Artificial Organs 14(5):387-398, Raven Press, Ltd., New York 0 1990 International Society for Artificial Organs
Thoughts and Progress It is the goal of this section to publish material that provides information regarding specific issues, aspects of artificial organ application, approach, philosophy, suggestions, andor thoughts for the future.
It has a potent chemotactic effect on the leukocytes (1,2), which is likely to contribute to the pulmonary leukostasis observed on the membranes’ activating complement system. It proves in the circulation the activation principally of the neutropolymorphonuclear leukocytes (neutrophils), and to a lesser extent the monocytes, due to there being fewer (2). It is involved in many cellular interactions and acts as a true inflammatory mediator (1,2). As a result it is certainly possible to make it part of the “inflammatory picture” associated with hemodialysis treatment. This possibly explains why repetitive treatments can cause degenerative lesions in the long term (3).
Leukotriene B4 in Hemodialysis A . Tridon, E . Albuisson, P . Deteix, A . Marquzs Verdier, G . Gaillard, G . Be‘tail, and J . C . Baguet, Laboratoire d’lrnrnunologie, Faculte‘ Me‘decine Pharrnacie, Clerrnont-Ferrand, France
Abstract: Leukotrienes are eicosanoids arising from arachidonic acid via 5 lipooxygenase, an enzyme essentially present in leukocyte cells. Leukotriene B4 might be an indicator of neutropolymorphonuclear leukocyte activation when there is contact with artificial membranes. The level of plasmatic leukotriene B4 was measured at three different times during the hemodialysis treatment in several patients undergoing dialysis on three different membranes (one cellulosic and two synthetics). A moderate increase of leukotriene B4 was observed early (at 15 min), comparable among the three membranes, but levels returned to baseline at 180 min. Leukotriene B4 production proved leukocyte activation and was probably related to a direct interaction with dialysis membrane. Nevertheless, complement intervention could not be excluded. Leukotriene B4 is one molecule more among the group of inflammatory mediators produced during hemodialysis treatment. Key Words: Leukotriene B L H e m o dialysis-Complement system-Biocompatibility.
MATERIAL AND METHODS Patients underwent hemodialysis on three different membranes: seven on a substituted cellulosic membrane, Cellulose Acetate (CA 110, Travenol Baxter), seven on Polysulfone (Spiraflo HFT 10, Sorin-Biomedica), and nine on Acrylonitrile AN69 (Filtral 10, Hospal). Among these patients, five underwent dialysis on the three membranes and were consequently paired. The dialysis conditions were very precisely defined: ultrafiltration flow, 200 ml/h during 3 h; blood flow, 200 ml/min; and dialysate flow, 500 ml/min in the extracorporeal system. Heparinization is a continuous process after a bolus at the start. Three samples are taken during treatment, one at the beginning (TO) in the arteriovenous fistula, just before commencing extracorporeal circulation; one at 15 min (T15); and the other at 180 min (T180) in the extracorporeal circulation, after the dialyzer. They were used for LTB4 measurement, and at the same time neutrophil counts (Technicon H6000) and the anaphylatoxin C3a determination (radioimmunoassay Amersham) were performed. The LTB4 was measured on 1 ml of plasma collected in EDTA and immediately frozen at - 80°C. An extraction with the Amprep column (Amer-
Biocompatibility evaluation of extracorporeal devices requires the establishment of sensitive indicators of blood cells/surface interactions such as eicosanoids. Hence leukotrienes, generated from arachidonic acid through the presence of 5lipooxygenase in leukocytes and macrophages (1,2), might be important indicators of activation of these cells. The study was interested in leukotriene B4 (LTB4) in the context of hemodialysis. -
Received August 1989; revised April 1990. Address correspondence and reprint requests to Dr. A. Tridon, Laboratoire d’immunologie, Facultt Mtdecine Pharmacie, BP 38, 63000 Clermont-Ferrand, France.
