James E. Olson

Optimal positioning for cervical immobilization.

STUDY OBJECTIVE We hypothesized that optimal positioning of the head and neck to protect the spinal cord during cervical spine immobilization can be determined with reference to external landmarks. In this study we sought to determine the optimal position for cervical spine immobilization using magnetic resonance imaging (MRI) and to define this optimal position in a clinically reproducible fashion. METHODS Our subjects were 19 healthy adult volunteers (11 women, 8 men). In each, we positioned the head to produce various degrees of neck flexion and extension. This positioning was followed by quantitative MRI of the cervical spine. RESULTS The mean ratio of spinal canal and spinal cord cross-sectional areas was smallest at C6 but exceeded 2.0 at all levels from C2 to T1 (P < .05). At the C5 and C6 levels, the maximal area ratio was most consistently obtained with slight flexion (cervical-thoracic angle of 14 degrees) (P < .05). For a patient lying flat on a backboard, this corresponds to raising the occiput 2 cm. More extreme flexion or extension produced variable results. CONCLUSION In healthy adults, a slight degree of flexion equivalent to 2 cm of occiput elevation produces a favorable increase in spinal canal/spinal cord ration at levels C5 and C6, a region of frequent unstable spine injuries.

Relation of taurine transport and brain edema in rats with simple hyperammonemia or liver failure

Taurine (Tau), an amino acid that abounds in brain, has been implicated in inhibitory neuromodulation and osmoregulation, the latter function being manifested by Tau release along with osmotically obligated water in response to brain tissue edema. A previous study (Hilgier and Olson: J. Neurochem. 62:197–204, 1994) had shown that simple hyperammonemia (HA) induced in rats by daily administration of ammonium acetate resulted in a decrease of both tissue specific gravity indicative of edema and Tau content, in basal ganglia (BG) but not in cerebral cortex (CC). By contrast, rats with hepatic encephalopathy (HE) following administration of a hepatotoxin, thioacetamide, were characterized by CC edema and an increased Tau content in both BG and CC. In the present study, we tested the following parameters that may potentially have affected Tau distribution in the two models: a) spontaneous, and stimulated (hypoosmolarity‐induced) release of loaded [3H] Tau in vitro from CC and BG slices; b) blood Tau content; and c) uptake of [14C] Tau in vivo from blood to brain corrected for [3H] water passage—the so‐called brain uptake index (BUI).

Taurine Release by Astrocytes Modulates Osmosensitive Glycine Receptor Tone and Excitability in the Adult Supraoptic Nucleus

Cells can release the free amino acid taurine through volume-regulated anion channels (VRACs), and it has been hypothesized that taurine released from glial cells is capable of inhibiting action potential (AP) firing by activating neuronal glycine receptors (GlyRs) (Hussy et al., 1997). Although an inhibitory GlyR tone is widely observed in the brain, it remains unknown whether this specifically reflects gliotransmission because most neurons also express VRACs and other endogenous molecules can activate GlyRs. We found that VRACs are absent in neurons of the rat supraoptic nucleus (SON), suggesting that glial cells are the exclusive source of taurine in this nucleus. Application of strychnine to rat hypothalamic explants caused a depolarization of SON neurons associated with a decrease of chloride conductance and could excite these cells in the absence of fast synaptic transmission. This inhibitory GlyR tone was eliminated by pharmacological blockade of VRACs, by cellular taurine depletion, by metabolic inactivation of glia with fluorocitrate, and after retraction of astrocytic processes that intercalate neuronal somata and dendrites. Finally, GlyR tone varied inversely with extracellular fluid tonicity to mediate the osmotic control of AP firing by SON neurons. These findings establish taurine as a physiological gliotransmitter and show that gliotransmission is a spatially constrained process that can be modulated by the morphological rearrangement of astrocytes.

