Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain

Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.     

Allometry of the perivascular spaces of the putamen in aging

Summary The equation of simple allometry was used to study the relative growths of the diameters of the perivascular spaces (PSs), the diameters of the arterial vessels and their wall thicknesses in specimens of the putamen from 24 subjects aged 62 to 90 years. Our findings supported the view that the diameters of the PSs were mainly related to the dimensions of their arteries, especially in the elderly, and that they grew isometrically with arterial diameters. In addition, the diameters of the PSs were basically independent of age.     

AAV2-mediated gene delivery to monkey putamen: evaluation of an infusion device and delivery parameters

In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic l-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson's disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 muL/min to 1 muL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 muL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated.     

The development of an implantable catheter system for chronic or intermittent convection-enhanced delivery

Convection-enhanced delivery (CED) is a promising technique for the administration of therapeutic agents such as cytotoxics, neurotrophins and enzymes to the brain. In this study we describe the development of an implantable catheter system that is compatible with long-term intermittent CED. Catheters made from fused silica, PEEK or carbothane, and of various internal and external diameters were implanted in the striatum of rats and assessed for patency at 21 or 28 days. A high-rate of catheter blockage was observed with all fused silica and PEEK catheters. Carbothane catheters with an outer diameter of 0.6 mm and an inner diameter of 0.35 mm had significantly lower rates of blockage (P ≤ 0.01). Carbothane catheters were then implanted into 4 Large White/Landrace pigs and 4 NIH miniature pigs and infusions undertaken at monthly intervals to evaluate catheter patency and infusate distribution. Catheter patency was demonstrated for a maximum period of 163 days in one animal. Widespread and reproducible intraputamenal CED could be achieved with intermittent drug delivery at flow-rates as high as 5 μl/min. Problems were encountered using the pig model due to catheter distortion from rapid animal growth. In conclusion, it is possible to achieve intermittent high-flow CED with a chronic implanted carbothane catheter with a low rate of catheter blockage.     

短暂性脑缺血发作和轻型卒中患者血管周围间隙扩大和眼底血管病变的相关性研究

目的:探讨轻型卒中和短暂性脑缺血发作(transient ischemic attacks,TIA)患者血管周围间隙扩大(enlarged perivascular space,EPVS)与眼底血管病变之间的关系。方法:连续收集2019年3—8月在常州市第二人民医院住院治疗的TIA或轻型卒中患者(美国国立卫生研究院卒中量表评分≤3分)。对每例患者完善磁共振成像和眼底照相检查,半自动测量视网膜动静脉直径,评估视网膜动脉硬化程度、血管弯曲度,观察眼底出血、微血管瘤、硬性渗出、软性渗出、动静脉交叉、静脉串珠的情况。根据TIA和轻型卒中患者有无EPVS分成两组,比较两组患者的基线资料,使用多因素Logistic回归分析眼底病变与EPVS之间的关系。对TIA和轻型卒中患者的EPVS进行计数及程度分级,并对EPVS的数量进行ln对数转换为正态分布数据,进一步分析EPVS的等级及数量与眼底血管病变之间的关系。结果:共纳入123例患者,其中脑梗死患者99例,TIA患者24例;无EPVS组52例,EPVS组71例。EPVS组患者在年龄[(68.61±12.71)岁与(63.37±13.53)岁,t=-2.198,P=0.030]、高血压病史[52例(73.2%)与25例(48.1%),χ2=8.118,P=0.004]、眼底血管瘤[17例(23.9%)与5例(9.6%),χ2=4.196,P=0.041]、眼动静脉交叉征比例[50例(70.4%)与8例(15.4%),χ2=36.488,P<0.05]、眼底动脉硬化程度[1(1,2)级与0(0,1)级,Z=-7.454,P<0.05]方面均高于非EPVS组;EPVS组患者视网膜中央动脉直径[central retinal artery equivalent,CRAE;(106.31±15.02)mm与(113.89±11.86)mm,t=3.014,P=0.003]、视网膜动静脉直径比值(arteriole-to-venule ratio,AVR;0.54±0.07与0.59±0.05,t=4.553,P<0.05)均小于非EPVS组。回归分析发现眼底动脉硬化程度(OR=7.781,95%CI 2.876~21.055,P<0.05)和高血压病(OR=3.203,95%CI 1.049~9.777,P=0.041)是TIA和轻型卒中患者有无EPVS的独立相关因素。EPVS的严重程度与眼底动脉硬化程度呈正相关(r=0.764,P<0.05),与CRAE呈负相关(r=-0.287,P<0.05),与AVR呈负相关(r=-0.422,P<0.05)。ln转换后的EPVS数量与EPVS严重程度高度相关(r=0.972,P<0.05)。进一步校正年龄、性别、高血压病、糖尿病等因素后,ln转换后的EPVS数量与动静脉交叉征呈正相关(B=0.556,95%CI 0.203~0.910,P=0.003),与眼底动脉硬化呈正相关(B=0.417,95%CI 0.259~0.576,P<0.05),与AVR呈负相关(B=-4.213,95%CI-6.712^-1.714,P=0.001)。结论:高血压病和眼底动脉硬化是TIA和轻型卒中患者EPVS的独立相关因素;TIA和轻型卒中患者EPVS的等级及数量与眼底病变有相关性,EPVS的数量越多,动静脉交叉征出现比例越高,眼底动脉硬化程度越严重,CRAE越小,AVR越小。     

