Study of laser fluorescence spectroscopy in livers of rats with hypothermic ischemia.

PURPOSE: After partial hepatectomy (PH), the remaining liver (RL) undergoes regenerative response proportional to the host. Limited literature exists on hepatic viability after tissue injury during hypothermic preservation. Spectroscopy measures cellular fluorescence and is explored for tissue characterization and parameter investigation. This study aimed to assess fluorescence analysis (spectroscopy) in evaluating liver viability and its relationship with hepatic tissue regeneration 24 hours after PH. Additionally, we analyzed liver regeneration in RL after 70% partial hepatectomy under hypothermic conditions with laser irradiation. METHODS: Fifty-six Wistar rats were divided into four groups: total non-perfused liver (control), total perfused liver, partial hepatectomy "in situ", and partial hepatectomy "ex situ". Tissue analysis was performed at 0 and 24 hours using spectroscopy with laser devices emitting at 532 (green) and 405 nm (violet). RESULTS: Spectroscopy identified tissue viability based on consistent results with Ki67 staining. The fluorescence spectra and Ki67 analysis displayed similar patterns, linking proliferative activity and absorption intensity. CONCLUSIONS: Fluorescence spectroscopy proves to be promising for real-time analysis of cellular activity and viability. Metabolic activity was observed in groups of live animals and hypothermically preserved samples, indicating cellular function even under blood deprivation and hypothermic conditions.

Study of laser fluorescence spectroscopy in livers of rats with hypothermic ischemia

Purpose: After partial hepatectomy (PH), the remaining liver (RL) undergoes regenerative response proportional to the host. Limited literature exists on hepatic viability after tissue injury during hypothermic preservation. Spectroscopy measures cellular fluorescence and is explored for tissue characterization and parameter investigation. This study aimed to assess fluorescence analysis (spectroscopy) in evaluating liver viability and its relationship with hepatic tissue regeneration 24 hours after PH. Additionally, we analyzed liver regeneration in RL after 70% partial hepatectomy under hypothermic conditions with laser irradiation. Methods: Fifty-six Wistar rats were divided into four groups: total non-perfused liver (control), total perfused liver, partial hepatectomy "in situ", and partial hepatectomy "ex situ". Tissue analysis was performed at 0 and 24 hours using spectroscopy with laser devices emitting at 532 (green) and 405 nm (violet). Results: Spectroscopy identified tissue viability based on consistent results with Ki67 staining. The fluorescence spectra and Ki67 analysis displayed similar patterns, linking proliferative activity and absorption intensity. Conclusions: Fluorescence spectroscopy proves to be promising for real-time analysis of cellular activity and viability. Metabolic activity was observed in groups of live animals and hypothermically preserved samples, indicating cellular function even under blood deprivation and hypothermic conditions.

Evaluation of Indigo carmine on hepatic ischemia and reperfusion injury.

PURPOSE: To evaluate the effects of treatment with Indigo Carmine (IC) on rat livers subjected to ischemia-reperfusion injury. METHODS: The animals were subdivided into 4 groups: 1.SHAM group(SH) - saline; 2.SHAM group with IC-2mg/Kg(SHIC); 3.IR group - rats submitted to ischemia and reperfusion with saline(IR); 4.IR group with IC-2mg/Kg(IRIC). The IR protocol consists of liver exposure and administration of drug or saline intravenously, followed by 60 minutes of ischemia and 15 of reperfusion. Liver samples were collected for biochemical analysis. RESULTS: State 3 of mitochondrial respiration showed a significant worsening of the IRIC group in relation to all others. State 4 showed a difference between IRIC and SHIC. The Respiratory Control Ratio showed statistical decrease in IR and IRIC versus Sham. The osmotic swelling showed significant difference between SHxIR; SHICxIRIC and SHxIRIC. There was a significant increase in ALT in the IRIC group in relation to all the others. Concerning the nitrate dosage, there was a decrease in the group treated with IC(IRxIRIC). There was no difference regarding the dosage of Malondialdehyde. CONCLUSION: IC was not able to protect mitochondria from IR injury and proved to be a potentiating agent, acting in synergy with the IR injury promoting damage to the hepatocyte membranes.

Comparative study between fructose 1-6 bisphosphate and histidine-tryptophan-ketoglutarate in liver preservation in rats submitted to total cold ischemia.

