Dihydrofolate reductase-activity in brain tissue. Effect of X-irradiation

The mechanism responsible for the toxic late effects of cranial irradiation, followed by the administration of systemic methotrexate (MTX) on brain tissue, is still under discussion. We studied the influence of X-irradiation on dihydrofolate reductase (E.C. 1.5.1.3) activity (DHFR), the enzyme inhibited by MTX. New Zealand white rabbits, 6-9 weeks old, underwent 24 Gy fractionated or 20 Gy single-dose brain irradiation using a 60Co source. Before, immediately following, and 1, 2, 4, 12 weeks after irradiation, DHFR was measured in brain and liver tissue by a photometric assay. DHFR in brain tissue was 11.9 +/- 2.9 mU/g wet weight (ww) X h and in liver tissue 121.8 +/- 24.2 mU/g ww X h. Fractionated brain irradiation with 2 Gy per day produced no significant changes in brain DHFR. Single-dose cranial irradiation significantly decreased brain DFHR (7.3 +/- 0.6 mU/g ww X h). Suppression of the developmental increase of DHFR by X-irradiation in young rabbits could be excluded by determining the unchanged brain-to-liver ratios of DHFR in the animals with fractionated brain irradiation.     

Long-term infusions of p-boronophenylalanine for boron neutron capture therapy: evaluation using rat brain tumor and spinal cord models

Rat 9L gliosarcoma cells infiltrating the normal brain have been shown previously to accumulate only approximately 30% as much boron as the intact tumor after administration of the boronated amino acid p-boronophenylalanine (BPA). Long-term i.v. infusions of BPA were shown previously to increase the boron content of these infiltrating tumor cells significantly. Experiments to determine whether this improved BPA distribution into infiltrating tumor cells after a long-term i.v. infusion improves tumor control after BNCT in this brain tumor model and whether it has any deleterious effects in the response of the rat spinal cord to BNCT are the subjects of the present report. BPA was administered in a fructose solution at a dose of 650 mg BPA/kg by single i.p. injection or by i.v. infusion for 2 h or 6 h, at 330 mg BPA/kg h(-1). At 1 h after the end of either the 2-h or the 6-h infusion, the CNS:blood (10)B partition ratio was 0.9:1. At 3 h after the single i.p. injection, the ratio was 0.6:1. After spinal cord irradiations, the ED(50) for myeloparesis was 14.7 +/- 0.4 Gy after i.p. administration of BPA and 12.9 +/- 0.3 Gy in rats irradiated after a 6-h i.v. infusion of BPA; these values were significantly different (P < 0.001). After irradiation with 100 kVp X rays, the ED(50) was 18.6 +/- 0.1 Gy. The boron compound biological effectiveness (CBE) factors calculated for the boron neutron capture dose component were 1.2 +/- 0.1 for the i.p. BPA administration protocol and 1.5 +/- 0.1 after irradiation using the 6-h i.v. BPA infusion protocol (P < 0.05). In the rat 9L gliosarcoma brain tumor model, the blood boron concentrations at 1 h after the end of the 2-h infusion (330 mg BPA/kg h(-1); n = 15) or after the 6-h infusion (190 mg BPA/kg h(-1); n = 13) were 18.9 +/- 2.2 microg 10B/g and 20.7 +/- 1.8 microg 10B/g, respectively. The irradiation times were adjusted individually, based on the preirradiation blood sample, to deliver a predicted 50% tumor control dose of 8.2 Gy ( approximately 30 photon-equivalent Gy) to all tumors. In the present study, the long-term survival was approximately 50% and was not significantly different between the 2-h and the 6-h infusion groups. The mode of BPA administration and the time between administration and irradiation influence the 10B partition ratio between the CNS and the blood, which in turn influences the measured CBE factor. These findings underline the need for clinical biodistribution studies to be carried out to establish 10B partition ratios as a key component in the evaluation of modified administration protocols involving BPA.     

