The authors would like to thank Zeiss Mexico for the unrestricted support to use the Cirrus 6000 with AngioPlex equipment.

This protocol was approved by Sala Uno&#39;s human research ethics committee. See Video 1 for sections 1 and 2 and the Table of Materials for details about the equipment used in this study.
1. Retinal analysis in the OCTA device
<ol>
	<li>Select the menu for retinal analysis in the OCTA device.</li>
	<li>Select a 3 x 3 mm retinal map; select superficial if the OCTA device measures different capillary plexuses.</li>
	<li>Select vessel length density (or its equivalent, e.g., skeletonized vascular density).</li>
	<li>Measure vessel length density in mm-1 in a 3 x 3 mm retinal map. 
	NOTE: The map is divided into two regions: center (within a 1 mm circle, concentric to the foveal center) and inner (outside the 1 mm center circle, Figure 3). The equipment also measures a full density (within the 3 mm circle) and subdivides the inner region into four fields: superior, inferior, temporal, and nasal (Figure 4). Each region is specified so that the vessel length densities are measured automatically. The instruments display the values for center, inner, and full densities and for superior, temporal, inferior, and nasal fields of the inner density.</li>
	<li>Return to the menu for retinal analysis.</li>
	<li>Select a 3 x 3 mm retinal map; select superficial if the OCTA device measures different capillary plexuses.</li>
	<li>Select perfusion density (or its equivalent, e.g., vessel density).</li>
	<li>Measure perfusion density in % in a 3 x 3 mm retinal map. 
	NOTE: The map is divided into two regions: center (within a 1 mm circle, concentric to the foveal center) and inner (outside the 1 mm center circle). The equipment also measures a full density (within the 3 mm circle) and subdivides the inner region into four fields: superior, inferior, temporal, and nasal. Each region is specified so that the perfusion densities are measured automatically. The instruments display the values for center, inner, and full densities and for superior, temporal, inferior, and nasal fields of the inner density.</li>
	<li>Verify that the density maps have a signal strength &#62; 7; then, verify that the maps have no measurement errors resulting from artifacts or eye movements.</li>
	<li>Register the values of center vessel length density, center perfusion density, inner vessel length density, inner perfusion density, superior vessel length density, superior perfusion density, inferior vessel length density, inferior perfusion density, temporal vessel length density, temporal perfusion density, nasal vessel length density, and nasal perfusion density in a spreadsheet.</li>
</ol>
2. Calculation of the coefficients of determination using a spreadsheet
<ol>
	<li>Select the variables to be evaluated (e.g., center vessel length density and center perfusion density). Select the values of both variables for a defined group (e.g., group 1).</li>
	<li>In the toolbar, click on insert.</li>
	<li>Click on the recommended charts button in the graphs section. Wait for a scatter chart to appear as a suggestion in a window. Click the OK button to accept the suggestion.</li>
	<li>Inspect the scatter chart of the data. Right-click on the series to display an options menu.</li>
	<li>Select the add trendline option. Wait for a linear trendline to be added to the chart and for a menu on the right side of the screen.</li>
	<li>Displace the menu downwards to find the Display R-squared value on chart option. Select this option to display the R-squared value on the chart. Select the R-squared-value.</li>
	<li>Select Home on the toolbar and then click on the copy button.</li>
	<li>Prepare a chart of coefficients of determination on a new page.</li>
	<li>Select a destination cell (e.g., center coefficient of determination for group 1). Click on the right mouse button. Select paste with keep source formatting.</li>
	<li>Prepare a new chart to show the percentage of perfusion density changes explained by changes in vessel density.</li>
	<li>Select the cell with the coefficient of determination in the previous chart. Click on the right mouse button. Select copy.</li>
	<li>Select a destination cell in the new chart (e.g., center in group 1). Click on the right mouse button. Select paste.</li>
	<li>Select the cell with the pasted value; then, in the toolbar, select home | percent style in the number menu.</li>
	<li>Select increase decimal in the number menu and click it once. 
	NOTE: The resulting number is the percentage of changes in perfusion density explained by the changes in vessel density.</li>
	<li>Prepare another table to show the percentage of perfusion density explained by the changes in vessels larger than capillaries.</li>
	<li>Select a destination cell (e.g., center in group 1). Subtract the last result from 1.</li>
	<li>Select this cell. Select home in the toolbar.</li>
	<li>Select percent style in the number menu.</li>
	<li>Click once on increase decimals in the number menu.</li>
	<li>Format the charts to display the contribution of capillaries (vessel density) and vessels larger than capillaries to the changes in perfusion density.</li>
	<li>Repeat the procedure to obtain the values of inner vessel/perfusion densities and superior, inferior, temporal, and nasal vessel/perfusion densities in group 3.</li>
</ol>
3. Comparison of the coefficients of determination
<ol>
	<li>Compare the coefficients of determination in three groups: 1, healthy people; 2, patients with arterial hypertension without hypertensive retinopathy; and 3, patients with type 2 diabetes mellitus without diabetic retinopathy. In group 3, also compare the coefficients of determination between fields: superior, inferior, temporal, and nasal.</li>
</ol>
4. Compare the percentage differences in the contribution of capillaries and vessels larger than capillaries to perfusion density, between groups and between fields in group 3

