Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.     

Twenty-four-hour variation of L-arginine/nitric oxide/cyclic guanosine monophosphate pathway demonstrated by the mouse visceral pain model

The L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L-arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light-dark cycle. Animals were injected s.c. with saline, 2 mg/kg L-arginine (a NO precursor), 75 mg/kg L-N(G)-nitroarginine methyl ester (L-NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p-benzoquinone (PBQ)-induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L-arginine or L-NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time-dependent differences in PBQ-induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L-NAME, methylene blue, or sodium nitroprusside, but not saline or L-arginine. These results suggest that components of L-arginine/NO/cGMP pathway exhibit biological time-dependent effects on visceral nociceptive process.     

Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model

Apelin, as the endogenous ligand of the APJ receptor, is a novel identified neuropeptide whose biological functions are not fully understood. APJ receptor mRNA was found in several brain regions related to descending control system of pain, such as amygdala, hypothalamus and dorsal raphe nucleus (DRN). The present study was designed to determine whether supraspinal apelin-13 may produce antinociceptive effect observed in the acetic acid-induced writhing test, a model of visceral pain. Apelin-13 not only significantly produced preemptive antinociception at the dose of 0.3, 0.5, 1 and 3μg/mouse when injected intracerebroventricularly (i.c.v.) before acetic acid, but also significantly induced antinociception at a dose of 0.5, 1 and 3μg/mouse when injected i.c.v. after acetic acid. And i.c.v. apelin-13 did not influence 30-min locomotor activity counts in mice. Intrathecal (i.t.) administration of apelin-13 (1 and 3μg/mouse) significantly decreased the number of writhes, however, intraperitoneal (i.p.) injection of apelin-13 (10-100μg/mouse) had no effect on the number of writhes in the writhing test. The specific APJ receptor antagonist apelin-13(F13A), no-specific opioid receptor antagonist naloxone and μ-opioid receptor antagonist β-funaltrexamine hydrochloride (β-FNA) could significantly antagonize the antinociceptive effect of i.c.v. apelin-13, suggesting APJ receptor and μ-opioid receptor are involved in this process. Central low dose of apelin-13 (0.3μg/mouse, i.c.v.) could significantly potentiate the analgesic potencies of modest and even relatively ineffective doses of morphine administrated at supraspinal level. This enhanced antinociceptive effect was reversed by naloxone, suggesting that the potentiated analgesic response is mediated by opioid-responsive neurons.     

Regulation in the neural plate of Xenopus laevis demonstrated by genetic markers

To follow the subsequent history of grafted tissue in experiments designed to study regulation and commitment in the amphibian neural plate, previous workers have relied on graft scars, vital dyes applied externally to cells, or xenoplastic grafts. Each of these methods has been criticized on the grounds that they do not indicate unambiguously the origins of individual cells within the operated host. To overcome these difficulties, homoplastic, genetically marked embryonic grafts were taken from the prospective spinal neuroectoderm of triploid and tetraploid Xenopus laevis frogs and transplanted to presumptive eye and prosencephalic regions of the neural plate of diploid X. laevis embryos. Orthotopic presumptive eye grafts also were done. Marked cells were scored in section either by nucleolar number or computerized nuclear size analysis. Of 28 heterotopically grafted embryos that survived to stage 41, when the retina has differentiated, prospective spinal cord neuroectoderm in eight animals gave rise to cell types unique to the eye. The remaining 20 survivors appeared to be mosaic. These results substantiate claims of regulation in the neural plate and extend these observations to the level of individual cell types, a level of resolution not previously obtained in other studies.     

Persistent pain model reveals sex difference in morphine potency

Central or systemic administration of agonists directed at the mu or delta opiate receptors generally produce a greater degree of analgesia in males than in females. To date, most studies examining sex-based differences in opioid analgesia have used acute noxious stimuli (i.e., tail-flick and hot plate test); thus the potential dimorphic response of centrally acting opiates in the alleviation of persistent inflammatory pain is not well established. In the present study, right hind paw withdrawal latency (PWL) to radiant thermal stimuli was measured in intact male and cycling female Sprague-Dawley rats before and after unilateral hind paw injection of the inflammatory agent complete Freund's adjuvant (CFA). Control animals received intraplantar injection of saline. Twenty four hours after CFA or saline injection, animals received either saline or morphine bisulfate (0.5-15 mg/kg sc). Separate groups of control or inflamed animals were tested on their responsiveness to morphine at 7, 14, and 21 days post-CFA or saline. No sex differences were noted for baseline PWLs, and females displayed slightly less thermal hyperalgesia at 24 h post-CFA. At all morphine doses administered, both the antihyperalgesic effects of morphine in the inflamed animals and the antinociceptive effects of morphine in control animals were significantly greater in males compared with females. Similarly, in males, the antihyperalgesic effects of morphine increased significantly at 7-21 days post-CFA; no significant shift in morphine potency was noted for females. These studies demonstrate sex-based differences in the effects of morphine on thermal hyperalgesia in a model of persistent inflammatory pain.     

