Combined Ligation Research Articles

Nanobodys: Strategien zur chemischen Funktionalisierung und intrazelluläre Anwendungen

AbstractNanobodys sind ein modernes Mittel zur Erkennung und Modulation von Antigenen, die für herkömmliche Antikörper unzugänglich sind. Aufgrund ihrer kompakten Form und ihrer hohen Stabilität werden sie häufig in der Grundlagenforschung verwendet. In diesem Aufsatz werden zentrale Aspekte der Funktionalisierung von Nanobodys nebst ausgewählten Anwendungen in der Molekularbiologie dargestellt. Während ältere Konjugationsstrategien auf der zufälligen Modifikation natürlicher Aminosäuren beruhen, basieren neuere Methoden auf ortsspezifischer Modifikation mit funktionellen Einheiten. Solche Techniken umfassen chemoenzymatische Ansätze, Ligation exprimierter Proteine und die Unterdrückung des Amber‐Codons in Kombination mit bioorthogonalen Modifikationsstrategien. Mit einer ständig wachsenden Auswahl an Methoden aus der Proteinsynthese und ‐konjugation sind auch die Anwendungen auf dem Vormarsch. Diese reichen von hochentwickelter Bildgebung und Massenspektrometrie bis hin zum Einschleusen von Nanobodys in lebende Zellen, um intrazelluläre Antigene zu visualisieren und zu manipulieren.

Combined perventricular septal defect closure and patent ductus arteriosus ligation via the lower ministernotomy approach.

Over the past decade, minimally invasive approaches have been advocated for surgical correction of congenital defects to reduce costs related to hospitalization and for improved cosmesis. Minimal skin incisions and partial sternotomy reduce surgical trauma, however these techniques might not be successful in treating a number of congenital pathological conditions, particularly for combined congenital defects. We focused on cases with a combined presentation of ventricular septal defect and patent ductus arteriosus. We studied 12 infants who successfully underwent surgical treatment for a combined single-stage ventricular septal defect and patent ductus arteriosus closure through a lower ministernotomy without using cardiopulmonary bypass and X-rays. No intraoperative and early postoperative complications or mortality were noted. Postoperative echocardiography did not reveal residual shunts. The proposed technique is safe and reproducible in infants.

Tips and pitfalls in direct ligation of large spontaneous splenorenal shunt during liver transplantation.

Patients with large spontaneous splenorenal shunts (SRSs) prove challenging during liver transplantation (LT), regardless of organizing portal vein (PV) thrombosis. Here, we detail the clinical outcomes of 26 patients who underwent direct ligation of large SRSs during LT. Direct ligation of large SRS was applied in poor portal flow during LT. We performed temporary test clamping of the SRS before direct ligation and applied PV pressure monitoring in patients who showed signs of portal hypertension, such as bowel edema. We retrospectively reviewed and evaluated their clinical outcomes. Among 843 patients who underwent LT between 2010 and 2015, 26 (3.1%) underwent direct ligation of SRS without any intraoperative event. Mean preoperative Model for End-Stage Liver Disease score was 16.7 ± 9.0. The main PV diameter on preoperative computed tomography was 8.3 ± 3.4 mm (range, 3.0-14.0 mm). SRS was easily identified at just below the distal pancreas and beside the inferior mesenteric vein in all patients. Accompanying PV thrombectomy was done in 42.3% of patients. Among 26 patients, massive and prolonged ascites was evident in 15.4% (n = 4) postoperatively. They were all living donor LT recipients with a small PV diameter (4.0-6.7 mm). Except for 1 patient who underwent splenic artery embolization, ascites was tolerable and well controlled by conservative management. There was a 7.7% rate of major complications related to direct ligation, including reoperation due to combined ligation of SRS along with a left renal vein at the confluence. Except for 1 hospital mortality due to sepsis, 25 patients (96.2%) are alive with no evidence of further PV complications. In conclusion, direct ligation of large SRS during LT is a safe and feasible method to overcome the effects of a large SRS. Liver Transplantation 23 899-906 2017 AASLD.

