German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

The use of human tissues must be reviewed and approved by an institutional review board or the equivalent. The work described here was approved by the local ethics committee and had approval for scientific examination. This work was performed according to the Declaration of Helsinki. The human scleral specimens were obtained from the eyes of globe donors (maximum post-mortem time 24 hr) at the Eye Bank of the Department of Ophthalmology, University of Cologne, Germany.
1. Experimental Preparation
<ol>
	<li>Prepare 96% ethanol and phosphate buffered saline (PBS) in different tubes. Prepare the primary antibodies in the recommended dilution of PBS containing 2% bovine serum albumin (BSA) and keep all solutions on ice. Prepare 100 &#956;l per sample.</li>
	<li>Clean all instruments and use sharp scalpels. After repeated cutting with the same scalpel, change the scalpel.</li>
	<li>Obtain 1-2 colibri forceps, straight micro dissecting scissors, a #10 scalpel or ophthalmic scalpel micro feather, and four 26 G needles.</li>
	<li>Wrap aluminum foil around a polystyrene plate or use a cork plate to affix the tissue. Work underneath a binocular stereo microscope. Work sterile underneath a laminar airflow if needed.</li>
</ol>
2. Prepare the Sclera
<ol>
	<li>Gently hold the bulb and perform a perforating trepanation on the corneoscleral part, then rotate the trepan. Rotate the trepan carefully and evenly onto the surface to perform an equally round cut. Use a 15.5 mm sized trepan. Cut the remaining attachments with curved scissors. Remove the corneoscleral trepanation. This will result in an anteriorly opened bulb.
	<ol>
		<li>Put the sclera on a swab with the open part upwards. Remove the retina and uvea, using colibri forceps to pull off both layers (retina and uvea) from the sclera. Do not leave the pigmented uvea on the inner sclera. Use curved scissors to remove the retina and uvea from the papilla. Remove the remaining conjunctiva, extraocular muscles, and tenon&#39;s capsule from the superficial sclera.</li>
	</ol>
	</li>
	<li>Remove the scleral sample in the required size by using straight-type scissors and the colibri forceps. Take about 2 cm2 sized scleral samples from different locations. Because of the small size, hold the sclera gently and cut out the required size as squares using the straight-type scissors. The corneoscleral trepanation defines the anterior margin of the scleral samples.
	<ol>
		<li>Avoid repeated grabbing with the forceps as the area where the tissue was grabbed is not suitable for confocal microscopy analyses. Prepare the required number of samples depending on the experiment type (e.g. anteriorly vs. posteriorly, compare Figure 1) and the amount of antibodies used.</li>
	</ol>
	</li>
	<li>Put the samples in 1.5 ml tubes for later use. Fix the samples in 1.5 ml 96% ethanol for 15 min, and then wash them three times each for 5 min in 1.5 ml PBS on the shaker. If working with frozen tissue, perform this step before snap freezing.</li>
	<li>Use fresh tissue. However, if this is not possible, snap freeze the sclera in liquid nitrogen and keep it at -20 &#176;C for later use. If working with frozen tissue, thaw before use. Avoid repeated thawing and freezing cycles.</li>
	<li>Keep each sample in PBS containing 1.5 ml 5% BSA at room temperature for 2 hr. This step induces a swelling of the sample that is helpful for the laminating and also prevents unspecific binding.</li>
	<li>After the samples have swelled, prepare the scleral squares for lamination. Work underneath a microscope, e.g. a binocular stereo microscope. Affix the posterior scleral samples to the polystyrene membrane wrapped in aluminum foil or the cork plate using the 26 G needles.</li>
	<li>Bend the edges of the needles so they do not interfere with the visual field underneath the microscope.</li>
	<li>Laminate the full-thickness scleral samples.
	<ol>
		<li>First, hold the anterior scleral edge using colibri forceps. Then carefully cut a thin layer of the superficial sclera using the #10 scalpel, the ophthalmic scalpel micro feather, or a spatula blade. Hold the scalpel horizontally and cut the superficial layer as thinly as possible from the underlying layer.</li>
		<li>Dissect a thin scleral layer from the scleral square. This method is comparable to standard surgical techniques to prepare a flap during trabeculectomy and should result in 30-80 &#181;m thick layers (compare Figure 2).</li>
	</ol>
	</li>
	<li>Prevent drying of the sclera by adding small amounts of PBS to the tissue, e.g. 50 &#181;l PBS using a pipette.</li>
	<li>Put the cut layers into 100 &#181;l of PBS in the 96 well plate and label both the orientation (towards extern and intern) and the layer (superficial and profound) e.g. with a waterproof color.</li>
	<li>Repeat steps 2.7-2.9 until the tissue is fully laminated.</li>
</ol>
3. Perform Immunohistochemistry
<ol>
	<li>Add 100 &#181;l of the required primary antibodies into the recommended dilution. Here, e.g. use CD 31 (monoclonal mouse anti human) and LYVE1 antibody (rabbit anti human), both in a dilution of 1:100 in PBS (containing 2% BSA) and incubate at 4 &#176;C overnight. To change the medium in the 96 well plate, hold the pipette to the wall of the well and remove the fluid.</li>
	<li>The next day, wash the samples three times each for 5 min with 200-300 &#181;l PBS on the shaker. Add 100 &#181;l of the corresponding secondary antibodies; here use goat anti mouse fluorescein isothiocyanate (FITC) and goat anti rabbit cyanine dyes 3 (Cy3), and incubate the samples at room temperature for 1-2 hr. Dilute the secondary antibodies 1:300 in PBS containing 2% goat serum.</li>
	<li>Rinse the samples again three times each for 5 min in 200-300 &#181;l PBS on the shaker.</li>
	<li>Perform nucleus staining, e.g. 4&#39;,6-diamidino-2-phenylindole (DAPI) (dilution 1:2,000, 100 &#181;l in each well and incubate 10 min at room temperature), then wash again 2 times in 200-300 &#181;l PBS on the shaker.</li>
	<li>Transfer the samples onto microscope slides, embed them in 1-2 drops of fluorescent mounting medium, add a coverslip, seal it by covering the edges with transparent varnish, and store at 4 &#176;C for later use or directly examine the slides with the confocal microscope.</li>
	<li>Use a confocal microscope (or equivalent) to analyze the samples. Use the desired magnification, e.g. 10-40X magnification.</li>
	<li>To verify the thickness of the samples, measure with confocal microscopy using Z-Stacks. The amount of possible scleral sections depends on the location of the sclera, as the thickness differs equatorially and posteriorly (compare introduction).</li>
</ol>

