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Background: Chronic degeneration of the zygapophyseal joints in the cervical or lumbar spine are common causes of axial back pain. Radiofrequency (RF) ablation is a treatment modality in the denervation of facet joint-related pain. Although multiple factors have been theorized to contribute to the size of the optimal RF lesion, the addition of hypertonic saline solution has been posited to create larger RF lesion sizes., Objectives: This study compares lesion of 20-gauge RF monopolar probe using 2% lidocaine, 0.9% normal saline solution, and 3% saline solution administered through the RF needle prior to ablation, with subsequent lesion sizes recorded., Study Design: Randomized, double-blinded, ex vivo study using clinically relevant conditions., Setting: Procedural laboratory in an academic institution., Methods: RF ablation lesions were reproduced in room temperature (21°C ± 2°C) chicken breast specimens with 20-gauge monopolar RF probes inserted. RF was applied for 90 seconds at 80°C after injection of 1 mL of either 2% lidocaine, 2% lidocaine and 0.9% normal saline solution in a 1:1 ratio, or 2% lidocaine and 3% saline solution in a 1:1 ratio. Tissues were dissected, measured, and ellipsoid volumes of burn calculated. Homogeneity of variances was assessed via the Bartlett's test, and heteroskedasticity with the studentized Breusch-Pagan test. One-way analysis of variance (ANOVA) (alpha of 0.05) was used to evaluate statistical significance between volume means across groups. When the null hypothesis of no difference in burn volume between samples could not be rejected, a predefined equivalence volume of ± 0.05 cm3 was used with Welch's 2 one-sided t-tests (TOST) with a Bonferroni adjusted alpha of 0.0167 to evaluate for null acceptance., Results: The mean lesion volume for monopolar RF with 1 mL 2% lidocaine was 0.16 cm3. Monopolar RF with 1 mL 2% lidocaine + 0.9% normal saline solution had a mean lesion volume of 0.15 cm3, and treatment with 1 mL 2% lidocaine + 3% saline solution measured 0.17 cm3. ANOVA failed to reject the null, and TOST accepted as equivalent all 3 comparisons., Limitations: In vivo anatomy and physiology of a human organism was not used for this study. Samples were not warmed to physiologic temperature. Randomization resulted in slightly unequal sample sizes, although all groups were of sufficient size that the central limit theorem should apply., Conclusions: Three commonly used solutions were found to have equivalent lesion sizes from monopolar probe RF ablation., Key Words: Radiofrequency, ablation, lesion shape, lesion size, monopolar RF, hypertonic saline solution.

Zebrafish is an excellent model that can be utilized as an adjunct to current rodent models for studies of eye diseases because the anatomy and ultrastructural characterization of its cornea show much similarity with the human cornea. Therefore, we developed a behavioral model of corneal nociception using the adult zebrafish (Danio rerio). We analyzed the nociceptive effect of hypertonic saline (0.15-5.0 M sodium chloride [NaCl]) applied to the surface of the right or left cornea, on the animals' gender and locomotor activity through the open-field test. The behavioral model of corneal nociception was characterized by the antinociceptive effect of morphine (8.0 or 16 mg/kg; intraperitoneally [i.p.]), an opioid analgesic, and capsazepine, an antagonist of transient receptor potential vanilloid type 1 channels. We also tested whether the corneal antinociceptive effect of morphine could be modulated by naloxone, an opioid antagonist. Finally, we used the light and dark test to assess the anxiolytic effect of hypertonic saline (5.0 M NaCl; 5 μL) applied to the right or left cornea of the animals. As a result, hypertonic saline significantly increased (p < 0.01 vs. control) the corneal nociceptive behavior of adult zebrafish (D. rerio). Morphine significantly inhibited (p < 0.01 vs. 5.0 M NaCl) the hypertonic saline-induced corneal nociception and this effect was blocked by naloxone. Capsazepine (20 mg/kg; i.p.) significantly inhibited (p < 0.05 vs. control) the corneal nociception induced by hypertonic saline. Hypertonic saline, applied to the surface of the right or left cornea of the animals, induced nociception and did not cause a presumptive anxiolytic effect. Gender and site of application did not affect the profile of response to hypertonic saline. The results suggest that the adult zebrafish can also be used as a behavioral model of corneal nociception, with the advantages of significant homology with the human genome and low cost.

The surface of the cornea contains the highest density of nociceptive nerves of any tissue in the body. These nerves are responsive to a variety of modalities of noxious stimuli and can signal pain even when activated by low threshold stimulation. Injury of corneal nerves can lead to altered nerve morphology, including neuropathic changes which can be associated with chronic pain. Emerging technologies that allow imaging of corneal nerves in vivo are spawning questions regarding the relationship between corneal nerve density, morphology, and function. We tested whether noxious stimulation of the corneal surface can alter nerve morphology and neurochemistry. We used concentrations of menthol, capsaicin, and hypertonic saline that evoked comparable levels of nocifensive eye wipe behaviors when applied to the ocular surface of an awake rat. Animals were sacrificed and corneal nerves were examined using immunocytochemistry and three-dimensional volumetric analyses. We found that menthol and capsaicin both caused a significant reduction in corneal nerve density as detected with β-tubulin immunoreactivity 2 hr after stimulation. Hypertonic saline did not reduce nerve density, but did cause qualitative changes in nerves including enlarged varicosities that were also seen following capsaicin and menthol stimulation. All three types of noxious stimuli caused a depletion of CGRP from corneal nerves, indicating that all modalities of noxious stimuli evoked peptide release. Our findings suggest that studies aimed at understanding the relationship between corneal nerve morphology and chronic disease may also need to consider the effects of acute stimulation on corneal nerve morphology., (© 2017 Wiley Periodicals, Inc.)

Pain is associated with changes in the neural drive to muscles. For the upper trapezius muscle, surface electromyography (EMG) recordings have indicated that acute noxious stimulation in either the cranial or the caudal region of the muscle leads to a relative decrease in muscle activity in the cranial region. It is, however, not known if this adaption reflects different recruitment thresholds of the upper trapezius motor units in the cranial and caudal region or a nonuniform nociceptive input to the motor units of both regions. This study investigated these potential mechanisms by direct motor unit identification. Motor unit activity was investigated with high-density surface EMG signals recorded from the upper trapezius muscle of 12 healthy volunteers during baseline, control (intramuscular injection of isotonic saline), and painful (hypertonic saline) conditions. The EMG was decomposed into individual motor unit spike trains. Motor unit discharge rates decreased significantly from control to pain conditions by 4.0 ± 3.6 pulses/s (pps) in the cranial region but not in the caudal region (1.4 ± 2.8 pps; not significant). These changes were compatible with variations in the synaptic input to the motoneurons of the two regions. These adjustments were observed, irrespective of the location of noxious stimulation. These results strongly indicate that the nociceptive synaptic input is distributed in a nonuniform way across regions of the upper trapezius muscle., (Copyright © 2016 the American Physiological Society.)

