The authors would like to express our gratitude for the support and technical experience shared by the personnel at the Biomedical Laboratory, Aalborg University Hospital, Denmark.

All animal experiments described were performed at Aalborg University Hospital, Denmark, in accordance with existing laws and under the approval of the Danish Animal Experiments Inspectorate (license no. 2020-15-0201-00401). Domestic swine, female, approximately 40 kg and 3 months of age, were used for this study. The details regarding the reagents and equipment used are listed in the Table of Materials.
1. Subject housing
<ol>
	<li>House subjects in groups with 12 h light/dark cycles in approved pens for at least 7 days before the surgical procedure to ensure acclimation.</li>
</ol>
2. Anesthesia and monitoring
<ol>
	<li>Sedate the animal with an intramuscular injection of 2 mL/10 kg of a mixture containing Tiletamine 6.25 mg/mL, Ketamine 6.25 mg/mL, Zolazepam 6.25 mg/mL, Butorphanol 1.25 mg/mL, and Xylazine 6.25 mg/mL.</li>
	<li>Place the animal in a supine position on a heating blanket to ensure optimal thermoregulation.</li>
	<li>Intubate the subject with a size 6.5 tube7 and ventilate it mechanically with non-humidified air, a tidal volume of 8 mL/kg, and a respiratory rate of 16-20 breaths/min according to the expiratory end-tidal CO2 concentrations &#60;6.0 kPa. 
	NOTE: This also ensures correct placement of the tube within the trachea.</li>
	<li>Maintain anesthesia by inhalation of 1% to 2% sevoflurane.</li>
	<li>Apply ophthalmic ointment carefully to both eyes of the subject to prevent dryness during anesthesia.</li>
	<li>Ensure the degree of anesthesia by checking for ciliary reflexes and adjusting the sevoflurane accordingly.</li>
	<li>Insert a bladder catheter with a thermometer into the subject&#39;s bladder through the urethra to monitor temperature and collect urine in a catheter bag.</li>
	<li>Monitor the animal&#39;s vital signs throughout the procedure. 
	NOTE: Vital signs include pulse, continuous arterial blood pressure, temperature, and end-tidal CO2.</li>
	<li>Insert a central venous catheter (6 Fr sheath) in the right jugular vein by percutaneous puncture and use it for continuous saline (NaCl, 0.9%) infusion, drug infusion, and euthanasia at the end of the study.</li>
</ol>
3. Animal positioning
<ol>
	<li>Place the subject in a prone position with the head raised and stabilized with sandbags until the frontal bone is nearly horizontal to ensure optimal positioning. 
	NOTE: Ensure the head is somewhat immobilized to prevent unwanted movement.</li>
	<li>Shave the hair from the surgical site using a trimmer or razor.</li>
</ol>
4. Preparation of surgical equipment
<ol>
	<li>Prepare the surgical equipment as listed in the Table of Materials.</li>
</ol>
5. Exposing the frontal bone
<ol>
	<li>Identify the nuchal prominence and the caudal aspect of each superior orbital crest (Figure 1) to define the expected sagittal midline.</li>
	<li>Begin with a midline incision using scalpel no. 24 to cut through both the skin and the galea aponeurotica onto the periosteum of the frontal bone.</li>
	<li>Wipe away the blood with surgical swabs.</li>
	<li>Gradually separate the galea aponeurotica from the underlying frontal bone around the incision using a 12 mm flattened rongeur.</li>
	<li>Use a surgical retractor to separate the galea aponeurotica and expose the underlying frontal bone (Figure 2).</li>
</ol>
6. Identifying anatomical landmarks of the exposed frontal bone
<ol>
	<li>Identify the sagittal suture as a reference for the anatomical midline and the coronal suture (Figure 2).</li>
	<li>Identify the three bone structures through manual palpation: the nuchal prominence and the caudal aspect of both superior orbital crests (Figure 1 and Figure 3). 
	NOTE: These landmarks form a triangle within which the craniectomy is performed (Figure 1 and Figure 4A).</li>
</ol>
7. Access to the dura mater
<ol>
	<li>Using a high-speed pneumatic drill with a rounded diamond-coated burr (Figure 3A), define each corner of a rectangle within the borders of the previously defined triangle.</li>
	<li>Connect each corner with a 4 mm rounded diamond burr to ensure the correct location of the opening (Figure 4B).</li>
	<li>Gradually thin the frontal bone with the 4 mm rounded diamond burr until the dura mater is exposed. 
