Exploring the Utility of Brain Natriuretic Peptide Measurement in Vascular Surgery.

BACKGROUND: The Canadian Cardiovascular Society 2016 guidelines recommend pre-operative measurement of brain natriuretic peptide (BNP) to risk-stratify patients for a 30-day composite outcome of death, myocardial infarction, or asymptomatic myocardial injury after noncardiac surgery (MINS). Whether this practice affects outcomes is unclear. The aim of this study was to examine the clinical utility of brain natriuretic peptide and myocardial injury after noncardiac surgery. METHODS: Analysis of a prospectively maintained database identified all elective open vascular surgery cases at an academic teaching hospital from January 2015 to December 2018. Pre-operative BNP values were available from June 2018 onward after becoming institutionally mandated. Co-morbidities were also collected to stratify patients using the Revised Cardiac Risk Index. The composite outcome of 30-day mortality, myocardial infarction, or MINS was determined. RESULTS: Prior to BNP becoming an institutionally required test, data was available from 1176 open cases. The 30-day mortality was 1.3% (15/1176) and post-operative myocardial infarction rate was 2.3% (27/1176). BNP measurements were collected in 91 consecutive patients. Ten patients (11%) experienced the composite outcome of mortality, myocardial infarction, or MINS. Elevated BNP was associated with increased odds of the composite outcome (P = 0.04), but not with mortality or myocardial infarction. Revised Cardiac Risk Index score was not predictive of outcomes. The majority of patients who qualified for the composite outcome experienced only an asymptomatic troponin rise (80%). Two patients met the universal definition of myocardial infarction, one of whom died. No other deaths occurred within 30 days. Detection of MINS did not result in any significant changes to patient management. CONCLUSIONS: Elevated BNP correlates with increased MINS. An asymptomatic troponin rise is the most commonly observed event, with unclear clinical implications. BNP may over-estimate surgical risk. Further studies on the long-term outcomes of patients with elevated BNP and MINS are required before widely adopting this strategy in vascular surgery patients.

Molecular and Signaling Mechanisms for Docosahexaenoic Acid-Derived Neurodevelopment and Neuroprotection.

The neurodevelopmental and neuroprotective actions of docosahexaenoic acid (DHA) are mediated by mechanisms involving membrane- and metabolite-related signal transduction. A key characteristic in the membrane-mediated action of DHA results from the stimulated synthesis of neuronal phosphatidylserine (PS). The resulting DHA-PS-rich membrane domains facilitate the translocation and activation of kinases such as Raf-1, protein kinase C (PKC), and Akt. The activation of these signaling pathways promotes neuronal development and survival. DHA is also metabolized in neural tissues to bioactive mediators. Neuroprotectin D1, a docosatriene synthesized by the lipoxygenase activity, has an anti-inflammatory property, and elovanoids formed from DHA elongation products exhibit antioxidant effects in the retina. Synaptamide, an endocannabinoid-like lipid mediator synthesized from DHA in the brain, promotes neurogenesis and synaptogenesis and exerts anti-inflammatory effects. It binds to the GAIN domain of the GPR110 (ADGRF1) receptor, triggers the cAMP/protein kinase A (PKA) signaling pathway, and activates the cAMP-response element binding protein (CREB). The DHA status in the brain influences not only the PS-dependent signal transduction but also the metabolite formation and expression of pre- and post-synaptic proteins that are downstream of the CREB and affect neurotransmission. The combined actions of these processes contribute to the neurodevelopmental and neuroprotective effects of DHA.

Midterm Performance of a Guided-Motion Bicruciate-Stabilized Total Knee System: Results From the International Study of Over 2000 Consecutive Primary Total Knee Arthroplasties.

