GABAerge Interneurontypen des Gyrus Dentatus der Ratte: eine morpho-physiologische Klassifizierung mittels Cluster-Analyse

Background: Cortical GABAergic interneurons (INs) have been shown to play a crucial role in regulating local cell excitability which is required for a coordinated ensemble activity and ultimately information processing. However, despite their importance for local circuit function, the morphological and physiological diversity of cortical GABAergic INs in many brain areas is still not fully understood. In the dentate gyrus (DG), the main entry point of spatial information to the hippocampus, several IN types with diverse morphological and physiological characteristics have been described, but a comprehensive morpho-physiological classification is still lacking. Methods: Whole-cell patch-clamp recordings were performed in acute hippocampal slices from vGAT-YFP rats from a total of 80 DG GABAergic INs, including 7 DG principal cells (4 dentate granule cells, 3 mossy cells) as reference, to assess their electrophysiological characteristics. Intracellularly labeled neurons were then visualized using 3-dimensional confocal microscopy followed by neuronal reconstructions and morphological analysis. Neurochemical marker expression was assessed in 64 of the 80 included GABAergic INs using immunohistochemistry. Finally, based on 38 morphological and 15 physiological parameters post hoc cluster analyses following Ward´s minimum variance method combined with preceding kernel principal component analyses were employed to classify GABAergic IN types of the DG. Results: GABAergic INs of the DG fall into distinct classes based on their morphological and physiological characteristics with 12 and 8 types identified by our analyses, respectively. However, a combined morpho-physiological classification revealed the greatest diversity with a total of 13 distinct GABAergic IN types comprising previously described and 6 novel IN classes. Neurochemical marker expression of morphophysiological types was non-uniform in some classes likely reflecting their diverse embryonic origins. Conclusion: Our results show that GABAergic INs of the DG divide into at least 13 distinct morpho-physiological types offering us deeper insights into circuit structure and function.Kortikale GABAerge Interneurone (IN) spielen eine zentrale Rolle in der Regulation lokaler neuronaler Aktivität, die für eine koordinierte Informationsweitergabe und schlussendlich Informationsverarbeitung unerlässlich ist. Trotz dieser zentralen Bedeutung für neuronale Netzwerke, ist die morphologische und physiologische Diversität kortikaler GABAerger IN noch über weite Strecken unerforscht. Im Gyrus dentatus, der Haupteintrittspforte des Hippocampus für räumliche Information, sind bislang einige GABAerge IN-Typen mit charakteristischen morphologischen und physiologischen Eigenschaften beschrieben worden, doch eine umfassende morphophysiologische Klassifizierung ist zum jetzigen Zeitpunkt noch ausstehend. Methodik: In Gehirnschnitten von vGAT-YFP positiven Ratten wurden insgesamt 80 GABAerge IN und 7 Hauptzellen (4 Körnerzellen und 3 Mooszellen) des Gyrus dentatus mittels „Patch-clamp“- Technik in „Whole-cell“- Konfiguration auf ihre physiologischen Eigenschaften hin untersucht. Anschließend erfolgte die Visualisierung der simultan intrazellulär angefärbten Neurone mittels 3-dimensionaler konfokaler Mikroskopie mit darauffolgenden neuronalen Rekonstruktionen und letztlich morphologischen Analysen. Immunhistochemische Untersuchungen in 64 der 80 eingeschlossenen GABAergen IN dienten der Feststellung der Expression neurochemischer Marker. Basierend auf insgesamt 38 morphologischen und 15 physiologischen Parametern erfolgten schließlich, verbunden mit vorausgegangenen Kernel Hauptkomponentenanalysen, hierarchische Cluster Analysen nach der „Ward´s minimum variance“- Methode zur Klassifikation GABAerger IN-Typen des Gyrus dentatus. Ergebnisse: GABAerge IN bilden, basierend auf ihren morphologischen, aber auch physiologischen Eigenschaften, charakteristische IN-Klassen mit insgesamt 12 morphologisch und 8 physiologisch identifizierten Typen. Die größte Diversität zeigte sich jedoch nach Integration morphologischer und physiologischer Eigenschaften mit insgesamt 13 morpho-physiologischen Typen. Dabei wurden bereits bekannte INKlassen bestätigt, aber auch 6 neue IN-Klassen identifiziert. Die Expression neurochemischer Marker zeigte sich in manchen IN-Klassen uneinheitlich, was möglicherweise auf unterschiedliche embryonale Ursprünge hinweist. Schlussfolgerung: GABAerge IN des Gyrus dentatus bilden mindestens 13 verschiedene morpho-physiologische IN-Typen, die dazu beitragen unser Verständnis bezüglich hippocampaler neuronaler Netzwerke und im weiteren Sinne Informationsverarbeitung weiter zu vertiefen

