The comparisons on total RNA from different source-original neurons applied in LMPC.

Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods (1) Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; (2) Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; (3) Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results (1) Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC; relative short amplicons such as GAPDH, NSE, MCH and MC4R were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; (2) In mangocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two; (3) The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.

[Interaction of 5-HT2 and 5-HT3 receptor subtype in 5-HT-induced nociceptive responses in peripheral primary sensory nerve ending].

AIM: To study the correlation between 5-HT-induced pain response and the contribution by individual 5-HTR subtypes including 5-HT1R, 5-HT2R and 5-HT3R at the level of peripheral primary afferent. METHODS: The experiments were done on acutely isolated trigeminal ganglion (TG) neurons using whole-cell patch clamp technique and the nociceptive effect was observed on behavior experiments by intraplantar injection of test drugs. RESULTS: The majority of cells examined responded to 5-HT in a manner of concentration dependence (10(-6) - 10(-3) mol/) (61.4%, 54/88) and with a fast activating and rapid desensitizing inward current (I(5-HT)), which was thought to be mediated by the activation of 5-HT3R, since it could be blocked by 5-HT3R antagonist ICS 205930 and mimicked by 5-HT3R agonist 2-methyl-5-HT. It was found that I(5-HT) was potentiated by 5-HT2R agonist alpha-methyl-5-HT markedly, while 5-HT1R agonist R-(+)-UH 301 did not. In behavioral experiment performed on conscious rats, intraplantar injection of 5-HT(10(-5), 10(-4) and 10(-3) mol/L) induced an increment of cumulative lifting time first 20 min in a manner of concentration dependence. By dissociating 5-HTR subtypes using their corresponding antagonists (ICS and CYP) the potency order of hindpaw lifting time was identified as follows: 5-HT > 5-HT + ICS > 5-HT + CYP. CONCLUSION: The results suggest that in 5-HT-induced nociceptive response at the primary sensory level 5-HT3R may play a role of initiation, but 5-HT2R mediates maintaining and modulatory effect in the processes of nociceptive information convey.

[Effect of electroacupuncture at "Neiguan" (PC 6) on expression of c-fos gene in myocardium of the rat with acute myocardial ischemia].

OBJECTIVE: To explore the mechanism of electroacupuncture at "Neiguan" (PC 6) improving acute myocardial ischemia. METHODS: The rats were randomly divided into a sham operation group, a myocardial ischemia model group and a myocardial ischemia model plus electroacupuncture group. The acute myocardial ischemia model was developed byligation of the descending anterior branch of the coronary artery, and electroacupuncture was given at bilateral "Neiguan" (PC 6). Serum myocardial enzymes was determined by biochemical method and the expression of c-fos mRNA in myocardium was detected by using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The activities of serum myocardial enzymes and the expression of c-fos mRNA in ischemic myocardium were significantly increased as compared with those in the sham operation group (P < 0.05), and after electroacupuncture they were significantly decreased (P < 0.05). CONCLUSION: The mechanism of electroacupuncture at "Neiguan" (PC 6) improving acute myochadial ischemia is possibly related with down-regulation of expression of c-fos mRNA in myocardium.

Opposite modulatory effects of substance P on GABA-and 5-HT-activated currents in the same sensory neurons.

The modulation by substance P of gamma-aminobutyric acid (GABA)- and 5-hydroxytryptamine (5-HT)-activated currents (I(GABA) and I(5-HT)) was studied by using patch-clamp technique in rat trigeminal ganglion (TG) neurons. The majority of neurons examined responded to GABA and 5-HT with inward currents in the same cells (63.8%, 30/47). In 22 out of 30 neurons sensitive to both GABA and 5-HT, pretreatment with substance P (SP, 0.01 micromol/L) suppressed I(GABA) by (35.7 +/-6.1)% and enhanced I(5-HT) by (65.2 +/- 8.7)%. GR 82334, a potent and specific antagonist of NK1 tachykinin receptor, reversibly blocked the modulatory effects of SP. The SP modulation on I(GABA) and I(5-HT) was also abolished by intracellular dialysis of GDP-beta-S, a non-hydrolyzable GDP analog, or GF 109203X, a selective protein kinase C inhibitor. These results suggest that SP exerts opposite modulatory actions on GABA(A) receptor and 5-HT3 receptor activity of the same primary sensory neuron via the same intracellular signal transduction pathway.

