Brain Death In Rats Research Articles

Extracorporeal membrane oxygenation ameliorate hepatic injury in brain death rat donors with hemodynamic instability.

Donation after brain death (DBD) serves as the primary source for liver transplantation. However, livers obtained through DBD often incur damage due to unstable hemodynamics, potentially impacting transplantation outcomes. Extracorporeal Membrane Oxygenation (ECMO) emerges as an optimal technique for donor liver retrieval and has found application in clinical settings. Despite its clinical implementation, the precise mechanisms through which ECMO enhances liver functions remain elusive. This study aims to investigate the mechanisms underlying how ECMO ameliorates liver function in brain-dead donors. We randomly assigned 18 male Sprague-Dawley (SD) rats (350 ± 50 g) into three groups: Con (n = 6), DBD-assisted drug (n = 6), and DBD-assisted ECMO (n = 6). After 3 h of ECMO, the rats were sacrificed. We assessed and compared changes in heart rate, blood pressure, cumulative liver damage (evaluated through HE and TUNEL staining), serum levels of AST and ALT, alterations in serum oxidative stress factors (MDA, H2O2, SOD, and 8-OHdG), and serum concentrations of related inflammatory factors (interleukin [IL]-1β, IL-6, IL-8, and TNF-α) among rats in the Con, DBD-assisted drug, and DBD-assisted ECMO groups. Subsequently, we established a rat orthotopic liver transplantation (OLT) model and transplanted livers obtained through the aforementioned methods. The post-transplantation status of the livers was observed. After 3 h of brain death, liver injury worsened, accompanied by a significant increase in serum transaminases, inflammatory responses, oxidative stress, and TUNEL staining. Strikingly, ECMO not only stabilized hemodynamics after DBD but also mitigated liver damage, leading to an alleviated status post liver transplantation. ECMO stabilizes hemodynamics, attenuates inflammatory responses and oxidative stress, thereby enhancing the quality of liver grafts for transplantation.

Targeting Kidney Inflammation After Brain Death and Cold Storage: Investigating the Potential of an NLRP3 Inflammasome Inhibitor (MCC950) for Preconditioning Donor Kidneys.

Brain death (BD) and cold storage (CS) are critical factors that induce inflammation in donor kidneys, compromising organ quality. We investigated whether treating kidneys from BD rats with an inflammasome Nod-like receptor family pyrin domain containing 3 (NLRP3) inhibitor (MCC950) followed by CS could reduce kidney inflammation. BD rats were assigned to MCC950-treated or nontreated (NT) groups. Kidneys were evaluated immediately before CS (T0) and after 12 h (T12) and 24 h (T24) of CS. Mean arterial pressure, serum creatinine, gene/protein expression, and histology were evaluated. At T0, MCC950 treatment did not affect mean arterial pressure but tended to reduce serum creatinine and ameliorated the histological score of acute tubular necrosis. However, MCC950 reduced NLRP3, caspase-1, interleukin (IL)-1β, IL-6, Kim-1, nuclear factor kappa B, tumor necrosis factor alpha, and caspase-3 gene expression while increasing IL-10 cytokine gene expression. After 12 h of CS, only the expression of the NLRP3 and caspase-1 genes decreased, and after 24 h of CS, no further changes in the gene expression profile were observed. The levels of the inflammasome proteins NLRP3, caspase-1, and IL-1β consistently decreased across all time points (T0, T12, and T24). These findings suggest that MCC950 treatment holds promise for mitigating the proinflammatory state observed in kidneys after BD and CS.

366.8: Microcirculatory effects of alteplase associated to methylpredisolone after brain death in rats.

366.8: Microcirculatory effects of alteplase associated to methylpredisolone after brain death in rats.

366.6: Males and females respond differently to 17beta-estradiol and methylprednisolone associated treatment during ex vivo lung perfusion of brain death rats.

366.6: Males and females respond differently to 17beta-estradiol and methylprednisolone associated treatment during ex vivo lung perfusion of brain death rats.

MG132 protects against lung injury following brain death in rats.

