OG-P foi apoiada por CONACyT s subvenção (CB-2008-101476) e Fraba (686/10). AQ-H apoiado pelo Instituto Nacional de Saúde, o Howard Hughes Medical Institute, da Robert Wood Johnson Foundation e da Fundação Maryland células-tronco.

<ol><li> Fazer agulhas de vidro antes da cirurgia mouse: <ol class="lalpha"><li> Coloque o tubo de vidro capilar em um extrator de micropipeta. </li><li> O calor do meio do tubo de vidro para suavizar o tubo de vidro na área localizada. </li><li> Estique o tubo de vidro ao longo de seu eixo longitudinal por uma distância inicial suficiente para causar uma redução no diâmetro do tubo de vidro na área localizada. </li><li> Mantenha o tubo de vidro que se estende até que quebre. Desta forma, dois idênticos agulhas único barril são obtidos. </li></ol></li><li> Coloque a agulha de vidro em uma microforge. Um ângulo de 30 ° é suficiente para chanfrar a ponta. </li><li> Verifique sob o microscópio que cada agulha tem o diâmetro correto interior. Para a maioria das drogas hidrofílicas um diâmetro 30-50 mM interior é ideal (figura 1). </li><li> Encha-se a agulha com óleo mineral a partir da maior final de agulha de vidro. Deixe que o óleo mineral (MSDS, Cat. M7700) entra por capilaridade, até atingir ~ 50% do comprimento do tubo capilar. Coloque a vácuo de alta gordura (Dow Corning, Cat. 05054-AB) em todo o êmbolo para selar o maior ponta da agulha. </li><li> Insira o êmbolo pelo maior ponta da agulha e empurre delicadamente o óleo mineral até que uma pequena gota de óleo é visto saindo da ponta da agulha de vidro. </li><li> Segura a agulha de vidro no suporte do microinjetor. </li><li> Anestesiar o rato com Avertin 2,5% (2,2,2-tribromoetanol + terc-amil álcool, 01:01 w / v). Dose de 25 30μL por grama intraperitoneal. </li><li> Coloque uma almofada de aquecimento no aparelho estereotáxico para manter a temperatura corporal do animal a 37 ° C. </li><li> Lugar e segura a cabeça do rato no dispositivo estereotáxico usando barras de ouvido e um suporte de dentes. </li><li> Limpe a cabeça do rato com a solução de gluconato de clorexidina 0,1%, seguido por etanol 70% e gluconato de clorexidina 0,1% pela segunda vez. </li><li> Inciso a pele com uma lâmina cirúrgica # 15 de orelhas de rato para a linha do crânio Lamba. </li><li> Polonês do crânio com um cotonete para secar o produto e puxar a pele para fora do campo cirúrgico. </li><li> Use a ponta da agulha de vidro para apontar para o vértice da sutura bregma. Lá você tem que definir a posição inicial do injetor (a coordenada &quot;zero&quot;). </li><li> Definir o ponto a ser perfurado, movendo o &quot;X&quot; e &quot;Y&quot; eixo do dispositivo estereotáxico sobre o crânio. </li><li> Broca com muito cuidado buracos na coordenada selecionada. Microdrills e ferramentas de outros metais foram previamente esterilizado a 250 ° C por 60 segundos com um pano seco de contas de vidro esterilizador (Cole Palmer, Cat. No. EW-10779-00). </li><li> Com uma pinça fina remover com cuidado a camada mais profunda do osso e romper a membrana duramatter (você vai ver algum fluido cérebro-espinhal vazando). </li><li> Confirmar que a agulha pode passar por buracos perfurados. </li><li> Pipetar 1 mL da droga você quer injetar e colocá-lo em um pequeno pedaço de parafilme. </li><li> Coloque o parafilme sobre o crânio. </li><li> Sugam a droga girando a roda microinjetor, enquanto verifica sob o microscópio que o fluido está subindo para dentro da agulha de vidro. </li><li> Remover o parafilme e re-definir a coordenar zero. </li><li> Mover a agulha para as coordenadas selecionado, o porta até que a ponta da agulha de vidro tocar suavemente na superfície do cérebro e definir a &quot;Z&quot; coordenar a zero. </li><li> Introduzir a agulha de vidro para o parênquima cerebral em profundidade selecionada. </li><li> Injetar a droga lentamente a uma taxa de ~ 1 nl / sec. </li><li> Quando o volume desejo é injetado, deixe o difuso de drogas dentro do parênquima por 2 min. Em seguida, avance para remover a agulha suavemente e lentamente. </li><li> Cola a ferida cirúrgica e remover o animal a partir do dispositivo estereotáxico e colocar o animal em uma gaiola pré-aquecido contendo uma cama limpa para a recuperação da anestesia. </li><li> Para fornecer analgesia pós-operatória todos os animais receberam 5 mg / kg subcutânea Ketorolac cada hora de 12 para 24 h. </li></ol> Resultados representativos: Ao seguir este protocolo, uma injeção muito precisa é obtida e uma faixa muito estreita agulha minimiza lesão cerebral. Como resultado representante deste método, injetamos lysophosphatidyl colina (lysolecithin) no corpo caloso, que produz desmielinização de tratos de substância branca 1-4. Para minimizar a lesão cerebral produzido pela agulha de vidro, injetou apenas 20 nl de lysolecithin no corpo caloso, mas se necessário maior volume de até 200 nl pode ser injetado com o mesmo método. Desmielinização é detectada pela ausência de mielina expressão da proteína básica na tratos de substância branca (Figura 2). <img src="/files/ftp_upload/2082/2082fig1.jpg" alt="Figura 1" /> Figura 1. Uma agulha de vidro com um diâmetro de 50 mícrons. Distância entre dois ticks curtana escala representa um comprimento de 50 mícrons. <img src="/files/ftp_upload/2082/2082fig2.jpg" alt="Figura 2" /> Figura injeção Lysolecithin 2. No corpo caloso. Desmielinização é mostrado como um não a expressão da proteína básica de mielina (área pontilhada). Observe o pequeno tamanho do vidro do trato agulha (setas). Bar = 100 mm

