Free Saline Research Articles

Effect of different extenders and cryoprotectants on sperm viability of mud crab, Scylla tranquebarica for short term storage

The aim of the present study was to determine the extenders such as calcium-free saline, Ringer’s solution and phosphate buffer and to determine the effect of different concentrations of cryoprotectants with different duration of exposure for short term storage of mud crab, Scylla tranquebarica sperm cells. A total of 30 mud crabs were used in the present study. In the present study, three treatments with three replicates were tested to determine the suitable extender. Besides, three treatments (dimethyl sulfoxide, glycine and glycerol) were tested to determine the suitable cryoprotectants. Eight treatments (25, 20, 16, 4, 2, -4, -20 and -80 °C) were assessed to determine the optimal cooling rate. The result showed the phosphate buffer solution produced the highest sperm viability (82.36 ± 5.84 %) after 60 min compared the Calcium free saline and Ringer’s solution. The results for sperm viability at -4 °C, -20 C and -80 °C after 24 hours were 95.63 0.13 % (5 % glycine), 91.51 ± 1.91 % (5 % DMSO) and 93.97 ± 0.43 % (5 % glycine). By using the 5 % glycine as the best cryoprotectant, the results for the sperm viability in 6 h, 12 h, 24 h, day 7, day 14 and day 21 at 80 °C were 95.17 ± 0.92 %, 94.11 ± 0.64 %, 93.31 ± 0.51 %, 90.84 ± 2.05 % and 82.82 ± 0.41 %. In a nutshell, the phosphate buffer and 5 % glycine were the best extender and cryoprotectant in the present study.

In Vivo Evaluation of Self-assembled nano-Saikosaponin-a for Epilepsy Treatment.

Saikosaponin-a (SSa) exhibits antiepileptic effects. However, its poor water solubility and inability to pass through the blood-brain barrier greatly limit its clinical development and application. In this study, SSa-loaded Methoxy poly (ethylene glycol)-poly(ε-caprolactone) (MePEG-SSa-PCL) NPs were successfully prepared and characterized. Our objective was to further investigate the effect of this composite on acute seizure in mice. First, we confirmed the particle size and surface potential of the composite (51.00 ± 0.25nm and - 33.77 ± 2.04 mV, respectively). Further, we compared the effects of various MePEG-SSa-PCL doses (low, medium, and high) with those of free SSa, valproic acid (VPA - positive control), and saline only (model group) on acute seizure using three different acute epilepsy mouse models. We observed that compared with the model group, the three MePEG-SSa-PCL treatments showed significantly lowered seizure frequency in mice belonging to the maximum electroconvulsive model group. In the pentylenetetrazol and kainic acid (KA) acute epilepsy models, MePEG-SSa-PCL increased both clonic and convulsion latency periods and shortened convulsion duration more effectively than equivalent SSa-only doses. Furthermore, hematoxylin-eosin and Nissl staining revealed considerably less neuronal damage in the hippocampal CA3 area of KA mice in the SSa, VPA, and three MePEG-SSa-PCL groups relative to mice in the model group. Hippocampal gamma-aminobutyric acid-A (GABA-A) receptor and cleaved caspase-3 expression levels in KA mice were significantly higher and lower, respectively, in the three MePEG-SSa-PCL treatment groups than in the model group. Thus, MePEG-SSa-PCL exhibited a more potent antiepileptic effect than SSa in acute mouse epilepsy models and could alleviate neuronal damage in the hippocampus following epileptic seizures, possibly via GABA-A receptor expression upregulation.

A rare complication after endoscopic neurolysis of the sciatic nerve: а clinical case

