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Objective To investigate the role of protein kinase C (PKC) in induction of vascular endothelial growth factor (VEGF) secretion by isoflurane in primary cultured rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 6 groups ( n = 6 each): control group (group C), 3 different concentration isoflurane groups (group Ⅰ1-3 ), PKC inhibitor calphostin C group (group P), and PKC inhibitor + isoflurane group (group PI). The cells were exposed to 0.7%, 1.4% and 2.1% isoflurane for6 h in group Ⅰ1-3 respectivly. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L in group P. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L, then the cells were exposed to 1.4% isoflurane for 6 h in group PI. VEGF concentrations and expression of PKC isoforms were determined by ELISA and Western blot respectively. Results Compared with group C, the VEGF concentration was significantly increased in group Ⅰ2 and Ⅰ3, and PKCε expression was down-regulated in the cytoplasm while upregulated in the cytomembrane in group Ⅰ2 ( P ＜ 0.01 ), but no significant change was found in the parameters mentioned above in group Ⅰ2 ( P ＞ 0.05). PKCα, PKCδ and PKCζ expression was significantly higher in the cytoplasm than in the cytomembrane in group C and Ⅰ2. VEGF concentrations were gradually increased with the increase in isoflurane concentrations ( P ＜ 0.05). VEGF concentrations were significantly lower in group PI than in Ⅰ2 ( P ＜0.05) .Conclusion Isoflurane induces VEGF secretion in primary cultured rat cardiomyocytes through translocation of PKCε from the cytoplasm to the cytomembrane, suggesting that it is a mechanism of the cardioprotective effects of isoflurane.
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Objective To investigate the efficacy of percutaneous laser disc decompression(PLDD)combined with injection of collagenase through a target location for treatment of lumbar intervertebral disc protrusion.Methods Ninety patients with lumbar intervertebral disc protrusion scheduled for discolysis,aged 31-52 yr,weighing 58-70 kg,were randomly divided into 3 groups: PLDD group(group P,n = 29),collagenase injection group(group C,n = 31),PLDD combined with injection of collagenase through a target location group(group PC,n = 30).The puncture was performed under the guidance of CT.Group P was treated using PLDD.Group C was treated with collagenase injection.Group PC was treated with injection of collagenase after PLDD was completed.The therapeutic effect was assessed before operation and on day 7,30,60 and 90 after operation using M-JOA score.Results M-JOA grade was significantly higher at the each time point after operation in group P and PC,and on day 30,60 and 90 after operation in group C than that before operation(P ＜ 0.05).M-JOA grade was significantly lower on day 30 after operation in group P,while higher on day 30,60 and 90 after operation in group C and PC than that on day 7 after operation(P ＜ 0.05).M-JOA grade was significantly lower at the each time point after operation in group P and C than in group PC.Conclusion The therapeutic effect of PLDD combined with collagenase injection through a target location is stable for treatment of lumbar intervertebral disc herniation and better than that of PLDD or collagenase injection alone.
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BACKGROUND: Based on previous technique prepared for encapsulating living cells with alginate-polysine- alginate (APA) microcapsules, it has been confirmed that microencapsulated chromaffin cells have good analgesic effects. The immunoisolated effects of such microcapsule materials need to be evaluated. OBJECTIVE: This study aimed to investigate the immunological rejections of APA microencapsulated chromaffin cells transplanted into rat anterior chamber of eyes and tendon of feet, and to evaluate the immunoisolated effect of microencapsulation.DESIGN: A randomized controlled animal experiment. SETTING: Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Forty-eight female SD rats, with the age of 3 months, were provided by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. Alginate and polylysine used in the experiment were the products of Sigma Company, USA. Microcapsule generator was gifted by Germany. METHODS: This study was performed at the Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from September 2002 to September 2003. Suprarenal medulla was taken from 6 healthy adult cadavers of brain death. After isolated, digested and cultured, suprarenal medulla was prepared into chromaffin cell suspension. Written informed consents were obtained from the family members of donors, and the protocol was given approval by the Ethics Committee of the hospital. Empty microcapsules and microencapsulated cells were prepared by APA. The 48 rats were randomly divided into the human chromaffin cell (HCC) group, the empty microcapsule group and the microencapsulated HCC (ME-HCC) group. In each group, there were two transplanted regions of anterior chamber of eyes and tendon of feet, with 8 rats used for each region. Each rat in the HCC group was perfused 2×1010 L-1 cell suspension into the anterior chamber of eyes and tendon of feet. Those in the empty microcapsule group and the ME-HCC group were perfused 100 empty capsules and ME-HCCs (100 microcapsules, 400-500 HCCs per microcapsule) into the same regions, respectively. MAIN OUTCOME MEASURES: On day 7 after transplantation, serum interleukin (IL)-2 level was determined by ELISA. Serum IgG and IgM levels were determined with a laser turbidimeter. On day 28 after transplantation, rat right eyeball and left feet were harvested, routinely sliced and stained by haematoxylin-eosin (HE). Histo-morphological structure was observed under a 40×light microscope. RESULTS: Forty-eight rats were included in the final analysis. Serum IL-2, IgG and IgM levels were significantly lower in the empty microcapsule group and ME-HCC group than in the HCC group (t=8.544-21.64, P < 0.01). A lot of lymphocyte and neutrophile infiltration could be found in the anterior chamber of eyes and tendon of feet of rats in the HCC group, but a little seen in that of the empty microcapsule group and ME-HCC group. CONCLUSION: APA microencapsulation has an effective immunoisolated effect on immunological rejection due to its good biocompatibility and mechanical stability.
