Selective chemiluminescent TURN-ON quantitative bioassay and imaging of intracellular hydrogen peroxide in human living cells.

Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 µM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

The Kullback-Leibler autodependogram.

The autodependogram is a graphical device recently proposed in the literature to analyze autodependencies. It is defined computing the classical Pearson χ2-statistics of independence at various lags in order to point out the presence lag-depedencies. This paper proposes an improvement of this diagram obtained by substituting the χ2-statistics with an estimator of the Kullback-Leibler divergence between the bivariate density of two delayed variables and the product of their marginal distributions. A simulation study, on well-established time series models, shows that this new autodependogram is more powerful than the previous one. An application to a well-known financial time series is also shown.

Cervical spinal demyelination with ethidium bromide impairs respiratory (phrenic) activity and forelimb motor behavior in rats.

Although respiratory complications are a major cause of morbidity/mortality in many neural injuries or diseases, little is known concerning mechanisms whereby deficient myelin impairs breathing, or how patients compensate for such changes. Here, we tested the hypothesis that respiratory and forelimb motor functions are impaired in a rat model of focal dorsolateral spinal demyelination (ethidium bromide, EB). Ventilation, phrenic nerve activity and horizontal ladder walking were performed 7-14 days post-C2 injection of EB or vehicle (SHAM). EB caused dorsolateral demyelination at C2-C3 followed by significant spontaneous remyelination at 14 days post-EB. Although ventilation did not differ between groups, ipsilateral integrated phrenic nerve burst amplitude was significantly reduced versus SHAM during chemoreceptor activation at 7 days post-EB but recovered by 14 days. The ratio of ipsi- to contralateral phrenic nerve amplitude correlated with cross-sectional lesion area. This ratio was significantly reduced 7 days post-EB versus SHAM during baseline conditions, and versus SHAM and 14-day groups during chemoreceptor activation. Limb function ipsilateral to EB was impaired 7 days post-EB and partially recovered by 14 days post-EB. EB provides a reversible model of focal, spinal demyelination, and may be a useful model to study mechanisms of functional impairment and recovery via motor plasticity, or the efficacy of new therapeutic interventions to reduce severity or duration of disease.

Excimer Laser Assisted Non-occlusive Anastomosis (ELANA). Our experience with a training model in vivo.

AIM: In this study the ELANA Technique has been reproduced in our experimental laboratory in order to verify its feasibility and reproducibility, the percentage of patent anastomosis in acute at different steps along the learning curve of the surgical team, specific problems related to the surgical technique. METHODS: In 20 rabbits New Zealand 4kg body weight the training model in vivo proposed by Tulleken and coworkers has been reproduced, realizing 40 ELANA anastomosis. The model consists in the realization of two different end-to-side anastomosis on the abdominal aorta of each experimental animal, assisted by a special designed suction/excimer laser catheter, then connected by an end-to-end suture. After a few hours the animals are sacrificed and the by-pass site withdrawn and examined in order to verify the percentage of patency in acute. RESULTS: In the first 5 animals (group A), the anastomosis were realized using a jugular vein graft and the procedure results successful in only 3 cases out of ten (30%). For the following experiments - groups B, C and D where an aorta artery graft was used, the percentage of arterial flap retrieval was respectively 50%, 60% and 80%. CONCLUSIONS: ELANA is a feasible fascinating microsurgical technique for the realization of high flow, non-occlusive anastomosis. The rate of success results progressively higher along the learning curve of the surgical team. In our opinion, before the application of the ELANA technique on humans, a period of propaedeutic training in vivo on laboratory animals is essential for the dedicated team.

[Psychic disorders. An unknown complication of surgery of the posterior cranial fossa]. / Disturbi psichici. Una complicanza misconosciuta della chirurgia in fossa cranica posteriore.

