The yield of diagnostic laparoscopy with peritoneal lavage in gastric adenocarcinoma: A retrospective cohort study.

INTRODUCTION: Diagnostic laparoscopy (DL) with peritoneal lavage has been adopted as a standard staging procedure for patients with gastric cancer (GC). Evaluation of the value of DL is important given ongoing improvements in diagnostic imaging and treatment. As contemporary data from European centres are sparse, this retrospective cohort study aimed to assess the yield of DL in patients with potentially curable gastric cancer, and to identify predictive factors for peritoneal metastases. METHODS: Patients with adenocarcinoma of the stomach, treated between January 2016 and December 2018, were identified from institutional databases of two high volume European Upper-GI centres. Patients who underwent a DL with peritoneal lavage for potentially curable disease after clinical staging with imaging (cT1-4N0-3M0) were included. The primary outcome was the proportion of patients with a positive DL, defined as macroscopic metastatic disease, positive peritoneal cytology washings (PC+) or locally irresectable disease. RESULTS: Some 80 of 327 included patients (24.5%) had a positive DL, excluding these patients from neoadjuvant treatment (66 of 327; 20.2%) and/or surgical resection (76 of 327; 23.2%). In 34 of 327 patients (10.3%), macroscopic metastatic disease was seen, with peritoneal deposits in 30 of these patients. Only 16 of 30 patients with peritoneal disease had positive cytology. Some 41 of 327 patients (12.5%) that underwent DL had PC+ in the absence of macroscopic metastases and five patients (1.5%) had an irresectable primary tumour. Diffuse type carcinoma had the highest risk of peritoneal dissemination, irrespective of cT and cN categories. CONCLUSION: The diagnostic yield of staging laparoscopy is high, changing the management in approximately one quarter of patients. DL should be considered in patients with diffuse type carcinoma irrespective of cT and cN categories.

Adjuvant Therapy for Patients with a Tumor-Positive Resection Margin After Neoadjuvant Chemoradiotherapy and Esophagectomy.

BACKGROUND: Approximately 4-9% of patients have a tumor-positive resection margin after neoadjuvant chemoradiotherapy (nCRT) and esophagectomy. Although it is associated with decreased survival, Western guidelines do not recommend adjuvant treatment. OBJECTIVE: The aim of this study was to assess the proportion of patients who received adjuvant therapy, and to evaluate overall survival (OS) after esophagectomy in patients with a tumor-positive resection margin. METHODS: Patients diagnosed with resectable (cT2-4a/cTxN0-3/NxM0) esophageal cancer between 2015 and 2022, and treated with nCRT followed by irradical esophagectomy, were selected from the Netherlands Cancer Registry. The primary outcome was the proportion of patients with a tumor-positive resection margin who started adjuvant treatment ≤16 weeks after esophagectomy, including chemotherapy/radiotherapy, immunotherapy, or targeted therapy. OS was calculated from the date of surgery until the date of death or last day of follow-up. RESULTS: Overall, 376 patients were included in our study, of whom 357 were treated with nCRT. Of these 357 patients, 98.3% had a microscopically irradical resection and 1.7% had a macroscopically irradical resection. Approximately 72.3% of tumors showed a partial response (Mandard 2-3) and 11.8% showed little/no pathological response (Mandard 4-5) to nCRT. One of 357 patients underwent adjuvant chemoradiotherapy and 39 patients (61%) underwent adjuvant immunotherapy (nivolumab). The median and 5-year OS rate of all patients was 16.4 months (95% confidence interval 13.1-19.8) and 21%, respectively. CONCLUSION: Real-world population-level data showed that no patients with a tumor-positive resection margin underwent adjuvant therapy following nCRT and esophagectomy prior to 2021. Interestingly, 61% of patients were treated with adjuvant nivolumab in 2021-2022. OS after irradical esophagectomy is poor and long-term data will explore the added value of nivolumab.

Robot-assisted minimally invasive thoraco-laparoscopic esophagectomy versus minimally invasive esophagectomy for resectable esophageal adenocarcinoma, a randomized controlled trial (ROBOT-2 trial).