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Objective To investigate the effects of mediastinal block(MB)on coronary atherogenesis and hemodynamics in rabbits with hyperlipoidemia.Methods Forty-eight male New Zealand white rabbits were randomly divided into 4 groups(n=12 each):control group received normal diet 150 g/d for 16 weeks,hypercholesterol group received hypercholesterol diet 150 g/d for 16 weeks,thoracic epidurial block(TEB)group received hypercholesterol diet 150 g/d for 16 weeks and TEB was performed from 13th to 16th week with 2% lidocaine 2 mg/kg twice a day,and MB group received hypercholesterol diet 150 g/d for 16 weeks and MB was performed from 13th to 16th week with 2% lidocaine 2 mg/kg twice a day.MAP was measured before and after 1st block was performed.The serum levels of total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)were measured on 1st day,and on4th,6th,8th and 16th week during the experiment.At the end of 16 th week,all rabbits were killed by air embolism.Heart was removed and kept in 10% formalin for a week.The ventricles were transversely sectioned at the level of papillary muscle and slices from the cross section of the ventricles were obtained for determination of the degree of atherosclerosis by microscopy.Results MAP was decreased significandy after TEB in TEB group,while there was no significant changes in MAP after MB in MB group(P＜0.05).The serum levels of TC,TG and LDL-C were significandy higher in hypercholesterol.TEB and MB groups than in control group(P＜0.05 or 0.01).The ratios of atherogenesis and intimal thickening were significantly lower in TEB and MB groups than in hyperoholesterol group(P＜0.01),there was no significant difference between TEB and MB groups.Conclusion Mediastinal block can inhibit the development of coronary atherogenesis in rabbits with hyperlipoidemia to a great degree similar to that of thoracic epidural block,but has no effect on hemodynamics.
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Objective To investigate the changes in TNF? content of sciatic nerve induced by chronic constriction injury (CCI) of sciatic nerve to determine the role of TNFa in the development of neuropathic pain. Methods Eight-four female health SD rats weighing 250-300g were anesthetized with sodium barbiturate. Unilateral sciatic nerve was exposed and ligated at the middle of thigh. Three ligatures (chromic catgut 4.0) were placed around the sciatic nerve and tied. The distance between the two ligatures was about 1 mm. Sham operation was performed on the contralateral thigh. The sciatic nerve was exposed and mobilized but not ligated. The thermal nociceptive threshold was determined by measuring the withdrawal latency of hindpaw placed on a 58℃ hot plate on days 0.5, 1, 3, 5, 7, 9, 11, 13 and 14 after surgery. Animals were sacrificed on days 0.5, 1, 3, 7, 10 and 14 after surgery. Sciatic nerves were removed from both thighs and frozen at - 80℃ for determination of TNF? content. Sciatic nerve from healthy animals was used as control. The percentage of maximal possible response (% MPR) , was determined for each group (CCI, sham operation, control) % MPR= (new withdrawal latency- average baseline latency)/( 15-average baseline latency) . The distribution of TNF? between supernatant and sediment was also determined. Results The average baseline nociceptive threshold (withdrawal latency) was (7.9?0.2)s. There was significant different in %MPR between the two hindpaws on days 1, 3, 5, 7, 9 and 11 after surgery. The TNF? content of sciatic nerve from healthy rats was (40.62? 0.24) pg/mg protein. The TNF? content of the ligated sciatic nerve was elevated abruptly in 12h after ligation, then abruptly declined to a plateau but was still significantly higher than that of sham-operated side on days 1 and 3. There was no significant difference in TNF? content of sciatic nerve between control group and sham-operation group. The relative content of TNF? content in the sediment of ligated sciatic nerve gradually increased and reached the peak on day 7 and then gradually decreased. Conclusion The TNF? content of peripheral nerves plays an important role in the development of neuropathic pain. Membrane-combined TNF? is involved in the process of nerve repairing.