Effects of chronic reserpine treatment on development of maturity of the putamen in fetal rabbits

Developing nigrostriatal axons and their perikarya have substantial quantities of dopamine (DA) before the axons reach their postsynaptic target. In order to investigate possible developmental effects of these stores of DA, we have depleted DA chronically during critical periods in the ontogeny of the nigrostriatal system. Reserpine (0.04–0.14 mg/kg/day) was given repeatedly to maternal rabbits for various periods starting before neuroblasts of the substantia nigra first exhibit fluorescence until 2 days before term when the fetuses were sacrificed. Reserpine crossed the placenta and depleted DA in the fetal putamens. Control fetuses had widespread fluorescent axons and terminals. Counts of mature axonal boutons in electron micrographs of the putamen of reserpine-treated fetuses showed that there were 4.3 ± 0.6 SE/100 μm², which is less than the control value of 10.2 ± 0.6 SE/100 μm² (p < 0.001). The neuropil of the putamen of the reserpine-treated fetus was also less mature; the relative volume occupied by growth cones (40.5% ± 5.7 SE) was twice that of controls (20.6% ± 2.4 SE) (p < 0.005). Although it remains to be shown that the delayed development of both pre- and postsynaptic elements of the nigrostriatal system is specifically related to the known ability of reserpine to deplete DA, the results are consistent with the hypothesis that early stores of DA may be important in developing dopaminergic systems.     

渗透泵强化对流释放给药治疗恶性脑胶质瘤的裸小鼠模型

目的 探寻渗透泵强化对流释放给药治疗脑肿瘤的裸小鼠模型,为新药的临床前期应用提供良好的实验动物模型.方法 14只裸小鼠基底节区种植4×105个人胶质瘤U87细胞,并安置渗透泵强化对流释放给药系统.裸小鼠按体重和活体荧光信号强度分为二组:治疗组(n=7)的泵内注入1 00μl(1μg)的DTATEGF,对照组(n=7)泵内注入100μl(1 μg)的无关毒素DT.观察动物的生长特征,Kaplan- Meier生存曲线分析和脑内肿瘤的病理学特征.结果 实验动物没有与渗透泵及外科手术相关的病残和病死发生.动物对渗透泵给药系统耐受好,无抓搔及渗透泵脱位,伤口无感染及裂开等情况.Kaplan - Meier生存曲线显示治疗组动物的中位生存期较对照组明显延长,差异有显著性.(治疗组126天vs.对照组68天,P =0.0002).治疗组中有两只小鼠生存期超过180天.病理学检测裸小鼠脑内种植生长的肿瘤与人类胶质母细胞瘤的病理特征极为相似.结论 该裸小鼠实验动物模型简便、实用、可靠,适用于新的药物包括单克隆抗体、靶向小分子、肿瘤疫苗等通过强化对流投递治疗脑内恶性肿瘤的临床前期应用.     

