Name: symmetric bihemispheric postmortem brain cutting to study healthy and pathological brain conditions in humans
Uploaded: 2016-12-18T14:00:00
Description: Watch this Scientific Journal Video about Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans at JoVE.com

We thank the thousands of brain donors, patients, families, and neuroscientists around the world who, during the last two centuries and through their generous gifts and intellectual efforts, helped to discover how the human brain works, to understand devastating brain diseases, and to develop treatments thereof. We particularly thank Mrs. Cecilia V. Feltis for editing and reviewing this manuscript.

Procedures involving postmortem human tissues have been reviewed by the institutional review board and exempted under 45 CFR (Code of Federal Regulations).
NOTE: The protocol describes a symmetric bihemispheric brain cutting procedure for postmortem brain assessment finalized for neuropathological studies in humans. Detailed descriptions of the apparatuses, instruments, materials, and supplies necessary to perform human brain cutting will be excluded. Materials and supplies for brain dissections are selected at the discretion of the s...

This brain cutting method can be adapted to the specific needs of each neuropathology lab (for example, by reducing the number of cerebral regions to assess for each hemisphere) while still retaining the bihemispheric symmetric cutting procedure as one of its main features. This proposed protocol could be used for routine procedures (research-oriented neuropathological centers) or only when necessary (specific clinically oriented studies). It can be selectively used only for specific types of investigations (i.e.,</e...

Neuropathologists have the scientific privilege, intellectual honor, and diagnostic obligation to assess human brains. For many decades, detailed clinical descriptions of brain diseases and major efforts to individuate their possible neurohistological correlates in human postmortem brains have been undertaken. Historically, those efforts represented the most productive modality by which the medical sciences, and neurology in particular, advanced in the modern era. Thanks to previous eminent neuropathologists and their dedication, determination, scholarship, and astonishing capacity to discriminate between normal and abnormal brain tissues (often using very rudimental tools), we can now investigate and target diseases such as Alzheimer-Perusini&#39;s disease (unfairly only called Alzheimer&#39;s disease; APD/AD)1, Parkinson&#39;s disease (PD)2, Creutzfeldt-Jakob disease (CJD)3, Lou-Gehrig&#39;s disease/Amyotrophic Lateral Sclerosis (ALS)4, and Guam disease5, to mention a few.
Advanced techniques of neuroimaging, such as high-definition computerized tomography (i.e., multisection spiral CT scan; CT angiography), functional and morphological magnetic resonance imaging (i.e., fMRI, diffusion-MRI, tractography-MRI, etc.), Positron Emission Tomography (PET), ultrasound-based imaging, and others, have certainly modified our general approach on how to diagnose and cure neurological and psychiatric patients. Nonetheless, although neuroimaging techniques are capable of visualizing a person&#39;s brain when alive, they do not offer the opportunity, at the occurring moment, to directly analyze the highly intricate cellular and subcellular structures of cells, such as neurons; or to visualize, mark, and quantify specific types of intracellular lesions; or to precisely indicate their neuroanatomical or subregional localization at circuital and sub-circuital anatomical levels. For example, neuroimaging techniques cannot identify or localize Lewy Bodies (LB) in pigmented neurons of the Substantia Nigra (SN), a common pathologic feature associated with PD, or neurofibrillary tangles (NFT) in the entorhinal cortex, a classical feature of AD and other brain pathologies. Neuropathological investigations combined with advanced digital microscopy are still unreplaceable for detailed clinicopathological correlations and, thus, for definitive diagnoses.
Due to the peculiar anatomo-functional properties of the human brain, and especially to its anatomical localization (that is, inside the skull, a natural protective system that does not allow the direct examination of its content), the introduction of in vivo neuroimaging techniques have extraordinarily helped clinicians and investigators to find initial answers to some of the mysteries of this complex tissue. However, there is no clinical or neuroimaging methodology that can replace the unique opportunity to directly analyze brain tissue during an autopsy. Only the organized collection, preservation, and categorization of human brains can allow direct and systematic investigations of neuronal and non-neuronal cells, their subcellular constituents, intracellular and extracellular pathological lesions, and any type of abnormality inside the brain to confirm, modify, or redefine clinical diagnoses and to discover new clinicopathological correlations. One of the apparent limitations concerning the assessment of the brain at autopsy has been the fact that this procedure is a cross-sectional methodology. There will always be a delay between an ongoing neuropathological process (clinically manifested or not) and the chance, if any, to define it at the neurohistological level. This is mainly due to the incapacity of the human brain to regenerate itself. It is not currently possible to obtain brain tissue in vivo without creating permanent damage. Consequently, it is not possible to longitudinally and neuropathologically assess the same brain/person. However, standardized brain banking procedures and an increased awareness for brain donation among the general public could greatly contribute to the resolution of brain-autopsy timing issues by consistently increasing the number of cases to collect and analyze. In this manner, more adequate numbers of postmortem brains could be obtained to define constant patterns of pathological origin and progression for each specific type of brain lesion associated with each human brain disease. This would require donation and collection of as many brains as possible from patients affected by any neuropsychiatric disorder, as well as from healthy control subjects across all ages. One possible method could be collecting as many postmortem brains as possible from general and specialized medical centers as a standard routine. The need for brain donations has been recently expressed by those who study dementia and normal aging6. The same necessity should be expressed by the neuropsychiatric field as whole.
For the abovementioned and for other reasons, an update of ongoing brain cutting procedures is necessary. Moreover, brain cutting procedures should be universally standardized across different neuropathology research centers around the world, also taking in account the possibility to employ current and future biotechnological techniques to better investigate and, hopefully, to definitively understand, the causes and mechanisms of brain diseases in humans.
Here, mainly for research purposes, we describe a symmetric methodology for postmortem brain cutting in humans. This procedure proposes collecting more cerebral regions than normally done and from both cerebral and cerebellar hemispheres. A symmetric bi-hemispheric brain cutting procedure will fit much better with our current knowledge of human neuroanatomy, neurochemistry, and neurophysiology. This method also allows the possibility to neuropathologically analyze the unique features of the human brain, such as hemispheric specialization and lateralization that are associated with higher cognitive and non-cognitive functions typically or exclusively present in our species. Whether there are specific pathogenetic relationships between hemispheric specialization/lateralization and specific types of brain lesions, or whether a peculiar neuropsychiatric pathogenetic event is initially, prevalently, or exclusively associated with a specific hemisphere and function is not currently known. By describing this symmetric brain cutting procedure, we aim to propose an updated method of human brain dissection that could help to better understand normal and pathological conditions in a highly specialized tissue, the brain. This method also takes into consideration those morpho-functional hemispheric aspects that exist only in humans.

