Effect of a commercial anionic dietary supplement on urinary pH and concentrations of electrolytes and pH in blood of horses.

Aims: To compare urine urinary pH, blood pH and concentration of electrolytes in blood of healthy horses fed an anionic salt supplement to achieve diets with a dietary cation-anion difference (DCAD) of -40 or 0 mEq/kg DM, with horses a fed a diet with a DCAD of 85 mEq/kg DM.Methods: Eight healthy horses received each of three diets in a randomised crossover design. Diets consisted of grass hay and concentrate feed, with a varying amount of an anionic supplement to achieve a DCAD of 85 (control), 0 or -40 mEq/kg DM. They were fed for 14 days each with a washout period of 7 days between. Urine pH was measured daily and blood samples were collected on Days 0, 7 and 14 of each study period for the measurement of pH and concentration of electrolytes.Results: Four horses voluntarily consumed the anionic supplement with their feed, but four horses required oral supplement administration via dose syringe. During the study period mean urine pH was lower in horses fed diets with a DCAD of 0 (6.91; SD 0.04) and -40 (6.83; SD 0.04) mEq/kg DM compared to the control diet (7.30; SD 0.04). Compared with horses fed the control diet, mean urine pH was lower in horses fed the 0 and -40 mEq/kg DM diets on Days 1-12 and 14 (p < 0.05) of the study period. On Day 13 it was only lower in horses fed the -40 mEq/kg DM diet (p < 0.01). Urine pH was similar for horses fed the 0 and -40 mEq/kg DM diets (p = 0.151). The DCAD of the diet had no effect on blood pH, ionised Ca or anion gap. Mean concentrations of bicarbonate in blood were affected by diet (p = 0.049); they were lower when horses were fed the 0 mEq/kg diet relative to the control diet on Day 14.Conclusions and clinical relevance: The anionic supplement reduced urine pH in horses fed diets with a DCAD of 0 or -40 mEq/kg DM compared with 85 mEq/kg DM. However as urinary pH did not fall below pH 6.5, the pH below which calcium carbonate uroliths do not form, this reduction in urine pH is unlikely to be clinically significant. The supplement was variably palatable and showed minimal promise as an effective urinary acidifier at the doses administered in this study.

Evaluation of updated sepsis scoring systems and systemic inflammatory response syndrome criteria and their association with sepsis in equine neonates.

BACKGROUND: The original equine sepsis score provided a method of identifying foals with sepsis. New variables associated with sepsis have been evaluated, but the sepsis score has not been updated. OBJECTIVES: To evaluate the sensitivity and specificity of 2 updated sepsis scores and the systemic inflammatory response syndrome (SIRS) criteria in regard to detecting sepsis in foals. ANIMALS: Two-hundred and seventy-three ill foals and 25 healthy control foals. METHODS: Historical, physical examination, and clinicopathologic findings were used to calculate the original sepsis score and 2 updated sepsis scores. SIRS criteria were also evaluated. Sepsis scores and positive SIRS scores were statistically compared to foals with sepsis. RESULTS: One-hundred and twenty-six foals were septic and 147 sick-nonseptic. The original and updated sepsis scores were significantly higher in septic foals as compared to sick-nonseptic and healthy foals. The sensitivity and specificity of the updated sepsis scores to predict sepsis were not significantly better than those of the original sepsis score. One-hundred and twenty-seven of 273 (46.5%) foals met the original SIRS criteria and 88/273 (32%) foals met the equine neonatal SIRS criteria. The original SIRS criteria had similar sensitivity and specificity for predicting sepsis as did the 3 sepsis scores in our study. CONCLUSIONS AND CLINICAL IMPORTANCE: The updated sepsis scores did not provide improved ability in predicting sepsis. Fulfilling the original SIRS criteria provided similar sensitivity and specificity in predicting sepsis as the modified sepsis score and might serve as a diagnostic aid in identifying foals at risk for sepsis.

Mechanistic basis for unexpected bioavailability enhancement of polyelectrolyte complexes incorporating BCS class III drugs and carrageenans.