BACKGROUND: The JOURNEY II Bi-Cruciate Stabilizing Total Knee System (BLINDED) is a second-generation guided-motion knee implant that has been used in over 100,000 primary total knee arthroplasties (TKAs) worldwide. However, performance information is limited. METHODS: Data for 2059 primary TKAs were abstracted at 7 US and 3 European sites. Estimates of cumulative incidence of revision were compared with registry data for cemented posterior-stabilized implants. RESULTS: Average age was 64.3 years (range, 18-91); 58.5% were females; and 12.3% TKAs were in subjects younger than 55 years. Patellae were resurfaced in 95.9%. Median time since primary TKA was 4.2 years; longest was 6.1 years; and 78.9% were 3 years or more since primary TKA. Of 67 revisions (3.2%), 20 (30%) involved femoral or tibial component removal compared to 42% in the Australian Joint Registry (Australian Orthopedic Association National Joint Replacement Registry). All-component revisions accounted for 15 of 67, femoral component only for 2 of 67, tibial component only for 3 of 67, patellar component with/without tibial insert exchange for 17 of 67, and isolated tibial insert exchange for 30 of 67. In addition, there were 18 reoperations without component exchange. Component revision indications were infection (33%), mechanical loosening (21%), fracture of bone around the joint (16%), and instability (15%). Kaplan-Meier revision estimate was 3.1 and 3.6 per 100 TKAs at 3 and 5 years, respectively, compared to Australian Orthopedic Association National Joint Replacement Registry estimates of 3.1 and 4.1 per 100 TKAs. CONCLUSION: The revision rate for the second-generation implant was similar to cemented posterior-stabilized registry controls.

Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry.

INTRODUCTION: Medicinal plants have played an important role in maintaining human health for thousands of years. However, the interactions between the active components in medicinal plants and some certain biological targets during a disease are still unclear in most cases. OBJECTIVE: To conduct the high-throughput screening for small active molecules that can interact with biological targets, which is of great theoretical significance and practical value. METHODOLOGY: The ultrafiltration mass spectrometry (UF-LC/MS) is a powerful bio-analytical method by combining affinity ultrafiltration and liquid chromatography-mass spectrometry (LC/MS), which could rapidly screen and identify small active molecules that bind to biological targets of interest at the same time. Compared with other analytical methods, affinity UF-LC/MS has the characteristics of fast, sensitive and high throughput, and is especially suitable for the complicated extracts of medicinal plants. RESULTS: In this review, the basic principle, characteristics and some most recent challenges in UF-LC/MS have been demonstrated. Meanwhile, the progress and applications of affinity UF-LC/MS in the discovery of the active components from natural medicinal plants and the interactions between small molecules and biological target proteins are also briefly summarised. In addition, the future directions for UF-LC/MS are also prospected. CONCLUSION: Affinity UF-LC/MS is a powerful tool in studies on the interactions between small active molecules and biological protein targets, especially in the high-throughput screening of active components from the natural medicinal plants.

Integration of phosphoproteomic, chemical, and biological strategies for the functional analysis of targeted protein phosphorylation.

Reversible phosphorylation, tightly controlled by protein kinases and phosphatases, plays a central role in mediating biological processes, such as protein-protein interactions, subcellular translocation, and activation of cellular enzymes. MS-based phosphoproteomics has now allowed the detection and quantification of tens of thousands of phosphorylation sites from a typical biological sample in a single experiment, which has posed new challenges in functional analysis of each and every phosphorylation site on specific signaling phosphoproteins of interest. In this article, we review recent advances in the functional analysis of targeted phosphorylation carried out by various chemical and biological approaches in combination with the MS-based phosphoproteomics. This review focuses on three types of strategies, including forward functional analysis, defined for the result-driven phosphoproteomics efforts in determining the substrates of a specific protein kinase; reverse functional analysis, defined for tracking the kinase(s) for specific phosphosite(s) derived from the discovery-driven phosphoproteomics efforts; and MS-based analysis on the structure-function relationship of phosphoproteins. It is expected that this review will provide a state-of-the-art overview of functional analysis of site-specific phosphorylation and explore new perspectives and outline future challenges.

Post-translational modification by ß-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).

Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ß-lysyl-lysine. Here we present biochemical evidence for ß-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The ß-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for ß-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing ß-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ∼4-fold over the preparations containing unmodified EF-P and/or α-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the ß-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.

Protocol for identifying physiologically relevant binding proteins of G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners. For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1.

Interaction between GPR110 (ADGRF1) and tight junction protein occludin implicated in blood-brain barrier permeability.