Tissue Distribution, Metabolism, Anticonvulsant Efficacy, and Effect on Brain Amino Acid Levels of the Glia-Selective γ-Aminobutyric Acid Transport Inhibitor 4,5,6,7-Tetrahydroisoxazolo[4,5-c]pyridin-3-ol in Mice and Chicks

Using tritium-labelled 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) its tissue distribution and metabolism were investigated in adult mice and 4-day-old chicks after systemic administration of the drug. It was found not to be significantly metabolized in the brain since metabolites of THPO corresponding to only approximately 8% of the parent compound could be detected 30 min after administration of the drug intramuscularly in mice. In the liver, however, THPO was found to be metabolized to a considerable extent. In chicks THPO metabolites were found in the brain but they accounted for less than 35% of the radioactivity. The brain concentration of THPO in mice and chicks corresponded to respectively 10 and 50% of the dose injected intramuscularly and the tissue level was essentially constant for at least 3 h after injection. Following systemic administration of THPO to mice and chicks the contents of aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) in whole brain and in synaptosomes was determined. It was found that only GABA contents were affected being increased in synaptosomes from mice and decreased in whole brain in chicks. Doses of THPO, which in chicks but not in mice led to brain levels that were sufficient to inhibit glial GABA uptake, were found to protect chicks but not mice against isonicotinic acid hydrazide-induced seizures. The findings are compatible with the notion that THPO exerts its anticonvulsant activity by inhibition of astrocytic GABA uptake.     

Differential radiation effect in tumor and normal tissue after treatment with ramipril, an angiotensin-converting enzyme inhibitor

The angiotensin-converting enzyme inhibitor, ramipril, has been shown to mitigate radiation injury in normal tissues. Using A549 cell xenografts grown in athymic mice, we measured the effect of ramipril on radiation damage to tumors. Ramipril did not alter tumor response to radiation despite different times of drug administration with respect to radiation delivery (drug started 2 weeks before or immediately after irradiation). In contrast, using the same dose, ramipril reduced normal tissue radiation injury (30 Gy x 2 or 6 Gy x 10) as assessed by a semi-quantitative scale of skin damage and relative leg contraction. The results indicate that ramipril could offer therapeutic gain due to its different effect on normal tissues and tumors.     

Dextran retention in the rat brain following release from a polymer implant.

Intracranial controlled release polymers may improve drug administration to the brain, where therapy is frequently limited due to the low permeability of brain capillaries to therapeutic agents. On the basis of drug transport and elimination rates, we proposed that high molecular weight, water-soluble molecules would be retained in the brain space following release from an intracranial implant. To test this hypothesis, solid particles of different molecular weight fractions of fluorescein isothiocyanate labeled dextran (FITC-dextran; 4 x 10(3) Da (4 kDa) < weight-averaged molecular weight (Mw) < 150 kDa) or fluorescein were uniformly dispersed in matrices of a polyanhydride copolymer synthesized from a fatty acid dimer and sebacic acid in a 50:50 ratio, P(FAD:SA). When incubated in buffered saline, FITC-dextran fractions of 70 kDa Mw were released from the polymer within 48 h; 4 kDa Mw FITC-dextran and fluorescein were released more slowly. Following implantation of P(FAD:SA) matrices containing either 70 kDa Mw FITC-dextran, 4 kDa Mw FITC-dextran, or fluorescein into the brains of normal rats, fluorescent tracers were continuously released into the brain tissue for 30 days. Tracer concentrations within the brain were significantly higher for large molecular weight tracers (70 kDa Mw FITC-dextran > 4 kDa Mw FITC-dextran > fluorescein). The rate of elimination, kapp, of each tracer from the brain was determined by comparing experimental data with a model describing tracer diffusion/elimination in the brain extracellular space; kapp decreased with increasing molecular weight (fluorescein > 4 kDa Mw FITC-dextran > 70 kDa Mw FITC-dextran).     

Vascular permeability and late radiation fibrosis in mouse lung

It has been suggested that fibrosis which develops after irradiation is caused by increases in vascular permeability. Plasma proteins leak into irradiated tissue where fibrinogen may be converted into fibrin which is gradually replaced by fibrous tissue. Vascular and fibrotic changes in mouse lung were investigated after X irradiation of the right hemithorax. Blood volume and accumulation of extravascular proteins were measured using indium (111In)-labeled red cells, iodinated (131I) albumin, and iodinated (125I) fibrinogen. Tracers were injected 1-47 weeks after irradiation and lungs were excised 24 or 96 hr later to determine radioactivity. The amount of collagen was estimated by measuring the hydroxyproline content. During the first few months after X rays, lung blood volume decreased to a plateau which depended on radiation dose (10-25 Gy). Small increases in extravascular albumin and fibrinogen occurred at 1-12 weeks after 10-25 Gy. Subsequently, protein returned to normal after 10 Gy, remained elevated after 15 Gy, and increased after 20 and 25 Gy. Hydroxyproline per gram of dry irradiated lung was increased at 18 weeks after 15-25 Gy. Subsequently it showed little change although both total hydroxyproline content and dry weight decreased after 20 and 25 Gy. Support for the hypothesis was that hydroxyproline per gram only increased after X-ray doses which caused marked extravasation of protein. There was no evidence, however, for deposition of 125I-fibrin or for a gradual increase in fibrosis corresponding to the prolonged excess of extravascular protein.     

