Early life stress induces sex-dependent increases in phosphorylated extracellular signal-regulated kinase in brains of mice with neuropathic pain.

BACKGROUND: Both early life stress and neuropathic pain induce morphological and functional abnormalities of the nervous system that are associated with emotional regulation. In our previous study, early life stress enhanced nerve injury-induced hyperalgesia in adult male and female mice. In the present study, using phosphorylated extracellular signal-regulated kinase (p-ERK) as a marker of neuronal activation, we examined the effect of early life stress on neuronal function following partial sciatic nerve ligation (PSL). METHODS: Early life stress was induced by maternal separation from 2 to 3 weeks of age and by social isolation after weaning (MSSI). Neuropathic pain was induced by PSL at 9 weeks of age, and p-ERK expression after light touch stimulation to the ipsilateral paw was measured using immunohistochemistry 1 week after nerve injury. RESULTS: Although MSSI increased p-ERK expression in the paraventricular nucleus (PVN) and amygdala of male mice, PSL did not affect p-ERK expression in control and MSSI mice. In female mice, increased p-ERK expression was observed in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Furthermore, p-ERK expression in the PVN and amygdala was increased in MSSI-PSL mice. CONCLUSIONS: The present data suggest that early life stress sex-dependently and site-specifically increases neuronal activity in the brain. In addition, increased neuronal activity in multiplebrain regions of mice subjected to early life stress may enhance hyperalgesia after nerve injury. WHAT DOES THIS STUDY ADD?: Maternal separation and social isolation (MSSI) increased p-ERK in the paraventricular nucleus (PVN) and amygdala of male mice. MSSI increased p-ERK in the medial prefrontal cortex and nucleus accumbens of female mice. Neuropathic pain increased p-ERK in the PVN and amygdala of female MSSI mice.

The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system.

BACKGROUND AND PURPOSE: The ω-3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid-induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons. EXPERIMENTAL APPROACH: Formalin-induced pain behaviours were measured in mice. Antinociception induced by FFA1 receptor agonists was examined by intrathecal injections of a catecholaminergic toxin, 5-HT lowering drug or these antagonists. The expression of FFA1 receptor protein and c-Fos was estimated by immunohistochemistry, and the levels of noradrenaline and 5-HT in the spinal cord were measured by LC-MS/MS. KEY RESULTS: FFA1 receptors colocalized with NeuN (a neuron marker) in the medulla oblongata and with tryptophan hydroxylase (TPH; a serotonergic neuron marker) and dopamine ß-hydroxylase (DBH; a noradrenergic neuron marker). A single i.c.v. injection of GW9508, a FFA1 receptor agonist, increased the number of c-Fos-positive cells and the number of neurons double-labelled for c-Fos and TPH and/or DBH. It decreased formalin-induced pain behaviour. This effect was inhibited by pretreatment with 6-hydroxydopamine, DL-p-chlorophenylalanine, yohimbine or WAY100635. Furthermore, GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord. In addition, GW1100, a FFA1 receptor antagonist, significantly increased formalin-induced pain-related behaviour. CONCLUSION AND IMPLICATIONS: Activation of the FFA1 receptor signalling pathway may play an important role in the regulation of the descending pain control system.

Analysis of excitatory synapses in the guinea pig inferior colliculus: a study using electron microscopy and GABA immunocytochemistry.