Activation of adhesion receptor GPR110 by the endogenous ligand synaptamide promotes neurogenesis, neurite growth, and synaptogenesis in developing brains through cAMP signal transduction. However, interacting partners of GPR110 and their involvement in cellular function remain unclear. Here, we demonstrate using chemical crosslinking, affinity purification, and quantitative mass spectrometry that GPR110 interacts with the tight junction adhesion protein occludin. By removing non-specific partners by comparing the binding proteins of GPR110 WT and an inactive mutant exhibiting impaired surface expression, occludin was distinguished as a true binding partner which was further confirmed by reciprocal co-immunoprecipitation assay. Deletion of GPR110 in mice led to the disruption of blood-brain barrier (BBB) and reduced occludin phosphorylation at Y285 in the brain. The Y285 phosphorylation increased upon the ligand-induced activation of GPR110. These data suggest an important role of GPR110-occludin interaction in BBB function and association of previously unknown GPR110-dependent occludin phosphorylation at Y285 with BBB integrity.

N-Docosahexaenoylethanolamide promotes development of hippocampal neurons.

DHA (docosahexaenoic acid, C22:6,n-3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n-3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.

Effects of docosahexaenoic acid on mouse brain synaptic plasma membrane proteome analyzed by mass spectrometry and (16)O/(18)O labeling.

Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC-ESI-MS/MS and (16)O/(18)O labeling. The DHA level in mouse brains was lowered by dietary depletion of n-3 fatty acids, and SPM was prepared by differential centrifugation followed by osmotic shock. SPM proteins from DHA-adequate and depleted brains were analyzed by nanoLC-ESI-MS/MS after SDS-PAGE, in-gel digestion, and differential O(18)/O(16) labeling. This strategy allowed comparative quantitation of more than 200 distinct membrane or membrane-associated proteins from DHA-adequate or depleted brains. We found that 18 pre- and postsynaptic proteins that are relevant to synaptic physiology were significantly down-regulated in DHA-depleted mouse brains. The protein network analysis suggests involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency.

Risk Factors for Herpes Zoster Infection: A Meta-Analysis.

BACKGROUND: The burden of herpes zoster (HZ) is significant worldwide, with millions affected and the incidence rising. Current literature has identified some risk factors for this disease; however, there is yet to be a comprehensive study that pools all evidence to provide estimates of risk. Therefore, the purpose of this study is to identify various risk factors, excluding immunosuppressive medication, that may predispose an individual to developing HZ. METHODS: The literature search was conducted in MEDLINE, EMBASE, and Cochrane Central, yielding case control, cohort, and cross-sectional studies that were pooled from January 1966 to September 2017. Search terms included the following: zoster OR herpe* OR postherpe* OR shingle* AND risk OR immunosupp* OR stress OR trauma OR gender OR ethnicity OR race OR age OR diabetes OR asthma OR chronic obstructive pulmonary disease OR diabetes. Risk ratios (RRs) for key risk factors were calculated via natural logarithms and pooled using random-effects modeling. RESULTS: From a total of 4417 identified studies, 88 were included in analysis (N = 3, 768 691 HZ cases). Immunosuppression through human immunodeficiency virus/acquired immune deficiency syndrome (RR = 3.22; 95% confidence interval [CI], 2.40-4.33) or malignancy (RR = 2.17; 95% CI, 1.86-2.53) significantly increased the risk of HZ compared with controls. Family history was also associated with a greater risk (RR = 2.48; 95% CI, 1.70-3.60), followed by physical trauma (RR = 2.01; 95% CI, 1.39-2.91) and older age (RR = 1.65; 95% CI, 1.37-1.97). A slightly smaller risk was seen those with psychological stress, females, and comorbidities such as diabetes, rheumatoid arthritis, cardiovascular diseases, renal disease, systemic lupus erythematosus, and inflammatory bowel disease compared with controls (RR range, 2.08-1.23). We found that black race had lower rates of HZ development (RR = 0.69; 95% CI, 0.56-0.85). CONCLUSIONS: This study demonstrated a number of risk factors for development of HZ infection. However, many of these characteristics are known well in advance by the patient and clinician and may be used to guide discussions with patients for prevention by vaccination.

Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.

Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.

Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano-electrospray ionization mass spectrometry.

Amide hydrogen exchange coupled to nano-electrospray ionization mass spectrometry (nano-ESI-MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D(2)O buffer, digested with pepsin, and analyzed by nano-ESI-MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four beta-strand (beta1, beta2 beta5 and beta6) regions containing membrane-binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these beta-strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning alpha-helix J region and the C-terminal locus (T450-470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival.

Multiple pathways involved in the biosynthesis of anandamide.

Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5' phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD(-/-) mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.

Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt.

Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50 = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.

Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.

Mass spectrometry with chemical cross-linking was used to probe the conformational changes of HSA (human serum albumin) in solution on interaction with monounsaturated OA (oleic acid) or polyunsaturated AA (arachidonic acid) or DHA (docosahexaenoic acid). Fatty acid-free or -bound HSA was modified with lysine-specific cross-linkers and digested with trypsin. Cross-linked peptides were analysed by nano-electrospray ionization MS to localize the sites of cross-linking. Our data indicated that a local conformational change involving movement of the side chains of Lys-402 of subdomain IIIA or Lys-541 of subdomain IIIB occurred upon binding of all three fatty acids. Our data also indicated that the side chains of Lys-205 (IIA) and Lys-466 (IIIA) moved closer towards each other upon binding AA or DHA, but not OA, suggesting that the conformations of HSA when bound to mono- and poly-unsaturated fatty acids are distinctively different. While these observations agreed with previous X-ray crystallographic studies, the distances between epsilon-amino groups of most cross-linked lysine pairs were shorter than the crystal structure predicted, possibly reflecting a discrepancy between the solution and crystal structures. This method can serve as a useful complement to X-ray crystallography, particularly in probing the structure of a protein in solution.

Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function.

Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function.

Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.

Topical high-molecular-weight hyaluronan and a roofing barrier sheet equally inhibit postlaminectomy fibrosis.

