Physiological and pathological characterization of capsaicin-induced reversible nerve degeneration and hyperalgesia.

BACKGROUND: The study aimed to investigate the physiology, psychophysics, pathology and their relationship in reversible nociceptive nerve degeneration, and the physiology of acute hyperalgesia. METHODS: We enrolled 15 normal subjects to investigate intraepidermal nerve fibre (IENF) density, contact heat-evoked potential (CHEP) and thermal thresholds during the capsaicin-induced skin nerve degeneration-regeneration; and CHEP and thermal thresholds at capsaicin-induced acute hyperalgesia. RESULTS: After 2-week capsaicin treatment, IENF density of skin was markedly reduced with reduced amplitude and prolonged latency of CHEP, and increased warm and heat pain thresholds. The time courses of skin nerve regeneration and reversal of physiology and psychophysics were different: IENF density was still lower at 10 weeks after capsaicin treatment than that at baseline, whereas CHEP amplitude and warm threshold became normalized within 3 weeks after capsaicin treatment. Although CHEP amplitude and IENF density were best correlated in a multiple linear regression model, a one-phase exponential association model showed better fit than a simple linear one, that is in the regeneration phase, the slope of the regression line between CHEP amplitude and IENF density was steeper in the subgroup with lower IENF densities than in the one with higher IENF densities. During capsaicin-induced hyperalgesia, recordable rate of CHEP to 43 °C heat stimulation was higher with enhanced CHEP amplitude and pain perception compared to baseline. CONCLUSIONS: There were differential restoration of IENF density, CHEP and thermal thresholds, and changed CHEP-IENF relationships during skin reinnervation. CHEP can be a physiological signature of acute hyperalgesia. SIGNIFICANCE: These observations suggested the relationship between nociceptive nerve terminals and brain responses to thermal stimuli changed during different degree of skin denervation, and CHEP to low-intensity heat stimulus can reflect the physiology of hyperalgesia.

Pitfalls in the use of brain slices.

In vitro brain slices are the preparation of choice for the detailed examination of local circuit properties in mammalian brain. However it is the investigator's responsibility to verify that the circuits under investigation are indeed confined within the boundaries of the functional region of the slice used. The medium in which the slice is maintained is under the full control of the investigator. This places the burden on the investigator to ensure that: (1) the properties of the medium are fully under control; (2) the effects of the medium on the slice are known; (3) the conditions under which the slice is being maintained bear some reasonable relation to those it enjoys (or endures) in vivo. Generalizations to in vivo conditions must be made with caution. If at all possible, similar studies (perhaps less extensive, due to the greater technical difficulties) should be done in vivo to provide a basis for comparison. Investigators using drugs should be aware of, and respect, the basic pharmacological principles cited in the text. In particular, the substantial freedom the investigator has in defining the extracellular medium should not be abused.

Doxorubicin-induced ultrastructural and growth changes in primary cultures of 7,12-dimethylbenz[a]anthracene-induced mammary tumors.

For the determination of their response to doxorubicin (Dx) (adriamycin), monodispersed mammary tumor cells (from female outbred Sprague-Dawley rats) were maintained as monolayer primary culture. Dose-response curves and [3H]thymidine labeling indices showed the antimitotic and cytocidal effects of the drug varied in a dose-dependent fashion. Dose-response curves revealed that the median lethal dose concentration was 10(-4) M. A 24-hour treatment at concentrations of 10(-4) to 10(-5) M completely arrested DNA synthesis of the tumor cells. Surviving cells exhibited chromatin abnormalities, accumulation of cytoplasmic myelin bodies, vacuolization of endoplasmic reticulum, and increased density of mitochondrial matrix. This study showed 1) 7,12-dimethylbenz[a]anthracene-induced mammary tumor cells were highly sensitive to Dx; 2) Dx induced specific ultrastructural effects on the nuclei, mitochondria, and membranes of the cells; and 3) the in vitro response of the primary culture may be useful for prediction of the response of the source tumor to chemotherapy.

Ultrastructure of the hormone-dependent N-nitrosomethylurea-induced mammary carcinoma of the rat.

