Experimental Study On The Anesthetic Management Of Establishment Of Brain-dead Model And Lung Injury In The Brain-dead State And Its Protection

Part â…  Anesthetic management of establishment and the maintenance of brain-dead model with Ba-Ma mini pigsObjective:To investigate the anesthetic management and integral physiological functional regulation of establishment and the maintenance of brain-dead model with Ba-Ma mini pigs, and to examine the hemodynamic changes during the course of establishment of brain-dead model and the course of maintenance of brain death for a long time so as to provide experience of pigs anesthetic management and the hemodynamic maintenance of brain-dead model. Materials and Methods:26 healthy Ba-Ma mini pigs were randomly divided into 5 groups: control group (group C, n=5) was maintained anesthesia for 24h but not created brain-dead model; Under intravenous mixed anesthesia brain-dead group (group B, n=6) was established brain-dead model by increasing intracranial pressure in a modified, slow andintermittent way, and maintained brain-dead state for 24h by respiration and circulation support; long-term brain-dead group (group L, n=5), was induced brain-death as group B and maintained for 48h, medicine pretreatment group1 (group Ml, n=5), medicine pretreatment group2(group M2, n=5), was induced brain-death as group B and maintained for 24h but had medicine pretreatment . When the mean artery pressure (MAP)≥60mm Hg, saturation oxygen(SPO₂)≥95%, P_aO₂≥100mm Hg, body temperature≥35℃, central venous pressure(CVP)≤10¹2mm Hg, and hemodynamic was stable, the brain-dead models were considered suitable as the brain-dead donor. MAP, HR and CVP of 5 groups were monitored, the parameters for judging brain death in group B, group L, group Ml and group M2 were recorded at time of after anesthesia and before increasing intracranial pressure (T₁), peak value of MAP and HR time in the course of increasing intracranial pressure (T2), the first time of confirmation of brain death (T3), after the first time of confirmation of brain death 3h(T₄), 6h (T₅), 9h (T₆), 12h (T₇), 18h (T₈) and 24h (T₉). Changes of the above-mentioned indexes of corresponding time in the course of experiment of group C were monitor and recorded too. Results:1. Establishment of Ba-MA mini pig brain-dead model: in group B, 5 out of 6 Ba-Ma mini pig brain-dead models were established according to the criteria, and 1 out of 6 developed sudden ventricular premature beat, ventricular fibrillation, cardiac arrest, and died of cardiac and pulmonary death. Animals in group L, Ml and M2 were all successfully made brain-dead models.2. Hemodynamic changes during the experiment: after anesthesia, MAP of group C, group B, group L, group Ml and group M2 were (108.20±8.35 mmHg), (108.00 ±12.21 mmHg), (108.40±8.96 mmHg), (108.00±13.56 mmHg) and (108.00±6.36 mmHg),and HR of group C, group B, group L, group Ml and group M2 were (96.20 ±10.06 beats/min), (93.80±7.33beats/min), 94.60 ±6.19 beats/min), (95.80 ± 6.83 beats/min) and (96.20 ±6.99 beats/min), respectively. MAP and HR among 5 groups had no significant difference (P>0.05). MAP and HR of group C during the whole experiment had no significant changes, while those for group B, group L, group Mland group M2 were significant increase after increasing intracranial pressure: a) after slowly and intermittently increasing the intracranial pressure via Foley balloon catheter, the intracranial pressure increased promptly, higher than MAP, ranged 282.81 to 392.81mmHg. Animals presented Cushing reflex, that was HR and MAP first decreased slowly then increased abruptly, respiration frequency slowed, with deeper breath, and the peak MAP and HR were significantly higher than before, (P<0.05); b ) after flattening of brain waves, MAP and HR began to decrease instead of increasing; c) then the intracranial pressure decreased to (118.28¹43.05 mmHg), however, still higher than MAP; d ) after the confirmation of brain death, MAP decreased constantly till MAP^60mmHg or systolic blood pressure ^90mmHg, and the self-regulation of the animals could not maintain stable MAP