Caloric restriction overcomes pre-diabetes and hypertension induced by a high fat diet and renal artery stenosis

Background

Calorie restriction (CR) is a type of dietary intervention that is essential in weight loss through modulation of critical metabolic control pathways, is well established and understood in cases of systemic arterial hypertension, however, its role in renovascular hypertension is still unclear.

Methods

Rats were divided into three groups: SHAM, and two groups that underwent surgery to clip the left renal artery and induce renovascular hypertension (OH and OHR). The SHAM diet was as follows: 14 weeks normolipidic diet; OH: 2 weeks normolipidic diet?+?12 weeks hyperlipidic diet, both ad libitum; OHR, 2 weeks normolipidic diet?+?8 weeks ad libitum high-fat diet?+?4 weeks 40% calorie-restricted high-fat diet.

Results

Rats in the OHR group had decreased blood pressure, body weight, and glucose levels. Reductions in insulinemia and in lipid and islet fibrotic areas in the OHR group were observed, along with increased insulin sensitivity and normalization of insulin-degrading enzyme levels. The expression of nicotinamide phosphoribosyltransferase (NAMPT), insulin receptor (IR), sirtuin 1 (SIRT1), and complex II proteins were increased in the liver tissue of the OHR group. Strong correlations, whether positive or negative, were evaluated via Spearman’s model between SIRT1, AMPK, NAMPT, PGC-1α, and NNMT expressions with the restoration of normal blood pressure, weight loss, glycemic and lipid panel, and mitochondrial adaptation.

Conclusion

CR provided short-term beneficial effects to recover the physiological parameters induced by a high-fat diet and renal artery stenosis in obese and hypertensive animals. These benefits, even in the short term, can provide physiological benefits in the long term.

     

Determination of naringenin and its glucuronide conjugate in rat plasma and brain tissue by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method with ultraviolet detection was utilized for the investigation of the pharmacokinetics of naringenin and its glucuronide conjugate in rat plasma and brain tissue. Plasma and brain tissue were deproteinized by acetonitrile, then centrifuged for sample clean-up. The drugs were separated by a reversed-phase C₁₈ column with a mobile phase consisting of acetonitrile–orthophosphoric acid solution (pH 2.5–2.8) (36:64, v/v). The detection limits of naringenin in rat plasma and brain tissue were 50 ng/ml and 0.4 μg/g, respectively. The glucuronide conjugate of naringenin was evaluated by the deconjugated enzyme β-glucuronidase. The naringenin conjugation ratios in rat plasma and brain tissue were 0.86 and 0.22, respectively, 10 min after naringenin (20 mg/kg, i.v.) administration. The mean naringenin conjugation ratio in plasma was approximately four fold that in brain tissue.     

不同剂量的X-射线全身照射对小鼠健康状况及涎腺的影响

目的:通过直线加速器全身照射昆明小鼠建立辐射损伤模型,探索不同放射剂量对小鼠健康状况及涎腺功能和结构的影响。方法:选取八种不同剂量对昆明小鼠行体外全身照射,于照射后一个月内观察小鼠生长情况、体重变化;照射后一周、一个月检测各组小鼠血象的变化;测定放射半数致死剂量;照射后两个月,测定各组小鼠的唾液流量及唾液淀粉酶含量,并对下颌下腺组织切片行HE染色。结果:13Gy和15Gy照射组小鼠的体重逐渐下降,一周后死亡,其余组小鼠体重最终呈增加趋势。X-射线全身照射的半数致死量为10Gy。照射后一周,照射组小鼠的白细胞数目明显降低,与对照组比较有明显统计学差异(P0.01);在其他血象方面,除了7Gy组外,其他照射组与对照组比较也均有统计学差异(P0.05)。照射一个月后,各照射组小鼠的血象均恢复正常。照射后两个月,9Gy组和11Gy组小鼠的唾液流量及唾液淀粉酶含量均显著低于0Gy组,且11Gy组较9Gy组亦明显降低,差异均有统计学意义(P0.05)。随照射剂量的增加,小鼠的下颌下腺腺泡细胞数目逐步减少,结构排列紊乱,组织损伤逐渐加重。结论:X-射线全身照射引起小鼠健康状况受损,免疫功能减低,损伤程度与放射线强度呈剂量依赖性,小鼠半数致死量为10Gy,该剂量适合建立全身放射损伤模型。     