We have recently shown that intramuscular infusion of hypertonic saline, causing pain lasting ~60min, increases muscle sympathetic nerve activity (MSNA) in one group of subjects, yet decreases it in another. Across subjects these divergent sympathetic responses to long-lasting muscle pain are consistent over time and cannot be foreseen on the basis of baseline MSNA, blood pressure, heart rate or sex. We predicted that differences in anxiety or attitudes to pain may account for these differences. Psychometric measures were assessed prior to the induction of pain using the State and Trait Anxiety Inventory (STAI), Pain Vigilance and Awareness Questionnaire (PVAQ), Pain Anxiety Symptoms Scale (PASS) and Pain Catastrophising Scale (PCS); PCS was also administered after the experiment. MSNA was recorded from the common peroneal nerve, before and during a 45-minute intramuscular infusion of hypertonic saline solution into the tibialis anterior muscle of 66 awake human subjects. Forty-one subjects showed an increase in mean burst amplitude of MSNA (172.8±10.6%) while 25 showed a decrease (69.9±3.8%). None of the measured psychological parameters showed significant differences between the increasing and the decreasing groups. We conclude that inter-individual anxiety or pain attitudes do not determine whether MSNA increases or decreases during long-lasting experimental muscle pain in healthy human subjects., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10-100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na(+), since substituting excess Na(+) with choline reduced cell death to control levels, whereas gluconate instead of excess Cl(-) did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na(+)] caused reduction in intracellular free [Ca(2+)], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca(2+)] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia., (Copyright © 2016. Published by Elsevier B.V.)

Injection of hypertonic saline via episcleral veins toward the limbus in laboratory rats can produce elevated intraocular pressure (IOP) by sclerosis of aqueous humor outflow pathways. This article describes important anatomic characteristics of the rat optic nerve head (ONH) that make it an attractive animal model for human glaucoma, along with the anatomy of rat aqueous humor outflow on which this technique is based. The injection technique itself is also described, with the aid of a supplemental movie, including necessary equipment and specific tips to acquire this skill. Outcomes of a successful injection are presented, including IOP elevation and patterns of optic nerve injury. These concepts are then specifically considered in light of the use of this model to assess potential neuroprotective therapies. Advantages of the hypertonic saline model include a delayed and relatively gradual IOP elevation, likely reproduction of scleral and ONH stresses and strains that may be important in producing axonal injury, and its ability to be applied to any rat (and potentially mouse) strain, leaving the unmanipulated fellow eye as an internal control. Challenges include the demanding surgical skill required by the technique itself, a wide range of IOP response, and mild corneal clouding in some animals. However, meticulous application of the principles detailed in this article and practice will allow most researchers to attain this useful skill for studying cellular events of glaucomatous optic nerve damage., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs). Tumor necrosis factor α (TNFα) signaling has been linked to RGC damage, however, the mechanism by which TNFα promotes neuronal death remains poorly defined. Using an in vivo rat glaucoma model, we show that TNFα is upregulated by Müller cells and microglia/macrophages soon after induction of ocular hypertension. Administration of XPro1595, a selective inhibitor of soluble TNFα, effectively protects RGC soma and axons. Using cobalt permeability assays, we further demonstrate that endogenous soluble TNFα triggers the upregulation of Ca(2+)-permeable AMPA receptor (CP-AMPAR) expression in RGCs of glaucomatous eyes. CP-AMPAR activation is not caused by defects in GluA2 subunit mRNA editing, but rather reflects selective downregulation of GluA2 in neurons exposed to elevated eye pressure. Intraocular administration of selective CP-AMPAR blockers promotes robust RGC survival supporting a critical role for non-NMDA glutamate receptors in neuronal death. Our study identifies glia-derived soluble TNFα as a major inducer of RGC death through activation of CP-AMPARs, thereby establishing a novel link between neuroinflammation and cell loss in glaucoma., Significance Statement: Tumor necrosis factor α (TNFα) has been implicated in retinal ganglion cell (RGC) death, but how TNFα exerts this effect is poorly understood. We report that ocular hypertension, a major risk factor in glaucoma, upregulates TNFα production by Müller cells and microglia. Inhibition of soluble TNFα using a dominant-negative strategy effectively promotes RGC survival. We find that TNFα stimulates the expression of calcium-permeable AMPA receptors (CP-AMPAR) in RGCs, a response that does not depend on abnormal GluA2 mRNA editing but on selective downregulation of the GluA2 subunit by these neurons. Consistent with this, CP-AMPAR blockers promote robust RGC survival supporting a critical role for non-NMDA glutamate receptors in glaucomatous damage. This study identifies a novel mechanism by which glia-derived soluble TNFα modulates neuronal death in glaucoma., (Copyright © 2015 the authors 0270-6474/15/3512088-15$15.00/0.)

We assessed the effects of simulated weightlessness, hindlimb unloading (HU) by 7 days of tail suspension, on noxious mechanically and heat evoked spinal withdrawal reflexes and spinal Fos expression during muscle nociception elicited by intramuscular (i.m.) injection of hypertonic (HT; 5.8%) saline into gastrocnemius muscle in rats. In HU rats, i.m. HT saline-induced secondary mechanical hyperalgesia was enhanced, and secondary heat hypoalgesia was significantly delayed. After 7 days of HU, basal Fos expression in spinal L4-6 segments was bilaterally enhanced only in superficial (I-II) but not middle and deep laminae (III-VI) of the spinal dorsal horn, which finding was not influenced by tail denervation. Unilateral i.m. HT saline injection increased spinal Fos expression bilaterally in both the control rats and 7 days of HU rats. The HT saline-induced bilateral increase of spinal Fos occurred within 0.5h and reached its peak within 1h, after which it gradually returned to the control levels within 8h. Spatial patterns of spinal Fos expression differed between the control group and 7 days of HU group. In superficial laminae, the HT saline-induced increases in Fos expression were higher and in the middle and deep laminae V-VI lower in the 7 days of HU than control rats. It is suggested that supraspinal mechanisms presumably underlie the effects of HU on spinally-organized nociception. Simulated weightlessness may enhance descending facilitation and weaken descending inhibition of nociception., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purpose: Although tear hyperosmolarity is assumed to play a major role in dry eye disease, correlation between the level of hyperosmolarity and inflammation remains unclear. The purpose of this study was to examine the effect of short-time hyperosmolarity exposure in the production of inflammatory cytokines in corneal epithelial cells in vitro., Methods: Human corneal epithelial (HCE) cells were cultured under different osmotic conditions [310 (control), and 400-1000 mOsm]. Lactate dehydrogenase (LDH) release after short-term (10 minutes) or long-term (24 hours) hyperosmotic stress exposure was evaluated to determine HCE cell cytotoxicity. Production of inflammatory cytokines, including IL-6, IL-1β, IL-8, IL-23, and TGF-β1, due to hyperosmotic stress was also measured by enzyme-linked immunosorbent assay and semiquantitative real-time polymerase chain reaction., Results: After a 24-hour culture, exposures above 700 mOsm caused all HCE cells to die, 500 and 600 mOsm damaged the cells, whereas 400 mOsm caused no morphological changes. However, there was a significant increase in the release of LDH after 24-hour cultures, even in 400 mOsm. In contrast, LDH examination showed that there was no cytotoxicity for the 10-minute exposures, even at above 800 mOsm. The significant increases in IL-6 production and mRNA expression at 700 mOsm during the short-time exposures were both dependent on the osmolarity. Other cytokines such as IL-1β, IL-8, IL-23, and TGF-β1 were not detected., Conclusions: Short-time hyperosmolarity exposure may activate IL-6 expression and production in HCE cells without cytotoxicity. These observations suggest that hyperosmolarity could cause inflammation on the ocular surface in dry eye disease.