BACKGROUND: For patients with esophageal adenocarcinoma or cancer of the gastroesophageal junction, radical esophagectomy with 2-field lymphadenectomy is the cornerstone of the multimodality treatment with curative intent. Both conventional minimally invasive esophagectomy (MIE) and robot assisted minimally invasive esophagectomy (RAMIE) were shown to be superior compared to open transthoracic esophagectomy considering postoperative complications. However, no randomized comparison exists between MIE and RAMIE in the Western World for patients with esophageal adenocarcinoma. METHODS: This is an investigator-initiated and investigator-driven multicenter randomized controlled parallel-group superiority trial. All adult patients (age ≥ 18 and ≤ 90 years) with histologically proven, surgically resectable (cT1-4a, N0-3, M0) esophageal adenocarcinoma of the intrathoracic esophagus or adenocarcinoma of the gastroesophageal junction and with European Clinical Oncology Group performance status 0, 1 or 2 will be assessed for eligibility and included after obtaining informed consent. Patients (n = 218) with resectable esophageal adenocarcinoma of the intrathoracic esophagus or adenocarcinoma of the gastroesophageal junction are randomized to either RAMIE (n = 109) or MIE (n = 109). The primary outcome of this study is the total number of resected abdominal and mediastinal lymph nodes specified per lymph node station. CONCLUSION: This is the first randomized controlled trial designed to compare RAMIE to MIE as surgical treatment for resectable esophageal adenocarcinoma or adenocarcinoma of the gastroesophageal junction in the Western World. The hypothesis of the proposed study is that RAMIE will result in a higher abdominal and mediastinal lymph node yield specified per station compared to conventional MIE. Short-term results and the primary endpoint (total number of resected abdominal and mediastinal lymph nodes per lymph node station) will be analyzed and published after discharge of the last randomized patient within this trial. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04306458 . Registered 13th March 2020, https://clinicaltrials.gov/ct2/show/NCT04306458; Date of first enrolment 18.01.2021; Target sample size 218; Recruitment status: Recruiting; Protocol version 2; Issue date 10.03.2020; Rev. 02.02.2021; Authors ET, PCvdS, PPG.

Meta-analysis of randomized controlled trials and individual patient data comparing minimally invasive with open oesophagectomy for cancer.

BACKGROUND: Minimally invasive oesophagectomy (MIO) for oesophageal cancer may reduce surgical complications compared with open oesophagectomy. MIO is, however, technically challenging and may impair optimal oncological resection. The aim of the present study was to assess if MIO for cancer is beneficial. METHODS: A systematic literature search in MEDLINE, Web of Science and CENTRAL was performed and randomized controlled trials (RCTs) comparing MIO with open oesophagectomy were included in a meta-analysis. Survival was analysed using individual patient data. Random-effects model was used for pooled estimates of perioperative effects. RESULTS: Among 3219 articles, six RCTs were identified including 822 patients. Three-year overall survival (56 (95 per cent c.i. 49 to 62) per cent for MIO versus 52 (95 per cent c.i. 44 to 60) per cent for open; P = 0.54) and disease-free survival (54 (95 per cent c.i. 47 to 61) per cent versus 50 (95 per cent c.i. 42 to 58) per cent; P = 0.38) were comparable. Overall complication rate was lower for MIO (odds ratio 0.33 (95 per cent c.i. 0.20 to 0.53); P < 0.010) mainly due to fewer pulmonary complications (OR 0.44 (95 per cent c.i. 0.27 to 0.72); P < 0.010), including pneumonia (OR 0.41 (95 per cent c.i. 0.22 to 0.77); P < 0.010). CONCLUSION: MIO for cancer is associated with a lower risk of postoperative complications compared with open resection. Overall and disease-free survival are comparable for the two techniques. LAY SUMMARY: Oesophagectomy for cancer is associated with a high risk of complications. A minimally invasive approach might be less traumatic, leading to fewer complications and may also improve oncological outcome. A meta-analysis of randomized controlled trials comparing minimally invasive to open oesophagectomy was performed. The analysis showed that the minimally invasive approach led to fewer postoperative complications, in particular, fewer pulmonary complications. Survival after surgery was comparable for the two techniques.