387
THOUGHTS AND PROGRESS
388
sham) takes place before the LTB4 determination, using a radioimmunological method (Amersham). We have obtained a coefficient of variation of 7-8% in 90% of the measures (each plasma sample was used for two readings). The statistical analysis was carried out using the Friedman’s test (and KendaLl’s coefficient of concordance). They are nonparametric tests dependent only on the ranks of the observations. They allow us to compare dependent variables and to have a distribution-free approach, which is desired there because of the small number of patients. Differences with p values less than 0.05 were considered significant. RESULTS The data are expressed as the means k SEM (Tables 1 and 2). (The results of the LTB4 test at the start of the dialysis correspond to an average of 223.10 ? 13.52 pg/ml, with 21 results). There are no other data at this moment for the plasmatic levels of LTB4 in other contexts, neither normal nor pathological. To show the contribution due to time, the study compared for each membrane and for each parameter (LTB4, C3a, and neutrophil count) the results at 0, 15, and 180 min (Table 1). The study made use of all the data obtained on the three membranes. The LTB4 level varied significantly during the treatment, that is to say, an increase at 15 min followed by a decrease to the initial values at 180 min (p < 0.05 on the three membranes). To show the contribution due to membrane, the study compared in the paired group two “relative variations” for each parameter (Table 2). This comparison failed to indicate statistically significant dif-
ferences. Parameter variations were studied according to time, using this reduced size (Table 2). Considering this reduced size and at a lesser degree, we observed in results and tendencies a good consistency with previous conclusions (Table 1). DISCUSSION We have shown a moderate and transient increase of LTB4 during hemodialysis on all three membranes studied. Historically, the complement system is the first biological system whose activation was recognized in the field of extracorporeal circulation (4). The appearance of anaphylatoxins in the patients’ circulation induced activation of other biological systems (4-6). The study tried to investigate the importance of complement activation in the production of LTB4. The anaphylatoxin C5a, generated in the same way as C3a, does not seem to be a stimulus of LTB4 neosynthesis (7). The activation of the production of LTB4 by means of complexes CSb-C9 deposited on neutrophils is possible (7); however, during hemodialysis these complexes are not detected, even on membranes that are high activators of the alternative pathway of complement system (8). The study could not demonstrate a significant difference between the three membranes for C3a, neutrophil counts, and LTB4 variations (Table 2), which might have been explained by the small number of patients. According to means values, we could see that C3a levels at T15 were higher on Cellulose Acetate than on Polysulfone and Acrylonitrile AN69 (Tables 1 and 2). Nevertheless, LTB4 means appeared homogeneous on the three membranes.
TABLE 1. LTB4, C3a, and neutrophil count variations according to time, means
* (SEM)
Min Membrane Cellulose Acetate LTB4,“ pg/rnl C3a, ng/ml PN/kI Polysulfone LTB4, pg/ml C3a, ngiml PN/kl Acrylonitrile AN69 LTB4, pg/ml C3a, ng/rnl PN/pI ( I
Size
0
15
180
Contribution to time (Friedman’s test)
6 7 7
214.17 (22.56) 347.14 (64.20) 4,880.00 (527.97)
331.67 (30.89) 1,907.71 (524.13) 3,634.29 (632.83)
250.83 (3 1.45) 745.43 (118.52) 5,595.71 (864.86)
p < 0.05 p < 0.01 p < 0.02
6 7 7
235.00 (27.72) 355.71 (33.71) 4,754.29 (501.20)
304.17 (14.28) 880.00 (122.32)
4,141.43 (553.63)
234.17 (31.63) 517.86 (46.82) 4,650.00 (638.21)
p < 0.05 p < 0.01 p < 0.05
9 9 9
221.11 (22.71) 255.78 (20.51) 4,748.89 (440.07)
297.22 (18.41) 607.56 (149.52) 4,490.00 (438.17)
224.44 (13.00) 316.44 (37.86) 4,937.78 (525.60)
p < 0.05 p < 0.01
LTB4, leukotriene B4; PN, polymorphonuclear leukocyte.
