Name: cannula implantation into the cisterna magna of rodents
Uploaded: 2018-05-23T14:00:00
Description: Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

Este trabalho foi financiado pelo Novo Nordisk Foundation e Instituto Nacional de Disorders Neurological e curso, NINDS/NIH (M.N.). A.L.R.X. e Samões-R são beneficiários de uma bolsa de pós-doutorado e uma bolsa de doutoramento da Fundação Lundbeck, respectivamente.

Todos os procedimentos foram realizados em conformidade com a Directiva 2010/63/UE Europeu para pesquisas com animais e foram aprovados pelo Conselho de experimentos animais sob o Ministério do ambiente e comida (2015-15-0201-00535).
1. procedimento para canulação
<ol><li>Preparação de cânula Nota: Evite tocar a cânula com luvas não estéreis.<ol>
<li>Quebre a ponta de metal chanfrada com uma agulha de 30g dental usando um porta-agulha.</li>
		<li>Usando um porta-agulha, preparar ...

<ol>
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Nós apresentamos um protocolo que descreve um procedimento detalhado para canulação de magna cisterna (CMc), que oferece um método simples para entregar rotulado de moléculas para o compartimento do CSF. CMc permite a visualização posterior da dinâmica do CSF, tanto in vivo e ex vivo, usando diferentes modalidades de imagem ou histologia.
Uma das principais vantagens da técnica da CMc reside no seu acesso direto para o espaço subaracnoide sem a necessidade de expor ...

Líquido cefalorraquidiano (CSF) banha o sistema nervoso central (SNC) em todo o sistema ventricular e ao longo dos espaços subaracnoide, um espaço anatomicamente definido no continuum com os ventrículos, que envolve o cérebro e a medula espinhal. Uma das principais funções do QCA é fornecer uma rota para apuramento dos metabolitos e solutos do parênquima cerebral. Acesso é facilitado através da glymphatic recentemente descoberto sistema1, o cérebro analógico para o sistema linfático periférico. Neste documento, podemos descrever e discutir a canulação cisterna magna (CMc), um método minimamente invasivo para a entrega direta de moléculas para o CSF. CMc é o método principal para estudar a função glymphatic. Além disso, CMc também pode ser aplicado para o estudo da dinâmica do CSF e para uma entrega rápida, todo o cérebro de moléculas permeável do cérebro não-sangue barreira (BBB) para o parênquima cerebral, ao longo do espaço perivascular.
O CMc explora princípios fisiológicos da dinâmica de movimento CSF através do CNS para entregar o palpador rotulado moléculas ou drogas para o espaço cheio de CSF da cisterna magna (CM). Moléculas são injetadas através de uma cânula implantada para a cobertura de dura-máter membrana atlanto-occipital, que as moléculas CM. em seguida são transportadas por fluxo de massa CSF para o parênquima cerebral através do paravascular espaço1. Agente de marcador ou contraste injetado através do CMc segue o movimento do CSF, que permite a avaliação da circulação do CSF e influxo de glymphatic através da quantificação de níveis de intensidade de moléculas rotulados que insira o parênquima cerebral. CMc é compatível com diferentes técnicas de imagem, incluindo epifluorescência, 2-fotão microscopia e ressonância magnética (MRI). Além disso, esta avaliação pode ser realizada tanto em vivo ou ex vivo. Importante, CMc permite a visualização do sistema de glymphatic sob anestesia ou durante o sono natural, assim como em animais acordados, andando livremente.
A técnica de CMc pode ser utilizada para estudar os diferentes aspectos da dinâmica dos fluidos no CSF, mas provou para ser particularmente útil para estudar o sistema de glymphatic. Glymphatic atividade impulsiona o fluxo convectivo de LÍQUOR do espaço periarterial através de canais de água aquaporina-4 (AQP-4), que são amarrados na membrana dos hamartomas vasculares quebra automática de endfeet. O fluxo convectivo permite o intercâmbio de CSF e fluido intersticial (ISF) dentro do parênquima cerebral. CSF/ISF contendo solutos e resíduos metabólicos é então removido o parênquima cerebral através do espaço de perivenous2,3. Finalmente, a CSF/ISF alcança a periferia através de vasos linfáticos dural recentemente descrito4,5. O sistema glymphatic foi mostrado crucial para a habilitação de metabolitos resíduos prejudiciais tais como β-amiloide2. Além disso, glymphatic afastamento é prejudicado no envelhecimento6, depois de lesão cerebral traumática7e em modelos animais de diabetes8 e a doença de Alzheimer9. Nomeadamente, glymphatic atividade é estado dependente, mostrando atividade significativamente maior durante o sono ou anestesia em comparação com a vigília1. De fato, animais anestesiados jovens apresentam a mais alta atividade de glymphatic. Assim, a quantificação experimental da atividade glymphatic é crítica quando estudando o seu papel na saúde e na doença.
Vários estudos têm abordado CSF dinâmica e seu intercâmbio com o líquido intersticial (ISF) no parênquima cerebral. No entanto, os métodos pelos quais moléculas etiquetadas são entregues são bastante invasivos, provocando dano do parênquima cerebral e alterações na pressão intracraniana (ICP) (ver revisão10). Alguns exemplos são intraventricular ou injeções difuso que envolvem craniotomia ou perfuração de uma rebarba do furo no crânio. Esses procedimentos foram mostrados para alterar o ICP, rompendo assim a glymphatic função2. Além disso, tais métodos invasivos induzem astrogliosis e aumentam imunorreatividade AQP-4 na área danificada de parênquima cerebral e seus arredores11,12. Como astrócitos e AQP-4 são elementos-chave do sistema de glymphatic, o CMc é o método de escolha para seus estudos. As principais vantagens do CMc em comparação com procedimentos invasivos mais são a manutenção de uma parênquima de crânio e cérebro intacta, evitando alterações de ICP e astrogliosis, respectivamente. Assim, o CMc em conjunto com diferentes ferramentas de imagem abre para uma ampla gama de possibilidades para estudar não só o sistema de glymphatic, mas também a dinâmica e os mecanismos de fluxo de fluido na homeostase, bem como em modelos animais de doenças neurológicas.
O procedimento de cateterização (CMc) cisterna magna permite acesso fácil e direto para o líquido cefalorraquidiano (CSF). Através da injeção de moléculas diferentes (por exemplo, marcadores fluorescentes, agentes de contraste MRI) o experimentador pode acompanhar o seu movimento no interior do compartimento do CSF e avaliar a atividade do sistema glymphatic. O protocolo seguinte descreve ambos o CMc aguda, para injeções imediatamente após a cirurgia e implantação crônica da cânula, em que o animal se recupera do procedimento cirúrgico para uma posterior injeção. A diferença mais importante entre a implantação aguda e crônica é que a implantação crônica permite o estudo da atividade glymphatic em ratos acordados.