Purpose To compare Fructose-1,6-Bisphosphate (FBP) to Histidine-Tryptophan-Ketoglutarate (HTK) in liver preservation at cold ischemia. Methods Male rats (Sprague-Dawley: 280-340g) divided into three groups (n=7): Control; Fructose-1,6-bisphosphate (FBP); Histidine-Tryptophan-Ketoglutarate (HTK). Animals underwent laparotomy-thoracotomy for perfusion of livers with saline. Livers were removed and deposited into solutions. Mitochondria were isolated to determine State 3 (S3), State 4 (S4), Respiratory Control Ratio (RCR) and Swelling (S). Liver enzymes (AST, ALT, LDH) were determined in solution. At tissue, Malondialdehyde (MDA) and Nitrate (NOx) were determined. All parameters were analyzed at 0.6 and 24 hours of hypothermic preservation. Statistics analysis were made by Mann-Whitney test (p<0.05). Results Regarding ALT, there was a difference between FBP-6h/HTK-6h, lower in HTK. Regarding AST, there was a significant difference between FBP-24h/HTK-24h, lower in FBP. Regarding NOx, there was a difference between 0h and 6h, as well as 0h and 24h for both solutions. Regarding S3, there was a significant difference in 24h compared to Control-0h for both solutions, and a significant difference between FBP-6h/FBP-24h. Regarding S4, there was a difference between Control-0h/HTK-24h and FBP-24h/HTK-24h, higher in HTK. There was a difference between Control-0h/FBP-24h for Swelling, higher in FBP. Conclusion Fructose-1,6-Bisphosphate showed better performance at nitrate and aspartate aminotransferase compared to histidine-tryptophan-ketoglutarate.

Ischemia/Reperfusion Injury Revisited: An Overview of the Latest Pharmacological Strategies.

Ischemia/reperfusion injury (IRI) permeates a variety of diseases and is a ubiquitous concern in every transplantation proceeding, from whole organs to modest grafts. Given its significance, efforts to evade the damaging effects of both ischemia and reperfusion are abundant in the literature and they consist of several strategies, such as applying pre-ischemic conditioning protocols, improving protection from preservation solutions, thus providing extended cold ischemia time and so on. In this review, we describe many of the latest pharmacological approaches that have been proven effective against IRI, while also revisiting well-established concepts and presenting recent pathophysiological findings in this ever-expanding field. A plethora of promising protocols has emerged in the last few years. They have been showing exciting results regarding protection against IRI by employing drugs that engage several strategies, such as modulating cell-surviving pathways, evading oxidative damage, physically protecting cell membrane integrity, and enhancing cell energetics.

Chronic Liver Disease Questionnaire as a Tool to Evaluate the Quality of Life in Liver Transplant Candidates.

INTRODUCTION:: The advanced stage of liver disease causes impairments in the quality of life due to the physiological symptoms, besides the social and emotional stress. The aim of this study was to evaluate the quality of life of candidates for liver transplantation in specialized center in the interior of the state of São Paulo, Brazil. METHODS:: An observational study was carried out, with a quantitative approach. The sample was of convenience with the participation of 50 candidates for liver transplantation. Demographic characterization data, clinical data, and the Chronic Liver Disease Questionnaire were used to evaluate the quality of life. RESULTS:: The majority of the participants were male (68%), married (76%), with an average age of 55.7 years and average income of 2 to 3 minimum wages (42%). The main cause of liver disease was alcoholism (34%), mean time on the waiting list was 247.8 days, and mean model for end-stage liver disease was 20.5 points. The mean quality of life score was 2.9 points (standard deviation = 1.0) and the analysis of the 6 domains showed greater impairment of fatigue (2.2), activity (2.7), and concern (2.9) and better evaluation of systemic symptoms (3.4) and emotion (3.3). CONCLUSION:: Quality of life is impaired in most of the participants, indicating the need for greater attention and evaluation in the physical, mental, and social scope of this clientele.

The cardiopulmonary bypass and cancer dissemination puzzle1.