Oesophagectomy for cancer is associated with a high risk of complications. A minimally invasive approach might be less traumatic, leading to fewer complications and may also improve oncological outcome. A meta-analysis of randomized controlled trials comparing minimally invasive to open oesophagectomy was performed. The analysis showed that the minimally invasive approach led to fewer postoperative complications, in particular, fewer pulmonary complications. Survival after surgery was comparable for the two techniques.

The Circular Stapled Esophagogastric Anastomosis in Esophagectomy: No Differences in Anastomotic Insufficiency and Stricture Rates Between the 25 mm and 28 mm Circular Stapler.

BACKGROUND: For patients undergoing an Ivor Lewis esophagectomy with a circular stapled anastomosis, the optimal diameter of the used circular stapler to restore continuity is unknown. The aim of this study was to compare the 25 mm stapled versus the 28 mm stapled esophagogastric anastomosis after Ivor Lewis esophagectomy, focusing on anastomotic insufficiency and postoperative anastomotic strictures. METHODS: Between February 2008 and June 2019, 349 consecutive patients underwent Ivor Lewis esophagectomy with gastric conduit reconstruction and circular stapled anastomosis. Patient characteristics and postoperative results, such as anastomotic insufficiency rates, postoperative anastomotic stricture rates, time to anastomotic stricture rate, and the number of dilatations, were recorded in a prospective database and analyzed. RESULTS: In 222 patients (64%), the 25 mm circular stapler was used and in 127 patients (36%) the 28 mm circular stapler was used. There were no differences in baseline characteristics. Anastomotic insufficiency rates were comparable between the 25 mm (12%) and the 28 mm groups (11%) (p = 0.751). There were no differences between postoperative anastomotic strictures in the 25 mm (14%) and the 28 mm groups (14%) (p = 0.863). Within patients with postoperative anastomotic strictures, a median number of 2 dilatations were observed in each group (p = 0.573) without differences in the time to first diagnosis (p = 0.412). CONCLUSION: There were no differences in anastomotic insufficiency and postoperative anastomotic stricture rates between the 25 mm and the 28 mm circular stapled esophagogastric anastomosis after Ivor Lewis esophagectomy. Both the 25 mm and 28 mm stapler can be safely used to create a circular stapled esophagogastric anastomosis to restore continuity after esophagectomy.

Recurrent laryngeal nerve monitoring during totally robot-assisted Ivor Lewis esophagectomy.

PURPOSE: The robot-assisted approach for Ivor Lewis esophagectomy offers an enlarged, three-dimensional overview of the intraoperative situs. The vagal nerve (VN) can easily be detected, preserved, and intentionally resected below the separation point of the recurrent laryngeal nerve (RLN). However, postoperative vocal cord paresis can result from vagal or RLN injury during radical lymph node dissection, presenting a challenge to the operating surgeon. METHODS: From May to August 2019, 10 cases of robot-assisted minimally invasive esophagectomy (RAMIE) with extended 2-field lymphadenectomy, performed at the University Medical Center Mainz, were included in a prospective cohort study. Bilateral intermittent intraoperative nerve monitoring (IONM) of the RLN and VN was performed, including pre- and postoperative laryngoscopy assessment. RESULTS: Reliable mean signals of the right VN (2.57 mV/4.50 ms) and the RLN (left 1.24 mV/3.71 ms, right 0.85 mV/3.56 ms) were obtained. IONM facilitated the identification of the exact height of separation of the right RLN from the VN. There were no cases of permanent postoperative vocal paresis. Median lymph node count from the paratracheal stations was 5 lymph nodes. CONCLUSION: IONM was feasible during RAMIE. The intraoperative identification of the RLN location contributed to the accuracy of lymph node dissection of the paratracheal lymph node stations. RLN damage and subsequent postoperative vocal cord paresis can potentially be prevented by IONM.

Robot-assisted minimally invasive esophagectomy (RAMIE) compared to conventional minimally invasive esophagectomy (MIE) for esophageal cancer: a propensity-matched analysis.

