High-intensity laser for acupuncture-like stimulation.

The insertion of needles into specific parts of the body was shown to provide analgesic and therapeutic effects. In this study, we tested the analgesic effects of high-intensity infrared laser for acupuncture-like stimulation. Twelve adult Sprague-Dawley rats weighing 230 to 250 g were randomly assigned to laser, needle, or restraint groups. Stimulation was directed to the meridian point Taixi (KI 3) for 10 min. For laser stimulation, a pulsed Er:YAG system was used. The laser settings were adjusted to provide a focal raise in the skin temperature to about 45 degrees C. The anti-nociceptive effect was evaluated by the tail-flick test. Both needling and laser stimulation significantly increased the tail-flick latency. Peak needling effect was observed immediately after treatment, while laser stimulation was effective both immediately and 45 min after treatment. High-intensity laser stimulation may be used alternatively or in combination with conventional acupuncture needling for pain relief.

Hypergravity modulates behavioral nociceptive responses in rats.

Hypergravity (2G) exposure elevated the nociceptive threshold (pain suppression) concomitantly with evoked neuronal activity in the hypothalamus. Young Wistar male rats were exposed to 2G by centrifugal rotation for 10 min. Before and after 2G exposure, the nociceptive threshold was measured as the withdrawal reflex by using the von Frey type needle at a total of 8 sites of each rat (nose, four quarters, upper and lower back, tail), and then rats were sacrificed. Fos expression was examined immunohistochemically in the hypothalamic slices of the 2G-treated rats. When rats were exposed to 2G hypergravity, the nociceptive threshold was significantly elevated to approximately 150 to 250% of the 1G baseline control levels in all the examination sites. The 2G hypergravity remarkably induced Fos expression in the paraventricular and arcuate nuclei of the hypothalamus. The analgesic effects of 2G hypergravity were attenuated by naloxone pretreatment. Data indicate that hypergravity induces analgesic effects in rats, mediated through hypothalamic neuronal activity in the endogenous opioid system and hypothalamo-pituitary-adrenal axis.

Gravity stress elevates the nociceptive threshold level with immunohistochemical changes in the rat brain.

Young Wistar male rats were exposed to 2G hypergravity by continuous centrifugation for 15 minutes. The nociceptive threshold was measured by using the von Frey type filament on the rat skin surfaces after hypergravity exposure. Following the hypergravity exposure, rats were sacrificed with anesthesia, then perfused and fixed for immunohistochemical examination. The 2G hypergravity elevated the nociceptive threshold up to 2-fold and induced analgesic effects on rats that remained for 2 hours after termination of centrifugation. Expression of Fos-immunoreactive proteins was prominently induced by 2G hypergravity in the arcuate nucleus and the paraventricular nucleus of the hypothalamus. The 15-minute flash exposure to 2G hypergravity induced pain suppression in rats, which might be attributed to change of neuronal activity in rat hypothalamus.

Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol.

Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19).

Aromatase P450 (CYP19) is an enzyme responsible for conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeting disruption of the CYP19 gene and observed that the ArKO males exhibited a complete loss of aggressive behaviour against intruder mice when examined using a resident-intruder paradigm. The defect in the behaviour of ArKO males was reinstated when the mice received supplements of 17beta-oestradiol soon after birth. Nevertheless, the cumulative duration of the behaviour displayed by the treated mice during the test period of 15 min was 19+/-10 s, which was much shorter than that displayed by wild-type males, 90+/-17 s. When the supplementation was started at 7 days after birth, the defect was not restored. These findings illustrate an absolute requirement for oestrogen during the neonatal stage of a male's life for the development of the potential for aggression observed in adulthood. Furthermore, the present study demonstrates that ArKO males are a useful model in which to investigate the neural mechanisms by which aggressive behaviour is controlled.

Long-term local hyperthermia in the treatment of advanced breast cancer (case report).