Retinoids and TIMP1 prevent radiation-induced apoptosis of capillary endothelial cells

Radiation-induced changes in capillaries constitute a basic injury in the pathogenesis of chronic radiation damage to the heart, lung, liver, kidney and brain. It is important to identify new radioprotectors for capillary endothelial cells for use during radiotherapy to minimize normal tissue damage and possibly to increase the deliverable dose. Previously we demonstrated that exposure to ionizing radiation (10 Gy) results in death of bovine adrenal capillary endothelial cells in confluent monolayers by apoptosis. We also showed that retinoids inhibit the growth of endothelial cells, induce their differentiation, down-regulate matrix metalloproteinase (MMP) production, and up-regulate tissue inhibitors of matrix metalloproteinases (TIMPs). In the present studies, we demonstrated that radiation (10 Gy) induced an immediate increase in the amounts and activation of MMP1 and MMP2 in the cell fraction and medium of bovine capillary endothelial cells followed by an incidence of apoptosis. We also obtained data indicating that radiation-induced apoptosis can be inhibited by exposing bovine capillary endothelial cells to all-trans-retinol or all-trans-retinoic acid for 6 days before irradiation, even when the vitamins were removed 24 h before irradiation. Finally, we determined that inhibition of MMPs by TIMP was sufficient to block radiation-induced apoptosis, suggesting that the mechanism of protection by retinoids is through the alteration of levels of MMPs and TIMPs produced by the cells.     

Irradiation to the immature brain attenuates neurogenesis and exacerbates subsequent hypoxic‐ischemic brain injury in the adult

Cranial radiotherapy is common in pediatric oncology. Our purpose was to investigate if irradiation (IR) to the immature brain would increase the susceptibility to hypoxic‐ischemic injury in adulthood. The left hemisphere of postnatal day 10 (P10) mice was irradiated with 8 Gy and subjected to hypoxia‐ischemia (HI) on P60. Brain injury, neurogenesis and inflammation were evaluated 30 days after HI. IR alone caused significant hemispheric tissue loss, or lack of growth (2.8 ± 0.42 mm³, p < 0.001). Tissue loss after HI (18.2 ± 5.8 mm³, p < 0.05) was synergistically increased if preceded by IR (32.0 ± 3.5 mm³, p < 0.05). Infarct volume (5.1 ± 1.6 mm³) nearly doubled if HI was preceded by IR (9.8 ± 1.2 mm³, p < 0.05). Pathological scoring revealed that IR aggravated hippocampal, cortical and striatal, but not thalamic, injury. Hippocampal neurogenesis decreased > 50% after IR but was unchanged by HI alone. The number of newly formed microglia was three times higher after IR + HI than after HI alone. In summary, IR to the immature brain produced long‐lasting changes, including decreased hippocampal neurogenesis, subsequently rendering the adult brain more susceptible to HI, resulting in larger infarcts, increased hemispheric tissue loss and more inflammation than in non‐irradiated brains.     

High-Precision Radiosurgical Dose Delivery by Interlaced Microbeam Arrays of High-Flux Low-Energy Synchrotron X-Rays

Microbeam Radiation Therapy (MRT) is a preclinical form of radiosurgery dedicated to brain tumor treatment. It uses micrometer-wide synchrotron-generated X-ray beams on the basis of spatial beam fractionation. Due to the radioresistance of normal brain vasculature to MRT, a continuous blood supply can be maintained which would in part explain the surprising tolerance of normal tissues to very high radiation doses (hundreds of Gy). Based on this well described normal tissue sparing effect of microplanar beams, we developed a new irradiation geometry which allows the delivery of a high uniform dose deposition at a given brain target whereas surrounding normal tissues are irradiated by well tolerated parallel microbeams only. Normal rat brains were exposed to 4 focally interlaced arrays of 10 microplanar beams (52 µm wide, spaced 200 µm on-center, 50 to 350 keV in energy range), targeted from 4 different ports, with a peak entrance dose of 200Gy each, to deliver an homogenous dose to a target volume of 7 mm³ in the caudate nucleus. Magnetic resonance imaging follow-up of rats showed a highly localized increase in blood vessel permeability, starting 1 week after irradiation. Contrast agent diffusion was confined to the target volume and was still observed 1 month after irradiation, along with histopathological changes, including damaged blood vessels. No changes in vessel permeability were detected in the normal brain tissue surrounding the target. The interlacing radiation-induced reduction of spontaneous seizures of epileptic rats illustrated the potential pre-clinical applications of this new irradiation geometry. Finally, Monte Carlo simulations performed on a human-sized head phantom suggested that synchrotron photons can be used for human radiosurgical applications. Our data show that interlaced microbeam irradiation allows a high homogeneous dose deposition in a brain target and leads to a confined tissue necrosis while sparing surrounding tissues. The use of synchrotron-generated X-rays enables delivery of high doses for destruction of small focal regions in human brains, with sharper dose fall-offs than those described in any other conventional radiation therapy.     