Overly rapid correction of chronic hyponatremia can cause osmotic demyelination syndrome (ODS). Minocycline protects ODS associated with overly rapid correction of chronic hyponatremia with hypertonic saline infusion in rats. In clinical practice, inadvertent rapid correction frequently occurs due to water diuresis, when vasopressin action suddenly ceases. In addition, vasopressin receptor antagonists have been applied to treat hyponatremia. Here the susceptibility to and pathology of ODS were evaluated using rat models developed to represent rapid correction of chronic hyponatremia in the clinical setting. The protective effect of minocycline against ODS was assessed. Chronic hyponatremia was rapidly corrected by 1 (T1) or 10 mg/kg (T10) of tolvaptan, removal of desmopressin infusion pumps (RP), or administration of hypertonic saline. The severity of neurological impairment in the T1 group was significantly milder than in other groups and brain hemorrhage was found only in the T10 and desmopressin infusion removal groups. Minocycline inhibited demyelination in the T1 group. Further, immunohistochemistry showed loss of aquaporin-4 (AQP4) in astrocytes before demyelination developed. Interestingly, serum AQP4 levels were associated with neurological impairments. Thus, minocycline can prevent ODS caused by overly rapid correction of hyponatremia due to water diuresis associated with vasopressin action suppression. Increased serum AQP4 levels may be a predictive marker for ODS.

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.

We recently showed that long-lasting muscle pain, induced by intramuscular infusion of hypertonic saline, evoked two patterns of cardiovascular responses across subjects: one group showed parallel increases in muscle sympathetic nerve activity (MSNA), blood pressure, and heart rate, while the other group showed parallel decreases. Given that MSNA is consistent day to day, we tested the hypothesis that individuals who show increases in MSNA during experimental muscle pain will show consistent responses over time. MSNA was recorded from the peroneal nerve, together with blood pressure and heart rate, during an intramuscular infusion of hypertonic saline causing pain for an hour in 15 subjects on two occasions, 2-27 weeks apart. Pain intensity ratings were not significantly different between the first (5.8 ± 0.4/10) and second (6.1 ± 0.2) recording sessions. While four subjects showed significant decreases in the first session (46.6 ± 9.2% of baseline) and significant increases in the second (159.6 ± 8.9%), in 11 subjects, there was consistency in the changes in MSNA over time: either a sustained decrease (55.6 ± 6.8%, n = 6) or a sustained increase (143.5 ± 6.1%, n = 5) occurred in both recording sessions. There were no differences in pain ratings between sessions for any subjects. We conclude that the changes in MSNA during long-lasting muscle pain are consistent over time in the majority of individuals, reflecting the importance of studying interindividual differences in physiology.

Injection of hypertonic saline into deep tissues of the back (subcutis, muscle, or the surrounding fascia) can induce acute low back pain (LBP). So far, no study has analyzed differences in temporal, qualitative, and spatial pain characteristics originating from these tissues. The current study aimed to investigate the role of the thoracolumbar fascia as a potential source of LBP. In separate sessions, 12 healthy subjects received ultrasound-guided bolus injections of isotonic saline (0.9%) or hypertonic saline (5.8%) into the erector spinae muscle, the thoracolumbar fascia (posterior layer), and the overlying subcutis. Subjects were asked to rate pain intensity, duration, quality, and spatial extent. Pressure pain thresholds were determined pre and post injection. Injections of hypertonic saline into the fascia resulted in significantly larger area under the curve of pain intensity over time than injections into subcutis (P<0.01) or muscle (P<0.001), primarily based on longer pain durations and, to a lesser extent, on higher peak pain ratings. Pressure hyperalgesia was only induced by injection of hypertonic saline into muscle, but not fascia or subcutis. Pain radiation and pain affect evoked by fascia injection exceeded those of the muscle (P<0.01) and the subcutis significantly (P<0.05). Pain descriptors after fascia injection (burning, throbbing, and stinging) suggested innervation by both A- and C-fiber nociceptors. These findings show that the thoracolumbar fascia is the deep tissue of the back that is most sensitive to chemical stimulation, making it a prime candidate to contribute to nonspecific LBP but not to localized pressure hyperalgesia., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Proteostasis is the maintenance of the proper function of cellular proteins. Hypertonic stress disrupts proteostasis and causes rapid and widespread protein aggregation and misfolding in the nematode Caenorhabditis elegans. Optimal survival in hypertonic environments requires degradation of damaged proteins. Inhibition of protein synthesis occurs in response to diverse environmental stressors and may function in part to minimize stress-induced protein damage. We recently tested this idea directly and demonstrated that translation inhibition by acute exposure to cycloheximide suppresses hypertonicity-induced aggregation of polyglutamine::YFP (Q35::YFP) in body wall muscle cells. In this article, we further characterized the relationship between protein synthesis and hypertonic stress-induced protein damage. We demonstrate that inhibition of translation reduces hypertonic stress-induced formation and growth of Q35::YFP, Q44::YFP, and α-synuclein aggregates; misfolding of paramyosin and ras GTPase; and aggregation of multiple endogenous proteins expressed in diverse cell types. Activation of general control nonderepressible-2 (GCN-2) kinase signaling during hypertonic stress inhibits protein synthesis via phosphorylation of eukaryotic initiation factor-2α (eIF-2α). Inhibition of GCN-2 activation prevents the reduction in translation rate and greatly exacerbates the formation and growth of Q35::YFP aggregates and the aggregation of endogenous proteins. The current studies together with our previous work provide the first direct demonstration that hypertonic stress-induced reduction in protein synthesis minimizes protein aggregation and misfolding. Reduction in translation rate also serves as a signal that activates osmoprotective gene expression. The cellular proteostasis network thus plays a critical role in minimizing hypertonic stress-induced protein damage, in degrading stress-damaged proteins, and in cellular osmosensing and signaling.

Intravenous hyperosmotic NaCl infusion is an effective treatment for circulatory shock. However, a fast infusion rate (2 mL/kg at the rate of 1 mL/s) induces transient hypotension. This response has been reported to be due to decreased total peripheral resistance and/or decreased cardiac performance. Although the hypotension is transient and recovers within 2 min without detrimental consequences, it is important to understand the associated hemodynamics and mechanisms. We found that the hypotensive effect was larger with intravenous NaCl infusion than with intra-aortic infusion, indicating that change in cardiac performance played a more significant role than change in peripheral resistance. NaCl infusion induced an increase in pulmonary vascular resistance and central venous pressure and a decrease in right ventricular dP/dt max, suggesting acute cor pulmonale. Diastolic ventricular crosstalk-induced left ventricular failure was also observed. Hyperosmotic NaCl-induced hypotension was therefore mainly due to a combination of acute cor pulmonale and left ventricular failure.

