Cell Biology

Protocols in Current Issue
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1 Q&A 3112 Views Jun 20, 2021

Basal forebrain cholinergic neurons (BFCNs) regulate circuit dynamics underlying cognitive processing, including attention, memory, and cognitive flexibility. In Alzheimer’s disease and related neurodegenerative conditions, the degeneration of BFCNs has long been considered a key player in cognitive decline. The cholinergic system thus represents a key therapeutic target. A long-standing obstacle for the development of effective cholinergic-based therapies is not only the production of biologically active compounds but also a platform for safe and efficient drug delivery to the basal forebrain. The blood-brain barrier (BBB) presents a significant challenge for drug delivery to the brain, excluding approximately 98% of small-molecule biologics and nearly 100% of large-molecule therapeutic agents from entry into the brain parenchyma. Current modalities to achieve effective drug delivery to deep brain structures, such as the basal forebrain, are particularly limited. Direct intracranial injection via a needle or catheter carries risks associated with invasive neurosurgery. Intra-arterial injection of hyperosmotic solutions or therapeutics modified to penetrate the BBB using endogenous transport systems lack regional specificity, which may not always be desirable. Intranasal, intrathecal, and intraventricular administration have limited drug distribution beyond the brain surface. Here, we present a protocol for non-invasively, locally, and transiently increasing BBB permeability using MRI-guided focused ultrasound (MRIgFUS) in the murine basal forebrain for delivery of therapeutic agents targeting the cholinergic system. Ongoing work in preclinical models and clinical trials supports the safety and feasibility of MRIgFUS-mediated BBB modulation as a promising drug delivery modality for the treatment of debilitating neurological diseases.