The inferior colliculus (IC) integrates ascending auditory input from the lower brainstem and descending input from the auditory cortex. Understanding how IC cells integrate these inputs requires identification of their synaptic arrangements. We describe excitatory synapses in the dorsal cortex, central nucleus, and lateral cortex of the IC (ICd, ICc and IClc) in guinea pigs. We used electron microscopy (EM) and post-embedding anti-GABA immunogold histochemistry on aldehyde-fixed tissue from pigmented adult guinea pigs. Excitatory synapses were identified by round vesicles, asymmetric synaptic junctions, and gamma-aminobutyric acid-immunonegative (GABA-negative) presynaptic boutons. Excitatory synapses constitute ∼60% of the synapses in each IC subdivision. Three types can be distinguished by presynaptic profile area and number of mitochondrial profiles. Large excitatory (LE) boutons are more than 2 µm(2) in area and usually contain five or more mitochondrial profiles. Small excitatory (SE) boutons are usually less than 0.7 µm(2) in area and usually contain 0 or 1 mitochondria. Medium excitatory (ME) boutons are intermediate in size and usually contain 2 to 4 mitochondria. LE boutons are mostly confined to the ICc, while the other two types are present throughout the IC. Dendritic spines are the most common target of excitatory boutons in the IC dorsal cortex, whereas dendritic shafts are the most common target in other IC subdivisions. Finally, each bouton type terminates on both gamma-aminobutyric acid-immunopositive (GABA+) and GABA-negative (i.e., glutamatergic) targets, with terminations on GABA-negative profiles being much more frequent. The ultrastructural differences between the three types of boutons presumably reflect different origins and may indicate differences in postsynaptic effect. Despite such differences in origins, each of the bouton types contact both GABAergic and non-GABAergic IC cells, and could be expected to activate both excitatory and inhibitory IC circuits.

Chronic microstimulation of cat auditory cortex effective to evoke detection behaviors.

The purpose of this study was to clarify stimulus pulse parameters effective to elicit behaviors of cats trained to detect electric pulse stimuli through chronically implanted electrodes in the primary auditory cortex. One or two pulse parameters were systematically shifted from the standard stimulus consisting of constant-current pulses of amplitude 80 µA, duration 0.2 ms, number of pulses 33, and rate 200 Hz (compatible with interpulse interval 5 ms). Interaction between the pulse amplitude and pulse duration was investigated: although the proportion of stimulus detection responses increased with increasing phase charge (pulse amplitude×pulse duration), a combination of relatively high amplitude during short pulse duration elicited a higher proportion of detection responses when phase charge was constant. Interaction between the number of pulses and interpulse intervals was investigated. We found that the proportion of detection responses is explained by the linear function of two factors, overall charge (phase charge×the number of pulses) and train duration: the proportion of detection responses increased with increasing overall charge and decreasing train duration. Interaction between pulse amplitude and the number of pulses was investigated. We again found that the proportion of detection responses is explained by the linear function of overall charge and train duration in the amplitude-number shift paradigm. Thus, the behavior performance (proportion of detection responses) is a linear time function of overall charge and train duration regardless of the stimulus paradigm. We believe that the findings will contribute to the development of auditory cortex implants for transfer of auditory information directly to the brain.

Responses of neurons in the cat primary auditory cortex to sequential sounds.

In the natural acoustic environment sounds frequently arrive at the two ears in quick succession. The responses of a cortical neuron to acoustic stimuli can be dramatically altered, usually suppressed, by a preceding sound. The purpose of this study was to determine if the binaural interaction evoked by a preceding sound is involved in subsequent suppressive interactions observed in auditory cortex neurons. Responses of neurons in the primary auditory cortex (AI) exhibiting binaural suppressive interactions (EO/I) were studied in barbiturate-anesthetized cats. For the majority (72.5%) of EO/I neurons studied, the response to a monaural contralateral stimulus was suppressed by a preceding monaural contralateral stimulus, but was not changed by a preceding monaural ipsilateral stimulus. For this subset of EO/I neurons, when a monaural contralateral stimulus was preceded by a binaural stimulus, the level of both the ipsilateral and the contralateral component of the binaural stimulus influenced the response to the subsequent monaural contralateral stimulus. When the contralateral level of the binaural stimulus was constant, increasing its ipsilateral level decreased the suppression of the response to the subsequent monaural contralateral stimulus. When the ipsilateral level of the binaural stimulus was constant, increasing its contralateral level increased the suppression of the response to the subsequent monaural contralateral stimulus. These results demonstrate that the sequential inhibition of responses of AI neurons is a function of the product of a preceding binaural interaction. The magnitude of the response to the contralateral stimulus is related to, but not determined by the magnitude of the response to the preceding binaural stimulus. Possible mechanisms of this sequential interaction are discussed.