Total Chemical Synthesis and Folding of All-l and All-d Variants of Oncogenic KRas(G12V).

The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered "undruggable" due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all-l- and all-d-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-d peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target.

Randomized controlled trial of scleroligation versus band ligation alone for eradication of gastroesophageal varices

Gastric varices develop in 5% to 33% of patients with portal hypertension. Their most common form is concomitant gastroesophageal varices. Scleroligation (combined sclerotherapy and band ligation) has been used successfully in management of esophageal varices but has not been evaluated previously in the management of gastroesophageal varices. The aim of this work was evaluation of a new scleroligation technique for management of bleeding gastroesophageal varices regarding efficacy, adverse events, variceal recurrence, and survival. This study was conducted on 120 cirrhotic patients with bleeding gastroesophageal varices, whom we divided randomly into 2 groups of 60 patients each-a band ligation group and a scleroligation group. The mean number of sessions was lower in the scleroligation group than in the band ligation group (2.22 ± 0.92 and 3.43 ± 0.67, respectively) (P< .001), as were the duration of treatment and total number of bands used. Cost and survival were comparable in the 2 groups. There was no significant difference between the 2 maneuvers regarding adverse events, recurrence rates, or rebleeding rates after obliteration. Recurrence was significantly higher in patients with larger varices, ulceration, and postprocedure pyrexia. Rebleeding was significantly higher among those who experienced postprocedure pyrexia and developed or had worsening of gastric antral vascular ectasia. Scleroligation appears to achieve a faster rate of eradication with fewer treatment sessions and total number of bands deployed to achieve variceal obliteration than band ligation and is comparable in cost and in adverse event and recurrence rates. (Clinical trial registration number: NCT02646202.).

Combined transcatheter aortic valve implantation and type II endoleak repair after endovascular repair for abdominal aortic aneurysm

This report describes an elderly severe aortic stenosis (AS) patient, who had a history of coronary artery bypass grafting and endovascular repair for an abdominal aortic aneurysm (AAA). Type II endoleak with enlargement of AAA was diagnosed and ligation of inferior mesenteric artery (IMA) was recommended. Because aortic valve replacement (AVR) was high risk, we planned transcatheter aortic valve implantation (TAVI). Considering risks of IMA ligation under dual antiplatelet therapy, increased blood pressure after TAVI, and general anesthesia, we performed combined TAVI and IMA ligation. TAVI could be useful for AS patients who are at high risk for AVR before non-cardiac surgery.

Enabling N-to-C Ser/Thr Ligation for Convergent Protein Synthesis via Combining Chemical Ligation Approaches.

In this article, Ser/Thr ligation(on/off) has been realized to enable N-to-C successive peptide ligations using a salicylaldehyde semicarbazone (SAL(off)) group by in situ activation with pyruvic acid of the peptide SAL(off) ester into the peptide salicylaldehyde (SAL(on)) ester. In addition, a peptide with a C-terminal thioester and N-terminal Ser or Thr as the middle peptide segment can undergo one-pot Ser/Thr ligation and native chemical ligation in the N-to-C direction. The utility of this combined ligation strategy in the N-to-C direction has been showcased through the convergent assembly of a human cytokine protein sequence, GlcNAcylated interleukin-25.

A two-color fluorogenic carbene complex for tagging olefins via metathesis reaction

We describe a fluorogenic ruthenium (II) carbene complex in which the chromophore is directly connected to the metal center. The compound introduces a boron dipyrromethene (BODIPY) moiety into target double bonds by metathesis. Tagging of terminal double bonds is demonstrated on immobilized styrene units on a glass surface. We also show that two compounds with distinguishable fluorescence properties are formed in the model reaction with styrene. The outcome of the metathesis reaction is characterized by 19F-NMR, optical spectroscopy, and, finally, single-molecule trajectories. This labeling scheme, in our perception, is of particular interest in the fields of interfacial science and biorthogonal ligation in combination with super-resolution imaging.

