Impact of hypotension and global hypoperfusion in postoperative delirium: a pilot study in older adults undergoing open colon surgery / Impacto da hipotensão e hipoperfusão global sobre o delírio pós-operatório: um estudo piloto com idosos submetidos à cirurgia aberta do cólon

Abstract Background: Post-operative delirium is a serious complication in patients undergoing major abdominal surgery. It remains unclear whether peri-operative hemodynamic and perfusion variables affect the risk for postoperative delirium. The objective of this pilot study was to evaluate the association between perfusion and hemodynamics peri-operative with the appearance of post-operative delirium. Methods: Prospective cohort study of adults 60 years or older undergoing elective open colon surgery. Multimodal hemodynamic and perfusion variables were monitored, including central venous oxygenation (ScvO2), lactate levels, and non-invasive cerebral oxygenation (rSO2), according to a standard anesthesia protocol. Fisher's exact test or Student's t-test were used to compare patients who developed post-operative delirium with those who did not (p < 0.05). Results: We studied 28 patients, age 73 ± 7 years, 60.7% female. Two patients developed post-operative delirium (7.1%). These two patients had fewer years of education than those without delirium (p = 0.031). None of the peri-operative blood pressure variables were associated with incidence of post-operative delirium. In terms of perfusion parameters, postoperative ScvO2 was lower in the delirium than the non-delirium group, without reaching statistical significance (65 ± 10% vs. 74 ± 5%; p = 0.08), but the delta-ScvO2 (the difference between means post-operative and intra-operative) was associated with post-operative delirium (p = 0.043). Post-operative lactate and rSO2 variables were not associated with delirium. Conclusions: Our pilot study suggests an association between delta ScvO2 and post-operative delirium, and a tendency to lower post-operative ScvO2 in patients who developed delirium. Further studies are necessary to elucidate this association.

Resumo Justificativa: O delírio pós-operatório é uma complicação séria em pacientes submetidos à cirurgia abdominal de grande porte. Ainda não está claro se as variáveis hemodinâmicas e de perfusão no período perioperatório afetam o risco de delírio pós-operatório. O objetivo deste estudo piloto foi avaliar a associação entre perfusão e hemodinâmica no perioperatório com o surgimento de delírio pós-operatório. Métodos: Estudo prospectivo de coorte de adultos com 60 anos ou mais, submetidos à cirurgia eletiva aberta do cólon. As variáveis multimodais de hemodinâmica e perfusão foram monitoradas, inclusive oxigenação venosa central (ScvO2), níveis de lactato e oxigenação cerebral não invasiva (rSO2), de acordo com um protocolo-padrão de anestesia. O teste exato de Fisher ou o teste t de Student foram usados para comparar os pacientes que desenvolveram delírio pós-operatório com aqueles que não desenvolveram p < 0,05. Resultados: Avaliamos 28 pacientes, 73 ± 7 anos, 60,7% do sexo feminino. Dois pacientes desenvolveram delírio pós-operatório (7,1%). Esses dois pacientes tinham menos anos de escolaridade do que aqueles sem delírio pós-operatório (p = 0,031). Nenhuma das variáveis de pressão arterial no perioperatório foi associada à incidência de delírio. Quanto aos parâmetros de perfusão, ScvO2 foi menor no grupo que apresentou delírio pós-operatório do que no grupo que não apresentou delírio, sem atingir significância estatística (65 ± 10% vs. 74 ± 5%; p = 0,08), mas o delta-ScvO2 (a diferença entre as médias no pós-operatório e intraoperatório) foi associado ao delírio (p = 0,043). As variáveis de lactato e rSO2 no pós-operatório não foram associadas ao delírio. Conclusões: Nosso estudo piloto sugere uma associação entre delta-ScvO2 e delírio e uma tendência à diminuição da ScvO2 no pós-operatório de pacientes com delírio. Estudos adicionais são necessários para elucidar essa associação.

[Impact of hypotension and global hypoperfusion in postoperative delirium: a pilot study in older adults undergoing open colon surgery]. / Impacto da hipotensão e hipoperfusão global sobre o delírio pós­operatório: um estudo piloto com idosos submetidos à cirurgia aberta do cólon.