The effect of alpha-tocopherol on the nitration of gamma-tocopherol by peroxynitrite

It has been proposed (S. Christen et al. Proc. Natl. Acad. Sci. USA 94, 3217-3222, 1997) that although alpha-tocopherol (alpha-TH) is an efficient antioxidant, the presence of gamma-tocopherol (gamma-TH) may be required to scavenge peroxynitrite-derived reactive nitrogen species. To investigate the reactions between alpha-TH, gamma-TH, and peroxynitrite, endogenous levels of both alpha-TH and gamma-TH were monitored when low-density lipoprotein was oxidized in the presence of the peroxynitrite generator 5-amino-3-(4-morpholinyl)-1, 2,3-oxadiazolium (SIN-1). SIN-1 oxidized alpha-TH while gamma-TH levels remained constant. The sparing of gamma-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing alpha-TH and gamma-TH were incubated with either SIN-1 or peroxynitrite. Our data show that alpha-TH inhibits peroxynitrite-mediated gamma-TH nitration, i.e., 5-NO2-gamma-tocopherol formation. The rate constants for the reactions between both alpha-TH and gamma-TH with peroxynitrite suggest that the sparing of gamma-TH by alpha-TH does not occur by competitive scavenging, but may be due to the formation of a transient gamma-TH intermediate. Nitration of gamma-TH becomes significant only after alpha-TH levels have been depleted. We conclude alpha-TH alone is sufficient to remove any peroxynitrite-derived reactive nitrogen species, as the presence of alpha-TH attenuates nitration of both gamma-TH and tyrosine. The present results also indicate that a bolus addition of peroxynitrite or SIN-1 to liposomes containing gamma-TH forms 5-NO2-gamma-tocopherol in similar yields. This is in contrast to their reaction profile with tyrosine in aqueous solution. Under these conditions, SIN-1 does not form nitrotyrosine at detectable yields.     

Autophagy impairment in a mouse model of neuropathic pain

Background

High frequency electrical stimulation (HFS) of primary nociceptive afferents in humans induce a heightened sensitivity in the surrounding non-stimulated skin area. Several studies suggest that this heterotopic effect is the result of central (spinal) plasticity. The aim of this study is to investigate HFS-induced central plasticity of sensory processing at the level of the brain using the electroencephalogram (EEG). To this end we measured evoked potentials in response to noxious electrical pinprick-like stimuli applied in the heterotopic skin area before, directly after and 30 minutes after HFS.

Results

We observed potential cortical electrophysiological correlates of heterotopic facilitation. Two different cortical correlates were found; the first one was a lateralized effect, i.e. a larger N100 amplitude on the conditioned arm than the control arm 30 minutes after end of HFS. This was comparable with the observed lateralized effect of visual analogue scale (VAS) scores as response to the mechanical punctate stimuli. The second correlate seems to be a more general (non-lateralized) effect, because the result affects both arms. On average for both arms the P200 amplitude increased significantly 30 minutes after end of HFS with respect to baseline.

Conclusions

We suggest that for studying heterotopic nociceptive facilitation the evoked brain response is suitable and relevant for investigating plasticity at the level of the brain and is perhaps a more sensitive and reliable marker than the perceived pain intensity (e.g. VAS).     

Fluid mechanics in dentinal microtubules provides mechanistic insights into the difference between hot and cold dental pain

Dental thermal pain is a significant health problem in daily life and dentistry. There is a long-standing question regarding the phenomenon that cold stimulation evokes sharper and more shooting pain sensations than hot stimulation. This phenomenon, however, outlives the well-known hydrodynamic theory used to explain dental thermal pain mechanism. Here, we present a mathematical model based on the hypothesis that hot or cold stimulation-induced different directions of dentinal fluid flow and the corresponding odontoblast movements in dentinal microtubules contribute to different dental pain responses. We coupled a computational fluid dynamics model, describing the fluid mechanics in dentinal microtubules, with a modified Hodgkin-Huxley model, describing the discharge behavior of intradental neuron. The simulated results agreed well with existing experimental measurements. We thence demonstrated theoretically that intradental mechano-sensitive nociceptors are not "equally sensitive" to inward (into the pulp) and outward (away from the pulp) fluid flows, providing mechanistic insights into the difference between hot and cold dental pain. The model developed here could enable better diagnosis in endodontics which requires an understanding of pulpal histology, neurology and physiology, as well as their dynamic response to the thermal stimulation used in dental practices.     

