Effect of non-alcoholic liver disease on recurrence rate and liver regeneration after liver resection for colorectal liver metastases.

BACKGROUND: Resection of metastases is the only potential cure for patients with liver metastasis from colorectal cancer (crc-lm). But despite an improved overall 5-year survival, the recurrence rate is still as high as 60%. Non-alcoholic fatty liver disease (nafld) can decrease the liver's capacity to regenerate after resection and might also affect cancer recurrence, potentially by elevating transforming growth factor ß, levels of specific metalloproteinases, and oxidative stress. The objective of the present work was to determine the effect of the histologic features of nafld on cancer recurrence and liver regeneration. METHODS: This retrospective analysis considered 60 patients who underwent an R0 hepatectomy for crc-lm. Volumetric analysis of the liver was calculated using axial view, portovenous phase, 2.5 mm thickness, multiphasic computed tomography images taken before and after surgery. The histologic features of nafld (steatosis, inflammation, and ballooning) were scored using the nafld activity score, and the degree of fibrosis was determined. RESULTS: The hepatic recurrence rate was 38.33%. Median overall survival duration was 56 months. Median disease-free survival duration was 14 months, and median hepatic disease-free survival duration was 56 months. Multivariate analysis revealed significant correlations of hepatic disease-free survival with hepatocyte ballooning (p = 0.0009), lesion diameter (p = 0.014), and synchronous disease (p = 0.006). Univariate and multivariate analyses did not reveal any correlation with degree of steatosis or recurrence rate. CONCLUSIONS: This study reveals an important potential negative effect of hepatocyte ballooning on hepatic disease-free survival.

Combined sorafenib and yttrium-90 radioembolization for the treatment of advanced hepatocellular carcinoma.

BACKGROUND AND AIMS: In this pilot study, we assessed the safety and tolerability of combining sorafenib with 90Y radioembolization for the treatment of unresectable hepatocellular carcinoma (hcc). METHODS: The study, conducted prospectively during 2009-2012, included eligible patients with unresectable hcc and a life expectancy of at least 12 weeks. Each patient received sorafenib (400 mg twice daily) for 6-8 weeks before 90Y treatment. Safety and tolerability were assessed. RESULTS: Of the 40 patients enrolled, 29 completed treatment (combined therapy). In the initial cohort, the most common cause of hcc was hepatitis C (32.5%), and most patients were staged Child A (82.5%). The 29 patients who completed the study had similar baseline characteristics. Grades 1 and 2 toxicities accounted for 77.8% of all adverse events reported. The most common toxicities reported were fatigue (19.0%), alteration in liver function (7.9%), and diarrhea (6.3%). There were 12 grade 3 and 2 grade 4 toxicity events reported. One patient died of liver failure within 30 days after treatment. During the study, the sorafenib dose was reduced in 6 patients (20.7%), and sorafenib had to be interrupted in 4 patients (13.8%) and discontinued in 4 patients (13.8%). The disease control rate was 72.4% per the modified Response Evaluation Criteria in Solid Tumors, and tumour necrosis was observed in 82.8% of patients. Overall survival in patients undergoing combined therapy was 12.4 months. CONCLUSIONS: Preliminary results demonstrate the safety and tolerability of combining 90Y radioembolization and sorafenib for advanced hcc. A larger prospective study is needed to determine the extent of the survival benefit.

Portal vein embolization and its effect on tumour progression for colorectal cancer liver metastases.

BACKGROUND: The aim of this study was to evaluate the long-term outcomes of patients with colorectal cancer liver metastasis (CRCLM) exhibiting disease progression after portal vein embolization (PVE). METHODS: Patients with CRCLM requiring PVE before hepatectomy between 2003 and 2014 were included. Clinical variables, and liver and tumour volumes determined by three-dimensional CT volumetry were assessed before and after PVE. Overall and disease-free survival data were obtained. Univariable and multivariable logistic regression analyses were performed to identify predictors of tumour progression after PVE. RESULTS: Of 141 patients who underwent PVE, 93 (66.0 per cent) had tumour progression and 17 (12.1 per cent) developed new contralateral lesions. Significantly fewer patients had resectable disease in the group with disease progression than among those with stable disease: 43 (46 per cent) of 93 versus 36 (75 per cent) of 48 respectively (P = 0.001). Median survival was similar in patients with and without tumour growth after PVE: 22.5 versus 26.0 months for patients with unresectable tumours (P = 0.706) and 46.2 versus 52.2 months for those with resectable disease (P = 0.953). However, disease-free survival for patients with tumour progression after PVE was shorter than that for patients with stable disease (6.0 versus 20.2 months; P = 0.045). Response to neoadjuvant chemotherapy was the only significant factor associated with tumour progression in multivariable analysis. CONCLUSION: Tumour progression after PVE did not affect overall survival, but patients with resected tumours who had tumour growth after embolization experienced earlier recurrence. A borderline response to neoadjuvant chemotherapy seemed to be associated with tumour progression after PVE.