Potentiation of 5-HT3 receptor function by the activation of coexistent 5-HT2 receptors in trigeminal ganglion neurons of rats.

5-HT receptor subtypes are widely expressed in primary sensory neurons, yet so far little is known about the interaction among them. This study aimed to investigate whether the activation of 5-HT2 and 5-HT1 receptors could modulate 5-HT3 receptor mediated current in rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The majority of TG neurons examined responded to 5-HT (10(-7)-10(-3) M) with a fast activating and rapid desensitizing inward current (77.2%, 71/92). This 5-HT activated current (I(5-HT)) was blocked by ICS 205-930 and mimicked by 2-methyl-5-HT, indicating that it was mediated by 5-HT3 receptor. With alpha-methyl-5-HT applied prior to 5-HT application, I(5-HT) was potentiated in a concentration-dependent manner, with the maximal modulatory effect at 10(-9) M of alpha-methyl-5-HT. The concentration-response curve for I(5-HT) pretreated with alpha-methyl-5-HT shifts upwards compared with that for I(5-HT) without alpha-methyl-5-HT pretreatment, the maximal I(5-HT) value having increased by (60.3 +/- 5.7)% of its control while the EC50 values of the two curves being very close, i.e. (2.0 +/- 0.3) x 10(-5) M vs (1.7 +/- 0.2) x 10(-5) M, respectively. The alpha-methyl-5-HT potentiation of I(5-HT) was removed by intracellular dialysis of either GDP-beta-S, a non-hydrolyzable GDP analog, or GF109203X, a selective PKC inhibitor, almost completely. Preapplication of R-(+)-UH-301, a selective agonist of 5-HT(1A) receptor, had no modulatory effect on I(5-HT). These results suggest that in the membrane of TG neurons, the activation of 5-HT2 receptors can exert an enhancing effect on the function of coexistent 5-HT3 receptors while that of 5-HT(1A) receptors cannot.

Substance P potentiates 5-HT3 receptor-mediated current in rat trigeminal ganglion neurons.

The present study aimed to investigate the interaction between the coexistent SP receptor and 5-HT3 receptor in trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The majority of the neurons examined responded to 5-HT with an inward current (I5-HT) (78.2%, 79/101) that could be blocked by 5-HT3 receptor antagonist, ICS-205,930. The I5-HT was potentiated by preapplication of SP (10(-10) to 10(-8) M) in most 5-HT-sensitive cells(78.5%, 62/79). Coapplication of SP and GR-82334, antagonist of NK1 receptor, had no enhancing effect on I5-HT. The concentration-response curves for 5-HT with and without SP preapplication show that: (1) the threshold 5-HT concentrations with and without SP preapplication are basically the same, while SP preapplication increased the maximal value of I5-HT by 38.0% of its control; (2) the EC50 values of the curves with and without SP pretreatment are very close, i.e. 1.89 x 10(-5) M and 2.08 x 10(-5) M (P > 0.1; n = 9), respectively. Intracellular dialysis of GDP-beta-S, a non-hydrolyzable GDP analog, and GF-109203X, a selective protein kinase C inhibitor, removed the SP potentiation of I5-HT. These results may offer a clue to understanding the mechanism underlying the generation and/or regulation of peripheral pain caused by tissue damage inflammation, etc.

Effects of tanshinone on neuropathological changes induced by amyloid beta-peptide(1-40) injection in rat hippocampus.

