The role of regional anaesthesia and multimodal analgesia in the prevention of chronic postoperative pain: a narrative review.

Effective prevention of chronic postoperative pain is an important clinical goal, informed by a growing body of studies. Peri-operative regional anaesthesia remains one of the most important tools in the multimodal analgesic toolbox, blocking injury-induced activation and sensitisation of both the peripheral and central nervous system. We review the definition and taxonomy of chronic postoperative pain, its mechanistic basis and the most recent evidence for the preventative potential of multimodal analgesia, with a special focus on regional anaesthesia. While regional anaesthesia targets several important aspects of the mechanistic pathway leading to chronic postoperative pain, evidence for its efficacy is still mixed, possibly owing to the heterogeneity of risk profiles within the surgical patient, but also to variation in techniques and medications reported in the literature.

Protein kinase C isoform expression and activity in the mouse heart.

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.

Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury.

Antagonists of glutamate receptors of the N-methyl-d-aspartate subclass (NMDAR) or inhibitors of nitric oxide synthase (NOS) prevent nervous system plasticity. Inflammatory and neuropathic pain rely on plasticity, presenting a clinical opportunity for the use of NMDAR antagonists and NOS inhibitors in chronic pain. Agmatine (AG), an endogenous neuromodulator present in brain and spinal cord, has both NMDAR antagonist and NOS inhibitor activities. We report here that AG, exogenously administered to rodents, decreased hyperalgesia accompanying inflammation, normalized the mechanical hypersensitivity (allodynia/hyperalgesia) produced by chemical or mechanical nerve injury, and reduced autotomy-like behavior and lesion size after excitotoxic spinal cord injury. AG produced these effects in the absence of antinociceptive effects in acute pain tests. Endogenous AG also was detected in rodent lumbosacral spinal cord in concentrations similar to those previously detected in brain. The evidence suggests a unique antiplasticity and neuroprotective role for AG in processes underlying persistent pain and neuronal injury.

A farnesyltransferase inhibitor attenuates cardiac myocyte hypertrophy and gene expression.

The overexpression of either oncogenic ras or calmodulin in cardiac myocytes can elicit a hypertrophic response, albeit their recruitment by physiologically relevant stimuli remains unresolved. The present study utilized a pharmacological approach to examine the role of ras and calmodulin in norepinephrine- and endothelin-1-stimulated hypertrophy of neonatal rat cardiac myocytes. The pretreatment of cardiac myocytes with the farnesyltransferase inhibitor BMS-191563 (25 microM) increased the level of unfarnesylated ras in the cytosolic fraction, and caused a concomitant 42 +/- 2% decrease in immunodetectable farnesylated ras in the particulate fraction. In parallel, BMS-191563 pretreatment inhibited norepinephrine-mediated 3H-leucine uptake (80 +/- 10% decrease: n = 6; P<0.01), whereas a significant but less pronounced effect on the endothelin-1 response (46 +/- 6% decrease: n = 6; P<0.05) was observed. The calmodulin inhibitor W7 caused a 50 +/- 10% decrease (n = 8; P<0.05) of norepinephrine stimulated protein synthesis, whereas the endothelin-1 response was unaffected. Consistent with the recruitment of ras, BMS-191563 pretreatment attenuated norepinephrine and endothelin-1-stimulated extracellular signal-regulated kinase (ERK) activity. However, PD098059-mediated inhibition of MEK-dependent stimulation of ERK did not alter the hypertrophic response of either agonist. At the molecular level, the pretreatment with either BMS-191563 or W7 attenuated the norepinephrine-mediated increase of prepro-ANP and -BNP mRNA. Likewise, BMS-191563 caused a significant decrease of endothelin-1-mediated expression of the natriuretic peptide mRNAs, but to a lesser extent, as compared to norepinephrine. Thus, the present study has shown the treatment of neonatal rat cardiac myocytes with a farnesyltransferase inhibitor can attenuate the hypertrophic phenotype in response to physiologically relevant stimuli, thereby supporting a role of the small GTP-binding protein ras. Moreover, these data further suggest alternative ras-independent signaling pathways are also implicated in the hypertrophic response, albeit, there appears to exist a stimulus-specific heterogeneity in their recruitment.