Members of the genus Sphingomonas are important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degrading Sphingomonas sp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.

Ethnopharmacological Relevance: A major physiological role of the kidney is to regulate body water and urine concentration. Aquaporin-2 (AQP2), a family of water channels, plays an important role in the urinary concentrating process and regulation of water balance in the kidney. The dried sclerotia of Poria cocos Wolf has been known to have a diuretic effect and used for the treatment of chronic edema and nephrosis., Aim of the Study: This study was conducted to evaluate the inhibitory effect of the sclerotia of Poria cocos (WPC) on hypertonic stress-induced AQP2 expression and apoptosis in inner medullary collecting duct cell lines (IMCD-3)., Materials and Methods: Hypertonic stress was induced by 175mM NaCl. Inhibitory effect of WPC on hypertonic stress-induced AQP2 expression and apoptosis were determined by western blot, RT-PCR, and immunofluorescence., Results: Hypertonic stress (175mM NaCl) increased in the levels of AQP2 expression by hypertonicity in IMCD-3 cells. WPC attenuated the hypertonicity-induced increase in protein and mRNA levels of AQP2 in a concentration-dependent manner. Pretreatment with WPC attenuated hypertonicity-induced cell death. Hypertonicity increased serum- and glucocorticoid-inducible protein kinase (Sgk1) phosphorylation, however, WPC attenuated the hypertonicity-induced Sgk1 activation. Tonicity-responsive enhancer binding protein (TonEBP) mRNA was also recovered by WPC under hypertonic stress. Pretreatment with WPC presented the similar effect of PKA inhibitor which decreased hypertonic stress-induced AQP2 expression. Hypertonicity increased cAMP levels and the changes were blocked by WPC. On the other hand, hypertonic stress-induced Bax or caspase-3 expression was decreased by WPC, resulting in anti-apoptotic effect., Conclusions: These results provided evidence that the beneficial effect of WPC in water balance against in vitro hypertonic stress of renal collecting ducts. In addition, WPC exhibits anti-apoptotic property response to hypertonic stress. Thus, these data suggests that WPC has benefit for the therapeutic approach to the inhibition of renal disorder., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The genus Dermamoeba unifies oblong, flattened amoebae of lingulate morphotype, possessing a thick multilayered cell coat. It includes two species, D. granifera and D. minor. In this paper we describe a third species of this genus, D. algensis n. sp. This species is algivorous; engulfing a large algal cell, it destroys part of the cell coat liberating the plasma membrane, which forms the food vacuole. Thus the glycocalyx never appears inside the phagosome. This observation confirms that some of the thick-coated amoebae may use this way to avoid energetically costly digestion of their own glycocalyx. Studies of the physiology of this organism show that it feeds most actively at a temperature of 22-25 °C. Below and above this temperature the feeding intensity drastically decreases. The new species can survive NaCl concentrations up to 5%, which roughly corresponds to 50 ppt salinity. Accordingly, D. algensis has a wide range of salinity tolerance., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Background and Objectives: Epidural injections are popular therapies for sciatica and low back pain. Local anesthetics and corticosteroids are commonly used for most injections techniques, but some treatments use a specific combination of several agents. The epidural lysis of adhesions procedure (Racz) uses a combination of bupivacaine, hyaluronidase, a corticosteroid, and hypertonic saline. Because severe complications, some with permanent neurologic deficits, have been observed, we considered the possibility that individual agents or a combination thereof might be capable of damaging or destroying cells in primarily the epidural tissues., Methods: We used monolayer cell cultures of human fibroblasts in Dulbecco modified Eagle medium to study these pharmacological agents alone or in combination. Cell viability and proliferation were assessed by Trypan blue staining, cell counts, and the WST-1 assay. Time and concentration series were performed., Results: With the corticosteroid, we observed the previously described proliferation-retarding effects. Hyaluronidase was not found to have a relevant effect on fibroblast proliferation. Bupivacaine and hypertonic saline were found to have a time- and concentration-dependent effect on cell viability and proliferation. Both were found to be toxic at concentrations well below the ones used clinically., Conclusions: We identified a potential for harm caused by commonly used pharmacological agents when applied epidurally. Animal studies will have to show whether the same can be observed in living tissues., (Copyright © 2011 by American Society of Regional Anesthesia and Pain Medicine)

Hyperosmolar factors induce the neurogenic inflammatory response, leading to bladder overactivity (OAB). The aim of the study was to compare the bladder motor activity in a hyperosmolar and acute cyclophosphamide (CYP)-induced model of OAB. Furthermore, we set our sights on defining the most physiological model of OAB in experimental practice. Forty-two female rats were divided randomly into 5 groups. All animals underwent cystometry with the usage of isotonic saline or saline of increasing concentration. Acute chemical cystitis was induced by CYP to elicit OAB. The following cystometric parameters were analyzed: basal pressure, threshold pressure, micturition voiding pressure, intercontraction interval, compliance, functional bladder capacity, motility index, and detrusor overactivity index. CYP and hypertonic saline solutions induced OAB. Having been compared with CYP OAB, none of the rats infused with hypertonic solution exhibited macroscopic signs of bladder inflammation. The comparison of CYP and hyperosmolar models of OAB revealed that the greatest similarity existed between the 2080 mOsm/L OAB model and the acute CYP-induced model. We postulate that the 2080 mOsm/L model of OAB can be established as being a less invasive and more physiological model when compared with the CYP-induced OAB model. Additionally, it may also be a more reliable experimental tool for evaluating novel therapeutics for OAB as compared with CYP-induced models.

Ocular exposure to ultraviolet irradiation (UVR) induces photokeratitis, a common environmental concern that inflames ocular tissues and causes pain. The central neural mechanisms that contribute to the sensory aspects of photokeratitis after UVR are not known. In awake male rats, ocular surface application of hypertonic saline evoked eye wipe behavior that was enhanced 2-3 days after UVR and returned to control levels by 7 days. Similarly, under isoflurane anesthesia, hypertonic saline-evoked activity of ocular neurons in superficial laminae at the trigeminal subnucleus caudalis/cervical (Vc/C1) region was enhanced 2 days, but not 7 days, after UVR. By contrast, the response of neurons at the interpolaris/caudalis (Vi/Vc) transition region to hypertonic saline was not affected by UVR. The background activity and convergent cutaneous receptive field areas of Vc/C1 or Vi/Vc neurons were not affected by UVR. Aqueous humor protein levels were elevated 2 and 7 days after UVR. UVR enhanced nociceptive behavior, after a latent period, with a time course similar to that of ocular neurons in superficial laminae at the Vc/C1 region. The Vc/C1 region plays a key role in primary hyperalgesia induced by UVR, whereas the Vi/Vc region likely mediates other aspects of ocular function., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

The authors describe the case of a 63-year-old, female nursing home inhabitant suffering from trisomy 21, who accidentally ingested the anti-epileptic medication of another nursing home inhabitant. After telephone instructions from a specialist in internal medicine, caregivers forced the woman to vomit by means of saline solution and digital manipulation. This caused not only substantial hypernatriaemia but also aspiration pneumonia, from which the woman died after short hospitalization. The potential toxicity by major electrolyte shifts in terms of hypernatriaemia following administration of sodium chloride solution is well known; this measure is medically contraindicated for the induction of vomiting. The mechanisms leading to death in this case are presented, differentiated and discussed against the background of the literature.