Early cancer diagnosis, new therapies that increased survival of patients, besides the increasingly elderly population are some factors would be associated with possible cancer dissemination in patients under cardiopulmonary bypass (CPB) cardiac surgery. Also, the benefits, and risks, regarding long-term survival, have not yet been established. Therefore, cardiac surgery morbimortality may be superior in patients with cancer disease. Also, immunologic and inflammatory changes secondary to CPB can also increase tumor recurrence. After a brief introduction and CPB immunologic the two main topic subjects included: 1) Combined heart surgery and lung resection and; 2) Possible influence of neoplasia type. After observing the relative literature scarcity, we keep the opinion that "CPB has a modest association with cancer progression" and that "CPB and cancer dissemination should be a logical but unlikely association."

Evaluation of the therapeutic effect of methylene blue on the liver of rats submitted to ischemia and reperfusion

Abstract Purpose: To analyze the effect of methylene blue (MB) therapy during the liver ischemia-reperfusion injury (I/R) process. Methods: Thirty-five male Wistar rats were used, (70%) submitted to partial ischemia (IR) or not (NIR) (30%) were obtained from the same animal. These animals were divided into six groups: 1) Sham (SH), 2) Sham with MB (SH-MB); 3) I/R, submitted to 60 minutes of partial ischemia and 15 minutes of reperfusion; 4) NI/R, without I/R obtained from the same animal of group I/R; 5) I/R-MB submitted to I/R and MB and 6) NI/R-MB, without I/R. Mitochondrial function was evaluated. Osmotic swelling of mitochondria as well as the determination of malondialdehyde (MDA) was evaluated. Serum (ALT/AST) dosages were also performed. MB was used at the concentration of 15mg/kg, 15 minutes before hepatic reperfusion. Statistical analysis was done by the Mann Whitney test at 5%. Results: State 3 shows inhibition in all ischemic groups. State 4 was increased in all groups, except the I/R-MB and NI/R-MB groups. RCR showed a decrease in all I/R and NI/R groups. Mitochondrial osmotic swelling showed an increase in all I/R NI/R groups in the presence or absence of MB. About MDA, there was a decrease in SH values in the presence of MB and this decrease was maintained in the I/R group. AST levels were increased in all ischemic with or without MB. Conclusions: The methylene blue was not able to restore the mitochondrial parameters studied. Also, it was able to decrease lipid peroxidation, preventing the formation of reactive oxygen species.

The cardiopulmonary bypass and cancer dissemination puzzle

Abstract Early cancer diagnosis, new therapies that increased survival of patients, besides the increasingly elderly population are some factors would be associated with possible cancer dissemination in patients under cardiopulmonary bypass (CPB) cardiac surgery. Also, the benefits, and risks, regarding long-term survival, have not yet been established. Therefore, cardiac surgery morbimortality may be superior in patients with cancer disease. Also, immunologic and inflammatory changes secondary to CPB can also increase tumor recurrence. After a brief introduction and CPB immunologic the two main topic subjects included: 1) Combined heart surgery and lung resection and; 2) Possible influence of neoplasia type. After observing the relative literature scarcity, we keep the opinion that "CPB has a modest association with cancer progression" and that "CPB and cancer dissemination should be a logical but unlikely association."

Rivastigmine prevents injury induced by ischemia and reperfusion in rat liver.

PURPOSE: To evaluate whether pre-treatment with rivastigmine is able to attenuate the I/R induced lesions in rat liver. METHODS: SHAM animals or those submitted to I/R, non-treated or pre-treated with rivastigminine (2mg/kg) either 50 or 15 minutes before ischemia, were used. After I/R protocol, these animals were killed and their livers were harvested to measurement of the mitochondrial swelling as well as the malondialdehyde (MDA), nitrite and nitrate tissue concentration. Blood was also harvested for serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) determinations. RESULTS: I/R promoted a significant increase of mitochondrial swelling in the studied animals. This increase of mitochondrial swelling was partially prevented by rivastigmine, but only if administered 50 minutes before ischemia. No significant modification of MDA, nitrite or nitrate tissue concentrations was observed in consequence of I/R, followed or not by rivastigmine treatments. In addition, I/R elevated both AST and ALT. These elevations of serum enzymes were not reversed by the different rivastigmine treatments. CONCLUSIONS: Rivastigmine administered 50 minutes before ischemia attenuates I/R-induced mitochondrial swelling, that indicates liver injury. This protective effect may be related to a greater stimulation of α7nAChR present in the Kupffer cells by the non-methabolized ACh, leading to an attenuation of I/R-induced inflammation.