Distant upstream regulatory domains direct high levels of beta -myosin heavy chain gene expression in differentiated embryonic stem cells.

Eukaryotic gene transcription takes place in the context of chromatin. In order to study the expression of the beta -myosin heavy chain (MyHC) gene in its appropriate cardiac environment in vitro, embryonic stem cell lines were generated and induced to differentiate into the cardiac lineage. We show that the upstream region of the beta -MyHC gene (-5518 to -2490 relative to the transcriptional start site) directed high levels of transcriptional activity only when stably integrated, but not when expressed extrachromosomally in transient assays. These results are consistent with earlier findings using an in vivo transgenic approach. The expression of beta -MyHC reporter gene constructs was strictly correlated to differentiation status and coincided with the expression of endogenous cardiac marker genes and with morphological differentiation of embryoid bodies in vitro. Using populations of stably transfected cell clones, two domains important for high level expression were identified. The analysis of individual cell clones suggested that the positive regulatory domains act according to the graded model of enhancement. These results show that chromosomal integration is necessary for the appropriate function of the beta -MyHC gene's upstream regulatory region.

Misfolding of HLA-B27 as a result of its B pocket suggests a novel mechanism for its role in susceptibility to spondyloarthropathies.

The MHC class I protein HLA-B27 is strongly associated with susceptibility to spondyloarthropathies and can cause arthritis when expressed in rats and mice, implying a direct role in disease pathogenesis. A prominent hypothesis to explain this role suggests that the unique peptide binding specificity of HLA-B27 confers an ability to present arthritogenic peptides. The B pocket, a region of the peptide binding groove that is an important determinant of allele-specific peptide binding, is thought to be critical for arthritogenicity. However, this hypothesis remains unproven. We show that in addition to its role in peptide selection, the B pocket causes a portion of the pool of assembling HLA-B27 heavy chains in the endoplasmic reticulum to misfold, resulting in their degradation in the cytosol. The misfolding phenotype is corrected by replacing the HLA-B27 B pocket with one from HLA-A2. Our results suggest an alternative to the arthritogenic peptide hypothesis. Misfolding and its consequences, rather than allele-specific peptide presentation, may underlie the strong link between the HLA-B27 B pocket and susceptibility to spondyloarthropathies.

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70-Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Aglycosylated and phosphatidylinositol-anchored MHC class I molecules are associated with calnexin. Evidence implicating the class I-connecting peptide segment in calnexin association.

The endoplasmic reticulum resident protein calnexin interacts with several glycoproteins including class I MHC molecules. Calnexin is thought to retain free class I heavy chains and/or promote their folding and assembly with beta 2-microglobulin and peptide ligand. Whereas with other glycoproteins, Asn-linked glycans seem to be involved in calnexin association, with class I molecules the transmembrane region has been implicated. To critically define the structures on class I molecules that determine their interaction with calnexin, we have studied carbohydrate-deficient and transmembrane-variant class I molecules. Carbohydrate-deficient class I molecules were found to accumulate intracellularly in an open, non-beta 2-microglobulin-associated conformation. However, open as well as conformed class I molecules showed significant calnexin association whether they were aglycosylated or fully glycosylated. Thus, carbohydrate moieties may be necessary for efficient class I folding, but are not required for calnexin association. Calnexin was also found associated with a soluble class I molecule that has a truncated transmembrane segment, demonstrating that membrane attachment of class I is not required for interaction with calnexin. Finally, two isoforms of the class Ib molecule Q7b were compared. Unexpectedly, the glycosylphosphatidylinositol-anchored Q7b isoform was found associated with calnexin, whereas the soluble Q7b isoform was not calnexin associated. These comparisons of Q7b isoforms implicate the class I-connecting peptide segment and not the transmembrane region as a site of interaction with calnexin.

Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Ak molecules. Ak beta TM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Ak conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Ak beta chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Ak beta chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to assess a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the Ak alpha Cy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.

Molecular mechanisms that control expression of the B lymphocyte antigen receptor complex.