Convection-enhanced delivery of AAV2 in white matter—A novel method for gene delivery to cerebral cortex

Background

Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge.

New method

We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model.

Results

Infusion volumes of up to 700 μl at flow rates as high as 10 μl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter.

Comparison with existing methods

AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures.

Conclusions

AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration.     

Dilated perivascular space is related to reduced free-water in surrounding white matter among healthy adults and elderlies but not in patients with severe cerebral small vessel disease

Perivascular space facilitates cerebral interstitial water clearance. However, it is unclear how dilated perivascular space (dPVS) affects the interstitial water of surrounding white matter. We aimed to determine the presence and extent of changes in normal-appearing white matter water components around dPVS in different populations. Twenty healthy elderly subjects and 15 elderly subjects with severe cerebral small vessel disease (CSVD, with lacunar infarction 6 months before the scan) were included in our study. And other 28 healthy adult subjects were enrolled under a different scanning parameter to see if the results are comparable. The normal-appearing white matter around dPVS was categorized into 10 layers (1 mm thickness each) based on their distance to dPVS. We evaluated the mean isotropic-diffusing water volume fraction in each layer. We discovered a significantly reduced free-water content in the layers closely adjacent to the dPVS in the healthy elderlies. however, this reduction around dPVS was weaker in the CSVD subjects. We also discovered an elevated free-water content within dPVS. DPVS played different roles in healthy subjects or CSVD subjects. The reduced water content around dPVS in healthy subjects suggests these MR-visible PVSs are not always related to the stagnation of fluid.     

血管周围间隙与多发性硬化的相关性研究

目的研究血管周围间隙与多发性硬化的相关性。方法对38例多发性硬化患者(多发性硬化组)及50名健康体检者(对照组)进行头颅MRI检查,计数其血管周围间隙并测量其直径。对结果进行组间比较。结果多发性硬化组患者血管周围间隙检出率(42.1%,16/38)明显高于对照组(16.0%,8/50)(P0.01)。多发性硬化组患者血管周围间隙的数目[(5.4±2.4)个/例]明显多于对照组[(2.7±2.0)个/例](P0.01)。多发性硬化组患者血管周围间隙的直径[(3.0±1.0)mm]明显大于对照组[(2.2±0.7)mm](P0.05)。线性回归分析显示,对照组中血管周围间隙的数目、直径均与年龄呈正相关(r1=0.857,r2=0.956,均P0.01)。结论血管周围间隙与多发性硬化有一定的相关性。     

Effects of melatonin on pulmonary and coronary vessels are exerted through perivascular nerves

The effects of melatonin on porcine pulmonary and coronary vessels have been studied. Vessels were isolated from normal pigs, placed in a tissue bath, and precontracted with 30 mM KCl. The biophysical responses to cumulative doses of melatonin were then assessed. In the pulmonary artery, melatonin caused a dose-dependent relaxation which was blocked by vasoactive intestinal peptide antagonists or prior 6-hydroxydopamine treatment of the vessels. In the coronary artery, melatonin caused the reverse, a dose-dependent contraction which was blocked by the alpha antagonist prazosin or prior 6-hydroxydopamine treatment. These experiments indicate that melatonin has different mechanisms of action in the coronary and pulmonary circulation, although both seem to depend on the integrity of perivascular 6-hydroxydopamine sensitive nerves. Such differing mechanisms may provide insight into pathophysiological events in the lungs (such as nocturnal asthma) and heart (early morning coronary infarction).     