<ol>
	<li>Ong, J. X., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+diabetic+retinopathy+from+advanced+retinal+vascular+imaging.">Perspectives on diabetic retinopathy from advanced retinal vascular imaging.</a> Eye. , (2022).</li><li>Tan, A. C. 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=An+overview+of+the+clinical+applications+of+optical+coherence+tomography+angiography.">An overview of the clinical applications of optical coherence tomography angiography.</a> Eye. 32 (2), 262-286 (2018).</li><li>Elnahry, A. G., Ramsey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+angiography+imaging+of+the+retinal+microvasculature+is+unimpeded+by+macula+xanthophyll+pigment.">Optical coherence tomography angiography imaging of the retinal microvasculature is unimpeded by macula xanthophyll pigment.</a> Clinical and Experimental Ophthalmology. 48 (7), 1012-1014 (2020).</li><li>Elnahry, A. G., Ramsey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image+alignment+for+comparing+microvascular+changes+detected+by+fluorescein+angiography+and+optical+coherence+tomography+angiography+in+diabetic+retinopathy.">Automated image alignment for comparing microvascular changes detected by fluorescein angiography and optical coherence tomography angiography in diabetic retinopathy.</a> Seminars in Ophthalmology. 36 (8), 757-764 (2021).</li><li>Rosenfeld, P. J., 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=Zeiss+AngioPlex+spectral+domain+optical+coherence+tomography+angiography:+technical+aspects.">Zeiss AngioPlex spectral domain optical coherence tomography angiography: technical aspects.</a> Developments in Ophthalmology. 56, 18-29 (2016).</li><li>Nesper, P. 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=Hemodynamic+response+of+the+three+macular+capillary+plexuses+in+dark+adaptation+and+flicker+stimulation+using+optical+coherence+tomography+angiography.">Hemodynamic response of the three macular capillary plexuses in dark adaptation and flicker stimulation using optical coherence tomography angiography.</a> Investigative Ophthalmology and Visual Science. 60 (2), 694-703 (2019).</li><li>Zhang, Y. S., Lee, H. E., Kwan, C. C., Schwartz, G. W., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+delays+retinal+neurovascular+coupling+during+dark+to+light+adaptation+in+healthy+eyes+revealed+by+optical+coherence+tomography+angiography.">Caffeine delays retinal neurovascular coupling during dark to light adaptation in healthy eyes revealed by optical coherence tomography angiography.</a> Investigative Ophthalmology and Visual Science. 61 (4), 37 (2020).</li><li>Barraso, 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=Optical+coherence+tomography+angiography+in+type+1+diabetes+mellitus.+Report+1:+Diabetic+Retinopathy.">Optical coherence tomography angiography in type 1 diabetes mellitus. Report 1: Diabetic Retinopathy.</a> Translational Vision Science and Technology. 9, 34 (2020).</li><li>Xu, Q., Sun, H., Huang, X., Qu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+microvascular+metrics+in+untreated+essential+hypertensives+using+optical+coherence+tomography+angiography.">Retinal microvascular metrics in untreated essential hypertensives using optical coherence tomography angiography.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 259 (2), 395-403 (2021).</li><li>Yeh, R. Y., Nischal, K. K., LeDuc, P., Cagan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Written+in+blood:+applying+grammars+to+retinal+vasculatures.">Written in blood: applying grammars to retinal vasculatures.</a> Translational Vision Science &amp; Technology. 9, 36 (2020).</li><li>Corvi, F., Sadda, S. R., Staurenghi, G., Pellegrini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thresholding+strategies+to+measure+vessel+density+by+optical+coherence+tomography+angiography.">Thresholding strategies to measure vessel density by optical coherence tomography angiography.</a> Canadian Journal of Ophthalmology. 55 (4), 317-322 (2020).</li><li>Pathak, P., Panday, S. B., Ahn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+neural+network+model+effectively+estimates+muscle+and+fat+mass+using+simple+demographic+and+anthropometric+measures.">Artificial neural network model effectively estimates muscle and fat mass using simple demographic and anthropometric measures.</a> Clinical Nutrition. 41 (1), 144-152 (2022).</li><li>OFee, K., Deych, E., Ciani, O., Brown, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+nonfatal+myocardial+infarction+as+a+surrogate+for+all-cause+and+cardiovascular+mortality+in+treatment+or+prevention+of+coronary+artery+disease:+a+meta-analysis+of+randomized+clinical+trials.">Assessment of nonfatal myocardial infarction as a surrogate for all-cause and cardiovascular mortality in treatment or prevention of coronary artery disease: a meta-analysis of randomized clinical trials.</a> JAMA Internal Medicine. 181 (12), 1575-1587 (2021).</li><li>Kushner-Lenhoff, S., Ashimatey, B. S., Kashani, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+reactivity+as+assessed+by+optical+coherence+tomography+angiography.">Retinal vascular reactivity as assessed by optical coherence tomography angiography.</a> Journal of Visualized Experiments: JoVE. (157), e60948 (2020).</li><li>Sousa, D. C., 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=A+protocol+to+evaluate+retinal+vascular+response+using+optical+coherence+tomography+angiography.">A protocol to evaluate retinal vascular response using optical coherence tomography angiography.</a> Frontiers in Neuroscience. 13, 566 (2019).</li><li>Falavarjani, K. G., 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=Effect+of+segmentation+error+correction+on+optical+coherence+tomography+angiography+measurements+in+healthy+subjects+and+diabetic+macular+oedema.">Effect of segmentation error correction on optical coherence tomography angiography measurements in healthy subjects and diabetic macular oedema.</a> British Journal of Ophthalmology. 104 (2), 162-166 (2020).</li><li>Warner, R. 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=Full-field+flicker+evoked+changes+in+parafoveal+retinal+blood+flow.">Full-field flicker evoked changes in parafoveal retinal blood flow.</a> Scientific Reports. 10 (1), 16051 (2020).</li><li>Zhang, Y. 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=Reversed+neurovascular+coupling+on+optical+coherence+tomography+is+the+earliest+detectable+abnormality+before+clinical+diabetic+retinopathy.">Reversed neurovascular coupling on optical coherence tomography is the earliest detectable abnormality before clinical diabetic retinopathy.</a> Journal of Clinical Medicine. 9 (11), 3523 (2020).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