Artif Organs. Vol. 14, No. 5 , 1990
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THOUGHTS AND PROGRESS
389
TABLE 2. LTB4, C3a, and neutrophil count variations in paired patients, means
&
(SEM)
~~
Minutes Membrane Cellulose Acetate LTB4,” pg/ml C3a, ng/ml PN/pI Pol ysulfone LTB4, pg/rnl C3a, ng/ml PN/p1 Acrylonitrile AN69 LTB4, pg/ml C3a, ng/ml PN/pI
Size
0
15
180
Contribution to time (Friedman’s test)
192.50 (25.04) 378.00 (85.94) 5,022.00 (662.51)
300.00 (26.06) 1,923.60 (697.71) 3,932.00 (831.38)
225.00 (42.18) 760.00 (163.65) 6,096.00 (1,078.28)
NS p < 0.02 p < 0.05
257.00 (20.65) 368.80 (47.11) 5,058.00 (621.90)
315.00 (11.40) 774.00 (116.04) 4,560.00 (647.27)
232.00 (38.65) 530.60 (66.29) 4,900.00 (866.58)
p < 0.05
223.00 (37.30) 264.00 (33.94) 5,622.00 (483.88)
294.00 (15.36) 760.00 (256.98) 5,360.00 (448.85)
245.00 (10.12) 290.80 (63.30) 6,008.00 (555.54)
p < 0.05
NS
NS NS
NS
For the contribution to membrane (Friedman’s test), there were two “relative variations”: (T rnax - T min)/T min (T15 - TO)/TO. LTB4, NS; C3a, NS; PN, NS. ” LTB4, leukotriene B4; PN, polymorphonuclear leukocyte.
Then complement system activation did not appear to be a determining factor in the generation of LTB4. An activation of leukocytes by means of a mechanical interaction with the dialysis membrane (5) followed by neosynthesis is possible. This is being considered by authors working on cyclo-oxygenase products release: prostaglandin PGE, and thromboxane TXB, (9,lO). These molecules increase 10 min after blood contact with membranes in vitro. This production is not connected to the activation of the complement system, but it varies according to the membranes (9,lO). The in vitro kinetics of the occurrence and then the decrease of LTB4 have been studied, mostly on isolated neutrophils. The time delay in the release is several minutes after the action of various stimuli (2,7,11). The neutrophils, which are subjected to mechanical stresses in the presence of platelets that have been previously shaken, released the LTB4 very quickly (12). The in vitro metabolism of LTB4, either endogenous or exogenous, by isolated neutrophils occurs rapidly by means of omega oxidation (13,14). However, in vitro experiments on blood or on inflammatory synovial fluids show a relative stability of the LTB4 amount after a synthesis stimulus (14). Consequently, the transient increase of LTB4 could be explained by an early leukocyte activation when leukocytes came into contact with the membranes, without predicting the duration of this phenomenon. Preliminary studies have been directed toward LTB4 in hemodialysis. One of these (15) points to a significant increase
of LTB4 at 10 min during the hemodialysis treatment on Cuprophan, followed by a drop to baseline levels at the end of the dialysis treatment. The LTB4 could be one of the factors of the pulmonary leukostasis on this high activator of the complement system (15). The other study (16) was concerned with exogenous arachidonic acid metabolism in isolated leukocytes, preincubated or not with the dialysis membranes. The production of LTB4 in these conditions was not modified, without or with any membrane (16). This in vitro model, which estimated the potential lipooxygenase activities, did not show the influence of membranes on LTB4 synthesis. CONCLUSION
The study demonstrated a moderate LTB4 production by leukocytes during hemodialysis treatment, occurring probably independently of complement system activation. The LTB4 is part of the group of inflammatory mediators that are released during hemodialysis treatment, as are anaphylatoxins (4), interleukin 1 (3,and histamine (6), whose importance for hemodialysis patients can be seen in the long term (3). Acknowledgment: We thank Hospal for financial support.
References 1. Henderson W. Lipid-derived and other chemical mediators of inflammation in the lung. J Allergy Clin Immunol
1987;79:543-53. 2. Lee T, Austen F. Arachidonic acid metabolism by the 5lipooxygenase pathway and the effects of alternative dietary fatty acids. Adv Immunol 1986;39:145-70.