VCAM-1、TLR2在亚急性1-甲基-4-苯基-1,2,3,6-四氢吡啶帕金森病小鼠模型纹状体中表达的研究

目的研究亚急性1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)帕金森病小鼠模型纹状体中VCAM-1、TLR2的表达。方法腹腔注射MPTP制作亚急性(20mg/kg,每天1次,共7d)帕金森病小鼠模型。实验分为3组,8～10周龄小鼠组、8月龄中年小鼠组、亚急性小鼠模型组,每组6只C57BL/6小鼠。对照组小鼠(8～10周龄组、8月龄中年小鼠组)腹腔注射等量的生理盐水。模型组小鼠在末次注射MPTP 1d后取脑组织。用胶质细胞纤维酸性蛋白(GFAP)免疫组织化学染色,观察亚急性小鼠模型纹状体中炎性标志细胞——星型胶质细胞改变的情况。用RT-PCR方法检测VCAM-1 mRNA、TLR2 mRNA的基因表达水平。用Western blot方法检测VCAM-1蛋白表达水平。结果亚急性小鼠模型纹状体GFAP阳性细胞明显增多。亚急性模型组纹状体VCAM-1 mRNA表达水平明显增高,高于正常对照8～10周龄小鼠组和8月龄中年小鼠组(P0.05),TLR2 mRNA表达各组间没有显著性差异(P0.05)。亚急性模型组纹状体VCAM-1蛋白表达水平亦增高,高于正常对照组和中年小鼠组(P0.05)。结论 VCAM-1在MPTP帕金森病小鼠纹状体中表达增高,可能与炎症反应有关,从而参与神经损伤和帕金森病病理生理过程。     

Potential role of kallikrein in diurnal rhythms and perivascular distribution in rat pineal glands

Kallikrein hydrolyzes various biologically active peptides, other than kininogens, such as vasoactive intestinal polypeptide (VIP), in vitro. Since kallikrein and VIP have been immunohistochemically shown to be present in the perivascular areas of the pineal gland, this study was designed to determine their topographic proximity in these glands, using immunohistochemical and immunoelectron microscopic double staining methods. Furthermore, since this gland is well-known to have a circadian rhythm, the kallikrein content was measured every 4 h, using a synthetic substrate, Pro–Phe–Arg–MCA, and an enzyme-linked immunosorbent assay (ELISA) to determine whether kallikrein has a circadian rhythm. The immunoreactivities of kallikrein and VIP were highly localized in the perivascular extracellular spaces and were virtually identical in distribution. The kallikrein content changed every 4 h and was high under light and low under dark conditions. The change was more evident when the synthetic substrate was used, and this rhythm was subtle on ELISA. VIP is also said to have a circadian rhythm in the pineal glands, being low under light and high under dark conditions, i.e., opposite to that of kallikrein. Since kallikrein degrades VIP in vitro, it is reasonable to speculate that pineal gland kallikrein is involved in the processing of VIP and possibly other biologically active peptides in the perivascular areas with a discernible circadian rhythm.     

Characterization of the perivascular reticulin network in a case of primary brain lymphoma

Summary The character of the silver positive reticulin network was analyzed with immunofluorescence and immunoperoxidase methods in an intra vitam diagnosed case of primary brain lymphoma. The network was shown to contain connective tissue proteins rich in hexose-sugars, such as type III collagen (classical reticulin), basal lamina constituents type IV collagen and laminin, pericellular type V collagen, as well as fibronectin (protein involved in cell adhesion). On the other hand, very little of the fibrous type I collagen was discernible. Similarly as the silver positive network, the immunohistochemically demonstrable reticulum seemed to hold the cells in the perivascular location, and once it was broken diffuse spread into the tissue occurred.Since malignant cells of B-lymphocyte origin are not known to synthesize so-called reticulin, it is suggested that the network in primary brain lymphomas is produced by cells in the brain parenchyma (possibly pericytes or astrocytes) as a protective attempt to restrict the spread of foreign cells into the brain.     