The B cell receptor for antigen (BCR) is a complex of membrane immunoglobulin (mIg) and at least two other proteins, Ig alpha (mb-1) and Ig beta (B29). This complex promotes surface expression of the BCR and acts to transduce an activation signal. We have used a system of mu heavy chain constructs transfected into murine B cell lines to probe structure-function relationships in the BCR complex. One mutant mu chain, in which two polar transmembrane residues (Tyr587, Ser588) are replaced with valine, fails to associate with Ig alpha and Ig beta and is incapable of transducing signals as a result of mIg cross-linking. This mutant is expressed on the surface at high levels when transfected into a plasmacytoma line that lacks Ig alpha, whereas wild-type mu is retained in this cell line in the endoplasmic reticulum. Pulse-chase and immunoprecipitation analyses indicate that the mutant is more rapidly released from calnexin than the wild-type mu. Further, transfection of Ig alpha into this Ig alpha-negative cell line allows release of the mu chain from calnexin and surface expression of the BCR. These results identify the transmembrane residues of mu heavy chain that control binding to calnexin and Ig alpha, and suggest that calnexin-dependent intracellular retention is an important control mechanism for expression of the BCR complex.

Class II histocompatibility molecules associate with calnexin during assembly in the endoplasmic reticulum.

Class II histocompatibility antigens are composed of polymorphic alpha and beta polypeptides which associate in the endoplasmic reticulum (ER) with a third, non-polymorphic invariant polypeptide (Ii). The alpha beta Ii complexes are subsequently transported through the Golgi to the endosomes, where the Ii chain dissociates before the alpha beta complex is transported to the cell surface. Results from transport-defective class II expression variant studies suggest that class II intracellular transport is regulated in multiple intracellular compartments. Consistent with this, a large number of studies have demonstrated that protein folding and/or oligomerization is facilitated in the ER by a class of proteins collectively known as molecular chaperones. In this report, we show that the ER-resident protein calnexin associates with human and murine class II antigens. Specifically, calnexin associates in the ER with free Ii polypeptides and partially assembled wild-type class II complexes, including A alpha and/or A alpha Ii complexes, as well as with alpha beta dimers isolated from class II transport-defective cells. Calnexin also physically associates with alpha beta Ii complexes, but not with mature alpha beta dimers. These results suggest that calnexin may regulate class II intracellular transport by facilitating the production of transport competent molecules out of the ER. In addition, we report that the nucleotide sequence of the gene encoding murine calnexin shows a high degree of homology to human IP90 and dog calnexin at both the nucleotide and deduced amino acid sequence level. The isolation of cDNA fragments encoding murine calnexin will allow us to further evaluate the functional consequences of calnexin-class II interaction.

Effects of bone marrow-derived natural suppressor activity on B cell responses to lipopolysaccharide.

Natural suppressor (NS) activity is detected in tissues undergoing intense hematopoietic regeneration. This includes the spleens of mice recovering from total lymphoid irradiation or cyclophosphamide treatment, or after induction of chronic graft-versus-host disease against minor histocompatibility antigens. NS cells are thought to act via an antiproliferative mechanism, based on the observation that NS cells inhibit thymidine ([3H]TdR) uptake by mitogen- or antigen-activated lymphocytes. In the present study, the mechanism of B cell inhibition by NS activity present in normal adult bone marrow is analyzed. [3H]TdR uptake of LPS-stimulated B cells is inhibited by the presence of bone marrow cells (BMC). Consistent with the decrease in DNA synthesis, cell cycle analysis reveals that the majority of B cells fail to exit G0. To determine whether cells in G1 are also susceptible to inhibition by BMC, we tested the ability of cells with NS activity to inhibit the LPS response of either low-density B cells or B cells preactivated by LPS. Both populations of cells were readily inhibited in their uptake of [3H]TdR. Direct analysis of B cell growth in suppressed cultures demonstrates that B cell numbers remain constant, with only 6-30% of the control number of B cells present in cultures containing BMC. Taken together, these results indicate that the antiproliferative effect of NS cells is a result of not only resting B cells being inhibited from entering the cell cycle, but also the inhibition of B cells already in G1.