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Objective To compare the catecholamine (CA ) and M enkephalin(M ENK) release from human chromaffin cells (HCC) and microencapsulated HCC (ME HCC ) and investigate the effects of microencapsulation on the growth and activity of chromaffin cells Methods Adrenal glands were taken from brain death healthy adults The chromaffin cells were isolated and primarily cultured in vitro During culture the chromaffin cells were HE stained and assessed by labelling the cells with tyrosine hydroxylase monoclonal antibody The percentage of tyrosine positive cells were counted under microscope (1) The chromaffin cells were microencapsulated with 2% alginate and cultured in vitro (experimental group) HCC which were not microencapsulated were used as control The culture media of both groups were replaced every 48h and collected and stored under -20℃ for determination of CA and M ENK concentration (2) On the 6th day of culture, nicotine was added to HCC and MC HCC suspension After 30 min incubation, the suspension was centrifuged and the supernate collected and stored under -20 ℃ for determinatoin of CA and M ENK concentration with radioimmunoassay Results (1) ME HCC grew fairly well in vitro in the culture medium and was morphologically similar to HCC (2) There was no significant difference in CA and M ENK concentration in HCC and ME HCC culture media (3) CA and M ENK concentrations in supernate were increased by nicotine stimulation and there was no difference in the CA and M ENK concentration in the supernate between the two groups Conclusions Alginate and microencapsulation technique are not harmful to HCC, ME HCC has fairly good activity and release function and can be effectively used for transplantation
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Objective To evaluate the analgesic effect and safety of subarachnoid chromaffin cell allograft for terminal cancer pain Methods Ten patients with intractable cancer pain despite traditional treatments were randomly divided into two groups In test group(n=4), 2ml of the suspension chromaffin cells cultured in vitro for 3 days was injected into the subarachnoid space through lumber puncture The same amount of cell free culture solution was injected intrathecally in control group(n=6) Opioids were administered continuously after transplantation The intensity of pain was assessed by VAS, the dose of opioids taken was recored,and the catecholamine and enkephalin concentrations in cerebrospinal fluid and immune function were measured before and after transplantation Results The VAS scores declined markedly in both groups after transplantation (P
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Objective:To investigate the efficacy and characteristics of subarachnoid xenogenetic adrenal medullary transplants on neuropathic pain. Method: Eighty rats were assigned to 4 different groups before their right sciatic nerves were tied according to the method described by Bennet. Each rat received subarachnoidly xenogenetic medullary pieces or isolated chromaffin cells according to the groups. The basal pain thresholds to thermal and electrical stimuli were determined before nerve ligations,and the analgesiometric tests were repeated at weekly intervals following transplantation for 10 weeks. Behavioral indications of spontaneous pain were recorded concomitantly. The effects of naloxone, phentolamine and fentanyl on the antinociception of chromaffin cells were evaluated. Result:The pain thresholds to noxious thermal and electrical stimuli increased after transplantation of medullary pieces or isolated chromaffin cells subarachnoidly. The Behavioral indications of spontaneous pain and hyperalgesia to thermal and electrial were also eliminated after the transplantation. These antinociceptive effects can be reversed by naloxone and phentolamine. Conclusion:Transplanting xenogenetic chromaffin cells into subarachnoid space can reduce neuropathic pain effectively,and this antinociception is conducted via adrenoreceptors and opiate receptors.