Interneuron Diversity in the Rat Dentate Gyrus: An Unbiased In Vitro Classification

Information processing in cortical circuits, including the hippocampus, relies on the dynamic control of neuronal activity by GABAergic interneurons (INs). INs form a heterogenous population with defined types displaying distinct morphological, molecular, and physiological characteristics. In the major input region of the hippocampus, the dentate gyrus (DG), a number of IN types have been described which provide synaptic inhibition to distinct compartments of excitatory principal cells (PrCs) and other INs. In this study, we perform an unbiased classification of GABAergic INs in the DG by combining in vitro whole‐cell patch‐clamp recordings, intracellular labeling, morphological analysis, and unsupervised cluster analysis to better define IN type diversity in this region. This analysis reveals that DG INs divide into at least 13 distinct morpho‐physiological types which reflect the complexity of the local IN network and serve as a basis for further network analyses

Sacral nerve stimulation in patients with ileal pouch-anal anastomosis

Purpose: Functional results after proctocolectomy and ileal pouch-anal anastomosis (IPAA) are generally good. However, some patients suffer from high stool frequency or fecal incontinence. Sacral nerve stimulation (SNS) may represent a therapeutic alternative in these patients, but little is known about indication and results. The aim of this study was to evaluate incontinence after IPAA and demonstrate SNS feasibility in these patients. Methods: This retrospective study includes patients who received a SNS between 1993 and 2020 for increased stool frequency or fecal incontinence after proctocolectomy with IPAA for ulcerative colitis. Proctocolectomy was performed in a two- or three-step approach with ileostomy closure as the last step. Demographic, follow-up data and functional results were obtained from the hospital database. Results: SNS was performed in 23 patients. Median follow-up time after SNS was 6.5 years (min. 4.2-max. 8.8). Two patients were lost to follow-up. The median time from ileostomy closure to SNS implantation was 6 years (min. 0.5-max. 14.5). Continence after SNS improved in 16 patients (69%) with a median St. Marks score for anal incontinence of 19 (min. 4-max. 22) before SNS compared to 4 (0-10) after SNS placement (p = 0.012). In seven patients, SNS therapy was not successful. Conclusion: SNS implantation improves symptoms in over two-thirds of patients suffering from high stool frequency or fecal incontinence after proctocolectomy with IPAA. Awareness of the beneficial effects of SNS should be increased in physicians involved in the management of these patients

Compartmental distribution of GABA-B receptor-mediated currents along the somato-dendritic axis of hippocampal principal cells

Activity of cortical principal cells is controlled by the GABAergic system providing inhibition in a compartmentalized manner along their somatodendritic axis. While GABAAR-mediated inhibitory synaptic transmission has been extensively characterized in hippocampal principal cells, little is known about the distribution of postsynaptic effects of GABABRs. In the present study, we have investigated the functional localization of GABABRs and their effector inwardly rectifying potassium (Kir3) channels by combining electrophysiological recordings in acute rat hippocampal slices, high-resolution immunoelectron microscopic analysis and single cell simulations. Pharmacologically-isolated slow inhibitory postsynaptic currents were elicited in the three major hippocampal principal cell types by endogenous GABA released by electrical stimulation, photolysis of caged-GABA, as well as the canonical agonist baclofen, with the highest amplitudes observed in the CA3. Spatially restricted currents were assessed along the axis of principal cells by uncaging GABA in the different hippocampal layers. GABABR-mediated currents were present along the entire somatodendritic axis of principal cells, but non-uniformly distributed: largest currents and the highest conductance densities determined in the simulations were consistently found on the distal apical dendrites. Finally, immunocytochemical localization of GABABRs and Kir3 channels showed that distributions overlap but their densities diverge, particularly on the basal dendrites of pyramidal cells. GABABRs current amplitudes and the conductance densities correlated better with Kir3 density, suggesting a bottlenecking effect defined by the effector channel. These data demonstrate a compartmentalized distribution of the GABABR-Kir3 signaling cascade and suggest differential control of synaptic transmission, dendritic integration and synaptic plasticity at afferent pathways onto hippocampal principal cells

