Sueli Gomes Ferreira

Intrathecal injection of human umbilical cord blood stem cells attenuates spinal cord ischaemic compromise in rats

OBECTIVES Spinal cord ischaemia with resulting paraplegia remains a devastating and unpredictable complication after thoraco-abdominal aortic surgery. With the advent of stem cell therapy and its potential to induce nervous tissue regeneration processes, the interest in the use of these cells as a treatment for neurological disorders has increased. Human stem cells, derived from the umbilical cord, are one of the strong candidates used in cell therapy for spinal cord injury because of weak immunogenicity and ready availability. We sought to evaluate the use of human umbilical cord blood stem cells (HUCBSCs) to attenuate the neurological effects of spinal cord ischaemia induced by high thoracic aorta occlusion. METHODS Forty Wistar rats were randomized to receive intrathecal injection of 10 µl phosphate buffered saline (PBS) solution containing 1 × 10(4) HUCBSCs, 30 min before (Tpre group: n = 10) and 30 min after (Tpos group: n = 10) descending thoracic aorta occlusion by intraluminal balloon during 12 min. Control groups received only PBS solution (Cpre group: n = 10; and Cpos group: n = 10). During a 28-day observational period, motor function was assessed by a functional grading scale (Basso, Beattie and Bresnahan). Segments of thoracolumbar spinal cord specimens were analysed for histological and immunohistochemical assessment for detection and quantification of human haematopoietic cells (CD45(+)) and apoptosis (transferase-mediated deoxyuridine triphosphate-biotin nick-end labelling). RESULTS Overall mortality was 12 animals (30%). Therefore, the observational sample was composed of 28 animals. All groups showed similar incidence of paraplegia and mortality. The mean motor function scores showed no difference during time between the animals of each group, excepting for the Tpos group, which improved from 8.14 (±8.6) to 14.28 (±9.8) (P < 0.01). A treatment-by-time interaction was detected among animals that received HUCBSCs 30 min after ischaemia, with BBB scores higher from Days 14 to 28 compared with the first observational day with statistical difference (P = 0.01). Number of viable neurons was higher in the Tpos group (P = 0.14) and the incidence of apoptosis was lower in the same animals (P = 0.048), but showed no difference with its respective control. We confirmed the presence of CD45(+) cells 4 weeks after intrathecal injection in both therapeutic groups but mainly in the Tpos group. CONCLUSIONS Intrathecal transplantation of HUCBSCs is feasible, and it improved spinal cord function, when they were delivered 30 min after spinal cord ischaemia, in a model of endovascular descending thoracic aorta occlusion in rats. Human umbilical cord blood is one of the potentially useful sources of stem cells for therapy of spinal cord ischaemia.

Effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation during early sepsis treatment.

OBJECTIVES: Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. This study aimed to investigate the effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation in a live Escherichia coli-induced sepsis model in rats. METHODS: Male Wistar rats were administered an intravenous suspension of E. coli bacteria or were subjected to a sham procedure. Three hours after bacterial infusion, the rats were randomized into the following groups: a control group without treatment, a group treated with lactated Ringers solution (4 mL/kg, i.v.), and a group treated with lactated Ringers solution (4 mL/kg, i.v.) plus ethyl pyruvate (50 mg/kg). At 24 h after bacterial infusion, leukocyte-endothelial interactions were investigated using intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule-1 was evaluated via immunohistochemistry. White blood cell and platelet counts were also determined at baseline and 3 h and 24 h after E. coli inoculation. RESULTS: The non-treated and lactated Ringers solution-treated groups exhibited increases in the numbers of rolling leukocytes (∼2.5-fold increase), adherent cells (∼3.0-fold), and migrated cells (∼3.5-fold) compared with the sham group. In contrast, treatment with Ringers ethyl pyruvate solution reduced the numbers of rolling, adherent and migrated leukocytes to the levels observed in the sham group. Additionally, the expression of P-selectin and intercellular adhesion molecule-1 was significantly increased on mesenteric microvessels in the non-treated group compared with the sham group (p<0.001). The expression of both adhesion molecules was reduced in the other groups, with ethyl pyruvate being more effective than lactated Ringers solution. Infusion of bacteria caused significant leukopenia (3 h), followed by leukocytosis with granulocytosis (24 h). There was also an intense and progressive reduction in the number of platelets. However, no differences were observed after treatment with the different solutions. CONCLUSIONS: The presented data suggest that ethyl pyruvate efficiently reduces the inflammatory response in the mesenteric microcirculation in an experimental model of sepsis induced by live E. coli and is associated, at least in part, with down-regulation of P-selectin and intercellular adhesion molecule-1.