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			<td>Copy of signed informed consent allowing autopsy and brain donation for research use.</td>
			<td></td>
			<td></td>
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			<td>Detailed clinical history of the subject which should include a detailed description of any neurologic and psychiatric symptoms and signs.</td>
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			<td>Medical or not-medical video-recordings when available (especially useful in movement disorders field). Next-of-kin&#8217;s consent required.</td>
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			<td>Genetic and family history data. Genetic reports review, if neurogenetic diseases were diagnosed.</td>
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			<td>Histology Container</td>
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			<td>Accu-Punch 2 mm</td>
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			<td>Formalin solution, neutral buffered, 10%</td>
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			<td>HT501128 SIGMA</td>
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			<td>Hematoxylin Solution, Gill No. 2</td>
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			<td>Eosin Y solution, aqueous</td>
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			<td>anti-beta-amyloid</td>
			<td>Covance, Princeton, NJ</td>
			<td>SIG-39220</td>
			<td>1&#160; 500</td>
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			<td>anti-tau</td>
			<td>Thermo Fisher Scientific</td>
			<td>MN1020</td>
			<td>1&#160; 500</td>
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			<td>anti-alpha-synuclein</td>
			<td>Abcam</td>
			<td>ab27766</td>
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			<td>anti-phospho-TDP43</td>
			<td>Cosmo Bio Co.</td>
			<td>TIP-PTD-P02</td>
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			<td>Digital Camera</td>
			<td>Any</td>
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			<td>Head Impulse Sealing machine&#160;</td>
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symmetric bihemispheric postmortem brain cutting to study healthy and pathological brain conditions in humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Protocol Length
The time spent for a single symmetric bihemispheric fixed brain cutting procedure is estimated at 1 h (excluding the time spent setting up the dissection table, tools, and cutting surfaces; labeling; etc.). The time required for a single symmetric bi-hemispheric alternating frozen and fixed brain cutting procedure is estimated to take 2 h. It can take at least between 4 - 6 weeks to obtain definitive histol...

