Flow cytometry as a method for studying effects of stressors on primary rat neurons

The mechanisms associated with cell death have been an important focus for neurobiology research. In the present study, the methodology of flow cytometry was used to optimize quantification of the toxic effects of tumor necrosis factor-alpha (TNF-alpha), trans-4-hydroxy-2-nonenal (4-HNE), and aged amyloid-beta (Abeta1-42) on rat primary cortical neurons. The fluorescent dyes annexin V-FITC and propidium iodide (PI) were used to identify populations of viable, early apoptotic, necrotic and late apoptotic cells by flow cytometry. Prior to exposure, the primary cultures showed 83% cell viability. Flow cytometry following labeling of cells with a specific neuronal marker, TUJ-1, revealed 82% pure neuronal populations, whereas approximately 7% were astrocytic as shown by glial fibrillary acidic protein positivity. Exposure of primary cultures to TNF-alpha, 4-HNE, and aged Abeta1-42 gave an increased number of early apoptotic cells. We show that flow cytometry is a suitable method for quantifying effects of different stressors on neurons in primary cultures. This technique could be useful for screening and testing of pharmacological compounds relevant to neurodegenerative disorders.     

Long-term effects of a ketogenic diet in obese patients

BACKGROUND:

Although various studies have examined the short-term effects of a ketogenic diet in reducing weight in obese patients, its long-term effects on various physical and biochemical parameters are not known.

OBJECTIVE:

To determine the effects of a 24-week ketogenic diet (consisting of 30 g carbohydrate, 1 g/kg body weight protein, 20% saturated fat, and 80% polyunsaturated and monounsaturated fat) in obese patients.

PATIENTS AND METHODS:

In the present study, 83 obese patients (39 men and 44 women) with a body mass index greater than 35 kg/m², and high glucose and cholesterol levels were selected. The body weight, body mass index, total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides, fasting blood sugar, urea and creatinine levels were determined before and after the administration of the ketogenic diet. Changes in these parameters were monitored after eight, 16 and 24 weeks of treatment.

RESULTS:

The weight and body mass index of the patients decreased significantly (P<0.0001). The level of total cholesterol decreased from week 1 to week 24. HDL cholesterol levels significantly increased, whereas LDL cholesterol levels significantly decreased after treatment. The level of triglycerides decreased significantly following 24 weeks of treatment. The level of blood glucose significantly decreased. The changes in the level of urea and creatinine were not statistically significant.

CONCLUSIONS:

The present study shows the beneficial effects of a long-term ketogenic diet. It significantly reduced the body weight and body mass index of the patients. Furthermore, it decreased the level of triglycerides, LDL cholesterol and blood glucose, and increased the level of HDL cholesterol. Administering a ketogenic diet for a relatively longer period of time did not produce any significant side effects in the patients. Therefore, the present study confirms that it is safe to use a ketogenic diet for a longer period of time than previously demonstrated.     

Pathological outcomes of men eligible for active surveillance after undergoing radical prostatectomy: are results predictable?

Introduction

To analyze pathological results in patients with prostate cancer eligible for active surveillance (AS) after radical prostatectomy and available prediction systems.

Methods

A retrospective analysis was performed of 612 patients who underwent radical prostatectomy during a 14-year period. Subsequently, we selected those patients who would have been eligible for AS according to 2 different published criteria. Group AS-A matched the following criteria: ≤T2a; Gleason Score ≤6; and prostate-specific antigen <10 ng/mL, while group AS-B applied to different criteria: ≤T2a; Gleason Score <7; and prostate-specific antigen ≤15 ng/mL. Pathological outcomes were compared with results of the 2001 Partin tables.

Results

Altogether, 125 (20.4%) patients were included in group AS-A and 159 (25.9%) in group AS-B. We detected 32 cases of >pT2c (25.6%) for group AS-A and 47 cases (29.6%) for AS-B, respectively. Gleason score upgrading was recorded in 34.4% (AS-A) and 38.3% (AS-B). Results of the Partin tables showed good discrimination among patients at risk for positive lymph nodes but limited discrimination for organ-confined disease, seminal vesicle.

Conclusions

Overall >25% of patients eligible for AS showed either upstaging or Gleason score upgrading, which could not be measured with the examined predictive tools. Patients should be informed about the risks of inaccurate preoperative diagnostic.     

Glutamate and dopamine synaptic terminals in extended amygdala after 14-week chronic alcohol drinking in inbred alcohol-preferring rats.

Alcohol has been shown to affect glutamate (GLU) and dopamine (DA) release and their correlated receptors in the key reward center--extended amygdala--which includes the shell of nucleus accumbens (sNAc) and central nucleus of amygdala (cAmg). It is unclear to date whether there is an alteration in the number of presynaptic GLU/DA nerve terminals. In this study, we investigated the number of GLU and DA terminals in the extended amygdala of alcohol-preferring (P) rats that chronically drank ethanol. P rats have a propensity to drink ethanol to intoxication and develop an alcohol dependency. The P rats were divided into (1) Water group given ad libitum chow and water for 14 weeks; (2) Continuous alcohol group (C-Alc) given ad libitum chow and choice of 15 or 30% (v/v) ethanol or water for 14 weeks; and (3) Repeated deprivation (RD-Alc) group given the same choice of ethanol or water for 6 weeks, followed by a twice repeated cycle of 2 weeks without ethanol followed by 2 weeks with ethanol. Two subpopulations of GLU terminals were labeled by immunostaining for the vesicular GLU transporter 1 (vGLUT1) and vesicular GLU transporter 2 (vGLUT2). DA terminals were labeled by immunostaining for tyrosine hydroxylase (TH). The GLU and DA immunostained (im) varicosities were quantified and analyzed using stereological methods. We found that chronic alcohol did not alter the number of TH-im terminals in the extended amygdala in either the C-Alc or RD-Alc drinking paradigms. Thus, the increases in extracellular levels of DA previously reported following chronic alcohol are likely due to a change in the efficiency of DA release rather than a change in the number of DA terminals. The number of vGLUT1-im terminals was also unchanged in the extended amygdala; however, the number of vGLUT2-im terminals, which represent the greater population of GLU terminals, was increased in the sNAc of the RD-Alc group compared to the Water group. Chronic alcohol is known to affect GLU release, and our findings indicate that repeated alcohol deprivation may preferentially increase GLU terminals in the sNAc bearing the vGLUT2, which are primarily afferents from the thalamus. Our results further indicate that repeated deprivation of alcohol can change the ratio of GLU to DA innervation in the sNAc, a key region of the reward circuitry.