Automated measurement of spontaneous pain-associated limb movement and drug efficacy evaluation in a rat model of neuropathic pain.

BACKGROUND: The withdrawal response elicited by a nociceptive stimulus, i.e., evoked pain measure, is commonly used as an efficacy endpoint in neuropathic pain animal models. It, however, has several limitations, which highlight the importance of examining spontaneous pain. The present study describes an automated method for measuring spontaneous pain behaviour in a rat model of neuropathic pain caused by chronic constriction injury (CCI) of sciatic nerve. METHODS: After CCI surgery, a small magnet was implanted into the operated limb. The rat was placed in a test chamber that was surrounded by wire coil. Limb movements, including lifting/guarding, flinching/shaking, licking and walking in the operated limb, caused changes in the electromagnetic field, including a change in voltage and transformed into a signal via an amplifier. RESULTS: CCI rats consistently showed more frequent limb movement than sham rats. There was no significant correlation between the frequency of spontaneous pain behaviour and the evoked pain symptoms. Treatment with duloxetine (30 mg/kg p.o.) and amitriptyline (30 and 100 mg/kg p.o.) significantly reduced this frequency. Pregabalin at 30 mg/kg p.o. tended to reduce the frequency, and diclofenac up to 10 mg/kg p.o. had no effect. CONCLUSION: A non-subjective automated method for measuring spontaneous pain behaviour in an animal model of neuropathic pain was established. It is expected that the current system will greatly enhance the analysis of spontaneous pain-related behaviour, which is a predominant symptom in patients with neuropathic pain. The current system may also be valuable in the screening of potential analgesic treatments.

Evaluation of combination therapy using aciclovir and corticosteroid in adult patients with herpes simplex virus encephalitis.

OBJECTIVE: Herpes simplex virus encephalitis (HSVE) is associated with significant morbidity and mortality, even with appropriate antiviral therapy. In the present investigation, the first to assess efficacy of corticosteroid treatment with aciclovir therapy in HSVE, multiple logistic regression analysis was performed of predictors of outcome in adult patients with HSVE. METHODS: A non-randomised retrospective study of 45 patients with HSVE treated with aciclovir was conducted. The patients were divided into poor and good groups based on outcome at three months after completion of aciclovir treatment. The variables evaluated were: clinical variables (sex, age, days after onset at initiation of aciclovir, Glasgow Coma Scale (GCS) at initiation of aciclovir, initial and maximum values for the cell numbers and protein concentration in the cerebrospinal fluid, and corticosteroid administration); neuroradiological variables (detection of lesions by initial cranial computed tomography and by initial magnetic resonance imaging); and one neurophysiological variable (detection of periodic lateralised epileptiform discharges on the initial electroencephalogram). Single variable logistic regression analysis was performed followed by multiple logistic regression analysis. The best set of predictors for the outcome of HSVE was estimated by stepwise logistic regression analysis. RESULTS: A poor outcome was evident with older age, lower GCS score at initiation of aciclovir, and no administration of corticosteroid. Patient age, GCS at initiation of aciclovir, and corticosteroid administration were found to be significant independent predictors of outcome on multiple logistic regression analysis, and these three variables also formed the best set of predictors (R(2) = 0.594, p<0.0001). CONCLUSION: Combination therapy using both aciclovir and corticosteroid represents one of the predictors of outcome in HSVE.

FR226807: a potent and selective phosphodiesterase type 5 inhibitor.