BACKGROUND: It is difficult to control non-resectable locally advanced primary and recurrent breast cancer by conventional modalities. Recently, hyperthermia (HT) has been recognized as an effective adjuvant to radiotherapy (RT) and chemotherapy (CT) in treatment of various malignancies, including breast cancer. PATIENT AND METHODS: The patient was a 58-year-old female Japanese, with breast cancer, T4N2M0, stage IIIb (papillo-tubular carcinoma). Previous treatment included RT and neoadjuvant CT Local HT was performed with a total number of 87 sessions given over 12 months. The mean time of each session was 40 minutes. Elevation of temperature to a tumoricidal level of 43 degrees C was confirmed. The patient received cyclophosphamide (50 mg p.o./day) and tamoxifen (20 mg p.o./day) during the whole period of HT. Due to the decreased amount of WBC, further CT was not possible, except for one course of CMF performed 3 months after the start of HT. RESULTS: The patient had a decrease in the intensity of pain even after the first 3 sessions. In one month, movement in the right shoulder became possible in an anterio-posterior direction. By 5 months, the healing of ulceration became evident. At present, the patient is in continuous CR for 15 months after HT. The movement in the shoulder joint is markedly improved in all directions. In addition, HT did not cause any notable complications. CONCLUSION: Long-term HT may be useful in the management of locally advanced breast cancer and these results should encourage further clinical study.

Hypergravity and opioid-mediated pain suppression in rats.

It is known that pain suppression in animals is induced by certain environmental stimulus. However, little is known about the effects of gravitational alteration on the nociceptive responses in rats. A recent study indicated that Fos protein expression was strongly induced in the vestibular-related brainstem regions of rats that were exposed to 2 G hypergravity (Gustave Dit Duflo et al., 2000). A number of studies indicate that Fos expression is induced in the brain by various kinds of stress. We showed that either long-term exposure or short-term exposure to 2 G hypergravity elevated the nociceptive threshold in the rat skin surfaces, in concomitant with Fos induction in the hypothalamus including the arcuate nucleus and paraventricular nucleus (Kumei et al., 2000). We have examined the possible involvement of beta-endorphin, an endogenous opioid, in the hypergravity-induced analgesic effects on rats and its counteraction by naloxone, an opioid receptor antagonist.

[A case of amebic colitis cured with multiple cicatricial strictures].

A 53-year-old male was admitted to Gifu Red Cross Hospital with the complaints of diarrhea, abdominal pain, and fever. He had a temperature of 38.4 degrees C and diffuse abdominal tenderness without guarding or rebound. Plain film of the abdomen showed marked dilatation of the transverse colon. Sigmoidoscopy showed multiple aphthoid erosions and pseudomorphic ulcers, and mucosal biopsies demonstrated numerous trophozoites of Entamoeba histolytica. The patient was treated with oral metronidazole with rapid improvement. Barium enema and colonoscopy after improvement showed multiple cicatricial strictures. Although prompt diagnosis and therapy prevented fulminant changes, the patient was cured with multiple cicatricial strictures, a rare complication of amebic colitis. It is important to keep in mind severe amebic colitis in the differential diagnosis of patients with diarrhea and high fever.

Neoadjuvant intra-arterial infusion chemotherapy combined with hormonal therapy for locally advanced breast cancer.

To determine the efficacy of combined neoadjuvant intra-arterial infusion chemotherapy and hormonal therapy for treating locally advanced breast cancer, we compared the outcomes of patients with or without this therapy, and also assessed histologic response. Ninety-four patients with locally advanced breast cancer (stage IIIa, 56; stage IIIb, 38). Nineteen stage IIIa and 17 stage IIIb patients received intra-arterial plus hormonal therapy while 37 stage IIIa and 21 stage IIIb patients with similar ages and follow-up durations did not. Treated patients received intra-arterial epirubicin plus oral medroxy-progesterone. Five-year disease-free survival rates were 77.5% for intra-arterially treated and 33.0% for other patients in stage IIIa, and 70.5% for intra-arterially treated and 38.1% for other patients in stage IIIb. Five-year overall survival rates were 94.4% for intra-arterially treated and 61.7% for other patients in stage IIIa, and 90.9% for intra-arterially treated and 56.3% for other patients in stage IIIb. Ten-year overall survival rates in stage IIIb were 90.9% for treated and 22.5% for other group patents. All differences were statistically significant (p<0.05). Good histologic response to intra-arterial therapy was seen in 75% of the primary tumors and 71% of involved lymph nodes. Neoadjuvant intra-arterial therapy with hormonal therapy yielded better survival rates than no intra-arterial therapy or our previous intra-arterial regimen.