(18)F-Fluorodeoxyglucose positron-emission tomography could have a prognostic role in patients with advanced hepatocellular carcinoma.

INTRODUCTION: We set out to evaluate the prognostic value of (18)F-fluorodeoxyglucose positron-emission tomography (pet) in patients with advanced (non-transplant-eligible) hepatocellular carcinoma (hcc) and to evaluate the correlation between standardized uptake values (suvs) and survival outcomes. METHODS: We identified patients with hcc who, from 2005 to 2013, underwent pet imaging before any treatment. This retrospective study from our hcc database obtained complete follow-up data for the 63 identified patients. RESULTS: Of the 63 patients, 10 underwent surgical resection, and 59 underwent locoregional therapy. In this cohort, 28 patients were pet-positive (defined as any lesion with a suv ≥ 4.0) before any therapy was given, and 35 patients were pet negative (all lesions with a suv < 4.0). On survival analysis, median survival was greater for the pet-negative than for the pet-positive patients: 29 months (range: 16.3-41.1 months) versus 12 months (range: 4.0-22.1 months) respectively, p = 0.0241. The pet-positive patients more often had large tumours (≥5 cm), poor differentiation, and extrahepatic disease, reflecting more aggressive tumours. On multivariate analysis, only pet positivity was associated with poor survival (p = 0.049). CONCLUSIONS: Compared with pet-positive patients, pet-negative patients with hcc experienced longer survival. Imaging by pet can be of value in early prognostication for patients with hcc, especially patients receiving locoregional therapy for whom pathologic tumour differentiation is rarely available. This potential role for pet requires further validation in a prospective study.

The role of interventional radiology in the management of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (hcc) is one of the most common causes of cancer-related death worldwide. Overall, liver transplantation and resection are the only available treatments with potential for cure. Various locoregional therapies are widely used to manage patients with advanced hcc or as a bridging therapy for patients with early and intermediate disease. This article reviews and evaluates the role of interventional radiology in the management of such cases by assessing various aspects of each method, such as effect on rates of survival, recurrence, tumour response, and complications. METHODS: A systemic search of PubMed, medline, Ovid Medline In-Process, and the Cochrane Database of Systematic Reviews retrieved all related scientific papers for review. RESULTS: Needle core biopsy is a highly sensitive, specific, and accurate method for hcc grading. Portal-vein embolization provides adequate expansion of the future liver remnant, making more patients eligible for resection. In focal or multifocal unresectable early-stage disease, radiofrequency ablation tops all other thermoablative methods. However, microwave ablation is preferred in large tumours and in patients with Child-Pugh B disease. Cryoablation is preferred in recurrent disease and in patients who are poor candidates for anesthesia. Of the various transarterial modalities-transarterial chemoembolization (tace), drug-eluting beads, and transarterial radio-embolization (tare)-tace is the method of choice in Child-Pugh A disease, and tare is the method of choice in hcc cases with portal vein thrombosis. CONCLUSIONS: The existing data support the importance of a multidisciplinary approach in hcc management. Large randomized controlled studies are needed to provide clear indication guidelines for each method.

Randomized clinical trial of the impact of insulin therapy on liver function in patients undergoing major liver resection.