Psychic trouble is not generally recognised as a possible complication of posterior fossa surgery. Reviewing a series of 63 pediatric patients operated on for posterior fossa intraxial tumors, the Authors found 21 cases of psychic troubles in the post-operative period. The incidence was higher in: a) medulloblastoma; b) rostral vermis location; c) supracerebellar infratentorial and transvermian approach. The neuropathophysiological basis of the phenomenon is discussed.

Surgical indications for intracranial arachnoid cysts.

Diagnostic work-up and management of intracranial arachnoid cysts are still controversial. The authors have standardized a therapeutic protocol based on the information derived from CSF contrast flow studies. The report concerns 16 cases of intracranial arachnoid cysts treated according to their protocol.

Trigeminal neuralgia treated by percutaneous thermocoagulation. Comparative analysis of percutaneous thermocoagulation and other surgical procedures.

The authors analyze the results they obtained by percutaneous radiofrequency technique for trigeminal neuralgia. The clinical material consists of 605 cases observed from 1977 to 1986 at their Institute. There was a female preponderance (62%) and an average age of 65 years. Idiopathic, atypical and symptomatic trigeminal neuralgia has been diagnosed respectively in 568, 21 and 16 cases. From 1977 to 1980 the working temperature was above 65 C, thereafter a lower temperature has been employed to coagulate the Gasserian ganglion. The rate of pain relief was 97% for idiopathic trigeminal neuralgia, 75% for symptomatic and 21% for atypical. The loss of facial sensation accounts for 80% of side effects of this procedure in their series. The recurrence of pain was observed in 16% of cases with a follow-up ranging from 2 to 10 years. It is noteworthy that there is a correlation between the coagulation temperature and the rate of recurrence, the higher the former, the lower the latter. The authors compare their results (rate of pain relief, morbidity, mortality, rate of recurrence) with those of major reports in the literature concerning either percutaneous or other surgical procedures (mircosurgical decompression of the trigeminal nerve, glycerol injection into the trigeminal cistern and percutaneous microcompression).

Giant aneurysm of the calloso-marginal artery.

A giant aneurysm of the right callosomarginal artery is reported in a 3-month-old child. This location is rare: including our case reported here, only three cases have been described. Clinicoradiological findings are presented and the surgical procedure is illustrated.

Neutrophil chemotactic activity in human gastric secretions.

To study levels of neutrophil chemotactic activity in human gastric secretions, these secretions were collected via the endoscope during elective esophagogastroduodenoscopy. Fresh samples were then prepared for the neutrophil chemotactic assay with the use of human peripheral neutrophils in modified Boyden chambers. Mean maximum chemotactic activity was 45.8% +/- 11.2% in patients with gastric inflammation compared with only 10.1% +/- 4.2% in patients with normal results of endoscopic examination. The chemotactic factors responsible for this chemotactic activity may play a role in the recruitment of neutrophils to areas of gastric mucosal injury.

Simplified, noninvasive PET measurement of blood-brain barrier permeability.

Blood-brain barrier (BBB) permeability to [68Ga]EDTA was measured by positron emission tomography (PET) in four normal volunteers and in 11 patients with brain tumors. A unidirectional transfer constant, Ki, was calculated applying multiple-time graphical analysis (MTGA). This method allows the detection of backflux from brain to blood and, by generalization, the measurement of the constant Kb (brain to blood). Furthermore, the need for an independent measurement of the intravascular tracer is obviated: MTGA itself provides an estimate of the cerebral plasma volume (Vp). In the four normal volunteers the Ki was 3.0 +/- 0.8 X 10(-4) ml g-1 min-1 (mean +/- SD) and the Vp 0.034 +/- 0.007 ml g-1. A net increase in Ki up to a maximum of 121.0 X 10(-4) ml g-1 min-1 (correspondent value of Kb = 0.025 min-1) as well as an increase of Vp was observed in malignant tumors. The input function was calculated using both the [68Ga]EDTA concentration in sequential arterial blood samples and, noninvasively, the activity derived from the superior sagittal sinus image. The values of Ki and Vp from these two calculations were in good agreement. The application of MTGA to PET permits the evaluation of passage of substances across the BBB without making assumptions about the compartments in which the tracer distributes.