Background: Еndoscopic neurolysis of the sciatic nerve has proven itself as a low traumatic and effective technique of treatment for deep gluteal pain syndrome and sciatic nerve neuropathy. Until now, there have been no publications dedicated to the parietal peritoneum damage after endoscopic sciatic nerve decompression. Clinical case description: A 70-year-old female patient with deep gluteal pain syndrome and sciatic nerve neuropathy on the right side and failure of a conservative treatment. According to the VAS scale, the severity of pain syndrome was 10 cm. The severity of the motor dysfunction, according to the BMRC scale, was 4 points. The severity of the sensitive dysfunction, according to the Seddon scale, was 3 points. The functional activity of the lower limb, according to the LEFS scale, was 48 points. The instrumental investigation of the sciatic nerve included MRI, ultrasound, and ENMG. Endoscopic sciatic nerve decompression was performed in the prone position with saline irrigation using the technique published before. After the surgery, an ultrasound study and a CT scan were performed, which determined the presence of a free fluid in the abdominal cavity, and edema of the right retroperitoneal space. The patient underwent laparoscopy, which revealed the area of the parietal peritoneum damage in the lower floor, with a free saline fluid detected in the abdominal cavity, which was evacuated. The patient was discharged from the hospital on the 10th day after the surgery. 6 months after the surgery, the functional activity of the lower extremity, according to the LEFS scale, was 52 points. Pain syndrome, according to the VAS scale, was 8 cm. The severity of the motor dysfunction, according to the BMRC scale, was 4 points. The severity of the sensitive dysfunction, according to the Seddon scale, was 3 points. The performed surgical endoscopic treatment was rendered ineffective. Conclusion: The method of endoscopic sciatic nerve decompression with saline pumping has a risk of the following complication: damaging the parietal peritoneum, saline penetration into the abdominal cavity, edema of the retroperitoneal space. To decrease the risk of this complication, it is necessary to observe the specific surgical conditions such as: controlled hypotension in a patient and a low pressure in the arthroscopic pump, the time of surgery not exceeding 1 hour, and the saline consumption not exceeding 10 liters. It is also crucial not to perform the dissection and neurolysis too proximal to the piriformis area.

#6578 ACUTE KIDNEY INJURY IN PATIENTS WITH VENTRICULAR SEPTAL RUPTURE: A SINGLE CENTER STUDY

Abstract Background and Aims Ventricular septal rupture (VSR) is an infrequent but dreaded complication of acute myocardial infarction(AMI). Acute kidney injury(AKI) is one of the major determinants of morbidity and mortality in these patients. Renal replacement therapy (RRT) is, in this set of patients of high-risk nature and can be prolonged. Focused data on the management of AKI in patients with VSR from developing countries is scarce. We present a large cohort of patients presenting with VSR from a tertiary care Centre in South India, evaluating the incidence of AKI, RRT, and short-term outcomes in this cohort. Method Observational study of all adult patients diagnosed with VSR following AMI over 5 years in a single centre in Chennai, India. Demographic characteristics, co-morbidities,clinical and laboratory,Serial renal function tests and urine output was noted. eGFR was calculated using the CKD-EPI formula.AKI definition and staging -KDIGO clinical Practice Guidelines. Data analysis was done using the IBM SPSS version 24. P-value less than 0.05 is used to indicate statistical significance. Results During the study period, 3024 patients presented to our hospital with acute coronary syndrome (ACS) and seventy patients presented with VSR (2.3%). Of these, forty-seven (67.1%) developed AKI. A nephrology consult was obtained for thirty-three (70%) patients. Older age, higher total count and lower systolic blood pressure were significantly associated with the occurrence of AKI. The mortality in the AKI group was significantly higher (63.8%). VSR closure was done in 12.7% and 21.7% of the AKI and non-AKI groups respectively. Surgical VSR repair was done in 61.7% and 60.8% of the patients with and without AKI respectively. Most of the patients (26, 55.3%) presented with KDIGO stage 3 AKI and all were oliguric. 10 and 11 patients were staged into KDIGO stages 1 and 2 respectively. 26 patients with Stage 3 AKI, only 14 (29.7%%) of them received nephrology consult.. RRT was initiated in fifteen patients (31.9%). The indications for RRT were oliguria, pulmonary oedema, metabolic acidosis, and worsening electrolyte disturbance.Acute peritoneal dialysis was initiated in 11 patients (73.3%) using a 43cm swan neck double cuffed tunnelled Tenckhoff peritoneal dialysis catheter placed at the bedside. Percutaneous peritoneal dialysis catheter insertion by a nephrologist was done in and the rest was inserted by open laparotomy technique by a trained general surgeon. All 11 were initiated on urgent low volume supine exchanges. Of these ten patients had manual exchanges done by a trained peritoneal dialysis nurse and one patient was put on automated peritoneal dialysis. The average volume per dwell was 500 ml±250 ml which was gradually increased if there was no leak. The total average peritoneal dialysis volume per day was 10L±2 L. The strength of bags (1.36%, 2.25%, 3.86%) dextrose was determined based on volume status and patients’ hemodynamics. No patient had a leak or peritoneal dialysis-related peritonitis. All patients’ peritoneal dialysis catheter was immobilized with a tight abdominal dressing. One patient continued continuous ambulatory peritoneal dialysis at home as there was no recovery of renal function and was lost to follow up. Sustained low-efficiency dialysis was performed on four patients. Duration of Sustained low-efficiency dialysis ranged from 6-8 hours with average blood flows of 150 ml/min and average fluid removal between 1.5-3L. Unfractionated heparin was used in three patients, and one was heparin free saline dialysis. Discharge eGFR in the AKI group was significantly lower as compared to the non-AKI group. (38 vs 104ml/min/1.73m2, p<0.001). Follow up was done on 14 at 3 months, The mean eGFR was 57.8ml/min/1.73 m2 when compared to 81.8 ml/min/1.73 m2. Conclusion The incidence of AKI is high and is associated with a high rate of mortality in patients with VSR. Peritoneal dialysis is a safe and effective option for those patients requiring RRT. Follow up of AKI patients at three months did not show a significant decline in renal function