	NOTE: Avoid making the first point of contact with the dura mater in the midline, corresponding to the sagittal suture (Figure 1), as the large dorsal sagittal sinus is located here. Perforation at this point can cause significant venous hemorrhage.</li>
	<li>Use the first point of contact with the dura to visually assess the thickness of the remaining frontal bone.</li>
	<li>Continue carefully thinning the frontal bone in the defined rectangle using the 4mm rounded diamond burr. 
	NOTE: Save the bone dust for later hemostasis of bleeding from veins or exposed spongiosa of the frontal bone.</li>
	<li>Slide a 3 mm dissector under the sufficiently thinned bone around the bone plate and chip it off with gentle manual pressure to expand the opening to the dura mater. 
	NOTE: Alternate between drilling and chipping off the bone until multiple points of the dura mater around the bone plate are exposed.</li>
	<li>Apply sterile saline with a syringe to clear the view.</li>
</ol>
8. Removal of the bone plate
<ol>
	<li>Insert the 3 mm dissector under the bone plate and apply gradual downward pressure to its handle to break off the bone plate. 
	NOTE: The underlying dura mater should now be exposed, revealing the contours of both cerebral hemispheres (Figure 5 and Figure 6).</li>
	<li>Assess the integrity of the dura mater by visually inspecting for cerebrospinal fluid (CSF) leakages. 
	NOTE: Both hemispheres will elevate in synchronous pulsation if there are no significant defects in the dura mater.</li>
	<li>Stop minor venous bleedings using saved bone dust or by carefully applying monopolar or bipolar coagulation with a cautery at low voltage. 
	NOTE: Minor venous bleedings from emissary or diploic veins are expected.</li>
</ol>
9. Protecting exposed dura mater
<ol>
	<li>Cover the exposed dura mater with a sterile surgical swab soaked in sterile saline to prevent drying out of the underlying tissue.</li>
</ol>
10. Insertion of microdialysis catheters (MDC)
<ol>
	<li>Use the dorsal sagittal sinus as a reference for the midline. Place a microdialysis catheter (MDC) within the lumen of a disposable 18 G needle. Penetrate the dura mater 10 mm laterally from the dorsal sagittal sinus with the needle at a 10&#176;-15&#176; angle in a cranial-rostral direction until 20 mm of the needle is within the tissue.</li>
	<li>Slowly withdraw the needle while ensuring the MDC remains at the same location within the superficial cerebral cortex.</li>
	<li>Securely fasten the catheter to nearby skin to prevent dislocation of the MDC. 
	NOTE: Ensure the tip of the MDC does not contact the sharp tip of the needle to avoid damage.</li>
	<li>Repeat the procedure using the dorsal sagittal sinus as a midline reference. Place an MDC within a needle as described above and penetrate the dura mater on the contralateral hemisphere 15 mm from the midline with the needle perpendicular (90&#176; angle).
	<ol>
		<li>Insert the needle 20 mm before slowly withdrawing it as described, leaving the MDC within the subcortical cerebral tissue. Secure the MDC as described above.</li>
	</ol>
	</li>
	<li>Prepare a 2 mL disposable plastic syringe with an 18 G disposable needle. Fill the syringe with 0.5 mL of sterile saline.</li>
	<li>Penetrate the dura mater 20 mm laterally from the midline while keeping the needle at a 45&#176; angle aimed towards the midline. Slowly introduce the needle 1 mm at a time while gently aspirating with each step of insertion.</li>
	<li>Observe the syringe during each aspiration until cerebrospinal fluid (CSF) is obtained.</li>
	<li>Separate the needle from the syringe while keeping the needle in the same anatomical location. Insert an MDC through the needle into the lateral ventricle until resistance is felt.</li>
	<li>Securely fasten the MDC with surgical tape as described above.</li>
</ol>
11. Microdialysis (MD)
<ol>
	<li>Connect each microdialysis catheter (MDC) to a separate microdialysis pump.</li>
	<li>Initiate the microdialysis process by pumping sterile saline through the MDC and collecting it in a suitable sample. Ensure flow through each MDC by observing saline in each sample.</li>
	<li>Start a tissue-calibration period of 30 min of continuous microdialysis at a flow rate of 1 &#181;L/min.</li>
</ol>
12. Euthanaziation
<ol>
	<li>Administer a bolus of intravenous pentobarbital (50 mg/kg) through the central venous catheter (following institutionally approved protocols).</li>
	<li>Observe the pulse, blood pressure, and end-tidal CO2 curves on the respirator for a flatline as confirmation of cardiac arrest.</li>
</ol>