<table border="0" cellpadding="0" cellspacing="0" width="1153">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">SOPIRA Carpule 30G 0.3 x 12mm</td>
			<td style="width:245px;">Kulzer</td>
			<td style="width:257px;">AA001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Polyethylene Tubing 0.024&#8221; OD x 0.011&#8221; ID</td>
			<td style="width:245px;">Scandidact</td>
			<td>PE10-CL-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G x &#189;&#8221; 0.3 x 12 mm Luer-Lock</td>
			<td style="width:245px;">Chirana T. Injecta</td>
			<td>CHINS01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:485px;">Chlorhexidine 0.5% (chlorhexidine digluconate)</td>
			<td style="width:245px;">Meda AS</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.meda.dk/behandlingsomrader/desinfektion/desinfektion-af-hud/klorhexidin-sprit-medic-05/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Swab 70% Isopropyl Alcohol 30 x 60mm</td>
			<td>Vitrex Medical A/S</td>
			<td>520213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viskoese Oejendraeber Ophtha</td>
			<td>Ophtha</td>
			<td>145250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Wooden applicator, Double cotton bud (&#216; appr. 4 - 5 mm, length appr. 12 mm)</td>
			<td style="width:245px;">Heinz Herenz</td>
			<td style="width:257px;">1032018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye spears</td>
			<td>Medicom</td>
			<td>A18005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric chloride 10% solution</td>
			<td style="width:245px;">Algeos</td>
			<td style="width:257px;">NV0382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimtech Science Precision Wipes Tissue Wipers</td>
			<td style="width:245px;">Kimberly Clark Professional</td>
			<td style="width:257px;">05511</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Loctite Super Glue Precision 5g</td>
			<td style="width:245px;">Loctite</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.loctite-consumer.dk/da/produkter/superglue-liquid.html&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insta-Set CA Accelerator</td>
			<td style="width:245px;">Bob Smith Industries</td>
			<td style="width:257px;">BSI-152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Powder</td>
			<td style="width:245px;">A-M Systems</td>
			<td style="width:257px;">525000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical weld&#160;</td>
			<td style="width:245px;">Kent Scientific Corporation</td>
			<td style="width:257px;">INS750391</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Hamilton syringe GASTIGHT&#174; , 1700 series, 1710TLL, volume 100 &#956;L, PTFE Luer lock</td>
			<td>Hamilton syringes</td>
			<td style="width:257px;">1710TLL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">LEGATO 130 Syringe pump</td>
			<td style="width:245px;">KD Scientific</td>
			<td style="width:257px;">788130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Paraformaldehyde powder, 95%</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Phosphate buffered saline (PBS; 0.01M; pH 7.4)</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">P3813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Ovalbumin, Alexa Fluor 647 Conjugate</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">O34784</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;"> 
			DAPI (diamidino-2-phenylindole) Solution (1 mg/mL)</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Dextran, Fluorescein, 3000 MW, Anionic</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">D3305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-Z Anesthesia EZ-7000 Classic System</td>
			<td style="width:245px;">E-Z Systems</td>
			<td>EZ-7000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Attane Isofluran 1000 mg/g</td>
			<td style="width:245px;">ScanVet</td>
			<td>55226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Euthanimal 200mg/mL (sodium pentobarbital)</td>
			<td style="width:245px;">ScanVet</td>
			<td style="width:257px;">545349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Ketaminol Vet 100 mg/mL (ketamine)</td>
			<td>Intervet International BV</td>
			<td style="width:257px;">511519</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Rompin Vet 20 mg/mL (xylazin)</td>
			<td style="width:245px;">KVP Pharma + Veterin&#228;r Produkte GmbH</td>
			<td style="width:257px;">148999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Xylocain 20 mg/mL (lidocain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">158543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Marcain 2.5 mg/mL (bupivacain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">123918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Bupaq Vet 0.3 mg/mL (buprenorphine)</td>
			<td style="width:245px;">Richter Pharma AG</td>
			<td style="width:257px;">185159&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

