NaCl Injection Research Articles

The Effect of CRH, Dexamethasone and Naltrexone on the Mu, Delta and Kappa Opioid Receptor Agonist Binding in Lamb Hypothalamic-Pituitary-Adrenal Axis.

The aim of the study was to evaluate changes in the opioid receptor binding (mu, delta and kappa) in the hypothalamus, anterior pituitary and adrenal cortex (HPA) of lambs treated in vivo with corticotrophin releasing hormone (CRH), naltrexone, an opioid receptor antagonist (NAL), and dexamethasone, a potent cortisol analog (DEX). Experiment was carried out on 3 months old female lambs of polish mountain strain. Lambs received a single i.v. injection of NaCl (control), CRH (alone or in combination with naltrexone), naltrexone or dexamethasone. One hour later animals were decapitated under anaesthesia, tissues were dissected out and receptor binding assays were performed with radioligands for each type of opioid receptors--3H-DAGO, 3H-DPDPE and 3H-EKC for mu, delta and kappa receptor, respectively. Coexistence of specific binding sites for each type of opioid receptor was demonstrated in all levels of HPA axis of control lambs, however their distribution was uneven. Acute treatment with CRH, DEX and NAL caused downregulation or upregulation of mu, delta, kappa receptor binding in each level of HPA axis. CRH effects on mu, delta and kappa opioid receptor binding varied within the HPA axis and were modulated by naltrexone. Treatment with naltrexone increased in vitro mu, delta and kappa receptor binding in most tested structures except delta receptor binding in adrenal (decrease by 52%) and kappa receptor binding in pituitary (decrease by 41%). Dexamethasone significantly decreased the mu, delta and kappa opioid receptor binding in adrenal cortex but differentially affected opioid receptor binding in hypothalamus and pituitary. It seems probable that endogenous opioid peptides acting through mu, delta and kappa receptors interact with the hormones released from the hypothalamic-pituitary-adrenal axis in physiological and pathophysiological situations.

Increased brain L-arginine availability facilitates cutaneous heat loss induced by running exercise.

The effects of increased brain availability of L-arginine (L-arg), a precursor for nitric oxide synthesis, on core body temperature (Tcore ) and cutaneous heat loss were evaluated in running rats. One week prior to the experiments, adult male Wistar rats received the following implants: a chronic guide cannula in the lateral cerebral ventricle and a temperature sensor in the abdominal cavity. On the day of the experiments, the rats were assigned to receive a 2-μL intracerebroventricular injection of either NaCl (0.15 mol/L) or L-arg solution (0.825, 1.65 or 3.30 mol/L); Tcore and tail skin temperature were measured while the rats ran at a speed of 18 m/min until they were fatigued. L-arginine induced a dose-dependent reduction in the threshold Tcore required for cutaneous heat loss (38.09 ± 0.20°C for 3.30-mol/L L-arg vs 38.61 ± 0.10°C for saline; P < 0.05), which attenuated the exercise-induced hyperthermia. Although the rats treated with L-arg presented a lower Tcore at the end of exercise (~0.7°C lower after treatment with the highest dose), no changes in the time to fatigue were observed relative to the control trial. These results suggest that brain L-arg controls heat loss during exercise, most likely by modulating the sympathetic vasoconstrictor tonus to skin vessels. Furthermore, despite facilitating cutaneous heat loss mechanisms, increased brain L-arg availability did not enhance physical performance.

Anatomical organization of the rat organum vasculosum laminae terminalis.

