Claire A. Sheldon

Sodium Influx Pathways during and after Anoxia in Rat Hippocampal Neurons

Mechanisms that contribute to Na+ influx during and immediately after 5 min anoxia were investigated in cultured rat hippocampal neurons loaded with the Na+-sensitive fluorophore sodium-binding benzofuran isophthalate. During anoxia, an influx of Na+ in the face of reduced Na+,K+-ATPase activity caused a rise in [Na+]i. After the return to normoxia, Na+,K+-ATPase activity mediated the recovery of [Na+]i despite continued Na+ entry. Sodium influx during and after anoxia occurred through multiple pathways and increased the longer neurons were maintained in culture. Under the experimental conditions used, Na+ entry during anoxia did not reflect the activation of ionotropic glutamate receptors, TTX- or lidocaine-sensitive Na+ channels, plasmalemmal Na+/Ca2+ exchange, Na+/H+ exchange, or \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HC}\mathrm{O}_{3}^{-}\) \end{document}-dependent mechanisms; rather, contributions were received from a Gd3+-sensitive pathway activated by reactive oxygen species and Na+/K+/2Cl- cotransport in neurons maintained for 6-10 and 11-14 d in vitro (DIV), respectively. Sodium entry immediately after anoxia was not attributable to the activation of ionotropic glutamate receptors, voltage-activated Na+ channels, or Na+/K+/2Cl- cotransport; rather, it occurred via Na+/Ca2+ exchange, Na+/H+ exchange, and a Gd3+-sensitive pathway similar to that observed during anoxia; 11-14 DIV neurons received an additional contribution from an \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HC}\mathrm{O}_{3}^{-}\) \end{document}-dependent mechanism(s). The results provide insight into the intrinsic mechanisms that contribute to disturbed internal Na+ homeostasis during and immediately after anoxia in rat hippocampal neurons and, in this way, may play a role in the pathogenesis of anoxic or ischemic cell injury.

The Tumor Invasion Inhibitor Dihydromotuporamine C Activates RHO, Remodels Stress Fibers and Focal Adhesions, and Stimulates Sodium–Proton Exchange

The motuporamines are macrocyclic alkaloids that inhibit tumor cell invasion by an, as yet, unknown mechanism. A structure–activity study recently identified dihydromotuporamine C (dhMotC) as a highly active and readily synthesized analogue. Here, we show that dhMotC causes subtle cytoskeletal alterations in highly invasive MDA231 breast tumor cells that include an increase in the thickness and number of cytoplasmic actin stress fibers. Experiments with serum-starved Swiss 3T3 fibroblasts showed that micromolar concentrations of dhMotC that inhibit tumor cell invasion induce the formation of new stress fibers and large focal adhesion complexes that are dispersed around the entire cell periphery. dhMotC treatment of Swiss 3T3 cells also initiates a strong, long-lived activation of the small GTP-binding protein Rho, and it stimulates Rho kinase-dependent sodium–proton exchanger activity. Liposome-mediated cell loading of C3 exoenzyme prevents dhMotC-mediated Rho activation and stress fiber formation in 3T3 cells. C3 exoenzyme loading also reestablishes elongated MDA231 breast tumor cell invasion in the presence of dhMotC. Taken together, these results indicate that the ability to activate Rho is one important determinant of the anti-invasive activity of dhMotC.

The word-length effect in acquired alexia, and real and virtual hemianopia.

A word-length effect is often described in pure alexia, with reading time proportional to the number of letters in a word. Given the frequent association of right hemianopia with pure alexia, it is uncertain whether and how much of the word-length effect may be attributable to the hemifield loss. To isolate the contribution of the visual field defect, we simulated hemianopia in healthy subjects with a gaze-contingent paradigm during an eye-tracking experiment. We found a minimal word-length effect of 14 ms/letter for full-field viewing, which increased to 38 ms/letter in right hemianopia and to 31 ms/letter in left hemianopia. We found a correlation between mean reading time and the slope of the word-length effect in hemianopic conditions. The 95% upper prediction limits for the word-length effect were 51 ms/letter in subjects with full visual fields and 161 ms/letter with simulated right hemianopia. These limits, which can be considered diagnostic criteria for an alexic word-length effect, were consistent with the reading performance of six patients with diagnoses based independently on perimetric and imaging data: two patients with probable hemianopic dyslexia, and four with alexia and lesions of the left fusiform gyrus, two with and two without hemianopia. Two of these patients also showed reduction of the word-length effect over months, one with and one without a reading rehabilitation program. Our findings clarify the magnitude of the word-length effect that originates from hemianopia alone, and show that the criteria for a word-length effect indicative of alexia differ according to the degree of associated hemifield loss.