Hormone dependency of the N-nitrosomethylurea-induced mammary tumor in rats has been demonstrated by ovariectomy. Tumors showing a clear reduction in size in response to ovariectomy have been used in an ultrastructural study. Histologically, the tumor is an adenocarcinoma. The multilayered tumor parenchyma contains myoepithelial cells and highly and less-well-differentiated cells. The adluminal cells tended to be the most differentiated and were secretory in nature. After ovariectomy, junctional complexes of the luminal cells showed little change, but intercellular adhesion among the less-well-differentiated cells appeared weakened by the altered endocrine milieu; consequently, the parenchyma appeared less compact. Cellular degeneration occurred randomly and affected all cell types. However, the least affected cells were the poorly differentiated cells with a high nuclear-cytoplasmic ratio and few cytoplasmic organelles. The findings suggest that the latter may be the hormone-independent cell subpopulation in the N-nitrosomethylurea-induced mammary tumor.

Peroxidase content in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats.

Peroxidase has been investigated as a potential marker protein for the prediction of response to hormonal therapy in tumors of steroid-sensitive tissues, but the cellular origin of the enzyme has been questioned. To localize the observed peroxidase activity, in this study cell subpopulations were isolated from mammary tumors induced by 7,12-dimethylbenz(a)anthracene and from mammary tissue of virgin and lactating female rats. Cells from each of the four cell bands, regularly obtained by isopyknic velocity sedimentation after mechanical and enzymatic dispersion of these tissues, were assayed biochemically and histochemically for peroxidase activity. In addition, cells from each subpopulation were examined at both the light and electron microscopic levels. Elevated enzyme levels were observed in each of the cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced tumors when these were compared with tissue in either of the physiological states assayed. In each tissue type, the levels of peroxidase increased from the lighter cell bands to the heavier cell bands. Light and electron microscopic examination revealed the highest proportion of epithelial cells in the lighter bands and an increase in granulocytes and fibroblasts in the heavier bands, suggesting a nonepithelial contribution. Histochemical examination of intact tissue revealed most peroxidase activity in the stromal compartment with limited activity in parenchymal cells.

Light, fluorescent, and electron microscopic analysis of cultured breast tumor cells (T-47D) treated with 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride.

The influence of bisantrene on T-47D human breast tumor cells was assessed by colony-forming assay in soft agar and by light, fluorescence, and electron microscopy. Test solutions of bisantrene solubilized in distilled water or dimethyl sulfoxide were added to cultures at final concentrations between 0.01 and 60 micrograms/ml. Brightly fluorescent particles appeared in a concentration-dependent fashion after cultures were treated with water-soluble bisantrene at concentrations greater than 0.1 microgram/ml. Similar fluorescent crystals appeared in culture media when concentrations of the dimethyl sulfoxide-dissolved drug exceeded 10 micrograms/ml. Clonogenic survival as defined by soft agar assay indicated significant reproductive impairment in cells treated with concentrations greater than 1 micrograms/ml (p less than 0.01). Nuclear and cytoplasmic fluorescence was evident in treated cells. Cells that survived 24-hr drug treatment displayed round nuclei with watery nucleoplasm when examined under the light microscope. Under the electron microscope, nuclei of these cells revealed fragmentation of the nucleolar complex and a highly electron-lucent nucleoplasm. Cytoplasmic responses, which seem to be relatively innocuous, include incorporation of the fluorescent crystals into lysosomes and some mitochondrial abnormalities. Crystalline inclusions engulfed by lysosomes were found in cells obtained from cultures showing bisantrene precipitates.

Effects of homoharringtonine on protein glycosylation in human bladder carcinoma cell T-24.

Rates of [3H]glucosamine and mannose incorporation into glycoproteins and dolichol-linked oligosaccharides in exponentially growing T-24 bladder cancer cells were examined after exposure to homoharringtonine (HHT). Two-h treatment of HHT (10 ng/ml) reduced [3H]glucosamine and mannose incorporation into the glycoproteins to 61% and 32% of controls. Concomitantly, respective sugar incorporation into dolichol-linked oligosaccharides was elevated 29% and 30% above control. The maximal inhibition of glycoprotein biosynthesis and stimulation of the lipid-linked oligosaccharides occurred within 2 to 4 h after exposure to 50 ng/ml of the drug. Prolonged drug exposure (greater than 8 h) resulted in generalized suppression of glycoprotein biosynthesis and lipid-linked oligosaccharide formation. The kinetic study indicated that the time course on reduction of glycoprotein biosynthesis and accumulation of dolichol-linked oligosaccharides paralleled the decline in protein synthesis. Further, the inhibition of glycoprotein synthesis and stimulation of dolichol-linked oligosaccharides were reversible 4 h after drug withdrawal. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of the [3H]mannose-labeled glycoprotein revealed no pronounced difference between HHT-treated and control cells. These data suggest that the inhibition of glycosylation results from combined decrease of acceptors for glycoprotein biosynthesis with a simultaneous accumulation of the dolichol-linked oligosaccharides. Collectively these data may account for many of the HHT-induced bioresponses.