Efficacy of dexmedetomidine and magnesium sulphate as adjuvant to bupivacaine among patients undergoing lower extremity surgeries

In this prospective randomised double blind study, an attempt was made to compare the effects of adding dexmedetomidine 10 mcg or magnesium sulphate 50mg to 0.5% hyperbaric bupivacaine on subarachnoid block characteristics and postoperative analgesia in patients undergoing elective lower extremity surgeries. Randomization of the participants was done in three equal groups (n=30). A total of 90 patients of age between 18-60 years of either sex and good physical status posted for elective lower extremity surgery were included. Group B received 15 mg hyperbaric bupivacaine(3ml) with 0.5ml preservative free normal saline,Group D received 15 mg hyperbaric bupivacaine (3ml) with 10 mcg dexmedetomidine in 0.5 ml preservative free normal saline intrathecally and Group M received 15 mg hyperbaric bupivacaine (3ml) with 50mg magnesium sulphate (0.5ml) intrathecally for subarachnoid block. Different parameters of sensory block, motor block and analgesia effect were assessed as outcomes. Results showed that the groups were similar in demographic parameters like age, sex, height, weight, physical status. Bupivacaine with dexmedetomidine as adjuvant shows early onset of sensory and motor block, early onset of peak block height, prolonged sensory block, prolonged two segment regression time.

Comparison of Outcomes of External Dacryocystorhinostomy and Transcanalicular Laser-Assisted Dacryocystorhinostomy in Patients with Primary Acquired Nasolacrimal Duct Obstruction.

This study aimed to compare anatomical and functional outcomes of external dacryocystorhinostomy (EX-DCR) and transcanalicular multidiode laser dacryocystorhinostomy (TDL-DCR) in patients with primary acquired nasolacrimal duct obstruction (PANDO). This study was conducted on 60 eyes of 60 patients with PANDO. The study subjects were randomly divided into two groups. Group 1 included 30 patients who underwent EX-DCR, and group 2 included 30 patients who underwent TDL-DCR. Surgery success rates were determined by patency of the neo-ostium, with free saline flow on irrigation and the absence of epiphora. There were 23 women (76.7%) and 7 men (23.3%) with a mean age of 47.33 ± 12.44 years in group 1, and 18women (60%) and 12 men (40%) with a mean age of 46.2 ± 19.4 years in group 2. There was no significant difference between groups 1 and 2 with respect to age or gender (p = 0.801, p = 0.267, respectively). The mean duration of symptoms was 3.2 years, ranging from 1.5 to 5 years. The mean postoperative follow-up was 12.3 ± 2.44 months. At the end of the follow-up period, the surgery success rates were 96.7% (29/30 eyes) in group 1, and 90% (27/30 eyes) in group 2. There was no statistical difference in the surgery success rates between groups (p = 0.612). TDL-DCR is a minimally invasive and safe procedure. The lack of bleeding and incision scar and shorter operation time suggest that TDL-DCR can be a good alternative in PANDO patients.

