Studies on the intracerebral injection of bleomycin free and entrapped within liposomes in the rat.

Reverse phase prepared liposomes of defined composition (dipalmitoyl phosphatidyl choline, cholesterol and dipalmitoyl phosphatidic acid; 7:2:1; DPC) when injected intracerebrally in the rat produced no tissue damage beyond that of the penetrating wound, or altered behaviour pattern over 1 week of observation. The cerebral tissue response and behaviour of rats injected with bleomycin of increasing concentration, free and entrapped within liposomes was studied in short and long term experiments. In separate experiments blood, urine and tissue levels of bleomycin were measured after intracerebral injection of free and liposome entrapped bleomycin in the rat. These studies demonstrated that bleomycin when entrapped within liposomes and injected intracerebrally was of low toxicity to normal cerebral tissue, and was cleared more slowly from the injection site than when the free drug was injected. The results obtained indicated a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of cerebral gliomas.     

Studies on the use of antimitotic drugs entrapped within liposomes and of their action on a human glioma cell line

The optimal conditions have been determined for producing the maximum incorporation within liposomes of two cell cycle-specific antimitotic drugs (bleomycin and vincristine) used in the treatment of human cerebral gliomas. Under these conditions the subsequent rate of release of these two drugs was studied in vitro. Neither cell fusion nor endocytosis could be demonstrated between liposomes of the size distribution used in these experiments (0.1-15 micron) and a human glioma cell line (U.251-MG) using a variety of marker techniques. Dose-response curves were similar for the two drugs whether free or entrapped within liposomes when applied to this cell line. These results suggest that drugs entrapped within liposomes may be useful as a potential depot preparation in the treatment of human gliomas.     

A potential application for the intracerebral injection of drugs entrapped within liposomes in the treatment of human cerebral gliomas.

After intravenous injection of therapeutic doses of bleomycin only small amounts could be measured in glioma tissue obtained at operation in patients with malignant gliomas and the drug was rapidly cleared from the blood (T1/2 = 2 hrs). Negatively charged liposomes injected through Ommaya reservoirs into the glioma tumour bed were tolerated without observable side effect. The appearance of bleomycin in the blood and urinary clearance after intracerebral injection of bleomycin entrapped within negatively charged liposomes through an Ommaya reservoir in patients with malignant gliomas was decreased as compared with levels when free bleomycin was injected. These preliminary observations suggest a role of drugs entrapped within liposomes as a depot preparation in the treatment of human cerebral gliomas.     

Efficiency of liposomal ATP in cerebral ischemia: Bioavailability features

This study was performed to elucidate the mechanism by which adenosine triphosphate (ATP) encapsulated into liposomes was able to protect against experimental brain ischemia in the rat. After intracarotidal administration of liposomally entrapped ATP, the ATP blood level increased dramatically whereas no change was observed after administration of free ATP. This suggested that liposomes may protect ATP from its degradation by endothelial ectonucleotidases. On the other hand, it was observed that after administration of liposomally entrapped carboxyfluorescein (CF) to ischemic rats, the distribution of the brain fluorescence under the form of numerous punctiform structures was completely different from the diffuse fluorescence obtained with free CF injections. These data suggest that under certain hypoxic conditions the blood-brain barrier is open allowing the liposomes to reach the cerebral parenchyma. The mechanism of brain uptake is, however, still unclear: endothelial tight junctions opening or endothelial transcytosis.     

Delivery of antisense oligonucleotides to neuroblastoma cells

pH-Sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesterol hemisuccinate (3:2 mol/mol) were applied in delivery of antisense oligodeoxynucleotides (asODN) into NG 108-15 neuroblastoma and glioma cells. Fluorescently labelled asODN were entrapped in liposomes by a modified freeze-thawing method (20% encapsulation efficiency). The uptake of asODN (free or entrapped in liposomes) by NG 108-15 cells was monitored by fluorescence-activated cell sorting and confocal microscopy. Delivery of asODN was significantly improved when antisense were entrapped in liposomes as compared to free (nonliposomal) asODN. The uptake was dose-dependent and optimum was achieved after 2 h incubation.     