We describe the pharmacological characteristics of a novel phosphodiesterase type 5 inhibitor FR226807, N-(3,4-dimethoxybenzyl)-2-[[(1R)-2-hydroxy-1-methylethyl]amino]-5-nitrobenzamide. FR226807 inhibited phosphodiesterase type 5 isolated from human platelets with an IC(50) value of 1.1 nM. FR226807 also inhibited phosphodiesterase type 6 with an IC(50) of 20 nM; however, the IC(50) value for phosphodiesterase type 6 was 18-fold higher than that for phosphodiesterase type 5. The IC(50) values of FR226807 for other phosphodiesterases (phosphodiesterase type 1, phosphodiesterase type 2, phosphodiesterase type 3, and phosphodiesterase type 4) were 1000-fold higher than that for phosphodiesterase type 5. FR226807 increased the cyclic guanosine monophosphate (cGMP) content in corpus cavernosum isolated from rabbit, an effect associated with relaxation of the muscle. FR226807 enhanced the relaxation response induced by electrical field stimulation of corpus cavernosum isolated from the rabbit. In an anesthetized dog model for the evaluation of erectile function, intravenous administration of FR226807 prolonged the time to return to 75% of maximal intracavernosal pressure after cessation of electrical stimulation of the pelvic nerve. In summary, FR226807 is a potent and highly selective phosphodiesterase type 5 inhibitor with an augmentative effect on penile erection and will be useful for the treatment of erectile dysfunction.

Glycyrrhizin increases survival of mice with herpes simplex encephalitis.

We examined the effect of glycyrrhizin (GR), a component of licorice root extract, on herpetic encephalitis that was inflicted on mice by inoculation of herpes simplex virus 1 (HSV-1) onto their cornea. Intraperitoneal (i.p.) administration of GR to mice suffering from herpetic encephalitis increased their survival rate in average about 2.5 times (from 37.5-29.0% to 81.8-83.3%; mean values from 2 experiments) while it reduced HSV-1 replication in the brain to 45.6% of the control. These results demonstrate a stimulative effect of GR on the mouse defense system(s) against HSV-1 infection.

Effects of sodium nitroprusside on renal functions and NO-cGMP production in anesthetized dogs.

Although the renal nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) system plays an important role in maintaining urinary sodium and water excretion, effects of an authentic NO donor sodium nitroprusside (SNP) on urine formation have been controversial. In this study, we examined whether SNP increases renal NO release and cGMP production and induces natriuresis in the denervated kidney of anesthetized dogs. The intrarenal arterial infusion of SNP at 10, 30, and 100 ng/kg/min did not affect renal function or NO-cGMP production. The higher dose of SNP (1,000 ng/kg/min) reduced systemic blood pressure and urine flow rate. The antidiuresis was observed also in the contralateral control kidney, the degree of which was larger than that observed in the ipsilateral SNP-infused kidney. During the SNP infusion, reductions in urinary Na+ excretion, fractional Na+ excretion, and urinary nitrite + nitrate excretion occurred in the control kidney but not in the SNP-infused kidney. Urinary cGMP excretion and renal venous plasma cGMP concentration were significantly increased during the SNP infusion in the SNP-infused kidney but not in the control kidney. These renal effects of SNP were similar to those obtained by intrarenal arterial infusion of a specific NO donor, NOC 7 (300 ng/kg/min). These results suggest that SNP can produce nitric oxide and increase cGMP levels in the kidney and suppress sodium reabsorption, but the natriuretic property of SNP may be masked by its counteracting effects including the systemic hypotension in anesthetized dogs.

Cerebrospinal fluid interleukin 6 in amyotrophic lateral sclerosis: immunological parameter and comparison with inflammatory and non-inflammatory central nervous system diseases.

We assayed IL-6 in 105 cerebrospinal fluid (CSF) samples from patients with ALS, MS, HTLV-1 associated myelopathy (HAM), and controls. There was considerable overlap in IL-6 levels in all patient groups. The mean IL-6 in 27 patients with ALS was significantly higher than in 21 patients in the other neurological disease (OND) group (P=0.0075). There were no significant differences in MS or HAM and the OND control group. Overall, CSF IL-6 correlated with protein concentration but not with percentage IgG or IgG-albumin index. Patients with CSF oligoclonal bands were no more likely to have detectable IL-6 than patients without oligoclonal bands. Similarly, IL-6 did not correlate with clinical disease activity in MS when subgroups of patients were compared or when an individual patient was followed over time. The elevated IL-6 in ALS may reflect an ongoing humoral immune response, or IL-6 may be non-specifically expressed in these patients as a putative neurotrophic factor in response to nerve cell degeneration.

