ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

Iatrogenic Impairment of Hearing During Surgery for Acoustic Neuroma

Isolated or combined labyrinthine, neural, and vascular damage account for failure to preserve hearing during removal of acoustic neuromas. However, the specific mechanisms of auditory impairment remain unclear unless surgical maneuvers can be related to peri- and postoperative hearing on the basis of intraoperative monitoring of auditory function.

Among the different auditory monitoring techniques, recording of cochlear nerve action potentials (CNAPs) from the intracranial portion of the nerve has proven particularly useful for identifying the mechanisms of iatrogenic auditory injury.

The present investigation analyzes intra- and postoperative auditory impairment in relation to surgical steps in a group of 38 subjects with acoustic neuroma (size ranging from 5 to 24 mm) undergoing removal via a retrosigmoid approach.

Coagulation close to the cochlear nerve, drilling of the internal auditory canal, and removal of the intrameatal portion of the acoustic neuroma have prove to be the most critical surgical steps in hearing preservation.

Changes were correlated with intra- and extrameatal tumor size, the relationship between the internal auditory canal and vestibule, and internal auditory canal enlargement, anatomic involvement of the cochlear nerve, preoperative auditory level, and ABR and ENG test findings.

Changes in CNAP morphology and latency are detailed, and mechanisms of injury are analyzed and discussed as a function of these variables.

     

Endoscopic treatment of benign tumors of the nose and paranasal sinuses.

OBJECTIVE: The endoscopic approach can be successfully employed for the treatment of benign tumors such as fibroosseus and vascular lesions, pleomorphic adenomas, gliomas, and schwannomas. STUDY DESIGN AND SETTING: Nineteen patients diagnosed with benign tumors of the nasal cavity and paranasal sinuses and treated using an endoscopic approach were retrospectively reviewed. The endoscopic approach was used exclusively in 15 cases and was associated with an external approach in 4 cases (due to an intracranial extension of the tumor in 3 patients and its location at the level of the anterior wall of the frontal sinus in the last case). RESULTS: The resection of the lesions was radical in 18 patients and subtotal in 1 case. The mean follow-up was 27 months and only 1 recurrence (5%) was observed in the juvenile angiofibroma group 20 months postoperatively. CONCLUSION: and significance In selected cases, endoscopic surgery can be considered an effective treatment for the resection of benign tumors involving the sinonasal tract.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Migraine and depression

Neurological Sciences - Investigations of migraine comorbidity have confirmed its association with diverse psychiatric conditions. This association appears to be stronger for major depression and...     

Evaluation of myocardial metabolism with microdialysis during bypass surgery with cold blood- or Calafiore cardioplegia.

BACKGROUND: For the first time, microdialysis was used to investigate in vivo and online the myocardial metabolism during and after cardiac surgery in patients treated with two different methods of myocardial protection. METHODS: Thirty patients underwent standard CABG with one of two different methods of myocardial protection. The patients were randomised to receive either cold blood (COLD group) or warm modified Calafiore cardioplegia (WARM group). Microdialysis probes were implanted into the myocardium of left ventricular apical region of the heart. Cardioplegia was given antegrade only. Microdialysis measurements were performed at time intervals before, during and 24 h after cardiopulmonary bypass and analysed for glucose, lactate, pyruvate and glycerol. RESULTS: Myocardial lactate concentrations were significantly higher in the WARM group compared with that of the COLD group, while serum lactate was comparable. Glycerol was significantly higher at the end of the clamping time in the WARM group. At the same time the glucose-lactate ratio as a marker of nutritional disorder had significantly lower levels in the WARM group. The cumulative CK-MB release over 24 h was significantly higher in those hearts protected with warm blood. CONCLUSIONS: The oxidative stress measured was significantly higher in patients undergoing CABG using modified Calafiore cardioplegia, whereas the cold cardioplegia minimised the effects of aortic clamping. The results indicate that cold cardioplegia offers superior protection of the heart, in terms of more rapid normalisation of myocardial metabolism. In elective myocardial revascularisation, intermittent antegrade warm blood cardioplegia is a comparable safe method of myocardial protection. However, in patients referring to a long clamping time, advantages of cold cardioplegia for myocardial revascularisation may be magnified.