Nuclear Charge Radius of

Collinear laser spectroscopy was performed on the isomer of the aluminium isotope ^{26m}Al. The measured isotope shift to ^{27}Al in the 3s^{2}3p ^{2}P_{3/2}^{○}→3s^{2}4s ^{2}S_{1/2} atomic transition enabled the first experimental determination of the nuclear charge radius of ^{26m}Al, resulting in R_{c}=3.130(15) fm. This differs by 4.5 standard deviations from the extrapolated value used to calculate the isospin-symmetry breaking corrections in the superallowed ß decay of ^{26m}Al. Its corrected Ft value, important for the estimation of V_{ud} in the Cabibbo-Kobayashi-Maskawa matrix, is thus shifted by 1 standard deviation to 3071.4(1.0) s.

Discovery of an Exceptionally Strong ß-Decay Transition of

A significant fraction of stars between 7 and 11 solar masses are thought to become supernovae, but the explosion mechanism is unclear. The answer depends critically on the rate of electron capture on ^{20}Ne in the degenerate oxygen-neon stellar core. However, because of the unknown strength of the transition between the ground states of ^{20}Ne and ^{20}F, it has not previously been possible to fully constrain the rate. By measuring the transition, we establish that its strength is exceptionally large and that it enhances the capture rate by several orders of magnitude. This has a decisive impact on the evolution of the core, increasing the likelihood that the star is (partially) disrupted by a thermonuclear explosion rather than collapsing to form a neutron star. Importantly, our measurement resolves the last remaining nuclear physics uncertainty in the final evolution of degenerate oxygen-neon stellar cores, allowing future studies to address the critical role of convection, which at present is poorly understood.

Reduced expression of lipopolysaccharide-induced CXC chemokine in Porphyromonas gingivalis-induced experimental periodontitis in matrix metalloproteinase-8 null mice.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase-8 (MMP-8) is a central mediator in chronic periodontitis. Recently developed MMP-8-deficient mice show an impaired polymorphonuclear neutrophil response and more severe alveolar bone loss in Porphyromonas gingivalis-induced experimental periodontitis. The main mediators involved in neutrophil and monocyte/macrophage recruitment and in bone loss include lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), stromal-derived factor-1/CXC chemokine ligand 12 (SDF1/CXCL12) and RANKL. Therefore, the aim of this study was to characterize the expression of LIX/CXCL5, SDF1/CXCL12 and RANKL in Porphyromonas gingivalis-induced experimental periodontitis in MMP-8⁻/⁻ (knockout) and wild-type mice. MATERIAL AND METHODS: MMP-8 null and WT P. gingivalis-infected and uninfected mice were included. Histopathological changes were assessed and LIX/CXCL5, SDF1/CXCL12 and RANKL were immunodetected and quantified. RESULTS: Typical histopathological features of chronic periodontitis were seen in P. gingivalis-infected groups. LIX/CXCL5 expression was restricted to the gingival papilla in all four groups. Significantly lower expression of LIX/CXCL5 was seen in the knockout group compared with the wild-type infected group (p < 0.05). SDF1/CXCL12 and RANKL expression was mainly localized to the alveolar crest, including inflammatory leukocytes, vascular endothelium, osteoblasts and osteoclasts. Significant increases of SDF1/CXCL12 and RANKL were seen in both knockout and wild-type P. gingivalis-infected groups compared with uninfected groups (p < 0.05). CONCLUSION: RANKL and SDF1/CXCL12 are up-regulated in P. gingivalis-induced periodontitis and they appear to be associated with the pathogenesis of the disease. MMP-8 is associated with a reduced expression of LIX/CXCL5 in the P. gingivalis-induced experimental periodontitis model.

Evidence of a sudden increase in the nuclear size of proton-rich silver-96.

