Behavioral and neuropathological changes in animal models of chronic painful scar.

BACKGROUND: Long-lasting limb pain or back pain after surgery occasionally develops into chronic pain that leads to lower activity and a poorer quality of life for many patients. To determine the histopathological and neuropathological mechanisms that cause persistent post-operative pain, we developed an original animal model with sustained painful scars and then examined pain-related behavior and the pathological alteration of peripheral tissues and spinal nerves associated with the model. METHODS: The animal model (Scar group) was prepared in rats by extensively stripping subcutaneous tissue from the plantar in the hind paw followed by subsequent examination of pain-related behavior over the next 12 weeks. Thereafter, we conducted histological staining of the scar tissues, immunohistochemical staining of c-Fos (L5 dorsal horn), and electron microscopic analysis of the L5 spinal nerve fibers/dorsal roots. RESULTS: The mechanical pain threshold decreased specifically in the ipsilateral plantar in animals with scar. This state was maintained for 12 weeks. A collagen layer developed from fibers derma to the muscular layer in the scar tissue in which many fibroblasts were observed. No statistical differences were found for the areas of the c-Fos-immunoreactive (c-Fos-IR) neurons in the ipsilateral and contralateral sides of the L5 level of the dorsal horn in both the Scar group and Pinhole (sham operation) group. However, myelin sheath fragmentation of the nerve fibers was observed in the ipsilateral dorsal root at the L5 position. CONCLUSIONS: We created a persistent painful scar model through extensive injury of the peripheral tissues. Fibrotic thickening of the cutaneous tissues, possible sensitization, and partial degradation of the spinal nerve related to the painful scar were observed. This model should enable us to better understand the mechanism of sensitization caused by painful scar and investigate new methods for treating painful scars in humans.

Diverging mechanisms of activation of chemokine receptors revealed by novel chemokine agonists.

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions.

Low-dose gabapentin as useful adjuvant to opioids for neuropathic cancer pain when combined with low-dose imipramine.

PURPOSE: Painful neuropathic conditions of cancer pain often show little response to nonopioid and opioid analgesics but may be eased by antidepressants and anticonvulsants. Although gabapentin is effective in the treatment of neuropathic pain in patients with cancer, some patients experience intolerable side effects sufficient to warrant discontinuation. The aim of this study was to see whether low-dose gabapentin is effective in treating cancer-related neuropathic pain when combined with low-dose imipramine. METHODS: Fifty-two cancer patients diagnosed as having neuropathic pain were allocated into four groups: G400-I group took gabapentin 200 mg and imipramine 10 mg every 12 h orally; G400 group took gabapentin 200 mg every 12 h orally; G800 group took gabapentin 400 mg every 12 h orally; I group took imipramine 10 mg every 12 h orally. RESULTS: Low-dose gabapentin-imipramine significantly decreased the total pain score and daily paroxysmal pain episodes. Several patients developed mild adverse symptoms in the four groups, and three patients discontinued treatment due to severe adverse events in the G800 group. CONCLUSION: Low-dose gabapentin-antidepressant combination with opioids was effective in managing neuropathic cancer pain without severe adverse effects.

Incidence and risk factors of postoperative delirium in cervical spine surgery.

STUDY DESIGN: Retrospective clinical review and prospective report of postoperative delirium after cervical spine surgeries. OBJECTIVE: To investigate factors contributing to the development of delirium after cervical surgery and see whether amended therapeutic protocols could improve or alter postoperative outcomes. SUMMARY OF BACKGROUND DATA: Important consequences of postoperative delirium for the orthopedic patients include impaired recovery and increased morbidity and mortality. Although its risk factors have been reported in orthopedic surgery, there are a very few reports regarding postoperative delirium in spine surgery. METHODS: Eighty-one cervical myelopathy patients were retrospectively examined about the incidence of postoperative delirium and the risk factors. Similarly, 41 patients who received postoperative care under modified protocols were prospectively examined. RESULTS: Postoperative delirium occurred more commonly in patients over 70 years and those with hearing impairment. Patients who received high-dose methylprednisolone (>1000 mg) demonstrated an increased incidence of postoperative delirium. Under modified protocol, we reduced the usage of methylprednisolone and encouraged free body movement with cervical orthosis immediately after surgery. The incidence of postoperative delirium was significantly lower under the modified protocol. CONCLUSION: Early commencement of mobilization after cervical spine surgery would be crucial to the prevention of postoperative delirium in the elderly.

Intradermal administration of magnesium sulphate and magnesium chloride produces hypesthesia to mechanical but hyperalgesia to heat stimuli in humans.

