This work was supported by the Juvenile Diabetes Research Foundation International (grant 17-2008-1031) and the National Institutes of Health (grant 1R01DK077903-01A1).

Introduction: 
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. 
HRT III- Rostock Corneal Module (RCM)
Manual of Operation

1. Preparation of the Camera

The initial step in examination using HRT is preparation of the objective lens tip. 
<ol>
<li>First at the objective tube of the laser scanning camera, set the refraction to +12 diopters and then adjust the camera to the lowest position.</li>
<li>Apply a large homogenous, bubble free, pea sized, drop of Viscotears on the lens tip.</li>
<li>Remove a TomoCap from its sterile container and mount it over the lens tip such that the gel forms a meniscus between the objective lens and the cap. Push it as far as possible over the holder. Be careful not to touch the front surface of TomoCap during mounting. 
<img src="/files/ftp_upload/2194/2194fig1.jpg" alt="Figure 1" /> 
Figure 1. Preparation of the objective lens tip (top) applying viscotear gel (bottom) Applying the TomoCap.</li>
<li>Move the laser scanning camera as far backwards as possible on the camera mount.</li>
<li>The inner and outer surface of the TomoCap both appear as a bright laser reflection. The focal plane adjustment wheel should be adjusted until a bright reflection is observed, indicating that the lens is focused within the front of the cap. The depth value shown in the focus position display should now be between -150 &mu;m and +150 &mu;m.</li>
<li>Reset the depth setting to zero. 
<img src="/files/ftp_upload/2194/2194fig2.jpg" alt="Figure 2" /> 
Figure 2. Adjustment of the focal plane.</li>
</ol>
2. Preparing the Patient 
The subject's eyes are anaesthetised using a drop of 0.4% benoxinate hydrochloride, and Viscotears is used on the front of the eye for lubrication. 
The subject is seated comfortably, with the chin on the chin rest and asked to press their forehead firmly against the forehead bar.
<img src="/files/ftp_upload/2194/2194fig3.jpg" alt="Figure 3" /> 
Figure 3. Preparing the patient
3. Aligning the Camera 
It makes the examination easier if you instruct the patient to fix on the outer fixation light with the eye not being examined. The compact size of the microscope head means that the subject's contralateral eye is not obscured, enabling the use of distance targets.
<img src="/files/ftp_upload/2194/2194fig4.jpg" alt="Figure 4" /> 
Figure 4. Objective lens of the corneal module.
<ol>
<li>Position the CCD camera such that its optical axis runs perpendicular to the optical axis of the laser scanning camera. The camera should be on the patient's right side when imaging the right eye and vice versa. 
At this position, you should see the front surface of the TomoCap in the centre of the CCD camera live image.</li>
<li>Move the laser scanning camera towards the patient until the patient's cornea is at a distance of about 5 to 10 mm from the TomoCap, then move the laser scanning camera up/down and left/right using the black knobs on the camera mount until the TomoCap is positioned in the centre of the patient's cornea. At this stage you can check that the red laser light is in the centre of the cornea and for fine adjustment, observe the reflection of the laser beam from the cornea, which is visible in the CCD camera image. This reflection must occur exactly at the anterior pole of the cornea. 
<img src="/files/ftp_upload/2194/2194fig5.jpg" alt="Figure 5" /> 
Figure 5. Alignment of the laser beam on the anterior pole of the cornea to capture central corneal images.</li>
<li>Ask the patient to open his/her eye as wide as possible. Move the laser scanning camera slowly forwards towards the patient until the TomoCap contacts the patient's cornea. If it is required to correct the position of the laser scanning camera, then move the camera away from the patient first, do the correction, then move it forward again to contact the cornea. 