Superior protection in orthotopic rat lung transplantation with cyclic adenosine monophosphate and nitroglycerin-containing preservation solution.

BACKGROUND: Primary lung graft failure is common, and current lung preservation strategies are suboptimal. Because the decline in lung levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate during preservation could enhance adhesiveness of endothelial cells for leukocytes as well as increase vascular permeability and vasoconstriction, we hypothesized that buttressing these levels by means of a preservation solution would significantly improve lung preservation. METHODS: An orthotopic rat left lung transplantation model was used. Lungs were harvested from male Lewis rats and preserved for 6 hours at 4 degrees C with (1) Euro-Collins solution (n = 8); (2) University of Wisconsin solution (n = 8); (3) low-potassium dextran glucose solution (n = 8); (4) Columbia University solution (n = 8), which contains a cyclic adenosine monophosphate analog (dibutyryl cyclic adenosine monophosphate) and a nitric oxide donor (nitroglycerin) to buttress cyclic guanosine monophosphate levels; or (5) Columbia University solution without cyclic adenosine monophosphate or nitroglycerin (n = 8). PaO2, pulmonary vascular resistance, and recipient survival were evaluated 30 minutes after left lung transplantation and removal of the nontransplanted right lung from the pulmonary circulation. RESULTS: Among all groups studied, grafts stored with Columbia University solution demonstrated the highest Pa O2 (355 +/- 25 mm Hg for Columbia University solution versus 95 +/- 22 mm Hg for Euro-Collins solution, P <.01, 172 +/- 55 mm Hg for University of Wisconsin solution, P <.05, 76 +/- 15 mm Hg for low-potassium dextran glucose solution, P <.01, and 82 +/- 25 mm Hg for Columbia University solution without cyclic adenosine monophosphate or nitroglycerin, P <.01) and the lowest pulmonary vascular resistances (1 +/- 0.2 mm Hg * mL-1 * min-1 for Columbia University solution versus 12 +/- 4 mm Hg * mL-1 * min-1 for Euro-Collins solution, P <.01, 9 +/- 2 mm Hg * mL-1 * min-1 for University of Wisconsin solution, 14 +/- 6 mm Hg * mL-1 * min-1 for low-potassium dextran glucose solution, P <.01, and 8 +/- 2 mm Hg * mL-1 * min-1 for Columbia University solution without cyclic adenosine monophosphate and nitroglycerin). These functional and hemodynamic improvements provided by Columbia University solution were accompanied by decreased graft leukostasis and decreased recipient tumor necrosis factor alpha and interleukin 1alpha levels compared with the other groups. In toto, these improvements translated into superior survival among recipients of Columbia University solution-preserved grafts (100% for Columbia University solution, 37% for Euro-Collins solution, P <.01, 50% for University of Wisconsin solution, P <.05, 50% for low-potassium dextran glucose solution, P <.05, and 13% for Columbia University solution without cyclic adenosine monophosphate and nitroglycerin, P <.01). CONCLUSION: Nitroglycerin and cyclic adenosine monophosphate confer beneficial vascular effects that make Columbia University solution a superior lung preservation solution in a stringent rat lung transplantation model.

Establishment and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium.

The behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear. Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results. To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern. In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors. The cell aggregation activity of F-2C in the presence of Ca2+ was much greater than that of F-2. The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis. These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity. We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.

Reversal of altered intestinal mucosal immunity in rats fed elemental diet by supplementation of oleic acid.

