Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.     

中枢疲劳研究与思考

中枢疲劳既可以作为独立疾病影响人们的日常工作和学习,又可以作为症状出现于多种慢性疾病,其定义和机制国内外说法不尽相同。中枢疲劳是由于中枢神经系统发生退行性或其他不良变化,从而导致躯体、神经、包括心理一系列的疲劳样反应。其机制涉及到中枢神经系统和外周传导系统等多个维度、多个节点的变化,充分把握中枢疲劳的概念本质及潜在生物学机制对其临床防治有着重要理论和实践意义。此外,动物模型作为基础研究的前提和必要工具是中枢疲劳研究过程中又一重要问题。本文在文献整理的基础上,先从定义的角度出发由疲劳引申到中枢疲劳,将现阶段对中枢疲劳的不同概念阐述做一分析,并从机制和动物模型两个方面展开对国外研究进展进行综述。     

Lipid Peroxides in the Free Radical Pathophysiology of Brain Diseases

1. Polyunsaturated fatty acids are essential for normal neural cell membrane functioning because many membrane properties, such as fluidity and permeability, are closely related to the presence of unsaturated and polyunsaturated side chains. Lipid peroxidation results in loss of membrane polyunsaturated fatty acids and oxidized phospholipids as polar species contributing to increased membrane rigidity.2. Polyunsaturated fatty acids are released from membrane phospholipids by a number of enzymic mechanisms involving the receptor-mediated stimulation of phospholipase A₂ and phospholipase C/diacylglycerol lipase pathways.3. The overstimulation of excitatory amino acid (EAA) receptors stimulates the activities of lipases and phospholipases, and this stimulation produces changes in membrane phospholipid composition, permeability, and fluidity, thus decreasing the integrity of plasma membranes.4. Alterations in properties of plasma membranes may be responsible for the degeneration of neurons seen in neurodegenerative diseases. Two major processes may be involved in neuronal injury caused by the overstimulation of EAA receptors. One is a large Ca²⁺ influx and the other is an accumulation of free radicals and lipid peroxides as a result of neural membrane phospholipid degradation. It is suggested that calcium and free radicals act in concert to induce neuronal injury in acute trauma (ischemia and spinal cord injury) and in neurodegenerative diseases.     

T-Cell Tolerance: Central and Peripheral

Somatic recombination of TCR genes in immature thymocytes results in some cells with useful TCR specificities, but also many with useless or potentially self-reactive specificities. Thus thymic selection mechanisms operate to shape the T-cell repertoire. Thymocytes that have a TCR with low affinity for self-peptide–MHC complexes are positively selected to further differentiate and function in adaptive immunity, whereas useless ones die by neglect. Clonal deletion and clonal diversion (Treg differentiation) are the major processes in the thymus that eliminate or control self-reactive T cells. Although these processes are thought to be efficient, they fail to control self-reactivity in all circumstances. Thus, peripheral tolerance processes exist wherein self-reactive T cells become functionally unresponsive (anergy) or are deleted after encountering self-antigens outside of the thymus. Recent advances in mechanistic studies of central and peripheral T-cell tolerance are promoting the development of therapeutic strategies to treat autoimmune disease and cancer and improve transplantation outcome.T cells recognize pathogen fragments in the context of surface MHC molecules on host cells. As such, they have the potential to do enormous damage to healthy tissue when they are not appropriately directed, that is, when they respond to self-antigens as opposed to foreign antigens. T lymphocyte tolerance is particularly important, because it impacts B-cell tolerance as well, through the requirement of T cell help in antibody responses. Thus, failure of T-cell tolerance can lead to many different autoimmune diseases. The tolerance of T cells begins as soon as a T-cell receptor is formed and expressed on the cell surface of a T-cell progenitor in the thymus. Tolerance mechanisms that operate in the thymus before the maturation and circulation of T cells are referred to as “central tolerance.” However, not all antigens that T cells need to be tolerant of are expressed in the thymus, and thus central tolerance mechanisms alone are insufficient. Fortunately, additional tolerance mechanisms exist that restrain the numbers and or function of T cells that are reactive to developmental or food antigens, which are not thymically expressed. These mechanisms act on mature circulating T cells and are referred to as “peripheral tolerance.”     