The objective of this study was to investigate the potential of λ-carrageenan to work as an absorption modifying excipient in combination with formulations of BCS class 3 substances. Trospium chloride was used as a model BCS class 3 substance. Polyelectrolyte complexes of trospium and λ-carrageenan were produced by layer-by-layer complexation. A λ-carrageenan-containing formulation was administered either in capsules size 9 to rats by gavage or directly into ligated intestinal loops of rats. Exceptionally strong variations were observed in the plasma concentrations of the rats that received λ-carrageenan compared to the control group, but enhanced plasma concentrations were observed only in some of the rats. In vitro permeability studies were performed across Caco2-monolayers and across excised segments of rat jejunum in a modified Ussing chamber to learn more about the mechanism of absorption enhancement. The complex did not show any effect in Caco2-cells, but led to a major enhancement of permeability across excised segments in modified Ussing chambers. Carrageenan did not lead to alterations of tight junctions. The bioavailability enhancing effect thus was most likely due to an interaction of the polyelectrolyte-drug complex with the mucus, which provided an intimate contact between the drug and the absorbing surface. A similar effect was also achievable with other types of carrageenan and was also transferable to other compounds. In conclusion, λ-carrageenan-drug complexes show interesting excipient-drug-epithelium interactions - however, for full utilization of the permeation enhancing potential, an intimate and reproducible contact between absorbing epithelia and the complex is needed.

When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain.

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cystophora cristata remain much longer active in hypoxic conditions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cytochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O(2) affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclusively in neurons, whereas in seals neuroglobin is mainly located in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives.

Massive pulmonary thromboembolism in six horses.

This report involves 6 cases in which medical records and post mortem findings were reviewed leading to the diagnosis of massive pulmonary thromboembolism (MPTE). All horses were mature and MPTE has not been recognised previously as a sequel to generalised systemic illness in mature horses. The clinical data and pathological findings of the cases are reported and the authors conclude that MPTE is an uncommon but important complication of medical and surgical disorders in mature horses. In 3 of the cases, the condition was nonfatal suggesting that some horses having developed PTE survive and the condition may not be recognised in such cases. The incidence of the condition might be higher than supposed.

Suspension packaging cell lines for the simplified generation of T-cell receptor encoding retrovirus vector particles.

The transfer of T-cell receptor (TCR) genes into primary human T-cells to endow their specificity toward virus-infected and tumor cells is becoming an interesting tool for immunotherapy. TCR-modified T cells are mainly generated by retrovirus-mediated gene transfer. To produce TCR-retrovirus particles, fibroblast packaging cell lines are the most common tool. We constructed two packaging cell lines based on the human suspension T-cell lymphoma line Deltabeta-Jurkat, which lacks endogenous TCRbeta-chains and is therefore unable to express CD3 complexes on the cell surface. After supply of gag-pol (murine leukemia virus (Mo-MLV)) and env (GALV or MLV-10A1) genes, a green fluorescent protein (GFP)-encoding retrovirus vector was transduced into both packaging cell clones, which then stably produced GFP-retroviruses with titers of up to 4 x 10(5) infectious particles (IP)/ml. After transfer of a TCRalpha/beta-encoding retrovirus vector, Deltabeta-Jurkat/GALV and Deltabeta-Jurkat/10A1 cells expressed CD3 molecules on the cell surface. CD3-high expressing packaging cells were enriched by fluorescence-activated cell sorter sorting. In these cells, the CD3 expression level directly correlated with the titer of vector particles. TCR-retroviruses efficiently transduced human T-cell lines and primary T cells. In conclusion, the method allowed the fast and easy generation of high virus titer supernatants for TCR gene transfer.

Contribution of Ca2+-activated K+ channels to hyperpolarizing after-potentials and discharge pattern in rat supraoptic neurones.

The contribution of Ca(2+)-activated K(+) channels to hyperpolarizing after-potentials (HAP) of action potentials, to spike-frequency adaptation and thus to the shaping of discharge pattern, was examined in rat supraoptic magnocellular neurosecretory cells. In addition, the expression of BK channels and SK3 subunits of SK channels was studied using double immunofluorescence detection. The presence of BK channels and SK3 subunits was detected in many supraoptic neurones containing either vasopressin or oxytocin. Current-clamp recordings of current-induced spike trains revealed that HAPs comprise a fast and a slow HAP (fHAP and sHAP). Correlation analyses revealed that the increase of the fHAP in amplitude and spike broadening were correlated to a moderate gradual increase of the interspike interval and thus to weak spike-frequency adaptation. By contrast, marked prolongation of the interspike interval and strong spike-frequency adaptation depended on the appearance and on the amplitude of the sHAP. The sHAP and spike-frequency adaptation were blocked by cadmium, as well as by the SK channel antagonist apamin. The fHAP was attenuated by the BK channel antagonist iberiotoxin (IbTX), by the BK/IK channel antagonist charybdotoxin (ChTX) and by apamin. ChTX attenuated fHAPs throughout the entire spike train. By contrast, the IbTX-induced attenuation of the fHAP was restricted to the initial part of the spike train, while the apamin-induced attenuation slowly increased with the progression of the spike train. These results suggest that strong spike-frequency adaptation in supraoptic neurones essentially depends on the generation of the sHAP by activation of SK channels. Comparison of effects of IbTX, ChTX and apamin suggests a complementary contribution of SK-, BK- and IK-channels to fHAPs.