Understanding the evolution of the nuclear charge radius is one of the long-standing challenges for nuclear theory. Recently, density functional theory calculations utilizing Fayans functionals have successfully reproduced the charge radii of a variety of exotic isotopes. However, difficulties in the isotope production have hindered testing these models in the immediate region of the nuclear chart below the heaviest self-conjugate doubly-magic nucleus 100Sn, where the near-equal number of protons (Z) and neutrons (N) lead to enhanced neutron-proton pairing. Here, we present an optical excursion into this region by crossing the N = 50 magic neutron number in the silver isotopic chain with the measurement of the charge radius of 96Ag (N = 49). The results provide a challenge for nuclear theory: calculations are unable to reproduce the pronounced discontinuity in the charge radii as one moves below N = 50. The technical advancements in this work open the N = Z region below 100Sn for further optical studies, which will lead to more comprehensive input for nuclear theory development.

Involvement of a disintegrin and a metalloproteinase 8 (ADAM8) in osteoclastogenesis and pathological bone destruction.

OBJECTIVES: The eventual role of a disintegrin and a metalloproteinase 8 (ADAM8) in osteoclastogenesis was studied in erosive rheumatoid arthritis (RA) and in vitro. METHODS: ADAM8 protein and mRNA expression was measured in RA pannus and synovitis and compared to osteoarthritic (OA) synovial membrane. Human monocytes were isolated and stimulated with proinflammatory cytokines and their ADAM8 expression and surface ADAM8 were measured. Human peripheral blood monocytes and RAW 264.7 mouse monocyte/macrophage cells were stimulated to osteclast like-cells, and their expression of ADAM8 and osteoclastic markers (calcitonin receptor, integrin beta 3, cathepsin K, TRAP) were analysed. Transfection and small interfering RNA (siRNA) were used to assess the role of ADAM8 in formation of polykaryons. RESULTS: Increased numbers of ADAM8 positive cells were shown particularly in the pannus-cartilage/bone junction close or adjoining to TRAP positive multinucleate cells under formation (60 (2)% in pannus, 47 (2)% in synovitis vs 10 (1)% in OA, p<0.001). Human pannus contained high ADAM8 mRNA copy numbers (23 (7) in pannus, 14 (4) in synovitis vs 1.7 (0.3) in OA, p<0.001). Functional studies in vitro disclosed ADAM8 mRNA and protein, which was first converted to a proteolytically active and then to fusion-active form. Gene transfection and siRNA experiments enhanced and inhibited, respectively, expression of osteoclast markers and maturation of multinuclear cells. CONCLUSIONS: ADAM8 may be involved in bone destruction in RA because it is upregulated in RA pannus adjacent to developing erosions and enhances maturation of osteoclast-like cells.

Immunohistochemical localization of Toll-like receptors 1-10 in periodontitis.

BACKGROUND/AIM: In periodontitis, bacteria and pathogen-associated molecular patterns are sensed by Toll-like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR-1 to TLR-10) were immunohistochemically detected in gingival epithelium and connective tissue. METHODS: Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR-positive cells were determined. RESULTS: Both healthy and periodontitis gingival tissues expressed all TLRs except TLR-10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group. CONCLUSIONS: For the first time, the cellular expression and distribution of TLR-1 to TLR-10 have been studied in periodontitis, indicating that TLR-1 to TLR-9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR-7 and TLR-8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.

MMPs, IL-1, and TNF are regulated by IL-17 in periodontitis.

Periodontitis is characterized by periodontal tissue destruction. Since interleukin-17 (IL-17) has been reported to up-regulate IL-1beta and tumor necrosis factor-alpha (TNF-alpha), it was hypothesized that it is increased in periodontitis and up-regulates these cytokines and tissue-destructive matrix metalloproteinases (MMP) in local migrant and resident cells. Immunocytochemistry disclosed elevated IL-1beta, TNF-alpha, and IL-17 levels in periodontitis. These cytokines induced proMMP-1 and especially MMP-3 in gingival fibroblasts, whereas MMP-8 and MMP-9 were not induced. IL-17 was less potent as a direct MMP inducer than IL-1beta and TNF-alpha, but it induced IL-1beta and TNF-alpha production from macrophages, and IL-6 and IL-8 from gingival fibroblasts. In accordance with these findings, immunocytochemistry disclosed that MMP-1 and MMP-3 were increased in periodontitis. Gingival fibroblasts may play an important role in tissue destruction in periodontitis via cytokine-inducible MMP-1 and MMP-3 production, in which IL-17 plays a role as a key regulatory cytokine.