Little impact of donor/recipient major mismatch for neutrophil-specific antigen NA2 on neutrophil recovery after allogeneic SCT.

The Fcgamma receptor IIIb (FcgammaRIIIb), a receptor for the Fcgamma region of IgG, is specifically expressed on neutrophils. It has two allelic polymorphisms, NA1 and NA2, which are highly immunogenic and act as targets in alloimmune or autoimmune neutropenia. Thus, neutrophil antigens (NA) compatibility of donor/recipient pairs might be expected to affect the engraftment of neutrophils after allogeneic SCT (allo-SCT). Here, the impact of NA compatibility of 17 patients and their donors undergoing allo-SCT with a myeloablative regimen was determined. Leukocyte depletion filters were used for all transfusions before and post-SCT; most patients received G-CSF after transplant. Major mismatches for NA1 and NA2 were present in 1 and 7 patient/donor pairs, respectively. These eight patients receiving NA major-mismatched allo-SCT were compared with nine patients who received NA compatible allo-SCT. Engraftment of neutrophils and the incidence of post-engraftment neutropenia were found to be identical in the two groups. Despite the limitations in statistical power because of the small number of patients analyzed, these observations suggest that the major mismatching for NA2 antigen has little impact on the engraftment of neutrophils after myeloablative allo-SCT, at least in patients transfused using leukocyte depletion filters and receiving G-CSF after transplantation.

Some investigations into non-passive listening.

Our knowledge of the function of the auditory nervous system is based upon a wealth of data obtained, for the most part, in anaesthetised animals. More recently, it has been generally acknowledged that factors such as attention profoundly modulate the activity of sensory systems and this can take place at many levels of processing. Imaging studies, in particular, have revealed the greater activation of auditory areas and areas outside of sensory processing areas when attending to a stimulus. We present here a brief review of the consequences of such non-passive listening and go on to describe some of the experiments we are conducting to investigate them. In imaging studies, using fMRI, we can demonstrate the activation of attention networks that are non-specific to the sensory modality as well as greater and different activation of the areas of the supra-temporal plane that includes primary and secondary auditory areas. The profuse descending connections of the auditory system seem likely to be part of the mechanisms subserving attention to sound. These are generally thought to be largely inactivated by anaesthesia. However, we have been able to demonstrate that even in an anaesthetised preparation, removing the descending control from the cortex leads to quite profound changes in the temporal patterns of activation by sounds in thalamus and inferior colliculus. Some of these effects seem to be specific to the ear of stimulation and affect interaural processing. To bridge these observations we are developing an awake behaving preparation involving freely moving animals in which it will be possible to investigate the effects of consciousness (by contrasting awake and anaesthetized), passive and active listening.

Resistance of infant leukemia with MLL rearrangement to tumor necrosis factor-related apoptosis-inducing ligand: a possible mechanism for poor sensitivity to antitumor immunity.

Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.

Measurement of soluble Fcgamma receptor type IIIa derived from macrophages in plasma: increase in patients with rheumatoid arthritis.