Acute moderate hyponatraemia and its rapid correction: effects on striatal and pontine ultrastructure in an animal model of the TURP syndrome.

BACKGROUND AND OBJECTIVE: To investigate the effects of moderate hyponatraemia, induced by intravenous application of an electrolyte-free irrigation fluid, as a model of the human transurethral prostate resection syndrome and of its rapid correction by hypertonic saline infusion in rats. METHODS: Experimental animals received irrigation fluid (Purisole SM) 20 mL kg(-1) body weight, intravenously. In one group, hyponatraemia was subsequently rapidly corrected by infusion of hypertonic saline (NaCl 5.85%), while rats of group two were 'sham-corrected' by infusion of a balanced salt crystalloid solution. Plasma sodium concentrations were analysed during and at the end of the experiments. After 10 days, experimental and untreated control animals were killed humanely, fixed by perfusion and the brains were prepared for electron microscopic investigation of myelin sheets and glial cell numbers in the striatum and pons. RESULTS: The myelin appearance was unaltered in experimental groups compared to controls, but glial cell numbers were distinctly altered in the pons but not in the striatum. In the pons, oligodendrocytes were significantly reduced in number upon rapid correction of hyponatraemia, while astrocyte numbers were increased in rats with uncorrected hyponatraemia. CONCLUSIONS: Our electron microscopic data demonstrate that the effects of hyponatraemia and of its rapid correction are multifarious in animals. This may also apply for human patients during transurethral prostate resection.

Expression and regulation of mPer1 in immortalized GnRH neurons.

Hypothalamic GnRH (gonadotropin-releasing hormone) neurons play a critical role in the initiation and maintenance of reproduction competence. Using the mouse GnRH neuronal cell line, GT1-7, we have characterized the expression of the gene mPer1, a recognized key element of the mammalian circadian clockwork. Both mPer1 transcripts and the 136 kDa mPER1 gene product could be detected in these cells. Immunocytochemical analysis also confirmed expression of mPER1 both in vitro and in vivo in GnRH neurons. Activation of cyclic AMP signalling pathways in vitro elevated GnRH secretion as well as mPer1 expression and nuclear mPER1 immunoreactivity. As mPER1 is known to feedback on transcriptional activities in many cell models, the data presented here point to a role for mPER1 in the regulation of gene expression in GnRH neurons, and thus in the control of neuroendocrine activities.

Expression analysis of neuroglobin mRNA in rodent tissues.

Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the first detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting differential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is sufficient for its putative primary function as an oxygen-supplying protein.

Nitric oxide synthase neurons in the rodent spinal cord: distribution, relation to Substance P fibers, and effects of dorsal rhizotomy.