Thermogelling and Chemoselectively Cross-Linked Hydrogels with Controlled Mechanical Properties and Degradation Behavior.

Chemoselectively cross-linked hydrogels have recently gained increasing attention for the development of novel, injectable biomaterials given their limited side reactions. In this study, we compared the properties of hydrogels obtained by native chemical ligation (NCL) and its recently described variation termed oxo-ester-mediated native chemical ligation (OMNCL) in combination with temperature-induced physical gelation. Triblock copolymers consisting of cysteine functionalities, thermoresponsive N-isopropylacrylamide (NIPAAm) units and degradable moieties were mixed with functionalized poly(ethylene glycol) (PEG) cross-linkers. Thioester or N-hydroxysuccinimide (NHS) functionalities attached to PEG reacted with cysteine residues of the triblock copolymers via either an NCL or OMNCL pathway. The combined physical and chemical cross-linking resulted in rapid network formation and mechanically strong hydrogels. Stiffness of the hydrogels was highest for thermogels that were covalently linked via OMNCL. Specifically, the storage modulus after 4 h reached a value of 26 kPa, which was over a 100 times higher than hydrogels formed by solely thermal physical interactions. Endothelial cells showed high cell viability of 98 ± 2% in the presence of OMNCL cross-linked hydrogels after 16 h of incubation, in contrast to a low cell viability (13 ± 7%) for hydrogels obtained by NCL cross-linking. Lysozyme was loaded in the gels and after 2 days more than 90% was released, indicating that the cross-linking reaction was indeed chemoselective as the protein was not covalently grafted to the hydrogel network. Moreover, the degradation rates of these hydrogels under physiological conditions could be tailored from 12 days up to 6 months by incorporation of a monomer containing a hydrolyzable lactone ring in the thermosensitive triblock copolymer. These results demonstrate a high tunability of mechanical properties and degradation rates of these in situ forming hydrogels that could be used for a variety of biomedical applications.

Sequential Percutaneous LAA Ligation and Pulmonary Vein Isolation in Patients with Persistent AF: Initial Results of a Feasibility Study.

Left atrial appendage (LAA) ligation results in LAA electrical isolation and a decrease in atrial fibrillation (AF) burden. This study assessed the feasibility of combined percutaneous LAA ligation and pulmonary vein isolation (PVI) in patients with persistent AF. A total of 22 patients with persistent AF underwent LAA ligation with the LARIAT device followed by PVI. PVI was confirmed with the demonstration of both entrance and exit block. Patients (n = 10) in sinus rhythm pre- and post-LAA ligation underwent P-wave analysis. Monitoring for AF was performed at 1, 3, and 6 months postablation. LAA ligation was successful in 21 of 22 (95%) patients. The procedure was aborted in one patient due to pericardial adhesions. PVI was performed in 20 of 21 patients. One patient converted to atrial flutter with a controlled ventricular response after LAA ligation and refused subsequent PVI. Demonstration of entrance and exit block was achieved in 19 of 20 patients. At 3 months, 13 of 19 (68.4%) patients were in sinus rhythm. Four patients underwent a second PVI. At 6 months, 15 of 20 (75%) patients were in sinus rhythm. There was a significant decrease in P-wave duration and P-wave dispersion after LAA ligation. Complications with LAA ligation included pericarditis, a delayed pleural effusion, and a late pericardial effusion. Staged LAA ligation and PVI is feasible and decreases P-wave dispersion. Randomized studies are needed to assess the efficacy of LAA ligation as adjunctive therapy to PVI for maintaining sinus rhythm in patients with persistent AF.