The organum vasculosum of the laminae terminalis (OVLT) is a circumventricular organ located along the ventral part of the anterior wall of the third ventricle. Because it lacks a complete blood-brain barrier (BBB), blood-borne signals detected in the OVLT provide the brain with information from the periphery and contribute to the generation of centrally mediated responses to humoral feedback and physiological stressors. Experimental studies on the rat OVLT are hindered by a poor understanding of its precise anatomical dimensions and cellular organization. In this study, we use histological techniques to characterize the spatial outline of the rat OVLT and to examine the location of neurons, astrocytes, tanycytes, and ependymocytes within its confines. Our data reveal that OVLT neurons are embedded in a dense network of tanycyte processes. Immunostaining against the neuronal marker NeuN revealed that neurons are distributed throughout the OVLT, except for a thick midline septum, which comprises densely packed cells of unknown function or lineage. Moreover, the most ventral aspect of the OVLT is devoid of neurons and is occupied by a dense network of glial cell processes that form a thick layer between the neurons and the pial surface on the ventral aspect of the nucleus. Lastly, combined detection of NeuN and c-Fos protein following systemic injection of hypertonic NaCl revealed that neurons responsive to this stimulus are located along the entire midline core of the OVLT, extending from its most anterior ventral aspect to the more caudally located "dorsal cap" region.

Two-color Dye-swap DNA Microarray approach toward confident gene expression profiling in PMCAO mouse model for ischemia-related and PACAP38-influenced genes.

Toward twin goals of identifying molecular factors in brain injured by ischemic stroke, and the effects of neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain, we have established the permanent middle cerebral artery occlusion (PMCAO) mouse model and utilized the Agilent mouse whole genome 4 × 44 K DNA chip. PACAP38 (1 pmol) injection was given intracerebroventrically in comparison to a control saline (0.9% NaCl) injection, to screen genes responsive to PACAP38. Two sets of tissues were prepared, whole hemispheres (ischemic and non-ischemic) and infract core and penumbra regions at 6 and 24 h. In this study, we have detailed the experimental design and protocol used therein and explained the quality controls for the use of total RNA in the downstream DNA microarray experiment utilizing a two-color dye-swap approach for stringent and confident gene identification published in a series of papers by Hori and coworkers (Hori et al., 2012–2015).

Unraveling the Specific Ischemic Core and Penumbra Transcriptome in the Permanent Middle Cerebral Artery Occlusion Mouse Model Brain Treated with the Neuropeptide PACAP38.

Our group has been systematically investigating the effects of the neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain. To do so, we have established and utilized the permanent middle cerebral artery occlusion (PMCAO) mouse model, in which PACAP38 (1 pmol) injection is given intracerebroventrically and compared to a control saline (0.9% sodium chloride, NaCl) injection, to unravel genome-wide gene expression changes using a high-throughput DNA microarray analysis approach. In our previous studies, we have accumulated a large volume of data (gene inventory) from the whole brain (ipsilateral and contralateral hemispheres) after both PMCAO and post-PACAP38 injection. In our latest research, we have targeted specifically infarct or ischemic core (hereafter abbreviated IC) and penumbra (hereafter abbreviated P) post-PACAP38 injections in order to re-examine the transcriptome at 6 and 24 h post injection. The current study aims to delineate the specificity of expression and localization of differentially expressed molecular factors influenced by PACAP38 in the IC and P regions. Utilizing the mouse 4 × 44 K whole genome DNA chip we show numerous changes (≧/≦ 1.5/0.75-fold) at both 6 h (654 and 456, and 522 and 449 up- and down-regulated genes for IC and P, respectively) and 24 h (2568 and 2684, and 1947 and 1592 up- and down-regulated genes for IC and P, respectively) after PACAP38 treatment. Among the gene inventories obtained here, two genes, brain-derived neurotrophic factor (Bdnf) and transthyretin (Ttr) were found to be induced by PACAP38 treatment, which we had not been able to identify previously using the whole hemisphere transcriptome analysis. Using bioinformatics analysis by pathway- or specific-disease-state focused gene classifications and Ingenuity Pathway Analysis (IPA) the differentially expressed genes are functionally classified and discussed. Among these, we specifically discuss some novel and previously identified genes, such as alpha hemoglobin stabilizing protein (Ahsp), cathelicidin antimicrobial peptide (Camp), chemokines, interferon beta 1 (Ifnb1), and interleukin 6 (Il6) in context of PACAP38-mediated neuroprotection in the ischemic brain. Taken together, the DNA microarray analysis provides not only a great resource for further study, but also reinforces the importance of region-specific analyses in genome-wide identification of target molecular factors that might play a role in the neuroprotective function of PACAP38.