Inhibition of Autonomic Storm by Epidural Anesthesia Does Not Influence Cardiac Inflammatory Response After Brain Death in Rats

BACKGROUND After brain death (BD) donors usually experience cardiac dysfunction, which is responsible for a considerable number of unused organs. Causes of this cardiac dysfunction are not fully understood. Some authors argue that autonomic storm with severe hemodynamic instability leads to inflammatory activation and myocardial dysfunction. OBJECTIVES To investigate the hypothesis that thoracic epidural anesthesia blocks autonomic storm and improves graft condition by reducing the inflammatory response. METHODS Twenty-eight male Wistar rats (250-350 g) allocated to four groups received saline or bupivacaine via an epidural catheter at various times in relation to brain-death induction. Brain death was induced by a sudden increase in intracranial pressure by rapid inflation of a ballon catheter in the extradural space. Blood gases, electrolytes, and lactate analyses were performed at time zero, and 3 and 6 hours. Blood leukocytes were counted at 0 and 6 hours. After 6 hours of BD, we performed euthanasia to measure vascular adhesion molecule (VCAM)-1, intracellular adhesion molecule (ICAM)-1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, Bcl-2 and caspase-3 on cardiac tissue. RESULTS Thoracic epidural anesthesia was effective to block the autonomic storm with a significant difference in mean arterial pressure between the untreated (saline) and the bupivacaine group before BD (P < .05). However, no significant difference was observed for the expressions of VCAM-1, ICAM-1, TNF-α, IL-1β, Bcl-2, and caspase-3 (P > .05). CONCLUSION Autonomic storm did not seem to be responsible for the inflammatory changes associated with BD; thoracic epidural anesthesia did not modify the expression of inflammatory mediators although it effectively blocked the autonomic storm.

Effect of N-acetylcysteine on pulmonary cell death in a controlled hemorrhagic shock model in rats

PURPOSE To evaluate the effect of N-acetylcysteine (NAC) combined with fluid resuscitation on pulmonary cell death in rats induced with controlled hemorrhagic shock (HS). METHODS Two arteries (MAP calculation and exsanguination) and one vein (treatments) were catheterized in 22 anesthetized rats. Two groups of male albino rats were induced with controlled HS at 35mmHg MAP for 60 min. After this period, the RL group was resuscitated with Ringers lactate and the RL+NAC group was resuscitated with Ringers lactate combined with 150mg/Kg NAC. The control group animals were cannulated only. The animals were euthanized after 120 min of fluid resuscitation. Lung tissue samples were collected to evaluate the following: histopathology, TUNEL and imunohistochemical expression of caspase 3. RESULTS RL showed a greater number of cells stained by TUNEL than RL + NAC, but there was no change in caspase 3 expression in any group. CONCLUSION N-acetylcysteine associate to fluid resuscitation, after hemorrhagic shock, decreased cell death attenuating lung injury.

Sex-related differences in lung inflammation after brain death

BACKGROUND Donor sex has been suggested to be a factor influencing organ transplantation outcome. Sex hormones possess inflammatory and immune-mediating properties; therefore, immune responses may differ between males and females. Brain death (BD) affects organ function by numerous mechanisms including alterations in hemodynamics, hormonal changes, and increased systemic inflammation. In this study, we investigated sex-dependent differences in the evolution of lung inflammation in a rat model of BD. MATERIALS AND METHODS BD was induced by a sudden increase in intracranial pressure by rapidly inflating a balloon catheter inserted into the intracranial space. Groups of male, female, and ovariectomized (OVx) female rats were used. Lung vascular permeability, inducible nitric oxide synthase, and intercellular adhesion molecule 1 expression were analyzed 6 h after BD. Serum female sex hormones, vascular endothelial growth factor, and cytokine-induced neutrophil chemoattractant 1 levels were also quantified. Lung sections were analyzed by histology. RESULTS After 6 h of BD, serum estradiol and progesterone concentrations in female rats were significantly reduced. Lung microvascular permeability was increased in females compared to males. Cytokine-induced neutrophil chemoattractant 1 and vascular endothelial growth factor concentrations were increased in female rats compared to males. Furthermore, female rats showed higher levels of leukocyte infiltration and inducible nitric oxide synthase expression in the lung parenchyma. CONCLUSIONS Our results indicate that the more severe lung inflammation in female animals after BD might be related to acute estradiol reduction. Based on our findings, we believe that, in a future study, a group of female treated with estradiol after BD could indicate a possible therapy for the control of lung inflammation in the female donor.