BACKGROUND: Postoperative liver dysfunction is the major source of morbidity and mortality in patients undergoing partial hepatectomy. This study tested the benefits of a metabolic support protocol based on insulin infusion, for reducing liver dysfunction following hepatic resection. METHODS: Consecutive consenting patients scheduled for liver resection were randomized to receive preoperative dextrose infusion followed by insulin therapy using the hyperinsulinaemic normoglycaemic clamp protocol (n = 29) or standard therapy (control group, n = 27). Patients in the insulin therapy group followed a strict dietary regimen for 24 h before surgery. Intravenous dextrose was started at 2 mg per kg per min the night before and continued until surgery. Hyperinsulinaemic therapy for a total of 24 h was initiated at 2 munits per kg per min at induction of anaesthesia, and continued at 1 munit per kg per min after surgery. Normoglycaemia was maintained (3.5-6.0 mmol/l). Control subjects received no additional dietary supplement and a conventional insulin sliding scale during fasting. All patients were tested serially to evaluate liver function using the Schindl score. Liver tissue samples were collected at two time points during surgery to measure glycogen levels. RESULTS: Demographics were similar in the two groups. More liver dysfunction occurred in the control cohort (liver dysfunction score range 0-8 versus 0-4 with insulin therapy; P = 0.031). Median (interquartile range) liver glycogen content was 278 (153-312) and 431 (334-459) µmol/g respectively (P = 0.011). The number of complications rose with increasing severity of postoperative liver dysfunction (P = 0.032) CONCLUSION: The glucose-insulin protocol reduced postoperative liver dysfunction and improved liver glycogen content. REGISTRATION NUMBER: NCT00774098 (http://www.clinicaltrials.gov).

Bevacizumab-based therapy for colorectal cancer: experience from a large Canadian cohort at the Jewish General Hospital between 2004 and 2009.

BACKGROUND: Before its regulatory approval in Canada, bevacizumab to treat patients with colorectal cancer (crc) was accessed through the Bevacizumab Expanded Access Trial and a special-access program at the Jewish General Hospital. We retrospectively evaluated patient outcomes in that large cohort. METHODS: All patients (n = 196) had metastatic crc, were bevacizumab-naïve, and received bevacizumab in combination with chemotherapy at the Jewish General Hospital between 2004 and 2009. We collected patient demographics and clinical characteristics; relevant medical history, disease stage and tumour pathology at diagnosis; type, duration, and line of therapy; grades 3 and 4 adverse events (aes), time to disease progression (ttp), and overall survival (os) from diagnosis. RESULTS: Median follow-up was 36.0 months. Median ttp was 8.0 months [95% confidence interval (ci): 7.0 to 9.0 months). Median os was 41.0 months (95% ci: 36.0 to 47.0 months). Of the 40 grades 3 and 4 bevacizumab-related aes experienced by 38 patients (19.4%), the most common were thrombocytopenia (n = 17), deep-vein thrombosis (n = 6), pulmonary embolism (n = 4), and hypertension (n = 3). CONCLUSIONS: In an expanded access setting, our data reflect the efficacy and safety of bevacizumab-based therapy in the controlled post-registration clinical trial setting.

Magnitude of change in alpha-fetoprotein in response to transarterial chemoembolization predicts survival in patients undergoing liver transplantation for hepatocellular carcinoma.

BACKGROUND: Downsizing strategies are often attempted for patients with hepatocellular carcinoma (hcc) before liver transplantation (lt). The objective of the present study was to determine clinical predictors of favourable survival outcomes after transarterial chemoembolization (tace) before lt for hcc outside the Milan criteria, so as to better select candidates for this strategy. METHODS: In this retrospective study, patients with hcc tumours either beyond Milan criteria (single lesion > 5 cm, 3 lesions with 1 or more > 3 cm) or at the upper limit of Milan criteria (single lesions between 4.1 cm and 5.0 cm), with a predicted waiting time of more than 3 months, received carboplatin-based tace treatments. Exclusion criteria for tace included Child-Pugh C cirrhosis or the presence of portal vein invasion or extrahepatic disease on imaging. Only patients without tumour progression after tace underwent lt. RESULTS: Of 160 hcc patients who received liver grafts between 1997 and 2010, 35 were treated with tace preoperatively. The median of the sum of tumour diameters was 6.7 cm (range: 4.8-8.5 cm), which decreased with tace to 5.0 cm (range: 3.3-7.0 cm) at transplantation (p < 0.0004). The percentage drop in alpha-fetoprotein (αfp) was a positive predictor (p = 0.0051) and the time from last tace treatment to transplantation was a negative predictor (p < 0.0001) for overall survival. CONCLUSIONS: The percentage drop in αfp and a shorter time from the final tace treatment to transplantation significantly predicted improved overall survival after lt for hcc downsized with tace. As a serum marker, αfp should be followed when tace is used as a strategy to stabilize or downsize hcc lesions before lt.