ArtifOrgons, Vol. 14, N o . 5 , 1990
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THOUGHTS AND PROGRESS
3. Yukihiko N. Long-term compatibility of artificial kidneys. Artif Organs 1988;12:1. 4. Chenoweth D. Complement activation produced by biomaterials. Artif Organs 1988;12:5024. 5. Vanholder R, Ringoir S . Biocompatibility: an overview. Int J Artif Organs 1989;12:356-65. 6. Tridon A, Deteix P, Coves G , et al. Evaluation de la biocompatibilitt d’une nouvelle membrane cellulosique par I’ttude du systeme du compltment e t de I’histaminolibtration. Nephrology 1987;8:217-21. 7. Seeger W, Suttorp N, Hellwig A, Bhakdi S . Non cytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes. J Zmunol 1986;137:1286-93. 8. Kazatchkine MD, Carreno MP. Activation of the complement system at the interface between blood and artificial surfaces. Biomaterials 1988;9:30-5. 9. Mahiout A, Jorres A, Schultze G , Meinhold H, Kessel M. Eicosanoid release as laboratory indicator of biocompatibility. Artif Organs 1989;13:251-4. 10. Schultze G , Wagner K, Neumayer H, Fitzner R, Molzahn M. Effect of dialyser membranes on in vitro generation of eicosanoids. Int J Artif Organs 1987;10:275-8. 11. Dessein A, Lee T, Elsas P, et al. Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated by calcium ionophore A23 187. J Immunol 1986;136:3829-38. 12. MacIntire LV, Frangos JA, Rhee G , Eskin SG, Hall E. The effect of fluid mechanical stress on cellular arachidonic acid metabolism. Ann NY Acad Sci 1987;516:513-24. 13. Brom J, Schonfeld W, Konig W. Metabolism of leukotriene B4 by activated human polymorphonuclear granulocytes. Immunology 1988 ;64:50%18. 14. MacMillan R, Foster S , Dieppe P. Leukotriene B4 metabolism in human leukocytes: fact or artefact? Agents actions 1987;21:355-7. 15. Schiffl H, Strasser T, Holnoser J, Weber P. Rapid generation of leukotriene B4 by Cuprophane membranes during hemodialysis [Abstract]. Nephrol Dial Transplant 1988; 3:549. 16. Vincent D, Charmes JP, Benzakour M, Gualde N, Rigaud M, Leroux RC. A simple in vitro test to evaluate biocompatibility of dialysis membranes. Nephrol Dial Transplant 1989;4:306-8.
Glued Carbon Fiber Electrodes for Diaphragm Pacing Motohiko Kimura, *Toshifumi Sugiura, Yoshihito Fukui, Morio Togawa, and f Yukio Harada, Graduate School of Electronic Science and Technology and *Research Institute of Electronics, Shizuoka University, and ?First Department of Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan
Abstract: Carbon fibers with fibrin glue were used as electrodes for diaphragm pacing. The electrodes were applied to three mongrel dogs and the effectiveness was tested. The carbon leads were glued to phrenic nerves by means of the fibrinogen and thrombin bilaterally. The tidal volumes and threshold current level for stimulation
Artif Organs, Vol. 14. N o . 5 , 1990
were measured at various time up to 9 weeks after implantation. Effective contraction of diaphragm were observed for 9 weeks. By using this electrode, the exfoliation of the nerve is not necessary, the nerve can be maintained in an intact state, and the risk of the implanting operation can be minimized. Key Words: Diaphragm pacing-Electrical stimulation-Pacemaker-Electrode.
Since the first clinical application of the long-term stimulation in 1968 ( l ) , diaphragm pacing has been applied to only 600-plus patients. One of the significant reasons for this low number is the fact that even if physicians pay meticulous attention to phrenic nerves, the implant operation of the stimulating electrodes runs a major risk of nerve injury. In order to implant the present electrode, which is constructed from platinum and silastic cuff, exfoliation of the phrenic nerves is necessary (2). A stimulating electrode was developed that could easily be used by gluing a carbon lead with the phrenic nerve. By using this electrode, the exfoliation of the nerve is not necessary and the risk of the operation could be minimized. The effectiveness of the proposed electrode was tested by three mongrel dogs. METHODS The proposed electrode is made with a carbon lead and fibrin glue. The carbon lead is electrically insulated except for 6 mm from the tip of it. Fibrin glue was generated by mixing the fibrinogen solution (50 mg/500 mg water) and thrombin (500 U) at the site of attaching. Under pentbarbital anesthesia (20 mg/kg), the right side third intercostal space of three mongrel dogs (case 1, 11.5 kg; case 2, 13.0 kg; case 3, 10.5 kg) were opened with a 4 cm incision, and the upper mediastinum was opened. The carbon leads were glued along the phrenic nerves bilaterally. After 10 min, the counter end of the carbon leads were passed through the chest wall and the incision was closed. As a counter electrode, a platinum plate (25 mm x 20 mm x 0.1 mm) was subcutaneously implanted. The change in maximum tidal volume VT,,,and threshold current was measured for 9 weeks. The nerves were stimulated only when these parameters Received February 1990; revised May 1990. Address correspondence and reprint requests to Dr. Motohiko Kimura, Graduate School of Electronic Science and Technology, Shizuoka University, 3-5-1 Johoku, Hamamatsu 432, Japan.