By adjusting the mixing order of ingredients in traditional formula, an innovative bread making process was developed. The effect of salt-stressed Baker's yeast on bread dough of different sugar levels was investigated. Baker's yeast was stressed in 7% salt solution then mixed into dough, which was then evaluated for fermentation time, dough fermentation producing gas, dough expansion, bread specific volumes, and sensory and physical properties. The results of this study indicated that salt-stressed Baker's yeast shortened fermentation time in 16% and 24% sugar dough. Forty minutes of salt stress produced significant amount of gas and increased bread specific volumes. The bread was softer and significantly improved sensory properties for aroma, taste, and overall acceptability were obtained.

Ethanol is a chemical irritant able to induce a large variety of effects in the airways. It has been reported that ethanol sensitizes the transient receptor potential vanilloid-1 (TRPV1) to various stimuli and inhalation of ethanol enhances the cough mediated by TRPV1 activation (capsaicin) in patients suffering of airway sensory hyperreactivity. Here, we set out to investigate whether ethanol sensitizes the cough induced by TRPV1 activation in a guinea pig model and the possible mechanism of such exacerbating effect. Aerosolized resiniferatoxin (RTX, 0.5 microM) and hypertonic saline (7%) produced a cough response dependent and independent of TRPV1 activation, respectively. Ethanol (3%, 10 min) inhalation, that per se did not cause any tussive response, significantly increased the number of coughs evoked by RTX inhalation without affecting hypertonic saline (7%) induced cough. Potentiation by ethanol of the tussive response to RTX was prevented by the PKC inhibitor, GF109203X (GFX). In conclusion, ethanol selectively exaggerates, via a PKC-dependent pathway, the cough response evoked by TRPV1 stimulation. The present results may contribute to explain respiratory distresses sometimes associated to alcohol consumption, including cough and asthma.

Human studies conducted more than half a century ago have suggested that superficial pain induces excitatory effects on the sympathetic nervous system, resulting in increases in blood pressure (BP) and heart rate (HR), whereas deep pain is believed to cause vasodepression. To date, no studies have addressed whether deep or superficial pain produces such differential effects on muscle sympathetic nerve activity (MSNA). Using microneurography we recorded spontaneous MSNA from the common peroneal nerve in 13 awake subjects. Continuous blood pressure was recorded by radial arterial tonometry. Deep pain was induced by intramuscular injection of 0.5 ml hypertonic saline (5%) into the tibialis anterior muscle, superficial pain by subcutaneous injection of 0.2 ml hypertonic saline into the overlying skin. Muscle pain, with a mean rating of 4.9 +/- 0.8 (S.E.M.) on a 0-10 visual analog scale (VAS) and lasting on average 358 +/- 32 s, caused significant increases in MSNA (43.9 +/- 10.0%), BP (5.4 +/- 1.1%) and HR (7.0 +/- 2.0%) - not the expected decreases. Skin pain, rated at 4.9 +/- 0.6 and lasting 464 +/- 54 s, also caused significant increases in MSNA (38.2 +/- 12.8%), BP (5.1 +/- 2.1%) and HR (5.6 +/- 2.0%). The high-frequency (HF) to low-frequency (LF) ratio of heart rate variability (HRV) increased from 1.54 +/- 0.25 to 2.90 +/- 0.45 for muscle pain and 2.80 +/- 0.52 for skin pain. Despite the different qualities of deep (dull and diffuse) and superficial (burning and well-localized) pain, we conclude that pain originating in muscle and skin does not exert a differential effect on muscle sympathetic nerve activity, both causing an increase in MSNA and an increase in the LF:HF ratio of HRV. Whether this holds true for longer lasting experimental pain remains to be seen.

Glaucoma (GL) is an optic neuropathy characterized by progressive loss of visual field due to retinal cell death and optic nerve (ON) degeneration, usually in response to abnormal elevated intraocular pressure (EIOP). It has previously demonstrated that cells of the ON express nerve growth factor (NGF) and NGF-receptors. Relatively little is known, however, about their role on the ON of the glaucomatous eye. The aim of the study was to elucidate this aspect. Using a rat model of GL we investigated the response of NGF and NGF-receptors in the ON of subjects with experimentally induced EIOP. Our results show that EIOP significantly impairs the presence of NGF and NGF-receptor proteins and TrkA gene expression in the ON of glaucomatous eye. These findings suggest that NGF and NGF-receptor might be important signals for the ON response in the EIOP.

In conscious rats implanted with an intracerebroventricular (icv) cannula, effect of icv injections of histamine, chlorpheniramine (H(1)-receptor antagonist) and ranitidine (H(2)-receptor blocker) was investigated in a rat model of acute trigeminal pain. Acute trigeminal pain was induced by putting a drop of 5 M NaCl solution on the corneal surface of the eye and the numbers of eye wipes were counted during the first 30 s. Histamine (20, 40 microg) and chlorpheniramine (80 microg) significantly decreased the numbers of eye wipes. Ranitidine alone had no effect. Pretreatment with chlorpheniramine did not change the histamine-induced analgesia, whereas the histamine effect on pain was inhibited with ranitidine pretreatment. These results indicate that the brain histamine, through central H(2) receptors, may be involved in the modulation of the acute trigeminal pain in rats.

Purpose: To evaluate the effects of hyperosmolar stress on expression of cornified envelope (CE) precursors and transglutaminases (TGs) by primary cultured human corneal epithelial (PCHCE) cells and the regulatory effects of JNK MAPK on this process., Methods: Expression of CE precursors and TGs were evaluated in PCHCE cells exposed to media of increasing osmolarity (350-450 mOsM) for 24, 48, and 72 hours. JNK1 and -2 MAPKs were inhibited by addition of short interfering (si)RNA. Relative levels of mRNA transcripts and proteins were evaluated. TG activity, cell viability, and apoptosis were detected in PCHCE cells, with or without siRNA-JNKs., Results: Exposure of PCHCE cells to hyperosmolar medium increased TG activity at 3 hours, levels of the CE precursors SPRR1b and -2a and membrane-associated TG1 mRNA at 6 hours, and tissue-type TG2 mRNA at 24 hours. Osmotic stress decreased corneal epithelial cell viability, which was due in part to stimulation of apoptosis and cornification death. Inhibiting JNK2 production by siRNA in osmotically stressed PCHCE cells prevented the stimulation of SPRR and membrane-associated TG1 production and TG activity, and improved cell viability, whereas inhibition of JNK1 prevented early apoptosis., Conclusions: Osmotic stress promotes production of certain CE proteins and cross-linking membrane-associated TG1 and decreases cell viability via JNK MAPK-mediated pathways. Strategies that inhibit JNK production downregulate the cornification response of PCHCE cells to osmotic stress. These findings have potential therapeutic implications for preventing cornification of the corneal epithelium in response to the hyperosmolar tear film in dry eye disease.