Robot-assisted minimally invasive esophagectomy (RAMIE) is increasingly being applied as treatment for esophageal cancer. In this study, the results of 50 RAMIE procedures were compared with 50 conventional minimally invasive esophagectomy (MIE) operations, which had been the standard treatment for esophageal cancer prior to the robotic era. Between April 2016 and March 2018, data of 100 consecutive patients with esophageal carcinoma undergoing modified Ivor Lewis esophagectomy were prospectively collected. All operations were performed by the same surgeon using an identical intrathoracic anastomotic reconstruction technique with the same perioperative management and pain control regimen. Intra-operative and postoperative complications were graded according to definitions stated by the Esophagectomy Complications Consensus Group. Data analysis was carried out with and without propensity score matching. Baseline characteristics did not show significant differences between the RAMIE and MIE group. Propensity score matching of the initial group of 100 patients resulted in two equal groups of 40 patients for each surgical approach. In the RAMIE group, the median total lymph node yield was 27 (range 13-84) compared to 23 in the MIE group (range 11-48), P = 0.053. Median intensive care unit (ICU) stay was 1 day (range 1-43) in the RAMIE group compared to 2 days (range 1-17) in the MIE group (P = 0.029). The incidence of postoperative complications was not significantly different between the two groups (P = 0.581). In this propensity-matched study comparing RAMIE to MIE, ICU stay was significantly shorter in the RAMIE group. There was a trend in improved lymphadenectomy in RAMIE.

Innovative fully robotic 4-arm Ivor Lewis esophagectomy for esophageal cancer (RAMIE4).

Nowadays robotic surgery is established for abdominal and thoracic surgery. It has been shown that complex procedures are feasible using robotic systems, e.g., da Vinci Xi, with a huge benefit in precision. Different techniques for esophageal cancer surgery are reported; however, only a few robotic and partial robotic procedures are described. Therefore, a fully robotic (abdominal and thoracic) Ivor Lewis esophageal resection using four robotic arms-RAMIE4-the standard technique used for lower esophageal cancer, is presented in this paper. The technique shown in the video was performed successfully in 100 cases in 24 months. The reconstruction is performed with a gastric conduit pull-up and intrathoracic manually inserted 28-mm circular end-to-side stapled anastomosis. This video demonstrates the feasibility of RAMIE4 in the abdomen and thorax and reveals advantages of the robotic assistance.

Preoperative Chemoradiotherapy Versus Perioperative Chemotherapy for Patients With Resectable Esophageal or Gastroesophageal Junction Adenocarcinoma.

BACKGROUND: This study compares neoadjuvant chemoradiotherapy (nCRT) with perioperative chemotherapy (pCT) for patients with resectable esophageal or gastroesophageal junction (GEJ) adenocarcinoma in terms of toxicity, postoperative complications, pathologic response, and survival. METHODS: This study retrospectively analyzed and compared 313 patients with resectable esophageal or GEJ adenocarcinoma treated with either nCRT (carboplatin/paclitaxel 41.4 Gy, n = 176) or pCT (epirubicin, cisplatin and capecitabine, n = 137). RESULTS: The baseline and tumor characteristics were similar in both groups. The ability to deliver all planned preoperative cycles was greater in the nCRT group (92.0 vs. 76.6%). Whereas nCRT was associated with a higher rate of grades 3 and 4 esophagitis, pCT was associated with a higher rate of grades 3 and 4 thromboembolic events, febrile neutropenia, nausea, vomiting, diarrhea, hand-foot syndrome, mucositis, cardiac complications, and electrolyte imbalances. Two patients in the pCT group died during neoadjuvant treatment due to febrile neutropenia. More postoperative cardiac complications occurred in the nCRT group. All other postoperative complications and the in-hospital mortality rate (nCRT, 4.7%; pCT, 2.3%) were comparable. The pathologic complete response (pCR) rate was 15.1% after nCRT and 6.9% after pCT. Radicality of surgery was comparable (R0: 93.0 vs. 91.6%). The median overall survival was 35 months after nCRT versus 36 months after pCT. CONCLUSION: For patients with esophageal or GEJ adenocarcinoma, chemoradiotherapy with paclitaxel, carboplatin and concurrent radiotherapy, and perioperative chemotherapy with epirubicin, cisplatin, and capecitabin lead to equal oncologic outcomes in terms of radical resection rates, lymphadenectomy, patterns of recurrent disease, and (disease-free) survival. However, neoadjuvant chemoradiotherapy is associated with a considerably lower level of severe adverse events and should therefore be the preferred protocol until a well-powered randomized controlled trial provides different insights.