0 Q&A 2866 Views Oct 5, 2020
We describe a protocol for preparing acute brain slices which can produce robust hippocampal sharp wave-ripples (SWRs) in vitro. The protocol is optimized for its simplicity and reliability for the preparation of solutions, slicing, and recovery incubation. Most slices in almost every mouse prepared though the protocol expressed vigorous spontaneous SWRs for ~24 h, compared to the 20-30% viability from "standard" low sodium slicing protocols. SWRs are spontaneous neuronal activity in the hippocampus and are essential for consolidation of episodic memory. Brain slices reliably expressing SWRs are useful for studying memory impairment and brain degeneration diseases in ex vivo experiments. Spontaneous expression of SWRs is sensitive to conditions of slicing and perfusion/oxygenation during recording. The amplitude and abundance of SWRs are often used as a biomarker for viable slices. Key improvements include fast circulation, a long recovery period (3-6 h) after slicing, and allowing tissue to recover at 32 °C in a well perfused incubation chamber. Slices in our custom-made apparatus can express spontaneous SWRs for many hours, suggesting a long period with balanced excitation and inhibition in the local networks. Slices from older mice (~postnatal 180 days) show similar viability to younger (postnatal 21-30) mice.
0 Q&A 2765 Views Sep 20, 2020
Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain, infertility and a reduced quality of life, and it lacks adequate treatment. Several rodent models of endometriosis have been developed through heterologous and homologous transplantation of endometrial tissue into the abdominal compartment. Here we describe a surgical procedure to generate a syngeneic model of endometriosis in immunocompetent mice with intact uterine and ovarian tissues. In this model, four uterine fragments from a donor mouse at diestrus are sutured to the abdominal wall of a recipient mouse. One month after surgeries, endometrial implants develop into cysts with glandular epithelium and stroma, mimicking the endometriotic lesions observed in women with endometriosis. Therefore, this mouse model provides a valuable tool to study the pathophysiology of endometriosis and the efficacy of potential treatments.
0 Q&A 4435 Views Aug 5, 2020
In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological conditions can disrupt this barrier, causing BBB permeability and subsequent CNS dysfunction. Understanding the mechanisms involved in BBB permeability are essential to elucidating the pathology of various neurological disorders as well as identifying methods for drug delivery to the CNS. Here, we describe several in vivo methods to measure BBB permeability in mice using an array of diverse sized tracers including exogenous 376 Da fluorescein salt, 66.5 kDa bovine serum albumin, and 70 kDa dextran as well as endogenous 160 kDa mouse IgG. When administered intravenously, these substances are excluded from a healthy brain by the BBB. However, BBB dysfunction can allow entry of these tracers into the brain and this accumulation can be measured using spectrophotometry, fluorescent microscopy, and immunohistochemistry. We also describe a method to induce BBB permeability using Clostridium perfringens epsilon toxin. Finally, we include a short discussion about the advantages and disadvantages of each method and their appropriate downstream applications.
0 Q&A 4509 Views May 5, 2019
Alcohol use disorder (AUD) is a significant public health and economic burden and is often characterized by repeated bouts of alcohol intoxication and withdrawal. Virtually all organ systems are impacted by chronic alcohol exposure. These effects can be investigated using the rat as a model organism; however, rats typically will not self-administer alcohol to levels necessary to achieve physiological and behavioral aspects of dependence. The protocol described herein can be utilized to induce alcohol dependence in rats by administering alcohol vapor to the homecage for an extended period of time. This method allows the researcher to control the level, duration, and pattern of intoxication, and it reliably induces physiological and behavioral aspects of alcohol dependence, allowing for the study of biology and behavior with relevance for AUD in humans.
0 Q&A 5669 Views Mar 5, 2019
Skin cells are constantly exposed to environmental influences such as air pollution, chemicals, pathogens and UV radiation. UV radiation can damage different biological structures, but most importantly cellular DNA. Mitochondria contain their own genome and accumulate UV-induced DNA mutations to a large extent. This can result, e.g., in accelerated skin aging. Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on the development of age-related diseases. Previous studies have been carried out on cell cultures derived from primary cells, which does not fully represent the real situation in the skin, while the mitochondrial parameters were considered barely or not at all. Here we describe a method to measure mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using an Agilent Seahorse XF24 Flux Analyzer. Before the assay, epidermis and dermis are separated enzymatically, we then used the XF24 Islet capture microplates to position the epidermis samples to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). In these plates, small nets can be fixed to the plate bottom. The epidermis was placed with the vital–basal–side on the net. Active ingredients in the three ports were injected consecutively to determine the effect of each compound. This allows determining the efficiency of the individual complexes within the respiratory chain. This protocol enables the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.
0 Q&A 6465 Views Jan 20, 2019
Insulin resistance is a pathophysiological state defined by impaired responses to insulin and is a risk factor for several metabolic diseases, most notably type 2 diabetes. Insulin resistance occurs in insulin target tissues including liver, adipose and skeletal muscle. Methods such as insulin tolerance tests and hyperinsulinaemic-euglycaemic clamps permit assessment of insulin responses in specific tissues and allow the study of the progression and causes of insulin resistance. Here we detail a protocol for assessing insulin action in adipose and muscle tissues in anesthetized mice administered with insulin intravenously.
1 Q&A 4560 Views Nov 5, 2018
Eye drop treatments are typically used to apply drugs to the anterior structures of the eye. Recently, however, studies have demonstrated that eye drops can reach the retina in the back of the eye if pharmacological agents are carried in appropriate vehicles. Here, we introduce an eye drop procedure to deliver a drug (PNU-282987), in combination with BrdU, to stimulate cell cycle re-entry and label dividing cells in the retinas of adult rodents. This procedure avoids potential systemic complications of repeated intraperitoneal injections, as well as the retinal damage that is induced by repeated intravitreal injections. Although the delivery of PNU-282987 and BrdU is the focus of this article, many different proliferating compounds could be delivered to the retina using this procedure.
0 Q&A 5557 Views Oct 20, 2018
Coronary artery disease remains an important cause of morbidity and mortality. Previous work, including ours, has focused on the role of intraplaque hemorrhage, particularly from immature microvessel angiogenesis, as an important contributor to plaque progression via increases in vascular permeability leading to further intraplaque hemorrhage, which increases red cell membrane-derived free cholesterol in plaque content and inflammatory cell recruitment. Evans Blue Dye (EBD) assay is widely used as a standard assay for vasculature permeability. However, the method has not been established in fresh human coronary artery autopsy samples to evaluate intraplaque microvessel permeability and angiogenesis. In this protocol, we describe a method to evaluate human coronary samples for microvascular permeability, including procedures to perfuse coronary arteries, collection of artery samples for histological analysis and immunostaining as well as the use of appropriate methodology to analyze the images. An optional procedure is also provided for the use of FITC-dextran in mouse model to evaluate vascular permeability. These Evans Blue Dye procedures may be useful in providing functional measure of the endothelium integrity and permeability in both human samples and animal models in various pathological conditions.
0 Q&A 4443 Views Sep 20, 2018
Research in the area of in vivo olfactory physiology benefits from having direct access to the nasal airways through which odorants can be presented. Ordinarily, the passage of odorants through the airways is controlled by respiratory rhythm. This fact makes it difficult to control the timing and strength of an olfactory stimulus, since animals must breathe regularly, and the act of breathing itself also controls odorant presentation. However, using an artificial inhalation preparation allows us to decouple breathing from olfaction. With this technique we present oxygen and anesthetic (if desired) to the lungs directly and independently control odorant access to the nasal passages. This technique allows for direct control of odorant presentation in vivo, enabling more precise control of parameters of stimulation when investigating olfactory processing. This technique may have additional applications, for example in aerosolized drug delivery.

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