Abstract 10631: Exosome-Encapsulated Adeno-Associated Viruses as a Novel Cardiotropic Vector for Gene Therapy in Heart Failure

Adeno-associated viruses (AAVs) are the vectors of choice for long-term delivery of genes to the heart and due to their safety in clinics. Despite its many favorable features, a notable drawback is the presence of preexisting neutralizing antibodies (NAb), which bind to free AAVs and impair their clinical effect. Here, we describe delivery of AAVs encapsulated in extracellular vesicles (eAAV), as a superior cardiac gene delivery system that offers higher NAb resistance. Methods: Ultracentrifuge-isolated eAAVs were characterized using TEM, DLS, qNano, nanoflow cytometry, ExoView, qPCR and Western blot. Gene delivery efficacy of free AAV and eAAVs were compared with and without NAbs using human cardiomyocytes in vitro, and in mice in vivo, using flow cytometry (with mCherry) and bioluminescence imaging (with luciferase). Therapeutic efficacy of gene delivery by free AAV and eAAV in presence and absence of NAb was determined using a cardiac contractile gene SERCA2a in a mouse model of myocardial infarction (MI). eAAV uptake and trafficking was studied both in vitro and in vivo using fluorescence dye-labeled eAAV. Results: eAAV outperformed free AAV in delivering genes even in the presence of NAbs, both in vitro and in vivo. SERCA2a delivered by eAAV significantly improved cardiac function (ejection fraction, %EF, ~60%) compared to when delivered by free AAV (~40%) or saline control (~20%), in intramyocardially injected MI mice with NAb. Fluorescence-tagged eAAVs injected to mouse hearts were internalized into all cell types (CMs and non-CMs), but, eAAV-mCherry was primarily expressed in CMs, indicating that eAAV-mediated gene delivery is cardiotropic. eAAVs were uptaken in to acidic endosomal (Rab5 & 7 positive) compartments of cultured cardiomyocytes and delivered significantly higher amounts of luciferase to the nucleus of human CMs compared to free AAVs. This uptake of eAAVs was inhibited by the proton pump inhibitor in endosomes, Bafilomycin A1. Conclusion: Our study demonstrated that eAAV-mediated gene delivery can circumvent neutralizing antibody (NAb) issue for gene therapy. eAAVs are cardiotropic and deliver genes to the nucleus via uptake into acidic endosomes. eAAV is a superior gene delivery vector and a new platform to treat heart failure.

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na+ transport across the anterior midgut of the vector of Chagas disease, Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect's hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and anti-diuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e.crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na+ channels. Incubation in Na+-free, but not Cl--free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na+-K+-2Cl- cotransporter (NKCC) and Na+-Cl- cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na+/K+-ATPase with ouabain inhibited Isc but the H+-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channel and a basolateral ouabain-sensitive Na+/K+-ATPase, while Cl- is transported through a passive paracellular pathway.

Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine–orexin A interaction in nucleus accumbens neurons.

A COMPARATIVE STUDY OF INTRATHECAL BUPIVACAINEHEAVY (0.5%) WITH BUPIVACAINE HEAVY (0.5%) PLUS DEXMEDETOMIDINE FOR LOWER ABDOMINAL SURGERIES

Introduction: Several adjuncts like adrenaline, opioids and alpha-2 adrenergic agonists are being used with local anaesthetics intrathecally for prolongation of intra-operative and post-operative analgesia and to reduce the side-effects of high doses of local anaesthetics.
 Aim: The present study was done to evaluate the onset and duration of sensory and motor block, hemodynamic effects, post-operative analgesia and adverse effects of Dexmedetomidine given intrathecally with hyperbaric 0.5% Bupivacaine.
 Materials and methods: Sixty inpatients of ASA class I and II scheduled for various lower abdominal surgeries under Sub-Arachnoid Block were randomly divided into two groups of 30 each namely C (Control), D(Dexmedetomidine) . All received 12.5mg hyperbaric bupivacaine plus 0.5 ml Normal Saline in Group C (Control),10µg Dexmedetomidine (diluted in preservative free Normal saline of 0.5ml) in Group D(Dexmedetomidine) . The onset time to reach peak sensory and motor level, the regression time for sensory and motor block, hemodynamic changes and side-effects were noted.
 Results: The duration of sensory and motor block, rescue analgesia was significantly prolonged in Dexmedetomidine group when compared to that of Control group.
 Conclusion: Dexmedetomidine 10 µg seems to be a better neuraxial adjuvant to hyperbaric Bupivacaine.
 Keywords: Bupivacaine; Dexmedetomidine; intrathecal.