Comparison of anticonvulsant effects of valproic acid entrapped in positively and negatively charged liposomes in amygdaloid-kindled rats

Intraperitoneal injection of free valproic acid (VPA) suppressed amygdaloid-kindled seizure 1 h after injection in rats, but had no effect at 24 h. VPA entrapped in positively charged liposomes showed a prolonged anticonvulsant effect lasting for 2 days, while the effect evaluated at 1 h was not different from that with free VPA. VPA entrapped in negatively charged liposomes exerted a significantly stronger effect at 1 h than did free VPA, while it had no significant effect at 24 h. These results suggest that surface charges on liposomes play an important role in modifying the anticonvulsant effect of VPA.     

CDP-choline liposomes provide significant reduction in infarction over free CDP-choline in stroke

Cytidine-5'-diphosphocholine (CDP-choline, Citicoline, Somazina) is in clinical use (intravenous administration) for stroke treatment in Europe and Japan, while USA phase III stroke clinical trials (oral administration) were disappointing. Others showed that CDP-choline liposomes significantly increased brain uptake over the free drug in cerebral ischemia models. Liposomes were formulated as DPPC, DPPS, cholesterol, GM(1) ganglioside; 7/4/7/1.57 molar ratio or 35.8/20.4/35.8/8.0 mol%. GM(1) ganglioside confers long-circulating properties to the liposomes by suppressing phagocytosis. CDP-choline liposomes deliver the agent intact to the brain, circumventing the rate-limiting, cytidine triphosphate:phosphocholine cytidylyltransferase in phosphatidylcholine synthesis. Our data show that CDP-choline liposomes significantly ( P < 0.01) decreased cerebral infarction (by 62%) compared to the equivalent dose of free CDP-choline (by 26%) after 1 h focal cerebral ischemia and 24 h reperfusion in spontaneously hypertensive rats. Beneficial effects of CDP-choline liposomes in stroke may derive from a synergistic effect between the phospholipid components of the liposomes and the encapsulated CDP-choline.     

Tissue affinity of the infusate affects the distribution volume during convection-enhanced delivery into rodent brains: implications for local drug delivery

Convection-enhanced delivery (CED) is a recently developed technique for local delivery of agents to a large volume of tissue in the central nervous system (CNS). We have previously reported that this technique can be applied to CNS delivery of nanoparticles including viruses and liposomes. In this paper, we describe the impact of key physical and chemical properties of infused molecules on the extent of CED-mediated delivery. For simple infusates, CED distribution was significantly increased if the infusate was more hydrophilic or had less tissue affinity. Encapsulation of tissue-affinitive molecules by neutral liposomes significantly increased their tissue distribution. The poorer brain distribution observed with cationic liposomes, due to their greater tissue affinity, was completely overcome by PEGylation, which provides steric stabilization and reduced surface charge. Finally, liposomal encapsulation of doxorubicin reduced its tissue affinity and substantially increased its distribution within brain tumor tissue. Taken together, the physical and chemical properties of drugs, small molecules and macromolecular carriers determine the tissue affinity of the infusate and strongly affect the distribution of locally applied agents. Thus, an increased and more predictable tissue distribution can be achieved by reducing the tissue affinity of the infusate using appropriately engineered liposomes or other nanoparticles.     

Anticonvulsant effect of liposome-entrapped superoxide dismutase in amygdaloid-kindled rats.

Amygdaloid-kindled rats received intravenous human copper-zinc superoxide dismutase (CuZn-SOD) either in free form or entrapped within liposomes (SOD-L), at 5, 10 or 20 mg/kg. The animals were stimulated at the generalized seizure-triggering threshold 5 min, 2 h and then every 24 h after the drug was given, until 5 consecutive stage 5 seizures were induced. Free CuZn-SOD had little or no effect. However, SOD-L, particularly at 10 mg/kg, had a prolonged anticonvulsant effect, although there was great individual variation in the onset and duration of seizure suppression. This effect of SOD-L may be due to the ability of liposomes to act as a depot for the sustained release of drugs.     