On a role of liquid intermediates in nucleation of gold sulfide nanoparticles in aqueous media.

Previously, we found a series of fluid nanoscale intermediates preceding nucleation of gold sulfide in the reaction between aqueous HAuCl4 and sodium sulfide. Here, the effects of temperature, addition of an inert electrolyte and some other factors on characteristics of the "dense liquid" intermediates and the formation of solid nuclei were studied using UV-vis absorption spectroscopy, DLS, zeta-potential measurements, and SAXS. It was revealed, in particular, that the negatively-charged interfaces of the dense liquid species critically impede their fusion into large enough dense droplets in which nucleation can take place. The nucleation and following coagulation of poorly crystalline gold sulfide proceed not instantly but progressively as the dense liquid droplets arise, with the process being sharply accelerated by the injection of NaCl or temperature increase.

Reduced cerebellar neurodegeneration after combined therapy with cyclodextrin/allopregnanolone and miglustat in NPC1: a mouse model of Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disease characterized by a deficiency of NPC1 gene function. The malfunction of protein results in a progressive accumulation of lipids in many organs. A combined approach with substrate-reduction therapy (SRT) and byproduct therapy (BPT) has been shown to ameliorate the disease course in a mutant mouse model (NPC1(-/-)). The present study examines the morphological parameters underlying these changes. For the combined SRT/BPT treatment, NPC1(-/-) mutant mice (NPC1(-/-SRT/BPT)) were injected with allopregnanolone/cyclodextrin weekly, starting at postnatal day (P) 7. Starting at P10, a miglustat injection was administered daily until P23. Thereafter, miglustat was added to the powdered chow. For the sham treatment, both mutant NPC1(-/-) (NPC1(-/-sham)) and wild-type (NPC1(+/+sham)) mice received an NaCl injection and were fed powdered chow without miglustat. Analysis was performed on cerebellar slices by histology and immunohistochemistry. The volumes and cell counts of cerebellar structures were quantified. Additionally, ultrastructural analysis was performed with transmission electron microscopy. In agreement with previous studies, the current study demonstrates Purkinje cell degeneration in the mutant mice, which was partially abrogated by SRT/BPT. The volumes of cerebellar white matter and molecular layer were reduced as well. Also, the number of neurons was reduced in granular and molecular layers. However, only the molecular layer benefited from the therapy, as shown by an increase in the volume and the amount of neurons. The volume and number of neurons of the deep cerebellar nuclei were significantly decreased in mutant mice; an appreciable therapeutic benefit could be demonstrated for the nucleus interpositus.

Subacromial triamcinolone acetonide, hyaluronic acid and saline injections for shoulder pain an RCT investigating the effectiveness in the first days

BackgroundSubacromial impingement is a common cause of shoulder complaints in general practice. When the initial treatment with acetaminophen and low dose Non Steroidal Anti Inflammatory Drugs fails, triamcinolone acetonide injections are commonly used. Triamcinolone acetonide injections are effective at four to six weeks. Little is known about the pain relief effect of triamcinolone acetonide injections in the first days after injection and the effect of repeated injection. In this study we investigate the effect of triamcinolone acetonide injections compared to hyaluronic acid and NaCl injections using a pain diary.Methods159 Patients recruited for an RCT comparing the effect of subacromial injections of triamcinolone acetonide, hyaluronic acid and sodium chloride (NaCl) were used in this study. They were blinded for their treatment and could receive up to three injections. Primary outcome consisted of the patient perceived pain on a VAS score recorded on a daily basis during 21 days following injection. Secondary outcome consisted of the amount of taken escape medication following injection and adverse effects.ResultsAll patients received the first injection. 150 patients also received the second and third injections. 97% Of the paper and pencil pain diaries were returned for data analysis.The triamcinolone acetonide group showed the largest decrease in pain on the VAS scores after injection compared to the hyaluronic acid and NaCl group in the first week after injection. The reduction in pain was best achieved after the first injection, the second triamcinolone acetonide injection showed a further reduction in pain. The third triamcinolone acetonide injection only showed a slight improvement in pain reduction.ConclusionsIn this study we could show a booster effect in pain reduction after repeated triamcinolone acetonide injection. The triamcinolone acetonide group showed a faster reduction in pain after injection compared to the hyaluronic acid and NaCl group. The effect was best seen after the first and second triamcinolone acetonide injection, it is therefore questionable whether it is necessary to repeat triamcinolone acetonide injections more than two times.Trial registrationISRCTN51511455. Registered 20 December 2005Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-352) contains supplementary material, which is available to authorized users.