BACKGROUND: Although magnesium ions (Mg2+) are known to display many similar features to other 2+ charged cations, they seem to have quite an important and unique role in biological settings, such as NMDA blocking effect. However, the role of Mg2+ in the neural transmission system has not been studied as sufficiently as calcium ions (Ca2+). To clarify the sensory effects of Mg2+ in peripheral nervous systems, sensory changes after intradermal injection of Mg2+ were studied in humans. METHODS: Magnesium sulphate, magnesium chloride and saline were injected into the skin of the anterior region of forearms in healthy volunteers and injection-induced irritating pain ("irritating pain", for short), tactile sensation, tactile pressure thresholds, pinch-pain changes and intolerable heat pain thresholds of the lesion were monitored. RESULTS: Flare formation was observed immediately after magnesium sulphate or magnesium chloride injection. We found that intradermal injections of magnesium sulphate and magnesium chloride transiently caused irritating pain, hypesthesia to noxious and innocuous mechanical stimulations, whereas secondary hyperalgesia due to mechanical stimuli was not observed. In contrast to mechanical stimuli, intolerable heat pain-evoking temperature was significantly decreased at the injection site. In addition to these results, spontaneous pain was immediately attenuated by local cooling. CONCLUSION: Membrane-stabilizing effect and peripheral NMDA-blocking effect possibly produced magnesium-induced mechanical hypesthesia, and extracellular cation-induced sensitization of TRPV1 channels was thought to be the primary mechanism of magnesium-induced heat hyperalgesia.

Expression of brain-type fatty acid-binding protein (fabp7) in medaka during development.

Fatty acid-binding proteins (FABPs) belong to a multigene family of small intracellular proteins that bind hydrophobic ligands. Recent studies have indicated that FABP7 plays important roles in neurogenesis or neuronal migration in vertebrates. In this study, we isolated cDNA and the genomic fragment containing the fabp7 gene for medaka fish and examined the expression of the medaka fabp7 gene through the development of their central nervous system (CNS). The medaka fabp7 gene consists of four exons in approximately 1 kb of the genomic region. Its deduced amino acid sequence exhibits over 80% identity with those of other higher vertebrates. In situ hybridization analysis demonstrated that fabp7-positive cells first appear at stage 22 in a small dorsal domain of the retina, dorsal diencephalon, and rhombencephalon, then expand to the entire CNS including the retina and the spinal cord. In addition, we generated two lines of transgenic medaka with 1.7 kb upstream of the fabp7 gene combined with the enhanced-green fluorescence protein (EGFP) gene. The spatio-temporal expression patterns of EGFP in these animals were consistent with the results of in situ hybridization analysis. The result of our reporter assays with a series of truncated fabp7 promoters suggested that POU elements play a role in fabp7 expression in medaka as well as in other vertebrates. Our transgenic animal will contribute to clarifying the role of FABP7 in the development of CNS.

Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans.

Microcephaly is a malformation associated with in utero exposed atomic bomb survivors and can be induced in mice by fetal exposure to ionizing radiation (IR). The pathogenesis of IR-induced microcephaly, however, has not been fully understood. Our analyses of high-coverage expression profiling (HiCEP) demonstrated that the abnormal spindle-like microcephaly associated gene (ASPM) was down-regulated in irradiated human diploid fibroblasts. ASPM was recently reported as the causative gene for MCPH-5, the most common type of congenital microcephaly in humans. Here, we show that the expression of the Aspm gene was significantly reduced by IR in various human and murine cells. Additionally, Aspm was found downregulated in the irradiated fetal mouse brain, particularly in the ventricular zones. A similar suppression was observed in the irradiated neurosphere cultures. This is the first report suggesting that the suppression of Aspm by IR could be the initial molecular target leading to the future microcephaly formation.

Chemokine signaling specificity: essential role for the N-terminal domain of chemokine receptors.

Chemokine IL-8 (CXCL8) binds to its cognate receptors CXCR1 and CXCR2 to induce inflammatory responses, wound healing, tumorogenesis, and neuronal survival. Here we identify the N-loop residues in IL-8 (H18 and F21) and the receptor N-termini as the major structural determinants regulating the rate of receptor internalization, which in turn controlled the activation profile of ERK1/2, a central component of the receptor/ERK signaling pathway that dictates signal specificity. Our data further support the idea that the chemokine receptor core acts as a plastic scaffold. Thus, the diversity and intensity of inflammatory and noninflammatory responses mediated by chemokine receptors appear to be primarily determined by the initial interaction between the receptor N-terminus and the N-loop of chemokines.

Mcl-1 depletion in apoptosis elicited by ionizing radiation in peritoneal resident macrophages of C3H mice.

Remarkably, apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice, to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins, Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1, but not Bcl-2, Bcl-X(L), Bax, A1, or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand, Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis.

Radio-sensitivity of the cells from amyotrophic lateral sclerosis model mice transfected with human mutant SOD1.