Brain death (BD) results in injury to organs and induces lung donor dysfunction. Since the 20S proteasome abnormality is associated with a variety of diseases, the present study investigated whether it was involved in lung injury following BD in rats, and the effects of the proteasome inhibitor MG132 on lung injury was also assessed. Rats were assigned to a BD group or a control sham group. The BD group of rats were sacrificed at different time points after BD. Administration of MG132 was performed intraperitoneally 30 min before BD. Arterial blood was drawn to measure the oxygenation index [partial artery pressure of oxygen (PaO2)/fractional concentration of inspired oxygen (FiO2)]. The right lung was used for staining with hematoxylin and eosin, immunohistochemistry, immunofluorescence, western blotting and RT-qPCR analysis. The left lung was used to measure the wet and dry weights. Rat alveolar macrophages (NR8383) were treated with MG132 and hypoxia/reoxygenation (H/R) and used for western blotting and flow cytometry. The PaO2/FiO2 ratio decreased after BD; the wet/dry weight ratio, histological lung injury score and protein expression of 20S proteasome β1 and inducible nitric oxide synthase (iNOS) gradually increased in rats after BD. Colocalization in the immunofluorescence between 20S proteasome β1 and iNOS was observed. MG132 treatment increased the PaO2/FiO2 ratio and decreased the wet/dry weight ratio, histological lung injury score and protein expression of 20S proteasome β1 and iNOS in rats after BD. MG132 was revealed to increase NR8383 apoptosis after H/R and to upregulate the protein expression levels of p-JNK and cleaved-caspase 3. Overall, the proteasome inhibitor MG132 could effectively reduce lung injury, which may be associated with its ability to inhibit the expression of the proteasome and promote the apoptosis of alveolar macrophages.

Blocking protease-activated receptor 4 alleviates liver injury induced by brain death

Brain death (BD) induces a systemic inflammatory response that influences donor liver quality. Protease-activated receptor 4 (PAR4) is a thrombin receptor that mediates platelet activation and is involved in inflammatory and apoptotic processes. Therefore, we investigated the role of PAR4 blockade in liver injury induced by BD and its associated mechanisms. In this study, we constructed a BD rat model and treated rats with TcY-NH2, a selective PAR4 antagonist, to block PAR4 signaling at the onset of BD induction. Our results revealed that PAR4 protein expression increased in the livers of rats with BD. PAR4 blockade alleviated liver injury induced by BD, as indicated by lower serum ALT/AST levels and an improvement in histomorphology. Blood platelet activation and hepatic platelet accumulation in BD rats were reduced by PAR4 blockade. Additionally, PAR4 blockade attenuated the inflammatory response and apoptosis in the livers of BD rats. Moreover, the activation of NF-κB and MAPK pathways induced by BD was inhibited by PAR4 blockade. Thus, our results suggest that PAR4 contributes to liver injury induced by BD by regulating inflammation and apoptosis through the NF-κB and MAPK pathways. Thus, PAR4 blockade may provide a feasible approach to improve the quality of organs from BD donors.

Caspase-1 Inhibitor Reduces Pyroptosis Induced by Brain Death in Kidney.

Brain death (BD) induces an organ-level inflammatory response. However, the underlying mechanisms have not been fully elucidated. Here, we investigated the role of caspase-1-mediated pyroptosis in BD-induced kidney injury in rats. A BD model was established in Sprague-Dawley rats. The rats were intravenously injected with Z-YVAD-FMK 1 h before BD, and sham-operated rats served as controls. After 0, 1, 2, 4, and 6 h of BD, renal injury, and renal expression of the nod-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, caspase-11, gasdermin D (GSDMD), IL-1β, and IL-18 were assessed using quantitative reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Blood urea nitrogen and serum creatinine levels were measured. Additionally, renal tubular epithelial cells (NRK-52E) were subjected to 3 h of hypoxia followed by 6 h of reoxygenation and incubated with Z-YVAD-FMK before hypoxia and reoxygenation. Caspase-11 was knocked-down using small interfering RNA technology. Cell viability and levels of pyroptosis-associated proteins were assessed thereafter. NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 expression levels were upregulated in BD rats. Treatment with Z-YVAD-FMK reduced mRNA and protein levels of caspase-1, GSDMD, IL-1β, and IL-18, improved renal function, and alleviated renal injury. Z-YVAD-FMK efficaciously reduced pyroptosis effects in kidneys in BD rats. Thus, it could be considered as a therapeutic target for BD-induced kidney injury.