Characterization of human primary osteoblast response on bioactive glass (BaG 13-93)- coated poly-L,DL-lactide (SR-PLA70) surface in vitro.

Bioabsorbable polylactide-based polymers are commonly used for bone reconstruction. Although these polymers have proven successful in many applications, they do not have the capacity to induce osteoconduction. Therefore, several strategies have been developed to manufacture osteoconductive polylactide-based composites. In this study, we have investigated in vitro response of human primary osteoblasts for self-reinforced poly-L,DL-lactide 70/30 (SR-PLA70) plates coated with spheres of bioactive glass 13-93 (SR-PLA70 + BaG). Osteoblasts were cultured on SR-PLA70 and SR-PLA70 + BaG plates for 2, 7, or 14 days. By day 7, both materials induced a reduction in total cell population. However, by day 14 the proliferative response of osteoblasts on SR-PLA70 + BaG surface was such that the cell population had regained similar levels as that of day 2 controls. Alkaline phosphatase activity was higher on SR-PLA70 at day 7 but declined to control levels by day 14. There were no significant time-dependent variations in alkaline phosphatase activity on SR-PLA70 + BaG. After in vitro hydrolysis for 7 days, the elemental analysis of SR-PLA70 + BaG surface showed the presence of mineral precipitates that were confirmed as crystalline hydroxyapatite. This was accompanied by osteoblast spreading, protrusions of microvilli adhered to BaG 19-39 surface, cuboidal phenotype and cell surface associated formation of hydroxyapatite microspheres. In conclusion, the SR-PLA70 + BaG composite is capable of inducing a proliferative response of human primary osteoblasts, and appears to support the development of mature osteoblast phenotype. Therefore, the SR-PLA70 + BaG composites appear as promising osteoconductive scaffold candidates for reconstruction and regeneration of bone matrix.

Chondrocyte-mediated collagenolysis correlates with cartilage destruction grades in osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is associated with destruction of type II collagen-rich hyaline articular cartilage. We hypothesized that classical interstitial collagenases cleave collagen type II, leading to the increased expression of the 3/4 native type II collagen fragment (COL2-3/4C) and the corresponding denatured type II collagen fragment (COL2-3/4M), which could correlate with different cartilage destruction grades. In addition, we assessed whether these fragments could be measured in joint fluid and serve as diagnostic markers. METHODS: Cartilage specimens were obtained from the femoral heads of hip joints from total hip replacement operations. Articular gliding surfaces of the cartilage were categorized into normal (G0), fibrillated (G1), superficiallyfissured (G2) and deeplyfissured (fissures that reach to the subchondral bone) (G3). A histological scoring of the cartilage was also used. COL2-3/4C and COL2-3/4M were detected by immunohistochemical staining. Dot blotting was used to detect these fragments in joint fluid. RESULTS: COL2-3/4C and COL2-3/4M were found in the perichondrocyte matrix around lacunae. Such COL2-3/4C (p < 0.05) and COL2-3/4M (p < 0.05) immunoreactivity was significantly increased in G3 and G2 compared to GO and G1. A positive correlation (n = 35, Spearman rank correlation) was observed between the histological score and the percentage of COL2-3/4C positive lacunae (r = 0.43, p = 0.01) and COL2- 3/4M positive lacunae (r = 0.53, p = 0.001). All 7/7 joint fluid samples contained COL2-3/4C in dot blots whereas only 4/7 contained COL2-3/4M. CONCLUSION: Collagenase-cleaved collagen--both native and denatured--increases as the severity of OA increases, assessed using a macroscopic clinical and microscopic histological grading system. Collagen degradation was always most apparent around chondrocytes. Furthermore, the native COL2-3/4C fragment has potential as a joint fluid marker for OA.

Pannus invasion and cartilage degradation in rheumatoid arthritis: involvement of MMP-3 and interleukin-1beta.

OBJECTIVE: Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA. METHODS: Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR. RESULTS: MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1. CONCLUSION: This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.

Developmental regulation of nitric oxide synthase expression in rat skeletal bone.