In order to clarify the possible involvement of oxidative damage induced by ionizing radiation in the onset and/or progression of familial amyotrophic lateral sclerosis (ALS), we studied radio-sensitivity in primary cells derived from ALS model mice expressing human mutant SOD1. The primary mouse cells expressed both mouse and the mutant human SOD1. The cell survival of the transgenic mice (with mutant SOD1), determined by counting cell numbers at a scheduled time after X-irradiation, is very similar to that of cells from wild type animals. The induction and repair of DNA damage in the transgenic cells, measured by single cell gel electrophoresis and pulsed field gel electrophoresis, are also similar to those of wild type cells. These results indicate that the human mutant SOD1 gene does not seem to contribute to the alteration of radio-sensitivity, at least in the fibroblastic cells used here. Although it is necessary to consider the difference in cell types between fibroblastic and neuronal cells, the present results may suggest that ionizing radiation is not primarily responsible for the onset of familial ALS with the SOD1 mutation, and that the excess risks are probably not a concern for radiation diagnosis and therapy in familial ALS patients.

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice.

Gamma ray-radiation induced significant apoptosis in peritoneal resident macrophages (PRMs) of C3H/HeJ (C3H) mice, but not in other strains of mice. To investigate the role of DNA damage in the apoptosis, DNA damage was quantified in PRMs by use of the alkaline single-cell gel electrophoresis (Comet) assay. No significant difference was found between C3H and C57Black/6 mice in either radiation-induced DNA damage or repair. Radiation induced apoptosis at the same levels in PRMs of p53 knockout mice and atm knockout mice as those of wild-type C3H mice; however radiation-induced apoptosis was significantly less extensive in the thymocytes of these mutant mice than in those of wild-type mice. Apoptosis was also induced at the same level by an irradiation in PRMs of C3H scid mice as in those of wild-type C3H mice. Therefore it was suggested that radiation-induced DNA damage and TP53, ATM, or DNA-PK-mediated cellular responses occurring downstream thereof were not involved in the radiation-induced apoptotic cell death in C3H mouse PRMs.

Identification of a signal transduction switch in the chemokine receptor CXCR1.

Chemokine receptors belong to the superfamily of G protein-coupled receptors, which regulate the trafficking and activation of leukocytes, and operate as coreceptors in the entry of HIV-1. To investigate the early steps in the signal transmission from the chemokine-binding site to the G protein-coupling region we engineered metal ion-binding sites at putative extracellular sites in the chemokine receptor CXCR1. We introduced histidines into sites located in the second and third putative extracellular loops of CXCR1, creating single, double, and triple mutant receptors: R199H, R203H, D265H, R199H/R203H, R199H/D265H, R203H/D265H, R203H/H207Q, and R199H/R203H/D265H. Cells expressing the double mutants R199H/D265H and R203H/D265H and the triple mutant R199H/R203H/D265H failed to trigger interleukin 8-dependent calcium responses. Interestingly, calcium responses mediated by the single mutant R203H and the double mutants R199H/R203H and R203H/H207Q were blocked by Zn(II), indicating the creation of a functional metal ion-binding site. On the other hand, cells expressing all single, double, or triple histidine-substituted CXCR1 demonstrated high affinity binding to interleukin 8 in the presence and absence of metal ions. These findings indicate that occupation of the engineered metal-binding site uncouples the chemokine-binding site from the activation mechanism in CXCR1. Most importantly, we identify for the first time elements of an early signal transduction switch of chemokine receptors before the activation of cytoplasmic G proteins.

Structural biology of chemokine receptors

Chemokine receptors are G protein-coupled receptors that mediate migration and activation of leukocytes as an important part of a protective immune response to injury and infection. In addition, chemokine receptors are used by HIV-1 to infect CD4 positive cells. The structural bases of chemokine receptor recognition and signal transduction are currently being investigated. High-resolution X-ray diffraction and NMR spectroscopy of chemokines indicate that all these peptides exhibit a common folding pattern, in spite of its low degree of primary-sequence homology. Chemokines' functional motifs have been identified by mutagenesis studies, and a possible mechanism for receptor recognition and activation is proposed, but high-resolution structure data of chemokine receptors is not yet available. Studies with receptor chimeras have identified the putative extracellular domains as the major selectivity determinants. Single-amino acid substitutions in the extracellular domains produce profound changes in receptor specificity, suggesting that motifs in these domains operate as a restrictive barrier to a common activation motif. Similarly HIV-1 usage of chemokine receptors involves interaction of one or more extracellular domains of the receptor with conserved and variable domains on the viral envelope protein gp 120, indicating a highly complex interaction. Elucidating the structural requirements for receptor interaction with chemokines and with HIV-1 will provide important insights into understanding the mechanisms of chemokine recognition and receptor activation. In addition, this information can greatly facilitate the design of effective inmunomodulatory and anti-HIV-1 therapeutic agents