Thoughts and Progress It is the goal of this section to publish material that provides information regarding specific issues, aspects of artificial organ application, approach, philosophy, suggestions, andor thoughts for the future.
It has a potent chemotactic effect on the leukocytes (1,2), which is likely to contribute to the pulmonary leukostasis observed on the membranes’ activating complement system. It proves in the circulation the activation principally of the neutropolymorphonuclear leukocytes (neutrophils), and to a lesser extent the monocytes, due to there being fewer (2). It is involved in many cellular interactions and acts as a true inflammatory mediator (1,2). As a result it is certainly possible to make it part of the “inflammatory picture” associated with hemodialysis treatment. This possibly explains why repetitive treatments can cause degenerative lesions in the long term (3).
Leukotriene B4 in Hemodialysis A . Tridon, E . Albuisson, P . Deteix, A . Marquzs Verdier, G . Gaillard, G . Be‘tail, and J . C . Baguet, Laboratoire d’lrnrnunologie, Faculte‘ Me‘decine Pharrnacie, Clerrnont-Ferrand, France
Abstract: Leukotrienes are eicosanoids arising from arachidonic acid via 5 lipooxygenase, an enzyme essentially present in leukocyte cells. Leukotriene B4 might be an indicator of neutropolymorphonuclear leukocyte activation when there is contact with artificial membranes. The level of plasmatic leukotriene B4 was measured at three different times during the hemodialysis treatment in several patients undergoing dialysis on three different membranes (one cellulosic and two synthetics). A moderate increase of leukotriene B4 was observed early (at 15 min), comparable among the three membranes, but levels returned to baseline at 180 min. Leukotriene B4 production proved leukocyte activation and was probably related to a direct interaction with dialysis membrane. Nevertheless, complement intervention could not be excluded. Leukotriene B4 is one molecule more among the group of inflammatory mediators produced during hemodialysis treatment. Key Words: Leukotriene B L H e m o dialysis-Complement system-Biocompatibility.
MATERIAL AND METHODS Patients underwent hemodialysis on three different membranes: seven on a substituted cellulosic membrane, Cellulose Acetate (CA 110, Travenol Baxter), seven on Polysulfone (Spiraflo HFT 10, Sorin-Biomedica), and nine on Acrylonitrile AN69 (Filtral 10, Hospal). Among these patients, five underwent dialysis on the three membranes and were consequently paired. The dialysis conditions were very precisely defined: ultrafiltration flow, 200 ml/h during 3 h; blood flow, 200 ml/min; and dialysate flow, 500 ml/min in the extracorporeal system. Heparinization is a continuous process after a bolus at the start. Three samples are taken during treatment, one at the beginning (TO) in the arteriovenous fistula, just before commencing extracorporeal circulation; one at 15 min (T15); and the other at 180 min (T180) in the extracorporeal circulation, after the dialyzer. They were used for LTB4 measurement, and at the same time neutrophil counts (Technicon H6000) and the anaphylatoxin C3a determination (radioimmunoassay Amersham) were performed. The LTB4 was measured on 1 ml of plasma collected in EDTA and immediately frozen at - 80°C. An extraction with the Amprep column (Amer-
Biocompatibility evaluation of extracorporeal devices requires the establishment of sensitive indicators of blood cells/surface interactions such as eicosanoids. Hence leukotrienes, generated from arachidonic acid through the presence of 5lipooxygenase in leukocytes and macrophages (1,2), might be important indicators of activation of these cells. The study was interested in leukotriene B4 (LTB4) in the context of hemodialysis. -
Received August 1989; revised April 1990. Address correspondence and reprint requests to Dr. A. Tridon, Laboratoire d’immunologie, Facultt Mtdecine Pharmacie, BP 38, 63000 Clermont-Ferrand, France.