The indirect immunofluorescent method was employed to investigate the distribution of neuronal nitric oxide synthase-like immunoreactivity(nNOS-LI) in the spinal cord of the golden hamster and to compare it to data obtained from rats. Immunoreactive neurons were found throughout the cervico-sacral extent in the dorsal horn (mainly in laminae I-III) and in the preganglionic autonomic regions, i.e., the sympathetic intermediolateral nucleus (IML), lateral funicle (LF), intercalated region (IC), the area surrounding the central canal (CA), and the sacral preganglionic parasympathetic cell group. While the distribution of immunoreactive cells was generally similar in both species, some differences were observed. For example in the hamster LF, a higher percentage of stained neurons was seen than in the IML, while the situation was rather inverse in the rat. In order to study the coincidence of nNOS-LI in the population of preganglionic sympathetic neurons (PSN) that innervate the superior cervical ganglion (SCG), these were identified by retrograde axonal transport of fluoro-gold (FG) following unilateral injection into the SCG. PSN were localized ipsilateral to the injection site mainly in the IML and LF of spinal segments C7-Th4. The portion of double-labeled neurons of the IML were lower in hamster (17% in C7, 34% in C8) of FG-labeled cells) than in rat (47% in C8, 77% in Th2), while in the LF of segments C8-Th2 in both species the majority of FG-neurons contained nNOS. While only very few double-labeled neurons were detected in the IC in hamster and rat, a striking difference was observed in the CA, where no double-labeled neurons were found in hamster, but up to 50% in rat. Double immunofluorescence detection of nNOS and substance P (SP) showed that in both the autonomic regions and the dorsal horn, SP-LI fibers and puncta were present in close spatial relationship to nNOS-LI cell bodies. These results were basically identical in the hamster and rat. Unilateral transection of the dorsal roots of segments C6-Th2 in rats resulted in a clear reduction of SP-LI structures in the dorsal horn 5 days after rhizotomy, but not in the autonomic regions. Compared to the unlesioned side, the numbers of nNOS-LI neurons in the superficial laminae of the dorsal horn were reduced to 32-46% in the lesioned segments, and to 53% and 87%, respectively, in the two segments cranial to the rhizotomized segments but remained unchanged caudally to the lesion. Numbers of nNOS-LI cell bodies in the autonomic regions were not altered following dorsal root transection. The present study provides data on the widespread distribution of nNOS in the spinal cord of golden hamster and describes the partial coincidence of the enzyme in PSN. The effects of dorsal rhizotomy on nNOS-LI neurons in the dorsal horn reveal that primary-afferent fibers provide a stimulatory influence on neurons of the dorsal horn to generate the gaseous neuroactive substance, nitric oxide.

Distribution and projections of nitric oxide synthase neurons in the rodent superior olivary complex.

The superior olivary complex (SOC), a group of interrelated brainstem nuclei, sends efferents to a variety of neuronal structures including the cochlea and the inferior colliculus. The present review describes data obtained from rodents providing evidence that the gaseous, short-living neuroactive substance nitric oxide (NO) is produced in the SOC. The NO-synthesizing enzyme neuronal NO-synthase (nNOS) has been localized by means of several methods including histochemistry and immunohistochemistry. Perikarya containing nNOS were found in several nuclei of the SOC. Their largest numbers and percentages of total cells were observed in the medial nucleus of the trapezoid body. Stained terminals were observed mainly in the lateral superior olivary nucleus and in the superior paraolivary nucleus. While retrograde neuronal tracing identified a considerable number of nNOS-immunoreactive neurons as to be part of the olivo-cochlear pathway, the projection patterns of other nNOS-immunoreactive SOC cell groups remain to be investigated. We also review other putative sources of cochlear NO, and discuss the possible role of NO in the lower auditory brainstem and organ of Corti with regard to physiological and pathophysiological mechanisms.

Anterograde tracing of retinal afferents to the tree shrew hypothalamus and raphe.

The anterograde neuronal transport of Cholera toxin B subunit (CTB) was used in this study to label the termination of retinal afferents in the hypothalamus of the tree shrew Tupaia belangeri. Upon pressure-injection of the substance into the vitreous body of one eye, a major projection of the retinohypothalamic tract (RHT) was found to the hypothalamic suprachiasmatic nuclei (SCN). Although the innervation pattern was bilateral, the ipsilateral SCN received a somewhat stronger projection. Labeling was also found in the supraoptic nucleus and its perinuclear zone, respectively, mainly ipsilaterally as well as in the bilateral para- and periventricular hypothalamic regions without lateral predominance. In the raphe region, scattered fibers and terminals were seen in the dorsal and median raphe nuclei. CTB-immunoreactive structures were observed neither in the locus ceruleus nor in vagal nuclei. Our results, partly in contradiction to earlier studies using different tracing techniques in another tree shrew species (Tupaia glis), reveal that hypothalamic nuclei, in particular the SCN, are contacted by retino-afferent fibers which are thought to mediate the effects of light to the endogenous 'clock' and to parts of the neuroendocrine system.

Induced hypothermia in experimental pneumococcal meningitis.