An Efficient High-Speed Pneumatic Drill Craniectomy to Access the Porcine Brain

<ol>
	<li>Mariager, T., Bjarkam, C., Nielsen, H., Bodilsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+animal+models+for+brain+abscess:+a+systematic+review.">Experimental animal models for brain abscess: a systematic review.</a> Br J Neurosurg. , 1-8 (2022).</li><li>Bassols, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+as+an+animal+model+for+human+pathologies:+A+proteomics+perspective.">The pig as an animal model for human pathologies: A proteomics perspective.</a> Proteomics Clin Appl. 8, 715-731 (2014).</li><li>Meurens, F., Summerfield, A., Nauwynck, H., Saif, L., Gerdts, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig:+A+model+for+human+infectious+diseases.">The pig: A model for human infectious diseases.</a> Trends Microbiol. 20 (1), 50-57 (2012).</li><li>Swindle, M. M., Makin, A., Herron, A. J., Clubb, F. J., Frazier, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swine+as+models+in+biomedical+research+and+toxicology+testing.">Swine as models in biomedical research and toxicology testing.</a> Vet Pathol. 49 (2), 344-356 (2012).</li><li>Lind, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pigs+in+neuroscience:+Modeling+brain+disorders.">The use of pigs in neuroscience: Modeling brain disorders.</a> Neurosci Biobehav Rev. 31 (5), 728-751 (2007).</li><li>Hoffe, B., Holahan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pigs+as+a+translational+model+for+studying+neurodegenerative+diseases.">The use of pigs as a translational model for studying neurodegenerative diseases.</a> Front Physiol. 10, 838 (2019).</li><li>Ettrup, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+surgical+techniques+in+the+g&#246;ttingen+minipig:+Intubation,+bladder+catheterization,+femoral+vessel+catheterization,+and+transcardial+perfusion.">Basic surgical techniques in the g&#246;ttingen minipig: Intubation, bladder catheterization, femoral vessel catheterization, and transcardial perfusion.</a> J Vis Exp. (52), e2652 (2011).</li><li>Bjarkam, C. R., Glud, A. N., Orlowski, D., S&#248;rensen, J. C. H., Palomero-Gallagher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telencephalon+of+the+G&#246;ttingen+minipig,+cytoarchitecture+and+cortical+surface+anatomy.">The telencephalon of the G&#246;ttingen minipig, cytoarchitecture and cortical surface anatomy.</a> Brain Struct Funct. 222 (5), 2093-2114 (2017).</li><li>Hou, N., Du, X., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+pig+models+of+human+diseases.">Advances in pig models of human diseases.</a> Animal Model Exp Med. 5 (2), 141-152 (2022).</li><li>Munk, M., Poulsen, F. R., Larsen, L., Nordstr&#246;m, C. H., Nielsen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+metabolic+changes+related+to+oxidative+metabolism+in+a+model+of+bacterial+meningitis+induced+by+lipopolysaccharide.">Cerebral metabolic changes related to oxidative metabolism in a model of bacterial meningitis induced by lipopolysaccharide.</a> Neurocrit Care. 29 (3), 496-503 (2018).</li><li>Kyllar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiography,+computed+tomography+and+magnetic+resonance+imaging+of+craniofacial+structures+in+pig.">Radiography, computed tomography and magnetic resonance imaging of craniofacial structures in pig.</a> J Vet Med C: Anat Histol Embryol. 43 (6), 435-452 (2014).</li><li>Mariager, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+evaluation+of+single-dose+moxifloxacin+concentrations+in+brain+extracellular+fluid,+cerebrospinal+fluid,+and+plasma:+A+novel+porcine+model.">Continuous evaluation of single-dose moxifloxacin concentrations in brain extracellular fluid, cerebrospinal fluid, and plasma: A novel porcine model.</a> J Antimicrobial Chemother. , (2024).</li></ol>

The authors have no conflicts of interest to declare.

The demonstrated procedure involves several critical steps. Firstly, the accurate planning of the craniectomy&#39;s location is crucial due to the composition of the porcine skull. Since the thickness of the porcine frontal bone increases at the lateral edges, placing the opening too laterally11 can make it difficult to reach the dura mater during drilling. Additionally, locating the opening correctly within the midline is important to reduce the risk of unintended damage to the underlying dorsal ...