FcgammaRIII (CD16) is found in two alternative forms, a transmembrane FcgammaRIIIa expressed on NK cells and macrophages, and a glycosylphosphatidylinositol-linked FcgammaRIIIb present on neutrophils. Previously, we measured soluble FcgammaRIIIa (sFcgammaRIIIa) in plasma of NA(1 +, 2-) phenotyped donors with the anti-FcgammaRIII monoclonal antibody (MoAb) GRM1, which recognizes NA2-FcgammaRIIIb and FcgammaRIIIa. The level of sFcgammaRIIIa, as well as the total sFcgammaRIII (sFcgammaRIIIa plus sFcgammaRIIIb) in patients with rheumatoid arthritis (RA) was significantly higher than that in healthy controls. In this study, we measured sFcgammaRIIIa(M)(phi) in plasma with a newly developed anti-FcgammaRIII MoAb, MKGR14 (mIgM), which recognizes FcgammaRIIIa(M)(phi) specifically. From the recovery of purified sFcgammaRIIIa(M)(phi), the amount of sFcgammaRIIIa(M)(phi) present was about half that of sFcgammaRIIIa(NK), and that of sFcgammaRIIIa was about 50 times lower than that of sFcgammaRIIIb in pooled plasma from healthy NA(1 +, 2-) phenotyped donors. The level of sFcgammaRIIIa(M)(phi) in RA patients was about four times higher than that in healthy controls. In RA patients, both the sFcgammaRIIIa(M)(phi) and sFcgammaRIIIa levels were increased as proportionally as the Lansbury Index. The sFcgammaRIIIa, but not sFcgammaRIIIa(M)(phi) levels, were increased directly proportional to C-reactive protein. sFcgammaRIIIa(M)(phi) may be a novel marker of disease activity in RA.

Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.

F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

Synthesis of sialyl Lewis X-polysaccharide conjugates.

Sialyl Lewis X (SLeX) is well known as a ligand of the cell adhesion molecule E-selectin which is specifically expressed at inflammatory lesion sites. We have synthesized several SLeX-polysaccharide conjugates and examined their potential for drug delivery to inflammatory lesions. The AUC (area under the blood concentration-time curve) 0-24 h of SLeX-CMCht (1), SLeX-CMPul (2) and SLeX-DSH (3) at the inflammatory lesion was about 60-, 300-, and 30-fold higher than that of the monovalent SLeX (7), respectively. Moreover, 1 showed 2-fold higher accumulation in the inflammatory lesion than SLN-CMCht (4), and 2 showed 2.5-fold higher accumulation than SLN-CMPul (5).

Annexin VII as a novel marker for invasive phenotype of malignant melanoma.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti-annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis-negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.

The response of Parp knockout mice against DNA damaging agents.

Gene-disruption studies involving poly(ADP-ribose) polymerase (Parp) have identified the various roles of Parp in cellular responses to DNA damage. The partial rescue of V[D]J recombination process in SCID/Parp(-/-) double mutant mice indicates the participation of Parp in the repair of DNA strand break. Parp(-/-) mice are more sensitive to the lethal effects of alkylating agents. Parp is also thought to be involved in base-excision repair after DNA damage caused by alkylating agents. On the other hand, resistance of Parp(-/-) mice to DNA damage induced by reactive oxygen species implicates the contribution of Parp to cell death through NAD depletion. Parp(-/-) mice with two different genetic backgrounds also show enhanced sensitivity to the lethal effects of gamma-irradiation. Parp(-/-) mice show more severe villous atrophy of the small intestine compared to the wild-type counterpart in a genetic background of 129Sv/C57BL6. Other forms of enhanced tissue damage have been identified in Parp(-/-) mice with a genetic background of 129Sv/ICR. For example, Parp(-/-) mice exhibit extensive hemorrhage in the glandular stomach and other tissues, such as the testes, after gamma-irradiation. Severe myelosuppression is also observed in both Parp(+/+) and Parp(-/-) mice, but Parp(+/+) mice show extensive extramedullary hematopoiesis in the spleen during the recovery phase of post-irradiation, whereas the spleen of Parp(-/-) mice exhibits severe atrophy with no extramedullary hematopoiesis. The absence of extramedullary hematopoiesis in the spleen is probably the underlying mechanism of hemorrhagic tendency in various tissues of Parp(-/-) mice. These findings suggest that loss of Parp activity could contribute to post-irradiation tissue hemorrhage.

Clinical significance of serum soluble IL-2R levels in patients with adult T cell leukaemia (ATL) and HTLV-1 carriers.