Multi-endpoint biological monitoring of phosphine workers

5-Aminosalicylic acid (5ASA), a prescribed drug for ulcerative colitis, is a potent scavenger of oxygen-derived free radicals. The present study was undertaken to ascertain its ability to protect against radiation-induced damage. The drug dose-dependent effect, optimum time of drug administration and radiation dose-dependent effect (0-4 Gy) on in vivo radiation protection against micronuclei induction in polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were studied in the bone marrow of mice. Intraperitoneal injection of 10-125 mg/kg of the drug 30 min before whole body irradiation with 3 Gy produced a significant reduction in the frequency of micronucleated erythrocytes at 24 h after exposure. The optimum dose for protection without drug toxicity was 25 mg/kg body weight. Injection of 25 mg/kg of the drug 60 or 30 min before or within 15 min after 3 Gy whole body gamma-irradiation resulted in a significant decrease in the radiation-induced PCE and NCE with micronuclei (MPCE and MNCE) and an increase in the ratio of PCE to NCE (P/N), at 24 h post-irradiation. Maximum effect was seen when the drug was administered 30 min before irradiation. Therefore, to study the radiation dose-response, mice were pre-treated with 25 mg/kg of 5ASA 30 min before 1-4 Gy of gamma-irradiation. Radiation increased the MN frequency linearly (r(2)=0.99) with dose. Pre-treatment with 5ASA significantly reduced the MN counts to 40-50% of the radiation (RT) alone values, giving a dose modification factor (DMF) of 2.02 (MPCE) and 2.53 (MNCE). Irradiation resulted in a dose-dependent decline in the P/N ratio at all the doses of radiation studied. 5ASA produced a significant increase in the P/N ratio from that of irradiated controls, at all doses of radiations tested. These results show that 5ASA protect mice against radiation-induced MN formation and mitotic arrest.     

Anaerobic shift of energy metabolism in the mouse brain during the recovery period in acute radiation sickness

Three months after whole-body irradiation of mice with a sublethal dose of 5 Gy a study was made of some indices of energy metabolism like tissue respiration, oxidative phosphorylation, and formation of lactic acid in the survived brain homogenate. Revealed were the diminution of coupling of tissue respiration of oxidative phosphorylation, the rate of oxygen consumption and the level of cyano-resistant respiration being constant, the increase in the rate of glycolysis in anaerobic and particularly, in aerobic conditions, and reduction of the Pasteur and Crabtree effects. The above mentioned changes in the brain energy metabolism seem to be a manifestation of the process of the reduced metabolism formation in the nervous tissue at the remote times after irradiation.     

MDA, oxypurines, and nucleosides relate to reperfusion in short-term incomplete cerebral ischemia in the rat.

Short-term incomplete cerebral ischemia (5 min) was induced in the rat by the bilateral clamping of the common carotid arteries. Reperfusion was obtained by removing carotid clamping and was carried out for the following 10 min. Animals were sacrificed either at the end of ischemia or reperfusion. Controls were represented by a group of sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. Neutralized perchloric acid extracts of brain tissue were analyzed by a highly sensitive high-performance liquid chromatography (HPLC) method for the direct determination of malondialdehyde, oxypurines, nucleosides, nicotinic coenzymes, and high-energy phosphates. In addition, plasma concentrations of malondialdehyde, hypoxanthine, xanthine, inosine, uric acid, and adenosine were determined by the same HPLC technique. Incomplete cerebral ischemia induced the appearance of a significant amount (8.05 nmol/g w.w.; SD = 2.82) of cerebral malondialdehyde (which was undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde (53.30 nmol/g w.w.; SD = 17.77) and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia (0.050 mumol/L; SD = 0.015), significantly increased after 5 min of ischemia (0.277 mumol/L; SD = 0.056) and was strikingly augmented after 10 min of reperfusion (0.682 mumol/L; SD = 0.094). A similar trend was observed for xanthine, uric acid, inosine, and adenosine, while hypoxanthine reached its maximal concentration after 5 min of incomplete ischemia, being significantly decreased after reperfusion. From the data obtained, it can be concluded that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism, thus representing early biochemical markers of short-term incomplete brain ischemia and reperfusion in the rat. In particular, these results suggest the possibility of using the variation of malondialdehyde, oxypurines, and nucleosides in peripheral blood as a potential biochemical indicator of reperfusion damage occurring to postischemic tissues.     