101. Peripheral inflammation impairs motivation through modulation of fundamental gain and loss associated prediction error signals in ventral striatum and anterior insula

Inflammation reduces reward-seeking behaviour and increases avoidance of aversive stimuli. However, how this is neurally mediated remains unclear. Here we addressed this using a Typhoid vaccine experimental inflammation model together with computational modelling of an instrumental learning task performed during fMRI (1). Twenty-two healthy participants were each tested 3-h after blindly administered typhoid vaccine (0.025 mg) and placebo (0.5 ml 0.9% NaCl) injection. Typhoid but not placebo was associated with a threefold increase in plasma IL-6 ( t (21) = 5.20, p F (21,1) = 6.41, p = 0.019. Computational modelling of individuals’ choices using a Q-learning model (free parameters alpha (learning-rate), beta (choice-randomness) and R (reward value)) demonstrated significant reduction in reward cue value and increase in value of punishment cues following inflammation F (21,1) = 3.63, p p p

Inhibition of α5β1‐integrin significantly improves the surgical outcome of glaucoma surgery in mice compared to MMC

AbstractPurpose The aim of this study was to evaluate the therapeutic potential of α5β1‐integrin inhibitor (CLT‐28643) to improve the outcome of filtering surgery in a mouse model. Different dose regimen and administration routes of the inhibitor were compared to mitomycin C (MMC), the gold standard in clinical practice.Methods The efficacy of CLT‐28643 on surgical outcome was studied in a mouse model for filtering surgery (n=40 eyes from 20 mice per group). Group 1 received a single subconjunctival (SC) injection of 2 µg of the integrin inhibitor immediately after surgery, whereas repeated SC injections were administered on day 0, 3, 7, 14 and 21 in groups 2 and 3 (2 and 1 µg, respectively). Group 4 received topical eye drops containing 10 µg of the compound 3 times daily. MMC 0.02% was applied for 2 minutes in group 5 and repeated SC injections of NaCl were given in the last group. Treatment outcome was studied by a masked observer every other day by clinical investigation of the bleb until postoperative day 28.Results Surgical outcome was improved in all treatment groups compared to NaCl treated eyes (P&lt;0.001). A dose‐response curve was observed in the efficacy of the subconjunctival injections, with the repetitive administration of 1µg being significantly inferior to, the single injection of 2 µg or topical administration (10 µg) comparable to, and the repetitive injection of 2 µg significantly superior to MMC.Conclusion These data suggest that administration of the α5β1‐integrin inhibitor CLT‐28643 has therapeutic potential as an adjunct to glaucoma surgery, possibly with a superior efficacy to MMC when used at the optimal dose.