Experimental Lung Donor Models for Transplant

<h3>Purpose</h3> The main types of accepted donations may be divided into: Donation after Brain Death and Donation after Circulatory Death. In addition to that, some important events are frequently associated to these two types of donors, such as Hemorrhagic Shock (HS) and Cold Ischemia (CI). These events also guide the standardization of the various experimental models developed by research groups for the employment of specific treatments: brain death (BD), circulatory death (CD), HS and CI. In this study, we aimed to describe and compare the lung mechanics and inflammatory process related to these four experimental models of lung donors. <h3>Methods</h3> Forty Sprague-Dawley rats were divided into 4 groups: BD (intracranial Fogarty balloon inflation), HS (hypovolemic protocol - 40 mmHg), CD (circulatory death + 3 h warm ischemia) and CI (6 h cold ischemia). Inflammatory responses and edema were assessed. In addition to these, as the BD and HS rats were kept hemodynamically stable for six hours, monitoring of lung mechanics was possible. <h3>Results</h3> Six hours after the induction of the HS, increase in elastance and resistance were observed (fig. 1). In lung tissue, BD and HS presented high levels of IL-1β and IL-6 in comparison to the CD and CI groups. In contrast, IL-10 levels were higher in the HS group, being significantly different when compared to the CD group. Finally, the BD group had the highest lung wet-to-dry ratio (fig. 2). <h3>Conclusion</h3> The experimental model of HS cause dysfunction of the lung mechanics. BD and HS lead to increased levels of cytokines and lung edema. These differences should be carefully considered to choose the ideal experimental model for developing specific therapeutic strategies.

Sex differences in the coagulation process and microvascular perfusion induced by brain death in rats.

Brain death (BD) leads to a systemic inflammation associated with the activation of coagulation, which could be related to decreased microcirculatory perfusion. Evidence shows that females exhibit higher platelet aggregability than males. Thus, we investigated sex differences in platelets, coagulation and microcirculatory compromise after BD. BD was induced in male and female (proestrus) Wistar rats. After 3h, we evaluated: (i) intravital microscopy to evaluate mesenteric perfusion and leucocyte infiltration; (ii) platelet aggregation assay; (iii) rotational thromboelastometry; and (iv) Serum . Female rats maintained the mesenteric perfusion, whereas male reduced percentage of perfused vessels. Male BD presented higher platelet aggregation than the controls. In contrast, female BD had lower platelet aggregation than the control. Thromboelastometry indicated a reduction in clot firmness with increased clotting time in the female group compared with the male group. Serum level in female BD was higher than that in the male BD and female control. There is sex dimorphism in platelet function and clotting process, which are altered in different ways by BD. Thus, it is possible to connect the reduction in microcirculatory perfusion in males to intravascular microthrombi formation and the maintenance of perfusion in females to a higher inflammatory response and NO synthesis.

17β-Estradiol as a New Therapy to Preserve Microcirculatory Perfusion in Small Bowel Donors.

Intestine graft viability compromises retrieval in most brain-dead donors. Small bowel transplantation is a complex procedure with worse outcomes than transplantation of other abdominal organs. The hormone 17β-estradiol (E2) has shown vascular protective effects in lung tissue of brain death (BD) male rats. Thus, estradiol might be a treatment option to improve the quality of intestinal grafts. Male Wistar rats were divided into 3 groups (n = 10/group): rats that were trepanned only (sham-operated), rats subjected to rapid-onset BD, and brain-dead rats treated with E2 (280 µg/kg, intravenous) (BD-E2). Experiments performed for 180 minutes thereafter are included: (a) laser-Doppler flowmetry and intravital microscopy to evaluate mesenteric perfusion; (b) histopathological analysis; (c) real-time polymerase chain reaction of endothelial nitric oxide synthase (eNOS) and endothelin-1; (d) immunohistochemistry of eNOS, endothelin-1, P-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 expression; and (e) ELISA for cytokines and chemokines measurement. 17β-Estradiol improved microcirculatory perfusion and reduced intestinal edema and hemorrhage after BD. The proportions of perfused small vessels were (mean ± scanning electron microscope) BD rats (40% ± 6%), sham-operated rats (75% ± 8%), and BD-E2 rats (67% ± 5%) (P = 0.011). 17β-Estradiol treatment was associated with 2-fold increase in eNOS protein (P < 0.0001) and gene (P = 0.0009) expression, with no differences in endothelin-1 expression. BD-E2 rats exhibited a reduction in vascular cell adhesion molecule 1 expression and reduced cytokine-induced neutrophil chemoattractant 1 and interleukina-10 serum levels. 17β-Estradiol was effective in improving mesenteric perfusion and reducing intestinal edema and hemorrhage associated with BD. The suggestion is that E2 might be considered a therapy to mitigate, at least in part, the deleterious effects of BD in small bowel donors.