A Chemoselective and Modular Post‐Synthetic Multi‐Functionalization of NHC–Platinum Complexes

AbstractWe report oxime ligation in combination with metal ligand exchange as a novel orthogonal and practical approach to the multifunctionalization of NHC–platinum complexes. This strategy, which enables strong diversity enhancement of metallodrug candidates, could also be applied to selective bioconjugation.

Abstract T P238: Evaluating the Stent-Wall Interaction in a Hemodynamically Induced Swine Model of Carotid Atherosclerosis: Correlation of Pre-Treatment Black-Blood 3T MRI with Post-Stenting Histopathology

Introduction: We evaluated the histopathologic characteristics of the stented arterial segments in a swine model of carotid atherosclerosis. Methods: Atherosclerotic plaque was created by inducing 80% stenosis via partial surgical ligation (without endothelial balloon injury) in combination with dietary hypercholesterolemia in a single Yucatan minipig (Fig A). High-resolution black-blood 3T MRI of both carotid arteries was obtained at 6 months. Stenting was performed immediately after imaging. The animal was sacrificed 4 weeks after stenting. Histopathologic and morphometric analysis was performed. Results: MRI: Figure A. Contrast-enhanced MRA. B. Cross-sectional 2mm views of proton density (PD), T2, T1, T1 post contrast, and time-of-flight (TOF) sequences of the stenotic segments of the R (top) and L (bottom) carotid arteries. The image characteristics of the R carotid stenosis are consistent with an American Heart Association Lesion Type IV/V. No athermoatous features are seen in the L carotid. Histopathology: Moderate lymphocyte proliferation was noted in the narrowest mid-section (N-Mid) from the right carotid, corresponding to the depicted lesion on MRI. This segment had the smallest neointimal/thrombus thickness (Table). Only mild residual atherosclerosis was appreciated within the stent. EEL - external elastic lamina IEL - internal elastic lamina Conclusions: Our findings indicate histologic reversibility of the MRI-defined carotid atherosclerotic lesion four weeks after stenting, possibly due to improved flow within the hemodynamically-induced plaque. Unlike prior studies, neointimal proliferation was least prominent at the pre-existing atheroma.

Latex ligation in treatment of chronic hemorrhoids

We analyzed the results of treatment of 432 patients with chronic hemorrhoids using different variants of latex ligation. New technique including ligation of mucosa and submucosa of low-ampullar rectum providing ligation of hemorrhoidalvessels, lifting and recto-anal repair is developed and suggested. This method is advisable to use in case of chronic internal hemorrhoids stages I and II. The authors recommend simultaneous combined ligation of mucosa of low-ampullar rectum and internal hemorrhoids for stages III and IV. Different variants of latex ligation with external hemorrhoids excision were used in 103 patients. Pointed variants of latex ligation preserve important advantages including mini-invasiveness, simplicity and wide availability, low cost. Good remote results were obtained after these procedures in 87.3% of observations. Suggested tactics extends use of latex ligation and increases its effectiveness in treatment of different stages and forms of chronic hemorrhoids.

Complete genome of a Puumala virus strain from Central Europe.

Puumala virus (PUUV) is one of the predominant hantavirus species in Europe causing mild to moderate cases of haemorrhagic fever with renal syndrome. Parts of Lower Saxony in north-western Germany are endemic for PUUV infections. In this study, the complete PUUV genome sequence of a bank vole-derived tissue sample from the 2007 outbreak was determined by a combined primer-walking and RNA ligation strategy. The S, M and L genome segments were 1,828, 3,680 and 6,550 nucleotides in length, respectively. Sliding-window analyses of the nucleotide sequences of all available complete PUUV genomes indicated a non-homogenous distribution of variability with hypervariable regions located at the 3'-ends of the S and M segments. The overall similarity of the coding genome regions to the other PUUV strains ranged between 80.1 and 84.7 % at the level of the nucleotide sequence and between 89.5 and 98.1 % for the deduced amino acid sequences. In comparison to the phylogenetic trees of the complete coding sequences, trees based on partial segments revealed a general drop in phylogenetic support and a lower resolution. The Astrup strain S and M segment sequences showed the highest similarity to sequences of strains from geographically close sites in the Osnabrück Hills region. In conclusion, a primer-walking-mediated strategy resulted in the determination of the first complete nucleotide sequence of a PUUV strain from Central Europe. Different levels of variability along the genome provide the opportunity to choose regions for analyses according to the particular research question, e.g., large-scale phylogenetics or within-host evolution.