α-Lipoic acid attenuates x-irradiation-induced oxidative stress in mice

The development of nontoxic but effective radioprotectors is needed because of the increasing risk of human exposure to ionizing radiation. We have reported that α-lipoic acid confers considerable radio-protective effect in mouse tissues when given prior to x-irradiation. In the present study, α-lipoic acid supplementation prior to x-irradiation with 4 and 6 Gy significantly inhibited the radiation-induced decline in total antioxidant capacity (TAC) of plasma. Radiation-induced decline in non-protein sulfhydryl content (NPSH) of different tissues, namely, brain, liver, spleen, kidney, and testis, was also ameliorated significantly at both 4 and 6 Gy doses. Maximal augmentation of radiation-induced protein carbonyl content was observed in spleen followed by brain, kidney, testis, and liver. Maximal protection in terms of carbonyl content was observed in spleen (116%) at 6 Gy dose, and minimal protection was found in liver (22.94%) at 4 Gy dose. Maximal increase in MDA (malondialdehyde) content was observed in brain, followed by testis, spleen, kidney, and liver. Protection by α-lipoic acid pretreatment in terms of MDA content was maximal in brain (51.67%) and minimal in spleen. The findings support the idea that α-lipoic acid is a free-radical scavenger and a potent antioxidant.     

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

The kinetics of Toxoplasma gondii infection reactivation in the brain and muscles was analyzed in this study to determine the preferred tissue by the parasite during immunosuppression. Two groups of Wistar rats (G1 and G2) were inoculated with 10⁴ bradyzoites of BTU10 strain (genotype I), p.o., and other two groups (G3 and G4) were inoculated with 0.9% saline solution. G2 and G4 were immunosuppressed with dexamethasone (DXM) and hydrocortisone sodium succinate (HSS). The presence of antibodies was researched in all groups through modified agglutination test (MAT) on days 0 and 21 p.i., and brain and muscle tissues of the rats were bioassayed in mice. G2 rats died at approximately 19.2 days after drug treatment, while G1 rats survived. The reactivation was initially observed in G1 brain and G2 muscles. Thus, the initial reactivation in muscles after immunosuppression allows doctors to save precious time to control the evolution of reactivated infection, preventing brain damage to the host.     

Incomplete cerebral ischemia in the rat provokes increase of tissue and plasma malondialdehyde

Short-term incomplete cerebral ischemia was induced in the rat by bilaterally clamping for 5 min the common carotid arteries; subsequent reperfusion of 10 min was obtained by removing carotid occlusion. At the end of ischemia or reperfusion, animals were sacrificed by decapitation. A control group was represented by sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. A highly sensitive HPLC method for the direct determination of malondialdehyde, oxypurines, and nucleosides was used on 200 μL of brain tissue and plasma extracts. Incomplete cerebral ischemia induced the, appearance of a significant amout of tissue malondialdehyde (undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia, significantly increased after 5 min of ischemia, being strikingly augmented after 10 min of reperfusion. A similar trend was observed for oxypurines and nucleosides. From these data, it can be affirmed that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism thus representing early biochemical markers of short-term incomplete brain ischemia, and reperfusion in the rat.     

Comparative assessment of mutations in mtDNA of x-irradiated mice using mismatch-specific endonuclease and temporal temperature gradient electrophoresis

Mutations in brain mtDNA of mice X-irradiated to 5 Gy were determined using a mismatch-specific endonuclease (CEL I) and by temporal temperature gradient gel electrophoresis (TTGE). The CEL I nuclease method allows simultaneous analysis of multiple DNA samples and proves to yield better results than TTGE; it is more sensitive, quite reproducible, and economical, requiring no complicated equipment. The CEL I nuclease assay for mtDNA mutations in the brain of x-irradiated mice has shown that the amount of mutant mtDNA copies is markedly reduced (2–3 times) from the 8th to the 28th day after irradiation, while the overall amount of mtDNA copies in the brain tissue remains fairly constant over this period, though lower than in the control. It can be suggested that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the postirradiation period.