The contribution of vessels larger than capillaries to perfusion density changes in retinal vascular diseases before the development of retinopathy. It decreased in the inner region of patients with arterial hypertension and varied between fields in patients with diabetes. There are direct methods for measuring vascular reactivity in the retina, which depend on the exposure to a stimulus14,15. The measurement proposed in this paper uses two metrics, automatically...

Retinal circulation is the combination of arteriolar, capillary, and venular flow, whose contribution can vary to meet the oxygen needs of the different retinal layers. This circulation does not depend on the autonomous nervous system regulation and has been traditionally evaluated with fluorescein angiography, an invasive method that uses intravenous contrast to delineate retinal vessels. Sequential photographs allow the evaluation of arterial, arteriolar, venular, and venous circulation, as well as sites of capillary damage in retinal vascular diseases1.
A current method to measure the macular circulation is optical coherence tomography angiography (OCTA), which uses interferometry to obtain retinal images and can outline capillaries and larger retinal vessels2. Unlike fluorescein angiography, OCTA imaging is not influenced by macular xanthophyll pigment shadowing, allowing superior imaging of macular capillaries3. Other advantages of OCTA over fluorescein angiography are its noninvasiveness and higher resolution4.
OCTA devices measure the superficial capillary plexus at the parafovea in a 3 x 3 mm map, concentric to the foveal center (Figure 1). The equipment automatically measures vessel length density (the length of capillaries with circulation in the measured area) and perfusion density (the percentage of the measured area with circulation), which includes that of vessels larger than capillaries (Figure 2)5. Vessel density has a substantial contribution to perfusion density under physiological conditions. Some devices measure vessel density as a &#34;skeletonized vascular density&#34; and perfusion density as &#34;vessel/vascular density.&#34; Regardless of the device, there is usually a measurement for length (measured in mm/mm2 or mm-1) and another for the area with circulation (measured in %), which are generated automatically.
Vessel density can change in healthy people when exposed to darkness, flicker light6, or caffeinated drinks7 because of the neurovascular coupling that redistributes blood flow between the superficial, middle, and deep capillary plexuses according to the retinal layer with the highest activity. Any decrease in vessel density caused by this redistribution returns to baseline values after the stimulus ceases and does not represent capillary loss, a pathological change reported before retinopathy appears in vascular diseases such as diabetes8 or arterial hypertension9.
The decrease in capillaries could be compensated partially by arteriolar vasodilatation. Measuring only a percentage or perfused area does not provide any insight into whether there is vasodilatation, which can appear when capillaries reach a minimum threshold. Measuring vessel density would not help detect an increased circulation area resulting from vasodilatation. The contribution of arteriolar circulation to perfusion density can be estimated indirectly using a coefficient of determination between vessel density and perfusion density, and defining the percentage of the area with circulation that corresponds to capillaries or other vessels.
The rationale behind this technique is that regression analysis can identify the extent to which the changes of an independent numeric value result in changes of a dependent numeric value. In macular vessel imaging using OCTA, capillary circulation is an independent variable that influences the area with circulation because there are few larger vessels in the evaluated region. However, the parafovea has larger vessels that can dilate and change the percentage of the area with circulation, which cannot be identified directly by the current automated OCTA metrics. The advantage of using a coefficient of determination is that it measures a relationship between two existing metrics to produce two more: the percentage of the area with circulation that corresponds to capillaries, and the percentage that corresponds to other vessels. Both percentages can be measured directly using a pixel count with imaging software. However, the coefficient of determination can be calculated for a sample with the numbers that the OCTA devices generate automatically10,11.