The effect of chronic graft-versus-host disease on B cell development.

Chronic graft-versus-host disease often results in a combined deficiency of humoral and cell-mediated immunity. Clinical and experimental studies have suggested that the decrease in B cell responsiveness is due to a failure of B cell production in the bone marrow, intrinsic B cell defects, excessive suppressor T cell activity, and deficient T helper activity. In the present study, we analyze the basis of B cell immunodeficiency in C.B-20-->(C.B-20 x B10.D2)F1 animals afflicted with chronic GVHD. The initial decline in B cell production in the BM accounts for the early reduction in the number of B cells in the spleen and BM. Later, as B cells appear in near-normal numbers in the BM, the spleen and lymph node are repopulated by the newly derived B cells. Associated with the appearance of B cells in the BM and spleen is the ability to respond to lipopolysaccharide. In contrast, both B cell populations are severely diminished in their ability to proliferate in response to agar-derived mitogens to form colonies (CFU-B). The reduction in the CFU-B response is most likely a consequence of an inherent B cell defect, since purification of C.B-20-->F1 splenic B cells does not restore the colony-forming potential. Unlike BM and splenic B cells, LN B cells are unable to respond to either mitogen. Taken together, these results imply that a population of B cells derived from a distinct lineage and/or B cell maturation is defective in mice undergoing GVHD.

Effect of graft-versus-host disease on anti-tumor immunity.

BCL1, a spontaneous B cell leukemia of BALB/c origin, is rejected by C.B-20 (Ighb, H-40b) but not BALB/c (Igha, H-40a) mice. Adoptive transfer of C.B-20 anti-BCL1 effector cells specific for the minor histocompatibility Ag H-40a protects irradiated C.B-20 but not BALB/c recipients. Because C.B-20 donor cells could potentially generate graft-vs-host disease (GVHD) in BALB/c recipients, we investigated the possibility that GVHD prevents the anti-tumor effect. GVHD was induced in (C.B-20 X B10.D2)F1 [H-2d, H-40b X H-2d,H-40b] recipients after injection of B10.D2-primed C.B-20 donor cells. GVHD was indicated by the histologic appearance of tissue sections from C.B-20----F1 livers, target organs of GVHD, which showed a marked mononuclear cell infiltrate around the portal tracts and central veins. In addition, splenic lymphocytes from these mice had altered CD4/CD8 ratios and were unable to respond to the polyclonal activators Con A and LPS. The mitogen unresponsiveness was at least partially due to the presence of a suppressor cell, because proliferation of normal spleen cells to Con A and LPS was suppressed upon addition of C.B-20----F1 spleen cells. Further immune dysfunction was evident by the inability of T cells from mice with GVHD to generate a CTL response to H-2 alloantigens. Addition of C.B-20----F1 spleen cells to F1 responder cells at the induction of culture did not prevent generation of CTL, indicating that a suppressor cell was not responsible for the lack of CTL activity. In this setting of GVHD, F1 recipients were able to reject BCL1 upon adoptive transfer of C.B-20 anti-BCL1 effector cells. These data indicate that GVHD-induced immune dysfunction does not inhibit the activity of antileukemia T cells.

Developmental coupling of expression of the Igh-linked minor antigen H-40 to membrane immunoglobulin expression.

C.B-20 (Ighb) but not BALB/c (Igha) mice are able to reject BCL1, a spontaneous B cell leukemia of BALB/c origin. The protective immune response is directed toward the minor histocompatibility (H) antigen, H-40, which can be detected by cytotoxic T lymphocytes and is expressed on surface (s) immunoglobulin positive tumor cells including BCL1 as well as sIg+ lymphoblasts activated by lipopolysaccharide. The fact that only sIg+ tumor cells and lymphoblasts express H-40 suggests that sIg itself may be a component involved in CTL recognition of this antigen. However, this possibility was ruled out by demonstrating that removal of sIg from target cells did not prevent H-40-specific CTL recognition. To determine whether H-40 expression was coordinately regulated with that of membrane Ig, we determined whether H-40- sIg- cells acquire H-40 when induced to express sIg. We also transfected a functional mu c and nu region gene into a sIg- lymphoma so that it acquired a sIg+ phenotype. In neither case did such sIg+ cells acquire H-40. Together, these data indicate that H-40 is expressed on B cells representing a particular stage of differentiation that is coincidental with sIg. We previously showed that H-40 and Igh loci are linked on the 12th murine chromosome. In this study we further localized H-40 to a point beyond Aat (formerly Pre-1) near Lm-1, a locus previously described to encode minor H-antigens expressed on lymphomyeloid cells. Thus, H-40 and Lm-1 may represent a gene cluster encoding antigens expressed on subsets of lymphoid and myeloid cells.