This article highlights the experimental and clinical data, controversies and postulated mechanisms surrounding osmotherapy with hypertonic saline (HS) solutions in the neurocritical care arena and builds on previous reviews on the subject. Special attention is focused on HS therapy on commonly encountered clinical paradigms of acute brain injury including traumatic brain injury (TBI), post-operative "retraction edema", intracranial hemorrhage (ICH), tumor-associated cerebral edema, and ischemia associated with ischemic stroke.

The present study aimed to investigate whether tonic cutaneous pain exerts any effect on the cortical processing of nociceptive input and if this effect may involve only body parts in pain. Tonic cutaneous pain was obtained in nine healthy human subjects by infusion of a hypertonic saline (5%) in the s.c. tissue over the hypothenar muscles (10 ml/h for 20 min). Nociceptive cutaneous CO2 laser-evoked potentials were recorded after stimulation of the right hand dorsum, which was adjacent to the painful area, and the right perioral region, corresponding to the adjacent cortical sensory area. Laser-evoked potentials were obtained before saline injection, at the peak pain and 20 min after pain disappeared. During saline infusion, the laser-evoked pain to right hand stimulation was reduced and the vertex laser-evoked potentials (N2a-P2, mean latency 181 ms and 319 ms for the N2a and the P2 potentials, respectively), which are generated in the anterior cingulate cortex, were significantly decreased in amplitude compared with the baseline. Moreover, the topography of these potentials was modified by cutaneous pain, shifting from the central toward the parietal region. Dipolar modeling showed that the dipolar source in the anterior cingulate cortex moved backward during saline infusion. This result suggests that cutaneous pain may modify the relative activities of the anterior and posterior anterior cingulate cortex parts, which are thought to be devoted to encode different aspects of pain sensation. No laser-evoked potential change was observed after stimulation of the right perioral region, suggesting that functional changes in the nociceptive system are selective for the painful regions and not for areas with cortical proximity.

Intramuscular injection of hypertonic saline (4-6% NaCl) is widely used to induce muscle pain in volunteers. The quality of the pain is comparable to clinical muscle pain with localised and referred pain. The objective was to evaluate the muscle toxicity of hypertonic saline by characterisation of 1) cytotoxicity in vitro, 2) local muscle toxicity in rabbits and 3) number of spinal dorsal horn neurones expressing c-fos after intramuscular injection in pigs as an indicator of nociception. Rat myocyte cultures and erythrocyte suspensions were treated with hypertonic NaCl solutions. The creatine kinase activity remaining in the myocytes and haemolysis were measured. Groups of six rabbits were given an intramuscular injection of 0.5 ml of 0.9, 3 or 6% NaCl. Three days later, creatine kinase activity was determined in injection site muscle tissue and normal contralateral muscle. The amount of injection site muscle tissue totally depleted of creatine kinase was calculated. Groups of two pigs were given an intramuscular injection of 3.0 ml of 6% NaCl. The spinal cord was sampled 1, 2 or 3 hr later and processed for stereological quantification of the number of dorsal horn neurones expressing c-fos. Saline was not toxic in vitro at 0.9-6%, but toxic to erythrocytes at 7% or higher and rat myocytes at 15% or higher. No muscle toxicity was seen in rabbits. The number of dorsal horn neurones expressing c-fos was not above basal level. In conclusion, 6% saline caused no in vitro or in vivo toxicity in sensitive models. Consequently, the pain caused by intramuscular injection of hypertonic saline is most likely not related to tissue damage. Consistently, intramuscular injection of 6% NaCl did not activate dorsal horn neurones in pigs to express c-fos beyond basal level.

Hypotonic shock rapidly inhibits Cl(-) secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKC alpha and beta subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKC alpha and PKC beta subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 micromol l(-1)) inhibited Cl(-) secretion that was high, increased Cl(-) secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca(2+) and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting on FAK to set levels of NKCC activity.

This study compares the effect of experimental muscle pain on deep tissue sensitivity and force attenuation in the wrist extensors of patients with lateral epicondylalgia (n=20), and healthy controls (n=20) with experimentally induced sensori-motor characteristics simulating lateral epicondylalgia. Delayed onset muscle soreness (DOMS) in wrist extensors of healthy controls was induced by eccentric exercise in one arm 24h prior to injection (Day 0). Saline-induced pain intensity (visual analogue scale, VAS), distribution, and quality were assessed quantitatively in both arms for both groups. Pressure pain thresholds (PPT) were assessed at three different sites in the wrist extensors. Maximal grip force and wrist extension force were recorded. In response to saline-induced pain in the extensor carpi radialis brevis, regardless of arm, the patient group demonstrated a significantly quicker pain onset (P<0.01), mapped larger pain areas and more referred pain areas, compared to healthy controls (P<0.03). Pain persisted significantly longer in the sore arm of the patient group, compared with all other arms (P<0.02). Patients demonstrated significant bilateral hyperalgesia at extensor carpi radialis brevis during and post saline-induced pain compared to pre-injection and healthy controls (P<0.04). The sore arm in patients and the DOMS arms in healthy subjects showed significantly reduced maximal force (P<0.0001), at all Day 1 times compared with the control arms. In patients, the bilateral increase in deep tissue sensitivity and enlarged referred pain areas during saline-induced pain might suggest involvement of central sensitisation.

Tension-type headache is the most common type of primary headaches but no conclusive concept of pathophysiology exists. This may be due to a lack of an appropriate animal model. This study addressed the hypothesis that noxious neck muscle input induces central sensitization of orofacial sensorimotor processing. The effect of hypertonic saline injection into the semispinal neck muscle on the jaw-opening reflex (JOR) was investigated in anaesthetized mice (n = 11). Hypertonic saline injection into the neck muscle facilitated the JOR for at least one hour: integral (+94.5%) and duration (+18.7%) increased, latency decreased (-7.5%). The reflex threshold decreased to 61% after injection. Isotonic saline injection into the neck muscle (n = 11) or hypertonic saline injection into a hindpaw muscle (n = 10) did neither change the reflex integral nor the threshold. Long-term potentiation of the JOR by noxious neck muscle input may be an appropriate model to investigate tension-type headache pathophysiology.

Objective: Saline is not an ideal storage solution. It has a low pH, no buffering capacity, and lacks other ions and nutrients. The objective was to explore the effects of storing cardiac muscle in saline., Design: Guinea pig papillary muscles and ventricular myocytes were exposed to saline. The effects on action potential, membrane current, contraction and cell shortening were recorded in vitro at 35-37 degrees C., Results: Saline caused transient hyperpolarization of the resting potential (-140 mV), prolonged duration of the action potential, and increased contraction amplitude, which was later reversed. The membrane resting potential depolarized after a few minutes to about -15 mV and the preparations became unexcitable. The depolarized preparations remained slightly contracted. Upon reperfusion both papillary muscles and cells became unstable and spontaneously active. Storing myocytes in saline for only 2 h resulted in excessive cell death., Conclusion: Saline is disastrous for the function of the heart muscle and leads to depolarization, sustained contraction and unexcitable tissue. Saline should not be used as a storage medium, even for short periods of time.