Activation of Old Water Wells by Seawater and Use of Solar Pool Network

In order to purify water, studies have been conducted in different countries, and for this purpose, they use free saline water and inject this water into dry wells so that the water can be purified and reused after passing through and contacting the ground layers or wells. This method depends on the soil type of the area and has been done successfully in some areas but has failed in some areas. Therefore, in this study, we propose the use of a solar pool network that uses the properties of solar pools and land layers for both seawater treatment. Of course, to activate the wells, it is better to perform an initial treatment to remove floating organisms and bacteria that are not compatible with the environment near the wells and take some of the seawater salts that do not cause salinization of nearby lands.

Monitoring Anticoagulation with Unfractionated Heparin on Renal Replacement Therapy. Which is the Best aPTT Sampling Site?

BackgroundControlled anticoagulation is key to maintaining continuous blood filtration therapies. Objective: The study aimed to compare different blood sampling sites for activated partial thromboplastin time (aPTT) to evaluate anticoagulation with unfractionated heparin (UFH) in continuous renal replacement therapy (CRRT) and identify the most appropriate sampling site for safe patient anticoagulation and increased filter life span.MethodThe study was a prospective observational single-centre investigation targeting intensive care unit (ICU) patients on CRRT using an anticoagulation protocol based on patient characteristics and a weight-based modified nomogram. Eighty-four patients were included in the study. Four sampling sites were assessed: heparin free central venous nondialysis catheter (CVC), an arterial line with heparinised flush (Artery), a circuit access line (Access), and a circuit return line (Postfilter). Blood was sampled from each of four different sites on every patient, four hours after the first heparin bolus. aPTT was determined using a rapid clot detector, point of care device.ResultsA high positive correlation was obtained for aPTT values between CVC and Access sampling sites (r (84) =0.72; p <0 .05) and a low positive correlation between CVC and Arterial sampling site (r (84) =0.46, p < 0.05). When correlated by artery age, the young Artery (1-3 day old) correlates with CVC, Access and Postfilter (r (45) = 0.74, p >0.05). The aPTT values were significantly higher at Postfilter and Arterial sampling site, older than three days, compared to the CVC sampling site (p<0.05).ConclusionConsidering patient bleeding risks and filter life span, the optimal sampling sites for safe assessment of unfractionated heparin anticoagulation on CRRT during CVVHDF were the central venous catheter using heparin free lavage saline solution, a heparinised flushed arterial catheter not older than three days, and a circuit access line.

P1128INTERMITTENT SALINE FLUSH VS CONTINUOUS SALINE INFUSION IN HEPARIN FREE DIALYSIS, A COMPARATIVE ANALYSIS

Abstract Background and Aims Proper anticoagulation during dialysis for the patients with bleeding risk or contraindication for heparin is a challenge. Regional anticoagulation with heparin and protamine or citrate and calcium though accepted has well known complications and has logistic and monitoring problem. Intermittent saline flush is another well accepted techniques. But it is labour intensive and often leads to fiber loss or coagulation of the dialyser. Keeping these in mind we hypothesised continuous prepump saline infusion may be another alternative technique in these situations. In this study We compared the outcome between intermittent saline flush versus continuous saline infusion while dialysing patients without heparine. Method Patients &amp;gt;18 year of age, on mHD @3/wk atleast for 3 months, having mature Avfistula and Hb between 8.5-11.5gm/dl with standard EPO and decided for heparin free dialysis were included for the study. Exclusion criteria were Intradialytic weight gain more than 3kg receiving any blood component transfusion or albumin,parenteral nutritional product infusion, any need for UF /Na/HCO3 profiling. Patients on antiplatelet drug or P-time prolonged &amp;gt; 6sec, APTT &amp;gt; 2 times normal, and platelet count less than 80,000 were also excluded. After inclusion, for each patient 8 session were studied systematically as per the preset protocol. Session 1,3,5,7 was subjected to intermittent saline flush(ISF) where as session 2,4,6,8 were subjected to continuous saline infusion(CSIF). The blood flow during dialysis was kept in the range of 250ml/min to 300ml/min and dialysate flow was kept fixed at 500ml/min. and UF goal was kept between 2.5 to 3.5lit and treatment duration was fixed at 4hrs.In ISF, every 15min 80ml (=priming volume of the dialyser, F6, Polysulphone, Fresenius)0.9%NS was flushed in the dialyser clamping the arterial line where as in CSIF 0.9%NS bottle is connected to the prepump arterial line and saline is infused through saline infusion pump @320ml/hr. The each dialysis session outcome were recorded and analysed. Potential clotting in the bubble trap was visually observed in each hour and graded on a 4 point scale. 1=normal, 2= fibrinous ring,3=clot formation and 4= coagulated system. In either group the total saline infusion volume added in the net UF goal. The dialyser was inspected visually at the end of each session 1= normal,2= a few blood stripes affecting less than 10% of the surface area,3= many blood stripes more than 10% of surface area, 4= coagulated dialyser. URR(as per KDOQI guideline) and kt/v(by diascan software of Gambro AK96 machine) was calculated in each session. Data were analysed with spss v23. Results Total 8 patients(5men 3 women) were included for the study. After exclusion total 54 session of dialysis were finally analysed. The incidence of the of blood clot in the whole dialysis system leading to stopping of dialysis was 3 in ISF and 1 in CSIF (p=0.001).The multiple linear regression analysis with repeated measurememt for the time and UF rate adjusted clot incidence of different grade in bubble trap was significantly low in the CSIF group than the ISF group.(p=0.0001). When adjusted for total body water,net UF,PreHD Urea, blood flow the difference in URR and Kt/v both the group was not significantly different (p=0.8). Among the two group the operational comfort for nurses were significantly better(p=0.3) in the CSIF group than the ISF group. Conclusion While considering heparine free dialysis Continuous saline infusion @320ml/hr is easy less labourintensive method than intermittent saline flush technique with significantly less clotting incidence and without any adverse effect on the dialyser clearance.