Regulation of Cl−−Hco3− exchangers by cAMP‐dependent protein kinase in adult rat hippocampal CA1 neurons

The contributions of HCO3−‐dependent, DIDS‐sensitive mechanisms to the maintenance of steady‐state pHi, and the regulation of their activities by cAMP‐dependent protein kinase (PKA), were investigated in CA1 neurons with the H+‐sensitive fluorophore, BCECF. The addition of HCO3−/CO2 to neurons with ‘low’ (pHi≤ 7.20) and ‘high’ (pHi > 7.20) initial pHi values under Hepes‐buffered conditions, increased and decreased steady‐state pHi, respectively. Conversely, under HCO3−/CO2‐buffered conditions, DIDS caused pHi to decrease and increase in neurons with low and high initial pHi values, respectively. In the presence, but not the absence, of HCO3−, the PKA inhibitor Rp‐adenosine‐3′,5′‐cyclic monophosphorothioate (Rp‐cAMPS; 50 μm) evoked DIDS‐sensitive increases and decreases in pHi in neurons with low and high initial pHi values, respectively. In contrast, in neurons with low initial pHi values, activation of PKA with the Sp isomer of cAMPS (Sp‐cAMPS; 25 μm) elicited increases in pHi that were smaller in the presence than in the absence of HCO3−, whereas in neurons with high initial pHi values, Sp‐cAMPS‐evoked rises in pHi were larger in the presence than in the absence of HCO3−; the differences between the effects of Sp‐cAMPS on pHi under the different buffering conditions were attenuated by DIDS. Consistent with the possibility that changes in the activities of HCO3−‐dependent, DIDS‐sensitive mechanisms contribute to the steady‐state pHi changes evoked by the PKA modulators, in neurons with initial pHi values ≤ 7.20, Rp‐cAMPS concurrently inhibited Na+‐independent Cl−‐HCO3− exchange and stimulated Na+‐dependent Cl−‐HCO3− exchange; in contrast, Sp‐cAMPS concurrently stimulated Na+‐independent Cl−‐HCO3− exchange and inhibited Na+‐dependent Cl−‐HCO3− exchange. Data from a limited number of neurons with initial pHi values > 7.20 suggested that the directions of the reciprocal changes in anion exchange activities (inhibition or stimulation) evoked by Rp‐ and Sp‐cAMPS may be opposite in cells with low vs. high resting pHi values. Taken together, the results indicate that the effects of modulating PKA activity on steady‐state pHi in rat CA1 neurons under HCO3−/CO2‐buffered conditions reflect not only changes in Na+‐H+ exchange activity but also changes in Na+‐dependent and Na+‐independent Cl−‐HCO3− exchange activity that, in turn, may be dependent upon the initial pHi.

Reading words, seeing style : The neuropsychology of word, font and handwriting perception

The reading of text is predominantly a left hemisphere function. However, it is also possible to process text for attributes other than word or letter identity, such as style of font or handwriting. Anecdotal observations have suggested that processing the latter may involve the right hemisphere. We devised a test that, using the identical stimuli, required subjects first to match on the basis of word identity and second to match on the basis of script style. We presented two versions, one using various computer fonts, and the other using the handwriting of different individuals. We tested four subjects with unilateral lesions who had been well characterized by neuropsychological testing and structural and/or functional MRI. We found that two prosopagnosic subjects with right lateral fusiform damage eliminating the fusiform face area and likely the right visual word form area were impaired in completion times and/or accuracy when sorting for script style, but performed better when sorting for word identity. In contrast, one alexic subject with left fusiform damage showed normal accuracy for sorting by script style and normal or mildly elevated completion times for sorting by style, but markedly prolonged reading times for sorting by word identity. A prosopagnosic subject with right medial occipitotemporal damage sparing areas in the lateral fusiform gyrus performed well on both tasks. The contrast in the performance of patients with right versus left fusiform damage suggests an important distinction in hemispheric processing that reflects not the type of stimulus but the nature of processing required.

Concurrent measurements of the free cytosolic concentrations of H+ and Na+ ions with fluorescent indicators.

We report a method for the concurrent measurement of intracellular [Na+] ([Na+]i) and pH (pHi) in cells co-loaded with SBFI, a Na+-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H+-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na+]i and pHi in rat hippocampal neurons. Neither the Na+ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na+]i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower pKa in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na+]i and pHi observed in response either to intracellular acid loads imposed by the NH4+ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na+]i and pHi.

An integrated mechanism of pediatric pseudotumor cerebri syndrome: evidence of bioenergetic and hormonal regulation of cerebrospinal fluid dynamics

Pseudotumor cerebri syndrome (PTCS) is defined by the presence of elevated intracranial pressure (ICP) in the setting of normal brain parenchyma and cerebrospinal fluid (CSF). Headache, vision changes, and papilledema are common presenting features. Up to 10% of appropriately treated patients may experience permanent visual loss. The mechanism(s) underlying PTCS is unknown. PTCS occurs in association with a variety of conditions, including kidney disease, obesity, and adrenal insufficiency, suggesting endocrine and/or metabolic derangements may occur. Recent studies suggest that fluid and electrolyte balance in renal epithelia is regulated by a complex interaction of metabolic and hormonal factors; these cells share many of the same features as the choroid plexus cells in the central nervous system (CNS) responsible for regulation of CSF dynamics. Thus, we posit that similar factors may influence CSF dynamics in both types of fluid-sensitive tissues. Specifically, we hypothesize that, in patients with PTCS, mitochondrial metabolites (glutamate, succinate) and steroid hormones (cortisol, aldosterone) regulate CSF production and/or absorption. In this integrated mechanism review, we consider the clinical and molecular evidence for each metabolite and hormone in turn. We illustrate how related intracellular signaling cascades may converge in the choroid plexus, drawing on evidence from functionally similar tissues.