Allopurinol pretreatment improves evoked response recovery following global cerebral ischemia in dogs

The reperfusion of previously ischemic tissue may lead to the formation of highly reactive free radicals that promote tissue injury. Xanthine oxidase has been implicated as one source of these free radicals. We examined the role of xanthine oxidase in brain injury using a cerebrospinal fluid compression model of global cerebral ischemia with 15 minutes of ischemia and 4 hours of reperfusion. Seven dogs were pretreated with the xanthine oxidase inhibitor allopurinol (50 mg/kg for 5 days). Neurophysiological recovery was monitored with cortical somatosensory evoked potentials. As an attempt to correlate brain recovery with the mechanism of protection, free brain malondialdehyde was measured at the end of reperfusion by high-performance liquid chromatography. Brain water content was measured by wet-dry weights. Compared with seven untreated control dogs, allopurinol pretreatment significantly improved recovery of somatosensory evoked potentials after 4 hours of reperfusion. However, the amount of free malondialdehyde in the allopurinol-treated dogs was 32% greater than that in the controls. Brain water content was similar in the two groups. These results suggest that xanthine oxidase contributes to brain injury after ischemia and reperfusion. However, tissue damage caused by xanthine oxidase may be mediated through mechanisms other than free radical production.     

首乌延寿丹对大鼠局灶性脑缺血的保护作用

目的研究首乌延寿丹对大鼠局灶性脑缺血的保护机制。方法采用改良的线栓法制作大鼠大脑中动脉栓塞模型(MCAO)。将健康成年大鼠随机分为5组:假手术组、缺血模型组、缺血前首乌延寿丹低、中、高处理组。测定丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化氢酶(CAT),并进行病理组织学检查。结果首乌延寿丹能明显提高缺血脑组织中SOD、CAT活性,降低MDA含量,减轻脑水肿,首乌延寿丹能改善脑缺血造成的大鼠脑组织损伤。结论首乌延寿丹对大鼠局灶性脑缺血有明显的保护作用,其机制可能是通过有效的拮抗自由基损伤,提高脑组织抗氧化能力来实现的。     

Role of brain tissue leukotriene in brain oedema following cerebral ischaemia: effect of a 5-lipoxygenase inhibitor, AA-861

It has been postulated that lipoxygenase metabolites of arachidonic acid play a role in the pathogenesis of cerebral ischaemia. Severe forebrain ischaemia in rats was induced by four-vessel occlusion with mild hypotension. After 30 min of ischaemia, circulation was restored by removing the arterial clamps and increasing blood pressure to preischaemic levels. During 30 min of cerebral ischaemia, free arachidonic acid increased by approximately 8.5 times compared with the preischaemic level. This accumulation was reversed within 60 min of reperfusion. The concentration of leukotriene C4 in brain tissue increased significantly during reperfusion: treatment with a 5-lipoxygenase inhibitor, AA-861, decreased the increase of brain water content associated with reperfusion. This study demonstrated that the increased arachidonic acid resulting from cerebral ischaemia in rats is metabolized to leukotrienes via the lipoxygenase pathway once circulation is restored, and these leukotrienes may play some role in the development of postischaemic cerebral oedema.     

Thrombotic thrombocytopenic purpura in the brain: possible prognostic impact of von Willebrand factor physiology

We report three patients with CNS involvement in thrombotic thrombocytopenic purpura (TTP). The first two cases presented with fluctuating levels of consciousness and lateralized neurologic deficits. Repeated brain CT scans were normal in case 1, and showed small parietal lucencies in case 2. Both patients recovered to only minor residues after combined treatment of plasma exchange, corticosteroids, immunoglobulins, vincristine (case 1), and cyclophosphamide (case 2), respectively. Case 3 with multiple infarctions in brain CT died of heart failure. Autopsy showed myocardial infarction and multiple hemorrhages in various organs including cerebral cortex. Excess deposition of von Willebrand factor antigen within hyaline thrombi in small and medium-sized arteries of the cerebral cortex is shown for the first time immunohistologically.     