Effects of zaprinast on renal nerve stimulation-induced anti-natriuresis in anaesthetized dogs.

1. We examined whether zaprinast, a putative cGMP-specific phosphodiesterase inhibitor, affects neural control of renal function in pentobarbital-anaesthetized dogs. 2. Renal nerve stimulation (1 Hz, 1 ms duration) reduced urine flow rate, urinary Na+ excretion (UNaV) and fractional excretion of Na+ (FENa) with little change in either renal blood flow (RBF) or glomerular filtration rate (GFR). 3. Intrarenal arterial infusion of zaprinast (10 and 100 micrograms/kg per min) increased basal urine flow rate, UNaV and FENa but not RBF or GFR. Zaprinast infusion (100 micrograms/kg per min) also increased renal venous plasma cGMP concentration and urinary cGMP excretion. 4. Renal nerve stimulation-induced reductions in UNaV and FENa were attenuated during zaprinast infusion, whereas the reduction in urine flow rate was resistant to zaprinast. 5. Renal nerve stimulation increased the renal venous plasma noradrenaline concentration and renal noradrenaline efflux, which remained unaffected during infusion of zaprinast (100 micrograms/kg per min). 6. The results of the present study suggest that zaprinast induces natriuresis and counteracts adrenergically induced antinatriuresis by acting on renal tubular sites in the dog kidney in vivo.

Root canal system of the mandibular incisor.

To better assess the efficiency of the mechanical preparation of root canals, 1085 transparent specimens of extracted mandibular incisors were examined for canal configuration, thickness and curvature of the root canals, condition of any accessory canals, and location of the apical foramen. Greater than 85% of the root canals possessed a single canal (Type I). Of specimens in which furcation was observed, only 3% possessed two separate canals (Type III and IV). Fewer than 30% of the specimens showed accessory canals that were mechanically impossible to clean. The majority of the lateral branches were small, greater than 80% of the specimens were smaller than a #15 reamer, and none of the branches were larger than a #30 reamer. Although apical foramina located distal to the apex were observed in about 50% of the specimens, 83.6% of all apical foramina were within 0.5 mm of the apex, and 99.5% were within 1.0 mm. Data on the thickness of the root and main canal in the apical portion and curvature of the root canal suggest that for adequate apical preparation, a #40 reamer must be able to reach the apical constriction.

[Risk factors of cerebro-cardiovascular events in treated hypertensive male workers in the fifth decade].

The relationship between cerebro-cardiovascular events (CCE) and work-related factors was examined in a cohort of 899 treated hypertensive men who were aged 50-59 yr and working more than 7 portal to portal hours (PPH). During the follow-up of 2.8 yr (2,513 person-years), 27 cases of CCEs occurred, which were classified into 18 cases of stroke, 7 cases of acute myocardial infarction, and 2 cases of others. Using univariate analysis, it was found that managerial position and long PPH (more than 11 h) were significantly related to CCE (relative risk of 3.0 and 2.2, respectively) as well as risk factors such as emaciation, left ventricular hypertrophy, excessive sleeping hours, obesity, cigarette smoking, and inadequate control of systolic blood pressure. Using Cox proportional hazards general model, both managerial position and long PPH remained independently related to the risk of CCE (hazards ratio and 95% confidence interval, 4.1; 1.7-10.0 and 2.7; 1.1-6.2, respectively), after adjustment for other risk factors. These findings suggested that work-related factors, such as managerial position and long PPH, are independent risk factors of CCE among treated hypertensive male workers in the fifth decade.

Interleukin-6 in cerebrospinal fluid of HTLV-I-associated myelopathy.