Bevacizumab Revisited: Its Use in Different Mouse Models of Ocular Pathologies

ABSTRACTPurpose: Previous reports have yielded conflicting data on the activity of bevacizumab (Avastin), a recombinant humanized monoclonal antibody against VEGF-A, in the mouse. The current study was designed to further explore the use of this VEGF inhibitor in various murine models of ocular diseases and compare it to the widely used murine anti-VEGF-R2 neutralizing antibody (DC101).Methods: Murine models of laser-induced choroidal neovascularization (CNV), oxygen-induced retinopathy (OIR) and glaucoma filtration surgery (GFS) were used to investigate the effect of bevacizumab. Mice either received an intravitreal (CNV–OIR) or subconjunctival (GFS) injection. In all models, they were divided in two groups (n = 10 per group). In the first group, one eye was injected with bevacizumab (1 µl; 25 µg) and the other eye was used as a negative control and received an injection of NaCl (1 µl; 0.9%). In the second group, one eye was injected with DC101 (1 µl; 6.2 µg), whereas an isotype-matched control antibody (1C8; 4.8 µg) was administered in the contralateral eye. Treatment outcome was studied by clinical investigation (GFS) and immunohistological analysis of angiogenesis (CD31/FITC-dextran/H&E) and fibrosis (Sirius Red).Results: Analysis of blood vessel density (CNV) and blood vessel growth (OIR) showed a comparable decrease after intravitreal administration of bevacizumab or DC101. Furthermore, in the mouse model of GFS, clinical investigation of the bleb and a CD31 staining on sections demonstrated that subconjunctival injection of both antibodies similarly improved the surgical outcome (bleb area and survival) by reducing angiogenesis. Moreover, morphometric analysis after Sirius Red staining showed a comparable reduction in collagen deposition after administration of the inhibitors.Conclusion: Our findings consistently demonstrate that bevacizumab is as effective as the murine anti-VEGF-R2 antibody (DC101) in mouse models of CNV, OIR and GFS, thus confirming its suitability for translational ophthalmological research.

Inhibitors of endopeptidase and angiotensin-converting enzyme lead to an amplification of the morphological changes and an upregulation of the substance P system in a muscle overuse model.

BackgroundWe have previously observed, in studies on an experimental overuse model, that the tachykinin system may be involved in the processes of muscle inflammation (myositis) and other muscle tissue alterations. To further evaluate the significance of tachykinins in these processes, we have used inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), substances which are known to terminate the activity of various endogenously produced substances, including tachykinins.MethodsInjections of inhibitors of NEP and ACE, as well as the tachykinin substance P (SP), were given locally outside the tendon of the triceps surae muscle of rabbits subjected to marked overuse of this muscle. A control group was given NaCl injections. Evaluations were made at 1 week, a timepoint of overuse when only mild inflammation and limited changes in the muscle structure are noted in animals not treated with inhibitors. Both the soleus and gastrocnemius muscles were examined morphologically and with immunohistochemistry and enzyme immunoassay (EIA).ResultsA pronounced inflammation (myositis) and changes in the muscle fiber morphology, including muscle fiber necrosis, occurred in the overused muscles of animals given NEP and ACE inhibitors. The morphological changes were clearly more prominent than for animals subjected to overuse and NaCl injections (NaCl group). A marked SP-like expression, as well as a marked expression of the neurokinin-1 receptor (NK-1R) was found in the affected muscle tissue in response to injections of NEP and ACE inhibitors. The concentration of SP in the muscles was also higher than that for the NaCl group.ConclusionsThe observations show that the local injections of NEP and ACE inhibitors led to marked SP-like and NK-1R immunoreactions, increased SP concentrations, and an amplification of the morphological changes in the tissue. The injections of the inhibitors thus led to a more marked myositis process and an upregulation of the SP system. Endogenously produced substances, out of which the tachykinins conform to one substance family, may play a role in mediating effects in the tissue in a muscle that is subjected to pronounced overuse.