Deep white matter hyperintensity is associated with the dilation of perivascular space

Understanding the pathophysiology of white matter hyperintensity (WMH) is necessary to reduce its harmfulness. Dilated perivascular space (PVS) had been found related to WMH. In the present study, we aimed to examine the topological connections between WMH and PVS, and to investigate whether increased interstitial fluid mediates the correlation between PVS and WMH volumes. One hundred and thirty-six healthy elder subjects were retrospectively included from a prospectively collected community cohort. Sub-millimeter T2 weighted and FLAIR images were acquired for assessing the association between PVS and WMH. Diffusion tensor imaging and free-water (FW) analytical methods were used to quantify white matter free water content, and to explore whether it mediates the PVS-WMH association. We found that most (89%) of the deep WMH lesions were spatially connected with PVS, exhibiting several interesting topological types. PVS and WMH volumes were also significantly correlated (r = 0.222, p < 0.001). FW mediated this association in the whole sample (β = 0.069, p = 0.037) and in subjects with relatively high WMH load (β = 0.118, p = 0.006). These findings suggest a tight association between PVS dilation and WMH formation, which might be linked by the impaired glymphatic drainage function and accumulated local interstitial fluid.     

Migration of perivascular cells into the neuropil and their involvement in β-amyloid plaque formation

Summary Our recent ultrastructural studies of amyloid angiopathy in biopsy specimens from Alzheimer's disease patients showed that perivascular cells and perivascular microglia are involved in the production of amyloid fibrils. Further examination of the walls of the vessels with and without amyloid deposits presented in this report reveals numerous mononuclear cells with a broad spectrum of morphological appearances. Some of these cells produce amyloid in the vascular wall and migrate into the neuropil. Others do not produce amyloid in this location but also migrate through the vascular basal lamina and position themselves on the external surface of basal lamina or in the neuropil outside the vascular astrocytic end-feet processes. The presence of clusters or rows of six or more of these cells in the position of perivascular microglial cells suggests their proliferation in the perivascular region. After leaving the perimeter of the vessel wall, perivascular cells become the perivascular, neuropil, and satellite microglia cells. Migrating perivascular cells become the microglia, which are engaged in amyloid fibril formation and development of classical and primitive plaques.Supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities and a grant from the National Institutes of Health, National Institute of Aging No. PO1-AGO-4220     

Evidence for rapid fluid flow from the subarachnoid space into the spinal cord central canal in the rat

Horseradish peroxidase (HRP) was used as a CSF tracer in Sprague-Dawley rats. One group of rats received an injection of HRP in the cistema magna and a second group was injected in the thoracic spinal subarachnoid space. The animals were sacrificed 0, 10 or 30 min after HRP injection by rapid perfusion with paraformaldehyde and glutaraldehyde. In both groups, there was rapid HRP labeling of brain and spinal cord perivascular spaces. HRP was present in the central canal in a pattern that was not consistent with flow from the fourth ventricle: in both groups there were segments of unlabeled central canal between the fourth ventricle and central canal segments containing HRP. HRP-labeled perivascular spaces were seen in the central gray matter adjacent to the central canal. There was a distinctive pattern of interstitial HRP between perivascular spaces and the central canal. The results suggest that there is a normal flow of fluid from the subarachnoid space, into the perivascular spaces, across the interstitial space and into the central canal. The function of this flow may be to clear metabolites from the interstitial space. The existence of such a flow would add considerable support to the theory that non-communicating syringomyelia develops in segments of central canal isolated by occlusion or stenosis at each end.     

Decreased densities of dopamine D1 receptors in the putamen and hippocampus in senile dementia of the Alzheimer type

The density and distribution of dopamine D1 receptors as labeled with [3H]SCH 23390 was analyzed in post-mortem brain tissue from patients with senile dementia of the Alzheimer type (SDAT) and in controls using quantitative autoradiography. In SDAT patients D1 receptor densities were markedly decreased in parts of the hippocampus, with reductions of up to 89% compared to the control values in the molecular layer of the dentate gyrus, 57% in the strata oriens and pyramidalis of the CA1 and 74% in the CA3 subfields. Significant decreases in D1 receptors were also observed in the putamen (23%) but not in the caudate and substantia nigra. A slight but not significant decrease of D1 binding was observed in most external layers of the temporal and occipital cortices.