<ol>
	<li>Menn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+oligodendrocytes+in+the+subventricular+zone+of+the+adult+brain.">Origin of oligodendrocytes in the subventricular zone of the adult brain.</a> J Neurosci. 26, 7907-7918 (2006).</li><li>Gonzalez-Perez, O., Romero-Rodriguez, R., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Growth+Factor+Induces+the+Progeny+of+Subventricular+Zone+Type+B+Cells+to+Migrate+and+Differentiate+into+Oligodendrocytes.">Epidermal Growth Factor Induces the Progeny of Subventricular Zone Type B Cells to Migrate and Differentiate into Oligodendrocytes.</a> Stem Cells. 27, 2032-2043 (2009).</li><li>Hall, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+aspects+of+remyelination+after+demyelination+produced+by+the+intraneural+injection+of+lysophosphatidyl+choline.">Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidyl choline.</a> J Cell Sci. 13, 461-477 (1973).</li><li>Webster, G. R., Thompson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+lysolecithin+in+nervous+tissues.">Observations on the presence of lysolecithin in nervous tissues.</a> Biochim Biophys Acta. 63, 38-45 (1962).</li><li>Doetsch, F., Caille, I., Lim, D. A., Garc&#237;a-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+Zone+Astrocytes+Are+Neural+Stem+Cells+in+the+Adult+mammalian+Brain.">Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult mammalian Brain.</a> Cell. 97, 1-20 (1999).</li><li>Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+converts+transit-amplifying+neurogenic+precursors+in+the+adult+brain+into+multipotent+stem+cells.">EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.</a> Neuron. 36, 1021-1034 (2002).</li><li>Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106, 15472-15477 (2009).</li><li>Richardson, R. M., Barbaro, N. M., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+cell+transplantation+for+temporal+lobe+epilepsy.">Developing cell transplantation for temporal lobe epilepsy.</a> Neurosurg Focus. 24, E17-E17 (2008).</li><li>Alvarez-Dolado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26, 7380-7389 (2006).</li></ol>

Não há conflitos de interesse declarados.

O método mostrou neste vídeo é muito útil para entregar a maioria das drogas ou vetores virais em lugares muito precisos para o cérebro. Algumas das principais vantagens desta técnica são a confiabilidade do ponto de focalização, a precisão das injeções eo pequeno tamanho da lesão cerebral e danos trato 1, 2, 5, 6. Uma vez que a técnica é padronizada a faixa de 50 mm deve mistargeting ou menos 1, 2. Transplantes de células também pode ser feito usando mais amplo, 100-150 mM 7-9, agulhas. Entrega eficiente de células, portanto, pode ser depositado em muito específicas minimizar danos colaterais lesão induzida por transplante de células. Há três passos cruciais nesta técnica: <ol><li> Recomendamos sempre arranhando suavemente o crânio com uma agulha de insulina para rotular as coordenadas selecionado antes de perfuração. </li><li> Introduzir a agulha de vidro com um movimento rápido para penetrar rapidamente na superfície do cérebro. </li><li> Uma vez que a profundidade ideal é atingido você deve introduzir a agulha 0,1 milímetros mais profundamente no parênquima cerebral para fazer um &quot;canal&quot; que deixe de drogas livremente difusa no cérebro. </li></ol>