Hypertonic saline reduces cell infiltration into the lungs after brain death in rats

BackgroundLung transplantation is a treatment method for end stage lung disease, but the availability of donor lungs remains a major constraint. Brain death (BD) induces hemodynamic instability with microcirculatory hypoperfusion and increased inflammation, leading to pulmonary dysfunction. Hypertonic saline solution (HSS) is a volume expander possessing immunomodulatory effects. This study evaluated the influence of HSS on pulmonary dysfunction and inflammation in a rat model of BD. MethodsBD was induced by inflation of an intracranial balloon catheter. Rats were divided into [1]: Sham, without BD [2]; NS, NaCl treatment (0.9%, 4 mL/kg, i.v.) immediately after BD [3]; HSS1, HSS treatment (NaCl 7.5%, 4 mL/kg, i.v.) immediately after BD; and [4] HSS60, HSS treatment 60 min post BD. All groups were analyzed after 360 min. ResultsAnimals subjected to BD exhibited increased exhaled O2 and decreased CO2.The number of leukocytes in the lungs was significantly increased in the NS group (p = 0.002) and the HSS treatment was able to reduce it (HSS1, p = 0.018 and HSS60 = 0.030). In parallel, HSS-treated rats showed reduced levels of ICAM-1 expression, which was increased in the NS compared to Sham group. Lung edema was found increased in the NS group animals compared to Sham and no effect of the HSS treatment was observed. There were no differences among the groups in terms of TNF-α, VEGF, and CINC-1 lung concentrations. ConclusionsHSS is capable of reducing inflammatory cell infiltration into the lung after BD induction, which is associated with the reduction of ICAM-1 expression in organ vessels.

Conditioned Medium from Mesenchymal Stem Cells Protects Vascular Grafts of Brain-Dead Rats against In Vitro Ischemia/Reperfusion Injury

Purpose The endothelium has a pivotal role in the maintenance of cardiac function after heart transplantation, mainly by controlling the coronary circulation. Brain death (BD) as well as ischemia/reperfusion (IR) injury compromise vascular endothelium and thereby myocardial protection. Beneficial effects of conditioned medium (CM) from mesenchymal stem cells (MSCs) against IR injury have been demonstrated. We hypothesized that physiological saline supplemented CM could protect vascular grafts of BD rats from IR injury. Methods Rat MSCs derived CM was used for conservation purposes. We detected 28 factors involved in inflammation, oxidative stress and apoptosis. Donors were either subjected to sham operation or BD by inflation of a subdural balloon for 5.5h. Then, thoracic aortic rings from BD rats were isolated and immediately mounted after preparation in an organ bath chamber (BD group) or underwent 24h cold ischemic preservation in physiological saline supplemented either with a vehicle (BD-IR group) or CM (BD-IR+CM group). Immunohistochemical staining for myeloperoxidase (MPO), nitrotyrosine (NT), caspase-8, -9, and -12 was performed. Results Characterization of BD donors: Compared to the sham group, BD leads to significantly impaired hemodynamic parameters, higher aortic MPO, NT, caspase-8, -9, and -12 contents. Organ bath experiments: The preservation of BD-IR aortic rings with CM significantly improved IR-induced decreased maximum endothelium dependent vasorelaxation (Rmax) to acetylcholine compared to the vehicle treated BD-IR group (Rmax to acetylcholine: BD 81±2 vs BD-IR 50±3 vs BD-IR+CM 72±2%, p Conclusion Endothelial dysfunction during IR injury is improved by preservation of vascular grafts from BD rats with CM. This protective effect may partly arise from a reduction of inflammation and nitro oxidative stress responses, and inhibition of caspase-8 and -9 mediated apoptosis.