Nitric oxide (NO) has been implicated in bone growth and remodeling by studies showing that inhibition of NO-synthase (NOS) activity retards normal gain in bone mineral density both during skeletal development and after sexual maturity. In the present study, we aimed to assess the level of expression and cellular localization of the three NOS isoforms during skeletal bone development from neonatal to sexual maturity in female Wistar rats. Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the presence of NOS1 (neuronal), NOS2 (inducible), and NOS3 (endothelial) transcripts in femoral bone from neonatal, 4-, 8-, and 12-week-old rats. RT-PCR amplified NOS1, NOS2, and NOS3 transcripts of 472-, 807-, and 289-bp, respectively. There were no detectable differences in the levels of NOS1 mRNA between the groups; however, NOS2 mRNA was more abundant in the neonatal group compared with 4-, 8-, and 12-week groups. Expression of NOS1 protein could not be detected in bones by either Western blotting or immunocytochemistry in any of the age groups investigated. Western blots for NOS2 revealed expression in the neonatal group only and it was not detected in any of the older age groups. Immunostaining for NOS2 was also most evident in the neonatal group and was localized specifically to trabecular osteoblasts and osteoclasts. In all age groups studied, NOS3 mRNA and protein were found in bone-resorbing osteoclasts, cuboidal active osteoblasts, and osteocytes. Semiquantitative RT-PCR provided evidence of down-regulation of NOS3 transcripts during the skeletal development. This was confirmed using in situ hybridization, which showed higher expression in neonatal and 4-week groups than in other groups. Western blots and counting the ratio of trabecular osteoblasts that were NOS3 immunoreactive showed parallel down-regulation of NOS3 protein during skeletal development. Taken together, these data show that there is regulation of NOS2 and in particular NOS3 expression during skeletal development and this may be significant to trabecular bone growth and remodeling.

Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes.

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non-load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.

Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity.

Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast metabolism. The present study was performed to investigate the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the expression of inducible NO-synthase (iNOS) and to measure high-output production of NO by primary rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 and MG-63. In addition, we have investigated if NO may mediate some of the effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA and protein expression in primary rat osteoblasts in response to cytokine treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for authentic rat iNOS. Nitrite accumulation in culture medium was induced by IFN-gamma in a time- and dose-dependent manner and inhibited by cotreatment with inhibitors of NOS activity and by dexamethasone. IL-1 beta, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surprisingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite and induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cytokine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show that the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteoblast-derived NO may have an important role in mediation of localized bone destruction associated with inflammatory bone diseases such as rheumatoid arthritis.

Decreased nitric oxide levels and bone turnover in amenorrheic athletes with spinal osteopenia.

Amenorrheic athletes have been likened to postmenopausal women, with low estrogen levels and osteopenia. It has been suggested that estrogen exerts its antiresorptive actions on bone via a nitric oxide (NO)-dependent mechanism. This study investigated whether the mechanism of bone loss in amenorrheic athletes is similar to that of postmenopausal women with reduced NO levels and high bone turnover. Eleven amenorrheic athletes, 15 eumenorrheic athletes, and 10 sedentary controls were studied. Spine and hip bone mineral density was measured using dual-energy x-ray absorptiometry. Bone turnover was assessed by biochemical markers of formation (osteocalcin and bone-specific alkaline phosphatase) and resorption (deoxypyridinoline). NO metabolites were measured from 24-h urine samples using a chemiluminescence assay. Spine, but not hip, bone mineral density was reduced in the amenorrheic group, compared with the eumenorrheic (P = 0.0001) and control (P = 0.04) groups. Osteocalcin, bone-specific alkaline phosphatase, and deoxypyridinoline were similar in all groups. NO metabolites were lower in the amenorrheic group, compared with controls (P = 0.035), despite a higher dietary intake of nitrates. Unlike postmenopausal women, amenorrheic athletes do not have raised bone turnover but do have reduced NO metabolites and spinal osteopenia. The results show, however, that reduced NO production is a common denominator in both conditions and further support the importance of NO in estrogen-mediated protection of skeletal mass and strength.

Effect of nitric oxide donor nitroglycerin on bone mineral density in a rat model of estrogen deficiency-induced osteopenia.