4362 The Utilization of Polyethylene Glycol Fusion to Improve Facial Reanimation

OBJECTIVES/GOALS: This study’s goal is to determine whether intraoperative treatment of facial nerves with polyethylene glycol (PEG) fusion technology improves facial paralysis outcomes. Improved facial nerve regeneration in facial paralysis patients would lead to improved recovery time and effectiveness. METHODS/STUDY POPULATION: 30 rats were utilized; 15 underwent facial nerve regeneration without PEG fusion, and 15 with PEG fusion. Facial paralysis was initiated on the left by transection of the buccal and marginal mandibular branches of facial nerve. The buccal branch was repaired though microsuture technique. Neurorrhaphy sites of rats in the PEG group were exposed to calcium free saline, methylene blue, and polyethylene glycol. Nerve continuity was assessed post-operative in 5 animals in each group through electron microscopy. Functionality was assessed in the other 10 per group by EMG and whisker analysis after surgery, and weekly for 8 weeks. At 8 weeks, nerves and distal muscles were histologically analyzed. RESULTS/ANTICIPATED RESULTS: PEG fusion technology immediately restored axonal continuity following surgery, demonstrated by electron microscopy. Electrophysiology was also similarly restored across the site immediately, determined through intraoperative nerve stimulation, in the PEG fusion group. The nonintervention group showed dramatically reduced functional recovery than the PEG fusion group following surgery, shown by lower whisking activity and poor electrophysiology outcomes. Furthermore, the PEG fusion group showed statistically significant higher fascicle counts, myelination diameter, axonal diameter, and distal muscle fibers histologically. DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates that polyethylene fusion technology may improve facial reanimation outcomes. PEG is already a FDA-approved drug, and thus the pathway to translational clinical application of this work may thus be streamlined, bringing new options to patients with facial paralysis.

A subset of taste receptor cells express biocytin‐permeable channels activated by reducing extracellular Ca2+ concentration