Liver ischemia and reperfusion injury. Pathophysiology and new horizons in preconditioning and therapy.

It is well known that during hepatic operative procedures, it is often critical that the irrigation is interrupted to avoid possible bleeding, blood transfusions, variable intensities, and their short and long-term consequences. It was believed in the past that the flow interruption should not exceed 20 minutes, which limited the use of this maneuver. However, it has been postulated that ischemia could be maintained for more than 60 minutes in healthy livers. The present paper review includes: 1) A brief introduction to justify the rationale of the review design; 2) Aspects of the pathophysiology of the three stages of the liver ischemia-reperfusion injury; 3) The innate and acquired immunity; 4) Oxidative stress; 5) Apoptosis and autophagy, Some essential biomarkers (Tumor Necrosis Factor-α, nitric oxide, metalloproteinases); and, finally; 6) Preventive ("cheating") strategies, non-pharmacological and pharmacological options to treat the liver IR injury.

Rivastigmine prevents injury induced by ischemia and reperfusion in rat liver

Abstract Purpose: To evaluate whether pre-treatment with rivastigmine is able to attenuate the I/R induced lesions in rat liver. Methods: SHAM animals or those submitted to I/R, non-treated or pre-treated with rivastigminine (2mg/kg) either 50 or 15 minutes before ischemia, were used. After I/R protocol, these animals were killed and their livers were harvested to measurement of the mitochondrial swelling as well as the malondialdehyde (MDA), nitrite and nitrate tissue concentration. Blood was also harvested for serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) determinations. Results: I/R promoted a significant increase of mitochondrial swelling in the studied animals. This increase of mitochondrial swelling was partially prevented by rivastigmine, but only if administered 50 minutes before ischemia. No significant modification of MDA, nitrite or nitrate tissue concentrations was observed in consequence of I/R, followed or not by rivastigmine treatments. In addition, I/R elevated both AST and ALT. These elevations of serum enzymes were not reversed by the different rivastigmine treatments. Conclusions: Rivastigmine administered 50 minutes before ischemia attenuates I/R-induced mitochondrial swelling, that indicates liver injury. This protective effect may be related to a greater stimulation of α7nAChR present in the Kupffer cells by the non-methabolized ACh, leading to an attenuation of I/R-induced inflammation.

Liver ischemia and reperfusion injury. Pathophysiology and new horizons in preconditioning and therapy

Abstract It is well known that during hepatic operative procedures, it is often critical that the irrigation is interrupted to avoid possible bleeding, blood transfusions, variable intensities, and their short and long-term consequences. It was believed in the past that the flow interruption should not exceed 20 minutes, which limited the use of this maneuver. However, it has been postulated that ischemia could be maintained for more than 60 minutes in healthy livers. The present paper review includes: 1) A brief introduction to justify the rationale of the review design; 2) Aspects of the pathophysiology of the three stages of the liver ischemia-reperfusion injury; 3) The innate and acquired immunity; 4) Oxidative stress; 5) Apoptosis and autophagy, Some essential biomarkers (Tumor Necrosis Factor-α, nitric oxide, metalloproteinases); and, finally; 6) Preventive ("cheating") strategies, non-pharmacological and pharmacological options to treat the liver IR injury.

Prophylactic application of laser light restores L-FABP expression in the livers of rats submitted to partial ischemia.

OBJECTIVES: The objective of the present study was to evaluate the protective effect of pre-conditioning treatment with laser light on hepatic injury in rats submitted to partial ischemia using mitochondrial function and liver fatty acid binding protein as markers. METHODS: Rats were divided into four groups (n=5): 1) Control, 2) Control + Laser, 3) Partial Ischemia and 4) Partial Ischemia + Laser. Ischemia was induced by clamping the hepatic pedicle of the left and middle lobes of the liver for 60 minutes. Laser light at 660 nm was applied to the liver immediately prior to the induction of ischemia at 22.5 J/cm2, with 30 seconds of illumination at five individual points. The animals were sacrificed after 30 minutes of reperfusion. Blood and liver tissues were collected for analysis of mitochondrial function, determination of malondialdehyde and analysis of fatty acid binding protein expression by Western blot. RESULTS: Mitochondrial function decreased in the Partial Ischemia group, especially during adenosine diphosphate-activated respiration (state 3), and the expression of fatty acid binding protein was also reduced. The application of laser light prevented bioenergetic changes and restored the expression of fatty acid binding protein. CONCLUSION: Prophylactic application of laser light to the livers of rats submitted to partial ischemia was found to have a protective effect in the liver, with normalization of both mitochondrial function and fatty acid binding protein tissue expression.