BACKGROUND CONTEXT: The relevance of epidural fibrosis to failed back surgical outcomes remains controversial. Previous studies on the correlation between epidural fibrosis and clinical outcome after laminectomy are inconclusive, and clinical approaches applied to reduce postlaminectomy spinal canal scarring have produced mixed outcomes. PURPOSE: Improved preclinical models are required to address the fundamental question of the relationship between postlaminectomy fibrosis and chronic pain. This study is directed at establishing small animal postlaminectomy models characterized by significantly reduced scar within the spinal canal postoperatively. Such preclinical models are offered as a platform for future studies to explore the potential relationship between postlaminectomy epidural fibrosis and persistent neuropathy with its potential for altered spinal mechanisms for pain processing, so-called spinal facilitation. Such experiments could be constructed in these models for comparison of pain behavior and its underlying neurochemistry both in the presence and absence of extensive postlaminectomy epidural scar. STUDY DESIGN/SETTING: A modified rat laminectomy model was employed to assess epidural fibrosis using a quantitative biochemical collagen assessment approach along with correlative histology. This group served as the control for comparison with groups in which antifibrotic measures were employed. We compared antifibrotic efficacy of a bioabsorbable roofing barrier sheet placed over the laminectomy defect with topical high-molecular-weight hyaluronan (HMW HA) gel, each applied postoperatively to prevent proliferative epidural scarring. Routine biomechanical tensile strength testing was employed to assess wound-healing strength. METHODS: A bilateral laminectomy (L5 and L6) with associated unilateral disc injury (L5-L6) was performed in 98 male Harlan Sprague-Dawley rats. The laminectomy models described incorporated a unilateral disc injury at L5-L6 because herniated disc material has been shown to contribute proinflammatory cytokines in the postoperative wound. Five groups were employed for the study: 1) normal controls without surgery; 2) a laminectomy-disc injury group without treatment; 3) a laminectomy-disc injury group treated with topical HMW HA gel; 4) a laminectomy-disc injury group treated with 0.2-mm thick bioabsorbable roofing barrier sheet in which a protected space was maintained between overlying paraspinous muscles and the dura and 5) a 0.02-mm thin barrier sheet treatment group in which the sheet was placed directly on the dura. The animals were sacrificed at 3- and 8-week postoperative intervals for analysis. The dissected specimens were studied biochemically for hydroxyproline content to estimate total collagen within the canal and on the dura between L4 and L7. Additional specimens were prepared histologically and stained with Masson-Goldner Trichrome stain to confirm presence of proliferative collagen and to describe the presence or absence of wound-healing scar adherence to the dura. The surgical incisions were studied biomechanically by uniaxial tensile testing to determine ultimate force, strain and prefailure stiffness. Statistics were performed using analysis of variance. RESULTS: Gross appearance and histology studies showed that the untreated laminectomy group demonstrated postoperative scar formation that is adherent between the wound and the dorsum of the dura mater in both 3- and 8-week groups. Proliferative scar was substantially increased grossly between the 3- and 8-week intervals. By gross observation there was adherence of the L5 spinal nerve to the underlying disc and adjacent pedicle on the disc injury side. Gross observation of treatment groups, in contrast, disclosed that both the 0.2-mm thick roofing barrier sheet and topical HMW HA gel each prevented scar attachment to the dural sleeve at both the 3- and 8-week postoperative intervals. Furthermore, both the HMW HA gel and 0.2-mm thick roofing barrier sheet treatment groups had significant reduction of total collagen content in the laminectomy specimens measured biochemically at the two time periods compared with the untreated controls. Histologically, the HMW HA gel and the 0.2-mm thick barrier sheet findings were consistent with the gross observations concerning lack of adherence between scar of the overlying wound and the dura. Notably, both the 0.2- and the 0.02-mm barrier sheets became enveloped by a fibrotic envelope consistent with a foreign body reaction. In the group in which the 0.02-mm thin sheet was placed within the canal on top of the dura, there was an increase of fibrosis around the sheet within the canal leading to a space-occupying mass within the canal. Although the 0.2-mm thick roofing barrier placed external to the canal became enveloped by scar, it appeared to attract proliferative scar away from the epidural space, leaving the dura relatively free of scarring or adherence to overlying tissues. The mechanical properties of the incisional wound increased significantly between 3 and 8 weeks. The ultimate strength, stress, strain and stiffness of the several groups were similar at each time point. CONCLUSION: These results provide two preclinical rat laminectomy models of potential usefulness for the future study of the relevance of epidural fibrosis to behaviorally defined pain states, and for the study of the potential of an altered neurochemical signature in postlaminectomy pain conditions. Such preclinical models have become standard in studies of pain behavior and its neurochemistry in preclinical sciatic nerve and spinal nerve injury models, and should be of utility in the studies of postlaminectomy fibrosis. There was progressive scar proliferation and maturation in the untreated postlaminectomy group in the postoperative interval between 3 and 8 weeks. HMW HA gel applied topically and a 0.2-mm thick bioabsorbable Macropore sheet used as a roofing barrier each significantly reduced postlaminectomy proliferative scar without affecting the integrity of incisional wound healing. However, if the 0.02-mm thin barrier sheet used in this study is placed within the canal in contact with the dura and adjacent to the pedicles, the process of reabsorption results in a fibrotic mass within the canal. The preferred barrier sheet placement for this model is clearly in a roofing position bridging over the open epidural space. It must be placed in a manner to block off the paraspinous muscle healing response and still leave a gap between the sheet and the dura.

Topical high molecular weight hyaluronan reduces radicular pain post laminectomy in a rat model.