Brain Death-Induced Donor Heart Injury Diminished by Melatonin Treatment via Suppressing DAMP Signaling

Purpose We test the hypothesis that melatonin diminishes brain death (BD)-induced donor heart injury by suppressing inflammatory damage-associated molecular pattern (DAMP) signaling. Methods H9C2 cells or BD-derived brain tissue extract (BDEX)-treated H9C2 cells were co-cultured with BDEX (BDEX), healthy brain tissue extracts (sham control, SC) or reagent (NC) and Sprague-Dawley rats were divided into NC (healthy brain), BD, BD + Mel (BD rats treated with melatonin), and BD + Mel + Luz (BD rats treated with melatonin and its antagonist, luzindole) to check DAMP proteins, the downstream proinflammatory cytokines and the impact of melatonin on these inflammatory signaling protein levels. Additional rats were categorized into groups 1 NC (only left thoracotomy), 2 SC (healthy brain tissue extracts implanted into the LV myocardium (LVM)), 3 BDEX (BDEX implanted into the LVM), 4 BDEX-Mel (BDEX plus melatonin treatment) and 5 BDEX-Mel-Luz (BDEX plus melatonin and luzindole treatments) to assess the protective effect of melatonin against BD-induced myocardial injury by detecting cardiac inflammatory and immune cell infiltration, DNA damage, fibrosis, oxidative-stress, apoptosis, and transthoracic echocardiographic findings 5 days after BDEX implantation. Results Levels of HMGB1 and inflammatory markers were higher in BDEX-treated H9C2 cardiac myoblasts than in cells treated with healthy brain tissue extract. Levels of a DAMP component (HMGB1) and inflammatory markers (TLR-2, TLR-4, MYD88, IκB, NF-κB, IL-1β, IFN-γ, TNF-α and IL-6) were higher in brain tissue from BD animals than controls. These increases were diminished by melatonin but re-induced with luzindole (all P Conclusion Brain death-induced donor heart injury might be diminished by melatonin suppressing the DAMP inflammatory axis.

Renal effects of exendin-4 in an animal model of brain death

Organ transplantation is the gold standard therapy for the majority of patients with terminal organ failure. However, it is still a limited treatment especially due to the low number of brain death (BD) donors in relation to the number of waiting list recipients. Strategies to increase the quantity and quality of donor organs have been studied, and the administration of exendin-4 (Ex-4) to the donor may be a promising approach. Male Wistar rats were randomized into 3 groups: (1) control, without central nervous system injury; (2) BD induced experimentally, and (3) BD induced experimentally + Ex-4 administered immediately after BD induction. After BD induction, animals were monitored for 6h before blood collection and kidney biopsy. Kidney function was assessed by biochemical quantification of plasma kidney markers. Gene and protein expressions of inflammation- and stress-related genes were evaluated by RT-qPCR and immunoblot analysis. Animals treated with Ex-4 had lower creatinine and urea levels compared with controls. BD induced oxidative stress in kidney tissue through increased expression of Ucp2, Sod2 and Inos, and Ex-4 administration reduced the expression of these genes. Ex-4 also induced increased expression of the anti-apoptotic Bcl2 gene. Nlrp3 and Tnf expressions were up-regulated in the BD group compared with controls, but Ex-4 treatment had no effect on these genes. Our findings suggest that Ex-4 administration in BD rats reduces BD-induced kidney damage by decreasing the expression of oxidative stress genes and increasing the expression of Bcl2.

Effect of Methylprednisolone on Liver Injury and Endotoxin Levels Following Brain Death in Rats

Background and aimBrain death impairs liver function in potential donors and is associated with inflammatory activation. Methylprednisolone treatment after brain death has been shown to reduce inflammatory activity. The aim of the present study was to evaluate the effects of methylprednisolone on liver injury and endotoxin levels in brain-dead rats. MethodsThirty-two rats were randomly divided into 4 groups: a sham-operation group (Sham group), a brain death with methylprednisolone treatment group (Methy group), a brain death with saline treatment group (Saline group), and a brain death group (BD group). The rats were anesthetized and induced by gradually increasing the intra-cranial pressure using a Fogarty catheter balloon for brain death. All of the animals were observed and ventilated for 6 h prior to being euthanized. Hepatic pathologic histology (Knodell histology activity index), liver inflammatory cytokine levels, liver function and endotoxin levels were assessed. ResultsAfter brain death, methylprednisolone markedly alleviated the Knodell histology activity index of liver injury (P < .05). Additionally, significant reductions in the levels of TNF-α, IL-1β, and IL-10 were observed in the Methy group compared to those in the Saline and BD groups (P < .01), whereas no significant differences were found between the Saline and BD groups (P > .05). Interestingly, although the rate of liver injury after brain death in the methylprednisolone treatment group improved, the endotoxin level did not decline in the Methy group compared to the levels in the Saline and BD groups (P > .05). ConclusionThe present study verified that methylprednisolone was protective for liver injury in rats subjected to brain death. This protection appeared to be due to reduced inflammatory activity with no influence on the endotoxin level.