Alterations of hepatic enzyme levels and of the acinar distribution of glutamine synthetase in response to experimental liver injury in the rat.

Glutamine synthetase shows a striking heterogeneous distribution in normal rat liver as consistently revealed by immunohistochemistry using a specific antiserum against the rat liver enzyme or a cross-reacting antiserum. The effects of zonal liver injury induced by allylformate or CCl4 on this distribution and on the activity of glutamine synthetase as well as of enzymes with different acinar distribution were investigated. Treatment with allylformate or CCl4 at appropriate concentrations led to severe hepatocyte necrosis in the periportal and perivenous zone, respectively, as revealed by histological examination and by the levels of serum marker enzymes. Exposure to allylformate (50 to 100 microliter per kg) for less than 1 day did not change the distribution and activity of glutamine synthetase but reduced the specific activities of the urea cycle enzymes. In contrast, treatment with CCl4 (1,000 microliter per kg) strongly reduced the activity and the acinar region covered by glutamine synthetase but not, for instance, the activities of the urea cycle enzymes. These results in conjunction with the data obtained for other enzymes indicate that a short exposure to these hepatotoxins affects different enzyme activities in close accord with their preferential acinar localization. During prolonged exposure this initial response was often modified due to adaptation. In the case of glutamine synthetase, however, no adaptive appearance of glutamine synthetase in other parts of the acinus could be detected even if the cell population originally expressing this phenotype was destroyed. This extremely inflexible distribution suggests that glutamine synthetase expression is a matter of cell differentiation rather than of modulation by nutritional and hormonal factors (or their acinar gradients) as found for many other hepatic enzymes.

Morphometry of canine coronary arteries, arterioles, and capillaries during hypertension and left ventricular hypertrophy.

The mechanisms responsible for the increase in minimal coronary vascular resistance per unit mass of myocardium in animals with chronic hypertension and left ventricular hypertrophy remain unidentified. Because increases in wall thickness of resistance vessels in some vascular beds in response to hypertension may decrease luminal diameter, we hypothesized that similar changes may occur in the coronary vasculature. To test this hypothesis, we performed hemodynamic and morphometric studies on eight dogs with renovascular hypertension (one kidney, one clip) of 6 weeks' duration, and in six normotensive dogs. Hypertension evoked a 27% increase in left ventricular mass and was associated with a 67% increase in left ventricular minimal coronary vascular resistance per 100 g calculated from coronary perfusion measured with microspheres during adenosine infusion. The vasculature was fixed via perfusion of glutaraldehyde and tissue samples from the left ventricle were embedded in Epon. Wall:lumen ratios, determined by light microscopy, of coronary arteries and arterioles were similar in hypertensive and normotensive dogs. Lumen diameters of large epicardial arteries (greater than 640 microns) of hypertensive dogs increased significantly so that wall:lumen ratios were normal despite an increased medial thickness. Ultrastructural analysis, however, showed an enhancement of the relative extracellular compartment of the tunica media of large coronary arteries of hypertensive dogs: 36.4 +/- 3.4% vs. 26.5 +/- 1.6% (mean +/- SEM). Capillary numerical density and surface area (surface area:tissue volume) were significantly lower in the endomyocardium, while capillary volume density (volume:tissue volume) was lower in the midmyocardium and endomyocardium of hypertensive dogs compared to normotensives.(ABSTRACT TRUNCATED AT 250 WORDS)