A new method of quantitatively assessing the opening of the blood-brain barrier in murine animal models.

The blood-brain barrier (BBB) restricts the delivery of drugs into the brain. Different strategies have been developed to circumvent this obstacle. One such approach, the osmotic BBB disruption (BBBD), has been under pre-clinical study since the 70's. Typically, qualitative ex vivo assessment of the extent of BBBD has been performed using Evan's blue staining technique. In this study, we describe a simple quantitative technique based on albumin indirect immunohistochemistry to measure the extent of BBB breach. Thirty Fischer rats were assigned to one of 6 groups: a control group, and BBBD groups with escalation in IA mannitol infusion rate: 0.06, 0.08, 0.10, 0.12 and 0.15 cc/s. Fifteen minutes after the BBBD procedure, the animals were sacrificed, brain harvested and sections stained for albumin. Using an image analysis software, isolated albumin staining pixels were expressed as a fraction of the treated hemisphere. This ratio was used as a percentage value in the intensity of the BBB permeabilization. All sections studied harbored staining, averaging 0.37% for the controls (group 1), 5.69% for group 2 (0.06 cc/s), 10.44% for group 3 (0.08 cc/s), 6.99% for group 4 (0.1 cc/s), 18.50% for group 5 (0.12 cc/s) and reaching 61.70% for group 6 (0.15 cc/s). Important variations were observed between animals. A threshold effect was observed, and animals in group 6 presented a significant increase in BBB permeabilization compared to the other groups. We hereby detail a simple technique that can be applied to quantitatively measure the extent of the BBB breach notwithstanding the pathological process.

Changes of the cerebral mannitol concentrations in the course of brain death of a rodent model.

Determining the time of brain death is one of the critical issues in forensic examinations. Few authors have attempted to determine the time of brain death using pharmacokinetic approaches. We investigated cerebral concentrations of mannitol of which a single dose (1 g/kg) was administered in the course of brain death. The inflation of an epidural balloon was adopted as a rodent model of brain death. Brain death was determined using ordinary tests. Specimens were collected 4 h after brain death. Brain water content was higher in brain dead (BD) groups than those in control groups. Cerebral concentrations of mannitol in the BD group were significantly higher than those in the control group (P<0.01). In all areas of brain the concentration was the highest at the time when mannitol was administered during balloon inflation. Interhemispheric difference in the cerebrum was observed, followed by balloon inflation (P<0.05). Significant differences were observed in the average concentration of administered mannitol before and after brain death in the contralateral hemisphere (P<0.01) and in the brainstem (P<0.01). As the concentrations of mannitol in the brain are affected by cerebral trauma and brain death follows, mannitol can be used to determine the time of brain death at forensic examinations.

[Effects of metabolic substrates and mannitol on efficiency of cardioplegic protection in isolated rat heart]. / Vliianie metabolicheskikh substratov i mannita na éffektivnost' kardioplegicheskoi zashchity izolirovannogo serdtsa krysy.

The aim of this work was to study rationality of addition of aspartic acid, phosphocreatine, mannitol and tris(bydroxymethyl) aminomethane (trisamine) to a sanguineous cardioplegic solution. Isolated perfused rat hearts were subjected to 40-min normothermic total ischemia and 30-min reperfusion. Cardioplegic solutions were infused for 5 min prior to ischemia. A modified Ringer solution with 25 mM KCI was used as control. Osmolarity and pH of cardioplegic solutions were 340+/-5 mOms and 7.6+/-0.1 at 22 degreesC, respectively. Efficiency of myocardial protection was evaluated by recovery of contractile and pump function during reperfusion. The optimal solution contained aspartic acid (21.5 mM), mannitol (20.0 mM) and trisamine (5 mM). By the end of reperfusion the heart protected by this solution showed almost complete recovery of coronary flow (98+/-3% of the initial value vs. 77+/-3% in the control), and 2.6-fold higher recovery of stroke volume compared to the control. As a result, recovery of external cardiac work index, calculated as cardiac output-mean perfusion pressure, was 64+/-1% of the initial value vs. 24+/-5% in the control. Increase in buffer capacity of this cardioplegic solution by trisamine (up to 20.0 mM) as well as addition of phosphocreatine (10.0 mM) did not result in further augmentation of cardiac function recovery. The results suggest promising perspectives for development of medicinal form of this solution.

HPLC simultaneous determination of glycerol and mannitol in human tissues for forensic analysis.

A method for simultaneous determination of glycerol and mannitol in various human tissues was devised and for this we used high-performance liquid chromatography (HPLC). Specimens were homogenized in a mixture of chloroform and methanol, phosphate buffer (pH 7.0) and pentaerythritol (IS) solution. After centrifugation, an aliquot of the aqueous layer was evaporated to dryness and derivatized with p-nitrobenzoyl chloride at 50 degrees C for 1h, then applied to HPLC with analytical conditions of: column, CAPCELL PAK C18 MG (250 mm x 3.0 mm i.d., 5 microm, Shiseido Co. Ltd., Tokyo, Japan); column temperature, 1-2 degrees C; mobile phase, 75% acetonitrile-distilled water containing 0.05% trifluoroacetic acid, 0.05% heptafluoro-n-butyric acid and 0.1% triethylamine; flow rate, 0.5 ml/min; wavelength, 260 nm. Calibration curves for both substances were linear in concentration ranges from 1 to 500 microg/0.1g and correlation coefficients exceeded 0.99. The relative standard deviation (R.S.D.) of the method was evaluated at concentrations of 10 and 100 microg/0.1g, and ranged from 0.84 to 10.6%. Using this method, we determined the regional distribution levels of glycerol and mannitol in various tissues from an autopsied brain dead man.

[The pharmacokinetics of poliosm]. / Farmakokinetika poliosma.

Experiments were conducted on cats to study the pharmacokinetics of polyosm possessing diuretic and antiedemic properties whose primary acting component is polyethyleneoxide 400. Biexponential dependence of the blood drug concentration on the time with T1/2 values of 41 min and 4.8 h was revealed. In the first 5 h after intravenous infusion of the drug 60.8% of the introduced dose of polyethyleneoxide 400 (1 g/kg) was excreted through the kidneys.