KAHA ligations that form aspartyl aldehyde residues as synthetic handles for protein modification and purification.

Aldehydes are widely recognized as valuable synthetic handles for the chemoselective manipulation of peptides and proteins. In this report, we show that peptides and small proteins containing the aspartic acid semialdehyde (Asa) side chain can be easily prepared by a chemoselective amide-forming ligation that results in the formation of the Asa residue at the ligation site. This strategy employs the α-ketoacid-hydroxylamine (KAHA) ligation in combination with a new isoxazolidine monomer that forms a side-chain aldehyde upon ligation. This monomer is easily prepared on a preparative scale by a catalytic, enantioselective approach and is readily introduced onto the N-terminus of a peptide segment by solid phase peptide synthesis. The ligated product can be further functionalized by bioorthogonal reactions between the aldehyde residue and alkoxyamines or hydrazides. We demonstrated that glucagon aldehyde, an unprotected 29-mer peptide prepared by KAHA ligation, can be site specifically and chemoselectively modified with biotin, dyes, aliphatic oximes, and hydroxylamines. We further describe a simple and high recovery one-step purification process based on the capture of a 29-mer glucagon aldehyde and a 76-mer ubiquitin aldehyde by an alkoxyamine-functionalized polyethylene glycol resin. The peptide or protein was released from the resin by addition of a hydroxylamine to provide the corresponding oximes.

Magnetic resonance imaging characteristics of hepatocyte apoptosis (induced by right portal vein ligation) and necrosis (induced by combined right portal vein and right hepatic artery ligation) in rats.

Using magnetic resonance imaging (MRI), this study aimed to demonstrate the appearance of hepatocytes following ligation of the right portal vein or combined ligation of the right portal vein and right hepatic artery, in a rat model. Ninety adult Sprague-Dawley rats (body weight 250-300 g) were divided into three groups (n = 30 per group): ligation of the right portal vein (Group A); combined ligation of the right portal vein and right hepatic artery (Group B); no intervention to obstruct blood supply (control group). Rats were then randomly subdivided into five groups that underwent examination at 3 h and 1, 3, 7 and 14 days postprocedure (n = 6 per group). Livers were examined by routine MRI, diffusion-weighted imaging (DWI) and magnetic resonance spectroscopy (MRS). After examination, each group of rats was sacrificed. Right hepatic lobes were removed and examined by pathology in six rats per timepoint; transmission electron microscopy (TEM) was undertaken in up to three rats per timepoint. Hepatocyte apoptosis and necrosis, by right portal vein ligation and combined right portal vein and right hepatic artery ligation respectively, were confirmed by pathology and TEM. In Group A, there were slight increases in intensity of T1- and T2-weighted images and in the apparent diffusion coefficient (ADC). In addition there were larger areas under the lactic acid and lipid peaks on MRS. In Group B, there were increased T1 and T2 signals, a decrease in the ADC and an increase of the area under the lactic acid peaks on MRS. Hepatocyte apoptosis and necrosis induced by ligation procedures exhibited specific changes that were evident on MRI, DWI and MRS, and could be detected in vivo by MRI, in a rat model.

Comprehensive chronic lymphocytic leukemia diagnostics by combined multiplex ligation dependent probe amplification (MLPA) and interphase fluorescence in situ hybridization (iFISH).