We demonstrated significant titers of interleukin-6 (IL-6) in the CSF from 6 of 11 patients with HTLV-I-associated myelopathy (HAM). The patients positive for IL-6 generally had more severe clinical symptoms and signs than those negative for IL-6. There was no correlation between the value of IL-6 and inflammatory findings in the HAM CSF.

Early loss of astrocytes in herpes simplex virus-induced central nervous system demyelination.

Immunohistochemistry was used to study herpes simplex virus type 1-induced central nervous system demyelination in the trigeminal root entry zone of mice inoculated with herpes simplex virus type 1 by the corneal route. There was no change in peripheral nervous system myelin as shown by immunostaining for P0 glycoprotein. Double immunoperoxidase staining for herpes simplex virus type 1 antigens and glial fibrillary acidic protein showed that most of the infected cells were astrocytes. Glial fibrillary acidic protein immunostaining was completely lost in the inferior medial portion of the trigeminal root entry zone at 6 days after herpes simplex virus type 1 inoculation, a time when central nervous system myelin was preserved as indicated by immunostaining for myelin basic protein. The pattern of glial fibrillary acidic protein staining did not change and herpes simplex virus type 1 antigens were no longer detected after day 8. There was a progressive loss of myelin basic protein staining within the area unstained by glial fibrillary acidic protein antisera on days 8 to 14. This pattern of astrocyte loss before central nervous system demyelination is strikingly different from the reactive astrocytosis seen in other demyelinating lesions, such as acute experimental allergic encephalomyelitis, progressive multifocal leukoencephalopathy, or acute multiple sclerosis. Herpes simplex virus type 1 infection in mice provides an unusual model of acute central nervous system demyelination preceded by a loss of astrocytes.

[The detection and quantitative analysis of viral antigens, infiltrating mononuclear cells, IFN-gamma, LT and IL-6 bearing cells in the frozen brain tissue sections from a patient with herpes simplex encephalitis by immunocytochemistry].

To identify viral antigens, the types of infiltrating mononuclear cells and cytokine bearing cells, the frozen brain tissue sections form a patient with herpes simplex encephalitis who died on 12th hospital days, were examined by immunocytochemistry methods and combined immunocytochemistry and in situ hybridization. The avidin-biotin peroxidase complex (ABC) techniques were applied for the detection of antigens. All monoclonal antibodies to Leu series and polyclonal antisera to lymphotoxin (LT), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) were purchased form Becton Dickinson Co., and Genzyme Co., (USA) respectively. A large number of neurons and glial cells staining positively HSV-1 antigens were found in the gray matter. Moreover, although a moderate number of HLA-DR (Ia) positive cells were found in the parenchyma, there were few cells displaying positively for Leu-3a, Leu-2a and Leu-7 respectively. To evaluate the number of positive cells appeared in the brain tissues, Leu stain for 4, 2a, 3a, 7, 12 and HLA-DR demonstrated 1.6%, 0.4%, 0.9%, 0.7% and 10% respectively. In addition, numerous number of IFN-gamma positive cells were detected around the lesion and randomly distributed thoroughly the lesion. IL-6 positive cells and LT positive cells were also similar in distribution to IFN-gamma positive cells. Moreover, in simultaneous detection of HLA-DR and HSV-1 mRNA by the combined immunocytochemistry and in situ hybridization, there were seen glial cells staining positively for HLA-DR (Ia) and several cells with mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

[Simultaneous in situ detection of viral antigens and RNA in brain tissues from a patient with herpes simplex encephalitis by immunocytochemistry and in situ hybridization].