Acute hypernatremia promotes anxiolysis and attenuates stress-induced activation of the hypothalamic–pituitary–adrenal axis in male mice

Previous investigation by our laboratory found that acute hypernatremia potentiates an oxytocinergic tone that inhibits parvocellular neurosecretory neurons in the paraventricular nucleus of the hypothalamus (PVN), attenuates restraint-induced surges in corticosterone (CORT), and reduces anxiety-like behavior in male rats. To investigate the neural mechanisms mediating these effects and extend our findings to a more versatile species, we repeated our studies using laboratory mice. In response to 2.0M NaCl injections, mice had increased plasma sodium concentrations which were associated with a blunted rise in CORT subsequent to restraint challenge relative to 0.15M NaCl injected controls. Immunofluorescent identification of the immediate early gene product Fos found that 2.0M NaCl treatment increased the number of activated neurons producing oxytocin in the PVN. To evaluate the effect of acute hypernatremia on PVN neurons producing corticotropin-releasing hormone (CRH), we used the Cre-lox system to generate mice that produced the red fluorescent protein, tdTomato, in cells that had Cre-recombinase activity driven by CRH gene expression. Analysis of brain tissue from these CRH-reporter mice revealed that 2.0M NaCl treatment caused a dramatic reduction in Fos-positive nuclei specifically in CRH-producing PVN neurons. This altered pattern of activity was predictive of alleviated anxiety-like behavior as mice administered 2.0M NaCl spent more time exploring the open arms of an elevated-plus maze than 0.15M NaCl treated controls. Taken together, these results further implicate an oxytocin-dependent inhibition of CRH neurons in the PVN and demonstrate the impact that slight elevations in plasma sodium have on hypothalamic–pituitary–adrenocortical axis output and anxiety-like behavior.

True colors – experimental identification of hydrological processes at a hillslope prone to slide

Abstract. This study investigated runoff formation processes of a pre-alpine hillslope prone to slide. The experimental pasture plot (40 m × 60 m) is located in the northern front range of the Swiss Alps on a 30° steep hillslope (1180 m a.s.l., 1500 + mm annual precipitation). A gleysol (H-Go-Gr) overlies weathered marlstone and conglomerate of subalpine molasse. We conducted sprinkling experiments on a subplot (10 m × 10 m) with variable rainfall intensities. During both experiments fluorescein line-tracer injections into the topsoil, and sodium chloride (NaCl) injections into the sprinkling water were used to monitor flow velocities in the soil. The observed flow velocities for fluorescein in the soil were 1.2 and 1.4 × 10−3 m s−1. The NaCl breakthrough occurred almost simultaneously in all monitored discharge levels (0.05, 0.25 and 1.0 m depth), indicating a high-infiltration capacity and efficient drainage of the soil. These initial observations suggested "transmissivity feedback", a form of subsurface stormflow, as the dominant runoff process. However, the results of a brilliant blue dye tracer experiment completely changed our perceptions of the hillslope's hydrological processes. Excavation of the dye-stained soils highlighted the dominance of "organic layer interflow", a form of shallow subsurface stormflow. The dye stained the entire H horizon, vertical soil fractures, and macropores (mostly worm burrows) up to 0.5 m depth. Lateral drainage in the subsoil or at the soil–bedrock interface was not observed, and thus was limited to the organic topsoil. In the context of shallow landslides, the subsoil (Go/Gr) acted as an infiltration and exfiltration barrier, which produced significant lateral saturated drainage in the topsoil (H) and possibly a confined aquifer in the bedrock.

A Nicotinic Acetylcholine Receptor Agonist Prevents Loss of Retinal Ganglion Cells in a Glaucoma Model

The purpose of this study was to analyze the neuroprotective effect of an α7 nAChR agonist, PNU-282987, using an in vivo model of glaucoma in Long Evans rats. One eye in each animal was surgically manipulated to induce glaucoma in control untreated animals and in animals that were treated with intravitreal injections of PNU-282987. To induce glaucoma-like conditions, 0.05 mL of 2 M NaCl was injected into the episcleral veins of right eyes in each rat to create scar tissue and increase intraocular pressure. The left eye in each rat acted as an internal control. One month following NaCl injection, rats were euthanized, retinas were removed, flatmounted, fixed, and nuclei were stained with cresyl violet or RGCs were immunostained with an antibody against Thy 1.1 or against Brn3a. Stained nuclei in the RGC layer and labeled RGCs in NaCl-injected retinas were counted and compared with cell counts from untreated retinas in the same animal. NaCl injections into the episcleral veins caused a significant loss of cells by an average of 27.35% (± 2.12 SEM) in the RGC layer within 1 month after NaCl injection, which corresponded to a significant loss of RGCs. This loss of RGCs was eliminated if 5 μL of 100 μM PNU-282987 was injected into the right eye an hour before NaCl injection. The results from this study support the hypothesis that the α7 agonist, PNU-282987, has a neuroprotective effect in the rat retina. PNU-282987 may be a viable candidate for future therapeutic treatments of glaucoma.