cannula implantation into the cisterna magna of rodents

Canulação de cisterna magna (CMc) é um procedimento simples que permite acesso direto para o líquido cefalorraquidiano (CSF) sem danos operativo para o crânio ou o parênquima cerebral. Em roedores anestesiados, a exposição da dura-máter por dissecção romba dos músculos do pescoço permite a inserção de uma cânula para a cisterna magna (CM). A cânula, composta por uma agulha fina chanfrada ou borosilicato capilar, está ligada através de um tubo de polietileno (PE) com uma seringa. Usando uma bomba de seringa, moléculas podem então ser injetadas em taxas controladas diretamente o CM, que é contínua com o espaço subaracnoideo. Partir do espaço subaracnoideo, poderemos rastrear fluxos CSF pelo fluxo convectivo no espaço perivascular em torno de arteríolas penetrantes, onde ocorre a troca de soluto com o fluido intersticial (ISF). CMc pode ser realizada por injeções agudas, imediatamente após a cirurgia, ou para implantação de crônica, com posterior injeção em anestesiados ou acordado, movimentando-se livremente roedores. Quantificação da distribuição do traçador no parênquima cerebral pode ser executada por epifluorescência, 2-fotão microscopia e ressonância magnética (MRI), dependendo das propriedades físico-químicas das moléculas injetadas. Assim, o CMc em conjunto com várias técnicas de imagem oferece uma poderosa ferramenta para avaliação do sistema de glymphatic e dinâmica do CSF e função. Além disso, a CMc pode ser utilizada como um canal para entrega rápida, todo o cérebro de sinalização moléculas e substratos metabólicos que caso contrário não atravessar a barreira hemato-encefálica (BBB).

Mediante a fixação de ratos ou ratos em uma armação estereotáxica, os músculos do pescoço em torno da região de crista occipital sem rodeios são dissecados para expor a cisterna magna (CM). A estrutura triangular do CM é facilmente reconhecida entre a porção caudal do cerebelo e a medula (figura 1A-1 C). A cânula é inserida 1-2mm a CM perfurando delicadamente a membrana atlanto-occipital (Figura 1)....

Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

Cannula Implantation into the Cisterna Magna of Rodents

Cânula de implantação para a Cisterna Magna de roedores

Aqui descrevemos um protocolo para realizar a cateterização da cisterna magna (CMc), uma forma minimamente invasiva para entregar traçadores, substratos e moléculas sinalizadoras no fluido cerebrospinal (CSF). Combinado com diferentes modalidades de imagem, CMc permite sistema de glymphatic e avaliação de dinâmica do CSF, bem como a entrega de todo o cérebro de vários compostos.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

The overall goal of this surgical procedure is to enable direct delivery of tracers, substrates and signaling molecules into the cerebrospinal fluid. This method can help to answer key questions in the neuroscience filed regarding CSF and ISF dynamics in the brain. This method allows the delivery of different molecules, tracers, active metabolic substrates into the CSF, cerebrospinal fluid, compartment without damaging the skull or the brain parenchyma.The implications of this technique extend towards neurological diseases because it allows the evaluation of CSF-ISF fluxes in the brain and also the clearance of metabolites and toxic proteins from the brain parenchyma. So this method provides insight into the dynamics of the CSF-ISF fluxes and additionally we can use it as a fast, brain-wide delivery route for signaling molecules and molecular substrates that could not otherwise pass the blood-brain barrier. To prepare the cannula, break of the beveled metal tip of a 30 gauge dental needle using a needle holder.Then, insert the beveled metal tip into a 30 centimeter length of PE-10 tubing filled with aCSF. Next, use a one milliliter syringe fitted with a 30 gauge needle to flush the cannula with artificial cerebrospinal fluid. The cannula is then ready for use in the surgery.After anesthetizing the mouse, position the animal over a heating pad on a stereotaxic apparatus. Check for a lack of pedal reflex with a toe pinch, and wait for the animal's respiration to become slow and steady. Next, shave the head and neck.Then, wipe away the fur and sterilize the exposed skin. First, scrub the skin with alcohol. Then make two applications of 0.5 per cent chlorohexidine or two per cent iodine solution.For chronic cannulations, also administer analgesics at the cannulation site. Now, fix the mouse in the stereotaxic frame, making use of either the intraneural arch or the zygomatic arch. Then, tilt the head slightly so that it forms an angle of 120 degrees to the body.Next, apply ophthalmic ointment and reapply it as needed during the surgery. Find the occipital crest, which is the part of the skull protruding immediately above the neck muscles. Lift the overlying skin using a pair of tweezers and cut an almond-shaped piece of skin of approximately one centimeter along the midline.Then, using the occipital crest as a reference point, pull apart the superficial connective tissue to expose the neck muscles below. Use cotton swabs or eye spears to control any resultant bleeding. To proceed, separate the muscles at the midline by carefully running the tweezers down the middle of the incision site in the anterior to posterior axis.Then, with a pair of curved forceps in each hand, join the tips in the middle near the bottom of the skull and pull the muscles aside to expose the cisterna magna. The cisterna magna looks like a tiny inverted triangle, outlined by the cerebellum above and medulla below. Using a surgical eye spear or cotton swab, wipe the dural membrane covering the CM.Next, with curved tweezers in the dominant hand, grasp the cannula near the tube-covered needle.Then, place the middle finger of the other hand on the ear bar to create a stable rest for the cannula. Now, at the center of the CM, puncture the dura at 45 degrees relative to the mouse head. Only insert the needle to where the bevel is under the dura to avoid penetrating into the cerebellum or medulla.If necessary, clean off any leaked CSF with a spear or swab. Then, apply a few drops of cyanoacrylate onto the dural membrane surrounding the cannula and add a drop of glue accelerator to cure the glue immediately. Next, combine dental cement and cyanoacrylate glue.Then, cover the entire incision site and quickly apply a drop of glue accelerator to cure. For injection in mice with acute cisterna magna cannulation, proceed directly to injection of CSF tracers with the aid of an injection pump. For chronic cannulation, cut the tubing down to two or three centimeters and seal it with a surgical weld to retain the inter-cranial pressure levels.Then, administer carprofen. Now, house the mouse alone and keep the cage over a heating pad until the mouse has regained sternal recumbency. To prepare the cannula, break off the beveled metal tip of a 30 gauge dental needle using a needle holder.Then, insert the beveled metal tip into a 30 centimeter length of PE-10 tubing filled with aCSF. Using a 30 gauge needle, flush the tubing. Attach the 30 gauge needle to a 100 microliter syringe filled with distilled water and then attach this to a syring pump.Using the pump, withdraw enough air to create a one centimeter bubble in the cannula. Then, dip the needle into the CSF tracer solution and withdraw 12 microliters into the cannula. Cut the tubing attached to the cannula up to three to four centimeters length and quickly connect the CSF tracer-filled tubing with the implanted cannula with the aid of fine forceps.To inject CSF tracers into the CM of anesthetized animals, use a syringe pump to deliver tracers at one microliter per minute for five to ten minutes. Then allow the tracers to circulate for 30 minutes without moving the cannula. Gently restrain the animal and cut off approximately one centimeter of the tubing from the implanted cannula.Then, quickly connect the two cannulas, using the aid of a fine forceps and a pair of curved tweezers if necessary. Now, inject one microliter of tracer per minute over seven to twelve minutes, using a syring pump. After the injection, let the tracer circulate for 30 minutes while taking the necessary measures to ensure that the cannula remains undisturbed and the tubing remains attached.Then proceed with harvesting the brain. Mouse brains were injected with CSF tracers into the CM as described. A macroscopic dorsal view shows the distribution of the tracers in the subarachnoid cisterns of the cerebellopontine region in the olfactory bulb and in the paravascular space along the middle cerebral arteries.In the ventral portion of the brain, a macroscopic view shows the CSF tracer distribution along the Circle of Willis. Histological sections of CM-injected brains further reveal the paravascular distribution of tracers within the brain parenchyma. Mice injected under anesthesia or while asleep had much more tracer distribution than mice injected while awake and freely moving in their home cage.After watching this video, you should gain a good knowledge of how to perform cisterna magna cannulation that will allow you to deliver chemicals or molecules of interest into the cerebrospinal fluid compartment without any operative damage to the skull or to the brain parenchyma. Once mastered, this technique can be performed in between 20 to 30 minutes if performed properly. While doing this procedure, it is important to apply the correct amount of pressure when puncturing the dura mater to avoid major CSF leakage that could interfere with the intercranial pressure.After its development, this cisterna magna cannulation has paved the way for studies in the CSF-ISF clearance in the brain via the so-called lymphatic system that nowadays is widely used in different animal models of neurological diseases.