We have previously demonstrated that elemental diet (ED) induces decreased lymphocyte transport in intestinal lymph and significant changes in T cell subsets and the number of IgA-containing cells in gut-associated lymphoid tissues of rats. In order to examine whether the low fat content contributes to the induction of immunological changes in gut-associated lymphoid tissues, the effects of additional fatty acid in the ED were investigated. Rats were divided into four groups: elemental diet alone, elemental diet supplemented with 5% oleic acid (OA), elemental diet with 10% OA and conventional diet as a control. These diets were given at the same daily calorie intake for 4 weeks. The flow rate of intestinal lymph showed no significant difference between the four groups. However, lymphocyte flux as well as the percentage of CD3+ and CD4+ cells were significantly greater in the control and the 10% OA groups than in the ED and 5% OA groups. Intestinal lymph showed decreased concentrations of IgG and IgA in the ED group, whereas the addition of 10% OA significantly attenuated the decrease in these levels. In mesenteric lymph nodes, the CD4+/CD8+ ratio was significantly decreased in the ED group, but 10% OA reversed this change. Immunohistochemical analysis of the ileal mucosa showed that in the ED group the population of CD4+ cells was decreased, while the number of CD8+ cells was increased. Supplementation of OA to ED produced similar stepwise attenuation of the changes in lymphocyte subpopulations in the lamina propria, while the 10% OA group reached levels that were not statistically different from controls. In the elemental diet group, there was a significant decrease in immunoglobulin-containing cells of the IgA class in the lamina propria of the intestine. Similarly, the addition of OA induced dose-dependent recovery in the number of IgA-containing cells. These results suggest that a low dietary concentration of fat may be closely related to changes in lymphocyte transport in intestinal lymph and mucosal immunity of intestinal mucosa induced by the feeding of a long-term ED.

Ipsilateral hemiplegia caused by right internal capsule and thalamic hemorrhage: demonstration of predominant ipsilateral innervation of motor and sensory systems by MRI, MEP, and SEP.

A patient with a right internal capsule and thalamic hemorrhage showed ipsilateral hemiplegia. MRI at 10 months after the cerebral hemorrhage demonstrated Wallerian degeneration, which could be traced to the ipsilateral anterior funiculus at the cervical level. The findings of motor evoked potentials and somatosensory evoked potentials indicate a predominantly ipsilateral innervation of motor and sensory systems in this particular patient.

[The significance of intra-arterial infusion therapy for neoadjuvant chemotherapy].

Arterial infusion therapy with anticancer drugs is now attracting attention as a valuable modality for locally advanced breast cancer. Since 1977, we have used this therapy in 122 patients with primary breast cancer. The present report mainly discusses the clinical and histological response as well as the prognosis. The anticancer drugs were mainly given by two routes, infusion into the internal mammary artery and the subclavian artery. Continuous infusion of 5-FU and intermittent injections of MMC, ADR, 4'-epi-ADR and THP-ADR were jointly or individually made in each artery. Clinical response, defined as CR + PR, was noted in 48.4% of 5-FU group and 72.7% of ADR-MMC group. Histological response according to Shimosato Criteria, defined as grade IIb or better, appeared in 45.2% of main tumors and 25.4% of metastatic lymph nodes in the 5-FU group, and 70.9% main tumors and 46.3% of metastatic lymph nodes in the ADR-MMC group. The non-infusion group contained 27.7% of stage IIIb, against 72.2% in the infusion group. The 5-year overall survival rates were non-infusion group 62%, 5-FU group 34.1% and ADR-MMC group 66.2%. A significant difference was seen between the 5-FU infusion group and the ADR-MMC group (p = 0.03). Administration of high dose medroxy progesterone acetate for two weeks and 4'-epi-ADR infusion chemotherapy resulted in an excellent histological response. This combination therapy is a promising neoadjuvant chemo-endocrine therapy for advanced breast cancer.

Changes of proliferative activity and phenotypes in spontaneous differentiation of a colon cancer cell line.