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L., 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=Absence+of+lymphatic+vessels+in+the+developing+human+sclera.">Absence of lymphatic vessels in the developing human sclera.</a> Exp Eye Res. 125, 203-209 (2014).</li><li>Hos, D., Cursiefen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+vessels+in+the+development+of+tissue+and+organ+rejection.">Lymphatic vessels in the development of tissue and organ rejection.</a> Adv Anat Embryol Cell Biol. 214, 119-141 (2014).</li><li>Hos, D., Schlereth, S. L., Bock, F., Heindl, L. 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S., Sainz de la Maza, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The sclera. , (2012).</li><li>Kiss, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Der+Blutkreislauf+des+Auges.">Der Blutkreislauf des Auges.</a> Ophthalmologica. 106, 225 (1943).</li><li>Ashton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+study+of+Schlemm's+canal+and+aqueous+veins+by+means+of+neoprene+casts.+Part+I.+Aqueous+veins.">Anatomical study of Schlemm's canal and aqueous veins by means of neoprene casts. Part I. 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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

Laminating the human sclera is a method for performing confocal microscopy on this tissue. A critical step in this process is the use of ethanol instead of formalin for affixing the tissue. In our experience, better results are obtained when using ethanol instead of formalin for fixation. Blunt scalpels aggravate the procedure and should be avoided. Similarly, the drying up of the sclera should be avoided, as it complicates the procedure and reduces the quality of the images.
Regarding the imm...

The sclera is the rigid outer layer that covers the eye, which is made of dense connective tissue. It helps to protect intraocular structures and to maintain intraocular pressure. Thus, the sclera is essential for clear vision. It is devoid of lymphatic vessels 1,2 and thereby forms an outer lymphatic-free border between it and the lymphatic-free inner eye 3-7. It also provides attachment sites for extraocular muscles, thereby sharing anatomical similarities with tendons. Because the sclera mainly consists of dense bundles of type I collagen and has smaller numbers of collagen types III, IV, V, VI, VIII 8,9 and elastin 10,11, this tissue is not easy to use for immunohistochemistry.
Anatomically, the sclera can be separated into three main layers: (1) the superficial vascularized episclera, found underneath the conjunctiva and tenon&#39;s capsule and towards the sides and the back of the eye facing the orbit; (2) the scleral stroma, the main part of the sclera; and (3) the lamina fusca, which is a thin, pigmented layer located directly above the uvea. Our anatomical knowledge about the sclera stems mainly from the first half of the 20th century. At that time, researchers studied the anatomy of vasculature mainly by using India ink injections 12 and vascular casting 13-15. Later, it was researched in angiographic studies 16-19.
Since that time, older techniques have been improved and new ones have been developed that have allowed us to supplement previous anatomical knowledge. For example, it has only been about one decade since we have had such reliable lymphatic markers as lymphatic vascular endothelium specific hyaluronan receptor-1 (LYVE1) 20 or podoplanin 21. Confocal microscopy offers new possibilities for studying the anatomical features of the different tissues of the eye. It allows for multiple stains to be used for differentiating markers of cells or for the localization of cells in relation to blood vessels and other anatomical structures. It provides an overview when the sample is of a larger size and allows us to scan through a sample when in search of a specific cell type. With Z-Stack technology, confocal microscopy can be used for samples up to 100-200 &#181;m. The sclera differs in thickness between 0.3 mm behind the muscle insertions and 1 mm at the posterior pole 11. Due to both its thickness and opaqueness, the sclera is not suitable for confocal microscopy using traditional methods.
To remedy this, scleral tissues were laminated to allow for their analysis with confocal microscopy. This technology is useful for gaining a better understanding of both physiological and pathological situations in the human sclera.

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.

In the representative experiments performed here, there are demonstrable benefits derived from the use of this particular laminating technique. The first experiment illustrates the diverse network of the episcleral blood vessel plexus in three representative pictures (Figure 3). The vessels are positive for CD31.
The second experiment shows immune cells, in particular LYVE1 + cells of the episclera and their rel...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

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7.5K Views.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

Video: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...