furthermore, with respiration and circulation support, the animals can just maintain MAP^ 60mmHg . comparison of MAP at T4 , T5 , T6 , T7, T8 and T9 time points , MAP of group B, group L, group Ml and group M2 were lower than that of group C ,and the difference was significant (P<0.05). e) HR of group B, group L, group Ml and group M2 were significant faster at T2, T3, T4, T5 and T6 time points than group C (P<0.05). There were no significant difference of HR among group B, group L, group Ml and group M2 at T2, T3, T4, T5 and T6 time points, f) At T7 , Tgtime points HR of group B and group L were significant faster than group C (i^O.OS). At T7, T8 time points HR of group Ml and group M2 drop to normal basically, had no significant difference among organizing compared with group C. g) At T9 time point, in group B and group L, HR still very fast, however, had no significance different among organizing compared with group C, group Ml and group M2 (P>0.05). h) HR and MAP change violently during the course of establishment of brain-dead model, The peak values of MAP and HR might not appear at the same time, but HR is faster while MAP is higher. After confirming the brain death for the first time, MAP and HR began to decrease, but out of step, it is very fast for MAP to drop, HR drop gradually, HR of medicine pretreatment group drop to normal faster than group B and group L relatively. After flattening of brain waves, MAP began to decrease, with respiration and circulationsupport, MAP^ 60mmHg could be maintained for more than 48h steadily.3. Peculiar sign during establishment of brain death: animals in group B, group L, group Ml and group M2 all presented transient, rhythmless systematic twitch with HR and MAP prompt increase and turbulence of brain wave before the initial confirmation of brain death; after the confirmation of brain death, spontaneous or reflex movements can be seen, such as undulating leg flexion movements or waving of pigtail etc. Conclusion:1. Intravenous mixed anaesthesia is optimal for establishment of brain-dead model with Ba-Ma mini pigs. It is necessary to maintain circulation stable, correct imbalance of acid base and serum electrolytes, and maintain normal body temperature. The prevention and treatment of diabetes insipidus and lung hydroncus are very-crucial in addition.2. The assay of establishing Ba-MA mini pigs by increasing intracranial pressure in a modified, slow and intermittent way can effectively mimic the clinical brain death; during the course of establishment of brain-dead model the hemodynamic change violently; with respiration and circulation support, the brain-dead state could be maintained for 24h⁴8h.Part II Experimental study on how brain-dead state affects the lung morphology and function of Ba-Ma mini Pigs and the protective effects of N-acetylcysteine on lung injuryObjective:To investigate the effects and mechanism on lung function and morphology and NF- k B (Nuclear factor- k appaB) mRNA and its protein expression of BA-Ma mini pigs with brain-dead state and to investigate the protective effects of NAC(N-acetylcysteine) on lung injury. Materials and Methods:Fifteen Ba-Ma mini pigs were randomly divided into 3 groups: brain-dead group (group B, n=5), NAC pretreatment group (group N, n=5) and control group (group C, n=5). The brain-dead models were eatablished according to the methods of Part I. NAC at 150mg/kg was injected via the vein in group N immediately after the establishment of the brain-death model. At time after anesthesia and 3, 6, 12, 18 and 24h after the initial brain death, serum TNF- a (Tumor necrosis factor- a ) , IL-1 0 (Interleukin-1 P), and IL-6(Interleukine-6) were determined. At 24h after the initial brain death, lung tissues were taken, the changes of lung tissues were observed by HE staining, the expression of NF- k B by immnohistochemistry, and NF- k B mRNA by RT-PCR(Reverse transcriptase polymase chain reaction). The ultrastructure changes of lung were observed under electron microscope. Total protein content in the BALF(Bronchoalveolar lavage fluid), lung index, lung water content were measured too. Results:1. Changes