<img src="/files/ftp_upload/2194/2194fig6.jpg" alt="Figure 6" /> 
Figure 6. Demonstration of the thin gel bridge between the TomoCap and the cornea.</li>
<li>Minimal contact between the camera and the cornea is enough. In an optimal adjustment, a thin gel bridge between the TomoCap and the cornea is visible on the CCD camera live image.</li>
</ol>
If you press the TomoCap or move the cornea too close to the camera then you will see pressure on the cornea with a flattened appearance in the cornea via the CCD camera live image. Also in the images obtained a line showing the pressure will appear, therefore do not apply too much pressure to the cornea. Once contact has been established, do not move the position of the headrestto avoid sliding the cornea on the TomoCap.
<img src="/files/ftp_upload/2194/2194fig7.jpg" alt="Figure 7" /> 
Figure 7. (left) Appearance of a straie due to application of excess pressure on the cornea by the TomoCap; (right) An image with normal appearance without excess pressure.
4. Examining the Patient 
After the field of view has been selected, the HRT III laser camera is turned on and the image acquisition window appears on the screen. Use the CCD image to position the TomoCap and laser light on the patient's cornea. Examination using the selected mode may then be commenced.
<img src="/files/ftp_upload/2194/2194fig8.jpg" alt="Figure 8" /> 
Figure 8. Viewing mode on monitor during acquisition of images.
The mode of acquisition 
In the section mode, one single image is acquired and stored each time you press the foot switch. We use this mode for capturing images from the whole corneal layer including Bowman's layer in the centre of cornea. And then after capturing sufficient numbers of images, one can try the other modes including sequence and volume.
<img src="/files/ftp_upload/2194/2194fig9.jpg" alt="Figure 9" /> 
Figure 9. Display with choice for different image acquisition modes.
With the Sequence scan, one can acquire a sequence of up to 100 images with an adjustable frame rate. You can choose a frame rate between 30 frames and 1 frame per second (fps) and based on this mode it allows you to acquire a movie with a duration of 3 to 100 seconds.
In volume scan mode, the camera automatically acquires a series of 40 images at consecutive focal planes. A total depth of 85 &mu;m can be scanned with the "FOV 400 &mu;m" field lens and the focal distance between two consecutive images will be approximately 2.1 &mu;m.
After a few scans the focus plane of lens can be changed to measure the depth of a corneal cell layer relative to the corneal surface by bringing the epithelial cell layer into focus and then clicking the Reset button to set the depth value to 0.
For the purpose of our present study, we obtain images from the central cornea at different depths. Therefore we do not move the scanning laser camera around the cornea horizontally or vertically, we just move backward and forward and capture images from all depths of Bowman's layer at the same corneal point. The distance between each image is approximately 1 &mu;m; therefore if the depth of Bowman's is 10 &mu;m, we will have 10 images. However for final analysis, we select images with a distance of approximately 2-3 &mu;m between them.
After the examination is complete turn the camera off by clicking the camera power button in the acquisition window. Make the patient aware that he or she should not rub his or her eyes until the local anesthesia has worn off. As a precaution, you may wish to perform a slit lamp examination of the patient's cornea after the examination.
Remove the TomoCap from the camera and dispose it and then clean the microscope lens with a cotton swab moistened with distilled water.
5. Analyzing an Examination 