Nitric oxide (NO) may modulate estrogen's anabolic effects on bone homeostasis by restraining osteoclast-mediated bone resorption and stimulation of osteoblast activity. Accordingly, NO donated by organic nitrates, including nitroglycerin, is thought to protect against bone loss associated with estrogen deficiency. In this study, we have explored this phenomenon. Thirty-two 12-week-old female Wistar rats were divided into four groups prior to bilateral ovariectomy or a sham operation. The ovariectomised rats received (1). vehicle control (OVX control), (2). 17-beta-estradiol (OVX+E2), or (3). transdermal nitroglycerin (OVX+NG) for 4 weeks. Femoral and tibial bone mineral density (BMD), serum alkaline phosphatase and urine deoxypyridinoline and NO metabolites were analysed at the end of the study period together with failure torque and torsional rigidity of the tibiae and cellular localisation of the NO-synthase (NOS) isoforms. In OVX+E2 group, proximal and distal femoral and proximal tibial BMD exceeded that of the Sham controls. Nitroglycerin prevented BMD loss at these three sites at levels comparable to that of the Sham controls. Deoxypyridinoline excretion did not change except in the OVX-E2 group that showed an expected reduction when compared to the Sham and OVX controls. There were no treatment-related differences in total alkaline phosphatase or urinary NO metabolites. Tibial failure torque was comparable between the groups but both OVX+E2 and OVX+NG groups showed decreased torsional rigidity compared with the OVX controls. Endothelial and inducible NOS were found in osteoblast-like cells associated with calcifying cartilage spicules in the distal femoral metaphysis. These data confirm previous findings and show that nitroglycerin counteracts the estrogen deficiency-induced osteopenia in the ovariectomised rat model. Organic nitrates may thus be beneficial in conditions where bone turnover is compromised such as in osteoporosis.

Neural involvement in post-traumatic osteopenia: an experimental study in the rat.

The effect of sciatic nerve resection on post-traumatic bone loss and mechanical strength of the ipsilateral (IL) and contralateral (CL) femoral shafts and necks was studied 25 days after a tibial fracture. We subjected 45 male rats to a standardized tibial fracture, stabilized it with a modular intramedullary nail and then randomly allocated the animals to two groups: right sciatic nerve resection (SNR group) or sham operation (sham group). All of the operated hindlimbs were further immobilized in a plaster cast to avoid unequal loadbearing between the two groups. After 25 days of healing, 85Sr incorporation in the IL femora was 10% lower in the SNR group compared to the sham group, indicating a lower bone mineralization after sciatic nerve resection. The bone mineral content was 15% higher in the SNR group ipsilaterally. Accordingly, the bending moment and energy absorption in the femoral midshaft were higher in the SNR group compared to the sham group. The sciatic nerve resection protected the femoral shaft against the normally occurring post-traumatic bone loss after a tibial fracture. This protective effect of the neurectomy also occurred in the femoral neck, but not to the same extent. A protective effect was also present in the CL femur, suggesting additional systemic effects of the sciatic nerve resection.

The influence of chemical sympathectomy on pain responsivity and alpha 2-adrenergic antinociception in neuropathic animals.