Reduced contribution from Na+/H+ exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons

The effect of anoxia on Na+/H+ exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+‐sensitive fluorophore, BCECF. Five‐minute anoxia imposed under nominally HCO3–/CO2‐free conditions induced a fall in pHi, the magnitude of which was smaller following prolonged exposure to medium in which N‐methyl‐d‐glucamine (NMDG+) was employed as an extracellular Na+ (Na+o) substitute. Also consistent with the possibility that Na+/H+ exchange becomes inhibited soon after the induction of anoxia, rates of Na+o‐dependent pHi recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na+o‐dependent pHi recovery observed prior to anoxia. At the time at which rates of pHi recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na+/H+ exchange. In support, incubation of neurons with 2‐deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na+o‐dependent rates of pHi recovery from imposed acid loads; conversely, pre‐treatment with 10 mm creatine attenuated the effects of anoxia to reduce both ATP levels and Na+o‐dependent rates of pHi recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na+/H+ exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia‐induced falls in intracellular ATP.

Correlation of clinical and pathologic diagnoses of corneal disease in penetrating keratoplasties in Vancouver: A 10-year review

OBJECTIVES The objectives of the present study were: (i) to examine the leading clinical indications and pathologic diagnoses of penetrating keratoplasties in Vancouver, BC; and (ii) to evaluate the correlation between the clinical and pathologic diagnoses. DESIGN Retrospective chart review of all pathologic records of corneal tissue submitted to the Department of Pathology & Laboratory Medicine, Vancouver General Hospital, during an 11-year period. METHODS For each penetrating keratoplasty, information regarding the clinical indication and pathologic features identified on histopathologic analysis was compiled. RESULTS The top 5 clinical indications for transplant were failed graft (21%); bullous keratopathy (20%); keratoconus (17%); scarring, with or without inflammation (14%); and Fuchs dystrophy (11%). The top 5 pathologic diagnoses corresponded to these clinical diagnoses. In the majority of cases, there was agreement between clinical and pathologic diagnoses; however, in 6% of records, the clinical and pathologic diagnoses did not correlate. This situation occurred most often when considering grafts with clinical diagnoses of bullous keratopathy, Fuchs dystrophy, or keratoconus. A review of the grafts with infectious keratitis illustrated the potential for histopathologic evaluation of corneal buttons to identify unsuspected infectious agents. CONCLUSION Routine histopathologic evaluation of penetrating keratoplasty clarifies the clinical diagnosis in a proportion of cases and aids in identifying infectious agents.

Overweight and Obesity in Pediatric Secondary Pseudotumor Cerebri Syndrome

PURPOSE To examine the clinical, demographic, and anthropometric patient characteristics of secondary pseudotumor cerebri syndrome in children and adolescents based on the recently revised diagnostic criteria. DESIGN Retrospective observational case series. METHODS Patients seen at a tertiary childrens hospital for pseudotumor cerebri syndrome were classified as having either primary idiopathic (n = 59) or secondary pseudotumor cerebri syndrome (n = 16), as rigorously defined by recently revised diagnostic criteria. Outcomes included body mass index Z-scores (BMI-Z), height and weight Z-scores, demographics, and clinical features at presentation, such as headache, sixth nerve palsy, and cerebrospinal fluid (CSF) opening pressure. RESULTS In this cohort, the associated conditions and exposures seen in definite secondary pseudotumor cerebri syndrome included tetracycline-class antibiotics (n = 11), chronic kidney disease (n = 3), withdrawal from chronic glucocorticoids (n = 1), and lithium (n = 1). Other associations observed in the possible secondary pseudotumor cerebri syndrome group included Down syndrome, vitamin A derivatives, and growth hormone. In comparison with primary pseudotumor cerebri syndrome, definite secondary pseudotumor cerebri syndrome patients were on average older (15.0 vs 11.6 years; P = .003, Mann-Whitney test). According to US Centers for Disease Control (CDC) classifications, 79% of children with secondary pseudotumor cerebri syndrome were either overweight or obese (36% overweight [n = 5] and 43% obese [n = 6]), as compared to 32% nationally. CONCLUSIONS Even when a potential inciting exposure is identified for pediatric pseudotumor cerebri syndrome, the possible contribution of overweight and obesity should be considered.