of inflammatory factors: at the points of after anesthesia and 3, 6,12, 18, and 24h after the initial brain death, TNF- a for group C were 13.78±0.52, 14.97±0.27, 15.02±0.36, 14.94±0.28, 15.00±0.11 and 14.87+0.31 pg/ml respectively, those for group B were 13.76 ±0.79,19.52 ±0.16,21.77 ±0.24,24.27 ±0.88,27.74+ 0.27 and 31.35 ±0.13 pg/ml respectively, and those for group N werel3.68± 0.81,18.49 ± 0.09,20.31 ± 0.31,23.39 ± 0.96,25.87 ± 0.64 and 28.37 ± 0.29 pg/ml respectively; IL-1 P for group C were 5.89±0.16, 5.92±0.14, 5.89±0.13, 5.93±0.09, 5.97±0.11 and 6.06+0.14pg/ml respectively, those for group B was 5.91+0.12,8.38±0.08, 9.15±0.08, 10.28±0.19, 12.58±0.25 and 16.37±0.11 pg/ml respectively, and those for group N were 5.90±0.18, 8.09+0.10, 8.57+0.27, 9.14±0.11, 9.49±0.23 and 10.29±0.46 pg/ml respectively; IL-6 for group C were 15.10±0.49, 17.45±0.48, 18.13±0.24, 18.24+0.52, 18.35±0.50 and 17.39±0.69pg/ml respectively, those for group B were 15.O4±O.5O, 29.34±1.56, 38.05±1.28, 43.92±2.46, 56.10+2.00 and 66.42±1.76pg/ml respectively, and those for group N were 14.89+0.58,20.41±0.82, 22.47±1.00, 30.25+1.48, 34.17±1.81 and 42.32±2.41pg/ml respectively. TNF- a , IL-1 3 and IL-6 for group C , group B and group N at time point after anesthesia had no significant difference; since 3h after the innial brain death, these parameters for group B and group N began to increase. TNF- a , IL-1 P and IL-6 at 3, 6, 12, 18, and 24h after the initial brain death, had significant differences (P<0.05); and compared with group N, these parameters for group B were significantly higher at each time point (P<0.05).2. Changes of NF- k B mRNA in lung tissues: NF- k B mRNA expression of group C was 0.648±0.171, that of group B was 2.036±0.102, and that of group N was 0.958+0.089. NF- k B mRNA expression of group B and N was significantly increased than that of group C (P<0.05),and which was significantly higher for group B than that of group N (P<0.05). Expression of NF- k B: the expression of NF- k B protein for group C was 0.0910+0.0213, that for group B was 0.7189±0.0616, and that for group N was 0.3109±0.0232. Expression of NF- k B protein for group B and N was significantly increased than that for group C (P<0.05),and which was significantly higher for group B than that for group N (P<0.05).3. Morphological changes of lung tissues: In group B , By light microscope ,broadened lung alveolar septum, edematous and congestive with localized bleeding, alveolar edema and partial pulmonary collapse, infiltration of lymphocytes, microvascular congestion were observed, Electronmicroscopic examination found that the mitochondria of the type- II epithelial cells swelled, the mitochondria cristae fragmented, lamellar bodies in alveolar type- II epithelial cells reduce, and their structures were destroyed. NAC group have slight pathology disordered, similar to normal both Under light microscope and electron microscopic examination. In group C, lung tissues appearances were normal.4.Total protein content in the BALF, lung index, lung water content of group B and group N were significant higher compared with group C (P<0.05),and whichwere significant higher for group B than that for group N (P<0.05). Conclusion:1. Brain death may evoke lung functional and morphological injury of Ba-mamini pigs.2. The release of TNF- a , IL-1 P and IL-6 and the levels of NF- k B mRNA transcription and protein translation in the lung tissues can be enhanced by the brain death.3.The lung injury changes of brain-dead models not only caused by the hemodynamic changes, inflammatory mediators and the decrease of alveolar fluid transportation also play the important role.4. NAC has preventive effect against the lung injury caused by brain death. The protective mechanism of NAC may be related with inhibition of NF- k. B mRNA transcription and protein translation in the lung tissues and diminishing the release of TNF- a , IL-1 3 and IL-6.5. Diminishing the injury changes of AT II cell, enhance the function of alveolar fluid clearance and transportation play the important role of the preventive effect against the lung injury of NAC.