Lens thiol depletion by peroxynitrite. Protective effect of pyruvate

Pyruvate (PY) is known to be a potent scavenger of H₂O₂ by undergoing its peroxidative decarboxylation. While doing so, it also inhibits ^· OH generation, in addition to its direct ^· OH scavenging effect. We now hypothesize that PY would also be decarboxylated by cleaving the -O-O- bond in peroxynitrite (ONOO⁻) (PN), with the effect of protecting tissues against NO_x induced damage. We have verified this by measuring ¹⁴CO₂ formation on incubation of 1-¹⁴C-PY with 3-morpholinosydnonimine (SIN-1). Its protective effect against PN induced thiol depletion was initially assessed by determining its ability to inhibit oxidation of pure GSH. This was further evaluated by incubating lens homogenate with SIN-1 with or without PY. As conceived, PY did inhibit PN induced loss of protein as well as non-protein -SH. The findings therefore appear potentially useful to protect against nitrite induced damage to the lens and other tissues known to occur with aging and certain diseases such as diabetes.     

Growth plate abnormalities in a new dwarf mouse model: tich

Growth plate cartilage calcification has been examined in a recently described mouse mutant, tich, which is co-isogenic with the A.TL strain. Long bones were studied from 1-day-old and 1-month-old mice which carried a homozygous recessive gene mutation making them short limbed and dumpy. Specimens were studied by routine histology, scanning electron microscopy and radiography. In 1-day-old tich mice the front of calcified cartilage was recessed behind the advancing periosteum and bone. No similar recess was seen in control mice. At 1 month of age, a number of the long bone growth plates were irregularly thickened, particularly in the central area. This produced a central tongue of non-calcified cartilage (particularly prominent in the proximal tibia) which gave rise to a corresponding pit in the calcified cartilage layer, in macerated specimens. This was accompanied by poor resorption of calcified cartilage. At both ages the presence of the respective defects was radiographically confirmed. At present it is not known whether this is primarily a defect of calcification or resorption but its presence, apparently from a single mutation in a genetically defined mouse strain, makes it a potentially valuable model.     

Augmentation of analgesic effect of ibuprofen by alprazolam in experimental model of pain

The reports of analgesic effects of benzodiazepines are inconsistent. There is evidence of a hyperalgesic effect induced by activation of supraspinal GABAA receptors and an antinociceptive effect induced by activation of receptors located in the spinal cord (dorsal horns). The aim of the study was to discover whether the systemic administration of a benzodiazepine agent alprazolam increases the systemic analgesic efficacy of non-opioid analgesic ibuprofen. Experimental studies combining these agents have not yet been published. We used three experimental methods - writhing test (with acetic acid), tail-flick test and plantar test to assess analgesic action. The drugs were administered orally. Augmentation of the analgesic effect of ibuprofen by alprazolam was proved for the writhing test at a dose of 30 mg/kg of ibuprofen and alprazolam 1 mg/kg. The reaction time of the combination was significantly prolonged in comparison with ibuprofen alone. The results of the tail-flick test and plantar test were negative. The effect of ibuprofen was not enhanced by alprazolam in tests of acute thermal pain. Our results have demonstrated that the analgesic action of ibuprofen is only weakly enhanced by alprazolam.     

Beneficial effects of FeTSPP, a peroxynitrite decomposition catalyst, in a mouse model of spinal cord injury

The aim of the present study was to assess the contribution of peroxynitrite formation in the pathophysiology of spinal cord injury (SCI) in mice. To this purpose, we used a peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTSPP). Spinal cord trauma was induced by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of inflammatory mediators, tissue damage, and apoptosis. FeTSPP treatment (10-100 mg/kg, i.p.) significantly reduced in dose-dependent manner 1 and 4 h after the SCI (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and poly-(ADP-ribose) polymerase activation, (4) proinflammmaory cytokines expression, (5) NF-kappaB activation, and (6) apoptosis (TUNEL staining, Bax and Bcl-2 expression). Moreover, FeTSPP significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. Taken together, our results clearly demonstrate that FeTSPP treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma similarly to dexamethasone, a well-known antiinflammatory agent which we have used as positive control.     