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Capillary glass tube</td>
<td>&nbsp;</td>
<td>Wiretrol I</td>
<td>5-000-1001</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model 730</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Model 48000</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>MSDS</td>
<td>M7700</td>
<td>&nbsp;</td>
<tr>
<td>High-vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>05054-AB</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia: 2.5% Avertin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2,2,2-tribromoethanol + tert-amyl alcohol, 1:1 w/v</td>
</tr>
<tr>
<td>Heater pad</td>
<td>&nbsp;</td>
<td>Mastex</td>
<td>Model 900</td>
<td>&nbsp;</td>
<tr>
<td>Stereotactic device</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model Kopf-900</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scalpel blade # 15</td>
<td>&nbsp;</td>
<td>Medi-Cut</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro driller</td>
<td>&nbsp;</td>
<td>Ideal Micro Drill</td>
<td>67-1000</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Fine Scientific Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-&mu;L Micropipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm M.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Vasiorkop with Contraves system</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>Model MO-10</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants

Microinjeções no parênquima cerebral são procedimentos importantes para entregar drogas, vetores virais ou transplantes de células. A lesão cerebral que uma agulha de injeção produz durante sua trajetória é uma grande preocupação, especialmente no cérebro do rato, não só para o cérebro é pequeno, mas às vezes também de múltiplas injeções são necessárias. Mostramos aqui um método para produzir agulhas capilar de vidro com um lúmen 50 mícrons, que reduz significativamente os danos cerebrais e permite uma segmentação precisa no cérebro de roedores. Este método permite uma entrega de pequenos volumes (20-100 nl), reduz os riscos de sangramento, e minimiza a difusão passiva de drogas dentro do parênquima cerebral. Usando diferentes tamanhos de tubos capilares de vidro, ou mudando o lúmen da agulha, vários tipos de substâncias e células pode ser injetado. Microinjeções com um tubo de vidro capilar representam uma melhoria significativa nas técnicas de injeção e cerebral profunda segmentação com danos colaterais mínimos em pequenos roedores.

Watch this Scientific Journal Video about Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants at JoVE.com

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Neste artigo, vamos mostrar um método para fazer agulhas capilar de vidro com um lúmen 50 mícrons. Esta técnica reduz significativamente os danos cerebrais, minimiza difusão passiva de drogas e permite uma segmentação precisa no cérebro de roedores.

Segmentação de estruturas cerebrais profundas com Microinjeções para entrega de drogas, vetores virais, ou Transplantes de Células

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

targeting-deep-brain-structures-with-microinjections-for-delivery

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.5K visualizações.  University of Colima. Neste artigo, vamos mostrar um método para fazer agulhas capilar de vidro com um lúmen 50 mícrons. Esta técnica reduz significativamente os danos cerebrais, minimiza difusão passiva de drogas e permite uma segmentação precisa no cérebro de roedores.

Vídeo: Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Escalável Fluidic Arrays Injector para Viral Segmentação de Intact Circuitos 3-D do cérebro

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Neurociência

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Injeção intracraniana da adeno-associado vetores virais

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

Estimulação Cerebral Profunda com fMRI simultâneo em Roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Não restrição EEG Radiotelemetria: Epidural e Deep intracerebral estereotáxica EEG Colocação de eletrodos

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Agulhas personalizadas para alevinos do cérebro de roedor

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Explorando o espaço profundo - descobrindo a anatomia das estruturas periventriculares para revelar os ventrículos laterais do cérebro humano

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Biologia celular quantitativa de neurodegeneração em Drosophila , através de uma análise imparcial das proteínas fluorescente etiquetadas usando o ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Produção, purificação e controle de qualidade para vetores de baseados em vírus Adeno-associado

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Modelagem e análise tridimensional da forma de estruturas do cérebro

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

In vivo reprogramação direta de células gliais residentes em interneurônios por injeção intracerebral de vetores virais

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...