a. Selecting the frames

To review and analyze existing examinations, the appropriate patient record(s) must be selected in the database window. The image viewing window provides an overview of existing cornea examinations for the selected patient. Examinations performed on different days are stored in separate visit tabs. The three different scan types are represented by three different icons in the HRT III cornea visit tab of the image viewing window of the Heidelberg Eye Explorer.
To survey the cornea image, double-click the appropriate image icon and the "Show" function opens the following window:
<img src="/files/ftp_upload/2194/2194fig10.jpg" alt="Figure 10" /> 
Figure 10. Display of all captured images after examination.
<img src="/files/ftp_upload/2194/2194fig11.jpg" alt="Figure 11" /> 
Figure 11. Display of selected image before export with CCD camera view of eye to show correct alignment.
To export the appropriate images for further analysis, click "Export" on the toolbar and then select the folder you want to save the images to.

We obtain and capture images from all layers of the cornea for each patient, but as the main focus of this research is on Bowman's layer and the corneal nerves, only the images from this layer will be analyzed. We select 5-6 frames from Bowman's layer for each subject at different depths of the cornea.
<img src="/files/ftp_upload/2194/2194fig12.jpg" alt="Figure 12" /> 
Figure 12. Display of all corneal layers from a healthy subject.
On average 5-6 frames from Bowman's layer in the centre of cornea at different depths are selected randomly for quantitative measurements and analysis.
b. Analyzing images
For analyzing the images captured from Bowman's layer of the central cornea with HRT III, we use specialized software that has been developed within our group called "CCM Image Analysis Tools v 0.6".
Four main parameters for corneal nerves are measured with this software:
<ul>
<li>Nerve fibre density (NFD) which is defined as the number of main nerves/frame.</li>
<li>Nerve branch density (NBD) number of main branches/frame.</li>
<li>Nerve fibre length (NFL) which is the total length of all nerves/frame.</li>
<li>Nerve fibre tortuosity (NFT) of the main nerves/frame.</li>
</ul>

To open CCM image, go to the file &gt; open. 
<ul>
<li>Once the image is loaded, its size and type is displayed in the Image Information Box on the top left hand-side of the Control Panel.</li>
<li>The default scale is displayed in the edit line of the image Information. If the scale is different, modify the number in the edit line.</li>
<li>To start analyzing the image, select the parameter you want to measure from the Metric Box in the top right-hand side of the control panel.</li>
<li>At the result box at the bottom of the page the number of NFD, NBD is presented in actual number, NFL in mm and tortuosity as a Tortuosity Coefficient (TC).</li>
<li>However after exporting the results to an excel sheet, the results will be presented in no/mm2 for NFD and NBD and mm/mm2 for NFL.</li>
<li>For this purpose after finishing measurements click on Add measurements, then go to File &gt; Save as and then open the saved results with Excel.</li>
</ul>
<img src="/files/ftp_upload/2194/2194fig13.jpg" alt="Figure 13" /> 
Figure 13. Display of corneal nerve image with tracing in analysis tool using "CCM Image Analysis Tool".
6. Representative Results:
We have demonstrated a progressive increasing in corneal nerve degeneration with increasing severity of diabetic neuropathy 1 and early nerve damage in diabetes which precedes loss of intraepidermal nerve fibres in skin biopsies 2 together with stratification of neuropathic severity and repair following pancreas transplantation 3 in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy 4 and Fabry disease 5.
<img src="/files/ftp_upload/2194/2194fig14.jpg" alt="Figure 14" /> 
Figure 14. Corneal confocal microscopy images with HRT III of the corneal Bowman's layer (a) Control subject compared to (b) Patient with neuropathy and severe nerve damage.

<ol>
	<li>Abbott, C. A., Kallinikos, P., Marshall, A., Finnigan, J., Morgan, P., Efron, N., Boulton, A. J. M., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Confocal+Microscopy:+A+Novel+Non-invasive+Test+to+Diagnose+and+Assess+Progression+of+Human+Diabetic+Neuropathy.">Corneal Confocal Microscopy: A Novel Non-invasive Test to Diagnose and Assess Progression of Human Diabetic Neuropathy.</a> Diabetes Care. 33, 1792-1797 (2010).</li><li>Quattrini, C., Tavakoli, M., Jeziorska, M., Kallinikos, P., Tesfaye, S., Marshall, A., Finnigan, J., Boulton, A. J. M., Efron, N., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surrogate+Markers+of+Small+Fiber+Damage+in+Human+Diabetic+Neuropathy.">Surrogate Markers of Small Fiber Damage in Human Diabetic Neuropathy.</a> Diabetes. 56, 2148-2154 (2007).</li><li>Mehra, S., Tavakoli, M., Efron, N., Boulton, A. J. M., Augustine, T., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Confocal+Microscopy+Detects+Early+Nerve+Regeneration+After+Pancreas+Transplantation+in+Patients+with+Type+1+Diabetes.">Corneal Confocal Microscopy Detects Early Nerve Regeneration After Pancreas Transplantation in Patients with Type 1 Diabetes.</a> Diabetes Care. 30, 2608-2612 (2007).</li><li>Tavakoli, M., Marshall, A., Pitceathly, R., Fadavi, H., Gow, D., Roberts, M. E., Efron, N., Boulton, A. J. M., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+confocal+microscopy:+A+novel+means+to+detect+nerve+fibre+damage+in+patients+with+impaired+glucose+tolerance+and+idiopathic+small+fibre+neuropathy.">Corneal confocal microscopy: A novel means to detect nerve fibre damage in patients with impaired glucose tolerance and idiopathic small fibre neuropathy.</a> Experimental Neurology. 223, 245-250 (2010).</li><li>Tavakoli, M., Marshall, A., Thompson, L., Kenny, M., Waldek, S., Efron, N., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+confocal+microscopy:+A+novel+technique+to+detect+small-fibre+neuropathy+in+patients+with+Fabry+disease.">Corneal confocal microscopy: A novel technique to detect small-fibre neuropathy in patients with Fabry disease.</a> Muscle &#38; Nerve. 40, 976-984 (2009).</li></ol>