Mesenteric hypoperfusion and inflammation induced by brain death are not affected by inhibition of the autonomic storm in rats

OBJECTIVES: Brain death is typically followed by autonomic changes that lead to hemodynamic instability, which is likely associated with microcirculatory dysfunction and inflammation. We evaluated the role of the microcirculation in the hemodynamic and inflammatory events that occur after brain death and the effects of autonomic storm inhibition via thoracic epidural blockade on mesenteric microcirculatory changes and inflammatory responses. METHODS: Male Wistar rats were anesthetized and mechanically ventilated. Brain death was induced via intracranial balloon inflation. Bupivacaine (brain death-thoracic epidural blockade group) or saline (brain death group) infusion via an epidural catheter was initiated immediately before brain death induction. Sham-operated animals were used as controls (SH group). The mesenteric microcirculation was analyzed via intravital microscopy, and the expression of adhesion molecules was evaluated via immunohistochemistry 180 min after brain death induction. RESULTS: A significant difference in mean arterial pressure behavior was observed between the brain death-thoracic epidural blockade group and the other groups, indicating that the former group experienced autonomic storm inhibition. However, the proportion of perfused small vessels in the brain death-thoracic epidural blockade group was similar to or lower than that in the brain death and SH groups, respectively. The expression of intercellular adhesion molecule 1 was similar between the brain death-thoracic epidural blockade and brain death groups but was significantly lower in the SH group than in the other two groups. The number of migrating leukocytes in the perivascular tissue followed the same trend for all groups. CONCLUSIONS: Although thoracic epidural blockade effectively inhibited the autonomic storm, it did not affect mesenteric hypoperfusion or inflammation induced by brain death.

Acute administration of oestradiol or progesterone in a spinal cord ischaemia–reperfusion model in rats

OBJECTIVES Despite research into protective pharmacological adjuncts, paraplegia persists as a dreaded complication after thoracic and thoracoabdominal aortic interventions. Reports on gender-related neurological outcomes after ischaemic and traumatic brain injuries have led to increased interest in hormonal neuroprotective effects and have generated other studies seeking to prove the neuroprotective effects of the therapeutic administration of 17β-oestradiol and of progesterone. We hypothesised that acute administration of oestradiol or progesterone would prevent or attenuate spinal cord ischaemic injury induced by occlusion of the descending thoracic aorta. METHODS Male rats were divided into groups receiving 280 µg/kg of 17β-oestradiol or 4 mg/kg of progesterone or vehicle 30 min before transitory endovascular occlusion of the proximal descending thoracic aorta for 12 min. Hindlimb motor function was assessed by a functional grading scale (that of Basso, Beattie and Bresnahan) for 14 days after reperfusion. On the 14th day, a segment of the thoracolumbar spinal cord was harvested and prepared for histological and immunohistochemical analyses. RESULTS There was significant impairment of the motor function of the hindlimb in the 3 study groups, with partial improvement noticed over time, but no difference was detected between the groups. On Day 1 of assessment, the 17β-oestradiol group had a functional score of 9.8 (0.0-16.5); the progesterone group, a score of 0.0 (0-17.1) and the control group, a score of 6.5 (0-16.9); on the 14th day, the 17β-oestradiol group had a functional score of 18.0 (4.4-19.4); the progesterone group had a score of 7.5 (0-18.5) and the control group had a score of 17.0 (0-19.9). Analysis of the grey matter showed that the number of viable neurons per section was not different between the study groups on the 14th day. Immunostaining of the spinal cord grey matter was also similar among the 3 groups. CONCLUSIONS Acute administration of oestradiol or of progesterone 30 min before transitory occlusion of the proximal descending thoracic aorta of male rats could not prevent or attenuate spinal cord ischaemic injury based on an analysis of functional and histological outcomes.

Sex differences on solid organ histological characteristics after brain death1.