387
THOUGHTS AND PROGRESS
388
sham) takes place before the LTB4 determination, using a radioimmunological method (Amersham). We have obtained a coefficient of variation of 7-8% in 90% of the measures (each plasma sample was used for two readings). The statistical analysis was carried out using the Friedman’s test (and KendaLl’s coefficient of concordance). They are nonparametric tests dependent only on the ranks of the observations. They allow us to compare dependent variables and to have a distribution-free approach, which is desired there because of the small number of patients. Differences with p values less than 0.05 were considered significant. RESULTS The data are expressed as the means k SEM (Tables 1 and 2). (The results of the LTB4 test at the start of the dialysis correspond to an average of 223.10 ? 13.52 pg/ml, with 21 results). There are no other data at this moment for the plasmatic levels of LTB4 in other contexts, neither normal nor pathological. To show the contribution due to time, the study compared for each membrane and for each parameter (LTB4, C3a, and neutrophil count) the results at 0, 15, and 180 min (Table 1). The study made use of all the data obtained on the three membranes. The LTB4 level varied significantly during the treatment, that is to say, an increase at 15 min followed by a decrease to the initial values at 180 min (p < 0.05 on the three membranes). To show the contribution due to membrane, the study compared in the paired group two “relative variations” for each parameter (Table 2). This comparison failed to indicate statistically significant dif-
ferences. Parameter variations were studied according to time, using this reduced size (Table 2). Considering this reduced size and at a lesser degree, we observed in results and tendencies a good consistency with previous conclusions (Table 1). DISCUSSION We have shown a moderate and transient increase of LTB4 during hemodialysis on all three membranes studied. Historically, the complement system is the first biological system whose activation was recognized in the field of extracorporeal circulation (4). The appearance of anaphylatoxins in the patients’ circulation induced activation of other biological systems (4-6). The study tried to investigate the importance of complement activation in the production of LTB4. The anaphylatoxin C5a, generated in the same way as C3a, does not seem to be a stimulus of LTB4 neosynthesis (7). The activation of the production of LTB4 by means of complexes CSb-C9 deposited on neutrophils is possible (7); however, during hemodialysis these complexes are not detected, even on membranes that are high activators of the alternative pathway of complement system (8). The study could not demonstrate a significant difference between the three membranes for C3a, neutrophil counts, and LTB4 variations (Table 2), which might have been explained by the small number of patients. According to means values, we could see that C3a levels at T15 were higher on Cellulose Acetate than on Polysulfone and Acrylonitrile AN69 (Tables 1 and 2). Nevertheless, LTB4 means appeared homogeneous on the three membranes.
TABLE 1. LTB4, C3a, and neutrophil count variations according to time, means
* (SEM)
Min Membrane Cellulose Acetate LTB4,“ pg/rnl C3a, ng/ml PN/kI Polysulfone LTB4, pg/ml C3a, ngiml PN/kl Acrylonitrile AN69 LTB4, pg/ml C3a, ng/rnl PN/pI ( I
Size
0
15
180
Contribution to time (Friedman’s test)
6 7 7
214.17 (22.56) 347.14 (64.20) 4,880.00 (527.97)
331.67 (30.89) 1,907.71 (524.13) 3,634.29 (632.83)
250.83 (3 1.45) 745.43 (118.52) 5,595.71 (864.86)
p < 0.05 p < 0.01 p < 0.02
6 7 7
235.00 (27.72) 355.71 (33.71) 4,754.29 (501.20)
304.17 (14.28) 880.00 (122.32)
4,141.43 (553.63)
234.17 (31.63) 517.86 (46.82) 4,650.00 (638.21)
p < 0.05 p < 0.01 p < 0.05
9 9 9
221.11 (22.71) 255.78 (20.51) 4,748.89 (440.07)
297.22 (18.41) 607.56 (149.52) 4,490.00 (438.17)
224.44 (13.00) 316.44 (37.86) 4,937.78 (525.60)
p < 0.05 p < 0.01
LTB4, leukotriene B4; PN, polymorphonuclear leukocyte.