In human subjects the triceps surae of one leg was exercised eccentrically by asking subjects to walk backwards on an inclined treadmill. Before the exercise controlled local pressure, applied to the muscle with an electromagnet, produced mild soreness, which was reduced when the pressure was combined with vibration. When delayed-onset muscle soreness (DOMS) had set in, 24-48 h after the exercise, vibration increased pain from local pressure. Vibrating at different frequencies suggested 80 Hz as the optimal frequency. During 2-h testing post-exercise, evidence of a change in character of the effects of vibration was first detected at 6 h. It persisted up to 72 h post-exercise. When muscle pain was generated in an unexercised triceps by injection of hypertonic (5%) saline, controlled local pressure applied to the sore area increased pain levels by 32% while pressure plus vibration reduced this to 11%. In a subject with DOMS, local pressure again increased pain from saline by 32% but combining it with vibration increased pain further by an additional 20%. The effect of vibration on DOMS could be abolished with a large nerve fibre block applied to the sciatic nerve. It is concluded that the vibration effects are the result of stimulation of large-diameter mechanoreceptive afferents in the muscle which, it is speculated, play a role in generating DOMS.

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant "resurrection plants," trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3-10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mgg fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.

Osmotic and ionic balance in the central nervous system is especially regulated by solutes and water transport across the blood-brain barrier. The aim of this study was to investigate the effect of systemic hyperosmolarity on the blood-brain barrier permeability in both sexes after experimentally induced seizures. Eight groups of rats were studied: Group I: female control; Group II: male control; Group III: hyperosmotic female; Group IV: hyperosmotic male; Group V: non-hyperosmotic female + seizure; Group VI: non-hyperosmotic male + seizure; Group VII: hyperosmotic female + seizure; Group VIII: hyperosmotic male + seizure. In female rats with pentylenetetrazol-induced seizures, the extravasation of Evans-blue was greater in the brains of hyperosmotic animals than that in normal rats (P < 0.02). However, in male rats, the extravasation of Evans-blue was similar in the brains of hyperosmotic male rats and normal rats after seizure. Our results concluded that hyperosmotic female rats were shown to have a large increase in blood-brain barrier permeability in comparison to hyperosmotic male rats after pentylenetetrazol-induced seizures., (Copyright 2001 Academic Press.)

Fast-repairing potentially lethal damage (PLD) in seven osteosarcoma cell lines was analyzed after treatment with a hypertonic 0.5 M NaCl solution for 20 min and compared to that in seven human fibroblast strains. Fixation of PLD after exposure to ionizing radiation was observed without exception in both the osteosarcoma cells and the fibroblast strains. The percentages by which the D(o)'s of the osteosarcoma cells decreased were significantly higher than the percentage decreases in the C(o)'s of the fibroblast strains (P < 0.01). Hypertonic treatment resulted in radiosensitization due to fixation of PLD in all of the osteosarcoma cell lines, demonstrating that osteosarcoma cells can repair PLD better than normal fibroblast cells. The radiobiological response of the osteosarcoma cells, with enhanced killing after hypertonic treatment, was similar to that of normal untreated fibroblast cells.

Purpose: To determine the diural intraocular pressure (IOP) response of Brown Norway rat eyes after sclerosis of the aqueous humor outflow pathways and its relationship to optic nerve damage., Methods: Hypertonic saline was injected into a single episcleral vein in 17 animals and awake IOP measured in both the light and dark phases of the circadian cycle for 34 days. Mean IOP for light and dark phases during the experimental period were compared with the respective pressures of the uninjected fellow eyes. Optic nerve cross sections from each nerve were graded for injury by five independent masked observers., Results: For fellow eyes, mean light- and dark-phase IOP was 21 +/- 1 and 31 +/- 1 mm Hg, respectively. For four experimental eyes, mean IOPs for both phases were not altered. Six eyes demonstrated significant mean IOP elevations only during the dark phase. Of these, five showed persistent, large circadian oscillations, and four had partial optic nerve lesions. The remaining seven eyes experienced significant IOP elevations during both phases, and all had extensive optic nerve damage., Conclusions: Episcleral vein injection of hypertonic saline is more likely to increase IOP during the dark phase than the light. This is consistent with aqueous outflow obstruction superimposed on a circadian rhythm of aqueous humor production. Because these periodic IOP elevations produced optic nerve lesions, both light- and dark-phase IOP determinations are necessary for accurate correlation of IOP history to optic nerve damage in animals housed in a light- dark environment.

Salt loading on pigeons (C. livia) had stimulatory effects on brain amines (dopamine and 5-hydroxytryptamine), corticosterone, norepinephrine and epinephrine contents of adrenal gland. Conjoint administration of dopamine with hypertonic saline restored the brain amines and corticosterone of adrenal gland, but had no effect on catecholamine (CAM) contents of adrenal medulla. The excessive release of CAM in the plasma indicates sympathetic stimulation after both the treatments.

Objective/hypothesis: Physiologic saline (NaCl 0.9%) is commonly used in treating acute and chronic rhinosinusitis. Moreover, physiologic saline is used as a control medium, vehicle, or solvent in studies on ciliary beat frequency (CBF). Hypertonic saline (NaCl 7% and 14.4%) has been applied in attempts to enhance mucociliary transport in patients with cystic fibrosis or asthma and in healthy subjects. Therefore the objective of this study is to document in vitro effects of saline solutions in different concentrations on CBF., Study Design: Experimental, in vitro., Methods: The effects on CBF of cryopreserved mucosa of the sphenoidal sinus was measured by a photoelectrical method. Initial frequencies, measured in Locke-Ringer's solution (LR), were compared with CBF after exposure to NaCl in concentrations of 0.9%, 7.0%, and 14.4% (w/v)., Results: NaCl 0.9% has a moderately negative effect on CBF. The 7% solution leads to a complete ciliostasis within 5 minutes, although this effect turns out to be reversible after rinsing with LR. A hypertonic solution of 14.4% has an irreversible ciliostatic effect., Conclusion: LR is an isotonic solution that has no effect on CBF. Therefore it is probable that this solution is more appropriate than saline for nasal irrigation and nebulization or antral lavage. Moreover, the results of this study suggest that mucolytic effects induced by hyperosmolarity should be attained preferably with hypertonic saline 7% in patients with cystic fibrosis or asthma. At this concentration, the ciliostatic effect is reversible, whereas irreversible changes are to be expected at higher concentrations.