Evaluation of the therapeutic effect of methylene blue on the liver of rats submitted to ischemia and reperfusion.

PURPOSE: To analyze the effect of methylene blue (MB) therapy during the liver ischemia-reperfusion injury (I/R) process. METHODS: Thirty-five male Wistar rats were used, (70%) submitted to partial ischemia (IR) or not (NIR) (30%) were obtained from the same animal. These animals were divided into six groups: 1) Sham (SH), 2) Sham with MB (SH-MB); 3) I/R, submitted to 60 minutes of partial ischemia and 15 minutes of reperfusion; 4) NI/R, without I/R obtained from the same animal of group I/R; 5) I/R-MB submitted to I/R and MB and 6) NI/R-MB, without I/R. Mitochondrial function was evaluated. Osmotic swelling of mitochondria as well as the determination of malondialdehyde (MDA) was evaluated. Serum (ALT/AST) dosages were also performed. MB was used at the concentration of 15mg/kg, 15 minutes before hepatic reperfusion. Statistical analysis was done by the Mann Whitney test at 5%. RESULTS: State 3 shows inhibition in all ischemic groups. State 4 was increased in all groups, except the I/R-MB and NI/R-MB groups. RCR showed a decrease in all I/R and NI/R groups. Mitochondrial osmotic swelling showed an increase in all I/R NI/R groups in the presence or absence of MB. About MDA, there was a decrease in SH values in the presence of MB and this decrease was maintained in the I/R group. AST levels were increased in all ischemic with or without MB. CONCLUSIONS: The methylene blue was not able to restore the mitochondrial parameters studied. Also, it was able to decrease lipid peroxidation, preventing the formation of reactive oxygen species.

Use of Methylene Blue to Treat Hypovolemic Shock Followed by Ischemia-Reperfusion Injury in the Postoperative Orthotopic Liver Transplant Patient: A Case Report.

A 57-year-old female patient received elective liver transplant due to nonalcoholic steatohepatitis complicated by hepatocellular carcinoma. Her preoperative Model for End-Stage Liver Disease score was 11. The total transplant ischemic time was 10 hours and 35 minutes, and the warm ischemic time was 35 minutes. Even with aggressive fluid overload and use of high concentrations of vasoactive amines, the patient developed possible primary graft dysfunction with poor response to fluids and vasopressor support, suggesting vasoplegic syndrome. On the basis of the hypothesis of vasoplegic syndrome, the patient received methylene blue intravenously (100 mg bolus for 12 h/1.5 mg/kg). The catastrophic situation was controlled. The patient's urine output markedly improved, she was subsequently weaned from vasoactive support, and mechanical ventilation was discontinued 2 days later. The patient was discharged on the 20th postoperative day.

Prophylactic application of laser light restores L-FABP expression in the livers of rats submitted to partial ischemia

OBJECTIVES: The objective of the present study was to evaluate the protective effect of pre-conditioning treatment with laser light on hepatic injury in rats submitted to partial ischemia using mitochondrial function and liver fatty acid binding protein as markers. METHODS: Rats were divided into four groups (n=5): 1) Control, 2) Control + Laser, 3) Partial Ischemia and 4) Partial Ischemia + Laser. Ischemia was induced by clamping the hepatic pedicle of the left and middle lobes of the liver for 60 minutes. Laser light at 660 nm was applied to the liver immediately prior to the induction of ischemia at 22.5 J/cm2, with 30 seconds of illumination at five individual points. The animals were sacrificed after 30 minutes of reperfusion. Blood and liver tissues were collected for analysis of mitochondrial function, determination of malondialdehyde and analysis of fatty acid binding protein expression by Western blot. RESULTS: Mitochondrial function decreased in the Partial Ischemia group, especially during adenosine diphosphate-activated respiration (state 3), and the expression of fatty acid binding protein was also reduced. The application of laser light prevented bioenergetic changes and restored the expression of fatty acid binding protein. CONCLUSION: Prophylactic application of laser light to the livers of rats submitted to partial ischemia was found to have a protective effect in the liver, with normalization of both mitochondrial function and fatty acid binding protein tissue expression.