Peptide Signatures for Prognostic Markers of Pancreatic Cancer by MALDI Mass Spectrometry Imaging

Simple Summary: Pancreatic cancer remains one of the most lethal tumor entities worldwide given its overall 5-year survival after diagnosis of 9%. Thus, further understanding of molecular changes to improve individual prognostic assessment as well as diagnostic and therapeutic advancement is crucial. The aim of this study was to investigate the feasibility of Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify specific peptide signatures linked to established prognostic parameters of pancreatic cancer. In a patient cohort of 18 patients with exocrine pancreatic cancer after tumor resection, MALDI imaging analysis additional to histopathological assessment was performed. Applying this method to tissue sections of the tumors, we were able to identify discriminative peptide signatures corresponding to nine proteins for the prognostic histopathological features lymphatic vessel invasion, lymph node metastasis and angioinvasion. This demonstrates the technical feasibility of MALDI-MSI to identify peptide signatures with prognostic value through the workflows used in this study. Abstract: Despite the overall poor prognosis of pancreatic cancer there is heterogeneity in clinical courses of tumors not assessed by conventional risk stratification. This yields the need of additional markers for proper assessment of prognosis and multimodal clinical management. We provide a proof of concept study evaluating the feasibility of Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify specific peptide signatures linked to prognostic parameters of pancreatic cancer. On 18 patients with exocrine pancreatic cancer after tumor resection, MALDI imaging analysis was performed additional to histopathological assessment. Principal component analysis (PCA) was used to explore discrimination of peptide signatures of prognostic histopathological features and receiver operator characteristic (ROC) to identify which specific m/z values are the most discriminative between the prognostic subgroups of patients. Out of 557 aligned m/z values discriminate peptide signatures for the prognostic histopathological features lymphatic vessel invasion (pL, 16 m/z values, eight proteins), nodal metastasis (pN, two m/z values, one protein) and angioinvasion (pV, 4 m/z values, two proteins) were identified. These results yield proof of concept that MALDI-MSI of pancreatic cancer tissue is feasible to identify peptide signatures of prognostic relevance and can augment risk assessment

Survival rates and prognostic factors in right- and left-sided colon cancer stage I–IV: an unselected retrospective single-center trial

Purpose: Colorectal cancer revealed over the last decades a remarkable shift with an increasing proportion of a right- compared to a left-sided tumor location. In the current study, we aimed to disclose clinicopathological differences between right- and left-sided colon cancer (rCC and lCC) with respect to mortality and outcome predictors. Methods: In total, 417 patients with colon cancer stage I-IV were analyzed in the present retrospective single-center study. Survival rates were assessed using the Kaplan-Meier method and uni/multivariate analyses were performed with a Cox proportional hazards regression model. Results: Our study showed no significant difference of the overall survival between rCC and lCC stage I-IV (p = 0.354). Multivariate analysis revealed in the rCC cohort the worst outcome for ASA (American Society of Anesthesiologists) score IV patients (hazard ratio [HR]: 16.0; CI 95%: 2.1-123.5), CEA (carcinoembryonic antigen) blood level > 100 mu g/l (HR: 3.3; CI 95%: 1.2-9.0), increased lymph node ratio of 0.6-1.0 (HR: 5.3; CI 95%: 1.7-16.1), and grade 4 tumors (G4) (HR: 120.6; CI 95%: 6.7-2179.6) whereas in the lCC population, ASA score IV (HR: 8.9; CI 95%: 0.9-91.9), CEA blood level 20.1-100 mu g/l (HR: 5.4; CI 95%: 2.4-12.4), conversion to laparotomy (HR: 14.1; CI 95%: 4.0-49.0), and severe surgical complications (Clavien-Dindo III-IV) (HR: 2.9; CI 95%: 1.5-5.5) were identified as predictors of a diminished overall survival. Conclusion: Laterality disclosed no significant effect on the overall prognosis of colon cancer patients. However, group differences and distinct survival predictors could be identified in rCC and lCC patients

Rethinking the TNM Classification Regarding Direct Lymph Node Invasion in Pancreatic Ductal Adenocarcinoma