Successful salvage of kidney allografts threatened by ureteral stricture using pyelovesical bypass.

Ureteral stricture is the most common urologic complication after renal transplantation. When endourologic management fails, open ureteral reconstruction remains the standard treatment. The complexity of some of these procedures makes it necessary to explore other means of repair. This study evaluated the intermediate-term outcome of subcutaneous pyelovesical bypass graft (SPBG) on renal transplant recipients. We reviewed 8 patients (6 male and 2 female; mean age 52 years) with refractory ureteral strictures postrenal transplantation, who received SPBG as salvage therapy. All patients failed endourologic management and half failed open management of their strictures. After a mean follow-up of 19.4 months, 7 out of 8 renal grafts have good function with mean GFR of 58.5 mL/min/1.73 m(2), without evidence of obstruction or infection. One patient lost his graft due to persistent infection of the SPBG and one patient developed a recurrent urinary tract infection managed with long-term antibiotics. SPBG offers a last resort in the treatment of ureteral stricture after renal transplantation refractory to conventional therapy.

Delayed graft function has an equally bad impact on deceased donor renal graft survival in both standard criteria donors and expanded criteria donors.

INTRODUCTION: The use of expanded criteria donors (ECDs) is still limited because of inferior graft survival compared to standard criteria donors (SCDs). We assessed the impact of immediate graft function (IGF) on renal graft survival among recipients of SCD and ECD grafts to determine whether these kidneys performed equally well under "ideal" conditions favoring IGF. METHODS: We included all cadaveric renal transplants performed from 1990 to 2002 (n = 335). Delayed graft function (DGF) was defined as the need for dialysis in the first 7 days posttransplant. Slow graft function (SGF) and IGF were defined as a serum creatinine fall by <20% versus >20% in the first 24 hours posttransplant, respectively. Non-death censored actual graft survivals are reported herein. RESULTS: Seventy-two of the 335 subjects (21.5%) received organs from ECDs and displayed IGF in 54.7%, SGF 16.2%, and DGF 29.1%. Among SCDs, the SGF and DGF rates were 15.3% and 23.4%, respectively. In ECD, the SGF and DGF rates were 19.4% and 50% (P < .02). Actual graft survivals at 1 and 5 years was 86.3% and 70.4%, respectively. Patients with IGF had higher actual graft survival at 5 years compared to SGF and DGF (83.5% vs 74.1% vs 45.4%). DGF had an equally bad impact on actual 5-year graft survival in SCDs and ECDs (42.6% vs 50%). CONCLUSION: DGF has a strong detrimental impact on 5-year graft survival. There is a higher rate of DGF in ECD versus SCD kidneys. The detrimental impact on 5-year actual graft survival is equal in SCD and ECD kidneys. Minimizing DGF should be our goal.

Recovery of graft function early posttransplant determines long-term graft survival in deceased donor renal transplants.

INTRODUCTION: Because kidneys show remarkable resilience and can recover function, we examined the impact on long-term graft survival in deceased donor renal transplants of both immediate graft function (IGF) and the rate of renal function recovery over the first 3 months after transplantation. METHODS: We included all cadaveric renal transplants from 1990 to 2007 (n = 583). Delayed graft function (DGF) was defined as the need for dialysis in the first 7 days posttransplant. Slow graft function (SGF) and IGF were defined by serum creatinine falls of <20% or >20% in the first 24 hours posttransplant respectively. Recovery of renal function was expressed as either the best creatinine clearance (CrCl) in the first 3 months post-renal transplantation (BCrCl-3mos) as calculated using the Cockcroft-Gault formula or as a percentage of actual versus expected value (as calculated from the donors' CrCl at procurement). RESULTS: There were 140 (23.6%) subjects who received extended criteria donor (ECD) organs. The overall graft survival at 1 and 5 years was 87.8% and 74%, respectively. The 5-year graft survivals for patients with IGF, SGF, and DGF were 85%, 76%, and 54%, respectively (P < .02). ECD kidneys showed twice the DGF rate (49% vs 23%, P < .001). BCrCl-3mos of <30 mL/min displayed a 5-year graft survival of 34%; 30 to 39 mL/min, 72%; 40 to 49 mL/min, 85%; and >50 mL/min, 82% (P < .001). Similarly, a recovery within 90% of expected CrCl in the first 3 months posttransplant correlated with 5-year graft survival of 81%; a recovery of 70% to 90%, with 65%; and a recovery of <70%, with 51% (P < .001). CONCLUSION: Early graft function in the first 3 months showed a significant impact on long-term graft survival after deceased donor renal transplantation.