Watch this Scientific Journal Video about Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans at JoVE.com

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

The overall goal of this procedure is to systematically collect and analyze a constant series of cerebral regions symmetrically dissected from both hemispheres of the human brain in order to assess severity, clinical pathologic staging and hemispheric distribution of various brain diseases. This method can help answer key questions in the neuropathology field providing a systematically organized collection of qualitative and quantitative neuropathologic data for detailed clinical pathologic and imaging pathologic correlations. The main advantage of this technique consists in yielding relevant data related to the pathologic processes per se as well as the histofunctional specialized aspect of each cerebral hemisphere.After fixing post-mortem human brain tissue according to the text protocol, place the brain on a flat surface with the frontal poles directed opposite the investigator. Orient the brain to allow full and clear visualization of all cortical gyri and sulci of the entire cerebrum. Examine the tissue for meningeal anomalies, macroscopic hemispheric asymmetries, macroscopic tissue allegiance, congenital malformations, vessel abnormalities or any other abnormalities or unusual presentations of the cerebral surface.With the frontal poles oriented away from the investigator and the superficial aspects of the hemispheres facing the investigator, take as many digital pictures as necessary to document possible macro anomalies and to account for possible clinic, neural, anatomical and post-cutting considerations. Take digital pictures perpendicularly to the brain to capture the entire cortical surface. Then using ink or colored needles, mark pre and post central cortical gyri.Next, with the frontal poles still facing away, flip the brain 180 degrees then carefully inspect the base of the brain paying special attention to the cerebrovascular systems and the cranial nerves at the brain stem exit and entrance levels. Manage the olfactory bulbs and tracts with special care to avoid tissue laceration due to their extreme frailty. Take digital photographs perpendicularly to the brain to capture the entirety of the cortical and brain stem surfaces.Facing the base of the brain and using a scalpel, cut the brain stem transversely at the upper portion of the pons as close as possible to the base of the cerebrum. Carefully inspect the substantia nigra for pallor and inspect other neighboring structures. Using an audio recording device, take note of any unusual appearance of the brain in comparison to a normal brain.Here, the olfactory bulb and tract of the left and then right hemispheres are being dissected. Flip the brain back over and using a sharp knife, follow a fronto-occipital direction to separate the two hemispheres by cutting the corpus callosum centrally through the medial longitudinal fissure. Inspect each side of each hemisphere for possible anomalies.After taking as many pictures as necessary, including perpendicularly to the brain to capture the entire cortical surface, place the two hemispheres flat laying on their medial aspects with the frontal lobes facing away from the investigator and with their centers touching. Using a sharp knife, manually cut through both cerebral hemispheres starting at the frontal poles and move towards the occipital poles through the entire length of the hemispheres to obtain two series of one centimeter thick slabs of brain tissue. Position the brain slabs in an anatomically organized and symmetric sequence on a flat white surface with a ruler printed on it for photographing.Ensure that the slab's coronal surfaces are visible for direct eye inspection and digital photography. Use cutting surfaces with printed millimetric grids on both sides to localize brain structures, sizes, and possible abnormalities in a more accurate manner. With the coronal surfaces visible, take digital photographs perpendicularly to capture the entire cortical surface.Take notes of any unusual aspect of the brain in comparison to a normal brain. Next, using a sharp scalpel, manually dissect small rectangular blocks of brain tissue for each established cerebral region. Follow the proposed cerebral region collection scheme described in this table.Create the identifying codes to label the histo cassettes by generating random or semi-random numbers for each case where AD stands for Alzheimer's Disease Study, 16 stands for the year the autopsy was performed and 0001 is a progressive accession specimen number. Place each tissue block in separate histo cassettes. Take as many digital pictures as described earlier of the entire cut brain and the associated histo cassettes.After taking a complete set of pictures, punch two to five millimeter pieces of tissue for DNA extraction and genetic analysis. Finally, immerse all histo cassettes containing brain tissue blocks in the same type of fixative solution as previously used and process the tissue according to the text protocol. This table describes some preliminary semi-quantitative data using a symmetric bihemispheric brain cutting procedure performed on a series of human brains collected in the brain bank.Preliminary data show that most control brains from older subjects were actually positive for beta-A neuritic plaques and tau-neurofibrillary tangles and that levels of insoluble of beta-A neuritic plaques were higher in the left entorhinal cortex and hippocampus in comparison to the same brain regions on the right side. Once mastered, this technique can be completed in an hour for fixed brains and in two hours for fresh brains if it's properly performed. While attempting this procedure, it's important to remember following the proposed cutting scheme, collect each cerebral region from both hemispheres based upon precise human anatomical knowledge.Following this procedure, other methods like DNA, RNA or protein extraction can be performed in order to answer additional questions such as what are the activation levels of a specific gene in a specific brain region in a specific hemisphere in a specific brain disease. With each diffusion, this technique will pave the way for researchers in the field of neuropathology, clinical pathology and imaging pathology correlation to explore the hemispheric related pathologic aspects of various neuropsychiatric diseases. After watching this video, you should have a good understanding of how to collect specific anatomical regions from both hemispheres of the human brain to assess severity of the disease, pathologic stage of the disease and hemispheric distribution of different neuropathologic processes.Don't forget that working with the human brain is working with the history of a person with complex neuroanatomical circuits and possibly with different diseases and that blades, scalpels, formalin must be handled with extreme precaution.

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Research

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Medicine

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...