RPE hemichannels speeds both cell division and proliferation in the neural retina.

P.2.16 The influence of spontaneous blood pressure elevations on the QT dispersion in patients with hypertension and after myocardial infarction

P.2.16 The influence of spontaneous blood pressure elevations on the QT dispersion in patients with hypertension and after myocardial infarction

P.2.16 The influence of spontaneous blood pressure elevations on the QT dispersion in patients with hypertension and after myocardial infarction

P.2.16 The influence of spontaneous blood pressure elevations on the QT dispersion in patients with hypertension and after myocardial infarction

P.2.16 The influence of spontaneous blood pressure elevations on the QT dispersion in patients with hypertension and after myocardial infarction

P.2.16 The influence of spontaneous blood pressure elevations on the QT dispersion in patients with hypertension and after myocardial infarction

Early effects of metabolic inhibition on intracellular Ca2+ in toad pacemaker cells: involvement of Ca2+ stores.

The early effects of metabolic inhibition on intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) current, and sarcoplasmic reticulum (SR) Ca(2+) content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca(2+)](i) (Ca(2+) transients) was reduced after 5-min exposure to 2 mM NaCN from 338 +/- 30 to 189 +/- 37 nM (P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 +/- 2 to 12 +/- 4 beats/min (P < 0.002, n = 9). It has been proposed that CN(-) acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca(2+) current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca(2+) transients and firing rate. CN(-) caused an insignificant fall of Ca(2+) current (23 +/- 11%) but a substantial reduction of SR Ca(2+) content (by 65 +/- 5%), whereas clotrimazole produced a larger reduction of Ca(2+) current and did not affect the SR Ca(2+) content. Thus the main effect of CN(-) does not seem to be through inhibition of cytochrome P-450. In conclusion, CN(-) appears to reduce Ca(2+) release from the SR mainly by reducing SR Ca(2+) content. A likely cause of the decreased SR content is reduced Ca(2+) uptake by the SR pump.

Extracellular amino acid levels measured with intracerebral microdialysis in the model of posttraumatic epilepsy induced by intracortical iron injection

An iron induced model of posttraumatic chronic focal epilepsy in rats was studied with respect to extracellular amino acids, electrophysiology, and morphology, approx. 6 months after intracortical injection of ferrous chloride. Twenty-six of the twenty-eight (93%) rats developed spontaneous epileptiform EEG-activity and electrical cortical stimulation done in eight animals evoked seizure activity in five animals (62.5%). Epileptic brain tissue displayed significantly higher extracellular interictal levels of aspartate (ASP), compared to normal brain, measured with intracerebral microdialysis. The interictal levels of serine (SER) were significantly higher at the lesion side compared to the contralateral cortex in epileptic animals. Spontaneous elevations of ASP and glutamate (GLU) levels up to 8 times the basal level were found in 4/5 (80%). There was no consistent amino acid pattern following the electrically induced seizures, but in association with more intense seizure activity ASP and GLU were elevated. Histopathologically, the necrotic lesions in the cortex contained small vessels and iron pigment loaded astrocytes. Scattered eosinophilic neurons were found in the hippocampus, bilaterally in 37% of the animals. The results show that a focal epileptiform activity developed in a high percentage of animals that received an intracortical iron injection. The observed amino acid changes in epileptic animals may be involved in the development of seizures in this model of posttraumatic epilepsy.

Continuous intrathecal fluid infusions elevate nerve growth factor levels and prevent functional deficits after spinal cord ischemia

Continuous intracerebroventricular or intrathecal infusions of neurotrophic factors have been reported to prevent neuronal degeneration, stimulate axonal sprouting and ameliorate behavioral deficits in various models of CNS injury and aging. In the present study, the ability of intrathecal infusions of recombinant human nerve growth factor (NGF) to reduce functional deficits following spinal cord ischemia was investigated. Adult rabbits underwent intrathecal cannulation and continuous infusions of either 300 μg/ml recombinant human NGF or artificial CSF (vehicle) at a rate of 143 μl/day for 7 days prior to induction of spinal cord ischemia. Continuous infusions were maintained after induction of ischemia. Four days later, both NGF-treated and vehicle-infused subjects showed a significant amelioration of functional motor deficits compared to lesioned, non-infused subjects ( P<0.05). The average duration of tolerated ischemia increased from 23.4±1.8 min in lesioned, non-infused subjects to 35.5±3.1 min in lesioned, artificial CSF-infused subjects and 35.6±4.7 min in NGF-infused subjects (mean±S.E.M.). Significantly elevated NGF protein levels were attained within the spinal cords of both NGF-treated subjects and artificial CSF-infused subjects, although levels were substantially higher in NGF-treated subjects (9.8±3.8 ng/g in NGF-infused vs. 2.0±0.4 ng/g in vehicle-infused and only 0.4±0.2 ng/g in lesioned, non-infused animals). These findings indicate that the process of intrathecal cannulation and fluid infusion elicits alterations in the spinal cord environment that are neuroprotective, including spontaneous elevations in NGF levels.

An Antagonist of cADP-ribose Inhibits Arrhythmogenic Oscillations of Intracellular Ca2+ In Heart Cells

Oscillations of Ca2+ in heart cells are a major underlying cause of important cardiac arrhythmias, and it is known that Ca2+-induced release of Ca2+ from intracellular stores (the sarcoplasmic reticulum) is fundamental to the generation of such oscillations. There is now evidence that cADP-ribose may be an endogenous regulator of the Ca2+ release channel of the sarcoplasmic reticulum (the ryanodine receptor), raising the possibility that cADP-ribose may influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose, an antagonist of cADP-ribose, suppressed oscillatory activity associated with overloading of intracellular Ca2+ stores in cardiac myocytes exposed to high doses of the beta-adrenoreceptor agonist isoproterenol or the Na+/K+-ATPase inhibitor ouabain. The oscillations suppressed by 8-amino-cADP-ribose included intracellular Ca2+ waves, spontaneous action potentials, after-depolarizations, and transient inward currents. Another antagonist of cADP-ribose, 8-bromo-cADP-ribose, was also effective in suppressing isoproterenol-induced oscillatory activity. Furthermore, in the presence of ouabain under conditions in which there was no arrhythmogenesis, exogenous cADP-ribose was found to be capable of triggering spontaneous contractile and electrical activity. Because enzymatic machinery for regulating the cytosolic cADP-ribose concentration is present within the cell, we propose that 8-amino-cADP-ribose and 8-bromo-cADP-ribose suppress cytosolic Ca2+ oscillations by antagonism of endogenous cADP-ribose, which sensitizes the Ca2+ release channels of the sarcoplasmic reticulum to Ca2+.