Effects of disrupted ghrelin receptor function on fear processing, anxiety and saccharin preference in mice.

BACKGROUND: Obesity is a risk factor for stress-related mental disorders such as post-traumatic stress disorder. The underlying mechanism through which obesity affects mental health remains poorly understood but dysregulation of the ghrelin system may be involved. Stress increases plasma ghrelin levels, which stimulates food intake as a potential stress-coping mechanism. However, diet-induced obesity induces ghrelin resistance which in turn may have deleterious effects on stress-coping. In our study, we explored whether disruption of ghrelin receptor function though high-fat diet or genetic ablation affects fear processing, anxiety-like behavior and saccharin preference in mice. METHODS: Adult male C57BL6/J mice were placed on a standard diet or high-fat diet for a total period of 8 weeks. We first established that high-fat diet exposure for 4 weeks elicits ghrelin resistance, evidenced by a blunted hyperphagic response following administration of a ghrelin receptor agonist. We then carried out an experiment in which we subjected mice to auditory fear conditioning after 4 weeks of diet exposure and evaluated effects on fear extinction, anxiety-like behavior and saccharin preference. To explore whether fear conditioning as such may influence the effect of diet exposure, we also subjected mice to auditory fear conditioning prior to diet onset and 4 weeks later we investigated auditory fear extinction, anxiety-like behavior and saccharin preference. In a final experiment, we further assessed lack of ghrelin receptor function by investigating auditory fear processing, anxiety-like behavior and saccharin preference in ghrelin receptor knockout mice and their wild-type littermates. RESULTS: High-fat diet exposure had no significant effect on auditory fear conditioning and its subsequent extinction or on anxiety-like behavior but significantly lowered saccharin preference. Similarly, ghrelin receptor knockout mice did not differ significantly from their wild-type littermates for auditory fear processing or anxiety-like behavior but showed significantly lower saccharin preference compared to wild-type littermates. CONCLUSION: Taken together, our data suggest that disruption of ghrelin receptor function per se does not affect fear or anxiety-like behavior but may decrease saccharin preference in mice.

Low predictive value of intrinsic fibroblast radiosensitivity for fibrosis development following radiotherapy for breast cancer.

PURPOSE: To test whether the intrinsic radiosensitivity of skin fibroblasts from breast cancer patients correlates with the degree of breast fibrosis after breast conserving therapy. METHODS: In a systematic study design, 79 patients were selected from an earlier study group of 385 patients based on observed fibrosis and seven identified clinical risk factors for fibrosis development. In vitro radiosensitivity of patients' dermal fibroblasts was determined by clonogenic assay of early passage cultures. Survival was determined after irradiation at 0, 2 and 4 Gy, given in two fractions of 2 Gy with a 6 h interval. RESULTS: There was a significant inter-patient variation for SF2 values (coefficient of variation = 40%). The ratio of SF2 values for fibroblasts from patients with breast fibrosis versus those without was 0.80 (95% CI: 0.60-1.07). This was a statistically non-significant trend (p = 0.13). The same ratio for a derived value for SF2 ((SF2 + square root of SF4)/2) was 0.88 (p = 0.19). CONCLUSIONS: A significant variation in intrinsic radiosensitivity of breast cancer patients' dermal fibroblasts was observed. However, the degree of radiosensitivity did not show a significant correlation with fibrosis development. This indicates that the use of fibroblast radiosensitivity will have a limited usefulness for predicting fibrosis following breast irradiation.

Radiation-induced differentiation of human skin fibroblasts: relationship with cell survival and collagen production.

The aim of this study was to determine whether significant inter-individual differences exist between skin fibroblast strains obtained from radiotherapy patients in both radiation-induced differentiation and collagen production in vitro, for use as potential parameters for a predictive assay for fibrosis following radiotherapy in patients. Morphological cell differentiation was determined 7 days after irradiation in seven early-passage primary human fibroblast cell strains and correlated with cell survival. Collagen production was measured in two cell strains by flow cytometry and incorporation of 3H-proline. There was a wide variation in the extent of radiation-induced differentiation for the seven cell strains, each showing a dose-related increase. The correlation between induced differentiation and cell survival was poor (r = 0.64) but statistically significant (p < 0.01). Collagen synthesis increased 7 days after irradiation for one cell strain (HF-48), as measured by incorporation of 3H-proline, but not in radiation sensitive AT-1 cells. The collagen I content of the two cell strains was assessed by flow cytometry but no significant differences were observed between the strains tested or with increasing dose. In conclusion, marked variations in radiation-induced fibroblast differentiation were observed between patients, this being an important criterion for a predictive assay.