Silver Impregnation of Peripheral and Central Axons

A silver impregnation method suitable for peripheral and central nervous system axons is described. Essential features are the use of reagent grade chemicals only, a pretreatment solution to ensure optimal impregnation of different organs from different animals and species, and an unvarying procedure. The results are compared to those obtainable with a number of current impregnation methods and with modern immunocytochemical reactions.     

Peripheral Nerve Injury Modulates Neurotrophin Signaling in the Peripheral and Central Nervous System

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75^NTR and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.     

Cerebrovascular Diseases in West Central India

Cerebral angiographic findings in ischaemic stroke are described and discussed in detail. Though the Indian patients studied had altogether different social customs, living standards, and dietary habits from Western people, the relative incidence of various cerebral vascular lesions did not differ significantly. Irrespective of the poor nutritional status of the patients, thrombosis associated with atherosclerosis was chiefly responsible for a non-embolic cerebral infarction. Atherothrombosis in the young normotensive persons not showing any evidence of arteritis, diabetes mellitus, or hypercholesterolaemia was also identified.The grave risks involved in cerebral angiography in cases of acute stroke are re-emphasized.As to prognosis, the nutritional status, the type and territory of an ictal lesion, and the blood levels of sugar and cholesterol had no significant influence on the immediate survival-after a non-embolic cerebral infarction. However, a significantly greater number of deaths were encountered in the hypertensive patients. Female patients and patients with a large cerebral infarction had a poor prognosis.     

Central and Systemic Responses to Methionine-Induced Hyperhomocysteinemia in Mice

Hyperhomocysteinemia has been considered a risk factor for neuropsychiatric disorders, but the mechanisms involved in this process have not been completely elucidated. The aim of this study was to analyze the influence of hyperhomocysteinemia induction by methionine supplementation considering different levels and periods of exposure in mice. For this purpose, methionine supplementation at concentrations of 0.5 and 1% were administered in water to increase homocysteinemia in male C57BL/6 mice, and was maintained for 3 time periods (2, 4 and 6 months of treatment). The results from one-carbon metabolism parameters, brain-derived neurotrophic factor (BDNF) concentrations and behavioral evaluation were compared. The 0.5% supplementation was efficient in increasing plasma homocysteine levels after 2 and 6 months. The 1% supplementation, increased plasma homocysteine after 2, 4 and 6 months. Little influence was observed in cysteine and glutathione concentrations. Frontal cortex BDNF levels showed a lack of treatment influence in all periods; only the expected decrease due to increasing age was observed. Moreover, the only behavioral alteration observed using a novel object recognition task was that which was expected with increasing age. We found that responses to hyperhomocysteinemia varied based on how it was reached, and the length of toxicity. Moreover, hyperhomocysteinemia can affect the normal pattern of one carbon metabolism during age increase in mice. These findings allow the establishment of a reliable animal model for studies in this field.     

Salivary Biomarkers for Detection of Systemic Diseases

Background and Objective

Analysis of inflammatory biomarkers in saliva could offer an attractive opportunity for the diagnosis of different systemic conditions specifically in epidemiological surveys. The aim of this study was to investigate if certain salivary biomarkers could be used for detection of common systemic diseases.

Materials and Methods

A randomly selected sample of 1000 adults living in Skåne, a county in the southern part of Sweden, was invited to participate in a clinical study of oral health. 451 individuals were enrolled in this investigation, 51% women. All participants were asked to fill out a questionnaire, history was taken, a clinical examination was made and stimulated saliva samples were collected. Salivary concentrations of IL-1β, -6, -8, TNF-α, lysozyme, MMP-8 and TIMP-1 were determined using ELISA, IFMA or Luminex assays.

Results

Salivary IL-8 concentration was found to be twice as high in subjects who had experience of tumour diseases. In addition, IL-8 levels were also elevated in patients with bowel disease. MMP-8 levels were elevated in saliva from patients after cardiac surgery or suffering from diabetes, and muscle and joint diseases. The levels of IL-1β, IL-8 and MMP-8, as well as the MMP-8/TIMP-1 ratio were higher in subjects with muscle and joint diseases.

Conclusion

Biomarkers in saliva have the potential to be used for screening purposes in epidemiological studies. The relatively unspecific inflammatory markers used in this study can not be used for diagnosis of specific diseases but can be seen as markers for increased systemic inflammation.     