Current status of laparoscopic transhiatal esophagectomy for esophageal cancer patients: a systematic review of the literature.

Minimally invasive techniques in transhiatal esophagectomy (THE) were introduced to reduce morbidity and enhance postoperative recovery. Aim of this study was to systematically review the current status and possible beneficial effects of the minimally invasive approach in THE. A systematic search was performed in PubMed, the Cochrane Library, and Embase to identify English articles published on laparoscopic THE. Comparative cohort studies were included for critical appraisal. Data describing perioperative and oncological outcomes were analyzed. A total of four comparative cohort studies that compared laparoscopic THE (n = 122) with open THE (n = 144) and four noncomparative cohort studies reporting on laparoscopic THE (n = 212) were included in this review. Median blood loss was significantly lower in the laparoscopic group in all studies (100-500 vs. 526-900 mL). Length of hospital stay was also significantly shorter for the laparoscopic approach in all studies (9-13 vs. 12-16 days). One study reported less major postoperative complications after laparoscopic THE (12 vs. 23%), in the other studies no differences were found. Also no differences were found with regard to operating time, postoperative morbidity, radicality, and lymph node retrieval. Based on these pioneer studies, laparoscopic THE was demonstrated to be safe and feasible with evidence of reduced blood loss and shorter hospital stays. However, level 1 evidence is lacking and further research is warranted to confirm these findings and also to evaluate long-term oncologic outcomes.

Laparoscopic gastrectomy in Western European patients with advanced gastric cancer.

BACKGROUND: The advantage of laparoscopic gastrectomy compared to open gastrectomy has been established in Asian patient series with early gastric cancer. However, its feasibility in Western European patients with locally advanced gastric cancer is unknown. METHODS: Between 2006 and 2014 70 consecutive patients with advanced gastric cancer underwent laparoscopic gastrectomy with D2 lymph node dissection. A Billroth II reconstruction was performed after distal gastrectomy. In case of total gastrectomy a jejunal J-pouch reconstruction was performed. RESULTS: Total gastrectomy was performed in 56 patients and distal gastrectomy in 14 patients. Perioperative chemotherapy was administered in 45/70 (64%) patients. A radical resection was achieved in 63/70 (90%). The median number of dissected lymph nodes was 17 (2-62). The median intraoperative blood loss was 305 (30-2700) milliliters. The median postoperative hospital stay was 11 (5-91) days. The 30-day mortality was 4.3%. CONCLUSIONS: Laparoscopic gastrectomy can be performed in Western European patients with advanced gastric cancer and meets the oncologic standard with low intraoperative blood loss and short hospital stay.

Prophylactic Laparoscopic Total Gastrectomy with Jejunal Pouch Reconstruction in Patients Carrying a CDH1 Germline Mutation.

BACKGROUND: For patients with an identified germline E-cadherin-1 (CDH1) mutation, prophylactic gastrectomy is the treatment of choice to eliminate the high risk of developing diffuse gastric cancer. Laparoscopic total gastrectomy with jejunal pouch reconstruction is a novel approach that may be especially suitable in these patients. METHODS: Patients with a germline CDH1 mutation who underwent prophylactic laparoscopic total gastrectomy with jejunal pouch were included in our prospective database. RESULTS: A total of 11 patients with a median age of 40 (22-61) years were included. The average operative time was 4:26 ± 0:49 h and the average blood loss was 219 ± 155 ml. Median length of hospital stay was 10 (7-27) days. In two patients, an esophagojejunal anastomotic leakage occurred (grade 4). The leakages were seen in patient numbers 2 and 3, which may be a result of a learning curve. The latter eight patients did not develop anastomotic leakage. Pulmonary complications occurred in one patient with atelectasis and in one patient with pneumonia (grade 2). The 60-day mortality rate was 0 %. Multiple foci of intramucosal diffuse gastric signet ring cell carcinoma were found in the resection specimen of 9/11 (82 %) patients. All 11/11 (100 %) resections were microscopically radical. CONCLUSIONS: Prophylactic laparoscopic total gastrectomy with jejunal pouch reconstruction in patients with a CDH1 germline mutation is feasible and safe. In 82 % of patients, foci of intramucosal diffuse gastric signet ring cell carcinoma in the resection specimen were found.