An Improved Classification of Kidney Function Recovery Using Estimated Glomerular Filtration Rate Slope Post-transplantation.

BACKGROUND: The impact of renal function recovery on graft survival was examined using estimated glomerular filtration rate (eGFR) slope after kidney transplantation (GAP classification); this was compared to the conventional classification of immediate graft function (IGF), slow graft function (SGF), and delayed graft function (DGF). MATERIALS AND METHODS: Overall, 541 cases of cadaveric renal transplants were reviewed from a prospective transplant database. eGFR and its slope were measured using the harmonic mean over the first week post-transplantation. Next, 495 kidney transplant recipients from an independent institution were assessed to determine the prognostic value of graft function based on the eGFR slope. RESULTS: The main discrimination of eGFR slopes occurred within the first 7 days. Three groups in the GAP classification (Good graft function, Average graft function, Poor graft function) were defined based on eGFR slope tertiles: good graft function (GGF), average graft function (AGF), and poor graft function (PGF) were defined based on the ΔCrCL per day over the first 7 days: <1 mL/min, 1-4 mL/min, and >4 mL/min, respectively. When applied to the validation cohort, the 5-year graft failure was 20% for the PGF group, 4% for the AGF group, and 3% for the GGF group. Multivariable Cox regression analysis demonstrated better prediction of long-term graft function with the new classification (C statistic 0.49 [old)] vs 0.61 [new]). CONCLUSION: The new GAP criteria were better at predicting long-term graft survival and renal function compared to the conventional classification system, and deserve further consideration in future studies.

Effect of Insulin Therapy using Hyper-insulinemic Normoglycemic Clamp on Inflammatory Response in Brain Dead Organ Donors.

BACKGROUND: Brain death is a major stress that is associated with a massive inflammatory response and systemic hyperglycemia. Severe inflammation leads to increased graft immunogenicity and risk of graft dysfunction; while acute hyperglycemia aggravates the inflammatory response and increases the risk of morbidity and mortality. Insulin therapy not only controls hyperglycemia but also suppresses inflammation. The present study is to investigate the anti-inflammatory properties and the normoglycemia maintenance of high dose insulin on brain dead organ donors. DESIGN: 15 brain dead organ donors were divided into 2 groups, insulin treated (n=6) and controls (n=9). Insulin was provided for a minimum of 6 h using the hyperinsulinemic normoglycemic clamp technique. The changes of serum cytokines, including IL-6, IL-10, IL-1ß, IL-8, TNFα, TGFα and MCP-1, were measured by suspension bead array immunoassay and glucose by a glucose monitor. RESULTS: Compared to controls, insulin treated donors had a significant lower blood glucose 4.8 (4-6.9) vs. 9 (5.6-11.7) mmol/L, p<0.01); the net decreases of pro-inflammatory cytokines, such as IL-6 and MCP-1, and the net increase of anti-inflammatory cytokine, such as IL-10, reached significant level in insulin treated donors compared with those in controls. CONCLUSION: High dose insulin therapy decreases the concentrations of inflammatory cytokines in brain dead donors and preserves normoglycemia. High dose of insulin may have anti-inflammatory effects in brain dead organ donors and therefore, improve the quality of donor organs and potentially improve outcomes.

Cytokine modulation of defensive rage behavior in the cat: role of GABAA and interleukin-2 receptors in the medial hypothalamus.