Pneumococcal meningitis resulting from Streptococcus pneumoniae has a death rate of 28% in adults. In severe head injury and stroke, inflammatory changes and intracranial hypertension are improved by induced hypothermia, which also is neuroprotective. We hypothesized that moderate hypothermia ameliorates inflammatory changes in experimental pneumococcal meningitis. Wistar rats were cooled systemically, and meningitis was induced by pneumococcal cell wall components. The increase of regional cerebral blood flow in the meningitis animals was blocked by hypothermia at 6 hours. The reduction of intracranial pressure correlated with temperature. The influx of leukocytes into the cerebrospinal fluid and levels of tumor necrosis factor alpha in the cerebrospinal fluid were decreased. Cooling the animals 2 hours after meningitis induction to 30.5 degrees C was also protective. We conclude that hypothermia is a new adjuvant approach to reduce meningitis-induced changes, in particular intracranial pressure, in the early phase of the disease.

Evidence for increased nitric oxide production in the auditory brain stem of the aged dwarf hamster (Phodopus sungorus): an NADPH-diaphorase histochemical study.

Age-related changes of the auditory system such as presbyacusis are believed to be due, at least in part, to alterations of central structures. The superior olivary complex (SOC), a group of interrelated brain stem nuclei, projects to a variety of neuronal structures including the cochlea and the inferior colliculus (IC). The soluble gas nitric oxide (NO), believed to function as a neuroactive substance within the SOC and cochlea, is thought to be involved in ageing processes. Since it is unknown whether NO-production is altered in the ageing auditory system, the present study was conducted to investigate whether the number of NO-producing cells in the SOC is changed with increasing age. The histochemical detection of NADPH-diaphorase activity (NADPH-d), a marker for neurons containing NOS, was utilized to investigate the numbers of NO-producing cells in the SOC of adult and senile Djungarian dwarf hamsters (Phodopus sungorus). Our results demonstrate that the number of stained neurons was almost doubled in the SOC of senile hamsters. The most distinct changes were observed in the medial nucleus of the trapezoid body. In contrast, NO-producing preganglionic sympathetic neurons of the spinal intermediolateral nucleus, which was studied for comparison, did not exhibit significant differences between adult and senile animals. It is concluded that the increase of NO-production in the ageing auditory brain stem, as revealed by our data, may be related to hearing impairments with increasing age.

Nitric oxide synthase in trigeminal ganglion cells projecting to the cochlea of rat and guinea pig.

Nitric oxide (NO) influences electrophysiological and morphological parameters of the mammalian cochlea. Recently, the isoform of the NO-producing enzyme neuronal NO synthase (nNOS) has been demonstrated in spiral ganglion cells and olivocochlear neurons. The cochlea is also innervated by fibers stemming from the trigeminal ganglion (TG) and superior cervical ganglion (SCG). Whether these ganglion cells contain nNOS is not known yet. We therefore identified TG and SCG cells upon injection of Fluoro-Gold (FG) into the cochlea and retrograde neuronal transport of FG in rat and guinea pig. These ganglion cells were investigated for neuronal NOS immunohistochemically. Perikarya labeled by FG were found in the ipsilateral TG and SCG. In both species investigated, a considerable number of FG-labeled TG cells were also nNOS-immunoreactive whereas SCG cells were not. These data, demonstrating the existence of nNOS-containing TG cells that project to the cochlea, provide evidence that these neurons are further sources of nitric oxide in the cochlea.

Nitric oxide synthase in identified olivocochlear projection neurons in rat and guinea pig.

Nitric oxide (NO) is thought to be involved in the effects of amino acids at the level of cochlear hair cell afferents. Recently, the isoform of the NO-producing enzyme, neuronal NO synthase (nNOS), has been demonstrated in neuronal structures of the cochlea in rats and guinea pigs histochemically and immunohistochemically. To investigate the sources of cochlear NO, we injected Fluoro-Gold (FG) into the cochlea of rats and guinea pigs. Upon terminal uptake of the tracer and neuronal transport we observed FG in terminals at the base of inner (IHC) and outer hair cells (OHC) and in neurons of the spiral ganglion. Ganglion cells and terminals at the IHC were clearly nNOS-positive, while terminals at the OHC exhibited nNOS-immunoreactivity to a minor degree. The immunohistochemical investigation of the auditory brainstem showed that about one-fourth of the neurons of the superior olivary complex contained nNOS. The comparison with retrogradely labeled neurons showed that perikarya in the lateral superior olivary nucleus and, in particular, the medial nucleus of the trapezoid body were double-labeled. These results were similar in both, rat and guinea pig. Our data reveal that neurons of the superior olivary complex are likely to be additional sources of neuronal NOS in the cochlea.