Kupffer cell depletion by gadolinium chloride aggravates liver injury after brain death in rats.

Brain death (BD) impairs liver function in potential donors, and is associated with hormonal and metabolic changes or molecular effects with pro-inflammatory activation. Resident macrophages in the liver named Kupffer cells (KCs) undergo pro- or anti-inflammatory pathway activation, which affects liver function. However, the role of the KCs in liver dysfunction following BD has not been fully elucidated. The aim of the present study was to investigate the role of KCs in liver dysfunction in the context of BD and the effects of their inhibition by gadolinium chloride (GdCl3). Rats were randomly divided into the following groups: Control, BD with GdCl3 pretreatment and BD with normal saline pretreatment. Liver function, hepatic pathological histology and cytokine levels in the liver were assessed. Apoptosis and apoptosis-related proteins [cleaved caspase-3, caspase-3 and apoptosis regulator Bcl-2 (Bcl-2)] were evaluated. GdCl3 significantly aggravated liver injury by elevating alanine aminotransferase and aspartate aminotransferase levels (P<0.05) by inhibiting KCs. Interleukin (IL)-1β and tumor necrosis factor α levels in the GdCl3 group were significantly increased compared with those in the control and saline groups (P<0.01). However, IL-10 levels in the GdCl3 group were significantly reduced compared with those in the saline group (P<0.05). Caspase-3 and cleaved caspase-3 activation, and apoptosis induction in the context of BD were also significantly aggravated by the depletion of KCs, whereas Bcl-2 was significantly suppressed by the administration of GdCl3. The present study indicated that GdCl3 efficiently inhibits the activity of KCs, and is involved in the onset of liver injury through its effects on pro-inflammatory and anti-inflammatory activation. KCs are protective in the liver in the context of BD. This protection appears to be due to KCs secretion of the potent anti-inflammatory cytokine IL-10, suggesting that KCs are an attractive target for the prevention and treatment of liver injury in the context of BD in rats.

Inhibition of Endoplasmic Reticulum Stress Alleviates Lung Injury Induced by Brain Death.

Brain death (BD) can induce inflammation and injury of organs. Endoplasmic reticulum (ER) stress is associated with a variety of diseases. However, little is known about how ER stress is implicated in brain death (BD)-induced lung injury. In this study, a stable BD rat model was constructed to investigate the role of ER stress on BD-induced lung injury. H&E staining demonstrated that BD can induce lung injury in rats. The results of Western blot and immunohistochemistry showed that apoptosis was observed in the lung tissues of BD rats. And the level of GRP78, p-PERK, p-eIF2α, CHOP, and Caspase-12 was highly expressed in BD rats compared with the control group. Inhibition of ER stress with salubrinal reduced the BD-induced lung inflammation. Moreover, BD-induced increase of NF-κB activity was lowered by inhibition of ER stress. These results suggested that inhibition of ER stress alleviates BD-induced lung inflammation by regulating NF-κB signaling pathway.

Evidence of bone marrow downregulation in brain-dead rats.

Experimental findings support the evidence of a persistent leucopenia triggered by brain death (BD). This study aimed to investigate leucocyte behaviour in bone marrow and blood after BD in rats. BD was induced using intracranial balloon catheter inflation. Sham-operated (SH) rats were trepanned only. Thereafter bone marrow cells were harvested every six hours from the femoral cavity and used for total and differential counts. They were analysed further by flow cytometry to characterize lymphocyte subsets, granulocyte adhesion molecules expression and apoptosis/necrosis [annexin V/propidium iodide (PI) protocol]. BD rats exhibited a reduction in bone marrow cells due to a reduction in lymphocytes (40%) and segmented cells (45%). Bone marrow lymphocyte subsets were similar in BD and SH rats (CD3, P=0.1; CD4, P=0.4; CD3/CD4, P=0.4; CD5, P=0.4, CD3/CD5, P=0.2; CD8, P=0.8). Expression of L-selectin and beta2 -integrins on granulocytes did not differ (CD11a, P=0.9; CD11b/c, P=0.7; CD62L, P=0.1). There were no differences in the percentage of apoptosis and necrosis (Annexin V, P=0.73; PI, P=0.21; Annexin V/PI, P=0.29). In conclusion, data presented suggest that the downregulation of the bone marrow is triggered by brain death itself, and it is not related to changes in lymphocyte subsets, granulocyte adhesion molecules expression or apoptosis and necrosis.