AIM: To investigate the effect of tanshinone (Tan) on the neuropathological changes induced by amyloid beta-peptide1-40 (Abeta1-40) injection in hippocampus in rats. METHODS: Abeta1-40 10 microg was injected bilaterally into the dorsal blade of the dentate gyrus in the hippocampus. The level of acetylcholinesterase (AChE) in hippocampus was evaluated by histochemistry. The expressions of neuronal nitric oxide synthase (nNOS) and inducible form of NOS (iNOS) were detected by immunohistochemistry and Western blot. Abeta1-40-injected rats were treated ig with Tan, the major active ingredient from Salvia miltiorrhiza of Chinese herb extract. RESULTS: The level of AChE positive fibers of each subfield in Abeta1-40-injected hippocampus decreased significantly compared with those of control (P<0.01). The expression of nNOS was down-regulated whereas the iNOS was up-regulated. After treatment with Tan (50 mg/kg, ig), the changes mentioned above were significantly improved. Moreover, the correlation analysis revealed a significant negative correlation between the area percentage of AChE positive fibers and the number of iNOS positive neural cells in CA1, CA2 to CA3 (CA2-3), and dentate gyrus (DG) subfields (P<0.01). CONCLUSION: Tan can protect the neuropathological changes induced by Abeta1-40 injection in hippocampus.

Enzymes activity of intestinal grafts after liver small bowel transplantation in rats.

OBJECTIVES: To investigate the activity alterations of enzymes in intestine grafts after liver/small bowel transplantation in rats and the relations of these changes to immune rejection of grafts. METHODS: A model of liver/small bowel transplantation (LSBT) was established in closed colony SD and Wistar rats. The activity of enzymes including triphosphatase (ATPase), alkalinophosphatase (AKP), acytelcholinesterase (AchE), oxidesynthase (NOS) and monoamine oxidase (MAO) in bowel grafts was studied histochemically at regular postoperative intervals. RESULTS: The activity of enzymes in the wall of the grafts disappeared eventually in isolated small bowel transplantation (SBT) rats. In contrast, the activity in LSBT rats remained and recovered postoperatively. CONCLUSIONS: The rejection in grafted intestine could be prevented or delayed in LSBT rats. The changes in the activity of enzymes and neurons might be used to detect the rejection and function of the graft.

Expressions of PCNA, p53, p21(WAF-1) and cell proliferation in fetal esophageal epithelia: comparative study with adult esophageal lesions from subjects at high-incidence area for esophageal cancer in Henan, North China.

AIM: To characterize the expression of p53, p21(WAF-1) and proliferation-cell-nuclear-antigen (PCNA) in fetal esophageal epithelia and to determine the role of these genes in proliferation of fetal and adult esophageal epithelial cells. METHODS: Immunohistochemical avdin-biotin peroxidase complex (ABC) method was applied to 31 cases of fetal esophageal specimens and 194 cases of adult esophageal specimens to detect the expression of p53, p21(WAF-1) and PCNA in fetal and adult esophageal epithelia. RESULTS: Both the PCNA positive immunostaining cell number and PCNA positive immunostaining rate in fetal esophageal epithelia (506+/-239) were significantly higher those that in adults, including normal epithelia (200+/-113) and epithelia with basal cell hyperplasia (BCH) (286+/-150) (P<0.05, t test). However, the number of PCNA positive immunostaining cells in adult esophageal dysplasia (719+/-389) and squamous cell carcinoma(SCC) (1261+/-545) was apparently higher than that in fetal esophageal epithelia (506+/-239) (P<0.05, t test). The positive immunostaining rate of P53 was 10 % (3/31) in fetal esophageal epithelia, which was significantly lower than that in adult normal esophageal epithelia (50 %), adult epithelia with basal cell hyperplasia (62 %), dysplasia (73 %) and squamous cell carcinoma (86 %) (P<0.05, Fisher's exact test). No p21(WAF-1) positive immunostaining cells were observed in fetal esophageal epithelia. However, p21(WAF-1) positive immunostaining cells were observed in adult esophagus with 39 % (11/28) in normal, 38 % (14/37) in BCH, 27 % (3/11) in DYS and 14 % (1/7) in SCC. CONCLUSION: PCNA could act as an indicator accurately reflecting the high proliferation status of fetal esophageal epithelium. p53 may play an important role in growth and differentiation of fetal esophageal epithelium. p21(WAF-1) may have no physiological function in development of fetal esophageal epithelium.