Artif Organs. Vol. 14, No. 5 , 1990
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TABLE 2. LTB4, C3a, and neutrophil count variations in paired patients, means
&
(SEM)
~~
Minutes Membrane Cellulose Acetate LTB4,” pg/ml C3a, ng/ml PN/pI Pol ysulfone LTB4, pg/rnl C3a, ng/ml PN/p1 Acrylonitrile AN69 LTB4, pg/ml C3a, ng/ml PN/pI
Size
0
15
180
Contribution to time (Friedman’s test)
192.50 (25.04) 378.00 (85.94) 5,022.00 (662.51)
300.00 (26.06) 1,923.60 (697.71) 3,932.00 (831.38)
225.00 (42.18) 760.00 (163.65) 6,096.00 (1,078.28)
NS p < 0.02 p < 0.05
257.00 (20.65) 368.80 (47.11) 5,058.00 (621.90)
315.00 (11.40) 774.00 (116.04) 4,560.00 (647.27)
232.00 (38.65) 530.60 (66.29) 4,900.00 (866.58)
p < 0.05
223.00 (37.30) 264.00 (33.94) 5,622.00 (483.88)
294.00 (15.36) 760.00 (256.98) 5,360.00 (448.85)
245.00 (10.12) 290.80 (63.30) 6,008.00 (555.54)
p < 0.05
NS
NS NS
NS
For the contribution to membrane (Friedman’s test), there were two “relative variations”: (T rnax - T min)/T min (T15 - TO)/TO. LTB4, NS; C3a, NS; PN, NS. ” LTB4, leukotriene B4; PN, polymorphonuclear leukocyte.
Then complement system activation did not appear to be a determining factor in the generation of LTB4. An activation of leukocytes by means of a mechanical interaction with the dialysis membrane (5) followed by neosynthesis is possible. This is being considered by authors working on cyclo-oxygenase products release: prostaglandin PGE, and thromboxane TXB, (9,lO). These molecules increase 10 min after blood contact with membranes in vitro. This production is not connected to the activation of the complement system, but it varies according to the membranes (9,lO). The in vitro kinetics of the occurrence and then the decrease of LTB4 have been studied, mostly on isolated neutrophils. The time delay in the release is several minutes after the action of various stimuli (2,7,11). The neutrophils, which are subjected to mechanical stresses in the presence of platelets that have been previously shaken, released the LTB4 very quickly (12). The in vitro metabolism of LTB4, either endogenous or exogenous, by isolated neutrophils occurs rapidly by means of omega oxidation (13,14). However, in vitro experiments on blood or on inflammatory synovial fluids show a relative stability of the LTB4 amount after a synthesis stimulus (14). Consequently, the transient increase of LTB4 could be explained by an early leukocyte activation when leukocytes came into contact with the membranes, without predicting the duration of this phenomenon. Preliminary studies have been directed toward LTB4 in hemodialysis. One of these (15) points to a significant increase
of LTB4 at 10 min during the hemodialysis treatment on Cuprophan, followed by a drop to baseline levels at the end of the dialysis treatment. The LTB4 could be one of the factors of the pulmonary leukostasis on this high activator of the complement system (15). The other study (16) was concerned with exogenous arachidonic acid metabolism in isolated leukocytes, preincubated or not with the dialysis membranes. The production of LTB4 in these conditions was not modified, without or with any membrane (16). This in vitro model, which estimated the potential lipooxygenase activities, did not show the influence of membranes on LTB4 synthesis. CONCLUSION
The study demonstrated a moderate LTB4 production by leukocytes during hemodialysis treatment, occurring probably independently of complement system activation. The LTB4 is part of the group of inflammatory mediators that are released during hemodialysis treatment, as are anaphylatoxins (4), interleukin 1 (3,and histamine (6), whose importance for hemodialysis patients can be seen in the long term (3). Acknowledgment: We thank Hospal for financial support.
References 1. Henderson W. Lipid-derived and other chemical mediators of inflammation in the lung. J Allergy Clin Immunol
1987;79:543-53. 2. Lee T, Austen F. Arachidonic acid metabolism by the 5lipooxygenase pathway and the effects of alternative dietary fatty acids. Adv Immunol 1986;39:145-70.