BackgroundBanding-karyotyping and metaphase-directed-fluorescence-in-situhybridization (FISH) may be hampered by low mitotic index in leukemia. Interphase FISH (iFISH) is a way out here, however, testing many probes at the same time is protracted and expensive. Here multiplex-ligation-dependent-probe-amplification (MLPA) was used retrospectively in chronic lymphocytic leukemia (CLL) samples initially studied by banding cytogenetics and iFISH. Detection rates of iFISH and MLPA were compared and thus a cost-efficient scheme for routine diagnostics is proposed.ResultsBanding cytogenetics was done successfully in 67/85 samples. DNA was extracted from all 85 CLL samples. A commercially available MLPA probe set directed against 37 loci prone to be affected in hematological malignancies was applied. Besides, routine iFISH was done by commercially available probes for following regions: 11q22.3, 12p11.2-q11.1, 13q14.3, 13q34, 14q32.33 and 17p13.1. MLPA results were substantiated by iFISH using corresponding locus-specific probes.Aberrations were detected in 67 of 85 samples (~79%) applying banding cytogenetics, iFISH and MLPA. A maximum of 8 aberrations was detected per sample; however, one aberration per sample was found most frequently. Overall 163 aberrations were identified. 15 of those (~9%) were exclusively detected by banding cytogenetics, 95 were found by MLPA (~58%) and 100 (~61%) by routine iFISH. MLPA was not able to distinguish reliably between mono- and biallelic del(13)(q14.3q14.3), which could be easily identified as well as quantified by routine iFISH. Also iFISH was superior to MLPA in samples with low tumor cell load. On the other hand MLPA detected additional aberrations in 22 samples, two of them being without any findings after routine iFISH.ConclusionsBoth MLPA and routine iFISH have comparable detection rates for aberrations being typically present in CLL. As MLPA can detect also rare chromosomal aberrations it should be used as an initial test if routine cytogenetics is not possible or non-informative. Still iFISH should be used additionally to distinguish mono- from biallelic deletions and also to determine rate of mosaicism for 13q14.2 to 13q14.3. In case MLPA is negative the corresponding CLL samples should be tested at least by iFISH using the standard probe set to.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-014-0079-2) contains supplementary material, which is available to authorized users.

Laparoscopic Tubal Ligation Performed at the Time of Bariatric Surgery

Study Objective: To evaluate all patients who underwent laparoscopic tubal ligation in combination with laparoscopic bariatric surgery. Design: Case controlled retrospective study performed between January 2009 and March 2014. Setting: University based tertiary care medical center. Patients: Four reproductive age women undergoing bariatric surgery with desire for permanent surgical sterilization. Intervention: Laparoscopic vertical gastric sleeve procedure and laparoscopic tubal ligation. Measurements and Main Results: Four combined laparoscopic tubal ligations and gastric sleeve procedures were attempted. All four cases were successfully completed using a minimally invasive approach. The mean age of the patients was 44.25 years; the mean BMI was 49.25 kg/m (mean weight 303.75 lbs). The mean parity was 2.25 and none of the patients were nulliparous. Two patients had one prior cesarean section. Co –morbid conditions included hypertension, sleep apnea, polycystic ovarian disorder, type II diabetes mellitus, and gastro esophageal reflux disease. All patients successfully underwent laparoscopic tubal ligation as well as laparoscopic vertical gastric sleeve procedure. There were no intraoperative complications were noted in any of the cases. Post-surgical complications were reported in one of the four patients. Complications were related to intraabdominal adhesive disease and development of a paraesophageal hernia. This patient underwent lysis of adhesions and paraesophageal hernia repair with mesh. This complications were not thought to be related to the tubal ligation. Conclusion: Performing tubal occlusion at the time of bariatric surgery is a safe and effectiveway to eliminate the risk of future pregnancy. Fifty percent of pregnancies are undesired despite adequate and available contraception. Pregnancy presents additional risk to obese patients and those who have undergone bariatric surgery. It is therefore reasonable to screen patients for their desire for future child bearing and offer combined surgical tubal ligation in patients planning to undergo bariatric weight loss surgery.