We examined the alterations of proliferative activity and c-myc expression of a colon cancer cell line (Caco-2) during its spontaneous differentiation. Caco-2 cells were cultured in various types of media and the degree of differentiation was monitored in terms of dome formation in cell monolayers and expression of alkaline phosphatase (ALP) activity. In Caco-2 cells cultured with Eagle's minimum essential medium (EMEM) containing 10% fetal calf serum (FCS), dome formation was demonstrated and ALP activity was markedly increased after the cells reached confluence. Five-fold reduction of c-myc mRNA and a marked decrease in S-phase cells were observed in the differentiated cells. These changes were not induced in FCS-free EMEM. The addition of insulin and transferrin to FCS-free EMEM did not induce cell differentiation or reduction of c-myc mRNA expression. When Caco-2 cells were cultured with three different serum-free media, the induction of dome formation and the increase of ALP activity were observed to varying degrees. Expression of c-myc mRNA in the cells cultured with one serum-free medium decreased to a level similar to that in fully differentiated cells cultured with EMEM containing 10% FCS. These results suggest that a spontaneous switch from a proliferative state with high c-myc expression to differentiated phenotype occurs after cells reach confluence and depends on the culture conditions.

[Effect, recurrence and prognosis of intra-arterial infusion chemotherapy with anticancer agents for advanced breast cancer--especially, the significance of tumor blood vessels].

Between January 1977 and March 1991, 86 patients of Stage III and Stage IV breast cancer were treated by intra-arterial infusion chemotherapy (IAIC) in Sapporo Medical College. The 5- and 10-year overall survival rates of Stage III b were: infusion group 53.1% and 53.1%, non infusion group 50.1% and 16.7%, respectively. A significant difference was seen in the 10-year survival rate (p less than 0.05). The 5- and 10-year overall survival rate of Stage III b histologically effective group was 67.3% and non-effective group 31.8%. A significant difference was seen between these two groups (p = 0.0241). A significant difference was seen between n2 positive Stage III b infusion group and non-infusion group in 7-year survival rate (p less than 0.01). The ischemic effect on tumor cells by degeneration changes of the blood vessels was important for the histological effect. Pre-operative angiography helped in the choice of anticancer agents. The sufficient adjuvant chemo-endocrine therapy will assure a good prognosis for IAIC-treated Stage III b breast cancer patients.

An analysis of histaminergic efferents of the tuberomammillary nucleus to the medial preoptic area and inferior colliculus of the rat.

The efferent projections of the five histaminergic neuronal subgroups in the tuberomammillary nucleus to the medial preoptic area (MPO) and inferior colliculus (IC) were examined by immunocytochemistry with antihistidine decarboxylase (HDC) antibodies combined with retrograde axonal tracing with Fast Blue (FB). The term "E groups" were used for the histaminergic neuronal subgroups. About 10% of the HDC-immunoreactive (HDCI) neurons were retrogradely labeled after FB injection into the MPO. The labeled neurons were not concentrated in any particular area, but were diffusely distributed bilaterally in all the subgroups. About two-thirds of the labeled neurons were observed on the side ipsilateral to the injection site and one-third on the contralateral side. The percentages of labeled neurons (double-labeled neurons/HDCI neurons) in the five subgroups were not significantly different with each other. The percentages in group E1 and E2 were particularly close, while that in group E4 resembled that in group E5. About 4% of the HDCI neurons were retrogradely labeled after the dye injections into the IC, and about half of the labeled neurons were detected on the ipsilateral side. The percentage of the double-labeled neurons in the five groups were not significantly different. Furthermore, those in E1 and E2, and in E4 and E5 were almost identical, respectively, to the situation following injection of FB into the MPO. These results indicate that each subgroup of histaminergic neurons in the tuberomammillary nucleus has similar efferent projections to the MPO and IC.

Ultraviolet radiation suppresses mouse-ear edema induced by topical application of arachidonic acid.

The effect of ultraviolet (UV) radiation on arachidonic acid (AA) cascade was examined using an in vivo model. Mouse ear lobes were painted with 1 mg AA, and the maximum response of ear swelling was measured 1 h after challenge. Arachidonic-acid-induced ear swelling was significantly suppressed by preexposure to topical psoralen plus noninflammatory doses of long-wave UV radiation (PUVA) or middle-wave UV (UVB) radiation. Ultraviolet radiation may interfere with AA pathways to suppress ear swelling since AA-induced ear swelling is considered to be mediated by metabolites derived from exogenous AA. The results may relate to the therapeutic mechanisms of UV radiation in psoriasis in which the eicosanoid cascade is involved.