Taste receptor cells (type II cells) transmit taste information to taste nerve fibres via ATP-permeable channels, including calcium homeostasis modulator (CALHM), connexin and/or pannexin1 channels, via the paracrine release of adenosine triphosphate (ATP) as a predominant transmitter. In the present study, we demonstrate that extracellular Ca2+ -dependent biocytin-permeable channels are present in a subset of type II cells in mouse fungiform taste buds using biocytin uptake, immunohistochemistry and in situ whole-cell recordings. Type II cells were labelled with biocytin in an extracellular Ca2+ concentration ([Ca2+ ]out )-sensitive manner. We found that the ratio of biocytin-labelled type II cells to type II cells per taste bud was approximately 20% in 2mM Ca2+ saline, and this ratio increased to approximately 50% in nominally Ca2+ -free saline. The addition of 300µM GdCl3 , which inhibits various channels including CALHM1 channels, significantly inhibited biocytin labelling in nominally Ca2+ -free saline, whereas the addition of 20µM ruthenium red did not. Moreover, Cs+ -insensitive currents increased in nominally Ca2+ -free saline in approximately 40% of type II cells. These increased currents appeared at a potential of above -35mV, reversed at approximately +10mV and increased with depolarization. These results suggest that biocytin labels type II cells via ion channels activated by [Ca2+ ]out reduction, probably "CALHM-like" channels, on the basolateral membrane and that taste receptor cells can be categorized into two groups based on differences in the expression levels of [Ca2+ ]out -dependent biocytin-permeable channels. These data indicate electrophysiological and pharmacologically relevant properties of biocytin-permeable channels and suggest their contributions to taste signal transduction.

Hydrogen Peroxide Gates a Voltage-Dependent Cation Current in Aplysia Neuroendocrine Cells.

Nonselective cation channels promote persistent spiking in many neurons from a diversity of animals. In the hermaphroditic marine-snail, Aplysia californica, synaptic input to the neuroendocrine bag cell neurons triggers various cation channels, causing an ∼30 min afterdischarge of action potentials and the secretion of egg-laying hormone. During the afterdischarge, protein kinase C is also activated, which in turn elevates hydrogen peroxide (H2O2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase. The present study investigated whether H2O2 regulates cation channels to drive the afterdischarge. In single, cultured bag cell neurons, H2O2 elicited a prolonged, concentration- and voltage-dependent inward current, associated with an increase in membrane conductance and a reversal potential of ∼+30 mV. Compared with normal saline, the presence of Ca2+-free, Na+-free, or Na+/Ca2+-free extracellular saline, lowered the current amplitude and left-shifted the reversal potential, consistent with a nonselective cationic conductance. Preventing H2O2 reduction with the glutathione peroxidase inhibitor, mercaptosuccinate, enhanced the H2O2-induced current, while boosting glutathione production with its precursor, N-acetylcysteine, or adding the reducing agent, dithiothreitol, lessened the response. Moreover, the current generated by the alkylating agent, N-ethylmaleimide, occluded the effect of H2O2 The H2O2-induced current was inhibited by tetrodotoxin as well as the cation channel blockers, 9-phenanthrol and clotrimazole. In current-clamp, H2O2 stimulated burst firing, but this was attenuated or prevented altogether by the channel blockers. Finally, H2O2 evoked an afterdischarge from whole bag cell neuron clusters recorded ex vivo by sharp-electrode. H2O2 may regulate a cation channel to influence long-term changes in activity and ultimately reproduction.SIGNIFICANCE STATEMENT Hydrogen peroxide (H2O2) is often studied in a pathological context, such as ischemia or inflammation. However, H2O2 also physiologically modulates synaptic transmission and gates certain transient receptor potential channels. That stated, the effect of H2O2 on neuronal excitability remains less well defined. Here, we examine how H2O2 influences Aplysia bag cell neurons, which elicit ovulation by releasing hormones during an afterdischarge. These neuroendocrine cells are uniquely identifiable and amenable to recording as individual cultured neurons or a cluster from the nervous system. In both culture and the cluster, H2O2 evokes prolonged, afterdischarge-like bursting by gating a nonselective voltage-dependent cationic current. Thus, H2O2, which is generated in response to afterdischarge-associated second messengers, may prompt the firing necessary for hormone secretion and procreation.

GPR55-mediated effects on brain microvascular endothelial cells and the blood–brain barrier