Robot-assisted minimally invasive esophagectomy for esophageal cancer: A systematic review.

This paper describes the technique of robot-assisted minimally invasive esophagectomy. (RAMIE) Also, a systematic literature search was performed. Safety and feasibility of RAMIE was demonstrated in all reports. Short term oncologic results show radical resection rates of 77-100% and 18-43 lymph nodes harvested. RAMIE offers great visualization of the mediastinum and enables meticulous dissection in the mediastinum from diaphragm to thoracic inlet.

Oncologic Long-Term Results of Robot-Assisted Minimally Invasive Thoraco-Laparoscopic Esophagectomy with Two-Field Lymphadenectomy for Esophageal Cancer.

BACKGROUND: Open transthoracic esophagectomy is the worldwide gold standard in the treatment of resectable esophageal cancer. Robot-assisted minimally invasive thoraco-laparoscopic esophagectomy (RAMIE) for esophageal cancer may be associated with reduced blood loss, shorter intensive care unit (ICU) stay, and less cardiopulmonary morbidity; however, long-term oncologic results have not been reported to date. METHODS: Between June 2007 and September 2011, a total of 108 patients with potentially resectable esophageal cancer underwent RAMIE at the University Medical Centre Utrecht, with curative intent. All data were recorded prospectively. RESULTS: Median duration of the surgical procedure was 381 min (range 264-636). Pulmonary complications were most common and were observed in 36 patients (33 %). Median ICU stay was 1 day, and median overall postoperative hospital stay was 16 days. In-hospital mortality was 5 %. The majority of patients (78 %) presented with T3 and T4 disease, and 68 % of patients had nodal-positive disease (cN1-3). In 65 % of patients, neoadjuvant treatment (chemotherapy 57 %, chemoradiotherapy 7 %, radiotherapy 1 %) was administered, and in 103 (95 %) patients, a radical resection (R0) was achieved. The median number of lymph nodes was 26, median follow-up was 58 months, 5-year overall survival was 42 %, median disease-free survival was 21 months, and median overall survival was 29 months. Tumor recurrence occurred in 51 patients and was locoregional only in 6 (6 %) patients, systemic only in 31 (30 %) patients, and combined in 14 (14 %) patients. CONCLUSION: RAMIE was shown to be oncologically effective, with a high percentage of R0 radical resections and adequate lymphadenectomy. RAMIE provided good local control with a low percentage of local recurrence at long-term follow up.

Safety, efficacy, and long-term follow-up evaluation of perioperative epirubicin, Cisplatin, and capecitabine chemotherapy in esophageal resection for adenocarcinoma.

BACKGROUND: Perioperative epirubicin, cisplatin, and capecitabine (ECC) chemotherapy was evaluated in patients who underwent esophageal resection for adenocarcinoma of the esophagus or gastroesophageal junction (GEJ). METHODS: A cohort of 93 consecutive patients was analyzed. The median follow-up period was 60 months. Source data verification of adverse events was performed by two independent observers. RESULTS: All three planned preoperative chemotherapy cycles were administered to 65 patients (69.9 %). Only 27 % of the patients completed both pre- and postoperative chemotherapy. The reasons for not receiving postoperative adjuvant chemotherapy could be separated in two main problems: toxicity of the preoperative chemotherapy and postoperative problems involving difficulty in recovery and postoperative complications. Finally, 25 patients (27 %), completed three preoperative and three postoperative cycles. Grades 3 and 4 nonhematologic adverse events of preoperative chemotherapy mainly consisted of thromboembolic events (16.2 %) and cardiac complications (7.5 %). A history of cardiac and vascular disease was independently associated with discontinuation of preoperative chemotherapy and the occurrence of grade 3 or higher adverse events. Surgery was performed for 94 % of all the patients who started with ECC chemotherapy. A radical resection (R0) was achieved in 93 % of the patients. A complete pathologic response was observed in 8 % of the patients. During a median follow-up period of 60 months, the median disease-free survival time was 28 months, and the median overall survival time was 36 months. The 3-year overall survival rate was 50 %, and the 5-year overall survival rate was 42 %. CONCLUSION: For patients with adenocarcinoma of the esophagus or GEJ, six cycles of ECC-based perioperative chemotherapy is associated with a relatively high number of adverse events. Although this toxicity did not affect the esophageal resectability rate, this regimen should be used with caution in this patient population.