BACKGROUND: Post-operative delirium is a serious complication in patients undergoing major abdominal surgery. It remains unclear whether peri-operative hemodynamic and perfusion variables affect the risk for postoperative delirium. The objective of this pilot study was to evaluate the association between perfusion and hemodynamics peri-operative with the appearance of post-operative delirium. METHODS: Prospective cohort study of adults 60 years or older undergoing elective open colon surgery. Multimodal hemodynamic and perfusion variables were monitored, including central venous oxygenation (ScvO2), lactate levels, and non-invasive cerebral oxygenation (rSO2), according to a standard anesthesia protocol. Fisher's exact test or Student's t-test were used to compare patients who developed post-operative delirium with those who did not (p<0.05). RESULTS: We studied 28 patients, age 73±7 years, 60.7% female. Two patients developed post-operative delirium (7.1%). These two patients had fewer years of education than those without delirium (p=0.031). None of the peri-operative blood pressure variables were associated with incidence of post-operative delirium. In terms of perfusion parameters, postoperative ScvO2 was lower in the delirium than the non-delirium group, without reaching statistical significance (65±10% vs. 74±5%; p=0.08), but the delta-ScvO2 (the difference between means post-operative and intra-operative) was associated with post-operative delirium (p=0.043). Post-operative lactate and rSO2 variables were not associated with delirium. CONCLUSIONS: Our pilot study suggests an association between delta ScvO2 and post-operative delirium, and a tendency to lower post-operative ScvO2 in patients who developed delirium. Further studies are necessary to elucidate this association.

The pro-inflammatory cytokines, IL-1beta and TNF-alpha, inhibit intestinal alkaline phosphatase gene expression.

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.

Thyroid hormone positively regulates the enterocyte differentiation marker intestinal alkaline phosphatase gene via an atypical response element.

Thyroid hormone (T3) is a critical regulator of intestinal epithelial development and homeostasis, but its mechanism of action within the gut is not well understood. We have examined the molecular mechanisms underlying the T3 activation of the enterocyte differentiation marker intestinal alkaline phosphatase (IAP) gene. RT-PCR and Western blotting showed that thyroid hormone receptors TRalpha1 and TRbeta1 were expressed in human colorectal adenocarcinoma Caco-2 cells. Northern blotting detected expression of two IAP transcripts, which were increased approximately 3-fold in response to T3. Transient transfection studies with luciferase reporter plasmids carrying various internal and 5' deletion mutations of the IAP promoter localized a putative thyroid hormone response element (TRE) to a region approximately 620 nucleotides upstream (-620) of the ATG start codon. EMSAs using TRalpha1-retinoid X receptor alpha (RXRalpha) on sequential 5' and 3' single nucleotide deletions defined the TRE between -632 and -612 (5'-TTGAACTCAgccTGAGGTTAC-3'). Compared with the consensus TRE, the IAP-TRE is novel in that it contains an everted repeat of two nonamers (not hexamers) separated by three nucleotides. Neither TRalpha1 nor RXRalpha binds to the IAP-TRE; however, TRbeta1 binds to this TRE with minimal affinity. In the presence of TR and RXRalpha, only the TR-RXRalpha heterodimer binds to the IAP-TRE. Mutagenesis of either nonamer abolishes the biological activity of IAP promoter. We have thus identified a novel response element that appears to mediate the T3-induced activation of the enterocyte differentiation marker, intestinal alkaline phosphatase.

Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor.

We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.

Convergence of the thyroid hormone and gut-enriched Krüppel-like factor pathways in the context of enterocyte differentiation.

The gut-enriched Krüppel-like factor (KLF4) and the ligand-bound thyroid hormone receptor (TR) have each been shown to play a critical role in mammalian gut development and differentiation. We investigated an interrelationship between these two presumably independent pathways using the differentiation marker gene, intestinal alkaline phosphatase (IAP). Transient transfections were performed in Cos-7 cells using luciferase reporter plasmids containing a 2.5 kb segment of the proximal human IAP 5' regulatory region, as well as multiple deletions. Cells were cotransfected with TR and/or KLF4 expression vectors and treated+/-100 nmol/L thyroid hormone (T3). IAP reporter gene transactivation was increased independently by KLF4 (ninefold) and ligand-bound TR beta 1 (sevenfold). Cells cotransfected with KLF4 and TR beta 1 in the presence of T3 showed synergistic activation (70-fold). A similar pattern was seen with the other T3 receptor isoform, TR alpha 1. The synergistic effect was lost with deletions of the T3 and KLF4 response elements in the IAP promoter and was completely or partially abolished in the case of mutant KLF4 expression vectors. The thyroid hormone receptor complex and KLF4 synergistically activate the enterocyte differentiation marker gene IAP, suggesting a previously unrecognized interrelationship between these two transcription factor pathways.

Transcriptional activation of the enterocyte differentiation marker intestinal alkaline phosphatase is associated with changes in the acetylation state of histone H3 at a specific site within its promoter region in vitro.

Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between -378 and -303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between -1378 and -1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program.