Taurine synthesis and cysteine metabolism in cultured rat astrocytes: effects of hyperosmotic exposure

We investigated mechanisms controlling taurine synthesis in cultured rat cerebral astrocytes. The mean ± SE rate of taurine synthesis from extracellular cysteine was 21.2 ± 2.0 pmol ⋅ mg protein-1 ⋅ min-1, whereas taurine degradation was <1.3% of this rate. Eliminating cellular glutathione and inhibiting glutathione biosynthesis increased taurine synthesis from extracellular cysteine by 39%. In cell homogenates, cysteine dioxygenase (CDO) and cysteine-sulfinate decarboxylase activities were 2.4 ± 0.2 and 8.3 ± 2.8 nmol ⋅ mg protein-1 ⋅ min-1, respectively. CDO activity was strongly dependent on cysteine concentration over physiological and pathophysiological ranges of intracellular cysteine concentration. Growth in hyperosmotic medium caused a greater increase in culture medium taurine content than that measured from cells in isosmotic growth medium. Hyperosmotic treatment transiently increased the rate of cysteine accumulation and cellular cysteine and glutathione contents but had no effect on the synthesis rate of taurine from extracellular cysteine. Thus cysteine is accumulated and then metabolized to taurine through CDO, whose activity depends on the intracellular cysteine concentration and appears to be rate limiting for taurine synthesis. Hyperosmotic exposure increases net taurine production yet has no effect on taurine synthesis from exogenously applied cysteine. Availability of substrate from intracellular pools must contribute to maintenance of high intracellular taurine during hyperosmotic exposure.We investigated mechanisms controlling taurine synthesis in cultured rat cerebral astrocytes. The mean +/- SE rate of taurine synthesis from extracellular cysteine was 21.2 +/- 2.0 pmol.mg protein-1.min-1, whereas taurine degradation was < 1.3% of this rate. Eliminating cellular glutathione and inhibiting glutathione biosynthesis increased taurine synthesis from extracellular cysteine by 39%. In cell homogenates, cysteine dioxygenase (CDO) and cysteine-sulfinate decarboxylase activities were 2.4 +/- 0.2 and 8.3 +/- 2.8 nmol.mg protein-1.min-1, respectively. CDO activity was strongly dependent on cysteine concentration over physiological and pathophysiological ranges of intracellular cysteine concentration. Growth in hyperosmotic medium caused a greater increase in culture medium taurine content than that measured from cells in isosmotic growth medium. Hyperosmotic treatment transiently increased the rate of cysteine accumulation and cellular cysteine and glutathione contents but had no effect on the synthesis rate of taurine from extracellular cysteine. Thus cysteine is accumulated and then metabolized to taurine through CDO, whose activity depends on the intracellular cysteine concentration and appears to be rate limiting for taurine synthesis. Hyperosmotic exposure increases net taurine production yet has no effect on taurine synthesis from exogenously applied cysteine. Availability of substrate from intracellular pools must contribute to maintenance of high intracellular taurine during hyperosmotic exposure.

Increased potassium, chloride, and taurine conductances in astrocytes during hypoosmotic swelling.

Membrane conductances during hypoosmotic swelling were characterized in rat astrocytes in primary tissue culture. Using whole cell patch clamp techniques, mean ± SEM cell conductance in isoosmotic phosphate‐buffered saline (PBS) was 55.6 ± 5.8 pS/pF. Cell conductance (mean ± SEM) increased from this initial value to 187 ± 46%, 561 ± 188%, and 1216 ± 376% within 9 min of exposure to 220 mOsm, 190 mOsm, and 145 mOsm PBS, respectively. With each of these hypoosmotic exposures, no change occurred in membrane capacitance. When CsCl replaced KCl in the microelectrode solution, a similar conductance increase was obtained at each osmolality. However, when gluconate salts were used in place of chloride salts in the electrode solution, no significant conductance increase was observed with 190 mOsm PBS. With a KCl microelectrode solution, all conductance increase which occurred in 190 mOsm PBS was inhibited by 200 μM niflumic acid, but not by 5 mM BaCl2. Both niflumic acid and BaCl2 inhibited 60–80% of the conductance increase of cells in 145 mOsm PBS. Using a microelectrode solution containing taurine as the major anion, membrane conductance increased 5‐fold when cells were placed in 250 mOsm medium. This conductance increase was completely inhibited by 200 μM niflumic acid. Thus, independent chloride and potassium conductances are activated by hypoosmotic swelling of cultured astrocytes while plasma membrane area is unaltered. The chloride conductance pathway is activated at a significantly lower degree of hypoosmotic exposure than that which activates the potassium pathway and may be permeable to anionic taurine. These conductance pathways may mediate diffusive loss of potassium, chloride, and taurine from these cells during volume regulation following hypoosmotic swelling. GLIA 20:254–261, 1997.