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Research

JoVE Journal

Neuroscience

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Isolando Nasal Células-Tronco olfativo dos roedores ou humanos

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Neurociência

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

Um sistema totalmente automatizado e altamente versátil para testes de várias funções cognitivas e registro das atividades neuronais em roedores

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Um método para fazer uma craniotomia no crânio ventral de neonatos Roedores

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Microeletrodos guiada implantação de eletrodos no subtalâmica Núcleo de Ratos por longo prazo Estimulação Profunda do Cérebro

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

Operante protocolos para avaliar o custo-benefício durante a tomada de decisão reforçada por roedores

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

O poder do intervalo de Interstimulus para a avaliação do processamento Temporal em roedores

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Dupla injeção direta de sangue na Cisterna Magna como modelo de hemorragia subaracnóide

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

Implantação direta de cânula na Cisterna Magna dos Porcos

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Abordagem Cirúrgica Estereotaxic para Microinpor o Tronco Cerebral Caudal e Medula Espinhal Cervical Superior através da Cisterna Magna em Camundongos

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

Surgical Implantation of the Micro-Drive on the Mouse Skull

After preparing the mouse for surgery and exposing the skull, place the bone screws in the extreme lateral, rostral or caudal portions of the skull where the bone is thickest and the bone screws are sufficiently far away from the micro drive implant. Use a scalpel blade or drill bit to score the skull near the bone screw hole locations, providing a rough surface for the liquid cyanoacrylate to bind. Then thread each bone screw into place, careful not to pierce the underlying dura.Using a sterile 30-gauge needle, apply liquid cyanoacrylate around each bone screw, thickening the skull where the bone screws have been attached. Ensure that no cyanoacrylate enters the exposed dura above the recording sites.