Ischemic brain edema and the osmotic gradient between blood and brain

The relationship of the osmotic pressure gradient between blood and brain, and the development of ischemic brain edema was studied. Focal cerebral ischemia was produced by left middle cerebral artery occlusion in rats. Brain osmolality was determined with a vapor pressure osmometer, brain water content by wet-dry weight, and tissue sodium and potassium contents by flame photometry. Permeability of the BBB was tested by Evans blue. Measurements were made from the ischemic cortex within 14 days of occlusion. Brain osmolality increased from 311 +/- 2 to 329 +/- 2 mOsm/kg by 6 h after occlusion. Serum osmolality did not change significantly. The osmotic gradient between blood and brain peaked at approximately 26 mOsm/kg. Brain osmolality then decreased to 310 +/- 2 mOsm/kg by 12 h after occlusion and remained at about that same level. Water content increased progressively within 1 day of occlusion, then gradually decreased by 14 days. Brain tissue sodium plus potassium content did not increase within 6 h of occlusion, and Evans blue extravasation was not seen within that time. These findings indicate that an osmotic pressure gradient contributes to the formation of edema only during the early stage of cerebral ischemia. Furthermore, the increase in brain osmolality is not related to tissue electrolyte change or BBB disruption to protein.     

Time-dependent changes in cerebral choline and acetylcholine induced by transient global ischemia in rats

We occluded the carotid and vertebral arteries of 12 rats for 15 minutes to measure the brain concentrations of choline and acetylcholine and cerebral blood flow at the end of the ischemic period or 15, 30, or 150 minutes after circulation was reestablished. The animals were sacrificed with microwave radiation focused to the head immediately after a brief infusion of [14C]iodoantipyrine with rapid sampling of arterial blood. Brain tissue samples were extracted with ether to separate the tracer, which was subsequently measured by liquid scintillation counting and used to calculate local cerebral blood flow. The aqueous phase was then processed for the measurement of choline and acetylcholine concentrations by gas chromatography/mass spectrometry. The results showed a large increase in tissue choline content and a decrease in tissue acetylcholine content during ischemia. During recirculation, choline levels progressively declined, reaching levels lower than those in four control rats after 150 minutes of recirculation for most brain regions. A reciprocal relation between the brain choline concentration and local cerebral blood flow was found. Acetylcholine levels showed an initial rebound to greater than control during recirculation, with subsequent normalization. Brain acetylcholine concentration was positively correlated with brain choline concentration, provided that cerebral blood flow was greater than 0.3 ml x g-1 x min-1. Because tissue free choline was depleted in most brain regions 150 minutes after transient ischemia, we speculate that prolonged ischemia may produce a greater depletion of tissue free choline with a resulting decline in tissue acetylcholine. This could play an important role in the cognitive deficit associated with vascular dementia.     

3-Hydroxyglutaric acid induces oxidative stress and decreases the antioxidant defenses in cerebral cortex of young rats

Glutaryl-CoA dehydrogenase deficiency (GDD) is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of glutaric, 3-hydroxyglutaric (3-OHGA) and glutaconic acids and clinically by severe neurological symptoms and cerebral atrophy whose pathophysiology is poorly known. In the present study we investigated the effect of 3-OHGA, considered the main neurotoxin in GDD, on the lipoperoxidation parameters chemiluminescence and thiobarbituric acid-reactive species (TBA-RS), and on the amount of nitric oxide metabolites in cerebral cortex of young rats. Total radical-trapping antioxidant potential (TRAP), which reflects the tissue antioxidant defenses, was also examined. We observed that 3-OHGA significantly increased chemiluminescence, TBA-RS and nitric oxide metabolites, in contrast to TRAP, which was decreased by the metabolite. The data indicate a stimulation of lipid peroxidation and free radical production, and a reduction of the tissue antioxidant defenses caused by the metabolite. In case these findings also occur in the human condition, it may be presumed that oxidative stress is involved in the brain damage observed in GDD.     

Temporal profile of the superoxide dismutase and the ascorbic acid in focal cerebral ischemia]

It has been proposed that free radical reactions are involved in ischemic brain damage. Since irreversible pathological changes occurs very early phase of the focal ischemia and the ischemic brain edema reaches its peak at about 2 days of ischemia, the free radical reactions must take place before these changes. Superoxide dismutase is a famous enzyme that dismutase superoxide anion, which is believed to be one of the initiator of the free radical reactions. If superoxide anion plays a pivotal role in the genesis of pathological ischemic brain damage and edema, the activity of the enzyme may decrease in the early phase of ischemia. Ascorbic acid is also known to be a scavenger of superoxide anion, and brain tissue contains it in a high concentration. We investigated the changes in superoxide dismutase activity and concentration of reduced ascorbate in focal ischemia. Focal ischemia was produced in rats by permanent occlusion of the left middle cerebral artery. The animals were decapitated 30 minutes, 4, 24, and 48 hours after the operation. Middle cerebral artery territory of each cerebral hemisphere was homogenized and centrifuged with phosphate buffer. The supernatant was divided into two aliquots; one was dialyzed to remove ascorbate and the other was not. The SOD activity was measured by electron-spin-resonance (ESR) spin trapping method, and the ascorbic acid concentration was measured by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein concentration was measured by Lowry's method. The enzyme activity was expressed as unit/mg protein, and the ascorbic acid concentration was expressed as microgram/g tissue. The SOD activity decreased markedly by dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortical tissue pressure gradients in early ischemic brain edema