针刺外周神经对中枢神经的电生理变化

目的:探索电针外周神经是否促进中枢神经损伤修复,为电针促进神经再生修复研究提供一点基础性参考.方法:24只SD成年雌性大鼠随机分成3组,即正常组,脊髓T11全横断损伤21天组和脊髓T11全横断后电针治疗2l天组.电针穴位选取外周足三里、悬钟和伏兔、三阴交,两组穴位隔日交替进行电针.2l天后,应用临床体感诱发仪比较三者的皮层体感诱发电位(CSEP).结果:CSEP检测发现电针21天与损伤21天比较,P1潜伏期及P1-N1波幅无统计学差异(P＞0.05);但二者与正常组相比,除P1-N1波幅未见明显差异外(P＞0.05),P1潜伏期存在明显的统计学差异(P＜0.05).结论:电针促进神经损伤修复的关键是根据损伤类型选择相应的电针穴位,同时说明针刺外周神经不利于中枢神经损伤修复.     

The Magnitude of Peripheral Muscle Fatigue Induced by High and Low Intensity Single-Joint Exercise Does Not Lead to Central Motor Output Reductions in Resistance Trained Men

Purpose

To examine quadriceps muscle fatigue and central motor output during fatiguing single joint exercise at 40% and 80% maximal torque output in resistance trained men.

Method

Ten resistance trained men performed fatiguing isometric knee extensor exercise at 40% and 80% of maximal torque output. Maximal torque, rate of torque development, and measures of central motor output and peripheral muscle fatigue were recorded at two matched volumes of exercise, and after a final contraction performed to exhaustion. Central motor output was quantified from changes in voluntary activation, normalized surface electromyograms (EMG), and V-waves. Quadriceps muscle fatigue was assessed from changes in the size and shape of the resting potentiated twitch (Q._pot.tw). Central motor output during the exercise protocols was estimated from EMG and interpolated twitches applied during the task (VA_sub).

Results

Greater reductions in maximal torque and rate of torque development were observed during the 40% protocol (p<0.05). Maximal central motor output did not change for either protocol. For the 40% protocol reductions from pre-exercise in rate and amplitude variables calculated from the Q._pot.tw between 66.2 to 70.8% (p<0.001) exceeded those observed during the 80% protocol (p<0.01). V-waves only declined during the 80% protocol between 56.8 ± 35.8% to 53.6 ± 37.4% (p<0.05). At the end of the final 80% contraction VA_sub had increased from 91.2 ± 6.2% to 94.9 ± 4.7% (p = 0.005), but a greater increase was observed during the 40% contraction where VA_sub had increased from 67.1 ± 6.1% to 88.9 ± 9.6% (p<0.001).

Conclusion

Maximal central motor output in resistance trained men is well preserved despite varying levels of peripheral muscle fatigue. Upregulated central motor output during the 40% contraction protocol appeared to elicit greater peripheral fatigue. V-waves declines during the 80% protocol suggest intensity dependent modulation of the Ia afferent pathway.     

Central and Peripheral Cytokines Mediate Immune-Brain Connectivity

The immune system is a homeostatic system that contributes to maintain the constancy of the molecular and cellular components of the organism. Immune cells can detect the intrusion of foreign antigens or alteration of self-components and send information to the central nervous system (CNS) about this kind of perturbations, acting as a receptor sensorial organ. The brain can respond to such signals by emitting neuro/endocrine signals capable of affecting immune reactivity. Thus, the immune system, as other physiologic systems, is under brain control. Under disease conditions, when priorities for survival change, the immune system can, within defined limits, reset brain-integrated neuro-endocrine mechanisms in order to favour immune processes at the expenses of other physiologic systems. In addition, some cytokines initially conceived as immune products, such as IL-1 and IL-6, are also produced in the “healthy” brain by glial cells and even by some neurons. These and other cytokines have the capacity to affect synaptic plasticity acting as mediators of interactions between astrocytes and pre- and post-synaptic neurons that constitute what is actually defined as a tripartite synapse. Since the production of cytokines in the brain is affected by peripheral immune and central neural signals, it is conceivable that tripartite synapses can, in turn, serve as a relay system in immune-CNS communication.