In general, animal models are used when practical and/or ethical limitations prohibit the use of human patients to examine diseases or test surgical methods. Novel animal models are generally established to provide new knowledge with translational value to human conditions. Rodents are often utilized due to practical and financial considerations, but they have limited translational value to humans, especially due to substantial anatomical differences1. Pigs, however, offer several advantages compared to rodents. Not only do pigs share several key anatomical, physiological, metabolic, and genetic features with humans, but the size of the porcine organ systems can be weight-matched to resemble human organs2,3. This gives pigs a unique role among surgical animal models and in procedural training4. Although the use of porcine models requires certain practical and financial capabilities compared to the use of rodents, pigs offer both a financially and ethically more acceptable option compared to the use of non-human primates.
The porcine brain is of particular interest in translational neuroscience research. Firstly, the architecture of the pig brain is similar to that of the human brain, as both are white matter-predominant and gyrencephalic3,5,6. Secondly, the larger brain size in pigs compared to rodents permits the use of surgical equipment and various imaging modalities equivalent to those used in clinical settings7,8. Consequently, various porcine models have been used extensively in neuroscience research over recent decades9. The majority of these porcine CNS models, however, require direct analysis of brain tissue, which can be obtained in various ways (e.g., implantation of catheters or electrodes, tissue biopsies, etc.)10. Since most of these modalities require some degree of instrumentalization and direct access to the brain, different approaches for surgical access must be considered.
This method involves performing an anterior craniectomy through the frontal bone on a sedated 3-month-old female Danish Landrace pig. The overall purpose of this manuscript is to describe a method for exposing a large proportion of the ventral porcine brain through a craniectomy using a pneumatic high-speed drill. The first step is to place the subject in a suitable position with an elevated head. Since the porcine cranium is quite different from that of humans, the second step involves planning the placement of the craniectomy using various anatomical landmarks. The third step is to access the underlying dura mater covering both hemispheres without damaging it.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL plastic syrringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>303219</td>
			<td></td>
		</tr>
		<tr>
			<td>107 Microdialysis pump</td>
			<td>M Dialysis</td>
			<td>P000127</td>
			<td>&#160;107 Microdialysis Pump</td>
		</tr>
		<tr>
			<td>2 mL plastic syrringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>300928</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm, 18 G needles</td>
			<td>Becton, Dickinson and Company</td>
			<td>304100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bair Hugger heater</td>
			<td>3M</td>
			<td>B5005241003</td>
			<td></td>
		</tr>
		<tr>
			<td>Bair Hugger heating blanket</td>
			<td>3M</td>
			<td>B5005241003</td>
			<td></td>
		</tr>
		<tr>
			<td>Batery for microdialysis pump</td>
			<td>M Dialysis</td>
			<td>8001788</td>
			<td>Battery 6V, 106 &#38; MD Pump</td>
		</tr>
		<tr>
			<td>Dissector</td>
			<td>Karl Storz</td>
			<td>223535</td>
			<td>Flattended 3 mm dissector</td>
		</tr>
		<tr>
			<td>Endotracheal tube size 6.5</td>
			<td>DVMed</td>
			<td>DVM-107860</td>
			<td>Cuffed endotracheal tube</td>
		</tr>
		<tr>
			<td>Euthasol Vet</td>
			<td>Dechra Veterinary Products A/S</td>
			<td>380019</td>
			<td>phentobarbital for euthanazia, 400 mg/mL</td>
		</tr>
		<tr>
			<td>Farabeuf Rougine</td>
			<td>Mahr Surgical</td>
			<td></td>
			<td>Flat headed rougine (12 mm)</td>
		</tr>
		<tr>
			<td>Foley Catheter 12 F</td>
			<td>Becton, Dickinson and Company</td>
			<td>D175812E</td>
			<td>Catherter with in-built thermosensor</td>
		</tr>
		<tr>
			<td>Intravenous sheath</td>
			<td>Coris Avanti</td>
			<td></td>
			<td>Avanti Cordis Femoral Sheath 6 F</td>
		</tr>
		<tr>
			<td>Microdialysis brain catheters</td>
			<td>M Dialysis</td>
			<td>P000050</td>
			<td>membrane length 10 mm -shaft 100 mm 4/pkg</td>
		</tr>
		<tr>
			<td>Microdialysis syringe</td>
			<td>M Dialysis</td>
			<td>8010191</td>
			<td>&#160;106 Pump&#160;Syringe 20/pkg</td>
		</tr>
		<tr>
			<td>Microvials for microdialysis sampling</td>
			<td>M Dialysis</td>
			<td>P000001</td>
			<td>Microvials 250/pkg</td>
		</tr>
		<tr>
			<td>Operating table</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic high-speed drill</td>
			<td>Medtronic</td>
			<td></td>
			<td>Medtronic Midas Rex 7 drill</td>
		</tr>
		<tr>
			<td>Primus respirator</td>
			<td>Dr&#228;ger</td>
			<td></td>
			<td>Respirator with in-built vaporiser for supplementary Sevofluran anesthesia</td>
		</tr>
		<tr>
			<td>Rounded diamond drill</td>
			<td>Medtronic</td>
			<td>7BA40D-MN</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-retaining retractor</td>
			<td>World Precission Instruments</td>
			<td>501722</td>
			<td>Weitlander retractor, self-retaining, 14 cm blunt</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Fresnius Kabi</td>
			<td>805541</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td>Sterile surgical swaps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scalpel no 24</td>
			<td>Swann Morton</td>
			<td>5.03396E+12</td>
			<td>Swann Morton Sterile Disposable Scalpel No. 24</td>
		</tr>
		<tr>
			<td>Zoletil Vet</td>
			<td>Virbac</td>
			<td></td>
			<td>Medical mixture for induction of anesthesia</td>
		</tr>
	</tbody>
</table>