In order to elucidate the correlation between herpes simplex virus (HSV-1) and the central nervous system tissue, we performed the simultaneous detection of viral antigens and RNA in the brain tissue sections from a patient with herpes simplex virus (HSV) encephalitis using immunocytochemistry and in situ hybridization. In the present study the hybridization protocol reported by Brahic M et al. in 1984 were applied for the simultaneous detection of viral RNA and antigens with a few modification. The sections were first immunocytochemically stained to detect HSV-1 antigens by ABC method, and then hybridized with 3H-labelled HSV-1 cDNA probe for the detection of RNA after the acetylation of slides for the prevention of nonspecific bindings of isotope to slides. In the present study, viral antigens were immunocytochemically stained to brown-colored deposits located in the cytoplasm and nucleus whereas viral RNA were detected as the accumulation of many silver grains over the nuclei or cytoplasm. In this case the light microscopic findings in a part of temporal lobe showed multiple areas of necrosis mainly involving the gray matter and a few inflammatory changes such as perivascular cell cuffings. HSV-1 infected Vero cells as positive control demonstrated both antigens and RNA as shown in Fig.1 a. However, no hybridization signals and color deposits were observed in uninfected Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of latent ganglionic infection with herpes simplex virus via maxillary gingiva and viral re-activation in vivo after trauma.

Herpes simplex virus can remain latent for months or years in sensory and automatic ganglia of animals and man, and can be re-activated in vivo by several procedures such as neurectomy, irritation of epithelial surfaces, and administration of immunosuppressive agents. The objective of this study was to determine whether dental stimuli can cause re-activation of the latent herpes simplex virus. Homogenization and explanation of ganglia from mice showed that herpes simplex virus (type 1) traveled from maxillary gingiva to trigeminal ganglia, and remained latent. It was also shown that mice passively immunized with rabbit antibody to herpes simplex virus, following the inoculation of herpes simplex virus by the maxillary gingiva route, developed a latent infection in the trigeminal ganglia. Neutralizing antibody was cleared from the circulation and could not be detected in most of these animals after five weeks. A neutralizing test showed that antibody-negative mice with latent infection were able to produce antibody to re-infection with herpes simplex virus, suggesting that re-activation can be identified by measurement of serum antibody. By use of this mouse model system, it was shown that when maxillary gingiva was traumatized with dry ice, viral re-activation occurred in 58% of these animals, as demonstrated by the appearance of neutralizing antibody. Irradiation by a Stomalaser beam had no effect on the re-activation of latent herpes simplex virus. Our mouse model system may serve as a useful model for obtaining new information on re-activating or inhibitory factors in dentistry.

Biphasic effects of chlorpromazine on cell viability in a neuroblastoma cell line.

Although chlorpromazine was shown to greatly inhibit a Ca2+-mediated cell death at favorable concentrations (10(-6)-10(-5) M), it caused a drastic decrease in cell viability at higher concentrations (10(-4)-10(-3) M) in a human neuroblastoma cell line. The toxic effect of chlorpromazine also occurred in Ca2+-free medium and was not parallel to the amount of thiobarbituric acid-reactive substances produced. These results indicate that chlorpromazine has biphasic effects on cell viability according to the concentrations added, i.e. a protective effect against cell damage caused by Ca2+, and a direct toxic effect independent of extracellular Ca2+ or of lipid peroxidation.

Significant increase in immunosuppressive acidic protein (IAP) in serum of patients with multiple sclerosis and other inflammatory neurological disorders.

IAP, a type of alpha 1-acid glycoprotein, is mainly produced by macrophages when stimulated in the presence of circulating immune complexes or some inflammatory substances. We assayed the serum levels of IAP by a single radial immunodiffusion method. The normal level of IAP is below 500 micrograms/ml (385 +/- 73). In multiple sclerosis patients, however, IAP increased during exacerbation (630 +/- 191) and decreased during the inactive stage (433 +/- 170). Eighty-five percent of patients with neuro-Behcet's disease also had high levels of IAP, correlating well with disease activity. In some patients with Guillain-Barré syndrome or Miller Fisher syndrome, IAP increased during the acute phase. In patients with herpes simplex encephalitis, IAP levels remained abnormally high for more than 60 days after onset. The mean value of IAP in patients with amyotrophic lateral sclerosis did not differ from that of normal controls. An increase in IAP in the serum of patients seems to reflect the activity of an inflammatory or immunopathological process.