Main-chain dominated amyloid structures demonstrated by the effect of high pressure

It has been suggested that, while the globular native forms of proteins are a side-chain-dominated compact structure evolved by pursuing a unique fold with optimal packing of amino acid residues, amyloid fibrils are a main-chain-dominated structure with an extensive hydrogen bond network. To address this issue, the effects of hydrostatic pressure on amyloid fibrils of beta2-microglobulin (beta2-m), involved in dialysis-related amyloidosis, were studied. A systematic analysis at various pressures and concentrations of guanidine hydrochloride conducted by monitoring thioflavin T fluorescence, light-scattering, and tryptophan fluorescence revealed contrasting conformational changes occurring consecutively: first, a pressure-induced reorganization of fibrils and then a pressure-induced unfolding. The changes in volume as well as the observed structural changes indicate that the beta2-m amyloid fibrils under ambient pressure are less tightly packed with a larger number of cavities, consistent with the main-chain-dominated amyloid structure. Moreover, the amyloid structure without optimal packing will enable various isoforms to form, suggesting the structural basis of multiple forms of amyloid fibrils in contrast to the unique native-fold.     

Heart-protective effect of n-3 PUFA demonstrated in a rat model of diabetic cardiomyopathy

This study was designed to examine in vivo functional changes of the heart in the early stages of streptozotocin (STZ)-induced diabetic cardiomyopathy and to evaluate the effects of n-3 PUFA intake. Moreover, we investigated whether modulation of diabetes-related abnormalities of myocardial connexin-43 (Cx43), β-myosin heavy chain (β-MHC), and β1-adrenergic receptors (β1-AR) might be implicated in the cardioprotective mechanism of n-3 PUFA. Our results showed significantly reduced cardiac output and ejection fraction (using the microtip pressure–volume catheter technique) as well as stroke volume and stroke work, 4 weeks after STZ-induced diabetes, with improvement of these parameters due to n-3 PUFA consumption. Myocardial expression of Cx43 mRNA estimated by real-time polymerase chain reaction did not change in diabetic rats regardless of n-3 PUFA consumption (100 mg/100 g b.w./day). In contrast, the total and functional phosphorylated form of Cx43 protein increased significantly, and its cardiomyocyte-related distribution was disordered in the diabetic heart, but these changes normalized because of n-3 PUFA intake. Furthermore, acute diabetes was accompanied by decrease of myocardial β1-AR mRNA expression and mild yet nonsignificant increase of β-MHC mRNA. These alterations were not significantly affected by n-3 PUFA. In conclusion, the results point out that STZ-diabetic rats benefit from n-3 PUFA consumption particularly because of the attenuation of myocardial Cx43 abnormalities that most likely contributes to improvement of cardiac function.     

Effects of a metalloporphyrinic peroxynitrite decomposition catalyst,ww-85, in a mouse model of spinal cord injury

The aim of the present study was to assess the effect of a metalloporphyrinic peroxynitrite decomposition catalyst, ww-85, in the pathophysiology of spinal cord injury (SCI) in mice. Spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, production of inflammatory mediators, tissue damage and apoptosis. ww-85 treatment (30–300 µg/kg, i.p. 1 h after the SCI) significantly reduced in a dose-dependent manner: (1) the degree of spinal cord inflammation and tissue injury, (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and PARP activation, (4) pro-inflammatory cytokines expression, (5) NF-κB activation and (6) apoptosis. Moreover, ww-85 significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. The results demonstrate that ww-85 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture.

Cell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus. The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow. We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM). In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL [Terminal deoxynucleotidyl transferase mediated d-UTP nick end-labelling] labelling at the early blastocyst stage. This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM. The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001). Blastocysts from MF1 x MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM. Blastocysts from C57BL6/CBA x C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM. When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 x MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005). MF1 x MF1 embryos and C57BL6/CBA x MF1 embryos of 60-79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA x C57BL6/CBA. When strain was compared MF1 x MF1 embryos of 60-79 cells had a significantly greater apoptotic index than C57BL6/CBA x MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA x C57BL6/CBA in M16 (P = 0.019). Our data suggest that genetic make-up and the chemical composition of simple medium are equally important in determining the level of apoptosis.