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels.

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.

Effects of taurine and ketamine on bovine retinal membrane lipid peroxidation.

Lipid peroxidation disrupts membrane integrity and causes structural and functional alterations in ischemic tissues. Taurine and ketamine are putative ischemic protectants that affect Ca2+ influx. Here we report the influence of these compounds on lipid peroxidation in subcellular fractions, isolated cells and intact tissue from bovine retinas. P2 membrane fractions and isolated cells were exposed to the lipid peroxidation inducers cadmium chloride (200 microM) or L-ascorbic acid (1 mM) in the presence of 0-50 mM taurine, 0-10 mM ketamine, 1 mM kynurenic acid or 1 mM dextromethorphan. The latter compounds are N-methyl-D-aspartate receptor antagonists. Lipid peroxidation in isolated eyes reperfused after 1 h of ischemia either with or without protectants was determined by thiobarbituric acid assay. Glutathione was measured in isolated retinas subjected in vitro to simulated ischemia (no glucose or oxygenation) for 60 min either alone or in the presence of taurine or ketamine. Ketamine inhibited chemical- or ischemia-induced lipid peroxidation as well as ischemic glutathione depletion. Under the same conditions, taurine failed to affect lipid peroxidation or glutathione. The data show a direct effect of ketamine on lipid peroxidation and point to separate mechanisms of action for ketamine and taurine.

Inhibition of protein synthesis and cell proliferation in cultured human breast cancer cells treated with mitoxantrone.

Mitoxantrone suppresses cell proliferation, inhibits protein synthesis and induces ultrastructural alterations in the T-47D and MDA-MB-231 breast cancer cell lines. After 24 h treatment with 10(-9), 10(-7) and 10(-5) M drug and 8 h incubation with [35S]methionine, protein synthesis declined rapidly. While a 10-15% decrease in protein synthesis at 10(-9) M was observed, more than 95% inhibition of protein synthesis occurred at 10(-5) M mitoxantrone in both cell lines. Sodium dodecylsulfate (SDS) gel electrophoresis of labeled proteins revealed no qualitative changes in either cell line. However, only trace amounts of several proteins were present in T-47D cells treated with 10(-5) M drug. At 10(-9) M mitoxantrone had little effect on cell proliferation. At 10(-7) M, 25% and 35% growth inhibition in T-47D and MDA-MB-231 cells was observed, respectively. Cell growth at 10(-5) M was abolished. Cytotoxicity was evident at drug concentrations above 10(-5) M. Ultrastructural alterations in the nucleoli of both cell lines included disintegration and segregation of granular and fibrillar components and the disappearance of nucleolar organizers at 10(-7)-10(-5) M mitoxantrone.

Acute effects of mitoxantrone on the template activity of isolated nuclei from the T-47D human breast tumor cell line.

The effect of mitoxantrone on the template activity of nuclei isolated from the T-47D human breast tumor cell line was investigated. The results suggest that mitoxantrone significantly inhibits total RNA synthesis of these nuclei in a concentration-dependent manner. At low drug concentrations (10(-9) M and 10(-7) M) RNA synthesis was inhibited by 21.9% and 41% compared to control values, respectively. Greater inhibition was observed when the mitoxantrone concentration was increased to 10(-5) M or 10(-4) M (56% and 77%, respectively). Experiments utilizing alpha-amanitin revealed that mitoxantrone inhibits RNA polymerase II activity in a concentration-dependent manner.

Effects of luteinizing hormone releasing hormone on the ultrastructure of gonadotrophs in the foetus of the rhesus monkey near term.