We examined the role of ischemic brain edema, tissue pressure gradients, and regional CBF (rCBF) in adjacent regions of cerebral cortex in cats with middle cerebral artery (MCA) occlusion (MCAO). Tissue pressure, rCBF, and water content were measured from gray matter in the central core and the peripheral margin of the MCA territory over 6 h after MCAO. Ventricular fluid pressure and CSF pressure were recorded. Tissue pressure in the ischemic core, with a flow of approximately 5 ml/100 g/min, increased more than that in the periphery where flow was approximately 19 ml/100 g/min. Tissue pressure rose progressively to 14.8 +/- 1.0 mm Hg in the core over 6 h after MCAO, establishing a significant pressure gradient between that tissue and the lateral ventricle nearby or the subarachnoid space in the middle fossa within the first 3 h. The increase in tissue pressure was linearly related to the amount of edema fluid that developed until the edema reached a severe degree. This study shows that a hydrostatic tissue pressure gradient within ischemic cortex is associated with ischemic brain edema. The magnitude of the gradient that develops is related to the severity of ischemic edema in that tissue.     

Oral coenzyme Q10 administration prevents the development of ischemic brain lesions in a rabbit model of symptomatic vasospasm

Treatment with oral coenzyme Q₁₀ (CoQ₁₀, 10 mg/kg per day for 6 days) was compared with no treatment in a previously described rabbit model of symptomatic cerebral vasospasm [Endo et al. (1988) Stroke 19: 1420–1425]. The treatment was initiated within 1–2 h after injection of autologous blood into the subarachnoid space. In CoQ₁₀-untreated rabbits, moderate to severe neurological deficits developed, and multiple focal ischemic lesions were found in the brain regions with compromised blood supply, i.e., in the regions normally supplied by common carotid arteries which are subject to ligation in this model. CoQ₁₀ treatment prevented the development of both the neurological deficits and histologically detectable brain tissue damage. In both CoQ₁₀-treated and -untreated rabbits, infiltration of mononuclear cells was evident in the brain stem, although this region did not show signs of ischemic damage. The findings indicate that the histological and neurological correlates of brain tissue damage in this rabbit model of symptomatic cerebral vasospasm develop via mechanism(s) involving free radical-mediated oxidation of plasma lipoproteins. Similar mechanisms may play a role in the development of brain damage attributed to cerebral atherosclerosis. Received: 11 December 1996 / Revised, accepted: 10 April 1997     

卒中合并脑微出血的影像学研究

目的 探讨ICH和脑梗死患者合并脑微出血（cerebral microbleeds，CMBs）数量分级及部位分布差异。 方法 连续选取2017年8月-2018年12月在北京市大兴区人民医院神经内科病房住院且资料完整的 卒中患者，分为ICH和脑梗死两组。收集患者发病2周内头颅MRI磁敏感加权成像影像，统计ICH与脑梗 死患者中CMBs数量分级及部位分布特点。 结果 共入组患者234例，其中ICH组79例，脑梗死组155例。ICH组合并CMBs 65例（82.3%），其中 CMBs＞5个的患者41例（51.9%）；脑梗死组合并CMBs 73例（47.1%），其中CMBs＞5个的患者21例 （13.6%），两组差异有统计学意义（P＜0.001）。ICH组中，CMBs同时累及脑叶和深部脑组织的患者40例 （50.6%）；脑梗死组中，CMBs同时累及脑叶和深部脑组织的患者25例（16.1%），两组差异有统计学意 义（P＜0.001）。 结论 CMBs病灶＞5个多见于ICH患者；CMBs同时累及脑叶及深部脑组织多见于ICH患者。