Increased vasopressin immunoreactivity in the rat brain after a postmortem interval of 6 hours.

Enhanced immunocytochemical staining of vasopressin-containing neurons was observed after incubation of rat brain slices in Ringer medium for 6 h at room temperature, as compared to brain tissue fixed immediately after death. Hypothalamic vasopressin neurons in the supraoptic nucleus, the paraventricular nucleus and the suprachiasmatic nucleus stained more intensely after a postmortem interval of 6 h. Extrahypothalamic vasopressin neurons (VP) in the bed nucleus of the stria terminalis, the medial amygdala and the locus coeruleus proved to be stained as well. Extrahypothalamic VP neurons in the locus coeruleus could, until now, only be visualized after in vivo pretreatment with colchicine. In addition, staining was observed at two new sites, the dorsal raphe nucleus and the lateral septum. Staining of VP was corroborated by application of different antibodies directed against the intact vasopressin molecule as well as by antibodies directed against the other parts of the vasopressin precursor molecule, i.e. neurophysin and glycopeptide. The specificity of the VP-staining was validated by using pre-immune serum and using Brattleboro rat brain tissue, resulting in a negative staining in both cases. Furthermore, homogenated punches of the suprachiasmatic nucleus were submitted to iso-electric focussing on polyacrylamide gel, followed by press blotting and subsequent immunocytochemical staining for vasopressin. Iso-electric foccussing enabled us to characterize and quantify peptides in the suprachiasmatic nucleus. The vasopressin content increased 6 h postmortem, while c-terminal glycopeptide and neurophysin levels remained stable. Similar results were observed in the suprachiasmatic nucleus from decapitated rats whose brains were left intact in the skull for 6 h at room temperature.

Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing.

A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.

Press-blotting on gelatin-coated nitrocellulose membranes. A method for sensitive quantitative immunodetection of peptides after gel isoelectric focusing.

A method is presented for the fixation of peptides in nitrocellulose membranes after isoelectric focusing on thin polyacrylamide gels. Focusing gels are covered with gelatin-coated nitrocellulose membrane. Using glutaraldehyde, focused peptides are covalently fixed onto this membrane. Fixed peptides are stained using the peroxidase-anti-peroxidase method and the immunoreaction is quantified by rendering the membrane transparent and measuring the optical density of the precipitated chromogen in each band. The effect of pore size and gelatin content of the membrane, glutaraldehyde concentration and fixation time on fixation efficiency and immunostaining has been investigated. Gelatin coating considerably increases the efficiency of glutaraldehyde fixation of peptides and greatly enhances antibody-binding. Consequently, sensitive quantitative immunodetection is possible and, depending on the antiserum, peptides are readily detected in quantities down to 10 pg.

Propressophysin is present in neurones at multiple sites in Wistar and homozygous Brattleboro rat brain.

Vasopressin (VP) is synthesized as propressophysin, containing also neurophysin (NP) and C-terminal glycopeptide (CPP), within the hypothalamo-neurohypophyseal system (HNS). Recently, VP and NP-immunoreactive cells were demonstrated in other rat brain nuclei. Here we report CPP immunoreactivity in perikarya in these nuclei. Within the homozygous Brattleboro rat, known to be deficient in neuronal VP production, no CPP immunoreactivity was seen in these nuclei. However, intense VP and CPP immunoreactivity was present in solitary cells (52.2 +/- 3.3 per rat) and fibres within the HNS.