Acute hyperoxia increases lipid peroxidation and induces plasma membrane blebbing in human U87 glioblastoma cells

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H(2)O(2)) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O(2), normobaric hyperoxia (0.95 ATA O(2)) or hyperbaric hyperoxia (HBO(2), 3.25 ATA O(2)) for 60 min. H(2)O(2) (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H(2)O(2). Average membrane roughness (R(a)) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The R(a) of the plasma membrane was 34+/-3, 57+/-3 and 63+/-5 nm in 0.20 ATA O(2), 0.95 ATA O(2) and HBO(2), respectively. R(a) was 56+/-7 and 138+/-14 nm in 0.2 and 2 mM H(2)O(2). Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H(2)O(2) and correlated with O(2)-induced membrane blebbing (r(2)=0.93). Coapplication of antioxidant, Trolox-C (150 microM), significantly reduced membrane R(a) and MDA levels during hyperoxia. Hyperoxia-induced H(2)O(2) production increased 189%+/-5% (0.95 ATA O(2)) and 236%+/-5% (4 ATA O(2)) above control (0.20 ATA O(2)). We conclude that MLP and membrane blebbing increase with increasing O(2) concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage.

Calcium/calmodulin-modulated chloride and taurine conductances in cultured rat astrocytes.

Osmotically swollen rat cerebral astrocytes develop an increased anion conductance which can mediate chloride and taurine release. We used whole cell patch clamp to study mechanisms that modulate this conductance. Astrocyte chloride conductance increased within 4 min of exposure to 200 mOsm medium and was 670+/-123% of its initial value after 15 min (mean+/-S.E.M.). This conductance was substantially reduced in 0.1 mM extracellular calcium with 20 mM BAPTA added to the electrode solution and was completely inhibited with calcium-free perfusion solution containing 1 mM EDTA (n=4). The conductance increase in 200 mOsm medium also was inhibited in a dose-dependent manner by nimodipine with a calculated K(i) of 0.31+/-0.4 microM and mean+/-S.E.M. inhibition of 84.4+/-4% at 100 microM nimodipine. In the presence of 100 microM W-7, a calmodulin antagonist, the mean+/-S.E.M. conductance increase after 15 min was 223+/-40% of the initial value while 300 microM W-7 or 100 microM trifluoperazine inhibited the conductance increase completely (n=6). With taurine as the major anion in electrode and perfusion solutions, a significant conductance increase was observed in 200 mOsm medium. This conductance increase was inhibited by 300 microM W-7 or 100 microM nimodipine. We conclude extracellular calcium influx via L-type calcium channels leads to increased astrocyte anion conductance in 200 mOsm conditions via calmodulin-dependent activation of anion channels. Efflux of anionic taurine from swollen astrocytes also may be affected by calcium influx through a similar calcium/calmodulin-dependent process.

Taurine enhances volume regulation in hippocampal slices swollen osmotically.

Cell volume regulation has been studied in neuronal and glial cultures but little is known about volume regulation in brain tissue with an intact extracellular space. We investigated volume regulation in hippocampal slices maintained in an interface chamber and exposed to hypo-osmotic medium. Relative changes in intracellular and extracellular volume were measured respectively as changes in light transmittance and extracellular resistance. Slices exposed to hypo-osmotic medium (200-240 mOsm/L) showed a decrease in light transmittance, which occasionally was preceded by a brief transient increase. However, hypo-osmotic exposure was always accompanied by a monotonic increase in extracellular resistance. Peak changes in light transmittance and extracellular resistance occurred at 15-20 min following exposure to hypo-osmotic medium. Optical evidence of volume regulation (RVD) was observed in six of 12 slices and occurred over the next 60-90 min. We hypothesized that the relatively low incidence of RVD was related to depletion of taurine, an osmolyte known to play an important role in volume regulation, during preparation of the slices. Indeed, taurine levels in freshly prepared slices were <50% of those reported in intact hippocampus. Incubation of slices in 1 mM taurine restored taurine to levels observed in situ and increased both the likelihood and magnitude of RVD in hypo-osmotic medium. Inhibition of taurine flux with 100 microM 5-nitro-2-(3 phenylpropylamino) benzoic acid blocked both RVD and the transient undershoot of volume commonly associated with return of swollen slices to iso-osmotic medium. Taurine treatment had no effect on levels of several other amino acids but preserved slice potassium content. The results indicate a critical role for cellular taurine during hypo-osmotic volume regulation in hippocampal slices. Inconsistencies between optical measurements of cellular volume changes and electrical measurements of extracellular space are likely to result from the complex nature of light transmittance in the interface slice preparation.