PURPOSE To investigate gender differences in the evolution of the inflammatory process in rats subjected to brain death (BD). METHODS Adult Wistar rats were divided into three groups: female; ovariectomized female; and male rats. BD was induced using intracranial balloon inflation and confirmed by maximal pupil dilatation, apnea, absence of reflex, and drop of mean arterial pressure. Six hours after BD, histological evaluation was performed in lungs, heart, liver and kidneys, and levels of inflammatory proteins, estrogen, progesterone, and corticosterone were determined in plasma. RESULTS In the lungs, females presented more leukocyte infiltration compared to males (p<0.01). Ovariectomized female rat lungs were more hemorrhagic compared to other groups (p<0.001). In the heart, females had higher leukocyte infiltration and tissue edema compared to males (p<0.05). In the liver and kidneys, there were no differences among groups. In female group estradiol and progesterone were sharply reduced 6 hours after BD (p<0.001) to values observed in ovariectomized females and males. Corticosterone levels were similar. CONCLUSIONS Sex hormones influence the development of inflammation and the status of organs. The increased inflammation in lungs and heart of female rats might be associated with the acute reduction in female hormones triggered by BD.

17β-Estradiol protects against lung injuries after brain death in male rats

BACKGROUND Brain death elicits microvascular dysfunction and inflammation, and thereby compromises lung viability for transplantation. As 17β-estradiol was shown to be anti-inflammatory and vascular protective, we investigated its effects on lung injury after brain death in male rats. METHODS Wistar rats were assigned to: sham-operation by trepanation only (SH, n = 7); brain death (BD, n = 7); administration of 17β-estradiol (280 μg/kg, iv) at 60 minutes after brain death (BD-E2, n = 7). Experiments were performed 180 minutes thereafter. Histopathological changes in the lung were evaluated by histomorphometry. Gene expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and endothelin-1 was measured by real-time polymerase chain reaction. Protein expression of NO synthases, endothelin-1, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), BCL-2, and caspase 3 was assessed by immunohistochemistry. Cytokines were quantified by enzyme-linked immunosorbent assay. RESULTS Treatment with 17β-estradiol after brain death decreased lung edema and hemorrhage (p < 0.0001), and serum levels of cytokine-induced neutrophil chemoattractant-1 (CINC-1; p = 0.0020). iNOS (p < 0.0001) and VCAM-1 (p < 0.0001) also diminished at protein levels, while eNOS accumulated (p = 0.0002). However, gene expression of iNOS, eNOS, and endothelin-1 was comparable among groups, as was protein expression of endothelin-1, ICAM-1, BCL-2, and caspase 3. CONCLUSIONS 17β-Estradiol effectively reduces lung injury in brain-dead rats mainly due to its ability to regulate NO synthases. Thus, the drug may improve lung viability for transplantation.

Differential Effects of Brain Death on Rat Microcirculation and Intestinal Inflammation: Female Versus Male

Brain death (BD) affects organs by multiple mechanisms related to hemodynamic effects, hormonal changes, and the systemic inflammatory response, which reduce organ function and viability. BD reduces microcirculatory perfusion in rat mesentery; this disturbance is also observed in the pancreas and lungs. Sex hormones can affect microcirculatory function, altering tissue perfusion and influencing the inflammatory process. Here, we present differences between sexes in the microcirculatory alterations generated by BD and in inflammatory infiltrate. Male, female, and ovariectomized-female Wistar rats were submitted to BD by intracranial balloon catheter sudden inflation. BD was confirmed by maximally dilated and fixed pupils, apnea, absence of reflexes, and a drop in mean arterial pressure. Perfusion and flow of the mesenteric microcirculation were analyzed. Intestinal myeloperoxidase activity and leukocyte infiltration were quantified. ELISA quantified serum estradiol, corticosterone, and inflammatory mediators, whereas expression of eNOS, endothelin, and endothelial adhesion molecule was measured by immunohistochemistry. Male rats presented lower percentages of mesenteric perfused microvessels and reduced blood flow compared to females. The female group presented higher eNOS and endothelin expression. Leukocyte infiltration into intestinal walls was higher in females in comparison to that in males. Moreover, the female group showed higher mesenteric vessel ICAM-1 expression than males, whereas serum TNF-α, IL-1β, and IL-10 levels did not differ between sexes. The high estradiol concentration before BD and high eNOS expression apparently favored the maintenance of microvascular perfusion/flow; however, BD caused an acute reduction of female sex hormone concentration and higher ICAM-1 level; thus, the proinflammatory organ status after BD is favored.