The modifying effects of Chelidonium majis L. (Papaveraceae) herb extract (CH), an analgesic traditionally prescribed for gastric and duodenal ulcer patients, on gastric tumor development were studied in rats given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sixty-four male 6-week-old Wistar rats were used. Group 1 rats were initially given MNNG (200 mg/kg b.w.) by gavage at days 0 and 14 as well as saturated sodium chloride solution (S-NaCl, 1 ml per rat) every 3 days during weeks 0-3 (six times), and then placed on basal diet containing 0.1 or 0.2% CH for 16 weeks from week 4. Rats of Group 2 and 3 were treated with MNNG together with S-NaCl or saline (0.9% NaCl, 1 ml per rat), respectively, timed as in Group 1 but without further treatment. All surviving animals were killed at week 20 and histopathologically investigated. In the glandular stomach, the number of preneoplastic pepsinogen 1 altered pyloric glands (PAPGs) in the MNNG + S-NaCl-->CH (0.1%) group (Group 1) was significantly smaller than in the MNNG + S-NaCl group (Group 2) (P < 0.02). The incidences of forestomach neoplastic lesions (papillomas and squamous cell carcinomas) also showed a tendency to decrease with the CH treatment. The results thus indicate that CH exerts inhibitory effects on glandular stomach carcinogenesis in the rat, so that it may have potential as a chemopreventive agent for stomach cancer in man.

The effect of aerosol challenge with distilled water, and with isotonic and hypertonic saline on the respiratory system of the anaesthetized rabbit was investigated. Nebulisation of hypertonic (3.6%) saline caused an increase in the extravascular lung water without altering the total body weight. Morphometrical investigations revealed an increase of the subepithelial tissue compartment (connective tissue and smooth muscle) of the airway wall. X-ray microanalysis showed higher content of Na, K, and Cl in this compartment already 10 min after nebulisation of hypertonic saline. The formation of oedema was associated with a significant decrease in both compliance and gas exchange.

The present study evaluated the induction of micronuclear potency of indigenously developed bone wax saline extract. The experiment was designed with 2 batches, each consisting of 8 groups with 3 male Swiss albino mice each. In each batch, the first 3 groups received Chitra's bone wax saline extract and the second 3 groups received Johnson & Johnson bone wax saline extract at 12.5, 25.0 or 50.0 ml/kg body weight ip for 2 d. The remaining 2 groups were vehicle (saline) and positive (cyclophosphamide) controls. Animals were sacrificed 24 and 36 h after the second dosing and bone marrow smears were prepared and evaluated for micronuclei. The Chitra's and Johnson & Johnson's bone wax extracts failed to induce micronuclei in bone marrow erythrocytes of mice.

Background & Aims: Gastric mucosal hyperemia is a protective response mediated at least in part by the response of sensory afferent neurons to hydrogen ions. The aim of this study was to determine if other pathways to the hyperemic response are present and if these neurons have an effect exclusive of hyperemia on mucosal protection and repair., Methods: Rat sensory afferent neurons were ablated by capsaicin treatment. Chambered stomachs were damaged by hypertonic saline followed by either acidic or neutral isotonic saline. Blood flow was measured by laser Doppler velocimetry, and mucosal morphology was quantitatively evaluated by microscopy., Results: Mucosal damage alone evoked a strong hyperemic response in both control and ablated rats. Ablated rats lost gastric protection despite this hyperemic response. Acid exposure after damage sustained the hyperemic response. Rapid epithelial restitution occurred faster (even over hemorrhagic lesions) in control rats., Conclusions: The hyperemic response to mucosal damage alone is not mediated by sensory neurons. Protection of the stomach by sensory afferent neurons occurs by mechanisms also unrelated to their elicitation of hyperemia. Restitution during acid challenge is enhanced by the sustained hyperemic response mediated through sensory afferent neurons.

Syngeneically-pregnant CBA mice were stressed by injecting them with saline, sc twice daily, on days 10-18 of their pregnancy. Pups born to these mothers exhibited disturbances in their immuno-regulatory cell development. Splenocytes harvested from these pups, when one day old, failed to suppress xenogeneic Graft-versus-Host reaction and proliferative response of syngeneic mature cells to concanavalin-A, as control splenocytes did. Also, thymocytes of 4-6-day-old mice, born to stressed mothers, did not exert an inhibitory effect on neonatal suppression, as control thymocytes did. These results demonstrate that neonatal stress is an useful physiological model for studying interactions between some regulatory systems of the organism, in its early stages of development.

Intragastric administration of 3.4 M NaCl damages the gastric mucosa and increases the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Polyamines are essential for the repair of gastric erosions. Little is known about the restitution of damaged mucosa except that cell migration is essential. Actin is the principal cytoskeletal protein and is essential for migration. This investigation determines the relationship between polyamines, actin, and gastric healing. Rats were fasted for 22 h and given 1.0 ml of 3.4 M NaCl intragastrically and killed 1, 2, 4, 8, and 10 h later. The mucosa was assayed for ODC activity and stained for G- and F-actin. F-actin was concentrated below the damaged mucosa at 1.5, 2, and 4 h. There was no increase in F-actin distribution at any time point, when NaCl-treated animals were given alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC. In addition, DFMO significantly prevented the healing of the mucosal lesions. Spermidine treatment after DFMO + NaCl significantly prevented the effects of DFMO. Cytochalasin D, a potent actin-disrupting drug, significantly delayed normal gastric mucosal healing. The endogenous polyamines increased significantly in NaCl animals. Data indicate that increases in polyamine synthesis after damage influence the distribution of F-actin in vivo, which may play a part in the healing of mucosal erosions.

Background: Gastric mucosal adaptation to injury induced by repeated application of aspirin (ASA) or stress is a well-documented phenomenon, but it is known whether such adaptation affects the mucosal tolerance to other strong irritants., Methods: In this study gastric adaptation was induced by repeated daily administration of acidified ASA for 4 consecutive days (Series A) or by 3.5H of water immersion and restraint stress (WRS) applied every other day for up to 8 days (series B). When the adaptation to ASA or WRS was fully developed, rats of series A and B were challenged with strong irritants such as 100% ethanol, 200 mM acidified taurocholate (TC), or 25% NaCl for 1 h or with WRS for 3.5 h., Results: ASA or WRS applied once produced numerous gastric lesions and deep histologic necrosis accompanied by a decrease in gastric blood flow. With repeated application of ASA or stress the mucosal adaptation to ASA and WRS developed; the area of gastric lesions was reduced by 86% and 56%, respectively, and this was accompanied by a marked decrease of superficial and deep necrosis, and increase in gastric blood flow (GBF) and the enhancement of mucosal regeneration. An increase in mucosal and luminal contents of epidermal growth factor (EGF) and in mucosal expression of EGF receptors was also observed in the mucosa adapted to ASA or stress. In rats adapted to ASA or stress and then challenged with 100% ethanol, 200 mm TC, 25% NaCl, stress or ASA, the areas of macroscopic gastric lesions and deep histologic necrosis were remarkable reduced as compared with those in non-adapted vehicle-treated rats. This was also accompanied by a significant decrease in (GBF), a marked increase of mucosal and luminal contents of EGF and expression of its receptors, and enhanced mucosal cell proliferation., Conclusions: Gastric adaptation to ASA or stress enhances mucosal resistance to the injury induced by strong irritants, and this appears to be mediated by mucosal regeneration, probably resulting from increased luminal and mucosal contents of EGF and excessive expression of its receptors.