ArtifOrgons, Vol. 14, N o . 5 , 1990
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3. Yukihiko N. Long-term compatibility of artificial kidneys. Artif Organs 1988;12:1. 4. Chenoweth D. Complement activation produced by biomaterials. Artif Organs 1988;12:5024. 5. Vanholder R, Ringoir S . Biocompatibility: an overview. Int J Artif Organs 1989;12:356-65. 6. Tridon A, Deteix P, Coves G , et al. Evaluation de la biocompatibilitt d’une nouvelle membrane cellulosique par I’ttude du systeme du compltment e t de I’histaminolibtration. Nephrology 1987;8:217-21. 7. Seeger W, Suttorp N, Hellwig A, Bhakdi S . Non cytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes. J Zmunol 1986;137:1286-93. 8. Kazatchkine MD, Carreno MP. Activation of the complement system at the interface between blood and artificial surfaces. Biomaterials 1988;9:30-5. 9. Mahiout A, Jorres A, Schultze G , Meinhold H, Kessel M. Eicosanoid release as laboratory indicator of biocompatibility. Artif Organs 1989;13:251-4. 10. Schultze G , Wagner K, Neumayer H, Fitzner R, Molzahn M. Effect of dialyser membranes on in vitro generation of eicosanoids. Int J Artif Organs 1987;10:275-8. 11. Dessein A, Lee T, Elsas P, et al. Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated by calcium ionophore A23 187. J Immunol 1986;136:3829-38. 12. MacIntire LV, Frangos JA, Rhee G , Eskin SG, Hall E. The effect of fluid mechanical stress on cellular arachidonic acid metabolism. Ann NY Acad Sci 1987;516:513-24. 13. Brom J, Schonfeld W, Konig W. Metabolism of leukotriene B4 by activated human polymorphonuclear granulocytes. Immunology 1988 ;64:50%18. 14. MacMillan R, Foster S , Dieppe P. Leukotriene B4 metabolism in human leukocytes: fact or artefact? Agents actions 1987;21:355-7. 15. Schiffl H, Strasser T, Holnoser J, Weber P. Rapid generation of leukotriene B4 by Cuprophane membranes during hemodialysis [Abstract]. Nephrol Dial Transplant 1988; 3:549. 16. Vincent D, Charmes JP, Benzakour M, Gualde N, Rigaud M, Leroux RC. A simple in vitro test to evaluate biocompatibility of dialysis membranes. Nephrol Dial Transplant 1989;4:306-8.
Glued Carbon Fiber Electrodes for Diaphragm Pacing Motohiko Kimura, *Toshifumi Sugiura, Yoshihito Fukui, Morio Togawa, and f Yukio Harada, Graduate School of Electronic Science and Technology and *Research Institute of Electronics, Shizuoka University, and ?First Department of Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan
Abstract: Carbon fibers with fibrin glue were used as electrodes for diaphragm pacing. The electrodes were applied to three mongrel dogs and the effectiveness was tested. The carbon leads were glued to phrenic nerves by means of the fibrinogen and thrombin bilaterally. The tidal volumes and threshold current level for stimulation
Artif Organs, Vol. 14. N o . 5 , 1990
were measured at various time up to 9 weeks after implantation. Effective contraction of diaphragm were observed for 9 weeks. By using this electrode, the exfoliation of the nerve is not necessary, the nerve can be maintained in an intact state, and the risk of the implanting operation can be minimized. Key Words: Diaphragm pacing-Electrical stimulation-Pacemaker-Electrode.
Since the first clinical application of the long-term stimulation in 1968 ( l ) , diaphragm pacing has been applied to only 600-plus patients. One of the significant reasons for this low number is the fact that even if physicians pay meticulous attention to phrenic nerves, the implant operation of the stimulating electrodes runs a major risk of nerve injury. In order to implant the present electrode, which is constructed from platinum and silastic cuff, exfoliation of the phrenic nerves is necessary (2). A stimulating electrode was developed that could easily be used by gluing a carbon lead with the phrenic nerve. By using this electrode, the exfoliation of the nerve is not necessary and the risk of the operation could be minimized. The effectiveness of the proposed electrode was tested by three mongrel dogs. METHODS The proposed electrode is made with a carbon lead and fibrin glue. The carbon lead is electrically insulated except for 6 mm from the tip of it. Fibrin glue was generated by mixing the fibrinogen solution (50 mg/500 mg water) and thrombin (500 U) at the site of attaching. Under pentbarbital anesthesia (20 mg/kg), the right side third intercostal space of three mongrel dogs (case 1, 11.5 kg; case 2, 13.0 kg; case 3, 10.5 kg) were opened with a 4 cm incision, and the upper mediastinum was opened. The carbon leads were glued along the phrenic nerves bilaterally. After 10 min, the counter end of the carbon leads were passed through the chest wall and the incision was closed. As a counter electrode, a platinum plate (25 mm x 20 mm x 0.1 mm) was subcutaneously implanted. The change in maximum tidal volume VT,,,and threshold current was measured for 9 weeks. The nerves were stimulated only when these parameters Received February 1990; revised May 1990. Address correspondence and reprint requests to Dr. Motohiko Kimura, Graduate School of Electronic Science and Technology, Shizuoka University, 3-5-1 Johoku, Hamamatsu 432, Japan.