Ischemia-Induced Brain Damage is Enhanced in Human Renin and Angiotensinogen Double Transgenic Mice

To investigate the role of brain angiotensin II (ANG II) in the pathogenesis of injury following ischemic stroke, mice overexpressing renin and angiotensinogen (R+A+) and their wild-type control animals (R-A-) were used for experimental ischemia studies. Focal brain ischemia was induced by middle cerebral artery occlusion (MCAO). The severity of ischemic injury was determined by measuring neurological deficits and histological damage at 24 and 48 h after MCAO, respectively. To exclude the influence of blood pressure and local collateral blood flow, brain slices were used for oxygen and glucose deprivation (OGD) studies. The severity of OGD-induced damage was determined by measuring indicators of tissue swelling and cell death, the intensity of the intrinsic optical signal (IOS), and the number of propidium iodide (PI) staining cells, respectively. Results showed 1) R+A+ mice showed higher neurological deficit score (3.8 +/- 0.5 and 2.5 +/- 0.3 for R+A+ and R-A-, respectively, P < 0.01) and larger infarct volume (22.2 +/- 1.6% and 14.1 +/- 1.2% for R+A+ and R-A-, respectively, P < 0.01); 2) The R+A+ brain slices showed more severe tissue swelling and cell death in the cortex (IOS: 140 +/- 6% and 114 +/- 10%; PI: 139 +/- 20 cells/field and 39 +/- 9 cells/field for R+A+ and R-A-, respectively, P < 0.01); 3) treatment with losartan (20 micromol/l) abolished OGD-induced exaggeration of cell injury seen in R+A+ mice. The data indicate that activation of ANG II/AT(1) signaling is harmful to brain exposed to ischemia.

Brain water content brain blood volume blood chemistry and pathology in a model of cerebral edema

STUDY OBJECTIVES The objective was to correlate regional changes during brain water content with alterations in blood chemistry and cerebral pathology during hypo-osmotic edema. PARTICIPANTS Sprague-Dawley male adult rats were used in these studies. DESIGN Animals were block-randomized to receive either an intraperitoneal distilled water injection equivalent to 5% or 15% of their body weight or no injection (controls). Rats were sacrificed 15 or 60 minutes after water injection or at an equivalent time for controls. INTERVENTIONS No interventions were performed. MEASUREMENTS AND MAIN RESULTS Water content of cerebral cortical gray and white matter was calculated from measurements of tissue specific gravity. Blood plasma osmolality and sodium and potassium concentrations were determined at various times after water injection. An index of blood-brain barrier permeability was obtained by measuring brain red blood cell and plasma volumes. A qualitative assessment of edema was made from light and electron micrographs of the cerebral cortex. We found that water injection produced a dose-dependent decrease in plasma osmolality and sodium concentration within 15 minutes. Cortical water content was unchanged after this period. An influx of water into cerebral gray, and, less readily, into cerebral white matter occurred during the next 15 minutes. Whole blood specific gravity and brain blood content were unchanged and thus did not confound the measurement of cerebral water content. Hematocrit was increased 60 minutes after a 15% water injection. The blood-brain barrier remained intact throughout this period. Microscopy revealed astrocytic swelling with slight extracellular fluid accumulation 60 minutes after the water injection. CONCLUSIONS Homeostatic mechanisms in the cerebral cortex can maintain constant water content for at least 15 minutes during maintained intravascular hypo-osmolality. Fluid that subsequently moves into the tissue primarily enters an intracellular compartment. This model will be useful in investigating physiological mechanisms of brain water regulation and the pathogenesis of brain edema, a common clinical entity in emergency conditions.