Mechanisms of lymph node invasion seem to play a prognostic role in pancreatic ductal adenocarcinoma (PDAC) after resection. However, the 8th edition of the TNM classification of the American Joint Committee on Cancer (AJCC) does not consider this. The aim of this study was to analyse the prognostic role of different mechanisms of lymph node invasion on PDAC. One hundred and twenty-two patients with resected PDAC were examined. We distinguished three groups: direct (per continuitatem, Nc) from the main tumour, metastasis (Nm) without any contact to the main tumour, and a mixed mechanism (Ncm). Afterwards, the prognostic power of the different groups was analysed concerning overall survival (OS). In total, 20 patients displayed direct lymph node invasion (Nc = 16.4%), 44 were classed as Nm (36.1%), and 21 were classed as Ncm (17.2%). The difference in OS was not statistically significant between N0 (no lymph node metastasis, n = 37) and Nc (p = 0.134), while Nm had worse OS than N0 (p < 0.001). Direct invasion alone had no statistically significant effect on OS (p = 0.885). Redefining the N0 stage by including Nc patients showed a more precise OS prediction among N stages (p = 0.001 vs. p = 0.002). Nc was more similar to N0 than to Nm; hence, we suggest a rethinking of TNM classification based on the mechanisms of lymph node metastases in PDAC. Overall, this novel classification is more precise

Accuracy of various criteria for lymph node staging in ductal adenocarcinoma of the pancreatic head by computed tomography and magnetic resonance imaging

Background: Lymph node staging of ductal adenocarcinoma of the pancreatic head (PDAC) by cross-sectional imaging is limited. The aim of this study was to determine the diagnostic accuracy of expanded criteria in nodal staging in PDAC patients. Methods: Sixty-six patients with histologically confirmed PDAC that underwent primary surgery were included in this retrospective IRB-approved study. Cross-sectional imaging studies (CT and/or MRI) were evaluated by a radiologist blinded to histopathology. Number and size of lymph nodes were measured (short-axis diameter) and characterized in terms of expanded morphological criteria of border contour (spiculated, lobulated, and indistinct) and texture (homogeneous or inhomogeneous). Sensitivities and specificities were calculated with histopathology as a reference standard. Results: Forty-eight of 66 patients (80%) had histologically confirmed lymph node metastases (pN+). Sensitivity, specificity, and Youden’s Index for the criterion “size” were 44.2%, 82.4%, and 0.27; for “inhomogeneous signal intensity” 25.6%, 94.1%, and 0.20; and for “border contour” 62.7%, 52.9%, and 0.16, respectively. There was a significant association between the number of visible lymph nodes on preoperative CT and lymph node involvement (pN+, p = 0.031). Conclusion: Lymph node staging in PDAC is mainly limited due to low sensitivity for detection of metastatic disease. Using expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivity remaining low. Combining specific criteria yields improved sensitivity with specificity and PPV remaining high

Survival rates and prognostic factors in right- and left-sided colon cancer stage I–IV: an unselected retrospective single-center trial

Purpose!#!Colorectal cancer revealed over the last decades a remarkable shift with an increasing proportion of a right- compared to a left-sided tumor location. In the current study, we aimed to disclose clinicopathological differences between right- and left-sided colon cancer (rCC and lCC) with respect to mortality and outcome predictors.!##!Methods!#!In total, 417 patients with colon cancer stage I-IV were analyzed in the present retrospective single-center study. Survival rates were assessed using the Kaplan-Meier method and uni/multivariate analyses were performed with a Cox proportional hazards regression model.!##!Results!#!Our study showed no significant difference of the overall survival between rCC and lCC stage I-IV (p = 0.354). Multivariate analysis revealed in the rCC cohort the worst outcome for ASA (American Society of Anesthesiologists) score IV patients (hazard ratio [HR]: 16.0; CI 95%: 2.1-123.5), CEA (carcinoembryonic antigen) blood level &amp;gt; 100 µg/l (HR: 3.3; CI 95%: 1.2-9.0), increased lymph node ratio of 0.6-1.0 (HR: 5.3; CI 95%: 1.7-16.1), and grade 4 tumors (G4) (HR: 120.6; CI 95%: 6.7-2179.6) whereas in the lCC population, ASA score IV (HR: 8.9; CI 95%: 0.9-91.9), CEA blood level 20.1-100 µg/l (HR: 5.4; CI 95%: 2.4-12.4), conversion to laparotomy (HR: 14.1; CI 95%: 4.0-49.0), and severe surgical complications (Clavien-Dindo III-IV) (HR: 2.9; CI 95%: 1.5-5.5) were identified as predictors of a diminished overall survival.!##!Conclusion!#!Laterality disclosed no significant effect on the overall prognosis of colon cancer patients. However, group differences and distinct survival predictors could be identified in rCC and lCC patients