Pharmacokinetics in melanoma-bearing mice of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a candidate compound for boron neutron capture therapy.

Blood pharmacokinetics and tissue distribution of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a boron carrier with postulated melanin-seeking properties for boron neutron capture therapy, were determined in C57/BL mice with subcutaneous pigmented or non-pigmented B16 melanomas. Borocaptate sodium (BSH) was used as a boron compound without melanin-seeking properties in a comparative biodistribution study in the same animal tumour models. Administration of single doses showed that BPTU was retained better in the pigmented B16 tumour than in the non-pigmented variant. BPTU was found in large concentrations in kidney and liver. Brain boron was approximately 10-fold lower than tumour boron. On a molar basis, BPTU demonstrated higher affinity for B16 tumours than BSH. Owing to solubility limits, tumour boron concentrations in this mouse study were too low for effective application of BNCT. However, the high tumour-to-blood and tumour-to-normal tissues ratios indicate that, with appropriate formulation, BPTU could be a promising candidate for clinical BNCT.

Reduction of systemic exposure and toxicity of cisplatin by encapsulation in poly-lactide-co-glycolide.

The tissue distribution and normal tissue toxicity of cisplatin (cDDP) administered as poly-lactide-co-glycolide (PLAGA) microspheres, developed for loco-regional administration of cDDP to the liver, were studied in Wag/Rij rats. Venoportal administration of this formulation resulted in a reduction in total systemic and renal toxicity, which correlated with a decrease in normal tissue exposure to cDDP while maintaining high liver platinum levels. Liver-to-kidney platinum level ratios were 28 times higher after 4 h and 19 times higher after 24 h with PLAGA-cDDP microspheres than with free cDDP. Liver-to-blood platinum ratios at these times were 38 times and 36 times higher using PLAGA-cDDP. In a CC531 colon carcinoma liver micrometastases model, cytotoxicity of microsphere-released cDDP was confirmed in vivo by equal inhibition of tumor growth by PLAGA-cDDP and free cDDP over a period of 26 days. Free cDDP, however, caused significantly more histological renal damage and total body weight loss. The results were supported by the finding of higher plasma creatinine and urea concentrations 26 days after administration of free cDDP. Kidney platinum levels were 7 times lower when PLAGA-cDDP was used. These findings indicate a sparing effect on normal tissues when cDDP is targeted to the liver by formulation in PLAGA. PLAGA-cDDP microspheres may, therefore, be a useful and effective addition to current techniques of loco-regional chemotherapy for disseminated hepatic tumors.

Polymer-coated albumin microspheres as carriers for intravascular tumour targeting of cisplatin.

We used a poly-lactide-co-glycolide polymer (PLAGA 50:50) to formulate cisplatin (cDDP) into microspheres designed for intravascular administration. Two systems were developed. PLAGA-coated albumin microspheres and microspheres consisting of PLAGA only. PLAGA-coated microspheres displayed a mean diameter of 31.8 +/- 0.9 microns and a payload of 7.5% cDDP (w/w). Solid PLAGA microspheres exhibited a mean diameter of 19.4 +/- 0.6 microns and a payload of 20% cDDP. Release characteristics and in vitro effects on L1210 leukemia and B16 melanoma cell lines were investigated. Both types of microsphere overcame the initial rapid release of cDDP (burst effect), and PLAGA-coated albumin microspheres also showed a lag phase of approximately 30 min before cDDP release began. PLAGA-coated albumin microspheres released most of their payload through diffusion, and the coating eventually cracked after 7 days' incubation in saline supplemented with 0.1% Tween at 37 degrees C, enabling the release of any cDDP remaining. Effects of platinum, pre-released from PLAGA-coated albumin microspheres on the in vitro growth of L1210 cells were comparable with those of standard formulations (dissolved) of cDDP. Material released from non-drug-loaded PLAGA microspheres had no effect on L1210 cell growth, suggesting the absence of cytotoxic compounds in the matrix. The colony-forming ability of B16 cells was also equally inhibited by standard cDDP and pre-released drug. These studies show that formulation of cDDP in PLAGA-based microspheres prevents the rapid burst effect of cDDP seen in previous preparations and offers an improved system of administration for hepatic artery infusion or adjuvant therapy, enabling better clinical handling and the promise of a higher ratio of tumour tissue to normal tissue.