Figures 4 - 6: Courtesy of Heidelberg Engineering

Corneal confocal microscopy (CCM) is a noninvasive clinical technique that may be used to detect early nerve damage and quantify small fibre pathology in peripheral neuropathies including diabetes, Fabry disease and idiopathic small fibre neuropathies (ISFN). The immediate clinical impact of this technique represents a huge leap forward in terms of our ability to diagnose, follow progression and assess therapeutic response in patients with diabetic neuropathy and other peripheral neuropathies and will have an immediate i...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HRT III CCM Machine - The Rostock Cornea Module</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Heidelberg Engineering</td>
</tr>
<tr>
<td>CCM image analysis software v0.6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>University of Manchester</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies at JoVE.com

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

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. ...

corneal-confocal-microscopy-novel-non-invasive-technique-to-quantify

Research

JoVE Journal

Medicine

26.3K Views.  University of Manchester. 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.

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

A Novel Capsulorhexis Technique Using Shearing Forces with Cystotome

Purpose:
To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye.
Method:
Our technique involves creating the initial anterior capsular tear with a cystotome to raise a flap. The flap left unfolded on the lens surface. The cystotome tip is tilted horizontally and is engaged on the flap near the leading edge of the tear. The cystotome is moved in a circular fashion to direct the vector forces. The loose flap is constantly swept towards the centre so that it does not obscure the view on the tearing edge.
Results:
Our technique has the advantage of reducing corneal wound distortion and subsequent anterior chamber collapse. The capsulorhexis flap is moved away from the tear leading edge allowing better visualisation of the direction of tear. This technique offers superior control of the capsulorhexis by allowing the surgeon to change the direction of the tear to achieve the desired capsulorhexis size.
Conclusions:
The EYESI Surgical Simulator is a realistic training platform for surgeons to practice complex capsulorhexis techniques. The shearing forces technique is a suitable alternative and in some cases a far better technique in achieving the desired capsulorhexis.

This video demonstrates a novel capsulorhexis technique in modern cataract surgery. Capsulorhexis is the most difficult and important part of phacoemulsification surgery.

Purpose:
To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye....

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

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.

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 ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

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 ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

A Novel Non-invasive Method for the Detection of Elevated Intra-compartmental Pressures of the Leg

Acute Compartment Syndrome is a devastating consequence of musculoskeletal trauma. Currently the diagnosis is based on clinical signs and symptoms, and while adjuncts such as invasive intra-compartmental pressure measurements are often used to corroborate the physical exam findings, there remains no reliable objective test to aid in the decision to perform a decompressive fasciotomy. In a cadaver model of compartment syndrome, an ultrasound (US) based method has been shown to be a reliable measurement of increased intra-compartmental pressure. An absolute pressure of &#62;100 mbar or a difference of 50 mbar in the CFFP between the legs can be considered pathologic. Using an ultrasound transducer, coupled with a pressure sensor, the pressure needed to flatten the superficial fascia of the anterior compartment of lower legs (Compartment Fascia Flattening Pressure [CFFP]) can be measured. The CFFP of the injured leg is compared to the CFFP of the uninjured leg. This US measured index can then serve as an adjunct to the physical exam in evaluating injured lower extremities and assessing the need for decompressive fasciotomy. The advantages of this protocol include: being a non-invasive method and an easily reproducible technique.

An ultrasound probe coupled with a pressure sensor is used to assess the intra-compartmental pressure of the leg by directly measuring the compartment fascia flattening pressure (CFFP). This non-invasive protocol will provide reliable assessment of the pressure inside the anterior muscular compartment of the lower leg.

Acute Compartment Syndrome is a devastating consequence of musculoskeletal trauma. Currently the diagnosis is based on clinical signs and symptoms, and ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...