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Objective To investigate the changes in small-diameter sensory neuron (SNS)/ peripheral nerve type 3(PN3) Na+ channel transcript and in tetrodotoxin-resistant (TTX-R) Na+ current in dorsal root ganglion (DRG) neurons in a chronic constriction injury (CCI) model of neuropathic pain. Methods Eighteen rats were divided into 6 groups of 3 animals each. Chronic constriction injury model was established after Dib-Hajj et al. Pain threshold was significantly lowered after CCI as compared with that in control group. The animals were deeply anesthetized and rapidly decapitated 14 days after surgery. The L4-5 DRG of the operated side was removed and crushed and total RNA was extracted with trizol reagent. The DRG of the contralateral side was used as control. The change in SNS/PN3 Na + channel expression was determined by semi-reverse transcriptase-PCR. The DRG neurons were isolated enzymatically and the change in voltage-gated TTX-R Na+ current was recorded using whole-cell patch clamp technique. Results Sensory neuron specific TTX-R Na+ channel transcript SNS/PN3 was down-regulated by 60% 14 days after CCI as compared with that in control group. TTX-R Na+ current density was significantly reduced but its activation and steady state inactivation were unchanged. Conclusions Na+ channel SNS/PN3 is involved in the hyperextability of the primary sensory neurons after CCI.
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Objective To compare the survival of isolated chromaffin cells with that of adrenal medullary pieces implanted into the subarachnoid space .Methods Forty male Sprague Dawley rats, 180 220g, were randomly allocated to be implanted into the subarachnoid space with the homologous adrenal medullary pieces washed with PBS(group A,n=20) or the isolated chromaffin cells 5?107 in 10?l suspension (group B,n=20), respectively, after the implants were in vitro cultured in three days . The thermal threshold was determined before ,5 and 10 weeks after the transplantation. Five or ten weeks after the transplantation, 6 rats of either group were randomly selected to obtain the adrenal medullary grafts from the subarachnoid space or the sediments of cerebrospinal fluid ,observed with an electron microscopy.Results Five weeks after transplantation, the thermal threshold in both groups increased markedly,but without significant difference between them ,and there were intact chromaffin cells in the sections of both groups. Ten weeks after the operation, the thermal threshold in group A decreased to basaline, and was significantly lower than in group B; no intact chromaffin cells were found ,with the infiltration of a large number lymphocytes in the sections of group A , and in the sections of group B, the intact chromaffin cells were found with the infiltration of lymphocytes in lower amount and density. Conclusions The isolated chromaffin cells implantation is superior to the adrenal medulla pieces implantation for analgesia.
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The effects of clonidine as a premedicant were studied during isoflurane-indueed hypotension(IIH). Twenty-four adult patient, ASA grade Ⅰ-Ⅱ, scheduled for elective cerebral surgery, were randomly allocated into two groups. Atropine 0.5mg and luminal sodium 100mg were given I. M. as premedication in group Ⅰ 30 mins before induction, and clonidine 5?g?kg~(-1)P. O. was combined in group Ⅱ 90 mins prior to induction. The methods of both groups were similar in induction and maintenance of anesthesia. The status of hypotension was induced and kept by inhaltion of isoflurane, with MAP being decreased by 25% or so. The arterial plasma Concentrations of adrenaline and noradrenaline were measured by electrochemistry method, at preoperation, prior to hypotension, 15 and 30 mins following IIH and 30 mins after MAP recovery, respectively. The results revealed that as compared with the values in group Ⅰ, the inspired and end-tidal concentrations of isoflurane reduced significantly during induction and maintenance of controlled hypotension (P0.05). It is suggested that clonidine premedication can increase the efficiency of isoflurane for controlled hypotension and do not affect the plasma catecholamine concentration and airway function.
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Objective To determine the changes in expression of Ca2+ channel ?1B subunit mRNA in dorsal root ganglion (DRG) neurons in a rat model of neuropathic pain. Methods Twenty-four SD rats weighing 160-220g were randomly divided into 3 groups with 8 animals in each group: (1) neuropathic pain group; (2) sham-operated group and (3) control group. In neuropathic pain group the animals were anesthetized with intraperitoneal phenobarbital 50 mg?kg-1 . The left L5 spinal nerve was exposed and ligated and cut. In sham-operated the left L5 spinal nerve was exposed but not ligated and cut. Control group underwent no operation. Bilateral paw withdrawal threshold to von Frey hair stimulation was measured before and 7, 14 days after operation. The animals were killed on the 14th day after operation and the left L5 DRGs were obtained for determination of Ca2+ channel ?1B subunit mRNA by in situ hybridization. Results Mechanical allodynia developed in neuropathic pain group but not in sham-operated group. The expression of Ca2+ channel ?1B subunit mRNA was significantly lower in neuropathic pain group (0.033 ? 0.011) than in sham-operated group (0.065 ? 0.042) and control group (0.066 ? 0.034) (P