Eighty micrograms of synthetic luteinizing hormone releasing hormone (LH-RH) were infused systemically into male and female foetuses of rhesus monkeys near term. Control animals were given infusions of saline. Morphologically, the control gonadotrophs varied from cells filled with secretory granules to highly stimulated cells with numerous cytoplasmic vesicles. In the LH-RH-treated animals, however, many cells showed depletion of secretory granules, dilatation of the endoplasmic reticulum and condensed nuclear chromatin. It is concluded, therefore, that foetal gonadotrophs can respond to administration of synthetic LH-RH.

Oestrogen dependence of lactate dehydrogenase and peroxidase in cell subpopulations of 7,12-dimethylbenz[alpha]anthracene-induced mammary tumours in the rat.

Recent investigations of certain enzymes as markers for predicting the response of breast tumours to hormonal therapy have neglected the possible differential contribution of cell subpopulation(s) within a solid tumour to enzyme activity. In this investigation, lactate dehydrogenase (LDH) and peroxidase activities in density-defined cell subpopulations from autotransplanted 7,12-dimethylbenz[alpha]anthracene-induced mammary tumours were determined. The effects of ovariectomy and subsequent oestrogen administration on the activity of these enzymes were also examined. Five cell subpopulations (cell bands) were routinely obtained from each mammary tumour. The highest LDH activity was found in cell band 4. The highest level of peroxidase activity was found in cell band 5. These two cell bands with high levels of enzyme activity consisted mainly of poorly differentiated cells. After bilateral ovariectomy, a significant (P less than 0.001) decrease in the level of LDH activity in cell bands 3, 4 and 5 was observed. The enzyme activity was reduced to 20, 2.1 and 12% of the preovariectomy levels respectively. Significant (P less than 0.05) decreases between baseline and postovariectomy peroxidase values were evident in each cell band. In the presence of oestradiol-17 beta, significant increases in the LDH activity of band 4 (P less than 0.001) and the peroxidase activity of band 5 (P less than 0.05) were observed. Our data suggest that, given the existence of multiple cell types in hormone-responsive tumour tissue, the actual cell subpopulation(s) responsible for any enzyme response may be a more precise indicator of hormone dependence.

Prediction of chemotherapy response in human leukemia using in vitro chemosensitivity test.

We performed the dye exclusion assay (DEA) and the MTT dye reduction assay to determine the drug sensitivity of acute leukemia using short-term microplate cultures. The in vitro results were compared with the clinical response of 31 patients with acute leukemia treated by combination chemotherapy. The true-positive rates of the DEA and MTT assays were 86.7 and 91.3%, respectively; the true-negative rates were 33.3 and 77.8%, respectively; and the predictive accuracy was 62.5 and 87.5%, respectively. The DEA and MTT assays gave comparable results in drug sensitivity testing of leukemic blast cells. Our data suggest that MTT assay is the more suitable for assessing chemosensitivity in acute leukemia.

Comparison of university of wisconsin, euro-collins, low-potassium dextran, and krebs-henseleit solutions for hypothermic lung preservation.

OBJECTIVE: We sought to test the effectiveness of 4 different solutions for hypothermic rat lung preservation. METHODS: One hundred ninety-two rats were used. The rats were divided into 4 groups, and University of Wisconsin, Euro-Collins, low-potassium dextran, or Krebs-Henseleit solution was used in each group. They were further divided into 6 subgroups of 8 rats each. The lungs were preserved at 4 degrees C for 0, 4, 6, 8, 12, or 24 hours, respectively, and lung function was studied by using a living rat perfusion model. RESULTS: Pulmonary arterial flow decreased in each group after 4 to 6 hours of preservation; the low-potassium dextran group decreased the least and the Krebs-Henseleit group decreased the most. Pulmonary vascular resistance increased in each group after 6 hours of preservation; the Krebs-Henseleit group increased the most. Although airway pressure increased, static lung compliance and gas exchange capacity decreased after 8 hours of preservation; the Krebs-Henseleit group exhibited the worst values. Lung tissue wet/dry weight ratio increased gradually during preservation; the University of Wisconsin group exhibited the least increase. An ultrastructural study indicated the least morphologic changes in the low-potassium dextran group at 24 hours. CONCLUSIONS: At 4 degrees C, all solutions preserved rat lungs for 4 hours with acceptable function. However, 6 hours of preservation resulted in damaged pulmonary function in some lungs, and this damage increased when preservation time was extended. The lungs preserved in low-potassium dextran solution had the best overall function, but the lungs preserved in University of Wisconsin solution had less edema.