The levels of soluble IL-2Ralpha (sIL-2Ralpha) in serum were measured in HTLV-1 carriers and ATL patients in order to evaluate their possible correlation with clinical status. Mean sIL-2R levels in ATL patients were found to be 9704 U/ml for the acute/lymphoma type, 1961 U/ml for the chronic type and 788 U/ml for the smouldering type. The level for asymptomatic HTLV-1 carriers was 475 U/ml, and 165 U/ml for healthy young adult HTLV-1- controls. The serial measurement of sIL-2R in ATL patients, healthy HTLV-1 carriers, and HTLV-1 carriers with diseases other than ATL showed a good correlation between serum levels of sIL-2R and the pathophysiological status of disease. Furthermore, an increase in the sIL-2Ralpha level in serum indicated the exacerbation of HTLV-1 infection and autoimmune diseases. The measurement of sIL-2Ralpha levels is therefore a very useful parameter for determining disease status.

[Is more really up?: experimental evidence for orientational metaphor].

The purpose of this study was to provide experimental evidence for orientational metaphors based on the UP-DOWN image schema. Two experiments were conducted to examine the effects of the image schema on the picture-word comparison tasks. In the experiments, subjects were required to judge if the location of kanji-target matched the word printed at the center of a reference square. Under the condition in which the location and the metaphorical orientation of the kanji-target disagree, it took longer time for judgment. These results are interpreted in terms of accessibility and availability of the image schema.

[Stent intubation for the airway stenosis under PCPS].

Placement of stents for the tracheal or carinal stenosis have a meaning of maintaining the airway. Failure of the stenting causes death. In cases of severe airway stenosis and low pulmonary function, the pulmonary support method should be performed instead of intubation and mechanical ventilation. Generally, PCPS (percutaneous cardio-pulmonary support system) is used as a pulmonary support. This method was very useful to place stents for airway stenosis. We concluded that PCPS was useful in emergency cases, and in cases of severe fixed type airway stenosis and low pulmonary function it had be on stand-by.

Isolation and sequence of the GCR3 homologue from Candida albicans by complementation of (delta)gcr3 strain of Saccharomyces cerevisiae.

To study the function of GCR3, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Candida albicans gene which complements the growth defect of the (delta)gcr3 mutant was isolated. Transformants of this gene also recovered the glycolytic enzyme activities. Its DNA sequencing predicted an 886 amino acid protein with 30.4% identity to the Gcr3p of S. cerevisiae.

Distribution and excretion of boron after intravenous administration of disodium mercaptoundecahydro-closo-dodecaborate to rats.

Disodium mercaptoundecahydro-closo-dodecaborate (BSH) is an important compound for boron neutron capture therapy. The pharmacokinetics of boron by BSH were studied in normal rats after rapid intravenous injection at three doses (30, 100, 300 mg/kg) or continuous infusion (100 mg/kg/30 min). The boron concentration in biological samples was measured by inductively coupled plasma atomic emission spectroscopy. The blood half-lives of boron in the elimination phase (t1/2 beta) after rapid injection of BSH at doses of 30, 100 and 300 mg/kg were 1.7, 17 and 19 hr, respectively. AUC (32, 219 and 4030 micrograms.hr/ml) increased with the dose, but there was no proportionality among the values. Total clearance decreased drastically from 233 ml/hr/kg (100 mg/kg) to 38 ml/hr/kg (300 mg/kg). As boron was excreted mainly into urine, these results suggest that renal function failure might occur with dosing of 300 mg/kg. In the case of continuous infusion of 100 mg/kg of BSH for 30 min, the pharmacokinetic parameters were similar to those of rapid injection of 100 mg/kg. The highest boron concentration was observed in the kidney and the lowest in the brain. After multiple dosing of BSH at 100 mg/kg/day x 14 days, the boron concentrations in blood, liver, lung and kidney at 24 hr after the last dosing were higher than those after single dosing and were similar to those of simulated values calculated from the single dosing parameters. These results clearly indicated that boron does not accumulate unexpectedly in any tissue with multiple dosing of 100 mg/kg of BSH for two weeks.