Defensive rage behavior is a form of aggressive behavior occurring in nature in response to a threatening stimulus. It is also elicited by stimulation of the medial hypothalamus and midbrain periaqueductal gray (PAG) and mediated through specific neurotransmitter-receptor mechanisms within these regions. Since interleukin (IL)-2 modulates the release of neurotransmitters linked to aggression and rage, we sought to determine whether IL-2 microinjected into the medial hypothalamus would modulate defensive rage. Microinjections of relatively low doses of IL-2 into the medial hypothalamus significantly suppressed defensive rage elicited from the PAG in a dose-dependent manner and in the absence of signs of sickness behavior. Pre-treatment with an antibody directed against IL-2Ralpha or a GABA(A) receptor antagonist blocked IL-2's suppressive effects upon defensive rage. Since the suppression of defensive rage is also mediated by 5-HT(1) receptors in the medial hypothalamus, a 5-HT(1) antagonist was microinjected into this region as a pretreatment for IL-2; however, it did not block IL-2's suppressive effects. Immunocytochemical data provided anatomical support for these findings by revealing extensive labeling of IL-2Ralpha on neurons in the medial hypothalamus. IL-2 microinjected into the medial hypothalamus did not modulate predatory attack elicited from the lateral hypothalamus. In summary, we provide evidence for a novel role for IL-2 in the medial hypothalamus as a potent suppressor of defensive rage behavior. These effects are mediated through an IL-2-GABA(A) receptor mechanism.

Potentiating role of interleukin-1beta (IL-1beta) and IL-1beta type 1 receptors in the medial hypothalamus in defensive rage behavior in the cat.

Recently, this laboratory provided evidence that interleukin-1beta (IL-1beta), an immune and brain-derived cytokine, microinjected into the medial hypothalamus, potentiates defensive rage behavior in the cat elicited from the midbrain periaqueductal gray (PAG), and that such effects are blocked by a 5-HT2 receptor antagonist. Since this finding represents the first time that a brain cytokine has been shown to affect defensive rage behavior, the present study replicated and extended these findings by documenting the specific potentiating role played by IL-1beta Type 1 receptor (IL-1RI), and the anatomical relationship between IL-1beta and 5-HT2 receptors in the medial hypothalamus. IL-1beta (10 ng) microinjected into the medial hypothalamus induced two separate phases of facilitation, one at 60 min and another at 180 min, post-injection. In turn, these effects were blocked with pretreatment of the selective IL-1 Type I receptor antagonist (IL-1ra) (10 ng), demonstrating the selectivity of the effects of IL-1beta on medial hypothalamic neurons upon PAG-elicited defensive rage behavior. The next stage of the study utilized immunohistochemical methods to demonstrate that IL-1beta and 5-HT2 receptors were present on the same neurons within regions of the medial hypothalamus where IL-1beta and the IL-1beta receptor antagonists were administered. This provided anatomical evidence suggesting a relationship between IL-1RI and 5-HT2 receptors in the medial hypothalamus that is consistent with the previous pharmacological observations in our laboratory. The overall findings show that activation of IL-1RI in the medial hypothalamus potentiates defensive rage behavior in the cat and that these effects may also be linked to the presence of 5-HT2 receptors on the same groups of neurons in this region of hypothalamus.

Interleukin-1 beta in the hypothalamus potentiates feline defensive rage: role of serotonin-2 receptors.

The neurochemistry of aggression and rage has largely focused on the roles played by neurotransmitters and their receptor mechanisms. In contrast, little attention has been given to the possible functions of other substances. Interleukin-1beta is an immune and brain-derived cytokine that is present in the hypothalamus. Functionally, interleukin-1 has been shown to induce the release of serotonin (5-HT), a neurotransmitter known to potently affect aggression and rage behavior. Thus, the goal of the present study was to test the hypothesis that interleukin-1beta in the medial hypothalamus could modulate defensive rage behavior in the cat. In the first experiment, electrical stimulation of sites in the medial hypothalamus from which defensive rage could be elicited and where microinjections of specific compounds were later placed, facilitated defensive rage elicited from the periaqueductal gray (PAG), thus demonstrating the functional relationship between these two regions. In the second experiment, microinjections of relatively low doses of interleukin-1beta into the medial hypothalamus potentiated defensive rage behavior elicited from the midbrain periaqueductal gray in a dose-related manner. In the third experiment, pretreatment with a selective 5-HT2 receptor antagonist, LY-53857, blocked the facilitating effects of interleukin-1beta upon defensive rage. These findings reveal for the first time that brain cytokines can dramatically alter aggressive behavior. In particular, interleukin-1beta in the medial hypothalamus potentiates defensive rage behavior elicited from the periaqueductal gray in the cat, and the potentiating effects of interleukin-1beta on this form of emotional behavior are mediated via a 5-HT2 receptor mechanism.