Administration of a second generation perfluorochemical in combination with hyperbaric oxygenation does not provide additional benefit in a model of permanent middle cerebral artery occlusion in rats.

ObjectiveBoth, second generation perfluorochemicals (Oxycyte®) and hyperbaric oxygen (HBO) have been shown to reduce necrotic tissue volume if administered early after experimental cerebral ischemia. With the idea of exponentiation of oxygen delivery to ischemic tissue, this study was conducted to investigate the combined effect of both treatment modalities on the extent of ischemic brain damage.MethodsPermanent focal cerebral ischemia was induced in rats by middle cerebral artery occlusion (MCAO). Animals were assigned randomly to one of the following treatment groups: Control (0.9% NaCl, 1 ml/100 g i.v.), Oxycyte® (1 ml/100 g i.v.), HBO (1 bar hyperbaric oxygenation for 1 h) and HBO + Oxycyte® (1 ml/100 g i.v. combined with 1 bar hyperbaric oxygenation for 1 h). Injection of NaCl or Oxycyte® was performed following MCAO. After injection, breathing was changed to 100% oxygen in Oxycyte®-, HBO- and HBO + Oxycyte®-groups. After eight hours the necrotic volume was calculated from serial coronal sections stained with silver-nitrate and corrected for the extent of swelling.ResultsHemodynamic and metabolic parameters were not affected by infusion of Oxycyte®. Total necrosis volume was significantly reduced in HBO-treated animals (223 ± 70 mm3), when compared to control animals (335 ± 36 mm3). In animals after Oxycyte®-treatment alone (299 ± 33 mm3) or combined HBO + Oxycyte®-treatment (364 ± 50 mm3) did not show a significantly smaller necrosis volume compared to control animals (necrosis volumes are given as mean ± SD).DiscussionThese results suggest that combination of hyperbaric oxygenation and Oxycyte® administered immediately after onset of vascular occlusion does not provide an additional neuroprotective effect in the early phase of brain ischemia.

Influence of ovarian hormones on cortical spreading depression and its suppression by L-kynurenine in rat.

Migraine is sexually dimorphic and associated in 20–30% of patients with an aura most likely caused by cortical spreading depression (CSD). We have previously shown that systemic L-kynurenine (L-KYN), the precursor of kynurenic acid, suppresses CSD and that this effect depends on the stage of the estrous cycle in female rats. The objectives here are to determine the influence of ovarian hormones on KCl-induced CSD and its suppression after L-KYN by directly modulating estradiol or progesterone levels in ovariectomized rats. Adult female rats were ovariectomized and subcutaneously implanted with silastic capsules filled with progesterone or 17β-estradiol mixed with cholesterol, with cholesterol only or left empty. Two weeks after the ovariectomy/capsule implantation, the animals received an i.p. injection of L-KYN (300 mg/kg) or NaCl as control. Thirty minutes later CSDs were elicited by applying KCl over the occipital cortex and recorded by DC electrocorticogram for 1 hour. The results show that both estradiol and progesterone increase CSD frequency after ovariectomy. The suppressive effect of L-KYN on CSD frequency, previously reported in normal cycling females, is not found anymore after ovariectomy, but reappears after progesterone replacement therapy. Taken together, these results emphasize the complex role of sex hormones on cortical excitability. The CSD increase by estradiol and, more surprisingly, progesterone may explain why clinically migraine with aura appears or worsens during pregnancy or with combined hormonal treatments.