We studied the effect of chemical sympathectomy by 6-hydroxydopamine (6-OHDA) on pain behavior and alpha(2)-adrenergic antinociception in rats with a spinal nerve ligation-induced neuropathy. For assessment of alpha(2)-adrenergic antinociception, the rats were treated systemically with two alpha(2)-adrenoceptor agonists, one of which only poorly (MPV-2426) and the other very well (dexmedetomidine) penetrates the blood-brain barrier. Moreover, the effect of MPV-2426 on spontaneous activity of dorsal root nerve fibers proximal to the nerve injury was determined. Systemic treatment with 6-OHDA produced a marked decrease in immunocytochemical labeling of sympathetic nerve fibers in the skin but it produced no marked change in basal pain sensitivity to mechanical stimulation either in neuropathic or sham-operated animals. Systemic administration of MPV-2426 and dexmedetomidine produced a dose-dependent tactile antiallodynic effect in neuropathic animals. Intraplantar injection of MPV-2426 had an identical antiallodynic effect independent of whether it was injected into the neuropathic or contralateral hindpaw. In a test of mechanical nociception and hyperalgesia, dexmedetomidine markedly attenuated pain responses in all experimental groups, whereas MPV-2426 had a weak but significant pain attenuating effect only in neuropathic animals. In the tail flick test, both alpha(2)-adrenoceptor agonists had a significant antinociceptive effect. The pain attenuating effect of MPV-2426 was enhanced by pretreatment with 6-OHDA, except in a test of tactile allodynia. MPV-2426-induced modulation of spontaneous activity was not a general property of dorsal root fibers proximal to the injury. The results indicate that a chemical destruction of sympathetic postganglionic nerve fibers innervating the skin does not markedly influence cutaneous pain sensitivity nor is it critical for the alpha(2)-adrenoceptor agonist-induced attenuation of pain behavior in neuropathic or non-neuropathic animals. Chemical sympathectomy, independent of neuropathy, enhanced the pain attenuating effect by MPV-2426, probably due to a peripheral action, whereas in non-sympathectomized control and neuropathic animals peripheral mechanisms have only a minor, if any, role in the alpha(2)-adrenoceptor agonist-induced antinociception.

Prevention by lamotrigine, MK-801 and N omega-nitro-L-arginine methyl ester of motoneuron cell death after neonatal axotomy.

Motoneuron cell death was analysed in the rat facial motor nucleus after neonatal facial nerve transection. In situ DNA fragmentation labelling showed that axotomized motoneurons die by an apoptotic mechanism. In order to investigate the existence of excitotoxic mechanisms in this type of neuronal death, rats were treated with several agents known to possess neuroprotective action through a variety of mechanisms. The Na+ channel inhibitor lamotrigine and the antagonist for the N-methyl-D-aspartate-type glutamate receptor, dizocilpine maleate (MK-801) were found to be able to rescue motoneurons from cell death induced by axotomy. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester was also able to protect motoneurons from death, but to a lesser extent. The distribution of constitutive and inducible isoforms of nitric oxide synthase was investigated by immunocytochemistry in the facial motor nucleus. No changes were detected in constitutive nitric oxide synthase immunoreactivity in the facial motor nucleus after axotomy. However, in the axotomized facial motor nucleus, inducible nitric oxide synthase showed a positive immunolabelling specifically located in activated astrocytes, but not in microglia. Nitric oxide derived from activated astrocytes may have a role in promoting excitotoxic mechanisms in axotomized motoneurons. We conclude that excitotoxic mechanisms involving apoptotic cell death are present when immature motoneurons die as a consequence of target disconnection.

Rapid proliferation of calcitonin gene-related peptide-immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling.

The nervous system may be actively involved in bone repair and in remodelling of callous tissue in bone fractures, as well as in the regulation of nociceptive impulses from the site of the trauma. The aim of this study was to assess the distribution and nature of the periosteal innervation of normal control bone and during bone healing subsequent to fracture of rat tibiae at seven, 14 and 21 days after experimental fracture using immunocytochemistry and image analysis quantification of the neuronal marker protein gene product 9.5 and sensory neuropeptide calcitonin gene-related peptide. At seven days, periosteal protein gene product 9.5- and calcitonin gene-related peptide-immunoreactive fibres showed dense ramifications and terminal sprouting. In addition to periosteum, the nerve fibres were found in the middle of the callus interspersed with inflammatory cells and penetrating into secondary minor fractures. At days 14 and 21 many tortuous nerves were found in the periosteum but not in mid callus. Image analysis quantification revealed a uniform increased proliferation of nerves after seven days. At 21 days, the intercept countings showed in excess of a three-fold increase of calcitonin gene-related peptide-immunoreactive nerve fibres compared with the normal control group (P > or = 0.0001) and were almost as numerous as protein gene product 9.5-immunoreactive fibres (P < 0.005). It is postulated that calcitonin gene-related peptide-containing sensory innervation may have a potential importance in the fracture vascular control, angiogenesis and osteogenesis in addition to a protective role against excessive fracture movement. The results are consistent with the neural involvement in bone growth and remodelling.