Information needs of family caregivers regarding liver transplant candidates. / Necessidades de informação do cuidador familiar de candidatos ao transplante de fígado.

Objective To assess the information needs of family caregivers of candidates on the waiting list for a liver transplant. Methods It is a cross-sectional study conducted in a transplant center in São Paulo State in the period between April and October of 2012. For the assessment of information needed, an instrument submitted to face and content value was used. The caregivers put 10 subjects in order according to their importance and the amount of interest they had in learning about each, prior to the transplant their family member would be subjected to. Sociodemographic characteristics were also recorded. For data analysis, descriptive statistics were used. Results 42 families participated in the study. The information need about liver disease complications, complications after transplantation and care needed after surgery had higher averages. Conclusions Knowing the information needs of caregivers is important to plan teaching-learning strategies aimed at improving assistance to patients and families in transplant programs.

Necessidades de informação do cuidador familiar de candidatos ao transplante de fígado / Necesidades de información de cuidador familiar de los candidatos a trasplante de hígado / Information needs of family caregivers regarding liver transplant candidates

RESUMO Objetivo Avaliar as necessidades de informação do cuidador familiar de candidatos, que aguardam em fila de espera a realização do transplante de fígado. Métodos Trata-se de estudo transversal, realizado em centro transplantador do interior paulista, no período de abril a outubro de 2012. Para a avaliação das necessidades de informação foi utilizado instrumento submetido à validade de face e conteúdo, o cuidador ordenou por importância, 10 assuntos que gostaria de aprender antes da realização do transplante pelo seu ente familiar, além do registro das características sociodemográficas. Para análise dos dados, utilizou-se a estatística descritiva. Resultados Participaram do estudo 42 familiares. As necessidades de informação sobre complicações da doença do fígado, complicações após o transplante e cuidados necessários no pós-operatório obtiveram maiores médias. Conclusões Conhecer as necessidades de informação dos cuidadores é relevante para planejar estratégias de ensino-aprendizagem, visando a melhoria da assistência aos pacientes e familiares em programas de transplantes.

RESUMEN Objetivo Evaluar las necesidades de información de los cuidadores familiares delos candidatos en la lista de espera para trasplante hepático. Método Se trata de un estudio transversal realizado en el centro de trasplante en São Paulo, de abril a octubre de 2012. Para evaluar las necesidades de información, se aplicó un cuestionario sometido a la validación de apariencia y contenido y pedimos la orden de la familia por orden de importancia, los 10 temas que le gustaría aprender antes de un trasplante de hígado para su único familiar, así como el registro de las características socio-demográficas. Para el análisis de datos, se utilizó la estadística descriptiva. Resultados Se recogieron datos de 42 familias. Las necesidades de información de las complicaciones de la enfermedad hepática, complicaciones después del trasplante y el cuidado necesario después de la cirugía fueron las que tuvieron el mayor promedio. Conclusión El conocimiento de las necesidades de información de los cuidadores es valioso para la planificación de estrategias para la enseñanza y el aprendizaje, con el fin de mejorar la atención al paciente y la familia en los programas de trasplantes de órganos.

ABSTRACT Objective To assess the information needs of family caregivers of candidates on the waiting list for a liver transplant. Methods It is a cross-sectional study conducted in a transplant center in São Paulo State in the period between April and October of 2012. For the assessment of information needed, an instrument submitted to face and content value was used. The caregivers put 10 subjects in order according to their importance and the amount of interest they had in learning about each, prior to the transplant their family member would be subjected to. Sociodemographic characteristics were also recorded. For data analysis, descriptive statistics were used. Results 42 families participated in the study. The information need about liver disease complications, complications after transplantation and care needed after surgery had higher averages. Conclusions Knowing the information needs of caregivers is important to plan teaching-learning strategies aimed at improving assistance to patients and families in transplant programs.