The Effects of Sirolimus on Pancreatic Fibrosis and Apoptosis in Experimental Chronic Pancreatit Model

Purpose: Sirolimus reduces hepatic fibrosis by inhibiting hepatic stellat cells. We aimed to show the similar effect by inhibiting the pancreatic stellat cells in experimental chronic pancreatitis. Methods: In our study 55 male, Sprague-Dawley rats weighing between 200 and 400 grams randomized into four groups. Chronic pancreatitis is induced by injection of 0,4 ml, 2% trinitrobenzene sulfanic acid (TNBS) into major pancreatic duct. Group A (n=15): chronic pancreatitis + sirolimus; TNBS induction, 4 weeks later, 2 mg/kg/day oral sirolimus for 2 weeks; Group B (n=15): Chronic pancreatitis + placebo; TNBS induction, 4 weeks later 2 ml/kg/day oral water for 2 weeks. Group C (n=15): Chronic pancreatitis; TNBS induction, 4 weeks later sacrified. Group D (n=10): Pancreatic cannulation + sirolimus; 0,4 ml, % 0,9 NaCl injection into pancreatic duct, 4 weeks later 2 mg/kg/day oral sirolimus for 2 weeks. At the end of the ninth week in group of A, B and D all rats have been sacrifi ced, and rat pancreatic tissues, oxidative stress markers and progression of fibrosis and apoptsosis have been examined Results: Tissue and blood malondialdehyde (MDA) levels has been found significantly lower in sirolimus group compared to placebo group (p<0,001) and superoxide dismutase (SOD) and glutation peroxidase (GSH-Px) activities have been significantly higher. (Respectively p<0,001 and p<0,001). Tissue and blood MDA, SOD, GSH Px levels was no different significantly in the sirolimus group compared to the pancreatic cannulation group (p>0,05) (Table 1). Histopathological analysis of the tissues revealed that score of sirolimus group was significantly higher at respect to other groups (p<0,05). The levels of apoptotic cell of sirolimus group was higher at respect to placebo group, but this was not statistically significant (Table 2).Table: Table. Levels of oxidative stress markers in tissue and bloodTable: Table. Histopathological FindingsConclusion: Administration of sirolimus decreases oxidative stress in experimental chronic pancreatitis, but hasn't beneficial effects on fibrosis and apoptosis.

Combined therapy with cyclodextrin/allopregnanolone and miglustat improves motor but not cognitive functions in Niemann–Pick Type C1 mice

Niemann–Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy.NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data.The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy.At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy.CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.

What is the most optimal administration route of bevacizumab after glaucoma filtration surgery in mice?

AbstractPurpose Glaucoma filtration surgery (GFS) frequently leads to surgical failure due to excessive postoperative wound healing. The current study was designed to investigate the effect on postoperative scarring of a single subconjunctival (SC), intracameral (IC) or intravitreal (IV) injection of bevacizumab.Methods The effect of bevacizumab (AvastinTM, Genentech) was investigated in a model of GFS in C75Bl/6 mice. Immediately after surgery, mice were divided in 3 groups (n=10 per group) and received a SC, IC or IV injection. In all groups, one eye was injected with bevacizumab (1 µl; 25 µg) and the other eye was used as a control and received an injection of NaCl (1 µl; 0.9%). Treatment outcome was studied by clinical investigation of bleb area and bleb survival every other day. Mice were killed on postoperative day 14 and immunohistological analysis of angiogenesis and collagen deposition were performed.Results In the mouse model of GFS, treatment using a SC, IC, IV injection of bevacizumab significantly improved surgical outcome by increasing bleb area with 53%; with 45% and with 49%, respectively, compared to negative control. Bleb survival significantly improved only after SC injection with 37,5%. In all groups, blood vessel density was significantly reduced after bevacizumab treatment with 36%; with 31% and with 34%, respectively. All administration routes of bevacizumab significantly diminished collagen deposition with 27%, compared to negative control.Conclusion This study shows that a subconjunctival injection of bevacizumab had the largest effect on surgical outcome compared to intracameral and intravitreal injection.