(583) - Hypertonic Saline Solution Preserves Left Ventricular Function and Inhibit Myocardial Injury Triggered by Brain Death in Rats

Brain death (BD) induces hemodynamic instability with microcirculation hypoperfusion leading to increased heart tissue inflammation and dysfunction. This study investigates the effects of 7.5% hypertonic saline solution administration on the course of myocardial and left ventricular (LV) function changes observed after BD induction in rats. Male Wistar rats were anesthetized and mechanically ventilated. BD was induced by inflation of a balloon catheter placed into intracranial cavity. Rats were randomly divided into three groups: NS - rats maintained with normal saline immediately after BD induction (0.9% NaCl, 4 mL/Kg); HS - rats treated with hypertonic saline (7.5% NaCl, 4 mL/Kg) immediately after BD; HS60 - rats treated with hypertonic saline (7.5% NaCl, 4 mL/Kg) 60 minutes after BD. LV function was evaluated by conductance microcatheter technique for 6 hours after induction of BD. Immunohistochemistry was performed to investigate endothelial intercellular adhesion molecules and apoptotic proteins expressions in LV samples collected after animal’s euthanasia. All groups presented similar hypertensive peak followed by hypotension after BD induction. Six hours after induction of BD, preload recruitable stroke work and dp/dt(max) were significantly reduced in comparison to baseline in NS group, whereas these parameters were preserved at similar baseline levels in both HS and HS60 groups. LV ejection fraction was also reduced in NS group (70% of baseline) at 6 hours, but in contrast it was preserved in HS60 group, which presented significantly elevated values in comparison to NS group (72 ± 6 vs. 41 ± 6, p<0.001). Tau was significantly elevated versus baseline 6 hours after induction of BD especially in NS and HS60 groups. Both treatments, HS and HS60, reduced the expression of vascular cell adhesion molecules (VCAM-1) and increased the levels of BCL-2 anti-apoptotic proteins, when compared to NS group. Data presented suggest that treatment of BD rats with hypertonic saline solution improves LV function and reduces myocardial injury, even when performed in the course of BD progression.

Protective role of SP600125 in apoptosis of cardiomyocytes in brain death rats

Objective To investigate the protective role of SP600125, a selective c-Jun N-terminal kinase inhibitor,in the apoptosis of cardiomyocytes in brain death rats. Methods Twenty Sprague-Dawley(SD) rats were randomly divided into:Sham group(n= 5), only given intracranial insertion of Fogarty tube after anesthesia; brain- death group(n= 5), given brain death for 6 h; SP600125 group (n= 5), given peritoneal injection of SP600125(10 mg/kg)1 h before brain death for 6 h; and DMSO group(n= 5), given peritoneal injection of placebo solution DMSO(10 mg/kg)1 h before brain death for 6 h.At 6 h, the apical myocardial samples were obtained for detection of Cysteinyl aspartate-specific protease(Caspase)- 3 and cytochrome- C(Cyt- C) mRNA and protein expression by real- time quantitative polymerase chain reaction(real- time PCR)and Western blotting respectively.Apoptosis of cardiac muscle cells was determined by terminal deoxynucleotidyl transferase d UTP nick end labelling(TUNEL) assay. Results (1)Real-time PCR showed that the mRNA expression of Caspase- 3 and Cyt- C mRNA in brain death group was(2.37±0.12, 2.03±0.08) times higher than that in sham group(P 0.05). The mRNA expression of Caspase-3 and Cyt- Cin SP600125 group(1.21±0.05 and 1.23±0.24)was significantly lower than that in brain death group(2.38±0.11 and 2.06±0.09)(P 0.05), and that in SP600125 group(39.45±5.73 and 20.34±5.69) was decreased significantly as compared with that in brain death group(P 0.05],and that in SP600125 group[(7.63±3.09)%]was significantly reduced as compared with that in brain death group(P< 0.05). Conclusion SP600125 played protective roles in the apoptosis of cardiomyocytes in brain death rats through specifically inhibiting the activity of JNK signaling pathway. Key words: Donor heart; Brain death; Apoptosis; c-Jun N-terminal kinase inhibitor