Ligation of mouse L4 and L5 spinal nerves produces robust allodynia without major motor function deficit

Spinal nerve L5/L6 ligation (SNL) in rats has become the standard for mechanistic studies of peripheral neuropathy and screening for novel analgesics. Conventional SNL in our hybrid mice resulted in a wide range of allodynia. Anatomical evaluation indicated that a variable number of lumbar vertebrae existed, resulting in L4/L5 or L5/L6 being ligated. Surprisingly, L4/L5 ligation did not result in ipsilateral hind limb paralysis and produced robust allodynia. Following a recent report that the mouse L4 neural segment is homologous with rat L5 we generated L4, L5 or both L4 and L5 (L4/L5) ligations in C57 mice after establishing a modified set of surgical landmarks. In contrast to rats, L4 ligation in these mice did not result in hind limb paralysis. Robust allodynia was observed in all three ligation groups. Nerve degeneration confirmed that L4 and L5, respectively, are primary contributors to the tibial and sural branches of the sciatic nerve in mice. A larger von Frey sensitive area reflected the wider distribution of Wallerian degeneration in the hindlimb of L4- compared to L5-ligated mice. Ligation of mouse L4 and L5 spinal nerves produces consistent, robust neuropathic pain behaviors and is suitable as a model for investigating mechanisms of neuropathic pain and for testing of novel analgesics. Gabapentin, used as a validation drug in neuropathic pain models and as a reference compound for novel analgesics, significantly reduced allodynia in the mice tested (L4/L5 ligations). Given the ease of surgery, robust allodynia, and larger von Frey sensitive area, we conclude that combined ligation of spinal nerves L4 and L5 optimizes the SNL model in mice.

High-Gd-Payload P22 protein cage nanoparticles for imaging vascular inflammation

Methods 1) P22: The P22 protein cage (60 nm) is bioengineered with an internal polymer network with amine functional groups allowing incorporation of ~9100 Gd-DTPA molecules per cage via the amine groups (Figure 1: [1]). This provides a per cage relaxivity of 70000 mMs, superior to Gd-DTPA for the equivalent Gd concentration. 2) Atherosclerosis Models: Both ApoE-deficient (ApoE) and FVB mice were used. ApoE mice develop atherosclerosis enhanced by high-fat diet. FVB mice develop macrophage-rich carotid lesions with carotid ligation in combination with high-fat diet and diabetes induction [2]. 3) P22-polymer-Gd in vivo MR imaging: Mice were injected intravenously with P22-polymer-Gd (N=5, 20 μmol Gd/kg, one-fifth the typical clinical dose) or Magnevist (N=1, 20 μmol Gd/kg). Vascular MRA at 1T was performed (Aspect M2, 500 mT/m, 2500 T/m/s) using 3D-SPGR (TR/TE=12 ms/2.1 ms, slice thickness=1 mm, FOV=5 cm, matrix=128x128, FA=45). Vessel wall MRI at 3T was performed (Signa HDx, GE Healthcare, 50mT/m, 150 T/m/s) with a phased-array mouse coil (RAPID MR International), using a double inversion recovery fast spin echo sequence (TR/TE= 400 ms/15 ms, slice thickness=1mm, FOV=3 cm, matrix= 256x256) up to 24 hours after injection. 4) RGD-targeted P22 ex vivo fluorescence imaging: Molecular targeting of P22 was evaluated by attaching RGD peptides externally, which targets the aVb3 integrin, upregulated on activated macrophages. ApoE mice (N=4) were injected intravenously with RGDP22 or RGD P22 (labeled with Cy5.5, 4 nmol/mouse). Forty-eight hours later, ex vivo fluorescence imaging was performed using Maestro imaging system (Cri, Woburn, MA). Maximum plaque signal intensities were measured and compared.