GPR55, an atypical cannabinoid receptor activated by lysophosphatidylinositol (LPI) has been involved in various physiological and pathological processes. We examined the effect of GPR55 activation on rat brain microvascular endothelial cells (RBMVEC), an essential component of the blood–brain barrier (BBB). GPR55 was detected in RBMVEC by western blot and immunocytochemistry. Treatment of RBMVEC with LPI increased cytosolic Ca2+ concentration, [Ca2+]i, in a concentration-dependent manner; the effect was abolished by the GPR55 antagonist, ML-193. Repetitive application of LPI induced tachyphylaxis. LPI-induced increase in [Ca2+]i was not sensitive to U-73122, a phospholipase C inhibitor, but was abolished by the blockade of voltage-gated Ca2+ channels or in Ca2+-free saline, indicating that Ca2+ influx was involved in this response. LPI induced a biphasic change in RBMVEC membrane potential: a fast depolarization followed by a long-lasting hyperpolarization. The hyperpolarization phase was prevented by apamin and charibdotoxin, inhibitors of small- and intermediate-conductance Ca2+-activated K+ channels (KCa). Immunofluorescence studies indicate that LPI produced transient changes in tight and adherens junctions proteins and F-actin stress fibers. LPI decreased the electrical resistance of RBMVEC monolayer assessed with Electric Cell-Substrate Impedance Sensing (ECIS) in a dose-dependent manner. In vivo studies indicate that systemic administration of LPI increased the permeability of the BBB, assessed with Evans Blue method. Taken together, our results indicate that GPR55 activation modulates the function of endothelial cells of brain microvessels, produces a transient reduction in endothelial barrier function and increases BBB permeability.

Ameliorating DSS-induced Colitis in Mice Using a Lactobacillus Salivarius Li01 Encapsulated Delivery System (P20-026-19)

ObjectivesThe beneficial role probiotics in lowering colonic inflammation has been demonstrated previously. However, most probiotics are very susceptible to the harsh conditions during GI transit, which significantly affect their functions in the body. Here, we encapsulated the Lactobacillus salivarius Li01 (Li01) in a layer-by-layer MgO doped delivery system (LbL). MethodsLi01 LbL was prepared using alginate, chitosan and MgO, and lyophilized by freeze-drying. The encapsulated Li01 was determined in vitro under simulated GI conditions. The ameliorative effect of the prepared Li01 LbL on the dextran sulfate sodium (DSS)-induced colitis in mice was investigated by oral gavage. Meanwhile, gut microbial variation were also detected through 16 S rRNA genes by Miseq sequencing in order to determine the correlations among probiotics, colitis and gut microbiota. ResultsOur results demonstrated that the Li01 LbL developed in this study improved the viability of Li01 in the GI tract as well as facilitate mucosadhesion on the intestinal surfaces, compared to non-encapsulated Li01. Notably, oral administrations of Li01 LbL reduced DSS-induced mucosal inflammation, as defined by colonic histology and expression of pro-inflammatory markers such as IL-6, TNF-α and IL-10. However, no significant difference was found between the model mice fed with free Li01 and saline buffer, which may be due to low viability of the probiotics. The 16 S rRNA sequencing results also showed an obvious increase of the bacterial abundance in the model fed with Li01 LbL, suggesting a mechanism dependent on the modulation of gut microbiota. ConclusionsThe encapsulation approach plays a critical role for improving efficacy of probiotics in prevention or treatment of gut microbiota-related disease. Funding SourcesNational Key Science and Technology Project of China (2018YFC2000500).

Calcium-calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions.

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

Lithospheric Signature of Late Cenozoic Extension in Electrical Resistivity Structure of the Rio Grande Rift, New Mexico, USA

AbstractWe present electrical resistivity models of the crust and upper mantle from two‐dimensional (2‐D) inversion of magnetotelluric (MT) data collected in the Rio Grande rift, New Mexico, USA. Previous geophysical studies of the lithosphere beneath the rift identified a low‐velocity zone several hundred kilometers wide, suggesting that the upper mantle is characterized by a very broad zone of modified lithosphere. In contrast, the surface expression of the rift (e.g., high‐angle normal faults and synrift sedimentary units) is confined to a narrow region a few tens of kilometers wide about the rift axis. MT data are uniquely suited to probing the depths of the lithosphere that fill the gap between surface geology and body wave seismic tomography, namely the middle to lower crust and uppermost mantle. We model the electrical resistivity structure of the lithosphere along two east‐west trending profiles straddling the rift axis at the latitudes of 36.2 and 32.0°N. We present results from both isotropic and anisotropic 2‐D inversions of MT data along these profiles, with a strong preference for the latter in our interpretation. A key feature of the anisotropic resistivity modeling is a broad (~200‐km wide) zone of enhanced conductivity (&lt;20 Ωm) in the middle to lower crust imaged beneath both profiles. We attribute this lower crustal conductor to the accumulation of free saline fluids and partial melt, a direct result of magmatic activity along the rift. High‐conductivity anomalies in the midcrust and upper mantle are interpreted as fault zone alteration and partial melt, respectively.