accessing the porcine brain via high-speed pneumatic drill craniectomy

The use of pigs as an experimental animal model is especially relevant in neuroscience research, as the porcine and human central nervous systems (CNS) share many important functional and architectural properties. Consequently, pigs are expected to have an increasingly important role in future research on various neurological diseases. Here, a method to perform an anterior craniectomy through the porcine frontal bone is described. After a midline incision and subsequent exposure of the porcine frontal bone, anatomical landmarks are used to ensure the optimal location of the craniectomy. By careful and gradual thinning of the frontal bone with a rounded drill, a rectangular opening to the dura mater and underlying cerebral hemispheres is achieved. The presented method requires certain surgical materials, including a pneumatic high-speed drill, and some degree of surgical experience. Potential complications include unintended lesions of the dura mater or dorsal sagittal sinus. However, the method is simple, time-efficient, and offers a high degree of reproducibility for researchers. If performed correctly, the technique exposes a large portion of the unaffected pig brain for various neuromonitoring or analyses.

The prone position of the pig&#39;s head provides optimal access for the surgeon during the procedure, and the use of stabilizing sandbags reduces the risk of unintended shifts in the pig&#39;s head position while drilling.
During this demonstration, the superficial anatomical landmarks of the pig&#39;s superior skull (both superior orbital crests and the nuchal crest) (Figure 1 and Figure 3) were used to precisely identify the center...

Author Spotlight: A Reproducible and Efficient Method for Accessing Porcine Brain via Craniectomy

This protocol describes performing a craniectomy using a high-speed pneumatic drill on a 3-month-old Danish Landrace pig. The access is made through the frontal bone and reveals the ventral dura mater and underlying cerebral hemispheres. This procedure allows for access to a large portion of the pig brain.

Accessing the Porcine Brain via High-Speed Pneumatic Drill Craniectomy

The use of pigs as an experimental animal model is especially relevant in neuroscience research, as the porcine and human central nervous systems (CNS) ...

accessing-the-porcine-brain-via-high-speed-pneumatic-drill-craniectomy

Research

JoVE Journal

Neuroscience

278 Views.  Aalborg University Hospital. This protocol describes performing a craniectomy using a high-speed pneumatic drill on a 3-month-old Danish Landrace pig. The access is made through the frontal bone and reveals the ventral dura mater and underlying cerebral hemispheres. This procedure allows for access to a large portion of the pig brain.

Video: Author Spotlight: A Reproducible and Efficient Method for Accessing Porcine Brain via Craniectomy

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined curved surfaces, and images of real-world objects (with and without disparity) in a similar manner as described in the ventral visual stream. In addition, parieto-frontal areas are believed to convert visual object information into appropriate motor outputs, such as the pre-shaping of the hand during grasping. To better characterize object selectivity in the cortical network involved in visuomotor transformations, we provide a battery of tests intended to analyze the visual object selectivity of neurons in parieto-frontal regions.

A detailed protocol to analyze object selectivity of parieto-frontal neurons involved in visuomotor transformations is presented.

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...