Cavernous sinus meningiomas.

Analysing 12 cases of cavernous sinus meningiomas the authors conclude that: the clinical history is characteristic; CT scan has proved to be more sensitive than carotid angiography in detecting small lesions. However in large lesions the vascular supply pattern is the only feature that exactly identifies the site of the lesion. Both angiography and CT scan allow a near definitive diagnosis of meningioma; because of their location it is almost always impossible to remove these tumours completely. Actually the best treatment is a piecemeal subtotal microsurgical removal via a subtemporal approach. Radiotherapy is indicated for partially resected lesions. On the basis of the revision of the anatomy of this region and looking forward to a widespread use of the Cavitron in neurosurgery, a more radical approach to this lesion is foreseen.

Intracellular pH changes during neutrophil activation: Na+/H+ antiport.

Activation of the Na+/H+ antiport mechanism was studied in human neutrophils by monitoring intracellular pH with a carboxyfluorescein derivative. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phospholipase C (PLC) induced biphasic pH changes. Amiloride, which inhibits the antiport, completely blocked alkalinization but enhanced acidification. Polymyxin B, which inhibits protein kinase C, only blocked alkalinization. Activation with phorbol 12-myristate 13-acetate (PMA) led to alkalinization only; this was inhibited by amiloride or polymyxin B. Thus, during polymorphonuclear leukocyte (PMN) activation, intracellular alkalinization appears to be mediated by an amiloride-sensitive Na+/H+ antiport. Antiport activity can also be blocked indirectly by inhibition of protein kinase C activity. Early intracellular acidification does not appear to require kinase activity but is observed when phospholipids are remodeled with PLC. The antiport was also activatable by hypertonic buffered media. This response did not appear to be mediated by protein kinase C because it was unaffected by polymyxin B. Finally, superoxide generation was investigated. It is affected by, but not soley controlled by, either antiport or protein kinase C activity.

Cerebral pan-angiography in rabbit. Technical note.

An open technique of cerebral four-vessel angiography via femoral artery "in vivo" of the rabbit is described. This method allows a good visualization of extracranial and intracranial circulation without direct damage to the cerebral vessels.

Surgical management of acute cerebellar infarction.

After reviewing the literature, a personal series of 10 adult patients with cerebellar infarction diagnosed by CT scan is described. The clinical picture in young adult men is characterized by rapid onset of headache, vomiting, vertigo, ataxia and blurred vision. After this sudden onset the patients may present a stable course or a rapid or delayed onset of brain stem compression, revealed by impairment of consciousness. CT scan is the diagnostic method of choice. The correlation between angiographic and CT localization of the infarction is not good. For therapy the following policy is suggested: in alert and clinically stable patients: medical treatment (mannitol, glycerol, dexamethason), ICP and serial CT monitoring; in alert patients with hydrocephalus or mass effect: medical treatment and monitoring as mentioned before; ventricular drainage if ICP surpasses 350 mm H2O; in patients with impaired consciousness and hydrocephalus or mass effect: immediate ventricular drainage. If it is not followed by prompt improvement of the level of consciousness, an emergency suboccipital craniectomy with removal of the infarcted tissue should be done.

Ceftazidime in the treatment of infective complications of spinal cord lesions.

The use and the abuse of the antibiotics are certainly a subject of great concern, particularly for the increasing incidence of multiresistant bacteria. Since these resistant organisms may be sensitive to cephalosporin, we have tested the effectiveness of a new cephalosporin, ceftazidime, in the treatment of resistant infection in spinal cord injured patients. A randomized study of ceftazidime versus amikacin was carried out on twenty patients. The preliminary results are presented.