Micromanipulation of retinal neurons by optical tweezers.

Micromanipulation by optical tweezers has been tested in cultures of mature isolated retinal cells to determine its potential for use in creating synaptic circuits in vitro. Rod and cone photoreceptors as well as other retinal nerve cell types could be optically trapped with a 980 nm diode laser mounted on an inverted light microscope using a 40x oil immersion objective numerical aperture of 1.3. Manipulation was done under sterile conditions using transparent culture dishes. To form cell groups, one half of a culture dish was made less adhesive by application of a thin layer of silicone elastomer. Unattached cells were trapped and relocated next to cells lying on an adhesive culture substrate. Optical trapping did not affect the ability of neurons to subsequently attach to the culture substrate. Up to 60% of trapped cells survived for 2 or more days. The pattern and rate of process outgrowth for manipulated cells was comparable to unmanipulated cells and by 2 days, cell-cell contacts were observed. Cultures were fixed at 2 and 5 days for electron microscopy. Organelle, nuclear and cytoplasmic structure of manipulated cells was completely normal and in photoreceptors, synaptic vesicles and ribbons were intact. Optical tweezers, therefore, provide a benign technique with which to micromanipulate whole neurons. The procedures also bestow increased precision to the study of cell-cell interactions by allowing the selection of potentially interacting cell types at a single cell level.

Oxidative stress increases ubiquitin--protein conjugates in synaptosomes.

Synaptosomes were incubated in the presence of FeSO4 to test the hypothesis that iron-catalyzed oxidative damage causes an increase in the ubiquitination of synaptosomal proteins. Incubation with 10 or 50 microM FeSO4 caused concentration-dependent increases in carbonyl groups (an indication of protein oxidation) and ubiquitinated proteins (determined by probing Western blots with a monoclonal antibody to ubiquitin). Differences in protein ubiquitination occurred within 5 min of incubation, indicating a rapid response to oxidative stress. Results of experiments with MG-132, an inhibitor of the degradation of ubiquitinated proteins, suggested that oxidative damage stimulated ubiquitination rather than inhibited degradation of ubiquitinated proteins. The data are consistent with the hypothesis that synaptic terminals utilize the ubiquitin/proteasome proteolytic pathway to degrade oxidatively damaged proteins.

Differential modulation of feline defensive rage behavior in the medial hypothalamus by 5-HT1A and 5-HT2 receptors.

Previous studies have established that the expression of defensive rage behavior in the cat is mediated over reciprocal pathways that link the medial hypothalamus and the dorsolateral quadrant of the midbrain periaqueductal gray matter (PAG). The present study was designed to determine the roles played by 5-HT(1A) and 5-HT(2C) receptors in the medial hypothalamus on the expression of defensive rage behavior elicited from electrical stimulation of the PAG. Monopolar stimulating electrodes were placed in the midbrain PAG from which defensive rage behavior could be elicited by electrical stimulation. During the course of this study, defensive rage was determined by measuring the latency of the "hissing" component of this behavior. Cannula-electrodes were implanted into sites within the medial hypothalamus from which defensive rage behavior could also be elicited by electrical stimulation in order that serotonergic compounds could be microinjected into behaviorally identifiable regions of the hypothalamus at a later time. Microinjections of the 5-HT(1A) receptor agonist 8-OHDPAT (0.1, 1.0 and 3.0 nmol) into the medial hypothalamus suppressed PAG-elicited hissing in a dose-dependent manner. Administration of the 5-HT(1A) antagonist p-MPPI (3.0 nmol) blocked the suppressive effects of 8-OHDPAT upon hissing. The suppressive effects of 8-OHDPAT were specific to defensive rage behavior because this drug (3 nmol) facilitated quiet biting attack. Microinjections of the 5-HT(2C) receptor agonist (+/-)-DOI hydrochloride into the medial hypothalamus (0.5, 1.0, and 3.0 nmol) facilitated the occurrence of PAG-elicited hissing in a dose-dependent manner. In turn, these facilitating effects were blocked by pretreatment with the selective 5-HT(2) antagonist, LY-53,857, which was microinjected into the same medial hypothalamic site. The findings of this study provide evidence that activation of 5-HT(1A) and 5-HT(2) receptors within the medial hypothalamus exert differential modulatory effects upon defensive rage behavior elicited from the midbrain PAG of the cat.