Pathak et al. used a coefficient of determination to estimate lean muscle and fat mass from demographic and anthropometric measures using an artificial neural network. Their study found that their model had an R2 value of 0.92, which explained the variability of a large portion of their dependent variables12. O&#39;Fee and colleagues used a coefficient of determination to rule out nonfatal myocardial infarction as a surrogate for all-cause and cardiovascular mortality because they found an R2 of 0.01 to 0.21. Those results showed that the independent variable explained less than 80% of the changes of the dependent variables, set as a criterion of surrogacy (R2= 0.8)13.
The coefficient of determination is used to assess the effect of changes of a variable, a group of variables, or a model over the changes of an outcome variable. The difference between 1 and the R2 value represents the contribution of other variables to the changes of the outcome variable. It is uncommon to attribute the difference to a single variable because there are usually more than two contributing to the outcome. However, the proportion of the macular area that has circulation can only originate from the area covered by capillaries and from that covered by larger vessels, as larger vessels dilate more than capillaries. Moreover, reactive vasodilation is considered to most probably originate from retinal arterioles, because a reduced capillary circulation could decrease oxygen supply.
Only two sources contribute to a percentage of area with circulation in the macula: capillaries and vessels larger than them. The coefficient of determination between vessel density and perfusion density determines the contribution of capillaries to the area with circulation, and the remaining changes (the difference between 1 and the R2 value) represent the contribution of the only other variable that represents an area with circulation (that within larger retinal vessels). This paper describes the method of measuring this contribution in healthy people (group 1) and how it changes in patients with retinal vascular diseases: arterial hypertension without hypertensive retinopathy (group 2) and diabetes mellitus without diabetic retinopathy (group 3).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cirrus 6000 with Angioplex</td>
			<td>Carl Zeiss Meditec Inc., Dublin CA</td>
			<td>N/A</td>
			<td>3 x 3 vessel and perfusion density maps</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>spreadsheet</td>
		</tr>
		<tr>
			<td>Personal computer</td>
			<td>Generic</td>
			<td>N/A</td>
			<td>for running the calculations on the spreadsheet</td>
		</tr>
	</tbody>
</table>

evaluation of capillary and other vessel contribution to macular perfusion density measured with optical coherence tomography angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

There were 45 subjects in group 1, 18 in group 2, and 36 in group 3. Table 1 shows the distribution of age and densities by group; only vessel and perfusion densities in group 1 were lower than in group 2. The coefficients of determination of center vessel and perfusion densities are shown in Figure 5. There was no significant difference between the groups.
The coefficient of determination between the inner vessel and perfusion densities was 0.818...

Watch this Scientific Journal Video about Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography at JoVE.com

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

evaluation-capillary-other-vessel-contribution-to-macular-perfusion

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1.7K Views.  Sala Uno Opthalmology Clinic. We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Video: Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&amp;#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient&#8217;s microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.1 This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...