A comparison of the effects of photodynamic therapy on normal and tumor blood vessels in the rat microcirculation.

The effects of light activation of the tumor photosensitizer dihematoporphyrin ether (DHE) were studied in the microcirculation of the rat cremaster muscle. Arterioles and venules in an implanted chondrosarcoma were studied by in vivo television microscopy and were compared to normal vessels of the same size elsewhere in the preparation and in control preparations. Activation with green light (530-560 nm, 200 mW/cm2, 120 J/cm2) 48 h after intraperitoneal injection of DHE (10 mg/kg body wt) resulted in significant narrowing of diameters of red blood cell columns in tumor arterioles and venules. The response in normal and control arterioles and venules was not significantly different from that seen in the tumor vessels except that the control arterioles did not remain significantly constricted during the treatment period. Treatment resulted in stasis of blood flow in 90% of tumor and normal arterioles at the completion of light activation. In venules, stasis of blood flow was observed in 75% of tumor and 70% of normal vessels. Vasoconstriction was the primary response in arterioles, while thrombosis predominated in venules. Morphologic assessment of light-activated vessels in the cremaster preparation by transmission electron microscopy revealed platelet aggregation with damage to endothelial cells and smooth muscle cells. Perivascular effects observed included interstitial edema and damage to skeletal muscle cells. In the tumor-bearing preparation, no direct cytotoxic effect on the tumor cells was shown. The surrounding vessels exhibited similar vascular stasis, but the lining cells appeared minimally affected. Photoactivation of DHE results in significant changes in the microcirculation which lead to stasis of blood flow. In this model, the response was similar for the normal microvasculature and for the microcirculation of an implanted chondrosarcoma. These effects may account, in part, for the mechanism of action of photodynamic therapy.

A case study of ligation induced calcification in middle cerebral artery in rat.

A 90 min ligation of the middle cerebral artery (MCA) followed by 72-hour reperfusion appeared to cause calcium deposition in vascular myocytes of the tunica media and the perivascular tissue of the Sprague Dawley rat. The presence of small ovoid to large irregularly shaped intracellular opaque deposits were demonstrated by light and electron microscopy. Using X-ray elemental analysis the chemical nature of the deposits was found to be calcium phosphate. The functional significance of this first demonstration of acute calcification following transient ligation of the rodent MCA invites further studies.

The glucose paradox in cerebral ischemia. New insights.

The present in vivo findings that lactate, accumulated during an ischemic episode, is an essential aerobic energy substrate during the initial postischemic period are in full agreement with out in vitro findings. Moreover, the beneficial effects of hyperglycemia are also in agreement with our and others' in vitro results that have demonstrated a neuroprotective effect of glucose against hypoxic change. The aggravation of ischemic delayed neuronal damage by glucose loading 15 min prior to the ischemic insult is likely the result of glucose induction of a short-acting (30 to 60 min) systemic factor (hormonal?) that, when combined with an ischemic insult, potentiates the ischemic damage.

Ultrastructural localization of hippocampal TNF-alpha immunoreactive cells in rats following transient global ischemia.

Using a polyclonal antibody against rat TNF-alpha, we have identified specific intracellular immunoreactive sites in hippocampal pyramidal cells, astroglia, and in microglia within 72 h after a period of ischemia. Electron opaque immunoreactive products in pyramidal cells were found mainly in somata and dendrites. Astrocytes and microglia were nearly devoid of such complexes. These findings demonstrate the presence of TNF-alpha in hippocampal neurons and its enhancement by ischemic stress.

The stability of the hippocampal slice preparation: an electrophysiological and ultrastructural analysis.

The viability and stability of the in vitro rat hippocampal slice preparation were assessed using electrophysiological and electron microscopical means. Slices exhibited lifetimes of 6-19 h. Correlation between the duration of electrical activity and changes in ultrastructure of these slices was found. Possible reasons for the wide variability in lifetime of the hippocampal slice are suggested.