BACKGROUND CONTEXT: A controversy exists about the mechanism of causation of the post-laminectomy pain syndrome. Some believe that epidural scarring, and attendant spinal nerve and nerve root scarring and tethering to the disc or pedicle at the site of surgery contributes to post-laminectomy pain in such patients. However, clinical outcome studies on this question are inconclusive and the assertion remains controversial. Definitive studies to help resolve the question are needed. Previously our laboratory has reported on a preclinical post-laminectomy model that mimics the postoperative proliferative fibrotic response grossly, as well as by biochemical assessment of the collagen content within the spinal canal. The post-laminectomy fibrotic response was attenuated in that study by application of a topical antifibrotic (high molecular weight hyaluronan gel) or by insertion of an absorbable roofing barrier (0.2-mm-thick Macropore sheet material) over the laminectomy defect before wound closure. The question remains of relevance of the attenuation of the fibrotic response to post-laminectomy chronic pain syndromes. PURPOSE: The purpose of this study is to evaluate the effect of therapeutic attenuation of proliferative scar within the spinal canal post laminectomy on the pain-related behavioral response in a preclinical rat model. STUDY DESIGN/SETTING: An established L5-L6 rat laminectomy model with a unilateral L5-6 disc injury was employed to assess postoperative proliferative fibrosis of the L5 spinal nerves using quantitative biochemical hydroxyproline assessment of the collagen content in four experimental groups. These observations were correlated with gross descriptions of spinal nerve scarring or tethering. Associated manifestations of a sensory pain-related response in the L5 spinal nerve receptor area of the hind paws was studied using standard tactile allodynia assessment with the von Frey hair technique. The tactile allodynia findings were supplemented by weekly descriptors of behavioral pain manifestations. METHODS: Bilateral laminectomies at L5 and L6 and a unilateral right disc injury (L5-6) were performed on 35 male adult Sprague-Dawley rats, weighing 400+ grams (approved by the VA Institutional Animal Care Use Committee). The study consisted of four groups: 1) normal nonoperative control; 2) a sham-operated group; 3) an untreated laminectomy-disc injury group; and 4) a laminectomy-disc injury treatment group in which 0.1 cc topical high molecular weight hyaluronan (HMW HA) gel was layered over the dura and into the laminectomy canal before closure. Before animals were entered into the study, they were checked for the presence of abnormal response to the tactile testing procedure of the L5 sensory receptor area. Animals exhibiting anomalous responses were excluded from the study. Behavioral testing for tactile allodynia was performed at weekly intervals post laminectomy beginning at 3 weeks. Pain-related behavior was characterized at weekly intervals. A behavioral test cage with a wire mesh floor allowed for tactile allodynia testing. Graduated von Frey hairs whose stiffness increased logarithmically from 0.41 to 15 g were used for tactile allodynia tests. The animals were killed 8 weeks postoperatively for analysis. The dissected spinal nerve and nerve root specimens were studied biochemically for hydroxyproline content to estimate total collagen in and around the L5 neural structures. Statistical analyses were performed using analysis of variance and a Fisher comparison t test. RESULTS: The major observations on the untreated preclinical post-laminectomy rat model previously described by this laboratory were confirmed. All untreated animals developed a tail contracture concave toward the right (disc injury side) consistent with asymmetrical lumbar muscle spasm. Only one animal in the HA gel treatment group had a tail contracture. It was of mild degree and occurred in an animal that demonstrated slightly increased right L5 tactile sensitivity. Gross inspection of the dissected specimens demonstrated spinal nerve scarring and tethering to the disc and pedicle greater on the right than the left in untreated animals, findings that were markedly reduced in the treatment group. Collagen content of the L5 spinal nerve and nerve roots with attached scar were significantly lower in the HA gel treatment group than in the untreated laminectomy group (p=.0014). Pain behavioral testing of the L5 receptor area of the right hind paw in the untreated laminectomy group showed markedly increased sensitivity to tactile allodynia testing compared with the corresponding limb of the control group (p=.0001), to the corresponding limb of the sham group (p=.0001), and compared with the HMW HA gel treatment group (p=.0010). Comparisons of the pain behavioral data between the sham and the post-laminectomy HA gel treatment group and the control animals lacked statistical significance. CONCLUSION: This study supports the concept of a relationship between perineural fibrosis and radicular neuropathy in the model described, and emphasizes the role of disc injury and spinal nerve retraction in the post-laminectomy fibrotic process. Furthermore, it shows promise for preliminary assessment of interventions with other anti-inflammatory agents, for characterization of the neurochemical profile of the post-laminectomy pain state, and for exploration of newer pharmaceutical agents potentially useful in the prevention or management of the post-laminectomy syndrome. Post-laminectomy scar is but one of many potential causes of the post-laminectomy pain syndrome. Furthermore, a cautionary note must be emphasized as in all studies using preclinical models, conclusions drawn from the studies cannot be extended directly to patients without confirmatory clinical follow-up studies.